Application of ADAP2 as epithelial ovarian cancer prognosis evaluation biomarker and target and application of ADAP2 detection reagent

Abstract

The invention belongs to the field of oncomolecular biology, and discloses application of ADAP2 as an epithelial ovarian cancer prognosis evaluation biomarker and a target spot and application of a reagent for detecting ADAP2. The reagent for detecting the content or expression quantity of the ADAP2 protein in the sample is used for preparing the kit for evaluating the epithelial ovarian cancer prognosis, has the advantages of convenience, simplicity, rapidness, accuracy and the like, can timely and rapidly evaluate the epithelial ovarian cancer prognosis, can provide effective guidance for clinical decisions, and is beneficial to improving the survival and prognosis of patients.

Description

Technical Field

[0001] The present invention belongs to the field of tumor molecular biology, and specifically relates to an application of ADAP2 as a biomarker and target for prognosis assessment of epithelial ovarian cancer and an application of a reagent for detecting ADAP2. Background Art

[0002] Ovarian cancer is the malignant tumor with the highest mortality rate in the female reproductive system. Among them, epithelial ovarian carcinoma (EOC) accounts for about 90% of ovarian malignant tumors. The latest statistics show that there are nearly 300,000 new cases of ovarian cancer and more than 180,000 deaths worldwide each year. Because the ovaries are located deep in the pelvic cavity, the onset is relatively hidden and difficult to detect in the early stage. About 2 / 3 of patients have metastases at the time of diagnosis, resulting in poor results of tumor reduction surgery and chemotherapy. Traditional methods for evaluating the prognosis of EOC patients mainly rely on clinical parameters, which cannot well reflect the intrinsic molecular biological characteristics of the tumor. The prognosis of a considerable number of patients cannot be accurately evaluated, and existing studies lack effective molecular biological markers to guide treatment strategies. Metabolic disorders in the tumor microenvironment (TME) have recently been shown to play a decisive role in tumor formation and progression. Therefore, it is of great significance to explore new prognostic markers related to the immune microenvironment of epithelial ovarian cancer and explore therapeutic targets.

[0003] ADAP2 is a member of the protein family with an ADP-ribosylation factor-GTPase activating protein (Arf-GAPs) domain. It can bind to β-tubulin, increase the stability of microtubules, and play an important role in cardiac development. Studies have also shown that ADAP2 can act as an interferon-stimulated gene, blocking viral invasion by changing Arf6-mediated transport, and exerting an antiviral effect. In recent years, studies have shown that ADAP2 is related to the prognosis of tumor patients and can promote biological behaviors such as tumor invasion and metastasis. However, there are currently no reports on the prognostic evaluation and therapeutic potential of ADAP2 in EOC. Summary of the invention

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

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides a use of a reagent for detecting the content or expression of ADAP2 protein in a sample in the preparation of a kit for evaluating the prognosis of epithelial ovarian cancer.

[0006] The second aspect of the present invention provides a use of a reagent for detecting the content or expression of ADAP2 protein in a sample in a kit for preparing a prognosis and treatment plan for epithelial ovarian cancer.

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

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

[0009] Through the above technical scheme, the present invention found that the expression level of GTPase-related protein ADAP2 has a significant effect on the prognosis of patients with epithelial ovarian cancer, and high expression of ADAP2 is closely related to tumor microenvironment disorders, suggesting that patients with high expression of ADAP2 may have better therapeutic effects on immunotherapy, and thus ADAP2 becomes a potential target for the treatment of epithelial ovarian cancer.

