Use of urine microvesicle proteins as diagnostic markers for kidney cancer

Abstract

This invention discloses the application of urinary microvesicle proteins as diagnostic markers for renal cell carcinoma. The protein markers derived from urinary extracellular vesicles associated with clear cell renal carcinoma are characterized by being selected from one or more of the following proteins: CSPG4, AQP1, PLIN2, CA9, and HAVCR1. The invention also describes the application of reagents for detecting these protein markers in the preparation of auxiliary diagnostic reagents for clear cell renal carcinoma. The inventors, using flow cytometry, analyzed 200 patients and found that five proteins derived from urinary extracellular vesicles could effectively distinguish patients with clear cell renal carcinoma from controls (including healthy individuals, patients with benign renal tumors, patients with benign renal cysts, and patients with chronic kidney disease).

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostics and relates to the application of urinary microvesicle proteins as diagnostic markers for renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Its global incidence is rising, accounting for approximately 4% of all malignant diseases in adults, and it ranks first in annual mortality among urological tumors. Many RCCs progress to advanced stages without obvious symptoms; only about 20% of patients present with the classic three symptoms of RCC (hematuria, abdominal mass, and pain). 50% of patients are discovered incidentally during routine physical examinations via imaging, and approximately 16% of RCCs have already metastasized at diagnosis. Most patients can only receive palliative treatment, resulting in a poor prognosis, with a 5-year survival rate of less than 10%. Therefore, actively searching for sensitive biomarkers for RCC is crucial for the diagnosis of early and small RCCs.

[0003] Extracellular vesicles (EVs) are membrane-bound vesicles released by cells, rich in various bioactive substances such as proteins, cholesterol, and phospholipids. The bioactive substances contained in EVs not only reflect the cells of origin but also change according to the physiological or pathological state of the source cells. EVs can be released by cells into various body fluids, such as blood, urine, ascites, amniotic fluid, and bronchoalveolar fluid. Their outer membrane structure has a protective function, preventing the contents from being hydrolyzed by proteases and nucleases, thus making EVs ideal biomarkers for disease diagnosis. Urine, being a fluid, non-invasive fluid, is an ideal source of biological biomarkers for kidney disease due to its ease of obtaining large samples. Studies have shown that EVs in urine mainly originate from intrinsic cells of various stages of nephrons and epithelial cells of the urinary pathway. The proteins, nucleic acids, and other bioactive substances in urinary EVs can serve as biomarkers for kidney disease and other urinary system diseases. In this invention, we developed a method for enriching extracellular vesicles from urine and identified a group of proteins derived from urinary extracellular vesicles associated with clear cell renal cell carcinoma using high-throughput proteomic ... Summary of the Invention

[0004] The purpose of this invention is to provide a protein biomarker derived from extracellular vesicles in urine for the diagnosis of clear cell renal cell carcinoma.

[0005] Another object of the present invention is to provide the application of the marker.

[0006] Another object of the present invention is to provide a reagent for detecting the marker and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] Proteins derived from extracellular vesicles in urine associated with clear cell renal cell carcinoma, including any one or more of the following proteins: Chondroitin sulfate proteoglycan 4 (CSPG4), Aquaporin-1 (AQP1), Perilipin-2 (PLIN2), Carbonic anhydrase-9 (CA9), and Kidney injury molecule-1 (HAVCR1).

[0009] The urinary extracellular vesicle-derived protein markers of the present invention preferably include a combination of Chondroitinsulfate proteoglycan 4 (CSPG4), Aquaporin-1 (AQP1), Perilipin-2 (PLIN2), Carbonicanhydrase-9 (CA9), and Kidney injury molecule-1 (HAVCR1).

[0010] The extracellular vesicles described in this invention are extracted from urine.

[0011] The detection reagent for detecting protein markers derived from extracellular vesicles in urine as described in this invention.

[0012] The detection reagent is preferably a primer or chip for detecting protein markers derived from extracellular vesicles in urine associated with clear cell renal cell carcinoma as described in this invention.

[0013] The present invention relates to the application of urinary extracellular vesicle-derived protein markers associated with clear cell renal cell carcinoma as detection targets in the preparation of diagnostic reagents for clear cell renal cell carcinoma.

