Fig. 5

CircNFIB1 acts as a sponge for miR-486-5p in PDAC. a and b Representative FISH images (a) and subcellular fractionation assays (b) showed the subcellular distribution of circNFIB1 in Capan-2 cells. U6 was used as a nuclear control and 18S rRNA was used as a cytoplasmic control. Scale bar: 100 μm. c the potential target miRNAs of circNFIB1 were predicted using CircInteractome. d and e RNA pulldown assays revealed that miRNAs directly interact with circNFIB1 in PANC-1 (d) and Capan-2 (e) cells. f The secondary structure of circNFIB1 was predicted by RNAalifold. g Schematic illustrations showed the alignment of circNFIB1 with miR-486-5p and the red part indicates the mutagenesis nucleotides. h Dual luciferase reporter assays show the luciferase activity of wild-type or mutant circNFIB1 following co-transfection with either the miR-486-5p or control mimics. Relative firefly luciferase expression was normalized to that of Renilla luciferase. i RNA pulldown assays showed the RNAs captured by biotinylated miR-486-5p. j Representative FISH images showed the colocalization of circNFIB1 and miR-486-5p. Scale bar: 100 μm. Significance level was assessed using two-tailed Student t-tests and one-way ANOVA followed by Dunnett’s tests for multiple comparisons. Figures with error bars showed standard deviations of three independent experiments. *p < 0.05 and **p < 0.01