Use of the SPON2 gene or its protein as a target in the diagnosis or treatment of endometriosis

Abstract

This invention belongs to the field of biotechnology, and particularly relates to the use of the SPON2 gene or its protein as a target in the diagnosis or treatment of endometriosis. Supported by multi-omics and single-cell level data and tested with clinical samples, this invention, for the first time, elucidates the correlation between SPON2 and endometriosis (EM), revealing that high expression of SPON2 in eutopic and ectopic endometrial fibroblasts in endometriosis patients enhances their proliferation, invasion, and migration abilities. Inhibiting SPON2 gene expression or inhibiting SPON2 protein function reduces the proliferation and invasion abilities of endometrial tissue, as well as lesion migration, invasion, and fibrosis, thus improving the condition of endometriosis. This suggests that the SPON2 gene or its protein can serve as a diagnostic or therapeutic target, providing new ideas and directions for the clinical diagnosis and targeted therapy of endometriosis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the use of the SPON2 gene or its protein as a target in the diagnosis or treatment of endometriosis. Background Technology

[0002] Endometriosis (EM) is a gynecological condition where endometrial tissue (containing glands and stroma) grows outside the uterine cavity. It is also known as endometriosis and is estrogen-dependent. Common clinical symptoms of EM include progressively worsening dysmenorrhea, as well as abnormal menstrual bleeding, dyspareunia, and infertility. Different lesion locations can lead to different symptoms; for example, intestinal endometriosis can cause abdominal pain and bowel problems, while respiratory endometriosis can cause hemoptysis and pneumothorax, severely impacting patients' quality of life. The incidence rate in women of reproductive age is approximately 10%-15%, and it is showing a significant upward trend.

[0003] Estrogen-induced endometriosis (EM) has long lacked effective therapeutic targets, and its treatment has primarily relied on medication and surgery. However, surgery is often ineffective and prone to recurrence, thus medication remains crucial. Commonly used and effective drugs for treating endometriosis include danazol, gestrinone, gonadotropin-releasing hormone analogs or agonists, progestins, and oral contraceptives. These drugs inhibit estrogen-dependent endometrial growth, but they suffer from high recurrence rates, significant side effects (such as osteoporosis caused by hormone therapy), and a lack of targeted therapy. Therefore, novel diagnostic and treatment strategies based on pathological mechanisms are urgently needed.

[0004] The pathogenesis of endometriosis (EM) is complex, involving biological processes such as inflammatory responses, angiogenesis, extracellular matrix adhesion, and invasion. Genetic factors play a significant role in its development. Certain gene mutations and dysregulations, such as overexpression of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) related to invasiveness, overexpression of vascular endothelial growth factor (VEGF) related to angiogenesis, abnormal or excessive expression of c-kit and DAD-1 related to anti-apoptosis, abnormal expression of aromatase and insufficient expression of 17β-hydroxysteroid dehydrogenase (17βHSD) related to local estrogen excess, abnormal progesterone receptor α / β ratio related to progesterone responsiveness, and overexpression of cyclooxygenase 2 (COX-2) related to inflammatory responses, are associated with an increased risk of endometriosis. However, research in these areas is still in its early stages and primarily focuses on traditional areas such as hormonal regulation and immune inflammation, lacking definitive diagnostic methods and specific treatments. Therefore, exploring the potential molecular mechanisms of endometriosis, deeply analyzing the multidimensional pathogenesis and candidate genes of EM, and finding reliable and specific diagnostic and targeted therapy biomarkers are key to advancing precision medicine, reducing the recurrence of EM, and are of great significance for the clinical diagnosis, disease monitoring, prevention and treatment of EM, as well as improving the quality of life of patients. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides the use of the SPON2 gene or its protein as a target in the diagnosis or treatment of endometriosis. This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of this invention provides the use of reagents for detecting the SPON2 gene or its protein in the preparation of diagnostic reagents or kits for endometriosis.

[0007] This invention, supported by multi-omics and single-cell level data and tested with clinical samples, for the first time elucidates the correlation between SPON2 and endometriosis (EM). SPON2 gene methylation levels are negatively correlated with the incidence of endometriosis, while mRNA and protein expression levels are positively correlated with the incidence of endometriosis. This suggests that SPON2 can serve as a diagnostic biomarker for EM, filling a long-standing technological gap in this field due to the lack of specific diagnostic molecular targets. It provides new ideas and directions for the clinical diagnosis and disease monitoring of endometriosis, possessing significant scientific value and potential clinical application value. Furthermore, the expression characteristics of SPON2 can be used to develop non-invasive detection methods (such as SPON2 protein / nucleic acid detection kits in blood, menstrual blood, uterine lavage fluid, or urine) to improve the early diagnosis rate of EM. Combined with indicators such as CA125 and IL-6, it can also optimize EM risk screening models.

[0008] In actual testing, SPON2 gene mRNA levels, SPON2 protein levels, and SPON2 gene methylation levels can all serve as independent diagnostic factors. Elevated mRNA and / or protein expression levels suggest an individual at risk of endometriosis (EM), while decreased SPON2 gene methylation levels suggest an individual at risk of EM. Using SPON2 as a diagnostic biomarker for EM can significantly improve the sensitivity and specificity of disease diagnosis.

[0009] Optionally, the reagents for detecting the SPON2 gene or its protein include at least one of the reagents for detecting the SPON2 gene methylation level, the SPON2 gene mRNA level, and the SPON2 protein level.

[0010] Optionally, the reagents for detecting the SPON2 gene mRNA level include primer pairs that specifically amplify the SPON2 gene, and the detection methods include, but are not limited to, quantitative real-time PCR (qRT-PCR). The nucleotide sequences of the upstream and downstream primers of the primer pair are shown below:

[0011] SPON2-F: ACAGCATCACCTTCACGGGCAA (see SEQ ID NO.4);

[0012] SPON2-R: CTGACGTACTGGTTCTTCCTCC (see SEQ ID NO. 5).

[0013] Optionally, reagents for detecting SPON2 protein levels include antibodies that specifically target the SPON2 protein, which are commercially available. Detection methods include, but are not limited to, Western blotting, immunohistochemistry, and immunofluorescence.

[0014] The second aspect of this invention provides the use of a reagent that inhibits the expression level of the SPON2 gene in the preparation of a drug for treating endometriosis.