[0014] The application of the detection reagent described in this invention in the preparation of diagnostic reagents for clear cell renal cell carcinoma.

[0015] A diagnostic reagent for clear cell renal cell carcinoma, comprising a detection reagent for protein markers derived from extracellular vesicles in urine as described in this invention.

[0016] Beneficial effects:

[0017] The inventors discovered, using high-throughput proteomic proteomic analysis, that five proteins from urinary extracellular vesicles are expressed only in patients with clear cell renal cell carcinoma, while they are almost not expressed in control groups (including healthy individuals, patients with benign renal tumors, and patients with benign renal cysts). Further analysis using flow cytometry in 200 patients revealed that these five proteins from urinary extracellular vesicles effectively distinguished patients with clear cell renal cell carcinoma from control groups (including healthy individuals, patients with benign renal tumors, patients with benign renal cysts, and patients with chronic kidney disease). Overall, these proteins from urinary extracellular vesicles can serve as diagnostic biomarkers for the diagnosis, staging, and prognosis of clear cell renal cell carcinoma. Attached Figure Description

[0018] Figure 1 A schematic diagram showing the capture of urinary extracellular vesicles by lectin-coupled magnetic beads and the determination of urinary extracellular vesicle proteins by flow cytometry.

[0019] Figure 2 Urinary extracellular vesicle protein expression profile. A. Heatmap of differentially expressed proteins in urinary extracellular vesicles. Blue dots represent proteins that decrease in urinary extracellular vesicles in clear cell renal cell carcinoma; red dots represent proteins that increase in urinary extracellular vesicles in clear cell renal cell carcinoma. B. Volcano plot. Blue dots represent proteins that decrease in urinary extracellular vesicles in clear cell renal cell carcinoma; red dots represent proteins that increase in urinary extracellular vesicles in clear cell renal cell carcinoma; black dots represent proteins that remain unchanged in urinary extracellular vesicles in clear cell renal cell carcinoma.

[0020] Figure 3 The expression levels of Chondroitin sulfate proteoglycan 4 (CSPG4) protein A, Aquaporin-1 (AQP1) protein B, Perilipin-2 (PLIN2) protein C, Carbonicanhydrase-9 (CA9) protein D, and Kidney injury molecule-1 (HAVCR1) protein E in urinary microvesicles of patients with clear cell renal cell carcinoma and controls (including healthy individuals, patients with benign renal tumors, patients with benign renal cysts, and patients with chronic kidney disease) are shown using flow cytometry.

[0021] Figure 4The ROC curves of five proteins—Chondroitin sulfate proteoglycan 4 (CSPG4) protein A, Aquaporin-1 (AQP1) protein B, Perilipin-2 (PLIN2) protein C, Carbonic anhydrase-9 (CA9) protein D, Kidney injury molecule-1 (HAVCR1) protein E, and the combined use of these five indicators (F) in urinary microvesicles were shown to differentiate renal clear cell carcinoma from control samples.

[0022] Table 1. Proteins that are detectable only in urinary microvesicles of patients with clear cell renal cell carcinoma, but almost undetectable in urinary microvesicles of control groups (including healthy individuals, benign renal tumors, and benign renal cysts).

[0023] Detailed Implementation

[0024] Example 1: Construction of condensate-coupled magnetic beads

[0025] 1. Purchase 2μm NHS magnetic beads (Solarbio, catalog number M2450). Take 500μL of the magnetic bead suspension and place it in a 1.5mL EP tube. Place the EP tube in a magnetic separator to enrich the magnetic beads and remove the supernatant. Add 1mL of pre-cooled (4℃) Washing Buffer A to the 1.5mL EP tube and vortex for 15s to ensure the magnetic beads are thoroughly mixed. Place the EP tube in a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0026] 2. Weigh the lyophilized wheat germ lectin powder and resuspend it in Coupling Buffer (Solarbio, catalog number M2450) to obtain a protein solution of 3.0 mg / mL. Add 500 μL of the wheat germ lectin protein solution to the EP tube from step 1 and vortex for 30 seconds to mix thoroughly. Vortex the EP tube for 15 seconds and place it on a mixer to mix at room temperature for 2 hours. If the mixture is not uniform, remove the EP tube and vortex for 15 seconds every 5 minutes for the first 30 minutes of the reaction. Thereafter, remove the EP tube and vortex for 15 seconds every 15 minutes.

