Nuclear Receptor Subfamily 4 Group A Member 3: A Potential Marker of Endometriosis

Nuclear receptor subfamily 4 group A member 3 ( NR4A3) is lowly expressed in ectopic endometrium and can be degraded by ubiquitination in vascular endothelial cells. Murine double minute 2 ( MDM2) is predicted to be the ubiquitin ligase of NR4A3. Hence, we investigated the effects of NR4A3 and MDM2 on endometriosis and clarified corresponding regulatory mechanisms.

The ubiquitin ligase of NR4A3 was predicted using bioinformatics and validated by immunoprecipitation. The effects of NR4A3 and MDM2 on the migration and proliferation of human endometrial stromal cells (hESCs) were examined by Transwell assay and 5-ethynyl-2 ′-deoxyuridine (EdU) staining. NR4A3 and MDM2 expressions were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. An endometriosis model was constructed in Sprague-Dawley rats, followed by body weight analysis, ultrasonic imaging of ectopic cysts, and Western blot.

Overexpression of NR4A3 inhibited, but siNR4A3 boosted hESC migration and proliferation. MDM2 promoted NR4A3 ubiquitination and degradation. MDM2 overexpression enhanced hESC migration and proliferation and partially reversed the inhibitory effect of NR4A3 overexpression. Overexpression of NR4A3 reduced ectopic cysts in endometriotic rats, which was offset by MDM2 overexpression.

NR4A3, which is promoted to ubiquitination and degradation by MDM2, inhibits the proliferation and migration of hESCs in vitro, and reduces the growth of ectopic endometrial cysts in vivo, thereby inhibiting the progression of endometriosis.

endometriosis; NR4A3; MDM2; ubiquitination

Endometriosis is an estrogen-dependent chronic gyne- cological disease [ 1], with the presence and growth of en- dometrial glands and stroma outside the uterine cavity as the hallmark [2]. Endometriosis has a variety of clinical man- ifestations such as dysmenorrhea, pelvic mass, infertility, and cancer, which seriously affect the quality of life of pa- tients [ 3]. Currently, the diagnosis of endometriosis relies on laparoscopic surgical evaluation as the gold standard and lacks non-invasive markers [4,5]. Therefore, there is an ur- gent need to explore the pathogenesis of endometriosis and find a non-invasive biomarker for diagnosis and treatment. Nuclear receptors (NRs) are a class of eukaryotic tran- scription factors widely distributed in cells [ 6]. By regu- lating the transcription and expressions of numerous key genes, NRs participate in various pathophysiological pro- cesses such as inflammation and immune response in the human body [ 6]. Nuclear receptor subfamily 4 group A (NR4A) is a special class of NRs whose endogenous ligands have not yet been found [7]. The NR4A protein family con- sists of three well-characterized members: nuclear receptor subfamily 4 group A member 1 (NR4A1), nuclear receptor subfamily 4 group A member 2 (NR4A2), and nuclear re- ceptor subfamily 4 group A member 3 (NR4A3) [ 8]. The existing study has shown that the expression of NR4A1 is reduced in the ectopic endometrium of patients with adeno- myosis, leading to impaired endometrial function through interaction with Forkhead Box O1 (FOXO1A) and reduced female fertility [9]. Moreover, a study also pointed out that NR4A3 in ectopic endometrium of adenomyosis patients shares the same expression trend with NR4A1 [9]. How- ever, the effect of NR4A3 on endometriosis and the associ- ated regulatory mechanism, which are not well understood, are the focus of this study. It is worth noting that NR4A3 regulates endothelial cell injury, and can be degraded by ubiquitination in vas- cular endothelial cells [ 10]. Therefore, we speculate that the function of NR4A3 is also related to its ubiquitination level in endometrial cells. Through the UbiBrowser web- 2377 site, it was found that Murine double minute 2 (MDM2) may be the ubiquitin ligase that mediates the ubiquitination of NR4A3. MDM2 has been reported to be highly expressed in endometriosis and to further promote the progression of endometriosis through ubiquitination [ 11–13]. Based on this, we set out to investigate the effect of NR4A3 on en- dometriosis through in vitro and in vivo experiments and to further explore whether MDM2 mediates the ubiquitination of NR4A3 in endometriosis.

