长链非编码RNA CASC15促进小胶质细胞活化参与急性脊髓损伤的机制研究

It is well known that the hyper-activation of microglia is an important pathological basis of damage after spinal cord injury. The molecular mechanism of microglia activation after spinal cord injury remains unclear. Our preliminary results showed that CASC15 expression was significantly upregulated in lesion of spinal cord injured mice and BV-2 cells with the treatment of LPS. Down-regulation of CASC15 contributed to the decreased expression of IL-1β and TNFα. In addition, the level of CASC15 was closely related the expression of miR-199b/IKKβ/NF-κB and positively controlled by C/EBPβ in BV-2 cells exposed to LPS. We therefore speculated that the activation of C/EBPβ led to the increase of CASC15 expression, resulting in the regulation of miR-199b/IKKβ/NF-κB pathway, which promotes the activation of microglia and its mediated neuronal apoptosis in the development of spinal cord injury. In this study, in vitro and in vivo models will be established and a series of molecular biology techniques are used to investigate the role and molecular mechanism of CASC15 in the activation of microglia, aiming to providing potential target for the prevention and treatment of spinal cord injury.

小胶质细胞过度活化是脊髓损伤的重要病理基础，然而脊髓损伤后小胶质细胞过度活化的分子机制尚不清楚。本课题前期研究显示，急性脊髓损伤小鼠损伤局部及LPS刺激的小胶质细胞BV-2中CASC15表达显著上调，干扰CASC15降低LPS刺激的BV-2中IL-1β、TNFα表达，此外CASC15表达与活化的BV-2内miR-199b-5p/IKKβ/NF-κB轴表达密切相关，且受C/EBPβ正调控，因此推测，脊髓损伤后，C/EBPβ上调CASC15表达，CASC15通过调节miR-199b-5p/IKKβ/NF-κB通路促进小胶质细胞炎性反应，诱发神经元凋亡，参与脊髓损伤发展。本项目拟通过建立急性脊髓损伤小鼠模型及LPS刺激的BV-2细胞模型，结合多种分子生物学技术，从分子、细胞和模型动物多层面研究CASC15调节小胶质细胞活化参与脊髓损伤的作用及其分子机制，旨在为脊髓损伤的防治提供新的潜在作用靶点。

本课题主要探究长链非编码RNA（lncRNA）通过调节小胶质细胞M1极化和神经炎症进而影响急性脊髓损伤（SCI）进展的作用机制。本研究发现，lncRNA MEG3在SCI小鼠脊髓组织和LPS处理的小胶质细胞中表达均显著下调，而且lncRNA MEG3过表达可以通过调控NF-κB信号抑制小胶质细胞M1极化和神经炎症。通过文献挖掘发现NF-κB信号通路抑制蛋白A20可发挥炎症抑制作用，并在MEG3过表达的小胶质细胞中表达上调，进一步干扰A20表达则会逆转MEG3对NF-κB信号通路的调控，说明MEG3通过上调A20表达抑制NF-κB信号。为了进一步明确MEG3调控A20表达的分子机制，通过生物信息学分析发现MEG3可能与HuR蛋白结合，其中HuR蛋白被证实能够促进A20 mRNA的降解作用。采用RIP和RNA pull-down实验验证MEG3与HuR蛋白的相互作用，并进一步证实MEG3能够促进HuR蛋白的泛素化降解，进而抑制HuR蛋白对A20 mRNA的降解作用，上调A20表达。进一步实验结果表明，MEG3通过HuR/A20/NF-κB信号轴调控小胶质细胞M1型极化及神经炎症，且MEG3过表达能够促进SCI小鼠的运动功能恢复，并抑制脊髓组织中的小胶质细胞M1型极化，缓解神经炎症。由此可得，MEG3通过HuR/A20/NF-κB信号轴调控抑制小胶质细胞M1型极化及神经炎症，进而缓解急性脊髓损伤。