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

Abstract

The present application relates to the medical technical field, especially to the application of FYB1 gene as a marker in preparation of gastric cancer diagnosis or prognosis judging reagent. The present application research finds that FYB1 gene is highly expressed in gastric cancer tissue and negatively correlated with the prognosis of patients, and can be used as a gastric cancer marker for gastric cancer detection and efficacy evaluation. In addition, FYB1 is positively correlated with the expression of Treg cell marker FOXP3 and co-localized in tissues, suggesting that FYB1 may promote gastric cancer tumor immune escape; the expression of FYB1 gene can significantly inhibit the proliferation, migration and invasion ability of gastric cancer cells, and inhibit tumor growth, indicating that FYB1 gene can be used as a gastric cancer treatment target and play an important role in the treatment of gastric cancer patients.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to a... FYB1 Application of genes as biomarkers in the preparation of reagents for gastric cancer diagnosis or prognosis. Background Technology

[0002] In recent years, gastric cancer remains one of the most common malignant tumors of the digestive tract worldwide. Currently, the main treatments for gastric cancer include surgery and chemotherapy. However, due to the low early diagnosis rate, many patients are already in advanced or locally advanced stages, or even have distant metastases, when they seek medical attention, making traditional treatments ineffective. Although immunotherapy research has made many breakthroughs in recent years, its effectiveness in treating gastric cancer remains unsatisfactory. Therefore, finding new biomolecular markers or targets for immunotherapy to improve clinical efficacy and survival rates for gastric cancer patients is crucial.

[0003] FYN binding protein 1 (FYB1), as an adaptor protein, plays an important role in T cell activation, immune synapse formation, and signal transduction. Recent studies suggest that... FYB1 Genes may participate in tumor progression by regulating processes such as cell migration and proliferation. FYB1 The encoded protein is a linker protein in the signaling cascade between FYN protein in T cells and lymphocyte cytosolic protein 2 (LCP2). It is essential for T cell activation, and its influence on tumor pathogenesis and metastasis has been gradually studied in recent years. It has now been confirmed to be related to the pathogenesis and progression of tumors such as breast cancer and lung adenocarcinoma. FYB1 There are few studies on its correlation with gastric cancer, and its mechanism of action in gastric cancer, such as expression patterns, function, and its relationship with the immune microenvironment, has not been reported.

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

[0005] To solve the above-mentioned technical problems, the present invention provides a FYB1 Application of genes as biomarkers in the preparation of reagents for gastric cancer diagnosis or prognosis.

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

[0007] In a first aspect, the present invention provides FYB1 Application of genes and expressed proteins as biomarkers in the preparation of reagents for gastric cancer diagnosis or prognosis.

[0008] Preferably, FYB1Gene expression is higher in gastric cancer tissues than in normal tissues; and / or, in gastric cancer patients FYB1 The high gene expression group had worse prognostic and survival statistics than the low expression group, and it is used in pathological immunohistochemistry as a predictive marker of immune escape and poor prognosis in gastric cancer.

[0009] Secondly, the present invention provides FYB1 The application of genes and expressed proteins as biomarkers in the preparation of products for evaluating the efficacy of anti-gastric cancer drugs, wherein the products are reagents, kits, or devices.

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

[0011] The detection module is used for detection. FYB1 The content of genes in the sample to be tested;

[0012] The input module is used to obtain the detection results from the detection module;

[0013] The judgment module is used to compare the detection results obtained by the input module with the detection results of samples from non-gastric cancer individuals to determine whether the individual has gastric cancer or whether the gastric cancer has been cured.

[0014] The output module is used to output the diagnostic results.

[0015] Preferably, the judgment criteria of the judgment module include: if the sample to be tested contains... FYB1 If the gene content is significantly higher than that of samples from individuals without gastric cancer, then the individual from whom the sample was taken is determined to have gastric cancer or gastric cancer that has not been cured.

[0016] Thirdly, the present invention provides FYB1 Application of gene inhibitors in the preparation of anti-gastric cancer drugs.

[0017] This invention has found that by inhibiting FYB1 Gene expression can significantly inhibit the proliferation, migration, and invasion of gastric cancer cells, thus suppressing tumor growth, indicating that... FYB1 Genes can serve as therapeutic targets for gastric cancer. This invention will... FYB1 Genes, as targets for the design of drugs for the treatment of gastric cancer, have significant clinical translational and application value.

[0018] The present invention relates to the above. FYB1 The specific type and source of gene inhibitors are not particularly limited; those conventionally selected in this field that comply with relevant drug market regulations are acceptable. FYB1 Gene inhibitors are all acceptable. Preferably, the... FYB1Gene inhibitors are 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] Fourthly, the present invention provides an anti-gastric cancer drug, comprising: FYB1 Gene inhibitors.

[0022] Preferably, the anti-gastric cancer drug further includes a targeted agent that can target gastric cancer cells or gastric cancer tissue. This invention does not particularly limit the specific type and source of the targeted agent; any targeted agent conventionally selected in the art that conforms to relevant drug market regulations is acceptable.

[0023] Preferably, the anti-gastric cancer drug further includes a delivery carrier, the delivery carrier being capable of delivering... FYB1 Gene inhibitors are delivered to gastric cancer cells or gastric cancer tissue. This invention does not specifically limit the type or source of the delivery vector; any delivery vector conventionally selected in the art that conforms to relevant pharmaceutical market regulations is acceptable. For example, nucleic acid lipid nanoparticles (LNPs) can be selected as the delivery vector.

[0024] Beneficial effects:

[0025] This invention provides FYB1 The application of genes as biomarkers in the preparation of reagents for gastric cancer diagnosis or prognosis. This invention has discovered... FYB1 The gene is highly expressed in gastric cancer tissue and is negatively correlated with patient prognosis, and can be used as a gastric cancer biomarker for gastric cancer detection and treatment evaluation. Furthermore, by inhibiting... FYB1 Gene expression can significantly inhibit the proliferation, migration, and invasion of gastric cancer cells, thus suppressing tumor growth, indicating that... FYB1 Genes can serve as therapeutic targets for gastric cancer and play an important role in the treatment of gastric cancer patients. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

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

[0028] Figure 2 for FYB1 Differences in expression between gastric cancer and cancerous tissues, and results of survival analysis based on the TCGA database. Figure 2 Figure A is based on the TCGA database. FYB1 Differences in expression levels between gastric cancer tissue (Tumor = 410) and normal tissue (Normal = 36) P <0.05); Figure 2 Figure B in the middle is based on transcriptome and survival data from the TCGA database. FYB1 Survival analysis between high and low expression groups in gastric cancer ( P <0.05).

[0029] Figure 3 for FYB1 Results of clinical relevance analysis. Figure 3 Figure A shows gastric cancer patients based on the TCGA database. FYB1 Heatmap of expression level and clinical correlation analysis ( P <0.05); Figure 3 Figure B is FYB1 Box plot of correlation analysis between expression level and T stage ( P <0.05).

[0030] Figure 4 for FYB1 ESTIMATE immune score ( P <0.05, P <0.01, P <0.001).

[0031] Figure 5 for FYB1 Scatter plot of correlation analysis. Figure 5 Figure A is FYB1 and FOXP3 Correlation analysis scatter plot ( P <0.05); Figure 5 Figure B is FYB1 and CD4 Correlation analysis scatter plot ( P <0.05).

[0032] Figure 6 The results are the immunohistochemical staining of FYB1 protein. Figure 6 Figure A shows the immunohistochemical staining results of FYB1 in gastric cancer tissue. It is mainly located in the cytoplasm of cells, and is positive in gastric cancer and adjacent infiltrating cells (scale bar: 2 mm, × 4). Figure 6 Image B in the middle shows the results of FYB1 immunohistochemical staining in gastric cancer tissue after magnification (scale bar: 50 μm, × 20). Figure 6 The middle image (C) shows the immunohistochemical staining results of FYB1 in adjacent normal tissue (scale bar: 2 mm, × 4). Figure 6 The middle D figure shows the magnified immunohistochemical staining results of FYB1 in adjacent normal tissue (scale bar: 50 μm, × 20).

[0033] Figure 7 Immunohistochemical statistical analysis of FYB1 in gastric cancer. Figure 7 Figure A shows the statistical analysis results of FYB1 expression levels in 60 paired gastric cancer and adjacent non-cancerous tissues. P <0.05); Figure 7 Figure B shows the results of a comparative analysis of FYB1 expression levels in cancerous and adjacent tissues. P <0.0001).

[0034] Figure 8 This image shows the localization of FYB1, CD4, and FOXP3 in serially sliced ​​gastric cancer tissue via immunohistochemistry. Figure 8 Image A: The left image shows the immunohistochemical staining results of CD4 in gastric cancer tissue, mainly located 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 Image B: The left image shows the immunohistochemical staining results of FOXP3 in gastric cancer tissue, mainly located 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 Image C: The left image shows the immunohistochemical staining results of FYB1 in gastric cancer tissue, mainly located in the cytoplasm (scale bar: 5 mm, × 4). The right image shows the magnified immunohistochemical staining results of FYB1 in gastric cancer tissue (scale bar: 100 μm, × 20).

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

[0036] Figure 10 This shows the immune co-localization expression of FYB1 and FOXP3. Figure 10 Figure A shows the co-localization of FYB1 (green) and FOXP3 (pink) in gastric cancer tissue using immunofluorescence. Figure 10 Figure B shows the co-localization of FYB1 (green) and FOXP3 (pink) immunofluorescence in adjacent normal tissue (scale bar: 20 μm, ×20).

