目的:探讨miR-194对肝癌细胞增殖和细胞周期的影响及其潜在的作用机制.方法:实时定量聚合酶链式反应检测肝癌细胞系HepG2和正常肝细胞系L-O2中miR-194的表达水平.构建miR-194过表达质粒,采用MTT法检测细胞增殖活力,流式细胞术检测细胞周期;双荧光素酶报告基因分析法预测和验证miR-194可能的靶基因.结果:实时定量PCR结果显示,miR-194在肝癌细胞中的表达明显低于肝脏正常细胞.在肝癌细胞中过表达miR-194抑制细胞生长.而流式细胞术检测发现细胞周期进程减慢,G1期比例增加,S期比例相应的减少.靶基因筛选得到DNMT3A为miR-194的候选靶基因.荧光报告载体实验证实,miR-194能够通过作用于靶基因3＇非翻译区的特定位点,对其表达在转录后进行负性调节.而在miR-194 表达增加的肝癌细胞中,靶基因的mRNA表达水平和蛋白表达水平都有明显降低.在肝癌细胞HepG2中,敲除靶基因DNMT3A后,细胞的增殖能力减弱,相反,当把DNMT3A过表达后,细胞的增殖能力增强,可以挽救miR-194对细胞的表型影响.结论:miR-194可通过靶定DNMT3A基因抑制肝癌细胞的生长.%Objective:To explore effect of miR-194 on proliferation and cell cycle of the human hepatocellular carcinoma cell and its potential mechanism.Methods:Expressions of miR-194 in hepatoma cell line HepG2 as well as normal liver cell L-O2 were detected by RT-PCR.The plasmid with over-expression of miR-194 was con-structed.MTT and FACS were respectively used to measure proliferation ability of the cells and cell cycle.Possible target gene of miR-194 was forecasted and verified with dual luciferase report gene assay.Results:Compared with normal liver cell,miR-194 is low -expressed in HepG2.Over -expression of miR -194 decreased cell growth.FACS showed miR-194 arrested cells cycle in G1phase.The DNMT3A was identified to be a putative target gene,whose mRNA 3＇-untranslated region (3＇UTR)contains the potential binding site of miR-194.The fluorescent re-porter experiment also confirmed that miR-194 can directly bind to the target genes mRNA 3＇UTR.The mRNA and protein level of DNMT3A in HepG2 gave the clue that miR-194 can negatively regulate the genes expression through mRNA decay.Knockdown of DNMT3A by RNA interference can decrease the cell proliferation.Finally,when cells were transfected with DNMT3A and miR-194,the cell proliferation ability was significantly recovered comparing to the cells transfected with pcDNA and miR-194.Conclusion:miR-194 could inhibit proliferation and cell cycle of the hepatocellular carcinoma cell through targeting DNMT3A.
展开▼
