{"paper_id":"8ae74d3a-c0bb-4bb2-9d55-cd4d69a7798f","body_text":"R E S E A R C H Open Access\nMelatonin inhibits 17 β-estradiol-induced\nmigration, invasion and epithelial-\nmesenchymal transition in normal and\nendometriotic endometrial epithelial cells\nShasha Qi 1,2,3, Lei Yan 1,2,3, Zhao Liu 4, Yu-lan Mu 5, Mingjiang Li 5, Xingbo Zhao 5, Zi-Jiang Chen 1,2,3,6,7\nand Hui Zhang 5*\nAbstract\nBackground: Melatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear.\nHere, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic\nendometrial epithelial cells and explored the pathway that might be involved.\nMethods: This hospital-based study included 60 women of reproductive age using the endometrium for\nimmunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with\nendometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the\nexpression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot\nanalysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by\nCCK8 assays.\nResults: Compared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of\nNotch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by\nspecific inhibitor blocked 17β-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both\nnormal and endometriotic epithelial cells.\nConclusions:Our data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present\nin endometriotic endometrium. Melatonin may block 17 β-estradiol-induced migration, invasion and EMT in\nnormal and endometriotic epithelial cells by upregula ting Numb expression and decreasing the activity of\nthe Notch signaling pathway.\nKeywords: Endometrial epithelial cells, Melatonin, 17β-estradiol, Migration and invasion, Epithelial-mesenchymal\ntransition\nBackground\nEndometriosis is a chronic disease that affects approxi-\nmately 10% of reproductive-age women and is character-\nized by chronic pelvic pain and infertility [1]. Endometriosis\nis a benign disease, but its tendency to progression and re-\ncurrence causes disability and distress [ 2]. In recent\ndecades, much effort has been focused on developing new\ndrugs to relieve the clinical symptoms and prevent the\nrecurrence of the disease.\nEpithelial-mesenchymal transition (EMT), which is\ncharacterized by an increased rate of cellular migration,\ninvasion properties and increased resistance to apoptosis,\nis also considered essential for the formation and progres-\nsion of endometriosis [ 3–5]. Key transcription factors in-\ncluding Snail, Slug and Twist drive the EMT process,\nwhich along with cleaved and then degraded E-cadherin\nand increased expression of mesenchymal-related\n* Correspondence: huizhang1218@126.com\n5Department of Obstetrics and Gynecology, Shandong Provincial Hospital\nAffiliated to Shandong University, 324 Jingwu Road, Jinan 250021, People ’s\nRepublic of China\nFull list of author information is available at the end of the article\n© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 \nhttps://doi.org/10.1186/s12958-018-0375-5\n\nproteins, including N-cadherin, Vimentin [ 6]. Snail also\nrepresses E-cadherin transcription by binding to the\nE-box site in the promoter of E-cadherin [ 7]. In previous\nstudies, we found that the expression of both Notch1, a\nkey signaling factor involved in EMT regulation, and\nEMT-related proteins was upregulated in the ectopic\nendometrium of adenomyosis compared with those in\nnormal endometrium [8].\nMelatonin (N-acetyl-5-methoxy-tryptamine), a scavenger\nof free radicals and a broad-spectrum antioxidant, is the\nmain pineal hormone synthesized from tryptophan in re-\nsponse to darkness [9]. A series of studies have shown that\nmelatonin has a potential therapeutic effect on endometri-\nosis [10–16]. In experimental rat models, melatonin causes\nt h er e g r e s s i o na n da t r o p h yof endometriotic lesions [ 11],\nand the combination of letrozole and melatonin causes a\nsignificant regression in lesion volumes but not in the histo-\npathological scores of en dometriotic lesions [ 15]. Higher\ndoses of melatonin treatment have been reported to be\nmore effective in inducing the regression of implants and in\nimproving histologic scores [10]. Moreover, compared with\nletrozole, melatonin causes a more pronounced regression\nof endometriotic foci and lower recurrence [ 16]. It was re-\nported that pinealectomy increased the progression of\nendometriosis explants, and that melatonin reversed the ef-\nfects of pinealectomy [12]. Melatonin is effective in treating\nexperimental endometriosis induced by implanting human\nendometriotic cells in SCID mice [ 14]. Kocadal et al. [ 13]\nreported that melatonin caused a regression of endometrio-\ntic lesions and an improvement in their histopathological\nscores in an oophorectomized rat endometriosis model.