The Expression Of MIR-146A And Its Target Gene FBXL10 In Patients With Ischemic Stroke

Although advances in preclinical researches that have found mechanisms of apoptosis in ischemic stroke (IS) and identified potential strategies to prevent or reduce brain damage after stroke, few of these advances can be translated into clinical practice. More recent researchers have focused on novel target for endogenous neuroprotection against stroke, which is more relevant to the clinical presentation in IS patients. MiRNA has been becoming a hot topic of the international field in IS.The purpose of this study was to investigate the abnormal expression of miRNAs in blood of IS, and to explore the function and regulation mechanism of these miRNAS in IS.Part1 Differential Regulation of microRNAs in Patients with Ischemic StrokeObjective:To investigate the abnormal expression of miRNAs in blood of patients with IS and its potential role as a diagnostic biomarker.Methods:We enrolled 60 patients with IS in the acute or subacute phase and 30 healthy controls. We divided the patients into two groups, acute ischemic stroke (AIS) and subacute ischemic stroke (SIS). We measured circulating miRNAs expression by miRNA microarray and Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).Results: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 AIS compared with healthy individuals; moreover, we found that miR-146a levels were downregulated in AIS but upregulated in SIS.Conclusion:The abnormal expressions of miR-146a in blood of patients with IS can be used as potential markers, which have values for diagnosing AIS.Part2 MiR-146a and its target gene FBXL10 in the mechanism of neuronal apoptosis after OGD/RObjective:At present, miR-146a has been described to be involved in IS, but its role is not clear yet. In particular, miR-146a is a strong pro-apoptotic factor in some biological systems. FBXL10 and BCL2L2, two active anti-apoptotic factos, are the predicted targets of miR-146a. We hypothesized that dysregulation of miR-146a contributes to ischemic injury through targeting FBXL10 and/or BCL2L2.Methods:Targets of miR-146a were predicted using bioinformatics and then confirmed by dual luciferase reporter assay. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) SK-N-SH cell model to mimic cerebral ischemia/reperfusion (I/R) conditions. The expression levels of miR-146a, FBXL10 mRNA, BCL2L2 mRNA, FBXL10 protein and BCL2L2 protein were verified by qRT-PCR and Western blot. The effects of miR-146a mimic or miR-146a inhibitor on neuronal cell apoptosis were evaluated with flow cytometric analysis.Results:Dual-luciferase reporter assay showed that FBXL10, but not BCL2L2, is the target of miR-146a. Treatment with mimics of miR-146a promotes apoptosis in SK-N-SH cell line and significantly reduced the expression of FBXL10 (P<0.05). Conversely, inhibition of miR-146a resulted in attenuation of OGD/R-induced neuronal cell death and significantly upregulated the levels of FBXL10 (P<0.05)Conclusion:FBXL10 was identified as a target of miR-146a. Moreover, our results indicate that up-regulation of FBXL10, targeted by miR-146a likely protects against ischemic neuronal death.