{"paper_id":"d03f3f07-2461-476d-a586-86054506f3a1","body_text":"Vol.:(0123456789)1 3\nReproductive Sciences (2023) 30:2715–2727 \nhttps://doi.org/10.1007/s43032-023-01189-w\nADENOMYOSIS: ORIGINAL ARTICLE\nThe Inactivation of Hippo Signaling Pathway Promotes \nthe Development of Adenomyosis by Regulating EMT, Proliferation, \nand Apoptosis of Cells\nTingting Jin1,3 · Mengqi Li1,3 · Ting Li1,3 · Simiao Yan1,3 · Qingzhen Ran2 · Wanqun Chen1,3 \nReceived: 22 November 2022 / Accepted: 30 January 2023 / Published online: 20 March 2023 \n© The Author(s) 2023\nAbstract\nAdenomyosis is a benign gynecological disease. The pathogenesis of adenomyosis is still unclear. The Hippo signaling \npathway is highly conserved in vivo and associated with endometriosis and various cancers. Our objective was to study \nthe expression of Hippo signaling pathway–related proteins in the uterus of mice with and without adenomyosis. We also \nsought to determine the relationship between the Hippo signaling pathway and cell migration, invasion, proliferation, and \napoptosis in adenomyosis. The inactivation of Hippo signaling pathway and abnormal expression of EMT-related proteins \nwere observed in mice with adenomyosis. In vitro, the YAP inhibitor verteporfin can inhibit the proliferation and migration of \nIshikawa cells and promote apoptosis, while inhibiting the EMT process. In addition, intraperitoneal injection of verteporfin \ninhibits EMT process and proliferation and promotes apoptosis of cells in the uterus of adenomyosis mice. It suggests that \nthe Hippo signaling pathway participates in the EMT, proliferation, and apoptosis of cells in adenomyosis. In conclusion, \nthese results suggest that Hippo signaling pathway may be involved in the development of adenomyosis by regulating EMT, \nproliferation, and apoptosis of cells, which provide a potential target for the treatment of adenomyosis.\nKeywords Adenomyosis (ADM) · YAP (Yes-associated protein) · Hippo signaling pathway · Epithelial-mesenchymal \ntransition (EMT)\nIntroduction\nAdenomyosis is a common benign chronic gynecological \ndisease [1, 2], which is characterized by abnormal growth of \nglands and stroma invading into the myometrium, accompa-\nnied with hypertrophy and hyperplasia of the myometrium \n[3]. Common clinical symptoms include increased uterine \ndiffusion, dysmenorrhea, pelvic pain, abnormal uterine \nbleeding (AUB), and infertility [4 ], which seriously affect \nthe quality of life of the patients. Currently, the pathogen-\nesis of adenomyosis is still unclear, but one of the gener -\nally accepted theories is the invagination theory, in which \nendometrial cells invade the myometrium by acquiring \ninvasiveness [5, 6]. Previous studies have demonstrated that \nepithelial-mesenchymal transition (EMT) plays an important \nrole in cell migration and invasion and is associated with the \ndevelopment of adenomyosis [7, 8].\nEMT refers to the process in which epithelial cells undergo \nphenotypic transformation under specific conditions and \nacquire a mesenchymal phenotype with stronger migra -\ntion ability [9 ]. The occurrence of EMT will promote the \nenhancement of cell invasion and migration ability, and the \nexistence of EMT can be found in various malignant tumors \n[10]. The typical molecular feature of EMT is that epithelial \ncells lose the expression of the epithelial marker (E-cadherin) \nand acquire the expression of mesenchymal markers (such as \nN-cadherin and Vimentin) [9]. In addition, a variety of tran-\nscription factors can also promote the occurrence of EMT by \n * Qingzhen Ran \n 1902518646@qq.com\n * Wanqun Chen \n tchwanq@jnu.edu.cn\n1 Department of Biochemistry and Molecular Biology, \nSchool of Medicine, Jinan University, Guangzhou 510632, \nGuangdong, China\n2 Department of Gynecology, Guangdong Provincial Hospital \nof Chinese Medicine, Guangzhou 510120, Guangdong, \nChina\n3 Engineering Technology Research Center of Drug \nDevelopment for Small Nucleic Acids, Guangzhou 510632, \nGuangdong, China\n\n2716 Reproductive Sciences (2023) 30:2715–2727\n1 3\ninhibiting the expression of E-cadherin, such as twist basic \nhelix-loop-helix transcription factor (Twist), snail family zinc \nfinger 2 (Slug), and snail family zinc finger 1 (Snail) [11]. \nSeveral studies have reported the role of EMT in adenomyosis. \nChen et al. [8] reported for the first time that estrogen-induced \nEMT is involved in the development of adenomyosis. In addi-\ntion, HGF-induced EMT may also be involved in the invagi-\nnation of glands into the myometrium in adenomyosis [12].\nThe Hippo signaling pathway was originally discovered to \nregulate the size of tissues and organs in Drosophila [13]. The \ncore components of the pathway include mammalian Ste20-\nlike 1/2 (Mst1/2) and large tumor suppressor 1/2 (Lats1/2) \nregulating transcriptional coactivators, yes-associated protein \n(YAP), and transcriptional coactivator with PDZ-binding \ndomain (TAZ) [14]. As a key protein of Hippo signaling path-\nway, the abnormal regulation of YAP is related to the occur-\nrence and development of various cancers [15, 16]. When YAP \nis phosphorylated, it is sequestered in the cytoplasm by bind-\ning to 14–3-3 protein and then degraded [17]; when YAP is \nnot phosphorylated, it can enter the nucleus and combine with \nthe transcription factor TEA domain family members (Teads) \nto promote gene transcription to regulate cell proliferation, \nmigration, and invasion [18]. Although overexpression of YAP \nhas been found in uterine endometrial-myometrial junctional \nzone (JZ) tissues of patients with adenomyosis [19], there is \nstill a lack of corresponding research on its molecular mecha-\nnism in the pathogenesis of adenomyosis. Previous studies \nhave shown that the occurrence of EMT is often accompanied \nwith the up-regulation of YAP expression and the increase of \nYAP content in the nucleus [20]. Therefore, we speculate that \nYAP is involved in the pathological development of adeno-\nmyosis by inducing the occurrence of EMT.\nCurrently, we aimed to study the change of Hippo signal-\ning pathway and its effect on proliferation, apoptosis, and \nEMT of cells in adenomyosis. On the basis of verifying \nthat activating the Hippo signaling pathway can inhibit the \nproliferation, migration, and EMT of endometrial cells and \npromote apoptosis, we further demonstrated in vivo experi -\nments that abnormal inactivation of the Hippo signaling \npathway occurred in the uterus of mice with adenomyosis \nand demonstrated that the YAP inhibitor verteporfin can \ninhibit EMT and proliferation and promote apoptosis of cells \nin the uterus of mice with adenomyosis. Therefore, our study \nmay provide a new idea for exploring the pathogenesis of \nadenomyosis.\nMaterials and Methods\nCell Culture and Drug Treatment\nThe Ishikawa cells (Human Asia endometrial adenocarci -\nnoma cell line) were derived from a 39-year-old female with \nendometrial adenocarcinoma. They are adherent epithelioid \ncells expressing ER and PR. Prominent gene expression \nchanges in Ishikawa cells mimic physiological processes in \nnormal endometrial gland cells, and this cell line is often \nused as a model of reproductive disorders including adeno-\nmyosis [21]. The Ishikawa cells were cultivated in DMEM/\nF12 containing 10% fetal bovine serum (FBS). Verteporfin \n(MedChemExpress), a YAP inhibitor, was dissolved in \ndimethyl sulfoxide (DMSO) and stored at − 20 °C. In vitro, \nIshikawa cells were treated with fresh medium or different \nconcentrations of verteporfin.\nCCK‑8 Assay\nWe used CCK-8 to detect cell proliferation. Briefly, 100 μl \nof cell suspension containing 5 ×  103 cells was seeded in \n96-well plate and cultured for 24 h. Cells were treated with \ndifferent concentrations of verteporfin for 24 h. Then, 10 μl \nof CCK-8 solution was added to each well and incubated at \n37 °C for 4 h. The absorbance at 450 nm was measured with \na microplate reader.\nColony Formation Assay\nIshikawa cells were seeded in six-well plates at a density \nof 500 cells/well. Thereafter, the cells were cultured for \n14 days to form colonies, during which time the medium \nwas changed every 3 days. After that, cells were fixed with \n4% paraformaldehyde for 30 min and stained with 0.1% \ncrystal violet.\nWound Healing Assay\nWound healing assay was used to detect cell migration. Ishi-\nkawa cells were seeded in six-well plates at a density of \n5 ×  105 cells/well. After culturing the cells until the conflu-\nence reaches 80–90%, 200 μl pipette tips were used to draw \na straight line. Excess cells were washed with phosphate \nbuffered saline (PBS). The cells were cultured in serum-\nfree medium. The scratch position was observed and photo-\ngraphed under the microscope at 0, 6, and 12 h, respectively. \nImages were analyzed with Image J.\nTranswell Migration Assay\nTranswell migration assay was used to detect cell migration. \nFive hundred microliters of DMEM/F12 medium contain-\ning 10% FBS was added to a 24-well plate. After placing \nthe chamber, 5 ×  104 cells suspended in serum-free medium \nwere seeded in the chamber. After culturing for 24 h, the \ncells remaining in the upper chamber were gently removed \nwith a cotton swab. After fixation with 4% paraformalde-\nhyde, the cells that penetrated the membrane were stained \n\n2717Reproductive Sciences (2023) 30:2715–2727 \n1 3\nwith 0.1% crystal violet solution. Stained cells were counted \nunder a light microscope.\n5‑Ethynyl‑2′‑Deoxyuridine (EdU) Assay\nCell proliferation was probed by EdU detection kit (KeyGen \nBiotech, China). Verteporfin-treated Ishikawa cells were \nincubated with 10 μM EdU reagent for 3 h. Cells were then \nfixed with 4% paraformaldehyde and treated with 0.5% Tri-\nton-X-100 to permeabilize cells. Nucleic acids were stained \nwith DAPI after 0.5 h staining with the Click-iT reaction \nmix. Finally, EdU positive cells were counted and photo-\ngraphed with a fluorescence microscope (Leica, Germany).\nFlow Cytometry Analysis\nApoptotic cells were detected by using the Annexin-V-\nAPC/PI double staining kit (KeyGen Biotech, China). After \ndetaching the verteporfin-treated cells from the six-well \nplate with 0.25% trypsin (without EDTA) (Gibco, USA), \nthey were washed twice with PBS and centrifuged (2000 g, \n5 min). Cells were then resuspended in binding buffer and \nstained with Annexin-V-APC and PI for 30 min in the dark. \nFinally, we detected stained cells by using flow cytometry \nand analyzed apoptosis with FlowJo X software.\nAnimals and Experiment Protocol\nAll experiments were performed in accordance with the \nguidelines of the National Research Council’s Guide for the \nCare and Use of Laboratory Animals and approved by the \nLaboratory Animal Review Committee of Jinan University \n(Approval number: IACUC-20200905–01).\nWe established the mouse model of adenomyosis by using \ntamoxifen [22]. Six female (7-week-old) and three male \n(8-week-old) ICR mice were purchased from Beijing Huafu-\nkang Company. Under controlled conditions (20 °C, 12-h \nlight–dark cycle), two female mice and one male mouse were \nhoused in the same cage with ad libitum access to water and \nfood. After the female mice became pregnant, each female \nmouse was housed in a separate cage, and the newborn mice \nwere used for subsequent modeling experiments.\nThe neonatal female mice were randomly divided into \ntwo groups: tamoxifen (TAM) and control group. Mice in \nthe TAM group were given oral tamoxifen to induce adeno-\nmyosis, and the control group was given only the vehicle \nin the same way. Mice in the TAM group were fed 1 mg/kg \nof tamoxifen suspended in a mixture of peanut oil/lecithin/\ncondensed milk (2:0.2:3 by volume) at a dose of 5 µl/g body \nweight per day from day 2 to 5 of birth. At the same time, \nthe control group was fed the same dose of solvent without \ntamoxifen. When female mice were grown to day 21, they \nwere weaned and separated from dams.\nMice were housed under normal conditions for 60 days. \nThree mice were randomly selected from each of the control \ngroup and the TAM group and sacrificed. The mouse uterus \nwere collected for paraffin embedding to make paraffin sec-\ntions. Histological examination was performed by hematoxy-\nlin and eosin (H&E) staining to verify whether the modeling \nwas successful.\nAfter successful modeling, five mice were randomly \nselected from each of the control group and the TAM group \nas the control group and the adenomyosis group in the next \nphase of the study. The remaining mice were randomly \ndivided into three groups: control group ( n = 5), adeno-\nmyosis group (n  = 5), and verteporfin group (n  = 5). Mice \nin the verteporfin group were intraperitoneally injected \nwith 50 mg/kg of verteporfin, and the control group and \nadenomyosis group were given the same dose of solvent. \nAll mice were administered consecutively for 7 days and \nsacrificed by cervical dislocation on the first day after the \nlast administration.