[0010] On the other hand, the present invention found that the expression level of ADAP2 in EOC is closely related to immune infiltration, and ADAP2 induces ovarian cancer cells to secrete cytokine IL6, revealing that ADAP2 can accurately evaluate the patient's prognosis by combining CD163 and / or IL6 indicators. The present invention provides an application of ADAP2 combined with CD163 and / or IL6 in the prognosis evaluation and treatment of epithelial ovarian cancer, which can formulate an individualized treatment plan based on the patient's gene expression characteristics, provide effective guidance for clinical decision-making, and help improve the patient's survival and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0013] Figure 2 Description CD163 + The value of cell infiltration level in the clinical prognosis of epithelial ovarian cancer: Figure 2 A is the immunohistochemical staining of CD163 expression 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 correlation bubble chart showing the correlation analysis results of 11 candidate molecules including ADAP2 and M2 macrophage markers; Figure 2 C is the difference of CD163 in EOC patients in validation cohort 1 (n=132). + Kaplan-Meier OS curve and PFS curve of cell infiltration level; Figure 2 D is the difference in CD163 in validation cohort 2 (n=138) + OS curves of Kaplan-Meier survival analysis of cell infiltration level and CD163 + Box plot of cell infiltration level, 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 the gene heat map in THP-1-Mφ cells after knockdown of ADAP2 in the co-culture system; Figure 3 B is the GO enrichment analysis of differentially expressed genes in THP-1-Mφ cells after knockdown of ADAP2 in the co-culture system; Figure 3 C is a bar graph showing the expression levels of M1 markers and M2 markers in THP-1-Mφ cells after knockdown of ADAP2 in the co-culture system.

[0015] Figure 4 Explain the value of IL6 in the clinical prognosis of epithelial ovarian cancer: Figure 4 A is a bar graph of the secretion levels of downstream cytokines regulated by ADAP2 screened by Luminex liquid phase chip; Figure 4B is a bar graph showing the IL6 secretion levels in the supernatants of different SKOV3 (si-NC and si-ADAP2) cells detected by ELISA;

[0016] Figure 4 C is a bar graph of 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 diagram of IL6 expression in different pathological types of tissues in EOC patients in validation cohort 1 (n=132) and the Kaplan-Meier OS curve and PFS curve of different IL6 expression levels; Figure 4 E is the immunohistochemical staining of IL6 expression in different pathological types of tissues in EOC patients in validation cohort 2 (n=138) and the Kaplan-Meier OS curves of different IL6 expression levels.

[0017] Figure 5 The value of ADAP2 / CD163 / IL6 expression levels in predicting the survival prognosis of EOC patients in validation cohort 1 (n=132) is illustrated as follows: Figure 5 A is the immunohistochemical staining of ADAP2 / CD163 / IL6 expression in epithelial ovarian cancer tissues in validation cohort 1 (n=132); Figure 5 B is the correlation between ADAP2 and IL6 protein levels and CD163 in epithelial ovarian cancer tissues + Spearman correlation analysis between cell infiltration levels; Figure 5 C is CD163 at different ADAP2 protein levels in EOC in validation cohort 1 (n=132). + Stacked column plots of differential analysis of cell infiltration and CD163 at different IL6 protein levels + Stacked column plots for differential analysis of cell infiltration levels; Figure 5 D is the OS curve and PFS curve of the Kaplan-Meier survival analysis of different ADAP2 / CD163 / IL6 expression levels in validation cohort 1 (n=132).

[0018] Figure 6 The value of ADAP2 / CD163 / IL6 expression level in predicting the survival prognosis of EOC patients in validation cohort 2 (n=138) is shown as follows: Figure 6 A is the immunohistochemical staining of ADAP2 / CD163 / IL6 expression in epithelial ovarian cancer tissues in validation cohort 2 (n=138); Figure 6 B is the correlation between ADAP2 and IL6 protein levels and CD163 in epithelial ovarian cancer tissues + Spearman correlation analysis between cell infiltration levels; Figure 6 C is CD163 with different ADAP2 protein levels in EOC in validation cohort 2 (n=138). + Stacked column plots of differential analysis of cell infiltration and CD163 at different IL6 protein levels + Stacked column plots for differential analysis of cell infiltration levels; Figure 6 D is the OS curve of Kaplan-Meier survival analysis of different ADAP2 / CD163 / IL6 expression levels in validation cohort 2 (n=138). DETAILED DESCRIPTION

[0019] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

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

[0021] In the present invention, data analysis: GraphPad Prism 8.0 software or R software (version 4.3.1) was used for statistical analysis, Student's t-test or Wilcoxon test method was used for significance analysis, Spearman analysis method was used for correlation analysis, and the figures were marked as statistically significant difference analysis: * means P < 0.05, ** means P < 0.01, *** means P < 0.001, ns means no significant difference, and the difference is considered statistically significant when P < 0.05. Kaplan-Meier survival analysis was verified by Log-rank test.

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

[0023] In the present invention, the amino acid sequence of 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 in SEQ ID NO: 2, SEQ ID NO: 2:

[0027]

[0028] The present invention finds that the GTPase-related protein ADAP2 is highly expressed in ovarian cancer and has a significant effect on the prognosis of epithelial ovarian cancer. It has the advantages of being simple to operate and having high technical reliability when used as a biomarker to assess prognosis.

