Application of FABP5 / S100A9 protein complex in preparation of kit for rapidly detecting breast cancer metastasis

Abstract

The invention provides an application of an FABP5 / S100A9 protein complex in preparation of a kit for rapidly detecting breast cancer metastasis. According to the invention, the fact that the protein complex (FABP5 / S100A9) in the human serum exosome is related to breast cancer metastasis is found for the first time, the protein of the complex is obviously increased in the body of a patient with breast cancer metastasis, the metastasis of breast cancer is promoted, and the value of the protein complex in breast cancer metastasis detection is prompted. The invention also provides a kit for detecting the binding condition of the FABP5 / S100A9 complex in the serum of a patient and the protein expression level in the body of the patient. According to the expression level and coprecipitation condition of the complex protein, the metastatic breast cancer is measured, a basis is provided for formulation of a clinical precise treatment scheme and prognosis prediction of a patient, and wide application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of testing and diagnosis, and specifically relates to an application of a FABP5 / S100A9 protein complex in preparing a kit for rapid detection of breast cancer metastasis. Background Art

[0002] Breast cancer (BC) is the most common malignancy in women worldwide. It is a highly heterogeneous disease characterized by different molecular features and clinically divided into three major subtypes based on hormone receptor (ER and PR) and HER2 (ERBB2) status: luminal ER-positive and PR-positive, further subdivided into luminal A and B, HER2-positive, and triple-negative breast cancer (TNBC). Metastatic breast cancer affects approximately 30% of breast cancer patients and accounts for approximately 90% of breast cancer mortality. Common distant metastatic sites of breast cancer are bone (~70%), liver (~30%), brain (10%-30%), lung, and lymph nodes. As with other cancers, the pathogenesis of metastatic breast cancer requires the activation of important pathways to maintain proliferation, resistance to apoptosis, angiogenesis, immune resistance, and the migration and invasion capabilities of disseminated tumor cells (DTCs) required to reach distant metastatic sites.

[0003] Clinically, the main treatments for breast cancer include surgical resection, radiotherapy (hereinafter referred to as radiotherapy), chemotherapy and targeted therapy. Surgical treatment is mainly for patients with tumor volume within a certain range. The most commonly used method is breast-conserving surgery (accounting for 75%-85%), while multicenter invasive cancer, extensive intraductal carcinoma, etc. require total mastectomy. Some patients have achieved a remission rate and survival rate consistent with total mastectomy through a combination of breast-conserving local surgery + radiotherapy. Currently, the latest advances in molecular biology and immunology have enabled the development of highly targeted therapies for various forms of breast cancer. The main goal of targeted therapy is to inhibit specific targets / molecules that support tumor progression.

[0004] In multiple studies on different cancer types, FABP5 (Fatty acid-binding protein 5) inhibits apoptosis and promotes cancer cell proliferation, colony formation, epithelial-mesenchymal transition (EMT), migration, cell invasion, and angiogenesis in vitro. In vivo, this translates into increased tumor growth, metastasis, and decreased survival. The mechanism by which this occurs mainly involves FABP5-mediated activation of various TFs. This leads to increased expression of key proteins involved in cell survival, prevention of apoptosis, cell proliferation, migration and invasion, lipid storage, and angiogenesis.

[0005] S100A9 is upregulated in multiple cancer types, including breast cancer (invasive ductal carcinoma), colitis-associated colon cancer, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, colorectal cancer, breast apocrine carcinoma, non-small cell lung cancer, and squamous cervical cancer. S100A9 levels are also elevated in nasopharyngeal carcinoma stroma and bladder transitional cell carcinoma. S100A9 may have both tumor-promoting and tumor-suppressive roles in breast cancer. In invasive breast ductal carcinoma, S100A9 immunopositivity correlated with mitotic activity, MIB-1 proliferation index (recombinant Ki-67 monoclonal antibody), HER2 overexpression, poor tumor differentiation, vascular invasion, lymph node metastasis, and poor pathological stage. S100A9 was associated with high grade, negative ER and PR status, high Ki67 and p53 expression, and ERBB2 and EGFR expression. S100A9 expression was also closely associated with the 10-protein basic signature CK5 / 6, CD10, EGFR, CAV1, CD44, ETS1, MET, Moesin, GATA3, and CK19.

