Fig. 2

CircGARS functions as a sponge for miR-19a-5p. a The ceRNAs were identified using circMir1.0 software. b The circGARS probe could capture miR-19a-5p by RNA pull-down analysis. c FISH showed the colocalization between circGARS and miR-19a-5p in PBMCs (red: circGARS probes were labeled with Cy3; blue: nuclei were stained with DAPI; green: locked nucleic acid miR-19a-5p probes were labeled with Dig; scale bar: 10 μm). d The interaction of circGARS with miR-19a-5p was tested by RIP of AGO2 from HEK293T cells, and the IP efficiency of the AGO2 antibody is displayed by western blotting, which represents circGARS levels associated with AGO2 relative to an input control. IgG antibody served as a control. e CircGARS levels were quantified by qRT–PCR, and (IP)/input ratios were plotted by Student’s t test. f CircGARS contains one site complementary to miR-19a-5p, as analyzed by using the RNAhybrid bioinformatics program. Schematic illustration of circGARS-WT and circGARS-MUT luciferase reporter vectors. g, h Luciferase reporter assay was applied to verify the interaction between circGARS and miR-19a-5p. The Luciferase activity was normalized to the value obtained in cells transfected with NC oligonucleotides. NC, negative control; miRNA, microRNA; IgG, immunoglobulin G; RIP, RNA immunoprecipitation; qRT-PCR, quantitative real time polymerase chain reaction. All data are presented as the means ± SD of 3 independent experiments (n = 3). NS: no significance, **P < 0.01; ***P < 0.001