{"paper_id":"1031ef81-dfda-4639-8906-07cb69cad447","body_text":"Page 1/20\nDown-Regulating Hexokinase 2 Inhibits Proliferation\nof Endometrial Stromal Cells Through a\nNoncanonical Pathway Involving Phosphorylated-\nSTAT1 in Endometriosis\nShuhui Hou \nShanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine\nhttps://orcid.org/0000-0003-1706-8506\nShating Lei \nShanghai First Maternity and Infant Hospital, Tongji University School of Medicine\nHaiyan Peng \nShanghai First Maternity and Infant Hospital, Tongji University School of Medicine\nLichun Weng \nShanghai First Maternity and Infant Hospital, Tongji University School of Medicine\nSiji Lv \nShanghai First Maternity and Infant Hospital, Tongji University School of Medicine\nMingqing Li \nObstetrics and Gynecology Hospital of Fudan University\nDong Zhao  (  hendryz@gmail.com )\nShanghai Jiao Tong University School of Medicine https://orcid.org/0000-0002-2118-2910\nResearch\nKeywords: Endometriosis, endometrial stromal cells, HK2, glycolysis, STAT1\nPosted Date: September 2nd, 2021\nDOI: https://doi.org/10.21203/rs.3.rs-845266/v1\nLicense:   This work is licensed under a Creative Commons Attribution 4.0 International License.  \nRead Full License\n\nPage 2/20\nAbstract\nBackground: Endometriosis is a benign gynecologic disease that causes chronic pelvic pain,\ndysmenorrhea and infertility and shares several characteristics with malignant tumors, aicting women\nof reproductive age. Hexokinase 2 (HK2) plays a pivotal role as the rst rate-limiting enzyme in the\nmetabolic glycolysis pathway, and its abnormal elevation in tumors is associated with tumor genesis and\nmetastasis. However, the expression and role of HK2 in endometriosis remain unclear.\nMethods: We sequenced the primary endometrial stromal cells from patients with endometrioma and\nadopted immunohistochemistry, quantitative real-time PCR and western blot to determine the expression\nof HK2. Then wound healing assays, cell invasion assays, cell proliferation assays were performed to\nexplore the functions of HK2 in endometrial stromal cells. Furthermore, mice models of endometriosis\nwere recruited to observe the effects of HK2 inhibitors in vivo. Lastly, glycolysis metabolism detection and\ntranscriptome sequencing were carried out in HK2-knockdown endometrial stromal cells to analyze the\nmechanism of HK2 affecting cell function.\nResults: Endometriotic stromal cells displayed active glycolysis metabolism and elevated expression of\nHK2. Downregulating HK2 reduced the migration, invasion and proliferation capacity of endometrial\nstromal cells. Knockdown of HK2 induced upregulation of signal transducer and activator of transcription\n1 (STAT1) and their phosphorylation to attenuate the proliferation of endometrial stromal cells.\nConclusions: HK2 was associated with the migration, invasion and proliferation of endometrial stromal\ncells, which might provide new insights into the pathogenesis and treatment of endometriosis.\nBackground\nEndometriosis is one of the most common gynecologic diseases, characterized by ectopic growth of the\nendometrium. It aicts approximately 6% -10% of reproductive-age women (1) with manifestations\ninvolving chronic pelvic pain, dysmenorrhea, infertility, and malignant features such as invasion and\nmetastasis (2). However, the pathogenesis has not been elucidated yet (3). Besides the widely accepted\nretrograde menstruation hypothesis (4), factors from genetics(5, 6), immunology (7), endocrinology (8),\nhypoxic environment and metabolism (9) might contribute to the pathogenesis of endometriosis. Stromal\ncells were the major cell type of endometrial tissue, secreting matrix and ber in the lamina propria of the\nendometrium (10). They proliferate and differentiate with the transformation of the menstrual cycle and\nmay play roles in the establishment and progression of endometriosis (11), while our understanding is far\nfrom sucient yet.\nHexokinase (HK), the rst key rate-limiting enzyme in the glycolysis pathway, catalyzes the formation of\nglucose 6 phosphates (12). There are ve isoforms in mammalian tissues: HK1, HK2, HK3, HK4 and\nHexokinase Domain Containing 1 (HKDC1) (13). HK2 is insulin-sensitive, mainly exists in fat, bone, and\nheart tissue (12). More importantly, it has been found that the high aberrant expression of HK2 in various\n\nPage 3/20\ntumors is closely related to cell survival (14), and HK2 is regarded as a potential therapeutic target of\ntumors (15, 16). However, the expression of HK2 and its role in endometriosis remain indistinct.\nSignal transducers and activators of transcription (STATs) are a family of proteins, locating at the\ncytoplasm before being activated. Once activated, STATs form dimers translocate into the nucleus and\nbind to the promoter of the target genes (17). Signal transducer and activator of transcription 1 (STAT1)\nis alternately spliced into two protein forms, STAT1a (91 kDa) and STAT1b (84 kDa) (18).\nPhosphorylation of tyrosine 701 (Y701) loci, situates in the transcriptional activation domain of STAT1, is\nnecessary for the translocation and functions of STAT1 homodimer and heterodimer (19). The classical\nrole of STAT1 is a tumor suppressor depending on the activation of Y701 (20).\nIn this study, we aimed to investigate the metabolic disorder and the role of HK2 in endometrial stromal\ncells endometriosis. Furthermore, we explored the non-catalytic effect of HK2, upregulating\nphosphorylation of STAT1. These ndings unveil the noncanonical function of HK2 and provide a\npromising therapeutic target for endometriosis.\nMethods\nPatients and samples\nForty-six participants were enrolled in Shanghai First Maternity and Infant Hospital from April 2019 to\nSeptember 2020. The collection of the samples was approved by the Ethics Committee and obtained\ninformed consent from all patients. The experimental group included 23 cases of ectopic cyst wall of the\novary endometrioma conrmed by pathology. The endometrium was obtained from 23 donors who\nreceived hysteroscopy for benign gynecological diseases, excluding endometriosis in the control group.\nNone of the patients had other immune diseases, acute inammation, or estrogen-dependent diseases,\nand no hormone medications were used within three months before surgery. Specimens from both the\nexperimental and control groups were collected during surgery. The specimen was achieved and\nimmediately placed into the specimen preservation solution and then transferred to the laboratory.\nPrimary cells isolation and culture\nEndometrial stromal cells were isolated and cultured according to the methods described previously (44,\n45) and with slight modication. Specimen tissues were washed with phosphate buffered saline (PBS)\nand cut into fragments. 1 mg/mL collagenase type IV (Sigma, USA) was added and digested for 20-40\nmin in a shaker at 37°C. Tissue homogenate was ltered through a 40 μ m cell strainer (Falcon, USA), and\nthe cell suspension was collected and centrifuged for 10 min to collect cells. The isolated primary cells\nwere resuspended with complete medium DMEM/F12 (Hyclone, USA) containing 10% fetal bovine serum\n(FBS, Sciencell, USA) and cultured in an incubator at 37°C in a humidied atmosphere with 5% CO2.\nCell transfection\n\nPage 4/20\nHESCs were purchased from American Type Culture Collection and cultured under the same conditions as\nprimary cells. Three designed HK2-short hairpin RNAs (shRNAs) cloned into pGMLV-SC5-puro vector\nplasmids and scrambled control vector plasmids were transfected respectively with the lentiviral\npackaging mix into 293T cells to produce the virus for 48 h. The supernatant containing the virus was\nltered through a 0.45 μ m lter and used to infect HESCs. Seventy-two hours later, the infected HESCs\nwere selected with 30 μ g/mL puromycin (Invitrogen, USA), and the knockdown eciency was detected.\nThe sequences of shRNAs were listed in Supplementary Table 1.\nRNA extraction and quantitative real-time PCR (qRT-PCR) analysis\nAccording to the reagent instructions, total RNA was extracted from tissues and cells using RNAiso Plus\nand transcribed into cDNA (Takara, Japan). Then qRT-PCR was carried out in triplicate on a 384-well plate\nusing a QuantStudio 5 Flex Real-time PCR system (Applied Biosystems, USA). The expression of the\ntarget gene relative to ACTB was determined using the 2- ΔΔ CT method. The sequences of primers were\nlisted in Supplementary Table 1.\nWestern Blotting\nTissue and cellular proteins were extracted with RIPA lysate mixed with phosphatase inhibitors and\nquantied by the BCA method. Following the manufacturer's instructions (Beyotime Biotechnology,\nChina), cytoplasm and nucleus proteins were extracted. The exact amount of protein was electrophoresis\nwith SDS-polyacrylamide gel and then transferred onto a piece of PVDF membrane (Millipore, USA). After\nblocking, the membrane was incubated with the primary antibody against the target protein on a shaking\ntable at 4 ℃  overnight. Then the membrane was incubated with a secondary antibody, and the protein\nbands were quantitated by electrochemiluminescence assay. The antibody details were listed in\nSupplementary Table 2.\nImmunohistochemistry, immunocytochemistry and hematoxylin-eosin (HE) staining\nTissue sections were deparanized, rehydrated, retrieved antigen and removed endogenous peroxidase\n(Biotech Well, China). After being blocked and washed, these sections were incubated with the primary\nantibody at 4°C overnight. The next day, secondary antibody incubation and chromogenic detection were\nperformed. The sections were then counterstained with hematoxylin, dehydrated, mounted, and\nexamined.\nAs for immunocytochemistry, cells were seeded in each compartment of slides (Millipore, USA). After\nxing with 4% paraformaldehyde and incubating with PBS containing 0.5% Triton X-100, endogenous\nperoxidase removal and subsequent steps were the same as immunohistochemistry. The antibody details\nwere listed in Supplementary Table 2.\nTissue sections for HE staining were deparanized and rehydrated. They were treated with hematoxylin\nfor 5 min, ethanol containing 1% hydrochloric for 45 s, and then immersed in eosin for 5 min. Dehydration\n\nPage 5/20\nand subsequent steps were similar to immunohistochemistry.\nWound healing assay\nCells were seeded in a 6-well plate cultured to 80-90% conuence and then scratched with a pipette tip\nvertical to the six-well plate. Cell debris was gently washed off with PBS and added FBS-free medium.\nImages of cells were recorded at 0 h, 24 h, and 48 h after scraping, and scratch areas were measured.\nCell invasion and migration assay\nFor the invasion assay, 50 μ L diluted Matrigel (BD Bioscience, USA) was added to each transwell\nchamber (Millipore, USA) and solidied at 37 ℃  before cells were seeded. For both invasion and\nmigration assay, 5×104 cells resuspended in 200 μ L of FBS-free medium were seeded in the upper\ncompartment of the chamber, and 600 μ L complete medium was added to the lower compartment. After\n48 h-incubation, cells on the upper compartment were wiped with a cotton swab. The invaded cells at the\nlower compartment were xed in 4% paraformaldehyde and stained with crystal violet. Then invaded cells\nwere photographed under a microscope.\nCell proliferation assay\nA total of 100 μ L suspension containing 3 000 cells were seeded in each well of a 96-well plate. After\ndifferent treatments for an appropriate time, 10 μ L cell counting kit-8 reagent (CCK8, Dojindo, Japan) was\nadded to the culture medium at specic time points of 0 h, 24 h, 48 h, 72 h. After incubating for 4 h at\n37°C in 5% CO2, the optical density was measured at 450 nm using a microplate reader.\nDetection of glucose consumption, lactic acid production and the activity of hexokinase \nCultured primary endometrial stromal cells were trypsinized and counted and then seeded into a 96-well\nplate. After 24 h, the experiments of glucose consumption, lactic acid production, the activity of\nhexokinase detection were performed following the manufacturer’s instructions (Solarbio, China).\nExtracellular acidication rate (ECAR) detection\n1×105 cells were seeded in each well of a 96-well plate. The XF96 extracellular ux kits were pretreated,\nand the essential medium was prepared. The next day, 175 μ L essential medium was added to each well\nof the 96-well plate which would be incubated in a CO2-free incubator at 37 ℃  for 1 h. In addition, 25 μ L\nD-glucose (10 mM), Oligomycin (1 μ M) and 2-DG (50 mM) per well were added into the upper part of\nXF96 extracellular ux kits, which were put into a seahorse XF96 cellular ux analyzer. After 30 min, the\nlower layer of the XF96 extracellular ux kits was replaced with the cell plate, and detection was\nproceeded according to the standard procedures.\nMice model of endometriosis\n\nPage 6/20\nThe intraperitoneal endometriosis model was constructed according to a previously described\nmethod (46) with minor alterations. The uteruses of donor mice were removed and minced, and then\ninjected into recipient mice intraperitoneally. The recipient mice were divided into experimental group and\ncontrol group randomly. Drug solutions and solvents were intraperitoneally injected into the mice of the\nexperimental group and control group, respectively. All recipient mice were sacriced 14 days after\nimplantation, and the endometriosis lesions were collected.\nStatistical analysis\nThe statistical analysis was carried out using GraphPad Prism version 6.02. Results are reported as the\nmean ± SEM from triplicate experiments. Student's t-test and one-way ANOVA were used to analyzed\nstatistics following correction. There was a statistical difference when P < 0.05.\nResults\nGlycolysis metabolism was enhanced in ESCs\nTo investigate the differences between ectopic stromal cells (ESCs) with normal stromal cells (NSCs), we\nperformed transcriptome sequencing (Figure 1A). Gene ontology (GO) enrichment of upregulated\ndifferently expressed genes showed that the metabolic processes and glucose metabolic processes vary\n(Figure 1B). Furthermore, we found that most glycolysis-related genes were upregulated in ESCs (Figure\n1C), suggesting that the glycolysis pathway might be vibrant in ectopic stromal cells. Interestingly, we\nnoted that HK2 was differently expressed merely among the four hexokinases in endometrial stromal\ncells (Figure 1D). To further investigate glycolysis metabolism in ESCs and NSCs, we detected the\nglucose consumption, lactic acid production and the activity of hexokinase. The results indicated that\nglucose consumption (Figure 1E) and lactic acid production (Figure 1F) are increased in ESCs compared\nto NSCs. Besides, the activity of hexokinase was also signicantly enhanced in ESCs (Figure 1G). Next,\nthe extracellular acidication rate (ECAR) was detected to evaluate the glycolysis metabolism (Figure 1H).\nStatistical results revealed that the basal glycolysis level of ESCs was elevated compared with NSCs\n(Figure 1I), and glycolysis capacity was enhanced (Figure 1J).\nHK2 is upregulated in ectopic tissue and ESCs\nTo explore the role of HK2 in endometriosis, we detected the expression of HK2 in tissues and primary\nstromal cells, respectively. Immunohistochemistry results indicated that HK2 mostly expressed in stromal\ntissues and distributed more in ectopic tissues (Figure 2A) than in the control group. The expression of\nmRNA (Figure 2B) and protein of HK2 (Figure 2C and 2D) was upregulated in ectopic lesions compared to\nthe control endometrium. Subsequently, primary stromal cells were isolated and stained for cytokeratin-7\nand vimentin for identication (Figure 2E). The results showed HK2 was overexpressed in ESCs (Figure\n2G and 2H).\nInhibiting HK2 suppressed proliferation, invasion and migration of HESCs\n\nPage 7/20\nNext, we studied the effect of HK2 through treating on Human endometrial stromal cells (HESCs) with its\ninhibitor. 2-Deoxy-D-glucose (2-DG) is a glucose analog that serves as a competitive inhibitor of glucose\nmetabolism, inhibiting glycolysis via acting on hexokinase (21). As shown in the wound healing assays,\nwe found that the migration of HESCs decreased when the 2-DG supplement (Figure 3A), and the\ntranswell migration experiment also proved this conclusion (Figure 3B). Notably, the invasion capacity of\nHESCs was inhibited (Figure 3C). Furthermore, the effect of the inhibitor on the proliferation of stromal\ncells was detected. The higher the inhibitor concentration was, the lower the cell proliferation ability was\n(Figure 3D).\nInhibiting HK2 alleviated endometriosis in vivo\n3-Bromopyruvate (3-BP), another inhibitor of HK2 (22), was utilized intraperitoneally into constructed\nmice models of endometriosis (Figure 4A). We found that the lesions of mice injected with 3-BP were\nsignicantly smaller than those in the solvent control group. Next, we measured the gross weight of each\nlesion, and the results showed a remarkable difference between the treatment and control group (Figure\n4B). HE staining of the lesions conrmed the distribution of stroma and glands, similar to the uterus\nendometrial tissue, indicating that the mice model was successfully established (Figure 4C). Ki67\nstaining demonstrated reduced proliferation in the 3-BP treatment group (Figure 4F).\nHK2 knockdown attenuated the migration, invasion and proliferation of HESCs\nTo further determine the effect of HK2 on cell function, we transfected HESCs with lentivirus to\ndownregulate HK2 (Figure 5A-5D), and we chose cell line transfected with shRNA2 for the following\nexperiment. Signicantly, the sh-HK2 cells appeared reduced ability of migration (Figure 5E and 5F),\ninvasion (Figure 5G and 5H) and proliferation (Figure 5I).\nHIF-1 α  upregulated HK2\nStudies have shown that endometriosis lesions are in anoxic environments, and the expression of\nhypoxia-inducible factor-1 α  (HIF-1 α ) was increased (23, 24). We validated it through an\nimmunohistochemistry assay (Figure 6A). According to the literature, we hypothesize that the\noverexpression of HK2 in ectopic lesions may attribute to the hypoxic environment. We treated HESCs\nwith dimethylallyl glycine (DMOG), inducing the accumulation and stabilization of HIF-1 α . The results\nindicated that with the increase of HIF-1 α , the expression of HK2 elevated (Figure 6B).\nHK2 knockdown reduced cell proliferation by affecting phosphorylation of STAT1 rather than glycolysis\npathway\nTo uncover how HK2 affected cell function, ECARs of sh-HK2 and sh-NC were determined (Figure 7A).\nSurprisingly, the results conrmed no signicant difference in basal glycolysis level and glycolysis ability\nof sh-HK2 cells compared with sh-NC (Figure 7B and 7C), suggesting HK2 regulated cell migration,\ninvasion, and proliferation without inuencing the glycolysis process. Furthermore, results revealed no\ndifferentially expressed genes of glycolysis pathways except HK2 (Supplementary Figure A) or\n\nPage 8/20\nenrichment information related to glycolysis in differential expressed genes of sh-HK2 and sh-NC cells\n(Supplementary Figure B and C).\nNevertheless, we found that knockdown of HK2 induced an increase of STAT1 expression and\nphosphorylation of its Y701 loci (Figure 7D). As reported, STAT1 enters the nucleus after being activated\nto inhibit cell proliferation and promote apoptosis as a tumor suppressor (25, 26). To unveil the\ndistribution of STAT1 under the impact of HK2, we extracted protein from the cell cytoplasm and nucleus,\nrespectively. HK2 was accidentally detected in the nucleus protein. Crucially, the phosphorylated-STAT1\n(p-STAT1) ratio to total STAT1 in the nucleus increased remarkably in sh-HK2 compared to sh-NC cells.\nMoreover, FBS treatment stimulated an increase of the ratio in the nucleus, while the cytoplasmic\nchanged inconspicuously (Figure 6E). These results suggest that HK2 may also play a non-canonical role\nas a signaling molecule other than participating in glycolysis as an enzyme.\nNext, we treated shHK2 cells and HESCs with udarabine, a phosphorylation inhibitor of STAT1, and\nobserved rescue changes in cell proliferation. The addition of STAT1 inhibitor recovered the reduced\nproliferation ability caused by knockdown of HK2 to a certain extent (Figure 6F). We concluded that the\neffect of HK2 on the proliferation of HESCs is partly dependent on the phosphorylation of STAT1.\nDiscussion\nCurrently, endometriosis therapy is limited to hormonal treatment and surgery, which cannot essentially\ncure the disorder (27). Kasvandik et al. reported metabolic reprogramming of ESCs by deep quantitative\nproteomics (28), and Young et al. reported the Warburg-like effects in ectopic lesions (29). Recently, Lee et\nal. conrmed the altered metabolism of ESCs and over-expressed pyruvate dehydrogenase kinase 1, a\nglycolysis enzyme (30). In this study, we further identied the abnormal glucose metabolism and\nincreased expression of HK2 in ESCs, the rst key enzyme of the glycolysis pathway. Moreover, the\nproliferation, migration, and invasion cell ability were reduced when HK2 was inhibited and knocked\ndown, and an inhibitor of HK2 could alleviate endometriosis in vivo. These results suggested that HK2\nmay be related to endometriosis progression and is a potential therapeutic target that needs further study.\nHIF-1 α  is closely associated with endometriosis, according to various studies (31). Retrograde\nendometrial fragments suffer from more hypoxic stress, and HIF-1 α  accumulates in ectopic areas (24).\nFurther, HIF-1 α  could affect epithelial to mesenchymal transition (32), growth of vascular endothelial cells\n(33), and adhesion (34), invasion (35), migration (36) of ESCs. Herein, we unveiled another working\npathway of HIF-1 α  via up-regulating HK2, a realistic and feasible target to treat endometriosis since\nanoxic state and HIF-1 α  accumulation are inevitable.\nConventionally, STATs were activated by cell surface receptor JAKs binding to the ligand and regulated\nthe immune system, inhibited cell proliferation, and promoted apoptosis by regulating genes expression\nassociated with the cell cycle (37, 38). In recent years, studies have found that STAT1 may act as a tumor\npromoter (39), suggesting that the function of STAT1 might change with the cellular and external\nenvironment (40). Elevation of p-STAT1 has previously been reported to promote apoptosis of ectopic\n\nPage 9/20\nendometrial stromal cells (41). We found that the effect of HK2 on cell proliferation was partially\ndependent on p-STAT1.\nStudies have shown that various metabolic enzymes in the cytoplasm and mitochondria could\ntranslocate into the nucleus to modify histones and DNA, regulating gene expression (42). A report stated\nHK2 could translocate into the nucleus as a coactivator participating in redox homeostasis (43). In our\nndings, HK2 affected the expression and distribution of phosphorylation of STAT1. So far, we have\nassociated HK2 with STAT1 and revealed the noncanonical function of HK2.\nConclusions\nIn summary, we demonstrate the vibrant glycolysis metabolism and overexpression of HK2 in ESCs.\nInhibiting HK2 reduces cell migration, invasion, and proliferation in vitro and alleviates endometriosis in\nvivo. HIF-1 α  upregulates the expression of HK2, and knockdown of HK2 attenuates cell proliferation by\nupregulating p-STAT1. The noncanonical role of HK2 is revealed, expecting to shed light on the\npathogenesis and potential treatment of endometriosis.\nAbbreviations\nHK2                         Hexokinase 2\nSTAT1                     Signal transducer and activator of transcription 1\nHK                           Hexokinase\nHKDC1                   Hexokinase Domain Containing 1\nY701                       Tyrosine 701\nESC                         Ectopic stromal cell\nNSC                         Normal stromal cell\nGO                           Gene Ontology\nECAR                      Extracellular acidication rate\nHESC                      Human endometrial stromal cell\n2-DG                       2-Deoxy-D-glucose\n3-BP                        3-Bromopyruvate\nHIF-1 α                      Hypoxia-inducible factor-1 α\n\nPage 10/20\nDMOG                    Dimethylallyl glycine\np-STAT1                  phosphorylated STAT1\nPBS                         Phosphate buffered saline\nFBS                         Fetal bovine serum\nshRNA                     short hairpin RNA\nqRT-PCR                 Quantitative real-time polymerase chain reaction\nHE                           Hematoxylin and eosin\nCCK8                      Cell counting kit-8\nDeclarations\nEthics approval and consent to participate\nThe study has been approved by the ethics committee of Shanghai First Maternity and informed consent\nwas obtained from all patients. The animal experiment was approved and monitored by the institutional\nanimal care.\nConsent for publication\nNot applicable.\nAvailability of data and materials\nAll data generated or analyzed during this study are included in this published article and its additional\nles.\nCompeting interests\nThe authors declare that they have no competing interests.\nFunding\nThis work was supported by the Natural Science Foundation of China (NSFC81771548 and\nNSFC82071615).\nAuthors' contributions\nSH designed the study, mainly performed the experiments, and wrote the manuscript under the direction\nof DZ; SL, HP, LW and SL participated in the experiments and analysis; ML participated in study design\n\nPage 11/20\nand advised the paper. 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STAT1 is a sex-specic tumor\nsuppressor in colitis-associated colorectal cancer. Mol Oncol. 2018;12(4):514–28.\n27. Berveiller P, Ropert S, Mir O. Endometriosis. N Engl J Med. 2020;383(2):193–4.\n2. Kasvandik S, Samuel K, Peters M, Eimre M, Peet N, Roost AM, et al. Deep Quantitative Proteomics\nReveals Extensive Metabolic Reprogramming and Cancer-Like Changes of Ectopic Endometriotic\nStromal Cells. J Proteome Res. 2016;15(2):572–84.\n29. Young VJ, Ahmad SF, Brown JK, Duncan WC, Horne AW. ID2 mediates the transforming growth\nfactor-beta1-induced Warburg-like effect seen in the peritoneum of women with endometriosis. Mol\nHum Reprod. 