Glucocorticoid Regulates The Synthesis Of Porcine Muscle Protein Through M~6A Modified Amino Acid Transporter SLC7A7

The occurrence of stress is unavoidable in the production process of livestock,and prolonged stress will cause the productivity of livestock to decrease.The stress response is mainly regulated by the hypothalamus-pituitary-adrenal(HPA)axis,which produces a large amount of stress hormones,namely glucocorticoid(GC),and has a greater impact on the energy metabolism of the animal body.It is reported that m6A modification plays an important role in the regulation of stress response and also participates in the process of muscle growth and development.The previous research of the research group showed that after cortisol was fed to piglets,the weight gain rate was extremely reduced,and the treatment of porcine skeletal muscle cells with the glucocorticoid analogue dexamethasone(DEX)would lead to the reduction of cell protein synthesis and Decomposition increases,and protein synthesis is related to the amount of amino acids transported by amino acid transporters.On the basis of these studies,this study continues to explore the changes in m~6A modification levels in porcine skeletal muscle cells under stress and the major amino acid transporters that affect their protein synthesis and their regulatory molecular mechanisms.The main findings are as follows:1.The effect of dexamethasone on protein synthesisThe SUn SET non-radioactive technique was used to detect protein synthesis rate after DEX treatment of porcine skeletal muscle cells for 48 hours.The results showed that DEX-treated cells would reduce the cell protein synthesis rate.After adding its antagonist RU486,the results showed that the protein synthesis rate was up-regulated.The conduction of m TOR signaling pathway plays an important role in the process of protein synthesis.After DEX is used to treat cells,the phosphorylation levels of m TOR signaling pathway and its downstream effectors S6K1 and 4EBP1 are detected.The results show that m TOR,S6K1 and The phosphorylation level of 4EBP1 was significantly reduced.2.Effect of dexamethasone on m~6A methylation modification in porcine skeletal muscle cellsAfter treating porcine skeletal muscle cells with dexamethasone,dot blot hybridization and high-performance liquid chromatography-mass spectrometry showed that the overall m~6A modification level in the cells was reduced.When the same concentration of RU486 was added to the cells,the m~6A modification level returned to the normal level,indicating that the stress regulated the m~6A level in pig skeletal muscle cells.The level of m~6A modification in cells is generally related to the expression of methylation modifying enzymes.At the same time,after DEX treatment of porcine skeletal muscle cells,the results showed that the expression of m~6A methylases METL3and METL14 were significantly reduced,while the expression of m~6A reading protein YTHDF2 Increase in volume.3.The effect of dexamethasone on differential amino acid transporter genesUsing q PCR to detect the expression of the amino acid transporter SLC7A7 in the muscle samples obtained after feeding piglets with cortisol in the early stage of the research group,the results showed that the expression of SLC7A7 was extremely significantly down-regulated in the samples fed with cortisol.After DEX treatment in porcine skeletal muscle cells,the expression of SLC7A7 gene was detected,and it was found that the expression of SLC7A7 was extremely significantly down-regulated.Me RIP-QPCR was used to detect the changes in the m~6A modification level on the SLC7A7 gene after DEX treatment.The results showed that the SLC7A7 gene m RNA m~6A modification was significantly down-regulated after DEX treatment.At the same time,when we treated the cells with the m~6A methylation modification inhibitor DAA,It was found that the expression of SLC7A7 was extremely significantly down-regulated,indicating that DEX mediates the expression of SL7A7 gene in a way that depends on m~6A modification.4.The effect of SLC7A7 on protein synthesisAfter overexpression of SLC7A7 in pig skeletal muscle cells,the results showed that after overexpression of SLC7A7,the protein synthesis rate was increased,and the m RNA expression levels of Atrogin-1 and MSTN genes,which promote skeletal muscle proteolysis,were significantly down-regulated.At the same time,the conduction of the m TOR signaling pathway was enhanced.The results showed that the phosphorylation levels of m TOR and S6K1 were extremely significantly up-regulated;when SLC7A7 was interfered,the protein synthesis rate was found to decrease,and the expression level of the IGF1 gene,which promotes protein synthesis,was significantly down-regulated.At the same time,m TOR and S6K1 and the phosphorylation level of 4EBP1 was significantly down-regulated.This shows that the amino acid transporter SLC7A7 controls protein synthesis by regulating the conduction of the m TOR signaling pathway.Conclusion:When the animal body is under stress,the level of glucocorticoids in the body increases,and the level of m~6A methylation modification in the cell is significantly down-regulated,which mediates the significant down-regulation of the m RNA m6A modification level of the amino acid transporter SLC7A7 gene,resulting in a significant expression of SLC7A7 Down-regulation results in decreased protein synthesis in pig skeletal muscle cells.