CN109975546B

CN109975546B - Screening of early breast cancer lung metastasis related urine protein marker and application thereof

Info

Publication number: CN109975546B
Application number: CN201711462691.7A
Authority: CN
Inventors: 高友鹤; 魏静
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2021-07-06
Anticipated expiration: 2037-12-28
Also published as: CN109975546A

Abstract

The invention relates to screening of a urinary protein marker related to early breast cancer lung metastasis and application thereof. In particular, the invention relates to a method for establishing an animal model for screening a urinary protein marker related to early breast cancer lung metastasis, a urinary protein marker related to early breast cancer lung metastasis obtained by using the animal model established by the method, and application of a reagent for detecting the urinary protein marker in preparing a kit for diagnosing early breast cancer lung metastasis of a subject.

Description

Screening of early breast cancer lung metastasis related urine protein marker and application thereof

Technical Field

The present invention relates to clinical medicine. In particular to a human breast cancer lung metastasis related urine protein marker. Specifically, the invention relates to a urine protein marker related to diagnosis and/or monitoring of lung metastasis of human breast cancer, which is obtained by utilizing a rat model of lung metastasis of breast cancer and a mass spectrometry proteomics technology, and application thereof.

Background

Urine proteomics is of great significance in disease marker research. Biomarkers are monitorable changes associated with pathophysiological processes and can be used to diagnose disease, monitor disease course, predict disease prognosis, and assess treatment efficacy, among other things.

Urine, as a product of the filtration of blood through the glomeruli, is an ideal source of disease markers: the urine is obtained simply and non-invasively, can be collected for a long time, and is more beneficial to observing the dynamic change of diseases; urine does not require regulation by homeostatic mechanisms and thus can accumulate and react to a greater extent to changes in the body that are potential disease markers. Meanwhile, the lack of homeostatic mechanism also determines that urine has great prospect in early diagnosis of diseases (see Gao Y. urine-an unsupported mineral for biorarer discovery. Sci China. Life Sci,2013,56(12): 1145; Wu J, Gao Y. physical considerations can be deflected in human urine protein and metabolome. expert review of proteomics,2015,12(6):623 636).

Urine proteomics as a novel, noninvasive and rapidly-developed analysis tool can be used for digging disease-specific biomarkers in urine so as to partially replace organ puncture pathological examination. At present, 6000 proteins can be deeply identified in the human uroproteome (ZHao, M et al, A complex urinary protein analysis: positional applications in disease biological discovery and identification. the Lancet,2015.386: p.S63). Urine protein has become a research direction of important attention of scientists and clinicians, and has important scientific significance and huge clinical application prospect.

Breast cancer is one of the most common cancers among women in the united states, with the incidence accounting for about one-third of all women suffering from cancer, and is also the second most lethal cancer immediately following lung cancer. About 6% of patients have distant metastasis at the time of initial visit, while 30% of patients diagnosed as early in breast cancer will have distant organ metastasis, with a five-year survival rate often less than 20% for metastatic breast cancer patients (see Giuliani J, Bonetiti A. trends in Survival for Patients with metastatic Breast cancer. 2015; 101(4): 347-52). The distant organ metastasis of breast cancer is often the main cause of death, and the common distant organ metastasis sites are mainly lung, pleura, liver, bone, etc., while the lung is the most common metastasis site (see Weigel B, Peterse JL, van't Veer LJ. Breast cancer patients: markers and models. Nat Rev cancer. 2005; 5(8): 591-. Breast cancer, a heterogeneous tumor, has not been routinely screened to predict the potential distant metastasis in early asymptomatic patients (see Cardoso F, Harbeck N, Fallowfield L, Kyrakides S, Senkus E, Group EGW. Locally recurrentor viral research cancer: ESMO Clinical Practice Guidelines for diagnosis, breast cancer and follow-up. Ann Oncol.2012; 23Suppl 7: vii 11-9); meanwhile, many female patients also receive unnecessary chemotherapy treatment due to imperfect prognostic criteria (see Weigelt B, Peterse JL, van't Veer LJ. Breast cancer patients: markers and models. Nat Rev cancer.2005; 5(8): 591-. Metastatic breast cancer is often treated clinically by methods for treating primary breast cancer, and often has no effect on controlling breast cancer metastasis (see Sharma R, Sharma R, Khaket TP, Dutta C, Chakraborty B, Mukherjee TK. Breast cancer metastasis: reactive thermal roller of cellular adsorption molecule-1.Cell Oncol (Dordr). 2017; 40(3): 199-208). Therefore, early diagnosis of breast cancer metastasis and timely monitoring of metastasis progress are of great significance for clinical treatment of metastatic breast cancer.

