Effects Of Exogenous SOCS-3on Cell Apoptosis And Proliferation In Ectopic Endometrial Stromal Cells

BackgroundEndometriosis (EMs) is a prevalent gynecological disorder which occurs in8%-10%of reproductive-aged women. The classic symptoms of EMs include dysmenorrhea, chronic pelvic pain, dyspareunia and infertility lead to a poor quality of life of affected women. Although EMs is a benign disease, it does have malignant behavior like tumors, such as proliferation, recurrence, metastasis et al. Several theories have been proposed to explain the development of EMs such as coelomic metaplasia, vascular and lymphatic metastasis, stem/progenitor cells theory and determinant of uterine eutopic endometrium, but the definite mechanism is still unclear. In order to form lesions outside the cavity and muscular layer of uterus, ectopic endometrial cells have to resist cell apoptosis, which is necessary to maintain the homeostasis of tissues. Many signaling pathways involved in cell proliferation and apoptosis can participate in the development of EMs, which provide us more new directions to study the pathogenesis and therapies of this disease.Janus kinase (JAK)/signal transducer and activator of transcription (STAT) is a widely studied signaling transduction, which plays vital role in proliferation, apoptosis, metastasis and angiogenesis of different cells. The expression and activity of Suppressor of cytokine signaling (SOCS) can negatively regulate JAK/STAT signaling pathway by acting as the main part of a negative feedback loop. Many researchers have found that aberrant and persistent activation of STAT-3and low-expression of SOCS-3are involved in various tumor cells. AG490, the specific inhibitor of JAK-2, or exogenous SOCS-3can restrain tumor cell proliferation and induce cell apoptosis through inhibition of STAT-3activity. Although EMs is a benign disease, it does have malignant behavior like tumors, such as over-proliferation, apoptosis resistance, recurrence, metastasis et al. Our previous work have determined that higher levels of p-STAT-3, the active form of STAT-3, and lower expression of SOCS-3exist in ectopic endometrium in comparison with eutopic and normal endometria, which indicate that significantly higher expression of p-STAT-3protein and lower expression of SOCS-3protein in the ectopic endometrium and the interaction between these two proteins are involved in the pathogenesis and progression of endometriosis. In present study, lentiviral vector expressing SOCS-3was used to infect ectopic endometrial stromal cells (EESCs) in order to investigate the effect of SOCS-3over-expression in JAK/STAT-3signaling pathways and explore the relationship between high level of SOCS-3and cell proliferation as well as cell apoptosis in EESCs.MethodEctopic endometrial stromal cells (n=12) were obtained from patients with histopathologically confirmed endometriosis and primary cultured in vitro. The purity of EESCs were verified by immunocytochemistry with specific monoclonal antibody Vimentin. Lentivirus which contains SOCS-3and GFP recombinant (LV-SOCS-3-GFP) or only GFP gene (LV-GFP) was used to infect two groups of EESCs from the same patient, respectively, which were defined as experimental group (EG) and negative control (NC). The infection efficiency of lentivirus was measured by GFP fluorescence expression observed under the microscope. Expression of SOCS-3, STAT-3and JAK-2mRNA and protein were analyzed by real-time PCR and western blot respectively. Moreover, phosphorylated JAK-2and STAT-3proteins (p-STAT-3and p-JAK-2) were also analyzed by western blot. Cell cycles and apoptosis were detected by flow cytometry.Results(1) High purity (>90%) of EESCs were confirmed by vimentin staining in our study.(2) The infection efficiency of lentivirus was greater than90%after72h of infection.(3) Compared with NC, EG showed a much higher expression of SOCS-3mRNA (P<0.001). No difference of JAK-2and STAT-3mRNA expression were observed between NC and EG.(4) Expression of SOCS-3protein was obviously higher in EG than that in NC. On the contrary, the expression of p-STAT-3protein was significantly lower in EG vs NC, while STAT-3, JAK-2and p-JAK-2proteins showed no difference between these two groups.(5) The percentage of apoptotic cells in EG were remarkably higher than that in NC (P<0.001). Moreover, EG cells showed a G0/G1arrest in comparison with NC (P<0.05).ConclusionExogenous over-expression of SOCS-3can inhibit the activity of STAT-3in EESCs, which can finally restrain cell growth by inducing cell apoptosis and cell cycles arrest. Moreover, we can further hypothesize that SOCS-3may be a new therapeutic target in EMs.