Application of bone metastasis-specific secreted protein dkk3 in early diagnosis of lung cancer bone metastasis

Abstract

The application belongs to the technical field of biological medicine, discloses application of bone metastasis specific secreted protein DKK3 in early diagnosis of lung cancer bone metastasis, and specifically discloses application of a substance for detecting DKK3 in preparation of a product for diagnosing or early screening of lung cancer bone metastasis. The application discloses the relationship between secreted protein DKK3 expression and lung cancer specific bone metastasis for the first time, and verifies that secreted protein DKK3 is specifically highly expressed in serum samples of lung cancer bone metastasis patients and lung cancer bone metastasis cells, which indicates that secreted protein DKK3 can be used as a new lung cancer bone metastasis diagnostic marker, and can significantly distinguish lung cancer patients with bone metastasis from lung cancer patients with metastasis to other organs and healthy people.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of bone metastasis-specific secreted protein DKK3 in the early diagnosis of lung cancer bone metastasis. Background Technology

[0002] Invasive metastasis is a characteristic of malignant tumors, and the tendency for recurrence and metastasis in malignant tumors greatly complicates cancer treatment. Tumor metastasis is not only a sign of poor prognosis but also a leading cause of death in cancer patients; statistics show that over 90% of cancer patients die from tumor metastasis. Lung cancer is the malignant tumor with the highest incidence and mortality rate globally, with approximately 1.8 million new cases and 1.6 million deaths annually. Due to the diverse clinical manifestations of lung cancer, the lack of obvious early symptoms, and the absence of effective early diagnostic targets, over 70% of lung cancer patients have already experienced malignant progression, including local invasion, lymph node metastasis, or distant organ metastasis, at the time of diagnosis. The median survival for lung cancer patients with distant metastases is less than 5 months, and bone is the most common distant metastatic organ for lung cancer patients.

[0003] Because bone metastases are often asymptomatic in their early stages, many patients only seek medical attention when they experience bone pain or pathological fractures, indicating the presence of large metastatic lesions and a poor prognosis. In clinical practice, bone imaging techniques such as radionuclide bone scans, PET, PET-CT, and MRI are commonly used to diagnose bone metastases in cancer patients. However, bone imaging techniques have drawbacks such as high cost and delayed diagnosis, and also have limitations in sensitivity, specificity, and operability.

[0004] In recent years, with more in-depth research on bone metastasis of tumors, some progress has been made in the study of biomarkers for the diagnosis of bone metastasis. Researchers have found that the serum ProGRP level is significantly higher in patients with bone metastasis compared to prostate cancer patients without bone metastasis. Through prospective studies, researchers have found that the presence of osteocalcin-positive circulating tumor cells indicates that patients are more prone to bone metastasis. In patients with advanced lung cancer, the expression levels of proteins such as CXCR4, BSP, OPN, and BMP4 in tumor cells are positively correlated with the risk of bone metastasis. However, the above studies are currently all in the preclinical research stage, and there are currently no bone metastasis-specific tumor markers used clinically. Therefore, exploring the molecular mechanisms of lung cancer bone metastasis and finding new, highly sensitive early diagnostic biomarkers for bone metastasis has important clinical value. Summary of the Invention

[0005] The first aspect of the present invention is to provide the use of a substance for detecting DKK3 in the preparation of products for the diagnosis or early screening of lung cancer bone metastases.

[0006] The second objective of this invention is to provide the application of DKK3 as a biomarker in the preparation of products for the early diagnosis of lung cancer bone metastases.

[0007] A third aspect of the present invention aims to provide the use of an inhibitory agent of DKK3 in the preparation of drugs for the prevention, inhibition and / or bone metastasis of lung cancer.

[0008] The fourth aspect of this invention aims to provide the application of DKK3 as a target in screening drugs for the treatment or prevention of bone metastases in lung cancer.

[0009] The fifth aspect of this invention aims to provide a method for constructing an animal model of lung cancer bone metastasis.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the invention provides the use of a substance for detecting DKK3 in the preparation of a product for the diagnosis or early screening of lung cancer bone metastases.