Animals Six-week-old female non-pregnant Sprague-Dawley rats (180–200 g, n = 32) were purchased from Hangzhou Medical College (China). All rats were housed in the laboratory with an automatic light control system of 12-h light/dark cycle, room temperature of 22 ± 0.5 °C, and rel- ative humidity of 40–60%. Cells, Culture and Transfection Human endometrial stromal cells (hESCs) (CP-H208) and corresponding medium (CM-H208) were obtained from Procell company (Wuhan, China), and hESCs were maintained in the culture medium at 37 °C with hu- midified air and 5% CO 2. NR4A3 or MDM2 over- expression plasmids were constructed by insertion of NR4A3 or MDM2 coding sequences (CDS, as Supple- mentary Materials ) into the pEX-3 vector (C05003, GenePharma, Shanghai, China). The pEX-3 vec- tor without insert was used as the negative control (NC). Small interfering RNAs (siRNAs) of NR4A3 or MDM2 (siNR4A3, 5 ′-GCAGAGCCTGAACCTTGA TA T- 3′; siMDM2, 5′-CTCTCGACTCAGAAGA TTA TA-3′) and siNC (5′-CAACAAGA TGAAGAGCACCAA-3′) were ob- tained from GenePharma (Shanghai, China). The siRNAs or overexpression plasmids were transfected into hESCs as per instructions of Lipofectamine 2000 (11668500, Invitro- gen, Carlsbad, CA, USA). The hESCs were routinely tested for mycoplasma contamination and were confirmed to be mycoplasma-free. Immunofluorescence Assay As for cell identification, hESCs (1 × 106) were fixed with 4% paraformaldehyde (441244, Sigma-Aldrich, St. Louis, MO, USA) for 15 min and permeabilized with 0.1% Triton X-100 (93443, Sigma-Aldrich, USA) for 10 min at room temperature. After washing with phosphate-buffered saline, the cells were incubated with 5% bovine serum al- bumin (V900933, Sigma-Aldrich, USA) at 37 °C for 30 minutes. The hESCs were then incubated overnight at 4 °C with Vimentin primary antibody (ab20346, Abcam, Cambridge, UK), followed by a 30-min incubation at 4 °C with a fluorescence-labeled secondary antibody (ab150115, Abcam, Cambridge, UK). Cell nuclei were stained with 4’,6-Diamidino-2’-phenylindole (DAPI, D9542, Sigma- Aldrich, USA) for 10 minutes in the dark. Observations were made using a confocal microscope (FV3000, Olym- pus, Tokyo, Japan) at ×200 magnification. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) A total RNA extraction kit (R1200, Solarbio, Bei- jing, China) was employed for total RNA collection. First-strand cDNA was synthesized using a first-strand cDNA synthesis kit (K1612, Thermo Fisher Scien- tific, Waltham, MA, USA). For the analysis of mRNA expression, PCR was conducted on the StepOnePlus Real-Time PCR system (4376600, Applied Biosystems, Foster City, CA, USA) with SYBR Green (HY -K0501A, MedChemExpress, Shanghai, China). Glyceraldehyde-3- phosphate dehydrogenase ( GAPDH) served as an internal reference. The PCR primer information was as follows (5′-3′): NR4A3: TGCGTCCAAGCCCAA TA TAGC (For- ward), GGTGTA TTCCGAGCTGTA TGTCT (Reverse); GAPDH: GGAGCGAGA TCCCTCCAAAA T (Forward), GGCTGTTGTCA TACTTCTCA TGG (Reverse). Western Blot Total proteins were harvested from rat ectopic cysts and hESCs with the help of radioimmunoprecipitation as- say (RIPA) lysis buffer (R0278, Sigma-Aldrich, USA) and quantified using a bicinchoninic acid (BCA) kit (ab102536, Abcam, UK). Proteins were separated via sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (IPVH08100, Millipore, Billerica, MA, USA) which were blocked with 5% nonfat milk. Membranes were then in- cubated with diluted primary antibodies at 4 °C overnight and then reacted with secondary antibodies for 1 h at room temperature. An Enhanced chemiluminescence (ECL) sub- strate kit (ECL-P-500) was obtained from Shanghai Y anxi Biological Technology Co., Ltd. (Shanghai, China) to vi- sualize blots. Antibody information is as follows: MDM2 (#51541, 90 kDa, 1:1000, Cell Signaling Technology, Boston, MA, USA); NR4A3 (sc-393902, 68 kDa, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA); GAPDH (ab181602, 36 kDa, 1:10,000, Abcam, UK); Goat Anti- Rabbit Immunoglobulin G Heavy and Light Chains (IgG H&L) (horseradish peroxidase (HRP)) (ab205719, 1:5000, Abcam, UK); Goat Anti-Mouse IgG (HRP) (ab97240, 1:5000, Abcam, UK). Transwell Assay The migration rate of hESCs was determined by Tran- swell assay. After 48-h transfection, hESCs (1 × 105) were inoculated in medium without fetal bovine serum (FBS) and then seeded into the upper Transwell chamber (CLS3412, Sigma-Aldrich, USA), whereas medium with 10% FBS was loaded into the lower chamber. After 24 h, migrating cells 2378 were fixed using a paraformaldehyde fixator (P885233, Macklin, Shanghai, China) and then stained by crystal vi- olet (C805209, Macklin, China) for 30 min, followed by observation using a microscope (IXplore Standard, OL YM- PUS, Tokyo, Japan) at ×250 magnification. Bioinformatics Analysis The ubibrowser site (http://ubibrowser.bio-it.cn/) was utilized to predict the E3 ubiquitin ligase of NR4A3. Briefly, on the homepage, the “substrate” and “H.sapiens” options were selected in the search bar, and then the analysis

