State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, China.
Invest Ophthalmol Vis Sci. 2023 Oct 3;64(13):18. doi: 10.1167/iovs.64.13.18.
N6-methyladenosine (m6A) is a commonly occurring modification of mRNAs, catalyzed by a complex containing methyltransferase like 3 (METTL3). Our research aims to explore how METTL3-dependent m6A modification affects the functions of retinal endothelial cells (RECs).
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% O2) or hypoxia (1% O2). Dot blot assay determined m6A 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 m6A modification in the target genes.
The m6A modification levels were significantly upregulated in the retinas and RECs of OIR mice. Exposure to hypoxia significantly elevated both METTL3 expression and m6A 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 m6A 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.
METTL3-mediated m6A 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.
N6-甲基腺苷（m6A）是一种常见的 mRNA 修饰，由包含甲基转移酶样 3（METTL3）的复合物催化。我们的研究旨在探索 METTL3 依赖性 m6A 修饰如何影响视网膜内皮细胞（REC）的功能。
建立氧诱导的视网膜病变（OIR）小鼠模型，使用磁珠法分离 REC。将人视网膜微血管内皮细胞（HRMEC）用常氧（21% O2）或缺氧（1% O2）处理。点印迹分析测定 m6A 修饰水平。定量 RT-PCR 和 Western blot 分别检测靶候选物的 mRNA 和蛋白表达水平。通过小干扰 RNA 转染敲低基因。基于 Matrigel 的血管生成和 Transwell 测定分别评估内皮管形成和迁移的能力。甲基化 RNA 免疫沉淀-qPCR 测定靶基因的 m6A 修饰水平。
OIR 小鼠的视网膜和 REC 中 m6A 修饰水平显著上调。HRMEC 暴露于缺氧环境中显著上调 METTL3 表达和 m6A 修饰水平。METTL3 敲低在常氧和缺氧条件下均显著抑制内皮管形成和迁移。同时，这种 HRMEC 中的敲低导致 MMP2 和 TIE2 转录物的 m6A 修饰水平降低，进而导致其各自蛋白表达减少。值得注意的是，MMP2 和 TIE2 的敲低也显著抑制了 HRMEC 的血管生成活性。
METTL3 介导的 m6A 修饰通过靶向 MMP2 和 TIE2 促进 REC 的血管生成行为，提示其在视网膜血管生成中的重要性和 METTL3 作为潜在的治疗靶点。
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