The Role Of GlyRS And Its Downstream Proteins NF?B1 And METTL3 On Autophagy Of Bovine Mammary Epithelial Cells

Bovine mammary epithelial cells(BMECs)are the basic units of milk protein and milk fat synthesis.During the regulation of breast lactation,the proliferation,apoptosis and autophagy of BMECs are all related to milk synthesis,but the detailed mechanism remains.For further study.Previous laboratory results showed that glycylated tRNA synthetase(GlyRS)regulates the function of milk synthesis in BMECs,GlyRS in the nucleus in the form of p-GlyRS and its downstream proteins nuclear transcription factor?B1(NF?B1)and m6 A methyltransferase(METTL3)Binding,further regulating the downstream target protein mTOR and further promote milk synthesis,studies have shown that mTOR is associated with regulatory cell autophagy,but whether the above three proteins regulate autophagy has not been reported.This study was to investigate whether GlyRS and its downstream proteins NF?B1 and METTL3 regulate BMECs autophagy.Previous studies have shown that p-GlyRS can activate NF?B1 to generate p-NF?B1,further intends to reveal whether p-GlyRS phosphorylates METTL3 and activates the molecular mechanism of METTL3.This study will help reveal the molecular mechanism of milk protein and milk fat regulation.Provides experimental data for milk synthesis regulation.In this study,purified BMECs were obtained using a tissue block method.The expression of CK18(Cytokeratin 18)and ?-casein in BMECs was identified by immunofluorescence staining and Western blot to identify cell purity.Transfected si-GlyRS,si-METTL3,si-NF?B1 and transfected NF?B1 overexpression plasmid,added Tre autophagy enhancer and methionine(Met),and detected the expression of autocrine marker protein LC3-II by Western blot.Amount;The above treatment groups were transfected with pCMV-C-EGFP-LC3 B plasmid at the same time,and the change of autophagic spot(LC3-II)was detected by immunofluorescence.Western blot was used to detect the interaction between p-GlyRS and METTL3 by co-immunoprecipitation(Co-IP).Prokaryotic expression and purification of GST-METTL3 was performed as a substrate for kinase reaction with p-GlyRS collected by Co-IP.The pCMV-C-Flag-GlyRS recombinant plasmid was transfected into cells,and the nuclear protein was purified.The purified plasmid p-GlyRS protein and the prokaryotic expressed GST-GlyRS were purified using the Flag purification kit and the phosphorylation kit.An in vitro kinase reaction was performed with GST-METTL3 to determine if there was phosphorylation of METTL3 protein.Western blot and immunofluorescence staining showed that interference with GlyRS gene,inhibited the expression of p-mTOR,promoted autophagy of BMECs;added Tre and Met and interfered with GlyRS gene expression,inhibited autophagy of BMECs.Interfering with NF?B1gene positively regulates autophagy of BMECs.After overexpression of NF?B1 and Tre addition,NF?B1 overexpression can reduce Tre-induced autophagy.Interference with METTL3 gene inhibits the expression of p-mTOR and promotes autophagy.These results indicate that GlyRS and its downstream proteins NF?B1 and METTL3 inhibit autophagy in BMECs.The in vitro kinase assay results showed that both the p-GlyRS collected by Co-IP and the purified p-GlyRS of nucleoprotein phosphorylate METTL3.Detection of GST-GlyRS and GST-METTL3 reaction groups revealed that GST-GlyRS failed to activate METTL3.This experiment obtained the following conclusions: G1 yRS inhibits BMECs autophagy by mediating Met-regulated mTOR signaling pathway.METTL3 inhibits autophagy of BMECs through mTOR signaling pathway,and NF?B1 inhibits autophagy of BMECs.In vitro kinase activity analysis showed that both Co-IP derived p-GlyRS and purified nucleoprotein p-GlyRS phosphorylate METTL3.This study provided experimental evidence for the regulation of autophagy in BMECs by GlyRS and its downstream proteins NF?B1 and METTL3;it also provided experimental data for revealing the molecular mechanism of p-GlyRS in regulating METTL3.