MicroRNA - Volume 8, Issue 1, 2019

Introduction: MicroRNAs (miRNAs), which are evolutionarily conserved, and endogenous non-coding RNAs, participate in the post-transcriptional regulation of eukaryotic genes. The biogenesis of miRNAs occurs in the nucleus. Then, in the cytoplasm, they are assembled along with some proteins in a ribonucleoprotein complex called RISC. miRNA component of the RISC complex binds to the complementary sequence of mRNA target depending on the degree of complementarity, and leads to mRNA degradation and/or inhibition of protein synthesis. miRNAs have been found in eukaryotes and some viruses play a role in development, metabolism, cell proliferation, growth, differentiation, and death. Objective: A large number of miRNAs and their targets were identified by different experimental techniques and computational approaches. The principal aim of this paper is to gather information about some miRNA databases and web-based tools for better and quicker access to relevant data. Results: Accordingly, in this paper, we collected and introduced miRNA databases and some webbased tools that have been developed by various research groups. We have categorized them into different classes including databases for viral miRNAs, and plant miRNAs, miRNAs in human beings, mice and other vertebrates, miRNAs related to human diseases, and target prediction, and miRNA expression. Also, we have presented relevant statistical information about these databases.

Preeclampsia (PE) continues to represent a worldwide problem and challenge for both clinicians and laboratory-based doctors. Despite many efforts, the knowledge acquired regarding its pathogenesis and pathophysiology does not allow us to treat it efficiently. It is not possible to arrest its progressive nature, and the available therapies are limited to symptomatic treatment. Furthermore, both the diagnosis and prognosis are frequently uncertain, whilst the ability to predict its occurrence is very limited. MicroRNAs are small non-coding RNAs discovered two decades ago, and present great interest given their ability to regulate almost every aspect of the cell function. A lot of evidence regarding the role of miRNAs in pre-eclampsia has been accumulated in the last 10 years. Differentially expressed miRNAs are characteristic of both mild and severe PE. In many cases they target signaling pathway-related genes that result in altered processes which are directly involved in PE. Immune system, angiogenesis and trophoblast proliferation and invasion, all fundamental aspects of placentation, are controlled in various degrees by miRNAs which are up- or downregulated. Finally, miRNAs represent a potential therapeutic target and a diagnostic tool.

Background: A small GTPase Protein, the Ras-related Protein 1 (RAP1), abundant in platelets is known to be activated following agonist-induced platelet activation, suggesting that RAP1 downregulation could, in turn, reduce platelet activation in storage. Our objective of this study is to identify RAP1 regulating miRNAs and their role in platelet activation during storage. Methods: We applied MS2-TRAP (tagged RNA affinity purification) methodology to enrich miRNAs that target the 3' untranslated region (3'UTR) of RAP1 mRNA in two mammalian cell lines followed by miRNA identification by microarray of total RNA samples enriched for miRNAs. Data analyses were done using different bioinformatics approaches. The direct miR:RAP1 3'UTR interaction was confirmed by using a dual luciferase reporter gene expression system in a mammalian cell line. Subsequently, platelets were transfected with one selected miR to evaluate RAP1 downregulation by this miRNA and its effect on platelet activation. Results: Six miRNAs (miR-320c, miR-181a, miR-3621, miR-489, miR-4791 and miR-4744) were identified to be enriched in the two cell lines tested. We randomly selected miR-320c for further evaluation. The luciferase reporter assay system confirmed the direct interaction of miR-320c with RAP1 3′UTR. Further, in platelets treated with miR-320c, RAP1 protein expression was decreased and concomitantly, platelet activation was also decreased. Conclusion: Overall, the results demonstrate that miRNA-based RAP1 downregulation in ex vivo stored platelets reduces platelet activation.

Background: Development of the mammalian palate is dependent on precise, spatiotemporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs, function as crucial modulators of cell and tissue differentiation, regulating expression of key downstream genes. Observations: Our laboratory has previously identified several developmentally regulated miRNAs, including miR-206, during critical stages of palatal morphogenesis. The current study reports spatiotemporal distribution of miR-206 during development of the murine secondary palate (gestational days 12.5-14.5). Result and Conclusion: Potential cellular functions and downstream gene targets of miR-206 were investigated using functional assays and expression profiling, respectively. Functional analyses highlighted potential roles of miR-206 in governing TGF and Wnt signaling in mesenchymal cells of the developing secondary palate. In addition, altered expression of miR-206 within developing palatal tissue of TGF-/- fetuses reinforced the premise that crosstalk between this miRNA and TGF is crucial for secondary palate development.

Background: Mural Granulosa Cells (MGCs) and Cumulus Cells (CCs) are two specialized cell types that differentiate from a common progenitor during folliculogenesis. Although these two cell types have specialized functions and gene expression profiles, little is known about their microRNA (miRNA) expression patterns. Objective: To describe the miRNA profile of mural and cumulus granulosa cells from human preovulatory follicles. Methods: Using small RNA sequencing, we defined the miRNA expression profiles of human primary MGCs and CCs, isolated from healthy women undergoing ovum pick-up for in vitro Fertilization (IVF). Results: Small RNA sequencing revealed the expression of several hundreds of miRNAs in MGCs and CCs with 53 miRNAs being significantly differentially expressed between MGCs and CCs. We validated the differential expression of miR-146a-5p, miR-149-5p, miR-509-3p and miR-182-5p by RT-qPCR. Analysis of proven targets revealed 37 targets for miR-146a-5p, 43 for miR-182-5p, 2 for miR-509-3p and 9 for miR-149-5p. Gene Ontology (GO) analysis for these 4 target gene sets revealed enrichment of 12 GO terms for miR-146a-5p and 10 for miR-182-5p. The GO term ubiquitin-like protein conjugation was enriched within both miRNA target gene sets. Conclusion: We generated miRNA expression profiles for MGCs and CCs and identified several differentially expressed miRNAs.

