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WO2020230957A1 - Composition for diagnosis of invasive brain cancer - Google Patents

Composition for diagnosis of invasive brain cancer Download PDF

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Publication number
WO2020230957A1
WO2020230957A1 PCT/KR2019/011921 KR2019011921W WO2020230957A1 WO 2020230957 A1 WO2020230957 A1 WO 2020230957A1 KR 2019011921 W KR2019011921 W KR 2019011921W WO 2020230957 A1 WO2020230957 A1 WO 2020230957A1
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Prior art keywords
brain cancer
protein
expression level
pcbp1
composition
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French (fr)
Korean (ko)
Inventor
강석구
박준성
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a composition for diagnosis of invasive brain cancer.
  • Cancer is one of the most common causes of death worldwide. About 10 million new cases occur every year, accounting for about 12% of all causes of death, making it the third-most cause of death.
  • brain cancer in particular occurs regardless of age, and has a higher incidence in children than other cancers.
  • the brain cancer is collectively referred to as primary brain cancer that occurs in the brain tissue and the meninges surrounding the brain, and secondary brain cancer that has metastasized from cancer that occurs in the skull or other parts of the body, and exhibits different characteristics from cancer that occurs in other organs.
  • Glioma is a tumor that accounts for 60% of primary brain tumors, and it is a malignant tumor with high incidence and difficult treatment, and there is still no special treatment other than radiation therapy.
  • glioblastoma multiforme GBM
  • GBM glioblastoma multiforme
  • Occupy In the case of glioblastoma, 2 to 3 cases per 100,000 people occur in Europe and North America.
  • glioblastoma exhibits an aggressive variant compared to other brain cancers, and if not treated as soon as possible, it can lead to fatal results within a few weeks. Furthermore, the glioblastoma has a very high tendency to metastasize outside the cranial cavity, and is more aggressive than other brain cancers.
  • glioblastoma has a very poor prognosis among central nervous system malignancies, and glioblastoma patients have an average survival rate of only 1 year, and only about 3% of patients survive for 3 years or more. Accordingly, there is a need for research on new diagnostic and therapeutic targets that can diagnose brain cancer early and predict the prognosis.
  • An object of the present invention is to provide a composition for diagnosis of invasive brain cancer and a kit including the same.
  • Another object of the present invention is to provide a method of providing information for diagnosis of invasive brain cancer.
  • Another object of the present invention is to provide a composition for predicting the prognosis of invasive brain cancer and a kit containing the same.
  • Another object of the present invention is to provide a method of providing information for predicting the prognosis of invasive brain cancer.
  • Another object of the present invention is to provide a method for screening a therapeutic agent for invasive brain cancer.
  • One embodiment of the present invention provides a composition for diagnosis of invasive brain cancer.
  • composition of the present invention includes an agent for measuring the expression level of the PCBP1 (Poly(RC) Binding Protein 1) protein or a gene encoding the same.
  • PCBP1 Poly(RC) Binding Protein 1
  • the PCBP1 protein of the present invention is a protein encoded by a gene located on human chromosome 2 (70.17-70.17 Mb), and has a function of forming a major cellular poly(rC)-binding protein.
  • the PCBP1 protein is present at a high level, the invasiveness of breast and ovarian cancer patients may be high ( IUBMB Life ., 2019 Feb;71(2):177-182.).
  • the expression level of the PCBP1 protein or the gene encoding it was significantly lowered in brain cancer patients with high invasiveness compared to brain cancer patients with low invasiveness (see FIG. 2).
  • the expression level of the PCBP1 protein or the gene encoding it is lowered compared to a normal control (an individual confirmed to be free from brain cancer or an individual with low invasive brain cancer) In some cases, it can be predicted as invasive brain cancer.
  • the PCBP1 protein of the present invention may consist of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.
  • the gene encoding the protein of the present invention may be composed of a nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.
  • the PCBP1 protein of the present invention is composed of an amino acid sequence having at least 70% sequence homology with the amino acid sequence represented by SEQ ID NO: 1, and includes all of the polypeptides that are naturally or artificially added, deleted, substituted or combined with the amino acid sequence.
  • amino acid sequence represented by SEQ ID NO: 1
  • the "homology" of the present invention is intended to indicate the degree of similarity between amino acid sequences, and includes sequences having the same percent or more of the same sequence as the amino acid sequence of the present invention. Such homology can be determined by visually comparing two sequences, but can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. Homology between the two amino acid sequences can be expressed as a percentage.
  • Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Alignment algorithms automated in the module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithms and homology determination for other useful alignments can be automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.
  • brain cancer refers to all tumors occurring in the cranial cavity, and may be, for example, a glioma, but is not limited thereto.
  • the "glioma” of the present invention is a tumor that accounts for 60% of primary brain tumors, has a high incidence and is difficult to treat, and is a malignant tumor that does not have any special treatment other than radiation therapy, for example, It may be an astrocytoma, glioblastoma, or oligodendrogliomas, preferably glioblastoma, but is not limited thereto.
  • the brain cancer of the present invention may be a cancer tumor cell, but is not limited thereto.
  • the "cancer tumor cell” of the present invention refers to a cell aggregate in which some cells having characteristics similar to stem cells among cells constituting cancer tissues can be formed under three-dimensional culture conditions.
  • the "invasive" of the present invention is a phenomenon in which tumor cells generated in a primary organ acquire a new genetic trait necessary for metastasis as cancer progresses, and then invasion into blood vessels and lymph glands.
  • tumor cells infiltrated by blood vessels and lymph glands circulate along the lymph, settling in tissues existing in other organs from the primary organ, and then proliferating, so that cancer can easily metastasize to other organs.
  • the preparation for measuring the expression level of the gene encoding the protein of the present invention refers to a preparation for measuring the expression level of the protein encoded from the target gene included in the sample.
  • the agent for measuring the expression level of the protein may be an antibody or aptamer capable of specifically binding to the protein.
  • the formulation is Western blot assay, ELISA (Enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket Rocket immunoelectrophoresis, Immunohistochemical staining, Immunoprecipitation Assay, Complement Fixation Assay, Immunofluorescence, Immunochromatography, FACS Antibodies or aptamers used in methods such as (Fluorescenceactivated cell sorter analysis) and protein chip technology assay may be included, but are not limited thereto.
  • the "antibody” of the present invention means a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule.
  • the form of the antibody is not particularly limited, and as long as it has a polyclonal antibody, a monoclonal antibody, or an antigen binding property, it may be included even if it is part of the antibody, and all kinds of immunoglobulin antibodies may be included.
  • special antibodies such as humanized antibodies may be included, and the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules.
  • the functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.
  • the "aptamer" of the present invention refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule.
  • the aptamer may have various three-dimensional structures according to its base sequence, and may have high affinity for a specific substance such as an antigen-antibody reaction.
  • the aptamer can inhibit the activity of a given target molecule by binding to a given target molecule.
  • the aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and the form may be linear or cyclic.
  • the antibody of the present invention can be prepared by a conventional method after obtaining a protein encoded by the gene by cloning the PCBP1 gene, for example, the nucleotide sequence represented by SEQ ID NO: 2 into an expression vector according to a conventional method.
  • the aptamer can be easily prepared according to a known method by a person of ordinary skill in the art with reference to the nucleotide sequence.
  • the agent capable of measuring the expression level of the gene of the present invention is a gene transcribed from the gene to be measured, for example, a gene transcribed from the gene to be measured, in order to confirm the expression level of the gene to be measured included in the sample. It refers to an agent that can be used in a method of measuring the level of mRNA.
  • the formulation is RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR (Competitive RT-PCR), real time quantitative RT-PCR (real time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blot assay, DNA chip analysis, and the like may include primer pairs or probes capable of specifically binding to the target mRNA to be measured, but are limited thereto. no.
  • the "primer” of the present invention is a base sequence having a short free 3'hydroxyl group and can form a complementary template strand and a base pair, and the start for template strand copying It refers to a short nucleotide sequence that functions as a point.
  • the primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.
  • the "probe” of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a base capable of specifically binding to the gene or mRNA transcribed from the gene, and such a probe is in the presence of a specific mRNA It may be labeled so that the presence or absence and level of expression (expression amount) can be confirmed.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, etc., but is not limited thereto.
  • the primer or probe of the present invention can be easily prepared by a method known by a person of ordinary skill in the art with reference to the known base sequence of PCBP1, for example, the base sequence represented by SEQ ID NO: 2.
  • kits for diagnosis of invasive brain cancer comprising the composition for diagnosis of invasive brain cancer of the present invention.
  • the diagnostic kit of the present invention uses the composition for diagnosis of invasive brain cancer, and when the expression level of the PCBP1 protein or a gene encoding it in a biological sample of a target individual is reduced compared to a normal control group, the invasiveness of brain cancer may be high. Can be predicted very effectively.
  • the PCBP1 protein and the gene encoding it are as described in the diagnostic composition. It is redundant, and it is omitted to avoid excessive complexity of the present specification.
  • the kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA (Enzyme-linked immunosorbent assay) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit, but is limited thereto. It is not.
  • kits of the present invention are for measuring the expression level of a gene
  • a test tube or other suitable container reaction buffer (various pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase and The same enzyme, DNase, RNAse inhibitor, DEPC-water, sterilized water, and the like may be further included, but are not limited thereto.
  • kit of the present invention is for measuring the expression level of a protein
  • a substrate a suitable buffer solution
  • a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, etc. may be included for the immunological detection of the antibody.
  • the substrate of the present invention may be a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin or a polystyrene resin, and a slide glass made of glass, and the coloring enzyme is peroxidase, alkaline phosphatase (Alkaline). Phosphatase) can be used, fluorescent materials can be used, such as FITC, RITC, etc., and the color developing substrate solution is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), OPD ( o-phenylenediamine), TMB (tetramethyl benzidine) may be used, but is not limited thereto.
  • the coloring enzyme is peroxidase, alkaline phosphatase (Alkaline). Phosphatase) can be used, fluorescent materials can be used, such as FITC, RITC, etc.
  • the color developing substrate solution is ABTS (2,2
  • Another embodiment of the present invention provides a method of providing information for predicting invasive brain cancer.
  • the information providing method of the present invention includes the step of measuring the expression level of the PCBP1 protein or a gene encoding it in a biological sample isolated from a target individual.
  • the expression level of the PCBP1 protein or the gene encoding it measured in a biological sample isolated from the object of the present invention is lower than that of the normal control, it can be predicted that the brain cancer of the object of interest has high invasiveness.
  • the "normal control" of the present invention may be an individual that has not been diagnosed with brain cancer or an individual that has not been confirmed to have invaded cancer cells to other organs, preferably, it has been confirmed that the invasion of cancer cells to other organs has not occurred. It may be an individual, but is not limited thereto.
  • the PCBP1 protein and the gene encoding it, invasiveness, brain cancer, and the like are overlapped with those described in the diagnostic composition, and are omitted in order to avoid excessive complexity of the present specification.
  • the step of measuring the expression level of the PCBP1 protein of the present invention can be performed by using the agent for measuring the expression level of the protein of the present invention, Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, It can be measured through at least one selected from the group consisting of immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, MRM analysis, and protein microarray.
  • the step of measuring the expression level of the PCBP1 gene of the present invention is a polymerase chain reaction, a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, and a Nuclease protection assay using the agent for measuring the expression level of the gene of the present invention. It may be measured through at least one selected from the group consisting of (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, and Northern blot.
  • the biological sample of the present invention refers to any substance, biological body fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells ( peripheral blood mononuclear cells), leukocyte buffy coat, blood including plasma and serum, sputum, tears, mucus, nasal washes, nasal cavity Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, Meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate aspirate), synovial fluid, joint aspirate, organ secretions, cells, cell extracts, cerebrospinal fluid, etc., but are limited thereto. no.
  • Another embodiment of the present invention provides a composition for predicting the prognosis of brain cancer.
  • composition for predicting prognosis of the present invention includes an agent for measuring the expression level of the PCBP1 protein or a gene encoding it.
  • the invasiveness of brain cancer when the expression level of the PCBP1 protein or the gene encoding it is decreased, the invasiveness of brain cancer is increased, and through this, the overall survival rate of the individual with brain cancer may be significantly reduced. It was confirmed (see Figs. 5 A and B).
  • the expression level of the PCBP1 protein or the gene encoding it when the expression level of the PCBP1 protein or the gene encoding it is decreased compared to the normal control, the invasiveness of brain cancer is increased, and accordingly, the overall survival rate of the individual with brain cancer is significantly reduced, and the prognosis It can be predicted very effectively that may be bad.
  • the PCBP1 protein and the gene encoding it are as described in the diagnostic composition. It is redundant, and it is omitted to avoid excessive complexity of the present specification.
  • Another embodiment of the present invention provides a screening method for a therapeutic agent for invasive brain cancer.
  • the screening method of the present invention comprises the steps of treating a target candidate material in a biological sample isolated from a brain cancer patient; And checking the expression level of the PCBP1 protein or a gene encoding it in the biological sample.
  • the step of selecting as a therapeutic agent for invasive brain cancer may be further included.
  • the step of measuring the expression level of the PCBP1 protein and the gene encoding it, brain cancer, and the protein, measuring the expression level of the gene, the biological sample, etc. are used for predicting the diagnostic composition and invasive brain cancer. As described in the method for providing information, it is omitted to avoid excessive complexity of the present specification.
  • the candidate substance of the present invention refers to a drug for testing whether there is an activity for treating invasive brain cancer, that is, an activity capable of increasing the expression of the PCBP1 protein or a gene encoding it, protein, oligopeptide, Includes any molecule such as organic molecules, polysaccharides, polynucleotides and a wide range of compounds. These candidate substances may include not only natural substances but also synthetic substances.
  • the composition of the present invention since the invasiveness of the target individual can be predicted very effectively, an appropriate treatment method, etc., is provided to the patient The cost can be significantly reduced. Further, when the expression level of the PCBP1 protein or the gene encoding it is decreased, the invasiveness may increase and the overall survival rate may be reduced, so that the prognosis of brain cancer can be predicted very effectively.
  • FIGS. 1A to 1C show the results of confirming the formation of glioblastoma tumor cells with a microscope according to an embodiment of the present invention (A and B of FIGS. 1) and the invasiveness of glioblastoma tumor cells through a Matrigel analysis method. It shows the confirmed result (FIG. 1C).
  • 2A and 2B show a gene profiling analysis method for gene expression levels between high invasive (High inv.) glioblastoma tumor cells and low invasive (Low inv.) glioblastoma tumor cells according to an embodiment of the present invention. It shows the result of comparison through.
  • 3A and 3B show the results of confirming the interaction of genes related to invasiveness and mutation rates of the genes according to an embodiment of the present invention.
  • FIG. 4A and 4B show the results of confirming the formation of glioblastoma tumor cells according to an embodiment of the present invention (Fig. 4A) and the results of confirming the invasiveness of glioblastoma tumor cells (Fig. 4B), and Fig. 4 C shows the result of confirming the expression level of the protein in the glioblastoma tumor cell according to an embodiment of the present invention through Western blot analysis.
  • 5A and B show the results of analyzing the survival rate of glioblastoma tumor cell transplant mice and glioblastoma patients.
  • a composition for diagnosing invasive glioblastoma comprising an agent for measuring the expression level of a PCBP1 (Poly(RC) Binding Protein 1) protein or a gene encoding the same; And it provides a composition for predicting the prognosis of glioblastoma.
  • PCBP1 Poly(RC) Binding Protein 1
  • kits for diagnosing invasive glioblastoma comprising a composition for diagnosing invasive glioblastoma.
  • Another preferred embodiment of the present invention provides a method of providing information for predicting invasive glioblastoma, comprising measuring the expression level of the PCBP1 protein or a gene encoding it in a biological sample isolated from a target individual.
  • Tissues of 23 newly diagnosed patients (15 low invasiveness, 8 high invasiveness) without treatment such as surgical operation, chemotherapy or radiotherapy were used in this study.
  • MR images were taken within 7 days using the Achieva 3.0T system (Philips Medical Systems) before proceeding with surgery to remove cancer tissue from each patient.
  • Axial images were taken by paralleling to the anterior and posterior edges of the corpus callosum. In the case of tumor tissue invasion, it was quantified as the area occupied by T2 FLAIR-T1 contrast enhanced (CE)/T1 CE.
  • CE contrast enhanced
  • TS tumor spheres
  • DMEM/F12 Mediatech, USA
  • 1 ⁇ B27 Invitrogen, USA
  • 20 ng/mL bFGF 20 ng/mL EGF (Sigma-Aldrich, USA)
  • 37° C. 5% CO 2 was cultured under conditions.
  • TS14-15 a low-invasive glioblastoma tumor cell, isolated in Preparation Example 2 in the right frontal lobe of the mouse to a depth of 4.5 mm (hereinafter,'TS14-15 ') or TS13-64 (hereinafter referred to as'TS13-64'), a highly invasive glioblastoma tumor cell, was transplanted.
  • TS13-64' a highly invasive glioblastoma tumor cell
  • D-luciferin (D-luciferin, 30 mg / mL; Promega) was injected intraperitoneally of the mouse, and anesthesia using 2.5% isoflurane It was carried out in the state. All of these experimental procedures were approved by the Animal Care and Use Committee of Yonsei University Medical School.
  • the case of glioblastoma tumor cells was divided into a case with low invasiveness (Low inv.) and a case with high invasiveness (High inv.).
  • TS13-03, TS13-20, TS13-30, TS13-64, TS13-71, TS15-67, TS15-88 and TS16-117 had high invasiveness, among which TS15-88 and TS13-64 Had the highest invasiveness.
  • ssGSEA Single sample gene set enrichment analysis
  • the gene expression profile of glioblastoma tumor cells was applied to ssGSEA using a set of transcriptional factor (TF) target genes retrieved from MSigDB c3.tft v5.1.
  • TF transcriptional factor
  • the enrichment scores calculated with the same transcription factor were averaged to obtain a single value for each transcription factor, and this averaged score was quantitatively standardized in all samples.
  • the difference between the low invasive glioblastoma tumor cell group and the high invasive glioblastoma tumor cell group (two-tailed Student's t-test) identified in Example 1 was compared.
  • the transcription factor identified as having a difference between the two groups was configured as a network map using Cytoscape and Reactome FI 9 plug-in, and functional interaction analysis was performed. .
  • STAT3 Signal transducer and activator of transcription 3
  • SPI1 high inv.
  • SBFA2T SBF activated by Bck2
  • PAX8 Paned box gene 8
  • BACH1/2 Transcription regulator protein BACH1
  • SRF Serum response factor
  • PTF1A Pancreas transcription factor 1 subunit alpha
  • PRRX2 Pigd Related Homeobox 2
  • TCF7 Transcription factor 7
  • SRY Sex-determining Region Y
  • SOX5 SRY-related HMG-box 5
  • PCBP1 Poly(RC) Binding Protein 1
  • the mutation (miss sense, deletion, and amplification) rate of the transcription factor was not high, so that glioblastoma invasion was prevented. It can be seen that the change of the involved transcription factor was changed by external factors.
  • the expression level of the PCBP1 transcription factor according to the present invention is reduced, the invasiveness of brain cancer, for example, glioblastoma, particularly glioblastoma, tumor cells, is high. Furthermore, it can be seen that the PCBP1 protein or a gene encoding it can induce an increase in the invasiveness of brain cancer.
  • STAT3 and PCBP1 genes were silenced in glioblastoma tumor cells, and then the invasiveness and protein expression levels were confirmed.
  • TS14-15 and TS13-64 were cultured for 24 hours. Then, the tumor cells were transformed with STAT3 or PCBP1 specific siRNA double strands (Bionia, Korea) using Lipofectamine 3000 (Invitrogen, USA). At this time, a random siRNA sequence was used as a negative control. After incubation for 72 hours after transformation, as in Example 1, the analysis of whether a sphere was formed and invasiveness was performed, and the results are shown in A and B of FIG. 4.
  • the protein isolated from the tumor cell was electrophoresed on a 10% tris-glycine gel, and then transferred to a nitrocellulose membrane, followed by STAT3 (Cell Signaling Technology), ⁇ -catenin (Cell Signaling Technology), PCBP1 (Abcam), N-cadherin (R&D Systems), Zeb1 (Sigma-Aldrich); Western blot analysis was performed using antibodies specific for SRF (Santa Cruz Biotechnology) and GAPDH (Santa Cruz Biotechnology), and the results are shown in C of FIG. 4.
  • STAT3 Cell Signaling Technology
  • ⁇ -catenin Cell Signaling Technology
  • PCBP1 Abcam
  • N-cadherin R&D Systems
  • Zeb1 Sigma-Aldrich
  • Western blot analysis was performed using antibodies specific for SRF (Santa Cruz Biotechnology) and GAPDH (Santa Cruz Biotechnology), and the results are shown in C of FIG. 4.
  • N-cadherin, ⁇ - which are proteins associated with invasion and mesenchymal transition in all glioblastoma tumor cells.
  • the expression levels of catenin and Zeb1 proteins were increased.
  • the PCBP1 protein or the gene encoding it according to the present invention has a function of inhibiting metastasis of brain cancer, for example, glioblastoma, particularly glioblastoma tumor cells, and the protein or protein encoding the same It can be seen that when the expression level is present at a lower level than that of the normal control, high invasiveness can be expected.
  • the composition of the present invention since the invasiveness of the target individual can be predicted very effectively, an appropriate treatment method, etc., is provided to the patient The cost can be significantly reduced. Further, when the expression level of the PCBP1 protein or the gene encoding it is decreased, the invasiveness may increase and the overall survival rate may be reduced, so that the prognosis of brain cancer can be predicted very effectively.
  • SEQ ID NO: 1 amino acid sequence
  • SEQ ID NO: 2 gene sequence

