Non-Coding RNA, Volume 10, Issue 1 (February 2024) – 15 articles

There are many articles on the quantitative analysis of miRNAs contained in a population of EVs of different sizes under various physiological and pathological conditions. For such analysis, it is important to correctly quantify the miRNA contents of EVs. It should be considered that quantification is skewed depending on the isolation protocol, and different miRNAs are degraded by nucleases with different efficiencies. In addition, it is important to consider the contribution of miRNAs coprecipitating with the EVs population, because the amount of miRNAs in the EVs population under study is skewed without appropriate enzymatic treatment. By studying a population of EVs from the blood plasma of healthy donors, we found that the absolute amount of miRNA inside the vesicles is commensurate with the amount of the same type of miRNA adhered to the outside of the EVs. The inside/outside ratio ranged from 1.02 to 2.64 for different investigated miRNAs. According to our results, we propose the hypothesis that high occupancy of miRNAs on the outer surface of EVs influence on the transporting RNA repertoire no less than the inner cargo received from the host cell. Full article

Tumors have high requirements in terms of nutrients and oxygen. Angiogenesis is the classical mechanism for vessel formation. Tumoral vascularization has the function of nourishing the cancer cells to support tumor growth. Vasculogenic mimicry, a novel intratumoral microcirculation system, alludes to the ability of cancer cells to organize in three-dimensional (3D) channel-like architectures. It also supplies the tumors with nutrients and oxygen. Both mechanisms operate in a coordinated way; however, their functions in breast cancer stem-like cells and their regulation by microRNAs remain elusive. In the present study, we investigated the functional role of microRNA-204 (miR-204) on angiogenesis and vasculogenic mimicry in breast cancer stem-like cells. Using flow cytometry assays, we found that 86.1% of MDA-MB-231 and 92% of Hs-578t breast cancer cells showed the CD44+/CD24− immunophenotype representative of cancer stem-like cells (CSCs). The MDA-MB-231 subpopulation of CSCs exhibited the ability to form mammospheres, as expected. Interestingly, we found that the restoration of miR-204 expression in CSCs significantly inhibited the number and size of the mammospheres. Moreover, we found that MDA-MB-231 and Hs-578t CSCs efficiently undergo angiogenesis and hypoxia-induced vasculogenic mimicry in vitro. The transfection of precursor miR-204 in both CSCs was able to impair the angiogenesis in the HUVEC cell model, which was observed as a diminution in the number of polygons and sprouting cells. Remarkably, miR-204 mimics also resulted in the inhibition of vasculogenic mimicry formation in MDA-MB-231 and Hs-578t CSCs, with a significant reduction in the number of channel-like structures and branch points. Mechanistically, the effects of miR-204 were associated with a diminution of pro-angiogenic VEGFA and β-catenin protein levels. In conclusion, our findings indicated that miR-204 abrogates the angiogenesis and vasculogenic mimicry development in breast cancer stem-like cells, suggesting that it could be a potential tool for breast cancer intervention based on microRNA replacement therapies. Full article

Plant species utilize a variety of regulatory mechanisms to ensure sustainable productivity. Within this intricate framework, numerous non-coding RNAs (ncRNAs) play a crucial regulatory role in plant biology, surpassing the essential functions of RNA molecules as messengers, ribosomal, and transfer RNAs. ncRNAs represent an emerging class of regulators, operating directly in the form of small interfering RNAs (siRNAs), microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs). These ncRNAs exert control at various levels, including transcription, post-transcription, translation, and epigenetic. Furthermore, they interact with each other, contributing to a variety of biological processes and mechanisms associated with stress resilience. This review primarily concentrates on the recent advancements in plant ncRNAs, delineating their functions in growth and development across various organs such as root, leaf, seed/endosperm, and seed nutrient development. Additionally, this review broadens its scope by examining the role of ncRNAs in response to environmental stresses such as drought, salt, flood, heat, and cold in plants. This compilation offers updated information and insights to guide the characterization of the potential functions of ncRNAs in plant growth, development, and stress resilience in future research. Full article

