RNA结合蛋白RALY调控甲基化CpG结合蛋白MBD1介导神经病理性疼痛的机制研究

Neuropathic pain, one of major clinical diseases, causes heavy burden on families and communities in healthcare expense because its treatment is ineffective. Understanding molecular mechanisms that underlie neuropathic pain genesis may open a new avenue in the management of this disorder. Recent studies showed that RNA binding proteins can regulate the expression of many signal transduction molecules and participate in the genesis of many diseases. Our preliminary study showed that RALY, a RNA binding protein, was up-regulated in the dorsal root ganglion (DRG) following peripheral nerve injury and that this up-regulation was positively correlated with hyperalgesia of mice with neuropathic pain. Knocking-down the expression of RALY in primary sensory neurons of DRG rescued this hyperalgesia. Further study revealed that RALY co-localized with methyl-CpG binding domain protein 1（MBD1）and the expression of MBD1 was regulated by RALY. Furthermore, eukaryotic translation initiation factor 4 gama 2 (EIF4G2) may contribute to the increased translation of MBD1. Therefore, we hypothesize that up-regulation of RALY in DRG induced by nerve injury activates EIF4G2. The latter enhances translation of the proteins including MBD1, resulting in MBD1-controlled down-regulation of MOR and Kv1.2 in the primary sensory neurons of DRG and neuropathic pain development. This project will clarify the role of RALY in neuropathic pain and elucidate the mechanism in vitro and in vivo. These studies are expected to explore novel molecular mechanism that underlies neuropathic pain and provide fundamental insight for RALY as the target of novel therapeutic options in the prevention and treatment of this disorder.

临床上针对神经病理性疼痛（NP）缺乏有效的治疗方法，究其原因是NP的机制尚未阐明。文献提示RNA结合蛋白调节多种信号传导分子表达，其表达异常与多种疾病相关。预实验发现NP小鼠背根神经节（DRG）中RNA结合蛋白RALY表达上调，且与痛敏行为正相关；在体敲减DRG中RALY表达可缓解NP小鼠痛觉敏化；进一步研究发现RALY与甲基化CpG结合蛋白MBD1在DRG神经元中共表达且MBD1蛋白表达受RALY调控；同时RALY可能调节翻译起始因子EIF4G2的表达。据此我们推测：神经损伤时，DRG中表达上调的RALY通过EIF4G2促进MBD1等蛋白翻译，进而参与NP的发生发展。本项目将从分子、细胞、组织和在体动物等层面明确DRG中RALY在NP发生发展中的作用，探索RALY通过EIF4G2调控MBD1表达的分子机制。研究结果有助于阐明NP发生发展的分子机制，为探索慢性疼痛的治疗方案提供实验依据。

目的: 探索RNA结合蛋白RALY调控神经病理性疼痛的机制。.方法: 在雄性CD1小鼠CCI模型建立后的第0天、第3天、第7天、第14天分别测定DRG中RALY蛋白和mRNA的表达。应用免疫荧光法检测RALY的细胞分布。分别应用AAV5-ShRNA-RALY和AAV5-RALY在小鼠背根神经节（DRG）内进行显微注射后并测定行为学改变。此外，检测甲基化cpG结合结构域蛋白(MBD1)和真核翻译起始因子4 gama2(EIF4G2)的表达。应用单细胞PCR技术研究RALY、MBD1和EIF4G2的共定位。用Co-IP方法验证了RALY与RNA 聚合酶II之间的相互作用。通过ChIP检测，研究 RALY/RNA聚合酶II与EIF4G2启动子的相互作用。.结果: 外周神经损伤后，RALY在DRG中呈时程性上调，这种上调与痛觉过敏呈正相关。RALY蛋白主要与β-tublinIII(一种特定的神经元标记物)共定位，但很少与GS(一种卫星神经胶质细胞标记物)共定位，表明RALY主要在DRG神经元中表达。RALY标记的神经元与神经丝200(NF200)、异凝集素B4(IB4)和降钙素基因相关肽(CGRP)共定位。通过抑制DRG中RALY的表达，缓解了CCI小鼠的痛觉过敏。DRG中RALY过表达诱导小鼠痛觉过敏。进一步的研究显示DRG中RALY 的敲低和过度表达调节了甲基-CpG 结合结构域蛋白MBD1和真核翻译起始因子4 gama2(EIF4G2)的表达。RALY与MBD1和EIF4G2共定位于DRG 神经元。CO-IP结果表明RALY与RNA聚合酶II相互作用。 RALY-RNA聚合酶II复合物可以与EIF4G2启动子相互作用，从而促进EIF4G2的转录。.结论: 神经损伤引起的DRG中RALY的上调转录激活EIF4G2。后者促进了包括MBD1在内的蛋白翻译，导致MBD1控制的DRG初级感觉神经元中的MOR和Kv1.2的下调并促进神经性疼痛的发生。RALY可能成为预防和治疗这种疾病的新的治疗选择的靶点。