CN114839375A - Lung cancer-related urine microvesicle protein and application thereof - Google Patents

Lung cancer-related urine microvesicle protein and application thereof Download PDF

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CN114839375A
CN114839375A CN202210559712.1A CN202210559712A CN114839375A CN 114839375 A CN114839375 A CN 114839375A CN 202210559712 A CN202210559712 A CN 202210559712A CN 114839375 A CN114839375 A CN 114839375A
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梁宏伟
王嘉凡
张衡
杨荣
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China Pharmaceutical University
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Abstract

The invention discloses a lung cancer-related urine microvesicle protein and application thereof. The application of a reagent for detecting protein markers derived from urine extracellular vesicles in the preparation of an auxiliary lung cancer diagnostic reagent is disclosed, wherein the protein markers derived from the urine extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15. The protein marker can effectively distinguish lung cancer patients from healthy people from benign lung nodule patients. In general, the protein in the urine extracellular vesicles can be used as a detection marker for the diagnosis, the staging and the prognosis of lung cancer.

Description

Lung cancer-related urine microvesicle protein and application thereof
Technical Field
The invention belongs to the field of medical diagnosis, and relates to a lung cancer-related urine microvesicle protein and an application thereof.
Background
Lung cancer is a malignant tumor that originates in the bronchial mucosa or glands of the lung and is most threatening the health and life of human beings, and the morbidity and mortality of the malignant tumor are the first of the malignant tumors. In recent years, the incidence and mortality of lung cancer are higher and higher due to the fact that air pollution is increasingly aggravated due to the continuous development of industrialization in China and the influence of factors such as aging and the like. In the next decades, lung cancer will be the most important factor in cancer prevention and treatment in China. The international early lung cancer action plan data show that the expected survival rate of the lung cancer in the first stage after reasonable treatment for 10 years can reach more than 90 percent, and the survival rate of the lung cancer in the later stage is lower than 5 percent. Therefore, the lung cancer is regularly screened and early treated by early detection, so that the cure rate can be obviously improved and the death rate can be reduced. The current lung cancer examination method mainly comprises serum marker examination, imaging examination and puncture examination. However, the above-mentioned examination methods have the reasons of low accuracy (serum marker examination), irradiation, high price, and difficult operation (imaging examination and puncture examination), which limit the application thereof in lung cancer screening.
Urine is an extremely valuable diagnostic medium, which is enriched with Extracellular Vesicles (EVs). The extracellular vesicles are rich in various bioactive substances such as proteins and nucleic acids, and can reflect physiological and pathological states of cells from which the extracellular vesicles are derived. Due to the protection of the outer membrane structure of the extracellular vesicles, the biologically active substances such as proteins and nucleic acids contained in the extracellular vesicles are very stable, and the proteins and nucleic acids contained in the extracellular vesicles have been considered as ideal markers for disease diagnosis. In the invention, specific protein fingerprints contained in the urine microvesicles of the lung cancer patients are identified, and noninvasive early diagnosis of the lung cancer can be performed by using the group of fingerprints.
Disclosure of Invention
The invention aims to provide the application of the protein marker for conveniently, quickly, accurately and efficiently diagnosing lung cancer.
Another objective of the invention is to provide an auxiliary diagnostic or prognostic reagent for lung cancer.
The purpose of the invention can be realized by the following technical scheme:
the application of a reagent for detecting protein markers derived from urine extracellular vesicles in the preparation of an auxiliary lung cancer diagnostic reagent is disclosed, wherein the protein markers derived from the urine extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15.
Preferably, the protein marker is a combination of MAPK1IP1L, STOM, LAP3 and RAB 33B.
Preferably, the reagent for detecting a protein marker derived from urine extracellular vesicles is a primer or a chip for detecting a gene encoding the protein marker.
Preferably, the reagent for detecting a protein marker derived from urine extracellular vesicles is an antibody, an antibody fragment or a combination thereof for detecting the protein marker.
The application of a reagent for detecting protein markers derived from urine extracellular vesicles in the preparation of a reagent for the prognosis of lung cancer is disclosed, wherein the protein markers derived from the urine extracellular vesicles are selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15.
Preferably, the protein marker is a combination of MAPK1IP1L, STOM, LAP3 and RAB 33B.
Preferably, the reagent for detecting a protein marker derived from urine extracellular vesicles is a primer or a chip for detecting a gene encoding the protein marker.
In a preferred embodiment of the present invention, the reagent for detecting a protein marker derived from urine extracellular vesicles is an antibody or a combination of antibodies for detecting the protein marker.
An auxiliary diagnosis or prognosis reagent for lung cancer, comprising a detection reagent for protein markers derived from urine extracellular vesicles; the protein marker derived from the urine extracellular vesicle is selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15.
Preferably, the protein marker is a combination of MAPK1IP1L, STOM, LAP3 and RAB 33B.
Has the advantages that:
the inventors found that proteins in urine extracellular vesicles are significantly different among healthy people, benign lung nodule patients and lung cancer patients by a high-throughput protein mass spectrometry technology. Through a series of screens, the final inventors selected 10 proteins that elevated in urine extracellular vesicles of lung cancer patients (TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5a2, RAB33B, STX7, CAPZA2, RAB 15). The inventor further analyzes and discovers by flow cytometry in 50 healthy people, 100 benign lung nodule patients and 200 lung cancer patients that the combination of the four proteins MAPK1IP1L, STOM, LAP3 and RAB33B derived from urine extracellular vesicles can effectively distinguish the lung cancer patients from the healthy lung nodule patients. In general, the protein in the urine extracellular vesicles can be used as a detection marker for the diagnosis, the staging and the prognosis of lung cancer.
Drawings
FIG. 1 urine extracellular vesicle protein expression profile. A urine extracellular vesicle differential protein heatmap. Blue represents reduced protein; red represents ascending protein. B t-SNE analysis. Urine extracellular vesicle proteins can distinguish lung cancer patients from healthy persons from benign lung nodule patients. LC is a lung cancer patient; ben for benign pulmonary nodule patients; ctl is healthy.
FIG. 2 shows the levels of TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB15 expressed in urine microvesicles of lung cancer patients (Lc) and healthy persons (Ctl) and benign lung nodule patients (Ben) as determined by flow cytometry.
Fig. 3 shows ROC curves for TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5a2, RAB33B, STX7, CAPZA2, RAB15 in urine microvesicles to identify lung cancer and healthy humans (Ctl) versus benign lung nodule patients (Ben). A ROC curve; AUC of the area under the B ROC curve, and 95% CI and p-value.
FIG. 4 shows ROC curves for MAPK1IP1L, STOM, LAP3, RAB33B in urine microvesicles to identify lung cancer (Lc) and healthy (Ctl) versus benign lung nodule patients (Ben). A ROC fitting curve parameters; b ROC curve.
FIG. 5 shows the change in MAPK1IP1L, STOM, LAP3, RAB33B content in urine microvesicles Before (Lc-Before) and one week after (Lc-after) surgery in lung cancer patients.
Table 1. high throughput protein mass spectrometry identifies proteins in urine extracellular vesicles that significantly rise in lung cancer patients (Lc) and healthy people (Ctl) and benign lung nodule patients (Ben), and the area under the ROC curve (AUC) for the corresponding proteins in identifying lung cancer (Lc) and healthy people (Ctl) and benign lung nodule patients (Ben): fold change; p is p-value.
Figure BDA0003656049290000031
Figure BDA0003656049290000041
Detailed Description
Example 1 Mass Spectrometry detection of urine microvesicle proteins
1. 20ml of each of 33 lung cancers (Lc) and 33 healthy persons (Ctl) and 40 patients with benign lung nodules (Ben) were collected, centrifuged at 3000 r.t. for 30 min to remove cell debris and the supernatant was retained.
2. Since extracellular vesicles are rich in glycosylation modified proteins on their surface, these glycosylation modified proteins can bind to lectins coupled to magnetic beads. Therefore, the supernatant of the urine obtained from the first step of separation is added with lectin-coupled magnetic beads and incubated at room temperature for 1 hour, at which time extracellular vesicles in the urine are captured by the magnetic beads.
3. And (3) placing the EP tube on a magnetic separation frame for magnetic separation to remove supernatant, and retaining the magnetic beads and the extracellular vesicles captured by the magnetic beads.
4. After adding 1000. mu.L of PBS solution (pH 7.2) containing 0.1% BSA to the magnetic beads obtained in the third step and the extracellular vesicle mixture captured by the magnetic beads, the magnetic beads were enriched, and the supernatant was discarded.
5. And collecting the magnetic beads obtained in the fourth step and extracellular vesicles captured by the magnetic beads, and extracting protein for protein mass spectrometry detection.
Results
Protein mass spectrometry detection shows that the urine microvesicle protein has significant difference in lung cancer (Lc) and healthy people (Ctl) and benign lung nodule patients (Ben), and a heat map shows that some proteins are highly expressed in urine microvesicles of lung cancer patients and some proteins are lowly expressed in urine microvesicles of lung cancer patients (fig. 1A). More interestingly, we found that urinary microvesicles protein could well distinguish lung cancer (Lc) and healthy people (Ctl) from benign lung nodule patients (Ben) using tSNE analysis (fig. 1B). Further analysis revealed that 25 proteins were significantly elevated in urine microvesicles of lung cancer patients (table 1). And the area under the ROC curve (AUC) of the proteins in the identification of lung cancer (Lc) and healthy people (Ctl) and benign lung nodule patients (Ben) is more than 0.5. We selected for further analysis 10 proteins (TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5a2, RAB33B, STX7, CAPZA2, RAB15) from which the area under the ROC curve (AUC) was greater than 0.7 in identifying lung cancer (Lc) and healthy humans (Ctl) versus benign lung nodule patients (Ben).
Example 2
Detection of urine microvesicle protein by flow cytometry as lung cancer diagnosis marker
1. Urine from lung cancer (Lc) and healthy (Ctl) and benign lung nodule patients (Ben) was collected, centrifuged at 3000 rpm for 30 minutes at room temperature to remove cell debris and the supernatant was retained.
2. Since extracellular vesicles are rich in glycosylation modified proteins on their surface, these glycosylation modified proteins can bind to lectins coupled to magnetic beads. Therefore, 0.5ml of prepared lectin-coupled magnetic beads was added to 1ml of the urine supernatant obtained from the first separation step, and incubated at room temperature for 1 hour, at which time extracellular vesicles in the urine were captured by the magnetic beads.
