E2‐mediated EMT by activation of β‐catenin/Snail signalling during the development of ovarian endometriosis

Abstract Endometriosis is an oestrogen‐dependent disease, and epithelial‐mesenchymal transition (EMT) is involved in the process of endometriosis. Whether oestrogen could induce EMT in endometriosis remains largely unknown. Here, we reported that up‐regulated expression of EMT markers in ovarian chocolate cyst is accompanied by high expression 17β‐hydroxysteroid dehydrogenase 1 (17β‐HSD1), and exposure of primary human endometrial epithelial cells to oestradiol conditions could promote EMT occurrence and activate both β‐catenin and Snail signalling. Furthermore, we found nuclear β‐catenin and Snail expression was closely linked in ovarian endometriosis, and β‐catenin knockdown abrogated oestrogen‐induced Snail mediated EMT in vitro. This is due to that β‐catenin/ TCF‐3 could bind to Snail promoter and activate its transcription. These results suggested that β‐catenin signalling functions as the Snail activator and plays a critical role in oestradiol‐induced EMT in endometriosis.

breast cancer cells, oestrogen could promote cell stemness and invasiveness by inducing EMT. 17 Some reports also indicated oestrogen participated in the EMT during the development of adenomyosis through the Slug signalling. 18,19 Whether oestrogen could induce EMT in endometriosis needs to explore.
Decreased E-cadherin expression in epithelial cells together with the increased mesenchymal proteins, such as N-cadherin and vimentin, is a hallmark of EMT. 20,21 The epithelial cells in human endometriosis showed decreased expression of E-cadherin. 22 Snail, promoting differentiation of epithelial cells into mesenchymal cells, functions as the transcriptional repressor of E-cadherin through interacting with the proximal E-boxes of its promoter. 23,24 Wnt/β-catenin signalling, known to play crucial roles in EMT, could also inhibit E-cadherin expression through transcription factors Twist and Slug.
Additionally, the loss of E-cadherin can further promote β-catenin release from cytomembrane and activation. 25,26 Our recent research discovered that oestrogen induced high expression of β-catenin in human endometrial stromal cells of ectopic endometrium, which increased cellular invasion and angiogenesis. 27,28 And the role of oestrogen on the EMT process of human endometrial epithelial cells remains unknown.
In the present research, we discovered the expression of E-cadherin, vimentin, Snail and β-catenin was correlated with oestradiol during EMT process in vitro and in vivo. And β-catenin signalling activating Snail promoter functions a crucial role in oestrogen-facilitated migration and invasion in endometrial epithelial cells. Primary endometrial epithelial cells were obtained from another 32 healthy women. Endometriosis was suspected from the results of either physical examination or ultrasonography. Endometriosis patients were confirmed by laparoscopic surgery. The staging of endometriosis is based on the revised American Society for Reproductive Medicine (rASRM). All of the patients did not receive any hormonal treatment for at least 6 months before specimen collection. All samples were histologically confirmed by the pathologist. In Table 1, the details of all patients are listed.

| Immunohistochemistry
The procedures of IHC were carried out as previously described. 29 The details of antibody can be found in Table S1. Immunohistochemical scores (IHS) and German immunoreactive score were used to analyse data. 30 The immunohistochemical scores were counted by the percentage of immunoreactive cells (PR) and the staining intensity (SI).
There are five levels of PR which range from 0-4 points: 0 point means no staining; 1 point means 1%-10% staining; 2 point means 11%-50% staining; 3 point means 51%-80% staining; and 4 point means 81%-100% staining. The points of staining intensity range from 0-3:0 point means no staining; 1 point means weak staining; 2 point means moderate staining; and 3 point means strong staining. A semiquantitative score was calculated with a range of 0-12. The IHS above 4 point was viewed as positive, and below 4 point was viewed as negative.

| Cell culture and drug treatment
The protocol of primary human endometrial epithelial cells (EECs) culture was confirmed as previously described. 31

| Real-time Quantitative PCR
RNA extractions and real-time quantitative PCR (RT-PCR) analyses were confirmed as previously described. 32 The sequences of primers are listed in Table S2. The △△Ct value was used to calculate the expression levels of target genes.

