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Introduction: We explored microRNA (miRNA) profiles correlated with the penumbra in three different phases of ischaemic stroke, using a permanent middle cerebral artery occlusion (p-MCAO) rat model., Materials and Methods: A 2-mm coronal section was cut from the optic chiasma in the caudal direction, and the penumbra was located in the area between a longitudinal line approximately 2 mm from the midline and a transverse diagonal line at the "2-o'clock" position. Total RNA was extracted from tissue specimens and peripheral blood samples, followed by deep sequencing analysis., Results: We identified nine novel miRNA candidates in tissues and evaluated their expression levels using real-time quantitative polymerase chain reaction. In situ hybridization was conducted to assess miRNA localization in the brain. Of these nine candidates, we identified and characterized a novel miRNA, rno-miR-686-3p, which was localized in cell nuclei of the cortex, and associated with the penumbra. rno-miR-686-3p was downregulated at 1 (p = 0.042), 3 (p = 0.032), and 4 h (p = 0.007) post-p-MCAO in the penumbra. A total of 297 potential target genes were predicted. Moreover, functional annotation clustering and pathway enrichment analysis predicted that rno-miR-686-3p participates in transcriptional regulation and the Wnt and cyclic adenosine monophosphate (cAMP) signalling pathways., Conclusion: rno-miR-686-3p is a novel miRNA associated with the ischaemic penumbra that is implicated in transcriptional regulation and modulation of the Wnt and cAMP signalling pathways. Furthermore, it may serve as a possible new biomarker with potential value for detecting the existence of the penumbra., (© 2022 S. Karger AG, Basel.)

Purpose: Ischemic penumbra is the main therapeutic target for acute ischemic stroke. The aim in this study is to investigate the potential serum biomarkers of penumbra that could fulfill a complementary role in the acute stroke clinical decision-making process., Experimental Design: An established focal cerebral ischemia model is applied in rats. Using isobaric tags for relative and absolute quantitation combined with liquid chromatography-tandem mass spectrometry, the global expression profiles of proteins in ischemic penumbra tissue and serum from rats subjected to different ischemic times are identified and quantified. Candidate biomarkers are screened out with bioinformatics analysis and further validated by western blotting., Results: Herein, a total of 4568 proteins in the penumbra and 1915 proteins in the serum are identified. Two proteins related to the penumbra, RHOA, and CDC42, are screened out through an integrative analysis. The expression levels of RHOA and CDC42 in the penumbra and serum gradually increase synchronously with the prolonged ischemia time., Conclusions and Clinical Relevance: The study provides the results of a proteomic analysis to identify serum biomarkers of the penumbra. Upregulation of RHOA and CDC42 may be useful for the early assessment of ischemic penumbra and could serve as potential serum biomarkers., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Early and accurate diagnosis of ischaemic stroke (IS) requires the use of an optimized biomarker. Exosomal microRNAs have the potential to serve as biomarkers owing to their stability and specificity. We investigated the expression levels of plasma-derived exosomal microRNA-21-5p and microRNA-30a-5p in the different phases of IS., Methods: One hundred forty-three patients with IS and 24 non-stroke controls were enrolled. The patients were divided into the following 5 groups: 1 group for the hyperacute phase IS (HIS, within 6 h); two for the acute phase IS (AIS, including days 1-3 and days 4-7); one for the subacute phase IS (SIS, days 8-14); and one for the recovery phase IS (RIS, days >14). Plasma exosomes were isolated using a QIAGEN exoRNeasy kit and examined by transmission electron -microscopy, nanoparticle tracking, and flow cytometry. The expression levels of miRNA-21-5p and miRNA-30a-5p were detected by quantitative real-time polymerase chain reaction., Results: The plasma exosomal miR-21-5p levels in SIS and RIS were significantly higher than that in controls (p < 0.05 and p < 0.01 respectively). The levels of miR-30a-5p in HIS were significantly higher (p < 0.05) and in AIS (days 1-3) were lower than that in controls (p < 0.05). In AIS (days 1-3), both miRNAs were decreased compared with the HIS group (p = 0.053 and 0.001, respectively). The area under the curve (AUC) of the miR-21-5p was 0.714 for SIS (95% CI 0.570-0.859, p = 0.007), 0.734 for RIS (95% CI 0.596-0.871, p = 0.003); the AUC of the miR-30a-5p was 0.826 for HIS (95% CI 0.665-0.988, p = 0.001), 0.438 for AIS (days 1-3; 95% CI 0.240-0.635, p = 0.516)., Conclusions: The plasma-derived exosomal miR-21-5p and miRNA-30a-5p in combination are promising biomarkers for diagnosing IS and distinguishing among HIS, SIS, and RIS, especially miRNA-30a-5p for the diagnosis of the HIS phase. Our results provide a new reference for clinicians to apply in early-stage diagnosis and identifies the possible value of biomarkers for IS thrombolysis therapy., (© 2018 S. Karger AG, Basel.)

