Circ_0000647 promotes cell injury by modulating miR-126-5p/TRAF3 axis in oxygen-glucose deprivation and reperfusion-induced SK-N-SH cell model

Abstract

Background: Emerging evidence has shown that circular RNAs (circRNAs) are involved in the pathogenesis of ischemic stroke (IS). Nonetheless, the function of circ_0000647 was not reported.

Methods: Oxygen-glucose deprivation and reperfusion (OGD/R)-treated SK-N-SH cells were used to mimic cerebral ischemia/reperfusion (I/R) conditions. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure the levels of circ_0000647, microRNA-126-5p (miR-126-5p) and TNF receptor associated factor 3 (TRAF3). Cell Counting Kit-8 (CCK-8) assay, 5'-ethynyl-2'-deoxyuridine (EDU) assay and flow cytometry analysis were employed to assess cell proliferation and apoptosis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the concentrations of IL-6 and TNF-α. Oxidative stress was assessed by determining malondialdehyde (MDA) level and superoxide dismutase (SOD) activity. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to estimate the relationships of circ_0000647, miR-126-5p and TRAF3. The morphology and size of exosomes were observed via transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) analysis.

Results: Circ_0000647 was elevated in OGD/R-treated SK-N-SH cells. OGD/R treatment suppressed the proliferation and promoted the apoptosis, inflammation and oxidative stress in SK-N-SH cells, while circ_0000647 knockdown reversed the effects. Circ_0000647 could sponge miR-126-5p, which directly targeted TRAF3. MiR-126-5p overexpression alleviated OGD/R-induced SK-N-SH cell damage and miR-126-5p inhibition reversed the effect of circ_0000647 knockdown on OGD/R-induced SK-N-SH cell damage. Moreover, TRAF3 elevation abated miR-126-5p-mediated effect on SK-N-SH cell injury. In addition, exosomal circ_0000647 level was increased in OGD/R-stimulated SK-N-SH cells.

Conclusion: Circ_0000647 interference relieved OGD/R-induced SK-N-SH cell damage by altering miR-126-5p/TRAF3 axis.

Keywords: Circ_0000647; Ischemic stroke; OGD/R; TRAF3; miR-126-5p.