[0015] This invention reveals for the first time that fibroblasts in both situ and ectopic endometrium of endometriosis patients can enhance their proliferation, invasion, and migration capabilities by overexpressing SPON2. Furthermore, fibroblasts that overexpress SPON2 can promote the proliferation, invasion, and migration capabilities of endometrial epithelial cells, as well as epithelial-mesenchymal transition, thereby driving the implantation and fibrosis of endometriosis lesions. By inhibiting the expression of the SPON2 gene or inhibiting the function of the SPON2 protein, the aforementioned phenomena can be reversed, reducing the proliferation and invasion capabilities of endometrial tissue, as well as lesion migration, invasion, and fibrosis, thus improving the condition of endometriosis. This suggests that SPON2 can serve as a therapeutic target, enabling precise regulation of the lesion microenvironment and more specific molecular targeted therapy, providing new ideas and strategies for the clinical prevention and / or treatment of endometriosis.

[0016] Optionally, the reagents used to inhibit SPON2 gene expression include, but are not limited to, SPON2 protein inhibitors and SPON2 gene-targeting interfering RNA (siRNA). The aforementioned reagents, such as siRNA, can be designed based on the SPON2 gene sequence, and the SPON2 protein inhibitors are conventional chemical inhibitors used in the art, which will not be elaborated further here.

[0017] Optionally, the interfering RNA targeting the SPON2 gene is selected from at least one of the following nucleotide sequences:

[0018] SPON2-Homo-450: GCCAAATACAGCATCACCTTC (see SEQ ID NO.6);

[0019] SPON2-Homo-555: GCGCATAGCTCCGACTACAGC (see SEQ ID NO. 7);

[0020] SPON2-Homo-640: GGGCGCTGATGAAGGAGATCG (see SEQ ID NO. 8). Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This invention provides a correlation analysis between SPON2 gene single nucleotide polymorphism sites and SPON2 gene methylation and expression levels.

[0023] Figure 2Correlation analysis of SPON2 gene methylation, mRNA, and protein levels with endometriosis in embodiments of the present invention;

[0024] Figure 3 This is a figure showing the results of SPON2 gene expression level analysis in situ endometrium and ectopic lesions of endometriosis patients in the single-cell transcriptome sequencing dataset GSE179640 of this invention.

[0025] Figure 4 This is a clustering diagram of different cells and a cell localization diagram of SPON2 gene expression in the single-cell transcriptome sequencing dataset GSE179640 of this invention.

[0026] Figure 5 This is a figure showing the analysis results of SPON2 gene expression level in different cells of endometriosis patients in the single-cell transcriptome sequencing dataset GSE213216 of this invention.

[0027] Figure 6 This is a clustering diagram of different cells and a cell localization diagram of SPON2 gene expression in the single-cell transcriptome sequencing dataset GSE213216 of this invention.

[0028] Figure 7 This is a figure showing the results of SPON2 gene expression level analysis in different tissues of endometriosis patients in the tissue RNA sequencing dataset GSE141549, an embodiment of the present invention.

[0029] Figure 8 This image shows the immunofluorescence staining results of SPON2 protein in endometrial tissue samples from women with and without endometriosis, according to an embodiment of the present invention.

[0030] Figure 9 This is an image showing the immunofluorescence staining results of primary endometrial fibroblasts in women with and without endometriosis, according to an embodiment of the present invention.

[0031] Figure 10 This figure shows the results of measuring the relative levels of SPON2 mRNA and the expression levels of its protein in endometrial fibroblasts of women with and without endometriosis, according to an embodiment of the present invention.

[0032] Figure 11 This is a diagram showing the proliferation capacity of primary endometrial fibroblasts in women with and without endometriosis, according to an embodiment of the present invention.

[0033] Figure 12 This is a graph showing the results of measuring the migration and invasion ability of primary endometrial fibroblasts in women with and without endometriosis, according to an embodiment of the present invention.

[0034] Figure 13 This is a graph showing the results of endometrial epithelial cell proliferation assays after co-incubation with primary endometrial fibroblasts in women with and without endometriosis, according to an embodiment of the present invention.

[0035] Figure 14 This figure shows the results of measuring the migration and invasion ability of endometrial epithelial cells after co-incubation with primary endometrial fibroblasts in women with and without endometriosis, according to an embodiment of the present invention.

[0036] Figure 15 This is a graph showing the results of endometrial epithelial cell proliferation assay after co-incubation with SPON2 protein in an embodiment of the present invention.

[0037] Figure 16 This is a graph showing the results of measuring the migration and invasion ability of endometrial epithelial cells after co-incubation with SPON2 protein in an embodiment of the present invention.

[0038] Figure 17 This figure shows the results of detecting the expression levels of E-cadherin and N-cadherin in endometrial epithelial cells after co-incubation with SPON2 protein using Western blotting, as an embodiment of the present invention.

[0039] Figure 18 The figure shows the results of measuring the relative levels of SPON2 mRNA and the expression levels of SPON2 protein in endometrial fibroblasts with SPON2 gene knocked down in an embodiment of the present invention.

[0040] Figure 19 This is a graph showing the results of the proliferation assay of endometrial fibroblasts with SPON2 gene knockdown in an embodiment of the present invention.

[0041] Figure 20 The figure shows the results of the migration and invasion ability determination of endometrial fibroblasts with overexpression and knockdown of the SPON2 gene in an embodiment of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are merely illustrative and not intended to limit the invention. Based on the information contained in this invention, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of this invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to the embodiments of this invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0043] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, etc., should in all cases be understood to be modified by the word "approximately". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and by conventional rounding methods. The meanings of words such as "comprising," "including," "containing," "having," etc., are non-limiting, allowing for the addition of other steps and other components that do not affect the result. "And / or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" would be considered to include (i) A, (ii) B, and (iii) A and B.

[0044] Endometriosis (EM) lesions are more inflammatory and fibrotic than normal endometrium. Ectopic menstrual fragments trigger inflammation, leading to platelet aggregation, immune cell recruitment, and the release of pro-inflammatory cytokines and mediators. In normal wound healing, inflammatory cytokines induce transient epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT), which are crucial for tissue remodeling and repair. However, in the chronic tissue damage of EM, persistent EMT and FMT lead to excessive extracellular matrix (ECM) deposition, resulting in fibrosis. Fibrosis is a key factor in EM progression and is associated with symptoms such as pain and infertility. Furthermore, ectopic endometrial cells undergo partial EMT under hormonal and inflammatory stimuli, enhancing their invasiveness and viability; adhesion and invasion between ectopic endometrial cells and the peritoneum are key steps in the formation of ectopic lesions. Existing studies have revealed the presence of EMT in ectopic endometrium, which is associated with disease progression and fibrosis (e.g., reference [1] "Zhu Tianhong, Zhang Xinmei. Research progress on epithelial-mesenchymal transition mediating the occurrence and development of endometriosis [J]. Journal of Zhejiang University: Medical Edition, 2016, 45(4):7." and reference [2] "Liu Chang, Liu Taihang, He Fan, et al. Screening of differential genes in endometriosis and verification of the expression of potential target growth arrest-specific protein 6 [J]. Chinese Journal of Biochemistry and Molecular Biology, 2021, 37(7):10.").

