Application of SPON2 gene or protein thereof as target spot in diagnosis or treatment of endometriosis

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an SPON2 gene or a protein thereof as a target spot in diagnosis or treatment of endometriosis. Through multi-omics and single cell level data support and clinical sample inspection, the correlation between SPON2 and endometriosis (EM) is analyzed for the first time, and it is revealed that proliferation, invasion and migration capacities of in-situ endometrial and ectopic endometrial fibroblasts of endometriosis patients can be enhanced through high expression of SPON2. By inhibiting the expression of the SPON2 gene or inhibiting the function of the SPON2 protein, the proliferation and invasion ability of endometrial tissues and the migration, invasion and fibrosis of lesions are reduced, and the condition of endometriosis is improved, which prompts that the SPON2 gene or the protein thereof can be used as a diagnosis or treatment target. And a new thought and direction are provided for clinical diagnosis and targeted therapy of endometriosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to use of SPON2 gene or its protein as a target in diagnosing or treating endometriosis. Background Art

[0002] Endometriosis (EM) is a gynecological disease in which 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, which may also include abnormal menstrual bleeding, dyspareunia, and infertility. Symptoms vary depending on the location of the lesion. For example, intestinal endometriosis can cause abdominal pain and defecation problems, while respiratory endometriosis may cause hemoptysis and pneumothorax, seriously affecting the patient's quality of life. The incidence rate among women of childbearing age is approximately 10%-15%, and is showing a clear upward trend.

[0003] EM has long lacked effective therapeutic targets, with treatment primarily relying on medication and surgery. However, surgery is difficult to cure and prone to recurrence. Therefore, medication remains crucial for the treatment of endometriosis. Commonly used and effective medications for endometriosis include danazol, gestrinone, gonadotropin-releasing hormone analogs or agonists, progestogens, and oral contraceptives. These drugs inhibit estrogen-dependent endometrial growth, but they are associated with high recurrence rates, significant side effects (such as osteoporosis caused by hormone therapy), and a lack of targeted therapy. New diagnostic and treatment strategies based on pathological mechanisms are urgently needed.

[0004] The pathogenesis of endometriosis is complex, involving biological processes such as inflammation, angiogenesis, extracellular matrix adhesion, and invasion. Genetic factors play a significant role in its pathogenesis. Certain gene mutations and dysregulations, such as overexpression of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) associated with invasiveness, overexpression of vascular endothelial growth factor (VEGF) associated with angiogenesis, abnormal or overexpression of c-kit and DAD-1 associated with anti-apoptosis, abnormal expression of aromatase and underexpression of 17β-hydroxysteroid dehydrogenase (17βHSD) associated with local estrogen excess, abnormal progesterone receptor α / β ratio associated with progesterone responsiveness, and overexpression of cyclooxygenase-2 (COX-2) associated with inflammation, have been associated with an increased risk of endometriosis. However, these studies are still in their early stages and primarily focus on traditional areas such as hormonal regulation and immune inflammation. Clear diagnostic methods and specific treatments are still lacking. 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 therapeutic biomarkers have become the key to promoting precision medicine and reducing the recurrence of EM disease. It is of great significance for the clinical diagnosis, disease monitoring, prevention and treatment of EM and improving the quality of life of patients. Summary of the Invention

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

[0006] In a first aspect, the present invention provides use of a reagent for detecting the SPON2 gene or its protein in the preparation of a diagnostic reagent or kit for endometriosis.

[0007] This study, supported by multi-omics and single-cell data and clinical sample testing, has for the first time analyzed 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 be used as a diagnostic marker for EM, filling a long-standing technical gap in the field of specific diagnostic molecular targets. This provides new insights and directions for the clinical diagnosis and disease monitoring of endometriosis, and has important scientific significance 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. In combination 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 be used as independent test factors. Elevated mRNA and / or protein expression levels indicate an individual's risk of developing endometriosis, while decreased SPON2 gene methylation levels indicate an individual's risk of developing endometriosis. Using SPON2 as a diagnostic marker for endometriosis, or in the diagnosis or auxiliary diagnosis of endometriosis, can significantly improve the sensitivity and specificity of disease diagnosis.

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

[0010] Optionally, the reagent for detecting the level of SPON2 gene mRNA includes a primer pair that specifically amplifies the SPON2 gene, and the detection method includes but is not limited to: fluorescent quantitative PCR (qRT-PCR). The nucleotide sequences of the upstream and downstream primers of the primer pair are as follows:

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

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

[0013] Optionally, the reagent for detecting the level of SPON2 protein includes an antibody specifically targeting the SPON2 protein, which can be obtained commercially. Detection methods include, but are not limited to, immunoblotting, immunohistochemistry, and immunofluorescence.

[0014] A second aspect of the present invention provides use of an agent for inhibiting the expression level of the SPON2 gene in the preparation of a drug for treating endometriosis.

