Nuclear factor IX promotes endometriosis progression through transcriptional activation of tetraspanin-2

Endometriosis is a benign yet aggressive disease characterized by enhanced proliferation and invasion of ectopic endometrial tissue. Identifying upstream regulators that co-regulate these processes will provide novel insights into endometriosis pathogenesis and potential therapeutic targets. In this study, by integrating public single-cell RNA-seq data with our own RNA sequencing data, we identified nuclear factor IX (NFIX) as predominantly enriched in endometriotic stromal cells (ESCs), correlating with enhanced proliferative and invasive capacities. However, the underlying molecular mechanisms remain to be elucidated. Using the Venny platform, we intersected NFIX target genes from the KnockTF2.0 database with differentially expressed genes from our RNA sequencing data. Among these overlapping genes, we further identified tetraspanin-2 (TSPAN2) as a target of NFIX and validated that increased TSPAN2 expression mediated the regulatory effects of NFIX on ESCs’ proliferation and invasion. Mechanistically, we found that NFIX exerts a significant stimulatory effect on TSPAN2 expression in ESCs. Luciferase reporter assays using serial deletion mutants confirmed that NFIX specifically binds to the −408 ~ −400 bp region of the TSPAN2 promoter, activating its transcription. Additionally, a chromatin immunoprecipitation (ChIP) assay revealed that the binding affinity of NFIX for the −408 ~ −400 bp region of the TSPAN2 promoter was higher in ESCs than in eutopic endometrial stromal cells (EMs). Moreover, NFIX knockdown in endometriosis mice downregulated TSPAN2 expression and inhibited ectopic lesion growth. Overall, this study demonstrated that NFIX promotes proliferation and invasion of ESCs by transcriptionally activating TSPAN2, suggesting NFIX as a potential therapeutic target. Key messages - NFIX expression was markedly higher in ESCs than in EMs, correlating with increased proliferation and invasion capabilities. - TSPAN2 was identified as a key mediator of NFIX-dependent regulation of proliferation and invasion of ESCs. - NFIX transcriptionally activated TSPAN2 by binding to the -408 to -400 bp region of its promoter. - In vivo knockdown of NFIX significantly inhibited the growth of endometriotic lesions in mice. Similar content being viewed by others

Endometriosis involves the existence and growth of endometrial tissue external to the uterine cavity. With a prevalence of approximately 10% in the reproductive-age female population, endometriosis is characterized by a symptomatic triad of dysmenorrhea, chronic pelvic pain, and dyspareunia, frequently compounded by infertility. This multifaceted symptomatology leads to profound quality-of-life deterioration and imposes significant socioeconomic costs [1]. Currently, the etiology of endometriosis remains incompletely understood, and patients undergoing treatments for endometriosis, including conservative surgery and hormone therapy, have high recurrence rates and can experience several complications [2, 3]. These limitations highlight the pressing need to delineate the molecular events driving lesion progression and reveal novel therapeutic targets for endometriosis. The pathogenesis of endometriosis is multifactorial and remains incompletely defined. Retrograde menstruation, as proposed by Sampson, is widely considered a primary initiating event. The onset and progression of endometriosis are complex processes and were reviewed by a previous study to occur as follows: endometrial cells displaced via retrograde menstruation successfully implant at ectopic sites and produce excessive estrogen in the local milieu; subsequently, cell proliferation is increased, resulting in lesion survival. Moreover, inflammatory factors released by macrophages trigger epithelial–mesenchymal transition, with a subsequent increase in invasion, resulting in lesion growth [4]. It can be seen that increased proliferation and invasion are critical steps in endometriosis progression. Additionally, numerous studies have reported higher levels of proliferation or invasion markers in human endometriotic tissues than in eutopic endometrial tissues [5,6,7,8]. Moreover, key signaling pathways—MAPK (ERK1/2, p38, JNK), IKKβ/NF-κB, and PI3K/AKT/mTOR—are activated in endometriotic tissues, driving cellular proliferation and invasion. Emerging evidence further highlights their regulatory roles in lesion hyperproliferation and invasiveness [9,10,11,12]. Hence, we strongly believe that exploring the co-regulatory mechanisms of aberrant proliferation and invasion is crucial for gaining a deeper insight into the pathogenesis of endometriosis and for developing therapeutic interventions for this disease. Nuclear factor IX (NFIX), a member of the NFI transcription factor family, contains canonical DNA-binding and dimerization domains that enable activation or repression of target genes through homo- or heterodimer formation [13]. NFIX regulates diverse biological functions—including proliferation, migration, differentiation, and tissue development—and is dysregulated in various malignancies, such as colorectal, lung, and endometrial cancers [14,15,16,17]. Given the malignant-like behavior of endometriosis and the paucity of studies investigating NFI family members in this context, the contribution of NFIX to endometriosis pathology remains an important unanswered question. Tetraspanins (TSPANs) are four-transmembrane proteins that act as membrane-associated scaffolds to modulate cell adhesion, migration, signaling, and vesicular trafficking by forming dynamic complexes with integrins, growth factor receptors, and intracellular signaling molecules [18, 19]. Although several TSPAN members have been implicated in cancer metastasis, inflammation, immune regulation, and reproductive biology [20, 21], TSPAN2 remains largely uncharacterized and has not been linked to endometriosis. This study aims to elucidate the transcriptional events governing the aberrant proliferative and invasive behavior of ESCs. We demonstrate that NFIX expression is substantially elevated in ESCs compared with EMs and is strongly associated with enhanced proliferative and invasive capacity. Mechanistically, we identify TSPAN2 as a critical downstream effector of NFIX and show that NFIX directly activates TSPAN2 transcription by binding to the −408 to −400 bp region of its promoter. Moreover, in vivo silencing of NFIX markedly suppresses ectopic lesion growth in a mouse model, supporting NFIX as a promising molecular target for therapeutic intervention in endometriosis.

Samples collection and cell culture This study recruited 15 patients who underwent hysterectomy surgery with laparoscopic excision of ovarian endometriomas, which was diagnosed by pathological examination. From each individual, self-paired ectopic and eutopic endometrium samples were simultaneously collected from ovarian endometrioma patients to minimize inter-individual variability. All participants were 25–40 years old and had regular menstrual cycles. Individuals were excluded if they had received any form of hormonal therapy within the preceding three months or were using intrauterine contraceptive devices, ensuring the restoration of endogenous hormone levels to baseline status. Participants with concurrent endometrial disorders—including endometrial polyps, endometrial hyperplasia, or endometrial cancer—were also excluded. These stringent criteria were implemented to eliminate confounding endocrine influences and avoid pathological heterogeneity, thereby ensuring the reliability and interpretability of downstream cellular and molecular analyses. The research protocol was given the green light by the Institutional Review Board of Peking University’s First Hospital (No.2021[446]), and each patient provided explicit consent, which was documented prior to the utilization of their specimens. A modified version of a previously established protocol was employed to isolate endometrial stromal cells (EMs) and endometriotic stromal cells (ESCs) from the acquired tissues [22]. Single-cell RNA sequencing data from two publicly available datasets (GSE179640 and GSE213216) were analyzed in this study [23, 24]. Immunofluorescence The process involved seeding ESCs and EMs on glass coverslips coated with laminin. They were then fixed using 4% paraformaldehyde (Solarbio, P1110) for a 10-min duration. Following fixation, the cells sequentially underwent permeabilization using 0.2% Triton X-100 (Solarbio, T8200) and were incubated using 5% bovine serum albumin (BSA; Solarbio, SW3015). Subsequently, cells were subjected to incubation with a primary antibody targeting vimentin at a dilution of 1:1000 (Servicebio, GB12192-100), and an anti-pan-cytokeratin antibody at a dilution of 1:500 (Santa Cruz, sc-8018). After washing, cells underwent incubation with secondary antibodies to detect the signals of the primary antibody. Finally, cells were stained using DAPI (Servicebio, C0065) and imaged using fluorescence microscopy after being coverslipped with an anti-fade reagent. Reverse transcription polymerase chain reaction (RT-PCR) The extraction of total RNA was performed with TRIzol (Invitrogen, A33251) in accordance with the provided instructions by the producer. The extracted RNA was subsequently assessed for its quality utilizing the NanoDrop 2000 instrument. 2 μg RNA was utilized for reverse transcription with the PrimeScript RT reagent Kit (Takara, RR047Q). To evaluate the levels of gene expression, RT-PCR was employed using Sybr Green (Lablead, R0202) and ABI 7500 System. Primers were designed and their functionality was verified for each target gene. The sequences of the primers used for human samples in the RT-PCR analysis are provided as follows: NFIX, forward 5′-AGCAGTCGAGCCCGTATTTC-3′, reverse 5′-GTCCGATGCTGACAAACCG-3′; TSPAN2, forward 5′-ACAGGTCCAACCTACATGCC-3′, reverse 5′-TGAGCTGGAGCTTAACACTGAT-3′; Cyclin B1, forward 5′-TTGGGGACATTGGTAACAAAGTC-3′, reverse 5′-ATAGGCTCAGGCGAAAGTTTTT-3′; MMP9, forward 5′-TTCCAAACCTTTGAGGGCGA-3′, reverse 5′-CTGTACACGCGAGTGAAGGT-3′; 18S, forward 5′-AGGAATTCCCAGTAAGTGCG-3′, reverse 5′-GCCTCACTAAACCATCCAA-3′. The primer sequences of mice samples used in RT-PCR were as provided: NFIX, forward 5′-ACTTTGTGCTAACCATCACGG-3′, reverse 5′-CACTGGGGCGACTTGTAGAG-3′; TSPAN2, forward 5′-CTAGCCGGATCAGCCGTTATT-3′, reverse 5′-AGCACCCGAAGAAACCCAC-3′; Cyclin B1, forward 5′-AGAGCTATCCTCATTGACTGGC-3′, reverse 5′-AACATGGCCGTTACACCGAC-3′; MMP9, forward 5′-GGACCCGAAGCGGACATTG-3′, reverse 5′-CGTCGTCGAAATGGGCATCT-3′; β-actin, forward 5′-GGCTGTATTCCCCTCCATCG-3′, reverse 5′-GCCTCACTAAACCATCCAA-3′. The method of comparative threshold cycle analysis, as previously outlined, was utilized for the assessment of relative quantification of all transcripts [25]. The expression levels of target genes were quantified and normalized against 18S rRNA or β-actin as endogenous controls, following methodological standards established in endometriosis research [26, 27]. Protein extraction and western blotting RIPA (Beyotime, P0013) incorporating 1% protease inhibitor (Sigma-Aldrich, P8340) and 1% phosphatase inhibitor (Sigma-Aldrich, P0044) was employed for extracting cell proteins. An Enhanced BCA Protein Assay Kit (Beyotime, P0010) was utilized to ascertain the protein concentration across all samples. Samples