Fig. 4

HES1-mediated down-regulation of miR-138–2 in ccRCC cells. a The putative HES1-binding site within the promoter region of miR-138–2. ChIP-seq data of K562 and MCF-7 cells obtained from the ENCODE project was shown. b Knockdown of HES1. 786-O (left) and CAKI-1 (right) cells were transfected with the control shRNA or with shRNA against HES1. After transfection, total RNA was prepared and analyzed for pre-miR-138–2, miR-138-5p and miR-138–2-3p by qPCR (n = 3). c Overexpression of HES1. 786-O (left) and CAKI-1 (right) cells were transfected with the empty plasmid or with the expression plasmid for HES1. After transfection, total RNA was prepared and analyzed for pre-miR-138–2, miR-138-5p and miR-138–2-3p by qPCR (n = 3). d Schematic diagram of the predicted HES1-binding sites within miR-138–2 genomic locus. e ChIP assay. Cross-linked CAK-1 genomic DNA was immunoprecipitated with the control IgG or with anti-HES1 antibody. The fragmented genomic DNA was purified from the immunoprecipitates and then subjected to qPCR with HES1P1- or with HES1P2-specific primers (n = 3). f Luciferase reporter assay. 786-O (upper) or CAK-1 (lower) cells were transfected with the luciferase reporter construct bearing 4-kb upstream sequence of miR-138–2 or its mutated sequence together with or without HES1 expression plasmid. After transfection, cells were lysed and their luciferase activities were measured (n = 3). g Mouse xenograft. Nude mice were injected with the control 786-O cells or with HES1-depleted 786-O cells. The representative pictures of the indicated tumors were shown. h qPCR. Total RNA was prepared from the indicated tumor tissues and analyzed for pre-miR-138–2, miR-138-5p and miR-138–2-3p by qPCR (n = 5)