Association Between GSTP1 Gene Promoter Region Rs6591256 Polymorphism And Risk,Biological Behavior And Prognosis Of Colorectal Cancer

Objective:Colorectal cancer（CRC）,one of the most common malignant tumors of the digestive tract,is a serious threat to human health.As a developing country of China,with the changes in dietary habits,the aging and environmental pollution,the incidence of colorectal malignant tumors has been increasing year by year.At present,the main treatment of colorectal cancer is still based on surgical radical surgery.At the same time,the role of adjuvant chemotherapy in perioperative period is increasingly prominent.More and more research has confirmed that adjuvant chemotherapy is beneficial for patients with colorectal cancer.However,there are significant individual differences in the efficacy and side effects of chemotherapy drugs,which ultimately affect the overall treatment outcome and prognosis.Molecular epidemiological studies have shown that genetic factors play an important role in the susceptibility of individuals to carcinogenic factors,especially the genetic polymorphisms of enzymes involved in the metabolism and biotransformation of carcinogens in the body become an important determinant of the degree of susceptibility to the same carcinogenic factors for different individuals.Glutathione S-transferase（GST）is one of the most important phase II metabolic enzymes for biotransformation in vivo,and it is the main detoxification system for cell damage resistance and cancer resistance.It plays an important role in detoxifying carcinogens,activating anti-tumor prodrugs,metabolizing chemotherapeutic agents,and participating in cell cycle and apoptosis regulation.Polymorphisms in the GST gene may affect the function of the encoded enzyme,causing changes in both gene expression and activity of the protein itself,and thus may have an effect on the risk of cancer development.Glutathione S-transferase P1（GSTP1）is located on chromosome 11q13and is 2.8 kb in length and consists of 7 exons and 6 introns.Through the PubMed database,the GeenBank search,there are SNP sites in the promoter region,exon region,and 3’UTR region of the GSTP1 gene.At present,most research focus on GSTP1 gene polymorphism is the SNP located in the 5th exon region of GSTP1 gene（rs1695）,its313th point occus A→G base substitution,lead to the 105th amino acid of protein peptide chain changed,ATC Isoleucine（Ile）becomes GTC proline（Val）,which in turn changes the volume and hydrophobicity of the amino acid,resulting in a decrease in the thermal stability of the encoded protein and a decrease in specific catalytic activity.Based on the biological functions of GSTP1 as a phase II metabolic enzyme,most of the current research on rs1695 focuses on the expression of its encoded protein and the sensitivity and prognosis of platinum-containing drugs,chemotherapeutic drug metabolism,and chemotherapeutic drug-related adverse reactions.Because the distribution frequency of different genotypes of GSTP1 has obvious racial and regional differences,its correlation with the risk of colorectal cancer remains controversial.Recently,we screened the potential functional tag SNP（tagSNP）of GSTP1 gene.In addition to the most frequently studied GSTP1Ile105Val polymorphism,there is rs6591256 SNP in the locus of GSTP1 promoter region,which has A-G polymorphism.Previous studies reported that rs6591256 polymorphism was associated with the risk of prostate cancer,and was associated with susceptibility to tics in Taiwan,at the same time,the chromosome 11 haplotype CTACGAGGCC associated with rs6591256 in the African-American population may increase the risk of asthma by nearly 5-fold.At present,the relationship between rs6591256 polymorphism and colorectal cancer has not been reported.As tumors are multi-gene,multi-step pathogenic diseases,which may affected by many factors such as genetics and environment,the recognition ability of single gene polymorphism is limited,and multi-factor joint detection has more obvious advantages.This study hopes to investigate the effect of rs6591256 polymorphism of GSTP1 gene and environmental factors on the risk and prognosis of colorectal cancer in Chinese by case-control study.Provide experimental basis and further research ideas for individualized diagnosis and prevention of colorectal cancer and further elucidation of the susceptibility mechanism of colorectal cancer.Methods:1.Selection of research cases.A total of 637 patients were admitted to the Department of Anorectal Surgery,the First Affiliated Hospital of China Medical University from December 2012 to June 2016.Among them,310 patients with colorectal cancer after operation were diagnosed as experimental group;327 patients with pathological diagnosis of rectal anal benign disease and after colonoscopy and rectum examination there were no colorectal malignant