[0027] 3. Place the EP tube in a magnetic separator to enrich the magnetic beads and remove the supernatant. Add 1 mL of Blocking Buffer (Solarbio, catalog number M2450) to the EP tube, vortex for 30 seconds, place the EP tube in a magnetic separator to enrich the magnetic beads, and discard the supernatant.

[0028] 4. Repeat step 3 four times. Add 1 mL of Blocking Buffer to the EP tube, vortex for 30 seconds, and place the EP tube in a mixer to react at room temperature for 2 hours. Place the EP tube in a magnetic separator to enrich the magnetic beads, and discard the supernatant. Add 1 mL of ultrapure water to the EP tube, mix thoroughly, enrich the magnetic beads using a magnetic separator, and discard the supernatant.

[0029] 5. Add 1 mL of PBS solution (pH 7.2) to an EP tube, mix thoroughly, enrich the magnetic beads using a magnetic rack, and discard the supernatant. Repeat this operation twice. Then, resuspend the prepared lectin-conjugated magnetic beads in 500 μL of PBS solution, mix thoroughly, and store at 4°C for later use. Note: The final concentration of the conjugated protein in the magnetic beads is 10 mg / mL.

[0030] Example 2: Detection of urinary microvesicle protein profiles

[0031] 1. Collect urine from patients with clear cell renal cell carcinoma and control groups (including healthy individuals, benign renal tumors, and benign renal cysts), centrifuge at 3000 rpm at room temperature for 30 minutes, remove cell debris, and retain the supernatant.

[0032] 2. Because the surface of extracellular vesicles is rich in glycosylated proteins, these glycosylated proteins can bind to lectins coupled to magnetic beads. Therefore, lectin-coupled magnetic beads are added to the urine supernatant obtained in the first step of separation and incubated at room temperature for 1 hour. During this time, extracellular vesicles in the urine will be captured by the magnetic beads.

[0033] 3. Place the EP tube on a magnetic separation rack to magnetically separate and remove the supernatant, retaining the magnetic beads and the extracellular vesicles they capture.

[0034] 4. Add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA to the mixture of magnetic beads and their captured extracellular vesicles obtained in step 3, wash 3 times, enrich the magnetic beads, and discard the supernatant.

[0035] 5. Collect the magnetic beads obtained in step 4 and the extracellular vesicles they capture, and extract proteins for protein proteomic analysis.

[0036] result

[0037] Proteomic analysis revealed significant differences in urinary microvesicle proteins between patients with clear cell renal cell carcinoma and controls (including healthy individuals, benign renal tumors, and benign renal cysts). Heatmaps and volcano plots showed that some proteins were highly expressed in urinary microvesicles of patients with clear cell renal cell carcinoma, while others were expressed at low levels. Figure 2More interestingly, we found that proteins such as Chondroitin sulfate proteoglycan 4 (CSPG4), Aquaporin-1 (AQP1), Perilipin-2 (PLIN2), Carbonic anhydrase-9 (CA9), and Kidney injury molecule-1 (HAVCR1) were detected only in urinary microvesicles of patients with clear cell renal cell carcinoma, while they were almost undetectable in urinary microvesicles of control groups (including healthy individuals, benign renal tumors, and benign renal cysts) (Table 1). These results suggest that proteins in urinary microvesicles can serve as biomarkers for the diagnosis of clear cell renal cell carcinoma.

[0038] Example 3

[0039] Detection of urinary microvesicle proteins using flow cytometry

[0040] 1. Collect urine from patients with clear cell renal cell carcinoma and control groups (including healthy individuals, benign renal tumors, and benign renal cysts), centrifuge at 3000 rpm at room temperature for 30 minutes, remove cell debris, and retain the supernatant.

