Fig. 5

miR-501-5p directly targets multiple repressors of wnt/β-catenin signaling pathway. a Predicted miR-501-5p target sequence in 3'UTRs of DKK1, NKD1 and GSK3B. The mutated miR-501-5p containing three altered nucleotides were indicated. b Western blots of DKK1, NKD1, GSK3β, p-GSK3β (Ser9) and β-catenin expression. α-Tubulin served as the loading control. c Luciferase assay of pGL3-DKK1-3′UTR, pGL3-NKD1-3'UTR or pGL3-GSK3B-3'UTR reporter in the miR-501-5p-, antagomiR-501-5p-, mutant miR-501-5p- and control-transfected MGC-803 cells. d MiRNP IP assay showing the association between miR-501-5p and DKK1, NKD1 and GSK3B transcripts in MGC-803 cells. GAPDH served as the negative control. e Individual silencing of DKK1, NKD1 and GSK3B potently rescued the TOP/FOP luciferase reporter activity and self-renewal ability in miR-501-5p-inhibited gastric cancer cells (Fig. 5e and f), demonstrating that these genes were functional effectors of miR-501-5p on regulating wnt/β-catenin signaling activation and stem cell-like phenotype in gastric cancer. Error bars represent the mean ± s.d. of three independent experiments. * P < 0.05