CCL21 promotes the development of endometriosis by activating NF-κB signaling pathway

CCL21 promotes the development of endometriosis by activating NF-κB signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CCL21 promotes the development of endometriosis by activating NF-κB signaling pathway Ning wang, Bo Liu, Yiyuan Zhang, Xiaoqian Man, Rujiao Jiang, Liang Yao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7558971/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Endometriosis, affecting approximately 10% of reproductive-aged women worldwide, is a typical chronic inflammatory disease. Inflammation-related genes (IRGs) play a crucial role in the occurrence and progression of various diseases, including endometriosis. However, identifying which IRGs affect the pathology of endometriosis and how to target them for treatment remains highly challenging. In the present study, the ectopic endometrium (EcE) and eutopic endometrium (EuE) from endometriosis patients were collected and a mouse model of endometriosis was established to investigate the expression levels of inflammatory factors. Three hub IRGs (CCL21, CFD, and ACKR1) in endometriosis were identified, which were able to accurately predict the occurrence of endometriosis. The expression of CCL21 was obviously increased in the EcE of endometriosis patients and mouse models. Knockdown of CCL21 inhibited the proliferation, migration, and invasion of 12z cells and promoted apoptosis. Mechanistically, reduced CCL21 alleviated the pathology of endometriosis through restraining the activation of the NF-κB signaling pathway and the downstream inflammatory factors (IL-6, IL-1β, and TNF-α). In conclusion, increased CCL21 promoted the development of endometriosis by activating the NF-κB signaling pathway, and this finding offers a novel therapeutic target for treatment. Endometriosis Inflammation-related genes CCL21 nomogram model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Endometriosis is characterized by the presence of endometrium-like tissue (including glands and stroma) outside the uterus, affecting approximately 10% of reproductive-age women worldwide [1]. This disease severely impairs the quality of life, commonly manifesting as pelvic pain and infertility. Notably, endometriosis is present in 25%-50% of infertile women, while 30%-50% of endometriosis patients experience infertility [2, 3, 4]. Additionally, it elevates the risk of epithelial ovarian cancer [5]. Although progestin-based therapy for endometriosis could cause the condition to regress in some patients, hormone treatment has unacceptable side effects, including menopausal symptoms, and is not effective in endometriosis patients with pelvic pain who are responsive to progesterone [6, 7]. Surgical removal of ectopic endometrial tissue is one of the effective treatments for endometriosis [8], but the recurrence rate of the surgery is high [9]. Despite advances in understanding its clinical burden, endometriosis incidence continues to rise. Therefore, elucidating the pathophysiology of endometriosis and its molecular mechanisms underlying infertility and pelvic pain represents a critical research imperative. Endometriosis has been well-established as a typical chronic inflammatory disease [10]. A series of changes occur in inflammatory cells, cytokines, and chemokines within endometriotic lesions and peritoneal fluid, forming an inflammatory microenvironment. This microenvironment interacts with endometriotic cells and plays a pivotal role in the development and progression of endometriosis [11]. During the formation of endometriotic lesions, inflammatory cells are recruited to the lesion site and release a variety of inflammation-related mediators such as cytokines and chemokines [12]. These inflammation-related mediators in turn act on the inflammatory cells, leading to the aggregation of more inflammatory cells within the endometriotic lesions [1]. This interaction between the inflammatory cells and substances forms a vicious cycle, resulting in the accumulation of endometriosis-related inflammation, which exacerbates the occurrence and progression of endometriosis. Inflammation also affects the carcinogenic process of endometriosis [13, 14]. Therefore, investigating the molecular and cellular mechanisms underlying the pathogenesis of endometriosis from the perspective of inflammation-related genes (IRGs) is of great significance for the development of advanced treatment approaches. In this study, we employed multifarious bioinformatics methods to identify potential hub IRGs and investigated their pathological roles in endometriosis. A novel nomogram based on the screened hub IRGs demonstrated good predictive value. Notably, CCL21 was significantly elevated in the lesion tissues of endometriosis. Using small interfering RNA technology, we established CCL21 knockdown cells to explore the impact of CCL21 on proliferation, migration, and invasion capabilities of 12z cells. Additionally, we attempted to investigate the mechanism by which CCL21 influences endometriosis through the NF-κB signaling pathway. Materials and methods Clinical samples Human EcE and EuE tissues (n = 8) were obtained from endometriosis patients undergoing surgical resections at the Second Affiliated Hospital of Dalian Medical University (Dalian, China) between June 2024 to December 2024. The age range of the patients was 28‑42 years. All patients have filled out the informed consent form before sample collection. The Ethics Committee of the Second Affiliated Hospital of Dalian Medical University approved the research protocol (Approval No. KY2024-174-01). Antibodies Primary antibodies used were anti-CCL21 (DF6681), anti-TNF-α (AF7014), anti-IL-6 (DF6087), anti-IL-1β (AF5103) and NF-κB (AF5006) from Affinity Biosciences; anti-β-Actin (20536-1-AP) from Proteintech. Secondary antibodies horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208), Alexa Fluor 488-/647- labeled goat anti-rabbit IgG(H+L) (A0423/A0468) from Beyotime. Data aquation and gene expression analysis RNA-seq data were retrieved from The Cancer Genome Atlas (TCGA) database, including GSE7305, GSE51981, GSE5108, GSE11691, GSE23339, GSE25628 datasets. Detailed information was shown in Supplementary Table 1. The “Limma” package was used to identify the differentially expressed genes (DEGs) of the control and endometriosis groups. All 737 protein-coding IRGs were selected and downloaded from the GeneCards database with a relevance score > 5. Functional enrichment analysis of DEGs Genes that exhibited increased expression in both GSE7305 and GSE51981 compared to control group, as well as genes that showed decreased expression in both GSE7305 and GSE51981, were referred to as common DEGs. Next, we applied “clusterProfiler” for Gene Ontology (GO) enrichment analysis, covering biological processes (BPs), cellular components (CCs), and molecular functions (MFs). Furthermore, we utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) to pinpoint the key pathways among DEGs. Weighted Gene Co-expression Network Analysis (WGCNA) To determine the module most relevant with endometriosis, we conducted WGCNA analysis in GSE51981 using the R package “WGCNA”. Specifically, we preprocessed the sample data, excluding any outliers, and then employed the “WGCNA” to construct a correlation matrix. The optimal soft threshold was chosen to transform the correlation matrix into an adjacency matrix, and then generated a Topological Overlap Matrix (TOM) from the adjacency matrix. Employing TOM-based phase dissimilarity metric, we classified genes with similar expression patterns into gene modules through average linkage hierarchical clustering. The module most strongly associated with endometriosis patients was selected for subsequent analysis. Nomogram construction and receiver operating characteristic (ROC) curve Intersecting genes of DEGs, WGCNA, and IRGs were considered candidate hub genes for endometriosis diagnosis. To assess the significance of hub genes in diagnosing endometriosis, we built a nomogram using “rms” R package. Additionally, we constructed the relevant calibration curve to evaluate the accuracy and reliability of the nomogram model’s predictions. We also assessed the prognostic value of hub genes and the nomogram through ROC curve. Gene Set Enrichment Analysis (GSEA) To identify the pathways enriched by CCL21, we divided the patients with endometriosis into high- and low-CCL21 expression groups based on the median CCL21 value. The CCL21-enriched KEGG pathways were identified using GSEA analysis with the “clusterProfiler” package (version 3.16.1). Construction of mice model of endometriosis BALB/c female mice, 8 week-old, were housed in specific pathogen-free experimental animal facilities (24 ± 1 ℃) with free food and water in Dalian Medical University. The experiment was divided into two groups: endometriosis model group and sham surgery control group, with 8 mice in each group. The entire surgical procedure was performed in a biosafety cabinet. Before surgery, the mice abdomen was depilated and disinfected sequentially with iodine and alcohol cotton balls. To established endometriosis model, a midline abdominal incision was performed on the female mice to expose the uterus under anesthesia with isoflurane, and followed by the excision of one uterine horn. The uterine horn was longitudinally split with scissors in a petri dish containing phosphate buffered saline (PBS). The excised uterine horn was cut into small fragments of approximately 2 mm 2 , which were then reimplanted into the mesentery of the same mice, followed by wound suturing[15, 16]. The control group (sham surgery) mice were anesthetized and opened using the same method to create a model. Without separating the uterus, the abdominal incision is directly sutured continuously, and the abdomen is routinely closed. After the surgery, the mice were carefully cared for. After 30 days of feeding, the mice were anesthetized using isoflurane and subsequently euthanized via cervical dislocation, and the endometriosis-like lesions that were removed were counted under a microscope for further experiments. The pre-transplant uterine tissues represent as EuE and endometriosis-like lesions refer to EcE in this study. Hematoxylin and eosin (H&E) staining After dewaxing and hydrating the sections, hematoxylin was used to stain the cell nucleus being deep blue. Following differentiation with 1% alcohol hydrochloride, eosin solution was used to stain the proteins pink, and finally, dehydration was achieved using a gradient of alcohol. Prussian blue (PB) staining The iron content within samples was measured using the PB staining kit (Yeasen, China) according to the manufacturer’s instructions. The iron-containing areas were stained with the reagent to exhibit a characteristic blue color. Cell culture and establishment of CCL21 knockdown Cells The 12z cells were acquired from the Cell Bank of the Shanghai Institute of Life Sciences (Chinese Academy of Sciences, Shanghai, China). Cells were cultured in DMEM/F12 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, China) and 100 U/ml penicillin-streptomycin (Seven, Beijing, China) in an incubator (Thermo Fisher Scientific, United States) at 37℃ with 5% CO 2 . Three targeting sequences of CCL21 small interfering (si) RNA were used to reduce the expression of CCL21 (Supplementary Table 2). 12z cells were transfected with 100 pmol of control siRNA or CCL21 siRNA, using 10 µl of Lipofectamine 3000 (L3000015, Invitrogen). Western blotting Protein samples form tissues or cells were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized using an enhanced chemiluminescence system (Beyotime, China). Quantification of the data was performed using ImageJ software. Real-time quantitative PCR (RT-qPCR) Total RNA was extracted using Trizol™ reagent (Invitrogen, USA) and converted to cDNA utilizing the Prime Script RT-PCR Kit (Seven Biotech, Beijing, China). cDNA amplification was performed using the Applied Biosystems Prism 7000 Sequence Detection System. The relative fold change of gene amplification was calculated according to the 2 -ΔΔCt analysis. The primer sequences used were provided in Supplementary Table 3. Immunofluorescence After dewaxing, hydrating and blocking the sections of ectopic endometrial tissues of patients and mice, they were incubated with primary antibody overnight and then incubated with fluorescent secondary antibody. The sections were ultimately examined under a fluorescence microscope (Nikon, Japan). Wound healing assay The si-CCL21 and control cells were individually seeded in each well of a 6-well plate to reach complete confluence. A scratch was created using a 200 µl sterile pipette. The cells were incubated in serum-deprived medium for 24- and 48-hrs, after which images of the same area were taken. Transwell The upper chamber of 24-well Transwell (Jet Biofil, China) was treated with 40 µl of Matrigel (Corning, USA) and incubated at 37℃ for 2 hrs. A total of 200 µl of si-CCL21 and control cells at a density of 1 × 10 4 in serum- deprived medium was respectively placed in the upper chamber, whereas 550 µl of medium containing FBS was supplemented to the lower chamber. Following a 48-hrs period, the upper chamber was washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Solarbio, China) for 20 minutes. The cells were then photographed and counted. Cell apoptosis assay The si-CCL21and control cells were digested and collected using trypsin without EDTA. The cells were treated with Annexin V-FITC and PI (Beyotime, China) in dark for 25 minutes. Subsequently, apoptosis rate detected using flow cytometry. Statistical analysis Data were presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis and visualization were conducted using GraphPad Prism 9.0, R 4.1.3, ImageJ, and FlowJo. A two-tailed Student’s t-test was used to assess the statistical differences between two groups. One-way ANOVA and two-way ANOVA were utilized to analyze statistical differences among multiple groups. P < 0.05 was considered statistically significant. Results The inflammation levels were increased in endometriotic lesions To assess levels of inflammation in endometriotic lesions, we investigated the expression of classical inflammatory factors in EuE and EcE of endometriosis patients. We first evaluated the expression of IL-6, IL-1β, and TNF-α in EcE from 8 endometriosis patients using immunofluorescence and RT-qPCR. Compared to EuE, the protein and mRNA levels of IL-6, IL-1β, and TNF-α were dramatically increased in EcE of endometriosis patients (Figure 1A-1B and Supplementary Figure 1A-1B). To further investigate the inflammation levels in EcE, we transplanted uterine tissue into the mesentery to construct mouse model of endometriosis. One month after the induction of endometriosis, the fluid-filled weight of endometriotic lesions was significantly increased compared to the baseline, indicating that we had successfully established mouse model of endometriosis (Figure 1C and 1D). Histological analysis revealed that the EcE in both endometriosis patients and the mouse model exhibited irregular shapes, abnormal arrangement, and increased cavitation (Figure 1A and 1E). Iron-overload is a characteristic feature of endometriosis [17, 18]. PB staining revealed that the iron-overloaded microenvironment resulted in localized iron accumulation in the mouse endometriotic lesions (Figure 1E). The levels of inflammatory factors (IL-6, IL-1β and TNF-α) were elevated in mouse endometriotic lesions (Figure 1F-1H and Supplementary Figure 1C-1D). These data reveal that increased inflammation levels positively correlate with the occurrence of endometriosis. Identification of DEGs between endometriosis and normal endometrium The microarray datasets GSE7305 and GSE51981 were obtained to screen the DEGs between endometriosis and normal endometrium. We identified 1,214 DEGs in the GSE7305 dataset and 1,984 DEGs in the GSE51981 dataset (Figure 2A and 2B). Among these, 49 genes were significantly upregulated in both datasets, while 158 genes were downregulated (Figure 2C). Subsequently, we conducted KEGG and GO enrichment analyses on common DEGs. KEGG enrichment findings indicated that these genes were predominantly involved in cell cycle, oocyte meiosis, cellular senescence, and P53 signaling pathway (Figure 2D). GO analysis showed that these genes were mainly enriched in organelle fission, spindle, and microtubule binding (Figure 2E-2G). Construction of WGCNA network of endometriosis To identify crucial genes associated with the pathogenesis of endometriosis more accurately, we performed WGCNA analysis on normal endometrium and endometriosis tissues from the GSE51981 dataset. Based on scale independence and mean connectivity, we set the soft threshold to 9 (Figure 3A). Through evaluating gene correlation, we assembled a hierarchical clustering dendrogram of genes, which enabled us to identify 6 distinct gene modules (Figure 3B-3D). To ascertain which module was closely associated with endometriosis, we conducted a correlation analysis comparing each module and phenotype. Our analysis indicated that the “lightcyan” module, which comprises 2,363 genes, exhibited the strongest correlation with endometriosis (Figure 3E). Additionally, a notable relationship between gene significance (GS) and module membership (MM) was observed within the “lightcyan” module (Figure 3F). Identification of hub genes in endometriosis Next, we conducted an intersection analysis on IRGs, DEGs, and “lightcyan” module genes from WGCNA, and we identified 3 hub genes, namely CCL21, CFD, and ACKR1 (Figure 4A). The expression of these three hub genes (CCL21, CFD, and ACKR1) was upregulated in endometriotic lesions of patients with endometriosis, as observed in GSE7305 and GSE51981 datasets (Figure 4B and 4C). The ROC curve indicated that the 3 hub genes had favorable diagnostic values for endometriosis within GSE7305 and GSE51981 datasets (Figure 4D and 4E). To evaluate the roles of these biomarkers (CCL21, CFD, and ACKR1) in the diagnosis of endometriosis, we developed a nomogram (Figure 4F). The calibration curve demonstrated the effectiveness of the predictive model (Figure 4G). Additionally, the nomogram model had accurate predictive capability (Figure 4H). Overall, the model constructed based on three biomarkers (CCL21, CFD, and ACKR1) can effectively predict the occurrence and progression of endometriosis. The expression of CCL21 was increased in endometriotic lesions To further investigate the importance of three hub genes in the development of endometriosis, we examined the levels of these genes across various datasets. It was observed that CCL21 was consistently upregulated across several datasets, including GSE5108, GSE11691, GSE23339, and GSE25628 (Figure 5A and Supplementary Figure 2A-2D). ROC curve results showed that CCL21 had good diagnostic value in endometriosis (Figure 5B). More importantly, we also found increased CCL21 expression in the endometriotic lesions of patients with endometriosis, as confirmed by our own transcriptome data (Figure 5C). CCL21 expression was elevated in endometriotic lesions of endometriosis patients and mouse model, as confirmed by Western blotting, immunofluorescence staining and RT-qPCR analysis (Figure 5D-5F and Supplementary Figure 3A). These results suggested that CCL21, a pivotal inflammation-related gene, was upregulated in the tissues of endometriotic lesions and contributed to the development and advancement of endometriosis. Knockdown of CCL21 attenuated endometriosis in vitro To clarify the relationship between CCL21 and endometriosis, we silenced the CCL21 gene in 12z cells. Initially, we designed specific CCL21 siRNA sequences targeting different regions of the CCL21 open reading frame. Among three siRNA constructs tested, siRNA-3 (217-237) was the most effective in inhibiting CCL21 expression (Figure 6A and 6B). Consequently, we selected siRNA-3 (217-237) to knockdown CCL21 expression in the following experimental procedures. In immunofluorescence staining, the expression of CCL21 was decreased in si-CCL21 group compared to control group (Figure 6C). In addition, knocking down CCL21 significantly inhibited the migration and invasion of 12z cells (Figure 6D and 6E). Furthermore, the proliferative ability of 12z cells in si-CCL21 group was obviously reduced (Figure 6F). In contrast, reducing CCL21 level promoted the apoptosis rate of 12z cells (Figure 6G). Knockdown of CCL21 inhibited inflammatory expression in endometriosis To investigate the molecular mechanisms by which CCL21 regulates endometriosis, based on the median value of CCL21, we divided endometriosis patients in the GSE51981 dataset into high- and low-CCL21 groups. Subsequently, we performed a differential expression analysis and revealed 2,654 DEGs, of which 1,756 genes were elevated, and 898 genes were reduced in high-CCL21 group (Figure 7A and Supplementary Figure 4A-4C). We investigated the differences in signaling pathways between two groups. The GSEA results revealed that NF-κB and chemokine signaling pathways were enriched in high-CCL21 group. In contrast, pathways such as Oocyte meiosis were enriched in low-CCL21 group (Figure 7B). Western blotting and immunofluorescence staining results showed that NF-κB level was elevated in endometriotic lesions of endometriosis patients and mouse model (Figure 7C-7D and Supplementary Figure 5A). In addition, knockdown of CCL21 significantly inhibited NF-κB and its downstream IL-6, IL-1β, and TNF-α expression (Figure 7E and 7F). Discussion Endometriosis is characterized as a typical chronic inflammatory disease, and inflammation is pivotal in the pathogenesis of endometriosis, even in the process of endometriosis-related carcinogenesis [19, 20]. Inflammatory mediators (IL-1β, IL-6, and TNF-α) up-regulate the production of haptoglobin in endometrium of endometriosis patients, which binds to macrophages and subsequently reduces their phagocytic function, thereby promoting the establishment of endometriosis [21]. The enhanced inflammation in endometriosis is associated with the inhibition of endothelial function [21]. A. Catarina Neto et al. used metformin to reverse infertility associated with endometriosis, possibly due to its anti-inflammatory properties during pregnancy[22]. Despite this knowledge, the pathogenicity and molecular mechanisms of inflammation in endometriosis remain to be clarified. Therefore, early identification of IRGs that contribute to the pathogenesis of endometriosis is essential to provide better treatment strategies. In this study, we screened for potential hub IRGs through comprehensive bioinformatics analysis and established a predictive model based on three hub genes (CCL21, CFD, and ACKR1), which showed good predictive ability. These findings might strongly support further comprehensive research into precision treatment of endometriosis. To investigate the potential functions of hub genes further, we collected ectopic tissues from endometriosis patients and established endometriosis mouse model. Among hub genes, CCL21 was increased in both endometriosis patients and mouse model. Knockdown of CCL21 significantly inhibited the proliferation, migration, and invasion of endometriotic cells and promoted apoptosis. Further analysis revealed that reduced CCL21 could inhibit endometriosis development via suppressing NF-κB signaling pathway. Endometriosis is a complex chronic inflammatory gynecological disorder associated with infertility and endometriosis-related pain [23]. A study on patient generated data found that fatigue, headache, mental confusion, gastrointestinal problems, and pain are common in endometriosis phenotypes, all of which are characteristics of low-grade inflammation[24]. Although its mechanisms are yet to be fully elucidated, current research suggests that inflammatory changes in endometriotic lesions are related to the advancement of endometriosis. Inflammation is considered a significant contributor to pain [25]. An elevated level of IL-1β has been detected in the peritoneal fluid and lesion tissues of women with endometriosis and pelvic pain [26, 27]. The increased IL-1β directly enhances the production of nerve growth factors, correlating with localized neurogenesis around endometriotic lesions and deep dyspareunia [26]. The number of activated macrophages in the peritoneal fluid of women with endometriosis increases, secreting more cytokines, including IL-6 [28, 29]. The elevated IL-6 then inhibits the cytolytic activity of NK cells [30]. Higher concentrations of TNF-α in peritoneal fluid and serum of patients with endometriosis play a pivotal role in activating