Application of ADAP2 as a prognostic biomarker and target for epithelial ovarian cancer and application of reagent for detecting ADAP2

Abstract

The application belongs to the field of tumor molecular biology, and discloses application of ADAP2 as a prognosis evaluation biomarker and target point of epithelial ovarian cancer and application of a reagent for detecting ADAP2. The reagent for detecting the content or expression amount of ADAP2 protein in a sample is used for preparing a prognosis evaluation kit of epithelial ovarian cancer, has the advantages of convenience, simplicity, rapidness and accuracy, can evaluate the prognosis of epithelial ovarian cancer in time and rapidly, can provide effective guidance for clinical decision-making, and is helpful to improve the survival and prognosis of patients.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular biology, specifically relating to the application of ADAP2 as a biomarker and target for prognostic assessment of epithelial ovarian cancer, and the application of reagents for detecting ADAP2. Background Technology

[0002] Ovarian cancer is the leading cause of death among malignant tumors of the female reproductive system. Epithelial ovarian carcinoma (EOC) accounts for approximately 90% of all ovarian malignancies. Recent statistics show that nearly 300,000 new cases of ovarian cancer are diagnosed globally each year, with over 180,000 deaths. Due to the deep location of the ovary in the pelvis, the onset of the disease is often insidious, making early detection difficult. About two-thirds of patients have already experienced metastasis at diagnosis, leading to poor outcomes from cytoreductive surgery and chemotherapy. Traditional methods for evaluating the prognosis of EOC patients mainly rely on clinical parameters, which do not accurately reflect the intrinsic molecular biological characteristics of the tumor. This results in inaccurate prognosis assessment for a significant proportion of patients, and current research lacks effective molecular biomarkers to guide treatment strategies. Metabolic disorders of the tumor microenvironment (TME) have recently been shown to play a decisive role in tumor formation and progression. Therefore, the discovery of novel prognostic biomarkers related to the immune microenvironment of epithelial ovarian cancer and the exploration of therapeutic targets are of great significance.

[0003] ADAP2 is a member of the protein family containing the ADP-ribosylation factor-GTPase activator (Arf-GAPs) domain. It binds to β-tubulin, increasing microtubule stability and playing a crucial role in heart development. Studies have also shown that ADAP2 can act as an interferon-stimulated gene, blocking viral invasion by altering Arf6-mediated transport and exerting antiviral effects. Recent research has indicated that ADAP2 is associated with prognosis in cancer patients, potentially promoting tumor invasion and metastasis. However, there are currently no reports on the prognostic assessment and therapeutic potential of ADAP2 in endocrine disorders (EOCs). Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of ADAP2 as a biomarker and target for prognostic assessment of epithelial ovarian cancer, as well as the application of reagents for detecting ADAP2. The application of the kit for assessing the prognosis of epithelial ovarian cancer based on ADAP2 protein content or expression level described in this invention has the advantages of simple operation and high technical reliability, and is suitable for clinical kit development.

[0005] To achieve the above objectives, the first aspect of the present invention provides the application of a reagent for detecting the content or expression level of ADAP2 protein in a sample in the preparation of a kit for assessing the prognosis of epithelial ovarian cancer.

[0006] A second aspect of the present invention provides the application of a reagent for detecting the content or expression level of ADAP2 protein in a sample in a kit for preparing a prognostic treatment regimen for epithelial ovarian cancer.

[0007] A third aspect of this invention provides the application of ADAP2 protein as a target in the preparation of a reagent for assessing the prognosis of epithelial ovarian cancer.

[0008] The fourth aspect of this invention provides the use of an ADAP2 protein inhibitor in the preparation of a medicament for treating the prognosis of epithelial ovarian cancer.

[0009] Through the above technical solutions, this invention has found that the expression level of GTPase-related protein ADAP2 has a significant impact on the prognosis of patients with epithelial ovarian cancer, and that high expression of ADAP2 is closely related to tumor microenvironment dysregulation, suggesting that patients with high expression of ADAP2 may have better efficacy in immunotherapy. Therefore, ADAP2 has become a potential target for the treatment of epithelial ovarian cancer.