[0037] Figure 11 This is a diagram showing the results of immune colocalization analysis of gastric cancer tissue. Figure 11 Figure A shows the analysis of the immune co-localization results of FYB1 and CD4 in gastric cancer tissue; Figure 11 Figure B in the middle is an analysis of the immune colocalization results of FYB1 and FOXP3 in gastric cancer tissue (FYB1 is located in the cytoplasm and infiltrating cells, while FOXP3 is located in the T cell nucleus).

[0038] Figure 12 This is a diagram showing the results of immune colocalization analysis of adjacent tissues in gastric cancer. Detailed Implementation

[0039] Previous differential proteomics studies have revealed that FYB1 expression is higher in gastric cancer than in the control group. However, its mechanism of action in gastric cancer remains unclear. The expression pattern, function, and relationship of FYB1 with the immune microenvironment in gastric cancer are still unknown. Therefore, exploring the immunomodulatory role of FYB1 in gastric cancer, and whether it activates regulatory T cells (Tregs) to promote immune escape, may provide a theoretical basis for revealing new therapeutic targets.

[0040] This invention aims to investigate the expression of FYB1 in gastric cancer tissues, its role and related mechanisms in gastric cancer, and to study its activation of Treg cells and promotion of immune escape from gastric cancer tumor cells. It seeks to explore the functional role of FYB1 in the development and progression of gastric cancer, its role in the immune microenvironment, and further elucidate the mechanism of action of FYB1 in gastric cancer, thus promoting the translation of FYB1 research from basic research to clinical application. In the future, combining FYB1-targeted intervention strategies (such as small molecule inhibitors or gene editing technology) may provide new insights into overcoming immunotherapy resistance and improving patient prognosis. Furthermore, the research results are expected to provide a scientific basis for improving the molecular subtyping system of gastric cancer and developing personalized treatment plans.

[0041] The research method of this invention includes:

[0042] The expression of FYB1 in pan-cancer was extensively investigated using the TIMER2.0 online database to look for evidence related to FYB1 and gastric cancer.

[0043] Based on the TCGA database, this study investigated the differential expression and survival analysis of FYB1 in gastric cancer, as well as the correlation between the gene expression level of FYB1 in gastric cancer tissue and clinicopathological data of gastric cancer patients.

[0044] Based on the TCGA database, this study investigated the immune infiltration of FYB1 in gastric cancer, its correlation with immune cells, and the correlation with the FYB1 gene.

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

[0046] Immunohistochemistry of serial sections was used to detect the localization of FYB1, CD4, and FOXP3 in gastric cancer tissue and adjacent normal tissue.

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

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

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

[0050] Using the TIMER2.0 online database FYB1 Pan-cancer analysis of expression levels showed the following results: FYB1 It is significantly highly expressed in cancer tissues such as esophageal cancer, glioblastoma, and renal cell carcinoma. P <0.05), FYB1 The expression level in gastric cancer tissues was higher than that in normal tissues, and the difference was statistically significant. P <0.05).

[0051] Based on the TCGA database, it was discovered that... FYB1 The expression level in gastric cancer tissues was higher than that in normal tissues, and this difference in expression level was statistically significant. P <0.05), in gastric cancer FYB1 The high-expression group had a worse prognosis, and the survival difference between the high-expression group and the low-expression group was statistically significant. P <0.05).

[0052] Based on the TCGA database, the ESTIMATE immune score results showed that high expression... FYB1This may be related to the poor prognosis of gastric cancer, and may promote tumor growth, invasion, and metastasis; FYB1 High expression of this substance was also accompanied by an increased proportion of immune cells and stromal cells in the tumor microenvironment. This verified that... FYB1 High expression of this substance is associated with the occurrence and development of gastric cancer and with the infiltration of immune cells. FYB1 The expression of [a specific substance] is significantly positively correlated with CD4+ T cells. P <0.05), verifying FYB1 It is positively correlated with the activation of regulatory T cells (Tregs).

[0053] Correlation analysis using the Person algorithm 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 development, maturation, and function of regulatory T cells (Tregs) and is currently the most sensitive biomarker for Tregs, this indicates a positive correlation between FYB1 and Treg cell differentiation, suggesting that FYB1 promotes Treg cell differentiation. P <0.05).

[0054] Immunohistochemical staining was performed 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] Based on the patients' clinical data, further analysis was conducted to determine the correlation between the expression level of FYB1 in gastric cancer tissue and the clinicopathological findings of gastric cancer patients. The results showed that the expression level of FYB1 was related to T stage, N stage, and differentiation degree, with statistically significant differences. P <0.05), and the differences with gender, age, and M stage were not significant.

[0056] Immunohistochemical staining of serial sections showed that FYB1 shared a common positive region with CD4 and FOXP3.

[0057] Immunofluorescence co-localization studies showed that FYB1 was expressed in gastric cancer tissue, and that FYB1 and CD4 were co-expressed in tissue-infiltrating T cells. FYB1 also co-localized with FOXP3+ Treg cells. Based on this, we can infer that FYB1 and Treg cells play a synergistic role in promoting cancer, either directly or indirectly.

