Application of IGSF3 as serum marker in breast cancer diagnosis

Abstract

The invention relates to the technical field of laboratory medicine, in particular to application of IGSF3 as a serum marker in preparation of medicines for breast cancer diagnosis. The invention discovers that the expression level of serum IGSF3 in a metastatic breast cancer patient is obviously increased for the first time. The IGSF3 can be used as a serum marker for diagnosing breast cancer metastasis, the expression level of the serum IGSF3 in a metastatic breast cancer patient is remarkably improved compared with a healthy control person and a non-metastatic breast cancer patient, and ROC curve analysis shows that AUC is 0.799. The non-invasive auxiliary diagnosis of the metastatic breast cancer is carried out by using a small amount of serum samples, so that large-scale breast cancer screening is facilitated, and early discovery, early intervention and early treatment of the breast cancer are facilitated.

Description

Technical Field

[0001] This invention relates to the field of laboratory medicine technology, specifically to the application of IGSF3 as a serum biomarker in the preparation of diagnostic reagents for breast cancer, particularly in the preparation of diagnostic reagents for metastatic breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, posing a significant threat to their health. Cancer metastasis, a key factor leading to patient mortality, involves a complex, multi-stage process. When primary tumor cells develop into distant metastases, it often indicates the disease has entered its terminal stage. This pathological process includes multiple biological behaviors such as tumor cells detaching from the primary site, invading surrounding tissues, evading immune clearance, and adapting to the new microenvironment. Clinical practice shows that early detection of breast cancer metastasis can significantly improve patient survival rates, but most cases are diagnosed after metastasis has already occurred. Because breast cancer has the characteristic of gradually developing from local lesions to lymph nodes or distant organs, clinical treatment faces enormous challenges, which is also a major reason for poor patient prognosis. Currently, the commonly used methods for clinical diagnosis of breast cancer are histopathological diagnosis and imaging diagnosis. These methods not only place a double burden on the diagnosticians psychologically and physically but also have technical limitations. Serum tumor marker testing is non-invasive and can be used to monitor treatment effectiveness, offering advantages in screening and early detection. Finding effective new methods for breast cancer diagnosis will greatly reduce the mortality rate of breast cancer.

[0003] Immunoglobulin superfamily member 3 (IGSF3) is an important cell adhesion molecule widely involved in intercellular interactions and signal transduction. Studies have shown that IGSF3 plays a crucial role in the development of the central nervous system, particularly in neural migration and synapse formation. In mouse models, the absence of IGSF3 did not significantly affect overall brain development, but its expression patterns in specific neuronal populations suggest that it may play a supporting role in neuronal maturation and functional integration.

[0004] Cancer biomarkers are any measurable molecular indicators of cancer occurrence, progression, transformation, and outcome. Currently, there are no studies on IGSF3 as a serum biomarker for diagnosing breast cancer. Summary of the Invention

[0005] This invention uses normal controls, patients diagnosed with non-metastatic breast cancer, and patients diagnosed with metastatic breast cancer as research subjects. The experiment found that the expression level of IGSF3 in the serum of patients with metastatic breast cancer was significantly higher than that in patients with non-metastatic breast cancer, while there was no significant difference in IGSF3 expression in the serum of normal control group and patients with non-metastatic breast cancer. Therefore, IGSF3 can be used as an effective serum biomarker for metastatic breast cancer.

[0006] The specific technical solution of this invention is as follows: This invention provides the application of IGSF3 as a serum biomarker in the preparation of breast cancer diagnostic products. The breast cancer is selected from non-metastatic and metastatic breast cancers identified clinically through histopathological diagnosis combined with multimodal imaging results.

[0007] In the above technical solution, the diagnostic product is further defined as a reagent or reagent kit.

[0008] In the above technical solution, the breast cancer is further described as metastatic breast cancer.

[0009] In the above technical solution, the diagnosis is further described as an auxiliary diagnosis, combined with histopathological diagnosis and multimodal imaging examination results.

[0010] The present invention provides a kit for diagnosing metastatic breast cancer, the kit comprising a reagent for detecting serum IGSF3 concentration.

