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Induced RNA Interference Can Suppress Persistent Drosophila A Virus (DAV) Infection in Cultured Drosophila Melanogaster S2 Cells -
Integrated Transcriptomic, In Silico, and In Vitro Characterization of lncRNA ENST00000615487.1 Reveals Epithelial-Specific Expression, Differential Subcellular Distribution Between Normal and Colorectal Cancer Cells, and Potential Regulatory Functions
Journal Description
Non-Coding RNA
Non-Coding RNA
is an international, peer-reviewed, open access journal on non-coding RNA research dealing with elucidating the structure, function and biology of regulatory non-coding RNAs, and published bimonthly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: JCR - Q1 (Genetics and Heredity) / CiteScore - Q2 (Genetics)
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 30.4 days after submission; acceptance to publication is undertaken in 4.6 days (median values for papers published in this journal in the first half of 2026).
- Recognition of Reviewers: Reviewers whose reports are timely and of high quality receive an APC discount voucher for a future publication in an MDPI journal. Become a reviewer.
- Journal Cluster of Biochemistry and Molecular Biology: Current Issues in Molecular Biology, International Journal of Molecular Sciences, Genes, Biomolecules, Biology, Cells, Proteomes, Non-Coding RNA, Epigenomes, Methods and Protocols, Journal of Molecular Pathology, BioChem, DNA, Stresses, Receptors and Kinases and Phosphatases.
Impact Factor:
4.2 (2025);
5-Year Impact Factor:
4.2 (2025)
Latest Articles
Skin-Derived Non-Coding RNAs as Potential Biomarkers and Therapeutic Targets
Non-Coding RNA 2026, 12(5), 38; https://doi.org/10.3390/ncrna12050038 - 28 Sep 2026
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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,
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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.
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Open AccessArticle
Long Noncoding RNA H19 Inhibits Differentiation and Function of Secretory Lineage Cells in the Intestinal Epithelium by Targeting ATOH1
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Min S. Kwon, Hee K. Chung, Hongxia Chen, Amy VanderStoep, Bridgette Warner, Haonan Zhao, Prathik Atluri, Emnet Tesfayohannes, Douglas J. Turner, Lan Xiao and Jian-Ying Wang
Non-Coding RNA 2026, 12(5), 37; https://doi.org/10.3390/ncrna12050037 - 15 Sep 2026
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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
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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.
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Open AccessArticle
A Transcriptome-Wide Analysis Nominates DIO3OS and C9orf139 as Survival-Associated Long Non-Coding RNAs in Glioblastoma
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Minseon Kim, Seung-Kyoon Kim and Jaeil Han
Non-Coding RNA 2026, 12(5), 36; https://doi.org/10.3390/ncrna12050036 - 7 Sep 2026
Abstract
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
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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.
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(This article belongs to the Special Issue ncRNAs in Human Diseases and Therapeutics)
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Open AccessArticle
Cerebrospinal Fluid miRNA Profiling as a Potential Liquid Biopsy for Vestibular Schwannomas
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Małgorzata Litwiniuk-Kosmala, Maria Makuszewska, Robert Bartoszewicz, Agnieszka Jasińska-Nowacka, Maciej Ołdak, Bartłomiej Gielniewski, Bartosz Wojtaś and Kazimierz Niemczyk
Non-Coding RNA 2026, 12(5), 35; https://doi.org/10.3390/ncrna12050035 - 1 Sep 2026
Abstract
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
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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.
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(This article belongs to the Special Issue ncRNAs in Human Diseases and Therapeutics)
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Open AccessReview
microRNAs as Regulators of the Immune Response and Their Potential Therapeutic Applications in Cancer
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Ezgi Biltekin and Bulent Ozpolat
Non-Coding RNA 2026, 12(5), 34; https://doi.org/10.3390/ncrna12050034 - 1 Sep 2026
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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
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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.
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Open AccessArticle
Circulating Cell-Free miR-25-3p Is Upregulated in Brazilian Patients with Prostate Cancer and Contributes to Tumor Aggressiveness
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Monyse Nobrega, Hellen Kuasne, Marilesia Ferreira de Souza, Mariana Bisarro dos Reis, Larissa Cristina Bastos de Oliveira, Paulo Emilio Fuganti, Morag Park and Ilce Mara de Syllos Cólus
Non-Coding RNA 2026, 12(5), 33; https://doi.org/10.3390/ncrna12050033 - 30 Aug 2026
Abstract
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
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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.
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(This article belongs to the Section Detection and Biomarkers of Non-Coding RNA)
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Open AccessReview
Exosomal lncRNAs in Cerebrovascular Diseases: Biomarkers, Pathological Mechanisms, and Therapeutic Potential
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Haiyu Su, Daiju Tao, Jia Teng, Li Zhang, Ying Shen, Renhua Yang, Jiarui Yang, Rongji Sun, Zhiqiang Shen, Peng Chen and Bo He
Non-Coding RNA 2026, 12(5), 32; https://doi.org/10.3390/ncrna12050032 - 24 Aug 2026
Abstract
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
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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.
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(This article belongs to the Special Issue ncRNAs in Human Diseases and Therapeutics)
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Open AccessArticle
Long-Read RNA Sequencing Reveals an Isoform Switching Pattern in Healthy Microglial Cell Lines Exposed to Radiotherapy
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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 and Giovanni Li Volti
Non-Coding RNA 2026, 12(4), 31; https://doi.org/10.3390/ncrna12040031 - 6 Aug 2026
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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
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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.
