Fig. 3

circRHOBTB3 acts as a sponge for miR-600. a The Venn diagram revealed the overlap of potential target miRNAs for circRHOBTB3 with prediction by miRanda, RNAhybrid and TargetScan. b The efficiency of circRHOBTB3 probe in PANC-1 and MiaPaca-2 cells were validated with qRT-PCR assays. c The 11 candidate miRNAs were selected to perform pull-down assays and their enrichment levels were detected with qRT-PCR. d Pull-down assays demonstrated the enrichment of circRHOBTB3 using biotin-labeled miR-600 probe. e RNA immunoprecipitation by AGO2 antibody showed that both circRHOBTB3 and miR-600 were significantly enriched in PANC-1 and MiaPaca-2 cell lines. f A schematic diagram of luciferase reporter plasmid with wild-type (WT) or mutant (MT) circRHOBTB3 sequence and showing its binding site for miRNA-600. g The luciferase activities of the circRHOBTB3 luciferase reporter plasmid (WT and MT) were detected in PANC-1 and MiaPaca-2 cells transfected with miR-600 mimics or mimic NC. h The relative expression of miR-600 in PDAC tissues and adjacent normal tissues were determined by qRT-PCR. i The miR-600 expression was detected in four PDAC cells lines and normal pancreatic epithelial cells (HPNE). j-k The FISH assay on both tissues and cell levels validated the colocalization of circRHOBTB3 and miR-600. The circRHOBTB3 probe was labeled with Cy3 (red), miR-600 probes were labeled with FAM (green), and nuclei were stained with DAPI (blue). The samples were imaged at 1000× magnification. Scale bar = 10 μm. l The Pearson correlation analysis showed that circRHOBTB3 expression is negatively associated with miR-600 expression in PDAC tissue. The miR-600 expression levels were determined by qRT-PCR upon circRHOBTB3 knockdown or overexpression. m The Kaplan-Meier curve showed that lower expression of miR-600 is correlated with shorter overall survival (OS) in PDAC patients (n = 110). All data are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001