WO2012011778A2 - Cancer diagnosis kit and pharmaceutical composition for prevention or treatment of cancer - Google Patents

Cancer diagnosis kit and pharmaceutical composition for prevention or treatment of cancer Download PDF

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WO2012011778A2
WO2012011778A2 PCT/KR2011/005433 KR2011005433W WO2012011778A2 WO 2012011778 A2 WO2012011778 A2 WO 2012011778A2 KR 2011005433 W KR2011005433 W KR 2011005433W WO 2012011778 A2 WO2012011778 A2 WO 2012011778A2
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cancer
gene
protein
c10orf81
expression level
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PCT/KR2011/005433
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French (fr)
Korean (ko)
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WO2012011778A3 (en
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정경숙
원미선
안지원
김영주
송은영
염영일
이희구
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한국생명공학연구원
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention provides a diagnostic composition for cancer comprising a preparation for measuring the expression level of mRNA or a protein of C10orf81 (Chromosome 10 open reading frame 81) gene, a kit for cancer diagnosis comprising the composition, mRNA expression level of the C10orf81 gene or the gene
  • a method for providing information for diagnosing cancer comprising measuring the level of expression of a protein encoded by the drug, screening a pharmaceutical composition for preventing or treating cancer, including a C10orf81 gene inhibitor or an activity inhibitor thereof It is about a method.
  • model organisms capable of simple function and morphology studies are increasing.
  • researches using model organisms, which have been used in various fields for a long time, for discovering targets for diagnosing or treating cancer are being conducted.
  • RNAi RNA interference
  • RNAi RNA interference
  • RNAi is a technique that induces the degradation of mRNA of the corresponding nucleotide sequence specifically in a cell using various types of oligonucleotides (miRNA, expressed dsRNA, synthetic siRNA) in a variety of forms to prevent the function of genes from appearing.
  • miRNA expressed dsRNA
  • siRNA synthetic siRNA
  • siRNA small interfering RNA
  • siRNA small interfering RNA
  • siRNA is a short 19-23 double ribonucleic acid chain, when introduced into the cell has the effect of suppressing the expression of a specific gene only without non-specific inhibition (promotes the production of cancer, inhibiting apoptosis)
  • siRNA has the effect of killing cancer cells by inhibiting the action of the gene in the cell and can be used to verify the therapeutic target gene. Therefore, siRNA research for the discovery of target genes for target diseases, validation of target genes, and development of therapeutic agents is very active.
  • C10orf81 gene chromosome 10 open reading frame 81, synonyms: FLJ23537, HEL185 (Epididymis luminal protein 185)
  • FLJ23537 chromosome 10 open reading frame 81
  • HEL185 Epidermal protein 1805
  • the present inventors have made diligent research efforts to find genes that are used for cancer diagnosis and drug screening and may be targets for cancer treatment. As a result, the inventors found that the expression of the C10orf81 gene in various cancer cells is significantly increased, and that there is an effect of cancer treatment when inhibited. By this, the present invention has been completed.
  • An object of the present invention is a cancer diagnostic composition selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer, comprising an agent for measuring the expression level of mRNA or protein of C10orf81 (Chromosome 10 open reading frame 81) gene To provide.
  • Another object of the present invention is to provide a cancer diagnostic kit selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, comprising the composition.
  • Another object of the present invention is to measure the mRNA expression level of the C10orf81 gene or the protein encoded by the gene from a biological sample of a cancer suspect patient; And (b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample, gastric cancer, liver cancer, colorectal cancer, It is to provide a method of providing information for cancer diagnosis selected from the group consisting of lung cancer and prostate cancer.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer comprising an inhibitor of expression of the C10orf81 gene or an activity inhibitor of a protein thereof. .
  • Another object of the present invention is to perform the steps of (a) analyzing the expression of the C10orf81 gene or the activity of the protein after treating the test substance; And (b) judging the expression of the C10orf81 gene or the activity of the protein thereof after the test substance is inhibited compared to the expression of the C10orf81 gene or the activity of the protein thereof without treatment of the test substance, the cancer treatment comprising: To provide a method for screening a cancer therapeutic agent selected from the group consisting of liver cancer, colon cancer, lung cancer and prostate cancer.
  • the present invention by providing genes specific for gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer and providing diagnostic markers for determining the metastasis and prognosis of the cancer, accurate cancer diagnosis is possible and treatment of cancer And it can be useful for prognosis management.
  • a cancer-specific therapeutic agent that inhibits the gene, it is possible to enable the development of bio-new drugs and customized anti-cancer agents with fewer side effects.
  • Figure 1a shows the results of analysis via RT-PCR changes in the amount of C10orf81 gene expression in human gastric cancer cell line, liver cancer cell line, colon cancer cell line and normal cells. GAPDH was used as a control gene.
  • Figure 1b shows the results of quantitative analysis of changes in the expression level of the C10orf81 gene in human gastric cancer cell line, liver cancer cell line, colon cancer cell line and normal cells. GAPDH was used as a control gene.
  • Figure 2 shows the results of the analysis and the data showing that the expression of the C10orf81 gene in 66 colorectal cancer clinical tissues through a microarray experiment using Illumina 48K chip.
  • Figure 3 shows the results of analyzing the change in the amount of expression of the C10orf81 gene in 10 colorectal cancer patients, 5 liver cancer tissues and gastric cancer tissues by RT-PCR. GAPDH was used as a control gene.
  • Figure 4 is a picture of the analysis of C10orf81 protein expression in colorectal cancer tissue using the C10orf81 antibody. Normal tissue was used as a comparison group.
  • 5 is a graph showing the change in cell growth rate due to overexpression of C10orf81 gene and the figure showing the growth promotion of cells by SRB staining.
  • Figure 6a shows the results of verifying the effect of C10orf81 siRNA by selecting the colorectal cancer cell line KM12C as a representative cell line.
  • Treatment of C10orf81 siRNA shows that the amount of actual C10orf81 mRNA is reduced, resulting in reduced protein.
  • 6B shows inhibition of cell growth according to treatment with siRNA of C10orf81.
  • FIG. 7 is a graph showing the effect of inhibiting cell growth rate by the treatment of siRNA in normal cells, gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer cell lines.
  • the present invention comprises gasoline, liver cancer, colorectal cancer, lung cancer and prostate, comprising an agent for measuring the expression level of the mRNA or protein thereof of the C10orf81 (Chromosome 10 open reading frame 81) gene It provides a cancer diagnostic composition selected from the group consisting of cancer.
  • the C10orf81 gene of the present invention also called FLJ23537 or HEL185 (Epididymis luminal protein 185), encodes a PH domain-containing protein.
  • the mRNA sequence of C10orf81 is as shown in SEQ ID NO: 1, and the ORF sequence is as shown in SEQ ID NO: 2.
  • SEQ ID NO: 3 shows the amino acid sequence of the protein expressed from the C10orf81 gene.
  • C10orf81 gene refers to DNA or mRNA of these genes, and is a concept that includes all or part of DNA or mRNA.
  • diagnosis refers to confirming the presence or characteristics of a pathological condition, and for the purposes of the present invention, the diagnosis is to identify whether cancer, gastric cancer, colon cancer, lung cancer, or prostate cancer develops.
  • the term "diagnostic marker, diagnostic marker, or diagnostic marker” is a substance capable of diagnosing cancer cells from normal cells, and increases or decreases cancer cells in comparison with normal cells.
  • Organic biomolecules such as visible polypeptides or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.
  • the cancer diagnostic markers of the present invention are encoded by the C10orf81 gene, which is expressed by gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer cells as compared to cells of normal colon tissue, Protein.
  • Gene expression levels in biological samples can be confirmed by identifying the amount of mRNA or protein.
  • the measurement of mRNA expression level is a process of confirming the presence and expression of mRNA of cancer marker genes in a biological sample to diagnose cancer, and can be known by measuring the amount of mRNA.
  • Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting or DNA chips, but are not limited thereto.
  • the agent for measuring the expression level of mRNA of the gene means a molecule that can be used for detection of the marker by confirming the expression level of C10orf81, a marker that increases expression in cancer cells, but is not limited thereto. It may be to include a primer or probe specifically binding to. Based on the C10orf81 mRNA sequence (SEQ ID NO: 1) and its ORF sequence (SEQ ID NO: 2), one of ordinary skill in the art can design primers or probes that specifically amplify specific regions of these genes.
