| [Background and Aims] MicroRNAs (miRNAs) are a class of small non-coding RNAs that include18-26nucleotides, which post-transcriptionally regulate gene expression. MiRNAs are involved in many physiological and pathological progress. Recent evidence indicates that many miRNAs function as oncogenes or tumor suppressors and play an important role in cancer initiation and progression by regulating their target genes negatively. Using oligonucleotide microarray, we found that microRNA-181b (miR-181b) and microRNA-182(miR-182) were significantly down-regulated in human gastric adenocarcinoma tissue samples. In this study, we focused on the effects of miR-181b and miR-182on the phenotypes of gastric adenocarcinoma cells as well as the identification of their direct target genes, in order to illuminate the molecular mechanisms of miR-181b and miR-182in the initiation and progression of gastric adenocarcinoma.[Methods] We detected the differencial expression of miR-181b and miR-182in human gastric adenocarcinoma and adjacent normal tissues by Real-time PCR assay. miR-181b and miR-182were overexpressed in three kinds of gastric adenocarcinoma cell lines respectively, and the changes of cell phenotypes were detected both with MTT assay and colony formation assay. Subsequently, we combined bioinformatics and cDNA microarray analysis and identified the candidate target genes for miR-181b and miR-182. Then fluorescent reporter assay was performed to confirm the reliability of the direct target genes. Furthermore, the mRNA levels and protein levels of target genes in miR-181b-and miR-182-overexpressed gastric cancer cells or tissues were detected with Real-time PCR and Western blot, in order to confirm the regulative role of miR-181b and miR-182on CREB1expression. Finally, the target gene was knocked down using RNA interference and changes of cell phenotypes were detected both with MTT assay and colony formation assay.[Results] Real-time PCR results showed that the expression of miR-181b and miR-182in human gastric adenocarcinoma tissues were lower than those in adjacent normal tissues. We report that after overexpression of miR-181b and miR-182, the proliferation activity and the colony formation activity of gastric cancer cells were both inhibited. Subsequently, we identified oncogene CREB1as congenerous candidate target genes for both miR-181b and miR-182. The CREB1mRNA3’ untranslated region (3’UTR) contains the potential binding site both of miR-181b and miR-182. The fluorescent reporter assay also confirmed that miR-181b and miR-182can directly bind to the specific site of CREB1mRNA3’UTR and negatively regulate the gene expression. When miR-181b and miR-182function was overexpressed in gastric cancer cells, mRNA level and protein level of CREB1were both depressed, and mRNA level of CREB1in human gastric adenocarcinoma tissues were higher than those in adjacent normal tissues. We also discoved that when the target gene was knocked down, the proliferation activity and the colony formation activity of gastric cancer cells were both inhibited apparently, consistent with the overexpression of miR-181b and miR-182.[Conclusions] Our results show that in gastric adenocarcinoma cells, miR-181b and miR-182functions as tumor suppressors and inhibit cell proliferation. The low-expression of miR-181b and miR-182in gastric adenocarcinoma cells lead to an abnormally high expression level of oncogene CREB1and result in the activation of cell proliferation. The elucidation of the mechanisms of miR-181b and miR-182in gastric adenocarcinoma helps us to further understand the mechanism of gastric adenocarcinoma initiation and progression, and pave a way for clinical diagnosis and therapy of gastric adenocarcinoma. |