systemic and local inflammation associated with the progression of endometriosis [31]. In the present study, we also validated the expression of IL-6, TNF-α, and IL-1β in lesion tissues of endometriosis patients and mouse model. The mouse model of endometriosis constructed by transplanting cornu uteri into mesentery is characterized by proliferation and inflammation, but without epithelial mesenchymal transformation and fibrosis. This model simulates some but not all features of human endometriosis[32]. Considering the complex mechanisms of endometriosis and the sensitivity of patients to different drugs, the search for new diagnostic markers and therapeutic targets is critical. We identified 207 commonly DEGs between the endometriotic lesions and normal endometrium of endometriosis patients through GSE7305 and GSE51981 datasets. By using WGCNA analysis, we screened out the “lightcyan” module, which is most significantly positively correlated with endometriosis. After intersecting IRGs, DEGs, and “lightcyan” module genes from WGCNA, three hub genes (CCL21, CFD, and ACKR1) were identified. Among these, CCL21 was increased in the lesion tissues of endometriosis across multiple GEO datasets, indicating that upregulated CCL21 might promote the occurrence and progression of endometriosis. Yanzhen Zhou et al. reported the identification of three potential key genes (MET, IL4R, and BST2) associated with endometriosis[33]. This result is inconsistent with our research findings and may be due to differences in databases, but it cannot be ruled out that the sample may be different. Anyway, our experiment confirms the importance of CCL21. CCL21 is a key chemokine of the CC subfamily, a small molecular weight protein that exhibits chemotactic properties for cell migration [34]. It mediates a range of physiological and disease-related processes, encompassing stress responses, infection, wound healing, and cell differentiation. The abnormal expression of CCL21 is involved in inflammation and immune regulation in various autoimmune diseases [35, 36]. Researchers have found that CCL21 is significantly increased in the ectopic tissue of endometriosis patients through laser capture microdissection analysis [37]. However, the pathological role of CCL21 in endometriosis remains unknown. In our study, we found that the expression of CCL21 was elevated not only in the lesion tissues of endometriosis across multiple datasets but also in the lesion tissues of endometriosis patients and mouse model, suggesting that CCL21 might also be involved in the development of endometriosis. While endometriosis is a benign disease, it exhibits biological characteristics similar to cancer, such as excessive proliferation, metastasis, and invasion [38]. To further investigate the impact of CCL21 on endometriosis, we used small interfering RNA methods to reduce CCL21 expression in 12z cells. Compared to the control group, knockdown of CCL21 suppressed the proliferation, migration, and invasion capabilities of 12z cells and promoted apoptosis. These results suggest that CCL21 could alleviate the pathology of endometriosis. To further investigate how CCL21 regulates the development of endometriosis, we divided patients with endometriosis into high- and low-CCL21 groups according to median CCL21 value. GSEA results showed that the high-CCL21 group was enriched in NF-κB and chemokine signaling pathway, while the low-CCL21 group was primarily enriched in oocyte meiosis. These results suggest that the inflammation in lesion tissues of patients in high-CCL21 group was increased, and the maturation of follicles was inhibited, which also indicated that patients in the high-CCL21 group might experience infertility. NF-κB regulates multiple genes in response to various stimuli, such as pro-inflammatory cytokines, thereby participating in the inflammatory response [39]. The activation of NF-κB leads to its translocation from cytoplasm to nucleus, where it activates the transcription of target genes, primarily encoding factors involved in inflammatory response. The abnormal level of NF-κB is associated with the pathogenesis of autoimmune diseases and autoinflammatory conditions [40, 41]. Our results indicated that NF-κB was elevated in lesion tissues of patients and mouse model with endometriosis, which also suggested that endometriosis patients were in a state of activated inflammation. Knocking down CCL21 in 12z cells significantly inhibited NF-κB and its downstream inflammatory factors (IL-6, TNF-α, and IL-1β). Overall, reducing CCL21 alleviated the progression of endometriosis by suppressing the activation of NF-κB signaling pathway and inhibiting the expression of IL-6, TNF-α, and IL-1β. There are still limitations in evaluating the severity and location of endometriosis, as well as diagnostic strategies[42]. More and more evidences suggest that endometriosis is a systemic disease, and there are complex interactions between angiogenesis, hormones, and immune changes caused by chronic inflammation in endometriosis. Developing new and safer anti-inflammatory and immunomodulatory drugs may become a long-term treatment option for endometriosis patients [43]. In summary, we have confirmed the important role of CCL21 in endometriosis, providing reference information for the diagnosis and treatment of endometriosis. Although there are still some issues that need to be addressed, such as the relationship between CCL21 and the severity of endometriosis, and how to develop targeted CCL21 drugs to avoid adverse reactions to other tissues, the results of this study may still help clarify the pathogenesis of endometriosis development and promote the establishment of treatment methods. Endometriosis is characterized as a typical chronic inflammatory disease, and inflammation is pivotal in the pathogenesis of endometriosis, even in the process of endometriosis-related carcinogenesis [19, 20]. Inflammatory mediators (IL-1β, IL-6, and TNF-α) up-regulate the production of haptoglobin in endometrium of endometriosis patients, which binds to macrophages and subsequently reduces their phagocytic function, thereby promoting the establishment of endometriosis [21]. The enhanced inflammation in endometriosis is associated with the inhibition of endothelial function [21]. A. Catarina Neto et al. used metformin to reverse infertility associated with endometriosis, possibly due to its anti-inflammatory properties during pregnancy[22]. Despite this knowledge, the pathogenicity and molecular mechanisms of inflammation in endometriosis remain to be clarified. Therefore, early identification of IRGs that contribute to the pathogenesis of endometriosis is essential to provide better treatment strategies. In this study, we screened for potential hub IRGs through comprehensive bioinformatics analysis and established a predictive model based on three hub genes (CCL21, CFD, and ACKR1), which showed good predictive ability. These findings might strongly support further comprehensive research into precision treatment of endometriosis. To investigate the potential functions of hub genes further, we collected ectopic tissues from endometriosis patients and established endometriosis mouse model. Among hub genes, CCL21 was increased in both endometriosis patients and mouse model. Knockdown of CCL21 significantly inhibited the proliferation, migration, and invasion of endometriotic cells and promoted apoptosis. Further analysis revealed that reduced CCL21 could inhibit endometriosis development via suppressing NF-κB signaling pathway. Endometriosis is a complex chronic inflammatory gynecological disorder associated with infertility and endometriosis-related pain [23]. A study on patient generated data found that fatigue, headache, mental confusion, gastrointestinal problems, and pain are common in endometriosis phenotypes, all of which are characteristics of low-grade inflammation[24]. Although its mechanisms are yet to be fully elucidated, current research suggests that inflammatory changes in endometriotic lesions are related to the advancement of endometriosis. Inflammation is considered a significant contributor to pain [25]. An elevated level of IL-1β has been detected in the peritoneal fluid and lesion tissues of women with endometriosis and pelvic pain [26, 27]. The increased IL-1β directly enhances the production of nerve growth factors, correlating with localized neurogenesis around endometriotic lesions and deep dyspareunia [26]. The number of activated macrophages in the peritoneal fluid of women with endometriosis increases, secreting more cytokines, including IL-6 [28, 29]. The elevated IL-6 then inhibits the cytolytic activity of NK cells [30]. Higher concentrations of TNF-α in peritoneal fluid and serum of patients with endometriosis play a pivotal role in activating systemic and local inflammation associated with the progression of endometriosis [31]. In the present study, we also validated the expression of IL-6, TNF-α, and IL-1β in lesion tissues of endometriosis patients and mouse model. The mouse model of endometriosis constructed by transplanting cornu uteri into mesentery is characterized by proliferation and inflammation, but without epithelial mesenchymal transformation and fibrosis. This model simulates some but not all features of human endometriosis[32]. Considering the complex mechanisms of endometriosis and the sensitivity of patients to different drugs, the search for new diagnostic markers and therapeutic targets is critical. We identified 207 commonly DEGs between the endometriotic lesions and normal endometrium of endometriosis patients through GSE7305 and GSE51981 datasets. By using WGCNA analysis, we screened out the “lightcyan” module, which is most significantly positively correlated with endometriosis. After intersecting IRGs, DEGs, and “lightcyan” module genes from WGCNA, three hub genes (CCL21, CFD, and ACKR1) were identified. Among these, CCL21 was increased in the lesion tissues of endometriosis across multiple GEO datasets, indicating that upregulated CCL21 might promote the occurrence and progression of endometriosis. Yanzhen Zhou et al. reported the identification of three potential key genes (MET, IL4R, and BST2) associated with endometriosis[33]. This result is inconsistent with our research findings and may be due to differences in databases, but it cannot be ruled out that the sample may be different. Anyway, our experiment confirms the importance of CCL21. CCL21 is a key chemokine of the CC subfamily, a small molecular weight protein that exhibits chemotactic properties for cell migration [34]. It mediates a range of physiological and disease-related processes, encompassing stress responses, infection, wound healing, and cell differentiation. The abnormal expression of CCL21 is involved in inflammation and immune regulation in various autoimmune diseases [35, 36]. Researchers have found that CCL21 is significantly increased in the ectopic tissue of endometriosis patients through laser capture microdissection analysis [37]. However, the pathological role of CCL21 in endometriosis remains unknown. In our study, we found that the expression of CCL21 was elevated not only in the lesion tissues of endometriosis across multiple datasets but also in the lesion tissues of endometriosis patients and mouse model, suggesting that CCL21 might also be involved in the development of endometriosis. While endometriosis is a benign disease, it exhibits biological characteristics similar to cancer, such as excessive proliferation, metastasis, and invasion [38]. To further investigate the impact of CCL21 on endometriosis, we used small interfering RNA methods to reduce CCL21 expression in 12z cells. Compared to the control group, knockdown of CCL21 suppressed the proliferation, migration, and invasion capabilities of 12z cells and promoted apoptosis. These results suggest that CCL21 could alleviate the pathology of endometriosis. To further investigate how CCL21 regulates the development of endometriosis, we divided patients with endometriosis into high- and low-CCL21 groups according to median CCL21 value. GSEA results showed that the high-CCL21 group was enriched in NF-κB and chemokine signaling pathway, while the low-CCL21 group was primarily enriched in oocyte meiosis. These results suggest that the inflammation in lesion tissues of