| Objective:Stroke is a common cerebrovascular disease with an increasing incidence rate and various complications.Even though current thrombolytic therapy and emerging endovascular interventional treatment can effectively open occluded blood vessels and rescue ischemic brain tissue to some extent,the effective prevention of stroke and reduction of ischemic injury after stroke are still clinical challenges.MicroRNAs（miRNAs）,which are 20-25 nucleotides in length,are a novel family of small nonprotein-coding RNAs that bind to target mRNAs and then lead to mRNA degradation or translation suppression.More than 20%of miRNA alterations are reported in the ischemic brain,suggesting that miRNAs act as promising mediators of ischemic stroke.Recently,a number of studies revealed that some miRNAs might play a neuroprotective role in the course of ischemic brain injuries.MiR-532-5p was dysregulated in some central nervous system diseases,such as multiple sclerosis,amyotrophic lateral sclerosis and intracerebral hemorrhage.In ischemic stroke,the level of miR-532-5p was found to be significantly decreased.However,the function of miR-532-5p in the central nervous system,especially in ischemic stroke,remains unknown.An in vitro report revealed that higher miR-532-5p expression suppressed hypoxia-induced H9c2 cell apoptosis.Whether miR-532-5p plays a protective role after hypoxic brain insults remains unclear.Phosphatase and tensin homolog deleted on chromosome 10（PTEN）acts as a tumor suppressor,which mainly depends on the dephosphorylation of phosphatidylinositol3,4,5-trisphosphate（PIP3）and the following inhibition of（PI3K）/Akt pathway.A number of studies have shown that PTEN is associated with the regulation of ischemic brain injury.Notably,some evidence has shown that miR-532-5p is related to the regulation of the PI3K/Akt signaling pathway.Interestingly,our bioinformatic analysis indicates that PTEN is a target gene of miR-532-5p.Therefore,it might be possible that miR-532-5p functions in ischemic stroke through the PTEN/PI3K/Akt signaling pathway.In the current study,we investigated the function of miR-532-5p in an in vivo middle cerebral artery occlusion（MCAO）model in mice.In vitro,the possible molecular mechanism of this effect was examined in mouse neuroblastoma cells（N2a）treated with oxygen-glucose deprivation and reperfusion（OGD/R）.In addition,the effect of miR-532-5p on PTEN and Akt was also detected in vivo and in vitro.Methods:1.Animals:Male C57BL/6 mice（～8 weeks old）were adaptively fed for 1 week.The mice were randomly divided into four groups:（A）sham-operated（Sham）;（B）MCAO;（C）MCAO+negative control mimic（MCAO+NC mimic）;and（D）MCAO+miR-532-5p mimic.2.Intracerebroventricular（ICV）administration:The injection location was determined according to the stereotactic map of the mouse brain,and the corresponding reagent was injected through the right ventricle according to different groups.3.Middle cerebral artery occlusion（MCAO）model:After 24 hours of intracerebroventricular injection,the MCAO model in mice was established by the intraluminal monofilament method.The mice in group A underwent the same procedure except for the filament insertion.4.Assessment of neurological function:The neurological function of each group was assessed 24 h after MCAO by an 18-point scoring system.5.2,3,5-Triphenyltetrazolium chloride（TTC）staining:The mice were sacrificed under deep anesthesia,and the brains were carefully removed.The brains slices were incubated in 2%TTC solution and digital images were then taken to calculate the infarct volume.6.Cell culture,transfection and oxygen-glucose deprivation（OGD）treatment:The N2a cells were seeded and cultured in four groups:（A）Control;（B）OGD;（C）OGD+negative control mimic（OGD+NC mimic）;and（D）OGD+miR-532-5p mimic.After transfection,N2a cells went through the OGD process.The control N2a cells were not exposed to OGD treatment.7.TUNEL assay and Nissl staining:The brain injury and necrotic cells were shown by Nissl staining.The apoptotic cells were shown by TUNEL assay.8.Total RNA extraction and real-time quantitative polymerase chain reaction（RT-qPCR）:The total RNA of the samples was extracted.Then,the purity and concentration of the total RNA were checked.The RNA samples were reverse transcribed into cDNA and real-time PCR was conducted.The levels of miR-532-5p and PTEN mRNA were calculated with the 2-ΔΔCt method.9.Western blot analysis:Levels of PTEN,p-Akt and Akt in ischemic penumbra of the mice were detected using Western blot analysis.Levels of PTEN,p-Akt,Akt,Bax,cleaved caspase-3,cleaved PARP and Bcl-2 in N2a cells were also measured by Western blot analysis.10.Detection of cell viability:Cells were seeded into 96-well plates.The cells were cultured and incubated with CCK-8 solution（10μl）for 2 h at 37°C.Then the optical density at 450 nm was measured using a microplate reader.11.Luciferase