{"paper_id":"9c4ffde1-4e77-419c-a1ab-06917c82971e","body_text":"He et al. BMC Molecular and Cell Biology           (2022) 23:37  \nhttps://doi.org/10.1186/s12860-022-00426-5\nRESEARCH\nEffects of an inhibitor of the SHH signaling \npathway on endometrial cells of patients \nwith endometriosis\nYanan He, J. Wang, Xinyan Jiang, Jianhua Gao, Yan Cheng, Tian Liang, Jun Zhou, Liyuan Sun and \nGuangmei Zhang* \nAbstract \nBackground: Endometriosis is one of the most common gynecological diseases, and seriously reduces the quality of \nlife of patients. However, the pathogenesis of this disease is unclear. Therefore, more studies are needed to elucidate \nits pathogenesis. Our previous publication found that the Sonic Hedgehog (SHH) signaling pathway was activated in \nendometriosis. This study tested whether SHH signaling in endometrial stromal cells (ESCs) was critical for the patho-\ngenesis of endometriosis.\nMethods: To examine the effect of inhibiting the SHH signaling pathway on endometriosis, we first isolated ESCs \nfrom eutopic endometrial tissues of patients with or without endometriosis and identified the extracted cells by \nmorphological observation and immunofluorescence. Then, we treated ESCs with the GLI inhibitor GANT61 and used \nCCK-8, wound healing and invasion assays to detect cell activities, such as proliferation, invasion and metastasis. Fur-\nthermore, we detected the expression of key proteins and proliferation markers of the SHH signaling pathway in the \nlesions of nude mice using immunochemistry.\nResults: We demonstrated that higher concentrations of GANT61 decreased the proliferation rate and migration \ndistance of ESCs. We observed that GANT61 inhibited the invasion of ESCs. In addition, blockage of the SHH signaling \npathway significantly reduced cell proliferation in vitro.\nConclusions: Our study suggested that inhibition of the SHH pathway is involved in cell proliferation and invasive \ngrowth in the pathogenesis of endometriosis.\nKeywords: Endometriosis, Sonic hedgehog, Signaling pathway\n© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line \nto the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory \nregulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this \nlicence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco \nmmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\nIntroduction\nEndometriosis is a common gynecologic disease with a \nhigh risk of recurrence, that is characterized by the exist -\nence of endometrial tissues outside of the uterus [1, 2]. \nThe abnormal growth of endometrial tissues can be \nfound in the peritoneal cavity, cervix and fallopian tubes, \nleading to pelvic pain, dysmenorrhea and infertility [3, \n4]. Although endometriosis seriously affects the qual -\nity of life of patients, the mechanism of the pathogenesis \nof endometriosis is largely unclear. To date, studies have \nfocused on retrograde menstruation, estrogen-dependent \nglycoproteins, cytokines, adhesion molecules and angio -\ngenic and growth factors, which are all associated with \nthe pathogenesis of endometriosis and the development \nof endometriotic lesions. Retrograde menstruation was \nproposed by Sampson but has been called into question \nand challenged [5, 6], thus, other causative factors likely \nOpen Access\nBMC Molecular and\nCell Biology\n*Correspondence:  guangmeizhang@126.com\nDepartment of Gynaecology, The First Affiliated Hospital of Harbin Medical \nUniversity, Harbin, China\n\nPage 2 of 9He et al. BMC Molecular and Cell Biology           (2022) 23:37 \nplay roles in the occurrence and development of this \ndisease.\nSonic Hedgehog (SHH), a mammalian member of the \nHedgehog family (SHH, Indian Hedgehog and Desert \nHedgehog) shares a common signaling pathway [7 , 8]. \nHH ligand binding to the Patched (Ptc) transmembrane \nprotein, activates the SHH pathway by relieving Patched1-\ndependent inhibition of Smoothened (SMO) signal trans-\nduction [9]. Subsequently, suppressor of fused (SUFU) and \nthe GLI zinc finger family members (GLI2, GLI3) transmit \nthe activated signal into the nucleus to regulate the expres-\nsion of target genes, such as GLI1 and vascular endothe -\nlial growth factor. Accumulating evidence has shown that \naberrant activation of the SHH signaling pathway plays a \nprotumorigenic role in various types of gynecological can-\ncers by enhancing cell proliferation, stem cell maintenance, \ncell differentiation and angiogenesis [10, 11]. Similar to \ncancers, endometriosis also develops through the dysregu-\nlation of cellular pathways regulating sensitivity to growth \nstimulation, cell proliferation and replication, angiogen -\nesis and tissue invasion and metastasis. However, the role \nof the SHH signaling pathway in endometriosis still needs \nto be elucidated. Matsumoto et al. confirmed that recom-\nbinant SHH protein could enhance the proliferation of \nmouse endometrial mesenchyme cells in  vitro [12]. Our \nprevious research indicated that the SHH pathway was \nupregulated in eutopic endometrial tissues of endome -\ntriosis. Furthermore, comparison of revised AFS scores \nshowed that SHH, SMO, GLI1 and GLI3 had significantly \nincreased expression levels in patients with advanced dis-\nease (III-IV) [13].