Regulation Of Hepatitis B Virus X Protein (HBx) On Human FABP1and NQO1Gene

Infection of human hepatitis B virus (HBV) resulted in acute and chronichepatitis, and closely related to cirrhosis and hepatocellular carcinoma (HCC). HBVX protein (HBx), a17kilodalton (KDa) protein encoded by the smallest open readingframe (ORF) of the HBV genome, is a pivotal protein in the progression ofHBV-associated pathogenesis. HBx binds dsDNA directly remains elusive but directlyinteracts with various nuclear transcription factors. Except for the stated above geneticmodels, mechanism of HBx regulating the transcriptional activity of cellular genes viaepigenetic modification have been identified by recent investigations. Previous studiesfrom our laboratory have shown that the protein level of FABP1is marked higher inHepG2cell line harboring1.2×unit-length of the HBV genome (HepG2-HBV3),while NQO1is significantly lower, compared with that in control cell line(HepG2-RepSal1) by using fluorescence two-dimensional difference gelelectrophoresis (2D-DIGE) and MALDI-TOF/TOF MS analysis. And on that basis,we investigated the effect of HBx protein on FABP1and NQO1expression, andsuggested a mechanism for HBV-associated pathogenesis.The first part of this study is to investigate whether HBV, expecially HBxaffected transciption and expression of FABP1and NQO1genes. We used realtimeRT-PCR and western blot to examine FABP1and NQO1expression at either mRNAlevel or protein level in human HBV-producing hepatoma cell lines, and the resultsindicated that FABP1expression is increased, while NQO1expression is decreased inHBV-producing cell lines as compared to control cells. Furthermore, it was alsodemonstrated that HBx enhanced FABP1expression and repressed NQO1expression.The second part of this study is to elucidate the mechanism of HBx-midiatedup-regulation of FABP1gene expression. Therefore, in the first part of this section, the full length FABP1promoter (nucleotides–2125to+50) and a series of truncatedpromoter regions were cloned. A luciferase reporter assay revealed that nucleotides–255to+50in the promoter region contained full of maximum FABP1promoteractivity compared with the full length promoter. Furthermore,high activity wasshown when the plasmid was transfected into liver-derived cells such as the humanhepatoblastoma cell line HepG2and the hepatoma cell line Huh7. TFSEARCH andTESS programs were used to predict potential transcription factor binding sites. Twoputative binding sites for the liver-enriched transcription factors hepatocyte nuclearfactor3β (HNF3β) and CCAAT/enhancer binding protein α (C/EBPα) were identifiedin the nucleotides–255to–155FABP1promoter region. Site-directed mutagenesis ofthese two sites reduced dramatically FABP1promoter activity. In addition, theelectrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitationassay (ChIP) revealed that these binding sites were recognized by HNF3β andC/EBPα respectively. Overexpression of HNF3β and C/EBPα enhanced thetranscription of FABP1and consequently improved the protein level of FABP1inHepG2cells, while knockdown of HNF3β and C/EBPα showed the inverse effects.In the second part of this section, we futher investigate the HBx-mediatedtransactivation of FABP1gene predominantly by liver-enriched transcription factorsHNF3β and C/EBPα. Site-directed mutagenesis of these two sites reduceddramatically HBx-induced FABP1promoter activity. Furthermore,high activity wasshown when the pGL3B-255was transfected into the human hepatoblastoma cell lineHepG2than that in colorectal cancer cell line HCT116which is found low-expressionof HNF3β and C/EBPα. Knockdown of HNF3β and C/EBPα repressed thetranscription of FABP1in HepG2cells. In addition, chromatin immunoprecipitationassay (ChIP) and co-immunoprecipitation revealed that HBx interacted with HNF3βand C/EBPα in different ways, HBx directly interacted with C/EBPα, while indirectlyinteracted with HNF3β in vivo to regulate FABP1gene transcription.The third part of this study is to elucidate the mechanism of HBx-midiateddown-regulation of NQO1gene expression. DNA methylation of NQO1genepromoter in hepatoma cells was detected by bisulfate sequencing, and the data revealed that CpG dinucleotide islands were significantly hypermethylated in Ad-HBxinfected cells. HBx-mediated protein binding to gene regulatory elements wasevaluated by chromatin immunoprecipitation (ChIP) and the result revealed that HBxchromatin immunoprecipitated the newly methylated region R1proximal totranscription start site of the NQO1gene and significantly enhanced DNMT3Abounding to R1region. HBx-mediated DNA methylation alterations was alsoconfirmed in HBV-related HCC specimens by methylation-specific PCR (MSP) andbisulfite sequencing and results demonstrated that transcription levels of NQO1andthe prevalence of the specific methylation abnormalities were significantly correlatedwith HBx expression. The amount of intracellular ROS level and H2O2-inducedcytotoxicity were detected. The results indicated that HBx-mediated down-regulatedNQO1expression could increase the generation of intracellular ROS and the level ofsensitivity to H2O2exposure.