Application of FYB1 gene as marker in preparation of gastric cancer diagnosis or prognosis reagent

Abstract

The invention relates to the technical field of medicines, in particular to application of an FYB1 gene as a marker in preparation of a reagent for gastric cancer diagnosis or prognosis. Researches find that the FYB1 gene is highly expressed in gastric cancer tissues and is in negative correlation with prognosis of patients, and the FYB1 gene can be used as a gastric cancer marker to be applied to gastric cancer detection and curative effect evaluation. In addition, the expression of the FYB1 and the expression of a Treg cell marker FOXP3 are in positive correlation and co-localization exists in tissues, which prompts that the FYB1 can promote the immune escape of the gastric cancer tumor; by inhibiting the expression of the FYB1 gene, the proliferation, migration and invasion capabilities of gastric cancer cells can be remarkably inhibited, and the tumor growth is inhibited, so that the FYB1 gene can be used as a gastric cancer treatment target and plays an important role in the treatment of gastric cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of an FYB1 gene as a marker in preparing a gastric cancer diagnosis or prognosis reagent. Background Art

[0002] In recent years, gastric cancer remains one of the most common digestive tract malignancies worldwide. Currently, the main treatments for gastric cancer include surgery and chemotherapy. However, due to the low rate of early diagnosis of gastric cancer, many patients are already in the late or locally advanced stages of the disease when they seek medical attention, or even have distant metastases, making them incurable with traditional treatments. Although immunotherapy research has made many breakthroughs in recent years, its effectiveness in treating gastric cancer is still not ideal. Therefore, it is crucial to identify new biomolecular markers or immunotherapy targets to improve clinical efficacy and increase the survival rate of gastric cancer patients.

[0003] FYB1, as an adaptor protein, plays a crucial role in T cell activation, immune synapse formation, and signal transduction. Recent studies suggest that the FYB1 gene may participate in tumor progression by regulating processes such as cell migration and proliferation. The protein encoded by FYB1 is an adaptor protein in the FYN protein and lymphocyte cytosolic protein 2 (LCP2) signaling cascade in T cells and is essential for T cell activation. Its role in tumor pathogenesis and metastasis has been increasingly studied in recent years, and has been shown to be associated with the pathogenesis and progression of tumors such as breast cancer and lung adenocarcinoma. However, studies on the association between FYB1 and gastric cancer are limited, and its mechanisms of action in gastric cancer, including its expression pattern, function, and relationship with the immune microenvironment, have not been reported.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a use of the FYB1 gene as a marker in the preparation of a reagent for diagnosing or prognosing gastric cancer.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of the FYB1 gene and its expressed protein as a marker in the preparation of a reagent for diagnosing or prognosing gastric cancer.

[0008] Preferably, the expression of the FYB1 gene in gastric cancer tissue is higher than that in normal tissue; and / or, the prognosis and survival statistics of the FYB1 gene high expression group in gastric cancer patients are worse than those of the low expression group, and it serves as a predictive marker for gastric cancer immune escape and poor prognosis in pathological immunohistochemistry.

[0009] In a second aspect, the present invention provides the use of the FYB1 gene and its expressed protein as markers in the preparation of a product for evaluating the efficacy of anti-gastric cancer drugs, wherein the product is a reagent, a kit or a device.

[0010] In an optional embodiment, the present invention provides a gastric cancer detection / therapy evaluation device, comprising:

[0011] A detection module, used to detect the content of the FYB1 gene in the sample to be tested;

[0012] An input module is used to obtain the detection results of the detection module;

[0013] A judgment module is used to compare the test results obtained by the input module with the test results of samples from individuals without gastric cancer to determine whether the patient has gastric cancer / whether the gastric cancer is cured;

[0014] Output module, used to output diagnosis results.

[0015] Preferably, the judgment criteria of the judgment module include: if the FYB1 gene content in the sample to be tested is significantly higher than the test result of the sample from the individual without gastric cancer, then the individual from whom the sample to be tested is judged to have gastric cancer / uncured gastric cancer.

[0016] In a third aspect, the present invention provides the use of a FYB1 gene inhibitor in the preparation of an anti-gastric cancer drug.

[0017] The present invention found that inhibiting FYB1 gene expression significantly inhibited gastric cancer cell proliferation, migration, and invasion, thereby suppressing tumor growth, indicating that the FYB1 gene could serve as a therapeutic target for gastric cancer. The present invention uses the FYB1 gene as a target for gastric cancer drug design, which has great clinical translation and application value.

[0018] The present invention does not particularly limit the specific type or source of the FYB1 gene inhibitor; any FYB1 gene inhibitor conventionally selected in the art that complies with relevant pharmaceutical market standards may be used. Preferably, the FYB1 gene inhibitor is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies.

[0019] Preferably, the anti-gastric cancer drug can inhibit the proliferation of gastric cancer cells.

[0020] Preferably, the anti-gastric cancer drug can inhibit the growth, invasion and metastasis of gastric cancer cells.

[0021] In a fourth aspect, the present invention provides an anti-gastric cancer drug comprising a FYB1 gene inhibitor.

[0022] Preferably, the anti-gastric cancer drug further comprises a targeting agent, which can target gastric cancer cells or gastric cancer tissues. The present invention does not particularly limit the specific type and source of the targeting agent, and any targeting agent conventionally selected in the art that complies with relevant drug market regulations can be used.

[0023] Preferably, the anti-gastric cancer drug further comprises a delivery vector capable of delivering the FYB1 gene inhibitor to gastric cancer cells or gastric cancer tissue. The present invention does not particularly limit the specific type or source of the delivery vector; any delivery vector conventionally selected in the art that complies with relevant pharmaceutical market standards may be used. For example, nucleic acid lipid nanoparticles (LNPs) may be used as the delivery vector.

