Use of reagents for detecting GALNT7 gene expression in the preparation of Luminal breast cancer screening kits

Abstract

This invention belongs to the field of diagnostic reagent kit technology, specifically relating to the use of a reagent for detecting GALNT7 gene expression in the preparation of a screening kit for Luminal breast cancer. This invention is the first to demonstrate that GALNT7 is upregulated in Luminal breast cancer patients, with an area under the ROC curve (AUC) of 0.846, indicating that GALNT7 has high clinical value as a non-invasive biomarker in Luminal breast cancer. Furthermore, using chemiluminescence immunoassay to detect CEA and CA125 in Luminal breast cancer patients and combining this with GALNT7 expression for diagnosis, it was found that the combined diagnosis of these three factors improves the diagnostic efficiency of Luminal breast cancer, exhibiting higher sensitivity and specificity. Moreover, this invention allows for serum-based testing, effectively reducing testing costs and minimizing harm to patients. Therefore, this invention has excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of diagnostic reagent kit technology, specifically relating to the use of a reagent for detecting GALNT7 gene expression in the preparation of a Luminal breast cancer screening kit. Background Technology

[0002] Breast cancer is the most common cancer worldwide and a leading cause of cancer-related deaths in women. Classifying breast cancer helps in accurate diagnosis and guides the selection of treatment methods and disease prognosis. Current technology classifies breast cancer into four molecular subtypes: Luminal A, Luminal B, HER2-positive, and triple-negative (TNBC).

[0003] Currently, molecular subtyping of breast cancer is mainly based on the different expression levels of two major types of receptors—hormone receptors (estrogen and progesterone receptors) and Her-2 receptors—in patient pathological specimens, as well as differences in Ki-67 levels. This method requires the collection of pathological tissue from the patient for testing, which is an invasive procedure and is not ideal in terms of testing costs and harm to patients.

[0004] Therefore, the search for new biomarkers that can be used for breast cancer subtyping and the development of non-invasive detection methods are of great significance in this field. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides the use of a reagent for detecting GALNT7 gene expression in the preparation of a Luminal breast cancer screening kit, with the aim of providing new diagnostic indicators for Luminal breast cancer and achieving accurate classification of Luminal breast cancer.

[0006] The use of the detection reagent in the preparation of a Luminal breast cancer screening kit, wherein the detection reagent is selected from at least one of the following reagents: a reagent for detecting GALNT7 gene expression, a reagent for detecting CEA level, or a reagent for detecting CA125 level.

[0007] Preferably, the Luminal type breast cancer includes Luminal A type breast cancer and Luminal B type breast cancer.

[0008] Preferably, the gene ID of the GALNT7 gene is 51809, the protein ID of the CEA is GenBank:CAE75559.1, and the protein ID of the CA125 is GenBank:AAL65133.2.

[0009] Preferably, the detection reagent is used to detect breast cancer tissue samples.

[0010] Preferably, the detection reagent is an immunohistochemical detection reagent for detecting the expression level of the GALNT7 gene.

[0011] Preferably, the test reagent is used to test urine or blood samples.

[0012] Preferably, the reagent is an ELISA assay, Western blot assay, protein chip assay, or chemiluminescence assay for detecting GALNT7 gene expression level, CEA level, or CA125 level.

[0013] Preferably, the detection reagents include: reagents for detecting GALNT7 gene expression levels, reagents for detecting CEA levels, and reagents for detecting CA125 levels.

[0014] This invention discovered that GALNT7 is upregulated in patients with lumens breast cancer, with an area under the ROC curve (AUC) of 0.846, indicating that GALNT7 has high clinical value as a non-invasive biomarker in lumens breast cancer. Furthermore, using chemiluminescence immunoassay to detect CEA and CA125 in lumens breast cancer patients and combining this with GALNT7 expression for diagnosis, it was found that the combined diagnosis of these three markers improves the diagnostic efficiency of lumens breast cancer, exhibiting higher sensitivity and specificity. In addition, this invention allows for detection using serum samples, effectively reducing testing costs and minimizing harm to patients. Therefore, this invention has excellent application prospects.

[0015] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0016] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0017] Figure 1 Figure A shows the expression analysis of GALNT7 in lumbar breast cancer. Figure B shows the expression level of GALNT7 in lumbar breast cancer tissues higher than that in normal tissues, based on GEPIA data. Figure B shows the expression analysis results of GALNT7 in lumbar breast cancer and adjacent cancer tissues based on the TCGA database.

[0018] Figure 2Figure 1 shows the prognostic and diagnostic value of GALNT7 in Luminal breast cancer. Figure 2 shows the impact of GALNT7 expression on overall survival in Luminal breast cancer patients using Kaplan-Meier analysis of TCGA data. Figure 3 shows the impact of GALNT7 expression on metastasis-free and recurrence-free survival in Luminal breast cancer patients. Figure 4 shows the impact of GALNT7 expression on overall survival in HER2+ breast cancer patients (no difference). Figure 5 shows the impact of GALNT7 expression on metastasis-free and recurrence-free survival in HER2+ breast cancer patients (no difference).

[0019] Figure 3 Figure A shows the clinical diagnostic value of GALNT7 in patients with Luminal breast cancer, and Figure B shows the expression level of GALNT7 in serum of Luminal breast cancer patients and healthy individuals detected by ELISA. Figure C shows the ROC curve analysis of serum GALNT7 levels in Luminal breast cancer patients and healthy individuals. Figure D shows the ROC curve analysis of the combined diagnostic value of serum GALNT7, CEA and CA125. Detailed Implementation

[0020] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0021] Example 1: A kit for detecting GALNT7 gene expression.

[0022] I. Components of the Reagent Kit

[0023] Test kit (48 test kits):

[0024] Components Dosage ELISA coated plate 1 board sealing film 2 pieces Standard (lyophilized) 2 Sample diluent 1 bottle, 20mL per bottle Biotinylated conjugates (100x) 1 animal, 120μL per animal Biotinylated conjugate diluent 1 bottle, 20mL per bottle Enzyme-labeled avidin (100x) 1 animal, 120μL per animal Enzyme-labeled avidin dilution solution 1 bottle, 20mL per bottle Concentrated detergent (25x) 1 bottle, 20mL per bottle TMB colorimetric solution 1 bottle, 12mL per bottle Washing solution (1×PBST) 1 bottle, 60mL per bottle Termination solution 1 bottle, 10mL per bottle

[0025] Among them, enzyme-labeled avidin is a specific biotin-labeled antibody GALNT7 antigen.

[0026] II. Instructions for using the reagent kit

[0027] 1) Warming: Remove the kit from the 4℃ refrigerator and allow it to warm to room temperature for 15 minutes;

[0028] 2) Sample addition: Set up standard wells and sample wells separately. Add 100 μL of standard or sample to be tested, being careful to avoid air bubbles. Add the sample to the bottom of the well of the ELISA plate, trying not to touch the well wall. Gently shake to mix. Cover the ELISA plate with a cap or membrane and incubate at 37°C for 2 hours.

[0029] 3) To ensure the validity of experimental results, please use a new standard solution for each experiment.

[0030] 4) After incubation for 2 hours, discard the liquid in the wells, spin dry, wash the plate 3 times, add 250 μL of washing solution (1x) to each well, soak for 1-2 minutes each time, and spin dry.

[0031] 5) Add 100 μL of biotinylated conjugate (1x) to each well (prepare by adding 1 μL of biotinylated conjugate to 99 μL of biotinylated conjugate diluent, mix gently, and prepare within 1 hour before use), and incubate at 37°C for 1 hour.

[0032] 6) After incubation for 1 hour, discard the liquid in the wells, spin dry, wash the plate 3 times, add 250 μL of washing solution (1x) to each well, soak for 1-2 minutes each time, and spin dry.

[0033] 7) Add 100 μL of enzyme-labeled avidin (1x) to each well (prepare by adding 1 μL of enzyme-labeled avidin to 99 μL of enzyme-labeled avidin dilution solution, mix gently, and prepare within 1 hour before use), and incubate at 37°C for 1 hour.

[0034] 8) After incubation for 1 hour, discard the liquid in the wells, spin dry, wash the plate 5 times, add 250 μL of washing solution (1x) to each well, soak for 1-2 minutes each time, and spin dry.

[0035] 9) Add 100 μL of TMB colorimetric reagent to each well in sequence, and develop the color at 37°C in the dark for 15-20 minutes (within 20 minutes, a clear gradient of blue should be visible to the naked eye in the first 3-4 wells of the standard, while the gradient should be less obvious in the last 3-4 wells, at which point the process can be stopped).

[0036] 10) Add 50 μL of stop solution to each well sequentially to stop the reaction (the blue color will immediately turn yellow). The order of adding the stop solution should be as similar as possible to the order of adding the substrate solution. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time has elapsed.

