Cerebral arteriovenous malformation (AVM) is a serious life-threatening congenital cerebrovascular disease. Specific anatomical features, such as nidus size, location, and venous drainage, have been validated to affect treatment outcomes. Until recently, molecular biomarkers and corresponding molecular mechanism related to anatomical features and treatment outcomes remain unknown. RNA N6-methyladenosine (m6A) Methyltransferase METTL3 was identified as a differentially expressed gene in groups with different lesion sizes by analyzing the transcriptome sequencing (RNA-seq) data. Tube formation and wound healing assays were performed to investigate the effect of METTL3 on angiogenesis. In addition, Methylated RNA Immunoprecipitation Sequencing technology (MeRIP-seq) was performed to screen downstream targets of METTL3 in endothelial cells and to fully clarify the specific underlying molecular mechanisms affecting the phenotype of cerebral AVM. In the current study, we found that the expression level of METTL3 was reduced in the larger pathological tissues of cerebral AVMs. Moreover, knockdown of METTL3 significantly affected angiogenesis of the human endothelial cells. Mechanistically, down-regulation of METTL3 reduced the level of heterodimeric Notch E3 ubiquitin ligase formed by DTX1 and DTX3L, thereby continuously activating the Notch signaling pathway. Ultimately, the up-regulated downstream genes of Notch signaling pathway dramatically affected the angiogenesis of endothelial cells. In addition, we demonstrated that blocking Notch pathway with DAPT could restore the phenotype of METTL3 deficient endothelial cells. Our findings revealed the mechanism by which m6A modification regulated the angiogenesis and might provide potential biomarkers to predict the outcome of treatment, as well as provide suitable pharmacological targets for preventing the formation and progression of cerebral AVM.

中文翻译：

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N6-甲基腺苷甲基转移酶METTL3通过调节Notch信号通路影响脑动静脉畸形的表型

脑动静脉畸形（AVM）是一种严重的威胁生命的先天性脑血管疾病。特定的解剖特征，如病灶的大小，位置和静脉引流，已被证实可影响治疗效果。直到最近，与解剖学特征和治疗结果相关的分子生物标志物和相应的分子机制仍然未知。通过分析转录组测序（RNA-seq）数据，RNA N6-甲基腺苷（m6A）甲基转移酶METTL3被鉴定为具有不同病变大小的组中的差异表达基因。进行管形成和伤口愈合测定以研究METTL3对血管生成的影响。此外，进行了甲基化RNA免疫沉淀测序技术（MeRIP-seq），以筛选内皮细胞中METTL3的下游靶标，并充分阐明影响脑AVM表型的特定潜在分子机制。在当前的研究中，我们发现METTL3在脑AVM的较大病理组织中的表达水平降低。此外，敲低METTL3显着影响人类内皮细胞的血管生成。从机制上讲，METTL3的下调降低了DTX1和DTX3L形成的异二聚体Notch E3泛素连接酶的水平，从而持续激活Notch信号通路。最终，Notch信号通路的下游基因的上调极大地影响了内皮细胞的血管生成。此外，我们证明了用DAPT阻断Notch通路可以恢复METTL3缺陷型内皮细胞的表型。我们的发现揭示了m6A修饰调节血管生成的机制，并可能提供潜在的生物标志物来预测治疗的结果，并提供合适的药理靶标来预防脑AVM的形成和发展。