{"paper_id":"59fb1d8a-3428-4098-8c3f-a21850dda1e1","body_text":"Article Discovery Medicine 2024; 36(191): 2376–2385\nhttps://doi.org/10.24976/Discov.Med.202436191.219\nCopyright: © 2024 The Author(s). Published by Discovery Medicine. This is an open access article under the CC BY 4.0 license .\nNote: Discovery Medicine stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nNuclear Receptor Subfamily 4 Group A Member 3: A\nPotential Marker of Endometriosis\nY unxiu Huang1,2, Yichuan Guo1,2, Xiaoyan Luo 1,2,3,*\n1Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, 610017 Chengdu, Sichuan, China\n2Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, Sichuan University, 610017 Chengdu, Sichuan,\nChina\n3Reproductive Endocrinology and Regulation Laboratory, West China Second University Hospital, Sichuan University, 610017 Chengdu, Sichuan,\nChina\n*Correspondence: luoxiaoyan_lxyan@163.com (Xiaoyan Luo)\nPublished: 20 December 2024\nBackground: Nuclear receptor subfamily 4 group A member 3 ( NR4A3) is lowly expressed in ectopic endometrium and can be\ndegraded by ubiquitination in vascular endothelial cells. Murine double minute 2 ( MDM2) is predicted to be the ubiquitin ligase\nof NR4A3. Hence, we investigated the effects of NR4A3 and MDM2 on endometriosis and clarified corresponding regulatory\nmechanisms.\nMethods: The ubiquitin ligase of NR4A3 was predicted using bioinformatics and validated by immunoprecipitation. The effects of\nNR4A3 and MDM2 on the migration and proliferation of human endometrial stromal cells (hESCs) were examined by Transwell\nassay and 5-ethynyl-2 ′-deoxyuridine (EdU) staining. NR4A3 and MDM2 expressions were detected by real-time quantitative\npolymerase chain reaction (RT-qPCR) and Western blot. An endometriosis model was constructed in Sprague-Dawley rats,\nfollowed by body weight analysis, ultrasonic imaging of ectopic cysts, and Western blot.\nResults: Overexpression of NR4A3 inhibited, but siNR4A3 boosted hESC migration and proliferation. MDM2 promoted NR4A3\nubiquitination and degradation. MDM2 overexpression enhanced hESC migration and proliferation and partially reversed the\ninhibitory effect of NR4A3 overexpression. Overexpression of NR4A3 reduced ectopic cysts in endometriotic rats, which was\noffset by MDM2 overexpression.\nConclusion: NR4A3, which is promoted to ubiquitination and degradation by MDM2, inhibits the proliferation and migration of\nhESCs in vitro, and reduces the growth of ectopic endometrial cysts in vivo, thereby inhibiting the progression of endometriosis.\nKeywords: endometriosis; NR4A3; MDM2; ubiquitination\nIntroduction\nEndometriosis is an estrogen-dependent chronic gyne-\ncological disease [ 1], with the presence and growth of en-\ndometrial glands and stroma outside the uterine cavity as the\nhallmark [2]. Endometriosis has a variety of clinical man-\nifestations such as dysmenorrhea, pelvic mass, infertility,\nand cancer, which seriously affect the quality of life of pa-\ntients [ 3]. Currently, the diagnosis of endometriosis relies\non laparoscopic surgical evaluation as the gold standard and\nlacks non-invasive markers [4,5]. Therefore, there is an ur-\ngent need to explore the pathogenesis of endometriosis and\nfind a non-invasive biomarker for diagnosis and treatment.