miR-136-5p靶向IKKβ/NF-κB/A20调控IL-17介导的脊髓损伤后炎症反应的分子机制

At the early stage of the spinal cord injury (SCI), inhibiting the reaction of immunologic cascade which is involved with inflammatory cytokines and chemokines as well as their effector cells is an effective way to reduce the secondary spinal cord injury. Our previous study has found that IL-17 is an important factor, which is involved in the initiation of the secondary spinal cord injury besides IL-1β and IL-6. Through with the high-throughput sequencing (HTS) technology and the Bioinformatics analysis, the spinal cord tissue in SCI rats and the whole miRNA expression profiles of astrocytes induced by IL-17 in the spinal cord of rats were respectively measured. And this is the first finding of our study that miR-136-5p may have the potential roles in regulating inflammatory responses after SCI and have relationships with NF-κB signaling pathways. In addition, there have no such related research at home and abroad. In order to make a thorough study of the exact mechanisms about how miR-136-5p regulates the IL-17-mediated inflammatory responses after SCI, we will use different experimental methods, such as dual-luciferase reporter (DLR) assay, RNA interfere (RNAi) and construction of lentiviral expression vectors on multiple levels of cell, gene, protein, signaling pathway and animal, to validate the molecular mechanism of IL-17-mediated inflammatory responses after SCI regulated by miR-136-5p-targeted IKKβ/NF-κB/A20 pathway.

脊髓损伤（SCI）早期,抑制炎性因子和趋化因子与其效应细胞参与的炎症免疫级联反应是减少脊髓继发性损伤的有效途径。本课题组前期研究发现IL-17是除IL-1β、IL-6之外诱导SCI后继发性炎症反应的重要因素，通过对SCI大鼠脊髓组织及IL-17刺激大鼠脊髓星形胶质细胞全基因组miRNA表达谱高通量测序及生物信息学分析，本课题组首次发现，miR-136-5p是调控SCI后炎症反应的潜在microRNA，并与NF-κB信号传导通路有关，国内外尚未有相关研究报道。为了深入研究miR-136-5p调控IL-17诱导SCI后炎症反应的精确机制，本课题组将采用双荧光素酶报告基因技术、RNAi、慢病毒载体构建等手段，从细胞、基因、蛋白质、信号通路、动物等多层次水平上明确miR-136-5p靶向IKKβ/ NF-κB/A20调控IL-17介导的脊髓损伤后炎症反应的分子机制。

项目背景：脊髓损伤（spinal cord injury, SCI）是由直接或间接外力作用导致损伤平面以下感觉、运动、括约肌功能完全或不完全丧失等为主要特征的损伤。miRNA 在细胞增殖、分化、凋亡等生物过程中起着重要的作用，同时，miRNA 参与了血细胞生成、神经系统构成、炎症反应、癌细胞生长等生命活动的过程。miR-136-5p 通过靶向 NF-κB调控 IL-17介导的脊髓损伤后炎症反应，且国内外尚未有其防治脊髓损伤的相关报道。因此，本课题组拟在前期研究有所发现的基础上系统深入的探索miR-136-5p 通过靶向 NF-κB 调控 IL-17介导的脊髓损伤后炎症反应的相关机制。.主要研究内容：（1）明确 miR-136-5p和IL-17在小鼠脊髓星形胶质细胞中的生物作用机制，探讨miR-136-5p 通过靶向IKKβ/NF-κB/A20调控IL-17介导的脊髓损伤后炎症反应的机制。（2）探讨miR-136-5p在IL-17刺激下的细胞模型和动物模型中的作用机制。（3）构建慢病毒表达载体，探讨miR-136-5p对IL-17刺激下的细胞模型和动物模型的影响，明确miR-136-5p在炎症反应中的分子机制。.重要结果：体外培小鼠脊髓星型胶质细胞，经IL-17刺激星形胶质细胞后，结果表现为对IL-17的时间和剂量依赖性；慢病毒表达载体（LV-miR-136-5p-inhibition）感染星形胶质细胞，计算转染效率约为92%；miR-136-5p抑制表达载体在细胞内能够有效抑制内源性的miR-136-5p的产生；IL -17 刺激星形胶质细胞后NF-κB、炎性因子（IL-6、TNF-α、MCP-1/5、MIP -2）的表达均显著增加，但在不同时间 (3、6、12、24、48 h) 表达量差异较大。A20 mR-NA 的表达随时间呈下降趋势。动物实验发现miR-136-5p靶向IKKβ/NF-κB/A20调控脊髓损伤后炎症反应，激活通路下游靶基因的表达。.科学意义：该项目以细胞和动物为研究模型，对具有调节细胞炎症免疫反应的miR-136-5p在炎症反应中所产生的生物学效应进行研究，探索其调控脊髓损伤后炎症反应的分子机制，通过体内外实验证实了miR-136-5p具有明显防治脊髓损伤的作用，为防治脊髓损伤提供潜在的治疗靶点和开辟新的研究思路。