CWC22调控Nnat前体RNA可变剪接介导的慢性炎性疼痛机制研究

Chronic pain pathological conditions can induce change in synaptic function through the alteration of transcription of many genes, resulting in the neurons membrane receptors, ion channels and intracellular signal transduction pathway disorders. However the molecular mechanisms underlying chronic pain development remain poorly understood. Several studies have shown that RNA alternative splicing act as a post-transcriptional regulator may be involved in the development of chronic pain through regulating expression of pain-related genes. We detected RNA express profiling in the spinal cord of persistent inflammatory pain model mice induced by complete Freund’s adjuvant (CFA) by high-throughput RNA sequencing ,we found some alternative splicing transcripts different expression, include Neuronatin(Nnat) encodes a membrane protein in the endoplasmic reticulum (ER), functions as triggering Ca2+ signaling. It's well know that Ca2+ acted as a key factor for chronic pain in many years.Also we found that CWC22, a RNA splicesome activating factor was significantly increased 3-days after CFA injection. Furthermore,intrathecal injection of aiRNA (one type of RNA interference) of CWC22 could significantly relieve sensitized pain behavior and decreased mRNA level of Nnat transcript variant 1(NnatV1). This project aim to elucidate the roles and mechanisms of CWC22 and target gene NnatV1 in chronic inflammatory pain.It would provide direct evidence RNA alternative splicing take part in chronic pain and provide potential therapeutic targets of chronic pain.

外周和中枢神经系统基因差异表达，引起神经元膜上受体、离子通道和胞内信号转导通路改变，导致神经元敏化是慢性疼痛发生发展的主要原因，少数研究发现RNA可变剪接与疼痛相关。前期研究证实在疼痛状态下，剪接体活化因子CWC22在脊髓腰膨大区差异表达，同时发现定位于内质网参与调节细胞内Ca2+浓度的neuronatin（Nnat）的剪接体之一NnatV1也发生显著差异表达，干扰CWC22后可逆转这一现象。本项目以CWC22为切入点，应用CFA慢性炎性疼痛小鼠模型，从整体、细胞和分子水平研究、阐明CWC22对Nnat前体RNA可变剪接的调节作用及机制，揭示CWC22通过调控Nnat可变剪接调节Ca2+浓度介导的疼痛信息传递功能，预期结果将揭示CWC22调控Nnat前体RNA可变剪接的新机制，为RNA可变剪接参与慢性疼痛的发生发展提供直接的科学证据，及基于RNA可变剪接的慢性疼痛治疗和药物研发提供新靶标。

慢性疼痛能影响身体的各个系统，引起严重的健康问题。RNA可变剪接介导的基因调控可产生功能不同的蛋白从而参与调控神经系统，而与神经系统关系很密切的慢性疼痛可能与RNA可变剪接相关，但具体调控机制仍不明确。研究表明CWC22是可变剪接的重要调控因子，本项目旨在探讨CWC2是否通过调控可变剪接参与慢性炎性疼痛。我们发现CFA小鼠脊髓腰膨大CWC22的表达水平明显高于正常小鼠，下调CFA小鼠CWC22表达可以缓解热疼痛反应，上调正常小鼠CWC22表达诱发热疼痛敏感。通过RNA芯片测序分析发现Spp1可变剪接体3和4（Spp1 V3和Spp1 V4）受CWC22调控，下调CFA小鼠Spp1蛋白表达同样可以延长热缩足潜伏期，缓解疼痛。同时我们发现只有上调正常小鼠Spp1 V4的表达可以介导疼痛反应。因此我们的研究表明CWC22可以通过介导Spp1的可变剪接参与调控慢性炎性疼痛。这一发现为为RNA可变剪接参与慢性炎性疼痛提供直接科学证据，同时丰富疼痛神经等相关学科基础理论内容，为开发新的药物及治疗方案提供靶标。