Association Study And Functional Validation Of SNPs In IL17A And IL17F With Gastric Cancer Susceptibility

Gastric Cancer（GC）is one of the malignant tumors with a high incidence rate and high mortality in the world,ranking second and third in the morbidity and mortality of malignant tumors in China.The five-year survival rate of GC in China is only 35%,which has caused serious disease burden and economic burden to patients.The etiology of GC is complex,which is usually considered to be the result of infection,genetic and environmental factors.Interleukin 17A（IL17A）and Interleukin17F（IL17F）are important pro-inflammatory cytokines secreted by Th17 cells to mediate the body’s inflammatory response.Studies have found that IL17A and IL17F are abnormally expressed in liver cancer,stomach cancer,colorectal cancer,and other cancers,which are involved in the occurrence and development of cancer.Single nucleotide polymorphisms（SNPs）of IL17A and IL17F are potentially associated with the genetic susceptibility of GC.ObjectiveThe purposes of this study were to explore the association between three SNPs of IL17A and IL17F（IL17A rs2275913,IL17A rs8193036,IL17F rs763780）and genetic susceptibility of GC,and to conduct a preliminary functional study on the associated SNPs to find effective biomarkers for GC.Methods（1）Bioinformatics screening of functional SNPs:Combined with NCBI,Ensembl,UCSC,and other databases to determine the gene location and promoter sequence,Haploview software was used to screen SNPs of IL17A and IL17F with potential functions.（2）Genotyping experiment:In this study,490 GC cases and 490 healthy controls were enrolled by a case-control study with frequency matching according to age and gender.DNA was extracted from the peripheral blood of research subjects,and the three SNPs（rs2275913,rs8193036,rs763780）were genotyped by improved multiple ligase detection reaction（i MLDR）method.（3）Dual-luciferase reporter gene assay:Wild-type plasmids and mutant plasmids of IL17A（rs2275913,rs8193036）were constructed and transfected into HEK-293T cells.Dual-luciferase reporting system was detected to determine the effect of SNPs in IL17A promoter on transcriptional activity of the promoter.（4）Transcription factor prediction and binding validation experiment:PROMO and JASPAR transcription factor prediction websites were used to predict the possible binding transcription factors near IL17A rs2275913.The eukaryotic expression vector of the predicted transcription factor was constructed and co-transfected with plasmids of different IL17A genotypes into HEK-293T cells.The effect of SNP in the promoter region of IL17A on the binding ability of promoter and transcription factor was detected by the dual-luciferase reporter system.（5）Statistical analysis:SPSS 22.0 software was used for statistical analysis.Hardy-Weinberg software was applied to analyze whether genotype frequency distribution met Hardy-Weinberg Equilibrium in the control group;multivariate unconditional logistic regression was tested to evaluate the relationship between SNPs and genetic susceptibility of GC.Haplotype analysis was analyzed on the online website SHEsis;MDR software was used to analyze the interaction between SNPs and environmental factors.Population attributable risk percentage（PARP）was calculated to evaluate the public health significance of SNPs.Results（1）For rs2275913,GA（OR:1.34,95%CI:1.01-1.77）,AA（OR:1.54,95%CI:1.10-2.24）and GA+AA（OR:1.39,95%CI:1.07-1.81）genotypes increased the risk of gastric cancer.For rs8193036,CT（OR:1.32,95%CI:1.00-1.74）and CT+TT（OR:1.35,95%CI:1.04-1.76）genotypes were associated with increased risk of gastric cancer.However,no statistical association of IL17F rs763780 with gastric cancer was found in all genetic models（P>0.05）.（2）The stratified analysis based on the dominant model showed that individuals with rs2275913 A or rs8193036 T increased the risk of gastric cancer in the subgroups of≥50 years old,male,with a history of alcohol consumption,and no family history of gastric cancer.Rs763780 C showed the potential risk of gastric cancer only in those younger than 50 years old.（3）Based on the dominant model,the gastric cancer risk in the whole population attributable to rs2275913 GA+AA genotype and rs8193036 CT+TT was 17.40%and15.19%,respectively.（4）The haplotype C_rs8193036G_rs2275913can reduce the risk of gastric cancer（OR:0.72,95%CI:0.60-0.87）.The frequency of T_rs8193036A_rs2275913in the case group was higher than that in the control group,which was associated with the increased risk of gastric cancer（OR:2.54,95%CI:1.56-4.14）.（5）The gene-environment interaction analysis showed the risk of gastric cancer in the population with a history of alcohol consumption,family history of gastric cancer,and rs2275913 A allele was 2.40 times higher than that in the population without these three factors（OR:2.40,95%CI:1.85-3.12）.（6）Dual-luciferase reporter gene assay showed that the transcriptional activity of mutant plasmid rs2275913 A and rs8193036 T were 1.326±0.030 and 1.197±0.022,respectively,which were higher than that of the wild-type plasmid promoters（P<0.01）.（7）The target transcription factor NFAT2 was predicted by the transcription factors prediction websites.The results of the dual-luciferase reporter gene assay showed that the transcription activity of cells co-transfected with IL17A-MUT1 and pc DNA-NFAT2（1.763±0.061）was significantly higher than that of other control and experimental groups（P<0.01）.Conclusions（1）Both rs2275913 A and rs8193036 T alleles of IL17A were associated with increased susceptibility to GC.The haplotype T_rs8193036A_rs2275913could increase the risk of GC.IL17A rs2275913 interacts with the history of alcohol consumption and family history of GC,which can increase the risk of GC.（2）The rs8193036 C of IL17F was only associated with increased risk of gastric cancer in the<50 years old population.（3）SNP rs2275913 may enhance the transcriptional activity of the promoter by enhancing the binding ability of NFAT2 to the promoter region,then regulate the expression of IL17A.