[0024] Beneficial effects:

[0025] The present invention provides the use of the FYB1 gene as a marker in the preparation of diagnostic or prognostic reagents for gastric cancer. Research conducted by the present invention has found that the FYB1 gene is highly expressed in gastric cancer tissues and negatively correlated with patient prognosis, making it useful as a gastric cancer marker for gastric cancer detection and therapeutic evaluation. Furthermore, inhibiting FYB1 gene expression significantly inhibits gastric cancer cell proliferation, migration, and invasion, thereby suppressing tumor growth. This suggests that the FYB1 gene could serve as a therapeutic target for gastric cancer and play an important role in the treatment of gastric cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0027] Figure 1 These are the pan-cancer analysis results of FYB in the TIMER2.0 online database (*P<0.05, **P<0.01, ***P<0.001).

[0028] Figure 2 The expression differences of FYB1 in gastric cancer and cancer tissues and the survival analysis results based on the TCGA database. Figure 2 Figure A in the middle shows the differential expression analysis of FYB1 in gastric cancer tissues (Tumor=410) and normal tissues (Normal=36) based on the TCGA database (P<0.05); Figure 2 Figure B in the middle is the survival analysis of FYB1 high expression group and low expression group in gastric cancer based on transcriptome and survival data of TCGA database (P<0.05).

[0029] Figure 3 These are the results of clinical relevance analysis of FYB1. Figure 3 Figure A in the middle is a heat map of the correlation analysis between FYB1 expression and clinical features in gastric cancer patients based on the TCGA database (*P<0.05); Figure 3 Figure B is a box plot of the correlation analysis between FYB1 expression and T stage (P<0.05).

[0030] Figure 4 This is the ESTIMATE immune score of FYB1 (*P<0.05, **P<0.01, ***P<0.001).

[0031] Figure 5 This is a scatter plot of the correlation analysis of FYB1. Figure 5 Figure A in the middle is a scatter plot of the correlation analysis between FYB1 and FOXP3 (P<0.05); Figure 5 Figure B is a scatter plot of the correlation analysis between FYB1 and CD4 (P<0.05).

[0032] Figure 6 The figure shows the immunohistochemistry results of FYB1 protein. Figure 6 Figure A in the middle shows the immunohistochemical staining results of FYB1 in gastric cancer tissue, which is mainly localized in the cytoplasm of cells, with positive staining in gastric cancer and adjacent infiltrating cells (scale bar: 2 mm, ×4); Figure 6 Middle B shows the magnified immunohistochemical staining results of FYB1 in gastric cancer tissue (scale bar 50 μm, ×20); Figure 6 Figure C in the middle shows the immunohistochemical staining results of FYB1 in adjacent paracancerous tissues (scale bar: 2 mm, ×4); Figure 6 Figure D in the middle shows the magnified immunohistochemical staining results of FYB1 in adjacent cancer tissues (scale bar: 50 μm, ×20).

[0033] Figure 7 Statistical analysis of FYB1 immunohistochemistry in gastric cancer. Figure 7 Figure A in the middle shows the statistical analysis results of FYB1 expression levels in 60 paired gastric cancer and adjacent adjacent tissues (P<0.05); Figure 7 Figure B in the middle shows the comparative analysis results of FYB1 expression levels in cancer and adjacent adjacent tissues (****P<0.0001).

[0034] Figure 8 The localization of FYB1, CD4, and FOXP3 in gastric cancer tissues by immunohistochemistry in serial sections. Figure 8Middle A: The left image shows the immunohistochemical staining results of CD4 in gastric cancer tissue, which is mainly localized in the cytoplasm of cells, with positive staining in gastric cancer and adjacent infiltrating cells (scale bar 5 mm, ×4). The right image shows the magnified immunohistochemical staining results of CD4 in gastric cancer tissue (scale bar 100 μm, ×20); Figure 8 Middle B: The left image shows the immunohistochemical staining results of FOXP3 in gastric cancer tissue, which is mainly localized in the cell nucleus (scale bar 5 mm, ×4). The right image shows the magnified immunohistochemical staining results of FOXP3 in gastric cancer tissue (scale bar 100 μm, ×20); Figure 8 Middle C: The left image shows immunohistochemical staining of FYB1 in gastric cancer tissue, which is mainly localized in the cytoplasm (scale bar 5 mm, ×4). The right image shows the magnified immunohistochemical staining of FYB1 in gastric cancer tissue (scale bar 100 μm, ×20).

[0035] Figure 9 The figure shows the immunofluorescence expression of FYB1 and CD4 co-localization. Figure 9 Figure A in the middle shows the immunofluorescence co-localization of FYB1 (green) and CD4 (red) in gastric cancer tissues; Figure 9 Figure B shows the immunofluorescence co-localization of FYB1 (green) and CD4 (red) in adjacent cancer tissues (scale bar: 20 μm, ×20).

[0036] Figure 10 The figure shows the immuno-colocalization expression of FYB1 and FOXP3. Figure 10 Figure A in the middle shows the immunofluorescence co-localization of FYB1 (green) and FOXP3 (pink) in gastric cancer tissues; Figure 10 Figure B shows the immunofluorescence co-localization of FYB1 (green) and FOXP3 (pink) in adjacent cancer tissues (scale bar: 20 μm, ×20).

[0037] Figure 11 This is the result of immune co-localization analysis of gastric cancer tissue. Figure 11 Figure A in the middle shows the analysis of the immunocolocalization results of FYB1 and CD4 in gastric cancer tissues; Figure 11 Figure B in the middle is an analysis of the immunological co-localization results of FYB1 and FOXP3 in gastric cancer tissues (FYB1 is localized in the cytoplasm and infiltrating cells, and FOXP3 is localized in the nucleus of T cells).

[0038] Figure 12 This is the result of immune co-localization analysis of adjacent gastric cancer tissues. DETAILED DESCRIPTION

[0039] Previous differential proteomics studies of the present invention revealed that FYB1 expression is higher in gastric cancer than in controls. However, its mechanism of action in gastric cancer remains unclear. The expression pattern and function of FYB1 in gastric cancer, as well as its relationship with the immune microenvironment, remain unclear. Therefore, exploring the immunoregulatory role of FYB1 in gastric cancer and whether it activates regulatory T cells (Tregs) to promote immune escape may provide a theoretical basis for uncovering new therapeutic targets.