[0011] Furthermore, in the above technical solution, the detection method of the reagent kit includes the following steps: (1) Separate serum; (2) Enzyme-linked immunosorbent assay (ELISA) was performed to determine the concentration of protein IGSF3; (3) Data analysis.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This study found that serum IGSF3 expression levels were significantly elevated in patients with metastatic breast cancer. IGSF3 can serve as a serum biomarker for diagnosing breast cancer metastasis. Compared to healthy controls and non-metastatic breast cancer patients, serum IGSF3 expression levels were significantly elevated in patients with metastatic breast cancer, with an AUC of 0.799 as shown in ROC curve analysis. Using a small number of serum samples for non-invasive auxiliary diagnosis of metastatic breast cancer facilitates large-scale breast cancer screening, enabling early detection, intervention, and treatment. Attached Figure Description

[0013] Figure 1 ELISA analysis was performed on the expression of IGSF3 in normal controls, patients with non-metastatic breast cancer, and patients with metastatic breast cancer.

[0014] Figure 2 ROC curves of serum IGSF3 in the normal control group and the non-metastatic breast cancer patient group.

[0015] Figure 3 ROC curves of serum IGSF3 in patients with non-metastatic breast cancer and patients with metastatic breast cancer.

[0016] Figure 4 RT-PCR was used to detect the expression of IGSF3 in normal breast cells and breast cancer cell lines.

[0017] Figure 5 Immunohistochemical assays were used to detect the expression of IGSF3 protein in breast cancer tumor tissue and adjacent normal breast tissue. Detailed Implementation

[0018] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0019] 1. Experimental design (IGSF3 can be used as a biomarker for diagnosing breast cancer metastasis) This experiment involved collecting serum samples from patients diagnosed with breast cancer at Liaoning Cancer Hospital and healthy individuals undergoing physical examinations. Serum was then separated. The expression of IGSF3 protein was detected using an ELISA kit, and the differences in its expression between healthy individuals and breast cancer patients were statistically analyzed to assess its diagnostic efficacy.

[0020] 2. Patients and samples involved in the experiment Breast cancer patients and healthy individuals who visited the hospital in November 2023 and June 2024 were selected. Breast cancer patients were defined as women who had not received any treatment, were pathologically diagnosed with breast cancer, and were all female. A total of 110 subjects participated, divided into a control group and 30 healthy individuals. The breast cancer group was further divided into non-metastatic and metastatic breast cancer, with 14 cases of non-metastatic breast cancer and 66 cases of metastatic breast cancer. All patients had not received any anti-tumor treatment or had any other endocrine, immune, or metabolic diseases before peripheral blood collection. Healthy individuals did not exhibit breast cancer or other tumors or definitively diagnosed diseases. Breast cancer patients were staged according to TNM staging principles and classified as non-metastatic or metastatic breast cancer. After admission, informed consent was obtained from the patients, and they were instructed to fast in the morning. Peripheral venous blood was collected using disposable vacuum blood collection tubes and centrifuged at 4000 rpm for 10 minutes at 4°C to obtain serum. The serum was then frozen and stored at -80°C for later use.

[0021] Table 1 shows the general clinical data of non-metastatic and metastatic breast cancer patients.

[0022] Table 1

[0023] 3. Enzyme-linked immunosorbent assay (ELISA) to detect serum IgG5 protein concentration: The expression level of IGSF3 in serum was determined using a human IGSF3 ELISA kit (YJ589307, Shanghai Enzyme-Linked Biotechnology, China). The experimental procedure was performed according to the kit's instructions, and dilutions were made according to the sample reagents.

[0024] (1) Dilution of standard: The kit provides one tube of original standard. Dilute it in a small test tube according to Table 2 below.

[0025] Table 2

[0026] (2) Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same), standard wells, and sample wells. Accurately add 50 μl of standard to the enzyme-labeled plate. Add 40 μl of sample diluent to the sample wells, and then add 10 μl of sample to the sample wells (the final sample dilution is 5 times). When adding the sample, place it at the bottom of the well, avoiding touching the well wall as much as possible, and gently shake to mix.