[0010] On the other hand, this invention has found that the expression level of ADAP2 in EOC is closely related to immune infiltration, and ADAP2 induces ovarian cancer cells to secrete the cytokine IL6. This reveals that ADAP2 can accurately assess the patient's prognosis by combining with CD163 and / or IL6 indicators. This invention provides an application of ADAP2 combined with CD163 and / or IL6 in the prognostic assessment and treatment of epithelial ovarian cancer. It can formulate individualized treatment plans based on the patient's gene expression characteristics, provide effective guidance for clinical decision-making, and help improve the patient's survival and prognosis. Attached Figure Description

[0011] Figure 1 Explaining the expression of ADAP2 in epithelial ovarian cancer and its value in clinical prognosis: Figure 1 A is a ring-shaped heatmap of EPIC algorithm scores for different immune cells in GSE9891 (n=271) ovarian cancer samples; Figure 1 B is a volcano plot for screening differentially expressed genes related to macrophage infiltration; Figure 1 C is the OS curve of Kaplan-Meier survival analysis of ADAP2 expression level in GSE9891;

[0012] Figure 1 D is an immunohistochemical staining image showing the expression of ADAP2 in epithelial ovarian cancer tissues of different pathological types in validation cohort 1 (n=132); Figure 1E is the OS curve and progression-free survival (PFS) curve of Kaplan-Meier survival analysis of EOC patients with different ADAP2 levels in validation cohort 1 (n=132); Figure 1 F is an immunohistochemical staining image showing the expression of ADAP2 in epithelial ovarian cancer tissues of different pathological types in cohort 2 (n=138); Figure 1 G is the Kaplan-Meier OS curve for different ADAP2 levels in validation queue 2 (n=138); Figure 1 H is a box plot of ADAP2 versus clinical stage and T stage in validation cohort 2 (n=138).

[0013] Figure 2 Explanation of CD163 + The value of cell infiltration level in the clinical prognosis of epithelial ovarian cancer: Figure 2 A is an immunohistochemical staining image showing the expression of CD163 in epithelial ovarian cancer tissues of different pathological types in validation cohort 1 (n=132) and validation cohort 2 (n=138); Figure 2 B is a bubble chart showing the correlation analysis results between 11 candidate molecules, including ADAP2, and M2 macrophage markers. Figure 2 C represents different CD163 values ​​in EOC patients in validation cohort 1 (n=132). + Kaplan-Meier OS and PFS curves for cell infiltration levels; Figure 2 D is the distinct CD163 in verification queue 2 (n=138). + OS curves of Kaplan-Meier survival analysis of cell infiltration levels, and CD163 + Box plot of cell infiltration level in relation to clinical stage and T stage.

[0014] Figure 3 This indicates that the expression level of ADAP2 in EOC is closely related to immune infiltration: Figure 3 A is a gene heatmap of THP-1-Mφ cells after ADAP2 knockout in a co-culture system; Figure 3 B is a graph showing the differential gene GO enrichment analysis in THP-1-Mφ cells after ADAP2 knockout in the co-culture system; Figure 3 C is a bar graph showing the expression levels of M1 and M2 markers in THP-1-Mφ cells after ADAP2 knockout in a co-culture system.

[0015] Figure 4 Explaining the value of IL-6 in the clinical prognosis of epithelial ovarian cancer: Figure 4 A is a bar chart showing the secretion levels of downstream cytokines regulated by ADAP2, as screened using Luminex liquid-phase microarrays. Figure 4B is a bar graph showing the IL6 secretion level in the supernatant of different SKOV3 (si-NC and si-ADAP2) cells detected by ELISA;

[0016] Figure 4 C is a bar graph showing the IL6 mRNA levels in different SKOV3 and HEY cells (si-NC and si-ADAP2) detected by qRT-PCR; Figure 4 D is the immunohistochemical staining image of IL6 expression in different pathological tissues of EOC patients in validation cohort 1 (n=132), and the Kaplan-Meier OS curve and PFS curve at different IL6 expression levels; Figure 4 E is an immunohistochemical staining image of IL6 expression in different pathological tissues and a Kaplan-Meier OS curve for different IL6 expression levels in cohort 2 (n=138) of EOC patients.

[0017] Figure 5 To demonstrate the value of combined ADAP2 / CD163 / IL6 expression levels in predicting survival outcomes in cohort 1 (n=132): Figure 5 A is an immunohistochemical staining image showing the expression of ADAP2 / CD163 / IL6 in epithelial ovarian cancer tissues in validation cohort 1 (n=132); Figure 5 B represents the levels of ADAP2 and IL6 proteins and CD163 in epithelial ovarian cancer tissue. + Spearman correlation analysis between cell infiltration levels; Figure 5 C represents CD163 at different ADAP2 protein levels in validation cohort 1 (n=132) EOC. + Bar packing plot for differential cell infiltration and CD163 at different IL6 protein levels + Stacked column plot for analysis of differences in cell infiltration degree; Figure 5 D is the OS and PFS curves of Kaplan-Meier survival analysis for different ADAP2 / CD163 / IL6 expression levels in validation cohort 1 (n=132).