\nProtein Extraction and Western Blot Analysis\nFresh tissues or cells were added to RIPA lysis buffer, \nsonicated, lysed on ice for 30 min, centrifuged at 12,000 g \nat 4 °C for 15 min to extract total protein, and quantified \nwith BCA protein detection kit (Beyotime, China). Twenty \nmicrograms of protein was loaded on an SDS-PAGE gel, \nwhich was then transferred to a PVDF membrane (Milli-\npore, USA). After blocking in 5% nonfat milk (BD, USA) \nfor 1 h at room temperature, the membrane was incubated \nwith primary antibody overnight at 4 °C. Then, after wash-\ning off the primary antibody, the membrane was incubated \nwith HRP-conjugated goat anti-rabbit or mouse secondary \nantibody for 1 h at room temperature. Protein expression was \ndetermined using a chemiluminescence system and analyzed \nwith Imaje J software.\nImmunohistochemical Analysis\nParaffin sections were subjected to routine dewaxing and \nrehydration procedures. The sections were boiled with \ncitric acid antigen retrieval solution (pH = 6.0) for 15 min \nfor antigen retrieval. Endogenous peroxidase activity was \nremoved by 3%  H2O2. Ten percent goat serum was used to \nblock nonspecific binding for 1 h at room temperature. Then, \nsections were incubated with primary antibodies overnight \nat 4 °C. After washing off the primary antibody, sections \nwere then incubated with the secondary antibody for 2 h \nat room temperature. Immunoreactive proteins were visual-\nized by using 3, 3-N-Diaminobenzidine tetrahydrochloride \n(Sangon Biotech, China). Sections were then counterstained \nwith hematoxylin. Finally, sections were observed and pho-\ntographed under an optical microscope (Nikon, TOKYO).\n\n2718 Reproductive Sciences (2023) 30:2715–2727\n1 3\nTUNEL Assay\nThe apoptosis of mouse uterine tissue cells was detected by \none-step TUNEL Apoptosis Detection Kit (Keygen Biotcch, \nChina). After routine deparaffinization and hydration of sec-\ntions, permeabilization of cells was increased with protease \nK (10 ×). Then, the sections were incubated with the TdT \nreaction mixture (Equilibration Buffer:biotin-11-Dutp:TdT \nEnzyme = 45:1:5) in a humidified chamber for 60 min at \n37 °C. After PBS solution washing, the sections were incu-\nbated with Streptavidin-Fluoresce solution (Streptavidin-\nFluoresce solution:Labeling Buffer  = 1:9) for 30 min at \n37 °C, away from light. The sections were then counter -\nstained with DAPI for 10 min before sample analysis by \nfluorescence microscopy (Leica, Germany).\nStatistical Analysis\nIn this experiment, three independent replicates were per -\nformed. GraphPad Prism 8.0 software was used to process \nand draw experimental data. Data of repeated experiments \nare expressed as mean  ± SD. Data statistics and analysis \nbetween two groups were compared using Student’s t -test, \nand three groups were analyzed and compared using one-\nway ANOVA test; p  < 0.05 was regarded as significant \ndifference.\nResults\nInactivation of Hippo Signaling Pathway \nand Occurrence of EMT in Adenomyosis Mice\nIn order to investigate the correlation of Hippo signal-\ning pathway and EMT with adenomyosis, we established \na mouse model of adenomyosis by instilling tamoxifen to \nnewborn ICR mice. As shown in Fig. 1a, the results of H&E \nstaining showed that the myometrium of the control group \nwas arrayed in bundles and had been well-spaced from the \nendometrium, whereas the endometrium stroma from the \nTAM group was immersed into the muscular layer that was \ndivided into multiple bundles by the stroma. In addition, in \nthe sample from the TAM group, the smooth muscle struc-\nture of the muscular layer had been notably disordered, and \nthe endometrium boundary appeared unclear. These find-\nings illustrate that the establishment of adenomyosis mice \nis successful.\nThen, we detected the expression levels of Hippo signal-\ning pathway–related proteins by western blot. As shown in \nFig.  1b, the expression level of YAP in adenomyosis mice \nwas significantly higher and the p-YAP level was lower than \nthat in control mice. In addition, the expression of other pro-\nteins of the Hippo signaling pathway, Mst1 and p-Lats1, was \ndecreased, and the expression of Tead and YAP target gene \nCyr61 was increased. These results suggest that abnormal \ninactivation of the Hippo signaling pathway occurs in mice \nwith adenomyosis.