Authors: Ma Yu, Tang Shaohua, Jiang Yanrong, Shi Xuan
Anti-VEGF drugs are generally applied in the treatment of ocular neovascular diseases.However, the therapy effect is unsatisfactory in some patients.Studing the effect of hypoxia-inducible factor-1 (HIF-1), a upstream regulatory gene of VEGF, and its limiting enzyme prolyl-4-hydroxylase domain proteins (PHDs) is of important clinical significance.
This study was to investigate the negtive regulation of exogeneous PHDs on HIF-1 pathway in human RPE cells.
pFLAG-PHD1, pFLAG-PHD2 and pFLAG-PHD3 plasmids were constructed by extracting RNA from Hela cell line and coloning PHD1, PHD2 and PHD3 using reverse transcription PCR with restriction enzyme.The plasmids were identified by gene sequencing.ARPE-19 cells were cultured at 21% O2 (normoxia group), 1% O2 (hypoxia group), or in hypoxia-mimicking agents (CoCl2, anoxia group), respectively, and then were transfected with plasmids encoding FLAG-tagged PHD1, PHD2, PHD3 and pFLAG-CMV2 transfected cells served as blank control.The expressional intensities of PHD1, PHD2 and PHD3 in the cells were detected and compared among different groups by using Western blot assay.The transcriptional activity of HIF-1 in the cells was evaluated with dual luciferase reporter assay.
Western blot assay showed that PHD1, PHD2 and PHD3 all were expressed in ARPE-19 cells in the normoxia group, hypoxia group and anoxia group.The expression was strong in PHD2 protein and was weak in PHD3protein, a statistically significant difference was found between PHD2 protein expression and PHD1 or PHD3expressions (all at P<0.05). Endogenous HIF-1 activity was elevated in pFLAG-CMX transfected cells in the hypoxia group and anoxia group than that in the normoxia group.Compared with pFLAG-CMX transfected cells, no obvious change was seen in the endogenous HIF-1 activity in the normoxia group, however, HIF-1 activity was declined in the hypoxia group and anoxia group after pFLAG-PHD1, pFLAG-PHD2 or pFLAG-PHD3 transfection.Under the same oxygen environment, HIF-1 activity was lower in the pFLAG-PHD2 transfected cells than that in the pFLAG-PHD1 or pFLAG-PHD3 transfected cells (both at P<0.05).
PHDs play a negative regulation to HIF-1 activating pathway in human RPE cells, especially in hypoxia and anoxia cells.Among PHDs proteins, PHD2 presents the strongest inhibition on HIF-1 activating pathway.