[0015] The present invention reveals for the first time that the proliferation, invasion and migration abilities of eutopic endometrial and ectopic endometrial fibroblasts in endometriosis patients can be enhanced by overexpressing SPON2, and that fibroblasts overexpressing SPON2 can promote the proliferation, invasion and migration abilities of endometrial epithelial cells and the 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, the proliferation and invasion abilities of endometrial tissue, as well as the migration, invasion and fibrosis of lesions can be reduced, and the condition of endometriosis can be improved. This suggests that SPON2 can be used as a therapeutic target to achieve 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 EM diseases.

[0016] Optionally, the reagents for inhibiting the expression level of the SPON2 gene include, but are not limited to, SPON2 protein inhibitors, interfering RNA (siRNA) targeting the SPON2 gene, etc. The aforementioned reagents such as siRNA can be designed based on the SPON2 gene sequence, and the SPON2 protein inhibitors can be conventional chemical inhibitors in the art, which will not be described in detail 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). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is the correlation analysis between the single nucleotide polymorphism sites of the SPON2 gene and the methylation and expression levels of the SPON2 gene;

[0023] Figure 2This is the correlation analysis between SPON2 gene methylation, mRNA, protein levels and endometriosis in the embodiment of the present invention;

[0024] Figure 3 This is a graph showing the analysis results of the SPON2 gene expression levels in eutopic endometrium and ectopic lesions of endometriosis patients in the single-cell transcriptome sequencing dataset GSE179640 in an embodiment of the present invention;

[0025] Figure 4 This is a cluster diagram of different cells and a cellular localization map of SPON2 gene expression in the single-cell transcriptome sequencing dataset GSE179640 of the present invention;

[0026] Figure 5 This is a graph showing the analysis results of the SPON2 gene expression levels in different cells from endometriosis patients in the single-cell transcriptome sequencing dataset GSE213216 according to an embodiment of the present invention;

[0027] Figure 6 This is a cluster diagram of different cells and a cellular localization map of SPON2 gene expression in the single-cell transcriptome sequencing dataset GSE213216 of the present invention;

[0028] Figure 7 This is a graph showing the analysis results of the SPON2 gene expression levels in different tissues of endometriosis patients in the tissue RNA sequencing dataset GSE141549 according to an embodiment of the present invention;

[0029] Figure 8 This is a graph showing the immunofluorescence staining results of SPON2 protein in endometrial tissue samples from women with endometriosis and women without endometriosis according to an embodiment of the present invention;

[0030] Figure 9 This is a diagram showing the immunofluorescence staining results of primary endometrial fibroblasts from women with endometriosis and women without endometriosis in an embodiment of the present invention;

[0031] Figure 10 Graph showing the results of determining the relative levels of SPON2 mRNA and protein expression in endometrial fibroblasts of women with endometriosis and women without endometriosis in an embodiment of the present invention;

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

[0033] Figure 12 This is a graph showing the results of assaying the migration and invasion abilities of primary endometrial fibroblasts from women with endometriosis and women without endometriosis in an example of the present invention;

[0034] Figure 13 This is a graph showing the results of measuring the proliferation capacity of endometrial epithelial cells after co-incubation with primary endometrial fibroblasts from women with endometriosis and women without endometriosis in an embodiment of the present invention;

[0035] Figure 14 This is a graph showing the results of assaying the migration and invasion abilities of endometrial epithelial cells after co-incubation with primary endometrial fibroblasts from women with endometriosis and women without endometriosis in an embodiment of the present invention;

[0036] Figure 15 This is a graph showing the results of measuring the proliferation capacity of endometrial epithelial cells after co-incubation with SPON2 protein according to an embodiment of the present invention;

[0037] Figure 16 This is a graph showing the results of assaying the migration and invasion abilities of endometrial epithelial cells after co-incubation with the SPON2 protein according to an embodiment of the present invention;

[0038] Figure 17 This is a graph showing the expression levels of E-cadherin and N-cadherin in endometrial epithelial cells co-incubated with SPON2 protein using immunoblotting in an embodiment of the present invention;

[0039] Figure 18 This is a graph showing the results of measuring the relative levels of SPON2 mRNA and protein expression in endometrial fibroblasts with SPON2 gene knockdown according to an embodiment of the present invention;

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

[0041] Figure 20 Graph showing the results of measuring the migration and invasion abilities of endometrial fibroblasts with overexpression and knockdown of the SPON2 gene according to an embodiment of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the examples. The examples described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are all within the scope of the appended claims.

