| Background & aimsColorectal cancer(CRC) is one of the most frequent malignancys in our country, which is on the rise due to change in diet custom and the progress of industrialization. In shanghai, for example, the incidence of colorectal cancer has doubled since the last 30 years, making colorectal cancer the second most common malignancy. It is now generally accepted that the carcinogenesis and progression of colorectal cancer is the result of aggressive gene-environment interactions, a multi-step process involving effects of multi-genes. The key to improve the result of colorectal cancer management lies in early diagnosis and early treatment. The identification of susceptibility genes contributing to colorectal cancer would help to clarify pathophysiologic mechanisms relevant to carcinogenesis and would assist in predicting individual and population risk of colorectal cancer development.Both experimental and epidemiologic studies have clarified that estrogen and its receptors or metabolic enzymes play important roles in carcinogenesis and development of colorectal cancer, suggesting some genotypes of estrogen reptors or metabolic enzyemes as candidate cancer susceptibility genes. Several studies reports downregulation of ERαand ERβexpression in colorectal cancer, which is also found to have a role in the development of APC dependent MSI colorectal cancer. In vitro, ERβis found to induce the apoptosis of colorectal cancer cells and suppress its proliferation through p53 pathway. In recent years, several lines of epidemiologic, clinical and experimental evidences have been reported showing that estrogen hormones may have a role in preventing the carcinogenesis of colorectal cancer. The role of estrogen in a certain individual is in part determined by different expression pattern of ER subtypes and functional status of estrogen synthetic and metabolic enzymes. It's our hypothesis that polymorphism of ER or estrogen synthetic and metabolic enzyme genes would have an association with colorectal cancer susceptibility.To validate these hypotheses, We investigated the association of the polymorphisms in estrogen receptor genes and estrogen-metabolizing enzymes genes with susceptibility to CRC in this case-control association study. The bio-function of the positive association SNP was also inspected preliminarily .MethodsThe case-control study included 433 patients with CRC and 790 control subjects. All CRC patients were unrelated ethnic adult Chinese and residents in Chongqing municipality and its surrounding regions, excluding FAP and HNPCC patients, treated at 3 affiliated hospital of the third military medical university. All control subjects were patients which were enrolled due to trauma with no signs of malignancies. At recruitment, informed consent was obtained from each subject, and personal information on demographic factors, medical history, tobacco and alcohol use, and family history of CRC were collected with structured questionnaire.1. Genotyping was performed by SNPlex assay for 5 SNPs in ERαgene, 5 SNPs in ERβ, and rs2486758 in CYP17, rs10046 in CYP19, rs676387 in HSD17B1, rs4680 in COMT. The data was collected with Data Collection and analyzed with Genemapper.2. statisticsThe Hardy-Weinberg equilibrium at each SNP site in control groups was analyzed with the goodness of fitχ2 test. unconditioned- Logistic regression analysis was used for the calculation of Odds ratio and 95% confidence interval, adjusted with sex, ages, and smoking and alcohol status . SAS astatistical package 9.0 and UNPHASE 3.07 was used to conducted the analyses. Analyes included evaluating the distribution of the alleles, genotypes , haplotypes and diplotypes in the population studied the independent associations of genetic polymorphisms with colorectal cancer risk , and the joint effect of genotypes on colorectal cancer risk.We carried out multiple hypothesis testing using the Benjamini-Hochberg method to control the false discovery rate (FDR) in the unconditional logistic regression analysis.Haplotypes were estimated using Haploview4.1. MDR and conditioned-Logestic regression analysis was used for gene-gene interaction analysis. Stratification and conditioned-logistic regression analysis were used for genetic-environmental interaction.All the analyses was two-side test.3. Bioinformatic software was used for prediction of function of positive genes, and IHC was used for protein expression analysis.Result:1. Both association analysis and FDR test confirmed that rs2071454 at the promoter region of ERαand rs2077647 at exon 1 were correlated with risk of CRC. As compared to carriers of TT genotype at rs2071454, carriers of GT or GG had a higher risk of CRC(GT vs TT:OR=1.472,95%CI=1.149~1.885; GG+GT vs TT: OR=1.408,95%CI=1.108~1.789)。