\nMelatonin has been proved to be involved in the\nmodulation of EMT. Lipopolysaccharide- induced EMT\nwas inhibited by melatonin in peritoneal mesothelial\ncells via the inactivation of the Toll-like receptor (TLR)\n4/c-Jun N-terminal kinase and TLR4/NF κB-Snail signal-\ning pathways [ 17]. It has also been reported that mela-\ntonin inhibits TGF β1-induced EMT in human lung\nalveolar epithelial cells [ 18]. In the process of\nbleomycin-induced pulmonary fibrosis, melatonin sig-\nnificantly attenuated endoplasmic reticulum\nstress-mediated EMT [ 19]. However, the effects of mela-\ntonin on the EMT in endometriosis are unclear.\nIn the current study, we assumed that melatonin\nmight be involved in the EMT regulation of endometri-\nosis. We investigated the effect of melatonin on the mi-\ngration, invasion and EMT of normal and endometriotic\nepithelial cells, and explored the possible signaling path-\nways that might be involved.\nMethods\nMaterials\nCollagenaseΙA, trypsin, Melatonin and Matrigel were\nobtained from Sigma-Aldrich (St. Louis, MO, USA).\nPenicillin, DMEM/F12 (1:1) media were obtained from\nHyClone (Logan, Utah, USA). Charcoal-stripped fetal\nbovine serum (FBS) was obtained from GIBCO (Invitro-\ngen, NY, USA). Rabbit anti-human E-Cadherin,\nN-Cadherin and Vimentin primary antibodies were ob-\ntained from Abcam (Cambridge, MA, USA). Rabbit\nanti-human Notch, Numb, Slug and Snail primary anti-\nbodies were obtained from Cell Signaling Technology\n(Danvers, MA, USA) for western blot and obtained from\nAbcam (Cambridge, MA, USA) for immunohistochemis-\ntry. Mouse anti-human β-Actin primary antibody and\nGoat anti-rabbit and Goat anti-mouse HRP-conjugated\nsecondary antibodies were obtained from ZSGB-BIO\n(Beijing, China). Mammalian Cell Protein Extraction Kit\nwas purchased from Beyotime (Shanghai, China). ECL\nPlus Western Blotting Detection System was obtained\nfrom Millipore Corporation (Billerica, MA, USA).\nTissue collection and immunohistochemistry analysis\nNormal endometria were obtained from 30 women of\nreproductive age undergoing bilateral tubal ligation (pro-\nliferative phase: n = 15; secretory phase: n = 15). Endo-\nmetriotic eutopic endometria were obtained from 30\npatients with endometriosis undergoing hysterectomy or\nsubtotal hysterectomy (proliferative phase: n = 15;\nsecretory phase: n = 15). Normal and endometriotic\nendometrial tissues were collected during the surgery.\nThe diagnosis of endometriosis was confirmed by histo-\nlogical examination. No patients had received any hor-\nmonal therapy in the 3 months prior to their surgery.\nImmunohistochemistry analysis was performed on\nnormal and endometriotic endometria as previously\nreported [ 8]. The primary antibodies used in this study\nincluded Rabbit anti-human E-Cadherin(4 μg/ml),\nN-Cadherin(2 μg/ml), Notch1(400 μg/ml), Numb(7 μg/\nml), Slug(7 μg/ml), and Snail(5 μg/ml) antibodies. Im-\nmunostaining was expressed as the immunoscore, i.e.,\nthe H-score, which was a semi quantitative product of\nthe quantity score and staining intensity. The quantity\nscore was estimated as reported previously [ 8].\nTissue collection and cells culture\nEutopic endometria were obtained from 6 patients with\nendometriosis. Normal endometria obtained from 6\nwomen of reproductive age undergoing bilateral tubal\nligation were used as controls. All participants had regu-\nlar menstrual cycles and had not received any hormonal\ntherapy in the 3 months prior to their surgery. Diagno-\nses were confirmed by histological examination. Endo-\nmetriotic eutopic epithelial cells (EEC) and normal\nendometrial epithelial cells (NEC) were isolated from\nfresh tissues. Isolation and culture of endometrial cells\nwere conducted as reported previously [ 20]. Briefly,\nspecimens obtained during surgery were placed\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 2 of 12\n\nimmediately in ice-cold sterile PBS and transported to\nthe laboratory. Tissues were washed twice with sterile\nPBS to remove the blood, minced into small pieces, and\nincubated with 0.25% collagenase type IA in a shaking\nwater bath for 1 h at 37 °C. The collagenase activity was\nterminated by adding three volumes of pre-warmed\nmedium containing 10% FBS. The cell suspension was\nsequentially filtered through a 154 μm monofilament\nnylon mesh and then through a 38.5 μm monofilament\nnylon mesh. The 38.5 μm monofilament was washed\nthoroughly upside down with medium to obtain epithe-\nlial cells. The resulting cell suspension was collected and\ncentrifuged at 110 g for 10 min. The pellet was\nre-suspended in DMEM/F12 (1:1) medium containing\n10% FBS and was incubated in cell culture dishes for 2 h\nat 37 °C in 95% air and 5% CO 2. The medium was then\nreplaced with fresh medium; non-attached cells were\ndiscarded, and the attached epithelial cells were cultured\nfurther. The culture medium was changed every 2 –\n3 days. The cultured cells were characterized by im-\nmunocytochemical staining with mouse anti-human\nCytokeratin antibodies, the purified epithelial cells were\npositive for Cytokeratin [ 21]. The purity for cells of 1\npassage was more than 98%. The primary cultured cells\nwere used in the western blot and morphology experi-\nments. The cells of passage 1 were used in the CCK-8\nand transwell experiments.