The Relationship Between The Polymorphism Of XRCC1 Gene And Susceptibility, Chemotherapy Sensitivity Of Colorectal Cancer

Introduction and Objectives:Colorectal cancer(CRC)is one of the most common malignant tumors with high clinical incidence.It is expected that the number of newly colorectal cancer in China will be top one throughout the world in 2025 and the pressure of tertiary prevention of colorectal cancer will be more and more prominent.Early diagnosis of colorectal cancer is the bottleneck constraints to the survival period of the colorectal cancer patients.However,the existing diagnostic imaging measures and the inspections of tumor markers are often difficult to diagnose the early stage andcolorectal cancer is no exception.Though we have carbohydrate antigen(CA199)and carcinoembryonic antigen(CEA)as tumor markers,they still can't diagnose the colorectal cancer in early stage.Therefore we need to develop more and better tumor markers.In the future,the early diagnosis will be expected to be processed from the molecular level and susceptibility gene will be discovered to assist the diagnosis.Both the etiology and pathogenesis of colorectal cancer are still not clear.It's a pathological process with multiple factors and multiple steps,which include environmental factors and genetic factors.The individual differences of susceptibility to colorectal cancer are related with certain genes nucleotide polymorphisms(single nucleotide polymorphism,SNP).Both Genetic predisposition and the interactions of environmental factors play an important role in the process of CRC.External environmental factors can affect or damage the stability of DNA,which contain related genes vector,leading to cell appearances or cell mutation then forming malignant tumors.DNA stability is closely related to tumorigenesis and the ability of DNA recovery is extremely important after the damage of the gene.Thus,there is a certain relationship between the polymorphisms of DNA repair genes and malignant tumor susceptibility.X-ray repair cross complementing gene1(XRCC1)is the first gene discovered in mammalian cells,which is sensitive to ionizing radiation and located on human chromosome 19q13.2-19q13.3 with a molecular weight of 33kb.XRCC1 participates broadly in the repair of DNA damage and plays an important role in base excision repair and single-strand break repair.As molecular scaffolding,XRCC1 is involved in chromosome movement,mitosis and cell apoptosis.The aberrant expression and distribution of XRCC1 may be closely related to the occurrence of tumor,uncontrolled cell proliferation and the malignant behavior of tumor cells,such as unlimited growth,invasion and metastasis.XRCC1 is always with the gene polymorphisms and likely affect the function of protein coded from XRCC1 gene polymorphism,thereby affecting the occurrence of malignant tumors susceptibility.XRCC1 polymorphisms have mainly three Loci:C26304T(Arg194Trp),G27466A(Arg280His),G28152A(Arg399Gln),herein referred to 194,280 and 399 Locus respectively.Currently the most studied locus is 399 Locus.Recent studies show that the XRCC1 gene polymorphisms is related to the development of esophageal cancer,cervical cancer,lung cancer,stomach cancer,breast cancer and other malignancies.In recent years,extensive studies have carried out on the correlation between XRCC1 gene polymorphisms and the susceptibility of colorectal cancer at home and abroad but there is no clear conclusion yet,the results of each locus is not entirely consistent,which is mainly due to the small sample capacity and the insufficient validity of statistical investigations.Therefore,we need to integrate all the samples with small scales and analyze them together-Meta Analysis or increase the sample capacity to study the relationship between XRCC1 gene and the susceptibility of CRC.This is the reason why we chose XRCC1 as our etiology and mechanism of CRC as a breakthrough.It will help to make a contribution to the field of molecular epidemiology on cancer and contribution to early diagnosis strategies of CRC.Although we have made a great progress on the level of clinical treatment of colorectal cancer,the cure rate of colorectal cancer remains low.Therefore,the research of the treatment of CRC and clinical effects is still hot issue so far in clinical studies.The treatments of colorectal cancer gradually form a comprehensive treatment including surgery,radiotherapy,chemotherapy and molecularly targeted therapy.Chemotherapy has played a very important role in the comprehensive treatment of colorectal cancer,especially metastatic colorectal cancer.Platinum-containing chemotherapy drugs can prolong the survival period in advanced colorectal cancer and