Fig. 5

circCDK13 upregulates E2F5 protein level by sequestering miR-221-5p/449a

a, RT-qPCR detected the expression of E2F5 mRNA in PC3 cells transfected with circCDK13, si-circCDK13 or their respective control. b, Venn diagram displaying potential microRNAs associated with circCDK13 sequence from three online target-prediction programs. c, circCDK13 or empty vector was transfected into PC3 cells, RT-qPCR detected the pulled down efficiency of circCDK13 by biotinylated probes against circCDK13. *P < 0.05, **P < 0.01 vs. con probe or empty vector. d, RT-qPCR detected the level of indicated miRNAs pulled down by biotinylated probes against circCDK13. *P < 0.05, **P < 0.01 vs. con probe. e, Biotin-labeled E2F5 3′-UTR RNA was transfected into PC3 cells followed by a biotin pull-down assay using Streptavidin-coupled Dynabeads. The miRNAs were extracted from the sedimented beads, and the relative levels of 11 candidate miRNAs were detected by RT-qPCR. *P < 0.05, **P < 0.01 vs. control E2F5 3′-UTR. f, PC3 cells were co-transfected with miR-449a mimic, miR-212-5p mimic or both and circCDK13-directed luciferase reporter. Luciferase activity was measured by dual-luciferase reporter assays. *P < 0.05, **P < 0.01 vs. miR-NC. g, RNA in situ hybridization detected the co-localization between miR-449a or miR-212-5p with circCDK13 (arrowheads) in PC3 cells co-transfected with circCDK13 expression vector and miR-449a or miR-212-5p mimic. Nuclei were counterstained with DAPI. Scale bars = 25 μm. h, PC3 cells were co-transfected with miR-449a, miR-212-5p or control mimic (miR-NC) and wild type or mutated (mut) E2F5 3’UTR-directed luciferase reporter. Luciferase activity was measured by dual-luciferase reporter assays. Graph bars are mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01 vs. miR-NC or E2F5 3’UTR mut. i, PC3 and 22RV1 cells were transfected with miR-212-5p, miR-449a mimic or both, and then Western blotting detected the CDK13 and E2F5 expression.