MiR-137-3p在撕脱伤运动神经元凋亡中的作用和机制

Spinal roots avulsion is a kind of trauma which induces motor functional loss because of the spinal motoneuron apoptosis. Up to now, we can not repair the hand movement in clinic even with a sucessful surgery to rebuild the connection between the spinal cord and the target muscles. The reason is because the regenerative velocity of the motor nerves is slower compared to the distance of the spianl motoneurons and the intrinsic hand muscles. So, it is necessary to explore the mechanism of avulsion-induced motoneurons apoptosis so that we can inhibit the velocity of the motoneurons apoptosis, meanwhile to enhance the regenerative capacity of the spinal motoneurons after surgery. We have focused on mechanism of motoneurons apoptosis for 15 years. We have proved that the avulsion-induced nNOS protein expressions in adult spianl motoneurons represent the beginnning of motoneurons apoptosis instead of regeneration. However, downregulate the nNOS mRNAs in the spianl cord could not stop but accelerate the avulsion-induced motoneurons apoptosis. It suggestes that the nNOS gene was manupulated posttranslationally after avulsion injury. During the recent 3 years, we have studied microRNAs expression patterns in the spinal cord after roots avulsion. We prvoed that miR-137-3p is obviously upregulated and it's targets gene, Calpain2 is downregulated inside injured motoneurons. Since Calpain2 acts on cleavage of NOS proteins, and both nNOS protein inside motoneurons and iNOS protein inside glial cells have been proved to upregulated during the apoptosis of injured motoneurons, we therefore hypothesis the mechanism of avulsion-induced motoneurons apoptosis is the "miR-137-3p upregulation-Calpain2 downregulation-nNOS, iNOS upregulation; and NO overdose in the injured spinal cord-oxidative stress". In the present study, we will construct the lentivirus vectors with miR-137-3p or with miR-137-3p sponges; we also wants to construct the miR-137-3p sponges transgene mice. The lentivirus vectors will be tranfused to the spinal cord and the motoneurons after brachial roots avulsion of the adult rats. After the knock-in and knock-down of the miR-137-3p genes in injured spianl cords, we will study whether these method can save or accelarates avulsion-induced motoneruons apoptosis, and whether manupulation of the miR-137-3p genes will block the "Calapin2/nNOS,iNOS/NO; Caspase-3/oxidative stress" pathway. Finally we will answer the question that whether miR-137-3p can be used as a molecular taget for the treatment of motoneurons diseases.

神经根撕脱伤导致运动神经元凋亡的机制不明，因此临床无法完全修复患者的运动功能。前期研究我们证实：创伤导致运动神经元内nNOS基因在转录后表达水平的改变是不可逆凋亡的关键，但不明了调控nNOS基因的机制。近期我们发现撕脱伤后脊髓miR-137-3p显著上调，其靶基因Calpain2蛋白显著下调。鉴于Calpain2能通过降解NOS蛋白下调nNOS基因并与Caspase-3的激活相关；本项目提出:"miR-137-3p↑→Calpain2↓→nNOS和iNOS积聚→NO过量→过氧化;Caspase-3激活→运动神经元凋亡"的假说；并拟构建慢病毒载体转染撕脱伤大鼠脊髓，同时建立miR-137-3p海绵转基因小鼠，通过上调和下调脊髓miR-137-3p基因水平，比较调控前后运动神经元内上述各基因的表达变化，定量创伤运动神经元的凋亡程度，最终确定miR-137-3p是能阻止运动神经元凋亡的关键分子。

臂丛根性撕脱伤会引起运动神经元的大量死亡，前期的研究表明运动神经元的凋亡涉及一系列分子事件。撕脱伤后mRNA芯片筛查发现促神经元存活和轴突再生的基因表达下降，而与凋亡和DNA损伤相关的基因表达上升；但是调控这些基因变化的分子机制、以及这些变化的基因在运动神经元凋亡进程中的作用并未完全明了。微小RNA（microRNA）是一类非编码RNA，通过翻译后抑制靶蛋白的表达水平而发挥作用。目前越来越多的研究证明：在脊髓损伤后miRNAs的表达发生了变化。但是对于臂丛撕脱伤和运动神经元凋亡进程中miRNAs表达变化的研究十分有限，本课题组长期开展臂丛根性撕脱伤的基础研究，发现撕脱伤后凋亡和再生基因表达变化具有时间特异性，损伤后3天内检测凋亡和再生基因均显著异常，而损伤后14天则以凋亡相关基因和过氧化反应异常为主，结合撕脱伤导致运动神经元的显著凋亡也从损伤后14d开始，本研究选择撕脱伤后3和14天的节点，研究损伤脊髓内在miRNA的生物学特性，以期揭示miRNAs在撕脱伤中的作用和机制。研究表明成年大鼠臂丛根性撕脱伤诱导脊髓内在miRNAs出现差异表达的miRNAs，且这些差异表达的miRNAs表达具有时空特异性 。其中miR-137-3p在撕脱伤后2周表达下降。体外培养的神经细胞模型中验证了miR-137-3p的靶基因Capn2，且下调Calpain-2基因的表达能抑制神经细胞中nNOS蛋白的表达水平。确定了miR-137-3p-calpain-2-nNOS通路在H2O2诱导PC12细胞凋亡中的作用机制，上调miR-137-3p能减少神经细胞的过氧化损伤。确定了脊髓内定位微注射miR-137-3p慢病毒载体，能转染外源性miR-137-3p入活体动物损伤的运动神经元胞体且持续作用3周，达到上调miR-137-3p表达水平的效果。确定了miR-137-3p-calpain-2-nNOS通路在臂丛根性撕脱伤运动神经元凋亡中的作用机制，上调miR-137-3p能减少撕脱伤运动神经元的凋亡。为以miR-137-3p-calpain-2-nNOS为分子靶标，治疗运动神经元疾病提供了新的研究思路。