[0045] Epithelial-mesenchymal transition (EMT) refers to the process by which epithelial cells lose their polarity and intercellular adhesion properties, transforming into mesenchymal-like cells with migratory and invasive capabilities. This phenomenon is widely involved in embryonic development, tissue repair, cancer metastasis, and fibrotic diseases. EMT is characterized by the downregulation of epithelial cell markers such as endothelial cadherin (E-Cadherin), platelet-endothelial cell adhesion molecule (Pecam-1 / CD31), and vascular endothelial growth factor (VEGFR), and the upregulation of mesenchymal cell markers such as α-smooth muscle actin (α-SMA), N-cadherin, vimentin, fibronectin, and fibroblast-specific protein 1 (FSP1). These changes in the expression of epithelial and mesenchymal cell markers lead to reduced adhesion between transitional cells and adjacent epithelial cells, while increasing the secretion of enzymes that degrade the extracellular matrix. During this process, significant changes occur in the polarity, morphology, and function of epithelial cells, resulting in enhanced migration, motility, and proliferation capabilities. Simultaneously, the epithelial phenotype is lost, gradually giving way to a mesenchymal phenotype. In endometrial cells (EM), endometrial cells induce EMT through multiple pathways, enhancing cell migration, invasion, and anti-apoptosis abilities, participating in the formation of local endometriotic lesions, and promoting the development and progression of endometriosis.

[0046] Spondin-2 (SPON2) is a secreted protein belonging to the spondin protein family, also known as Mindin. It exists primarily in the extracellular matrix in a secreted form and participates in various biological processes, including cell adhesion, migration, immune response regulation, and tumor microenvironment regulation. SPON2 (NCBI gene ID: 10417, GenBank ID: NM_012445.4, Ensembl gene ID: ENSG00000159674, NCBI protein ID: NP_036577.2) has been shown to drive disease progression in some tumors by promoting endogenous tumor metastasis (EMT), cell migration, and fibrosis; however, its role in endogenous tumors (EM) remains unclear. Currently, there is no literature systematically elucidating the mechanism of action of SPON2 in the development and progression of EM, nor are there any EM treatment strategies targeting SPON2.

[0047] The amino acid sequence of the SPON2 protein is shown below:

[0048] MENPSPAAALGKALCALLLATLGAAGQPLGGESICSARALAKYSITFTGKWSQTAFPKQYPLFRPPAQWSSLLGAAHSSDYSMWRKNQYVSNGLRDFAERGEAWALMKEIEAAGEALQSVHAVFSAPAVPSGTG QTSAELEVQRRHSLVSFVVRIVPSPDWFVGVDSLDLCDGDRWREQAALDLYPYDAGTDSGFTFSSPNFATIPQDTVTEITSSSPSHPANSFYYPRLKALPPIARVTLVRLRQSPRAFIPPAPVLPSRDNEIVDSASVPETPLDCEVSLWSSWGLCGGHCGRLGTKSRTRYVRVQPANNGSPCPELEEEAECVPDNCV (see SEQ ID NO. 1).

[0049] This invention employs multi-omics joint analysis, retrieving and downloading transcriptome datasets related to endometriosis from the GTEx and eQTLGen databases, and combining them with plasma proteome datasets from the UKB, Decode, Nature, and NK databases, as well as DNA methylome datasets from the GTEx database. Using Mendelian randomized inverse variance weighted method (MR-IVW) to exclude confounding factors, the causal relationship between SPON2 gene expression and EM was verified, confirming that SPON2 gene expression is significantly high in EM patients. Further, open-source single-cell sequencing data related to endometriosis expression were downloaded from the gene expression omnibus (GEO) database for cross-validation. The results showed that SPON2 gene mRNA expression levels in both eutopic and ectopic endometrial tissues of EM patients were higher than in healthy eutopic endometrial tissues. Single-cell analysis also showed high SPON2 expression in fibroblasts, suggesting a potential association with fibroblast activation and fibrosis, and potentially making it a target for EM treatment. In clinical samples of endometriosis, the expression level of SPON2 protein in the situ endometrium and ectopic lesions of endometriosis patients was diagnosed using immunofluorescence staining (IF). The clinical pathological diagnosis results were consistent with the results of gene-level bioinformatics analysis, confirming that the expression level of SPON2 protein in the ectopic endometrium of endometriosis patients was higher than that in the healthy endometrium of non-endometriosis patients in the control group, and the expression was highest in the situ endometrium of endometriosis patients. This invention clarifies the correlation between SPON2 and EM for the first time, suggesting that SPON2 can serve as a diagnostic biomarker for EM, filling the long-standing technical gap in this field of lacking specific diagnostic molecular targets. Using SPON2 as a diagnostic biomarker for endometriosis (EM), SPON2 mRNA and / or protein levels are key factors that are significantly upregulated during the pathogenesis of EM, while methylation levels are key factors that are significantly downregulated during the pathogenesis of EM. The detection of SPON2 gene mRNA levels, SPON2 protein levels, and SPON2 gene methylation levels provides new ideas and directions for the clinical diagnosis and disease monitoring of endometriosis. It can greatly improve the sensitivity and specificity of disease diagnosis and is also conducive to the development of non-invasive detection methods to improve the early diagnosis rate of EM.