  • the term "primer” refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, the PCR can be performed using the sense and antisense primers of C10orf81 polynucleotide to diagnose cancer through the generation of a desired product. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.
  • probe refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which is capable of specific binding with mRNA, and is labeled to indicate the presence or absence of a specific mRNA. You can check it. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, hybridization is carried out using a probe complementary to C10orf81 polynucleotide, and cancer can be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.
  • Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, "capsulation", substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, Phosphoramidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
  • the protein expression level measurement refers to a process of confirming the presence and expression level of a protein expressed from a cancer marker gene in a biological sample in order to diagnose cancer.
  • the antibody specifically binds to the protein of the gene. You can check the amount of protein using.
  • Assays for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoelectrophoresis.
  • Tissue immunity staining immunoprecipitation assay
  • immunoprecipitation Assay immunoprecipitation Assay
  • complement fixation assay Complement Fixation Assay
  • flow cytometry Fluorescence Activated Cell Sorter, FACS
  • protein chips protein chip
  • the agent for measuring the expression level of the protein refers to a molecule that can be used for detection of the marker by confirming the expression level of the protein expressed from the C10orf81 gene, which is a marker of increased expression in cancer cells, but is not limited thereto. May include an antibody specific for the protein. Based on the amino acid sequence of the protein expressed from the C10orf81 gene (SEQ ID NO: 3), those skilled in the art can design antibodies specific for the protein.
  • an antibody refers to a specific protein molecule directed to an antigenic site as it is known in the art.
  • an antibody refers to an antibody that specifically binds to a protein expressed from the C10orf81 gene, which is a marker of the present invention.
  • Such an antibody may be cloned into an expression vector according to a conventional method for the marker gene.
  • the protein encoded by is obtained and can be prepared by conventional methods from the obtained protein.
  • partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids.
  • the form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.
  • Antibodies used in the detection of cancer diagnostic markers of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.
  • the present invention provides a kit for diagnosing cancer selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, comprising the composition.
  • kits of the present invention can detect a marker by confirming the expression level of mRNA or a protein thereof in the expression level of the cancer diagnostic marker C10orf81.
  • Kits of the present invention may include primers, probes or optionally antibodies that recognize the expression level of a cancer diagnostic marker, as well as one or more other component compositions, solutions or devices suitable for the assay.
  • the kit may use the principle of diagnosing cancer by confirming a reaction between the C10orf81 gene and a sample obtained from a subject. Confirmation of the reaction between the C10orf81 gene and the sample obtained from the subject is a common method used to confirm the reaction between DNA-DNA, DNA-RNA, DNA-protein, such as DNA chip, protein chip, polymerase chain reaction (PCR). ), Northern blotting, Southern blotting, Enzyme Linked Immunosorbent assay (ELISA), yeast two-hybrid, 2-D gel analysis and in vitro binding assays can be used. .
  • all or part of the gene is used as a probe to hybridize with nucleic acid isolated from the body fluid of the subject, and then various methods known in the art, such as reverse transcription polymerase chain reaction and southern blotting. By detecting this by Northern blotting, the subject can determine whether the cancer has occurred by investigating whether the gene is in a high or low expression state.
  • the kit may use the principle of diagnosing cancer by confirming a reaction between a protein expressed from the C10orf81 gene and a sample obtained from a subject.
  • the C10orf81 gene is specifically increased in gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer cells, and thus not only to investigate whether the gene is overexpressed, but also to overexpress the protein expressed from the gene. Investigate whether the cancer can be diagnosed.
  • Confirmation of the reaction between the composition and the sample containing the protein in the cancer diagnostic kit of the present invention is a common method used to confirm the reaction between the DNA-protein, RNA-protein, protein-protein, such as DNA chips, protein chips Polymerase chain reaction (PCR), Northern blotting, Southern blotting, Western blotting, Enzyme Linked Immunosorbent assay (ELISA), specific immunostaining, yeast two-hybrid, 2- D gel analysis and in vitro binding assays can be used.
  • PCR Polymerase chain reaction
  • Northern blotting Northern blotting
  • Southern blotting Southern blotting
  • Western blotting Western blotting
  • Enzyme Linked Immunosorbent assay ELISA
  • specific immunostaining yeast two-hybrid
  • yeast two-hybrid 2- D gel analysis and in vitro binding assays
  • all or part of a protein expressed from the genes as a probe may be hybridized with a nucleic acid or protein isolated from a subject's body fluid
  • the kit of the present invention is not limited thereto, but may preferably be a microarray, a gene amplification kit or an immunoassay kit.
  • a probe is immobilized on the solid surface of the microarray.
  • the probe is used as a hybridizable array element and is immobilized on a substrate.
  • Preferred gases include suitable rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • Said hybridization array element is arranged and immobilized on said gas. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV.
  • the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface.
  • the hybridization array element may be coupled to the gas through a linker (eg, ethylene glycol oligomer and diamine).
  • sample DNA or RNA applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray.
  • Hybridization conditions may vary, and detection and analysis of the degree of hybridization may be carried out in various ways depending on the labeling substance.
  • the cancer diagnostic kit of the present invention can be carried out based on hybridization.
  • a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.
  • the label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides.
  • Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin And hapten with specific binding partners such as digoxigenin and chelating groups.
  • fluorophores eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)
  • chromophores eg fluorescein, phyco
  • Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham” (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology , 65: 499 (1986)).
  • the label provides a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.
  • Suitable hybridization conditions in the present invention can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). For example, among the stringent conditions, the higher stringency conditions were hybridized to 65 ° C.
  • high stringency conditions mean washing at 48 ° C. in 6 ⁇ SSC / 0.05% sodium pyrophosphate.
  • Low stringency means washing at 42 ° C. conditions, for example, at 0.2 ⁇ SSC / 0.1% SDS.
  • the hybridization signal coming out of the hybridization reaction is detected.
  • the hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe.
  • the probe is labeled by an enzyme
  • the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization.
  • Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium).
  • Nitrate resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate).
  • BCIP bromochloroindolyl phosphate
  • the kit of the present invention may preferably be a gene amplification kit.
  • amplification means a reaction that amplifies a nucleic acid molecule.
  • Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR.
  • real-time PCR differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.
  • DD-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR inverse polymerase chain reaction
  • TAIL-PCR thermal asymmetric interlaced PCR
  • the amplification reaction When carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel.
  • components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is desired to provide cofactors such as Mg 2+ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. The buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.
  • the kit of the present invention may be a kit for immunoassay, and may be performed using an antibody or aptamer specifically binding to the cancer marker of the present invention.
  • the antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
  • Antibodies may be commercially available and methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US patents) 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol. Biol. , 222: 58, 1-597 (1991)). It may be prepared by. General procedures for antibody preparation are described in Harlow, E.
  • Monoclonal antibodies are generally developed by using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a substrate thereof. It may be quantitatively analyzed, or may be directly quantitated using a conjugate of AP or HRP enzyme or the like to a monoclonal antibody directed against the protein.
  • an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a substrate thereof. It may be quantitatively analyzed, or may be directly quantitated using a conjugate of AP or HRP enzyme or the like to a monoclonal antibody directed against the protein.
  • Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.
  • a monoclonal antibody or a part of polyclonal antibody is also included in the antibody of the present invention as long as it has antigen binding, and all immunoglobulin antibodies are included.
  • the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the present invention provides a method for treating cancer, comprising the steps of: (a) measuring the mRNA expression level of the C10orf81 gene or the expression level of a protein encoded by a biological sample of a suspected cancer patient; And (b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample, gastric cancer, liver cancer, colorectal cancer, Provided is a method of providing information for diagnosing cancer selected from the group consisting of lung cancer and prostate cancer.
  • biological sample refers to tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in gene expression levels of cancer markers due to the development of gastric cancer, liver cancer, colon cancer, lung cancer, or prostate cancer. Samples such as, but not limited to.
  • Separation of mRNA or protein thereof from the biological sample may be performed using a known process, and the expression level of the mRNA or protein thereof may be measured by various methods.
  • the mRNA expression level is not limited thereto, but may be preferably by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. It may be more preferably by reverse transcriptase polymerase reaction or DNA chip.
  • the reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band by checking the mRNA expression and degree of the gene used as a cancer diagnostic marker and compare it with the control group, it is easy to determine whether cancer Diagnosis can be made.