\nIn this study, we used the GLI inhibitor GANT61 to \ninhibit the SHH pathway and explored the effect of this \npathway on the pathogenesis of endometriosis. Our pur -\npose was to demonstrate the role of the SHH pathway in \nthe proliferation, migration and invasion of endometrial \nstromal cells (ESCs) from eutopic endometrium derived \nfrom patients with endometriosis. Additionally, identify -\ning new pathway mechanisms and providing new targets \nfor clinical treatment are needed. Nevertheless, little is \nknown about whether activation of the SHH pathway pro-\nmotes the occurrence and development of endometriosis. \nMoreover, the SHH signaling pathway has also been sug -\ngested to regulate the cell cycle [14, 15], therefore, further \nelucidation of the underlying mechanisms of the SHH \npathway in the biological function of ESCs is needed.\nParticipants, materials and methods\nParticipants\nHuman endometrial tissues were obtained from ten \nwomen aged 29–36 (32.3 ± 2.7) years undergoing surgery \nfor idiopathic infertility in the First Affiliated Hospital of \nHarbin Medical University (Harbin, PR China). Eutopic \nendometrial tissues were obtained from ten patients aged \n26–42 (34.3 ± 7.6) years undergoing surgery for endo -\nmetriosis. None of the patients had received any hormo -\nnal therapy prior to surgery within six months. Women \nsuffering from cancers, benign ovarian cysts other than \nendometriomas, perioperative pelvic inflammatory dis -\nease, or endometrial polyps were excluded from this \nstudy. All patients signed an informed consent form prior \nto recruitment and the study protocol was approved by \nthe Ethics Committee of Harbin Medical University \n(202106). All experimental methods were carried out in \naccordance with the approved guidelines of Harbin Med-\nical University. All patients gave their written informed \nconsent prior to study inclusion.\nAnimal experiments\nAll animal experiments were conducted using female \nBalb/c nude mice aged approximately 5 weeks and weigh-\ning 17–19 g. These mice were purchased from Charles \nRiver Laboratories in Beijing (No. 11400700316857), and \nthe animal experiments were performed in strict accord -\nance with the guidelines for the Care and Use of Labo -\nratory Animals of the Harbin Medical University Ethics \nCommittee. All procedures were approved by the Com -\nmittee on the Ethics of Animal Experiments of Harbin \nMedical University. All efforts were made to minimize \nanimal suffering. All cell protocols were approved by the \nHarbin Medical University Ethics Committee.\nIsolation and culture of ESCs\nEndometrial tissues were collected and washed with ice-\ncold medium (DMEM/F-12 1:1) (Hyclone, USA) con -\ntaining 10% fetal bovine serum (Ausbian, USA) and 1% \npenicillin-streptomycin (Gibco, USA). The samples were \ntransported to the laboratory on ice within 2 h. The endo-\nmetrial tissues were cut into smaller pieces and digested \nin type IV collagenase (Life Technologies, Carlsbad, CA, \nUSA) at 37 °C for 60–90 minutes. The cell suspension \nwas passed once through a 70-μM sieve (HEAD, Beijing, \nChina) to remove debris and glandular epithelial cells. \nThe filtrates were then centrifuged at 800 rpm for 5 min-\nutes at room temperature. The isolated cells were main -\ntained in the medium mentioned above at 37 °C and 5% \n CO2. The medium was replaced after 2–3 days to remove \nnonadherent cells. Cells were subcultured on new plates \nat a 1:2 ratio and marked as passage 1 (P1) [16, 17]. Cells \nfrom P3-P5 were used for the experiments.\nObservation of ESC morphology\nThe morphology of ESCs from different passages was \nobserved by inverted light microscopy (Olympus, Japan).\n\nPage 3 of 9\nHe et al. BMC Molecular and Cell Biology           (2022) 23:37 \n \nImmunofluorescence\nSerum-starved ESCs were seeded on cover glass slides, \nfixed with 4% paraform aldehyde for 15 min, and permea-\nbilized with 0.1% Triton X-100. The purity of ESCs was \ndetected by separately immunostaining for the epithelial \nmarker cytokeratin 7 (CK 7) (33,060 M, Biosis, China) \nand stromal marker vimentin (VIM) (0756R, Biosis, \nChina). We used 4′,6′-diamidino-2-phenylindole (DAPI) \nimmunofluorescence to identify ESC nuclei. Cells were \nincubated with fluorescein isothiocyanate (FITC)-phal -\nloidin (for F-actin staining, Sigma, USA) at room temper-\nature for 40 minutes before incubation with the primary \nantibodies anti-CK 7 and anti-VIM overnight at 4 °C.Cell \nnuclei were stained with DAPI (Thermo Fisher Scien -\ntific). Immunofluorescence signals were observed using \nan inverted light microscopy (Olympus, Japan) [18]. The \nareas with CK 7 and VIM were computed using ImageJ \nsoftware. At least 100 cells were analyzed from tripli -\ncate cover slides in each sample, and experiments were \nrepeated with samples from five different individuals.