WO2006080193A1 - Oncogene et kit de diagnostic utilisant ce dernier - Google Patents

Oncogene et kit de diagnostic utilisant ce dernier Download PDF

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Publication number
WO2006080193A1
WO2006080193A1 PCT/JP2006/300243 JP2006300243W WO2006080193A1 WO 2006080193 A1 WO2006080193 A1 WO 2006080193A1 JP 2006300243 W JP2006300243 W JP 2006300243W WO 2006080193 A1 WO2006080193 A1 WO 2006080193A1
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WO
WIPO (PCT)
Prior art keywords
acid sequence
amino acid
deleted
amino acids
seq
Prior art date
Application number
PCT/JP2006/300243
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English (en)
Japanese (ja)
Inventor
Hideaki Shimda
Takeshi Tomonaga
Kazuyuki Matsushita
Takenori Ochiai
Fumio Nomura
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National University Corporation Chiba University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by National University Corporation Chiba University filed Critical National University Corporation Chiba University
Priority to JP2007500455A priority Critical patent/JP4677566B2/ja
Publication of WO2006080193A1 publication Critical patent/WO2006080193A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • G01N33/57419—Specifically defined cancers of colon
    • 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 the field of cancer. Specifically, it relates to an oncogene and a diagnostic kit using the oncogene. Background art
  • Colorectal cancer (rectal cancer and colon cancer), one of the typical examples of cancer, is a malignant tumor that increases with age and peaks in the 60s. It is lymphatic, hematogenous, peritoneal Metastasis and other metastasis occur, and in particular, hematogenous metastasis often progresses to liver cancer. As with other malignant tumors, early detection of carcinoma is the most important issue as a countermeasure for colorectal cancer. In particular, cancer that has developed in the upper part of the large intestine has few subjective symptoms, so there is a high risk that the disease has already progressed at the time of discovery.
  • colorectal cancer has been mainly screened by fecal occult blood, diagnosed by serum markers such as CEA or CA19_9, and the course of treatment.
  • serum markers such as CEA or CA19_9
  • the positive rate is extremely low for cancer at an early stage (early cancer), making accurate diagnosis difficult. It was.
  • Patent Document 2 is useful because it suggests the FIR gene related to colorectal cancer and its modified form, there is still room for improvement regarding the identification of the entire form of the modified form and the accuracy as a tumor marker. In cancers where early detection is extremely important, very high accuracy is required as a function of the tumor marker.
  • an object of the present invention is to provide an oncogene that is effective in diagnosing colorectal cancer and a cancer diagnostic kit using the same. Disclosure of the invention
  • the present inventors extract the protein and mRNA from the normal part and the cancer part of the surgical specimen of the colon cancer patient, extract the protein and mRNA, and analyze them to specifically express the colorectal cancer cell.
  • two antigenic peptides that function with extremely high accuracy as tumor markers were discovered and the present invention was conceived. Specifically, the following means are adopted.
  • the polynucleotide according to any one of the following (a) to (d) is used.
  • (b) the amino acid described in SEQ ID NO: 2 A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, added, or deleted from the amino acid sequence in which the 9th to 37th amino acids are deleted from the acid sequence
  • (d) the amino acid sequence according to SEQ ID NO: 4 Encodes a protein comprising an amino acid sequence in which one or more amino acids are substituted, added, or deleted from the amino acid sequence from which amino acid
  • nucleic acid molecule containing the polynucleotide in the first means is used.
  • a cancer diagnostic kit using the polynucleotide and the specific factor in the first means is provided.