\nNuclear receptors (NRs) are a class of eukaryotic tran-\nscription factors widely distributed in cells [ 6]. By regu-\nlating the transcription and expressions of numerous key\ngenes, NRs participate in various pathophysiological pro-\ncesses such as inflammation and immune response in the\nhuman body [ 6]. Nuclear receptor subfamily 4 group A\n(NR4A) is a special class of NRs whose endogenous ligands\nhave not yet been found [7]. The NR4A protein family con-\nsists of three well-characterized members: nuclear receptor\nsubfamily 4 group A member 1 (NR4A1), nuclear receptor\nsubfamily 4 group A member 2 (NR4A2), and nuclear re-\nceptor subfamily 4 group A member 3 (NR4A3) [ 8]. The\nexisting study has shown that the expression of NR4A1 is\nreduced in the ectopic endometrium of patients with adeno-\nmyosis, leading to impaired endometrial function through\ninteraction with Forkhead Box O1 (FOXO1A) and reduced\nfemale fertility [9]. Moreover, a study also pointed out that\nNR4A3 in ectopic endometrium of adenomyosis patients\nshares the same expression trend with NR4A1 [9]. How-\never, the effect of NR4A3 on endometriosis and the associ-\nated regulatory mechanism, which are not well understood,\nare the focus of this study.\nIt is worth noting that NR4A3 regulates endothelial\ncell injury, and can be degraded by ubiquitination in vas-\ncular endothelial cells [ 10]. Therefore, we speculate that\nthe function of NR4A3 is also related to its ubiquitination\nlevel in endometrial cells. Through the UbiBrowser web-\n\n2377\nsite, it was found that Murine double minute 2 (MDM2) may\nbe the ubiquitin ligase that mediates the ubiquitination of\nNR4A3. MDM2 has been reported to be highly expressed\nin endometriosis and to further promote the progression of\nendometriosis through ubiquitination [ 11–13]. Based on\nthis, we set out to investigate the effect of NR4A3 on en-\ndometriosis through in vitro and in vivo experiments and to\nfurther explore whether MDM2 mediates the ubiquitination\nof NR4A3 in endometriosis.\nMaterials and Methods\nAnimals\nSix-week-old female non-pregnant Sprague-Dawley\nrats (180–200 g, n = 32) were purchased from Hangzhou\nMedical College (China). All rats were housed in the\nlaboratory with an automatic light control system of 12-h\nlight/dark cycle, room temperature of 22 ± 0.5 °C, and rel-\native humidity of 40–60%.\nCells, Culture and Transfection\nHuman endometrial stromal cells (hESCs) (CP-H208)\nand corresponding medium (CM-H208) were obtained\nfrom Procell company (Wuhan, China), and hESCs were\nmaintained in the culture medium at 37 °C with hu-\nmidified air and 5% CO 2. NR4A3 or MDM2 over-\nexpression plasmids were constructed by insertion of\nNR4A3 or MDM2 coding sequences (CDS, as Supple-\nmentary Materials ) into the pEX-3 vector (C05003,\nGenePharma, Shanghai, China). The pEX-3 vec-\ntor without insert was used as the negative control\n(NC). Small interfering RNAs (siRNAs) of NR4A3 or\nMDM2 (siNR4A3, 5 ′-GCAGAGCCTGAACCTTGA TA T-\n3′; siMDM2, 5′-CTCTCGACTCAGAAGA TTA TA-3′) and\nsiNC (5′-CAACAAGA TGAAGAGCACCAA-3′) were ob-\ntained from GenePharma (Shanghai, China). The siRNAs\nor overexpression plasmids were transfected into hESCs as\nper instructions of Lipofectamine 2000 (11668500, Invitro-\ngen, Carlsbad, CA, USA). The hESCs were routinely tested\nfor mycoplasma contamination and were confirmed to be\nmycoplasma-free.\nImmunofluorescence Assay\nAs for cell identification, hESCs (1 × 106) were fixed\nwith 4% paraformaldehyde (441244, Sigma-Aldrich, St.