Application of protein in small extracellular vesicles as prostate cancer diagnostic marker

Abstract

The invention discloses application of small extracellular endovesicular protein as a prostatic cancer diagnostic marker, and is characterized in that the small extracellular endovesicular protein is plasma sample sEV protein LAMB1 and / or urine sample sEV protein Histone H4, and the plasma sample sEV protein LAMB1 is applied as a metastatic prostatic cancer diagnostic marker. The sEV protein LAMB1 of the plasma sample is combined with a prostate specific antigen to serve as metastatic prostate cancer; the application of the sEV protein Histone H4 in the urine sample as a prostate cancer grading diagnosis marker, the application of the sEV protein Histone H4 in the urine sample as a high-risk prostate cancer diagnosis marker and the application of the combination of the sEV protein Histone H4 in the urine sample and a prostate specific antigen as the high-risk prostate cancer diagnosis marker have the advantages of high sensitivity and strong specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of diagnostic markers, and in particular relates to the application of a small extracellular vesicle protein as a diagnostic marker for prostate cancer. Background Art

[0002] Prostate cancer is the second most common cancer among men, with nearly 500,000 men diagnosed with prostate cancer each year worldwide. The characteristic of prostate cancer is that it is difficult to detect in the early stages. Studies have shown that early detection of prostate cancer can help improve the prognosis of prostate cancer patients and ultimately reduce mortality. The current difficulty in prostate cancer lies in the lack of accurate grading and early diagnosis. The survival of prostate cancer patients is largely related to the grade of the tumor. Risk grading can be used to assess the overall risk of metastasis of in situ prostate cancer, evaluate the best treatment plan, and predict the possibility of recurrence after treatment. In addition, patients with metastatic prostate cancer have the worst prognosis and basically lose the chance of surgical cure. They can only rely on endocrine therapy or chemotherapy, which seriously affects the survival of patients. Therefore, accurate grading and early diagnosis of prostate cancer are crucial for prostate cancer.

[0003] The existing tumor markers used for prostate cancer diagnosis and auxiliary risk stratification are mainly prostate specific antigen (PSA). PSA levels in the diagnostic gray zone of 4.0-10.0 ng / mL show low specificity and poor sensitivity (25-40%), which are usually overdiagnosed and overtreated, and have little or no benefit on prostate cancer-specific survival rate. PSA cannot distinguish between prostate hyperplasia, indolent tumors and early and late prostate cancer. Clinically, 4 ng / mL serum PSA is usually used as the screening standard. However, data show that PSA diagnosis alone is inaccurate. A large number of men with PSA <4 ng / mL may not be detected with prostate cancer, while up to 75% of men with positive PSA tests (>4 ng / mL) do not have prostate cancer. Prostate cancer screening based solely on PSA will lead to excessive puncture of patients with benign prostate in the gray zone, causing symptoms such as pain, bleeding, urinary retention, permanent urinary incontinence and sexual dysfunction. In addition, due to the multifocality and heterogeneity of prostate cancer histology, even with the multi-point puncture biopsy method, there is still a certain false negative rate, resulting in the failure to detect prostate cancer in time and miss the best treatment time, and the inability to dynamically monitor the occurrence and progression of the tumor. Therefore, improving the early diagnosis of prostate cancer and assisting in the scientific staging and grading of prostate cancer are crucial to improving the prognosis of prostate cancer patients and prolonging their survival.

[0004] Small extracellular vesicles (sEVs) are rich in proteins and nucleic acids wrapped in a double-layer phospholipid protective membrane. The complex signaling pathway network between tumor cells and the tumor microenvironment mediated by sEVs is a key factor in all stages of cancer progression. Studies have found that small extracellular vesicles play an important role in tumor immune regulation, microenvironment reorganization, angiogenesis, invasion, metastasis and survival. Studies have shown that small extracellular vesicles carrying integrin α3 and integrin β1 can promote the migration and invasion of epithelial cells. At present, small extracellular vesicle protein markers for early diagnosis of prostate cancer have not been reported. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a small extracellular vesicle protein with high sensitivity and strong specificity for use as a diagnostic marker for prostate cancer.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: the application of small extracellular vesicle proteins as diagnostic markers for prostate cancer, wherein the small extracellular vesicle proteins are sEV protein LAMB1 in plasma samples and / or sEV protein Histone H4 in urine samples.