3. And (3) placing the EP tube on a magnetic separation frame for magnetic separation to remove supernatant, and retaining the magnetic beads and the extracellular vesicles captured by the magnetic beads. After washing 3 times with 1000. mu.L of PBS containing 0.1% BSA (pH 7.2), the beads were enriched and the supernatant discarded.
4. And (3) adding 200ul of 4% paraformaldehyde solution into the compound of the magnetic beads and the extracellular vesicles obtained in the third step, and fixing the captured extracellular vesicles at room temperature for 5 min. After washing 3 times with 1000. mu.L of PBS containing 0.1% BSA (pH 7.2), the beads were enriched and the supernatant discarded.
5. And (3) adding 200ul of PBS-Triton solution (membrane breaking agent, 0.1%) into the compound of the magnetic beads and the extracellular vesicles obtained in the fourth step, and breaking the membrane of the captured extracellular vesicles at room temperature for 5 min. After washing 3 times with 1000. mu.L of PBS containing 0.1% BSA (pH 7.2), the beads were enriched and the supernatant discarded.
6. Adding 100ul of a PBS (pH 7.2) containing 0.1% BSA to the magnetic beads obtained in the fifth step and the extracellular vesicle mixture captured by the magnetic beads, respectively adding TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2 and RAB15 antibodies with fluorescent labels, and incubating for 1 hour at room temperature in the absence of light.
7. To the solution of the sixth step, 1000. mu.L of PBS solution (pH 7.2) containing 0.1% BSA was added for washing, magnetic beads were enriched, and the supernatant was discarded.
8. After repeating the seventh step three times, enriching the magnetic beads and discarding the supernatant. The magnetic beads and their captured extracellular vesicles were resuspended in 300. mu.L of PBS containing 0.1% BSA and detected by flow cytometry.
Results
The method comprises the following steps of adsorbing vesicles in urine by utilizing magnetic beads coupled with lectin, and detecting the content of protein contained in the urine vesicles through a flow cytometer and fluorescent antibodies of corresponding protein. Flow cytometry results show that TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2 and RAB15 are remarkably increased in urine micro-vesicles of lung cancer patients (figure 2), and further analysis of a subject working characteristic curve (ROC curve for short) shows that TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2 and RAB15 can effectively distinguish Ctl lung cancer patients from healthy persons (Ben) and benign lung nodule patients (Ben) (figure 3). It was found by binary logistic regression analysis that when MAPK1IP1L, STOM, LAP3 and RAB33B were used in combination, the ability to distinguish lung cancer patients from healthy persons (Ctl) from benign lung nodule patients (Ben) was greatest, with an area under the curve of 0.96660 (95% CI:0.9104to 1.000) (FIG. 4) and an accuracy and precision of 0.9315 and 0.9697, respectively.
Example 3
Flow cytometry is utilized to detect changes of urine micro-vesicle protein before and after operation of lung cancer patients
1. Urine of a lung cancer (Lc) patient for one week before and after operation is collected, centrifuged at 3000 r.t. for 30 minutes, cell debris is removed, and the supernatant is retained.
2. Since extracellular vesicles are rich in glycosylation modified proteins on their surface, these glycosylation modified proteins can bind to lectins coupled to magnetic beads. Therefore, 0.5ml of prepared lectin-coupled magnetic beads was added to 1ml of the urine supernatant obtained from the first separation step, and incubated at room temperature for 1 hour, at which time extracellular vesicles in the urine were captured by the magnetic beads.
3. And (3) placing the EP tube on a magnetic separation frame for magnetic separation to remove supernatant, and retaining the magnetic beads and the extracellular vesicles captured by the magnetic beads. After washing 3 times with 1000. mu.L of PBS containing 0.1% BSA (pH 7.2), the beads were enriched and the supernatant discarded.
4. And (3) adding 200ul of 4% paraformaldehyde solution into the compound of the magnetic beads and the extracellular vesicles obtained in the third step, and fixing the captured extracellular vesicles at room temperature for 5 min. After washing 3 times with 1000. mu.L of PBS containing 0.1% BSA (pH 7.2), the beads were enriched and the supernatant discarded.
5. And (3) adding 200ul of PBS-Triton solution (membrane breaking agent, 0.1%) into the compound of the magnetic beads and the extracellular vesicles obtained in the fourth step, and breaking the membrane of the captured extracellular vesicles at room temperature for 5 min. After washing 3 times with 1000. mu.L of PBS containing 0.1% BSA (pH 7.2), the beads were enriched and the supernatant discarded.
6. To the magnetic beads obtained in the fifth step and the extracellular vesicle mixture captured by them, 100ul of a PBS solution (pH 7.2) containing 0.1% BSA was added to the resuspended mixture, and the fluorescently labeled MAPK1IP1L, STOM, LAP3 and RAB33B antibodies were added, respectively, and incubated for 1 hour at room temperature in the absence of light.
7. To the solution of the sixth step, 1000. mu.L of PBS solution (pH 7.2) containing 0.1% BSA was added for washing, magnetic beads were enriched, and the supernatant was discarded.
8. After repeating the seventh step three times, enriching the magnetic beads and discarding the supernatant. The magnetic beads and their captured extracellular vesicles were resuspended in 300. mu.L of PBS containing 0.1% BSA and detected by flow cytometry.
Results
The method comprises the following steps of adsorbing vesicles in urine by utilizing magnetic beads coupled with lectin, and detecting the content of protein contained in the urine vesicles through a flow cytometer and fluorescent antibodies of corresponding protein. Flow cytometry results show that MAPK1IP1L, STOM, LAP3 and RAB33B are remarkably reduced in urine microvesicles of lung cancer patients after operations (figure 5), and the results not only suggest that the combined use of MAPK1IP1L, STOM, LAP3 and RAB33B in the urine microvesicles can be used as a marker for lung cancer diagnosis, but also can be used as a marker for lung cancer prognosis.