[0037] 11) Measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using an ELISA reader. Perform the test within 5 minutes after adding the stop solution.

[0038] Example 2: Kit for Detecting CEA Levels

[0039] Test kit (48 test kits):

[0040] Components Dosage ELISA coated plate 1 board sealing film 3 pieces Standard products 0.5ml x 1 tube Standard diluent 1 bottle, 1.5mL per bottle Sample diluent 1 bottle, 3mL per vial enzyme-labeled reagents 1 bottle, 3mL per bottle Color developer solution A 1 bottle, 3mL per vial Color developer solution B 1 bottle, 3mL per bottle Concentrated detergent (20x) 1 bottle, 20mL per bottle Termination solution 1 bottle, 3mL per bottle

[0041] The enzyme-labeled reagent is a specific horseradish peroxidase-labeled CEA antigen.

[0042] II. Instructions for using the reagent kit

[0043] 1) Dilution of Standards: Set up 10 wells for standards on an enzyme-labeled plate. Add 100 μl of standard to the first and second wells, then add 50 μl of standard diluent to the first and second wells and mix well. Then, take 100 μl from each of the first and second wells and add it to the third and fourth wells respectively. Add 50 μl of standard diluent to each of the third and fourth wells and mix well. Then, take 50 μl from each of the third and fourth wells and discard it. Take 50 μl from each of the third and fourth wells and mix well. Add the solution to wells 5 and 6, then add 50 μL of standard diluent to each well and mix well. After mixing, take 50 μL from each well and add it to wells 7 and 8, respectively. Then add 50 μL of standard diluent to each well 7 and 8, and mix well. Then take 50 μL from each well 7 and 8 and add it to wells 9 and 10, respectively. Then add 50 μL of standard diluent to each well 9 and 10, and mix well. Discard 50 μL from each well 9 and 10. (The volume added to each well after dilution is 50 μL, with concentrations of 9 mmol / L, 6 mmol / L, 3 mmol / L, 1.5 mmol / L, and 0.75 mmol / L, respectively).

[0044] 2) Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. First, add 40 μl of sample diluent to the sample wells on the enzyme-labeled plate, then add 10 μl of the sample to be tested (the final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.

[0045] 3) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0046] 4) Solution preparation: Dilute the 30 (20 times that of 48T) times concentrated washing solution with distilled water for 30 (20 times that of 48T) times and set aside.

[0047] 5) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing liquid, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0048] 6) Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for blank wells.

[0049] 7) Incubation: Same procedure as 3.

[0050] 8) Washing: Same as 5.

[0051] 9) Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0052] 10) Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0053] 11) Measurement: Zero the sample with a blank control and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0054] Example 3: A kit prepared from reagents for detecting CA125 levels

[0055] Test kit (48 test kits):

[0056]

[0057]

[0058] The enzyme-labeled reagent is a specific horseradish peroxidase-labeled CA125 antigen.

[0059] II. Instructions for using the reagent kit

[0060] 1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag and return them to 4℃.

[0061] 2) Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well;

[0062] 3) Add 50 μL of the sample to be tested to the sample well; do not add any to the blank well.

[0063] 4) Except for the blank wells, add 100 μL of spicy enzyme-labeled reagent to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min.

[0064] 5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used).

[0065] 6) Add 50 μL each of colorimetric reagent A and B to each well and incubate at 37°C in the dark for 15 min.

[0066] 7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0067] The technical solution of the present invention will be further explained below through experimental examples.

[0068] Example 1: The ability of GALNT7 expression level to differentiate Luminal breast cancer

[0069] I. Clinical Data

[0070] Thirty-one breast cancer patients and 33 healthy controls were selected. Their basic information is as follows:

[0071] Basic Information Luminal breast cancer patients Health comparison Number of people 31 33 age 52.2±8.9 51.7±7.7

[0072] II. Experimental Methods

[0073] 1. Analyze the relationship between GALNT7 expression levels and breast cancer prognosis in the TCGA and GEPIA databases.

[0074] We collected TCGA data to obtain mRNA expression data of GALNT7 in breast cancer patients and corresponding clinical data. Based on the expression differences, the patients were divided into low expression group and high expression group, and survival rate analysis was performed on these two groups.

[0075] We used the breast cancer database GEPIA for integrated analysis to verify the relationship between high GALNT7 expression and prognosis.

