Linc-MD1竞争性结合microRNAs调控山羊肌卫星细胞成肌分化的分子机制

The myogenic differentiation of muscle satellite cell is an important basis for animal skeletal muscle development, renew and regeneration, however, its regulatory mechanism, especially the post-transcriptional regulation mechanism is not clear. According to the applicant's previous study on goat myogenic miRNAs, combined with the latest research, it is speculated that long noncoding RNA linc-MD1 can competitively bind to myogenic miRNAs, thereby regulating the expression of downstream transcription factors, further affects the differentiation of goat muscle satellite cells. In the study, we will analyze the regulation of linc-MD1 on myogenic differentiation of goat muscle cells, and use CHIRP-Seq and Luciferase assays to screen for the miRNAs which were competed by linc-MD1, and their downstream target genes. At the end of the study, the competition effect of linc-MD1 on specific miRNAs and the regulation of related gene expression will be studied by RNA interference, real-time PCR and western-blot，in order to investigate the molecular mechanism of linc-MD1 regulates myoblastic differentiation of goat muscle cells through competing for shared miRNAs.

肌卫星细胞成肌分化是动物肌肉发育、更新及再生的重要基础，而其调控机制，尤其是转录后调节机制尚不明确。根据申请人对山羊肌源性miRNAs前期试验，结合最新研究，推测在山羊肌卫星细胞成肌分化的过程中，长链非编码RNA linc-MD1介导了一个转录后调控新路径，即linc-MD1竞争性结合肌源性miRNAs，调控下游转录因子的表达，进而影响山羊肌卫星细胞的成肌分化。为此，本项目首先分析linc-MD1对山羊肌卫星细胞成肌分化的调控作用；进而利用CHIRP-Seq和Luciferase assays甄别其竞争性结合的miRNAs，及其下游靶标基因；最后以山羊肌卫星细胞为试验对象，利用RNA干扰、荧光定量PCR和western-blot等技术探明linc-MD1对特异miRNAs的竞争作用，及其对相关基因表达的调控过程。以期探明linc-MD1通过miRNAs调控山羊肌卫星细胞成肌分化的分子机制。

肌卫星细胞成肌分化是动物肌肉发育、更新及再生的重要基础，而其调控机制，尤其是转录后调节机制尚不明确。为此，本研究以组内前期高通量测序获得的在山羊肌肉组织中差异表达的lncRNAs和肌源性miRNAs信息为基础，选择linc-MD1为主要研究对象；利用生物信息学、CHIRP-Seq和Luciferase assays筛选并确定，用于后续实验的目标肌源性miRNAs为miR-133a-3p，下游靶基因为DCTN2。Linc-MD1和miR-133a-3p的表达谱显示两者均具有肌肉组织特异性，表明它们与山羊骨骼肌的生长发育相关且可能参与骨骼肌卫星细胞的分化。然后以山羊肌卫星细胞为试验材料，通过细胞瞬时转染技术，干扰Linc-MD1和过表达miR-133a-3p后，对骨骼肌卫星细胞分化标志基因的检测结果表明Linc-MD1能够促进山羊骨骼肌卫星细胞的分化，而miR-133a-3p则能够抑制骨骼肌卫星细胞的分化；si-DCTN2后，qPCR检测骨骼肌卫星细胞分化标志基因，表明DCTN2能促进骨骼肌卫星细胞的分化，该结果与miR-133a-3p对肌卫星细胞的作用相反。最后，利用qPCR和western-blot等技术检测出Linc-MD1的表达水平可以受到miR-133a-3p的调节，说明Linc-MD1通过竞争性靶向结合miR-133a-3p来调控骨骼肌卫星细胞的分化。该项研究在理论上可望在肌肉细胞定向分化分子机制等方面取得突破性发现，为进一步揭示骨骼肌生长发育相关基因调控研究提供新的探索途径和理论基础。