\nCell treatments\nThe cells were initially cultured without any estradiol,\nand were cultured about 48 h until passage. The culture\nmedia was changed every 2 to 3 days. The cells were\nabout 50% confluent before treatment. Melatonin was\ndissolved in ethanol at a stock concentration of 100 mM\nand was stored at − 20 °C. 17 β-estradiol was dissolved in\nethanol at a stock concentration of 10 mM and stored at\n− 20 °C. DAPT, a specific Notch inhibitor, was dissolved\nin DMSO at a stock concentration of 10 mM and stored\nat − 20 °C. The concentrations chosen for the used treat-\nments were determined according to published\nliteratures. The cells were treated with 1 mM melatonin\n[22, 23], 10 μM DAPT [ 24, 25] or/and 10 nM\n17β-estradiol [ 26, 27]. Mock treatments with an identical\nvolume of ethanol or DMSO were used as controls.\nTranswell assays\nThe transwell assays were performed using 24-well\nplates with 8- μm pore size inserts (Corning Life\nSciences, NY, USA) according to the manufacturer ’s in-\nstructions. The cells were treated with various agents at\nthe indicated concentration for 48 h before they were\nseeded into the inserts. The migration and invasion as-\nsays were performed as reported previously [ 6].\nIn the migration assay, the cells (normal and endome-\ntriotic epithelial cells 10 5cells/well) were added to the\nupper chamber in 200 μL of serum-free DMEM medium\nand were allowed to migrate to the bottom compart-\nment, which contained DMEM medium with 10% FBS,\nfor 24 h. Then, the non-migrated cells were wiped off\nwith a cotton swab.\nFor the invasion assay, Matrigel (1 mg/ml, BD\nBiosciences) was prepared in serum-free cold cell culture\nmedium, placed in the upper chamber, and incubated\nfor 5 h at 37 °C. Next, the cells (normal and endometrio-\ntic epithelial cells 2 × 10 5cells/well) were placed in the\nupper chamber of each insert in 200 μL of serum-free\nmedium, and were allowed to invade to the bottom\ncompartment, which contained medium with 10% FBS,\nfor 36 h. Then, the non-invaded cells were wiped off\nwith a cotton swab.\nFor quantification, transwell filters were fixed in 4%\nparaformaldehyde for 15 min, stained with haematoxylin\nfor 15 min, and mounted on a glass slide. The results\nwere expressed as the number of cells migrated per field,\nas viewed under a microscope (× 200 magnification), and\nthe numbers of cells in three randomly selected fields\nwere counted. All experiments were performed three\ntimes.\nCell proliferation assay\nCell proliferation was assessed using the Cell Counting\nKit (CCK)-8 (Dojindo, Japan). Briefly, the cells (normal\nand endometriotic epithelial cells 8 × 10 3 cells /well)\nwere plated on 96-well plates in 100 μL of medium and\nwere allowed to attach overnight for 24 h. Then, they\nwere exposed to the indicated concentrations of\nmelatonin and DAPT, with or without 17 β-estradiol, and\nwere cultured for an additional 0, 24, 48 and 72 h, re-\nspectively. After treatments, 10 μL of CCK-8 reagent\nwas added to each well, and the plates were incubated at\n37 °C for 4 h. The cell confluence was about 50 –60%\nconfluence at 0 h, and allowed to grow to about 90%\nconfluence at 72 h. The optical density (OD) at 450 nm\nwas measured in each well using a microplate reader.\nThe measurements were performed at the density of\n90% confluence approximately. The data were shown as\nfold change of control (Day 0). The experiments were\nrepeated three times, and each assay was performed in\ntriplicate.\nTotal protein extraction and western blot analysis\nCells were harvested by trypsinization and centrifuga-\ntion. Total protein was extracted, and western blot\nanalysis was performed as reported previously [ 20].\n50 μg of total protein extracted from cells after 72 h\ntreatments was applied to a 10% polyacrylamide gel, and\nPageRuler Prestained Protein Ladder (Fermentas) was\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 3 of 12\n\nused as the size marker. After the proteins were trans-\nferred to membranes and the membranes were blocked,\nthe membranes were incubated overnight at 4 °C with\nthe primary antibodies. The primary antibodies used in\nthis study included Rabbit anti-human E-Cadherin\n(0.3 μg/ml), N-Cadherin(1 μg/ml), Vimentin(0.2 μg/ml),\nNotch1(0.85 μg/ml), Numb(0.26 μg/ml), Slug(0.25 μg/\nml), Snail(0.2 μg/ml) and β-actin (0.5 μg/ml). After incu-\nbation with the Goat anti-rabbit HRP-conjugated sec-\nondary antibody (0.1 μg/ml) for 1 h at room\ntemperature, the protein bands were detected using the\nECL detection system (BD Biosciences). β-actin was used\nas the loading control.