RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial‐mesenchymal transition and proliferation in endometriosis

Abstract Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive‐age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial‐mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial‐mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up‐regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up‐regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.


| INTRODUC TI ON
Endometriosis is a complex gynaecological disease, which is identified as the presence of endometrium-like tissues outside the uterine cavity and impacts 6%-10% female of reproductive age. 1 Endometriosis is considered as an oestrogen-dependent and inflammatory disorder that is usually accompanied by dysmenorrhoea, chronic pelvic pain, dyspareunia and infertility. [2][3][4] Furthermore, the recurrence rate of endometriosis is pretty high that severely influences patients' health and quality of life. 5,6 Oestrogen fuels the cell proliferation, promotes inflammation and inhibits apoptosis, and it is regarded as one of the factors that promotes the development of endometriosis. [7][8][9] Studies found that aromatase and oestrogen kept high level in endometriosis tissues and survived the cells in ectopic locations. 10,11 Furthermore, other studies have reported the aberrant level of EMT is present in endometriosis and oestrogen induces cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells. 12 Zhang et al found that oestradiol promoted EMT via MALAT1/miR200s sponge function. 13 Therefore, the development of endometriosis is affected by oestrogen in many ways and numerous signalling pathways are involved in the process, but the in-depth mechanism deserves further exploration. On the other hand, it was reported that uterine receptivity defects were triggered by ERα down-regulation in the mid-secretory phase. 14 Consequently, ERα is extraordinary significant for the endometrium to perform normal physiological functions. Other study found that the polymorphisms of ERα are useful markers for predicting endometriosis susceptibility. 15 But there is not adequately revealed relationship and mechanism of ERα and oestrogen in development in endometriosis.
Rho family serve as molecular switches involved in the regulation of diverse cellular functions including cytoskeletal organization, growth and differentiation. 16 It is reported that RhoA, RhoC and ROCK1 were significantly higher in ectopic endometrial cells compared with the eutopic endometrial cells in endometriosis, which involved with promoted migration of endometrial cells of endometriosis. 17 Yuge et al claimed that the increased expression of RhoA, ROCK1 and ROCK2 proteins of endometriotic stromal cells may be involved in the pathogenesis of endometriosis-associated fibrosis. 18 Other report considered the increased RhoC expression detected in endometriotic lesions might be among the key elements involved in the origin and the maintenance of endometriosis. 19 However, all of these researches lack comprehensive cognition and mechanism exploration about the Rho family in endometriosis, and the factors affecting the function of Rho family are little known. Further study on the role and mechanism of Rho family in endometriosis may have profound significance as a treatment for endometriosis.
In the present study, we aimed to investigate the expression of Rho family and its function on EMT and proliferation in endometriosis. Further researches were employed to assess the association between Rho family and oestrogen pathway and the molecular mechanism. We also sought to examine whether all the results presented in endometriotic mouse model.   (Table 1). The normal patients without history of endometriosis were confirmed by ultrasonography. All patients had regular menstrual cycles and were without hormone treatment for more 3 months before the surgery. The endometriotic lesions were collected after surgery and examined by pathology. The use of samples gated permission from the ethics committee of hospital, and all patients signed the informed consent.

| Patients and primary cell culture
The separation of primary cells from eutopic endometrial tissues was executed as previous report. 20 Briefly, the endometrial tissues were cut into small pieces and then dissociated by Collagenase Ⅳ and deoxyribonuclease for an hour. Then, the

| Drug treatment
The separated human eutopic EECs were digested by trypsin and

| Database source
The gene expression of Rho/ROCK pathway was analysed referred to the GEO database (https://www.ncbi.nlm.nih.gov/geo/). GSE25628 was selected as target gene expression profile, which was based on the GPL571 platform ([HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array). All the data were freely available online, and this study did not involve any experiment on humans or animals performed with any of the authors.

| Western blot
The protein samples from eutopic EECs or tissues were exacted by Blots were scanned and analysed using ChemiDoc MP Imaging System (Bio-Rad; California, USA). ImageJ software (1.52u version) was used to calculate to greyscale value of immunoblot.

| Transwell assays
To evaluate the function of RhoA, transwell assays were performed to detect the ability of cell migration.