Background: miR-146a, a strong pro-apoptotic factor in some pathophysiological processes, is reported to be involved in ischemic stroke (IS), though its role remains unclear. Fbxl10 is an active anti-apoptotic factor and a predicted target of miR-146a. We hypothesized that dysregulation of miR-146a contributes to ischemic injury by targeting Fbxl10., Methods: Circulating miRNAs were detected by miRNA microarray and qRT-PCR. miR-146a targets were predicted using bioinformatics and confirmed with a dual luciferase reporter assay. We used an in vitro ischemic model of oxygen-glucose deprivation and reperfusion (OGD/R) to mimic cerebral ischemia/reperfusion (I/R) conditions. Expression of miR-146a, Fbxl10 and Bcl2l2 mRNAs, and Fbxl10 and Bcl2l2 proteins was verified by qRT-PCR and Western blotting. The effects of miR-146a on neuronal cell apoptosis were evaluated by flow cytometry., Results: A significant reduction in miR-146a expression was observed in acute ischemic stroke (AIS). A dual-luciferase reporter assay showed that Fbxl10, but not Bcl2l2, is a target of miR-146a. Transfection with miR-146a mimics promoted apoptosis in SK-N-SH cells and significantly reduced expression of Fbxl10. Conversely, miR-146a inhibition attenuated OGD/R-induced neuronal cell death and significantly up-regulated Fbxl10 expression., Conclusions: miR-146a expression was significantly down-regulated in AIS, and Fbxl10 was identified as a target of miR-146a. Moreover, up-regulation of Fbxl10, a miR-146a target, likely protects neurons from ischemic death., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: MircroRNA (MiRNA) levels are associated with disease pathophysiology and are high in plasma exosomes. Plasma exosomal miRNAs serve as potential therapeutic targets and diagnosis biomarkers in some diseases but few studies have examined them in Ischemic Stroke (IS). Therefore, we explored the potential predictive value of plasma exosomal miR-422a and miR-125b-2-3p in different IS phases (acute and subacute phases)., Methods: Fifty-five IS patients and 25 age and sex matched healthy controls were recruited. Patients were classified into two groups: 27 patients in acute phase (days 1-3) and 28 patients in subacute phase (days 4-14). The plasma exosomal levels of miR-422a and miR-125b-2-3p were examined via quantitative real-time polymerase chain reaction (qRT-PCR). The Areas Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve were constructed to evaluate the diagnostic accuracy of these miRNAs in IS., Results: The expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group (P<0.001, P<0.001, respectively), and the miR-422a levels were increased in the acute phase group (P<0.005) as compared to the controls. Additionally, the expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group than in the acute phase group (P<0.001, P<0.005, respectively). ROC analysis showed high AUC values for miR-422a and miR-125b-2-3p in the subacute phase group as compared to those in healthy controls: 0.971 and 0.889, respectively, and miR-422a in the acute phase group as compared to healthy controls were 0.769., Conclusion: Plasma exosomal miR-422a and miR-125b-2-3p may serve as blood-based biomarkers for monitoring and diagnosing in IS patients, with plasma exosomal miR-422a showing the best diagnostic value. The use of these two plasma exosomal miRNAs in combination may be powerful for determining IS stage., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

MicroRNAs have been discovered as regulators of gene expression and thus their potential in clinical disease diagnostics, prognosis and therapy is being actively pursued. MicroRNAs play an important role in atherosclerosis-related diseases, such as cerebrovascular and cardiovascular disease. However, the effect of miR-185 and miR-146a on patients with ischemic stroke (IS) in the different phases has not been reported. In this study, we investigated the amounts of three miRNAs, miR-185, miR-146a, and miR-145 in blood circulation of IS patients. We enrolled 60 patients with IS in the acute or sub-acute phase and 30 healthy controls. We divided the patients into two groups, patients with ischemic stroke in the acute phase (ISA) and patients with ischemic stroke in the subacute phase (ISS). We measured circulating miRNAs expression by miRNA microarray and real-time polymerase chain reaction (PCR) analysis. Testing by miRNA microarray and RT-PCR analyses showed that miR-145 levels in healthy subjects were similar to patients with IS, whereas miR-146a and miR-185 were present with quite low abundance in ISA compared with healthy individuals; moreover, we found that miR-146a levels were downregulated in ISA but upregulated in ISS which may help provide new insights into the diagnosis and therapy of IS.

