Molecular Mechanism Of Muscle Growth Of Broiler Chicken:A Role Of MiR-199-5p In Regulating Differentiation Of Skeletal Myoblasts

The broiler chicken is widely used for meat production and also served as an ideal animal model for muscle growth control.In contrast to layer chicken,the broilers displayed more and thicker myofibers whereby faster proliferation and slower differentiation of myoblast cells.What is the regulatory determinants underlying such a featured process remains largely unclear.To address this,we here investigated the possible involvement of microRNA in gene regulation of the muscle-associated proteins.With microarray and qRT-PCR analysis,we found that miR-199-5p expression elevated significantly in developing skeletal muscle of the broilers.Exogenous introduction of miR-199-5p led to suppression of myotube formation and cell cycle maintenance of the primary chicken myoblasts from both the broilers and layers.Conversely,down-regulation of miR-199-5p promoted muscle cells differentiation and exiting from cell cycle.This effect may be observed also in mouse C2C12 myosblast cells,a widely used in vitro model of myogenesis.Our results suggest that miR-199-5p may be an essential factor determining skeletal muscle development of the broilers and layers.To find the target gene of this miRNA,we performed bioinformatics analysis with on-line software TargetScan,we found non-muscle myosin ?A was the target of miR-199-5p.Sequentially,constructed 3'-untranslated region(3'-UTR)luciferase vector of predicted genes.By luciferase assay,we confirmed miR-199-5p could directly bind with 3'-UTR of Myh9 mRNA,which encoded myosin heavy chain 9,the essential component of non-muscle myosin IIA(NM IIA).Western blot showed the expression of NM IIA protein was negatively related to miR-199-5p abundance during myoblast differentiation,which was independent on the level of Myh9 mRNA.Knockdown of Myh9 in C2C12 cells by siRNA caused significant inhibition of myogenic process in a similar manner of introduction of miR-199-5p.We also detected a relative lower abundance of NM IIA protein along with higher level of miR-199-5p in embryonic muscle of the broilers.We thus concluded that non-muscle myosin IIA served as a target gene of miR-199-5p during muscle development.In order to test the role of miR-199-5 in vivo,we designed oligonucleotides of mature miR-199-5p,and injected it into chicken embryos.It was interesting to find that,either the broilers or the layers,down-regulation of the miRNA by oligonucleotides inhibitor resulted in a decrease in postnatal body weight.At last,we compared the level of miR-199-5p and NM ?A in developing skeletal muscle from different broiler and layer lines.Strikingly,in contrast to the layers,all broilers showed higher level of miR-199-5p and lower NM ?A protein.In summary,we revealed a molecular mechanism for broiler chicken muscle growth.Increase in miR-199-5p was an essential factor determining myoblasts differentiation by maintaining myoblast in cell cycle and hence producing more cells necessary for muscle mass growth.