Replication Of Genome Wide Association Studies On Hepatocellular Carcinoma Susceptibility Loci In A Chinese Population Structural And Functional Analysis Of The ATF6Promoter

Background:Genome-wide association studies (GWAS) have identified two loci (rs17401966in KIF1B, rs7574865in STAT4) as being associated with hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) in a Chinese population, two loci (rs2596542in MICA, rs9275572located between HLA-DQA and HLA-DQB) with hepatitis C virus-related HCC (HCV-related HCC) in a Japanese population. In the present study, we use a case-control study to determine whether these SNPs are predictive for HBV-related HCC development in other Chinese population as well.Methods: We genotyped4SNPs, rs2596542, rs9275572, rsl7401966, rs7574865, in506HBV-related HCC patients and772chronic hepatitis B (CHB) patients in Han Chinese by TaqMan methods to examine their associations with hepatocarcinogenesis.Results:In our case-control study, significant differences were observed in genotype distribution (P=0.02) and allele frequencies (P=0.01, OR=0.78,95%CI=0.64-0.95) of rs9275572between HCC patients and CHB patients. The association between rs9275572and HCC remains significant after adjusting for age and gender under additive model (P=0.02, OR=0.73,95%CI=0.56-0.95). The remaining3SNPs, rs2596542, rs17401966, rs7574865, did not significantly differ between the case and control groups. In the further haplotype analysis between rs2596542and rs9275572, the frequency of G-A haplotype was significantly lower in case group (P=0.0007), G-A might have a protective effect on HBV-related HCC occurrence (P<0.001, OR=0.66,95%CI=0.52-0.84).Conclusion:These findings provided convincing evidence that rs9275572*A allele is significantly associated with the protection of HBV-related HCC. Background: Endoplasmic reticulum stress (ERS) is a protective response in eukaryocyte. Disruption of protein folding causes ER stress and activates a signaling network called the unfolded proteins response (UPR) to restore normal function of the cell and maintain ER homeostasis. It has been revealed that UPR is highly activated in a variety of tumors types and could contribute to tumor survival and growth. Activating transcription factor6(ATF6) is an endoplasmic reticulum membrane-anchored transcription factor activated by intramembrane proteolysis in the ERS response. ATF6, the main molecules of the UPR signaling, can activates the transcription of ER molecular chaperones, which in turn enhance the cell’s capacity for productive folding, trafficking and degradation, respectively.Objective: To clone ATF6gene promoter and identify its core promoter region.Methods: Approximately1kb of the5’flanking sequence of ATF6was cloned from the genomic DNA of HeLa cells. After sequencing, the5’flanking sequence was directly subcloned into pGL3-Basic vector and then the dual-luciferase reporter assay system was employed to identify its transcriptional activity in HeLa cells. In the truncating analysis of ATF6promoter, we prepared5’truncations of1kb fragments and identified the core promoter essential for transcriptional activation. With deletion mutations, electrophoresis mobility shift assay (EMSA) and overexpression, we identified the location and features of the core promoter and its upstream regulatory region of ATF6.Results: In this study, the5’flanking sequence of ATF6is consistent with the record in Genbank. In HeLa cells, the ATF6promoter exhibited high transcriptional activities. The transcriptional activity gradually decreased with5’truncations. In HeLa,-65to+30truncated fragment exhibited the maximal transcriptional activity. E2F1sites were essential for maintaining the basal transcriptional activity of the human IRF-3promoter. EMSA demonstrate the occupancy of the ATF6promoter by E2F1in vitro. Overexpression of E2F1protein markedly transactivated the human ATF6promoter.Conclusion:The-65to+30region in ATF6promoter was the core promoter region. E2F1played a critical role in regulating basal ATF6promoter activity in HeLa cells.