The Effect Of AKT1 On Dairy Cow Mammary Epithelial Cells Using DNA Methylation-Directed Gene Editing Technology

DNA methylation usually occurs in the promoter region of genes and regulates gene expression mainly by mediating gene silencing.DNA methylation plays an important role in cell differentiation,embryonic development and the development of diseases.At present,most of the research on DNA methylation focuses on the relevance of cancer.With the development of the research on the lactation biology,epigenetic modification,especially the regulation of DNA methylation on lactation function has attracted more and more attention,but there are few studies in this respect.The current methods of methylation modification research is mainly to construct methyltransferase(DNMT)overexpression vectors or add 5-AZA,which is methyltransferase inhibitor,to change methylation level,but these methods are all for the whole genome.It is impossible to achieve methylation regulation at specific sites of specific genes.In recent years,some researchers constructed a method of target methylation modification based on the CRISPR-dCas9 technology to achieve specific sites of target genes.The method introduces a new research method for the epigenetic research of lactation regulation.In this experiment,dairy cow mammary epithelial cells(DCMECs)were used as the experimental model.Based on the CRISPR-dCas9 technology,6 sgRNAs were designed targeting AKT1 gene.We construct 6 dcas9-mediated targeted methylation plasmids and then transfected them to dairy cow mammary epithelial cells using liposomes.The methylation level of AKT1 gene promoter in dairy cow epithelial cells was detected by bisulfite sequencing(BSP).The mRNA expression of AKT,mTOR,S6K1,STAT5 and and the gene related to milk protein,milk fat,lactose in dairy cow mammary epithelial cells were detected by qRT-PCR.The protein expression of AKT1,mTOR,S6K1,STAT5 and the gene related to milk protein,milk fat,and lactose in dairy cow epithelial cells were detected by Western Blotting.The secretion of triglycerides in dairy cow mammary epithelial cells was detected by triglyceride detection kit.The secretion of lactose in dairy cow mammary epithelial cells was detected by ELISA kit.The effect of AKT1 promoter methylation on the proliferation of dairy cow mammary epithelial cells was detected by MTT.The experimental results are as follows: compared with the control group,the sgRNA expression vectors carrying sgRNA1,sgRNA2,sgRNA3,sgRNA4 and sgRNA5 all have methylation effect on the promoter of AKT1 gene.The methylation efficiency of the sgRNA expression vector carrying with sgRNA1,sgRNA3 and sgRNA4 is relatively higher,and the methylation efficiency of pdcas9-DNMT3A-EGFP-sgRNA4 is the highest,which reach 25.63%.At the same time,we transfected the sgRNA expression vectors together which carrying sgRNA1,sgRNA3 and sgRNA4 respectively and found that when the sgRNA expression vectors carrying sgRNA1,sgRNA3 and sgRNA4 were co-transfected,the methylation efficiency reached 35.61%.We then studied the effect of AKT1 promoter methylation on dairy cow mammary epithelial cells by transfecting with sgRNA4 expression vector alone and co-transfecting with expression vectors carrying with sgRNA1,sgRNA3,sgRNA4.The experimental results showed that comparing with the control group,after the methylation modification of the AKT1 promoter with the sgRNA4 expression vector,the level of mRNA of AKT1,mTOR,SREBP1,GLUT1 and CSN2 were significantly reduced,while the mRNA expression of S6K1 and STAT5 have no changes.The protein expression of AKT1,p-AKT1,mTOR,p-mTOR,CSN2,SREBP1 and GLUT1 also all significantly reduced,the protein expression of STAT5 has no obvious change.In contrast,the protein expression of S6K1 and p-S6K1 significantly reduced,and the protein expression of p-STAT5 also significantly reduced.At the same time,comparing with the control group,after the AKT1 promoter methylation modification guided by sgRNA4,the content of triglycerides and lactose in dairy cow mammary epithelial cells were also significantly reduced,and the proliferation of dairy cow mammary epithelial cells was also inhibited.Similar to the results obtained by the sgRNA4 expression vector group,the sgRNA expression vector co-transfection group(sgRNA1+3+4 group)has a more obvious reduction effect on the expression of the AKT1 gene and the lactation-related functional genes,and comparing with the control group,the protein expression of CSN2,SREBP1 and GLUT1 in the sgRNA1+3+4 group extremely significantly reduced.The content of triglycerides and lactose in dairy cow mammary epithelial cells also extremely significantly decreased.The proliferation of dairy cow mammary epithelial cells was also more significantly inhibited.In summary,comparing with the sgRNA4 group alone,when the expression vectors carrying sgRNA1,sgRNA3 and sgRNA4 were co-transfected,the methylation level of the AKT1 gene promoter increased more significantly,and the effect on dairy cow mammary epithelial cells was also more significant.In summary,the experimental results showed that by introducing dCas9 specific site-directed methylation technology,this study successfully constructed and screened efficient methylation modified vectors—pdcas9-DNMT3A-EGFP-sgRNA for the methylation level modification of AKT1 promoter.The results of the study on the effect of the AKT1 promoter methylation modification by these vectors on dairy cow mammary epithelial cells showed that after the AKT1 promoter methylation level increased,the levels of the milk protein,milk fat and lactose secreted by dairy cow mammary epithelial cells decreased,the proliferation of dairy cow mammary epithelial cells was inhibited,and the expression levels of lactation-related proteins basically significantly reduced.It is proved that AKT1 is a key regulator of epigenetic factors to regulate the development and lactation of dairy cows’ mammary glands.