Kit for diagnosis and prognosis of castration-resistant prostate cancer neuroendocrine subtype based on CEACAM6 index

Abstract

The invention discloses a kit for diagnosis and prognosis of castration-resistant prostate cancer neuroendocrine subtypes based on a CEACAM6 index. The reagent comprises a CEACAM6 antibody, a coating solution, an antibody diluent, a confining solution, a washing solution, a developing solution, a stop solution and an enzyme-labeled secondary antibody. By detecting the level of CEACAM6 in serum and combining biological experimental verification, the strong correlation between the CEACAM6 and the castration-resistant prostate cancer neuroendocrine subtype is determined. The invention provides an innovative diagnostic tool, which is helpful for clinicians to identify neuroendocrine subtypes of castration-resistant prostate cancer, so that more accurate treatment schemes and prognosis evaluation are provided for patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, relates to a new use of CEACAM6, and specifically relates to a use of CEACAM6 in a diagnostic and prognostic kit for neuroendocrine subtypes of castration-resistant prostate cancer. Background Art

[0002] CEACAM6, the full name of which is Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6, is a glycoprotein molecule that spans the cell membrane and belongs to the important carcinoembryonic antigen (CEA) family. The molecule is encoded by its gene and is characterized by having an extracellular region and a domain involved in cell adhesion and signal transduction, which is essential for maintaining cell-to-cell interactions. Studies have shown that CEACAM6 and its antibodies are involved in the occurrence and development of colorectal cancer, pancreatic cancer, and breast cancer.

[0003] Prostate cancer, as a common male malignancy, is showing an increasing trend in incidence and mortality in my country. The development of the disease depends largely on the signal transduction pathway guided by the androgen receptor. Therefore, in the treatment of advanced prostate cancer, the therapy that inhibits the action of androgens (i.e. androgen deprivation therapy, ADT) has become a core means. However, over time, some prostate cancer cells are able to adapt to the low androgen environment, regenerate by restoring the androgen receptor signal transduction pathway, and then evolve into castration-resistant prostate cancer (CRPC). In this process, a special type of CRPC-neuroendocrine CRPC (NEPC) is particularly noteworthy. Its characteristics include decreased or even disappeared expression of androgen receptors, no longer relying on androgen receptor signal transduction, and showing neuroendocrine characteristics.

[0004] There is no public research report on the association between CEACAM6 and the neuroendocrine transformation subtype (NEPC) of castration-resistant prostate cancer and its mechanism of action. The present invention studies the relationship between CEACAM6 and the neuroendocrine transformation subtype of castration-resistant prostate cancer through clinical data and basic biological experimental data. The results of the present invention can provide a new method for clinical diagnosis of neuroendocrine subtype of castration-resistant prostate cancer. Summary of the invention

[0005] The purpose of the present invention is to address the challenges faced in the prior art for the diagnosis and prognosis of neuroendocrine subtypes of castration-resistant prostate cancer (CRPC), especially in the absence of a deep understanding and technical means of the association between CEACAM6 and CRPC neuroendocrine subtypes.

[0006] The present invention provides a CEACAM6-based diagnostic and prognostic reagent for neuroendocrine subtypes of castration-resistant prostate cancer and a kit thereof, which fills the gap in this research. By integrating clinical case data with in-depth basic biological experimental data, the present invention explores the relationship between CEACAM6 and the neuroendocrine transformation subtype (NEPC) of castration-resistant prostate cancer. Through the kit of the present invention, the expression level of CEACAM6 in the sample can be quantitatively or qualitatively determined, thereby providing a basis for clinical diagnosis and prognostic evaluation. The purpose of the present invention is to provide a biomarker based on CEACAM6 for use in the preparation of reagents for diagnosing CRPC neuroendocrine subtypes and judging patient prognosis. Through the present invention, we aim to solve the problem of insufficient research on the role and mechanism of action of CEACAM6 in CRPC neuroendocrine subtypes in the prior art, and provide a new technical solution to assist in the diagnosis of CRPC neuroendocrine subtypes and to judge patient prognosis. The expected research results will fill the research gap in this field and provide clinicians with a new diagnostic tool to accurately identify the neuroendocrine subtype of CRPC, and may help assess the prognosis of patients with this subtype, thereby providing important guidance for treatment strategies.

[0007] The technical solution of the present invention:

[0008] The present invention firstly provides a CEACAM6-based castration-resistant prostate cancer neuroendocrine subtype diagnosis and prognosis judgment reagent and its application.

[0009] The reagents include CEACAM6 antibody, coating solution, antibody diluent, blocking solution, washing solution, color developing solution, stop solution and enzyme-labeled secondary antibody.

[0010] ① Coating solution: 0.05 mol / L carbonate buffer (pH = 9.6). 0.75 g sodium carbonate, 1.46 g sodium bicarbonate, add deionized water to make up to 500 ml.

[0011] ② Antibody diluent: 0.02mol / LPBS (pH=7.4) + 0.2% BSA. 0.2g BSA is dissolved in 0.02mol / L phosphate buffer to 100g. 0.02mol / L phosphate buffer (pH=7.4). 0.2g potassium dihydrogen phosphate, 2.90g disodium hydrogen phosphate, 8g sodium chloride, add deionized water to 1000ml.

[0012] ③ Blocking solution: 0.05 mol / L carbonate buffer (pH=9.6) + 2.0% BSA by weight. 2.0 g BSA was added to the prepared 0.05 mol / L carbonate buffer and dissolved to 100 g.

[0013] ④ Washing solution: 0.02 mol / L PBS (pH=7.4) + 0.05% Tween-20. Dissolve 50ul Tween-20 in 100ml 0.02mol / L phosphate buffer and mix by shaking.

[0014] ⑤ Color development solution: TMB-urea hydrogen peroxide solution. Solution A (3,3',5,5'-tetramethylbenzidine, TMB): weigh 20 mg of TMB and dissolve it in 10 ml of anhydrous ethanol. After it is completely dissolved, add double distilled water to 100 ml. Solution B (0.1 mol / L citric acid-0.2 mol / L sodium dihydrogen phosphate buffer, pH = 5.0-5.4): weigh 14.34 g of Na2HPO4·12H2O, 1.87 g of citric acid and dissolve it in 180 ml of double distilled water, add 1.28 ml of 0.75% urea hydrogen peroxide, make up to 200 ml, and adjust the pH to 5.0-5.4. Mix solution A and solution B at a volume ratio of 1:1 to form TMB-urea hydrogen peroxide (application) solution.

[0015] ⑥ Stop solution: 2 mol / L H2SO4 solution. Add 10 ml 98% concentrated sulfuric acid to 60 ml double distilled water, make up to 100 ml, and store at room temperature.

[0016] ⑦ Enzyme-labeled secondary antibody: HRP-labeled rabbit anti-human IgG, diluted 3000 times with antibody diluent when used.

[0017] The reagent can be used to detect the content of CEACAM6 in the patient's serum. The present invention uses enzyme-linked immunosorbent assay (ELISA) to detect CEACAM6.

[0018] The present invention also provides a kit for diagnosing and prognosing neuroendocrine subtypes of castration-resistant prostate cancer based on the CEACAM6 index, the kit comprising the above-mentioned reagents, auxiliary tools for sample processing and reagent distribution, and internal standards for stability and quality control.

[0019] In some specific embodiments of the present invention, the antibody in the reagent is an existing CEACAM6 antibody used in ELISA experiments.

[0020] According to some specific embodiments of the present invention, when the CEACAM6 level in serum is higher than the average normal level, that is, higher than the diagnostic threshold, the patient with the neuroendocrine subtype of castration-resistant prostate cancer being tested is diagnosed as a patient with the neuroendocrine subtype of castration-resistant prostate cancer; when the CEACAM6 level in serum is higher than the average normal level and higher than the prognostic threshold, the patient with the neuroendocrine subtype of castration-resistant prostate cancer being tested is diagnosed as having a poor prognosis.

[0021] Advantages and beneficial effects of the present invention:

[0022] Using the kit of the present invention, based on the strong correlation between CEACAM6 and neuroendocrine subtypes of castration-resistant prostate cancer in the clinical data and basic biological experimental data of the previous study, based on the correlation between the expression of CEACAM6 protein and neuroendocrine subtypes of castration-resistant prostate cancer, the protein is used as a molecular marker to detect its expression, which can be used to quickly guide the diagnosis of patients with neuroendocrine subtypes of castration-resistant prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Scatter plot showing CEACAM6 levels in serum of CRPC (castration-resistant prostate cancer), NEDPC (no evidence of disease progression within the first year after treatment), and NEPC (neuroendocrine subtype of castration-resistant prostate cancer);

[0024] Figure 2 Show the effect of siRNA interference on CEACAM6 receptor on cell invasion ability;

[0025] Figure 3 Show the effect of siRNA interference with CEACAM6 receptor on cell proliferation ability;

[0026] Figure 4 The results show that siRNA can interfere with CEACAM6 receptor expression. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are only used to explain the present invention and are not intended to limit the protection scope of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The present invention provides a method for detecting the correlation between CEACAM6 and castration-resistant prostate cancer neuroendocrine subtypes, and the specific operating steps of the method are as follows:

[0031] 1. Clinical testing

[0032] CEACAM6 levels were measured in the serum of patients with CRPC (castration-resistant prostate cancer), NEDPC (no evidence of disease progression within the first year after treatment), and NEPC (neuroendocrine subtype of castration-resistant prostate cancer).

[0033] 2. Basic Biological Experiments

[0034] (1) Use CEACAM6 to induce prostate cancer cells cultured in vitro and observe the changes in prostate cancer cells;

[0035] (2) After induction in step (1), the CEACAM6 signaling pathway is inhibited and the changes in prostate cancer cells are observed.

[0036] Through basic biological experiments, it was found that CEACAM6 can induce neuroendocrine transformation of prostate cancer cells, and when the CEACAM6 signaling pathway is inhibited, the neuroendocrine transformation process of prostate cancer cells is inhibited.

[0037] Preferably, the ELLSA method is used for clinical detection of the content of CEACAM6 in serum.

[0038] Preferably, the in vitro cultured prostate cancer cells used in the present invention are LNCaP cells.

[0039] The method of inhibiting the CEACAM6 signaling pathway includes adding a CEACAM6 antagonist, adding a CEACAM6 inhibitor, adding a CEACAM6 receptor blocker, adding an agent that interferes with the expression of the CEACAM6 receptor. In some specific embodiments of the present invention, the CEACAM6 signaling pathway is inhibited by adding siRNA targeting the CEACAM6 receptor.

[0040] The siRNA sequence information for CEACAM6 receptor is as follows: 5'-CCGGACAGTTCCATGTATACC-3'.

[0041] In some specific embodiments of the present invention, it was verified by Western blot experiments that when siRNA targeting CEACAM6 was introduced into prostate cancer cells, the expression of CEACAM6 protein in the cells was significantly inhibited.

[0042] In some specific embodiments of the present invention, the invasive ability of prostate cancer cells cultured in vitro induced by CEACAM6 is enhanced, while that of prostate cancer cells introduced with siRNA is weakened.

[0043] In some specific embodiments of the present invention, the proliferation ability of prostate cancer cells cultured in vitro induced by CEACAM6 is enhanced, while that of prostate cancer cells introduced with siRNA is weakened.

[0044] The construction of the castration-resistant prostate cancer neuroendocrine subtype of the present invention is achieved by the following method: culturing prostate cancer cells in vitro under androgen-free conditions and inducing prostate cancer cell lines using CEACAM6.

[0045] In the present invention, the term "subject" refers to a living human or non-human organism. Preferably, the subject is a prostate cancer patient.

[0046] In the present invention, the term "increased level" means a level above a certain threshold level.

[0047] Example 1: Study on the correlation between CEACAM6 and neuroendocrine subtypes of castration-resistant prostate cancer.

[0048] In this example, the level of CEACAM6 in serum is the fasting level of CEACAM6. The fasting level means that no food was taken 12 hours before the blood sample was collected.

[0049] 1. All research subjects were selected from 64 patients with prostate cancer who had undergone anti-androgenic treatment for varying periods of time and were pathologically confirmed by Tianjin Institute of Urology. These subjects underwent PSA monitoring examinations at Tianjin Institute of Urology from April 2023 to June 2023. Among them, 18 cases had no biochemical recurrence, and the remaining 46 cases had undergone neuroendocrine transformation (neuroendocrine subtype of castration-resistant prostate cancer, according to EAU Guideline).

[0050] 2. Research Methods

[0051] 2.1 Serum concentration detection of CEACAM6

[0052] 2.1.1 Steps: Collect serum samples from patients with prostate cancer. After collecting blood, remove the needle and slowly inject it into a sterilized conical flask. After solidification, use a sterile dropper to peel the blood along the edge, place it at room temperature for 1 hour, and then put it in a 4℃ refrigerator overnight (do not freeze, otherwise hemolysis will occur) to allow the serum to fully precipitate. Take it out the next day and centrifuge it at 4℃3000r / min for 30min, take the supernatant, and store it at -20℃ after aliquoting. Dilute the antigen to 0.01mg / ml with coating solution (0.05mol / L carbonate buffer, pH=9.6), and add 100μl of the diluted antigen solution to each well of the ELISA plate. Place the ELISA plate in a wet box and coat it at 4℃ overnight. Discard the coating solution, fill all ELISA wells with washing solution, and wrap them with gauze and toilet paper to dry. 1 time, and dry the water at the last time. Add 250μl of blocking solution (pH 9.6, 0.05mol / L carbonate buffer containing 2.0% BSA) to each well of the ELISA plate, place the ELISA plate in a wet box, and incubate overnight at 4℃. Alternatively, incubate at 37℃ for 2h. Discard the blocking solution and wash once as above. Add 10μl of CEACAM6 antibody to 990μl of antibody diluent to make a 1:100 antiserum. (Operate in a 1.5ml EP tube). Add 100μl of 1:100 serum to well 1 in row A of the ELISA plate. Add 100μl of antibody diluent to each of the 2nd to 12th wells of row A on the ELISA plate, then add 100μl of 1:100 antiserum to the second well of group A, pipette 10 times and then take 100μl of serum to the third well, pipette 10 times again and take 100μl to the fourth well, then add to the 12th well in sequence, and finally take out 100μl from the 12th well and discard. In this way, each well has 100μl, and after 11 times of double dilution, the serum dilution is 1:204800. The same operation is performed for rows B and C. Only 100μl of antibody diluent is added to the first two wells of row D as a blank control, 100μl of 100-fold diluted negative blood is added to wells 3-4, no antigen is added to wells 5-6, only blocking solution is used to block, and 100μl of 100-fold diluted antiserum is added to each well as a control. After adding, place the ELISA plate in a humidified box and incubate at 37℃ for 1h. Wash as above. Add enzyme-labeled secondary antibody, add 100 μl of enzyme-labeled secondary antibody (rabbit anti-human) diluted 1:3000 to each well, and incubate in a humidified box at 37°C for 1 hour. Wash as above. Add 100 μl of color development solution to each well and place the enzyme-linked plate in a humidified box away from light for about 3 minutes. Stop when the negative control well turns blue-green. Add 50 μl of stop solution to each well at the end. After termination, quickly measure the absorbance of each well solution at a wavelength of 450 nm using an enzyme-labeled instrument. Establish a standard curve and calculate the serum concentration of CEACAM6 in each sample.

[0053] 2.1.2 Statistical methods

[0054] The two-sided t test was used for statistics, and P < 0.05 was considered significant.

[0055] 2.1.3 Results

[0056] The results are as follows Figure 1 As shown, the level of CEACAM6 in the serum of patients with neuroendocrine subtype of castration-resistant prostate cancer (NEPC) was significantly increased compared with CRPC (castration-resistant prostate cancer), NEDPC (no evidence of disease progression within the first year after treatment).

[0057] 2.2 Effect of CEACAM6 on the invasive phenotype of castration-resistant prostate cancer neuroendocrine subtype

[0058] 2.2.1 Steps: First, CRPC and NEPC cell lines were cultured in a medium containing 10% fetal bovine serum to a density of 70-80%. Subsequently, siRNA targeting CEACAM6 was transfected into the cells using a transfection reagent, and a control group using non-targeted siRNA was set up. 48 hours after transfection, Matrigel matrix was prepared, mixed with culture medium in a certain proportion, coated on the polycarbonate membrane of the Transwell chamber, and incubated at 37°C to form a matrix layer. Next, the transfected cell suspension was adjusted to an appropriate concentration and inoculated into the Transwell chamber, placed in a 24-well plate containing culture medium, and incubated at 37°C and 5% CO2 for 24-48 hours. After the incubation, the chamber was washed with PBS, the invaded cells were fixed with a fixative, and then stained with a staining solution. The cells that passed through the matrix layer were observed and photographed using a microscope, and finally the number of cells that passed through the matrix layer was quantitatively analyzed by image analysis software to evaluate the effect of CEACAM6 on the invasion ability of NEPC cells.

[0059] 2.2.2 Results

[0060] The results are as follows Figure 2 As shown, CEACAM6 levels affect the invasive ability of the neuroendocrine subtype of castration-resistant prostate cancer, and there is a strong correlation between the two. That is, when the content of CEACAM6 in serum is higher than the normal level, the invasive ability of patients with the neuroendocrine subtype of castration-resistant prostate cancer is strong.

[0061] 2.3 Effect of CEACAM6 on the proliferative phenotype of the neuroendocrine subtype of castration-resistant prostate cancer

[0062] 2.3.1 Step: First, select CRPC and NEPC cell lines and culture them in a culture medium containing 10% fetal bovine serum to a density of 70-80%. Then, use a transfection reagent to transfect siRNA against CEACAM6 into the cells, and set up a control group for non-targeted siRNA. 24 hours after transfection, use a cell counter or manual counting method to determine the number of cells, and adjust to the appropriate inoculation concentration, and inoculate into a 96-well plate, with approximately 1x10^4 cells per well. 48 hours after transfection, add the corresponding detection reagent to each well according to the instructions of the selected proliferation detection reagent (such as CCK-8 or MTT). Return the 96-well plate to a 37°C, 5% CO2 incubator and incubate for 2-4 hours until MTT forms purple crystals or the CCK-8 reaction is complete. After the incubation, the absorbance of each well was measured using a microplate reader (usually 570 nm or 450 nm), and the cell proliferation rate at different time points was calculated based on the change in absorbance, thereby evaluating the effect of CEACAM6 on the proliferation ability of NEPC cells.

[0063] 2.3.2 Results

[0064] The results are as follows Figure 3 As shown, CEACAM6 levels affect the proliferation ability of the neuroendocrine subtype of castration-resistant prostate cancer, and there is a strong correlation between the two. That is, when the content of CEACAM6 in serum is higher than the normal level, the proliferation ability of patients with the neuroendocrine subtype of castration-resistant prostate cancer is strong.

[0065] Example 2 Detection of the interference efficiency of siRNA targeting CEACAM6 receptor

[0066] 1. Artificially synthesized siRNA

[0067] The gene sequence of CEACAM6 receptor was found in Genebank (GeneID: 4680). Designed and chemically synthesized by Shanghai Hanheng Biological Company, 200D (5nmol)*2, as follows:

[0068] The siRNA (siRNA) sequence information for CEACAM6 receptor 1 is as follows:

[0069] 5-CCGGACAGTTCCATGTATACC-3′;

[0070] 2. Cell culture: LNCaP cells were cultured in 1640 medium containing 10% FBS in a 37°C, 5% CO2 incubator.

[0071] 3. Establishment of a cell model of prostate cell neuroendocrine transformation

[0072] 3.1 Induction

[0073] The cells were seeded into a 24-well cell culture plate at 1×104 / well and cultured in an incubator at 37°C, 5% CO2 for 24 hours. CEACAM6 (prepared with sterile water) was added to the culture medium at a final concentration of 4 ng / ml to induce the prostate cancer cell line LNCaP for 3 generations, with each generation lasting one week.

[0074] 4. Transfection: The LNCaP cells that underwent neuroendocrine induction after CEACAM6 were inoculated into 24-well cell culture plates at 1×104 / well and cultured in a 37C, 5% CO2 incubator for 24 hours. The 1640 medium containing 10% FBS without double antibody was replaced, and transfection was performed according to the instructions of liposome transfection reagent 2000 (purchased from Invitrogen). The experiment was divided into a negative control group and an experimental group (the concentration of interfering RNA was 20 nM), wherein the siRNA in the negative control group was a universal control siRNA, i.e., siControl, which had no homology to the sequence of the CEACAM6 receptor gene; the experimental group was divided into two parallel groups. The negative control group and the experimental group were transfected at the same time.

[0075] 5. Detection of CEACAM6 receptor protein expression level by western blot: 48 hours after cell transfection, cells were collected with cell lysis buffer (150 mM sodium chloride + 1% NP-40 (detergent) + 0.1% SDS (detergent) + 2 μg / ml Aprotinin (protease inhibitor) (added before use) + 2 μg / ml Leupeptin (protease inhibitor) (added before use) or 1 mM PMSF (protease inhibitor) + 1.5 mM EDTA (protease inhibitor) + 1 mM Na Vanadate (phosphatase inhibitor)), and protein concentration was determined using a BCA protein detection kit. 50 μg of protein was taken from each experimental group for SDS-PAGE transfer and blocked with 5% skim milk powder. CEACAM6 receptor antibody and internal reference antibody were then added and incubated overnight at 4°C. The membrane was washed three times with TBST solution and then incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse (Zhongshan Jinqiao Co., Ltd., 1:5000) at room temperature for 1 hour. 5nin was incubated with ECL immunoblotting chemiluminescent reagent, then exposed, developed, and fixed. Housekeeping protein B-actin or GAPDH protein was used as internal reference protein.

[0076] 6. Results

[0077] like Figure 4 As shown, siRNA can significantly inhibit the expression of CEACAM6 receptor.

[0078] Although the invention has been described in detail above by means of general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made based on the invention. Therefore, these modifications or improvements made without departing from the spirit of the invention all fall within the scope of protection claimed by the invention.

Claims

1. A CEACAM6-based diagnostic and prognostic reagent for castration-resistant prostate cancer neuroendocrine subtypes, characterized in that: The reagents include CEACAM6 antibody, coating solution, antibody diluent, blocking solution, washing solution, color developing solution, stop solution and enzyme-labeled secondary antibody.

2. The reagent according to claim 1, characterized in that This reagent can be used to detect the content of CEACAM6 in patient serum.

3. The reagent according to claim 1, characterized in that This reagent detects CEACAM6 using an enzyme-linked immunosorbent assay (ELISA).

4. A kit for diagnosing and prognosticating neuroendocrine subtypes of castration-resistant prostate cancer based on the CEACAM6 index, characterized in that: The kit comprises the reagent according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that Aids for sample handling and reagent dispensing as well as internal standards for stability and quality control.

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