Fig. 2

CircCRIM1 binds directly to miR-422a in NPC. a Levels of circCRIM1 in the nuclear and cytoplasmic fractions of S18 cells. b RNA fluorescence in situ hybridization (FISH) for circCRIM1. The nuclei were stained with DAPI. Scale bar, 20 μm. c RNA immunoprecipitation (RIP) analysis of circCRIM1 in S18 and HONE1 cells using antibodies against AGO2. Western blotting analysis of immunoprecipitated AGO2 protein is shown. d Clustered heatmap of the differentially expressed miRNAs in S18 and S26 cells. e Overlapping of the target miRNAs of circCRIM1 based on miRNA microarray, miRanda and RNAhybrid algorithms, and StarBase. f Schematic of the predicted miR-422a binding site on circCRIM1. g Enrichment of circCRIM1 in S18 and HONE1 cells after pull-down assay with biotinylated miR-422a. Mean (n = 3) ± S.D. Student’s t-tests; *P < 0.05. h Luciferase activity of wild type or mutated circCRIM1 in NPC cells after cotransfection with miR-422a or miRNA control. i Colocalization between miR-422a and circCRIM1 was observed by RNA FISH in S18 cells. The nuclei were stained with DAPI. Scale bar, 10 μm. The data are presented as the mean (n = 3) ± S.D. Student’s t-test; *P < 0.05