[0018] Figure 6 To demonstrate the value of combined ADAP2 / CD163 / IL6 expression levels in predicting survival outcomes in cohort 2 (n=138) of patients with endocrine disorders (EOC): Figure 6 A is an immunohistochemical staining image showing the expression of ADAP2 / CD163 / IL6 in epithelial ovarian cancer tissues in validation cohort 2 (n=138); Figure 6 B represents the levels of ADAP2 and IL6 proteins and CD163 in epithelial ovarian cancer tissue. + Spearman correlation analysis between cell infiltration levels; Figure 6 C represents CD163 at different ADAP2 protein levels in validation cohort 2 (n=138) EOC. + Bar packing plot for differential cell infiltration and CD163 at different IL6 protein levels + Stacked column plot for analysis of differences in cell infiltration degree; Figure 6 D is the OS curve of Kaplan-Meier survival analysis for different ADAP2 / CD163 / IL6 expression levels in validation cohort 2 (n=138). Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] In this invention, the term "OS curve" refers to the overall survival curve, and the term "PFS curve" refers to the progression-free survival curve.

[0021] In this invention, data analysis was performed using GraphPad Prism 8.0 or R software (version 4.3.1). Significance analysis was conducted using Student's t-test or Wilcoxon test, and correlation analysis was performed using Spearman's method. The graphs indicate statistically significant differences: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference. A P < 0.05 was considered statistically significant. Kaplan-Meier survival analysis was validated using the Log-rank test.

[0022] The first aspect of this invention provides the application of a reagent for detecting the content or expression level of ADAP2 protein in a sample in the preparation of a kit for assessing the prognosis of epithelial ovarian cancer.

[0023] In this invention, the amino acid sequence of the ADAP2 protein is shown in SEQ ID NO: 1.

[0024] SEQ ID NO: 1:

[0025] MGDRERNKKRLLELLRAPDTGNAHCADCGAADPDWASYKLGIFICLNCCGVHRNFPDISRVKSVRLDFWDDSIVEFMIHNGNLRVKAKFEARVPAFYYIPQANDCLVLKEQWIRAKYERREFMADGETISLPGNREGFLWKRGRDNSQFLRRKFVLLAREGLLKYFTKEQGKSPKAVISIKDLNATFQTEKIGHPHGLQITYRRDGHTRNLFVYHESGKEIVDWFNALRAARLQYLKMAFPELPESELVPFLTRNYLKQGFMEKTGPKQKEPFKKRWFALDCHERRLLYYKNPLDAFEQGQVFLGNKEQGYEAYEDLPKGIRGNRWKAGLTIVTPERRFVLTCPSEKEQQEWLESLRGVLSSPLTPLNRLTASTESGRSSR。

[0026] In the present invention, the nucleotide sequence of the coding gene of the ADAP2 protein is shown as SEQ ID NO: 2, SEQ ID NO: 2:

[0027]

[0028] This invention discovers that the GTPase-associated protein ADAP2 is highly expressed in ovarian cancer and has a significant impact on the prognosis of epithelial ovarian cancer. As a biomarker for assessing prognosis, it has the advantages of simple operation and high technical reliability.

[0029] In this invention, the method for detecting the content or expression level of ADAP2 protein in the sample is at least one of immunohistochemistry, immunoblotting, immunomagnetic bead detection, chemiluminescence, or flow cytometry.

[0030] This invention does not impose any particular restrictions on the specific operation of immunohistochemistry, immunoblotting, immunomagnetic bead detection, chemiluminescence, or flow cytometry for detecting the content or expression of ADAP2 in samples. Those skilled in the art can select the appropriate technique based on known technologies to achieve excellent detection results.

[0031] Those skilled in the art can prepare corresponding kits for assessing epithelial ovarian cancer according to known techniques in the field of kit preparation based on the ADAP2 biomarker disclosed in this invention. These kits contain reagents capable of detecting the content or expression level of ADAP2 protein in a sample, such as test strips for detecting ADAP2 protein content.