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

[0030] The present invention has no particular limitation on the specific operation of immunohistochemistry, immunoblotting, immunomagnetic bead detection, chemiluminescence or flow cytometry for detecting the ADAP2 content or expression in a sample. Those skilled in the art can select according to known techniques to achieve excellent detection results.

[0031] A person skilled in the art can prepare a corresponding kit for evaluating epithelial ovarian cancer based on the ADAP2 biomarker disclosed in the present invention and the known techniques in the field of kit preparation, wherein the kit contains a reagent capable of detecting the ADAP2 protein content or expression in a sample, such as a test strip for detecting the ADAP2 protein content.

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

[0033] According to a preferred embodiment of the present invention, the sample is at least one of a cell or a tissue.

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

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

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

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

[0038] The present invention found that the expression level of ADAP2 in EOC is closely related to immune infiltration, and CD163 +High cell infiltration indicates a poor prognosis for patients; and ADAP2 induces ovarian cancer cells to secrete cytokine IL6. The overall survival (OS) and progression-free survival (PFS) of EOC patients with higher IL6 expression levels are significantly lower than those with low IL6 expression. The combined prediction of the survival prognosis of EOC patients by ADAP2 / CD163 / IL6 expression levels is not only conducive to prognostic judgment, but also can adjust the treatment strategy of high-risk patients in a targeted manner, which has important clinical value for the precise treatment of EOC.

[0039] The second aspect of the present invention provides a use of a reagent for detecting the content or expression of ADAP2 protein in a sample in a kit for preparing a prognosis and treatment plan for epithelial ovarian cancer.

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

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

[0042] In the present invention, the prognosis includes detection, prognosis assessment, recurrence monitoring and immunotherapy efficacy assessment.

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

[0044] In the present invention, ADAP2 expression level has a significant effect on the prognosis of patients with epithelial ovarian cancer. ADAP2 can be used as a biomarker for treating or predicting the prognosis of epithelial ovarian cancer. The application of a detection kit for evaluating the prognosis of epithelial ovarian cancer prepared based on the ADAP2 biomarker and ADAP2 target has the advantages of simple operation and high technical reliability, and is suitable for clinical kit development.

[0045] In order 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, rather than limiting the claims of the present invention.

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

[0047] In the following examples, human ovarian cancer cell lines SKOV3, HEY and human monocytes (acute monocytic leukemia cells) THP-1 were obtained from ATCC (American Type Culture Collection), wherein the THP-1 culture medium was induced into macrophages Mφ by adding 100 ng / ml PMA for 48 hours. Among them, SKOV3 was cultured in McCoy's 5A medium, HEY was cultured in DMEM medium, and THP-1 was cultured in RPMI1640 medium supplemented with 0.05 mM 2-mercaptoethanol, and all culture media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture, and cultured in a 5% CO2 incubator at 37°C.

[0048] In the following examples, Lipofectamine 3000 (Invitrogen, USA) was used to transfect siRNA into cells, wherein siRNAs were purchased from Ruibo Biotechnology Co., Ltd.

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

[0050] In the following examples, the expression levels of ADAP2 and IL6 were detected by immunohistochemical staining: ADAP2 and IL6 were evaluated for expression level using the H-score system, H-score = (∑IS × AP), where IS represents the staining intensity and AP represents the percentage of positively stained cells. IS depends on cell staining, with no staining being 0 points, weak staining being 1 point, moderate staining being 2 points, and strong staining being 3 points; AP depends on the percentage of stained cells, with 0% being 0 points, 1%-25% being 1 point, 26%-50% being 2 points, 51%-75% being 3 points, and 76%-100% being 4 points. H-score = 6 points 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 in the entire magnification field was recorded to determine the CD163 + The degree of cell infiltration was 0 points for no obvious infiltration, 1 point for a small amount of infiltration, 2 points for low infiltration, 3 points for moderate infiltration, and 4 points for high infiltration, with 2 as the cutoff value, and ≤2 defined as CD163 + Low cell infiltration group, defined as ≥3 CD163 + High cell infiltration group.

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

[0053] 1) Dewax the paraffin sections until they are hydrated: sequentially 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% alcohol for 5 min, 75% alcohol for 5 min, and wash with distilled water.

