WO2007034603A1 - Gène marqueur pour les tumeurs cérébrales malignes et utilisation de celui-ci - Google Patents

Gène marqueur pour les tumeurs cérébrales malignes et utilisation de celui-ci Download PDF

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WO2007034603A1
WO2007034603A1 PCT/JP2006/312267 JP2006312267W WO2007034603A1 WO 2007034603 A1 WO2007034603 A1 WO 2007034603A1 JP 2006312267 W JP2006312267 W JP 2006312267W WO 2007034603 A1 WO2007034603 A1 WO 2007034603A1
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brain tumor
tumor marker
marker gene
protein
expression level
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PCT/JP2006/312267
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English (en)
Japanese (ja)
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Ryuya Yamanaka
Kazuto Nishio
Masaru Sekijima
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Niigata University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G01N33/57407—Specifically defined cancers
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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

Definitions

  • the present invention relates to a marker gene that serves as an index of malignant brain tumor and use thereof.
  • Tumor cells proliferate out of order due to their own genetic abnormalities and lose their order, and therapies are primarily selected based on the molecular biological characteristics of these cells. Ideal. Even though the pathological characteristics of tumor cells are the same, their developmental forms, treatment sensitivity, and prognosis are very different, which is a major problem often encountered in daily clinical practice.
  • genetic abnormalities in tumor cells are related to abnormalities in multiple genes. Is essential.
  • the microarray method is an approach that simultaneously captures a large amount of changes in the transcription level of tumor cells, and can be applied to the diagnosis of individual tumor cells, sensitivity to treatment, resistance to drugs, prediction of side effects, identification of new target molecules, etc. . So far, molecular expression profiles using microarrays have been used to obtain molecular markers useful for therapeutic selection in various human cancer types. Examination of gene expression profiles using microarray technology has confirmed that many genes are differentially expressed in tumor histological types and grades.
  • Non-Patent Document 1 Freije WA, Castro- Vargas FE, Fang Z, Horvath S, Loughesy T, Liau
  • Non-Patent Document 2 Rickman DS, Bobek MP, Misek DE, Kuick R, Blaivas M, Kurnit DM,
  • Non-Patent Document 3 Sallinen SL, SallinenPK, Haapasalo HK, Helin HJ, Helen PT, Schraml P, KallioniemiOP, Kononen J. Cancer Res. 2000 Dec 1; 60 (23): 6617—22.
  • Non-Patent Document 4 Kim S, Dougherty ER, Shmulevich I, Hess KR, Hamilton SR, Trent J
  • Non-Patent Document 5 van den Boom J, Wolter M, Kuick R, Misek DE, Youkilis AS, Wechsle r DS, Sommer C, Reifenberger G, Hanash SM. Am J Pathol. 2003 Sep; 163 (3): 1033-
  • the search for new molecular targets for malignant brain tumors is expected to be developed by the microarray method, which is a systematic gene expression information analysis.
  • the results are expected to be applied to the development of new treatments and diagnostics for malignant brain tumors.
  • the present invention has been made in view of the circumstances as described above, and aims to provide a marker gene that serves as an index of malignant brain tumor for the development of a new therapeutic method and diagnostics, and its use. To do.
  • EAS guanyl releasing protein ⁇ (calcium 3 2 0 so-called -005825 and DAG-regul ated)
  • KIAA0537 gene product (AMP-auctivated 5 2 2 so-called —014840 protein kinase family member 5)
  • KIAA0449 protein (kinesin family member 1 2 2 9 ⁇ _371332 ⁇ 1 ⁇ )
  • sperm associated antigen 7 1 4 3 1 ⁇ _004890 spermine s nthase 1 5 3 2 ⁇ —004595
  • the present invention is as follows.
  • Claim 1 of the present invention is a brain tumor marker gene characterized by having the nucleotide sequence of any one of SEQ ID NOs: 1 to 17.
  • Claim 2 of the present invention is a probe for detecting a brain tumor marker gene, characterized in that it has a base sequence that is noblyzed under stringent conditions with any one of the brain tumor marker genes of claim 1. It is.
  • Claim 3 of the present invention is a primer for detecting a continuous brain tumor marker gene having a length of 15 to 30 bases for specifically amplifying any one of the brain tumor marker genes of claim 1. is there.