  • the DNA chip is a DNA chip in which the nucleic acid corresponding to the cancer marker gene or a fragment thereof is attached to a glass-like substrate at a high density. The DNA chip is isolated from a sample, and the terminal or the inside thereof is labeled with a fluorescent material. cDNA probes can be prepared, hybridized to DNA chips, and read for cancer.
  • the measurement of the protein expression level is not limited thereto, but preferably may be to use an antibody specific for the protein.
  • an antibody specific for the protein By comparing the amounts of antigen-antibody complexes formed using the antibodies, it is possible to diagnose whether cancer has occurred, and the antibodies have been described above.
  • the protein expression level is not limited thereto, but preferably, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement It may be using a fixed assay, FACS or protein chip.
  • ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support.
  • Various ELISA methods can be included, such as indirect sandwich ELISA using labeled secondary antibodies that recognize the antibody.
  • the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It can be detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops.
  • the entire protein is isolated from the sample, electrophoresed to separate the proteins according to size, and then transferred to a nitrocellulose membrane to react with the antibody.
  • the amount of the generated antigen-antibody complex can be confirmed by using a labeled antibody to check the amount of the protein produced by the expression of a gene, thereby determining whether the cancer has developed.
  • the detection method comprises a method of examining the expression level of the marker in the control group and the expression level of the marker in the cancer-causing cells.
  • the expression level can be expressed as an absolute (eg ⁇ g / ml) or relative (eg relative intensity of signal) difference of the marker protein.
  • normal colon epithelial tissue and tissue suspected of cancer are collected and fixed, followed by preparation of paraffin embedding blocks by methods well known in the art. These are sliced to a thickness of several ⁇ and attached to a glass slide, and then the antibodies are reacted by a known method. The unreacted antibody is then washed and labeled with a detection label to read whether the antibody is labeled on the microscope.
  • the protein When using a protein chip in which one or more antibodies against a cancer marker are arranged at a predetermined position on a substrate and immobilized at high density, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex. By reading this, the presence or expression of the protein can be checked to determine whether the cancer develops.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cancer selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer comprising an inhibitor of the expression of the C10orf81 gene or a protein inhibitor thereof. to provide.
  • the inhibitor of expression of the C10orf81 gene included as an active ingredient in the pharmaceutical composition of the present invention is not limited thereto, but may preferably be an antisense oligonucleotide, siRNA, shRNA or microRNA specific to the C10orf81 gene.
  • the antisense oligonucleotide is a short length DNA synthesis strand (or DNA analog) that is antisense (or complementary) to a specific DNA or RNA target, to achieve gene-specific inhibition in vivo as well as in vitro. It has been used successfully.
  • Antisense oligonucleotides have been proposed to prevent the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides have been successfully used in cell culture and animal models of disease.
  • Antisense oligonucleotides as used herein include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and to increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications to oligonucleotide backbones, modifications of sugar moieties or bases known in the art.
  • the siRNA small interfering RNA
  • the siRNA molecule is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and because it can suppress expression of a target gene, it is an efficient gene knockdown method or gene therapy method.
  • a double stranded structure is formed in which the sense strand (the sequence corresponding to the C10orf81 mRNA sequence (SEQ ID NO: 1)) and the antisense strand (the sequence complementary to the C10orf81 mRNA sequence) are positioned opposite to each other. It may have a single chain structure with self-complementary sense and antisense strands.
  • siRNAs are not limited to the complete pairing of double-stranded RNA pairs that pair with RNA, but include mismatches (corresponding bases are not complementary), expansion / bulge (bases corresponding to one chain). None) and the like may be included.
  • the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the C10orf81 gene can be inhibited by RNA interference (RNAi) effects.
  • RNAi RNA interference
  • the cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.
  • the siRNA molecule is not limited thereto, but may have a total length of 15 to 30 bases, preferably 19 to 21 bases.
  • siRNA is a very strong drug against the inhibition of expression of specific genes in vivo in terms of long lasting effects in cell culture and in vivo, the ability to transfect cells in vivo and resistance to serum degradation. Has the potential.
  • Delivery of siRNAs and expression constructs / vectors including siRNAs are known to those skilled in the art.
  • US applications 2004/106567 and 2004/0086884 disclose many expression consensus as well as delivery mechanisms including viral vectors, nonviral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates. Provide truck / vector.
  • the shRNA is a single-stranded RNA having a length of 45 to 70 nucleotides, an oligo linking 3-10 base linkers between the sense strand of the target gene siRNA sequence and the complementary antisense strand.
  • cloning into plasmid vectors or inserting and expressing shRNAs into retroviruses lentivirus and adenovirus results in loops of hairpin structured shRNAs and intracellular dicers. It is converted into siRNA by (dicer) to show RNAi effect.
  • microRNA regulates various biological processes such as development, differentiation, proliferation, conservation and apoptosis.
  • MicroRNAs generally regulate the expression of the gene encoding the target mRNA by destabilizing the target mRNA or disrupting translation.
  • RNAs eg., constitutive promoters, inducible promoters, tissue specific promoters or combinations thereof
  • the expression inhibitor of the gene may be any material that inhibits the expression of the gene in addition to the antisense oligonucleotide, siRNA, shRNA or microRNA.
  • the inhibitor of activity of the protein expressed from the C10orf81 gene included as an active ingredient in the pharmaceutical composition of the present invention is not limited thereto, but may preferably be an antibody specific for the C10orf81 protein.
  • the monoclonal antibody to the protein may be produced and used through a monoclonal antibody production method common in the art, or may be commercially available.
  • a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through an antiserum production method conventional in the art.
  • the pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including an additional pharmaceutically acceptable carrier in addition to the active ingredient described above for administration.
  • suitable pharmaceutically acceptable carriers can include, for example, water, saline, phosphate buffered saline, dextrin, glycerol, ethanol, as well as combinations thereof.
  • a pharmaceutically acceptable carrier may consist of a minimum amount of auxiliary material, such as a wetting or emulsifying agent, preservative or buffer, which increases the shelf life or effectiveness of the binding protein.
  • the carrier may be included differently depending on the method of administration, and oral administration may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like.
  • a binder a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like.
  • buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used.
  • bases, excipients, lubricants, preservatives and the like can be used for topical administration.
  • Formulations of the compositions of the present invention can be prepared in a variety of mixtures with the pharmaceutically acceptable carriers described above.
  • oral administration in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampoules or in multiple dosage forms.
  • the present invention provides a method for treating cancer, comprising administering the pharmaceutical composition to a cancer suspect selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer, and prostate cancer.
  • the cancer suspected subject refers to all animals including humans having or may develop cancer, and the pharmaceutical composition comprising an inhibitor of expression of the C10orf81 gene of the present invention or an activity inhibitor of a protein thereof is included in the cancer suspected subject.
  • the individual can be treated efficiently.
  • the term "administration" means introducing a composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the desired tissue.
  • the method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount.
  • the effective amount is defined as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently.
  • 0.01ng / kg-10mg / kg for siRNA, 0.01ng for the antisense oligonucleotide for mRNA of the gene / kg-10mg / kg, 0.1ng / kg-10mg / kg for the compound, 0.1ng / kg-10mg / kg for the monoclonal antibody to the protein can be administered.
  • the present invention provides a method for treating a C10orf81 gene or analyzing a protein thereof after treating a test substance; And (b) judging the expression of the C10orf81 gene or the activity of the protein thereof after the test substance is inhibited compared to the expression of the C10orf81 gene or the activity of the protein thereof without treatment of the test substance, the cancer treatment comprising:
  • the present invention provides a method for screening a cancer therapeutic agent selected from the group consisting of liver cancer, colorectal cancer, lung cancer and prostate cancer.
  • the step (a) is a step of analyzing the expression of the C10orf81 gene or the activity of the protein after the test material
  • the assay can be analyzed in cells or in vitro, but is not limited thereto.
  • it can be analyzed using RT-PCR, northern blotting, cDNA microarray hybridization reaction, in situ hybridization reaction, radioimmunoassay, immunoprecipitation, ELISA, western blotting and the like.
  • test substance refers to an unknown substance used in screening to test whether the expression of the C10orf81 gene or the activity of a protein thereof is affected.
  • the test substance may be an individual substance that is estimated or randomly selected as a cancer metastasis inhibitor according to a conventional selection method, and may include, but is not limited to, nucleic acids, proteins, chemicals, and natural extracts. Can be.
  • the test substance analyzed by the screening method of the present invention may be a single compound or a mixture of compounds, and may be obtained from a library of synthetic or natural compounds.
