Use of a reagent for detecting the expression level of the CBS gene in the manufacture of a product for the diagnosis and / or prognosis of breast cancer

Abstract

The application relates to the technical field of biological medicine, and discloses application of a reagent for detecting CBS gene expression level in preparation of a breast cancer diagnosis and / or prognosis product. The nucleotide sequence of the CBS gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded CBS protein is shown as SEQ ID NO. 2. The experimental results of the application show that the expression of the CBS protein in breast cancer tissues is significantly higher than that in paracancerous tissues, and the CBS protein can be used as a biomarker for breast cancer diagnosis. Meanwhile, the ROC curve evaluation of the CBS protein expression amount on the prognosis results of breast cancer patients shows that the AUC value reaches 0.841, which also shows that the CBS protein can be used as a marker for the prognosis of breast cancer patients, and the CBS protein has important diagnostic significance for the prognosis of breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of reagents for detecting CBS gene expression levels in the preparation of breast cancer diagnostic and / or prognostic products. Background Technology

[0002] Cancer is a major global health challenge that has a serious impact on human health and lives. Among women, breast cancer is the most frequently diagnosed cancer and a leading cause of cancer death. Breast cancer is a highly heterogeneous malignancy, exhibiting significant morphological and molecular heterogeneity between tumors and even within individual tumors. Molecular features include activation of human epidermal growth factor receptor 2 (HER2, encoded by ERBB2), activation of hormone receptors (estrogen receptor and progesterone receptor), and / or BRCA mutations.

[0003] Currently, the diagnosis of breast cancer mainly relies on methods such as clinical breast examination, mammography, ultrasound examination, and magnetic resonance imaging. Exploring new diagnostic and prognostic biomarkers for breast cancer will help in the early detection and diagnosis of breast cancer, promote early treatment, increase the chances of treatment success, and thus reduce mortality. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of a reagent for detecting CBS gene expression levels in the preparation of breast cancer diagnostic and / or prognostic products.

[0005] On the one hand, the present invention provides the application of a reagent for detecting the expression level of the CBS gene in the preparation of breast cancer diagnostic and / or prognostic products, wherein the nucleotide sequence of the CBS gene is shown in SEQ ID NO.1 and the amino acid sequence of the encoded CBS protein is shown in SEQ ID NO.2.

[0006] SEQ ID NO.1:

[0007] SEQ ID NO.2: .

[0008] Furthermore, the reagents for detecting CBS gene expression levels can be reagents based on immunohistochemical staining, reagents based on specific antibody detection, reagents based on mass spectrometry analysis, reagents based on metabolic marker detection, or reagents based on molecular biology detection methods.

[0009] The products further include chips, reagent kits, or test strips.

[0010] On the other hand, the present invention also provides the application of the CBS gene or CBS protein as a target in screening anti-breast cancer drugs, wherein the nucleotide sequence of the CBS gene is shown in SEQ ID NO.1 and the amino acid sequence of the encoded CBS protein is shown in SEQ ID NO.2.

[0011] On the other hand, the present invention also provides the application of the CBS gene or CBS protein in constructing a prognostic prediction model for breast cancer, wherein the nucleotide sequence of the CBS gene is shown in SEQ ID NO.1 and the amino acid sequence of the encoded CBS protein is shown in SEQ ID NO.2.

[0012] On the other hand, the present invention also provides a kit for the diagnosis and / or prognosis of breast cancer, the kit comprising reagents for detecting the expression level of the CBS gene, the nucleotide sequence of the CBS gene being shown in SEQ ID NO.1, and the amino acid sequence of the encoded CBS protein being shown in SEQ ID NO.2.

[0013] Furthermore, the reagents for detecting CBS gene expression levels can be reagents based on immunohistochemical staining, reagents based on specific antibody detection, reagents based on mass spectrometry analysis, reagents based on metabolic marker detection, or reagents based on molecular biology detection methods.

[0014] The advantages and positive effects of the reagent for detecting CBS gene expression levels described in this invention in the preparation of breast cancer diagnostic and / or prognostic products are as follows: Experimental results showed that CBS protein expression was significantly higher in breast cancer tissues than in adjacent normal tissues, making it a potential biomarker for breast cancer diagnosis. Furthermore, ROC curve analysis of the prognostic effect of CBS protein expression on breast cancer patients showed an AUC value of 0.841, further indicating that CBS protein can serve as a prognostic marker for breast cancer patients and thus possesses significant diagnostic value for breast cancer.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 The following are the expression results of CBS protein in breast cancer and adjacent normal tissues in the embodiments of the present invention, wherein... p < 0.001; Figure 2 This is the correlation analysis result between CBS protein and the prognosis of breast cancer patients in the embodiments of the present invention; Figure 3 This is the subject operating curve (ROC curve) of CBS protein in breast cancer patients in this embodiment of the invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0019] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments without specified sources are all commercially available materials. Tissue samples in this invention were all purchased from Shanghai Zhuohao Pharmaceutical Technology Co., Ltd.

