CN111289756A

CN111289756A - Urine marker related to lung metastasis and tumor formation of ovarian cancer

Info

Publication number: CN111289756A
Application number: CN201811502714.7A
Authority: CN
Inventors: 高友鹤; 魏静
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2020-06-16

Abstract

The invention discloses a urine marker related to lung metastasis and tumor formation of ovarian cancer. Specifically, the invention discloses the use of an identifying agent for a protein selected from the group consisting of: glypican-3, FAM151A protein, tyrosine protein phosphatase non-receptor type substrate 1, and the like. And the use of an agent for identifying a protein selected from the group consisting of: neutral and basic amino acid transporter rBAT, moesin, Na (+)/H (+) exchange-regulating cofactor NHE-RF3, malate dehydrogenase, fetuin-B, retinol-binding protein 4, etc. The protein disclosed by the invention can be used for early diagnosis of whether ovarian cancer lung metastasis is tumorous.

Description

Urine marker related to lung metastasis and tumor formation of ovarian cancer

Technical Field

The present invention relates to clinical medicine; in particular to a urine marker related to lung metastasis and tumor formation of ovarian cancer. In particular, the invention relates to a urine protein marker related to diagnosis and/or monitoring of whether metastasis is tumorous or not by using an ovarian cancer lung metastasis rat model and a mass spectrometry proteomics technology, and application thereof.

Background

Cancer markers refer to monitorable changes associated with the pathological course of cancer and can be used to diagnose cancer, monitor the progression of cancer, and assess the effectiveness of treatment. Urine, as a product of blood filtration through the glomeruli, lacks regulation of homeostatic mechanisms and can accumulate and respond to all pathological and physiological changes throughout the body, which may be potential markers, to a greater extent. Thus, urine is an ideal source of disease markers (see Gao YH. Urine- -an unsupported mineral for biomarker discovery. Sci. China. Life Sci,2013,56(12): 1145; Wu J, Gao Y. physiological considerations can be reflected in human urine protein and metabolism. expert review of properties 636, 2015,12(6): 623-.

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.

Uroproteomes are susceptible to a variety of factors, such as age, sex, diet, and medications, especially in clinical urine samples. To address this effect, corresponding animal models are often used to reduce the influencing factors and to search for urine proteins directly associated with the corresponding disease as potential markers.

Urine proteomics has been used in many different animal models to reflect Early changes of disease before Clinical symptoms appear, (1) urine proteins can detect Early changes of tumor growth before tumor mass formation in Walker-256 subcutaneous tumor model and can monitor tumor growth (Wu J, Guo Z, Gao Y. dynamic changes of urine protein a Walker Mecer Med.2017,6(11):2713-2722), and (2) urine proteins can detect Early fibrosis in bleomycin-induced Rat pulmonary fibrosis model and can evaluate pulmonary fibrosis Treatment (Wu J, Li X, ZHAO M, Huang H, Sun W, Gary. Early Detection of urine protein, Clinical diagnosis about Clinical markers of tumor, Clinical symptoms of diabetes mellitus, Clinical diagnosis about Clinical symptoms of tumor, Clinical diagnosis about Clinical symptoms of diabetes mellitus, Clinical diagnosis about Clinical specimen of diabetes mellitus, Clinical specimen, Clinical diagnosis about Clinical specimen, Clinical diagnosis about Clinical specimen, Clinical diagnosis about Clinical specimen, Clinical diagnosis about Clinical specimen, Clinical diagnosis about Clinical specimen, Clinical diagnosis about, Clinical diagnosis, Clinical.

Disclosure of Invention

In view of the above-described needs in the art, according to some embodiments of the present disclosure, there is provided use of an agent for identifying a protein selected from the group consisting of glypican-3, FAM151A protein, tyrosin phosphatase non-receptor type substrate 1, insulin-like growth factor binding protein 3, inhibin β C chain, pepsin, retinal dehydrogenase 1, prostaglandin receptor F2 negative regulator, CD320 antigen, calreticulin, sialidase-1, WAP four-disulfide core domain protein 2, atpase subunit S V type proton pump, connexon adhesion molecule a, amyloid 2, procollagen C endopeptidase enhancer 1, prostasin, reticulin 4 receptor analog 2, β defensin 1, cell surface glycoprotein MUC18, pyrophosphatase type pyrophosphate protein 24, secreted cathepsin Z, immunoglobulin γ C chain, fibrinogen plasminogen, neuropilin-1, guanylate binding protein subunit 3-2, phosphoprotein subunit 3, phosphoprotein kinase C-lyase C transferase, phosphoprotein kinase C-9, phosphoprotein kinase C-lyase C transferase protein kinase C-9, phosphoprotein kinase C protein kinase, phosphoprotein kinase C-9, phosphoprotein kinase C-protein kinase, phosphoprotein kinase C-9, phosphoprotein kinase-related protein kinase, phosphoprotein kinase protein kinase, phosphoprotein kinase-6, phosphoprotein kinase-6, phosphoprotein kinase-related protein kinase-protein kinase, phosphoprotein kinase-related protein kinase, phosphoprotein kinase-related protein kinase, phosphoprotein kinase-1, phosphoprotein kinase-related protein kinase, phosphoprotein kinase protein kinase, protein kinase, protein kinase, protein kinase, protein kinase, protein kinase.

In a preferred embodiment, the protein is selected from the group consisting of inhibin β C chain, pepsin, prostaglandin receptor F2 negative regulatory protein, glypican-3, amyloid 2, CD276 antigen, secreted pyrophosphate protein 24, insulin-like growth factor binding protein 3, FAM151A protein, biotinidase, lysine phosphate histidine inorganic pyrophosphate phosphatase, granulin protein, and combinations thereof.

In a specific embodiment, the early diagnosis is made when the lung has not yet developed a tumor.

In other embodiments, there is provided use of an agent for identifying a protein selected from the group consisting of neutral and basic amino acid transporter rBAT, moesin, Na (+)/H (+) exchange regulatory cofactor NHE-RF3, malate dehydrogenase, fetuin-B, retinol-binding protein 4, aminoacylase-1A, glutamate-cysteine ligase catalytic subunit, apolipoprotein A-I, vitamin D-binding protein, ubiquitin-60S ribosomal protein L40, ezrin, secretoglobin family 2A member 2, α -1-acid glycoprotein, leukemia inhibitory factor receptor, aminopeptidase N, zinc- α -2-glycoprotein, epidermal growth factor, serum amyloid P-component, protein 14B containing α/β hydrolase domain, protein AMBP, calpain homolog 71kDa protein, protein-glutamine gamma-glutamyltransferase, encephalospermase, neprilysin, NANHP-1/β hydrolase, phospholyase, phosphofructosyl transferase (+)/NHE-RF-1, phosphofructosyl-dehydrogenase, phosphofructosyl-transferase, NHE-1-6, phosphofructosyl-transferase, phosphofructosyl reductase, phosphofructosyl-fetoprotein, and phosphoenolase.

In a preferred embodiment, the protein is selected from fetuin-B, aminoacylase-1A, apolipoprotein A-I, vitamin D binding protein, leukemia inhibitory factor receptor, epidermal growth factor, calcium binding protein, kynurenine/α -aminoadipate aminotransferase, Na (+)/H (+) exchange regulatory cofactor NHE-RF1, and combinations thereof.

In a preferred embodiment, the early diagnosis is diagnosed at the same time period as the early diagnosis in the neoplasia group. That is, by early diagnosis, in the tumor formation group, diagnosis can be made when lung tumors have not formed, and the differential proteins used for early diagnosis of the tumor formation group can be identified even after lung tumors have formed, i.e., in the middle and late stages, suggesting that these differential proteins appear not only when lung tumors have not formed but also after formation, i.e., participate in the process of lung tumor formation. The significance of early diagnosis of the non-neoplastic group is whether tumors can form in the lung at an early stage in humans, as is known from early stage.

In particular embodiments, the protein is obtained by screening an animal model for a urine protein marker associated with early ovarian cancer lung metastasis to neoplasia. Optionally, the animal model is prepared from a rat model of lung metastasis of ovarian cancer obtained by tail vein injection of ovarian cancer cells. In a more specific embodiment, the ovarian cancer cells are NuTu-19 ovarian cancer epithelial cells.

In particular embodiments, an increased expression level of a protein selected from the group consisting of: calreticulin, immunoglobulin γ C chain, cytoplasmic aconitate hydratase, gelsolin, glutathione s-transferase-1, acid amidase, angiopoietin-related protein 4, polyubiquitin B, phosphoglycerate mutase 2, 3-hydroxyaminobenzoate 3, 4-dioxygenase, N-acyl-aromatic-L-amino acid amidohydrolase, sucrose-isomaltase, N (g) -dimethylarginine dimethylaminohydrolase 1, DnaJ cognate subfamily C member 5, neuronal membrane glycoprotein M6-a, brain acid soluble protein 1.

In particular embodiments, a decreased level of expression of a protein selected from the group consisting of glypican-3, FAM151A protein, tyrosinase-phosphatase non-receptor type substrate 1, insulin-like growth factor binding protein 3, inhibin β C chain, pepsin, retinal dehydrogenase 1, prostaglandin receptor F2 negative regulator, CD320 antigen, sialidase-1, WAP four-disulfide bond core domain protein 2, ATP subunit S1V type proton pump, adhesion molecule A, amyloid 2, Proossein C endopeptidase enhancer 1, prostatic protein, reticulin 4 receptor analogous protein 2, β defensin 1, cell surface glycoprotein MUC18, secreted pyrophosphate protein 24, cathepsin Z, plasminogen, neuropilin-1, guanylate binding protein β subunit-2, biotinylase, CD antigen, phospholipase 3, growth factor, phosphokinase, alkaline phosphatase receptor phosphatase, phosphoprotein kinase receptor kinase subunit S2, phosphoprotein kinase subunit S9, phosphoprotein kinase subunit S2, phosphoprotein kinase receptor kinase subunit S2, phosphoprotein kinase related to ATP receptor kinase, phosphoprotein kinase, ATP receptor kinase receptor subunit 2, ATP receptor kinase related to ATP receptor kinase, ATP receptor subunit 4, ATP receptor subunit 2, ATP receptor subunit related to ATP receptor subunit S4, ATP receptor subunit related protein kinase, ATP receptor subunit S4, ATP receptor subunit S9, ATP receptor subunit related protein kinase, ATP receptor subunit S4 negative regulator, ATP receptor.

