FIGURE 3:

TRAIL-induced miR-146a expression was NF-κB dependent. (A) Western blot assay for the activity of NF-κB in the mouse peritoneal macrophages treated with rsTRAIL for 24 h. The ratio of p-IκBα band intensity to GAPDH band intensity was normalized. (B) Dual-luciferase reporter assay of the miR-146a promoter. The 293T cells were transfected with miR-146a promoter-luciferase and pRL-TK as well as the NF-κB p65 subunit expression plasmid or the IκB-DN expression plasmid. After 48 h, the transfected cells were stimulated with rsTRAIL for 6 h, and the luciferase reporter assay was conducted. (C) RAW264.7 macrophages were transfected with si-P65 (siRNA of NF-κB P65 subunit) for 48 h, and miR-146a expression was measured by q-PCR and normalized relative to the expression of U6. (D) ChIP assay for NF-κB p65 binding to the miR-146a promoter in TRAIL-stimulated macrophages. Levels of the NF-κB p65 subunit at the miR-146a promoter were determined by q-PCR. Data are mean ± SD (n = 3) of three independent experiments. **, p < 0.01; *, p < 0.05 compared with control. ^##, p < 0.01 compared with NF-κB p65 expression plasmid–transfected cells in B.