[0058] The above findings indicate that FYB1 can serve as a potential prognostic marker and play a crucial role in the prognosis of gastric cancer. Furthermore, FYB1 can promote immune escape of gastric cancer tumor cells through the mechanism of action of Treg cells.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, 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 embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0060] In the following embodiments, 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. First, add 7,450 μL of antibody diluent, then add 50 μL of FYB1 concentrate, shake to mix, store at 4°C, and use immediately.

[0062] Preparation of sodium citrate antigen repair solution: To prepare a citrate antigen repair solution with pH=6.0, first add 990 mL of distilled water, then add 10 mL of citrate tissue antigen repair solution, mix well, and let stand at room temperature for later use.

[0063] Preparation of PBS phosphate buffer: First add 2,000 mL of distilled water, then add one packet of PBS phosphate buffer (powder), mix well and store at room temperature for later use.

[0064] Preparation of DAB chromogenic solution: Add 1 mL of stable DAB buffer, 50-100 μL of stable DAB substrate, and 50-100 μL of stable DAB chromogen to the provided small test tubes to prepare the DAB chromogenic solution. Store at 4°C after preparation; it is effective for 24 hours.

[0065] Preparation of FYB1 working solution for co-localization of fluorescent antibody: Prepare FYB1 working solution for co-localization of fluorescent antibody 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 at 4°C, and use immediately.

[0066] Preparation of fluorescent antibody co-localization CD4 working solution: Prepare the 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, vortex to mix, store at 4℃, and use immediately.

[0067] Preparation of FOXP3 working solution for co-localization of fluorescent antibody: Prepare FOXP3 working solution for co-localization of fluorescent antibody 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 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 4,975 μL of TSA buffer, then add 25 μL of concentrated TSA-520Plus fluorescent dye, mix well and store in the dark, and 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 4,975 μL of TSA buffer, then add 25 μL of concentrated TSA-520Plus fluorescent dye. Mix well and store in the dark. Prepare and use immediately.

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

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

[0072] Immunohistochemical Tissue Analysis: Ninety gastric cancer tissue samples were collected from patients who underwent radical gastrectomy at Wuxi Xishan People's Hospital between January 1, 2020, and December 31, 2024. None of these patients received preoperative radiotherapy or chemotherapy, and all were diagnosed with gastric cancer postoperatively. Complete clinicopathological data were available for all samples. The patients included 61 males and 29 females, aged 36-89 years. Fifty-one patients had lymph node metastasis, 39 did not, and 4 had distant metastasis. Differentiation grade: 31 cases were well- or moderately differentiated adenocarcinomas, and 59 cases were poorly differentiated adenocarcinomas. Sixty cases of normal mucosal tissue more than 5 cm from the tumor margin were used as controls. All samples were routinely embedded in paraffin and serially sectioned at a thickness of 2 mm. One HE section was taken from each tissue sample for re-examination. The inpatient medical records of each patient were retrieved based on their name and hospital number provided by the Medical Records Management Office of Wuxi Xishan People's Hospital, and the corresponding clinical information for each patient was compiled.

[0073] Immunofluorescence co-localization assay: Five gastric cancer tissue specimens were obtained from gastric cancer patients treated at Xishan People's Hospital in Wuxi City between June 1, 2024 and December 30, 2024. Gastric cancer specimens obtained during surgery were fixed in formalin, embedded in paraffin, and prepared into paraffin blocks for immunofluorescence co-localization. Three normal mucosal tissue samples located more than 5 cm from the tumor margin were selected as controls. None of the selected patients had undergone radiotherapy or chemotherapy prior to surgery.

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

[0075] Serum samples were collected from 48 gastric cancer patients treated at Xishan People's Hospital in Wuxi City between May 1, 2024 and September 30, 2024. All patients were diagnosed with gastric cancer via endoscopic biopsy or postoperative pathological diagnosis. Serum samples were also collected from 16 healthy individuals undergoing routine physical examinations as a control group. Serum sample collection: After centrifugation at 3000 rpm for 10 min, the supernatant was collected, aspirated using a micropipette, aliquoted, and stored at -80 ℃ to avoid repeated freeze-thaw cycles.

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

[0077] All data used in this study's online analysis platform came from The Cancer Genome Atlas (TCGA), including transcriptome data and clinical information. The samples used by each platform differed. Specifically, the dataset used for single-gene and immune invasion analysis consisted of raw transcriptome sequencing data from 446 gastric adenocarcinoma patients downloaded from the TCGA database, including 410 tumor samples and 36 adjacent normal tissue samples. The data used for analyzing clinically relevant characteristics were the clinical data corresponding to the 446 gastric adenocarcinoma patients downloaded earlier. The pan-cancer analysis in this study used the online analysis platform Tumor Immune Estimation Resource 2.0 (TIMER2.0) for TCGA data mining analysis.