[0076] 2. GEPIA Database Analysis

[0077] GEPIA (http: / / gepia.cancer-pku.cn / ) is a visualization website based on GTEx and TCGA data integration, containing differential gene expression between cancer and non-cancer patients. Searching for '...' in GEPIA...

[0078] Keywords 'GALNT7' and 'BRCA' were used to retrieve differential expression data of GALNT7 in breast cancer and normal breast tissue.

[0079] 3. Analyze the expression of the GALNT7 gene in the TCGA database.

[0080] PPEF1 expression profile data from 1108 breast cancer tissues and 112 adjacent normal tissues were collected from the TCGA database. The edgeR method was used for normalization, and differential analysis of GALNT7 in breast cancer tissues and adjacent normal tissues was performed. Dot plots were then plotted.

[0081] 4. Detection of serum GALNT7 levels and verification of the diagnostic efficiency of combining with traditional tumor markers.

[0082] ① 114 serum samples from the Affiliated Hospital of Southwest Medical University were included for combined diagnostic analysis of PPEF1 and traditional tumor markers, including 57 healthy controls and 57 untreated breast cancer patients;

[0083] ② The content of GALNT7 in serum samples was detected using a commercial enzyme-linked immunosorbent assay (ELISA) kit;

[0084] ③ Detect CEA and CA125 using an ELISA kit;

[0085] ④ Analyze the results of independent and combined diagnostic experiments using receiver operating characteristic curves (ROC) and logistic regression.

[0086] III. Experimental Results

[0087] Figure 1 Figure A shows the expression analysis of GALNT7 in multiple subtypes of breast cancer (Luminal, HER2+, and TNBC). Figure A shows that GEPIA data indicates that the expression level of GALNT7 in Luminal and HER2+ breast cancer tissues is higher than that in normal tissues. Figure B shows the expression analysis results of GALNT7 in Luminal and HER2+ breast cancer and adjacent cancer tissues based on the TCGA database.

[0088] Figure 2 Figure 1 shows the prognostic and diagnostic value of GALNT7 in Luminal and HER2+ breast cancer. Figure 2 shows the impact of GALNT7 expression on overall survival in Luminal breast cancer patients using Kaplan-Meier analysis of TCGA data. Figure 3 shows the impact of GALNT7 expression on metastasis-free and recurrence-free survival in Luminal breast cancer patients. Figure 4 shows the impact of GALNT7 expression on overall survival in HER2+ breast cancer patients (no difference). Figure 5 shows the impact of GALNT7 expression on metastasis-free and recurrence-free survival in HER2+ breast cancer patients (no difference).

[0089] The above data proves that:

[0090] 1. Luminal breast cancer and HER2+ breast cancer have an increased risk relative to healthy individuals, while triple-negative breast cancer has a decreased risk relative to healthy individuals.

[0091] 2. For prognostic analysis, GALNT7 is indicative of prognosis in Luminal breast cancer, but has no difference in prognostic impact in HER2+ breast cancer.

[0092] Figure 3 Figure A shows the clinical diagnostic value of GALNT7 in patients with lumbar breast cancer, as analyzed by ELISA in serum of patients with lumbar breast cancer and healthy individuals. Figure B shows the ROC curve analysis of serum GALNT7 levels in patients with lumbar breast cancer and healthy individuals. Figure C shows the ROC curve analysis of the combined diagnostic performance of serum GALNT7, CEA, and CA125. It can be seen that the combined diagnostic performance of GALNT7, CEA, and CA125 is superior to that of a single indicator.

[0093] Therefore, GALNT7 expression level can be used as an indicator for screening luminal breast cancer. In addition, combining it with the combined diagnosis of CEA and CA125 can further improve the accuracy of the diagnostic results.

[0094] As can be seen from the above embodiments and experimental examples, this invention provides a novel diagnostic indicator for lumbar breast cancer, exhibiting excellent sensitivity and specificity. Furthermore, this invention can use serum as a sample for detection, effectively reducing testing costs and minimizing harm to patients. Therefore, this invention has excellent application prospects.

Claims

1. The use of reagents for detecting serum GALNT7 protein expression levels in the preparation of Luminal breast cancer screening kits.

2. The use according to claim 1, characterized in that: The Luminal type of breast cancer includes Luminal A breast cancer and Luminal B breast cancer.

3. The use according to claim 1, characterized in that: The reagents used to detect the expression level of GALNT7 protein in serum are ELISA reagents, Western blot reagents, protein chip reagents, or chemiluminescence reagents.

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