Figure 5

Inhibition of KRAS and ANXA2 oncogenes mediates the observed miR-206 phenotypes in PDAC cells. (a) Basal NF-κB activity was determined in PANC-1 cells transfected with siRNAs against KRAS, ANXA2 or non-targeting control. NF-κB luciferase activity was normalized to the β-galactosidase activity. The experiment was performed with six replicates and in three independent experiments. (b) PANC-1 and PANC10.05 cells transfected with two individual siRNAs against KRAS or control were assessed for changes in mRNA level of the IL8, CXCL1, CCL2, CSF2, VEGFA and VEGFC genes as determined by qRT-PCR. The experiment was performed in triplicates and in three independent experiments. (c) Cell-cycle distribution of PANC-1 cells, previously transfected with siKRAS, siANXA2 or negative control, and growing in full-growth medium for 72 h was measured by 7-AAD staining and quantified by flow cytometry. Experiments were performed with three replicates. (d) Proliferation was evaluated in PANC-1 and PANC10.05 cells 3 days after transfection with two individual siRNAs against either KRAS or ANXA2 genes, using a WST-1 assay. Cell viability experiments were performed with six replicates and in three independent experiments. (e) Cell migration of PANC-1 cells previously transfected with two individual siRNAs against either ANXA2 or KRAS, as well as with the negative control was assessed by an RTCA (real-time cell analyzer) trans-well migration assay. Cells were seeded in 0.5% FCS medium and allowed to migrate for 10 h using full-growth medium as chemoattractant. Impedance measurements were performed in a time-resolved manner. Means of four replicates±s.d. are shown; a two-tailed upaired t-test was performed for the last time point. (f) End point matrigel invasion assay of PANC-1 and BxPC-3 cells transfected with siRNAs against either ANXA2 or KRAS, as well as with the negative control. Cells were transfected, seeded in 0.5% FCS medium and allowed to invade for 24 h (or 48 h in case of BxPC-3 cells) using full-growth medium as chemoattractant. The number of invaded cells was quantified by flow cytometry. Experiments were performed with four replicates and in three independent experiments. Data are shown as mean±s.d. (g) Plasmin production (activity) was determined in PANC-1 cells previously transfected with miR-206 mimic, siANXA2 or control. Cells were treated with t-PA (10 μg/ml), 0.4 μM of plasminogen and 10 μl of the plasmin substrate (Spectrozyme). Produced active plasmin was determined by measuring absorbance at 405 nm. Experiments were performed with four replicates and in three independent experiments. Data are shown as mean±s.d. All data were analyzed by a two-tailed unpaired t-test; *P<0.05, **P<0.01, ***P<0.001.