[0040] This study aims to investigate the expression of FYB1 in gastric cancer tissue, the role of FYB1 in gastric cancer, and its related mechanisms. The study also examines the role of FYB1 in activating Treg cells and promoting immune escape in gastric cancer cells. This study explores the role of FYB1 in the development and progression of gastric cancer and its role in the immune microenvironment. This study further reveals the mechanism of action of FYB1 in gastric cancer and promotes the translation of FYB1 from basic research to clinical practice. In the future, combining intervention strategies targeting FYB1 (such as small molecule inhibitors or gene editing technologies) may provide new approaches to overcome immunotherapy resistance and improve patient prognosis. Furthermore, the results are expected to provide a scientific basis for the improvement of gastric cancer molecular classification systems and the development of personalized treatment plans.

[0041] The research method of the present invention comprises:

[0042] The TIMER2.0 online database was used to extensively explore the expression of FYB1 in pan-cancer and to find evidence related to FYB1 and gastric cancer.

[0043] Based on the TCGA database, the differential analysis and survival analysis of FYB1 in gastric cancer and the correlation between the gene expression level of FYB1 in gastric cancer tissues and the clinicopathological data of gastric cancer patients were studied.

[0044] Based on the TCGA database, the immune infiltration analysis of FYB1 in gastric cancer, the correlation analysis with immune cells, and the correlation analysis of the FYB1 gene were studied.

[0045] Immunohistochemistry was used to detect the expression of FYB1 in gastric cancer tissues and adjacent normal tissues.

[0046] Serial section immunohistochemistry was used to detect the localization of FYB1, CD4 and FOXP3 in gastric cancer tissues and adjacent normal tissues.

[0047] The co-localization of FYB1 and CD4+ T cells was investigated by immunofluorescence co-staining.

[0048] The co-localization of FYB1 and FOXP3+Treg cells was further explored by immunofluorescence co-staining.

[0049] The research results of the present invention are as follows:

[0050] Pan-cancer analysis of FYB1 expression was performed using the TIMER2.0 online database. The results showed that FYB1 was significantly overexpressed in cancer tissues such as esophageal cancer, glioblastoma, and renal cell carcinoma (P<0.05). The expression of FYB1 in gastric cancer tissue was higher than that in normal tissue, and the difference was statistically significant (P<0.05).

[0051] Based on the TCGA database, it was found that the expression of FYB1 in gastric cancer tissues was higher than that in normal tissues, and the difference in expression was statistically significant (P<0.05). The FYB1 high expression group in gastric cancer had a worse prognosis, and the survival difference was statistically significant compared with the low expression group (P<0.05).

[0052] Based on the TCGA database, the ESTIMATE immune score results showed that high FYB1 expression may be associated with a poor prognosis in gastric cancer and may promote tumor growth, invasion, and metastasis. High FYB1 expression is also associated with an increased proportion of immune cells and stromal cells in the tumor microenvironment. This confirms that high FYB1 expression is associated with the development and progression of gastric cancer and is associated with immune cell infiltration. FYB1 expression was significantly positively correlated with CD4+ T cells (P<0.05), confirming a positive correlation between FYB1 and the activation of regulatory T cells (Tregs).

[0053] Correlation analysis using the Person algorithm based on gastric cancer transcriptome data from the TCGA database showed that FYB1 expression was significantly positively correlated with FOXP3 and CD4 expression. Since FOXP3 is essential for the maturation and function of regulatory T cells (Tregs) and is currently the most sensitive marker for Tregs, this suggests that FYB1 is positively correlated with Treg cell differentiation, promoting Treg cell differentiation (P<0.05).

[0054] Immunohistochemistry was used to detect the expression of FYB1 in gastric cancer tissues and adjacent normal tissues. The results showed that the expression of FYB1 in gastric cancer tissues was significantly higher than that in adjacent normal tissues, indicating a significant difference between gastric cancer tissues and adjacent normal tissues (P<0.05).

[0055] Combined with the patients' clinical data, the correlation between the expression level of FYB1 in gastric cancer tissue and the clinical pathology of gastric cancer patients was further analyzed. The results showed that the expression level of FYB1 was related to T stage, N stage, and degree of differentiation, with statistical differences (P<0.05), but there was no significant difference with gender, age, and M stage.

[0056] The immunohistochemical staining results of serial sections showed that FYB1, CD4 and FOXP3 had common positive areas.

[0057] Immunofluorescence colocalization studies showed that FYB1 is expressed in gastric cancer tissues and co-expressed with CD4 on tissue-infiltrating T cells. FYB1 colocalizes with FOXP3+ Treg cells, suggesting that FYB1 and Treg cells play a direct or indirect synergistic role in promoting gastric cancer.

[0058] The above research results indicate that FYB1 can serve as a potential prognostic marker and play a key role in the prognosis of gastric cancer; in addition, FYB1 can promote the immune escape of gastric cancer tumor cells through the mechanism of Treg cells.

[0059] The following examples provide a detailed description of the technical solutions provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are all commercially available.

[0060] In the following examples, the reagents and preparation methods used include:

[0061] Preparation of FYB1 working solution for immunohistochemistry: Prepare FYB1 working solution at a dilution ratio of 1:150 by first adding 7,450 μL of antibody diluent and then 50 μL of FYB1 concentrate. Vortex to mix thoroughly and store in a refrigerator at 4°C. Use immediately.

[0062] Preparation of sodium citrate antigen repair solution: Prepare citric acid antigen repair solution with pH = 6.0: first add 990mL of distilled water, then add 10mL of citric acid tissue antigen repair solution, mix well and place at room temperature for later use.

[0063] Preparation of PBS phosphate buffer: first add 2,000 mL of distilled water, then add 1 bag of PBS phosphate buffer (powder), mix well and place at room temperature for later use.

