Methane exerts antioxidant effects via a hsa_circ_0040806-miR-145-5p-NFE2L2 signaling axis in hypoxia-reoxygenation injury PC12 neuronal cells

Blood reperfusion after ischemic stroke is the first step of treatment but can result in serious brain injury, termed cerebral ischemia-reperfusion (IR) injury. Methane has shown protective effects against IR injury and we established an oxygen-glucose deprivation/reoxygenation (OGD/R) model in PC12 cells to investigate the mechanism of how this occurs. Methane treatment protected OGD/R PC12 cells from IR injury by reducing lactate dehydrogenase (LDH), reactive oxygen species (ROS) and malondialdehyde (MDA) while increasing Superoxide Dismutase type 1 (SOD1) and glutathione (GSH), which is indicative of oxidative stress. Methane treatment upregulated NFE2L2 expression and knockdown of NFE2L2 abrogated the protective effects of methane. We identified miR-145-5p as a candidate to interact with the 3′-untranslated region (3′-UTR) of NFE2L2 and used luciferase reporter assays to demonstrate a specific interaction between these two transcripts. Transfecting cells with a miR-145-5p mimic abrogated the protective effects of methane treatment while a miR-145-5p inhibitor enhanced the effects. We identified the circular RNA hsa_circ_0040806 as potentially having a sequence-specific interaction with miR-145-5p and luciferase reporter assays indicated a sequence-specific interaction between these two transcripts. Overexpression of hsa_circ_0040806 resulted in increased levels of NFE2L2, similar to treatment with methane. We propose a mechanism where methane causes increased expression of hsa_circ_0040806 , which binds to miR-145-5p . This prevents miR-145-5p from binding to the NFE2L2 mRNA transcript, resulting in increased protein levels. • Methane has been shown to have antioxidant effects in cerebral stroke. • NFE2L2 is a key gene influencing HO-1 expression in this effect. • Methane treatment could upregulate NFE2L2 expression. • hsa_circ_0040806 interacts with miR-145-5p. • miR-145-5p could bind to NFE2L2 mRNA and suppress its protein level.