patients in high-CCL21 group was increased, and the maturation of follicles was inhibited, which also indicated that patients in the high-CCL21 group might experience infertility. NF-κB regulates multiple genes in response to various stimuli, such as pro-inflammatory cytokines, thereby participating in the inflammatory response [39]. The activation of NF-κB leads to its translocation from cytoplasm to nucleus, where it activates the transcription of target genes, primarily encoding factors involved in inflammatory response. The abnormal level of NF-κB is associated with the pathogenesis of autoimmune diseases and autoinflammatory conditions [40, 41]. Our results indicated that NF-κB was elevated in lesion tissues of patients and mouse model with endometriosis, which also suggested that endometriosis patients were in a state of activated inflammation. Knocking down CCL21 in 12z cells significantly inhibited NF-κB and its downstream inflammatory factors (IL-6, TNF-α, and IL-1β). Overall, reducing CCL21 alleviated the progression of endometriosis by suppressing the activation of NF-κB signaling pathway and inhibiting the expression of IL-6, TNF-α, and IL-1β. There are still limitations in evaluating the severity and location of endometriosis, as well as diagnostic strategies[42]. More and more evidences suggest that endometriosis is a systemic disease, and there are complex interactions between angiogenesis, hormones, and immune changes caused by chronic inflammation in endometriosis. Developing new and safer anti-inflammatory and immunomodulatory drugs may become a long-term treatment option for endometriosis patients [43]. In summary, we have confirmed the important role of CCL21 in endometriosis, providing reference information for the diagnosis and treatment of endometriosis. Although there are still some issues that need to be addressed, such as the relationship between CCL21 and the severity of endometriosis, and how to develop targeted CCL21 drugs to avoid adverse reactions to other tissues, the results of this study may still help clarify the pathogenesis of endometriosis development and promote the establishment of treatment methods. Conclusion The levels of inflammatory factors in endometriotic lesions of endometriosis patients and mouse models were markedly increased. Through comprehensive bioinformatics methods, we have identified three hub IRGs (CCL21, CFD, and ACKR1) and established a predictive model that showed good predictive ability. The CCL21 was overexpressed in endometriotic lesions and that CCL21 depletion inhibited the proliferation, migration, and invasion of 12z cells and promote apoptosis. More importantly, reducing CCL21 suppressed the pathology of endometriosis by inhibiting the activation of NF-κB signaling pathway. Our research provided an innovative strategy of therapeutic intervention for endometriosis by targeting CCL21. Abbreviations IRGs Inflammation-related genes EcE ectopic endometrium EuE eutopic endometrium TCGA The Cancer Genome Atlas DEGs Differentially expressed genes GO Gene Ontology BPs Biological processes CCs Cellular components MFs Molecular functions KEGG Kyoto Encyclopedia of Genes and Genomes WGCNA Weighted Gene Co-expression Network Analysis TOM Topological Overlap Matrix ROC Receiver operating characteristic GSEA Gene Set Enrichment Analysis PBS Phosphate buffered saline H&E Hematoxylin and eosin PB Prussian blue FBS Fetal bovine serum SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis HRP Horseradish peroxidase RT-qPCR Real-time quantitative PCR GS Gene significance MM Module membership Declarations Acknowledgements Not applicable. Funding This study was funded by National Natural Science Foundation of China (No. 82201817); Basic Research Project of Liaoning Provincial Department of Education (No. LJKMZ20221290); Joint project of Liaoning Province Science and Technology Plan (No. 2024-BSLH-040); Youth Talent Cultivation Fund Project of Dalian Medical University (No. 508027 and No. 510016). Availability of data and materials The original data applied in this research are accessible from the corresponding author. Author’s contributions N.W., B.L. and Y.Y.Z. collected, analyzed, and interpreted data and wrote the manuscript. M.H.G., J.S. and X.Q.M. were responsible for conducting experiments in molecular biology and part of bio-information analysis. J.L.B., L.Y. and X.Y.D. were responsible for clinical sample collection and mouse model construction. J.S. and R.J.J. reviewed the article and make suggestions for the manuscript. N.W. and J.L.B. designed and developed the methodology, and the principal supervisor and funder of the study. All authors read and approved the final manuscript. Corresponding author Correspondence to Meihua Guo, Jie Sun or Jianlei Bi. Ethics declarations Ethics approval and consent to participate This study was conducted with the approval of the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University approved the research protocol (Approval No. KY2024-174-01). Animal care and experimental procedures were conducted following a protocol approved by the Animal Care and Use Committees of Dalian Medical University (Approval No. L250627113). Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Taylor H S, Kotlyar A M, Flores V A. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet (London, England). 2021; 397:839-52. 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Fertility and sterility. 2011; 95:437-40. Van Raemdonck K, Umar S, Palasiewicz K, Volkov S, Volin M V, et al. CCL21/CCR7 signaling in macrophages promotes joint inflammation and Th17-mediated osteoclast formation in rheumatoid arthritis. Cellular and molecular life sciences : CMLS. 2020; 77:1387-99. Van Raemdonck K, Umar S, Shahrara S. The pathogenic importance of CCL21 and CCR7 in rheumatoid arthritis. Cytokine & growth factor reviews. 2020; 55:86-93. Chand A L, Murray A S, Jones R L, Hannan N J, Salamonsen L A, et al. Laser capture microdissection and cDNA array analysis of endometrium i dentify CCL16 and CCL21 as epithelial-derived inflammatory mediators a ssociated with endometriosis. Reproductive biology and endocrinology : RB&E. 5:18. Wang M, Sun F, Zhang S, Zhang X, Sun Y, et al. NEK2 promotes the development of ovarian endometriosis and impairs decidualization by phosphorylating FOXO1. Cellular and molecular life sciences : CMLS. 2024; 81:237. Liu T, Zhang L, Joo D, Sun S-C. 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Scale bar, 100 µm. (B) RT-qPCR analysis of IL-6, IL-1β, and TNF-α genes expression in EuE and EcE from endometriosis patients. (C) The morphological size pre-transplant uterine tissues and endometriosis lesions, of which pre-transplant uterine tissues represent as EuE and endometriosis lesions refer to EcE. (D) The wet weight of EuE and EcE. (E) Representative images of EcE and EuE stained with H\u0026amp;E and prussian blue (PB) staining. Scale bar, 100 µm. (F) Representative images of immunofluorescence staining for IL-6 between EuE and EcE from endometriosis mouse model. Scale bar, 100 µm. (G) RT-qPCR analysis of IL-6, IL-1β, and TNF-α genes expression in EuE and EcE from endometriosis mouse model. (H) Western blotting was used to detect the protein expression of IL-6, IL-1β, and TNF-α in EuE and EcE from endometriosis mouse model.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/d6fa2d8bfb26cc622d890375.png"},{"id":94155378,"identity":"1c3c930e-78c6-4d32-a791-9e0b47a7cabf","added_by":"auto","created_at":"2025-10-23 02:45:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2957799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of DEGs in endometriosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe volcano shows DEGs between endometriosis and normal endometrium from GSE7305 (A) and GSE51981 (B). (C) The venn diagram of the intersection of up-regulated or down-regulated genes from GSE7305 and GSE51981. (D) The dot plot showed the enriched KEGG signaling pathways among DEGs. (E-G) The dot plot showed the enriched GO signaling pathways among DEGs, including biological processes (BPs), cellular components (CCs) and molecular functions (MFs).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/da321c4ea17ac80fe4e6380f.png"},{"id":94155383,"identity":"fb401eec-ef92-4b52-9c3e-ae697890e137","added_by":"auto","created_at":"2025-10-23 02:45:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3044460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of candidate key genes closely related to endometriosis based on WGCNA analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The soft threshold power (left) and mean connectivity (right) of WGCNA network. (B) Gene modules with different colors under the cluster dendrogram. (C) Clustering dendrogram of endometriosis and normal endometrium. (D) Heat map of eigengene adjacency. (E) The heatmap depicting the relationship between the modules form endometriosis and normal endometrium. (F) Correlation plot between module membership and the significance of genes within the “lightcyan” module.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/05b338f29c54bacbc254e4f9.png"},{"id":94155379,"identity":"c6a35fc9-10bb-44f6-857a-d69fa99794c5","added_by":"auto","created_at":"2025-10-23 02:45:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1942148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening for hub genes in endometriosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The venn diagram of the intersection of DEGs, “lightcyan” module genes from WGCNA and IRGs. The expression of CCL21, CFD, and ACKR1 in endometriotic lesions and normal endometrium from endometriosis patients in GSE7305 dataset (B) and GSE51981 dataset (C). (D) ROC analysis of three hub genes in GSE7305 dataset. (E) ROC analysis of three hub genes in GSE51981 dataset. (F) Nomogram was established by three hub genes for predicting the risk of endometriosis. (G) The verification of prediction model was tested by calibration curve. (H) ROC curve analysis of nomogram for diagnostic sensitivity.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/2fb34328584644f7fe78c6d9.png"},{"id":94155380,"identity":"f21632f6-4446-4abc-8427-92df1674c00a","added_by":"auto","created_at":"2025-10-23 02:45:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4837113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL21 expression was obviously increased in eutopic endometrium of endometriosis patients and mouse model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The expression of CCL21 in endometriotic lesions and normal endometrium from endometriosis patients in GSE5108, GSE11691, GSE23339, and GSE25628 datasets. (B) ROC analysis of CCL21 in GSE5108, GSE11691, GSE23339, and GSE25628 datasets. (C) The average FPKM value of CCL21, CFD, and ACKR1 in EuE and EcE from endometriosis patients. (D) Western blotting was used to detect the expression of CCL21 in EuE and EcE from endometriosis patients and mouse model. (E) Representative images of immunofluorescence staining for CCL21. Scale bar, 100 µm. (F) RT-qPCR analysis of CCL21 level in EuE and EcE of endometriosis mouse model.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/d049482216083145fa6020cd.png"},{"id":94155386,"identity":"84d3638d-fd0e-47cb-8f82-d227de901b38","added_by":"auto","created_at":"2025-10-23 02:45:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10965827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown CCL21 inhibited cell proliferation, migration and invasion \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The CCL21-siRNA fragments were designed to observe the knockdown efficiency of CCL21. The sequences of siRNA corresponded to nucleotides 127-147 (si-RNA-1), 155-175 (si-RNA-2) and 217-237 (si-RNA-3). The relative mRNA expression of CCL21 was normalized to GAPDH. (B) Western blotting detected the efficiency of knocking down CCL21 in 12z cells. (C) Representative images of immunofluorescence staining for CCL21 in control and si-CCL21 groups. Scale bar, 100 µm. (D) The migration ability of control and si-CCL21 cells was measured through wound healing assay. (E) The invasion ability of control and si-CCL21 cells was measured through Transwell assay. (F) Representative images of immunofluorescence staining for PCNA in control and si-CCL21 groups. Scale bar, 100 µm. (G) Representative images of cellular apoptosis in control and si-CCL21 groups.