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

作　　者：HE Hua LI Bin ZHANG Hong XIANG Nan LI Gui-gang 

机构地区：[1]Department of Ophthalmology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

出　　处：《Chinese Medical Journal》2011年第1期118-126,共9页中华医学杂志（英文版）

基　　金：This research was supported by grants fi-om the National Natural Science Foundation of China （No. 30901640）, New Teacher Foundation of China Science and Technology Development Center of Education Ministry （No. 20090142120068）, Natural Science Foundation of Hubei Provincial Government （No. 2008CDB155） and Chenguang Program of Wuhan Science and Technology Bureau （No. 200950431170）.

摘　　要：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 pmol/L CoOl2 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-duplexl 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-duplexl, 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 scrambledBackground 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 pmol/L CoOl2 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-duplexl 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-duplexl, 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

关 键 词：DLL4  siRNA choroidal neovascularization choroid-retinal endothelial cells hypoxia 

分 类 号：Q463[生物学—生理学] Q522