Micro-RNA-30a Regulates Ischemia-Induced Cell Death by Targeting Heat Shock Protein HSPA5 in Primary Cultured Cortical Neurons and Mouse Brain After Stroke
Micro-RNA-30a Regulates Ischemia-Induced Cell Death by Targeting Heat Shock Protein HSPA5 in Primary Cultured Cortical Neurons and Mouse Brain After Stroke
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Micro-RNA-30a 通过靶向原代培养的皮质神经元和中风后小鼠大脑中的热休克蛋白 HSPA5 来调节缺血诱导的细胞死亡
DOI：
10.1002/jnr.23637
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发表时间：
2015-11-01
影响因子：
4.2
通讯作者：
Li, Junfa
中科院分区：
文献类型：
--
作者：
Wang, Peng;Zhang, Nan;Li, Junfa
micro-RNAs（miRs）已成为许多疾病（包括中风）中的关键基因调控因子。我们最近报道了miR-30 a保护N2 A细胞免受缺血性损伤，部分是通过增强beclin 1介导的自噬。本研究进一步探讨了miR-30 a在原代皮层神经元和脑卒中小鼠脑缺血诱导的细胞凋亡中的作用及其可能机制。我们证明，miR-30 a水平显着降低皮质神经元后1小时氧-葡萄糖剥夺（OGD）/24小时复氧。通过3-（4，5-二甲基-2-噻唑基）-2，5-二苯基-2H-溴化四氮唑、乳酸脱氢酶、TUNEL和切割的caspase-3测定，miR-30 a的过表达加重了OGD诱导的神经元细胞死亡，而miR-30 a的抑制减弱了坏死和凋亡。通过TargetScan预测HSPA 5蛋白是miR-30 a的推定靶点，其量可被pre-miR-30 a减少，而被anti-miR-30 a增加。此外，荧光素酶报告基因测定证实miR-30 a直接结合到预测的hspa 5基因的30-UTR靶位点。HSPA 5 siRNA可加重OGD处理细胞中miR-30 a调控的细胞损伤。我们还观察到的相互作用的HSPA 5和caspase-12的免疫共沉淀，推测HSPA 5可能参与内质网应激诱导的细胞凋亡。在体内，减少miR-30 a增加了HSPA 5水平，并减轻了局灶性缺血中风小鼠的缺血性脑梗死。下调miR-30 a可通过上调HSPA 5蛋白表达来保护神经缺血损伤，减少内质网应激诱导的细胞凋亡可能是HSPA 5介导神经保护作用的机制之一。(C)2015 Wiley Periodicals，Inc.
Micro-RNAs (miRs) have emerged as key gene regulators in many diseases, including stroke. We recently reported that miR-30a protects N2A cells against ischemic injury, in part through enhancing beclin 1-mediated autophagy. The present study explores further the involvement of miR-30a in ischemia-induced apoptosis and its possible mechanisms in primary cortical neurons and stroked mouse brain. We demonstrate that miR-30a level is significantly decreased in cortical neurons after 1-hr oxygen-glucose deprivation (OGD)/24-hr reoxygenation. Overexpression of miR-30a aggravated the OGD-induced neuronal cell death, whereas inhibition of miR-30a attenuated necrosis and apoptosis as determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-di-phenyl-2H-tetrazolium bromide, lactate dehydrogenase, TUNEL, and cleaved caspase-3. The amount of HSPA5 protein, which is predicted to be a putative target of miR-30a by TargetScan, could be reduced by pre-miR-30a, whereas it was increased by anti-miR-30a. Furthermore, the luciferase reporter assay confirmed that miR-30a directly binds to the predicted 30-UTR target sites of the hspa5 gene. The cell injury regulated by miR-30a in OGD-treated cells could be aggravated by HSPA5 siRNA. We also observed an interaction of HSPA5 and caspase-12 by coimmunoprecipitation and speculate that HSPA5 might be involved in endoplasmic reticulum stress-induced apoptosis. In vivo, reduced miR-30a increased the HSPA5 level and attenuated ischemic brain infarction in focal ischemia-stroked mice. Downregulation of miR-30a could prevent neural ischemic injury through upregulating HSPA5 protein expression, and decreased ER stress-induced apoptosis might be one of the mechanisms underlying HSPA5-mediated neuroprotection. (C) 2015 Wiley Periodicals, Inc.