{"paper_id":"40f6e2fe-9fc0-4db5-b341-58bfaf168f04","body_text":"R E S E A R C H Open Access\nExpression of Talin-1 in endometriosis and\nits possible role in pathogenesis\nXian Tang 1, Qing Li 2, Lijie Li 2 and Jianfa Jiang 2*\nAbstract\nBackground: Endometriosis is a disease that involves active cell invasion and migration. Talin-1 can promote cell\ninvasion, migration and adhension in various cancer cells, but its role in endometriosis has not been investigated.\nThis study was to investigate the expression level of Talin-1 in endometriosis and the role of Talin-1 in the\nproliferation, adhesion, migration, and invasion of human endometrial stromal cells (ESCs).\nMethods: Ectopic and eutopic endometrial tissues were collected from women with endometriosis, and the\ncontrol endometrial tissues were obtained from patients without endometriosis. The expression level of Talin-1 was\ndetected in each sample using quantitative real-time polymerase chain reaction and immunohistochemistry. The\nexpression of Talin-1 was inhibited using RNA interference in ESCs, and its proliferation, apoptosis, adhesion,\nmigration, and invasion capacity were analyzed. Western blotting was performed to detect the expression of\nrelated molecules after the downregulation of Talin-1.\nResults: The results showed that the mRNA and protein expression of Talin-1 were significantly increased in the\nectopic endometrium and eutopic endometrial tissues compared with the controls. The knockdown of Talin-1 did not\naffect the proliferation and apoptosis of ESCs. The results indicated that the downexpression of Talin-1 inhibited the\nadhesion, invasion, and migration of ESCs. In addition, the expressions of N-cadherin, MMP-2, and integrin β3w e r e\nsignificantly lower after the deregulation of Talin-1, whereas the levels of E-cadherin were significantly increased.\nConclusions: The expression of Talin-1 was increased in the ectopic and eutopic endometrial tissues compared with\nthe control endometrium. The downregulation of Talin-1 inhibited the adhesion, invasion, and migration of ESCs.\nKeywords: Endometriosis, Talin-1, Adhesion, Migration, Invasion\nBackground\nEndometriosis is a commonly encountered benign gy-\nnecologic disease that affects 10% of reproductive-age\nwomen [ 1]. It can be classified into superficial or\nperitoneal, ovarian, and deep infiltrating endometriosis. It\ncauses chronic pelvic pain, dysmenorrhea, deep dyspar-\neunia, dysuria, dyschezia, fatigue, and infertility, all of\nwhich affect physical, mental, sexual, and social well-being\nas well as productivity [ 2]. Although endometriosis is a\nbenign disease, endometriotic cells exhibit many features\nsimilar to those of malignant cancer, such as migratory\nand invasive properties. A series of hypotheses that\nattempt to explain the genesis of endometriosis has been\nestablished. However, the etiology and pathogenesis of the\ndisease have not been fully elucidated [ 3].\nThe postulated origin of endometriotic tissue is consid-\nered retrograde menstruation [ 4]. However, retrograde\nmenstruation is a very common physiological phenomenon,\nand only 10% of women develop endometriosis. There\nmust be other factors that promote endometrial cells to\nadhere to ovaries, ligaments, and peritoneal surfaces and to\ndevelop endometriosis. Recent studies have suggested that\neutopic endometrium in patients with endometriosis is\n© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,\nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give\nappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if\nchanges were made. The images or other third party material in this article are included in the article's Creative Commons\nlicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons\nlicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain\npermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\nThe Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the\ndata made available in this article, unless otherwise stated in a credit line to the data.\n* Correspondence: 670561033@qq.com\n2Department of Gynecology, The Third Xiangya Hospital, Central South\nUniversity, NO.138 Tongzipo Road, Yuelu District, Changsha 410013, Hunan,\nChina\nFull list of author information is available at the end of the article\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 \nhttps://doi.org/10.1186/s12958-021-00725-0\n\ndifferent from normal endometrial cells, which facilitate the\nproliferation, implantation, and survival of endometrial tis-\nsue in the peritoneal cavity [ 5]. Recently, the involvement\nof adhesion molecules in endometriosis has gained much\nattention. Endometrial stromal cells from women with\nendometriosis exhibit adhesive capacity as a result of\naltered integrin profiles. Integrins may promote the attach-\nment of ectopic endometrial cells to the peritoneum [6].