Urine microvesicle proteins related to lung cancer and their applications

Abstract

The present invention discloses urine microvesicle proteins related to lung cancer and their applications. The application of a reagent for detecting protein markers derived from urine extracellular vesicles in the preparation of a reagent for the auxiliary diagnosis of lung cancer, wherein the protein markers derived from urine extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15. The protein markers of the present invention can effectively distinguish lung cancer patients and healthy individuals from patients with benign lung nodules. Generally speaking, the proteins in urine extracellular vesicles can be used as detection markers for the diagnosis, staging and prognosis of lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis, and relates to urine microvesicle proteins related to lung cancer and their applications. Background Art

[0002] Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs, which poses the greatest threat to the health and life of the population, and its incidence and mortality rate rank first among malignant tumors. In recent years, due to the continuous development of industrialization in China, the air pollution has become increasingly serious, and coupled with the influence of factors such as aging, the incidence and mortality rate of lung cancer are getting higher and higher. In the next few decades, lung cancer will be the top priority in cancer prevention and treatment in China. Data from the International Early Lung Cancer Action Program show that the 10-year expected survival rate after reasonable treatment of stage I lung cancer can reach more than 90%, while the survival rate of advanced lung cancer is less than 5%. Therefore, regular screening for lung cancer for early detection and early treatment can significantly improve the cure rate and reduce the mortality rate. At present, the examination methods for lung cancer mainly include serum marker examination, imaging examination and puncture examination. However, the above examination methods have limitations in their application in lung cancer screening due to reasons such as low accuracy (serum marker examination), radiation, high price, and difficult operation (imaging examination and puncture examination).

[0003] Urine is an extremely valuable diagnostic medium, which contains abundant extracellular vesicles (EVs). Extracellular vesicles are rich in various bioactive substances such as proteins and nucleic acids, and can reflect the physiological and pathological states of their source cells. Due to the protective effect of the outer membrane structure of extracellular vesicles, the bioactive substances such as proteins and nucleic acids contained in extracellular vesicles are very stable, and the proteins and nucleic acids contained in extracellular vesicles have been regarded as ideal markers for disease diagnosis. In the present invention, we identified the specific protein fingerprint contained in urine microvesicles of lung cancer patients, and this set of fingerprints can be used for non-invasive early diagnosis of lung cancer. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of a protein marker for convenient, fast, accurate and efficient diagnosis of lung cancer.

[0005] Another purpose of the present invention is to provide a reagent for auxiliary diagnosis or prognosis of lung cancer.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] Use of a reagent for detecting protein markers derived from urinary extracellular vesicles in the preparation of a reagent for the auxiliary diagnosis of lung cancer, wherein the protein markers derived from urinary extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15.

[0008] As a preference of the present invention, the protein markers are a combination of MAPK1IP1L, STOM, LAP3 and RAB33B.

[0009] As a preference of the present invention, the reagent for detecting protein markers derived from urinary extracellular vesicles is a primer or a chip for detecting the encoding genes of the protein markers.

[0010] As a preference of the present invention, the reagent for detecting protein markers derived from urinary extracellular vesicles is an antibody, an antibody fragment or a combination thereof for detecting the protein markers.

[0011] Use of a reagent for detecting protein markers derived from urinary extracellular vesicles in the preparation of a reagent for the prognosis of lung cancer, wherein the protein markers derived from urinary extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15.

[0012] As a preference of the present invention, the protein markers are a combination of MAPK1IP1L, STOM, LAP3 and RAB33B.

[0013] As a preference of the present invention, the reagent for detecting protein markers derived from urinary extracellular vesicles is a primer or a chip for detecting the encoding genes of the protein markers.

[0014] As a preference of the present invention, the reagent for detecting protein markers derived from urinary extracellular vesicles is an antibody or a combination of antibodies for detecting the protein markers.

[0015] A reagent for the auxiliary diagnosis or prognosis of lung cancer, comprising a reagent for detecting protein markers derived from urinary extracellular vesicles; the protein markers derived from urinary extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15.

