The Effect Of SND1Gene On Lactation Function Of Dairy Cow Mammary Epithelial Cells

Rate of low milk yield, milk fat and milk protein rate is not high mainly restrict the development of dairy industry in our country. How to improve the milk yield and milk quality become a problem we focus on. Methionine is the key substances to the synthesis of protein, previous studies mainly on methionine affect the production characteristics of ability, but in vitro dairy cow mammary gland epithelial cells, promote proliferation and lactation research is less. SND1is important gene transcription, but reports on SND1in the bovine mammary epithelial cells are rather less. In this research we aim to determine the function of SND1genes on milk synthesis, and reveal the important mechanism of this gene’s function in the synthesis of lactation, to build gene regulatory networks in cell signal transduction and regulation of lactating mammary gland.In this research we successfully constructed a model of the cow mammary gland epithelial cells in vitro, and determined the optimal dose of methionine (0.6mmoI/L), the best time was24h. CASY method was detected the cow mammary gland epithelial cells vitality and proliferation; Dairy cow mammary gland epithelial cell cycle distribution was detected by flow cytometry; Using the real-time fluorescent quantitative PCR we detected the expressions of SND1, β-casein and other related genes at mRNA expression level, and we also use Western blot to detect expression of lactation related proteins. After gene silencing and over-expression of SND1, we also detected the cell activity, proliferation ability, related gene mRNA expression and protein expression, we applicated immunofluorescence staining of cells to detect SND1, SREBP, STAT5and p-STAT5subcellular localization, to explore SND1role in lactation signaling pathways.The experimental results showed that the protein of SND1was located in the nucleus and cytoplasm of DCMECs, but mainly exist within the nucleus, only a small amount in the cell cytoplasm; SREBP mainly in the cytoplasm in dairy cow mammary gland epithelial cell; STAT5was mainly distributed in the cytoplasm, with a small amount distributed in the nucleus, p-STAT5mainly located in nucleus, less was distributed in the cytoplasm. After adding0.6mmol/L methionine, mRNA expression of P-casein and the related genes were increased significantly; Related lactation protein expressions were increased significantly by Western blot; CASY and flow cytometry analysis. SND1gene silencing significantly inhibited beta casein, STAT5and p-STAT5, mTOR, p-mTOR, SREBP, CyclinDl gene expression; also cell viability and cell proliferation; Overexpression of SND1promoted the expression of β-casein, STAT5, p-STAT5, mTOR, p-mTOR, SREBP, CyclinDl gene, also cell viability and cell proliferation. In summary, SNDl positively affects STAT5, mTOR and SREBP signaling pathway to promote milk protein and milk fat synthesis in the dairy cow mammary epithelial cells; SNDl also positively affects cell proliferation by stimulating the expression of CyclinDl; Methionine as a signal molecule promotes the expression of SND1, and also the expressions of STAT5, mTOR, and CyclinDl to promote milk protein synthesis and cell proliferation. This study provides an experimental basis for the research of SND1regulation on lactation, laying the theoretical foundation for future studies.