miR-122靶向调控RhoA基因影响乙型肝炎病毒复制与表达的机制研究  

Study on the mechanism of miR-122 targeted regulation of RhoA gene affecting the replication and expression of hepatitis B virus

作　　者：马红 李莉 周丽君[2] MA Hong;LI Li;ZHOU Lijun(Department of Clinical Laboratory,Urumqi Traditional Chinese Medicine Hospital,Urumqi,Xinjiang 830000,China;Department of Clinical Laboratory,Urumqi Blood Center,Urumqi,Xinjiang 830000,China)

机构地区：[1]乌鲁木齐市中医医院检验科,新疆乌鲁木齐830000 [2]乌鲁木齐市血液中心检验科,新疆乌鲁木齐830000

出　　处：《国际检验医学杂志》2023年第6期677-682,共6页International Journal of Laboratory Medicine

基　　金：乌鲁木齐市卫生健康委科技计划项目(202016)。

摘　　要：目的探讨miR-122通过调控小G蛋白超家族成员RhoA基因对隐匿性乙型肝炎病毒(HBV)复制与表达的影响及其作用机制。方法实时荧光定量PCR测定HBV(+)与HBV(-)的肝癌患者组织、HepG2细胞与HepG2.2.15细胞中miR-122表达差异,实时荧光定量PCR和Western Blot检测HBV(+)与HBV(-)的肝癌患者组织、HepG2细胞与HepG2.2.15细胞中RhoA mRNA和RhoA蛋白的表达差异;按照实验分组将pEGFP-C1-miR-122与pcDNA3.0-V14RhoA转染至HepG2.2.15细胞,实时荧光定量PCR测定miR-122与RhoA mRNA表达,Western Blot检测RhoA蛋白表达;生物信息学方法预测miR-122与RhoA基因的靶向结合位点,构建野生型RhoA 3′非翻译区(3′UTR)和突变型RhoA 3′UTR双荧光素酶表达载体,检测细胞荧光素酶活性变化;酶联免疫吸附试验测定细胞培养上清液中乙型肝炎表面抗原(HBsAg)与乙型肝炎e抗原(HBeAg)水平,实时荧光定量PCR检测细胞培养上清液中HBV DNA水平,Western Blot检测细胞内Rho相关蛋白激酶(ROCK)-1与ROCK-2相对表达水平。结果相较于HBV(-)肝癌患者组织,HBV(+)肝癌患者组织中miR-122表达下调,RhoA mRNA和RhoA蛋白相对表达水平上调(P<0.05);与HepG2细胞比较,HepG2.2.15细胞中miR-122表达也下调,RhoA mRNA和RhoA蛋白相对表达水平上调(P<0.05)。经预测发现RhoA mRNA 3′UTR与miR-122在特定区域存在碱基互补现象,miR-122 mimic和RhoA 3′UTR WT重组质粒共转染的细胞中相对荧光素酶活性降低(P<0.05)。此外,过表达miR-122的HepG2.2.15细胞中RhoA mRNA和RhoA蛋白相对表达水平下调(P<0.05)。与空白组比较,miR-122组HepG2.2.15细胞培养上清液HBsAg与HBeAg水平均降低,HBV DNA表达降低,ROCK-1与ROCK-2表达均上调(P<0.05);而V14RhoA组HBsAg与HBeAg水平则高于空白组,HBV DNA表达升高,ROCK-1与ROCK-2表达均上调(P<0.05)。结论miRNA-122可通过下调RhoA基因表达抑制HBV的复制和表达,其机制可能与抑制Rho/ROCK信号通路激活有关。Objective To explore the effect of miR-122 on the replication and expression of occult hepatitis B virus(HBV)and its mechanism by regulating RhoA gene,a member of the small G protein superfamily.Methods Real-time fluorescent quantitative PCR was used to determine the expression differences of miR-122 in HBV(+)and HBV(-)liver cancer patient tissues,HepG2 cells and HepG2.2.15 cells.Real-time fluorescent quantitative PCR and Western Blot detected differences in the expression of RhoA mRNA and RhoA protein in HBV(+)and HBV(-)liver cancer patient tissues,HepG2 cells and HepG2.2.15 cells;According to the experimental grouping,pEGFP-C1-miR-122 and pcDNA3.0-V14RhoA were transfected into HepG2.2.15 cells,Real-time fluorescent quantitative PCR measured the expression of miR-122 and RhoA mRNA,and Western Blot detected the expression of RhoA protein;Bioinformatics methods predicted the targeted binding site of miR-122 and RhoA gene,and constructed wild-type RhoA 3′UTR and mutant RhoA 3′UTR dual luciferase expression vectors,and detected the changes in cellular luciferase activity.Enzyme-linked immunosorbent assay measured the content of hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in the cell culture supernatant.Real-time fluorescent quantitative PCR detected the level of HBV DNA in the cell culture supernatant,Western Blot detected the expression protein levels of Rho-associated protein kinase(ROCK)-1 and ROCK-2 in the cells.Results Compared with the tissues of HBV(-)liver cancer patients,the expression of miR-122 was down-regulated and the expression of RhoA mRNA and RhoA protein were up-regulated in the tissues of HBV(+)liver cancer patients(P<0.05).Compared with HepG2 cells,the expression of miR-122 in HepG 2.2.15 cells was also down-regulated,and the expression of RhoA mRNA and RhoA protein were up-regulated(P<0.05).It is predicted that RhoA mRNA 3′UTR and miR-122 had base complementation in specific regions,and the relative luciferase activity of cells co-transfected with miR-122 mimic and Rh

关 键 词：乙型肝炎病毒 RHOA MIR-122 HEPG2.2.15细胞 

分 类 号：R512.63[医药卫生—内科学]