[0054] 2) Antigen repair: Place the tissue sections in a repair box filled with citric acid (pH 6.0) antigen repair solution in a microwave oven for antigen repair. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After natural cooling, place the slides in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time.

[0055] 3) Blocking endogenous peroxidase: The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time.

[0056] 4) BSA or serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), and then drop 3% BSA in the circle to evenly cover the tissue, and block at room temperature for 30 minutes.

[0057] 5) Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in a certain proportion with PBS on the slices, place the slices flat in a humidified box (add a small amount of water in the humidified box to prevent the antibody from evaporating) and incubate at 4°C overnight.

[0058] 6) Add secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP labeled) of the corresponding species to the primary antibody in the histochemistry kit to cover the tissue and incubate at room temperature for 50 minutes.

[0059] 7) DAB color development: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, and control the color development time under a microscope. The positive color is brown-yellow, and the slices are rinsed with tap water to stop the color development.

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

[0061] 9) Dehydration and sealing: Place the slices in 75% alcohol for 6 min - 85% alcohol for 6 min - anhydrous ethanol I for 6 min - anhydrous ethanol II for 6 min - xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

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

[0063] In the following examples, the real-time quantitative PCR experimental operation is as follows: after discarding the culture medium, slowly add an appropriate amount of PBS to wash the cell sample 3 times, add 1 ml of TRIzol to lyse the cells, and repeatedly blow with a 1 ml pipette to make it fully contact with the cells. After 10 minutes, use a 1 ml pipette to transfer all the liquid to a 1.5 ml EP tube, and then perform sample preparation and detection operations according to standard operating procedures. Use EZ-press RNA Purification Kit (EZBioscience, USA) according to the instructions to extract total cell RNA, and reverse transcribe using PrimeScript TM RT Master Mix kit (TaKaRa, Japan), qRT-PCR using the corresponding PCR kit (TaKaRa, Japan), data using 2 -ΔΔCt Method for analysis.

[0064] In the following examples, in validation cohort 1 (n=132), patients were divided into a high expression group (n=79) and a low expression group (n=53) according to 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) according to the median value of ADAP2 expression.

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

[0066] In the following examples, in validation cohort 1 (n=132), patients were divided into a high expression group (n=83) and a low expression group (n=49) according to 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) according to the median value of IL6 expression.

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

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

[0069] Example 1

[0070] The EPIC algorithm (EPIC is an algorithm that quantifies the level of immune cell infiltration in tumor samples based on gene expression data, which can reflect the diversity of immune cells and stromal cells) was used to determine the overall infiltration of 8 immune cells in epithelial ovarian cancer included in the GSE9891 dataset ( Figure 1 A, Figure 1 Only 6 of them are shown in A). The gene expression in the tissues of the macrophage high infiltration group and the low infiltration group in the EPIC algorithm was analyzed differentially (|logFC|>1, P<0.05), and the differential gene set was screened, and 355 differential genes were obtained ( Figure 1 B); Secondly, the 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 significant genes using the survival package and survminer package in R language. Eleven genes were screened to affect the prognosis of EOC patients: EVI2A, MSR1, DOCK2, GIMAP4, VSIG4, LY86, FPR3, CD37, ADAP2, FCGR1A, GPR65 ( Figure 2 B), ADAP2 was screened as a molecular marker, and Kaplan-Meier survival analysis was performed on the ADAP2 expression level in GSE9891. It was found that the progression-free survival of EOC patients with low ADAP2 expression was significantly improved compared with that of the ADAP2 high expression group (P=0.004, Figure 1 C), suggesting that high expression of ADAP2 is associated with poor prognosis of patients.

[0071] Example 2

[0072] According to the median expression of ADAP2, patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were divided into high expression group and low expression group. The effect of ADAP2 on the prognosis of EOC patients was discussed by Kaplan-Meier survival analysis. The overall survival and progression-free survival of validation cohort 1 (n=132) were as follows: Figure 1 As shown in E, the overall survival of the validation cohort 2 (n = 138) is as follows Figure 1 As shown in G, it is suggested that high expression of ADAP2 has a negative effect on the prognosis of EOC patients, indicating that ADAP2 is closely related to the progression of EOC.