[0064] Preparation of DAB colorimetric solution: In a small test tube, add 1 mL of stable DAB buffer, 50-100 μL of stable DAB substrate, and 50-100 μL of stable DAB chromogen to prepare DAB colorimetric solution. Store in a refrigerator at 4°C and it is valid for 24 hours.

[0065] Preparation of fluorescent antibody co-localization FYB1 working solution: Prepare fluorescent antibody co-localization FYB1 working solution at a dilution ratio of 1:150. First add 4,470 μL of antibody diluent, then add 30 μL of FYB1 concentrate, shake to mix, store in a refrigerator at 4°C, and use immediately.

[0066] Preparation of fluorescent antibody co-localization CD4 working solution: Prepare fluorescent antibody co-localization CD4 working solution at a dilution ratio of 1:150, add 4,470 μL of antibody diluent, then add 30 μL of FYB1 concentrate, shake to mix, store in a refrigerator at 4°C, and use immediately.

[0067] Preparation of fluorescent antibody co-localization FOXP3 working solution: Prepare fluorescent antibody co-localization FOXP3 working solution at a dilution ratio of 1:150, add 4,470 μL of antibody diluent, then add 30 μL of FOXP3 concentrate, shake to mix, store in a refrigerator at 4°C, and use immediately.

[0068] Preparation of TSA-520Plus fluorescent dye reaction solution: Prepare TSA-520Plus fluorescent dye solution at a dilution ratio of 1:200, add 4,975μL of TSA buffer, and then add 25μL of concentrated TSA-520Plus fluorescent dye. Mix well and place in the dark. Use immediately after preparation.

[0069] Preparation of TSA-570Plus fluorescent dye reaction solution: Prepare TSA-520Plus fluorescent dye solution at a dilution ratio of 1:200, add 4,975μL of TSA buffer, and then add 25μL of concentrated TSA-520Plus fluorescent dye. Mix well and place in the dark. Use immediately after preparation.

[0070] Preparation of TSA-690Plus fluorescent dye reaction solution: Prepare TSA-690Plus fluorescent dye solution at a dilution ratio of 1:200. Add 4,975μL of TSA buffer and 25μL of concentrated TSA-690Plus fluorescent dye. Mix well and store in a dark place. Use immediately after preparation.

[0071] In the following examples, the experimental tissues used include:

[0072] Immunohistochemistry: 90 gastric cancer tissue specimens were obtained from patients undergoing radical surgery at Wuxi Xishan People's Hospital between January 1, 2020, and December 31, 2024. None of the patients received preoperative chemotherapy or radiotherapy, and postoperative pathological diagnosis of gastric cancer was confirmed. Complete clinical and pathological data were available for all specimens. The gastric cancer patients included 61 males and 29 females, ranging in age from 36 to 89 years. Fifty-one of the patients had lymph node metastasis, 39 did not, and 4 had distant metastasis. Differentiation: 31 cases were well- or moderately differentiated adenocarcinoma, and 59 were poorly differentiated. Sixty normal mucosal tissue samples located more than 5 cm from the tumor margin were used as controls. All specimens used in this study were routinely paraffin-embedded and serially sectioned at 2 mm thickness. A corresponding HE section was obtained for each tissue sample for diagnostic review. The patient's medical records were retrieved using the patient name and hospitalization number provided by the Medical Records Management Office of Wuxi Xishan People's Hospital, and clinical information for each patient was compiled.

[0073] Immunofluorescence colocalization tissues: Five gastric cancer tissue specimens were obtained from patients admitted to Wuxi Xishan People's Hospital between June 1, 2024, and December 30, 2024. Gastric cancer specimens obtained during surgery were formalin-fixed, paraffin-embedded, and then prepared into wax blocks for immunofluorescence colocalization. Three normal mucosal tissues located more than 5 cm from the tumor margin were also collected as controls. None of the patients had received prior chemotherapy or radiotherapy.

[0074] In the following examples, the experimental serum used includes:

[0075] Serum samples were collected from 48 gastric cancer patients admitted to Wuxi Xishan People's Hospital between May 1, 2024, and September 30, 2024. All patients had gastric cancer confirmed by endoscopic tissue removal or postoperative pathological diagnosis. Serum samples were also collected from 16 healthy patients undergoing physical examinations as a control group. Serum samples were collected by centrifugation at 3000 rpm for 10 minutes. The supernatant was removed using a micropipette, aliquoted, and stored at -80°C to avoid repeated freezing and thawing.

[0076] In the following examples, the experimental data sources are as follows:

[0077] All data used in the online analysis platform in this study were from The Cancer Genome Atlas (TCGA), including transcriptome data and clinical information. The samples used by each platform vary. The datasets used for single-gene and immune infiltration analysis were raw transcriptome sequencing data from 446 gastric adenocarcinoma patients downloaded from the TCGA database, including 410 tumor samples and 36 adjacent adjacent samples. The data used for analysis of clinically relevant feature data were the clinical data corresponding to the 446 gastric adenocarcinoma patients downloaded previously. The TCGA project is a landmark cancer genomics project managed by the National Cancer Institute and the National Human Genome Research Institute of the U.S. government and is the largest online database for cancer genomics. The pan-cancer analysis in this study used the online analysis platform Tumor Immune Estimation Resource 2.0 (TIMER2.0) to perform TCGA data mining and analysis.

[0078] In the following examples, the inclusion and exclusion criteria are as follows:

[0079] Inclusion criteria: (1) Patients who underwent subtotal gastrectomy or total gastrectomy with lymph node dissection for gastric cancer during hospitalization and were diagnosed with gastric cancer by postoperative pathological examination; (2) Patients with primary gastric cancer; (3) Patients with complete clinical pathological data; (4) Patients who had not received radiotherapy, chemotherapy, targeted therapy, or immunotherapy before surgery.

[0080] Exclusion criteria: (1) Patients with other malignant tumors; (2) Patients with a family history of gastric cancer; (3) Patients with serious underlying medical diseases and long-term medication; (4) Patients and their families who do not give informed consent; (5) Patients with immune deficiency and autoimmune diseases.

