Figure 3
Effect of miR-136 on IL-6 expression in A549 cells.
(A) Schematic representation of mutant IL-6 3′UTR reporter constructs. Red and blue letters indicate nucleotides that were deleted or point mutated, respectively. (B) A miR-136 or inhibitor (20 nM, 40 nM, or 60 nM) were cotransfected with the pMIR-IL-6 3′UTR reporter plasmid into 293T cells. Luciferase 0 nM mimic or inhibitor were assigned a value of 1 for normalization purposes. (C) 293T cells were cotransfected with the indicated oligonucleotides and either the parental pmirGLO vector, or a pmirGLO construct encoding wild type, point mutant, or deletion mutant variants of the IL-6 3′UTR. Luciferase activities were measured at 24 hours post-transfection and the results shown reflect fold-inductions in luciferase activities relative to cells transfected with the empty pmirGLO vector. Cells were transfected with miR-136 or NC at a concentration of 60 nM. At 24 hours post-transfection, IL-6 mRNA levels (D) were detected by RT-qPCR and IL-6 protein expression in cell supernatants (E) and sediments (F) were measured by ELISAs and western blots, respectively. Dose-dependent inductions of IL6 mRNA (G) and protein (H) expression by the miR-136 were detected using RT-qPCR and ELISAs, respectively. Results are presented as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by one-way ANOVA with Bonferroni’s post hoc multiple comparison test (B,D,E,G,H) or Student’s t test (C). The blots were run under the same experimental conditions. Full-length blots/gels are presented in Supplementary Figure 5.