[0073] The validation cohort 2 (n = 138) was staged according to the clinical staging criteria and T staging criteria to explore the correlation between clinical staging, T staging and ADAP2. Figure 1As shown in Figure H, the expression level of ADAP2 in EOC was correlated with the clinical stage (P<0.01) and T stage (P<0.01), indicating that the expression level of ADAP2 was correlated with the progression of clinical stage and T stage, and 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] At the same time, immunohistochemical staining was performed to verify APAD2 in epithelial ovarian cancer tissues of different pathological types. The staining results of validation cohort 1 (n=132) are shown in Figure 1 D, Staining results of validation cohort 2 (n=138) are shown in Figure 1 F, This result shows that ADAP2 is generally highly expressed in epithelial ovarian cancer.

[0075] Example 3

[0076] The expression of ADAP2 in SKOV3 and HEY cells was knocked down by transfection of si-ADAP2. Furthermore, differentially expressed genes in THP-1-Mφ cells in the co-culture system were screened by micro-transcriptome sequencing ( Figure 3 A), and Gene Ontology (GO) enrichment analysis was performed on the differentially expressed genes. Figure 3 As shown in B, the results suggest that ADAP2 is involved in many important biological processes such as immune regulation, cytokine production regulation, monocyte proliferation and differentiation migration, etc.

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

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

[0079] Example 4

[0080] Observation of different CD163 by immunohistochemical staining + Relationship between cell infiltration level and epithelial ovarian cancer Figure 2 A) Patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were divided into CD163 + High cell infiltration group and CD163+ In the low cell infiltration group, the effect of CD163 expression level on the prognosis of EOC patients was discussed by Kaplan-Meier survival analysis, and the overall survival and progression-free survival of cohort 1 (n=132) were verified as follows: Figure 2 As shown in C, the overall survival of the validation cohort 2 (n = 138) is as follows Figure 2 As shown in D, CD163 + The overall survival of patients with high cell infiltration was significantly shortened, confirming that CD163 + High cell infiltration indicates a poor prognosis for the patient.

[0081] The correlation between clinical stage, T stage and CD163 was explored. Figure 2 D shows CD163 + The cell infiltration level was correlated with the clinical stage progression (P<0.05), and CD163 + The level of cell infiltration was correlated with T stage progression (P<0.05), indicating that CD163 + The level of cell infiltration can help predict the development of epithelial ovarian cancer.

[0082] Example 5

[0083] The present invention uses Luminex liquid phase chip to detect the levels of 26 cytokines in the supernatant of SKOV3 cells with ADAP2 knockdown to screen the downstream cytokines regulated by ADAP2 ( Figure 4 A), and found that IL6 expression was significantly different (P < 0.01), and further ELISA test ( Figure 4 B) and qRT-PCR ( Figure 4 C) was verified, and the results showed that the expression level of IL6 was significantly downregulated after knocking down ADAP2 (P<0.05), indicating that ADAP2 promoted the secretion of IL6 by ovarian cancer cells.

[0084] Immunohistochemical staining of IL6 in epithelial ovarian cancer tissues was performed to verify the immunohistochemical staining of cohort 1 (n = 132). Figure 4 D is shown in the figure. The immunohistochemical staining of validation cohort 2 (n=138) is shown in 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, the patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were divided into high expression group and low expression group, respectively. 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 Figure 4E, respectively. Figure 4 D, 4E, the above results all showed that the overall survival of patients with higher IL6 expression levels was significantly lower than that of patients with low IL6 expression levels (P<0.05).

[0085] Example 6

[0086] This example confirms the effect of combined use of ADAP2 / CD163 / IL6 on the prognosis assessment of epithelial ovarian cancer through two independent clinical validation cohorts.

[0087] Based on the results of immunohistochemical staining, the expression levels of ADAP2 / CD163 / IL6 in epithelial ovarian cancer tissues in validation cohort 1 (n=132) and validation cohort 2 (n=138) were studied, and correlation analysis was performed, showing that the expression levels of ADAP2, IL6 and CD163 were positively correlated ( Figure 5 A, 5B, Figure 6 A, 6B).

