Non-Coding RNA doi: 10.3390/ncrna12050039

Authors: Zhangchi Xu Rong Pan Tianzhou Ma

Background/Objectives: Non-coding RNAs (ncRNAs) play important roles in various biological processes, primarily via regulating gene expression at multiple levels. Nevertheless, compared to protein-coding genes, ncRNAs are relatively understudied. They are characterized by low expression, large variation, and more dynamic expression across different tissues and cell types, which creates unique challenges in differential expression (DE) analysis. Methods: We propose the CQRM, an adaptive multi-quantile testing framework for ncRNA count data that incorporates jittering to accommodate discrete counts, combines dependent evidence across quantiles, and selects the most informative quantile subset for distinguishing conditions. Results: The CQRM captures diverse expression differences, including changes in variability, skewness, and modality beyond mean shifts. Model-based simulations showed that the CQRM improved the detection of differentially expressed ncRNAs while controlling the false discovery rate relative to existing DE tools, especially when DE genes exhibited multimodal expression patterns and counts were low. Critical ncRNAs and biologically interpretable targeted pathways were identified when we further applied our method to a Smart-seq-total single-cell RNA-seq dataset and a human organ developmental RNA-seq dataset. Conclusions: CQRM complements mean-based DE methods by capturing heterogeneous ncRNA expression changes, supporting the identification of candidate ncRNA biomarkers and regulatory processes relevant to development and disease.

Non-Coding RNA doi: 10.3390/ncrna12050038

Authors: Jane L. Zen Quynh Mai Nguyen Le Shizhou Li George B. Hanna

Skin-derived extracellular nucleic acids—particularly non-coding RNAs (ncRNAs) such as miRNAs and lncRNAs—are a rapidly evolving frontier in molecular diagnostics. These molecules are present in skin tissue, dermal interstitial fluid, and sweat, whilst ncRNAs regulate essential processes including epidermal differentiation, wound healing, immune regulation, and tumourigenesis, positioning them as promising candidates for minimally invasive biomarker development across a broad spectrum of dermatological diseases. This narrative review summarises their biological functions and potential clinical applications. The available evidence provides a conceptual basis for an emerging cutaneous “surface liquid biopsy” framework, whilst highlighting the need for standardised sampling, analytical validation, and clinical evaluation before such approaches can be translated into clinical practice. Although ample disease-associated ncRNA signatures have been uncovered, many remain at exploratory or early translational stages, and the concordance of these ncRNA signatures with corresponding blood and tissue biomarkers needs to be established. Continued advances in sampling strategies, analytical technologies, and clinical validation are needed to translate these findings into clinically deployable platforms for dermatological diseases and, potentially, systemic diseases in the future.

Non-Coding RNA doi: 10.3390/ncrna12050037

Authors: Min S. Kwon Hee K. Chung Hongxia Chen Amy VanderStoep Bridgette Warner Haonan Zhao Prathik Atluri Emnet Tesfayohannes Douglas J. Turner Lan Xiao Jian-Ying Wang

Background/Objectives: Intestinal mucosal function relies on the integrated contribution of multiple cell lineages, including secretory cells. Long noncoding RNA H19 modulates many cell processes essential for human pathologies. Here, we investigated the role of H19 in development and function of secretory lineage cells and elucidated the implication of altered ATOH1 by H19 in dysfunction of the secretory cells. Methods: Studies were conducted in H19-transgenic (H19-Tg) and H19-knockout (H19−/−) mice, intestinal organoids, and Caco-2 cells. Secretory lineage cells were examined by fluorescent immunostaining with their specific cell markers, including Lysozyme, Mucin2, DCLK1, and Chromogranin A. Intestinal mucosal growth was measured by BrdU incorporation, and intestinal permeability was detected by paracellular tracer flux assay using FITC-Dextran. Single cell RNA-sequence, quantitative real-time PCR, and Western immunoblotting analyses were used to monitor changes in gene expression. Results: Tissue-specific transgenic expression of H19 in the intestinal epithelium caused defects in Paneth, goblet, tuft, and enteroendocrine cells in mice, whereas targeted deletion of the H19 gene increased the function of secretory lineage cells. Locally increasing the levels of H19 in the epithelium impaired gut barrier function in H19-Tg mice, although it failed to alter mucosal growth. The intestinal mucosal tissues of H19-Tg mice exhibited a remarkable decrease in the levels of differentiation-associated transcription factors including ATOH1. Ectopically expressed ATOH1 in the H19-enriched intestinal organoids derived from H19-Tg mice rescued development of the secretory lineage cells. Mechanistically, H19 inhibited ATOH1 expression via inhibition of its translation. Conclusions: These findings indicate that H19 functions as a negative regulator of differentiation and function of secretory lineage cells in the intestinal mucosa at least partially via the inhibition of ATOH1 translation.

Non-Coding RNA doi: 10.3390/ncrna12050036

Authors: Minseon Kim Seung-Kyoon Kim Jaeil Han

Background: Long non-coding RNAs (lncRNAs) are widely proposed as determinants of temozolomide response in glioblastoma (GBM), but many nominations rest on analyst-derived survival endpoints, screens unadjusted for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, and models evaluated with the same data used to select them. We asked which lncRNA–outcome associations remain when endpoint definition, confounder adjustment, and external validation are addressed explicitly. Methods: We analyzed 94 IDH-wildtype, temozolomide-treated primary GBM patients from The Cancer Genome Atlas (TCGA), using the rule-based progression-free interval (PFI) of the TCGA Pan-Cancer Clinical Data Resource and overall survival (OS) as separate endpoints. A joint transcriptome-wide discovery for OS was performed across TCGA and the identically processed CPTAC-3 cohort (n = 282, 209 deaths), the latter unselected for IDH status because IDH annotation is unavailable for CPTAC-3, with adjustment for MGMT promoter methylation status, cellular composition, and expression subtype. Nominated transcripts were tested for external validation in two independent CGGA cohorts, and incremental discrimination was assessed as the optimism-corrected change in C-index (ΔC-index), benchmarked against the gain expected from a transcriptome-wide search alone. Results: No lncRNA was associated with PFI at a transcriptome-wide false-discovery rate (FDR) below 0.05. The joint OS discovery nominated DIO3OS, the only transcript reaching transcriptome-wide significance (hazard ratio 1.38 per standard deviation, 95% confidence interval 1.20–1.58, FDR 0.022), and C9orf139 (FDR 0.065); both were independently significant in each discovery cohort and robust to adjustment for MGMT status, cellular composition, and expression subtype. Both, however, were null with reversed point estimates in the two CGGA cohorts, were unassociated with PFI, and added only modest optimism-corrected discrimination (ΔC-index 0.07–0.11), close to what a transcriptome-wide search alone generates. Replication was thus confined to two cohorts sharing a processing pipeline. Conclusions: We nominate DIO3OS and C9orf139 as candidate survival-associated lncRNAs in GBM, requiring prospective evaluation in cohorts with complete IDH annotation and progression endpoints, and show how endpoint definition, selection optimism, and external validation determine which lncRNA associations survive scrutiny.

Non-Coding RNA doi: 10.3390/ncrna12050035

Authors: Małgorzata Litwiniuk-Kosmala Maria Makuszewska Robert Bartoszewicz Agnieszka Jasińska-Nowacka Maciej Ołdak Bartłomiej Gielniewski Bartosz Wojtaś Kazimierz Niemczyk

Background/Objectives: This study aimed to identify a characteristic miRNA expression profile in the CSF of patients diagnosed with vestibular schwannoma and evaluate its potential for tumor assessment. Methods: In this prospective study, 17 CSF and corresponding tumor samples (seven small tumors—SVS and 10 large tumors—LVS) were collected from patients operated on for VS in a Tertiary Academic Center. The miRNA expression was analyzed using high-throughput RNA sequencing (NovaSeq 6000 Illumina). Data were normalized, and a comparative analysis of miRNA expression rankings was performed between VS patients and a public healthy donor dataset. Functional implications were explored using KEGG pathway enrichment analysis. Results: A total of 1633 miRNAs were identified in all CSF samples derived from VS patients. Comparison with healthy donors revealed a moderate ranking correlation (ρ = 0.39), with significant shifts for specific molecules like hsa-miR-766-3p and hsa-miR-182-5p. Only six miRNAs were found to correlate between CSF and tumor tissue, while 16 exhibited a negative correlation. No statistical correlation was found between tumor size and the CSF miRNA profile. KEGG analysis highlighted enriched pathways, including neurotrophin signaling and focal adhesion. Conclusions: The results of our study support the feasibility of miRNA-based CSF liquid biopsy for VS assessment. However, the results of miRNA expression profiling conducted in tumor tissue cannot be directly transferred into CSF sample analyses. Further studies are warranted to explain this phenomenon and to search for reliable miRNA markers of VS progression in the CSF liquid biopsy specimens.

Non-Coding RNA doi: 10.3390/ncrna12050034

Authors: Ezgi Biltekin Bulent Ozpolat

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as critical regulators of gene expression and key cellular processes in cancer, including tumor cell proliferation, survival, metastasis, and both innate and adaptive immune responses within the tumor microenvironment. Through their ability to modulate immune cell development, differentiation, polarization, antigen presentation, cytokine signaling and immune checkpoint expression, miRNAs play a vital role in shaping immune responses and represent attractive therapeutic candidates for remodeling the tumor immune microenvironment. This review provides a comprehensive overview of the mechanisms through which miRNAs regulate anti-tumor immunity and contribute to immune evasion with particular emphasis on their role in modulating major immune checkpoint pathways, including PD-1/PD-L1, CTLA-4, LAG-3, TIM-3 and CD28-mediated signaling. We further examine the contribution of miRNAs to therapeutic resistance and discuss their potential integration with immune checkpoint blockade, chemotherapy, PARP inhibitors and targeted therapies to enhance antitumor efficacy and overcome treatment resistance. Recent advances in spatial transcriptomics and computational approaches have expanded our understanding of miRNA-mediated regulatory networks at single-cell and tissue levels and provide new insights into tumor–immune interactions. In addition, we evaluate the current progress in the clinical development of miRNA-based therapeutics, including miRNA mimics and inhibitors and discuss the major challenges associated with efficient delivery, off-target effects, immunogenicity and patient heterogeneity. Collectively, the evidence highlights the growing potential of miRNAs as diagnostic biomarkers, therapeutic targets and therapeutic tools in cancer immunotherapy. Continued advances in RNA therapeutics, delivery technologies and multi-omics approaches are expected to accelerate the clinical translation of miRNA-based strategies for personalized cancer therapy.

Non-Coding RNA doi: 10.3390/ncrna12050033

Authors: Monyse Nobrega Hellen Kuasne Marilesia Ferreira de Souza Mariana Bisarro dos Reis Larissa Cristina Bastos de Oliveira Paulo Emilio Fuganti Morag Park Ilce Mara de Syllos Cólus

Background/Objectives: Prostate cancer (PCa) has a high incidence worldwide, yet reliable prognostic biomarkers to distinguish indolent from aggressive forms are lacking. MicroRNAs (miRNAs), a class of small non-coding RNAs, are stable molecules that play an important role in cancer development and can be detected in circulation by non-invasive methods. This study aimed to examine the influence of the cell-free (cf) miRNAs levels on PCa risk and aggressiveness using plasma samples and cell models. Methods: Four overexpressed miRNAs in prostate cancer tissue were selected from The Cancer Genome Atlas database: miR-25-3p, miR-92a-1-5p, miR-92a-2-5p, and miR-148a-3p. Cf-miRNA levels were validated via quantitative reverse transcription polymerase chain reaction (RT-qPCR) in plasma samples from 80 PCa patients and 40 controls in a Brazilian cohort. Clinicopathological associations were analyzed to determine prognostic value. Subsequently, functional analyses were performed using transient transfection of miR-25-3p in the LNCaP cell line, assessing proliferation, migration, invasion, and target gene regulation. Results: Among the miRNAs analyzed, only miR-25-3p was upregulated in the plasma of PCa patients compared to controls (p = 0.013). Patients with International Society of Urological Pathology (ISUP) grade ≥2 presented higher expression levels of miR-25-3p (p = 0.020) compared to patients with ISUP grade 1. Overexpression of miR-25-3p significantly increased cell proliferation, colony formation, migration, and invasion capacity and modulated the expression of BCL2L11, CDH1, CDKN1C, EHZ2, and TP53 genes. Conclusions: miR-25-3p had an oncogenic role in PCa, showing a potential marker to assess PCa development and progression using liquid biopsy.

Non-Coding RNA doi: 10.3390/ncrna12050032

Authors: Haiyu Su Daiju Tao Jia Teng Li Zhang Ying Shen Renhua Yang Jiarui Yang Rongji Sun Zhiqiang Shen Peng Chen Bo He

Background: Cerebrovascular diseases have complex pathogenesis and pose a serious threat to human health; thus, novel diagnostic and therapeutic strategies are needed. Small extracellular vesicles (sEVs), commonly referred to as exosomes, are 30–150 nm lipid-bilayer vesicles that shield long noncoding RNAs (lncRNAs) from degradation. Because exosomal lncRNAs are more stable than free lncRNAs in blood and cerebrospinal fluid and can cross the blood–brain barrier, they are promising as biomarkers and therapeutic vectors. This review summarizes the roles and mechanisms of exosomal lncRNAs in cerebrovascular diseases. Methods: This narrative review is based on the experimental literature and focuses on the biological functions and regulatory mechanisms of exosomal lncRNAs in cerebrovascular disorders. Results: As competing endogenous RNAs (ceRNAs), they sequester microRNAs (miRNAs), thereby derepressing downstream target-gene expression and modulating neuronal injury (oxidative stress, apoptosis, neuroinflammation) through the nuclear factor kappa-B (NF-κB) and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathways. They show altered profiles in acute stroke (ischemic/hemorrhagic) correlated with neurological deficits, and are relevant to the early diagnosis of chronic diseases (atherosclerosis, aneurysm) and vascular dementia. Conclusions: Exosomal lncRNAs demonstrate promising translational potential in preclinical studies because they combine exosome delivery capabilities with lncRNA regulatory functions, although clinical validation remains limited.

Non-Coding RNA doi: 10.3390/ncrna12040031

Authors: Tatiana Zuppelli Laura Orlando Giuseppe N. Conti Sofia P. Lombardo Lucia Longhitano Giulia Chisari Cesarina Giallongo Francesco Bellia Maria Gabriella Sabini Stefania Mele Ignazio A. Barbagallo Nunzio Vicario Rosalba Parenti Michelino Di Rosa Enrico La Spina Stefano Forte Daniele Tibullo Giovanni Li Volti

Background/Objectives: Radiotherapy (RT) is a fundamental treatment for brain tumors but often leads to long-term neurotoxicity, partly caused by microglial dysfunction. In this study, we used long-read RNA sequencing (LR-RNA-seq) to map the isoform-specific transcriptome of human microglial HMC3 cells after RT exposure. Methods: The HMC3 human microglial cell line was treated with standard radiotherapy and, after RNA extraction and library preparation, LR-RNA-seq was performed. Further bioinformatic analyses allowed to investigate on the isoform switching pattern after radiotherapy exposure. Results: Gene-level analysis found 591 differentially expressed genes, with positive enrichment of PI3K/AKT-related pathways and negative enrichment of translation-associated processes. Isoform-level analysis uncovered 827 significant isoform switching events across 420 genes, often involving shifts from protein-coding transcripts to non-coding variants. Functional annotation showed that most isoform switches resulted in the loss of open reading frames and protein domains, especially in ribosomal proteins and translation machinery components. Exploratory proteomic analysis identified 191 differentially abundant proteins, including a significant reduction in RPL7A, providing partial protein-level support for translation-associated transcriptomic remodeling. Conclusions: RT-induced stress causes qualitative transcriptomic remodeling beyond changes in overall gene expression. These findings indicate that radiotherapy induces coordinated gene-, transcript-, and isoform-level changes in HMC3 cells. The alterations observed in translation-associated genes, together with partial proteomic concordance, identify candidate mechanisms for future functional investigation.

Non-Coding RNA doi: 10.3390/ncrna12040030

Authors: Yogesh Vetriselvan Jayakumar Swetha Manoranjani Murugan Irisappan Ganesh Vishnu Bhat Ballambattu Pushpa Premanath Kotur Deepa Shanmugam Marcella Sherin Samuel Sambandam Ravikumar

Background: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that affects women of reproductive age, often associated with metabolic issues, causing infertility. Although it is common, the cause of PCOS remains unknown, and early diagnosis is challenging. MicroRNAs (miRNAs) serve as post-transcriptional regulators of gene expression, play a significant role in PCOS development, and have emerged as biomarkers for reproductive and metabolic disorders. However, research on miRNA signatures in the Indian population is limited. This pilot exploratory study aims to compare the candidate differentially expressed miRNAs (DE miRNAs) in serum samples from individuals with and without PCOS using high-throughput miRNA sequencing. Methods: In the study, patients with PCOS and age-matched controls were included; small RNAs were isolated from their serum, libraries were prepared, and the libraries were analyzed by next-generation sequencing. Bioinformatics analysis, including miRBase annotation, differential expression analysis, target prediction, GO/KEGG enrichment, and hub gene network analysis, was performed. Results: A total of 967 miRNAs were identified, with seven showing differential expression (log2FC > 1, p < 0.05). Among these, six miRNAs of hsa-miR-219a-2-3p, hsa-mir-384, hsa-miR-149-5p, hsa-miR-3182, hsa-miR-3960, and hsa-miR-4508 were upregulated, whereas hsa-mir-139 was downregulated. Functional enrichment and hub gene analyses identified key targets, including TP53, FOXO1, HIF1A, and HDAC1, that are crucial to the cell cycle, insulin signaling, and hypoxia. Conclusions: These preliminary findings identify candidate serum miRNA signatures in Indian women that may be associated with PCOS’s reproductive and metabolic issues. These pilot study results suggest potential miRNAs and their target pathway links with PCOS that need validation in larger cohorts for diagnostic or therapeutic applications.

Non-Coding RNA doi: 10.3390/ncrna12040029

Authors: Siddhartha Shah Tess Cherlin Yi Jing Stepan Nersisyan Benjamin Leiby Isidore Rigoutsos

Small non-coding RNAs (sncRNAs) have garnered considerable attention in recent years, following accumulating evidence of their critical roles in many cellular processes. Among sncRNAs, microRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), rRNA-derived fragments (rRFs), and Y RNA-derived fragments (yRFs) account for more than 95% of all sncRNAs found in cells. Despite their critical regulatory roles, most sncRNAs remain uncharacterized because their functionalization is a lengthy and challenging undertaking. Knowing an sncRNA’s abundance helps prioritize among the various choices, while knowing where it localizes in the cell greatly limits the number and identity of its potential targets and helps understand its function. Most studies to date have assumed that the subcellular localization of the various sncRNA classes is understood and remains unchanged across cell types. However, as we have recently demonstrated, the subcellular distribution of sncRNAs follows complex patterns that depend on the sequence of the sncRNA, the presence or absence of post-transcriptionally added non-templated nucleotides, and the cell type. To determine the subcellular localization and abundance of sncRNAs and aid the design of targeted experimental studies of sncRNA function, we developed “Fraction-seq.” The method combines an experimental and an analytical component to remove cross-fraction contamination and reconstruct the true abundance of sncRNAs in each considered fraction. Fraction-seq can be applied to any adherent cells from any organism without modifications and can reconstruct the abundance of all categories of sncRNAs. Accompanying this detailed protocol is a newly developed port of the original SAS codes to the widely used R programming language. The codes and an example are freely available through our center’s GitHub page.

