Figure 4. MiR-199a-3p inhibits cell proliferation, adhesion and invasion through the suppression of c-Met expression.

(A) Western blotting. SKOV3ip1 cells were transfected with pre-miR-199a-3p or negative control miR for 24 hours. Cell lysate were collected before and after HGF stimulation (40 ng/mL, 10 minutes). Immunoblotting was performed with antibodies against phosphorylated c-Met (p-c-Met), c-Met, phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated Akt (p-Akt), or β–actin. (B) Colony formation assay. Ovarian cancer cells (1 × 10⁵ cells; SKOV3ip1 and RMUG-S), cotransfected with miRNA (pre-miR-199a-3p or negative control miRNA) and an pIRES2-EGFP vector (containing MET or the empty control vector), were suspended in 0.33% agarose. After 10 days, colonies with a diameter greater than 100 μm were counted under a microscope at 40× magnification. Representative images are shown. Bar represents 400 μm. Data represent mean ± SEM; n = 10. (C) In vitro adhesion assay. SKOV3ip1 (5 × 10⁴) cells were cotransfected with miRNA (pre-miR-199a-3p or negative control miR) and the pIRES2-EGFP vector (containing MET or the empty control vector). The cells were plated onto 50 μg/mL fibronectin- or collagen type 1–coated 96-well plates. After being incubated for 75 minutes at 37°C, the plates were washed to discard nonadherent cells, and the number of adherent cells was counted. Representative images are shown. Bar represents 100 μm. Data represent mean ± SEM; n = 6. (D) Matrigel invasion assay. Ovarian cancer cells were cotransfected with miRNA (pre-miR-199a-3p or negative control miR) and an pIRES2-EGFP vector (containing MET or the empty control vector). The cells (5 × 10⁴) were plated in serum-free medium in modified Boyden chamber system that had been coated with 25 μg Matrigel and allowed to invade the lower chamber, which contained Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, for 24 hours. Noninvading cells were removed with a cotton swab, and invading cells on the underside of the filter were counted. Representative images are shown. Bar represents 200 μm. Data represent mean ± SEM; n = 10. (E) Western blotting. SKOV3ip1 cells were cotransfected with miRNA (pre-miR-199a-3p or negative control miR) and an pIRES2-EGFP vector (containing MET, or the empty control vector) for 4 hours. Cell lysates were then obtained, and immunoblotting was performed with antibodies against c-Met, various integrins (α1, α2, α5, αV, and β1), CD44, matrix metalloproteinase-2 (MMP-2), and β-actin (left). Densitometric ratios of the expression of c-Met, integrin β1, CD44, and MMP-2 (right). ^*P < 0.05; ^***P < 0.001; n.s., not significant. Densitometry ratios in each western blotting are shown below each blot.