[Effect of hypoxia-inducible factor-1alpha inhibition caused by RNA interference on permeability of hypoxic endothelial cells].
OBJECTIVE
To study the effect of hypoxia-inducible factor-1alpha (HIF-1alpha) inhibition caused by RNA interference on permeability of hypoxic vascular endothelial (VE) cells.


METHODS
Plasmid pcDNA6.2-GW/EmGFP-miR was applied to construct the RNA interference expression vector targeted to human HIF-1alpha gene. VE cells were divided into normal control group (NC), hypoxia group (H, cells were treated for hypoxia in mixed gas with 1% O(2) for 6 hours), transfection group (T), and transfection hypoxia group (TH, transfected with vector and treated with hypoxia). Expression of HIF-1alpha mRNA in NC and T groups were determined with RT-PCR. Expression of HIF-1alpha protein in each group was determined with Western blot. The permeability of VE cell monolayer was detected by fluorospectrophotometer. Another sample of VE cells were divided into dimethyloxallyl glycine (DMOG) group, transfected with DMOG group (TD), normal control group (NC), and transfection group (T), with 1 mmol/L DMOG (HIF-1alpha specific derivant) replacing hypoxia treatment. The expression of HIF-1alpha protein in each group was determined with Western blot. All data were recorded as density value ratio except for permeability data, which was recorded as fluorescence intensity value. Data were processed with t test (pairwise comparison among groups).


RESULTS
The relative content of HIF-1alpha mRNA of cells in NC group (0.765 +/- 0.069) was significantly higher than that of cells in T group (0.093 +/- 0.007, t = 16.696, P < 0.05). Content of HIF-1alpha protein of cells in TH group (0.591 +/- 0.029) was significantly lower than that of cells in H group (2.612 +/- 0.259, t = 13.415, P < 0.05). Content of HIF-1alpha protein of cells in TD group (0.566 +/- 0.008) was significantly lower than that of cells in DMOG group (3.243 +/- 0.551, t = 6.975, P < 0.05). The permeability of cell monolayer in H group (41.6 +/- 11.1) was significantly higher than that of cell monolayer in NC group (9.4 +/- 1.5, t = 6.238, P < 0.05). The permeability of cell monolayer in TH group (13.3 +/- 4.5) was markedly lower than that of cell monolayer in H group (t = 5.430, P < 0.05).


CONCLUSIONS
The expression of HIF-1alpha gene in vascular endothelial cells is effectively inhibited by specific RNA interference, which significantly prevents the hypoxia-induced increase in vascular endothelial cell permeability.
Liu
Chen
C
Institute of Burn Research, the Third Military Medical University, Chongqing 400038, China.
Wang
Pei
P
Li
Mu
M
Wang
Feng-jun
FJ
D001483:Base Sequence; D015687:Cell Hypoxia; D002460:Cell Line; D002463:Cell Membrane Permeability; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger