LncRNA-MEG3对猪骨骼肌细胞增殖的作用及调控机制

Cell proliferation is genetic basis for growth and development of animal and plant organism.Previous studies documented that there were significantly different proliferation activity of myoblast between pig breeds with variant phenotype, long no-coding RNA materally expressed gene 3 (lncRNA-MEG3) can inhibit cell proliferation in tumorigenesis. We firstly found that MEG3 was differently expressed during skeletal muscle development, and exhibited variant expression pattern between different breed pigs. Based on above facts, we supposed that lncRNA-MEG3 play an important role in skeletal muscle development, and maybe contribute to difference in cell proliferation activity and growth of skeletal muscle between pig breeds with different phenotype. In this study, we will analyze dynamic expression pattern of MEG3 during skeletal muscle development and its correlation with myoblast proliferation at molecular, cellular and animal level. Subsequently, the proliferation activity and cell cycle will be measured under over- and down-expression. Combining bioinformatics and experiment methods, we test regulation role of microRNA, transcript factor and DNA methylation in the expression of MEG3, and identify its interaction with target gene. These results will help us to explain the biological role in swine myoblast proliferation and skeletal muscle development for lncRNA-MEG3. Molecular marker and association analysis with meat production traits will be analyzed. This study provide candidate genes in molecular improvement of pigs.

细胞增殖是动植物机体生长发育的基础。研究表明不同类型猪骨骼肌细胞具有不同增殖能力，长链非编码RNA-MEG3（lncRNA-MEG3）在肿瘤细胞中具有抑制细胞增殖的作用。申请者首次发现MEG3在骨骼肌生长发育过程中差异表达，且在不同类型猪中具有不同表达模式。基于此，申请者推测lncRNA-MEG3参与骨骼肌的生长发育，可能对不同类型猪骨骼肌细胞的增殖能力和生长发育差异起重要调控作用。项目将在分子、细胞和个体水平分析MEG3在不同类型猪骨骼肌生长发育中的动态表达模式及其与猪肌细胞增殖的关系，研究过表达和敲低MEG3对肌细胞增殖能力和细胞周期的影响，结合生物信息学和c-KLAN等实验学方法，分析microRNA、转录因子及DNA甲基化对其表达的调控，并鉴定出MEG3的作用靶标，阐明其对猪肌细胞增殖和骨骼肌生长发育的调控机制。通过标记关联分析明确其与猪产肉性状的相关，将为猪分子育种提供候选基因。

细胞增殖是动植物机体生长发育的基础。研究表明不同类型猪骨骼肌细胞具有不同的增殖能力，长链非编码RNA-meg3(lncRNA-Meg3)在肿瘤细胞中具有抑制细胞增殖的作用。申请者首次发现Meg3在骨骼肌生长发育过程中差异表达，且在不同类型猪中具有不同表达模式。基于此，申请推测LncRNA-Meg参与骨骼肌的生长发育，可能对不同类型猪骨骼肌细胞的增殖能力和生长发育差异起重要调控作用。本项目中，申请者利用FISH和核质分离技术，发现lncRNA-Meg3主要在细胞质中表达并发挥功能，其特异性地在猪和小鼠的骨骼肌中高表达，在小鼠和猪骨骼肌发育过程中都表现在胚胎或出生早期高表达，CTX损伤模型分析结果表明Meg3与骨骼肌的损伤重建有关。在C2C12成肌细胞中敲低和过表达Meg3，发现过表达抑制成肌细胞增殖，而敲低则促进成肌细胞增殖，其表达受miRNA-378等调控。利用microarray分析敲低和过表达Meg3的基因表达表明，细胞增殖相关的基因在过表达条件显著下调，进一步证明Meg3对肌细胞的增殖起调控作用。在群体中进行SNP的扫描和基因分型，关联分析表明Meg3与达100公斤体重日龄显著相关。综上所述，lncRNA-Meg3主要通过调控胚胎和出生后早期骨骼肌细胞的增殖来影响骨骼肌的生长发育和肌肉表型，是猪产肉性状的一个候选基因。

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