\nTalin-1, which is located at the adhesion complex\nbetween cells and their extracellular matrix (ECM), has\nbeen reported to interact with multiple adhesion\nmolecules and to activate integrin and focal adhesion\nsignaling [ 7]. Recent studies have indicated that the\ndysregulation of Talin-1 can lead to cell spreading, mi-\ngration, and survival, and this has led to an extensive\ninvestigation into its role in cancer and other disorders\n[8–12]. Endometriosis is also a disease with active cell\nmigration and invasion. Our previous study demon-\nstrated that the level of Talin-1 was significantly higher\nin eutopic and ectopic endometrium in women with\nadenomyosis [ 13]. However, the relationship between\nTalin-1 and endometriosis has never been reported.\nThus, this study aimed to investigate the expression of\nTalin-1 in endometriosis and analyzed the potential role\nof Talin-1 in the development of endometriosis.\nMaterials and methods\nSubjects and sample collection\nMatched ectopic and eutopic endometrial tissues were\nobtained from women with ovarian endometriotic cysts\nundergoing laparoscopic surgery at the Gynecological\nDepartment of the Third Xiangya Hospital from January\nto June 2020. Endometriosis was confirmed through a\nhistological examination. The control endometrial tissues\nwere obtained from patients without endometriosis and\nwomen with histologically proven non-endometriotic be-\nnign ovarian cysts. All patients had regular menstruation\nand had not received any hormonal medication within the\nthree months prior to surgery. The samples were collected\nin proliferating phases of the menstrual cycle, which was\ndetermined by preoperative history and histological\nexamination.\nImmunohistochemistry\nImmunohistochemistry staining for Talin-1 was per-\nformed on 4 μm-thick paraffin sections. Following routine\ndeparaffinization, the sections were hydrated and then\nmicrowave-treated for antigen restoration. Endogenous\nperoxidase activity was eliminated using 3% hydrogen per-\noxide for 10 min. Then, polyclonal rabbit anti-human\nTalin-1 (1100 dilution, Bioss, Shanghai, China) was used\nto incubate overnight at 4 °C. The slides were incubated\nwith a horseradish peroxidase-conjugated antirabbit\nsecondary antibody for 30 min at room temperature. DAB\nstaining was performed. Finally, all sections were counter-\nstained with hematoxylin, dehydrated, and cover-slipped.\nImages were captured by a Leica DM4000B microscope\n(Leica, Wetzlar, Germany). The staining intensity was\ngraded as follows: 0 = none, 1 = weak, 2 = moderate, and\n3 = strong staining. The percentage of positive stained\ncells was graded as follows: 0 for no positive staining cells,\n1 for 25% positive staining cells, 2 for > 25% and < 50%,\nand 3 for > 50%. The immunoreactive score was calculated\nusing the following equation: immunoreactive score =\nstaining intensity multiplied percentage of positive cells.\nWestern blotting\nWestern blot analyses were performed as described pre-\nviously [ 14]. Briefly, protein was extracted from cells that\nwere lysed in radioimmunoprecipitation buffer and cen-\ntrifuged at 12,000×g for 15 min at 4 °C. The supernatant\nprotein was quantified by bicinchoninic acid assay\n(Thermo Fisher Scientific, Rockford, USA). Homogenate\nproteins were separated by 10% SDS-polyacrylamide gels\nand transferred onto polyvinylidene difluoride mem-\nbranes (Millipore, Billerica, MA, USA). The membranes\nwere blocked with 5% fat-free milk, washed, and then\nprobed with the following primary antibodies: β-actin (1:\n5000 dilution), integrin β3 (1:1000 dilution), N-cadherin\n(1:2000 dilution), MMP-2 (1:1000 dilution), E-cadherin\n(15,000 dilution), and Talin-1 (1500 dilution) (all from\nProteintech, Chicago, USA). After washing, the mem-\nbranes were incubated with horseradish peroxidase-\ncoupled goat anti-mouse secondary antibody at room\ntemperature for 80 min. Band intensity was quantified\nusing Quantity One software. β-actin served as the load-\ning control.\nQuantitative real-time polymerase chain reaction (qRT-\nPCR)\nQRT-PCR was performed as previously described [ 13].