| Objective:p53 gene is one of the earliest discovered cancer suppressor genes which is closely related with human malignant tumors.p53 gene is located at 17p13.1 section of the short arm of human chromosome with an overall length of 16~20Kb;it consists of 11 exons and 10 introns,which encodes the intranuclear phosphorylated protein consisting of 393 amino acids;its transcription product is 25 Kb m RNA.Wild-type p53(wtp53)is a cancer suppressor gene which plays a crucial role in multiple cellular processes including cell cycle,cell apoptosis,cell aging,gene stability and angiogenesis inhibition.As a key component of the anticancer defense mechanism,p53 is generally mutated in human tumors.Mutant-type p53(mtp53)can stimulate cell division and function as a cancer gene.Research has found that primary tumors in systems such as respiratory system,urinary system,reproductive system and digestive system are related to the mutation of p53 gene.Gene polymorphism is a common gene variation.As a result of the variation,products with the same genetic code may have different functions;individuals demonstrate susceptibility to diseases and sensitivity and resistance to drugs;clinical manifestations are diversified;disease development and prognosis and other genetic characters have different phenotypes.In addition to mutation,it has been reported that p53 gene has polymorphism.According to the existing literature,at least 8 polymorphic sites can be detected in the promoter region of p53 gene,the 1st,2nd,3rd,6th,7th and 10 th intron regions and the 7th exon region,including the Hae III restriction enzyme digestion site of the first intron,conversion between G and A of the second intron(at 38 bp of the lower course of the second exon),polymorphism of the repetitive sequence inserted in 16 bp of the third intron region(at the restriction enzyme digestion site of the digestion region of the 72 nd codon Bst U I),the restriction enzyme digestion site of MSP I of the sixth intron(at the 61 st nucleotide of the lower course of the sixth exon),the conversion between G and C at 37 bp of the upper courses of the fourth and seventh exons,the restriction enzyme digestion site of Apa I of the seventh intron,and the conversion between A and T at the tenth intron.Among these polymorphisms,three polymorphic sites are related to tumorgenesis: p53 gene CD72 Arg/Pro polymorphism is related to the genetic susceptibility to multiple tumors;the polymorphism of the repetitive sequence inserted in 16 bp of the third intron region of p53 is related to tumors;the polymorphism of the restriction enzyme digestion site of MSP I of the sixth intron of p53 gene is related to tumors.As one of the three common tumor types in the world,colorectal cancer(CRC)shows a high death rate in China,which occupies 7% of the total death rate related to cancer.Low rectal cancer(LRC)refers to the colorectal cancer located within 8cm around the anal verge.Compared with the middle and upper rectal cancer,LRC presents a big difference in operation difficulty,pathological type and patient outcome.In spite of the improvement in LRC treatment and the understanding of its biological mechanism,LRC is still a big challenge to human health due to a high local recurrent risk.Reports have been performed concerning p53 single nucleotide polymorphisms(SNPs)in CRC.However,in previous studies,most CRC specimens failed to be categorized on the basis of tumor site,or CRC was only divided into colon cancer and rectum cancer,which often leads to inconsistence,or even contradictory study results.For instance,rs1042522 is one of functional p53 SNPs that appears most commonly in studies in CRC,which can cause arginine of 72 codons to convert into proline.According to the studies of Gemignani,et al.,rs1042522 and CRC have no remarkable correlations.On the contrary,two study groups in Asia have verified that proline/rs1042522 proline genotype is associated with the increased CRC onset risk.However,they failed to get the totally same results.The study results of Song,et al.have shown that rs1042522 genotype was not remarkably correlated with smoking or drinking,while those of Zhu,et al.have shown that 72 P alleles may increase the CRC onset risk in drinkers.In the latest study,meta analysis was carried out on data from 32 documents issued previously,which has demonstrated that rs1042522 gene polymorphism fails to significantly increase the CRC onset risk of the total population.It should be noted that,if the study group is merely limited to rectal