Figure 2

ERβ1 induces the expression of E-cadherin by up-regulating members of the microRNA 200 family and repressing the expression of ZEB-1 and SIP-1. (A) E-cadherin mRNA levels in control (Lenti), ERα- and ERβ1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. The graph shows the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (B) Left panel: protein levels of EMT markers in control (Lenti), ERα- and ERβ1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. Right panel: E- and N-cadherin protein levels in control (Lenti) and ERβ1-expressing Hs578T cells. (C) ZEB-1 and SIP-1 protein levels in control (Lenti), ERα- and ERβ1-expressing MDA-MB-231 cells (upper panel) and in control and ERβ1-expressing Hs578T cells (lower panel). (D) Control, ERα- and ERβ1-expressing MDA-MB-231 cells were analyzed for miR-200a, miR-200b and miR-429 expression by qPCR. The graphs show data as fold change compared with the untreated Lenti cells (mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated). (E) MDA-MB-231 cells were transiently transfected with control or ERβ siRNA (3#) and analyzed for miR-200a, miR-200b and miR-429 expression by qPCR. The graphs show the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (F) ERβ1-expressing MDA-MB-231 cells were transfected with inhibitors of miR-200a, miR-200b and miR-429 or a negative control inhibitor. Cells were photographed and analyzed for E-cadherin expression by qPCR (scale bars, 50 μm). The level of functional knockdown of miR-200a-b-429 was examined by a miR-200a-b-429-regulated reporter assay. The graphs show the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated.