Figure 5.

MIR454 directly targets TSPAN1 and FAM83A. (A) Clustered heatmap of differentially expressed miRNAs. Each column indicates a tissue sample and each row indicates an individual gene. Red and green strips represent high and low miRNA expression, respectively. (B) Kaplan-Meier overall survival curves for MIR454 in pancreatic cancer patients. (C and D) MIR454 levels were inversely correlated with the levels of TSPAN1 (C) and FAM83A (D). (E) The relative mRNA level of MIR454 was detected in human normal pancreatic duct epithelial HPD E6-C7 cells and human pancreatic cancer PANC-1, ASPC-1, MIA-PACA-2, CAPAN-1 and SW1990 cells. (F) Relative mRNA levels of TSPAN1 and FAM83A after transfected with miR control, MIR454 mimics, inhibitor control and MIR454 inhibitor in PANC-1 cells. (G) Relative protein levels of TSPAN1 and FAM83A after transfected with miR control, MIR454 mimics, inhibitor control and MIR454 inhibitor in PANC-1 and ASPC-1 cells. (H) Schematic illustration of predicted miRNA binding sites within the 3′-UTR of TSPAN1 mRNA (position 150 to 157) and generation of the WT-TSPAN1 3′UTR and MT-TSPAN1 3′UTR luciferase reporter vectors. (I) Schematic illustration of predicted miRNA binding sites within the 3′-UTR of FAM83A mRNA (position 2008 to 2014), and generation of the WT-FAM83A 3ʹUTR and MT-FAM83A 3ʹUTR luciferase reporter vectors. (J) Luciferase activity was measured in lysates of HEK293T cells transfected with the wild-type 3′-UTR and mutant 3′-UTR of TSPAN1 luciferase constructs and a MIR454 mimic or a scrambled miRNA control. The luciferase activity was normalized to renilla luciferase activity. (K) Luciferase activity was measured in lysates of HEK293T cells transfected with the wild-type 3′-UTR and mutant 3′-UTR of FAM83A luciferase constructs and a MIR454 mimic or a scrambled miRNA control. The luciferase activity was normalized to renilla luciferase activity. Data were represented as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001