can be obtained by entering “ NR4A3” and clicking “explore”. 5-ethynyl-2′-deoxyuridine (EdU) Staining Assay The BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 594 (C0078L) for cell proliferation analysis was pro- vided by Beyotime Company (Shanghai, China). In brief, hESCs were seeded into 6-well plates, followed by the ad- dition of EdU working solution and incubation for 2 h. After being fixed and permeabilized, cells were cultivated with the Click reaction solution at room temperature for 30 min in the dark. After re-dyeing with 4’,6-Diamidino-2’- phenylindole (DAPI) solution (CC1162, G-CLONE, Bei- jing, China), EdU-positive cells were observed under a fluorescence microscope (STELLARIS 5, Leica, Wetzlar, Germany). Co-Immunoprecipitation (Co-IP) The interaction between MDM2 and NR4A3 in hESCs was determined via Co-IP assay with the help of a Co-IP kit (abs955, Absin, Shanghai, China). More specifically, IP lysis buffer (87787, Thermo Fisher Scientific, USA) was used to prepare hESCs, after which 500 µL cell lysate was incubated with 5 µg MDM2 antibody (ab259265, Abcam, Cambridge, UK), NR4A3 antibody (sc-393902, Santa Cruz Biotechnology, USA), or with control IgG (ab205718, Ab- cam, UK) at 4 °C overnight. Next, 5 µL Protein A and 5 µL Protein G were added into the cell lysate, followed by a 3-h incubation at 4 °C and 1-min centrifugation at 12,000 ×g. At the end, 0.5 mL Wash buffer was used to elute the protein complexes, followed by Western blot analysis. Ubiquitination Assay The hESCs were transiently transfected with Flag- NR4A3, hemagglutinin (HA)-MDM2, and HA-Histidine (His)-Ubiquitin (Ub) (3683524, Biovector NTCC, Beijing, China). 48 h after transfection, hESCs were treated with or without MG132 (HY -13259, 10 µM, MedChemExpress, China) for 6 h, and then cells were lysed in IP lysis buffer (87787, Thermo Fisher Scientific, USA) and incubated with anti-Flag-M2 affinity gel (HY -K0217, MedChemExpress, China) or anti-HA magnetic beads (HY -K0201, MedChem- Express, China) overnight at 4 °C, followed by SDS-PAGE, and the subsequent steps were consistent with Western blot. Antibodies used included polyclonal-Ubiquitin (poly-Ub, PA1-187, Invitrogen, USA), Flag (SAB4200071, Sigma- Aldrich, USA), and HA (H3663, Sigma-Aldrich, USA). Animal Assays Thirty-two Sprague-Dawley rats were used in the an- imal assays, and the establishment of the rat endometriosis model referred to a previous report [ 4]. SD rats were anes- thetized with 3% isoflurane (792632, Sigma-Aldrich, USA) using gas anesthesia machine (R500IP; RWD Life Technol- ogy Co., Shenzhen, China), and a small incision was made in the center of the abdomen. The left uterine horn was ex- cised to collect the endometrium which was later divided in half and placed on the left and right sides of the abdom- inal wall. On the first and tenth days, estradiol benzoate (HY -B1192, MedChemExpress, USA) was subcutaneously injected into rats to establish an endometriosis model. Mod- eling was performed in 26 rats and 24 modeled rats were obtained, which was verified by the ultrasonic imaging of cysts on the 20th day, with a success rate of 92.3%. Rats in the Sham group (n = 6) only had a small inci- sion in the abdomen without autologous endometrial trans- plantation. Immediately after modeling, rats were injected with NR4A3 overexpression vector, MDM2 overexpres- sion vector, NC, or an equivalent volume of normal saline (S0817, Sigma-Aldrich, USA) at the endometriotic lesions, and then fed for 20 days. During the feeding process, the body weight of the rats was measured and recorded every 5 days. On the 20th day, the rats were anesthetized (2% isoflurane) and underwent high-resolution ultrasound imaging (VisualSon- ics V evo770, VisualSonics, Toronto, ON, Canada) with the help of real-time microvisualization Scanhead (center fre- quency: 40 MHz; focal depth: 6 mm) [ 14]. After that, rats were euthanized via intraperitoneal administration of 1% pentobarbital sodium (P010, 150 mg/kg, Sigma-Aldrich, USA), and the ectopic endometrial cysts were removed to take photographs and measure the volume. Statistical Analysis Data were obtained from experiments performed three times and are presented as the mean ± standard deviation. The multi-group comparison was carried out using a one- way and follwed by Tukey post hoc test. All statistical anal- yses were conducted using GraphPad 8.0 software (Graph- Pad Software, San Diego, CA, USA), and p values < 0.05 were considered statistically significant.

