Fig. 5

NACC1 is a direct target of miR-600, upregulated in PDAC cells and tissues and serves as a predictor for poor OS. a Venn diagram showing 3 potential target genes predicted by four databases (miRWalk, TargetScan, miRDB, and miRTarbase). b The three candidate target genes of miR-600 were validated by evaluating their expression upon transfection with miR-600 mimics or tis inhibitor. c The schematic diagram of NACC1 3’UTR wild-type (WT) and mutant (MT) luciferase reporter plasmid and the corresponding circRHOBTB3 sequence. The relative luciferase activities of the reporter were analyzed in PANC-1 and MiaPaca-2 cells co-transfected with miR-600 mimics or its control. d The expression of NACC1 mRNA in PANC-1 and MiaPaca-2 cells upon circRHOBTB3 overexpression or knockdown were examined by qRT-PCR. e The expression levels of NACC1 mRNA were measured by qRT-PCR with co-transfecting circRHOBTB3 knockdown lentivirus and miR-600 inhibitor or circRHOBTB3 overexpression vector and miR-600 mimics. f The protein levels of NAC1 were measured by western blotting with co- transfecting circRHOBTB3 knockdown lentivirus and miR-600 inhibitor or circRHOBTB3 overexpression vector and miR-600 mimics. g The expression of NACC1 in PDAC cell lines were determined by qRT-PCR and western blotting. h The expression of NACC1 in PDAC tissues and adjacent normal tissues were validated by qRT-PCR. i IHC staining of NACC1 in cancerous and adjacent normal tissues from PDAC patients. The samples were imaged at 100× and 400× magnification. Scale bar = 100 and 25 μm. j The Pearson correlation analysis revealed that expression level of NACC1 was negatively correlated with miR-600 but positively correlated with circRHOBTB3. k The Kaplan-Meier survival curve showed that patients with high NACC1 expression had poorer overall survival than the low expression group. All data are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001