containing equal protein loads (at least 30 µg) were first subjected to SDS-PAGE separation. Subsequently, they were transferred onto nitrocellulose membranes and incubated with specific antibodies as indicated: anti-NFIX (1:500; Abclonal, A9390), anti-TSPAN2 (1:500; Proteintech, 20,463–1-AP), anti-Cyclin B1 (1:1000; Abcam, ab181593), anti-MMP9 (1:500; Abclonal, A23535), anti-GAPDH (1:1000; ZSGB-BIO, TA-08), and anti-β-actin (1:1000; ZSGB-BIO, TA-09). The visualization of protein bands was achieved using an ECL (Enhanced Chemiluminescence) solution (KeyGEN, KGP1127). The densitometric quantification for protein bands was conducted with the AlphaEaseFC. The ImageJ software was utilized to determine the relative expression of target proteins, with normalization to either GAPDH or β-actin for quantification purposes. Immunohistochemistry staining All samples were preserved using 4% paraformaldehyde (Solarbio, P1110) and embedded within a paraffin matrix (Solarbio, YA0012). These tissue specimens were then sliced into sections of dimensions 10 × 10 × 2 mm. Following the removal of paraffin, the sections underwent dehydration, antigen unmasking, and blocking procedures. Subsequently, the paraffin-coated sections were subjected to incubation using primary antibody against NFIX (1:500; Abclonal, A9390) and an antibody against TSPAN2 (1:200; Invitrogen, PA5-53355). Finally, all slides were treated with 3,3-diaminobenzidine tetrahydrochloride (DAB) as a substrate (Beyotime, P0203) and subsequently counterstained with hematoxylin (Beyotime, C0107). Microscopic visualization was employed to capture imagery of the stained slides. Small interfering RNA (siRNA) transfection ESCs were transfected with either a non-specific negative control siRNA (Ribobio, siN0000001-1–10) or siRNAs against human NFIX (si-NFIX) (Ribobio, SIGS0007249-1) and TSPAN2 (si-TSPAN2) (Ribobio, SIGS0009336-1) at 100 nmol/L when they reached an approximate confluence of 70%–80%. Adhering to the producer’s guidelines, Lipofectamine RNAiMAX (Invitrogen, 13778100) was utilized in conjunction with Opti-MEM low-serum medium (Invitrogen, 31985070) for the transfection experiment. Two days after the transfection procedure, the cells were collected and further analyzed through RT-PCR and Western blot. The siRNA sequences used were as follows: NFIX siRNA, 5′-GTACTTCAAGAAGCATGAA-3′; TSPAN2 siRNA, 5′-GCUCCAGCUCAUUGGAAUUTT-3′. Plasmid overexpression The pENTER-NFIX (NM_002501) and pENTER-TSPAN2 (NM_005725) overexpression plasmids and control plasmids were acquired from Vigene Biosciences (Shandong, China). All plasmids were verified by sequencing. ESCs were grown to a density of approximately 70–80% before the transfection. The plasmid transfection process involved the use of Lipofectamine 3000 (Invitrogen, L3000015) in conjunction with Opti-MEM reduced serum medium (Invitrogen, 31,985,070), following the instructions provided by the supplier. Forty-eight hours subsequent to the transfection, the cells were collected and further analyzed through RT-PCR and Western blotting. Matrigel invasion assay Cell invasion analysis was performed using matrigel-coated 24-well transwell chambers with 8 µm pore size and a diameter of 6.5 mm (Corning, USA). A total of 2 × 105 ESCs were seeded in the upper chamber using DMEM/F12 media supplemented with 2% FBS. The lower chamber was filled with 600 μl of DMEM/F12 media containing 20% FBS. Following incubation at a temperature of 37 °C for a duration of 48 h. Non-invading cells were gently removed from the top surface using a cotton-tipped swab. The cells that successfully invaded through the pores and reached the bottom side of the membrane were then fixed using a solution consisting of paraformaldehyde (4%) and stained utilizing 1% crystal violet (Beyotime, C0121). Subsequently, images capturing these stained cells were acquired employing a DP71 digital microscope camera. For each experiment, five fields were randomly selected and quantified. CCK8 assay The CCK8 assay was performed in accordance with the guidelines provided by the manufacturer (Lablead, CK001). Briefly, 24 h post transfection, ESCs were dissociated and plated in a 96-well dish at a concentration of 104 cells/well and then cultured at 37 °C for 4 days. The cells were treated with CCK8 reagent every 24 h, and the optical density was assessed at a wavelength of 450 nm using a microplate reader following a 4-h incubation period. Transient transfection and luciferase assay The JASPAR database (http://jaspar.genereg.net/) was utilized for the prediction of potential binding regions for NFIX within the TSPAN2 promoter, and 5 potential binding sites were identified. Accordingly, we designed 4 kinds of delete mutants for the TSPAN2 promoter-driven luciferase