[0020] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0021] Example 1 Analysis of CBS gene expression in breast cancer and adjacent normal tissues.

[0022] 1. Source of tissue samples: Tissue samples were randomly collected from 120 breast cancer patients between 2011 and 2016 (purchased from Shanghai Zhuohao Pharmaceutical Technology Co., Ltd.). All cases were diagnosed with breast cancer, and all patients were female, aged 31-66 years. All 120 samples contained cancerous tissue and adjacent tissue (1.5 cm away from the cancerous tissue).

[0023] 2. Fabrication of tissue microarrays, the method is as follows: All donor tissue paraffin blocks were routinely sectioned and stained with hematoxylin and eosin (HE). A pathologist performed a secondary diagnosis and marked typical pathological sites on the HE sections. Using a Beecher Instruments Inc. tissue microarray fabrication system, 1.5 mm diameter holes were drilled in blank recipient paraffin blocks. Target tissue cores were then extracted from the donor blocks according to the markings on the HE sections and placed into the array wells of the recipient blocks. This process was repeated to create matching dot array blocks C1 and N1 of breast cancer tissue and adjacent normal tissue. Serial sections were prepared using a Leica (Germany) paraffin microtome at a thickness of 4 μm and mounted on imported glass slides treated to prevent detachment.

[0024] 3. The immunohistochemical staining method is as follows: (1) Baking and dewaxing: Place the breast cancer tissue chip on a baking machine with the tissue side facing up and bake at 65°C for two hours. Then, dewax the tissue chip in a fume hood.

[0025] (2) Antigen retrieval: Place the slide in an immunohistochemistry box containing citric acid retrieval solution (pH 6.0), heat it in a microwave oven on high heat until boiling (about 3-4 minutes), then turn to medium-low heat and keep it simmering for 15 minutes. Let it cool naturally to room temperature (30-40 minutes), and rinse it three times with PBS for 5 minutes each time.

[0026] (3) Endogenous peroxidase blockade: add 3% H2O2 to cover the tissue, incubate at room temperature in the dark for 10 minutes, and then rinse with PBS 3 times, 5 minutes each time.

[0027] (4) Blocking: Use filter paper to absorb the liquid around the section, add blocking solution (5% normal goat serum) to completely cover the tissue, incubate in a humidified box at room temperature for 30 minutes, do not rinse, and directly aspirate the blocking solution.

[0028] (5) Primary antibody incubation: Anti-CBS Rabbit pAb is diluted with antibody diluent at a ratio of 1:500. Primary antibody working solution is added to cover the tissue and incubated overnight (16-18 hours) at 4°C in a humidified chamber.

[0029] (6) Primary antibody recovery and washing: Remove the wet box from 4°C, equilibrate at room temperature for 30 minutes, place it on a shaker and rinse with PBS 3 times, 10 minutes each time, and replace with fresh PBS each time.

[0030] (7) Secondary antibody incubation: Add HRP-labeled secondary antibody (ready-to-use type), incubate in a humidified chamber at room temperature for 30 minutes, and then rinse with PBS 3 times, 10 minutes each time.

[0031] (8) DAB staining: Prepare DAB staining solution at a ratio of 1:20. Use in the dark and prepare fresh solution immediately. Shake off the liquid on the tissue. Place the breast cancer tissue chip under a microscope and add DAB staining solution to the tissue for staining. When the specific staining is obvious and the background is clean, immediately immerse the breast cancer tissue chip in distilled water to stop the staining. Rinse with tap water for 5 minutes.

[0032] (9) Hematoxylin counterstaining and differentiation: Place the breast cancer tissue chip in hematoxylin staining solution for 2 minutes, rinse with tap water for 10 minutes, immerse the breast cancer tissue chip in 1% hydrochloric acid alcohol for 2-5 seconds, rinse with tap water immediately for 5 minutes, immerse the breast cancer tissue chip in dilute ammonia water for 30 seconds, and rinse with tap water for 5 minutes.

[0033] (10) Dehydration and clearing: Breast cancer tissue chip 75% ethanol, 2 minutes → 85% ethanol, 2 minutes → 95% ethanol I, 2 minutes → 95% ethanol II, 2 minutes → 100% ethanol I, 2 minutes → 100% ethanol II, 2 minutes (complete dehydration) → xylene I (fume hood operation), 5 minutes (clear) → xylene II (fume hood operation), 5 minutes (completely clear).

[0034] (11) Mount the slide: Remove the slide from xylene, let it dry slightly, add 1-2 drops of neutral resin, slowly cover with a coverslip to avoid air bubbles, and let it dry flat at room temperature for 24-48 hours.