\nCell proliferation assay\nCell proliferation was assessed by the Cell Counting Kit-8 \n(CCK-8; Dojindo, Japan) assays. To determine whether \nthe SHH signaling pathway effected ESC proliferation, we \napplied the SHH signaling pathway inhibitor GANT61. \nAfter 48 hours, cells were seeded in 96-well plates (4000 \ncells per well) stimulated with different concentrations \nof GANT61 (SIGMA, USA) in 100 μL of full culture \nmedium. The cells were tested in the absence (NC) and \npresence of 10 μmol/L, 20 μmol/L and 30 μmol/L inhibi -\ntor of GANT61. Ten microliters of CCK-8 solution was \nadded to each well. Absorbance was read at a wavelength \nof 450 nm by a microplate reader (ELX800; Bio-Tek, \nAmeria). The proliferation rate was derived from the cell \nindex, which was calculated as the difference between \nthe well with only cells minus the well with only culture \nmedia, divided by the nominal value. Three independent \nexperiments were performed in triplicate.\nWound healing assay\nTo examine the migratory capacity of ESCs, we con -\nducted a scratch wound assay. Cells cultured with \nGANT61 (10 μmol/L, 20 μmol/L and 30 μmol/L) were \nseeded in six-well culture plates with serum-contain -\ning medium and cultured until the cell density reached \n90–95% confluence. An artificial homogeneous wound \nwas created by scratching the monolayer with a sterile \n200 μL pipette tip. After scratching, the cells were washed \nwith PBS and then cultured with serum-free DMEM \nF12 1:1 media for 48 hours. Images of cells migrating \ninto the wound were captured at 0 and 48 hours using a \nmicroscope (EVOS, USA). The assay was performed in \ntriplicate [19]. Three independent experiments were per -\nformed in triplicate.\nTranswell invasion assay\nTranswell assays were used to assess cell invasive capac -\nity. Cell invasion assays were carried out using a Bio -\nCoat Matrigel Transwell chamber (BD, Franklin Lakes, \nNJ, USA) with a pore size of 8.0 μm. The inserts were \nplaced in 24-well plates containing 700 μL of DMEM F12 \n1:1 medium for 30 minutes in a humidified 37 °C incu -\nbator under 5%  CO2 before seeding the cells. GANT61 \n(30 umol/L) was used to block the SHH signaling path -\nway, and after 48 hours, 5 ×  104 cells in each group \nresuspended in DMEM F12 1:1 medium containing 5% \nFBS were placed in each chamber. The lower compart -\nment was loaded with full media containing 15% FBS as \nthe nutritional attractant. After incubated at 37 °C for \n48 hours, noninvaded cells were scraped off with a cotton \nswab. The translocated cells on the bottom of the upper \nchamber membrane were fixed with 5% formaldehyde \nand stained with 1% Giemsa stain. The number of cells \nthat penetrated the upper compartment of the Tran -\nswell chamber was determined under an inverted micro -\nscope. Five fields of fixed cells were randomly chosen and \ncounted under a light microscope [19].\nImmunohistochemistry\nAll tissues were fixed in 10% formaldehyde, embedded in \nparaffin and cut into 4 mm sections. Immunohistological \nstaining was conducted by boiling the sections in 10 mM \ncitric acid, pH 7.0. The slides were incubated with a poly -\nclonal rabbit antibody (1:200 dilution; Biosis) for 2 hours \nat 37 °C. The sections were washed in phosphate-buff -\nered saline (PBS) three times and then incubated with \nmouse anti-rabbit secondary antibody for 40 minutes at \n37 °C Peroxidase substrate containing 3,3′-diaminoben -\nzidine tetrahydrochloride chromogen was added to the \nsections for 2 minutes to develop the reaction. All slides \nwere examined and scored by two independent patholo -\ngists who were blinded to both the clinical and pathologi-\ncal data. The quantification of the selected proteins was \nperformed using Image-Pro Plus 6.0 (Media Cybernet -\nics). Scoring was carried out for the mean density (ratio \nof integrated optical density SUM/area) [19].\nESCs and eutopic endometrium tumourigenicity analysis\nMice were kept on a 12 h light/dark cycle and provided \nsterile food and water. The mice were allowed to accli -\nmate to specific pathogen-free (SPF) conditions before \nexperiments. The mice were randomly separated into \n2 tumorigenicity groups (n  = 5 per group), the ESC and \neutopic endometrium groups. For further study of the \n\nPage 4 of 9He et al. BMC Molecular and Cell Biology           (2022) 23:37 \nfunction of the SHH signaling pathway in vivo, a mouse \nmodel of experimental endometriosis was established by \ninjecting NS with 0.5  cm3 in size of eutopic endometrial \nfragments into the right subcutaneous scapular tissue \nand injecting the contralateral side with 0.2 ml of normal \nsaline (NS) as the negative control. Lesions were moni -\ntored daily in both groups. After 40 days, the mice were \nsacrificed by cervical dislocation. Both the left and right \nsubcutaneous scapular tissues were collected. Macro -\nscopic observation and H&E staining were used to assess \nlesion formation [20].