Fig. 3

CircFAT1 serves as a sponge for miR-375 in osteosarcoma cells. a Schematic illustration showed the conservation across 100 vertebrate species and AGO2 binding sites in the circFAT1 genomic region. b AGO2 RNA immunoprecipitation (RIP) assay for circFAT1 levels in HOS cells stably expressing shcircFAT1. Data represents the mean ± SD for three experiments. * P < 0.05. c Schematic illustration showing overlapping of the target miRNAs of circFAT1 predicted by miRanda, Targetscan, and RNAhybrid. d–e Lysates prepared from HOS and 143B cells stably transfected with circFAT1 or vector were subjected to RNA pull-down assay and tested by (d) RT–PCR and (e) real-time PCR. Relative level of circFAT1 was normalized to input. GAPDH was used as a negative control. Data represent the mean ± SD (n = 3). * P < 0.05 versus oligo probe (Student’s t-test). f The relative level of 16 miRNA candidates in the HOS and 143B lysates was detected by real-time PCR. g Luciferase reporter assay for the luciferase activity of LUC-circFAT1 or LUC-circFAT1-mutant in HEK-293 T cells co-transfected with miRNA mimics. Data represent the mean ± SD for three experiments. * P < 0.05. h Schematic illustration demonstrates complementary to the miR-375 seed sequence with circFAT1. Lowercase letters indicate mutated nucleotides. i 293T cells were co-transfected with miR-375 mimics and a luciferase reporter construct containing wild-type (WT) or mutated circFAT1. Data represent the mean ± SD (n = 3). * P < 0.01. j Fluorescence in situ hybridization (FISH) showing co-localization between circFAT1 and miR-375 in HOS and 143B cells. CircFAT1 probes were labeled with Alexa Fluor 488. Locked nucleic acid miR-375 probes were labeled with Cy3. Nuclei were stained with DAPI. Scale bar, 50 μm