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OBJECTIVEIt has been reported that microRNA-195 (miR-195) protects against chronic brain injury induced by chronic brain hypoperfusion. However, neither the expression profile of miR-195 nor its potential role during acute ischemic stroke has been investigated. In this study, the authors' aim was to verify the mechanism of miR-195 in acute ischemic stroke.METHODSThe plasma levels of miR-195 expression were assessed using real-time PCR in 96 patients with acute ischemic stroke, and the correlation with the National Institutes of Health Stroke Scale score was evaluated. In addition, cerebral infarct volume, neurological score, and levels of miR-195 and CX3CL1/CX3CR1 mRNA and protein expression were assessed in mice subjected to middle cerebral artery occlusion (MCAO) with or without intra-cerebroventricular infusion of lentiviral vector. The inflammatory cytokines tumor necrosis factor-α (TNFα), interleukin (IL)-1ß, and IL-6 of mouse brains after MCAO and BV2 cells treated with oxygen-glucose deprivation were measured using enzyme-linked immunosorbent assay, and apoptotic proteins were examined by Western blotting. Direct targeting of CX3CL1/CX3CR1 by miR-195 was determined by immunoblotting and dual luciferase assay.RESULTSIn ischemic stroke patients, miR-195 was significantly downregulated and expression levels of miR-195 in these patients negatively correlated with the National Institutes of Health Stroke Scale score. In mice after MCAO, miR-195 overexpression decreased infarct volume, alleviated neurological deficits, and most importantly, suppressed an inflammatory response. Meanwhile, miR-195 suppressed the expression of the inflammatory cytokines TNFα, IL-1ß, and IL-6 in vitro and in vivo. The authors further discovered that both CX3CL1 and CX3CR1 are direct targets of miR-195, but miR-195 exerts neuroprotective roles mainly through inhibiting CX3CR1-mediated neuroinflammation and subsequent neuronal cell apoptosis.CONCLUSIONSTaken together, these findings suggest that miR-195 promotes neuronal cell survival against chronic cerebral ischemic damage by inhibiting CX3CR1-mediated neuroinflammation. This indicates that miR-195 may represent a novel target that regulates neuroinflammation and brain injury, thus offering a new treatment strategy for cerebral ischemic disorders.

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OBJECTIVE: Ischemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke-related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke. METHODS: The JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)-induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3' untranslated region (3'UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase-9 [MMP-9], tumor necrosis factor-α [TNF-α], and interleukin-1ß [IL-1ß]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits. RESULTS: JAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3'UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1ß). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit. CONCLUSIONS: These findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.

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OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival signals in neuronal cells responsible for hypoxiainduced apoptotic insults through activation of a CREB/cIAP2-dependent mechanism.

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