Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways

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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.

ABBREVIATIONS 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; iNOS = inducible nitric oxide synthase; JAK = Janus tyrosine kinase; LDH = lactate dehydrogenase; MCAO = middle cerebral artery occlusion; miRNA = microRNA; miR-NC = miRNA negative control; 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; qRT-PCR = quantitative reverse-transcriptase polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis; siJAK2 = JAK2 short-interfering RNA; 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; 3ʹUTR = 3ʹ untranslated region.

Article Information

Correspondence Guo Zhong Li: Harbin Medical University, Harbin, Heilong Jiang Province, China. lgzhyd1962@163.com.

INCLUDE WHEN CITING Published online March 9, 2018; DOI: 10.3171/2017.5.JNS163165.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Changes in JAK2 and miRNA expression in OGD/R-induced neuronal cells. A: Scheme of 3 potential miRNAs that may target JAK2 through different species. B–D: The changes in miR-216a (B), miR-135 (C), and miR-433 (D) expression levels were measured using real-time qRT-PCR. The small nucleolar RNA (snRNA) U6 was used as an internal control for quantification of the miRNAs. E–G: The expression levels of JAK2 mRNA (E) and protein (F and G) were measured. GAPDH was used as an internal control.

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    miR-216a directly recognized the 3′UTR of JAK2 mRNAs in OGD-treated neuronal cells. A: Luciferase reporter assay results demonstrated that cotransfection of the luciferase reporter containing 3′UTR of JAK2 with miR-216a resulted in a decrease in luciferase activity. B: JAK2 mRNA levels were measured by using real-time qRT-PCR. GAPDH was used as an internal control for quantification of mRNA levels. There was decreased expression of JAK2 mRNA with miR-216a treatment. C: Quantitative analysis showed that miR-216a reduced JAK2 protein levels in neuronal cells. D: Representative results of Western blot analysis show the changes of JAK2 protein levels after being transfected with miR-NC and miR-216a mimics. *p < 0.05.

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    The effect of miR-216a and siJAK2 on PCN cells. A: Cell viability was detected by MTT assay. B: The LDH level was detected. C: Quantitative analysis showed that knockdown of JAK2 can decrease TUNEL-positive cells in OGD2/R24-treated neuronal cells. D: TUNEL assays. E and F: Quantitative analysis (E) and Western blot (F) showed that knockdown of JAK2 can decrease protein levels of p-STAT3 and cleaved caspase-3 (Cle caspase-3). G: ELISA analysis showed that the expression of p-STAT3 was promoted in the OGD/R group and inhibition of JAK2 partly decreased the upregulated p-STAT3 level induced by OGD/R. *p < 0.05; #p < 0.05 represents a comparison with the OGD/R group. Figure is available in color online only.

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    Overexpression of JAK2 can restore miR-216a–repressed JAK2/STAT3 activation and apoptosis in OGD2/R24-treated PCN cells. A: Quantitative analysis showed that miR-216a can decrease TUNEL-positive cells, while JAK2 can increase TUNEL-positive cells. Cotreatment with JAK2 and miR-216a can reverse this trend. B: Quantitative analysis showed that miR-216a can decrease protein levels of JAK2, p-STAT3, and cleaved caspase-3, while JAK2 can increase these protein levels. Cotreatment with miR-216a and JAK2 can reverse this trend. C: The levels of IL-1β and TNF-α were increased in the OGD/R group and pcDNA3-JAK2 group compared with normal cells, and the increase could be inhibited by introduction of miR-216a mimics. D: The ELISA results showed that the expression of p-STAT3 in the miR-216a group was increased in the pcDNA3-JAK2 group and transfection of miR-216a mimics can inhibit the increased p-STAT3 induced by overexpression of JAK2. E: TUNEL assays. F: Western blot was performed. G: miR-216a mimics reduced the upregulated expression of iNOS and MMP-9 induced by OGD/R and pcDNA3-JAK2, and the effect of miR-216a can be weakened by knockdown of JAK2. *p < 0.05; ^p < 0.05 represents a comparison with the pcDNA3-JAK2 group; #p < 0.05 represents a comparison with the OGD/R group. Figure is available in color online only.

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    Changes of JAK2 and miR-216a expression in the sham group, MCAO group, and miR-216a treatment group with 1 hour of ischemia following 24 hours of reperfusion at 3 hours, 12 hours, 1 day, 2 days, 3 days, and 7 days. A: The changes in miR-216a expression levels were measured using real-time qRT-PCR. U6 was used as an internal control for quantification; the expression of JAK2 protein was measured with immunohistochemistry. B: Representative immunohistochemical images of ischemia following 24 hours of reperfusion at 3 hours, 12 hours, 1 day, 2 days, 3 days, and 7 days. Figure is available in color online only.

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    p-STAT3, cleaved caspase-3, and iNOS levels were detected by immunohistochemistry in sham, MCAO, and miR-216a treatment groups with 1 hour of ischemia following 24 hours of reperfusion. Compared with the sham group, the protein levels of p-STAT3 (A), cleaved caspase-3 (B), and iNOS (C) of the MCAO group were increased and reached their highest levels on day 1, while miR-216a treatment reduced the protein levels. Figure is available in color online only.

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    miR-216a mimics attenuated infarct volumes, neurological dysfunction, and neuronal death. A: Ischemic injury was stained with TTC. B–D: Comparisons among the 3 groups for infarct volumes (B), neurological scores (C), and brain water content (D). E: Brain cell apoptosis was detected by TUNEL assay. I/R = ischemia/reperfusion. *p < 0.05. Figure is available in color online only.

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