miR-146a-5p 通过靶向 OTUD7B 抑制 NF-κB 信号通路抑制脉络膜新生血管形成。
Downregulation of miR-146a-5p Inhibits Choroidal Neovascularization via the NF-κB Signaling Pathway by Targeting OTUD7B.
机构信息
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University , Guangzhou, China.
出版信息
Curr Eye Res. 2020 Dec;45(12):1514-1525. doi: 10.1080/02713683.2020.1772831. Epub 2020 Jun 8.
PURPOSE
Choroidal neovascularization (CNV) is the key pathological change caused by irreversible blindness resulting from neovascular AMD (nAMD). However, the pathological mechanisms underlying CNV remain largely unknown. Here, we aimed to investigate the role of miR-146a-5p in CNV formation.
MATERIALS AND METHODS
At the cellular level, we overexpressed or downregulated miR-146a-5p in an umbilical vein endothelial cell line (EA.hy926) by transfecting cells with either a miR-146a-5p mimic or an inhibitor. CCK8, wound healing, and Matrigel assays were performed to examine the proliferation, migration, and tube formation of endothelial cells (EA.hy926). Target relationship between miR-146a-5p and OTUD7B was verified using a double luciferase reporter experiment. An experimental CNV model was established by treating fundi of male C57BL/6 J mice with 810 nm laser. Fundus fluorescein angiography (FFA) was performed to evaluate the leakage of CNV on day 7 after miR-146a-5p antagomir intravitreal injection. The CNV volume was measured using Choroidal Flatmounts in a confocal study. The expression levels of VEGF, ICAM1, and NF-κB (p50 and p65) were detected both in vitro and in vivo.
RESULTS
The expression of miR-146a-5p was increased in LPS-stimulated endothelial cells and in experimental CNV RPE-choroidal complexes in mouse models. LPS-induced proliferation, migration, and tube formation were inhibited by the miR-146a-5p inhibitor. The miR-146a-5p antagomir attenuated CNV formation and fluorescent leakage in the vivo CNV model. In the LPS-stimulated endothelial cells and the CNV mouse model, the NF-κB signaling pathway was activated and the expression of VEGF and ICAM1 increased. Conversely, downregulation of miR-146a-5p inactivated the NF-κB signaling pathway and reduced the expression of VEGF and ICAM1.
CONCLUSIONS
Our results indicated that downregulation of miR-146a-5p inhibited experimental CNV formation via inactivation of the NF-κB signaling pathway.
目的
脉络膜新生血管（CNV）是由新生血管性年龄相关性黄斑变性（nAMD）引起的不可逆失明的关键病理变化。然而，CNV 的病理机制在很大程度上仍不清楚。本研究旨在探讨 miR-146a-5p 在 CNV 形成中的作用。
材料和方法
在细胞水平上，通过转染 miR-146a-5p 模拟物或抑制剂，在脐静脉内皮细胞系（EA.hy926）中过表达或下调 miR-146a-5p。CCK8、划痕愈合和 Matrigel 测定用于检测内皮细胞（EA.hy926）的增殖、迁移和管形成。通过双荧光素酶报告实验验证 miR-146a-5p 和 OTUD7B 之间的靶关系。通过用 810nm 激光处理雄性 C57BL/6J 小鼠的眼底建立实验性 CNV 模型。在 miR-146a-5p 反义寡核苷酸玻璃体腔内注射后第 7 天进行眼底荧光血管造影（FFA）评估 CNV 的渗漏。在共聚焦研究中使用脉络膜扁平片测量 CNV 体积。在体外和体内检测 VEGF、ICAM1 和 NF-κB（p50 和 p65）的表达水平。
结果
LPS 刺激的内皮细胞和小鼠模型中的实验性 CNV RPE-脉络膜复合物中 miR-146a-5p 的表达增加。miR-146a-5p 抑制剂抑制 LPS 诱导的增殖、迁移和管形成。miR-146a-5p 反义寡核苷酸在体内 CNV 模型中减轻了 CNV 的形成和荧光渗漏。在 LPS 刺激的内皮细胞和 CNV 小鼠模型中，NF-κB 信号通路被激活，VEGF 和 ICAM1 的表达增加。相反，下调 miR-146a-5p 使 NF-κB 信号通路失活，降低了 VEGF 和 ICAM1 的表达。
结论
我们的结果表明，下调 miR-146a-5p 通过使 NF-κB 信号通路失活来抑制实验性 CNV 的形成。
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