\nStatistical analysis\nThe statistical analyses were performed using SPSS 19.0\n(SPSS, Chicago, USA). The values are expressed as the\nmeans ± SD. The differences between the two groups\nwere determined by one-way ANOVA. A p value < 0.05\nwas considered statistically significant.\nResults\nAberrant expression of notch/numb signaling and EMT\nmarkers in endometriotic endometrium\nThe expression of Notch/Numb signaling and EMT\nmarkers in normal endometria and in endometriotic\neutopic endometria were determined by immunohisto-\nchemical analysis. We take Notch1 as the representative\nof Notch family. As shown in Fig. 1, in normal endome-\ntria, the staining of Notch1 (NICD) (Fig. 1A),\nN-cadherin (Fig. 1B), and Slug (Fig. 1C) were weakly\npositive or positive and were concentrated in the cyto-\nplasm of endometrial epithelial cells. In stromal cells,\nthe immunostainings of Notch1, N-cadherin, and Slug\nwere very weak. In endometriotic eutopic endometria,\nthe immunostaining of Notch1 (Fig. 1D), N-cadherin\n(Fig. 1E), and Slug (Fig. 1F) was strongly positive and\nwas restricted to the cytoplasm of epithelial cells,\nwhereas weak immunostaining patterns were observed\nin stromal cells. Endometriotic eutopic endometria\nshowed significantly increased Notch1 (Fig. 1a, p< 0.05),\nN-cadherin (Fig. 1b, p < 0.05), and Slug (Fig. 1c, p <\n0.05) expression levels compared to normal endometria.\nNo significant differences in Notch1 (Fig. 1d, p > 0.05),\nN-cadherin (Fig. 1e, p > 0.05), or Slug (Fig. 1f, p > 0.05)\nexpression were observed between endometriotic endo-\nmetria in the proliferative and secretory phases.\nIn normal endometria, the staining of Snail was weakly\npositive or positive and was restricted to the nucleus of\nendometrial epithelial cells (Fig. 1G). In stromal cells,\nthe immunostaining of Snail was very weak. In endome-\ntriotic eutopic endometria, the immunostaining of Snail\nwas strongly positive and was restricted to the nucleus of\nepithelial cells (Fig. 1H), whereas weak immunostaining\nwas observed in stromal cells. Endometriotic eutopic en-\ndometria showed significantly increased Snail expression\ncompared to normal endometria (Fig. 1g, p <0 . 0 5 ) . N o\nsignificant difference of Snail expression was observed be-\ntween endometriotic endometria in the proliferative and\nsecretory phases (Fig. 1h, p >0 . 0 5 ) .\nIn normal endometria, the immunostaining of Numb\n(Fig. 1I) and E-cadherin (Fig. 1J) was strongly positive,\nand the staining was concentrated in the cytoplasm of\nendometrial epithelial cells. In stromal cells, the immu-\nnostaining of Numb and E-cadherin was very weak. In\nendometriotic eutopic endometria, the immunostaining\nof Numb (Fig. 1K) and E-cadherin (Fig. 1L) was weakly\npositive and was restricted to the cytoplasm of epithelial\ncells. Endometriotic eutopic endometria showed signifi-\ncantly decreased Numb (Fig. 1i, p < 0.01) and E-cadherin\n(Fig. 1j, p < 0.01) expression compared to normal\nendometria. No significant difference in Numb (Fig. 1k,\np > 0.05) and E-cadherin (Fig. 1l, p > 0.05) expression\nwas observed between endometriotic endometria in the\nproliferative and secretory phases.\nMelatonin abolished 17 β-estradiol-induced proliferation\nin normal and endometriotic epithelial cells\nCCK-8 assays were performed to determine the prolifer-\nation of EEC and NEC. 17 β-estradiol significantly in-\ncreased the growth of EEC and NEC on days 2 –3\n(Fig. 2a, b , p < 0.05). DAPT, a specific inhibitor of Notch\nsignaling, significantly decreased the growth of both\nEEC and NEC (Fig. 2a, b , p < 0.05). DAPT also abolished\n17β-estradiol-induced cell growth (Fig. 2a, b , p < 0.05).\nIn CCK-8 assays, melatonin significantly decreased the\ngrowth of EEC and NEC at day 3 (Fig. 2a, b , p < 0.05).\nMelatonin also abolished 17 β-estradiol-induced cell\ngrowth in both cells (Fig. 2a, b , p < 0.05).\nMelatonin abolished 17 β-estradiol-induced migration and\ninvasion in normal and endometriotic epithelial cells\nThe migration and invasion of EEC (Fig. 3) and NEC\n(Fig. 4) were determined using transwell assays. In mi-\ngration assays, 17 β-estradiol significantly increased the\nmigration of EEC ( p < 0.01) and NEC ( p < 0.01) after\ntreatment for 24 h. DAPT significantly decreased the mi-\ngration of EEC ( p < 0.05) and NEC (p < 0.05). DAPT also\nabolished 17 β-estradiol-induced cell migration ( p < 0.01).\nSimilar data were obtained in invasion assays.\n17β-estradiol significantly increased the invasion of EEC\n(p < 0.01) and NEC (p < 0.01) after treatment for 36 h,\nwhereas DAPT significantly decreased the invasion and\n17β-estradiol-induced invasion in EEC ( p < 0.01) and\nNEC (p < 0.01).