Effects Of MiR-24-3p On Cell Proliferation And Milk Protein Synthesis Of Mammary Epithelial Cells In Dairy Cattle Through Cooperatively Acting With MEN1 Gene

Understanding the development process of mammary gland and improving the milk yield and quality is the key purpose of geneticists and breeders in dairy cattle field.Previous studies in our laboratory have shown that the expression level of MEN1/menin play s an important role in the proliferation of mammary gland epithelial cells and the synthesis of milk protein,but the up-steam regulating factor of MEN1/menin expression is not known at present.It was predicted that mi R24-3p could interact with the 3’UTR of MEN1 by using Targetscan software,therefore regulate its expression level.This study was designed to investigate whether miR-24-3p modulates the expression of MEN1 and explain its function in mammary epithelial cells.In this study,we found miR24-3p expression change can modulate the proliferationof mammary epithelial cells and milk protein synthesisthrough the two known important signaling pathways PI3K/AKT/m TOR and JAK2/STAT5.The study is of great significance to the further research of milk protein synthesis in mammary glands of dairy cows.The main results are as follows:（1）Target Scan6.2 and RNAhybrid2.2 software to screen out miR-24-3 that regulated MEN1 gene expression,Co transfection of recombinant plasmid pMIR-REPORT-bMEN1-3’UTR and bta-miR-24-3p mimics/inhibitor in MAC-T cells revealed that bta-miR-24-3p could specifically target MEN1 gene mRNA 3’UTR.（2）Transient transfection of bta-miR-24-3p mimics/inhibitor was found that the expression of 24h was the best in all the two groups after transfection,and can negatively regulate the expression of MEN1 gene;transient transfection of recombinant plasmid pEGFP-C2-bMEN1 and MEN1 gene-specificsiRNA was found that can positively regulate bta-miR-24-3p expression（3）The cell proliferation was detected by cell counting and CCK-8,which found that bta-miR-24-3p mimics could promote the proliferation of MAC-T cells,but bta-miR-24-3p inhibitor had no significant effect;The cell cycle was detected by flow cytometry,and it was found that bta-miR-24-3p mimics could promote MAC-T cell cycle from C₀/G₁ phase to S phase;qRT-PCR detection of cell cycle factor showed that CyclinD1 and CDK6 were significantly increased（P<0.01）,CDK4（P<0.01）and p27（P<0.05）significantly decreased,but no significant change were found in p18（P>0.05）;bta-mi R-24-3p inhibitor had no significant effect on cell cycle,qRT-PCR detection of cell cycle factor found that CyclinD1（P<0.05）,CDK6（P<0.01）significantly reduced,and CDK4,p18,p27 was not statistically significant（P>0.05）.（4）The detection of qRT-PCR found that bta-miR-24-3p mimics can significantly reduce the expression of genes related to milk protein synthesis in mRNA level,such as AKT（P<0.01）,mTOR（P<0.01）,4E-BP1（P<0.05）,STAT5（P<0.01）,and can reduce the expression of CSNK（P<0.05）,but the bta-mi R-24-3p inhibitor showed the opposite regulation.In summary,bta-mi R-24-3p can target MEN1 gene in MAC-T cells;bta-miR-24-3p can negatively regulate the expression of MEN1 gene in mammary epithelial cells,while bta-miR-24-3p is positively regulated by MEN1 gene;bta-miR-24-3p can increase the proliferation of MAC-T cells by promoting the transition of cell phase from G0/G1 to S.In addition,bta-miR-24-3p can negatively regulate the expression of genes which were involved in PI3K/AKT/mTOR and JAK2/STAT5 signaling pathways,and also genes involved in the synthesis of milk protein in mammary epithelial cells of dairy cows.