[0050] Based on the aforementioned research revealing SPON2 as a pathogenic target, this invention also explores the feasibility of SPON2 as a therapeutic target. Overexpression of SPON2 in endometrial fibroblasts showed that fibroblasts with high SPON2 expression exhibited stronger proliferation, invasion, and migration abilities, consistent with the characteristics of endometriosis pathological cells. Treatment of endometrial epithelial cells with SPON2 protein also enhanced their proliferation, invasion, and migration abilities, and showed significant EMT transformation. These biological behaviors are conducive to the implantation and fibrosis of endometriosis lesions. This invention reveals for the first time that eutopic and ectopic endometrial fibroblasts in endometriosis patients drive the implantation and fibrosis of endometriosis lesions through high SPON2 expression. Inhibiting SPON2 gene expression or SPON2 protein function can reverse these phenomena, significantly reducing lesion migration, invasion, and fibrosis. This demonstrates that SPON2 can serve as a therapeutic target, providing new ideas and strategies for the clinical prevention and / or treatment of endometriosis.

[0051] For a long time, endometriosis (EM) research has mainly focused on traditional areas such as hormone regulation and immune inflammation, with insufficient attention paid to the function of fibroblasts in EM. This invention breaks through this inherent research perspective. Based on systematic multi-omics data and cell subset analysis, it proposes the core role of fibroblast activation and its key regulator SPON2 in the EM process, overcoming existing technological path dependence and research bias, and opening up a new direction for EM research and treatment. Using SPON2 as a therapeutic target, by intervening in or downregulating the expression level of the SPON2 gene or protein, it can inhibit fibroblast activation, suppress the fibrosis process of EM lesions, inhibit epithelial-mesenchymal transition (EMT) of endometrial cells, reduce the proliferation and invasiveness of endometrial tissue, improve the condition of endometriosis, achieve precise regulation of the lesion microenvironment and more specific molecular targeted therapy, and reduce interference with the systemic hormonal axis. Efficacy can be improved and side effects reduced through local administration (such as intrauterine injection or sustained-release gel). The SPON2-based targeted intervention strategy is expected to break through the limitations of current treatment methods and develop new drugs with higher efficacy and lower side effects. Given that EM is prevalent among women of childbearing age and has a significant impact on their quality of life, this invention has broad prospects for clinical translation and huge market potential, and is expected to bring considerable economic and social benefits to the field of women's health.

[0052] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or conditions recommended by the manufacturer.

[0053] 1. Multi-omics combined analysis to screen potential targets for endometriosis (EM)—SPON2

[0054] 1.1 Proteomics Analysis

[0055] Candidate druggable proteins were obtained by intersecting proteomic data (UKB, NC2022, Nature2018, and Decode) with 5355 drugable genes.

[0056] Acquisition of the protein library: The UK Biobank (UK Biobank) proteomics data collected a large number of participants’ biological samples and detailed health information. The participants were approximately 500,000 UK residents aged 40 to 69. Plasma samples from more than 54,000 participants were analyzed. Using the Olink Explore platform, nearly 3,000 proteins were measured in each sample (see reference [3] SUN BB, CHIOU J, TRAYLOR M, et al. Plasma proteomicas associations with genetics and health in the UK Biobank [J]. Nature, 2023, 622 (7982): 329-38.). The NC2022 (Nature Cardiovascular Research 2022) proteome data (see reference [4] FERKINGSTAD E, SULEM P, ATLASON BA, et al. Large-scale integration of the plasma proteome with genetics and disease[J].Nat Genet,2021,53(12):1712-21.) explored the association between blood protein levels and various cardiovascular diseases. The participants were more than 4,700 Icelandic people from the deCODE genetics study cohort, and about 4,900 plasma proteins were measured in the study. The Nature 2018 proteome data (see reference [5] SUN BB, MARANVILLE JC, PETERS JE, et al. Genomic atlas of the human plasma proteome[J]. Nature, 2018, 558(7708):73-9.) conducted a genome-wide association study (GWAS) on the plasma proteome of a large population, identifying thousands of genetic loci (pQTLs) that affect protein levels. Participants included multiple European population cohorts, and nearly 3,000 plasma proteins were measured using the SomaScan platform. The deCODEGenetics (deCODE genetics / Amgen) proteome data utilized Iceland's unique and relatively homogeneous population resources to establish a large genetic and health database; participants were mainly Icelandic population cohorts, and in its landmark study (see reference [4]), approximately 5,000 plasma proteins from plasma samples of nearly 36,000 Icelanders were analyzed.

[0057] Screening and establishment of a druggable gene library: ① Screening drug target genes from CHEMBL+UNIPROT. Screening criteria: human origin, protein type (Single protein, selectivity group, protein-protein interaction, protein complex group, protein complex, chimeric protein, protein family), and the corresponding drugs of the drug target protein are clinically approved drugs or clinical candidate drugs. 2613 drug target genes were screened. ② Screening drug target genes from existing literature, including literature [6] "Finan C, Gaulton A, Kruger FA, et al. The druggable genome and support for target identification and validation in drug development[J]. Science Translational Medicine, 2017, 9(383): eaag1166. (reported 4479 drug target gene Ensembl IDs)" and literature [7] "Gaziano, L., Giambartolomei, C., Pereira, AC et al. Actionable and druggable genome-wide Mendelian randomization identifies repurposing opportunities for COVID-19. Nat Med 27, 668–676 (2021). (reported 1384 drug target gene Ensembl IDs)". ③ The drug target genes from the above two sources were combined to obtain a total of 5355 drug target gene Ensembl IDs.

[0058] Candidate proteins were used as risk factors (exposure variables). Data from 16,588 endometriosis cases and 111,583 non-endometriosis female controls provided by the Finngen r10 biobank were used as outcome variables. Using genetic variations (such as single nucleotide polymorphisms, SNPs) as instrumental variables, Mendelian randomization (MR) analysis with inverse variance weighting (IVW) was employed to explore the causal relationship between risk factors and the disease (outcome). At least 24 druggable plasma proteins highly expressed in endometriosis were screened: RSPO3, MGAT2, SLC14A1, SCGB1A1, ST3GAL1, LCT, GFRA1, EPHA5, TNFRSF12A, CHGA, SERPINE1, DDC, IL1RN, HTN1, STX1A, BRPF1, MSR1, SIGLEC11, GPLD1, CBLN1, CD33, TNC, SPON2, and PARP1. Among them, a highly significant causal relationship was found between the heritable elevation of SPON2 protein levels and the risk of endometriosis (beta = 0.2386, SE = 0.066, p = 0.0003). For every standard deviation increase in SPON2 protein levels, the hazard ratio (Odds Ratio) for endometriosis increased by e. 0.2386 (Approximately 1.27 times).