[0035] 4. Quantitative analysis is as follows: The staining results were photographed, and the positive area and staining intensity within each section were converted into corresponding H-Score values ​​to achieve semi-quantitative analysis of tissue staining. The histochemical H-Score ranged from a minimum of 0 to a maximum of 8, with a median of 4. Patients were grouped according to their H-Score: those above the median were classified as high-expression, and those below or equal to the median were classified as low-expression.

[0036] 5. Statistical analysis is as follows: The expression of CBS protein in breast cancer and adjacent normal tissues was analyzed using a t-test. The correlation between CBS protein expression and clinical indicators in breast cancer patients was analyzed using a chi-square test. The correlation between CBS protein and the prognosis of breast cancer patients was analyzed using Kaplan-Meier survival analysis and log-rank statistical tests for univariate survival. A p-value < 0.05 was considered statistically significant.

[0037] 6. Results Analysis: (1) Expression analysis of CBS protein in breast cancer and adjacent normal tissue.

[0038] like Figure 1 As shown, the expression level of CBS protein in cancer tissue was significantly higher than that in adjacent normal tissue (p < 0.001).

[0039] (2) Correlation analysis between CBS protein and clinical indicators.

[0040] The above immunohistochemical staining results were analyzed using Fisher's test to examine the correlation between CBS protein expression in breast cancer tissue and clinical indicators. A p < 0.001 result was considered statistically significant. The results are shown in Table 1. The results showed that CBS protein expression was correlated with TNM subtype of breast cancer (p < 0.001) but had no significant statistical relationship with age (p > 0.05).

[0041] Table 1. Correlation between CBS protein expression and clinical indicators in breast cancer patients.

[0042] (3) Correlation analysis between CBS protein and patient prognosis.

[0043] The prognostic value of CBS in breast cancer was analyzed using Cox regression statistical methods. Patients in the CBS high expression group had a lower 20-year survival rate than those in the CBS low expression group, indicating that high CBS expression is associated with poor prognosis, and the survival rate of patients in the high expression group was significantly lower than that of the low expression group. Figure 2 ).

[0044] Example 2 Validation of the CBS gene in the diagnosis of breast cancer.

[0045] 1. Tissue Sample Source: Tissue samples were randomly selected from 100 breast cancer patients (purchased from Shanghai Zhuohao Pharmaceutical Technology Co., Ltd., excluding the 120 samples in Example 1). All 100 samples contained cancerous tissue and adjacent tissue (1.5 cm away from the cancerous tissue).

[0046] 2. Fabrication of tissue microarrays, the method is as follows: All donor tissue paraffin blocks were routinely sectioned and stained with hematoxylin and eosin (HE). A pathologist performed a secondary diagnosis and marked typical pathological sites on the HE sections. Using a Beecher Instruments Inc. tissue microarray fabrication system, 1.5 mm diameter holes were drilled in blank recipient paraffin blocks. Target tissue cores were then extracted from the donor blocks according to the markings on the HE sections and placed into the array wells of the recipient blocks. This process was repeated to create matching dot array blocks C1 and N1 of breast cancer tissue and adjacent normal tissue. Serial sections were prepared using a Leica (Germany) paraffin microtome at a thickness of 4 μm and mounted on imported glass slides treated to prevent detachment.

[0047] 3. The immunohistochemical staining method is as follows: (1) Baking and dewaxing: Place the breast cancer tissue chip on a baking machine with the tissue side facing up and bake at 65°C for two hours. Then, dewax the tissue chip in a fume hood.

[0048] (2) Antigen retrieval: Place the slide in an immunohistochemistry box containing citric acid retrieval solution (pH 6.0), heat it in a microwave oven on high heat until boiling (about 3-4 minutes), then turn to medium-low heat and keep it simmering for 15 minutes. Let it cool naturally to room temperature (30-40 minutes), and rinse it three times with PBS for 5 minutes each time.

[0049] (3) Endogenous peroxidase blockade: add 3% H2O2 to cover the tissue, incubate at room temperature in the dark for 10 minutes, and then rinse with PBS 3 times, 5 minutes each time.

[0050] (4) Blocking: Use filter paper to absorb the liquid around the section, add blocking solution (5% normal goat serum) to completely cover the tissue, incubate in a humidified box at room temperature for 30 minutes, do not rinse, and directly aspirate the blocking solution.

[0051] (5) Primary antibody incubation: Anti-CBS Rabbit pAb is diluted with antibody diluent at a ratio of 1:500. Primary antibody working solution is added to cover the tissue and incubated overnight (16-18 hours) at 4°C in a humidified chamber.

[0052] (6) Primary antibody recovery and washing: Remove the wet box from 4°C, equilibrate at room temperature for 30 minutes, place it on a shaker and rinse with PBS 3 times, 10 minutes each time, and replace with fresh PBS each time.

