CN113759113A - Urine protein marker for diagnosing medulloblastoma and application thereof - Google Patents

Abstract

The invention relates to a urine protein marker for diagnosing medulloblastoma and application thereof. In particular, the invention relates to the use of a reagent for detecting the amount of a protein in urine of a subject, wherein the protein in the urine is selected from one or more of the following: CADH1, FIBB, FGFR4, A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB 4.

Description

Urine protein marker for diagnosing medulloblastoma and application thereof

Technical Field

The present invention relates to the technical field of clinical diagnosis. In particular, the present invention relates to urine protein markers for the diagnosis of medulloblastoma. In particular, the invention relates to a urine protein marker related to human, especially child medulloblastoma, obtained by using a mass spectrometry proteomics technology and application thereof.

Background

Medulloblastoma (MB) is a malignant brain tumor that is common in children, accounting for about 30% of malignant brain tumors in children. The tumor is clinically advanced rapidly and is very recurrent, with approximately 75% of patients having recurrence of the tumor within 2 years after surgery [ see, A. Y. Minn, B.H. Pollock, L.Garzarella, G.V.Dahl, L.E.Kun, J.M.Ducore, A.Shibata, J.Kepner, P.G.Fisher, Surveillance neuroimaging to detect playback in bone graft turbines: a Pediatric Oncolog Group study, J.Clin. Oncolol.19 (2001) 4135-4140 htps:// doi.org/10.1200/JCO.2001.19.21.4135 ]. Headache, nausea and vomiting are the most common first symptoms [ see, s.wilne, j.collier, c.kennedy, a.jenkins, j.grout, s.mackie, k.koller, r.grundy, d.walker, progress from first system to diagnosis in heart bright blue bugs, eur.j.pediatr.171(2012) 87-93. https:// doi.org/10.1007/s00431-011 and 1485-7], but these symptoms are not characteristic for children and are often misdiagnosed by primary hospitals as "cold, enteritis".

Magnetic Resonance Imaging (MRI) is the primary method of diagnosing this tumor and is used for imaging monitoring after surgery. To obtain high quality images, younger children often need to take tranquilizers, which exposes them to the risk of airway obstruction, aspiration, hypoxia and even death, and thus MRI is not easy to implement for children. For the above reasons, missed diagnosis and misdiagnosis of the disease often occur in children.

Currently there are no reliable biomarkers (biomarkers) for clinical screening and post-operative monitoring. Therefore, for children, the research on biomarkers for early clinical screening and postoperative monitoring from samples obtained by non-invasive methods is of high research value.

In order to quickly search for specific biomarkers of tumors, proteomics analysis based on mass spectrometry detection is a very effective method. The method has been successfully used for detecting the Proteome configuration of various body fluids [ see, C.Zhang, W.leng, C.Sun, T.Lu, Z.Chen, X.Men, Y.Wang, G.Wang, B.Zhen, J.Qin, Urine protein Profiling Predicts Lung Cancer from Control Cases and Other turbines, EBioMedicine.30(2018) 120-128. https:// doi.org/10.1016/j.ebiom.2018.03.009; and M.Frantzi, J.Metzger, R.E.Bank, H.Husi, J.Klein, M.Dakna, W.Mullen, J.J.Cartledge, J.P.Schanstra, K.Brand, M.A.Kuczyk, H.Mischak, A.Vlahou, D.Theoderescu, A.S.Merseburger, Discovery and validation of waste biomarkers for detection of secondary cell carcinosa, J.Proomics.98 (2014) 44-58. https:// doi.org/10.1016/j.jprot.2013.12.010 ]. Cerebrospinal fluid, blood and urine are important body fluids commonly used for proteomic analysis. For the sick children, the waist is worn difficultly, and the compliance of patients and family members is poor. Therefore, the difficulty of obtaining cerebrospinal fluid from a child patient in this invasive manner may limit the clinical utility of this fluid testing. The heterogeneity of proteins in blood, the large dynamic fluctuations in concentration, and the rapid changes in proteome configuration make the use of blood for proteomic analysis a difficult task. In comparison, the urine can be obtained in a non-invasive manner for multiple times and in a large amount, which is very beneficial to analyzing the specific biomarkers of the diseases through mass spectrum detection. Urine has been shown to contain Biomarkers associated with tumors and to allow long term monitoring of efficacy [ see, m.an, y.gao, Urinary Biomarkers of Brain Diseases, Genomics biologics 13(2015) 345-354. https:// doi.org/10.1016/j.gpb.2015.08.005 ]. In addition, larger fluctuations in constituents in urine may be more likely to reflect Changes in the human body [ see, M.Li, M.ZHao, Y.Gao, Changes of proteins induced by antimicrobial agents can be more sensitive in urine than in plasma, Sci China Life Sci.57(2014) 649-656. https:// doi.org/10.1007/s 11427-014-. Thus, it is believed by the scholars that urine is a better, more sensitive sample of bodily fluid than plasma that can be used to detect biomarkers [ see e.r. smith, d.zuakawski, a.saad, r.m.scott, m.a.moses, urea biolakers precursor blood specimen present and response to heat, clin.cancer res.14(2008) 2378-2386. https:// doi.org/10.1158/1078-0432.CCR-07-1253 ]. Although there are many proteomic studies on urine, there are no reports on urine proteomes or biomarkers associated with medulloblastoma in children.

In order to find a biomarker capable of quickly, conveniently and reliably diagnosing the specificity of medulloblastoma of children, the inventor uses the urine of normal children, the preoperative urine and the postoperative urine of medulloblastoma patients to perform mass spectrometry so as to find a urine proteomic marker related to the tumor.

Disclosure of Invention

In one aspect, the present invention provides the use of a reagent for detecting the amount of a protein in urine of a subject, wherein the protein in the urine is selected from one or more of the following: CADH1, FIBB, FGFR4, A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB 4; preferably, wherein the subject is a human subject; more preferably, wherein the subject is a pediatric subject with an age of 0-16 years.