Disclosure of Invention

The present inventors have unexpectedly found that there is a significant difference in the urine protein profile in a rat model with early breast cancer lung metastasis from control rats. According to this unexpected discovery by the inventors, in one aspect, the present invention provides a method of establishing an animal model for screening for a urine protein marker associated with early breast cancer lung metastasis, the method comprising the steps of:

i) obtaining a rat model with lung metastasis of breast cancer by tail vein injection of breast cancer cells;

ii) collecting rat urine before and after establishing a rat model of breast cancer lung metastasis; and

iii) identification of early breast cancer lung metastasis by mass spectrometry rat models protein spectra in rat urine before and after establishment.

Preferably, the urine of the rat is collected before tail vein injection of the breast cancer cells and at

days

2, 4, 6 and 9 after tail vein injection of the breast cancer cells in step ii) of the above-described method of the present invention.

Further preferably, in step iii) of the method of the present invention, the mass spectrum of proteins in rat urine before tail vein injection of breast cancer cells, and 2, 4, 6 and 9 days after tail vein injection of breast cancer cells is identified by LC-MS/MS tandem mass spectrometry.

The method according to the present invention as described above, further comprising the step of iv) comparing the protein profile obtained in step iii) before tail vein injection of breast cancer cells with the protein profiles at

days

2, 4, 6 and 9 after tail vein injection of breast cancer cells.

The method according to the present invention as described above, wherein said breast cancer cells are walker256 breast cancer sarcoma cells.

In another aspect, the present invention provides the use of a rat model with lung metastasis of breast cancer obtained by tail vein injection of breast cancer cells in the preparation of an animal model for screening urine protein markers associated with early lung metastasis of breast cancer.

In another aspect, the present invention provides the use of a reagent for detecting one or more proteins in urine, wherein the one or more proteins are selected from urine protein markers associated with early breast cancer lung metastasis obtained by using the animal model established by the method of the present invention described above, in the preparation of a kit for diagnosing early breast cancer lung metastasis in a subject.

Use of a reagent for detecting one or more proteins in urine according to the present invention for the preparation of a kit for diagnosing early breast cancer lung metastasis in a subject, wherein the one or more proteins are selected from the group consisting of: aminoacylase-1A, aminopeptidase N, EH structure threshold protein 1, insulin growth factor binding protein 3, pancreatic secretory granule membrane glycoprotein 2, tubulin beta-4B chain, glyceraldehyde triphosphate dehydrogenase, sodium-hydrogen ion exchange regulatory cofactor 1, annexin A2, NKG2-D class II membrane proteins, endothelial cell protein C receptor, ezrin, charged vesicle protein 5, heat shock homologous 71kDa protein, inhibin beta C chain, ester hydrolase C11orf54 homolog, beta 2-glycoprotein 1, CD48 antigen, methyldopa beta subunit, pyruvate kinase isomerase M1/M2, fructose-diphosphate aldolase B, granule protein, transforming protein RhoA, cell division control protein 42 homolog, milk lectin, LIM and SH3 domain protein 1, monoglyceride lipase, hydroxyacyl hydrolase, glutathione hydrolase, beta-glucosidase, Glutamine ammonia synthase, synegenin-forbidden 2, 14-3-3 protein theta, secretoglobin 2A, cerebro-acid-soluble 1, 14-3-3 protein gamma, tyrosine protein kinase B1, sulfotransferase 1C2, dissociation-inhibiting protein 1, CD59 glycoprotein, histone H4, protein with a molecular weight of 16kDa subunit of type V proton ATPase, myosin light chain polypeptide 6, L-lactate dehydrogenase A chain, histidine nucleotide-binding protein 1, amidase NIT2, ketohexokinase, guanine nucleotide-binding protein G (i) subunit alpha, peroxiredoxin 6, mitoprotein-1, heat shock protein 90 beta subunit, podocyte marker protein, adenylate ribosylation factor, CD9 antigen, adenylate kinase, aspartate aminotransferase, cardiac fatty acid-binding protein, pyridoxal kinase, amyloid 2 pyridoxine, pyridoxine, Peroxiredoxin 1, calmodulin, immunoglobulin gamma chain C region, fructose-diphosphate aldolase A, prostaglandin H2D isomerase, neuronal membrane glycoprotein M6-a, cystatin-C, Di-N-acetyl chitobiase, glutamyl peptidase, polyubiquitin B, leukemia inhibitory factor receptor, nerve cell adhesion molecule 1, glutamyl hydrolase, alpha-enolase, prolactin-inducible protein homolog, cytosolic ferrithioprotein assembly protein ciao1, solute family 22, voltage-dependent anion-selective channel protein 1, translation control tumor protein, intracellular chloride channel protein 1, actin 1, aquaporin 1, glutathione s-transferase, protein-glutamine gamma-glutamyltransferase 4, synapse-associated protein 23, trefoil factor 1, protein-glutamine gamma-glutamyltransferase 4, and protein, Fructose-1, 6-bisphosphatase 1, intracellular chloride channel protein 4, cystatin B, protein RoBo-1, malate dehydrogenase, elongation factor alpha 1, glutamyl peptidase, EGF-containing fibular extracellular matrix protein 1 and serum transferrin.