[0059] 1.2 Transcriptomics Analysis

[0060] First, the plasma mRNA databases of eQTLGen and GTEx were used to verify whether the 24 candidate proteins screened were also highly expressed at the plasma mRNA level. eQTLGen (eQTLGen Consortium) identifies genetic loci (eQTLs) affecting gene expression levels in blood by integrating data from multiple large cohort studies worldwide. This project integrated data from 37 different study cohorts, with a total sample size of 31,684 individuals, primarily whole blood or peripheral blood mononuclear cells (PBMCs), providing genetic regulatory information on the expression of 19,960 genes, and successfully locating significant cis-eQTLs (cis-eQTLs) for 16,987 of these genes. GTEx (Genotype-Tissue Expression) explores gene function and regulatory mechanisms in specific tissues and is widely used to identify blood-related biomarkers and disease pathways. Participants were deceased tissue donors, covering a wide age range and diverse ethnic backgrounds. The latest GTExV8 version includes whole blood samples from 755 donors. Comprehensive RNA sequencing was performed on each tissue sample (including whole blood), analyzing the expression levels of over 20,000 protein-coding genes and long non-coding RNAs. Plasma mRNA analysis showed that SPON2 was highly expressed in both mRNA databases.

[0061] Secondly, GTEx plasma DNA methylation data were used to determine whether the high expression of the target gene was affected by DNA methylation modification. Using mRNA and DNA methylation data as exposure variables and endometriosis and control data provided by the Finngen r10 biobank as outcome variables, the impact of the target gene on endometriosis was assessed through MR analysis and Mendelian randomization (SMR) analysis based on abstract data. Transcriptomic SMR analysis of the SPON2 gene revealed multiple single nucleotide variants (SNPs), including rs73067776, rs28856334, and rs6836335. Among these, the top SNP (rs73067776) was significantly associated with endometriosis (EM) and showed significant association with EM and its multiple subtypes (such as pelvic peritoneal endometriosis, ovarian endometriosis, and ASRM stage 3 / 4 endometriosis), suggesting that SPON2 may play an important role in the pathogenesis of EM.

[0062] In the methylation status analysis, two SNPs with significant p-values ​​(p-value < 0.05) (rs28856334, rs6836335) were both associated with hypomethylation at the cg03250742 site of SPON2 (see [link to analysis]). Figure 1The left-middle figure shows the SNP sites on the vertical axis and the methylation level at the cg03250742 site of the SPON2 gene on the horizontal axis. This causes the gene's "expression switch" to be turned on. Further analysis showed that the genotypes of these two SNPs were significantly correlated with the expression of the SPON2 gene (p value < 0.05) (see [reference needed]). Figure 1 The right-hand side of the figure shows that the vertical axis represents the whole blood SPON2 expression level, and the horizontal axis represents the SNP locus genotype. This demonstrates that SPON2 is hypomethylated in endometriosis, leading to its high gene expression.

[0063] In summary, SPON2 at methylation, mRNA, and protein levels was statistically significantly associated with the risk of endometriosis (p < 0.05), expressed as adjusted hazard ratios (HR) and 95% confidence intervals (CI). SPON2 mRNA and protein expression levels were positively correlated with the occurrence of endometriosis, while methylation status was negatively correlated with the incidence of endometriosis. See the summary of related results below. Figure 2 The vertical axis of the figure represents SPON2 methylation, mRNA levels from different databases, and protein levels from different databases, while the horizontal axis represents the adjusted HR value and its 95% confidence interval. This analysis, combined with proteomics analysis, suggests that SPON2 is a potential diagnostic and therapeutic target significantly associated with endometriosis.

[0064] 1.3 Single-cell transcriptome analysis

[0065] This invention further introduces single-cell transcriptome analysis technology, using open-source single-cell transcriptome sequencing data (GEO accession numbers GSE179640 and GSE213216) for analysis, to accurately locate the source of disease from the cellular level.

[0066] The GSE179640 dataset (published in reference [8] "Tan, Y., Flynn, WF, Sivajothi, S. et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis acrosseutopic and ectopic tissues. Nat Cell Biol 24, 1306–1318 (2022)") contains biopsy single-cell sequencing data from 14 individuals, including 11 EM patients with situ endometrial and ectopic ovarian lesions, peritoneal lesions and their adjacent areas, and 3 non-endometriosis patients with normal endometrial tissue (control, Ctrl). Transcriptome sequencing data of eutopic endometrium (EuE), ectopic peritoneum (EcP), and adjacent regions (EcPA) and ectopic ovary lesions (EcO) were collected from GSE179640. SPON2 gene expression levels were analyzed. Results showed that SPON2 expression in eutopic endometrium (EuE), ectopic peritoneum (EcP), and ectopic ovary lesions (EcO) was significantly higher than in the control (Ctrl) eutopic endometrium (see...). Figure 3 The vertical axis represents SPON2 expression level, and the horizontal axis represents disease type, indicating that SPON2 is highly expressed in both situ endometrium and ectopic lesions in patients with endometriosis (EM). Further analysis of SPON2 expression at the cellular level revealed that, regardless of whether it is ectopic lesions or situ endometrium, SPON2 is mainly expressed in fibroblasts (see...). Figure 4 The left image is a UMAP map of SPON2 expression levels, with red indicating SPON2 expression sites and darker colors indicating higher SPON2 expression levels. The right image is a UMAP map of cell type clusters (different colors represent different cell types). This suggests that SPON2 may promote the formation and development of fibrosis in endometriosis lesions through specific signaling pathways (such as inflammatory responses and extracellular matrix remodeling). Therefore, we can investigate whether targeting SPON2 can improve the pathological state of endometriosis, such as slowing down fibrosis or reducing inflammation levels.

[0067] The GSE213216 dataset (published in the literature [9] "Tan Y, Flynn WF, Sivajothi S, Luo D, Bozal SB, Davé M, Luciano AA, Robson P, Luciano DE, Courtois ET. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol. 2022 Aug; 24(8):1306-1318. doi:10.1038 / s41556-022-00961-5. Epub 2022 Jul 21. Erratumin: Nat Cell Biol. 2022 Nov; 24(11):1679.") contains three groups of samples: situ endometrium from endometriosis patients, ectopic lesions from endometriosis patients, and endometrium from healthy controls. The same method described above was used to analyze this dataset, and the relevant results are shown below. Figure 5 (The vertical axis represents SPON2 expression level, and the horizontal axis represents disease type) and Figure 6 (UMAP plot of SPON2 expression level, the right figure is a clustered UMAP plot of cell types), which also shows that SPON2 is highly expressed in the situ endometrium of endometriosis patients and is mainly expressed in fibroblasts.

