MiR-222 Involved In Genistein On C2C12 Myoblasts Differentiation

Genistein,a kind of phytoestrogen,is a natural tyrosine kinase inhibitor,which has many biological functions,such as anti-tumor,anti-osteoporosis,anti-oxidation,anti-depressant,endocrine regulation and animal growth and development.It exists widely in leguminous plants.In recent years,as a functional composition of food,some positive effects have been obtained in animal production by adding genistein.However,there are few studies on the specific mechanism of genistein,especially the study of genistein in promoting the meat performance of livestock and poultry.Previous studies have independently found that genistein can promote or inhibit the differentiation of myoblasts,but the effective dose and regulation mechanism are not clear.miRNAs are small non-coding RNAs(approximately 22 nucleotides in length)that silence gene expression at the post-transcriptional level by specifically binding to the 3?-UTR to control mRNA stability and translational efficiency.Many miRNAs have been reported to be closely related to the development of skeletal muscle.In this study,C2C12 myoblasts and mice were used as experimental models to explore the effect of genistein on skeletal muscle differentiation and the development of skeletal muscle in mice,in which we also studied the genetic regulation of miR-222.The main results of the study are as follows:1.During pregnancy fed mice feed with genistein,which could cause the down regulation of miR-222 in skeletal muscle tissue(P<0.01)and increased the expression of MyoD(P<0.01)and MyoG(P=0.09);2.Bidirectional regulation of genistein on the differentiation of C2C12 myoblasts in a dose-dependent manner,10?M/L genistein could promote C2C12 myoblast differentiation,in which the number of multinucleated myotubes increased(P<0.05)and MyHC,MyoD,MyoG and MRF4 increased in expression(P<0.01);however,at treatment concentrations higher than 10?M/L,the effect on myoblast differentiation was rapidly inhibited as the concentration increased,in which the number of multinucleated myotubes reduced(P<0.01)and the expression level of MyHC decreased significantly;3.Genistein could affect the expression of microRNAs related to myogenesis.10?M/L genistein could down-regulated miR-222(P<0.01)and up-regulated MyoG,MyoD and ER?,the target genes of miR-222(P<0.01),which promoted the differentiation of myoblast;4.The effect of miR-222 on the differentiation of C2C12 myoblast cells was further verified by MyHC immunofluorescence staining and quantitative analysis of the genes related to the myogenic differentiation;the double luciferase reporter system showed that BTG2 may be a new target gene for miR-222 in myoblasts;5.Genistein combined with miR-222 mimics and inhibitors to deal with C2C12 cells.The results showed that miR-222 mimics could significantly resist the downregulation of miR-222 caused by genistein(P<0.01)and significantly attenuate the effect of genistein on the differentiation of C2C12 myoblasts(P<0.01);miR-222 inhibitors further promoted the downregulation of miR-222 caused by genistein(P<0.01)and enhanced the effect of genistein on C2C12 myoblast differentiation(P<0.01).