Figure 2

miR-181b directly targets IL-6 gene. (A) miR-181b and its putative binding sequence in the 3′-UTR of IL-6. Mutant sequences were shown in bold type. (WT: wild type; MUT: mutant type). (B, C) Analysis of luciferase activity. (B) HEK-293 cells and (C) RAW264.7 cells were cotransfected with miR-181b inhibitors or negative control inhibitors, pRL-TK and firefly luciferase reporter plasmid containing 3′ UTR of IL-6 gene. pRL-TK was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of negative control inhibitors was set as relative luciferase activity 1 respectively. (D) Effects of miR-181b inhibitors on the endogenous IL-6 mRNA levels. RAW264.7 cells were cotransfected with miR-181b inhibitors or negative control inhibitors. 24 h after transfection, cells were primed with 100 ng/ml LPS continuously for 6 h and 12 h, and then cells were isolated, the expression of IL-6 was analyzed by qPCR. (E) Effects of miR-181b inhibitors on the endogenous IL-6 protein levels. 24 h after transfection with the miR-181b inhibitors or negative control inhibitors, cells were primed with 100 ng/ml LPS continuously for 6 h and 12 h, and then cells were isolated. RAW264.7 cells were analyzed by ELISA. NC: negative control; I: inhibitors. The data were subjected to Student’s t-test. *p < 0.05, **p < 0.01.