improve quality of life,but the effects of the chemotherapy have large individual differences.The effect of chemotherapy is mainly due to the sensitivity of tumor cells to anticancer drugs.XRCC1 gene polymorphisms may be associated with the sensitivity to chemotherapy of colorectal cancer and with the toxicity of chemotherapy but we still have different opinions on this issue.Hence,we made a further observation in order to assess the estimated value of the gene polymorphism to the effect of the CRC chemotherapy.In short,in order to observe the relationship between XRCC1 gene polymorphisms and colorectal cancer susceptibility and the relationship between XRCC1 gene polymorphisms and chemotherapy sensitivity to the CRC,we intend to carry out researches from the following three aspects:The first part is to check a large amount of literatures about the relationship between XRCC1 Arg399Gln gene polymorphisms and susceptibility to colorectal cancer and analyze the relationship between XRCC1 Arg399Gln gene polymorphisms and colorectal cancer susceptibility by systematic evaluation and Meta-analysis.The second part is to observe the relationships between the three locus of XRCC1 genes(Arg194Trp?Arg280His?Arg399Gln)polymorphism and colorectal cancer susceptibility by experiments.The third part is to observe the effect of the XRCC1 Arg399Gln gene polymorphism and the reaction of the CRC to the chemotherapy drugs and toxicity.Part ?:XRCClArg399Gln gene polymorphism and susceptibility to colorectal cancer:Meta-analysisObjective:Quantitative summary of the correlation of XRCC1 Arg399Gln polymorphism and CRC susceptibility.Methods:1.Literature-Search Strategy:We conducted a comprehensive search from the PubMed(Jan,1966-Sep,2012),Embase(Jan,1988-Sep,2012),and China National Knowledge Infrastructure(1981-Sep,2012)databases for all articles on the association between XRCC1 Arg399Gln polymorphism and CRC risk from their inception up to 31th,September 2012.The following key words and MeSH terms were used:"XRCC1";"X-ray repair cross complementing group 1";"rs25487","Arg399Gln",or "G399A";"colon cancer","colon carcinoma","rectum cancer","rectal cancer","rectal carcinoma","colorectal cancer","colorectal carcinoma",or"CRC";and "polymorphism or polymorphisms".The literature search was without restriction to the language.The references of the retrieved articles were also hand-searched at the same time to identify additional published articles.2.Statistical analysis:All the analysis are processed through Stata version 12.0(StataCorpLP,College Station TX)and the P value is hibateral.On the basis of using the Hardy-Weinberg(HWE)to evaluate the quality of the literatures,we estimate the distribution of the gene polymorphisms among the control groups by goodness-of-fit test.If P<0.05,it was considered statistically significant.The pooled OR with its corresponding 95%CI was calculated to assess the strength of the association between XRCC1 Arg399Gln polymorphism and CRC risk.The significance of the pooled OR was evaluated by the Z test,and a P value less than 0.05 was considered significant.The chi-square statistic test(Cochran's Q statistic)was used to test for heterogeneity.If P<0.05,it was considered statistically significant.The I2 statistic is to assess the balance of the total variation for all the original literatures.When evaluating the heterogeneity,data was collected by using the DerSimonian and Laird random effects model or Mantel and Haenszel fixed-effects model based on the results of heterogeneity analysis.According to the ethnicity of the control group,we use the subgroup analysis to evaluate the heterogeneity of the original literature data.Sensitivity analysis was performed by sequential omission of individual studies to assess the stability of the results in this meta-analysis.Both the Begg's funnel plot and Egger's weighted regression tests were used to assess the publication bias.Results:Meta Analysis to the correlation of the XRCC1 Arg399Gln Gene polymorphisms and the susceptibility of CRC:1.Characteristics of Original Literatures:According to the inclusion criteria,we got 26 case control trials with a total of 6,979 patients and 11,470 control groups.There were 22 literatures in English and four in Chinese.Studies were conducted in China,Austria,Egypt India,Italy,Japan,Korea,Mexico,Norway,Poland,Singapore,Spain,Turkey,Taiwan,and USA etc,.Among these 26 studies were as follows:13 studies with 3,202 CRC patients and 5,564 controls were from Caucasians,11 studies with 3,626 CRC patients and 5,564 controls were from Asians,one study with 48 CRC patients and 48 controls was from Africans,and one study was unknown due to the lack of information.The genotype