[0032] To quickly detect the content or expression level of ADAP2 in a sample, according to a preferred embodiment of the present invention, the method for detecting the content or expression level of ADAP2 in the sample is immunohistochemistry.

[0033] According to a preferred embodiment of the present invention, the sample is at least one of cells and tissues.

[0034] In this invention, the kit also includes reagents for detecting the content or expression level of IL6 protein in a sample.

[0035] In this invention, the method for detecting the IL6 protein content or expression level in the sample is at least one of ELISA, immunohistochemistry, Western blotting, immunomagnetic bead detection, chemiluminescence, or flow cytometry.

[0036] In this invention, the kit also includes reagents for detecting the content or expression level of CD163 protein in a sample.

[0037] In this invention, the method for detecting the CD163 protein content or expression level in the sample is at least one of immunohistochemistry or flow cytometry.

[0038] This invention found that the expression level of ADAP2 in EOC is closely related to immune infiltration, and CD163 +High cellular infiltration suggests a poor prognosis for patients; furthermore, ADAP2 induces ovarian cancer cells to secrete the cytokine IL6, and EOC patients with higher IL6 expression levels had significantly lower overall survival (OS) and progression-free survival (PFS) than those with low IL6 expression. Predicting the survival prognosis of EOC patients using the combined expression levels of ADAP2, CD163, and IL6 is not only beneficial for prognostic assessment but also allows for targeted adjustments to treatment strategies for high-risk patients, possessing significant clinical value for precision treatment of EOC.

[0039] A second aspect of the present invention provides the application of a reagent for detecting the content or expression level of ADAP2 protein in a sample in a kit for preparing a prognostic treatment regimen for epithelial ovarian cancer.

[0040] A third aspect of this invention provides the application of ADAP2 protein as a target in the preparation of a reagent for assessing the prognosis of epithelial ovarian cancer.

[0041] The fourth aspect of this invention provides the use of an ADAP2 protein inhibitor in the preparation of a medicament for treating the prognosis of epithelial ovarian cancer, wherein the medicament targets the ADAP2 protein.

[0042] In this invention, the prognosis includes detection, prognostic assessment, relapse monitoring, and evaluation of the efficacy of immunotherapy.

[0043] According to a preferred embodiment of the present invention, the prognostic assessment includes: assessing the prognosis of epithelial ovarian cancer based on the content or expression level of ADAP2 protein, wherein a high content or high expression level of ADAP2 protein indicates a poor prognosis.

[0044] In this invention, the expression level of ADAP2 has a significant impact on the prognosis of patients with epithelial ovarian cancer. ADAP2 can be used as a biomarker for the treatment or prediction of the prognostic development of epithelial ovarian cancer. The application of the ADAP2 biomarker and ADAP2 target preparation and evaluation kit for the prognosis of epithelial ovarian cancer has the advantages of simple operation and high technical reliability, and is suitable for clinical kit development.

[0045] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention.

[0046] In the following examples, 271 EOC patients included in dataset GSE9891 from the GEO platform (https: / / www.ncbi.nlm.nih.gov / geo / ) were used as the training cohort. RNA expression data and related clinical data were obtained from this gene expression microarray. Human epithelial ovarian cancer tissue microarrays were purchased from a specialized company and used as validation cohort 1 (n=132) and validation cohort 2 (n=138).

[0047] In the following examples, human ovarian cancer cell lines SKOV3 and HEY, and human monocytes (acute monocytic leukemia cells) THP-1 were obtained from ATCC (American Type Culture Collection). THP-1 cells were induced into macrophages (Mφ) by culturing in medium with 100 ng / ml PMA for 48 hours. SKOV3 cells were cultured in McCoy's 5A medium, HEY cells in DMEM medium, and THP-1 cells in RPMI 1640 medium with 0.05 mM 2-mercaptoethanol added. All media were supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin and cultured in a 37°C, 5% CO2 incubator.

[0048] In the following examples, siRNAs were transfected into cells using Lipofectamine 3000 (Invitrogen, USA), and the siRNAs were purchased from Ribobio Biotechnology Co., Ltd.

[0049] In the following examples, the macrophage M1 and M2 type-related biomarkers are derived from existing research findings. Macrophage M1 type-related biomarkers include: NOS2, TNF, and IL-1β; M2 type-related biomarkers include: CD163, CD204, ARG1, and IL-10.

