Fig. 6: MYCN directly interacts with the PKIB promoter in BLCA cells.

A Correlation of MYCN and PKIB mRNA expression in BLCA tissues. The X- and Y-axes represent the log2-transformed FPKMs of MYCN and PKIB mRNA in BLCA tissues, respectively. B mRNA expression level of MYCN in BLCA and normal tissues in the TCGA database. (http://gepia2.cancer-pku.cn/#index). ^*P < 0.05. C Kaplan-Meier survival curves (http://kmplot.com/analysis/) of bladder cancer patients (n = 408) with high or low expression levels of MYCN. The log-rank test was used to analyse the difference between two groups. D qRT–PCR and Western blot analyses of PKIB mRNA and protein levels in MYCN-silenced T24 cells. qRT–PCR data are shown as the mean ± SD (n = 3). ^***P < 0.0001 according to unpaired Student’s t test. E The conserved MYCN binding site (MBS) is shown (https://jaspar.genereg.net/matrix/MA0104.4/). F Schematic showing two predicted binding sites for MYCN in the PKIB promoter (upper left panel). Boxed areas indicate several PKIB promoter segments containing wild-type (PKIB-WT) or mutant MBS-1/2 (PKIB-Mut1, PKIB-Mut2, or PKIB-Mut1*2; lower left panel), which were subcloned and inserted into the pGL3-Luc reporter vector. The numbers indicate the ___location of the nucleotides of the PKIB promoter. The constructs were transiently transfected into 293 T cells overexpressing MYCN, after which luciferase activities were determined (right panel). The data are shown as the mean ± SD (n = 3). PKIB-WT vs PKIB-Mut1 ^***P = 0.0003, others ^***P < 0.0001, ns = not significant according to one-way ANOVA with Tukey’s multiple comparison tests. G Different fragments of the PKIB promoter (PKIB-WT, PKIB-2 and PKIB-3) were subcloned and inserted into the pGL3-Luc reporter vector (left panel), which was transiently transfected into 293 T cells overexpressing MYCN, after which luciferase activity was determined (right panel). The data are shown as the mean ± SD (n = 3). ^**P = 0.0018, PKIB-WT ^***P < 0.0001, PKIB-2 ^***P = 0.0018, ns = not significant according to one-way ANOVA with Tukey’s multiple comparison tests. H The ChIP assay was performed on T24 cells using an anti-MYCN antibody. An anti-IgG antibody was used as a negative control. The immunoprecipitated DNA fragment was subjected to PCR and analysis to determine the enrichment of MYCN.