miR-194通过靶定DNMT3A基因调控肝癌细胞的生长  

Regulation of miR-194 on growth of hepatocellular carcinoma cell through targeting DNMT3A

作　　者：马一栋[1] 杨茜[1] 霍月红[2] 尉继林[3] 丁志明[3] 席海英[4] Ma Yidong;Yang Qian;Huo Yuehong;Yu Jilin;Ding Zhiming;Xi Haiying(Department of Oncology,The Fifth Peoples Hospital of Datong, Shanxi Datong 037009 , China;Department of Rheumatology and Immunology,The Fifth Peoples Hospital of Datong, Shanxi Datong 037009 , China;Department of Clinical Laboratory,The Fifth Peoples Hospital of Datong, Shanxi Datong 037009 , China;Department of Dermatology ,The Fifth Peoples Hospital of Datong, Shanxi Datong 037009 , China)

机构地区：[1]大同市第五人民医院肿瘤科,山西大同037009 [2]大同市第五人民医院风湿免疫科,山西大同037009 [3]大同市第五人民医院检验科,山西大同037009 [4]大同市第五人民医院皮肤科,山西大同037009

出　　处：《现代肿瘤医学》2019年第2期187-192,共6页Journal of Modern Oncology

基　　金：大同市基础研究计划项目(编号:2015103)

摘　　要：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,G_1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 constructed. 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 G1 phase. 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 reporter 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 pc DNA and miR-194. Conclusion: miR-194 could inhibit proliferation and cell cycle of the hepatocellular carcinoma cell through targeting DNMT3A.

关 键 词：微小RNA-194 HEPG2细胞 增殖 细胞周期 甲基化转移酶3A 

分 类 号：R735.7[医药卫生—肿瘤]