cancer patients,the rs1042522 CC genotype is remarkably correlated with the onset risk of rectal cancer,implying that the primary site of tumor may have a big impact on the relation between p53 SNPs and the CRC onset risk.To sum up,a great number of studies have been conducted on the relation of p53 gene loss,mutation and protein overexpression with tumor,but studies on p53 gene polymorphism are in the preliminary stage and the experimental results are still in dispute.Study on association between p53 gene polymorphism and differential expression,and LRC onset risk,biological behavior and prognosis,have not been reported.By case-control studies on population in North China,this study systematically discussed the correlations between five polymorphic sites of p53 gene and LRC onset risk,biological behavior and prognosis,the existence of interaction between the five polymorphic sites and LRC environmental factors such as smoking and drinking and the relation between the five polymorphic sites of p53 gene and its protein expression and between p53 protein expression and LRC biological behavior and prognosis,so as to provide a theoretical basis for realizing the LRC preoperative evaluation and individualized treatment.Methods: 1.Selection of research cases.The patients diagnosed as rectal cancer with the tumor distance within 8cm from the anal verge during December,2011 to June,2016 were selected as 350 research objects from Department of Anus and Intestine Surgery of the First Hospital of China Medical University,which were named as low rectal cancer patient group.In addition,350 patients without low rectal malignant tumor through digital rectal examination and related examinations as benign control group being hospitalized during the same time period,diagnosed as benign anus diseases.Before the operation,all the patients are collected with fasting venous blood and stored for tests at-20? after separating serum from sludged blood.Inclusion criteria of experimental group:1.clearly diagnosed as rectal cancer and the distance of tumor from anal verge within 8cm2.Selecting adults with the age more than 18Exclusion criteria of experimental group:1.excluding patients suffering from immune system diseases2.excluding patients suffering from infectious diseases such as tuberculosis3.excluding patients accompanied with primary tumors on other visceral organs before operation4.excluding patients who received neoadjuvant chemoradiotherapy before operation2.Selection of p53 gene candidate polymorphic sites: according to latest data published by NCBI db SNP and Hap Map Database,software forecasting methods were adopted to select promising sites.Some certain frequency distribution among Chinese population according to polymorphic sites and research target spots are selected according to tag SNPs with the minimum allele frequency(MAF)>5%.Related literatures are further referred to select the polymorphisms which are related with diseases without being reported in tumors and low rectal cancer.5 tag SNPs including p53 rs1042522,p53 rs12947788,p53 rs1625895,p53 rs2909430 and p53 rs12951053 are finally established to do relevant researches with onset risks of low rectal cancer,clinical biological behaviors and patient prognosis.3.Research case p53 gene SNP sequencing-based typing: 5 sites including p53 rs1042522,p53 rs12947788,p53 rs1625895,p53 rs2909430 and p53 rs12951053 were sequenced through Kasp Method for 347 low rectal cancer patient group and 353 control group.4.For the correlation analysis for p53 gene SNP typing and onset risks of low rectal cancer,we adopted Hardy–Weinberg Poise(HWP)in chi-square test calculation control group to inspect expected frequency.Chi-square tests are adopted to assess the correlation between polymorphism of p53 gene and onset risks of low rectal cancer.Logistic regression model was used to calculate odds ratio between each genotype and genome model(ORs)and its 95% confidence interval(CI).Logistic regression model is adopted to assess SNPs and classical hazards,including interaction between gender,age,smoking state,and drinking state.5.Collection of various clinicopathological parameters for patient group.Information of pathological parameters were collected according to postoperative pathological sections,immuno histochemistry and other methods,including lymphatic metastasis,TNM-staging,depth of invasion,growth pattern,pattern of organization,vessel carcinoma embolus,ganglion invasion,extranodal plantation,peri-cancerous lymphocytes infiltration