There were gene-environment interaction between rs2071454 and age, sex, smoking and drinking.A higher CRC risk was also seen among carriers of AG or GG genotype at rs2077647 as compared to AA carriers,(AG vs AA:OR=1.460,95%CI=1.125~1.895; AG+GG vs AA: OR=1.453,95%CI=1.127~1.873)。There were gene-environment interaction between rs2077647 and age, sex, smoking and drinking. A linkage disequilibrium was found between rs2071454and rs2077647. There was three haplotypes in our subjects as these two SNPs were concerned, TA(60.2%),GG(29.2%)and TG(10.0%). Carriers of GG haplotype is more likely to be susceptible to CRC(OR=1.231,95%CI=1.018~1.488).2. There was no significant association between 5 SNPs in ERβand CRC susceptibility.3. The gene-gene interaction analysis suggested a Multiplicative effect between rs2077647 in ERαand rs3829768 in ERβ, which was confirmed by logistic regression analysis. (GG+AG)/AA vs AA/AA: OR=1.837,95%CI=1.374~2.455; AA/(GG+AG)vs AA/AA: OR=OR=1.947, 95%CI=1.177~3.219。4. Polymorphisms at rs2486758 in CYP17, rs10046 in CYP19, rs676387 in HSD17B1, rs4680 in COMT was analyzed, rs676287 in HSD17B1 and rs10046 in CYP19 were found to be associated to CRC risk after FDR analysis. For rs676387 in HSD17B1: GT vs GG: OR=3.280,95%CI=2.326~4.625; TT vs GG : OR=3.018,95%CI=2.109~4.318; TT+GT vs GG: OR=2.002,95%CI=1.330~3.013。There were gene-environment interaction between rs676387 and age, sex, smoking and drinking.For rs10046 in CYP19: CT vs CC :OR=0.594 ,95%CI=0.442~0.797 ; TT+CT vs CC::OR=0.638,95%CI=0.480~0.849。There were gene-environment interaction between rs10046 and age, sex, smoking and drinking.There was no association between CRC risk and polymorphisms at rs2486758 in CYP17 or rs4680 in COMT .Gene-gene interactions exist between the following pairs of SNPs: (1) CYP17 rs2486758 and CYP19 rs10046;(2)HSD17B1 rs676387 and CYP19 rs10046; (3)ERαrs2071454 and CYP19 rs10046;(4)ERαrs2071454 and HSD1B1 rs676387;(5)ERαrs2077647 and HSD1B1 rs676387;(6) ERαrs2077647 and CYP19 rs10046。5. Bioinformation software was employed for alle function prediction, Mutation from T to G at rs2071454 in ERαwould would lead to the loss of two transcription factor binding site(RAP1 and CAP). IHC detection observed that the expression of ERαin cancer tissue was lower than in normal tissue. A lower expression of ERαwas observed in CRC cancer patients with GT or GG at rs2071454 in ERα. The expression of ERαwas downregulated both in cancer tissue.Conclusion1. There is a correlation between CRC susceptibility and polymorphisms at rs2071454 in 5'end of ERαand rs2077647 in exon1of ERα. The difference in CRC risk is also observed among different haplotypes or diplotypes at rs2071454 and rs2077647. A genetic environmental interaction exists between rs2071454 or rs2077647 polymorphisms and age, sex, smoking and drinking.2. There is no asscociation between ERβpolymorphisms and CRC. A Multiplicative interaction between rs2077647 in ERαand rs3829768 in ERβis also observed.3. Two SNPs rs676287 and rs10046 in two rate-limiting enzyme of estrogen synthesis HSD17B1 and CYP19 respectively have correlation with CRC susceptibility. No correlation between CRC risk and rs248658 in CYP17 or rs4680 in COMT is found. rs676387 or rs10046 polymorphisms had a gene-gene interaction with age, sex, smoking and drinking.4. Gene-gene interactions lie between the following pairs of SNPs: (1) CYP17 rs2486758 and CYP19 rs10046;(2)HSD17B1 rs676387 and CYP19 rs10046; (3)ERαrs2071454 and CYP19 rs10046;(4)ERαrs2071454 and HSD1B1 rs676387;(5)ERαrs2077647 and HSD1B1 rs676387;(6) ERαrs2077647 and CYP19 rs10046。5. Mutation in rs2071454 will cause the loss of two transcripting factor binding site, RAP1 and CAP, and thus likely leading to the downregulation ERαin colorectal mucosa. This hypothesis needs further genetic functional test. |
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