The Role Of DNA Methylation Modification In Promoter Region Of DDR2 Gene On Breast Cancer Cells

DNA methylation is an important epigenetic modification,which is involved in many biological processes,such as cell differentiation,early embryonic development,genomic imprinting,X chromosome inactivation and tumorigenesis.Aberrant DNA methylation causes genes dysfunction,and is closely related to the occurrence and development of tumor.Therefore,identifying the aberrant DNA methylation in tumor cell,modifying accordingly may provide a new therapeutic approach for cancer diagnosis and treatment.In this study,RNA-seq data were used to screen and verify candidate genes related to breast cancer,then the correlation between candidate genes and DNA methylation were detected,and the effects of DNA methylation editing in DDR2 gene promoter region by CRISPR/dCas9 on the gene expression and the malignant phenotype of cancer cells were discussed.1.The regulation of gene expression by DNA methylation in different breast cancer cell linesIn this study,RNA-seq data from NCBI database were used to analyze the differential expression genes between breast cancer cell lines MCF7,HS578 T and breast epithelial cell line MCF10 A,and 62 common differential genes were obtained.After retrieved the existing literature,10 of these genes related to breast cancer were further verified by qPCR in MCF7 and HS578 T cells,results showed that the expression trend was consistent with RNA-seq data.Among the 10 selected genes,previous data shown that DDR2 plays an important role in the migration and invasion of breast cancer.In this study,the expression of DDR2 gene was further examined in different breast cancer cell lines MCF7,HS578 T and BCAP37,results showed that the expression of DDR2 mRNA and protein was significantly higher in three breast cancer cells than that in breast epithelial cells,and the highest expression was ovserved in the triple negative breast cancer cell lines HS578 T.The DNA methylation level in the promoter region of DDR2 gene was detected by bisulfite sequencing,results showed that the methylation level of DDR2 gene was significantly lower in three breast cancer cell lines than that in breast epithelial cells,with the most differences in HS578 T cell line.These results suggest that DDR2 gene is highly expressed in breast cancer cell lines,and its expression is positive correlation with the DNA methylation level in its promoter region.2.Effect of CRISPR/dCas9-mediated DNA methylation editing on DDR2 gene expression and tumor malignant phenotypeIn order to investigate the regulation of DNA methylation on the expression ofDDR2 gene,CRISPR/dCas9 technique was used to edit the DNA methylation in the promoter region of DDR2 gene.The results showed that the methylation level of DDR2 gene was significantly increased in all breast cancer cell lines after transfected with gRNA-DDR2 and dCas9-Dnmt3 a,and the expression of DDR2 mRNA was significantly decreased.The most significant change were observed in the triple negative breast cancer cell line HS578 T,both for DNA methylation and gene expression inhibition.The effect of decreased expression of DDR2 gene on malignant phenotype of breast cancer cells was further detected.The results showed that the proliferation,migration,invasion and cloning ability of cancer cells were significantly decreased,and the most significant inhibition effect were observed in HS578 T cells.Subcutaneous injected the transfected BCAP37 cells into nude mice,the results showed that the volume and the mass of tumor formed were decreased significantly,the density of tumor tissue were decreased,the expression of DDR2 protein were decreased significantly.The effect of decreased DDR2 expression on other differentially expressed genes screened by RNA-seq data analysis were also detected in HS578 T cells.The results showed that after transfected,the expression of oncogenes ERBB4,KIT,MAP4K3 and CDK14 were decreased significantly,and the expression of tumor suppressor genes BRCA2 and APC were increased significantly.The expression of matrix metalloproteinases MMP1 and MMP8 related to distant metastasis of cancer were decreased significantly.In conclusion,DDR2 was highly expressed in all breast cancer cell lines detected,and the expression was negatively correlated with DNA methylation.CRISPR/dCas9 mediated DNA methylation editing can significantly increase DNA methylation level and inhibit gene expression,accordingly,the malignant phenotype of cancer cells,such as proliferation,invasion,migration,clone formation and tumorigenesis ability,were significantly inhibited.These results implied that DDR2 can be used as a potential target for diagnosis and treatment of breast cancer,and this study provides a research basis for tumor therapy by CRISPR/dCas9-mediated specific site DNA methylation editing.