{"paper_id":"7f9649b0-e554-4372-8bef-a2ad41a4a88c","body_text":"Laboratory Investigation (2021) 101:760 –774\nhttps://doi.org/10.1038/s41374-021-00544-2\nARTICLE\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial\ncancer cells by targeting the Rac1/NF- κB/MMP2 signal pathway\nXishuang Wang 1 ●\nZengtao Wei 1,2 ●\nZhongyun Tang 3 ●\nChenyue Xue 3 ●\nHuayun Yu 3 ●\nDerui Zhang 3 ●\nYulan Li 1 ●\nXihong Liu 4 ●\nYongyu Shi 1 ●\nLining Zhang 1 ●\nGuoling Chen 5 ●\nHuaiyu Zhou 6 ●\nJianing Wang 1 ●\nXiaoyan Wang 1\nReceived: 3 August 2020 / Revised: 16 January 2021 / Accepted: 21 January 2021 / Published online: 22 March 2021\n© The Author(s), under exclusive licence to United States and Canadian Academy of Pathology 2021\nAbstract\nEndometrial carcinoma is one of the most common malignancies in the female reproductive system. Interleukin-37 (IL-37) is\na newly discovered anti-inflammatory factor belonging to the IL-1 family. IL-37 has five different isoforms, and IL-37b is the\nmost biologically functional subtype. In recent years, the protective roles of IL-37 in different cancers, including lung and\nliver cancers, have been successively reported. IL-37 also plays an important role in some gynecological diseases such as\nendometriosis, adenomyosis, and cervical cancer. However, the role and mechanism of IL-37b, especially the mature form of\nIL-37b, in endometrial carcinoma have not been elucidated. The present study demonstrated that IL-37 protein was\ndownregulated in endometrial carcinoma cells compared with the control endometrium. IL-37b did not affect the proliferation\nand colony-forming ability of endometrial cancer cells. A mature form of IL-37b (IL-37b Δ 1-45) effectively suppressed the\nmigration and invasion of endometrial cancer cells by decreasing the expression of matrix metalloproteinase 2 (MMP2) via\nRac1/NF-κB signal pathway. However, it did not affect epithelial –mesenchymal transition (EMT) or filamentous actin\n(F-actin) depolymerization of endometrial cancer cells. IL-37b Δ 1-45 attenuated tumor metastasis in a peritoneal metastatic\nxenograft model of endometrial cancer. To sum up, these results suggested IL-37b could be involved in the pathogenesis of\nendometrial carcinoma and provide a novel target for the diagnosis and treatment of endometrial carcinoma.\nIntroduction\nEndometrial carcinoma is one of the most common malig-\nnancies in the female reproductive system [ 1, 2]. The vast\nmajority (>90%) of endometrial carcinomas are found in\nwomen aged over 50 years; only very few women suffered\nunder the age of 35 [ 3]. According to pathogenesis and bio-\nlogical behavior characteristics, endometrial carcinomas can\nbe divided into two types (Type I and Type II) [ 4, 5]. Among\nthem, more than 80% of endometrial carcinomas are endo-\nmetrioid adenocarcinomas, which are estrogen-dependent\n(type I endometrial carcinomas) [ 1, 6–9]. Type II endo-\nmetrial carcinomas are estrogen-independent, such as uterine\npapillary serous carcinoma and clear cell carcinomas [ 7].\nThese authors contributed equally: Xishuang Wang, Zengtao Wei\n* Xiaoyan Wang\nwxy990809@163.com\n1 Department of Immunology, School of Basic Medical Sciences,\nCheeloo College of Medicine, Shandong University,\nJinan, Shandong, PR China\n2 Department of Gynecology and Obstetrics, Clinical Medical\nSchool, Cheeloo College of Medicine, Shandong University,\nJinan, Shandong, PR China\n3 Department of Gynecology and Obstetrics, Jinan Central Hospital,\nCheeloo College of Medicine, Shandong University,\nJinan, Shandong, PR China\n4 Department of Pathology, The Fourth People ’s Hospital of Jinan,\nJinan, Shandong, PR China\n5 Department of Ophthalmology, The Second Hospital, Cheeloo\nCollege of Medicine, Shandong University, Jinan, Shandong, PR\nChina\n6 Department of Microbiology, School of Basic Medical Sciences,\nCheeloo College of Medicine, Shandong University,\nJinan, Shandong, PR China\nSupplementary information The online version contains\nsupplementary material available at https://doi.org/10.1038/s41374-\n021-00544-2.\n1234567890();,:\n1234567890();,:\n\nNowadays, the etiology of endometrial carcinoma is unclear,\nand its risk factors may be relevant to fat, diabetes, hyper-\ntension, and menstrual disorder, etc. With the development\nof modern treatment strategies, the survival rates of patients\nwith endometrial carcinoma have signi ficant improvements.\nHowever, many patients are not sensitive to traditional treat-\nment methods and easy to be recurrence and metastasis.\nTherefore, understanding the underlying mechanism of tumor\ncell proliferation, migration, and invasion will be of great\nvalue for exploring new targets to treat endometrial carcinoma\npatients.\nInterleukin-37 (IL-37) is a novel anti-in flammatory cyto-\nkine discovered by bioinformatics analysis in 2000 [ 10]. IL-\n37 is one of the IL-1 family member, which includes pro-\ninflammatory factors (IL-1α and IL-1β,I L - 1 8 ,I L - 3 3 ,I L - 3 6α,\nIL-36β,I L - 3 6γ) and anti-inflammatory factors (IL-37 and IL-\n38). IL-37 plays a critical inhibitory role in innate and\nadaptive immunity by directly decreasing pro-in flammatory\ncytokines’ production. The gene encoding IL-37 is located\non human chromosome 2 [ 11, 12]. Interestingly, there is no\nmouse-derived IL-37 gene so far [ 13, 14]. The human IL-37\ngene needs to undergo a process of variable splicing [ 15],\nthus forming different subtypes: IL-37a (21.55kD), IL-37b\n(24.13kD), IL-37c (19.61kD), IL-37d (21.95kD), IL-37e\n(17.46kD). Among them, IL-37b is the biggest subtype and\ncontains exons 1, 2, 4, 5, and 6. Therefore, it may be the most\nbiologically functional subtype. Two mature forms of IL-\n37b, D20-218 (IL-37b Δ 1-20), and V46-218 (IL-37b Δ 1-45),\nare generated through protease cleavages. Mature D20-218 is\ncleaved in the cytoplasm by activated caspase-1 at residue 20\n(Asp). Another mature form, V46-218 is possibly cleaved by\nneutrophil secreted protease in the supernatants of IL-37\ngene-transfected human embryonic kidney 293 cells. This\nform shows higher biologic activity than the longer (amino\nacids 20 –218) [ 16]. IL-37 expresses in a variety of tissues\nand organs and has evident tissue speci ficity [ 17]. It can be\nfound in the cytoplasm or nucleus of cells, and it is also\nsecreted [18]. In recent years, the protective roles of IL-37 in\ndifferent cancers [ 19], including lung and liver cancer, have\nbeen successively reported [ 20, 21]. IL-37 also plays a vital\nrole in some gynecological diseases such as endometriosis\n[22–26], adenomyosis [ 27], and cervical cancer [ 28]. How-\never, the role and mechanism of IL-37b, especially the\nmature form of IL-37b, in endometrial carcinoma have not\nbeen reported.\nIn the present study, we detected the expression of IL-37\nin the endometrial carcinoma tissues and explored the effect\nand molecular mechanism of IL-37b overexpression or\nknockdown on proliferation, migration, and invasion of\nendometrial cancer cells. The impact of IL-37b on metastasis\nof endometrial cancer cells in nude mice was also studied.\nThe results demonstrated that IL-37 protein expression was\ndecreased in endometrial carcinoma cells compared with the\ncontrol endometrium. A mature form of IL-37b (IL-37b Δ 1-\n45) inhibited the migration, invasion, and metastasis of\nendometrial cancer cells in vitro and in vivo, and the\nmechanism was related to Rac1/NF- κB/MMP2 signal path-\nway, suggesting IL-37b was a new target for treating\nendometrial carcinoma.\nMaterials and methods\nSample collection\nForty paraf fin-embedded specimens and sixteen freshly\nfrozen (stored in the −80 °C) specimens were obtained from\nendometrioid adenocarcinoma patients aged 44 –74 years\nwho underwent primary surgeries in the Department of\nGynecology, Jinan Central Hospital, Shandong University.\nThe patients had not received any hormone therapy,\nradiotherapy, or chemotherapy before surgery. The clinical-\nstage was assessed according to the International Federation\nof Gynecology and Obstetrics (FIGO) system (2009) [ 29].\nTumor differentiation degree, depth of myometrial invasion\n(MI), the expression of estrogen receptor (ER), and pro-\ngesterone receptor (PR) were also evaluated. The essential\ncharacteristics of endometrioid adenocarcinoma patients\nwere listed in Table 1. Forty-six paraf fin-embedded control\nTable 1 Association analysis of IL-37 expression in endometrioid\nadenocarcinoma tissues and clinicopathologic parameters.\nClinical and\npathological Features\nIL-37 expression\nn Negative Weak-moderate P value\nAge\n>55 23 10 13 0.0006***\n≤55 15 10 5\nMyometrial invasion (%)\n>50% 19 8 11 0.0046**\n≤50% 19 12 7\nFIGO stage\nIA-IB 22 12 10 0.8872\nIC-II 17 9 8\nTumor differentiation degree\nWell-moderate\ndifferentiation\n33 18 15 0.0896\nPoor differentiation 7 3 4\nEstrogen receptor\nNegative-weak 10 7 3 0.0137*\nModerate-strong 21 11 10\nProgestin receptor\nNegative-weak 11 8 3 0.0001***\nModerate-strong 20 9 11\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial cancer cells by targeting the. . . 761\n\nendometrial specimens were obtained from surgical patients\nwith non-endometrial diseases and divided into proliferative\n(n = 26) or secretory phases ( n = 20) according to the\npatients’ menstrual history and histopathological examina-\ntion [ 30], and 16 fresh control endometrial specimens were\ncollected and frozen in the −80 °C. The Institutional Ethics\nCommittee of Shandong University approved this study and\nthe collection of all human samples, and all of the patients\ngave their informed consent.\nCell culture\nHuman endometrial cancer cell line (Ishikawa, ISK) was\nkindly gifted by Qilu Hospital, Shandong University, and\nmaintained in high glucose DMEM (Hyclone, Logan City,\nUtah, USA) supplemented with 10% fetal bovine serum\n(Gibco Carlsbad, CA, USA). Human endometrial cancer\ncell lines (HEC-1-A, AN3CA, and RL95-2) were purchased\nfrom the China Center for Type Culture Collection (Wuhan,\nHubei, China). HEC-1-A cells were grown in McCoy ’s5 A\nmedium (Gibco, Carlsbad, CA, USA) with 10% fetal\nbovine serum (Gibco, Carlsbad, CA, USA). AN3CA cells\nwere cultured in MEM containing 1% non-essential amino\nacid and 1% sodium pyruvate supplemented with 10% fetal\nbovine serum (Gibco, Carlsbad, CA, USA). RL95-2 cells\nwere cultured in DMEM/F12 supplemented with 10% fetal\nbovine serum (Gibco, Carlsbad, CA, USA). All of these cell\nlines were routinely cultured at 37 °C in a humidi fied\natmosphere with 5% CO\n2.\nHormones treatment\nIshikawa and HEC-1-A cells were respectively treated with\n0, 0.1, 1, 10, 100, 1000 nM of 17 β-estradiol (E2, E2758-\n250MG, Sigma-Aldrich, St Louis, MO, USA) or 0, 0.01,\n0.1, 1, 10, 20 μM of progesterone (P4, P0130-25G, Sigma-\nAldrich, St Louis, MO, USA). The protein from all treated\ncells was collected to detect the expression of IL-37 using\nwestern blot.\nAntibodies, Plasmids, siRNA, and transient\ntransfection\nThe primary antibody against IL-37 (PA5-28817) was from\nThermo Scienti fic (Waltham, MA, USA). The primary\nantibody against IL-37 (60296-1-Ig) was from proteintech\n(Wuhan, Hubei, China). Primary antibodies against MMP2\n(ab92536), MMP9 (ab76003) and Rac1 (ab33186) were\nfrom Abcam (Cambridge, UK). Primary antibodies against\nFlag (F1804-50UG), Myc (M4439-100UL) were from\nSigma- Aldrich (St Louis, MO, USA). Primary antibodies\nagainst E-Cadherin (3195), N- Cadherin (13116), Vimentin\n(5741), p-PAK1/2/3 (2604), p-I κκα/β (2697), p-I κBα\n(9246), p-AKT (4060), AKT (4691), p-P65 (3033), P65\n(8242), p-SAPK/JNK (4668), SAPK/JNK (9252), p-p44/\n42MAPK (ERK1/2) (4370), p44/42MAPK (ERK1/2)\n(4695), p-p38MAPK (4511), p38MAPK (8690) were from\nCell Signaling Technology (Danvers, MA, USA). The\nprimary antibody against β-actin was from ZSGB-Bio\n(Beijing, China).\nAll plasmid vectors for pcDNA3.1/HisC/IL-37 (Full-\nlength human IL-37b, IL-37b Δ 1-45, and IL-37b Δ 1-20)\nwith a C-terminal 3× flag and PRK5/Rac1 (Rac1- 61L) with\nan N-terminal Myc were kindly gifted by the Team of\nZhang Lining in Department of Immunology, School of\nBasic Medical Sciences, Shandong University. Speci fic\nsiRNAs for IL-37 were as follows: siIL-37-1: 5 ′-GCAU\nUAGCCUCAU CCUUGAUU-3 ′ and 5 ′-UCAAGGAUG\nAGGCUAAUGCUU-3′; siIL-37-2: 5 ′-GAG AACAGGA\nAACACAUUGUU-3′ and 5 ′-CAAUGUGUUUCCUGUU\nCUCUU-3′; siIL-37-3: 5 ′-UCUACUGUGACAAGGAUA\nAUU-3′ and 5 ′-UUAUCCUUGUCAC AGUAGAUU-3 ′;\nnegative control (NC): 5 ′-UUCUCCGAACGUGUCAC\nGUTT-3′ and 5 ′-ACGUGACACGUUCGGAGAATT-3′.