tumors,were diagnosed as control group.2.Case group clinical follow-up and information collection.The patients in the experimental group were followed up by telephone every six months after operation.The patients were followed up for distant metastasis,overall survival（OS）,and follow-up data were collected.The basic information of each case group and control group was collected by questionnaire.The basic information includes gender,age,and smoking and drinking status.Detailed clinical information of patients with colorectal cancer was collected from medical records,including pathological information and blood lipid analysis results.In the subgroup analysis,subjects with missing data for any of the above variables were excluded.3.GSTP1gene rs6591256 polymorphism SNP sequence typing.GSTP1 rs6591256 polymorphism were sequenced for all 637 samples using KASP method.4.Association analysis of GSTP1 rs6591256 polymorphisms SNP genotypes and risk of colorectal cancer;the interaction between SNP genotypes and environmental factors such as smoking and drinking and risk of colorectal cancer.Multivariate logistic regression（LR）was used to assess the association between the GSTP1 rs6591256 polymorphisms and the risk of colorectal cancer.The logistic regression model was used to calculate the odds ratio（ORs）and 95%confidence interval（CI）for each genotype and genome model.Logistic regression models were used to assess the interaction between SNPs and classical risk factors,including gender,age,smoking status,and drinking status.Adjusted by gender and age,the total factor P was used to calculate the total P value of the interaction between genotypes and smoking and drinking.Statistical analysis was performed using SPSS software version 22.0（SPSS Inc.Chicago,IL,USA）.The significant level of all tests was set to bilateral P<0.05.5.Association analysis of GSTP1 rs6591256 polymorphism SNP genotypes and pathobiological behavior of colorectal cancer;the interaction between SNP genotypes and environmental factors such as smoking and drinking and pathobiological behavior of colorectal cancer.Multivariate logistic regression analysis was used to calculate the risk of association between GSTP1 rs6591256 polymorphism and clinical pathobiological behaviors of colorectal cancer.Using the full factor model to calculate the total P value of the interaction between genotypes and smoking and drinking.6.Association analysis of GSTP1 rs6591256 polymorphism SNP genotypes and abnormal metabolism of blood lipid of colorectal cancer;the interaction between SNP genotypes and environmental factors such as smoking and drinking and abnormal metabolism of blood lipid of colorectal cancer.Multivariate logistic regression analysis was used to calculate the risk of association between GSTP1 rs6591256 polymorphism and abnormal metabolism of blood lipid of colorectal cancer.Using the full factor model to calculate the total P value of the interaction between genotypes and smoking and drinking.7.Association analysis of GSTP1 rs6591256 polymorphism SNP genotypes and prognosis of colorectal cancer;the interaction between SNP genotypes and environmental factors such as smoking and drinking and prognosis of colorectal cancer.Cox proportional hazards mode was used to do univariate and multivariate analysis of death-related risks.The survival curve of each genotype was depicted using COX regression curve.The Log-rank test was used to assess the significance of differences in overall survival（OS）of different patients in the subgroup.Adjusted by gender and age,using the full factor model to calculate the total P value of the interaction between genotypes and smoking and drinking.8.GSTP1 protein immunohistochemical staining and scoringThe expression of GSTP1 protein in 310 colorectal cancer specimens was detected by immunohistochemical staining.The concreteness is in accordance with the principle of double-blind method.Two pathological researchers above associate professors will grade independently.If the scores are inconsistent,superior doctors will reach a consensus by consultation.They adopted count method to judge the results.The results of immunohistochemistry were determined by the HSCORE method（Histological score）.9.Association analysis between the expression of GSTP1 protein and biological behavior of colorectal cancerThe relationship between GSTP1 protein expression and clinical pathological parameters of colorectal cancer was tested by chi-square test.The comparison between groups of survival time was performed by Log-rank test.10.Association analysis between the expression of GSTP1 protein and prognosis of colorectal cancerUnivariate and multivariate analysis of death-related risk of GSTP1 differential expression using the Cox proportional hazards model.11.Association analysis of GSTP1 rs6591256 polymorphism SNP genotypes and GSTP1protein expressionThe relationship between rs6591256 polymorphism SNP genotypes and GSTP1 protein expression was analyzed by chi-square test.13.GSTP1 promoter activity assay1.The GSTP1 promoter region fragment containing rs6591256A allele and G allele was synthesized separately using chemical synthesis method（Shanghai Sequential Biotechnology Co.,Ltd.）,and sent to Shanghai Jikai Company for sequencing and identification.2.Construction of the pGL3-Basic rs6591256 reporter plasmid.（1）Amplification,extraction,digestion of pGL3-Basic plasmid（2）pGL3-Basic plasmid and synthetic DNA fragment single digestion（3）Connection reaction（4）Transformation competence（5）Identification3.Prepare the target cells4.Target cell plasmid transfection5.Luciferase detection14.Effect of rs6591256 SNP genotypes on promoter activityThe fluorescence value of each well is the ratio of Firefly luciferase activity（F value）to Renilla luciferase activity（R value）.The average ratio of each well was divided by the average ratio of each well of the pGL3-Basic empty plasmid group to obtain the value of luciferase.All data were expressed as X±s,and independent samples were analyzed by independent sample T test for activity comparison between groups.Results:1.The association between GSTP1 promoter region rs6591256 polymorphism,SNP–smoking and drinking interaction and colorectal cancer risk.We analysis the association between rs6591256 and risk of colorectal cancer found the rs6591256G allele carrier was associated with a decreased risk of colon cancer(GA/AA（OR=0.48,95%CI 0.28-0.82,P=0.007）,and（GG+GA vs.AA）（OR=0.47,95%CI0.28-0.78,P=0.003）.When study group was stratified according to risk factors,it was found to be associated with a decreased risk of colorectal cancer.Risk-decreased populations were concentrated in populations younger than 60 years of age,and rs6591256GA/AA genotype carriers had a reduced risk of colorectal cancer（OR=0.58,95%CI 0.34-0.92,P=0.020）,rs6591256（GG+GA vs.AA）had a reduced risk of colorectal cancer（OR=0.55,95%CI 0.35-0.86,P=0.009）.Analizing the SNP–smoking and drinking interaction and colorectal cancer risk,there was no obvious statistical significance(P_interaction>0.05).2.The association between GSTP1 rs6591256 polymorphism,SNP–smoking and drinking interaction and clinicopathological parameters of colorectal cancer.In stratified analysis,rs6591256GA/AA genotype carriers（P=0.014）,rs6591256（GG+GA vs.AA）（P=0.025）were associated with tumor infiltration depth in male colorectal cancer patients,rs6591256GG/AA genotype carriers were associated with tumor infiltration depth of patients with CRC without smoking history（P=0.021）;rs6591256GA/AA genotype carriers（P=0.039）;（GG+GA vs.AA）（P=0.043）were associated with tumor infiltration depth in patients with normal TC serum TC level.Analizing the SNP–smoking and drinking interaction and clinicopathological parameters of colorectal cancer,there was no obvious statistical significance(P_interaction>0.05)3.The association between GSTP1 rs6591256 polymorphism,SNP–smoking and drinking interaction and abnormal metabolism of blood lipid in patients with colorectal cancer.It was found that rs6591256 GG/AA genotype carriers had an increased risk of TG metabolic abnormalities（OR=4.92,95%CI 1.02-23.62,P=0.047）,rs6591256 GA/AA genotype carriers（OR=0.32,95%CI 0.12-0.82,P=0.017）,and（GG+GA vs.AA）（OR=0.39,95%CI 0.16-0.91,P=0.029）had a decreased risk of metabolic abnormalities in HDL-C.Stratified analysis for the data by age,gender,smoking,and drinking,we found that rs6591256 was significantly associated with decreased risk of HDL-C metabolic abnormalities in patients with CRC.The rs6591256GA/AA genotype carrier was associated with the decreased risk of HDL-C metabolic abnormalities in patients with CRC more than 60 years old（GA/AA:OR=0.19,95%CI 0.04-0.94,P=0.042）,patients with no smoking history（GA/AA:OR=0.16,95%CI 0.04-0.74,P=0.019）,patients with no drinking history（GA/AA:OR=0.33,95%CI 0.12-0.93,P=0.037）.The decreasing effect of the risk of HDL-C metabolic abnormalities was more significant in patients with CRC of males（GG+GA vs.AA:OR=0.39,95%CI 0.15-0.99,P=0.048）,and patients with no smoking history（GG+GA vs.AA:OR=0.30,95%CI 0.09-0.96,P=0.043）.Analizing the SNP–smoking and drinking interaction and abnormal metabolism of blood lipid in patients with colorectal cancer,there was no obvious statistical significance（Pinteraction>0.05）4.The association between GSTP1 rs6591256 polymorphism,SNP–smoking and drinking interaction and prognosis of colorectal cancer.It was found that rs6591256（GG+GA vs.AA:HR=0.48,95%CI 0.23-0.97,P=0.040）was co-related with better prognosis in patients with CRC.And the association between this polymorphism and the better prognosis is more pronounced in patients with rectal cancer（GG+GA vs.AA:HR=0.37,95%CI 0.16-0.88,P=0.025）.Analizing the SNP–smoking and drinking interaction and prognosis of colorectal cancer,there was no obvious statistical significance(P_interaction>0.05)5.The association between GSTP1 protein expression and pathological biological behavior of colorectal cancerThe GSTP1 protein is positively expressed in cancer cells and localized to the cytoplasm of tumor cells,showing a brown-yellow granule.The GSTP1 protein expression was not found to be significantly associated with the pathological biological behavior of colorectal cancer in the overall analysis.We further evaluated the association of the GSTP1 protein expression and clinicopathological parameters of CRC based on epidemiological risk factors.We found that in patients with colorectal cancer over 60years old,the expression of GSTP1 protein in patients with positive perineural invasion was higher than that in the negative group,the difference was statistically significant（Negative:Positive P=0.010,Expression level P=0.017）.In male patients with colorectal cancer,the expression of GSTP1 protein in patients with positive peritumor lymphocyte infiltration was higher than that in negative group,the difference was statistically significant（P=0.019）.In patients with colorectal cancer with smoking history,the expression of GSTP1 protein in patients with positive peritumor lymphocyte infiltration was higher than that in negative group（P=0.020）,the expression of GSTP1 protein in patients with negative vascular cancer embolus was higher than that in positive group（P=0.038）.In patients with colorectal cancer with no smoking history,the expression of GSTP1 protein in patients with highly histopathological grade was higher than that in poorly group（P=0.014）.In patients with colorectal cancer with no drinking history,the expression of GSTP1 protein in patients with positive perineural invasion was higher than that in the negative group（Negative:Positive P=0.028,Expression level P=0.020）.6.The association between GSTP1 protein expression and prognosis of colorectal cancer There was no association between the level of GSTP1 protein expression and the overall survival time of colorectal cancer.According to stratified analysis of the classic risk factors（sex,smoking,and drinking status）,it was found that in patients with colorectal cancer with no smoking history,patients with low expression of GSTP1 protein had longer survival than those with high expression（HR=0.42,95%CI 0.21-0.86,P=0.018）.7.The association between rs6591256 and GSTP1 protein expression.There was no association between GSTP1 rs6591256 SNP genotypes and GSTP1 protein expression in overall analysis.The stratified analysis of this group of data by sex,smoking,drinking risk factors found that in patients with colorectal cancer with smoking history,the rs6591256 polymorphism was associated with GSTP1 protein expression（AG vs AA P=0.022,GG+AG vs AA P=0.026）.8.The effect of GSTP1gene protmer region rs6591256 polymorphism promoter activity The promoter activity of GSTP1_rs6591256 carrying the G allele was lower than that carrying the A allele（0.620±0.056 vs 0.806±0.052,P=0.014）.Conclusion:1.GSTP1 promoter region rs6591256 polymorphism was associated with the decreased risk of colon cancer and associated with the decreased risk of CRC in people younger than 60 years of age.2.The interaction between GSTP1 promoter region rs6591256 polymorphism and environmental factors such as smoking and drinking have no significant association with the risk of colorectal cancer.3.GSTP1 promoter region rs6591256 polymorphism was associated with peritumor lymphocyte infiltration status and depth of invasion in colorectal cancer,the interaction between this polymorphism and environmental factors such as smoking and drinking has no significant effect on the pathological biological behavior of colorectal cancer.4.GSTP1 promoter region rs6591256 polymorphism was associated with increasing risk of TG metabolic abnormalities and decreasing risk of metabolic abnormalities in HDL-C in patients with colorectal cancer,the interaction between this polymorphism and environmental factors such as smoking and drinking has no significant effect on the lipid metablism of colorectal patients with cancer.5.GSTP1 promoter region rs6591256 GG+GA genotype carriers has better prognosis of patients with colorectal cancer,especially in patients with rectal cancer.6.GSTP1 protein expression was associated with histological type,peritumor lymphocyte infiltration status,perineural invasion status,vascular cancer embolus status in patients with colorectal cancer.7.Low expression of GSTP1 protein is associated with a good prognosis in patients with colorectal cancer with no smoking history.8.In CRC patients with no smoking history,GSTP1 promoter region rs6591256polymorphism affects the expression of GSTP1 protein in colorectal cancer.9.GSTP1 promoter region rs6591256 polymorphism may affect GSTP1 protein expression by decreasing promoter activity.