In particular embodiments, an increased level of expression of a protein selected from the group consisting of neutral and basic amino acid transporter rBAT, moesin, Na (+)/H (+) exchange regulator cofactor NHE-RF3, malate dehydrogenase, aminoacylase-1A, glutamate-cysteine ligase catalytic subunit, ubiquitin-60S ribosomal protein L40, ezrin, secretoglobin family 2A member 2, aminopeptidase N, protein 14B containing the α/β hydrolase domain, heat shock homolog 71kDa protein, protein-glutamine gamma-glutamyltransferase 4, cerebropeptidase, gamma-glutamyltransferase 1, triosephosphate isomerase, alcohol dehydrogenase [ NADP (+) ], ribonuclease 4, programmed cell death 6 interacting protein, glycan, fructose bisphosphate aldolase B, peroxidase-1, kynurenine/α -aminoadipate/H (+) regulator, NHE (+) ribonuclease, UK1, as compared to the protein level in a healthy control, is indicative of the absence of early damage to a cancer lung metastasis.

In particular embodiments, a decreased level of expression of a protein selected from the group consisting of fetuin-B, retinol binding protein 4, apolipoprotein A-I, vitamin D binding protein, α -1-acid glycoprotein, leukemia inhibitory factor receptor, zinc- α -2-glycoprotein, epidermal growth factor, serum amyloid P-component, protein AMBP, calbindin, ceruloplasmin, serum albumin, as compared to the level of protein in a healthy control, is indicative of the absence of early injury to the cancer lung metastasis.

In a specific embodiment, a healthy control refers to an individual who does not have cancer.

Identification reagents suitable for use in the present disclosure are mass spectrometry identification reagents, antibodies, or antigen binding fragments thereof. In a specific embodiment, the antibody is a monoclonal antibody. The species source of the monoclonal antibody is not limited by the present disclosure, and any antibody capable of binding to the above-described protein can be used.

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 particular embodiments, protein markers according to the present disclosure can be used to diagnose whether ovarian cancer metastasizes to the lung.

In a specific embodiment, the expression level is selected from the group consisting of nucleic acid level and protein level, in particular protein level.

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

In a specific embodiment, the expression level is determined in a urine sample.

In a specific embodiment, the protein is a urine protein.

In another aspect, the present invention provides a kit or chip for early diagnosis of whether a subject with ovarian cancer develops a lung metastasis tumor, comprising an identifying agent for a protein selected from the group consisting of:

phosphatidylinositol proteoglycan-3, FAM151A protein, tyrosine phosphatase non-receptor type substrate 1, insulin-like growth factor binding protein 3, inhibin β C chain, pepsin, retinal dehydrogenase 1, prostaglandin receptor F2 negative regulator protein, CD320 antigen, calreticulin, sialidase-1, WAP four-disulfide bond core domain protein 2, ATPase subunit S1V type proton pump, adhesion molecule A, amyloid 2, procollagen C endopeptidase enhancer 1, prostasin, reticulin 4 receptor-like protein 2, β defensin 1, cell surface glycoprotein MUC18, secreted pyrophosphate protein 24, cathepsin Z, immunoglobulin gamma C chain, plasminogen, neuropilin-1, guanylate binding protein β subunit-2, biotinylase, CD antigen, phospholipase D3, growth factor precursor, alkaline phosphatase, granulin receptor type 1, hyaluronidase-1, cytoplasmic aconitase, membrane protein, neurotrypsin, glutathione transferase, laminin-transferase, histone transferase, phosphoprotein kinase A-binding protein-2, phosphoprotein kinase-binding protein G-9, phosphoprotein kinase-6, phosphoprotein kinase-binding protein, phosphoprotein kinase-binding protein subunit protein, phosphoprotein-2, phosphoenolpyruvate-binding protein kinase, phosphoprotein-III, phosphoprotein-IV receptor D-6, phosphoprotein-I-D, phosphoprotein-I-D, phosphoprotein-I, phosphoprotein I-I, phosphoprotein (G-I, phosphoprotein II, phosphoprotein I, phosphoprotein I-I, phosphoprotein II-I, phosphoprotein II-I, phosphoprotein I-I, protein II, protein I-I, protein II-I, protein II, protein I, protein II, protein I, protein II, protein I, protein II

Neutral and basic amino acid transporter rBAT, moesin, Na (+)/H (+) exchange regulatory cofactor NHE-RF3, malate dehydrogenase, fetuin-B, retinol binding protein 4, aminoacylase-1A, glutamate-cysteine ligase catalytic subunit, apolipoprotein A-I, vitamin D binding protein, ubiquitin-60S ribosomal protein L40, ezrin, secretoglobin family 2A member 2, α -1-acid glycoprotein, leukemia inhibitory factor receptor, aminopeptidase N, zinc- α -2-glycoprotein, epidermal growth factor, serum amyloid P-component, protein 14B containing α/β hydrolase domain, protein AMBP, calbindin, heat shock homolog 71kDa protein, protein-glutamine-glutamyltransferase 4, enkephalinase, gamma-glutamyltranspeptidase 1, triose phosphate isomerase, alcohol dehydrogenase [ ribonuclease (+) ], nuclease 4, programmed cell death 6 interacting protein, ceruloplasmin, plasma phycoerythrin, NHE-phosphofructosyl-L-folate reductase, NHE-RF-1, urokinase, and combinations thereof.

