Panoply™ Human MSTN Knockdown Stable Cell Line

Myostatin (MSTN) is an important negative regulator of muscle growth and development. Here, researchers used shotgun-based tandem mass spectrometry (TMT) six-fold labeling to perform comparative proteomic analysis on the gluteal muscle tissues of MSTN+/- Mongolian cattle (MG.MSTN+/-) and wild-type Mongolian cattle (MG.WT) to explore the regulatory mechanism of MSTN on bovine skeletal muscle growth and development. Bioinformatics analysis showed that knocking down the MSTN gene increased the expression of extracellular matrix and ribosome-related proteins and activated focal adhesion, PI3K-AKT, and ribosome pathways. Western blot experiments on muscle tissue and in vitro MSTN gene knockdown experiments validated the proteomic analysis results, revealing that knocking down MSTN gene expression promoted the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells (BSMSCs). Simultaneously, co-immunoprecipitation (CO-IP) experiments showed that the MSTN gene interacts with the extracellular matrix-associated protein type I collagen α1 chain (COL1A1). Knockdown of COL1A1 expression inhibited the activity of focal adhesion, the PI3K-AKT pathway, and the ribosome pathway, thereby suppressing the proliferation of bone marrow mesenchymal stem cells (BSMSCs). These results suggest that the MSTN gene regulates focal adhesion, the PI3K-AKT pathway, and the ribosome pathway through the COL1A1 gene. In conclusion, this study provides new insights into the regulatory mechanism of MSTN in muscle growth and development.

To investigate the mechanism by which the MSTN gene affects bovine skeletal muscle development, researchers studied the effects of MSTN gene knockdown on the proliferation and myogenic differentiation of bovine skeletal muscle mesenchymal stem cells (BSMSCs). They constructed MSTN-knockdown GM and DM3 cells (Figure 1A). Simultaneously, the expression of proliferation-related markers Pax7 and MyoD was examined to assess whether cell proliferation was affected. The results showed that in MSTN-knockdown cells, the expression level of Pax7 mRNA did not change significantly, while the expression level of MyoD mRNA was significantly upregulated (Figure 1B); the expression level of Pax7 protein did not change significantly, while the expression level of MyoD protein was significantly upregulated (Figure 1C). The results of the 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assay showed that when MSTN was knocked down, the number of EDU-positive cells (Figure 1D) and the EDU labeling index (Figure 1E) were both upregulated.

Figure 1. Knockdown of MSTN expression promotes the maintenance of bovine skeletal muscle satellite cells (BSMSCs) proliferation. (Sheng H, et al., 2021)