\nThe total RNA was extracted using Trizol reagent (Life\nTechnologies, CA, USA). Reverse transcription was con-\nducted using SuperScript III Transcript (Life Technolo-\ngies, CA, USA) in accordance with the manufacturer ’s\nprotocol. Reactions were performed using the 7500 Real-\nTime PCR System (Applied Biosystems Inc., Foster City,\nCA, USA). The primer sequences of Talin-1 and beta-\nactin are listed in Supplementary Table 1. The relative\ngene expression of Talin-1 was calculated using the\n2−△△Ct method.\nCell isolation and culture\nEndometriotic stromal cells (ESCs) from the eutopic\nendometrial samples of women with endometriosis\nculture were processed as described previously [ 14, 15].\nIn brief, the samples were collected under sterile condi-\ntions, washed, and transferred to the laboratory on ice.\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 2 of 10\n\nFollowing isolation, the ESCs were passed by using the\nstandard method of trypsinization, plated in culture\ndishes, and resuspended in phenol red-free Dulbecco ’s\nModified Eagle ’s Medium (DMEM; Gibco; Thermo\nFisher Scientific, Inc., Waltham, MA, USA) supplemented\nwith 10% fetal bovine serum (FBS; Sigma-Aldrich, St.\nLouis, MO, USA) at 37 °C in a humidified atmosphere\nwith 5% CO 2. The primary ESCs were examined by im-\nmunostaining for anti-vimentin (Abcam, Cambridge, MA,\nUSA), a specific marker of stromal cells. Only cultures\nwith more than 96% purity were included in our study.\nTransfection experiments\nRNA interference was performed by small interfering\nRNA (siRNA) transfection. Three different siRNAs\ntargeting Talin-1 and the negative control siRNA were\nsynthesized in GenePharma (Changsha, China). The\nsequences are listed in Supplementary Table 1. A quan-\ntity of10 5 ESCs was seeded in a six-well plate the day be-\nfore transfection. The transfection of the control siRNA\nor siRNA against Talin-1 was conducted using Lipofec-\ntamine 2000 (Invitrogen, Carlsbad, CA, USA) according\nto the manufacturer ’s protocol. After 24 h, the cells were\nharvested for Western blot to confirm the gene silence.\nThe cells were harvested after 48 h for other assays.\nCell proliferation assay\nThe proliferation of ESCs was determined using Cell\nCounting Kit (CCK)-8 assays. After being transfected\nwith siRNAs, the ESCs were seeded in 96-well plates at a\ndensity of 1 × 10 4 cells in 100 μL of culture medium per\nwell. After a culture time of 0, 24, 36, and 48 h, 20 μLo f\nCCK-8 (Dojindo, Kyushu, Japan) was added for an\nadditional 4 h of incubation. A spectrophotometric plate\nreader (Thermo Fisher Scientific, Inc., MA, USA) was\nused to read the absorbance at 450 nm. This experiment\nwas performed with triplicate wells and independently\nrepeated at least three times.\nApoptosis assay by flow cytometry\nApoptotic cell death was measured by a fluorescein\nisothiocyanate-annexin V/PI apoptosis detection kit\n(KeyGEN BioTECH, Nanjing, China) according to the\nmanufacturer’s protocol after transfection with siRNAs\nfor 48 h. The cells were washed twice with phosphate\nbuffered saline (PBS) and resuspended in a binding\nbuffer at a concentration of 1 × 10 5 cells/mL. The cells\nwere stained with annexin V-APC and propidium iodide\nand detected by flow cytometry. The cells labelled with\nannexin V were considered apoptotic, and propidium\niodide was used to detect dead cells. Fluorescence-\nactivated cells were quantified using a flow cytometer.\nThe apoptotic rate was the sum of annexin V-positive/\npropidium iodide-negative and annexin V-positive/\npropidium iodide-positive cells in two quadrants as a\nproportion of the total number of cells.\nAdhesion assay\nA CCK-8-based adhesion assay was used to determine\nthe adhesion capacity of the ESCs. A 96-well plate was\nused to perform this assay. Matrigel of 50 μL in a serum-\nfree medium (at 1:8 dilution) was added to each well.\nAfter transfection for 48 h, 1 × 10 4 cells/100 μL were\ncultured in each incubated well. After incubation for 30\nmin, the non-adherent cells were rinsed off, and 10 μL\nCCK-8 (Dojindo, Kyushu, Japan) was added for an\nadditional 4 h of incubation. A spectrophotometric plate\nreader was used to read the absorbance at 450 nm. The\nnumber of adhesion cells was characterized by the OD\nvalue.\nWound-healing assay\nA wound-healing assay was performed to evaluate the\ncells’ ability to migrate. Briefly, the cells transfected with\nTalin-1 siRNA were seeded into six-well culture plates\nat 90% confluency. A wound was made on a monolayer\nof cells using a standard 1000 μL plastic pipette tip. The\ncells were washed using PBS to clear away the cell deb-\nris. For each well, pictures were taken at 0, 24, and 48 h\nafter the wound. The wound width was calculated using\nImage-Pro Plu software. The migration rate was calcu-\nlated as [Cell-free area (0 h)- Cell-free area (24 h or 48\nh)]/Cell-free area (0 h).