Regulation Of TMEM8C And CTRP3in C2C12Myoblast Differentiation

The understanding of the molecular mechanism of skeletal muscle growth and development is important to animal breeding and genetics research. It is meaningful to reveal molecular mechanism for improving the skeletal growth rate of farm animal. Previous studies show that many genes like Pax3/7, Myf5, MyoD, MEF2, are crucial to regulate the skeletal muscle development, however, many underlying mechanisms remain unclear. Some new genes also have potential in regulating skeletal muscle development. A recent report suggests that TMEM8C involves in the fusion of myoblasts, which are skeletal precursor cells. In our study, we identified TMEM8C and CTRP3which was related to skeletal development, explored their expression regularity and their transcriptional regulation, and analyzed their role in myoblasts differentiation. The results was as shown below:(1) TMEM8C was specifically expressed in skeletal muscle tissue using RT-PCR test. During C2C12myogenic differentiation. TMEM8C showed up-regulated expression using Q-PCR test. We detected that TMEM8C showed low expression at C2C12proliferation phase, up-regulated expression during1-4d of myogenic differentiation; and the6th day’s expression was slightly reduced compared to the4th day’s.(2) SiRNA-mediated knockdown of TMEM8C inhibited C2C12myogenic differentiaion, and changed the early differentiated cell morphology of C2C12, which resulted in elongated cells with filopodia.(3) TMEM8C was located in the Golgi apparatus of the proliferative C2C12cells.(4) Two MyoD binding sites (3787bp and-3752bp) were found located in the upstream of TMEM8C start coden. Decreased activity of TMEM8C promoter after deletion of either of the two sites proved that MyoD was a positive regulator of TMEM8C. The GA microsatellites located in-287bp also played a positive role in regulating TMEM8C transcription.(5) The result of RT-PCR testified that CTRP3showed up-regulated expression during1-4d of C2C12myogenic differentiation, and the6th day’s expression was slightly reduced compared to the4th day’s.(6) Overexpression of CTRP3enhanced the mRNA level of Myogenin, one of marker genes of myoblast differentiaton. This result indicated that CTRP3can facilitate C2C12myogenic differentiation.