Extracellular vesicles (EVs) are defined as subcellular structures limited by a bilayer lipid membrane that function as important intercellular communication by transporting active biomolecules, such as proteins, amino acids, metabolites, and nucleic acids, including long non-coding RNAs (lncRNAs). These cargos can effectively be delivered to target cells and induce a highly variable response. LncRNAs are functional RNAs composed of at least 200 nucleotides that do not code for proteins. Nowadays, lncRNAs and circRNAs are known to play crucial roles in many biological processes, including a plethora of diseases including cancer. Growing evidence shows an active presence of lnc- and circRNAs in EVs, generating downstream responses that ultimately affect cancer progression by many mechanisms, including angiogenesis. Moreover, many studies have revealed that some tumor cells promote angiogenesis by secreting EVs, which endothelial cells can take up to induce new vessel formation. In this review, we aim to summarize the bioactive roles of EVs with lnc- and circRNAs as cargo and their effect on cancer angiogenesis. Also, we discuss future clinical strategies for cancer treatment based on current knowledge of circ- and lncRNA-EVs. Full article

Noncoding RNAs (ncRNAs), including circular RNAs (circRNAs) and N6-methyladenosine (m6A), have been shown to play a critical role in the development of various diseases including obesity and metabolic disorder-associated fatty liver disease (MAFLD). Obesity is a chronic disease caused by excessive fat accumulation in the body, which has recently become more prevalent and is the foremost risk factor for MAFLD. Causes of obesity may involve the interaction of genetic, behavioral, and social factors. m6A RNA methylation might add a novel inspiration for understanding the development of obesity and MAFLD with post-transcriptional regulation of gene expression. In particular, circRNAs, microRNAs (miRNAs), and m6A might be implicated in the progression of MAFLD. Interestingly, m6A modification can modulate the translation, degradation, and other functions of ncRNAs. miRNAs/circRNAs can also modulate m6A modifications by affecting writers, erasers, and readers. In turn, ncRNAs could modulate the expression of m6A regulators in different ways. However, there is limited evidence on how these ncRNAs and m6A interact to affect the promotion of liver diseases. It seems that m6A can occur in DNA, RNA, and proteins that may be associated with several biological properties. This study provides a mechanistic understanding of the association of m6A modification and ncRNAs with liver diseases, especially for MAFLD. Comprehension of the association between m6A modification and ncRNAs may contribute to the development of treatment tactics for MAFLD. Full article

Non-coding RNAs (ncRNAs) are RNA molecules that do not code for protein but play key roles in regulating cellular processes. NcRNAs globally affect gene expression in diverse physiological and pathological contexts. Functionally important ncRNAs act in chromatin modifications, in mRNA stabilization and translation, and in regulation of various signaling pathways. Non-alcoholic fatty liver disease (NAFLD) is a set of conditions caused by the accumulation of triacylglycerol in the liver. Studies of ncRNA in NAFLD are limited but have demonstrated that ncRNAs play a critical role in the pathogenesis of NAFLD. In this review, we summarize NAFLD’s pathogenesis and clinical features, discuss current treatment options, and review the involvement of ncRNAs as regulatory molecules in NAFLD and its progression to non-alcoholic steatohepatitis (NASH). In addition, we highlight signaling pathways dysregulated in NAFLD and review their crosstalk with ncRNAs. Having a thorough understanding of the disease process’s molecular mechanisms will facilitate development of highly effective diagnostic and therapeutic treatments. Such insights can also inform preventive strategies to minimize the disease’s future development. Full article

Biological research has demonstrated the significance of identifying miRNA–disease associations in the context of disease prevention, diagnosis, and treatment. However, the utilization of experimental approaches involving biological subjects to infer these associations is both costly and inefficient. Consequently, there is a pressing need to devise novel approaches that offer enhanced accuracy and effectiveness. Presently, the predominant methods employed for predicting disease associations rely on Graph Convolutional Network (GCN) techniques. However, the Graph Convolutional Network algorithm, which is locally aggregated, solely incorporates information from the immediate neighboring nodes of a given node at each layer. Consequently, GCN cannot simultaneously aggregate information from multiple nodes. This constraint significantly impacts the predictive efficacy of the model. To tackle this problem, we propose a novel approach, based on HyperGCN and Sørensen-Dice loss (HGSMDA), for predicting associations between miRNAs and diseases. In the initial phase, we developed multiple networks to represent the similarity between miRNAs and diseases and employed GCNs to extract information from diverse perspectives. Subsequently, we draw into HyperGCN to construct a miRNA–disease heteromorphic hypergraph using hypernodes and train GCN on the graph to aggregate information. Finally, we utilized the Sørensen-Dice loss function to evaluate the degree of similarity between the predicted outcomes and the ground truth values, thereby enabling the prediction of associations between miRNAs and diseases. In order to assess the soundness of our methodology, an extensive series of experiments was conducted employing the Human MicroRNA Disease Database (HMDD v3.2) as the dataset. The experimental outcomes unequivocally indicate that HGSMDA exhibits remarkable efficacy when compared to alternative methodologies. Furthermore, the predictive capacity of HGSMDA was corroborated through a case study focused on colon cancer. These findings strongly imply that HGSMDA represents a dependable and valid framework, thereby offering a novel avenue for investigating the intricate association between miRNAs and diseases. Full article