\nTranswell invasion assay\nTranswell invasion assay was performed to assess the in-\nvasion of the ESCs. In the invasion assay, the upper\nchamber was first coated with 60 μL matrigel (1:2 matri-\ngel and DMEM without phenol red) and incubated for 1\nh at 37 °C. The ESCs (10 5 cells/well) were plated into the\nupper chambers, and the lower chambers were filled\nwith phenol red-free DMEM plus 10% FBS. After 72 h of\nincubation, the cells in the upper chamber were re-\nmoved, and the transwell filters were fixed with 4%\nparaformaldehyde for 30 min, washed with PBS twice,\nstained with 0.5% hematoxylin for 5 min, and counted in\nthree representative fields under a light microscope\n(Olympus). A spectrophotometric plate reader was used\nto read the absorbance at 550 nm. The experiments were\nperformed in triplicate.\nStatistical analysis\nAll statistical analyses were performed using SPSS soft-\nware version 22.0 (SPSS, Inc., Chicago, USA). All data\nare presented as means ± standard deviation. One-way\nanalysis of variance was used to analyze the differences\namong multiple groups. P values < 0.05 was considered\nstatistically significant.\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 3 of 10\n\nResults\nIncreased Talin-1 protein expression in endometriosis\nEctopic and eutopic end ometrium tissues were\nobtained from 26 patients with endometriosis (mean\nage 36.65 ± 6.99 years), of whom 15 had AFS stage\nIII disease and 11 had stage IV disease. Samples of the\ncontrol endometrium were obtained from 15 women\nwithout endometriosis (mean age 35.53 ± 5.40 years).\nImmunohistochemical staining of the tissue showed\nthat Talin-1 was present in both epithelial and stromal\ncells, and that the staining was mostly cytoplasmic\n(Fig. 1a). Compared with that in the normal endomet-\nrium (2.73 ± 0.80), the Talin-1 protein expression in the\neutopic (3.35 ± 0.69) and ectopic endometrium (6.73 ±\n2.01) of ovarian endometriosis was significantly in-\ncreased ( P = 0.014; P < 0.001, respectively). The protein\nFig. 1 The mRNA and protein levels of Talin-1 were upregulated in human endometriotic tissues. a Representative images of different tissues\nshowing the Talin-1 immunohistochemical staining. The images of eutopic EM and ectopic EM were from the same patient. The original\nmagnification was 100×. b Immunostaining score analysis of talin1 protein expression in different tissues. c QRT-PCR analysis of talin1 mRNA\nexpression in human endometriotic tissues. * P<0.05\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 4 of 10\n\nlevels of Talin-1 in the ectopic endometria was\nsignificantly increased compared with the eutopic\nlevels ( P <0 . 0 0 1 ) ( F i g .1b).\nIncreased Talin-1 mRNA expression in endometriosis\nCompared with those in the control group (0.014 ±\n0.005), the mRNA levels of Talin-1 in the ectopic\nendometrium (0.037 ± 0.014) and eutopic endomet-\nrium tissues (0.018 ± 0.005) of patients were signifi-\ncantly higher ( P <0 . 0 0 1 a n d P = 0.013, respectively)\n(Fig. 1c). When tests were performed on the matched\nsamples of eutopic and ect opic endometrium tissues\nof women with endometriosis, the expression of\nTalin-1 was significantly increased in the ectopic\nendometrium ( P < 0.001).\nSilencing of Talin-1 had no effect on the proliferation and\napoptosis of ESCs\nAs the Talin-1 level was upregulated in human\nendometriotic tissues, we knocked down its expres-\nsion in the ESCs to investigate its functional roles.\nThe ESCs were transfected with three siRNA\nsequences targeting Talin-1. Western blot analysis\nshowed that the protein expression of Talin-1 was\nsignificantly decreased in the si-Talin-1 group com-\npared with the negative control (NC) group and\nblank control (BC) group (Fig. 2a). According to the\nresults of the Western blot analysis, the first siRNA\n(siRNA1) was selected for further investigations.\nTo determine the effect of Talin-1 on the proliferation\nand apoptosis of the ESCs, the cells were transfected\nwith siRNA1 or si-NC for 48 h, followed by CCK-8 and\nflow cytometry. The CCK-8 assays revealed that the\nsilencing of Talin-1 did not affect the proliferation of the\nE S C sc o m p a r e dw i t ht h eN Cg r o u pa n dB Cg r o u p\n(Fig. 2b). Flow cytometry showed that the downregu-\nlation of Talin-1 did not promote the apoptosis of\nthe ESCs compared with the controls (Fig. 2c).