\nLouis, MO, USA) for 15 min and permeabilized with 0.1%\nTriton X-100 (93443, Sigma-Aldrich, USA) for 10 min at\nroom temperature. After washing with phosphate-buffered\nsaline, the cells were incubated with 5% bovine serum al-\nbumin (V900933, Sigma-Aldrich, USA) at 37 °C for 30\nminutes. The hESCs were then incubated overnight at\n4 °C with Vimentin primary antibody (ab20346, Abcam,\nCambridge, UK), followed by a 30-min incubation at 4 °C\nwith a fluorescence-labeled secondary antibody (ab150115,\nAbcam, Cambridge, UK). Cell nuclei were stained with\n4’,6-Diamidino-2’-phenylindole (DAPI, D9542, Sigma-\nAldrich, USA) for 10 minutes in the dark. Observations\nwere made using a confocal microscope (FV3000, Olym-\npus, Tokyo, Japan) at ×200 magnification.\nReal-Time Quantitative Polymerase Chain Reaction\n(RT-qPCR)\nA total RNA extraction kit (R1200, Solarbio, Bei-\njing, China) was employed for total RNA collection.\nFirst-strand cDNA was synthesized using a first-strand\ncDNA synthesis kit (K1612, Thermo Fisher Scien-\ntific, Waltham, MA, USA). For the analysis of mRNA\nexpression, PCR was conducted on the StepOnePlus\nReal-Time PCR system (4376600, Applied Biosystems,\nFoster City, CA, USA) with SYBR Green (HY -K0501A,\nMedChemExpress, Shanghai, China). Glyceraldehyde-3-\nphosphate dehydrogenase ( GAPDH) served as an internal\nreference. The PCR primer information was as follows\n(5′-3′): NR4A3: TGCGTCCAAGCCCAA TA TAGC (For-\nward), GGTGTA TTCCGAGCTGTA TGTCT (Reverse);\nGAPDH: GGAGCGAGA TCCCTCCAAAA T (Forward),\nGGCTGTTGTCA TACTTCTCA TGG (Reverse).\nWestern Blot\nTotal proteins were harvested from rat ectopic cysts\nand hESCs with the help of radioimmunoprecipitation as-\nsay (RIPA) lysis buffer (R0278, Sigma-Aldrich, USA) and\nquantified using a bicinchoninic acid (BCA) kit (ab102536,\nAbcam, UK). Proteins were separated via sodium dodecyl-\nsulfate polyacrylamide gel electrophoresis (SDS-PAGE)\nand transferred onto polyvinylidene fluoride membranes\n(IPVH08100, Millipore, Billerica, MA, USA) which were\nblocked with 5% nonfat milk. Membranes were then in-\ncubated with diluted primary antibodies at 4 °C overnight\nand then reacted with secondary antibodies for 1 h at room\ntemperature. An Enhanced chemiluminescence (ECL) sub-\nstrate kit (ECL-P-500) was obtained from Shanghai Y anxi\nBiological Technology Co., Ltd. (Shanghai, China) to vi-\nsualize blots. Antibody information is as follows: MDM2\n(#51541, 90 kDa, 1:1000, Cell Signaling Technology,\nBoston, MA, USA); NR4A3 (sc-393902, 68 kDa, 1:1000,\nSanta Cruz Biotechnology, Dallas, TX, USA); GAPDH\n(ab181602, 36 kDa, 1:10,000, Abcam, UK); Goat Anti-\nRabbit Immunoglobulin G Heavy and Light Chains (IgG\nH&L) (horseradish peroxidase (HRP)) (ab205719, 1:5000,\nAbcam, UK); Goat Anti-Mouse IgG (HRP) (ab97240,\n1:5000, Abcam, UK).\nTranswell Assay\nThe migration rate of hESCs was determined by Tran-\nswell assay. After 48-h transfection, hESCs (1 × 105) were\ninoculated in medium without fetal bovine serum (FBS) and\nthen seeded into the upper Transwell chamber (CLS3412,\nSigma-Aldrich, USA), whereas medium with 10% FBS was\nloaded into the lower chamber. After 24 h, migrating cells\n\n2378\nwere fixed using a paraformaldehyde fixator (P885233,\nMacklin, Shanghai, China) and then stained by crystal vi-\nolet (C805209, Macklin, China) for 30 min, followed by\nobservation using a microscope (IXplore Standard, OL YM-\nPUS, Tokyo, Japan) at ×250 magnification.\nBioinformatics Analysis\nThe ubibrowser site (http://ubibrowser.bio-it.cn/) was\nutilized to predict the E3 ubiquitin ligase of NR4A3.