| Objectives:(1)This study aimed to systematically characterize distinctive expression signatures of serum microRNAs(miRNAs)in patients with ischaemic stroke(IS)and transient ischaemic attack(TIA),and to investigate the clinical application values of specific miRNAs in IS and TIA patients.(2)This study also aimed to confirm the predicted target genes of miR-23b-3p and miR-29b-3p,and to elucidate their molecular regulatory networks and mechanisms involved in ischemic cerebrovascular diseases.Methods:(1)Taq Man Low Density Array screening in pooled serum samples followed by the quantitative reverse-transcription PCR(q RT-PCR)validation in individual serum sample was initially employed to identify the distinctive expression profiles of serum miRNAs in IS patients.Markedly altered miRNAs levels in TIA patients were also determined.The relationships of specific miRNAs to the neurological deficit severity of IS and the subsequent stroke risk after TIA were analyzed by multiple statistical evaluations,such as the analyses of variance,Spearman correlation analyses,receiver operating characteristic curve(ROC)and multivariate Logistic regression analyses.(2)Through bioinformatics analyses,the possible target genes of miR-23b-3p and miR-29b-3p were predicted,and their possible molecular regulatory networks were also constructed.In vivo experiments,the consistency of the brain tissues and serum expression levels in the same rats following transient global cerebral ischemia/reperfusion were analyzed,and the molecular mechanisms of miR-23b-3p and miR-29b-3p involved in ischemic cerebrovascular disease were also preliminarily validated.Further in vitro experiments,the q RT-PCR,Western blotting,dual luciferase reporter gene assays and the transfection technique of miRNA agomir were used to elucidate the molecular mechanisms of miR-23b-3p and miR-29b-3p underlying the neuronal apoptosis induced by oxygen-glucose deprivation(OGD).Results: Serum miR-23b-3p,miR-29b-3p,miR-181a-5p and miR-21–5p levels were found to be significantly increased in IS patients compared with controls(all P<0.001).Of the IS patients,miR-23b-3p,miR-29b-3p,miR-181a-5p and miR-21–5p levels in severe IS group(NIHSS>13)were significantly higher than those in moderate IS(6?NIHSS?13)and mild IS(NIHSS<6)groups(all P<0.05);miR-23b-3p levels in moderate IS group were higher than those in mild IS group.Furthermore,the Spearman rank correlation analyses showed that miR-23b-3p and miR-29b-3p levels in IS patients were positively related with admission NIHSS scores(all P<0.01).Serum miR-23b-3p,miR-29b-3p and miR-181a-5p levels were also significantly increased in TIA patients compared with controls(all P<0.01).Moreover,miR-23b-3p,miR-29b-3p and miR-21–5p levels in IS patients were significantly higher than those in TIA patients(all P<0.05).Of the TIA patients,miR-23b-3p,miR-29b-3p and miR-181a-5p levels in high-risk group(6?ABCD3?9)were significantly higher than those in moderate-risk(4?ABCD3?5)and low-risk(0?ABCD3?3)groups(all P<0.05);miR-23b-3p levels in moderate-risk group were higher than those in low-risk group(all P<0.05).Furthermore,the Spearman rank correlation analyses showed that miR-23b-3p and miR-181a-5p levels in TIA patients were positively related with admission ABCD3 scores(all P<0.05).(2)Bioinformatics analyses demonstrated that miR-23b-3p and miR-29b-3p may jointly regulate the expression of phosphatase and tensin homolog(PTEN),and miR-29b-3p may also regulate the expression of orkhead transcriptional factor 3(FOXO3A).In vivo experiments,levels of miR-23b-3p and miR-29b-3p were found to be significantly increased in serum,prefrontal cortex and hippocampus from rats following transient global cerebral ischemia/reperfusion(all P<0.05),and serum levels of miR-23b-3p and miR-29b-3p were positively correlated with their levels in prefrontal cortex,respectively(all P<0.01).Levels of PTEN,FOXO3 A and phosphorFOXO3A(p-FOXO3A)were significantly down-regulated in prefrontal cortex and hippocampus from rats following transient global cerebral ischemia/reperfusion,while levels of pro-apoptotic Bim and Cleaved Caspase-3 were significantly up-regulated(all P<0.05).In vitro experiments,levels of miR-23b-3p and miR-29b-3p were found to be significantly increased in cortical neurons following OGD(all P<0.05).Levels of PTEN,FOXO3 A,p-FOXO3 A as well as their mRNA were significantly downregulated in cortical neurons following OGD(all P<0.05).Dual luciferase reporter assays confirmed the targeting relationships between miR-23b-3p/miR-29b-3p and PTEN and between miR-29b-3p and FOXO3 A,respectively.Further in transient transfection experiments,levels of PTEN,Bim and Cleaved Caspase-3 were found to be significantly down-regulated in cortical neurons after transient transfection of miR-23b-3p agomir,and levels of PTEN,FOXO3 A,p-FOXO3 A,Bim and Cleaved Caspase-3 were significantly down-regulated in cortical neurons after transient transfection of miR-29b-3p agomir(all P<0.05).Levels of Bim and Cleaved Caspase-3 were also significantly down-regulated in cortical neurons after transient transfection of miR-23b-3p/miR-29b-3p agomir following OGD compared with their levels in cortical neurons following OGD(all P<0.05).Conclusions: Serum miR-23b-3p,miR-29b-3p,miR-181a-5p and miR-21-5p may function as predictive and discriminative biomarkers for IS and TIA,and their distinctive expression signatures may contribute to assessing neurological deficit severity of IS and subsequent stroke risk after TIA.miR-23b-3p and miR-29b-3p may participate in the processes of neuronal apoptosis induced by hypoxia-ischemia through the PTEN-AKT-FOXO3 A signaling pathway.These above findings may provide a new theoretical basis for the application of specific miRNAs in the monitoring,prognosis,and treatment of ischemic cerebrovascular diseases. |
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