The microRNA miR-181c controls microglia-mediated neuronal apoptosis by suppressing tumor necrosis factor

Abstract

Background: Post-ischemic microglial activation may contribute to neuronal damage through the release of large amounts of pro-inflammatory cytokines and neurotoxic factors. The involvement of microRNAs (miRNAs) in the pathogenesis of disorders related to the brain and central nervous system has been previously studied, but it remains unknown whether the production of pro-inflammatory cytokines is regulated by miRNAs.

Methods: BV-2 and primary rat microglial cells were activated by exposure to oxygen-glucose deprivation (OGD). Global cerebral ischemia was induced using the four-vessel occlusion (4-VO) model in rats. Induction of pro-inflammatory and neurotoxic factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide (NO), were assessed by ELISA, immunofluorescence, and the Griess assay, respectively. The miRNA expression profiles of OGD-activated BV-2 cells were subsequently compared with the profiles of resting cells in a miRNA microarray. BV-2 and primary rat microglial cells were transfected with miR-181c to evaluate its effects on TNF-α production after OGD. In addition, a luciferase reporter assay was conducted to confirm whether TNF-α is a direct target of miR-181c.

Results: OGD induced BV-2 microglial activation in vitro, as indicated by the overproduction of TNF-α, IL-1β, and NO. Global cerebral ischemia/reperfusion injury induced microglial activation and the release of pro-inflammatory cytokines in the hippocampus. OGD also downregulated miR-181c expression and upregulated TNF-α expression. Overproduction of TNF-α after OGD-induced microglial activation provoked neuronal apoptosis, whereas the ectopic expression of miR-181c partially protected neurons from cell death caused by OGD-activated microglia. RNAinterference-mediated knockdown of TNF-α phenocopied the effect of miR-181c-mediated neuronal protection, whereas overexpression of TNF-α blocked the miR-181c-dependent suppression of apoptosis. Further studies showed that miR-181c could directly target the 3'-untranslated region of TNF-α mRNA, suppressing its mRNA and protein expression.

Conclusions: Our data suggest a potential role for miR-181c in the regulation of TNF-α expression after ischemia/hypoxia and microglia-mediated neuronal injury.

Figure 1

Hypoxia/ischemia induces microglial activation and the release of pro-inflammatory cytokines and neurotoxic factors. (A-C) BV-2 cells were exposed to oxygen-glucose deprivation (OGD), and the mRNA levels of the pro-inflammatory cytokines (A)tumor necrosis factor (TNF)-α, (B) interleukin (IL)-1β and (C)inducible nitric oxide synthase (iNOS) were evaluated using real-time PCR at 3 hoursafter OGD treatment. (D-E) The release of(D) TNF-α (E) and IL-1β into the medium of BV-2 cells was measured by ELISA at 48 hours after OGD. (F) The release of NO into the medium of BV-2 cells was measured by the Griess assay. Results are presented as the mean ± SE from three independent experiments. (G) Brain homogenates were obtained from the hippocampus 3 daysafter transient global cerebral ischemia/reperfusion (I/R) injury. The supernatant concentrations of IL-1β and TNF-α were tested by ELISA. (H) Sections from rat brain taken 3 daysafter I/R injury were incubated with primary antibodies against CD11b and iNOS. Representative immunoreactivities in the hippocampal CA1 region are shown. Photomicrographs are shown at ×400 magnification. ‘Ctrl’ (control) represents the microglial cells that were not subjected to OGD treatment. *P < 0.05.

Figure 2

Oxygen-glucose deprivation (OGD) upregulates tumor necrosis factor (TNF)-α expression and downregulates microRNA (miR)-181c expression in BV-2 cells. (A) BV-2 cells were exposed to OGD for 3 hours. The miRNA was isolated, and the expression profiles of OGD-activated BV-2 cells were compared with those of resting cells. A heat map of the 26 differentially expressed miRNAs (9 upregulated and 17 downregulated) is shown. (B) Alignment of the predicted miRNA binding sites in the 3′-UTR of the TNF-α mRNA. (C) Relative expression levels of BV-2 TNF-α mRNA and miR-181c at different time points after OGD treatment. Control cells were not subjected to OGD (0 hours). The results are presented as the mean ± SE from three independent experiments. ‘Ctrl’ (control) represents the control microglial cells that were not subjected to OGD treatment.

Figure 3

tumor necrosis factor-α is a direct target of microRNA (miR)-181c. (A) A mouse TNF-α 3′-UTR fragment containing wild-type or mutated miR-181c-binding sites was cloned downstream of the luciferase reporter gene. Mutations were generated in the TNF-α 3′-UTR sequences complementary to the seed region of miR-181c, as indicated. (B-C) Luciferase activity assays using reporters with wild-type or mutant mouse TNF-α 3′-UTRs were performed after co-transfection with miR-181c mimics or NC in HEK293 (B) or BV-2 (C) cells. The luciferase activity of the NC transfection in each experiment was used to normalize the data; the luciferase activity of the NC transfection was set to 1. (D) BV-2 cells were transfected with miR-181c mimics or NC. After 48 hours, cells were harvested, and the Expression levels of TNF-α mRNA and protein were evaluated by real-time PCR and western blotting. The results are presented as the mean ± SE from three independent experiments. Ctrl represents the control microglial cells that were not subjected to OGD treatment; NC represents the negative control for the miR-181c mimics. *P < 0.05. NS, not significant.

