458nt～1308nt乙型肝炎病毒剪接特异性蛋白对Huh7细胞基因表达的影响

Effects of the splicing-specific novel protein encoded by 458 nt-1308 nt spliced variants of the hepatitis B virus genome on the gene expression profile in Huh7 hepatocytes

目的研究458nt^1 308nt乙型肝炎病毒剪接特异性蛋白TSR′r′对Huh7细胞基因表达的影响,探讨其可能的致病机制。方法PCR扩增TSR′r′编码序列并克隆入pcDNA3.1/HisC。以FuGENE6将重组表达载体及相应空载体分别瞬时转染Huh7细胞,Trizol抽提细胞总蛋白与总RNA;以融合表达多肽表位抗体为一抗,Western blot检测目的蛋白的表达;用Affymetrix Genechip U133 plus 2.0人类基因表达谱芯片检测Huh7肝细胞基因转录的变化,并以半定量RT-PCR进一步验证。结果成功构建重组真核表达载体pcDNA3.1/HisC-TSR′r′,转染48 h后在Huh7细胞中表达TSR′r′蛋白。TSR′r′导致Huh7细胞载脂蛋白H等27个基因转录上调,其中包括5种代谢相关基因,7种免疫相关基因,7种胶原及胞外基质基因,5种干扰素诱导蛋白基因;TSR′r′还导致Huh7细胞核受体共激活物1基因在内的7种基因转录下降。结论458nt^1 308nt乙型肝炎病毒剪接特异性蛋白可能多方面影响肝细胞功能,具有重要的致病意义。

Objective To investigate the effects of the splicing-specific novel protein （denoted here as TSR＇r＇ ） encoded by the 458 nt-1 308 nt spliced variants of the hepatitis B virus on the gene expression profile in Huh7 hepatocytes. Methods The coding region for TSR＇r＇ was amplified by means of PCR and cloned to vector peDNA3.1/HisC. The recombinant construct or empty vector was separately transfeeted into Huh7 hepatocytes by FuGENE6 reagent. Total protein and RNA of transfeeted Huh7 cells were extracted, the expressed target protein was detected by Western blotting using the first antibody against the tag epitope, and the altered gene expression of the transfeeted Huh7 cells was evaluated by gene expression array assay followed by semi-quantitative RT-PCR. Results Recombinant vectors of pcDNA3. 1- TSR＇r＇ expressing the TSR＇r＇ protein in Huh7 hepatocytes at high levels were successfully constructed. Microarray assay revealed that TSR＇r＇ up-regulates 27 genes including 5 metabolism-related genes, 7 immunity-related genes, 7 collagen and extracellular matrix genes, and 5 interferon-induced genes; TSR＇r＇ also down-regulates 7 genes including nuclear receptor coactivator 1. Conclusion The splicing-specific novel protein encoded by the 458 nt-1 308 nt spliced variant of HBV may effect cellular function and may be closely correlated with the pathogenicity of hepatitis 13 virus.