MicroRNAs are small, non-coding RNA molecules that regulate gene expression, and miR-124 is the most abundant miRNA in the brain. Studies have shown that miR-124 is clearly reduced in the ischemic brain after stroke; however, the role of miR-124 after stroke is less well studied. Using TargetScan, MicroCosm Targets version 5, and microRNA.org databases, we identified miR-124 as a possible regulator of the DNA repair protein Ku70. We validated that Ku70 is a target for miR-124 with a luciferase reporter activity assay. Moreover, adult rats subjected to focal cerebral ischemia exhibited a substantial reduction of miR-124 expression, which was inversely upregulated by Ku70 expression. In vivo treatment with miR-124 antagomir effectively enhanced Ku70 mRNA and protein levels in the ischemic region. Furthermore, knockdown of cerebral miR-124 reduced cell death and infarct size and improved neurological outcomes. Our data demonstrate that miR-124 is an endogenous regulator of Ku70 that improves ischemia/reperfusion (I/R)-induced brain injury and dysfunction.

Objective: To study the effects of cerebellar fastigial nucleus (FN) electrical stimulation on telomerase reverse transcriptase expression and mitochondrial apoptotic pathway in rats with focal cerebral ischemia and reperfusion., Methods: A total of 100 adult male Wistar rats were randomly divided into 3 groups: sham operation group, modeling group (2-hour cerebral ischemia, followed by 24, 48 & 72-hour reperfusion) and FN-stimulating group (electrical stimulation of FN for 1-hour one day before 2-hour cerebral ischemia, followed by 24, 48 & 72-hour reperfusion). HE (hematoxylin and eosin) and TTC (triphenyl tetrazolium chloride) staining were used to observe the morphological changes in rat brain and measure the ischemic lesion volumes. The expressions of TERT (telomerase reverse transcriptase) and Bax were detected by immunohistochemical methods and apoptotic cells by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). The co-expression of TERT and Bax was detected by immunofluorescence double-labeling plus laser confocal microscopy., Results: The morphological changes in rat brain were less greater in the FN-stimulating group than those in the modeling group. And the size of the cerebral infarct was significantly smaller in the FN-stimulating group (78.1 ± 2.9, 83.1 ± 4.5, 83.7 ± 4.8) than that in the modeling group (120.9 ± 8.2, 137.0 ± 4.2, 141.1 ± 3.3) (P < 0.05) at all reperfusion time points. As compared with the modeling group (16.1 ± 2.7, 16.9 ± 2.4, 11.6 ± 3.5), the FN-stimulating group (31.1 ± 3.5, 30.0 ± 3.4, 18.9 ± 3.3) had a significantly larger number of TERT-positive cells (P < 0.05) and a significantly reduced number of TUNEL-positive cells (49.6 ± 2.8, 67.0 ± 3.7, 46.8 ± 3.2 vs 40.2 ± 3.1, 54.8 ± 2.8, 37.3 ± 2.4) (P < 0.05). The number of Bax-positive cells at different reperfusion time points in the FN-stimulating group was not significantly different from those in the modeling group (P > 0.05). TERT partially co-localized with Bax in the cytoplasm. The number of double-labeled cells was significantly higher in the FN-stimulating group than that in the modeling group (14.1 ± 1.3, 12.9 ± 2.4, 9.0 ± 2.0 vs 8.2 ± 1.1, 6.3 ± 2.4, 6.0 ± 2.9) (P < 0.05)., Conclusion: The expression of TERT significantly increases after a stimulation of FN. TERT may bind to Bax and inhibit Bax-mediated apoptosis by suppressing the mitochondrial relocalization of Bax from cytosol.

Objective: To investigate the relationship between polymorphisms in paraoxonase1 (PON1) gene Gln192Arg (Q192R) and arterial ischemic stroke in young adults., Methods: The Q192R genotype was analyzed by polymerase chain reaction in 131 young adults with ischemic stroke and 135 age- and gender-matched controls. The plasma lipids were also determined in patients and controls respectively. Furthermore, carotid artery intima-media thickness (IMT) in patients were measured by carotid ultrasonography., Results: The distributions of Q192R genotype frequency were significantly different between patients with ischemic stroke and control individuals. And the patients had more RR genotypes than control individuals (P < 0.05). Odds ratio (OR) for stroke were 1.743 (95% confidence interval [CI], 1.032-2.943) in subjects with RR genotype. We also studied the relationship between the polymorphisms and the lipid concentration in patients and control individuals. However, no significant association was detected between Q/R192 genotype and any of lipid measurements. Further, the prevalence of cigarette smoking, hypertension and diabetes showed no significant difference between RR and non-RR genotypes in patients. Body mass index (BMI) in two groups did not differ significantly. But IMT of patients with RR genotype obviously increased in comparison to those without RR genotype (P < 0.05)., Conclusion: The PON1 gene Q192R polymorphism may be associated with the susceptibility of ischemic stroke in young adults. RR genotype is a genetic risk for young adults with ischemic stroke through an increased carotid artery intima-media thickness and an accelerated atherosclerotic process.