reporter plasmids to clarify the specific transcriptional regulatory regions by NFIX. On the day before transfection, human endometrial cancer cell line ECC-1 was seeded in twelve-well culture plates at a confluence of 70–80%. Transfections were carried out using Lipofectamine 3000 following the manufacturer’s protocol. Each transfection involved the use of 2 μg of pCDNA3.1(+)-NFIX overexpression or control pCDNA3.1(+) plasmids (Genomeditech), 1 μg of pGL3 plasmids containing site mutants designed for the TSPAN2 promoter (Genomeditech) and carrying the firefly luciferase reporter construct, along with 90 ng of Renilla luciferase reporter plasmid pRL-TK (Promega, E2241) as an internal control. Cells were gathered 48 h post transfection, and the luciferase assay system (Promega, E1500) was used to measure the activity of luciferase. The activity of firefly luciferase was standardized based on the activity of Renilla luciferase in each well. Chromatin immunoprecipitation (ChIP) assay ChIP assay was conducted with a ChIP assay kit (Pierce Chromatin Prep Module; Thermo Scientific, 26,156) following the guidelines provided by the manufacturer. In short, the cells were treated with 1% formaldehyde for cross-linking and subsequently gathered in chilled PBS with 1% protease inhibitors. The cells that had been cross-linked were subsequently broken down, and the genomic DNA was fragmented through enzymatic digestion using micrococcal nuclease. The fragmented chromatin was referred to as “input.” Ten percent of the fragmented chromatin was extracted and preserved at −20 °C for use as an “input” control. This sample was employed for IP elution and subsequent retrieval of DNA. The remaining digested chromatin was subjected to immunoprecipitation using anti-NFIX antibody (Novus, NBP2-15039) and IgG antibody. The protein/DNA complexes were subsequently released from the beads. The purified DNA was then analyzed by real-time PCR. The following primers were used: 5′-CCCTGGACACTCAGTCAGGT-3′ (forward) and 5′-ACTTGGTCTCGTGGCTATGG-3′ (reverse) for the predictive DNA binding site on the TSPAN2 promoter. Endometriosis mouse model The mouse model of endometriosis was established following the method described in a previous study by Han et al. [12]. Eighteen female C57BL/6J mice, aged 8 weeks, were sourced from Beijing Vital River Laboratory. Two days before auto-transplantation, estrogen (Sigma) was injected intramuscularly. On the day of the surgical procedure, the mice were administered anesthesia. One side of the uterine horn was isolated and a longitudinal incision was made, the muscle layer was removed, and the remainder of the uterine horn was cut into two small pieces of approximately 4 × 4 mm2. These pieces were sutured to the abdominal wall in each flank, with a rich blood supply. After the operation, intramuscular injections of estrogen were administered to maintain the endometriotic lesions. Two weeks after establishment of the endometriotic lesions, the mice were randomly allocated into three separate groups: (1) the PBS group, (2) the negative control siRNA (in vivo siRNA) group, and (3) the si-NFIX (in vivo siRNA) group. Subsequently, intraperitoneal injection of PBS alone or PBS-compatible in vivo negative control small interfering RNA (in vivo si-NC) or NFIX-targeting siRNA (in vivo si-NFIX) (RiboBio, China) was conducted every 3 days. The siRNA sequences used were as follows: In vivo NFIX siRNA, 5′-TAACCATCACGGGCAAGAA-3′. On the 15th day following the initial injection, the mice were euthanized for sample collection. The sizes of the tissue implants were determined using the following method: volume = 0.5 × length × width2. We gathered the implanted endometrial lesions and extracted RNA and protein for quantitative analysis. HE staining The implanted endometrial lesions of the mouse model were gathered and preserved in 4% paraformaldehyde for a period of one night. The specimens underwent dehydration in a sequence of ethanol and xylene before being encased in paraffin. Subsequently, 5 μm sections were obtained from the samples. The HE staining (HE; Beyotime, C0105) was carried out following established procedures. Statistical analyses All trials were conducted on a minimum of three occasions. The two-tailed Student’s t-test was employed to determine the comparison between two groups, while one-way analysis of variance (ANOVA) was used for comparisons involving multiple groups. The data were presented as mean values accompanied by standard errors, and significance was defined as a P-value below 0.05. The SPSS 17.0 software was used to conduct statistical analysis on the experimental data. Data graphs were drawn using GraphPad Prism 5. The results of western blot were analyzed using the ImageJ software.

Identification of ESCs and EMs In this study, we collected self-paired eutopic and ectopic endometrial tissues from ovarian endometriomas and isolated primary endometrial stromal cells (EMs) and endometriotic stromal cells (ESCs). As shown in Fig. 1A, the cultured EMs and ESCs exhibited a fibroblast-like morphology. Immunofluorescence analysis of the cultured stromal cells showed vimentin-positive staining, indicating the presence of a stromal cytoskeletal marker, while cytokeratin staining was negative, suggesting the absence of an epithelial marker (Fig. 1B). The stromal cell characteristics of the EMs and ESCs were confirmed. NFIX expression is highly elevated in ESCs We analyzed our previous RNA sequencing data [28] together with publicly available single-cell RNA sequencing data from endometriosis patients and healthy controls (GSE179640 and GSE213216). Among all cell types, we identified that NFIX was mainly localized and highly expressed in ESCs in patients with endometriosis (Fig. 2A). Endometriosis is recognized as a benign disease with malignant behaviors. Recently, NFIX has been shown to exist as an oncogene in multiple cancers. However, very few studies have reported the effects of NFIX on endometriosis. For verification, we investigated the difference in the expression of NFIX in paired EMs and ESCs by real-time PCR and western blotting and found that the expression of NFIX at both the mRNA level (Fig. 2B, n = 15, P < 0.001) and the protein level (Fig. 2C, n = 15, P < 0.0001) was significantly higher in ESCs than in EMs. In addition, the immunohistochemical analysis indicated that the NFIX protein was present in both endometrial glandular epithelial cells and stromal cells, with notably stronger staining in the stromal compartment of endometriotic tissues (Fig. 2D, n = 6). The above results suggest that the level of NFIX expression exhibits a notable increase in the ESCs of patients with endometriosis, supporting its potential involvement in the pathophysiology of endometriosis. NFIX regulates the proliferation and invasion of ESCs To explore whether highly expressed NFIX in endometriotic lesions can regulate the proliferation and invasion of ESCs, NFIX was knocked down and overexpressed in ESCs via si-NFIX and an NFIX overexpression plasmid (pENTER-NFIX) transfection, respectively. The knockdown efficiency (Fig. 3A, n = 3; P < 0.01) and overexpression efficiency (Fig. 3B, n = 3; P < 0.01) were verified by western blotting. As shown by real-time PCR, the mRNA levels of Cyclin B1 and MMP9 decreased after NFIX was knocked down in ESCs (Fig. 3C, n = 5; P < 0.05, P < 0.05). The expression of Cyclin B1 and MMP9 proteins exhibited consistent reductions (Fig. 3D, n = 3; P < 0.05, P < 0.01). NFIX knockdown resulted in a notable decrease in the proliferation and invasion capacities of ESCs, as evidenced by the outcomes of CCK8 cell proliferation and Transwell invasion assays (Fig. 3E, n = 3; P < 0.05, P < 0.0001). In contrast, real-time PCR and western blot analyses showed that transfection of the pENTER-NFIX plasmid resulted in increased expression of Cyclin B1 and MMP9 at both the mRNA level (Fig. 3F, n = 3; P < 0.05, P < 0.05) and protein level (Fig. 3G, n = 3; P < 0.05, P < 0.001). The CCK8 cell proliferation and Transwell invasion assays yielded consistent results (Fig. 3H, n = 3; P < 0.05, P < 0.05). These results indicate that NFIX acts as a positive regulator of ESC proliferation and invasion. The downstream mechanisms mediating this regulatory effect warrant further investigation. TSPAN2 is highly expressed in ESCs, and is regulated by NFIX in endometriosis The transcription factor NFIX is a DNA-binding protein that can recognize and bind to a TTGGC(N5)GCCAA sequence or a TGCCA sequence in its target genes, thereby regulating expression. The KnockTF2.0 database (http://www./KnockTF/search.php) was utilized for the prediction of target genes under the regulation of the transcription factor NFIX. The intersection of NFIX target genes retrieved from the KnockTF2.0 database and differentially expressed genes identified in our RNA sequencing dataset was analyzed using the Venny platform. Among these overlapping genes, we further identified tetraspanin-2 (TSPAN2) as a target of NFIX. Subsequently, 33 overlapping targets were identified through the intersection of NFIX target genes obtained from the KnockTF2.0 database (GSE45492, q < 0.1) and differentially expressed genes (DEGs) derived from our RNA sequencing dataset comparing eutopic and ectopic endometrial tissues (q < 0.1) [28]. This analysis was performed using the Venny platform (Fig. 4A) and visualized as a heatmap (Fig. 4B). Among the top 10 upregulated intersecting genes, TSPAN2 emerged as a strong candidate target. Consistently, real-time PCR and western blot analyses confirmed that TSPAN2 expression was markedly higher in ESCs than in EMs at both the mRNA (Fig. 4C, n = 15; P < 0.001) and protein (Fig. 4D, n = 15; P < 0.01) levels. Moreover, as shown by immunohistochemical staining, TSPAN2 was found to be present in both stromal cells and glandular epithelial cells, with a higher concentration detected specifically in stromal cells of endometriotic tissues (Fig. 4E, n = 6). Furthermore, we investigated the impact of NFIX on the regulation of TSPAN2 expression. Transfection with si-NFIX led to notable reductions in the levels of TSPAN2 mRNA and protein expression in ESCs (Fig. 4F, n = 5; P < 0.05, P < 0.01). In contrast, NFIX overexpression upregulated TSPAN2 mRNA and protein expression in ESCs (Fig. 4G, n = 3; P < 0.001, P < 0.001). These findings identify TSPAN2 as a direct downstream target