METTL3 (Methyltransferase Like 3)-Dependent N6-Methyladenosine Modification on Braf mRNA Promotes Macrophage Inflammatory Response and Atherosclerosis in Mice

Abstract

BACKGROUND: Atherosclerosis is a chronic inflammatory disease, in which macrophages determine the progression of atherosclerotic plaques. However, no studies have investigated how METTL3 (methyltransferase like 3) in macrophages affects atherosclerotic plaque formation in vivo. Additionally, whether Braf is modified by METTL3-dependent N6-methyladenosine (m6A) methylation remains unknown. METHODS: We analyzed single-cell sequencing data of atherosclerotic plaques in mice fed with a high fat diet for different periods. Mettl3 fl/fl Lyz 2 cre Apoe –/– mice and littermate control Mettl3 fl/fl Apoe –/– mice were generated and fed high fat diet for 14 weeks. In vitro, we stimulated peritoneal macrophages with ox-LDL (oxidized low-density lipoprotein) and tested the mRNA and protein expression levels of inflammatory factors and molecules regulating ERK (extracellular signal-regulated kinase) phosphorylation. To find METTL3 targets in macrophages, we performed m6A-methylated RNA immunoprecipitation sequencing and m6A-methylated RNA immunoprecipitation-qPCR. Further, point mutation experiments were used to explore m6A-methylated adenine. Using RNA immunoprecipitation assay, we explored m6A methylation-writing protein bound to Braf mRNA. RESULTS: In vivo, METTL3 expression in macrophages increased with the progression of atherosclerosis. Myeloid cell–specific METTL3 deletion negatively regulated atherosclerosis progression and the inflammatory response. In vitro, METTL3 knockdown or knockout in macrophages attenuated ox-LDL-mediated ERK phosphorylation rather than JNK (c-Jun N-terminal kinase) and p38 phosphorylation and reduced the level of inflammatory factors by affecting BRAF protein expression. The negative regulation of inflammation response caused by METTL3 knockout was rescued by overexpression of BRAF. In mechanism, METTL3 targeted adenine (39725126 in chromosome 6) on the Braf mRNA. Then, YTHDF1 could bind to m6A-methylated Braf mRNA and promote its translation. CONCLUSIONS: Myeloid cell–specific Mettl3 deficiency suppressed hyperlipidemia-induced atherosclerotic plaque formation and attenuated atherosclerotic inflammation. We identified Braf mRNA as a novel target of METTL3 in the activation of the ox-LDL-induced ERK pathway and inflammatory response in macrophages. METTL3 may represent a potential target for the treatment of atherosclerosis.

Transcriptome-wide N6-methyladenosine methylation profile of atherosclerosis in mice

“…Our results showed that the expression of METTL3 was significantly increased in the aorta of AS mice, which is consistent with a previous study [ 18 ]. Furthermore, METLL3 silencing could hamper atherosclerotic plaque formation by targeting JAK2/STAT3 pathway or Braf mRNA, indicating the critical role of RNA methylation in AS progression [ 19 , 20 ], although the underlying molecular mechanism, deserves further investigation. Another study has demonstrated that the expression of FTO was significantly decreased in the human carotid artery plaques of early atherosclerosis [ 1 ].…”

The role of RNA methylation in tumor immunity and its potential in immunotherapy

“…Furthermore, lactic acid facilitated M2 polarization by activating METTL3 via the Trib1/ERK/STAT3 pathway [ 209 ]. Knockdown of METTL3/METTL14 significantly inhibited macrophage activation and secretion and slowed the progression of liver fibrosis [ 210 , 211 ]. Additionally, WTAP and RBM15 interact with M1 macrophages and mediate downstream inflammatory responses [ 212 ] (Fig.…”

N6-Methyladenosine (m6A) Modification in Natural Immune Cell-Mediated Inflammatory Diseases

“…Similarly, METTL3 promoted oxLDL-induced macrophage inflammatory response via the m6A modification of Stat1 and Braf mRNA, explaining the observation that oxLDL enhanced m6A modification of macrophage mRNA. METTL3 also interacted with STAT1 protein to promote transcription of macrophage inflammatory factors [33,34]. However, decreased m6A modification of macrophage RNA in response to oxLDL has been reported, mediated by Matrin-3 (Matr3) which enhanced the formation of the METTL3-METTL14 complex.…”

“…The diverse reported patterns in oxLDL-stimulated macrophage m6A may be attributed to the different cell models (RAW264.7 and THP-1) and detection techniques (dot blot and flow cytometry) used [33][34][35]. Further investigations, including assessment of cell models and detection techniques, are necessary to establish whether m6A modification promotes or inhibits AS and molecular mechanisms involved.…”