\nAfter demonstrating abnormal inactivation of Hippo sign-\naling pathway in adenomyosis mice, we further investigated \nthe expression of EMT-related proteins in adenomyosis by \nwestern blot and IHC analysis. As shown in Fig.  1c, the \nresults of western blot showed that the expression of the \nepithelial marker E-cadherin decreased, and the expres -\nsion of the mesenchymal markers N-cadherin and Vimen-\ntin increased in adenomyosis mice compared with control \nmice. Our study also detected the expression of multiple \ntranscription factors that inhibit E-cadherin protein level. \nThe expression of Twist and Snail was increased in adeno-\nmyosis mice compared with control mice. In addition, the \nexpression of proteins matrix metallopeptidase 2 (MMP-2) \nand matrix metallopeptidase 9 (MMP-9) related to invasion \nand migration was elevated. Consistently, the expression lev-\nels of E-cadherin, N-cadherin, Vimentin, and Twist detected \nby IHC further indicated that EMT occurred in adenomyosis \nmice (Fig.  1d). Thus, our findings demonstrate that inacti-\nvation of the Hippo signaling pathway and EMT occurs in \nmice with adenomyosis.\nVerteporfin Affects Viability, Proliferation, \nand Apoptosis of Ishikawa Cells\nIshikawa cells are a well-differentiated endometrial adeno-\ncarcinoma cell line and a good model for studying normal \nendometrial epithelial cells [8 , 23–26]. We further used \nIshikawa cells to study the regulatory relationship between \nthe Hippo signaling pathway and EMT. Verteporfin is a \nspecific YAP inhibitor that can inhibit tumor development \nby inhibiting the expression of YAP [27]. CCK-8, colony \nformation, EdU assay, and flow cytometry were used to \nanalyze the viability, proliferation, and apoptosis of Ishi-\nkawa cells after verteporfin treatment. As shown in Fig.  2a, \nthe cell viability gradually decreased with the increase of \nverteporfin concentration. The data also showed that the pro-\nliferation rate and the number of colony formation of cells \nwere inhibited by verteporfin compared to the control group \n(Fig.  2b). In addition, we also found that the expression of \nanti-apoptotic marker B cell leukemia 2 (Bcl2) decreased \nand the expression of pro-apoptotic protein Bcl2-associated \nX protein (Bax) increased with the increase of verteporfin \nconcentration (Fig.  2c). EdU staining also showed that inhi-\nbition of YAP reduced cell proliferation (Fig.  2d). Flow \ncytometry showed that the percentage of apoptotic cells was \nsignificantly increased in verteporfin-treated Ishikawa cells \n(Fig.  2e). These data suggest that activation of Hippo sign-\naling pathway leads to reduced proliferation of endometrial \nepithelial cells and promotion of apoptosis.\n\n2719Reproductive Sciences (2023) 30:2715–2727 \n1 3\nFig. 1  Inactivation of Hippo signaling pathway and occurrence \nof EMT in adenomyosis mice. a H&E staining of the successful \nestablishment of adenomyosis mice. b Protein expression of Mst1, \np-Lats1, YAP, p-YAP, Tead, and Cyr61 in uterine tissue was deter -\nmined by western blot. c Protein expression of E-cadherin, N-cad-\nherin, Vimentin, Twist, Snail, MMP-2, and MMP-9 in uterine tis-\nsue was determined by western blot. d The expression levels of \nE-cadherin, N-cadherin, Vimentin, and Twist in uterine tissue were \ndetected by IHC assay. Data were presented as mean ± SD. n = 5. \n*P < 0.05; **P < 0.01; ns, no significance\n\n2720 Reproductive Sciences (2023) 30:2715–2727\n1 3\nVerteporfin Inhibits the Migration Ability \nof Ishikawa Cells\nTo examine the role of the Hippo signaling pathway in the \ndevelopment of adenomyosis, we sought to determine the effect \nof verteporfin on cell migration in vitro. As shown in Fig.  3, \nboth wound healing assay and transwell assay results showed \nthat the migration of Ishikawa cells was significantly reduced \nafter verteporfin treatment, indicating that the Hippo signaling \npathway plays an important role in mediating cell migration.