Figure 4

The attenuation of miRNA-146 enhances Toll-like receptor signaling. A: HGFs were transfected with a Cy™ 3 labeled dye as a negative control and anti-miRNA-146a/anti-miRNA-146b-5p inhibitors. After 24 hours of stimulation with 1 μg/ml of P.g LPS, the expression levels of IRAK1 and TRAF6 were analyzed by real-time PCR (A) and immunoblotting (B). The immunoblot shown in the figure is representative of three independent experiments. C: We amplified the IRAK1 3’-UTR (1352 bp starting from TGA) and cloned the sequence into the 3’ site of the luc2 reporter gene on the pGL4 plasmid. We co-transfected 200 ng of the reporter plasmid, 20 ng of pRL-TK-Renilla-luciferase and indicated miRNA mimic (final concentration 40 nM) into the HGFs. The cells were stimulated with 1 μg/ml of P.g LPS after transfection, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions. Medium: 24 hour stimulation with 1 μg/ml of P.g LPS without the miRNA inhibitor; control: 24 hour stimulation with 1 μg/ml of P.g LPS after being transfected with a negative control. The expression levels are shown as the mean ± SD. *: p < 0.05.