Fig. 3

CircTADA2A acts as a sponge for miR-203a-3p in osteosarcoma cells. a RIP assay for circTADA2A levels in HEK-293 cells transfected with Ago2 (RIP, AGO2 RNA immunoprecipitation). b Schematic illustration exhibiting overlapping of the target miRNAs of circTADA2A predicted by miRanda, circbank, TargetScan, and RNAhybrid. c & d The relative levels of 20 miRNA candidates in the HOS and 143B lysates were examined by qRT-PCR. Multiple miRNAs can be pulled down by circTADA2A. e Luciferase activities of luc-circTADA2A or luc-circTADA2A-mutant in HEK-293 cells cotransfected with 6 selected miRNA mimics or mimics N.C. were determined by a luciferase reporter assay. f Apoptosis assay with Annexin V-FITC/PI staining was performed to analyze the apoptotic rates of HOS and 143B cells transfected with 6 selected miRNA mimics or mimics N.C. g Luciferase reporter assay was performed to detect the luciferase activities of HEK-293 cells cotransfected with a luciferase reporter construct containing wild-type (or mutant) circTADA2A and miR-203a-3p mimics (or mimics N.C.). h Colocalization between miR-203a-3p and circTADA2A was observed via FISH in both HOS and 143B cells. CircTADA2A probes were labeled with Alexa Fluor 555. MiR-203a-3p probes were labeled with Alexa Fluor 488. Nuclei were stained with DAPI; scale bar, 50 μm. Data are from three independent experiments (mean ± SEM) (a and c-g) or are representative of three independent experiments with similar results (h) (*P < 0.01 vs control or as indicated by Student’s t-test)