Stary Creed M, Xu Lijun, Sun Xiaoyun, Ouyang Yi-Bing, White Robin E, Leong Jason, Li John, Xiong Xiaoxing, Giffard Rona G
From the Department of Anesthesia, Stanford University School of Medicine, CA (C.M.S, L.X., X.S., Y.-B.O., J. Leong, J. Li, X.X., R.G.G.); and Department of Biology, Westfield State University, MA (R.E.W.).
Stroke. 2015 Feb;46(2):551-6. doi: 10.1161/STROKEAHA.114.007041. Epub 2015 Jan 20.
MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin.
Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot.
Pretreatment with miR-200c antagomir decreased post-middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3' untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection.
These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.
短暂性脑缺血后，大脑中微小RNA（miR）-200c迅速增加，但其在中风后脑损伤中的作用尚不清楚。Reelin是神经元迁移和突触形成的调节因子，是miR-200c的预测靶点。我们假设miR-200c通过靶向Reelin导致短暂性脑缺血损伤。
在接受1小时大脑中动脉闭塞的小鼠中，评估脑梗死体积、神经功能评分以及miR-200c、Reelin mRNA和Reelin蛋白水平，这些小鼠接受或未接受脑室内注射miR-200c拮抗剂、模拟物或错配对照。通过双荧光素酶测定和免疫印迹在体外评估miR-200c对Reelin的直接靶向作用。
用miR-200c拮抗剂预处理可降低大脑中动脉闭塞后脑内miR-200c水平，导致梗死体积和神经功能缺损显著减少。脑内miR-200c水平的变化与Reelin蛋白表达呈负相关。通过双荧光素酶测定验证了miR-200c对Reln 3'非翻译区的直接靶向作用。抑制miR-200c导致体外氧化损伤后细胞存活率增加。这种作用被Reelin mRNA敲低所阻断，而应用Reelin蛋白则提供保护。
这些发现表明，中风后miR-200c的增加通过抑制Reelin表达导致脑细胞死亡，降低中风后miR-200c是减轻中风诱导脑损伤的潜在靶点。