[0006] Currently, there is no relevant research on the FABP5 / S100A9 protein complex in breast cancer metastasis. In this patent, a series of experiments prove that the FABP5 / S100A9 complex is upregulated in patients with breast cancer metastasis and the two are co-precipitated in the patient's body. Summary of the invention

[0007] The purpose of the present invention is to solve the problem of diagnosing metastatic breast cancer and to provide an application of a FABP5 / S100A9 protein complex in preparing a kit for rapidly detecting breast cancer metastasis.

[0008] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides application of FABP5 / S100A9 protein complex in preparing a kit for rapid detection of breast cancer metastasis.

[0009] The rapid detection kit for breast cancer metastasis uses an immunoprecipitation method to detect the expression amount and coprecipitation of the FABP5 / S100A9 protein complex in the serum of patients with breast cancer metastasis.

[0010] The rapid detection kit for breast cancer metastasis comprises a rabbit-derived FABP5 polyclonal antibody, a rabbit-derived S100A9 polyclonal antibody and an immunoprecipitation detection reagent.

[0011] The immunoprecipitation detection reagent includes Protein A / G magnetic beads, IP Lysis / Wash Buffer, PBS (pH=7.2-7.4) and 5× SDS-PAGE protein loading buffer.

[0012] Compared with the prior art, the present invention has the following beneficial effects: By jointly detecting the protein expression levels of FABP5 and S100A9, the binding of FABP5 and S100A9 in the serum of breast cancer patients was detected and analyzed, and metastatic breast cancer was screened out, providing important basis for the precise diagnosis and treatment of tumors and prognosis prediction. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the mass spectrum of the ELGVGIALR peptide of FABP5 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0014] Figure 2 This is the mass spectrum of the FEETTADGR peptide of FABP5 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0015] Figure 3 This is the mass spectrum of the FEETTADGRK peptide of FABP5 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0016] Figure 4 This is the mass spectrum of the DLQNFLK peptide of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0017] Figure 5 This is the mass spectrum of the LGHPDTLNQGEFK peptide of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0018] Figure 6 This is the mass spectrum of the LTWASHEK peptide of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0019] Figure 7 This is the mass spectrum of the MHEGDEGPGHHHKPGLGEGTP peptide of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0020] Figure 8 Western Blot verification of FABP5 / S100A9 protein expression levels in serum exosomes of healthy subjects, breast cancer patients without metastasis, and breast cancer patients with metastasis; n=10, **** indicates P<0.0001.

[0021] Fig. 9 The binding and expression of FABP5 / S100A9 complex in patients with breast cancer metastasis.

[0022] Fig.10The binding and expression of FABP5 / S100A9 complex in clinical breast cancer metastasis patients. DETAILED DESCRIPTION

[0023] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiment. The specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0024] Example 1 (1) Collection and processing of serum samples: Serum exosomes were extracted from the patient's serum and used for the detection of metastatic breast cancer. The patient's serum exosomes were extracted using the Exosome Extraction and Purification Kit (Blood) (Umibio, UR52136). The specific steps are as follows: First, take the serum sample frozen at -80℃ out of the refrigerator and thaw it in a 25℃ water bath. Place the completely thawed sample on ice. Take 200ul of the sample into a centrifuge tube, centrifuge it at 10000g for 10min at 4℃ to remove impurities in the sample, and transfer the supernatant after centrifugation to a new centrifuge tube. First, add 1ml of 1×PBS pre-cooled on ice to dilute the centrifuged supernatant after removing impurities, then add Blood PureExo Solution (BPS), immediately cover the centrifuge tube, mix it on a vortex oscillator for 1min, and then let it stand at 4℃ for 2h. Then centrifuge it at 10000g for 60min at 4℃, aspirate the supernatant as much as possible, and the precipitate is rich in exosome particles. Take 100ul 1×PBS and blow the centrifuged precipitate evenly. After it dissolves, transfer the resuspended liquid to a new 1.5ml centrifuge tube, centrifuge at 12000g for 2min at 4℃, transfer the supernatant to the upper chamber of ExosomePurification Filter (EPF column), centrifuge at 3000g for 10min at 4℃, collect the liquid at the bottom of the EPF column after centrifugation, and this liquid is the purified exosome particles. If the purified exosomes are not used immediately, they can be stored in a -80℃ refrigerator for subsequent experiments.