In a preferred embodiment of the invention, the CADH1 has the amino acid sequence as shown in SEQ ID NO: 10-13, or an amino acid sequence represented by SEQ ID NO: 10-13, or a pharmaceutically acceptable salt thereof;

in a further preferred embodiment, the FIBB has the sequence as set forth in SEQ ID NO: 4, or an amino acid sequence represented by SEQ ID NO: 4;

further preferably, said FGFR4 has the amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence represented by SEQ ID NO: 15, and (b) the amino acid sequence shown in the specification;

preferably, said A1BG has the amino acid sequence as shown in SEQ ID NO: 5-6, or an amino acid sequence represented by any one of SEQ ID NOs: 5-6, or a pharmaceutically acceptable salt thereof; the 1433B has the sequence shown in SEQ ID NO: 16, or an amino acid sequence represented by SEQ ID NO: 16; the VSIG4 has the sequence shown in SEQ ID NO: 22-23, or an amino acid sequence represented by any one of SEQ ID NOs: 22-23; the LYVE1 has the sequence shown in SEQ ID NO: 24-25, or an amino acid sequence represented by any one of SEQ ID NOs: 24-25, or a pharmaceutically acceptable salt thereof; the KVD28 has the sequence shown in SEQ ID NO: 3, or an amino acid sequence represented by SEQ ID NO: 3, and the amino acid sequence shown in the specification; the GELS has the sequence shown in SEQ ID NO: 7-8, or an amino acid sequence represented by any one of SEQ ID NOs: 7-8, or a pharmaceutically acceptable salt thereof; the PGRP1 has the sequence shown in SEQ ID NO: 1-2, or an amino acid sequence represented by SEQ ID NO: 1-2, or a pharmaceutically acceptable salt thereof; the LV321 has the nucleotide sequence shown in SEQ ID NO: 17, or an amino acid sequence represented by SEQ ID NO: 17; the SAP3 has an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence represented by SEQ ID NO: 14; the IGLC2 has the sequence as shown in SEQ ID NO: 9, or an amino acid sequence represented by SEQ ID NO: 9; the PCOC2 has the sequence set forth in SEQ ID NO: 21, or an amino acid sequence represented by SEQ ID NO: 21; the GGT6 has the sequence shown in SEQ ID NO: 18, or the amino acid sequence represented by SEQ ID NO: 18; the OGFD3 has the nucleotide sequence shown in SEQ ID NO: 19, or an amino acid sequence represented by SEQ ID NO: 19; the LIRB4 has the sequence shown in SEQ ID NO: 20, or an amino acid sequence represented by SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method of diagnosing and/or prognosing medulloblastoma in a subject, the method comprising detecting in the urine of the subject the amount of one or more urine proteins selected from the group consisting of: 1433B, A1BG, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, FGFR4, PCOC2, CADH1, GGT6, OGFD3, LIRB4, and FIBB;

wherein the subject has an increased or decreased, preferably greater than or equal to 1.3-fold, protein content in urine prior to treatment as compared to a healthy control group or a non-neoplastic neurological disease control group; indicating that the subject has medulloblastoma; and/or

Diagnosing that the urine protein content of the subject's urine is restored after treatment (including but not limited to surgery, radiation therapy and/or chemotherapy) of the subject with medulloblastoma, the prognosis of the subject is good; preferably, the restoration of protein content in the urine of the subject refers to an increase or decrease in urine protein content of greater than or equal to 1.2-fold after treatment as compared to before treatment; preferably, the non-neoplastic neurological disease includes, but is not limited to, epilepsy, congenital malformations, and other neurological diseases, except for neoplastic or neurological infections.

In another aspect, the present invention provides use of a reagent for detecting the amount of a protein in urine of a subject, wherein the protein in the urine is a combination of CADH1 and FIBB, in the preparation of a reagent for the diagnosis and/or prognosis of medulloblastoma in the subject; preferably, wherein the proteins in the urine further comprise FGFR 4; more preferably, the urine protein further comprises one or more of 1433B, A1BG, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB 4.

The method or use of the invention as described above, wherein the reagent for detecting the protein content in the urine of the subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; preferably, the reagent for detecting the protein content in the urine of the subject is a monoclonal antibody.

In another aspect, the present invention provides a kit or chip for diagnosing and/or prognosing medulloblastoma, which comprises a reagent for detecting the content of protein in urine of a subject, wherein the protein in the urine is a combination of the following proteins: 1433B, A1BG, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, FGFR4, PCOC2, CADH1, GGT6, OGFD3, LIRB4, and FIBB.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in the specification are herein incorporated by reference. The following examples are set forth in order to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed in any way to limit the scope of the invention, which is defined in the claims.

Term(s) for

As used herein, the term "Medulloblastoma (MB)" is the most common malignant brain tumor in children (WHO grade IV). The peak age at diagnosis is about 6-8 years, but there are still patients who may develop medulloblastoma in the first year after birth or in adulthood. Histomorphologically, medulloblastoma is an embryonic tumor located in the cerebellum, which may originate from different neural stem cells or precursor cells in early life.

A number of key genetic events have been identified in medulloblastoma. High level MYC gene amplification is the change in the level of the first reported gene. MYC is now considered to be the most common mutation and most characteristic tumor gene in medulloblastoma. Current treatment modalities are not universally effective in infants with MYC-amplifying medulloblastoma. The information obtained from the familial tumor syndrome predisposed to medulloblastoma, i.e., Gorlin syndrome and Familial Adenomatous Polyposis (FAP), provides early clues to the genes and pathways involved in medulloblastoma pathogenesis. For example, germline mutations in PTCH1 or SUFU (negative regulators of SHH signaling pathway) predispose to Gorlin syndrome, and mutations in both genes in the germline and in somatic cells confirm the important role of SHH signaling in certain medulloblastoma patients. Furthermore, about 1% of patients present with APC germline mutations (predisposed to FAP) and about 7% of patients present with CTNNB1 somatic mutations, and thus WNT signaling defects are associated with medulloblastoma pathogenesis 1.

As used herein, the term "Proteomics" is an "omics" technology rapidly developed after "genome", which forms the subject of system biology with metabolomics and genomics, thereby helping human beings comprehensively and multi-dimensionally recognize diseases from genes, proteins and metabolites.