| Western blot analysis
The extractions of protein and Western blot protocol were confirmed as previously described. 32 The details of antibody can be found in Table S1.

| Transwell migration and invasion assays
Polycarbonate filters with 8 μm pore size membranes (Corning Costar) combined with 24-well culture plates were used for migration (plain) and invasion (matrigel-coated) assay.

| Small interfering RNAs (siRNAs) and transfection
Transfection was used by Lipofectamine 2000 (Invitrogen Life Technologies). A certain proportion of siRNA and Lipofectamine 2000 were mixed in accordance with the instruction manual.
Ishikawa cells were transfected with the mixture, and cell culture medium was changed to DMEM/F-12 supplemented with 5% FBS after 6 hours. Then, the transfected cells were cultivated in 5% CO 2 at 37°C.

| Dual immunofluorescence
EECs were cultured on cover sheet placed inside 6-well plates and stimulated with 10 −6 mol/L E 2 (Sigma-Aldrich) or the control DMSO for 48 hours. EECs were fixed by 4% cold paraformaldehyde for 20 minutes when grown on a cover sheet and fused to 95%-100%.

| Chromatin immunoprecipitation assay
Before the chromatin immunoprecipitation assay (ChIP), cells were dealt with E 2 or DMSO for 48 hours. ChIP was carried out on the basis of the product manual for the ChIP kit (Millipore). Antibody against β-catenin (1:25, #8480, CST) was used. PCR was then performed by using a set of primers designed to amplify the Snail promoter TCF binding sites (TBS). The primer sequences are listed: 5′-ACTATGCCCACCGACCCT-3′ and 5′-CCAGACCTTTCCCACCTT-3.

| Statistical analysis
All values were shown as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Fisher's exact tests were applied for the comparison of dichotomous variables. Unpaired t test or one-way analysis of variance was applied for the comparison of different groups. Spearman's rank correlation coefficient was applied for rank correlation. Statistical significance was set at P-value <.05. All experiments were performed in triplicate.

| The expression of EMT markers and 17β-HSD1 in ovarian endometriosis
IHC analysis of EMT markers in eutopic endometrium (EU) and ovarian chocolate cyst (OC) was performed in tissues from 21 patients with endometriosis and 21 normal control (NC) cases. Representative IHC results are shown in Figure 1A, and the expression profiles of the markers in the NC, EU and OC are shown in Table S3.  Figure 1G-I). The detailed immunostaining score for E-cadherin, vimentin, β-catenin and Snail is shown in Table   S4, respectively. 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) is a predominant enzyme that catalyse the estrone to oestradiol (E 2 ). 33,34 In Figure 1B, we also detected 17β-HSD1 mRNA levels were higher in OC compared with EU and NC.
Collectively, these phenomena indicated that EMT occurs during ovarian endometriosis, which mainly owing to down-reg-

| Oestradiol induces EMT and increases the invasion ability of endometrial epithelial cells
Endometriosis is known as oestrogen-dependent disease. 35,36 Next, we explored the effect of oestradiol on the EMT of primary Immunostaining scores for E-cadherin in EECs were apparently decreased in the 10 −8 mol/L E 2 group (3.333 ± 2.082) compared with the control (8.667 ± 2.082), and most obviously in the 10 −6 mol/L E 2 group (2.667 ± 1.528). During EMT process, epithelial cells obtain the ability of distant migration and metastasis and show morphological changes. 37 Transwell assays showed that E 2 -treated human endometrial epithelial cells were more migratory and invasive compared with control cells (Figure 2B, 2). Also, E 2 (10 −6 mol/L) induced EECs to shift from a cobblestone-like appearance (epithelial phenotype) to an elongated spindle-shaped cell (mesenchymal phenotype).
And cytokeratin expression of the EECs was apparently decreased in the E 2 treated group ( Figure 2D).