\nIn migration and invasion assays, melatonin\nsignificantly decreased the migration and invasion of\nEEC (Fig. 3, p < 0.05) and NEC (Fig. 4, p < 0.05).\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 4 of 12\n\nMelatonin also abolished 17 β-estradiol-induced cell mi-\ngration and invasion ( p < 0.05).\nMelatonin abolished 17 β-estradiol-induced EMT in normal\nand endometriotic epithelial cells\nThe expression of EMT markers was determined using\nwestern blot analysis. As shown in Fig. 5,1 7 β-estradiol\nsignificantly increased the expression of N-cadherin,\nSlug and Snail, and decreased the expression of Numb\nand E-cadherin in EEC ( p < 0.05). In NEC, 17 β-estradiol\nsignificantly increased the expression of N-cadherin and\ndecreased the expression of Numb (Fig. 6, p < 0.05), but\nshowed no significant effect on the expression of\nNotch1 (NICD), Vimentin, E-cadherin, Slug, or Snail\n(Fig. 6, p > 0 . 0 5 ) .I nE E C ,D A P Ts i g n i f i c a n t l y\ndecreased the expression of Notch1 (NICD), Vimen-\ntin, Slug, and Snail and increased the expression of\nNumb and E-cadherin (Fig. 5, p < 0.05). DAPT also\nabolished 17 β-estradiol-induced expression of Notch1\n(NICD), Vimentin, Slug, and Snail and the downregulation\nFig. 1 Aberrant expressions of Notch1/Numb signaling and EMT markers in endometrium of endometriosis. A, B, C, G, I, J : The expression of\nNotch1, N-Cadherin, Slug, Snail, Numb and E-Cadherin in normal endometrium ( n = 15); D, E, F, H, K, L : The expression of Notch1, N-Cadherin,\nSlug, Snail, Numb and E-Cadherin in eutopic endometria of endometriosis ( n = 15); a, b, c, g, i, j : Immunoscore of Notch1, N-Cadherin, Slug, Snail,\nNumb and E-Cadherin in normal endometrium ( n = 30) and eutopic endometria of endometriosis ( n = 30); d, e, f, h, k, l : Immunoscore of Notch1,\nN-Cadherin, Slug, Snail, Numb and E-Cadherin in the proliferative phases ( n = 15) and secretory phases ( n = 15) of endometriotic endometria;\n1,2,3,4,5,6: negative controls. Magnification: × 200. * p < 0.05\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 5 of 12\n\nFig. 2 Melatonin abolishes 17 β-estradiol-induced proliferation in EEC and NEC. a: EEC were treated with MLT, DAPT, or E2 with or without MLT/\nDAPT, cell numbers were measured by CCK-8 assays at the indicated times. b: NEC were treated with MLT, DAPT, or E2 with or without MLT/\nDAPT, cell numbers were measured by CCK-8 assays at indicated times. Data are presented as the mean ± SD. E2: 17 β-estradiol; MLT: melatonin\nFig. 3 Melatonin abolishes 17 β-estradiol-induced migration and invasion in EEC. a: Transwell migration assays of EEC after treatment. EEC were\ntreated with MLT, DAPT, or E2 with or without MLT/DAPT b: Transwell invasion assays of EEC after treatment. Representative images were\nobtained at 200× magnification. Graphs show the relative number of migrating and invading cells for each treatment group (averaged across\nfour random images). Scale bar: 50 μm. Data are presented as the mean ± SD. E2: 17 β-estradiol; MLT: melatonin. * p < 0.05, ** p < 0.01 vs.\nuntreated cells. # p < 0.05, ##p < 0.01 vs. E2-treated cells\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 6 of 12\n\nFig. 4 Melatonin abolishes 17β-estradiol-induced migration and invasion in NEC.a: Transwell migration assays of NEC after treatment. NEC were treated with\nM L T ,D A P T ,o rE 2w i t ho rw i t h o u tM L T / D A P Tb: Transwell invasion assays of NEC after treatment. Representative images were obtained at 200× magnification.\nGraphs show the relative number of migrating and invading cells for each treatment group (averaged across four random images). Scale bar: 50μm. Data are\npresented as the mean ± SD. E2: 17β- e s t r a d i o l ;M L T :m e l a t o n i n .*p <0 . 0 5 ,* *p < 0.01 vs. untreated cells. #p < 0.05, ##p < 0.01 vs. E2-treated cells\nFig. 5 Melatonin reverses 17 β-estradiol-induced EMT in EEC involving Notch1 signaling pathway. EEC were treated with different drugs for 72 h.\na The protein expression levels of Notch1 (NICD), E-cadherin, N-cadherin, Vimentin, Slug, Snail, Numb and Actin were determined by western\nblot. β-actin was used as a loading control. b The ratios of Notch1/E-cadherin/N-cadherin/Vimentin/Slug/Snail/Numb to β-actin were analyzed.\nData are presented as the mean ± SD. E2: 17β-estradiol; MLT: melatonin. *p < 0.05, ** p < 0.01 vs. untreated cells. #p < 0.05, ##p < 0.01 vs. E2-treated cells\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 7 of 12\n\nof Numb and E-cadherin (Fig. 5, p <0 . 0 5 ) .I n N E C , N o t c h\ninhibition by DAPT significantly increased the expression\nof Numb, and 17 β-estradiol-caused downregulation of\nNumb (Fig. 6, p <0 . 0 5 ) .