The Roles And Mechanisms Of NEDD9 And Its Involvement Of TGF-?1/smads Pathway In Endometriosis

Endometriosis is a chronic disease characterized by the growth of endometrial-like tissue outside the uterus and the sites of endometriotic implants are usually in the surface of ovaries and between rectum and vagina.About 10%to 15%women of reproductive age suffer from endometriosis,and 40%to 60%of women with chronic pelvic pain or infertility.With more and more cellular and molecular mechanisms are uncovered,the studies on endometriosis from local to complex and systemic,thus,theories like cultivation of endometrial theory,induction theory,aberrant immunologic response,endocrine dysfunction and stem cells theory were proposed.The exact pathogenesis remained unclear by now,though some theories have been proposed.Neural precursor cell expressed developmentally down-regulated 9(NEDD9)is a cytoplasmic docking protein of the Crk-associated substrate(CAS)family.As a scaffolding protein,NEDD9 coordinates the Src-FAK(focal adhesion kinase),Rac,and AURKA(aurora A kinase)signaling cascades,and thus plays an important role in cell adhesion,migration and chemotaxis.In recent years,NEDD9 expression has been found elevated in several cancer types and indicate poor prognosis.Endometriosis is a benign gynecologic disease with malignant behaviors such as invasive and distant metastasis.Our previous research has demonstrated that NEDD9 is expressed in both normal,eutopic and ectopic endometrial tissues,and is overexpressed in ectopic endometrial tissues significantly,which indicate NEDD9 plays a role in the pathogenesis of endometriosis.However,the function of NEDD9 in the pathogenesis of endometriosis remains unclear.Transforming growth factor beta 1(TGF-?1)is a multi-effect factor,which is a kind of signaling molecule closely related to tumor formation.TGF-?1 interacts with its receptor,promoting Smad2/3 carboxyl terminal SSXS sequence phosphorylation,phosphorylation of Smad2/3 and Smad4 form complexes,transfer to the nucleus,interacts with other transcription regulatory factor to regulate target gene,thus further participation in cell proliferation,apoptosis,differentiation,migration and other cells reconstruction process.Research suggested,in more than one normal cells and tumor cell lines,TGF-?1 participates in the regulation of expression of NEDD9,in turn,affects the cells in the proliferation,invasion,metastasis and other abnormal biological behavior.Our preliminary experimental results show that the TGF-?1 expression in ectopic endometrium tissue is significantly higher than that in eutopic tissue and the control endometrial tissue,which speculate that TGF-?1 may participate in the abnormal expression of NEDD9,which is involved in the pathogenesis of endometriosis.The purpose of this study is to detect the expression of NEDD9 within non-endometriosis endometrial tissues,eutopic and ectopic endometrium tissues of ovarian endometriosis patients.At the same time to explore the effect of abnormal expression of NEDD9 on cell proliferation,invasion and migration in primary cultured ectopic endometrial stromal cells,and to further explore TGF-?1 and its involvement of NEDD9 in the regulation of cell and molecular mechanism in the development of endometriosis.Part ? The expression of NEDD9 and TGF-?1 in differentendometrial tissuesObjective:To study the expression of NEDD9 and TGF-?1 in the non-endometriosis endometrial tissues,eutopic and ectopic endometrium tissues of endometriosis patients.Methods:Using western blot and immune histochemical method to detect the protein expression level of NEDD9 and TGF-?1 compare the expression and localization in the three groups.Results:1.The immune histochemical results showed that NEDD9 and TGF-?1 were expressed in both endometrial glandular epithelium and stroma,the staining levels were:ectopic endometrium tissue>eutopic tissue>control tissue,the difference between groups has statistical significance(p<0.05).2.Western blot results showed that NEDD9 and TGF-?1 were expressed in both three groups,the expression levels were:ectopic endometrium tissue>eutopic tissue>control tissue,the difference between groups has statistical significance(p<0.05).Conclusion:High expression level of NEDD9 and TGF-?1 probably have a certain correlation with the development of endometriosis.Part ? The effects of NEDD9 on the proliferation,invasion andmigration of ectopic endometrial stromal cellsObjective:To investigate the effect of NEDD9 on the proliferation,invasion and migration of endometrial stromal cells.Methods:The endometiral tissue of the patients with endometriosis was collected to culture primary ectopic endometrial stromal cells,and the cells were purified and identified.The siRNA-NEDD9 was used to knock down NEDD9,qRT-PCR and western blot were used to detect and identify the interferences efficiency.Using the cck-8 method to detect the cell proliferation after NEDD9 siRNA interference.Transwell assay was used to detect changes in the invasion and migration capacity of endometrial stromal cells,and the expression of MMP2 and MMP9 was also detected.Results:1.Successfully purificate and cultivate the primary ectopic endometrial stromal cells from patients with endometriosis and endometrial stromal cells from patients without endometriosis,these two groups of cells were similar in form,present long spindle in shape,state stable and can be passage.2.SiRNA-NEDD9 was successfully constructed and used for endometrial stromal cells,and the expression of NEDD9 in mRNA and protein levels was significantly reduced after 48 hours with qRT-PCR and Western blot detection(p<0.0001).3.CCK-8 assay showed that silence NEDD9 expression significantly restricted the proliferation ability of ectopic endometrial stromal cells(p<0.001).4.Transwell results showed that knockdown the expression of NEDD9 could inhibit the invasion and migration of ectopic endometrial stromal cells.5.Silence NEDD9 expression reduced the mRNA and protein expression levels of MMP2 and MMP9.Conclusion:1.The abnormal expression of NEDD9 was associated with the proliferation,invasion and migration of ectopic endometrial stromal cells.Reducing the expression of NEDD9 can inhibit the proliferation,invasion and migration of ectopic endometrial stromal cells.2.NEDD9 may participate in regulating the invasion and migration of endometrial stromal cells through MMP2 and MMP9.Part? The mechanism of TGF-?1 regulates NEDD9 in thedevelopment of endometriosisObjective:To determine whether TGF-?1 is involved in regulating NEDD9 expression in endometriosis.Methods:Collect 30 cases of eutopic and ectopic endometrial tissues from patients with endometriosis,30 cases of control endometrial tissues from patients without endometriosis,extracting RNA for qRT-PCR assay to detect the expression of TGF-?1 and NEDD9,and Pearson correlation analysis was conducted.Fresh ectopic endothelial tissue of the patients with endometriosis was collected for primary ectopic endometrial stromal cells culture.TGF-?1 of 2.5ng/ml concentration was used for ectopic endometrial stromal cells culture,and total proteins were extracted after 48 hours co-culture.Western blot was used to detect the expression of NEDD9,TGF-?1,smad2/3 and p-smad2/3.Results:1.The qRT-PCR of tissue samples showed that TGF-?1 and NEDD9 expression levels were significantly higher in ectopic tissues than in control endometrial tissues(p<0.0001),Pearson correlation analysis suggested TGF-?1 was positively correlated with NEDD9 in mRNA expression levels.2.The protein levels of NEDD9(p<0.01)and p-smad2/3(p<0.001)were significantly increased after 48 hours of high concentration TGF-?1 stimulation,and no significant change was found in total smad2/3.Conclusion:1.TGF-?1 and NEDD9 were significantly high expressed and positively correlated in the ectopic endometrial tissues.2.The high expression of TGF-?1 can increase the expression of NEDD9 and phosphorylated smad2/3,which suggested that NEDD9 may participate in the TGF-?1/smad signaling pathway and play a role in the development of endometriosisso.