[0025] (2) Mass spectrometry detection Firstly, serum exosomes were extracted from the serum of healthy people, breast cancer patients without metastasis and breast cancer patients with metastasis, and then subjected to mass spectrometry detection. Each group of samples contained blood samples from 10 people. Figure 1-3 and Figure 4-7 This is the mass spectrometry of FABP5 and S100A9, confirming the accuracy of these two proteins. The full length of FABP5 amino acids is: MATVQQLEGRWRLVDSKGFDEYMK EL GVGIALRKMGAMAKPDCIITCDGKNLTIKTESTLKTTQFSCTLGEK FEETTADGRK TQTVCNNFTDGALVQHQEWDGKESTITRKLKDGKLVVECVMNNVTCTRIYEKVE, Figure 1 , Figure 2 and Figure 3 The peptide segments are ELGVGIALR, FEETTADGR, and FEETTADGRK, which correspond to the underlined positions in the above FABP5 amino acid full length, so this protein is determined to be FABP5. The amino acid full length of S100A9 is: MTCKMSQLERNIETIINTFHQYSVK LGHPDTLNQGEFK ELVRK DLQNFL K KENKNEKVIEHIMEDLDTNADKQLSFEEFIMLMAR LTWASHEKMHEGDEGPGHKP GLGEGTP , Figure 4-Figure 7 The peptide segments are DLQNFLK, LGHPDTLNQGEFK, LTWASHEK, and MHEGDEGPGHHHKPGLGEGTP, which correspond to the underlined positions in the full length of the above-mentioned S100A9 amino acids, so this protein is determined to be S100A9. Table 1 shows that the expression levels of FABP5 and S100A9 proteins in the serum of patients with breast cancer metastasis are significantly increased compared with healthy people and patients with breast cancer without metastasis. This indicates that FABP5 and S100A9 play an important role in the metastasis of breast cancer, and the two may be combined.

[0026] Western Blot was used to verify the expression level of FABP5 / S100A9 protein in serum exosomes of healthy people, breast cancer patients without metastasis, and breast cancer patients with metastasis. The extracted serum exosomes were mixed with exosome protein lysis buffer (Umibio, UR333101) in a ratio of 1:1, lysed on ice for 10 minutes, centrifuged at 12000g for 5 minutes, and the supernatant was taken for BCA quantification and Western Blot experiment. First, 30ug of sample was taken from each group in a new EP tube, and an appropriate amount of 5× protein loading buffer was added to make the final concentration 1×, incubated at 95℃ for 5 minutes to fully denature the protein sample, and cooled at room temperature. After loading, SDS-PAGE gel electrophoresis was performed, first running the electrophoresis at 60V voltage until the end of the concentrated gel, and then running the separation gel at 120V voltage. Then the membrane was wet-transferred, and the PVDF membrane was activated with methanol before use. The transfer conditions were ice bath, 100V, 35min. After transfer, wash the membrane with TBS for 10 minutes, block with TBST containing 5% skim milk powder for 1.5 hours, wash the membrane with TBST 3 times, 10 minutes each time; place the membrane in the primary antibody working solution of FABP5 (1:500) and S100A9 (1:500), incubate overnight at 4°C; wash the membrane with TBST 3 times, 10 minutes each time; place the membrane in the corresponding secondary antibody working solution, incubate at room temperature for 1.5 hours; wash the membrane with TBST 3 times, 10 minutes each time; wash the membrane with TBS for 10 minutes; place the membrane in the 1:1 ECL chemiluminescent colorimetric solution, incubate at room temperature for 2 minutes away from light. Preheat the chemiluminometer, place the membrane on the chemiluminometer panel, and observe the color development.

[0027] The results are as follows Figure 8 As shown, the FABP5 and S100A9 proteins in the serum exosomes of patients with breast cancer metastasis were significantly increased compared with healthy people and patients with breast cancer without metastasis, verifying the accuracy of the mass spectrometry results.

[0028] Example 2 In this embodiment, the kit for detecting the expression level of FABP5 and S100A9 proteins in serum includes rabbit FABP5 and S100A9 polyclonal antibodies and immunoprecipitation detection reagents. The ordering companies and product numbers of the antibodies for the two protein detections are: FABP5 (Proteintech Group, 12348-1-AP) and S100A9 (Proteintech Group, 26992-1-AP).

[0029] Immunoprecipitation detection reagents include: PBS (pH = 7.2-7.4), Protein A / G magnetic beads (Medchemexpress, HY-K020), IP Lysis / Wash Buffer (Biolinkedin, L-7101), PMSF (Beyotime, ST507-10ml), Phosphatase inhibitor cocktail Ⅱ (Medchemexpress, HY-K0022), and SDS-PAGE protein loading buffer (5×) (Beyotime, P0015).

