miR-183在肌萎缩侧索硬化中的作用及机制研究

Amyotrophic lateral sclerosis (ALS）is a fatal neurodegenerative disease that mainly affects pyramidal upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. Accumulative evidences suggest dysregulation of microRNAs has emerged as a new mechanism involved in ALS pathogenesis, although the accurate pathogenic mechanism of ALS remains unclear. In our previous study, compared with PD and healthy controls, we found the expression of miR-183 was specifically decreased in ALS. In addition, the lower-expression of miR-183 was also found in the leukocytes and gastrocnemius of the pre-symptomatic (60 days after born) and early symptomatic (90 days after born) stage SOD1G93A mice than that in wild type mice. In cell experiment, we found over-expression of miR-183 promoted NSC34 cells proliferating, and suppressed NSC34 cells apoptosis by transfected the miR-183 mimics. Combination the results of the mRNA microarray by transfecting miR-183 mimics or miR-183 inhibitor and bioinformatics analysis, we speculate miR-183/EGR1/PI3K-Akt and miR-183/EGR1/p53 axis play an important role in the development of ALS. In the present project, we plan to fully explore the role and pathogenesis of miR-183 in amyotrophic lateral sclerosis by cell and animal experiments. In the NSC34 cell and primary mice neuron, we will analyze the cell cycles, proliferation and apoptosis after over-expression or down-expression of miR-183 or EGR1, and detect the expression of the relevant mRNA/protein of PI3K-Akt and p53 signaling pathway, in order to certify the pathogenic mechanism of miR-183-EGR1 involve in the neuronal death. In addition, we will prepare over-expression or down-expression of miR-183 of SOD1G93A mice by medullary cavity injection, the loss of neuron and proliferation of neuroglia will be tested in the different stage of these mice after born, such as 30 days, 60 days and 90 days, in order to reveal the role of miR-183 are related to the development and progression of ALS. Overall, the results of the present project will provide novel insights into the mechanisms of miR-183 contribute to ALS, and identify genes and pathways as potential targets for further development of the therapeutic reagents.

肌萎缩侧索硬化（ALS）是一种严重致死性神经变性疾病，研究表明miRNA 异常表达参与了ALS的发病，然而其具体机制不清。申请者前期工作发现 ALS 患者和SOD1G93A小鼠miR-183表达降低，细胞实验发现miR-183影响了NSC34细胞的增殖和凋亡，结合基因表达谱结果，推测miR-183可能通过EGR1介导的PI3K-Akt和p53通路在ALS运动神经元的死亡中发挥重要作用。本研究拟通过干扰NSC34细胞或小鼠原代神经元中miR-183或EGR1的表达，观察这两种细胞生物行为学的改变，检测PI3K-Akt和p53通路相关分子的表达变化，明确miR-183-EGR1在神经元凋亡坏死中的作用机制；同时在动物水平，检测不同时期miR-183过表达/表达抑制的SOD1G93A小鼠脊髓、大脑皮层运动神经元的丢失，胶质细胞的增生，探讨miR-183对ALS发生发展的影响。

肌萎缩侧索硬化(ALS)是一种病因不明，发病后进展迅速，而又缺乏有效治疗手段的严重致死性神经系统变性疾病。目前该病发病机制仍不十分清楚，因此进一步深入探讨该病新的可能的发病机制，对于开发新的有效药物或探究新的治疗方法显得十分迫切和重要。在既往的研究中，通过临床样本我们发现4个miRNA的异常表达可能参与了ALS的发病，进一步分析发现相较于其他3个miRNA，miR-183可能作为一个特异性的miRNA在ALS中表达下调。因此，本项目拟在前期的研究基础上进一步探讨miR-183在ALS发生发展中的作用及其机制。. 在本研究中，我们从四个方面进行了研究：1）收集ALS患者及ALS小鼠模型（SOD1G93A小鼠），在不同的病程中外周血白细胞，肌肉和腰段脊髓组织，检测miR-183的表达情况，比较miR-183在ALS发生发展过程中的表达模式及中枢与外周的可比性；2）利用运动神经元样细胞（NSC34），通过miR-183的模拟物和抑制物探讨miR-183对细胞增殖、凋亡和周期的影响；3）结合细胞表达谱、细胞生物学功能分析及既往的研究，挑选miR-183导致细胞凋亡最可能的靶基因及通路进行机制研究。4）通过SOD1G93A小鼠过表达或表达抑制miR-183，检测miR-183对SOD1G93A小鼠的影响。.我们发现miR-183在ALS发生的早期表达升高，而在疾病的进展期及晚期，miR-183表达进行性降低，同时发现中枢的miR-183表达变化与外周miR-183的表达变化一致。通过在NSC34细胞过表达miR-183或者使miR-183表达抑制，发现miR-183能够减少NSC34细胞的凋亡，增强细胞活力，缩短细胞周期。进一步机制研究发现，miR-183靶向调控EGR1的表达，通过miR-183/EGR1/PTEN/PI3K-Akt和miR-183/EGR1/p53通路是miR-183导致运动神经元损伤。.因此，我们的研究结果提示：miR-183在ALS的发展早期表达的上调可能是生物体的一种诱导的保护机制；在ALS的发展中晚期，这种反馈机制破坏，通过下调miR-183的表达，而抑制PI3K-Akt信号通路，增加p53的表达，从而促进细胞凋亡以增加神经元的损伤。因此，通过对疾病中晚期干预miR-183的表达，对延缓ALS疾病的进展提供新的靶点。