[0012] Preferably, the substance includes a substance for the quantitative detection of DKK3.

[0013] Preferably, the substance for detecting DKK3 includes a substance for detecting DKK3 at the gene level and / or protein level.

[0014] Preferably, the substance used to detect DKK3 is a substance selected from one or more detection techniques or methods chosen from the group consisting of: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, immunoblotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin assay.

[0015] Preferably, the substance used to detect DKK3 is selected from: substances specific to DKK3, DKK3-specific probes, gene chips, PCR primers, etc.

[0016] Preferably, the substance specific to DKK3 is any one of (b1) to (b3):

[0017] (b1) Antibodies that specifically bind to DKK3;

[0018] (b2) Ligand proteins or peptides that specifically bind to DKK3;

[0019] (b3) Specific recognition of non-protein compounds of DKK3.

[0020] Preferably, the antibody includes at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody.

[0021] Preferably, the product comprises at least one of reagents, reagent kits, test strips, and chips.

[0022] Preferably, the subject's sample for the product can be selected from body fluid samples, tumor samples, and cell samples isolated from the subject.

[0023] Preferably, the body fluids include, but are not limited to, blood, plasma, serum, lymph, cerebrospinal fluid, synovial fluid, urine, saliva, and mucus; the tumor samples are in the form of, but are not limited to, living tissue, paraffin-embedded tissue, and frozen tissue; the cell samples can be selected from cells that can be isolated from the subject, such as peripheral blood mononuclear cells, T lymphocytes, B lymphocytes, and circulating tumor cells.

[0024] Preferably, when the DKK3 level of the subject is significantly elevated relative to a reference level, the subject is diagnosed with lung cancer bone metastasis; the reference level is the level of subjects of the same age who do not have lung cancer bone metastasis.

[0025] Preferably, the subjects in the same age group are healthy individuals and / or lung cancer patients without bone metastases.

[0026] In the medical field, the term "no bone metastasis" refers to the absence of bone metastasis. Generally speaking, when analyzing collected tumor samples, if the tumor sample does not have bone metastasis, it is classified as a tumor sample without bone metastasis. Those skilled in the art should know that a tumor sample without bone metastasis may be a tumor sample that has not metastasized at all, or it may be a tumor sample that has metastasized to other non-bone sites (such as the liver, brain, or lungs).

[0027] Preferably, the lung cancer includes non-small cell lung cancer and small cell lung cancer.

[0028] Preferably, the non-small cell lung cancer includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.

[0029] Preferably, the DKK3 is upregulated in patients with lung cancer bone metastases.

[0030] A second aspect of the present invention provides the use of DKK3 as a biomarker in the preparation of products for the early diagnosis of lung cancer bone metastases.

[0031] Preferably, the product includes at least one of reagents, reagent kits, test strips, and chips.

[0032] A third aspect of the invention provides the use of an inhibitory agent of DKK3 in the preparation of drugs for the prevention, inhibition, and / or bone metastasis of lung cancer.

[0033] Preferably, the DKK3 inhibitory agent comprises at least one of the following: a substance that inhibits DKK3 activity, a substance that degrades DKK3, and a substance that reduces the expression level of DKK3.

[0034] Preferably, the DKK3 inhibitory agent includes substances that can completely or partially inhibit the expression of the DKK3 gene, and / or substances that inhibit the function of the DKK3 protein.

[0035] Preferably, the inhibitory agent for PDKK3 is selected from: proteins, oligonucleotides, oligonucleotide expression vectors, and small molecule compounds.

[0036] Preferably, the expression vector includes a eukaryotic cell expression vector, which includes a plasmid expression vector or a viral expression vector.

[0037] A fourth aspect of the invention provides the use of DKK3 as a target in screening drugs for the treatment or prevention of bone metastases in lung cancer.

[0038] A fifth aspect of the present invention provides a method for constructing an animal model of lung cancer bone metastasis, comprising the following steps: placing an expression cassette, vector, or cell line overexpressing DKK3 into an animal to obtain an animal model of lung cancer bone metastasis.

[0039] Preferably, the vector includes a plasmid vector, a viral vector, or a eukaryotic expression vector.

[0040] Preferably, the plasmid vector can be an optional plasmid, and the eukaryotic expression vector does not include propagation material.

[0041] Preferably, the viral vector can be any suitable vector, including but not limited to retroviral vectors, adenovirus vectors, adenovirus-associated virus vectors, herpesvirus (e.g., herpes simplex virus, vaccinia virus, and EB virus) vectors, and alphavirus vectors.

[0042] Preferably, the eukaryotic expression vector can be any suitable expression vector, including but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEF Bos expression vector, pTet expression vector, pTRE expression vector, or a vector modified based on a known expression vector, such as pBin438, pCAMBIA1301, etc.

[0043] The beneficial effects of this invention are:

[0044] This invention discloses for the first time the relationship between the expression of secretory protein DKK3 and lung cancer-specific bone metastasis, and verifies that secretory protein DKK3 is specifically highly expressed in serum samples of lung cancer patients with bone metastasis and in lung cancer bone metastasis cells, suggesting that secretory protein DKK3 can serve as a novel diagnostic biomarker for lung cancer bone metastasis, and can significantly distinguish lung cancer patients with bone metastasis from lung cancer patients with metastasis to other organs and healthy individuals.

[0045] In clinical applications, ELISA can be used to detect DKK3 levels in patients' blood samples to assess the risk of bone metastasis and enable earlier intervention. Compared with traditional methods for diagnosing bone metastasis, such as tissue biopsy or imaging screening, ELISA detection reveals significantly elevated DKK3 expression levels in the serum of lung cancer patients, suggesting a high probability of bone metastasis and allowing for more timely treatment, leading to better prognosis. Furthermore, it is more convenient and easier to perform, provides more sensitive results, and has lower experimental costs. From the patient's perspective, the non-invasive nature of serum ELISA significantly reduces both psychological and financial burdens. Attached Figure Description

[0046] Figure 1 The expression of DKK3 in lung cancer bone metastasis cells and serum of patients with lung cancer bone metastasis is shown in Figure 1. A represents the expression of DKK3 in lung cancer bone metastasis cells, B represents the expression of DKK3 in serum of patients with lung cancer bone metastasis, and C represents the ROC curve of DKK3 for the diagnosis of lung cancer bone metastasis.

[0047] Figure 2 The figure shows the results of an in vivo mouse experiment to detect the effect of DKK3 expression on lung cancer bone metastasis.

[0048] Figure 3 The expression level of DKK3 in lung cancer cells after treatment with DKK3 RNAi.

[0049] Figure 4 The figure shows the results of in vivo experiments in mice to detect the effect of targeted inhibition of DKK3 on lung cancer bone metastasis. Detailed Implementation

[0050] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0051] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Experimental methods without specific conditions are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0052] Example 1: Expression of DKK3 in lung cancer bone metastases and serum of patients with lung cancer bone metastases

[0053] The inventors' team screened and extracted PC9 bone metastasis (PC9-BM) cells and PC9 lung metastasis (PC9-LM) cells in mice. They found that DKK3 was significantly highly expressed in PC9-BM cells, suggesting that the expression of the secreted protein DKK3 is associated with lung cancer bone metastasis and could serve as a hematological molecular diagnostic marker for early lung cancer bone metastasis. The inventors used enzyme-linked immunosorbent assay (ELISA) to detect DKK3 expression in the supernatant of lung cancer-specific organ metastasis subcell lines and in the serum of lung cancer bone metastasis patients, verifying the specific secretion of DKK3 in bone metastasis cells and the serum of lung cancer bone metastasis patients. The specific steps included:

[0054] 1. Collect cell supernatant / extract patient serum

[0055] Collect cell supernatant: at 2 × 10 6 PC9 parental cells (PC9-PR), PC9-BM, and PC9-LM were seeded at different densities in 100×20mm culture dishes. After cell adhesion at 37℃ and 5% CO2, the culture medium was discarded, and the cells were washed twice with PBS, which was then discarded. 8 mL of serum-free culture medium (Opti-MEM, Gibco, Catalog: #31985070) was added for further culture. After 48 hours, the cell supernatant was collected and filtered through a 0.45μm filter to remove cell debris. The cell filtrate was collected and used immediately for subsequent experiments or stored at -40℃ for later use.