Further, the use of the reagent for detecting one or more proteins in urine according to the present invention in the preparation of a kit for diagnosing early breast cancer lung metastasis in a subject, wherein the reagent for detecting one or more proteins in urine is a specific antibody of the protein or an antigen binding fragment thereof, preferably the specific antibody is a monoclonal antibody or an antigen binding fragment thereof.

In particular embodiments, antigen-binding fragments include, but are not limited to: fab, Fab ', (Fab')₂Fv, ScFv, bispecific antibody, trispecific antibody, tetraspecific antibody, bis-scFv, mimi antibody. Any antibody fragment that retains antigen binding activity is suitable for use in the present disclosure.

In a particular embodiment, the subject is a mammal, preferably a human.

In another aspect, the present invention also provides a kit or a chip for diagnosing early breast cancer lung metastasis, which comprises a reagent for detecting one or more proteins in urine, wherein the one or more proteins are selected from the group consisting of: aminoacylase-1A, aminopeptidase N, EH structure threshold protein 1, insulin growth factor binding protein 3, pancreatic secretory granule membrane glycoprotein 2, tubulin beta-4B chain, glyceraldehyde triphosphate dehydrogenase, sodium-hydrogen ion exchange regulatory cofactor 1, annexin A2, NKG2-D class II membrane proteins, endothelial cell protein C receptor, ezrin, charged vesicle protein 5, heat shock homologous 71kDa protein, inhibin beta C chain, ester hydrolase C11orf54 homolog, beta 2-glycoprotein 1, CD48 antigen, methyldopa beta subunit, pyruvate kinase isomerase M1/M2, fructose-diphosphate aldolase B, granule protein, transforming protein RhoA, cell division control protein 42 homolog, milk lectin, LIM and SH3 domain protein 1, monoglyceride lipase, hydroxyacyl hydrolase, glutathione hydrolase, beta-glucosidase, Glutamine ammonia synthase, synegenin-forbidden 2, 14-3-3 protein theta, secretoglobin 2A, cerebro-acid-soluble 1, 14-3-3 protein gamma, tyrosine protein kinase B1, sulfotransferase 1C2, dissociation-inhibiting protein 1, CD59 glycoprotein, histone H4, protein with a molecular weight of 16kDa subunit of type V proton ATPase, myosin light chain polypeptide 6, L-lactate dehydrogenase A chain, histidine nucleotide-binding protein 1, amidase NIT2, ketohexokinase, guanine nucleotide-binding protein G (i) subunit alpha, peroxiredoxin 6, mitoprotein-1, heat shock protein 90 beta subunit, podocyte marker protein, adenylate ribosylation factor, CD9 antigen, adenylate kinase, aspartate aminotransferase, cardiac fatty acid-binding protein, pyridoxal kinase, amyloid 2 pyridoxine, pyridoxine, Peroxiredoxin 1, calmodulin, immunoglobulin gamma chain C region, fructose-diphosphate aldolase A, prostaglandin H2D isomerase, neuronal membrane glycoprotein M6-a, cystatin-C, Di-N-acetyl chitobiase, glutamyl peptidase, polyubiquitin B, leukemia inhibitory factor receptor, nerve cell adhesion molecule 1, glutamyl hydrolase, alpha-enolase, prolactin-inducible protein homolog, cytosolic ferrithioprotein assembly protein ciao1, solute family 22, voltage-dependent anion-selective channel protein 1, translation control tumor protein, intracellular chloride channel protein 1, actin 1, aquaporin 1, glutathione s-transferase, protein-glutamine gamma-glutamyltransferase 4, synapse-associated protein 23, trefoil factor 1, protein-glutamine gamma-glutamyltransferase 4, and protein, Fructose-1, 6-bisphosphatase 1, intracellular chloride channel protein 4, cystatin B, protein RoBo-1, malate dehydrogenase, elongation factor alpha 1, glutamyl peptidase, EGF-containing fibular extracellular matrix protein 1 and serum transferrin.

