K2p1.1基因甲基化参与化疗诱导神经病理性疼痛的表观遗传机制研究

Chemotherapy induced neuropathic pain (CINP) is closely related to peripheral neurotoxicity and there is barely successful pharmacological treatment by now. Previously, we found that chemotherapy agents can lead to an increase of DNA methyltransferase 3a (DNMT3a) and a decrease of K2p1.1 in DRG neurons. K2p1.1 plays a crucial role in the maintenance of the resting membrane potential of dorsal root ganglion (DRG) neurons and the downregulation of K2p1.1 expression contributes to neuronal hyperexcitability. However its detailed transcriptional regulation is still unknown. We hypothesize that DNMT3a may participate the downregulation of K2p1.1 in DRG via methylation of its gene promoter. To prove this hypothesis, we will use DRG-specific DNMT3a conditional knockout mice and check whether DNMT3a expression changes can affect the expression of K2p1.1, DRG neuronal electrophysiology and the pain-like behavior of animal, in both cellular and animal levels. We will use molecular biotechnology methods to verify the detailed mechanism underlying DNMT3a-mediated DNA methylation of K2p1.1 gene. The goal of this project is to clarify the role of down-regulation of DRG K2p1.1 via DNA methylation in the development of CINP and provide theoretical basis for the treatment of CINP.

化疗诱导神经病理性疼痛（化疗痛）与外周神经毒性损伤密切相关，目前仍无有效治疗方法。在前期研究中，我们发现化疗药物可引起背根神经节（DRG）神经元内DNA甲基转移酶3a（DNMT3a）增高和K2p1.1表达下调。K2p1.1维持DRG神经元膜静息电位，其表达下降可增加神经元兴奋性，是病理性疼痛的潜在治疗靶点，但是其转录调控机制尚不明确。我们提出假说，K2p1.1表达降低可能是由于DNMT3a甲基化K2p1.1基因启动子区，抑制基因转录所致。本课题拟利用DRG特异性DNMT3a敲除小鼠，从细胞和动物水平观察DNMT3a对K2p1.1的表达影响，以及DRG神经元电生理和动物行为学的相应改变；利用分子生物学技术验证DNMT3a对K2p1.1基因的甲基化调控；旨在阐明DNMT3a介导的K2p1.1表达下调在化疗痛发病过程中的作用，为临床治疗提供理论依据。

项目背景：目前临床上缺乏治疗化疗诱导神经病理性疼痛（化疗痛）的有效手段，因此探索化疗痛发病机制对优化临床治疗策略具有重要意义。在前期研究中，我们发现化疗药物可引起背根神经节（DRG）神经元内DNMT3a增高、K2p1.1降低；特异性敲除DRG神经元内的DNMT3a，可以阻断化疗药物引起的K2p1.1表达下调，缓解化疗痛；染色体免疫共沉淀实验发现，DNMT3a可特异性结合K2p1.1基因启动子区。我们推测DNMT3a可能是通过甲基化K2p1.1基因启动子区，降低其在DRG内表达并诱发疼痛。.主要研究内容和结果：1、通过siRNA敲低DRG内K2p1.1表达，发现K2p1.1下调可以引起背根神经节神经元兴奋性增加和小鼠疼痛行为；而病毒逆转DRG内K2p1.1表达下调可以缓解化疗痛模型的疼痛行为。2、在DRG内过表达DNMT3a，发现DNMT3a表达增加可以引起K2p1.1下调；条件基因敲除DRG神经元内DNMT3a，发现DNMT3a表达减少可以引起K2p1.1上调，并缓解化疗痛。3、通过离体培养神经元和DRG在体实验证实，化疗痛模型背根神经节内DNMT3a增加可引起K2p1.1基因启动子区甲基化水平增加，DNMT3a敲除可以引起K2p1.1基因启动子区甲基化水平降低。此外我们还对化疗痛小鼠背根神经节进行全转录组测序，筛选了可能参与K2p1.1表达调控的circRNA。.结论：通过上述研究结果，我们基本阐明DNMT3a介导的K2p1.1减少致DRG神经元兴奋性增加是化疗痛的发病机制之一，揭示K2p1.1基因表达的表观遗传学机制，为临床治疗化疗痛提供新理论和新靶点。