Study on suppression of hypoxia inducible factor-1 alpha and vascular endothelial growth factor expression by siRNA targeting hypoxia inducible factor-1 alpha

Abstract

To evaluate suppression efficiency of hypoxia inducible factor-1 alpha (HIF-1 alpha) specific siRNA derived from recombinant plasmid (pSUPER(H1-siHIF-1 alpha)) on both HIF-1 alpha mRNA and protein expression and concomitant downregulation of expression of downstream angiogenic factor vascular endothelial growth factor (VEGF). Stable pSUPER(H1-siHIF-1 alpha) expression cell lines were constructed by transient transfection of pSUPER(H1-siHIF-1 alpha) eukaryotic expression vector, followed by puromycin selection. Stable expression pSUPER(H1-siHIF-1 alpha) cell line with highest HIF-1 alpha inhibition efficiency determined by reverse transcription-polymerase chain reaction (RT-PCR) was cultured under normoxia (20% O(2)) and hypoxia conditions (1% O(2)) together with control cells. RT-PCR, western blot and ELISA were used to measure inhibition ability of pSUPER(H1-siHIF-1 alpha) on HIF-1 alpha and VEGF expression. Compared to the control cells, both mRNA and protein level of HIF-1 alpha and VEGF were dramatically decreased by pSUPER(H1-siHIF-1 alpha) under hypoxia conditions. Under sufficient oxygen supply situation, HIF-1 alpha mRNA level was downregulated by pSUPER(H1-siHIF-1 alpha), but pSUPER(H1-siHIF-1 alpha) did not cause suppression of VEGF expression. pSUPER(H1-siHIF-1 alpha) could decrease HIF-1 alpha expression under both normoxia and hypoxia conditions. VEGF expression was downregulated under hypoxia conditions only. Consequently, pSUPER(H1-siHIF-1 alpha) might be a powerful tool for the inhibition of retinal neovascularization.