Department of Ophthalmology and Clinical Centre of Optometry, Peking University People's Hospital, Eye Diseases and Optometry Institute, Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases, College of Optometry, Peking University Health Science Center, Beijing, China.
Department of Emergency, Peking University People's Hospital, Beijing, China.
J Proteomics. 2024 Oct 30;309:105282. doi: 10.1016/j.jprot.2024.105282. Epub 2024 Aug 22.
This research aimed to identify the function of fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), and explore its possible mechanisms in uveal melanoma (UVM).
We performed quantitative real-time PCR (qPCR), Western blotting and gene correlation analysis with GEPIA2 to assess FTO expression and identify its potential targets in UVM. CCK-8, colony formation, cell cycle, cell apoptosis, wound healing and Transwell invasion assays were utilized to assess cell viability, cell cycle distribution, apoptosis, migration and invasion. Western blotting, qPCR and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were carried out to explore the underlying mechanism of FTO in 2 UVM cell lines.
FTO, a key m6A demethylase, was found to be upregulated in human UVM tissues compared with normal choroid tissues. Knockdown of FTO in Mel270 and OMM2.3 cells significantly promoted proliferation and migration and suppressed apoptosis. Mechanistically, knockdown of FTO decreased the expression of ATG5, an autophagy-related gene, leading to attenuation of autophagosome formation, thereby inhibiting autophagy. Upon FTO knockdown, increased levels of methylated ATG5 and decreased ATG5 stability were detected. Furthermore, ATG5 dramatically alleviated FTO downregulation-induced tumor growth and metastasis.
Our research highlights the importance of the m6A demethylase FTO in UVM by demonstrating that it direct regulates ATG5-induced autophagy in an m6A-dependent manner. These findings suggest that FTO may serve as a potential therapeutic target for UVM.
本研究旨在确定脂肪量和肥胖相关蛋白（FTO）的功能，FTO 是 N6-甲基腺苷（m6A）的清除剂，并探索其在葡萄膜黑色素瘤（UVM）中的可能机制。
我们通过 GEPIA2 进行定量实时 PCR（qPCR）、Western blot 和基因相关性分析，以评估 FTO 在 UVM 中的表达并确定其潜在靶点。CCK-8、集落形成、细胞周期、细胞凋亡、划痕愈合和 Transwell 侵袭实验用于评估细胞活力、细胞周期分布、凋亡、迁移和侵袭。Western blot、qPCR 和甲基化 RNA 免疫沉淀-qPCR（MeRIP-qPCR）用于探索 FTO 在 2 种 UVM 细胞系中的潜在机制。
与正常脉络膜组织相比，FTO（一种关键的 m6A 去甲基化酶）在人 UVM 组织中上调。在 Mel270 和 OMM2.3 细胞中敲低 FTO 可显著促进增殖和迁移，并抑制凋亡。机制上，敲低 FTO 降低了自噬相关基因 ATG5 的表达，导致自噬体形成减少，从而抑制自噬。在 FTO 敲低后，检测到甲基化 ATG5 水平增加和 ATG5 稳定性降低。此外，ATG5 显著缓解了 FTO 下调诱导的肿瘤生长和转移。
我们的研究通过证明 FTO 以 m6A 依赖的方式直接调节 ATG5 诱导的自噬，强调了 m6A 去甲基酶 FTO 在 UVM 中的重要性。这些发现表明 FTO 可能成为 UVM 的潜在治疗靶点。
