野生型和自然缺失突变型HBx蛋白对肝癌细胞生物学行为的影响

Effects of wild-type and natural deletion mutant HBx protein on biological behavior of hepatoma cells

目的探讨野生型和自然缺失突变型乙型肝炎病毒X(HBx)蛋白对肝癌细胞生物学行为的影响。方法取人肝癌细胞系SMMC-7721,常规培养、传代。取对数生长期、生长状态良好的SMMC-7721细胞,随机分为空载体组、HBx-FJ组、HBx-128w组,分别转染pcDNA3.1、pcDNA3.1-HBx-FJ和pcDNA3.1-HBx-128w。Western blotting法检测HBx蛋白表达。采用MTT法检测细胞增殖活性,平板克隆实验检测细胞增殖能力,流式细胞术检测细胞周期变化,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,裸鼠成瘤实验检测体外成瘤能力。结果HBx-FJ组和HBx-128w组细胞增殖活性、增殖能力、侵袭能力及体外成瘤能力均高于空载体组(P均<0.05);HBx-128w组细胞增殖活性、增殖能力、迁移能力、侵袭能力及体外成瘤能力均高于HBx-FJ组(P均<0.05);与空载体组相比,HBx-FJ组和HBx-128w组G1期细胞减少,S期细胞增多(P均<0.05)。结论野生型和自然缺失突变型HBx蛋白均具有促进肝癌细胞恶性进程的作用,但自然缺失突变型HBx-128w较野生型HBx具有更强的致癌作用。HBx可通过缺失突变影响其生物学功能,促进肝癌的进展。

Objective To investigate the effects of wild-type and natural deletion mutant hepatitis B virus X(HBx)protein on the biological behavior of hepatocellular carcinoma(HCC)cells SMMC-7721.Methods HCC cell line SMMC-7721 was taken for routine culture and passage.SMMC-7721 cells in the logarithmic phase with good condition were randomly divided into the empty vector group,HBX-FJ group and HBX-128w group,which were transfected with pcDNA3.1,pcDNA3.1-HBX-FJ,and pcDNA3.1-HBX-128w,respectively.Western blotting was used to detect the expression of HBx protein in each group.The cell proliferation activity was detected by MTT assay.The cell proliferation ability was detected by Flat-plate colony assay.The cell cycle changes were detected by flow cytometry.The cell migration ability was detected by Scratch assay.The cell invasion ability was detected by Transwell assay.The tumorigenic ability in vitro was detected by tumorigenesis assay in nude mice.Results The proliferation activity,proliferation ability,invasion ability,and tumorigenic ability of cells in the HBX-FJ group and HBX-128w group were significantly higher than those in the empty vector group(all P<0.05).The proliferation activity,proliferation ability,migration ability,invasion ability and tumorigenic ability in vitro of cells in the HBx-128w group were significantly higher than those in the HBX-FJ group(all P<0.05).Compared with the empty vector group,the cells in the G1 phase decreased significantly while the cells in the S phase increased significantly in the HBX-FJ group and HBX-128w group(all P<0.05).Conclusion Both wild-type and natural deletion mutant HBx protein can promote the process of cell malignancy,but natural deletion mutant HBx-128w has a stronger carcinogenic effect than wild-type HBx;HBx can affect its biological function by deletion mutation and thus promote the progression of HCC.