Recently, post-transcriptional regulation of mRNA mediated by N⁶-methyladenosine (m⁶A) has been found to have profound effects on transcriptome regulation during plant responses to various abiotic stresses. However, whether this RNA modification can affect an oxidative stress response in plants has not been studied. To assess the role of m⁶A modifications during copper-induced oxidative stress responses, m⁶A-IP-seq was performed in Arabidopsis seedlings exposed to high levels of copper sulfate. This analysis revealed large-scale shifts in this modification on the transcripts most relevant for oxidative stress. This altered epitranscriptomic mark is known to influence transcript abundance and translation; therefore we scrutinized these possibilities. We found an increased abundance of copper-enriched m⁶A-containing transcripts. Similarly, we also found increased ribosome occupancy of copper-enriched m⁶A-containing transcripts, specifically those encoding proteins involved with stress responses relevant to oxidative stressors. Furthermore, the significance of the m⁶A epitranscriptome on plant oxidative stress tolerance was uncovered by assessing germination and seedling development of the mta (N⁶-methyladenosine RNA methyltransferase A mutant complemented with ABI3:MTA) mutant exposed to high copper treatment. These analyses suggested hypersensitivity of the mta mutant compared to the wild-type plants in response to copper-induced oxidative stress. Overall, our findings suggest an important role for m⁶A in the oxidative stress response of Arabidopsis. Full article

Recently, myriad studies have defined the versatile abilities of gasotransmitters and their synthesizing enzymes to play a “Maestro” role in orchestrating several oncological and non-oncological circuits and, thus, nominated them as possible therapeutic targets. Although a significant amount of work has been conducted on the role of nitric oxide (NO) and carbon monoxide (CO) and their inter-relationship in the field of oncology, research about hydrogen sulfide (H₂S) remains in its infancy. Recently, non-coding RNAs (ncRNAs) have been reported to play a dominating role in the regulation of the endogenous machinery system of H₂S in several pathological contexts. A growing list of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are leading the way as upstream regulators for H₂S biosynthesis in different mammalian cells during the development and progression of human diseases; therefore, their targeting can be of great therapeutic benefit. In the current review, the authors shed the light onto the biosynthetic pathways of H₂S and their regulation by miRNAs and lncRNAs in various oncological and non-oncological disorders. Full article

The analysis of small RNA sequencing data across a range of biofluids is a significant research area, given the diversity of RNA types that hold potential diagnostic, prognostic, and predictive value. The intricate task of segregating the complex mixture of small RNAs from both human and other species, including bacteria, fungi, and viruses, poses one of the most formidable challenges in the analysis of small RNA sequencing data, currently lacking satisfactory solutions. This study introduces sRNAflow, a user-friendly bioinformatic tool with a web interface designed for the analysis of small RNAs obtained from biological fluids. Tailored to the unique requirements of such samples, the proposed pipeline addresses various challenges, including filtering potential RNAs from reagents and environment, classifying small RNA types, managing small RNA annotation overlap, conducting differential expression assays, analysing isomiRs, and presenting an approach to identify the sources of small RNAs within samples. sRNAflow also encompasses an alternative alignment-free analysis of RNA-seq data, featuring clustering and initial RNA source identification using BLAST. This comprehensive approach facilitates meaningful comparisons of results between different analytical methods. Full article

A characteristic of the cellular response to stress is the production of RNAs generated from a readthrough transcription of genes, called downstream-of-gene-(DoG)-containing transcripts. Additionally, transcription inhibitor drugs are candidates for fighting cancer. In this work, we report the results of a bioinformatic analysis showing that one of the responses to transcription inhibition is the generation of DoGs in cancer cells. Although some genes that form DoGs were shared between the two cancer lines, there did not appear to be a functional correlation between them. However, our findings show that DoGs are generated as part of the cellular response to transcription inhibition like other types of cellular stress, suggesting that they may be part of the defense against transcriptional stress. Full article

