乙型肝炎病毒X蛋白和p14^ARF依赖性、非依赖性途径对肝癌细胞生长的影响  

Hepatitis B virus X protein inhibits hepatoma cell growth in vitro through p14^(ARF)-dependent and p14^(ARF)-independent pathways

作　　者：余党会[1] 林静[1,2] 曲建慧[3] 祝峙[1] 李芳梅[1] 倪灿荣[1] 朱明华[1] 

机构地区：[1]第二军医大学长海医院病理科,上海200433 [2]福建医科大学基础医学院组胚教研室,福建福州350004 [3]济南军区总医院传染科,山东济南250031

出　　处：《南方医科大学学报》2009年第6期1089-1093,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30070344,30070839)

摘　　要：目的探讨乙型肝炎病毒X蛋白(HBx)是否通过p14ARF途径影响肝癌细胞生长及其作用机制。方法将HBx及p14ARF单转染及共转染含野生型p53但不表达p14ARF的肝癌细胞株HepG2。实验分为pcDNA3、pcDNA3HBx、pcDNA3p14ARF、pcDNA3HBx+pcDNA3p14ARF4组。通过流式细胞仪比较各转染组HepG2细胞凋亡、细胞周期的变化情况。用含p53结合位点p21WAF1启动子荧光素酶活性检测和Western blotting观察各转染组细胞p14ARF、MDM2、p53、p21WAF1蛋白表达水平的变化。结果HBx及p14ARF单转染及共转染含野生型p53但无p14ARF表达的HepG2细胞,单转染HBx及p14ARF组其细胞凋亡率(14.11%、13.72%)、G0/G1期阻滞细胞数(63.62%、61.75%)、p21WAF1启动子荧光素酶活性(1.25±0.05、1.09±0.06)及p53、p21WAF1蛋白的表达较对照组(10.66%、57.42%、0.77±0.03)明显升高,而共转染组与单转染p14ARF组相比其p14ARF蛋白表达及上述各项指标(18.61%、66.74%、3.53±0.43)又进一步升高。结论HBx通过依赖及非依赖p14ARF途径诱导p53表达从而导致激活p21WAF1,进而引起细胞周期G0/G1期的阻滞及细胞凋亡的增加。Objective To explore the effects of hepatitis B virus X protein （HBx） on hepatoma cell growth through p14^ARF-dependent and p14^ARF-indcpendent pathways. Methods HBx and p14^ARF were transfected either separately or in combination into HepG2 cells containing wt-p53 but not expressing p14^ARF. The cells were divided into 4 groups, namely pcDNA3 （control）, pcDNA3HBx, pcDNA3p14^ARF, and pcDNA3HBx ＋ pcDNA3p14^ARF groups. Flow cyt0metry was used to examine the apoptosis rates and cell cycle progression of HepG2 cells in different groups. The expression ofp14^ARF, MDM2, p53, and p2 ^WAF1 proteins were investigated by detecting the activity of p21 ^WAF1 promoter-luciferase and using Western blotting. Results The apoptosis rates of HepG2 cells in pcDNA3HBx and pcDNA3pl4^ARF groups were significantly higher than that in the control group （14.11%, 13.72 % vs 10.66%）. Compared with the control group, pcDNA3 HBx an d pcDNA3p 14^ARF groups also showed significantly higher cell percentages arrested at G0/G1 phase （63.62%, 61.75% vs 57.42%）, luciferase activity of p21 promoter （1.25±0.05, 1.09±.06 vs 0.77±0.03） and expressions of p53 and p21^WAF1. The cell apoptosis rate, percentage of cells in G0/GI phase and expression level of p 14ARF were even higher in pcDNA3HBx＋pcDNA3p 14^ARF group （18.61%, 66.74%, and 3.53 _＋0.43, respectively） than in either p14^APF or HBx group. Conclusion HBx induces p53 expression through p14^ARF-dependent and independent pathways to activate p21^WAF1 promoter, leading to G0/G1 arrest and apoptosis of HepG2 cells.

关 键 词：肝肿瘤 p53 P14^ARF P21^WAF1 乙型肝炎病毒X蛋自 细胞周期 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]