[0027] (3) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0028] (4) Solution preparation: Dilute the 30-fold (20 times that of 48T) concentrated washing solution with 30 times (20 times that of 48T) distilled water and set aside. (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.

[0029] (6) Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells. (7) Incubation: The procedure is the same as (3).

[0030] (8) Washing: Same as (5).

[0031] (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 color at 37°C in the dark for 10 minutes.

[0032] (10) Termination: Add 50 μl of stop solution to each well to terminate the reaction (at this time, the blue color will immediately turn yellow).

[0033] (11) Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0034] Test results as follows Figure 1 As shown in Table 3, compared with healthy individuals and patients with non-metastatic breast cancer, the serum IGSF3 expression level was significantly increased in patients with metastatic breast cancer.

[0035] Table 3. Number of participants in the normal control group, non-metastatic breast cancer patient group, and metastatic breast cancer patient group, and expression of IGSF3 protein in each group.

[0036]

[0037] 4. Statistical Analysis This study primarily used SPSS software (IBM SPSS Statistics 27, USA) for statistical analysis of the experimental data and Graphpad Prism 10 (Graphpad Prism 10, USA) for data visualization. For normally distributed measurement data, the mean ± standard deviation (x̄ ± s) was used, and independent samples t-tests were employed for comparisons between groups. Non-normally distributed data were expressed as median (range), and Mann-Whitney U test (for two groups) or Kruskal-Wallis test (for multiple groups) were used for comparisons between groups. A p-value less than 0.05 was considered statistically significant.

[0038] The diagnostic value of serum IGSF3 was systematically evaluated using receiver operating characteristic (ROC) curves. The optimal diagnostic cut-off value was determined by calculating the maximum value of the Youden index (J = sensitivity + specificity - 1), thus deriving the optimal sensitivity and specificity of IGSF3 in diagnosing breast cancer. Simultaneously, the overall diagnostic efficacy of IGSF3 was comprehensively evaluated by quantifying the area under the ROC curve (AUC): an AUC value closer to 1 indicates higher diagnostic accuracy; an AUC in the 0.7-0.9 range suggests moderate diagnostic value, while an AUC above 0.9 indicates high diagnostic value.

[0039] like Figure 2 As shown, the area under the ROC curve (AUC) of IGSF3 was 0.432 (95% CI = 0.245–0.620) in both the normal control group and the non-metastatic breast cancer patient group. p =0.473), which does not have diagnostic efficacy, while Figure 3 The results showed that the area under the ROC curve (AUC) was 0.799 (95% CI = 0.685–0.912) in patients with non-metastatic breast cancer and patients with metastatic breast cancer. p The result (<0.001) indicates good diagnostic efficacy. These results suggest that serum IGSF3 can serve as a diagnostic marker for metastatic breast cancer with good efficacy.

[0040] 5. RT-PCR (1) Total RNA extraction: 1) Place the culture dish in a ventilated area, discard the culture medium, wash the cell surface twice with 1×PBS, discard the PBS, add 1 mL of RNAiso Plus, use a pipette to blow the cells until they are completely dissolved in RNAiso Plus, and transfer all the liquid in the culture dish to a sterile 1.5 mL centrifuge tube without enzymes. 2) Let stand at room temperature for 5 minutes; 3) Add 200 μL of chloroform and shake vigorously for 15 s to mix. 4) Let stand at room temperature for 15 minutes; 5) Transfer to a pre-cooled centrifuge at 4°C and centrifuge at 12,000 rpm for 15 min; 6) Carefully transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol to the supernatant, and gently tumble to mix. 7) Let stand at room temperature for 10 minutes; 8) Transfer to a pre-cooled centrifuge at 4°C and centrifuge at 12,000 rpm for 10 min; 9) Carefully aspirate the supernatant, add 1 mL of 75% ethanol to the precipitate, and gently invert the centrifuge tube so that the precipitate at the bottom of the tube floats in the liquid; 10) Transfer to a pre-cooled centrifuge at 4°C and centrifuge at 5000 rpm for 3 min; 11) Carefully aspirate the supernatant, keep the tube cap open, and allow the liquid to air dry in a fume hood until the white precipitate becomes transparent; 12) Add 30 μL of ddH2O to completely dissolve the precipitate; 13) Measure and record the RNA concentration using a spectrophotometer. Store at -80℃.