In some embodiments, the kit comprises an agent for identifying the above-described protein. In other embodiments, the chip has immobilized thereon an identifying agent for the protein. In some embodiments, the identification agent is an antibody or antigen-binding fragment thereof.

According to some embodiments, there is provided a method for early diagnosis of the presence or absence of metastasis of cancer lung in a subject, comprising the steps of:

1) urine samples were obtained from the subjects and healthy controls,

2) determining the expression level of a protein selected from the group consisting of:

3) Comparing the expression level of the protein in the subject to the expression level of the protein in a healthy control,

4) determining whether the subject has early stage damage of cancer lung metastasis.

In particular embodiments, an increased expression level of a protein selected from the group consisting of:

neutral and basic amino acid transporter rBAT, moesin, Na (+)/H (+) exchange regulation cofactor NHE-RF3, malate dehydrogenase, aminoacylase-1A, glutamate-cysteine ligase catalytic subunit, ubiquitin-60S ribosomal protein L40, ezrin, secretoglobin family 2A member 2, aminopeptidase N, protein 14B containing α/β hydrolase domain, heat shock homolog 71kDa protein, protein-glutamine gamma-glutamyltransferase 4, enkephalinase, gamma-glutamyltranspeptidase 1, triose phosphate isomerase, alcohol dehydrogenase [ NADP (+) ], ribonuclease 4, programmed cell death 6 interacting protein, glycan, fructose bisphosphate aldolase B, peroxidase-1, kynurenine/α -aminoadipate aminotransferase, Na (+)/H (+) exchange regulation cofactor NHE-RF1, ribonuclease UK 114.

In particular embodiments, a decreased level of expression of a protein selected from the group consisting of fetuin-B, retinol binding protein 4, apolipoprotein A-I, vitamin D binding protein, α -1-acid glycoprotein, leukemia inhibitory factor receptor, zinc- α -2-glycoprotein, epidermal growth factor, serum amyloid P-component, protein AMBP, calbindin, ceruloplasmin, serum albumin, as compared to the level of protein in a healthy control, is indicative of the absence of a cancer metastasis lesion in the subject.

In specific embodiments, the expression level is determined using mass spectrometry, ELISA, or Western methods.

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.

In another aspect, the present invention provides a method of establishing an animal model for screening for a urine protein marker associated with early ovarian cancer lung metastasis, the method comprising the steps of:

i) obtaining a rat model of ovarian cancer lung metastasis tumor formation and non-tumor formation by tail vein injection of ovarian cancer cells;

ii) collecting urine from the rat before establishing the rat model of ovarian cancer lung metastasis, wherein the urine is from the rat before forming tumor and before forming tumor; and

iii) identifying protein mass spectra in urine of tumor-forming and non-tumor rat before establishing the rat model of early ovarian cancer lung metastasis by mass spectrometry;

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

Drawings

FIG. 1 shows the results of weight monitoring of rats in the tail vein injection rat tumorigenic group, non-tumorigenic group and control group, ■ represents tumorigenic group, ▲ represents non-tumorigenic group, and ● represents normal control group.

Fig. 2A to 2E: lung tissue section HE staining results of mice in 60 days of control group (FIG. 2A) and lung metastasis and tumor formation group model (FIG. 2B-FIG. 2E).

Fig. 2F to 2J: lung tissue section HE staining results of rats on day 60 in control group (fig. 2F), lung metastasis non-tumorigenic group model (fig. 2G-fig. 2J).

Fig. 3A to 3D: control group (fig. 3A), lung metastasis model day 35 (fig. 3B), day 45 (fig. 3C), day 52 (fig. 3D), rat lung tissue section HE staining results.

FIGS. 4A to 4L ovarian cancer lung metastasis tumorigenesis group validation of differential proteins amyloid 2 (FIG. 4A), pepsin (FIG. 4B), inhibin β C chain (FIG. 4C), prostaglandin receptor F2 negative regulator protein (FIG. 4D), FAM151A protein (FIG. 4E), biotinases (FIG. 4F), glypican-3 (FIG. 4G), insulin-like growth factor binding protein 3 (FIG. 4H), secreted pyrophosphate protein 24 (FIG. 4I), CD276 antigen (FIG. 4J), lysine phosphate histidine inorganic pyrophosphate phosphatase (FIG. 4K), and granulin protein (FIG. 4L).

FIGS. 5A to 5I ovarian cancer Lung metastasis non-tumorigenic group verified the differential proteins aminoacylase-1A (FIG. 5A), kynurenine/α -aminoadipate aminotransferase (FIG. 5B), fetuin-B (FIG. 5C), apolipoprotein A-I (FIG. 5D), leukemia inhibitory factor receptor (FIG. 5E), epidermal growth factor (FIG. 5F), Na (+)/H (+) exchange regulatory cofactor NHE-RF1 (FIG. 5G), vitamin D binding protein (FIG. 5H), calbindin (FIG. 5I).

