EZH2基因通过多重表观调控Semaphorin3D激活施万细胞的功能促进周围神经再生的机制研究

Schwann cells play a key role in the process of peripheral nerve regeneration. To explore the effective way to activate function of Schwann cell will contribute to the repair of peripheral nerve injury. Based on the systematic analysis and preliminary experimental results, we found that EZH2 was significantly up-regulated after transection of sciatic nerve in rats, which was related to the the activation of Schwann cells function. Therefore, EZH2 may be an important speed-limiting molecule for the regeneration of peripheral nerve injury. We have screened that Semaphorin3D (Sema3D) is the target gene of EZH2 by silencing of EZH2 in Schwann cells, combined transcriptome sequencing, ChIP-seq and MeDIP-seq technology. So we put forward the hypothesis: after sciatic nerve injury, the upregulated EZH2 may target the function of its down-stream gene (Sema3D) by histone methylation and DNA methylation, and further promote proliferation, migration of Schwann cell and axon guidance, which promote the recovery of nerve function. This project intends to deeply investigate the EZH2 function, its target Sema3D gene in Schwann cells and the process of peripheral nerve regeneration and their regulatory mechanism that clarify the repairing mechanism of peripheral nerve injury from the regulatory level of epigenetics. The expected results of this project wil provide new overviews for the research and treatment of peripheral nerve injury.

施万细胞在周围神经再生过程中起着关键作用，探寻有效途径激活施万细胞功能将有助于周围神经的损伤修复。我们在前期系统性分析和预实验结果中发现：大鼠坐骨神经离断后EZH2的表达显著上调，并与施万细胞功能的激活相关。因而EZH2可能是周围神经损伤再生的重要限速分子。基于EZH2的多重表观调控机制，我们在施万细胞中沉默EZH2后联合转录组测序、ChIP-seq及MeDIP-seq技术筛选出EZH2下游的靶基因Semaphorin3D（Sema3D）。为此我们提出假说：坐骨神经损伤后上调的EZH2可能通过组蛋白甲基化及DNA甲基化来调控靶基因Sema3D的功能，进而促进施万细胞增殖、迁移及轴突导向功能，有利于神经功能的恢复。本课题拟通过深入研究EZH2与Sema3D在施万细胞及周围神经再生过程中的功能和调控机制，从表观调控层面阐明周围神经损伤修复的机制，为周围神经损伤的研究和治疗提供新的思路。

EZH2是一种组蛋白甲基转移酶，其在周围神经损伤再生过程中的作用及相关调控机制尚不清楚。本项目研究发现EZH2在周围神经损伤后表达显著上调，并与施万细胞功能的激活相关。EZH2基因沉默后抑制了施万细胞的增殖、迁移及分化能力。在大鼠坐骨神经桥接模型中过表达EZH2促进髓鞘再生。通过转录组测序、H3K27me3 ChIP-seq、EZH2 ChIP-seq及MeDIP-seq技术联合分析，结合施万细胞的功能，筛选出EZH2/H3K27me3直接调控的靶基因Sema3D并验证。结果表明EZH2介导了组蛋白甲基化及DNA甲基化，进而调控Sema3D的表达。通过施万细胞与神经元共培养模型发现施万细胞中EZH2、Sema3D的表达影响神经元轴突生长。结合体内大鼠坐骨神经桥接模型的结果，提示EZH2/Sema3D调控途径对周围神经再生起到调控作用。通过对EZH2/Sema3D上游调控因子核心信号流分析，找到EZH2上游调控miR-26a并得到验证。以上通过深入研究EZH2与Sema3D在施万细胞及周围神经再生过程中的功能和调控机制，从表观调控层面阐明周围神经损伤修复的机制，为周围神经损伤的研究和治疗提供新的靶点。