Figure 4

Inhibition of Zeb1 mediates the tumour suppressor functions of miR-144.

(A, B) Endogenous Zeb1 expression was efficiently repressed by siRNA. A549 and 95D cells were transfected with non-specific siRNA control (sicontrol) or Zeb1-specific siRNA (siZeb1) for 48 h. The expression of Zeb1 were measured by qRT-PCR (A) and Western blotting (B). Inhibition of Zeb1 expression led to up-regulation of E-cadherin and down-regulation of Vimentin (B). (C) Inhibition of Zeb1 expression reduced the number of migrated cells in the Transwell assay. (D) Inhibition of cell migration by siZeb1 was confirmed by the wounding healing assay. Data represent the means ± SD from three independent experiments. (E) Ectopic expression of Zeb1 rescued the inhibition of cell migration associated with the re-expression of miR-144 in A549, 95D and A549-luciferase-miR-144 cells. Transwell assays were performed after the co-transfection of the miR-144 mimic and the plasmid containing Zeb1. (F) Zeb1 re-expression abrogated miR-144-induced up-regulation of E-cadherin and down-regulation of Vimentin. (G–J) Six-week-old SCID Beige female mice were administered with 1 × 10⁶ A549-luciferase cells stably expressing miR-144 or the miR-control via the tail veins. (G) One month later, tumours in the lungs were surveyed with the IVIS Spectrum Imaging System. (H) Body weights of the mice were measured. (I) Immunohistochemistry (IHC) staining of Zeb1 in lung tissues. (J) The expression of Zeb1, E-cadherin and Vimentin was detected by Western blotting.