The Roles And Mechanisms Of PRL-3 And Its Involvement Of SOCS3/JAK2/STAT3 Pathway In Endometriosis

Characterized by the presence of endometrium outside the uterine cavity, endometriosis is a major cause of infertility and chronic pain, affecting about 10% women of reproductive age. With more and more celluar and molecular mechanism are uncovered, the studies pay attention to endometriosis from local to complex and systemic. There are three distinct forms of endometriosis according to the sites of endometriotic implants:on the surface of pelvic peritoneum (peritoneal endometriosis), residing between rectum and the vagina, usually blended with adipose and fibromuscular tissue (rectovaginal endometriotic nodule), and endometrioid mucosa in ovarian cysts (endometrioma). The three clinical types may be caused by different mechanisms or the same pathologic process. The exact etiology of the causality is still unclear by now, though some theories have been proposed. Here we focused on endometrioma because of the high morbidity and the feasibility of obtaining the specimens.Phosphatase of regenerating liver-3 (PRL-3) belongs to the family of protein tyrosine phosphatases with unique C-terminal prenylation motif, which determines the function and cellular localization of this protein. Up-regulated PRL-3 expression has been reported to be associated with metastatic phenotype in colorectal carcinoma. In the past decade, elevated PRL-3 expression has also been detected in many gynecological malignancies, such as breast, ovarian, cervical and endometrial cancer. PRL-3 has been reported to regulate Rho family GTPases, increase cell migratory and invasive capability in many cell types. Similar to cancer, endometriosis also displays features such as progression, invasion and recurrence. Our previous research has demonstrated that increased PRL-3 expression is associated with the advanced clinical stages of endometriosis and a higher recurrence rate, suggesting that PRL-3 may play a role in the disease progression. However, to date, the function of PRL-3 in the pathogenesis of endometriosis is still unknown.Janus kinase (JAK)/signal transducer and activator of transcription (STAT) is one of the classical studied signaling pathways, and the role in proliferation, apoptosis, migration, invasion and angiogenesis in cancer cells is widely studied. As the one part of the negative feedback loop, Suppressor of cytokine signaling (SOCS) can inhibit JAK/STAT signaling pathway. Aberrant or persistent phosphorylation of STAT3 with deficient expression of SOCS3 were detected in some malignant diseases, in turn, overexpression of SOCS3 in cells have reduced activation of STAT3. Our previous studys have confirmed the decreased expression of SOCS3 in endometrioma compare to the eutopic endometrium from both endometriosis and endometriosis-free women by immunohistochemistry.The aim of our study is to examine and compare the expression of PRL-3 and SOCS3 in stromal cells derived from eutopic and ectopic endometrial tissue of women with or without ovarian endometriosis.We studied the role of PRL-3 in regulating cytoskeleton remodeling, cell migration and invasion of endometrial stromal cells (ESCs) from ectopic and eutopic ovarian endometriosis. Then the activation of SOCS3/JAK2/STAT3 and the interaction with PRL-3 in OvESCs are detected and analyzed in order to explore the relevant cellular and molecular mechanism.Part I The expression of PRL-3 in different type of endometrial stromal cellsObjective:To compare the expression of PRL-3 in the primary endometrial or endometriotic stromal cells (ESCs) derived from eutopic endometrium of women without endometriosis (EuCo), with histologically proven endometrioma (EuEM) and the cyst wall of ovarian endometriosis (OvEM); To explore the fluctuation of PRL-3 in the menstrual cycle.Methods:ESCs were primarily cultured, purified and separated after collecting fresh clinical specimens of EuCo, EuEM and OvEM. The purity of ESCs was confirmed by fluorescence immunocytochemistry. The protein expression of PRL-3 was tested by western blot, and the mRNA expression of PRL-3 was detected by RT-qPCR.Results:1. Endometrial stromal cells derived from eutopic endometrium of women without endometriosis (CoESCs) or with endometrioma (EuESCs) and endometriotic stromal cells derived from ovarian endometriosis (OvEM) were successfully cultured and purified.2. The characteristics of 3 groups of ESCs were stable and similar, which showed elongated, spindle-like mesenchymal phenotype and could be sub-cultured.3. Both protein and mRNA levels of PRL-3 were significantly higher in OvESCs compared with CoESCs and EuESCs, whereas no significantly difference of protein or mRNA expression was observed between CoESCs and EuESCs.4. No significant difference was detected in PRL-3 protein or mRNA levels between proliferative and secretory phases in CoESCs, EuESCs or OvESCs.Conclusion:1. The primary cultures of ESCs provide the basic research of endometriosis idealized models in vitro.2. Elevated expression of PRL-3 may be relevant to endometriosis.3. PRL-3 may not associate with the steroid-dependence of endometriosis.Part II Elevated PRL-3 promotes cytoskeleton reorganization, cell migration and invasion in endometriotic stromal cellsObjective:To explore the roles and mechanisms of PRL-3 in the cytoskeleton and cell motility of ESCs derived from endometriosis.Methods:Transient or stable knockdown of PRL-3 in OvESCs were tansfected with siRNA (siRNA-Ctrl/siRNA-PRL-3) or infected with fluorescent