| Colorectal cancer(CRC)is one of the most common malignancies in digestive system.Globally,the incidence of colorectal cancer in the developed countries is higher than in the developing nations.Recently,as the change in diet structure and transformation of life style in Chinese,the incidence of colorectal cancer is rising each year.It has been seriously damaging physical health and quality of life of residents.With the development of medical technology,the detection of colorectal cancer has been remarkably rising due to the colonoscopy and fecal occult blood test.The mortality of colorectal cancer has been significantly declining on account of medical interventions and treatments.Nevertheless,because of onset concealment of colorectal cancer,more than half of patients have been detected at advanced stage and suffering from poor therapeutic effect and unfavorable prognosis.Additionally,the overall survival rate of patients with distant metastasis could be as low as 12.5%.As other malignant tumors,the pathogenesis of colorectal cancer is complex.The process of carcinogenesis is a multistep process involving environmental and genetic factors and their interactions.The epidemiological studies provide evidence that diet structure,consumption of cigarette and alcohol,obesity and inadequate physical activity could result in the incidence of colorectal cancer.Whereas,among those who were exposed to the same environmental risk factors,only part of them suffered from colorectal cancer,indicating that genetic factors play an important role in the individual susceptibility to colorectal cancer.Human genome project(HGP),the attempt to identify human chromosome nucleotide sequence and map genetic code,is a powerful instrument in genetics research.Among different individuals' sequence,there are only 0.1%hereditary difference defined as genetic polymorphism,most of them are single nucleotide polymorphisms(SNPs).Additionally,since the development and improvement of high throughput sequencing technology promotes the measurement of genome-wide genetic variations,genome-wide association studies(GWAS)has been a powerful tool for scaning cancer susceptibility genes.Since 2007,a series of GWAS have identified about 20 colorectal cancer susceptibility loci,such as 8q24 and 18q21.However,because of racial difference,some genetic variations such as chromosome 6q26 region,identified by GWAS among Europeans,are not associated with colorectal cancer among the Chinese population.Recently,a GWAS for colorectal cancer conducted in East Asian population has identified 11 new susceptibility loci,which can only account for a small part of colorectal eancer oecurrence.Therefore,Identification of susceptibility loci is of great significance for pathogenesis of colorectal cancer.In this study,based on a three-stage case-control study,we performed a GWAS for colorectal cancer to screen additional susceptibility SNPs and further verify SNPs among Europeans.Futhernore,we conducted a series of molecular cell biology experiments to reveal potential biological mechanism of susceptibility SNPs involved in the development of colorectal cancer.Part 1 Identification of colorectal cancer susceptibility gene ETV6GWAS is a powerful method in genetic predisposition studies of complicated diseases such as malignant tumors.To date,several colorectal cancer GWAS have reported a sdes of susceptible SNPs,which could explain a small part of colorectal cancer incidence.This study comprising three stages:GWAS stage followed by two-stage validations.In the GWAS stage,based on Illumina Human Omni ZhongHua chip,we performed a genome-wide scan in 1,023 colorectal cancer cases and 1,306 controls.Those SNPs with P<1.0×10-3 were chosen for further validation that consist of 855 colorectal cancer cases and 1,258 controls based on Sequenom Mass ARRAY chip in the first stage validation.A total of 4,462 colorectal cancer cases and 5,629 controls were further genotyped by TaqMan method in the second stage validation.Logistic regression model was used to analyze the association between genetic variation genotypes and colorectal cancer risk.Strong evidence was found for rs2238126 in ETV6(P=2.67×10-10)located at 12pl3.2 at genome-wide significant level(P<5.0×10-8).Combing all subjects in the GW AS and validation stages,we found individuals carrying rs2238126 G allele suffering higher risk of colorectal cancer,compared with those with A allele(P2.67×10-10,OR=1.17,95%CI=1.11-1.23).After filling up 12pl3.2 region,37 additional SNPs were related to colorectal cancer risk(P<0.05).Howevere,adjusting rs2238126 resulted in the disappearance of association between other SNPs and colorectal cancer risk,indicating that rs2238126 contributes to colorectal cancer risk most at 12p13.2 region.When analyzing onset age of colorectal cancer patients,we found that subjects carrying rs2238126 GG alleles had 2.2 years in advance than those carrying rs2238126 AA genotypes.Logistic regression model also showed a significant associaiton between rs2238126 G allele and colorectal cancer early onset.We further validated rs2238126 in a European population comprising 1,046 colorectal cancer cases and 1,076 controls.The rs2238126 showed significantly related with colorectal cancer risk in the consistent direction among Europeans(P=0.034,OR=1.19).Therefore,this study revealed that genetic variation of ETV6 located at chromosome 12ql3.2 may contribute to the susceptibility to colorectal cancer.Part 2 The biological function of genetic variation in ETV6 involved in the development and progression of colorectal cancerA large number of studies have confirmed that SNPs can influence the transcriptional activity and expression level of host gene.We hypothesized that genetic variation of ETV6 could effect the expression of ETV6 and involve in the development and progression of colorectal cancer.In this study,we predicted the regulatory effect of rs2238126 on ETV6 by bioinformatics tools and utilized a series of experiments including dual-luciferase reporter gene assay,electrophoretic mobility shift assay,chromatin Immunoprecipitation and real-time polymerase chain reaction to further verify this function.We have detected the expression level of ETV6 mRNA in colorectal cell lines and tissues.Besides,we have conducted ETV6 plasmid and siRNA,transiently transfecting colorectal cell lines to perform phenotype experiments and explore the pathogenic mechanism of ETV6.Our study indicated that rs2238126 located in the intron of ETV6,which mRNA expression had tissue specificity.In colon tissues,the expression levels of ETV6 were expressed.Bioinformatics,dual-luciferase reporter gene assay,electrophoretic mobility shift assay and chromatin Immunoprecipitation revealed that different alleles of rs2238126 had different combining capacity with transcription factor MAX,significantly affecting the transcriptional activity and expression level of ETV6.Futhermore,rs2238126 had an eQTL effect on ETV6 in human colonic adenocarcinoma tissue.TCGA and immunohistochemistry of 67 pairs of colorectal cancer tissues indicated that ETV6 significantly expressed lower in tumor than in normal tissue.In the edl phenotype experiments,overexprssion of ETV6 remarkably increased the proliferation,but not cell cycle progression or apoptosis in cell lines.Cumulative effects of colorectal cancer susceptibility loci revealed that more individuals carried risk alleles,more they suffered colorectal cancer(OR=1.41-5.09,Ptrnd=2.34×10-24),suggesting a cumulative effect of associated loci on colorectal cancer risk,GRAIL analysis showed that rs2238126 in ETV6 presented a weak connection with other reported colorectal cancer GWAS susceptibility loci,suggesting rs2238126 is an independent susceptibility locus.Our results suggest that different combining capacity of rs2238126 alleles with transcription factor MAX affected the expression and function of ETV6 involved in the development and progression of CRC. |
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