[0068] 1.4 Analysis of Tissue RNA Sequencing Data

[0069] To further validate the high expression of SPON2 in endometriosis patients, a pooled GEO dataset (GSE141549) containing RNA sequencing data from 115 patients and 53 controls was included in the analysis. The GSE141549 dataset is published in the literature

[10] "Gabriel M, Fey V, Heinosalo T, Adhikari P, The dataset contains over 24,000 genes and clinical characteristics, such as age, disease stage, hormone use, menstrual cycle stage, and different types of endometriosis lesions. It can be interactively analyzed using the EndometDB online software (access link https: / / endometdb.utu.fi / ). (References: K, Komulainen T, Huhtinen K, Laajala TD, Siitari H, Virkki A, Suvitie P, Kujari H, Aittokallio T, Perheentupa A, Poutanen MA, Relational database to identify differentially expressed genes in the endometrium and endometriosis lesions. Sci Data. 2020 Aug 28;7(1):284.”)

[0070] The analysis results for GSE141549 were consistent with those for single-cell data GSE179640 and GSE213216. Compared with the control, SPON2 expression was upregulated in both the situ endometrium and peritoneum of endometriosis patients, and SPON2 expression was also increased in multiple ectopic lesions in the pelvis (peritoneum, deep tissue, ovary) (see [link to analysis]). Figure 7 (The vertical axis represents the SPON2 expression level, and the horizontal axis represents the tissue location).

[0071] The above results indicate that endometrial-associated fibroblasts are an important source of SPON2, and their high expression makes endometrial tissue more susceptible to implantation and metastasis, leading to the formation and progression of endometriosis lesions. The mechanism may be that SPON2 derived from these fibroblasts is secreted into the extracellular matrix (ECM), promoting epithelial-mesenchymal transition (EMT) of endometrial cells by regulating EMT remodeling. EMT enhances the proliferation and invasion of endometrial tissue, promoting cell adhesion, migration, and invasion, thus leading to the implantation and progression of endometriosis lesions.

[0072] 1.5 Immunofluorescence analysis of clinical samples

[0073] Embedded tissues from surgical patients at Xiangya Hospital (all of whom signed informed consent forms) were selected, namely, control endometrial tissue and endometrial tissue in situ with endometriosis. Multiplex immunofluorescence staining of SPON2 protein was performed. The anti-SPON2 protein primary antibody (purchased from abclonal, catalog number A12077), anti-a-SMA (purchased from abcam, catalog number Ab5694), and the corresponding immunofluorescence secondary antibody (purchased from AKOYA, catalog number NEL861001KT) were all commercially available.

[0074] See results Figure 8 SPON2 protein stained red after binding with primary and secondary antibodies, and blue after DAPI staining, indicating nuclear localization. α-Smooth muscle actin (α-SMA), as a marker of mesenchymal fibroblasts, stained green after binding with primary and secondary antibodies against α-SMA protein. The merged image shows a red-blue-green fusion. It is evident that SPON2 is expressed in mesenchymal fibroblasts (marker α-SMA) in endometrial tissue, and its expression in the situ endometrium of patients is higher than that in the endometrium of non-endometriosis controls.

[0075] 2. The effect of SPON2 gene expression level on biological behavior in endometrial fibroblasts of patients with endometriosis.

[0076] 2.1 Isolation and identification of primary human endometrial fibroblasts

[0077] Extraction of primary fibroblasts from human endometrium: Endometrial tissue was minced to a diameter of less than 2 mm, washed three times with ice-cold PBS containing 200 U / mL penicillin / streptomycin, and minced again to a diameter of less than 1 mm. 10 mL of tissue digestion solution (1 mg / mL collagenase IV / II) was added, and the mixture was incubated at 37°C on a shaker at 110 rpm for 40-50 min. Digestion was stopped by adding 20 mL of serum-containing DMEM / F12 medium. Undissociated tissue was initially removed using a 70 μm sieve, and the cells were then passed through a 40 μm sieve to separate fibroblasts and epithelial cells. The cell suspension was collected and centrifuged at 4°C, 400 × g for 5 min, and the supernatant was discarded. 5 mL of erythrocyte lysis buffer was added, and the cells were lysed at room temperature for 10 min. The supernatant was then removed by centrifugation, and the washing steps were repeated twice. The cell pellet was resuspended in 3 mL of culture medium and incubated in T25 culture flasks at 37°C for 48 h.

[0078] (2) Immunofluorescence staining (IF) identification of primary fibroblasts: Cells in the logarithmic growth phase were digested, resuspended, and plated on glass slides in a chamber. After 12 hours of cell adhesion, the culture medium was discarded, and 4% paraformaldehyde was added for fixation at room temperature for 30 minutes. After washing twice with PBS, the cell membrane was permeated with 0.5% Triton-100. After blocking with 3% FBS at room temperature for 10 minutes, anti-α-SMA antibody (1:1000 dilution) and anti-pan-CK antibody (1:500 dilution) were added respectively. Incubate overnight at 4℃; discard the antibody, wash three times with PBST for 5 min each time, add the corresponding species' fluorescently labeled secondary antibody (1:2000 dilution), and incubate at room temperature in the dark for 30 min; discard the antibody, wash twice with PBST for 5 min each time, wipe the slide dry, add anti-fluorescence quenching mounting solution containing DAPI (4',6-diamidino-2-phenylindole) (purchased from Beyotime, catalog number P0131) to mount the slide, and observe under a fluorescence microscope.

[0079] Figure 9 The image shows the IF results of characteristic markers of isolated primary fibroblasts. The top image shows endometrial fibroblasts (NF cells) from women without endometriosis, and the bottom image shows endometrial fibroblasts (EMF cells) from women with endometriosis. It can be seen that the primary cells all express the characteristic marker α-SMA and do not express the epithelial cell marker pan-CK, confirming that the cells are fibroblasts.

[0080] 2.2 Determination of SPON2 gene expression levels in primary fibroblast NF and EMF

[0081] SPON2 protein levels were detected using Western blotting (WB). Total RNA was extracted from primary fibroblasts (NF) and EMF cells and reverse transcribed into cDNA using a reverse transcription kit (Takara, catalog number rr047a). Tubin was used as an internal control gene. SPON2 gene expression levels in NF and EMF cells were detected using a quantitative real-time PCR (qRT-PCR) kit (Vazyme, catalog number Q712-02). The primer sequences for the internal control gene and SPON2 gene detection are as follows:

[0082] Tubin-F: CACCATTGGCAATGAGCGGTTC (see SEQ ID NO. 2);

[0083] Tubin-R: AGGTCTTTGCGGATGTCCACGT (see SEQ ID NO.3);

[0084] SPON2-F: ACAGCATCACCTTCACGGGCAA (see SEQ ID NO.4);

[0085] SPON2-R: CTGACGTACTGGTTCTTCCTCC (see SEQ ID NO. 5).