distributions in the control groups among all the studies followed HWE except four studies.Therefore,there were 22 studies with high quality and four with low quality according to the quality criteria.2.Forest Plots:This plot shows that the association of XRCC1 Arg399Gln polymorphism with CRC susceptibility.Overall,meta-analyses of total studies showed that the correlation of the variant allele A of XRCC1 Arg399Gln with the CRC was increased compared with the allele G(ORA vs G=1.13,95%CI 1.03-1.23,POR=0.008).The pooled results of total studies suggested that XRCC1 Arg399Gln polymorphism was significantly associated with CRC susceptibility in three genotypic contrast models(ORGln/Gln vs.Arg/Arg=1.24,95%CI 1.04-1.46,POR=0.015;ORGln/Gln vs.Arg/Gln + Arg/Arg=1.19,95%CI 1.03-1.38,POR=0.021;ORGln/Gln + Arg/Gln vs.Arg/Arg=1.14,95%CI 1.02-1.28,POR=0.022),whereas no significant association was found in the additive contrast model(ORArg/Gln vs.Arg/Arg=1.11,95%CI 0.99-1.25,POR=0.064).3.Stratified Analyses:According to stratified analyses by ethnicities and HWE in control groups,the pooled ORs show that there was not significant relevant in Caucasian between XRCC1 Arg399Gln polymorphism and CRC susceptibility.However,all five genetic models in Asian population indicated that XRCC1 Arg399Gln polymorphism was associated with an increasing susceptibility of CRC.(OR A vs.G=1.19,95%CI 1.04-1.37,POR=0.013;ORGln/Gln vs.Arg/Arg=1.35,95%CI 1.04-1.76,POR=0.026;ORArg/Gln vs.Arg/Arg=1.25,95%CI 1.03-1.50,POR=0.021;ORGln/Gln vs.Arg/Gln + Arg/Arg=1.24,95%CI 1.06-1.45,POR=0.007;ORGln/Gln + Arg/Gln vs.Arg/Arg=1.26,95%CI 1.05-1.53,POR=0.014).What's more,meta-analyses of studies with high quality showed that XRCC1 Arg399Gln polymorphism was modestly associated with CRC risk in three genetic models(ORA vs.G=1.11,95%CI 1.01-1.22,POR=0.036;ORGln/Gln vs.Arg/Arg=1.22,95%CI 1.00-1.49,POR=0.048;ORGln/Gln vs.Arg/Gln + Arg/Arg=1.20,95%CI 1.01-1.43,POR=0.041).4.Heterogeneity and Sensitivity Analysis:Significant heterogeneity was found in the overall studies among the allelic genome,hybrid mutation genome,and the homozygous mutation genome(A vs G,I2=62.1%,PH<0.001;Arg/Gln vs.Arg/Arg,I2=57.8%,PH<0.001;Gln/Gln + Arg/Gln vs.Arg/Arg,I2=61.7%,PH<0.001).The heterogeneity was significant in Asian population but not in Caucasian population and this data suggested that ethnicity was one main source of heterogeneity in different studies.5.Publication Bias:Begg's funnel plot and Egger's tests were performed to estimate the publication bias of the published studies about all the homozygous genes model research(PEgger= 0.135).Furthermore,the shape of all the funnel plots for all the reference models did not reveal any obvious evidence of asymmetry and all P values of Egger's tests were more than 0.05,providing statistical evidence of symmetry of the funnel plots..Conclusions:In general analysis,three genetic models of XRCC1 Arg399Gln polymorphism was modestly associated with CRC susceptibility..The statistically significant association between the XRCC1 Arg399Gln polymorphism and CRC risk was observed in Asians among studies with high quality,but not in Caucasians.In order to get a better deep understanding about the relationship between the polymorphisms of XRCC1 Arg399Gln and the susceptibility of CRC,the future studies need to investigate the correlation between the risk of CRC and the interaction of gene-to-gene and gene-to-environment so that we can provide some certain theoretical basis on the etiology,relevant prevention and treatment of CRC.Part ?:Experimental study of XRCC1 gene polymorphisms and the susceptibility of colorectal cancerObjective:To observe the relationship between the three XRCC1 gene(Arg194Trp,Arg280His,Arg399Gln)polymorphisms and the susceptibility of colorectal cancer respectively.Methods:1.Case-Control Study:This study is comprised of 200 cases of colorectal cancers and 248 cases of healthy control groups.Peripheral blood samples from them were experimented as following:DNA extraction,PCR amplification,RT reverse transcription kit synthetic cDNA,digestion,etc,,Protein detection by Western Blot,measuring and analyzing the optical density value by gel analysis system.PCR products extracting from XRCC1 gene 194,280 and 399 locus were processed through sequence analysis.2.Statistical Treatment:Statistical software IBM SPSS20.0 was used for Statistical analysis.MassARRAY method was used to detect the distribution of genotype of XRCC1 polymorphisms.Chi-square tests were used to analysis the difference of the frequency distribution of genotypes between the colorectal cancer groups and control groups.T-test or rank sum test was applied to analyze whether there are differences in colorectal