Correlation Between Loss Of DCC Gene Expression And Methylation Status Of DCC Gene Promoter In Colorectal Carcinoma

IntroductionColorectal carcinoma is one of the most common tumor types in our country.In present,scientists focus their studies on cancer surppressor genes to find out the moleculor mechanisms of tumor oncogenesis.The found of Deleted in Colorectal Carcinoma gene and its fuctions in the colorectal caner were regarded importantly. Researches showed that loss of DCC expression was found in more than 50% colorectal carcinomas.Scientists have studied loss of heterozygosity and mutations a lot about the mechanisms of loss of DCC expression in colorectal cancer.As an important pattern of epgenetics--methylation of DNA promoter may also participate in the mechanisms of loss of DCC expression in colorectal cancer,but this have not been reported.Our study investigated DCC expression and methylation status of DCC promoter in 71 pairs of cancerous and correponding non-cancerous colorectal carcinoma tissues,and also analysed the correlation between DCC expression,methylation of DCC promoter and clinic pathological parameters of colorectal cancers.Materials and Methods1.Colorectal cancer tissues 71 pairs of cancerous and corresponding non-cancerous tissues were surgically obtained from colorectal cancer patients in the First Hospital of China Medical University between 2005.4 and 2007.3.All the cancerous tissues had clear pathological diagnosis.The non-cancerous tissues were far away more than 5cm from cancerous mucoses,and also had clear pathological diagnosis:Normal mucoses 39 cases,Inflammatory mucoses 22 cases,Atypical hyperplasia mucoses 10 cases.All the tissues were divided into two parts for storing, one part was immediately frozen and stored at -80℃for MSP,and the other part was embedded in paraffin for immunohistochemistry.2.Immunohistochemistry Paraffin-embedded sections were evaluated immunohistochemically with a purified rabbit anti-human DCC monoclonal antibody. Individual tissue sections of 4μm were deparaffinized and heated in citric acid monophosphate buffer(pH=6.0)in a microwave oven for antigen retrieval.The primary antibody was used at a dilution of 1:100,4℃overnight,second antibody signed with biotin was used at a dilution of 1:200,37℃30 min,SP complex 37℃30 min.Sections were stained by DAB,than counter-stained,dehydrate,lucidity, envelop.Result determination:Take count of positive tumor cells and total tumor cells from 5 eyeshots in the hightimes lens,than calculated the percentage of positive cells,Immunoreactivity was judged as positive when at least 10%of tumor cells were immunoreactive with the DCC monoclonal antibody,otherwise judged as negative.3.MSP DNA was extracted from the 71 colorectal cancers and corresponding non-cancerous mucosae using phenol-chloroform extraction,than treated with bisulfite,briefly,1μg DNA was denatured with NaOH and modified with sodium bisulfite.DNA samples were then purified using Wizard DNA purification resin, treated again with NaOH,precipitated with ethanol,and resuspended in water for template to PCR.Sss-I methylase treated normal human peripheral blood DNA served as positive controls after bisulfite-modification,and ddH₂O was negative control.10μl samples of each PCR reaction product were loaded directly onto 2.5%agarose gels, stained with ethidium bromide,and visualized under UV illumination.Result determination:Only when PCR product was amplified with methylated primer,then the promoter of DCC gene could be regarded as methylation.4.Statistical analysis All the data were analyzed with SPSS13.0 statistics software.The statistical significance was defined as P＜0.05.Results1.The expression of DCC in cancerous and corresponding non-cancerous tissues in colorectal cancers The positive immunostaining rate of DCC in in colorectal cancer tissues(35/71,49.3%)was significantly lower than in normal mucosaes of corresponding non-cancerous tissues(34/39,87.2%,P＜0.001).2.The methylation status of DCC promoter in cancerous and corresponding non-cancerous tissues in colorectal cancers Methylated rate of DCC in cancer tissues(54/71,76.1%)was higher than normal mucosaes(14/39,35.9%,P＜0.001)and inflammatory mucosaes(10/22,45.5%,P=0.007)of corresponding non-cancerous tissues.3.The relationship between loss of DCC expression and methylation of DCC promoter in cancerous and corresponding non-cancerous tissues in colorectal cancers According to DCC expression whether or not,we divided cancerous and non-cancerous tissues into two group,the methylation rate of DCC promoter in the group of DCC negative expression(34/36,94.4%)was siginificantly higher than that in the group of DCC positive expression(20/35,57.1%,P＜0.001).4.The correlation between DCC expression,methylation of DCC promoter and pathologic parameters in clinic of colorectal carcinomas DCC expression was correlated with Dukes staging in clinic(P=0.023);Methylation of DCC promoter associated to tumor position,methylated rate in rectum cancers(28/31,90.3%)was higher than that in colon cancers(27/40,67.5%,P=0.022)DiscussionIn the progress from normal mucosae,inflammatory mucosae and atypical hyperplasia mucose to cancerous mucosae,the positive expression rate of DCC expression gradually decreased,the level of DCC expression in cancerous mucosaes was siginificantly lower than that in normal mucosaes of non-cancerous tissues.These indicate that loss of DCC expression exactly exists in colorectal cancer,and which may accelerate the oncogenesis of colorectal carcinoma.In addition,loss of DCC expression was correlated with Dukes staging,the worse of Dukes staging,the more obviously of DCC negative expression,from what we can persume that loss of DCC expression may accelerate the invasion and metastasis of colorectal carcinoma. From the study of methylation status of DCC promoter,we found that the methylated rate of DCC promoter had a tendency of increase,which was siginificantly higher in cancerous mucosae than that in normal and inflammatory mucosae of non-cancerous tissues.These indicate that methylation of DCC promoter probably participate in the progression of colorectal cancer.On the side methylation status of DCC promoter was correlated with tumor position of colorectal cancer, which shows that DCC promoter methylation may have more relation with oncogenesis of recum.Connecting the methylation status of DCC promoter and the level of DCC expression,we found that DCC promoter methylation rate of DCC positive expression group was siginificantly higher than that of DCC negative expression group.These show that there is correlation between loss of DCC expression and DCC promoter methylation.Methylation of DCC promoter may be one of mechanisms about frequent loss of DCC expression in colorectal cancer.Conclusion1.Loss of DCC expression exactly exists in colorectal carcinomas,and correlates with Dukes staging;2.Methylation of DCC promoter is a frequent event in colorectal carcinomas, and has a tendency to rectum cancers.3.Frequent loss of DCC expression in colorectal cancers is probably due to the methylation of DCC promoter...