30 January 2020 : Laboratory Research
MicroRNA-668-3p Protects Against Oxygen-Glucose Deprivation in a Rat H9c2 Cardiomyocyte Model of Ischemia-Reperfusion Injury by Targeting the Stromal Cell-Derived Factor-1 (SDF-1)/CXCR4 Signaling Pathway
Zhan Gao1ABCE, Qiang Gao1BDEF, Xiaodong Lv2AF*DOI: 10.12659/MSM.919601
Med Sci Monit 2020; 26:e919601
Abstract
BACKGROUND: Ischemia-reperfusion injury (IRI) results from the restoration of blood supply to ischemic organs, including the heart. Expression of microRNA-668-3p (miR-668-3p) is known to protect the kidney from IRI. This study aimed to investigate the role of miR-668-3p in oxygen-glucose deprivation (OGD) in a rat H9c2 cardiomyocyte model of IRI.
MATERIAL AND METHODS: Rat H9c2 cardiomyocytes were cultured in glucose-free Dulbecco’s modified Eagle’s medium (DMEM) under anaerobic conditions, followed by oxygenation, to create the OGD model of IRI. The luciferase reporter assay evaluated the interaction between stromal cell-derived factor-1 (SDF-1), or CXC motif chemokine 12 (CXCL12), and miR-668-3p. Protein and mRNA levels of SDF-1, CXCR4, Bcl2, Bax, cleaved caspase-3, endothelial nitric oxide synthase (eNOS), and phosphorylated endothelial nitric oxide synthase (p-eNOS) were analyzed by Western blot and quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and apoptosis were assessed by flow cytometry. Enzyme-linked immunosorbent assay (ELISA) measured reactive oxygen species (ROS), including malondialdehyde (MDA), nitric oxide (NO), p-eNOS, and the inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 (MCP-1) in H9c2 cell supernatants.
RESULTS: In the OGD rat H9c2 cardiomyocyte model of IRI, miR-668-3p levels were reduced. Overexpression of miR-668-3p inhibited SDF-1, CXCR4, the expression of inflammatory cytokines, markers of oxidative stress, and p-eNOS. The overexpression of SDF-1 reversed these findings. Overexpression of SDF-1 promoted cell apoptosis, which was reduced by miR-668-3p.
CONCLUSIONS: In the OGD rat H9c2 cardiomyocyte model of IRI, miR-668-3p suppressed mediators of inflammation and oxidative stress and enhanced cell viability through the SDF-1/CXCR4 signaling pathway.
Keywords: Myocardial Ischemia, Base Sequence, Cell Line, Glucose, Models, Biological, Nitric Oxide, Nitric Oxide Synthase Type III, Oxygen
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