Mirna-23a Regulatory Role In Myoblast C2c12 Cell Differentiation Process

Vertebrate skeletal muscle differentiation is regulated by an intricate network of transcription factors. Numerous genes, including growth factors, regulatory proteins, receptors, and transcription factors have been identified to participate in the regulation of myogenesis.Several signal transduction pathways are involved in positive or negative regulation of myogenesis. Recently, microRNAs (miRNAs) are reported to finely regulate the development of skeletal muscle at a new layer.MiRNAs are a class of small non-coding RNAs that negatively regulate gene expression post-transcriptionally, which are about 22-25 nucleotides. Several microRNAs have been implicated in regulating the intermediate processes during myogenesis, such as miRNA-133, miRNA-206 and miRNA-214. We found through bioinformatics that there are pairing sequences of the seed sequence of miRNA-23a in the 3'-UTR fragments of the mRNAs of the three myosin heavy chain gene (Myhl, Myh2 and Myh4). We therefore performed following experiments:1. We constructed the expression plasmid of pIRES2-EGFP-miRNA-23a, and the luciferase reporter plasmids by inserting the 3'-UTR fragments of the mRNAs of Myh1, Myh2 or Myh4 genes into the 3'-end of the luciferase gene in pGL-3-promoter plasmid. The pIRES2-EGFP-miRNA-23a plasmid and the pGL-3-promoter plasmids containing 3'-UTRs of Myh1, Myh2 and Myh4 mRNAs were co-transfected into HEK293 cells. The results from luciferase activity analysis showed that miRNA-23a significantly inhibited the expression of luciferase, indicating that the three genes are direct targets of miRNA-23a.2. C2C12 myoblasts can be induced to terminal differentiate to myotubes. We examined the expression of mature miRNA-23a in C2C12 cells during the differentiate process by real time RT-PCR. There was a low level of miRNA-23a at the initial of differentiation, upon C2C12 myoblast differentiation, miRNA-23a expression reduced progressively till the 3rd day of induction, and then dramatically elevated at the 5th day of induction when the myotubes were well-formed. The increase expression of myosin heavy chains was corresponding to the decline of miRNA-23a level, suggesting that miRNA-23a may negatively regulate the differentiation of C2C12 cells through inhibition the expression of myosin heavy chain.3. The regulatory effects of miRNA-23a on myogenic differentiation in C2C12 cells were confirmed by use of mimic and inhibitor. The expression of myosin heavy chain proteins and the differentiation were both inhibited C2C12 cells transfected mimic, inverse effects were observed with inhibitor transfection. However, the mRNA levels of Myh were not declined. The above data suggests that miRNA-23a regulates the expression of myosin heavy chain at translation but transcription level.4. C2C12 cells proliferate normally in growth medium, they exit from cell cycle and switch to differentiation when the medium changed to differention medium. BrdU is a analogous of nucleotide which can be incorporated into DNA chain in S phase of cell cycle. We found that the positive staining cell of BrdU was significant increased in miRNA-23a mimic transfected cells compared with that of control, and a inverse effect was observed in inhibitor treated cells, indicating that miRNA-23a promote the proliferation of C2C12 cells, which was consistent with its inhibition effects on differentiation.5. It has been reported that miRNA-23a play distinct roles in different type of cells by targeting respect mRNAs. We found that there were pairing sequence of miRNA-23a in the 3'-UTR of mRNAs of some myogenic regulatory factors including MEF-2C and MurF through bioinformatics analysis. The expression of MEF-2C was decreased in mimic transfected cells, while no alteration was observed in MurF expression level. The luciferase analysis data revealed that MEF-2C was not the target gene of miRNA-23a, while the changed expression may be due to other mechanism.In conclusion, we found that miRNA-23a directly regulates myogenic differentiation of C2C12 myoblasts through inhibition the expression of myosin heavy chain protein. Our data suggests a novel mechanism under the regulatory effects on myogenesis by miRNAs.