Department of Ophthalmology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
Department of Pathology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
Invest Ophthalmol Vis Sci. 2024 Oct 1;65(12):34. doi: 10.1167/iovs.65.12.34.
This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV).
A mouse CNV model was established through corneal alkali burns. Total m6A levels were measured using an m6A RNA methylation quantification kit. The mRNA expression of candidate m6A-related enzymes was quantified by quantitative RT-PCR. Small interfering RNA targeting ALKBH5 was injected subconjunctivally into alkali-burned mice. The CNV area, corneal epithelial thickness, and pathological changes were evaluated. Protein expression was detected by western blot and immunofluorescence. Human umbilical vein endothelial cells (HUVECs) were treated with IL-6. Plasmid transfection knocked down ALKBH5 or overexpressed FOXM1 in IL-6-induced HUVECs. The assays of CCK8, wound healing, and tube formation evaluated the cell proliferation, migration, and tube formation abilities, respectively. The dual-luciferase assay examined the binding between ALKBH5 and FOXM1. Methylated RNA immunoprecipitation-qPCR detected the m6A levels of FOXM1.
Significant CNV was observed on the seventh day. Total m6A levels were reduced, and ALKBH5 expression was increased in CNV corneas and IL-6-induced HUVECs. ALKBH5 knockdown alleviated corneal neovascularization and inflammation and countered IL-6-induced promotion of cell proliferation, migration, and tube formation in HUVECs. ALKBH5 depletion increased m6A levels and decreased VEGFA and CD31 expression both in vivo and in vitro. This knockdown in HUVECs elevated m6A levels on FOXM1 mRNA while reducing its mRNA and protein expression. Notably, FOXM1 overexpression can reverse ALKBH5 depletion effects.
ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.
本研究旨在探讨 ALKBH5 介导的 N6-甲基腺苷（m6A）去甲基化修饰在角膜新生血管（CNV）中的调控作用及潜在机制。
通过角膜碱烧伤建立小鼠 CNV 模型。采用 m6A RNA 甲基化定量试剂盒检测总 m6A 水平。采用定量 RT-PCR 检测候选 m6A 相关酶的 mRNA 表达。通过结膜下注射 ALKBH5 小干扰 RNA 处理碱烧伤小鼠。评估 CNV 面积、角膜上皮厚度和病理变化。采用 Western blot 和免疫荧光检测蛋白表达。用白细胞介素-6（IL-6）处理人脐静脉内皮细胞（HUVEC）。用质粒转染敲低 IL-6 诱导的 HUVEC 中的 ALKBH5 或过表达 FOXM1。用 CCK8、划痕愈合和管形成实验分别评估细胞增殖、迁移和管形成能力。双荧光素酶报告基因检测 ALKBH5 与 FOXM1 之间的结合。甲基化 RNA 免疫沉淀-qPCR 检测 FOXM1 的 m6A 水平。
第 7 天观察到明显的 CNV。CNV 角膜和 IL-6 诱导的 HUVEC 中总 m6A 水平降低，ALKBH5 表达增加。ALKBH5 敲低减轻了角膜新生血管和炎症，并拮抗了 IL-6 诱导的 HUVEC 中细胞增殖、迁移和管形成的促进作用。ALKBH5 敲低在体内和体外均增加了 FOXM1 mRNA 的 m6A 水平，降低了 VEGFA 和 CD31 的表达。该敲低还增加了 HUVECs 中 FOXM1 mRNA 的 m6A 水平，同时降低了其 mRNA 和蛋白表达。值得注意的是，FOXM1 的过表达可以逆转 ALKBH5 敲低的作用。
ALKBH5 调节 FOXM1 m6A 去甲基化，影响 CNV 进展，提示其作为治疗靶点的潜力。
