Association Of Wnt Pathy Gene Polymorphisms With Occurrence And Development Of Hepatocellular Carcinoma In Guangxi

Part 1 Relationship of Wnt pathway gene polymorphisms with genetic susceptibility to hepatocellular carcinoma familyObjective: The Wnt pathway is closely related to the occurrence and development of hepatocellular carcinoma(HCC).A total of 24 polymorphic sites of 12 important genes in the Wnt pathway were selected.To investigate the correlation between Wnt pathway polymorphisms and the family clustering genetic susceptibility to HCC in Guangxi.Methods: Using Hap Map database,dbSNP database and 1000 Genomes database,searching for potential functional sites and identify 24 tag single nucleotide polymorphisms in 12 genes.We genotyped 24 SNPs in 12 genes of 119 hepatocellular carcinoma family members(including 27 HCC patients and 92 family members)and 82 control family members from Fusui County in Guangxi.The SNPs included rs2228946 of Wnt2 gene,rs752109,rs41270175 and rs41270177 of Wnt3 A gene,rs10737462 and rs3765351 of Wnt4 gene,rs566926 of Wnt5 A gene,rs833842 and rs374162 7of Wnt10 B gene,rs9870255,rs2293303 and rs4135387 of CTNNB1 gene,rs12928797 and rs7198290 of Axin1 gene,rs7591 of Axin2 gene,rs414098,rs397768 and rs62364017 of APC gene,rs6808874 and rs1546753 of GSK3?gene,rs1261084,rs12457949,and rs13381608 s of TCF4 gene and rs4918791 of TCF7L2 gene.We extracted genomic DNA from blood samples and genotyped the SNP by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)on the MassARRAY IPLEX platform.We designed the PCR primers using MassARRAY 3.0 software.The SPSS18.0 software was used for data analysis.The odds ratio(OR)and 95% confidence interval(95% CI)were calculated using non-conditional Logistic regression analysis to show the correlation and relative risk of various SNP genotypes and HCC risk.Results: The rs2228946 of Wnt2 gene,rs833842 and rs3741627 of Wnt10 B gene,rs414098 of APC gene,rs12457949 and rs13381608 of TCF4 gene were removed,because they were not consistent with the Hardy-Weinberg equilibrium(HWE)law.In the normal families,the risk of HCC for members with GT genotype/ G allele were 2.93(95%CI:1.10-7.82,P=0.03)and 2.63(95%CI: 1.33-5.18,P=0.01)times higher than that of members with TT genotype/T allele in rs566926 of Wnt5 A gene.In the hepatocellular carcinoma families,the risk of HCC for members with TC genotype/ T allele were 0.29(95%CI: 0.10-0.83,P=0.02)and 0.34(95%CI: 0.13-0.92,P=0.03)times higher than that of members with CC genotype/ C allele in rs2293303 of CTNNB1 gene.In the normal families,the risk of HCC for members with T allele was 0.34(95%CI: 0.13-0.91,P=0.03)times higher than that of members with C allele in rs2293303 of CTNNB1 gene.In the hepatocellular carcinoma families,the risk of HCC for members with C allele was 0.42(95%CI: 0.19-0.88,P=0.02)times higher than that of members with G allele in rs9870255 of CTNNB1 gene.In the hepatocellular carcinoma families,the risk of HCC for members with T allele was 0.42(95%CI: 0.19-0.92,P=0.03)times higher than that of members with C allele in rs4135387 of CTNNB1 gene.In the hepatocellular carcinoma families,the risk of HCC for members with GT genotype/ T allele were 12.49(95%CI: 3.03-51.49,P<0.01)and(95%CI: 2.68-41.12,P<0.01)times higher than that of members with GG genotype/ G allele in rs12928797 of Axin1 gene.In the hepatocellular carcinoma families,the risk of HCC for members with AG genotype/ G allele were 4.31(95%CI: 1.61-11.53,P=0.01)and 2.43(95%CI: 1.28-4.61,P=0.01)times higher than that of members with AA genotype/ A allele in rs1261084 of Axin1 gene.The CTNNB1 gene,in the HBV positive carrier,the high risk of HCC of CG+CC genotype in the core members of the family is 0.16(95% CI: 0.04-0.57,P= 0.01)times that of the GG genotype in rs9870255 locus.The risk of HCC in the core members of the HCC family carrying the CT+TT genotype is 0.17(95% CI: 0.054-0.597,P=0.01)times that of the CC genotype in the rs2293303 locus.The risk of HCC in the core members of the high-risk family carrying AT+ AA genotype was 0.19(95% CI: 0.06-0.59,P=0.004)times higher than that of the TT genotype in the rs4135387 locus.At the rs12928797 locus of Axin1 gene,in the AFP negative individual or male individual,the risk of HCC in the core members of the HCC family with the GT+TT genotype were 8.00(95% CI: 1.43 to 4.64,P = 0.02)and 11.25(95% CI: 2.09-60.49,P = 0.01)times higher than that of the GG genotype.In the rs1261084 locus of TCF4 gene,the risk of HCC in the core members of HCC family carrying the AG+GG genotype were 8.76(95% CI: 1.85-41.44,P=0.01),4.83(95% CI: 1.41-11.13,P=0.01)and 6.45-fold(95% CI: 1.74-23.913,P=0.01)times that of the AA genotype in AFP negative individuals,male individuals,and non-smoking individuals,respectively.Conclusions: Wnt5 A gene rs566926 locus,Axin1 gene rs12928797 locus and TCF4 gene rs1261084 locus are associated with increased HCC risk.CTNNB1 locus rs2293303 locus,rs9870255 locus and rs4135387 locus are associated with reduced HCC risk.Stratified analysis found that the susceptibility of HCC to the rs9870255,rs2293303 and rs4135387 sites of the CTNNB1 gene was associated with HBV infection.The