摘要
目的:探索甲基转移酶3(METTL3)介导的N6-甲基腺苷(m6A)甲基化修饰在脉络膜新生血管发病中对血管内皮细胞生物学活性的调控作用及机制。方法:将体外培养的人脐静脉内皮细胞(HUVEC)分为:对照组(正常培养)、低密度脂蛋白(LDL)组、荧光标记LDL(Dil-LDL)组、12.5μg/mL、25μg/mL氧化低密度脂蛋白(ox-LDL)组、12.5μg/mL、25μg/mL荧光标记ox-LDL(Dil-ox-LDL)组、DMSO组、STM2457(METTL3抑制剂)组、DAPT组;将体外培养的猴视网膜-脉络膜内皮细胞(RF/6A)分为:对照组、DMSO组、12.5μg/mL ox-LDL组、DAPT组。荧光显微镜观察细胞内吞脂蛋白水平,dot blot检测m6A甲基化水平,Western blot检测METTL3及血管形成相关蛋白的表达水平,实时荧光定量聚合酶链反应(RT-qPCR)检测METTL3及血管形成相关标志物的mRNA表达水平,免疫荧光检测METTL3表达水平及定位,transwell实验检测细胞迁移能力,体外成管实验检测细胞血管形成能力。结果:与对照组相比,Dil-LDL组、12.5μg/mL、25μg/mL Dil-ox-LDL组细胞内荧光标记的脂蛋白含量显著升高,12.5μg/mL、25μg/mL ox-LDL组m6A甲基化水平显著升高(均P<0.05),METTL3蛋白表达水平显著升高(均P<0.01),细胞迁移及血管形成能力显著上升(均P<0.01),12.5μg/mL ox-LDL组METTL3 mRNA表达水平显著上调(P<0.05);与DMSO组相比,STM2457组m6A甲基化水平显著降低(P<0.05),METTL3的蛋白及mRNA表达水平无显著差异(均P>0.05),血管内皮生长因子(VEGF)等血管形成相关标志物表达水平显著下降(均P<0.05),细胞迁移及血管形成能力显著下降(均P<0.01),NICD表达水平显著下降(P<0.05);与DMSO组相比,DAPT组NICD、VEGF表达水平显著下降(均P<0.05),HUVEC及RF/6A细胞迁移及血管形成能力显著下降(均P<0.01)。结论:METTL3介导的m6A甲基化修饰在脉络膜新生血管发病中可经Notch通路促进血管内皮细胞的血管形成。
AIM:To investigate the role and mechanism of methyltransferase-like 3(METTL3)-mediated N6-methyladenosine(m 6 A)methylation modification in regulating biological activity of vascular endothelial cells in the pathogenesis of choroidal neovascularization.METHODS:Human umbilical vein endothelial cells(HUVEC)cultured in vitro were divided into the following groups:control group(normal culture),low density lipoprotein(LDL)group,fluorescence-labelled LDL(Dil-LDL)group,12.5μg/mL and 25μg/mL oxidized LDL(ox-LDL)groups,12.5μg/mL and 25μg/mL fluorescence-labelled ox-LDL(Dil-ox-LDL)groups,DMSO group,STM2457(METTL3 inhibitor)group,DAPT group;and monkey retina-choroidal endothelial cells(RF/6A)cultured in vitro were divided into control group,DMSO group,12.5μg/mL ox-LDL group,and DAPT group.Endocytosed lipoprotein level was examined through fluorescence microscopy.RNA m 6 A methylation level was detected through a dot blot assay.Protein and RNA levels of METTL3 or angiogenesis-related markers were measured through Western blot assays and real-time quantitative polymerase chain reaction(RT-qPCR),respectively.METTL3 expression and localization were investigated through immunofluorescence.Cell migratory and tube formation capacities were assessed through transwell migration and tube formation assays,respectively.RESULTS:Endocytosed lipoprotein levels in HUVECs exposed to Dil-LDL,12.5μg/mL and 25μg/mL Dil-ox-LDL groups were significantly higher than those in the control group.12.5μg/mL and 25μg/mL ox-LDL groups significantly increased m 6 A methylation(all P<0.05),METTL3 protein expression(all P<0.01),and cell migration and angiogenesis capacities(all P<0.01).METTL3 mRNA level was significantly unregulated in the 12.5μg/mL ox-LDL group(P<0.05).In comparison to the DMSO group,the addition of STM2457 caused significant decrease in m 6 A methylation level(P<0.05),expression of VEGF and other angiogenesis-related markers(all P<0.05),cell migration and angiogenesis capacities(all P<0.01)and the expression of NICD(P<0.05).However,there were no significant differences in METTL3 protein and mRNA levels(all P>0.05).The expression of VEGF and NICD(all P<0.05),as well as the ability of cell migration and angiogenesis of RF/6A,was all significantly decreased in the DAPT group compared to the DMSO group(all P<0.01).CONCLUSION:METTL3-mediated m 6 A methylation modification promotes angiogenesis in vascular endothelial cells via the Notch signaling pathway in the pathogenesis of choroidal neovascularization.
作者
唐韵
陈思
叶巍
王文喆
高颖
葛轶睿
黄振平
Yun Tang;Si Chen;Wei Ye;Wen-Zhe Wang;Ying Gao;Yi-Rui Ge;Zhen-Ping Huang(Department of Ophthalmology,Affiliated Jinling Hospital,Medical School,Nanjing University(General Hospital of Eastern Theater Command),Nanjing 210002,Jiangsu Province,China)
出处
《国际眼科杂志》
CAS
北大核心
2023年第5期723-730,共8页
International Eye Science
