miR-615调控LINGO-1基因促进移植神经干细胞定向分化修复脊髓损伤的研究

Neural stem cells (NSCs) transplantation is considered as one of the most promising strategies for clinical treatment of spinal cord injury. The challenge is how to make proportionally more neural stem cells differentiate into neurons rather than glial cells in the injured spinal cord microenvironment. In our earlier research, antagonism of LINGO-1 has been confirmed that promoted rat embryonic NSCs directed differentiating into neurons, but the mechanism remains unclear. Further studies showed that miR-615 plays a negative regulation role on LINGO-1 expression in NSCs. Based on the previous experiments we hypothesize that the directional differentiation of NSCs may be associated with miR-615 regulation on LINGO-1 gene. Accordingly, we want to adopt dual luciferase report system and Western blotting verify the above regulation, analyzes its influence on development and differentiation of NSCs in vitro, and NSCs carrying the EGFP reporter gene and miR-615 mimics or inhibitor co-transplanted into spinal cord transected rats, to observe the effect of LINGO-1 gene regulation of miR-615 on the directed differentiation of transplanted NSCs in vivo and repairing of the nerve regeneration and functional recovery after spinal cord injury. This research is helpful to deepen the understanding of the mechanism of NSCs transplantation for repairing spinal cord injury.

神经干细胞（NSCs）移植被认为是临床治疗脊髓损伤最具前景的手段之一。面临的挑战是如何在损伤脊髓微环境让比例更多的移植NSCs分化为神经元而非胶质细胞。申请者前期研究证实：拮抗LINGO-1能促进大鼠胚胎NSCs向神经元定向分化，但机制未明。进一步的研究表明miR-615对LINGO-1在NSCs的表达具有负调控作用，结合前期实验，推测NSCs定向神经元分化可能与miR-615对LINGO-1基因的调控有关。据此，我们拟采用双荧光报告系统和Western blotting等验证以上调控作用，分析其对体外NSCs发育分化的影响，并构建携带EGFP报告基因的NSCs和miR-615 agomir/antagomir联合移植脊髓全横断大鼠，观察miR-615调控LINGO-1基因对移植NSCs体内定向分化的作用及对损伤后神经再生和功能恢复的影响。研究有助于深化理解NSCs移植修复脊髓损伤的机制。

LINGO-1（LRR and Ig domain containing NOGO receptor interacting protein 1）是脊髓损伤（spinal cord injury, SCI）修复的重要治疗靶点，其在神经元及少突胶质细胞存活、轴突再生和髓鞘形成及运动功能恢复中起关键调控作用。然而，LINGO-1潜在的胞内调控机制仍不清楚。MicroRNAs (MiRNAs)是一类短链、非编码RNA，其可在转录后水平通过靶向结合mRNA来调控基因表达。通过生物信息学分析，我们发现miR-615可潜在调控LINGO-1表达。进一步的双荧光素酶报告分析显示，miR-615能够靶向结合LINGO-1 3’-UTR，从而抑制LINGO-1翻译过程。在NSCs分化过程中，miR-615与LINGO-1表达呈大致相反趋势，提示miR-615在此过程中可负性调控LINGO-1表达。 MiR-615 mimics体外转染可明显抑制NSCs中LINGO-1表达并促进其增殖和神经元性分化。随后的LINGO-1相关信号蛋白检测显示，NSCs 中RhoA、EGFR及JAK/STAT3等信号蛋白水平在MiR-615 mimics转染后发生差异性改变，提示miR-615介导的LINGO-1调控可能通过影响这些信号蛋白相关通路来干预NSCs增殖分化。此外，miR-615 agomir 移植能有效降低SCI 动物体内LINGO-1蛋白水平，从而促进神经轴突的延伸、髓鞘形成及后肢运动功能的恢复。以上研究表明，miR-615在LINGO-1的内源性调控中起重要的作用，有望成为LINGO-1基础疗法的新型治疗靶点，为脊髓损伤修复提供新的治疗思路和实验依据。