[0056] Patient serum extraction (separation of serum from freshly collected whole blood): Whole blood was collected from 20 healthy individuals, 33 patients with early-stage lung cancer without distant metastasis, and 45 patients with lung cancer with bone metastasis (from Sun Yat-sen University Cancer Center) in anticoagulant tubes (containing EDTA) and incubated overnight at 4°C. After incubation, stratification was observed, with plasma at the bottom and serum at the top. The clear yellow serum layer at the top was aspirated into centrifuge tubes and centrifuged at 2500 rpm for 30 minutes at 4°C. The precipitate was discarded, and the serum component at the top was aspirated to obtain patient serum, which was immediately used for subsequent experiments or stored at -80°C for later use.

[0057] 2. ELISA was used to detect the expression level of DKK3 in cell supernatant and serum samples.

[0058] The DKK3 protein in cell supernatant and serum samples was detected using the Duoset series ELISA kit (R&D, Catalog: #DY1118) from R&D Corporation. The specific procedures are as follows:

[0059] (1) Dilute the capture antibody (R&D, Catalog: #842225) with PBS solution to the working concentration (2 mg / mL), add 100 μL to each well of an ELISA 96-well plate, and incubate overnight at room temperature (20-30°C);

[0060] (2) Discard the capture antibody in the ELISA 96 wells, wash each well 3 times with 400 μL of ELISA wash buffer, and after the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0061] (3) Add 300 mL of Reagent Diluent to each well, incubate at room temperature for 1 hour, and then seal the well.

[0062] (4) Discard the Reagent Diluent in the wells and wash each well three times with 400 μL ELISA wash buffer each time. After the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0063] (5) DKK3 protein standard (R&D, Catalog: #842227) was diluted to concentrations of 31.3 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, 500 pg / mL, 1000 pg / mL and 2000 pg / mL according to the instructions. The test sample and the diluted DKK3 protein standard were added to 100 μL of each well of an ELISA 96-well plate and incubated at room temperature for 2 hours.

[0064] (6) Discard the sample in the well and wash each well 3 times with 400 μL ELISA wash buffer; after the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the well.

[0065] (7) Add 100 μL of 100 ng / mL detection antibody (Detection antibody, R&D, Catalog: #842225) to each well and incubate at room temperature for 2 hours;

[0066] (8) Discard the liquid in the wells and wash each well three times with 400 μL ELISA wash buffer; after the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0067] (9) Add 100 μL Streptavidin-HRP (200×) to each well and incubate at room temperature in the dark for 20 minutes;

[0068] (10) Discard the liquid in the wells and wash each well three times with 400 μL ELISA wash buffer. After the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0069] (11) Add 100 μL of substrate solution to each well for color development. After adding the substrate solution, incubate at room temperature in the dark for 20 minutes.

[0070] (12) Add 50 μL of colorimetric stop solution to each well to stop the reaction. After adding the stop solution, gently tap the plate to mix it completely.

[0071] (13) Immediately after termination, use a multi-functional microplate reader to detect the absorbance value at 450 nm, calculate the regression curve based on the OD450 nm reading of the standard, and calculate the protein concentration of the sample to be tested.

[0072] The results are as follows Figure 1 As shown, DKK3-specific expression is upregulated in lung cancer bone metastases ( Figure 1 The expression of DKK3 in the serum of lung cancer patients with bone metastases was upregulated compared to that in healthy individuals and patients with early-stage lung cancer without bone metastases (p<0.001). Figure 1 (B) and was statistically significant (p<0.001).

