肌肉增强子因子2对猪肌肉生长抑制素启动子活性的调节

Regulation of myostatin promoter activity by myocyte enhancer factor 2

肌肉生长抑制素(Myostatin,Mstn)是转化生长因子-β超家族的成员,在哺乳动物的骨骼肌生长和分化过程中起负调控作用,其转录调控受到多个基因的影响,其中肌肉增强子因子2(MEF2)是重要的调控因子之一。因此,对猪Mstn启动子上MEF2位点及其作用方式的探讨具有重要意义。首先,通过PCR方法扩增了猪Mstn基因上游1 969 kb的启动子序列,利用生物信息学方法分析该序列包含3个MEF2的结合位点;其次,采用逐步删除的方法获得5个长度不等的启动子,用荧光素酶报告系统评估了它们在小鼠成肌细胞C2C12中的活性。其次,转入含有MEF2结合位点的启动子片段和MEF2C表达载体,可以显著增强启动子活性2～6倍,高表达另一亚型MEF2A则启动子活性没有明显改变。最后,转入MEF2C表达载体,用实时定量PCR和Western blotting方法检测Mstn的转录和蛋白水平的变化,结果发现mRNA升高了2～4倍;在肌管细胞中,蛋白翻译水平也有显著升高。这些结果显示,MEF2C可以通过激活Mstn参与猪肌肉生长和发育阶段的调节。研究为Mstn基因的转录调控提供了有效的作用靶点和效应分子,为进一步探讨Mstn的功能调控提供了一种新的思路。

Myostatin（Mstn） is a member of the transforming growth factor-superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals.The transcriptional regulation of Mstn is controlled by multiple genes including MEF2,which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying.In this study,we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells.Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs.Using a serial deletion strategy,we tested the activity of several promoter fragments by luciferase assay.Overexpression of MEF2C,but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds,in both C2C12 myoblasts and myotubes.When we transfected exogenous MEF2C,Mstn mRNA level was also upregulated in C2C12 cells,but the protein level was only significantly increased in myotubes.Thus,we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine.Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.