牛骨骼肌卫星细胞成肌分化相关miRNAs的鉴定和功能研究

Some studies indicated miRNAs plays important role in muscle growth and development. However, its functions in muscle satellite cell proliferation and differentiation remain unclear. In this study, bovine skeletal muscle satellite cells will be isolated, purified and identified, and myogenic differentiation model was established in vitro. The expression changes of miRNAs in the process of myogenic differentiation were detected by miRNAs microarray tests, followed by real-time PCR analysis, and candidate miRNAs related to bovine muscle satellite cell differentiation were selected. The target genes of candidate miRNAs were predicted by bioinformatics software, and then validated by gene expression regulating test and dual luciferase reporter assay. When the differentiation related miRNAs were confirmed, miRNA mimics, miRNA inhibitors and lentivirus loading the specific miRNA were used to change miRNAs expression level in bovine muscle satellite cells, and its functions were evaluated by cell proliferation and differentiation detection. Through these studies, the relationship between miRNAs and bovine muscle satellite cell will be clarified, which can provide theoretical support and experimental basis for further research on mechanism of miRNAs regulating muscle proliferation and differentiation, and may offer new idea for beef cattle breeding.

本研究拟分离培养牛骨骼肌卫星细胞，进行纯化和鉴定，建立体外成肌分化模型，然后通过miRNAs芯片筛选肌卫星细胞分化前后差异表达的miRNAs，通过real-time PCR验证芯片筛选结果，选出肌卫星细胞成肌分化相关候选miRNAs，利用生物信息学软件预测候选miRNAs的靶基因，分析整合后确认候选靶基因，接下来通过表达调控实验和双荧光素酶实验对靶基因进行验证，确定分化相关miRNAs，根据以上研究结果选用miRNA mimics、miRNA inhibitors或构建真核表达载体转染肌卫星细胞上调或下调miRNAs的表达丰度，通过细胞增殖及分化检测，研究分化相关miRNAs对肌卫星细胞增殖和分化的调控作用。通过这些研究，系统地阐明肌卫星细胞与miRNAs表达的相互作用机制，为肌细胞发育分化的miRNAs调控机制积累基础资料，并为肉牛新品种培育提供新的思路和有益的线索。

本项目分离培养牛骨骼肌卫星细胞，进行纯化和鉴定，建立了体外成肌诱导分化模型，然后采用miRNAs芯片技术对牛肌卫星成肌分化过程中783个牛成熟miRNAs的表达模式进行检测，芯片筛选出了17个可信度较高的差异表达miRNAs，通过定量PCR技术对芯片分析结果进行验证，最终得到15个在肌卫星细胞成肌分化过程中显著差异表达的miRNAs。. 利用生物信息学分析方法对筛选出的成肌分化相关miRNAs进行靶基因预测，共预测出3077个靶基因，对靶基因进行了进一步的GO和KEGG分析，构建出了成肌分化相关miRNAs及其靶基因的调控网络，分析结果显示这些靶基因主要富集在转录调控、信号转导、蛋白转运、多细胞生物发育和细胞分化等有关的生物学过程中。结果表明，这些成肌分化相关miRNAs可能在牛成肌分化过程中发挥了重要的调控作用。. 综合分析后选出5个差异表达miRNAs，预测并验证靶基因，改变miRNAs表达丰度，研究分化相关miRNAs对肌卫星细胞增殖和分化的调控作用，并探讨其作用机制。研究结果表明miR-1 和miR-206可以通过调控Pax7和HDAC4的表达来促进牛肌卫星细胞的成肌分化过程；miR-139可以通过调控N-ras和IGF1R的表达，进而通过调控MAPK和PI3K/Akt信号通路来调控牛肌卫星细胞的成肌分化过程；miR-128可通过靶向Sp1进而调控CDKN1A和MyoD的表达，从而调控牛肌卫星细胞增殖与分化过程；miR-143可以靶向IGFBP5，可能通过调控IGF1R介导的信号通路的活性进而调控了牛肌卫星细胞增殖与分化过程；研究结果提示它们可能都参与了对生肌调节因子MRFs家族成员的调控，同时对成肌分化相关信号通路可能具有协同调控作用；失神经肌萎缩模型中的实验也表明miR-139和miR-128在体内也发挥了重要的调控作用。本项目研究结果可以为骨骼肌的发育分化和肌损伤修复研究提供一定的参考。. 进一步通过高通量测序技术筛选出了在肌卫星细胞成肌分化前后差异表达的1ncRNA 301条，并将其与差异表达miRNAs进行了关联分析，绘制出了差异表达miRNAs和1ncRNA的调控网络图，为后续深入挖掘分析miRNAs和1ncRNA的相互调控作用及其在肌肉发育分化过程中的作用奠定了基础。