Effects of the splicing-specific novel protein encoded by 458 nt-1 308 nt spliced variants of the hepatitis B virus genome on the gene expression profile in Huh7 hepatocytes.

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 pcDNA3.1/HisC.The recombinant construct or empty vector was separately transfected into Huh7 hepatocytes by FuGENE6 reagent.Total protein and RNA of transfected 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 transfected 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 B virus.