乙型肝炎病毒X蛋白对阿霉素诱导的肝癌细胞凋亡及p53、PTEN表达的影响

Hepatitis B virus X protein suppresses adriamycin-induced apoptosis of hepatocellular carcinoma cells and expression of p53 and PTEN

目的探讨乙型肝炎病毒(HBV)X蛋白对阿霉素诱导的肝癌细胞凋亡及p53、PTEN表达的影响。方法用阿霉素(2.5μg/ml)分别处理HepG2及稳定表达GFP、GFP-HBx融合蛋白的细胞系HepG2/GFP、HepG2/GFP-HBx,处理后不同时间在显微镜下观察细胞形态变化,流式细胞术检测细胞凋亡;RT-PCR检测p53、PTENmRNA水平;Westernblotting检测p53、PTEN蛋白水平。结果流式细胞术检测显示阿霉素处理后36h,HepG2/GFP-HBx细胞凋亡率为3.94%,明显低于HepG2(59.03%)、HepG2/GFP细胞(61.38%)(P<0.001),而与未处理对照组细胞(2.12%、2.78%、2.55%)无显著差别(P>0.05)。RT-PCR分析显示HepG2/GFP-HBx细胞PTENmRNA水平低于HepG2及HepG2/GFP细胞,而p53mRNA水平无明显差别。Westernblotting检测显示HepG2/GFP-HBx细胞PTEN蛋白明显低于HepG2及HepG2/GFP细胞,而p53蛋白无明显差别。结论 HBVX蛋白能够抑制阿霉素诱导的细胞凋亡及PTEN表达,HBVX蛋白对其细胞凋亡的抑制可能与其对p53-PTEN的抑制有关。

Objective To investigate the effect of hepatitis B virus X protein （HBx） on adriamycin-induced apoptosis of hepatocellular carcinoma cells and the expressions of p53 and PTEN. Methods HepG 2 , HepG 2 /GFP, and HepG 2 /GFP-HBx cells were treated with adriamycin （2.5 μg/ml）, and the apoptotic cell death was determined by observing the morphological changes and flow cytometry. The expressions of p53 and PTEN mRNA in the 3 cells were detected by RT-PCR, and the expressions of p53 and PTEN protein were analyzed by Western blotting. Results Adriamycin induced significant cell death in HepG 2 and HepG 2 /GFP cells, which became rounded, shrunk, and detached after the treatment; but no significant cell death occurred in HepG 2 /GFP-HBx cells. Flow cytometry analysis showed that the apoptotic rate was significantly lower in HepG 2 /GFP-HBx cells （3.94%） than in HepG 2 （59.03%） and HepG 2 /GFP cells （61.38%） at 36 h after the treatment （P〈0.001）, while no significant difference was observed between HepG 2 /GFP-HBx （3.94%） and the control cells （2.12%, 2.78%, and 2.55%） （P〉0.05）. RT-PCR showed lowered expression of PTEN mRNA in HepG 2 /GFP-HBx cells as compared to that in HepG 2 and HepG 2 /GFP cells, while no significant difference was noted in p53 mRNA. Western blot analysis showed that PTEN protein decreased while p53 protein remain unchanged in HepG 2 /GFP-HBx cells. Conclusion HBx suppresses adriamycin-induced apoptosis of HepG 2 cells and PTEN expression. The inhibitory effect of HBx on the cell apoptosis may be related to the inhibition of p53-PTEN pathway.