miRNA reporter assay of PLAG1-miRNA interaction. PLAG1-3′UTR was cloned to pIS1-vector containing a Renilla luciferase construct. PLAG1-3′UTR construct was cotransfected with pIS0 firefly luciferase control vector into HEK-293 cells (n = 5). For detection of miRNA binding, synthetic miRNAs were cotransfected as indicated. miR-181a, -181b, -424, and -107 are demonstrated to significantly reduce luciferase activity via PLAG1-3′UTR interaction. Specificity of miRNA-PLAG1 interaction was proven by site-directed mutagenesis of miRNA-binding sites in PLAG1 3′UTR. Concerning the miR-181 family, 2 binding sites were mutated, revealing abrogated inhibitory effects of miR-181a and -181b.