[0043] In order to better understand the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express dosage, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. The meaning of "including", "comprising", "containing", "having" and similar words is non-restrictive, that is, other steps and other ingredients that do not affect the result can be added. "And / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0044] Endometriosis (EM) lesions are more inflammatory and fibrotic than normal endometrium. Ectopic menstrual debris triggers inflammation, leading to platelet aggregation, immune cell recruitment, and the release of proinflammatory 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 deposition of extracellular matrix (ECM), which in turn triggers fibrosis. Fibrosis is a key factor in the progression of EM and is associated with symptoms such as pain and infertility. In addition, ectopic endometrial cells undergo partial EMT in response to hormonal and inflammatory stimuli, which can enhance cell invasiveness and survival. 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 lesion progression and fibrosis (for example, literature [1] "Zhu Tianhong, Zhang Xinmei. Research progress on the occurrence and development of endometriosis mediated by epithelial-mesenchymal transition [J]. Journal of Zhejiang University: Medical Edition, 2016, 45(4):7." and literature [2] "Liu Chang, Liu Taihang, He Fan, et al. Screening of differentially expressed genes in endometriosis and expression verification 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 the ability to migrate and invade. This phenomenon is widely involved in embryonic development, tissue repair, cancer metastasis, and fibrotic diseases. EMT is characterized by 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 upregulation of mesenchymal cell markers such as α-smooth muscle actin (α-SMA), N-cadherin, vimentin, fibronectin, and fibroblast-specific protein 1 (FSP1). Changes in the expression of these epithelial and mesenchymal 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, epithelial cells undergo significant changes in polarity, morphology, and function, acquiring enhanced migration, motility, and proliferation capabilities, while simultaneously losing their epithelial phenotype and gradually acquiring a mesenchymal phenotype. In endometriosis, endometrial cells induce EMT through multiple pathways, enhancing their migration, invasion, and anti-apoptosis abilities, contributing to the formation of localized endometriosis lesions and promoting the development and progression of endometriosis.

[0046] Spondin-2 (SPON2) is a secreted protein belonging to the spondin family, also known as Mindin. It is primarily secreted into the extracellular matrix and participates in a variety of 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 certain tumors by promoting EMT, cell migration, and fibrosis. However, its role in endometrial cancer remains unclear. Currently, there is no literature systematically elucidating the mechanism of action of SPON2 in the development and progression of endometrial cancer, nor are there any therapeutic strategies targeting SPON2 for endometrial cancer.

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

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

[0049] This study used a multi-omics analysis to retrieve and download endometriosis-related transcriptome datasets from the GTEx and eQTLGen databases. This was combined with plasma proteome datasets from the UKB, Decode, Nature, and NK databases, and DNA methylome datasets from the GTEx database. Using Mendelian randomization with inverse variance weighting (MR-IVW) to eliminate confounding factors, the study validated the causal relationship between SPON2 gene expression and endometriosis (EM) and confirmed that SPON2 is significantly overexpressed in EM patients. Further cross-validation was performed using open-source single-cell sequencing data related to endometriosis expression downloaded from the Gene Expression Omnibus (GEO). The results showed that SPON2 mRNA levels were higher in both eutopic and ectopic endometrial tissues of EM patients than in healthy eutopic endometrium. Single-cell analysis revealed that SPON2 is highly expressed in fibroblasts, suggesting a possible association with fibroblast activation and fibrosis, making it a potential target for EM treatment. In endometriosis clinical samples, immunofluorescence staining (IF) was used to pathologically diagnose SPON2 protein expression levels in the eutopic endometrium and ectopic lesions of endometriosis patients. The clinical pathological diagnosis results were consistent with the results of genetic bioinformatics analysis, confirming that SPON2 protein expression levels in the ectopic endometrium of endometriosis patients were higher than in the healthy endometrium of non-endometriosis patients in the control group, with the highest expression in the eutopic endometrium of endometriosis patients. This study, for the first time, clearly defines the correlation between SPON2 and endometriosis, suggesting that SPON2 can be used as a diagnostic marker for endometriosis, filling a long-standing technical gap in this field, which has lacked specific diagnostic molecular targets. Taking SPON2 as a diagnostic marker for EM, SPON2 mRNA and / or protein levels are core factors that are significantly upregulated during the pathogenesis of EM, and methylation levels are core 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, which can greatly improve the sensitivity and specificity of disease diagnosis, and is conducive to the development of non-invasive detection methods to improve the early diagnosis rate of EM.

[0050] Based on the above studies revealing SPON2 as a pathogenic target, the present invention also explores the feasibility of SPON2 as a therapeutic target. By overexpressing SPON2 in endometrial fibroblasts, it was observed that fibroblasts with high SPON2 expression had stronger proliferation, invasion and migration abilities, which is consistent with the performance of endometriosis pathological cells. By treating endometrial epithelial cells with SPON2 protein, it was observed that the proliferation, invasion and migration abilities of epithelial cells were also enhanced, and there was obvious EMT transformation. The occurrence of these biological behaviors is conducive to the implantation and fibrosis of endometriosis lesions. The present invention reveals for the first time that endometriosis patients' eutopic and ectopic endometrial fibroblasts drive the implantation and fibrosis of endometriosis lesions by high expression of SPON2. By inhibiting the expression of the SPON2 gene or inhibiting the function of the SPON2 protein, the above phenomenon can be reversed, significantly reducing lesion migration, invasion and fibrosis, revealing that SPON2 can be used as a therapeutic target, and providing new ideas and strategies for the clinical prevention and / or treatment of EM diseases.