2016;22(9):648–54.\n30. Lee HC, Lin SC, Wu MH, Tsai SJ. Induction of Pyruvate Dehydrogenase Kinase 1 by Hypoxia Alters\nCellular Metabolism and Inhibits Apoptosis in Endometriotic Stromal Cells. Reprod Sci.\n\nPage 13/20\n2019;26(6):734–44.\n31. Zhan L, Wang W, Zhang Y, Song E, Fan Y, Wei B. 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Hypoxia Promotes Invasion of Endometrial Stromal Cells via\nHypoxia-Inducible Factor 1alpha Upregulation-Mediated beta-Catenin Activation in Endometriosis.\nReprod Sci. 2016;23(4):531–41.\n3. Liu H, Zhang Z, Xiong W, Zhang L, Xiong Y, Li N, et al. Hypoxia-inducible factor-1alpha promotes\nendometrial stromal cells migration and invasion by upregulating autophagy in endometriosis.\nReproduction. 2017;153(6):809–20.\n37. Su Q, Wang F, Dong Z, Chen M, Cao R. IFNgamma induces apoptosis in human melanocytes by\nactivating the JAK1/STAT1 signaling pathway. Mol Med Rep. 2020;22(4):3111–6.\n3. Zenke K, Muroi M, Tanamoto KI. IRF1 supports DNA binding of STAT1 by promoting its\nphosphorylation. Immunol Cell Biol. 2018;96(10):1095–103.\n39. Hou Y, Li X, Li Q, Xu J, Yang H, Xue M, et al. STAT1 facilitates oestrogen receptor alpha transcription\nand stimulates breast cancer cell proliferation. J Cell Mol Med. 2018;22(12):6077–86.\n40. Zhang Y, Liu Z. STAT1 in cancer: friend or foe? Discov Med. 2017;24(130):19–29.\n41. Li J, Abudula M, Fan X, Wang F, Chen Y, Liu L. Sunitinib induces primary ectopic endometrial cell\napoptosis through up-regulation of STAT1 in vitro. J Clin Lab Anal. 2020;34(11):e23482.\n42. Xu D, Shao F, Bian X, Meng Y, Liang T, Lu Z. The Evolving Landscape of Noncanonical Functions of\nMetabolic Enzymes in Cancer and Other Pathologies. Cell Metab. 2021;33(1):33–50.\n43. Sheikh T, Gupta P, Gowda P, Patrick S, Sen E. Hexokinase 2 and nuclear factor erythroid 2-related\nfactor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells. J\nBiol Chem. 2018;293(13):4767–77.\n44. Ryan IP, Schriock ED, Taylor RN. Isolation, characterization, and comparison of human endometrial\nand endometriosis cells in vitro. J Clin Endocrinol Metab. 1994;78(3):642–9.\n45. Lei S, Cao Y, Sun J, Li M, Zhao D. H2S promotes proliferation of endometrial stromal cells via\nactivating the NF-kappaB pathway in endometriosis. Am J Transl Res. 2018;10(12):4247–57.\n\nPage 14/20\n4. Lai ZZ, Yang HL, Shi JW, Shen HH, Wang Y, Chang KK, et al. Protopanaxadiol improves endometriosis\nassociated infertility and miscarriage in sex hormones receptors-dependent and independent\nmanners. Int J Biol Sci. 2021;17(8):1878–94.\nFigures\nFigure 1\n\nPage 15/20\nGlycolysis metabolism was enhanced in ESCs. (A) Heatmap of the differentially expressed genes\nbetween ectopic stromal cells (ESC) vs. normal stromal cells (NSC) groups. (B) Top 20 GO items enriched\nfrom upregulated differentially expressed genes in ESC vs. NSC groups. (C) Expression of genes related\nto glycolysis process in NSCs and ESCs. (D) Expression of four hexokinases in NSCs and ESCs. (E-G)\nDetermination of glucose uptake, lactic acid production and hexokinase activity in NSCs and ESCs using\nkits, respectively. (H-J) Extracellular acidication rates of NSCs and ESCs were shown in gure 1H. The\nresponse inducing by the addition of glucose (GLU) reects cellular basal glycolysis level, and statistical\nresults were shown in gure 1I. The response inducing by the addition of oligomycin (OLI) reects cellular\nmaximal glycolysis capacity, and statistical results were shown in gure 1J. The data are expressed as\nthe mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001; ns, no statistical difference.\nFigure 2\nHK2 is upregulated in ectopic tissue and ESCs. (A) Immunohistochemistry revealed the expression of HK2\nin tissues from negative control (NC) and endometriosis (EMS) groups. (B) The mRNA level of HK2 in NC\nand EMS groups was determined by qRT-PCR. (C, D) The protein level of HK2 in NC and EMS groups was\nanalyzed by western blotting and quantied normalized to α -tubulin. (E) Primary endometrial stromal\n\nPage 16/20\ncells were identied with vimentin and cytokeratin 7 (CK7) staining. (F) The mRNA level of HK2 in NSCs\nand ESCs determined by qRT-PCR. (G, H) The protein level of HK2 in NSCs and ESCs groups was\nanalyzed by western blotting and quantied normalized to α -tubulin. The data are expressed as the mean\n± S.E.M. *p<0.05, **p<0.01, ***p<0.001; ns, no statistical difference.