Fig. 3

miR-190 reduces p27 mRNA stability by binding to its 3′UTR. a, b real-time PCR was performed to determine CDKN1B mRNA expression levels in UROtsa(miR-190) and UMUC3(miR-190 inhibitor) and their vector control cells. GAPDH was used as an internal control. Bars represent mean ± SD from three independent experiments. The symbol (*) indicates a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001). c UMUC3(LacZ) and UMUC3(miR-190 inhibitor) cells were treated with Act D (20 µg/mL) for the indicated time periods. Total RNA was isolated and subjected to real-time PCR analysis for CDKN1B mRNA expression. d The CDKN1B 3′UTR reporters were co-transfected with pRL-TK into the indicated cells. Twenty-four hours post-transfection, the transfectants were extracted for determination of the luciferase activity, and TK was used as the internal control. The results are shown as CDKN1B 3’UTR activity relative to vector control transfectant, and each bar indicates the mean ± SD from three independent experiments. The symbol (*) indicates a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001). e Potential miR-190 targeting sequences of the CDKN1B mRNA 3′-UTR were analyzed using TargetScan software. Schematics of the CDKN1B mRNA 3′-UTR luciferase reporter and its mutants (MUT) are shown. f WT and mutant CDKN1B 3′-UTR reporters were co-transfected with pRL-TK into the indicated UMUC3 (miR-190 inhibitor) transfectants. At 24 h after transfection, transfectants were extracted to assess luciferase activity; TK was used as the internal control. Each bar indicates the mean ± SD of three assays; *significant difference (*P < 0.05, **P < 0.01, ***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001)