Figure 4

Ectopic expression of microRNA (miR)-181c attenuates oxygen-glucose deprivation-activated BV2-induced neuronal apoptosis. (A-B) BV-2 cells were transfected with miR-181c mimics or NC 24 hoursbefore activation by OGD. After 48 hours, the production levels of TNF-α (A) and NO (B, top) were determined by ELISA and the Griess assay, respectively. The cells were harvested at 48 hours after OGD, and iNOS protein expression was evaluated by western blotting (B, lower). (C-D) BV-2 cells were transfected with miR-181c mimics or NC 24 hoursbefore activation by OGD. After 48 hours, the conditioned media were harvested. Cultured neurons were grown in the microglia-conditioned medium (MCM) and then analyzed for apoptosis using Hoechst 33342 staining. The percentage of apoptotic cells in the total neuronal population was calculated (C). Representative photographs are shown in (D). The results are presented as the mean ± SE from three independent experiments. NC represents thenegative control for the miR-181c mimics. *P < 0.05.

Figure 5

microRNA (miR)-181c controls microglia-mediated neuronal apoptosis dependent on tumor necrosis factor (TNF)-α. (A) Neurons were treated with vehicle or soluble (s)TNF-α as indicated. After 48 hours, the percentage of apoptotic cells in the total neuronal population was calculated. (B) BV-2 cells were transfected with negative control (NC) or miR-181c as indicated before being subjected to OGD. After 48 hours, the conditioned medium (CM) was harvested. sTNF-α was added to the CM at the indicated concentration before cultured neurons were grown in microglia-conditioned medium (MCM) for 48 hours, and neuronal apoptosis was detected using Hoechst 33342 staining. (C-D) BV-2 cells were transfected with NC or small interfering TNF before they were subjected to OGD. (C) TNF-α protein expression levels were determined by western blotting after 24 hours, and TNF-α release levels were determined by ELISA after 48 hours. (D) Cultured neurons were then grown in the MCM for 48 hours, and neuronal apoptosis was detected using Hoechst 33342 staining. (E, F) BV-2 cells were transfected with NC, miR-181c or TNF-α (open reading frame without the 3′-untranslated region) expression vectors as indicated, before being subjected to OGD. (E) TNF-α protein expression levels were determined by western blotting after 24 hours, and TNF-α release levels were determined by ELISA after 48 hours. (F) Cultured neurons were then grown in MCM for 48 hours, and neuronal apoptosis was detected using Hoechst 33342 staining. The results are presented as the mean ± SE from three independent experiments.NC represents the negative control for the miR-181c mimics. *P < 0.05. NS, not significant.

Figure 6

microRNA (miR)-181c controls tumor necrosis factor-α production in primary rat microglial cells. (A-B) Primary rat microglia cells were transfected with miR-181c mimics or NC. After 48 hours, cells were harvested, and the TNF-α mRNA (A) and protein (B) expression levels were evaluated by real-time PCR and western blotting. (C-D) Primary rat microglial cells were transfected with miR-181c mimics or NC 24 hoursbefore activation by OGD. After 48 hours, the production of TNF-α was determined by ELISA (C). Cultured neurons were then grown in the microglia-conditioned medium (MCM) for 48 hours, and neuronal apoptosis was detected using Hoechst 33342 staining (D). (E-F) Primary rat microglia cells were transfected with miR-181c mimics or NC 24 hoursbefore activation by OGD. After 48 hours, the production of NO was determined by the Griess assay (E). The cells were harvested, and iNOS protein expression was evaluated by western blotting (F). The results are presented as the mean ± SE from three independent experiments. Ctrl represents the microglial cells that were not treated with oligonucleotide transfection; NC represents the negative control for the miR-181c mimics. *P < 0.05. NS, not significant.

Figure 7

A schematic of the role for microRNA (miR)-181c-tumor necrosis factor (TNF)-α in microglia-mediated neuronal injury. Hypoxia-ischemia resulted in microglia activation and miR-181c downregulation. Downregulation of miR-181c leads to increased TNF-α production, as TNF-α is a direct target of miR-181c. TNF-α can activate receptor-mediated proapoptotic pathways within the neuron. TNF-α can also stimulate microglia activation in the form of inducible nitric oxide synthase (iNOS) induction, which leads to production of nitric oxide (NO) Finally, the interaction between NO and superoxide anion (O2⁻) forms toxic peroxynitrite (ONOO⁻), which also causes neuron damage.