[0051] For a long time, 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 and, based on systematic multi-omics data and cell subpopulation analysis, proposes the core role of fibroblast activation and its key regulatory factor SPON2 in the EM process. It overcomes the existing technical path dependence and research bias and opens up a new direction for EM research and treatment. Using SPON2 as a therapeutic target, it is possible to intervene or downregulate the expression level of SPON2 gene or protein to inhibit fibroblast activation, inhibit the fibrosis process of EM lesions, inhibit the epithelial-mesenchymal transition (EMT) of endometrial cells, reduce the proliferation and invasion ability 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 hormone axis; it can improve drug efficacy and reduce side effects through local administration (such as intrauterine injection, sustained-release gel) and other methods. Targeted intervention strategies based on SPON2 are expected to break the limitations of current treatment methods and develop new drugs with higher efficiency and lower side effects. Considering that EM is highly 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 in the field of women's health.

[0052] The present invention is further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or conditions recommended by manufacturers.

[0053] 1. Multi-omics combined analysis to screen SPON2, a potential target for endometriosis (EM)

[0054] 1.1 Proteomic Analysis

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

[0056] Acquisition of protein library: UKB (UK Biobank) proteomic data collected biological samples and detailed health information of a large number of participants, including approximately 500,000 UK residents aged 40 to 69. The plasma samples of more than 54,000 participants were analyzed, and nearly 3,000 proteins were measured in each sample using the Olink Explore platform (see reference [3] SUN BB, CHIOU J, TRAYLOR M, et al. Plasma proteomicassociations with genetics and health in the UK Biobank [J]. Nature, 2023, 622(7982): 329-38.). The NC2022 (Nature Cardiovascular Research 2022) proteomic 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 approximately 4,900 plasma proteins were measured in the study. The Nature 2018 proteomic 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 and discovered thousands of genetic loci (pQTLs) that affect protein levels. The participants included multiple European population cohorts, and nearly 3,000 plasma proteins were measured using the SomaScan platform. The deCODE Genetics (deCODE genetics / Amgen) proteomic data used Iceland's unique and relatively homogeneous population resources to establish a large genetic and health database; the participants were mainly Icelandic population cohorts, and in its landmark study (see reference [4]), approximately 5,000 plasma proteins in plasma samples of nearly 36,000 Icelanders were analyzed.

[0057] Screening and establishment of a druggable gene library: ① Screening of drug target genes from CHEMBL+UNIPROT. The screening criteria were: 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 proteins were clinically approved drugs or clinical candidate drugs. A total of 2,613 drug target genes were screened. ② Screen 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 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: a total of 5355 drug target gene Ensembl IDs were obtained.

[0058] Candidate proteins were used as risk factors (exposure variables), and data from 16,588 endometriosis cases and 111,583 non-endometriosis female controls provided by the Finnish Biobank (Finngen r10) were used as outcome variables. By using genetic variants (such as single nucleotide polymorphisms, SNPs) as instrumental variables and inverse variance weighted (IVW) Mendelian randomization (MR) analysis, the causal relationship between risk factors and disease (outcome) was explored. A total of at least 24 druggable plasma proteins that are highly expressed in endometriosis were screened out: 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, there is a highly significant causal relationship between the genetic increase in 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 odds ratio of endometriosis will increase by e 0.2386 (approximately 1.27) times.

[0059] 1.2 Transcriptomic Analysis

[0060] First, the plasma mRNA databases of eQTLGen and GTEx were used to verify whether the 24 candidate proteins screened out were also highly expressed at the plasma mRNA level. eQTLGen (eQTLGen Consortium) identifies genetic loci (eQTLs) that affect gene expression levels in the blood by integrating and analyzing data from multiple large-scale cohort studies worldwide. The project integrated data from 37 different research cohorts with a total sample size of 31,684 people. The sample types were mainly whole blood or peripheral blood mononuclear cells (PBMCs), providing expression genetic regulatory information for 19,960 genes and successfully locating significant cis-eQTLs (cis-eQTLs) for 16,987 of them. GTEx (Genotype-Tissue Expression) explores the functions and regulatory mechanisms of genes in specific tissues and is widely used to identify blood-related biomarkers and disease pathways. Participants are deceased tissue donors, covering a wide age range and different ethnic backgrounds. The latest GTExV8 release 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 revealed that SPON2 was highly expressed in both mRNA databases.

[0061] Secondly, using GTEx plasma DNA methylation data, we determined whether the elevated expression of target genes was influenced by DNA methylation modification. Using mRNA and DNA methylation data as exposure variables and endometriosis and control data from the Finnish Biobank (Finngen r10) as outcome variables, we evaluated the impact of target genes on endometriosis using MR analysis and summary-based Mendelian randomization (SMR) analysis, respectively. Transcriptome SMR analysis of the SPON2 gene revealed multiple single nucleotide variants (SNPs) in the SPON2 gene, including rs73067776, rs28856334, and rs6836335. The top SNP (rs73067776) was significantly associated with endometriosis (EM), particularly with EM and its multiple subtypes (e.g., pelvic peritoneal EM, ovarian EM, and ASRM stage 3 / 4 EM), suggesting that SPON2 may play an important role in the pathogenesis of EM.

