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结节性硬化复合物 2(TSC2)基因的过表达抑制山羊成肌细胞的增殖和分化,以了解肌肉发育的潜在机制。

Overexpression of the Tuberous sclerosis complex 2 (TSC2) gene inhibits goat myoblasts proliferation and differentiation in understanding the underlying mechanism of muscle development.

机构信息

Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.

Department of Xinjiang Institute for Cancer Research, Xinjiang Cancer Hospital of Xinjiang Medical University, Urumqi 830011, China.

出版信息

Gene. 2020 Oct 5;757:144943. doi: 10.1016/j.gene.2020.144943. Epub 2020 Jul 9.

Abstract

The growth of animal skeletal muscle is mainly determined by the synthesis processes of total proteins in skeletal muscle cells, which has a significant impact on the postnatal growth of young animals. An increasing number of studies are focusing on the functions of Tuberous sclerosis complex 2 (TSC2) during the process of cell protein synthesis and growth. However, it is still unclear the effect of whether and how TSC2 on goat myoblasts proliferation and differentiation. Here, we found that TSC2 gene has opposite expression patterns in proliferation and differentiation of myoblasts. An expression vector containing goat TSC2 cDNA sequences linked with pcDNA3.1 plasmid was constructed. Myoblasts proliferation activity was significantly inhibited and cell cycle transition slowed down after the transfection of pcDNA3.1-TSC2 plasmid into goat primary myoblasts by EdU staining, CCK-8 and flow cytometry. Mechanically, we further confirmed that the overexpression TSC2 was able to down-regulate the mRNA and protein expression of mechanistic target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 (p70S6K) and some cell cycle related genes. In addition, the expression of myogenic genes and myotube formation were attenuated. Collectively, all our results of the experiment demonstrate that TSC2 could regulate myoblasts cells proliferation and differentiation via the activation of the mTOR/p70S6K signaling pathway.

摘要

动物骨骼肌的生长主要取决于骨骼肌细胞中总蛋白的合成过程,这对幼小动物的出生后生长有重要影响。越来越多的研究集中在结节性硬化复合物 2(TSC2)在细胞蛋白质合成和生长过程中的作用。然而,TSC2 是否以及如何影响山羊成肌细胞的增殖和分化尚不清楚。在这里,我们发现 TSC2 基因在成肌细胞的增殖和分化中具有相反的表达模式。构建了一个含有山羊 TSC2 cDNA 序列与 pcDNA3.1 质粒相连的表达载体。通过 EdU 染色、CCK-8 和流式细胞术,我们发现转染 pcDNA3.1-TSC2 质粒后,山羊原代成肌细胞的增殖活性显著受到抑制,细胞周期转换减慢。在机制上,我们进一步证实,过表达 TSC2 能够下调雷帕霉素靶蛋白(mTOR)、p70 核糖体 S6 激酶 1(p70S6K)和一些细胞周期相关基因的 mRNA 和蛋白表达。此外,成肌基因的表达和肌管形成减弱。总之,我们实验的所有结果表明,TSC2 可以通过激活 mTOR/p70S6K 信号通路来调节成肌细胞的增殖和分化。

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