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  • br Fig Activin A downregulates E cadherin

    2020-03-24


    Fig. 1. Activin A downregulates E-cadherin in human ovarian cancer cells.
    Y. Yi, et al. Experimental Cell Research xxx (xxxx) xxx–xxx
    Fig. 2. Activin A downregulates E-cadherin through the ALK4 receptor in SKOV3 cells.
    3.3. Activin a upregulates SNAIL and SLUG expression in human ovarian cancer cells
    E-cadherin expression is regulated by several transcriptional re-pressors including SNAIL [17] and SLUG [18]. Given the suppressive effect of activin A on E-cadherin expression, we next examined the ef-fect of activin A on SNAIL and SLUG expression. As shown in Fig. 3A, treatment with activin A upregulated both SNAIL and SLUG mRNA levels in a time-dependent manner in SKOV3 cells, with significant ef-fects observed after 1–3 h of treatment. Similarly, Western blot analyses confirmed the stimulatory effects of activin A on SNAIL and SLUG protein levels (Fig. 3B). Moreover, the effects of activin A on SNAIL and SLUG mRNA and protein levels were abolished by pretreatment with SB431542 (Fig. 3C and D), suggesting activin type I receptor is required for activin A-induced SNAIL and SLUG expression. 
    3.4. SNAIL and SLUG are required for activin A-induced downregulation of E-cadherin
    To further investigate whether SNAIL and SLUG mediate the sup-pressive effect of activin A on E-cadherin, SKOV3 and OVISE Minocycline HCl were transfected with SNAIL or SLUG siRNA for 48 h prior to activin A treatment for 24 h. RT-qPCR analyses showed that siRNA transfection specifically downregulated both endogenous and activin A-induced SNAIL or SLUG mRNA levels (Fig. 4A–D). In addition, Western blot analyses confirmed the knockdown efficiency of siRNAs specific to SNAIL or SLUG at the protein level in SKOV3 cells (Fig. 4E and F). Importantly, knockdown of either SNAIL or SLUG was able to attenuate the downregulation of E-cadherin mRNA and protein levels induced by activin A (Fig. 4A–F). To further assess the regulatory role of SNAIL and SLUG on E-cadherin expression, we exogenously overexpressed SNAIL or SLUG in SKOV3 cells by transfecting the cells with expression vectors for human SNAIL (SNAIL1) or SLUG (SNAIL2). As shown in Fig. 4G, forced-expression of either SNAIL or SLUG downregulated E-cadherin protein levels in SKOV3 cells. These results demonstrate that SNAIL and
    Y. Yi, et al. Experimental Cell Research xxx (xxxx) xxx–xxx
    Fig. 3. Activin A upregulates SNAIL and SLUG expression in SKOV3 cells. A-B.
    SLUG suppress E-cadherin expression and mediate activin A-induced E-cadherin downregulation in human ovarian cancer cells.
    3.5. SMAD2/3-SMAD4 signaling is required for activin A-induced downregulation of E-cadherin
    To explore the signaling pathways that are involved in activin A-downregulated E-cadherin expression, we first examined the activation of canonical SMAD2/3 signaling. Treatment with 100 ng/mL activin A for 10, 30 and 60 min significantly increased the levels of phosphory-lated SMAD2 and SMAD3 (Fig. 5A). Interestingly, activin A has been shown to stimulate ovarian cancer cell migration through non-cano-nical SMAD signaling (Dean et al., 2017). Thus, we also examined the effects of activin A on PI3K-AKT and MEK-ERK1/2 signaling, which have been reported to be activated by activin A in various cell types (Loomans and Andl 2014). As shown in Fig. 5B, the levels of phos-phorylated AKT and ERK1/2 were not altered by activin A at any of the time points tested in SKOV3 cells.
    To examine the involvement of canonical SMAD signaling, we first used specific siRNA to knockdown common SMAD4 in SKOV3 and OVISE cells and found the suppressive effects of activin A on E-cadherin 
    mRNA and protein levels were abolished by SMAD4 knockdown (Fig. 6A–D). Next, we performed knockdown of SMAD2 and SMAD3 to confirm their individual roles because they have been documented to compensate, cooperate or antagonize each other in various biological contexts [19]. Knockdown of either SMAD2 or SMAD3 blocked the ef-fects of activin A on E-cadherin mRNA and protein levels in both SKOV3 (Fig. 7A–D) and OVISE (Fig. 7E–H) cells, suggesting that SMAD2 and SMAD3 are both required for the suppression of E-cadherin expression by activin A in ovarian cancer cells. Taken together, these results in-dicate that activin A downregulates E-cadherin through activation of the canonical SMAD2/3-SMAD4 signaling pathway in ovarian cancer cells.