  • the step (b) is a step of judging the expression of C10orf81 gene or the activity of the protein thereof after treatment with the test substance is inhibited compared to the expression of the C10orf81 gene or the activity of the protein thereof without treatment with the test substance.
  • the expression of the C10orf81 gene or the activity of the C10orf81 protein is down-regulated and measured as a cancer drug.
  • test substance that has a function of enhancing the expression of a cancer high expression gene or a protein activity obtained through the screening method of the present invention, and vice versa, a function of inhibiting the expression of a cancer high expression gene or inhibiting the activity of a protein.
  • the test substance in the former case, may be a cancer drug candidate by developing an inhibitor for the test substance, and in the latter case, it may be a cancer drug candidate.
  • Such candidates for cancer treatment will act as leading compounds in the development of cancer treatments in the future, and by modifying and optimizing the structure so that the leading substances can exhibit the inhibitory effect of the gene or protein expressed therefrom. Can develop new cancer therapies.
  • Example 1 Confirmation of the effect of increasing the amount of C10orf81 gene in cancer cell line
  • a simple way of estimating the cancer's relevance to a gene is to compare changes in the amount of expression of that gene in cancer tissue or cancer cell lines.
  • the relationship between the expression level according to the cancer production and progression should be investigated by comparing whether the cancer cell line is expressed or reduced in a large amount compared to normal cells. To do this, extract the total RNA from the cancer cell line to see how the expression of the gene is actually expressed in the cancer cell line, perform RT-PCR and Realtime-PCR using the oligonucleotide of the gene, and compare the amount of each gene with the normal cell line. The expression level of the gene in the cell is confirmed.
  • the expression level of the C10orf81 gene was analyzed for mRNA levels in human gastric cancer, liver cancer, colorectal cancer cell lines and normal cells.
  • RPMI1640 (GIBCO) cultures containing 10% fetal bovine serum (Fetal Bovine Serum, GIBCO), penicillin (10000 U / mL) and streptomycin (10 mg / mL) were used.
  • SNU620, SNU484 and SNU638 (all of Seoul National University Cell Line Bank) as gastric cancer cell lines, Huh7 (ATCC), Chang (ATCC) and SNU354 (Seoul National University Cell Line Bank) as liver cancer cell lines, KM12C (Seoul National University Cell Line Bank), Colo205 as colon cancer cell lines (Seoul National University Cell Line Bank), SW480 (ATCC) and HT29 (ATCC) were used.
  • IMR90 human fetal lung fibroblasts, ATCC # CCL-186 was used as a normal cell line. Cancer cell lines were inoculated with a cell number of 1 ⁇ 10 6 and cultured in an incubator at 37 ° C. with 5% CO 2 .
  • Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantified using Experion RNA StdSens (Bio-Rad) chip. The cells were collected by centrifugation and lysed by adding 10 ⁇ l of beta mercapto ethanol to 1 ml of digestion buffer RLN (50 mM TrisCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP-40) in the kit. . 1 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After two washes, total RNA was isolated by adding 100 ⁇ l of RNase-free water.
  • RNA / primer 2 ⁇ g of total RNA of each sample, 1 ⁇ l of 50 ⁇ M oligo (dT) primer and 2.5 ⁇ l of 10 mM dNTP were added and sterile water containing RNase inhibitor DEPC (diethyl pyrocarbonate) was added to make 25 ⁇ l total RNA / primer. A mixed solution was made. After reacting at 65 ° C. for 5 minutes, the mixture was transferred to 55 ° C. and stored.
  • RNase inhibitor DEPC diethyl pyrocarbonate
  • each cDNA was diluted 20-fold, and then PCR reaction was performed using 2 ⁇ l of the diluted sample.
  • PCR was performed using 15 ⁇ l of 2X PCR premix (Hot start), 2 ⁇ l of GAPDH 5 'primer, 2 ⁇ l of 3' primer, and 11 ⁇ l of distilled water, and 20, 23 and 25 cycles were performed.
  • PCR reaction conditions were performed at 94 °C 30 seconds, 50 °C 30 seconds and 72 °C 1 minute, the product size was 457 bp.
  • Each product was loaded into a 2% agarose gel, electrophoresed, electrophoresed, and gel images were taken. The images were quantified using the TotalLab v1.0 program (Nonlinear Dynamix), and then corrected again to perform PCR for quantification. The concentration of was equally corrected.
  • CDNA diluted to the same amount was PCR using the sense and antisense primers of the C10orf81 gene.
  • cDNA was mixed with 3 ⁇ l, 10 ⁇ l of 2X premix, 2 ⁇ l of primer each (20 pmole) and 2 ⁇ l of distilled water to make 20 ⁇ l of total solution.
  • the PCR reaction was performed at 94 ° C for 1 minute, 54 ° C for 30 seconds and 72 ° C for 1 minute. The cycle was performed.
  • it was electrophoresed using 2% agarose gel and analyzed using an imaging apparatus.
  • Realtime RT-PCR was used with DNASYBRI reagent from Qiagen (CA, USA) and LightCycler (Roche). Melt Curve analysis was used to assess the quality of PCR products and gene expression was analyzed using LightCycler version 3.5 software (Roche).
  • the C10orf81 specific primers used in the RT-PCR are as follows. N-terminal primer 5'-AAGAGGAACCCCAGACCCTT-3 '(SEQ ID NO: 4), C-terminal primer 5'-TCTCCACTTGGCTTTCATCG-3' (SEQ ID NO: 5). GAPDH primers for RT-PCR are N-terminal 5'-TCATGACCACAGTCCATGCC-3 '(SEQ ID NO: 6), C-terminal 5'-TCCACCACCCTGTTGCTGTA-3' (SEQ ID NO: 7), GAPDH used as control of Realtim-PCR is Qiagen Primer (QT00079247) was used.
  • Colorectal cancer and normal tissue from 66 patients with colorectal cancer were obtained from Samsung Medical Center (Seoul, South Korea). Each tissue was surgically removed from the patient and stored in liquid nitrogen until analysis.
  • QIAGEN kit RNeasy Maxi kit: cat # 75162
  • Experion RNA StdSens Bio-Rad chip.
  • the cancer clinical tissues and normal tissues were cut to an appropriate size, and then dissolved in 15 ml of a dissolution buffer in a kit containing 150 ⁇ l of beta mercapto ethanol. 15 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After washing twice, total RNA was isolated by adding 1.2 ml of RNase-free distilled water.
  • Fluorescently labeled DNA chips were scanned using a confocal laser scanner (Illumina, Inc.) to obtain fluorescence data present in each spot and stored as image files in TIFF form.
  • TIFF image files were quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence values of each spot. Quantified results were corrected using the 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.
  • FIG. 2 is a C10orf81 gene expression profile measured in cancerous and normal tissues of colorectal cancer patients, and is shown in red when high expressed and green when low expressed.
  • the C10orf81 gene increased the mean amount of expression 6 times in cancer tissues.
  • Example 2 Ten pairs of colorectal cancer patient samples (cancer tissue (T1-T66) and normal tissues (N1-N66) taken from 66 pairs of cancer patients in Example 2) , N1-N10)), four pairs of gastric cancer clinical samples (Samsung Medical Center), five liver cancer clinical samples (Samsung Medical Center) using the RT-PCR method to determine the expression level of the C10orf81 gene identified in Example 1 Analyzed through.
  • Total RNA was isolated by the method of Example 2 and cDNA was synthesized by the same method as in Example 1, and the concentration correction process and RT-PCR of the template was performed in the same manner as in Example 1.
  • N means normal tissue (non tumor tissue)
  • T means the corresponding cancer tissue.
  • Tissue slides were immunostained to confirm the presence and expression of proteins in normal colon epithelial and colon cancer tissues.
  • paraffin embedding blocks were made by removing colonic epithelial cells and colon cancer cells from colon cancer patients through surgical resection. This was cut to a thickness of 5 ⁇ m using a microtome and attached to a glass slide to form a tissue slide. These were stained by a known immunostaining method to confirm the presence and location of proteins in the tissue under a microscope.
  • the antibody used for immunization is an anti C10orf81 antibody (Novus, 1: 1000). As a result, in immunohistochemical staining, C10orf81 protein was expressed in cell membrane and cytoplasm, and was stronger than normal mucosa (FIG. 4).
  • the C10orf81 gene was amplified using PCR technique from Invitrogen's human brain c-DNA library.