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

[0079] Inclusion criteria: (1) The patient underwent partial gastrectomy or total gastrectomy with lymph node dissection for gastric cancer during hospitalization, and the postoperative pathological examination confirmed gastric cancer. (2) The patient had primary gastric cancer. (3) The patient had complete clinical and pathological data. (4) The patient had not undergone 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 conditions requiring long-term medication; (4) Patients and their families who do not give informed consent; (5) Patients with immune deficiencies and autoimmune-related diseases.

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

[0082] Example 1

[0083] This embodiment provides experimental methods for immunohistochemistry, immunofluorescence colocalization, bioinformatics analysis, and statistical analysis, respectively.

[0084] (a) Immunohistochemistry.

[0085] 1) Use a slicer to make continuous slices with a thickness of 2 mm.

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

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

[0088] 4) Add water to the pressure cooker and prepare citrate buffer (pH=6.0) according to 10 mL citric acid + 1,000 mL distilled water, and heat to boiling.

[0089] 5) Place the slide on a stainless steel or heat-resistant plastic slide holder, immerse it in boiling buffer solution, close the pressure valve, cook on the rice cooking mode for 8 minutes, turn off the power and let it sit for 7 minutes, then place it in a cold water bath at room temperature. After cooling to room temperature, rinse the slide with water, place it in a humidified chamber, and rinse twice with PBS.

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

[0091] 7) Shake off the PBS, wipe away any water around the specimen, add 1 drop of reagent R1 (polymer enhancer), incubate for 20 min, and rinse 3 times with PBS.

[0092] 8) Shake off the PBS, wipe away any water around the sample, add 1 drop of reagent R2 (enzyme-labeled anti-mouse / rabbit polymer), incubate for 30 minutes, and then rinse 3 times with PBS.

[0093] 9) Shake off the PBS, wipe away any water around the specimen, add DAB chromogenic reagent (1 ml of solution A, 1 drop of solution B, and 1 drop of solution C), and develop the color within 10 minutes. Once the color has noticeably deepened to 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, blued with running water for 10 min, and dehydrated with a gradient of alcohols (95% ethanol for 3 min, 100% anhydrous ethanol I for 3 min, 100% anhydrous ethanol II for 3 min).

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

[0096] 12) Result Interpretation: Immunohistochemical results were independently evaluated and recorded by two pathologists using a blinded method. The staining intensity and cell positivity rate of each sample were scored separately. The criteria for positive expression were: 1. By percentage of positive cells: <5% = 0 points, 5%~25% = 1 point, 26%~50% = 2 points, 51%~75% = 3 points, >75% = 4 points; 2. By staining intensity: colorless = 0 points, light yellow = 1 point, brownish-yellow = 2 points, brownish-red = 3 points. The final score was equal to the staining intensity score of each sample multiplied by the percentage of positive cells score, with 0-4 points indicating low expression, 6-8 points indicating medium expression, and greater than 8 points indicating high expression.

[0097] Precautions: 1) The chromogenic reagent should be prepared fresh and used within 30 minutes of preparation. 2) The chromogenic process requires protection from light. 3) The procedure for co-localization analysis of FYB1, CD4, and FOXP3 on serial sections is the same as above.

[0098] (ii) Immunofluorescence co-localization.

[0099] 1) Use a slicer to make continuous slices of the wax block at a thickness of 2 mm.

[0100] 2) Routine dewaxing of paraffin sections to water: The sections were baked in a 68 ℃ oven for 30 min. Then the sections were dewaxed with xylene (xylene I 10 min, xylene II 5 min) and hydrated with a gradient of alcohols (100% anhydrous ethanol 3 min, 95% ethanol 3 min, 75% ethanol 3 min, 50% ethanol 3 min). The treated sections were rinsed with distilled water and then immersed in distilled water for 5 min.

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

[0102] 4) Dissolve the EDTA antigen retrieval solution powder in distilled water and bring the volume to 2 L to prepare the EDTA retrieval working solution (pH 9.0). Immerse the slide in the EDTA retrieval solution, heat it in the microwave until it boils, then turn off the power. Repeat the retrieval 1-2 times after 5-10 minutes, and then cool to room temperature.

[0103] 5) After drying the sections, draw circles around the tissue with an immunohistochemical pen, add 5% BSA blocking solution, incubate at 37 ℃ for 30 min, dry, and do not wash.

[0104] 6) Add FYB1 primary antibody diluted 1:150 and incubate at 37°C for 1.5 h. Rinse with PBS 3 times, 5 min each time.

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

[0106] 8) Dilute the concentrated fluorescent dye TSA-520Plus 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 min. Rinse with PBS 3 times for 5 min each time.

[0107] 9) Immerse the slides in antigen retrieval solution in a 37°C water bath for 25-40 minutes. Rinse with PBS, 3 times for 5 minutes each time.