\nIn western blot analyses, melatonin significantly de-\ncreased the expression of Notch1 (NICD), Vimentin,\nSlug, and Snail and increased the expression of Numb\nand E-cadherin in EEC (Fig. 5, p < 0.05). Melatonin also\nabolished the 17 β-estradiol-induced expression of\nNotch1 (NICD), Vimentin, Slug, and Snail and the\ndownregulation of Numb and E-cadherin in EEC (Fig. 5,\np < 0.05). In NEC, melatonin significantly increased the\nexpression of Numb (Fig. 6, p < 0.05) but had no signifi-\ncant effect on the expression of Notch1 (NICD), Vimen-\ntin, N-cadherin, E-cadherin, Slug or Snail. Melatonin\nalso abolished the 17 β-estradiol-induced expression of\nVimentin and downregulation of Numb in NEC (Fig. 6,\np < 0.05).\nWe then examined the effect of 17 β-estradiol and\nmelatonin on the morphology of normal and endome-\ntriotic epithelial cells. The cells were treated with recom-\nbinant transforming growth factor- β1 (TGF- β1), which\nis known to be an EMT inducer. As expected, after\nstimulation with 0.78 nM of recombinant TGF- β1 for\nFig. 6 The expression of EMT and Notch signaling pathway-related markers in NEC. NEC were treated with different drugs for 72 h. a The protein\nexpression of Notch1 (NICD), E-cadherin, N-cadherin, Vimentin, Slug, Snail, Numb and Actin were determined by western blot. β-actin was used\nas a loading control. b The ratios of Notch1/ E-cadherin/ N-cadherin/Vimentin/Slug/ Snail/Numb to β-actin were analyzed. Data are presented as\nthe mean ± SD. E2: 17 β-estradiol; MLT: melatonin. * p < 0.05, ** p < 0.01 vs. untreated cells. # p < 0.05, ##p < 0.01 vs. E2-treated cells\nFig. 7 The morphology of EEC treated with E2, TGF- β1, Melatonin, DAPT, a combination of E2 and Melatonin, or a combination of E2 and DAPT\nfor 48 h. The cells were observed using phase contrast microscopy at 400× magnification. Scale bar: 20 μm.E2: 17β-estradiol; MLT: Melatonin\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 8 of 12\n\n48 h, both EEC (Fig. 7) and NEC (Fig. 8) showed reduc-\ntion of the epithelial “cobblestone” morphology, became\nscattered, acquired a spindle-shaped morphology, and\nlost cell-cell contacts, all of which are characteristics of a\nmesenchymal-like morphology. 17 β-estradiol exhibited\nsimilar effects as TGF- β1 in the cells. Treatment with\n1 mM melatonin or 10 μM DAPT for 48 h abolished the\nTGF-β1o r1 7 β-estradiol-induced morphological changes\nin EEC and NEC.\nDiscussion\nAlthough endometriosis is a benign disease, it exhibits a\nseries of biological behaviors similar to malignant\ntumors, including adhesion, invasion, and implantation\n[28]. In the current study, we found that aberrant ex-\npression of EMT-related markers existed in endometrio-\ntic eutopic endometrium, and estrogen promoted the\nmigration, invasion and EMT phenotype in normal and\nendometritic eutopic epithelial cells, while melatonin\nand the blocking of Notch signaling inhibited\n17β-estradiol-induced migration, invasion and\nepithelial-mesenchymal transition in normal and endo-\nmetriotic endometrial epithelial cells.\nThe Notch signaling pathway is thought to be critical\nfor the induction of EMT and is involved in the progres-\nsion of a series of diseases [ 29, 30]. Notch signaling can\npromote TGF- β1-induced EMT via the induction of\nSnai1 [ 30]. Jagged1-mediated Notch signaling activation\ncan elevate the expression of Snail and Slug, resulting in\nthe repression of E-cadherin in various disease models\n[29]. Numb is an inhibitory regulator of Notch1 signal-\ning that acts by promoting the ubiquitination and deg-\nradation of the Notch1 intracellular domain [ 31]. In the\ncurrent study, decreased epithelial marker expression\nand elevated mesenchymal marker expression indicate\nthat the phenotype of EMT exists in endometriosis. In\naddition, elevated expression of EMT inducer, Snail and\nSlug, was also noted in endometriotic eutopic endomet-\nrium, suggesting an essential role of EMT in the\ndevelopment and pathogenesis of endometriosis. Immu-\nnohistochemistry analysis also showed increased Notch1\n(NICD) expression and decreased Numb expression in\nendometriotic eutopic epithelial cells, indicating that\nNotch1/Numb signaling might be involved in the regula-\ntion of EMT in the endometriotic eutopic endometrium.\nFurthermore, there is no significant difference between\nproliferative and secretory phases observed in endomet-\nriosis. In our previous studies, we found that the expres-\nsion of Numb, Slug and E-cadherin showed no\nsignificant difference, but Snail, N-cadherin and Notch1\nshowed cycle changement between proliferative and\nsecretory phases in normal endometria. This might be\ndue to the aberrant level of hormones in endometriosis.