MicroRNA-132 protects hippocampal neurons against oxygen-glucose deprivation-induced apoptosis

Abstract

Hypoxic-ischemic brain injury (HIBI) results in death or long-term neurologic impairment in both adults and children. In this study, we investigated the effects of microRNA-132 (miR-132) dysregulation on oxygen-glucose deprivation (OGD)-induced apoptosis in fetal rat hippocampal neurons, in order to reveal the therapeutic potential of miR-132 on HIBI. MiR-132 dysregulation was induced prior to OGD exposure by transfection of primary fetal rat hippocampal neurons with miR-132 mimic or miR-132 inhibitor. The effects of miR-132 overexpression and suppression on OGD-stimulated hippocampal neurons were evaluated by detection of cell viability, apoptotic cells rate, and the expression of apoptosis-related proteins. Besides, TargetScan database and dual luciferase activity assay were used to seek a target gene of miR-132. As a result, miR-132 was highly expressed in hippocampal neurons following 2 h of OGD exposure. MiR-132 overexpression significantly increased OGD-diminished cell viability and reduced OGD-induced apoptosis at 12, 24, and 48 h post-OGD. MiR-132 overexpression significantly down-regulated the expressions of Bax, cytochrome c, and caspase-9, but up-regulated BCl-2. Caspase-3 activity was also significantly decreased by miR-132 overexpression. Furthermore, FOXO3 was a direct target of miR-132, and it was negatively regulated by miR-132. To conclude, our results provide evidence that miR-132 protects hippocampal neurons against OGD injury by inhibiting apoptosis.

Keywords: FOXO3; apoptosis; hippocampal neuron cells; hypoxic-ischemic brain injury; microRNA-132; oxygen-glucose deprivation.

Figure 1.

MiR-132 was highly expressed in hippocampal neurons following OGD exposure. (a) Primary hippocampal neurons were exposed to OGD conditions for 2 h and then the expression of miR-132 in cells was detected by qRT-PCR at 48 h post-OGD. (b) Primary hippocampal neurons were transfected with miR-132 mimic or miR-132 inhibitor and then exposed to OGD conditions for 2 h. Expression of miR-132 was analyzed by qRT-PCR at 48 h post-OGD. Data represent the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001 compared to control group.

Figure 2.

Overexpression of miR-132 increased OGD-diminished viability in hippocampal neurons. Primary hippocampal neurons were transfected with miR-132 mimic or miR-132 inhibitor. Following exposure to OGD for 2 h, cell viability was analyzed by CCK-8 assay at 12, 24, and 48 h post-OGD. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared to control group.

Figure 3.

Overexpression of miR-132 inhibited OGD-induced apoptosis in hippocampal neurons. (a and b) Primary hippocampal neurons were transfected with miR-132 mimic or miR-132 inhibitor. Following exposure to OGD for 2 h, apoptotic cells rate were detected by flow cytometry at 12, 24, and 48 h post-OGD. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.

Figure 4.

Overexpression of miR-132 altered the expression of apoptosis-related proteins in hippocampal neurons following OGD. (a–d) Primary hippocampal neurons were transfected with miR-132 mimic or miR-132 inhibitor before exposure to OGD for 2 h. Expression of apoptosis-related proteins in hippocampal neurons was analyzed by Western blotting at 12, 24, and 48 h post-OGD. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.

Figure 5.

Overexpression of miR-132 inhibited caspase-3 activity in hippocampal neurons following OGD. Primary hippocampal neurons were transfected with miR-132 mimic or miR-132 inhibitor before exposure to OGD for 2 h. Caspase-3 activity was analyzed using a colorimetric assay at 12, 24, and 48 h post-OGD. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.

Figure 6.

FOXO3 was a direct target of miR-132. (a) TargetScan database was used to predict whether FOXO3 was a target gene of miR-132. (b) Dual luciferase activity assay was performed to verify the prediction from TargetScan. (c and d) Primary hippocampal neurons were transfected with miR-132 mimic or miR-132 inhibitor and then the mRNA and protein expression levels of FOXO3 were assessed by qRT-PCR and western blot analysis. Data represent the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001 compared to control group.