[0050] In the following examples, the expression levels of ADAP2 and IL6 were detected by immunohistochemical staining. The expression levels of ADAP2 and IL6 were assessed using the H-score system: H-score = (∑IS × AP), where IS represents staining intensity and AP represents the percentage of positively stained cells. IS depends on cell staining: no staining = 0 points, weak staining = 1 point, moderate staining = 2 points, and strong staining = 3 points. AP depends on the percentage of stained cells: 0% = 0 points, 1%-25% = 1 point, 26%-50% = 2 points, 51%-75% = 3 points, and 76%-100% = 4 points. An H-score of 6 was set as the cutoff value; H-score ≥ 6 points was defined as the high expression group, and H-score < 6 points was defined as the low expression group.

[0051] In the following examples, the expression level of CD163 was detected by immunohistochemical staining: the number of all corresponding CD163-positive cells was recorded in the entire microscopic field of view to determine the expression level of CD163. + The degree of cell infiltration is scored as follows: no obvious infiltration is 0 points, a small amount of infiltration is 1 point, low-level infiltration is 2 points, moderate infiltration is 3 points, and high-level infiltration is 4 points. A cutoff value of 2 is used, and ≤2 is defined as CD163. + The low cell infiltration group, ≥3 was defined as CD163 + High cell infiltration group.

[0052] In the following examples, the immunohistochemical staining method includes:

[0053] 1) Dewaxing paraffin sections to hydration: Place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with distilled water.

[0054] 2) Antigen retrieval: Tissue slides were placed in a retrieval box filled with citric acid (pH 6.0) antigen retrieval solution and microwaved for antigen retrieval. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be dried out. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0055] 3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0056] 4) BSA or serum blocking: After slightly drying the slide, draw a circle around the tissue with a histochemical pen (to prevent antibody from flowing away), and add 3% BSA evenly to the circle to cover the tissue. Block at room temperature for 30 minutes.

[0057] 5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, place the slide flat in a humidified chamber (add a small amount of water to the humidified chamber to prevent antibody evaporation) and incubate overnight at 4°C.

[0058] 6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the sections, add the secondary antibody (HRP-labeled) of the same species as the primary antibody from the histochemistry kit to the inside of the slide and incubate at room temperature for 50 minutes.

[0059] 7) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop the staining process.

[0060] 8) Counterstaining cell nuclei: Harris hematoxylin counterstain for about 3 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, return to blue with ammonia water, and rinse with running water.

[0061] 9) Dehydration and mounting: Place the sections in 75% alcohol for 6 min, 85% alcohol for 6 min, anhydrous ethanol I for 6 min, anhydrous ethanol II for 6 min, and xylene I for 5 min in sequence to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0062] 10) Microscopic examination, image acquisition and analysis.

[0063] In the following examples, the real-time quantitative PCR experimental procedure was as follows: After discarding the culture medium, the cell samples were slowly washed three times with an appropriate amount of PBS. 1 ml of TRIzol was added to lyse the cells, and the mixture was repeatedly pipetted with a 1 ml pipette to ensure full contact with the cells. After 10 minutes, the liquid was transferred entirely to a 1.5 ml EP tube using a 1 ml pipette. Sample preparation and detection were then performed according to standard operating procedures. Total RNA was extracted from the cells using the EZ-press RNA Purification Kit (EZBioscience, USA) according to the manufacturer's instructions. Reverse transcription was performed using PrimeScript. TM RT Master Mix kit (TaKaRa, Japan) was used, and qRT-PCR was performed using the corresponding PCR kit (TaKaRa, Japan). Data were analyzed using 2... -ΔΔCt The method is used for analysis.

[0064] In the following embodiments, in validation cohort 1 (n=132), patients were divided into a high expression group (n=79) and a low expression group (n=53) based on the median value of ADAP2 expression; in validation cohort 2 (n=138), patients were divided into a high expression group (n=86) and a low expression group (n=52) based on the median value of ADAP2 expression.

[0065] In the following examples, in verification queue 1 (n=132), according to CD163 + The level of cell infiltration classifies patients into CD163 groups. + High cell infiltration group (n=81) and CD163 + In the low-infiltration cell group (n=51); in validation cohort 2 (n=138), based on CD163... +The level of cell infiltration classifies patients into CD163 groups. + High cell infiltration group (n=96) and CD163 + Low cell infiltration group (n=42).

[0066] In the following embodiments, in validation cohort 1 (n=132), patients were divided into a high expression group (n=83) and a low expression group (n=49) based on the median value of IL6 expression; in validation cohort 2 (n=138), patients were divided into a high expression group (n=75) and a low expression group (n=63) based on the median value of IL6 expression.

