Fig. 1
CDDP induces miR-424 in bladder cancer cells in a HIF-1α-dependent manner. a FACS analysis. Bladder cancer 5637, J82, BIU87 and T24 cells were treated with the increasing concentrations of CDDP. Twenty-four hours after treatment, cells were subjected to FACS analysis. b CDDP-mediated cleavage of PARP. The indicated cells were exposed to 10 μM of CDDP. Twenty-four hours after exposure, cell lysates were prepared and analyzed by Western blotting. β-actin was used as a loading control. c CDDP-mediated induction of HIF-1α. Five thousand six hundred thirty-seven (left panels) and T24 (right panels) cells were treated with 10 μM of CDDP or left untreated. Twenty-four hours after treatment, cell lysates were analyzed for HIF-1α by Western blotting. β-actin was used as a loading control. d CDDP specifically potentiates HIF-1α transcriptional activity. T24 cells were treated with 10 μM of CDDP or left untreated. Twenty-four hours after treatment, total RNA was prepared and analyzed for CA9 and RPL13A by qRT-PCR. e CDDP-dependent up-regulation of miR-424. T24 (left panels) and 5637 (right panels) cells were treated as in (c). Twenty-four hours after treatment, total RNA was prepared and analyzed for pri-miR-424 (upper panels) and miR-424 (lower panels) by qRT-PCR. f Chromatin immunoprecipitation (ChIP). T24 cells were exposed to 10 μM of CDDP or left untreated. Cross-linked chromatin was immunoprecipitated with the control IgG or with anti-HIF-1α antibody, and the immunoprecipitated DNA was subjected to qPCR using primers that flanked the putative HIF-1α-binding site. The results were normalized to T24 cells without CDDP treatment, in which ChIP was carried out with the control IgG (mean ± SEM; n = 3). The nucleotide sequence of the HIF-1α-binding site is also shown. g HIF-1α-dependent induction of miR-424 in response to CDDP. Five thousand six hundred thirty-seven cells were transfected with the scramble siRNA or with siRNA against HIF-1α and treated with or without 10 μM of CDDP. Knockdown efficiency of HIF-1α siRNAs was validated by qRT-PCR (left panel). Twenty-four hours after CDDP treatment, total RNA was prepared and subjected to qRT-PCR. *P < 0.05, **P < 0.01