Integrated MicroRNA-mRNA Analysis Reveals miR-204 Inhibits Cell Proliferation in Gastric Cancer by Targeting CKS1B, CXCL1 and GPRC5A.

Gastric cancer (GC) is the second most frequent cause of cancer-related deaths worldwide. MicroRNAs are single-stranded RNA molecules of 21-23 nucleotides that regulate target gene expression through specific base-pairing interactions between miRNA and untranslated regions of targeted mRNAs. In this study, we generated a multistep approach for the integrated analysis of miRNA and mRNA expression. First, both miRNA and mRNA expression profiling datasets in gastric cancer from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) identified 79 and 1042 differentially expressed miRNAs and mRNAs, respectively, in gastric cancer. Second, inverse correlations between miRNA and mRNA expression levels identified 3206 miRNA-mRNA pairs combined with 79 dysregulated miRNAs and their 774 target mRNAs predicted by three prediction tools, miRanda, PITA, and RNAhybrid. Additionally, miR-204, which was found to be down-regulated in gastric cancer, was ectopically over-expressed in the AGS gastric cancer cell line and all down-regulated targets were identified by RNA sequencing (RNA-seq) analysis. Over-expression of miR-204 reduced the gastric cancer cell proliferation and suppressed the expression of three targets which were validated by qRT-PCR and luciferase assays. For the first time, we identified that CKS1B, CXCL1, and GPRC5A are putative targets of miR-204 and elucidated that miR-204 acted as potential tumor suppressor and, therefore, are useful as a promising therapeutic target for gastric cancer.

Honeysuckle aqueous extract and induced let-7a suppress dengue virus type 2 replication and pathogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Honeysuckle (Lonicera japonica Thunb.), a traditional Chinese herb, has widely been used to treat pathogen infection. However, the underlying-mechanism remains elusive. AIMS OF THE STUDY: To reveal the host microRNA (miRNA) profile with the anti-viral activity after honeysuckle treatment. MATERIALS AND METHODS: Here we reveal the differentially expressed miRNAs by Solexa® deep sequencing from the blood of human and mice after the aqueous extract treatment. Among these overexpressed innate miRNAs both in human and mice, let-7a is able to target the NS1 region (nt 3313-3330) of dengue virus (DENV) serotypes 1, 2 and 4 predicated by the target predication software. RESULTS: We confirmed that let-7a could target DENV2 at the predicated NS1 sequence and suppress DENV2 replication demonstrated by luciferase-reporter activity, RT-PCR, real-time PCR, Western blotting and plaque assay. ICR-suckling mice consumed honeysuckle aqueous extract either before or after intracranial injection with DENV2 showed decreased levels of NS1 RNA and protein expression accompanied with alleviated disease symptoms, decreased virus load, and prolonged survival time. Similar results were observed when DENV2-infected mice were intracranially injected with let-7a. CONCLUSION: We reveal that honeysuckle attenuates DENV replication and related pathogenesis in vivo through induction of let-7a expression. This study opens a new direction for prevention and treatment of DENV infection through induction of the innate miRNA let-7a by honeysuckle.

miRTarBase 2016: updates to the experimentally validated miRNA-target interactions database.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides, which negatively regulate the gene expression at the post-transcriptional level. This study describes an update of the miRTarBase (http://miRTarBase.mbc.nctu.edu.tw/) that provides information about experimentally validated miRNA-target interactions (MTIs). The latest update of the miRTarBase expanded it to identify systematically Argonaute-miRNA-RNA interactions from 138 crosslinking and immunoprecipitation sequencing (CLIP-seq) data sets that were generated by 21 independent studies. The database contains 4966 articles, 7439 strongly validated MTIs (using reporter assays or western blots) and 348 007 MTIs from CLIP-seq. The number of MTIs in the miRTarBase has increased around 7-fold since the 2014 miRTarBase update. The miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) are integrated to provide an effective overview of this exponential growth in the miRNA experimental data. These improvements make the miRTarBase one of the more comprehensively annotated, experimentally validated miRNA-target interactions databases and motivate additional miRNA research efforts.

Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

BACKGROUND: MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. The development of Argonaute cross-linked immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) provides a system to effectively determine miRNA target genes. Likewise, the accuracy of dissecting the transcriptional regulation of miRNA genes has been greatly improved by chromatin immunoprecipitation of the transcription factors coupled with sequencing (ChIP-Seq). Elucidation of the miRNA target repertoire will provide an in-depth understanding of the functional roles of microRNA pathways. To reliably reconstruct a miRNA-mediated regulatory network, we established a computational framework using publicly available, sequence-based transcription factor-miRNA databases, including ChIPBase and TransmiR for the TF-miRNA interactions, along with miRNA-target databases, including miRTarBase, TarBase and starBase, for the miRNA-target interactions. We applied the computational framework to elucidate the miRNA-mediated regulatory network in the Mir122a⁻/⁻ mouse model, which has an altered transcriptome and progressive liver disease. RESULTS: We applied our computational framework to the expression profiles of miRNA/mRNA of Mir122a⁻/⁻ mutant mice and wild-type mice. The miRNA-mediated network involves 40 curated TFs contributing to the aberrant expression of 65 miRNAs and 723 curated miRNA target genes, of which 56% was found in the differentially-expressed genes of Mir122a--mice. Hence, the regulatory network disclosed previously-known and also many previously-unidentified miRNA-mediated regulations in mutant mice. Moreover, we demonstrate that loss of imprinting at the chromosome 12qF1 region is associated with miRNA overexpression in human hepatocellular carcinoma and stem cells, suggesting initiation of precancerous changes in young mice deficient in miR-122. A group of 9 miRNAs was found to share miR-122 target genes, indicating synergy between miRNAs and target genes by way of multiplicity and cooperativity. CONCLUSIONS: The study provides significant insight into miRNA-mediated regulatory networks. Based on experimentally verified data, this network is highly reliable and effective in revealing previously-undetermined disease-associated molecular mechanisms. This computational framework can be applied to explore the significant TF-miRNA-miRNA target interactions in any complex biological systems with high degrees of confidence.

MethHC: a database of DNA methylation and gene expression in human cancer.

We present MethHC (http://MethHC.mbc.nctu.edu.tw), a database comprising a systematic integration of a large collection of DNA methylation data and mRNA/microRNA expression profiles in human cancer. DNA methylation is an important epigenetic regulator of gene transcription, and genes with high levels of DNA methylation in their promoter regions are transcriptionally silent. Increasing numbers of DNA methylation and mRNA/microRNA expression profiles are being published in different public repositories. These data can help researchers to identify epigenetic patterns that are important for carcinogenesis. MethHC integrates data such as DNA methylation, mRNA expression, DNA methylation of microRNA gene and microRNA expression to identify correlations between DNA methylation and mRNA/microRNA expression from TCGA (The Cancer Genome Atlas), which includes 18 human cancers in more than 6000 samples, 6548 microarrays and 12 567 RNA sequencing data.

A systematic review of microRNA expression profiling studies in human gastric cancer.

Gastric cancer (GC) is the second leading cause of global cancer mortality. Most GC patients are diagnosed with advanced-stage disease and show extremely poor prognosis. All of the GC research has a common interest to search for the specific and sensitive biomarkers for early diagnosis of GC. Number of microRNAs play important role in GC. We carried out a systematic review of published miRNA profiling studies that compared the miRNA expression profiles between GC tissues and paired noncancerous gastric tissue. A vote-counting strategy was followed with the collection of information like total number of studies reporting differential expression of miRNA, total number of tissue samples used in the studies, direction of differential expression and fold change. A total of 352 differentially expressed microRNAs were reported in the 14 microRNA expression profiling studies that compared GC tissues with normal tissues with 120 microRNAs reported at least in two studies. In the group of consistently reported microRNAs, miR-21 was reported upregulated in 10 studies followed by miR-25, miR-92, and miR-223 upregulated in eight studies. MiR-375 and miR-148a were found downregulated in six and five studies, respectively, followed by miR-638 in four studies. MiR-107 and miR-103 were reported in nine and eight studies, respectively, but their expression were inconsistent. From this study, the most consistently reported upregulated microRNA was found to be miR-21. This systematic review study of human GC microRNA expression profiling studies would provide information on microRNAs with potential role as the biomarkers in gastric cancer.

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis.

MicroRNA-122 (miR-122), which accounts for 70% of the liver's total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

miRNAMap 2.0: genomic maps of microRNAs in metazoan genomes.

MicroRNAs (miRNAs) are small non-coding RNA molecules that can negatively regulate gene expression and thus control numerous cellular mechanisms. This work develops a resource, miRNAMap 2.0, for collecting experimentally verified microRNAs and experimentally verified miRNA target genes in human, mouse, rat and other metazoan genomes. Three computational tools, miRanda, RNAhybrid and TargetScan, were employed to identify miRNA targets in 3'-UTR of genes as well as the known miRNA targets. Various criteria for filtering the putative miRNA targets are applied to reduce the false positive prediction rate of miRNA target sites. Additionally, miRNA expression profiles can provide valuable clues on the characteristics of miRNAs, including tissue specificity and differential expression in cancer/normal cell. Therefore, quantitative polymerase chain reaction experiments were performed to monitor the expression profiles of 224 human miRNAs in 18 major normal tissues in human. The negative correlation between the miRNA expression profile and the expression profiles of its target genes typically helps to elucidate the regulatory functions of the miRNA. The interface is also redesigned and enhanced. The miRNAMap 2.0 is now available at http://miRNAMap.mbc.nctu.edu.tw/.