CircNFIB attenuates hypoxia/reoxygenation-induced ferroptosis in AC16 cardiomyocytes with involvement of the miR-338-3p/GPX4 pathway.

Abstract

Cardiomyocyte ferroptosis contributes to myocardial ischemia/reperfusion injury, but the upstream noncoding RNA mechanisms remain incompletely defined. This study examined whether nuclear factor I B-derived circular RNA (circNFIB) regulates hypoxia/reoxygenation (H/R)-induced ferroptosis in human ventricular cardiomyocyte-derived AC16 cells and whether the miR-338-3p/GPX4 pathway contributes to this effect. The circNFIB back-splice junction was verified by Sanger sequencing. Cell viability, malondialdehyde, intracellular Fe2+, boron-dipyrromethene (BODIPY) 581/591 C11 oxidation, and glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), and transferrin receptor 1 (TfR1) protein abundance were evaluated. H/R decreased circNFIB expression, cell viability, and GPX4 abundance, while increasing lipid peroxidation, intracellular Fe2+ accumulation, and ACSL4 abundance; the reference ferroptosis inhibitor ferrostatin-1 partially reversed these changes. circNFIB overexpression improved cell viability, reduced intracellular Fe2+ accumulation and lipid peroxidation, partially restored GPX4 protein abundance, and lowered ACSL4 and TfR1 abundance. circNFIB was predominantly cytoplasmic. In reporter assays, miR-338-3p decreased the activity of reporters containing the wild-type circNFIB sequence or GPX4 3' untranslated region (3'UTR), whereas no significant reductions were observed with the corresponding mutant reporters. H/R increased miR-338-3p expression, whereas circNFIB overexpression reduced it. miR-338-3p overexpression aggravated the ferroptosis-associated phenotype and partially counteracted the protection associated with circNFIB overexpression. These findings indicate that circNFIB attenuates H/R-induced ferroptosis in AC16 cells, with the miR-338-3p/GPX4 pathway contributing to this effect.