[0067] In the following examples, in validation cohort 2 (n=138), patients were classified into stage I (n=54), stage II (n=16), stage III (n=58), and stage IV (n=10) according to clinical staging criteria (FIGO 2014 staging based on surgery and pathology); and into pT1 (n=56), pT2 (n=16), and pT3 (n=66) according to T staging criteria (AJCC 2017 8th edition TNM staging).

[0068] In the following embodiments, in validation queue 1 (n=132), based on the expression levels of ADAP2 and IL6 and CD163... + Patients were divided into three groups based on their level of cell infiltration: ADAP2 high +CD163 high +IL6 high (n=47), ADAP2 low +CD163 low +IL6 low (n=25), others (n=60, not labeled in the figure); in validation cohort 2 (n=138), based on ADAP2 and IL6 expression levels and CD163 + Patients were divided into three groups based on their level of cell infiltration: ADAP2 high +CD163 high +IL6 high (n=50), ADAP2 low +CD163 low +IL6 low (n=17), others (n=71, not labeled in the figure).

[0069] Example 1

[0070] The overall infiltration of eight immune cell types in epithelial ovarian cancer included in the GSE9891 dataset was determined using the EPIC algorithm (EPIC is an algorithm that quantifies the level of immune cell infiltration in tumor samples based on gene expression data, reflecting the diversity of immune cells and stromal cells). Figure 1 A, Figure 1 Only 6 types are shown in A). Differential gene expression analysis was performed on the macrophage high-infiltration and low-infiltration groups using the EPIC algorithm (|logFC|>1, P<0.05), and a differential gene set of 355 genes was obtained. Figure 1 B); Secondly, differentially expressed genes were ranked by p-value. We used the GSE9891 dataset (n=271) as the training cohort and performed univariate Cox regression analysis on the top 50 genes with significant p-values ​​using the survival and survminer packages in R. This revealed 11 genes influencing the prognosis of EOC patients: EVI2A, MSR1, DOCK2, GIMAP4, VSIG4, LY86, FPR3, CD37, ADAP2, FCGR1A, GPR65 ( Figure 2 B) ADAP2 was selected as a molecular marker. Kaplan-Meier survival analysis was performed on the expression level of ADAP2 in GSE9891 cells. It was found that EOC patients with low ADAP2 expression had significantly better progression-free survival than those with high ADAP2 expression (P = 0.004). Figure 1 C) suggests that high ADAP2 expression is associated with poor patient prognosis.

[0071] Example 2

[0072] Based on the median ADAP2 expression, patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were divided into high-expression and low-expression groups. Kaplan-Meier survival analysis was used to discuss the impact of ADAP2 on the prognosis of EOC patients. Overall survival and progression-free survival in validation cohort 1 (n=132) are as follows: Figure 1 As shown in E, the total survival of verification queue 2 (n=138) is as follows: Figure 1 As shown in G, all results indicate that high ADAP2 expression has a negative effect on the prognosis of EOC patients, suggesting that ADAP2 is closely related to the progression of EOC.

[0073] The validation cohort 2 (n=138) was staged according to clinical staging criteria and T-staging criteria to explore the association between clinical staging, T-staging, and ADAP2. The results are as follows: Figure 1As shown in Figure H, the expression level of ADAP2 in EOC was correlated with clinical stage (P<0.01) and T stage (P<0.01), suggesting that the expression level of ADAP2 is related to the progression of clinical stage and T stage. Furthermore, the expression level of ADAP2 in the tissues of patients with advanced EOC was significantly higher than that in patients with early stage, indicating that the expression level of ADAP2 can help predict tumor development.

[0074] Simultaneously, immunohistochemical staining was performed to validate APAD2 in epithelial ovarian cancer tissues of different pathological types. The staining results of validation cohort 1 (n=132) are shown below. Figure 1 D, the staining results for validation cohort 2 (n=138) are shown in [reference]. Figure 1 F, this result demonstrates that ADAP2 is generally highly expressed in epithelial ovarian cancer.

[0075] Example 3

[0076] By transfecting si-ADAP2 to knock down ADAP2 expression in SKOV3 and HEY cells, and further screening for differentially expressed genes in THP-1-Mφ cells in a co-culture system using micro-transcriptome sequencing, the following methods were employed. Figure 3 A) and perform gene ontology (GO) enrichment analysis on the differentially expressed genes, such as Figure 3 As shown in B, the results suggest that ADAP2 is involved in a variety of important biological processes, including immune regulation, regulation of cytokine production, and monocyte proliferation, differentiation, and migration.

