Fig. 5

TET1 was a potential direct target gene of miR-190. a, b Cell lysates from indicated cells were evaluated for DNMT3a, DNMT3b, TETI, and TET2 expression. PARP was used as a protein loading control. c TET1 was transfected into UROtsa(miR-190)cells, and cell lysates from the indicated cells were subjected to western blot to analyze TET1 and p27 protein expression. β-Actin was used as a protein loading control. d Schematic diagram of CDS and 3’UTR of TET1 mRNA binding to miR-190. e, f Predicted miR-190 binding site region downstream of the firefly luciferase gene (pMIR-report Vector) was cloned (e), and pMIR-TET1-CDS were co-transfected with pRL-TK into the indicated UMUC3 (miR-190 inhibitor) and UMUC3(LacZ) transfectants. At 24 h after transfection, transfectants were extracted to assess luciferase activity ( f); 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). g Potential miR-190 targeting sequences of the TET1 CDS were analyzed using TargetScan software. Schematics of the TET1 CDS luciferase reporter and its mutants (MUT) are shown. h WT and mutant pMIR-TET1-CDS reporters were co-transfected with pRL-TK into the indicated UMUC3 (miR-190 inhibitor) and UMUC3(LacZ) 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)