Genetic Susceptibility Study Of HBV-related Diseases And Function Verification

BackgroundsApproximately350million people are chronic hepatitis B virus (HBV) carriers over the world, some of who are at risk of developing end-stage liver including liver cirrhosis (LC) and hepatocellular carcinoma (HCC). LC is a consequence of a sustained wound-healing response to chronic liver disease. HCC is one of the most common malignancies worldwide. Factors such as viruses, environment, and the host genetic factors can affect the development of HBV infection. Now a number of researches have focused on the abnormalities in several critical cell signaling pathways.Current evidence indicates that the transforming growth factor beta (TGF-B) signaling pathway plays a crucial role in different cellular processes, including proliferation, differentiation, apoptosis, fibrosis, tumorigenesis, and immunological responses, etc. The members of TGF-β superfamily, TGF-β1, the type I TGF-β receptor (TBRI), type II TGF-β receptor (TBRII), and type III TGF-B receptor (TBRIII), play an extremely important role in the development of chronic liver disease. Moreover, vascular endothelial growth factor A (VEGFA) has been proved to mediate angiogenesis and increase the vascular permeability. Therefore, VEGFA play a crucial role in regulating tumor cell proliferation, angiogenesis, invasion and metastasis. In the present study, case-control study was used to investigate the association between the host genetic factors and the susceptibility of HBV-related disease, with TGF-β1, TβRⅠ, TβRⅡ, and TβRⅢ as the candidate genes. Furthermore, functional assays were performed to verify the biological significances of the associated single nucleotide polymorphisms (SNPs) in VEGFA promoter with the susceptibility to HCC.MethodsA total of2179northern Chinese Han participants were enrolled in the study, who were divided into four groups:708patients with chronic hepatitis B (CHB),243with LC,347patients with HCC, and the control group (CON) consisted of individuals who had never been infected with HBV, in whom all HBV serum markers were negative. Sixteen SNPs (rs2317130, rs4803457, rs56719111, rs1800469, and rs1800470in TGF-β1; rs334348, rs334349, rs3739798, and rs1590in TβRⅠ; rs2228048in TβRⅡ; rs1805109, rs1805110, rs2810904, rs1805112, rs284878, and rs1804506in TβRⅢ) were genotyped by the PCR/RFLP (restricted fragment length polymorphisms, RFLP) and TaqMan methods to examined the association between the SNPs and susceptibility to HBV-related liver dieases. What’s more, functional analyses, including dual-luciferase reporter assay, electrophoretic mobility shift assay (EMSA), and real-time PCR, were conducted to verify the biological significances of the associated SNPs in VEGFA promoter with the susceptibility to HCC.Results1. Case-control studies were performed to exam the association of TGF-β1and its receptor genes genetic polymorphisms with the susceptibility to the development of chronic liver diseases.1) Case-control study performed in CHB group and CON groupSingle marker association analysis showed that no significant association of TGF-β1and its receptor genes genetic polymorphisms with the susceptibility to CHB.2) Case-control study performed in LC group and CON groupSingle marker association analysis showed that no significant association of TGF-β1and its receptor genes genetic polymorphisms with the susceptibility to LC.3) Case-control study performed in HCC group and CON groupSingle marker association analysis showed that the frequency of the rs1805110T allele was significantly higher in the case group than in the control group (P=0.034). After stratification, the results for rs1805110remained significant in male participants (P=0.005), but there was no statistical difference in females. In males, the frequency of the C-C-G-C-A haplotype resulting from SNPs in TβRⅢ was significantly lower in the case group than in the control group (P=0.001), whereas the reverse was true for the T-C-G-C-A haplotype (P=0.036).2. Functional analyses on the HCC susceptibility gene VEGFA1) The influence of VEGFA promoter SNPs on the promoter activityAfter the4generated luciferase reporter vectors, spanning-1661to+279bp of the VEGFA promoter region, were transiently transfected HepG2cells, the dual-luciferase reporter assay result showed that rs833061C-rs1570360A haplotype had2.59fold higher luciferase activity compared with rs833061T-rs1570360G haplotype (P=0.00005).2) The interaction between the18bp insert allele in VEGFA and transcription factorThe electrophoretic mobility shift assay (EMSA) indicated that the18bp insert allele had created a binding site of transcriptional factor Spl.3) The mRNA expression of VEGFAThe real-time PCR result showed that HCC patients had6.95-fold and5.16-fold higher VEGFA mRNA expression than HBsAg-negative healthy individuals and CHB patients (P=1.15e-10).Conclusion1. Case-control study showed that the rs1805110T allele is associated with susceptibility to HBV-related HCC in males.2. Functional study indicates that VEGFA promoter SNPs may contribute to susceptibility of HCC by altering promoter activity.