Musclin基因在骨骼肌营养代谢中的分子调控机制研究

Musclin is a muscle-derived cytokine. The expression of musclin is regulated by nutrition, hormone and cell differentiation, but the specific regulatory mechanisms about the expression and function of musclin gene are not clear on the international level. In our previous experiment, the 5' regulatory region, the coding region and the 3'UTR of musclin gene in sheep were cloned, and the putative motifs and factors along the 5' regulatory region were predicted by bioinformatical method. Now we are doing some researches about the transcriptional regulation of musclin gene in sheep. On the basis of it, sheep will still be selected as the main research object, and the molecular and cellular technologies such as site-directed mutagenesis, EMSA, RNA interference and biochip will be used. The project aims to clarify the molecular regulatory mechanisms of musclin gene by screening and indentifying some motifs, upstream molecules and microRNA which regulate the expression of musclin gene and some downstream molecules which are regulated by musclin in skeletal muscle (cell), and by investigating the effects of musclin on the nutritional metabolism and ultrastructure of skeletal muscle (cell). The project will lay a foundation for uncovering the molecular mechanisms of growth/development and nutritional metabolism of skeletal muscle, improving the growth performance of domestic animals or avians and preventing/curing the muscle diseases of human beings.

Musclin是一种肌源性细胞因子，该基因的表达受营养、激素和细胞分化状态等影响，但具体的表达调控和作用机制在国际上尚未阐明。在前期试验中，我们已经克隆了绵羊Musclin基因5'调控区、编码区和3'UTR序列，并对5'调控区的潜在元件和因子进行了生物信息学预测，目前正在进行转录调控的部分研究。本项目旨在已有基础上，继续以绵羊为主要研究对象，利用定点突变、EMSA、RNA干扰、芯片等分子和细胞生物学技术在骨骼肌（细胞）筛选并鉴定影响Musclin基因表达的元件、上游分子和microRNA以及Musclin调控的下游分子，并分析Musclin对骨骼肌（细胞）营养代谢和超微结构的影响，以阐明Musclin基因在骨骼肌表达和作用的分子调控机制，为揭示骨骼肌生长发育和营养代谢的分子机制奠定基础，也为改进畜禽生长性能和防治人类肌肉相关疾病提供依据。

Musclin是一种肌源性细胞因子，其表达受营养和激素影响，作用与糖代谢有关。本项目在分子、细胞和动物水平系统研究了Musclin基因在骨骼肌营养代谢中的表达调控和作用机制。结果表明：① 鉴定了对绵羊Musclin基因转录起重要作用的系列调控元件如E-box、GA-box、HMTB、VTBP、MEF2、MEF3、SMAD和FOXO等及其相应的转录因子或激素。葡萄糖和血清等营养状态、分化状态、转录因子、胰岛素、糖皮质激素和孕激素对绵羊Musclin启动子活性具有明显的调控作用，其中糖皮质激素和孕激素及Foxo、Mef2和Smad3等因子的作用与启动子区的相应元件有关。生物信息学分析发现绵羊Musclin基因5'UTR内含子区也存在许多潜在调控元件，3'UTR区存在不少潜在microRNA，通过实验鉴定了miR-609、307、302c、67和29。绵羊禁食状态下，Musclin表达下调，GLUT4、IRS-1、mTOR、AKT、S6K1的表达与其呈正相关，而AMPK、MuRF1、atrogin-1、UCP2、UCP3的表达与其呈负相关，表达谱测序已经完成，结果即将分析完毕。② 实现了小鼠Musclin基因的高效过表达和siRNA沉默，发现生理水平Musclin有促进成肌细胞增殖的作用，生理和过表达水平Musclin可抑制葡萄糖摄取并促进氨基酸分解，并影响肌肉相关基因Smad3、Myostatin和LXRα的表达。进一步实现了绵羊Musclin基因的高效过表达，发现其可以抑制成肌细胞增殖，抑制葡萄糖摄取和促进氨基酸分解，并使GLUT4、IRS-1、mTOR、AKT和Smad3表达下调而LXRα、Myostatin和Myomaker表达上调。糖皮质激素对绵羊Myomaker基因5′调控区活性的调控作用与其剂量及细胞状态有关，作用机制至少部分是通过糖皮质激素受体与（n）GRE元件结合而介导的，暗示了Musclin过表达促进肌细胞融合分化基因Myomaker表达的原因至少部分是通过Musclin影响糖代谢而介导的。另外，采用Cas9/gRNA技术在人诱导性多能干细胞中进行了Musclin基因敲除并获得较高效率，为进一步深入研究奠定了基础。本项目为构建骨骼肌生长发育和营养代谢的分子调控网络奠定了理论基础，为保障动物肌肉组织的健康发育和良好代谢提供了科学依据。