| Oestradiol could induce EMT and stimulate activation of β-catenin signalling, which were blocked by ICI
A classic genomic mechanism of oestradiol is to stimulate oestrogen receptors (ERs) to induce their target genes expression. 38 ICI 182.780 (ICI) is an oestrogen receptor antagonist that could abolish the effect of oestradiol on ERs. 39 To address whether the above oestradiol-induced EMT changes are dependent on ER, EECs were treated with DMSO, E 2 without or with ICI (ERs antagonist, 10 −6 mol/L).
RT-PCR revealed that decreased E-cadherin mRNA expression in E 2 group was reversed by ICI ( Figure 3A). Meanwhile, we also observed that E 2 -up-regulated expression of vimentin, β-catenin and Snail

| Relative expression of β-catenin and Snail was evaluated by immunostaining
β-Catenin and Snail could both regulate the expression of the above EMT markers, and we want to make clear their relationship. 40,41 The analysis for nuclear β-catenin and nuclear Snail is shown in  (Table S5). In EU samples, nuclear accumulation of β-catenin was 23.8%, and nuclear Snail protein was detected in 19.0% of tissues (Table   S5). In OC samples, nuclear accumulation of β-catenin and Snail was detected in 61.9% and 57.1% of cases, respectively (Table S5)  that the positive relationship between β-catenin and Snail signalling pathways in the ovarian endometriosis.

