Fig. 6

IRAK1 and TRAF6 are functional targets of miR-146a. a Predicted miR-146a binding sites in 3’ UTRs of IRAK1 and TRAF6. CDS, coding sequence. b and c HEK-293T cells were co-transfected with miR-146a mimics or miR-ctrl, along with the wild-type or mutated IRAK1 3’ UTR or TRAF6 3’ UTR luciferase reporter plasmids, and assessed for luciferase activity 24 h after transfection. d and e The mRNA and protein levels of IRAK1 and TRAF6 were determined in U251 cells after 24 h of transfection with miR-146a, anti-miR-146a, or their corresponding control oligonucleotides. f–h The mRNA and protein levels of IRAK1 and TRAF6 were determined in PCN033-infected U251 cells. i U251 cells were transfected with anti-miR-146a or anti-miR-ctrl and then infected with PCN033 for 3 h. The expression of IRAK1 and TRAF6 was determined by Western blotting. j and k The mRNA and protein levels of IRAK1 and TRAF6 were determined in U251 cells after 24 h of transfection with siRNA-IRAK1, siRNA-TRAF6, or negative control (NC). l U251 cells were transfected with siRNA-IRAK1, siRNA-TRAF6, or NC and then infected with PCN033; the protein levels of NF-κB p65 and phosphorylated p65 were determined by Western blotting. m U251 cells were transfected with anti-miR-ctrl, anti-miR-146a, or anti-miR-146a, along with siRNA-IRAK1, or anti-miR-146a, along with siRNA-TRAF6, and then infected with PCN033 for 3 h. The expression of IL-1β, IL-6, TNF-α, MCP-1, and MIP-2 was determined by qPCR. All data were represented as mean + SD (n = 3/group). Statistical analysis was carried out by Student’s t test or one-way ANOVA. p < 0.05 was considered statistically significant; p < 0.01 and p < 0.001 indicated extremely significant differences