ALKBH5 通过介导 FOXM1 M6A 去甲基化调控角膜新生血管形成

IF 5 2区 医学 Q1 OPHTHALMOLOGY
Wei Wang, Hua Li, Yiyong Qian, Min Li, Manli Deng, Dexi Bi, Jun Zou
{"title":"ALKBH5 通过介导 FOXM1 M6A 去甲基化调控角膜新生血管形成","authors":"Wei Wang, Hua Li, Yiyong Qian, Min Li, Manli Deng, Dexi Bi, Jun Zou","doi":"10.1167/iovs.65.12.34","DOIUrl":null,"url":null,"abstract":"<p><strong>Purpose: </strong>This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV).</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.</p>","PeriodicalId":14620,"journal":{"name":"Investigative ophthalmology & visual science","volume":null,"pages":null},"PeriodicalIF":5.0000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11512564/pdf/","citationCount":"0","resultStr":"{\"title\":\"ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation.\",\"authors\":\"Wei Wang, Hua Li, Yiyong Qian, Min Li, Manli Deng, Dexi Bi, Jun Zou\",\"doi\":\"10.1167/iovs.65.12.34\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Purpose: </strong>This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV).</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.</p>\",\"PeriodicalId\":14620,\"journal\":{\"name\":\"Investigative ophthalmology & visual science\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":5.0000,\"publicationDate\":\"2024-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11512564/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Investigative ophthalmology & visual science\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1167/iovs.65.12.34\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"OPHTHALMOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Investigative ophthalmology & visual science","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1167/iovs.65.12.34","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"OPHTHALMOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

目的: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)中的调控作用和潜在机制:方法:通过角膜碱烧伤建立小鼠CNV模型。方法:通过角膜碱烧伤建立小鼠 CNV 模型,使用 m6A RNA 甲基化定量试剂盒测定 m6A 总含量。通过定量 RT-PCR 对候选 m6A 相关酶的 mRNA 表达进行定量。向碱烧伤小鼠结膜下注射靶向 ALKBH5 的小干扰 RNA。评估了 CNV 面积、角膜上皮厚度和病理变化。蛋白表达通过 Western 印迹和免疫荧光进行检测。用 IL-6 处理人脐静脉内皮细胞(HUVECs)。质粒转染敲除 ALKBH5 或过表达 FOXM1。CCK8、伤口愈合和管形成试验分别评估了细胞的增殖、迁移和管形成能力。双荧光素酶试验检测了 ALKBH5 与 FOXM1 的结合情况。甲基化 RNA 免疫沉淀-qPCR 检测了 FOXM1 的 m6A 水平:结果:第七天观察到显著的 CNV。在 CNV 角膜和 IL-6 诱导的 HUVEC 中,m6A 总含量降低,ALKBH5 表达增加。敲除 ALKBH5 可减轻角膜新生血管和炎症,并抵消 IL-6 诱导的促进 HUVECs 细胞增殖、迁移和管形成的作用。在体内和体外,ALKBH5 的耗竭会增加 m6A 的水平,降低 VEGFA 和 CD31 的表达。这种在 HUVECs 中的敲除会提高 FOXM1 mRNA 上的 m6A 水平,同时降低其 mRNA 和蛋白表达。值得注意的是,FOXM1的过表达可以逆转ALKBH5的耗竭效应:结论:ALKBH5 可调节 FOXM1 m6A 的去甲基化,从而影响 CNV 的进展并突显其作为治疗靶点的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation.

Purpose: This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV).

Methods: 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.

Results: 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.

Conclusions: ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
6.90
自引率
4.50%
发文量
339
审稿时长
1 months
期刊介绍: Investigative Ophthalmology & Visual Science (IOVS), published as ready online, is a peer-reviewed academic journal of the Association for Research in Vision and Ophthalmology (ARVO). IOVS features original research, mostly pertaining to clinical and laboratory ophthalmology and vision research in general.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信