Non-Coding RNA, Volume 8, Issue 5 (October 2022) – 10 articles

Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to load multiple miRNAs in HEK293T EVs. Combinations of miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst the tested groups. Full article

Long noncoding RNAs (lncRNAs) play critical regulatory roles in human development and disease. Although there are over 100,000 samples with available RNA sequencing (RNA-seq) data, many lncRNAs have yet to be annotated. The conventional approach to identifying novel lncRNAs from RNA-seq data is to find transcripts without coding potential but this approach has a false discovery rate of 30–75%. Other existing methods either identify only multi-exon lncRNAs, missing single-exon lncRNAs, or require transcriptional initiation profiling data (such as H3K4me3 ChIP-seq data), which is unavailable for many samples with RNA-seq data. Because of these limitations, current methods cannot accurately identify novel lncRNAs from existing RNA-seq data. To address this problem, we have developed software, Flnc, to accurately identify both novel and annotated full-length lncRNAs, including single-exon lncRNAs, directly from RNA-seq data without requiring transcriptional initiation profiles. Flnc integrates machine learning models built by incorporating four types of features: transcript length, promoter signature, multiple exons, and genomic location. Flnc achieves state-of-the-art prediction power with an AUROC score over 0.92. Flnc significantly improves the prediction accuracy from less than 50% using the conventional approach to over 85%. Flnc is available via GitHub platform. Full article

Circular RNAs (circRNAs) have recently been implicated in impaired β-cell function in diabetes. Using microarray-based profiling of circRNAs in human EndoC-βH1 cells treated with pro-inflammatory cytokines, this study aimed to investigate the expression and possible regulatory roles of circRNAs in human β cells. We identified ~5000 β-cell-expressed circRNAs, of which 84 were differentially expressed (DE) after cytokine exposure. Pathway analysis of the host genes of the DE circRNAs revealed the enrichment of cytokine signaling pathways, indicative of circRNA transcription from inflammatory genes in response to cytokines. Multiple binding sites for β-cell-enriched microRNAs and RNA-binding proteins were observed for the highly upregulated circRNAs, supporting their function as ‘sponges’ or ‘decoys’. We also present evidence for circRNA sequence conservation in multiple species, the presence of cytokine-induced regulatory elements, and putative protein-coding potential for the DE circRNAs. This study highlights the complex regulatory potential of circRNAs, which may play a crucial role during immune-mediated β-cell destruction in type 1 diabetes. Full article

Long noncoding RNAs (lncRNAs) undergo splicing and have multiple transcribed isoforms. Nevertheless, for lncRNAs, as well as for mRNA, measurements of expression are routinely performed only at the gene level. Metformin is the first-line oral therapy for type 2 diabetes mellitus and other metabolic diseases. However, its mechanism of action remains not thoroughly explained. Transcriptomic analyses using metformin in different cell types reveal that only protein-coding genes are considered. We aimed to characterize lncRNA isoforms that were differentially affected by metformin treatment on multiple human cell types (three cancer, two non-cancer) and to provide insights into the lncRNA regulation by this drug. We selected six series to perform a differential expression (DE) isoform analysis. We also inferred the biological roles for lncRNA DE isoforms using in silico tools. We found the same isoform of an lncRNA (AC016831.6-205) highly expressed in all six metformin series, which has a second exon putatively coding for a peptide with relevance to the drug action. Moreover, the other two lncRNA isoforms (ZBED5-AS1-207 and AC125807.2-201) may also behave as cis-regulatory elements to the expression of transcripts in their vicinity. Our results strongly reinforce the importance of considering DE isoforms of lncRNA for understanding metformin mechanisms at the molecular level. Full article

Digestive tract cancers represent a serious public health issue. In recent years, evidence has accumulated that microRNA miR-185 is implicated in the pathogenesis of this group of highly malignant tumors. Its expression variations correlate with clinical features, such as tumor size, lymph node metastasis, tumor node metastatic stage, survival, recurrence and response to adjuvant therapy, and have diagnostic and prognostic potential. In this review, we compile, evaluate and discuss the current knowledge about the roles of miR-185 in digestive tract cancers. Interestingly, miR-185 is apparently involved in regulating both tumor suppressive and oncogenic processes. We look at downstream effects as well as upstream regulation. In addition, we discuss the utility of miR-185 for diagnosis and its potential concerning novel therapeutic approaches. Full article

Enhancers are distal cis-acting elements that are commonly recognized to regulate gene expression via cooperation with promoters. Along with regulating gene expression, enhancers can be transcribed and generate a class of non-coding RNAs called enhancer RNAs (eRNAs). The current discovery of abundant tissue-specific transcription of enhancers in various diseases such as cancers raises questions about the potential role of eRNAs in disease diagnosis and therapy. This review aimed to demonstrate the current understanding of eRNAs in cancer research with a focus on the potential roles of eRNAs as prognostic and diagnostic biomarkers in cancers. Full article