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 rat tumor cell tail vein injection model

The rat tail vein injection model is a classic animal model for researching the progress of cancer lung metastasis tumor, and is suitable for researching the change of pathophysiology and morphology in the progress process of lung metastasis tumor. The animal model is used for simulating the cancer lung metastasis process, observing the integral changes of the cancer lung metastasis from non-metastasis, early metastasis, metastasis progress and late metastasis, and the pathological process and the pathological characteristics of the animal model are similar to those of the cancer lung metastasis of human beings, so that the animal model has important guiding significance for clinically diagnosing the cancer lung metastasis at early stage and monitoring the disease progress of the lung metastasis.

In the present application, a rat lung metastasis animal model was prepared by tail vein injection of ovarian cancer cells (NuTu-19 ovarian cancer epithelial cells purchased from shanghai meixuan biotechnology limited), and urine of lung metastasis at early stage (days 12 and 27) and middle and late stage (days 39 and 52) was collected. The animal model is used for simulating cancer lung metastasis, and has important guiding significance for early diagnosis of whether cancer lung metastasis is tumorous or not 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

The frozen ovarian cancer epithelial cells are rapidly recovered at 37 ℃, and then cultured in a DMEM culture solution at 37 ℃. Cell activity was identified by 0.4% trypan blue staining.

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

Establishing a lung metastasis ovarian cancer rat model: tumor cells were diluted 2X 10 with sterile physiological saline⁷Mu.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 rats are placed in a metabolism cage to collect normal urine, and the rats are placed in the metabolism cage to collect urine samples on the 12 th day, the 27 th day, the 39 th day and the 52 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 the 35 th, 45 th, 52 th, and 60 th days of molding, a part of the rats in example 1 were euthanized and rat lung tissues were 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.

Results of the experiment

1. Weight change:

the body weight of rats at different time points is measured, and the body weight of a tumor forming group of NuTu-19 tail vein injection rats is found to be obviously reduced at the 47 th day and has obvious statistical difference with the body weight of the control group; while the body weight of the non-tumorigenic group was reduced compared to the control group, there was no statistical difference (fig. 1).

HE staining results:

fig. 2A to 2E, HE staining results show: at day 60, the lungs began to develop a transspecular tumor (fig. 2B-2E) in the neoplastic rats compared to the control group (fig. 2A). 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.

Fig. 2F to 2J, HE staining results show: compared with the control group (FIG. 2F), no metastatic tumor appeared in the lung, and a large amount of lymphocytes were accumulated in the vicinity of the bronchi.

Fig. 3A to 3D, HE staining results show: metastatic tumors appeared on day 45 of tail vein injection of NuTu-19 tumor cells (figure 3C) as compared to the control group (figure 3A), indicating that days 12 and 27 were early stages of tumor metastasis progression.

Test example 2 protein analysis

1. Extracting and storing urine protein: centrifuging urine at 4 deg.C for 30 min at 2000g, collecting supernatant, placing in new EP tube, and centrifuging at 4 deg.C for 30 min at 12000 g; 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 60 minutes and the column flow rate was 0.3. mu.l/min. The elution gradient was 5% to 30% mobile phase B (mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid + 79.9% acetonitrile + 20% water). The eluted peptide fragments were analyzed using a Thermo Orbitrap Lumos mass spectrometer. A cationic mode is used. The spraying voltage is 2.4kV, and the temperature of an ion transmission tube is 320 ℃. First-stage full scanning: 350 + 1550m/z, 120000 resolution, automatic gain control (AGC 1e5), ion implantation time 100 ms. The secondary scan is a data dependent acquisition mode, cycle time 3s, maximum speed mode, automatic gain control (AGC 5e4), ion implantation time 50ms, resolution 30000. Other parameters: HCD fragmentation mode, 30% fragmentation energy, scan start 110 m/z. Each sample was subjected to 2 technical replicates.

4. Database retrieval:

all mass spectra results were database retrieved using Mascot software (version 2.5.1). The database used was Swissprot _ rat (data up to 2017, 2 months, containing 7992 sequences). The retrieval conditions are as follows: cutting with pancreatin; 2 leaky cleavage sites were allowed; fixed modification by carbamoylation of cysteine; oxidation of methionine to a variable modification; the mass spectrum data retrieval tolerance is as follows: parent ion 10ppm, daughter ion 0.02 Da.

5. Relative quantification of protein:

the results of the Mascot library search were subjected to proteomic data quantitative analysis by Progenetics QI LC/MS software (version 4.1) and Scaffold software (version 4.7.5), respectively. For Progenetics QI LC/MS, the software automatically selected the most appropriate data for reference and corrected the retention time for the other mass spectral data. Parent ions with charges of 2+, 3+, 4+ were selected for subsequent quantification. Peptide fragments with Mascot score >30 and P <0.01 were used for subsequent quantification. At least 2 unique (unique) polypeptides were retained per protein. For the Scaffold software, the FDR value of the protein was set to 1%, the peptide FDR was set to 1%, and the protein possessed at least 2 unique polypeptides.

