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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: Chemokine CXCL12, MicroRNAs, Myocardial Ischemia, Myocytes, Cardiac, Receptors, CXCR4, Apoptosis, Base Sequence, Cell Line, Glucose, Models, Biological, Nitric Oxide, Nitric Oxide Synthase Type III, Oxidative Stress, Oxygen, Reperfusion Injury, Signal Transduction

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