The Roles Of Cell Migration And Invasion Mediated By Twist Promoter Methylationin Endometriosis

Endometriosis(EMs)is a disorder in which abnormal growth of tissue,histologically resembling the endometrium,present in locations other than the uterine lining.It's a common disease.There is 10%-15%of women in reproductive age suffering from it,especially who aged between 25 and 40 years old,with an increasing trend in recent years.The symptoms of EMs,such as chronic pelvic pain,dysmenorrhea and infertility,significantly impacted women's health and life quality.EMs was firstly described by Von Rokitansky in 1860,but its pathogenesis remains to be unclarified.The hypothesis suggested by Sampson,"retrograde menstruation and implantation",has been widely accepted.However,retrograde menstruation was common among reproductive-age women with an occurrence rate of 76%-90%,while the incidence of EMs is only 10%-15%.This suggests that retrograde menstruation might be only a precipitating factor,while the biological behaviors of ectopic endometrium is key in pathogenesis of EMs,such as the migration and invasion of the EMs,adhesion and infiltration to pelvic organs like ovary or peritoneum.In recent years,a number of studies have confirmed that epigenetic changes are essential for the pathogenesis of EMs.Meanwhile gene methylation is an important part of epigenetics.Twist,a highly conserved protein which can suppress apoptosis,whose methylation involving the embryonic development and human diseases.The functions of Twist include inducing epithelial-mesenchymal transition(EMT)and enhancing migration and invasion of tumor cells,inhibiting cell apoptosis,promoting tumor angiopoiesis,as well as causing chromosome instability.Generally,methylation of gene will result in gene silence,while the demethylation of Twist gene upregulates the protein expression.However,the relationship between methylation of the Twist gene promoter and EMs has not reported yet.Twist can regulate EMT through inhibiting E-cadherin and increasing N-cadherin.EMT is an efficient way through which epithelial cells gain migratory and invasive capacity,and plays an important role in tumor initiation and invasion.And EMT may be involved in the pathogenesis of EMs,which shares some characteristics with malignant tumors.The objective of this study is to investigate and compare the expression of Twist,E-cadherin and N-cadherin in ectopic endometrium and eutopic endometrium of ovarian EMs and normal endometrium of non-EMs patients.Meanwhile,it aims to study the effects of overexpressed Twist on migration and invasion of eutopic stromal endometrium cells.It may provide new ideas on targeted therapy and preventing recurrence of EMs with clarifying the role of cell migration of invasion mediated by aberrant Twist methylation in EMs pathogenesis.Part ? The expression of Twist,N-cadherin and E-cadherin in different kinds of endometrial tissuesObjective:To investigate the differences of Twist,N-cadherin and E-cadherin expression on translation and transcription levels in ectopic endometrium and eutopic endometrium of ovarian endometriosis,and normal endometrium of non-endometriosis patients.Methods:The protein levels and locations of Twist,N-cadherin and E-cadherin were measured by Western blot and immunohistochemistry in ectopic endometrium and eutopic endornetrium of ovarian endometriosis as well as normal endometrium of non-endometriosis patients.The mRNA expression of Twist,N-cadherin and E-cadherin in the these tissues was measured by quantitative RT-PCR.Results:1.Results from immunohistochemistry showed that Twist,N-cadherin and E-cadherin were expressed in both stromal cells and glandular epithelium.Twist and N-cadherin showed the highest expression in ectopic endometrium of ovarian endometriosis,while lowest in normal endometrium of non-endometriosis patients.On the contrary,the expression of E-cadherin showed highest in normal endometrium of non-endometriosis patients.The differences between different groups were significant(P<0.05).Spearman analysis showed positive correlation between N-cadherin and Twist,while negative correlation between E-cadherin and Twist.2.The expression of Twist and N-cadherin protein measured by Western blot decreased stepwise in ectopic endometrium of ovarian endometriosis,eutopic endometrium of ovarian endometriosis,and normal endometrium of non-endometriosis patients(P<0.05).The expression of E-cadherin protein measured by Western blot increased stepwise in ectopic endometrium of ovarian endometriosis,eutopic endometrium of ovarian endometriosis,and normal endometrium of non-endometriosis patients(P<0.05)3.Results from RT-PCR showed the mRNA levels of Twist and N-cadherin in ectopic endometrium and eutopic endometrium of ovarian endometriosis was significantly higher when compared with normal endometrium of non-endometriosis patients(P<0.05).However,E-cadherin was expressed highest in normal endometrium of non-endometriosis patients.There were signifant differences among groups(P<0.05).Conclusions:Twist gene may be associated with the incidence of endometriosis.It may cause endometriosis by mediating epithelial-mesenchymal transition(EMT).Part ? Overexpression of Twist promote the migration and invasion of endometrial stroma cellsObjective:To investigate the roles of Twist in the migration and invasion of eutopic endometrial stromal cells.Methods:Endometrial stromal cells were primarily cultured.Stable overexpression of Twist in eutopic endometrial stromal cells was transfected with a plasmid-mediated delivery system(cmv-mcs-3flag-sv40-Neom-Twist).The protein expression was detected by western blot.The mRNA expression was tested by RT-PCR.The changes of migration and invasion of endometrial stromal cells were explored by transwell.The cellular morphology,protein expression and location of Twist,N-cadherin and E-cadherin in different endometrial stromal cells were evaluated by immunofluorescence.Results:1.The overexpression of Twist after transfection significantly up-regulated the protein and mRNA expression of N-cadherin,while down-regulated the protein and mRNA expression of E-cadherin.There is significant difference between groups.2.For transwell,the overexpression of Twist in eutopic endometrial stromal cell significantly promoted cell migration and invasion.The difference is significant.3.For immunofluorescence,expressions of Twist,N-cadherin and E-cadherin were found in the endometrial stromal cells of all three groups with more polymorphic phenotypes in ectopic endometrial stromal cells.Twist and N-cadherin were expressed highest in eutopic endometrial stromal cells.However,E-cadherin was expressed highest in non-endometriosis stromal cell.Conclusion:Twist overexpression resulted in the increased migratory and invasive capability of eutopic endometrial stromal cells of ovarian endometriosis.The expression of Twist upregulated the expression of N-cadherin,while downregulated the expression of E-cadherin.It suggests that the pathogenesis of endometriosis may be related with the increase of migration and invasion in endometrial stromal cells,mediated by EMT.Part ? The difference of Twist promoter methylation among different kinds of endometrial tissuesObjective:To study the difference of Twist promoter methylation among ectopic endometrium and eutopic endometrium of ovarian endometriosis and normal endometrium of non-endometriosis patients.Methods:Six pairs of Twist gene promoter were designed,and then the the levels of Twist promoter methylation were measured by pyrosequencing method for methylation-specific PCR(MSP)in the three kinds of endometrial tissues.Results:Hypomethylation of Twist gene promoter was found in some areas of ectopic endometrium and eutopic endometrium of ovarian endometriosis.Conclusions:Local hypomethylation of Twist gene promoter was found both in ectopic endometrium and eutopic endometrium of ovarian endometriosis,which can result in Twist protein overexpression and may be a direct precipitating factor for endometriosis.