Correlation And Molecular Mechanism Between TNFAIP2 Gene Polymorphism And Prediction/prognosis Of Gastric Cancer

Objective: Gastric cancer(GC)is considered to be one of the most common malignant tumors in the world,which is completely asymptomatic or mild symptoms in the early days and prone to recurrence and metastasis leading to poor prognosis.The incidence of GC has obvious regional difference in the world,and is higher in East Asia,Eastern Europe,the Andes region of South America and parts of Western Asia.China is a country with a high incidence of GC,accounting for about 16% of adults' malignant tumors.At present,although the pathogenesis of GC is not completely clear,some etiological studies have found that the changes of some specific genes and specific loci will have an important impact on the occurrence and development of GC.In recent years,it was found that the TNF-? induced tumor necrosis factor alpha induced proteins(TNFAIPs)may be involved in apoptosis,cell differentiation,immune response,inflammation response,signal transduction,material transport and so on.TNFAIP2,as an important member of TNFAIPs,may play multiple roles in the occurrence and development of malignant tumors.By analyzing the gene polymorphism of TNFAIP2,current studies have confirmed that it is correlated with the occurrence and development of nasopharyngeal cancer,esophageal cancer,GC,breast cancer,lymphoma and other tumors.In addition,TNFAIP2 functional polymorphic site is mainly located in the 3'non-coding region(3'UTR),which may regulate gene expression by changing the target binding ability of mi RNA,and eventually lead to the difference in tumor susceptibility.So far,there are few reports on the correlation between TNFAIP2 gene polymorphism and the occurrence,development and prognosis of GC,especially the lack of data in Asian or Chinese population.This project aims to explore the correlation between TNFAIP2 gene polymorphism and GC risk,clinical pathophysiological behavior and prognosis and its molecular mechanism,which may provide theoretical basis for the diagnosis and individualized treatment of GC.Methods: From December 1997 to December 2013,640 patients with GC and 639 cases with non-cancer from multiple medical centers in northern China were enrolled in this study.All patients with GC had pathological diagnosis.Cases were extracted fasting venous blood,and serum samples was isolated and saved under 20 ? below zero.The epidemiological information and clinicopathological parameters of cases were recorded,and GC patients were followed up by telephone every six months.The main follow-up contents were overall survival(OS)and the deadline for data collection is June 30,2017.This study was approved by the ethics committee of the first hospital of China medical university,and all subjects have signed the informed consent.According to the NCBI db SNP database and Hap Map database,the functional tag SNPs of TNFAIP2 gene were screened by Haploview software and NIH Snpinfo website,and need to meet: A.Chinese han population(CHB);B.Minimum allele frequency(MAF)>5%;C.Frequency distribution r2>0.8.Human whole blood genomic DNA was extracted by phenol chloroform method and analyzed by KASP SNP genotyping and sequencing method.The expression of TNFAIP2 protein in serum samples was determined by enzyme-linked immunosorbent assay(ELISA).The effect of TNFAIP2 3'UTR rs8126 polymorphism on TNFAIP2 protein expression were analyzed by different TNFAIP2 3'UTR rs8126 genotyping.Micro RNA(mi RNA)binding to TNFAIP2 3'UTR rs8126 was predicted by Mir SNP database and Target Scan database,respectively.NCBI db SNP database and mir BASE database were used to compare the base sequence of TNFAIP2 3'UTR rs8126 and predicted mi RNA,and to determine the mi RNA that might bind to rs8126 in 3'UTR region of TNFAIP2 gene.The binding ability of TNFAIP2 functional SNPs to the target mi RNA was verified by dual-luciferase reporter assay.SPSS 20.0 software(SPSS Inc.,Chicago,USA)was used for statistical analysis,and methods included chi-square test,T test,Logistic regression analysis,Kaplan-Meier analysis and COX regression analysis.Bilateral P<0.05 was considered statistically significant.Results: In this study,1279 peripheral blood samples were collected and 1247 eligible samples including 622 in GC group and 625 in non-cancer group were analyzed.Four functional polymorphic sites of TNFAIP2 gene were screened,including mi RNA binding site(rs8126 and rs710100)and transcription factor binding sites(rs3759571 and rs3759573).1.Correlation between TNFAIP2 gene polymorphism and GC risk There is a correlation between TNFAIP2 rs8126 polymorphism and GC risk in general population,and GC risk in heterozygous patients is higher than that in wild-type patients(P=0.001,OR=1.557).In the dominant model,GC risk in TNFAIP2 rs8126 polymorphic carriers is 1.419 times higher(P=0.007).There was no correlation between TNFAIP2 rs710100,TNFAIP2 rs3759571 and TNFAIP2 rs3759573 polymorphisms and GC risk,and TNFAIP2 rs3759573 were excluded in the subsequent analysis due to don't match Hardy Weinberg's genetic linkage balance(PHWE<0.05).The subgroup analysis revealed that among male cases,GC risk in TNFAIP2 