CRISPR-dCas9-mediated DNA Methylation Editing In Lung Cancer

CRISPR-dCas9 which derived from the CRISPR-Cas9 system,has been used in many fields such as gene regulation,epigenetic regulation,and high-throughput screening.DCas9 lacks nuclease activity but maintains its ability to bind both the sgRNA and targeted DNA,dCas9 can guide intermolecular interactions by being fused to various regulating molecules.In this study,CRISPR-dCas9 system was used to target demethylation of CDH1 gene promoter region in lung cancer H460 cell,then the changes of DNA methylation,the effect of demethylation on CDH1 gene expression and the malignant phenotype of lung cancer were detected.In addition,DNA demethylating agent 5-Aza-2?-deoxycytidine(5-Aza-dC)was used to compare the effect of modification.In this study,the RNA-seq data of lung epithelial cell HBE and non-small cell lung cancer cell H460 were downloaded from NCBI database,with a p value of 0.0001,total of 18007 differentially expressed genes were obtained.After analyzed by KEGG pathway analysis and GSEA gene set enrichment analysis,CDH1 genes were selected for further study,which is associated with cancer EMT pathways,migration and drug resistance.The expression of CDH1 gene and the DNA methylation in the promoter region were detected by real-time quantitative PCR(qPCR)and Bisulfite PCR sequencing,respectively.The results showed that the expression of CDH1 gene was significantly lower in H460 cells than that in HBE cells,while the DNA methylation was higher in H460 cells.This indicates that there is negative correlation between the expression of CDH1 gene and the DNA methylation in the promoter region.To further verify the correlation between CDH1 gene expression and promoter methylation,CRISPR-dCas9-Tet1 were used to demethylate the promoter region of CDH1 gene,then the gene expression and the malignant phenotype of lung cancer cells H460 were examined.Results showed that the transfection of dCas9-Tet1 significantly decreased the methylation level of CDH1 gene in the promoter region,the mRNA expression of CDH1 gene was significantly increased,and the protein expression was also increased.The malignant phenotype of H460 cells was significantly inhibited,and the proliferation,migration,invasion and in vitro tumorigenic ability of the cells were decreased,the cell apoptosis was significantly increased.In addition,DNA demethylase inhibitor 5-Aza-dC were used to treat H460 cells with 40 ?M,5-Aza-dC significantly decreased the promoter methylation and increased the mRNA expression of CDH1 gene,meanwhile cell proliferation and migration ability were inhibited.The results showed that CRISPR-dCas9-mediated demethylation on tumors has the same effect as clinical drugs 5-Aza-dC.In summary,the CDH1 gene is low expressed in lung cancer cell H460,and the expression level is negatively correlated with DNA methylation;CRISPR-dCas9-Tet1-mediated gene editing technology can target DNA methylation modification,reduce DNA methylation in CDH1 promoter region,promote gene expression,and inhibit tumor malignant phenotype,and has similar effects as broad-spectrum demethylation drugs.This study provides a basis for the study of tumor therapy by CRISPR/d-Cas9-mediated specific site DNA methylation editing.