Oestrogen induces epithelial‐mesenchymal transition in endometriosis via circ_0004712/miR‐148a‐3p sponge function

Abstract Endometriosis is a common, chronic gynaecologic disease affecting up to 10% of women in their reproductive age and leading to pain and infertility. Oestrogen (E2)‐induced epithelial‐mesenchymal transition (EMT) process has been considered as a key factor of endometriosis development. Recently, the dysregulated circular RNAs (circRNAs) have been discovered in endometriosis tissues. However, the molecular mechanism of circRNAs on the E2‐induced EMT process in endometriosis is still unknown. Here, we demonstrated that circ_0004712 up‐regulated by E2 treatment in endometrial epithelial cells. Knock‐down the expression of circ_0004712 significantly suppressed E2‐induced cell migration activity. Meanwhile, we identified miR‐148a‐3p as a potential target miRNA of circ_0004712. Inhibited the expression of miR‐148a‐3p could recovered the effect of circ_0004712 knock‐down in E2‐treated endometrial epithelial. Furthermore, Western blot assay showed that E2 treatment could increase the expression and activity of β‐catenin, snail and N‐cadherin and reduce the expression of E‐cadherin. The expression and activity of β‐catenin pathway were recovered by circ_0004712 knock‐down or miR‐148a‐3p overexpression. Altogether, the results demonstrate that circ_0004712/miR‐148a‐3p plays an important role in E2‐induced EMT process in the development of endometriosis, and the molecular mechanism may be associated with the β‐catenin pathway. This work highlighted the importance of circRNAs in the development of endometriosis and provide a new biomarker for diagnosis and therapies.


| INTRODUC TI ON
Endometriosis is a chronic disease in reproductive age women, which characterized as the ectopic growth of endometrial-like tissues outside the uterine cavity, resulted in chronic pelvic pain and infertility. 1 Due to the lack of effective biomarkers and treatment options, this chronic disease severely impairs patients' quality of life and imposes a lot of economic burden. 2 Therefore, exploring the effective markers for identifying the mechanisms related to exact pathogenesis of endometriosis is very important for improving the diagnosis and therapies.
Previous studies have demonstrated that abnormal oestrogen (E 2 ) secretion is associated with the pathogenesis of endometriosis. [3][4][5] Using aromatase inhibitor to suppress the activity of aromatase, which is a key enzyme to produce oestrogen, has been demonstrated to be an effective treatment for this disease. 6 However, the molecular mechanism of E 2 on the development of endometriosis has not been fully clarified. Recent study found that E 2 could induce epithelial-mesenchymal transition (EMT) process during the development of endometriosis. 7 Epithelial-mesenchymal transition is a biological process that promotes the polarized epithelial cell to process a mesenchymal phenotype, in which the epithelial cell obtain the ability of migration, invasion and re-localization. 8 The abnormal activation of EMT programs is considered as a key factor in tumour invasiveness and metastasis, and other pathological processes. 9 Many studies revealed that an enhanced EMT-like process was occurred in the establishment of ovarian endometriosis, [10][11][12] but the molecular mechanism of oestrogen on inducing EMT process of endometrial epithelial cells still unknown. Circular RNAs (circRNAs) are a number of non-coding RNAs, which have been considered as a gene regulator at transcriptional or post-transcriptional level. 13 Because of its structure, it can stable expressed in cells and often act as miRNAs inhibitors by sponging miRNAs. Therefore, circRNAs could be potential biological regulators for recognizing the molecular mechanisms of disease and finding effective diagnostic biomarkers or therapeutic targets.

| Western blot assay
Treated cells were lysed in radio-immunoprecipitation assay (RIPA) antibodies at room temperature for 1 hour. After that, the band was exposed by ECL system (Thermo Fisher Scientific, USA) and analysed by Quantity One software (Bio-Rad, San Diego, CA, USA).