Non-Coding RNA doi: 10.3390/ncrna12040028

Authors: Wiebke Kochendörfer Klaus Förstemann

Background: The RNA interference (RNAi) pathway is a highly conserved antiviral mechanism in eukaryotes, including insects such as fruit flies. Many viruses have therefore evolved mechanisms that protect their transcripts and/or genomes; how effectively RNAi can act is thus a question that must be answered case-by-case. Methods: We demonstrate that the RNAi machinery can successfully combat Drosophila A virus (DAV) in persistently infected Drosophila melanogaster S2 cell lines, but only when supported with exogenous triggers. Since such an infection poses a challenge to the fitness of cells and the reproducibility of results obtained with them, our approach provides a convenient method to recover precious experimentally modified cell lines from infection through the application of exogenous double-stranded RNA (dsRNA) targeting the DAV sequence. The in vitro transcribed dsRNAs were applied to infected S2 cells via direct “soaking,” utilizing the cells’ natural endocytic uptake. Results: We monitored treatment efficacy via RT-PCR-based detection of viral sequences, and our results demonstrate that continued application of DAV-specific dsRNAs led to a reduction in viral abundance and all eight tested cell lines appeared DAV-negative within several weeks of treatment. While a set of cell lines exhibited viral recurrence several weeks after the cessation of treatment, others remained virus-free for at least 16 weeks, suggesting that a permanent “cure” can indeed be achieved. Notably, while RNAi activation effectively cleared DAV in some cell lines, no beneficial effect was observed for a concomitantly applied Drosophila X-virus (DXV) treatment in the co-infected cultures. Conclusions: This suggests that DXV has more efficient evasion mechanisms but cannot protect DAV in trans. Our protocol thus provides a robust, scalable approach for clearing persistent DAV infections, but may not be effective against all viruses known to infect Drosophila cell cultures.

Non-Coding RNA doi: 10.3390/ncrna12040027

Authors: Nicolò Salvi Mariangela Morlando

Circular RNAs (circRNAs) have recently emerged as a class of abundant and remarkably stable non-coding RNAs preferentially enriched in the nervous system. In neurons, the fine-tuned spatial regulation of gene expression is critical for proper synaptic function; accordingly, several studies have demonstrated that circRNAs exhibit highly compartmentalized localization, specifically within dendrites, axons, and synapses. These spatial localization properties imply the presence of active transport mechanisms, which control the intracellular trafficking of circRNAs. This review highlights the current understanding of circRNA transport in neurons, focusing on the molecular machinery driving synaptic enrichment. We explore the potential role of ribonucleoprotein-based transport as a primary mechanism driving circRNA localization and examine how such spatial distribution influences synaptic plasticity and post-transcriptional gene regulation. Finally, we discuss the clinical implications of these processes, exploring the link between dysregulated RNA transport and the development of neuronal abnormalities.

Non-Coding RNA doi: 10.3390/ncrna12040026

Authors: Neelu Batra Mei-Juan Tu Su Guan Jonathan W. Riess Ai-Ming Yu

Background/Objectives: Recent advances in immunotherapy have revolutionized cancer treatment, as exemplified by multiple monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1). Nevertheless, immunotherapeutic antibodies exhibit certain limitations, which drives the development of alternative approaches, such as small interfering RNA (siRNA)-based therapeutics. The aim of this study was to design and produce new biological PD-L1 siRNA (BioRNA/PD-L1-siRNA) molecules and further define their immunotherapeutic efficacy against non-small cell lung cancer (NSCLC) in vitro. Methods: A novel RNA molecular bioengineering platform was employed to produce new BioRNA/PD-L1-siRNA agents. The functions of BioRNA/PD-L1-siRNAs were determined by quantitative PCR, Western blot, immunofluorescence confocal imaging, flow cytometry, and PD-1/PD-L1 blockade assays in human NSCLC cells, alone and co-cultured with human peripheral blood mononuclear cells (PBMCs). Results: After heterologous overexpression and purification of five BioRNA molecules, one siRNA named BioRNA/PD-L1-siRNA-1 was identified as the most effective to selectively suppress human PD-L1 mRNA and protein levels in H460 and H1975 cells. Disruption of PD-1/PD-L1 interactions by BioRNA/PD-L1-siRNA-1 was further demonstrated via a PD-1/PD-L1 blockade bioassay. In addition, the immunomodulatory effectiveness of BioRNA/PD-L1-siRNA-1 was established in co-culture models, as indicated by the induction of T-cell and natural killer cell populations and an increase in specific cytokines and cytotoxic granules, and subsequent enhancement of apoptosis and greater inhibition of NSCLC cell viability. Conclusions: Overall, these findings demonstrate the potential of bioengineered PD-L1 siRNA entities for NSCLC immunotherapy.

Non-Coding RNA doi: 10.3390/ncrna12040025

Authors: Ilyas M. Kabdesh Albert A. Rizvanov Yana O. Mukhamedshina

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is caused in most cases by pathogenic variants in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Despite substantial progress in the development of gene therapy, restoring MECP2 expression remains challenging because MeCP2 is highly dosage-sensitive. Both deficiency and excessive expression of this protein are associated with severe neurological abnormalities. This makes simple viral vector-mediated replacement of MECP2 potentially unsafe and underscores the need for multilayered systems that control transgene expression. This review discusses current and emerging strategies for regulating MeCP2 expression in RTT, with an emphasis on non-coding RNA-based and epigenetic mechanisms. Particular attention is given to the limitations of conventional AAV-mediated gene therapy, the use of cell-specific and endogenous promoters, miRNA-regulated elements, autoregulatory systems, and post-transcriptional control of MECP2 expression. Strategies for reactivating the inactive X chromosome are also discussed, including XIST-dependent regulation and epigenome editing. In addition, the review considers CRISPR-mediated regulation, selective epigenetic activation, and combined therapeutic platforms that integrate viral delivery, RNA-dependent post-transcriptional control, and endogenous gene regulation. Overall, clinically applicable gene therapy for RTT will likely need to move beyond simple MECP2 replacement and instead rely on precise cell- and dose-dependent regulation of its expression. Non-coding RNA and epigenetic mechanisms represent important layers of such control and may contribute to the development of safer gene therapy strategies for RTT.

Non-Coding RNA doi: 10.3390/ncrna12040024

Authors: Nataša Đokić Anastasija Bubanja Jelena Karanović Jovana Despotović

Background/Objectives: Long non-coding RNAs (lncRNAs) are important regulators of tumor biology through their interactions with DNA, proteins, and non-coding RNAs. Although ENST00000615487.1 (also known as CTD-2396E7.11/AC010503.4) has been associated with multiple malignancies, its biological role in colorectal cancer (CRC) remains poorly characterized. This study aimed to investigate the expression pattern, cellular and subcellular localization, and potential functional role of ENST00000615487.1 in CRC using integrated in vitro and in silico approaches. Methods: Molecular characteristics of the transcript were obtained with the CPC2 and RNA Analyzer 3 tools. Differential expression of ENST00000615487.1 across 10 tumor types was analyzed using the UCSC Xena Browser. Transcript expression was experimentally evaluated in normal, tumor, and fibroblastic colon cell lines by PCR, while subcellular localization was assessed through the lncATLAS, lncLocator, and iLoc-LncRNA tools, and experimentally confirmed by qRT-PCR. Single-cell RNA sequencing data from the GSE161277 dataset were analyzed to determine cell type-specific expression patterns. Potential interactions with DNA, miRNAs, and proteins were investigated using Fasim-LongTarget, miRDB, and AnnoLnc2, followed by functional enrichment analyses using STRING and Enrichr. Results: ENST00000615487.1 was identified as a structurally stable non-coding transcript with a highly organized secondary structure. Differential expression analysis demonstrated significant downregulation in CRC compared with that in normal colon tissue. Single-cell transcriptomic analysis revealed predominantly epithelial-specific expression. In silico and experimental analyses demonstrated predominant nuclear localization in normal colon cells, whereas cytoplasmic enrichment was observed in CRC cells. Functional analyses identified potential interactions with HIP1R, RPH3AL, specific miRNAs, and proteins involved in transcriptional regulation and RNA processing pathways, as well as functional connections with proteins involved in vesicular transport. Conclusions: ENST00000615487.1 is a structurally stable lncRNA exhibiting context-dependent expression and localization patterns in CRC, suggesting a potential shift from nuclear transcriptional regulation toward cytoplasmic post-transcriptional functions during colorectal carcinogenesis.

Non-Coding RNA doi: 10.3390/ncrna12040023

Authors: Francisco J. Enguita Suresh K. Alahari Luca Agnelli Raphaël Zemmour Florent Hubé Patrick K. T. Shiu Sophia M. Zhang Mohammadjavad Mohammadi Shuxing Zhang Sreyasree Dhar Simon J. Conn Agnieszka Bronisz Jakub Godlewski Abhishek Kaushik Alexander Serganov Barbara Pardini André P. Gerber Mark W. Feinberg Eleonora Leucci Phoebe Philpott Andrea Caporali Toshiaki Takahashi Ajay Goel Ling Yang

We are delighted to share with you our fifteenth Journal Club and highlight some of the most interesting papers published recently [...]

Non-Coding RNA doi: 10.3390/ncrna12040022

Authors: Paul Flondor Mircea Olteanu Radu Stefan Corina Elena Minciuna Catalin Vasilescu

Background: MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally by binding to target messenger RNAs (mRNAs) and suppressing their expression. Competing endogenous RNAs (ceRNAs), including mRNAs and circular RNAs (circRNAs), modulate miRNA availability through competitive binding, forming regulatory networks that fine-tune gene expression. CircRNAs can act as miRNA sponges, reducing miRNA-mediated repression of other targets, a mechanism implicated in various pathophysiological processes, including oncogenesis. Methods: We propose a mathematical model describing the dynamics of miRNA–mRNA–protein interactions, extending existing frameworks for miRNA–mRNA regulation. A qualitative analysis of the associated nonlinear differential equations system is performed. Results: We prove the boundedness of all positive solutions, establish the existence of a unique positive attracting equilibrium, and provide a mathematical perspective on the crosstalk mechanism in protein production. Conclusions: The effectiveness of ceRNA interactions depends on the relative abundance of miRNAs and their targets. This highlights the ongoing debate regarding the biological impact of low-abundance RNA transcripts on miRNA-mediated regulation.

Non-Coding RNA doi: 10.3390/ncrna12040021

Authors: Sayema Naaz Haley A. Kominek Lydia A. Hayes-Guastella Autumn M. McDaniel Enas S. Alsatari Adeyeye I. Haastrup Olivia G. Clark Devin M. Katerski Francois O. Prinsloo Olivia R. Roberts Meredith A. Shaddix Bridgette N. Sullivan Isabella M. Swan Emily M. Hartsell Jeffrey D. DeMeis Suhas S. Patil Richard H. Pham Makala R. Cox Glen M. Borchert

Background/Objectives: Outer membrane vesicles (OMVs) are membrane-encapsulated spherical structures ~120 nm in diameter derived from Gram-negative bacterial cell envelopes. OMVs are primarily generated by outer membrane blebbing but contain proteins, DNA, and RNAs at concentrations distinct from that of the intracellular complement. OMVs have been associated with a number of different cellular functions including intercellular communication and resistance to phage. Methods: As bacterial small RNAs (sRNAs) also participate in bacteriophage defense and are specifically delivered to and enriched in OMVs, we recently elected to examine the effects of P22 infection on Salmonella cytosolic and OMV sRNA abundance by employing RNA sequencing. Results: We find that P22 infection triggers a global reduction in sRNAs (with Salmonella sRNA expression levels averaging only 15.6% those observed in noninfected cells) coupled with a reciprocal 72.7% global increase in Salmonella tRNA expression levels. Additionally, of note, while OMV small noncoding RNA (sncRNA) abundance is normally ~1/10 that found in the cytosol, we find that P22 infection triggers active OMV encapsulation and secretion of: (1) a subset of sRNAs, (2) all Salmonella tRNAs including one highly complementary to the P22 genome, and, much to our surprise, (3) ten distinct sRNAs expressed from P22. Conclusions: In summary, the work presented here identifies several Salmonella sncRNA cytosolic and/or OMV abundances significantly altered during P22 infection, and to our knowledge, this constitutes the first reported characterization of bacteriophage-encoded sRNAs being actively secreted within host OMVs.

Non-Coding RNA doi: 10.3390/ncrna12030020

Authors: Maria V. Lukashevich Margarita M. Rudenok Suzanna A. Partevian Maria I. Shadrina Petr A. Slominsky Anelya Kh. Alieva

Background/Objectives: MicroRNAs are key post-transcriptional regulators involved in various diseases. Despite its status as the gold standard, real-time RT-PCR faces challenges arising from high sequence homology among closely related microRNAs and the substantial biomaterial required to enrich small RNA fractions. This study aimed to develop an optimized protocol for simultaneous analysis of microRNA and mRNA expression from a single total RNA sample using mouse (Mus musculus) brain tissue, avoiding dependence on pre-designed commercial assay panels. Methods: We optimized a real-time RT-PCR workflow enabling simultaneous analysis of mature microRNAs and mRNAs from a single total RNA sample. Modifications include a redesigned universal reverse primer, LNA-modified TaqMan probes, and omission of the 65 °C denaturation step during reverse transcription. The method was validated for five microRNAs in mouse brain tissue. Results: The assay showed high specificity, discriminating closely related miR-125a-5p and miR-125b-5p with a ΔCt difference of 6.7 ± 1.2 cycles. Co-analysis with Oligo(dT)18 and Random hexamer primers did not interfere with microRNA detection. Conclusions: The developed approach enables reliable detection of closely related microRNAs and parallel analysis of different RNA types, which is particularly important for studying regulatory networks when working with limited amounts of biomaterial. This protocol provides a complementary, accessible option for targeted studies in resource-limited settings or for non-cataloged miRNA targets.

Non-Coding RNA doi: 10.3390/ncrna12030019

Authors: Mónica Alejandra Rosales-Reynoso Anilú Margarita Saucedo-Sariñana Clara Ibet Juárez-Vázquez César de Jesús Tovar-Jácome Rubria Alicia González-Sánchez Karen Guadalupe Mestas-Villagran Gustavo Andrés Torres-Sánchez José Elías García-Ortíz Efraín Salas-González Martha Patricia Gallegos-Arreola

Recent evidence has highlighted the crucial role of non-coding genetic elements in regulating gene expression and has been linked to a broad range of biological functions. Notably, dysregulation of long non-coding RNAs has been strongly associated with tumorigenesis and cancer progression. Background/Objectives: This study aimed to investigate the potential association between the H19 rs3741219 T>C, MEG3 rs7158663 G>A, POLR2E rs3787016 T>C, and ANRIL rs10757274 A>G variants and Breast Cancer (BC) susceptibility, as well as their relationship with clinicopathological characteristics in Mexican patients. Methods: DNA was obtained from peripheral blood samples of 505 women (254 patients and 251 control females). Genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methodology. Associations were calculated using odds ratios (ORs), with p-values adjusted by the Bonferroni test (p < 0.012). In silico analyses were conducted to predict the functional impact of the variants associated. Results: Patients carrying the C/C genotypes in H19 rs3741219 and POLR2E rs3787016 variants showed increased susceptibility to developing BC and with clinical and pathological characteristics (age at diagnosis, TNM stage, histologic type and molecular subtype) (p < 0.001). Conclusions: The results suggest that H19 rs3741219 and POLR2E rs3787016 variants significantly influence BC risk.

Non-Coding RNA doi: 10.3390/ncrna12030018

Authors: Saioa Arza-Apalategi Daan Gilissen Anne C. van der Grinten Seline N. van den Oever Erik B. van den Akker Marieke Griffioen Joop H. Jansen Joost H. A. Martens Anna E. Marneth Bert A. van der Reijden

Background/Objectives: Acute myeloid leukemia (AML) comprises genetic subclasses with distinct gene expression profiles. While AML gene expression studies have mainly focused on protein-coding genes, our understanding of expression patterns of long intergenic noncoding RNAs (lincRNAs) remains incomplete. This is due to limited sample sizes, as well as incomplete annotation of lncRNAs with context-dependent expression. Methods: To address this gap, we developed the bioinformatic pipeline LIRA (long intergenic noncoding RNA annotator) to identify novel lincRNAs using stringent criteria, including spliced and intergenic transcripts, and algorithms to exclude coding potential. Results: By applying LIRA to RNA-sequencing data from 878 pediatric and adult AML cases and 20 healthy controls, we identified 1560 novel lincRNAs, expanding the GENCODE v38 lincRNA catalog by 27%. Integration of in-house-generated CAGE- and ChIP-sequencing data from KMT2A::MLLT3 samples revealed that 80% of the novel lincRNAs are 5′ capped, and at least 67% harbor activating epigenetic marks at their transcription start sites. Unsupervised analysis of the 1000 most variable known and newly identified lincRNAs uncovered subclass-specific expression patterns, mirroring those observed for protein-coding genes. Weighted Gene Co-expression Network Analysis identified 17 lincRNA expression modules associated with AML subclasses. Notably, expression of these modules decreased upon degradation of the leukemogenic onco-fusion proteins KMT2A::MLLT3 and PML::RARA, indicating that lincRNA expression is responsive to oncogenic signaling. Conclusions: This comprehensive analysis shows that lincRNAs exhibit similar subclass-specific expression patterns as protein-coding genes and establishes a valuable resource for future studies on genetically defined AML subclasses, with potential implications for biomarker discovery and therapeutic targeting.

Non-Coding RNA doi: 10.3390/ncrna12030017

Authors: Raunak Sharda Valentina L. Kouznetsova Igor F. Tsigelny

Background/Objectives: Autism Spectrum Disorder (ASD) is a set of neurological and neurodevelopmental disorders characterized by difficulties in social communication and interaction, repetitive behaviors, and sensory processing differences. Recent studies have shown that circRNAs play a crucial role in the pathophysiology of ASD. In this study, we present an exploratory machine learning framework integrating circRNA sequence features, miRNA interactions, gene targets, and pathway enrichment analysis to investigate ASD-associated molecular signatures. Methods: Differential circRNAs were identified from human peripheral blood datasets, and informative features were selected using attribute-based filtering and Information Gain ranking. Machine learning models were developed using the WEKA platform. Results: The HyperPipes classifier achieved the highest performance (92.5% accuracy under cross-validation). Analysis using an independent ASD gene expression dataset showed consistent discriminative patterns of the derived gene-level signatures across multiple machine learning classifiers. The competitive endogenous RNA network and enriched gene pathways were also analyzed. Conclusions: Overall, this study provides a computational, preliminary framework for analyzing circRNA-associated molecular patterns in ASD. Findings should be interpreted in the context of limited sample size and dataset availability.