[0081] In addition, the collection of the experimental specimens mentioned above was approved by the Clinical Research Ethics Committee of Xishan People's Hospital, with the ethics approval number: xs2024ky027.

[0082] Example 1

[0083] This example provides experimental methods for immunohistochemistry, immunofluorescence co-localization, bioinformatics analysis, and statistical analysis.

[0084] (I) Immunohistochemistry.

[0085] 1) Use a microtome to make serial sections with a thickness of 2 mm.

[0086] 2) Bake the slices in a 68°C oven for 30 minutes.

[0087] 3) Dewax the sections in xylene (xylene I for 10 min, xylene II for 5 min) and hydrate with graded alcohol (100% anhydrous ethanol for 3 min, 95% ethanol for 3 min, 75% ethanol for 3 min, and 50% ethanol for 3 min). Rinse the sections with distilled water and soak them in distilled water for 5 min.

[0088] 4) Add water to a pressure cooker and prepare citrate buffer (pH 6.0) by mixing 10 mL of citric acid with 1,000 mL of distilled water. Bring to a boil.

[0089] 5) Place slides in a stainless steel or heat-resistant plastic slide rack in boiling buffer. Close the pressure valve and cook for 8 minutes. Turn off the power and simmer for 7 minutes. Place in a cold water bath at room temperature. After cooling to room temperature, rinse the slides with clean water, place in a wet chamber, and rinse twice with PBS.

[0090] 6) Shake off the PBS, wipe off the water marks around the specimen, add 1 drop of Boster FYB1 primary antibody, incubate at room temperature for 1 hour, and rinse with PBS 3 times.

[0091] 7) Shake off the PBS, wipe off the water marks around the specimen, add one drop of reagent R1 (polymer enhancer), incubate for 20 minutes, and rinse three times with PBS.

[0092] 8) Shake off the PBS, wipe off the water marks around the specimen, add one drop of reagent R2 (enzyme-labeled anti-mouse / rabbit polymer), incubate for 30 minutes, and rinse three times with PBS.

[0093] 9) Shake off PBS, wipe off water marks around the specimen, add DAB color developer (1 ml of solution A, 1 drop of solution B, 1 drop of solution C), and allow color to develop within 10 minutes. When the color is significantly darkened by the naked eye, rinse with distilled water.

[0094] 10) The sections were counterstained with hematoxylin for 1 min, rinsed with distilled water, differentiated with 0.5% hydrochloric acid-alcohol for 1 second, bluing with running water for 10 min, and dehydrated with graded alcohols (95% ethanol for 3 min, 100% anhydrous ethanol I for 3 min, and 100% anhydrous ethanol II for 3 min).

[0095] 11) After drying with a hair dryer, seal the slide with neutral resin.

[0096] 12) Result Assessment: Immunohistochemistry results were independently evaluated and recorded by two pathologists using a blinded method. Each sample was scored for staining intensity and cell positivity. The criteria for positive expression were: 1. Percentage of positive cells: <5% = 0, 5%-25% = 1, 26%-50% = 2, 51%-75% = 3, >75% = 4; 2. Staining intensity: colorless = 0, light yellow = 1, brownish yellow = 2, and brownish brown = 3. The final score was the staining intensity score for each sample multiplied by the percentage of positive cells. Scores of 0-4 indicate low expression, 6-8 indicate moderate expression, and greater than 8 indicate high expression.

[0097] Notes: 1) The colorimetric reagent must be prepared and used immediately and within 30 minutes of preparation. 2) Protect from light during the colorimetric development process. 3) Serial sections should be analyzed for FYB1, CD4, and FOXP3 colocalization using the same procedures as above.

[0098] (B) Immunofluorescence colocalization.

[0099] 1) Use a microtome to make serial sections of the wax block at a thickness of 2 mm.

[0100] 2) Paraffin sections were routinely dewaxed to hydrate: oven-bake the sections at 68°C for 30 minutes. Sections were then dewaxed in xylene (xylene I for 10 minutes, xylene II for 5 minutes) and hydrated with graded alcohols (100% anhydrous ethanol for 3 minutes, 95% ethanol for 3 minutes, 75% ethanol for 3 minutes, and 50% ethanol for 3 minutes). The sections were rinsed with distilled water and then immersed in distilled water for 5 minutes.

[0101] 3) Incubate in 3% H2O2 deionized water at room temperature for 5-10 minutes to eliminate endogenous peroxidase activity, then rinse with PBS for 5 minutes x 3 times.

[0102] 4) Prepare EDTA antigen retrieval solution (pH 9.0) by dissolving EDTA antigen retrieval solution powder in distilled water to 2 L. Immerse the sections in the EDTA retrieval solution and microwave until boiling. Remove the microwave and repeat retrieval 1-2 times every 5-10 minutes before cooling to room temperature.

[0103] 5) After the slices are dried, draw a circle around the tissue with an immunohistochemistry pen, add 5% BSA blocking solution and incubate at 37°C for 30 minutes. Dried, do not wash.

[0104] 6) Add FYB1 primary antibody at a dilution of 1:150 and incubate at 37°C for 1.5 h. Rinse with PBS for 5 min three times.

[0105] 7) Add HRP goat anti-rabbit / mouse IgG secondary antibody and incubate at 37°C for 30 min. Rinse with PBS for 5 min x 3 times.

[0106] 8) Dilute the concentrated fluorescent dye TSA-520 Plus with TSA buffer at a ratio of 1:200. Add the corresponding TSA fluorescent dye reaction solution to the circle and incubate at room temperature in the dark for 1-15 minutes. Rinse with PBS for 5 minutes x 3 times.

[0107] 9) Immerse the sections in antigen retrieval solution in a 37°C water bath for 25-40 minutes. Rinse with PBS for 5 minutes three times.

[0108] 10) After the slices are dried, draw a circle around the tissue with an immunohistochemistry pen, add 5% BSA blocking solution, incubate at 37°C for 30 minutes, and then dry. Do not wash.

[0109] 11) Add CD4 primary antibody at a dilution of 1:150 and incubate at 37°C for 1.5 h. Rinse with PBS for 5 min x 3 times.

