电刺激促进神经轴突生长的新机制：miRNAs对DCLK1的调控作用

Electrical stimulation (ES) has long been used to accelerate neurite outgrowth in clinics, but its underlying molecular mechanism, especially the role of non-coding RNA has been unclear thus far. Our previous work has identified two microRNAs (miR-363-5p and miR-3102), which have been found to play a vital role in the enhanced neurite outgrowth by ES. In addition, miR-363-5p and miR-3102 potentially target the same gene DCLK1. Thus far, the role of miR-363-5p and miR-3102 in nervous system has not been investigated in previous publications. Moreover, studies on DCLK1 have been focused on central nervous system; the role of DCLK1 in peripheral nervous system has been unclear. Therefore, the present study was designed to investigate the regulatory effect of miR-363-5p and miR-3102 on DCLK1, and further clarify its involvement in the enhanced neurite outgrowth by ES. In addition, the potential value of miR-363-5p, miR-3102 and DCLK1 as neuroprotective targets is also evaluated. The present study: ① reveals novel molecular mechanisms for ES-enhanced neurite outgrowth；② identifies the role of miR-363-5p and miR-3102 in nervous system for the first time, which provides targets for the development of drugs for promoting nerve regeneration; ③ clarifies the role of DCLK1 in peripheral nervous system for the first time, which makes us better understand how electrical signals was translated into neurite outgrowth.

电刺激是临床上加快神经再生的有效技术，但其分子机制，尤其是非编码RNA在其中的作用尚不清楚。我们前期发现了2个新miRNA分子（miR-363-5p及miR-3102）发挥关键作用，且这两个miRNA可能同时作用于同一个基因DCLK1。既往研究发现DCLK1在中枢神经形成中发挥重要作用，但在周围神经再生中作用如何尚不清楚。因此，本项目拟进一步明确这两个miRNA分子对DCLK1的调控作用，并揭示其在电刺激促进神经再生中的作用。在此基础上，明确以这些分子为靶点建立促进神经再生新方法的可行性。本项目意义在于：①揭示了电刺激促进神经轴突生长的新机制；②首次揭示miR-363-5p及miR-3102在神经系统中的功能，为神经损伤修复药物的开发提供新靶点；③首次分析DCLK1分子在电刺激促进周围神经再生中的关键作用，为揭示物理信号（电刺激）转化生物学信号（轴突生长）的科学难题提供新思路。

电刺激是临床上加快神经再生的有效技术，但其分子机制，尤其是非编码RNA在其中的作用尚不清楚。本项目利用高通量microRNA测序技术，初步筛选鉴别在电刺激促进神经轴起生长中差异表达显著的miRNA列表，通过对16种显著变化的miRNA进行验证及功能分析，最终发现1个重要的miRNA分子（miR-363-5p）在电刺激加快神经轴突生长中发挥关键作用。进一步的研究发现，miR-363-5p可在体外培养环境下调控神经元突起生长并影响靶基因DCLK1启动子活性，进而影响其转录表达。DCLK1作为关键靶基因，可明显参与调节神经轴突生长过程，而靶基因的表达改变可影响单纯miRNA分子改变引起的突起生长调节效应。我们进一步在体内坐骨神经模型中进行了深入研究，发现miR-363-5p可以通过调控DCLK1促进神经轴突早期再生。通过干预miR-363-5p及DCLK1可以促进神经再生，并促进感觉、运动等功能恢复，抑制靶肌肉萎缩。本项目首次通过活体基因转染技术在体内验证了电刺激促进神经再生中关键miRNA分子的促进神经损伤再生作用，揭示上述调控作用在电刺激促进神经轴突生长中的关键作用，为神经损伤修复药物的开发提供新靶标，具有重要意义。本项目培养博士研究生2名，硕士研究生1名，发表SCI论文5篇。