Fig. 4

Cir2047 maintains activation of the NF-κB pathway and the secretion of MMPs through sponging miR-1931 in F4/80-positive cells. A The top 30 biological pathways, including the NF-κB pathway, generated by the predicted targets of miR-1931. B The effects of miR-1931 on TRAF6 expression were validated using luciferase reporter assays. The 3'-UTR of Traf6 and sequences containing mutated variants of miRNA-binding sites (M) were examined to determine their effect on miRNA expression. Results are expressed as relative luciferase activity and represent the mean ± standard deviation of at least three replicates. C Western blot showing TRAF6 expression levels in F4/80-positive cells at 48 h after transfection with specific siRNAs. The levels of NF-κB (p65) and NF-κB1 (p50) in nuclei were also decreased when Traf6 was knocked down. D Western blot showing that miR-1931 decreased the expression of NF-κB (p65) and NF-κB1 (p50) in the nucleus by targeting TRAF6 following treatment with an miRNA mimic. The relative density of bands is shown under each immunoblot after normalization to β-actin or histone levels. Representative blots from three independent experiments are shown. E qPCR showing miR-1931 levels in F4/80-positive cells after transgenesis. F Western blotting revealed expression of TRAF6 in cells and NF-κB (p65) and NF-κB1 (p50) in nuclei after transgenesis. The relative band density is shown under each immunoblot after normalization to β-actin or histone levels. Representative blots from three independent experiments are shown. G Immunofluorescence detected the activation of the NF-κB pathway in F4/80-positive cells after transgenesis. Fluorescence images used to evaluate the localization of FITC-labeled NF-κB1 (p50) and Cy5-labeled NF-κB (p65); FQ, fluorescence quantitation; a.u., arbitrary unit. H and I Flow cytometry of M1 (iNOS) and M2 (CD206) macrophage markers in F4/80-positive cells after transgenesis and quantification of iNOS and CD206 expression levels in F4/80-positive cells (n = 3). J mRNA levels of Mmp9, Mmp2, and Mmp13 in F4/80-positive M1 polarized cells after transgenesis. K ELISA of MMP-9, MMP-2, and MMP-13 expression in cellular supernatants derived from F4/80-positive M1 polarized cells after transgenesis; *p < 0.05; **p < 0.01; ns, not significant