\nTalin-1 knockdown inhibited the adhesion of ESCs\nThe effect of Talin-1 knockdown on the adhesion ability\nof the ESCs was examined in vitro. In the adhesion\nassay, the downregulation of Talin-1 effectively sup-\npressed the adhesion of the ESCs (Fig. 2d) compared\nwith the control cells.\nFig. 2 The effect of Talin-1 on the proliferation, apoptosis and adhension capacity of ESCs. a Down-regulation of Talin-1 assessed by western\nblotting after transfection with three short interfering RNA (siRNA) or the negative control (NC). The first siRNA (siRNA1) was selected for further\ninvestigations. BC, blank control without siRNA. b Knockdown of Talin-1 had no effect on the proliferation of ESCs. c Knockdown of Talin-1 had\nno effect on the apoptosis of ESCs. d Down-regulation of Talin-1 inhibited the adhesion of ESCs. * P<0.05\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 5 of 10\n\nDownregulation of Talin-1 inhibited the migration and\ninvasion of ESCs\nTo investigate the effects of Talin-1 downexpression on\nESC migration, wound-healing assay was performed. As\nshown in Fig. 3, the silencing of Talin-1 led to a signifi-\ncant decrease in ESC migration ability compared with\nthat in the NC group and BC group.\nTalin-1 knockdown inhibited the invasion of ESCs\nThe relationship between Talin-1 knockdown and cell\ninvasion was analyzed using siRNA technology in the\nESCs. The results showed that the number of ESCs that\nhad invaded through the Matrigel-precoated transwell\nfilters was significantly reduced in the siRNA1 group\n(Fig. 4). The negative control siRNA did not significantly\ninterfere with the invasion ability of the ESCs compared\nwith the BC group.\nEffects of Talin-1 on the related molecule expression in\nESCs\nThe effect of Talin-1 on the expressions of adhesion-\nrelated molecule integrin β3, migration-related molecule\nN-cadherin and E-cadherin, and invasion-related mol-\necule MMP-2 on ESCs was assessed using Western blot\nafter transfection with siRNA1 for 48 h. The results\nshowed that the downregulation of Talin-1 clearly\ndecreased the expression of integrin β3, N-cadherin, and\nMMP-2 in the ESCs compared with the BC group and\nFig. 3 Downregulation of Talin-1 inhibited the migration of ESCs a Migration of ESCs transfected with siRNA1 was assessed in wound healing\nassays. BC, blank control without siRNA. NC, negative control. Magnification, 400×. b Quantification results of the wound-healing assays\n(mean ± SD). *P<0.05\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 6 of 10\n\nFig. 4 Downregulation of Talin-1 inhibited the invasion of ESCs. a Invasion of ESCs transfected with siRNA1 was assessed in transwell invasion\nassays. BC, blank control without siRNA. NC, negative control. Magnification, 400×. b Quantification results of the transwell invasion assays\n(mean ± SD). *P<0.05\nFig. 5 Effects of Talin-1 down-regulation on related molecule expression. a Representative western blots analysis, with values normalized to β-\nactin. b Quantification results of Talin-1 down-regulation on related molecule expression. The results showed that down-regulation of Talin-1\nobviously decreased the expression of integrin β3, N-cadherin, MMP-2 in ESCs compared to BC group and NC group, while the expression of E-\ncadherin was increased. * P<0.05\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 7 of 10\n\nNC group, whereas the expression of E-cadherin in-\ncreased (Fig. 5).\nDiscussion\nTo the best of our knowledge, this study is the first re-\nport on the expression of Talin-1 in endometriosis and\nits possible role in pathogenesis. We identified that the\nmRNA and protein of Talin-1 were highly expressed in\nthe ectopic and eutopic endometrial tissues of patients\nwith endometriosis compared to the control endomet-\nrium tissues. The downregulation of the expression of\nTalin-1 in the ESCs showed decreased adhesion, migration,\nand invasion ability, confirming the involvement of\nTalin-1 in the pathological endometriotic process. The\nknockdown of Talin-1 affected the expression of the\nadhesion-related molecule integrin β3, migration-\nrelated molecules E-cadherin and N-cadherin, and\ninvasion-related molecule MMP-2. The present findings\nprovide novel insights into the role of Talin-1 in\nendometriosis.