Further, the kit or chip according to the present invention, wherein the reagent for detecting one or more proteins in urine is a specific antibody of the protein or an antigen-binding fragment thereof, preferably the specific antibody is a monoclonal antibody. Further, the kit is used in a quantitative mass spectrometry method, an ELISA method or a Western method.

When mass spectrometry is used to determine the protein and its expression level, a digestion step may also be included after the step of obtaining a urine sample. In a specific embodiment, the protein in the urine sample is digested with a protease.

Drawings

FIG. 1: breast cancer lung rats and control rats were monitored for body weight. ● represents a model set; ■ represents the normal control group. Denotes p < 0.05; denotes p < 0.01; denotes p < 0.001.

Fig. 2A to 2E: results of HE staining rat lung tissue sections from the control group (fig. 2A), breast cancer lung metastasis model group 2 (fig. 2B), 4 (fig. 2C), 6 (fig. 2D) and 9 day group (fig. 2E).

Detailed Description

The present application will be further illustrated by the following non-limiting examples. It will be apparent to those skilled in the art that many changes can be made in this application without departing from the spirit thereof, and such changes are within the scope of the application. The experimental materials used are all available from commercial companies, unless otherwise specified.

Examples

Example 1: establishment of breast cancer lung metastasis rat model

The rat walker256 lung metastasis tumor model of breast cancer is a classical animal model for researching the progress of the lung metastasis tumor of the breast cancer, and is suitable for researching the change of pathophysiology and morphology in the progress process of the lung metastasis tumor. The animal model is used for simulating the lung metastasis process of the breast cancer, observing the integral changes of the lung metastasis of the breast cancer from non-metastasis, early-stage metastasis, metastasis progress and late-stage metastasis, and has a pathological process and pathological characteristics similar to those of the lung metastasis of the breast cancer of human beings, thereby having important guiding significance for clinically early diagnosing the lung metastasis of the breast cancer and monitoring the disease progress of the breast cancer.

In the present application, a rat breast cancer lung metastasis animal model was prepared using the tail vein annotation walker256 cell method, and urine of breast cancer lung metastasis at an early stage (days 2, 4) and a mid-late stage (days 6, 9) was collected. The animal model is used for simulating the lung metastasis of the breast cancer, and has important guiding significance for the early diagnosis, treatment and prognosis of the lung metastasis of the breast cancer clinically.

1. Materials and reagents

1) The instrument comprises the following steps:

rat metabolic cage: purchased from Beijing Jiayuan industry science and technology, Inc. Thermo orbitrap fusion lumos mass spectrometer: purchased from Thermo Fisher Scientific; thermo EASY-Nlc1200 high performance liquid chromatograph: purchased from Thermo Fisher Scientific; MilliQ RG ultrapure water system: purchased from Millipore corporation; c18 reverse phase analytical column (RP column, 0.1 × 150mm,3 μm,

): purchased from Michrom biosources company.

2) The main reagents are as follows:

the deionized water is from a MilliQ RG ultrapure water system; chromatographic grade acetonitrile, formic acid and methanol are produced by Fisher corporation; acetylammonium Iodide (IAA), ammonium bicarbonate, Dithiothreitol (DTT) were purchased from Sigma; sequencing grade pancreatin was purchased from Promega corporation.

