Abstract：Myostatin (MSTN) is a negative regulator of muscle development. Studies have shown that it can regulate cell proliferation. In order to explore the further mechanism of MSTN mutant in regulating the proliferation and differentiation of muscle-derived satellite cells (MDSCs), in this study, the MSTN mutant (MT) and wild type (WT) bovine (Bos taurus) MDSCs were used to detect the cell cycle and gene expression in the process of proliferation and differentiation. Results of EdU proliferation assay showed that the proliferation index of MSTN mutant cells (0.78±0.0559) was significantly higher than that of wild-type cells (0.57±0.0366), indicating that MSTN mutant promoted the proliferation of MDSCs. When myogenic differentiation was induced, the MSTN mutant cells began to differentiate and formed the myotubes after 1day of induction, while the wild-type cells began to differentiate after 2 days of induction. Flow cytometry was used to further analyze the effect of myogenic induction on the cell cycle. Results also showed that the MT cells began to stagnate in the G1/S phase after 1 day of induction, and the cell cycle of WT cells stagnated after 2 days of induction. These results indicated that when cells were induced to differentiate, MSTN mutant cells exited from the cell cycle and entered to a differentiated state before wild-type cells. The expression of cell cycle-related genes and proteins was detected by qPCR and Western blot. Results showed that in MSTN mutant cells, the expression of CyclinA was significantly up-regulated (Relative expression=19.5), while the expression of cyclin-dependent kinase inhibitor 1C (CDKN1C) was significantly inhibited (Relative expression=0.009). These results showed that CyclinA and CDKN1C may play important roles in the regulation of MSTN mutant to the proliferation and differentiation of MDSCs. Online prediction through the JASPAR database showed that the key transcription factors downstream of MSTN, SMAD2/ SMAD3 may bind to the promoter of CDKN1C. ChIP-qPCR results demonstrated that the SMAD2/SMAD3 transcription factor combined with the promoter of CDKN1C thus to increase the expression of CDKN1C. In conclusion, MSTN mutant may inhibit the expression of CDKN1C gene by down-regulating the combination of SMAD2/SMAD3 transcription factor and CDKN1C promoter, thereby up-regulating the expression of CyclinA-CDK2, and promote DNA synthesis and cell cycle progress.This study discovered a new mechanism for MSTN mutant to promote muscle development, and provides a certain theoretical basis for the preparation of muscular gene-edited animals.
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