\nVerteporfin Inhibits EMT Process of Ishikawa Cells\nTo verify the regulation of Hippo signaling pathway on EMT \nin adenomyosis, we performed western blot to detect the \nexpression of EMT marker proteins in verteporfin-treated \nFig. 2  Verteporfin inhibits proliferation and promotes apoptosis of \nIshikawa cells. a, b, and d CCK-8, colony formation, and EdU assay \nof the proliferation of verteporfin-treated Ishikawa cells. c Protein \nexpression of bcl2 and bax in verteporfin-treated Ishikawa cells was \ndetermined by western blot. e Flow cytometry analysis of the apop-\ntosis of verteporfin-treated Ishikawa cells. Data were presented as \nmean ± SD. ***P < 0.001; ****P < 0.0001\n\n2721Reproductive Sciences (2023) 30:2715–2727 \n1 3\nIshikawa cells. As shown in Fig.  4a, verteporfin acti-\nvates the Hippo signaling pathway, which is manifested \nin the increased expression of Lats1 and p-YAP, and the \ndecreased expression of YAP and Tead. At the same time, \nthe expression of YAP target genes CTGF and Cyr61 \ndecreased. Figure  4b shows that with the increase of verte-\nporfin concentration, the EMT process was inhibited, which \nwas reflected in the increased expression of E-cadherin and \nFig. 3  Verteporfin inhibits the migration ability of Ishikawa cells \nin vitro. a Wound healing assay of the migration ability of verteporfin-\ntreated Ishikawa cells. b  Transwell assay of the migration ability of \nverteporfin-treated Ishikawa cells. Data were presented as mean ± SD. \n*P < 0.05; **P < 0.01; ***P < 0.001\nFig. 4  Verteporfin inhibits EMT \nprocess in Ishikawa cells. a Pro-\ntein expression of Lats1, YAP, \np-YAP, Tead, CTGF, and Cyr61 \nin the verteporfin-treated Ishi-\nkawa cells was determined by \nwestern blot. b Protein expres-\nsion of E-cadherin, N-cadherin, \nVimentin, Slug, Snail, and \nTwist in the verteporfin-treated \nIshikawa cells was determined \nby western blot\n\n\n2722 Reproductive Sciences (2023) 30:2715–2727\n1 3\n\n\n2723Reproductive Sciences (2023) 30:2715–2727 \n1 3\ndecreased expression of N-cadherin, Vimentin, Slug, Snail, \nand Twist. Collectively, these findings suggest that activation \nof Hippo signaling pathway suppresses the EMT process of \nIshikawa cells.\nInhibition of Hippo Signaling Pathway Partially \nReverses EMT in Mice with Adenomyosis\nTo verify the regulatory relationship between the Hippo \nsignaling pathway and EMT in adenomyosis, we injected \nthe YAP inhibitor verteporfin into adenomyosis mice. As \nshown in Fig.  5a, the western blot results showed that the \nexpression of Mst1, p-Lats1, and p-YAP was increased, and \nthe expression of YAP, Tead, and Cyr61 was decreased \nin the verteporfin group compared with the adenomyosis \ngroup. These results demonstrate that verteporfin activates \nthe Hippo signaling pathway in mice with adenomyosis.\nAfter verifying that the Hippo signaling pathway was \nactivated in the verteporfin group mice, we detected the \nexpression of EMT-related proteins in the verteporfin group \nmice by western blot and IHC assay, and further studied \nthe relationship between the Hippo signaling pathway and \nEMT in adenomyosis. As shown in Fig.  5b, compared with \nthe adenomyosis group, the expression of E-cadherin was \nincreased and the expression of N-cadherin, Twist, Snail, \nMMP-2, and MMP-9 was decreased in mice of verteporfin \ngroup. And the expression of EMT-related proteins detected \nby IHC further indicated that verteporfin inhibited the pro-\ngression of EMT in adenomyosis mice (Fig. 5c). These data \nsuggest that activation of the Hippo signaling pathway in \nadenomyosis can partially reverse the development of EMT, \nwhile reducing cell migration and invasion.\nActivation of Hippo Signaling Pathway Inhibits \nCell Proliferation and Promotes Apoptosis in Mice \nwith Adenomyosis\nThe development of adenomyosis is accompanied by abnormal \ncell proliferation and apoptosis. As shown in Fig. 6a, western \nblot results showed that compared with the control mice, the \nexpression of anti-apoptotic marker Bcl2 was increased and the \nexpression of apoptosis protein Bax was decreased. Quantita-\ntive analysis showed that Bcl2/Bax was increased. In addition, \nwe used the positive expression of proliferating cell nuclear \nantigen (PCNA) to identify cell proliferation in situ to detect \ncell proliferation. The level of PCNA positive staining in the \nuterine tissue of adenomyosis mice was significantly higher \nthan that of control mice (Fig. 6b). These results suggest that \nabnormal proliferation occurs in mice with adenomyosis. At \nthe same time, compared with the adenomyosis group, the \nexpression of Bcl2 was decreased, the expression of Bax was \nincreased, and Bcl2/Bax was decreased, while the expression \nof PCNA was decreased in the verteporfin group. These data \ndemonstrate that activation of Hippo signaling pathway by \nverteporfin inhibits abnormal proliferation in adenomyosis mice \nrelative to control mice.