[0030] The specific detection steps are as follows: (1) Take 200 µL of patient serum, add 300 ul PBS to make up to 500 µL, then add an equal amount of IP Lysis / Wash Buffer, 5 ul PMSF, and 10 ul Phosphatase inhibitor cocktail Ⅱ, mix well, place on ice for 20 min, centrifuge at 4°C, 12000 g for 10 min, and collect the supernatant. Keep 200 µl of the supernatant as the input group.

[0031] (2) Add 4 μl of FABP5 antibody and incubate overnight at 4°C to form an antigen sample / antibody mixture.

[0032] (3) Mix the Protein A / G magnetic beads by inverting repeatedly. Take 30 µL of magnetic beads into a new 1.5 mL centrifuge tube, add 600 µL of ice-cold PBS, mix gently, collect the magnetic beads with a magnetic rack, remove the supernatant and add 400 µL of IPLysis / Wash Buffer to the centrifuge tube. Gently invert the centrifuge tube for 1 min, collect the magnetic beads with a magnetic rack, and remove the supernatant.

[0033] (4) Add the antigen sample / antibody mixture to the centrifuge tube containing the magnetic beads, place it on a rotating mixer, and incubate at room temperature for 2 h.

[0034] (5) Collect the magnetic beads using a magnetic rack, remove the unbound sample, add 1000 µL IP Lysis / Wash Buffer to the centrifuge tube, gently mix the magnetic beads for 10 min, collect the magnetic beads using a magnetic rack, remove the supernatant, and repeat the operation twice.

[0035] (6) Take 20 µL of SDS-PAGE protein loading buffer (5×) and add 80 µL of PBS to dilute the SDS-PAGE protein loading buffer concentration to 1×.

[0036] (7) Add the diluted 1× SDS-PAGE protein loading buffer to the precipitate obtained in step (5), heat in a 100°C metal bath for 10 min, separate the magnetic beads using a magnetic rack, and retain the supernatant containing the target antigen, which is the IP group sample.

[0037] (8) Perform Western Blot detection on the obtained samples. FABP5 antibody (1:500) and S100A9 antibody (1:500) were incubated at the same time as the primary antibody. If FABP5 and S100A9 bands were found in both the Input group and the IP group during the final color development, it indicated that the two were bound together. If the FABP5 antibody was used for immunoprecipitation and the final result showed no S100A9 band in the IP group, it indicated that the two were not bound together. Similarly, if the S100A9 antibody was used for immunoprecipitation and the final result showed no FABP5 band in the IP group, it also indicated that the two were not bound together.

[0038] Fig. 9 The results show that FABP5 and S100A9 bind to each other in the serum of patients with breast cancer metastasis. FABP5 and S100A9 are highly expressed in the serum of patients with breast cancer, and the two co-precipitate, so as to screen for metastatic breast cancer.

[0039] After testing 10 random patient samples with this immunoprecipitation kit, it was found that the test results of 8 patients showed the presence of FABP5 / S100A9 complex. Some of the results are as follows: Fig.10 This result is consistent with the clinical diagnosis.

[0040] In summary, the kit for detecting FABP5 / S100A9 complex by immunoprecipitation of the present invention can be used for the identification of metastatic breast cancer by utilizing the characteristics that FABP5 and S100A9 are highly expressed at the same time and can form a complex.

[0041] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

[0042] Table 1 shows the mass spectrometry information of the protein complex (FABP5 / S100A9) in serum exosomes of healthy subjects, breast cancer patients without metastasis, and breast cancer patients with metastasis

[0043] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Application of FABP5 / S100A9 protein complex in the preparation of a rapid detection kit for breast cancer metastasis.

2. The use according to claim 1, characterized in that: The rapid detection kit for breast cancer metastasis uses an immunoprecipitation method to detect the expression amount and coprecipitation of the FABP5 / S100A9 protein complex in the serum of patients with breast cancer metastasis.

3. The use according to claim 1, characterized in that: The rapid detection kit for breast cancer metastasis comprises a rabbit-derived FABP5 polyclonal antibody, a rabbit-derived S100A9 polyclonal antibody and an immunoprecipitation detection reagent.

4. The use according to claim 3, characterized in that: The immunoprecipitation detection reagent comprises Protein A / G magnetic beads, IP Lysis / Wash Buffer, PBS buffer with a pH of 7.2-7.4 and 5× SDS-PAGE protein loading buffer.

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