cancer among the different genotypes.Logistic regression analysis was used to test the correlation between the XRCC1 gene polymorphisms and the susceptibility of colorectal cancer.Results:1.General Information of the Patients:There were 200 colorectal cancer patients,which consisted of 120 male patients,80 female patients,aged from 24 to 71 years old,average age 52.3 ± 5.34 years of age,including 81 cases of colon cancers,119 cases of rectal cancers.The ratio of male and female is 120:80.There were 248 cases of healthy control groups,149 male patients and 99 female patients,average age 52.5 ± 5.36 years old.There was no significant difference in gender and age between each group,P>0.05.2.Study on the Relationship between XRCC1 Gene Polymorphism and CRC and Phenotypic CRC.All subjects were detected by the extraction of DNA with a result of A260/A280>1.70,which indicated that detected DNA in the study is accorded with purity requirement of DNA for PCRAgarose gel electrophoresis results indicated that the extraction of DNA in peripheral blood were successful.There was a single and distinct DNA band in Agarose gel results.The integrity of DNA band showed a good quality of DNA,which could be used in the next experiment.The results by Western blot showed that the extraction of XRCC1 protein was successful with a single and distinct protein band in western blot band and this protein band indicated the uniformity of the expression of XRCC1 protein in the sample.After analyzing different genetic PCR products from XRCC1 194,280 and 399 locus,the results is coincident with locus gene sequence quoted in Pubmed.3.The correlation between the polymorphism of each gene locus and the susceptibility of CRC.The frequency of the XRCC1 Arg194Trp:CC?CT?TT in healthy control groups was respectively 51.60%?41.10%?7.30%while that in CRC groups was 50.00%?40.00%?10.00%respectively.Compared with CC,patients with CT and TT showed no significant relativity with the risk of CRC.(ORct/cc=0.995,95%CI:0.672-1.474,P=0.979 while ORtt/cc=1.411,95%CI;0.699-2.848,P=0.321).Meanwhile,compared with CC,the patients with T gene is also no significant relativity with the risk of CRC(ORct+tt vs cc=0.937,95%CI 0.645-1.360,P=0.731).Through statistical analysis,we confirmed that there's no significant difference between age and gender without OR adjustment.The frequency of the XRCC1 Arg280His:GG,GA,AA in healthy control groups were 79.0%.19.0%.2.0%respectively while that in CRC groups was 85,0%?13.5%?1.5%respectively..Compared with GG,patients with AG and AA showed no significant relativity with the risk of CRC(OR AG VS.GG=1.503,95%CI:0.896-2.520,P=0.123 while ORAA VS.GG=1.476,95%CI 0.345-6.308,P=0.599).Meanwhile,compared with GG,the patients with A gene is also no significant relativity with the risk of CRC(ORAA+AGVS.GG=1.500,95%CI 0.914-2.460,P=0.108).Through statistical analysis,we confirmed that there's no significant difference between age and gender without OR adjustment.The frequency of the XRCC1 Arg399Gln:GG,GA,AA in healthy controls was 48.0%?50.0%?2.0%respectively,while that in CRC groups was 44.0%?48.0%?8.0%respectively.Compared with GG,patients with GA showed no significant relativity with the risk of CRC(ORGG VS GA=0.949,95%CI 0.635-1.419,P=0.799).Compared with GG,patients with AA showed significant relativity with the risk of CRC(ORGG VS AA=O.228,95%CI 0.080-0.647,P=0.005).Meanwhile,compared with GG,the patients with A gene is no significant relativity with the risk of CRC(ORAA+GA VS.GG=0.832,95%CI 0.563-1.230,P=0.357).Through statistical analysis,we confirmed that there's no significant difference between age and gender without OR adjustmentConclusions:1.There was significant correlation between XRCCI Arg399Gln polymorphism and colorectal cancer susceptibility;2.There was no significant correlation between XRCC1 Arg194Trp,polymorphism and colorectal cancer susceptibility;3.There was no significant correlation between XRCC1 Arg280His polymorphism and colorectal cancer susceptibility;4.The future studies is needed to collect more samples.Through a large amount of samples,we will further testify and discuss deeply about the relationship between XRCC1 gene and the susceptibility of CRC.Other factors should be also discussed,such as environment factors,genetic factors,individual factors,life styles and racial differences.By making a synthesis of all those factors,we will establish a clear relationship between XRCC1 polymorphism and the susceptibility of CRC.Part ?:Studies on the effect of the XRCC1 Arg399Gln gene polymorphism and the reaction of the CRC to the chemotherapy drugs and toxicityObjectives:to discover the relationship between the XRCC1 Arg399Gln gene polymorphism and the