Regulation Of CDH1 Gene Promoter Methylation On The Expression Of E-cadherin And β-catenin In Human Colon Carcinoma

objective To explore the effects of CDH1 gene promoter methylation on the expression of E-cadherin and B-catenin and evaluate the correlation with clinicopathological characteristics of the colonic carcinoma.Methods Methylation specific PCR (MSP) was used to detect CDH1 gene promoter methylation of 68 cases of colonic adenocarcinoma, adjacent tissues and normal tissues. The expression of E-cadherin andβ-catenin was evaluated by immunohistochemistry straining method. MSP and RT-PCR methods were utilized to examine methylation status of CDH1 gene promoter and the changes of E-cadherin mRNA on colon carcinoma cell line HT-29 before and after the treatment with 5-Aza-CdR.Meanwhile,expression of E-cadherin and localization of B-catenin were labeled by immunofluorescence and analyzed with a laser scanning confocal microscope.Results The positive rates of CDH1 gene promoter methylation, in adjacent tissues were 32.4%,in adenocarcinoma were 57.4%.But no detectable methylation was found in all normal tissues.The difference were significant. The positive rates of E-cadherin decreased by turns in the normal tissues,adjacent tissues and adenocarcinoma tissues were 92.6%,66.2%,44.1%.Whereas the expression of B-catenin were translocated from membrane to cytoplasm and nucleus. The aberrant expression rate of B-catenin in adjacent tissues and adenocarcinoma tissues were 29.4% and 50.0%.In all normal tissues. B-catenin expressed at membrane. The positive rates of CDHl gene promoter methylation were negatively correlated with E-cadherin expression (r=-0.312,P=0.01) and significantly correlated withβ-catenin cytosolic/nucleus expression (r=0.309,P=0.018).E-cadherin expression was negatively correlated withB-catenin expression(in cytoplasm/nucleus)(r=- 0.296,P=0.014).Both aberrant expression of E-cadherin,β-catenin and promoter methylation of CDH1 were related to differentiation and metastasis of colonic carcinoma(P<0.05). CDH1 gene promoter methylation was positive and unmethylation was negative on HT-29 cell before the treatment with 5-Aza-CdR.After being treated with 5-Aza-CdR for 24 hours,methylation turned negative and unmethylation positive bands were detected.The E-cadherin mRNA failed to be amplified in cells before the treatment, however,it could be detected after the treatment,the mRNA expression level was correlated with the agent concentration.Before treated with 5-Aza-CdR,the expression of E-cadherin was not detected on HT-29 cell andB-catenin was detected in cytoplasm and nucleus, while both E-cadherin and B-catenin staining were positive on the cell membrane by immunofluorescence after the treatment. With E-cadherin was induced to express by 5-Aza-CdR,membranous expression of B-catenin was elevated while the nuclear expression reduced.immunofuorescence double staining displayed the distribution of B-catenin was corresponding with E-cadherin on the cell membrane.Conclusions 1).The low expression of E-cadherin and B-catenin cytosolic/nucleus expression were correlated with CDH1 gene promoter methylation.2). CDH1 gene promoter methylation was not related to gender, age,size and tumor location.But related to differentiation and lymph node metastasis of colonic carcinoma.3). CDH1 gene promoter methylation may lead to the loss of E-cadherin expression and the altered distribution ofβ-catenin in colon carcinoma cells.4).Membranous expression of E-cadherin andβ-catenin were elevated by 5-Aza-CdR,which showed that specific methylation inhibitors may provid an new method in tumor prevention and treatment.