miRNA-128调节脑缺血损伤细胞周期的作用及机制

The key problem of basic research on cerebral ischemia is to find new promising therapeutic target. MicroRNAs (MiRNAs) are considered to be biomarkers and therapeutic targets for a variety of diseases. Our previous clinical research displayed that, in acute ischemic stroke patients, miRNA-128 is in the center of immune function-regulating network in lymphocytes by miRNA microarray analysis, and the expression of miRNA-128 is notably decreased in the circulating lymphocytes, while significantly increased in the plasma. In the mouse model of focal cerebral ischemia/reperfusion injury, the expression of miRNA-128 is gradually reduced in the penumbra at 1h, 4h and 24h after reperfusion, while in infarction core, the expression of miRNA-128 is decreased at 1 h after reperfusion but increased at 4h, 24 h after reperfusion. In the hydrogen peroxide-stimulated Neuro2a cells and PC12 cells, miRNA-128 regulates the cell cycle and inhibits apoptosis. The above results indicated that miRNA-128 might directly or indirectly participate in the pathology of focal cerebral ischemia/reperfusion injury. Our present study is aiming to clarify the expression pattern of miRNA-128 in development and prognosis of ischemic stroke. Then further demonstrate the effect and possible mechanism of miRNA-128 in focal cerebral ischemia/reperfusion injury, including clarify the function of miRNA-128 on brain damage and neurological function by up-regulating or down-regulating the expression of miRNA-128, and its effect on cell cycle of neuron and microglia through its target genes, supplying a new therapeutic target for acute ischemic stroke.

寻找有前景的神经损伤保护新靶点，是目前缺血性脑卒中基础研究的核心问题。miRNAs是多种疾病的生物标志物和治疗靶点。我们前期对急性脑梗死患者外周血淋巴细胞的miRNA芯片研究发现miRNA-128位于调节免疫功能的网络中心，其表达在淋巴细胞中显著降低，在血浆中显著增高；在小鼠脑缺血再灌注模型中，miRNA-128表达在梗死周边区随时间逐渐降低，在梗死核心区先降低后逐渐增高；在Neuro2a、PC12氧化应激细胞模型中miRNA-128可调节细胞周期并抑制细胞凋亡，提示miRNA-128可能直接或间接参与了脑缺血-再灌注损伤病理过程。本项目拟明确miRNA-128在脑梗死发生发展及预后中的变化规律；证实miRNA-128对小鼠脑缺血神经细胞损伤和神经功能障碍的作用；从明确miRNA-128对脑缺血后神经细胞的细胞周期的作用入手，明确其靶基因，阐明其机制，为急性脑梗死的早期治疗提供新的靶点。

MiR-128是人脑中含量最高的miRNAs之一，已有报道miR-128通过抑制P38α MAPK来保护大脑中动脉栓塞模型（MCAO）小鼠。它是否参与了急性缺血性卒中患者的发病机制尚待确定。本课题集中在miR-128对缺血性卒中的临床重要性及其潜在机制上。我们采用RT-PCR方法检测急性缺血性脑卒中患者外周血淋巴细胞、中性粒细胞和血浆miR-128水平，发现急性缺血性脑卒中患者外周血淋巴细胞、中性粒细胞和血浆miR-128水平显著升高。此外，我们对miR-128与临床重要指标进行了相关性分析，发现循环淋巴细胞miR-128水平与脑梗死体积、7天NIHSS评分和90天mRS评分呈正相关。随后的芯片KEGG通路分析表明，MAPK信号通路和细胞周期是miR-128靶向的通路之一。我们构建了MCAO小鼠模型，发现miR-128在缺血半暗带区域减少，在同侧脑组织梗死核心区域增加。此外，体外研究我们分离了小鼠原代神经元，发现miR-128拮抗剂可加重OGD/R刺激诱导的原代神经元损伤和细胞周期再激活，其机制可能与增加细胞周期蛋白cyclin A2、PTEN和ERK的表达及促进PTEN和ERK的磷酸化有关。以上的临床和基础研究的结果提示，急性缺血性脑卒中患者外周血淋巴细胞miR-128的上调与脑卒中的严重程度有关，miR-128拮抗剂通过促进神经元细胞周期的激活而加重缺血再灌注诱导的神经元损伤。我们的研究结果为临床急性缺血性卒中的预后提供了一个新的标志物，并未急性缺血性卒中的治疗提供了一个新的表观调控靶点。