Research On The Gene Function Of ARID1B Inhibiting Met-activated MTOR Signaling Pathway In Mouse Mammary Epithelial Cells

Milk is rich in nutrients,including milk protein,milk fat,amino acids and vitamins,which are essential nutrients in human daily life,and is also an essential source of nutrition for mammalian babies.The content of milk protein and milk fat in milk is an important standard to measure the quality of milk.Therefore,improving the content of milk protein and milk fat in milk has become a focus in the current research field.Amino acids are one of the most important nutrients in living organisms,and also an important regulator of mammalian milk synthesis.Amino acids can promote milk synthesis by activating the mammalian target protein of rapamycin(mTOR),which is a key important signal molecule of milk synthesis in mammary epithelial cells(MECs)and its proliferation.Although a lot of studies have been carried out on the mTOR signal pathway involved in milk synthesis promotion,the specific regulatory mechanism is still not well understood.AT-rich interaction domain 1B(ARID1B)is one of the non-catalytic subunits of BAF chromatin remodeling complex.At present,the research on ARID1 B mainly focuses on cancer and neurological diseases,the molecular mechanism of ARID1 B is still unclear,and whether ARID1 B participates in amino acid regulation of milk synthesis is not known yet.In this study,we aim to explore the role and molecular mechanism of ARID1 B in Met-stimulated milk synthesis in MECs.The milk synthesis ability of MECs is determined by milk fat and protein synthesis and proliferation ability of MECs.The study on the molecular mechanism milk synthesis in MECs is helpful to provide a new basic theory basis to develop new technologies to promote milk synthesis.We previously found through proteomic analysis that ARID1 B might be associated with milk synthesis in MECs.To explore whether ARID1 B plays a regulatory role in milk synthesis,ARID1 B gene expression in HC11 cells was knocked down by ARID1 B si RNA transfection and ARID1 B gene expression was activated by the CRISPR/d Cas9 vector system.Western blotting was used to detect the expression of β-casein after ARID1 B gene inhibition.Triglyceride content and lipid droplet formation were detected using Triglyceride(TG)detection kit and fluorescence method to detect milk fat synthesis in HC11 cells.Ed U assay.In addition,CCK8 kit,Ed U kit,and cell counting plate were used to detect the cell proliferation ability after ARID1 B inhibition.The experimental results show that ARID1 B gene inhibition significantly promoted milk fat and protein synthesis and proliferation of HC11 cells.The results of ARID1 B gene activation experiment were all opposite to these of the inhibition experiment.Taken together,these above data demonstrate that ARID1 B plays a negative regulatory role in milk fat and protein synthesis and proliferation of HC11 cells.Further experiments were performed to show the effect of ARID1 B on mTOR m RNA expression and protein phosphorylation.ARID1 B gene inhibition significantly increased the expression of mTOR m RNA and protein phosphorylation,and the gene activation experiment has the opposite results.These results indicates that ARID1 B negatively regulates mTOR m RNA expression and protein phosphorylation.To study the influence of Met on ARID1 B and mTOR,different concentrations of Met(0,0.3,0.6,0.9,and 1.2 m M)were added to HC11 cells.ARID1 B protein level decreased to the peak and mTOR protein phosphorylation increased to the peak when Met concentration was at 0.6 m M.ARID1 B gene activation completely blocked Met’s stimulation on mTOR m RNA expression and protein phosphorylation.Ch IP-PCR results showed that ARID1 B bound only to the-4368 ~-4591 nt sequence in the-1 ~-4843 nt region of the mTOR promoter.Ch IP-q PCR results showed that Met reduced the binding of ARID1 B to the mTOR promoter.The above results indicate that ARID1 B inhibits Metstimulated mTOR gene transcription by binding to its promoter.Further studies were performed on the effects of Met on the degradation of ARID1 B and its molecular mechanism.q RT-PCR detected that Met addition reduced ARID1 B m RNA expression,whereas the inhibition of PI3K(LY 294002)treatment led to increased ARID1 B m RNA level in spite of Met treatment.Western blotting showed that LY 294002 completely blocked Met-stimulated decrease of ARID1 B protein level.We further explored whether Met can reduce ARID1 B protein level through the ubiquitination degradation pathway.HC11 cells were treated with protein synthesis inhibitor CHX and autophagy inhibitor CQ.CHX and CQ did not block Met-induced down-regulation of ARID1 B protein level.However,MG132 treatment completely restored the downregulation of ARID1 B protein level by Met.These data suggest that Met decreases ARID1 B protein level by PI3 K signaling pathway,and thereby inhibiting ARID1 B gene expression and promoting its ubiquitin degradation.To sum up,this paper reveals ARID1 B acts as an important negative regulator in the process of Met stimulation on mTOR m RNA expression and protein phosphorylation to promote milk fat and protein synthesis and cell proliferation.This study add a new theoretical basis for the application of Met to the healthy and safe production of mammary milk.