Fig. 8

miR-7 suppresses NF-κB transcriptional activity by reducing p65 and active NF-κB/p65 expression. a miR-7 suppressed NF-κB transcriptional activity while NF-κB activator LPS reversed this activity. NF-κB transcriptional activity was detected by dual-luciferase reporter assay. NF-κB activator LPS was used in indicated groups. The data were presented as fold inductions of the ratio was normalized to Renilla luciferase activity. b-c LPS treatment reverted the abilities of cell migration and cell invasion after miR-7 transfection. Indicated cells were treated with or without 50 ng/ul or 100 ng/ul LPS and then cell migration and cell invasion were analyzed in miR-7-transfected HGC-27 (b) and MKN-28 cells (c). Data are presented as mean ± SD of three independent experiments. Representative images are shown. d-e Restoration of miR-7 decreased total p65 and active p-p65(Ser536) levels in GC cells. p65 and p-p65(Ser536) proteins were detected by FACS analysis in miR-7-transfected HGC-27 (d) and MKN-28 cells (e). Representative images are shown. f-g Cellular distribution of p65 and p-p65(Ser536) in miR-7-transfected GC cells. Immunofluorescence (IF) staining was used to detect cellular distribution of p65 and p-p65(Ser536) in indicated GC cells (GFP). AF555 (Red) or AF647 (Pink)-conjugated secondary antibodies were used to detect primary antibodies. Nuclei were counterstained with DAPI (Blue). Images were captured using a confocal microscope (Scale bars: 25 μm). h-i Restoration of miR-7 reduced p65 and p-p65(Ser536) expression in metastatic tissues. p65 and p-p65(Ser536) were detected by IHC staining in metastatic lung and liver tissues. Representative IHC images are shown. Scale bars: (main) 50 μm; (inset) 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated two groups determined by paired student’s t test