Non-Coding RNA doi: 10.3390/ncrna12030016

Authors: Silvia Grassilli Andrea Baroni Marina Pierantoni Federica Brugnoli Nicola Lamberti Sofia Straudi Fabio Manfredini Valeria Bertagnolo

Background: MiRNAs within extracellular vesicles can encompass body barriers, reflecting stage, progression, and response to treatments of various diseases, including multiple sclerosis (MS)—a chronic immune-mediated disease of the central nervous system that causes progressive disability, with highly variable clinical courses. In this context, urinary exosomal miRNAs could be an appealing source of biomarkers, thanks to their non-invasive and easily repeatable collection. Methods: In this exploratory investigation, we tried to assess if profiling urinary exosomal miRNAs could reveal subtle differences within an apparently homogeneous MS population. The study involved 24 patients with primary or secondary progressive MS, whose urinary exosomes (UEs) were subjected to evaluation of a panel of 87 miRNAs variously correlated with neuroinflammation, cardiovascular functions, and/or involved in MS. Results: We revealed that the examined miRNAs were heterogeneously expressed across the patients, reflecting, as expected, their gender and/or hormonal status. Two miRNAs discriminated against primary or secondary progressive MS, and a panel of 14 commonly upmodulated miRNAs identified patients with longer disease duration and a greater degree of disability. Conclusions: Even if preliminary, these data represent the first relationship between UEs and MS features in humans and suggest that urine could constitute a non-invasive source of exosomal miRNAs, which could prove useful in complementing conventional monitoring to provide a more personalized management of MS patients.

Non-Coding RNA doi: 10.3390/ncrna12030015

Authors: Beshoy Armanios Jing Jin Ankit P. Laddha Le Tra Giang Nguyen Sherouk M. Tawfik Neha Mishra Jose E. Manautou Xiao-Bo Zhong

Background: Long noncoding RNAs (lncRNAs) have emerged as critical regulators of hepatic metabolism and disease progression. The hepatocyte nuclear factor 1 alpha antisense 1 (HNF1A-AS1) lncRNA modulates liver-specific transcription factors; however, its physiological role in diet-dependent lipid homeostasis remains poorly defined. Methods: In this study, we investigated the mouse ortholog, Hnf1a opposite strand 1 (Hnf1aos1), using AAV-mediated knockdown in C57BL/6J mice fed either a chow diet (10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) for 12 weeks. Metabolic phenotyping included hepatic lipid quantification, histological analysis, serum biochemistry, and quantitative gene expression profiling. Results: Loss of Hnf1aos1 produced distinct, diet-dependent alterations in hepatic lipid handling. Under chow conditions, knockdown mice exhibited selective hepatic cholesterol accumulation (6.10 ± 2.9 mg/g tissue vs. 3.51 ± 1.1 mg/g in controls), accompanied by dysregulation of cholesterol clearance pathways. In contrast, under HFD conditions, knockdown precipitated severe macrovesicular degeneration, with hepatic triglyceride levels approximately doubled relative to HFD-fed controls (51.72 ± 19.8 mg/g vs. 26.34 ± 11.9 mg/g) and a numerically elevated triglyceride-to-cholesterol ratio (TG:TC ≈ 6.1:1; p = 0.0621, trend). Chow/Kd mice gained significantly less weight than chow-fed controls, whereas HFD/Kd mice exhibited weight gain comparable to HFD controls despite severe hepatic steatosis. This paradoxical phenotype suggests impaired metabolic feedback at the post-transcriptional level, in which compensatory upregulation of Hnf1a mRNA is insufficient to suppress lipid-associated genes such as Cd36, despite profound lipid overload; however, HNF1A protein levels were not directly measured in this study. Conclusion: Collectively, these findings identify Hnf1aos1 as a regulator of hepatic lipid homeostasis whose loss produces a phenotype consistent with inappropriate lipid accumulation during nutrient excess, without defining the underlying molecular mechanism. Our results support a role for Hnf1aos1 in shaping hepatic metabolic plasticity and provide insight into lncRNA-associated MASLD phenotypes.

Non-Coding RNA doi: 10.3390/ncrna12030014

Authors: Hee Kyoung Chung Lan Xiao Jian-Ying Wang

The mammalian intestinal epithelium is a rapid self-renewal tissue in the body, serving as a critical interface between the host and the external environment. Maintaining the intestinal epithelium homeostasis requires precise coordination of cellular processes, including proliferation, migration, differentiation, autophagy, and cell-to-cell interaction. An increasing body of evidence has unveiled circular RNAs (circRNAs) as abundant and stable regulatory molecules that play pivotal roles in the intestinal epithelial biology and are intimately involved in many aspects of gut mucosal pathologies. Unlike linear RNAs, circRNAs form covalently closed loop structures through back-splicing events, conferring remarkable stability and resistance to exonucleolytic degradation. circRNAs regulate the growth of the intestinal mucosa, injury-induced epithelial regeneration, and gut barrier function via diverse mechanisms, including interactions with microRNAs and RNA-binding proteins. Deregulated circRNAs are implicated in the pathogenesis of various gut mucosal disorders such as inflammatory bowel disease and malignancies. In this review, we highlight pathobiological functions and mechanisms of intestinal epithelium-enriched circRNAs, particularly circHIPK3, Cdrlas, and circPABPN1, in the epithelium homeostasis and pathologies and also discuss potential clinical application of circRNAs as diagnostic biomarkers and therapeutic targets in patients with critical diseases.

Non-Coding RNA doi: 10.3390/ncrna12020013

Authors: Mario García-Domínguez

Neuropathic pain represents a complex, prolonged pain state arising from lesions within the somatosensory nervous system. Despite significant advances in elucidating its pathophysiology, current therapeutic approaches remain largely symptomatic and frequently inadequate. MicroRNAs, a class of small non-coding RNAs that regulate gene expression post-transcriptionally, have recently emerged as critical modulators of neuronal excitability, neuroinflammation, and synaptic plasticity, which are crucial processes in the development and maintenance of neuropathic pain. This review summarizes the current evidence linking specific miRNAs to the onset and maintenance of neuropathic pain, with an emphasis on their roles in peripheral and central sensitization. The potential of miRNA-based biomarkers for diagnosis and prognostic evaluation is also highlighted. A thorough understanding of the complex miRNA regulatory networks underlying neuropathic pain could facilitate the development of novel, mechanism-based therapies and ultimately improve clinical outcomes.

Non-Coding RNA doi: 10.3390/ncrna12020012

Authors: Graham H. Read Kristen McGreevy Hanny Issawi Tiffany Yang Cynthia Tsang Ihsan A. Turk Emily Rietdorf Whitaker Cohn David W. Salzman Julian P. Whitelegge Joanne B. Weidhaas

Background/Objectives: Canonical microRNAs possess a 5′ phosphate required for Argonaute binding and activity. However, prior work identified an unphosphorylated, inactive nuclear pool of the important radiation-responsive microRNA, miR-34, that is rapidly phosphorylated and activated in response to ionizing radiation (IR). Here, we extend this work and investigate the role of paraspeckles, a phase-separated nuclear sub-compartment, and their association with the localization of unphosphorylated miR-34a. Methods: Mass spectrometry was performed to identify interacting partners of unphosphorylated mir-34. CRISPR-mediated deletion of the paraspeckle NEAT1_2 triple helix motif was performed to create an A549 cell line lacking paraspeckles (dTH). Activity and expression of mir-34a post-irradiation were evaluated by qRT-PCR and luciferase assays comparing dTH and wild-type (WT) A549 cell lines. In situ hybridization (ISH) was performed to evaluate mir-34a localization before and after IR, comparing dTH and WT cell lines. Results: Mass spectrometry identified paraspeckle proteins as significantly enriched interacting partners of unphosphorylated mir-34 mimics. By qRT-PCR and luciferase assays, we found that paraspeckle loss prevented radiation-induced early activation of unphosphorylated mir-34a. We found no difference in radiation-induced transcription of pri-miR-34a, but early processing to pre-miR-34a appeared delayed. ISH confirmed that loss of paraspeckles altered the nuclear localization of miR-34a before and after IR. Conclusions: These data suggest that paraspeckles are associated with nuclear localization and early radiation-responsive activation of unphosphorylated miR-34a. This suggests a coordinated nuclear sequestration of this important miR in its unphosphorylated state to enable an enhanced radiation response.

Non-Coding RNA doi: 10.3390/ncrna12020011

Authors: Francesca Pia Carbone Stefania Hanau Nicoletta Bianchi

Background/Objectives: Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to sustain proliferation, survive under metabolic stress, and develop therapeutic resistance. While oncogenic signaling pathways regulating cancer metabolism have been extensively studied, increasing evidence indicates that non-coding RNAs (ncRNAs) play essential roles in coordinating metabolic adaptation. This review aims to synthesize current knowledge on long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) as important but relatively less characterized regulators of cancer metabolic adaptation and discuss their potential as biomarkers and therapeutic targets. Methods: We analyzed their roles across multiple types of cancer, prioritizing studies that integrate ncRNA profiling with metabolomics and mechanistic investigations, with particular attention to their diagnostic, prognostic, and predictive value. Results: LncRNAs and circRNAs regulate major metabolic pathways, including glycolysis, mitochondrial function, glutaminolysis, lipid metabolism, and redox balance. They act through transcriptional and epigenetic mechanisms, protein scaffolding, peptide encoding, and miRNA sponging, frequently converging on key regulators such as HIF-1α, c-Myc, p53, AMPK, and mTOR. However, many reported associations remain largely correlative, with limited integration of quantitative metabolic flux analyses and insufficient validation in physiologically relevant models. Conclusions: Although lncRNAs and circRNAs constitute an important context-dependent regulatory layer linking oncogenic signaling to metabolic reprogramming, future studies should combine ncRNA perturbation with stable isotope tracing, fluxomics, spatial metabolomics, long-read sequencing, and single-cell approaches to define causal and spatially resolved metabolic functions. Such integrative strategies may improve biomarker development and support ncRNA-informed, metabolism-oriented therapeutic interventions.

Non-Coding RNA doi: 10.3390/ncrna12020010

Authors: Daniel Angelo R. Mirador Jose Lorenzo M. Ferrer Kim Denyse Hao Lin Reynaldo L. Garcia

Background/Objectives: Lung cancer remains a major global health burden, largely driven by cigarette use. Although electronic cigarettes (e-cigarettes) are viewed as safer alternatives due to their reduced chemical load, growing evidence shows their vapor can disrupt cellular transcriptomes, including long noncoding RNAs (lncRNAs). In this study, we examined the regulation and function of vape-associated lncRNA transcript 1 (VALT1), a novel transcript upregulated in the oral transcriptomes of e-cigarette users and similarly elevated in non-small-cell lung cancer (NSCLC) tumors. Methods: Publicly available RNA-seq datasets were analyzed, and VALT1 was identified as an e-cigarette-responsive lncRNA. Its dose-dependent induction by e-cigarette smoke extract (eCSE) and cytoplasmic localization were confirmed via RT-qPCR. Its effects on cancer-associated phenotypes including proliferation, ROS detoxification, resistance to apoptosis, migration, cytoskeletal disorganization, and nuclear remodeling were assessed through overexpression and siRNA-mediated knockdown in A549 and BEAS-2B cells. Results: Acute eCSE exposure induced a biphasic, dose-dependent increase in VALT1 expression, accompanied by enhanced proliferation, ROS detoxification, apoptosis resistance, migration, cytoskeletal disorganization, and nuclear remodeling in A549 cells. VALT1 overexpression reproduced these phenotypes in both cell lines without eCSE treatment, whereas knockdown attenuated them. VALT1 promoted survival under cytotoxic stress in A549 but not BEAS-2B cells. Conclusions: These findings support an active role for VALT1 as an e-cigarette vapor-upregulated transcript that contributes to its phenotypic readout and enhances cellular survival under extracellular chemical stress—thereby aggravating tumorigenic phenotypes even in the absence of mutations that contribute to malignant transformation.

Non-Coding RNA doi: 10.3390/ncrna12020009

Authors: Caio Felipe Freire Thalles Souza-Lopes Murilo Sena Amaral Ana Carolina Tahira Sergio Verjovski-Almeida

Background/Objectives: Schistosomiasis is a neglected tropical disease affecting >200 million people worldwide. Praziquantel is the sole recommended drug against Schistosoma mansoni; however, it lacks activity against juvenile forms and cannot prevent reinfection. Thus, there is an urgent need to identify novel therapeutic targets. Long noncoding RNAs (lncRNAs) are known to regulate various biological processes in S. mansoni, including parasite pairing and fertility; therefore, screening for novel lncRNAs could reveal new potential targets. Methods: We compiled all publicly available RNA-seq data from the Sequence Read Archive (SRA) and performed a hierarchical transcriptome assembly using the multi-sample assembler Ryūtō, combined with version 10 of the S. mansoni genome. We applied HOMER for peak-calling and identification of histone marks and used weighted gene co-expression network analysis (WGCNA) to infer putative functions of lncRNAs in sexual dimorphism. Results: Using a robust pipeline, we identified 10,170 novel lncRNA genes comprising 16,990 novel lncRNA transcripts, including 8783 intergenic, 7918 antisense, and 289 intronic lncRNA transcripts. Most (78.7%) have histone regulatory marks (H3K4me3, H3K27me3, H3K27ac, or H4K20me1) near their transcription start sites, indicating potential expression regulation. Comparing male and female samples, we identified 1991 differentially expressed genes (FDR < 5%, |log2FC| ≥ 1.5), including 296 known lncRNAs and 339 novel lncRNAs. WGCNA identified hub lncRNAs within co-expression modules, and Gene Ontology enrichment analyses (FDR ≤ 5%) suggest that these lncRNAs are involved in cell differentiation and morphogenesis pathways. Conclusions: We provide a comprehensive catalog of S. mansoni lncRNAs. These findings offer opportunities to discover potential new therapeutic targets, advancing the future development of anti-schistosome therapies.

Non-Coding RNA doi: 10.3390/ncrna12020008

Authors: Monia Cecati Valentina Pozzi Valentina Schiavoni Giuseppina Barrasso Veronica Pompei Daniela Marzioni Nicoletta Bonci Stefania Fumarola Andrea Ballini Davide Sartini Roberto Campagna

Renal cell carcinoma (RCC) represents the most frequent kidney malignancy and remains a major clinical challenge due to its often silent onset, high metastatic potential, and limited responsiveness to conventional chemotherapy. Increasing evidence indicates that non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), are key regulators of RCC tumorigenesis, progression, and therapy resistance. Rather than providing a purely descriptive overview, this review focuses on emerging mechanistic paradigms through which ncRNAs actively shape tumor behavior and therapeutic response in RCC. This review summarizes current knowledge on the biological and clinical relevance of ncRNAs in RCC, highlighting their dual roles as oncogenic drivers or tumor suppressors through the modulation of pathways involved in proliferation, apoptosis, angiogenesis, invasion, immune evasion, metabolic reprogramming, and ferroptosis. Particular emphasis is placed on mechanistically defined ncRNA regulatory axes controlling ferroptosis, autophagy, metabolic reprogramming, and immune escape, as well as on ncRNA-mediated intercellular communication via extracellular vesicles, which promotes the dissemination of resistance to targeted therapies. The review also addresses ncRNA-based diagnostic and prognostic applications, including miRNA signatures capable of discriminating RCC subtypes and circulating ncRNAs as minimally invasive biomarkers. Moreover, the manuscript discusses ncRNA-mediated mechanisms of resistance to targeted therapies such as sunitinib, sorafenib, and axitinib, emphasizing regulatory networks involving miRNA targets, lncRNA–miRNA sponging, RNA-binding proteins, extracellular vesicle transfer, and epigenetic modulation. Emerging therapeutic opportunities are also addressed, including strategies aimed at inhibiting oncogenic ncRNAs or restoring tumor-suppressive ncRNAs to enhance drug sensitivity and improve patient stratification.

Non-Coding RNA doi: 10.3390/ncrna12010007

Authors: Alma D. Campos-Parra Jonathan Puente-Rivera César López-Camarillo Stephanie I. Nuñez-Olvera Nereyda Hernández Nava Gabriela Alvarado Macias Macrina Beatriz Silva-Cázares

Extracellular matrix (ECM) stiffening is a defining biophysical feature of solid tumors that reshape gene regulation through mechanotransduction. Increased collagen crosslinking and stromal remodeling enhance integrin engagement, focal-adhesion signaling and force transmission to the nucleus, where key hubs such as lysyl oxidase (LOX), focal adhesion kinase (FAK) and the Hippo co-activators YAP1 and TAZ (WWTR1) promote proliferation, invasion, stemness and therapy resistance. Here, we synthesize evidence that quantitative changes in matrix stiffness remodel the miRNome and lncRNome in both tumor and stromal compartments, including extracellular vesicle cargo that reprograms metastatic niches. To address heterogeneity in experimental support, we classify mechanosensitive ncRNAs into studies directly validated by stiffness manipulation (e.g., tunable hydrogels/AFM) versus indirect associations based on mechanosensitive signaling, and we summarize physiological versus pathophysiological stiffness ranges across tissues discussed. We further review competing endogenous RNA (ceRNA) networks converging on mechanotransduction nodes and ECM remodeling enzymes, and discuss translational opportunities and challenges, including targeting mechanosensitive ncRNAs, combining ncRNA modulation with anti-stiffening strategies, delivery barriers in dense tumors, and the potential of circulating/exosomal ncRNAs as biomarkers. Overall, integrating ECM mechanics with ncRNA regulatory circuits provides a framework to identify feed-forward loops sustaining aggressive phenotypes in rigid microenvironments and highlights priorities for validation in physiologically relevant models.

Non-Coding RNA doi: 10.3390/ncrna12010006

Authors: Christopher Klapproth Franziska Reinhardt Peter F. Stadler Sven Findeiß

Background/Objectives: The telomerase RNA (TR) is an indispensable part of the telomerase protein complex responsible for telomere elongation in most eukaryotic species. Although the telomere terminal repeat sequence (TTAGGC)n in Caenorhabditis elegans has been known for years, a telomerase RNA gene was not identified in the entire phylum of Nematoda until recently. Methods: In this exploratory study, we employ a combination of different approaches to identify likely telomerase RNA candidates among putative non-coding transcripts. Results: A detailed analysis of our prime candidate shows compelling evidence that it encodes the missing RNA element of the telomerase complex, which is notably located in an intron of the coding gene nmy-2. Using nmy-2 homologs in other nematodes as anchors, we annotate the conserved TR gene in 21 Caenorhabditis species. We furthermore show that the intronic localization of the TR gene is conserved in two distinct branching groups of the Caenorhabditis phylogeny and demonstrate that this property likely emerged from a single point of origin. Conclusions: While the intronic TR represents a very interesting evolutionary adaption that seems to have been successful in the Elegans and Japonica groups, the question regarding the macroscopic nematode TR evolution remains.

