BARD1-mediated stabilization of METTL14 promotes retinal neovascularization by m6A-modifying MXD1 mRNA on a YTHDF2-dependent manner.

Abstract

Retinal vascular diseases are typified by the proliferation of irregular and leaky microvessels, resulting in vision impairment. Although the etiology of retinal angiogenesis is not yet fully understood, it is evident that microglia play a pivotal role in promoting angiogenesis. Methods: In vivo, the METTL14 conditional knockout (cKO) mouse was constructed to investigate the role of METTL14 in oxygen-induced retinopathy (OIR). In vitro, a combination of methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-sequencing (RNA-seq), RNA Immunoprecipitation (RIP) assay, dual-luciferase reporter assays, and Chromatin immunoprecipitation-qPCR (ChIP-qPCR), was performed to explore the underlying mechanisms. Results: The proteomic analysis of hypoxic microglia has uncovered a pronounced enrichment in pathways related to RNA modification. Western blot has revealed that N6-methyladenosine (m6A) methyltransferase-like 14 (METTL14) exhibits the most significant increase among the RNA methylases. METTL14 cKO mice within an OIR model showed fewer neovascular formations. Additionally, in co-culture with sh-METTL14 HMC3 cells, HRMECs also exhibited reduced angiogenesis capabilities. Mechanically, E3 ubiquitin-protein ligase BARD1 can directly interact with METTL14, leading to an up-regulation of METTL14 protein level in hypoxic microglia. METTL14 could directly modifies and regulates the transcription factor MAX Dimerization Protein 1 (MXD1), which is subsequently recognized by the m6A "reader" YTH domain-containing family protein 2 (YTHDF2). Consequently, the modified MXD1 modulates the expression of VEGFA and VCAM1, promotes retinal neovascularization. Conclusion: Our study highlights the critical role of METTL14 in the OIR model and suggests a novel therapeutic target for addressing retinal vascular diseases.