MiRNAs play crucial roles in a broad spectrum of biological processes, both physiological and pathological. Different reports implicate miR-205 in the control of breast stem cell properties. Differential miR-205 expression has been observed in different stages of mammary gland development and maturation. However, a functional role in this process has not been clearly demonstrated. We generated an miR-205 knockout in the FVB/N mouse strain, which is viable and characterized by enhanced mammary gland development. Indeed, mammary glands of miR-205^−/− female mice at different ages (1.5 and 5.5 months) show increased outgrowth and branching. This evidence is consistent with our previously reported data demonstrating the direct miR-205-mediated targeting of HER3, a master regulator of mammary gland development, and the oncosuppressive activity of this microRNA in different types of breast cancer. Full article

Heart failure (HF) is a widespread cardiovascular condition that poses significant risks to a wide spectrum of age groups and leads to terminal illness. Although our understanding of the underlying mechanisms of HF has improved, the available treatments still remain inadequate. Recently, long non-coding RNAs (lncRNAs) have emerged as crucial players in cardiac function, showing possibilities as potential targets for HF therapy. These versatile molecules interact with chromatin, proteins, RNA, and DNA, influencing gene regulation. Notable lncRNAs like Fendrr, Trpm3, and Scarb2 have demonstrated therapeutic potential in HF cases. Additionally, utilizing lncRNAs to forecast survival rates in HF patients and distinguish various cardiac remodeling conditions holds great promise, offering significant benefits in managing cardiovascular disease and addressing its far-reaching societal and economic impacts. This underscores the pivotal role of lncRNAs in the context of HF research and treatment. Full article

MicroRNAs (miRNAs) are crucial post-transcriptional regulators of gene expression, and their deregulation contributes to many aspects of cancer development and progression. Thus, miRNAs provide insight into oncogenic mechanisms and represent promising targets for new therapeutic approaches. A type of cancer that is still in urgent need of improved treatment options is triple negative breast cancer (TNBC). Therefore, we aimed to characterize a novel miRNA with a potential role in TNBC. Based on a previous study, we selected miR-4646-5p, a miRNA with a still unknown function in breast cancer. We discovered that higher expression of miR-4646-5p in TNBC patients is associated with better survival. In vitro assays showed that miR-4646-5p overexpression reduces growth, proliferation, and migration of TNBC cell lines, whereas inhibition had the opposite effect. Furthermore, we found that miR-4646-5p inhibits the tube formation ability of endothelial cells, which may indicate anti-angiogenic properties. By whole transcriptome analysis, we not only observed that miR-4646-5p downregulates many oncogenic factors, like tumor-promoting cytokines and migration- and invasion-related genes, but were also able to identify a direct target, the GRAM domain-containing protein 1B (GRAMD1B). GRAMD1B is involved in cellular cholesterol transport and its knockdown phenocopied the growth-reducing effects of miR-4646-5p. We thus conclude that GRAMD1B may partly contribute to the diverse tumor-suppressive effects of miR-4646-5p in TNBC. Full article

The enteric nervous system (ENS) is an essential network of neurons and glia in the bowel wall. Defects in ENS development can result in Hirschsprung disease (HSCR), a life-threatening condition characterized by severe constipation, abdominal distention, bilious vomiting, and failure to thrive. A growing body of literature connects HSCR to alterations in miRNA expression, but there are limited data on the normal miRNA landscape in the developing ENS. We sequenced small RNAs (smRNA-seq) and messenger RNAs (mRNA-seq) from ENS precursor cells of mid-gestation Ednrb-EGFP mice and compared them to aggregated RNA from all other cells in the developing bowel. Our smRNA-seq results identified 73 miRNAs that were significantly enriched and highly expressed in the developing ENS, with miR-9, miR-27b, miR-124, miR-137, and miR-488 as our top 5 miRNAs that are conserved in humans. However, contrary to prior reports, our follow-up analyses of miR-137 showed that loss of Mir137 in Nestin-cre, Wnt1-cre, Sox10-cre, or Baf53b-cre lineage cells had no effect on mouse survival or ENS development. Our data provide important context for future studies of miRNAs in HSCR and other ENS diseases and highlight open questions about facility-specific factors in development. Full article