Study On Transregulatory Effects Of HBV X Protein

Hepatitis B virus (HBV) infection causes acute and chronic viral hepatitis, which is also considered to be a major etiological factor in the development of liver cirrhosis and hepatocellular carcinoma (HCC). Virus-encoded proteins can moderately stimulate transcription of many host cellular transcription elements and transregulate gene expression of host hepatocellular proteins, which may be one of the molecular mechanisms of HBV-associated diseases. HBV genome is characterized by four overlapping ORFs(S, C, P, X). The HBx protein encoded by X gene comprises of 154 amino acids, with a molecular mass of approximately 17.5 kDa. HBx acts as a transregulator and its transregulatory effects linked to the progression of HCC from different groups remain controversial. The exact role of HBx in viral replication and pathogenesis has yet to be elucidated. Comprehensive understanding the transregulatory roles of HBx using oligonucleotide DNA microarray and detecting the changes of gene expression simultanously may help partially clarify the molecular mechanism of HBV infection.First, to study the transregulatory effects of HBx protein, the recombinant expression plasmid pcDNA3.1(-)-X was constructed and transiently transfected into the human hepatoblastoma cell lines HepG2 cells, and pcDNA3.1(-) empty vector was used as control. Oligonucleotide DNA microarray and bioinformatics techniques were employed to screen and analyze the differentially expressed mRNAs between the two groups. Among the 21,329 genes, it was found that 426 genes were differentially expressed, 168 genes were up-regulated and 258 genes were down-regulated. These might be target genes transregulated by HBx protein, among which some genes encoding proteins were involved in cell cycle regulation, cell apoptosis, cell signal transduction, immune response, energy metabolism, tumor development and metabasis. These might explain the possible mechanism of HBx protein in vivo. In addition, we discovered some novel genes with unknown function transregulated by HBx and one of these novel genes was named as HBx transactivated protein 11 (XTP11).Second, to investigate the transcriptional regulatory role of HBx protein on the expression of p53 tumor suppression gene, the promoter sequence of the p53tumor suppression gene (p53p) was identified and the recombinant reporter gene expression plasmid pCAT3-p53p was constructed and transiently transfected and cotransfected with pcDNA3.1(-)-X into the HepG2 cells. The choloraphenical acetyltransferase (CAT) activity was detected by enzyme-linked immunosorbent assay (ELISA). Results showed that the p53 promoter could cis-activate the transcription of the CAT gene. The relative expression level of CAT gene in HepG2 cells cotransfected with pCAT3-p53p and pcDNA3.1(-)-X was lower than the control. The expression of p53 mRNA was further detected by RT-PCR with or without HBx protein expression. The findings indicated that HBx could transcriptionally inhibit the expression of p53 gene, which might be a possible molecular mechanism responsible for the development of HCC.Third, we investigated the biological functions of XTPll. ?The full length encoding sequence and promoter region were confirmed by bioinformatics. The amplified product of XTPll promoter was subcloned into pCAT3-basic reporter vector, named as pCAT3-XTPllp. After transfection, the CAT activity was detected. The results indicated that the XTPll promoter identified in this study had transcriptional activity and HBx had transactivating effect on XTPll promoter. ?The XTPll-encoding gene was directionally cloned into the reporter plasmid pEGFP-Cl. After transfection, fluorescent fusion protein could be observed steadily in cell plasma by fluorescent microscope. (3)We expressed successfully XTPll fusion proteins with His tag employing procaryote expressing vector pET32a(+) and the corresponding BL21 (DE3) strain. ?We expressed successfully XTPll fusion proteins with c-myc tag in yeast cells. The yeast cells transformed with pGBKT7-XTPll plasmid could grow well on both the synthetic dropout nutrient medium and turned blue on medium containing x-cc-gal, which suggested that the XTPll protein fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast. The detailed biological functions of XTPl 1 in pathogenesis of HBV need to be further elucidated.Taken together, the findings provide theoretical basis and research methods for the molecular biological mechanism on the transregulatory effects of HBx.