miRNA-424 调节细胞周期对缺血脑损伤的作用及机制

Ischemic stroke is burdened with a high morbidity and mortality in our society. The major target of clinical and basic research in cerebral ischemia is searching for new therapeutic target. MicroRNAs (miRNAs) are small (typically ≈22 nt in size) regulatory RNA molecules that function to modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes. The miRNAs are detectable and stable in human peripheral blood, and are considered to be new biomarkers and therapeutic targets for neurodegenerative diseases. However, only few miRNAs are functionally demonstrated in the cerebral ischemia. In our previous clinical research, the expression of miRNA-424 is significantly decreased in the circulating leukocytes of patients with acute cerebral infarction. And further in the mouse model of focal cerebral ischemia, miRNA-424 is also gradually reduced in the ipsilateral cortex at 4h, 8h and 24h after ischemia. Based on the above results and the in situ hybridization analysis of miR-424 on aldult mouse brain, we proposed that miRNA-424 might directly or indirectly participate in the pathology of acute cerebral ischemia. The present study is aiming to clarify the time-dependent expression pattern of miRNA-424 by using the animal and cell models, and further demonstrate the effect and possible mechanism of miRNA-424 in the pathological progress of cerebral ischemia,including clarify the function of miRNA-424 on brain damage and neurological function after acute cerebral ischemia by systemic and local overexpression of miRNA-424, and its effect on neuronal and microglial cell cycle through its target genes. We will provide a new therapeutic target for clinical intervention at an early stage after stroke.

脑卒中是高致残率、致死率的疾病，寻找其治疗靶点一直是缺血性卒中临床基础研究的主要目标。microRNAs在血清/血浆中稳定存在，近来已成为多种疾病的生物标志物和治疗靶点，但目前仅有少数miRNAs在脑缺血中的作用得到阐明。我们前期临床实验发现，miRNA-424在急性脑梗死患者外周血白细胞中显著减低；在小鼠脑缺血模型中发现，miRNA-424在缺血脑组织皮层中呈逐步降低的趋势；结合小鼠脑组织原位杂交结果分析，miRNA-424可能直接或间接参与了缺血脑损伤病理过程的调节。本项目拟利用动物和细胞模型，明确miRNA-424在缺血脑组织中表达和分布变化的时间规律；证实miRNA-424在脑缺血神经元损伤和神经功能障碍的作用；从明确miRNA-424对脑缺血后神经元、小胶质细胞的细胞周期的作用入手，明确其调节的靶基因，阐明其作用及机制，为急性缺血性卒中的早期治疗提供新的靶点。

研究背景：缺血性脑卒中是高致残率、致死率的疾病，寻找其治疗靶点一直是临床基础研究的主要目标。microRNAs在血液中稳定存在，是多种疾病的生物标志物和治疗靶点，但目前仅少数miRs 在脑缺血中的作用及机制得到阐明。研究内容：确定miR-424 与缺血脑损伤的关系，从细胞周期调控角度阐明其作用机制。研究结果：临床研究发现：急性缺血性卒中患者血浆中的miR-424水平显著下调，其表达与患者的barthel指数呈正相关，提示miR-424水平与神经功能损伤程度有关，血浆中miR-424水平高，行动能力可能会更好。进一步的基础研究发现：在持续脑缺血模型中，miR-424在缺血侧脑皮层、海马、基底节中均随着缺血时间的延长呈逐步降低的趋势；在脑缺血-再灌注模型中，miR-424在缺血1h/再灌注1h时表达显著高于假手术组，之后逐渐下降，在缺血1h/再灌注24h时候显著低于假手术组。在此基础上，结合动物实验与细胞实验，通过体内外转染miR-424高表达慢病毒和miR-424 mimics，证明miR-424对2种小鼠脑缺血模型（持续脑缺血、脑缺血-再灌注）神经损伤存在保护作用；其机制部分通过靶向下调细胞周期激活蛋白CDK6、Cyclin D1、CDC25A的表达，进而抑制小胶质细胞的增殖周期，减少炎症因子TNF-α、IL-1β在体内体外的表达（Stroke. 2013, 44(6): 1706-1713. IF=6.018，引用36次。论文获得2013年Brain & Brain PET的青年学者奖）。除了对小胶质细胞的活性调控作用外，miR-424还对神经元有直接的保护作用。miR-424通过上调缺血后脑组织中转录因子Nrf-2及其下游的抗氧化蛋白MnSOD、ecSOD的表达及活性，在体内减轻小鼠脑缺血-再灌注损伤，在体外抑制过氧化氢诱导的原代皮层神经元的氧化应激损伤（Stroke. 2015, 46(2):513-519. IF=6.018，引用5次）。此外，miR-424慢病毒侧脑室注射，可增加小鼠持续脑缺血脑组织中转录因子PU.1和 HIF-1a、及抗凋亡蛋白p53的表达，从而减轻神经细胞凋亡（中国比较医学杂志. 2013, 23 (6): 6-11.）。本课题证明了miR-424在脑缺血急性期神经损伤中的作用及机制，为缺血性脑卒中的治疗提供了新的潜在靶点。