\nVimentin is a mesenchymal cell marker and is in-\ncreased in cells that undergo EMT as known. In im-\nmunochemistry assays, cytokeratin immunostaining is\npositive in normal and endometriotic epithelial cells,\nwhile Vimentin immunostaining was negative [ 20]. In\nwestern blot experiments, we found that Vimentin\nshowed bands in both cells and showed differences in\ndrug intervention. Then, we searched other literature\nand found that Vimentin was also determined in endo-\nmetrial epithelial cells by western blot [ 32, 33]. We can-\nnot explain this phenomenon and will continue to\nexplore it in follow-up studies.\nEndometriosis is an estrogen-dependent disease [ 34].\nConsiderable biochemical evidence demonstrates that\naromatase activity and P450 aromatase mRNA\nexpression were noted in endometrial tissues from endo-\nmetriosis [ 35], suggesting that endometriotic tissues are\nable to produce estrogens locally. It has been reported\nthat estrogen receptor (ER α) signaling regulates\nE-cadherin and EMT through slug, and estrogen is\nproved to be involved in the process of EMT [ 36]. In hu-\nman ovarian and breast cancer cells, 17 β-estradiol can\ninduce EMT via the activation of the PI3K/AKT pathway\nby enhancing the expression of snail and slug [ 37, 38].\nIn prostate epithelium, the ER α-mediated enhanced\nFig. 8 The morphology of NEC treated with E2, TGF- β1, Melatonin, DAPT, a combination of E2 and Melatonin, or a combination of E2 and DAPT\nfor 48 h. The cells were observed using phase contrast microscopy at 400× magnification. Scale bar: 20 μm.E2: 17β-estradiol; MLT: Melatonin\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 9 of 12\n\nestrogenic effect is a crucial inductive factor of epithelial\ndedifferentiation, giving rise to the activation of an EMT\nprogram [ 39]. However, the role of estrogen in the EMT\nof endometriosis is rarely reported. In the current study,\nwe found that estrogen promoted the migration, inva-\nsion and mesenchymal phenotype in normal and endo-\nmetritic eutopic epithelial cells, suggesting that estrogen\nplays a role in the regulation of EMT in endometrial epi-\nthelial cells. What ’s more, 17 β-estradiol activated the\nNotch pathway, which is a key signaling pathway in\nEMT regulation, and downregulated the expression of\nNumb, an inhibitory regulator of Notch signaling. In\naddition, DAPT, a specific Notch inhibitor, abolished the\neffect of 17 β-estradiol in endometrial epithelial cells,\nsuggesting that Notch signaling might participate in the\neffect of 17 β-estradiol on migration, invasion and\nEMT-related markers.\nReversing the migration and invasion of eutopic\nendometrium should be meaningful for the preven-\ntion and treatment of endometriosis. In in-vivo stud-\nies, melatonin showed potential therapeutic effects on\nendometriosis in animal models [ 40–42]. However,\nthe molecular mechanism of the melatonin effect re-\nmains unclear. In animal studies, melatonin signifi-\ncantly inhibits ovarian aromatase expression and\nincreases the levels of uterine ER α and progesterone\nreceptor [ 43]. The genotoxic changes in the uterus\ncaused by estrogen might be prevented by giving\nmelatonin [ 44]. The addition of melatonin to estrogen\nreplacement treat ment is associated with a decrease in\nendometrial proliferation and prevents the appearance of\ncellular atypia [ 45]. In non-photoperiodic animals such as\nrats, melatonin positively affects the endometrial morph-\nology and improves embryo implantation [ 46]. These data\nindicate that the effect of 17 β-estradiol can be modified by\nmelatonin. In the current study, melatonin abolished the\n17β-estradiol-induced proliferation, migration, invasion\nand EMT phenotype in both normal and endometriotic\nepithelial cells, demonstrating the protective effect of\nmelatonin on the endometrium, especially on endo-\nmetriotic endometrium, which has locally elevated es-\ntrogen levels [ 35].\nIn the current in-vitro study, melatonin showed a\nsimilar effect on normal and endomotriotic epithelial\ncells. However, in the local high estrogen microenviron-\nment of endometriosis in vivo, melatonin may show\nanti-estrogen effects and therefore has a potential thera-\npeutic effect.\nNotch signaling pathway is a key signaling pathway for\nEMT regulation. Here, we found that Notch signaling\npathway is also involved in EMT regulation in endomet-\nriosis. We showed that a specific inhibitor of Notch sig-\nnaling pathway inhibited the proliferation, migration,\ninvasion and in endometriotic epithelial cells and normal\nendometrial cells, but EMT-like phenotype was not\ninhibited in normal endometrial cells. The data sug-\ngested that Notch signaling plays a key role in the regu-\nlation of EMT in endometriosis. Melatonin can inhibit\nthe activity of Notch1 signaling pathway in endometrio-\ntic epithelial cells, which is reflected in the decrease of\nNICD expression. This phenomenon was absent in nor-\nmal endometrial epithelial cells. There is insufficient evi-\ndence that the Notch signaling pathway is a direct\npathway for melatonin action or that it is the only down-\nstream pathway for melatonin action in endometriosis.