| BackgroundAdenomyosis is a benign disease characterized by the presence of active endometrial glands and stromal cells into the myometrium of uterus.Similar to endometriosis,adenomyosis usually causes dysmenorrhea,irregular menstruation,and women’s fertility declines,which seriously affected the health and quality of life of women.Endometriosis is an estrodial-dependent benign disease that affects approximately 10%of reproductive-age women,also characterized by chronic pelvic pain and infertility.Its tendency of progression and recurrence causes disability and distress,along with high economic impact.In recent decades,much effort has been focused on developing new drugs to relieving the clinical symptoms and preventing the recurrence of the disease.Endometrial carcinoma is the most common gynecologic malignancy worldwide among women which is mostly in peri-menopausal period and menopause period.Due to the environment,its incidence remains increasing in recent years.The majority of women suffered endometrial cancer present with early-stage symptom and the favorable endometrioid histologic subtype,approximately 85%-90%.Moreover,70%were diagnosed at stage I.Related to this,the majority of women with uterine cancer are treated with surgery.To the patients who are diagnosed at stage Ⅲ-Ⅳ,they are treated with surgery combined with radiotherapy or chemotherapy.Thus,it is important to elucidate the factors influencing endometrial carcinoma development.Notch signaling is defined as a highly evolutionary conservative pathway,which plays a crucial role in the fate determination,as well as implementation of differentiation,proliferation,and apoptosis programs.Notch signaling is also involved in the regulation of epithelial-mesenchymal transition(EMT).The member of Notch signaling pathway includes Notch receptors(Notch-1,2,3,4),DSL(Delta-Serrate-Lag2) ligands(Dll-1,3,4 and Jagged-1,2),CSL(CBF1/Su(H)/Lag-1)and other transcription factor.The Classic Notch signaling pathway also is known as CBF-1/RBPκ-J depending signaling way.The activation of Notch signaling in mammals requires three steps:Notch is cleaved to ECN subunit and TM subunit by a furin-like protease at the S1 cleavage site then produces a mature heterodimeric receptor intracellular;ligand binding induces conformational change of Notch and the exposure of S2 site for subsequently cleavage by a member of metalloproteinase family of proteases;the y-secretase complex acts on the S3 site to liberates the Notch Intracellular Domain(NICD)which transfers to the nucleus,binds with the EMT-related transcription factor-Snail,Slug and initiates an intercellular communication program.The role of Snail 1 and Snail2(Slug)as transcriptional repression mechanisms of E-cadherin have been intensively explored.Epithelial-mesenchymal transition is a process which epithelial cells change in cellular morphology,remodel cell construction,loss matrix adhesion,gain migratory,invasive capabilities and convert into mesenchymal phenotype.EMT occur during embryonic development,tissue regeneration,and are considered as major regulator for tumor progression.A mount of transcription factors have been identified exerting important influence on the initiation of EMT and metastatic process,such as Snail,Slug,Zebl/2,Twist and so on.EMT is also characterized with the down-regulation of E-cadherin and up-regulation of N-cadherin,Vimentin,along with the enhancement of motility ability.Melatonin(N-acetyl-5-methoxy-tryptamine)and its metabolite,a scavenger of free radicals and broad spectrum antioxidant,is the main pineal gland and retina hormone synthesized from tryptophan.Melatonin is diversely functional,and the most obvious characteristics of this hormone being circadian and seasonal rhythmicity,which could be in response to darkness.Series of studies have shown that melatonin has a potential therapeutic effect on various diseases,such as regulating circadian function,promote deeper sleep,immuno-regulation,biological responses,working as hormone,neurotransmitters and antioxidants.In experimental mice models,melatonin was examined as a role of prevention and therapeutic,causing regression and atrophy of the endometriotic lesions.Studies also suggest that melatonin effect on endometriosis via MMP3 and apoptosis-related pathway.What’s more,melatonin inhabits the EMT process in multiple tumor.However,in endometriosis and endometrial carcinoma,the influence of melatonin on EMT via Notch signaling pathway is not clear.Part I Aberrant expression of Notch1/numb/snail signaling in adenomyosisObjective:Adenomyosis is a common disease which characterized by the presence of active endometrial glands and stromal cells into the myometrium of uterus,usually causes dysmenorrhea,irregular menstruation,and infertility.Epithelial Mesenchymal Transition(EMT)is involved in the pathogenesis of adenomyosis and Notch signaling