MiR-23A Affects The Growth, The Apoptosis And The Invasion Ability Of Gastric Adenocarcinoma Cell Line MGC803

[Aims] Recently microRNAs (miRNAs) are discovered as a class of small non-coding RNAs which post-transcriptionally regulate gene expression.They can post-transcriptionlly inhibit genes expression by completely or non-completely binding to3’untranslated region (3’UTR) of its target mRNAs and negatively regulate their target genes. More and more evidence indicate that some miRNAs may function as oncogenes or tumor suppressors and play a critical role in cancer initiation and progression. Does miRNA participate in the initiation and the development of gastric adenocarcinoma? Using microarray assay, we had found that miR-23a was up-regulated in human gastric adenocarcinoma tissue samples camparing with normal human gastric tissue samples. In this research, we studied the effects of miR-23a on the growth, apoptosis and invasion of gastric adenocarcinoma cells.Then we identified the direct target genes of miR-23a, in order to elucidate the molecular mechanisms of miR-23a in the initiation and progression of gastric adenocarcinoma.[Methods] Firstly, in human gastric adenocarcinoma cell line MGC803, miR-23a ASO was used to block the function of endogenous miR-23a or pcDNA3/pri-23a was transfected to over-express miR-23a.Then the changes of cell growth were detected with MTT assay and colony formation assay. And the TUNEL assay or the transwell assay was adopted to detect the effects on the apoptosis or the invasion of MGC803. Then, we combined cDNA microarray analysis with bioinformation and identified the candidate target genes for miR-23a. The the direct target genes was verified by dual fluorescent reporter assay. Subsequently, the mRNA levels and/or protein levels of target genes in miR-23a-inhibited or over-expressed gastric adenocarcinoma cells or tissues were detected with semi-quantitative RT-PCR and quantitative real-time PCR and/or Western blot, in order to verify the regulating role of miR-23a in the target genes expression. After IL-6R,PPP2R5E or IRF1mRNA in MGC803was inhibited with the corresponding siRNA, the changes of cell growth were detected with MTT assay and plate colony formation assay.[Results] The result of miRNA microarray showed that miR-23a was significantly up-regulated in gastric adenocarcinom tissues compared with normal tissues.After suppression of miR-23a, the MGC803cell growth activity, the colony formation activity and non-anchoring growth activity were all inhibited. However, after over-expresssion of miR-23a, the MGC803cell growth activity, the colony formation activity and non-anchoring growth activity were all promoted. After ASO-23a was used to block the function of endogenous miR-23a or pcDNA3/pri-23a was transfected to over-express miR-23a,the apoptosis or the invasion of MGC8O3were enhanced or attenuated (attenuated or enhanced). Then, we identified IL-6R, PPP2R5E and IRF1as the candidate target genes for miR-23a. Their mRNAs3’UTR contain the potential binding sites of miR-23a. The dual fluorescent reporter assay also verified that miR-23a can directly bind to the specific sites of their mRNAs3’UTR and down regulate the genes expression. When miR-23a was inhibited in gastric cells or normal gastric tissues, mRNA level and protein level of IL-6R were both upgraded. Otherwise, in miR-23a over-expressed gastric adenocarcinoma cells or tissues, mRNA level and protein level of IL-6R were both inhibited. After IL-6R,PPP2R5E or IRF1mRNA in MGC803was inhibited respectively, the cell growth activity and colony formation activity were promoted.[Conclusions] The over-expressed miR-23a in gastric adenocarcinoma cells may directly down-regulate the expression level of IL-6R as well as PPP2R5E or IRF1, which leading to the activation of cell growth and invasion and inhibition of apoptosis.Our results indicate that in MGC803, miR-23a may functions as an oncogene. The illumination of the mechanisms of miR-23a in gastric adenocarcinoma helps us to further apprehend its initiation and progression, and offers new proofs to gastric adenocarcinoma diagnosis and prognosis. The results may have meaning for exploring new way to treat gastric adenocarcinoma.