Post-insult valproic acid-regulated microRNAs: potential targets for cerebral ischemia.
ABSTRACT: Stroke is a devastating brain injury that is a leading cause of adult disability with limited treatment options. Using a rat model of middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, we profiled microRNAs (miRNAs), small non-protein coding RNAs, in the ischemic cortex. Many miRNAs were confirmed by qPCR to be robustly upregulated 24 hours following MCAO surgery including miR-155, miR-297a, miR-466f, miR-466h, and miR-1224. In addition, we treated MCAO rats with valproic acid (VPA), a mood stabilizer and histone deacetylase inhibitor. This post-insult treatment was shown to improve neurological deficits and motor performance following MCAO. To provide mechanistic insight into the potential targets and pathways that may underlie these benefits, we profiled miRNAs regulated following this VPA treatment. Two promising post-insult VPA-regulated candidates were miR-331 and miR-885-3p. miR-331 was also regulated by VPA pre-treatment in rat cortical neuronal cultures subjected to oxygen-glucose deprivation, an in vitro ischemic model. The predicted targets of these miRNAs analyzed by Ingenuity Pathway Analysis (IPA) identified networks involved in hematological system development, cell death, and nervous system development. These predicted networks were further filtered using IPA and showed significant associations with neurological diseases including movement disorders, neurodegenerative disorders, damage to cerebral cortex, and seizure disorders among others. Collectively, these data support common disease mechanisms that may be under miRNA control and provide exciting directions for further investigations aimed at elucidating the miRNA mechanisms and targets that may yield new therapies for neurological disorders.
Project description:BACKGROUND AND PURPOSE:The migratory efficiency of mesenchymal stem cells (MSC) toward cerebral infarct after transplantation is limited. Valproate (VPA) and lithium enhance in vitro migration of MSC by upregulating CXC chemokine receptor 4 and matrix metalloproteinase-9, respectively. Ability of VPA and lithium to promote MSC homing and to improve functional recovery was assessed in a rat model of cerebral ischemia. METHODS:MSC primed with VPA (2.5 mmol/L, 3 hours) and/or lithium chloride (2.5 mmol/L, 24 hours) were transplanted into rats 24 hours after transient middle cerebral artery occlusion (MCAO). Neurological function was assessed via rotarod test, Neurological Severity Score, and body asymmetry test for 2 weeks. Infarct volume was analyzed by MRI. The number of homing MSC and microvessel density in the infarcted regions were measured 15 days after MCAO using immunohistochemistry. RESULTS:Priming with VPA or lithium increased the number of MSC homing to the cerebral infarcted regions, and copriming with VPA and lithium further enhanced this effect. MCAO rats receiving VPA-primed and/or lithium-primed MSC showed improved functional recovery, reduced infarct volume, and enhanced angiogenesis in the infarcted penumbra regions. These beneficial effects of VPA or lithium priming were reversed by AMD3100, a CXC chemokine receptor 4 antagonist, and GM6001, a matrix metalloproteinase inhibitor, respectively. CONCLUSIONS:Priming with VPA and/or lithium promoted the homing and migration ability of MSC, improved functional recovery, reduced brain infarct volume, and enhanced angiogenesis in a rat MCAO model. These effects were likely mediated by VPA-induced CXC chemokine receptor 4 overexpression and lithium-induced matrix metalloproteinase-9 upregulation.
Project description:Recently, long non-coding RNAs (lncRNAs) are considered as critical regulators in pathogenesis progression of cerebral ischemia. In present study, lncRNA-small nucleolar RNA host gene 14 (SNHG14) was found upregulated in middle cerebral artery occlusion/reperfusion (MCAO/R) treated brain tissues and oxygen-glucose deprivation and reoxygenation (OGD/R) treated PC-12 cells. Interference of SNHG14 by shRNA vector enhanced neuron survival and suppressed inflammation in response to OGD/R insult. SNHG14 positively regulated the expression of Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) via acting as a sponge of microRNA (miR)-136-5p. SNHG14 promoted neurological impairment and inflammatory response through elevating the expression of ROCK1 while decreasing miR-136-5p level in OGD/R induced damage. Collectively, we illustrated that SNHG14 could be a novel strategy for treatment ischemia stoke.