[0073] To further evaluate the diagnostic value of the secreted protein DKK3 in the early stage of lung cancer bone metastasis, ROC curves were used to predict the bone metastasis status of lung cancer patients. The results are as follows: Figure 1As shown in Figure C, the inventors found that the AUC of secreted protein DKK3 in distinguishing between bone metastasis and non-bone metastasis (including healthy individuals and early-stage non-metastasis) was 0.8138, with a 95% CI (0.7321–0.8956); the AUC in distinguishing between healthy individuals and early-stage non-bone metastasis was 0.9394, with a 95% CI (0.858–1.021); and the AUC in distinguishing between early-stage non-bone metastasis and bone metastasis was 0.7017, with a 95% CI (0.5869–0.8165). This indicates that secreted protein DKK3 has good accuracy and specificity in predicting early diagnosis of lung cancer patients.

[0074] Example 2: In vivo mouse experiment to detect the effect of DKK3 expression on lung cancer bone metastasis

[0075] 1. Establishment of a lung cancer cell line with stable high expression of DKK3 (PC9 DKK3 cells)

[0076] Viral solutions (cell culture medium containing viral plasmid particles, referred to as viral solutions) were prepared using lentiviral vectors. The viral transfection experimental method is described below using liposome transfection as an example.

[0077] Preparation before transfection: Prepare a cell count of approximately 2.0–3.0 × 10⁶ cells. 6 Personal embryonic kidney cells (293FT cells) were seeded into 100×20mm cell culture dishes. After 24 hours, the cell status was observed under a microscope to ensure that the cells were in good condition. The transfection system was prepared as follows: Solution A and Solution B. Solution A: 6μg psPAX2 (Addgene, Catalog:#12260), 4μg pMD2.G (Addgene, Catalog:#12269), 10μg DKK3 high expression plasmid (Vezhen Biotechnology Co., Ltd., Catalog:#CH804339), 15μL p3000 (Thermo, Catalog:#L3000-015), and 500μL Opti-MEM (Gibco, Catalog:#31985070). Solution B: 500μL Opti-MEM and 20μL lipofectamine 3000 (Thermo, Catalog:#L3000-015).

[0078] Transfection: Gently mix solution A and solution B thoroughly, let stand at room temperature for 15-20 minutes, and slowly and evenly add the mixed solution to the well-grown 293FT cells;

[0079] Viral fluid collection: 24 hours after transfection, the viral fluid was collected using a 10 mL syringe and filtered through a 0.45 μm pore size filter to remove cell debris. The filtrate was collected as the viral fluid. At the same time, fresh culture medium was added to the 293FT cells and the cells were cultured for another time.

[0080] Cell preparation for infection: On the day of infection, the cell count should be approximately 1.5 × 10⁻⁶. 5 The cells to be infected (PC9 cells) were evenly spread to a depth of 25 cm. 2 In cell culture flasks;

[0081] Cell infection: Add 40 mg / mL polybrene solution to the collected virus solution at a volume ratio of 1:1000, mix well, and then infect PC9 cells. Collect the virus solution every 8 to 12 hours for three consecutive days.

[0082] Screening for positive cells: After three consecutive days of infection, Puromycin solution (10 mg / mL) was added to the culture medium at a volume ratio of 1:1000 to screen for positive cells. The culture medium containing Puromycin was then added to the infected cells to screen for positive cells. After 5–7 days of screening, once the positive cells had stabilized, they were collected. RNA was extracted from the cells using the Trizol method, and protein was extracted using SDS protein lysis buffer (Beyotime, Catalog: #P0013G). The efficiency of the stable overexpression cell line was verified by qRT-PCR and Western blot experiments. Cells that successfully expressed the positive cells were then expanded or cryopreserved to obtain PC9 Vector cells and PC9 DKK3 cells.