Proteomics is the study and description of the expression of all protein components contained in an organism, organ, tissue, cell or organelle during a specific period of time. The comparative proteomics is used for researching the dynamic change of protein expression level of organisms in different states so as to discover key regulatory molecules and protein markers related to diseases, and finally can provide theoretical basis for prevention, diagnosis and treatment of the diseases and the like. Therefore, quantitative analysis of proteins is required, and thus quantitative proteomics technology is gradually developed. Currently, the most commonly used techniques include LC-MS/MS, CE-MS, and 2DE MALDI-TOF.

LC-MS/MS is a high resolution technique that can achieve more refined proteomic features using pre-separation prior to MS analysis. Although thousands of proteins can be identified by this technique, the analysis time is long, the complexity of the data analysis is high, and there is a risk of false positives. Unlabeled quantitation and stable isotope labeling quantitation are two commonly used quantitation techniques, the latter being more widely used. Since multiple samples can be analyzed simultaneously by running the MS once, the measurement error of the quantitative analysis of isotopic labeling is significantly reduced, but the number of samples analyzed at one time is limited by the number of labels. In contrast, using a non-labeled quantitative method, each sample is analyzed serially, and thus the resulting changes per run of the MS may affect the results. However, this method has no limitation on the number of samples.

CE-MS is used as a complementary high resolution technique to identify peptides and small molecule proteins originally present in body fluids (mainly urine). The reproducibility of the analysis was higher and the analysis run time was shorter (about 60 minutes) compared to LC-MS/MS. In addition, CE-MS has good technical properties and can be used for biomarker discovery, validation and clinical applications. However, subsequent sequencing of the peptide fragment requires a more sensitive mass spectrometer detector. Advances in MS-based technology will help overcome some of the limitations of classical gel-based proteomics technologies (e.g. 2DE/DIGE), namely low throughput and low resolution. For validation, techniques that allow targeted analysis of the selected protein are typically used. Although MS-based targeting techniques have been advanced, immunoassays (e.g., ELISA, WB) remain the most common. The immunoassay has high monitoring speed and is generally compatible with equipment in a clinical laboratory, but the separability of the immunoassay depends on the quality of an antibody, and synchronous analysis of a plurality of proteins at one time is difficult. When it is desired to develop a panel consisting of multiple biomarkers, the inability to simultaneously analyze multiple proteins will be the greatest impediment to the application of this technique. Thus, some MS-based platforms are applied for biomarker validation, including targeted (SRM/MRM/PRM) and non-targeted mass spectrometry (CE-MS). These methods do not require antibodies and can be used for more specific, sensitive assays.

In recent years, quantitative proteomics has become a hotspot in proteomics research, is an important way and means for discovering disease-related biomarkers, and has important significance in exploring pathogenic factors and pathogenesis of diseases and screening disease biomarkers. The overall change characteristics of the disease can be reflected better through the combination of the multi-molecular markers, and the sensitivity and the specificity to the disease diagnosis are superior to those of the traditional method.

Cerebrospinal fluid, blood and urine are important body fluids commonly used for proteomic analysis. Obtaining cerebrospinal fluid for testing requires lumbar puncture and is usually routinely performed 1-2 weeks after surgery. For the children with medulloblastoma, obstructive hydrocephalus exists before most of operations, the cranial pressure is high, and the lumbar puncture has the risk of causing cerebral hernia. Therefore, the difficulty of obtaining cerebrospinal fluid from a child patient in this invasive manner may limit the clinical utility of this fluid testing. The heterogeneity of proteins in blood, the large dynamic fluctuations in concentration, and the rapid changes in proteome configuration make the use of blood for proteomic analysis a difficult task. In comparison, the urine can be obtained in a non-invasive manner for multiple times and in a large amount, which is very beneficial to analyzing the specific biomarkers of the diseases through mass spectrum detection. It has been shown that urine contains biomarkers associated with tumors and that long term monitoring of therapeutic efficacy is possible. In addition, larger fluctuations in the constituents of urine may be more likely to reflect changes in the human body. Thus, it is believed by the scholarly that urine is a better, more sensitive sample of bodily fluid than plasma that can be used to detect biomarkers. Although there are many proteomics studies on urine, there is no study on urine proteomics and biomarkers of medulloblastoma in children.

The term "CADH 1" as used herein is a protein belonging to the Cadherin superfamily, a member of the classical type I cadherins, also known as E-cadherins (E-cadherins). It is encoded by the CHD1 gene. It cooperates with fibronectin-1 (nectin-1) to form cell-to-cell adhesive junctions [ see, K.Tachibana, H.Nakanishi, K.Mandai, K.Ozaki, W.Ikeda, Y.Yamamoto, A.Nagafuchi, S.Tsukuita, Y.Takai, Two cell adhesion molecules, nectin and cadherin, interracil through the cytokine cytological domain-associated proteins, J.cell biol.150(2000) 1161-1176. https:// doi. org/10.1083/jcb.150.5.1161 ]. Studies have shown that E-cadherin dysfunction or down-regulation of expression is associated with breast, nasopharyngeal, pancreatic, lung, gastric, digestive, kidney and prostate cancers [ see, P.Tiwari, A.Mrigwaii, H.kaur, P.Kaila, R.Kumar, P.Guptaarmama, Structural-Mechanical and Biochemical Functions of classic cadherin cell Junctions: A Review and Some superposes, adv.Exp.Med.biol.1112(2018) 107-138 https:// doi.org/10.1007/978-. In addition, CADH1 is thought to play a role in the development of malignant brain tumors. The expression of CADH1 was down-regulated in glioma patients and was considered to be significantly associated with poor overall survival. Overexpression of CADH1 in glioblastoma cells significantly prolonged survival of mice [ see, B.xu, R.Ma, L.Russell, J.Y.Yoo, J.Han, H.cui, P.Yi, J.Zhang, H.Nakashima, H.Dai, E.A.Chiocca, B.Kaur, M.A.Caligiuri, J.Yu, An interactive human virus expression E-calierdhn animal susvival in mouse modules of glioblotoma, Nat.Biotechnol. (2018). https:// doi.org/10.1038/nbt.4302 ].