  • Claim 4 of the present invention is a DNA chip characterized in that at least one of the probe for detecting a brain tumor marker gene according to claim 2 is immobilized.
  • Claim 5 of the present invention is a method for diagnosing malignant brain tumor, comprising the following steps (a) and (b): (a) A biological sample prepared from a subject. A step of measuring the expression level of at least one brain tumor marker gene of the brain tumor marker single gene according to claim 1, in the RNA or complementary polynucleotide in which the RNA force is also transcribed, and (b) the expression level is healthy. A process of diagnosing a malignant brain tumor in a large number of subjects as compared to that of.
  • Claim 6 of the present invention comprises a substance that suppresses the expression of a brain tumor marker gene according to claim 1 characterized in that it comprises the following steps (a), (b), and (c): Screening method: (a) express at least one of the test substance and the brain tumor marker gene according to claim 1! (B) contacting the cells to be injured, (b) measuring the expression level of the brain tumor marker gene in the cells in contact with the test substance and not contacting the test substance! / Corresponding to the above in the control cells A step of comparing with the expression level of the brain tumor marker gene, (c) selecting a test substance that decreases the expression level of the brain tumor marker gene based on the comparison result of (b) above.
  • Claim 7 of the present invention is a diagnostic kit for a malignant brain tumor, comprising at least one or more selected from the group forces consisting of the following (a) to (c). (A) at least one probe for detecting a brain tumor marker gene according to claim 2, (b) at least one primer for detecting a brain tumor marker gene according to claim 3, (c) according to claim 4. DNA chip.
  • Claim 8 of the present invention is a brain tumor marker protein comprising a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 18 to 34.
  • Claim 9 of the present invention is an antibody for detecting a brain tumor marker protein, characterized by specifically recognizing any one of the brain tumor marker proteins of claim 8.
  • Claim 10 of the present invention is a protein chip characterized in that at least one antibody for detecting a brain tumor marker protein selected also for the antibody for detecting a brain tumor marker protein according to claim 9 is immobilized. is there.
  • Claim 11 of the present invention is a method for diagnosing malignant brain tumor, comprising the following steps (a) and (b): (a) in a protein prepared from a biological sample of a subject. A step of measuring the expression level of at least one brain tumor marker protein of the brain tumor marker protein according to claim 8, and (b) diagnosing a subject having a higher expression level than that of a healthy subject as a malignant brain tumor.
  • Claim 12 of the present invention comprises the following steps (a), (b), and (c): A screen of a substance that suppresses the expression of a brain tumor marker protein according to claim 8 -A method of: (a) contacting the test substance with a cell expressing at least one of the brain tumor marker proteins according to claim 8, (b) the brain tumor in the cell contacted with the test substance Measuring the expression level of the marker protein and comparing it with the expression level of the brain tumor marker protein corresponding to the above in the control cells not contacted with the test substance, (c) based on the comparison result in (b) above, And selecting a test substance that reduces the expression level of the brain tumor marker protein.
  • Claim 13 of the present invention is a diagnostic kit for a malignant brain tumor, comprising at least one or more selected from the group forces consisting of the following (a) to (b): (a) The protein chip according to claim 10, wherein at least one of the antibodies for detecting a brain tumor marker protein according to claim 9, and (b).
  • the invention's effect is not limited to: (a) The protein chip according to claim 10, wherein at least one of the antibodies for detecting a brain tumor marker protein according to claim 9, and (b).
  • a brain tumor marker gene serving as a tumor marker for malignant brain tumor is provided.
  • various molecular biological materials related to this brain tumor marker gene are provided.
  • drug discovery such as a novel anticancer agent.
  • Detection for brain tumor marker gene For example, a brain tumor marker gene can be detected and a brain tumor can be diagnosed widely and reliably. It is also possible to screen for substances that suppress brain tumors and advance the development of new anticancer agents.
  • FIG. 1 shows the results of immunohistological analysis of protein level expression of selected genes in glioma tissue.
  • FIG. 2 is a graph showing a correlation between protein expression level and patient survival time.
  • FIG. 3 is a diagram showing the results of analysis of cell proliferation using Tetm cororone after introduction of siRNA for each gene into a Dario cell line.
  • FIG. 4 shows the results of introducing siRNA for each gene into a Dario cell line and analyzing the expression level of each gene by quantitative PCR.
  • the “brain tumor” of the present invention refers to all tumors that develop in the cranium.
  • a typical one with a high incidence is glioma, which further includes astrocytoma, glioblastoma and the like.
  • Other brain tumors include pituitary adenomas and schwannomas.
  • the “gene” in the present invention includes DNA and RNA.
  • DNA includes, for example, cDNA genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof.
  • Single-stranded DNA which may be double-stranded or single-stranded, may be a coding DNA that is a sense strand or an anti-coding strand that is an antisense strand.
  • the "polynucleotide” in the present specification is used to include RNA and DNA!
  • the DNA includes any of cDNA, genomic DNA, and synthetic DNA.
  • the above RNA includes total RNA, mRNA, rRNA and synthetic RNA!
  • the brain tumor marker gene of the present invention refers to a gene that is hardly expressed at all in benign brain tumors or normal brain tissues and is highly expressed only in malignant brain tumors.
  • the brain tumor marker gene of the present invention is characterized by having the base sequence described in any one of SEQ ID NOs: 1 to 17.
  • the gene accession numbers shown in Table 1 can be used in the NCBI gene database.
  • the brain tumor marker gene of the present invention can be obtained by a known genetic manipulation technique.
  • the probe for detecting a brain tumor marker gene of the present invention is any one of SEQ ID NOs: 1 to 17.
  • the selected partial sequence may be any portion, but is characterized in that it is a nucleotide sequence that is hybridized with or without stringent conditions with a brain tumor marker gene.
  • the brain tumor marker gene detection probe may be labeled with a radiolabel, a fluorescent label, or the like.
  • the probe for detecting a brain tumor marker gene can be DNA or RNA complementary to the selected sequence based on the selected partial sequence. For example, by preparing a DNA probe of an appropriate length from the nucleotide sequence of the brain tumor marker gene of the present invention, appropriately attaching a label such as a fluorescent label, and hybridizing it with a subject, the brain tumor marker gene To detect brain tumors.
  • the conditions for "nobbling with a brain tumor marker gene under stringent conditions" are, for example, 42 ° C. And 1 X SSC (0.15 M NaCl, 0.015 M sodium citrate), wash at 42 ° C with a buffer containing 0.1% SDS. More preferably, a washing treatment at 65 ° C with a buffer solution containing hybridization at 65 ° C and 0.1 X SSC, 0.1% SDS can be mentioned.
  • the primer for detecting a brain tumor marker gene of the present invention specifically amplifies any one of the brain tumor marker genes having the base sequence described in any one of SEQ ID NOs: 1 to 17. This is a primer for detecting a continuous brain tumor marker gene having a length of 15 to 30 nucleotides.
  • the target brain tumor marker gene can be amplified based on a known method such as PCR.
  • Primers can be easily designed and amplified by conventional methods such as using commercially available primer design software based on the nucleotide sequence (SEQ ID NOs: 1 to 17) of the brain tumor marker gene of the present invention.
  • the primer may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, etc.), or may be modified with piotin, phosphate, amine, or the like.
  • the DNA chip of the present invention is capable of detecting a brain tumor marker gene having a nucleotide sequence that is hybridized under stringent conditions with any one of the brain tumor marker genes having any one of the nucleotide sequences of SEQ ID NOS: 1 to 17. It is characterized in that at least one probe for use is immobilized.
  • a method for producing a DNA chip is known to those skilled in the art. By using a DNA chip, the expression level of the brain tumor marker gene of the present invention can be detected and measured.
  • the method for diagnosing malignant brain tumor of the present invention comprises the following steps (a) and (b): (a) RNA prepared from a biological sample of a subject or the RNA cartridge Measuring the expression level of at least one brain tumor marker gene of the brain tumor marker gene having the base sequence of any one of SEQ ID NOS: 1 to 17 in a complementary polynucleotide transcribed from (b) the expression level The process of diagnosing malignant brain tumors in many subjects compared to that of healthy individuals.
  • a "subject” is a patient who has been diagnosed as suspected of having a brain tumor, for example, by MRI examination, and the "biological sample” is a patient. It is the excised tissue or blood from the brain.
  • the biological sample to be evaluated can be any sample containing a marker gene.
  • RNA can be prepared from a biological sample from a patient's brain according to a conventional method.
  • a complementary polynucleotide transcribed from the RNA can be prepared from a tissue excised from the patient's brain or blood according to a conventional method. For example, cDNA is synthesized and PCR amplified using mRNA isolated from the subject's cell strength as a cage.
