Figure 3

miR-206 negatively regulates basal NF-κB signaling and inhibits the expression of pro-inflammatory and pro-angiogenic factors in PDAC. (a) Basal NF-κB activity was determined in PANC-1 and PANC10.05 cells transfected with miR-206 mimic or negative control. Thirty nM of mimics, 100 ng of 3 × KBL vector and 20 ng of pMIR-β-gal were co-transfected in 96-well plate format. Forty-eight hours later, NF-κB luciferase activity was determined and normalized to the β-galactosidase activity. All experiments were performed in six replicates and data are shown as mean±s.d. (b) EMSA of nuclear extracts from PANC-1 cells previously transfected with miR-206 mimic or negative control. Wild-type (WT) as well as mutated (MUT) NF-κB oligos were used as assay controls. The intensity of the bands was determined using ImageJ software. (c) Changes in mRNA expression levels of IL8, CXCL1, CCL2, CSF2, VEGFA, VEGFC and MMP9. PANC-1 or PANC10.05 cells transfected with miR-206 or control were assessed for changes in mRNA level of the NF-κB-dependent target genes, as determined by qRT-PCR. (d) VEGFC, CXCL1 and IL8 secretion (ELISA assay) in the supernatant of PANC-1 or PANC10.05 cells transfected with miR-206 mimic or negative control. All experiments were performed in triplicates and data are shown as mean±s.d. and analyzed by a two-tailed unpaired t-test; *P<0.05, **P<0.01, ***P<0.001.