miR-221 促进喉返神经损伤后修复机制的研究

Through post-transcriptional regulation, microRNA plays a vital role in the differentiation, proliferation and survival of nerve cell. Our previous studies have demonstrated that miR-221 was up-regulated in recurrent laryngeal nerve injury. To date, its role and mechanism on peripheral nerve repair still remains unknown. By using bioinformatics analysis, PTEN was identified as a potential target of miR-221. Downregulation of PTEN may promote the regeneration of peripheral nerve injury through PI3K /Akt signaling pathway，and their downstream effector GSK-3β. In addition, SOCS, as a known miR-221 target, may also be involved in the repair process by regulate JAK/STAT pathway. MiR-221 may serve as a mutual regulatory factor by various signaling pathways. To verify the hypothesis, miR-221 lentivirus transfection both in vivo and in vitro will be applied to investigate its role in laryngeal nerve injury repairing. Dual-luciferase report system and reverse blocking technique will be used to validate PTEN as its effector gene. The central role of miR-221 in nerve cell regeneration will be tested through the above two pathways. The study will clarify the mechanism that how miR-221 promotes the recurrent laryngeal nerve repair, and may offer a new target of laryngeal nerve injury gene therapy.

针对微小RNA通过转录后调控对神经细胞分化、增殖和存活发挥重要作用。课题组前期研究表明miR-221在喉返神经损伤后表达上调，但它在神经损伤修复中的作用及其机制尚不明确。我们已经通过生物信息学分析，预测miR-221可能的靶基因为PTEN，进而通过PI3K/Akt信号通路和其下游的GSK-3β促进周围神经损伤修复。同时，SOCS作为已知的miR-221靶基因，亦可能受其抑制而激活JAK/STAT通路参与周围神经损伤修复。即miR-221可能作为喉返神经修复不同信号通路的共同调节因子。为验证以上假说, 本课题拟通过体内、体外miR-221慢病毒转染实验，首度确认其在神经细胞修复中的功能；并使用双荧光酶报告系统验证PTEN为其作用的靶基因；并分别通过上述两条信号通路证明miR-221在神经细胞修复中的重要作用；深度揭示miR-221促进喉返神经修复的机制，为喉返神经损伤的基因治疗提供新的靶点。

前期研究发现大鼠喉返神经损伤后miR-221表达呈现一个逐步升高的趋势，我们猜测miR-221在外周神经损伤修复中可能扮演着很重要的作用，成功构建miR-221过表达和敲减的慢病毒质粒，感染PC12细胞，证实了miR-221体外促进神经生长增殖的作用并研究对下游PTEN，SOCS等靶点影响，骨髓间充质干细胞（BMSC）因具有向多系分化的潜能，在外周神经损伤修复的研究中颇受关注，成功提取BMSC并上调miR-221表达，在mRNA、蛋白以及细胞分泌水平都表明miR-221能进一步促进BMSC中BDNF和GDNF的表达，transwell共培养体系显示miR-221可以进一步增强BMSC趋化雪旺细胞迁移聚集的能力；通过NSE染色发现miR-221上调的BMSC出现部分突触样伸出，神经细胞分化趋势更明显。体内研究示miR-221上调的BMSC处理组和BMSC对照处理组相比PBS处理组喉返神经修复和相关功能恢复更好，但上调组和对照组间无显著性差异。结论表明miR-221表达水平与神经细胞的增殖呈现一定的正向关系，其水平上调对BMSC神经营养因子的表达有促进作用且能进一步增强BMSC趋化雪旺细胞迁移聚集的能力并增强BMSC向神经细胞分化的潜能，这为miR-221及BMSC参与喉返神经损伤修复的进一步研究提供了重要理论基础。体内研究证实了骨髓间充质干细胞对喉返神经挤压伤修复的积极作用，虽然miR-221并非独立影响其修复作用的因素，但通过细化评估手段和观察窗口仍很可能进一步确认miR-221的相关治疗优势。