\nTransfection with plasmids was performed using Lipo-\nfectamine2000 (Invitrogen, Carlsbad, CA, USA). Trans-\nfection with siRNAs was performed using INTERFERin\n(PolyPlus, Strasbourg, France).\nImmunohistochemistry (IHC)\nThe specimens were embedded in paraf fin and sectioned\n(5 μm) by Servicebio (Wuhan, Hubei, China). Tissue sec-\ntions were deparaf finized in xylene and rehydrated in gra-\nded ethanol routinely. Antigen retrieval was performed with\na high-pressure method. Endogenous peroxidase was\nblocked with 3% H\n2O2 for 10 min at room temperature, and\nnonspecific binding was blocked with 10% goat serum for\n15 min at 37 °C. Rabbit polyclonal antibody against IL-37\n(PA5-28817, 1:300, Thermo Scienti fic) was added to the\nslides separately, and the slides were incubated overnight at\n4 °C in a wet chamber. The next day, the slides were\nbalanced at room temperature for 30 min and incubated with\nHRP-conjugated goat anti-rabbit IgG for 1 h at 37 °C. The\nsections were then stained with 3,3 ′-diaminobenzidine\n(DAB, 1:20, ZSGB-Bio, Beijing, China). Finally, all sec-\ntions were counterstained with hematoxylin, differentiated\nwith 1% acid alcohol, backed to blue with 1% ammonium,\nand covered with glasses.\nThe intensity and extent of positive staining were eval-\nuated by the observers blinded to the clinical information.\nThe sum of staining intensity and positive area percentage\nwas used as the final score of IL-37. A total score of 0\nindicated no expression; a total score of 1 and 2 indicated\n762 X. Wang et al.\n\nweak expression; a total score of 3 and 4 indicated moderate\nexpression; a total score of 5 and 6 indicated strong\nexpression.\nQuantitative real-time PCR (qPCR)\nTotal RNA was extracted using Trizol Reagent (TIANGEN,\nBeijing, China) and reversely transcribed into cDNA with\nReverse Transcription System (Takara, Shiga, Japan).\nQPCR was performed on the LightCycler\n®96 system\n(Roche, Basle, Switzerland) using UltraSYBR Mixture\n(CWBIO, Beijing, China). The levels of gene expression\nwere normalized to GAPDH and analyzed by the 2\n−ΔΔ Ct\nmethod. Each sample was examined in triplicate. The pri-\nmer sequences were listed in Table 2.\nWestern blot\nThe samples were lysed using RIPA lysis buffer (Beyotime,\nBeijing, China) containing protease and phosphatase inhi-\nbitors (Bimake, Houston, TX, USA). After centrifugation,\nthe supernatant was quanti fied using a BCA assay kit\n(Thermo Scientific, Waltham, MA, USA). An equal amount\nof protein was separated using sodium dodecyl sulfate-\npolyacrylamide gel and then transferred onto the poly-\nvinylidene fluoride membranes (Millipore, Billerica, MA,\nUSA). The membranes were blocked with (TBST) con-\ntaining 5% bovine serum albumin (Sigma-Aldrich, St\nLouis, MO, USA) for 2 –3 h, and then respectively incu-\nbated with primary antibodies overnight at 4 °C. The next\nday, the membranes were incubated with HRP-conjugated\nsecondary antibodies (1:5000, Jackson Immuno Research,\nWest Grove, PA, USA) at room temperature for 1 h.\nThe signal was detected by the enhanced chemilumines-\ncence kit (Millipore, Billerica, MA, USA).\nCell counting Kit-8 (CCK-8) assay\nIL-37 overexpressing/silenced cells were seeded at a density\nof 1 × 10\n4 Ishikawa cells or 2 × 10 4 AN3CA cells per well in\n96-well plates. Cell viability was detected using CCK-8\n(Dojindo Laboratories, Japan) at the indicated time points\n(0, 24, 48, and 72 h). The absorbance was determined at\n450 nm wavelength. Triplicate was performed in each\nexperimental group.\nColony formation assay\nIL-37 overexpressing/silenced cells were seeded at a density\nof 1000 Ishikawa cells or 3000 AN3CA cells per well in 6-\nwell plates and cultured at 37 °C for 10 or 18 days, chan-\nging the medium every 3 days. At the end of the incubation,\nthe cells were fixed with methanol and stained with crystal\nviolet (Beyotime, Beijing, China). The number of colonies\ncontaining more than 50 cells was counted. Each sample\nwas performed in triplicate.\nTranswell migration and invasion assay\nFor migration assay, IL-37 overexpressing/silenced cells\nwere suspended in the medium with 0.1% FBS. After\ncounted, the same number of cells (150 μl) were added into\nthe upper chambers with 8 μm pore size (Greiner bio-one,\nFrickenhausen, Germany), and the medium containing 20%\nFBS (650 μl) was added into the lower chamber. For\ninvasion assay, the upper chamber was covered with\nMatrigel (Corning Incorporated, New York, USA) in\nadvance. After incubation for 24 or 48 h, the cells were\nfixed with methanol and then stained with crystal violet.\nThe cells on the upper side were wiped off, and the cells\nthat adherent to the underside of the membrane were\nremained. Five randomly selected fields were counted under\na light microscope at ×100 magni fications using an Olym-\npus DP72 digital camera and DP Controller software\n(Olympus, Tokyo, Japan). Each experiment was performed\nin triplicate.\nImmunofluorescence\nIL-37bΔ 1-45 overexpressing cells were seeded in 24-well\nplates with coverslips for 24 h at 37 °C. The cells on cover-\nslips were fixed for 10 min with 4% paraformaldehyde,\npermeabilized for 10 min with 1% Triton X-100 (T8200,\nsolarbio), and blocked for 1 h with 3% BSA. The cells were\nthen incubated with the phalloidin- Tetramethylrhodamine B\nisothiocyanate (TRITC-conjugated phalloidin) (P1951-1MG,\nTable 2 The primer sequences for qPCR.\nGene name Sequences\nf-hGAPDH 5 ′-AAC GGA TTT GGT CGT ATTGGG-3 ′\nr-hGAPDH 5 ′-CCT GGA AGA TGG TGA TGG GAT-3\nf-hIL-37 5 ′-TTCTTTGCATTAGCCTCATCCTT -3 ′\nr-hIL-37 5 ′-CGTGCTGATTCCTTTTGGGC -3 ′\nf-h E-cadherin 5 ′-TGATTCTGCTGCTCTTGCTG-3′\nr-h E-cadherin 5 ′-CTCTTCTCCGCCTCCTTCTT-3\nf-h N-cadherin 5 ′-CGTGAAGGTTTGCCAGTGT-3′\nr-h N-cadherin 5 ′-CAGCACAAGGATAAGCAGGA-3′\nf-h Twist 5 ′-AGCAAGATTCAGACCCTCAAG-3′\nr-h Twist 5 ′-ATCCTCCAGACCGAGAAGG-3′\nf-hMMP2 5 ′-AACTACAACTTCTTCCCTCGCAA-3′\nr-hMMP2 5 ′-CAAAGGCATCATCCACTGTCTCT-3′\nf-hMMP9 5 ′-CCACCCTTGTGCTCTTCCCTG-3′\nr-hMMP9 5 ′-TCTGCCACCCGAGTGTAACCA-3′\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial cancer cells by targeting the. . . 763\n\nSigma-Aldrich, St Louis, MO, USA) at 37 °C for 1 h in a\ndark place. The nuclei were stained for 5 min with 4 ′,6-dia-\nmidino-2-phenylindole (C1005, Beyotime). Images were\nobserved using a VS120 (Olympus, Tokyo, Japan).