METTL3 (Methyltransferase Like 3)-Dependent N6-Methyladenosine Modification on Braf mRNA Promotes Macrophage Inflammatory Response and Atherosclerosis in Mice
METTL3 (Methyltransferase Like 3)-Dependent N6-Methyladenosine Modification on Braf mRNA Promotes Macrophage Inflammatory Response and Atherosclerosis in Mice
复制标题
Braf mRNA上的METTL3 （Methyltransferase Like 3）依赖性n6 -甲基腺苷修饰促进小鼠巨噬细胞炎症反应和动脉粥样硬化
DOI：
10.1161/atvbaha.122.318451
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发表时间：
2023-05-01
影响因子：
8.7
通讯作者：
Zhang, Cheng
中科院分区：
文献类型：
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作者：
Li, Qian;Yu, Liwen;Zhang, Cheng
背景：动脉粥样硬化是一种慢性炎症性疾病，其中巨噬细胞决定动脉粥样硬化斑块的进展。然而，尚无研究调查巨噬细胞中的 METTL3（甲基转移酶样 3）如何影响体内动脉粥样硬化斑块的形成。此外，Braf mRNA 是否被 METTL3 依赖性 N6-甲基腺苷 (m6A) 甲基化修饰仍不清楚。方法：我们分析了不同时期高脂饮食小鼠动脉粥样硬化斑块的单细胞测序数据。生成Mettl3(fl/fl) Lyz2(cre) Apoe(-/-)小鼠和同窝对照Mettl3(fl/fl) Apoe(-/-)小鼠并饲喂高脂肪饮食14周。在体外，我们用ox-LDL（氧化低密度脂蛋白）刺激腹膜巨噬细胞，并测试炎症因子和ERK（细胞外信号调节激酶）磷酸化调节分子的mRNA和蛋白表达水平。为了寻找巨噬细胞中的 METTL3 靶标，我们进行了 m6A 甲基化 RNA 免疫沉淀测序和 m6A 甲基化 RNA 免疫沉淀 qPCR。此外，使用点突变实验来探索 m6A 甲基化腺嘌呤。使用 RNA 免疫沉淀测定，我们探索了与 Braf mRNA 结合的 m6A 甲基化写入蛋白。结果：在体内，巨噬细胞中METTL3的表达随着动脉粥样硬化的进展而增加。骨髓细胞特异性 METTL3 缺失负向调节动脉粥样硬化进展和炎症反应。在体外，巨噬细胞中的 METTL3 敲低或敲除会减弱 ox-LDL 介导的 ERK 磷酸化，而不是 JNK（c-Jun N 末端激酶）和 p38 磷酸化，并通过影响 BRAF 蛋白表达来降低炎症因子水平。 METTL3 敲除引起的炎症反应的负调节可以通过 BRAF 的过表达来挽救。在机制上，METTL3 靶向 Braf mRNA 上的腺嘌呤（6 号染色体上的 39725126）。然后，YTHDF1 可以与 m6A 甲基化的 Braf mRNA 结合并促进其翻译。结论：骨髓细胞特异性Mettl3缺陷可抑制高脂血症诱导的动脉粥样硬化斑块形成并减轻动脉粥样硬化炎症。我们确定 Braf mRNA 是 METTL3 激活 ox-LDL 诱导的 ERK 通路和巨噬细胞炎症反应的新靶标。 METTL3可能是治疗动脉粥样硬化的潜在靶点。
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 mRNA 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) Lyz2(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 promoted 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.