3) Animals:

male Wistar rats (weighing 150g) were purchased from Beijing Wittiglihua laboratory animal technology, Inc. and housed in a standard housing environment.

2. Experimental methods

1) Passage of tumor cells

After the frozen walker256 breast cancer sarcoma cells are rapidly recovered at 37 ℃, the rat is inoculated into the abdominal cavity, and the animal is sacrificed after one week to extract ascites. The ascites was diluted with physiological saline and quantitatively inoculated into the abdominal cavities of two other rats. One week later, the rats were sacrificed and ascites fluid was withdrawn.

2) Breast cancer lung metastasis rat model establishment and sample collection

Lung metastasis of breast cancer ratA model establishing step: diluting the ascites tumor cells with sterile normal saline 10⁶Mu.l/ml of tumor cell suspension was injected via tail vein into rats.

Establishing a rat model of a control group: the mixture was injected into rats via tail vein with 100. mu.l of sterile physiological saline.

Sample collection procedure: before modeling, the rat is placed in a metabolism cage to collect normal urine, and the rat is placed in the metabolism cage to collect urine samples on the 2 nd day, the 4 th day, the 6 th day and the 9 th day in the modeling process. Meanwhile, the body weight of the rat was measured in the morning after each urine collection.

Test example

Test example 1 Lung histopathological examination

On

days

2, 4, 6 and 9 of molding, a part of the rats of example 1 was euthanized and rat lung tissue was taken. Lungs were removed from each animal and fixed by immersion in 4% formaldehyde at 4 ℃. After sectioning, changes in lung tissue were observed by H & E staining.

Test example 2 protein analysis

1. Extracting and storing urine protein: centrifuging urine at 4 deg.C 2000g for 30 min, collecting supernatant, placing in new EP tube, and centrifuging at 4 deg.C 12000g for 30 min; the supernatant was taken and stored at-80 ℃.

2. Ethanol precipitation of urine proteins, Bradford method for protein concentration followed by on-membrane cleavage, see Wisniewski JR, Zougman a, Nagaraj N, man m. universal sample preparation method for protein analysis. nature methods 2009; 6:359-62. The BCA method measures the polypeptide concentration.

LC-MS/MS tandem mass spectrometry:

the polypeptide sample was diluted with 0.1% formic acid to 0.5. mu.g/. mu.l. The polypeptide sample is separated by an EASY-nLC1200 loading system of a Thermo liquid phase system. The elution time was 120 minutes and the column flow rate was 0.3. mu.l/min. The elution gradient was 5% to 40% mobile phase B (mobile phase A: 0.1% formic acid; mobile phase B: 89.9% acetonitrile). The eluted peptide fragments were analyzed using a Thermo Orbitrap Lumos mass spectrometer. In the cation mode, the resolution of parent and daughter ions was 120000.

The polypeptide sample was diluted to 1. mu.g/. mu.l with 0.1% formic acid. After the polypeptide sample was loaded onto the capture column by an autosampler, the column was washed at a flow rate of 5. mu.l/min for 5min to further remove impurities, and then reverse column gradient elution was performed at a flow rate of 0.3. mu.l/min. The elution gradient was 5% to 28% mobile phase (0.1% formic acid, 89.9% acetonitrile, 10% water) with an elution time of 45 minutes. Triple TOF 5600 mass spectral identification was then performed. The m/z scanning range of the parent ions is 350-1250, and the scanning time is 500 ms. And acquiring an MS/MS spectrum at the speed of 20Hz for data acquisition, wherein the m/z scanning range of the daughter ions is 250-1800, and the scanning time is 50 MS. The energy is set to rolling impact. The scan intensity is higher than 125counts/s per cycle, and the charge comprises 30 daughter ions from +2 to + 5.

4. Database retrieval:

all mass spectrometry results were database retrieved using mascot software. The database used is the Swissprot _ rat. The retrieval conditions are as follows: cutting with pancreatin; 2 leaky cleavage sites were allowed; fixed modification of cysteine +57 Da; the mass spectrum data retrieval tolerance is as follows: parent ion 0.05Da, daughter ion 0.05 Da.

5. Relative quantification of protein:

mascot search results proteomic data quantitative analysis was performed by Progenetics QI LC/MS software. The FDR value of the protein is set to 1%, and the protein has at least 2 unique polypeptides.