and so on.6.Analysis of relation between p53 gene SNP typing and low rectal cancer's biological behavior.The correlation between p53 gene SNP typing and LRC clinical case parameter was calculated by nonparametric rank and Mann-Whitney U test.Logistic regression model was adopted to assess SNPs and classic risk factors,and the interaction among gender,age,smoking and drinking.7.Clinical follow-up of patient group.Regular follow-up includes relapse free survival and overall survival,etc.The postoperative follow-up information was collected.Self-reported questionnaire was used to collect the basic information of every subject and his control group.The basic information includes gender,age,smoking and drinking(smoking history is defined as smoking at least one cigarette every day within at least one year.Drinking history is defined as drinking at least 50 g every day in average within at least one year).We also collected the detailed clinical information of low rectal cancer patients according to their medical history and surgery records,including tumor-node-metastasis system classification,depth of infiltration,growth way,lymph metastasis status,histological type,peritumoral lymphocyte's infiltration status,paraneural infiltration status,vessel carcinoma embolus condition and extranodal tumor status.The measured outcome variables include overall survival and death date in follow-up period by the end of August 21 st,2016.Owing to losing some patients during follow-up or being rejected by family members of patients,at last,only 304 subjects' death information is collected.8.Analysis of relation between p53 gene SNP typing and low rectal cancer's prognosis.In the subgroup analysis,subjects who lack data of above-mentioned variables were excluded.Cox proportional hazard model was used to analyze single factor and multiple factors of death risks,Kaplan-Meier curve to draw points for survivorship curve of every p53 gene polymorphism.The Log-rank test was used to assess the difference conspicuousness of overall survival for different patients in the subgroup.SPSS 20.0(SPSS Inc.U.S.Chicago IL)software was applied to carry out statistical analysis.All tests' level of significance is that p is less than 0.05 in the both sides.9.Analysis of relation among p53 gene polymorphism – environment interaction,low rectal cancer onset risk,biological behavior,prognosis.Illness case querying method was adopted to record the smoking and drinking conditions of patients in patient group.Sort them by gender and age and adopt full factorial model to calculate the total P value by the interaction among gene typing,smoking and drinking.10.P53 protein immunohistochemical staining and score.Immunohistochemical method was used to conduct P53 immunohistochemical staining on 347 low rectal cancer pathological slice and slice analysis as well as layering.The concreteness is in accordance with the principle of double-blind method.Two pathological researchers above associate professors will grade independently.If they give different results,superior doctors will reach a consensus by consultation.They adopted count method to judge the results.Every slice was fully observed under low-power lens at first.Cancer cell's positive staining area will be selected in the view of low-power lens.Then,it will be placed under high-power lends for observation.10 high-power lenses will be casually selected.Every view counts 100 cancer cells.The averaged numerical value of positive expression's cell number will be selected as the immunohistochemical positive expression rate of the sample.P53 protein has positive expression in cancer cells,located in cell nucleus of cancer cells.It shows a yellow and browned color under high-power lens.Its trait is granular.The judgment result of P53 is: the proportion of positive cell number of every sample is less than 10%.It is negative.10%~30%(+);30%~50%(++);50%~100%(+++).It has four layers.11.The statistical analysis of p53 gene polymorphism variation's influence on P53 protein expression.p53 gene polymorphism influence on P53 protein expression adopts chi-square test.A two-tailed P value<0.05 indicates statistical significance.12.The correlation analysis of P53 protein differential expression,low rectal cancer risk,biological behavior and prognosis conducts statistical analysis by utilizing SPSS 20.0(SPSS Inc.Chicago USA IL)software.The relationship between P53 genetic polymorphism and p53 protein expression,between p53 genetic expression and LRC clinical case parameter