Metformin Inhibits the Estrogen-mediated Epithelial-Mesenchymal Transition of Ectopic Endometrial Stromal Cells in Endometriosis

Abstract

Background/aim: Endometriosis is an estrogen-dependent disease characterized by the ectopic implantation and growth of endometrial tissue outside the uterus. Endometrial stromal cells (ESCs) play a crucial role in the pathogenesis of endometriosis. Epithelial-mesenchymal transition (EMT) has recently been described in endometriosis and was induced by estrogen. Metformin has been shown to inhibit EMT in various diseases, but its role in endometriosis remains unclear.

Materials and methods: We collected endometrial tissue samples from patients with endometriosis and healthy controls and isolated primary ESCs. We performed gene expression analysis using the Gene Expression Omnibus (GEO) dataset and validated the results by immunohistochemistry in tissue samples. We also assessed the effects of metformin on the proliferation, migration and invasion of ectopic ESCs (EESCs) by Cell Counting Kit-8 and Transwell migration and invasion assays, respectively. We analyzed the protein expression of EMT-related markers (N-cadherin, vimentin, twist, and snail) and β-catenin by Western blotting and immunohistochemistry.

Results: We found that vimentin was highly expressed in ectopic endometrial tissues compared to normal endometrial tissues. Metformin treatment inhibited the proliferation, migration and invasion of EESCs in a dose-dependent manner. Metformin treatment also downregulated the expression of EMT-related markers and reduced the expression and nuclear translocation of β-catenin in EESCs.

Conclusion: Our results suggest that metformin inhibits estrogen-induced EMT and regulates the expression of β-catenin in EESCs. This study provides new insights into the potential therapeutic role of metformin in endometriosis.

Keywords: Endometriosis; endometrial stromal cells; epithelial-mesenchymal transition; estrogen; metformin; β-catenin.

Figure 1. Ectopic endometrial tissue expresses high levels of EMT-related genes. (A) The analysis of EMT-related gene expression in endometriosis and normal endometrium from GEO database. (B): IHC analysis of vimentin expression in ectopic endometrial tissue and normal endometrial tissue. Scale bars represent 50 μm. *p<0.05.

Figure 2. Metformin inhibits estrogen-induced proliferation, migration, and invasion of EESCs and decreases the expression of EMT-related proteins. (A-C) The cell proliferation ability was detected by the CCK8 assay. (D) Transwell assay results after cells were treated with metformin (5 mM) and/or estrogen (10^–8 M) for 72 h. (E) Eutopic and ectopic EESCs were treated with 17β-Estradiol and/or metformin for 72 h and subjected to immunofluorescence staining for Vimentin. (F) Expression levels of EMT markers (N-Cad, Vimentin, Twist, and Snail) were detected in the EESCs in the different treatment groups. Data are presented as the mean±standard deviation and analyzed using one-way and two-way ANOVA. Tukey’s post hoc test was used to determine significance. *p<0.05, **p<0.01 and ****p<0.0001.

Figure 3. Metformin decreases β-catenin expression and nuclear translocation. (A) mRNA levels of ESR2 were determined in the cells in the different treatment groups. (B) Gene set enrichment analysis in endometriosis of pathway genes most closely related to EMT. (C) mRNA levels of CTNNB1 were determined in the cells in the different treatment groups. (D) Immunofluorescence staining for β-catenin of EESCs in the different treatment groups. (E) Cells were fractionated into the cytosol and nucleus, and the expression levels of total and nuclear β-catenin were detected in the ESCs in the different treatment groups. Scale bars represent 100 μm. Data are presented as the mean±standard deviation and analyzed using one-way ANOVA. Tukey’s post hoc test was used to determine significance. **p<0.01 and ****p<0.0001.