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/510742bc19d61a1667d3340e.png"},{"id":94155384,"identity":"386d44ff-3179-448d-be74-9cbfd56bb19d","added_by":"auto","created_at":"2025-10-23 02:45:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5845807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnocking down CCL21 inhibited inflammation in endometriosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The volcano plot represents DEGs (high-CCL21 versus low-CCL21), of which the red dots represent as up-regulated genes and blue dots refer to down-regulated genes in high-CCL21 group. (B) GSEA between endometriosis patients with high- and low-CCL21 expression in GSE51981 cohort. (C) Representative images of immunofluorescence staining for NF-κB in EcE and EuE from endometriosis patients. Scale bar, 100 µm. (D) Western blotting analysis of NF-κB expression in EcE and EuE from endometriosis patients and mice model. (E) Western blotting was used to detect the protein expression of NF-κB, IL-6, IL-1β, and TNF-α in control and si-CCL21 groups. (F) Representative images of immunofluorescence staining for IL-6 in control and si-CCL21 groups. Scale bar, 100 µm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/c8f8303daa58225a2df9af7c.png"},{"id":95653858,"identity":"f5187867-2d1a-4350-b134-76d1abb21e77","added_by":"auto","created_at":"2025-11-11 16:01:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":43128678,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/ecc72378-5926-4eaa-a543-ea87fee62b54.pdf"},{"id":94155385,"identity":"66feb277-c4cf-4b99-9a6e-d982661d7a76","added_by":"auto","created_at":"2025-10-23 02:45:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3106811,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7558971/v1/c84698f9330e09614d2c7a9f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CCL21 promotes the development of endometriosis by activating NF-κB signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is characterized by the presence of endometrium-like tissue (including glands and stroma) outside the uterus, affecting approximately 10% of reproductive-age women worldwide [1].\u0026nbsp;This disease severely impairs the quality of life, commonly manifesting as pelvic pain and infertility. Notably, endometriosis is present in 25%-50% of infertile women, while 30%-50% of endometriosis patients experience infertility\u0026nbsp;[2, 3, 4]. Additionally, it elevates the risk of epithelial ovarian cancer\u0026nbsp;[5]. Although progestin-based therapy for endometriosis could cause the condition to regress in some patients, hormone treatment has unacceptable side effects, including menopausal symptoms, and is not effective in endometriosis patients with pelvic pain who are responsive to progesterone\u0026nbsp;[6, 7]. Surgical removal of ectopic endometrial tissue is one of the effective treatments for endometriosis\u0026nbsp;[8], but the recurrence rate of the surgery is high\u0026nbsp;[9]. Despite advances in understanding its clinical burden, endometriosis incidence continues to rise. Therefore, elucidating the pathophysiology of endometriosis and its molecular mechanisms underlying infertility and pelvic pain represents a critical research imperative.\u003c/p\u003e\n\u003cp\u003eEndometriosis has been well-established as a typical chronic inflammatory disease [10]. A series of changes occur in inflammatory cells, cytokines, and chemokines within endometriotic lesions and peritoneal fluid, forming an inflammatory microenvironment. This microenvironment interacts with endometriotic cells and plays a pivotal role in the development and progression of endometriosis [11]. During the formation of endometriotic lesions, inflammatory cells are recruited to the lesion site and release a variety of inflammation-related mediators such as cytokines and chemokines [12]. These inflammation-related mediators in turn act on the inflammatory cells, leading to the aggregation of more inflammatory cells within the endometriotic lesions [1]. This interaction between the inflammatory cells and substances forms a vicious cycle, resulting in the accumulation of endometriosis-related inflammation, which exacerbates the occurrence and progression of endometriosis.\u0026nbsp;Inflammation also affects the carcinogenic process of endometriosis\u0026nbsp;[13, 14]. Therefore, investigating the molecular and cellular mechanisms\u0026nbsp;underlying\u0026nbsp;the pathogenesis of endometriosis from the perspective of inflammation-related genes (IRGs) is of great significance for the development of advanced treatment approaches.\u003c/p\u003e\n\u003cp\u003eIn this study, we employed multifarious bioinformatics methods to identify potential hub IRGs and investigated their pathological roles in endometriosis. A novel nomogram based on the screened hub IRGs demonstrated good predictive value. Notably, CCL21 was significantly elevated in the lesion tissues of endometriosis. Using small interfering RNA technology, we established CCL21 knockdown cells to explore the impact of CCL21 on proliferation, migration, and invasion capabilities of 12z cells. Additionally, we attempted to investigate the mechanism by which CCL21 influences endometriosis through the NF-\u0026kappa;B signaling pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eClinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman EcE and EuE tissues (n = 8) were obtained from endometriosis patients undergoing surgical resections at the\u0026nbsp;Second Affiliated Hospital of Dalian Medical University (Dalian, China) between June 2024 to December 2024. The age range of the patients was 28‑42 years. All patients have filled out the informed consent form before sample collection. The Ethics Committee of the Second Affiliated Hospital of Dalian Medical University approved the research protocol (Approval No. KY2024-174-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary antibodies used were anti-CCL21 (DF6681), anti-TNF-α (AF7014), anti-IL-6 (DF6087), anti-IL-1β (AF5103) and NF-κB (AF5006) from Affinity Biosciences; anti-β-Actin (20536-1-AP) from Proteintech. Secondary antibodies horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208), Alexa Fluor 488-/647- labeled goat anti-rabbit IgG(H+L) (A0423/A0468) from Beyotime.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData aquation and gene expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data were retrieved from The Cancer Genome Atlas (TCGA) database, including GSE7305, GSE51981, GSE5108, GSE11691, GSE23339, GSE25628 datasets.\u0026nbsp;Detailed information was shown in Supplementary Table 1. The “Limma” package was used to identify the differentially expressed genes (DEGs) of the control and\u0026nbsp;endometriosis groups. All 737 protein-coding IRGs were selected and downloaded from the GeneCards database with a relevance score \u0026gt; 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of DEGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenes that exhibited increased expression in both GSE7305 and GSE51981 compared to control group, as well as genes that showed decreased expression in both GSE7305 and GSE51981, were referred to as common DEGs. Next, we applied “clusterProfiler” for Gene Ontology (GO) enrichment analysis, covering biological processes (BPs), cellular components (CCs), and molecular functions (MFs). Furthermore, we utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) to pinpoint the key pathways among DEGs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeighted Gene Co-expression Network Analysis (WGCNA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the module most relevant with endometriosis, we conducted WGCNA analysis in GSE51981 using the R package “WGCNA”. Specifically, we preprocessed the sample data, excluding any outliers, and then employed the “WGCNA” to construct a correlation matrix. The optimal soft threshold was chosen to transform the correlation matrix into an adjacency matrix, and then generated a Topological Overlap Matrix (TOM) from the adjacency matrix. Employing TOM-based phase dissimilarity metric, we classified genes with similar expression patterns into gene modules through average linkage hierarchical clustering. The module most strongly associated with endometriosis patients was selected for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNomogram construction and receiver operating characteristic (ROC) curve\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntersecting genes of DEGs, WGCNA, and IRGs were considered candidate hub genes for endometriosis diagnosis. To assess the significance of hub genes in diagnosing endometriosis, we built a nomogram using “rms” R package. Additionally, we constructed the relevant calibration curve to evaluate the accuracy and reliability of the nomogram model’s predictions. We also assessed the prognostic value of hub genes and the nomogram through ROC curve.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Set Enrichment Analysis (GSEA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the pathways enriched by CCL21, we divided the patients with endometriosis into high- and low-CCL21 expression groups based on the median CCL21 value. The CCL21-enriched KEGG pathways were identified using GSEA analysis with the “clusterProfiler” package (version 3.16.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of mice model of endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALB/c female mice, 8 week-old, were housed in specific pathogen-free experimental animal facilities (24\u0026nbsp;±\u0026nbsp;1\u0026nbsp;℃) with free food and water in Dalian Medical University. The experiment was divided into two groups: endometriosis model group and sham surgery control group, with 8 mice in each group. The entire surgical procedure was performed in a biosafety cabinet. Before surgery, the mice abdomen was depilated and disinfected sequentially with iodine and alcohol cotton balls. To established endometriosis model, a midline abdominal incision was performed on the female mice to expose the uterus under anesthesia with isoflurane, and followed by the excision of one uterine horn. The uterine horn was longitudinally split with scissors in a petri dish containing phosphate buffered saline (PBS). The excised uterine horn was cut into small fragments of approximately 2 mm\u003csup\u003e2\u003c/sup\u003e, which were then reimplanted into the mesentery of the same mice, followed by wound suturing[15, 16]. The control group (sham surgery) mice were anesthetized and opened using the same method to create a model. Without separating the uterus, the abdominal incision is directly sutured continuously, and the abdomen is routinely closed. After the surgery, the mice were carefully cared for. After 30 days of feeding, the mice were anesthetized using isoflurane and subsequently euthanized via cervical dislocation, and the endometriosis-like lesions that were removed were counted under a microscope for further experiments. The pre-transplant uterine tissues represent as EuE and endometriosis-like lesions refer to EcE in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and eosin (H\u0026amp;E) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter dewaxing and hydrating the sections, hematoxylin was used to stain the cell nucleus being deep blue. Following differentiation with 1% alcohol hydrochloride, eosin solution was used to stain the proteins pink, and finally, dehydration was achieved using a gradient of alcohol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrussian blue (PB) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe iron content within samples was measured using the PB staining kit (Yeasen, China) according to the manufacturer’s instructions. The iron-containing areas were stained with the reagent to exhibit a characteristic blue color.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and establishment of CCL21 knockdown Cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 12z cells were acquired from the Cell Bank of the Shanghai Institute of Life Sciences (Chinese Academy of Sciences, Shanghai, China). Cells were cultured in DMEM/F12 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, China) and 100 U/ml penicillin-streptomycin (Seven, Beijing, China) in an incubator (Thermo Fisher Scientific, United States) at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. Three targeting sequences of\u0026nbsp;CCL21\u0026nbsp;small interfering\u0026nbsp;(si) RNA were used to reduce the expression of CCL21 (Supplementary Table 2). 