Construction of myostatin gene knockout C2C12 cell line and expression of related microRNA

Abstract

The strategy of blocking MSTN signal transduction has always been regarded as an entry point and breakthrough in the treatment of patients with muscle loss. However, blocking agents often face problems such as lacking strength, fatigue and poor muscle proliferation due to muscle hypertrophy and multi-receptors. To shed light on these matters a serous of experiments were carried out on a C2C12 cell line in this study. Firstly, the pX601-SaCas9-sgRNA/puro vector carrying a Cas9 encoded gene was constructed, and subsequently used to produce MSTN-knockout (MSTN-KO) C2C12 cell lines. The expression level of MSTN protein and the growth characteristics of the cell lines were verified. Moreover, the expression of muscle-growth-related miRNAs in the cell lines were analyzed by RT-PCR. These results indicate that we successfully established a method for constructing MSTN-KO cell lines with stable passage. No expression of MSTN protein and strong cell proliferation were observed in the cell lines. Moreover, RT-PCR experiments showed that the expression levels of miR-1, miR-431, miR-206 and miR-133a were extremely significant increased(p < 0.01), the expression levels of miR-23a was significant increased༈p < 0.05༉, while the expression level of miR-486 was significant decreased༈p < 0.05༉, indicating that multiple miRNAs are closely associated with MSTN’s regulation. This study lays a foundation for further study of the effect of Mstn gene on the physiological function of myoblasts and the development of drugs that block MSTN signal pathway.