Study On The Mechanism Of Mettl21c Regulating Myoblastic Differentiation Based On Autophagy Effect

Non-histone lysyl-methyltransferase-like 21C(Mettl21c)is one of the distant family members of the Methyltransferase superfamily.Studies in poultry have found that Mettl21c is expressed in all tissues of the body of poultry animals,especially in skeletal muscle tissues,which can participate in maintaining skeletal muscle homeostasis and regulating skeletal muscle growth and development of poultry animals.In this study,mice were used as research objects to build a mouse model of exercise to explore the morphological changes of skeletal muscle and the expression level of Mettl21c during exercise.The recombinant lentiviral interference vector was constructed with mouse C2C12 myoblast cells as the model,and the C2C12 cell line with stable interference of Mettl21c was obtained by lentivirus method to explore the role of Mettl21c in the process of myoblast differentiation,and the mechanism of Mettl21c 's regulation of myoblast differentiation was studied based on the autophagy effect of cells.To provide a reference for the analysis of the function of Mettl21c in the growth and development of mammals,and to provide theoretical value and clinical significance for the effective treatment of muscle-related diseases.The main experimental results of this study are as follows:1.Through the rotary type sports fatigue tester six-week run test,build sports model mice,according to the results of frozen section HE staining to promote muscle fiber hypertrophy,muscle bundle spacing shorten found in m RNA and protein level of mice after motion Mettl21c expression level rises in the calf muscles.At the same time,the expression level of Mettl21c was also found to be the highest in 8 tissues such as testis,groin,fat,heart,kidney,small intestine,brain,intestinal fat,gastrocnemius,which was significantly higher than other tissues.2.Two pairs of p LKO.1-TRC-Mettl21c shRNA(shRNA1 and shRNA2)interference vectors were constructed,and C2C12-NC?C2C12-shRNA1 and C2C12-shRNA2 cell lines were obtained by lentiviral packaging.Compared with the NC group,the expression level of Mettl21c in the shRNA1,sh RNA2 group was significantly decreased(**P<0.01),and shRNA1 group(83.9%)of jamming efficiency is higher than sh RNA2 group(59.9%)(**P<0.01).3.The temporal expression profile of Mettl21c in the process of proliferation and differentiation of mouse C2C12 cells was established.It was found that the gene expression of Mettl21 c was gradually up-regulated during the process of differentiation of mouse C2C12 cells,and it was significantly higher after 5d than that speculated at the early stage of differentiation that Mettl21c played a positive regulatory role in the process of proliferation and differentiation of skeletal muscle.The m RNA and protein levels of myoblast differentiation related genes were detected.It was found that the myoblast differentiation level was significantly decreased after the interference with Mettl21 c,and the expression of the slow muscle marker gene MYH7 was also significantly decreased.Preliminary results showed that the interference with Mettl21c inhibited myoblast differentiation and slow muscle formation.Interference with Mettl21c can reduce the dimethylation and trimethylation levels of lysine in various proteins.4.Exercise promotes skeletal muscle autophagy Through cell morphological observation and Western Blot analysis,25?g/mL of RAPA-induced autophagy was selected as the final concentration.Under the condition of RAPA-induced autophagy,Mettl21c interfered with the ratio of autophagy marker protein LC3B?/? and promoted the expression of P62 protein.At the same time,it was found that the expression level of Myog protein was also decreased,indicating that the interference of Mettl21c in the process of myoblast differentiation of C2C12 cells inhibited autophagy level and myoblast differentiation ability.Conclusion: Exercise promotes the expression of autophagy and Mettl21c in skeletal muscle of mice,and Mettl21c affects the level of autophagy in skeletal muscle cells,thereby promoting myoblast differentiation and slow muscle formation.