骨骼肌损伤纤维化中差异表达LncRNA的筛选及其功能与机制研究

Skeletal muscle fibrosis after injury is a key factor limiting the healing of injured skeletal muscle. In order to facilitate the rehabilitation following skeletal muscle injury, the research in the mechanism of skeletal muscle fibrosis is of vital importance. Our previous studies have confirmed that TGF-β / Smads and other signaling pathways and their complex interactions with related microRNAs play an important role in the skeletal muscle fibrosis. In order to explore more upstream regulatory mechanisms, LncRNA, which plays a significant regulatory role at different levels, including epigenetics, transcription and post-transcription, has aroused our concern. In this study, LncRNA microarray is used to identify LncRNAs significantly differentially expressed during the process of skeletal muscle fibrosis. The function of target LncRNAs in fibrosis will be verified by constructing and transferring overexpression and knockdown vectors. According to bioinformatics analysis and database prediction, combined with results of RNA Pulldown, RIP and other methods, we will identify the target of LncRNA and clarify its mechanism of action. Then we will further explore the role and mechanism of target LncRNAs in the regulation of fibrosis-related pathways, and finally illuminate the function of LncRNAs in skeletal muscle fibrosis and its specific mechanisms, hence providing novel ideas and scientific basis for possible new treatments.

骨骼肌损伤后纤维化是制约损伤骨骼肌修复的关键因素，损伤骨骼肌纤维化的机制研究是探讨促进骨骼肌功能康复的重要方向。课题组既往研究证实了TGF-β/Smads等信号通路以及其与相关microRNA的复杂相互作用在损伤骨骼肌中发挥了重要作用。为探讨更上游的调控机制，在表观遗传、转录及转录后多个层面均可发挥明显调控作用的LncRNA，引起了我们的关注。本研究拟筛选并验证骨骼肌纤维化中显著差异表达的目标LncRNA；以过表达和敲减载体对目标LncRNA在纤维化中的功能进行验证；根据生物信息分析及数据库预测，并结合RNA pulldown、RIP等方法明确其作用靶点及其作用方式；再进一步探究对TGF-β/Smads等纤维化关键通路的调节作用与机制。最终明确LncRNA调控骨骼肌纤维化的具体功能及作用机制，从而为新的治疗方法提供思路和科学依据。

骨骼肌损伤后纤维化是制约损伤骨骼肌修复的关键因素，损伤骨骼肌纤维化的机制研究是探讨促进骨骼肌功能康复的重要方向。课题组既往研究证实了TGF-β/Smads等信号通路以及其与相关microRNA的复杂相互作用在损伤骨骼肌中发挥了重要作用。为探讨更上游的调控机制，在表观遗传、转录及转录后多个层面均可发挥明显调控作用的LncRNA，引起了我们的关注。.在本研究中，我们首次报道一种新的在骨骼肌损伤后纤维化中高表达的lncRNA (lnc-MFAT1)。我们证明lnc-MFAT1基因敲除可减少TGFβ诱导的体外纤维化，并可减轻小鼠急性挫伤后体内骨骼肌纤维化。进一步研究表明，lnc-MFAT1能作为miR-135a-5p的竞争性内源性RNA。此外，抑制miR-135a-5p表达可以通过靶向Tgfbr2/Smad4分子，显著促进TGFβ诱导的体外纤维化。我们还发现lnc-MFAT1通过海绵吸附miR-135a-5p发挥竞争性内源性RNA功能来调控Tgfbr2/Smad4的表达，从而导致TGFβ通路的激活。.此外，我们还报道了lncRNA-H19能够促进骨骼肌纤维化。lnc-H19在体内、体外骨骼肌纤维化模型中均高表达，体外抑制lnc-H19表达可以减轻的纤维化。下调lnc-H19被证明可以通过海绵吸附miR-20a-5p，通过发挥竞争性内源性RNA功能调控Tgfbr2的表达，从而抑制C2C12成肌细胞中TGFβ/Smad通路的激活。.总之，本项目确定了lnc-MFAT1-miR-135a-Tgfbr2/Smad4轴和lnc-H19-miR-20a-Tgfbr2轴在骨骼肌纤维化中的关键作用，为骨骼肌损伤后纤维化提供了具有潜力的治疗选择。