| β-Catenin/snail signalling is required for E2induced EMT in vitro
Ishikawa cells are a well-differentiated endometrial adenocarcinoma cell line that retains the endometrial epithelial phenotype and displays apical adhesiveness and expression profiles of different molecules similar to those of the endometrium under the control of oestradiol and progesterone. 42,43 To investigate the relationship between β-catenin and Snail in E 2 -induced EMT, Ishikawa cells were transfected with β-catenin siRNA and Snail siRNA for 24 hours and were incubated with E 2 for another 24 hours. As shown in Figure 4A, the E 2 -induced overexpression of Snail was abrogated by β-catenin siRNA, and β-catenin protein expression was not changed by adding Snail siRNA, which demonstrated that the expression of Snail is relevant to the expression of β-catenin. playing a key role cell growth and development. Wnt signalling activation could lead to β-catenin dephosphorylation and accumulation in the nucleus. 44 Snail, as a transcriptional factor, also mainly locates in nucleus and induces its target genes expression. 45 So their localization in EECs was firstly explored. As shown in Figure 5A, dual immunofluorescence studies revealed intense nuclear colocalization of β-catenin and Snail in EECs after treatment with E 2 for 48 hours. We hypothesized that β-catenin might up-regulate Snail activity through binding to its promoter and activating its transcription. We cloned the human Snail promoter (~2.1 kb) spanning −2084 to +50 from the transcriptional start site into the VCG pGL3-Basic (pGL3B) luciferase vector. As shown in Figure 5C and D E2 treatment F I G U R E 5 E 2 stimulates β-catenin and Snail colocalization in the nucleus and induced Snail transcription through β-catenin. A, Representative confocal microscopy images of EECs immunostained for β-catenin(green) and Snail (red). B, The binding of β-catenin to the Snail proximal promoter in response to E 2 treatment was determined by ChIP assay and quantified by PCR.C-F, Snail promoter activity was measured under the following conditions. C, Ishikawa cells were transfected with different concentrations of β-catenin overexpression plasmid, ranging from 0 to 200 ng, and then incubated with DMSO or E 2 (*P < .05 compared to E 2 (-)+β-catenin(-)group, n = 3). D, Ishikawa cells were transfected with different concentrations of β-catenin siRNA, ranging from 0 to 100 pmol, and then incubated with DMSO or E 2 (*P < .05 compared to E 2 (-)+siβ-cat(-) group, #P < .05 compared to E 2 (+)+siβ-cat(-) group, n = 3). E, β-catenin/TCF-3 overexpression plasmid-transfected Ishikawa cells were cotransfected with different length wild-type Snail promoters (*P < .05 compared to Luc(−2084~+50)+pcDNA3.0 group, #P < .05 compared to Luc(−2084~+50)+β-catenin + TCF3 group, n = 3). F, β-catenin overexpression plasmid-or β-catenin siRNA-transfected Ishikawa cells were cotransfected with wild-type or mutant Snail promoters and then incubated with DMSO or E 2 (*P < .05 compared to WT group, #P < .05 compared to E 2 + MUT group, n = 3). The results are expressed as the mean ± SD. (WT = wild type; MUT = mutant; CON = control; siβ-cat = β-catenin siRNA) significantly enhanced Snail promoter luciferase activity in Ishikawa cells. Furthermore, β-catenin stimulated or inhibited Snail promoter in a dose-dependent manner by transfection of β-catenin expressing vector or siRNA.
Non-phosphorylated β-catenin, a co-transcription factor, translocates into the nucleus and binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Finally, the compound activates the transcription of their target genes. TCF3 is one of the most important members of TCF/LEF family. 44  Wang et al reported that oestrogen could induce EMT and promote tumour growth in breast cancer. 47 Chen et al reported that oestrogen-induced EMT was critical for the development of adenomyosis. 18,19 And, the acquisition of invasiveness is the main characteristic of EMT, in which epithelial cells miss their expression of epithelial markers and gain mesenchymal markers expression through activating some important protein such as NF-κB, Snail and TGF-β1. 48 Furthermore, our results indicated decreased epithelial marker E-cadherin, increased mesenchymal markers vimentin, increased β-catenin and Snail, and key regulators of EMT, in epithelial cells of endometriotic lesions. These EMT marker expressions were closely associated with local 17β-HSD1 expression, which catalyse estrone into oestradiol. So oestradiol may be a crucial primary activator in the process of EMT in ovarian endometriosis.
In order to explore the mechanism how oestradiol promote EMT in ovarian endometriosis, we extracted EECs from human endometrium and assessed our conjecture. Oestradiol could also apparently contribute to these changes in isolated primary epithelial cells in vitro. And this effect was reversed by ICI, further indicating oestrogen receptors involved in EMT process. ICI is the antagonist for oestrogen receptors, ERα and ERβ, and which one is responsible for the process needs more research.
Accumulating evidence shows that Wnt/β-catenin signalling plays a critical role in EMT regulation by down-regulating the expression of E-cadherin, which subsequently leads to β-catenin release and activation. 26 And previous studies have demonstrated that Snail transcription factor also participates in EMT process by repressing the transcription of E-cadherin. 49,50 A recent study suggested that Wnt signalling is related to the increased expression of Snail, mediating EMT in ovarian cancer cells. 51 Furthermore, Wnt signalling has been reported to increases Snail protein levels and activity by inhibit snail phosphorylation and eventually induced epithelial-mesenchymal transition in cancer cells. 52 However, how the Wnt/β-catenin signalling pathway to affect the expression pattern of snail has never been described.
In this study, we first confirmed a positive correlation between as evidenced by promoter activity assays and chromatin immunoprecipitation assays. These data prove a molecular mechanism to interpret a functional link between β-catenin and Snail in ovarian endometriosis.
It is the first to illustrate β-catenin/ Snail signalling in the development of ovarian endometriosis. We get a conclusion that oestradiol promotes β-catenin dephosphorylation and activation, which subsequently leads to the up-regulation of Snail transcription and expression. Overexpressed Snail interacts with the proximal E-boxes of the E-cadherin promoter, causing decreased expression of E-cadherin, which could further promote β-catenin release and activation in a positive feedback manner.
In conclusion, highlighting the crucial role of β-catenin/Snail in E 2 -induced EMT in ovarian endometriosis is the major purpose of our research. These results reveal new highlights on pathophysiology of endometriosis and provided direction to investigate potential therapeutic strategies.

ACK N OWLED G EM ENTS
We would like to thank nursing staff colleagues of Obstetrics and Gynecology of Union Hospital for sample collection.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

AUTH O R CO NTR I B UTI O N S
Yi Liu and Zhibing Zhang conceived and designed the experiments. WenqianXiong, Ling Zhang executed the experiments.
WenqianXiong and Ling Zhang analysed the data. WenqianXiong wrote the article, and all the other authors are contributed to the manuscript. Haitang He, Na Li and Yu Du are responsible for collecting specimens.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.