were all analyzed by chi-square test.The compassion among groups of survival time adopts Log-rank test.A two-tailed P value<0.05 indicates statistical significance.Results: 1.The association between p53 SNP genotypes and low rectal cancer risk.We analyzed the association between 5 candidate SNPs of p53 rs12947788,and the results found that carriers of variant A allele had a increase of risk of low rectal cancer(AA+GA vs.GG: OR = 1.393,95% CI 1.030-1.884,P=0.032).All other analyzed genotypes had no significant correlation with the risk of low rectal cancer.When study group was stratified according to risk factors,three SNPs among the five selected SNPs were found to be associated with an increased risk of low rectal cancer.In the recessive model,rs1042522 increased the risk of low rectal cancer in smokers(CC vs.CG+GG: OR = 2.561,95% CI 1.146-5.721,P=0.022).In the dominant model,rs12947788 significantly increased the risk of low rectal cancer in cases with drinking history(AA+GA vs.GG: OR = 3.235,95% CI 1.158-9.040,P=0.025)or under 60 years old(AA+GA vs.GG: OR = 1.749,95% CI 1.113-2.748,P=0.015).In the recessive model,compared with heterozygous and wild genotypes,rs12951053 CC genotype has an association with increased risk of male low rectal cancer(CC vs.CA+AA: OR = 2.473,95% CI 1.061-5.765,P=0.036).2.The association between p53 SNP genotypes and biological behavior of low rectal cancer riskWe further analyzed the association between 5 candidate SNPs of p53 genes and clinical parameters of low rectal cancer including Tumor-Lymph node-Distant metastasis(TNM)classification system,infiltration depth,lymph node metastasis status,histological type,peritumoral lymphocyte infiltration status,ganglion infiltration status,vessel embolus and extranodal tumor implantation.The results showed that in the dominant model,rs12947788 was obviously related to the histological type of low rectal cancer(P=0.044).In the subgroup analysis,rs12947788 was also obviously related to tumor histological type in male low rectal cancer patients(P=0.022)and those over 60 years old(P=0.043),without history of alcohol(P=0.048)and with smoking habit(P=0.039).3.The association between p53 SNP genotypes and low rectal cancer prognosis.When analyzed the association between 5 candidate SNPs of p53 genes and rectal cancer patients' prognosis,no significant association was found for 5 candidate SNPs of p53 genes in the general analysis.In the subgroup analysis on the basis of clinical features,compared to wild type gene carriers,heterozygous genotype carriers of some SNPs were found to be related to low rectal cancer patients' prognosis.Interestingly,for low rectal cancer patients with nest growth pattern,rs12947788 is a relevant factor for their good prognosis(GA vs.GG: HR = 0.204,95% CI 0.042-0.980,P=0.047),but rs1625895(GG vs.GA: HR = 26.189,95% CI 2.399-285.91,P=0.007)and rs2909430(AA vs.GA: HR =7.284,95% CI 1.361-38.996,P=0.020)are relevant factors for their poor prognosis.Furthermore,for female patients(AA vs.CA: HR = 4.170,95% CI 1.017-17.106,P=0.047),young patients under the age of 60(AA vs.CA: HR = 3.561,95% CI 1.192-10.638,P=0.023),patients with smoking history(AA vs.CA: HR = 4.940,95% CI 1.178-20.718,P=0.029)positive lymph node metastasis patients(AA vs.CA: HR = 2.367,95% CI 1.028-5.453,P=0.043),or patients with infiltrative growth pattern(AA vs.CA: HR = 3.007,95% CI 1.271-7.115,P=0.012),rs12951053 is a relative factor for their bad prognosis.4.The association between 5 SNP haplotypes of p53 gene and low rectal cancer t risk,biological behavior and prognosis.C-A-G-A-A haplotype was associated with increased risk of low rectal cancer(P=0.001,OR(95%CI)=4.15(1.63-10.58)).5.The association between SNP-environment interaction and low rectal cancer risk,biological behavior and prognosis.There was no obvious statistical significance(p>0.05)between the SNP-environment interaction and low rectal cancer risk,biological behavior and prognosis.6.The association between p53 SNP genotypes and P53 protein expression.p53_rs1042522 polymorphism was associated with P53 protein expression(CG vs GG P=0.027,CC+CG vs GG P=0.032).P53 protein expression positivity was obviously higher in p53_rs1042522 mutant carriers than other genotypes.7.The association between P53 protein differential expression and low rectal cancer biological behavior and prognosis.We analyzed the association between P53 protein expression and clinical parameters of low rectal cancer including Tumor-Lymph node-Distant metastasis(TNM)classification