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02 February 2020 : Laboratory Research  

Downregulation of MiR-218-5p Protects Against Oxygen-Glucose Deprivation/Reperfusion-Induced Injuries of PC12 Cells via Upregulating N-myc Downstream Regulated Gene 4 (NDRG4)

Huiying Zhu1BE, Xiaojing Wang2CF, Shaoyuan Chen2AG*

DOI: 10.12659/MSM.920101

Med Sci Monit 2020; 26:e920101

Abstract

BACKGROUND: Cerebral ischemia is a major player of acute ischemic stroke (AIS) and mainly caused by blood vessels obstruction-induced reduced blood flow. Furthermore, miR-218-5p level was elevated in patients with AIS compared with controls. The present study investigated the biochemical mechanisms underlying the role of miR-218-5p in AIS in vitro.

MATERIAL AND METHODS: PC12 cells were chosen to establish oxidative-glucose deprivation/re-oxygenation (OGD/R) injury model. The interaction between miR-218-5p and N-myc downstream regulated gene 4 (NDRG4) was evaluated by Luciferase reporter assay. The levels of NDRG4, endothelial nitric oxide synthase (eNOS) and protein related to cell apoptosis were quantitatively analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) or western blotting. Inflammatory cytokines, myeloperoxidase (MPO) and oxidative stress status were measured using specific commercial assay kits. Further, the cells apoptosis was analyzed with flow cytometry assay.

RESULTS: MiR-218-5p level was notably increased in OGD/R injured PC12 cells and directly targeted NDRG4. MiR-218-5p inhibitor significantly inhibited inflammatory cytokines release, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemotactic protein 1 (MCP-1). In addition, miR-218-5p downregulation ameliorated nitric oxide (NO) and eNOS levels and suppressed the inducible nitric oxide synthase (iNOS) expression and cell apoptosis. However, NDRG4 silencing abolished all corrective effects of miR-218-5p inhibitor in OGD/R injured PC12 cells.

CONCLUSIONS: Downregulation of miR-218-5p protect against OGDR-induced injuries of PC12 cells through reducing inflammatory cytokines secretion, oxidative stress status, apoptosis rate and maintenance of endovascular homeostasis via upregulating NDRG4. MiR-218-5p may serve as a novel effective biomarker to monitor AIS progression.

Keywords: Encephalitis, Hypoxia-Ischemia, Brain, biomarkers, Culture Media, Down-Regulation, Glucose, ischemic stroke, Muscle Proteins, Nerve Tissue Proteins, Oxygen, Reperfusion Injury, Up-Regulation

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750