[0083] 2. In vivo mouse assay to detect the effect of DKK3 on lung cancer bone metastasis

[0084] Preparation of tumor cell suspension: PC9 Vector and PC9 DKK3 cells were digested and counted, and the cells were resuspended in PBS solution to a cell concentration of 5 × 10⁻⁶. 5 / 100μL;

[0085] Ten 6-8 week old balb / c-nu / nu mice were selected and randomly divided into two groups: the PC9Vector group (injected with PC9 Vector cells) and the PC9 DKK3 group (injected with PC9 DKK3 cells). Each mouse was anesthetized by intraperitoneal injection of 100 μL of 4% chloral hydrate. Tumor cell suspension was injected into the left ventricle of the mice, 100 μL per mouse, taking care to avoid air bubbles entering the syringe. Blood was sprayed out of the syringe when the injection was accurate. After injection, the mice were routinely cultured and tumor metastasis was continuously observed for 2-6 weeks using small animal in vivo imaging.

[0086] The results are as follows Figure 2As shown, 4 weeks after tumor cell inoculation, the PC9 DKK3 group showed significantly enhanced bone metastasis ability of lung cancer cell lines compared to the PC9 Vector group, indicating that DKK3 plays an important role in the bone metastasis process of lung cancer.

[0087] Example 3: Effect of RNAi inhibition on DKK3 expression levels in bone metastatic lung cancer cells

[0088] 1. Treatment of lung cancer cells with DKK3 RNAi

[0089] (1) With 1.5×10 5 PC9 cells were seeded into 6-well plates at a density of 37°C and 5% CO2 until the cells adhered. The culture medium was then discarded, and the cells were washed twice with PBS. The PBS was then discarded. 2 mL of serum-free culture medium (Opti-MEM, Gibco, Catalog: #31985070) was added and the cells were cultured for another time.

[0090] (2) PC9 cells were transiently expressed with DKK3 RNAi using liposome transfection.

[0091] Prepare the transfection system: Solution A and Solution B. Solution A: 0.1 nmol of DKK3 RNAi (Raybot Biotechnology Co., Ltd., Catalog:#SIGS0010769-1) or negative control (NC, Raybot Biotechnology Co., Ltd., Catalog:#siN0000001-1-10), 10 μL of p3000 (Thermo, Catalog:#L3000-015), and 200 μL of Opti-MEM (Gibco, Catalog:#31985070). Solution B: 500 μL of Opti-MEM and 20 μL of lipofectamine 3000 (Thermo, Catalog:#L3000-015).

[0092] Transfection: Gently mix solution A and solution B evenly, let stand at room temperature for 15-20 minutes, and slowly and evenly add the mixed solution to the PC9 cells in (1); 4-6 hours later, remove the culture medium containing the transfection system and add fresh DMEM culture medium again;

[0093] Forty-eight hours after transfection, the cell supernatant was collected and filtered through a 0.45 μm pore size filter to remove cell debris. The filtrate was then transferred into a 10 kDa ultrafiltration tube (Catalog: UFC501096) for ultrafiltration. The supernatant was centrifuged at 4 °C for 30 minutes to obtain a concentrated cell supernatant with a final volume of about 100 mL. This supernatant was then used immediately for subsequent experiments or stored at -40 °C for later use.

[0094] 2. ELISA was used to detect the expression level of DKK3 in cell supernatant.

[0095] The expression level of DKK3 protein in the supernatant of PC9 cells after DKK3 RNAi treatment was detected using an ELISA kit from R&D (R&D, Catalog: #DY1118). The specific procedures are as follows:

[0096] (1) Dilute the capture antibody (R&D, Catalog: #842225) with PBS solution to the working concentration (2 mg / mL), add 100 μL to each well of an ELISA 96-well plate, and incubate overnight at room temperature (20-30°C);

[0097] (2) Discard the capture antibody in the ELISA 96 wells, wash each well 3 times with 400 μL of ELISA wash buffer, and after the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0098] (3) Add 300 mL of Reagent Diluent to each well, incubate at room temperature for 1 hour, and then seal the well.

