Cerebrovascular Diseases Research Institute, Xuanwu Hospital of Capital Medical University, Key Laboratory of Neurodegenerative Diseases (Capital Medical University), Ministry of Education, Beijing, China.
Stroke. 2013 Jul;44(7):1973-80. doi: 10.1161/STROKEAHA.111.000613. Epub 2013 May 21.
p53-mediated neuronal death is a central pathway of stroke pathophysiology, but its mechanistic details remain unclear. Here, we identified a novel microRNA mechanism that downregulation of inhibitory member of the apoptosis-stimulating proteins of p53 family (iASPP) by the brain-specific microRNA-124 (miR-124) promotes neuronal death after cerebral ischemia.
In a mouse model of focal permanent cerebral ischemia, the expression of iASPP and miR-124 was quantified by reverse transcription quantitative real-time polymerase chain reaction, immunofluorescence staining, and Western blot. Luciferase reporter assay was used to validate whether miR-124 can directly bind to the 3'-untranslated region of iASPP mRNA. To evaluate the role of miR-124, miR-124 mimic and its inhibitor were transfected into Neuro-2a cells and C57 mice.
There was no change in the iASPP mRNA level in cerebral ischemia. However, iASPP protein was remarkably decreased, with a concurrent elevation in miR-124 level. Furthermore, miR-124 can bind to the 3'-untranslated region of iASPP in 293T cells and downregulate its protein levels in Neuro-2a cells. In vivo, infusion of miR-124 decreased brain levels of iASPP, whereas inhibition of miR-124 enhanced iASPP levels and significantly reduced infarction in mouse focal cerebral ischemia.
These data demonstrate that p53-mediated neuronal cell death after stroke can be nontranscriptionally regulated by a novel mechanism involving suppression of endogenous cell death inhibitors by miR-124. Further dissection of microRNA regulatory mechanisms may lead to new therapeutic opportunities for preventing neuronal death after stroke.
p53 介导的神经元死亡是中风病理生理学的中心途径，但其机制细节仍不清楚。在这里，我们发现了一种新的 microRNA 机制，即脑特异性 microRNA-124（miR-124）下调凋亡刺激蛋白 p53 家族的抑制成员（iASPP）可促进脑缺血后神经元死亡。
在局灶性永久性脑缺血的小鼠模型中，通过逆转录定量实时聚合酶链反应、免疫荧光染色和 Western blot 定量 iASPP 和 miR-124 的表达。荧光素酶报告基因测定用于验证 miR-124 是否可以直接结合 iASPP mRNA 的 3'-非翻译区。为了评估 miR-124 的作用，将 miR-124 模拟物及其抑制剂转染到 Neuro-2a 细胞和 C57 小鼠中。
脑缺血时 iASPP mRNA 水平没有变化。然而，iASPP 蛋白显着减少，同时 miR-124 水平升高。此外，miR-124 可以与 293T 细胞中的 iASPP 3'-非翻译区结合，并下调 Neuro-2a 细胞中的蛋白水平。在体内，miR-124 的输注降低了脑内 iASPP 的水平，而 miR-124 的抑制增强了 iASPP 的水平，并显着减少了小鼠局灶性脑缺血的梗死。
这些数据表明，中风后 p53 介导的神经元细胞死亡可以通过一种新的机制进行非转录调节，该机制涉及 miR-124 抑制内源性细胞死亡抑制剂。进一步剖析 microRNA 调节机制可能为预防中风后神经元死亡提供新的治疗机会。
