野生型及羧基端缺失突变HBx对肝癌细胞增殖的影响

Effects of wild- type and mutant hepatitis B virus X protein with deletion at C-terminus on cell proliferation in hepatocellular carcinoma

目的明确氨基端或羧基端缺失突变50个氨基酸的HBx对肝癌细胞增殖的影响及可能机制。方法 PCR法分别扩增氨基端、羧基端缺失50个氨基酸的HBx基因片段(HBxn、HBxc),并定向插入绿色荧光蛋白(GFP)真核表达载体pEGFP-C1以构建重组体pGFP-HBxn及pGFP-HBxc。脂质体转染法将重组体转染Hep G2细胞,用G418筛选,荧光显微镜下观察。RT-PCR鉴定稳定表达GFP-HBxn、GFP-HBxc融合蛋白的HepG2细胞系。MTT法检测细胞增殖,流式细胞术检测细胞周期,Western blot法检测p16的表达。结果 RTPCR检测示HepG2/GFP-HBxn、Hep G2/GFP-HBxc细胞分别有HBxn、HBxc的转录和表达。MTT法检测示Hep G2/GFP-HBx、HepG2/GFP-HBxc细胞的增殖速度明显快于HepG2、HepG2/GFP、HepG2/GFP-HBxn细胞(P<0.05)。流式细胞术检测示HepG2/GFP-HBx、HepG2/GFP-HBxc细胞的G0/G1期百分比较HepG2、HepG2/GFP细胞明显减少(P<0.05),而HepG2/GFP-HBxn细胞与HepG2、HepG2/GFP细胞比较差异无统计学意义。Western blots显示HepG2/GFP-HBx、HepG2/GFP-HBxc细胞的P16表达量明显低于HepG2、HepG2/GFP细胞,HepG2/GFP-HBxn细胞P16的表达水平与HepG2、HepG2/GFP细胞无明显差异。结论 HBx及羧基端缺失突变50个氨基酸的HBx能促进肝癌细胞的增殖,其作用可能是通过抑制抑癌基因p16表达而调控细胞周期进而促进肝癌细胞的增殖所致;HBx氨基端可能是HBx调控细胞周期及促进肝癌增殖的重要功能区域。

Objective To investigate the biological function of HBV X deletion mutation in N- terminus or C-terminus and its roles in carcinogenesis of hepatocellular carcinoma. Methods HBV X gene fragment encoding mutant HBV X protein with deletion 50 amino acid residues at N- terminus or C- terminus（ HBxn or HBxc） was amplified from subtype amplification of HBV DNA by PCR. The purified HBxn and HBxc gene fragments were inserted into GFP expression vector,pEGFP- C1,to establish recombinant expression vector p GFP / HBxn and pGFP / HBxc. HepG2 cells were transfected with these recombinant constructs by lipofectamine reagent,and resistant clones expressing GFP- HBxn or GFP- HBxc fusion proteins were selected with G418 and observed under fluorescence microscope. The expression of deletion mutant HBV X gene was demonstrated by RT- PCR analysis. Growth rates and cell cycle were determined by MTT and flow cytometry analysis. The expression of p16 in these cells were analyzed by Western blot. Results RT- PCR analysis showed that HBxn and HBxc were expressed in GFP- HBxn and GFP- HBxc,respectively. MTT analysis showed that proliferation of HepG2 / GFP- HBx cells and HepG2 / GFP- HBxc cells were faster than that of HepG2,HepG2 / GFP and HepG2 / GFP- HBxn cells. Flow cytometry analysis showed that the percentage of G0/ G1 phase in HepG2 / GFP- HBx and HepG2 / GFP- HBxc cells were significantly decreased compared with HepG2 or HepG2 / GFP（ P 0. 05）. However,no significant differences were observed among Hep G2 /GFP- HBxn,HepG2 and HepG2 /GFP cells. Western blot analysis demonstrated that the levels of P16 in HepG2 / GFP- HBx and HepG2 / GFP- HBxc cells significantly decreased compared with HepG2 or HepG2 / GFP cells. However,level of p16 in HepG2 / GFP- HBxn cells were similar to that in HepG2 and HepG2 / GFP cells. Conclusion Wild- type and the C- terminus- deleted HBx promotes cell growth and cell cycle progression via down regulating p16 pathway. It is also indicated that the N- terminus of HBx is important in presenting biologic function of HBx in modulating cell cycle and carcinogenesis of hepatocellular carcinoma.