Collaborative Regulation Of Milk Protein Synthesis Between JAK2-STAT5and MTOR Signalling Pathways In Bovine Mammary Epithelial Cells

This study with milk protein synthesis of signaling pathways acted as the breakthroughpoint, chooseing STAT5gene is necessary for milk protein gene expression of JAK-STAT5,and AKT1and mTOR in the PI3K-AKT-mTOR signalling pathway, which is closely relatedwith milk protein translation, then analysis the interaction between them. To reveal theregulation of milk synthesis and signal coordination mechanism in the process of milkprotein in dairy cow mammary epithelial.This study selected Chinese Holstein cows as the research object, it cultured dairy cowmammary epithelial cells in vitro (DCMEC), serum-free medium DIP (hydrocortisone, nsulin,prolactin hormone), in which the addition of insulin-like growth factor-1(IGF-1) and growthhormone (GH), by using RT-PCR and Western blotting, it tested the effects of AKT1, STAT5and mTOR and their phosphorylated form. The RNA interference was used to knock downAKT1and STAT5gene, the expression of AKT1, STAT5, mTOR and their phosphorylationform in the mRNA and protein levels were subsequently examined, and it affected β-caseinexpression capacity.According to the above experiments, the addition of IGF-1, GH, promoted cellproliferation and cell viability. RT-PCR and Western blotting experimental results showedthat with time goes by, the expression of AKT1and mTOR and its phosphorylated form inthe mRNA and protein levels had an increasing trend, When grown to24h, the changes inexpression levels incresed higher than before treatment (P <0.05), IGF-1, GH groupcompared with the control group, the expression level of AKT1and mTOR and itsphosphorylated form was also increase significantly (P <0.05). During this time, but theexpression of STAT5, p-STAT5were relatively complex, tended to increase the amount ofexpression within24h. In short, IGF-1and GH had a promoting effect. Gene silenceexperimental was used to knock down AKT1or STAT5, the mRNA level of AKT1, STAT5and mTOR were decreased (P <0.05) and its protein level of p-STAT5, p-AKT1and p-mTOR protein also significant with reducing (P <0.05). Add addition of IGF-1, GH to control group,the expressions of these β-casein mRNA were significantly increased (P <0.05), but thecorresponding interference group compared with the control group, AKT1, STAT5andmTOR and its phosphorylated form was significantly decreased (P <0.05), the mRNA level ofβ-casein was remarkably decreased (P<0.05), cell proliferation and viability of cells werereduced significantly (P<0.05).In summary, in the dairy cow mammary epithelial cells, AKT/mTOR and JAK2/STAT5signalling pathways that affect the synthesis of β-casein. Both at the mRNA and protein level,when the negative regulation of AKT1or STAT5, the one will change as the other change,and regulate its downstream mTOR signalling pathway, which inhibit the expression of β-casein mRNA and cell proliferation. IGF-1or GH can enhance AKT/mTOR and theJAK2/STAT5, thus boost the expression β-casein mRNA.