Non-Coding RNA doi: 10.3390/ncrna12010005

Authors: Clara Claus Carla Borini Etichetti Bruno Costa Julieta B. Grosso Juan Pablo Tosar Uciel Chorostecki Silvana V. Spinelli

Background/Objectives: Human milk is a complex biological fluid containing not only macro- and micronutrients but also diverse bioactive molecules, including extracellular RNAs. Although RNA has been detected in milk for decades, only a subset of RNA species has been characterized in detail, and abundant families such as tRNA-, yRNA-, and rRNA-derived fragments remain underexplored. This study aimed to define the composition, fragmentation patterns, stability, and exploratory functional activity of these highly abundant RNAs in human milk. Methods: We performed small RNA sequencing on skim milk samples and analyzed the resulting profiles in comparison with publicly available milk and biofluid datasets. RNA stability assays, Northern blotting, and RT-qPCR were conducted to validate RNA abundance and degradation kinetics. Extracellular vesicles (EVs) and non-vesicular fractions were analyzed to determine the subcellular distribution of RNA species. Exploratory functional assays using synthetic RNA fragments were carried out to assess their ability to modulate cellular responses in vitro. Results: Human milk was found to be highly enriched in small RNA fragments derived from tRNA, yRNA, and rRNA, dominated by a limited set of discrete sequences. These profiles were highly reproducible across independent datasets and distinct biofluids. Orthologal validation assays confirmed their abundance and stability, with RNA levels exceeding those of serum by over two orders of magnitude. Full-length transcripts were enriched in EVs, whereas shorter fragments predominated in the non-vesicular fraction. Synthetic milk-derived exRNAs showed detectable pro-survival activity under stress conditions in vitro. Conclusions: This study reveals that human milk carries a limited set of highly abundant stable sRNA molecules, primarily derived from tRNAs, yRNAs, and rRNAs. These findings provide new insights into the RNA cargo of human milk and offer preliminary evidence that selected sRNA fragments can modulate cellular stress responses in in vitro models.

Non-Coding RNA doi: 10.3390/ncrna12010004

Authors: Samuel J. Gonzalez Edgardo Linares Allison M. Porman Swain Aaron M. Johnson

This review provides a thorough survey of long noncoding RNAs that bear the RNA modification N6-methyladenosine (m6A) and current work to understand the resulting mechanistic and biological consequences. We give an overview of lncRNA and m6A biology first, describing the writers, erasers, and readers of m6A and their targeting of lncRNAs. Next, we give an in-depth review of the field of nuclear lncRNAs that regulate chromatin and their regulation via m6A. We then describe the growing appreciation of liquid–liquid phase separation properties in lncRNA and m6A biology. Finally, we cover examples of cytoplasmic lncRNAs regulated by m6A. Overall, this review aims to emphasize how epitranscriptomics influences noncoding RNA mechanisms to provide additional layers of regulation, integrated into downstream biological processes.

Non-Coding RNA doi: 10.3390/ncrna12010003

Authors: Nozha Raguema Sylvie Dussault Kevin Sawaya Michel Desjarlais Eric Boilard Sylvain Chemtob Alain Rivard

Background/Objectives: In severe peripheral artery disease (PAD) with limb ischemia, hypercholesterolemia (HC) is associated with impaired neovascularization. Extracellular vesicles (EVs) are present within ischemic muscles, and they contain microRNAs (miRs) involved in several biological functions, including angiogenesis and neovascularization. Methods: We used a mouse model of PAD and compared the response to hindlimb ischemia in hypercholesterolemic ApoE−/− vs. normocholesterolemic mice. Next-generation sequencing (NGS) was used to perform full miR expression profiling in ischemic skeletal muscles and in EVs of varying sizes—large EVs (lEVs) and small EVs (sEVs)—within these muscles. Results: We identified several miRs with potential pro-angiogenic effects (angiomiRs) that are reduced by HC in lEVs (Let-7b-5p, miR-151-3p, Let-7c-5p) or sEVs (miR-21a-5p, miR-196b-5p, miR-340-5p). As proof of principle, we showed that the overexpression of Let-7b-5p in lEVs, or miR-21a-5p in sEVs, can significantly increase the angiogenic capacity of these EVs in vitro. HC also impaired the enrichment of specific angiomiRs in lEVs (miR-100-5p), sEVs (miR-142a-3p), or in both lEVs and sEVs (miR-146b-5p). In silico approaches, including the prediction of miR targets, pathway unions, and gene unions, identified the resulting predictive effects of HC-modulated miRs in EVs on processes with key roles in the modulation of angiogenesis and neovascularization, such as the regulation of the actin cytoskeleton and focal adhesion and the HIF-1, MAPK, AMPK, and PI3K-Akt signaling pathways. Conclusions: Our results constitute an important first step towards the identification of specific miRs that could be targeted to improve EV angiogenic function in hypercholesterolemic conditions and reduce tissue ischemia in patients with severe PAD.

Non-Coding RNA doi: 10.3390/ncrna12010002

Authors: Eirini Panteli Epameinondas Koumpis Vasileios Georgoulis Georgios Petros Barakos Evangelos Kolettas Panagiotis Kanavaros Alexandra Papoudou-Bai Eleftheria Hatzimichael

Diffuse large B-cell lymphoma (DLBCL) is the most common and clinically aggressive subtype of non-Hodgkin lymphoma (NHL). While novel therapies such as rituximab and polatuzumab vedotin have led to improved outcomes, approximately 35% of patients eventually develop relapsed or refractory disease. MicroRNAs (miRNAs), a class of endogenous single-stranded RNAs approximately 22 nucleotides in length, play a pivotal role in the regulation of gene expression at the post-transcriptional level through interactions with complementary target RNAs and contribute significantly to the development, progression, and treatment response of DLBCL. Oncogenic miRNAs, such as miR-155, miR-21, and the miR-17–92 cluster, promote proliferation, survival, immune evasion, and therapy resistance by modulating pathways including PI3K/AKT, NF-κB, and MYC. Conversely, tumor-suppressive miRNAs such as miR-34a, miR-144, miR-181a, and miR-124-3p inhibit oncogene activity and enhance apoptosis, with their loss often associated with adverse outcomes. Among these, miR-155 and miR-21 are particularly well studied, playing central roles in both tumor progression and remodeling of the tumor microenvironment. This review summarizes current evidence on the biological and clinical relevance of miRNAs in DLBCL, emphasizing their diagnostic and prognostic potential.

Non-Coding RNA doi: 10.3390/ncrna12010001

Authors: Vladimir Beljanski Maria J. Moreno Hollweg Renee Potens Tanner Blaylock Andres B. Irausquin Nikhila Paleati Lubov Nathanson

Background/Objectives: Bone marrow mesenchymal stromal cells (MSCs) are therapeutic cells that adopt an immunomodulatory phenotype when exposed to pro-inflammatory cytokines. Recent research efforts uncovered that many therapeutic benefits of MSCs can be attributed to the secretion of extracellular vesicles (EVs) such as exosomes, small membrane vesicles of endocytic origin present in the cellular secretome. EVs’ formation and release are impacted by the autophagy pathway, which recycles proteins and organelles via lysosomal degradation. Methods: To evaluate how modulation of autophagy affects properties of MSC EVs enriched in exosomes under pro-inflammatory conditions, we treated the cells with either tamoxifen (TX) or chloroquine (CQ), two drugs known to stimulate or inhibit autophagy, respectively, together with IFNγ. MSC EVs enriched in exosomes were then purified from serum-free media, and their immunoregulatory properties were evaluated ex vivo using activated CD4 T cells; small RNA sequencing was also conducted to determine EVs’ microRNA content. Results: Our data indicate that MSCs treated with CQ + IFNγ yield EVs that possess somewhat higher capacity to decrease T cell proliferation compared to other EVs. Small RNA sequencing revealed that, although similar microRNAs were found in EVs isolated from all treated cells, the treatments exerted more effect on the levels of multiple microRNAs that are known to regulate either cancer or inflammation-related biological pathways in target cells. Conclusions: Overall, we conclude that the co-treatment of MSCs with TX or CQ in the presence of pro-inflammatory cytokine IFNγ has the potential to modulate microRNA content of EVs, potentially affecting biological properties of such EVs and their effect on target cells.

Non-Coding RNA doi: 10.3390/ncrna11060081

Authors: Sergiu Chira Cornelia Braicu Stefan Strilciuc George Calin Ioana Berindan-Neagoe

Background: Transposable elements are normally silenced by epigenetic mechanisms; however, during malignant transformation, epigenetic alterations enable transposons to produce functional molecules like miRNAs. Among these, LINE-2 (L2) elements can generate miRNAs capable of regulating key genes, including tumor suppressors. Two L2-derived miRNAs, miR-28 and miR-708, have been linked to lung cancer, yet the mechanisms underlying their dysregulation remain poorly understood. Our study reveals how genomic context contributes to aberrant gene expression through comprehensive bioinformatic analyses. Methods: Using bioinformatics analysis, we evaluated the expression of miR-28 and miR-708 in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) datasets from TCGA. Further, we assessed the expression and methylation status of miR-28 and miR-708 host genes, LPP and TENM4, respectively, using computational tools. Finaly, we searched for potential candidate tumor suppressor genes targeted by miR-28 and miR-708, which are downregulated in LUAD and LUSC. Results: We found that intragenic L2-derived miR-28 and miR-708 are significantly upregulated in LUAD and LUSC. While TENM4 gene also displays a marked increase in expression in LUAD and LUSC, in tumor versus normal tissue, this difference is less obvious for the LPP gene. We suggest that such dysregulations in expression might be linked to specific methylation patterns of their genomic locations. Furthermore, we emphasize that miR-28 and miR-708 might contribute to lung cancer pathogenesis by targeting key tumor suppressor genes. Conclusions: Alterations in the methylation status of L2-miRNAs genomic loci might result in elevated levels of miRNAs and subsequent targeting of tumor suppressor genes with potential implications in lung cancer pathogenesis.

Non-Coding RNA doi: 10.3390/ncrna11060080

Authors: Margherita Puppo

Bone metastasis is a common and severe complication in advanced stages of breast cancer (BC) that is characterised by limited treatment options and poor patient prognosis. MicroRNAs (miRNAs) are a large class of regulatory small non-coding RNAs (ncRNAs) expressed by cells. Moreover, miRNAs can be released by cells into the blood and lymphatic streams, acting as distant cell-to-cell communicators. Of note, miRNAs have pivotal roles in the metastatic progression of BC to bone. This review summarises the most recent findings on miRNAs and their mRNA targets in driving BC bone metastasis. Furthermore, the potential clinical uses of miRNAs as future therapeutic targets/agents or biomarkers for BC bone metastasis are discussed.

Non-Coding RNA doi: 10.3390/ncrna11060079

Authors: Morgane Gourvest Coen van Solingen

CARINH is an intriguing long noncoding RNA whose unique regulatory functions intersect the seemingly distinct processes of innate immunity and cancer development. Notably, CARINH is conserved across species, offering powerful experimental models for uncovering its mechanistic roles and physiological functions across diverse biological contexts. Stimulated by interferons and viral infections, CARINH stands out as a key player in the body’s antiviral defense mechanisms. Additionally, its dysregulation has been implicated in autoimmune disorders such as psoriasis, asthma, and inflammatory bowel disease, underscoring its broader role in maintaining immune homeostasis. Furthermore, alterations in CARINH expression have been connected to cancer progression, highlighting its dual role in immune response and tumor suppression. In this review, we delve into CARINH’s pivotal function in modulating interferon responses and influencing cancer development, with a focus on the molecular pathways that regulate its expression and contribute to its diverse roles. Understanding these pathways is crucial for evaluating CARINH’s significance as a biomarker and therapeutic target, potentially leading to groundbreaking advancements in medical research and treatment strategies.

Non-Coding RNA doi: 10.3390/ncrna11060078

Authors: Isabela Anda Komporaly Adelina Silvana Gheorghe Lidia Anca Kajanto Elena Adriana Iovănescu Bogdan Georgescu Raluca Ioana Mihăilă Andreea Mihaela Radu Daniela Luminița Zob Mara Mădălina Mihai Mihai Teodor Georgescu Dana Lucia Stănculeanu

Breast cancer remains a leading cause of cancer-related mortality worldwide, with treatment resistance and tumor heterogeneity posing major clinical challenges. MicroRNAs (miRNAs), small non-coding RNAs regulating gene expression, have emerged as key players in breast cancer biology, influencing tumor initiation, progression, and therapy resistance. This narrative review synthesizes recent evidence on the involvement of miRNAs in breast cancer subtypes and their impact on treatment response. Notably, miR-155, miR-503, and miR-21 have shown potential as non-invasive biomarkers and modulators of pathways such as PI3K-Akt, MAPK, and TNF signaling. Additionally, exosomal miRNAs may reflect chemoresistance profiles and predict pathological response to neoadjuvant therapy. Emerging data also support the use of specific miRNAs to sensitize tumors to radiotherapy or modulate immune checkpoints like PD-L1 in triple-negative breast cancer. However, challenges persist regarding standardization, sample types, and study heterogeneity. Further translational research is needed to validate miRNA signatures and their utility in guiding personalized treatment. By highlighting mechanistic insights and potential clinical applications, this review aims to contribute to the ongoing efforts of integrating miRNAs into precision oncology for breast cancer.

Non-Coding RNA doi: 10.3390/ncrna11060077

Authors: Akari Fukumoto Moeka Nakashima Satoru Matsuda

Food is a crucial component affecting the health of individuals, which may have the potential to expand lifespan. It has been shown that a long lifespan may be related to fine-tuned autophagy. In general, suitable autophagy could play a significant role in the anti-aging biological exertion of the host. AMPK, a member of serine and threonine kinases, could play vital roles within the autophagy signaling pathway in various cells. In addition, alterations in the kinase activity of AMPK have been shown to be connected to several pathologies of aging-related diseases. Therefore, autophagy could control the lifespan-related homeostasis within the host from cells to a body via the modification of AMPK. The design of the diet and/or nutrition targeting the AMPK would be a possibility to expand the lifespan. Some analyses of the molecular biology underlying the autophagy suggest that supplementation of accurate nutraceuticals, as well as dietary restriction, mild fasting, and/or appropriate physical exercise, could modulate AMPK signaling, which may be advantageous for life extension with the alteration of autophagy. Remarkably, it has been revealed that several non-coding RNAs (ncRNAs) might also play significant roles in the regulation of autophagy. In addition, the production of some ncRNAs may be associated with the alteration of gut microbiota with certain diets. Therefore, the modulation of AMPK action with ncRNAs through choosing the relevant diet could be a therapeutic tactic for promoting longevity, which is also accompanied by a reduced risk for several aging-related diseases.

Non-Coding RNA doi: 10.3390/ncrna11060076

Authors: Luděk Záveský Eva Jandáková Vít Weinberger Luboš Minář Radovan Turyna Adéla Tefr Faridová Veronika Hanzíková Ondřej Slanař

Background/Objectives: Breast cancer is a leading cause of cancer-related mortality among women worldwide. Small nucleolar RNAs (snoRNAs) represent a class of non-coding RNAs with potential as novel biomarkers applicable to improve diagnostic and prognostic applications. Methods: We performed a comprehensive evaluation of the snoRNA-related gene expression by qPCR using benign and tumor tissue samples associated with invasive breast carcinomas of no special type (NST). Selected candidate snoRNAs, i.e., SCARNA2, SCARNA3, SNORD15B, SNORD94, SNORA68, and SNHG1, along with RNU2-1 snRNA, were further validated and their associations with clinicopathological parameters were examined. External datasets and plasma samples were used for additional validation. Results: SCARNA2 was identified as the most promising snoRNA biomarker candidate, showing a positive association with better progression-free survival (PFS) in our data (13.3-month survival difference between low- and high-expression groups) and with both PFS and overall survival in external RNA-seq datasets. SNORD94, SNORD15B, SCARNA3, and RNU2-1 snRNA were also indicated as putative tumor suppressors. SNORD94 was associated with better progression-free survival (PFS) in our data as well (12.4-month survival difference between low- and high expression groups). Greater downregulation in the low-expression tumor subgroup compared to benign samples further supports the prognostic potential of SCARNA2 and SNORD94. Evidence for SNHG1 and SNORA68 as putative oncogenes was less conclusive. Conclusions: Several small nucleolar RNAs were found to be dysregulated in breast cancer specimens, supporting their further evaluation as potential biomarkers. In particular, SCARNA2, SNORD94, SNORD15B, SCARNA3, and RNU2-1 snRNA merit further investigation to determine their clinical relevance and biological roles in breast cancer.

Non-Coding RNA doi: 10.3390/ncrna11060075

Authors: El Cheima Mhamedi Florent Hubé Suresh K. Alahari Francisco J. Enguita Barbara Pardini Mark W. Feinberg Laura Poliseno Beshoy Armanios Jing Jin Xiao-Bo Zhong Nikolaos Sideris Salih Bayraktar Leandro Castellano Gaetano Santulli Stanislovas S. Jankauskas Will S. Plewa Simon J. Conn Ling Yang Patrick K. T. Shiu Abhishek Kaushik Alexander Serganov Massimo Gentile Giuseppe Viglietto Nicola Amodio Tijana Mitić Andrea Caporali

The field of non-coding RNA research is advancing at a breathtaking pace, continually uncovering new layers of regulatory complexity and functional diversity [...]

Non-Coding RNA doi: 10.3390/ncrna11060074

Authors: Marian T. Underwood Varsha Devarapalli Goodwin G. Jinesh John H. Lockhart Marco Napoli Nino Mtchedlidze Elsa R. Flores Andrew S. Brohl

Background/Objectives: Hepatocellular carcinoma (HCC) is one of the world’s deadliest cancers; however, the mechanisms that contribute to its aggressiveness are poorly understood. In the recent literature, overexpression of the Chromosome 19 MicroRNA Cluster (C19MC) has been associated with an aggressive phenotype and unfavorable prognosis in HCC. However, the molecular consequences of C19MC overexpression in HCC remain poorly understood. Methods: Here, we created a constitutive C19MC-overexpressing HCC model and used two different CRISPR-engineered C19MC-overexpressing HCC models to analyze phenotype and transcriptomic changes. Results: We observed that C19MC overexpression induces cancer stem cell (CSC) phenotypic features in vitro and analyzed transcriptomic changes in genes correlating with stemness, such as NFκB and EMT. Conclusions: C19MC induces changes in HCC that are consistent with stemness and aggression, which provides a better understanding of why C19MC could be a biomarker of poor prognosis.

Non-Coding RNA doi: 10.3390/ncrna11060073

Authors: Jessica Kreutz Tijana Mitić Andrea Caporali

The emergence of single-cell sequencing and computational analysis has dramatically improved our understanding of cellular diversity and gene expression dynamics. The rapid advancement of high-throughput omics technologies has led to an exponential growth in biological data. However, many gene regulatory processes at the single-cell level remain underexplored, especially those regulated by post-transcriptional mechanisms involving microRNAs (miRNAs). miRNAs are essential regulators of gene expression, affecting cellular functions in both normal and disease states. Recent innovations, such as single-cell gene expression profiling and bioinformatic analysis, have enabled comprehensive studies that uncover previously hidden miRNA profiles. In this context, we present experimental tools and computational methods for analysing cell-specific miRNA abundance and investigating their mechanisms. These approaches are expected to reveal the complex nature of miRNA biology and, more broadly, enhance our understanding of life sciences and diseases.

Non-Coding RNA doi: 10.3390/ncrna11050072

Authors: Javier Avalos Adrián Perera-Bonaño M. Carmen Limón

Long noncoding RNAs (lncRNAs) are transcripts generated by polymerase II, therefore subject to 5′ capping and 3′ polyadenylation, categorized as such when they are at least 200 nt in size and lack coding function. The lncRNAs were initially interpreted as spurious transcription products, but over the last two decades an increasing amount of evidence has accumulated for regulatory functions. They are found in all taxonomic groups, including bacteria, archaea, fungi, animals and plants. In fungi, global analyses anticipate their presence in higher numbers than initially expected considering the simplicity of these organisms. Except for the numerous studies performed in budding and fission yeast, relatively few lncRNAs have been investigated in sufficient detail in the rest of the fungi, but their number has increased steadily in recent years. The lncRNAs can be transcribed from intergenic regions or coincide totally or partially with protein-coding genes, in which case they are most frequently antisense transcripts. Their regulatory functions can be performed by a wide variety of mechanisms, both in cis on neighboring genes and in trans on distant genes or on proteins. Among the most frequent mechanisms are interference on the transcription of neighboring genes and generation of epigenetic modifications in the environment of target genes. Here, we review the most representative cases of global analyses of the presence of lncRNAs in fungal transcriptomes and describe the lncRNAs that have received more detailed attention.