Project description:Previous studies have demonstrated a close relationship between abnormal regulation of microRNA (miRNA) and various types of diseases, including epilepsy and other neurological disorders of memory. However, the role of miRNA in the memory impairment observed in epilepsy remains unknown. In this study, a model of temporal lobe epilepsy (TLE) was induced via pentylenetetrazol (PTZ) kindling in Sprague-Dawley rats. First, the TLE rats were subjected to Morris water maze to identify those with memory impairment (TLE-MI) compared with TLE control rats (TLE-C), which presented normal memory. Both groups were analyzed to detect dysregulated miRNAs in the hippocampus; four up-regulated miRNAs (miR-34c, miR-374, miR-181a, and miR-let-7c-1) and seven down-regulated miRNAs (miR-1188, miR-770-5p, miR-127-5p, miR-375, miR-331, miR-873-5p, and miR-328a) were found. Some of the dysregulated miRNAs (miR-34c, miR-1188a, miR-328a, and miR-331) were confirmed using qRT-PCR, and their blood expression patterns were identical to those of their counterparts in the rat hippocampus. The targets of these dysregulated miRNAs and other potentially enriched biological signaling pathways were analyzed using bioinformatics. Following these results, the MAPK, apoptosis and hippocampal signaling pathways might be involved in the molecular mechanisms underlying the memory disorders of TLE.
Project description:Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is known to protect against cerebral ischemia. The effects of VPA on blood-brain barrier (BBB) disruption were investigated in rats subjected to transient middle cerebral artery occlusion (MCAO). Postischemic VPA treatment remarkably attenuated MCAO-induced BBB disruption and brain edema. Meanwhile, VPA significantly reduced MCAO-induced elevation of matrix metalloproteinase-9 (MMP-9), degradation of tight junction proteins, and nuclear translocation of nuclear factor-κB (NF-κB). Sodium butyrate, another HDAC inhibitor, mimicked these effects of VPA. Our findings suggest that BBB protection by VPA involves HDAC inhibition-mediated suppression of NF-κB activation, MMP-9 induction, and tight junction degradation.
Project description:Objective:With the growing incidence of ischemic stroke worldwide, there is an urgent need to identify blood biomarkers for ischemic stroke patients. Thus, our aim was to identify potential circulating microRNA (miRNA) as a potential biomarker and to explore its potential mechanism for ischemic stroke in rats. Methods:The mRNA dataset GSE97537 and miRNA dataset GSE97532 were downloaded from the Gene Expression Omnibus (GEO) GSE97537 including 7 middle cerebral artery occlusion (MCAO) rat brain tissues and 5 sham-operated rat brain tissues GSE97532 including 6 MCAO rat blood samples and 3 sham-operated rat blood samples. Differentially expressed mRNAs and miRNAs with corrected p value ? 0.01 and fold change ?2 or ?0.05 were identified. To explore potential biological processes and pathways of differentially expressed mRNAs, functional enrichment analysis was performed. The target mRNAs of differentially expressed miRNAs were predicted using DNA Intelligent Analysis (DIANA)-microT tools. The target mRNAs and differentially expressed mRNAs were intersected. Results:1228 differentially expressed mRNAs in MCAO rat brain tissues were identified. Highly expressed mRNAs were mainly enriched in the inflammatory responses. Nine differentially expressed miRNAs were identified in MCAO rat blood samples. A total of 673 target mRNAs were predicted to significantly bind these differentially expressed miRNAs. Among them, 54 target mRNAs were differentially expressed in MCAO rat blood samples. Enrichment analysis results showed that these 54 target mRNAs were closely related to neurological diseases and immune responses. Among all miRNA-mRNA relationship, miR-3552-CASP3 interaction was identified, indicating that CASP3 might be mediated by miR-3552. Functional enrichment analysis revealed that CASP3 was involved in the apoptosis pathway, indicating that miR-3552 might participate in apoptosis by CASP3. Conclusion:Our findings reveal that circulating miR-3552 shows promise as a potential biomarker for ischemic stroke in rats.