[0110] 12) Add HRP goat anti-rabbit / mouse IgG secondary antibody and incubate at 37°C for 30 min. Rinse with PBS for 5 min x 3 times.

[0111] 13) Dilute the concentrated fluorescent dye TSA-690 Plus with TSA buffer at a ratio of 1:200. Add the corresponding TSA fluorescent dye reaction solution to the circle and incubate at room temperature in the dark for 1-15 minutes. Rinse with PBS for 5 minutes three times.

[0112] 14) Add DAPI staining solution and incubate at room temperature for 5-10 minutes. Rinse with PBS for 5 minutes x 3 times.

[0113] 15) After the sections are dried, they are mounted with anti-fluorescence attenuation mounting medium.

[0114] 16) Confocal microscopy observation: FYB1 excitation wavelength is 490 nm, emission wavelength is 520 nm; CD4 excitation wavelength is 630 nm, emission wavelength is 690 nm; FOXP3 excitation wavelength is 630 nm, emission wavelength is 690 nm.

[0115] Note: 1) Select the antigen retrieval method and intensity as needed. Heat retrieval, enzyme retrieval, or no retrieval are acceptable. 2) Keep sections moist throughout the entire process. 3) Immunofluorescence co-staining of FYB1 and FOXP3 is performed using the same method as above.

[0116] (3) Bioinformatics analysis

[0117] 1) Online database analysis.

[0118] Pan-cancer analysis of FYB1 (FYB) was performed using the "Gene" module of the TIMER2.0 online analysis database to evaluate the expression of FYB1 in different tumors.

[0119] 2) Transcriptome bioinformatics credits.

[0120] The gastric cancer transcriptome data and clinical information downloaded from the TCGA database were collated using R software (version 4.4.1). The downloaded gastric cancer transcriptome data were differentially analyzed using the "limma" package in the R package, and the differential expression of FYB1 in gastric cancer was analyzed. Survival information from the clinical data of gastric cancer patients was collated, and the optimal cutoff value of FYB1 expression was found to be 25.3 using the x-tile software. Survival analysis was performed using the optimal cutoff value of 25.3 using the "survival" package in the R package, and the data were visualized using the "survminer" package in the R package. .2 version) to organize the clinical-related characteristic data of gastric cancer patients downloaded from the TCGA database and match them with the transcriptome data for clinical correlation analysis. The correlation between FYB1 expression levels and these clinical data was analyzed. The ESTIMATE immune score was performed using the "limma" and "estimate" packages in the R package to analyze the relationship between FYB1 and tumor immune-infiltrating cells. Based on the gastric cancer transcriptome data from the TCGA database, the "limma", "ggplot2", "ggpubr", and "ggExtra" packages in the R package were used to perform correlation analysis based on the person algorithm and visualize the data.

[0121] (4) Statistical analysis

[0122] The collected data were organized using Excel 2021 software, and the experimental results were statistically analyzed and visualized using SPSS29, GraphPad Prism10, R4.4.1, coloc 2 and other software. The measurement data were expressed as mean ± standard deviation, and the count data and rank data were expressed as frequency and percentage. The significance analysis of the count data between the two groups was performed using t-test, and the analysis of variance was performed using χ2 test for the comparison of multiple groups. 2 The correlation between FYB1 and clinical factors (such as patient gender, age, tumor size, invasion depth, lymph node metastasis, and pathological stage) was analyzed using the Kaplan-Meier method and the log-rank test. A P value < 0.05 was considered statistically significant.

[0123] The experimental results are as follows:

[0124] (1) Bioinformatics analysis results

[0125] (1) Pan-cancer analysis results of FYB1 based on the online database TIMER2.0.

[0126] A pan-cancer analysis of FYB1 (FYB) expression was performed using the TIMER2.0 online database. The results showed that FYB1 was significantly overexpressed in esophageal cancer, glioblastoma, renal cell carcinoma and other cancer tissues (P<0.05), while the expression of FYB1 in gastric cancer tissue was higher than that in normal tissue, and the difference in expression was statistically significant (P<0.05). Figure 1 )

[0127] (2) Bioinformatics analysis results based on the TCGA database.

[0128] 1) Expression of FYB1 in gastric cancer and prognostic analysis results.

[0129] By performing differential analysis on the downloaded gastric cancer transcriptome data, we found that the expression of FYB1 in gastric cancer tissues was higher than that in normal tissues, and this difference in expression was statistically significant (P<0.05) ( Figure 2 Figure A). The optimal cutoff value of FYB1 expression for survival analysis was found to be 25.3 using x-tile. Survival was then used to perform survival analysis using the "survival" package in the R package. The data were visualized using the "survminer" package in the R package. It was found that the high-expression group of FYB1 in gastric cancer had a worse prognosis, and the survival difference was statistically significant compared with the low-expression group (P<0.05). ( Figure 2 (Figure B).

[0130] 2) Analysis of the expression level and clinical correlation of FYB1 in gastric cancer.

[0131] The clinical information of gastric cancer patients downloaded from the TCGA database was matched with the transcriptome data and the results of clinical correlation analysis were analyzed. It was found that the expression of FYB1 was significantly correlated with T stage (P<0.05) ( Figure 3 Figure A in the middle), and in the T stage, the expression of FYB1 gradually increased with the increase of stage, among which the expression differences between T1 and T2, T1 and T3, T1 and T4, T2 and T4, and T3 and T4 were statistically significant (P<0.05) ( Figure 3 (Figure B).

[0132] 3) Immune score analysis results of FYB1 in gastric cancer.