of NFIX and confirm that NFIX positively regulates TSPAN2 expression in ESCs. TSPAN2 mediates the regulatory effect of NFIX on the proliferation and invasion of ESCs Given that NFIX regulates TSPAN2 expression in ESCs, we next investigated whether TSPAN2 is required for NFIX-mediated control of ESC proliferation and invasion. First, we transfected ESCs with si-TSPAN2 and pENTER-TSPAN2 to specifically knock down and overexpress TSPAN2, respectively, and the efficiency of knockdown (Fig. 5A, n = 3; P < 0.01) and overexpression (Fig. 5B, n = 3; P < 0.01) of TSPAN2 was verified by western blotting. Decreased expression of Cyclin B1 and MMP9 was detected at both the mRNA level (Fig. 5C, n = 3; P < 0.05, P < 0.05) and the protein level (Fig. 5D, n = 3; P < 0.05, P < 0.05) following TSPAN2 knockdown, while elevated expression of Cyclin B1 and MMP9 was observed at both the mRNA level (Fig. 5E, n = 3; P < 0.01, P < 0.05) and the protein level (Fig. 5F, n = 3; P < 0.01, P < 0.001) following TSPAN2 overexpression. Moreover, the results of the CCK8 cell proliferation assay and Transwell invasion assay showed that knockdown of TSPAN2 inhibited the proliferation and invasion of ESCs (Fig. 5G, n = 3; P < 0.05, P < 0.05), while TSPAN2 overexpression promoted the proliferation and invasion of ESCs (Fig. 5H, n = 3; P < 0.05, P < 0.01). The above experimental results suggest that the highly expressed TSPAN2 in ESCs promotes cell proliferation and invasion. To further determine whether TSPAN2 mediates NFIX function, ESCs were transfected with si-NFIX, pENTER-TSPAN2, or both. As expected, si-NFIX alone decreased Cyclin B1 and MMP9 expression and impaired cell proliferation and invasion, while TSPAN2 overexpression alone elevated these parameters. Importantly, co-transfection with pENTER-TSPAN2 partially rescued the inhibitory effects of NFIX knockdown on Cyclin B1/MMP9 expression (Fig. 5I, n = 3; P < 0.05) as well as on proliferation and invasion (Fig. 5J, n = 3; P < 0.05). Collectively, these results indicate that TSPAN2 acts as a key downstream effector mediating the regulatory role of NFIX in promoting ESC proliferation and invasion. Identification of the critical promoter region through which NFIX regulates TSPAN2 expression Mechanistically, we utilized the JASPAR database (http://jaspar.genereg.net/) to predict potential binding sites of NFIX within the promoter region of TSPAN2, and identified five putative binding sites as follows: −1406 bp to −1398 bp (P1), −1289 bp to −1281 bp (P2), −1255 bp to −1247 bp (P3), −771 bp to −763 bp (P4), and −408 bp to −400 bp (P5) (Fig. 6A). In order to identify the specific and critical binding site through which NFIX regulates TSPAN2 expression in ESCs, we constructed a series of reporter plasmids. The full-length promoter region of TSPAN2 (−1947 bp to +52 bp) was cloned into the pGL3 vector to generate the wild-type reporter plasmid, designated as pGL3-WT. Additionally, multiple truncation mutants of the TSPAN2 promoter were generated as follows: pGL3-DEL1 (deletion of P1, −1334 bp to +52 bp), pGL3-DEL2 (deletion of P1–P3, −963 bp to +52 bp), pGL3-DEL3 (deletion of P1–P4, −589 bp to +52 bp), and pGL3-DEL4 (deletion of P1–P5, −378 bp to +52 bp). Given that the binding sites P2 and P3 within the TSPAN2 promoter are closely adjacent, it is difficult to construct a reporter plasmid containing only the truncated P2 sequence. The luciferase reporter plasmids containing these various promoter regions were transfected into ECC-1 cells with the Renilla luciferase reporter plasmid and the NFIX overexpression plasmid. Luciferase reporter assays showed that NFIX overexpression significantly enhanced the luciferase activity of the full-length promoter (pGL3-WT) compared with the control. Notably, deletion of P5 (pGL3-DEL4) completely abolished the NFIX-induced increase in luciferase activity, whereas the other truncated constructs retained NFIX responsiveness (Fig. 6B, n = 3; P < 0.01). These findings indicate that NFIX transcriptionally activates TSPAN2 and that the −408 to −400 bp region (P5, sequence CCTGCCAGC) is essential for this activation. Subsequently, we carried out chromatin immunoprecipitation (ChIP) assays using the self-paired EMs and ESCs and found that the binding of NFIX to the −408 bp to −400 bp region of the TSPAN2 promoter was greater in ESCs than in EMs, indicating that NFIX regulates the expression of TSPAN2 via direct binding (Fig. 6C, n = 3; P < 0.05). Knockdown of NFIX inhibits the growth of endometriotic grafts in vivo To confirm the effects of NFIX on endometriotic tissues in vivo, an endometriosis mouse model was established, and a randomized allocation was employed to divide the mice into 3 groups (Fig. 7A): the PBS control group, the PBS + si-NC (in vivo siRNA) group, and the PBS + si-NFIX (in vivo siRNA) group. Fifteen days after the first injection, the mice were sacrificed, endometriotic grafts were collected, and pathological examinations were conducted (Fig. 7B). The volume of ectopic lesions in the PBS + si-NFIX group was significantly reduced compared with that in the PBS + si-NC group (Fig. 7C, n = 6; P < 0.001). Real-time PCR and western blot analyses were used to evaluate the expression of target genes in the ectopic lesions of mice in each group, and the results showed that the mRNA (Fig. 7D, n = 6; P < 0.05, P < 0.05, P < 0.01, P < 0.01) and protein (Fig. 7E, n = 6; P < 0.05, P < 0.001, P < 0.05, P < 0.05) levels of NFIX, TSPAN2, Cyclin B1, and MMP9 in the PBS + si-NFIX group were lower than those in the PBS + si-NC group. These results suggested that NFIX knockdown suppresses the growth of endometriotic grafts.