Non-Coding RNA doi: 10.3390/ncrna11050071

Authors: Oenone Rodgers Anna De Beer Thomas Waterfield

Background: Distinguishing between bacterial and viral infections in children remains a significant challenge for clinicians. Traditional biomarkers have limited utility, often leading to antibiotic overprescription due to clinician uncertainty. With rising antimicrobial resistance, novel biomarkers are needed to improve diagnosis. This scoping review examines current host miRNA biomarkers for acute bacterial and viral infections in children (0–18), focusing on study methods, diagnostic metrics, and research gaps to support clinical translation. Results: Of the 1147 articles identified, 36 studies were included. Notably, 72.2% of the studies originated from Asia, and the distribution across the paediatric age groups was relatively even. A total of 17 miRNAs were validated in at least two independent studies. Three miRNAs, hsa-miR-182-5p, hsa-miR-363-3p, and hsa-miR-206, were consistently associated with bacterial infection in children. Meanwhile, nine miRNAs were associated with viral infections: hsa-miR-155, hsa-miR-29a-3p, hsa-miR-155-5p, hsa-miR-150-5p, hsa-miR-140-3p, hsa-miR-142-3p, hsa-miR-149-3p, hsa-miR-210-3p, and hsa-miR-34a-5p. Across the 12 studies reporting diagnostic accuracy metrics, miRNA biomarkers exhibited a sensitivity ranging from 70% to 100%, and a specificity ranging from 72% to 100%. The area under the curve across the studies demonstrated a range from 0.62 to 0.99. Conclusions: This scoping review highlights the potential of miRNA targets for diagnosing paediatric infections when studied rigorously. However, clinical translation is limited by poor adherence to STARD guidelines, lack of robust diagnostic metrics, and study heterogeneity. Many studies were set up with a case–control design, a design that, while highlighting differences, is more likely to identify non-specific biomarkers rather than those that are useful for novel clinical diagnostics.

Non-Coding RNA doi: 10.3390/ncrna11050070

Authors: Shailendra S. Maurya Sarita Maurya Sumit K. Chaturvedi

Acute myeloid leukemia (AML) is a highly heterogeneous disease, with significantly higher incidence and fatality rates in the elderly. Even with recent decades of research progress in AML, the exact etiology of this deadly disease is still not fully understood, with recent advancements in sequencing technologies highlighting the role of a growing number of non-coding RNAs (ncRNAs) that are intimately associated with AML leukemogenesis. These ncRNAs have been found to have a significant role in leukemia-related cellular processes such as cell division, proliferation, and death. A few of these non-coding RNAs exhibit potential as prognostic biomarkers. The three main groups of ncRNAs that contribute unique activities, especially in cancer, are microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Their existence or altered expression levels frequently offer vital information on the diagnosis, course of treatment, and follow-up of cancer patients. The identification of ncRNAs has opened up new avenues for the diagnosis, prognosis, and therapy of acute myeloid leukemia. In order to provide a clear understanding of the significant influence that lncRNAs have on prognostic predictions and diagnostic accuracy in AML, this review aims to provide a comprehensive and insightful understanding of how these molecules actively participate in the complex landscape of the disease.

Non-Coding RNA doi: 10.3390/ncrna11050069

Authors: Judit Danis Márta Széll

Psoriasis is a chronic inflammatory skin disorder affecting approximately 2% of the global population, characterized by abnormal keratinocyte proliferation and dysregulated immune responses. This review examines the emerging role of long non-coding RNAs (lncRNAs) in psoriasis pathogenesis, highlighting their significance as regulatory molecules in disease initiation, progression, and chronicity. LncRNAs demonstrate distinct expression patterns in psoriatic lesions, with upregulated transcripts such as MALAT1, XIST, MIR31HG, and HOTAIR promoting keratinocyte hyperproliferation, inhibiting apoptosis, and amplifying inflammatory cascades through mechanisms including microRNA sponging and transcription factor modulation. These molecules primarily target key signaling pathways including NF-κB, STAT3, and PI3K/AKT. Conversely, downregulated lncRNAs like NEAT1, MEG3, and PRINS normally function as tumor suppressor molecules that maintain epidermal homeostasis through pro-apoptotic and anti-inflammatory mechanisms. Their reduced expression contributes to the pathological hyperproliferative phenotype characteristic of psoriatic skin. Importantly, genetic variants within lncRNA loci have been identified as significant contributors to psoriasis susceptibility and treatment responses across different populations. Single- nucleotide polymorphisms in genes such as TRAF3IP2-AS1, HOTAIR, and CDKN2B-AS1 demonstrate population-specific associations with disease risk and therapeutic outcomes, suggesting their potential utility as pharmacogenomic markers. The complex regulatory networks involving lncRNAs provide new insights into psoriasis pathogenesis and offer promising avenues for personalized treatment strategies. Integration of lncRNA profiling into clinical practice may enhance our understanding of disease heterogeneity and improve therapeutic outcomes for psoriatic patients.

Non-Coding RNA doi: 10.3390/ncrna11050068

Authors: Bingning Xie Ann Dean

Inside the eukaryotic nucleus, various RNAs are associated with chromatin. These include protein-coding pre-mRNA and different types of non-coding RNAs that are referred to as chromatin-associated RNAs (caRNAs). Recent studies have revealed the important roles of these caRNAs in regulating gene expression and chromatin interactions. In this review, we discuss the recent advances in understanding caRNAs. We first focus on their mode of action, then we summarize the methods used to detect caRNAs and categorize them into three classes: RNA-centric, DNA-centric and protein-centric. Finally, we turn to the proteins that mediate their functions.

Non-Coding RNA doi: 10.3390/ncrna11050067

Authors: Dongning Chen Ikrame Naciri Jie Wu Sha Sun

Background/Objectives: The X-inactivation specific transcript (XIST) is a long noncoding RNA playing a crucial regulatory role in X chromosome inactivation (XCI)—a transcriptional regulatory process that silences one of the two X chromosomes in females to ensure proper dosage compensation between male and female mammals. The transcription of XIST is maintained throughout a female’s lifespan in all somatic cells, where XIST RNA binds to the X chromosome in cis and ensures chromosome-wide gene silencing. Disrupting XIST expression can lead to transcriptional reactivation of X-linked genes and epigenetic changes affecting cell development. The prevalence of XIST regulatory effects on mammalian transcription, however, remains unclarified. Methods: Here we performed a comparative expression analysis using RNA-sequencing datasets from recently published studies and examined the consequences of XIST-deletion on transcription at the whole genome, individual chromosome, and specific gene levels. We investigated the common differentially expressed genes (DEGs) and biological pathways following XIST loss across cell types, together with differential transcriptional analysis comparing the X chromosome and autosomes using cumulative distribution fractions. We analyzed the distribution of DEGs along the X chromosome with scatterplots and correlation analysis incorporating gene density and transposable elements. Results: Our findings indicate that the loss of XIST causes transcriptional changes in the X chromosome and autosomes that differ depending on cell type and state. XIST-deletion results in differential expression of genes subject to XCI-silencing as well as genes escaping XCI. In all the cell types we analyzed, X-linked genes show differential expression across the entire X chromosome in a cluster-like pattern according to gene density and, in certain cell types, correlate strongly with short interspersed nuclear element (SINE) distributions. Conclusions: Our results demonstrate that transcriptional roles of XIST can be highly associated with cell state: stem cells have different transcriptional responses compared to differentiated cells following XIST loss.

Non-Coding RNA doi: 10.3390/ncrna11050066

Authors: Parastoo Pourali Veronika Benson

Background/Objectives: Bio-produced gold nanoparticles (AuNPs) are effective carriers of short RNAs into specialized mammalian cells. Their potential application is still limited by scarce knowledge on their uptake and intracellular fate. Gold nanoparticles that are not biologically produced (NB-AuNPs) enter specialized cells primarily via clathrin-dependent endocytosis. Unlike the NB-AuNPs, the bio AuNPs possess natural surface coatings that significantly alter the AuNPs properties. Our research aimed to reveal the cellular uptake of the AuNPs with respect to delivering a functional RNA cargo. Methods: The AuNPs were conjugated with short inhibitory RNA specific to miR 135b. Mammary cancer cells 4T1 were pretreated with inhibitors of caveolin- and clathrin-mediated endocytosis and macropinocytosis. AuNPs’ uptake, fate, and miR 135b knock-down were assessed with TEM and qPCR. Results: The AuNPs-antimiR 135b conjugates entered 4T1 cells via all the tested pathways and could be seen inside the cells in early and late endosomes as well as cytoplasm. In contrast to the clathrin-dependent pathway, the caveolae-mediated endocytosis and the macropinocytosis of the AuNPs resulted in the effective targeting and reduction of the miR 135b. Conclusions: The bio-produced AuNPs can effectively enter mammalian cells simultaneously by different endocytic pathways but the delivery of functional cargo is not achieved via the clathrin-dependent endocytosis.

Non-Coding RNA doi: 10.3390/ncrna11050065

Authors: Shohei Takaoka Marcos E. Jaso-Vera Xiangbo Ruan

RNA-binding proteins (RBPs) play essential roles in all major steps of RNA processing. Genetic studies in human and mouse models support that many RBPs are crucial for maintaining homeostasis in key tissues/organs, but to what extent the function of RBPs is conserved between humans and mice is not clear. Our recent study using a chimeric humanized liver mouse model found that knocking down human HuR in human hepatocytes resulted in a broad upregulation of human genes involved in fatty acid catabolism. This regulation is human-specific, as the knocking down of mouse HuR in the liver of traditional mouse models did not show these effects. To further study this human-specific role of HuR, we co-overexpressed HuR with PPARα, a master transcription factor that promotes fatty acid catabolism, in cultured cells. We found that HuR suppressed the expression of PPARα-induced fatty acid catabolism genes in human cells but not in mouse cells. We provide evidence supporting that the human-specific suppressive effect of HuR is independent of PPARα expression or location. The regulatory effects of HuR are also independent of its role in regulating mRNA stability. Using the human HMGCS2 gene as an example, we found that the suppressive effect of HuR cannot be explained by decreased promoter activity. We further provide evidence supporting that HuR suppresses the pre-mRNA processing of HMGCS2 gene, leading to accumulated intron/pre-mRNA expression of HMGCS2 gene. Furthermore, overexpression of HuR blocked and knocking down of HuR sensitized PPARα agonist-induced gene expression. By analyzing published RNA-seq data, we found compromised pre-mRNA processing for fatty acid catabolism genes in patients with fatty liver diseases, which was not observed in mouse fatty liver disease models. Our study supports the model that HuR suppresses the expression of fatty acid catabolism genes by blocking their pre-mRNA processing, which may partially explain the mild effects of PPARα agonists in treating fatty liver diseases in humans as compared with studies in mice.

Non-Coding RNA doi: 10.3390/ncrna11050064

Authors: Manlio Fazio Fabio Stagno Giuseppa Penna Giuseppe Mirabile Alessandro Allegra

Under physiological and pathological conditions, all cells release extracellular vesicles named exosomes, which act as transporters of lipidic, protein, and genetic material from parent to recipient cells. Neoplastic cells can secrete higher number of exosomes to exert pro-tumoral effects such as microenvironmental changes, disease progression, immunosuppression and drug-resistance. This holds true for both organ-specific cancers and hematologic malignancies. One of the most important components of exosomal cargo are microRNAs which can mediate all the abovementioned effects. More specifically, microRNAs are small non-coding RNAs, routinely detected through quantitative real-time PCR, which act as translational suppressors by regulating protein-coding genes. Considering their high stability in all body fluids and viability in circulation, research is currently focusing on this type of RNAs for the so called “liquid biopsy”, a non-invasive tool for disease diagnosis and longitudinal monitoring. However, several issues remain to be solved including the lack of standardized protocols for exosome isolation and miRNA detection. Starting with this premise, our review aims to provide a wide description of the known microRNA panels employed in the prominent hematological malignancies, which will hopefully redefine the approach to these very challenging diseases in the near future.

Non-Coding RNA doi: 10.3390/ncrna11050063

Authors: Adesupo Adetowubo Sathyanarayanan Vaidhyanathan Andrey Grigoriev

Background/Objectives: Transfer RNA-derived fragments (tRFs) are small non-coding RNAs increasingly implicated in gene regulation and disease, yet their target specificity and disease relevance remain poorly understood. This is an exploratory study that investigates the phenomenon of identical tRF sequences reported in distinct disease contexts and evaluates the consistency between experimental findings and predictions from both target-based and abundance-based tRF databases. Methods: Five tRFs with identical sequences across at least two peer-reviewed disease studies were selected from a recent systematic review. Their validated targets and disease associations were extracted from the literature. Motifs and predicted targets were cross-referenced using three target-oriented databases: tatDB, tRFTar, and tsRFun. In parallel, the abundance enrichment of cancer-associated tRFs was assessed in OncotRF and MINTbase using TCGA-based abundance data. Results: Among the five tRFs, only LeuAAG-001-N-3p-68-85 showed complete alignment between experimental data and both tatDB and tRFTar predictions. Most of the other four displayed at least partial overlaps in motif/binding regions with some of validated targets. tRF abundance data from MINTbase and OncotRF showed inconsistent enrichment, with only AlaAGC-002-N-3p-58-75 exhibiting concordance with its experimentally validated cancer type. Most functionally relevant tRFs were not strongly represented in abundance-only databases. Conclusions: Given the limited number of tRFs analyzed, this study serves primarily as a pilot analysis designed to generate hypotheses and guide future in-depth research, rather than offering comprehensive conclusions. We did, however, illustrate how the analysis of tRFs can benefit from utilizing currently available databases. Target-based databases more closely reflected experimental evidence for mechanistic details when a tRF or a motif match is found. Yet all database types are incomplete, including the abundance-focused tools, which often fail to capture disease-specific regulatory roles of tRFs. These findings underscore the importance of using integrated data sources for tRF annotation. As a pilot analysis, the study provides insights into how identical tRF sequences might function differently across disease contexts, highlighting areas for further investigation while pointing out the limitations of relying on expression data alone to infer functional relevance.

Non-Coding RNA doi: 10.3390/ncrna11040062

Authors: Srinaath Narasimhan Stefan J. Erkeland

MicroRNAs (miRNAs) are key post-transcriptional regulators controlling gene expression across several cellular processes, including development, proliferation, and apoptosis. Their biogenesis involves a multi-step pathway, including the processing of primary transcripts and the assembly of the RNA-Induced Silencing Complex (RISC) with Argonaute (AGO) proteins at its core. This review provides a comprehensive overview of the molecular dynamics of miRNA-loaded RISC (miRISC), focusing on the post-translational modifications, the interactors of AGOs and the mechanisms that fine-tune and coordinate miRISC activity. The composition of miRISC influences AGO stability, localization, and silencing efficiency, thereby maintaining cellular homeostasis and development and mediating the response to various types of cellular stress. Uncommon regulatory mechanisms, including AGO modifications during, e.g., hypoxia or Type 2 T cell responses and miRISC functionality, with myriad RNA-binding proteins (RBPs), will be discussed. This review aims at highlighting the recent advances in the understanding of the intricate regulation of miRISC-driven gene silencing.

Non-Coding RNA doi: 10.3390/ncrna11040061

Authors: Ammar Ansari Aleksandra Szczesnowska Natalia Haddad Ahmed Elbediwy Nadine Wehida

Female cancers such as breast and gynaecological cancers contribute to a significant global health burden and are a leading cause of fatality among women. With current treatment options often limited by resistance to cytotoxic drugs, side effects and lack of specificity to the cancer, there is a pressing need for alternative treatments. Recent research has highlighted the promising role of non-coding RNAs (ncRNA) in regulating these issues and providing more targeted approaches to suppressing key cancer pathways. This review explores the involvement of the various types of non-coding RNAs in regulating key oncogenic pathways, namely, the MAPK, PI3K/Akt/mTOR, Wnt/β-catenin and p53 pathways, in a range of female cancers such as breast, cervical, ovarian and endometrial cancers. Evidence from a multitude of studies suggests that non-coding RNAs function as double-edged swords, serving as both oncogenes and tumour suppressors, depending on their expression and cellular interactions. By mapping and investigating these regulatory interactions, this review demonstrates the complexity and dual functionality of ncRNAs in cancer. Understanding these complex mechanisms is essential for the development of new and effective ncRNA-based diagnostic methods and targeted therapies in female cancer treatment.

Non-Coding RNA doi: 10.3390/ncrna11040060

Authors: Andrea Corsi Tonia De Simone Angela Valentino Elisa Orlandi Chiara Stefani Cristina Patuzzo Stefania Fochi Maria Giusy Bruno Elisabetta Trabetti John Charles Rotondo Chiara Mazziotta Maria Teresa Valenti Alessandra Ruggiero Donato Zipeto Cristina Bombieri Maria Grazia Romanelli

Background/Objectives: Non-coding microRNA-34a (miR-34a) regulates the expression of key factors involved in several cellular processes, such as differentiation, apoptosis, proliferation, cell cycle, and senescence. Deregulation of the expression of these factors is implicated in the onset and progression of several human diseases, including cancer, neurodegenerative disorders, and pathologies associated with viral infections and inflammation. Despite numerous studies, the molecular mechanisms regulated by miR-34a remain to be fully understood. The present study aimed to generate miR-34a knockout cell lines to identify novel genes potentially regulated by its expression. Methods: We employed the CRISPR-Cas9 gene editing system to knock out the hsa-miR-34a gene in HeLa and 293T cell lines, two widely used models for studying molecular and cellular mechanisms. We compared proliferation rates and gene expression profiles via RNA-seq and qPCR analyses between the wild-type and miR-34a KO cell lines. Results: Knockout of miR-34a resulted in a decreased proliferation rate in both cell lines. Noteworthy, the ablation of miR-34a resulted in increased expression of the long non-coding RNA MALAT1. Additionally, miR-34a-5p silencing in the A375 melanoma cell line led to MALAT1 overexpression. Conclusions: Our findings support the role of the miR-34a/MALAT1 axis in regulating proliferation processes.

