Objective: Ischemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke-related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke.
Methods: The JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)-induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3' untranslated region (3'UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase-9 [MMP-9], tumor necrosis factor-α [TNF-α], and interleukin-1β [IL-1β]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits.
Results: JAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3'UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1β). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit.
Conclusions: These findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.
Keywords: 3ʹUTR = 3ʹ untranslated region; CCA = common carotid artery; DMEM = Dulbecco’s modified Eagle’s medium; ELISA = enzyme-linked immunosorbent assay; FBS = fetal bovine serum; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; ICA = internal carotid artery; IL-1 = interleukin-1; JAK = Janus tyrosine kinase; JAK2/STAT3; LDH = lactate dehydrogenase; MCAO; MCAO = middle cerebral artery occlusion; MMP-9 = matrix metalloproteinase–9; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OGD = oxygen-glucose deprivation; OGD/R = oxygen-glucose deprivation/reoxygenation; OGD2/R24 = 2 hours of OGD and 24 hours of reoxygenation; PCN = primary cortical neuronal; SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis; STAT = signal transducer and activator of transcription; TNF-α = tumor necrosis factor–α; TTC = 2,3,5-triphenyltetrazolium chloride; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; iNOS = inducible nitric oxide synthase; ischemic injury; miR-216a; miR-NC = miRNA negative control; miRNA = microRNA; qRT-PCR = quantitative reverse-transcriptase polymerase chain reaction; siJAK2 = JAK2 short-interfering RNA.