Direct regulation of EPB41L3 expression by miR-223. (A) Schematic depicting the hypothetical duplexes formed by the interactions between the binding sites in the EPB41L3 3′-UTR (top) and miR-223 (bottom). The predicted free energy value of each hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species, including human, mouse and rat. (B) Quantitative RT-PCR analysis of miR-223 levels in A549 cells treated with pre-miR-control and pre-miR-223. (C) Firefly luciferase reporters containing wild-type (WT) or mutant (MUT) miR-223 binding sites in the EPB41L3 3′-UTR were co-transfected into A549 cells along with pre-miR-control, or pre-miR-223. he cells were assayed using a luciferase assay kit 24 h post-transfection. (D-E) Western blotting analysis of EPB41L3 protein levels in A549 cells treated with pre-miR-control, or pre-miR-223. D: representative image; E: quantitative analysis. (F-G) Western blotting analysis of EPB41L3 protein levels in A549 cells treated with pre-miR-control plus control plasmid, pre-miR-control plus EPB41L3 plasmid, pre-miR-223 plus control plasmid, or pre-miR-223 plus EPB41L3 plasmid. F: representative image; G: quantitative analysis. Data are presented as the mean ± SEM of five independent experiments (*, P < 0.05, **, P < 0.01,***, P < 0.001).