The Role And Mechanism Of MiR-199a-3p In C2C12Myoblast Differentiation

Recent studies have found that miRNA plays a crucial role in muscle development. SomemiRNAs can regulate the proliferation and differentiation of muscle cells, such as miR-1,miR-133, miR-26a, miR-27b, miR-29, miR-125b, miR-155, miR-181and miR-214. SomemiRNAs can regulate muscle fiber type transformation, such as miR-208. Additionally,miRNAs are also involved in regulating muscle cell hypertrophy, atrophy and musclediseases.MiR-199a-3p has two precursors, one is embedded in the intron15of Dynamin2,another in the anti-sense chain of intron14in Dynamin3. Dynamin is very important proteinfor muscle development and previous report showed that miR-199a-3p was markedlyincreased in Duchenne muscular dystrophy muscle. TGF-β, a main inhibitor of skeletalmuscle differentiation, could induce the expression of miR-199a-3p. Using TargetScan, wefound that miR-199a-3p may target several signal molecules of mTOR signal pathway,including IGF-1, PIK3r1(p85), mTOR and RPS6KA6. These results revealed thatmiR-199a-3p may have a role in myogenesis, however, it requires further experiments toconfirm this.Therefore, in this paper, RT-qPCR was to used to detect the expression of miR-199a-3pduring C2C12myogenic differentiation; C2C12myoblasts were transfected with mimic andinhibitor of miR-199a-3p and induced to differentiation, then immunofluorescence, RT-qPCRand WB were used to detect the changs of myotube formation, myogenic marker genes andmiR-199a-3p target genes; The3’UTRs luciferase reporter vector of miR-199a-3p targetinggenes were constructed and used to detect whether IGF-1, PIK3r1(p85), mTOR andRPS6KA6were the direct target gene of miR-199a-3p, further validated the mechanism ofmiR-199a-3p in muscle cells differentiation.The main results are as follows：(1) MiR-199a-3p was highly expressed in lung and skeletal muscle and had an increasingexpression during myogenic differentiation of C2C12myoblasts.(2) Overexpression of miR-199a-3p in C2C12myoblasts inhibited formation of myotubes, reduced the fusion index and muscle diameter, as well as the myogenic markergenes，such as Myf5, MyoD, MyoG and MyHC.(3) Interference of miR-199a-3p increased myotubes formation，as well as both mRNAand protein levels of MyoG and MyHC. The mRNA level of muscle atrophy marker gene,MuRF1were decreased after miR-199a-3p interference.(4) Overexpression of miR-199a-3p in C2C12myoblasts decreased the mRNA levels ofIGF-1, mTOR, RPS6KA6and inhibited the activation of mTOR signal pathway. Interferenceof miR-199a-3p increased the mRNA levels of IGF-1, mTOR and RPS6KA6.(5) The miR-199a-3p mimic was cotransfected into293T cells with the3′UTRdual-luciferase vectors of its targeting genes, miR-199a-3p overexpression dramaticallyinhibited the dual-luciferase reporter activity of IGF, PIK3r1mTOR and RKS6KA6.