  • the term “specific factor” means that the affinity for the biological factor (eg, polynucleotide) is other unrelated (especially less than 30% identity). Or, in certain cases, less than 99% identity, in yet another embodiment, such as those having only point mutation differences) than affinity for a biological factor (eg, a polynucleotide or polypeptide) Is also significantly higher.
  • affinity can be measured by, for example, a hybridization assay or a binding assay.
  • the specific factor is at least one of a nucleic acid molecule, a polypeptide, a lipid, a sugar chain, an organic small molecule, and a complex molecule thereof
  • the cancer diagnosed by the cancer diagnostic kit is: Desirably, it is at least one of rectal cancer and colon cancer.
  • the step of determining the expression level of the polynucleotide described in any of the following (a) to (d), and the ratio of the calculated expression levels A step of calculating.
  • one of the samples is a sample collected from a non-cancerous tissue, and the other is a sample collected from a tissue suspected of being a cancerous tissue, for example. If so, you can determine that you are a high-risk person suspected of having cancer.
  • the step of obtaining the expression of the polynucleotide comprises using PCR, and the step of calculating the expression level ratio is to examine the presence or absence of the expression.
  • one of the samples is a sample collected from a non-cancerous tissue
  • the other is a sample collected from a tissue suspected of being a cancerous tissue, for example. If so, you can determine that you are a high-risk person suspected of having cancer.
  • the step of determining protein expression is to use Western blot
  • one of the two collected samples is a normal sample
  • the step of calculating the ratio of expression levels is It is also desirable to check for the presence or absence of expression.
  • an oncogene capable of diagnosing colorectal cancer with extremely high accuracy and a cancer diagnostic kit using the same can be provided.
  • FIG. 1 is a diagram showing amino acid sequence structures of FIR 542, PUF 60 and their variants.
  • FIG. 2 is a diagram showing the results of RT-PCR in Example 1.
  • Figure 3 shows the results shown in Figure 2 more clearly.
  • FIR refers to an FBP interaction repressor, and specifically refers to a factor specified by SEQ ID NO: 1 or SEQ ID NO: 2.
  • SEQ ID NO: 1 shows the base sequence of FIR
  • SEQ ID NO: 2 shows the amino acid sequence of FIR.
  • the FIR identified by SEQ ID NOs: 1 and 2 is also referred to herein as FIR 542 and is a normal sequence.
  • the function of FIR has been reported to suppress the expression of.
  • c-Myc is a transcription factor that regulates cell proliferation, differentiation and apoptosis. c Both My c expression up-regulation and down-regulation can induce apoptosis under certain circumstances.
  • FIR FBP interaction repressor
  • c-myc FIR transcriptional targets
  • Evaluation of colorectal tumors revealed an increase in c-my c expression (although often observed in colorectal cancer) regardless of increases in FIR mRNA and FIR protein levels. Eliminating FIR-mediated c-myc inhibition Resistance to FIR-driven apoptosis probably represents an important step in multistage carcinogenesis that ultimately leads to malignant colorectal cancer.
  • This embodiment relates to one variant of FIR.
  • the nucleotide sequence of one of the conventionally known FIR variants is shown in SEQ ID NO: 3, and its amino acid sequence is shown in SEQ ID NO: 4.
  • the FIR identified by SEQ ID NOs: 3 and 4 is called FIR 559 or PUF 60 because it has a length of 559 amino acid sequence (hereinafter, collectively referred to as “PUF 60 J” in this specification).
  • PUF 60 is said to be involved in RNA splicing, and corresponds to 17 amino acids between the amino acid at position 98 and the amino acid at position 99 in the amino acid sequence of FIR 542 shown in SEQ ID NO: 2.
  • the structure of the amino acid sequence of the modified FIR referred to in this specification is shown in FIG. FIG.
  • the variant of FIR referred to in this specification has a length of 530 amino acid sequences