\nTUNEL apoptosis test showed that TUNEL positive \nstaining (green fluorescence) in adenomyosis mice was \nsignificantly lower than that in the control group, while \nthe fluorescence intensity of verteporfin group was sig-\nnificantly higher than that of adenomyosis group, indi-\ncating that the number of apoptotic cells in adenomyosis \nmice was much lower than that in control mice and verte-\nporfin treatment increased cell apoptosis in mouse uter -\nine tissue (Fig.  6c). Therefore, these results suggest that \nactivation of the Hippo signaling pathway by verteporfin \nin adenomyosis mice inhibits abnormal proliferation and \npromotes apoptosis of cells in adenomyosis mice.\nDiscussion\nAdenomyosis is an estrogen-dependent benign uterine dis-\nease with an incidence of about 20–30% worldwide [28], and \nthe age of onset tends to be younger. Although the develop-\nment of diagnostic techniques such as transvaginal ultra-\nsonography (TVUS) and magnetic resonance imaging (MRI) \nhas facilitated the clinical diagnosis of adenomyosis, the \nsystematic basis for its pathogenesis is still lacking. Except \nfor hysterectomy [29], there is currently no specific drug \nthat can cure the disease, which brings great inconvenience \nto patients with fertility needs. There are many hypotheses \nabout the pathogenesis of adenomyosis, one of which is gen-\nerally accepted is the invagination theory. According to the \ninvagination theory, endometrial tissue invades the myome-\ntrium mainly through the damaged endometrial-myometrial \njunction (JZ) [30]. Therefore, increased migratory and inva-\nsive abilities of endometrial cells may be a key factor in the \ndevelopment of adenomyosis. According to the available \nevidence, estrogen-induced epithelial-mesenchymal transi-\ntion (EMT) provides endometrial cells with the ability to \nmigrate and invade, which is critical for the pathogenesis of \nadenomyosis [12, 31]. In the current study, we found for the \nfirst time that the EMT occurring in adenomyosis may be \nregulated by the Hippo signaling pathway.\nIn recent years, YAP, as a key effector protein of the \nHippo signaling pathway, has been identified to play an \nimportant role in the regulation of cell proliferation and \nFig. 5  Verteporfin inhibits EMT process in mice with adenomyosis. \na Protein expression of Mst1, p-Lats1, YAP, p-YAP, Tead, and Cyr61 \nwas determined by western blot. b Protein expression of E-cadherin, \nN-cadherin, Twist, Snail, MMP-2, and MMP-9 was determined by \nwestern blot. c The expression levels of E-cadherin, N-cadherin, \nVimentin, and Twist in uterine tissue were detected by IHC assay. \nData were presented as mean ± SD. n = 5. *P < 0.05; **P < 0.01; \n***P < 0.001; ns, no significance\n◂\n\n2724 Reproductive Sciences (2023) 30:2715–2727\n1 3\n\n\n2725Reproductive Sciences (2023) 30:2715–2727 \n1 3\napoptosis, tissue metabolism, organ growth and tumori-\ngenesis, and development [ 32, 33]. Dysregulation of YAP \nhas been found in a variety of diseases. Previous studies \nhave shown that in various subtypes of breast cancer, the \nup-regulation of YAP expression can promote tumor cell \nproliferation, enhance the ability of tumor cells to metasta-\nsize, and make tumor cells resistant to chemotherapy [34]. \nZhang et al. [35] showed that in glioma cells, the expres-\nsion of mesenchymal markers N-cadherin and Twist was \ndecreased after YAP knockdown and significantly increased \nafter YAP overexpression. In addition, Zhang et al. [36] also \nreported that Furin can promote EMT through the Hippo \nsignaling pathway in pancreatic cancer cells. In our study, \nwe found that verteporfin inhibited the viability, prolifera-\ntion, and migration and promoted apoptosis of Ishikawa cells \nin a concentration-dependent manner. Song et al. [14] found \nabnormal expression of YAP in endometriosis, and knock -\ndown of YAP resulted in decreased cell proliferation and \nincreased apoptosis in ESCs, which is consistent with our \nstudy. To confirm the relationship between the Hippo sign-\naling pathway and EMT, the effect of verteporfin on EMT-\nrelated proteins in Ishikawa cells was investigated. Verte-\nporfin treatment increased the expression level of E-cadherin \nand decreased the expression levels of N-cadherin, Vimen-\ntin, Twist, Snail, and Slug. These in vitro experiments dem-\nonstrated that activation of the Hippo signaling pathway \ncould reduce the proliferation of endometrial epithelial cells, \nenhance cell apoptosis, and inhibit the occurrence of EMT.