Genetic Association Study Between Candidate Genes Polymorphisms And Hepatocellμlar Carcinoma In Chinese Population And Functional Study Of SNPs In The Promoter Of CD82

Background & AimsHepatocellular carcinoma (HCC) is one of the most common malignancy in our country. The pathogenesis of HCC is still poorly understood. In fact, hepatocarcinogenesis is a long-term multistage process with the involvement of hereditary and environmental factors. The identification of susceptibility genes contributing to HCC would help to clarify pathophysiologic mechanisms relevant to hepatocarcinogenesis and would assist in predicting individual and population risk of HCC development.On the one hand, both animal models and human epidemiologic studies have documented that estrogen act as tumor promoters and might induce hepatocarcinogenesis. Estrogens realize their biogenesis, bioavailability and degradation via estrogen-metabolizing enzymes and exert their biological effects through the estrogen receptor (ER) of target cells. So we hypothesize that the estrogen-metabolizing enzymes and estrogen receptor ? may be the excellent candidate susceptibility genes for HCC and the genetic polymorphisms within these genes would result in differences in susceptibility to HCC. On the other hand, an increased oxidant burden may result in nuclear or mitochondrial DNA damage, DNA methylation, lipid peroxidation and regulation of signal transduction pathways and gene expression, which has been implicated in hepatocarcinogenesis. Several antioxidant enzymes assure protection against oxidative damage. So the genetic variation within antioxidant enzymes could also result in genotype-dependent differences in risk of HCC. To test these hypotheses, we investigated the association of the polymorphisms in estrogen-metabolizing enzymes genes, estrogen receptor ? and antioxidant enzymes genes with susceptibility to HCC in chapter I and chapter II.In the past 4 years, our group has screened SNPs systematically in 128 functionally important genes and discovered 1,400 SNPs in regulatory region, coding region and splicing region, including two common SNPs in the tumor metastasis suppressor gene, KAI1/CD82, regulatory region (-1,572bp to +415bp). Clarifying the influence of these two SNPs on gene expression may highlight a novel therapeutic target for cancer. So we analyzed the function of these two SNPs in the chapterⅢ.MethodsThe case-control study included 434 patients with HCC and 480 control subjects. All subjects were unrelated ethnic adult Chinese and residents in Fusui County and its surrounding regions at Guangxi province, a well-known high-risk region for HCC located in southern China. At recruitment, informed consent was obtained from each subject, and personal information on demographic factors, medical history, tobacco and alcohol use, and family history of HCC were collected via structured questionnaire.Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for four SNPs in ESR2 gene (G1082A, A1730G, rs1256054 and T1057G ), eight in estrogen-metabolizing enzymes genes (CYP17 Msp AI, CYP19 Trp39Arg, CYP19 I/D, CYP1A1 MspI, CYP1A1 Ile462Val, CYP1B1 Val432Leu, COMT Ala72Ser and COMT Val158Met), and six in antioxidant enzymes genes (CuZnSOD A1934C, MnSOD Ala16Val, ECSOD Ala40Thr, ECSOD Arg213Gly, CAT C-262T and GPx-1 Pro198Leu). We also genotyped the (TTTA)n variable number of tandem repeats polymorphism in CYP19 gene through GeneScan technique.Electrophoretic mobility shift assay (EMSA) was conducted to identify potential transcription factors that bind to -262T or -262G probe. To determine whether the polymorphism in the human CD82 promoter will affect the transcription of this gene, PCR amplified promoter fragments of 2065bp with T/C at rs934178 site and T/G at T-262G site were obtained from four heterozygous individuals and subcloned into pGL3 basic vector. Transient transfection was performed in HepG2 and 293 cells using LipofectamineTM 2000 and luciferase activity was determined using a Dual-Luciferase Reporter Assay System.Associations between polymorphisms and risk of HCC were estimated by unconditional logistic regression analyses using SPSS software (version 14.0; SPSS Inc., Chicago, IL). The odds ratios (ORs) were calculated by logistic regression and adjusted for age, sex, status of smoking and drinking and pack-years of smoking where appropriate. Potential modification effects of the polymorphisms on HCC risk by the above risk factors were assessed by the addition of interaction