  • the forward primer 5'-TGC GGATCC ATGGAACCCAAACCTCAGAAGA-3 ' (BamHI enzyme cleavage site comprises - underline, SEQ ID NO: 8) and reverse primer 5'-ATG GATATC TCATATCCGTCCTGTGGCTTC-3' (EcoRV cleavage site include an enzyme-underline, SEQ ID NO: 9)
  • Gene was amplified by PCR using pfu premix (iNtRON, Inc.) at 37 cycles at 55 °C.
  • the DNA band of the C10orf81 gene was purified, cut with BamHI / EcoRV restriction enzyme, and cloned into the pENTR3C vector.
  • DNA sequencing confirmed the nucleotide sequence (1089 bp) of the C10orf81 gene, and was cloned according to the manufacturer's manual using the GATE Way system (Invitrogen Corp. www.invitrogen.com).
  • pENTR3C-C10orf81 was cloned by LR reaction with pDEST (Invitrogen Corp. www.invitrogen.com).
  • the plasmid pENTR3C-C10orf81 was mixed with the vector pDEST DNA, and then the LR clonase II enzyme mixture was added and incubated for 1 hour at room temperature. After culturing, proteinase K was added and incubated at 37 ° C. for 10 minutes to complete the LR reaction. E. coli DH5 ⁇ competent cells were transformed to obtain a pDEST-C10orf81 clone.
  • DMEM medium containing a mixture of 5% bovine calf serum and penicillin (10,000 units / ml) and streptomycin (10 mg / ml) in a normal cell line, NIH3T3 fibroblasts (ATCC), diluted 100-fold.
  • GEBCO NIH3T3 fibroblasts
  • C10orf81 plasmid DNA was transfected into the cultured cells.
  • the cells were transfected with pDEST vector DNA and treated the same as the NIH3T3 cells to be used as a control.
  • the results are shown in FIG. 5, the horizontal axis represents time, and the vertical axis represents cell growth rate.
  • PDEST represents a cell transfected with the vector without the C10orf81 gene, and pDEST-C10orf81 transfects the vector cloned with the C10orf81 gene to overexpress the C10orf81 protein. Means cells.
  • the growth rate was increased by about 35% in cells overexpressing the C10orf81 gene, indicating that the C10orf81 gene is associated with increasing the growth rate of cells characteristic of tumor cells (FIG. 5). These results indicate that increased expression level of the C10orf81 gene may be a diagnostic marker for tumor cells.
  • Example 7 Confirmation of the growth inhibitory effect of the cancer cell line according to the introduction of siC10orf81 in the cancer cell line
  • siRNA techniques can be used to observe whether there is a change in cell proliferation rate by inhibiting gene expression.
  • each siRNA for C10orf81 was designed and introduced into the cancer cell line, and then the growth rate of the cancer cell line was observed according to the decrease in the gene mRNA expression.
  • Gastric cancer cell lines SNU484 (Seoul National University Cell Line Bank), SNU638 (Seoul National University Cell Line Bank), Liver Cancer Cell Chang (ATCC) and SNU354 (Seoul National University Cell Line Bank), Colon Cancer Cell Lines KM12C (Seoul National University Cell Line Bank) and Colo205 (Seoul National University Cell Line) Bank), lung cancer cell line A549 (ATCC), prostate cancer cell line PC3 (ATCC).
  • IMR90 human fetal lung fibroblasts, ATCC # CCL-186) was used as a normal cell line.
  • siRNA to be used as a control were designed and synthesized (Samchully Pharmaceutical, Korea).
  • the synthesized siRNA consists of a structure in which two bases of a single chain are suspended in a double ribonucleic acid chain of 19 oligonucleotides designed based on gene sequences.
  • Control siRNA was used as a negative control of siRNA experiments that had no effect on cell proliferation.
  • siC10orf81 (sense sequence, SEQ ID NO: 10) and siControl (sense sequence, SEQ ID NO: 11) were introduced into cells by hyperfect method (Qiagen Co.) for 72 hours. And transformed.
  • RNA was extracted from each cell to confirm that gene mRNA was reduced by RT-PCR, and protein expression was analyzed by Western blot to confirm that gene expression was inhibited by siRNA ( 6a).
  • siControl represents the negative control.
  • siControl shows no siRNA targets for sequences that are not homologous to human genes.
  • SRB Sulfur rhodamine B, Sigma Co.
  • siRNA sense sequence, SEQ ID NO: 11
  • siC10orf81 sense sequence, SEQ ID NO: 10
  • Table 1 shows an example of siRNA for the C10orf81 gene.
  • the present invention by providing genes specific for gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer and providing diagnostic markers for determining the metastasis and prognosis of the cancer, accurate cancer diagnosis is possible and treatment of cancer And it can be useful for prognosis management.
  • a cancer-specific therapeutic agent that inhibits the gene, it is possible to enable the development of bio-new drugs and customized anti-cancer agents with fewer side effects.

Abstract

The present invention relates to a composition for cancer diagnosis having a formulation for measuring the expression level of Chromosome 10 open reading frame 81 (C10orf81) gene mRNA or protein thereof, a kit for cancer diagnosis having the composition, a method for providing information on cancer diagnosis including a step for measuring the expression level of C10orf81 gene mRNA or the expression level of protein coded by the gene, and a method for screening pharmaceutical compositions for the prevention or the treatment of cancer including an expression inhibitor for the C10orf81 gene or an activity inhibitor for the protein thereof, and therapeutic agents for cancer. The present invention enables an accurate diagnosis of cancer by providing genes specific to stomach cancer, liver cancer, colorectal cancer, lung cancer, or prostate cancer, and diagnostic markers for determining the metastasis and prognosis of the cancer, and can be useful in the diagnosis and prognosis management of cancer. Also, the present invention enables the development of new bio-medicines having fewer side effects and customized anticancer drugs by providing a cancer-specific therapeutic agent capable of inhibiting the gene.

Description

암 진단 키트 및 암 예방 또는 치료용 약제학적 조성물Cancer Diagnostic Kits and Pharmaceutical Compositions for Preventing or Treating Cancer
본 발명은 C10orf81(Chromosome 10 open reading frame 81) 유전자의 mRNA 또는 이의 단백질의 발현수준을 측정하는 제제를 포함하는 암 진단용 조성물, 상기 조성물을 포함하는 암 진단용 키트, C10orf81 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 측정하는 단계를 포함하는 암 진단을 위한 정보의 제공 방법, C10orf81 유전자의 발현 억제제 또는 이의 단백질의 활성 억제제를 포함하는 암의 예방 또는 치료용 약제학적 조성물 및 암 치료제 스크리닝 방법에 관한 것이다.The present invention provides a diagnostic composition for cancer comprising a preparation for measuring the expression level of mRNA or a protein of C10orf81 (Chromosome 10 open reading frame 81) gene, a kit for cancer diagnosis comprising the composition, mRNA expression level of the C10orf81 gene or the gene A method for providing information for diagnosing cancer, comprising measuring the level of expression of a protein encoded by the drug, screening a pharmaceutical composition for preventing or treating cancer, including a C10orf81 gene inhibitor or an activity inhibitor thereof It is about a method.
인간 유전체 프로젝트 연구 결과, 인체의 질환을 분자 수준에서 이해하고 새로운 질환 관련 표적을 발굴하고 이를 이용한 신약을 개발하고자 하는 신개념의 바이오 생명공학 기술의 시대가 열리고 있다. 이에 따라, 유전적 환경이 다른 환자 개개인에 맞추어 각종 다양한 질병을 진단, 치료, 예측하는 맞춤의학 시대가 현실로 다가오고 있다. 맞춤의학에는 유전체 진단용 바이오마커의 발굴, 표적화 치료기술, 질병 특이적 약물작용점의 발굴, 표적 치료용 신약, 정확한 임상 정보, 환자의 유전체 정보, 유전체적 역학결과, 그리고 이들을 분석 통합할 수 있는 바이오 인포매틱스 등이 상호보완적으로 구성된 약물 유전체기술 개발이 수반되어야 한다. 특히, 맞춤의학에서 경쟁력을 갖추기 위해서는 질환과 개인에 대한 약물 예측 시스템, 진단 바이오마커 발굴, 새로운 표적유전자 및 단백질을 발굴하는 기초연구 및 이를 이용한 치료제의 개발이 중요하다.As a result of research on the human genome project, a new era of bio-biotechnology is opening up to understand human diseases at the molecular level, to discover new disease-related targets, and to develop new drugs using them. Accordingly, the era of customized medicine for diagnosing, treating, and predicting various diseases according to individual patients with different genetic environments is coming to reality. Personalized medicine includes the discovery of genome diagnostic biomarkers, targeted therapeutic technology, discovery of disease-specific drug action points, new drugs for targeted treatment, accurate clinical information, patient genome information, genomic epidemiologic results, and bioinformation that can integrate them. The development of drug genome technology that complements Matics should be accompanied. In particular, in order to be competitive in personalized medicine, it is important to develop a drug prediction system for a disease and an individual, to discover a diagnostic biomarker, to discover new target genes and proteins, and to develop a therapeutic agent using the same.
