miR-655 Is an EMT-Suppressive MicroRNA Targeting ZEB1 and TGFBR2
Figure 2
EMT-suppressive effects of miR-655 on mesenchymal-like cancer cells having phenotypic plasticity at EMT/MET.
A, TaqMan real-time RT-PCR analysis of CDH1/E-cadherin and miR-655 in a panel of 23 pancreatic cancer cell lines and a breast cancer cell line, MDA-MB-231. Relative expression levels of transcripts of CDH1/E-cadherin and miR-655 were quantified in comparison to GAPDH and RNU6B, respectively, to normalize the initial input of total RNA. Bar graphs show the ratio of the expression level in these cell lines to that in normal pancreas (Ambion). B, Representative results of phase contrast images (Upper) and CDH1/E-cadherin protein expression level detected by immunofluorescence staining (Lower) in Panc1, KP1N, KP4-4 and MDA-MB-231 cells 96 hours after transfection with 10 nM of ds-NC or dsRNA mimicking miR-655 (ds-miR-655) (Ambion). C, TaqMan real-time RT-PCR analysis (Upper) and Western blot (Lower) analysis of mRNA and protein levels of CDH1/E-cadherin, respectively, in Panc1, KP1N, KP4-4 and MDA-MB-231 cells 96 hours after transfection of 10 nM of ds-NC or ds-miR-655. Asterisks (*), statistical analysis with the Mann-Whitney U test. D, Growth curves in Panc1, KP1N, KP4-4 and MDA-MB-231 cells after transfection of 10 nM of ds-NC or ds-miR-655. Each data point represents the mean of duplicate determinations (bars, SD) in these experiments. Asterisks (*), statistical analysis with the Mann-Whitney U test. E, Representative phase micrographs of Panc1, KP1N, KP4-4 and MDA-MB-231 cells transiently transfected with 10 nM of ds-NC or ds-miR-655 in cell migration and invasion assays in vitro using uncoated and Matrigel-coated transwell-chamber culture systems (Becton Dickinson), respectively. At 48 hours after transfection of dsRNA, cells were transferred into the upper chamber of the transwell (4×104 cells per well). The migrating or invading cells on the lower surface of filters were fixed and stained with the Diff-Quik stain 48 hours after cell transfer. F, Quantification of the cell migration (Left) and invasion (Right) shown in Figure 3B. Bar graphs show the percentage (%) of miR-655-transfectants migrating (Left) or invading (Right) through uncoated or Matrigel-coated filters, respectively, relative to control-transfectants. Asterisks (*), statistical analysis with the Mann-Whitney U test.