IRS1 Gene Regulates The Proliferation,Apoptosis And Casein Synthesis Of Mammary Epithelial Cells In Dairy Goats

China is one of the largest number of dairy goats in the world,but the low level of milk protein affects the quality of dairy products.The low level of milk protein has become a bottleneck problem restricting the development of dairy goat industry.Therefore,it’s an important scientific problem to explore the molecular mechanism of milk protein synthesis and build a precise and rapid method,which improve the milk quality of dairy goat in the basic research field of dairy goat.In order to further study the mechanism of IRS1 regulating the development of mammary gland and milk protein synthesis,si RNA and NT-157 were used to treat mammary epithelial cells.The effects of IRS1 on the proliferation,apoptosis and casein synthesis of milk goat mammary epithelial cells(GMECs)were studied by RT-q PCR and Western blotting.This research clarifies the regulatory effect of IRS1 on milk goat mammary epithelial cells and provides an experimental basis for milk regulation.Mainly as follows:1.Effects of interfering IRS1 on the proliferation and apoptosis of GMECs The results showed that down-regulation of IRS1 using si RNA significantly inhibited the proliferation and promoted the apoptosis of GMECs(P<0.05).At the same time,it was found that IRS1 interference could promote GMECs apoptosis by increasing Bax protein level and reducing caspase 3 and BCL2 protein level.2.Effects of NT-157 on the proliferation and apoptosis of GMECs After treatment with NT-157(a selective inhibitor of IRS1 that degrades IRS1 expression),the results showed that NT-157 significantly inhibited GMECs proliferation and promoted GMECs apoptosis(P<0.05).In addition,the protein expression levels of some genes related to apoptosis were analyzed.It was found that NT-157 promoted GMECs apoptosis by increasing caspase 3 protein level.3.Effects of IRS1 on casein synthesis in GMECs The expressions of β-casein,as-1 and κ-casein in GMECs were detected by ELISA Kit after IRS1 interference and the IRS1 inhibitor NT-157 treatment.The results showed that siIRS1 significantly reduced the synthesis of β-casein and significantly increased the synthesis of as-1 and κ casein in GMECs(P<0.05).However,NT-157,significantly promoted the synthesis of as-1 and κ-casein in GMECs(P<0.05),but had no significant effect on the synthesis of β-casein.The results showed that IRS1 could significantly improve the synthesis of as-1 and κ-casein casein in GMECs.Western blot was used to detect the protein expressions and phosphorylation levels of key genes in LKB1/AMPK/mTOR pathway.The results showed that IRS1 activated LKB1 /AMPK/mTOR signal pathway by phosphorylating LKB1,AMPK and mTOR to promote the synthesis of as-1 and κ-casein in GMECs.In conclusion,siIRS1 and NT-157 both significantly inhibited the proliferation and apoptosis of GMECs;siIRS1 significantly increased the synthesis of as-1 and κ-casein and reduced the synthesis of β-casein in GMECs;NT-157 significantly promoted the synthesis of as-1 and κ-casein,but had no significant effect on the synthesis of β-casein in GMECs.IRS1 promoted the synthesis of as-1 and κ-casein by promoting the phosphorylation of LKB1,AMPK and mTOR and activating the LKB1/AMPK/mTOR signaling pathway in GMECs.