MicroRNA-124-Mediated Regulation of Inhibitory Member of Apoptosis-Stimulating Protein of p53 Family in Experimental Stroke
MicroRNA-124-Mediated Regulation of Inhibitory Member of Apoptosis-Stimulating Protein of p53 Family in Experimental Stroke
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DOI：
10.1161/strokeaha.111.000613
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发表时间：
2013-07-01
期刊：
影响因子：
8.3
通讯作者：
Ji, Xunming
中科院分区：
文献类型：
--
作者：
Liu, Xiangrong;Li, Fang;Ji, Xunming
背景和目的 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p53家族凋亡刺激蛋白（iASPP）的抑制性成员促进脑缺血后神经元死亡。 方法-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印迹来定量iASPP和miR-124的表达。使用荧光素酶报告基因测定来验证 miR-124 是否可以直接结合 iASPP mRNA 的 3'-非翻译区。为了评价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。结果-脑缺血时iASPP mRNA水平没有变化。然而，iASPP 蛋白显着降低，同时 miR-124 水平升高。此外，miR-124 可以与 293T 细胞中 iASPP 的 3'-非翻译区结合，并下调 Neuro-2a 细胞中其蛋白水平。在体内，输注 miR-124 降低了 iASPP 的脑水平，而抑制 miR-124 增强了 iASPP 水平，并显着减少了小鼠局灶性脑缺血的梗塞。结论-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涉及 miR-124 抑制内源性细胞死亡抑制剂的新机制进行非转录调节。进一步剖析 microRNA 调节机制可能会为预防中风后神经元死亡带来新的治疗机会。
Background and Purpose-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.Methods-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.Results-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.Conclusions-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.