| EdU labelling assays
EdU labelling assays were performed as described in instructions of

| Immunochemistry (IHC)
The human and mouse tissue samples were fixed by formalin solution and embedded by wax. These tissue waxes were cut into slides, which were then routinely dewaxed, dehydrated and antigen-repaired using pressure cooker boiling by 10 mmol/L sodium citrate buffer. The slides were incubated with the primary anti- HE staining was similar to immunochemistry but without usage of antibodies.

| Endometriotic Mouse Model assay
The endometriotic mouse model was established referred to the previously described method with minor modifications. 21,22 Female BALB/c mice (6-8 weeks old and 15-20 g) were purchased and raised in Animal Research Laboratory of Xiamen University.
The operations on mice were in accordance with animal ethics. All the mice were ovariectomized to eliminate effect of endogenous oestrogen. The mice were divided into donor group (10 mice), control group (Ctrl group, 10 mice) endometriotic groups (EM group, 10 mice) and oestrogen-treated endometriotic groups (E2/EM group, 10 mice).
The donor mice were killed, and the uterine horns and myometrium were peeled off to obtain the endometrium-rich fragments.
And then, the endometria were cut into pieces and equally injected into the abdominal cavity of EM and E2/EM group mice. The mice of E2/EM group were abdominal injected 1mL PBS containing oestrogen (0.6 g/L), and EM group was injected with 1 mL PBS every day. The mice were monitored for endometriotic lesion growth for one month, and uterine and ectopic lesions were collected, respectively.

| Statistical analysis
All data were analysed by GraphPad Prism 7 software (San Diego, CA, USA). Comparison of data was performed with Student's t test or one-way analysis of variance (ANOVA). P value < 0.05 was deemed to be statistically significant.

| The expression of RhoA and ROCK1/2 was significantly increased in endometriosis
To explore the expression of Rho/ROCK pathway in endometriosis, GEO database was queried to assess the expression of RhoA, RhoB, RhoC, ROCK1 and ROCK2. As shown in Figure 1A and Figure Figure 1E). And RhoA, ROCK1 and ROCK2 were remarkably increased in the endometriotic lesions of mice ( Figure 1F).
Thus, we concluded that RhoA/ROCK pathway was intensively promoted in endometriosis.

| RhoA modulated the EMT and proliferation of eutopic EECs
To assess the effect of RhoA/ROCK pathway in endometriosis,

RhoA-overexpression (RhoAoe) and RhoA-knockdown (siRhoA)
human eutopic EECs were constructed ( Figure S1C). The RhoA was significantly increased in RhoAoe cells and that was enormously reduced in siRhoA cells (Figure 2A,B). As shown in Figure 2C, the enhancement of RhoA attenuated E-cadherin and raised vimentin and N-cadherin, and the reduction of RhoA exhibited opposite effect.
Moreover, the migration of RhoAoe cells was remarkably promoted, and that of siRhoA cells was obviously decreased ( Figure 2D,E). In addition, the results of Figure 2F indicated that RhoA obviously advanced the proliferation. And the augment of RhoA enhanced the EdU-positive cells, which presented that RhoA up-regulated the cell division to elevate the cell proliferation ( Figure 2G). Therefore, the

| RhoA/ROCK pathway mediated the effect of oestrogen on EMT and proliferation
Endometriosis is considered as an oestrogen-dependent disorder, and we hypothesized that oestrogen might be an upstream regulator of RhoA/ROCK pathway. The results of Figure 3A implied that oestrogen remarkably promoted the transition of activated RhoA and the protein expression of RhoA and ROCK1/2. Further explorations found that the effect of oestrogen promoting EMT was markedly strengthened by enhancement of RhoA and lessened by loss of RhoA ( Figure 3B and Figure S2A). As in Figure 3C, the effect of oestrogen regulating the proliferation of eutopic EECs was affected by the expression of RhoA. Consequently, these results suggested that RhoA/ROCK was responsible for the regulation of oestrogen on advancing EMT and proliferation in endometriosis.

| RhoA/ROCK pathway was modulated by ERα, which was involved with regulation of oestrogenic function
ERα, as one of important oestrogen receptors, mediates the effect and function of oestrogen. To further analyse the function of ERα on oestrogen signalling in endometriosis, the ERα-overexpression (ERαoe) and ERα-knockdown (siERα) cells were established by lentivirus engineering ( Figure S2B). And the expression of ERα was confirmed by Western blot ( Figure 3D). As Figure 3E and Figure Figure 3H and Figure S2D). Moreover, modulating the expression of RhoA could influence the regulation of ERα on proliferation ( Figure 3I). In conclusion, ERα might partially mediate the regulation of oestrogen and modulated RhoA/ROCK pathway to enhance the EMT level and proliferation.