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Abstract

The present invention relates to a composition for the diagnosis of invasive brain cancer. In the case of measuring an expression level of the PCBP1 protein or a gene encoding same by using the composition of the present invention, the composition enables very effective prediction of the invasiveness for a targeted individual, and thus provides appropriate treatment methods and the like to patients, and accordingly, can remarkably reduce social costs incurred by such treatments. Furthermore, when the expression level of the PCBP1 protein or a gene encoding same is reduced, invasiveness increases, resulting in a reduction in the overall survival rate, and thus the prognosis of brain cancer can be very effectively predicted.

Description

침윤성 뇌암의 진단용 조성물Composition for diagnosis of invasive brain cancer

본 발명은 침윤성 뇌암의 진단용 조성물에 관한 것이다.The present invention relates to a composition for diagnosis of invasive brain cancer.

암은 전세계적으로 가장 보편적인 사망원인 중의 하나이다. 약 천만 건의 새로운 케이스가 매년 발생하며, 전체 사망원인의 약 12%를 차지하여 세 번째로 많은 사망의 원인이 되고 있다. 여러 가지 종류의 암 중에서 특히 뇌암은 연령에 관계없이 발생되며, 소아에 발생 빈도가 다른 암에 비하여 높은 특징이 있다. 상기 뇌암은 뇌 조직과 뇌를 싸고 있는 뇌막에서 발생되는 원발성 뇌암과 두개골이나 신체의 다른 부위에서 발생된 암으로부터 전이된 이차성 뇌암을 통칭하여 일컫는 것으로, 다른 장기에서 발생되는 암과 다른 특징을 나타낸다.Cancer is one of the most common causes of death worldwide. About 10 million new cases occur every year, accounting for about 12% of all causes of death, making it the third-most cause of death. Among various types of cancer, brain cancer in particular occurs regardless of age, and has a higher incidence in children than other cancers. The brain cancer is collectively referred to as primary brain cancer that occurs in the brain tissue and the meninges surrounding the brain, and secondary brain cancer that has metastasized from cancer that occurs in the skull or other parts of the body, and exhibits different characteristics from cancer that occurs in other organs.

신경교종(Glioma)은 원발성 뇌 암(Primary brain tumor)의 60%를 차지하는 종양으로서, 발생 빈도가 높고 치료가 어려워, 현재까지도 방사선 치료 외엔 특별한 치료법이 없는 악성 종양에 해당한다. 신경교종 중에서 특히 교모세포종(GBM; Glioblastoma multiforme)은 인간에서 나타나는 120 가지의 다양한 유형의 원발성 뇌암 중에서 가장 보편적으로 나타나는 형태이며, 공격적인 악성 원발성 뇌암으로서, 신경교세포와 관련된 모든 두개내 종양 중에서 가장 많은 부분을 차지한다. 교모세포종의 경우 유럽 및 북아메리카에서 100,000명 당 2 ~ 3건 정도 발병된다.Glioma is a tumor that accounts for 60% of primary brain tumors, and it is a malignant tumor with high incidence and difficult treatment, and there is still no special treatment other than radiation therapy. Among gliomas, glioblastoma multiforme (GBM) is the most common form among 120 different types of primary brain cancer in humans, and is an aggressive malignant primary brain cancer, the largest part of all intracranial tumors related to glial cells. Occupy. In the case of glioblastoma, 2 to 3 cases per 100,000 people occur in Europe and North America.