6. Statistical analysis

The mean peak area/number of spectra of the proteins at different time points were used for statistical analysis. The statistical method is one-way analysis of variance. The proteins at day 12, 27, 39, 52 in the tumor/non-tumor groups were compared to the pre-model self-control. The screening conditions are as follows: the credible value is more than or equal to 200; the change multiple is more than or equal to 1.5 or less than or equal to 0.67; elevated/reduced protein was elevated/reduced in each mouse of the corresponding group; corrected P value < 0.05.

Protein identification results:

the rats in the tumor formation group (n ═ 4) and the rats in the non-tumor formation group (n ═ 4) and their respective control urine proteins were identified by 2 technical repeat mass spectra. At levels of FDR less than 1%, 532 and 505 proteins were identified based on two peptides or more, respectively. Comparing with the quantitative peak area/spectrogram number of the protein on the 0 th day, and screening out the differential protein of which the change times of 12 th, 27 th, 39 th and 52 th are more than or equal to 1.5 or less than or equal to 0.67 and the corrected P value is less than 0.05.

99 differential proteins in urine at day 12 and 120 differential proteins at day 27 in a rat animal model in the neoplasia group compared to day D0; 34 differential proteins in urine at day 12 and 84 differential proteins at day 27 in an animal model of rats in the non-neoplastic group. After removing the differential proteins identified in both the tumorigenic and non-tumorigenic groups, the Uniprot database was used to convert the rat protein to the corresponding human homologous protein from the differential proteins at day 12 and day 27.

TABLE 1 shows that 79 total proteins are selected in early tumorigenic stage (days 12 and 27) from the secretory protein receptor of reticulon, immunoglobulin gamma C chain, cytoplasmic aconitate hydratase, gelsolin, glutathione S-transferase-1, acid amidase, angiopoietin-related protein 4, polyubiquitin B, phosphoglycerate mutase 2, 3-hydroxyphenylformate 3, 4-dioxygenase, N-acyl-aromatic-L-aminoacid amidohydrolase, sucrose-isomaltase, N (G) -dimethylarginine-dimethylamido 1, DnaJ homologous subfamily C member 5, neuronal membrane glycoprotein M6-a, increased expression of brain acid soluble protein 1, phosphatidylinositol proteoglycan-3, FAM151A protein, tyrosine phosphatase non-receptor type 1, insulin-like growth factor binding protein 3, inhibin β C chain, pepsin, retinal dehydrogenase 1, prostaglandin receptor F2 negative regulator protein, CD antigen, core phosphatase, proteinase-1, laminin-receptor protein transferase, phosphoprotein kinase receptor protein kinase-9, phosphoprotein kinase-protein kinase-9, phosphoprotein kinase-protein kinase-9, protein kinase-protein kinase, protein kinase-protein kinase, protein kinase 9, protein kinase, protein kinase, protein kinase 9, protein kinase, protein kinase 9, protein kinase, protein kinase protein.

TABLE 1 early diagnosis of urinary protein markers for lung metastasis and neoplasia of cancer

Table 2 shows that a total of 38 differential proteins were selected in the non-neoplastic early stage (days 12 and 27) wherein neutral and basic amino acid transporter rBAT, moesin, Na (+)/H (+) exchange regulator cofactor NHE-RF3, malate dehydrogenase, aminoacylase-1A, glutamate-cysteine ligase catalytic subunit, ubiquitin-60S ribosomal protein L40, ezrin, secretoglobin family 2A member 2, aminopeptidase N, protein 14B containing α/β hydrolase domain, heat shock homolog 71kDa protein, protein-glutamine gamma-glutamyltransferase 4, enkephalinase, gamma-glutamyltranspeptidase 1, triosephosphate isomerase, alcohol dehydrogenase [ NADP (+) ], ribonuclease 4, programmed cell death 6 interacting protein, glycan, fructose bisphosphate aldolase B, peroxidase-1, kynurenine/α -aminoadipate aminotransferase, Na (+)/H (+) exchange regulator cofactor NHE-RF 2, protamine K-2, apolipoprotein K-binding protein, apolipoprotein A-64, apolipoprotein-binding protein, apolipoprotein A-binding protein, apolipoprotein-binding protein 3894, apolipoprotein-binding protein.

TABLE 2 early diagnosis of urinary protein markers for lung metastasis without neoplasia

Test example 3 verification of the protein markers screened

1. In the verification experiment of the test example, the protein to be targeted was verified in urine of 4 other rats in the tumor formation group and 3 other rats in the non-tumor formation group (day 0, day 12, day 27, day 39 and day 52) by a Parallel reaction detection (Parallel reaction monitoring) targeted quantitative mass spectrometry collection method.

2. In the tumor-forming group of rats, the targeted and quantified proteins were differential proteins that varied at all four time points, day 12, day 27, day 39 and day 52; in the non-neoplastic group of rats, the proteins targeted for quantification were those that varied at both day 12 and day 27 time points.

The detailed procedure of this test example is as follows:

1. histopathological examination of the lungs

On days 35, 45, 52, 60 of molding, the rat model prepared as in 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.

2. Protein analysis

1) Extracting and storing urine protein: centrifuging urine at 4 deg.C for 30 min at 2000g, collecting supernatant, placing in new EP tube, and centrifuging at 4 deg.C for 30 min at 12000 g; 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.