[0041] (2) cDNA synthesis Using the reverse transcription system from Nanjing Novizan Biotechnology Co., Ltd.: Add 1 pg-1 μg template RNA, 4 μL 4×g DNA wiper mix, and RNase-free ddH2O to a final volume of 16 μL. Gently mix by pipetting. Incubate at 42℃ for 2 min.

[0042] Add 4 μL of 5×HiScnpt III qRT SuperMix to 16 μL of the previous reaction solution to form a 20 μL reaction system. Incubate at 37℃ for 15 sec, then at 85℃ for 4 min. Store the synthesized cDNA at -20℃.

[0043] (3) RT-PCR 1) Design primers for IGSF3 and GAPDH. The primer sequences are shown in Table 2.4.

[0044] Table 4 Primer sequences for IGSF3 and GAPDH

[0045] 2) The PCR reaction system is shown in the table below: Table 5 PCR reaction system

[0046] 3) PCR reaction procedure:

[0047] 4) Detection of PCR products by agarose gel electrophoresis.

[0048] RT-PCR experiments concluded that IGSF3 expression was significantly increased in breast cancer cells compared to normal breast cells.

[0049] 6. Immunohistochemistry (1) Dewaxing The tissue microarray was immersed in xylene (I) and (II) solutions for 20 min in sequence.

[0050] (2) Rehydration The tissue chip was immersed in anhydrous ethanol, 95% ethanol, 90% ethanol and 80% ethanol in sequence for 5 min, and then transferred to ddH2O for 10 min.

[0051] (3) Staining 1) Wash the tissue chip twice with PBS for 5 min each time, then incubate it in citric acid solution at 96°C for 15 min; 2) After cooling to room temperature, wash the chip three times with PBS for 3 minutes each time, and then incubate it in a 3% H2O2 solution in a 37 ℃ water bath for 10 minutes. 3) Wash with PBS 3 times, 3 min each time, then incubate with 5-10% sheep serum for 15 min; 4) After discarding the sheep serum, add the primary antibody dilution solution and incubate overnight at 4°C; 5) Wash the chip with PBS, and add secondary antibody dilution solution and horseradish enzyme-labeled streptavidin in sequence. Incubate at 37°C for 30 min for each step. 6) Clean the chip, develop the color with DAB solution, then stop the reaction with ddH2O, then counterstain with hematoxylin, and wash with ddH2O 2-3 times. 7) Immerse the chip in 0.5% hydrochloric acid ethanol for 1 min to differentiate, then wash 2-3 times with ddH2O; 8) If the ammonia solution turns blue, wash with ddH2O 2-3 times.

[0052] (4) Dehydrated and transparent The tissue chip was immersed in 80% ethanol, 90% ethanol and 95% ethanol for 1 min in sequence, and then immersed in xylene for 10 min.

[0053] The slide was sealed and photographed under a microscope.

[0054] Immunohistochemical experiments revealed that IGSF3 expression in breast cancer tissue was significantly higher than that in adjacent normal tissue.

Claims

1. Application of IGSF3 as a serum biomarker in the preparation of breast cancer diagnostic products.

2. The application according to claim 1, characterized in that, The diagnostic product is a reagent or kit.

3. The application according to claim 1, characterized in that, The breast cancer mentioned is metastatic breast cancer.

4. The application according to claim 3, characterized in that, The diagnosis mentioned is an auxiliary diagnosis, which is combined with histopathological diagnosis and multimodal imaging examination results.

5. A reagent kit for diagnosing metastatic breast cancer, characterized in that, The kit includes reagents for detecting serum IGSF3 concentration.

6. The kit according to claim 5, characterized in that, The detection method of the kit includes the following steps: (1) Separate serum; (2) Enzyme-linked immunosorbent assay (ELISA) was performed to determine the concentration of protein IGSF3; (3) Data analysis.

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