[0016] As a preference of the present invention, the protein markers are a combination of MAPK1IP1L, STOM, LAP3 and RAB33B.

[0017] Beneficial effects:

[0018] The inventors found through high-throughput proteomic technology that there were significant differences in the proteins in urinary extracellular vesicles among healthy people, patients with benign lung nodules, and patients with lung cancer. Through a series of screenings, the inventors finally selected 10 proteins (TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15) that were up-regulated in the urinary extracellular vesicles of patients with lung cancer. The inventors further analyzed by flow cytometry technology in 50 healthy people, 100 patients with benign lung nodules, and 200 patients with lung cancer and found that the combination of four proteins, MAPK1IP1L, STOM, LAP3, and RAB33B, derived from urinary extracellular vesicles could effectively distinguish patients with lung cancer from healthy people and patients with benign lung nodules. Generally speaking, the proteins in urinary extracellular vesicles can be used as detection markers for the diagnosis, staging, and prognosis of lung cancer. Description of the drawings

[0019] Figure 1 Protein expression profile of urinary extracellular vesicles. A Heat map of differentially expressed proteins in urinary extracellular vesicles. Blue represents down-regulated proteins; red represents up-regulated proteins. B t-SNE analysis. The proteins in urinary extracellular vesicles can distinguish patients with lung cancer from healthy people and patients with benign lung nodules. LC: patients with lung cancer; Ben: patients with benign lung nodules; Ctl: healthy people.

[0020] Figure 2 Shows the expression levels of TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, and RAB15 in urinary microvesicles of patients with lung cancer (Lc) and healthy people (Ctl) and patients with benign lung nodules (Ben) measured by flow cytometry.

[0021] Figure 3 Shows the ROC curves of TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, and RAB15 in urinary microvesicles for differentiating lung cancer from healthy people (Ctl) and patients with benign lung nodules (Ben). A ROC curve; B Area under the ROC curve AUC and 95% CI and p-value.

[0022] Figure 4Shows the ROC curves of MAPK1IP1L, STOM, LAP3, and RAB33B in urine microvesicles for differentiating lung cancer (Lc) from healthy individuals (Ctl) and patients with benign lung nodules (Ben). A ROC fitting curve parameters; B ROC curves.

[0023] Figure 5 Shows the changes in the levels of MAPK1IP1L, STOM, LAP3, and RAB33B in urine microvesicles before surgery (Lc - Before) and one week after surgery (Lc - after) in lung cancer patients.

[0024] Table 1. Proteins significantly up - regulated in urine extracellular vesicles identified by high - throughput proteomic technology in lung cancer patients (Lc), healthy individuals (Ctl), and patients with benign lung nodules (Ben), and the area under the ROC curve (AUC) of the corresponding proteins for differentiating lung cancer (Lc) from healthy individuals (Ctl) and patients with benign lung nodules (Ben). FC: fold change; p: p - value.

[0025]

[0026] Detailed implementation methods

[0027] Example 1 Detection of urine microvesicle proteomics

[0028] 1. Collect 20 ml of early morning urine from 33 lung cancer patients (Lc), 33 healthy individuals (Ctl), and 40 patients with benign lung nodules (Ben). Centrifuge at 3000 rpm at room temperature for 30 minutes to remove cell debris and retain the supernatant.

[0029] 2. Since the surface of extracellular vesicles is rich in glycoprotein - modified proteins, these glycoprotein - modified proteins can bind to lectins conjugated to magnetic beads. Therefore, add lectin - conjugated magnetic beads to the urine supernatant obtained in the first step and incubate at room temperature for 1 hour. At this time, the extracellular vesicles in the urine will be captured by the magnetic beads.

[0030] 3. Place the EP tube on a magnetic separation rack for magnetic separation to remove the supernatant, and retain the magnetic beads and the extracellular vesicles they capture.

[0031] 4. Add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA to the mixture of magnetic beads and the extracellular vesicles they capture obtained in the third step, wash 3 times, enrich the magnetic beads, and discard the supernatant.

[0032] 5. Collect the magnetic beads and the extracellular vesicles they capture obtained in the fourth step, extract proteins for proteomic detection.

