Kuo-Wang Tsai

Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer.

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′‐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.

Epigenetic control of the expression of a primate-specific microRNA cluster in human cancer cells

Many of the known microRNAs (miRNAs) are encoded by polycistronic transcripts and are thought to be co-expressed. In this study, we discovered that the expression of a large miRNA cluster (C19MC) on human chromosome 19 is upregulated by the demethylating agent, 5-aza-2-deoxycytidine (5-Aza-dC), in AGS gastric cancer cells. We found that C19MC was rarely expressed in most cells, but its expression was restored through DNA demethylation. We confirmed that this miRNA cluster was mainly expressed in the placenta, as previously reported. Furthermore, its expression pattern was highly correlated with the methylation state of a distal CpG-rich region located about 17.6 kb upstream of the miRNA cluster. Using combined bisulfite restriction analysis (COBRA) and bisulfite-sequencing techniques, we determined that this CpG-rich region is hypermethylated in the AGS and HeLa cells, but hypomethylated in the placenta tissue. In conclusion, we demonstrated that the expression pattern of the C19MC was activated in human cancer cells through demethylation of a CpG-rich region.

Aberrant hypermethylation of miR-9 genes in gastric cancer

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly down-regulated in gastric cancers compared with adjacent normal tissues (P value < 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.

miRNA arm selection and isomiR distribution in gastric cancer

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.

Epigenetic regulation of miR-196b expression in gastric cancer

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.

Discovery and characterization of medaka miRNA genes by next generation sequencing platform

BackgroundMicroRNAs (miRNAs) are endogenous non-protein-coding RNA genes which exist in a wide variety of organisms, including animals, plants, virus and even unicellular organisms. Medaka (Oryzias latipes) is a useful model organism among vertebrate animals. However, no medaka miRNAs have been investigated systematically. It is beneficial to conduct a genome-wide miRNA discovery study using the next generation sequencing (NGS) technology, which has emerged as a powerful sequencing tool for high-throughput analysis.ResultsIn this study, we adopted ABI SOLiD platform to generate small RNA sequence reads from medaka tissues, followed by mapping these sequence reads back to medaka genome. The mapped genomic loci were considered as candidate miRNAs and further processed by a support vector machine (SVM) classifier. As result, we identified 599 novel medaka pre-miRNAs, many of which were found to encode more than one isomiRs. Besides, additional minor miRNAs (also called miRNA star) can be also detected with the improvement of sequencing depth. These quantifiable isomiRs and minor miRNAs enable us to further characterize medaka miRNA genes in many aspects. First of all, many medaka candidate pre-miRNAs position close to each other, forming many miRNA clusters, some of which are also conserved across other vertebrate animals. Secondly, during miRNA maturation, there is an arm selection preference of mature miRNAs within precursors. We observed the differences on arm selection preference between our candidate pre-miRNAs and their orthologous ones. We classified these differences into three categories based on the distribution of NGS reads. Finally, we also investigated the relationship between conservation status and expression level of miRNA genes. We concluded that the evolutionally conserved miRNAs were usually the most abundant ones.ConclusionsMedaka is a widely used model animal and usually involved in many biomedical studies, including the ones on development biology. Identifying and characterizing medaka miRNA genes would benefit the studies using medaka as a model organism.

Aberrant expression of miR-196a in gastric cancers and correlation with recurrence

MicroRNAs (miRNAs) are short noncoding RNAs (˜22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Here, we examined the relationship between miR‐196a and gastric cancer. By the analysis of 72 gastric cancer samples, we found that the expression level of miR‐196a microRNA significantly increased in primary gastric cancer tissues versus adjacent normal tissues. In addition, extracellular miR‐196a detected in conditioned medium was strongly correlated with its cellular expression status and increased circulating miR‐196a in patient serum was associated with gastric cancer disease status and relapse. Furthermore, ectopic expression of miR‐196a microRNA promoted the epithelial‐mesenchymal transition and migration/invasion capabilities of transfected cells, suggesting its oncogenic potential in gastric cancer progression. Altogether, our data demonstrate that miR‐196a exerts an oncogenic role in gastric cancer and miR‐196a may be a novel biomarker for detecting gastric cancer and for monitoring disease recurrence.

