title: Effect of shRNA-mediated knockdown of vascular endothelial growth factor on the proliferation of choroid-retinal endothelial cells under hypoxic conditions.
abstract: The aim of the present study was to investigate the role of vascular endothelial growth factor (VEGF) in cell proliferation under hypoxic conditions. Gene knockdown of VEGF was conducted in the choroid-retinal endothelial RF/6A cell line by transfection with short hairpin RNA (shRNA), in which a shRNA fragment against VEGF was synthesized and cloned into the vector, pSilencer 2.1-U6 neo. Subsequently, a model of hypoxia was established in the RF/6A cell line via treatment with CoCl2, into which the recombinant plasmids, containing the VEGF-targeting shRNA (p-shRNA), were transfected. The study included four treatment groups, namely a control group (normal group), a hypoxia group treated with CoCl2(CoCl2group), a control plasmid group that were subjected to CoCl2treatment and transfection with a pSilencer 2.1-U6 neo plasmid without the shRNA (CoCl2+ p-NC group), and a group treated with CoCl2and transfected with a pSilencer 2.1-U6 neo plasmid containing the VEGF-targeting shRNA (CoCl2+ p-shRNA group). Subsequent to treatment, the mRNA and protein expression levels of VEGF were evaluated using quantitative polymerase chain reaction and western blot analysis, respectively, In addition, cell proliferation was assessed. RF/6A cells treated with CoCl2reduced cell connectivity, irregular morphology and reduced thickness compared with the cells in the normal group. However, cells in the CoCl2+ p-shRNA group exhibited an improved morphology compared with the CoCl2and CoCl2+ p-NC groups. Cell proliferation in the CoCl2group was enhanced in a time-dependent manner. However, the hypoxia-induced increase in cell proliferation was significantly inhibited in the CoCl2+ p-shRNA group, with inhibition rates of 16, 32 and 38% at 24, 48 and 72 h, respectively. The mRNA and protein expression levels of VEGF were increased in the CoCl2group when compared with the normal group, and these hypoxia-induced increases in VEGF expression were reduced in the CoCl2+ p-shRNA group. Therefore, the results indicated that the targeted knockdown of VEGF in vascular endothelial cells may be effective for the treatment of retinal neovascularization diseases.
authors.affiliation: Department of Glaucoma and Fundus Diseases, Tianjin Medical University Eye Hospital, Tianjin 300384, P.R. China.
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Liu
authors.affiliation: Department of Glaucoma and Fundus Diseases, Tianjin Medical University Eye Hospital, Tianjin 300384, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Glaucoma and Fundus Diseases, Tianjin Medical University Eye Hospital, Tianjin 300384, P.R. China.
authors.forename: Y I
authors.initials: YI
authors.lastname: Shi
authors.affiliation: Department of Glaucoma and Fundus Diseases, Tianjin Medical University Eye Hospital, Tianjin 300384, P.R. China.
authors.forename: Guo-Ling
authors.initials: GL
authors.lastname: Sun
authors.affiliation: Department of Glaucoma and Fundus Diseases, Tianjin Medical University Eye Hospital, Tianjin 300384, P.R. China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
keywords: CoCl2; cell proliferation; retinal neovascularization diseases; short hairpin RNA; vascular endothelial growth factor