The Role And Mechanism Of All-Trans Retinoic Acid On Proliferation And Differentiation Of Sheep Muscle Precursor Cells In Vitro

Vitamin A(VA)plays an important role in organ formation and tissue development of mammals.All-trans retinoic acid(ATRA)is a key metabolite of VA,which is involved in many physiological processes,such as cell proliferation,differentiation and apoptosis.However,the effect of ATRA on the proliferation and differentiation of sheep skeletal muscle precursor cells and its related mechanisms have not been elucidated.The aim of this study was to investigate the effects and mechanisms of ATRA on the proliferation,differentiation and epigenetics of sheep skeletal muscle precursor cells.In this experiment,the hind limb skeletal muscle of the lamb was separated by type II collagenase digestion and differential adherent method.Sheep primary skeletal muscle precursor cells were cultured and treated with control group(0 nM)and 1 nM,10 nM,50 nM,100 nM,500 nM and 1000 nM ATRA treatment group.After 24 h,the effects of ATRA on the proliferation of skeletal muscle precursor cells were detected by MTT assay.10 nM and 100 nM suitable concentrations were selected for follow test.The effects of ATRA activation on the proliferation of sheep skeletal muscle precursor cells were further examined by Ed U labeling and scratch test.The effects of ATRA on the maturation of sheep skeletal muscle precursor cells during skeletal muscle precursor cells differentiation were observed by immunofluorescence staining.And the effect of histone modification on skeletal muscle precursor cells differentiation induced by ATRA on the promoter of myogenin was detected by chromatin immunoprecipitation(CHIP).The effect of ATRA on glucose uptake during skeletal muscle precursor cells differentiation was detected by 2-NBDG assay.To study the effect of ATRA on the activity of m TOR signaling pathway.The results were showed as below.1)The data showed that sheep skeletal muscle precursor cells had been successfully isolated and induced myogeninc differentiation.2)During the growth of sheep skeletal muscle precursor cells,the expression of PCNA m RNA was not affected by ATRA treatment,and the protein expression of PCNA,Cyclin D1 and CDK4 was significantly decreased(P < 0.01).The inhibitory role on the proliferation of skeletal muscle precursor cells was most significant in the group treated with 100 nM ATRA.3)10 nM and 100 nM ATRA increased the MHC protein content(P < 0.01),compared with the control group,the activation of ATRA significantly promoted the enrichment of H3K4me3(P < 0.01).And 100 nM ATRA significantly inhibited the enrichment of H3K27me3(P < 0.05),however,the 10 nM ATRA had no effect on the enrichment of H3K27me3.Thus,ATRA can increase the Myogenin m RNA and protein content(P < 0.01),and promote the myoblasts fusion.4)The expression of m RNA and protein of Glut4 was upregulated(P < 0.01)and glucose uptake can be increased(P < 0.05)by 100 nM ATRA treatment.The p38 MAPK and m TOR signaling pathway was activated by ATRA treatment,and the phosphorylation levels of Akt,m TOR,4EBP and p70 were significantly increased(P < 0.01).ATRA activated the Wnt/?-catenin signaling pathway by increasing the expression of ?-catenin m RNA and protein(P < 0.01).In conclusion,ATRA inhibited the proliferation of sheep skeletal muscle precursor cells and promoted their myogenic differentiation.The effect of 100 nM ATRA treatment was the most obvious.