Down-regulation of vascular endothelial growth factor in human retinal pigment epithelial cells by small hairpin loop RNA targeting hypoxia inducible factor-1 alpha under hypoxia condition

Abstract

To explore the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) gene on the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (hRPE) cells under hypoxia conditions by using small hairpin loop RNA (shRNA) to silence HIF-1 alpha. CoCl(2) (150 micromol/L) was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1 alpha mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected by this shRNA in vitro. Then, these cells were cultured under hypoxia conditions (150 micromol/L CoCl(2)). The mRNA expression of HIF-1 alpha and VEGF was measured by semi-quantitative reverse transcription PCR (RT-PCR). The protein level of HIF-1 alpha and VEGF was studied by western blot analysis. After hRPE cells were transfected by HIF-1 alpha-specific shRNA, RT-PCR showed that the expression of HIF-1 alpha mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1 alpha protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. Under hypoxia conditions, HIF-1 alpha-specific shRNA effectively keeps HIF-1 alpha gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1 alpha is one of the most important cytokines for retinal neovascularization.