Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway

Abstract

Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia-reperfusion injury.

Keywords: B-cell lymphoma-2; Spinal cord ischemia–reperfusion injury; hypoxia; hypoxia-inducible factor-1α; microRNA-204; neuronal cells.

Figure 1

Hypoxia induces apoptosis in neuronal cells and decreases the expression of BCL-2. (a) Exposed to hypoxia or normoxia for 18 h, cell apoptosis was tested by Annexin V/PI flow cytometry in AGE1.HN and PC12 cells. (b) Graphical representation of the data shown in a. (c) qRT-PCR analyses were performed to examine the mRNA expression of BCL-2 in AGE1.HN and PC12 cells. (d) Western blotting was performed to determine BCL-2 protein levels in AGE1.HN and PC12 cells. The data are the mean ± SD. **P < 0.01 vs. control. (A color version of this figure is available in the online journal.)

Figure 2

Hypoxia increases the expression of miR-204 in neuronal cells. (a) The expression of miR-204 in peripheral plasma of patients with spinal cord ischemia–reperfusion. (b) qRT-PCR analyses were performed to examine the expression of miR-204 in AGE1.HN and PC12 cells exposed to hypoxia or normoxia for different duration. The data are the mean ± SD. *P < 0.05 vs. control, **P < 0.01 vs. control

Figure 3

BCL-2 is the target of miR-204. (a) Sequence alignment of miR-204 and 3′-UTR of BCL-2 using mirco-RNA.org. (b) AGE1.HN and PC12 cells were transfected with miR-204 mimic or miR-204 inhibitor or controls under normal oxygen conditions. The expression of BCL-2 was detected with qRT-PCR. (c) Relative luciferase activity in cells transfected with miR-204 mimics (or miR-204 inhibitor) and luciferase expression vector of BCL-2 under normal oxygen conditions. (d) AGE1.HN and PC12 cells were transfected with miR-204 mimic or miR-204 inhibitor or controls under normal oxygen conditions. The protein expression of BCL-2 was detected with Western blotting. (e) AGE1.HN and PC12 cells were transfected with miR-204 inhibitor or controls and then exposed to hypoxia. The protein expression of BCL-2 was detected with Western blotting. The data are the mean ± SD. **P < 0.01 vs. control; ^#P < 0.05 vs. inhibitor control. (A color version of this figure is available in the online journal.)

Figure 4

Hypoxia increases the expression of miR-204 and reduces the expression of BCL-2 via HIF1-α pathway. (a) AGE1.HN and PC12 cells were treated with 1 µmol/L and 10 µmol/L YC-1 for 6 h and then exposed to hypoxia for 24 h or transfected with siHIF-1α. The protein expression of HIF1α was detected with Western blotting. (b) AGE1.HN and PC12 cells were treated with 1 µmol/L and 10 µmol/L YC-1 for 6 h and then exposed to hypoxia for 24 h or transfected with siHIF-2α. The protein expression of HIF-2α was detected with Western blotting. (c) The mRNA level of miR-204 was detected in cells transfected with YC-1 or siHIF-1α or siHIF-2α and exposed to hypoxia. (d) The mRNA level of BCL-2 was detected in cells transfected with YC-1 or siHIF-1α or siHIF-2α and exposed to hypoxia. (e) The protein level of BCL-2 was detected in AGE1.HN and PC12 cells transfected with YC-1 and siHIF-1α and exposed to hypoxia. (f) The percentage of apoptotic cells was detected by flow cytometry in AGE1.HN and PC12 cells transfected with YC-1 or siHIF-1α. The data are the mean ± SD. **P < 0.01 vs. control; ^#P < 0.05 vs. hypoxia

Figure 5

Hypoxia induces cell apoptosis via miR-204 and BCL-2. (a) AGE1.HN and PC12 cells were transfected with miR-204 inhibitor or controls and then exposed to hypoxia. Flow cytometry was performed to detect the levels of apoptosis. (b) AGE1.HN and PC12 cells were transfected with BCL-2 plasmid or controls and then exposed to hypoxia. Flow cytometry was performed to detect the levels of apoptosis. The data are the mean ± SD. **P < 0.01 vs. control; ^#P < 0.05 vs. hypoxia