NR4A3 Regulated hESC Migration and Proliferation The isolated cells exhibited prominent Vimentin stain- ing, confirming their identity as hESCs ( Supplementary Fig. 1 ). To better understand the function of NR4A3 in en- dometriosis, we transfected NR4A3 overexpression plasmid 2379 Fig. 1. Expression and regulation of nuclear receptor subfamily 4 group A member 3 ( NR4A3) in endometriosis. (A,B) NR4A3 overexpression plasmids and small interfering RNA (siRNA) were transfected into human endometrial stromal cells (hESCs), and transfection efficiency were determined by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference control. (C) The effect of NR4A3 overexpression or silencing on the migration of hESCs was assessed using Transwell assay. (D) 5-ethynyl-2 ′-deoxyuridine (EdU) staining was performed to test cell proliferation. ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3. DAPI, 4’,6-Diamidino-2’-phenylindole; NC, negative control. 2380 Fig. 2. Ubiquitination regulation of NR4A3 by MDM2. (A) The ubiquitin ligase of NR4A3 was predicted using the ubibrowser website (http://ubibrowser.bio-it.cn/). The capital letters represent different subfamilies of E3 ligases, with “U” for UBOX, “H” for HECT, “R” for RING, and “SO” for SINGLE_other. The thickness of the lines is related to the confidence score, with thicker lines indicating higher confidence scores. (B) The effect of MDM2 on NR4A3 expression was determined by Western blot. (C) The interaction between MDM2 and NR4A3 was determined by co-immunoprecipitation assay. (D) After cells were treated with or without MG132, ubiquitination assays were performed after Flag-NR4A3 and hemagglutinin-Histidine-Ubiquitin (HA-His-Ub) were co-transfected with or without HA- MDM2 into hESCs. ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3. MDM4, Murine double minute 4; MDM2, Murine double minute 2; SMURF1, SMAD-specific E3 ubiquitin protein ligase 1; SMURF2, SMAD-specific E3 ubiquitin protein ligase 2; BARD1, BRCA1 Associated RING Domain 1; RANBP2, RAN Binding Protein 2; STUB1, STIP1 Homology And U-Box Containing Protein 1; ARIH2, Ariadne RBR E3 Ubiquitin Protein Ligase 2; UBOX5, U-Box Domain Containing 5; BRCA1, BRCA1 DNA Repair Associated; ITCH, Itchy E3 Ubiquitin Protein Ligase; RBBP6, RB Binding Protein 6, Ubiquitin Ligase; TRIM24, tripartite Motif Containing 24; HECW1, HECT, C2 And WW Domain Containing E3 Ubiquitin Protein Ligase 1; LITAF, Lipopolysaccharide Induced TNF Factor; PIAS2, Protein Inhibitor Of Activated STA T2; PIAS3, Protein Inhibitor Of Activated STA T3; RBX1, Ring-Box 1; TOPORS, TOP1 Binding Arginine/Serine Rich Protein, E3 Ubiquitin Ligase; RCHY1, Ring Finger And CHY Zinc Finger Domain Containing 1; IgG, Immunoglobulin G; poly-Ub, polyclonal-Ubiquitin; IP , immunoprecipitation. 2381 or siRNA into hESCs to manipulateNR4A3 expression (p < 0.001, Fig. 1A,B). We observed thatNR4A3 overexpression significantly inhibited the migration of hESCs, whereas siNR4A3 promoted cell migration ( p < 0.01, Fig. 1C). Additionally, NR4A3 overexpression significantly reduced, whereas siNR4A3 increased the EdU-positive cells ( p < 0.001, Fig. 1D). MDM2 Mediated NR4A3 Ubiquitin Degradation to Regulate hESC Migration and Proliferation NR4A3 regulates endothelial cell damage and can be degraded by ubiquitination in vascular endothelial cells. Here, MDM4, MDM2, and SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) were found to be possible ubiq- uitin ligases of NR4A3 (Fig. 2A). Among them, MDM2 is highly expressed in endometriosis, and further accelerates the progression of endometriosis via ubiquitination [ 11– 13]. Overexpression of MDM2 increased the MDM2 pro- tein level but decreased the NR4A3 protein level, whereas siMDM2 transfection had the opposite effect ( p < 0.01, Fig. 2B). Importantly, we observed protein interaction be- tween MDM2 and NR4A3 (Fig. 2C), and found that MDM2 greatly enhanced NR4A3 ubiquitination (Fig. 2D). Next, we conducted rescue experiments and found that NR4A3 overexpression had no effect on MDM2 expres- sion, but reversed the inhibiting role of MDM2 upregula- tion on NR4A3 expression ( p < 0.01, Fig. 3A). In addi- tion, MDM2 overexpression promoted migration and pro- liferation of hESCs, which was offset by overexpression of NR4A3 (p < 0.001, Fig. 3B,C). MDM2 Partially Reversed the Effects of NR4A3 in a Rat Model of Endometriosis To further clarify the role of MDM2-NR4A3 interac- tion, we used a rat model of endometriosis. No significant difference was found in body weight between model rats and NR4A3 and/or MDM2 overexpression vector-injected rats compared with the sham-operated rats (Fig. 4A). Ultra- sonic imaging showed that there were no ectopic cysts in the sham group, but obvious ectopic cysts in the Model group and Model+NC group. Overexpression of NR4A3 reduced the cysts caused by the modeling, which was counteracted by to overexpression of MDM2 (p < 0.01, Fig. 4B–D). Additionally, we examined the expressions of MDM2 and NR4A3 in rat endometrial cyst tissue. As shown in Fig. 4E, NR4A3 upregulation did not significantly affect MDM2, but increased NR4A3 expression; whereas MDM2 upreg- ulation not only significantly promoted MDM2 expression but also reversed NR4A3 upregulation-induced promotion of NR4A3 protein level (p < 0.05).