[0062] In the methylation status analysis, two SNPs (rs28856334 and rs6836335) with significant p values ​​(p value less than 0.05) were associated with hypomethylation of the cg03250742 site of SPON2 (see Figure 1In the middle left figure, the vertical axis is the SNP site and the horizontal axis is the methylation level of the SPON2 gene cg03250742 site), which will cause the gene's "expression switch" to be turned on; further analysis of the genotypes of these two SNPs was significantly correlated with the expression of the SPON2 gene (p value less than 0.05) (see Figure 1 In the middle right figure, the vertical axis represents the expression level of SPON2 in whole blood, and the horizontal axis represents the genotype of the SNP site. This indicates that SPON2 is hypomethylated in endometriosis, leading to high expression of its gene.

[0063] In summary, SPON2 at the methylation, mRNA, and protein levels was associated with the risk of endometriosis, and was statistically significant (p value less than 0.05). The adjusted hazard ratio (HR) and 95% confidence interval (CI) were used to express the correlation. SPON2 mRNA and protein expression levels were positively correlated with the occurrence of endometriosis, while methylation status was negatively correlated with the occurrence of endometriosis. The relevant results are summarized in Figure 2 The vertical axis in the figure represents SPON2 methylation, mRNA levels in different databases, and protein levels in different databases, while the horizontal axis represents the adjusted HR value and its 95% confidence interval. This analysis, combined with the results of proteomics analysis, suggests that SPON2 is a potential diagnostic and therapeutic target significantly associated with endometriosis.

[0064] 1.3 Single-cell transcriptome analysis

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

[0066] The GSE179640 dataset (published in the literature [8] “Tan, Y., Flynn, WF, Sivajothi, S. et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol 24, 1306–1318 (2022)”) contains single-cell sequencing data of biopsies from 14 individuals, including 11 EM patients’ in situ endometrium and ectopic ovarian lesions, peritoneal lesions and their adjacent areas, and 3 normal endometrium (control) from non-endometriosis patients. Transcriptome sequencing data of eutopic endometrium (EuE), ectopic peritoneal lesions (EcP), adjacent areas (EcPA), and ectopic ovarian lesions (EcO) in GSE179640 were collected and the expression level of SPON2 gene was analyzed. The results showed that the expression of SPON2 gene in eutopic endometrium (EuE), ectopic peritoneal lesions (EcP), and ectopic ovarian lesions (EcO) tissues was higher than that in the control (Ctrl) eutopic endometrium (see Figure 3 , the vertical axis is the expression level of SPON2, and the horizontal axis is the disease type), indicating that SPON2 is highly expressed in the eutopic endometrium and ectopic lesions of EM patients. Further analysis of SPON2 expression at the cellular level revealed that SPON2 is mainly expressed in fibroblasts, regardless of ectopic lesions or eutopic endometrium (see Figure 4 The left picture is a UMAP diagram of SPON2 expression level, red shows the SPON2 expression site, the darker the color, the higher the SPON2 expression level, and the right picture is a clustered UMAP diagram of cell types, different colors represent different cell types), suggesting that SPON2 may promote the formation and development of fibrosis in endometriosis lesions through specific signaling pathways (such as inflammatory response and extracellular matrix remodeling). In this regard, we can study 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] "TanY, FlynnWF, Sivajothi S, Luo D, BozalSB, DavéM, LucianoAA, Robson P, Luciano DE, Courtois ET. Single-cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat CellBiol. 2022Aug;24(8):1306-1318.doi:10.1038 / s41556-022-00961-5.Epub 2022Jul21.Erratumin: Nat Cell Biol. 2022Nov;24(11):1679.") contains three groups of samples, namely the eutopic endometrium of endometriosis patients, the ectopic lesions of endometriosis patients and the endometrium of healthy controls. The same method as above was used to analyze the data set, and the relevant results are shown in Figure 5 (the vertical axis is the SPON2 expression level, the horizontal axis is the disease type) and Figure 6 (UMAP diagram of SPON2 expression levels, the right figure is a clustered UMAP diagram of cell types), which also shows that SPON2 is highly expressed in the eutopic endometrium of endometriosis patients, and is mainly expressed in fibroblasts.

[0068] 1.4. Tissue RNA Sequencing Data Analysis

[0069] To further verify the high expression of SPON2 in endometriosis patients, a GEO dataset (GSE141549) of RNA sequencing of 115 patients and 53 controls was included in the analysis. The GSE141549 dataset is publicly available in the literature

[10] “Gabriel M, Fey V, Heinosalo T, Adhikari P, 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." contains more than 24,000 genes and clinical characteristics such as age, disease stage, hormone use, menstrual cycle stage and different endometriosis lesion types. The dataset can be interactively analyzed using the EndometDB online software (access link https: / / endometdb.utu.fi / ).

