Protective mechanisms of microRNA-27a against oxygen-glucose deprivation-induced injuries in hippocampal neurons

Abstract

Hypoxic injuries during fetal distress have been shown to cause reduced expression of microRNA-27a (miR-27a), which regulates sensitivity of cortical neurons to apoptosis. We hypothesized that miR-27a overexpression attenuates hypoxia- and ischemia-induced neuronal apoptosis by regulating FOXO1, an important transcription factor for regulating the oxidative stress response. miR-27a mimic was transfected into hippocampal neurons to overexpress miR-27a. Results showed increased hippocampal neuronal viability and decreased caspase-3 expression. The luciferase reporter gene system demonstrated that miR-27a directly binded to FOXO1 3'UTR in hippocampal neurons and inhibited FOXO1 expression, suggesting that FOXO1 was the target gene for miR-27a. These findings confirm that miR-27a protects hippocampal neurons against oxygen-glucose deprivation-induced injuries. The mechanism might be mediated by modulation of FOXO1 and apoptosis-related gene caspase-3 expression.

Keywords: FOXO1; apoptosis; brain injury; caspase 3; cell survival; hippocampal neurons; hypoxic-ischemic; luciferase reporter gene system; miR-27a; nerve regeneration; neural regeneration; neuroprotection; oxygen-glucose deprivation.

Figure 1

Neuronal cell viability and miR-27a expression in OGD-induced primary cultured hippocampal neurons. (A) MTT assay was used to measure neuronal cell viability. (B) Expression of miR-27a was detected by qRT-PCR, and miR-27a expression was normalized to U6. Histogram shows cell viability or miR-27a expression in primary hippocampal neurons exposed to OGD/reoxygenation for 6, 12, and 24 hours, or normoxia, respectively. *P < 0.05, **P < 0.01, vs. normoxia control (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; qRT-PCR: quantitative real-time polymerase chain reaction; OGD: oxygen-glucose deprivation; h: hours.

Figure 2

The effects of miR-27a on viability and apoptosis of OGD-exposed primary cultured hippocampal neurons. (A) Expression of miR-27a was detected by qRT-PCR, and miR-27a expression was normalized to U6. (B) MTT assay was used to measure neuronal cell viability. (C) Flow cytometry was used to analyze neuronal cell apoptosis. Histogram shows miR-27a expression, neuronal cell viability, and cell apoptosis in primary hippocampal neurons transfected with miR-27a mimic, mimic control (ctrl), or untransfected (blank) exposed to OGD/reoxygenation for 12 hours or normoxia, respectively. *P < 0.05, **P < 0.01, vs. normoxia control, #P < 0.05, ##P < 0.01, vs. blank or mimic ctrl (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; qRT-PCR: quantitative real-time polymerase chain reaction; OGD: oxygen-glucose deprivation. Blank: Group with untransfected; mimic ctrl: group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

Figure 3

FOXO1 is a direct target of miR-27a. (A) Schematic representation of a section of FOXO1 3′-UTR showing the putative miR-27a target site. (B) The resulting histogram shows relative luciferase activities in each group. **P < 0.01, vs. mimic ctrl (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). 3′-UTR: 3′ Untranslated region. Mimic ctrl: Group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

Figure 4

Effect of miR-27a on caspase-3 activation in OGD-exposed primary cultured hippocampal neurons. Representative western blot image (the upper) and resulting histogram (the lower) show cleaved caspase-3 expression in primary hippocampal neurons transfected with miR-27a mimic, mimic ctrl, or untransfected (blank) after exposure to OGD/reoxygenation for 12 hours or normoxia, respectively. *P < 0.05, P < 0.01, vs. normoxia control; NRR-11-1285-g001 P < 0.01, vs. blank control or mimic ctrl (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). OGD: Oxygen-glucose deprivation; Blank: group with untransfected; Mimic ctrl: group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

Figure 5

Effect of miR-27a on FOXO1 protein expression in rat primary hippocampal neurons. Representative western blot image (the upper) and resulting histogram (the lower) show FOXO1 protein expressions in neurons transfected with miR-27a mimic, mimic ctrl, or untransfected, respectively. **P < 0.01, vs. untransfected ctrl and mimic ctrl (mean ± SEM; experiments were performed in triplicate; Student's t-test). Untransfected ctrl: Normal group; Mimic ctrl: group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.