Genome-wide Exons Single Nucleotide Polymorphism Characterization Screening And Functional Identification Of Codon Mutations At TP53 Gene Codon 249 In AFB1/HBV Double Exposure Of Primary Hepatocellular Carcinoma Subtype

Research content: This study was divided into three parts: 1,Analysis of mutation and clinical prognosis of TP53 gene codon 249 in AFB1/HBV double exposure of Guangxi primary hepatocellular carcinoma subtype;2,Polymorphism characterization screening of genome-wide exons which impact TP53 gene codon 249 in AFB1/HBV double exposure of Guangxi primary hepatocellular carcinoma subtype;3,Preliminary verification of the function of mutation susceptibility candidate gene which impact TP53 gene codon 249 in AFB1/HBV double exposure of Guangxi primary hepatocellular carcinoma subtype.PART ONE ANALYSIS OF MUTATION AND CLINICAL PROGNOSIS OF TP53 GENE CODON 249 IN AFB1/HBV DOUBLE EXPOSURE OF GUANGXI PRIMARY HEPATOCELLULAR CARCINOMA SUBTYPEObjective: To investigate the analysis of mutation and clinical prognosis of TP53 gene codon 249 in AFB1/HBV double exposure of Guangxi primary hepatocellular carcinoma subtype.Materials and methods: A total of 485 patients with hepatocellular Carcinoma(HCC)underwent hepatectomy from Hepatobiliary Surgery at the First Affiliated Hospital of Guangxi Medical University from 2001 to 2013 were enrolled.Serum hepatitis B surface antigen(HBV)of all patients was positive.Sanger sequencing was used to detect the mutations in the TP53 gene codon 249 in HCC tissues.The map of TP53 gene codon 249 of Hepatitis B virus-associated primary hepatocellular carcinoma in Guangxi region was drafted.Mutation of 249 th codon of TP53 gene was associated with follow-up data to analyze its prognostic effect.Univariate analysis was performed using chisquare test and Cox proportional hazards model,and multivariate analysis was conducted using Logistic regression and Cox risk ratio model.The statistical software is SPSS 18.0.P <0.05 was considered statistically significant.Results: 1.In the 485 cases of this study,we found that there were 165(34.0%)cases of TP53 gene 249 codon mutation,the main types of mutations were: AGG> AGT(Arg> Ser)(159 cases,32.8%)and AGG> AGC(Arg> Ser)(6 cases,1.2%).The mutation rate of Nanning area was 34.7%,Guilin area was 23.1%.2.we measured the exposure level of aflatoxin indirectly by the proportion of effective days of AFB1 production and analyzed the Spearman correlation between the proportion of effective days of AFB1 production and the mutation rate of codon 249 of TP53 gene in HBV-related HCC patients.The results show that there is no significant correlation between both(P = 0.302,R =-0.297).3.After propensity Score Matching(PSM),a total of 322 cases were included in the clinical prognosis analysis.Among them,there were 161 cases of TP53 gene codon 249 mutation,and 161 cases of TP53 gene 249 codon nonmutation.Multivariate Cox risk ratio model results showed that there was no significant difference in long-term survival and tumor-free survival between TP53 gene codon 249 mutation and non mutation group(P>0.05).There was statistically significant difference in tumor-free survival between the two groups(P = 0.039,HR=1.47,95% CI=1.02-2.18),the recurrence risk within 2years was relatively higher in mutation group.4.The combination analysis of TP53 gene codon 249 mutation and preoperative AFP levels showed that in the AFP level> 400ng/m L & TP53 gene codon 249 mutation group,the clinical prognosis was the worst,the recurrence within 2 years(P = 0.009,HR=1.90,95% CI=1.18-3.07)was higher than the other groups.5.Stratification analysis of TP53 gene 249 codon mutation and postoperative clinical prognosis of patients with HBV-associated primary hepatocellular carcinoma showed that in the TP53 gene codon 249 mutation hierarchical patients,radical resection,postoperative TACE treatment and postoperative antiviral therapy could not significantly prolong the patient's postoperative tumor-free 2 years,but radical resection(P = 0.012,HR=0.58,95% CI=0.38-0.89)and postoperative anti-virus therapy(P=0.022,HR=0.61,95% CI=0.40-0.93)could give better tumor-free survival within 2 years for TP53 gene codon 249 non-mutation patients.Conclusion:1.The proportion of days of aflatoxin production of Aspergillus flavus can not effectively and accurately reflect the level of aflatoxin exposure.2.TP53 