[0070] The results of the analysis of GSE141549 were consistent with the single-cell data GSE179640 and GSE213216. Compared with the controls, the expression of SPON2 in the endometrium and peritoneum of patients with endometriosis was upregulated, and the expression of SPON2 in multiple ectopic lesions in the pelvic cavity (peritoneum, deep, and ovary) was also increased (see Figure 7 , the ordinate is the SPON2 expression level, and the abscissa is the tissue location).

[0071] These results suggest that endometrial fibroblasts are an important source of SPON2. High SPON2 expression in endometrial tissue increases the likelihood of implantation and metastasis, leading to the formation and progression of endometriosis. The mechanism may be that fibroblast-derived SPON2 is secreted into the extracellular matrix (ECM), regulating ECM remodeling and promoting epithelial-mesenchymal transition (EMT) in endometrial cells. EMT enhances the proliferation and invasive capacity of endometrial tissue, promoting cell adhesion, migration, and invasion, and ultimately leading to the implantation and progression of endometriosis.

[0072] 1.5 Immunofluorescence analysis of clinical samples

[0073] Embedded tissues from surgical patients at Xiangya Hospital (all signed informed consent forms) were selected, including control endometrial tissue and eutopic endometrial tissue with endometriosis, for multiple immunofluorescence staining of SPON2 protein. Anti-SPON2 protein primary antibody (purchased from abclonal, product number A12077), anti-a-SMA (purchased from abcam, product number Ab5694) and corresponding immunofluorescence secondary antibody (purchased from AKOYA, product number NEL861001KT) are all commercially available.

[0074] See the results Figure 8 SPON2 protein stains red after the combination of primary and secondary antibodies, and DAPI stains blue, indicating nuclear localization. α-Smooth muscle actin (α-SMA), a marker for stromal fibroblasts, stains green after the combination of anti-α-SMA primary and secondary antibodies. (Merge) shows a fusion of red, blue, and green. This indicates that SPON2 is expressed in stromal fibroblasts (marker α-SMA) in endometrial tissue, and expression is higher in endometrium from patients than in non-endometriosis controls.

[0075] 2. Effect of SPON2 gene expression levels in endometrial fibroblasts of endometriosis patients on their biological behavior

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

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

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

[0079] Figure 9 The IF results of characteristic markers of isolated primary fibroblasts are shown. The upper figure shows endometrial fibroblasts (NF cells) from women without endometriosis, and the lower figure shows endometrial fibroblasts (EMF cells) from women with endometriosis. It can be seen that both primary cells express the characteristic marker α-SMA, and neither expresses the epithelial cell marker pan-CK, confirming that the cells are fibroblasts.

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

[0081] Immunoblotting (WB) was used to detect SPON2 protein levels. Total RNA was extracted from primary NF and EMF fibroblasts and reverse-transcribed into cDNA using a reverse transcription kit (purchased from Takara, Catalog No. rr047a). Tubin was used as an internal reference gene. Fluorescence quantitative PCR (qRT-PCR) kit (purchased from Vazyme, Catalog No. Q712-02) was used to detect SPON2 gene expression in NF and EMF cells. The primer sequences for the internal reference 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] Related results can be found in Figure 10 The left graph shows SPON2 mRNA levels, with the horizontal axis representing cell type. NF represents endometrial fibroblasts from women without endometriosis, and EMF represents endometrial fibroblasts from women with endometriosis. The vertical axis represents the relative expression level of SPON2 mRNA, and the right graph shows SPON2 protein levels. It can be seen that SPON2 gene expression levels in EMF endometrial fibroblasts from endometriosis are significantly higher than in NF endometrial fibroblasts from women without endometriosis.

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

[0088] The proliferation ability of NF and EMF cells is detected by the CCK8 method, including the following steps: S1. 2000 cells per well of the cell suspension are inoculated into a 96-well plate at a predetermined density, and the volume of each well is usually 100 μL. The cells are cultured at 37°C and 5% CO2 until the cells adhere to the wall; S2. The old culture medium is aspirated, fresh culture medium is added, and the culture is continued for the corresponding time (such as 24 hours, 48 ​​hours, 72 hours). The detection is performed at different time points according to the experimental design; S3. CCK-8 reagent is added at the predetermined detection time point, the culture medium in each well is aspirated, and a 10% (wt) CCK-8 solution diluted with fresh culture medium is added, 100 μL per well, and mixed thoroughly; S4. The 96-well plate is returned to the incubator and incubated in the dark for 2 hours; S5. The absorbance (OD value) of each well is measured at a wavelength of 450 nm.

