Fig. 6

circFIRRE acts as a sponge for miR-486-3p and miR-1225-5p (A-C) Anti-AGO2 RNA immunoprecipitation (RIP) assay was performed to estimate AGO2 binding to circFIRRE in MG63 and U2OS cells, followed by RT-qPCR and RT-PCR detection, using IgG as the negative control (n=3 in each group). D Schematic illustration exhibited overlapping of the target miRNAs of circFIRRE predicted by five algorithms. E The relative expression of miR-486-3p and miR-1225-5p declined in 35 paired OS samples relative to adjacent normal controls through RT-qPCR detection, respectively. F-G Representative images of FISH were shown to illustrated the declined miR-486-3p and miR-1225-5p expression in OS samples compare with the adjacent normal samples. Scale bars=100 μm and 50 μm. H-I Pearson correlation analysis indicated a negative correlation between circFIRRE and two miRNAs in 35 paired samples. J-K The relative expression of miR-486-3p and miR-1225-5p was detected by RT-qPCR after transfecting circFIRRE siRNA or overexpression vectors in both MG63 and U2OS cells (n=3 in each group). L-M circFIRRE-specific biotin-labelled probe could successfully capture miR-486-3p and miR-1225-5p in MG63 and U2OS, using the oligo probe as the negative control (n=3 in each group). N-O Specific biotin-labelled miR-486-3p and miR-1225-5p probes could successfully capture circFIRRE relative to biotin-NC probe in both MG63 and U2OS (n=3 in each group). P Dual luciferase assay was performed to estimate the binding between circFIRRE and two miRNAs, after co-transfecting miRNA mimics and luciferase reporter plasmids into HEK-293 T (n=3 in each group). Q-R Typical fields of FISH were showed to illustrate the co-localization of circFIRRE and two miRNAs in both MG63 and U2OS. Scale bar=50 μm. Values are presented as mean ± SD; the bar charts, error bars and dots represent the quantitative analysis of 3 independent experiments; two-way ANOVA were used; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant