The Role Of DNA Methylation In CDH1 Gene Promoter Region On Pancreatic Cancer Cells

Pancreatic cancer is a malignant tumor of the digestive system,characterized by high malignancy,difficulty in early diagnosis,poor prognosis,and lack of effective treatment.DNA methylation,as one of the major epigenetic modifications,is closely related to the occurrence and development of cancer.Therefore,identification the aberrant DNA methylation in tumor cell and modification accordingly,may provide a new therapeutic approach for cancer diagnosis and treatment.CDH1(Cadherin 1)gene,located on human chromosome 16q22,encodes E-cadherin which is involved in regulating cell adhesion,migration and epithelial cell proliferation.Mutations of CDH1 gene are closely related to gastric cancer,breast cancer,colorectal cancer,thyroid cancer and ovarian cancer.Studies have shown that DNA methylation plays a role in the expression regulation of CDH1 gene,but the role is different depend on tumor types.In this study,gene expression and promoter DNA methylation levels of CDH1 in different pancreatic cancer cells were detected.By transfecting of CRISPR/dCas9-Dnmt3 a and gRNA-CDH1 vectors,the effects ofDNA methylation on CDH1 gene expression and cell malignant phenotype were examined,and the potential application of specific DNA methylation modification in tumor treatment was discussed.1.Effects of DNA methylation on gene expression in pancreatic cancerTumor related genes EGFR,HOXB5,CDH1,DDR2 and RUNX3 were screened according to published papers.In order to detect the effect of DNA methylation in the promoter region on gene expression,real-time quantitative PCR was used to detect gene mRNA expression,and bisulfite-sequencing PCR(BSP)was carried out to analyze the methylation status of the CpGs in the promoter region of the above gene,and the two types of pancreatic cancer cells PANC-1 and CFPAC-1 were compared with normal pancreatic epithelial cells HPC-Y5.The results showed that EGFR gene expression was negatively correlated with DNA methylation in PANC-1 cells,but not in CFPAC-1 cells.The expressions of HOXB5,DDR2 and RUNX3 were not correlated with the DNA methylation of the detected region.CDH1 gene expression were significantly differences between tumor cells and normal cells,and its expression were negatively correlated with DNA methylation,therefore CDH1 gene was selected for further analysis.2.The role of DNA methylation modification in CDH1 gene promoter regionTo investigate the role of DNA methylation in the promoter region of CDH1 gene,the vectors CRISPR/dCas9-Dnmt3 a and gRNA-CDH1 were co-transfected with PANC-1 and CFPAC-1 cells,respectively,and the stable transfected cells were screened by flow cytometry.Results showed that in stable transfected cells,DNAmethylation in CDH1 promoter region was significantly increased,while mRNA expression was significantly decreased.Along with the increased DNA methylation,the malignant phenotype of PANC-1 and CFPAC-1 cells were significantly inhibited,such as proliferation,invasion,migration and clone formation ability.Subcutaneous injected the stable transfected PANC-1 and CFPAC-1 cells into nude mice,the results showed that the rate of tumor formation,tumor volume and tumor weight in vivo were inhibited.In summary,the expression of CDH1 mRNA was negatively correlated with DNA methylation level in the promoter region in pancreatic cancer cells PANC-1 ?CFPAC-1.DNA methylation in CDH1 promoter region was significantly increased by CRISPR/dCas9-Dnmt3 a,and the expression of CDH1 mRNA was significantly reduced accordingly,in the meantime,the proliferation,invasion,migration,clone formation ability and tumorigenic ability in vivo of PANC-1 and CFPAC-1 cells were significantly inhibited.This study explored the potential targets for early diagnosis and treatment of pancreatic cancer,and provided a research basis for tumor treatment by special modification the DNA methylation level of gene loci.