[0077] Validation was performed using qRT-PCR, such as Figure 3 As shown in C, after knocking down ADAP2 ovarian cancer cells in the co-culture system The expression of M1 markers (NOS2, TNF, IL-1β) in cells was significantly increased (P<0.01), while the expression of M2 markers (CD163, CD204, ARG1, IL-10) was significantly decreased (P<0.01), suggesting that ADAP2 in tumor cells induces macrophage Mφ to transform into the M2 pro-cancer phenotype.

[0078] The above results indicate that ADAP2 induces M2 polarization in macrophages in the co-culture system, and the expression level of ADAP2 in EOC is closely related to immune infiltration.

[0079] Example 4

[0080] Observation of different CD163 levels by immunohistochemical staining + The relationship between cell infiltration level and epithelial ovarian cancer ( Figure 2 A) Based on the staining results, patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were divided into CD163 groups. + High cell infiltration group and CD163+ In the low-infiltration cell group, the impact of CD163 expression level on the prognosis of EOC patients was discussed using Kaplan-Meier survival analysis. The overall survival and progression-free survival of cohort 1 (n=132) were validated. Figure 2 As shown in C, the total lifetime of verification queue 2 (n=138) is as follows: Figure 2 As shown in D, CD163 was found. + Patients with high cell infiltration have significantly shorter overall survival, confirming the presence of CD163. + High cellular infiltration suggests a poor prognosis for the patient.

[0081] The association between clinical stage, T stage, and CD163 was investigated, and the results are as follows: Figure 2 As shown in D, CD163 + Cell infiltration level was associated with clinical stage progression (P<0.05), CD163 + Cell infiltration levels were correlated with T stage progression (P<0.05), indicating that CD163... + Cellular infiltration levels can help predict the progression of epithelial ovarian cancer.

[0082] Example 5

[0083] This invention uses a Luminex liquid chromatography-mass spectrometry (LC-MS) chip to detect the levels of 26 cytokines in the supernatant of ADAP2-knockdown SKOV3 cells, in order to screen downstream cytokines regulated by ADAP2. Figure 4 A) revealed significant differential expression of IL6 (P<0.01), further confirmed by ELISA assay. Figure 4 B) and qRT-PCR Figure 4 C) The results showed that the expression level of IL6 was significantly downregulated after ADAP2 knockdown (P<0.05), indicating that ADAP2 promotes the secretion of IL6 by ovarian cancer cells.

[0084] Immunohistochemical staining was performed on IL6 in epithelial ovarian cancer tissue to verify the immunohistochemical staining of cohort 1 (n=132). Figure 4 As shown in Figure D, the immunohistochemical staining of validation cohort 2 (n=138) is shown in Figure 4E. Based on the immunohistochemical staining results, Kaplan-Meier survival analysis was performed on EOC patients with different IL6 expression levels. According to the staining results, patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were each divided into high-expression and low-expression groups. The overall survival and progression-free survival of validation cohort 1 (n=132) and the overall survival of validation cohort 2 (n=138) are shown in Figures 4E. Figure 4 Results D and 4E all showed that patients with higher IL6 expression levels had significantly lower overall survival than those with lower IL6 expression levels (P<0.05).

[0085] Example 6

[0086] This embodiment demonstrates the role of the combined application of ADAP2 / CD163 / IL6 in the prognostic assessment of epithelial ovarian cancer through two independent clinical validation cohorts.

[0087] Based on immunohistochemical staining results, the expression levels of ADAP2 / CD163 / IL6 in epithelial ovarian cancer tissues from validation cohort 1 (n=132) and validation cohort 2 (n=138) were investigated. Correlation analysis showed a positive correlation between the expression levels of ADAP2, IL6, and CD163. Figure 5 A, 5B Figure 6 A, 6B).

[0088] The high and low expression groups of ADAP2 and IL6 in validation cohort 1 (n=132) and validation cohort 2 (n=138) were compared with CD163. + Differential analysis of cell infiltration levels yielded the following results: Figure 5 C Figure 6 The column packing diagram shown in C represents CD163 in tissues with high ADAP2 expression. + Macrophage infiltration was significantly higher in the ADAP2 low expression group than in the IL6 high expression group, and CD163 in the IL6 high expression group was significantly higher. + Macrophage infiltration was significantly higher in the group with low IL6 expression than in the group with low IL6 expression, indicating that the expression levels of ADAP2 and IL6 are related to CD163. + The level of cell infiltration was significantly positively correlated (P<0.05).

[0089] The above examples suggest ADAP2, IL6, and CD163. + Cell infiltration is closely related to the tumor immune microenvironment; revealing the expression levels of ADAP2 / CD163 / IL6 and the immune microenvironment, the two work synergistically to play a unique role in the prognosis of EOC patients. This not only helps in prognostic assessment but also allows for targeted adjustment of treatment strategies for high-risk patients, which has important clinical value for precision treatment of EOC.

[0090] Example 7

[0091] Based on the expression levels of ADAP2 and IL6, as well as CD163 + The level of cell infiltration was used to divide patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) into three groups: ADAP2 high +CD163 high +IL6 high ADAP2 low +CD163 low +IL6 lowOthers (not labeled in the figure). Kaplan-Meier survival analysis was performed to validate the survival analysis results of cohort 1 (n=132). Figure 5 As shown in D, the survival analysis results of validation cohort 2 (n=138) are presented. Figure 6 As shown in D, ADAP2 was found in both verification queue 1 (n=132) and verification queue 2. high +CD163 high +IL6 high The worst prognosis is for ADAP2. low +CD163 low +IL6 low The best prognosis was observed, and the difference in prognosis between groups was statistically significant (P<0.01), suggesting that the expression levels of ADAP2 / CD163 / IL6 were negatively correlated with the overall survival and progression-free survival of patients. The synergistic high expression of the three strongly indicated a poor prognosis for patients.

[0092] This invention discovers that the GTPase-associated protein ADAP2 is associated with the tumor immune microenvironment that promotes cancer. Analysis of cell lines and clinical specimens reveals that ADAP2 is associated with poor prognosis in epithelial ovarian cancer, and its expression in early-stage EOC patients is significantly lower than that in late-stage EOC patients (clinical stage, T stage). Therefore, this invention applies ADAP2 as a prognostic biomarker to EOC prognostic prediction products.

[0093] This invention further discovered that the expression level of ADAP2 in EOC is closely related to immune infiltration, and that ADAP2 can regulate the secretion of IL6 cytokine by ovarian cancer cells, confirming that the expression levels of ADAP2 and IL6 are related to CD163. + The positive correlation between ADAP2, IL6, and CD163 indicates that... + Cellular infiltration is closely related to the tumor immune microenvironment. Further, through two independent clinical validation cohorts, the combined application of ADAP2 / CD163 / IL6 was confirmed for the prognostic assessment of ovarian cancer. The synergistic high expression of all three strongly suggests a poor prognosis, indicating that ADAP2 combined with CD163 and IL6 can serve as molecular markers for EOC prognostic prediction products. Its potential application in prognostic assessment is significant, enriching the predictive products and methods for survival prognosis in epithelial ovarian cancer patients and helping to guide clinical decision-making to improve the survival prognosis of ovarian cancer patients.

[0094] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Application of reagents for detecting the content or expression level of ADAP2 protein in samples in the preparation of a kit for assessing the prognosis of epithelial ovarian cancer.

2. The application according to claim 1, wherein, The amino acid sequence of the ADAP2 protein is shown in SEQ ID NO:

1.

3. The application according to claim 2, wherein, The nucleotide sequence of the gene encoding the ADAP2 protein is shown in SEQ ID NO:

2.

4. The application according to any one of claims 1-3, wherein, The method for detecting the content or expression level of ADAP2 protein in a sample is at least one of immunohistochemistry, Western blotting, immunomagnetic bead detection, chemiluminescence, or flow cytometry.

5. The application according to any one of claims 1-3, wherein, The sample is at least one of cells or tissues.

6. The application according to any one of claims 1-3, wherein, The kit also includes: reagents for detecting the content or expression level of IL6 protein in a sample; and / or A reagent used to detect the content or expression level of CD163 protein in a sample.

7. The application according to claim 6, wherein, The method for detecting the IL6 protein content or expression level in the sample is at least one of ELISA, immunohistochemistry, Western blotting, immunomagnetic bead detection, chemiluminescence, or flow cytometry; and / or The method for detecting the CD163 protein content or expression level in the sample is at least one of immunohistochemistry or flow cytometry.

8. Application of ADAP2 protein as a target in the preparation of reagents for assessing the prognosis of epithelial ovarian cancer.

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