[0007] Furthermore, the plasma sample sEV protein LAMB1 is used as a diagnostic marker for metastatic prostate cancer.

[0008] Furthermore, the plasma sample sEV protein LAMB1 is combined with prostate-specific antigen as a diagnostic marker for prostate cancer.

[0009] Furthermore, the plasma sample sEV protein LAMB1 is combined with prostate-specific antigen as metastatic prostate cancer.

[0010] Furthermore, the PRM verified peptide sequence of the plasma sample sEV protein LAMB1 is YFQMSLEAEER.

[0011] Furthermore, the sEV protein Histone H4 in the urine sample is used as a prostate cancer grading diagnostic marker.

[0012] Furthermore, the sEV protein Histone H4 in the urine sample is used as a diagnostic marker for high-risk prostate cancer.

[0013] Furthermore, the sEV protein Histone H4 in the urine sample is combined with prostate-specific antigen as a diagnostic marker for high-risk prostate cancer.

[0014] Furthermore, the PRM verified peptide sequence of the sEV protein Histone H4 in the urine sample is DAVTYTEHAK.

[0015] The above-mentioned small extracellular vesicle protein is used in the preparation of a drug for treating prostate cancer, wherein the small extracellular vesicle protein is the sEV protein LAMB1 in a plasma sample and / or the sEV protein Histone H4 in a urine sample.

[0016] Compared with the prior art, the advantages of the present invention are: the application of the small extracellular vesicle protein of the present invention as a diagnostic marker for prostate cancer, for the first time discovered that LAMB1 plays an important role in cancer cell communication and PCa metastasis mediated by sEV. The role of LAMB1 in basement membrane and tumor-derived sEV may indicate the communication between PCa cells and the tumor microenvironment. Combined with our results, this shows that the small extracellular vesicle protein LAMB1 used has a higher AUC value for diagnosing metastatic prostate cancer than the existing marker PSA, and has a higher specific sensitivity; and after combining with PSA, the AUC reached 0.9378, further improving the diagnostic value.

[0017] In addition, we also discovered for the first time the sEV protein Histone H4 that distinguishes low-risk PCa from high-risk PCa. The small extracellular vesicle protein Histone H4 has a higher AUC value for diagnosing high-risk prostate cancer than the existing marker PSA, and has higher specific sensitivity; and after combining with PSA, the AUC reached 0.9733, further improving the diagnostic value, which will help the early non-invasive diagnosis of prostate cancer and risk group stratification. In summary, our discovery provides a new way for the detection of early metastatic prostate cancer, risk stratification of prostate cancer, and treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Characterization of small extracellular vesicles extracted from cell supernatant and clinical plasma samples; A is the size distribution of small extracellular vesicles in cell supernatant (CCM, left), plasma (Plasma, right) and urine (Urine, bottom) measured by NTA; B is a representative TEM image of isolated small extracellular vesicles (red arrows) with lipid bilayer and cup-shaped structure; C is a Western blot of Flotilin-1, Syntenin-1, CD81 and Calnexin of small extracellular vesicles isolated from CCM, plasma and urine compared with whole cell lysate, whole plasma and whole urine lysate. The loading amount of whole cell lysate and sEV samples from cell supernatant is 2.5 μg protein; the loading amount of whole plasma lysate and sEV samples from plasma is 4.2 μg protein; the loading amount of whole urine lysate and sEV samples from urine is 1.7 μg protein; Figure 2To identify and discover sEV proteins in prostate cancer cell lines and normal prostate epithelial cell lines using a label-free proteomics approach; A is the identification and quantification results of proteomics, B is the number of proteins expressed in sEVs by five different cell lines, and C is the visualization of PCA principal component analysis of protein differences between samples from five different cell lines; Figure 3 The results of proteomic analysis based on sEVs, where A is a heat map of differential expression of sEV proteins in prostate cancer cell lines and normal prostate epithelial cell lines; B is the biological processes enriched in the top 10 GOs of sEVs secreted by 5 different cell lines; C is a Venn diagram of proteins detected in sEVs from 5 different source cell types, where the overlapping part shows the number of shared proteins, the side part shows the proteins expressed by single cell sEVs, the sEV proteins from each cell line are displayed in one color, and the boxes are EV protein markers found in the sEV samples; Figure 4 To quantify the expression of sEV plasma and urine proteins LAMB1, PXDN, URP2, DNAJA1, and Histone H4 using targeted PRM proteomics, A is LAMB1, B is PXDN, C is URP2, D is DNAJA1, and E is Histone H4, C indicates control, LR indicates low risk, H indicates high risk, M indicates metastasis, sEV-p indicates plasma-derived sEV, and sEV-u indicates urine-derived sEV; Figure 5 To verify the expression difference of LAMB1 protein in sEVs extracted from plasma samples of clinical control group and metastasis group by ELISA method and ROC curve evaluation of the diagnostic value of distinguishing control group from metastasis group. The red curve represents the diagnostic value of LAMB1 alone, the blue represents the diagnostic value of PSA alone, and the orange represents the diagnostic value of LAMB1 and PSA combined; Figure 6 To verify the expression difference of LAMB1 protein in sEVs extracted from plasma samples of clinical in situ prostate cancer group and metastasis group by ELISA method and to evaluate the ROC curve of the diagnostic value of distinguishing in situ group from metastasis group. The red curve represents the diagnostic value of LAMB1 alone, the blue curve represents the diagnostic value of PSA alone, and the orange curve represents the diagnostic value of LAMB1 and PSA combined; Figure 7 To verify the expression difference of LAMB1 protein in sEVs extracted from plasma samples of clinical control group and prostate cancer group by ELISA method and ROC curve evaluation of the diagnostic value of distinguishing control group from prostate cancer group. The red curve represents the diagnostic value of LAMB1 alone, the blue represents the diagnostic value of PSA alone, and the orange represents the diagnostic value of LAMB1 and PSA combined; Figure 8 To verify the expression difference of Histone H4 protein in sEV extracted from urine samples of low-risk and high-risk groups of prostate cancer by ELISA method and to evaluate the ROC curve of the diagnostic value of distinguishing the control group from the prostate cancer group. The yellow curve represents the diagnostic value of Histone H4 alone, the red curve represents the diagnostic value of PSA alone, and the purple curve represents the diagnostic value of Histone H4 and PSA combined; Fig. 9 The tissue microarray verifies the differential expression of protein markers in prostate cancer tissue and normal prostate tissue. A is the differential expression of LAMB1 protein on the tissue microarray; B is the differential expression of Histone H4 protein on the tissue microarray; Fig.10 To analyze the role of LAMB1 and Histone H4 in the prognosis of prostate cancer patients through database. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to the accompanying drawings. 1. Specific embodiments 1. Clinical sample collection Patients with histologically confirmed prostate cancer (PCa) or men aged 50 years or older without PCa were collected from the First Affiliated Hospital of Ningbo University. Peripheral blood, urine, and tissue samples were collected. 47 clinical blood samples and 46 urine samples were collected for PRM targeted proteomics, and 60 clinical blood samples and 60 urine samples were collected for ELISA protein marker validation. Immunohistochemistry was used to validate tissue microarrays (Bioaitech, China) for 79 tissue samples. All blood and urine clinical samples were approved by relevant ethics (ethics number: 2021-R106), and each subject filled in the informed consent form. The included clinical subjects were grouped according to pathological diagnosis, including in situ PCa group, metastatic PCa group, and non-tumor group (control group). According to the NCCN guidelines (Mohler et al., 2019), the in situ PCa group was further divided into extremely high, high, intermediate, and low risk groups. Considering the statistical significance and the balance of the number of patients in the groups, here we combined extremely high-risk and high-risk patients into the high-risk group; intermediate-risk and low-risk patients were combined into low-risk. All biological fluid samples were collected from fasting patients before any therapeutic intervention. Men with infectious diseases, taking medications that affect serum prostate-specific antigen (PSA) levels, a history of other cancers, or who had undergone surgical treatment within 3 months were excluded. The clinical characteristics of the patients participating in the study are shown in Table 1, and the basis for grouping is shown in Table 2.

