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.