miR-150靶向调控血管内皮生长因子影响脑梗死后神经康复和血管新生

Effect of miR-150 targeting vascular endothelial growth factor on neurological rehabilitation and angiogenesis after cerebral infarction

摘要:

目的：探讨miR-150对脑缺血大鼠血管结构的保护作用及促进血管生成的作用机制。方法：SD大鼠被分为4组（n=15）：假手术（Sham）组、中动脉闭塞（middle cerebral artery occlusion，MCAO）组、MCAO+miR-150组和MCAO+模拟物阴性对照（miR-NC）组。除Sham组外，其余组采用电凝法建立大鼠大脑MCAO模型。MCAO+miR-NC组和MCAO+miR-150组分别注射PEG脂质体包埋miR-NC或miR-150（2.4 mg/kg，iv）。建模后第14天，观察MCAO大鼠神经功能、神经损伤和血管生成的变化。建立缺氧葡萄糖剥夺（oxygen-glucose deprivation，OGD）脑微血管内皮细胞（brain microvascular endothelial cells，BMECs）模型，研究miR-150促进血管生成的作用及其可能机制。荧光素酶活性测定证实miR-150与血管内皮生长因子（vascular endothelial growth factor，VEGF）的结合位点。结果：体内实验结果表明，MCAO大鼠脑梗死组织中miR-150较Sham组明显下调（1.00±0.03 vs. 0.34±0.04，P<0.05）。miR-150上调可降低脑缺血大鼠的神经功能缺损评分（第7天：6.9±1.0 vs. 5.3±0.6，第14天：6.4±0.7 vs. 3.9±0.5，均P<0.01）和梗死体积[（13.8±1.6）% vs. （5.1±0.6）%，P<0.001]，促进血管生成。通过荧光素酶报告基因分析进一步证实VEGF基因是miR-150的靶基因，并且在miR-150处理的MCAO大鼠脑梗死组织中观察到VEGF表达上调。体外实验结果表明，miR-150上调能增加OGD诱导的BMECs中VEGF/VEGFR2共表达，并促进其管形成。机制分析显示miR-150促进血管生成作用与激活VEGF通路有关，SU1498可抑制miR-150的激活作用。结论：miR-150通过靶向VEGF调节MCAO大鼠的神经功能缺损和血管生成。

Abstract:

Objective：To investigate whether miR-150 promotes angiogenesis in cerebral ischemic rats and to identify its possible mechanisms. Methods：SD rats were divided into four groups（n=15）：Sham group，MCAO（middle cerebral artery occlusion） group，MCAO+miR-150 group and MCAO+miR-NC group. Except for the Sham group，the other groups established MCAO models by electrocoagulation method. MCAO+miR-NC group and MCAO+miR-150 group were injected with PEG liposome embedded miR-NC or miR-150（2.4 mg/kg，iv），respectively. On the 14th day after modeling，the changes of neurological function，nerve damage and angiogenesis of MCAO rats were observed. The oxygen-glucose deprivation（OGD） brain microvascular endothelial cells（BMECs） models were established to determine the roles and possible mechanisms of miR-150 in angiogenesis. The binding site between miR-150 and vascular endothelial growth factor（VEGF） was verified by luciferase activity assay. Results：In vivo，miR-150 was signifi－cantly down-regulated in MCAO rats compared with the Sham group（1.00±0.03 vs. 0.34±0.04，P<0.05）. Up-regulation of miR-150 reduced neurological deficit scores（day 7：6.9±1.0 vs. 5.3±0.6，day 14：6.4±0.7 vs. 3.9±0.5，both P<0.01） and infarct volume[（13.8±1.6）% vs. （5.1±0.6）%，P<0.001]，and promoted angiogenesis in cerebral ischemic rats. Luciferase reporter gene analysis confirmed that VEGF gene was the target gene of miR-150，and the up-regulation of VEGF expression was observed in MCAO rats treated with miR-150. In vitro，up-regulation of miR-150 increased the co-expression of VEGF/VEGFR2 in OGD induced BMECs and promoted the tube formation. Mechanism analysis showed that the effect of miR-150 on promoting angiogenesis was related to the activation of VEGF pathway，and the activating effects of miR-150 were inhibited by SU1498. Conclusion：miR-150 regulates neurologic impairment and angiogenesis in MCAO rats by targeting VEGF.