12z cells were transfected with 100 pmol of control siRNA or CCL21 siRNA, using 10\u0026nbsp;µl of Lipofectamine 3000 (L3000015, Invitrogen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein samples form tissues or cells were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized using an enhanced chemiluminescence system (Beyotime, China). Quantification of the data was performed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time quantitative PCR (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Trizol™ reagent (Invitrogen, USA) and converted to cDNA utilizing the Prime Script RT-PCR Kit (Seven Biotech, Beijing, China). cDNA amplification was performed using the Applied Biosystems Prism 7000 Sequence\u0026nbsp;Detection System. The relative fold change of gene amplification was calculated according to the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e analysis. The primer sequences used were provided in Supplementary Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter dewaxing, hydrating and blocking the sections of ectopic endometrial tissues of patients and mice, they were incubated with primary antibody overnight and then incubated with fluorescent secondary antibody. The sections were ultimately examined under a fluorescence microscope (Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe si-CCL21 and control cells were individually seeded in each well of a 6-well plate to reach complete confluence. A scratch was created using a 200 µl sterile pipette. The cells were incubated in serum-deprived medium for 24- and 48-hrs, after which images of the same area were taken.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe upper chamber of 24-well Transwell (Jet Biofil, China) was treated with 40 µl of Matrigel (Corning, USA) and incubated at 37℃ for 2 hrs. A total of 200 µl of si-CCL21 and control cells at a density of 1 × 10\u003csup\u003e4\u003c/sup\u003e in serum- deprived medium was respectively placed in the upper chamber, whereas 550 µl of medium containing FBS was supplemented to the lower chamber. Following a 48-hrs period, the upper chamber was washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Solarbio, China) for 20 minutes. The cells were then photographed and counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe si-CCL21and control cells were digested and collected using trypsin without EDTA. The cells were treated with Annexin V-FITC and PI (Beyotime, China) in dark for 25 minutes. Subsequently, apoptosis rate detected using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis and visualization were conducted using GraphPad Prism 9.0, R 4.1.3, ImageJ, and FlowJo. A two-tailed Student’s t-test was used to assess the statistical differences between two groups. One-way ANOVA and two-way ANOVA were utilized to analyze statistical differences among multiple groups. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe inflammation levels were increased in endometriotic lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess levels of inflammation in endometriotic lesions, we investigated the expression of classical inflammatory factors in EuE and EcE of endometriosis patients. We first evaluated the expression of IL-6, IL-1\u0026beta;, and TNF-\u0026alpha; in EcE from 8 endometriosis patients using immunofluorescence and RT-qPCR. Compared to EuE, the protein and mRNA levels of IL-6, IL-1\u0026beta;, and TNF-\u0026alpha; were dramatically increased in EcE of endometriosis patients (Figure 1A-1B and Supplementary Figure 1A-1B). To further investigate the inflammation levels in EcE, we transplanted uterine tissue into the mesentery to construct mouse model of endometriosis. One month after the induction of endometriosis, the fluid-filled weight of endometriotic lesions was significantly increased compared to the baseline, indicating that we had successfully established mouse model of endometriosis (Figure 1C and 1D). Histological analysis revealed that the EcE in both endometriosis patients and the mouse model exhibited irregular shapes, abnormal arrangement, and increased cavitation (Figure 1A and 1E). Iron-overload is a characteristic feature of endometriosis [17, 18]. PB staining revealed that the iron-overloaded microenvironment resulted in localized iron accumulation in the mouse endometriotic lesions (Figure 1E). The levels of inflammatory factors (IL-6, IL-1\u0026beta; and TNF-\u0026alpha;) were elevated in mouse endometriotic lesions (Figure 1F-1H and Supplementary Figure 1C-1D). These data reveal that increased inflammation levels positively correlate with the occurrence of endometriosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of DEGs between endometriosis and normal endometrium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microarray datasets GSE7305 and GSE51981 were obtained to screen the DEGs between endometriosis and normal endometrium. We identified 1,214 DEGs in the GSE7305 dataset and 1,984 DEGs in the GSE51981 dataset (Figure 2A and 2B). Among these, 49 genes were significantly upregulated in both datasets, while 158 genes were downregulated (Figure 2C). Subsequently, we conducted KEGG and GO enrichment analyses on common DEGs. KEGG enrichment findings indicated that these genes were predominantly involved in cell cycle, oocyte meiosis, cellular senescence, and P53 signaling pathway (Figure 2D). GO analysis showed that these genes were mainly enriched in organelle fission, spindle, and microtubule binding (Figure 2E-2G).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of WGCNA network of endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify crucial genes associated with the pathogenesis of endometriosis more accurately, we performed WGCNA analysis on normal endometrium and endometriosis tissues from the GSE51981 dataset. Based on scale independence and mean connectivity, we set the soft threshold to 9 (Figure 3A). Through evaluating gene correlation, we assembled a hierarchical clustering dendrogram of genes, which enabled us to identify 6 distinct gene modules (Figure 3B-3D). To ascertain which module was closely associated with endometriosis, we conducted a correlation analysis comparing each module and phenotype. Our analysis indicated that the \u0026ldquo;lightcyan\u0026rdquo; module, which comprises 2,363 genes, exhibited the strongest correlation with endometriosis (Figure 3E). Additionally, a notable relationship between gene significance (GS) and module membership (MM) was observed within the \u0026ldquo;lightcyan\u0026rdquo; module (Figure 3F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of hub genes in endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we conducted an intersection analysis on IRGs, DEGs, and \u0026ldquo;lightcyan\u0026rdquo; module genes from WGCNA, and we identified 3 hub genes, namely CCL21, CFD, and ACKR1 (Figure 4A). The expression of these three hub genes (CCL21, CFD, and ACKR1) was upregulated in endometriotic lesions of patients with endometriosis, as observed in GSE7305 and GSE51981 datasets (Figure 4B and 4C). The ROC curve indicated that the 3 hub genes had favorable diagnostic values for endometriosis within GSE7305 and GSE51981 datasets (Figure 4D and 4E). To evaluate the roles of these biomarkers (CCL21, CFD, and ACKR1) in the diagnosis of endometriosis, we developed a nomogram (Figure 4F). The calibration curve demonstrated the effectiveness of the predictive model (Figure 4G). Additionally, the nomogram model had accurate predictive capability (Figure 4H). Overall, the model constructed based on three biomarkers (CCL21, CFD, and ACKR1) can effectively predict the occurrence and progression of endometriosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe expression of CCL21\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewas increased\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in endometriotic lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the importance of three hub genes in the development of endometriosis, we examined the levels of these genes across various datasets. It was observed that CCL21 was consistently upregulated across several datasets, including GSE5108, GSE11691, GSE23339, and GSE25628 (Figure 5A and Supplementary Figure 2A-2D). ROC curve results showed that CCL21 had good diagnostic value in endometriosis (Figure 5B). More importantly, we also found increased CCL21 expression in the endometriotic lesions of patients with endometriosis, as confirmed by our own transcriptome data (Figure 5C). CCL21 expression was elevated in endometriotic lesions of endometriosis patients and mouse model, as confirmed by Western blotting, immunofluorescence staining and RT-qPCR analysis (Figure 5D-5F and Supplementary Figure 3A). These results suggested that CCL21, a pivotal inflammation-related gene, was upregulated in the tissues of endometriotic lesions and contributed to the development and advancement of endometriosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of CCL21\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;attenuated\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;endometriosis \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo clarify the relationship between CCL21 and endometriosis, we silenced the CCL21 gene in 12z cells. Initially, we designed specific CCL21 siRNA sequences targeting different regions of the CCL21 open reading frame. Among three siRNA constructs tested, siRNA-3 (217-237) was the most effective in inhibiting CCL21 expression (Figure 6A and 6B). Consequently, we selected siRNA-3 (217-237) to knockdown CCL21 expression in the following experimental procedures. In immunofluorescence staining, the expression of CCL21 was decreased in si-CCL21 group compared to control group (Figure 6C). In addition, knocking down CCL21 significantly inhibited the migration and invasion of 12z cells (Figure 6D and 6E). Furthermore, the proliferative ability of 12z cells in si-CCL21 group was obviously reduced (Figure 6F). In contrast, reducing CCL21 level promoted the apoptosis rate of 12z cells (Figure 6G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of CCL21 inhibited inflammatory expression in endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanisms by which CCL21 regulates endometriosis, based on the median value of CCL21, we divided endometriosis patients in the GSE51981 dataset into high- and low-CCL21 groups. Subsequently, we performed a differential expression analysis and revealed 2,654 DEGs, of which 1,756 genes were elevated, and 898 genes were reduced in high-CCL21 group (Figure 7A and Supplementary Figure 4A-4C). We investigated the differences in signaling pathways between two groups. The GSEA results revealed that NF-\u0026kappa;B and chemokine signaling pathways were enriched in high-CCL21 group. In contrast, pathways such as Oocyte meiosis were enriched in low-CCL21 group (Figure 7B). Western blotting and immunofluorescence staining results showed that NF-\u0026kappa;B level was elevated in endometriotic lesions of endometriosis patients and mouse model (Figure 7C-7D and Supplementary Figure 5A). In addition, knockdown of CCL21 significantly inhibited NF-\u0026kappa;B and its downstream IL-6, IL-1\u0026beta;, and TNF-\u0026alpha; expression (Figure 7E and 7F).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis is characterized as a typical chronic inflammatory disease, and inflammation is pivotal in the pathogenesis of endometriosis, even in the process of endometriosis-related carcinogenesis [19, 20]. Inflammatory mediators (IL-1β, IL-6, and TNF-α) up-regulate the production of haptoglobin in endometrium of endometriosis patients, which binds to macrophages and subsequently reduces their phagocytic function, thereby promoting the establishment of endometriosis [21]. The enhanced inflammation in endometriosis is associated with the inhibition of endothelial function [21]. A. Catarina Neto \u003cem\u003eet al.