Non-Coding RNA doi: 10.3390/ncrna11040059

Authors: Elizaveta Korunova B. Celia Cui Hao Ji Aliaksandra Sikirzhytskaya Srestha Samaddar Mengqian Chen Vitali Sikirzhytski Michael Shtutman

Stress granule formation is a type of liquid–liquid phase separation in the cytoplasm, leading to RNA–protein condensates that are associated with various cellular stress responses and implicated in numerous pathologies, including cancer, neurodegeneration, inflammation, and cellular senescence. One of the key components of mammalian stress granules is the DEAD-box RNA helicase DDX3, which unwinds RNA in an ATP-dependent manner. DDX3 is involved in multiple steps of RNA metabolism, facilitating gene transcription, splicing, and nuclear export and regulating cytoplasmic translation. In this study, we investigate the role of the RNA helicase DDX3’s enzymatic activity in shaping the RNA content of ribonucleoprotein (RNP) condensates formed during arsenite-induced stress by inhibiting DDX3 activity with RK-33, a small molecule previously shown to be effective in cancer clinical studies. Using the human osteosarcoma U2OS cell line, we purified the RNP granule fraction and performed RNA sequencing to assess changes in the RNA pool. Our results reveal that RK-33 treatment alters the composition of non-coding RNAs within the RNP granule fraction. We observed a DDX3-dependent increase in circular RNA (circRNA) content and alterations in the granule-associated intronic RNAs, suggesting a novel role for DDX3 in regulating the cytoplasmic redistribution of non-coding RNAs.

Non-Coding RNA doi: 10.3390/ncrna11040057

Authors: Gantsetseg Garmaa Stefania Bunduc Tamás Kói Péter Hegyi Dezső Csupor Dariimaa Ganbat Fanni Dembrovszky Fanni Adél Meznerics Ailar Nasirzadeh Cristina Barbagallo Gábor Kökény

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Non-Coding RNA doi: 10.3390/ncrna11040058

Authors: Leandro Teodoro Júnior Mari Cleide Sogayar

Background/Objectives: Increasing evidence suggests that lncRNAs are core regulators in the field of tumor progression, with context-specific functions in oncogenic tumorigenesis. LINC01133, a lncRNA that has been identified as both an oncogene and a tumor suppressor, remains largely unexplored in terms of its molecular mechanisms. The purpose of this study was to conduct an in silico analysis, incorporating literature research on various cancer types, to investigate the structural and functional duality of LINC01133. This analysis aimed to identify pathways influenced by LINC01133 and evaluate its mechanism of action as a potential therapeutic target and diagnostic biomarker. Methods: In silico analyses and a narrative review of the literature were performed to predict conserved structural elements, functional internal loops, and overall conservation of the LINC01133 sequence among different vertebrate organisms, summarizing the empirical evidence regarding its roles as a tumor suppressor and tumor-promoting roles in various types of tumors. Results: LINC01133 harbors the evolutionarily conserved structural regions that might allow for binding to relevant driver signaling pathways, substantiating its specific functionality. Its action extends beyond classical tumor mechanisms, affecting proliferation, migration, invasion, and epigenetic pathways in various types of tumors, as indicated by the in silico results and narrative review of the literature we present here. Clinical outcome associations pointed to its potential as a biomarker. Conclusions: The dual character of LINC01133 in tumor biology further demonstrates its prospective therapeutic value, but complete elucidation of its mechanisms of action requires further investigation. This study establishes LINC01133 as a multifaceted lncRNA, supporting context-specific strategies in targeting its pathways, and calls for expanded research to harness its full potential in oncology.

Non-Coding RNA doi: 10.3390/ncrna11040056

Authors: Raffaele Frazzi Enrico Farnetti Davide Nicoli

Background/Objectives: Krüppel-like factor 4 (KLF4) emerged as an epigenetically regulated gene in a variety of settings, including cell reprogramming and malignant cell proliferation. The aim of the present manuscript is to explore the relationship described in recent years between circular RNAs, miRNAs, and KLF4. These have been shown to be involved in cancers having diverse histological origins, including some of the most prevalent and deadly tumors for the human population. Expression and protein levels of this transcription factor correlate with invasiveness and prognosis in a context- and tissue-specific fashion. Methods: The literature was obtained through two main PubMed queries. The first is “miRNA and KLF4 and cancer” and is limited to the last 5 years. The second is “circRNA and KLF4”, which yielded publications between 2013 and 2024. The oncological publications were selected. Results: A number of circRNA/miRNA axes that regulate the downstream transcription factor KLF4 emerged in the last few years. circRNAs act as sponges for miRNAs and synergize with KLF4, which can function as either a tumor promoter or suppressor in different tumors. Conclusions: The axes represented by circRNA/miRNA/KLF4 emerged as a new layer of epigenetic regulation. These RNA-based modulators explain the complex regulation of this transcription factor and open the way to new therapeutic targeting possibilities.

Non-Coding RNA doi: 10.3390/ncrna11040055

Authors: Irma A. Jiménez-Ramírez Miguel A. Uc-Chuc Luis Carlos Rodríguez Zapata Enrique Castaño

Background: snoRNAs have traditionally been known for their role as guides in post-transcriptional rRNA modifications. Previously, our research group identified several RNAs that may bind to PIP2 with LIPRNA-seq. Among them, snR191 stood out due to its potential specific interaction with this lipid, distinguishing itself from other snoRNAs. However, a detailed study is needed to define the molecular interactions between RNA and lipids, which remain unknown but may serve as a mechanism for transport or liquid–liquid phase separation. This study aimed to determine the interaction between a snoRNA called snR191 and PIP2. Method: A novel methodology for RNA-PIP2 interaction was carried out. Total RNA from Saccharomyces cerevisiae was incubated with PIP2-bound nitrocellulose membranes and RT-PCR reactions. We performed the prediction of snR191-PIP2 interaction by molecular docking and in silico mutations of snoR191. Results: From LIPRNA-seq analysis, we identified that PIP2-bound RNAs were significantly enriched in diverse biological processes, including transmembrane transport and redox functions. Our RNA-PIP2 interaction approach was successful. We demonstrated that snR191 specifically interacts with PIP2 in vitro. The elimination of DNA ensured that the interaction assay was RNA-specific, strengthening the robustness of the experiment. PIP2 was docked to snR191 in a stem–loop–stem motif. Six hydrogen bonds across four nucleotides mediated the PIP2-snR191 interaction. Finally, mutations in snR191 affected the structural folding. Conclusions: In this study, we demonstrate the effectiveness of a new methodology for determining RNA–lipid interactions, providing strong evidence for the specific interaction between snR191 and PIP2. Integrating biochemical and computational approaches has allowed us to understand the binding of these biomolecules. Therefore, this work significantly broadens our understanding of snR191-PIP2 interactions and opens new perspectives for further research.

Non-Coding RNA doi: 10.3390/ncrna11040054

Authors: Lola Jaque-Cabrera Julia Buggiani Jérôme Bignon Patricia Daira Nathalie Bernoud-Hubac Jean-René Martin

Background/Objectives: Cancer research aims to understand the cellular and molecular mechanisms involved, in order to identify new therapeutic targets and provide patients with more effective therapies that generate fewer side undesirable and toxic effects. Previous studies have demonstrated the role of small nucleolar RNAs (snoRNAs) in many physiological and pathological cellular processes, including cancers. SnoRNAs are a group of non-coding RNAs involved in different post-transcriptional modifications of ribosomal RNAs. Recently, we identified a new snoRNA (jouvence), first in Drosophila, and thereafter, by homology, in humans. Methods: Here, we characterize the effect of the knockdown of jouvence by a sh-lentivirus on human primary patient-derived glioblastoma cells. Results: The sh-lentivirus anti-jouvence induces a significant decrease in cell proliferation and leads to cell death. EdU staining confirmed this decrease, while TUNEL also showed the presence of apoptotic cells. An RNA-Seq analysis revealed a decrease, in particular, in the level of BAALC, a gene known to potentiate the oncogenic ERK pathway and deregulating p21, leading to cell cycle blockage. Conclusions: Altogether, these results allow the hypothesis that the knockdown of jouvence could potentially be used as a new anti-cancer treatment (sno-Therapy), especially against glioblastoma and also, potentially, against acute myeloid leukemia (AML) due to the BAALC deregulation.

Non-Coding RNA doi: 10.3390/ncrna11040053

Authors: Raffaele Garraffo Manuel Beltran Nebot

Circular RNAs (circRNAs) are covalently closed RNA molecules generated through a non-canonical splicing event known as back-splicing. This particular class of non-coding RNAs has attracted growing interest due to its evolutionary conservation across eukaryotes, high expression in the central nervous system, and frequent dysregulation in various pathological conditions, including cancer. Traditionally, circRNAs have been characterised by their ability to function as microRNA (miRNA) and protein sponges. However, recent discoveries from multiple research groups have uncovered a novel and potentially transformative mechanism of action: the direct interaction of circRNAs with messenger RNAs (mRNAs) to regulate their fate. These interactions can influence mRNA stability and translation, revealing a new layer of post-transcriptional gene regulation. In this review, we present and analyse the latest evidence supporting the emerging role of circRNAs in diverse biological contexts. We highlight the growing body of research demonstrating circRNA-mRNA interactions as a functional regulatory mechanism and explore their involvement in key physiological and pathophysiological processes. Understanding this novel mechanism expands our knowledge of RNA-based regulation and opens new opportunities for therapeutic strategies targeting circRNA-mRNA networks in human disease.

Non-Coding RNA doi: 10.3390/ncrna11040052

Authors: Beshoy Armanios Jing Jin Holly Kolmel Ankit P. Laddha Neha Mishra Jose E. Manautou Xiao-Bo Zhong

Background/Objectives: Long non-coding RNAs (lncRNAs) play significant roles in tissue development and disease progression and have emerged as crucial regulators of gene expression. The hepatocyte nuclear factor alpha antisense RNA 1 (HNF1A-AS1) lncRNA is a particularly intriguing regulatory molecule in liver biology that is involved in the regulation of cytochrome P450 enzymes via epigenetic mechanisms. Despite the growing recognition of lncRNAs in liver disease, the comprehensive role of HNF1A-AS1 in liver function remains unclear. This study aimed to investigate the roles of the mouse homolog of the human HNF1A-AS1 lncRNA HNF1A opposite strand 1 (Hnf1aos1) in liver function, gene expression, and cellular processes using a mouse model to identify potential therapeutic targets for liver disorders. Methods: The knockdown of Hnf1aos1 was performed in in vitro mouse liver cell lines using siRNA and in vivo livers of AAV-shRNA complexes. Changes in the global expression landscapes of mRNA and proteins were revealed using RNA-seq and proteomics, respectively. Changes in the selected genes were further validated via real-time quantitative polymerase chain reaction (RT-qPCR). Phenotypic changes were assessed via histological and absorbance-based assays. Results: After the knockdown of Hnf1aos1, RNA-seq and proteomics analysis revealed the differential gene expression of the mRNAs and proteins involved in the processes of molecular transport, liver regeneration, and immune signaling pathways. The downregulation of ABCA1 and SREBF1 indicates their role in cholesterol transport and fatty acid and triglyceride synthesis. Additionally, significant reductions in hepatic triglyceride levels were observed in the Hnf1aos1-knockdown group, underscoring the impact on lipid regulation. Notably, the knockdown of Hnf1aos1 also led to an almost complete depletion of CYP7A1, the rate-limiting enzyme in bile acid synthesis, highlighting its role in cholesterol homeostasis and hepatotoxicity. Histological assessments confirmed these molecular findings, with increased hepatic inflammation, hepatocyte swelling, and disrupted liver architecture observed in the Hnf1aos1-knockdown mice. Conclusions: This study illustrated that Hnf1aos1 is a critical regulator of liver health, influencing both lipid metabolism and immune pathways. It maintains hepatic lipid homeostasis, modulates lipid-induced inflammatory responses, and contributes to viral immunity, indirectly affecting glucose and lipid metabolic balance.

Non-Coding RNA doi: 10.3390/ncrna11040051

Authors: Mario García-Domínguez

Chronic pain is a multifactorial and complex condition that significantly affects individuals’ quality of life. The underlying mechanisms of chronic pain involve complex alterations in neural circuits, gene expression, and cellular signaling pathways. Recently, ncRNAs, such as miRNAs, lncRNAs, circRNAs, and siRNAs, have been identified as crucial regulators in the pathophysiology of chronic pain. These ncRNAs modulate gene expression at both the transcriptional and post-transcriptional levels, affecting pain-related pathways like inflammation, neuronal plasticity, and sensory processing. miRNAs have been shown to control genes involved in pain perception and nociceptive signaling, while lncRNAs interact with chromatin remodeling factors and transcription factors to modify pain-related gene expression. CircRNAs act as sponges for miRNAs, thereby influencing pain mechanisms. siRNAs, recognized for their gene-silencing capabilities, also participate in regulating the expression of pain-related genes. This review examines the diverse roles of ncRNAs in chronic pain, emphasizing their potential as biomarkers for pain assessment and as targets for novel therapeutic strategies. A profound understanding of the ncRNA-mediated regulatory networks involved in chronic pain could result in more effective and personalized pain management solutions.

Non-Coding RNA doi: 10.3390/ncrna11040050

Authors: Roberto Piergentili Enrico Marinelli Gaspare Cucinella Alessandra Lopez Gabriele Napoletano Giuseppe Gullo Simona Zaami

There was an error in the original publication [...]

Non-Coding RNA doi: 10.3390/ncrna11040049

Authors: Weijun Yi Jason R. Miller Gangqing Hu Donald A. Adjeroh

Background: Long non-coding Ribonucleic Acids (lncRNAs) can be localized to different cellular compartments, such as the nuclear and the cytoplasmic regions. Their biological functions are influenced by the region of the cell where they are located. Compared to the vast number of lncRNAs, only a relatively small proportion have annotations regarding their subcellular localization. It would be helpful if those few annotated lncRNAs could be leveraged to develop predictive models for localization of other lncRNAs. Methods: Conventional computational methods use q-mer profiles from lncRNA sequences and train machine learning models such as support vector machines and logistic regression with the profiles. These methods focus on the exact q-mer. Given possible sequence mutations and other uncertainties in genomic sequences and their role in biological function, a consideration of these variabilities might improve our ability to model lncRNAs and their localization. Thus, we build on inexact q-mers and use machine learning/deep learning techniques to study three specific problems in lncRNA subcellular localization, namely, prediction of lncRNA localization using inexact q-mers, the issue of whether lncRNA localization is cell-type-specific, and the notion of switching (lncRNA) genes. Results: We performed our analysis using data on lncRNA localization across 15 cell lines. Our results showed that using inexact q-mers (with q = 6) can improve the lncRNA localization prediction performance compared to using exact q-mers. Further, we showed that lncRNA localization, in general, is not cell-line-specific. We also identified a category of LncRNAs which switch cellular compartments between different cell lines (we call them switching lncRNAs). These switching lncRNAs complicate the problem of predicting lncRNA localization using machine learning models, showing that lncRNA localization is still a major challenge.

Non-Coding RNA doi: 10.3390/ncrna11030048

Authors: Dalliane Oliveira Soares Lucas Yago Melo Ferreira Gabriel Victor Pina Rodrigues João Pedro Nunes Santos Ícaro Santos Lopes Lucas Barbosa de Amorim Conceição Tatyana Chagas Moura Isaque João da Silva de Faria Roenick Proveti Olmo Weyder Cristiano Santana Marco Antônio Costa Eric Roberto Guimarães Rocha Aguiar

MicroRNAs (miRNAs) are key post-transcriptional regulators involved in a wide range of biological processes in insects, yet little is known about their roles in stingless bees. Here, we present the first characterization of miRNAs in Melipona quadrifasciata using small RNAs (sRNAs) deep sequencing. A total of 193 high-confidence mature miRNAs were identified, including 106 M. quadrifasciata-exclusive sequences. Expression profiling revealed that mqu-miR-1 and mqu-miR-276 together accounted for over 70% of all miRNA reads, suggesting their central roles in development and reproduction. Comparative analyses showed a higher conservation of M. quadrifasciata miRNAs with other Hymenopterans, especially Apis mellifera and Bombus spp. Putative target genes were predicted using a consensus approach, and functional annotation indicated their involvement in diverse biological regulatory pathways. This work represents the first comprehensive identification of the miRNA repertoire in stingless bees using sRNAs and provides a valuable foundation for understanding miRNA-mediated gene regulation in this ecologically and economically important pollinator.

Non-Coding RNA doi: 10.3390/ncrna11030047

Authors: Sherien M. El-Daly Sahar S. Abdelrahman Amira Mohamed Abd El-Jawad Mahmoud A. Abdel-Monem Gamila S. M. El-Saeed

Background: Identifying liquid biopsy biomarkers with high efficacy is crucial for cancer diagnosis. Exosomal cargo, including miRNAs and proteins, offers enhanced stability in biofluids compared with their free circulating forms, but direct comparisons of their diagnostic performance remain limited. This study evaluates and compares the diagnostic value of selected miRNAs and protein markers in exosomal versus non-exosomal fractions across stages of lung carcinogenesis in a rat model. Methods: Lung cancer was induced in rats, and blood and lung tissue samples were collected at consecutive stages of tumor induction. We investigated the expression patterns of key miRNAs (miR-19b, miR-21, and miR-145) in exosomes, serum, and tissue and quantified levels of tumor biomarkers CEA and CYFRA 21-1 in exosomal and serum fractions. Results: Our results revealed distinct expression patterns of the evaluated miRNAs across exosomes, serum, and tissue, throughout different stages of tumor induction. The expression of exosomal miRNAs dynamically changed in parallel with the tumor induction process, demonstrating high diagnostic efficacy. Specifically, exosomal miR-19b and miR-21 were significantly upregulated from an early induction stage, whereas their serum and tissue forms increased only during the late stages of induction. On the other hand, miR-145 was consistently downregulated across all fractions at every stage. Both exosomal and serum CEA levels increased significantly during tumor induction, while serum CYFRA 21-1 outperformed its exosomal counterpart. Strong positive correlations linked exosomal miR-19b and miR-145 with their non-exosomal counterparts, while moderate correlations were seen for miR-21 and the protein markers. Conclusions: Our findings underscore the value of integrating exosomal biomarkers in liquid biopsies, highlighting their potential to improve early detection and monitoring of lung cancer development.

Non-Coding RNA doi: 10.3390/ncrna11030046

Authors: Caroline Eva Riedel Javier Ibáñez Annunziata Fragasso Angelika Schmitt Manuel Widmann Felipe Mattioni Maturana Andreas M. Niess Barbara Munz

Background: Physical activity induces a range of physiological and molecular adaptations, particularly affecting skeletal muscle and the cardiovascular system, regulating both tissue architecture and metabolic pathways. Emerging evidence suggests that PIWI-interacting RNAs (piRNAs) may serve as potential biomarkers for these adaptations. Here, we analyzed piRNA patterns in the context of exercise. Methods: This study selected eight participants of the iReAct study (DRKS00017446) for piRNA analysis. Baseline assessments included demographic profiling and fitness evaluation, particularly maximal oxygen uptake (V̇O2max) assessment. In addition, blood samples were collected pre- and (for six of the eight participants) post- standard reference training sessions. Subsequently, subjects underwent 6-week training protocols, employing standardized high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) regimens. Next, RNA sequencing was conducted to identify differentially expressed piRNAs, and correlation analyses were performed between piRNA expression patterns and training-associated changes in V̇O2max. Finally, to identify piRNAs potentially of interest in the context of exercise, different screening procedures were applied. Results: There were unique and specific changes in individual piRNA expression levels in response to exercise. In addition, we could define correlations of piRNA expression patterns, namely of piR-32886, piR-33151, piR-12547, and piR-33074, with changes in V̇O2max. These correlations did not reach significance in the small sample size of this pilot study, but might be verified in larger, confirming studies. Conclusions: This hypothesis-generating study identifies characteristic piRNA patterns in the context of exercise. Their significance as biomarkers is yet to be determined.

