HCV核心基因插入突变体编码蛋白对人肝癌细胞系(Huh-7)细胞基因表达的影响

Effect of hepatitis C virus core gene insertional mutein expressed in human hepatoma cell line on human gene expression

目的用基因表达分析法鉴定丙型肝炎病毒(HCV)核心(Core,C)区基因插入突变体编码蛋白在人肝癌细胞系(Huh7)表达,探讨该蛋白生物学功能及其基因表达改变与致病的关系。方法构建HCV1bC基因插入突变体编码蛋白重组表达质粒,建立表达C基因插入突变体编码蛋白Huh7细胞系,按Affymetrix公司实验程序制备探针、再与该公司HGU133A和HGU133B芯片杂交。对基因表达上调或下调≥3倍的基因,用NetAffx作进一步分析。并用半定量RTPCR对其中3个上调基因进行鉴定。结果Microarray分析显示,HCV1bC基因插入突变体编码蛋白比C蛋白引起更多的基因表达改变,主要集中在信号传导、蛋白酶活性、分子转运、免疫反应等,特别是免疫反应基因表达更加显著。C基因插入突变体编码蛋白表达可同时导致凋亡基因/抗凋亡基因表达上调或下调及致癌基因上调。半定量RTPCR对有趣的致癌基因FHL2、抗凋亡基因PRKCZ和凋亡基因LGALS1的鉴定结果表明,FHL2、PRKCZ和LGALS1基因的表达比空载体转染对照组相同基因明显上调。结论HCVC基因插入突变体编码蛋白在Huh7细胞表达对其基因表达有很大影响,其中对免疫反应基因的影响更明显,这一结果对理解HCVC基因插入突变体编码蛋白在HCV致病过程中的作用及其研制抗HCV药物均有重大的参考价值。

Objective To identify the effect of hepatitis C virus(HCV) core gene insertional mutein expressed in human hepatoma(Huh-7) cell line on human gene expression and to find its biological function, the relation between gene expression and pathogenesis. Methods The recombinants of expression of HCV genotype 1b core gene insertional mutein was constructed and Huh-7 cell line expressing core gene insertional mutein was established. Affymetrix human gene chip, the HG-U133 A and B were hold for identification gene expression in Huh-7 cell line. All of these genes were annotated by using NetAffx analysis and categorized based on their biological process. Three up-regulated genes expression were confirmed by semi-quantitative RT-PCR. Results The microarray analysis results suggested that core gene insertional mutein can improve expression up/down-regulated than that of core protein, such as signal transduction, protease activity, molecular transport, immune responses, especially in immune responses and that apoptosis or anti-apoptosis gene expression were up/down-regulated simultaneously and oncogenesis up-regulated in Huh-7 cell line expressed core shadow protein. It was also suggested that microarray hybridization results were similar to RT-PCR results. Conclusion There is a very significant effect on gene expression in Huh-7 cell line expressing core gene insertional mutein, especially in immune response gene. The result improved our understanding of novel function of HCV core gene insertional mutein in pathogenesis process and suggested a potential pathway for development anti-HCV drug.