  • PUF 6 It is a sequence in which amino acids from the 9th position to the 37th position of the 0 base sequence (SEQ ID NO: 4) are deleted (hereinafter referred to as “Short PUF 60” in this specification, see FIG. 1 (c)). .
  • the variant of FIR shown in Fig. 1 (d) has a length of 5 13 amino acid sequence, and not only the amino acid in which Short PUF 60 is deleted compared to PUF 60, but also FIR Compared to force S PUF 60, the amino acid that is deleted is also deleted. That is, for the amino acid sequence of UF 60, positions 9 to 37 (base positions 25 to 11 1 1), positions 99 to 1 15 (positions 295 and 295) The amino acid from the position to the position 345) is deleted (hereinafter referred to as “Short FIR” in this specification, see FIG. 1 (d)).
  • the Short PUF 60 and Short FIR referred to in can be used as indicators with extremely high accuracy. More specifically, S hort PUF 60, S ho For the two rt FIRs, only the presence or absence of these variants can be used as an indicator of cancer.
  • mRNA transcription product of a gene encoding FIR and ShortFIRR in cancer tissue and normal tissue was analyzed by RT-PCR.
  • frozen samples specimens were taken from cancer tissue and normal tissue immediately after surgery and frozen at 80 ° C and stored (hereinafter referred to as frozen samples) It is called a frozen specimen.) Then, an appropriate amount was separately taken out from the collected frozen specimen, and mRNA was extracted using RNeasyMinkit (manufactured by Qiagen).
  • This mRNA (5 g / 20 / z 1) is converted to cDNA by 1 st 'Strand cDNA Synthesis Kit for RT-PCR (AMV, catno. 1 483 1 88, Roche), This was amplified by PCR.
  • Specific conditions for the PCR method were 5′-g g c c c a c t a c a g a g c a c t c-3 ′ (SEQ ID NO: 7) as a forward primer, and 5, ⁇ g g g c t g g g c c a g g g t c a g-3 ′ (SEQ ID NO: 8) as a reverse primer. Also, after treating at 95 ° C for 1 minute, repeat the treatment at 94 ° ⁇ for 1 minute, 54 at 1 minute, 72 ° C for 10 minutes 30 times, and then at 72 ° C for 7 minutes. Done by processing.
  • frozen samples specimens were taken from cancer tissue and normal tissue immediately after surgery and frozen at 80 ° C and stored (hereinafter referred to as frozen samples) It is called a frozen specimen.)
  • the present invention can provide a gene for more reliably diagnosing colorectal cancer, and further provide a diagnostic kit that can be used to more accurately diagnose cancer.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Oncology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un oncogène capable de poser un diagnostic extrêmement précis du cancer du côlon, ainsi qu’un kit de diagnostic du cancer utilisant cet oncogène.
PCT/JP2006/300243 2005-01-06 2006-01-05 Oncogene et kit de diagnostic utilisant ce dernier WO2006080193A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2007500455A JP4677566B2 (ja) 2005-01-06 2006-01-05 癌遺伝子及びそれを利用した診断キット

Applications Claiming Priority (2)

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JP2005001765 2005-01-06
JP2005-001765 2005-01-06

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WO2006080193A1 true WO2006080193A1 (fr) 2006-08-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011129427A1 (fr) * 2010-04-16 2011-10-20 第一三共株式会社 Agent de diagnostic et agent thérapeutique pour les cancers

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018679A1 (fr) * 2002-08-23 2004-03-04 Japan Science And Technology Agency Methode et trousse de diagnostic du cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018679A1 (fr) * 2002-08-23 2004-03-04 Japan Science And Technology Agency Methode et trousse de diagnostic du cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 24 April 2006 (2006-04-24), XP003001146, accession no. NCBI Database accession no. (AF217197) *
LIU J. ET AL.: "The FBP interacting repressor targets TFITH to inhibit activated transcription", MOL. CELL, vol. 5, no. 2, 2000, pages 331 - 341, XP002986855 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011129427A1 (fr) * 2010-04-16 2011-10-20 第一三共株式会社 Agent de diagnostic et agent thérapeutique pour les cancers

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JP4677566B2 (ja) 2011-04-27
JPWO2006080193A1 (ja) 2008-06-19

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