Notch信号通路介导的microRNAs影响猪骨骼肌生长发育机理研究

Notch signaling pathway plays a key role in the regulation of the development of porcine skeletal muscle satellite cells (PSCs). However, its molecular regulatory mechanism, particularly the regulation in post transcription remains unclear. Previous studies have shown that the expression of a large subset of microRNAs changes with the Notch signaling. It is possible that there are some microRNAs take part in the process of the regulation of the development of PSCs mediated by Notch signaling pathway. In the current research, we set out to establish a PSCs cell model which stable express Notch intracellular domain (NICD), in which the differential expressed microRNAs are selected by sequencing and their target genes are speculated and verified. The influence of the proliferation on PSCs are confirmed by flow cytometry after transfecting microRNAs as well as their target genes mimics and inhibitor, respectively. RT-PCR, Westernblot and immunohistochemistry are applied to examine the regulation on the expression of Notch signaling pathway related genes, muscle specific genes and cyclins. In addition, to determine its function on the muscle tissue, the microRNAs lentivirus are injected into piglets hindlimb muscle. By these, to clarify the molecular mechanisms of Notch signaling pathway and mediated microRNAs on the development of porcine muscle,to lay the foundation of improving the meat production.

Notch信号通路对猪骨骼肌卫星细胞(PSCs)发育调控具有关键作用，其分子调控机制特别是转录后水平调控尚不明确。本团队前期发现多个microRNAs表达随着Notch信号的改变而改变，可能存在microRNAs参与Notch信号通路调控PSCs发育。本项目拟构建稳转NICD的PSCs模型，通过测序技术筛选Notch信号通路特异表达microRNAs，预测并验证靶基因，在PSCs中分别过表达和干扰microRNAs及靶基因，流式细胞仪检测细胞增殖的变化、荧光定量PCR、westernblot和免疫组化检测对Notch信号通路相关基因、肌特异性基因和细胞周期蛋白表达量调控；此外，拟包被microRNAs慢病毒注射仔猪后腿肌肉，研究其对猪肌肉发育的影响。从而明确Notch信号通路及介导的microRNAs对猪肌肉生长发育的作用及调控机理，为提高猪产肉力奠定基础。

骨骼肌卫星细胞对出生后骨骼肌的生长发育和损伤修复具有非常重要的作用。Notch信号通路和microRNA是调控细胞生长和功能的关键因子，但是二者在调控猪骨骼肌卫星细胞（PSCs）生长发育过程中的相互作用尚未有研究报道。.本研究通过构建稳定激活Notch信号通路的PSCs模型，选取增殖期1天和分化期第7天实验组和正常组样品进行数字基因表达谱和Small RNA测序，鉴定差异miRNA和基因并构建miRNA-mRNA调控网络，进一步解析Notch信号通路对PSCs增殖和分化的调控网络。共鉴定获得48个差异表达miRNA和295个差异表达基因。差异表达miRNA中，肌肉特异性ssc-miR-206和ssc-miR-133a-5p显著下调。下调的ssc-miR-10、ssc-miR-10a-5p、ssc-miR-125a、ssc-miR-125b和ssc-miR-214与MKI67和WHSC1形成关键miRNA-mRNA互作对；上调的ssc-miR-146a-5p、ssc-miR-221-3p与FGF2、RUNX1T1形成重要miRNA-mRNA互作对。基因互作网络和GO分析，发现11个上调核心hub基因，参与调控细胞周期过程；3个下调hub基因THBS2、ACTC1和FGF2，FGF2为该网络最核心hub基因，主要参与细胞粘附和迁移等生物过程。表明过表达N1ICD，主要参与促进细胞周期，抑制细胞外基质相关生物过程，进而促进PSCs增殖。针对关键候选miRNAs，研究发现miR- 34c通过Notch1形成调节环来抑制肌肉发育；miR-27b通过靶向MDFI促进PSCs生成；MiR-199b通过靶向Jag1促进PSCs增殖，并激活Notch信号通路及下游基因的转录；MiR-22抑制PSCs增殖和促进肌源性分化；miR-92a和miR-423促进PSCs增殖和分化，靶基因分别为MEF2D和NUMBL。针对筛选的关键基因，HEYL和RPL15都分别促进PSCs增殖和抑制分化；Stac3促进PSCs肌源性分化，myogenin抑制其启动子活性。.本研究明确了Notch 信号通路及介导的microRNAs 对猪肌肉生长发育的作用及调控机理，为生产实践中提高猪产肉力性状奠定了良好的基础。