\nHowever, the data suggest that Notch signaling pathway\nmay be a potential therapeutic target in endometriosis.\nStudies have reported that Numb completely prevents\nEMT by antagonizing Notch signaling [ 47, 48]. In the\npresent study, Melatonin and Notch-specific inhibitor\npromotes the expression of Numb in both normal and\nendometriotic endometrium, indicating that the effect of\nmelatonin on Notch signaling might be mediated by\nNumb upregulation. We also proved that Numb expres-\nsion was decreased in endometrium of endometriosis,\nimplying that Numb might be a potent therapeutic tar-\nget in endometriosis.\nConclusions\nIn summary, we confirm that aberrant expression of\nNotch1/Numb signaling and an active EMT process are\npresent in eutopic endometrium of endometriosis, and\nwe provide an experimental basis for considering mela-\ntonin as a potential treatment for endometriosis. In\naddition, we observed that Notch signaling pathway\nmight be involved in the progression of endometriosis.\nThe role of Notch signaling pathway in the effect of es-\ntrogen and melatonin in endometriosis need further in-\nvestigation in future studies.\nAbbreviations\nCCK-8: Cell Counting Kit-8; EEC: Endometriotic eutopic epithelial cells;\nEMT: Epithelial-mesenchymal transition; ER α: Estrogen receptor alpha;\nFBS: Charcoal-stripped fetal bovine serum; MMPs: Matrix metalloproteinases;\nNEC: Normal endometrial epithelial cells; NF κB: Nuclear factor- κB;\nNICD: Notch1 intracellular domain; NSC: Normal stromal cells; OD: Optical\ndensity; TLR: Toll-like receptor\nAcknowledgements\nWe are grateful to all the participants involved in this study. We thank\nAmerican Journal Experts for the language editing.\nFunding\nThis research was supported by grants from the National Natural Science\nFoundation of China (No.81300468; NO. 81671433), a grant from Shandong\nProvince key research and development projects (No. 2016GSF201086), a\ngrant from Shandong Province medical and health technology development\nproject (2016WS0442), a grant from Science and Technology Development\nProgram of Jinan (No. 201506012), and a grant from Shandong Province\nNatural Science Foundation (No. ZR2014HM008).\nAvailability of data and materials\nThe datasets used and analyzed during the current study are available from\nthe corresponding author on reasonable request.\nQi et al. Reproductive Biology and Endocrinology  (2018) 16:62 Page 10 of 12\n\nAuthors’ contributions\nHZ participated in the study design and coordination, and edited the\nmanuscript for submission. SQ carried out the experiments, participated in\nthe interpretation of the data and drafted the manuscript. LY and ZL helped\nto revise the manuscript and performed the statistical analysis. XZ, ML and\nZC participated in the design of the study, supervised the study and critically\nhelped to draft the manuscript. All authors read and approved the final\nmanuscript.\nEthics approval and consent to participate\nInformed consent was obtained from all participants prior to biopsy and the\nuse of human tissues was approved by the Institutional Research Ethics\nCommittees of Shandong Provincial Hospital Affiliated to Shandong\nUniversity. Written informed consent was obtained from all participants\nbefore being included in the study.\nCompeting interests\nThe authors declare that they have no competing interests.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in\npublished maps and institutional affiliations.\nAuthor details\n1Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to\nShandong University, Jinan 250021, People ’s Republic of China. 2National\nResearch Center for Assisted Reproductive Technology and Reproductive\nGenetics, Jinan 250021, People ’s Republic of China. 3The Key laboratory for\nReproductive Endocrinology, Shandong University, Ministry of Education,\nJinan 250021, People ’s Republic of China. 4Department of Urology, Qilu\nHospital of Shandong University, 107 Wenhua Xi Road, Jinan 250012,\nPeople’s Republic of China. 5Department of Obstetrics and Gynecology,\nShandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu\nRoad, Jinan 250021, People ’s Republic of China. 6Shanghai Key Laboratory for\nAssisted Reproduction and Reproductive Genetics, Shanghai 200030, People ’s\nRepublic of China. 7Center for Reproductive Medicine, Ren Ji Hospital, School\nof Medicine, Shanghai Jiao Tong University, Shanghai 200030, People ’s\nRepublic of China.\nReceived: 26 December 2017 Accepted: 4 June 2018\nReferences\n1. 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