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MicroRNAs (miRNAs) are small noncoding RNAs that play fundamental roles in diverse biological and pathological processes by targeting the expression of specific genes. Here, we identified 38 methylation-associated miRNAs, the expression of which could be epigenetically restored by cotreatment with 5-aza-2 0 -deoxycytidine and trichostatin A. Among these 38 miRNAs, we further analyzed miR-34b, miR-127-3p, miR-129-3p and miR-409 because CpG islands are predicted adjacent to them. The methylation-silenced expression of these miRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in a time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of mir-34b and mir-129-2 are frequently methylated in gastric cancer tissues compared to adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. The expression of miR-34b and miR-129-3p was downregulated by DNA hypermethylation in primary gastric cancers, and the low expression was associated with poor clinicopathological features. In summary, our study shows that tumor-specific methylation silences miR-34b and miR-129 in gastric cancer cells.

BackgroundMicroRNAs (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.MethodsWe 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.ResultsOur 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.ConclusionsArm 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.

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BackgroundMicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species.Methodology/Principal FindingsWe identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis.Conclusions/SignificanceTaking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.

MicroRNAs (miRNAs) are endogenous non-protein-coding RNAs of approximately 22 nucleotides. Thousands of miRNA genes have been identified (computationally and/or experimentally) in a variety of organisms, which suggests that miRNA genes have been widely shared and distributed among species. Here, we used unique miRNA sequence patterns to scan the genome sequences of 56 bilaterian animal species for locating candidate miRNAs first. The regions centered surrounding these candidate miRNAs were then extracted for folding and calculating the features of their secondary structure. Using a support vector machine (SVM) as a classifier combined with these features, we identified an additional 13,091 orthologous or paralogous candidate pre-miRNAs, as well as their corresponding candidate mature miRNAs. Stem-loop RT-PCR and deep sequencing methods were used to experimentally validate the prediction results in human, medaka and rabbit. Our prediction pipeline allows the rapid and effective discovery of homologous miRNAs in a large number of genomes.

Identification of microRNAs (miRNAs) is essential to studying their physiological functions. Due to the difficulties in discovering truly expressed miRNAs from genomic random hairpin secondary structure sequences, it is beneficial to predict them from expressed sequences--expressed sequence tags (ESTs) and intronic sequences. We used a modified scanning pipeline using criteria based on the features of known pre-miRNAs and phylogenetic conservation for predicting intronic miRNAs. Upon examination, we found that 25% of known human miRNAs belong to intronic regions of known protein-coding genes. About 50% of these intronic miRNAs reside in introns whose length is longer than 5,000 bps. It is likely that these intronic miRNAs can have their own independently regulated transcription units, which can be regulated by RNA polymerase II (Pol II) or RNA polymerase III (Pol III). It was recently demonstrated that RNA Pol III could transcribe human miRNAs through associated repetitive elements. Since various repetitive elements are often found to be present in the intronic regions, the distribution of intronic miRNAs and their possible transcription regulation are presented. Although the intronic miRNAs and their host genes could be regulated independently, it is possible that the intronic miRNA can still down-regulate its own host protein-coding gene by targeting the untranslated region (UTR) of the host gene. Another biological implication is that intronic miRNAs could play an important role as negative feedback regulators. We propose hypothetical models of such feedback regulation on host protein-coding genes by selecting the transcription factors as miRNA targets or by protein-protein interactions between intronic miRNA host gene product and miRNA target gene products.

MicroRNAs (miRNAs) are short noncoding RNAs that play important roles in cellular processes and disease pathogenesis via the control of specific targeted gene expression. The miR-196s miRNA is encoded at three paralogous loci in three HOX clusters and acts as an oncogenic miRNA in cancer progression. Recent studies have demonstrated that the expression of miR-196b increases cell proliferation and survival in leukemic cells. Here, we used a sequential methylation analysis to reveal that the methylation status correlated well with miR-196b expression in different cell lines. Treatment with the demethylating drug 5-Aza-dC reactivated miR-196b transcription in methylation-silenced cells. Using in vitro methylation approach, we further provide evidences that promoter hypermethylation represses miR-196b transcriptional activation tightly in human cancer cell lines. We also demonstrate that the expression of miR-196b is significantly elevated in gastric cancer and that hypomethylation status of miR-196b CpG islands frequently is observed in primary gastric tumors. Our results provide important information on miR-196s regulation and demonstrate that abnormal DNA hypomethylation induces overexpression of miR-196b in gastric cancer.