6. Statistical analysis:

and (3) performing statistical analysis on the proteome data of different time points, and screening out the protein with the change multiple of more than 1.5 times and the p value less than 0.05 as the differential protein from the comparison spectrogram number of 0 day before modeling.

Results of the experiment

1. Weight change:

the body weight of the walker256 rats was found to be significantly reduced on the sixth day compared to the control group, and the body weight of the experimental group was statistically different from that of the control group, as measured at different time points (fig. 1).

HE staining results:

as in fig. 2A to 2E, HE staining results show:

compared with the control group (fig. 2A), the tumor group rats began to develop subtopic metastatic nodules in the lung at day 4 (fig. 2C), and as the disease progresses, the metastatic foci gradually increase and increase (fig. 2D and 2E). The metastatic focus cells are closely arranged, are round, oval or irregular, are poorly differentiated, have large nuclei and are deeply dyed. When the lung is scattered in the lung parenchyma, tumor cells can grow in an invasive manner under an endoscope, part of the lung tissue structure is destroyed, and alveoli disappear.

3. Protein identification results:

urine from rats (n-7) in the control group and the breast cancer lung metastasis group was identified by mass spectrometry and each was subjected to 2 technical replicates. At levels of FDR less than 1%, 258 and 660 proteins were identified based on two peptides or more, respectively.

And comparing the protein with the quantitative spectrogram number of the protein at day 0, screening out other differential proteins with the time point change times of more than 1.5 times and the repeated measurement ANOVA p value of less than 0.05, and simultaneously converting the rat protein into the corresponding human homologous protein by using a Uniprot database.

Compared with the control group, the number of the differential proteins in the urine of the day 2 in the rat animal model with the breast cancer pulmonary metastasis is 73, and the number of the differential proteins in the urine of the day 4 in the rat animal model with the breast cancer pulmonary metastasis is 40, wherein the number of the differential proteins shared by the day 2 and the day 4 is 21.

Table 1 shows that a total of 21 common differential proteins were screened at early tumor stage (2, 4 days of modeling). After transformation to human homologous protein, in early breast cancer lung metastasis, the expression levels of aminoacylase-1A, aminopeptidase N, EH structural threshold protein 1, insulin growth factor binding protein 3, pancreatic secretory granular membrane glycoprotein 2, tubulin beta-4B chain, glyceraldehyde triphosphate dehydrogenase, sodium-hydrogen ion exchange regulatory cofactor 1, annexin A2, NKG2-D class II membrane protein, endothelial cell protein C receptor, ezrin, charged polypodopsin 5, heat shock homologous 71kDa protein, inhibin beta C chain, ester hydrolase C11orf54 homolog, beta 2-glycoprotein 1, CD48 antigen, methyldopa beta subunit, pyruvate kinase isomerase M1/M2, fructose-bisphosphate aldolase B are increased.

TABLE 1 diagnosis of early breast cancer pulmonary metastasis urine protein markers (identified on

day

2 and 4 together)

Table 2 shows that a total of 52 differential proteins were screened at the early stage of the tumor (day 2 of modeling), excluding the proteins identified on day 2 and day 4 in table 1. After conversion to human homologous protein, the granulin protein, the converted protein RhoA, the cell division controlling protein 42 homolog, lactadherin, LIM and SH3 domain protein 1, monoglyceride lipase, hydroxyacyl glutathione hydrolase, glutamine synthetase, paralogous protein 2, 14-3-3 protein theta, secretoglobin 2A, cerebro-acid soluble protein 1, 14-3-3 protein gamma, tyrosine protein kinase B1, sulfotransferase 1C2, dissociation-inhibiting protein 1, CD59 glycoprotein, histone H4, type V proton ATPase subunit molecular weight 16kDa protein lipid, myosin light chain polypeptide 6, L-lactate dehydrogenase A chain, histidine nucleotide binding protein 1, amidase NIT2, ketokinase, guanine nucleotide binding protein G (i) subunit alpha, peroxidase reductase 6, glutathione reductase, gamma, beta-gamma, the recombinant human insulin receptor is characterized by comprising the following components of silk cutting protein-1, heat shock protein 90 beta subunit, podocyte marker protein, adenylate ribosylation factor, CD9 antigen, adenylate kinase, aspartate aminotransferase, heart fatty acid binding protein, pyridoxal kinase, amyloid 2, peroxide reductase 1, calmodulin, immunoglobulin gamma chain C region, fructose-diphosphate aldolase A, prostaglandin H2D isomerase, neuronal membrane glycoprotein M6-a, cystatin-C, Di-N-acetyl chitobiase, glutamyl peptidase, polyubiquitin B, leukemia inhibitory factor receptor, nerve cell adhesion molecule 1, glutamyl hydrolase and alpha-enolase with increased expression levels.