[0021] Table 1 Clinical group samples and corresponding clinical parameters in this study

[0022] Table 2 Clinical groups of patients

[0023] 2. Plasma sample pretreatment Venous blood was collected from age-matched males using EDTA-treated blood collection tubes (BD bioscience, China). All blood samples were processed within 2 hours of collection from the subjects. Whole blood samples were centrifuged at 3,000 rpm for 15 minutes to collect raw plasma. The collected plasma was immediately transferred to a clean tube and centrifuged at 2,000 × g for 20 minutes at 4°C to remove cell residues. The supernatant was transferred to a new clean tube and centrifuged at 10,000 × g for 30 minutes at 4°C to remove large particles and aggregates, after which the pre-treated plasma was aliquoted and stored at -80°C.

[0024] 3. Cell culture Human normal prostate epithelial cell line (RWPE-1) and human prostate cancer (PCa) cell lines (PC3, DU145, LNCaP, 22Rv1) were purchased from Fuheng Biotechnology (Shanghai, China) and the ATCC cell bank in the United States, respectively. RWPE-1 cells were cultured in DefinedK-SFM (Life Technologies, China) medium, and PCa cell lines were cultured in RPMI1640 medium (Life Technologies, China). The cells were cultured to a density of 60-70%, then gently washed twice with DPBS and incubated in new exosome-free medium for 48 hours. All cell lines were free of mycoplasma contamination and were identified by STR.

[0025] 4. Pretreatment of cell supernatant The cell culture medium was centrifuged at 300 × g for 5 minutes at room temperature to remove live cells. The supernatant was collected and centrifuged at 2,000 × g at 4 °C for 20 minutes to remove dead cells, and then centrifuged at 10,000 × g for 30 minutes to remove aggregated cell debris and other biopolymers with a buoyant density higher than that of small extracellular vesicles (sEVs), and the supernatant was collected and filtered with a 0.22 μm pore size filter membrane.

[0026] 5. Isolation of small extracellular vesicles from cell culture medium sEVs from cell supernatants were isolated using the Total Exosome Isolation (Life Technologies, China) kit. Cell culture medium was incubated with the reagent at a ratio of 2:1 v / v at 4°C overnight (o / n). After incubation, the cell culture medium was centrifuged at 10,000 × g for 1 hour at 4°C, and all supernatant was carefully removed. The pellet was resuspended using filtered Dulbecco's phosphate buffered saline (DPBS). The sEV protein samples were aliquoted and stored in a -80°C refrigerator for later use.

[0027] 6. Ultracentrifugation to collect sEV proteins (referring to the proteins contained in small extracellular vesicles sEV) Fresh or thawed cell culture supernatant or plasma samples were diluted with cold PBS and centrifuged at 110,000 × g for 2 h at 4 ° C using a Beckman Coulter Type 70Ti fixed angle rotor (adjusted k factor 131, maximum acceleration, maximum deceleration). After the first ultracentrifugation, the sEV pellet was resuspended in cold PBS and then subjected to a second ultracentrifugation at 110,000 × g for 2 h at 4 ° C using a Beckman SW41Ti rotor. The final pellet was resuspended to a final volume of 100-200 μL with freshly filtered cold PBS and then subjected to protein quantification or frozen at -80 ° C.

[0028] 7. Protein blot experiment Total protein was extracted with RIPA lysis buffer (Cell Biolabs, China) containing 1×Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, China), and sEV protein was lysed 4 times for 15 seconds in an ice water bath with ultrasound, and incubated on ice for 10 seconds each time. The amount of protein was quantified using a BCA protein assay kit (ThermoFisher Scientific, China). Equal amounts of protein (1.7-20 μg) from the same type of sample were separated on a 4-12% Bis-Tris protein gel (Bio-Rad, China) and blotted on a 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). Blocked with 5% BSA for 1 hour at room temperature, followed by overnight incubation with the primary antibody at 4°C. The next day, after washing with TBST, the membrane was incubated with an HRP-conjugated secondary antibody for 1 hour at room temperature, then washed again with TBST and imaged by chemiluminescence. sEV characterization was performed by detecting related proteins (CD81, Syntenin-1, flotilin-1) and negative markers (Calnexin). The polyclonal antibody LAMB1 was purchased from Affinity Biosciences (China), and Histone H4 was purchased from Proteintech (China).

[0029] 8. Nanoparticle Tracking Analysis (NTA) Dilute the sEV sample to a suitable range, with a particle concentration of 10 8 -10 9 For each measurement sample, the parameters were set at Camera Level 13 and Threshold 5 (NTA3.4), and five 60 s videos were captured. The original particle concentration of the isolate was then calculated based on the measured concentration and dilution.

[0030] 9. LC-MS-based proteomics For the extracted sEV proteins, SDT (4% SDS, 100mM DTT, 150mM Tris-HCl pH 8.0) buffer was used for sample lysis and protein extraction. DTT, SDS and other low molecular weight components were removed by ultrafiltration (Millipore, 10kD) using UA buffer (8M urea, 150mM Tris-HCl pH 8.0). The samples were then treated in the dark with 100mL iodoacetamide (IAA) solution (containing 100mM IAA in UA buffer) for 30 minutes to block the reduced cysteine ​​residues, and then washed with UA buffer and NH 4 HCO 3The samples were then digested with trypsin overnight at 37°C. The digested peptides of each sample were 18 Spin Tips (Thermo Scientific, USA) were used for desalting, concentrated by SpeedVac centrifugal evaporator, and then dissolved in 0.1% FA. Tryptic digests were analyzed by LC-MS / MS using a hybrid quadrupole Orbitrap tandem mass spectrometer (Q-Exactive, Thermo Fisher Scientific, USA) coupled to Easy nLC. The MS raw data of each sample were identified and quantitatively analyzed using MaxQuant 1.5.3.17 software (Germany). Peptides and proteins were identified using the UniProt database.

[0031] 10. Targeted proteomics PRM detection sEV proteins were extracted by PCT-assisted lysis in a lysis buffer containing 6 M urea (Sigma, USA) and 2 M thiourea (Sigma, USA), followed by reduction in 200 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) at 32 °C for 30 min with gentle vortexing at 600 rpm. The proteins were then alkylated with 800 mM IAA for 30 min in the dark at 32 °C with gentle vortexing at 600 rpm. An additional 150 μL of ammonium bicarbonate (ABB) solution was added and digested with trypsin at a 1:4 enzyme-substrate ratio for 16 h at 32 °C. Finally, to terminate the digestion, 10% TFA was added to the solution at a final concentration of 1%. Desalting was performed by SOLAμHRP (Thermo Scientific, USA), followed by vacuum drying and keeping at -80 °C until MS analysis. The peptide precipitate was dissolved by 0.1% FA and 2% ACN, followed by vortexing in ice water for 1 min and sonication for 10 s. Peptide concentrations were measured using nanodroplets.

[0032] 11. Immunohistochemical analysis Prostate cancer tissue chip (M079Pr01) was purchased from Bioaitech (China). Immunohistochemistry (IHC) staining and analysis were performed as described below. In brief, tissue sections were dewaxed in xylene and rehydrated in graded ethanol (95%, 75%, 50% and distilled water). The slides were cleaned and immersed in 95°C 0.01M citrate buffer for 30 minutes to recover antigens, then sealed with goat serum for 1 hour at room temperature, and then incubated with the first antibody at 4°C (o / n). After incubation with the second antibody for 1 hour at room temperature the next day, the slides were developed using diaminobenzidine (Agilent, USA). Staining intensity was measured using an optical microscope (Leica, Germany). Intensity and histochemical scores (H-score) were both used to evaluate IHC results. Intensity is defined as follows: 0 = none, 1 = weak, 2 = medium, 3 = strong. H-score ranges from 0 to 300. Rabbit polyclonal antibodies LAMB1 (Affinity Biosciences, China) and Histone H4 were diluted at 1:400 and 1:2500, respectively.

[0033] 12. Statistical analysis Statistical analysis Data analysis and visualization were performed by using GraphPad Prism 8.0 (GraphPad Software, USA). Data are described in the form of mean and SEM. Parametric tests were performed by ANOVA and Turkey's post hoc test. Nonparametric tests were performed by Mann-Whitney test. P value < 0.05 was used to evaluate the significance of the data. The diagnostic ability of exosomal proteins and traditional tumor markers was tested by receiver operating characteristic (ROC) curves, and the area under the curve (AUC) was calculated. Kaplan-Meier survival curves were generated by log-rank test.

[0034] 2. Results Analysis 1. Identification and analysis of small extracellular vesicles (sEV) samples sEV samples isolated from cell supernatant (CCM), plasma, and urine were evaluated by nanoparticle tracking analysis (NTA). Figure 1 As shown in A. Figure 1B is a representative TEM image of isolated sEV (red arrow). Particles from different biological fluids are within the size range of sEV proteins (most particles in CCM are 105.3±6.6nm, plasma is 78.1±10.4nm, and urine is 139.9±4.1nm; the average particle size of CCM is 124.1±2.4nm, the average size of plasma is 110.9±1.7nm, and the average particle size of urine is 193.6±1.8nm), and the clear cup-shaped and lipid bilayer structures characteristic of sEVs are observed ( Figure 1 B), cup-shaped structures indicate intact bilipid membrane vesicles, but are dehydrated and therefore not perfectly spherical. Non-EV particles (i.e., lipid and protein aggregates) were also observed.

[0035] The protein expression of sEV samples from different biological fluids was also evaluated by Western blotting (WB). Three sEV markers (CD81, Syntenin-1, Flotillin-1) were used to define the presence of sEVs isolated from cell supernatants, plasma, and urine samples. Figure 1 As shown in C, sEV samples isolated from plasma and urine of different clinical groups (including control, low-risk, high-risk, and metastatic prostate cancer) were all positive for these sEV markers and negative for the non-sEV marker Calnexin, indicating that the cell lysate was minimally contaminated and a high concentration of pure sEV protein was obtained.

[0036] 2. Preliminary screening of protein markers in small extracellular vesicles of prostate cancer sEVs derived from four different prostate cancer cell lines (bone metastasis (PC3), brain metastasis (DU145), lymph node metastasis (LNCaP), and orthotopic tumor (22Rv1)) and a normal prostate epithelial cell line (RWPE-1) were evaluated by label-free proteomics, and a total of 1570 proteins were identified, of which 878 proteins were quantified, as shown in Figure 2 . Figure 2 As shown in A; 384 sEV proteins were expressed in the RWPE-1 cell line, 473 sEV proteins were expressed in PC3, 480 sEV proteins were expressed in DU145, 597 sEV proteins were expressed in LNCaP, and 570 sEV proteins were expressed in 22Rv1. Figure 2 B. In addition, principal component analysis (PCA) by qualitative comparison highlighted the different protein composition of normal RWPE-1 sEVs compared with prostate cancer sEVs, as shown in Figure 2 C. Analysis of sEV-based proteomics results is shown in Figure 3 As shown, the heat map shows that unsupervised hierarchical clustering shows that sEV proteins secreted by different prostate cancer cell lines and normal prostate epithelial cell lines can be clearly separated ( Figure 3A). Figure 3 As shown in B, GO enrichment analysis showed that when all differentially expressed sEV samples were compared, “extracellular exosomes” and “extracellular vesicles” were enriched in sEV samples secreted by all five different cell lines.

[0037] To further explore the association between sEVs from different cell lines, Figure 3 As shown in Figure C, we used a Venn diagram to illustrate the overlap of sEV proteins between different groups in. Among them, we identified 219 shared sEV proteins, 133 sEV proteins expressed only in prostate cancer, and 5 sEV proteins expressed only in metastatic prostate cancer cell lines. In addition, by comparing and analyzing with the EV database (Vesiclepedia), we identified a large number of EV proteins in samples isolated from different cell lines, further demonstrating our efficiency in EV extraction and providing a new research basis for subsequent studies on EV biological properties. Among the discovered sEV proteins, we further selected 20 of them for subsequent clinical sample verification based on comparisons between different prostate cancer groups, as shown in Table 3.

[0038] Table 3. Twenty potential sEV protein markers identified from prostate cancer cell lines As shown in Table 3, protein markers in prostate cancer sEVs: RTN4, LARS1, LAMB1, LAMB2, PXDN, NRP1, ABI3BP, FLNC, and SPON2 are highly expressed in sEVs of metastatic prostate cancer cell lines, but are absent or lowly expressed in sEVs of in situ prostate cancer or normal prostate epithelial cell lines; URP2, DNAJA1, ADK, Histone H4, CLTC, AHCY, MYH9, Histone H3.1, RAN, FLNA, and CD151 are highly expressed in sEVs of prostate cancer cell lines, but are absent or lowly expressed in sEVs of normal prostate epithelial cells.

[0039] 3. Targeted proteomic analysis of sEVs from biological fluids from different sources To validate the sEV proteins identified from the label-free proteomics, we also collected body fluids from clinical subjects, including blood and urine samples, and performed targeted quantification of the peptides of the target proteins by PRM targeted proteomics. Figure 4 As shown in Figure 2, we first found that the protein profiles of bulk sEVs in samples from different biological fluids were highly variable, or at least highly variable between cell supernatants, plasma, and urine. Figure 4As shown in AE, in plasma sEV (sEV-p, left figure) and urine sEV (sEV-u, right figure), the results of target proteins LAMB1, PXDN, URP2, DNAJA1 and Histone H4 were inconsistent in expression and trend, indicating that the results of sEV from different biological fluids cannot be generalized and are very different. The plasma sEV protein LAMB1 was expressed most highly in the metastatic group, while it was relatively low in the control group, low-risk group and high-risk group, and there were significant differences in the expression of the metastatic group to the high-risk group and the control group, while there was no similar trend or difference in the comparison of different urine groups. The urine sEV protein Histone H4 was expressed most highly in the high-risk group, and there was a significant difference in the comparison with the control group and the low-risk group, while there was no similar trend or difference in the comparison of the blood group. The PRM verified peptide sequences for LAMB1 and Histone H4 were YFQMSLEAEER and DAVTYTEHAK, respectively.

[0040] 4. ELISA was used to verify the differential expression of sEV protein LAMB1 in clinical plasma samples at different clinical stages. Figure 5 As shown in the figure, in the comparison between the metastatic group and the control group, the plasma sEV protein LAMB1 was significantly highly expressed in metastatic patients (left figure). For distinguishing metastatic prostate cancer and non-cancer populations, the AUC areas of the protein expression levels of sEV LAMB1 protein alone, traditional PSA protein, and combined sEV LAMB1+PSA were 0.9556, 0.9422, and 0.9911, respectively (right figure).

[0041] like Figure 6 As shown in the figure, in the comparison between the metastatic group and the in situ prostate cancer group, the plasma sEV protein LAMB1 was significantly highly expressed in metastatic patients (left figure). For distinguishing metastatic prostate cancer from in situ prostate cancer, the AUC areas of the protein expression levels of sEV LAMB1 protein alone, traditional PSA protein, and combined sEV LAMB1+PSA were 0.8733, 0.8178, and 0.9378, respectively (right figure).

[0042] like Figure 7 As shown in the figure, in the comparison of prostate cancer and clinical control group, plasma sEV protein LAMB1 was significantly overexpressed in metastatic patients (left figure). For distinguishing prostate cancer and non-cancer populations, the AUC areas of sEV LAMB1 protein alone, traditional PSA protein, and combined sEV LAMB1+PSA protein expression levels were 0.92, 0.8281, and 0.9363, respectively (right figure).

[0043] In summary, the sEV protein LAMB1 has a high accuracy in the diagnosis of metastatic prostate cancer, with higher specific sensitivity than traditional PSA, and higher diagnostic value than serum PSA. Importantly, the combined detection of sEV LAMB1 and PSA further improved the diagnostic value of metastatic prostate cancer for non-prostate cancer controls and primary prostate cancer (AUC = 0.9911 and 0.9378).

[0044] 5. ELISA was used to verify the differential expression of sEV protein Histone H4 in clinical urine samples at different clinical stages. Figure 8 As shown in the figure, in the comparison of the grading of low-risk prostate cancer and high-risk prostate cancer populations, the urine sEV protein Histone H4 was significantly highly expressed in high-risk patients (left figure). For distinguishing high-risk prostate cancer from low-risk prostate cancer populations, the AUC areas of the protein expression levels of sEV Histone H4 alone, traditional PSA, and combined sEV Histone H4+PSA were 0.9422, 0.7244, and 0.9733, respectively (right figure). It can be seen that Histone H4 protein shows a higher diagnostic value in the diagnosis of high-risk prostate cancer versus low-risk prostate cancer, and has a higher specific sensitivity than traditional PSA. The combined detection of sEV Histone H4 and PSA further improves the diagnostic value of high-risk prostate cancer versus low-risk prostate cancer, thereby helping clinical grading and treatment decisions for prostate cancer.

