miR-145靶向调控Nav1.8致糖尿病神经痛缓解的机制研究

miR-145 was specially low-expressed in the DRG's of painful diabetic neuropathy rat, a highly conserved microRNA identified from differential microRNA expression profile in dorsal root ganglion (DRG) neurons between normal and painful diabetic neuropathy rat. Bioinformatic analysis and experiment data indicated that Nav1.8 was the target gene of miR-145. Previous study reported that activation of Nav1.8 was the basis of abnormal neuronal excitability and pain production. In our previous study, overexpression of miR-145 downregulated the expression of Nav1.8, indicating that miR-145 could be involved in pain relief of Painful Diabetic Neuropathy. This study will confirm the role of miR-145 in pain production and pain relief of Painful Diabetic Neuropathy. Using overexpression and knock down rescue strategies in the Dorsal root ganglion cells to clarify the underlying mechanism between miR-145 and gene Nav1.8. By developing different animal models of pain, we assessed the role of miR-145-Nav1.8 in other pain reliefs. Up to now, there was no report about the function and mechanism of miR-145 by controlling Nav1.8 in regulating pain reliefs. This study may provide a new target for the gene targeted therapeutics of pain relief.

miR-145是我们通过基因芯片发现的、人鼠高度同源的、低表达于糖尿病神经痛大鼠DRG中的miRNA; 背根神经节中过表达miR-145可致大鼠糖尿病神经痛缓解。软件分析和实验证实Nav1.8是miR-145靶基因，已有研究报道Nav1.8活化是神经元兴奋异常和疼痛产生的基础，提示miR-145可能通过调控Nav1.8参与大鼠糖尿病神经痛缓解。本研究拟通过观察miRNA-145表达变化对糖尿病大鼠疼痛的影响，在体评估miRNA-145与糖尿病大鼠疼痛缓解的相关性；以Nav1.8为切入点，采用挽救实验策略，深入探讨miR-145调控Nav1.8表达缓解糖尿病神经痛的机制；通过构建不同疼痛动物模型，在体水平评估DRG组织中“miR-145-Nav1.8”调控机制在其他疼痛缓解中的作用。miRNA-145调控Nav1.8缓解糖尿病神经痛的机制未见报道，本研究有望为疼痛缓解防治提供新靶点。

目前临床对糖尿病神经痛药物治疗效果不佳，主要因为其发病机制不明确。本实验通过构建糖尿病神经痛大鼠模型，发现糖尿病神经痛大鼠背根神经节组织的miR-145mRNA表达降低伴Nav1.8表达上调，同时大鼠后肢的机械缩足阈值降低。利用Target Scan、PicTar和miRanda软件预测发现Nav1.8为miR-145潜在的作用靶点。为此我们构建miR-145过表达慢病毒，通过鞘内干预，发现在给药后3、7和14天，miR-145过表达组大鼠后肢机械缩足阈值升高，伴背根神经节组织的Nav1.8表达量下降。ji进一步通过构建Nav1.8干扰病毒，发现Nav1.8干扰组大鼠后肢机械缩足阈值明显升高，伴miR-145mRNA表达量增加。由此推断出miR-145可以通过抑制Nav1.8的表达，缓解糖尿病神经神经痛。Nav1.8是背根神经节上重要钠离子通道。为了探讨miR-145细胞水平调控机制，构建Nav1.8过表达慢病毒、Nav1.8干扰慢病毒、miR-145过表达慢病毒和miR-145感染慢病毒，分别感染DRG神经元，观察miR-145、Nav1.8表达变化和钠电流分布。结果提示Nav1.8过表达组中miR-145表达下降，而miR-145过表达组中Nav1.8表达降低；Nav1.8沉默组引起miR-145表达增加，而miR-145沉默组引起Nav1.8表达升高。 Nav1.8沉默组的总钠电流明显降低，即对总钠电流有明显的抑制率，而miR-145沉默组的总钠电流无明显降低；相反Nav1.8过表达组的电流则升高，计算总钠电流的抑制率呈现负值，而miR-145过表达组的电流明显降低。进一步证实miR-145可以通过抑制Nav1.8表达减低总钠电流，为糖尿病神经痛治疗提供一个新的思路。