Effect Of Down Regulating SUA39H1/2 On The Proliferation And STAT5a Expression Of Buffalo Mammary Epithelial Cells

Mammary gland is one of the corresponding periodic changes in mammalian age,estrus cycle and reproductive cycle,and its development and lactation are regulated by multiple factors.STAT5a,as a specific transcription factor of mammary glands,plays an important role in the regulation of mammary development and lactation.The formation of tri-methylation(H3K9me3)in lysine 9 of histone 3 that was catalyzed by the specific histone methyltransferase SUV39H1/2 has an important role in promoting chromatin concentration to form heterochromatin and regulating gene expression.In order to understand the role of SUV39H1/2 gene in mammary development and lactation,this study uses RNA interference to investigate the effect of down regulation of SUV39H1/2 gene expression on the growth of buffalo mammary epithelial cells,mammary development,lactation related genes and STAT5a expression,in order to clarify the function of H3K9me3 in the regulation of the development and lactation of the mammary glands of buffalo.The results of the study are as follows:1.Effects of 1.SUV39H1/2 on proliferation of buffalo mammary epithelial cells and expression of lactation related genesFirst,SUV39H1/2-shRNA lentivirus was used in 293T cell packaging to reduce the expression of SUV39H1/2 gene in the breast epithelial cells of Guangxi local buffalo through lentivirus.Compared with the control group,the experimental group(down regulation of SUV39H1/2 gene expression)could promote the proliferation and growth of mammary epithelial cells,and the flow cytometry was used to detect the cell cycle.It was found that the cell cycle of the experimental group was blocked in the S phase.The proportion of G0/G1 phase cells was not significantly different,and the proportion of G2 phase was significantly reduced(P<0.05).QRT-PCR results showed that the inhibition of SUV39H1/2 gene expression could significantly reduce the differentiation of mammary epithelial cells and the expression of SREBP1,FABP3,STATSa,mTOR,ELF5,CSN1S1 and milk fat synthesis related genes(P<0.05).2.Inhibition of 2.SUV39H1/2 gene expression on methylation level of STAT5a gene promoter in Buffalo mammary epithelial cellsFirst,the 2190 BP,355 BP,417 BP fragments of different length buffalo promoter region were cloned.The results of the double fluoro phosphor reporter gene system detection showed that the three fragments all had promoter activity and 2190 bp fragment activity was significantly higher than the other two bands(P<0.05),and the 2190 BP promoter was found through the analysis of the transcription factor binding site.The 2190 BP promoter was found.There are multiple transcription and binding sites,including SP1,P300,p53 and NF-kB.Secondly,the methylation degree of STAT5a promoter in the untreated group was 15.5%,and the degree of methylation in the STAT5a promoter region of the treated group was 31.5%after the treatment group was down regulated by lentivirus infection.These results suggest that inhibition of SUV39H1/2 gene expression can promote the.proliferation of buffalo mammary epithelial cells,and SUV39H1/2 gene may regulate the growth and differentiation of mammary epithelial cells,milk and milk protein synthesis related gene expression by regulating the level of STAT5a promoter methylation.