gene codon 249 mutation May be an independent risk factor for predicting recurrence within 2 years of HBV-related primary hepatocellular carcinoma patients.3.Combination analysis of preoperative serum AFP levels 400 ng / m L as transverse value and TP53 gene codon 249 mutation showed that AFP level>400ng/m L and TP53 gene codon 249 mutation might lead to a worse clinical prognosis.4.In the TP53 gene codon 249 mutation hierarchical patients,radical resection,postoperative TACE treatment and postoperative antiviral therapy could not significantly prolong the patient's postoperative tumor-free 2 years.PART TWO POLYMORPHISM CHARACTERIZATION SCREENING OF GENOME-WIDE EXON WHICH IMPACT TP53 GENE CODON 249 IN AFB1/HBV DOUBLE EXPOSURE OF GUANGXI PRIMARY HEPATOCELLULAR CARCINOMA SUBTYPEObjective: To study the genetic susceptibility of TP53 gene codon 249 mutation in the primary hepatocellular carcinoma subtype of AFB1/HBV double exposure in Guangxi by using genome-wide association analysis.Materials and methods: A total of 485 patients with hepatocellular Carcinoma(HCC)underwent hepatectomy from Hepatobiliary Surgery at the First Affiliated Hospital of Guangxi Medical University from 2001 to 2013 were enrolled.All patients were positive for hepatitis B surface antigen.Using a high throughput genome-wide exons genotyping platform Illumina exome beadchip12v1 to classify the single nucleotide polymorphisms in genome-wide exons.For the overall quality control of the crowd we tood the following aspects to achieve: to observe whether there is stratification in the population through the principal component analysis;samples with a sample size of> 95% was adopted;Adoption in the chip analysis,there was no fuzzy gender of the individual;In homology relationship identification the identity-by-descent <0.1875 group(ie,there is no genetic relationship).The quality control of the candidate sites we accepted the following program: Eliminated the SNP typing success rate <95%sites;Included site meed with Hiddh Weinberg equilibrium P <1×10-6;In order to ensure test effectiveness,the minimum allele frequency was adopted greater than 0.05.2.A total of 270 HBV-related HCC patients surgical specimens in Hepatobiliary Surgery at the First Affiliated Hospital of Guangxi Medical University from December 2013 to August 2016 were collected.3.Mass ARRAY SNP genotyping platform was used for independent sample validation of candidate SNP loci for whole genome exons scanning.3.Chip scan phase and independent sample repeat validation phase were adopted with Single Variant Test of EPACTS 3.2.6 software package(Logistic Score Test model was selected)to analyze the association between TP53 gene codon 249 mutation and SNP.Chi-square test and logistic regression analysis were used to analyze the relationship between genetic model and genotype.Linkage disequilibrium and recombination patterns analysis were accomplished by Locus Zoom software package.Results: 1.After quality control,a total of 459 samples,including 407 males and 52 females;a total of 21501 SNP loci meet the quality control requirements,the overall success rate was 98.35%.The results of the principal component analysis suggest that there is no stratification in the population(?=1.008).All samples did not have gender ambiguity and genetic relationship.Due to the limitation of the sample size,we selected the single nucleotide polymorphism sites with the smallest allele frequency greater than 0.05 to calculate.Verification of the chip classification results were carried out with a method of randomly direct sequencing of 10% samples,and the results showed that the classification and sequencing of the exon chips were consistent and the results were reliable.By calculation,no loci were statistically significant at the GWAS level(P<2×10-6,according to 21501 SNPs,?