[0133] The results of the ESTIMATE immune score show that high expression of FYB1 may be associated with a poor prognosis in gastric cancer and may promote tumor growth, invasion, and metastasis; high expression of FYB1 is also accompanied by an increase in the proportion of immune cells and stromal cells in the tumor microenvironment. It was found that high expression of FYB1 is associated with the occurrence and development of gastric cancer and is related to the infiltration of immune cells ( Figure 4 ). Previous studies by Sarah M. et al. have confirmed that the interaction between Treg cells and the matrix plays a very important role in tumor immune escape. Treg cell differentiation can promote matrix infiltration through mechanisms such as chemokine-mediated recruitment and the formation of an immunosuppressive microenvironment; at the same time, stromal cells such as tumor-associated fibroblasts (CAFs) can secrete cytokines such as TGF-β to promote the differentiation and infiltration of Treg cells. In addition, CAFs can also provide physical channels for the infiltration of Treg cells by regulating the composition of the extracellular matrix, while restricting the entry of effector T cells; matrix infiltration can also affect Treg cell differentiation through the induction of the tumor microenvironment, spatial distribution, and functional differentiation. For example, the TGF-β signaling pathway can transform Th1 cells into Treg cells with immunosuppressive functions in the tumor microenvironment. Therefore, the results of the immune score are also related to the prognosis of the tumor.

[0134] 4) Correlation analysis results of FYB1.

[0135] The results of the Person algorithm correlation analysis based on gastric cancer transcriptome data from the TCGA database showed that the expression of FYB1 was significantly positively correlated with the expression of FOXP3 and CD4. Since FOXP3 is an essential factor for the development and function of regulatory T cells (Treg) and is currently the most sensitive marker of Treg cells, it was verified that FYB1 was positively correlated with the activation of Treg cells (P<0.05) ( Figure 5 ). FYB1 may be related to the activation of Treg cells.

[0136] (2) Analysis of immunohistochemical results of FYB1 protein and clinical information.

[0137] (1) Immunohistochemical expression of FYB1 protein in gastric cancer tissues and normal tissues.

[0138] To verify the bioinformatics analysis results: whether FYB1 is highly expressed in gastric cancer is correct, we used immunohistochemistry to detect FYB1 protein in 90 gastric cancer tissues and 60 adjacent normal tissues. The immunohistochemical staining results are as follows: Figure 6As shown, we first statistically analyzed 60 pairs of gastric cancer tissues and adjacent tissues and found that the expression level of FYB1 in gastric cancer was also higher than that in adjacent tissues (P<0.05). Subsequently, we statistically analyzed all histochemical results and found that FYB1 was significantly overexpressed in gastric cancer tissues (P<0.05) ( Figure 7 ). It can be concluded that FYB1 is highly expressed in gastric cancer.

[0139] (2) The relationship between FYB1 expression level and clinical pathological characteristics.

[0140] To further explore the clinical significance of FYB1 in gastric cancer, we collected data on age, sex, degree of differentiation, depth of invasion, and the presence of lymph node metastasis from 90 gastric cancer patients based on the clinicopathological data of each gastric cancer specimen. FYB1 expression was then categorized into low- and high-expression groups based on the median of the corresponding immunohistochemical scores. We investigated whether FYB1 expression levels were correlated with these clinical characteristics, using a chi-square test for statistical significance. The results showed that FYB1 expression levels were statistically significantly associated with T stage, N stage, and degree of differentiation (P < 0.05), but not with sex, age, or M stage (Table 1).

[0141] Table 1. Correlation analysis between FYB1 expression and clinical characteristics.

[0142]

[0143]

[0144] (3) Correlation analysis between FYB1 expression level and some blood immune markers.

[0145] Based on the patients' untreated blood routine within one week of diagnosis, the patients' NLR, PLR, LMR, NPR, PAR, CAR, CLR, SII, SIRI, and CALLY indices were calculated to explore whether the FYB1 tissue expression level (divided into two groups, high and low, according to the median of the immunohistochemistry score) was correlated with the above-mentioned inflammatory indicators (serological related concentrations could not be used due to detection technical problems). The Pearson correlation coefficient was calculated using the pandas and numpy libraries. The results showed that the correlation coefficient between the FYB1 grouping and NLR (neutrophil-to-lymphocyte ratio) was 0.10, indicating a weak positive correlation. The correlation coefficient between the FYB1 grouping and PLR (platelet-to-lymphocyte ratio) was 0.13, indicating a weak positive correlation. The correlation coefficient between the FYB1 grouping and NPR (neutrophil-to-monocyte ratio) was 0.13, indicating a weak positive correlation. The correlation coefficient between the FYB1 grouping and CAR (CRP-to-albumin ratio) was 0.12, indicating a weak positive correlation. The correlation coefficient between the FYB1 grouping and CLR (CRP-to-lymphocyte ratio) was 0.13, indicating a weak positive correlation. The correlation coefficient was 0.06, indicating a weak correlation. The FYB1 grouping and SII (systemic immune inflammatory index) had a correlation coefficient of 0.22, indicating a moderate positive correlation. The FYB1 grouping and LMR (lymphocyte-to-monocyte ratio) had a correlation coefficient of -0.31, indicating a moderate negative correlation. The FYB1 grouping and CLR (CRP-to-lymphocyte ratio) had a correlation coefficient of 0.17, indicating a weak positive correlation. The FYB1 grouping and SIRI (systemic immune inflammatory index) had a correlation coefficient of 0.17, indicating a weak positive correlation. The FYB1 grouping and CALLY index had a correlation coefficient of 0.19, indicating a weak positive correlation (see Table 3-2). In summary, FYB1 had varying degrees of positive correlation with all validation indicators except LMR, and a moderate positive correlation with SII, suggesting that FYB1 has a certain positive correlation with inflammatory response.

[0146] Table 2. Correlation analysis between FYB1 expression and inflammatory indicators.

[0147]

[0148] (3) Verification of the correlation between FYB1 and Treg cells.

[0149] (1) Localization results of FYB1, CD4 and FOXP3 in serial sections.

[0150] In order to explore whether there is a positive correlation between FYB1 and Treg cells, the gastric cancer tissue paraffin blocks of the same patient were serially sectioned and immunohistochemically stained. The staining results are shown in Figure 2. Figure 8Based on this, we can find that FYB1 has positive expression in the same area with CD4 and FOXP3, and FYB1 has common localization with Treg cells, but whether the two have a synergistic effect and whether FYB1 can activate Treg cells still needs further verification.

