Effect Of DNA Methylation Level On Expression Related To Lactating Function Genes In Bovine Mammary Epithelial Cell

Milk is very important for the development of newborn mammals.It is the only ideal food that provides nutrition and immune defense factors before they can eat and digest solid food.Milk is also the main raw material of dairy products.Therefore,improving the yield and quality of milk has become one of the key purposes of lactation biology.The process of mammary lactation is regulated by both nerves and hormones.In terms of mammary development,lactation related genes and their signaling pathways and epigenetic regulation mechanism become more and more concerned with the development of lactation biology.As one of the epigenetic regulatory mechanism,DNA methylation is applied in the genetics and breeding experiments of agriculture and animal husbandry because of its controllable and heritable characteristics.In recent years,it has been found that the level of DNA methylation is related to the regulation of bovine milk quality,but its mechanism is still under exploration.The purpose of this study is to establish the model of DNA demethylation status of bovine mammary epithelial cells and the model of DNA methylation status of bovine mammary epithelial cells with the primary cultured bovine mammary epithelial cells(BMECs)to explore how DNA methylation level affect the function of BMECs and its mechanism.A methylation inhibitor 5-Aza was used to establish on DNA demethylation BMEC model.Then,the effect of demethylation reagent 5-Aza on the level of DNA methylation in BMECs was detected by cell viability test and methylation restriction enzyme cutting method.The results showed that 5-Aza could reduce the level of DNA methylation in BMECs genome.The overexpression of methyltransferase 3 alpha(DNMT3A)was used to establish the DNA methylation BMEC model.The expression of DNMT3 A mRNA in the BMECs increased significantly after the transfection of DNMT3 A overexpressed plasmid by the fluorescence quantitative detection technique.Then the promoter region of AKT1 gene was screened by the double luciferase reporter gene system,and the specificity of the methylation of the CpG site in the upstream of the AKT1 promoter region in different model cells was analyzed by bisulfite sequencing.The results showed that the methylation level of DNA promoter region decreased significantly in the DNA demethylation BMEC model compared with the blank control group,and the methylation level in the DNA promoter region of the DNA methylation BMEC model increased significantly.Further verify the establishment of DNA methylation / demethylation status BMEC model.The expression of mRNA level of lactating genes AKT1,RPS6KB1,STAT5 A,mTOR,CyclinD1 in DNA demethylation BMEC model,DNA methylation BMEC model and blank control group were detected by qRT-PCR technique.The changes in expression of above genes protein and their related phosphorylated protein in the DNA methylation BMEC model,DNA methylation BMEC model and blank control group were detected by Western blot technique.In the demethylated BMEC model compared with the blank control group,the results showed that the expression of proliferation of mammary gland related gene AKT1,CyclinD1 and their phosphorylated proteins and the expression of the milk quality related gene RPS6KB1,STAT5 A,mTOR and their phosphorylated protein were significantly increased.However,the expression of these proteins and their phosphorylated proteins in the DNA methylation BMEC model was significantly inhibited.Western blot was used to analyze the effect of DNA methylation level on BMECs synthesis of beta casein(CSN2),and the results showed that the level of DNA methylation in BMECs was negatively correlated with the expression of CSN2.The effects of methylation level on BMECs synthetic lactose and milk fat were detected by lactose and milk fat test kits.The results showed that the higher the level of DNA methylation in BMECs,the lower the lactose milk fat synthesis.The above results showed that the level of DNA methylation has an effect on the lactating function of BMECs.DNA methylation may regulate the lactating function of BMECs by regulating the expression of lactation related genes.