FHL2 Regulates Proliferation Differentiation and Autophagy of Bovine Skeletal Muscle Satellite Cells Through Wnt/β-catenin Signaling Pathway

The mechanism of physiological regulation of bovine skeletal muscle development is a complex process, and FHL2 has been studied in association with β-catenin activity and has previously been reported to play a role in skeletal muscle.However the mechanism of FHL2 action in regulating skeletal muscle development in bovine skeletal myosatellite is unclear. Here, we report that FHL2 can both promote the proliferation and differentiation of bovine myosatellite cells through the wnt signaling pathway and bovine skeletal muscle satellite cells through cellular autophagy. The results of western blotting, rt-qPCT, cell transfection assay showed that FHL2 gene expression was enhanced during the proliferation of skeletal muscle satellite cells, and FHL2 knockdown inhibited the proliferation and differentiation of bovine satellite cells and promoted the atrophy of myotubes. Furthermore, immunoprecipitation assays yielded that FHL2 knockdown decreased β-catenin activity in BMSCs and activated β-catenin-mediated wnt signaling pathway in combination with DVL-2, and that FHL2 knockdown induced autophagy in bovine satellite cells. Therefore, the FHL2 gene is a key gene in the regulation of bovine satellite cells.

A Novel miRNA Y-56 Mediates the Proliferation of Porcine Skeletal Muscle Satellite Cells by Targeting the IGF-1R-mediated AKT and ERK Pathways

Background: The skeletal muscle phenotype of the Bama Xiang pig (BM) is significantly different from the Landrace pig (LP). Uncovering the mechanism of porcine skeletal muscle growth will be of great significance to elucidate the mechanism of the different formation. As the key post-transcriptional regulators, miRNAs play an indispensable role in skeletal muscle development. The proliferation of skeletal muscle satellite cells not only maintain the muscle stem cell population, but also provide a large number of muscle derived cells. Thus, the goal of this study is to explore the effects of a novel miRNA Y-56 on the porcine skeletal muscle satellite cells (PSCs). Results: Firstly, we found that Y-56 was highly expressed in porcine muscle tissues, and its expression was higher in the BM than the LP. The EdU staining and CCK-8 assays results showed that increased levels of Y-56 suppressed cell proliferation, whereas decreased levels of Y-56 resulted in the opposite consequences. Furthermore, flow cytometry results showed that overexpression of Y-56 significantly reduced the percentage of S-phase cells, and the qRT-PCR and western blotting results showed that the expression levels of cyclin dependent kinase 2 (CDK4), proliferating cell nuclear antigen (PCNA) and Cyclin D1 were significantly inhibited. Moreover, downregulation of Y-56 increased the number of S-phase cells and the expression of CDK, PCNA and Cyclin D1. Furtherly, we identified that insulin like growth factor-1 receptor (IGF-1R) was a direct target of Y-56. Consistently, overexpression of IGF-1R promoted the cell proliferation of the PSCs, and increased the number of S-phase cells, as well as up-regulated the expressions of CDK4, PCNA and Cyclin D1. Meanwhile, knock-down of IGF-1R was associated with the opposite tend. What’s more, overexpression of IGF-1R partially reversed the inhibition of cell proliferation of the PSCs, the decrease of the percentage of S-phase cells and down-regulation of the expression levels of CDK4, PCNA and Cyclin D1, which caused by overexpression of Y-56. Finally, there was a significant decrease in the expression level of p-AKT and p-ERK with transfecting Y-56 mimics in the PSCs, and overexpression of IGF-1R reversed the decrease. Conclusion: Collectively, our findings suggested that Y-56 represses proliferation and cell cycle process of the PSCs through targeting IGF-1R that activated the AKT and ERK pathways.

A Novel miRNA Y-56 Mediates the Proliferation of Porcine Skeletal Muscle Satellite Cells by Targeting IGF-1R

Background: The skeletal muscle phenotype of the Bama Xiang pig (BM) is significantly different from the Landrace pig (LP). Uncovering the mechanism of porcine skeletal muscle growth will be of great significance to elucidate the mechanism of the different formation. As the key post-transcriptional regulators, miRNAs play an indispensable role in skeletal muscle development. The proliferation of skeletal muscle satellite cells not only maintain the muscle stem cell population, but also provide a large number of muscle derived cells. Thus, the goal of this study is to explore the effects of a novel miRNA Y-56 on the porcine skeletal muscle satellite cells (PSCs). Results: Firstly, we found that Y-56 was highly expressed in porcine muscle tissues, and its expression was higher in the BM than the LP. The EdU staining and CCK-8 assays results showed that increased levels of Y-56 suppressed cell proliferation, whereas decreased levels of Y-56 resulted in the opposite consequences. Furthermore, flow cytometry results showed that overexpression of Y-56 significantly reduced the percentage of S-phase cells, and the qRT-PCR and western blotting results showed that the expression levels of cyclin dependent kinase 2 (CDK4), proliferating cell nuclear antigen (PCNA) and Cyclin D1 were significantly inhibited. Moreover, downregulation of Y-56 increased the number of S-phase cells and the expression of CDK, PCNA and Cyclin D1. Furtherly, we identified that insulin like growth factor-1 receptor (IGF-1R) was a direct target of Y-56. Consistently, overexpression of IGF-1R promoted the cell proliferation of the PSCs, and increased the number of S-phase cells, as well as up-regulated the expressions of CDK4, PCNA and Cyclin D1. Meanwhile, knock-down of IGF-1R was associated with the opposite tend. Finally, overexpression of IGF-1R partially reversed the inhibition of cell proliferation of the PSCs, the decrease of the percentage of S-phase cells and down-regulation of the expression levels of CDK4, PCNA and Cyclin D1, which caused by overexpression of Y-56. Conclusion: Collectively, our findings suggested that Y-56 represses proliferation and cell cycle process of the PSCs through several biological mechanisms involving downregulation of IGF-1R.

miR-21-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting KLF3 in Chicken

The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.