susceptibility of HCC to the rs12928797 site of the Axin1 gene was related to AFP and sex.The susceptibility of HCC to the rs1261084 locus of the TCF4 gene was associated with sex,smoking and the AFP.Part 2 Association of Wnt pathway gene polymorphisms with clinical features and survival prognosis of HCCObjective: According to the first part,the Wnt pathway gene polymorphisms closely related to the HCC high-incidence family were selected,including Wnt5 A gene rs566926 locus,CTNNB1 gene rs9870255 locus,rs2293303 locus and rs4135387 locus,Axin1 gene rs12928797 locus and TCF4 gene rs1261084 locus.To further study the relationship between SNPs in Wnt pathway and clinical features and survival prognosis of HCC,and to screen the SNPs closely related to the clinical features and prognosis of HCC.Methods: We extracted genomic DNA from blood samples and tissues and genotyped the SNP by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)on the Mass ARRAY IPLEX platform.The SPSS18.0 software was used for data analysis.The odds ratio(OR)and 95% confidence interval(95% CI)were calculated using non-conditional Logistic regression analysis to show the association and relative risk of various SNP genotypes with clinical indicators of HCC.Kaplan-Meier method was used to describe the survival curve and Log-rank method to test the difference.The Cox proportional hazards model's lineage ratio(HRs)and 95% confidence interval(95%CI)were used to analyze the correlation between genotype and HCC survival.Results: Patients carrying CC genotype of rs9870255 in CTNNB1 gene had a 3.81(95% CI: 1.42-10.24,P=0.01)times increased HCC risk of multiple lesions of HCC compared with the carrying GG genotype.Patients carrying CC genotype of rs4135387 in CTNNB1 gene had a 0.28(95% CI: 0.10-0.80,P=0.02)times increased HCC risk of HBV-positive infection compared with carrying the TT genotype.The risk of distant metastasis in the rs1261084 locus of the TCF4 gene carrying the AG genotype was 0.11(95% CI: 0.01-0.89,P=0.04)times than that of the AA genotype.No correlation was found between the SNP genotypes and the size of HCC lesions,portal vein invasion,and BCLC stage(P>0.05).None of the SNP genotypes was found to be associated with the survival and prognosis of HCC patients(P>0.05).Conclusions: The CC genotype of rs9870255 in CTNNB1 gene may increase the risk of relatively more tumors in HCC patients.The AA genotype of rs4135387 in CTNNB1 gene is associated with a decreased risk of HBV positive infection in HCC patients.TCF4 gene rs1261084 AG genotype can reduce the risk of distant metastasis in patients with HCC.No relationship between gene polymorphisms and survival prognosis of HCC patients was found.Part 3 Correlation analysis of CTNNB1 m RNA and protein expression with SNPs in hepatocellular carcinomaObjective: To analyze the expression of CTNNB1 m RNA and protein in HCC tissues and adjacent normal tissues,and to analyze the relationship between CTNNB1 m RNA or protein expression and clinical parameters or survival prognosis.To explore the relationship between polymorphisms of CTNNB1 gene rs9870255 locus,rs2293303 locus and rs4135387 locus and CTNNB1 m RNA or protein expression.Methods: We extracted genomic DNA from blood samples.The expression of CTNNB1 m RNA in 123 cases of HCC and adjacent normal tissues was detected by real-time fluorescence quantitative PCR.Immunohistochemical SP method was used to detect the expression of ?-catenin protein in 50 cases of HCC and adjacent normal tissues.The Phonorrelation analyses the relationship between expression and genotype.The Kaplan-Meier method plots the survival curve Log-rank to assess differences in survival curves.The correlation between the expression level and HCC survival risk was analyzed using single factor and multifactor COX proportional hazards.Results: The expression of CTNNB1 mRNA in HCC tissues was higher than that in adjacent normal tissues(3.71±4.93 Vs.1.74±1.47,P<0.01).The expression of ?-catenin protein in HCC tissues with portal vein tumor thrombus was higher than that without portal vein tumor thrombus(5.42±6.01 Vs.2.66±3.79,P=0.01).No difference in CTNNB1 m RNA expression between rs9870255,rs2293303 and rs4135387 genotypes and HCC was found(P>0.05).The expression level of ?-catenin protein in HCC tissues was higher than that in adjacent normal tissues(64.0% Vs.26.0%,P=0.000).The expression of ?-catenin protein in HCC tissues with portal vein tumor thrombus was higher than that without portal vein tumor thrombus(100% Vs.52.6%,P=0.002).No correlation was found between ?-catenin protein expression and survival and prognosis of HCC(P>0.05).No difference was found between rs9870255,rs2293303 and rs4135387 genotypes with ?-catenin protein expression in HCC(P>0.05).Conclusions: The expression of CTNNB1 mRNA and ?-catenin protein in HCC tissues is higher than that in adjacent normal tissues.The expression of CTNNB1 m RNA and ?-catenin protein in HCC with portal vein tumor thrombus was higher than that without portal vein tumor thrombus.No differences were found between the rs9870255,rs2293303 and rs4135387 genotypes and CTNNB1 m RNA and ?-catenin protein expression in HCC.