Non-Coding RNA doi: 10.3390/ncrna11030045

Authors: Verena Gisa Md Rezaul Islam Dawid Lbik Raoul Maximilian Hofmann Tonatiuh Pena Dennis Manfred Krüger Susanne Burkhardt Anna-Lena Schütz Farahnaz Sananbenesi Karl Toischer Andre Fischer

Background: Heart failure (HF) is associated with an increased risk of cognitive impairment and hippocampal dysfunction, yet the underlying molecular mechanisms remain poorly understood. This study aims to investigate the role of microRNA (miRNA) networks in hippocampus-dependent memory recovery in a mouse model of HF. Methods: CaMKIIδC transgenic (TG) mice, a model for HF, were used to assess hippocampal function at 3 and 6 months of age. Memory performance was evaluated using hippocampus-dependent behavioral tasks. Small RNA sequencing was performed to analyze hippocampal miRNA expression profiles across both time points. Bioinformatic analyses identified miRNAs that potentially regulate genes previously implicated in HF-induced cognitive impairment. Results: We have previously shown that at 3 months of age, CaMKIIδC TG mice exhibited significant memory deficits associated with dysregulated hippocampal gene expression. In this study, we showed that these impairments, memory impairment and hippocampal gene expression, were no longer detectable at 6 months, despite persistent cardiac dysfunction. However, small RNA sequencing revealed a dynamic shift in hippocampal miRNA expression, identifying 27 miRNAs as “compensatory miRs” that targeted 73% of the transcripts dysregulated at 3 months but reinstated by 6 months. Notably, miR-181a-5p emerged as a central regulatory hub, with its downregulation coinciding with restored memory function. Conclusions: These findings suggest that miRNA networks contribute to the restoration of hippocampal function in HF despite continued cardiac pathology and provide an important compensatory mechanism towards memory impairment. A better understanding of these compensatory miRNA mechanisms may provide novel therapeutic targets for managing HF-related cognitive dysfunction.

Non-Coding RNA doi: 10.3390/ncrna11030044

Authors: Emma R. Blaustein Coen van Solingen

CHROMR is a primate-specific long noncoding RNA with emerging roles in homeostasis and pathophysiology. Elevated blood levels of CHROMR have been observed in patients with cardiovascular disease and several cancers, where it is correlated with poor clinical outcomes. Like many lncRNAs, CHROMR accumulates in both the nucleus and the cytoplasm, and it assumes distinct functions in each of these cellular compartments. In the nucleus, CHROMR sequesters a transcriptional repressor complex to activate interferon-stimulated gene expression and antiviral immunity. In the cytoplasm, CHROMR competitively inhibits microRNAs involved in cholesterol efflux and cell cycle regulation, thereby impacting gene pathways involved in reverse cholesterol transport, HDL biogenesis, and tumor growth. In this review, we detail the multifaceted functions of CHROMR in cholesterol metabolism, innate immunity, and cancer progression. We also explore the potential molecular mechanisms that govern its expression and dynamic subcellular localization, which may be key to its functional versatility. Advancing our understanding of the regulatory networks and cellular environments that shape CHROMR activity will be critical for assessing its promise as a therapeutic target and diagnostic biomarker.

Non-Coding RNA doi: 10.3390/ncrna11030043

Authors: Daniel J. García Marco A. Pulpillo-Berrocal José L. Ruiz Eduardo Andrés-León Laura C. Terrón-Camero

Introduction: Anal squamous cell carcinoma (ASCC) is a rare but increasingly common gastrointestinal malignancy, mainly associated with oncogenic human papillomaviruses (HPVs). The role of non-coding RNAs (ncRNAs) in tumorigenesis is recognized, but the impact of viral ncRNAs on host gene expression remains unclear. Methods: We re-analyzed total RNA-Seq data from 70 anal biopsies: 31 low-grade squamous intraepithelial lesions (LGSIL), 16 high-grade SIL (HGSIL), and 23 ASCC cases. Microbial composition was assessed taxonomically. Novel viral miRNAs were predicted using vsRNAfinder and linked to host targets using TargetScan and expression correlation analyses. Results: Microbial profiling revealed significant differences in abundance, with Alphapapillomaviruses types 9, 10, and 14 enriched across lesion grades. We identified 90 novel viral miRNAs and 177 significant anti-correlated miRNA–mRNA interactions. Target genes were enriched in pathways related to cell cycle, epithelial–mesenchymal transition, lipid metabolism, immune modulation, and viral replication. Discussion: Our findings suggest that HPV-derived miRNAs, including those from low-risk types, may contribute to neoplastic transformation by modulating host regulatory networks. Conclusion: This study highlights viral miRNAs as potential drivers of HPV-related anal cancer and supports their utility as early biomarkers and therapeutic targets in ASCC.

Non-Coding RNA doi: 10.3390/ncrna11030042

Authors: Anindita Sarma Parul Suri Megan Justice Raja Angamuthu Samuel Pushparaj

The poultry farming industry encounters considerable obstacles stemming from viral diseases, resulting in elevated mortality rates and substantial economic losses. Current research highlights the significant involvement of long non-coding RNAs (lncRNAs) in the interactions between hosts and pathogens by enhancing antiviral responses at different levels, such as the activation of pathogen recognition receptors, as well as through epigenetic, transcriptional, and post-transcriptional modifications. Specific long non-coding RNAs (lncRNAs), including ERL lncRNA, linc-GALMD3, and loc107051710, have been recognized as significant contributors to the antiviral immune response to multiple avian viral pathogens. Understanding the mechanisms by which long non-coding RNAs (lncRNAs) act offers valuable insights into prospective diagnostic and therapeutic approaches aimed at improving disease resistance in poultry. Differentially expressed lncRNAs may also be utilized as biomarkers for both prognosis and diagnosis of avian viral diseases. This review delves into the various roles of long non-coding RNAs (lncRNAs) in the context of viral diseases in chickens, such as avian leukosis, Marek’s disease, infectious bursal disease, avian influenza, infectious bronchitis, and Newcastle disease. It highlights the pivotal role of lncRNAs in the complex dynamics between the host and viral pathogens, particularly their interactions with specific viral proteins. Understanding these interactions may provide valuable insights into the spatial and temporal regulation of lncRNAs, aid in the identification of potential drug targets, and reveal the expression patterns of lncRNA and coding gene transcripts in response to different viral infections in avian species.

Non-Coding RNA doi: 10.3390/ncrna11030041

Authors: Carolyn M. Klinge Julia H. Chariker Kellianne M. Piell Belinda J. Petri Eric C. Rouchka Matthew C. Cave

Background/Objectives: Exposure of high-fat diet (HFD)-fed mice to polychlorinated biphenyls (PCBs) results in metabolic dysfunction-associated steatotic liver disease (MASLD) and progression to metabolic dysfunction-associated steatohepatitis (MASH). The mechanisms by which HFD diet and PCBs increase MASLD are unclear. Previously, we identified differences in HFD-fed mouse liver tRNA modifications with single oral exposures to the dioxin-like PCB126, the non-dioxin-like PCB mixture Aroclor 1260 (Ar1260), or the combination of Ar1260 + PCB126. Methods: Here, we used small RNA sequencing and the tRNA analysis of expression (tRAX) pipeline to examine if PCB exposures alter the tRNA transcriptome, including tRNA-derived fragments (tRFs), in the livers of the PCB-exposed mice. Results: Each PCB exposure produced distinct hepatic tRNA transcriptomes with more tRNAs decreased than increased. Only tRNA-Glu-TTC-1 was reduced with all three PCB exposures. More changes in tRFs were identified with Ar1260 alone or in combination with PCB126 than with PCB126 alone. Four tRF-3s were upregulated in both PCB126 and Ar1260 + PCB126 co-exposed mice, suggesting PCB126 as responsible for this increase. We previously reported that PCB126 exposure increased hepatic Angiogenin (ANG) protein which generates tRF-3s. Four previously reported tRNA modifications corresponded to positions of PCB-associated tRNA modifications identified by tRAX: m1A, m6A, ms2t6A, and Ψ. Conclusions: Overall, the differences in hepatic tRNAs and tRFs with three different PCB exposures suggest that PCB exposures play an unexplored role in regulating translation in mouse liver.

Non-Coding RNA doi: 10.3390/ncrna11030040

Authors: Verena Schlösser Helen Louise Lightfoot Christine Leemann Seyedeh Elnaz Banijamali Aathma Merin Bejoy Shashank Tiwari Jeffrey L. Schloßhauer Valentina Vongrad Andreas Brunschweiger Jonathan Hall Karin J. Metzner Jochen Imig

Seyedeh Elnaz Banijamali was not included as an author in the original publication [...]

Non-Coding RNA doi: 10.3390/ncrna11030039

Authors: Olga R. Borodulina Sergey A. Kosushkin Ilia G. Ustyantsev Nikita S. Vassetzky Dmitri A. Kramerov

Background/Objectives: SINEs (short interspersed elements) are eukaryotic non-autonomous retrotransposons. They are transcribed by RNA polymerase III (pol III) and generate non-coding RNAs. The 3′ end of many mammalian SINEs contains a polyadenylation signal (AATAAA), a pol III transcription terminator, and an A-rich tail. Studies have shown that, in human HeLa cells that have been transiently transfected with such SINEs, short pol III-generated SINE transcripts undergo polyadenylation, resulting in the addition of a long poly(A)-tail. Notably, this AAUAAA-dependent polyadenylation is not characteristic of any other transcripts synthesized by pol III. B2 SINEs, found in the genomes of mouse-like rodents, exemplify all these features. Methods: In this study, we implemented a novel approach to sequencing pol III-generated B2 transcripts from mouse cell cultures (L929 and 4T1) and organs (brain and testis). Results: Transcription occurred in 16,000–20,000 B2 copies in each cell type, 51–62% of which were transcribed in all four cell types. Effective transcription terminators (e.g., TCT>3 and T≥4) were found in approximately 40% of the transcribed B2 copies. The transcripts of these B2 copies contained a truncated terminator sequence, as pol III transcriptional arrest is known to occur within the terminator, with a poly(A)-tail immediately downstream. Such a tail could only have formed through RNA polyadenylation. Conclusions: These results demonstrate that B2 transcripts synthesized by pol III are capable of polyadenylation in mouse cells. We discuss the transcription of B2 copies with and without moderately efficient pol III terminators (TCTTT) and provide examples of the polyadenylation of such transcripts.

Non-Coding RNA doi: 10.3390/ncrna11030038

Authors: Mafalda Antunes-Ferreira Ilias Glogovitis Diogo Fortunato Silvia D’Ambrosi Mariona Colom Saborit Galina Yahubyan Vesselin Baev Michael Hackenberg Natasa Zarovni Thomas Wurdinger Danijela Koppers-Lalic

Background: Platelet-derived Extracellular Vesicles, or “Platelet Dust” (PD), are reported as the most-abundant extracellular vesicles in plasma. However, the PD molecular content, especially the small RNA profile, is still poorly characterized. This study aims to characterize PD and other extracellular vesicles (EVs) in patients with non-small-cell lung cancer (NSCLC), focusing on their small RNA signatures and diagnostic potential. Methods: The EVs were isolated directly from the plasma of healthy donors and patients with NSCLC using the surface markers CD9, CD63, CD81 (overall EVs), and CD61 (PD). Small RNA sequencing was then performed to comprehensively profile the miRNAs. Results: Our analysis revealed distinct small RNA profiles in the EVs and the PD from the patients with NSCLC. The EVs (CD9-, CD63-, and CD81-positive) showed the enrichment of four miRNAs and the depletion of ten miRNAs, while the PD (CD61-positive) exhibited a more complex profile, with nineteen miRNAs enriched and nine miRNAs depleted in the patients with NSCLC compared to those of the healthy controls. Conclusions: This exploratory study enhances our understanding of miRNA composition within different plasma vesicle populations, shedding light on the biology of plasma vesicles and their contents. Furthermore, utilizing an extracellular vesicle isolation method with potential clinical applicability offers the prospect of improved cancer characterization and detection by selecting the most informative subpopulation of plasma vesicles.

Non-Coding RNA doi: 10.3390/ncrna11030037

Authors: Montiel Guerrero-Sabater María Cosín-Villanueva Pedro Almiñana-Pastor Andrés López-Roldán

Objectives: Periodontitis is a chronic inflammatory disease that could influence the pathophysiology of cardiovascular diseases through immunoinflammatory and epigenetic mechanisms. MicroRNAs (miRNAs) could be key mediators in this interaction, regulating gene expression and the synthesis of inflammatory molecules. The objective of this systematic review was to evaluate the relationship between periodontitis and cardiovascular diseases by analyzing the expression of miRNAs involved in both pathologies. Methods: A systematic search was performed in the PubMed, Scopus, Embase, and Web of Science databases following the PRISMA guidelines. A total of 320 studies were identified, of which seven were included after applying eligibility criteria. Data on study design, sample characteristics, periodontal and cardiovascular diagnostic methodology, and the analyzed miRNAs were extracted. Results: The included studies were observational case-control studies in humans (n = 5) and experimental studies in animal models (n = 3). The miRNAs selected by the studies to link both pathologies were miR-155, miR-155-5p, miR-146a, miR-143, miR-145, and miR-23b. Most studies observed the overexpression of these miRNAs in patients with periodontitis and cardiovascular disease, with miR-146a being the most frequently associated. Conclusions: The findings suggest that certain miRNAs, particularly miR-146a, may play a key role in the connection between periodontitis and cardiovascular disease. Its overexpression in patients with both pathologies reinforces the hypothesis of its involvement in the inflammatory processes associated with both conditions. It would be interesting to conduct studies to validate their clinical applicability as biomarkers of susceptibility to cardiovascular disease.

Non-Coding RNA doi: 10.3390/ncrna11030036

Authors: Rajdeep Banerjee

Bacterial pathogens have evolved diverse strategies to infect hosts, evade immune responses, and establish successful infections. While the role of transcription factors in bacterial virulence is well documented, emerging evidence highlights the significant contribution of small regulatory RNAs (sRNAs) in bacterial pathogenesis. These sRNAs function as posttranscriptional regulators that fine-tune gene expression, enabling bacteria to adapt rapidly to challenging environments. This review explores the multifaceted roles of bacterial sRNAs in host–pathogen interactions. Firstly, it examines how sRNAs regulate pathogenicity by modulating the expression of key virulence factors, including fimbriae, toxins, and secretion systems, followed by discussing the role of sRNAs in bacterial stress response mechanisms that counteract host immune defenses, such as oxidative and envelope stress. Additionally, this review investigates the involvement of sRNAs in antibiotic resistance by regulating efflux pumps, biofilm formation, and membrane modifications, which contribute to multi-drug resistance phenotypes. Lastly, this review highlights how sRNAs contribute to intra- and interspecies communication through quorum sensing, thereby coordinating bacterial behavior in response to environmental cues. Understanding these regulatory networks governed by sRNAs is essential for the development of innovative antimicrobial strategies. This review highlights the growing significance of sRNAs in bacterial pathogenicity and explores their potential as therapeutic targets for the treatment of bacterial infections.

Non-Coding RNA doi: 10.3390/ncrna11030035

Authors: Maria Jose Navarro-Cobos Carolyn J. Brown

Dimorphism of sex chromosomes often leads to a need for dosage compensation. In eutherian mammals, XIST, a long non-coding RNA, is expressed from the X chromosome that will be silenced, triggering X-chromosome inactivation (XCI). XIST originated from the ancestral protein-coding Lnx3 gene with contributions from various mobile elements that contributed to the striking domains of tandem repeats within the first and sixth exons. Modular domains of XIST are now involved in recruiting heterochromatic marks and proteins essential for XCI initiation and maintenance. This review presents a comparative analysis of human XIST with five other eutherian mammals—chimpanzees, cats, pigs, sheep, and mice—examining conservation across exons as well as the tandem repeats. Notably, repeats exhibited higher conservation than exons, underscoring their functional importance. Additionally, a species-specific G repeat, previously described in pigs, was also identified in sheep and cats. These findings provide insights into the domains of XIST, a cis-acting silencer that has been used to proposed to alleviate the impact of a supernumerary chromosome in Down syndrome.

Non-Coding RNA doi: 10.3390/ncrna11030034

Authors: Roberto Piergentili Stefano Sechi Lina De Paola Simona Zaami Enrico Marinelli

Background/Objectives: Competing endogenous RNAs (ceRNA) are molecules that compete for the binding to a microRNA (miR). Usually, there are two ceRNA, one of which is a protein-coding RNA (mRNA), with the other being a long non-coding RNA (lncRNA). The miR role is to inhibit mRNA expression, either promoting its degradation or impairing its translation. The lncRNA can “sponge” the miR, thus impeding its inhibitory action on the mRNA. In their easier configuration, these three molecules constitute a regulatory axis for protein expression. However, each RNA can interact with multiple targets, creating branched and intersected axes that, all together, constitute what is known as a competing endogenous RNA network (ceRNET). Methods: In this systematic review, we collected all available data from PubMed about experimentally verified (by luciferase assay) regulatory axes in endometrial cancer (EC), excluding works not using this test; Results: This search allowed the selection of 172 bibliographic sources, and manually building a series of ceRNETs of variable complexity showed the known axes and the deduced intersections. The main limitation of this search is the highly stringent selection criteria, possibly leading to an underestimation of the complexity of the networks identified. However, this work allows us not only to hypothesize possible gap fillings but also to set the basis to instruct artificial intelligence, using adequate prompts, to expand the EC ceRNET by comparing it with ceRNETs of other cancers. Moreover, these networks can be used to inform and guide research toward specific, though still unidentified, axes in EC, to complete parts of the network that are only partially described, or even to integrate low complexity subnetworks into larger more complex ones. Filling the gaps among the existing EC ceRNET will allow physicians to hypothesize new therapeutic strategies that may either potentiate or substitute existing ones. Conclusions: These ceRNETs allow us to easily visualize long-distance interactions, thus helping to select the best treatment, depending on the molecular profile of each patient, for personalized medicine. This would yield higher efficiency rates and lower toxicity levels, both of which are extremely relevant factors not only for patients’ wellbeing, but also for the legal, regulatory, and ethical aspects of miR-based innovative treatments and personalized medicine as a whole. This systematic review has been registered in PROSPERO (ID: PROSPERO 2025 CRD420251035222).