  • the malignant brain tumor diagnosis method of the present invention is carried out by detecting and measuring the expression level of any of the brain tumor marker genes of the present invention in the biological sample.
  • the base according to any one of SEQ ID NOS: 1 to 17 in the biological sample of the subject based on the expression level of the brain tumor marker gene having the base sequence described in any one of SEQ ID NOS: 1 to 17 in the biological sample of a healthy person If the expression level of the brain tumor marker gene having the sequence is higher than the above criteria, a malignant brain tumor is determined.
  • the detection method of the present invention includes the expression level of the brain tumor marker gene by a known method such as RT-PCR, Northern blotting, DNA microarray analysis, in situ hybridization analysis, etc. Measure.
  • the probe for detecting a brain tumor marker gene or the primer for detecting a brain tumor marker gene of the present invention can be used as appropriate.
  • a screening method for a substance that suppresses the expression of a brain tumor marker gene having any one of the nucleotide sequences of SEQ ID NOS: 1 to 17 of the present invention comprises the following steps (a), (b) and (c): (A) contacting a test substance with a cell expressing at least one of the brain tumor marker genes having the nucleotide sequence of any one of SEQ ID NOS: 1 to 17, (b) ) Measuring the expression level of the brain tumor marker gene in the cells contacted with the test substance, and comparing the expression level of the brain tumor marker gene corresponding to the above in the control cells without contact with the test substance; (C) A step of selecting a test substance that decreases the expression level of the brain tumor marker gene based on the comparison result of (b) above.
  • the screening method for a substance that suppresses the expression of a brain tumor marker gene of the present invention provides a candidate substance that suppresses a malignant brain tumor by searching for a substance that decreases the expression level of the brain tumor marker gene. .
  • Examples of cells used in the screening of the present invention include all cultured cells that express a brain tumor marker gene having the base sequence described in any one of SEQ ID NOs: 1 to 17. Whether or not these brain tumor marker genes are expressed in cultured cells can be easily confirmed by detecting them by a known Western plot method or the like. Specifically, for example, a living tissue or blood cell-derived cell isolated or prepared from a cancer patient, or the brain tumor marker gene of the present invention was introduced as a cultured cell. A cell etc. can be mentioned.
  • the conditions for bringing the test substance into contact with the cells are not particularly limited, but the culture conditions (temperature, pH) in which the cells do not die and can express the brain tumor marker gene of the present invention. It is preferable to select a medium composition).
  • Cell force RNA expressing at least one of the brain tumor marker genes is prepared, and the brain tumor marker gene is measured.
  • a transcribed complementary polynucleotide transcribed from the RNA cartridge may be prepared to measure the brain tumor marker gene.
  • the expression level of the brain tumor marker gene is measured by a known method such as RT-PCR method, Northern blot method, DNA microarray analysis method, in situ hybridization analysis method.
  • the probe for detecting a brain tumor marker gene or the primer for detecting a brain tumor marker gene of the present invention can be used as appropriate. Use the DNA chip of the present invention.
  • the diagnostic kit for malignant brain tumor of the present invention is characterized by comprising at least one or more of the following (a) to (c) force group force: (a) SEQ ID NOs: 1 to 17 At least one of the probes for detecting a brain tumor marker gene having a base sequence that hybridizes under stringent conditions with any one of the brain tumor marker genes having the base sequence described in any one of the above, (b) SEQ ID NOs: 1 to 17 Any one of the brain tumor marker genes having a nucleotide sequence according to any one of the above, wherein at least one of the primers for detecting a continuous brain tumor gene having a length of 15 to 30 bases, c) A probe for detecting a brain tumor marker gene having a nucleotide sequence that is hybridized under stringent conditions with any one of the brain tumor marker genes having the nucleotide sequence of any one of SEQ ID NOS: 1 to 17 A DNA chip with at least one of them immobilized.
  • the brain tumor marker protein of the present invention refers to a protein that is hardly or not expressed in benign brain tumors or normal brain tissues and is highly expressed only in malignant brain tumors.
  • the brain tumor marker protein of the present invention is characterized by having a polypeptide consisting of the amino acid sequence described in any one of SEQ ID NOs: 18 to 34. Moreover, it can be approached by Gene Accession Number in Table 1 in NCBI gene database.
