MicroRNA-124–Mediated Regulation of Inhibitory Member of Apoptosis-Stimulating Protein of p53 Family in Experimental Stroke

Abstract

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.

MicroRNA-424 Protects Against Focal Cerebral Ischemia and Reperfusion Injury in Mice by Suppressing Oxidative Stress

“…35 The efficacy of miR-424 through intravenous injection or intranasal delivery in a mouse model of stroke will be the subject of future investigations. 19 …”

“…Samples were processed for western blot analysis as previously described. 19 Primary antibodies were as follows: rabbit monoclonal antimanganese (Mn)SOD (1:800), mouse monoclonal antiextracellular (Ec) SOD (1:500), and rabbit polyclonal anti-Nrf2 (1:800; all from Abcam, Cambridge, UK) and rabbit polyclonal antiβ-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), which was used as a loading control. Bands were detected using horseradish peroxidase-conjugated secondary (n=10).…”

“…MiR-424 angomir and the negative control were purchased from GenePharma (Shanghai, China) and administered as previously described 15 : 9 μL of miR-424 angomir or the control (100 μM) were mixed with 2.5 μL Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA) and incubated at 37°C for 30 minutes; 7 μL of the mixture was administered immediately by right intracerebroventricular injection, 19 and the needle was kept in place for 10 minutes before MCAO.…”

“…Ангомир микроРНК-424 и отрицательный контроль приобрели в GenePharma (Шанхай, Китай) и вводи-ли способом, описанным ранее [15] [19], иглу удерживали в месте инъекции в течение 10 минут до ОСМА.…”

“…Образцы ткани обрабатывали для проведения вестерн-блоттинга мето-дами, описанными ранее [19]. Использовали следую-щие первичные антитела: кроличьи моноклональные анти Mn-СОД (1:800), мышиные моноклональные антиэкстрацеллюлярные (Ес) СОД (1:500) и кроли-чьи поликлональные анти-Nrf2 (1:800; все из Abcam, Кембридж, Великобритания), а также кроличьи поли-клональные антитела antiβ-актин (1:1000; Santa Cruz Biotechnology, Санта Круз, Калифорния) в качест-ве контроля загрузки.…”