乙型肝炎病毒X蛋白抑制p16蛋白表达及其促进HepG2肝癌细胞生长

Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulalion expression of p16protein

目的探讨乙型肝炎病毒x蛋白（HBx）对肝癌细胞生长的影响及其机制。方法以HepG2细胞和稳定表达GFP／HBx融合蛋白的HepG2／GFP—HBx为实验细胞，四甲基偶氮唑盐（MTT）法检测细胞吸光度值爿伽，分析细胞生长增殖；流式细胞术检测细胞周期；甲基化PCR检测细胞p16INK4A基因的甲基化；Westernblot检测p16蛋白表达水平。结果72h时HeDG2／GFP-HBx细胞组的A490为3．225±0．038，分别与HepG2和HepG2-GFP细胞组的2．012±0．022、2．038±0．029比较，增殖能力明显增强，t值分别为46．86和42．51，P值均〈0．001，差异均有统计学意义。流式细胞术检测示HepG2／GFP—HBx细胞组的G0／G1期细胞占16．45％±0．45％，明显低于HepG2和HepG2／GFP细胞组的44．81％±1．36％、42．76％±1．58％，f值分别为-34．22和-28．88，尸值均〈0．001，差异均有统计学意义。而5-氮-2’-脱氧胞苷（5-Aza-CdR）处理的HepG2／GFP—HBx细胞Go／G，期细胞比例为33．25％±0．79％，较未处理HepG2／GFP-HBx细胞组的16．45％±0．45％显著增加，两组比较，t=31．85，JD〈0．001，差异有统计学意义。甲基化PCR检测显示HepG2／GFP—HBx细胞组发生了p16INK4A基因甲基化，而HepG2和HepG2-GFP细胞组及5-Aza—CdR处理的HepG2／GFP—HBx细胞组未检测到p16INK4A基因启动子甲基化。Westernblot检测示HepG2／GFP—HBx细胞组p16蛋白水平低于HepG2和HepG2／GFP细胞组，而5-Aza—CdR处理HepG2／GFP—HBx细胞后，p16蛋白水平高于未处理的HepG2／GFP-HBx细胞。结论HBx蛋白可通过缩短HepG2细胞生长的G0／G1期而加快细胞周期进程，从而促进肝癌细胞生长增殖，其机制可能与HBx蛋白诱导p161INK4A基因启动子甲基化及抑制p16蛋白表达有关。

Objective To investigate the effects and related mechanisms of hepatitis B virus X （HBx） protein on cell cycle and growth in hepatocellular carcinoma. Methods A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein （GFP）-tagged HBx （HepG2/GFP-HBx cells） was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2＇-deoxycytidine （5-Aza-CdR; 5 pmol/L）. Effect of HBx expression on promoter methylation status of the p 16lNK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting. Results The HeoG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs ofculture （3.225 ± 0.038 A490） than either control （HepG2：2.012± 0.022 A490, t = -46.86, P 〈 0.001; HepG2/ GFP： 2.038 ± 0.029 A490, t = 42.5 l, P 〈 0.001）. The HepG2/GFP-HBx cells also showed significantly lower proportion of ceils in the G0/G1 phase （16.45% ± 0.45%） than either control （HepG2： 44.81%± 1.36%, t = -34.202, P 〈 0.001; HepG2/GFP： 42.76% ± 1.58%, t = -28.88, P 〈 0.001）. However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/GI phase （33.25% ± 0.79%, t = 31.85, P 〈 0.001）. The p16rNK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demithylation after treatment with 5-Aza-CdR. However, no methylation of p 16INKaA promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx （vs. HepG2 and HepG2/GFP cells） and this level increased after treatment with 5-Aza-CdR. Conclusion HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16mK4A promoter methylation and downregulating p 16 protein expression.