\nFigure 3\nInhibiting HK2 suppressed proliferation, invasion and migration of HESCs. (A, B) Human endometrial\nstromal cells (HESCs) were treated with HK2 inhibitors, 2-Deoxy-D-glucose (2-DG, 0.25mM), and DMSO, a\nnegative control to observe the effect on cell migration by wound healing assay. The wound area at 12 h\nand 24 h relative to 0 h of the two groups were calculated and compared. (C, D) HESCs were treated with\n2-DG (0.1 mM, 0.25mM) and DMSO to observe the effect on cell migration and invasion by transwell\nassay. The invaded cells of the three groups were counted and compared. (E) HESCs were treated with 2-\nDG (0.1 mM, 0.25mM) and DMSO as a negative control to observe the effect on cell proliferation. The\ndata are expressed as the mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001; ns, no statistical difference.\n\nPage 17/20\nFigure 4\nInhibiting HK2 alleviated endometriosis in vivo. (A) The diagram showed the procedures of establishing\nthe endometriosis mice model and the experimental scheme. (B) The ectopic lesions in the mouse\nperitoneal cavity of the solvent group and two treatment groups (1 mg/kg and 2.5 mg/kg) were collected\nand weighed. (C) HE staining of the ectopic lesions showed that stromal cells and glands were distributed\nin the lesion tissue similar to uterus endometrium. (D) Ki67 staining of the ectopic lesions from the\ncontrol and treatment group. The data are expressed as the mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001;\nns, no statistical difference.\n\nPage 18/20\nFigure 5\nHK2 knockdown attenuated the migration, invasion and proliferation of HESCs (A) The transfection\neciency of sh-NC and sh-HK2 lentivirus was showed by green uorescence. (B-D) The HK2 knockdown\neciency of negative control (NC), sh-NC, and three designed sh-RNA targeted HK2 were conrmed by\nqRT-PCR, Western Blotting and immunocytochemistry. (E, F) The wound healing assay was performed to\nobserve the migration of sh-NC and sh-HK2 cells. The wound area at 12 h and 24 h relative to 0 h of the\ntwo groups were calculated and compared. (G, H) Transwell assay was performed to observe the\nmigration and invasion of sh-NC and sh-HK2 cells. The invaded cells of the two groups were counted and\ncompared. (I) Comparison of cell proliferation between sh-NC and sh-HK2 cells. The data are expressed\nas the mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001; ns, no statistical difference.\n\nPage 19/20\nFigure 6\nHK2 was upregulated by HIF-1 α . (A) Immunohistochemistry exhibited the expression of HIF-1 α  in tissues\nfrom NC and EMS groups. (B) HESCs were treated with the indicated concentrations of Dimethyl\nsulfoxide (DMOG), and the protein level of HIF-1 α  and HK2 was detected. The data are expressed as the\nmean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001; ns, no statistical difference.\nFigure 7\nHK2 knockdown reduced cell proliferation by affecting phosphorylation of STAT1 rather than glycolysis\npathway. (A-C) Extracellular acidication rates of sh-NC and sh-HK2 cells were shown in gure 7A. The\nresponse inducing by the addition of glucose (GLU) reects cellular basal glycolysis level, and statistical\n\nPage 20/20\nresults were shown in gure 7B. The response inducing by the addition of oligomycin (OLI) reects\ncellular maximal glycolysis capacity, and statistical results were shown in gure 7C. (D) The protein level\nof signal transducer and activator of transcription 1 (STAT1) and phosphorylation of Tyrosine701\n(Tyr701) loci in blank control, sh-NC and sh-HK2 cells detected by Western Blotting, and VINCULIN acted\nas an internal reference. (E) The protein level of HK2, STAT1, p-STAT1 in nucleus and cytoplasm of sh-NC\nand sh-HK2 cells with FBS or not detected by Western Blotting. Histone H3 and α -tubulin acted as the\ninternal reference of the nucleus and cytoplasm, respectively. (F) sh-HK2 cells were treated with\nudarabine (5 μ M), inhibiting phosphorylation of STAT1 and PBS as a negative control to observe the\neffect on cell proliferation. The data are expressed as the mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001;\nns, no statistical difference.\nSupplementary Files\nThis is a list of supplementary les associated with this preprint. Click to download.\nAdditionalle1.pdf\nAdditionalle2.pdf","source_license":"CC0","license_restricted":false}