Project description:Acute ischemic stroke (AIS) is a major public health problem in China. Impaired angiogenesis plays crucial roles in the development of ischemic cerebral injury. Recent studies have identified that microRNAs (miRNAs) are important regulators of angiogenesis, but little is known the exact effects of angiogenesis-associated miRNAs in AIS. In the present study, we detected the expression levels of angiogenesis-associated miRNAs in AIS patients, middle cerebral artery occlusion (MCAO) rats, and oxygen-glucose deprivation/reoxygenation (OGD/R) human umbilical vein endothelial cells (HUVECs). MiR-191 was increased in the plasma of AIS patients, OGD/R HUVECs, and the plasma and brain of MCAO rats. Over-expression of miR-191 promoted apoptosis, but reduced the proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs OGD/R model. Mechanically, vascular endothelial zinc finger 1 (VEZF1) was identified as the direct target of miR-191, and could be regulated by miR-191 at post-translational level. In vivo studies applying miR-191 antagomir demonstrated that inhibition of miR-191 reduced infarction volume in MCAO rats. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1. Therefore, miR-191 may serve as a biomarker or a therapeutic target for AIS.
Project description:Background: Evidences indicate that exosomes-mediated delivery of microRNAs (miRNAs or miRs) is involved in the neurogenesis of stroke. This study was to investigate the role of exosomal miRNAs in non-drug therapy of electro-acupuncture (EA) regulating endogenous neural stem cells for stroke recovery. Methods: The model of focal cerebral ischemia and reperfusion in rats were established by middle cerebral artery occlusion (MCAO) and treated by EA. The exosomes were extracted from peri-ischemic striatum and identified by exosomal biomarkers, and detected differentially expressed miRNAs with microarray chip. Primary stem cells were cultured, and oxygen-glucose deprivation and reperfusion (OGD/R) was used to mimic vitro ischemic injury. Results: The levels of exosomal biomarkers TSG101 and CD81 were increased in peri-ischemic striatum after EA treatment, and we revealed 25 differentially expressed miRNAs in isolated exosomes, of which miR-146b was selected for further analysis, and demonstrated that EA increased miR-146b expression and its inhibitors could block the effects. Subsequently, we confirmed that EA upregulated miR-146b expression to promote neural stem cells differentiation into neurons in peri-ischemic striatum. In vitro, it was verified that OGD/R hindered neural stem cells differentiation, and miR-146b inhibitors furtherly suppressed its differentiation, simultaneously NeuroD1 was involved in neural stem cells differentiation into neurons. Moreover, in vivo we found EA promoted NeuroD1-mediated neural stem cells differentiation via miR-146b. In addition, EA also could improve neurological deficits through miR-146b after ischemic stroke. Conclusion: EA promotes the differentiation of endogenous neural stem cells via exosomal miR-146b to improve neurological injury after ischemic stroke.
