FIGURE 3. C3 expression negatively correlates with E-cadherin expression in vitro and in vivo.

(A) After stable transduction of lentivirus carrying C3 cDNA (pC3) into low-C3 HeyA8, C3 expression (A, upper panels) or E-cadherin expression (A, lower panels) was detected by immunofluorescence microscopy, (B) by Western-blotting or (C) by quantitative RT-PCR, respectively. HeyA8 cells stably transduced by pC3 showed increase in C3 expression (B, left panel, n=3, p=0.002) and reduction in E-cadherin expression (B, right panel, n=3, p=0.008). (D) The functional effect of C3 overexpression in HeyA8 cells was studied by in vitro migration and invasion assays, using HeyA8 cells stably transfected with pC3. (E) HeyA8 cells overexpressing C3 showed 2.3 folds increase in migration ability (E, left panel, n=15, p=0.001) and 1.8 folds increase in invasion ability (E, right panel, n=15, p=0.001) as compared with those transfected with an empty control plasmid. (F) Ovarian (SKOV3 and OVCAR5) and endometrial (Ishikawa and KLE) cancer cell lines were transfected with C3 siRNA. C3 mRNA level was decreased by 55-75% in ovarian cancer cells and by 52-60% in endometrial cancer cells (n=3, p=0.001) as compared to scrambled siRNA transfected cells. (G) After C3 knockdown E-cadherin mRNA increased by 2.5-19 folds in ovarian cancer cells and by 1.3-2 folds in endometrial cancer cells (n=3, p=0.001). Cell lines transfected with scrambled siRNA served as controls. (H) After transient transfection of C3 siRNA into C3-high SKOV3, C3 and E-cadherin protein expression was determined by Western- and immunoblotting. Scrambled siRNA was used as a control. (I) After stable transfection of HeyA8 cells with plasmid containing TWIST1 cDNA (pTWIST1) and transfection of SKOV3 cells with TWIST1 siRNA, protein expression of TWIST1, C3, and E-cadherin was determined by Western-blotting. (J) Expression of E-cadherin and C3 in sections of ovarian tumors resected from tumor-bearing mice were studied using immunofluorescence microscopy. Tumors induced by ID8-VEGF cells transduced by C3 shRNA showed reduced expression of C3 and increased expression of E-cadherin as compared to tumors induced by ID8-VEGF cells transduced by scrambled shRNA.