METTL3-Mediated RNA m(6)A Modification Regulates the Angiogenic Behaviors of Retinal Endothelial Cells by Methylating MMP2 and TIE2

PURPOSE: N(6)-methyladenosine (m(6)A) is a commonly occurring modification of mRNAs, catalyzed by a complex containing methyltransferase like 3 (METTL3). Our research aims to explore how METTL3-dependent m(6)A modification affects the functions of retinal endothelial cells (RECs). METHODS: An oxygen-induced retinopathy (OIR) mouse model was established, and RECs were isolated using magnetic beads method. Human retinal microvascular endothelial cells (HRMECs) were treated with normoxia (21% O(2)) or hypoxia (1% O(2)). Dot blot assay determined m(6)A modification levels. Quantitative RT-PCR and Western blot detected the mRNA and protein expression levels of the target candidates, respectively. Genes were knocked down by small interfering RNA transfection. Matrigel-based angiogenesis and transwell assays evaluated the abilities of endothelial tube formation and migration, respectively. Methylated RNA immunoprecipitation–qPCR determined the levels of m(6)A modification in the target genes. RESULTS: The m(6)A modification levels were significantly upregulated in the retinas and RECs of OIR mice. Exposure to hypoxia significantly elevated both METTL3 expression and m(6)A modification levels in HRMECs. METTL3 knockdown curtailed endothelial tube formation and migration in vitro under both normoxic and hypoxic conditions. Concurrently, this knockdown in HRMECs resulted in reduced m(6)A modification levels of MMP2 and TIE2 transcripts, subsequently leading to a decrease in their respective protein expressions. Notably, knockdown of MMP2 and TIE2 also markedly inhibited the angiogenic activities of HRMECs. CONCLUSIONS: METTL3-mediated m(6)A modification promotes the angiogenic behaviors of RECs by targeting MMP2 and TIE2, suggesting its significance in retinal angiogenesis and METTL3 as a potential therapeutic target.