| Oestrogen promoted the development of endometriosis in mouse model
To verify the function of oestrogen and RhoA/ROCK pathway on the development of endometriosis in vivo, endometriosis mouse model was established. As shown in Figure 5A, the endometriotic lesions of the E2/EM group were much bigger than PBS-injected EM group, and the weight of lesion from E2/EM group was much heavier than the control EM group. Furthermore, the expression of RhoA, ROCK1 and ROCK2 mRNA was enhanced in E2/EM endometriotic lesions ( Figure 5B). And the RhoA and phosphorylated ERK were remarkably increased in E2-treated group ( Figure 5C). Thus, oestrogen promoted the development of endometriosis in mouse model and up-regulated RhoA and phosphorylated ERK.

| Discussions
In this research, we found that the protein expression of Rho/ROCK signalling pathway was abnormally changed in endometriosis and RhoA and ROCK1/2 were remarkably increased in eutopic and ectopic endometria. The mobility, EMT and proliferation of human Oestrogen significantly promotes inflammation, EMT and proliferation, which aggressively promoted endometriosis, which involved with multiple signal pathway such as MAPK pathway. Thus, comprehensive and thorough study on the mechanism of oestrogen is instructive to the search for the treatment of endometriosis.
ERα, as one of the most important oestrogen receptors, mediates the function of oestrogen. 26 It was reported that gonadotropin-releasing hormone agonist (GnRH-a) treatment especially decreased ERα mRNA levels in endometriotic cysts, which suggests reduction of ERα is an important step in the progression of endometriosis therapy by anti-oestrogen treatment. 27 Other research revealed that ERα gene polymorphism and recurrence of endometriosis were statistically significantly correlated. 28 In this study, we found ERα promoted the mobility, EMT and proliferation as a partial mediator of the oestrogen. However, the expression of ERα was decreased in endometriosis tissues comparing to eutopic and normal endometria in other reports. 29,30 And this interesting phenomenon highlights the complexity and particularity of endometriosis that is not as extreme like malignant tumours. Briefly, ERα partially mediates the effect of oestrogen promoting EMT and proliferation in endometriosis.
It is reported that the level of phosphorylated ERK was significantly higher in endometriotic lesions than in endometrial tissues. 31,32 And the activated ERK regulated over 160 proteins, which involved with the cell proliferation, apoptosis, angiogenesis and migration in endometriosis. 33  Optical: optical microscope. Data are expressed as mean ± SEM. NS = no significant difference, *P < 0.05, **P < 0.001, ***P < 0.0001 vs control group transcriptional activities in cells. 22,36 38 Another study found that ERα enhanced ROCK2/MMP2/9 pathway inducing cell migration and angiogenesis effects in endothelial cell. 39 These findings suggested that there may be involved many pathways in the process of oestrogen regulating RhoA/ROCK pathway. The expression of RhoA and ROCK1/2 was up-regulated by oestrogen, ERα and ERK. Furthermore, the activity of RhoA was increased accompanied by the enhancement of total RhoA, which implied that increasement of total RhoA might induce the activation of RhoA. However, whether the detailed mechanism of RhoA activation was involved with other factors needs further study.
Consequently, our present study concluded that oestrogen/ERα/ERK signal up-regulated the activation of RhoA and expression of RhoA and ROCK1/2 triggering the enhancement of EMT and proliferation.
In conclusion, this study specifically revealed RhoA/ROCK pathway was up-regulated by oestrogen/ERα/ERK signalling pathway promoting EMT and proliferation in endometriosis. Thus, RhoA/ ROCK pathway might be an attractive molecular target in anti-oestrogen treatment of endometriosis. These results provide a scientific basis for potential therapeutic strategies to treat endometriosis via RhoA/ROCK pathway.

ACK N OWLED G M ENTS
Thanks to Dr Q ian-Sheng Huang and Dr Wei-Dong Zhou for their valuable comments and excellent technical assistance in this research.

CO N FLI C T O F I NTE R E S T S
The author(s) announced there were no potential conflicts of interest in this article. Writing-review & editing (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.