[0088] The high and low expression groups of ADAP2 and the high and low expression groups of IL6 in the validation cohort 1 (n = 132) and validation cohort 2 (n = 138) were compared with CD163 + The cell infiltration level was analyzed differentially. Figure 5 C. Figure 6 The column stacking diagram shown in C shows CD163 in ADAP2-highly expressed tissues + Macrophage infiltration was significantly higher than that in the ADAP2 low expression group, and CD163 in IL6 high expression tissues + The infiltration of macrophages was significantly higher than that in the IL6 low expression group, indicating that the expression of ADAP2 and IL6 was related to CD163 + There was a significant positive correlation between the levels of cell infiltration and mitochondria (P<0.05).

[0089] The above examples suggest that ADAP2, IL6 and CD163 + There is a close relationship between cell infiltration and the tumor immune microenvironment; revealing the ADAP2 / CD163 / IL6 expression level and the immune microenvironment, the two synergistically play a unique role in the prognosis of EOC patients, which is not only conducive to prognostic judgment, but also can be used to adjust the treatment strategy of high-risk group patients in a targeted manner, which has important clinical value for the precise treatment of EOC.

[0090] Example 7

[0091] According to the expression levels of ADAP2 and IL6 and CD163 + The patients in validation cohort 1 (n=132) and validation cohort 2 (n=138) were divided into three groups: ADAP2 high +CD163 high +IL6 high , ADAP2 low +CD163 low +IL6 low, others (not marked in the figure). Kaplan-Meier survival analysis was performed to verify the survival analysis results of cohort 1 (n = 132). Figure 5 D shows the survival analysis results of validation cohort 2 (n = 138) Figure 6 D shows that ADAP2 was found in both validation cohort 1 (n=132) and validation cohort 2. high +CD163 high +IL6 high The worst prognosis, ADAP2 low +CD163 low +IL6 low The prognosis was the best, and the prognostic difference between the two groups was statistically significant (P<0.01), indicating that the expression level of ADAP2 / CD163 / IL6 was negatively correlated with the patient's overall survival and progression-free survival, and the synergistic high expression of the three strongly indicated a poor prognosis for the patient.

[0092] The present invention discovered that the GTPase-related protein ADAP2 is associated with a cancer-promoting tumor immune microenvironment. Analysis of cell lines and clinical specimens revealed that ADAP2 is associated with a poor prognosis of epithelial ovarian cancer, and that its expression in early EOC patients is significantly lower than that in late EOC patients (clinical stage, T stage). Therefore, the present invention applies ADAP2 as a prognosis prediction marker to EOC prognosis prediction products.

[0093] The present invention further found that the expression level of ADAP2 in EOC is closely related to immune infiltration, and ADAP2 can regulate the secretion of IL6 cytokine by ovarian cancer cells, confirming that the expression levels of ADAP2 and IL6 are closely related to CD163 + Cell infiltration was positively correlated, indicating that ADAP2, IL6 and CD163 + There is a close relationship between cell infiltration and the tumor immune microenvironment. The role of the combined application of ADAP2 / CD163 / IL6 in the prognosis assessment of ovarian cancer was further confirmed by two independent clinical validation cohorts. The high expression of the three strongly indicates a poor prognosis for patients, indicating that ADAP2 combined with CD163 and IL6 can be used as a molecular marker in EOC prognosis prediction products. Its potential application in prognosis assessment is of great significance, enriching the prediction products and means of survival prognosis of patients with epithelial ovarian cancer, and helping to guide clinical decision-making to improve the survival prognosis of patients with ovarian cancer.

[0094] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the ADAP2 protein content or expression in a sample in the preparation of a kit for evaluating the prognosis of epithelial ovarian cancer.

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

1.

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

2.

4. The use according to any one of claims 1 to 3, wherein: The method for detecting the ADAP2 protein content or expression in the sample is at least one of immunohistochemistry, immunoblotting, immunomagnetic bead detection, chemiluminescence or flow cytometry.

5. The use according to any one of claims 1 to 4, wherein: The sample is at least one of a cell and a tissue.

6. The use according to any one of claims 1 to 5, wherein: The kit also includes: a reagent for detecting the IL6 protein content or expression in the sample; and / or A reagent used to detect the CD163 protein content or expression in a sample.

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

8. Use of a reagent for detecting the ADAP2 protein content or expression level in a sample in a kit for preparing a prognosis and treatment plan for epithelial ovarian cancer.

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

10. Use of an ADAP2 protein inhibitor in the preparation of a drug for treating epithelial ovarian cancer prognosis, characterized in that: The drug targets the ADAP2 protein.

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