efficiency of curing the CRC based on oxaliplatin and toxical reactions.Methods:1.Peripheral blood samples are collected from 125 advanced CRC patients,of which 52 cases were colonic cancers and 73 cases were rectal cancers,of which 71 male patients and 54 female patients.CRC patients were experimented as following:94 cases are conducted with improved FOLFOX4 and 31 cases with XELOX.2.By checking the XRCC1 polymorphism from 125 colorectal cancers through PCR-LDR and analyzing different genetic PCR products from XRCC1 399 Locus,the result is consistent with the locus gene sequence quoted in Pubmed.3.Statistical Methods:Statistical software IBM SPSS20.0 was used for statistical analysis.MassARRAY method was used to detect genotype distribution of XRCC1 gene polymorphism.We use crosstabs analysis and chi-Square test to examine whether the correlation between genotypes by and the efficacy of chemotherapy has significant differences or not.Chi-square test was used to test the study association between XRCC1399 gene polymorphism and adverse reactions to the chemotherapy.Results:1.General information of the patients:There were 125 patients with colorectal cancer(71 males patients and 54 female patients),which consist of 52 colon cancers and 73 rectal cancers,aged from 26 to 73 years old,average age 55.2 years old.There were 96 patients with advanced colorectal cancer,of which 42 cases were colon cancers,54 cases were rectal cancers.In these 96 cases,there were 43 cases in stage III and 82 cases in stage IV according to the 2002 UICC TNM international Staging System.2.Chemotherapeutic Efficacy:There were 94 cases conducted by improved FOLFOX4 and 31 cases by XELOX.The results showed that there were 31 patients of XRCC1 399Arg/Arg with a rate of 68.89%.14 patients with XRCC1 399 Arg/Arg were in the progression of disease with a rate of 31.11%.There were 29 patients of XRCC1 399Arg/Gln with a rate of 45.31%.35 patients with XRCC1 399 Arg/Gln were in the progression of disease with a rate of 54.69%.There were 6 patients of XRCC1 399 Gln/Gln with a rate of 37.50%.10 patients with XRCC1 399 Gln/Gln were in the progression of disease with a rate of 62.5%.The results-Pearson x2=7.617,P=0.022 by crosstabs analysis showed that there were efficacy differences among these three genes.Checked by chi-square test,we found that there was a significant difference between Arg/Arg and Gln/Gln,x2=15.254,P=0.000.Arg/Gln and Gln/Gln,x2=28.800,P=0.000 and there was no significant difference between Arg/Arg and Arg/Gln,x2=2.604 and P=0.730.In conclusion,the effect of chemotherapy of CRC patients with Arg/Arg and Arg/Gln is better than that with Gln/Gln but the rate of disease progression with Arg/Arg and Arg/Gln is obvious lower than that with Gln/Gln.3,Adverse Effects:XRCC1 399 gene polymorphism was no statistic differences with hemameba and hemoglobin,P>0.005.Compared the numbers of the patients with Arg/Gln or Gln/Gln,those with Arg/Arg showed significantly differences when they are acting with glutamic-pyruvic transaminase,thrombocyte,nausea and vomiting,diarrhea,oral mucositis and neurotoxicity,P<0.005.Conclusions:1.There was correlation between Arg399Gln polymorphism in patients with CRC and the efficacy of FOLFOX4.The efficacy of patients with Arg/Arg genotype and Arg/Gln genotypes was significantly higher than that with Gln/Gln genotypes.The disease progression rate of patients with Gln/Gln genotype was significantly lower than those with Arg/Arg and Arg/Gln genotype.2.XRCC1 Arg399Gln gene polymorphism is relevant with some topical reactions.The Arg/Arg gene patients who were with glutamic-pyruvic transaminase,thrombocyte,nausea and vomiting,diarrhea,oral mucositis and neurotoxicity increased more than the Arg/Gln or Gln/Gln patients.But the Arg/Arg gene patients with the chance of decreasing blood cells are less than the Arg/Gln or Gln/Gln gene patients.There was no difference in the decrease of hemameba and hemoglobin.3.The studies need to expand the sample size to get more accurate research results.In the study XRCC1Arg399Gln gene polymorphism showed a statistical significance with adverse effects of chemotherapy on selected patients.However this study was just empirical medicine and need to be proved by evidence-base medicine.Summary:1,There was a significant correlation between XRCC1 Arg399Gln gene polymorphisms and colorectal cancer,which was expected to be the predictive index in the risk of colorectal cancer.The correlation between XRCC1 Arg194Trp,XRCC1 Arg280His and colorectal cancer need further study.2,XRCC1 Arg399Gln gene polymorphism will be one of the predictive indexes for both the efficacy of chemotherapy on colorectal cancer and toxical adverse effects.