Ma Qingyi, Dasgupta Chiranjib, Li Yong, Huang Lei, Zhang Lubo
Center for Neonatal Biology, Division of Pharmacology, Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
Int J Mol Sci. 2017 Jun 24;18(7):1356. doi: 10.3390/ijms18071356.
Cerebral edema, primarily caused by disruption of the blood-brain barrier (BBB), is one of the serious complications associated with brain injury in neonatal hypoxic-ischemic encephalopathy (HIE). Our recent study demonstrated that the hypoxic-ischemic (HI) treatment significantly increased microRNA-210 (miR-210) in the neonatal rat brain and inhibition of miR-210 provided neuroprotection in neonatal HI brain injury. The present study aims to determine the role of miR-210 in the regulation of BBB integrity in the developing brain. miR-210 mimic was administered via intracerebroventricular injection (i.c.v.) into the brain of rat pups. Forty-eight hours after the injection, a modified Rice-Vannucci model was conducted to produce HI brain injury. Post-assays included cerebral edema analysis, western blotting, and immunofluorescence staining for serum immunoglobulin G (IgG) leakage. The results showed that miR-210 mimic exacerbated cerebral edema and IgG leakage into the brain parenchyma. In contrast, inhibition of miR-210 with its complementary locked nucleic acid oligonucleotides (miR-210-LNA) significantly reduced cerebral edema and IgG leakage. These findings suggest that miR-210 negatively regulates BBB integrity i n the neonatal brain. Mechanistically, the seed sequences of miR-210 were identified complementary to the 3' untranslated region (3' UTR) of the mRNA transcripts of tight junction protein occludin and adherens junction protein β-catenin, indicating downstream targets of miR-210. This was further validated by in vivo data showing that miR-210 mimic significantly reduced the expression of these junction proteins in rat pup brains. Of importance, miR-210-LNA preserved the expression of junction proteins occludin and β-catenin from neonatal HI insult. Altogether, the present study reveals a novel mechanism of miR-210 in impairing BBB integrity that contributes to cerebral edema formation after neonatal HI insult, and provides new insights in miR-210-LNA mediated neuroprotection in neonatal HI brain injury.
脑水肿主要由血脑屏障（BBB）破坏引起，是新生儿缺氧缺血性脑病（HIE）脑损伤相关的严重并发症之一。我们最近的研究表明，缺氧缺血（HI）处理显著增加了新生大鼠脑中的微小RNA-210（miR-210），抑制miR-210可对新生HI脑损伤提供神经保护作用。本研究旨在确定miR-210在发育中大脑血脑屏障完整性调节中的作用。将miR-210模拟物通过脑室内注射（i.c.v.）注入幼鼠脑内。注射后48小时，采用改良的Rice-Vannucci模型制造HI脑损伤。检测后分析包括脑水肿分析、蛋白质印迹法以及血清免疫球蛋白G（IgG）渗漏的免疫荧光染色。结果显示，miR-210模拟物加剧了脑水肿以及IgG向脑实质的渗漏。相反，用其互补的锁核酸寡核苷酸（miR-210-LNA）抑制miR-210可显著减轻脑水肿和IgG渗漏。这些发现表明，miR-210对新生脑血脑屏障的完整性起负向调节作用。从机制上来说，已确定miR-210的种子序列与紧密连接蛋白闭合蛋白和黏附连接蛋白β-连环蛋白的mRNA转录本的3'非翻译区（3'UTR）互补，表明这是miR-210的下游靶点。体内数据进一步证实了这一点，该数据显示miR-210模拟物显著降低了幼鼠脑中这些连接蛋白的表达。重要的是，miR-210-LNA可使闭合蛋白和β-连环蛋白的表达免受新生HI损伤的影响。总之，本研究揭示了miR-210损害血脑屏障完整性从而导致新生HI损伤后脑水肿形成的新机制，并为miR-210-LNA介导的新生HI脑损伤神经保护作用提供了新见解。