[0033] Results

[0034] Using proteomic detection, it was found that there were significant differences in urinary microvesicle proteins among lung cancer (Lc), healthy individuals (Ctl), and patients with benign lung nodules (Ben). The heatmap showed that some proteins were highly expressed in the urinary microvesicles of lung cancer patients, while some were lowly expressed in the urinary microvesicles of lung cancer patients ( Figure 1 A). More interestingly, we found by tSNE analysis that urinary microvesicle proteins could well distinguish lung cancer (Lc) from healthy individuals (Ctl) and patients with benign lung nodules (Ben)( Figure 1 B). Further analysis revealed that 25 proteins were significantly increased in the urinary microvesicles of lung cancer patients (Table 1). And the area under the ROC curve (AUC) of these proteins in differentiating lung cancer (Lc) from healthy individuals (Ctl) and patients with benign lung nodules (Ben) was greater than 0.5. We selected 10 proteins (TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15) with an AUC greater than 0.7 in differentiating lung cancer (Lc) from healthy individuals (Ctl) and patients with benign lung nodules (Ben) for further analysis.

[0035] Example 2

[0036] Using flow cytometry to detect urinary microvesicle proteins as lung cancer diagnostic markers

[0037] 1. Collect urine from lung cancer (Lc), healthy individuals (Ctl), and patients with benign lung nodules (Ben), centrifuge at 3000 rpm at room temperature for 30 minutes to remove cell debris, and retain the supernatant.

[0038] 2. Since the surface of extracellular vesicles is rich in glycosylated proteins, these glycosylated proteins can bind to lectins conjugated to magnetic beads. Therefore, 0.5 ml of the prepared lectin-conjugated magnetic beads was added to 1 ml of the urine supernatant obtained in the first step, and incubated at room temperature for 1 hour. At this time, the extracellular vesicles in the urine were captured by the magnetic beads.

[0039] 3. Place the EP tube on the magnetic separation rack for magnetic separation to remove the supernatant, and retain the magnetic beads and the extracellular vesicles they captured. Subsequently, add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then enrich the magnetic beads and discard the supernatant.

[0040] 4. Add 200 μl of 4% paraformaldehyde solution to the magnetic bead and extracellular vesicle complex obtained in the third step, and fix the captured extracellular vesicles at room temperature for 5 min. Subsequently, add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then enrich the magnetic beads and discard the supernatant.

[0041] 5. Add 200 μL of PBS-Triton solution (membrane-breaking agent, 0.1%) to the magnetic bead and extracellular vesicle complex obtained in the fourth step, and break the membrane of the captured extracellular vesicles at room temperature for 5 min. Subsequently, add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then enrich the magnetic beads and discard the supernatant.

[0042] 6. Add 100 μL of PBS solution (pH 7.2) containing 0.1% BSA to the magnetic beads and the mixture of captured extracellular vesicles obtained in the fifth step to resuspend the mixture, and respectively add fluorescently labeled antibodies against TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, and RAB15, and incubate in the dark at room temperature for 1 hour.

[0043] 7. Add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA to the solution in the sixth step, wash, enrich the magnetic beads, and discard the supernatant.

[0044] 8. After repeating the seventh step 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 detect using a flow cytometer.

[0045] Results

[0046] Adsorb vesicles in urine using lectin-conjugated magnetic beads, and then detect the protein content contained in urine vesicles by flow cytometry and fluorescent antibodies of corresponding proteins. The flow cytometry detection results show that TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, and RAB15 are significantly increased in urine microvesicles of lung cancer patients ( Figure 2 ). Further analysis using the receiver operating characteristic curve (ROC curve) found that TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, and RAB15 can effectively distinguish lung cancer patients from healthy individuals (Ctl) and patients with benign lung nodules (Ben) ( Figure 3 ). Through binary logistic regression analysis, it was found that when the four proteins MAPK1IP1L, STOM, LAP3, and RAB33B are used in combination, the ability to distinguish lung cancer patients from healthy individuals (Ctl) and patients with benign lung nodules (Ben) is the strongest, and the area under the curve is 0.96660 (95% CI: 0.9104 to 1.000) ( Figure 4), and the accuracy and precision are 0.9315 and 0.9697 respectively.