As used herein, the term "FGFR 4" belongs to the family of growth factor receptors and is involved in many key cellular processes, such as cell proliferation, differentiation, migration, angiogenesis, and tissue repair. The FGFR4 signaling pathway is an important oncogenic pathway in a variety of tumors (e.g., breast, liver, prostate, and colon cancers) [ see, s.tang, y.hao, y.yuan, r.liu, q.chen, Role of fibroplast growth factor receptor 4in Cancer, Cancer sci.109(2018) 3024-3031. https:// doi.org/10.1111/cas.13759 ]. Expression of FGFR4 at the mRNA level was observed in the Medulloblastoma Cell line HD-MBO3 and in part of primary Medulloblastoma tissues [ see, k.santhana Kumar, a.neve, a.s.guerreiro Stucklin, c.m.kuzan-Fischer, e.j.rushing, m.d.taylor, d.tripolitisti, l.behrmann, d.kirschenbaum, m.a.grotzer, m.baumgartner, TGF- β determins the Pro-migratory patent of bFGF Signaling in medullolastoma, rep.23(2018)3798-3812. e.8. htps:// doi.org/10.1016/j.cell 2018.05.05.083 ].

As used herein, the term "FIBB" is a subunit of fibrinogen, identified as a Urine biomarker, useful for detecting bladder, prostate, pancreatic and Lung Cancer as well as renal cell carcinoma [ see, c.zhang, w.leng, c.sun, t.lu, z.chen, x.men, y.wang, g.wang, b.zhen, j.qin, urea protein Profiling precursors Lung Cancer cell Control Cases and oxygen turbines, ebiomedicine.30(2018) 120-.https://doi.org/10.1016/j.ebiom.2018.03.009；M.Frantzi,J.Metzger,R.E.Banks,H.Husi,J.Klein,M.Dakna,W.Mullen,J.J.Cartledge,J.P.Schanstra,K.Brand,M.A.Kuczyk,H.Mischak,A.Vlahou,D.Theodorescu,A.S.Merseburger,Discovery and validation of urinary biomarkers for detection of renal cell carcinoma,J Proteomics.98(2014)44–58.https://doi.org/10.1016/j.jprot.2013.12.010；M.Frantzi,K.E.van Kessel,E.C.Zwarthoff,M.Marquez,M.Rava,N.Malats,A.S.Merseburger,I.Katafigiotis,K.Stravodimos,W.Mullen,J.Zoidakis,M.Makridakis,M.Pejchinovski,E.Critselis,R.Lichtinghagen,K.Brand,M.Dakna,M.G.Roubelakis,D.Theodorescu,A.Vlahou,H.Mischak,N.P.Anagnou,Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study,Clin.Cancer Res.22(2016)4077–4086.https:// doi.org/10.1158/1078-0432.CCR-15-2715(ii) a And I.Belczacka, A.Latosinska, J.Siwy, J.Metzger, A.S.Merseburger, H.Mischak, A.Vlahou, M.Frantzi, V.Jankowski, Urinary CE-MS peptide marker pattern for detection of solid tumors, Sci Rep.8(2018)5227.https://doi.org/10.1038/s41598-018-23585-y]. Fibrinogen is one of the major components that links tumor cells to the extracellular matrix. Fibrinogen also promotes tumor angiogenesis and tumor progression [ see, M.W. Mosesson, Introduction: fibrinogen as a derivative of the metastatic potential of tumor cells, blood.96(2000)3301]。

As used herein, the term "A1 BG," also known as alpha-1-B glycoprotein, is a plasma glycoprotein of unknown function. The protein exhibits sequence similarity to the variable regions of certain immunoglobulin supergene family member proteins.

As used herein, the term "VSIG 4" (V-set and Ig domain-associating 4) VSIG4, a member of the B7 superfamily, is a complement receptor for C3B/iC3B, can inhibit activation of cytotoxic T lymphocytes and may play an important role in the development of gliomas [ see, Xu, Tao et al, "VSIG 4 is high expressed and corrected with a pore promoter of high-grade gliomas effect." American juourcel of relational research.7, 61172-80.15 Jun.2015 ].

As used herein, the term "1433B" (14-3-3protein beta/alpha), encoded by the YWHAB gene, blocks nuclear translocation of phosphorylated SRPK2(AKT1), antagonizes its stimulatory effect on cyclin D1 expression, and thereby blocks SRPK 2-induced neuronal apoptosis.

As used herein, the term "LYVE 1" (Lymphatic vessel endothionic acid receptor 1) is a ligand-specific transporter between an intracellular organelle (TGN) and a plasma membrane. Can be used as Hyaluronic Acid (HA) transporter for mediating the catabolic absorption in lymphatic endothelial cells or transporting it to the lumen of afferent lymph vessels for subsequent reabsorption and degradation in lymph nodes.

As used herein, the term "KVD 28" (Immunoglobulin kappa variable 2D-28), an Immunoglobulin light chain variable region V region involved in antigen recognition. In the recognition stage of humoral immunity, membrane-bound immunoglobulin serves as a receptor, and after binding to a specific antigen, B lymphocytes are triggered to clonally expand and differentiate into immunoglobulin-secreting plasma cells. The antigen binding site is composed of the variable domain of one heavy chain and the variable domain of its associated light chain. Thus, each immunoglobulin has two antigen binding sites with significant affinity for a particular antigen. After antigen exposure and selection, the variable domains are assembled by a process called V- (D) -J rearrangement, which allows affinity maturation of specific antigens.

As used herein, the term "GELS" (Gelsolin), calponin, actin-modulating protein, binds to the positive (or barbed) end of an actin monomer or filament, preventing monomer exchange. It may facilitate assembly of the monomers into filaments and the formed plurality of filaments.

As used herein, the term "PGRP 1" (Peptidoglycan replication protein 1) which has a bactericidal effect on gram-positive bacteria. It is possible to kill gram-positive bacteria by interfering with peptidoglycan biosynthesis. Combined with gram-negative bacteria, has bacteriostatic action on the gram-negative bacteria.

As used herein, the term "LV 321" (Immunoglobulin lambda variable 3-21), an Immunoglobulin light chain variable region V region involved in antigen recognition.

