The Interplay between FTO-Mediated m6A Demethylation and Ferroptosis in the Development of Diabetic Retinopathy

Abstract

Background: Diabetic vascular complications are mainly caused by endothelial dysfunction, which is influenced by inflammation and epigenetic modifications. In this study, we aimed to investigate the role of fat-, mass- and obesity-associated (FTO) protein, an N6-methyladenosine (m6A) demethylase, in diabetes-induced retinal vascular endothelial dysfunction. Methods: Human retinal vascular endothelial cells (RECs) were treated with high glucose (HG). The effect of HG on RECs was examined by various methods. The cell proliferation and apoptosis rate were determined using cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) methods. The oxidative stress represented by the levels of Fe²⁺, malondialdehyde (MDA), and reduced glutathione (GSH) was assessed using commercially available kits. Moreover, the levels of m6A methylation and FTO protein, and the levels of ferroptosis-related proteins were evaluated using corresponding kits and Western blot analysis, respectively. The cell transfection method was used to regulate FTO in RECs and to determine the consequent effect on HG-treated RECs. Additionally, the levels of reactive oxygen species (ROS), MDA, 4-hydroxynonenal (4-HNE), and the structural changes in mitochondria were observed using Transmission electron microscopy. Furthermore, Erastin was used to induce ferroptosis in RECs with FTO knockdown and the consequent effects on ferroptosis, m6A methylation, oxidative stress, proliferation and apoptosis of HG-treated RECs were evaluated. Results: HG treatment decreased proliferation (p < 0.01) and m6A methylation (p < 0.01), while increased apoptosis (p < 0.01), oxidative stress (p < 0.01), and ferroptosis (p < 0.01) in RECs. Furthermore, these HG-induced changes were ameliorated by FTO (p < 0.01). Additionally, it was found that the protective effect of FTO knockdown was disrupted by ferroptosis inducer (p < 0.01). Conclusion: This study revealed the pathological role of FTO-dependent RNA demethylation in the retinal endothelial homeostasis induced by diabetes. Our findings suggest that FTO mediates diabetes-induced vascular endothelial changes by regulating ferroptosis. This finding could provide a new strategy for intervening in FTO and its related inflammatory pathways to treat diabetic vascular complications.