reporter assay:Luciferase reporter plasmids were constructed and transfected into 293T cells together with mir-532-5p mimic or NC mimic.Luciferase activity was then detected.12.Statistical analysis:The data are expressed as the means±standard deviations（SDs）.Two-tailed unpaired Student’s t-test was used for comparing differences between groups.One-way ANOVA followed by Tukey’s post hoc test was used for multiple comparisons.A p-value less than 0.05 was considered statistically significant.Results:1.The expression levels of miR-532-5p and PTEN in the mouse MCAO model:Real-time qPCR revealed that the level of miR-532-5p was reduced in the MCAO mice compared with the mice in Sham group（p<0.001）.Conversely,the PTEN expression level was elevated in the MCAO mice（p<0.001）.2.MiR-532-5p attenuated ischemic injury after stroke:Compared with the sham group,the MCAO group displayed significantly decreased neurological scores.However,the miR-532-5p mimic improved neurological dysfunction in the MCAO mice（p<0.001）.Moreover,TTC staining showed that the miR-532-5p mimic reduced the infarct volume in the MCAO mice（p<0.001）.The results of Nissl staining revealed that the miR-532-5p mimic in the MCAO mice attenuated the neuronal injury induced by MCAO（p<0.001）.Moreover,the TUNEL assay demonstrated that MCAO promoted neuronal apoptosis,whereas the miR-532-5p mimic in the MCAO mice inhibited MCAO-induced neuronal apoptosis（p<0.001）.3.MiR-532-5p promoted the activation of the PI3K/Akt signaling pathway in MCAO mice:MCAO remarkably elevated PTEN protein levels and suppressed p-Akt protein levels.However,the miR-532-5p mimic significantly diminished the MCAO-induced PTEN protein levels and improved the MCAO-induced p-Akt protein levels（p<0.001）.The miR-532-5p mimic elevated the miR-532-5p level in the MCAO mice（p<0.001）.MiR-532-5p mimic decreased the PTEN expression level in the MCAO mice（p<0.001）.4.MiR-532-5p decreased the OGD-induced apoptosis of N2a cells:Real-time PCR showed that OGD suppressed miR-532-5p levels,whereas N2a cells transfected with the miR-532-5p mimic had raised miR-532-5p levels compared with those transfected with the NC mimic after OGD（p<0.001）.A CCK-8 assay revealed that miR-532-5p overexpression obviously ameliorated the reduced cell viability in N2a cells treated with OGD（p<0.01）.The results of TUNEL assay suggested that OGD generated notable apoptosis in N2a cells.In contrast,the miR-532-5p mimic protected against apoptosis in OGD-treated N2a cells（p<0.001）.Western blot analysis also showed that OGD treatment increased Bax,cleaved caspase-3 and cleaved PARP protein levels and reduced Bcl-2 protein levels in N2a cells（p<0.001）.Conversely,miR-532-5p overexpression ameliorated these protein level alterations in OGD-treated N2a cells（p<0.05）.5.MiR-532-5p promoted the activation of the PI3K/Akt signaling pathway following OGD in N2a cells:Western blot analysis showed that the PTEN protein levels were elevated in OGD-treated N2a cells,whereas the miR-532-5p mimic repressed PTEN protein levels in OGD-treated N2a cells（p<0.001）.In contrast,the p-Akt protein levels were decreased after OGD,while the overexpression of miR-532-5p alleviated the decrease in the p-Akt protein levels in OGD-treated N2a cells（p<0.001）.6.PTEN was verified as a downstream target gene of miR-532-5p:The luciferase reporter assay showed that the miR-532-5p mimic inhibited the luciferase activity of PTEN-WT-seed1 and PTEN-WT-seed2 compared with the NC mimic（p<0.05）.However,the miR-532-5p mimic showed no effect on the luciferase activity of PTEN-MUT-seed1 or PTEN-MUT-seed2（p>0.05）.Real-time PCR confirmed that the miR-532-5p mimic elevated miR-532-5p levels,and the miR-532-5p inhibitor suppressed miR-532-5p levels（p<0.001）.Moreover,real-time PCR and western blot analysis demonstrated that the miR-532-5p mimic decreased PTEN levels and that the miR-532-5p inhibitor increased PTEN levels（p<0.001）.7.PTEN overexpression reduced the protective effect of miR-532-5p on OGD-treated N2a cells:The CCK-8 assay and TUNEL detection confirmed that PTEN overexpression inhibited cell viability and promoted cell apoptosis in miR-532-5p mimic-treated N2a cells following OGD（p<0.05）.Further,we found that PTEN overexpression increased the PTEN protein levels and suppressed the p-Akt protein levels induced by the miR-532-5p mimic in OGD-treated N2a cells（p<0.001）.Conclusion:1.In the present study,we found that miR-532-5p reduced infarct volume,improved ischemic stroke outcomes and protected against neuronal injury and apoptosis.2.MiR-532-5p decreased the OGD-induced apoptosis of N2a cells.3.PTEN was verified as a downstream target gene of miR-532-5p.4.These findings reveal that miR-532-5p might protect against ischemic stroke by the suppression of PTEN expression and the activation of the PI3K/Akt signaling pathway. |
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