Gong Guo-zhong, Jiang Yong-fang, He Yan, Lai Li-ying, Xu Yun, Su Xian-shi
The Center for Liver Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhonghua Gan Zang Bing Za Zhi. 2005 Jul;13(7):505-8.
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.
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.
The AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14322+/-2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13843+/-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 (14582+/-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.
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.
探讨丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）对p53蛋白抑制甲胎蛋白（AFP）表达作用的影响及其可能的分子机制。
采用质粒转染和微粒子酶免疫分析法（MEIA）观察p53对Huh7细胞AFP表达的抑制作用以及HCV NS5A对p53功能的影响。采用蛋白质免疫印迹法（Western blot）检测HCV NS5A是否影响p53蛋白表达，采用谷胱甘肽巯基转移酶沉降试验（GST pull down assay）检测HCV NS5A与p53之间的相互作用。
用pRc/CMV转染的Huh7细胞培养上清中AFP浓度为（14322±2412）ng/ml，用pCNS5A转染的Huh7细胞培养上清中AFP浓度为（13843±3218）ng/ml，两组比较差异无统计学意义（t = 1.42，P > 0.05）。转染pC53-NS3后，AFP水平降至（10241±1326）ng/ml，与上述两组比较差异有统计学意义（t值分别为2.41和2.38，P < 0.05）。当pCNS5A与pC53-NS3共转染时，AFP表达（14582±1238）ng/ml恢复到pRc/CMV转染时的水平，与pC53-NS3转染细胞比较差异有极显著统计学意义（t = 3.12，P < 0.01）。蛋白质免疫印迹实验显示HCV NS5A对p53蛋白表达无作用。在谷胱甘肽巯基转移酶沉降试验中，加入GST-p53后出现一条HCV NS5A蛋白带，而仅加入GST时未检测到。
发现p53对Huh7细胞AFP表达有抑制作用，HCV NS5A可使p53的这一功能减弱。HCV NS5A不影响p53蛋白表达，但能与p53形成复合物，从而使p53功能失活。
