Figure 3

Overexpression of miR-99a did not influence cell morphology and subtly affected the expression of EMT-related proteins and metalloproteinases. (A) Ectopic miR-99a expression had no obvious effects on cell morphology when OEC-M1 and CGHNC9 cells with ectopic miR-99a expression (OEC-M1 miR-99a and CGHNC9 miR-99a) compared with their corresponding controls with lentiviral non-silencing microRNA (OEC-M1 NS and CGHNC9 NS) using phase contrast microscopy with 100× magnification (left panel). Cytoskeleton F-actin proteins were stained with Alex Fluro 488 phalloidin and viewed under fluorescence microscope with 630× magnification (right panel, shown in grey mode). (B) Immunoblot analysis of epithelial (E-cadherin and α-catenin) and mesenchymal (N-cadherin and vimentin) proteins in OEC-M1 NS, OEC-M1 miR-99a, CGHNC9 NS, and CGHNC9 miR-99a cells. (C) Immunoblot analysis of EMT-related transcription factors, including Snail, Slug, and Twist proteins in OEC-M1 NS, OEC-M1 miR-99a, CGHNC9 NS and CGHNC9 miR-99a cells. (D) Immunoblot analysis of pro- and active forms of metalloproteinase 2 (MMP2) and metalloproteinase 9 (MMP9) in OEC-M1 NS, OEC-M1 miR-99a, CGHNC9 NS, and CGHNC9 miR-99a cells. The protein levels were normalized against an internal control β-actin or α-tubulin. Ratios were determined by dividing the normalized protein levels in miR-99a expressing cells with that in control cells. The means of ratio in the graphs were measured by averaging the ratios from independent blots. Bar, SE; *p < 0.05.