受miRNA-1224靶向剪接调控的circleRNA-5252介导慢性炎性疼痛的分子机制研究

Chronic pain is a frequently-occurring and refractory disease, and lacks specific effective drugs in treatment. Central sensitization of spinal cord plays a critical role in the pathogenesis of chronic pain. The induction and maintenance of central sensitization are dependent on maladaptive alterations in the expression, distribution, and activity of receptors and ion channels on spinal neuronal membrane. Aberrant pain-related gene expression is one of most prominent contributors to these maladaptive processes. Therefore, the unraveling of the genetic basis and its regulatory mechanisms involved in central sensitization will improve our understanding of chronic pain and provide potential targets for developing novel therapeutic strategies. CircleRNA (cirRNA), a new regulatory mechanism of gene expression, attracts widespread attention on their potential role in complicated biological processes and human diseases. CirRNA is enriched in central nervous system, however, whether cirRNA could regulate chronic pain keeps unclear. In the preliminary experiment, the following critical results had been found. Firstly, cirRNA-5252 (cirR-5252) was significantly increased in spinal cord of Complete Freunds Adjuvant (CFA)-induced chronic inflammatory pain mice via high-throughput sequencing analysis. Knockdown of cirR-5252 with siRNA alleviated the pain sensitivity to the thermal or mechanical stimulus; on contrary, overexpression of cirR-5252 induced the production of the pain-like behavior. Secondly, cirR-5252 was predictively bound to the promoter of Ubr5, a ubiquitin ligase. Downregulation of cirR-5252 reversed the increased Ubr5 protein in spinal cord of CFA-induced pain mice. Additionally, knockdown of cirR-5252 with anti-RNA inhibited the pain behavior, suggesting Ubr5 is a possible positive regulatory target of cirR-5252. Thirdly, the decreased Kcnd2 protein, a voltage-gated potassium channel, was reversed by downregulation of Ubr5 in spinal cord of CFA-induced pain mice. Finally, miRNA-1224 was predicted to bind to the splicing point of cirR-5252 precursor. Overexpression of spinal miRNA-1224 inhibited the expression of spinal cirR-5252, and significantly alleviated pain induced by CFA. Therefore, the purpose of this study is to investigate whether cirR-5252 spliced by miRNA-1224 could modulate chronic inflammatory pain through ubiquitination-modified Kcnd2 by Ubr5 at the spinal cord level. This study will reveal a new functional regulatory mechanism underlying chronic pain process, which will provide a possible target for the development of analgesic drugs.

慢性疼痛是多原因引起的高发顽固性病症，且缺乏特异性治疗措施。脊髓神经元可塑性改变被认为是慢性痛发生的关键环节，该改变很大程度上依赖于基因调控引起的膜上受体和离子通道变化，但慢性疼痛基因调控机制仍不清楚。cirRNA丰富表达于中枢神经系统，是基因表达调控新方式。预试验发现：慢性痛下，小鼠脊髓cirR-5252增加显著，下调cirR-5252能减轻疼痛；泛素连接酶Ubr5启动子有1个cirR-5252结合位点，下调cirR-5252能抑制Ubr5表达；Ubr5降低则能逆转钾离子通道Kcnd2表达；cirR-5252前体剪接点能被miRNA-1224结合，过表达miRNA-1224能降低cirR-5252表达但其前体无变化。因此，本项目拟用CFA模型小鼠，阐明受miRNA-1224靶向剪接调控的cirR-5252通过Ubr5泛素化修饰Kcnd2介导慢性炎性痛机制，以期为研发理想镇痛药提供新思路。

1.项目研究背景：慢性疼痛是多原因诱发的高发和顽固性病症，临床上仍缺乏特异治疗措施，其原因主要是慢性痛基因调控机制仍不清楚。cirRNA丰富表达于中枢神经系统，是基因表达调控新方式。.2.主要研究内容：该课题从整体、细胞及分子水平围绕"miRNA-1224/cirRNA-5252（即cirRNA-Pilip1l）通过Ubr5脊髓神经元可塑性和疼痛调控机制"的科学假设，主要研究了：（1）目标cirRNA-Pilip1l的筛选、表征及其调控慢性炎性疼痛；（2）cirRNA-Pilip1l通过泛素连接酶Ubr5及其下游靶标调控疼痛；（3）miRNA-1224以Ago2依赖方式介导cirRNA-Pilip1l前体剪接及其调控疼痛。.3.重要结果、关键数据及其科学意义：（1）该研究发现，脊髓外周炎性损伤时，脊髓背角胞核内miRNA-1224表达降低，减轻了miRNA-1224（以Ago2依赖方式）对cirRNA-Pilip1l前体形成的抑制，增加了成熟cirRNA-Pilip1l表达；继而引起cirRNA-Pilip1l下游靶标Ubr5-mRNA转录增强和Ubr5蛋白表达增多。总之，该课题阐明了受miRNA-1224以Ago2依赖方式调控的cirRNA-Pilip1l通过Ubr5介导慢性炎性疼痛发生和维持的新机制。（2）该课题主要结果发表于The Journal of Neuroscience（2019，39(11): 2125–2143）。此外，课题相关延伸研究还探讨并揭示了DNA去甲基化（Anesthesiology ，2017, 127:147-63）、miRNA-23a（Journal of Neuroinflammation, 2018, 15(29): 1-19.）及RNA的m6A甲基化（2020年12月4日Pain接收）调控慢性炎性疼痛的机制。（3）2018年还获得课题相关国家发明专利授权3项（ZL201510587183.3、ZL201510587336.7和ZL201510354106.6）。（4）该课题不仅在学术上拓展了cirRNA神经生物学领域，而且为基于cirRNA及表观遗传修饰用于慢性疼痛临床治疗和药物研发提供新的靶标。