丙型肝炎病毒非结构蛋白5A影响p53抑制肝癌细胞甲胎蛋白表达的分子机制

Molecular mechanism of HCV NS5A on p53's inhibition of AFP expression in hepatocellular carcinoma cells

目的了解丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)对p53抑制甲胎蛋白(AFP)基因表达的影响及分子机制。方法采用质粒转染技术及微粒体酶免疫法观察p53蛋白对Huh7肝癌细胞AFP表达的抑制作用及HCV NS5A对其抑制作用的影响;蛋白印迹实验观察HCV NS5A对p53蛋白表达的影响;谷胱甘肽转移酶沉淀实验鉴定HCV NS5A与p53蛋白能否相互作用形成复合物。结果转染pRc/CMV空质粒的Huh7细胞上清液AFP浓度为(14 322±2412)ng/ml,转染pCNS5A质粒的Huh7细胞上清液的AFP 浓度为(13 843±3218)ng/ml,两组问t=1.42,P>0.05;转染pC53-NS3质粒的Huh7细胞上清液的AFP 浓度为(10 241±1326)ng/ml,与上述两组比较,t值分别为2.41及2.38,P值均<0.05;pCNS5A和pC53- NS3共转染者AFP浓度为(14 582±1238)ng/ml,与pC53-NS3单独转染组比较,t=3.12,P<0.01; pCNS5A和pC53-NS3共转染者和pC53-NS3单独转染者Huh7细胞p53蛋白表达无变化。在谷胱甘肽转移酶沉淀实验中,加入谷胱甘肽-p53融合蛋白后出现HCV NS5A蛋白条带,而仅加入谷光甘肽者则未出现此条带。结论p53蛋白能抑制Huh7细胞AFP的表达,HCV NS5A能减轻p53蛋白对AFP表达的抑制作用。HCV NS5A不影响p53蛋白的表达但能与p53蛋白结合形成复合物是使p53功能失活的分子机制。

Objectives To explore hepatitis C virus (HCV) non-structural protein 5A (NS5A)'s influence on inhibition of AFP expression executed by p53 protein and its possible molecular mechanism. Methods Plasmid transfection and MEIA were employed to observe p53's inhibitive effect on AFP expression of Huh7 cells and the HCV NS5A's influence on p53 function. Western blot was employed to find out if HCV NS5A affects p53 protein expression and GST pull down assay was applied to examine the interaction between HCV NS5A and p53. Results The AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14 322±2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13 843±3218) ng/ml; no significant difference existed between these two groups (t = 1.42, P > 0.05). After transfection with pC53-NS3, the AFP level was decreased to (10 241±1326) ng/ml, and in comparison to the above two groups it had a statistically significant difference (t values were 2.41 and 2.38, P < 0.05). When co-transfected with pCNS5A and pC53-NS3, the AFP expression (14 582±1238) ng/ml returned to the level of pRc/CMV transfected, and there was a remarkably significant difference between this and that of the pC53-NS3 transfected cells (t = 3.12, P < 0.01). HCV NS5A had no function on the p53 protein expression with Western blot experiment. In the GST pull down assay, an HCV NS5A protein band was found after GST-p53 was added, but not detected with GST only. Conclusion We found that p53 has an inhibitive function on the AFP expression in Huh7 cells and HCV NS5A minimized this p53 function. HCV NS5A did not affect p53 protein expression, but was able to form a complex with p53, by which HCV NS5A inactivated this p53 function.