Effects Of The Splicing-specific Novel Proteins Encoded By The 2.2Kb Spliced Variants Of Hepatitis B Virus Genome On Gene Expression Profile In Huh7 Hepatocytes

Spliced variants of hepatitis B virus genomes are a group of subgenomic length DNA generated from 3.5Kb pregenomic RNA by splicing and reverse transcription. More than 80% of these spliced variants are 2.2Kb doubly or singly spliced variants of hepatitis B virus genome, which could encode splicing-specific novel proteins and closely related with persistent infection and pathogenicity of HBV with unclear mechanism. Inorder to explore the potential pathogenicity of the novel proteins encoded by the 2.2Kb doubly or singly spliced variants of HBV genome, we investigated the effection of the novel proteins on Huh7 hepatocytes by gene expression profile array assay.The first part of this study was aimed to construct the splicing-specific proteins recombinant eukaryotic expression vectors and confirm their expression in Huh7 hepatocytes. To address this issue, the splicing-specific coding regions were cloned separately into the eukaryotic expression vector pcDNA3.1/HisC to construct the recombinant eukaryotic expression vectors pcDNA3.1/HisC-TPds(harboring the double spliced splicing-specific novel protein) and pcDNA3.1/HisC-TPss (harboring the signle spliced splicing-specific novel protein). The recombinant plasmids were transiently transfected into Hhu7 hepatocytes separately, and Western Blot using anti-Xpress antibody was used to confirm the expression of TPds and TPss.In the second part of this study , total RNA of cells transiently transfected with pcDNA3.1/HisC , pcDNA3.1/HisC-TPds and pcDNA3.1/HisC-TPss were extracted respectively and used for gene expression profile array assay. Semi-quantitative RT-PCR were introduced to validate the array assay. The result demonstrated that the splicing-specific novel proteins encoded by the 2.2Kb spliced variants of HBV genome may disturb the re-orgnazation of the cystoskeleton and the metabolism of Huh7 hepatocytes , which result in the abnormality of cell proliferation , mobility and metabolism, and eventually attributed to the carcinogenesis of Hepatocellular Carcinoma.