乙型肝炎病毒X蛋白羧基端30个氨基酸缺失突变体转染Huh7细胞的基因表达谱和蛋白质组差异分析

Gene expression profiles and proteomics analysis of Huh7 cells after transfection with mutant hepatitis B virus X with C-terminal 30 amino acid deletion

目的:本课题组既往研究显示乙型肝炎病毒X蛋白(HBx)羧基端30个氨基酸的缺失突变雄(△HBx)具有明显不同于野生型HBx(wtHBx)的细胞生物学效应,因此,本研究进一步探讨△HBx和wtHBx导致Huh7细胞差异生物学效应的分子基础。方法:采用cDNA芯片和双向凝胶电泳方法,从基因和蛋白质组水平,对分别转染了△HBx和wtHBx的Huh7细胞进行检测。结果:cDNA芯片检测结果显示,对比wtHBx组,△HBx组存在193个基因(0．95％)的显著性改变,154和39个基因分别显示表达上调和下调。双向凝胶电泳结果显示,对比wtHBx组,△HBx组存在147个蛋白差异点。基因表达谱和质谱分析结果显示,△HBx组差异表达的基因和蛋白主要涉及宿主细胞的基因转录、细胞连接、信号转导和代谢、免疫应答等过程及癌基因和抑癌基因。结论:HBx缺失突变体对细胞基因和蛋白质的影响是广泛的,涉及肝组织特异性的代谢基因的变化。这些差异表达的基因和蛋白质在HBx突变致癌过程中是否发挥了重要的作用,需要进一步的研究证实。

Objective：The hepatitis B virus X gene, which encodes the HBx protein, has multiple functions and is involved in hepatocarcinogenesis. It has been described that deletion in the distal COOH-terminal region of HBx and the double substitution at K130M/V131I may be characterized as a common feature of HBx in tumor tissue. Our previous studies have shown that △HBx（an engineered deletion mutant lacking the last 30 C-terminal amino acids） had different biological impacts on Huh7 cells compared with wild type HBx （wtHBx）. So the aim is to investigate the molecular mechanism for the different biological effects of △HBx and wtHBx. Methods： Huh7 cells were transfected with wtHBx or △HBx, respectively. A cDNA microarray containing more than 21 000 human genes and expressed sequence tags （ESTs） and two-dimensional gel electrophoresis （2-DE） were used to examine the different gene and protein expression profiles of Huh7 cells. Results： We identified 193 （0.95%） candidate genes and ESTs which had aberrant expression under △HBx induction. Among them 154 genes were up-regulated and 39 genes were down-regulated. 2-DE analysis demonstrated that there were 147 different protein spots in △HBx group compared with wtHBx group. Of them, 5 significantly different protein spots were identified using mass spectrometry and matched by protein database. Most different expression genes and proteins were related with gene transcription, cell adhesion, signal transduction and metabolism, immune response, and carcinogenesis, etc. Conclusion： This study shows that the mutant HBx affected cell genes and proteins expression in various processes, especially in the regulation of liver metabolism. However, whether these genes and proteins play a key role for the mutant HBx gene in carcinogenesis needs to be further elucidated.