is crucial in EMT.The objective of the study was to explore the expression of Notch1/Numb/Snail signaling in the adenomyosis.Material and Method:The expression levels of the members of the Notchl/Numb/Snail signaling cascade in normal endometria(proliferative phase:n =15;secretory phase:n = 15;postmenopausal phase:n = 15)and adenomyotic endometria(proliferative phase:n = 15;secretory phase;n = 15)were determined by immunohistochemistry analysis.Result:We found that the expressions of Notchl and EMT-related proteins,N-Cadherin,Snail and Slug were upregulated in ectopic endometrium of adenomyosis compared with normal endometrium.Numb.negative regulator of Notch signaling,was significantly decreased in adenomyosis.In addition,a reduced immunoexpression of E-Cadherin was noted in adenomyosis.Conclusion:We conclude that Notch1/Numb/Snail signaling plays an important role in the pathogenesis and development of adenomyosis.The current study may provide new insight into the diagnosis and treatment of adenomyosis.Part II Melatonin inhibits 17β-estradiol-induced migration,invasion and epithelial mesenchymal transition in normal and endometriotic endometrial epithelial cellsObjective:Endometriosis is a benign disease that exhibits as malignant tumor.Melatonin is a potential therapeutic agent for endometriosis,but its molecular mechanism is unclear.Here,we invested the effect of melatonin on the epithelial-mesenchymal transition(EMT)in endometrial epithelial cells of endometriosis and explored the pathway that might be involved.Material and Method:This hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry,6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis,respectively.We examined the expression of Notchl/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis,the invasion and migration of endometrial epithelial cells by transwell assays,and the cell proliferation by CCK8 assays.We examined the expression of Notchl/Numb signaling and EMT markers using immunohistochemistry analysis and western blot analysis,the invasion and migration of endometrial epithelial cells using transwell assays,and the cell growth using CCK8 assays.Result:Compared with normal endometrium,the endometriotic eutopic endometrium showed increased expression of Notch1,Slug,Snail,and N-cadherin,and decreased expression of E-cadherin and Numb.Melatonin or Notch inhibition by specific inhibitor blocked 17β-estradiol-induced cell proliferation,invasion,migration and EMT-related markers in both normal and endometriotic epithelial cells.Results shows increased expressions of Notchl,Slug,Snail,and N-cadherin were present in eutopic endometrium of endometriosis,while decreased expressions of E-cadherin and Numb were noted.Melatonin or Notch1 inhibition by specific inhibitor inhibited cell proliferation,invasion,migration,EMT with/without 17β-estradial in endometriotic epithelial cells.Conclusion:Aberrant expression of Notch1/Numb signaling and EMT were present in endometrium of endometriosis.Melatonin blocked 17β-estradiol-induced EMT in endometriotic epithelial cells via upregulating Numb expression and decreasing the activity of Notch1 signaling pathway.Part Ⅲ Melatonin inhibits 17β-estradiol-induced epithelial mesenchymal transition in endometrial carcinoma cells via Notch/Numb signalingObjective:Endometrial adenocarcinoma is one of the most common gynecological cancer worldwide.However,the role of melatonin on endometrial carcinoma via Notch/Numb signaling pathway has not been reported.Here,we invested the effect of melatonin on the epithelial-mesenchymal transition(EMT)in Ishikawa(ISK)cells and explored the pathway that might be involved.Material and Method:After treatment with melatonin,Notch signaling inhibitor and 17β-estrodial in ISK and KLE cells,we examined the cell proliferation by CCK8 assay;the invasion and migration of endometrial carcinoma cells by transwell assay and the expression of Notch1/Numb signaling and EMT markers by western blot analysis.Knockdown of Numb expression in ISK and KLE cells were obtained through transfected with lentivirus targeting to Numb.Then,we examined the cells(before and after transfected)proliferation by CCK8 and cell scratch assay;the invasion and migration by transwell assays.Result:Melatonin or Notch1 inhibition by specific inhibitor inhibited cell proliferation,invasion,migration,EMT with/without 17β-estradial in endometrial carcinoma cells.Campared with untransfected ISK and KLE cells,the proliferation,invasion,migration ability of Numb-knockdown cells was increased.Conclusion:Melatonin blocked 17β-estradiol-induced EMT in endometrial carcinoma cells via upregulating Numb expression and decreasing the activity of Notch1 signaling pathway.The absence of Numb expression accelerates proliferation,invasion and migration of endometrial carcinoma cells. 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