摘要
背景:年龄相关性黄斑变性(AMD)是老年人不可逆失明的主要原因。视网膜色素上皮细胞(retinal pigment epithelium,RPE)中的氧化应激被认为在AMD的发病机制中起着举足轻重的作用。 miR-25在几种细胞类型中作为响应氧化应激的基本调节剂起作用,但其在RPE细胞中的功能尚不清楚。 目的:探讨miR-25在RPE细胞中的作用及其在AMD发生发展中的作用。 方法:用碘酸钠(SI)诱导大鼠视网膜变性模型。进行视网膜下注射antagomiR-25进行干预,而争夺作为对照。用视网膜电图(ERG)记录视觉反应。进行TUNEL分析以检测细胞凋亡。进行体内吞噬体定量以评估RPE细胞功能。进行氧 - 葡萄糖剥夺治疗以模拟体外氧化应激。通过定量聚合酶链式反应(qPCR)和蛋白质印迹分别进行mRNA水平和蛋白质水平的基因表达。酶联免疫吸附试验(ELISA)测定培养液中色素上皮衍生因子(PEDF)水平。双荧光素酶检测法检测miR-25与整合素αV(IGTAV)/ PEDF 3''UTR之间的相互作用。进行染色质免疫沉淀(ChIP)测定以检查其miR-25的转录调控。 结果:氧化应激在RPE细胞的早期阶段上调了miR-25,伴随吞噬作用减弱,生长因子分泌减少。这种变化发生在经过处理的大鼠的RPE细胞凋亡和视力损害之前。此外,antagomiR-25干预有效地挽救了RPE细胞在这种模型中的退化。通过直接靶向IGTAV和PEDF证实增加的miR-25介导RPE变性。另一方面,在上游,在体内和体外模型中,发现miR-25在氧化应激下被STAT3信号传导上调。 结论:我们的研究结果表明,在SI治疗的大鼠中,氧化应激在非常早的阶段激活上调miR-25表达的STAT3信号传导。然后增加的miR-25抑制ITGAV和PEDF表达,导致RPE吞噬功能障碍,然后发生在AMD患者中观察到的RPE凋亡和视觉障碍。这些发现使我们更好地了解AMD的发病机制,并提示miR-25可能是氧化应激相关RPE疾病(如AMD)的潜在治疗靶点。
关键词: 视网膜变性,miR-25,氧化应激,RPE细胞,ITGAV,PEDF,STAT3。
Current Molecular Medicine
Title:miR-25 Mediates Retinal Degeneration Via Inhibiting ITGAV and PEDF in Rat
Volume: 17 Issue: 5
关键词: 视网膜变性,miR-25,氧化应激,RPE细胞,ITGAV,PEDF,STAT3。
摘要: Background: Age-related macular degeneration (AMD) is the main cause of irreversible blindness in the elderly. Oxidative stress in retinal pigment epithelium (RPE) is deemed to play a pivotal role in the pathogenesis of AMD. miR-25 functions as an essential modulator in response to oxidative-stress in several cell types, but its function in RPE cells is poorly understood.
Objective: To explore the roles of miR-25 in RPE cells and in the development of AMD.
Methods: A rat model of retinal degeneration was induced by sodium iodate (SI). Subretinal injection of antagomiR-25 was performed for the intervention while the scramble as control. Visual responses were recorded with Electroretinogram (ERG). TUNEL assay was performed to detect apoptosis. Phagosome quantification in vivo was performed to evaluate RPE cell function. Oxygen-glucose deprivation treatment was performed to mimic in vitro oxidative stress. Gene expression at mRNA level and protein level were performed by quantitative polymerase chain reaction (qPCR) and Western Blot, respectively. The pigment epithelium derived factor (PEDF) level in the cultured medium was measured by Enzyme-linked immunosorbent assay (ELISA). The interaction between miR-25 and integrin αV (IGTAV) / PEDF 3’UTR was examined by dual luciferase assay. Chromatin immunoprecipitation (ChIP) assay was performed to examine its transcriptional regulation of miR-25.
Results: Oxidative stress up-regulated miR-25 in RPE cells in very early stage, accompanied by decreased phagocytosis and reduced growth factor secretion in those cells. Such changes preceded RPE cell apoptosis and visual impairment in the SItreated rats. Furthermore, antagomiR-25 intervention effectively rescued RPE cells from degeneration in such model. The increased miR-25 was confirmed to mediate RPE degeneration through direct targeting IGTAV and PEDF. On the other hand, upstream, miR-25 was found to be up-regulated by STAT3 signaling under oxidative stress in both in vivo and in vitro models.
Conclusion: Our findings demonstrate that, in SI-treated rats, oxidative stress activates STAT3 signaling which up-regulates miR-25 expression, in a very early stage. The increased miR-25 then inhibits ITGAV and PEDF expressions, resulting in RPE phagocytosis dysfunction and then RPE apoptosis and visual impairment as observed in patients with AMD. These findings lead us to a better understanding of AMD pathogenesis, and suggest that miR-25 could be a potential therapeutic target for oxidative stress related RPE diseases, like AMD.
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miR-25 Mediates Retinal Degeneration Via Inhibiting ITGAV and PEDF in Rat, Current Molecular Medicine 2017; 17 (5) . https://dx.doi.org/10.2174/1566524018666171205122540
DOI https://dx.doi.org/10.2174/1566524018666171205122540 |
Print ISSN 1566-5240 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5666 |
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