이와 같이, 암의 형성은 다양한 유전자들과 이들 유전자들의 발현 및 조절 기작이 복합적으로 연관되어 진행되므로 최근 들어 다량의 유전자를 사용하는 올리고 칩을 이용한 암 관련 유전자들의 발현률을 비교하여 암의 새로운 진단이나 치료의 마커를 발굴하기 위한 연구들이 이루어지고 있다. 암세포에서 발현이 증가하거나 감소하는 유전자들은 세포분열, 세포신호전달, 세포 골격, 세포 운동, 세포 방어, 유전자 및 단백질의 발현 그리고 세포내 물질 대사등 여러 부분에 관여하는 것으로 환자 조직들에 따라 동일한 발현변화를 보이는 유전자가 있는 반면 다른 발현 변화를 보이는 유전자들이 많다. 이것은 각각 환자들이 특이성 때문일 가능성이 크므로 연구하는 대상의 환자 조직들의 정확한 병리학적 소견과 분류에 따라야 하며 정확한 유전자를 이용한 진단에는 보다 많은 새로운 유전자들의 검색과 확인이 필요하다. 특정 세포내에서 특정 유전자의 발현 빈도를 조사함으로서 암 관련된 유전자의 발굴이 가능하며, 이를 통하여 암 진행의 분자적 메카니즘을 이해하게 되고, 나아가 암 진단 및 치료 타겟으로의 사용이 가능하게 될 것이다.As such, the formation of cancer proceeds by a combination of various genes and the expression and regulation mechanisms of these genes. In recent years, the expression rate of cancer-related genes using oligo chips using a large amount of genes is compared to a new diagnosis of cancer. Research is being done to uncover markers of treatment. Genes whose expression increases or decreases in cancer cells are involved in various parts of cell division, cell signaling, cytoskeleton, cell movement, cell defense, gene and protein expression, and intracellular metabolism. While there are genes that change, many genes show other changes in expression. This is likely due to the specificity of each patient, so the exact pathologic findings and classification of the patient's tissues under study should be followed. By investigating the frequency of expression of specific genes in specific cells, it is possible to discover cancer-related genes, thereby understanding the molecular mechanism of cancer progression, and furthermore, to be used as cancer diagnosis and treatment targets.
최근, 전 세계적으로 난치병인 암의 생성 및 치료와 관련된 유전자의 기능 연구를 통해 치료용 표적을 발굴하고 이들을 진단 및 치료제 개발에 이용하기 위한 치열한 경쟁이 불붙고 있다. 게놈 연구의 활성화와 함께 인간 유전자 DNA 칩 또는 프로테옴 분석 연구가 활발하게 이루어지고 암과 관련된 유전자가 대량 발굴됨에 따라 많은 유전자들의 목록과 관련 데이터베이스는 구축되어 있으나 대부분 이들 유전자들에 대한 세포 내에서의 구체적 생물학적 기능 및 암 관련성은 아직 연구되지 않았거나 불확실하여 실제 암 관련성 또는 진단 및 표적 유전자로 활용하기에 상당한 어려움이 있다.Recently, there has been a fierce competition for discovering therapeutic targets and using them in diagnosis and development of therapeutics through the study of genes related to the generation and treatment of cancers, which are incurable diseases worldwide. As human genome DNA chip or proteome analysis studies are actively conducted along with the activation of genomic research and a large number of genes related to cancer have been discovered, a list of many genes and related databases have been established, but most of these genes are specific within cells. Biological function and cancer relevance have not yet been studied or uncertain and there are significant difficulties in using them as actual cancer relevance or diagnosis and target genes.
또한, 수많은 인간 유전자들의 기능에 대한 연구의 필요성이 부각됨에 따라 단순한 기능 및 형태 연구가 가능한 모델 생물체에 대한 활용도 및 관심은 더욱 높아지고 있다. 이에, 세포 내에서 일어나는 기본적이고 진화적으로 잘 보존되어 있는 기작을 이해하기 위하여 오랫동안 여러 분야에서 사용되어 왔던 모델 생물체 (model organism)를 암 진단 또는 치료용 타겟 발굴에 이용하는 연구가 진행되고 있다.In addition, as the necessity of research on the function of numerous human genes is highlighted, the utilization and interest of model organisms capable of simple function and morphology studies are increasing. In order to understand the basic and evolutionarily well-preserved mechanisms occurring in cells, researches using model organisms, which have been used in various fields for a long time, for discovering targets for diagnosing or treating cancer are being conducted.
암세포에서 대량 발현된 유전자에 대해 RNAi(RNA interference) 실험 기법을 이용하면 치료용 암 타겟으로의 사용 가능성을 확실히 확인할 수 있다. RNAi는 다양한 형태의 올리고 이중 리보핵산(miRNA, expressed dsRNA, synthetic siRNA)을 이용하여 세포 내에서 특이적으로 해당 염기서열의 mRNA의 분해를 유도하여 유전자의 기능이 나타나지 못하도록 하는 기법이다. 이렇게 유전자의 발현이 저해된 세포의 표현 현상을 관찰함으로써, 해당 유전자의 기능 연구 및 신호전달 경로(pathway) 분석이 가능하여 암 표적으로서의 검증(target validation) 등을 통한 신약 개발 연구가 수행되고 있다. RNAi 기법에 사용 가능한 올리고 이중 리보핵산 중 최근 각광을 받고 있는 siRNA(small interfering RNA)를 이용한 기법은 2002년 Science Journal에서 "breakthrough of the year"라고 선정할 정도로 획기적인 방법이다. siRNA는 짧은 19-23개의 이중 리보핵산쇄로 세포내 도입하면 비특이적 저해(non-specific inhibition) 없이 특정 유전자의 발현만을 억제하는 효과를 나타내므로 암의 생성을 촉진, 세포사멸을 억제하는 유전자에 대한 siRNA는 세포 내에서 해당 유전자 작용을 억제시켜 암세포를 죽이는 효과를 나타내며 이를 이용하여 치료용 표적유전자를 검증할 수 있다. 따라서 최근 질병에 대한 표적 유전자 발굴, 표적 유전자 검증, 치료제 개발을 위한 siRNA 연구가 매우 활발하다.The use of RNAi (RNA interference) experimental techniques on genes expressed in cancer cells can confirm the potential use as therapeutic cancer targets. RNAi is a technique that induces the degradation of mRNA of the corresponding nucleotide sequence specifically in a cell using various types of oligonucleotides (miRNA, expressed dsRNA, synthetic siRNA) in a variety of forms to prevent the function of genes from appearing. As a result of observing the expression of cells in which the expression of genes is inhibited, it is possible to study the function of the genes and analyze the signaling pathways, thereby developing new drugs through target validation. Among the oligo double ribonucleic acids available for RNAi technique, siRNA (small interfering RNA), which has recently been in the spotlight, is a breakthrough method that was selected as "breakthrough of the year" in the 2002 Science Journal. siRNA is a short 19-23 double ribonucleic acid chain, when introduced into the cell has the effect of suppressing the expression of a specific gene only without non-specific inhibition (promotes the production of cancer, inhibiting apoptosis) siRNA has the effect of killing cancer cells by inhibiting the action of the gene in the cell and can be used to verify the therapeutic target gene. Therefore, siRNA research for the discovery of target genes for target diseases, validation of target genes, and development of therapeutic agents is very active.