PRM (parallel Reaction monitoring) target quantitative mass spectrometry data acquisition

Establishing a spectrogram database for data acquisition: the urine samples used for the validation were enzymatically digested into polypeptide samples (time points: day 0, day 12, day 27, day 39, day 52). Wherein 4 rats are in the tumor formation group, 3 rats are in the non-tumor formation group, and 1 mu g of peptide fragment is taken from each sample to be combined with one mixed sample. Taking 900ng of mixed peptide fragment for chromatographic separation (Thermo EASY-nLC 1200): elution time 120min, column flow 0.3 μ L/min, elution gradient 4% -28% mobile phase B (0.1% formic acid + 79.9% acetonitrile + 20% water). The polypeptides eluted from the reverse phase column (C18, length 25cm, inner diameter 50um) were identified on an Orbitrap Fusion Lumos mass spectrometer. The spraying voltage is 2.1kV, and the temperature of an ion transmission tube is 300 ℃. First-stage full scanning: 350 + 1550m/z, 60000 resolution. The secondary scanning is a data-dependent acquisition mode, a cycle time of 3s and a highest speed mode. Other parameters: HCD fragmentation, 30% fragmentation energy, 30000 resolution, start of scan 110 m/z. And 6 technical repeated identifications are carried out.

PRM targeted data acquisition: the urine samples used for the validation were enzymatically decomposed into polypeptide samples (time points: day 0, day 12, day 27, day 39, day 52; 4 tumor-forming groups; 3 non-tumor-forming groups), and 900ng of peptide fragments were taken from each sample and subjected to chromatographic separation (Thermo EASY-nLC 1200): elution was performed for 120min, column flow 0.3 μ L/min, and elution gradient 4% -28% mobile phase B (0.1% formic acid + 79.9% acetonitrile + 20% water). The polypeptides eluted from the reverse phase column (C18, length 25cm, inner diameter 50um) were identified on an Orbitrap Fusion Lumos mass spectrometer. The spraying voltage is 2.1kV, and the temperature of an ion transmission tube is 300 ℃. First-stage full scanning: 350-1550m/z, 60000 resolution; secondary scanning: isolation window width 2m/z, HCD fragmentation, 30% fragmentation energy, 30000 resolution. The retention time window of the peptide fragment for secondary targeting quantification was 4 min.

PRM Targeted quantitation

The data processing of the PRM adopts Skyline software, and comprises the following specific steps:

a: establishing a spectrogram library: combining the 6 library-establishing mass spectrum raw data, and searching the library by using PD2.1 software: the species is rat; cutting with pancreatin; allowing a maximum of 2 leaky cleavage sites; the fixed modification is carbamido methylation of cysteine, protein nitrogen terminal acetylation modification and methionine oxidation modification are variable modification; the mass deviation of the parent ion is 10ppm, and the mass deviation of the daughter ion is 0.02 Da. The library search result is imported into Skyline together with the original file, and the protein FDR is set to 1%.

B: screening and quantifying peptide fragments: and (3) importing the differentially expressed protein sequence fasta file selected from the interested discovery stage into a Skyline spectrogram library, and deleting the unidentified protein in the spectrogram library. At least 2 specific peptide segments of each protein are reserved, the length of the peptide segment is 8-18 amino acids, the peptide segment of which cysteine is subjected to ureido methylation modification is reserved, and the signal peptide is removed. Setting the retention time window of the peptide fragment to be 4min, and deriving the m/z and the retention time window of the peptide fragment corresponding to the differential protein.

C: and importing the acquired PRM original data into Skyline. Screening quantitive ions: the valence of the parent ion is +2/+3, the valence of the daughter ion is +1, the daughter ion is b, y, p type, the mass deviation of the fragment ion is 0.02Da from the third ion to the penultimate ion.

D: the peak area of the secondary fragment ion of each peptide was extracted manually and derived.

5. Statistical analysis

The sum of the ion peak areas of different protein fragments at different time points was used for statistical analysis. The statistical method is one-way analysis of variance. The proteins at day 12, 27, 39, 52 in the tumor/non-tumor groups were compared to the pre-model self-control. The screening conditions are as follows: p value <0.05 compared to day 0 and trend consistent with the screening phase.

Results of the experiment

1. Ovarian cancer lung metastasis tumor formation group rat verification result

Another 4 tumor-forming groups of rats and their respective self-control urine proteins were used for differential protein validation.

The selected differential proteins are lung metastasis and tumorigenesis early diagnosis urine protein markers of ovarian cancer in Table 1, and the total number of the selected differential proteins is 79, after PRM verification, 12 of the differential proteins can be quantified in early stage (D12 and/or D27) and middle and late stage (D39 and/or D52) and have potential to be used as the lung metastasis and tumorigenicity early diagnosis urine protein markers of ovarian cancer, see Table 3 and FIGS. 4A to 4L, and in the early stage of lung metastasis, the inhibin β C chain, pepsin, prostaglandin receptor F2 negative regulatory protein, glypican-3, amyloid 2, CD276 antigen, secreted pyrophosphate protein 24, insulin-like growth factor binding protein 3, FAM151A protein, biotinase, lysine phosphate histidine inorganic pyrophosphate phosphatase and granulin-expressed amount are reduced.