\u003c/em\u003e used metformin to reverse infertility associated with endometriosis, possibly due to its anti-inflammatory properties during pregnancy[22]. Despite this knowledge, the pathogenicity and molecular mechanisms of inflammation in endometriosis remain to be clarified. Therefore, early identification of IRGs that contribute to the pathogenesis of endometriosis is essential to provide better treatment strategies. In this study, we screened for potential hub IRGs through comprehensive bioinformatics analysis and established a predictive model based on three hub genes (CCL21, CFD, and ACKR1), which showed good predictive ability. These findings might strongly support further comprehensive research into precision treatment of endometriosis. To investigate the potential functions of hub genes further, we collected ectopic tissues from endometriosis patients and established endometriosis mouse model. Among hub genes, CCL21 was increased in both endometriosis patients and mouse model. Knockdown of CCL21 significantly inhibited the proliferation, migration, and invasion of endometriotic cells and promoted apoptosis. Further analysis revealed that reduced CCL21 could inhibit endometriosis development via suppressing NF-κB signaling pathway.\u003c/p\u003e\n\u003cp\u003eEndometriosis is a complex chronic inflammatory gynecological disorder associated with infertility and endometriosis-related pain [23]. A study on patient generated data found that fatigue, headache, mental confusion, gastrointestinal problems, and pain are common in endometriosis phenotypes, all of which are characteristics of low-grade inflammation[24]. Although its mechanisms are yet to be fully elucidated, current research suggests that inflammatory changes in endometriotic lesions are related to the advancement of endometriosis. Inflammation is considered a significant contributor to pain [25]. An elevated level of IL-1β has been detected in the peritoneal fluid and lesion tissues of women with endometriosis and pelvic pain [26, 27]. The increased IL-1β directly enhances the production of nerve growth factors, correlating with localized neurogenesis around endometriotic lesions and deep dyspareunia [26]. The number of activated macrophages in the peritoneal fluid of women with endometriosis increases, secreting more cytokines, including IL-6 [28, 29]. The elevated IL-6 then inhibits the cytolytic activity of NK cells [30]. Higher concentrations of TNF-α in peritoneal fluid and serum of patients with endometriosis play a pivotal role in activating systemic and local inflammation associated with the progression of endometriosis [31]. In the present study, we also validated the expression of IL-6, TNF-α, and IL-1β in lesion tissues of endometriosis patients and mouse model. The mouse model of endometriosis constructed by transplanting cornu uteri into mesentery is characterized by proliferation and inflammation, but without epithelial mesenchymal transformation and fibrosis. This model simulates some but not all features of human endometriosis[32]. Considering the complex mechanisms of endometriosis and the sensitivity of patients to different drugs, the search for new diagnostic markers and therapeutic targets is critical. We identified 207 commonly DEGs between the endometriotic lesions and normal endometrium of endometriosis patients through GSE7305 and GSE51981 datasets. By using WGCNA analysis, we screened out the “lightcyan” module, which is most significantly positively correlated with endometriosis. After intersecting IRGs, DEGs, and “lightcyan” module genes from WGCNA, three hub genes (CCL21, CFD, and ACKR1) were identified. Among these, CCL21 was increased in the lesion tissues of endometriosis across multiple GEO datasets, indicating that upregulated CCL21 might promote the occurrence and progression of endometriosis. Yanzhen Zhou et al. reported the identification of three potential key genes (MET, IL4R, and BST2) associated with endometriosis[33]. This result is inconsistent with our research findings and may be due to differences in databases, but it cannot be ruled out that the sample may be different. Anyway, our experiment confirms the importance of CCL21.\u003c/p\u003e\n\u003cp\u003eCCL21 is a key chemokine of the CC subfamily, a small molecular weight protein that exhibits chemotactic properties for cell migration [34]. It mediates a range of physiological and disease-related processes, encompassing stress responses, infection, wound healing, and cell differentiation. The abnormal expression of CCL21 is involved in inflammation and immune regulation in various autoimmune diseases [35, 36]. Researchers have found that CCL21 is significantly increased in the ectopic tissue of endometriosis patients through laser capture microdissection analysis [37]. However, the pathological role of CCL21 in endometriosis remains unknown. In our study, we found that the expression of CCL21 was elevated not only in the lesion tissues of endometriosis across multiple datasets but also in the lesion tissues of endometriosis patients and mouse model, suggesting that CCL21 might also be involved in the development of endometriosis. While endometriosis is a benign disease, it exhibits biological characteristics similar to cancer, such as excessive proliferation, metastasis, and invasion [38]. To further investigate the impact of CCL21 on endometriosis, we used small interfering RNA methods to reduce CCL21 expression in 12z cells. Compared to the control group, knockdown of CCL21 suppressed the proliferation, migration, and invasion capabilities of 12z cells and promoted apoptosis. These results suggest that CCL21 could alleviate the pathology of endometriosis.\u003c/p\u003e\n\u003cp\u003eTo further investigate how CCL21 regulates the development of endometriosis, we divided patients with endometriosis into high- and low-CCL21 groups according to median CCL21 value. GSEA results showed that the high-CCL21 group was enriched in NF-κB and chemokine signaling pathway, while the low-CCL21 group was primarily enriched in oocyte meiosis. These results suggest that the inflammation in lesion tissues of patients in high-CCL21 group was increased, and the maturation of follicles was inhibited, which also indicated that patients in the high-CCL21 group might experience infertility. NF-κB regulates multiple genes in response to various stimuli, such as pro-inflammatory cytokines, thereby participating in the inflammatory response [39]. The activation of NF-κB leads to its translocation from cytoplasm to nucleus, where it activates the transcription of target genes, primarily encoding factors involved in inflammatory response. The abnormal level of NF-κB is associated with the pathogenesis of autoimmune diseases and autoinflammatory conditions [40, 41]. Our results indicated that NF-κB was elevated in lesion tissues of patients and mouse model with endometriosis, which also suggested that endometriosis patients were in a state of activated inflammation. Knocking down CCL21 in 12z cells significantly inhibited NF-κB and its downstream inflammatory factors (IL-6, TNF-α, and IL-1β). Overall, reducing CCL21 alleviated the progression of endometriosis by suppressing the activation of NF-κB signaling pathway and inhibiting the expression of IL-6, TNF-α, and IL-1β.\u003c/p\u003e\n\u003cp\u003eThere are still limitations in evaluating the severity and location of endometriosis, as well as diagnostic strategies[42]. More and more evidences suggest that endometriosis is a systemic disease, and there are complex interactions between angiogenesis, hormones, and immune changes caused by chronic inflammation in endometriosis. Developing new and safer anti-inflammatory and immunomodulatory drugs may become a long-term treatment option for endometriosis patients [43]. In summary, we have confirmed the important role of CCL21 in endometriosis, providing reference information for the diagnosis and treatment of endometriosis. Although there are still some issues that need to be addressed, such as the relationship between CCL21 and the severity of endometriosis, and how to develop targeted CCL21 drugs to avoid adverse reactions to other tissues, the results of this study may still help clarify the pathogenesis of endometriosis development and promote the establishment of treatment methods.\u003c/p\u003e\n\u003cp\u003eEndometriosis is characterized as a typical chronic inflammatory disease, and inflammation is pivotal in the pathogenesis of endometriosis, even in the process of endometriosis-related carcinogenesis [19, 20]. Inflammatory mediators (IL-1β, IL-6, and TNF-α) up-regulate the production of haptoglobin in endometrium of endometriosis patients, which binds to macrophages and subsequently reduces their phagocytic function, thereby promoting the establishment of endometriosis [21]. The enhanced inflammation in endometriosis is associated with the inhibition of endothelial function [21]. A. Catarina Neto \u003cem\u003eet al.\u003c/em\u003e used metformin to reverse infertility associated with endometriosis, possibly due to its anti-inflammatory properties during pregnancy[22]. Despite this knowledge, the pathogenicity and molecular mechanisms of inflammation in endometriosis remain to be clarified. Therefore, early identification of IRGs that contribute to the pathogenesis of endometriosis is essential to provide better treatment strategies. In this study, we screened for potential hub IRGs through comprehensive bioinformatics analysis and established a predictive model based on three hub genes (CCL21, CFD, and ACKR1), which showed good predictive ability. These findings might strongly support further comprehensive research into precision treatment of endometriosis. To investigate the potential functions of hub genes further, we collected ectopic tissues from endometriosis patients and established endometriosis mouse model. Among hub genes, CCL21 was increased in both endometriosis patients and mouse model. Knockdown of CCL21 significantly inhibited the proliferation, migration, and invasion of endometriotic cells and promoted apoptosis. Further analysis revealed that reduced CCL21 could inhibit endometriosis development via suppressing NF-κB signaling pathway.\u003c/p\u003e\n\u003cp\u003eEndometriosis is a complex chronic inflammatory gynecological disorder associated with infertility and endometriosis-related pain [23]. A study on patient generated data found that fatigue, headache, mental confusion, gastrointestinal problems, and pain are common in endometriosis phenotypes, all of which are characteristics of low-grade inflammation[24]. Although its mechanisms are yet to be fully elucidated, current research suggests that inflammatory changes in endometriotic lesions are related to the advancement of endometriosis. Inflammation is considered a significant contributor to pain [25]. An elevated level of IL-1β has been detected in the peritoneal fluid and lesion tissues of women with endometriosis and pelvic pain [26, 27]. The increased IL-1β directly enhances the production of nerve growth factors, correlating with localized neurogenesis around endometriotic lesions and deep dyspareunia [26]. The number of activated macrophages in the peritoneal fluid of women with endometriosis increases, secreting more cytokines, including IL-6 [28, 29]. The elevated IL-6 then inhibits the cytolytic activity of NK cells [30]. Higher concentrations of TNF-α in peritoneal fluid and serum of patients with endometriosis play a pivotal role in activating systemic and local inflammation associated with the progression of endometriosis [31]. In the present study, we also validated the expression of IL-6, TNF-α, and IL-1β in lesion tissues of endometriosis patients and mouse model. The mouse model of endometriosis constructed by transplanting cornu uteri into mesentery is characterized by proliferation and inflammation, but without epithelial mesenchymal transformation and fibrosis. This model simulates some but not all features of human endometriosis[32]. Considering the complex mechanisms of endometriosis and the sensitivity of patients to different drugs, the search for new diagnostic markers and therapeutic targets is critical. We identified 207 commonly DEGs between the endometriotic lesions and normal endometrium of endometriosis patients through GSE7305 and GSE51981 datasets. By using WGCNA analysis, we screened out the “lightcyan” module, which is most significantly positively correlated with endometriosis. After intersecting IRGs, DEGs, and “lightcyan” module genes from WGCNA, three hub genes (CCL21, CFD, and ACKR1) were identified. Among these, CCL21 was increased in the lesion tissues of endometriosis across multiple GEO datasets, indicating that upregulated CCL21 might promote the occurrence and progression of endometriosis. Yanzhen Zhou et al. reported the identification of three potential key genes (MET, IL4R, and BST2) associated with endometriosis[33]. This result is inconsistent with our research findings and may be due to differences in databases, but it cannot be ruled out that the sample may be different. Anyway, our experiment confirms the importance of CCL21.