HBsAg对肝细胞癌中DNMT3B表达及增殖的影响

Effects of HBsAg on Proliferation and DNMT3B Expression in Hepatocellular Carcinoma

目的探讨HBsAg对肝细胞癌中DNMT3B表达及增殖的影响。方法Western Blot检测LO2细胞与SMMC7721细胞中DNMT3B蛋白的表达水平。构建PIRES2-EGFP-HBsAg,与空载体质粒分别转染SMMC7721细胞,亚硫酸氢盐测序法检测转染细胞中抑癌基因MTSS1、RASSF1A、P16启动子甲基化水平。采用Western Blot检测转染细胞中抑癌基因MTSS1、RASSF1A、P16的表达差异。划痕、侵袭、迁移实验检测转染细胞的运动能力。CCK8、EDU实验检测转染细胞的增殖能力。结果LO2细胞中DNMT3B蛋白表达水平低于SMMC7721细胞(P<0.05);与空载体对照组比较,转染p-HBsAg后SMMC7721细胞中DNMT3B水平明显降低(P<0.05),MTSS1、RASSF1A、APC启动子甲基化下降而表达水平上升,细胞的增殖及运动能力均减弱(P<0.05)。结论HBsAg可能通过下调DNMT3B的表达抑制肝癌细胞增殖。

Objective To investigate the effects of HBsAg on proliferation and DNMT3B expression in hepatocellular carcinoma.Methods The expression of DNMT3B was detected by Western blot in LO2 and SMMC7721 cells.PIRES2-EGFP-HBsAg plasmid was constructed and transfected into SMMC7721 cells.The promoter methylation levels of tumor suppressor genes MTSS1,RASSF1A and APC were detected by bisulfite sequencing PCR in the transfected cells.The expression of MTSS1,RASSF1A and APC was measured by Western blot.The motor capacity of cells was determined by scratch,invasion and migration assays.The proliferation of cells was detected by CCK8 and EDU assays.Results The expression of DNMT3B in LO2 cells was lower than that in SMMC7721 cells(P<0.05).Compared with the empty plasmid group,p-HBsAg transfection decreased DNMT3B expression,inhibited MTSS1,RASSF1A and APC promoter methylation,increased MTSS1,RASSF1A and APC expression,and reduced proliferation and motor ability in SMMC7721 cells(P<0.05).Conclusion HBsAg may inhibit the proliferation of hepatocellular carcinoma by down-regulating the expression of DNMT3B.