\nPAK-PBD pull-down assay\nFlag-IL-37bΔ 1-45 plasmid transfected Ishikawa cells were\nincubated at 37 °C for 24 h. The protein was collected using\nRac1 Activation Assay Biochem Kit (20 Rxns, BK035-S,\nCytoskeleton, Denver, CO, USA). An equal amount of cell\nlysate was incubated with 20 μg of PAK-PBD protein beads\nfor 1 h at 4 °C with rotation. Active GTP-bound Rac1 could\nbe pulled down by PAK-PBD protein beads. The pull-down\nfractions and total protein were analyzed by western blot.\nCo-IP assay\nFlag-IL-37bΔ 1-45 and Myc-Rac1-61L plasmids were co-\ntransfected in Ishikawa cells at 37 °C for 24 h. The protein\nwas extracted with 500 μl IP buffer. After centrifuged,\n400 μl of prepared cell lysate (IP group) was incubated with\n1.5 μl mouse-derived monoclonal antibody (anti-Flag) for\n1 h at 4 °C with rotation. After that, 40 μl re-suspended\nProtein A/G Plus-Agarose (Sc-2003, Santa Cruz, Dallas,\nTX, USA) was added and incubated for 12 –16 h at 4 °C\nwith rotation. The bound fractions were washed three times\nwith 500 μl IP buffer, then 40 μl of 2× Laemmli buffer was\nadded. Eight microliters of 6× Laemmli buffer were added\nto 40 μl of prepared cell lysate (Input group). The proteins\nin the IP group and the Input group were boiled for 5 min in\na metal bath. The IP group and input group were analyzed\nby western blot.\nEndometrial cancer peritoneal metastatic\nxenograft model\nCV146/luciferase/puro lentiviral vectors with IL-37b Δ 1-45\nor NC were constructed by Genechem Company (Shanghai,\nChina). Lentivirus infection was performed according to the\nmanufacturer’s protocol (Ishikawa cell line, MOI = 20; 25×\nHiTransGA). After infection, puromycin (3 μg/ml) was used\nfor screening, qPCR and western blot were performed for\nidentification. The stably expressed cells (ISK-LV-IL-\n37bΔ 1-45 and ISK-LV-NC) were established.\nFour-week-old female BALB/c nude mice were pur-\nchased from Beijing Vital River Laboratories (Beijing,\nChina) and housed under Speci fic pathogen Free conditions.\nThe mice were randomly divided into two groups ( n = 10/\neach group). The peritoneal metastatic xenograft models of\nendometrial cancer were established by intraperitoneal\ninjection of ISK-LV-IL-37b Δ 1-45 cells or ISK-LV-NC\ncells (2 × 10\n6 in 200 μl of normal saline/each mouse).\nThirty-eight days later, all mice were injected intraper-\nitoneally with a substrate D-luciferin (abs42017256, Absin\nBioscience, Shanghai, China). Ten minutes later, the mice\nwere injected intraperitoneally with 0.3% pentobarbital\nsodium solution. Five minutes later, in vivo biolumines-\ncence imaging was performed in the above mice using the\nIn Vivo Imaging System spectrum (PerkinElmer, Santa\nClara, CA, USA). Then the mice were killed, and the organs\n(liver, lung, and kidney) were removed out for imaging.\nAfter that, the part of tumors and the above organs were\nfixed in formalin and embedded in paraf fin, and the rest was\nfrozen at −80 °C for HE staining, IHC staining, and western\nblot. All animal care and experiments were approved by the\nAnimal Ethics Committee of Shandong University and\naccorded with the Guidelines for the Care and Use of\nLaboratory Animals of Shandong University (Jinan, Shan-\ndong, China).\nStatistics analysis\nAll statistical analyses were performed using GraphPad\nPrism 7.0 software (La Jolla, CA, USA). All data were\npresented as means ± standard deviations. A two-tailed\nunpaired student ’s t-test was performed to evaluate the\ntest and control groups ’ statistical signi ficance. The Chi-\nsquare test was used to analyze the results from IHC. After a\none way ANOVA, a Dunnett ’s post-hoc test was used to\ncompare different test groups with one control group. P <\n0.05 was considered a statistically signi ficant difference.\nResults\nThe expression of IL-37 mRNA and protein was\nupregulated in endometrioid adenocarcinoma\ntissues detected by qPCR and western blot\nTo investigate the expression status of IL-37 in endome-\ntrioid adenocarcinomas, we firstly detected the IL-37\nmRNA and protein expression in control endometrium\nand endometrioid adenocarcinoma tissues using qPCR and\nwestern blot. We found that IL-37 mRNA and protein levels\nwere upregulated in endometrioid adenocarcinoma tissues\ncompared with control endometrium ( p < 0.01, Supple-\nmentary Fig. 1A, B).\nThe expression of IL-37 protein was downregulated\nin endometrioid adenocarcinoma cells detected\nby IHC\nTo explore the expression sites and levels of IL-37 protein,\nwe detected the expression of IL-37 in endometrioid ade-\nnocarcinoma tissues and control endometrium using IHC.\n764 X. Wang et al.\n\nThe results showed that IL-37 positive staining was loca-\nlized in the cytoplasm of glandular epithelial cells, while no\nevident positive staining could be observed in endometrial\nstromal cells (Fig. 1A). Statistical analysis results con firmed\nthat the levels of IL-37 protein were signi ficantly reduced in\nendometrioid adenocarcinoma cells compared with the\ncontrol endometrium ( p < 0.0001, Fig. 1B), which sug-\ngested that the occurrence of endometrioid adenocarcino-\nmas might have a relationship with IL-37.\nAmong 40 cases of endometrial carcinoma tissues, we\nfurther analyzed the relationship between IL-37 expression\nlevels and clinicopathological parameters. Table 1 showed\nno signi ficant correlations between IL-37 and tumor dif-\nferentiation degree, FIGO stage ( p > 0.05). However, we\nfound that the expression of IL-37 was signi ficantly related\nto age, myometrial invasion, ER, or PR ( p < 0.05).\nEstrogen and progesterone did not affect the\nexpression of IL-37 protein in endometrial cancer cells\nIt has been reported that the expression of IL-37 could be\nregulated by corticosteroids [31, 32]. To explore whether the\nexpression of IL-37 protein could be affected by ovarian steroid\nhormones, we compared the diff erences in IL-37 expression\nbetween the proliferative and se cretory phases of the control\nendometrium. It was found that the expression of IL-37 protein\nin the proliferative phase ( n = 26) was signi ficantly reduced\ncompared with that in the secretory phase ( n = 2 0 )o ft h e\ncontrol endometrium (p < 0.01, Fig. 1C). However, Ishikawa\nand HEC-1-A cells were treated with different estrogen or\nprogesterone concentrations, and the results showed that the\nexpression of IL-37 was not regulated by estrogen and pro-\ngesterone at protein levels (Fig. 1D, E).