한편, 상기 뇌암의 경우, 뇌혈관 장벽(Brain Blood Barrier)이 존재하기 때문에 치료를 위한 약물 전달이 목적하는 뇌 부위로 전달되기 어려울 뿐만 아니라, 상대적으로 뇌신경 생물학에 대한 이해가 부족하여 치료제의 개발이 활발하지 못한 것이 현실이다. 더욱이, 교모세포종은 다른 뇌암과 비교해볼 때 공격적 변이(Aggressive variant)를 나타내어, 이를 빠른 시일 내에 치료하지 않으면 몇 주 이내에 치명적인 결과를 초래할 수 있다. 나아가, 상기 교모세포종의 경우 두개강 외에 전이되는 경향성이 매우 높아 다른 뇌암에 비하여 더욱 공격적이라는 특징을 갖는다.On the other hand, in the case of the brain cancer, because the brain blood barrier exists, it is difficult to deliver drugs for treatment to the target brain region, and development of a therapeutic agent is difficult due to a relatively lack of understanding of cranial nerve biology. The reality is that they are not active. Moreover, glioblastoma exhibits an aggressive variant compared to other brain cancers, and if not treated as soon as possible, it can lead to fatal results within a few weeks. Furthermore, the glioblastoma has a very high tendency to metastasize outside the cranial cavity, and is more aggressive than other brain cancers.

현재까지 상기 교모세포종의 치료 방법으로는 동시 또는 순차적인 화학요법, 방사선요법, 항혈관형성 요법, 면역요법, 감마 나이프 방사선 수술 및 코르티코 스테로이드를 이용한 대증 치료뿐만 아니라, 절제술과 같은 치료 방법이 존재한다. 그럼에도 불구하고 교모세포종의 경우 중추 신경계 악성 종양 중에서 예후가 매우 나쁘며, 교모세포종 환자의 경우 평균 생존율이 1년 밖에 되지 않을 뿐만 아니라, 환자의 약 3%만이 3년 이상 생존한다는 한계점이 존재한다. 이에 따라, 뇌암을 조기에 진단하고 예후를 예측할 수 있는 새로운 진단 및 치료 표적에 대한 연구가 필요한 실정이다.Until now, treatment methods for glioblastoma include simultaneous or sequential chemotherapy, radiation therapy, antiangiogenic therapy, immunotherapy, gamma knife radiation surgery, and symptomatic treatment using corticosteroids, as well as treatment methods such as resection. . Nevertheless, glioblastoma has a very poor prognosis among central nervous system malignancies, and glioblastoma patients have an average survival rate of only 1 year, and only about 3% of patients survive for 3 years or more. Accordingly, there is a need for research on new diagnostic and therapeutic targets that can diagnose brain cancer early and predict the prognosis.

본 발명의 일 목적은 침윤성 뇌암의 진단용 조성물 및 이를 포함하는 키트를 제공하는 것이다.An object of the present invention is to provide a composition for diagnosis of invasive brain cancer and a kit including the same.

본 발명의 다른 목적은 침윤성 뇌암의 진단을 위한 정보 제공 방법을 제공하는 것이다.Another object of the present invention is to provide a method of providing information for diagnosis of invasive brain cancer.

본 발명의 또 다른 목적은 침윤성 뇌암의 예후 예측용 조성물 및 이를 포함하는 키트를 제공하는 것이다.Another object of the present invention is to provide a composition for predicting the prognosis of invasive brain cancer and a kit containing the same.

본 발명의 또 다른 목적은 침윤성 뇌암의 예후 예측을 위한 정보 제공 방법을 제공하는 것이다.Another object of the present invention is to provide a method of providing information for predicting the prognosis of invasive brain cancer.

본 발명의 또 다른 목적은 침윤성 뇌암의 치료제를 스크리닝하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening a therapeutic agent for invasive brain cancer.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned may be clearly understood by those of ordinary skill in the art from the following description.

본 발명의 일 구현 예예서는 침윤성 뇌암의 진단용 조성물을 제공한다.One embodiment of the present invention provides a composition for diagnosis of invasive brain cancer.

본 발명의 상기 조성물은 PCBP1(Poly(RC) Binding Protein 1) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 포함한다.The composition of the present invention includes an agent for measuring the expression level of the PCBP1 (Poly(RC) Binding Protein 1) protein or a gene encoding the same.

본 발명의 상기 PCBP1 단백질은 인간의 2번 염색체(70.17-70.17 Mb) 상에 위치하는 유전자에 의해 암호화되는 단백질로서, 주요 세포성 폴리(rC)-결합 단백질을 형성하는 기능을 갖는다. 상기 PCBP1 단백질이 높은 수준으로 존재하는 경우, 유방암 및 난소암 환자의 침윤성이 높을 수 있다(IUBMB Life., 2019 Feb;71(2):177-182.). 반면, 침윤성이 낮은 뇌암 환자와 비교하여, 침윤성이 높은 뇌암 환자에서 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 현저하게 낮아져 있는 것을 확인하였다(도 2 참고). 본 발명의 목적상 상기 조성물을 이용하여 목적하는 개체에서, 정상 대조군(뇌암이 없는 것으로 확인된 개체 또는 침윤성이 낮은 뇌암이 있는 개체)과 비교하여 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 낮아져 있는 경우에는 침윤성 뇌암으로 예측할 수 있다.The PCBP1 protein of the present invention is a protein encoded by a gene located on human chromosome 2 (70.17-70.17 Mb), and has a function of forming a major cellular poly(rC)-binding protein. When the PCBP1 protein is present at a high level, the invasiveness of breast and ovarian cancer patients may be high ( IUBMB Life ., 2019 Feb;71(2):177-182.). On the other hand, it was confirmed that the expression level of the PCBP1 protein or the gene encoding it was significantly lowered in brain cancer patients with high invasiveness compared to brain cancer patients with low invasiveness (see FIG. 2). For the purposes of the present invention, in an individual of interest using the composition, the expression level of the PCBP1 protein or the gene encoding it is lowered compared to a normal control (an individual confirmed to be free from brain cancer or an individual with low invasive brain cancer) In some cases, it can be predicted as invasive brain cancer.

본 발명의 상기 PCBP1 단백질은 서열번호 1로 표시되는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.The PCBP1 protein of the present invention may consist of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

본 발명의 상기 단백질을 암호화하는 유전자는 서열번호 2로 표시되는 염기 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.The gene encoding the protein of the present invention may be composed of a nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

본 발명의 상기 PCBP1 단백질은 서열번호 1로 표시되는 아미노산 서열과 최소한 70%의 서열 상동성을 갖는 아미노산 서열로 구성되어, 아미노산 서열의 자연적 또는 인위적인 부가, 결실, 치환 또는 조합된 폴리 펩타이드도 모두 포함되는 것으로서, 예를 들면, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 또는 99%의 서열 상동성을 갖는 아미노산 서열일 수 있으나, 이에 제한되는 것은 아니다.The PCBP1 protein of the present invention is composed of an amino acid sequence having at least 70% sequence homology with the amino acid sequence represented by SEQ ID NO: 1, and includes all of the polypeptides that are naturally or artificially added, deleted, substituted or combined with the amino acid sequence. As to be, for example, may be an amino acid sequence having sequence homology of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, but is limited thereto no.

본 발명의 상기 "상동성"이란, 아미노산 서열 간의 유사한 정도를 나타내기 위한 것으로서, 본 발명의 아미노산 서열과 상기와 같은 퍼센트 이상의 동일한 서열을 가지는 서열을 포함한다. 이러한 상동성은 두 서열을 육안으로 비교하여 결정할 수도 있으나, 비교대상이 되는 서열을 나란히 배열하여 상동성 정도를 분석해 주는 생물정보 알고리즘(Bioinformatic algorithm)을 사용하여 결정할 수 있다. 상기 두 개의 아미노산 서열 사이의 상동성은 백분율로 표시될 수 있다. 유용한 자동화된 알고리즘은 Wisconsin Genetics Software Package(Genetics Computer Group, Madison, W, USA)의 GAP, BESTFIT, FASTA와 TFASTA 컴퓨터 소프트웨어 모듈에서 이용 가능하다. 상기 모듈에서 자동화된 배열 알고리즘은 Needleman & Wunsch와 Pearson & Lipman과 Smith & Waterman 서열 배열 알고리즘을 포함한다. 다른 유용한 배열에 대한 알고리즘과 상동성 결정은 FASTP, BLAST, BLAST2, PSIBLAST 및 CLUSTAL W를 포함하는 소프트웨어에서 자동화될 수 있다.The "homology" of the present invention is intended to indicate the degree of similarity between amino acid sequences, and includes sequences having the same percent or more of the same sequence as the amino acid sequence of the present invention. Such homology can be determined by visually comparing two sequences, but can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. Homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Alignment algorithms automated in the module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithms and homology determination for other useful alignments can be automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

본 발명의 상기 "뇌암"이란, 두개강 내에서 발생된 모든 종양을 의미하는 것으로서, 예를 들면, 신경교종일 수 있으나, 이에 제한되는 것은 아니다.The term "brain cancer" of the present invention refers to all tumors occurring in the cranial cavity, and may be, for example, a glioma, but is not limited thereto.

본 발명의 상기 "신경교종"은 원발성 뇌 종양(primary brain tumor)의 60%를 차지하는 종양으로서, 발생 빈도가 높고 치료가 어려워, 현재까지도 방사선 치료 외엔 특별한 치료법이 없는 악성 종양으로서, 예를 들면, 성상세포종, 교모세포종 또는 핍지교종 등 일 수 있고, 바람직하게는 교모세포종일 수 있으나, 이에 제한되는 것은 아니다.The "glioma" of the present invention is a tumor that accounts for 60% of primary brain tumors, has a high incidence and is difficult to treat, and is a malignant tumor that does not have any special treatment other than radiation therapy, for example, It may be an astrocytoma, glioblastoma, or oligodendrogliomas, preferably glioblastoma, but is not limited thereto.

본 발명의 상기 뇌암은 암 종양구인 것일 수 있으나, 이에 제한되는 것은 아니다.The brain cancer of the present invention may be a cancer tumor cell, but is not limited thereto.

본 발명의 상기 "암 종양구"란, 암 조직을 구성하는 세포 중 줄기세포(Stem cell)와 유사한 특성을 가지는 일부의 세포가 3차원 배양 조건에서 형성될 수 있는 세포 응집체를 의미한다.The "cancer tumor cell" of the present invention refers to a cell aggregate in which some cells having characteristics similar to stem cells among cells constituting cancer tissues can be formed under three-dimensional culture conditions.

본 발명의 상기 "침윤성"이란, 원발 장기에서 발생된 종양 세포가 암이 진행됨에 따라 전이(metastasis)에 필요한 새로운 유전 형질을 획득한 뒤, 혈관과 림프선으로 침윤(Invasion)되는 현상으로서, 이와 같은 침윤성 높은 경우에는 혈관과 림프선으로 침윤된 종양 세포가 림프를 따라 순환함으로써 원발 장기로부터 다른 장기에 존재하는 조직에 정착한 뒤 증식됨으로써 궁극적으로 다른 장기에 암이 쉽게 전이될 수 있다.The "invasive" of the present invention is a phenomenon in which tumor cells generated in a primary organ acquire a new genetic trait necessary for metastasis as cancer progresses, and then invasion into blood vessels and lymph glands. In the case of high invasiveness, tumor cells infiltrated by blood vessels and lymph glands circulate along the lymph, settling in tissues existing in other organs from the primary organ, and then proliferating, so that cancer can easily metastasize to other organs.

본 발명의 상기 단백질을 암호화하는 유전자의 발현 수준을 측정하는 제제는 시료에 포함된 측정 대상이 되는 유전자로부터 암호화되는 단백질의 발현 수준을 측정하기 위한 제제를 의미한다.The preparation for measuring the expression level of the gene encoding the protein of the present invention refers to a preparation for measuring the expression level of the protein encoded from the target gene included in the sample.

상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합할 수 있는 항체 또는 앱타머일 수 있다. 구체적으로, 상기 제제는 웨스턴 블럿 분석(Western blot assay), ELISA(Enzyme linked immunosorbent assay), 방사선면역분석 (RIA: Radioimmunoassay), 방사 면역 확산법(Radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트 면역전기영동(Rocket immunoelectrophoresis), 면역조직화학염색법(Immunohistochemical staining), 면 역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 면역형광법 (Immunofluorescence), 면역크로마토그래피법(Immunochromatography), FACS(Fluorescenceactivated cell sorter analysis) 및 단백질 칩 분석법(protein chip technology assay) 등의 방법에 사용되는 항체 또는 앱타머를 포함할 수 있으나, 이에 제한되는 것은 아니다.The agent for measuring the expression level of the protein may be an antibody or aptamer capable of specifically binding to the protein. Specifically, the formulation is Western blot assay, ELISA (Enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket Rocket immunoelectrophoresis, Immunohistochemical staining, Immunoprecipitation Assay, Complement Fixation Assay, Immunofluorescence, Immunochromatography, FACS Antibodies or aptamers used in methods such as (Fluorescenceactivated cell sorter analysis) and protein chip technology assay may be included, but are not limited thereto.

본 발명의 상기 “항체”는 단백질 또는 펩티드 분자의 항원성 부위에 특이적으로 결합할 수 있는 단백질 분자를 의미한다. 상기 항체의 형태는 특별히 제한되지 않으며 폴리 클로날 항체, 모노 클로날 항체 또는 항원 결합성을 갖는 것이라면, 항체의 일부인 경우라도 포함될 수 있고, 모든 종류의 면역 글로불린 항체가 포함될 수 있다. 또한, 인간화 항체 등의 특수 항체가 포함될 수 있고, 상기 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합 기 능을 보유하고 있는 단편을 의미하며 Fab, F(ab'), F(ab') 2, Fv 등이 될 수 있으나, 이에 제한되는 것은 아니다.The "antibody" of the present invention means a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited, and as long as it has a polyclonal antibody, a monoclonal antibody, or an antigen binding property, it may be included even if it is part of the antibody, and all kinds of immunoglobulin antibodies may be included. In addition, special antibodies such as humanized antibodies may be included, and the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. The functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

본 발명의 상기 “앱타머”는 단일 가닥 올리고 뉴클레오티드를 의미하는 것으로, 소정의 표적 분자에 대한 결합 활성을 갖는 핵산 분자를 말한다. 상기 앱타머는 그 염기 서열에 따라 다양한 3차원 구조를 가질 수 있으며, 항원-항체 반응과 같이 특정 물질에 대하여 높은 친화력을 가질 수 있다. 앱타머는 소정의 표적 분자에 결합함으로써 소정의 표적 분자의 활성을 저해할 수 있다. 상기 앱타머는 RNA, DNA, 변형된(Modified) 핵산 또는 이들의 혼합물일 수 있으며, 그 형태가 직쇄상 또는 환상일 수 있다. The "aptamer" of the present invention refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may have various three-dimensional structures according to its base sequence, and may have high affinity for a specific substance such as an antigen-antibody reaction. The aptamer can inhibit the activity of a given target molecule by binding to a given target molecule. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and the form may be linear or cyclic.

본 발명의 상기 항체는, PCBP1 유전자, 예를 들면 서열번호 2로 표시되는 염기 서열을 통상적인 방법에 따라 발현 벡터에 클로닝하여 상기 유전자에 의해 암호화되는 단백질을 얻은 뒤에 통상적인 방법에 의해 제조될 수 있고, 상기 앱타머는 상기 염기 서열을 참조하여 당해 기술 분야에서 통상의 지식을 가진 자가 공지의 방법에 따라 쉽게 제작할 수 있다.The antibody of the present invention can be prepared by a conventional method after obtaining a protein encoded by the gene by cloning the PCBP1 gene, for example, the nucleotide sequence represented by SEQ ID NO: 2 into an expression vector according to a conventional method. In addition, the aptamer can be easily prepared according to a known method by a person of ordinary skill in the art with reference to the nucleotide sequence.

본 발명의 상기 유전자의 발현 수준을 측정할 수 있는 제제는 시료에 포함된 측정 대상이 되는 유전자의 발현 수준을 확인하기 위하여, 상기 측정 대상이 되는 유전자로부터 전사된 유전자, 예를 들면 유전자로부터 전사된 mRNA의 수준을 측정하는 방법에 사용될 수 있는 제제를 의미한다. 구체적으로, 상기 제제는 RT-PCR, 정량 실시간 PCR(quantified real time PCR), 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR(real time quantitative RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블럿 분석(Northern blot assay), DNA 칩 분석법 등의 방법에 사용되는 측정 대상이 되는 mRNA에 특이적으로 결합할 수 있는 프라이머 쌍 또는 프로브를 포함할 수 있으나, 이에 제한되는 것은 아니다.The agent capable of measuring the expression level of the gene of the present invention is a gene transcribed from the gene to be measured, for example, a gene transcribed from the gene to be measured, in order to confirm the expression level of the gene to be measured included in the sample. It refers to an agent that can be used in a method of measuring the level of mRNA. Specifically, the formulation is RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR (Competitive RT-PCR), real time quantitative RT-PCR (real time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blot assay, DNA chip analysis, and the like may include primer pairs or probes capable of specifically binding to the target mRNA to be measured, but are limited thereto. no.

본 발명의 상기 “프라이머”는 짧은 자유 3말단 수산화기(free 3'hydroxyl group)를 가지는 염기 서열로 상보적인 주형 가닥(template)와 염기쌍(base pair)을 형성할 수 있고, 주형 가닥 복사를 위한 시작 지점으로서 기능을 하는 짧은 염기 서열을 의미한다. 상기 프라이머는 적절한 완충 용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재 하에서 DNA 합성이 개시될 수 있다.The "primer" of the present invention is a base sequence having a short free 3'hydroxyl group and can form a complementary template strand and a base pair, and the start for template strand copying It refers to a short nucleotide sequence that functions as a point. The primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

본 발명의 상기 “프로브”는 상기 유전자, 또는 상기 유전자로부터 전사되는 mRNA와 특이적으로 결합을 이룰 수 있는 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하고, 이와 같은 프로브는 특정 mRNA의 존재 유무, 발현되는 수준(발현량)을 확인할 수 있도록 라벨링되어 있을 수 있다. 상기 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single strand DNA) 프로브, 이중쇄 DNA(double strand DNA)프로브, RNA 프로브 등의 형태로 제작될 수 있으나, 이에 제한되는 것은 아니다.The "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a base capable of specifically binding to the gene or mRNA transcribed from the gene, and such a probe is in the presence of a specific mRNA It may be labeled so that the presence or absence and level of expression (expression amount) can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, etc., but is not limited thereto.

본 발명의 상기 프라이머 또는 프로브는 공지된 PCBP1의 염기 서열, 예를 들면 서열번호 2로 표시되는 염기 서열을 참조하여 당해 기술 분야에서 통상이 지식을 가진 자가 공지된 방법에 의해 쉽게 제작될 수 있다.The primer or probe of the present invention can be easily prepared by a method known by a person of ordinary skill in the art with reference to the known base sequence of PCBP1, for example, the base sequence represented by SEQ ID NO: 2.

본 발명의 다른 구현 예에서는 본 발명의 상기 침윤성 뇌암의 진단용 조성물을 포함하는 침윤성 뇌암의 진단용 키트를 제공한다.Another embodiment of the present invention provides a kit for diagnosis of invasive brain cancer comprising the composition for diagnosis of invasive brain cancer of the present invention.

본 발명의 상기 진단용 키트는 상기 침윤성 뇌암의 진단용 조성물을 사용하여 정상 대조군에 비하여 목적하는 개체의 생물학적 시료에서 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 감소되어 있는 경우, 뇌암의 침윤성이 높을 수 있음을 매우 효과적으로 예측할 수 있다.The diagnostic kit of the present invention uses the composition for diagnosis of invasive brain cancer, and when the expression level of the PCBP1 protein or a gene encoding it in a biological sample of a target individual is reduced compared to a normal control group, the invasiveness of brain cancer may be high. Can be predicted very effectively.

본 발명의 상기 진단용 키트에서, PCBP1 단백질 및 이를 암호화하는 유전자, 침윤성, 뇌암, 단백질의 발현 수준을 측정할 수 있는 제제, 유전자의 발현 수준을 측정할 수 있는 제제 등은 상기 진단용 조성물에서 기재한 바와 중복되어, 이하 본 명세서의 과도한 복잡성을 피하기 위하여 생략한다.In the diagnostic kit of the present invention, the PCBP1 protein and the gene encoding it, invasiveness, brain cancer, a formulation capable of measuring the expression level of the protein, a formulation capable of measuring the expression level of the gene, etc. are as described in the diagnostic composition. It is redundant, and it is omitted to avoid excessive complexity of the present specification.

본 발명의 상기 키트는 RT-PCR 키트, DNA 칩 키트, ELISA(Enzyme-linked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(Rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트 등 일 수 있으나, 이에 제한되는 것은 아니다.The kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA (Enzyme-linked immunosorbent assay) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit, but is limited thereto. It is not.

본 발명의 상기 키트가 유전자의 발현 수준을 측정하기 위한 경우에는 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNAse 억제제, DEPC-수(DEPC-water), 멸균수 등이 더 포함될 수 있으나, 이에 제한되는 것은 아니다.When the kit of the present invention is for measuring the expression level of a gene, a test tube or other suitable container, reaction buffer (various pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase and The same enzyme, DNase, RNAse inhibitor, DEPC-water, sterilized water, and the like may be further included, but are not limited thereto.

본 발명의 상기 키트가 단백질의 발현 수준을 측정하기 위한 경우에는 항체의 면역학적 검출을 위하여 기재, 적당한 완충 용액, 발색 효소 또는 형광물질로 표지된 2차 항체, 발색 기질 등이 포함될 수 있다. When the kit of the present invention is for measuring the expression level of a protein, a substrate, a suitable buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, etc. may be included for the immunological detection of the antibody.

본 발명의 상기 기재는 니트로셀룰로오스 막, 폴리비닐 수지 또는 폴리스티렌 수지로 합성된 96웰 플레이트 및 유리로 된 슬라이드글라스 등이 사용될 수 있고, 상기 발색 효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(Alkaline Phosphatase)가 사용될 수 있으며, 형광물질은 FITC, RITC 등이 사용될 수 있고, 발색 기질액은 ABTS(2,2'-아지노-비스(3-에틸벤조티아졸린-6-설폰산)), OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘)가 사용될 수 있으나, 이에 제한되는 것은 아니다.The substrate of the present invention may be a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin or a polystyrene resin, and a slide glass made of glass, and the coloring enzyme is peroxidase, alkaline phosphatase (Alkaline). Phosphatase) can be used, fluorescent materials can be used, such as FITC, RITC, etc., and the color developing substrate solution is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), OPD ( o-phenylenediamine), TMB (tetramethyl benzidine) may be used, but is not limited thereto.

본 발명의 또 다른 구현 예에서는 침윤성 뇌암을 예측하기 위한 정보 제공 방법을 제공한다.Another embodiment of the present invention provides a method of providing information for predicting invasive brain cancer.

본 발명의 상기 정보 제공 방법은 목적하는 개체로부터 분리된 생물학적 시료에서, PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 단계를 포함한다.The information providing method of the present invention includes the step of measuring the expression level of the PCBP1 protein or a gene encoding it in a biological sample isolated from a target individual.

본 발명의 상기 목적하는 개체로부터 분리된 생물학적 시료에서 측정된 상기 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 정상 대조군에 비하여 낮은 경우, 상기 목적하는 개체의 뇌암이 침윤성이 높을 것으로 예측할 수 있다.When the expression level of the PCBP1 protein or the gene encoding it measured in a biological sample isolated from the object of the present invention is lower than that of the normal control, it can be predicted that the brain cancer of the object of interest has high invasiveness.

본 발명의 상기 "정상 대조군"은 뇌암으로 진단받지 않은 개체 또는 타 기관으로 암 세포의 침습이 일어나지 않은 것으로 확인된 개체일 수 있고, 바람직하게는 타 기관으로 암 세포의 침습이 일어나지 않은 것으로 확인된 개체일 수 있으나, 이에 제한되는 것은 아니다.The "normal control" of the present invention may be an individual that has not been diagnosed with brain cancer or an individual that has not been confirmed to have invaded cancer cells to other organs, preferably, it has been confirmed that the invasion of cancer cells to other organs has not occurred. It may be an individual, but is not limited thereto.

본 발명의 상기 침윤성 뇌암을 예측하기 위한 정보 제공 방법에서, PCBP1 단백질 및 이를 암호화하는 유전자, 침윤성, 뇌암 등은 상기 진단용 조성물에서 기재한 바와 중복되어, 이하 본 명세서의 과도한 복잡성을 피하기 위하여 생략한다.In the method of providing information for predicting invasive brain cancer of the present invention, the PCBP1 protein and the gene encoding it, invasiveness, brain cancer, and the like are overlapped with those described in the diagnostic composition, and are omitted in order to avoid excessive complexity of the present specification.

본 발명의 상기 PCBP1 단백질의 발현 수준을 측정하는 단계는 본 발명의 상기 단백질의 발현 수준을 측정하는 제제를 이용하여, 웨스턴블랏, ELISA, 방사선면역분석, 면역확산법, 면역 전기영동, 조직 면역염색, 면역침전 분석법, 보체 고정 분석법, FACS, 질량분석, MRM 분석 및 단백질 마이크로어레이로 구성된 군으로부터 선택되는 적어도 하나를 통해 측정할 수 있다.The step of measuring the expression level of the PCBP1 protein of the present invention can be performed by using the agent for measuring the expression level of the protein of the present invention, Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, It can be measured through at least one selected from the group consisting of immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, MRM analysis, and protein microarray.

본 발명의 상기 PCBP1 유전자의 발현 수준을 측정하는 단계는 본 발명의 상기 유전자의 발현 수준을 측정하는 제제를 이용하여, 중합효소연쇄반응, 역전사 중합효소연쇄반응, 경쟁적 중합효소연쇄반응, Nuclease 보호 분석(RNase, S1 nuclease assay), 인 시츄(in situ) 교잡법, 핵산 마이크로어레이 및 노던블랏으로 구성된 군으로부터 선택되는 적어도 하나를 통해 측정하는 것일 수 있다.The step of measuring the expression level of the PCBP1 gene of the present invention is a polymerase chain reaction, a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, and a Nuclease protection assay using the agent for measuring the expression level of the gene of the present invention. It may be measured through at least one selected from the group consisting of (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, and Northern blot.

본 발명의 상기 생물학적 시료는 개체로부터 얻어지거나 개체로부터 유래된 임의의 물질, 생물학적 체액, 조직 또는 세포를 의미하는 것으로, 예를 들면, 전혈(whole blood), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 혈장(plasma) 및 혈청(serum)을 포함하는 혈액, 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 골반 내 유체액(pelvic fluids), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 또는 뇌척수액(cerebrospinal fluid) 등 일 수 있으나, 이에 제한되는 것은 아니다.The biological sample of the present invention refers to any substance, biological body fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells ( peripheral blood mononuclear cells), leukocyte buffy coat, blood including plasma and serum, sputum, tears, mucus, nasal washes, nasal cavity Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, Meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate aspirate), synovial fluid, joint aspirate, organ secretions, cells, cell extracts, cerebrospinal fluid, etc., but are limited thereto. no.

본 발명의 또 다른 구현 예에서는 뇌암의 예후 예측용 조성물을 제공한다.Another embodiment of the present invention provides a composition for predicting the prognosis of brain cancer.

본 발명의 상기 예후 예측용 조성물은 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 포함한다.The composition for predicting prognosis of the present invention includes an agent for measuring the expression level of the PCBP1 protein or a gene encoding it.

본 발명의 일 실시예에서는 상기 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 감소되어 있는 경우, 뇌암의 침윤성이 증가되고 이를 통해 궁극적으로 뇌암이 발생된 개체의 전체 생존율이 현저하게 감소될 수 있음을 확인하였다(도 5 A 및 B 참고). 본 발명의 목적상 상기 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 정상 대조군에 비하여 감소되어 있는 경우에는 뇌암의 침윤성이 증가되어 있고, 이에 따라 뇌암이 발생된 개체의 전체 생존율이 현저하게 감소되어 예후가 나쁠 수 있음을 매우 효과적으로 예측할 수 있다.In one embodiment of the present invention, when the expression level of the PCBP1 protein or the gene encoding it is decreased, the invasiveness of brain cancer is increased, and through this, the overall survival rate of the individual with brain cancer may be significantly reduced. It was confirmed (see Figs. 5 A and B). For the purpose of the present invention, when the expression level of the PCBP1 protein or the gene encoding it is decreased compared to the normal control, the invasiveness of brain cancer is increased, and accordingly, the overall survival rate of the individual with brain cancer is significantly reduced, and the prognosis It can be predicted very effectively that may be bad.

본 발명의 상기 예후 예측용 조성물에서, PCBP1 단백질 및 이를 암호화하는 유전자, 뇌암, 단백질의 발현 수준을 측정할 수 있는 제제, 유전자의 발현 수준을 측정할 수 있는 제제 등은 상기 진단용 조성물에서 기재한 바와 중복되어, 이하 본 명세서의 과도한 복잡성을 피하기 위하여 생략한다.In the composition for predicting prognosis of the present invention, the PCBP1 protein and the gene encoding it, brain cancer, an agent capable of measuring the expression level of the protein, an agent capable of measuring the expression level of the gene, etc. are as described in the diagnostic composition. It is redundant, and it is omitted to avoid excessive complexity of the present specification.

본 발명의 또 다른 구현 예에서는 침윤성 뇌암 치료제의 스크리닝 방법을 제공한다.Another embodiment of the present invention provides a screening method for a therapeutic agent for invasive brain cancer.

본 발명의 상기 스크리닝 방법은 뇌암 환자로부터 분리된 생물학적 시료에 목적하는 후보물질을 처리하는 단계; 및 상기 생물학적 시료에서 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 확인하는 단계를 포함한다.The screening method of the present invention comprises the steps of treating a target candidate material in a biological sample isolated from a brain cancer patient; And checking the expression level of the PCBP1 protein or a gene encoding it in the biological sample.

본 발명의 상기 후보물질을 처리한 뒤, 상기 생물학적 시료에서 측정된 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 증가되는 경우, 침윤성 뇌암의 치료제로 선별하는 단계를 더 포함할 수 있다.After treatment of the candidate material of the present invention, when the expression level of the PCBP1 protein or the gene encoding the PCBP1 protein measured in the biological sample is increased, the step of selecting as a therapeutic agent for invasive brain cancer may be further included.

본 발명의 상기 스크리닝 방법에서, PCBP1 단백질 및 이를 암호화하는 유전자, 뇌암, 단백질의 발현 수준을 측정하는 단계, 유전자의 발현 수준을 측정하는 단계, 생물학적 시료 등은 상기 진단용 조성물 및 침윤성 뇌암을 예측하기 위한 정보를 제공하는 방법에서 기재한 바와 중복되어, 이하 본 명세서의 과도한 복잡성을 피하기 위하여 생략한다.In the screening method of the present invention, the step of measuring the expression level of the PCBP1 protein and the gene encoding it, brain cancer, and the protein, measuring the expression level of the gene, the biological sample, etc. are used for predicting the diagnostic composition and invasive brain cancer. As described in the method for providing information, it is omitted to avoid excessive complexity of the present specification.

본 발명의 상기 후보물질은 침윤성 뇌암을 치료하기 위한 활성, 즉 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현을 증가시킬 수 있는 활성이 존재하는지 여부에 대해 테스트하기 위한 약제를 의미하며, 단백질, 올리고 펩타이드, 유기 분자, 다당류, 폴리뉴클레오티드 및 광범위한 화합물 등의 임의의 분자를 포함한다. 이러한 후보물질은 천연물질뿐만 아니라, 합성 물질도 모두 포함하는 것일 수 있다.The candidate substance of the present invention refers to a drug for testing whether there is an activity for treating invasive brain cancer, that is, an activity capable of increasing the expression of the PCBP1 protein or a gene encoding it, protein, oligopeptide, Includes any molecule such as organic molecules, polysaccharides, polynucleotides and a wide range of compounds. These candidate substances may include not only natural substances but also synthetic substances.

본 발명의 조성물을 이용하여 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 경우에는 목적하는 개체의 침윤성을 매우 효과적으로 예측할 수 있기 때문에, 환자에 적절한 치료 방법 등을 제공하여 이와 같은 치료로 인한 사회적 비용을 현저하게 감소시킬 수 있다. 나아가, 상기 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 감소되어 있는 경우에는 침윤성이 높아져 전체 생존율이 감소될 수 있기 때문에 뇌암의 예후를 매우 효과적으로 예측할 수 있다.In the case of measuring the expression level of the PCBP1 protein or the gene encoding it by using the composition of the present invention, since the invasiveness of the target individual can be predicted very effectively, an appropriate treatment method, etc., is provided to the patient The cost can be significantly reduced. Further, when the expression level of the PCBP1 protein or the gene encoding it is decreased, the invasiveness may increase and the overall survival rate may be reduced, so that the prognosis of brain cancer can be predicted very effectively.

도 1의 A 내지 C는 본 발명의 일 실시예에 따른 교모세포종 종양구 형성을 현미경으로 확인한 결과(도 1의 A 및 B)와, 교모세포종 종양구의 침윤성을 마트리겔(Matrigel) 분석 방법을 통해 확인한 결과를 나타낸 것이다(도 1의 C).1A to 1C show the results of confirming the formation of glioblastoma tumor cells with a microscope according to an embodiment of the present invention (A and B of FIGS. 1) and the invasiveness of glioblastoma tumor cells through a Matrigel analysis method. It shows the confirmed result (FIG. 1C).

도 2의 A 및 B는 본 발명의 일 실시예에 따른 침윤성이 높은(High inv.) 교모세포종 종양구와, 침윤성이 낮은(Low inv.) 교모세포종 종양구 간의 유전자 발현 수준을 유전자 프로파일링 분석 방법을 통해 비교한 결과를 나타낸 것이다.2A and 2B show a gene profiling analysis method for gene expression levels between high invasive (High inv.) glioblastoma tumor cells and low invasive (Low inv.) glioblastoma tumor cells according to an embodiment of the present invention. It shows the result of comparison through.

도 3의 A 및 B는 본 발명의 일 실시예에 따른 침윤성과 관련된 유전자의 상호작용과, 상기 유전자의 돌연변이 비율을 확인한 결과를 나타낸 것이다.3A and 3B show the results of confirming the interaction of genes related to invasiveness and mutation rates of the genes according to an embodiment of the present invention.

도 4의 A 및 B는 본 발명의 일 실시에에 따른 교모세포종 종양구 형성을 확인한 결과와(도 4의 A), 교모세포종 종양구의 침윤성을 확인한 결과를 나타내며(도 4의 B), 도 4의 C는 본 발명의 일 실시예에 따른 교모세포종 종양구에서 단백질의 발현 수준을 웨스턴 블롯 분석을 통해 확인한 결과를 나타낸 것이다.4A and 4B show the results of confirming the formation of glioblastoma tumor cells according to an embodiment of the present invention (Fig. 4A) and the results of confirming the invasiveness of glioblastoma tumor cells (Fig. 4B), and Fig. 4 C shows the result of confirming the expression level of the protein in the glioblastoma tumor cell according to an embodiment of the present invention through Western blot analysis.

도 5의 A 및 B는 교모세포종 종양구 이식 쥐 및 교모세포종 환자의 생존율 분석 결과를 나타낸 것이다.5A and B show the results of analyzing the survival rate of glioblastoma tumor cell transplant mice and glioblastoma patients.

본 발명의 일 바람직한 구현 예에서는 PCBP1(Poly(RC) Binding Protein 1) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 포함하는 침윤성 교모세포종의 진단용 조성물; 및 교모세포종의 예후 예측용 조성물을 제공한다.In a preferred embodiment of the present invention, a composition for diagnosing invasive glioblastoma comprising an agent for measuring the expression level of a PCBP1 (Poly(RC) Binding Protein 1) protein or a gene encoding the same; And it provides a composition for predicting the prognosis of glioblastoma.

본 발명의 다른 바람직한 구현 예에서는 침윤성 교모세포종의 진단용 조성물을 포함하는 침윤성 교모세포종의 진단용 키트를 제공한다.Another preferred embodiment of the present invention provides a kit for diagnosing invasive glioblastoma comprising a composition for diagnosing invasive glioblastoma.

본 발명의 또 다른 바람직한 구현 예에서는 목적하는 개체로부터 분리된 생물학적 시료에서 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 단계를 포함하는 침윤성 교모세포종을 예측하기 위한 정보 제공 방법을 제공한다.Another preferred embodiment of the present invention provides a method of providing information for predicting invasive glioblastoma, comprising measuring the expression level of the PCBP1 protein or a gene encoding it in a biological sample isolated from a target individual.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실시예Example

[준비예 1] [Preparation Example 1] 환자 정보 및 MR(Magnetic resonance) 이미지 촬영Patient information and magnetic resonance (MR) imaging

외과적 수술, 화학 요법 또는 방사선 치료와 같은 처치 없이 새롭게 진단받은 23명(침윤성 낮음 15명, 침윤성 높음 8명)의 환자 조직을 본 연구에서 사용하였다. 각각의 환자에서 암 조직을 제거하기 위한 수술을 진행하기 전, 7일 내에 Achieva 3.0T 시스템(Philips Medical Systems)을 이용하여 MR 이미지를 촬영하였다. 뇌들보(Corpus callosum)의 전부(Anterior) 및 후부(Posterior) 가장자리에 평행하도록 하여 축상(Axial) 이미지를 촬영하였다. 종양 조직의 침윤의 경우, T2 FLAIR - T1 contrast enhanced (CE)/T1 CE에 의해 점유된 영역으로 정량화 하여 나타내었다.Tissues of 23 newly diagnosed patients (15 low invasiveness, 8 high invasiveness) without treatment such as surgical operation, chemotherapy or radiotherapy were used in this study. MR images were taken within 7 days using the Achieva 3.0T system (Philips Medical Systems) before proceeding with surgery to remove cancer tissue from each patient. Axial images were taken by paralleling to the anterior and posterior edges of the corpus callosum. In the case of tumor tissue invasion, it was quantified as the area occupied by T2 FLAIR-T1 contrast enhanced (CE)/T1 CE.

본 연구에 대해서는 연세대학교 의과대학 세브란스 병원의 제도 검토위원회(4-2012-0212, 4-2014-0649)를 통해 승인 받았으며, 연구에서 사용된 조직의 공여자에게 모두 서면 동의를 받았다.This study was approved by the institutional review committee (4-2012-0212, 4-2014-0649) of Severance Hospital, Yonsei University College of Medicine, and written consent was obtained from all donors of the tissue used in the study.

특징Characteristic 침윤성 낮음(n = 15)Low invasiveness (n = 15) 침윤성 높음(n = 8)High invasiveness (n = 8) 나이 (평균)Age (average) 6161 54.554.5 성별 (남성/여성)Gender (male/female) 9/69/6 5/35/3 IDH1 유전자 돌연변이IDH1 gene mutation 00 00 1p19q 코-결실(co-deletion)1p19q co-deletion 00 00 MGMT 유전자 프로모터에 메틸화(methylation in promoter)Methylation in promoter of MGMT gene 44 22 EGFR 증폭(amplification)EGFR amplification 1111 44 Ki-67 발현 수준 (평균)Ki-67 expression level (mean) 17.517.5 3535 전체 생존율 (평균)Overall survival rate (average) 11.9211.92 7.527.52

[[ 준비예Preparation example 2] 2] 교모세포종(Glioblastoma ( GlioblastomaGlioblastoma multiformemultiforme ; ; GBMGBM ) ) 종양구의Tumorigenic 분리 및 배양 Isolation and culture

상기 준비예 1의 환자로부터 GBM 세포를 분리한 뒤, 종양구(Tumor sphere; TS)를 생성하는 세포만을 선별하여, 교모세포종 종양구를 최종적으로 분리하였다(도 1의 A).After separating GBM cells from the patient of Preparation Example 1, only cells that generate tumor spheres (TS) were selected, and glioblastoma tumor cells were finally isolated (Fig. 1A).

상기 교모세포종 종양구의 경우, 1 × B27 (인비트로젠, 미국), 20 ng/mL의 bFGF 및 20 ng/mL의 EGF (시그마알드리치, 미국)가 포함되어 있는 DMEM/F12(미디어테크, 미국)에서 37 ℃, 5 %의 CO2 조건에서 배양하였다.For the glioblastoma tumor cells, DMEM/F12 (Mediatech, USA) containing 1 × B27 (Invitrogen, USA), 20 ng/mL bFGF and 20 ng/mL EGF (Sigma-Aldrich, USA) At 37° C., 5% CO 2 was cultured under conditions.

[준비예 3][Preparation Example 3] 교모세포종 이식 동물 모델 제작 및 사육Production and rearing of glioblastoma transplanted animal models

4 내지 8주령의 수컷 흉선 누드 마우스(athymic nude mice)(Central Lab. Animal Inc., 한국)를 마이크로-아이솔레이터 케이지(micro-isolator cages)에 넣고, 균이 존재하지 않는 조건에서 연구 시작 1주일 전부터 사육하였다. 이때, 조명, 온도 및 습도는 중앙에서 제어될 수 있도록 하였다.4 to 8 weeks old male athymic nude mice (Central Lab. Animal Inc., Korea) were placed in micro-isolator cages, from 1 week before the start of the study in the absence of bacteria. Breed. At this time, lighting, temperature, and humidity were centrally controlled.

가이드-스크류 시스템(Guide-Screw system)을 이용하여, 깊이 4.5 mm의 깊이로 상기 마우스의 오른쪽 전두엽에 상기 준비예 2에서 분리된 침윤성이 낮은 교모세포종 종양구인 TS14-15(이하, 'TS14-15'라 함) 또는 침윤성이 높은 교모세포종 종양구인 TS13-64(이하, 'TS13-64'라 함)를 이식하였다. 이때, 이식이 완료된 상기 마우스의 체중이 최대치에 비하여 15 % 이상 감소된 경우에는 승인된 프로토콜에 따라 마우스를 안락사 시켰다. IVIS 이미징 시스템 및 Living Image v4.2 소프트웨어 (Caliper Life Sciences)를 사용하여 Bioluminescence 수집 및 분석을 수행하였다. 아래의 실시예를 수행하기 15분 전에 100 μL D-루시퍼린(D-luciferin, 30 mg / mL; Promega)을 상기 마우스의 복강 내 주사하였으며, 2.5 %의 이소플루렌(Isoflurane)을 이용하여 마취된 상태에서 수행하였다. 이와 같은 모든 실험 절차는 연세대 의과대학 기관 동물 관리 및 사용위원회의 승인을 받았다.Using a guide-screw system, TS14-15, a low-invasive glioblastoma tumor cell, isolated in Preparation Example 2 in the right frontal lobe of the mouse to a depth of 4.5 mm (hereinafter,'TS14-15 ') or TS13-64 (hereinafter referred to as'TS13-64'), a highly invasive glioblastoma tumor cell, was transplanted. At this time, when the weight of the mouse, which has been transplanted, was reduced by more than 15% compared to the maximum, the mouse was euthanized according to an approved protocol. Bioluminescence collection and analysis were performed using the IVIS imaging system and Living Image v4.2 software (Caliper Life Sciences). 15 minutes before performing the following examples, 100 μL D-luciferin (D-luciferin, 30 mg / mL; Promega) was injected intraperitoneally of the mouse, and anesthesia using 2.5% isoflurane It was carried out in the state. All of these experimental procedures were approved by the Animal Care and Use Committee of Yonsei University Medical School.

[실시예 1] [Example 1] 교모세포종 종양구의 3D 침윤성 분석3D invasive analysis of glioblastoma tumor cells

교모세포종 종양구의 침윤성 분석을 확인하기 위하여, 마트리겔(Matrigel) 및 1형 콜라겐(Type I Collagen)을 96웰 플레이트에 넣고, 상기 마트리겔이 겔화되기 전에 상기 준비예 2의 교모세포종 종양구를 분주한 뒤, 상기 준비예 2의 배지를 첨가하였다. 그런 다음 0시간 내지 72시간 동안 배양하고, 현미경을 통해 구형 형성을 확인하여 그 결과를 도 1의 B에 나타내었다. 또한, 0시간 때에 상기 교모세포종 종양구가 존재하는 지역을 기준으로 침윤성 정도를 수치화하여, 그 결과를 도 1의 C에 나타내었다.To confirm the invasiveness analysis of glioblastoma tumor cells, Matrigel and Type I Collagen were placed in a 96-well plate, and the glioblastoma tumor cells of Preparation Example 2 were dispensed before the Matrigel gelled. After that, the medium of Preparation Example 2 was added. Then, it was cultured for 0 to 72 hours, and spherical formation was confirmed through a microscope, and the results are shown in B of FIG. 1. In addition, at 0 hours, the degree of invasiveness was quantified based on the area where the glioblastoma tumor cells were present, and the results are shown in FIG. 1C.

도 1의 B 및 C에서 보는 바와 같이, 교모세포종 종양구의 경우 침윤성이 낮은 경우(Low inv.)와 침윤성이 높은 경우(High inv.)로 나뉘었다. 구체적으로, TS13-03, TS13-20, TS13-30, TS13-64, TS13-71, TS15-67, TS15-88 및 TS16-117의 경우 침윤성이 높았으며, 그 중에서 TS15-88 및 TS13-64의 침윤성이 가장 높았다.1B and C, the case of glioblastoma tumor cells was divided into a case with low invasiveness (Low inv.) and a case with high invasiveness (High inv.). Specifically, TS13-03, TS13-20, TS13-30, TS13-64, TS13-71, TS15-67, TS15-88 and TS16-117 had high invasiveness, among which TS15-88 and TS13-64 Had the highest invasiveness.

상기 결과를 통해 본 발명에서 환자로부터 분리된 교모세포종 종양구의 경우에도 침윤성이 높은 경우 및 침윤성이 낮은 경우로 분리될 수 있음을 알 수 있다.From the above results, it can be seen that in the case of glioblastoma tumor cells isolated from patients in the present invention, it can be separated into cases with high invasiveness and low invasiveness.

[실시예 2] [Example 2] 교모세포종 종양구 침윤성과 관련된 유전자 동정Identification of genes related to glioblastoma tumor cell invasion

교모세포종 종양구의 침윤성과 관련된 전사 조절 네트워크를 확인하기 위하여 단일 샘플 유전자 세트 응축 분석(Single sample gene set enrichment analysis; ssGSEA)을 수행하였다. 퀴아젠 RNeasy 플러스 미니 키트(Qiagen RNeasy Plus Mini kit, Qiagen, 미국)을 이용하여 제조사가 제공하는 방법에 따라, 상기 준비예 2에서 분리된 교모세포종 종양구로부터 전체 RNA를 추출하였다. 상기 추출된 전체 RNA를 일루미나 휴먼HT-12 v4 발현 비드칩(Illumina HumanHT-12 v4 Expression BeadChip, Illumina, 미국)에 로딩하였다. 데이터 변형의 안정화 및 표준화를 위해 R/ Bioconductor lumi 패키지를 사용하는 분위 일반화(quantile normalization) 방법을 사용하였다. GENE-E 소프트웨어를 사용하여 피어슨의 상관관계를 거리 메트릭로서 평균 연결 계층적 클러스터링(average linkage hierarchical clustering)을 수행하고, 각각의 유전자의 발현 수준을 히트 맵으로 나타내었다.Single sample gene set enrichment analysis (ssGSEA) was performed to confirm the transcriptional regulatory network associated with the invasion of glioblastoma tumor cells. Total RNA was extracted from the glioblastoma tumor cells isolated in Preparation Example 2 using the Qiagen RNeasy Plus Mini kit (Qiagen, USA) according to the method provided by the manufacturer. The extracted total RNA was loaded onto an Illumina HumanHT-12 v4 Expression BeadChip (Illumina, USA). For the stabilization and standardization of data transformation, a quantitative normalization method using the R/Bioconductor lumi package was used. Using GENE-E software, average linkage hierarchical clustering was performed as a distance metric for Pearson's correlation, and the expression level of each gene was expressed as a heat map.

MSigDB c3.tft v5.1에서 검색된 전사인자(Transcriptional factor; TF) 표적 유전자 세트를 사용하여 교모세포종 종양구의 유전자 발현 프로파일을 ssGSEA에 적용하였다. 여기서, 동일한 전사인자로 계산된 농축 점수(Enrichment scores)는 각 전사인자마다 단일 값을 얻기 위해 평균화하였으며, 이 평균화된 점수를 모든 표본에서 양적 표준화 하였다. 그런 다음, 상기 실시예 1에서 확인된 침윤성이 낮은 교모세포종 종양구 그룹과 침윤성이 높은 교모세포종 종양구 그룹(two-tailed Student's t-test) 간의 차이를 비교하였다. 상기 두 그룹 간의 차이가 있는 것으로 확인된 전사인자를 사이토스케이프(Cytoscape) 및 리엑톰 FI 9 플러그-인(Reactome FI 9 plug-in)을 이용하여 네트워크 맵으로 구성하여, 기능적 상호작용 분석을 수행하였다. 또한, 교모세포종 환자에서 선택된 전사인자의 게놈 변이 분석을 위해 TCGA 데이터 세트(n=273)를 검색하여, 그 결과를 도 2의 A 및 B와, 도 3의 A 및 B에 나타내었다.The gene expression profile of glioblastoma tumor cells was applied to ssGSEA using a set of transcriptional factor (TF) target genes retrieved from MSigDB c3.tft v5.1. Here, the enrichment scores calculated with the same transcription factor were averaged to obtain a single value for each transcription factor, and this averaged score was quantitatively standardized in all samples. Then, the difference between the low invasive glioblastoma tumor cell group and the high invasive glioblastoma tumor cell group (two-tailed Student's t-test) identified in Example 1 was compared. The transcription factor identified as having a difference between the two groups was configured as a network map using Cytoscape and Reactome FI 9 plug-in, and functional interaction analysis was performed. . In addition, TCGA data sets (n=273) were searched for analysis of genomic variation of transcription factors selected in glioblastoma patients, and the results are shown in A and B in Figs.

