Application of CKAP4 reagent in detection specimens and bladder cancer detection kit

Abstract

The present invention discloses an application of a CKAP4 reagent in a detection specimen and a bladder cancer detection kit. CKAP4 is not only highly expressed in bladder cancer cell lines and the exosomes they secrete, but more importantly, clinically CKAP4 is significantly highly expressed in the urine exosomes of bladder cancer patients, while the expression level in urinary exosomes of urinary cancers such as prostate cancer and renal cell carcinoma, urinary system inflammation, and healthy donors is very low, with significant specificity. Therefore, the detection method based on urine exosome CKAP4 is a cheap, simple, non-invasive liquid biopsy method that can meet multiple tests, and is particularly suitable for the diagnosis and recurrence monitoring of bladder cancer patients.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor molecular biology and relates to an application of a CKAP4 reagent in a detection specimen and a bladder cancer detection kit. Background Art

[0002] Bladder cancer is a malignant urinary system disease that develops in the bladder epithelium. According to data from 2020, the incidence and mortality of bladder cancer rank in the top 10 worldwide, with 573,000 new cases and 213,000 deaths each year. Among them, approximately 75% are non-muscle-invasive bladder cancer and 25% are muscle-invasive bladder cancer. However, regardless of whether muscle invasion occurs, an important feature of bladder cancer is its high recurrence, and the metastasis of bladder cancer can cause rapid progression of the disease and cause the death of patients in a short period of time. Therefore, establishing a fast, simple, inexpensive method that meets high-frequency detection requirements is a key technology to deal with bladder cancer recurrence and improve treatment outcomes.

[0003] Among the existing detection methods, cystoscopic tissue biopsy is the gold standard for clinical diagnosis. This technology is an invasive and invasive examination method that inserts a cystoscope into the bladder through the urethra to directly observe the lesions in the bladder and urethra, and cuts suspected lesion tissue for pathological diagnosis. It has a high accuracy rate. However, this invasive examination method not only requires doctors to have rich operating experience, but also has many complications and is prone to missing flat urothelial carcinoma in situ. Patients have a low level of acceptance and it is difficult to meet the needs of large-scale promotion. Imaging examinations based on ultrasound, CT, MRI and other technologies are a type of detection method that is widely used in clinical practice. For bladder cancer, especially early non-invasive bladder cancer or small lesions that recur after surgery, this type of imaging detection method often leads to misdiagnosis and missed diagnosis due to the limitation of instrument resolution, and has great limitations. In addition, the performance indicators of urine-based non-invasive detection technologies currently used in clinical practice, such as urine exfoliative cytology, nuclear matrix protein 22 (NMP22) and chromosome fluorescence in situ hybridization (FISH), such as sensitivity and specificity, are difficult to meet the needs of clinical diagnosis, especially for the detection of low-grade bladder cancer, and it is even more impossible to achieve early screening of bladder cancer.

[0004] Exosomes are tiny vesicles with a size of 30-100nm secreted by cells. They carry a lot of biological information inside cells and can accurately reflect the molecular biological characteristics of their source cells. Due to the size limitation of glomerular filtration, exosomes in the blood usually cannot directly enter the urinary system. Therefore, the exosomes in urine are all derived from urinary system cells, which can effectively exclude the interference of exosomes secreted by other systemic cells. Studies have shown that compared with normal cells, cancer cells have a stronger exosome secretion ability due to their vigorous proliferation, and can secrete more exosomes containing cell biological information into the extracellular environment. In terms of separation and purification, due to the size effect of exosomes themselves, interfering impurities such as inorganic salts with small molecular weight, small biological molecules, impurity proteins and shed cells with large molecular weight will be greatly excluded. Therefore, as a disease that occurs in the urinary system, bladder cancer can be detected non-invasively through the biological characteristics of exosomes in patients' urine. It is a very promising new molecular detection method.

[0005] At present, in the research of exosome biomarkers, TACSTD2, AAT, Filamin-A and H2B1K were found to be high in tumor-derived exosome samples, but the signal was poor when detected by specific antibody blotting technology, and still could not reach the statistical difference between each group of patients and control samples. In addition, the lack of detection sensitivity makes it difficult to translate it into clinical application results. Therefore, the discovery of urine exosome biomarkers that can be used for clinical detection of bladder cancer has significant application advantages and market demand. Summary of the invention

[0006] The present invention discloses a protein molecule CKAP4 that is specific for bladder cancer. The molecule is not only highly expressed in bladder cancer cell lines and their exosomes, but also specifically highly expressed in the exosomes of urine of bladder cancer patients. The present invention also finds that CKAP4 regulates the mechanical properties of cell surfaces through exosomes, thereby promoting cell migration and metastasis in mice, emphasizing the important role of CKAP4 in bladder cancer exosomes.

[0007] The primary purpose of the present invention is to provide an application of a CKAP4 reagent in a detection specimen for preparing a bladder cancer diagnostic preparation, wherein the specimen comprises at least one of tissue, cells, cell exosomes, blood, urine and urine exosomes.

[0008] Furthermore, the expression level of CKAP4 in bladder cancer patient specimens was higher than that in healthy subjects, patients with urinary tract inflammation, or other cancers.

[0009] Furthermore, the reagent for detecting CKAP4 in a sample includes a reagent for detecting the content of CKAP4 protein in a sample.

[0010] Furthermore, the reagent for detecting CKAP4 in a specimen also includes a reagent for extracting CKAP4 protein in the specimen.

[0011] Furthermore, the specimen includes: at least one of urine and urine exosomes, especially urine exosomes.

[0012] The second object of the present invention is to provide a bladder cancer detection kit, comprising a reagent for detecting the content of CKAP4 protein in a specimen.

[0013] Furthermore, the kit also includes a reagent for extracting CKAP4 protein from a specimen.

[0014] The specimen includes at least one of tissue, cells, cell exosomes, blood, urine and urine exosomes.

[0015] Furthermore, the specimen includes: at least one of urine and urine exosomes, especially urine exosomes.

[0016] The present invention also provides the application of CKAP4 in the study of regulating the mechanical properties of the cell surface through the transport of exosomes and maintaining the high hardness gradient from the center to the edge of the cell surface. This property can significantly promote the metastasis of bladder cancer.

[0017] Clinically, the present invention collects morning urine samples from patients with different grades of bladder cancer before and after treatment, extracts exosomes by ultra-high-speed centrifugation, verifies the expression of CKAP4 in exosomes by werstern blot, and determines that CKAP4 is specifically highly expressed in urine exosomes of patients with bladder cancer, and the expression level in samples after surgical removal of the tumor is significantly reduced, while the expression level of CKAP4 in urine exosomes of patients with urinary inflammation and healthy donors is very low. Among the 32 samples involved in the present invention, the sensitivity, specificity, and accuracy of exosome CKAP4 detection were 93.8% (15 of 16 bladder cancer-positive patients were detected), 100% (CKAP4 expression was not detected in 16 non-bladder cancer patient negative samples), and 96.8% (31 of all 32 samples met the detection expectations) (e.g. Figure 7 ), its excellent selectivity and sensitivity are suitable for clinical detection standards.

[0018] The exosome protein molecules for detecting bladder cancer also include the following applications:

[0019] (1) Clinical detection methods based on exosomal CKAP4 in the urine of bladder cancer patients, such as test strips, kits, devices, instruments, etc.

[0020] (2) Application in the preparation of carrier reagents for the treatment of bladder cancer.

[0021] (3) Application in studying the differences between bladder cancer cells and normal cells.

[0022] (4) Application in the study of in vivo imaging of bladder tumors.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] 1. The sensitivity, specificity and accuracy of the detection of exosome CKAP4 in the urine of bladder cancer patients were 93.8%, 100% and 96.8% respectively, which are much greater than the existing detection methods based on free proteins and exfoliated cells;

[0025] 2. The exosomes collected by the present invention can come from bladder cancer cells at different disease sites, which can reflect the overall condition of the patient's disease site and avoid possible missed detection in detection methods such as cystoscopy;

[0026] 3. The detection method of the present invention can well avoid the influence of large exfoliated cells, precipitation, and metabolites in clinical samples, and can also avoid the influence of free impurity proteins that cannot be removed;

[0027] 4. The detection method of the present invention based on the exosomal protein CKAP4 can be used for applications such as initial onset detection and follow-up inspection.

[0028] The above advantages make the exosomal CKAP4 a bladder cancer-specific recognition molecular probe for diagnosis and targeted treatment of bladder cancer with great potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :The expression of CKAP4 in bladder cancer tissues;

[0030] a) Compared with normal tissues, CKAP4 is highly expressed in bladder cancer patients; BLCA(N-) represents bladder cancer without lymph node metastasis; BLCA(N+) represents bladder cancer with lymph node metastasis; NMIBC represents non-invasive bladder cancer; MIBC represents invasive bladder cancer;

[0031] b) Among the 176 samples, 165 samples were positive for CKAP4 in bladder cancer patients (samples with CKAP4 expression scores higher than those in normal tissues), with a positive rate of 93.75%;

[0032] c) High expression of CKAP4 is associated with the progression of bladder cancer, NMIBC: non-invasive bladder cancer; MIBC: invasive bladder cancer, *P<0.05; As shown in the figure, as the invasiveness of bladder cancer increases, the expression of CKAP4 increases accordingly;

[0033] d) CKAP4 expression is associated with bladder cancer metastasis, N0: no metastasis; N1-N2: bladder cancer metastasis, *P<0.05; As shown in the figure, with the deepening of bladder cancer lymph node metastasis, CKAP4 expression increased;

[0034] e) Forest plot evaluating the risk factors associated with bladder cancer progression. As shown in the figure, bladder cancer invasion and metastasis are more significant risk factors than age and gender.

[0035] Figure 2 : CKAP4 is highly expressed in bladder cancer cells and their secreted exosomes;

[0036] a) Western blot of CKAP4 expression in bladder cancer 5637 cells and normal cell line SV-HUC-1, with GAPDH as internal reference;

[0037] b) Figure 2 a) Quantitative analysis of protein blot; by using the principle of specific recognition and binding of antigen and antibody, the protein is transferred to PVDF membrane and then the specific antibody of Anti-CKAP4 is used to recognize and bind CKAP4, and then CKAP4 can be detected and quantitatively analyzed by chemiluminescence. As can be seen from the figure, compared with normal cells SV-HUC-1, CKAP4 is highly expressed in bladder cancer cell line 5637;

[0038] c) Transmission electron microscopy of exosomes isolated from cell culture supernatants of 5637 cells and SV-HUC-2 cells, scale bar 100 nm;

[0039] d) Particle size analysis of 5637 and SV-HUC-1 exosomes;

[0040] e) Western blot of CKAP4 expression in 5637 and SV-HUC-1 exosomes;

[0041] f) Figure 2 e) Quantitative analysis of Western blot; As can be seen from the figure, CKAP4 is highly expressed in the exosomes secreted by bladder cancer cell line 5637 cells, while the expression level in the exosomes of normal cells SV-HUC-1 is low.

[0042] gh) CKAP4 expression on the surface of exosomes labeled with AuNPs modified with CKAP4 aptamer;

[0043] g) Scale bar: 100 nm; and quantitative analysis h), ****P<0.0001.

[0044] Figure 3 : CKAP4 is a differentially expressed protein in 5637 and SV-HUC-1 exosomes;

[0045] a) Protein mass spectrometry analysis of 5637 and SV-HUC-1 exosomes; the results showed that compared with the SV-HUC-1 exosome protein derived from normal cells, CKAP4 was a highly expressed protein in bladder cancer exosomes, and its expression difference was the largest, and it was a protein that was highly expressed specifically in bladder cancer cells;

[0046] b) Protein mass spectrometry data of CKAP4; the data in the figure show the characteristic peptide information of CKAP4.

[0047] Figure 4 : Intracellular CKAP4 and exosome-derived CKAP4 promote changes in cell surface mechanical properties;

[0048] Among them, OE means Overexpression, i.e., overexpression of CKAP4, shNC means the control group of knockdown experiment; shCKAP4 means knockdown of CKAP4;

[0049] a) Schematic diagram of measuring cell surface hardness using atomic force probe;

[0050] b) Force curve characteristics of the softness and hardness of the cell surface;

[0051] c) Changes in cell morphology (top) and surface hardness (bottom) of bladder cancer 5637 cells after knockdown or overexpression of CKAP4;

[0052] d) The hardness and softness curve of the cell surface along the direction indicated by the arrow;

[0053] e) Figure 4 d) Statistical data, where each group contains more than 15 cells; statistics using student's ttest, **P<0.01, ****P<0.0001; Figure ce shows that CKAP4 in bladder cancer cells maintains a high hardness gradient from the center to the edge of the cell surface. When CKAP4 is knocked down, the gradient decreases, and when it is overexpressed, the gradient increases;

[0054] f) Exosomal CKAP4 enhances the surface stiffness gradient of recipient cells;

[0055] gh) Change curves of surface hardness of 5637 cells (g) and SV-HUC-1 cells (h) along the direction indicated by the arrows;

[0056] ij) Statistical differences in the cell mechanical properties of 5637 cells (i) and SV-HUC-1 cells (j), each group contained at least 15 cells, using student's t test, *P<0.05, ****P<0.0001, ns, no significant difference; As shown in Figure fj, under the action of bladder cancer exosomes CKAP4, the hardness gradients on the surface of 5637 cells and SV-HUC-1 cells increased, while the exosomes alone did not significantly change the cell surface mechanical properties.

[0057] Figure 5 : Exosomal CKAP4 improves bladder cancer cell motility and enhances cell migration;

[0058] No Exo: no exosomes were added; shNC Exo: exosomes extracted from the control group cells in the knockdown experiment were added; shCKAP4 Exo: exosomes extracted from the CKAP4 knockdown cells were added;

[0059] 5637shNC: control group for knockdown experiments in 5637 cells; 5637shCKAP4: knockdown of CKAP4 expression in 5637 cells;

[0060] a) Intracellular CKAP4 and exosomal CKAP4 promote the improvement of cell motility, each circle represents 10 minutes, scale 20μm; the motility of SV-HUC-1 cells with low CKAP4 expression is higher than that of 5637 cells expressing CKAP4; knocking down CKAP4 in 5637 cells weakened the cell motility; then exosomes containing CKAP4 and exosomes without CKAP4 were added to the control group and experimental group respectively, and it was found that the cell motility of the experimental group was enhanced after adding exosomes containing CKAP4;

[0061] b) Cell trajectory tracking; The cell trajectory was analyzed, and the range of movement of SV-HUC-1 cells with low CKAP4 expression was smaller than that of 5637 cells with high CKAP4 expression; the range of cell movement decreased after knocking down CKAP4 in 5637 cells; then exosomes containing CKAP4 and exosomes without CKAP4 were added to the control group and the experimental group respectively, and it was found that the range of cell movement in the experimental group increased after adding exosomes containing CKAP4;

[0062] c) Statistical data of the enhancement of cell motility by intracellular CKAP4 and exosomal CKAP4, more than 50 cells were counted in each group, using student's t test, **P<0.01, ****P<0.0001, ns, no significant difference; Figure 4a) Statistical analysis of the movement capacity, i.e., movement distance, of each cell and condition revealed that the movement distance of the control group with exosomes containing CKAP4 was significantly different from that of the control group with no exosomes or with exosomes without CKAP4, and this difference was more obvious in the experimental group. The same result was also observed in SV-HUC-1 cells with low CKAP4 expression.

[0063] d) Scratch test; in the control group, the wound healing ability, i.e., the cell migration ability, was worse than that of the group with exosomes containing CKAP4 added when no exosomes or exosomes containing CKAP4 were added; in the experimental group, the cell migration ability was weakened after knocking down CKAP4, and the migration ability was restored after adding exosomes containing CKAP4;

[0064] e) Figure 5 Statistical analysis in d) was performed using student's t test, **P<0.01, ****P<0.0001, ns, no significant difference; in the control group, there was a significant difference in the migration ability of cells without exosomes and cells with CKAP4 exosomes; in the experimental group, there was also a significant difference in the migration ability of cells without exosomes and cells with CKAP4 exosomes;

[0065] f) Transwell assay to detect the cell metastasis ability under the action of CKAP4; in the control group, the cell metastasis ability of the group without or without CKAP4 was poorer than that of the group with CKAP4 exosomes; in the experimental group, the cell metastasis ability was weakened after knocking down CKAP4, and the cell metastasis ability was restored after adding exosomes containing CKAP4;

[0066] g) Figure 5 f) Statistical analysis was performed using student's t test, ***P<0.001, ns, no significant difference. In the control group or the experimental group, the cell migration ability after adding exosomes containing CKAP4 was significantly different from that of the group without adding exosomes.

[0067] Figure 6 :The role of exosomal CKAP4 in the metastasis of bladder cancer cells in mice;

[0068] a) Schematic diagram of the metastasis model of bladder cancer cells in mice;

[0069] b) Schematic diagram of each group of information on the effects of cells and exosomes;

[0070] c) Metastasis of bladder cancer cells in mouse liver. Information of each group is as follows: Figure 6b); As can be seen from the figure, high expression of CKAP4 promotes the colonization of bladder cancer cells in the liver, and the metastatic lesions show obvious HE staining, Ki67 staining, and deepening of CKAP4 staining; CKAP4 further aggravates the metastasis to the liver under the action of exosomes; while exosomes without CKAP4 have no significant changes in the liver metastasis of bladder cancer cells.

[0071] df) Statistical analysis of bladder cancer cell metastasis sites in the liver;

[0072] d) number of tumor nodules;

[0073] e) Ki-67 index;

[0074] f) Expression of CKAP4, using student's t test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Data in Figure df) showed that high expression of CKAP4 in bladder cancer cells promoted their liver metastasis; under the action of exosomal CKAP4, the metastatic ability of bladder cancer cells was enhanced; exosomes that did not express CKAP4 had no obvious promoting effect on the liver metastasis of bladder cancer cells.

[0075] Figure 7 : Expression of CKAP4 in urine exosomes of bladder cancer patients;

[0076] a) Representative Western blot of CKAP4 expression in urine exosome samples from healthy subjects (HD), bladder cancer (high-grade hBLCA; low-grade lBLCA), postoperative bladder cancer (AS), prostate cancer (PRAD), kidney cancer (KIRC), and benign bladder tumors (bBL).

[0077] b) Heat map analysis of the relative content of CKAP4 in all tested urine exosome samples, where non-bladder tumors such as prostate cancer and kidney cancer are collectively referred to as non-bladder cancer (nBLCA);

[0078] c) Statistical analysis of the relative content of CKAP4 in all tested urine exosome samples, *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0079] The following specific embodiments are intended to further illustrate the present invention, but not to limit the present invention.

[0080] 1. Materials and Instruments

[0081] 1. Materials

[0082] 1.1 Cell lines

[0083] 5637 (human bladder cancer cells), SV-HUC-1 (human bladder epithelial immortalized cells).

[0084] 1.2 Culture medium and serum

[0085] DPBS, serum-free 1640, serum-free DMEM, PS, trypsin, Zeta serum (Thermo Fisher Scientific).

[0086] 1.3 Antibodies

[0087] Rabbit anti-CKAP4 polyclonal antibody (Shanghai Sangon Biotechnology Co., Ltd., D261564); CD81 antibody (D-4) sc-166028, CD63 (E-12): sc-365604, Alix (1A12): sc-53540 (SANTA CRUZ BIOTECHNOLOGY); mouse secondary antibody, rabbit secondary antibody (Merck).

[0088] 1.4 Clinical samples

[0089] The clinical samples were all from the Department of Urology of the Second Affiliated Hospital of Xiangya, and the healthy samples were donated by volunteers, all in compliance with bioethics.

[0090] 1.5 Other reagents

[0091] ECL chemiluminescent solution (Advansta); 1.5 M Tris-HCl pH 8.8, 1.0 M Tris-HCl pH 6.8 and 10% SDS (Shanghai Weiao Biotechnology Co., Ltd.).

[0092] 2. Instruments

[0093] Low-temperature ultrahigh-speed centrifuge and matching ultracentrifuge tubes (Beckman); disposable syringe filter 0.22um33mm (Millipore); centrifugal concentrator (LABCONCO); programmable five-stage metal bath (Tiangen); vertical electrophoresis apparatus, electrophoresis tank and other electrophoresis accessories, Biorad ChemiDoc XRS+ chemiluminescence gel imaging system (Biorad).

[0094] Example 1: Immunohistochemistry

[0095] The tissue microarrays containing normal, malignant and metastatic tissues were purchased from Avilabio (DC-Bla11021, Xi'an, China). The slides were soaked in sodium citrate antigen retrieval solution (pH 6.0). 2 O 2After blocking endogenous peroxidase and serum blocking, the sections were incubated with primary antibody against CKAP4 overnight at 4°C and labeled with HRP secondary antibody for 50 min at room temperature. Finally, the sections were developed in freshly prepared DAB color reagent until the nuclei appeared brownish yellow under the microscope and then counterstained with hematoxylin staining solution.

[0096] The expression level of CKAP4 in the tissue microarray was reviewed and scored by two independent experienced pathologists in a blinded manner, and the average percentage value was taken. CKAP4 expression was scored according to the proportion and intensity of cytoplasmic and nuclear staining as follows: 0-10% was scored as 0; 11-50% was scored as 1; 50-75% was scored as 2; and more than 75% was scored as 3. The nuclei stained with hematoxylin appeared blue. The positive cells cultured with DAB reagent had brown-yellow nuclei. The results are shown in Figure 1 .

[0097] Example 2: Collecting exosomes from cell samples using gradient centrifugation.

[0098] The specific steps of the experiment are as follows:

[0099] (1) Resuscitation of 5637 (human bladder cancer cells) and SV-HUC-1 (human bladder epithelial immortalized cells). When the cells proliferate to the logarithmic growth phase, subculture them to super-large dishes. When the cells in the super-large dishes grow to 60%, collect the culture medium supernatant and subculture them. Repeat the collection of cell culture medium supernatant until the volume reaches 300-500 mL.

[0100] (2) Transfer the culture supernatant to a 50 mL centrifuge tube, incubate at 4000 rpm for 30 min at 4°C; collect and transfer the supernatant to a new 50 mL centrifuge tube.

[0101] (3) Filter with a disposable syringe filter 0.22um 33mm and collect the supernatant.

[0102] (4) The supernatant was dispensed into Beckman ultracentrifuge tubes (55654) and the low-temperature ultracentrifuge (Beckman_L-100XP) was programmed to 110,000 g, 1 h, and 4°C.

[0103] (5) Discard the supernatant, resuspend the pellet with pre-cooled DPBS, and centrifuge at 110,000 g for 1 h at 4°C.

[0104] (6) Discard the supernatant, use the remaining liquid to resuspend the exosomes, and use 200-400 μl of pre-cooled DPBS to thoroughly collect the exosomes.

[0105] (7) The collected exosomes were frozen and concentrated into dry powder using a centrifugal concentrator, and 100 ul of ddHO was added. 2 O re-dissolved.

[0106] (8) Western Blot was used to detect the content of CKAP4 in exosomes, and CD81 was used as an internal reference. Sample preparation: Take an appropriate amount of exosomes, add 2ul 5× Protein Loading Buffer, and place in a 100℃ metal bath for 10min. After denaturation, place on ice immediately. When the temperature drops to room temperature, centrifuge the sample slightly.

[0107] Sample loading: Add the prepared sample to SDS-PAGE gel, and run the electrophoresis at 70V for 35min, 120V for 90min. Transfer to membrane at 300mA for 90min.

[0108] (9) The cells were blocked with 5% skim milk at room temperature. After blocking, the corresponding protein bands CKAP4 (63 kDa) and CD81 (22-26 kDa) were subtracted and incubated with the corresponding antibodies at 4°C overnight.

[0109] (10) Recover the antibody and wash three times with TBST, each time for 10 min. Incubate the mouse antibody and rabbit antibody at room temperature for 1 h. Recover the antibody and wash three times with TBST, each time for 10 min.

[0110] (11) The membrane surface was covered with ECL chemiluminescent liquid, and the corresponding bands were detected using the Bio-Rad ChemiDoc XRS+ chemiluminescent gel imaging system.

[0111] Preparation of relevant solutions:

[0112] (1) 5× electrophoresis buffer: 288 g Gly; 60.6 g Tris; 20 g SDS, and finally add deionized water to make up to 4 L. Use: 100 mL 5× electrophoresis buffer + 400 mL deionized water

[0113] (2) Transfer solution: 14.4 g glycine; 3.03 g Tris; 200 m methanol, and finally add deionized water to make up to 1 L.

[0114] (3) 10×TBS: 48.46 g Tris; 160.21 g NaCl; first add deionized water to 1600-1800 mL, adjust the pH to 7.6±0.1 with concentrated hydrochloric acid, and finally add deionized water to make up to 2000 mL.

[0115] (4) TBST: 50 mL 10×TBS + 450 mL deionized water + 500 ul Tween-20.

[0116] SDS-PAGE gel preparation:

[0117] 10% separation gel and 5% concentration gel. 10% SDS-PAGE protein separation gel (5mL): In a 50mL centrifuge tube, add 2.0mL ddH 2 O, 1.7mL 30% Acr-Bis (29:1), 1.25mL 1.5M Tris-HCl (pH8.8), 0.05mL 10% SDS, 0.05mL 10% APS, 0.002mL TEMED, mix well and add to the gel plate and remove bubbles with anhydrous ethanol. Leave at room temperature, add 5% SDS-PAGE protein concentration gel after the gel solidifies, and absorb 3.4mL ddH 2 O, 0.85mL 30% Acr-Bis (29:1), 0.625mL 1.0M Tris-HCl (pH6.8), 0.05mL 10% SDS, 0.05mL 10% APS, 0.005mL TEMED, mix well and add to the gel plate and insert the comb. It can be used after solidification.

[0118] Characterization of exosomal CKAP4 using colloidal gold AuNPs

[0119] The exosomes of 5637 and SV-HUC-1 cells were observed by transmission electron microscopy (TEM). First, the nucleic acid aptamer spl3c that can be specifically recognized was connected to the colloidal gold particles AuNPs with a size of 5nm, and then spl3c-AuNPs were incubated with exosomes at room temperature for 30 minutes. After that, 10μL of the above exosome sample was dropped on a copper grid for adsorption for 20 minutes, stained with 2% phosphotungstic acid for 10 minutes, and then washed 2-3 times with 100μL of pure water to remove excess dye and unbound AuNPs. Finally, it was dried at room temperature and TEM imaging (Hitachi H-7000NAR) was performed.

[0120] Results Figure 2 .

[0121] Example 3: Protein mass spectrometry of exosomes

[0122] Protein mass spectrometry was performed on the exosomes collected from 5637 and SV-HUC-1 cells, respectively, and the protein abundance scores and protein fold changes in the two groups of exosome samples were compared. Proteins with both high scores and large differences were selected, namely, significantly different proteins. Figure 3 As shown, CKAP4 is a protein characteristically expressed in bladder cancer exosomes.

[0123] The amino acid sequence of this protein is:

[0124] MPSAKQRGSKGGHGAASPSEKGAHPSGGADDVAKKPPPAPQQPPPPPAPHPQQHPQQHPQNQAHGKGGHRGGGGGGGKSSSSSSASAAAAAAAASSSASCSRRLGRALNFLFYLALVAAAAFSGWCVHHVLEEVQQVRRSHQDFSRQREE LGQGLQGVEQKVQSLQATFGTFESILRSSQHKQDLTEKAVKQGESEVSRISEVLQKLQNEILKDLSDGIHVVKDARERDFTSLENTVEERLTELTKSINDNIAIFTEVQKRSQKEINDMKAKVASLEESEGNKQDLKALKEAVKEIQTSAK SREWDMEALRSTLQTMESDIYTEVRELVSLKQEQQAFKEAADTERLALQALTEKLLRSEESVSRLPEEIRRLEEELRQLKSDSHGPKEDGGFRHSEAFEALQQKSQGLDSRLQHVEDGVLSMQVASARQTESLESLLSKSQEHEQRLAAL QGRLEGLGSSEADQDGLASTVRSLGETQLVLYGDVEELKRSVGELPSTVESLQKVQEQVHTLLSQDQAQAARLPPQDFLDRLSSLDNLKASVSQVEADLKMLRTAVDSLVAYSVKIETNENNLESAKGLLDDLRNDLDRLFVKVEKIHEKV

[0125] Example 4: Determination of cell surface mechanical properties

[0126] The cell surface mechanical map was measured on a JPK Nanowizard 4 atomic force spectrometer using a DNP-10 probe (0.06 N / m), and the cells were observed under bright field using an Olympus confocal microscope. Before measuring the Young's modulus of the cells, the spring constant of the cantilever was first calibrated by the thermal noise signal, and the force curve was collected on a cell culture dish filled with ultrapure water to correct the probe parameters. After that, the cells seeded in a 35 mm cell culture dish were washed three times with 1 mL of PBS, and finally the cells were placed in RPMI-1640 cell culture medium without FBS. The measurement parameters were set as follows: the down force was 2.0 nN, the Z length was 2000 nm, the Z scanning speed was 60 μm / s, the scanning area was 60×60 μm, and the pixel was 256. In order to fit the experimental data, the Herz model was selected, and the force curve was fitted using the JPKSPM data processing software.

[0127]

[0128] Where F is the force generated by the force curve, E is Young's modulus, ν is Poisson's ratio (usually 0.2-0.5), R is the tip radius, and δ is the indentation depth. Figure 4 .

[0129] Example 5: Cell migration ability assay

[0130] 1) Single cell movement tracking

[0131] The cells were seeded in a 35 mm glass-bottom culture dish. After the cells adhered, they were washed with PBS and then stained with Hoechst at a concentration of 4 μg / mL for 10 minutes at room temperature. The cells were washed three times with PBS and stored in complete culture medium for use. The changes in the relative position of the cells were observed under a 60× objective lens, and the cell position was recorded every 10 minutes for a total of 1 hour. The images were analyzed using ImageJ software, and more than 50 cells were counted for each experiment. (For the exosome effect experiment, the culture medium containing exosomes and the cells were first incubated overnight before cell inoculation to allow the exosomes to enter the cells.) The results are shown in Figure 5 .

[0132] 2) Scratch test

[0133] 5637NC and 5637shCKAP4 cells were seeded in 96-well plates overnight to allow the cells to adhere. After the cells attached, the original culture medium was replaced with RPMI-1640 medium without FBS and cultured for 24 hours. After the cell density reached 80-90%, a scratch instrument (BioTek) was used to evenly scratch uniformly sized marks on the monolayer of cells and washed three times to remove suspended cells and cell debris. The scratch healing was then monitored at 0, 4, 8 and 12 hours using an inverted microscope. The scratch area was measured and recorded using ImageJ software. (For the exosome effect experiment, the exosomes and cells were first incubated overnight before cell inoculation to allow the exosomes to enter the cells.) Results are shown in Figure 5 .

[0134] 3) Transwell assay

[0135] 2×10 4 The cells were suspended in 200 μL complete cell culture medium and seeded in the upper chamber of a 24-well plate (8 μm pore size, Corning) overnight to adhere to the wall. The next day, the culture medium in the upper chamber was replaced with fresh cell culture medium without FBS, and 500 μL of culture medium containing 20% ​​fetal bovine serum was added to the lower chamber to induce cell migration. Incubate at 37°C for 3 days. Subsequently, the cells were washed 3 times with PBS and then fixed with 4% paraformaldehyde and 1% crystal violet at room temperature for 10 min respectively. The cells were fixed with ddH 2O washing, wiping non-migrating cells with cotton swabs, acquiring images with the BioTek Cytation1Cell Imaging Multi-Mode Reader imaging system, and counting the number of migrating cells with ImageJ. (For the exosome effect experiment, the exosomes and cells were first incubated overnight before cell inoculation to allow the exosomes to enter the cells.) The results are shown in Figure 5 .

[0136] Example 6: Liver metastasis of bladder cancer cells in mice

[0137] In the tumor model of bladder cancer cells, female thymus-deficient BALB / c (BALB / c-nude mice) mice were purchased from Hunan SJA Experimental Animal Co., Ltd., and 6-week-old female BALB / c mice were used for the study. For the no exosome group, mice were intravenously injected with 200 μL of 5×10 6 For the exosome-treated group, 5637 cells with shNC and shCKAP4 were co-incubated with exosomes with or without CKAP4 for 12 h, and then the cells were collected and made into 200 μL of 5 × 10 6 The cell suspension was injected into mice, and then 5637 shNC or shCKAP4 exosomes were intravenously injected every 2 weeks until the 8th week. The liver was fixed in 4% paraformaldehyde and embedded in paraffin, and the liver metastasis of bladder cancer cells was analyzed using hematoxylin-eosin (H&E), Ki-67 and CKAP4 staining. The results are shown in Figure 6 .

[0138] Example 7: Collecting exosomes from urine samples using gradient centrifugation.

[0139] The specific steps of the experiment are as follows:

[0140] (1) Collect morning urine of 100-200 mL from healthy subjects (HD), bladder cancer (high-grade hBLCA; low-grade lBLCA), postoperative (AS), prostate cancer (PRAD), kidney cancer (KIRC), non-urinary tract cancer (nBLCA), and benign bladder tumors (bBL) patients. Transfer the urine to a 50 mL centrifuge tube at 4000 rpm for 30 min at 4°C. Collect and transfer the supernatant to a new 50 mL centrifuge tube at 20000 rpm for 40 min at 4°C. Collect and transfer the supernatant to a new 50 mL centrifuge tube.

[0141] (2) Filter with a disposable syringe filter 0.22um 33mm and collect the supernatant.

[0142] (3) The supernatant was dispensed into Beckman ultracentrifuge tubes (55654) and the low-temperature ultracentrifuge (Beckman_L-100XP) was programmed to 109,000 g, 1 h, 4°C.

[0143] (4) Discard the supernatant, resuspend the pellet with pre-cooled DPBS, and centrifuge at 110,000 g, 1 h, 4°C.

[0144] (5) Discard the supernatant, use the remaining liquid to resuspend the exosomes, and use 200-400 μl of pre-cooled DPBS to thoroughly collect the exosomes.

[0145] (6) The collected exosomes were frozen and concentrated into dry powder using a centrifugal concentrator, and 100 ul of ddHO was added. 2 O re-dissolved.

[0146] (7) Western Blot was used to detect the content of CKAP4 in exosomes, and CD81 was used as an internal reference. Sample preparation: Take an appropriate amount of exosomes, add 4ul Protein Loading Buffer, and place in a 100℃ metal bath for 10min. After denaturation, place on ice immediately. When the temperature drops to room temperature, centrifuge the sample slightly.

[0147] Sample loading: Add the prepared sample to SDS-PAGE gel, and run the electrophoresis at 70V for 35min, 120V for 90min. Transfer to membrane at 300mA for 90min.

[0148] (8) The cells were blocked with 5% skim milk at room temperature. After blocking, the corresponding protein bands CKAP4 (63 kDa) and CD81 (22-26 kDa) were subtracted and incubated with the corresponding antibodies at 4°C overnight.

[0149] (9) Recover the antibody and wash three times with TBST, each time for 10 min. Incubate the mouse antibody and rabbit antibody at room temperature for 1 h. Recover the antibody and wash three times with TBST, each time for 10 min.

[0150] (10) The membrane surface was covered with ECL chemiluminescent liquid, and the corresponding bands were detected using the Bio-rad ChemiDoc XRS+ chemiluminescent gel imaging system.

[0151] Results Figure 7 . Sequence Listing <110> Hunan University <120> Application of CKAP4 reagent in detection specimens and bladder cancer detection kit <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 602 <212> PRT <213> Homo sapiens <400> 1 Met Pro Ser Ala Lys Gln Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1 5 10 15 Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Val 20 25 30 Ala Lys Lys Pro Pro Pro Ala Pro Gln Gln Pro Pro Pro Pro Pro Ala 35 40 45 Pro His Pro Gln Gln His Pro Gln Gln His Pro Gln Asn Gln Ala His 50 55 60 Gly Lys Gly Gly His Arg Gly Gly Gly Gly Gly Gly Gly Lys Ser Ser 65 70 75 80 Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser 85 90 95 Ser Ala Ser Cys Ser Arg Arg Leu Gly Arg Ala Leu Asn Phe Leu Phe 100 105 110 Tyr Leu Ala Leu Val Ala Ala Ala Ala Phe Ser Gly Trp Cys Val His 115 120 125 His Val Leu Glu Glu Val Gln Gln Val Arg Arg Ser His Gln Asp Phe 130 135 140 Ser Arg Gln Arg Glu Glu Leu Gly Gln Gly Leu Gln Gly Val Glu Gln 145 150 155 160 Lys Val Gln Ser Leu Gln Ala Thr Phe Gly Thr Phe Glu Ser Ile Leu 165 170 175 Arg Ser Ser Gln His Lys Gln Asp Leu Thr Glu Lys Ala Val Lys Gln 180 185 190 Gly Glu Ser Glu Val Ser Arg Ile Ser Glu Val Leu Gln Lys Leu Gln 195 200 205 Asn Glu Ile Leu Lys Asp Leu Ser Asp Gly Ile His Val Val Lys Asp 210 215 220 Ala Arg Glu Arg Asp Phe Thr Ser Leu Glu Asn Thr Val Glu Glu Arg 225 230 235 240 Leu Thr Glu Leu Thr Lys Ser Ile Asn Asp Asn Ile Ala Ile Phe Thr 245 250 255 Glu Val Gln Lys Arg Ser Gln Lys Glu Ile Asn Asp Met Lys Ala Lys 260 265 270 Val Ala Ser Leu Glu Glu Ser Glu Gly Asn Lys Gln Asp Leu Lys Ala 275 280 285 Leu Lys Glu Ala Val Lys Glu Ile Gln Thr Ser Ala Lys Ser Arg Glu 290 295 300 Trp Asp Met Glu Ala Leu Arg Ser Thr Leu Gln Thr Met Glu Ser Asp 305 310 315 320 Ile Tyr Thr Glu Val Arg Glu Leu Val Ser Leu Lys Gln Glu Gln Gln 325 330 335 Ala Phe Lys Glu Ala Ala Asp Thr Glu Arg Leu Ala Leu Gln Ala Leu 340 345 350 Thr Glu Lys Leu Leu Arg Ser Glu Glu Ser Val Ser Arg Leu Pro Glu 355 360 365 Glu Ile Arg Arg Leu Glu Glu Glu Leu Arg Gln Leu Lys Ser Asp Ser 370 375 380 His Gly Pro Lys Glu Asp Gly Gly Phe Arg His Ser Glu Ala Phe Glu 385 390 395 400 Ala Leu Gln Gln Lys Ser Gln Gly Leu Asp Ser Arg Leu Gln His Val 405 410 415 Glu Asp Gly Val Leu Ser Met Gln Val Ala Ser Ala Arg Gln Thr Glu 420 425 430 Ser Leu Glu Ser Leu Leu Ser Lys Ser Gln Glu His Glu Gln Arg Leu 435 440 445 Ala Ala Leu Gln Gly Arg Leu Glu Gly Leu Gly Ser Ser Glu Ala Asp 450 455 460 Gln Asp Gly Leu Ala Ser Thr Val Arg Ser Leu Gly Glu Thr Gln Leu 465 470 475 480 Val Leu Tyr Gly Asp Val Glu Glu Leu Lys Arg Ser Val Gly Glu Leu 485 490 495 Pro Ser Thr Val Glu Ser Leu Gln Lys Val Gln Glu Gln Val His Thr 500 505 510 Leu Leu Ser Gln Asp Gln Ala Gln Ala Ala Arg Leu Pro Pro Gln Asp 515 520 525 Phe Leu Asp Arg Leu Ser Ser Leu Asp Asn Leu Lys Ala Ser Val Ser 530 535 540 Gln Val Glu Ala Asp Leu Lys Met Leu Arg Thr Ala Val Asp Ser Leu 545 550 555 560 Val Ala Tyr Ser Val Lys Ile Glu Thr Asn Glu Asn Asn Leu Glu Ser 565 570 575 Ala Lys Gly Leu Leu Asp Asp Leu Arg Asn Asp Leu Asp Arg Leu Phe 580 585 590 Val Lys Val Glu Lys Ile His Glu Lys Val 595 600

Claims

1. Use of a reagent for detecting CKAP4 in a specimen in the preparation of a bladder cancer diagnostic preparation, wherein the specimen comprises: At least one of tissue, cell, cell exosome, blood, urine and urine exosome; The expression level of CKAP4 in bladder cancer patient specimens is higher than that in healthy people, patients with urinary tract inflammation, or other cancers.

2. The use according to claim 1, characterized in that: The reagent for detecting CKAP4 in a sample includes a reagent for detecting the content of CKAP4 protein in a sample.

3. The use according to claim 2, characterized in that: The reagent for detecting CKAP4 in a specimen also includes a reagent for extracting CKAP4 protein in the specimen.

4. The use according to claim 1, characterized in that: The specimen includes: at least one of urine and urine exosomes.

5. The use according to claim 4, characterized in that: The specimen is urine exosomes.

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