SMYD3过表达对人肝内胆管癌细胞miR-124表达及细胞增殖的影响

摘要

目的:探讨肝内胆管癌细胞HCCC-9810中SET和MYND结构域含有蛋白3(SET and MYND domain-containing protein 3,SMYD3)过表达对miR-124表达及细胞增殖能力的影响.方法:瞬时转染SMYD3真核表达质粒后,Western blotting检测细胞中SMYD3蛋白水平的变化；qRT-PCR检测细胞中SMYD3mRNA和miR-124表达；甲基化特异性PCR检测miR-124-1、miR-124-2和miR-124-3基因启动子甲基化水平；CCK-8和平板克隆形成实验检测细胞增殖能力.结果:以空白组为对照,肝内胆管癌细胞HCCC-9810在转染pEGFP-SMYD3质粒后,SMYD3蛋白及mRNA表达均显著上升(P＜0.05),miR-124-1、miR-124-2和miR-124-3基因启动子甲基化显著增加(P＜0.05),miR-124表达明显下降(P＜0.05),细胞增殖能力显著提高(P＜0.05).结论:过表达SMYD3可引起miR-124基因启动子甲基化增加,导致胆管癌细胞中miR-124的表达下降,同时过表达SMYD3可增强细胞增殖能力.%AIM: To explore the effect of SET and MYND domain-containing protein 3 (SMYD3) over-expression on miR-124 expression and proliferation ability of human intrahepatic cholangiocarcinoma cells. METHODS: Transient transfection of SMYD3 eukaryotic expression plasmid into human intrahepatic cholangiocarcinoma cell line HCCC-9810 were performed. The expression of SMYD3 at mRNA and protein levels was measured by qRT-PCR and Western blotting, respectively. The expression of miR-124 was detected by qRT-PCR, and the methylation status of miR-124 gene was determined by methylation-specific PCR. Cell proliferation was examined by CCK-8 assay and colony formation experiment. RESULTS: After transfected with SMYD3 eukaryotic expression plasmid, the over-expression of SMYD3 in HCCC-9810 cells was observed. Compared with the blank cells, the expression level of miR-124 was significantly decreased and miR-124 gene promoter methylation was significantly increased. In addition, SMYD3 over-expression significantly promoted the proliferation of HCCC-9810 cells. CONCLUSION: The transient transfection of SMYD3 plasmid increases the methylation of miR-124 gene promoter and induces under-expression of miR-124. Over-expression of SMYD3 promotes the proliferation of cholangiocarcinoma cells.