\nFig. 1 The expression of IL-37 protein was downregulated in\nendometrioid adenocarcinoma cells detected by IHC. A Repre-\nsentative IHC staining for IL-37 protein in endometrioid adenocarci-\nnoma tissues and control endometrium (Scale bar, 100 μm). (a) The\nproliferative phase of control endometrium; (b) The secretory phase of\ncontrol endometrium; (c) The well differentiation of endometrioid\nadenocarcinoma; (d) The moderate differentiation of endometrioid\nadenocarcinoma; (e) The poor differentiation of endometrioid adeno-\ncarcinoma. B Statistical analysis of IL-37 protein expression detected\nby IHC showed that the levels of IL-37 protein were signi ficantly\nreduced in endometrioid adenocarcinoma cells compared with the\ncontrol endometrium ( p < 0.0001). C In the control endometrium, the\nexpression of IL-37 protein in the proliferative phase was signi ficantly\nreduced compared with that in the secretory phase ( p < 0.01). Pro-\ngesterone (D) and 17β-estradiol (E) did not affect the expression of IL-\n37 protein in Ishikawa and HEC-1-A cells detected by western blot.\nP4, progesterone; E2, 17 β-estradiol. ** p < 0.01; **** p < 0.0001.\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial cancer cells by targeting the. . . 765\n\nIL-37b overexpression or knockdown increased or\ndecreased the expression of IL-37 at mRNA and\nprotein levels\nTo choose the suitable cell lines, we detected the expression\nof IL-37 in different endometrial cancer cell lines at mRNA\nand protein levels. As shown in Fig. 2A, B, Ishikawa cells\nhad a relatively low IL-37 expression, while AN3CA cells\nhad a relatively high expression. Therefore, the over-\nexpression ef ficiency was detected after transfecting IL-\n37bΔ 1-45, IL37bΔ 1-20, and IL-37b full-length plasmids in\nIshikawa cells. The identi fication of IL-37 inference ef fi-\nciency was tested after transfecting three speci fic siRNAs\ntargeting IL-37 in AN3CA cells. The results showed that\nIL-37bΔ 1-45, IL37b Δ 1-20, and IL-37b were expressed at\nmRNA and protein levels in Ishikawa cells (Fig. 2C).\nMoreover, siIL-37-1 and siIL-37-3 could effectively\ndecrease the expression of IL-37 mRNA and protein ( p <\n0.05, Fig. 2D, E).\nIL-37b did not affect the proliferation and colony\nformation ability of endometrial cancer cells\nIL-37 can inhibit the proliferation of human cervical cancer\ncells [28] and hepatocellular carcinoma cells [ 21]. Here, we\nexamined the effects of IL-37b on proliferation and colony\nformation ability after transfecting respectively MOCK, IL-\n37bΔ 1-45, IL-37bΔ 1-20, and IL-37b full-length plasmids in\nIshikawa cells. We also detected the effects of IL-37 speci fic\nsiRNAs on proliferation and colony formation ability in\nAN3CA cells. The results from CCK-8 and colony forma-\ntion assay showed that IL-37b had no impact on the pro-\nliferation and colony formation ability of endometrial cancer\ncells ( p > 0.05, Fig. 3A–D). Cell proliferation is related to\nthe activation of MAPK and PI3K/AKT signal pathways. To\nfurther con firm the effect of IL-37b on cell proliferation in\nendometrial cancer cells, we examined the expression of\nproliferation-related signal molecules (p-ERK1/2, p-P38, p-\nJNK1/2, and p-AKT) after transfecting respectively MOCK,\nFig. 2 IL-37b overexpression or knockdown increased or\ndecreased the expression of IL-37 at mRNA and protein levels. The\nrelative expression of IL-37 mRNA ( A) and IL-37 protein ( B)i n\nhuman endometrial cancer cells (Ishikawa, HEC-1-A, RL95-2,\nAN3CA). The human hepatocellular carcinoma cell (Bel-7402) was\nused as a positive control. C Ishikawa cells transfected with IL-37\noverexpression plasmids (IL-37b Δ 1-45, IL-37b Δ 1-20, and IL-37b\nfull-length) showed higher expression of IL-37 than MOCK group\ndetected by qPCR and western blot. D AN3CA cells transfected\nwith siIL-37-1, siIL-37-2, and siIL-37-3 showed lower expression\nof IL-37 mRNA than NC group detected by qPCR ( p < 0.05).\nE AN3CA cells transfected with siIL-37-1 and siIL-37-3 showed\nlower expression of IL-37 protein than NC group detected by western\nblot ( p < 0.01). NC negative control, ns no signi ficant. * p < 0.05;\n**p < 0.01; *** p < 0.001.\n766 X. Wang et al.\n\nIL-37bΔ 1-45, IL-37b Δ 1-20, and IL-37b full-length plas-\nmids in Ishikawa cells. The results showed that these\nmolecules had no visible changes between the test group and\nthe control group (Fig. 3E).\nIL-37b suppressed the migration and invasion\nability of endometrial cancer cells\nMigration and invasion of endometrial cells are the main\ncharacteristics of endometrial cancer [ 33]. Previous reports\nhave shown that IL-37 plays an inhibitory role in the\nmigration and invasion of some cancer cells, such as A549\ncells [ 20] and SMMC-7721 cells [ 21]. To determine the\nroles of IL-37 in the migration and invasion of endometrial\ncancer cells, we detected the effect of IL-37b overexpression\non the migration or invasion ability in Ishikawa cells using\ntranswell migration assay or matrigel invasion assay. The\nresults showed that IL-37b Δ 1-45 overexpression in Ishi-\nkawa cells resulted in a signi ficant reduction in the number\nof cells passing through the chambers than the MOCK group\n(p < 0.05, Fig. 4A, B, Supplementary Fig. 2A, B). We also\ndetected the effect of IL-37 knockdown on the migration and\ninvasion ability in AN3CA cells using transwell migration\nassay and matrigel invasion assay. As we expected, IL-37\nknockdown in AN3CA cells increased the number of cells\nmigrating or invading the transwell membrane ’ lower sur-\nface ( p < 0.05, Fig. 4C, D). These data indicated that IL-\n37bΔ 1-45 could suppress the migration and invasion ability\nof endometrial cells.