[0089] Data analysis and calculation of cell viability / proliferation rate: First calculate the average OD value of each sample, then calculate the cell proliferation rate and draw a growth curve. Figure 11 The horizontal axis represents the culture time, and the vertical axis represents the cell viability. It can be seen that the proliferation ability of the fibroblasts EMF in the endometrium of endometriosis is stronger.

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

[0091] The migration and invasion abilities of NF and EMF cells were detected by Transwell assay, which included the following steps: ① Migration: The cells were digested, resuspended and counted in serum-free medium, 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-containing 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. The cells were then stained with crystal violet for 30 min, rinsed twice with tap water, and gently wiped with a cotton swab. After drying, the cells were photographed and counted. ② Invasion: The same steps were followed as before, except that 15% Matrigel was added to the upper chamber and incubated for 30 min before adding 5,000 cells to each well.

[0092] Related results can be found in Figure 12 The top image, from left to right, shows crystal violet staining of migrating cells and a statistical graph of the number of migrated cells in NF and EMF cells. The bottom image, from left to right, shows crystal violet staining of invasive cells and a statistical graph of the number of migrated cells in NF and EMF cells. This shows that compared to NF, endometrial fibroblasts in situ from endometriosis, EMF, have stronger invasion and migration capabilities.

[0093] 2.5 Changes in biological behavior after co-culture of primary fibroblasts NF and EMF with endometrial epithelial cells Ishikawa

[0094] NF and EMF cells were co-cultured with human endometrial cancer Ishikawa (an epithelial cell line, ISK cells) cells, respectively, comprising the following steps: NF and EMF cells were cultured in serum-free medium for 24 hours, the medium was collected, and the cells in the medium were filtered through a 0.22 μm filter, and then the medium was added to a 96-well plate or chamber at a ratio of 1:1 with complete medium to culture Ishikawa cells, with 2000 cells per well of the 96-well plate. At a set time, 10% CCK8 reagent was prepared in empty medium for incubation, and the OD was detected by a microplate reader. 450 In the migration and invasion assays, 30,000 cells were plated per well in transwell chambers and allowed to migrate and invade for 24 h using the same method as above.

[0095] Figure 13 The figure shows the proliferation capacity of different primary fibroblast types after co-culture with endometrial epithelial cells (ISK). The vertical axis represents cell viability, and the horizontal axis represents the 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 can be seen that the proliferation capacity of ISK cells was significantly enhanced after co-culture with NF and EMF, and the proliferation capacity of ISK cells was even higher after co-culture with EMF than with NF.

[0096] Figure 14 The figure shows the migration and invasion abilities of different primary fibroblast types after co-incubation with endometrial epithelial cells. The top image (left to right) shows crystal violet staining of migrating cells after co-incubation with NF and EMF cells, and the bottom image (left to right) shows crystal violet staining of invasive cells after co-incubation with NF and EMF cells. It can be seen that compared to NF, EMF co-cultured ISK cells exhibited enhanced migration and invasion abilities.

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

[0098] Ishikawa epithelial cells (ISK) were treated with 200 ng / mL or 400 ng / mL of SPON2 human recombinant protein (purchased from MCE, catalog number: HY.P70142) as follows: SPON2 recombinant protein was diluted to a concentration of 400 ng / mL or 200 ng / mL in DMEM F12 medium (Ishikawa cell culture medium). 2000 Ishikawa cells were then added to each well of a 96-well plate. The cells were incubated with 10% CCK8 reagent in empty culture medium for a set time, and the absorbance at OD420 was measured using a microplate reader. The effect of co-incubation with SPON2 protein on the proliferation of Ishikawa endometrial epithelial cells was determined. The results are shown in Table 1. Figure 15 The horizontal axis represents culture time, and the vertical axis represents cell viability. CTR represents ISK cells untreated with SPON2, and SPON2 represents ISK cells treated with 400 ng / mL SPON2. This indicates that treatment with recombinant SPON2 protein enhances the proliferation of Ishikawa endometrial epithelial cells, suggesting that SONP2 promotes ISK proliferation.

[0099] In addition, the migration and invasion abilities of endometrial epithelial cells ISK treated with 400 ng / mL SPON2 recombinant protein for 24 h were determined. Figure 16 The top images, from left to right, show crystal violet staining of migrating cells and statistical graphs of the number of migrating cells in ISK cells without SPON2 treatment (ISKCTR) and ISK cells co-incubated with SPON2 protein (ISK+SPON2). The bottom images, from left to right, show crystal violet staining of invasive cells and statistical graphs of the number of invasive cells in ISK cells co-incubated with SPON2 protein. It can be seen that treatment with recombinant SPON2 protein promotes the migration and invasion of ISK cells.