[0053] (7) Secondary antibody incubation: Add HRP-labeled secondary antibody (ready-to-use type), incubate in a humidified chamber at room temperature for 30 minutes, and then rinse with PBS 3 times, 10 minutes each time.

[0054] (8) DAB staining: Prepare DAB staining solution at a ratio of 1:20. Use in the dark and prepare fresh solution immediately. Shake off the liquid on the tissue. Place the breast cancer tissue chip under a microscope and add DAB staining solution to the tissue for staining. When the specific staining is obvious and the background is clean, immediately immerse the breast cancer tissue chip in distilled water to stop the staining. Rinse with tap water for 5 minutes.

[0055] (9) Hematoxylin counterstaining and differentiation: Place the breast cancer tissue chip in hematoxylin staining solution for 2 minutes, rinse with tap water for 10 minutes, immerse the breast cancer tissue chip in 1% hydrochloric acid alcohol for 2-5 seconds, rinse with tap water immediately for 5 minutes, immerse the breast cancer tissue chip in dilute ammonia water for 30 seconds, and rinse with tap water for 5 minutes.

[0056] (10) Dehydration and clearing: Breast cancer tissue chip 75% ethanol, 2 minutes → 85% ethanol, 2 minutes → 95% ethanol I, 2 minutes → 95% ethanol II, 2 minutes → 100% ethanol I, 2 minutes → 100% ethanol II, 2 minutes (complete dehydration) → xylene I (fume hood operation), 5 minutes (clear) → xylene II (fume hood operation), 5 minutes (completely clear).

[0057] (11) Mount the slide: Remove the slide from xylene, let it dry slightly, add 1-2 drops of neutral resin, slowly cover with a coverslip to avoid air bubbles, and let it dry flat at room temperature for 24-48 hours.

[0058] 4. Quantitative analysis is as follows: The staining results were photographed, and the positive area and staining intensity within each section were converted into corresponding H-Score values ​​to achieve semi-quantitative analysis of tissue staining. The histochemical H-Score ranged from a minimum of 0 to a maximum of 8, with a median of 4. Patients were grouped according to their H-Score: those above the median were classified as high-expression, and those below or equal to the median were classified as low-expression.

[0059] 5. Statistical analysis is as follows: The efficacy of CBS protein expression intensity in the prognostic diagnosis of breast cancer patients was analyzed using ROC curve analysis.

[0060] 6. ROC curve as shown Figure 3 As shown, the AUC value was 0.841, indicating that CBS protein can serve as a biomarker for diagnosing breast cancer.

[0061] Therefore, this invention utilizes the reagents described above for detecting CBS gene expression levels in the preparation of breast cancer diagnostic and / or prognostic products. Experimental results show that CBS protein expression in breast cancer tissue is significantly higher than in adjacent normal tissue, making it a potential biomarker for breast cancer diagnosis. Furthermore, ROC curve analysis of the prognostic effect of CBS protein expression on breast cancer patients showed an AUC value of 0.841, further indicating that CBS protein can serve as a prognostic marker for breast cancer patients and demonstrating its significant diagnostic value for breast cancer prognosis.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of a reagent for detecting CBS gene expression levels in the preparation of breast cancer diagnostic and / or prognostic products, characterized in that: The nucleotide sequence of the CBS gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded CBS protein is shown in SEQ ID NO.

2.

2. The application of the reagent for detecting CBS gene expression levels according to claim 1 in the preparation of breast cancer diagnostic and / or prognostic products, characterized in that: The reagents for detecting CBS gene expression levels can be based on immunohistochemical staining, specific antibody detection, mass spectrometry, metabolic marker detection, or molecular biology detection.

3. The application of the reagent for detecting CBS gene expression levels according to claim 1 in the preparation of breast cancer diagnostic and / or prognostic products, characterized in that: The products include chips, reagent kits, or test strips.

4. The application of CBS gene or CBS protein as a target in screening anti-breast cancer drugs, characterized by: The nucleotide sequence of the CBS gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded CBS protein is shown in SEQ ID NO.

2.

5. The application of the CBS gene or CBS protein in constructing a prognostic prediction model for breast cancer, characterized by: The nucleotide sequence of the CBS gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded CBS protein is shown in SEQ ID NO.

2.

6. A kit for the diagnosis and / or prognosis of breast cancer, characterized in that: The kit contains reagents for detecting the expression level of the CBS gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of which encodes the CBS protein is shown in SEQ ID NO.

2.

7. The reagent kit according to claim 6, characterized in that: The reagents for detecting CBS gene expression levels can be based on immunohistochemical staining, specific antibody detection, mass spectrometry, metabolic marker detection, or molecular biology detection.

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