\nEMT is known to promote the development of adenomyosis \n[37–39]. The result of EMT is often that cells acquire high \nmigration and invasiveness. We found the occurrence of EMT \nin the uterine tissue of mice with adenomyosis in an in vivo \nstudy. Specifically, decreased expression of the epithelial \nmarker E-cadherin and increased abundance of mesenchy -\nmal markers (i.e., N-cadherin, Vimentin, Twist, Snail) were \nrecorded. At the same time, we also observed inactivation of \nthe Hippo signaling pathway in adenomyosis mice, which is \nmanifested in the high expression of the key protein YAP in \nadenomyosis. The study by Huang et al. [19] found that YAP \nis overexpressed in adenomyosis, which provides support for \nour findings. Subsequently, the inhibition of YAP by verteporfin \ninjection in adenomyosis mice increased the expression level of \nE-cadherin and decreased the expression levels of N-cadherin, \nTwist, and Snail. Therefore, our findings suggest that EMT in \nadenomyosis is regulated by the Hippo signaling pathway.\nUnder the condition that EMT is regulated by Hippo signal-\ning pathway in adenomyosis mice, changes in cell proliferation \nand apoptosis are the most intuitive phenotypes in the develop-\nment of adenomyosis. The current study showed that the use of \nverteporfin to activate the Hippo signaling pathway increased \napoptosis and inhibited proliferation of cells. This observation \nsuggests that abnormal inactivation of the Hippo signaling path-\nway may promote cell proliferation and inhibit apoptosis, which \nin turn accelerates the development of adenomyosis.\nIn conclusion, both in vitro and in vivo experiments indi-\ncated that EMT and abnormal proliferation and apoptosis of \ncells in adenomyosis may be related to the inactivation of Hippo \nsignaling pathway. Specifically, inactivation of the Hippo sign-\naling pathway in adenomyosis has been proposed. Inhibition of \nYAP expression inhibits cell proliferation and promotes apopto-\nsis in vitro and in vivo. EMT in adenomyosis may be related to \nthe inactivation of Hippo signaling pathway, providing a refer-\nence for the underlying mechanism of adenomyosis.\nAuthor Contribution We thank all authors for the following contribu-\ntions. WQC and QZR conceived and designed the experiments. TTJ \nand MQL performed the experiments. TTJ, TL, and SMY analyzed the \ndata. WQC and QZR contributed reagents/materials/analysis tools. TTJ \nwrote the paper. WQC critically reviewed the manuscript. We thank the \nLaboratory Animal Center of Jinan University for caring for animal.\nFunding We greatly appreciate the fund support from the Guangdong \nProvincial Hospital of Chinese Medicine-Weixian Li famous doctor \nstudio (E43719).\nData Availability The data and material that support the findings of \nthis study are available from the corresponding author upon reason-\nable request.\nCode Availability Codes for data analysis are available upon request.\nDeclarations \nEthics Approval All animal experiments were approved by the Labora-\ntory Animal Review Committee of Jinan University (Approval number: \nIACUC-20200905–01).\nConsent to Participate All authors had final approval of the submitted \nversions.\nConsent for Publication All authors read the manuscript and agreed \nto its publication.\nConflict of Interest The authors declare no competing interests.\nOpen Access This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article's Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article's Creative Commons licence and your intended use is not \nFig. 6  Verteporfin inhibits cell proliferation and promotes apopto-\nsis in mice with adenomyosis. a Protein expression of Bcl2 and Bax \nwas determined by western blot. b The expression level of PCNA \nin uterine tissue was detected by IHC assay. c The cell apoptosis in \nuterine tissue was detected by TUNEL assay. Data were presented as \nmean ± SD. n = 5. *P < 0.05; **P < 0.01; ns, no significance\n◂\n\n2726 Reproductive Sciences (2023) 30:2715–2727\n1 3\npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\nReferences\n 1. 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Reprod Sci. 2020;27(9):1698–706. \nhttps:// doi. org/ 10. 1007/ s43032- 020- 00139-0.\nPublisher's Note Springer Nature remains neutral with regard to \njurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}