乙型肝炎病毒X蛋白通过miR-122调节肝癌细胞增殖及细胞周期的研究

Hepatitis B Virus X Protein Regulates Proliferation and Cell Cycle of Hepatocellular Carcinoma Cells Through miR-122

乙型肝炎病毒中的功能蛋白乙肝病毒X蛋白(Hepatitis B virus X protein,HBx)在促进肝细胞恶性改变中起到重要作用,但目前HBx调控肝癌细胞生长的具体机制仍未完全阐明。miR-122是具有抑癌特性的一类miR,在乙肝相关肝癌中表达减少。为了研究HBx通过微小RNA(microRNA,miR)-122调节肝癌细胞增殖及细胞周期的作用,本研究培养肝癌HepG2细胞株并进行分组,NC组转染NC慢病毒载体、HBx组转染HBx慢病毒载体、HBx+NC模拟物组转染HBx慢病毒载体及NC模拟物、HBx+miR-122模拟物组转染HBx慢病毒载体及miR-122模拟物、NC模拟物组转染NC模拟物、miR-122模拟物组:转染miR-122模拟物。通过MTS法检测细胞增殖活力,流式细胞术检测细胞周期,PCR检测miR-122表达量,western blot检测细胞周期蛋白G1(CyclinG1)、X连锁凋亡抑制蛋白(XIAP)、β-连环蛋白(β-catenin)的表达量。结果显示HBx组细胞的OD值、细胞周期G2/M期比例及细胞中CyclinG1、XIAP、β-catenin的表达量均明显高于NC组(P<0.05),细胞周期G0/G1期、S期比例及细胞中miR-122表达量均明显低于NC组(P<0.05);HBx+miR-122模拟物组细胞的OD值、细胞周期G2/M期比例及细胞中CyclinG1、XIAP、β-catenin的表达量均明显低于HBx+NC模拟物组(P<0.05),细胞周期G0/G1期、S期比例及细胞中miR-122表达量均明显高于HBx+NC模拟物组(P<0.05);miR-122模拟物组CyclinG1、XIAP、β-catenin荧光素酶报告基因的荧光活性明显低于NC模拟物组(P<0.05)。本研究结果充分说明HBx能够增强肝癌细胞的增殖活力及明显加速细胞周期,且该作用部分由miR-122的下调所介导。本研究首次阐明了HBx调节肝癌细胞生长的分子机制,也初步探明了具有抑癌活性的miR-122在肝癌细胞中可能靶向CyclinG1、XIAP、β-catenin等基因。

Hepatitis B virus X protein(HBx),a functional protein of hepatitis B virus,plays an important role in promoting the malignant changes of liver cells. However,the mechanism of HBx in regulating the growth of liver cancer cells has not been fully elucidated. microRNA(mi-R)-122 is a kind of miR with anti-tumor properties,and its expression is decreased in hepatitis B-related liver cancer. To study the role of HBx in regulating the proliferation and cell cycle of hepatocellular carcinoma cells(HCCs)through miR-122,HepG2 cell lines were cultured and grouped. The NC group was transfected with the negative control(NC)lentivirus vector. The HBx group was transfected with the HBxlentivirus vector. The HBx+NC mimic group was transfected with the HBxlentivirus vector and NC mimic. The HBx+miR-122 mimic group was transfected with the HBxlentivirus vector and miR-122 mimic. The NC mimic group was transfected with the NC mimic.The miR-122 mimic group was transfected with the miR-122 mimic. Then cell proliferation was detected by MTS,cell cycle was detected by flow cytometry,expression of miR-122 was detected by PCR,cyclin G1,Xlinked inhibitor of apoptosis protein(XIAP)and beta-catenin were detected by Western Blot. Results showed that the optical density(OD),ratio of the G2/M phase of the cell cycle and expression of cyclinG1,XIAP,β-catenin in cells of the HBx group were significantly higher than those of the NC group. The ratio of the G0/G1 phase to S phase and expression of miR-122 in cells of the HBx group were significantly lower than those of the NC group. The OD,ratio of G2/M phase of the cell cycle and expression of cyclinG1,XIAP and β-catenin in cells of the HBx+mR-122 mimic group were significantly lower than those of the HBx+NC mimic group. The ratio of the G0/G1 phase to S phase and expression of miR-122 in cells of the HBx+mR-122 mimic group were significantly higher than those of the HBx+NC mimic group. The fluorescence activity of cyclin G1,XIAP andβ-catenin luciferase reporter genes in the miR-122 mimic group were significantly lower than those in the NC mimic group. The results showed clearly that HBx could enhance the proliferation and cell cycle of HCCs,and this effect was mediated(at least in part)by down-regulation of miR-122 expression. The present study firstly elucidated the molecular mechanism of HBx regulating the growth of hepatocellular carcinoma cells,and also identified the target genes of anti-cancer activity of miR-122 in hepatocellular carcinoma cells are CyclinG1,XIAP and β-catenin.