도 2의 A 및 B에서 보는 바와 같이, 침습성이 낮은 교모세포종(Low inv.) 그룹에 비하여, 침습성이 높은 교모세포종(High inv.) 그룹에서 STAT3(Signal transducer and activator of transcription 3), SPI1(Transcription factor PU.1), SBFA2T(SBF activated by Bck2), PAX8(Paired box gene 8), BACH1/2(Transcription regulator protein BACH1), SRF(Serum response factor), 및 PTF1A(Pancreas transcription factor 1 subunit alpha)가 높은 농축 점수를 갖고, PRRX2(Paired Related Homeobox 2), TCF7(Transcription factor 7), SRY(Sex-determining Region Y), SOX5(SRY-related HMG-box 5) 및 PCBP1(Poly(RC) Binding Protein 1)가 낮은 농축 점수를 갖는 것으로 확인되었다. 이와 같은 전사인자 중에서 특히 PCBP1의 경우, 침습성이 낮은 경우에 비하여 침습성이 높은 교모세포종 그룹에서 전반적으로 낮은 수준으로 존재하였다.As shown in Fig. 2A and B, STAT3 (Signal transducer and activator of transcription 3), SPI1 (high inv.) group, compared to the low invasive glioblastoma (Low inv.) group, Transcription factor PU.1), SBFA2T (SBF activated by Bck2), PAX8 (Paired box gene 8), BACH1/2 (Transcription regulator protein BACH1), SRF (Serum response factor), and PTF1A (Pancreas transcription factor 1 subunit alpha) Has a high concentration score, PRRX2 (Paired Related Homeobox 2), TCF7 (Transcription factor 7), SRY (Sex-determining Region Y), SOX5 (SRY-related HMG-box 5) and PCBP1 (Poly(RC) Binding Protein 1) was found to have a low concentration score. Among these transcription factors, PCBP1, in particular, was present at an overall lower level in the highly invasive glioblastoma group than in the case of low invasiveness.

도 3의 A 및 B에서 보는 바와 같이, 상기 확인된 전사인자들의 기능적인 상호작용과 돌연변이 정도를 확인한 결과, 전사인자의 돌연변이(미스센스, 결실 및 증폭)율이 높지 않은 것으로 보아 교모세포종 침윤에 관여하는 전사인자의 변화는 외부 요인에 의해 변경된 것을 알 수 있다.3A and 3B, as a result of confirming the functional interaction and the degree of mutation of the identified transcription factors, the mutation (miss sense, deletion, and amplification) rate of the transcription factor was not high, so that glioblastoma invasion was prevented. It can be seen that the change of the involved transcription factor was changed by external factors.

상기 결과를 통해, 본 발명에 따른 PCBP1 전사인자의 발현 수준이 감소되어 있는 경우 뇌암, 예를 들면 교모세포종, 특히 교모세포종 종양구의 침윤성이 높은 것을 알 수 있다. 나아가, 이와 같이 PCBP1 단백질 또는 이를 암호화하는 유전자가 뇌암의 침윤성이 증가되도록 유도할 수 있음을 알 수 있다. From the above results, it can be seen that when the expression level of the PCBP1 transcription factor according to the present invention is reduced, the invasiveness of brain cancer, for example, glioblastoma, particularly glioblastoma, tumor cells, is high. Furthermore, it can be seen that the PCBP1 protein or a gene encoding it can induce an increase in the invasiveness of brain cancer.

[실시예 3] [Example 3] 교모세포종 종양구의 침윤성과 관련된 전사인자 확인Identification of transcription factors related to the invasiveness of glioblastoma tumor cells

상기 실시예 2에서 확인된 전사인자와 교모세포종 종양구의 침윤성과의 관계를 확인하기 위하여, 교모세포종 종양구에서 STAT3 및 PCBP1 유전자를 침묵(Silencing)시킨 뒤, 침윤성 및 단백질 발현 수준을 확인하였다.In order to confirm the relationship between the transcription factor identified in Example 2 and the invasiveness of glioblastoma tumor cells, STAT3 and PCBP1 genes were silenced in glioblastoma tumor cells, and then the invasiveness and protein expression levels were confirmed.

구체적으로, TS14-15와 TS13-64를 24시간 동안 배양하였다. 그런 다음, 상기 종양구 각각에 STAT3 또는 PCBP1에 특이적인 siRNA 이중가닥(바이오니아, 한국)을 리포펙타민 3000(인비트로젠, 미국)을 이용하여 형질전환시켰다. 이때, 무작위 siRNA 서열을 음성 대조군으로 사용하였다. 형질전환 후 72시간 동안 배양한 뒤, 상기 실시예 1에서와 같이 구형 형성 여부 및 침윤성 분석을 수행하여, 그 결과를 도 4의 A 및 B에 나타내었다.Specifically, TS14-15 and TS13-64 were cultured for 24 hours. Then, the tumor cells were transformed with STAT3 or PCBP1 specific siRNA double strands (Bionia, Korea) using Lipofectamine 3000 (Invitrogen, USA). At this time, a random siRNA sequence was used as a negative control. After incubation for 72 hours after transformation, as in Example 1, the analysis of whether a sphere was formed and invasiveness was performed, and the results are shown in A and B of FIG. 4.

또한, 상기 교모세포종 종양구에서 단백질의 발현 수준을 확인하기 위해, 상기 종양구로부터 분리된 단백질을 10% 트리스-글리신 겔에 전기영동 한 뒤, 니트로셀룰로스 막에 옮긴 뒤에 STAT3(Cell Signaling Technology), β-카테닌(β-catenin, Cell Signaling Technology), PCBP1(Abcam), N-카드헤린(N-cadherin, R&D Systems), Zeb1(Sigma-Aldrich); SRF(Santa Cruz Biotechnology) 및 GAPDH (Santa Cruz Biotechnology)에 특이적인 항체를 이용하여 웨스턴 블롯 분석을 수행하여, 그 결과를 도 4의 C에 나타내었다.In addition, in order to confirm the expression level of the protein in the glioblastoma tumor cell, the protein isolated from the tumor cell was electrophoresed on a 10% tris-glycine gel, and then transferred to a nitrocellulose membrane, followed by STAT3 (Cell Signaling Technology), β-catenin (Cell Signaling Technology), PCBP1 (Abcam), N-cadherin (R&D Systems), Zeb1 (Sigma-Aldrich); Western blot analysis was performed using antibodies specific for SRF (Santa Cruz Biotechnology) and GAPDH (Santa Cruz Biotechnology), and the results are shown in C of FIG. 4.

도 4의 A 및 B에서 보는 바와 같이, 음성 대조군(Control)과 비교하여, 침습성이 높은 그룹에서 증가되어 있는 STAT3에 특이적인 siRNA가 형질전환된 TS13-64의 경우(TS13-64, siSTAT3) 침습 지역이 6배 정도 감소되었다. 반면, 침습성이 높은 그룹에서 감소되어 있는 PCBP1에 특이적인 siRNA가 형질전환된 TS14-15(TS14-15, siPCBP1) 및 TS13-64(TS13-64, siPCBP1)에서 모두 침습 지역이 약 2배 증가되었다.As shown in A and B of Figure 4, compared to the negative control (Control), the case of TS13-64 transformed with siRNA specific to STAT3 increased in the highly invasive group (TS13-64, siSTAT3) invasion The area was reduced by 6 times. On the other hand, in the highly invasive group, both TS14-15 (TS14-15, siPCBP1) and TS13-64 (TS13-64, siPCBP1) transfected with a reduced PCBP1-specific siRNA increased approximately 2 times. .

또한, 도 4의 C에서 보는 바와 같이, PCBP1에 특이적인 siRNA가 형질전환된 경우, 모든 교모세포종 종양구에서 침윤 및 중간엽 전이(Mesenchymal transition)와 관련되어 있는 단백질인 N-카드헤린, β-카테닌 및 Zeb1 단백질의 발현 수준이 증가되었다.In addition, as shown in Fig. 4C, when the PCBP1-specific siRNA is transformed, N-cadherin, β-, which are proteins associated with invasion and mesenchymal transition in all glioblastoma tumor cells. The expression levels of catenin and Zeb1 proteins were increased.

상기 결과를 통해, 본 발명에 따른 PCBP1 단백질 또는 이를 암호화하는 유전자는 뇌암, 예를 들면 교모세포종, 특히 교모세포종 종양구의 전이를 억제할 수 있는 기능을 갖는 것으로서, 이와 같은 단백질 또는 이를 암호화하는 단백질의 발현 수준이 정상 대조군에 비하여 낮은 수준으로 존재하는 경우에는 침윤성이 높은 것으로 예상할 수 있음을 알 수 있다.Through the above results, the PCBP1 protein or the gene encoding it according to the present invention has a function of inhibiting metastasis of brain cancer, for example, glioblastoma, particularly glioblastoma tumor cells, and the protein or protein encoding the same It can be seen that when the expression level is present at a lower level than that of the normal control, high invasiveness can be expected.

[실시예 4] [Example 4] 교모세포종 종양구의 침윤성과 예후와의 관계 확인Confirmation of the relationship between invasion and prognosis of glioblastoma tumor cells

상기 실시예 1 내지 3의 결과를 토대로 교모세포종 환자, 특히 교모세포종 종양구를 갖는 환자에서 교모세포종의 침윤성과 예후와의 상관관계가 있는지 확인하였다.Based on the results of Examples 1 to 3, it was confirmed whether there was a correlation between the invasiveness of glioblastoma and the prognosis in glioblastoma patients, particularly those with glioblastoma tumor cells.

Kaplan-Meier 곡선을 기반으로 상기 준비예 3에서 제작된 동물 모델과, 상기 준비예 2에서 분리된 교모세포종 종양구와 일치되는 환자의 전반적인 생존율을 분석하여, 그 결과를 도 5의 A 및 B에 나타내었다. Based on the Kaplan-Meier curve, the animal model prepared in Preparation Example 3 and the overall survival rate of patients matching the glioblastoma tumor cells isolated in Preparation Example 2 were analyzed, and the results are shown in A and B of FIG. 5. Done.

도 5의 A 및 B에서 보는 바와 같이, 침윤성이 낮은 경우(Low)의 생존율과 비교하여, 침윤성이 높은 경우(High)의 생존율이 현저하게 낮았다.5A and 5B, compared with the survival rate of low invasiveness (Low), the survival rate of high invasiveness (High) was significantly lower.

상기 결과를 통해, 본 발명에서와 같이 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 낮은 경우에는 침윤성이 높으며, 나아가 이와 같이 침윤성이 높은 경우에는 예후가 나쁠 수 있음을 알 수 있다.From the above results, it can be seen that when the expression level of the PCBP1 protein or the gene encoding it is low as in the present invention, the invasiveness is high, and further, when the invasiveness is high, the prognosis may be poor.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

본 발명의 조성물을 이용하여 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 경우에는 목적하는 개체의 침윤성을 매우 효과적으로 예측할 수 있기 때문에, 환자에 적절한 치료 방법 등을 제공하여 이와 같은 치료로 인한 사회적 비용을 현저하게 감소시킬 수 있다. 나아가, 상기 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 감소되어 있는 경우에는 침윤성이 높아져 전체 생존율이 감소될 수 있기 때문에 뇌암의 예후를 매우 효과적으로 예측할 수 있다.In the case of measuring the expression level of the PCBP1 protein or the gene encoding it by using the composition of the present invention, since the invasiveness of the target individual can be predicted very effectively, an appropriate treatment method, etc., is provided to the patient The cost can be significantly reduced. Further, when the expression level of the PCBP1 protein or the gene encoding it is decreased, the invasiveness may increase and the overall survival rate may be reduced, so that the prognosis of brain cancer can be predicted very effectively.

