Fig. 4

IRAK4 and EphA2 were functional targets of miR-302b. a IRAK4 3′ UTRs and EphA2 3′ UTRs contain one predicted miR-302b binding. Predicted duplex formations between IRAK4 3′ UTR and miR-302b (upper), and EphA2 3′ UTR and miR-302b (lower). b, c Real-time qPCR analysis of IRAK4 and EphA2 mRNA levels in NS-m and 302b-m-transfected THP-1 cells with/without treatment of MSU. d Normalized luciferase activity of a reporter containing wild-type or point-mutated 3′ UTR reporter constructs (WT UTR or mutant UTR) of IRAK4 (left) and EphA2 (right) in THP-1 cells cotransfected with NS-m or 302b-m. e–j THP-1 cells transfected with a negative control siRNA (si-NC), IRAK4 siRNA (si-IRAK4), or EphA2 siRNA (si-EphA2) for 48 h. e Real-time qPCR analysis of IRAK4 mRNA level in THP-1 cells transfected with negative control siRNA si-NC or si-IRAK4. f si-NC or si-IRAK4-transfected THP-1 cells treated with MSU for 8 h. ELISA of IL-1β protein levels in cell culture medium. g Western blot analysis of p-NF-κB in THP-1 cells treated with MSU treatment for 3 h after transfection with si-NC or si-IRAK4. h Real-time qPCR analysis of EphA2 mRNA level in THP-1 cells transfected with negative control siRNA si-NC or si-EphA2. i si-NC or si-EphA2-transfected THP-1 cells treated with MSU for 8 h. ELISA of IL-1β protein levels in cell culture medium. j Western blot analysis of cleaved caspase-1 p20 in THP-1 cells treated with MSU treatment for 3 h after transfection with si-NC or si-EphA2. Densitometric quantification of western blotting gel shown as fold change under the band. Data represent three experiments, shown as means ± SEMs (**p < 0.01, *p < 0.05 by t test). IRAK-4 interleukin-1 receptor-associated kinase 4, EphA2 EPH receptor A2, CTRL control, MSU monosodium urate, NS-m negative control mimic, 302b-m miR-302b mimic, IL-1β interleukin-1β