Transcriptional regulation of miR-196b by ETS2 in gastric cancer cells

E26 transformation-specific sequence (ETS)-2 is a transcriptional modulator located on chromosome 21, alterations in its expression have been implicated with a reduced incidence of solid tumors in Down syndrome patients. MicroRNAs (miRNAs) are thought to participate in diverse biological functions; however, the regulation of miRNAs is not well characterized. Recently, we reported that miR-196b is highly expressed in gastric cancers. Herein, we demonstrate that miR-196b expression was significantly repressed by ETS2 during gastric cancer oncogenesis. We demonstrate that knockdown of endogenous ETS2 expression increases miR-196b expression. A genomic region between −751 and −824 bp upstream of the miR-196b transcriptional start site was found to be critical for the repression activity. This putative regulatory promoter region contains three potential ETS2-binding motifs. Mutations within the ETS2 binding sites blocked the repression activity of ETS2. Furthermore, knockdown of ETS2 or overexpression of miR-196b significantly induced migration and invasion in gastric cancer cells. In addition, alterations in ETS2 and miR-196b expression in gastric cancer cell lines affected the expression of epithelial–mesenchymal transition-related genes. The levels of vimentin, matrix metalloproteinase (MMP)-2 and MMP9 were drastically induced, but levels of E-cadherin were decreased in shETS2- or miR-196b-transfected cells. Our data indicate that ETS2 plays a key role in controlling the expression of miR-196b, and miR-196b may mediate the tumor suppressor effects of ETS2. We demonstrated that miR-196b was transcriptionally regulated by ETS2 and there was an inverse expression profile between miR-196b and ETS2 in clinical samples. This finding could be beneficial for the development of effective cancer diagnostic and alternative therapeutic strategies.

MetaMirClust: Discovery of miRNA cluster patterns using a data-mining approach

Recent genome-wide surveys on ncRNA have revealed that a substantial fraction of miRNA genes is likely to form clusters. However, the evolutionary and biological function implications of clustered miRNAs are still elusive. After identifying clustered miRNA genes under different maximum inter-miRNA distances (MIDs), this study intended to reveal evolution conservation patterns among these clustered miRNA genes in metazoan species using a computation algorithm. As examples, a total of 15-35% of known and predicted miRNA genes in nine selected species constitute clusters under the MIDs ranging from 1kb to 50kb. Intriguingly, 33 out of 37 metazoan miRNA clusters in 56 metazoan genomes are co-conserved with their up/down-stream adjacent protein-coding genes. Meanwhile, a co-expression pattern of miR-1 and miR-133a in the mir-133-1 cluster has been experimentally demonstrated. Therefore, the MetaMirClust database provides a useful bioinformatic resource for biologists to facilitate the advanced interrogations on the composition of miRNA clusters and their evolution patterns.

Interrogation of rabbit miRNAs and their isomiRs

Rabbit (Oryctolagus cuniculus) is the only lagomorph animal of which the genome has been sequenced. Establishing a rabbit miRNA resource will benefit subsequent functional genomic studies in mammals. We have generated small RNA sequence reads with SOLiD and Solexa platforms to identify rabbit miRNAs, where we identified 464 pre-miRNAs and 886 mature miRNAs. The brain and heart miRNA libraries were used for further in-depth analysis of isomiR distributions. There are several intriguing findings. First, several rabbit pre-miRNAs form highly conserved clusters. Second, there is a preference in selecting one strand as mature miRNA, resulting in an arm selection preference. Third, we analyzed the isomiR expression and validated the expression of isomiR types in different rabbit tissues. Moreover, we further performed additional small RNA libraries and defined miRNAs differentially expressed between brain and heart. We conclude also that isomiR distribution profiles could vary between brain and heart tissues.