miR-29家族调控神经丛蛋白Plexins表达在视网膜神经节细胞损伤修复中的作用机制研究

Glaucoma, the second leading cause of blindness in the world, is a chronic optic neuropathy characterized by progressive retinal ganglion cell (RGC) axons degeneration and death leading to distinctive visual field defects. Although the pathogenesis of glaucoma is still largely unknown, it is hypothesized that RGCs become damaged through various mechanisms, including oxidative stress, excitotoxicity, defective axonal transport, trophic factor withdrawal, and neuroinflammation. Our preliminary study, observed that miR-29 family decreased significantly in development and optic nerve injury model, whereas increased Plexins expression in the retina. Overexpression of miR-29 into PC12 cells, observed reduced protein levels of Plexin A4 and activation of Akt-mTOR pathway. During induced pluripotent stem cells (iPSCs)-derived neuronal cells, we also observed that the antagomir-29 improved neuronal differentiation efficiency and maintained longer survive. Consequently, we intended to combine the genetically-modified mice, primary RGCs and iPSCs-derived neurons, exploring the biological functions of miR-29 family in the retinal development and restoration and regeneration after optic nerve injury. Aims to discover new target genes and determine the signaling pathways. Attempts to study the possibility and feasibility for the treatment of neurodegenerative diseases by targeting to miR-29 provide an important scientific basis for the development of new methods of diagnosis and treatment.

青光眼是全球第二位致盲性眼病，以进行性视网膜神经节细胞（RGC）死亡与轴突变性以及特异性视野缺损为主要特征的慢性视神经病变。目前认为RGC损害机制包括氧化应激、轴突运输障碍、营养因子缺乏和神经炎症等，但其发病机制尚不够清楚。我们发现，发育期和视神经损伤模型视网膜中miR-29家族的表达量降低，Plexins表达量却增多。PC12过表达miR-29发现，下调Plexin A4蛋白表达量，激活Akt-mTOR信号通路。诱导多能干细胞（iPS）定向分化神经元过程中转染miR-29抑制剂，发现神经元分化效率更高、存活更久。鉴于此，我们拟联合基因修饰鼠和原代RGCs、iPS分化神经元，进行miR-29家族在视网膜神经发育以及视神经损伤后修复再生中的功能研究，探索和验证新的靶基因，明确其调控信号通路。尝试通过调控miR-29的表达而治疗神经变性疾病的可能性及可行性，为开发诊疗新方法提供重要科学依据。

青光眼是全球首位不可逆致盲性眼病，是因为病理性高眼压等各种原因导致视神经萎缩、视野缺损，最终导致失明的一种神经退行性病变。以进行性视网膜神经节细胞（RGC）死亡与轴突变性为主要特征。目前认为RGC损伤机制包括氧化应激、轴突运输障碍、营养因子缺乏和神经炎症等，但分子机制复杂尚不够清楚。MiRNA是一种内源性的具有调控功能的非编码小分子RNA，通过引导沉默复合体降解mRNA或阻碍其翻译。MiRNA可以影响数百个基因的表达和翻译，一个基因也可受到多个miRNAs的靶向调控，通过多条信号通路共同参与其同一病理生理过程。本研究旨在探索miR-29家族在视网膜神经生长发育以及视神经损伤保护中的功能作用，并验证miR-29是否通过调控靶基因Plexin A4，介导下游信号通路发挥其生物学功能。研究结果显示，miR-29家族在视网膜发育和神经分化过程中持续上调，预测靶基因Plexin A4在视网膜发育过程中下调；miR-29a敲除小鼠视网膜miR-29a缺失影响视网膜多种神经细胞发育，视网膜功能紊乱；miR-29b-3p过表达抑制NPC增殖，促进体外神经元的诱导分化效率；慢性高眼压和视神经夹伤模型小鼠视网膜均表现为miR-29家族表达下调和Plexin A4上调；视网膜过表达miR-29b-3p和miR-29b2/c 基因敲入小鼠具有显著的视神经损伤保护作用，Plexin A4敲除小鼠与miR-29b-3p过表达相似，同样具有显著的视神经损伤保护作用；进一步机制研究证明，Plexin A4确定为miR-29b-3p的靶基因，miR-29b-3p通过抑制其靶基因Plexin A4，一方面激活mTOR信号通路，另一方面调控DNA甲基化水平，从而发挥视神经损伤的保护作用。本研究的完成，为青光眼等视神经退行性疾病的发病机制研究拓宽了理论边界，为新的视神经退行性病变治疗药物靶点的开发提供了坚实的数据支持。