\nIL-37b had no clear impact on\nepithelial–mesenchymal transition (EMT) or\nfilamentous actin (F-actin) depolymerization of\nendometrial cancer cells\nIt has been reported that IL-37 suppresses the migration and\ninvasion of gallbladder cancer cells through the inhibition of\nHIF-1α induced EMT [ 34]. To examine IL-37 and EMT ’s\nrelationship, we examined the markers of epithelial cells (E-\ncadherin) and stromal cells markers (N-cadherin, Vimentin)\nand Twist at mRNA and protein levels. The results showed\nthat IL-37b Δ 1-45 overexpression did not affect EMT ( p >\n0.05, Supplementary Fig. 3A, B). Cell migration is criti-\ncal to cancer cell invasion and metastasis and relates to\nFig. 3 IL-37b did not affect the proliferation and colony formation\nability of endometrial cancer cells. IL-37b overexpression in Ishi-\nkawa cells ( A) and IL-37 knockdown in AN3CA cells ( B) did not\naffect the proliferation of endometrial cancer cells detected by CCK8\nassay ( p > 0.05). IL-37b overexpression in Ishikawa cells ( C) and IL-\n37 knockdown in AN3CA cells ( D) did not affect the colony-forming\nability of endometrial cancer cells detected by the colony formation\nassay ( p > 0.05). E The expression of p -ERK1/2, p-P38, p-JNK1/2,\nand p-AKT had no obvious changes after transfecting respectively\nMOCK, IL-37bΔ 1-45, IL-37bΔ 1-20, and IL-37b full-length plasmids.\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial cancer cells by targeting the. . . 767\n\ncytoskeletal remodeling [ 35, 36]. We used immuno-\nfluorescence to examine the effect of IL-37 Δ 1-45 over-\nexpression on F-actin in AN3CA cells. We found that\nF-actin depolymerization was not signi ficantly changed in\nIL-37bΔ 1-45 overexpressed group compared with the\nMOCK group ( p > 0.05, Supplementary Fig. 3C). PAK\n(p21-activated kinases) enables actin filaments to continue\ngrowing [ 37]. We further tested the expression of p-PAK\nusing western blot after transfecting IL-37b Δ 1-45 and\nfound that the expression of p-PAK in Ishikawa cells and\nAN3CA cells had no signi ficant differences between IL-\n37bΔ 1-45 overexpressed group and MOCK group (Sup-\nplementary Fig. 3D).\nIL-37b decreased the expression of MMP2 by Rac1/\nNF-κB signal pathway in endometrial cancer cells\nExtracellular matrix (ECM) degradation contributes to\ncancer cell invasion [ 38]. MMP2 and MMP9 are the key\nmolecules that degrade the basement membrane [ 39].\nTherefore, qPCR and western blot were performed to detect\nthe effect of IL-37 on the expression of MMP2 and MMP9\nin endometrial cancer cells. The qPCR results showed that\nIL-37b overexpression in Ishikawa cells did not affect\nMMP2 and MMP9 mRNA ( p > 0.05, Fig. 5A). However,\nwestern blot results showed that IL-37b overexpression in\nIshikawa cells resulted in a signi ficant reduction of MMP2\nprotein ( p < 0.05), the expression of MMP9 protein had no\nobvious change ( p > 0.05, Fig. 5B). Inversely, IL-37\nknockdown in AN3CA cells increased the expression of\nMMP2 protein ( p < 0.05) but did not affect the level of\nMMP9 protein ( p > 0.05, Fig. 5C).\nThe NF- κB signal pathway is closely related to the\nexpression of MMP2. Previous reports showed that IL-37\ncould participate in the above signal pathway [ 40, 41]. To\nfurther explore the mechanism of IL-37b affecting the\nexpression of MMP2 protein, we detected the expression of\nrelative molecules (p-P65, p-I κBα,p - Iκκα/β) in the NF- κB\nsignal pathway after transfecting IL-37b plasmids in Ishi-\nkawa cells, and we found that IL-37b only reduced the\nFig. 4 IL-37b suppressed the migration and invasion ability of\nendometrial cancer cells. IL-37bΔ 1-45 overexpression in Ishikawa\ncells resulted in a signi ficant reduction in the number of cells migrating\n(A) or invading ( B) the lower surface of chambers compared with the\nMOCK group ( p < 0.05). IL-37 knockdown in AN3CA cells resulted\nin a signi ficant increase in the number of cells migrating ( C) and\ninvading (D) the lower surface of the chamber compared with the NC\ngroup ( p < 0.05). * p < 0.05; ** p < 0.01; **** p < 0.0001.\n768 X. Wang et al.\n\nexpression of p-P65 ( p < 0.05, Fig. 5D). On the contrary,\nIL-37 silence could upregulate the expression of p-P65 ( p <\n0.05, Fig. 5E).\nIt has been reported that intracellular mature IL-37 sup-\npresses tumor metastasis via inhibiting Rac1 activation [ 42].\nHere, we examined the effect of IL-37b Δ 1-45 on the acti-\nvation of Rac1 using PAK-PBD pull-down assay and found\nthat IL-37b Δ 1-45 overexpression inhibited the expression\nof GTP-Rac1 in Ishikawa cells (Fig. 5F). Furthermore,\nthe interaction between IL-37b Δ 1-45 and Rac1-61L was\nconfirmed using Co-IP after co-transfecting Flag-IL-37b Δ 1-\n45 and Myc-Rac1-61L plasmids in Ishikawa cells. The\nresult showed that IL-37b Δ 1-45 could bind to Rac1-61L\n(Fig. 5G).\nIL-37b suppressed the migration and invasion\nability of endometrial cancer cells by the Rac1/NF-\nκB/MMP2 signal pathway\nIL-37bΔ 1-45 is the most ef ficient in inhibiting the migration\nand invasion of endometrial cancer cells. Therefore, we\nconstructed IL-37b Δ 1-45-stable expressed cell line and\ncontrol cell line by infecting IL-37 Δ 1-45 lentivirus or NC\nlentivirus into Ishikawa cells. The qPCR and western blot\nresults con firmed that IL-37b Δ 1-45-stable expressed cell\nline and control cell line were successfully constructed, and\nstable IL-37bΔ 1-45 expression reduced the levels of MMP2\nprotein (Fig. 6A). Moreover, the migration and invasion\nability of IL-37b Δ 1-45-stable expressed cell line were\nFig. 5 IL-37b decreased the expression of MMP2 by Rac1/NF- κB\nsignal pathway in endometrial cancer cells. A IL-37b over-\nexpression in Ishikawa cells did not affect the MMP2 and MMP9\nmRNA levels detected by qPCR ( p > 0.05). B IL-37b overexpression\nin Ishikawa cells downregulated the levels of MMP2 protein detected\nby western blot ( p < 0.05). However, the expression of MMP9 protein\nhad no signi ficant change ( p > 0.05). C IL-37 knockdown in AN3CA\ncells increased the expression of MMP2 protein detected by western\nblot ( p < 0.05). However, the expression of MMP9 protein had no\nsignificant change ( p > 0.05). D IL-37b overexpression decreased the\nexpression of p-P65 detected by western blot ( p < 0.05), but the\nexpression of p-I κκα/β and p-IκBα had no signi ficant change. E IL-37\nknockdown increased the expression of p-P65 detected by western blot\n(p < 0.05). F IL-37bΔ 1-45 overexpression in Ishikawa cells inhibited\nRac1 activation detected by western blot. G IL-37bΔ 1-45 binds to\nRac1-61L after Myc-tagged Rac1-61L and 3×Flag-tagged IL-37b Δ 1-\n45 plasmids were co-transfected into Ishikawa cells. The immuno-\nprecipitation was performed with anti-Flag antibody, and the pre-\ncipitates and lysates were analyzed by immunoblotting with antibodies\nfor Myc and Flag. * p < 0.05; ** p < 0.01.