Prolactin-inducing protein homolog and cytosolic ferritin assembly protein ciao1 are expressed at reduced levels compared to protein levels in healthy controls.

TABLE 2 early Breast cancer Lung metastasis diagnostic urine protein markers (identified separately on day 2)

Table 3 shows that a total of 19 differential proteins were screened at the early stage of the tumor (4 days of modelling), excluding the proteins identified jointly at day 2 and day 4 as described in table 1. After conversion to human homologous protein, the expression level of solute carrier family 22, voltage-dependent anion selective channel protein 1, translational control tumor protein, intracellular chloride channel protein 1, actin, aquaporin 1, glutathione s-transferase, protein-glutamine gamma-glutamyltransferase 4, synapse-associated protein 23, trefoil factor 1, fructose-1, 6-bisphosphatase 1, intracellular chloride channel protein 4, cystatin B, protein RoBo-1, malate dehydrogenase, elongation factor alpha 1, glutamylpeptidase, EGF-containing fibular extracellular matrix protein 1 was increased compared to the protein level in healthy controls.

Serum transferrin expression levels are reduced compared to the protein levels in healthy controls.

TABLE 3 early Breast cancer Lung metastasis diagnostic urine protein markers (identified separately on day 4)

Claims

1. Use of reagents for detecting a set of proteins in urine for the manufacture of a kit for diagnosing early breast cancer lung metastasis in a subject, wherein the set of proteins consists of: aminoacylase-1A, aminopeptidase N, EH structure threshold protein 1, insulin growth factor binding protein 3, pancreatic secretory granule membrane glycoprotein 2, tubulin beta-4B chain, glyceraldehyde triphosphate dehydrogenase, sodium-hydrogen ion exchange regulatory cofactor 1, annexin A2, NKG2-D class II membrane proteins, endothelial cell protein C receptor, ezrin, charged vesicle protein 5, heat shock homologous 71kDa protein, inhibin beta C chain, ester hydrolase C11orf54 homolog, beta 2-glycoprotein 1, CD48 antigen, methyldopa beta subunit, pyruvate kinase isomerase M1/M2, fructose-diphosphate aldolase B, granule protein, transforming protein RhoA, cell division control protein 42 homolog, milk lectin, LIM and SH3 domain protein 1, monoglyceride lipase, hydroxyacyl hydrolase, glutathione hydrolase, beta-glucosidase, Glutamine ammonia synthase, synegenin-forbidden 2, 14-3-3 protein theta, secretoglobin 2A, cerebro-acid-soluble 1, 14-3-3 protein gamma, tyrosine protein kinase B1, sulfotransferase 1C2, dissociation-inhibiting protein 1, CD59 glycoprotein, histone H4, protein with a molecular weight of 16kDa subunit of type V proton ATPase, myosin light chain polypeptide 6, L-lactate dehydrogenase A chain, histidine nucleotide-binding protein 1, amidase NIT2, ketohexokinase, guanine nucleotide-binding protein G (i) subunit alpha, peroxiredoxin 6, mitoprotein-1, heat shock protein 90 beta subunit, podocyte marker protein, adenylate ribosylation factor, CD9 antigen, adenylate kinase, aspartate aminotransferase, cardiac fatty acid-binding protein, pyridoxal kinase, amyloid 2 pyridoxine, pyridoxine, Peroxiredoxin 1, calmodulin, immunoglobulin gamma chain C region, fructose-diphosphate aldolase A, prostaglandin H2D isomerase, neuronal membrane glycoprotein M6-a, cystatin-C, Di-N-acetyl chitobiase, glutamyl peptidase, polyubiquitin B, leukemia inhibitory factor receptor, nerve cell adhesion molecule 1, glutamyl hydrolase, alpha-enolase, prolactin-inducible protein homolog, cytosolic ferrithioprotein assembly protein ciao1, solute family 22, voltage-dependent anion-selective channel protein 1, translation control tumor protein, intracellular chloride channel protein 1, actin 1, aquaporin 1, glutathione s-transferase, protein-glutamine gamma-glutamyltransferase 4, synapse-associated protein 23, trefoil factor 1, protein-glutamine gamma-glutamyltransferase 4, and protein, Fructose-1, 6-bisphosphatase 1, intracellular chloride channel protein 4, cystatin B, protein RoBo-1, malate dehydrogenase, elongation factor alpha 1, glutamyl peptidase, EGF-containing fibular extracellular matrix protein 1 and serum transferrin.

