Myostatin Gene Knockdown by Myostatin-specific Short Interfering Hairpin RNAs Increases MyoD Expression in C2C12 Myoblasts*

Abstract

Myostatin is a TGF-β superfamily member that negatively regulates the growth of the skeletal muscle mass. Remarkable muscle increase was observed in myostatin-knockout mice. Injection and electroporation of myostatin-targeting shRNA into rat tibialis anterior resulted in an increase in its weight, fiber size, and MHCⅡ expression. Two siRNAs targeting mouse myostatin were identified to block mouse myostatin expression upon co-transfection with a myostatin-expressing plasmid into HEK293 cell culture. These siRNAs were cloned into shRNA expression vectors and transferred into C2C12 myoblasts. ShRNA-positive cells were screened by neomycin selection and flow cytometry. By using real-time PCR, it was determined that the endogenous myostatin mRNA expression decreased by 10.2% and 35.5% in Mst-shRNA1-treated and Mst-shRNA2-treated C2C12 myoblasts, respectively. Western blot analysis indicated that the myostatin protein expression level decreased by 29.3% and 64.7%, respectively, in the two groups. it was also demonstrated that downstream MyoD pathway was affected by myostatin blockade, as evidenced by the 24.4% and 40.4% upregulation of MyoD expression in shRNA-treated cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently downregulate myostatin expression. This RNAi-based method of increasing muscle mass could provide an alternative strategy to gene knockout methods for genetic breeding and may be useful in improving the economic properties of livestock.