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.