Fig. 1
Identification and validation of upregulated circFIRRE in OS tissues and cells. A A Volcano plot illustrating the differential expression of circRNA in RNA-sequencing, where the red and blue points represent upregulated and downregulated circRNAs that met screening criteria as described. B Validation of differentially expressed circular RNAs by RT-qPCR (n=3 in each group). C The relative expression of circFIRRE in 104 paired clinical OS samples and normal controls were analyzed by RT-qPCR. D RNA–fluorescence in situ hybridization assays (FISH) were conducted in clinical OS and normal tissues to demonstrate circFIRRE expression using Cy3-labeled probes (red); DAPI-stained nuclei (blue). Scale bars=100 μm and 50 μm. E The relative expression of circFIRRE in human OS cell lines and normal osteoblasts (n=3 in each group). F-G RT-qPCR indicated the abundance of circFIRRE in cytoplasm of MG63 and U2OS cells. GAPDH and U6 were used as positive controls for cytoplasm and nucleus (n=3 in each group). H FISH assay demonstrated that circFIRRE was predominantly localized in the cytoplasm of MG63 and U2OS cells. Red indicates circFIRRE; DAPI-stained nuclei (blue). Scale bar=50 μm. I Schematic illustration presents the formation of circFIRRE. The trans-splicing site of circFIRRE validated by Sanger sequencing is highlighted by the red underline. J-K The relative expression of circFIRRE and linear FIRRE was analyzed by RT-qPCR at different time points after Actinomycin D treatment in MG63 and U2OS cells (n=3 at each time point). L-O The relative expression of circFIRRE and linear FIRRE was detected by RT-PCR and RT-qPCR in MG63 and U2OS cells in the presence of RNase R. P-Q The presence of circFIRRE was validated in MG63 and U2OS cells by RT-PCR. circFIRRE was amplified by divergent primers in cDNA, but not in genomic DNA. Values are presented as mean ± SD; the bar charts, line charts, error bars and dots represent the quantitative analysis of 3 independent experiments; (B, C, 2-tailed Student t test; E, one-way ANOVA; J, K, M and O, two-way ANOVA); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant