{"paper_id":"63aeacfc-a50e-4baf-8dd4-e6f6120ad208","body_text":"Vol:.(1234567890)\nReproductive Sciences (2023) 30:2932–2944\nhttps://doi.org/10.1007/s43032-023-01253-5\nENDOMETRIOSIS: ORIGINAL ARTICLE\nMicroRNA‑210‑3p Regulates Endometriotic Lesion Development \nby Targeting IGFBP3 in Baboons and Women with Endometriosis\nKentaro Kai1,2  · Niraj R. Joshi1  · Gregory W. Burns1 · Samantha M. Hrbek1 · Erin L. Vegter1 · \nMaria Ariadna Ochoa‑Bernal1 · Yong Song1 · Genna E. Moldovan1 · Lorenzo F. Sempere3 · Eduardo H. Miyadahira4 · \nPaulo C. Serafini5 · Asgerally T. Fazleabas1 \nReceived: 8 September 2022 / Accepted: 24 April 2023 / Published online: 15 May 2023 \n© The Author(s) 2024\nAbstract\nMicroRNAs (miRs) play an important role in the pathophysiology of endometriosis; however, the role of miR-210 in endo-\nmetriosis remains unclear. This study explores the role of miR-210 and its targets, IGFBP3 and COL8A1, in ectopic lesion \ngrowth and development. Matched eutopic (EuE) and ectopic (EcE) endometrial samples were obtained for analysis from \nbaboons and women with endometriosis. Immortalized human ectopic endometriotic epithelial cells (12Z cells) were utilized \nfor functional assays. Endometriosis was experimentally induced in female baboons (n = 5). Human matched endometrial \nand endometriotic tissues were obtained from women (n = 9, 18–45 years old) with regular menstrual cycles. Quantitative \nreverse transcript polymerase chain reaction (RT-qPCR) analysis was performed for in vivo characterization of miR-210, \nIGFBP3, and COL8A1. In situ hybridization and immunohistochemical analysis were performed for cell-specific localization. \nImmortalized endometriotic epithelial cell lines (12Z) were utilized for in vitro functional assays. MiR-210 expression was \ndecreased in EcE, while IGFBP3 and COL8A1 expression was increased in EcE. MiR-210 was expressed in the glandular \nepithelium of EuE but attenuated in those of EcE. IGFBP3 and COL8A1 were expressed in the glandular epithelium of EuE \nand were increased compared to EcE. MiR-210 overexpression in 12Z cells suppressed IGFBP3 expression and attenuated \ncell proliferation and migration. MiR-210 repression and subsequent unopposed IGFBP3 expression may contribute to \nendometriotic lesion development by increasing cell proliferation and migration.\nKeywords Endometriosis · MicroRNA-210 · IGFBP3 · COL8A1 · Cell proliferation · Glandular epithelium\nIntroduction\nEndometriosis is an estrogen-dependent chronic inflamma-\ntory and fibrotic disease, characterized by the presence of \nextra-uterine implantation of endometrial-like tissue mainly \nin the peritoneal cavity [ 1, 2]. The disease affects approxi -\nmately 190 million women worldwide [3]. Endometriosis is \nassociated with approximately 75% of chronic pelvic pain \nin adolescents [4 ], up to 50% of infertility in reproductive-\naged women [3], and approximately 10% of epithelial ovar-\nian cancers in perimenopausal women [5]. Nevertheless, its \npathophysiology remains unclear due to cellular heteroge-\nneity in human samples and limited access to physiologi-\ncally relevant in vivo models [6 ]. Endometriosis is divided \ninto three subtypes: peritoneal endometriosis (PE), ovarian \nendometriosis (OE), and deep infiltrating endometriosis \n(DIE). Only PE has an established in vivo model that sup-\nports Sampson’s theory, in which endometriosis is caused by \nthe ectopic implantation of retrograde menstrual blood [7 ]. \nThe molecular profiles of endometriosis differ depending on \nthe localization of lesions [8 ], cell subtypes [9 ], menstrual \nfluctuation [6], and individual disease duration [10].\n * Asgerally T. Fazleabas \n fazleaba@msu.edu\n1 Department of Obstetrics and Gynecology, and Reproductive \nBiology, College of Human Medicine, Michigan State \nUniversity, Grand Rapids, MI 49503, USA\n2 Department of Obstetrics and Gynecology, Oita University \nFaculty of Medicine, Yufu, Japan\n3 Department of Radiology, Precision Health Program, \nMichigan State University, East Lansing, MI, USA\n4 Clínica Vida Bem Vinda, São Paulo, Brazil\n5 Department of Gynecology, Faculdade de Medicina da \nUniversidade de São Paulo, São Paulo, Brazil\n\n2933Reproductive Sciences (2023) 30:2932–2944 \nMicroRNAs (miRs) play an important role in the patho-\nphysiology of endometriosis [11]. Using the baboon model \nof endometriosis, we have previously reported that miR-451 \nand miR-29c expressions are altered in baboons and women \nwith endometriosis [ 12, 13]. MicroRNA-210 (miR-210) is \none of the miRs differently expressed between normal endo-\nmetrial stromal cells (NESCs) from women with no disease \nand endometriotic cyst stromal cells (ECSCs) [14]. Dai et al. \nreported that aberrantly increased expression of miR-210-3p \nduring the proliferative phase may promote endometriosis \nby targeting BRCA1-associated RING domain 1 (BARD1) \n[15]. While miR-210 has attracted particular attention for its \npro- and anti-tumoral effects in different cancer types [16, \n17], no study has investigated the anti-proliferative effect of \nmiR-210 in endometriosis. Additionally, the profile and role \nof miR-210 and its downstream targets during the secretory \nphase have yet to be determined, even though gene altera-\ntions related to progesterone resistance that reduces endo-\nmetrial receptivity and causes implantation failure in women \nwith endometriosis have been mostly observed during the \nsecretory phase [18]. Further, the expression of miR-210 in \nwomen is physiologically upregulated in the secretory phase \ncompared with the proliferative phase [19].\nPreviously, Okamoto et al. identified 29 potential down-\nstream targets of miR-210-3p using gene expression microar-\nray analysis and Ingenuity Pathway Analysis of NESCs with \nor without miR-210 overexpression [20]. Aoyagi et al. reported \na total of 186 potential downstream targets of miR-30a-5p and \nmiR-210-3p that were differently expressed between NESCs \nand ECSCs after in vitro decidualization [21]. miR-210, insu-\nlin-like growth factor (IGF)-binding protein 3 (IGFBP3), and \ncollagen type VIII alpha 1 chain (COL8A1) were shared by \nthe target list of both Okamoto et al. and Aoyagi et al. [20, \n21]. This manuscript explores the mechanisms involved in the \npathogenesis of endometriosis via these molecules.\nIGFBP3 is a member of the IGFBP family that has two \nglycosylated forms and plays a pivotal role in regulating the \nphysiological functions of IGFs (IGF-dependent actions) [22]. \nHowever, recent studies showed that IGFBP3 modulated angi-\nogenesis, and inhibited migration, survival, and proliferation \nindependent of IGF [23]. The pro-tumor effects of IGFBP3 \nwere reported in esophageal carcinoma, breast cancer, and \noral squamous cell carcinoma [24]. Further, IGFBP3 was vali-\ndated as a direct target gene of miR-210 [25, 26].\nIn addition, COL8A1 is a type VIII short-chain non-fibril-\nlar collagen present in microvascular endothelial cells [27]. \nType VIII collagen is secreted by human mast cells, and \nits functions include angiogenesis, tissue remodelling, and \nfibrosis in chronic inflammatory, immunologic, and fibrotic \nstates [28]. Vascular remodelling often occurs before devel-\noping fibrosis in other fibrotic diseases, such as idiopathic \npulmonary fibrosis and liver fibrosis [29]. In endometrio-\nsis, repeated tissue injury and repair (ReTIAR) promotes \nfibroproliferation and deposition of the extracellular matrix, \nresulting in pathological fibrosis [30].\nThis study tests the hypothesis that altered miR-210-3p \nexpression results in the development of endometriotic \nlesions by regulating IGFBP3 or COL8A1. We profiled the \nexpression of our target genes in a baboon (Papio anubis) \nendometriosis model, in which the onset of endometriosis \ncan be precisely determined [31], and in human samples. We \nfurther validated the functional consequences of miR-210 \noverexpression in vitro using immortalized human ectopic \nendometriotic epithelial cells (12Z cells).\nMaterials and Methods\nBaboon Endometriosis Model\nAll experimental procedures were approved by the Institu-\ntional Animal Care and Use Committee of the University \nof Illinois, Chicago, and Michigan State University. Endo-\nmetriosis was experimentally induced in female baboons \n(Papio anubis) as previously described [32]. Briefly, in the \ncycle before the induction of endometriosis, the animals were \nexamined for spontaneously occurring disease and, if found \n(n = 4), eutopic endometrium (EuE) and ectopic endome-\ntrium (EcE) were collected. If not (n = 8), the control endo-\nmetrium (Ctrl) was obtained via laparotomy on day 10 post-\novulation (mid-secretory phase). We determined the phase \nof the cycle by monitoring the pre-ovulatory estradiol surge. \nPeritoneal endometriosis was then induced in the same 8 ani-\nmals by intraperitoneal inoculation of autologous menstrual \ntissue over two consecutive cycles. Following laparoscopic \nconfirmation of endometriosis at the second inoculation, the \nanimals were sampled at 3-month intervals post-inoculation \nand euthanized under intravenous anesthesia after 15 months. \nAt necropsy, EuE and EcE were collected. Samples were \neither snap-frozen in liquid nitrogen for RNA/protein extrac-\ntion or fixed in 10% formalin for morphological and immu-\nnohistochemical analysis. Following microscopic evaluation \nof endometriotic lesions, we selected 5 out of 8 matched EuE \nand EcE from baboon with induced endometriosis for further \nexperiments to keep the same duration of disease (EuE and \nEcE from baboons with spontaneous endometriosis were not \nused because the duration of disease varied).\nHuman Endometrial and Endometriotic Samples\nWe obtained Institutional Review Board (IRB) approval \nfrom the School of Medicine of the University of São Paulo. \nThe patients who have a regular menstrual cycle and have \nprimarily been treated for infertility were recruited. Other \ninclusion criteria were: (i) independently of symptoms, all \n\n2934 Reproductive Sciences (2023) 30:2932–2944\nthe patients were submitted to transvaginal ultrasound with \nbowel preparation (TVUS-BP) evaluation; (we do not have \nthe data of each patient’s symptoms) (ii) body mass index < \n30 kg/m2; and (iii) absence of other significant systemic dis-\neases (e.g., hypertension and diabetes). Exclusion criteria \nwere: (i) infection with HIV, hepatitis B or C, (ii) presence \nof abnormal vaginal bleeding, and (iii) consumption of ille-\ngal drugs or hormones. Informed consent was obtained from \npatients. Patients and their phase of cycle were based on the \nresults of TVUS-BP (n  = 26). Fifteen women suspected of \nhaving DIE underwent laparoscopic resection of the ectopic \ntissue, and 11 patients not suspected of having endometri-\notic lesions (PE, OE, and DIE) served as the control group. \nNo statistical difference between the groups was observed \nin age, body mass index, basal follicle stimulating hormone, \nbasal oestradiol, previous in vitro fertilization failure, and \nrepeated abortion (Supplementary Table 1). Endometrial \nsamples were collected from both groups. In the endome-\ntriosis, group samples were obtained before (EuE pre-op) \nand after the surgery (EuE post-op). Further, ectopic tis-\nsue (EcE) was collected during the surgical intervention. \nEndometrial samples were obtained with a Pipelle curette \n(Pipelle de Cornier, Laboratoire C. C. D., Paris, France) and \nstored in RNA later at −80 °C. Among the DIE group, we \nincluded 9 cases in which matched EuE (pre-op) and EcE \ncould be collected during the mid-secretory phase.\nRNA Isolation and Quantitative Reverse \nTranscription Polymerase Chain Reaction (RT‑qPCR)\nWe utilized all 9 matched EuE and EcE from women with \nendometriosis and 5 matched EuE and EcE from baboons \nwith induced endometriosis. Total RNA was isolated using \nthe Trizol reagent (Invitrogen, Waltham, MA, USA), and \nRNA concentration was checked using the NanoDrop 2000 \n(Thermo Fisher Scientific, Waltham, MA, USA). We per -\nformed  TaqManTM assay for miR-210 expression analysis \nand  SYBRTM Green assay for IGFBP1, COL8A1, and HIF1A \nusing the ViiA7 qPCR System (Applied Biosystems). For the \nmicroRNA analysis, 100 ng of total RNA was reverse tran-\nscribed to cDNA using the  TaqManTM MicroRNA Reverse \nTranscription Kit (4366596, Applied Biosystems, Foster City, \nCA, USA). RT-qPCR was performed to assess the expression \nof miR-210 using the  TaqManTM Universal Master Mix II \nwith UNG (4440038, Applied Biosystems). The  TaqManTM \nMicroRNA Assays (4427975, Applied Biosystems) with \nmiR-210-3p (000512, Applied Biosystems) and U6 (001973, \nApplied Biosystems) snRNA were used for microRNA-spe-\ncific RT-qPCR. For mRNA analysis, 1000 ng of total RNA was \nreverse transcribed to cDNA using the High-Capacity cDNA \nReverse Transcription Kit (4368814, Applied Biosystems). \nRT-qPCR was performed to assess the expression of the target \ngene expression using the  PowerUpTM  SYBRTM Green Master \nMix (A25742, Applied Biosystems). The primer sequences \nfor target genes analyzed using RT-qPCR are listed in Supple-\nmentary Table 2. We utilized primers not only for IGFBP3 and \nCOL8A1 but also for hypoxia-inducible factor 1 subunit alpha \n(HIF1A), which was a well-known master regulator of miR-\n210, for the validation of miR-210 expression. The expression \ndata were normalized to U6 in the microRNA-specific RT-\nqPCR and by RPL17 or 18S in the quantitative RT-qPCR. All \nquantitative reverse transcription-polymerase chain reactions \nwere run for 40 cycles, and the fold change was calculated \nusing the  2−ΔΔCt  method [33].\nMultiplex In Situ Detection and Image Analysis\nWe utilized 5 matched EuE and EcE from baboons with \ninduced endometriosis. Multiplex in situ hybridization assay \nfor the co-detection of miR-210 and small nuclear U6 was \nperformed as previously described [34]. Briefly, 5 μm forma-\nlin-fixed paraffin-embedded baboon tissue was processed for \nthe in situ hybridization assay on a Leica Bond Rx automated \nstainer. We custom-designed a locked nucleic acid-modified \nprobe with 5′ and 3′ terminal FAM moieties for the detection \nof miR-210 and a DNA probe with 5′ and 3′ terminal biotin \nmoieties for the detection of U6 (Supplementary Table 3). The \nprobes were purchased from Integrated DNA Technologies \n(IDT; Coralville, IA, USA) or Eurogentec (Seraing, Belgium). \nProbes were sequentially detected with tyramide signal ampli-\nfication (TSA) reaction. First, miR-210 probe was detected \nwith primary anti-FITC rabbit antibody (DAKO, P5100) and \nsecondary goat anti-rabbit antibody (Biorad, 170-6515) conju-\ngated to horseradish peroxidase (HRP), followed by TSA reac-\ntion with FITC-tyramide (Thermo Scientific, 46410). Then, \nU6 probe was detected with streptavidin conjugated to HRP \n(Thermo Scientific, 21140), followed by TSA with rhodamine-\ntyramide (Thermo Scientific, 46406). Tissue sections were \ncounterstained with 4,6-diamidino-2-phenylindole (DAPI), \nand whole-slide images were acquired using the Aperio Versa \nimaging system with 20× objective (OBJ HC PL APO 20×, \nLeica No: 23OBJ020PAPDRY) with customized narrow-width \nband excitation and emission filter cubes: standard set (Chroma \nTechnology, 49000) for DAPI, standard set (Chroma Technol-\nogy, 49020) for fluorescein, and custom set (Chroma Technol-\nogy, ET546/10×, T555lpxr, ET570/20×) for rhodamine. The \nnuclear DAPI signal was used for automated cell enumera-\ntion and segmentation using the Aperio Cellular IF Algorithm \n(Leica Biosystems, No: 23CIFWL). The cell classification was \nbased on the levels of miR-210 expression, which depended on \nthe signal threshold (low vs. high).\nHistology and Immunohistochemistry\nWe utilized 3 matched EuE and EcE from women with \nendometriosis and 3 out of 5 matched EuE and EcE \n\n2935Reproductive Sciences (2023) 30:2932–2944 \nfrom baboons with induced endometriosis. Tissues were \nfixed in 10% buffered formalin or 4% paraformaldehyde, \nembedded in paraffin, and sectioned at a thickness of 6 \nμm. The sections were then deparaffinized and rehydrated \nin a graded alcohol series. After antigen retrieval and \nhydrogen peroxide treatment (antigen unmasking solu-\ntion, H-3300, Vector Laboratories, Burlingame, CA, \nUSA), sections were blocked and incubated with anti-\nIGFBP3 (goat polyclonal, 1:50 dilution, AF675, R&D \nSystems, Minneapolis, MN, USA) and anti-COL8A1 \n(rabbit polyclonal, 1:100 dilution, HPA053107, Sigma-\nAldrich, St. Louis, MO, USA) overnight at 4 °C. On the \nfollowing day, the sections were incubated with bioti-\nnylated secondary antibodies, followed by incubation with \nhorseradish peroxidase-conjugated streptavidin. Immuno-\nreactivity was detected using the DAB substrate kit (Vec-\ntor Laboratories). Normal baboon placenta (IGFBP3) and \nlung (COL8A1) were used as positive controls. Normal \ngoat IgG (IGFBP3) and rabbit IgG (COL8A1) were used \nas negative controls. The digital H-score method using \nImageJ software 1.52a (National Institutes of Health, \nBethesda, MD, USA) was performed by a single-blinded \nobserver (K.K.) to semi-quantitate the expression levels \nof these proteins as previously described [35]. Images \nwere taken at ×20 magnification using an upright micro-\nscope (Ni-U, Nikon Instruments, Melville, NY).\nCell Culture\nImmortalized human ectopic endometriotic epithelial \n(12Z) cells [36] were cultured using Dulbecco’s modi-\nfied Eagle’s medium (DMEM)/F-12 (11330-032, Gibco, \nWaltham, MA, USA), which was supplemented with \n10% heat-inactivated fetal bovine serum (16000-044, \nGibco, Dublin, Ireland), 100 U/mL of penicillin (15140-\n122, Gibco, Dublin, Ireland), 100 μg/mL of streptomy -\ncin (15140-122, Gibco, Dublin, Ireland), and 0.1 mM of \nsodium pyruvate (11360-070, Gibco, Dublin, Ireland) at \n37 °C under 5%  CO2 and 95% air [36, 37]. According to \noriginal protocols, 12Z cells were established from perito-\nneal endometriosis biopsies and characterized as cytoker -\natin-positive, E-cadherin-negative, invasive cells in vitro \nby immunofluorescence and Matrigel assay [36]. Follow -\ning the optimization of parameters, the Lipofectamine \nRNAiMAX (13778-150, Invitrogen) was used to transfect \n12Z cells with 5 pmol of hsa-miR-210 mimic (4464066, \nLife Technologies, Foster City, CA, USA) or with 5 pmol \nof non-targeting negative controls (464058, Life Tech-\nnologies). RNA and protein were isolated after 24 and \n48 h. To check the expression of miR-210, IGFBP3, and \nCOL8A1 transcripts, RT-qPCR was performed. IGFBP3 \nand COL8A1 protein levels in the same cells were ana -\nlyzed using western blotting.\nWestern Blotting\n12Z cells were rinsed with ice-cold phosphate buffer saline \non ice and were lysed with Pierce® RIPA lysis buffer \n(89901, Thermo Fischer Scientific) supplemented with \n HaltTM protease inhibitors (78430, Thermo Fischer Scien -\ntific) and  HaltTM phosphatase inhibitors (78420, Thermo \nFischer Scientific). Protein concentration was measured \nusing the  PierceTM BCA Protein Assay Kit (23227, Thermo \nFischer Scientific). Equal amounts of protein extracts (10 \nμg for IGFBP3 and 30 μg for COL8A1) were separated on \n4–20% Tris-Glycine gels (XP04205BOX, Invitrogen) and \nwere transferred onto polyvinylidene fluoride membranes \n(1620177, Bio-Rad Laboratories, Hercules, CA, USA). The \nmembranes were incubated for 1 h at room temperature \nin 5% bovine serum albumin tris-buffered saline with a \n0.1% Tween 20 detergent buffer. The membranes were then \nincubated at 4 °C in blocking buffer overnight with pri -\nmary antibodies against IGFBP3 (1:1000 dilution, #25864, \nCell Signalling, Danvers, MA, USA), COL8A1 (1:2000 \ndilution, ab236653, Abcam, Cambridge, UK), or β-actin \n(1:10,000 dilution, #4967, Cell Signalling). The next day, \nthe membranes were incubated with their respective sec-\nondary antibodies, which were labeled with horseradish \nperoxidase (1:10,000 dilution for IGFBP3 and 1:50,000 \ndilution for COL8A1, #7074, Cell Signalling), for 1 h at \nroom temperature. Immunocomplexes were visualized \nusing enhanced chemiluminescence (RPN2232; GE Life \nSciences, Marlborough, MA, USA). Densitometry of pro-\ntein bands was performed using ImageJ software 1.52a \n(National Institutes of Health, Bethesda, MD, USA). Pro-\ntein levels were normalized to that of β-actin, which was \nthe internal control.\nCell Proliferation Assay\nThe 12Z cells were seeded into 96-well plates at a density of \n10,000 cells per well in six replicates. At 10 h after transfec-\ntion, the transfection media was changed and was set at 0 h. \nAt 1-, 2-, 3-, 4-, and 5-day post-transfection with the miR-\n210 mimic or with the non-targeting negative control, 20 μl \nof MTS (G3580, Promega, Madison, WI, USA) reagent was \nadded to each well, and the mixture was incubated for 1 h at \n37 °C. After incubation, the optical density (OD) was meas-\nured at 490 nm. The rate of proliferation was calculated as \nthe percentage of the mean OD of the control group.\nScratch Wound Healing Assay\nThe 12Z cells were seeded at 80–90% confluency per well in \n6-well plates 12 h before transfection with miR-210 mimics \nor a negative control. Approximately 24 h after the indicated \ntransfections, a cell scraper apparatus (08-100-241, Fisher \n\n2936 Reproductive Sciences (2023) 30:2932–2944\nScientific, Hampton, NH) was used to generate a wound \nscratch approximately 1 mm wide in the center of each well. \nWound repair was manually measured by a single-blinded \nobserver (K.K.) by calculating the repaired area in square \nmicrometers between the cell edges at 0 h and 24 h using \nImage J 1.52a.\nStatistical Analyses\nData are shown as the mean ± standard deviation. We used \nStudent’s t-test to compare the means of the two groups \n(Figs.  1, 3 and 7 C) and two-way analysis of variance \n(ANOVA) with Turkey’s post hoc test to determine how \na response was affected by two factors: miR-210 overex-\npression and different time points (Figs.  4, 5, 6 and 7 A). \nStatistical significance was set at *P  < 0.05 or **P < 0.01. \nAll tests were two-sided. GraphPad Prism 9.3.1 (GraphPad \nSoftware, San Diego, CA, USA) was used for data analysis.\nResults\nDownregulation of MiR‑210 in Ectopic Endometrium \nof Both Women and Baboons with Endometriosis\nA study workflow diagram is shown in Supplementary Fig-\nure 1. To explore the characterization of miR-210 in vivo, \nwe first performed RT-qPCR on matched mid-secretory EuE \nand EcE from women with DIE (n  = 9) and baboons with \ninduced disease (n  = 5). The RT-qPCR analysis revealed \nthat the expression of miR-210 was significantly decreased \nin EcE compared to EuE in both women (P < 0.05; Fig.  1A) \nand baboons (P < 0.05; Fig.  1B) with endometriosis. Like-\nwise, the expression profile of HIF1A in baboons with \ninduced endometriosis was consistent with those of miR-\n210 (Supplementary Figure 2).\nTo identify the cell-specific localization of miR-210 \nin vivo, we next performed in situ hybridization on matched \nmid-secretory EuE and EcE in the baboon endometriosis \nmodels. In situ hybridization analysis revealed that miR-210 \nwas predominantly expressed in the glandular epithelium \nof EuE and was attenuated in EcE (Fig.  2A). This finding \nis supported in computer-assisted image analysis and figure \ncomposition in which cell segmentation and cell classifica-\ntion were performed based on expression levels of DAPI, \nmiR-210, and U6 (Fig.  2B).\nUpregulation of IGFBP3 and COL8A1 in Ectopic \nEndometrium of Both Women and Baboons \nwith Endometriosis\nTo explore the characterization of IGFBP3 and COL8A1  \nin vivo, we performed RT-qPCR on matched mid-secretory \nEuE and EcE from baboons and women with endometrio-\nsis. In contrast to the attenuation of miR-210 expression \nin EcE, RT-qPCR analysis revealed that the expression of \nIGFBP3 was significantly increased in EcE compared to \nEuE in women (P < 0.05; Fig.  3A) and baboons (P < 0.01; \nFig.  3B) with endometriosis. The expression of COL8A1 was \nalso significantly increased in EcE compared to EuE in both \ngroups (P < 0.01; Fig.  3C, D).\nTo identify the cell-specific localization of IGFBP3 and \nCOL8A1 in vivo, we performed immunohistochemistry on \nmatched mid-secretory EuE and EcE of women with endo-\nmetriosis. As shown in Fig.  4A (upper panels), immuno-\nhistochemistry analysis revealed that IGFBP3 in EuE was \npredominantly expressed in the glandular epithelium rather \nthan in the stroma and was significantly increased in EcE \ncompared to EuE (P < 0.01); IGFBP3 in the stroma showed \nno significant change in expression between EuE and EcE. In \ncontrast, as shown in Fig.  4A (lower panels), COL8A1 was \npredominantly expressed in the glandular epithelium rather \nFig. 1  Attenuation of miR-210 in mid-secretory ectopic endome-\ntrium from women and baboons with endometriosis. A A significant \ndecrease in the expression of miR-210 was observed in ectopic endo-\nmetrium (EcE) compared to eutopic endometrium (EuE) of women \n(n = 9, biological replicate) with spontaneous endometriosis (Eosis). \nB RT-qPCR analysis showed that miR-210 expression significantly \ndecreased in EcE compared with the EuE of baboons (n = 5, biologi-\ncal replicate) at 15 months (15M) following endometriosis induction. \nMean (SD) is shown. Student’s t-test. *P < 0.05. RT-qPCR, quantita-\ntive reverse transcript polymerase chain reaction; SD, standard devia-\ntion\n\n2937Reproductive Sciences (2023) 30:2932–2944 \nthan in the stroma in EuE but was significantly increased in \nboth the glandular epithelium and the stroma in EcE (P  < \n0.01). These changes were also evident in the EuE and EcE \nof baboons with endometriosis (Fig.  4B).\nMiR‑210 Regulation of IGFBP3 Expression \nin Endometriotic Epithelial Cells\nFollowing in vivo characterization and cell-specific localiza-\ntion of miR-210, IGFBP3, and COL8A1 in ectopic lesions, \nwe performed in silico analysis to predict miR-210 targets \nusing miRWalk 3.0 and RNA22 v2 (Supplementary Fig-\nure 3). We confirmed that IGFBP3 and COL8A1 are pre-\ndicted targets of miR-210. In general, miRNAs regulate \ngene function by degradation and/or direct translational \nrepression of the target transcripts. To determine whether \nmiR-210 downregulates IGFBP3 and COL8A1 at the tran-\nscriptional level, we performed RT-qPCR on immortal-\nized human ectopic endometriotic epithelial (12Z) cells. \nThe 12Z cells were transfected with a miR-210 mimic as \nsemi-quantitative analysis of the in situ hybridization data \nsuggested that miR-210 was predominantly localized to the \neutopic glandular epithelium. The RT-qPCR data confirmed \nthe overexpression of miR-210 following the transfection \n(Fig.  5A) compared to non-targeting negative controls (P \n< 0.01). MiR-210 overexpression led to significant inhibi-\ntion of IGFBP3 expression at 24 and 48 h after transfection \n(Fig. 5 B; P < 0.01); however, miR-210 overexpression had \nno significant effect on COL8A1 expression (Fig. 5C). These \nresults suggested that miR-210 downregulated IGFBP3  \nbut not COL8A1  at the transcriptional level. To determine \nwhether miR-210 overexpression directly represses IGFBP3 \nand COL8A1 at the translational level, we performed west-\nern blotting on protein extracts from the miR-210-trans-\nfected 12Z cells. As illustrated in Fig.  6A, western blot \nanalysis demonstrated that IGFBP3 protein expression was \nsignificantly decreased at 24 h ( P < 0.01) and 48 h ( P < \n0.01) after transfection, whereas COL8A1 protein expres-\nsion was considerably reduced at 24 h after transfection, but \nnot at 48 h (Fig.  6B). These results suggested that miR-210 \ndownregulated IGFBP3 but not COL8A1 at the translational \nlevel. To establish a functional link between the attenuation \nFig. 2  In situ miR-210 expression was greater in the eutopic glandu-\nlar epithelium compared to the ectopic glandular epithelium. A In a \nbaboon endometriosis model, in situ hybridization showed that miR-\n210, labeled with FITC, was expressed in glandular epithelium from \nthe mid-secretory in the eutopic endometrium (EuE) (upper panels) \nand decreased in that of ectopic endometrium (EcE) (lower panels). \nOriginal magnification ×20. Scale bars, 100 μm. FITC, fluorescein \nisothiocyanate; DAPI, 4,6-diamidino-2-phenylindole. B Computer-\nassisted image analysis of cell segmentation and cell classification \nbased on the expression of miR-210 and U6. DAPI signal was used as \nnuclear counterstaining to segment cells and the intensity of miR-210 \nand U6 was used to classify cells as miR-210 positive (green color) \nor negative (brown color). miR-210 positive cells in glandular epithe-\nlium decreased in EcE. Representative images are shown. Original \nmagnification ×5. Scale bars, 100 μm\n\n2938 Reproductive Sciences (2023) 30:2932–2944\nof miR-210 followed by the amplification of IGFBP3 and \nectopic lesion development, we next focused on the effects \nof IGFBP3 on cell growth.\nInhibition of Cell Proliferation and Migration \nby MiR‑210 Overexpression in Endometriotic \nEpithelial Cells\nTo evaluate the effects of miR-210-IGFBP3 signalling on \ncell growth, we performed cell proliferation and scratch \nassays on miR-210-transfected 12Z cells because these \ntwo functions are well-established hallmarks of endome-\ntriosis leading to the development of ectopic lesions. The \ncell proliferation assay demonstrated that overexpression of \nmiR-210 significantly inhibited cell proliferation 3, 4, and \n5 days after transfection compared to the negative control \n(P < 0.01; Fig.  7A). The scratch assay demonstrated that \noverexpression of miR-210 significantly reduced migration \nactivity compared to the negative control (P < 0.05; Fig. 7B, \nC). These results suggested that miR-210 overexpression \ninhibited cell proliferation and migration in the ectopic \nglandular epithelium. Taken together, attenuated miR-210 \nexpression and consequent amplification of IGFBP3 in the \nglandular epithelium at the mid-secretory phase contributed \nto the development of ectopic lesions through increased cell \nproliferation and migration.\nDiscussion\nPrevious microarray studies on gene expression profiles \nhave shown that miR-210 and its downstream targets \n(IGFBP3 and COL8A1) play a role in pathophysiology \nof endometriosis, but the underlying mechanisms remain \nunclear [20 , 21]. Therefore, this validation study builds \non these prior reports, and further focuses on how miR-\n210 and its downstream targets contribute to the devel-\nopment and growth of endometriotic lesions. A series \nof experiments provided four main findings: First, the \nexpression of miR-210 was significantly decreased in \nFig. 3  Amplification of IGFBP3 and COL8A1 in mid-secretory \nectopic endometrium from women and baboons with endometrio-\nsis. A, B RT-qPCR analysis showed that IGFBP3 expression is sig-\nnificantly increased in the ectopic endometrium (EcE) compared \nwith eutopic endometrium (EuE) both in women (n = 9, biological \nreplicate) with spontaneous Eosis and in baboons (n = 4, biological \nreplicates) at 15 months (15M) after endometriosis (Eosis) induction. \nC, D COL8A1 expression is significantly increased in EcE compared \nwith EuE both in women (n = 9, biological replicate) with spontane-\nous Eosis and in baboons (n = 4, biological replicate) at 15M after \nEosis induction. Mean (SD) is shown. Student’s t-test. *P < 0.05; \n**P < 0.01. RT-qPCR, quantitative reverse transcript polymerase \nchain reaction; SD, standard deviation; IGFPBP3, insulin-like growth \nfactor-binding protein 3; COL8A1, collagen type VIII alpha 1 chain\n\n2939Reproductive Sciences (2023) 30:2932–2944 \nEcE compared to EuE in the secretory phase, which \nwas likely associated with the reduced expression of \nmiR-210 in the glandular epithelium of EcE. Second, \nthe expression of IGFBP3 was inversely increased in \nEcE compared to EuE during the same phase, which \nwas likely associated with the increased expression \nof IGFBP3 in the glandular epithelium of EcE. Third, \nthe expression of COL8A1 was also increased in EcE \n\n\n2940 Reproductive Sciences (2023) 30:2932–2944\ncompared to EuE, which was probably associated with \nthe increased expression of COL8A1 in the glandular \nepithelium and the stroma of EcE. Finally, the overex-\npression of miR-210 in the 12Z cells attenuated IGFBP3  \nand its protein expression while decreasing cell prolif-\neration and migration.\nThe expression of IGFBP3 in the glandular epithe-\nlium was increased in EcE compared to EuE of women \nand baboons with endometriosis in the secretory phase. \nIGFBP3 in endometriosis has been investigated in the \nperitoneal fluid (PF), and consequently expanded to the \neutopic endometrium and endometriotic lesions previ-\nously only in human samples. Koutsilieris et al. [38] \nfirst reported the mitogenic effect of IGFBP3 purified \nfrom PF of women without endometriosis on endome-\ntrial epithelial cells, suggesting its possible implica -\ntion in the ectopic growth of endometriotic lesions \n[39]. Later, Akoum et al. [40] performed an immuno-\nhistochemical analysis using Ctrl, EuE, and EcE and \nshowed that IGFBP3 was predominantly localized in \nthe glandular epithelium in the secretory phase. Addi -\ntionally, IGFBP3 expression was increased in EuE and \nEcE compared to Ctrl in the secretory phase (with no \ndirect comparison between EuE and EcE). More recently, \nLembessis et al. [41] conducted an RT-PCR analysis and \nfound that IGFBP3 expression was increased three- to \ntenfold in EcE compared to EuE. Increased expression \nof IGFBP3 in the glandular epithelial cells during the \nsecretory phase was also confirmed in our baboon model \nof endometriosis.\nThe expression of COL8A1 was increased in the glan-\ndular epithelium and stroma of EcE compared to EuE. \nHowever, miR-210 overexpression in 12Z cells had no \nsignificant effect on the COL8A1 expression at the tran -\nscriptional and translational levels. A possible reason \nmight be that extracellular matrix (ECM) production and \nfibrotic changes occur mainly in the stroma of endometri -\notic lesions [42]. The ECM is composed of collagen, pro-\nteoglycans, hyaluronic acid, and chondroitin, and is asso-\nciated with tissue injury and repair, fibrosis, and tumors \n[43]. Recently, some studies demonstrated that upregula-\ntion of COL8A1 is associated with poor survival in gastric \ncancer [44], renal cell carcinoma [45], and breast cancer \n[43]. Furthermore, in colon adenocarcinoma, COL8A1 \nwas shown to play a role in the tumor progression pos-\nsibly by mediating focal adhesion-related pathways [46]. \nPrevious studies revealed the role of collagen I in EuE \n[47] and EcE [42]; hence, this study was the first to show \nthe in vivo characterization of COL8A1 in endometriosis. \nGiven the consistent upregulation of COL8A1 in endo-\nmetriotic lesions from humans and baboons with endo-\nmetriosis, the role of COL8A1 in endometriosis will be \nworth exploring.\nOverexpression of miR-210 in 12Z cells, an ectopic \nglandular epithelium-derived cell line from PE, attenu-\nated IGFBP3 expression, cell proliferation, and cell \nmigration. MiR-210 is prototypical hypoxia-associated \nmiR, and a marker of poor prognosis in solid tumors \nbecause hypoxia is a hallmark of the tumor microenvi-\nronment [48]. Although paradoxically opposing results \nwere documented regarding whether miR-210 is an onco-\ngene or a tumor suppressor, a reasonable explanation is \nthat miR-210 acts differentially, depending on the cel-\nlular context, the extent, and duration of hypoxia, and \nthe target mRNAs available in specific cells [17]. In \nnon-cancerous conditions, overexpression of miR-210 \nwas reported to attenuate cell proliferation and migra-\ntion in human extra-villous trophoblast cell lines target-\ning NOTCH1 [49] or fibroblast growth factor 1 [50]. We \nillustrated a proposed schematic representation of the \nmiR-210-IGFBP3 interaction in the pathophysiology of \nendometriosis (Fig.  8). MiR-210 expression was aber -\nrantly downregulated in EcE, resulting in the upregu-\nlation of IGFBP3, which subsequently enhances cell \nproliferation and migration which contributes to ectopic \nlesion development. One possible mechanism explain-\ning this is the ability of IGFBP3 to activate sphingo-\nsine kinase which is involved in IGF-independent sphin-\ngolipid signalling pathways [23]. Sphingosine kinase, \nactivated by IGFBP3, is an enzyme that converts sphin-\ngosine to sphingosine 1 phosphate, sphingosine inhibits \ncell growth, and sphingosine 1 phosphate stimulates cell \ngrowth [51].\nThe expression of miR-210 in the glandular epithelium \nwas significantly decreased in EcE compared to EuE of \nwomen and baboons with endometriosis. This finding \nseemingly contradicts a report by Dai et al. [15] in which \nthe expression of miR-210 significantly increased in EcE \ncompared to matched EuE of women with DIE. However, \nmenstrual cycle fluctuation of miR-210 observed in the \nbaboon endometriosis model could explain this discrep-\nancy; miR-210 expression is significantly higher in the \nmid-secretory phase than in the proliferative phase in the \nFig. 4  Amplification of IGFBP3 in the ectopic granular epithelium \nand of COL8A1 in both ectopic glandular epithelium and stroma. A \nWhen comparing eutopic endometrium (EuE) with ectopic endome-\ntrium (EcE) in women with Eosis (n = 3, biological replicate), immu-\nnohistochemical staining and H-score analysis showed that IGFBBP3 \nprotein expression was significantly increased in the ectopic glandular \nepithelium (GE) but not in the stroma (ST); COL8A1 protein expres-\nsion was significantly increased both in ectopic GE and ST. B These \nfindings were replicated in baboons 15 months (M) after endome-\ntriosis (Eosis) induction (n = 3, biological replicate). Mean (SD) is \nshown. Two-way ANOVA. *P < 0.05; **P < 0.01; ns, not significant. \nRepresentative images are shown. Original magnification ×20. Scale \nbars, 50 μm. IGFPBP3, insulin-like growth factor-binding protein 3; \nCOL8A1, collagen type VIII alpha 1 chain; SD, standard deviation\n◂\n\n2941Reproductive Sciences (2023) 30:2932–2944 \nbaboon (Supplementary Figure 2). We collected human \nand baboon samples in the mid-secretory phase, but Dai \net al. obtained those samples in the proliferative phase. \nThe other thing to note is that altered downregulation of \nmiR-210 in endometriotic lesions should depend on the \nlocalization of lesions. In women with OE, the expres -\nsions of miR-210 and HIF1A were increased in parallel \nin EcE (n  = 10) compared to Ctrl (n  = 10) [52]. Further -\nmore, Filippi et al. [53] studied 15 healthy women and \n11 patients with DIE and found that HIF1A expression \nincreased in OE but not in DIE. We performed ancillary \nRT-qPCR analysis and confirmed parallel downregulation \nof HIF1A and miR-210 in baboons with endometriosis \n(Supplementary Figure 2).\nSome limitations of our study should be considered. \nFirst, obtaining well-defined matched endometrial clini-\ncal tissues is a challenge; this study was conducted using \na limited number of clinical samples from 9 patients with \nFig. 5  Overexpression of miR-210 in ectopic epithelial cells inhibits \nthe transcription of IGFBP3. A RT-qPCR analysis showed that trans-\nfection of miR-210 mimic into ectopic epithelial cells (12Z cells) \nmarkedly elevated miR-210 expression at 24 and 48 h after transfec-\ntion (n = 3, technical replicate). B The overexpression of miR-210 \nsignificantly decreased IGFBP3 mRNA expression at 24 and 48 h \nafter transfection; C COL8A1 mRNA expression was unchanged. \nMean (SD) is shown. Two-way ANOVA. **P < 0.01; ns, not sig-\nnificant. RT-qPCR, quantitative reverse transcript polymerase chain \nreaction; IGFPBP3, insulin-like growth factor-binding protein 3; \nCOL8A1, collagen type VIII alpha 1 chain; SD, standard deviation\nFig. 6  Overexpression of miR-210 in ectopic epithelial cells \ndecreased IGFBP3 protein levels. A  Western blot analysis and \nsemi-quantification by densitometry showed that IGFBP3 pro-\ntein expression, forming a doublet as two glycosylation forms of \nIGFBP3 protein core, was significantly attenuated at 24 and 48 h \nafter miR-210 transfection into 12Z cells (n  = 3, technical repli-\ncate); B whereas COL8A1 protein expression was significantly \nattenuated at 24 h after miR-210 transfection but was unchanged