miRNA arm selection and isomiR distribution in gastric cancer

BACKGROUND: MicroRNAs (miRNAs) are small non-protein-coding RNAs. miRNA genes need several biogenesis steps to form function miRNAs. However, the precise mechanism and biology involved in the mature miRNA molecules are not clearly investigated. In this study, we conducted in-depth analyses to examine the arm selection and isomiRs using NGS platform. METHODS: We sequenced small RNAs from one pair of normal and gastric tumor tissues with Solexa platform. By analyzing the NGS data, we quantified the expression profiles of miRNAs and isomiRs in gastric tissues. Then, we measured the expression ratios of 5p arm to 3p arm of the same pre-miRNAs. And, we used Kolmogorov-Smirnov (KS) test to examine isomiR pattern difference between tissues. RESULTS: Our result showed the 5p arm and 3p arm miRNA derived from the same pre-miRNAs have different tissue expression preference, one preferred normal tissue and the other preferred tumor tissue, which strongly implied that there could be other mechanism controlling mature miRNA selection in addition to the known hydrogen-bonding selection rule. Furthermore, by using the KS test, we demonstrated that some isomiR types preferentially occur in normal gastric tissue but other types prefer tumor gastric tissue. CONCLUSIONS: Arm selections and isomiR patterns are significantly varied in human cancers by using deep sequencing NGS data. Our results provided a novel research topic in miRNA regulation study. With advanced bioinformatics and molecular biology studies, more robust conclusions and insight into miRNA regulation can be achieved in the near future

IsomiRs: A New Approach to Cancer Study

microRNAs are regulatory non-coding RNA molecules that containing about 18-24 nucleotides which involved in critical steps of many cellular processes, specifically gene expression, therefore, their deregulation can lead to human tumorigenesis. some kinds of variants of microRNAs are isomiRs that their length or sequence varies from miRNAs and they commonly araised from diverse cleavage by the ribonucleases Drosha and Dicer. Recent reports confirm that some of isomiRs may be yielded from a miRNA locus, and these physiological mature isomers have important roles in miRNA evolution. In this research, we reviewed new field of miRNAs structural /sequence diversity that can be involved in cancer biology. Also describe the evolutionary approaches and the functional significance of the miRNAs isoforms; however, there are lots of isomiR/miRNA molecular mechanisms that remain unclear. More studies to reveal more insights of functionality isomiRs into the carcinogenesis will provide informative strategies to be used in prognosis, diagnosis or treatment

3’ isomiR species and DNA contamination influence reliable quantification of microRNAs by stem-loop quantitative PCR

MicroRNAs (miRNAs) are ∼20-24 nucleotide-long regulatory RNAs that have been proven to play important roles in many cellular processes. Since their discovery, a number of different techniques have been developed to detect and accurately quantify them. For individual mature miRNA measurements, quantitative stem-loop real-time PCR represents a widely used method. Although there are some data on optimization of this technique, there are still many factors that have not been investigated yet. In this study, we have thoroughly optimized this technique and pointed out several important factors that influence reliable quantification. First, we found that total RNA input can affect the measurements. Second, our data showed that carryover DNA contamination could also mislead the detection in a sequence-specific manner. Additionally, we provided evidence that different 3' isomiR species of a particular miRNA can be reverse transcribed and cross-detected even by specifically targeted assays. Besides these, we have investigated the measurement of reaction efficiencies from total RNA samples and the accuracy of simultaneous reverse transcription reactions for increasing reliability and cost effectiveness without the loss of sensitivity and specificity. In summary, we provide a detailed, refined protocol for reliable detection of microRNA species by quantitative stem-loop PCR

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype

isomiR-SEA: An RNA-Seq analysis tool for miRNAs/isomiRs expression level profiling and miRNA-mRNA interaction sites evaluation

>Background: Massive parallel sequencing of transcriptomes, revealed the presence of many miRNAs and miRNAs variants named isomiRs with a potential role in several cellular processes through their interaction with a target mRNA. Many methods and tools have been recently devised to detect and quantify miRNAs from sequencing data. However, all of them are implemented on top of general purpose alignment methods, thus providing poorly accurate results and no information concerning isomiRs and conserved miRNA-mRNA interaction sites. >Results: To overcome these limitations we present a novel algorithm named isomiR-SEA, that is able to provide users with very accurate miRNAs expression levels and both isomiRs and miRNA-mRNA interaction sites precise classifications. Tags are mapped on the known miRNAs sequences thanks to a specialized alignment algorithm developed on top of biological evidence concerning miRNAs structure. Specifically, isomiR-SEA checks for miRNA seed presence in the input tags and evaluates, during all the alignment phases, the positions of the encountered mismatches, thus allowing to distinguish among the different isomiRs and conserved miRNA-mRNA interaction sites. >Conclusions: isomiR-SEA performances have been assessed on two public RNA-Seq datasets proving that the implemented algorithm is able to account for more reliable and accurate miRNAs expression levels with respect to those provided by two compared state of the art tools. Moreover, differently from the few methods currently available to perform isomiRs detection, the proposed algorithm implements the evaluation of isomiRs and conserved miRNA-mRNA interaction sites already in the first alignment phases, thus avoiding any additional filtering stages potentially responsible for the loss of useful information