Fig. 4

CircNEIL3 serves as a sponge for miR-432-5p. a. Venn diagram showing the overlap of the target miRNAs of circNEIL3 predicted by miRanda, TargetScan and RNAhybrid. b. The efficiency of the circNEIL3 probe in PDAC cells was validated using RT-qPCR after the RNA pull-down assay. A random sequence NC probe served as a negative control. c. The relative levels of 11 miRNA candidates in CFPAC-1 and MiaPaca-2 lysates were detected by RT-qPCR. d. Biotinylated miRNA pull-down in PDAC cells, and RT-qPCR results showing circNEIL3 expression levels. A random sequence NC probe served as a negative control. e. A schematic of the wild-type (WT) and mutant (MUT) circNEIL3 luciferase reporter vectors. f. The luciferase activities of the circNEIL3 luciferase reporter vector (WT or MUT) in CFPAC-1 and MiaPaca-2 cells transfected with miR-432-5p mimics or mimic NC. g. Anti-Ago2 RIP was performed using PDAC cells followed by RT-qPCR to detect circNEIL3 and miR-432-5p. h. The colocalization of circNIEL3 and miR-432-5p in PDAC cells was detected using a FISH assay. The circNIEL3 probe was labeled with Cy3 (red), miR-432-5p probes were labeled with FAM (green), and nuclei were stained with DAPI (blue). The samples were imaged at 1000× magnification. Scale bar = 10 μm. i. RT-qPCR analysis of the relative expression levels of miR-432-5p in pancreatic epithelial cells (HPNE) and PDAC cell lines. j-k. The relative expression of miR-432-5p was detected in 104 paired PDAC tissues and adjacent normal tissues by RT-qPCR. l. FISH results showing the colocalization of circNIEL3 and miR-432-5p in PDAC and adjacent normal tissues from patients. The samples were imaged at 400× magnification. Scale bar = 25 μm. m. The relative expression of miR-432-5p in cells was detected by RT-qPCR after transfection with indicated vectors. All data are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001