Background: Globally, colorectal cancer (CRC) is the third most common cancer in women and the fourth most common cancer in men. Dysregulation of small non-coding miRNAs have been correlated with colon cancer progression. Since there are increasing reports of candidate miRNAs as potential biomarkers for CRC, this makes it important to explore common miRNA biomarkers for colon cancer. As computational prediction of miRNA targets is a critical initial step in identifying miRNA: mRNA target interactions for validation, we aim here to construct a potential miRNA network and its gene targets for colon cancer from previously reported candidate miRNAs, inclusive of 10 up- and 9 down-regulated miRNAs from tissues; and 10 circulatory miRNAs. Methods: The gene targets were predicted using DIANA-microT-CDS and TarBaseV7.0 databases. Each miRNA and its targets were analyzed further for colon cancer hotspot genes, whereupon DAVID analysis and mirPath were used for KEGG pathway analysis. Results: We have predicted 874 and 157 gene targets for tissue and serum specific miRNA candidates, respectively. The enrichment of miRNA revealed that particularly hsa-miR-424-5p, hsa-miR-96-5p, hsa-miR-1290, hsa-miR-224, hsa-miR-133a and has-miR-363-3p present possible targets for colon cancer hallmark genes, including BRAF, KRAS, EGFR, APC, amongst others. DAVID analysis of miRNA and associated gene targets revealed the KEGG pathways most related to cancer and colon cancer. Similar results were observed in mirPath analysis. A new insight gained in the colon cancer network pathway was the association of hsa-mir-133a and hsa-mir-96-5p with the PI3K-AKT signaling pathway. In the present study, target prediction shows that while hsa-mir-424-5p has an association with mostly 10 colon cancer hallmark genes, only their associations with MAP2 and CCND1 have been experimentally validated. Conclusion: These miRNAs and their targets require further evaluation for a better understanding of their associations, ultimately with the potential to develop novel therapeutic targets.

Background: Spinocerebellar ataxia type 3 (SCA3), the most common spinocerebellar ataxia, is caused by a polyglutamine (polyQ) expansion in the protein ataxin-3 (ATXN3). Silencing the expression of polyQ-expanded ATXN3 rescues the cellular disease phenotype. Objective: This study investigated the differential expression of microRNAs (miRNAs), small noncoding RNAs targeting gene expression, in lymphoblastoid cells (LCs) from SCA3 patients and the capability of identified deregulated miRNAs to target and alter ATXN3 expression. Methods: MiRNA profiling was performed by microarray hybridization of total RNA from control and SCA3-LCs. The capability of the identified miRNAs and their target sites to suppress ATXN3 expression was analyzed using mutagenesis, reverse transcription PCR, immunoblotting, luciferase reporter assays, mimics and precursors of the identified miRNAs. Results: SCA3-LCs showed significantly decreased expression levels of ATXN3 and a significant upregulation of the ATXN3-3'UTR targeting miRNAs, miR-32 and miR-181c and closely related members of the miR-25 and miR-181 family, respectively. MiR-32 and miR-181c effectively targeted the 3'UTR of ATXN3 and suppressed the expression of ATXN3. Conclusions: The simultaneous upregulation of closely related miRNAs targeting the 3'UTR of ATXN3 and the significantly reduced ATXN3 expression levels in SCA3-LCs suggests that miR-25 and miR-181 family members cooperatively bind to the 3'UTR to suppress the expression of ATXN3. The findings further suggest that the upregulation of miR-25 and miR-181 family members in SCA3- LCs reflects a cell type-specific, protective mechanism to diminish polyQ-mediated cytotoxic effects. Thus, miRNA mimics of miR-25 and miR-181 family members may prove useful for the treatment of SCA3.

Background and Objective: Accurately evaluating the severity of liver cirrhosis is essential for clinical decision making and disease management. This study aimed to evaluate the value of circulating levels of microRNA (miR)-26a and miR-21 as novel noninvasive biomarkers in detecting severity of cirrhosis in patients with chronic hepatitis B. Methods: Thirty patients with clinically diagnosed chronic hepatitis B-related cirrhosis and 30 healthy individuals were selected. The serum levels of miR-26a and miR-21 were quantified by qRT-PCR. Receiver operating characteristic curve analysis was performed to evaluate the sensitivity and specificity of the miRNAs for detecting the severity of cirrhosis. Results: Serum miR-26a and miR-21 levels were found to be significantly downregulated in patients with severe cirrhosis scored at Child-Pugh class C in comparison to healthy controls (miR-26a p<0.01, and miR-21 p<0.001, respectively). The circulating miR-26a and miR-21 levels in patients were positively correlated with serum albumin concentration but negatively correlated with serum total bilirubin concentration and prothrombin time. Receiver operating characteristic curve analysis revealed that both serum miR-26a and miR-21 levels were associated with a high diagnostic accuracy for patients with cirrhosis scored at Child-Pugh class C (miR-26a Cut-off fold change at ≤0.4, Sensitivity: 84.62%, Specificity: 89.36%, P<0.0001; miR-21 Cut-off fold change at ≤0.6, Sensitivity: 84.62%, Specificity: 78.72%, P<0.0001). Conclusion: Our results indicate that the circulating levels of miR-26a and miR-21 are closely related to the extent of liver decompensation, and the decreased levels are capable of discriminating patients with cirrhosis at Child-Pugh class C from the whole cirrhosis cases.