at \n48 h. MCF7 was used as a positive control for IGFBP3 and HeLa \ncells for COL8A1, respectively. Mean (SD) is shown. Two-way \nANOVA. *P  < 0.05; **P  < 0.01; ns, not significant. IGFPBP3, \ninsulin-like growth factor-binding protein 3; COL8A1, collagen \ntype VIII alpha 1 chain; SD, standard deviation\n\n2942 Reproductive Sciences (2023) 30:2932–2944\nDIE, and we could not collect matched paired samples \nin the remaining 6 patients with DIE. Second, absence \nof endometriotic lesions was confirmed only by trans -\nvaginal ultrasound, which is commonly used to evalu-\nate women with suspected endometriosis, but subject to \nfalse negatives compared with exploratory laparoscopy. \nAdditionally, there could be some potential heterogeneity \nin clinical symptoms among study participants. There-\nfore, caution is needed when interpreting our data. Third, \nexpression data is proportional to the sum of the sample \nwhich potentially contains not only endometriotic epithe-\nlium and stroma but also other tissue components (adi-\npose tissue and blood vessels). Fourth, we did not assess \nthe cellular uptake and transfection efficiency of miR-210 \nmimic using confocal laser scanning and flow cytome-\ntry. Fifth, we focused on the proliferation and migration \npotential of IGFBP3 but not on the cell cycle. Further \nin vitro assays involving cell cycle regulation to support \nthe observed miR-210 effects on endometriotic cell prolif-\neration are warranted. Finally, although we demonstrated \nchanges in the expression of COL8A1, the molecular \nmechanisms underlying its role in endometriosis remain \nunclear. Expanding the analysis of the in vitro studies to \nstromal compartments using an appropriate ectopic stro-\nmal cell lines is essential for understanding the role of \nmiR-210 in a heterogenous endometriotic cell population.\nOur present study showed that miR-210 was localized \nto the glandular epithelium of EuE, and its expression was \naberrantly decreased in the EcE, while its target IGFBP3 was \nincreased. Overexpression of miR-210 inhibited IGFBP3  \nexpression, cell proliferation, and migration of ectopic glan-\ndular epithelial cells. These results suggest that repression of \nmiR-210 and a corresponding increase in IGFBP3 expres-\nsion could contribute to the development of endometriotic \nlesions. In future studies, we will explore the possibility of \nmiR-210-IGFBP3 signalling as a potential diagnostic or \ntherapeutic marker for endometriosis.\nSupplementary Information The online version contains supplemen-\ntary material available at https:// doi. org/ 10. 1007/ s43032- 023- 01253-5.\nFig. 7  Overexpression of miR-210 inhibited ectopic epithelial cell \nproliferation and migration. A MTS assay showed that transfection \nof miR-210 mimic into 12Z cells significantly inhibited cell prolifera-\ntion compared to that of non-targeting negative control on days 2, 3, \n4, and 5 after transfection (n = 6, technical replicate). Mean (SD) is \nshown. Two-way ANOVA. **P < 0.01. B, C Scratch assay showing \nthat the transfection of miR-210 mimic in 12Z cells (n = 3, technical \nreplicate) significantly attenuated cell migration capability compared \nwith that of non-targeting negative control 24 h after transfection. \nMean (SD) is shown. Student’s t-test. *P < 0.05. SD, standard devia-\ntion\nFig. 8  Proposed schematic representation of miR-210-IGFBP3 molec-\nular interaction in the pathophysiology of endometriosis. Upregula-\ntion of miR-210 at the mid-secretory phase controls cell proliferation \nand migration through the suppression of IGFBP3, whereas in ectopic \nlesions, this suppression is decreased due to the aberrant downregula-\ntion of miR-210, promoting endometriotic lesion development\n\n2943Reproductive Sciences (2023) 30:2932–2944 \nAcknowledgements We would like to thank Dr. John I. Risinger and \nDr. Hong Im Kim from Michigan State University for their technical \nassistance and for providing samples for antibody validation.\nAuthors’ Contributions KK, NJ, and AF contributed to the study \ndesign, interpretation of the data, and wrote the manuscript. KK, YS, \nMO, GM, SH, and EV performed the experiments. KK, GB, and NJ \nanalyzed the data and prepared graphs and tables. LS performed and \nanalyzed ISH data. EM and PS contributed to sample collection. KK, \nNJ, and AF performed the review, editing, and preparation of the manu-\nscript. We thank Dr. Eduardo H. Miyadahira and Paulo C. Serafini \n(Clínica Vida Bem Vinda, São Paulo, Brazil) for supplying the human \nendometriosis sample.\nFunding This work was supported by the Eunice Kennedy Shriver \nNational Institute of Child Health & Human Development/National Insti-\ntute of Health R01 HD083273 grant to ATF, by Nakatsu City Grand-in-Aid \nfor Perinatal Care Doctors to KK, and by Oita University Grand-in-Aid \nfor Study Abroad to KK. In addition, GWB, MAO-B and GEM were also \nsupported by a grant from the Eunice Kennedy Shriver National Institute of \nChild Health & Human Development of the National Institutes of Health \nunder Award Number T32HD087166, and Michigan State University.\nData Availability Not applicable.\nCode Availability Not applicable.\nDeclarations \nEthics Approval Appropriate ethical approvals were obtained for the study.\nConsent to Participate Informed consent was obtained from the study \nparticipants.\nConsent for Publication All the authors agree and consent to the pub-\nlication of this study.\nConflict of Interest The authors declare no competing interests.\nOpen Access This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. 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Reprod Sci. 2016;23(4):492–7.\nPublisher’s Note Springer Nature remains neutral with regard to \njurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}