한편, 현재 C10orf81(chromosome 10 open reading frame 81, synonyms: FLJ23537, HEL185(Epididymis luminal protein 185)) 유전자는 PH 도메인-포함 단백질로 그 기능은 전혀 알려져 있지 않다.Meanwhile, the C10orf81 gene (chromosome 10 open reading frame 81, synonyms: FLJ23537, HEL185 (Epididymis luminal protein 185)) is a PH domain-containing protein and its function is not known at all.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 암 진단 및 약물 스크리닝에 사용되고 암 치료 타겟이 될 수 있는 유전자의 발견을 위하여 예의 연구 노력한 결과, 각종 암 세포 내 C10orf81 유전자의 발현이 현저히 증가되며 이를 억제하는 경우 암 치료 효능이 있다는 것을 알아냄으로써, 본 발명을 완성하게 되었다.The present inventors have made diligent research efforts to find genes that are used for cancer diagnosis and drug screening and may be targets for cancer treatment. As a result, the inventors found that the expression of the C10orf81 gene in various cancer cells is significantly increased, and that there is an effect of cancer treatment when inhibited. By this, the present invention has been completed.
본 발명의 목적은 C10orf81(Chromosome 10 open reading frame 81) 유전자의 mRNA 또는 이의 단백질의 발현수준을 측정하는 제제를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단용 조성물을 제공하는 것이다.An object of the present invention is a cancer diagnostic composition selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer, comprising an agent for measuring the expression level of mRNA or protein of C10orf81 (Chromosome 10 open reading frame 81) gene To provide.
본 발명의 다른 목적은 상기 조성물을 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a cancer diagnostic kit selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, comprising the composition.
본 발명의 또 다른 목적은 (a) 암 의심 환자의 생물학적 시료로부터 C10orf81 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 측정하는 단계; 및 (b) 상기 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 정상 대조구 시료의 해당 유전자의 mRNA 발현수준 또는 단백질 발현수준과 비교하는 단계를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단을 위한 정보의 제공 방법을 제공하는 것이다.Another object of the present invention is to measure the mRNA expression level of the C10orf81 gene or the protein encoded by the gene from a biological sample of a cancer suspect patient; And (b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample, gastric cancer, liver cancer, colorectal cancer, It is to provide a method of providing information for cancer diagnosis selected from the group consisting of lung cancer and prostate cancer.
본 발명의 또 다른 목적은 C10orf81 유전자의 발현 억제제 또는 이의 단백질의 활성 억제제를 포함하는 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암의 예방 또는 치료용 약제학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer comprising an inhibitor of expression of the C10orf81 gene or an activity inhibitor of a protein thereof. .
본 발명의 또 다른 목적은 (a) 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성을 분석하는 단계; 및 (b) 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성이 시험물질을 처리하지 않은 C10orf81 유전자의 발현 또는 이의 단백질의 활성에 비하여 억제되면 암 치료제로 판단하는 단계를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 치료제의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to perform the steps of (a) analyzing the expression of the C10orf81 gene or the activity of the protein after treating the test substance; And (b) judging the expression of the C10orf81 gene or the activity of the protein thereof after the test substance is inhibited compared to the expression of the C10orf81 gene or the activity of the protein thereof without treatment of the test substance, the cancer treatment comprising: To provide a method for screening a cancer therapeutic agent selected from the group consisting of liver cancer, colon cancer, lung cancer and prostate cancer.
본 발명에 따라 위암, 간암, 대장암, 폐암 또는 전립선암에 특이적인 유전자를 제공하고 상기 암의 전이 및 예후를 판단할 수 있는 진단 마커를 제공함으로써, 정확한 암 진단을 가능하게 하며, 암의 치료 및 예후관리에 유용하게 사용할 수 있다. 또한, 상기 유전자를 억제하는 암 특이적인 치료제를 제공함으로써, 부작용이 적은 바이오 신약 및 맞춤형 항암제의 개발을 가능하게 할 수 있다.According to the present invention, by providing genes specific for gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer and providing diagnostic markers for determining the metastasis and prognosis of the cancer, accurate cancer diagnosis is possible and treatment of cancer And it can be useful for prognosis management. In addition, by providing a cancer-specific therapeutic agent that inhibits the gene, it is possible to enable the development of bio-new drugs and customized anti-cancer agents with fewer side effects.
도 1a는 인간 위암 세포주, 간암 세포주, 대장암 세포주 및 정상 세포에서 C10orf81 유전자의 발현양의 변화를 RT-PCR을 통해 분석한 결과를 나타낸다. GAPDH를 대조 유전자로 사용하였다.Figure 1a shows the results of analysis via RT-PCR changes in the amount of C10orf81 gene expression in human gastric cancer cell line, liver cancer cell line, colon cancer cell line and normal cells. GAPDH was used as a control gene.
도 1b는 인간 위암 세포주, 간암 세포주, 대장암 세포주 및 정상 세포에서 C10orf81유전자의 발현양의 변화를 Realtime-PCR 을 이용하여 정량적으로 분석한 결과를 나타낸다. GAPDH를 대조 유전자로 사용하였다.Figure 1b shows the results of quantitative analysis of changes in the expression level of the C10orf81 gene in human gastric cancer cell line, liver cancer cell line, colon cancer cell line and normal cells. GAPDH was used as a control gene.
도 2는 Illumina 48K chip을 이용한 마이크로어레이 실험을 통해 66명의 대장암 임상 조직에서 C10orf81 유전자의 발현이 증가되었음을 나타내는 그림과 데이터 분석 결과를 나타낸다. Figure 2 shows the results of the analysis and the data showing that the expression of the C10orf81 gene in 66 colorectal cancer clinical tissues through a microarray experiment using Illumina 48K chip.
도 3은 대장암 환자 10명, 간암환자 조직 5명 및 위암환자 조직에서 C10orf81 유전자의 발현양의 변화를 RT-PCR을 통해 분석한 결과를 나타낸다. GAPDH를 대조 유전자로 사용하였다.Figure 3 shows the results of analyzing the change in the amount of expression of the C10orf81 gene in 10 colorectal cancer patients, 5 liver cancer tissues and gastric cancer tissues by RT-PCR. GAPDH was used as a control gene.
도 4는 대장암조직에서의 C10orf81 단백질 발현량을 C10orf81 항체를 이용하여 분석한 그림이다. 정상조직을 비교군으로 사용하였다.Figure 4 is a picture of the analysis of C10orf81 protein expression in colorectal cancer tissue using the C10orf81 antibody. Normal tissue was used as a comparison group.
도 5는 C10orf81 유전자 과다발현에 의한 세포 성장 속도 변화를 나타낸 그래프와 세포의 성장 촉진을 SRB 염색으로 조사한 그림이다.5 is a graph showing the change in cell growth rate due to overexpression of C10orf81 gene and the figure showing the growth promotion of cells by SRB staining.
도 6a는 대표세포주로 대장암 세포주 KM12C를 선택하여 C10orf81 siRNA의 효과를 검증한 결과를 나타낸다. C10orf81의 siRNA를 처리하여 실제 C10orf81 mRNA의 양이 감소되어 단백질이 감소됨을 보여준다.Figure 6a shows the results of verifying the effect of C10orf81 siRNA by selecting the colorectal cancer cell line KM12C as a representative cell line. Treatment of C10orf81 siRNA shows that the amount of actual C10orf81 mRNA is reduced, resulting in reduced protein.
도 6b는 C10orf81의 siRNA를 처리한 경우 그에 따른 세포 성장의 억제를 보여준다.6B shows inhibition of cell growth according to treatment with siRNA of C10orf81.
도 7은 정상세포, 위암, 간암, 대장암, 폐암 및 전립선암 세포주에서 siRNA의 처리에 의한 세포 성장 속도 저해 효과를 나타내는 그래프이다. 7 is a graph showing the effect of inhibiting cell growth rate by the treatment of siRNA in normal cells, gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer cell lines.
상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 C10orf81(Chromosome 10 open reading frame 81) 유전자의 mRNA 또는 이의 단백질의 발현수준을 측정하는 제제를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단용 조성물을 제공한다.As one embodiment for achieving the above object, the present invention comprises gasoline, liver cancer, colorectal cancer, lung cancer and prostate, comprising an agent for measuring the expression level of the mRNA or protein thereof of the C10orf81 (Chromosome 10 open reading frame 81) gene It provides a cancer diagnostic composition selected from the group consisting of cancer.
