{"paper_id":"a4cedf10-7cd2-4836-b13a-4952abd529a7","body_text":"Endometriosis, a common contributor for infertility and chronic pelvic pain,\nis characterized by the extra-uterine growth of endometrial glands and stroma ( Giudice and Kao 2004 ). Among the numerous\ntheories regarding the pathogenesis of endometriosis, the most commonly accepted one\nis Sampson’s hypothesis of retrograde menstruation, which states that the\nendometrial tissues shed from uterine cavity during menses exit the uterus through\nfallopian tubes ( Sampson 1927 ). Although\nendometriosis is generally assumed to be a benign disease, it has the similar\nmalignant biological behavior like cancer, such as aggressive migration and invasion\n( Bassi,  et al . 2009a ),\nwhich is crucial for the development of endometriosis.\nAccumulating evidence has suggested that hypoxia played important roles in\nendometriosis ( Hsiao,  et al .\n2014 ,  Lin,  et al .\n2012 ). Hypoxia-inducible factor (HIF)-1, a heterodimeric transcriptional\nfactor mediating the cellular response to hypoxia, is made up of α and\nβ subunits ( Majmundar,  et\nal . 2010 ). Unlike the constitutively expressed\nHIF-1β, HIF-1α is highly regulated by cellular oxygen tension. Under\nnormoxic conditions, the HIF-1α subunit is rapidly degraded; whereas under\nhypoxic conditions, HIF-1α is stabilized and translocated to the nucleus\nwhere it heterodimerized with HIF-1β to cause target gene transcription\n( Semenza 2009 ). HIF-modulated genes have\nbeen identified to contribute to invasion, angiogenesis and autophagy of different\ntypes of tumor cells ( Cheng,  et al .\n2013 ,  Zhao,  et al .\n2010 ). Moreover, the expression level of HIF-1α in serum and\nectopic endometrium of patients with endometriosis was elevated ( Karakus,  et al . 2016 ,  Wu,  et al . 2007 ).\nAutophagy is an evolutionary conserved process responsible for the bulk\ndegradation of cytoplasmic components, such as long-lived proteins and damaged\norganelles ( Hale,  et al .\n2013 ). During autophagy, double-membrane vesicles surround and deliver\nthe cytoplasmic material to lysosomes for degradation ( Yang and Klionsky 2010 ). Autophagosomes formation\nrequires two ubiquitin-like protein conjugation pathways: autophagy-related gene\n(Atg)12-Atg5 conjugation system and the microtubule-associated protein light chain 3\nbeta (LC3) - lipid phosphotidylethanolamine (PE) conjugation system ( Ohsumi and Mizushima 2004 ). LC3, a mammalian homolog of\nyeast Atg8, is known to be present on autophagosomes, and the conversion of LC3 I to\nLC3 II is a widely used marker to monitor this process ( Mizushima, et al. 2010 ). Beclin1 (also named ATG6) is\nanother key regulator, which play essential roles in autophagy activation ( Kang, et al. 2011 ). It is now generally\naccepted that autophagy is a protective mechanism for cells adaptation to stress\nconditions like hypoxia ( Bhogal,  et\nal . 2012 ,  Hu,  et\nal . 2012 ). Interestingly, autophagy upregulation was\nobserved in ectopic endometrium of patients with ovarian endometriosis ( Allavena,  et al . 2015 ).\nHowever, it is still unclear whether autophagy was activated in human endometrial\nstromal cells (HESCs) under hypoxia environment and few studies have elucidated the\ncorrelation between HIF-1α and autophagy in the pathogenesis of\nendometriosis.\nTherefore, in the present study, we aimed to investigate: (a) whether\nexpression levels of HIF-1α and autophagy were changed in ectopic\nendometrium; (b) the molecular mechanism of autophagy activation by hypoxia in human\nendometrial stromal cells (HESCs); (c) the possible role of autophagy in\nHIF-1α induced migration and invasion of HESCs.\n\nThe tissue samples were obtained with full and informed patient consent.\nEthics approval for this study was obtained from the local Ethics Committee of\nTongji Medical College, Huazhong University of Science (IORG No:\nIORG0003571).\nThe patients recruited for the study were non-pregnant women of\nchildbearing age (22–48 years) attending the Department of Obstetrics\nand Gynecology, Union Hospital, Tongji Medical College, Huazhong University of\nScience and Technology between October 2013 and October 2015. All patients had\nregular menstrual cycles and were not taking any combination hormonal\ncontraception for at least six months prior to surgery. The endometrial samples\nwere collected during the proliferative stage, which was confirmed based on\nclinical or histologic criteria.\nAs controls, eleven cases of normal endometrium were obtained from\npatients with tubal infertility. Ten cases of eutopic endometrium (from another\ngroup of women with ovarian endometriosis) and ten cases of ectopic endometrium\n(from ovarian endometriotic cysts) were obtained from patients who underwent\nlaparoscopic surgery or hysterectomy. All of the ectopic endometrium were\nclassified as revised American Fertility Society stage III or IV ( 1997 ). The collected endometrial tissues was divided\ninto two parts: the first part was used for immunohistochemistry (IHC) analysis\naccording to the criteria of Noyes  et al  ( Noyes,  et al . 1975 ), and the second\npart was used for western blot analysis. Besides, another thirty cases of\neutopic endometrium of patients with endometriosis were collected for isolation\nand cultivation of endometrial stromal cells. The endometrial tissues were\ncollected using the Nowak’s curette just before the surgical procedure,\nand immediately transported to the laboratory.\nAll fresh surgical specimens were fixed in 10 % formaldehyde for\n24 hours, then embedded in paraffin blocks. Formalin-fixed and paraffin-embedded\nendometrial tissues were sectioned at 5 μm and mounted on\nalcohol-cleaned glass slides. The sections were dewaxed in xylene and rehydrated\nby passing through a graded series of alcohol to water, and antigen retrieval\nwas performed by heating sections in citrate buffer at pH 6.0. Endogenous\nnon-specific peroxidase activity was quenched by incubating the section in\n50% ethanol solution containing 3% H2O2 for 30 min. The sections\nwere sequentially blocked with protein block for 30 minutes followed by blocking\nin bovine serum albumin for 30 min and then incubated with primary antibodies\nagainst HIF-1α (1:1000; Affinity, USA) and LC3B (1:1000; Abcam,\nCambridge, UK) overnight at 4 °C. After washing in PBS, the sections\nwere incubated with peroxidase-labelled anti-rabbit IgG (1:500; Wuhan Boster\nBiotechnology Co., Ltd, China) for 30 min. Finally, all slides were incubated\nwith DAB-Substrate (Beyotime, China) and counterstained in haematoxylin before\ndehydrated and mounted. After the immunohistochemical analysis, IPP software\n(image-pro plus 6.0) was used to analyze the optical density of the\nrepresentative images (see  Supplemental Table. 1 ).\nThe collected tissues were washed with PBS for three times, then minced\ninto 1mm pieces with a sterile surgical scissors and digested in PBS containing\n2 mg/mL of type II collagenase (0.1%, Sigma-Aldrich) at 37°C for\n45–60 minutes with constant agitation. Stromal cells were isolated from\nthe epithelial cells and debris by use of 150 and 37.4μm sieves, and the\nfiltered stromal cells were plated in T25 flasks. After overnight culture, the\nstromal cells attached, and the contaminated blood cells and debris that were\nsuspended in the culture medium were removed by aspiration, and the stromal\ncells were washed with PBS. The stromal cells were subsequently cultured in\nDulbecco’s modified Eagle’s/F12 medium (DMEM/F12; HyClone)\nsupplemented with 20% fetal bovine serum (FBS; HyClone), 100 U/mL\npenicillin, and 100 mg/mL streptomycin (HyClone) in humidified atmosphere with\n5% CO2 at 37°C. The purity of isolated stromal cells was\n>95% and stromal cells were contaminated by less than 1% of\nepithelial cells, as determined by diffuse and strong cytoplasmic immunostaining\nfor vimentin (diluted 1:100; Cell Signaling Technology, USA) and negative\ncellular staining for E-cadherin (diluted 1:150; Cell Signaling Technology, USA)\nin immunocytochemistry (see  Supplemental Fig. 1 ). Endometrial stromal cells were cultured in\nDMEM/F12 medium with the addition of either 10 mM 3-methyladenine (3-MA) or 500\nμM Chloroquine to inhibit autophagy.\nUpon reaching confluence, the endometrial stromal cells (4 ×\n10 5 ) were seeded in 60 mm culture dishes and fresh medium was\nused to keep the cells healthy by providing fresh nutrients before hypoxia\ntreatment. The culture dishes were incubated in a modular incubator chamber\n(Thermo Scientific, USA) containing humidified hypoxic air (1% O2,\n5% CO2, 94% N2) for 0, 4, 8, 16 and 24 hours at 37 °C.\nStromal cells cultured under normoxic condition (20% O2, 5% CO2\nand 75% N2) were used as controls.\nHypoxia treated cells were collected at the indicated time points and\nprepared for Western blot analysis. After cultured under hypoxic conditions for\n24 hours, monodansylcadaverine (MDC) staining and acridine orange (AO) staining\nwas performed to detect the accumulation of autophagic vacuoles. In addition,\nthe ultrastructure of autophagosomes in hypoxia treated cells was observed by\ntransmission electron microscopy.\nThe immunocytochemistry were performed to detect mesenchymal marker\nvimentin and epithelial marker E-cadherin. HESCs were plated into a 6-well plate\nat a density of 2 × 10 4  cells/well and grown until\napproximately 80% confluent. The medium was removed, and the cells were\nwashed three times in PBS. After fixation with 4% paraformaldehyde at\n4°C for 15 min, the cells were soaked in 0.3% Triton X-100\n(Sigma, Belgium) for 15 min to increase their permeability to antibodies. For\nblocking unspecific binding site of antigens, the cells were rinsed with\n10% bovine serum albumin in PBS for 60 min, and incubated overnight at\n4°Cwith the primary antibodies that mentioned previously. The cells were\nthen washed three times in PBS and incubated with horseradish\nperoxidase-conjugated secondary antibody for two hours, followed by washing in\nPBS. Images were collected using an Eclipse TE2000-S microscope system (Nikon UK\nLtd, Surrey) and Image-Pro Plus (Media Cybernetics UK, Berkshire).\nCollected endometrial tissues and cultured HESCs were washed three times\nwith ice cold PBS and lysed in radio immunoprecipitation assay (RIPA) buffer\n(Beyotime Biotechnology, China) containing protease inhibitors (Sigma, USA). The\ncells were scraped in this lysis buffer, kept on ice for at least 30 minutes,\ncentrifuged at 12,000 g at 4°C for 15 minutes, and diluted in 5x sample\nbuffer (Beyotime Biotechnology, China). BCA protein assay kit (Beyotime, China)\nwas used to determine the protein concentrations. Equal amounts of proteins\n(30ug) were mixed with the sample buffer (4% SDS, 10%\nbeta-mercaptoethanol, and 20% glycerol in 0.125 M Tris, pH 6.8)\ncontaining bromophenol blue, and were boiled for10 minutes at 95°C. The\nsamples were loaded and separated by 12% sodium dodecyl\nsulfate–polyacrylamide gel electrophoresis gels (PAGE) with running\nbuffer. The proteins separated by SDSPAGE were transferred to polyvinylidene\ndifluoride (PVDF) membranes (Immobilon-P transfer membrane). The membranes were\nincubated with 5% fat-free milk in Tris-buffered saline containing\n0.05% Tween 20 for 1 hours, and were then incubated overnight at\n4°C with the following primary antibodies: HIF-1α (diluted\n1:1000; Affinity, USA), LC3B (diluted 1:1000,Abcam, Cambridge, UK), Beclin1\n(diluted 1:1,000, Abcam, Cambridge, UK) and GAPDH (diluted 1:1000; Affinity,\nUSA). The membranes were washed three times with TBST for 15 minutes, and then\nincubated with an HRP-labeled secondary Ab at room temperature for 1 hour. The\nmembranes were washed again and treated with ECL-Western blot detecting reagent\n(Millipore. USA) according to the manufacturer’s recommendations. The\nprotein bands intensity were observed by imaging system (Gel Doc 2000; Bio-Rad,\nUSA) and analysis with Image J software (NIH) (version 1.5, USA).\nAcridine orange (AO) is a fluorescent cationic dye used to detect acidic\nvesicular organelles (lysosomes) within cells. It can interact with DNA emitting\ngreen fluorescence or accumulate in acidic organelles in which it becomes\nprotonated forming aggregates that emit bright yellow-to-orange fluorescence\n( Pierzynska-Mach,  et al .\n2014 ). The cytoplasm and nucleus showed bright green fluorescent\nsignal, while the acidic vesicular organelles showed bright yellow-to-orange\nfluorescent signal. Briefly, 5 × 10 4  cells were stained with\n1 μg/mL acridine orange (AO) (Sigma-Aldrich, St. Louis, MO, USA) in PBS\nand incubated for 15 min at 37 °C in the dark. After incubation, cells\nwere washed with PBS for three times and immediately observed using an inverted\nfluorescence microscope (IX51, Olympus, Tokyo, Japan). The autophagy was\nmeasured by quantification of the rate of AO positive stained vacuoles in five\nrandom fields (a field containing at least 40 cells) for each experimental\ncondition.\nTo detect autophagic vacuoles, monodansylcadaverine (MDC), a fluorescent\ndye known as specific marker for autophagic vacuoles, was used. 5 ×\n10 4  cells were grown on coverslips in 6-well plate, and cultured\nunder hypoxic conditions for the indicated time, followed by washing three times\nwith PBS and fixed in 10% formalin solution for 10 min. Then cells were\nstained with 0.05 mM MDC (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes at\n37°C in the dark. The following procedures were the same as AO staining.\nThe autophagy was measured by quantification of the rate of MDC positive stained\nvacuoles in five random fields (a field containing at least 40 cells) for each\nexperimental condition.\nTo identify autophagosomes at the ultrastructural level, HESCs were\ncultured under hypoxic or normoxic conditions for 24h. After the indicated\ntreatment, HESCs were washed three times with PBS and incubated with trypsin for\n2 min. Cells were collected by centrifugation at 1,000 × g for 5 min.\nThe cell pellets were suspended and fixed with 2.5% glutaraldehyde in\n0.1 M Na-phosphate buffer (pH 7.4) at 4°C overnight, and then washed in\n0.1 M Na-phosphate buffer (pH 7.4) twice for 15 min each and post-fixed with\n1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) for 3 hours. After being\nwashed by 0.1 M Na-phosphate buffer, the cells were then dehydrated at\n25°C with a graded series of ethanol and gradually infiltrated with\nepoxy resin mixture (812 resin embedding kit). The samples were sequentially\npolymerized at 37°C for 12h, 45°C for 12 h, and 60°C for\n24 h. Ultrathin sections (50–70 nm) were cut by using LKB microtome and\nmounted on single-slot copper grids. THE sections were subjected to double\nstaining with uranyl acetate and lead citrate and examined using a transmission\nelectron microscope (Philips CM-120).\nHIF-1α siRNA, Beclin1 siRNA and scrambled negative control siRNA\nwere purchased from Shanghai GenePharma (China). The siRNA sequences included\nHIF-1α siRNA (sense, 5′-GCUGGAGACAAUCAUAUTT-3′,\nantisense, 5′-AUAUGAUUGUGUCUCCAGCTT-3′), Beclin1 siRNA (sense,\n5′-CGGGAAUACAGUGAAUUUATT-3′, antisense,\n5′-UAAAUUCACUGUAUUCCCGTT-3′) and scrambled negative control\nsiRNA (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense,\n5′-ACGUGACACGUUCGGAGAATT-3′). HIF-1α overexpression\nplasmid (pG/CMV/HIF-1α/IRES/EGFP) and negative control (NC) plasmid were\npurchased from Gemma Pharmaceutical Technology (China). For knockdown, HESCs\n(2x10 5  cells/well) were seeded in 6-well plates and grown to\n60–80% confluence, followed by transfected with the above\nplasmids or siRNA using lipofectamine2000 (Invitrogen Life Technologies, USA)\naccording to the manufacturer’s protocol. Transfection mixture was\nreplaced 6 hours later with DMEM/F-12 with 20% FBS. Then HESCs were\nincubated in normoxic or hypoxic conditions for another 24 hours and subjected\nto western blot analysis and GFP-LC3 adenoviral vector transfection.\nThe indicated cells were seeded on coverslips in a 24-well plates and\nallowed to reach 50% – 70% confluence at the time of\ntransfection. GFP-LC3 adenoviral vectors were purchased from Beyotime\nBiotechnology Co. Ltd. (Beyotime Biotechnology, China). Adenoviral infection was\nperformed according to the manufacturer’s instructions. HESCs were\nincubated in growth medium with the adenoviruses at a MOI of 50 for 24 h at 37\n°C. The cells in the control group and HIF-1α overexpression\ngroup were cultured under normoxic condition for another 24 h; the cells in the\nsiHIF-1α and hypoxia groups were cultured under hypoxic condition for\nanother 24h. After treatment, cells were washed with ice-cold PBS for three\ntimes and fixed with 4% paraformaldehyde for 15 min at room temperature.\nThen, the cells were washed three times with PBS and cover slips were mounted on\nthe slides. Autophagy was observed immediately observed using a laser scanning\nconfocal microscope (Olympus America Inc, Center Valley, PA). Autophagic level\nwas determined by evaluating the number of GFP –LC3 puncta (puncta/cell\nwere counted).\nThe indicated cells were seeded and grown on coverslips in a 6-well\nplates. The cells in the control group and HIF-1α overexpression group\nwere cultured under normoxic condition for 24 h; the cells in the\nsiHIF-1α and hypoxia groups were cultured under hypoxic condition for\n24h. After treatment, cells were washed with ice-cold PBS and fixed with\n4% paraformaldehyde for 15 min. Then cells were incubated with\n5% BSA (bovine serum albumin) for 1 hour to block non-specific binding\nat room temperature and incubated with a LC3B antibody (1:300; Abcam, USA) at\n4°C overnight. The next day, the cells were incubated with goat\nFITC-conjugated anti-rabbit IgG (1:100, Abcam, Cambridge, UK) for 2 hours at\ndark room and then incubated with 4′, 6-diamidino-2 phenylindole (DAPI)\nfor 15 min at room temperature. Finally, the cells were washed three times with\nPBS, and immediately observed using a laser scanning confocal microscope\n(Olympus America Inc, Center Valley, PA).\nMigration and invasion assays were performed using transwell 24-well\nplates with 8-μm diameter filters (Corning Costar, Tewksbury, MA, USA).\nFor invasion assay, microfilters were precoated with 40 μl of working\nmatrigel (1:3 diluted with FBS-free DMEM) (Becton, Dickinson and Company, USA)\nand were matained at 37°C for at least 5 h. The following procedures\nwere the same for migration and invasion assays. Approximately 2x10 5 \ncells in 200μl of serum-free medium containing 500 μM\nchloroquine or 10 mM 3-Methyladenine (3-MA) were loaded in the upper matrigel\ncoated chamber and 500μl of medium containing contain 20% fetal\nbovine serum was placed in the lower chamber. The cells cultured in normoxic\ncondition were used as the control groups. The cells cultured under hypoxic\ncondition with or without 500 μM chloroquine or 10 mM 3-MA were used as\nexperimental groups. To evaluate the migration potential, cells were allowed to\nmigrate towards medium over a period of 24h. For the invasion assay, after\nseeded, cells were allowed to invade for 48h. After the indicated treatment,\ncells were fixed in methanol for 20 min and stained with 0.1% crystal\nviolet for another 20 min. Then the cells on the upper surface of the filters\nwere wiped off with cotton swabs, and the filters were washed three times with\nPBS. The cells on the underside of the filters were observed and counted under\nan inverted microscope at x200 magnification. Duplicate wells per condition were\ntested in three independent experiments.\nStatistical analysis software Graphpad Prism (version 6.01; GraphPad\nSoftware Inc., CA, USA) was used to carry out the statistical analyses. The\nKruskal–Wallis test were used for statistical significance of\ndifferences in variables with non-normal distribution. The Student’s t\ntest and one-way analysis of variance followed by Tukey’s post hoc test\nwere used to measure comparisons between groups in normal distribution. All data\nsets were shown as mean ± standard deviations (SD) from at least three\nindependent experiments. Differences with P values of <0.05 were considered\nstatistically significant.\n\nTo determine the autophagy activity in endometriosis tissues and its\nrelationship with HIF-1α, immunohistochemical staining was performed to\ndetect the expression of HIF-1α and autophagy marker LC3. Representative\nstaining examples are shown in  Figure 1  and\nimmunostaining score are depicted in  Supplemental Table 1 . As shown in\n Figure. 1A , HIF-1α was\nexpressed in the nuclei of epithelial and stromal cells, while LC3 was localized\nwithin the cytoplasm of both cells. The expression levels of HIF-1α and\nLC3 in the ectopic endometrium were significantly greater than those in normal\nendometrium and eutopic endometrium from women with endometriosis.\nHIF-1α expression levels in ectopic endometrium significantly correlated\nwith the levels of LC3 ( Supplemental Tab.1 ). Moreover, western blot analysis revealed the\nsimilar trend for HIF-1α and LC3-II proteins expression ( Fig. 1B and 1C ). However, no significant difference\nwas observed between normal endometrium and eutopic endometrium from the\npatients with endometriosis ( Fig. 1B and\n1C ). Taken together, these results suggested that autophagy is\nupregulated in ectopic endometrium and HIF-1α may play a vital role in\nthis event.\nTo determine whether autophagy is activated by hypoxia stress, we\nperformed a series of investigations to evaluate it. At first, acridine orange\n(AO) and monodansylcadaverine (MDC) staining were used to detect acidic\nvesicular organelles (AVOs), which reflecting autophagosomes. As shown in  Figure. 2A and 2B , exposure of HESCs to\nhypoxic conditions resulted in accumulation of autophagic vacuoles in a time\ndependent manner. Electron microscopy remains to be one of the most accurate\nmethods to detect autophagy and quantify autophagic accumulation ( Swanlund,  et al . 2010 ).\nThe ultrastructural results showed that numerous cytoplasmic phagolysosomes were\npresent after hypoxia treatment ( Fig. 2C ,\nright panel). However, few phagolysosomes was observed in the control group\n( Fig. 2C , left panel). Furthermore,\nwestern blot analysis displayed that the protein expression levels of\nHIF-1α, Beclin 1 and LC3-II and were increased in a time dependent\nmanner after hypoxia treatment ( Figure. 2D and\n2E ). As the elevation of LC3 protein level could be resulted from\nincreased autophagosomes formation or decreased autophagosome degradation ( Mizushima and Yoshimori 2007 ), autophagy\nflux was evaluated in the presence and absence of lysosomal degradation\ninhibitor chloroquine. As shown in  Figure. 2F and\n2G , a blockade of the autophagosome-lysosome fusion by using\nchloroquine significantly increased the accumulation of endogenous LC3-II\nprotein, and hypoxia apparently augmented this effect, indicating that\nhypoxia-induced elevation of LC3-II is due to autophagy activation, rather than\nblockage of lysosomal degradation. Altogether, these results demonstrate that\nhypoxia is able to induce autophagy in HESCs.\nTo examine whether HIF-1α has an effect on autophagy, HESCs were\ntransfected with a HIF-1α expression plasmid under normoxia conditions.\nWestern blot results showed that HIF-1α overexpression caused increased\nexpression levels of HIF-1α and Beclin1 and LC3, ( Fig. 3A and 3B ). In addition, we found that compared\nwith the negative control group, the HESCs transfected with HIF-1α\noverexpression plasmid showed typically dense accumulation of GFP-LC3 puncta in\nthe perinuclear region under normoxia condition ( Fig. 3C ).\nTo further corroborate the role of HIF-1α in hypoxia induced\nautophagy, HESCs were transfected with specific siRNA targeting HIF-1α\nunder hypoxic conditions. Compared with HESCs transfected with control siRNA,\ndecreased expression of HIF-1α and Beclin1 and LC3 were observed in\nHESCs transfected with HIF-1α siRNA under hypoxia condition ( Fig. 4A and 4B ). Moreover, GFP-LC3 puncta\naccumulation was significant decreased in HESCs transfected with HIF-1α\nsiRNA compared to that of negative control group under hypoxia condition ( Fig. 4C ). These, together with the above\nresults, suggest that autophagy upregulation under hypoxic condition was\ndependent on the status of HIF-1α.\nTo explore the effect of hypoxia on cellular motility, transwell\nmigration and invasion assays were conducted. Treatment with hypoxia\nsignificantly enhanced the migration and invasion ability of HESCs, when\ncompared with the untreated control cells ( Figure.\n5A and 5B ). On the contrary, the hypoxia triggered invasive ability\ncould be attenuated by HIF-1α siRNA. The number of cells that crossed\nthe lower chamber decreased upon HIF-1α siRNA treatment ( Figure. 5A and 5B ). These results demonstrated hypoxia\ncan augment the ability migration and invasion of HESCs in vitro, and this event\nwas dependent on HIF-1α.\nTo examine whether autophagy has an effect on migration and invasion of\nHESCs under hypoxia condition, transwell migration and invasion assays were\nconducted. Here, two types of autophagy inhibitors, 3-MA and Chloroquine, were\nused to inhibit autophagy. 3-MA inhibits autophagy at early stage by blocking\nautophagosome formation via the inhibition of type III Phosphatidylinositol\n3-kinases, involved in the initiation of autophagosome formation ( Wu,  et al . 2010 ).\nChloroquine inhibits autophagy at late stage by inhibiting lysosomal proteases\nand preventing autophagosome-lysosome fusion ( Geng,  et al . 2010 ). Our data revealed that HESCs\ntreated with hypoxia displayed significantly increased migration and invasion\nabilities comparison to those untreated, which was nevertheless markedly\nreversed by the addition of 3-MA or chloroquine ( Fig 6A and 6B ). The pro-invasion role of autophagy in HESCs was\nfurther validated by genetically impairing the autophagy pathway using siRNA to\nBeclin1. As shown in  Fig 7A and 7B , the\nBeclin1 siRNA suppressed expression of the Beclin1 protein. Similar to autophagy\ninhibition by 3-MA and Chloroquine, the genetic inhibition of autophagy by\nBeclin1 siRNA also attenuated HESCs migration and invasion abilities under\nhypoxia condition ( Fig 7C and 7D ).\nCollectively, these results demonstrated that autophagy facilitates the hypoxia\ntriggered migration and invasion of HESCs in vitro.\n\nAlthough endometriosis is recognized as a benign disease, its behavior is\ncharacterized by certain biological features that also are seen in malignancy ( Bassi,  et al . 2009b ). The\nmigration, invasion and angiogenesis of viable endometrial tissues outside the\nuterine cavity is a crucial step for the progression of endometriosis ( Moggio,  et al . 2012 ).\nSampson’s retrograde menstruation hypothesis is the most widely accepted\ntheory. However, this theory does not fully explain why most women suffer from\nretrograde menstruation but only 10 percent of them finally develop endometriosis.\nResearchers have found that other factors like local hypoxia microenvironment may\ncontributes to the development of endometriosis. When shed endometrial tissue\nfragments retrogrades to the pelvic cavity, the first stress faced is the local\naltered hypoxic microenvironment. Accumulating evidence reported that HIF-1α\nwas upregulated in ectopic endometrium and possibly involved in the invasion process\nof HESCs ( Filippi,  et al .\n2016 ,  Lu,  et al .\n2014 ,  Zhan,  et al .\n2016 ). Therefore, a better understanding of the molecular mechanisms of\nHIF-1α regulated endometrial cells migration and invasion is helpful for\ntreatment of this disease.\nAutophagy is important in keeping cellular homeostasis, and its\ndysregulation is closely linked to numerous human pathophysiological processes,\nincluding cancer, myopathy, neurodegeneration disease ( Levine and Kroemer 2008 ). Stressful conditions, like\nhypoxia, can trigger the activation of autophagy ( Wu, et al. 2015b ). Recently, regulation of autophagy by HIF-1α\nhas been reported. Hypoxia leads to HIF-1α stabilization, which subsequently\nactivate the downstream gene BNIP3 that competes with Bcl-2 and Bcl-XL for\ninteraction with Beclin to trigger autophagy ( Bellot,\n et al . 2009 ). Autophagy is generally considered as a\nmechanism of cellular protection. In addition, autophagy has also been shown to be\ninvolved in modulating cancer cell motility and invasion ( Mowers, et al. 2016 ). For example, autophagy could\nfacilitates TLR3 and TLR4-triggered invasion of lung cancer cells ( Zhan,  et al . 2014 ) and contributes to\nsalivary adenoid cystic carcinoma cells invasion under hypoxia environment ( Wu, et al. 2015a ). In recent years increasing\nresearch efforts have investigated the possible regulatory mechanism of autophagy in\nhypoxia triggered cell migration. Autophagy induction during intermittent hypoxia\ncould facilitates the invasiveness of pancreatic cancer cell through activation of\nepithelial–mesenchymal transition ( Zhu, et\nal. 2014 ). Moreover, autophagy upregulated by HIF-1α\noverexpression could supports extra villous trophoblasts invasion by supplementation\nof cellular adenosine triphosphate (ATP) ( Yamanaka-Tatematsu, et al. 2013 ). Interestingly, autophagy activation in\nMDA-MB-231 cells resulted in attenuated invasiveness through HIF-1α\ndegradation by autophagic pathway ( Indelicato, et\nal. 2010 ). These apparently disparate conclusions in the field suggests\nthat autophagy may play more complicated roles in tumor invasion, which will be an\ninteresting area for future study.\nPrevious study indicated that autophagy is upregulated in ovarian\nendometriosis and possibly contributes to survival of endometriotic cells and to\nlesion maintenance in ectopic sites ( Allavena,\n et al . 2015 ). However, there have been controversial\nresults regarding the expression level of autophagy in endometriosis. JongYeob Choi\n et al.  reported that autophagy level was decreased in ectopic\nendometrium together with activation of p70S6K phosphorylation (signature of mTOR\nactivation) ( Choi,  et al .\n2014 ). These contrasting results may be explained by the fact that a\ncomplex signaling networks involved in the regulation of autophagy. The study have\nfound that akt-mammalian target of rapamycin (mTOR) signaling was activated in\novarian endometriosis ( Leconte,  et\nal . 2011 ,  Yagyu,  et\nal . 2006 ). As a negative regulator of autophagy, mTOR\nactivation may resulted in autophagy inhibition in endometriosis. In fact, besides\nthe canonical PI3K-AKT-mTOR signaling ( Wu,\n et al . 2009 ), autophagy can be also induced through\nnon-canonical signaling like ammonia pathway ( Polletta,  et al . 2015 ) and hypoxia-inducible factor\n(HIF)-dependent pathways ( Bellot,  et\nal . 2009 ). Based on the abovementioned correlations between\nHIF-1α, autophagy and endometriosis, we hypothesized that autophagy\nupregulation in endometriosis may due to local hypoxia and autophagy play a role in\nHIF-1α induced HESCs migration and invasion. To elucidate these questions,\nwe designed and conducted a series of investigations.\nIn our present study, our results from immunohistochemical staining and\nwestern blots showed that both HIF-1α and autophagy related protein LC3\nexpression level were elevated in ectopic endometrium compared with normal and\neutopic endometrium of endometriosis patients, which indicated that autophagy was\nupregulated and HIF-1α may correlated with this event. After hypoxia\ntreatment for different time points, the protein expression level of HIF-1α,\nBeclin1 and LC3 were upregulated. Meanwhile, increased autophagic vacuoles and\nautophagosome accumulation were observed under hypoxic conditions. To elucidate the\nregulatory role of HIF-1α on autophagy, we transfected HESCs with\nHIF-1α overexpression plasmid or HIF-1α siRNA. The results showed\nthat overexpression of HIF-1α resulted in upregulated autophagy under\nnormoxic condition and HIF-1α siRNA abrogated hypoxia induced autophagy.\nFurthermore, in order to investigate the effect of HIF-1α and autophagy on\ncell migration and invasion, transwell assays were performed. We observed that\nhypoxia was able to enhance migration and invasion of HESCs, while transfected with\nHIF-1α siRNA reversed this effect, suggesting that hypoxia promotes HESCs\ncell migration and invasion through HIF-1α. Furthermore, the application of\nautophagy inhibitors and specific Beclin1 siRNA significantly reversed the\nhypoxia-stimulated migration and invasion of HESCs.\nThere are three limitations in the present study: (a) the sample size is\nrelatively small; (b) the expression of autophagy has not been detected in different\nphases of the menstrual cycle; and (c) the exact molecular mechanisms underlying\nautophagy in HESCs invasion under hypoxia environment remains to be established.\nThus, future research is needed to gain deeper insight into these questions.\nIn conclusion, we demonstrated in this study that HIF-1α is able to\nenhance the migration and invasion of HESCs through upregulating autophagy. It is\nworth noting that autophagy inhibitor Chloroquine has been applied to a series of\nclinical trials targeting malignant diseases like melanoma ( Rangwala,  et al . 2014 ) and lung cancer\n( Goldberg,  et al . 2012 ).\nMoreover, a study using murine endometriosis model revealed that inhibition of\nautophagy by hydroxychloroquine effectively promotes apoptosis of human\nendometriotic cells and decreases the number of endometriotic lesions ( Ruiz,  et al . 2016 ). Taken\ntogether, these findings reinforce the view that inhibition of autophagy might act\nas a therapeutic tool in the prevention and treatment of endometriosis.\n\nImmunocytochemistry microscopy staining of (A) E-cadherin and (B)\nVimentin. Blue signal represent nuclear DNA staining by DAPI.\nRepresentative immunofluorescence image of LC3 in HESCs transfected\nwith negative control (NC) plasmid or HIF-1α expression plasmid\nunder normoxic condition for 24 hours. Photographs were taken at\nmagnifications of 200× (left panels) and 800× (right panels)\nrespectively. Blue signal represent nuclear DNA staining by DAPI.\nImmunostaining score for HIF-1α and autophagy marker LC3.\nAll data are expressed as mean±SD. Statistical significance (One way\nANOVA analysis).","source_license":"CC0","license_restricted":false}