DNMT1 methylates E3 ligase FBXO32 to regulate Myc stability and glycolytic reprogramming in diabetic retinopathy associated endothelial cells.

F-box protein 32 (FBXO32), an E3 ubiquitin ligase, has been implicated in various cellular processes, but its role and regulatory network in DR-associated endothelial dysfunction are unclear. Here, we aimed to investigate the function of FBXO32 in high glucose (HG)-induced vascular endothelial cell injury and the underlying molecular mechanisms mediated by DNMT1 and Myc. Human retinal microvascular endothelial cells (HRMECs) were exposed to HG to mimic DR in vitro. The expression of FBXO32 and DNMT1 was detected by qRT-PCR and Western blot. Gain-of-function and loss-of-function experiments were performed to manipulate FBXO32, DNMT1, and Myc expression. Cell migration was evaluated by wound healing assay. Inflammatory cytokines (IL-1β, IL-6, TNF-α) were measured by ELISA. Glycolytic metabolism was assessed by detecting glucose consumption, lactate production, ATP level, and extracellular acidification rate (ECAR). Co-immunoprecipitation (Co-IP), ubiquitination assay, protein stability assay and GST pull down assay were used to verify the interaction between FBXO32 and Myc. Methylation-specific PCR (MSP) was performed to detect the methylation status of FBXO32 promoter. HG treatment significantly downregulated FBXO32 expression in HRMECs. Overexpression of FBXO32 inhibited HG-induced cell migration, inflammation (decreased IL-6 and TNF-α levels), and glycolytic metabolism (reduced glucose consumption, lactate production, ATP level, and ECAR). Treatment with 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, abolished the effects of FBXO32 overexpression on cell migration and inflammation, indicating that FBXO32 exerts its function by regulating glycolysis. The interaction between FBXO32 and Myc were confirmed by Co-IP assay and GST pull down assay. Ubiquitination and protein stability assays showed that FBXO32 promotes the ubiquitination and degradation of Myc, thereby reducing Myc protein stability. Overexpression of Myc reversed the inhibitory effects of FBXO32 on HG-induced cell migration, inflammation, and glycolysis. Furthermore, we identified DNMT1 as an upstream regulator of FBXO32. DNMT1 binds to the promoter region of FBXO32 and induces its methylation, leading to the downregulation of FBXO32. Knockdown of DNMT1 upregulated FBXO32 expression, inhibited HG-induced cell migration, inflammation, and glycolysis, while co-knockdown of DNMT1 and FBXO32 reversed these effects. Our findings demonstrate that DNMT1-mediated methylation downregulates FBXO32 expression in HG-induced vascular endothelial cells. FBXO32 inhibits HG-induced endothelial cell migration, inflammation, and glycolytic reprogramming by promoting the ubiquitination and degradation of Myc. This DNMT1-FBXO32-Myc regulatory axis provides a novel therapeutic target for the treatment of DR.