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. 2019 Mar 1:14:377-387.
doi: 10.1016/j.omtn.2018.12.011. Epub 2018 Dec 22.

The Attenuation of Trophoblast Invasion Caused by the Downregulation of EZH2 Is Involved in the Pathogenesis of Human Recurrent Miscarriage

Shijian Lv  1 , Na Wang  2 , Hong Lv  1 , Jieqiong Yang  1 , Jianwei Liu  1 , Wei-Ping Li  3 , Cong Zhang  4 , Zi-Jiang Chen  5
Affiliations

The Attenuation of Trophoblast Invasion Caused by the Downregulation of EZH2 Is Involved in the Pathogenesis of Human Recurrent Miscarriage

Shijian Lv et al. Mol Ther Nucleic Acids. .

Abstract

Recurrent miscarriage (RM) is currently defined as two or more losses of a clinically established intrauterine pregnancy. Despite years of research, RM continues to be a clinically frustrating challenge for patients and physicians, and its etiology remains poorly understood. Accumulating evidence has suggested that epigenetic modifications are involved in early embryogenesis, and defects in epigenetic patterning contribute to the development of RM. Here, we studied the role of enhancer of zeste homolog 2 (EZH2) in the pathogenesis of RM and found that the EZH2 expression was significantly decreased in the villi from women with RM compared with that in control villi. EZH2 promoted the invasion of trophoblast cells. Moreover, EZH2 could promote epithelial-mesenchymal transition by epigenetically silencing CDX1. Both chromatin immunoprecipitation (ChIP)-PCR and dual-luciferase report assays demonstrated that EZH2 repressed CDX1 transcription via direct binding to its promoter region and then trimethylating Histone3-Lysine27. Furthermore, we discovered that progesterone, which is used extensively in the treatment of miscarriage and RM, increased the expression of EZH2 via the extracellular signaling-regulated kinase (ERK1/2) pathway. These findings revealed that EZH2 may regulate trophoblast invasion as an epigenetic factor, suggesting that EZH2 might be a potential therapeutic _target for RM.

Keywords: CDX1; EMT; EZH2; H3K27me3; recurrent miscarriages; trophoblast.

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Figures

Figure 1
Figure 1
EZH2 Expression Was Decreased in Villi of Women with RM (A) EZH2 mRNA expression levels in the villous tissue of recurrent miscarriage (RM) patients (n = 21) and healthy controls (HCs) (n = 22) were determined by qPCR. The relative RNA amount was calculated with the 2−ΔΔCt method and normalized to the internal control ACTB. (B) Expression of EZH2 protein relative to β-actin (n = 12 for each group). The data are shown as the mean ± SEM. *p < 0.05; **p < 0.01. (C) Representative images of EZH2 protein expression in the villous tissues from HCs and RM patients after immunohistochemistry. Brown staining represents the _target protein EZH2. HC, healthy pregnancy control; NC, negative control; RM, recurrent miscarriage. Scale bars, 50 μm.
Figure 2
Figure 2
EZH2 Promoted Trophoblast Invasion In Vitro (A) Knockdown of EZH2 by siRNA decreased H3K27me3 and western blot analysis of EZH2 and H3K27me3 expressions in trophoblast cells transfected with siEZH2 or NC after 48 h. H3 and β-actin were used as internal controls relative to H3K27me3 and EZH2, respectively. (B) Statistical analysis results of (A). (C) EZH2 knockdown or an EZH2 inhibitor EPZ005687 significantly decreased trophoblast cell invasion compared with that of the controls. (D) Statistical analysis results of (C). (E) EZH2 overexpression significantly increased trophoblast cell invasion. After the transfection of EZH2 and vector plasmids for 48 h, the overexpression of EZH2 enhanced the invasion of trophoblast cells in comparison with that of the control group. (F) Trophoblast cell proliferation was measured after transient transfection of siEZH2 at the indicated times using a Cell Counting Kit-8 (CCK-8) assay kit. The scale bars represent 200 μm. All data are reported as the mean ± SEM. *p < 0.05; **p < 0.01. NC, scrambled siRNA.
Figure 3
Figure 3
Silencing EZH2 Increased E-Cadherin and CDX1 Expressions in the Trophoblasts (A) The effects of EZH2 knockdown on the expression of 15 invasion suppressor genes in trophoblast cells detected by qPCR. (B) Knockdown of EZH2 increased the expression of E-cadherin and reduced N-cadherin levels by western blot analysis. (C) Statistical analysis of the western blot results in (B). (D) Inhibition of EZH2 enzymatic activity by GSK343 and EPZ005687 increased the expression of CDX1 mRNA, as detected by qPCR. (E) Knockdown of EZH2 increased the expression of CDX1 by western blot analysis. (F) CDX1 mRNA levels in the villous tissue of RM patients (n = 21) and HCs (n = 22) were determined by qPCR. (G) The CDX1 mRNA levels in the villous tissues were measured using qPCR and correlated with the EZH2 mRNA level in the same cohort (n = 40). All data are reported as the mean ± SEM. *p < 0.05; **p < 0.01. Ctrl, DMSO control; NC, scrambled siRNA.
Figure 4
Figure 4
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.
Figure 5
Figure 5
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.
Figure 6
Figure 6
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.
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