National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, People's Republic of China.
J Cell Biochem. 2012 Aug;113(8):2652-60. doi: 10.1002/jcb.24140.
Myostatin, a secreted growth factor highly expressed in skeletal muscle, negatively regulates skeletal muscle growth and differentiation. Recently, myostatin is emerged as a potential target for anti-atrophy and anti-fibrotic therapies. Therefore, to investigate the regulation of myostatin in sheep adult fibroblasts, we used the RNA interference mediated by lentiviral vector to gene silence myostatin. Simultaneously, we also had constructed the sheep myostatin overexpression vector to further explore the function of myostatin in fibroblasts. The results here demonstrated that the lentiviral vector could significantly reduce myostatin gene both at mRNA and protein level by 71% and 67%, respectively (P < 0.01). Inhibition of myostatin also resulted in a remarkable increase of activin receptor 2B (ACV2B), p21, PPARγ, leptin, C/EBPβ, and MEF2A expression, and a decrease of Akt1, CDK2, MEF2C, and Myf5 expression. Ectopic myostatin mRNA and protein were also present in the fibroblasts transfection. Furthermore, we observed that overexpression of myostatin contributed to an increase of Akt1, CDK2, Myf5 and PPARγ, and a decrease of p21, C/EBPα and leptin at the transcript level. These results suggested that myostatin positively regulated Akt1, CDK2, Myf5, leptin, and C/EBPα, but negatively regulated p21 mRNA expression in adult fibroblasts, and it also expanded our understanding of the regulation mechanism of myostatin. Moreover, the lentiviral system inactivated myostatin gene in fibroblasts would be used to generate transgenic sheep and to ameliorate muscle fibrosis and atrophy by gene therapy in the future.
肌肉生长抑制素（Myostatin）是一种在骨骼肌中高度表达的分泌生长因子，它负调控骨骼肌的生长和分化。最近，肌肉生长抑制素被认为是抗萎缩和抗纤维化治疗的潜在靶点。因此，为了研究绵羊成纤维细胞中肌肉生长抑制素的调节作用，我们使用慢病毒载体介导的 RNA 干扰技术使肌肉生长抑制素基因沉默。同时，我们还构建了绵羊肌肉生长抑制素过表达载体，以进一步探索肌肉生长抑制素在成纤维细胞中的功能。结果表明，慢病毒载体可使肌肉生长抑制素基因在 mRNA 和蛋白水平上分别显著降低 71%和 67%（P<0.01）。肌肉生长抑制素的抑制也导致激活素受体 2B（ACV2B）、p21、PPARγ、瘦素、C/EBPβ 和 MEF2A 的表达显著增加，Akt1、CDK2、MEF2C 和 Myf5 的表达显著减少。转染的成纤维细胞中也存在外源性肌肉生长抑制素 mRNA 和蛋白。此外，我们观察到肌肉生长抑制素过表达导致 Akt1、CDK2、Myf5 和 PPARγ 的增加，以及 p21、C/EBPα和瘦素转录水平的降低。这些结果表明，肌肉生长抑制素正向调节 Akt1、CDK2、Myf5、瘦素和 C/EBPα，但负向调节成纤维细胞中 p21 mRNA 的表达，这也扩展了我们对肌肉生长抑制素调节机制的认识。此外，未来慢病毒系统失活成纤维细胞中的肌肉生长抑制素基因将用于生成转基因绵羊，并通过基因治疗改善肌肉纤维化和萎缩。
