Roles of HIF1α- and HIF2α-regulated BNIP3 in hypoxia-induced injury of neurons

Abstract

Background: To explore the roles of HIF1α- and HIF2α-regulated BNIP3 in hypoxia-induced injury of neurons.

Methods: The sera of neonates with hypoxic-ischemic encephalopathy (HIE) within 24 h after birth and full-term healthy newborns (n = 40) were collected. The BNIP3 levels were detected by ELISA. AGE1.HN cells were cultured in 1% O₂ at 37 °C. The apoptosis of cells treated with 1, 5 and 10 ng/ml BNIP3 for 48 h was detected by flow cytometry. The proliferation of cells transfected with siBNIP3 was detected by CCK-8 assay. The mRNA level of BNIP3 in cells under hypoxic conditions was measured by RT-PCR. The protein level of BNIP3 in cells cultured under hypoxic conditions after pretreatment with HIF1α or HIF2α inhibitor was measured by Western blot.

Results: The serum BNIP3 concentration of HIE neonates ((4.5 ± 2.1) ng/ml) was significantly higher than that of healthy neonates ((1.2 ± 0.5) ng/ml) (P < 0.001). Compared with untreated group, the number of apoptotic AGE1.HN cells treated with BNIP3 significantly increased (P < 0.05). Under hypoxic conditions (1%), the mRNA and protein levels of BNIP3 increased significantly with prolonged time. After pretreatment with HIF1α or HIF2α inhibitor and hypoxic culture, BNIP3 expression was significantly lower than that of cells hypoxically cultured only. Inhibiting the expression of HIF1α or HIF2α or transfecting with siBNIP3 before hypoxic treatment significantly reduced the number of apoptotic cells. Under hypoxic conditions, HIF1α or HIF2α bound BNIP3 promoter, which did not occur under normal culture conditions. HIF1α or HIF2α was significantly enriched near the hypoxia response element (HRE) site of BNIP3 promoter.

Conclusions: BNIP3 was involved in the apoptosis of cells undergoing HIE. The HRE site of BNIP3 promoter bound HIF to promote its transcription.

Keywords: Apoptosis; BNIP3; HIF; Hypoxic-ischemic encephalopathy; Neonate.