\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial cancer cells by targeting the. . . 769\n\ndecreased compared with the control cell line ( p < 0.05,\nFig. 6B, C).\nBesides, Rac1/NF-κB/MMP2 signal pathway was further\nidentified by transfecting Myc-Rac1-61L plasmid in IL-\n37bΔ 1-45-stable expressed cell line. The result showed that\nthe expression of p-P65 and MMP2 were upregulated after\ntransfecting Rac1-61L (Fig. 6D), and the migration and\ninvasion ability of IL-37b Δ 1-45-stable expressed cell line\nwere also increased ( p < 0.05, Fig. 6E, F).\nIL-37b attenuated tumor metastasis in a peritoneal\nmetastatic xenograft model of endometrial cancer\nTo provide in vivo evidence that IL-37 suppressed endo-\nmetrial cancer cells ’ metastasis, we established a peritoneal\nmetastatic xenograft model of endometrial cancer in female\nBALB/c nude mice using an intraperitoneal injection of IL-\n37b Δ 1-45-stable expressed endometrial cancer Ishikawa\ncells and the parental endometrial cancer Ishikawa cells.\nThirty-eight days later, the results from an in vivo imaging\ntechnique showed that the LV-IL-37b Δ 1-45 group had a\nsignificantly reduced tumor metastasis compared with the\nLV-NC group (Fig. 7A, B). Image results of liver, kidney,\nand lung con firmed that IL-37b Δ 1-45 could substantially\ndecrease liver metastasis (Fig. 7C). However, the weight of\nthe mice was monitored every week, and the results showed\nno significant differences between the two groups (Fig. 8A).\nThe results from an anatomic diagram in the abdomen and\nHE staining further con firmed that the peritoneal metastatic\nxenograft model of endometrial cancer had been success-\nfully established (Fig. 8B, C). The results of IHC showed\nthat IL-37 levels in the LV-IL-37b Δ 1-45 group were higher\nthan those in the LV-NC group (Fig. 8D). Western blot\nresults con firmed that the expression of MMP2 was\ndownregulated in the LV-IL-37b Δ 1-45 group compared\nwith the LV-NC group (Fig. 8E).\nFig. 6 IL-37b suppressed the migration and invasion ability of\nendometrial cancer cells by the Rac1/NF- κB/MMP2 signal path-\nway. A Ishikawa cells infected with IL-37b Δ 1-45 lentivirus (LV-IL-\n37bΔ 1-45) showed higher expression of IL-37 than that infected with\nnegative control lentivirus (LV-NC) detected by qPCR and western\nblot ( p < 0.0001). The expression of MMP2 protein was down-\nregulated in Ishikawa cells with IL-37b Δ 1-45 lentivirus detected by\nwestern blot. LV-IL-37b Δ 1-45 decreased signi ficantly the number of\ncells migrating ( B) or invading ( C) the lower surface of chambers\ncompared with the LV-NC group ( p < 0.05). D The inhibitory effect of\nIL-37bΔ 1-45 on MMP2 and p-P65 was reversed after the Rac1-61L\noverexpression plasmid was used to transfect LV-IL-37b Δ 1-45 stably\nexpressed cells (ISK-LV-IL-37b Δ 1-45). Rac1-61L overexpression\nsignificantly increased the number of IL-37b Δ 1-45 stably expressed\ncells migrating ( E) or invading ( F) the lower surface of chambers\ncompared with the MOCK group (PRK5 control plasmid) ( p < 0.05).\nLV Lentivirus. * p < 0.05; ** p < 0.01; *** p < 0.001.\n770 X. Wang et al.\n\nDiscussion\nIL-37 is a newly discovered immune negative regulator. It\nhas anti-in flammatory properties in immune responses\nthrough the downregulation of pro-in flammatory molecules\n[43] and plays an important role in tumorigenesis [ 19]. The\npresent study is the first illustration of the functions of IL-37\nin endometrial carcinoma. Our results con firmed the\nmetastasis of endometrial carcinoma was inhibited by\nheightening the expression of IL-37 in vitro and in vivo.\nUnderstanding mechanisms by which IL-37 inhibits endo-\nmetrial carcinoma ’s metastasis is likely to reveal speci fic\nand effective targets for endometrial carcinoma treatment.\nIn the present study, qPCR and western blot results\nshowed that IL-37 mRNA and protein were upregulated in\nendometrioid adenocarcinoma tissues compared with control\nendometrium. However, IHC results confirmed that the levels\nof IL-37 protein were signi ficantly lower in endometrioid\nadenocarcinoma cells than those in the control endometrium.\nOsborne et al. [ 44] reported that IL-37 mRNA was upregu-\nlated in lymphocytes (T, B, and natural killer cells) of the\nmelanoma patients’ blood, suggesting IL-37 could be highly\nexpressed in in flammatory cells. The inconsistency between\nthe results of qPCR, western blot, and IHC may be the reason\nthat qPCR and western blot could not exclude the expression\nof IL-37 in in flammatory cells. The results from IHC were\nmeaningful because IHC could be used to determine the\nexpression of IL-37 protein in endometrioid adenocarcinoma\ncells. The decreased expression of IL-37 protein in endo-\nmetrioid adenocarcinoma cells suggested IL-37 could play\ninhibitory roles in endometrial cancer. Besides, the relation-\nship between decreased IL-37 protein expression and clin-\nicopathological parameters showed that the expression of IL-\n37 was signi ficantly related to age, myometrial invasion,\nestrogen, or progestin receptor. Yan et al. [ 45]r e p o r t e dt h a t\nthe downregulation of IL-37 was significantly correlated with\ncancer stage, nodal involvement, invasion depth, distant\nmetastasis, differentiation, and it was also shown to be an\nFig. 7 IL-37b attenuated tumor metastasis in the peritoneal\nmetastatic xenograft model of endometrial cancer. A , B In vivo\nimaging technique showed that the LV-IL-37b Δ 1-45 group had a\nsignificantly reduced tumor metastasis compared with the LV-NC\ngroup. The bioluminescence was quanti fied by determining the total\nflux (photons/sec; p/s) in each ROI. Data acquired from supine ( A) and\nprone ( B) views were shown. Data were represented as mean ± SD.\nn = 10 for each group ( p < 0.05). C Image results of liver, kidney, and\nlung con firmed that IL-37b Δ 1-45 could signi ficantly decrease liver\nmetastasis. The bioluminescence quanti fication represented that IL-\n37bΔ 1-45 inhibited liver metastasis ( p < 0.05). * p < 0.05; ** p < 0.01.