Effect Of CircNEB On The Proliferation And Differentiation Of Myoblast Cells And The Transformation Of Skeletal Muscle Fiber Types

Skeletal muscle,as the largest secretory tissue in the body,coordinates body functions and its structure consists of several different physicochemically and metabolically distinct fiber types.CircNEB is highly expressed in muscle tissue and has an important role in the proliferation and differentiation of myogenic cells and in the transformation of skeletal muscle fiber types in mice.RT-q PCR results showed that circNEB was differentially expressed in different muscle tissues of mice.Therefore,in this study,C2C12 myoblasts and C57 BL/6J mice were used to investigate the regulatory role of circNEB on myofiber type transformation and its downstream ce RNA mechanism.In this paper,we firstly screened circNEB highly expressed in skeletal muscle on circ Atlas,and interfered with circNEB in mouse C2C12 myogenic cell line to detect changes in myogenic cell proliferation,differentiation and myofiber type;then we constructed adeno-associated virus inhibiting circNEB and injected it into tibialis anterior muscle of1-month-old mice to detect body condition,locomotor ability,myofiber cross-sectional area and myofiber type transformation;finally,we investigated the miRNAs and downstream target genes of circNEB regulating myofiber type transformation and obtained the main results as follows:1.Interfere with circNEB to promote the proliferation and differentiation of muscle into C2C12 myogenic cells.circNEB was highly expressed in skeletal muscle tissue;the expression of circNEB was inhibited by si RNA in mouse C2C12 myogenic cells,and compared with the control group,the cell proliferation-related genes PCNA,cyclin D1,cyclin E and differentiation-related genes My OD,My OG,My HC were significantly increased in the test group(P<0.05).The m RNA levels and protein levels of My OG and My HC were significantly elevated in the test group compared with the control group(P<0.05).2.Interfering with circNEB promotes the formation of fast type myotubes in C2C12 myogenic cells.By interfering with circNEB expression in mouse C2C12 myogenic cells,the expression levels of the fast contractile type myofiber marker genes Tnni2,Tnnt3 and related proteins were highly significantly increased(P<0.01).The expression levels of the slow contractile type myofiber marker genes Tnni1,Tnnt1 and related proteins were significantly decreased(P<0.01).3.Interference with circNEB in mouse skeletal muscle did not affect the normal physiological condition of mice,but altered the muscle fiber type of mouse tibialis anterior muscle.Adeno-associated viral vectors for muscle-specific interference with circNEB were constructed and injected intramuscularly into one-month-old mice at multiple points.The body weight and the ratio of muscle,fat,liver and heart to body weight in the test group were not significantly different from those in the control group(P>0.05);the results of the forced swimming experiment showed that the duration of endurance exercise in the test group was significantly lower compared with the control group(P<0.05);the expression levels of genes and proteins related to different muscle fiber types in the tibialis anterior muscle were measured,and the results showed that the slow contraction muscle in the test group was significantly lower than that in the control group(P<0.05).The results showed that the m RNA and protein levels of genes related to slow-contracting muscle decreased significantly in the test group(P<0.05),and the m RNA and protein levels of genes related to fast-contracting muscle increased significantly(P<0.05);the HE staining results showed that the area of muscle fibers in the control group was significantly smaller than that in the test group(P<0.05).4.Predicted and validated that circNEB promotes slow-type myotube type formation via miR-6538/AMPK signaling pathway in C2C12 cells.The miRNA targeted by circNEB was predicted to be miR-6538 by Circ Mir software online,and the predicted results showed that the two were tightly bound by RNAhybrid test;the dual luciferase activity assay showed that the test group could reduce luciferase activity extremely significantly(P<0.01);by overexpressing miR-6538 in mouse C2C12 adult myoblasts by overexpressing miR-6538 in mouse C2C12 myogenic cells,the expression levels of fast contractile myofiber marker genes Tnni2,Tnnt3 and related proteins were significantly increased(P<0.05);the m RNA levels of slow contractile myofiber marker genes Tnni1 and Tnnt1 and related protein levels were significantly decreased(P<0.05);the protein levels of P-AMPK/SIRT1/PGC-1α were significantly reduced(P<0.05).In summary,circNEB was found to be highly expressed in skeletal muscle;circNEB can promote C2C12 myogenic cell proliferation,myogenic differentiation and fast-type myotubes formation,while circNEB regulates myofiber type conversion in mice and affects skeletal muscle myofiber cross-sectional area and motor performance.The results enriched the circRNA regulatory network that regulates myogenesis and myofiber type transformation,and also provided theoretical support for improving meat quality.