  • the antibody for detecting a brain tumor marker protein of the present invention specifically recognizes any one of the brain tumor marker proteins having a polypeptide having an amino acid sequence ability described in any one of SEQ ID NOs: 18 to 34, And Since the antibody for detecting brain tumor marker protein of the present invention has a property of specifically binding to the brain tumor marker protein, the brain tumor marker protein expressed in the tissue of a subject by using the antibody for detecting brain tumor marker protein is used. Can be detected specifically
  • the method for producing an antibody is known, and the antibody for detecting a brain tumor marker protein of the present invention can also be produced according to a conventional method.
  • the antibody for detecting a brain tumor marker protein of the present invention is a polyclonal antibody
  • the brain tumor marker protein of the present invention expressed and purified in Escherichia coli or the like according to a conventional method, or according to a conventional method.
  • the oligopeptide having the partial amino acid sequence of the brain tumor marker protein of the invention can be synthesized to immunize non-human animals such as rabbits and obtained from the sera of the immunized animals according to a conventional method.
  • a monoclonal antibody it is obtained by immunizing a non-human animal such as a mouse with the brain tumor marker protein of the present invention or an oligopeptide having a partial amino acid sequence of the protein expressed and purified in Escherichia coli according to a conventional method.
  • the obtained spleen cells and myeloma cells can be obtained from the cells prepared from cell fusion of the spleen cells and myeloma cells.
  • the antibody may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, etc.), or may be appropriately modified with piotin or the like!
  • a brain tumor marker protein used as an immunizing antigen for producing an antibody for detecting a brain tumor marker protein is a DNA cloning based on the sequence information (SEQ ID NOs: 1 to 17) of the gene provided by the present invention, It can be obtained by constructing each plasmid, transfection into the host, culturing the transformant and recovering the protein from the culture. These operations can be performed according to methods known to those skilled in the art.
  • the protein chip of the present invention is an antibody for detecting a brain tumor marker protein that specifically recognizes any one of brain tumor marker proteins having a polypeptide having the amino acid sequence strength described in any one of SEQ ID NOs: 18 to 34. It is characterized by fixing at least one or more. Methods for producing protein chips are known to those skilled in the art. [0047]
  • the method for diagnosing malignant brain tumor of the present invention is characterized by comprising the following steps (a) and (b): (a) SEQ ID NOs: 18 to 34 in a protein whose biological sample force of a subject is also adjusted.
  • a step of measuring the expression level of at least one brain tumor marker protein of the brain tumor marker protein having a polypeptide having an amino acid sequence ability according to any one of (1), and (b) the expression level is higher than that of a healthy subject The process of diagnosing many subjects with malignant brain tumors.
  • a "subject” is a patient who is diagnosed as having a suspected brain tumor by, for example, MRI examination, and the "biological sample” is a patient. It is the excised tissue or blood from the brain. Proteins can also be prepared from biological specimens from excised tissues from the patient's brain or blood according to conventional methods.
  • the malignant brain tumor diagnosis method of the present invention is carried out by detecting and measuring the expression level of any of the brain tumor marker proteins in the biological sample. Based on the expression level of the brain tumor marker protein having the base sequence described in any one of SEQ ID NOS: 1 to 17 in a healthy subject's biological sample, the base sequence described in any one of SEQ ID NOS: 1 to 17 in the subject's biological sample is used. If the expression level of the brain tumor marker protein possessed is higher than the above criteria, a malignant brain tumor is determined. Specifically, a part of a patient's tissue is collected based on a known method, and the protein is prepared according to a conventional method.
  • the expression level of the brain tumor marker protein can be detected based on a known detection method such as Western blotting, ELISA, or fluorescent antibody method.
  • a known detection method such as Western blotting, ELISA, or fluorescent antibody method.
  • the above-mentioned antibody for detecting brain tumor protein can be appropriately used.
  • the expression level of the brain tumor marker protein may be detected by using the protein chip.
  • a screening method for a substance that suppresses the expression of a brain tumor marker protein having a polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 to 34 of the present invention includes the following steps (a), (b) and (c) comprising: (a) a test substance and a cell expressing at least one of brain tumor marker proteins having a polypeptide having an amino acid sequence ability according to any one of SEQ ID NOs: 18 to 34; (B) measuring the expression level of the brain tumor marker protein in the cell contacted with the test substance, and comparing it with the expression level of the corresponding brain tumor marker protein in the control cell not contacted with the test substance Step (c) Based on the comparison result of (b) above, brain tumor marker protein Selecting a test substance that reduces the expression level of the quality.