[0045] 6. Clinical association analysis between LAMB1 and Histone H4 like Fig. 9 As shown in Figure 2, through tissue microarray and immunohistochemistry experiments, we first found that LAMB1 ( Fig. 9 A) and Histone H4( Fig. 9 B) Significantly higher expression in prostate cancer tissues than in normal tissues, indicating consistency in protein expression of LAMB1 and Histone H4 in sEVs and tissues.

[0046] Further, if Fig.10 As shown, through clinical database analysis, it was found that high expression of LAMB1 and Histone H4 in tumors was closely associated with low OS rate and poor prognosis of prostate cancer patients, indicating the potential therapeutic value of sEV LAMB1 and Histone H4.

[0047] In summary, this study constructed a PCa-related circulating sEV detection system based on sEV proteomics. sEV protein was quantified by targeted proteomics and ELISA, and the expression levels of sEV proteins in blood and urine were compared in different disease groups (non-tumor group, low-risk prostate cancer group, high-risk prostate cancer group, in situ prostate cancer group, and metastatic prostate cancer group). We found that the expression level of LAMB1 in plasma sEVs in the metastatic prostate cancer group was significantly changed compared with the in situ prostate cancer group and the control subject group, and had a higher diagnostic value than PSA to distinguish metastatic prostate cancer. The combination of LAMB1 and PSA had a higher AUC value than a single marker. Compared with the low-risk prostate cancer group, the expression level of Histone H4 in urine sEVs in the high-risk prostate cancer group was significantly changed, and had a higher diagnostic value than PSA to distinguish low-risk and high-risk prostate cancer, thereby helping to grade prostate cancer. The combination of Histone H4 and PSA had a higher AUC value than a single marker.

[0048] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Use of small extracellular vesicle proteins as diagnostic markers for prostate cancer, characterized in that: The small extracellular vesicle protein is the sEV protein LAMB1 in plasma samples and / or the sEV protein Histone H4 in urine samples.

2. The use of the small extracellular vesicle protein as a diagnostic marker for prostate cancer according to claim 1, characterized in that: The application of the plasma sample sEV protein LAMB1 as a diagnostic marker for metastatic prostate cancer.

3. The use of the small extracellular vesicle protein as a diagnostic marker for prostate cancer according to claim 1, characterized in that: The plasma sample sEV protein LAMB1 is used in combination with prostate-specific antigen as a diagnostic marker for prostate cancer.

4. The use of the small extracellular vesicle protein as a diagnostic marker for prostate cancer according to claim 3, characterized in that: The plasma sample sEV protein LAMB1 is used in combination with prostate-specific antigen as a diagnostic marker for metastatic prostate cancer.

5. Use of a small extracellular vesicle protein according to any one of claims 1 to 4 as a diagnostic marker for prostate cancer, characterized in that: The PRM verified peptide sequence of the plasma sample sEV protein LAMB1 is YFQMSLEAEER.

6. The use of the small extracellular vesicle protein as a diagnostic marker for prostate cancer according to claim 1, characterized in that: The application of the sEV protein Histone H4 in the urine sample as a prostate cancer grading diagnostic marker.

7. The use of the small extracellular vesicle protein as a diagnostic marker for prostate cancer according to claim 6, characterized in that: The application of sEV protein Histone H4 in the urine sample as a diagnostic marker for high-risk prostate cancer.

8. The use of the small extracellular vesicle protein as a diagnostic marker for prostate cancer according to claim 1, characterized in that: The sEV protein Histone H4 in the urine sample is used in combination with prostate-specific antigen as a diagnostic marker for high-risk prostate cancer.

9. Use of a small extracellular vesicle protein as a diagnostic marker for prostate cancer according to any one of claims 6 to 8, characterized in that: The PRM verified peptide sequence of the sEV protein Histone H4 in the urine sample is DAVTYTEHAK.

10. Use of small extracellular vesicle protein in the preparation of a drug for treating prostate cancer, characterized in that: The small extracellular vesicle protein is the sEV protein LAMB1 in plasma samples and / or the sEV protein Histone H4 in urine samples.

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