Targeting RNA Methyltransferase METTL3 Alleviates Doxorubicin-Induced Vascular Injury

This BioProject focuses on investigating the role of RNA N6-methyladenosine (m6A) modification mediated by methyltransferase METTL3 in doxorubicin (DOX)-induced vascular injury, and exploring the therapeutic potential of an endothelium-targeted nano-delivery system.DOX, a widely used anthracycline chemotherapeutic, causes severe vascular endothelial toxicity (e.g., drug-induced phlebitis, accelerated atherosclerosis) that impairs patient quality of life. While DOX disrupts vascular homeostasis via endothelial senescence, inflammation, and oxidative stress, its underlying epigenetic regulatory mechanisms remain unclear. m6A modification, catalyzed by METTL3, is a key epitranscriptional regulator in cardiovascular diseases, but whether METTL3 participates in DOX-induced vascular injury and its potential as a therapeutic target need further exploration.In this study, we first validated the effects of DOX on METTL3 expression and m6A modification levels in vascular endothelial cells using in vitro and in vivo models, combined with SA-beta-gal staining, monocyte adhesion assays, and Western blot to analyze cell senescence, inflammation, and adhesion molecule expression. We then constructed endothelial-specific METTL3 knockout mice (METTL3cKO) to evaluate DOX-induced vascular senescence and plaque formation via aortic Oil Red O (ORO) staining, HE staining, and beta-GAL staining. Additionally, we developed a biomimetic nanoparticle (ETMN, Endothelium-Targeted METTL3 Nano-inhibitor) using mesoporous silica (MSN) loaded with the METTL3 inhibitor STM2457, modified with platelet membrane fusion and CD31 antibody for endothelial targeting, and functionalized with beta-cyclodextrin/adamantane for drug sustained release. The physicochemical properties of ETMN were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FTIR); its targeting ability and therapeutic efficacy were verified by in vivo small animal imaging and pharmacodynamic experiments.For m6A profiling, m6A-IP was performed using a GenSeq RNA methylation kit, followed by RNA library construction with the NEBNext Ultra II Directional RNA Library Prep Kit (quality-controlled via Agilent 2100 Bioanalyzer) and high-throughput sequencing on an Illumina NovaSeq 6000 platform. Raw reads were quality-filtered (Q30), adapter-trimmed with cutadapt (v1.9.3) to obtain clean reads, aligned to the Mouse reference genome (MM10) using HISAT2 (v2.0.4), and m6A-modified genes were identified via MACS. Differential m6A-modified genes were detected using difReps, followed by annotation of peaks located on mRNA exons (via in-house scripts) and GO/Pathway enrichment analysis.This project aims to clarify the mechanism of METTL3-m6A modification in DOX-induced vascular injury and provide a novel endothelium-targeted nano-therapeutic strategy to alleviate DOX-associated vascular toxicity.