Small hairpin loop RNA targeting HIF-1α down-regulates VEGF and up-regulates PEDF in human retinal pigment epithelial cells under hypoxic condition

Abstract

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.

Keywords

shRNA / hypoxia-inducible factor 1α / vascular endothelial growth factor / pigment epithelium derived factor / hypoxia / retinal pigment epithelium

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