\nTalin-1 is a focal adhesion protein that binds to\nmultiple adhesion molecules and is an essential mediator\nof cell –ECM adhesion [ 16, 17]. The N-terminal head\ndomain of Talin-1 binds to the integrin β subunit cyto-\nplasmic domain, which causes integrin activation and\nstimulates integrin binding to the ECM [ 18, 19]. More-\nover, Talin-1 can act independent of integrins by sup-\npressing the expression of E-cadherin, which is a cell –\ncell adhesion molecule [ 20]. Accumulating evidence sug-\ngests that the expression of Talin-1 is dysregulated in\nvarious malignant neoplasms, such as colorectal cancer\n[21], hepatocellular carcinoma [ 22], prostate cancer [ 16],\nand oral squamous cell carcinoma [ 23]. Although endo-\nmetriosis is a benign disease, it exhibits many cancer-\nlike features, such as proliferation, anti-apoptosis, and\ncell migration. Whether Talin-1 is associated with the\npathophysiology of endometriosis remains unclear. In\nthe current study, we found that Talin-1 expression at\nboth mRNA and protein levels was significantly upregu-\nlated in the eutopic and ectopic endometrial tissues of\nendometriosis compared with the controls, suggesting\nthat Talin-1 could be associated with the genesis and\nprogress of endometriosis. But it ’s worth noting that the\nincrease in the expression of Talin-1 in the ectopic\nendometrial tissues of patients with endometriosis was\nhigher than that in eutopic endometrium. The specific\nreason is unclear, which may be related to stronger ad-\nhesive behavior of ectopic endometrial stromal cells [ 24].\nHowever, the role of Talin-1 in endometriosis is not\nclear. Some studies have indicated that endometriotic\ncells have aggressive ability. Talin-1 has been described\nas an oncogene, and it mediates cell adhesion, prolifera-\ntion, tumorigenesis, and metastasis. Talin-1 overexpres-\nsion markedly enhanced the migration and invasion\npotential of human prostate cancer cells by activating\nECM–integrin-mediated signaling and promoting anoi-\nkis resistance [ 16]. Talin-1 can also significantly promote\nhepatocellular carcinoma cell proliferation and metasta-\nsis. Integrin signaling has been shown to be crucial in\ncell invasion and migration not only by physically tether-\ning cells to the matrix but also by sending and receiving\nmolecular signals [ 25]. Based on the above findings, the\nhigh expression of Talin-1 could be associated with\nendometrial tissue adherence and migration at ectopic\nsites to form endometriotic lesions. In the current study,\nwe found that the downregulation of Talin-1 could in-\nhibit the adhesion, migration, and invasion of endomet-\nrial stromal cells.\nTalin-1 regulates integrin and focal adhesion signaling.\nA recent study showed that Talin-1 played an important\nrole in integrin activation, cell adhesion, migration, inva-\nsion, and anoikis of prostate cancer cells, and promotion\nof prostate cancer bone metastasis [ 12]. At the initial\nstages of endometriosis, the attachment of retrograde\nendometrial tissues onto the pelvic mesothelium is a\ncritical step [ 26]. Several integrins, including αv, β3, β4,\nand β1, have been reported to mediate the attachment\nof endometrial cells to the mesothelium [ 27, 28]. The ex-\npression of these integrins is tightly regulated by diverse\nmolecules. The results of the current study showed that\nthe knockdown of Talin-1 affected the expression of in-\ntegrin β3, indicating that Talin-1 could promote adhe-\nsion and migration by regulating integrin β3.\nThe pathogenesis of endometriosis also requires ECM\nbreakdown. The involvement of MMPs in the develop-\nment of endometriosis has been confirmed. MMP-2 is\none of the members of the MMP family proteins that\nplay an important role in the formation of endometri-\nosis. It can degrade ECM and increase activity in endo-\nmetriosis as well as mediate the migration and invasion\nof endometriotic cells [ 29, 30]. The current study\nshowed that the expression of MMP-2 was positively re-\nlated with Talin-1, suggesting that Talin-1 could be in-\nvolved in the invasion process by regulating MMP-2\nexpression. However, the underlying mechanism remains\nto be clarified.\nThis study has several limitations, including lacking\nESCs from women without endometriosis in parallel,\nlack of an established physiologic level of Talin-1 in\nESCs, lacking ectopic ESCs experiments, and the possi-\nbility of off-target effects from siRNAs. We plan to con-\nduct further studies to determine which parts of Talin-1\nprotein are responsible for the observed effects. We also\nwant to know what is downstream of Talin-1 and how\ndoes it relate to integrins. In the further studies, we will\ninclude ESCs from women without endometriosis and\nectopic ESCs from patients with endometriosis in paral-\nlel to firmly establish our findings.