Figure 3

MiR-188 arrests cell cycle at G ₁ /S transition through negative regulation of Rb-E2F axis. (A) CNE cells transfected with miR-188 or miR-NC were synchronized at G1/S boundary by hydroxyurea treatment. Cells were released from hydroxyurea block for 6 h, fixed and stained with propidium iodide (PI) for flow cytometry analysis. (B) miR-188 inhibits BrdU incorporation. BrdU Elisa Assay of CNE cells released from hydroxyurea block for 4 h. The BrdU incorporation was quantified by measuring the chemiluminescence. Data shown as means ± SD from three independent experiments (Student t test, ***p < 0.001). (C) Confocal immunofluorescence analysis of CNE cells stained with EdU (green) and DAPI (blue). Scar bar: 10 μm. (D) Quantification of EdU positive cells in CNE cells transfected with miR-188 or miR-NC released from hydroxyurea block for 2 h. Data are presented as means ± SD from three independent experiments (Student t test, **p < 0.01). (E) Immunoblot analysis of phosphorylated and total Rb expression in CNE cells transfected with miR-NC, miR-188, (F) Ant-NC or Ant-188 respectively. GAPDH were used as internal control. (G) E2F promoter activity was determined directly by use of a pE2F-TA-Luc luciferase reporter plasmid which contains four E2F binding sites upstream of TA promoter. Data shown as means ± SD of three independent experiments (Student t test, *p < 0.05).