Endometriosis is a common benign gynecological dis- ease, but it has similar biological behaviors to tumors and has the ability to adhere, invade, and metastasize with a high recurrence rate [15]. This study provided new evidence that MDM2 mediates the ubiquitination of NR4A3 to affect the proliferation and migration of hESCs. NR4A3 has been reported to be a master gene involved in various physiopathologies [16]. Lee et al. [ 17] suggested that NR4A3 acts as an oncogene in acinar cell carcinoma by interacting with MYB proto-oncogene, transcription factor (MYB). Deutsch et al. [ 18] reported that NR4A3 represses lymphomagenesis by inducing pro-apoptotic genes. NR4A3 also promotes the inflammatory response of osteoarthri- tis through the nuclear factor kappa-B (NF- κB) pathway [19]. However, NR4A3 suppresses inflammatory responses through the Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STA T3)/NF-κB pathway in acute myocardial infarction [20]. The paradoxical roles of NR4A3 in various diseases make us more interested in exploring its role and mechanism in endometriosis. Endometrial stromal cells are an important component of endometriosis progres- sion [ 21,22]. Studying the migration and proliferation of endometrial stromal cells contributes to the diagnosis and treatment of endometriosis at the cellular level. Here, we found that overexpressed NR4A3 inhibited hESC migration and proliferation, whereas knockdown of NR4A3 had the opposite effect, suggesting that NR4A3 may play a protec- tive role in endometriosis progression. Ubiquitination, as one of the most prevalent post- translational modifications in the proteome, has also been widely explored in endometriosis [ 23–25]. Wang et al . [23] revealed that tripartite motif containing 59 (TRIM59) ubiquitination degrades protein phosphatase, Mg 2+/Mn2+ dependent 1A (PPM1A) and activates the transforming growth factor- β (TGF-β)/Smad pathway to promote en- dometriosis progression. SMURF1-mediated ubiquitina- tion of SH2 domain-containing phosphatase 1 (SHP-1) ac- celerates the invasion and proliferation of endometrial stro- mal cells in endometriosis [ 24]. Wu et al . [ 25] pointed out that TRIM65, highly expressed in ectopic endometrial tissues, inhibits dual specificity phosphatase 6 (DUSP6) through ubiquitination and activates the ERK1/2/C-myc signaling pathway to promote the invasion of ectopic endometrial stromal cells. Here, we demonstrated that MDM2 may mediate NR4A3 degradation through ubiquiti- nation, further providing evidence for ubiquitination in en- dometriosis. MDM2, a vital E3 ligase, can ubiquitinate a variety of substrates and participates in many cellular physiological and pathological processes [ 26–28]. MDM2 interacts with immediate early response 3 (IER3) and promotes its ubiq- uitination to reduce apoptosis of cervical cancer cells [ 28]. p53 is a common ubiquitination substrate of MDM2 and has been repeatedly reported to be inhibited by MDM2 via MDM2-driven ubiquitination [29–31]. p53 can directly in- duce transcription of NR4A3 by binding to the promoter of NR4A3 and thus exert a tumor-suppressing effect [ 32]. The 2382 Fig. 3. MDM2 regulated hESC migration and proliferation through NR4A3 ubiquitin degradation. (A) The expressions of MDM2 and NR4A3 were determined by Western blot. (B) The effects of MDM2 and NR4A3 overexpression on the migration of hESCs were detected by Transwell assay. (C) EdU staining was employed to reveal the impacts of MDM2 and NR4A3 on cell proliferation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3. 2383 Fig. 4. Effects of MDM2 and NR4A3 on endometriosis rat model. (A–E) Six-week-old female non-pregnant Sprague-Dawley rats were used to construct a rat model of endometriosis, with six rats in each group (Sham, Model, Model+NC, Model+NR4A3, and Model+NR4A3+MDM2 groups). (A) Changes in rat body weight were recorded. (B–D) Ultrasonic imaging of removed cysts and volume detection on day 20. (E) Expressions of MDM2 and NR4A3 in rat endometrial cyst tissue were determined by Western blot. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 3. interaction of MDM2, p53, and NR4A3 is intriguing and can be further explored in the future. The role of MDM2 in endometriosis has been reported in various ways [ 11–13]. For example, Li et al. [ 12] found through bioinformatics analysis that MDM2 is one of the central genes in endometriosis and may mediate the ubiq- uitination of p27. Sang et al . [ 11] revealed high expres- sion of MDM2 in endometriosis, consistent with our results. 2384 Chen et al. [ 13] demonstrated that silencing MDM2 hinders the development of endometriosis in mice through loss-of- function experiments. Here, we found through gain-of- function experiments that overexpression of MDM2 pro- moted the proliferation and migration of hESCs and re- versed the inhibitory effect of NR4A3 overexpression on rat endometriosis, implying that MDM2 regulated NR4A3 in endometriosis by inducing NR4A3 ubiquitination.