\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 8 of 10\n\nConclusion\nWe showed that the expression of Talin-1 was elevated\nin women with endometriosis. The knockdown of Talin-\n1 could decrease cell adhesion, migration, and invasion\nin eutopic endometriotic stromal cells from women with\novarian endometrioma. These effects were mediated by\nregulating MMP-2 and integrin β3. Our findings can\nprovide insights into the possible role of Talin-1 in the\ngenesis and progress of endometriosis.\nAbbreviations\nESCs: Endometrial stromal cells; ECM: Extracellular matrix; qRT-PCR\n: Quantitative real-time polymerase chain reaction; siRNA: Small interfering\nRNA; PBS: Phosphate buffered saline; CCK: Cell Counting Kit; NC: Negative\ncontrol; BC: Blank control\nSupplementary Information\nThe online version contains supplementary material available at https://doi.\norg/10.1186/s12958-021-00725-0.\nAdditional file 1: Supplementary Table 1. The sequences of Talin-1\nand siRNA targeted to Talin-1.\nAcknowledgements\nNot applicable.\nDisclosure statement\nThe authors report no conflicts of interests.\nAuthors’ contributions\nXT contributed to cell experiment and data collection. QL contributed to\ntissue sample collection. LJL contributed to data collection and data analysis.\nJFJ contributed to project design, data analysis and manuscript writing. All\nauthors read and approved the final manuscript.\nFunding\nNational Natural Science Foundation of China (No. 81801422) and the New\nXiangya Talent Project of The Third Xiangya Hospital of Central South\nUniversity (No. JY201706).\nAvailability of data and materials\nThe datasets used and/or analyzed during the current study are available\nfrom the corresponding author on reasonable request.\nDeclaration\nEthics approval and consent to participate\nThis study was approved by the ethics committee and institutional review\nboard of the Third Xiangya Hospital of Central South University\n(No.2020S530). Written informed consent was obtained from all human\nsubjects.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors report no conflicts of interests.\nAuthor details\n1Department of Obstetrics and Gynecology, Loudi Central Hospital of Hunan\nProvince, Loudi, Hunan Province, China. 2Department of Gynecology, The\nThird Xiangya Hospital, Central South University, NO.138 Tongzipo Road,\nYuelu District, Changsha 410013, Hunan, China.\nReceived: 24 December 2020 Accepted: 23 February 2021\nReferences\n1. Shafrir AL, Farland LV, Shah DK, Harris HR, Kvaskoff M, Zondervan K, Missmer\nSA. Risk for and consequences of endometriosis: a critical epidemiologic\nreview. Best Pract Res Clin Obstet Gynaecol. 2018;51:1 –15.\n2. Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med. 2020;\n382(13):1244–56.\n3. Vercellini P, Vigano P, Somigliana E, Fedele L. Endometriosis: pathogenesis\nand treatment. Nat Rev Endocrinol. 2014;10(5):261 –75.\n4. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Vigano P.\nEndometriosis. Nat Rev Dis Primers. 2018;4(1):9.\n5. Baranov V, Malysheva O, Yarmolinskaya M. Pathogenomics of endometriosis\ndevelopment [J]. Int J Mol Sci. 2018;19(7):1852.\n6. Chen Y, Li H, Cheng HY, Rui-Qiong M, Ye X, Cui H, Hong-Lan Z, Chang XH.\nFibrinogen alpha chain is up-regulated and affects the pathogenesis of\nendometriosis. Reprod BioMed Online. 2019;39(6):893 –904.\n7. Goult BT, Yan J, Schwartz MA. Talin as a mechanosensitive signaling hub. J\nCell Biol. 2018;217(11):3776 –84.\n8. Pulous FE, Carnevale JC, Al-Yafeai Z, Pearson BH, Hamilton JAG, Henry CJ,\nWayne Orr A, Petrich BG. Talin-dependent integrin activation is required for\nendothelial proliferation and postnatal angiogenesis. Angiogenesis. 2020.\nhttps://doi.org/10.1007/s10456-020-09756-4.\n9. Wei X, Sun Y, Wu Y, Zhu J, Gao B, Yan H, Zhao Z, Zhou J, Jing Z.\nDownregulation of Talin-1 expression associates with increased proliferation\nand migration of vascular smooth muscle cells in aortic dissection. BMC\nCardiovasc Disord. 2017;17(1):162.\n10. Chen P, Zheng X, Zhou Y, Xu Y, Zhu L, Qian Y. Talin-1 interaction network\npromotes hepatocellular carcinoma progression. Am J Respir Cell Mol Biol.\n2017;8(8):13003–14.\n11. Kang W, Kim SH, Cho HJ, Jin J, Lee J, Joo KM, Nam DH. Talin1 targeting\npotentiates anti-angiogenic therapy by attenuating invasion and stem-like\nfeatures of Glioblastoma multiforme. Oncotarget. 