The Regulatory Mechanism Of METTL3 On Milk Protein And Milk Fat Synthesis In Bovine Mammary Epithelial Cells

The mechanism of milk protein and milk fat synthesis is one of the important basic theories in life science.In recent years,obvious progress on this research has been made.At present,it has been found that mTOR(the mammalian target of rapamycin)mainly regulates milk protein synthesis,whereas SREBP-1c promotes milk fat synthesis in bovine mammary epithelial cells(BMECs).But the detailed biochemical mechanism remains unclear.Studies have found that METTL3(Methyltransferase-like protein 3)controls the m6 A methylation levels of m RNA and plays an important role in the regulation of translation,but whether it can regulate milk protein and milk fat synthesis is still unknown.In this study,we investigated the regulatory role and molecular mechanism of METTL3 on milk protein and milk fat synthesis in BMECs.BMECs were cultured and purified by tissue culture method.The purity of these cells were identified by western blotting and immunofluorescence(IF)analysis of the expression of keratin 18(CK18)and CSN2.We detected the impact of METTL3 on milk protein and milk fat synthesis of BMECs after overexpression or inhibition of METTL3 by Western blotting analysis.The results showed that,the expressions of mTOR,Gly RS,CSN2,and SREBP-1c were increased or decreased,the expressions of p-mTOR and p-Gly RS were increased or decreased.Those results indicated that METTL3 could affect milk fat and protein synthesis by modulating the expression of related signaling pathway genes.We established estrogen(E)and methionine-stimulating BMEC models,and detected the expression and localization of METTL3 by IF observation.The results showed that,METTL3 are mainly distributed in the nucleus,and E and Met could promote METTL3 expression and localization in nucleus.The interaction between METTL3 and Gly RS in the BMECs were identified by co-immunoprecipitation(Co-IP).The results showed that,METTL3 binds to Gly RS in the nucleus.We further inhibited Gly RS with si RNA after Met or E stimulation,we also overexpressed Gly RS together with knocking down METTL3 with si RNA.The results showed that,the stimulation of Met and E on the expressions of mTOR,CSN2,SREBP-1c,and METTL3 and the level of p-mTOR were completely abolished after GlyRS was inhibited.The expression of mTOR,CSN2,SREBP-1c,and METTL3 and the level of p-mTOR were significantly increased after Gly RS was overexpressed,but when METTL3 was further inhibited,the stimulation of Gly RS overexpression on the expression of mTOR,CSN2,SREBP-1c,and METTL3 and the level of p-mTOR were partially diminished.These results reveal that METTL3 partially mediates Met or E-induced Gly RS-dependent mTOR and SREBP-1c activation for milk protein and fat synthesis.In summary,METTL3 was an important signal molecule.It can positively regulate milk protein and fat synthesis in BMECs.When Gly RS was stimulated by Met and E,it can promote the expressions of METTL3 and stimulate the mTOR and SREBP-1c pathways to trigger milk protein and fat synthesis in BMECs.