Fig. 4

CircCYP24A1 serves as a sponge for miR-1301-1p. A Venn diagram showing the overlap of the eight candidate target miRNAs of circCYP24A1 predicted by miRanda, RNAhybird and TargetScan. B The efficiency of the circCYP24A1 probe in DU145-DR and DU145 cells. A random sequence probe served as a negative control. C-D The fold enrichment of the target miRNAs in pull-down complex of DU145-DR cells (C) and DU145 cells (D). E-G The expression of those candidate miRNAs after down−/up-regulation of circCYP24A1 in DU145-DR (E), 22Rv1 (F) and DU145 cells (G). H Schematic view of RNA pull down assays by biotin-labeled miRNA. I Biotin-labeled miRNA pull-down in DU145 and DU145-DR cells, and qRT-PCR results showing circCYP24A1 enrichment in miRNA probe. A random sequence probe served as a negative control. J The luciferase activities of the circCYP24A1 luciferase reporter in HEK293T cells transfected with miR-1301-3p mimics or mimic NC (upper) and the schematic of the wild-type (WT) and mutant (MUT) circCYP24A1 luciferase reporter vectors (bottom). K Schematic view of anti-AGO2 RIP assays. L-M. Anti-AGO2 RIP assays were performed in DU145 cells, and the enrichment of circCYP24A1 (L) and miR-1301-3p (M) was detected by qRT-PCR. N The colocalization of circCYP24A1 and miR-1301-3p in DU145-DR cells was detected by FISH assay. The circCYP24A1 probe was labeled with Cy3 (red), miR-1301-3p probes were labeled with FAM (green), and nuclei were stained with DAPI (blue). O FISH score of miR-1301-3p in DTX-sensitive (n = 30) and DTX-resistant (n = 40) PCa biopsy tissues. P Representative FISH images of miR-1301-3p in three DTX-resistant/−sensitive patients, respectively. The miR-1301-3p probe was labeled with FAM (green), and the nuclei were stained with DAPI (blue). All data are presented as the means ± SD of three independent experiments. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001