[0151] (2) Immunofluorescence co-localization results of FYB1 protein and Treg cells in gastric cancer patients.

[0152] To explore whether FYB1 has a synergistic effect with Treg cells, we first used fluorescence immunofluorescence colocalization to find that FYB1 was expressed in the cytoplasm and infiltrating cells of gastric cancer cells ( Figure 9 ), the fluorescence signals of FYB1 and CD4 have a high overlap rate. The results of laser confocal microscopy analysis fully show that FYB1 and CD4 are closely related in gastric cancer tissue ( Figure 11 Middle A) and adjacent tissues ( Figure 12 ) are co-expressed in gastric cancer. Based on this, we can infer that FYB1 and T cells play a synergistic role in promoting cancer in gastric cancer, either directly or indirectly. CD4 is only a marker in Treg cells. To further clarify whether there is a synergistic effect between FYB1 and Treg cells, we then performed immunofluorescence co-localization of FYB1 and FOXP3, the most sensitive marker of Treg, and found that FOXP3 is expressed in the nucleus of T cells ( Figure 10 (Figure A in the middle section) Through fluorescence immunofluorescence colocalization, we found a high overlap between the fluorescence signals of FYB1 and FOXP3. This preliminarily confirms the hypothesis that FYB1 and Treg cells play a synergistic role in promoting gastric cancer. We will further verify how FYB1 activates Treg cells to promote immune evasion of gastric cancer cells in subsequent in vitro and in vivo studies.

[0153] (3) Analysis of immunofluorescence colocalization results.

[0154] 1) Analysis of co-localization results in gastric cancer tissues.

[0155] First, the colocalization analysis of FYB1 and CD4 in gastric cancer tissues was performed: coloc2 software was used for immune colocalization analysis, and bisection threshold regression (Bisection) was used to analyze the Pearson correlation coefficient: the Pearson correlation coefficient without threshold was 0.67, indicating that there was a strong positive correlation between the two channels. The Pearson correlation coefficient above the threshold was 0.40, indicating that there was a moderate positive correlation between the pixels above the threshold. Li's ICQ value: 0.374. It showed a moderate degree of colocalization. Spearman rank correlation coefficient: 0.71844681, indicating that there was a strong positive correlation between the two. Kendall's Tau-b rank correlation coefficient: 0.6399, indicating that there was a strong positive correlation between the two channels. The Costes randomized mean was 0.00 and the standard deviation was 0.01, indicating that there was a correlation. Then, the co-localization analysis of FYB1 and FOXP3 in gastric cancer tissues was performed: in the data above the threshold, the Pearson correlation coefficient was 0.73, indicating that there was a strong positive linear correlation between the two variables in these data. FYB1 and FOXP3 are correlated, and FYB1 co-localizes with Treg cells in gastric cancer tissues. Since FYB1 is located in the cytoplasm and infiltrating cells, and FOXP3 is located in the nucleus of T cells, although the two are located on the same cell, due to their different positions in the same cell, two-dimensional analysis shows weak co-localization. The results are shown in Figure 11 .

[0156] 2) Analysis of the co-localization results of FYB1 and CD4 in adjacent cancer tissues.

[0157] We then analyzed the colocalization results of FYB1 and CD4 in adjacent adjacent tissues. Immunolocalization analysis was performed using coloc 2 software, and bisection threshold regression was used to analyze the Pearson correlation coefficient. The Pearson's R value above the threshold was 0.14, indicating a weak positive correlation between the two channels in pixels above the threshold. The Kendall's Tau-b rank correlation value was 0.4264, indicating a moderate positive correlation between the two channels. A Costes P value of 1.00 indicated that no colocalization was observed in the randomized images that was stronger than that observed in the actual images, thus indicating that colocalization was not significant. The Costes randomized mean was 0.00, indicating that the mean value of colocalization in the randomized images was 0, and the Costes randomized standard deviation was 0.01, indicating that the variability of colocalization in the randomized images was minimal. The proportion of randomized Pearson's R values greater than or equal to the actual Pearson's R value was 0.00, indicating that no colocalization was observed in the randomized images that was stronger than that observed in the actual images. In summary, there is a weak correlation between FYB1 and CD4 in adjacent tissues. Figure 12 .

[0158] The above-described embodiments merely illustrate several implementation methods of the present invention, and are provided to facilitate a specific and detailed understanding of the technical solutions of the present invention. They should not be construed as limiting the scope of the invention patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the scope of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. Application of FYB1 gene and expressed protein as markers in the preparation of diagnostic or prognostic reagents for gastric cancer.

2. The application according to claim 1, characterized in that The expression of the FYB1 gene in gastric cancer tissue is higher than that in normal tissue; and / or, the prognosis and survival statistics of the gastric cancer patients in the high-expression group of the FYB1 gene are worse than those in the low-expression group, and it serves as a predictive marker for gastric cancer immune escape and poor prognosis in pathological immunohistochemistry.

3. Application of the FYB1 gene and its expressed protein as markers in the preparation of products for evaluating the efficacy of anti-gastric cancer drugs, wherein the products are reagents, kits or devices.

4. Application of FYB1 gene inhibitors in the preparation of anti-gastric cancer drugs.

5. The use according to claim 4, characterized in that The FYB1 gene inhibitor is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies.

6. The application according to claim 4, characterized in that: The anti-gastric cancer drug can inhibit the proliferation of gastric cancer cells.

7. The use according to claim 4, characterized in that The anti-gastric cancer drug can inhibit the growth, invasion and metastasis of gastric cancer cells.

8. An anti-gastric cancer drug, characterized in that: Including FYB1 gene inhibitors.

9. The anti-gastric cancer drug according to claim 8, characterized in that: Also included is a targeting agent that can target gastric cancer cells or gastric cancer tissues.

10. The anti-gastric cancer drug according to claim 8 or 9, characterized in that: Also included is a delivery vector capable of delivering the FYB1 gene inhibitor to gastric cancer cells or gastric cancer tissues.

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