\nBriefly, on the homepage, the “substrate” and “H.sapiens”\noptions were selected in the search bar, and then the analysis\nresults can be obtained by entering “ NR4A3” and clicking\n“explore”.\n5-ethynyl-2′-deoxyuridine (EdU) Staining Assay\nThe BeyoClick EdU Cell Proliferation Kit with Alexa\nFluor 594 (C0078L) for cell proliferation analysis was pro-\nvided by Beyotime Company (Shanghai, China). In brief,\nhESCs were seeded into 6-well plates, followed by the ad-\ndition of EdU working solution and incubation for 2 h.\nAfter being fixed and permeabilized, cells were cultivated\nwith the Click reaction solution at room temperature for 30\nmin in the dark. After re-dyeing with 4’,6-Diamidino-2’-\nphenylindole (DAPI) solution (CC1162, G-CLONE, Bei-\njing, China), EdU-positive cells were observed under a\nfluorescence microscope (STELLARIS 5, Leica, Wetzlar,\nGermany).\nCo-Immunoprecipitation (Co-IP)\nThe interaction between MDM2 and NR4A3 in hESCs\nwas determined via Co-IP assay with the help of a Co-IP kit\n(abs955, Absin, Shanghai, China). More specifically, IP\nlysis buffer (87787, Thermo Fisher Scientific, USA) was\nused to prepare hESCs, after which 500 µL cell lysate was\nincubated with 5 µg MDM2 antibody (ab259265, Abcam,\nCambridge, UK), NR4A3 antibody (sc-393902, Santa Cruz\nBiotechnology, USA), or with control IgG (ab205718, Ab-\ncam, UK) at 4 °C overnight. Next, 5 µL Protein A and 5\nµL Protein G were added into the cell lysate, followed by\na 3-h incubation at 4 °C and 1-min centrifugation at 12,000\n×g. At the end, 0.5 mL Wash buffer was used to elute the\nprotein complexes, followed by Western blot analysis.\nUbiquitination Assay\nThe hESCs were transiently transfected with Flag-\nNR4A3, hemagglutinin (HA)-MDM2, and HA-Histidine\n(His)-Ubiquitin (Ub) (3683524, Biovector NTCC, Beijing,\nChina). 48 h after transfection, hESCs were treated with\nor without MG132 (HY -13259, 10 µM, MedChemExpress,\nChina) for 6 h, and then cells were lysed in IP lysis buffer\n(87787, Thermo Fisher Scientific, USA) and incubated with\nanti-Flag-M2 affinity gel (HY -K0217, MedChemExpress,\nChina) or anti-HA magnetic beads (HY -K0201, MedChem-\nExpress, China) overnight at 4 °C, followed by SDS-PAGE,\nand the subsequent steps were consistent with Western blot.\nAntibodies used included polyclonal-Ubiquitin (poly-Ub,\nPA1-187, Invitrogen, USA), Flag (SAB4200071, Sigma-\nAldrich, USA), and HA (H3663, Sigma-Aldrich, USA).\nAnimal Assays\nThirty-two Sprague-Dawley rats were used in the an-\nimal assays, and the establishment of the rat endometriosis\nmodel referred to a previous report [ 4]. SD rats were anes-\nthetized with 3% isoflurane (792632, Sigma-Aldrich, USA)\nusing gas anesthesia machine (R500IP; RWD Life Technol-\nogy Co., Shenzhen, China), and a small incision was made\nin the center of the abdomen. The left uterine horn was ex-\ncised to collect the endometrium which was later divided\nin half and placed on the left and right sides of the abdom-\ninal wall. On the first and tenth days, estradiol benzoate\n(HY -B1192, MedChemExpress, USA) was subcutaneously\ninjected into rats to establish an endometriosis model. Mod-\neling was performed in 26 rats and 24 modeled rats were\nobtained, which was verified by the ultrasonic imaging of\ncysts on the 20th day, with a success rate of 92.3%.\nRats in the Sham group (n = 6) only had a small inci-\nsion in the abdomen without autologous endometrial trans-\nplantation. Immediately after modeling, rats were injected\nwith NR4A3 overexpression vector, MDM2 overexpres-\nsion vector, NC, or an equivalent volume of normal saline\n(S0817, Sigma-Aldrich, USA) at the endometriotic lesions,\nand then fed for 20 days.\nDuring the feeding process, the body weight of the\nrats was measured and recorded every 5 days. On the\n20th day, the rats were anesthetized (2% isoflurane) and\nunderwent high-resolution ultrasound imaging (VisualSon-\nics V evo770, VisualSonics, Toronto, ON, Canada) with the\nhelp of real-time microvisualization Scanhead (center fre-\nquency: 40 MHz; focal depth: 6 mm) [ 14]. After that, rats\nwere euthanized via intraperitoneal administration of 1%\npentobarbital sodium (P010, 150 mg/kg, Sigma-Aldrich,\nUSA), and the ectopic endometrial cysts were removed to\ntake photographs and measure the volume.\nStatistical Analysis\nData were obtained from experiments performed three\ntimes and are presented as the mean ± standard deviation.\nThe multi-group comparison was carried out using a one-\nway and follwed by Tukey post hoc test. All statistical anal-\nyses were conducted using GraphPad 8.0 software (Graph-\nPad Software, San Diego, CA, USA), and p values < 0.05\nwere considered statistically significant.\nResults\nNR4A3 Regulated hESC Migration and Proliferation\nThe isolated cells exhibited prominent Vimentin stain-\ning, confirming their identity as hESCs ( Supplementary\nFig. 1 ). To better understand the function of NR4A3 in en-\ndometriosis, we transfected NR4A3 overexpression plasmid\n\n2379\nFig. 1. Expression and regulation of nuclear receptor subfamily 4 group A member 3 ( NR4A3) in endometriosis. (A,B)\nNR4A3 overexpression plasmids and small interfering RNA (siRNA) were transfected into human endometrial stromal cells (hESCs),\nand transfection efficiency were determined by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, with\nGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference control. (C) The effect of NR4A3 overexpression or silencing on\nthe migration of hESCs was assessed using Transwell assay. (D) 5-ethynyl-2 ′-deoxyuridine (EdU) staining was performed to test cell\nproliferation. ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3. DAPI, 4’,6-Diamidino-2’-phenylindole; NC, negative control.\n\n2380\nFig. 2. Ubiquitination regulation of NR4A3 by MDM2. (A) The ubiquitin ligase of NR4A3 was predicted using the ubibrowser website\n(http://ubibrowser.bio-it.cn/). The capital letters represent different subfamilies of E3 ligases, with “U” for UBOX, “H” for HECT, “R”\nfor RING, and “SO” for SINGLE_other. The thickness of the lines is related to the confidence score, with thicker lines indicating higher\nconfidence scores. (B) The effect of MDM2 on NR4A3 expression was determined by Western blot. (C) The interaction between MDM2\nand NR4A3 was determined by co-immunoprecipitation assay. (D) After cells were treated with or without MG132, ubiquitination\nassays were performed after Flag-NR4A3 and hemagglutinin-Histidine-Ubiquitin (HA-His-Ub) were co-transfected with or without HA-\nMDM2 into hESCs. ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3. MDM4, Murine double minute 4; MDM2, Murine double minute 2; SMURF1,\nSMAD-specific E3 ubiquitin protein ligase 1; SMURF2, SMAD-specific E3 ubiquitin protein ligase 2; BARD1, BRCA1 Associated\nRING Domain 1; RANBP2, RAN Binding Protein 2; STUB1, STIP1 Homology And U-Box Containing Protein 1; ARIH2, Ariadne\nRBR E3 Ubiquitin Protein Ligase 2; UBOX5, U-Box Domain Containing 5; BRCA1, BRCA1 DNA Repair Associated; ITCH, Itchy E3\nUbiquitin Protein Ligase; RBBP6, RB Binding Protein 6, Ubiquitin Ligase; TRIM24, tripartite Motif Containing 24; HECW1, HECT, C2\nAnd WW Domain Containing E3 Ubiquitin Protein Ligase 1; LITAF, Lipopolysaccharide Induced TNF Factor; PIAS2, Protein Inhibitor\nOf Activated STA T2; PIAS3, Protein Inhibitor Of Activated STA T3; RBX1, Ring-Box 1; TOPORS, TOP1 Binding Arginine/Serine Rich\nProtein, E3 Ubiquitin Ligase; RCHY1, Ring Finger And CHY Zinc Finger Domain Containing 1; IgG, Immunoglobulin G; poly-Ub,\npolyclonal-Ubiquitin; IP , immunoprecipitation.\n\n2381\nor siRNA into hESCs to manipulateNR4A3 expression (p <\n0.001, Fig. 1A,B). We observed thatNR4A3 overexpression\nsignificantly inhibited the migration of hESCs, whereas\nsiNR4A3 promoted cell migration ( p < 0.01, Fig. 1C).\nAdditionally, NR4A3 overexpression significantly reduced,\nwhereas siNR4A3 increased the EdU-positive cells ( p <\n0.001, Fig. 1D).\nMDM2 Mediated NR4A3 Ubiquitin Degradation to\nRegulate hESC Migration and Proliferation\nNR4A3 regulates endothelial cell damage and can be\ndegraded by ubiquitination in vascular endothelial cells.\nHere, MDM4, MDM2, and SMAD-specific E3 ubiquitin\nprotein ligase 1 (SMURF1) were found to be possible ubiq-\nuitin ligases of NR4A3 (Fig. 2A). Among them, MDM2 is\nhighly expressed in endometriosis, and further accelerates\nthe progression of endometriosis via ubiquitination [ 11–\n13]. Overexpression of MDM2 increased the MDM2 pro-\ntein level but decreased the NR4A3 protein level, whereas\nsiMDM2 transfection had the opposite effect ( p < 0.01,\nFig. 2B). Importantly, we observed protein interaction be-\ntween MDM2 and NR4A3 (Fig. 2C), and found that MDM2\ngreatly enhanced NR4A3 ubiquitination (Fig. 2D).\nNext, we conducted rescue experiments and found that\nNR4A3 overexpression had no effect on MDM2 expres-\nsion, but reversed the inhibiting role of MDM2 upregula-\ntion on NR4A3 expression ( p < 0.01, Fig. 3A). In addi-\ntion, MDM2 overexpression promoted migration and pro-\nliferation of hESCs, which was offset by overexpression of\nNR4A3 (p < 0.001, Fig. 3B,C).\nMDM2 Partially Reversed the Effects of NR4A3 in a\nRat Model of Endometriosis\nTo further clarify the role of MDM2-NR4A3 interac-\ntion, we used a rat model of endometriosis. No significant\ndifference was found in body weight between model rats\nand NR4A3 and/or MDM2 overexpression vector-injected\nrats compared with the sham-operated rats (Fig. 4A). Ultra-\nsonic imaging showed that there were no ectopic cysts in the\nsham group, but obvious ectopic cysts in the Model group\nand Model+NC group. Overexpression of NR4A3 reduced\nthe cysts caused by the modeling, which was counteracted\nby to overexpression of MDM2 (p < 0.01, Fig. 4B–D).\nAdditionally, we examined the expressions of MDM2 and\nNR4A3 in rat endometrial cyst tissue. As shown in Fig. 4E,\nNR4A3 upregulation did not significantly affect MDM2,\nbut increased NR4A3 expression; whereas MDM2 upreg-\nulation not only significantly promoted MDM2 expression\nbut also reversed NR4A3 upregulation-induced promotion\nof NR4A3 protein level (p < 0.05).\nDiscussion\nEndometriosis is a common benign gynecological dis-\nease, but it has similar biological behaviors to tumors and\nhas the ability to adhere, invade, and metastasize with a high\nrecurrence rate [15]. This study provided new evidence that\nMDM2 mediates the ubiquitination of NR4A3 to affect the\nproliferation and migration of hESCs.