2. The use of claim 1, wherein the reagent for detecting a set of proteins in urine is an antibody or antigen-binding fragment thereof specific for the proteins.

3. The use of claim 2, wherein the specific antibody is a monoclonal antibody or an antigen-binding fragment thereof.

4. The use of any one of claims 1-3, wherein the subject is a mammal.

5. The use of claim 4, wherein the subject is a human.

6. A kit or chip for diagnosing early breast cancer lung metastasis, comprising reagents for detecting a set of proteins in urine, wherein the set of proteins consists of: aminoacylase-1A, aminopeptidase N, EH structure threshold protein 1, insulin growth factor binding protein 3, pancreatic secretory granule membrane glycoprotein 2, tubulin beta-4B chain, glyceraldehyde triphosphate dehydrogenase, sodium-hydrogen ion exchange regulatory cofactor 1, annexin A2, NKG2-D class II membrane proteins, endothelial cell protein C receptor, ezrin, charged vesicle protein 5, heat shock homologous 71kDa protein, inhibin beta C chain, ester hydrolase C11orf54 homolog, beta 2-glycoprotein 1, CD48 antigen, methyldopa beta subunit, pyruvate kinase isomerase M1/M2, fructose-diphosphate aldolase B, granule protein, transforming protein RhoA, cell division control protein 42 homolog, milk lectin, LIM and SH3 domain protein 1, monoglyceride lipase, hydroxyacyl hydrolase, glutathione hydrolase, beta-glucosidase, Glutamine ammonia synthase, synegenin-forbidden 2, 14-3-3 protein theta, secretoglobin 2A, cerebro-acid-soluble 1, 14-3-3 protein gamma, tyrosine protein kinase B1, sulfotransferase 1C2, dissociation-inhibiting protein 1, CD59 glycoprotein, histone H4, protein with a molecular weight of 16kDa subunit of type V proton ATPase, myosin light chain polypeptide 6, L-lactate dehydrogenase A chain, histidine nucleotide-binding protein 1, amidase NIT2, ketohexokinase, guanine nucleotide-binding protein G (i) subunit alpha, peroxiredoxin 6, mitoprotein-1, heat shock protein 90 beta subunit, podocyte marker protein, adenylate ribosylation factor, CD9 antigen, adenylate kinase, aspartate aminotransferase, cardiac fatty acid-binding protein, pyridoxal kinase, amyloid 2 pyridoxine, pyridoxine, Peroxiredoxin 1, calmodulin, immunoglobulin gamma chain C region, fructose-diphosphate aldolase A, prostaglandin H2D isomerase, neuronal membrane glycoprotein M6-a, cystatin-C, Di-N-acetyl chitobiase, glutamyl peptidase, polyubiquitin B, leukemia inhibitory factor receptor, nerve cell adhesion molecule 1, glutamyl hydrolase, alpha-enolase, prolactin-inducible protein homolog, cytosolic ferrithioprotein assembly protein ciao1, solute family 22, voltage-dependent anion-selective channel protein 1, translation control tumor protein, intracellular chloride channel protein 1, actin 1, aquaporin 1, glutathione s-transferase, protein-glutamine gamma-glutamyltransferase 4, synapse-associated protein 23, trefoil factor 1, protein-glutamine gamma-glutamyltransferase 4, and protein, Fructose-1, 6-bisphosphatase 1, intracellular chloride channel protein 4, cystatin B, protein RoBo-1, malate dehydrogenase, elongation factor alpha 1, glutamyl peptidase, EGF-containing fibular extracellular matrix protein 1 and serum transferrin.