Experimental Study On Expression Of IAP-2 And HIF-1 In Human Hepatocellular Carcinoma HepG-2 Induced By Hypoxia

Aim:To investigate the alteration of apoptosis inhibitory protein-2 (IAP-2) in human hepatocellular carcinoma HepG-2 under severe hypoxia and explore its relation with hypoxia inducible factor-1 (HIF-1).Methods:HepG-2 cells were cultured in different groups: normoxia cell group, 4 h of hypoxia cell group (cultured in 2%O2+5%CO2+93%N2), 1, 3, 5 h of sever hypoxia cell group (cultured in 95%N2+5%CO2), reoxygenation after 1 h of severe hypoxia, normoxia cell added colalt chloride (300μmol/l). Immunocytochemistry was used to qualitatively detect IAP-2 protein expression. After extraction of cytoplasmic proteins and nuclear proteins, Western blot was used to quantitatively analyze IAP-2 protein expression, and the expression of HIF-1 protein was measured simultaneously to compare the expression consistency of the two protein. Then reverse transcription-polymerase chain reaction (RT-PCR) was used to estimate the mRNA expression of IAP-2.Results: The immunocytochemical stain showed the positive expression of IAP-2 protein in HepG-2 cell cytoplasm. Western blot revealed there was no significant difference between the expression levels of IAP-2 protein under normoxia and 4 h of hypoxia. Whereas the expression of IAP-2 protein could be markedly induced (t=5.723, P<0.05) by 1 h of severe hypoxia and remain high after 3 or 5 h. There was no significant difference among hours. Reoxygenation made the expression of IAP-2 protein return to basal level. Our results suggested that expression of IAP-2 and HIF-1 protein was inconsistent. HIF-1 protein was undetectable in normoxic HepG-2 cell, but induced by hypoxia. Under sever hypoxia HIF-1 protein expression was moderate but IAP-2 protein expression was abundant. After reoxygenation HIF-1 was degraded and IAP-2 returned to basal level. Colalt chloride activated HIF-1 expression but not IAP-2.These results implied sever hypoxia induced IAP-2 up-regulation in HepG-2 through HIF-1-independent pathways. RT-PCR analysis found the expression of IAP-2 mRNA after 1 h of sever hypoxia apparently higher than normoxia (t=7.097, P<0.05), and remain high after 3 or 5 h. Reoxygenation led the expression of IAP-2 mRNA to basal level. The results were consistant with above, which primarily showed IAP-2 up-regulation by sever hypoxia at transcriptional level.Conclusion:This study provided the first demonstration that sever hypoxia induced the up-regulation of IAP-2 in HepG-2 through HIF-1-independent pathways.