\u003c/p\u003e\n\u003cp\u003eCCL21 is a key chemokine of the CC subfamily, a small molecular weight protein that exhibits chemotactic properties for cell migration [34]. It mediates a range of physiological and disease-related processes, encompassing stress responses, infection, wound healing, and cell differentiation. The abnormal expression of CCL21 is involved in inflammation and immune regulation in various autoimmune diseases [35, 36]. Researchers have found that CCL21 is significantly increased in the ectopic tissue of endometriosis patients through laser capture microdissection analysis [37]. However, the pathological role of CCL21 in endometriosis remains unknown. In our study, we found that the expression of CCL21 was elevated not only in the lesion tissues of endometriosis across multiple datasets but also in the lesion tissues of endometriosis patients and mouse model, suggesting that CCL21 might also be involved in the development of endometriosis. While endometriosis is a benign disease, it exhibits biological characteristics similar to cancer, such as excessive proliferation, metastasis, and invasion [38]. To further investigate the impact of CCL21 on endometriosis, we used small interfering RNA methods to reduce CCL21 expression in 12z cells. Compared to the control group, knockdown of CCL21 suppressed the proliferation, migration, and invasion capabilities of 12z cells and promoted apoptosis. These results suggest that CCL21 could alleviate the pathology of endometriosis.\u003c/p\u003e\n\u003cp\u003eTo further investigate how CCL21 regulates the development of endometriosis, we divided patients with endometriosis into high- and low-CCL21 groups according to median CCL21 value. GSEA results showed that the high-CCL21 group was enriched in NF-κB and chemokine signaling pathway, while the low-CCL21 group was primarily enriched in oocyte meiosis. These results suggest that the inflammation in lesion tissues of patients in high-CCL21 group was increased, and the maturation of follicles was inhibited, which also indicated that patients in the high-CCL21 group might experience infertility. NF-κB regulates multiple genes in response to various stimuli, such as pro-inflammatory cytokines, thereby participating in the inflammatory response [39]. The activation of NF-κB leads to its translocation from cytoplasm to nucleus, where it activates the transcription of target genes, primarily encoding factors involved in inflammatory response. The abnormal level of NF-κB is associated with the pathogenesis of autoimmune diseases and autoinflammatory conditions [40, 41]. Our results indicated that NF-κB was elevated in lesion tissues of patients and mouse model with endometriosis, which also suggested that endometriosis patients were in a state of activated inflammation. Knocking down CCL21 in 12z cells significantly inhibited NF-κB and its downstream inflammatory factors (IL-6, TNF-α, and IL-1β). Overall, reducing CCL21 alleviated the progression of endometriosis by suppressing the activation of NF-κB signaling pathway and inhibiting the expression of IL-6, TNF-α, and IL-1β.\u003c/p\u003e\n\u003cp\u003eThere are still limitations in evaluating the severity and location of endometriosis, as well as diagnostic strategies[42]. More and more evidences suggest that endometriosis is a systemic disease, and there are complex interactions between angiogenesis, hormones, and immune changes caused by chronic inflammation in endometriosis. Developing new and safer anti-inflammatory and immunomodulatory drugs may become a long-term treatment option for endometriosis patients [43]. In summary, we have confirmed the important role of CCL21 in endometriosis, providing reference information for the diagnosis and treatment of endometriosis. Although there are still some issues that need to be addressed, such as the relationship between CCL21 and the severity of endometriosis, and how to develop targeted CCL21 drugs to avoid adverse reactions to other tissues, the results of this study may still help clarify the pathogenesis of endometriosis development and promote the establishment of treatment methods.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe levels of inflammatory factors in endometriotic lesions of endometriosis patients and mouse models were markedly increased. Through comprehensive bioinformatics methods, we have identified three hub IRGs (CCL21, CFD, and ACKR1) and established a predictive model that showed good predictive ability. The CCL21 was overexpressed in endometriotic lesions and that CCL21 depletion inhibited the proliferation, migration, and invasion of 12z cells and promote apoptosis. More importantly, reducing CCL21 suppressed the pathology of endometriosis by inhibiting the activation of NF-\u0026kappa;B signaling pathway. Our research provided an innovative strategy of therapeutic intervention for endometriosis by targeting CCL21.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eIRGs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eInflammation-related genes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eEcE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eectopic endometrium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eEuE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eeutopic endometrium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eTCGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eThe Cancer Genome Atlas\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eDEGs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eDifferentially expressed genes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eGene Ontology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eBPs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eBiological processes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eCCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eCellular components\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eMFs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eMolecular functions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eWGCNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eWeighted Gene Co-expression Network Analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eTOM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eTopological Overlap Matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eROC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eReceiver operating characteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eGSEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eGene Set Enrichment Analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003ePhosphate buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eH\u0026amp;E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eHematoxylin and eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003ePB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003ePrussian blue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eFBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eFetal bovine serum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eSDS-PAGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eHRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eHorseradish peroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eRT-qPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eReal-time quantitative PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eGene significance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8698%;\"\u003e\n \u003cp\u003eMM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76.1302%;\"\u003e\n \u003cp\u003eModule membership\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by National Natural Science Foundation of China (No. 82201817); Basic Research Project of Liaoning Provincial Department of Education (No. LJKMZ20221290); Joint project of Liaoning Province Science and Technology Plan (No. 2024-BSLH-040); Youth Talent Cultivation Fund Project of Dalian Medical University (No. 508027 and No. 510016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original data applied in this research are accessible from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.W., B.L. and Y.Y.Z. collected, analyzed, and interpreted data and wrote the manuscript. M.H.G., J.S. and X.Q.M. were responsible for conducting experiments in molecular biology and part of bio-information analysis. J.L.B., L.Y. and X.Y.D. were responsible for clinical sample collection and mouse model construction. J.S. and R.J.J. reviewed the article and make suggestions for the manuscript. N.W. and J.L.B. designed and developed the methodology, and the principal supervisor and funder of the study. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Meihua Guo, Jie Sun or Jianlei Bi.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted with the approval of the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University approved the research protocol (Approval No. KY2024-174-01). Animal care and experimental procedures were conducted following a protocol approved by the Animal Care and Use Committees of Dalian Medical University (Approval No. L250627113).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTaylor H S, Kotlyar A M, Flores V A. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet (London, England). 2021; 397:839-52.\u003c/li\u003e\n\u003cli\u003eChapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. 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Reprod Biomed Online. 2024; 49:104292.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, Inflammation-related genes, CCL21, nomogram model","lastPublishedDoi":"10.21203/rs.3.rs-7558971/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7558971/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Endometriosis, affecting approximately 10% of reproductive-aged women worldwide, is a typical chronic inflammatory disease. Inflammation-related genes (IRGs) play a crucial role in the occurrence and progression of various diseases, including endometriosis. However, identifying which IRGs affect the pathology of endometriosis and how to target them for treatment remains highly challenging. In the present study, the ectopic endometrium (EcE) and eutopic endometrium (EuE) from endometriosis patients were collected and a mouse model of endometriosis was established to investigate the expression levels of inflammatory factors. Three hub IRGs (CCL21, CFD, and ACKR1) in endometriosis were identified, which were able to accurately predict the occurrence of endometriosis. The expression of CCL21 was obviously increased in the EcE of endometriosis patients and mouse models. Knockdown of CCL21 inhibited the proliferation, migration, and invasion of 12z cells and promoted apoptosis. Mechanistically, reduced CCL21 alleviated the pathology of endometriosis through restraining the activation of the NF-κB signaling pathway and the downstream inflammatory factors (IL-6, IL-1β, and TNF-α). In conclusion, increased CCL21 promoted the development of endometriosis by activating the NF-κB signaling pathway, and this finding offers a novel therapeutic target for treatment.","manuscriptTitle":"CCL21 promotes the development of endometriosis by activating NF-κB signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 02:45:46","doi":"10.21203/rs.3.rs-7558971/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3cef8d1e-8ddd-4511-907d-89a01fef5a15","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-06T13:23:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 02:45:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7558971","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7558971","identity":"rs-7558971","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}