[0108] 10) After drying the sections, draw circles around the tissue with an immunohistochemical pen, add 5% BSA blocking solution, incubate at 37 ℃ for 30 min, dry, and do not wash.

[0109] 11) Add CD4 primary antibody diluted 1:150 and incubate at 37°C for 1.5 h. Rinse with PBS 3 times, 5 min each time.

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

[0111] 13) Dilute the concentrated TSA-690Plus fluorescent dye 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 min. Rinse with PBS 3 times for 5 min each time.

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

[0113] 15) After sectioning and drying, mount the sections with anti-fluorescence attenuation mounting medium.

[0114] 16) Confocal microscopy observation. The excitation wavelength of FYB1 was 490 nm and the emission wavelength was 520 nm; the excitation wavelength of CD4 was 630 nm and the emission wavelength was 690 nm; the excitation wavelength of FOXP3 was 630 nm and the emission wavelength was 690 nm.

[0115] Note: 1) Select the antigen retrieval method and intensity as needed. Thermal retrieval, enzyme retrieval, or no retrieval may be used. 2) Keep the slides moist throughout the process. 3) The immunofluorescence co-staining method for FYB1 and FOXP3 is the same as above.

[0116] (III) Bioinformatics analysis.

[0117] 1) Online database analysis.

[0118] The "Gene" module of the TIMER2.0 online analysis database is used for analysis. FYB1 Pan-cancer analysis assessment of (FYB) FYB1 Expression in different tumors.

[0119] 2) Transcriptome bioinformatics credits.

[0120] The gastric cancer transcriptome data and clinical information downloaded from the TCGA database were organized using R software (version 4.4.1). The "limma" package in R was used to perform differential analysis on the downloaded gastric cancer transcriptome data. FYB1 Differential expression in gastric cancer; survival information from clinical data of gastric cancer patients was analyzed, and x-tile software was used to identify... FYB1 The optimal cutoff value for expression level was 25.3, and survival analysis was performed using the "survival" package in R with the optimal cutoff value of 25.3. The data were visualized using the "survminer" package in R. Clinically relevant characteristics of gastric cancer patients downloaded from the TCGA database were compiled using Excel 2021 and R software (version 4.2.2) and matched with transcriptome data for clinical relevance analysis. FYB1 The correlation between expression levels and these clinical data was analyzed; the ESTIMATE immune score was calculated using the "limma" and "estimate" packages in the R package. FYB1 The relationship between tumor immune infiltrating cells and the tumor; based on the TCGA database, the correlation analysis of gastric cancer transcriptome data was performed using the PEN algorithm based on the R packages “limma”, “ggplot2”, “ggpubr”, and “ggExtra”, and then visualized.

[0121] (iv) Statistical analysis.

[0122] The collected data were processed using Excel 2021. SPSS 29, GraphPad Prism 10, R 4.4.1, and coloc 2 were used for statistical analysis and visualization of the experimental results. Quantitative data were expressed as mean ± standard deviation, while categorical and ordinal data were expressed as frequency and percentage. The significance of categorical data between two groups was analyzed using the t-test, and analysis of variance was used for comparisons among multiple groups. 2 The correlation between FYB1 and clinical factors (such as patient gender, age, tumor size, depth of invasion, lymph node metastasis, pathological stage, etc.) was analyzed, and the Kaplan-Meier method and log-rank test were used for survival analysis.P <0.05 indicates a statistically significant difference.

[0123] The experimental results are as follows:

[0124] (a) Bioinformatics analysis results.

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

[0126] Using the TIMER2.0 online database FYB1 Pan-cancer analysis of (FYB) expression levels showed that... FYB1 It is significantly highly expressed in esophageal cancer, glioblastoma, renal cell carcinoma and other cancer tissues. P <0.05), and FYB1 The expression level in gastric cancer tissues was higher than that in normal tissues, and this difference in expression level was statistically significant. P <0.05). Figure 1 )

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

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

[0129] By performing differential analysis on the downloaded gastric cancer transcriptome data, we found... FYB1 The expression level in gastric cancer tissues was higher than that in normal tissues, and this difference in expression level was statistically significant. P <0.05)( Figure 2 (Figure A). Find it using x-tiles. FYB1 The optimal cutoff value for expression levels in survival analysis was 25.3. Survival analysis was performed using the "survival" package in R with this optimal cutoff value of 25.3, and the data was visualized using the "survminer" package in R. FYB1 High expression in gastric cancer was associated with a poorer prognosis, and the survival difference between the high expression group and the low expression group was statistically significant. P <0.05)( Figure 2 (Figure B in the middle)

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

[0131] The results of a clinical correlation analysis were conducted by matching clinical information and transcriptome data of gastric cancer patients downloaded and compiled from the TCGA database. The conclusions are as follows: FYB1 The expression level was significantly correlated with the T stage.P <0.05)( Figure 3 (Figure A in the middle), and in the T-period, FYB1 The expression level of [a substance] gradually increased with the increase of stage, and the expression differences in stages T1 and T2, T1 and T3, T1 and T4, T2 and T4, and T3 and T4 were all statistically significant. P <0.05)( Figure 3 (Figure B in the middle)

[0132] 3) FYB1 Results of immune scoring analysis in gastric cancer.