[서열목록][Sequence List]

서열번호 1: 아미노산 서열SEQ ID NO: 1: amino acid sequence

10 20 30 40 50 10 20 30 40 50

MDAGVTESGL NVTLTIRLLM HGKEVGSIIG KKGESVKRIR EESGARINIS MDAGVTESGL NVTLTIRLLM HGKEVGSIIG KKGESVKRIR EESGARINIS

60 70 80 90 100 60 70 80 90 100

EGNCPERIIT LTGPTNAIFK AFAMIIDKLE EDINSSMTNS TAASRPPVTL EGNCPERIIT LTGPTNAIFK AFAMIIDKLE EDINSSMTNS TAASRPPVTL

110 120 130 140 150 110 120 130 140 150

RLVVPATQCG SLIGKGGCKI KEIRESTGAQ VQVAGDMLPN STERAITIAG RLVVPATQCG SLIGKGGCKI KEIRESTGAQ VQVAGDMLPN STERAITIAG

160 170 180 190 200 160 170 180 190 200

VPQSVTECVK QICLVMLETL SQSPQGRVMT IPYQPMPASS PVICAGGQDR VPQSVTECVK QICLVMLETL SQSPQGRVMT IPYQPMPASS PVICAGGQDR

210 220 230 240 250 210 220 230 240 250

CSDAAGYPHA THDLEGPPLD AYSIQGQHTI SPLDLAKLNQ VARQQSHFAM CSDAAGYPHA THDLEGPPLD AYSIQGQHTI SPLDLAKLNQ VARQQSHFAM

260 270 280 290 300 260 270 280 290 300

MHGGTGFAGI DSSSPEVKGY WASLDASTQT THELTIPNNL IGCIIGRQGA MHGGTGFAGI DSSSPEVKGY WASLDASTQT THELTIPNNL IGCIIGRQGA

310 320 330 340 350 310 320 330 340 350

NINEIRQMSG AQIKIANPVE GSSGRQVTIT GSAASISLAQ YLINARLSSE NINEIRQMSG AQIKIANPVE GSSGRQVTIT GSAASISLAQ YLINARLSSE

KGMGCSKGMGCS

서열번호 2: 유전자 서열SEQ ID NO: 2: gene sequence

1 gaaagcgggc ttcccgcccc gcccagaccg ccgaggctgc cgccggagtc gccaccgccg1 gaaagcgggc ttcccgcccc gcccagaccg ccgaggctgc cgccggagtc gccaccgccg

61 cgccctcgcc cacccgcccg cccgccgctc ccggccccgc tcgccccctc cgccgccgcc61 cgccctcgcc cacccgcccg cccgccgctc ccggccccgc tcgccccctc cgccgccgcc

121 gcccgcccct gcgactacgc tgcggcctcc cgcccgctcc cgctcgctcc cgcggccctc121 gcccgcccct gcgactacgc tgcggcctcc cgcccgctcc cgctcgctcc cgcggccctc

181 gctcgcctcg cgccggcagt tttgggccta cacctcccct ccccccgcca gccgccaaag181 gctcgcctcg cgccggcagt tttgggccta cacctcccct ccccccgcca gccgccaaag

241 acttgaccac gtaacgagcc caactccccc gaacgccgcc cgccgctcgc catggatgcc241 acttgaccac gtaacgagcc caactccccc gaacgccgcc cgccgctcgc catggatgcc

301 ggtgtgactg aaagtggact aaatgtgact ctcaccattc ggcttcttat gcacggaaag301 ggtgtgactg aaagtggact aaatgtgact ctcaccattc ggcttcttat gcacggaaag

361 gaagtaggaa gcatcattgg gaagaaaggg gagtcggtta agaggatccg cgaggagagt361 gaagtaggaa gcatcattgg gaagaaaggg gagtcggtta agaggatccg cgaggagagt

421 ggcgcgcgga tcaacatctc ggaggggaat tgtccggaga gaatcatcac tctgaccggc421 ggcgcgcgga tcaacatctc ggaggggaat tgtccggaga gaatcatcac tctgaccggc

481 cccaccaatg ccatctttaa ggctttcgct atgatcatcg acaagctgga ggaagatatc481 cccaccaatg ccatctttaa ggctttcgct atgatcatcg acaagctgga ggaagatatc

541 aacagctcca tgaccaacag taccgcggcc agcaggcccc cggtcaccct gaggctggtg541 aacagctcca tgaccaacag taccgcggcc agcaggcccc cggtcaccct gaggctggtg

601 gtgccggcca cccagtgcgg ctccctgatt gggaaaggcg ggtgtaagat caaagagatc601 gtgccggcca cccagtgcgg ctccctgatt gggaaaggcg ggtgtaagat caaagagatc

661 cgcgagagta cgggggcgca ggtccaggtg gcgggggata tgctgcccaa ctccaccgag661 cgcgagagta cgggggcgca ggtccaggtg gcgggggata tgctgcccaa ctccaccgag

721 cgggccatca ccatcgctgg cgtgccgcag tctgtcaccg agtgtgtcaa gcagatttgc721 cgggccatca ccatcgctgg cgtgccgcag tctgtcaccg agtgtgtcaa gcagatttgc

781 ctggtcatgc tggagacgct ctcccagtct ccgcaaggga gagtcatgac cattccgtac781 ctggtcatgc tggagacgct ctcccagtct ccgcaaggga gagtcatgac cattccgtac

841 cagcccatgc cggccagctc cccagtcatc tgcgcgggcg gccaagatcg gtgcagcgac841 cagcccatgc cggccagctc cccagtcatc tgcgcgggcg gccaagatcg gtgcagcgac

901 gctgcgggct acccccatgc cacccatgac ctggagggac cacctctaga tgcctactcg901 gctgcgggct acccccatgc cacccatgac ctggagggac cacctctaga tgcctactcg

961 attcaaggac aacacaccat ttctccgctc gatctggcca agctgaacca ggtggcaaga961 attcaaggac aacacaccat ttctccgctc gatctggcca agctgaacca ggtggcaaga

1021 caacagtctc actttgccat gatgcacggc gggaccggat tcgccggaat tgactccagc1021 caacagtctc actttgccat gatgcacggc gggaccggat tcgccggaat tgactccagc

1081 tctccagagg tgaaaggcta ttgggcaagt ttggatgcat ctactcaaac cacccatgaa1081 tctccagagg tgaaaggcta ttgggcaagt ttggatgcat ctactcaaac cacccatgaa

1141 ctcaccattc caaataactt aattggctgc ataatcgggc gccaaggcgc caacattaat1141 ctcaccattc caaataactt aattggctgc ataatcgggc gccaaggcgc caacattaat

1201 gagatccgcc agatgtccgg ggcccagatc aaaattgcca acccagtgga aggctcctct1201 gagatccgcc agatgtccgg ggcccagatc aaaattgcca acccagtgga aggctcctct

1261 ggtaggcagg ttactatcac tggctctgct gccagtatta gtctggccca gtatctaatc1261 ggtaggcagg ttactatcac tggctctgct gccagtatta gtctggccca gtatctaatc

1321 aatgccaggc tttcctctga gaagggcatg gggtgcagct agaacagtgt aggttccctc1321 aatgccaggc tttcctctga gaagggcatg gggtgcagct agaacagtgt aggttccctc

1381 aataacccct ttctgctgtt ctcccatgat ccaactgtgt aatttctggt cagtgattcc1381 aataacccct ttctgctgtt ctcccatgat ccaactgtgt aatttctggt cagtgattcc

1441 aggttttaaa taatttgtaa gtgttcagtt tctacacaac tttatcatcc gctaagaatt1441 aggttttaaa taatttgtaa gtgttcagtt tctacacaac tttatcatcc gctaagaatt

1501 taaaaatcac attctctgtt cagctgttaa tgctgggatc catatttagt tttataagct1501 taaaaatcac attctctgtt cagctgttaa tgctgggatc catatttagt tttataagct

1561 tttccctgtt tttagttttg ttttgggttt tttggctcat gaattttatt tctgtttgtc1561 tttccctgtt tttagttttg ttttgggttt tttggctcat gaattttatt tctgtttgtc

1621 gataagaaat gtaagagtgg aatgttaata aatttcagtt tagttctgta atgtcaagaa1621 gataagaaat gtaagagtgg aatgttaata aatttcagtt tagttctgta atgtcaagaa

1681 tttaagaatt aaaaaacgga ttggttaaaa aatgcttcat atttgaaaaa gctgggaatt1681 tttaagaatt aaaaaacgga ttggttaaaa aatgcttcat atttgaaaaa gctgggaatt

1741 gctgtcttaa aaaaaaaaaa aaaaaaaaaa aa1741 gctgtcttaa aaaaaaaaaa aaaaaaaaaa aa

Claims (17)

PCBP1(Poly(RC) Binding Protein 1) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 포함하는, 침윤성 뇌암의 진단용 조성물.PCBP1 (Poly(RC) Binding Protein 1) A composition for diagnosing invasive brain cancer, comprising an agent for measuring the expression level of a protein or a gene encoding it. 제 1항에 있어서,The method of claim 1, 상기 뇌암은 교모세포종(Glioblastoma)인 것인, 침윤성 뇌암의 진단용 조성물.The brain cancer will be glioblastoma (Glioblastoma), a composition for diagnosis of invasive brain cancer. 제 1항에 있어서,The method of claim 1, 상기 뇌암은 암 종양구인 것인, 침윤성 뇌암의 진단용 조성물.The brain cancer is a cancer tumor cell, a composition for diagnosis of invasive brain cancer. 제 1항에 있어서,The method of claim 1, 상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체 또는 앱타머인 것인, 침윤성 뇌암의 진단용 조성물.The agent for measuring the expression level of the protein is an antibody or aptamer that specifically binds to the protein, a composition for diagnosis of invasive brain cancer. 제 1항에 있어서,The method of claim 1, 상기 유전자의 발현 수준을 측정하는 제제는 프라이머 쌍 또는 프로브인 것인, 침윤성 뇌암의 진단용 조성물.The agent for measuring the expression level of the gene is a primer pair or a probe, a composition for diagnosis of invasive brain cancer. 제 1항 내지 제 5항의 조성물을 포함하는 침윤성 뇌암의 진단용 키트.A kit for diagnosis of invasive brain cancer comprising the composition of claims 1 to 5. 목적하는 개체로부터 분리된 생물학적 시료에서,In a biological sample isolated from the subject of interest, PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 단계를 포함하는, 침윤성 뇌암을 예측하기 위한 정보 제공 방법.A method of providing information for predicting invasive brain cancer, comprising the step of measuring the expression level of the PCBP1 protein or a gene encoding the same. 제 7항에 있어서,The method of claim 7, 상기 목적하는 개체로부터 분리된 생물학적 시료에서 측정된 상기 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 정상 대조군에 비하여 낮은 경우, 상기 목적하는 개체의 뇌암이 침윤성이 높을 것으로 예측하는 것인, 침윤성 뇌암을 예측하기 위한 정보 제공 방법.When the expression level of the PCBP1 protein or the gene encoding it measured in a biological sample isolated from the target individual is lower than that of the normal control, it is predicted that the brain cancer of the target individual will have high invasiveness, invasive brain cancer. How to provide information to predict. 제 7항에 있어서,The method of claim 7, 상기 단백질의 발현 수준을 측정하는 단계는 웨스턴블랏, ELISA, 방사선면역분석, 면역확산법, 면역 전기영동, 조직 면역염색, 면역침전 분석법, 보체 고정 분석법, FACS, 질량분석, MRM 분석 및 단백질 마이크로어레이로 구성된 군으로부터 선택되는 적어도 하나를 통해 측정하는 것인, 침윤성 뇌암을 예측하기 위한 정보 제공 방법.The steps of measuring the expression level of the protein include Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, MRM assay and protein microarray. The method of providing information for predicting invasive brain cancer is measured through at least one selected from the group consisting of. 제 7항에 있어서,The method of claim 7, 상기 유전자의 발현 수준을 측정하는 단계는 중합효소연쇄반응, 역전사 중합효소연쇄반응, 경쟁적 중합효소연쇄반응, Nuclease 보호 분석(RNase, S1 nuclease assay), in situ 교잡법, 핵산 마이크로어레이 및 노던블랏으로 구성된 군으로부터 선택되는 적어도 하나를 통해 측정하는 것인, 침윤성 뇌암을 예측하기 위한 정보를 제공하는 방법.The step of measuring the expression level of the gene includes polymerase chain reaction, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray and Northern blot. The method of providing information for predicting invasive brain cancer is measured through at least one selected from the group consisting of. PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 포함하는, 뇌암의 예후 예측용 조성물.A composition for predicting the prognosis of brain cancer, comprising an agent for measuring the expression level of the PCBP1 protein or a gene encoding it. 제 11항에 있어서,The method of claim 11, 상기 뇌암은 교모세포종인 것인, 뇌암의 예후 예측용 조성물.The brain cancer is glioblastoma, a composition for predicting the prognosis of brain cancer. 제 11항에 있어서,The method of claim 11, 상기 뇌암은 암 종양구인 것인, 뇌암의 예후 예측용 조성물.The brain cancer is a cancer tumor cell, a composition for predicting the prognosis of brain cancer. 제 11항 내지 제 13항 중 어느 한 항을 포함하는 뇌암의 예후 예측용 키트.A kit for predicting the prognosis of brain cancer comprising any one of claims 11 to 13. 뇌암 환자로부터 분리된 생물학적 시료에 목적하는 후보물질을 처리하는 단계; 및Processing a target candidate material in a biological sample isolated from a brain cancer patient; And 상기 생물학적 시료에서 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준을 확인하는 단계를 포함하는, 침윤성 뇌암 치료제의 스크리닝 방법.A screening method for a therapeutic agent for invasive brain cancer comprising the step of checking the expression level of the PCBP1 protein or a gene encoding the same in the biological sample. 제 15항에 있어서,The method of claim 15, 상기 후보물질을 처리한 뒤, 상기 생물학적 시료에서 측정된 PCBP1 단백질 또는 이를 암호화하는 유전자의 발현 수준이 증가되는 경우, 침윤성 뇌암의 치료제로 선별하는 단계를 더 포함하는 것인, 침윤성 뇌암 치료제의 스크리닝 방법.When the expression level of the PCBP1 protein or the gene encoding the PCBP1 protein measured in the biological sample increases after the treatment of the candidate substance, the method further comprising the step of selecting as a therapeutic agent for invasive brain cancer, . 제 15항에 있어서,The method of claim 15, 상기 뇌암은 교모세포종인 것인, 침윤성 뇌암 치료제의 스크리닝 방법.The brain cancer is glioblastoma, the screening method of the therapeutic agent for invasive brain cancer.
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