As used herein, the term "SAP 3" (Ganglioside GM2 activator) can bind gangliosides and stimulate degradation of Ganglioside GM 2. Stimulation of β -hexosaminidase a breakdown of ganglioside GM2 and glycolipid GA 2.

As used herein, the term "IGLC 2" (Immunoglobulin lambda constant 2) is an Immunoglobulin light chain constant region.

As used herein, the term "PCOC 2" (Procollagen C-endooptidase enhancer2) binds to the C-terminal propeptide of type I and type II procollagens and facilitates cleavage of the propeptide by BMP 1.

As used herein, the term "GGT 6" (glutaminone hydrosase 6), can break down Glutathione conjugates.

As used herein, the term "LIRB 4" (leukcyte immunoglobulin-like receptor subunit B member 4), MHC class I antigen receptor. Can widely identify the alleles of HLA-a and HLA-B, HLA-C, HLA-G. Participate in the down regulation of immune response and the generation of tolerance, inhibit receptor-mediated cellular protein phosphorylation and mobilization of intracellular calcium ions.

Drawings

FIG. 1 shows a flowchart of the medulloblastoma-associated biomarker screening of the present invention. In the discovery of biomarkers, urine of 9 cases of medulloblastoma patients before and after operation and 9 cases of healthy children is subjected to quantitative tandem mass spectrometry (TMT) analysis to determine the proteomic characteristics of the patients; in the biomarker verification, PRM (parallel response monitoring) verification was performed on 112 samples (58 urine samples from 29 infants with medulloblastoma before and after surgery, 26 urine samples from healthy children, and 28 urine samples from patients with non-neoplastic neurological disease).

Fig. 2 shows a score plot for an unsupervised PCA analysis showing that the healthy control group can be separated from the two disease groups, while the pre-medulloblastoma group can be partially separated from the post-medulloblastoma group.

Fig. 3 shows a heat map of 114 different proteins that were significantly altered between the pre-medulloblastoma group and the healthy control group, but returned to normal in the post-medulloblastoma group, indicating that the urine proteome may reflect the characteristics of medulloblastomas.

Fig. 4 shows the results of PRM validation of 17 different proteins (25 polypeptides) in pre-and post-operative, healthy and disease control groups.

Fig. 5A shows ROC curves for the combination of CADH1, FIBB, FGFR4 to differentiate pre-medulloblastoma from healthy controls; figure 5B shows ROC curves for the combination of CADH1 and FIBB used to differentiate pre-medulloblastoma from disease control.

Detailed Description

The inventor uses the urine of normal children and the preoperative and postoperative urine of patients with Medulloblastoma (MB) to carry out mass spectrometry, and further draws out the proteomics characteristics of the urine of children with tumors. And the levels of urinary protein in the three groups of samples were quantified. Parallel Reaction Monitoring (PRM) was used to verify the results obtained, and a new sample was used at this stage, including: control group, preoperative group, postoperative group, non-tumor nervous system disease group (fig. 1). The present study may provide a useful clue for determining urine protein markers in patients with medulloblastoma.

Example 1 Collection and preparation of urine samples for Mass Spectrometry

1. Group entry patients and sample Collection

In the biomarker discovery stage, 9 infants with medulloblastoma were collected with urine before and after tumor resection. These patients were diagnosed at the Beijing Temple Hospital, university of capital medicine, and surgery was performed at that hospital. The grouping standard of the children patients is as follows: age 16 years or less, postoperative pathology confirmed medulloblastoma, and no treatment (radiotherapy or chemotherapy) was received before and during sample collection. Urine before operation is collected before operation, and urine after operation is collected for 1-2 weeks after operation. At this stage, urine from 9 healthy children was collected as a control group. To verify the results, 29 infants with medulloblastoma were collected using the same standard for preoperative and postoperative urine, 26 matched healthy children urine, and 28 infants with non-tumor neurological disease (including epilepsy, congenital malformations, and other neurological diseases, except for tumor or neurological infections). The total amount of urine collected each time was 40 mL. Morning at 7:00-9:00, patient retained morning urine (midcourse urine) on fasting. Urine routine examination was performed on each sample to rule out potential disease. Centrifuging at 3000g × 15min to obtain supernatant, and storing in a refrigerator at-80 deg.C. The study was approved by the ethical committee and patients and legal guardians signed informed consent.

TABLE 1 overview of clinical samples participating in the study of the invention

2. Sample preparation

2.1 protein enrichment

Taking 10mL of urine sample, adding acetone at the temperature of minus 20 ℃ to precipitate protein in the urine sample, centrifuging for 14000g multiplied by 15min, removing supernatant, dissolving the precipitate in lysate containing 8mol/L urea to redissolve the protein, centrifuging for 14000g multiplied by 15min, and keeping the supernatant.

2.2 protein concentration determination

Bradford method for determining protein concentration of sample

BSA (bovine serum albumin) standard protein of 1mg/mL was diluted to different concentrations, a Bradford working solution quantification reagent was added, and the absorbance at 595nm was measured using a microplate reader, after which a standard curve was drawn. The absorbance value of the protein in the sample to be measured was measured by the same measurement method, and the protein concentration in the sample to be measured was calculated from the previously obtained standard curve.

2.3 reductive alkylation and digestion of proteins

1) Protein degradation Using FASP method

According to J.R.

A.Zougman, N.Nagaraj, M.Mann, Universal sample preparation method for protein analysis, nat. methods.6(2009) 359-362. https:// doi.org/10.1038/nmeth.1322 the procedure was first to add 20mM DTT (Dithiothreitol) to the urine protein sample and to water bath at 95 ℃ for 5 minutes (reduction). Thereafter, 50mM IAA (Iodoacetamide ) was added, and the mixture was left at room temperature in the dark for 45 minutes (alkylation). The reductively alkylated urine protein sample was then applied to a 30kd filter and washed twice with UA solution (containing 8mol urea) and 25mmol/L ammonium bicarbonate solution. And finally, mixing the trypsin and the protein according to the mass ratio of 1: trypsin was added at a ratio of 50 and the digestion was carried out overnight at 37 ℃.

