缺氧条件下 DLL4 siRNA 对脉络膜视网膜内皮细胞增殖、迁移及管腔形成的影响。

Effect of DLL4 siRNA on proliferation, migration and tube formation of choroid-retinal endothelial cells under hypoxic conditions.

机构信息

Department of Ophthalmology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

出版信息

Chin Med J (Engl). 2011 Jan;124(1):118-26.

PMID:21362319

Abstract

BACKGROUND

Delta-like 4 (DLL4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV.

METHODS

We utilized chemical hypoxia induced by 200 µmol/L CoCl2 to observe the expression of DLL4 in choroid-retinal endothelial cells (RF/6A cells), which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA (siRNA), mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used: one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated.

RESULTS

The results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA (P < 0.05).

CONCLUSIONS

DLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.

摘要

背景

Delta-like 4（DLL4）是一种内皮细胞特异性 Notch 配体，已被证明在生理和病理血管生成中作为调节因子发挥作用。据报道，DLL4-Notch 信号通路受缺氧调节，并可能通过抑制血管生成分支和触发血管成熟来防止过度血管生成。脉络膜新生血管（CNV）是一种病理性血管生成形式，其中缺氧被认为起着重要作用。本研究旨在评估 DLL4 在 CNV 发展中的作用。

方法

我们利用 200µmol/L CoCl2 诱导的化学缺氧来观察 DLL4 在脉络膜视网膜内皮细胞（RF/6A 细胞）中的表达，RF/6A 细胞是参与 CNV 的主要细胞。用 DLL4 小干扰 RNA（siRNA）转染后，通过 RT-PCR、实时 PCR 和 Western blot 分析研究缺氧 RF/6A 细胞中 DLL4 及其关键下游基因（包括 HES1 和 HEY1）的 mRNA 和蛋白表达。使用了三个对照：一个没有转染，一个有转染试剂，一个有乱序阴性对照 siRNA。研究了 DLL4 siRNA 对缺氧 RF/6A 细胞在血管生成过程中的生物学功能（包括细胞增殖、迁移和管形成）的影响。

结果

结果表明，体外缺氧条件导致 RF/6A 细胞中 DLL4 的表达上调。转染后，靶向 DLL4 的 siRNA 使 DLL4 mRNA 水平降低了 91.4%，与乱序 siRNA 对照组相比，DLL4 蛋白表达也同样受到影响。空白对照组、转染试剂对照组和乱序 siRNA 组之间 DLL4 表达无显著差异。此外，用 DLL4 siRNA 转染缺氧 RF/6A 细胞后，DLL4-Notch 信号下游的 HES1 和 HEY1 的 mRNA 水平分别降低了 75.1%和 86.3%，与乱序 siRNA 对照组相比。此外，DLL4 表达的下调显著促进了缺氧 RF/6A 细胞的增殖，并导致其在细胞周期的 S 期停滞。用 DLL4 siRNA 诱导缺氧 RF/6A 细胞的迁移和管形成明显增加，迁移细胞数量增加 1.6 倍，总管长度增加 82.3%，与乱序 siRNA 相比（P<0.05）。