2.2kb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 proteins encoded by the 2.2 kb spliced variants of hepatitis B virus genome on gene expression profile in Huh7 hepatocytes

目的研究单剪接及双剪接型2.2 kb乙型肝炎病毒基因编码剪接特异性蛋白(分别为TPss及TPds)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TPss及TPds编码序列并克隆入pcDNA3.1/HisC。采用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Westernblot检测目的蛋白的表达,通过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-TPss及pcDNA3.1/HisC-TPds,在Huh7细胞中能分别表达TPss(单剪接)及TPds(双剪接)蛋白。TPss蛋白导致Huh7细胞DGKβ(diacyl-glycerol kinase beta)等4个基因转录上调,ZNF652(zinc finger protein 652)等5个基因转录下降;TPds蛋白导致细胞MTSS1(Metastasis suppressor 1)等3个基因转录上调,WARS2基因(Tryptophanyl tRNA synthetase 2)转录下降。结论2.2 kb乙型肝炎病毒基因组剪接体编码剪接特异性蛋白可能影响肝细胞骨架的重塑、细胞内物质代谢等方面,与肝细胞的增生、迁移及代谢异常密切相关。

Objective To investigate the effects of the splicing-specific novel proteins encoded by the 2. 2 kb singly （termed as TPss） and doubly（termed as TPds） spliced variants of hepatitis B virus genome on gene expression profile in Huh7 hepatocytes. Methods Coding region of TPss or TPds were amplified by PCR and cloned into vector pcDNA3.1/ HisC. The recombinant vectors were separately transfected into Huh7 hepatocytes by FuGENE6 reagent. Total proteins and RNAs of transfected Huh7 cells were extracted, the expressed target proteins were detected by Western blot, and the gene expression alteration of the transfected Huh7 cells was evaluated by gene expression profile array assay followed by semi-quantitative RT-PCR. Results Recombinant vectors of pcDNA3. 1/HisC-TPss and pcDNA3. 1/HisC-TPds, which express well the TPss and TPds proteins respectively in Huh7 hepatocytes, were successfully constructed. Expression of TPss could up-regulate 4 genes including DGKβ, and down-regulate 5 genes including ZNF652, while the expression of TPds up-regulate 3 genes including MTSS1, and down-regulate WARS2 gene. Conclusion The splicing-specific novel proteins encoded by the 2.2 kb spliced variants of HBV may disturb cell function including the re-organization of the cystoskeleton and metabolism, which attribute to the abnormal cell proliferation, mobility and aberrant cellular metabolism.