  • the screening method for a substance that suppresses the expression of a brain tumor marker protein of the present invention provides a candidate substance that suppresses a malignant brain tumor by searching for a substance that decreases the expression level of the brain tumor marker protein.
  • Examples of the cells used in the screening of the present invention include all cultured cells that express a brain tumor marker protein having a polypeptide having an amino acid sequence ability described in any one of SEQ ID NOs: 18 to 34. Whether or not these brain tumor marker proteins are expressed in cultured cells can be easily confirmed by detecting them by a known Western plot method or the like. Specific examples of the cultured cells include cells derived from living tissue or blood cells isolated and prepared from cancer patients, or cells into which the brain tumor marker gene of the present invention has been introduced.
  • the conditions for bringing the test substance into contact with the cells are not particularly limited, but the culture conditions (temperature, pH) in which the cells do not die and can express the brain tumor marker protein of the present invention. It is preferable to select a medium composition).
  • a cell force protein that expresses at least one of the brain tumor marker proteins is prepared and a brain tumor marker gene is measured.
  • the expression level of the brain tumor marker gene is measured by a known method such as a Western plot method, an ELISA method, or a protein microarray analysis method.
  • the above-mentioned antibody for detecting brain tumor marker protein can be appropriately used.
  • the expression level of brain tumor marker protein may be detected by using the protein chip.
  • the diagnostic kit for malignant brain tumor of the present invention comprises at least one or more of the following (a) to (b) selected group powers: (a) SEQ ID NOs: 18 to 34 At least one of the antibodies for detecting a brain tumor marker protein that specifically recognizes any one of the brain tumor marker proteins having a polypeptide comprising the amino acid sequence according to any one of the above, (b) any one of SEQ ID NOs: 18 to 34 A brain tumor marker protein detection antibody that specifically recognizes any one of the brain tumor marker proteins having a polypeptide having the amino acid sequence ability described in 1 was immobilized. Protein chip. Configure the diagnostic kit for malignant brain tumor (a)-(b) as above By using it, a malignant brain tumor can be diagnosed.
  • the subjects were 52 gliomas (WHO gra dell: 16 cases, ⁇ : 14 cases, IV: 22 cases) that were surgically removed at Niigata University Brain Research Institute.
  • High-grade patients were treated with radiation therapy (local tumor 40Gy, irradiation 20Gy) and physiologic therapy centered on Trosourea.
  • the survival period was defined as the patient's death date or the last visit date from the date of diagnosis. Histological diagnosis was based on WHO brain tumor histology.
  • the experiments described in this specification obtained informed consent from all target patients in accordance with the rules of the Niigata University Ethics Committee.
  • RNA quality was measured by rRNA ratio [28sZl8s] using Bioanalyzer 2100 (Agilent). The purified total RNA was stored in 70% ethanol at -80 ° C until use.
  • Each mRNA of eclampsia tissue was from clontech.
  • cDNA was synthesized using an agilent fluorescent direct label kit. Using 10 g of purified total RNA and oligo dT primer with ⁇ 7 promoter sequence, 1.25 ImM FluoroLink dCTP (Cy3-dCTP or Cy5-dCTP), 2 After reacting with 00 units of SuperScriptll reverse transcriptase for 1 hour at 42 ° C to synthesize the first strand cDNA, the second strand cDNA was synthesized, extracted with phenol Z chloroform, and purified with Phase Loc gel. Tumor RNA was labeled with Cy5, and normal brain tissue RNA was labeled with Cy3. The obtained cDNA was recovered by ethanol precipitation and stored at -80 ° C in 70% ethanol until use.
  • the gene expression of the primary cancer of brain tumor patients was tested using an agilent microarray. Hybridization is in 25 1 solution containing 2X Deposition Control Buffer 12.51, Cot-1 DNA 2.51, nuclease-free water 7.5; zl, cDNA 2.51. At 65 ° C for 17 hours. After washing the chip with 0.5% SSC / 0.1% SDS solution, the fluorescence intensity of each pixel is measured with an HP laser scanner, imaged, and further, each gene is detected in tumor tissue against normal brain tissue. Numerical values were calculated for the expression ratio. This experiment measured the expression of approximately 12729 genes in human brain tumor patients.
