br Small interfering RNA siRNA
2.5. Small interfering RNA (siRNA) transfection and gene forced-expression
To knockdown endogenous SMAD2, SMAD3, SMAD4, ALK4, SNAIL and SLUG, Trichostatin A (TSA) were transfected for 48 h with 50 nM ONTARGETplus NON-TARGETINGpool siRNA (control) or ONTARGETplus SMARTpool siRNA (Dharmacon, Lafayette, CO) targeting specific genes using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific).
Forced-expression of E-cadherin was achieved by transiently trans-fecting cells with 1 μg empty vector pcDNA3.1 (#V79020, Invitrogen) or vector encoding full-length human E-cadherin (#45769, Addgene, MA) using Lipofectamine 3000 (Invitrogen). Empty vector pCMV-neo (#16440, Addgene) and vector encoding full-length of human SNAIL1 and SNAIL2 were used for SNAIL and SLUG forced-expression.
2.6. Migration assay
Transwell cell culture inserts (8-μm pore size, 24 wells, BD Biosciences, Mississauga, ON) were used for cell migration measure-ment. Cells (5 × 104 cells) in 250 μL medium supplemented with 0.1% FBS were seeded in the insert. 750 μL medium supplemented with 10% FBS was added to the lower chamber as the chemotactic agent. After incubation for 48 h, non-migrating cells were removed with a cotton swab from the upper side of the membrane. Cells that penetrated the membrane were fixed with cold methanol, and cell nuclei were stained with crystal violet (0.5%, Sigma) for 30 min and subsequently washed thoroughly with tap water. Each individual experiment was performed with triplicate inserts. In each insert, five microscopic fields were photographed under an optical microscope and the cell numbers were counted manually.
2.7. Statistical analysis
Results are presented as the mean ± SEM of at least three in-dependent experiments. Multiple group comparisons were analyzed by one-way ANOVA and Tukey's multiple comparison tests using PRISM software (GraphPad Software, CA). Means were considered sig-nificantly diﬀerent if p < 0.05 and are indicated by diﬀerent letters.
3.1. Activin a downregulates E-cadherin expression in human ovarian cancer cells
We first examined the eﬀects of activin A on E-cadherin and N-cadherin expression in human ovarian cancer cells. As shown in Fig. 1A, treatment with diﬀerent concentrations of activin A (1, 10 or 100 ng/
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mL) downregulated E-cadherin mRNA levels in a dose-dependent manner in SKOV3 cells, with the significant eﬀect observed at 10–100 ng/mL. Interestingly, N-cadherin mRNA levels were not altered by treatment with activin A at any of the concentrations tested (Fig. 1B). Western blot analysis confirmed the suppressive eﬀect of activin A on E-cadherin protein levels in SKOV3 cells (Fig. 1C). Simi-larly, treatment with activin A significantly downregulated E-cadherin protein levels in OVISE human ovarian cancer cells (Fig. 1D).
3.2. Activin a suppresses E-cadherin expression through ALK4
ALK4 has been shown to be a major activin type I receptor med-iating the cellular functions of activins. To investigate the involvement of ALK4 in activin A-induced E-cadherin downregulation, we used the specific activin type I receptor (ALK4/5/7) inhibitor SB431542 to block receptor activity. Pretreatment with 10 μM SB431542 for 1 h before activin A treatment completely blocked the suppressive eﬀects of ac-tivin A on E-cadherin mRNA levels in SKOV3 cells (Fig. 2A). Western blot analysis showed similar inhibitory eﬀects of SB431542 on activin A-induced downregulation of E-cadherin protein levels (Fig. 2B). To avoid possible non-specific eﬀects of pharmacological inhibition, we used specific siRNA targeting ALK4 to further confirm the involvement of ALK4 in activin A-induced downregulation of E-cadherin. As shown in Fig. 2C and D, siRNA-mediated ALK4 knockdown abolished the suppressive eﬀects of activin A on E-cadherin mRNA and protein levels in SKOV3 cells. Interestingly, basal E-cadherin mRNA and protein levels were increased following knockdown of ALK4 alone. Taken together, these results indicate that ALK4 is required for the activin A-induced downregulation of E-cadherin in human ovarian cancer cells.