[0041] 2. Because the surface of extracellular vesicles is rich in glycosylated proteins, these glycosylated proteins can bind to lectins coupled to magnetic beads. Therefore, 0.5 ml of the prepared lectin-coupled magnetic beads were added to 1 ml of urine supernatant obtained in the first step and incubated at room temperature for 1 hour. During this time, extracellular vesicles in the urine were captured by the magnetic beads.

[0042] 3. Place the EP tube on a magnetic separation rack for magnetic separation to remove the supernatant, retaining the magnetic beads and the extracellular vesicles they capture. Then, add 1000 μL of PBS solution containing 0.1% BSA (pH 7.2) and wash three times to enrich the magnetic beads, discarding the supernatant.

[0043] 4. Add 100 μL of PBS solution (pH 7.2) containing 0.1% BSA to the mixture of magnetic beads and their captured extracellular vesicles obtained in step 3 to resuspend the mixture. Add fluorescently labeled Chondroitin sulfate proteoglycan4 (CSPG4) antibody, Aquaporin-1 (AQP1) antibody, Perilipin-2 (PLIN2) antibody, Carbonic anhydrase-9 (CA9) antibody, and Kidney injury molecule-1 (HAVCR1) antibody. Incubate at room temperature in the dark for 1 hour.

[0044] 5. Add 1000 μL of PBS solution containing 0.1% BSA (pH 7.2) to the solution from step 4 for washing, enrich the magnetic beads, and discard the supernatant.

[0045] 6. After repeating step 5 three times, enrich the magnetic beads and discard the supernatant. Resuspend the magnetic beads and the captured extracellular vesicles in 300 μL of PBS solution containing 0.1% BSA and analyze using flow cytometry.

[0046] result

[0047] Glucosin-conjugated magnetic beads were used to adsorb vesicles in urine, and the content of proteins on the surface of urinary vesicle membranes was then detected by flow cytometry and fluorescent antibodies against the corresponding proteins. Figure 1 Flow cytometry results showed that Chondroitinsulfate proteoglycan 4 (CSPG4), Aquaporin-1 (AQP1), Perilipin-2 (PLIN2), Carbonicanhydrase-9 (CA9), and Kidney injury molecule-1 (HAVCR1) were significantly elevated in urinary microvesicles from patients with clear cell renal cell carcinoma. Figure 3 Further analysis using receiver operating characteristic (ROC) curves revealed that Chondroitin sulfate proteoglycan 4 (CSPG4), Aquaporin-1 (AQP1), Perilipin-2 (PLIN2), Carbonic anhydrase-9 (CA9), and Kidney injury molecule-1 (HAVCR1) effectively distinguished patients with clear cell renal cell carcinoma from controls (including healthy individuals, benign renal tumors, and benign renal cysts), with areas under the curves of 0.9200, 0.8400, 0.9200, 0.9600, and 0.8000, respectively. Figure 4 AE), through binary logistic regression analysis, when the five indicators Chondroitin sulfate proteoglycan 4 (CSPG4), Aquaporin-1 (AQP1), Perilipin-2 (PLIN2), Carbonic anhydrase-9 (CA9), and Kidney injury molecule-1 (HAVCR1) were used in combination to distinguish between patients with clear cell renal cell carcinoma and control groups (including healthy individuals, benign renal tumors, and benign renal cysts), the area under the ROC curve was 1.000 ( ). Figure 4 F).

Claims

1. Application of reagents for detecting CSPG4 derived from extracellular vesicles in urine in the preparation of auxiliary diagnostic reagents for clear cell renal cell carcinoma.

2. The application according to claim 1, characterized in that, The reagents for detecting CSPG4 derived from extracellular vesicles in urine are primers or chips for detecting the CSPG4 encoding gene.

3. The application according to claim 1, characterized in that, The reagent for detecting CSPG4 derived from extracellular vesicles in urine is an antibody or antibody fragment of CSPG4.

4. The application according to claim 1, characterized in that, A reagent for detecting CSPG4 derived from extracellular vesicles in urine is used to differentiate between patients with clear cell renal cell carcinoma and healthy individuals, patients with benign renal tumors, and patients with benign renal cysts.

Images