Rnai-mediated Blocking Of Ezrin Reduces Migration Of Ectopic Endometrial Cells In Endometriosis

Backgroud:Endometriosis is characterized by growth of endometrial tissue (including glands and stroma) outside the uterus. It causes abdominal pain and infertility in10-15%of women of reproductive age, A variety of theories have been proposed to account for the pathogenesis of this disease include retrograde transplantation theory, metaplasia of coelomic epithelium, hematogenic and lymphogenic spread, and remnants of the Mullerian duct. However, the etiopathology of endometriosis is still obscure. Recent research shows that endometrial cells in endometriosis have increased adhesion, proliferation and migratory capacity compared to normal endometrial cells,and exhibit features of malignant behaviour including proliferation, angiogenesis, abnormal apoptosis and invasion.Ezrin is a member of the ezrin-radixin-moesin (ERM) family of membrane cytoskeletal linkage proteins.It links actin filaments to cell membrane either directly, by binding to cytoplasmic tails of transmembrane proteins, or indirectly, via scaffolding proteins attached to transmembrane proteins.Previous research demonstrates that ezrin contributes to cytoskeletal processes underlying many cellular functions including cell division, adhesion and migration. To date, the role of ezrin in migration of cancer cells have been proved well and its roles in common gynecological diseases have gained lots of scholars’attention. However, the relationship between endometriosis and Ezrin has not extensively investigated.This study seeks to determine whether ezrin/Rho/ROCK pathways exert regulatory actions on migration of human endometrial cells in endometriosis.We obtained endometrial cells from endometriosis lesions (ectopic-EMs), and, homologous eutopic endometrium (eutopic-EMs) tissue in women with stage Ⅲ-Ⅳ endometriosis (according to revised AFS classification).Subjects included15women (mean age35.8years, parity23-52);9/15were in the proliferative phase of the menstrual cycle and6/15in the secretory phase. As a control group (non-EMs), we obtained endometrial tissues from14women without endometriosis undergoing surgical procedures for benign gynaecological diseases (mean age39.2years, parity25-49);9/14in proliferative and5/14in secretory phase).Then the primary cell culture of ectopic endometrial cells, siRNA interference, wound healing assays and Western blot analysis were used to confirm the role of ezrin in migration of ectopic cells and pathogenesis of endometriosis.Objective:To investigate the role of ezrin in the migration of ectopic endometrial cells and pathogenesis of endometriosis.Methods:We conducted real time quantitative RT-PCR, RNAi, wound healing assays and Western blot analysis of endometrial cells from women with, or without, endometriosis.Results: 1、We found significantly higher levels of mRNA expression of ezrin in ectopic endometrial cells, compared to eutopic endometrial cells in endometriosis(p<0.05).The equal expression level of ezrin in ectopic, eutopic and non-EMs group is0.428±0.187,0.272±0.105,0.194±0.066, respectively.2、mRNA expression of RhoA in ectopic, eutopic and non-EMs group is0.995±0.292,0.742±0.279,0.673±0.198,respectively, which is significant higher in ectopic endometrium(p<0.05).3、mRNA expression of RhoC and ROCK1in ectopic group are significantly higher than eutopic and non-EMs group(p<0.005).4、Neither of ezrin, RhoA, RhoC nor ROCK1has significant differences between secretory phase and proliferative phase among ectopic, eutopic and non-EMs group (p>0.05).5、Blocking ezrin with siRNA (mean interference efficiency of ezrin in protein level was66.35%±12%) significantly reduced migration of ectopic endometrial cells (declined by41.1%after48hr transfection), with decreased expression of RhoA (42.68±8%), RhoC (58.42±14%), and, ROCK1(59.88±13%),respectively.Conclusion:1、The mRNA expression of ezrin, RhoA, RhoC,and ROCK1is significantly higher in ectopic endometrium of women with endometriosis.2、Blocking expression of ezrin in ectopic endometrial cells in endometriosis reduces cell migration.3、High expression of ezrin in ectopic endometrium may play a role in the pathogenesis of endometriosis, which takes effect could through the RhoC/ROCK1pathway rather than the RhoA/ROCK1pathway.