The Expression And Regulation Of Slit2 In Endometriosis-derived Stromal Cells

Endometriosis is a common gynaecological condition affecting up to 10-15%of women of reproductive age, and is histologically characterized by the presence and growth of endometrial-like glands and stroma outside the uterine cavity. The condition is, in the majority of cases, associated with dysmenorrhea, dyspareunia and pelvic pain, and can significantly compromise the quality of life of the affected women. The prevalence in infertile women is up to 30%(Cramer and Missmer,2002). The real prevalence, however, may even be higher, because the disease is often not diagnosed due to its heterogeneous clinical manifestation.The etiology of the disease, in spite of its high prevalence, remains a challenging problem both clinically and scientifically. Now a majority of scholars are inclined to the retrograding-implantation theory. According to this theory the implanted tissues need oxygen and essential nutrients supply to grow into ectopic lesions (Groothuis et al.,2005). This interpretation highlights the angiogenesis as a decisive event in the formation of endometriosis. In fact during the last decade, it has become evident that angiogenesis does play a central role in the pathogenesis of endometriosis so that endometriosis is defined as a kind of angiogenesis-dependent disease. But the knowledge about the mechanism of angiogenesis in ectopic endometrium is very limited yet.At present the main treatment choices for endometriosis are surgery and GnRH-a medication. But there are problems such as high recurrence risk after surgery and side effects of medication with current therapies. Due to the angiogenesis-dependent nature of the disease, the anti-angiogenesis research has been developed in the recent years especially the anti-VEGF-a drugs for treating endometriosis. The anti-angiogenesis therapies show excellent short-term effect but poor long-range performance, which indicates compensation of angiogenesis via other unknown pathways.SLIT2 is a big secreted molecular found to be expressed in a series of malignant tumors. Recombinant human SLIT2 has been proved to attract endothelial cells. Then in vivo experiments were designed and proved that SLIT2 plays central role in the angiogenesis of neoplasm (Biao et al., 2003). Since endometriosis is similar in biological behavior to malignant tumors, that is, being highly dependent on angiogenesis, SLIT2 is also very likely to be expressed in ectopic endometrial lesions. The SLIT2 expression can help us interpreting the phenomenon of resistance to anti-angiogenesis therapy such as VEGF-A if SLIT2 was another pro-angionenesis factor beyond our current knowledge. Studies using immunohistochemical method demonstrated up-regulated SLIT protein expression in ectopic endometrium, and its expression is closely relevant to microvessel density (MVD). Since stromal cells play critical role in endometriotic lesion formation, this study mainly focus on the expression profile of SLIT2 and its receptor ROBO1 in endometriosis-derived stromal cells and their possible regulatory mechanism.The first part:Objective:To detect the SLIT2 mRNA expression in endometriosis-derived stromal cells and the relative level compared to that in normal endometrium-derived stromal cells.Methods:Using primary culture method to obtain endometriosis-derived stromal cells and normal endometrium-derived stromal cells; using RT-PCR method to detect SLIT2 mRNA expression in the primary-cultured cells; using relative quantitative Real-time PCR to quantify the SLIT2 mRNA expression and observe whether it changes within the menstrual cycle; using relative quantitative Real-time PCR method to compare the SLIT2 mRNA in endometriosis-derived stromal cells to that in the normal endometrium-derived stromal cells.Results:SLIT2, SLIT3, ROBO1 and ROBO2 mRNA are expressed in endometriosis-derived and normal endometrium-derived stromal cells and are closely relevant to each other; SLIT2 mRNA is up-regulated in endometriosis-derived stromal cells; SLIT2 and ROBO1 mRNA are fluctuated in menstrual cycle.Conclusion:SLIT2 mRNA is up-regulated in endometriosis-derived stromal cells.The second part:Objective:To find out the factors that can regulate the SLIT2 expression in endometriosis-derived stromal cells.Methods:Using relative quantitative Real-time PCR to detect whether estrogen, progesterone and TNF-a of various concentrations or exposure times regulate SLIT2 mRNA expression. Using Western blot to detect the SLIT2 protein expression change when exposed to 10-7 mol/L 17β-estrodial and 0.1ng/ml TNF-αResults:Estrogen and TNF-a regulate SLIT2 mRNA in a concentration-dependent mode; 10-7 mol/L 17β-estrodial and 0.1ng/ml TNF-αsignificantly induces SLIT2 mRNA and protein expression.Conclusion:Estrogen and TNF-αregulate the SLIT2 mRNA and protein expression. The third part:Objective:To investigate the mechanism how estrogen induces SLIT2 expression.Methods:Expose the endometriosis-derived stromal cells to estrogen receptor antagonist ICI182780 with or without 17β-estrodial, then detect the SLIT2 expression by Western blot to decide whether estrogen induces SLIT2 via estrogen receptor; expose the endometriosis-derived stromal cells to ER a agonist PPT and ERβagonist DPN with or without ICI 182780, to detect whether ER a and ERβare involved in the regulation of SLIT expression; expose the endometriosis-derives stromal cells to Akt inhibitor IV or LY294002 with or without estrogen, to detect whether estrogen induces SLIT2 via PI3K-Akt signaling pathway; transfect the Akt over-expression plasmid into endometrosis-derived stromal cells, the detect the SLIT2 expression by Western blot to decide whether Akt regulates SLIT2 expression; expose the endometriosis-derived stromal cells to eNOs inhibitor L-NAME with or without estrogen, to explore the molecular downstream the PI3K-Akt signaling pathway involved in estrogen-induced SLIT2 expression.Results:1μM ICI 182780, 100nM Akt inhibitor IV,10μM LY294002, 500μM L-NAME significantly reduce the background and 10-/mol/L 17β-estradiol induced SLIT2 and ROBO1 protein expression in endometriosis-derived stromal cells. Both the ER a agonist PPT and ERβagonit DPN up-regulate SLIT2 expression. When transfected with Akt over-expressing plasmid, total Akt, phosphorylated Akt, SLIT2 and ROBO1 protein expression in endometriosis-derived stromal cells were significantly up-regulated.Conclusion:Estrogen induces SLIT2 expression via ER a and ERβ, and PI3K-Akt-eNOs pathway.