[0047] Example 3

[0048] Detect the changes of urinary microvesicle proteins before and after surgery in lung cancer patients by flow cytometry

[0049] 1. Collect the urine of lung cancer (Lc) patients before surgery and one week after surgery, centrifuge at 3000 rpm at room temperature for 30 minutes to remove cell debris, and retain the supernatant.

[0050] 2. Since the surface of extracellular vesicles is rich in glycosylated proteins, these glycosylated proteins can bind to lectins conjugated to magnetic beads. Therefore, add 0.5 ml of the prepared lectin-conjugated magnetic beads to 1 ml of the urine supernatant obtained in the first step, and incubate at room temperature for 1 hour. At this time, the extracellular vesicles in the urine will be captured by the magnetic beads.

[0051] 3. Place the EP tube on the magnetic separation rack to magnetically separate and remove the supernatant, and retain the magnetic beads and the extracellular vesicles they capture. Subsequently, add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then enrich the magnetic beads and discard the supernatant.

[0052] 4. Add 200 μl of 4% paraformaldehyde solution to the magnetic bead and extracellular vesicle complex obtained in the third step, and fix the captured extracellular vesicles at room temperature for 5 min. Subsequently, add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then enrich the magnetic beads and discard the supernatant.

[0053] 5. Add 200 μl of PBS-Triton solution (membrane-breaking agent, 0.1%) to the magnetic bead and extracellular vesicle complex obtained in the fourth step, and break the membrane of the captured extracellular vesicles at room temperature for 5 min. Subsequently, add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then enrich the magnetic beads and discard the supernatant.

[0054] 6. Add 100 μl of PBS solution (pH 7.2) containing 0.1% BSA to resuspend the mixture of the magnetic beads and the extracellular vesicles they capture obtained in the fifth step, and add fluorescently labeled MAPK1IP1L, STOM, LAP3, and RAB33B antibodies respectively, and incubate in the dark at room temperature for 1 hour.

[0055] 7. Add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA to wash the solution in the sixth step, enrich the magnetic beads, and discard the supernatant.

[0056] 8. After repeating step 7 three times, enrich the magnetic beads and discard the supernatant. Resuspend the magnetic beads and the extracellular vesicles they captured in 300 μL of PBS solution containing 0.1% BSA and detect them using a flow cytometer.

[0057] Results

[0058] Adsorb the vesicles in urine using lectin-conjugated magnetic beads, and then detect the protein content contained in the urine vesicles by flow cytometry and fluorescent antibodies against the corresponding proteins. The flow cytometry results show that MAPK1IP1L, STOM, LAP3, and RAB33B are significantly decreased in the urine microvesicles of lung cancer patients after surgery ( Figure 5 ), and these results not only suggest that the combined use of the four proteins MAPK1IP1L, STOM, LAP3, and RAB33B in urine microvesicles can be used as a biomarker for lung cancer diagnosis, but also can be used as a biomarker for lung cancer prognosis.

Claims

1. Use of a reagent for detecting protein markers derived from urinary extracellular vesicles in the preparation of a reagent for auxiliary diagnosis of lung cancer, characterized in that, The protein markers derived from urinary extracellular vesicles described above are a combination of MAPK1IP1L, STOM, LAP3, and RAB33B.

2. The application according to claim 1, wherein The reagent for detecting protein markers derived from urinary extracellular vesicles described above is a primer or a chip for detecting the genes encoding the protein markers.

3. The application according to claim 1 or 2, characterized in that, The reagent for detecting protein markers derived from urinary extracellular vesicles described above is an antibody, an antibody fragment, or a combination thereof for detecting the protein markers.

4. An adjuvant diagnostic reagent for lung cancer, characterized in that, A detection reagent containing protein markers derived from urinary extracellular vesicles; the protein markers derived from urinary extracellular vesicles described above are a combination of MAPK1IP1L, STOM, LAP3, and RAB33B.

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