system,infiltration depth,lymph node metastasis status,histological type,peritumoral lymphocyte infiltration status,ganglion infiltration status,vessel embolus and extranodal tumor implantation.No significant association was discovered between clinicopathological parameters and P53 expression.After stratification analysis,correlation between lymphatic metastasis in the low rectal cancer patients who smoked and p53 protein expression was discovered(P=0.032).In low rectal cancer patient over 60,histological type,TNM-staging and tumour infiltration depth were associated with p53 expression level(P=0.002,P=0.049,P=0.034).P53 expression was obviously higher in poor differentiated tumour than that in well differentiated tumour;it was obviously higher in III-IV phase than that in I-II phase and it was higher in T3-4 phase than that in T1-2 phase.There was significant correlation between P53 expression level and TNM-staging in female patients.The P53 expression level was prominently higher in III-IV phase than that in I-II phase in female patients.There was correlation between P53 expression level and low rectal cancer histological types among non-smokers.The P53 expression level was prominently higher in poor differentiated tumour than that in well differentiated tumour.Log-rank test was used to analyze the survival of LRC patients in diverse groups.The univariate survival analysis result showed that there were obvious correlation between p53 protein expression and LRC overall survival time.The survival period of low expression patients was obviously longer than that of high expression patients(P=0.028,HR=2.071,95%CI=1.083-3.958).Multivariate survival analysis result indicated that there was no prominent correlation between P53 protein expression and LRC overall survival time(P=0.195,HR=1.580,95%CI: 0.791-3.154).Stratification analysis according to some classical hazard factors of low rectal cancer patients(sex,age,smoking and drinking status)was conducted.By univariate survival analysis,it was founded that the survival period of low expression patients of P53 protein were longer than that of high expression patients in female patients and non-smokers(P=0.042,HR=3.280,95%CI=1.043-10.311,P=0.014,HR=2.724,95%CI=1.223-6.066).Multivariate survival analysis found that in patients over 60,patients with low p53 expression had longer survival period than high expression patients(P=0.021,HR=3.425,95%CI=1.208-9.712).Conclusion: 1.For p53rs12947788,carriers of variant A allele had a increase of risk of low rectal cancer.In the recessive model,rs1042522 increased the risk of low rectal cancer for smokers.In the dominant model,rs12947788 obviously increased the risk for patients with drinking history or under 60 to suffer from low rectal cancer.In the recessive model,compared to heterozygosis genotype and wild type gene,rs12951053 CC genotype has an association with increased risk of male low rectal cancer rs12947788 is obviously relevant to the histological type of low rectal cancer.For rs1294778,it was prominently related to the tumour's histological type of low rectal cancer for patients over 60,male,without drinking history and with the smoking habit.P53 rs1625895 and rs2909430 were relevant factors of poor prognosis for LRC.For female patients,young patients under 60,patients with smoking history,patients with positive lymphatic metastasis or infiltrative growth pattern,rs12951053 was a relevant factor of poor prognosis.2.C-A-G-A-A haplotype was associated with increased risk of low rectal cancer.There was no obvious statistical significance between the SNP-environment interaction and low rectal cancer risk,biological behavior and prognosis.3.p53_rs1042522 polymorphism was associated with p53 protein expression.p53 protein expression positivity was obviously higher in p53_rs1042522 mutant carriers than other genotypes.4.P53 expression was obviously higher in poor differentiated tumour than that in well differentiated tumour.it was obviously higher in III-IV phase than that in I-II phase and it was higher in T3-4 phase than that in T1-2 phase.There was significant correlation between P53 expression level and TNM-staging in female patients.The survival period of low expression patients of P53 protein were longer than that of high expression patients in female patients and non-smokers.Among patients over 60,the survival periods were longer in patients with low P53 protein expression than those with high expression. |
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