[0086] See relevant results Figure 10 The left graph shows SPON2 mRNA levels, with the horizontal axis representing cell type (NF cells from non-endometriotic women and EMF cells from endometriotic women), and the vertical axis representing the relative expression level of SPON2 gene mRNA. The right graph shows SPON2 protein levels. It is evident that the SPON2 gene expression level in EMF cells of situ endometriotic endometrium is significantly higher than that in NF cells of non-endometriotic endometriotic women.

[0087] 2.3 Determination of NF and EMF proliferation capacity of primary fibroblasts

[0088] The proliferation capacity of NF and EMF cells was detected using the CCK-8 assay, comprising the following steps: S1, seeding 2000 cells per well into a 96-well plate at a predetermined density, typically 100 μL per well, and culturing at 37°C and 5% CO2 until cell adhesion; S2, removing the old culture medium and adding fresh culture medium, and continuing culturing for the corresponding time (e.g., 24h, 48h, 72h), performing detection at different time points according to the experimental design; S3, adding CCK-8 reagent at the predetermined detection time point, removing the culture medium from each well, adding 10% (wt) CCK-8 solution diluted with fresh culture medium, 100 μL per well, and mixing thoroughly; S4, returning the 96-well plate to the incubator and incubating in the dark for 2h; S5, measuring the absorbance (OD value) of each well at a wavelength of 450 nm.

[0089] Data analysis was performed to calculate cell viability / proliferation rate: First, the average OD value for each sample was calculated, then the cell proliferation rate was calculated, and growth curves were plotted. See the relevant results below. Figure 11 The graph shows that the horizontal axis represents culture time and the vertical axis represents cell viability. It is evident that fibroblasts (EMFs) in situ endometrium exhibit stronger proliferative capacity in endometriosis.

[0090] 2.4 Determination of the invasion and migration abilities of primary fibroblasts using NF and EMF

[0091] The migration and invasion abilities of NF and EMF cells were detected using the Transwell assay, including the following steps: ① Migration: Cells were digested, resuspended in serum-free medium for counting, diluted with serum-free DMEM F12 medium to obtain 50,000 cells per well and added to the upper chamber. 600 μL of 10% FBS DMEM F12 complete medium was added to the lower chamber. After 48 h, the chamber was removed, the upper chamber medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min, then stained with crystal violet for 30 min. After rinsing twice with tap water, the cells were gently wiped with cotton swabs, dried, photographed, and counted. ② Invasion: The steps were the same as before. Before adding cells, 15% Methylprednisolone was added to the upper chamber and incubated in a gel incubator for 30 min. Then, 5,000 cells were added to each well.

[0092] See relevant results Figure 12 The top image, from left to right, shows crystal violet staining of migrating cells and statistical data on the number of migrating cells in NF and EMF cells. The bottom image, from left to right, shows crystal violet staining of invasive cells and statistical data on the number of invasive cells in NF and EMF cells. It is evident that, compared to NF, endometriosis-related endothelial fibroblasts (EMF) exhibit stronger invasive and migratory capabilities.

[0093] 2.5 Changes in biological behavior of primary fibroblasts (NF and EMF) co-cultured with endometrial epithelial cells (Ishikawa).

[0094] NF and EMF cells were co-cultured with human endometrial cancer Ishikawa (an epithelial cell line, ISK cells), including the following steps: NF and EMF cells were cultured in serum-free medium for 24 hours, the medium was collected, and the cells were removed by filtration through a 0.22 μm filter. Then, Ishikawa cells were added to complete medium at a 1:1 ratio in 96-well plates or chambers, with 2000 cells per well in the 96-well plate. At a set time, the cells were incubated with 10% CCK8 reagent prepared from empty medium. OD was measured using a microplate reader. 450 The absorbance was measured. In the migration and invasion assays, 30,000 cells were placed in each well of the transwell chamber, and migration and invasion were performed for 24 hours, using the same method as before.

[0095] Figure 13 This figure shows the proliferative capacity of different types of primary fibroblasts co-cultured with endometrial epithelial cells (ISK). The vertical axis represents cell viability, and the horizontal axis represents co-culture time. ISK represents untreated ISK cells, CO-NF represents ISK co-cultured with NF cells, and CO-EMF represents ISK co-cultured with EMF cells. It is evident that co-culturing ISK with NF and EMF significantly enhances its proliferative capacity; compared to NF, co-culturing ISK with EMF results in stronger proliferative capacity.

[0096] Figure 14 This study illustrates the migration and invasion abilities of different types of primary fibroblasts co-incubated with endometrial epithelial cells. The top image, from left to right, shows crystal violet staining of migrating cells after co-incubation of NF and EMF cells with endometrial epithelial cells. The bottom image, from left to right, shows crystal violet staining of invasive cells after co-incubation of NF and EMF cells with endometrial epithelial cells. It is evident that, compared to NF, EMF co-cultured with ISK cells exhibits stronger migration and invasion abilities.

[0097] 3. Effects of SPON2 protein co-incubation on the biological behavior of Ishikawa endometrial epithelial cells.

[0098] Ishikawa (ISK) epithelial cells were treated with 200 ng / mL or 400 ng / mL SPON2 recombinant protein (purchased from MCE, catalog number: HY.P70142). The method was as follows: SPON2 recombinant protein was diluted to a concentration of 400 ng / mL or 200 ng / mL with Ishikawa cell culture medium DMEM F12. Then, 2000 Ishikawa cells were added to each well of a 96-well plate. Incubation was performed at a set time using 10% CCK8 reagent prepared with empty culture medium. The absorbance at OD420 was measured using a microplate reader. The effect of SPON2 protein co-incubation on the proliferation ability of Ishikawa endometrial epithelial cells was determined. The results are shown in […]. Figure 15 The x-axis represents culture time, and the y-axis represents cell viability. CTR represents ISK cells not treated with SPON2, and SPON2 represents ISK cells treated with 400 ng / mL SPON2. It is evident that SPON2 recombinant protein treatment enhanced the proliferative capacity of Ishikawa endometrial epithelial cells, and SONP2 protein promoted ISK cell proliferation.