and conclusion Although endometriosis is a non-malignant condition, it exhibits characteristics that resemble those found in malignancies. Intensive studies have indicated the regulatory effects of NFIX on cancer progression [15,16,17]. However, as far as we are aware, there has been no research conducted on the involvement of NFIX in endometriosis. This is the first study of NFIX in ESCs and EMs and provides the first demonstration of the role of NFIX in modulating cell proliferation and invasion in the context of endometriosis. Here, we revealed that NFIX is enriched in ESCs and that the highly expressed NFIX in ESCs transcriptionally activates TSPAN2 expression through directly binding to the −408 ~ −400 bp region of the TSPAN2 promoter, in turn further increasing proliferation and invasion. Moreover, inhibition of ectopic lesion growth was observed in endometriosis model mice after NFIX knockdown, suggesting NFIX as a potential target for endometriosis treatment. The dual function of NFIX has been identified in different cancer types. In pancreatic cancer, the growth and migration of cancer cells were observed to be restored by the overexpression of NFIX, counteracting the inhibitory effects caused by MAFG-AS1 knockdown [29]. In lung adenocarcinoma, the independent prediction of poor prognosis was associated with the downregulation of NFIX [30]. Conversely, in colorectal cancer and esophageal squamous cell carcinoma, reduced expression of NFIX was positively associated with cancer progression [15, 31]. The above evidence showed that NFIX has the potential to act as either a tumor promoter or a tumor suppressor, indicating a possible cancer-type dependent role of NFIX. In addition, NFIX is characterized as a transcription factor and is thought to function as either an activator or a repressor in transcriptional regulation. Indeed, Brun et al. revealed that in glioblastoma cells, NFIX negatively regulates the expression of Hes-related family BHLH transcription factor with YRPW motif 1 (HEY1) by binding to its promoter, resulting in decreased cell proliferation [32]. Rossi et al. showed that NFIX represses the Myostatin promoter, thus influencing muscle regeneration [33]. However, Messina et al. showed that in developing skeletal muscle, NFIX can activate the transcription of fetal-specific genes, such as beta-enolase [34]. Hence, the dual effects of NFIX on the transcription of its target genes, which may be linked to different bioactivities, may also account for the difference in its role in different contexts. In our study, we found that ESCs transfected with si-NFIX exhibited decreased proliferation and invasion, while ESCs transfected with the NFIX overexpression plasmid exhibited increased proliferation and invasion, indicating the promoting role of NFIX in endometriosis progression. In addition, we found that TSPAN2, a target gene of NFIX, mediated the effect of NFIX on the proliferation and invasion of ESCs. TSPAN2 is a newly identified TSPAN family member, and its precise functions in different tissue types are unclear. TSPAN2 was shown to induce the invasion of lung cancer cells, and knockdown of TSPAN2 in a mouse model inhibited lung metastasis [35]. In contrast, in vascular smooth muscle cells, TSPAN2 was reported to suppress cell proliferation and migration [36]. Thus, TSPAN2 also has dual impacts on cell proliferation and invasion, and the difference in its impact on these behaviors may be cell specific. The present study, for the first time, elucidated the promoting role of TSPAN2 in endometriosis. Moreover, reversal of the inhibitory impacts caused by NFIX knockdown on ESCs’ proliferation and invasion was observed through the overexpression of TSPAN2. Normally, unscheduled expression of cell cycle-related genes tends to result in uncontrolled cell proliferation. CyclinB1 is a critical factor controlling the transition from the G2 phase to mitosis (M phase) and thus causes cells to begin proliferating [37]. In brief, cyclinB1 expression progressively increases throughout the cell cycle and peaks in the G2 phase. Then, cyclin B1 forms a complex with cyclin-dependent kinase 1 (CDK1) and activates CDK1 on centrosomes, allowing the transition to M phase [37]. Lv et al. showed that cyclinB1 suppression resulted in G2/M arrest and subsequent inhibition of proliferation in hepatocellular carcinoma cells [38]. Moreover, cyclinB1 has been reported to be enriched in ectopic endometrium compared to eutopic endometrium [39]. In this study, we measured the mRNA and protein levels of cyclinB1 and performed a CCK8 assay following NFIX or TSPAN2 knockdown or overexpression to demonstrate the roles of NFIX and TSPAN2 in proliferation in the context of endometriosis. We found that elevated levels of NFIX and TSPAN2 induced cyclinB1 expression in ESCs, resulting in cell proliferation. Matrix metalloproteinases (MMPs) are enzymes that break down the extracellular matrix, thereby facilitating the spread of cancer cells. MMP9 has the ability to break down type IV collagen, which is a key constituent of the basement membrane, and is recognized as a critical MMP for cell invasion. MMP9 has been indicated to be implicated in tumor cell invasion and metastasis [40,41,42]. Numerous studies have indicated that MMP9 is upregulated in endometriosis, especially in ectopic stromal cells [43, 44]. Moreover, Han et al. observed a reduced volume of ectopic lesions in generated MMP9-/- endometriosis model mice, suggesting an essential role of MMP9 in endometriotic tissue growth [45]. Herein, we measured the mRNA and protein levels of MMP9 and performed Transwell assays following NFIX or TSPAN2 knockdown or overexpression to demonstrate the roles of NFIX and TSPAN2 in invasion in endometriosis, and we found that elevated levels of NFIX and TSPAN2 induced MMP9 expression in ESCs, resulting in cell invasion. Despite the limited knowledge of TSPANs, emerging evidence has indicated the regulatory mechanisms of TSPAN expression. Phosphorylation of TSPAN8 at Ser129 by the kinase AKT has been proven to contribute to its nuclear translocation in a membrane-free form, therefore regulating the transcription of downstream cancer-promoting genes [46]. TSPAN9 was found to be a target of miR-9-5p and can be negatively regulated by miR-9-5p via direct binding to its 3′-untranslated region (UTR) [47]. In addition, a previous study mentioned that decreased expression of TSPAN2 was strongly associated with methylation at cg23999170, which is located in the first intron of TSPAN2 [48]. Zhao et al. used luciferase reporter and ChIP assays in vascular smooth muscle cells to determine that TSPAN2 can be regulated by the MYOCD/SRF and TGF-β1/SMAD pathways via binding to the CC(A/T)6GG(CArG) box and Smad-binding element (SBE), respectively, in its promoter [36]. In the present study, for the first time, we identified the increased expression of TSPAN2 in ESCs and found that TSPAN2 can be upregulated by NFIX. However, the underlying regulatory mechanism needs to be elucidated. The JASPAR database, a transcription factor-binding site search tool, was used to predict five binding sites of NFIX, located at −408–400 bp, −771–763 bp, −1255–1247 bp, −1289–1281 bp, and −1406–1398 bp, in the promoter of TSPAN2 upstream of the TSPAN2 transcription initiation site (+1). Sequentially, we generated deletion mutants and performed luciferase assays and found that the −408–400 bp binding site is critical for transcriptional activation of TSPAN2 expression after binding of NFIX. Additionally, the ChIP assay revealed that the binding affinity of NFIX for the −408–400 bp binding site of TSPAN2 in ESCs, which exhibit elevated expression of NFIX, is higher than that in EMs, confirming the role of direct binding of NFIX in the regulation of TSPAN2 expression in endometriosis. In conclusion, our study uncovers a previously unrecognized NFIX–TSPAN2 axis that drives endometriosis progression. The highly expressed NFIX in ESCs transcriptionally activated TSPAN2 expression by binding to the −408–−400 bp region of its promoter, in turn further increasing the proliferation and invasion of ESCs. Furthermore, knockdown of NFIX in endometriosis model mice inhibited ectopic lesion growth, highlighting NFIX as a promising candidate for therapeutic intervention in endometriosis. Data availability The authors declare that all data supporting the findings of this study are available within the article or from the corresponding author upon reasonable request.

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Yuqi Liu and Jie Li performed the statistical analysis. Jing Shang, Qing Xue and Peili Wu critically revised the manuscript. Jingwen Zhu and Cheng Zeng contributed to experimental guidance. Xin Li contributed to clinical sample collection. All authors contributed to the article and approved the submitted version. Corresponding author Ethics declarations Ethics approval The cell experimental procedures were approved by the institutional review board of the First Hospital of Peking University (No. 2021[446]), and signed informed consents for use of the samples were obtained from each patient. The First Hospital of Peking University Animal Care Committee approved the use of mice for this study. Conflict of interest The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. About this article Cite this article Liu, Y., Xue, Q., Zhu, J. et al. Nuclear factor IX promotes endometriosis progression through transcriptional activation of tetraspanin-2. J Mol Med 104, 73 (2026). https://doi.org/10.1007/s00109-026-02665-x Received: Revised: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s00109-026-02665-x