TABLE 3 early verification markers for lung metastasis and neoplasia

2. Ovarian cancer lung metastasis non-tumorigenic group rat verification result

The other 3 lung metastasis non-tumorigenic groups of ovarian cancer rats and their respective control urine proteins were used for differential protein validation, the selected differential proteins were early diagnostic urine protein markers for lung metastasis non-tumorigenic ovarian cancer in Table 2, and 38 total differential proteins were validated by PRM, of which 19 differential proteins were able to be quantified in both early (D12 and/or D27) and middle (D39 and/or D52) and potentially as early diagnostic urine protein markers for lung metastasis non-tumorigenic ovarian cancer, see Table 4 and FIGS. 5A-5I showing that expression of aminoacylase-1A, kynurenine/α -aminoadipate aminotransferase, Na (+)/H (+) exchange regulation cofactor NHE-RF1 is elevated, fetuin-B, A-I, vitamin D binding protein, leukemia inhibitory factor receptor, epidermal growth factor, and calbindin is reduced.

TABLE 4 early verification marker for ovarian cancer lung metastasis non-tumorigenicity

Claims

1. Use of an agent for identifying a protein selected from the group consisting of:

phosphatidylinositol proteoglycan-3, FAM151A protein, tyrosine phosphatase non-receptor type substrate 1, insulin-like growth factor binding protein 3, inhibin β C chain, pepsin, retinal dehydrogenase 1, prostaglandin receptor F2 negative regulator protein, CD320 antigen, calreticulin, sialidase-1, WAP four-disulfide bond core domain protein 2, ATPase subunit S1V type proton pump, adhesion molecule A, amyloid 2, procollagen C endopeptidase enhancer 1, prostasin, reticulin 4 receptor-like protein 2, β defensin 1, cell surface glycoprotein MUC18, secreted pyrophosphate protein 24, cathepsin Z, immunoglobulin gamma C chain, plasminogen, neuropilin-1, guanylate binding protein β subunit-2, biotinylase, CD antigen, phospholipase D3, growth factor precursor, alkaline phosphatase, granulin receptor type 1, hyaluronidase-1, aconitase, membrane protein, neurotrypsin, glutathione adhesion protein kinase, phosphoprotein kinase A-2, phosphoprotein kinase-related protein kinase, phosphoprotein kinase protein G-9, phosphoprotein kinase-binding protein G-9, phosphoprotein kinase-binding protein subunit I, phosphoprotein kinase, phosphoprotein G-9, phosphoprotein kinase, phosphoprotein G-III receptor subunit I-9, phosphoprotein G-III receptor D-9, phosphoprotein G-III receptor D, phosphoprotein G-III, phosphoprotein III receptor D-III, phosphoprotein III receptor D-6, phosphoprotein III receptor D-6, phosphoprotein I-IV, phosphoprotein III receptor D-I, phosphoprotein III, phosphoprotein I-I, phosphoprotein III-I, phosphoprotein III receptor D-I, phosphoprotein III, VEGF-I, VEGF-I.

2. The use of claim 1, wherein the protein is selected from the group consisting of inhibin β C chain, pepsin, prostaglandin receptor F2 negative regulatory protein, glypican-3, amyloid 2, CD276 antigen, secreted pyrophosphate protein 24, insulin-like growth factor binding protein 3, FAM151A protein, biotinidase, lysine phosphate histidine inorganic pyrophosphate phosphatase, granulin protein, and combinations thereof.

3. Use of an agent for identifying a protein selected from the group consisting of:

4. The use of claim 3, wherein the protein is selected from fetuin-B, aminoacylase-1A, apolipoprotein A-I, vitamin D binding protein, leukemia inhibitory factor receptor, epidermal growth factor, calcium binding protein, kynurenine/α -aminoadipate aminotransferase, Na (+)/H (+) exchange regulatory cofactor NHE-RF1, and combinations thereof.

5. The use according to any one of claims 1 to 4, wherein the protein is obtained by screening an animal model for a urine protein marker associated with early stage ovarian cancer lung metastasis to neoplasia,

optionally, the animal model is prepared from a rat model of lung metastasis of ovarian cancer obtained by tail vein injection of ovarian cancer cells.

6. The use of any one of claims 1 to 4, wherein the identification reagent is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof,

preferably, the antibody is a monoclonal antibody;

optionally, the expression level of the protein determined in the urine sample of the subject by the identifying agent,

optionally, the expression level is a nucleic acid expression level or a protein expression level.

7. Use according to any one of claims 1 to 4, wherein the subject is a mammal, preferably a human.

8. A kit or chip for early diagnosis of whether a subject with ovarian cancer develops metastatic lung neoplasias, comprising an identifying agent for a protein selected from the group consisting of:

9. A method of establishing an animal model for screening for a urine protein marker associated with early ovarian cancer lung metastasis, the method comprising the steps of:

iii) identification of protein profiles in urine of tumorigenic and non-tumorigenic rats before establishment of a rat model of early ovarian cancer lung metastasis by mass spectrometry.

10. Use of a rat model with lung metastases of ovarian cancer obtained by tail vein injection of ovarian cancer cells for the preparation of an animal model for screening of urinary protein markers associated with early lung metastasis neoplasia of ovarian cancer.