< 0.05).But we carried out independent sample verification in the candidate sites with top 30 P values.3.After Mass ARRAY SNP genotyping platform quality control,a total of 258 samples,including 80 samples of TP53 gene 249 codon mutation samples,178 cases of TP53 gene 249 codon non-mutations,29 candidate SNP sites meet the quality control requirements.The results of independent samples showed that the frequency of rs8022091 allele distribution was statistically significant between TP53 gene 249 codon mutation and non-mutation groups(P = 0.0232).The difference in the allele frequency of the candidate SNP loci rs9930984 located on the ADAMTS18 gene of chromosome 16 in the two groups was also significant(P = 0.0255).The distribution of the gene frequency on the SNP locus rs75218075 on the WDR49 gene on chromosome 3 was also significantly associated with the TP53 gene 249 codon mutation(P = 0.0295).3.Two data were merged after correcting environmental exposure factors as ages,sex,ethnicity,smoking status,and drinking status,the frequency of allele distribution still showed significant differences within rs8022091(P Combined =0.042),rs9930984(P Combined = 4.84×10-6)and rs75218075(P Combined = 7.36×10-5)between TP53 gene 249 codon mutation and non-mutation groups.Among them,rs8022091 showed a significant decline in the difference after the merger,while rs9930984 and rs75218075 showed a significantly increase.Conclusion: We used the GWAS strategy to initially screen the genomewide exon polymorphism that affected TP53 gene codon 249 mutation in the primary hepatocellular carcinoma subtype of AFB1/HBV double exposure in Guangxi,the rs9930984,rs75218075 and rs8022091 sites were identified to be significantly associated with TP53 gene codon 249 mutation.PART THREE PRELIMINARY VERIFICATION OF THE FUNCTION OF MUTATION SUSCEPTIBILITY CANDIDATE GENE WHICH IMPACT TP53 GENE CODON 249 IN AFB1/HBV DOUBLE EXPOSURE OF GUANGXI PRIMARY HEPATOCELLULAR CARCINOMA SUBTYPEObjective: To preliminarily study the function of TP53 gene codon 249 mutation susceptibility candidate SNP loci,and to investigate the effect of candidate genes on TP53 gene codon 249 mutation under AFB1 exposure pressure.Materials and methods: 1.The mRNA expression of ADAMTS18,WDR49 and SLC8A3 m RNA in hepatocellular carcinoma tissues and adjacent tissues were detected by q RT-PCR and the association with rs9930984,rs75218075 and rs8022091 genotypes were analyzed.2.Lentivirus mediated ADAMTS18 gene silencing and overexpression;AFB1 was used to interval MHCC-97 H,Hep G2 and HL-7702 cell lines,and then detect the effect of ADAMTS18 gene silencing and overexpression on TP53 gene codon 249 mutation.The statistical software is SPSS 18.0.P <0.05 was considered statistically significant.Results: 1.The expression of ADAMTS18 mRNA in HBV-related HCC tissues was higher than that in adjacent tissues(P = 0.041);The expression of WDR49 m RNA in HBV-related HCC tissues was higher than that in adjacent tissues(P = 0.0011);But there was no significant difference in the expression of SLC8A3 gene between cancer and adjacent tissues.2.The genotype of rs9930984 locus and the mRNA expression of ADAMTS18 gene were significantly correlated in paracancerous tissue(P = 0.0028);GT genotype expression was higher than that of TT genotype,but there was no significant difference in cancer tissue.There was no significant correlation among rs75218075,rs8022091 and the corresponding gene expression levels in the corresponding groups(P> 0.05).The m RNA expression of ADAMTS18,WDR49 and SLC8A3 m RNA were not statistically significant(P> 0.05),both in cancer tissues and adjacent tissues between TP53 gene 249 codon mutation and non-mutation groups.3.The genotypes of rs9930984 in MHCC-97 H,Hep G2,SMMC-721,BEL-7404,QGY-7703 and HL-7702 cell lines were TT genotype,rs75218075 were TT genotype,rs8022091 loci were CC genotype.4.MHCC-97 H and Hep G2 cell lines were treated with AFB1 at a concentration of 2?M(6wells per concentration).HL-7702 cells were treated with 1?M AFB1(6 wells),and all wells were mutated,and all were G to T mutations.This result suggested that ADAMTS18 gene silencing and overexpression did not affect the TP53 gene 249 codon mutation at codon 249 of TP53 gene At a concentration of 1 or2?M AFB1 intervention.Conculsion: 1.The genotype of rs9930984 and the mRNA expression of ADAMTS18 gene were significantly correlated in paracancerous tissue(P =0.0028);GT genotype expression was higher than that of TT genotype,but there was no significant difference in cancer tissue.2.Under the treatment of AFB1 at a concentration of 2?M in MHCC-97 H and Hep G2 cell lines,ADAMTS18 gene silencing and overexpression did not affect the TP53 gene 249 codon mutation.It may be necessary to redesign the AFB1 intervention concentration and the intervention time to explore the effect of ADAMTS18 gene expression on TP53 gene 249 codon mutation.