Study On The Expression Of E-cadherin And The Regulation Mechanism Of CDH1 Gene In Breast Cancer

Objective:1)Study of E-cadherin expression in breast infiltrating ductal carcinoma and the influencing factors;to explore the relationship between the expression of E-cadherin protein and clinicopathological features,lymph node metastasis and prognosis of invasive ductal carcinoma of the breast.2)To investigate the methylation of CDH1gene promoter in breast infiltrating ductal carcinoma;Tostudy the methylation of CDH1gene promoter in invasive breast cancer and its adjacent tissues;To investigate the difference of CDH1 promoter methylation in breast invasive ductal carcinoma tissues and adjacent tissues,Relationship between methylation of CDH1 gene promoter andgene expression,Clinical significance of methylation of CDH1 gene promoter in invasive ductal breast cancer.3)To elucidate the effects of CDH1 gene on cell biological behaviors such as proliferation and invasion of breast cancer cells（MCF-7,SK-BR-3）,and to investigate the regulatory mechanism of CDH1 gene in breast cancer.Methods:1)the expression of E-cadherin protein in invasive ductal carcinoma（IDC）were detected by immunohistochemical（IHC）method in 450 cases of breast,analyze the relationship between the expression of E-cadherin protein and clinicopathological features,molecular classification,lymph node metastasis;the relationship between the expression of E-cadherin protein and prognosis of invasive ductal carcinoma of breast was analyzed by Kaplan-Meier method.2)Methylation and protein expression of CDH1 gene in breast invasive ductal carcinoma tissues and adjacent normal tissues were studied by methylationspecific polymerase chain reaction（MSP）and immunohistochemistry.Collect the relevant clinical and pathological features（genetic background,age,tumorsize,axillary lymph node metastasis,tumorgrade and clinicalstage andmolecular subtypes）,using x² test,Logistic regression and COX regression model to analyze thesignificance of methylation of CDH1 gene promoter.3)The expression of CDH1 gene and protein in breast cancer cell line was analyzed by fluorescence quantitative PCR and Western blot.Silencing the CDH1 gene,observed the cell proliferation,cell cycle and invasion of MCF-7 and SK-BR-3 cell lines,and further study the regulation mechanism of CDH1gene in breast cancer cell lines.Results:1)E-cadherin protein washighly expressed in paracancerous tissues,and it was low expression in breast cancer tissues,thehigh expression of E-cadherin protein ratio was 49.04%（77/157）in 157 cases without lymph node metastasis,and the high expression of E-cadherin protein ratio was 29.69%（87/293）in 293 cases with lymph node metastasis,the two groups had significant difference（x²=10.528,P<0.001）.E-cadherin immunohistochemical staining,There were significant differences in lymph node metastasis,age,tumor size,histological grading of tumor cells,ER expression and molecular typing inhigh expression group and low expression group（P<0.05）.The low expression of E-cadherin is related to metastasis lymph node metastasis.E-cadherin proteinhigh expression group and low expression group in lymph node metastasis group and Triple negative breast cancer group,survival analysis had statistical difference（x²=9.546 P=0.002,x²=4.480 P=0.03）,but in lymph node metastasis group,Luminal A,Luminal B,HER-2 positive group,there was no significant difference in prognosis（P>0.05）.2)in 150 cases of breast cancer were found in 68 cases of CDH1gene promoter methylation,the methylation rate was 45.3%（68/150）,compared with the non methylation group,the expression of E-cadherin protein in the CDH1 promoter methylation group was significantly lower than that in the non methylation group,and the difference between the two groups was statistically significant(x²⁼12.277,P<0.001).Univariate analysis showed that methylation of CDH1 gene in axillary lymph node metastasis group compared with non methylation group(x²⁼8.392,P=0.015),histological grade（x²=6.952,P=0.031）and CerbB-2 expression（x²=5.334,P=0.021）and molecular typing(x²⁼15.050.P=0.002)there were significant differences.COX regression analysis showed that there was astatistically significant difference in the 5 years survival rate between CDH1 promoter methylation and non methylation group（P<0.001）.3)CDH1gene silencing in MCF-7 and SK-BR-3 cells,the cell proliferation rate wassignificantly faster than the control group,the difference was statistically significant（P<0.05）;Knockdown of CDH1 gene could significantly enhance the migration and invasion ability of MCF-7 and SK-BR-3 cells.After CDH1 suppression,the expression of E-cadherin was decreased,while the expression of beta-catenin in MCF-7 CDH1-siRNA and SK-BR-3CDH1-siRNA cells was significantly increased compared with the SK-BR-3 and MCF-7cells control group,and the difference was statistically significant（P<0.05）.Conclusion:1)Low expression of E-cadherin is associated with axillary lymph node metastasis of invasive ductal carcinoma of the breast,Follow up analysis of 450 breast cancer cases revealed that The prognosis of E-cadherin low expression group is worse than that of high expression group in Triple negative breast cancer and axillary lymph node metastasis groups,the difference was Significance between the two groups.These results suggest that E-cadherin protein expression can be used as prognostic marker for lymph node metastasis and Triple negative breast cancer patients.2)Methylation of CDH1 gene promoter is one of the reasons for the decrease in expression of mRNA,methylation of CDH1 gene promoter is associated with axillary lymph node metastasis in breast cancer,suggesting that methylation of CDH1 promoter may play a role in the progression of breast cancer.3)After CDH1 gene silencing,the proliferation of MCF-7 cells and SK-BR-3 cells enhanced;cell cycle accelerated;cell migration and invasion increased,which suggested that CDH1played a role in the progression of breast cancer disease process.4)In the study of the regulation mechanism of CDH1 gene in breast cancer cells,we found that inhibition of CDH1 expression enhanced the expression of beta-catenin.CDH1 regulates the proliferation and invasion of breast cancer cells by regulating the beta-catenin to activate the Wntsignaling pathway.