The Molecular Mechanism Study Of Genetic Variants In SLC22A3 On The Susceptibility To Colorectal Cancer

Background:Colorectal cancer(CRC)is one of the most common digestive system malignant tumors.On a global scale,it ranks the third highest incidence rate among the men and the second among the women.Since the rapid development of economy,our living habits and diet structure has been changing gradually,the incidence of colorectal cancer in China is rising year by year.The development of colorectal cancer is a long-term and comprehensive effect of many complicated factors and the mechanism has not been well understood.A number of studies have shown that environmental and genetic factors play a very important role in the occurrence and development of CRC.Genetic variation,characterized by existing heterogeneity in different populations,is an important factor for the differences between individual susceptibility and clinical progression of CRC.Single nucleotide polymorphism(SNP)is the most common type of genetic variations.SNP is due to a single base inserted,missing,or alternative,which leads to changes of gene function by regulating gene expression,and even leads to many diseases including CRC.A genome-wide association studies(GWAS)from the Japanese population reported that chromosome 6q26-q27 region was related with CRC risk.We replicated in Chinese population and found that there may be a novel polymorphism loci related to CRC risk in this region.Moreover,our previous study showed that SLC22A3,which located in 6q26-q27 region,was significantly over-expressed in CRC subjects.However,the real causal site in this region has not yet been addressed.Methods:We selected potential functional SNPs in chromosome 6q26-q27 region.A case-control study was to performed validate the association between SNPs with susceptibility of CRC in a Chinese population.TaqMan genotype methods were taken to genotype these SNPs in DNA samples of 1147 CRC cases and 1201 controls.According to the principle of frequency matching,our cases and controls were matched on the age and gender.Moreover,all samples were unrelated han Chinese Han population.Univariate and multivariate logistic regression model were applied for odd ratio(ORs)by SAS and PLINk softwares.Besides,Expression Quantitative Trait Loci(eQTL)and Luciferase Assay were adopted to investigate the effect of rs420038 different genotypes on the expression of SLC22A3.Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),Western Blot and Immunocytochemistry were used to detect the mRNA and protein expression level of SLC22A3 in colorectal cancer and para-cancer tissues.The experiments of over-expression and down-expression of SLC22A3 in CRC cells was used to explore the function of SLC22A3 in proliferation,metastasis and invasion of cells.Results:In the case-control study,rs420038 of SLC22A3 was found to be associated with the risk of CRC in the Chinese population.In the dominant model,compared with GG genotype,GA/AA genotype was associated with a decreased risk of CRC(adjusted OR=0.79,95%CI=0.67 0.94,P=0.007).The result was consistent in the additive model.The functional studies indicated rs420038 A allele suppressed the activity of the promoter compared with G allele.The eQTL analysis showed that linear correlation existed between the expression of SLC22A3 and rs420038 different genotypes(P=0.040).SLC22A3 was significantly higher expressed in 94 cases of colorectal cancer tissue versus the tissues adjacent to carcinoma(P<0.001),which is consistent with the paired comparison result of 32 colorectal carcinoma and adjacent tissues from TCGA.In CRC cell SW620,we transfected SLC22A3-siRNA,found that the ability of proliferation,migration and invasion of CRC cell was significantly inhibited.In addition,apoptosis increased and the number of G1 phase cells increased,while S phase cells decreased.When DLD1 was transfected with over-expression plasmid of SLC22A3,migration and invasion of CRC cells was significantly improved.Conclusion:rs420038 of SLC22A3 is associated with genetic susceptibility of colorectal cancer in Chinese patients,individuals with AA genotype will suffer lower colorectal cancer risk.rs420038 may influence the expression level of SLC22A3 by regulating SLC22A3 promoter activity.SLC22A3 is extremely over expressed in colorectal cancer samples,and affects the some biology behaviors of colorectal cancer cells,such as proliferation,migration,and invasion.In conclusion,SLC22A3 is correlated significantly with CRC.Further investigations on the molecular mechanisms and clinical applications of SLC22A3 in CRC are warranted.