F I G U R E 3
Circ_0004712 directly suppresses the expression of miR-148a-3p. A, The target sites of circ_0004712 and miR-148a-3p. B, The expression of miR-148a-3p in E 2 -treated Ishikawa and End1/E6E7 cells was tested by qPCR. C, The expression of miR-148a-3p in E 2treated orE 2 -untreated Ishikawa and End1/E6E7 cells after for si-circ or NC transfection was tested by qPCR. D, Relative luciferase activity in Ishikawa and End1/E6E7 cells transfected with miR-NC or miR-mimics and circ-wild-type (wt) or circ-mutation (mut), respectively. Results show as mean ± SD. **Means compared with control group, P < 0.05. ##Means compared with E 2 -treated group, P < 0.05

F I G U R E 4
Effect of circ_0004712/miR-148a-3p on cell migration. A, Cell migration of E 2 -treated orE 2 -untreated ishikawa and End1/ E6E7 cells after NC, si-circ and/or miR mimics transfection was analysed by transwell assay, respectively. B, Protein expression of E-cadherin and N-cadherin in E 2 -treated orE 2 -untreated ishikawa and End1/E6E7 cells after NC, si-circ and/or miR mimics transfection was analysed by Western blot assay. C, Protein expression of β-catenin, p-β-catenin and Snail in E 2 -treated or E 2 -untreated ishikawa and End1/E6E7 cells after NC, si-circ and/or miR mimics transfection was analysed by Western blot assay. Results show as mean ± SD. **Means compared with control group, P < 0.05. ##Means compared with E 2 -treated group, P < 0.05

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc). Unpaired two-sided t test was performed for analysing the between-group differences. The data were expressed as mean ± SD P values of less than 0.05 were considered statistically significant. All experiments for statistical analyses were repeated for triple times.

| circ_0004712 up-regulated by E 2 treating in endometrial epithelial cells
To explore the relationship between E 2 -induced EMT and circ_0004712 expression, we preformed different concentration of was significantly up-regulated as a dose-dependent manner in both of Ishikawa and End1/E6E7 cells ( Figure 1A). Transwell assay showed E 2 treatment markedly induced the migration activity as a dose-dependent manner ( Figure 1B).

| circ_0004712 promotes cell migration in endometrial epithelial cells after E 2 treatment
To study the potential biological effects of circ_0004712 on E 2-induced EMT process, we synthesized a specific interference RNA oligonucleotide (si-circ) to knock down endogenous expression of circ_0004712 in endometrial cells after E 2 (10 -8 mol/L) treatment. qPCR results showed the expression of circ_0004712 were significantly decreased by si-circ transfection in E 2 -treated Ishikawa and End1/E6E7 cells ( Figure 2A). Transwell assay showed that knock-down circ_0004712 significantly suppressed E 2 -induced cell migration activity ( Figure 2B).
Overall, the results suggested that high expression of circ_00471 was related with E 2 -induced EMT progress.

| circ_0004712 targeted to miR-148a-3p
Many evidences revealed that circRNAs could sponge miRNAs to regulate the expression of the target genes. Thus, we predicted the potential target miRNAs of circ_0004712 by bioinformatics. MiR-148a-3p have a binding site to circ_0004712 ( Figure 3A), and the expression of miR-148a-3p was significantly down-regulated after E 2 treatment as a dose-dependent manner ( Figure 3B). Furthermore, the expression of miR-148a-3p was significantly reduced by circ-000471 knocking down ( Figure 3C). The dual-luciferase assay further demonstrated the directly binding site between circ_0004712 and miR-148a-3p ( Figure 3D). Compared with control group, miR-148a-3p significantly reduced the relative luciferase activity, and no significant effect on luciferase activity when co-transfected with mutated plasmids. These results suggested that circ_0004712 could bind to miR-148a-3p to modulate the E 2 -induced EMT process in endometrial epithelial cells.