\nGANT61 treatment of ESCs in the endometriosis model \nin vivo\nHuman eutopic endometrial tissues were obtained from \npatients with endometriosis as described above. Mice \nwere maintained on a 12 h light/dark cycle and were pro -\nvided with sterile food and water. The mice were allowed \nto acclimate to SPF conditions before experiments. \nTwenty mice received a single subcutaneous injection \nof a 0.5  cm3 eutopic endometrial fragment in 0.2 ml of \nNS into their back. Seven days later, when the endome -\ntriosis model was confirmed, the mice were randomly \ndivided into two groups (n = 10 per group), the GANT61 \ngroup and the control group. GANT61 was subcutane -\nously injected into experimental group mice, while NS \nwas injected into the controls. In the GANT61 group, 30 \numol GANT61 in 0.3 ml of NS was administered intra -\nvenously into the tail vein. The mice in the control group \nwere only injected with 0.3 ml of NS. The injections were \nperformed weekly. The animals were sacrificed one week \nafter the third injection, and the endometriotic lesions \nof the two groups were collected to detect the effect of \nGANT61 on lesion reduction [20].\nStatistical analysis\nAll statistical analyses were performed using SPSS 19.0 \n(SPSS, Inc., Chicago, IL). Continuous variables are \nexpressed as the mean ± standard deviation. Differences \nbetween groups were evaluated using the independent \nsamples Student’s t test. Completely random design anal-\nysis of variance was performed to test the significance of \nthe migration of ESCs in response to different GANT61 \ndoses. Data from the invasion assay were assessed by \npaired T tests. Differences were considered statistically \nsignificant at P < 0.05.\nResults\nIsolation, culture and immunofluorescence identification \nof ESCs\nESCs were successfully extracted from endometral tis -\nsues by the centrifugal adherent method. After 5 to \n7 days in primary culture, adherent cells began to form \ncell clones. Primary ESCs exhibited a short polygonal \nor fusiform morphology, which gradually became a \nfibroblast-like spindle shape with an increasing number \nof passages. Cells from passages 3–5 (P3-P5) showed a \nrelatively homogenous morphology, long spindle shape \nand a swirling arrangement (Fig. 1 A).\nCellular immunofluorescence detection was used to \nidentify the type and purity of cultured cells. CK 7 was \nmainly localized in the endometrial glandular epithelial \ncells, and VIM was in the endometriotic stromal cells. \nAs shown in the Fig.  1B, endometrial glandular epithe -\nlial cell cytoplasm was positive for CK 7, while endo -\nmetrial glandular epithelial cell nuclei were positive \nfor VIM. We observed a large amount of VIM-positive \nstaining and a small amount of CK 7-positive staining \nby inverted fluorescence microscopy. The experiments \nthat measured VIM-positive cells at more than 95% \nprovided clear evidence that ESCs were successfully \nisolated.\nInhibition of the SHH signaling pathway reduces \nthe proliferation of ESCs\nThe ESCs stimulated with different concentrations \nof GANT61 showed a decreased proliferation index \ncompared with the controls (Fig.  2A). A significant \ndifference was observed when GANT61 was added at \na concentration of 30 μmol/L. These results were sta -\ntistically significant. The CCK-8 assay demonstrated \nthat stimulation with GANT61 diminished the pro -\nliferation of ESCs, especially at a concentration of \n30 μmol/L.\nReducing the migratory and invasive capability of the ESCs \nby inhibiting the SHH signaling pathway\nWould healing assays showed that the inhibitor of the \nSHH signaling pathway reduced the migration of ESCs \n(P < 0.01) compared to the controls (Fig.  2B). As shown \nin Fig.  2C, 20 μM and 30 μM GANT61 resulted in a \nsmaller scratch area after 48 h than that of the controls. \nThe average values of the scratched areas with GANT61 \nat 20 μM and 30 μM were 44.457 and 21.77, respectively. \nIn addition, Transwell assay showed that the invasion \nrate was significantly reduced by the inhibitor GANT61 \n(Fig.  2D). The number of invading cells was statistically \nsignificantly higher in the control group than in the \nexperimental group. As shown in Fig.  2E, a difference in \nthe invasion rate was observed before and after applica -\ntion of GANT61. Similar to the results of the prolifera -\ntion assay, these findings demonstrated that stimulation \nwith GANT61 decreased the migratory and invasive \ncapacity of ESCs.\n\nPage 5 of 9\nHe et al. BMC Molecular and Cell Biology           (2022) 23:37 \n \nFig. 1 Isolation, culture and morphological observation of ESCs. A Isolation, culture and morphological observation of ESCs. Scale = 1000 μm. B \nImmunofluorescence identification of the type and purity of cultured cells. Scale = 200 μm\nFig. 2 Inhibition of the SHH signaling pathway reduces the ability of proliferation, migration and invasion of ESCs. A Effects of different \nconcentrations of GANT61 (10 μmol/L, 20 μmol/L and 30 μmol/L) on the proliferation of ESCs. B Effects of different concentrations of GANT61 on \nthe migration of ESCs. Scale = 1000 μm. C Effect of GANT61 (30 mol/L) on the invasive ability of ESCs. D Quantitative plots of the number of invasive \ncells in the control group and the GANT61 group\n\nPage 6 of 9He et al. BMC Molecular and Cell Biology           (2022) 23:37 \nConstruction and characterization of a nude mouse \nxenograft model\nTwo days after injection of eutopic endometrial frag -\nments, nude mice developed a soft rash at the injection \nsite. After 1 week, a slightly soft round mass was observed \nat the injection site of the right subcutaneous scapular \ntissue in the nude mice, and the visible lesions increased \nin size over time. All mice were sacrificed 21 days post-\ninjection by cervical dislocation and multiple lesions \nwere removed for further analysis. By macroscopic \nobservation (Fig.  3A), we confirmed the endometriotic \nlesions in the nude mice. Moreover, endometrial glands \nand stromal structures were observed in the HE-stained \nsections under a microscope (Fig.  3C). Through visual \nobservation (Fig.  3A-2) and H&E staining (Fig.  3B-3), we \nfound that the right side of the five nude mice had formed \nendometrioid tissue, so the nude mouse xenograft model \nwas successfully constructed.\nDetecting key SHH signaling pathway proteins and Ki67 \nin ectopic lesions in the GANT61 group and the control \ngroup\nThe protein expression levels of the experimental group, \nincluding those of SHH, SMO, GLI1 and GLI3, were sig -\nnificantly lower than those of the controls (Fig.  4A and \nB). Ki67 is a nuclear antigen expressed in the mid-G1, \nS, G2 and M phases of the cell cycle, and it serves as a \nmarker of cell proliferation. Ki67 is closely associated \nwith mitotic cellular chromosomes and centrally involved \nin cell proliferation [21, 22]. Through a series of in vitro \nexperiments, we found that blocking the SHH signaling \npathway decrease the proliferation of ESCs (Fig.  4C and \nD). The in vivo proliferation experiments were applied to \nfurther validate the effect of blocking SHH signaling on \nKi67 protein expression. In addition, using an in  vitro \nassay, we found that blockade of the SHH signaling path -\nway can significantly reduce cell proliferation and that \nthe SHH pathway is essential for cell proliferation in \nendometriosis.\nDiscussion\nEndometriosis has similar characteristics to malignan -\ncies, including excessive cell proliferation, invasion, \nmetastasis and recurrence. The SHH signaling pathway \nhas a diverse range of biological functions, including the \npromotion of cell proliferation and differentiation along \nwith the induction of angiogenesis and cell migration \n[23]. Understanding the molecular mechanism by which \nthe biological behaviors of ESCs are regulated in endo -\nmetriosis would improve our understanding of the etiol -\nogy and pathogenesis of endometriosis.\nSimilar to other pathways, the SHH signaling pathway \nwas significantly related to changes in hormone levels in \nfemales. Monsivais et al. [24] showed that the occurrence \nFig. 3 Subcutaneous xenografts in nude mice. A Macroscopic observation of subcutaneous xenograft tumor formation after 3 days. B Macroscopic \nobservation of subcutaneous xenograft tumor formation after 14 days. C H&E of subcutaneous xenograft tumor formation in control nude mice and \nGANT61 nude mice. H&E staining (400 ×)\n\nPage 7 of 9\nHe et al. BMC Molecular and Cell Biology           (2022) 23:37 \n \nof endometriosis was related to the steroid signaling \npathway, especially the estrogen and progesterone signal-\ning pathways. Zhang et  al. [25] found that this elevated \nexpression of SHH signatures was associated with pro -\ngesterone receptor positivity in human trophoblasts. This \nresult suggested that the SHH signaling pathway may \nplay an important role in endometriosis. In our previ -\nous study, we investigated the effect of the SHH signal -\ning pathway on the development of endometriosis. The \neutopic endometrium was compared with the normal \nendometrium using qRT-PCR and immunohistochemi -\ncal staining. SHH, SMO, GLI1 and GLI3 expression was \nstrongly increased with clinical stages in the eutopic \nendometrium, which suggested that the SHH signal -\ning pathway was abnormally activated in endometriosis \n[13]. Additionally, the SHH signaling pathway might have \nimportant implications for the development and prog -\nnosis of tumor diseases. Noman et  al. [26] confirmed \nthat elevated levels of the SHH signaling pathway were \nobserved in breast patients who had a significantly higher \nrisk of recurrence and metastasis and had worse survival \nthan patients with progressive metastatic breast cancer. \nGomes et al. [27] found that the group with craniophar -\nyngiomas had more rapid cancer progression and poorer \nfive-year survival outcomes compared to the control \ngroup. Therefore, we further investigated the mechanism \nof the SHH pathway in endometriosis.\nAccording to our results, immunohistochemistry \nshowed higher expression levels of nuclear GLI1 and \nGLI3 not only in endometrial stromal cells but also in \nendometrial glandular epithelial cells. Expression in \nendometrial glandular epithelial cells is a marker of stem \nor progenitor cells. Based on both the importance of \nthe SHH signaling pathway in various cancers and the \ntumoroid characteristic of eutopic endometrial cells, we \nspeculated that differential expression of key SHH signal-\ning pathway proteins may change the biological behavior \nof eutopic endometrial cells.\nInhibitors targeting the SHH signaling pathway \ncould decrease cell proliferation, migration, and inva -\nsion. Small molecule modulators of the SHH signal -\ning pathway have been extensively investigated. Few \nstudies have examined the terminal transcription fac -\ntor GLI as a SHH pathway inhibitor to intervene in \ndisease occurrence and development. Consequently, \nwe assessed regulation of the SHH signaling pathway \nwith the development of endometriosis. We examined \nthe influence of the GLI transcription factor inhibitor \nGANT61 on endometriosis. GANT61 is an available \nand potent inhibitor of the terminal transcription fac -\ntor GLI that not only induced the proliferation of tumor \ncells in vitro but also promoted cell apoptosis [28, 29]. \nIn  vitro, we demonstrated that the small molecule \ninhibitor GANT61 exerted inhibitory effects on the \nexpression of GLI downstream target genes and blocked \npathway transmission. We also showed that GANT61 \ninhibited the proliferation of ESCs by blocking the SHH \nsignaling pathway. GANT61 could suppress ESC prolif -\neration compared to that of the control group without \ntreatment. Heard et  al. demonstrated that KLF9 dele -\ntion increased proliferation and reduced apoptosis in a \nmouse model of endometriosis through the Hedgehog \nFig. 4 Expression of SHH, SMO and GLI1 and GLI3 in ESCs. A The protein expression of SHH, SMO, GLI1 and GLI3 in subcutaneous xenograft tumor \nlesions of nude mice in the control group and the GANT61 group. B Quantitative mean density of SHH, SMO, GLI1 and GLI3 in the control group \nand the GANT61 group. C The protein expression of Ki67 in subcutaneous xenograft tumor lesions of nude mice in the control group and the \nGANT61 group (400 ×). D Quantitative mean density of KI67 in the control group and the GANT61 group (400 ×)\n\nPage 8 of 9He et al. BMC Molecular and Cell Biology           (2022) 23:37 \nand Notch signaling pathways; however, there were no \nfurther studies on Hedgehog signaling in endometrio -\nsis [30]. Additionally, we explored this problem further \nthrough in  vivo experiments. The protein expression \nlevels of the experimental group after GANT61 injec -\ntion, including those of SHH, SMO, GLI1 and GLI3, \nwere significantly lower than those in the control group. \nBarricading the SHH signaling pathway, the expres -\nsion of the cell proliferation-related protein Ki67 in the \nexperimental group showed a stronger decrease than \nthat in the control group in the animal model of endo -\nmetriosis. In  vitro, the cell proliferation rate of ESCs \ndecreased as the dose of GANT61 increased. The stud -\nies above demonstrated that the SHH signaling path -\nway was activated, and the proliferation of endometrial \nstromal cells was weakened if the pathway was blocked. \nThus, we believe that blockade of the targeted factor of \nthe SHH signaling pathway will decrease the expression \nand activation of the pathway and will also inhibit the \ndevelopment of endometriosis.\nCell migration is required for various physiological \nand pathological processes, such as wound repair, angi -\nogenesis, inflammatory responses, immune cell phago -\ncytosis and invasion and metastasis of cancer cells [31, \n32]. The invasion and migration of endometrial cells \nplay an important role during endometriosis develop -\nment [33, 34]. We examined the effect of the SHH sign -\naling pathway on the migration and invasion of ESCs. \nIn our study, blockage of the SHH signaling pathway \nresulted in a shorter migration area of ESCs and a lower \nnumber of invading cells compared with the controls. A \nprevious study showed that the antimigratory effect was \nassociated with the inhibition of HH signaling by SMO \ninhibitors, such as NVP-LDE-225 and GDC-0449, both \nof which have been approved for clinical use [35, 36]. \nHowever, these drugs might be ineffective against non -\nclassical GLI activation pathways. In addition to SMO \ninhibitors, direct inhibitors of GLI and/or inhibitors of \nsignaling pathways involved in the noncanonical HH-\nGLI pathway may be required for effective treatment. \nAdditionally, Souzaki et al. showed that the SHH sign -\naling pathway advanced the progression from ductal \ncarcinoma in  situ (DCIS) to invasive ductal carcinoma \n(IDC) [37]. Su et al. [38] suggested that the HH signal -\ning pathway enhanced the viability and invasion of gas -\ntric cancer cells.\nThe present research further extended the mechanis -\ntic study of the SHH signaling pathway in endometriosis. \nThe results above provided a basis for an in depth under -\nstanding of the pathophysiology and a search for new \ntherapeutic targets. Therefore, the SHH signaling pathway \nmay serve as a novel target for endometriosis therapy in \nthe future. These findings have significant implications for \nidentification of nonhormone medications for the treat -\nment of endometriosis. This study had some limitations. \nDue to the poor passaging ability of endometrial glan -\ndular epithelial cells, the Ishikawa cell line was generally \nused in the experiment, so endometrial glandular epithe -\nlial cells were not further studied in this report. Addition-\nally, the number of experimental nude mice was relatively \nsmall. In the future, it will be necessary to expand the sam-\nple size of experimental nude mice to further study the \nclinical application value of GANT61 in the treatment of \nendometriosis.\nTaken together, our previous research showed that \nthe expression levels of SHH, SMO, GLI1 and GLI3 were \nhigher in the control tissues than in the eutopic endome -\ntrial tissues [13]. Our study would provides a basis for a ret-\nrospective analysis of the endometrosis mechanism, which \nwe expect to play a role in the treatment of endometriosis. \nThe results of in vitro experiments showed that GANT61 \nhad antiproliferative, anti-invasive and antimetastatic \neffects in ESCs by inhibiting the SHH signaling pathway. \nThis study increased the understanding of the pathogen -\nesis of endometriosis, and provided a molecular basis for \nfurther expanding experimental samples to conduct in vivo \nexperiments to study the therapeutic effect of GANT61. \nOverall, this study provides new evidence for understand-\ning the etiology of endometriosis and seeking novel clinical \ntreatments.\nAcknowledgements\nWe would like to thank all the colleagues in our research.\nAuthors’ contributions\nYH and JW wrote the main manuscript text; XJ, LS and JG prepared Figs. 1 and \n2; TL, YC and JZ prepared Figs. 3 and 4; All authors reviewed the manuscript. \nThe authors read and approved the final manuscript.\nFunding\nThe work was supported by the National Nature Science Foundation of \nChina (81971359), the Key Project of Science and Technology of Harbin \n(2017AB9BS039), the Heilongjiang Postdoctoral Program Foundation (LBH-\nZ19085) and the Outstanding Young Medical Talents Training Fund project of \nthe First Affiliated Hospital of Harbin Medical University (HYD2020YQ0021).\nAvailability of data and materials\nAll data for this study are included in this article.\nDeclarations\nEthics approval and consent to participate\nAll patients signed an informed consent form prior to recruitment and the \nstudy protocol was approved by the Ethics Committee of Harbin Medical Uni-\nversity (202106), and all experimental methods were carried out in accordance \nwith approved guidelines of ARRIVE.\nConsent for publication\nNot applicable.\nCompeting interests\nAll authors have no competing interests in this research.\n\nPage 9 of 9\nHe et al. BMC Molecular and Cell Biology           (2022) 23:37 \n \nReceived: 13 November 2021   Accepted: 21 June 2022\nReferences\n 1. Bulun SE. Endometriosis. N Engl J Med. 2009;360:268–79.\n 2. Garry R. The endometriosis syndromes: a clinical classification in the pres-\nence of aetiological confusion and therapeutic anarchy. Hum Reprod. \n2004;19:760–8.\n 3. Giudice LC, Kao LC. Endometriosis. Lancet. 2004;364:1789–99.\n 4. Honda R, Katabuchi H. Pathological aspect and pathogenesis of endome-\ntriosis. Endometriosis. 2014:9–18.\n 5. Sampson JA. Peritoneal endometriosis due to the menstrual dissemi-\nnation of endometrial tissue into the peritoneal cavity. Am J Obstet \nGynecol. 1927;14:93–4.\n 6. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde men-\nstruation in healthy women and in patients with endometriosis. Obstet \nGynecol. 1984;64:151–4.\n 7. Briscoe J, Thérond PP . The mechanisms of hedgehog signalling and its \nroles in development and disease. Nat Rev Mol Cell Biol, 2013;14:416–429.\n 8. Farooqi AA, Mukhtar S, Riaz AM, et al. Wnt and SHH in prostate cancer: \ntrouble mongers occupy the TRAIL towards apoptosis. Cell Prolif. \n2011;44:508–15.\n 9. Jakobs P , Exner S, Schurmann S, Pickhinke U, Bandari S, Ortmann C, et al. \nScube2 enhances proteolytic Shh processing from the surface of Shh-\nproducing cells. J Cell Sci. 2014;127:1726–37.\n 10. Doheny D, Manore SG, Wong GL, et al. Hedgehog signaling and trun-\ncated GLI1 in Cancer. Cells. 2020;9(9).\n 11. Zipeng X, Fang W, Lingqiang L, et al. An SGLT2 inhibitor modulates SHH \nexpression by activating AMPK to inhibit the migration and induce the \napoptosis of cervical carcinoma cells. Cancer Lett. 2020;495:200-10.\n 12. Hiromichi M, Xuemei Z, Das Sanjoy K, et al. Indian hedgehog as a proges-\nterone-responsive factor mediating epithelial-mesenchymal interactions \nin the mouse uterus. Dev Biol. 2002;245:280–90.\n 13. Yanan H, Qiuyan G, Yan C, et al. Abnormal activation of the sonic hedge-\nhog signaling pathway in endometriosis and its diagnostic potency. Fertil \nSteril. 2018;110:128–136.e2.\n 14. Ashley S, Desmond B, Hankyu L, et al. Cell cycle-related kinase (CCRK) \nregulates ciliogenesis and hedgehog signaling in mice. PLoS Genet. \n2017;13:e1006912.\n 15. Ho Emily K, Tsai Anaïs E. Stearns Tim,Transient Primary Cilia Mediate \nRobust Hedgehog Pathway-Dependent Cell Cycle Control. Curr Biol. \n2020;30:2829–2835.e5.\n 16. Dmowski WP , Ding J, Shen J, Rana N, Fernandez BB, Braun DP . Apoptosis \nin endometrial glandular and stromal cells in women with and without \nendometriosis. Hum Reprod. 2001;16:1802–8.\n 17. Gargett CE, Schwab KE, Zillwood RM, Nguyen HPT, Wu D. Isolation and \nculture of epithelial progenitors and mesenchymal stem cells from \nhuman endometrium. Biol Reprod. 2009;80(6):1136–45.\n 18. Jing G, Li C, Ning L, et al. LPS/TLR4-mediated stromal cells acquire an \ninvasive phenotype and are implicated in the pathogenesis of adeno-\nmyosis. Sci Rep. 2016;6:21416.\n 19. Liang T, Li L, Cheng Y, et al. MicroRNA-194 promotes the growth, \nmigration, and invasion of ovarian carcinoma cells by targeting protein \ntyrosine phosphatase nonreceptor type 12. OncoTargets and Therapy. \n2016;9:4307–15.\n 20. Cheng Y, Li L, Wang D, et al. Characteristics of human endometrium-\nderived mesenchymal stem cells and their tropism to endometriosis. \nStem Cells Int. 2017;2017:4794827.\n 21. Mariano C-C, Miosotis G, Adalberto M, et al. Human endometriosis \ntissue microarray reveals site-specific expression of estrogen receptors, \nprogesterone receptor, and Ki67. Appl Immunohistochem Mol Morphol. \n2019;27:491–500.\n 22. Jiang Peng, Jia Mingzhu, Hu Jing, et al. Prognostic value of Ki67 in \npatients with stage 1-2 endometrial Cancer: validation of the cut-\noff value of Ki67 as a predictive factor. Onco Targets Ther, 2020, 13: \n10841–10850.\n 23. Xueping L, Yihang Z, Lijuan H, et al. Identification of a novel tumor angio-\ngenesis inhibitor targeting Shh/Gli1 signaling pathway in non-small cell \nlung cancer. Cell Death Dis. 2020;11:232.\n 24. Diana M, Dyson Matthew T, Ping Y, et al. Estrogen receptor β regulates \nendometriotic cell survival through serum and glucocorticoid-regulated \nkinase activation. Fertil Steril. 2016;105:1266–73.\n 25. Yuhong Z, Yangjia Z, Forkhead. Box C2 promotes the invasion ability of \nhuman trophoblast cells through hedgehog (Hh) signaling pathway. Cell \nBiol Int. 2018;42:859–66.\n 26. Noman AS, Uddin M, Chowdhury AA, et al. Serum sonic hedgehog (SHH) \nand interleukin-(IL-6) as dual prognostic biomarkers in progressive meta-\nstatic breast cancer. Sci Rep. 2017;7:1796.\n 27. Gomes DC, Jamra SA, Leal LF, et al. Sonic hedgehog pathway is upregu-\nlated in adamantinomatous craniopharyngiomas. Eur J Endocrinol. \n2015;172:603–8.\n 28. Zhihua Z, Changlai H, Rongjuan Z, et al. A Gli inhibitor GANT61 sup-\npresses cell proliferation, promotes cell apoptosis and induces G1/\nG0 cycle retardation with a dose- and time-dependent manner \nthrough inhibiting notch pathway in multiple myeloma. Cell Cycle. \n2020;19:2063–73.\n 29. Andrea C, Valentina I, Bruno B, et al. Chemical, computational and \nfunctional insights into the chemical stability of the hedgehog pathway \ninhibitor GANT61. J Enzyme Inhib Med Chem. 2018;33:349–58.\n 30. Nanta R, Kumar D, Meeker D, Rodova M, Van Veldhuizen PJ, Shankar S, \net al. NVP-LDE-225 (Erismodegib) inhibits epithelial-mesenchymal transi-\ntion and human prostate cancer stem cell growth in NOD/SCID IL2Rγ null \nmice by regulating Bmi-1 and microRNA-128. Oncogenesis. 2013;2:e42.\n 31. Magistri P , Battistelli C, Strippoli R, Petrucciani N, Pellinen T, Rossi L, et al. \nSMO inhibition modulates cellular plasticity and invasiveness in colorec-\ntal Cancer. Front Pharmacol. 2018;8:956.\n 32. Li Lihong Y, Rui CT, et al. Effects of immune cells and cytokines on inflam-\nmation and immunosuppression in the tumor microenvironment. Int \nImmunopharmacol. 2020;88:106939.\n 33. Fengyu W, Haili W, Lei S, et al. TRIM59 inhibits PPM1A through ubiquit-\nination and activates TGF-β/Smad signaling to promote the invasion of \nectopic endometrial stromal cells in endometriosis. Am J Physiol Cell \nPhysiol. 2020;319:C392–401.\n 34. Jing L, Zhifang Z, Jiamei L, et al. LIM kinase 1 mediates estradiol effects \non the phosphorylation of Cofilin1 in Eutopic endometrial stromal cells \nduring the invasion and proliferation of endometriosis. Reprod Sci. \n2019;26:1499–505.\n 35. Heard, Christian D. Simmons, Frank A. Simmen, et al. Krüppel-like factor \n9 deficiency in uterine endometrial cells promotes ectopic lesion estab-\nlishment associated with activated notch and hedgehog signaling in a \nmouse model of endometriosis. Endocrinology, 2014;155: 1532–1546.\n 36. Xavier T, Zaozao C, Ken J, et al. Cell migration Compr Physiol. \n2012;2:2369–92.\n 37. Masae S, Makoto K, Masaya K, et al. Hedgehog signaling pathway medi-\nates the progression of non-invasive breast cancer to invasive breast \ncancer. Cancer Sci. 2011;102:373–81.\n 38. Su JL, Do IG, Lee J, Kim KM, Jang J, Sohn I. Gastric cancer (GC) patients \nwith hedgehog pathway activation: PTCH1 and GLI2 as independent \nprognostic factors. Target Oncol. 2013;8(4):271–80.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in pub-\nlished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}