\nNR4A3 has been reported to be a master gene involved\nin various physiopathologies [16]. Lee et al. [ 17] suggested\nthat NR4A3 acts as an oncogene in acinar cell carcinoma by\ninteracting with MYB proto-oncogene, transcription factor\n(MYB). Deutsch et al. [ 18] reported that NR4A3 represses\nlymphomagenesis by inducing pro-apoptotic genes. NR4A3\nalso promotes the inflammatory response of osteoarthri-\ntis through the nuclear factor kappa-B (NF- κB) pathway\n[19]. However, NR4A3 suppresses inflammatory responses\nthrough the Janus kinase 2-signal transducer and activator\nof transcription 3 (JAK2-STA T3)/NF-κB pathway in acute\nmyocardial infarction [20]. The paradoxical roles of NR4A3\nin various diseases make us more interested in exploring its\nrole and mechanism in endometriosis. Endometrial stromal\ncells are an important component of endometriosis progres-\nsion [ 21,22]. Studying the migration and proliferation of\nendometrial stromal cells contributes to the diagnosis and\ntreatment of endometriosis at the cellular level. Here, we\nfound that overexpressed NR4A3 inhibited hESC migration\nand proliferation, whereas knockdown of NR4A3 had the\nopposite effect, suggesting that NR4A3 may play a protec-\ntive role in endometriosis progression.\nUbiquitination, as one of the most prevalent post-\ntranslational modifications in the proteome, has also been\nwidely explored in endometriosis [ 23–25]. Wang et al .\n[23] revealed that tripartite motif containing 59 (TRIM59)\nubiquitination degrades protein phosphatase, Mg 2+/Mn2+\ndependent 1A (PPM1A) and activates the transforming\ngrowth factor- β (TGF-β)/Smad pathway to promote en-\ndometriosis progression. SMURF1-mediated ubiquitina-\ntion of SH2 domain-containing phosphatase 1 (SHP-1) ac-\ncelerates the invasion and proliferation of endometrial stro-\nmal cells in endometriosis [ 24]. Wu et al . [ 25] pointed\nout that TRIM65, highly expressed in ectopic endometrial\ntissues, inhibits dual specificity phosphatase 6 (DUSP6)\nthrough ubiquitination and activates the ERK1/2/C-myc\nsignaling pathway to promote the invasion of ectopic\nendometrial stromal cells. Here, we demonstrated that\nMDM2 may mediate NR4A3 degradation through ubiquiti-\nnation, further providing evidence for ubiquitination in en-\ndometriosis.\nMDM2, a vital E3 ligase, can ubiquitinate a variety of\nsubstrates and participates in many cellular physiological\nand pathological processes [ 26–28]. MDM2 interacts with\nimmediate early response 3 (IER3) and promotes its ubiq-\nuitination to reduce apoptosis of cervical cancer cells [ 28].\np53 is a common ubiquitination substrate of MDM2 and\nhas been repeatedly reported to be inhibited by MDM2 via\nMDM2-driven ubiquitination [29–31]. p53 can directly in-\nduce transcription of NR4A3 by binding to the promoter of\nNR4A3 and thus exert a tumor-suppressing effect [ 32]. The\n\n2382\nFig. 3. MDM2 regulated hESC migration and proliferation through NR4A3 ubiquitin degradation. (A) The expressions of MDM2\nand NR4A3 were determined by Western blot. (B) The effects of MDM2 and NR4A3 overexpression on the migration of hESCs were\ndetected by Transwell assay. (C) EdU staining was employed to reveal the impacts of MDM2 and NR4A3 on cell proliferation. ∗p <\n0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3.\n\n2383\nFig. 4. Effects of MDM2 and NR4A3 on endometriosis rat model. (A–E) Six-week-old female non-pregnant Sprague-Dawley rats\nwere used to construct a rat model of endometriosis, with six rats in each group (Sham, Model, Model+NC, Model+NR4A3, and\nModel+NR4A3+MDM2 groups). (A) Changes in rat body weight were recorded. (B–D) Ultrasonic imaging of removed cysts and\nvolume detection on day 20. (E) Expressions of MDM2 and NR4A3 in rat endometrial cyst tissue were determined by Western blot. ∗p\n< 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3.\ninteraction of MDM2, p53, and NR4A3 is intriguing and can\nbe further explored in the future.\nThe role of MDM2 in endometriosis has been reported\nin various ways [ 11–13]. For example, Li et al. [ 12] found\nthrough bioinformatics analysis that MDM2 is one of the\ncentral genes in endometriosis and may mediate the ubiq-\nuitination of p27. Sang et al . [ 11] revealed high expres-\nsion of MDM2 in endometriosis, consistent with our results.\n\n2384\nChen et al. [ 13] demonstrated that silencing MDM2 hinders\nthe development of endometriosis in mice through loss-of-\nfunction experiments. Here, we found through gain-of-\nfunction experiments that overexpression of MDM2 pro-\nmoted the proliferation and migration of hESCs and re-\nversed the inhibitory effect of NR4A3 overexpression on\nrat endometriosis, implying that MDM2 regulated NR4A3\nin endometriosis by inducing NR4A3 ubiquitination.\nConclusion\nOur results highlight the suppressing effects ofNR4A3\noverexpression on the proliferation and migration of hESCs\nin vitro and ectopic cysts in vivo . In addition, this\nstudy found that MDM2 can promote the ubiquitination of\nNR4A3, which provides new clues for the mutual regula-\ntion between MDM2 and NR4A3, and a novel breakthrough\npoint for research on the diagnosis and treatment of en-\ndometriosis. In the future, we will conduct additional ex-\nperiments to confirm the findings and explore the feasibility\nof clinical application.\nAvailability of Data and Materials\nThe datasets used and analyzed during the current\nstudy are available from the corresponding author upon rea-\nsonable request.\nAuthor Contributions\nSubstantial contributions to conception and design:\nYXH. Data acquisition, data analysis, and interpretation:\nYCG and XYL. Drafting the article and critically revising\nit for important intellectual content: All authors. Final ap-\nproval of the version to be published: All authors. Agree-\nment to be accountable for all aspects of the work in en-\nsuring that questions related to the accuracy or integrity of\nthe work are appropriately investigated and resolved: All\nauthors.\nEthics Approval and Consent to Participate\nAll animal procedures were approved by the Animal\nExperiment Ethics Committee of Zhejiang Center of Labo-\nratory Animals for Experimental Animals Welfare (Ethics\nApproval No. ZJCLA-IACUC-20040169).\nAcknowledgment\nNot applicable.\nFunding\nThis research received no external funding.\nConflict of Interest\nThe authors declare no conflict of interest.\nSupplementary Material\nSupplementary material associated with this article\ncan be found, in the online version, at https://doi.org/10.\n24976/Discov.Med.202436191.219.\nReferences\n[1] Taylor HS, Kotlyar AM, Flores V A. Endometriosis is a chronic\nsystemic disease: clinical challenges and novel innovations.\nLancet. 2021; 397: 839–852.\n[2] Falcone T, Flyckt R. 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