\nIL-37bΔ1-45 suppresses the migration and invasion of endometrial cancer cells by targeting the. . . 771\n\nindependent prognostic indicator for patients with colon\ncancer. The true clinical signi ficance of IL-37 in endometrial\ncancer needs further exploration.\nEndometrial cancer, one of the most common gyneco-\nlogic malignancies, is a hormonally regulated disease [ 46].\nHere, we found that the expression of IL-37 was different\nbetween the proliferative and secretory phases of the control\nendometrium. The previous study reports that progesterone\nand estradiol exert an inhibitory effect on anti-in flammatory\ncytokine (IL-10) production [ 47]. Like IL-10, IL-37 is also\na kind of anti-in flammatory cytokine [ 48]. It is identi fied\nthat IL-37 has some relationship with endocrine hormones\n[31, 32, 49]. The above results suggest that the expression\nof IL-37 may be regulated by ovarian hormones (estradiol\nand progesterone) in endometrial cancer cells. However, our\nresults suggested that IL-37 was not regulated by estradiol\nand progesterone. There may be other reasons why there are\ndifferences in IL-37 expression between the control endo-\nmetrium’s proliferative and secretory phases.\nIL-37 has five subtypes; among them, IL-37b may be the\nmost biologically functional subtype [ 16]. In the study, we\nfound that the mature form of IL-37b (IL-37b Δ 1-45) had\nsignificant inhibitory effects on the migration and invasion\nof endometrial carcinoma cells in vitro and effectively\ninhibited the metastasis of endometrial cancer cells in tumor-\nbearing nude mice. However, IL-37 did not affect the growth\nof endometrial carcinoma cells. Similarly, the expression of\nsignal molecules in cell proliferation-related MAPK [ 25, 50]\nand PI3K/AKT [ 41] signal pathways had no obvious chan-\nges. Regretfully, only one cell line (Ishikawa cell) was used\nfor the overexpression functional study, and only one cell\nline (AN3CA cell) was used for the knockdown study.\nTherefore, there are a few limitations. More endometrial\ncarcinoma cells need to be studied in the future.\nTumor cell migration and invasion are related to EMT,\ncell motility, and degradation of basement membranes. To\ninvestigate the mechanism of IL-37b Δ 1-45 affecting the\nmigration and invasion of endometrial cancer cells, we\ndetected the expression of EMT-related markers [ 34, 51],\ncell motility-related F-actin [ 35], and basement membrane\ndegradation-related MMPs [ 25]. In our study, IL-37 did not\naffect EMT and F-actin in endometrial cancer cells. The\nFig. 8 IL-37b inhibits tumor metastasis in vivo by the degradation\nof the basement membrane. A The weight curves of female BALB/c\nnude mice were shown after intraperitoneal injection with the stably\nexpressed cells (ISK-LV-NC and ISK-LV-IL-37b Δ 1-45), and the IL-\n37bΔ 1-45 had no relationship with mice weight ( p > 0.05). B The\nanatomical images of female BALB/c nude mice were represented\nafter intraperitoneal injection with the stably expressed cells (ISK-LV-\nNC and ISK-LV-IL-37b Δ 1-45). C HE staining results con firmed the\nperitoneal metastatic xenograft model of endometrial cancer had been\nsuccessfully established (Scale, 100 μm). D IL-37 levels in the LV-IL-\n37bΔ 1-45 group were higher than those in the LV-NC group detected\nby IHC. E The expression of MMP2 was downregulated in the LV-IL-\n37bΔ 1-45 group compared with the LV-NC group detected by western\nblot. * p < 0.05; ** p < 0.01.\n772 X. Wang et al.\n\ndiscrepancy between our study and others may be because\nthe function of IL-37 possesses tissue and cell speci ficity.\nDegradation of basement membranes is crucial for tumor\ncell invasion and metastasis [ 21, 52]. MMP-2 and -9, also\nknown as the gelatinases, have been long recognized as\nmajor contributors to the ECM ’s degradation during tumor\ninvasion [39]. Here, we detected the effect of IL-37b on the\nexpression of MMP2 and MMP9 in endometrial cancer\ncells. The results showed that the expression of MMP2 was\ndownregulated in endometrial cancer cells after transfecting\nIL-37bΔ 1-45 plasmid, and IL-37 knockdown could upre-\ngulate the levels of MMP2.\nMMP2 is implicated in tumor metastasis and primary\ntumor growth; therefore, targeting MMP2 appears to offer\nhighly speci fic means to inhibit basement membranes ’\ndegradation. In our study, the MMP2-related NF- κBs i g n a l\npathway [53]a n dR a c 1[54] were detected. The expression of\nthe MMPs could be regulated at the transcriptional level via\nthe NF- κB signal pathway. Rac1, a member of small\nGTPases, is well-characterized in the Rho family [ 55]. The\nGTP-bound form of Rac1 performs a mutual effect on\ndownstream molecules and operates multiple cellular pro-\ncesses [56]. Previous reports suggested that Rac1 participated\nin the NF-κB signal pathway to promote tumor cell migration\nand invasion [ 57]. Li et al. [ 42] reported intracellular mature\nIL-37 directly bound to the CAAX motif in the C-terminal\nhypervariable region of Rac1 and then inhibited Rac1\nmembrane translocation and subsequent downstream signal\npathway. Here, our result showed that IL-37b Δ 1-45 could\ncombine with Rac1-61L and inhibit the activation of Rac1,\nthen downregulate the expression of p-P65 and MMP2.\nRac1-61L overexpression in the IL-37b Δ 1-45 stable\nexpressed cells could reverse the inhibitory effect of IL-37,\nupregulating the levels of p-P65 and MMP2 and promoting\nthe migration and invasion of endometrial cancer cells.\nIn conclusion, our research showed that IL-37 protein\nwas signi ficantly reduced in endometrioid adenocarcinoma\ncells, enhanced IL-37b Δ 1-45 could inhibit the migration\nand invasion of endometrial cancer cells in vitro and in vivo\nby targeting Rac1/NF- κB/MMP2 signal pathway, suggest-\ning intracellular mature IL-37b is a novel therapeutic target\nin endometrial cancer.\nCompliance with ethical standards\nConflict of interest The authors declare no competing interests.\nPublisher’s note Springer Nature remains neutral with regard to\njurisdictional claims in published maps and institutional af filiations.\nReferences\n1. 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