terms in the logistic model and by separate analyses of subgroups of subjects determined by these factors. In view of the multiple testing, the correction factor n(m-1) (n loci with m alleles each) was applied to correct the significance level. Haplotype frequencies between cases and controls were compared by using theχ2 test. The comparisons between more than two groups were carried out using the analysis of variance (ANOVA) in chapter III.Results1. The ESR2 T1057G polymorphism and CYP19 (TTTA)n polymorphism did not exist in this Chinese population. For the other polymorphisms of ESR2 gene and estrogen-metabolizing enzymes genes, except for COMT Ala72Ser polymorphisms, there was no association between these polymorphisms and HCC susceptibility in overall sample. When the subjects were stratified by HBV carriers status, age, gender, family history, smoking status and drinking status, there were no associations observed between the above all polymorphisms and HCC risk. The haplotype and diplotype distribution of ESR2, CYP1A1 and COMT exhibited no significant difference between the cases and controls.2. The CuZnSOD A1934C polymorphism did not exist in this population and the minor allele frequency of ECSOD Arg213Gly polymorphism was too low. On the basis of logistic regression analysis with adjustment for age, sex, status of smoking and drinking, and pack-years of smoking, there were no significant associations between three polymorphisms (MnSOD Ala16Val, CAT C-262T, GPx-1 Pro198Leu) and HCC susceptibility in overall sample, HBV carriers and non-HBV carriers. When the subjects were stratified by age, gender, family history, smoking status and drinking status, there were also no associations observed. For the ECSOD Ala40Thr polymorphism, no significant association was found between this polymorphism and HCC risk in overall subjects and HBV carriers. However, in non-HBV carriers, individuals with one 40Thr allele (Ala/Thr genotype) (OR = 2.13, 95% CI = 1.25-3.64, P = 0.0057) or at least one 40Thr allele (Ala/Thr and Thr/Thr genotype) (OR = 1.90, 95% CI = 1.15-3.15, P = 0.012) showed significantly higher risk to HCC, compared with Ala/Ala genotype. Although there was no significant interaction between ECSOD Ala40Thr and MnSOD Ala16Val in overall sample, HBV carriers or non-HBV carriers, the combined effects of ECSOD and MnSOD were greater than those for either polymorphism alone in overall sample and non-HBV carriers. Individuals with at least one ECSOD 40Thr allele and two MnSOD 16Val alleles showed increased risk compared with individuals with two ECSOD 40Ala alleles and at least one MnSOD 16Ala allele in overall sample(OR = 2.32, 95% CI = 0.13-0.80, P = 0.0018) and in non-HBV carriers (OR = 9.02, 95% CI =2.04-39.92, P = 0.0037). No interactions between genotypes of four polymorphisms and risk factors were obtained. No gene-gene interactions in these four polymorphisms were obtained.3. EMSA result showed that both -262T and -262G probe could form protein-DNA complex with HepG2 cell extracts. The G variant gives a more intense complex compared to the T probe, which was entirely competed by 200-fold molar excess of unlabeled G and T probes, respectively. The results indicated that the same protein bound with different affinity to G and T variants, respectively. Expression studies with reporter constructs showed significantly higher transcriptional activity of the C-T haplotype compared with T-G haplotype in HepG2 and 293 cells (P < 0.01).Conclusions1. The G1082A, A1730G and rs1256054 polymorphisms in ESR2 did not contribute to the differences in susceptibility to HCC in this Chinese population and the haplotypes and diplotypes based on these three polymorphisms also did not influence susceptibility to HCC.72. The eight polymorphisms in estrogen-metabolizing enzymes genes may not play a major role in mediating susceptibility to HCC. The haplotypes and diplotypes of CYP1A1 and COMT also did do not significantly confer an increased risk of HCC.3. The MnSOD Ala16Val, CAT C-262T and GPx-1 Pro198Leu polymorphisms did not contribute to the differences in susceptibility to HCC. ECSOD genotypes may modify the risk of HCC in non-HBV carriers. ECSOD and MnSOD had combined effects in mediating susceptibility to HCC.4. G and T variants at T-262G site in CD82 bound the same protein with different affinity. The G variant had higher affinity than the T variant. The haplotypes based on rs934178 and T-262G polymorphisms modified the transcriptional activity of CD82 promoter.