lentivirus (LV-GFP-shRNA-Ctrl/LV-GFP-shRNA-PRL-3), respectively. Stable overexpression of PRL-3 in EuESCs was tansfected with a plasmid-mediated delivery system (pCEP4/ pCEP4-PRL3). The protein expression was tested by western blot, and the mRNA expression was detected by RT-qPCR. The cellular distribution of F-actin and a-tubulin were examined by immunocytochemistry. Cell proliferation was tested by WST1 assay. Cell motility was evaluated by a transwell migration/invasion assay.Results:1. Endogenous inhibition of PRL-3 in OvESCs significantly modified the distribution of F-actin and a-tubulin cytoskeleton, down-regulated the protein and mRNA expression of Ras homolog gene family members A and C (RhoA, RhoC) and Rho-associated coiledcoil-containing protein kinase 1 (ROCK1).2. Knockdown of PRL-3 in OvESCs did not change the cell proliferation, while significantly inhibited cell migration and invasion, attenuated the expression of matrix metalloproteinase (MMP) 9, but not MMP2.3. Overexpression of PRL-3 in EuESCs significantly up-regulated the protein and mRNA expression of RhoA, RhoC and ROCK1.4. Overexpression of PRL-3 in EuESCs did not change the cell proliferation, similarly, significantly promoted cell migration and invasion, increased the expression of MMP 9, but not MMP2. Meanwhile, either ROCK1 inhibitor or MMP9 inhibitor can block the enhanced cell migrative and invasive abilities by PRL-3.Conclusion:1. PRL-3 may reorganize cytoskeleton of OvESCs by the bidirectional regulation of RhoA/RhoC/ROCK1.2. PRL-3 may be not relevant to cell proliferation of OvESCs and EuESCs.3. PRL-3 may change the cell migration and invasion of endometriosis through Rho family and MMP9 but not MMP2.Part Ⅲ The effect of PRL-3 inhibitor on the ectopic lesion in vivoObjective:To set up animal models of endometriosis and explore the effect of PRL-3 inhibitor in ectopic lesion of endometriosis in vivo.Methods:21 female SCID mice were xenotransplanted by human endometrium to set up endometriosis models.40 female SD rats were autotransplanted by endometrium to induce endometriosis. Epithelial and stromal tissues were confirmed by immunohistochemistry in the ectopic lesions. The ectopic lesions were analyzed and compared after intraperitoneal injection of PRL-3 inhibitor.Results:1. The survival rate of xenotransplantation on SCID mice was 72.2%, while the success rate of lesions in vivo was 59.0%.2. Compare the size of lesions after treatment of PRL-3 inhibitor, high-dose group was siginificiantly smaller than control and low-dose group, while no significant difference between the latter two groups.3. The survival rate of autotransplantation on SD rats was 86.1%, while the success rate of lesions in vivo was 92.5%.4. Compare the pre-administration with post-administration, ectopic lesions of control group were significantly extended, while lesions of high-dose group were significantly atropic, and no obvious difference were observed in low-and mid-dose groups.Conclusion:1. SD rats model of endometriosis is suitable for the study of endometriosis in vivo, which is operable, reproducible and low cost.2. High dose (1mg/kg/day) of PRL-3 inhibitor can suppress the development of ectopic lesions of SCID mice model of endometriosis.3. High dose (2mg/kg/day) of PRL-3 inhibitor can suppress the development of ectopic lesions of SD rats model of endometriosis.Part Ⅳ The involvement of PRL-3 in SOCS3/JAK2/STAT3 pathway in endometriosisObjective:To detect the expression and function of SOCS3 in primary CoESCs, EuESCs and OvESCs; To explore the interaction effect and mechanism of PRL-3 and SOCS3 in endometriosis.Methods:CoESCs, EuESCs and OvESCs were primary cultured. Knockdown of PRL-3 in OvESCs was tansfected with siRNA (siRNA-PRL-3), while overexpression of SOCS3 in OvESCs was infected with fluorescent lentivirus (LV-GFP-SOCS3). The protein expression was tested by western blot, and the mRNA expression was detected by RT-qPCR. Cell apoptosis and cell cycle were analyzed by flow cytometry. Cell proliferation was tested by WST1 assay. Cell motility was evaluated by a transwell migration/invasion assay.Results:1. Both the protein and mRNA expression of SOCS3 were significantly higher while the phosphorylation of STAT3 in OvESCs is lower compared with CoESCs and EuESCs, whereas no significantly difference of protein or mRNA expression was observed between CoESCs and EuESCs.2. Exogenous SOCS3 in OvESCs inhibited the phosphorylation of STAT3 but only inhibited the phosphorylation of JAK2 with the down-regulation of PRL-3.3. Overexpression of SOCS3 or inhibition of PRL-3 increased the rate of cell apoptosis; although si-PRL-3 did not change the cell cycle or proliferation, but down-regulation of PRL-3 enhanced the degree of exogenous SOCS3 in capturing more cells in G0G1 phase and fewer cells in S or G2M phase, and decreasing the rate of cell proliferation.4. Exogenous SOCS3 enhanced the effect of inhibition of PRL-3 in decreasing of cell migration and invasion, with the suppression of MMP9, but no difference in the expression of RhoA/RhoC/ROCK pathway or MMP2.Conclusion:1. Reduced expression of SOCS3 and over-phosphorylation of STAT3 may be relevant to ovarian endometriosis.2. The lower apoptosis rate and higher growth rate of OvESCs may be relevant to the reduced expression of SOCS3, while the elevated PRL-3 may enhance the effect by inducing the phosphorylation of JAK2.3. The deletion or reduction of SOCS3 may be enhance the effect of PRL-3 induced cell migration and invasion by increasing the expression of MMP2.