[0099] (4) Discard the Reagent Diluent in the wells and wash each well three times with 400 μL ELISA wash buffer each time. After the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0100] (5) DKK3 protein standard (R&D, Catalog: #842227) was diluted to concentrations of 31.3 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, 500 pg / mL, 1000 pg / mL and 2000 pg / mL according to the instructions. The test sample and the diluted DKK3 protein standard were added to 100 μL of each well of an ELISA 96-well plate and incubated at room temperature for 2 hours.

[0101] (6) Discard the sample in the well and wash each well 3 times with 400 μL ELISA wash buffer; after the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the well.

[0102] (7) Add 100 μL of detection antibody (Detection antibody, R&D, Catalog: #842225) to each well and incubate at room temperature for 2 hours;

[0103] (8) Discard the liquid in the wells and wash each well three times with 400 μL ELISA wash buffer; after the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0104] (9) Add 100 μL Streptavidin-HRP (200×) to each well and incubate at room temperature in the dark for 20 minutes;

[0105] (10) Discard the liquid in the wells and wash each well three times with 400 μL ELISA wash buffer. After the last wash, invert the ELISA 96-well plate onto absorbent paper and gently tap the plate to completely remove any residual ELISA wash buffer from the wells.

[0106] (11) Add 100 μL of substrate solution to each well for color development. After adding the substrate solution, incubate at room temperature in the dark for 20 minutes.

[0107] (12) Add 50 μL of colorimetric stop solution to each well to stop the reaction. After adding the stop solution, gently tap the plate to mix it completely.

[0108] (13) Immediately after termination, use a multi-functional microplate reader to detect the absorbance value at 450 nm, calculate the regression curve based on the OD450 nm reading of the standard, and calculate the protein concentration of the sample to be tested.

[0109] The results are as follows Figure 3 As shown, compared with the negative control (nc) treatment, the use of DKK3 RNAi significantly inhibited the expression level of DKK3 in PC9 cells, and the effect was statistically significant (p = 0.0002), indicating that inhibiting the expression level of DKK3 using DKK3 RNAi can be a strategy for targeting DKK3.

[0110] Example 4: In vivo mouse experiment to detect the effect of DKK3 RNAi on lung cancer bone metastasis

[0111] PC9 cells were treated with DKK3 RNAi and a negative control, respectively, following the method described in Example 3. After 48 hours of treatment, PC9 cells treated with the negative control and DKK3 RNAi were digested and counted, and the cells were resuspended in PBS to a volume of 5 × 10⁶ cells / mL. 6 / mL. Ten 6-8 week old blb / c-nu / nu mice were selected and randomly divided into two groups: a control group (PC9 cells treated with negative control) and an experimental group (PC9 cells treated with DKK3 RNAi). PC9 cells were injected into the mice via ventricular injection, with 100 μL injected into each mouse. After injection, the mice were cultured routinely, and tumor metastasis was continuously observed for 2-6 weeks using small animal in vivo imaging.

[0112] The results are as follows Figure 4 As shown, DKK3 RNAi treatment significantly inhibited the progression of lung cancer bone metastasis in week 4, indicating that targeted inhibition of DKK3, a lung cancer-specific bone metastasis protein, can serve as a novel treatment for inhibiting lung cancer bone metastasis.

[0113] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of reagents for detecting DKK3 in the preparation of products for the diagnosis or early screening of lung cancer bone metastases; The reagent is a reagent for the specific quantitative detection of DKK3; The reagent for detecting DKK3 is a reagent that specifically detects DKK3 at the gene level and / or protein level.

2. The application according to claim 1, characterized in that, The reagents used to detect DKK3 are substances selected from one or more detection techniques or methods from the group consisting of: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, immunoblotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin assay.

3. The application according to claim 1 or 2, characterized in that, The product includes at least one of reagents, reagent kits, test strips, and chips.

4. The application according to claim 1 or 2, characterized in that, The subject samples for the product are selected from body fluid samples, tumor samples, and cell samples of the subjects to be tested.

5. A method for constructing an animal model of lung cancer bone metastasis, comprising the following steps: A cell line overexpressing DKK3 was placed in an animal to obtain a lung cancer bone metastasis animal model. The cell line was PC9 cells.

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