[0099] In addition, the migration and invasion abilities of ISK cells in endometrial epithelial cells treated with 400 ng / mL SPON2 recombinant protein for 24 h were measured, and the results are shown in [Figure number missing]. Figure 16 The top image, from left to right, shows crystal violet staining and a statistical chart of the number of migrating cells in ISK cells co-incubated with SPON2 protein (ISKCTR) and ISK cells co-incubated with SPON2 protein (ISK+SPON2). The bottom image, from left to right, shows crystal violet staining and a statistical chart of the number of invasive cells in ISK CTR cells and ISK cells co-incubated with SPON2 protein. It is evident that SPON2 recombinant protein treatment promotes ISK migration and invasion.

[0100] Further investigation was conducted on the EMT behavior of ISK endometrial epithelial cells treated with 200 ng / mL or 400 ng / mL SPON2 recombinant protein for 48 h. EMT is increasingly recognized as a pathogenesis of endometrial emphysema in the context of chronic inflammation, manifesting as an invasive stromal phenotype (such as the loss of E-cadherin and the acquisition of N-cadherin (also known as cadherin 2), promoting the expression of growth factor signaling and matrix metalloproteinases required for cell proliferation). The expression levels of E-cadherin and N-cadherin in ISK cells were measured using Western blotting, with β-actin protein as an internal control. The relevant results are shown in [link to relevant data]. Figure 17In the figure, "+" indicates the SPON2 recombinant protein treatment group, and "-" indicates the control group. As can be seen from the figure, ISK cells treated with SPON2 recombinant protein underwent significant EMT transformation, with decreased expression of the epithelial cell marker E-cadherin and increased expression of the mesenchymal cell marker N-cadherin. Moreover, the expression level further increased with increasing treatment concentration and treatment time.

[0101] 4. Effects of SPON2 gene overexpression and knockdown on the biological behavior of NF in primary endometrial fibroblasts.

[0102] This study investigated the effects of SPON2 gene overexpression or inhibition on the proliferation, migration, and invasion of endometrial fibroblasts by overexpressing or inhibiting SPON2 gene expression in NF cells. The SPON2 gene overexpression vector was constructed by Shanghai Jikai Biotechnology Co., Ltd. using the lentiviral vector GV492. The SPON2 gene (NM_012445.4) was inserted between the BamHI and AgeI restriction sites of the vector. The SPON2 gene knockdown plasmid was constructed by Gemma Biotechnology Co., Ltd. using the lentiviral vector LV3 (H1 / GFP&Puro). RNA interference technology was employed, and the sequence of the interfering RNA (siRNA) is shown below:

[0103] SPON2-Homo-450(sh#1): GCCAAATACAGCATCACCTTC (see SEQ ID NO.6);

[0104] SPON2-Homo-555(sh#2): GCGCATAGCTCCGACTACAGC (see SEQ ID NO.7);

[0105] SPON2-Homo-640(sh#3):GGGCGCTGATGAAGGAGATCG (see SEQ ID NO.8);

[0106] SPON2-Homo-763(shNC): GGCACTCGCTGGTCTCGTTTG (see SEQ ID NO. 9).

[0107] SPON2 knockdown (NF-sh) and SPON2 overexpression fibroblast (NF-OE) models were established using lentiviral infection. sh NC cells served as the knockdown control group, without interfering with SPON2 gene expression. Western blotting was used to detect SPON2 protein levels in NF cells infected with both SPON2-overexpressing and SPON2-knockdown lentiviral vectors. qRT-PCR was used to detect SPON2 gene expression levels, with Tubin as an internal control.

[0108] See results Figure 18 The left panel shows the SPON2 protein level, and the right panel shows the SPON2 mRNA level. The horizontal axis represents cell type, and the vertical axis represents the relative expression level of SPON2 gene mRNA. NF-Sh#1-3 represent three SPON2 gene knockdown fibroblast strains (obtained by transfecting sh#1-3 lentiviral vectors), and NF-shNC represents control fibroblast cells (obtained by transfecting shNC lentiviral vector). The results show that transfecting SPON2 knockdown lentiviral vectors significantly reduced the expression of SPON2 gene and protein using RNA interference technology.

[0109] Cell viability of NF cells overexpressing and knocking down the SPON2 gene was measured, and the results are shown in the figure. Figure 19 The x-axis represents culture time, and the y-axis represents cell viability. NF sh#1-3 represents three NF cell lines with SPON2 gene knockdown, and NF shNC represents control fibroblast cells. The results showed that NF proliferation was inhibited after SPON2 expression was suppressed. Furthermore, the cell invasion and migration abilities of NF cells with SPON2 gene overexpression and knockdown were measured. The results showed that SPON2 overexpression significantly enhanced NF invasion and migration, while inhibiting SPON2 gene expression significantly reduced lesion migration, invasion, and fibrosis. Related results can be found in [link to relevant results]. Figure 20 The top image, from left to right, shows crystal violet staining of migrating cells in control NF cells (NF-shNC) and NF cells with SPON2 gene knockdown (NF-shSPON2) transformed with sh NC lentiviral vector. The bottom image, from left to right, shows crystal violet staining of invading cells in control NF cells (NF-shNC) and NF cells with SPON2 gene knockdown (NF-shSPON2).

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting SPON2 gene or its protein in the preparation of a diagnostic reagent or kit for endometriosis, characterized in that, The reagent for detecting the SPON2 gene or the protein thereof includes at least one of a reagent for detecting the mRNA level of the SPON2 gene or a reagent for detecting the protein level of the SPON2 gene; The reagent for detecting the mRNA level of the SPON2 gene includes a primer pair for specifically amplifying the SPON2 gene, and the nucleotide sequences of the upstream and downstream primers of the primer pair are respectively shown as SEQ ID NO. 4-5. The reagent for detecting the protein level of the SPON2 gene includes an antibody specifically targeting the SPON2 protein.

2. Use of an agent that inhibits the expression level of the SPON2 gene in the manufacture of a medicament for the treatment of endometriosis, characterized in that, The reagent for inhibiting the expression level of the SPON2 gene is selected from an interfering RNA targeting the SPON2 gene, and the nucleotide sequence of the interfering RNA targeting the SPON2 gene is selected from at least one of SEQ ID NO. 6-8.

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