[0133] The ESTIMATE immune score results indicate that high expression... FYB1 This may be related to the poor prognosis of gastric cancer, and may promote tumor growth, invasion, and metastasis; FYB1 High expression of this substance is also accompanied by an increased ratio of immune cells to stromal cells in the tumor microenvironment. It was discovered that... FYB1 High expression of this substance is associated with the occurrence and development of gastric cancer and is also 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 crucial 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. Simultaneously, stromal cells such as tumor-associated fibroblasts (CAFs) can secrete cytokines such as TGF-β, promoting Treg cell differentiation and infiltration. Furthermore, CAFs can regulate the composition of the extracellular matrix, providing physical pathways for Treg cell infiltration while restricting the entry of effector T cells. Matrix infiltration can also influence Treg cell differentiation through the induction, spatial distribution, and functional differentiation of the tumor microenvironment; for example, the TGF-β signaling pathway in the tumor microenvironment can convert Th1 cells into immunosuppressive Treg cells. Therefore, the results of immune scoring are also related to tumor prognosis.

[0134] 4) FYB1 The correlation analysis results.

[0135] Correlation analysis using Person's algorithm on gastric cancer transcriptome data from the TCGA database yielded the following results: FYB1 The expression and FOXP3 , CD4 The expression of FOXP3 showed a significant positive correlation. Since FOXP3 is essential for the development, maturation, and function of regulatory T cells (Tregs) and is currently the most sensitive biomarker for Treg cells, this validated the finding. FYB1 It is positively correlated with the activation of Treg cells. P <0.05)( Figure 5 ). FYB1 It may be related to the activation of Treg cells.

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

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

[0138] To verify the bioinformatics analysis results—that FYB1 is highly expressed in gastric cancer—we detected FYB1 protein in 90 gastric cancer tissues and 60 adjacent normal tissues using immunohistochemistry. The immunohistochemical staining results are as follows: Figure 6 As shown, we first statistically analyzed 60 pairs of paired gastric cancer tissues and adjacent normal tissues, and found that the expression level of FYB1 in gastric cancer was also higher than that in adjacent normal tissues. (P <0.05), and subsequently, after statistical analysis of all histochemical results, we found that FYB1 was significantly highly expressed in gastric cancer tissues ( P <0.05)( Figure 7 Therefore, it can be concluded that FYB1 is highly expressed in gastric cancer.

[0139] (2) The relationship between FYB1 expression level and clinicopathological features.

[0140] To further explore the clinical significance of FYB1 in gastric cancer, data on age, sex, differentiation degree, depth of invasion, and presence of lymph node metastasis were extracted and compiled from the clinicopathological data of 90 gastric cancer patients corresponding to each gastric cancer specimen. FYB1 expression was divided into low-expression and high-expression groups based on the median of the corresponding immunohistochemical scores. The chi-square test was used to investigate whether FYB1 expression level was correlated with the above clinical characteristics. The results showed that FYB1 expression level was statistically significant and correlated with T stage, N stage, and differentiation degree (P<0.05), but not with sex, age, or M stage (Table 1).

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

[0142]

[0143] (3) Correlation analysis of FYB1 expression level with some blood immune markers.

[0144] Based on the patient's untreated blood routine within one week of diagnosis, the patient's NLR, PLR, LMR, NPR, PAR, CAR, CLR, SII, SIRI, and CALLY indices were calculated to explore whether the FYB1 tissue expression level (divided into high and low groups according to the median of the immunohistochemical score) was correlated with the above inflammatory markers (serological related concentrations could not be used due to detection technology issues). Pearson correlation coefficients were calculated using pandas and numpy libraries. The results showed the following: FYB1 group vs. NLR (neutrophil-to-lymphocyte ratio): 0.10 (weak positive correlation); FYB1 group vs. PLR (platelet-to-lymphocyte ratio): 0.13 (weak positive correlation); FYB1 group vs. NPR (neutrophil-to-monocyte ratio): 0.13 (weak positive correlation); FYB1 group vs. CAR (CRP-to-albumin ratio): 0.12 (weak positive correlation); FYB1 group vs. CLR (CRP-to-lymphocyte ratio): [further details needed]. The correlation coefficient between FYB1 and SII (systemic immune inflammation index) was 0.06, indicating a weak correlation. The correlation coefficient between FYB1 and LMR (lymphocyte to monocyte ratio) was -0.31, indicating a moderate negative correlation. The correlation coefficient between FYB1 and CLR (CRP to lymphocyte ratio) was 0.17, indicating a weak positive correlation. The correlation coefficient between FYB1 and SIRI (systemic immune inflammation index) was 0.17, indicating a weak positive correlation. The correlation coefficient between FYB1 and CALLY index was 0.19, indicating a weak positive correlation. In summary, FYB1 showed 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 the inflammatory response.