Claims (10)

1. The application of the reagent for detecting the protein marker derived from the urine extracellular vesicles in the preparation of the auxiliary diagnostic reagent for the lung cancer is characterized in that the protein marker derived from the urine extracellular vesicles is selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15.
2. The use of claim 1, wherein the protein marker is a combination of MAPK1IP1L, STOM, LAP3, and RAB 33B.
3. The use according to claim 1 or 2, wherein the reagent for detecting the protein marker derived from the urine extracellular vesicles is a primer or chip for detecting a gene encoding the protein marker.
4. The use according to claim 1 or 2, wherein the reagent for detecting the protein marker derived from the urine extracellular vesicles is an antibody, an antibody fragment or a combination thereof for detecting the protein marker.
5. The application of the reagent for detecting the protein marker derived from the urine extracellular vesicles in preparing the reagent for the prognosis of the lung cancer is characterized in that the protein marker derived from the urine extracellular vesicles is selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15.
6. The use of claim 1, wherein the protein marker is a combination of MAPK1IP1L, STOM, LAP3, and RAB 33B.
7. The use according to claim 5 or 6, wherein the reagent for detecting the protein marker derived from the urine extracellular vesicles is a primer or chip for detecting a gene encoding the protein marker.
8. The use according to claim 5 or 6, wherein the reagent for detecting the protein marker derived from the urine extracellular vesicles is an antibody or a combination of antibodies for detecting the protein marker.
9. An auxiliary diagnosis or prognosis reagent for lung cancer, which is characterized by comprising a detection reagent for a protein marker derived from urine extracellular vesicles; the protein marker derived from the urine extracellular vesicle is selected from any one or more of the following proteins: TRAP1, MAPK1IP1L, STOM, LAP3, FGB, SLC5A2, RAB33B, STX7, CAPZA2, RAB 15.
10. The reagent of claim 9, wherein the protein marker is a combination of MAPK1IP1L, STOM, LAP3 and RAB 33B.
CN202210559712.1A 2022-05-23 2022-05-23 Lung cancer-related urine microvesicle protein and application thereof Pending CN114839375A (en)

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