2) Collection of Polypeptides

Placing the ultrafiltration tube into a centrifuge, centrifuging for 14000g × 30min, setting the temperature at 18 ℃, and leaving the polypeptide solution in the ultrafiltration tube until the liquid is completely separated. And adding 10 mu L of 500mM NaCl solution on the filter membrane, repeatedly blowing and beating for 30 times by using a gun head, and carrying out vortex oscillation for 60s to clean residual polypeptide adhered on the membrane. Placing into ultracentrifuge again, centrifuging 14000g × 30min, setting temperature at 18 deg.C for about 30min, and allowing the solution containing polypeptide in the retention tube to be mixed with the polypeptide obtained by the first centrifugation.

3) Peptide sample extraction by Oasis C18 solid phase extraction column

A3 mL C18 solid phase extraction column was placed in a solid phase extraction apparatus. Adding 500 μ L100% acetonitrile into C18 solid phase extraction column, waiting for the upper layer liquid of the extraction column to naturally drip, and repeating the above steps again to activate C18 solid phase extraction column. Then, 500 μ L of 1% trifluoroacetic acid was added to the C18 solid phase extraction column, and the residual acetonitrile solution on the extraction column was washed clean and repeated again after the liquid was naturally dripped off, to allow the C18 solid phase extraction column to equilibrate. Subsequently, the collected polypeptide solution was applied to a C18 solid-state extraction column, and the solution was allowed to drip naturally. The desalting was washed seven times using 500. mu.L of 1% trifluoroacetic acid. mu.L of 100% acetonitrile was added and the eluate was collected in a microcentrifuge tube (EP tube) to complete the elution of the polypeptide. And finally, putting the collected eluent into a rotary vacuum drying instrument, setting the eluent not to be heated, and after vacuum pumping, putting the eluent into a refrigerator at the temperature of-20 ℃ for storage and standby.

4) The polypeptide concentration was measured by BCA method (bicinchoninic acid method).

Angiotensin II was weighed and made into protein standard solutions of 0. mu.g/. mu.L, 0.1. mu.g/. mu.L, 0.2. mu.g/. mu.L, 0.5. mu.g/. mu.L, 1. mu.g/. mu.L with 1% formic acid. The polypeptide sample was dissolved in 1% formic acid to prepare a solution having a concentration of 0.3. mu.g/. mu.L. 200. mu.L of the prepared BCA working solution was added to each well of a 96-well plate. Adding 10 μ L of protein standard solution or polypeptide sample with different concentrations into each well, making 3 parallel multiple wells of the same sample, shaking in shaking table, and placing the well plate in constant temperature water bath at 37 deg.C for 30 min. The absorbance value of the standard at 562nm was measured using a microplate reader, after which a concentration/absorbance standard curve was plotted. The absorbance of the sample was measured by the same measurement method, and the polypeptide concentration of the sample was calculated from the standard curve obtained previously.

Example 2 urine protein profiling

TMT (Tandem mass tag) labeled quantitative proteomics analysis

1.1 TMT labelling and off-line liquid phase separation

1) TMT quantitative labelling

The 3 pre-and corresponding post-operative samples, 3 healthy control group samples, for a total of 9 polypeptide samples, were randomly labeled 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131, respectively. Use of 10-plex Tandem Mass Tags (TMT) reagents was made according to the manufacturer's instructions (Thermo Fisher Scientific). The mixed samples of the three groups of preoperative, postoperative and healthy controls were labeled with 126 TMT.

2) Off-line high-PH reversed-phase high performance liquid chromatography separation of TMT (Tetramethylbenzidine) labeled polypeptide

TMT-labeled samples were mixed and fractionated using a reverse phase high performance liquid chromatography column (Waters,4.6 mm. times.250 mm, Xbridge C18,3 μm). The sample was placed on a chromatographic column into buffer a1 (99.9% deionized water, 0.1% ammonia, pH 10). Buffer B1 was split with a gradient of 5-25% (90% acetonitrile, PH 10) at a flow rate of 0.8mL/min for 60 min. The peptide fragments eluted every minute were used as one fraction, and the eluted peptide fragments were collected at a rate of one fraction/min for a total of 60 fractions. The 60 fractions after vacuum drying were reconstituted with one thousandth of FA and then combined into one fraction according to numbers 1, 21, 51, 2, 22, 52, and so on, giving a total of 20 fractions. The 20 fractions collected were placed in a rotary vacuum desiccator and vacuum dried and frozen at-20 ℃.

1.2 LC-MS/MS analysis

TMT-labeled sample mixtures were analyzed using a self-packed reverse phase C18 capillary liquid chromatography column (75 μm. times.100 mm,1.9 μm). The elution gradient for buffer B2 was 5-30% (0.1% formic acid, 99.9% acetonitrile) with a flow rate of 0.3. mu.L/min for a duration of 45 minutes. The polypeptide mixture was identified by analysis using an LTQ Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. The instrument parameters are set to full scan using the highest speed data correlation mode (3 s); full scan with Orbitrap, resolution 60, 00; the HCD collision cell was set to 32% normalized collision energy with a resolution of 15,000; charge state screening (excluding precursors of unknown charge states or charge states of + 1); dynamic elimination (elimination duration is 30 s).

1.3 data analysis

The resulting mass spectral data were analyzed by the Proteome scanner software suite (v2.1, Thermo Fisher Scientific) using the SwissProt human database of Uniprot (www.uniprot.org). The retrieval parameters are: trypsin selective cleavage specificity, 2 sites of miscut; aminomethylated cysteine and TMT 10-plex tags were set for fixed modification, and the N-terminus of oxidized methionine, deamidated aspartic acid, deamidated glutamine, carbamoyllysine and peptide fragments was set for dynamic modification. The peptide tolerance used in the search was 20ppm and the mass error tolerance was 0.05 Da. The screening criteria were set as: the false positive rate is less than 1% on the protein level, and each protein at least contains 1 specific peptide segment. After screening, peptide abundance of different indicator ion channels was normalized. Protein abundance ratio was estimated based on the specific peptide fragment results. Identified as a proteomic differential protein: compared with the preoperative group and the healthy control group, the differentiation multiple is more than or equal to 1.3, and compared with the preoperative group, the postoperative group has recovery, and the differentiation multiple is more than or equal to 1.2.