Ubiquitous eukaryotic non-coding circular RNAs regulate transcription and translation. We have reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica with esterified 3′ss and 5′ss. Their 5′ss GU-rich elements are essential for their biogenesis and their suggested role in transcription regulation. Here, we explored whether exonic, exonic-intronic, and intergenic circular RNAs are also part of the E. histolytica and E. invadens ncRNA RNAome and investigated their possible functions. Available RNA-Seq libraries were analyzed with the CIRI-full software in search of circular exonic RNAs (circRNAs). The robustness of the analyses was validated using synthetic decoy sequences with bona fide back splice junctions. Differentially expressed (DE) circRNAs, between the virulent HM1:IMSS and the nonvirulent Rahman E. histolytica strains, were identified, and their miRNA sponging potential was analyzed using the intaRNA software. Respectively, 188 and 605 reverse overlapped circRNAs from E. invadens and E. histolytica were identified. The sequence composition of the circRNAs was mostly exonic although different to human circRNAs in other attributes. 416 circRNAs from E. histolytica were virulent-specific and 267 were nonvirulent-specific. Out of the common circRNAs, 32 were DE between strains. Finally, we predicted that 8 of the DE circRNAs could function as sponges of the bioinformatically reported miRNAs in E. histolytica, whose functions are still unknown. Our results extend the E. histolytica RNAome and allow us to devise a hypothesis to test circRNAs/miRNAs/siRNAs interactions in determining the virulent/nonvirulent phenotypes and to explore other regulatory mechanisms during amoebic encystment. Full article

Small cell neuroendocrine carcinoma is most frequently found in the lung (SCLC), but it has been also reported, albeit with a very low incidence, in the ovary. Here, we analyze a case of primary small cell carcinoma of the ovary of pulmonary type (SCCOPT), a rare and aggressive tumor with poor prognosis, whose biology and molecular features have not yet been thoroughly investigated. The patient affected by SCCOPT had a residual tumor following chemotherapy which displayed pronounced similarity with neuroendocrine tumors and lung cancer in terms of its microRNA expression profile and mTOR-downstream activation. By analyzing the metabolic markers of the neoplastic lesion, we established a likely glycolytic signature. In conclusion, this in-depth characterization of SCCOPT could be useful for future diagnoses, possibly aided by microRNA profiling, allowing clinicians to adopt the most appropriate therapeutic strategy. Full article

Pseudouridylation is one of the most abundant modifications found in RNAs. To identify the Pseudouridylation sites (Psi) in RNAs, several techniques have been developed, but the most common and robust is the CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide) treatment, which consists in the addition of an adduct on Psi that inhibits the reverse transcription. Here, we describe a turnkey method and a tool to design the bridging oligo (DBO), which is somewhat difficult to design. Finally, we propose a trouble-shooting guide to help users. Full article

Long non-coding RNAs (lncRNAs) have emerged as key regulators in a wide range of biological processes. Here, we identified a mouse miRNA-host gene lncRNA (Lnc-Nr6a1) upregulated early during epithelial-to-mesenchymal transition (EMT). We show that when lncRNA is processed, it gives rise to two abundant polyadenylated isoforms, lnc-Nr6a1-1 and lnc-Nr6a1-2, and a longer non-polyadenylated microprocessor-driven lnc-pri-miRNA containing clustered pre-miR-181a2 and pre-miR-181b2 hairpins. Ectopic expression of the lnc-Nr6a1-1 or lnc-Nr6a1-2 isoform enhanced cell migration and the invasive capacity of the cells, whereas the expression of the isoforms and miR-181a2 and miR-181b2 conferred anoikis resistance. Lnc-Nr6a1 gene deletion resulted in cells with lower adhesion capacity and reduced glycolytic metabolism, which are restored by lnc-Nr6a1-1 isoform expression. We performed identification of direct RNA interacting proteins (iDRIP) to identify proteins interacting directly with the lnc-Nr6a1-1 isoform. We defined a network of interacting proteins, including glycolytic enzymes, desmosome proteins and chaperone proteins; and we demonstrated that the lnc-Nr6a1-1 isoform directly binds and acts as a scaffold molecule for the assembly of ENO1, ALDOA, GAPDH, and PKM glycolytic enzymes, along with LDHA, supporting substrate channeling for efficient glycolysis. Our results unveil a role of Lnc-Nr6a1 as a multifunctional lncRNA acting as a backbone for multiprotein complex formation and primary microRNAs. Full article