Aims: Ischemic stroke has become one of the main causes of death worldwide. MicroRNAs (miRNAs) have been implicated in cerebral ischemia-reperfusion (I/R) injury and could serve as therapeutic targets. 5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes and has been implicated in inflammatory central nerve system disorders. The objective of this study was to explore the neuroprotective effects of miR-193b-3p against focal cerebral I/R injury in rats by regulating 5-LOX expression.
Methods and materials: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion injury. The level of miR-193b-3p expression was observed in the rat cortical peri-infarct region after focal cerebral I/R injury. Bioinformatics analysis was used to predict the binding sites of miR-193b-3p, and a dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX mRNA and miR-193b-3p. Then, rats were injected with a miR-193b-3p agomir (modified and enhanced mimic) or antagomir (modified and enhanced inhibitor) in the right lateral ventricle of the brain. Neurological deficit scores, infarct volumes, neuron damage and 5-LOX enzymatic activity and expression were measured. In an in vitro experiment, cultured PC12 cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R). OGD/R-induced cells were treated with a miR-193b-3p mimic or inhibitor and 5-LOX siRNA. Cell viability, lactate dehydrogenase release, apoptosis rate and 5-LOX expression were evaluated.
Results: The level of miR-193b-3p expression was increased in the cortical peri-infarct region of rats with cerebral focal I/R injury. The results of the dual-luciferase reporter gene assay showed that a miR-193b-3p binding site was located in the 3' untranslated region (3'UTR) of 5-LOX mRNA. Neurological deficit scores, infarct volumes and neuronal injury were alleviated by miR-193b-3p agomir treatment but aggravated by miR-193b-3p antagomir. Furthermore, leukotriene B4, cysteinyl-leukotrienes and 5-LOX expression in the cortical peri-infarct region of rats with focal cerebral I/R injury were also downregulated by miR-193b-3p agomir treatment but upregulated by miR-193b-3p antagomir. In PC12 cells, miR-193b-3p mimic significantly decreased OGD/R-induced cell death and reduced lactate dehydrogenase release and 5-LOX expression. In contrast, miR-193b-3p inhibitor exacerbated OGD/R-induced injury in PC12 cells. Additionally, the in vitro effects of miR-193b-3p inhibitor on OGD/R-induced cell injury were partially reversed by 5-LOX siRNA treatment.
Conclusion: MiR-193b-3p has a potentially neuroprotective effect on focal cerebral I/R-induced injury by inhibiting 5-LOX expression.
Keywords: 5-LOX; Focal cerebral ischemia-reperfusion injury; Inflammation; Leukotrienes; miRNA-193b-3p.
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