1.4 identification of differentially expressed urine proteins Using TMT Mass Spectrometry

Randomly selecting urine samples of 9 healthy children and urine samples of 9 children with medulloblastoma before and after operation, and respectively marking by using TMT reagent. In the quantitative proteomic analysis of TMT markers, we identified 2041 proteins in the pre-operative group, 2041 proteins in the post-operative group, and 1908 proteins in the healthy control group, which proteins contained at least one specific peptide fragment. 2015 proteins in the pre-operation group of medulloblastoma can be quantified, 2018 proteins in the post-operation group can be quantified, and 1893 proteins in the healthy control group can be quantified. Median CVs of preoperative, postoperative, healthy controls were 22.9%, 24.5%, 23.7%, respectively. 1470 proteins are shared by the pre-operative group, the post-operative group and the healthy control group of medulloblastoma.

First, to explore the differences in proteomic analysis of three groups of samples, unsupervised PCA analysis was performed. The score plots show that the healthy control group can be separated from the two disease groups, while the pre-medulloblastoma group can be partially separated from the post-medulloblastoma group (fig. 2). Second, OPLS-DA was performed to further determine proteomic differences between the three groups. In the OPLS-DA model, healthy control groups, pre-medulloblastoma and post-medulloblastoma groups may be separated from each other. One hundred permutation tests showed that these models did not over-fit.

In previous humoral proteomics studies, differential proteins typically accounted for 10% to 20% of the total quantified protein [ see, L.Pang, Q.Li, Y.Li, Y.Liu, N.Duan, H.Li, Urine proteins of primary chromatography using nanoscaled liquid chromatography analysis, Clin proteomics.15(2018)5.https:// doi.org/10.1186/s 12014-018-; J. -Y.Cao, Y.P.Xu, X.Z.Cai, TMT-based qualitative proteomics and systems, temporal evolution feedback strategies mechanisms of Brassica napus, said estimating of the genetic coding of a diagnostic, J proteomics.143(2016) 265-277. https:// doi.org/10.1016/j.jprot.2016.03.006; and H.Xiao, Y.Zhang, Y.Kim, S.Kim, J.J.Kim, K.M.Kim, J.yoshizawa, L.Y.Fan, C.X.Cao, D.T.W.Wong, Differential genomic Analysis of Human Saliva using Tandem Mass Tags Quantification for scientific Cancer Detection, Sci Rep.6(2016)22165.https:// doi.org/10.1038/srep 22165; and L.Zou, X.Wang, Z.Guo, H.Sun, C.Shao, Y.Yang, W.Sun, Differential urinary proteomics analysis of muscular involvement using iTRAQ qualification, Mol medical Rep.19(2019) 3972-3988. https:// doi.org/10.3892/mmr.2019.10088 ]. To find all potential medulloblastoma markers, the fold change was normalized to ≧ 1.3, and a total of 300 (about 20%) differential proteins were identified between medulloblastoma and healthy controls. Urine proteins that changed in the pre-medulloblastoma sample, but returned to levels relative to healthy control samples in the post-medulloblastoma sample, were considered to be differential proteins associated with medulloblastomas. Considering that medulloblastoma-associated proteins may not be fully restored to normal levels post-operatively, a protein that simultaneously meets the following criteria is defined as a medulloblastoma-associated differential protein: the fold change between medulloblastoma and healthy control group is more than or equal to 1.3; the reverse fold change after operation is more than or equal to 1.2. Thus, a total of 114 medulloblastoma-associated differential proteins were identified (51 upregulated and 63 downregulated in the medulloblastoma group). The heat map of 114 different proteins (fig. 3) showed that these proteins changed significantly between the pre-medulloblastoma group and the healthy control group, but returned to normal in the post-medulloblastoma group, indicating that the urine proteome may reflect the characteristics of medulloblastomas.

Example 3 Pathway Analysis of differential proteins (IPA)

All differentially expressed proteins were analyzed using IPA software (informaity Systems, Mountain View, CA). These proteins are divided into two categories, disease-related and function-related, and are depicted in the biological pathways of Ingenuity and other databases. Sorted by z-score and p-value, respectively.

Functional analysis of differentially expressed proteins

These 114 differential proteins were analyzed by IPA and could be classified into two categories related to disease and function, such as tumor cell migration, apoptosis, central nervous system metastasis, stage III tumor, recurrent tumor, head and neck tumor, metastasis, suggesting that these proteins may be related to nervous system tumors. Proteins enriched on biological pathways are associated with the following pathways: the functional role of these urinary proteins is demonstrated by the correlation with clathrin-mediated endocytic signals, glycolytic I, the role of tissue factor in cancer, PPAR α/RXR α activation, neuroinflammatory signaling pathways, and HIF1 α signaling. These differential proteins detected from urine are consistent with their source and function.

Example 4 Parallel Reaction Monitoring (PRM) analysis

17 tumor-related and functionally-related proteins from the 114 differential proteins analyzed by LC-MS/MS were selected for PRM validation. Each sample is analyzed using a schedule mode. In order to ensure the quality of the data, quality control analysis is carried out on the mixed sample to ensure the stability of the instrument signal in the whole process. IRT standard peptide analysis was also added to each sample and the stability of the chromatographic retention time was assessed during the analysis. Two technical replicates were performed for each sample. To reduce systematic variation, randomly ordered mass spectrometry was performed on different sets of samples.

To verify the results, 29 infants with medulloblastoma were collected using the same standard for preoperative and postoperative urine, 26 matched healthy children urine (HC), and 28 infants with non-neoplastic neurological Disease (DC) (including epilepsy, congenital malformations, and other neurological diseases, except for tumor or neurological infections).

In the PRM analysis, the method of protein extraction and decomposition is the same as described above. The peptide fragments eluted by liquid chromatography were analyzed by Triple TOF 5600 mass spectrometer. MS data was obtained using a high-sensitivity mode (high-sensitivity mode), setting the parameters as follows: PRM mode, full scan with a resolution of 40000, MS/MS scan with a resolution of 20000, rolling collision energy, charge state screening (including precursors of +2 to +4 charge states), dynamic elimination (excluding elimination time 15s), MS/MS scan range 100-. Each sample was repeated twice.