2015;6(29):27239 –51.\n1 2 . J i nJ K ,T i e nP C ,C h e n gC J ,S o n gJ H ,H u a n gC ,L i nS H ,G a l l i c kG E .\nTalin1 phosphorylatio n activates beta1 integrins: a novel mechanism\nto promote prostate cancer bone metastasis. Oncogene. 2015;34(14):\n1811 –21.\n13. Jiang J, Sun A, Wang Y, Deng Y. Increased expression of Talin1 in the\neutopic and ectopic endometria of women with adenomyosis. Gynecol\nEndocrinol. 2016;32(6):469 –72.\n14. Jiang J, Yu K, Jiang Z, Xue M. IL-37 affects the occurrence and development\nof endometriosis by regulating the biological behavior of endometrial\nstromal cells through multiple signaling pathways. Biol Chem. 2018;399(11):\n1325–37.\n15. Zhan H, Ma J, Ruan F, Bedaiwy MA, Peng B, Wu R, Lin J. Elevated\nphosphatase of regenerating liver 3 (PRL-3) promotes cytoskeleton\nreorganization, cell migration and invasion in endometrial stromal cells\nfrom endometrioma. Hum Reprod. 2016;31(4):723 –33.\n16. Sakamoto S, McCann RO, Dhir R, Kyprianou N. Talin1 promotes tumor\ninvasion and metastasis via focal adhesion signaling and anoikis resistance.\nCancer Res. 2010;70(5):1885 –95.\n17. Nieves B, Jones CW, Ward R, Ohta Y, Reverte CG, LaFlamme SE. The NPIY\nmotif in the integrin beta1 tail dictates the requirement for Talin-1 in\noutside-in signaling. J Cell Sci. 2010;123:1216 –26.\n18. Manso AM, Okada H, Sakamoto FM, Moreno E, Monkley SJ, Li R, Critchley\nDR, Ross RS. Loss of mouse cardiomyocyte Talin-1 and Talin-2 leads to beta-\n1 integrin reduction, costameric instability, and dilated cardiomyopathy. Nat\nMethods. 2017;114(30):6250–9.\n19. Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda JM, Ginsberg\nMH, Calderwood DA. Talin binding to integrin beta tails: a final common\nstep in integrin activation. Science. 2003;302(5642):103 –6.\n20. Becam IE, Tanentzapf G, Lepesant JA, Brown NH, Huynh JR. Integrin-\nindependent repression of cadherin transcription by Talin during axis\nformation in drosophila. Nat Cell Biol. 2005;7(5):510 –6.\n21. Vafaei S, Saeednejad Zanjani L, Habibi Shams Z, Naseri M, Fattahi F,\nGheytanchi E, Alemrajabi M, Ebrahimi M, Madjd Z. Low expression of Talin1\nis associated with advanced pathological features in colorectal cancer\npatients. Sci Rep. 2020;10(1):17786.\n22. Kanamori H, Kawakami T, Effendi K, Yamazaki K, Mori T, Ebinuma H, Masugi\nY, Du W, Nagasaka K, Ogiwara A, Kyono Y, Tanabe M, Saito H, Hibi T,\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 9 of 10\n\nSakamoto M. Identification by differential tissue proteome analysis of Talin-1\nas a novel molecular marker of progression of hepatocellular carcinoma.\nOncology. 2011;80:406 –15.\n23. Lai MT, Hua CH, Tsai MH, Wan L, Lin YJ, Chen CM, Chiu IW, Chan C, Tsai FJ,\nJinn-Chyuan SJ. Talin-1 overexpression defines high risk for aggressive oral\nsquamous cell carcinoma and promotes cancer metastasis. J Pathol. 2011;\n224(3):367–76.\n24. Delbandi AA, Mahmoudi M, Shervin A, Akbari E, Jeddi-Tehrani M, Sankian M,\nKazemnejad S, Zarnani AH. Eutopic and ectopic stromal cells from patients\nwith endometriosis exhibit differential invasive, adhesive, and proliferative\nbehavior. Fertil Steril. 2013;100:761 –9.\n25. Giancotti FG, Ruoslahti E. Integrin signaling. Science. 1999;285(5430):\n1028–32.\n26. Chapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms,\ndiagnosis and management of endometriosis. Nat Rev Endocrinol. 2019;\n15(11):666–82.\n27. Choi HJ, Park MJ, Kim BS, Choi HJ, Joo B, Lee KS, Choi JH, Chung TW,\nHa KT. Transforming growth factor β1e n h a n c e sa d h e s i o no f\nendometrial cells to mesothelium by regulating integrin expression.\nBMB Rep. 2017;50(8):429 –34.\n28. Ota H, Tanaka T. Integrin adhesion molecules in the endometrial glandular\nepithelium in patients with endometriosis or adenomyosis. J Obstet\nGynaecol Res. 1997;23(5):485 –91.\n29. Samartzis EP, Fink D, Stucki M, Imesch P. Doxycycline reduces MMP-2\nactivity and inhibits invasion of 12Z epithelial endometriotic cells as well as\nMMP-2 and -9 activity in primary endometriotic stromal cells in vitro.\nReprod Biol Endocrinol. 2019;17(1):38.\n30. Ahn JH, Choi YS, Choi JH. Leptin promotes human endometriotic cell\nmigration and invasion by up-regulating MMP-2 through the JAK2/STAT3\nsignaling pathway. Mol Hum Reprod. 2015;21(10):792 –802.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in\npublished maps and institutional affiliations.\nTang et al. Reproductive Biology and Endocrinology           (2021) 19:42 Page 10 of 10","source_license":"CC0","license_restricted":false}