Non-Coding RNA doi: 10.3390/ncrna11030033

Authors: Valeria Pecci Melissa Borsa Aurora Aiello Sara De Martino Luca Cis Cristian Ripoli Dante Rotili Francesco Pierconti Francesco Pinto Claudio Grassi Carlo Gaetano Antonella Farsetti Simona Nanni

Background/Objectives: Metastatic prostate cancer (PCa) remains a major clinical challenge with limited therapeutic options. The long non-coding RNA H19 has been implicated in regulating cell adhesion molecules and collective migration, key features of metastatic dissemination. This study investigates the role of the Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 in the H19-dependent transcriptional regulation of cell adhesion molecules. Currently, the major effects of BET inhibitors require androgen receptor (AR) expression. Methods: H19 was stably silenced in PC-3 (AR-null) and 22Rv1 (AR-positive) castration-resistant PCa cells. The cells were treated with the pan-BET inhibitors JQ1 and OTX015 or the BET degrader dBET6. In vivo, the effects of JQ1 were evaluated in xenograft mouse models. Chromatin immunoprecipitation (ChIP) and RNA-ChIP were used to assess BET protein recruitment and interaction with cell adhesion gene loci and H19. Organotypic slice cultures (OSCs) from fresh PCa surgical specimens were used as ex vivo models to validate transcriptional changes and BRD4 recruitment. Results: BET inhibition significantly reduced the expression of β4 integrin and E-cadherin and cell proliferation in both basal conditions, and following H19 knockdown in PC-3 and 22Rv1 cells. These effects were mirrored in JQ1-treated tumor xenografts, which showed marker downregulation and tumor regression. ChIP assays revealed that BRD4, more than BRD2/3, was enriched on β4 integrin and E-cadherin promoters, especially in regions marked by H3K27ac. H19 silencing markedly enhanced BRD4 promoter occupancy. RNA-ChIP confirmed a specific interaction between BRD4 and H19. These findings were validated in OSCs, reinforcing their clinical relevance. Conclusions: Our study demonstrates that BRD4 epigenetically regulates the H19-mediated transcriptional control of adhesion molecules involved in collective migration and metastatic dissemination. Importantly, these effects are independent of AR status, suggesting that targeting the H19/BRD4 axis may represent a promising therapeutic avenue for advanced PCa.

Non-Coding RNA doi: 10.3390/ncrna11030031

Authors: Nihan Aktas Pepe Busra Acar Gozde Erturk Zararsiz Serife Ayaz Guner Alaattin Sen

Background/Objectives: X-inactive-specific transcript (XIST) is a factor that plays a role in neuroinflammation. This study investigated the role of XIST in neuronal development, neuroinflammation, myelination, and therapeutic responses within cerebral organoids in the context of Multiple Sclerosis (MS) pathogenesis. Methods: Human cerebral organoids with oligodendrocytes were produced from XIST-silenced H9 cells, and the mature organoids were subsequently treated with either FTY720 or DMF. Gene expression related to inflammation and myelination was subsequently analyzed via qRT-PCR. Immunofluorescence staining was used to assess the expression of proteins related to inflammation, myelination, and neuronal differentiation. Alpha-synuclein protein levels were also checked via ELISA. Finally, transcriptome analysis was conducted on the organoid samples. Results: XIST-silenced organoids presented a 2-fold increase in the expression of neuronal stem cells, excitatory neurons, microglia, and mature oligodendrocyte markers. In addition, XIST silencing increased IL-10 mRNA expression by 2-fold and MBP and PLP1 expression by 2.3- and 0.6-fold, respectively. Although XIST silencing tripled IBA1 protein expression, it did not affect organoid MBP expression. FTY720, but not DMF, distinguished MBP and IBA1 expression in XIST-silenced organoids. Furthermore, XIST silencing reduced the concentration of alpha-synuclein from 300 to 100 pg/mL, confirming its anti-inflammatory role. Transcriptomic and gene enrichment analyses revealed that the differentially expressed genes are involved in neural development and immune processes, suggesting the role of XIST in neuroinflammation. The silencing of XIST modified the expression of genes associated with inflammation, myelination, and neuronal growth in cerebral organoids, indicating a potential involvement in the pathogenesis of MS. Conclusions: XIST may contribute to the MS pathogenesis as well as neuroinflammatory diseases such as and Alzheimer’s and Parkinson’s diseases and may be a promising therapeutic target.

Non-Coding RNA doi: 10.3390/ncrna11030032

Authors: Ebtihal Kamal

Background: Single-nucleotide polymorphisms (SNPs) are associated with multiple disorders and various cancer types. In the context of cancer, alterations within non-coding regions, specifically 3′ untranslated regions (3′ UTR), have proven substantially important. Methods: In this study, we utilized various bioinformatics tools to examine the effect of SNPs in the 3′ UTR. We retrieved the 3′ UTR SNPs of the Signal Transducer and Activator of Transcription 1 (STAT1) gene from the National Centre for Biotechnology Information (NCBI) website. Next, we employed the Polymorphism in miRNAs and their corresponding target sites (PolymiRTS) database to predict the 3′ UTR SNPs that create new microRNA (miRNA) binding sites and their respective miRNAs. The effect of the 3′ UTR SNPs on the messenger RNA structure was studied using RNAfold server. We used Cscape tool to predict the oncogenic 3′ UTR SNPs. Then, we submitted the miRNAs to the miRNet database to visualize the miRNA-miRNAs’ target genes interaction, for which gene enrichment analysis was performed using ShinyGO. Protein–protein interactions were conducted using the STRING database. We conducted miRNA enrichment analysis utilizing miRPathDB, subsequently performing miRNA differential expression analysis through oncoMIR, and the StarBase database. The survival analysis of the upregulated miRNAs in cancer was investigated using the Kaplan–Meier Plotter. Result: Twelve SNPs were predicted to create new miRNA binding sites. Two of them, rs188557905 and rs190542524, were predicted to destabilize the mRNA structures. We predicted rs190542524, rs11305, rs186033487, and rs188557905 to be oncogenic 3′ UTR SNPs, with high-confidence predictions and scores > 0.5. Using miRNAs’ target genes enrichment analysis, this study indicated that the miRNA target genes were more likely to be involved in cancer-related pathways. Our comprehensive analysis of miRNAs, their functional enrichment, their expression in various types of cancer, and the correlation between miRNA expression and survival outcome yielded these results. Our research shows that the oncogenic 3′ UTR SNP rs190542524 creates a new binding site for the oncogenic miRNA hsa-miR-136-5p. This miRNA is significantly upregulated in BLCA, LUSC, and STAD and is linked to poor survival. Additionally, rs114360225 creates a new binding site for hsa-miR-362-3p, influencing LIHC. Conclusions: These analyses suggest that these 3′ UTR SNPs may have a functional impact on the STAT1 gene’s regulation through their predicted effect on miRNA binding sites. Future experimental validation could establish their potential role in the diagnosis and treatment of various diseases, including cancer.

Non-Coding RNA doi: 10.3390/ncrna11030030

Authors: Lea Sleiman Sorina Dinescu

Adipocyte differentiation is a complex process in which pluripotent mesenchymal stem cells (MSCs) differentiate and develop into mature fat cells, also known as adipocytes. This process is controlled by various transcription factors, hormones, and signaling molecules that regulate the development of these cells. Recently, an increasing number of non-coding RNAs (ncRNAs), especially microRNAs (miRNAs), have been established to be involved in the regulation of many biological processes, including adipocyte differentiation, development, metabolism, and energy homeostasis of white and brown adipose tissue. Several in vitro and in vivo studies reported the significant role of ncRNAs in either promoting or inhibiting adipocyte differentiation into white or brown fat cells by targeting specific transcription factors and regulating the expression of key adipogenic genes. Identifying the function of ncRNAs and their subsequent targets contributes to our understanding of how these molecules can be used as potential biomarkers and tools for therapies against obesity, diabetes, and other diseases related to obesity. This could also contribute to advancements in tissue-engineering based treatments. In this review, we intended to present an up-to-date comprehensive literature overview of the role of ncRNAs, including miRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), focusing particularly on miRNAs, in regulating the differentiation and development of cells into white and brown adipose tissue. In addition, we further discuss the potential use of these molecules as biomarkers for the development of novel therapeutic strategies for future personalized treatment options for patients.

Non-Coding RNA doi: 10.3390/ncrna11020029

Authors: Annanda Lyra Ribeiro Bruno Dallagiovanna

The human genome sequencing revealed a vast complexity of transcripts, with over 80% of the genome being transcribed into non-coding RNAs. In particular, long non-coding RNAs (lncRNAs) have emerged as critical regulators of various cellular processes, including embryonic development and stem cell differentiation. Despite extensive efforts to identify and characterize lncRNAs, defining their mechanisms of action in state-specific cellular contexts remains a significant challenge. Only recently has the involvement of lncRNAs in human endoderm differentiation of pluripotent stem cells begun to be addressed, creating an opportunity to explore the mechanisms by which lncRNAs exert their functions in germ layer formation, lineage specification, and commitment. This review summarizes current findings on the roles of lncRNAs in endoderm differentiation, highlighting the functional mechanisms and regulatory aspects underlying their involvement in cell fate decisions leading to endoderm development. The key lncRNAs implicated in endoderm differentiation are discussed, along with their interaction with transcription factors and RNA-binding proteins and modulation of signaling pathways essential for endoderm development. Gaining insight into the regulatory roles of lncRNAs in endoderm differentiation enhances the understanding of developmental biology and provides a foundation for discovering novel lncRNAs involved in cell fate determination.

Non-Coding RNA doi: 10.3390/ncrna11020028

Authors: Subhadeep Das Sampad Basak Soumyadev Sarkar

Oral cancer (OC) ranks among the most prevalent head and neck cancers, becoming the eleventh most common cancer worldwide with ~350,000 new cases and 177,000 fatalities annually. The rising trend in the occurrence of OC among young individuals and women who do not have tobacco habits is escalating rapidly. Surgical procedures, radiation therapy, and chemotherapy are among the most prevalent treatment options for oral cancer. To achieve better therapy and an early detection of the cancer, it is essential to understand the disease’s etiology at the molecular level. Saliva, the most prevalent body fluid obtained non-invasively, holds a collection of distinct non-coding RNA pools (ncRNAomes) that can be assessed as biomarkers for identifying oral cancer. Non-coding signatures, which are transcripts lacking a protein-coding function, have been identified as significant in the progression of various cancers, including oral cancer. This review aims to examine the role of various salivary ncRNAs (microRNA, circular RNA, and lncRNA) associated with disease progression and to explore their functions as potential biomarkers for early disease identification to ensure better survival outcomes for oral cancer patients.

Non-Coding RNA doi: 10.3390/ncrna11020027

Authors: Aliaa Amr Alamoudi

Metabolic reprogramming is a hallmark of cancer, crucial for supporting the rapid energy demands of tumor cells. MYC, often deregulated and overexpressed, is a key driver of this shift, promoting the Warburg effect by enhancing glycolysis. However, there remains a gap in understanding the mechanisms and factors influencing MYC’s metabolic roles. Recently, non-coding RNAs (ncRNAs) have emerged as important modulators of MYC functions. This review focuses on ncRNAs that regulate MYC-driven metabolism, particularly the Warburg effect. The review categorizes these ncRNAs into three main groups based on their interaction with MYC and examines the mechanisms behind these interactions. Additionally, we explore how different types of ncRNAs may collaborate or influence each other’s roles in MYC regulation and metabolic function, aiming to identify biomarkers and synthetic lethality targets to disrupt MYC-driven metabolic reprogramming in cancer. Finaly, the review highlights the clinical implications of these ncRNAs, providing an up-to-date summary of their potential roles in cancer prognosis and therapy. With the recent advances in MYC-targeted therapy reaching clinical trials, the exciting potential of combining these therapies with ncRNA-based strategies holds great promise for enhancing treatment efficacy.

Non-Coding RNA doi: 10.3390/ncrna11020026

Authors: Sofia Fathi Oussama Aazzane Salma Guendaoui Nezha Tawfiq Souha Sahraoui Fadila Guessous Mehdi Karkouri

Colorectal cancer (CRC) is the third most diagnosed cancer and a leading cause of cancer-related mortality in Morocco, often detected at late stages. Circulating microRNAs (miRNAs) have emerged as promising non-invasive biomarkers for CRC detection, with miR-21, miR-29a, and miR-92a showing significant diagnostic potential. This study aimed to evaluate the expression levels of these miRNAs in a Moroccan population and their efficacy as diagnostic biomarkers. Methods: A prospective study was conducted using blood samples from 50 CRC patients and 50 healthy controls. Circulating miRNA expression levels were quantified through reverse transcription quantitative PCR (RT-qPCR), with normalization to miR-1228-3p. Statistical analyses, including the Mann–Whitney U test, Receiver Operating Characteristic (ROC) curve analysis, sensitivity (Sen), and specificity (Spe) evaluations, were performed to assess the diagnostic accuracy of individual miRNAs and their combined performance as panels. Results: The expression levels of miR-21, miR-29a, and miR-92a were significantly elevated in CRC patients compared to healthy controls (all p < 0.001). ROC analysis demonstrated that miR-92a exhibited the highest individual diagnostic performance (AUC: 0.938), followed by miR-21 (AUC: 0.907) and miR-29a (AUC: 0.898). Sensitivity and specificity were 88% and 90%, 92% and 56%, and 76% and 94%, respectively. Combinatorial analysis revealed that the miR-29a and miR-92a panel achieved the highest diagnostic accuracy (AUC: 0.976), surpassing individual miRNAs and other combinations, highlighting its potential as a robust, non-invasive biomarker panel for CRC. Conclusions: This study highlights the potential of the miR-29a and miR-92a combination, which achieved excellent diagnostic efficiency (AUC: 0.976). These findings underscore miRNA utility in enhancing early detection and reducing CRC-related mortality in Morocco.

Non-Coding RNA doi: 10.3390/ncrna11020025

Authors: Ronaldo C. Augusto Thomas Quack Christoph G. Grevelding Christoph Grunau

In this study, we employed a total of eight distinct modifications of histone proteins (H3K23ac, H3K27me3, H3K36me3, H3K4me3, H3K9ac, H3K9me3, H4K12ac, and H4K20me1) to discern the various chromatin colors encompassing lncRNA genes in both mature and immature gonads of the human parasite Schistosoma mansoni. Our investigation revealed that these chromatin colors exhibit a tendency to aggregate based on the similarities in their metagene shapes, leading to the formation of less than six distinct clusters. Moreover, these clusters can be further grouped according to their resemblances by shape, which are co-linear with specific regions of the genes, and potentially associated with transcriptional stages.

Non-Coding RNA doi: 10.3390/ncrna11020024

Authors: Mahnoor Naseer Gondal Hafiz Muhammad Umer Farooqi

Non-coding RNAs (ncRNAs) play crucial roles in colorectal cancer (CRC) development and progression. Recent developments in single-cell transcriptome profiling methods have revealed surprising levels of expression variability among seemingly homogeneous cells, suggesting the existence of many more cell types than previously estimated. This review synthesizes recent advances in ncRNA research in CRC, emphasizing single-cell bioinformatics approaches for their analysis. We explore computational methods and tools used for ncRNA identification, characterization, and functional prediction in CRC, with a focus on single-cell RNA sequencing (scRNA-seq) data. The review highlights key bioinformatics strategies, including sequence-based and structure-based approaches, machine learning applications, and multi-omics data integration. We discuss how these computational techniques can be applied to analyze differential expression, perform functional enrichment, and construct regulatory networks involving ncRNAs in CRC. Additionally, we examine the role of bioinformatics in leveraging ncRNAs as diagnostic and prognostic biomarkers for CRC. We also discuss recent scRNA-seq studies revealing ncRNA heterogeneity in CRC. This review aims to provide a comprehensive overview of the current state of single-cell bioinformatics in ncRNA CRC research and outline future directions in this rapidly evolving field, emphasizing the integration of computational approaches with experimental validation to advance our understanding of ncRNA biology in CRC.

Non-Coding RNA doi: 10.3390/ncrna11020023

Authors: Mohammad Mohebbi Amirhossein Manzourolajdad Ethan Bennett Phillip Williams

(1) Background: MicroRNAs are non-coding RNA sequences that regulate cellular functions by targeting messenger RNAs and inhibiting protein synthesis. Identifying their target sites is vital to understanding their roles. However, it is challenging due to the high cost and time demands of experimental methods and the high false-positive rates of computational approaches. (2) Methods: We introduce a Multi-Input Neural Network (MINN) algorithm that integrates diverse biologically relevant features, including the microRNA duplex structure, substructures, minimum free energy, and base-pairing probabilities. For each feature derived from a microRNA target-site duplex, we create a corresponding image. These images are processed in parallel by the MINN algorithm, allowing it to learn a comprehensive and precise representation of the underlying biological mechanisms. (3) Results: Our method, on an experimentally validated test set, detects target sites with an AUPRC of 0.9373, Precision of 0.8725, and Recall of 0.8703 and outperforms several commonly used computational methods of microRNA target-site predictions. (4) Conclusions: Incorporating diverse biologically explainable features, such as duplex structure, substructures, their MFEs, and binding probabilities, enables our model to perform well on experimentally validated test data. These features, rather than nucleotide sequences, enhance our model to generalize beyond specific sequence contexts and perform well on sequentially distant samples.

Non-Coding RNA doi: 10.3390/ncrna11020022

Authors: Elena Golovina Cory Eaton Virginia Cox Jozef Andel Karina Savvulidi Vargova

Circular RNAs (circRNAs) make up approximately 10% of the human transcriptome. CircRNAs belong to the broad group of non-coding RNAs and characteristically are formed by backsplicing into a stable circular loop. Their main role is to regulate transcription through the inhibition of miRNAs’ expression, termed miRNA sponging. CircRNAs promote tumorigenesis/lymphomagenesis by competitively binding to miRNAs at miRNA binding sites. In diffuse large B-cell lymphoma (DLBCL), several circRNAs have been identified and their expression is related to both progression and response to therapy. DLBCL is the most prevalent and aggressive subtype of B-cell lymphomas and accounts for about 25% to 30% of all non-Hodgkin lymphomas. DLBCL displays great heterogeneity concerning histopathology, biology, and genetics. Patients who have relapsed or have refractory disease after first-line therapy have a very poor prognosis, demonstrating an important unmet need for new treatment options. As more circRNAs are identified in the future, we will better understand their biological roles and potential use in treating cancer, including DLBCL. For example, circAmotl1 promotes nuclear translocation of MYC and upregulation of translational targets of MYC, thus enhancing lymphomagenesis. Another example is circAPC, which is significantly downregulated in DLBCL and correlates with disease aggressiveness and poor prognosis. CircAPC increases expression of the host gene adenomatous polyposis coli (APC), and in doing so inactivates the canonical Wnt/β-catenin signaling and restrains DLBCL growth. MiRNAs belong to the non-coding regulatory molecules that significantly contribute to lymphomagenesis through their target mRNAs. In DLBCL, among the highly expressed miRNAs, are miR-155-5p and miR-21-5p, which regulate NF-ĸB and PI3K/AKT signaling pathways. The aim of this review is to describe the function and mechanism of regulation of circRNAs on miRNAs’ expression in DLBCL. This will help us to better understand the regulatory network of circRNA/miRNA/mRNA, and to propose novel therapeutic targets to treat DLBCL.

Non-Coding RNA doi: 10.3390/ncrna11020021

Authors: Cristina-Sorina Cătană Monica Mihaela Marta Daniel Ungureanu Cătălina-Angela Crișan

Major depressive disorder (MDD) is one of the most prevalent psychiatric disorders, with an increasing incidence each year and an important socioeconomic burden. Although new treatments are continuously being developed, there is no effective monitoring method to determine the suitability of treatment and ensure positive outcomes. Therefore, patients often struggle with ineffective antidepressants and their potential adverse effects, which halts any future progress in managing the disorder. Considering the potential of microRNAs (miRNAs) as biomarkers for various pathologies and the increasing evidence of the modulation of several genes involved in MDD, this minireview aimed to evaluate the literature data on the impact of miRNAs in MDD and their usefulness in monitoring treatment response. The correlations between antidepressants and the expression of several miRNAs support the existence of a common epigenetic mechanism of antidepressants and explain the epigenetic differences influencing treatment efficacy in MDD.