Project description:Because our recent studies have demonstrated that miR-122 decreased in whole blood of patients and in whole blood of rats following ischemic stroke, we tested whether elevating blood miR-122 would improve stroke outcomes in rats. Young adult rats were subjected to a temporary middle cerebral artery occlusion (MCAO) or sham operation. A polyethylene glycol-liposome-based transfection system was used to administer a miR-122 mimic after MCAO. Neurological deficits, brain infarction, brain vessel integrity, adhesion molecule expression and expression of miR-122 target and indirect-target genes were examined in blood at 24?h after MCAO with or without miR-122 treatment. miR-122 decreased in blood after MCAO, whereas miR-122 mimic elevated miR-122 in blood 24?h after MCAO. Intravenous but not intracerebroventricular injection of miR-122 mimic decreased neurological deficits and brain infarction, attenuated ICAM-1 expression, and maintained vessel integrity after MCAO. The miR-122 mimic also down-regulated direct target genes (e.g. Vcam1, Nos2, Pla2g2a) and indirect target genes (e.g. Alox5, Itga2b, Timp3, Il1b, Il2, Mmp8) in blood after MCAO which are predicted to affect cell adhesion, diapedesis, leukocyte extravasation, eicosanoid and atherosclerosis signaling. The data show that elevating miR-122 improves stroke outcomes and we postulate this occurs via downregulating miR-122 target genes in blood leukocytes.
Project description:Ischemic strokes carry a significant risk of mortality and recurrent vascular events. Recent studies suggest that changes in microRNAs (miRNAs or miRs) may affect the development of the stroke. However, few studies have investigated the role of miRNAs in behavioral disorder in early stroke. In the present study, animal models of middle cerebral artery occlusion (MCAO) are used, as well as a cell model of neurite outgrowth to further investigate the role of miRNAs in targeting synapse-associated proteins expression in early stroke. The authors used miRNA expression microarrays on RNA extracted from the cortex tissue samples from the rats of MCAO and control rats. Reverse transcription?quantitative polymerase chain reaction was conducted to verify the candidate miRNAs discovered by microarray analysis. Data indicated that miR?125a was significantly increased in the cortex of the model of MCAO, which were concomitant with that rats of MCAO at the same age displayed significant behavioral deficits. Bioinformatics analysis predicted the cell adhesion molecule 2 (Cadm2, mRNA) neurite outgrowth-associated protein is targeted by miR?125a. Overexpression of miR?125a reduced the level of Cadm2 expression in PC12 cell injury induced by free-serum. In contrast, inhibition of miR?125a using miR?125a inhibitors significantly resulted in higher levels of Cadm2 expression. In conclusion, miR?125a is involved in the behavioral disorder of animal models of MCAO by regulation of Cadm2.
Project description:BACKGROUND AND PURPOSE:d-Amino acid oxidase (DAAO) is a flavine adenine dinucleotide-containing flavoenzyme and specifically catalyses oxidative deamination of d-amino acids. This study aimed to explore the association between increased cerebral DAAO expression or enzymic activity and the development of cerebral ischaemia. EXPERIMENTAL APPROACH:A mouse model of transient (90 min) middle cerebral artery occlusion (MCAO) was established, and western blotting, enzymic activity assay, and fluorescent immunostaining techniques were used. KEY RESULTS:The expression and enzymic activity of DAAO increased over time in the cortical peri-infarct area of the mice subjected to transient MCAO. The DAAO was specifically expressed in astrocytes, and its double immunostaining with the astrocytic intracellular marker, glial fibrillary acidic protein, in the cortical peri-infarct area was up-regulated following ischaemic insult, with peak increase on Day 5 after MCAO. Single intravenous injection of the specific and potent DAAO inhibitor Compound SUN reduced the cerebral DAAO enzymic activity and attenuated neuronal infarction and neurobehavioural deficits with optimal improvement apparent immediately after the MCAO procedure. The neuroprotective effect was dose dependent, with ED50 values of 3.9-4.5 mg·kg-1 . Intracerebroventricular injection of the DAAO gene silencer siRNA/DAAO significantly reduced cerebral DAAO expression and attenuated MCAO-induced neuronal infarction and behavioural deficits. CONCLUSIONS AND IMPLICATIONS:Our results, for the first time, demonstrated that increased cerebral astrocytic DAAO expression and enzymic activity were causally associated with the development of neuronal destruction following ischaemic insults, suggesting that targeting cerebral DAAO could be a potential approach for treatment of neurological conditions following cerebral ischaemia.