Fig 2: Progesterone Upregulated EZH2 Expression through the ERK1/2 Pathway(A and B) After treatment with progesterone for 24 h, EZH2 protein was upregulated, whereas CDX1 expression was reduced, as determined by western blotting. (B) is the statistical result of (A). (C and D) p-ERK1/2 protein levels, but not p-AKT levels, increased the following treatment with progesterone for 24 h. (D) is the statistical result of (C). (E and F) Inhibition of the ERK1/2 signaling pathway using U0126 for 24 h attenuated the protein level of EZH2; meanwhile, the CDX1 level was upregulated. (F) is the statistical result of (E). (G) The schematic roles of EZH2 in regulating the invasion behavior of trophoblasts. All data are shown as the mean ± SEM. *p < 0.05; **p < 0.01. P4, progesterone.

Fig 3: EZH2 Regulated CDX1 Expression via Direct Binding to Its Promoter(A) Schematic diagram of the CDX1 promoter region showing the location of CpG islands, the putative EZH2-binding region, and the spanning region of primers used for ChIP assay and PCR analysis. (B–E) Knockdown of EZH2 decreased the binding of both EZH2 and H3K27me3 on the promoter of CDX1. Immuno-precipitated DNA fragments were analyzed by qPCR with specific primer sets. Chromatin obtained from trophoblast cells was immune-precipitated using antibodies against EZH2, histone H3, and IgG. Each ChIP experiment was repeated three times. MYOD1 (B) was positive control; Primer1 (C) and Primer2 (D) were for EZH2; and GAPDH (E) was negative control. (F) Overexpression of EZH2 decreased the transcriptional activity of CDX1 promoter, as detected by dual-luciferase report assays. Approximately 2 kb upstream of the transcriptional start site (TSS) on the promoter of CDX1 was cloned into pGL3 vector. Three plasmids were cotransfected into trophoblast cells for 48 h. Fluorescence measurement was performed using dual-luciferase report assays. RLuc was used for the internal control. All data are reported as the mean ± SEM. *p < 0.05; **p < 0.01. FLuc, firefly luciferase; GAPDH, negative control; MYOD1, positive control; RLuc, Renilla luciferase.

Fig 4: CDX1 Overexpression Attenuated EMT in Trophoblasts(A) Overexpression of CDX1 increased the expression of E-cadherin and reduced the N-cadherin level by western blot analysis. (B) Statistical analysis of the western blot results in (A). (C and D) CDX1 overexpression significantly decreased trophoblast cell invasion. After transfection for 48 h, the overexpression of CDX1 attenuated the invasion of trophoblast cells in comparison with that of the control group. CDX1 overexpression also enhanced EZH2 siRNA-mediated deficient cell invasion. (D) is the statistical result of (C). The scale bar represents 200 µm. All data are reported as the mean ± SEM. *p < 0.05; **p < 0.01. EMT, epithelial-mesenchymal transition.