[0145] Table 2. Correlation analysis between FYB1 expression and inflammatory markers.

[0146]

[0147] (III) Validation of the correlation between FYB1 and Treg cells.

[0148] (1) Location results of FYB1, CD4, and FOXP3 in continuous slices.

[0149] To investigate whether there is a positive correlation between FYB1 and Treg cells, serial sections of gastric cancer tissue from the same patient were prepared and immunohistochemically stained. The staining results are as follows: Figure 8Based on this, we can find that FYB1, CD4, and FOXP3 share a common region of positive expression, and FYB1 and Treg cells have a common localization. However, whether the two have a synergistic effect and whether FYB1 can activate Treg cells still need further verification.

[0150] (2) Immunofluorescence colocalization results of FYB1 protein and Treg cells from gastric cancer patients.

[0151] To investigate whether FYB1 has a synergistic effect with Treg cells, we first used fluorescence immunoassay to find that FYB1 was expressed in both the cytoplasm and infiltrating cells of gastric cancer cells. Figure 9 The fluorescence signals of FYB1 and CD4 show a high degree of overlap. Laser confocal microscopy analysis clearly demonstrates the similarity between FYB1 and CD4 in gastric cancer tissue. Figure 11 (Figure A) and adjacent normal tissue ( Figure 12 Co-expression was observed in FYB1 and T cells. Based on this, we can infer that FYB1 and T cells play a synergistic role in promoting 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 on FOXP3, the most sensitive marker between FYB1 and Treg cells. We found that FOXP3 is expressed in the nucleus of T cells (…). Figure 10 (Figure A) Through fluorescence immunoassay, we found a high overlap in fluorescence signals between FYB1 and FOXP3. Based on this, we can preliminarily confirm that FYB1 and Treg cells play a synergistic role in promoting cancer development in gastric cancer. Further verification of how FYB1 activates Treg cells to promote immune escape from gastric cancer tumor cells will be conducted in subsequent in vivo and in vitro experiments.

[0152] (3) Analysis of immunofluorescence colocalization results.

[0153] 1) Analysis of colocalization results in gastric cancer tissue.

[0154] First, co-localization analysis of FYB1 and CD4 in gastric cancer tissue was performed: Immune co-localization analysis was conducted using coloc 2 software, and Pearson correlation coefficients were analyzed using bisection threshold regression. The Pearson correlation coefficient without a threshold was 0.67, indicating a strong positive correlation between the two channels. Above the threshold, the Pearson correlation coefficient was 0.40, indicating a moderate positive correlation between pixels above the threshold. The ICQ value of Li was 0.374, indicating moderate co-localization. The Spearman rank correlation coefficient was 0.71844681, indicating a strong positive correlation. The Kendall's Tau-b rank correlation coefficient was 0.6399, indicating a strong positive correlation between the two channels. The Costes randomized mean was 0.00, and the standard deviation was 0.01, suggesting a correlation. Next, co-localization analysis of FYB1 and FOXP3 in gastric cancer tissue was performed: In the data above the threshold, the Pearson correlation coefficient was 0.73, indicating a strong positive linear correlation between the two variables in these data. FYB1 and FOXP3 were correlated, and FYB1 co-localized with Treg cells in gastric cancer tissue. Since FYB1 is located in the cytoplasm and infiltrating cells, while FOXP3 is located in the nucleus of T cells, although both are located on the same cell, their different positions within the same cell resulted in weak co-localization in two-dimensional analysis. Results are shown below. Figure 11 .

[0155] 2) Analysis of co-localization results of FYB1 and CD4 in adjacent normal tissue.

[0156] We then analyzed the colocalization results of FYB1 and CD4 in adjacent normal tissues: Immune colocalization analysis was performed using coloc 2 software, and Pearson correlation coefficients were analyzed using bisection threshold regression. 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. The Costes P-value was 1.00, indicating that no stronger colocalization was found in the randomized images than observed in the actual images, therefore the colocalization was not significant. The Costes randomization mean was 0.00, indicating that the mean colocalization in the randomized images was 0, and the Costes randomization standard deviation was 0.01, indicating very low variability in colocalization in the randomized images. 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 stronger colocalization was found in the randomized images than observed in the actual images. In conclusion, FYB1 and CD4 showed a weak correlation in adjacent normal tissues. The results are as follows: Figure 12 .

[0157] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of the FYB1 gene or its expressed protein as a biomarker in the preparation of gastric cancer diagnostic reagents, characterized in that, The expression level of the FYB1 gene is higher in gastric cancer tissues than in normal tissues, and the expression level of FYB1 protein is higher in gastric cancer tissues than in normal tissues.

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