Our results highlight the suppressing effects ofNR4A3 overexpression on the proliferation and migration of hESCs in vitro and ectopic cysts in vivo . In addition, this study found that MDM2 can promote the ubiquitination of NR4A3, which provides new clues for the mutual regula- tion between MDM2 and NR4A3, and a novel breakthrough point for research on the diagnosis and treatment of en- dometriosis. In the future, we will conduct additional ex- periments to confirm the findings and explore the feasibility of clinical application. Availability of Data and Materials The datasets used and analyzed during the current study are available from the corresponding author upon rea- sonable request. Author Contributions Substantial contributions to conception and design: YXH. Data acquisition, data analysis, and interpretation: YCG and XYL. Drafting the article and critically revising it for important intellectual content: All authors. Final ap- proval of the version to be published: All authors. Agree- ment to be accountable for all aspects of the work in en- suring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved: All authors. Ethics Approval and Consent to Participate All animal procedures were approved by the Animal Experiment Ethics Committee of Zhejiang Center of Labo- ratory Animals for Experimental Animals Welfare (Ethics Approval No. ZJCLA-IACUC-20040169). Acknowledgment Not applicable. Funding This research received no external funding. Conflict of Interest The authors declare no conflict of interest. Supplementary Material Supplementary material associated with this article can be found, in the online version, at https://doi.org/10. 24976/Discov.Med.202436191.219.

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