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Open AccessArticle
An Exploratory Study on Transcriptomic Profiling of Circulating miRNA Associated with Hub Genes in the Development of Polycystic Ovary Syndrome
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Yogesh Vetriselvan, Jayakumar Swetha, Manoranjani Murugan, Irisappan Ganesh, Vishnu Bhat Ballambattu, Pushpa Premanath Kotur, Deepa Shanmugam, Marcella Sherin Samuel and Sambandam Ravikumar
Non-Coding RNA 2026, 12(4), 30; https://doi.org/10.3390/ncrna12040030 - 5 Aug 2026
Abstract
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
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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.
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(This article belongs to the Section Small Non-Coding RNA)
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Open AccessProtocol
Fraction-Seq: An Integrated Experimental and Computational Workflow for Determining the Localization and Abundance of Small Non-Coding RNAs in Subcellular Compartments
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Siddhartha Shah, Tess Cherlin, Yi Jing, Stepan Nersisyan, Benjamin Leiby and Isidore Rigoutsos
Non-Coding RNA 2026, 12(4), 29; https://doi.org/10.3390/ncrna12040029 - 5 Aug 2026
Abstract
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
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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.
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(This article belongs to the Section Small Non-Coding RNA)
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Open AccessArticle
Induced RNA Interference Can Suppress Persistent Drosophila A Virus (DAV) Infection in Cultured Drosophila Melanogaster S2 Cells
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Wiebke Kochendörfer and Klaus Förstemann
Non-Coding RNA 2026, 12(4), 28; https://doi.org/10.3390/ncrna12040028 - 30 Jul 2026
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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
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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.
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Open AccessReview
Circular RNAs in Neurons: From Transport to Function
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Nicolò Salvi and Mariangela Morlando
Non-Coding RNA 2026, 12(4), 27; https://doi.org/10.3390/ncrna12040027 - 29 Jul 2026
Abstract
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
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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.
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(This article belongs to the Section Long Non-Coding RNA)
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Open AccessArticle
Efficacy of Bioengineered PD-L1 siRNA for Immunotherapy Against Non-Small Cell Lung Cancer Cells
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Neelu Batra, Mei-Juan Tu, Su Guan, Jonathan W. Riess and Ai-Ming Yu
Non-Coding RNA 2026, 12(4), 26; https://doi.org/10.3390/ncrna12040026 - 27 Jul 2026
Abstract
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
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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.
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(This article belongs to the Section Small Non-Coding RNA)
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Open AccessReview
MeCP2 Dosage Control in Rett Syndrome: Non-Coding RNA-Based and Epigenetic Strategies for Safer Gene Therapy
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Ilyas M. Kabdesh, Albert A. Rizvanov and Yana O. Mukhamedshina
Non-Coding RNA 2026, 12(4), 25; https://doi.org/10.3390/ncrna12040025 - 22 Jul 2026
Abstract
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
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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.
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(This article belongs to the Section Clinical Applications of Non-Coding RNA)
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Integrated Transcriptomic, In Silico, and In Vitro Characterization of lncRNA ENST00000615487.1 Reveals Epithelial-Specific Expression, Differential Subcellular Distribution Between Normal and Colorectal Cancer Cells, and Potential Regulatory Functions
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Nataša Đokić, Anastasija Bubanja, Jelena Karanović and Jovana Despotović
Non-Coding RNA 2026, 12(4), 24; https://doi.org/10.3390/ncrna12040024 - 17 Jul 2026
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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.
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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.
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Open AccessEditorial
The Non-Coding RNA Journal Club: Highlights on Recent Papers—15
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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 and Ling Yangadd
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Non-Coding RNA 2026, 12(4), 23; https://doi.org/10.3390/ncrna12040023 - 17 Jul 2026
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We are delighted to share with you our fifteenth Journal Club and highlight some of the most interesting papers published recently [...]
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(This article belongs to the Collection The Non-Coding RNA Journal Club: Highlights on Recent Papers)
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A Mathematical Approach on the Limits of ceRNA Hypothesis Through an Ordinary Differential Equations (ODE) Model of mRNA-microRNA Interactions
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Paul Flondor, Mircea Olteanu, Radu Stefan, Corina Elena Minciuna and Catalin Vasilescu
Non-Coding RNA 2026, 12(4), 22; https://doi.org/10.3390/ncrna12040022 - 29 Jun 2026
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Abstract
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
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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.
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(This article belongs to the Section Computational Biology)
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P22 Small Noncoding RNAs Are Actively Secreted in Salmonella Outer Membrane Vesicles During Bacteriophage Infection
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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 and Glen M. Borchert
Non-Coding RNA 2026, 12(4), 21; https://doi.org/10.3390/ncrna12040021 - 26 Jun 2026
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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.
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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.
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An Optimized RT-qPCR Protocol for Comprehensive Analysis of microRNAs and mRNAs in Mus musculus Brain Tissues
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Maria V. Lukashevich, Margarita M. Rudenok, Suzanna A. Partevian, Maria I. Shadrina, Petr A. Slominsky and Anelya Kh. Alieva
Non-Coding RNA 2026, 12(3), 20; https://doi.org/10.3390/ncrna12030020 - 11 Jun 2026
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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
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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.
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Investigation of Long Non-Coding RNAs H19 rs3741219, MEG3 rs7158663, POLR2E rs3787016, and ANRIL rs10757274 with Breast Cancer Susceptibility and Clinicopathological Characteristics in a Mexican Population
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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 and Martha Patricia Gallegos-Arreola
Non-Coding RNA 2026, 12(3), 19; https://doi.org/10.3390/ncrna12030019 - 4 Jun 2026
Abstract
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:
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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.
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(This article belongs to the Section Long Non-Coding RNA)
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