背根神经节Nrcam基因选择性剪接参与神经病理痛的外周机制研究

Neuropathic pain is one of the most refractory and painful diseases in clinic. It is lack of effective drug therapy due to unclear mechanisms. The plasticity of the primary sensory afferent neurons located in the dorsal root ganglion (DRG) constitutes the biological basis of neuropathic pain. We did RNA sequencing in DRG from spinal nerve ligation (SNL) induced mouse neuropathic pain model, and found that SNL led to decreased exon skipping of Nrcam gene exon 10. The expression of Nrcam+10 RNA with exon 10 was increased in injuried DRG. Suppression of exon 10 could reduce SNL-induced pain hypersensitivity. The involvement of Nrcam+10 in neuropathic pain may be mediated by epidermal growth factor receptor (EGFR)-activated PI3K/Akt intracellular signaling pathway, and hnRNP F may act as an upstream regulatory element by inhibiting Nrcam alternative splicing in neuropathic pain. In this study, we will investigate hnRNP F regulating Nrcam exon 10 skipping involve in the peripheral mechanisms of neuropathic pain by affecting the activation of EGFR and its cellular signaling pathway PI3K/Akt in DRG using molecular biology, cell biology, immunohistochemistry, animal pain behavior test and etc. This study will provide new strategies and ways for clinical analgesia.

神经病理痛是临床上最为难治的疼痛疾病之一，由于机制不清，尚缺乏有效药物治疗。位于背根神经节（DRG）的初级传入感觉神经元可塑性变化构成了神经病理痛发生的生物学基础。我们对脊神经结扎（SNL）小鼠受损DRG进行RNA测序，发现SNL抑制 Nrcam基因10号外显子遗漏性剪接，含有10号外显子的RNA异构体Nrcam+10表达增加，抑制其表达能够减轻SNL引起的疼痛超敏反应。表皮生长因子受体（EGFR）激活的PI3K/Akt细胞内信号通路可能是Nrcam+10调控神经病理痛的关键途径，hnRNP F则可能作为上游调控元件通过抑制Nrcam选择性剪接参与神经病理痛。本研究利用分子生物学、细胞生物学、免疫组织化学和动物痛行为学等多种技术在DRG水平研究了Nrcam选择性剪接受hnRNP调控并通过影响EGFR/PI3K/Akt信号通路参与神经病理痛的发生机制，为临床镇痛提供了新的策略和方法。

神经病理痛是临床上最为难治的疼痛疾病之一，由于机制不清，尚缺乏有效药物治疗。本项目针对位于背根神经节（Dorsal root ganglion, DRG）的外周感觉神经元应用脊神经结扎（Spinal nerve ligation, SNL）的小鼠神经病理痛模型研究了编码细胞粘附分子NrCAM的基因Nrcam的可变剪接参与神经病理痛的外周机制。重要结果包括：1. RNA测序数据分析发现SNL导致Nrcam基因10号外显子可变剪接改变，具有10号外显子的Nrcam+10 mRNA表达量增加，而不具有10号外显子的Nrcam-10 mRNA 表达量减少，针对10号外显子的Nrcam 反义寡核苷酸Nrcam ASO能够减少Nrcam+10 mRNA表达量而增加Nrcam-10 mRNA表达量。Nrcam ASO能够减轻SNL、CCD等神经病理痛模型小鼠的痛觉过敏行为。2. Co-IP实验显示NrCAM蛋白与表皮生长因子受体（Epidermal growth factor receptor, EGFR）存在结合，而DRG神经元原代培养实验也显示Nrcam ASO能够影响EGFR受体的表达，说明Nrcam ASO 能够调控EGFR受体表达。3. DRG神经元原代培养实验显示Nrcam ASO能够抑制各种类型DRG神经元的轴突生长。4. SNL、CCD等创伤性神经病理痛小鼠模型DRG 的EGFR及其磷酸化形式p-EGFR表达上升，抑制EGFR活性或小干扰RNA技术敲低EGFR能够反转神经病理痛模型小鼠的病理痛行为，同时，敲低EGFR导致其细胞内mTOR信号通路的激活被抑制。这些结果表明NrCAM及其下游EGFR信号通路是神经病理痛镇痛研究的靶点，Nrcam ASO具有临床镇痛应用的潜力，可变剪接是神经病理痛机制研究的新方向。