  • PTTGl pituitary tumor-transforming l
  • RAS guanylreleasing protein 2 calcium and DAG-
  • the target cases were examined. 5 micron sections were prepared, and after deparaffinization, they were heated in 121 mM sodium citrate (pH 6.0) at 121 ° C. for 10 minutes. 0. 3% H O
  • Washed Avidin-biotin-peroxidase system (Vectasm elite AB kit, VectorLabs, Burlingame, CA) reacted, 0.01% 3,3-Diamaminobenzidine (DAB) (Sigma) and PBS 0.1% Color was developed with hydrogen peroxide.
  • DAB 3,3-Diamaminobenzidine
  • siRNAs for each gene were from AMBION and QIAGEN.
  • KIF3C ID # 11118; PTTG, ID # 41900; ARK5, ID # 964; STAC, ID # 12720; RASGRP2, ID # 16857 (both AMBION), and s iRNA for KIF1C is YAMA-1 (sense: GGAGAAUCAGUACCGGAAA; Antisense: UU UCCGGUACUGAUUCUCC, QIAGEN) was used.
  • RNAiHuman / Mouse Control kit Qiagen was used for these transformations including non-silencing control, and experiments were performed according to the protocol.
  • PCR was performed at 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 15 minutes, 55 ° C for 5 minutes, and 72 ° C for 10 minutes.
  • the reaction product passed through a post-PCR melting cycle, and the fluorescence intensity was measured at each cycle.
  • the following primers were used for PCR amplification.
  • KIFlC Hs01034140-gl.
  • KIF3C Hs00158482-ml: PTTGl, Hs00851754-gl,; ARK5, H s00934230— ml,
  • STAC Hs01070510— ml,
  • RASGRP2 Hs01057113— ml ,; (Applied Bio systems Japan Company).
  • the quantitative PCR method shows that by introducing siRNA, the expression of ARK5, RAS, PTTG1, STAC, KI F1C, and KIF3C mRNA can be suppressed to 20-30% compared to control siRNA. ( Figure 4).

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Abstract

La présente invention concerne un gène marqueur pouvant servir d’indicateur d’une tumeur cérébrale maligne ainsi que l’utilisation de ce gène marqueur. L’invention concerne un gène marqueur de tumeur cérébrale ayant une quelconque des séquences de nucléotides présentées à la SEQ ID NOs:1-17; une protéine marqueur de tumeur cérébrale ayant un polypeptide comprenant la séquence d’acides aminés présentée dans une quelconque des SEQ ID NOs:18-34; un procédé de diagnostic de tumeur cérébrale maligne qui utilise l’expression du gène marqueur ou de la protéine marqueur en tant qu'indicateur; un procédé de criblage d’une substance capable d’inhiber l’expression du gène marqueur ou de la protéine marqueur; et un kit destiné au diagnostic d’une tumeur cérébrale maligne utilisant le gène marqueur ou la protéine marqueur.
PCT/JP2006/312267 2005-09-20 2006-06-20 Gène marqueur pour les tumeurs cérébrales malignes et utilisation de celui-ci WO2007034603A1 (fr)

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CN105219856A (zh) * 2015-09-29 2016-01-06 北京泱深生物信息技术有限公司 骨性关节炎特异性甲基化标记基因及其应用
CN105734147A (zh) * 2016-03-31 2016-07-06 北京泱深生物信息技术有限公司 Muc21基因及其表达产物在垂体瘤诊疗中的用途
CN108884463A (zh) * 2016-04-01 2018-11-23 东丽株式会社 恶性脑肿瘤的检测试剂盒或器件和检测方法

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JP6367802B2 (ja) * 2012-08-30 2018-08-01 トゥルン イリオピスト 個体別の脳がん療法を選択する方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105219856A (zh) * 2015-09-29 2016-01-06 北京泱深生物信息技术有限公司 骨性关节炎特异性甲基化标记基因及其应用
CN105734147A (zh) * 2016-03-31 2016-07-06 北京泱深生物信息技术有限公司 Muc21基因及其表达产物在垂体瘤诊疗中的用途
CN108884463A (zh) * 2016-04-01 2018-11-23 东丽株式会社 恶性脑肿瘤的检测试剂盒或器件和检测方法

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