[0100] We further examined the EMT behavior of endometrial epithelial cells (ISKs) treated with 200 ng / mL or 400 ng / mL SPON2 recombinant protein for 48 hours. EMT is increasingly recognized as the pathogenesis of EM in the context of chronic inflammation, characterized by an invasive mesenchymal phenotype (such as the loss of E-cadherin and the gain of N-cadherin (also known as cadherin 2), which promotes the expression of growth factor signals and matrix metalloproteinases required for cell proliferation. The expression levels of E-cadherin and N-cadherin in ISK cells were determined by immunoblotting, with β-actin protein as the internal reference protein. The relevant results are shown in Figure 2. Figure 17In the figure, "+" indicates the SPON2 recombinant protein-treated group, and "-" indicates the control group. As can be seen from the figure, ISK cells treated with SPON2 recombinant protein underwent a significant EMT transformation, with reduced expression of the epithelial cell marker E-cadherin and enhanced expression of the mesenchymal cell marker N-cadherin. This expression level further increased with increasing treatment concentration and duration.

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

[0102] SPON2 gene expression was overexpressed or inhibited in NF cells to investigate the effects of targeting SPON2 on the proliferation, migration, invasion, and other biological behaviors of endometrial fibroblasts. The SPON2 gene overexpression vector was constructed by Shanghai Jikai Company 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 Genema Company using the lentiviral vector LV3 (H1 / GFP & Puro). RNA interference technology was used, and the interfering RNA (siRNA) sequence 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 overexpression (NF-OE) fibroblast models were established by lentiviral infection. Sh NC served as a knockdown control, which did not interfere with SPON2 gene expression. SPON2 protein levels in NF cells infected with SPON2-overexpressing and knockdown lentiviral vectors were detected by Western blotting. SPON2 gene expression was determined by qRT-PCR, using Tubin as an internal control.

[0108] See the results Figure 18 The left graph shows SPON2 protein levels, and the right graph shows SPON2 mRNA levels. The horizontal axis represents cell type, and the vertical axis represents the relative expression level of SPON2 gene mRNA. NF-Sh#1-3 represents three strains of fibroblasts with knockdown of the SPON2 gene (transfected with sh#1-3 lentiviral vectors, respectively), and NF-shNC represents control fibroblasts (transfected with shNC lentiviral vector). The results showed that by introducing the knockdown SPON2 lentiviral vector, RNA interference technology significantly reduced the expression of the SPON2 gene and protein.

[0109] The cell activity of NF cells with overexpression and knockdown of SPON2 gene was measured, and the results are shown in Figure 19 , where the horizontal axis represents the culture time, the vertical axis represents the cell activity (Cell Viability), NF sh#1-3 represents three strains of NF cells with knockdown of the SPON2 gene, and NF shNC represents the control fibroblast cells. The results showed that the proliferation of NF was inhibited after inhibiting the expression of SPON2. In addition, the cell invasion and migration ability of NF cells with overexpression and knockdown of the SPON2 gene were measured. The results showed that the invasion and metastasis of NF were significantly enhanced after overexpression of SPON2, and inhibiting the expression of the SPON2 gene could significantly reduce the migration, invasion and fibrosis of lesions. Related results can be found in Figure 20 The upper figures, from left to right, are crystal violet staining images of migrating cells in the control group NF cells (NF-shNC) and NF cells with SPON2 gene knockdown (NF-shSPON2) transformed with the sh NC lentiviral vector. The lower figures, from left to right, are crystal violet staining images of invasive cells in the control group 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 in the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the SPON2 gene or its protein in the preparation of a diagnostic reagent or kit for endometriosis.

2. Use of the reagent for detecting SPON2 gene or protein thereof according to claim 1 in the preparation of a diagnostic reagent or kit for endometriosis, characterized in that: The reagent for detecting the SPON2 gene or its protein includes at least one of the reagents for detecting the methylation level of the SPON2 gene, the mRNA level of the SPON2 gene, and the protein level of the SPON2 gene.

3. Use of the reagent for detecting SPON2 gene or protein thereof according to claim 2 in the preparation of a diagnostic reagent or kit for endometriosis, characterized in that: The reagent for detecting the level of SPON2 gene mRNA 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 shown as SEQ ID NO. 4-5 respectively.

4. Use of the reagent for detecting SPON2 gene or protein thereof according to claim 2 in the preparation of a diagnostic reagent or kit for endometriosis, characterized in that: The reagent for detecting the level of SPON2 protein includes an antibody that specifically targets the SPON2 protein.

5. Use of reagents that inhibit the expression level of SPON2 gene in the preparation of drugs for the treatment of endometriosis.

6. Use of the reagent for inhibiting the expression level of SPON2 gene according to claim 5 in the preparation of a drug for treating endometriosis, characterized in that: The agent for inhibiting the expression level of the SPON2 gene is selected from at least one of a SPON2 protein inhibitor and an interfering RNA targeting the SPON2 gene.

7. Use of the reagent for inhibiting the expression level of SPON2 gene according to claim 6 in the preparation of a drug for treating endometriosis, characterized in that: The nucleotide sequence of the interfering RNA targeting the SPON2 gene is selected from at least one of SEQ ID NOs. 6-8.

Images