rs8126 heterozygous type was higher than that in wild-type patients(P=0.005,OR=1.573),and GC risk was 1.443 times higher in TNFAIP2 rs8126 polymorphic carriers in the dominant model(P=0.018).Among subjects aged over 60 years,GC risk in TNFAIP2 rs8126 heterozygous type was higher than that in wild-type patients(P=0.005,OR=1.816),and GC risk was 1.693 times higher in TNFAIP2 rs8126 polymorphic carriers in the dominant model(P=0.010).Among subjects younger than 60 years old,TNFAIP2 rs8126 heterozygous type has a higher risk of GC than wild-type patients(P=0.049,OR=1.440).In subjects without H.pylori infection,GC risk in TNFAIP2 rs8126 heterozygous type was higher than that in wild-type patients(P=0.006,OR=1.560),and GC risk in polymorphic carriers with TNFAIP2 rs8126 in dominant model was 1.440 times higher(P=0.017).Among non-smoking subjects,GC risk in TNFAIP2 rs8126 heterozygous type was higher than that in wild-type patients(P=0.038,OR=1.701),and GC risk was 1.643 times higher in TNFAIP2 rs8126 polymorphic carriers in the dominant model(P=0.038).Among subjects who did not drink alcohol,TNFAIP2 rs8126 heterozygous type had a higher risk of GC than wild-type patients(P=0.045,OR=1.630).2.The interaction between TNFAIP2 gene polymorphism and environmental factors The interaction between TNFAIP2 polymorphism and environmental factors(H.pylory infection,smoking and drinking)on GC risk was analyzed,and the results showed that rs8126,rs710100 and rs3759571 polymorphism had no interaction with environmental factors(Pinteraction>0.05).3.Correlation between TNFAIP2 gene polymorphism and GC prognosis The prognosis of 299 patients with GC was analyzed,and it was found that there were significant differences in Borrmann typing,depth of invasion,growth pattern,lymphatic infiltration,lymph node metastasis and TNM stage.However,after SNP genotyping,univariate analysis showed no statistical differences between GC prognosis and TNFAIP2 rs8126,TNFAIP2 rs710100 and TNFAIP2 rs3759571(P>0.05).Due to the limited sample size for the registration of lymphatic infiltration and growth pattern,the multivariate analysis was performed using Borrmann typing,depth of invasion,lymph node metastasis and TNM stage as covariables,and it was found that TNFAIP2 polymorphisms were not related to GC prognosis in this group(P>0.05).In addition,TNFAIP2 rs8126 polymorphisms and GC prognosis were analyzed by gender,age and H.pylory infection,respectively,and no statistically significant difference was found in either univariate or multivariate analysis(P>0.05).4.The difference of TNFAIP2 protein expression in GC patients and healthy people ELISA was performed on 202 subjects,including 103 patients with GC and 99 healthy subjects.The results showed that the expression of TNFAIP2 protein in GC group was significantly higher than that in control group,and there was a significant difference between the two groups(P=0.029).5.Correlation between TNFAIP2 protein expression and clinicopathological parameters/ prognosis in GC patients TNFAIP2 protein expression was correlated with Borrmann typing.Compared with GC patients with Borrmann ?-?,serum TNFAIP2 protein expression of GC patients with Borrmann ?-? was higher and the difference was statistically significant(P=0.031).In addition,serum TNFAIP2 protein expression was not associated with the prognosis of patients with GC.6.Correlation between TNFAIP2 3'UTR rs8126 polymorphism and TNFAIP2 protein expression The effects of TNFAIP2 3'UTR rs8126 polymorphisms on TNFAIP2 protein expression in GC group were analyzed.The results indicated that TNFAIP2 protein expression was significantly different between TNFAIP2 3'UTR rs8126 polymorphic genotypes(P<0.001).7.Bioinformatics predicted mi RNAs that could bind to TNFAIP2 3'UTR rs8126 Five mi RNAs including hsa-mir-3191-3p,hsa-mir-711,hsa-mir-4668-5p,hsa-mir-92b-5p and hsa-mir-184 were found to be candidate mi RNAs after the prediction of TNFAIP2 3'UTR rs8126 by Mir SNP database and Target Scan database.The NCBI db SNP database and mir BASE database were further applied to compare rs8126 base sequence and predicted mi RNAs base sequence,and the results suggested that hsa-mir-3191-3p might be the target mi RNA binding to TNFAIP2 3'UTR rs8126 site.8.Validation of the binding ability of has-mir-3191-3p to TNFAIP2 3'UTR rs8126 The binding ability of TNFAIP2 3'UTR rs8126 to has-mir-3191-3p was verified by dual-luciferase reporter assay.The results indicated that compared with the 3'UTR-NC+mi RNA group,there was no significant decrease in luciferase activity in 3'UTR+mi RNA group(P>0.05),suggesting that has-mir-3191-3p could not effectively bind to TNFAIP2 3'UTR rs8126.Conclusion: 1.TNFAIP2 gene polymorphism is associated with GC risk.2.TNFAIP2 3'UTR rs8126 heterozygous or mutated carriers have an increased GC risk in male,aged 60 years or older,no H.pylori infection,no smoking or no drinking.3.Serum TNFAIP2 protein expression was increased in GC patients,and TNFAIP2 3'UTR rs8126 polymorphism could affect the expression of TNFAIP2 protein.4.The hsa-mir-3191-3p failed to bind to TNFAIP2 3'UTR rs8126,thus affecting the expression of TNFAIP2 protein.The mechanism of action needed to be further confirmed.