The Roles Of MEF2A In The Regulation Of Skeletal Muscle Myoblasts Proliferation And Differentiation In Qinchuan Beef Cattle

Skeletal muscle is an important constituent in indicating livestock muscle quality and maintaining metabolic homeostasis.Myocyte enhancer factor 2A(MEF2A)is an important transcription factor required for cardiomyocyte proliferation,skeletal muscle development and regeneration.Developing myoblasts in mice lacking MEF2 A are unable to differentiate,which causes severe disruption for skeletal muscle regeneration.Despite those findings,a limited number of studies have determined whether and how MEF2 A regulates myoblast proliferation.Furthermore,during myoblasts differentiation,the downstream genes regulated by MEF2 A and the underlying mechanisms are far from well-studied till now.Based on these research backgrounds,in this study,we attempt to investigate the roles of MEF2 A in regulating bovine skeletal muscle myoblasts proliferation and differentiation.The main findings of this study are listed below:1.In this study,by using collagenase ?/Dispase ? digestion and selective growth medium for cell isolation and culture,primary myoblasts with high purity and differentiation potential were successfully isolated from bovine skeletal muscle.RT-PCR results showed that the mRNA levels of MYOD1 was higher in the early stage of differentiation and then gradually decreased.The mRNA levels of MRF4,MYOG and MYH1 were continuously elevated during myoblast differentiation.These results indicated that the isolated primary myoblasts were satisfactory for the subsequent experiments.2.During development of beef cattle from 6 months of age to 24 months of age,the mRNA expression level of MEF2 A was continuously elevated.Under the conditions of in vitro cell culture,both the mRNA and protein expression levels of MEF2 A were all upregulated during myoblasts differentiation.These results indicated that there might be a specific role for MEF2 A in bovine skeletal muscle myogenesis.3.During myoblasts proliferation,overexpression of MEF2 A induced a noticeable increase in the number of proliferating myoblasts.Cell cycle and expression analysis showed that MEF2 A is a positive regulator for myoblasts proliferation through triggering cell cycle progression from G1 to S phase by activating CDK2?CCNA2?CCNE1 and CCNE2 expression.4.During myoblasts differentiation,MEF2 A knocked-down resulted in severely impaired myotube formation and reduced expression of MYOZ2.Through performing luciferase test,we found that MEF2 A promoted MYOZ2 expression by directly promoting transcriptional activity of the MYOZ2 promoter.Being consistent with the inhibitory effect of MEF2 A on myoblasts differentiation,interference of MYOZ2 also resulted in severely impaired myotube formation.These results indicated that MEF2 A was required for myoblasts differentiation through regulation of MYOZ2.Inversely,overexpression of MEF2 A in differentiating myoblasts resulted in severe apoptosis.5.During myoblasts differentiation,inhibition of MEF2 A repressed expression of MEG3 and its downstream miRNAs miR-758 and miR-543.The blocked expression of miR-758 and miR-543 released PPP2R2 C expression from the post-transcriptional inhibition,which activated PP2 A signaling and repressed myoblasts differentiation.Taken together,we report that MEF2 A is a key modulator for skeletal muscle myogenesis.MEF2 A can not only promote myoblasts proliferation,but its precious regulation is also necessary for myoblasts differentiation.Both overt expression or interference of MEF2 A inhibited myoblasts differentiation.In this study,we also identify two different regulatory pathways through MYOZ2 and MEG3/DIO3-PP2 A for MEF2 A to regulate myoblasts differentiation.These findings will provide valuable insights for the studies of skeletal muscle growth and regeneration.