Small Hairpin Loop RNA Targeting HIF-1α Down-regulates VEGF and Up-regulates PEDF in Human Retinal Pigment Epithelial Cells under Hypoxic Condition

• Published in: Journal of Huazhong University of Science and Technology. Medical sciences Vol. 28; no. 4; pp. 460 - 464
• Main Author: 肖青 曾水清 吕明良 凌士奇
• Format: Journal Article
• Language: English
• Published: Heidelberg Huazhong University of Science and Technology 01.08.2008; Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China%Department of Ophthalmology, People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China%Department of Ophthalmology, Guangzhou Children Hospital, Guangzhou 510120, China
• Subjects: Medicine; Medicine & Public Health; shRNA; 内皮细胞; 缺氧因素; 血管内皮生长因子; hypoxia-inducible factor 1α; hypoxia; retinal pigment epithelium; shRNA; pigment epithelium derived factor; vascular endothelial growth factor
• ISSN: 1672-0733; 1993-1352
• DOI: 10.1007/s11596-008-0419-8

The aim of this study was to explore the effect of small hairpin loop RNA （shRNA） silencing hypoxia-induced factor 1α （HIF-1α） gene on the expression of vascular endothelial growth factor （VEGF） and pigment epithelium derived factor （PEDF） in human retinal pigment epithelium （RPE） cells under hypoxic condition. Two target sites of HIF-1α mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 μmol/L CoCl2. The mRNA expressions of HIF-1α, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR （RT-PCR）. The protein levels of HIF-1α, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1α-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1α mRNA and the levels of HIF-1α protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1α-specific shRNA can effectively silence the HIF-1α gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.