Figure 5: Inhibition of exosome synthesis attenuates the induction of GEM resistance by miR-155.

(A) The expression of RAB27B in Panc1 cells transfected with a negative control oligonucleotide (NC) or with siRAB27B (90 nM siRNA). mRNA expression were assessed by qRT-PCR. (B) Immunocytochemistry shows RAB27B expression as a green signal in the cytoplasm of Panc1 cells. The nuclei were stained with Hoechst (blue; scale bar, 100 μm). (C and D) The amount of exosomes that was isolated from Panc1 cells after transfection with NC or with siRAB27B for 48 hours. The amount of exosomes was evaluated by the protein level by the Bradford method (C) or by the concentration of particles of a certain size (D). (E) Alterations of the EC50 of GEM in PDAC cells with siRAB27B or NC transfection. All PDAC cell lines showed ameliorated GEM resistance after transfection with siRAB27B, even cells transfected with pre-miR-155. (F and G) Alterations in apoptosis after 72 hours of GEM treatment in PDAC cells transfected with siRAB27B or with NC. Caspase-3/7 activity is represented as the level of luminescence (F), and the ratio of apoptotic cells was determined using the Annexin V assay (G). (E–G) Concentrations of transfected pre-miR-155: Panc1, 10 nM; MiaPaCa2, 5 nM; PSN1, 1 nM. Concentration of siRAB27B in the three cell lines, 90 nM). Concentration of GEM: Panc1 and MiaPaCa2, 50 μg/ml; and PSN1, 10 μg/ml. Columns in (A,C), and (E–G) show values that are the averages of triplicate measurements; bars show SD values. Data are representative of three experiments. *P < 0.05; **P < 0.01.