PRM data analysis

PRM data analysis was performed using Skyline 3.6 software. All data were imported into Skyline 3.6, the correct peaks were manually picked, and the resulting polypeptides in all samples were exported. The total ion intensity of +2- +5 charges in each sample was extracted using Progenesis software. The mass spectra of each peptide fragment in each sample were normalized using the sample total ion intensity to correct for errors in sample size and mass signal intensity. Each peptide fragment was quantitatively analyzed, and differential proteins among different groups were screened and compared with the TMT results.

Statistical analysis

Pattern recognition analysis (principal component analysis, PCA; orthogonal partial least squares discriminant analysis, OPLS-DA) was performed using SIMCA 14.0(Umetrics, Sweden) software.

The peptide fragments monitored between groups were compared by paired or unpaired t-test analysis. P values less than 0.05 are considered statistically significant. Using Metabionalyst software, establishing receiver operating characteristic curves (ROC) of each biomarker to be selected. And (5) performing combined ROC analysis by adopting a linear SVM algorithm. And (3) taking the medulloblastoma as a dependent variable, calculating the area under the ROC curve (AUC), and determining the diagnostic marker to be selected.

Results of differential protein PRM validation

In order to determine the biomarkers associated with tumors, 17 tumor-associated and functionally-related proteins were selected from the differential proteins analyzed by LC-MS/MS, and PRM-verified to determine whether these proteins had specificity for diagnosing medulloblastoma. To ensure system stability and limit systematic bias during analysis, a mixture of all urine samples was used as Quality Control (QC).

Each time

QC samples were injected before, after and during each sample. QC samples were tightly pooled by PCA analysis of QC samples and individual urine samples. The correlation plot for the QC samples also showed a high positive correlation for the QC samples, with an average pearson correlation coefficient of 0.926. The above results indicate the reproducibility of QC. These results indicate the stability of the MS platform during the analysis.

Compared with the healthy control group (HC), the PRM verification results of these 17 proteins (25 polypeptides) have the same variation trend as that of TMT, wherein 1 protein is up-regulated and 16 proteins are down-regulated (fig. 4, table 2). The diagnostic value of these 17 proteins was further evaluated using ROC analysis. Among 13 proteins that showed better discrimination (AUC >0.75) when used alone for diagnostic evaluation were A1BG, VSIG4, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, FGFR4, PCOC2, CADH1, GGT6, and FIBB, where the combination of CADH1, FIBB, FGFR4 significantly improved the diagnostic accuracy, with an AUC value of 0.973 (95% CI, 0.923-1, fig. 5A). These biomarkers can be used for the diagnosis of medulloblastoma.

Compared with disease control group (DC), 13 polypeptides were found to have significant changes, corresponding to 9 down-regulated proteins (fig. 4, table 2), which 9 down-regulated proteins were PGRP1, FIBB, A1BG, IGLC2, CADH1, FGFR4, LV321, LIRB4, VSIG 4. ROC analysis showed that the three proteins PGRP1, CADH1, FIBB, when evaluated alone, showed good discrimination (AUC >0.75, data not shown). The combination of CADH1 and FIBB showed higher sensitivity and specificity, with AUC values of 0.884 (95% CI, 0.75-0.978, fig. 5B). These biomarkers can be used to distinguish medulloblastoma from benign nervous system diseases.

Sequence listing

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Claims (10)

1. Use of an agent for detecting the amount of a protein in urine of a subject in the manufacture of a reagent for the diagnosis and/or prognosis of medulloblastoma in said subject, wherein the protein in said urine is selected from one or more of the following: CADH1, FIBB, FGFR4, A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB4, preferably, the urine protein has the amino acid sequence as set forth in SEQ ID NO: 1-25, or an amino acid sequence represented by any one of SEQ ID NOs: 1-25, or a pharmaceutically acceptable salt thereof.

2. The use of claim 1, wherein the subject is a human subject, preferably wherein the subject is a pediatric subject with an age of 0-16 years.

3. The use of claim 1 or 2, wherein the subject has an increased or decreased level of one or more urine proteins in the urine as compared to a healthy control group or a non-neoplastic neurological disease control group selected from the group consisting of: CADH1, FIBB, FGFR4, A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB4, diagnosing the subject as having medulloblastoma; and/or

Diagnosing a subject with medulloblastoma who, upon treatment, has a restored level in urine of one or more urine proteins selected from the group consisting of: CADH1, FIBB, FGFR4, A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB4, the prognosis of the subject is good.

4. The use of claim 3, wherein the increase or decrease in urine protein content in the subject's urine is an increase or decrease in urine protein content of greater than or equal to 1.3 fold compared to a healthy control group or a non-neoplastic neurological disease control group prior to treatment; and/or the recovery of protein content in the urine of said subject means that the increase or decrease in urine protein content after treatment is greater than or equal to 1.2 fold compared to before treatment.

5. The use of any one of claims 1-4, wherein the protein in urine is a combination of CADH1 and FIBB, preferably wherein the CADH1 has the amino acid sequence as set forth in SEQ ID NO: 10-13, or an amino acid sequence represented by SEQ ID NO: 10-13, or a pharmaceutically acceptable salt thereof; further preferably, wherein the FIBB has the sequence as set forth in SEQ ID NO: 4, or an amino acid sequence represented by SEQ ID NO: 4, and (b) the amino acid sequence shown in the figure.

6. The use of claim 5, wherein the protein in urine further comprises FGFR4, preferably wherein the FGFR4 has the amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence represented by SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

7. The use of claim 5, wherein the urine protein further comprises one or more of A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB 4.

8. The use of any one of claims 1 to 7, wherein the reagent that detects the amount of protein in the urine of a subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof.

9. The use of claim 8, wherein the reagent for detecting the amount of protein in the urine of a subject is a monoclonal antibody.

10. A kit or chip for diagnosing and/or prognosing medulloblastoma comprising a reagent that detects the amount of protein in the urine of a subject, wherein the protein in the urine is a combination of: CADH1, FIBB, FGFR4, A1BG, 1433B, VSIG4, LYVE1, KVD28, GELS, PGRP1, LV321, SAP3, IGLC2, PCOC2, GGT6, OGFD3, and LIRB4 FIBB.

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