| circ_0004712/miR-148a-3p regulated EMT process via β-catenin pathway
To further study the molecular mechanism of circ_0004712/miR-148a-3p on E 2 -induced EMT process, cells were treated with E 2 and transfected with si-circ and/or miR-148a-3p mimics, respectively. Transwell assay revealed that inhibiting the expression of circ_0004712 or increasing the expression of miR-148a-3p could significantly suppress the migration capacity in E 2 -treated endometrial epithelial cells ( Figure 4A). Furthermore, the effect of circ_0004712 knock-down on cell migration could be recovered by miR-148a-3p inhibitor. Western blot assay showed that E 2 treatment could increase the expression and activity of β-catenin pathway and N-cadherin and reduce the expression of E-cadherin ( Figure 4B). The expression and activity of β-catenin pathway were recovered by si-circ or miRNA mimics transfecting ( Figure 4C). These results suggested that circ_0004712 and miR-148a-3p had opposite effects on EMT process, and circ_0004712 could suppress the expression of miR-148a-3p. The molecular mechanism of circ_0004712/miR-148a-3p on the E 2 -induced EMT process may be associated with the β-catenin pathway.
In these three genes, SOS2 (Son of sevenless 2) was associated with EMT process. 15  treatment as a dose-dependent manner ( Figure 5C). The dual-luciferase assay further demonstrated the directly binding site between miR-148a-3p and SOS2 ( Figure 5B). Transwell assay revealed that knock-down the expression of SOS2 could significantly suppress the migration capacity in E 2 -treated endometrial epithelial cells and recovered the effect of miR-148a-3p inhibitor on the cell migration ( Figure 5D). To further explore the effect of miR-148a-3p/SOS2 on the regulating the EMT process, we tested the expression of N-cadherin and E-cadherin. The results of Western blot showed that the expression of N-cadherin and E-cadherin were recovered by si-SOS2 transfecting ( Figure 5E). These results suggested that SOS2 was the target of miR-148a-3p involved in the circ_0004712mediated E 2 -induced EMT process in endometrial cells.

| D ISCUSS I ON
Endometriosis is a common, chronic gynaecologic disease affecting women in their reproductive age and leading to pain and infertility. 16 Identifying the accrue biomarkers and specific therapeutic targets for the early diagnosis and treatment is immediately needed.
Increasing evidences have highlighted circRNAs as important molecular biomarkers and gene regulators. Recently, the expression profile of circRNAs in endometriosis has been reported, 14,17 and these studies indicated that dysregulated circRNAs might be potential molecular targets for clinical diagnosis and therapy. However, the molecular mechanism of these circRNAs is still unclear. Here, we studied the roles of an up-regulated circRNAs (circ_0004712) on the E 2 -induced EMT process in endometrial cells.
Epithelial-mesenchymal transition in endometrial cells is important for endometriosis establishment. 11 As a reproductive tissue, endometrial cells located in high level of oestrogen. Thus, endometriosis has been considered as an oestrogen-dependent disease. 18 Many studies demonstrated that oestrogen could induce in many cancers and adenomyosis. [19][20][21] Recent studies also suggested that oestrogen promotes EMT process during the development of endometriosis. 7 In our study, we demonstrated SOS2 was a target gene of miR-148a-3p involved in the E 2 -induced EMT process. SOS2 is a number of SOS (Son of sevenless) family, which is the Ras-specific guanine nucleotide-exchange factor. SOS2 enhances the activation of Ras and regulates many cellular process, such as cell proliferation and migration. 27 Previous study suggested that inhibiting the expression of SOS2 could suppress the cell proliferation and EMT process by down-regulating MAPK/Erk pathway in non-small-cell lung cancer cells. 15 In our study, we also found the expression of SOS2 increased in endometrial cells after E 2 treatment and demonstrated the binding ship between SOS2 and miR-148a-3p. Furthermore, inhibiting the expression of SOS2 could suppress cell migration after E 2 treatment. Thus, SOS2 acted as the target gene of miR-148a-3p involved in the E 2 -induced EMT process in endometrial cells.
The up-regulation of N-cadherin and Vimentin and down-regulation of E-cadherin are the most influential biomarkers of EMT. 8 E 2 have been demonstrated to reduce the expression of E-cadherin 28 and activate β-catenin signalling to promote EMT process. 29 Our results also revealed E 2 could activate β-catenin signalling.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

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