본 발명의 C10orf81 유전자는 FLJ23537 또는 HEL185(Epididymis luminal protein 185)라고도 불리는 유전자로서, PH 도메인-포함 단백질(PH domain-containing protein)을 코딩한다. C10orf81의 mRNA 서열은 서열번호 1에 표시된 것과 같고, 그 ORF 서열은 서열번호 2에 표시된 것과 같다. 서열번호 3은 C10orf81 유전자로부터 발현되는 단백질의 아미노산 서열을 표시한 것이다. 본 명세서에서 'C10orf81 유전자'라 함은 이들 유전자의 DNA 또는 mRNA를 말하며, DNA 또는 mRNA의 전부 또는 일부를 모두 포함하는 개념이다.The C10orf81 gene of the present invention, also called FLJ23537 or HEL185 (Epididymis luminal protein 185), encodes a PH domain-containing protein. The mRNA sequence of C10orf81 is as shown in SEQ ID NO: 1, and the ORF sequence is as shown in SEQ ID NO: 2. SEQ ID NO: 3 shows the amino acid sequence of the protein expressed from the C10orf81 gene. As used herein, the term “C10orf81 gene” refers to DNA or mRNA of these genes, and is a concept that includes all or part of DNA or mRNA.
본 발명에서 용어, "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미하며, 본 발명의 목적상 진단은 위암, 간암, 대장암, 폐암 또는 전립선암 발병 여부를 확인하는 것이다. As used herein, the term "diagnosis" refers to confirming the presence or characteristics of a pathological condition, and for the purposes of the present invention, the diagnosis is to identify whether cancer, gastric cancer, colon cancer, lung cancer, or prostate cancer develops.
본 발명에서 용어, "진단용 마커, 진단하기 위한 마커 또는 진단 마커(diagnosis marker)"란 암 세포를 정상 세포와 구분하여 진단할 수 있는 물질로서, 정상 세포에 비하여 암을 가진 세포에서 증가 또는 감소를 보이는 폴리펩타이드 또는 핵산(예: mRNA 등), 지질, 당지질, 당단백질 또는 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다. 본 발명의 목적상, 본 발명의 암 진단 마커는 정상 대장 조직의 세포에 비하여, 위암, 간암, 대장암, 폐암 또는 전립선암 세포에서 특이적으로 높은 수준의 발현을 보이는 C10orf81 유전자 및 이에 의해 코딩되는 단백질이다.As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is a substance capable of diagnosing cancer cells from normal cells, and increases or decreases cancer cells in comparison with normal cells. Organic biomolecules such as visible polypeptides or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For the purposes of the present invention, the cancer diagnostic markers of the present invention are encoded by the C10orf81 gene, which is expressed by gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer cells as compared to cells of normal colon tissue, Protein.
생물학적 시료 중의 유전자 발현수준은 mRNA 또는 단백질의 양을 확인함으로써 확인할 수 있다.Gene expression levels in biological samples can be confirmed by identifying the amount of mRNA or protein.
본 발명에서 mRNA 발현수준 측정이란 암을 진단하기 위하여 생물학적 시료에서 암 마커 유전자들의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로서, mRNA의 양을 측정함으로써 알 수 있다. 이를 위한 분석 방법으로는 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting) 또는 DNA 칩 등이 있으나, 이에 제한되는 것은 아니다.In the present invention, the measurement of mRNA expression level is a process of confirming the presence and expression of mRNA of cancer marker genes in a biological sample to diagnose cancer, and can be known by measuring the amount of mRNA. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting or DNA chips, but are not limited thereto.
상기 유전자의 mRNA의 발현수준을 측정하는 제제는 암 세포에서 발현이 증가하는 마커인 C10orf81의 발현수준을 확인함으로써 마커의 검출에 사용될 수 있는 분자를 의미하며, 이에 제한되지는 않으나 바람직하게는 상기 유전자에 특이적으로 결합하는 프라이머 또는 프로브를 포함하는 것일 수 있다. C10orf81의 mRNA 서열(서열번호 1) 및 그 ORF 서열(서열번호 2)을 바탕으로 당업자는 이들 유전자의 특정 영역을 특이적으로 증폭하는 프라이머 또는 프로브를 디자인할 수 있다.The agent for measuring the expression level of mRNA of the gene means a molecule that can be used for detection of the marker by confirming the expression level of C10orf81, a marker that increases expression in cancer cells, but is not limited thereto. It may be to include a primer or probe specifically binding to. Based on the C10orf81 mRNA sequence (SEQ ID NO: 1) and its ORF sequence (SEQ ID NO: 2), one of ordinary skill in the art can design primers or probes that specifically amplify specific regions of these genes.
본 발명에서 용어, "프라이머"는 짧은 자유 3말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍(base pair)을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다. 본 발명에서는 C10orf81 폴리뉴클레오타이드의 센스 및 안티센스 프라이머를 이용하여 PCR 증폭을 실시하여 원하는 생성물의 생성 여부를 통해 암을 진단할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 것을 기초로 변형할 수 있다.As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, the PCR can be performed using the sense and antisense primers of C10orf81 polynucleotide to diagnose cancer through the generation of a desired product. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.
본 발명에서 용어, "프로브"는 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며, 라벨링되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 C10orf81 폴리뉴클레오타이드와 상보적인 프로브를 이용하여 혼성화를 실시하여, 혼성화 여부를 통해 암을 진단할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당업계에 공지된 것을 기초로 변형할 수 있다.As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which is capable of specific binding with mRNA, and is labeled to indicate the presence or absence of a specific mRNA. You can check it. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, hybridization is carried out using a probe complementary to C10orf81 polynucleotide, and cancer can be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.
본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, "캡화", 천연 뉴클레오타이드 하나 이상의 동족체로의 치환, 및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, "capsulation", substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, Phosphoramidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
본 발명에서 단백질 발현수준 측정이란 암을 진단하기 위하여 생물학적 시료에서 암 마커 유전자로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 바람직하게는 상기 유전자의 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있다. 이를 위한 분석 방법으로는 웨스턴 블랏, ELISA(enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 조직면역 염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS), 단백질 칩(protein chip) 등이 있으나, 이에 제한되는 것은 아니다.In the present invention, the protein expression level measurement refers to a process of confirming the presence and expression level of a protein expressed from a cancer marker gene in a biological sample in order to diagnose cancer. Preferably, the antibody specifically binds to the protein of the gene. You can check the amount of protein using. Assays for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoelectrophoresis. , Tissue immunity staining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chips (protein chip) and the like, but is not limited thereto.
상기 단백질의 발현수준을 측정하는 제제는 암 세포에서 발현이 증가하는 마커인 C10orf81 유전자로부터 발현되는 단백질의 발현수준을 확인함으로써 마커의 검출에 사용될 수 있는 분자를 의미하며, 이에 제한되지는 않으나 바람직하게는 상기 단백질에 특이적인 항체를 포함하는 것일 수 있다. C10orf81 유전자로부터 발현되는 단백질의 아미노산 서열(서열번호 3)을 바탕으로 당업자는 상기 단백질에 특이적인 항체를 디자인할 수 있다.The agent for measuring the expression level of the protein refers to a molecule that can be used for detection of the marker by confirming the expression level of the protein expressed from the C10orf81 gene, which is a marker of increased expression in cancer cells, but is not limited thereto. May include an antibody specific for the protein. Based on the amino acid sequence of the protein expressed from the C10orf81 gene (SEQ ID NO: 3), those skilled in the art can design antibodies specific for the protein.
본 발명에서 용어, "항체"는 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 마커인 C10orf81 유전자로부터 발현되는 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함되며, 본 발명의 부분 펩티드로는, 최소한 7개 아미노산, 바람직하게는 9개 아미노산, 보다 바람직하게는 12개 이상의 아미노산을 포함한다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다. 본 발명의 암 진단 마커의 검출에 사용되는 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 기능을 보유하고 있는 단편을 뜻하며 Fab, F(ab'), F(ab') 2 및 Fv 등이 있다.As used herein, the term “antibody” refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed from the C10orf81 gene, which is a marker of the present invention. Such an antibody may be cloned into an expression vector according to a conventional method for the marker gene. The protein encoded by is obtained and can be prepared by conventional methods from the obtained protein. Also included are partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody. Antibodies used in the detection of cancer diagnostic markers of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.
다른 하나의 양태로서, 본 발명은 상기 조성물을 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단용 키트를 제공한다.In another aspect, the present invention provides a kit for diagnosing cancer selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, comprising the composition.
본 발명의 키트는 암 진단 마커인 C10orf81의 발현수준을 mRNA 또는 이의 단백질의 발현수준을 확인함으로써 마커를 검출할 수 있다. 본 발명의 키트에는 암 진단 마커의 발현수준을 측정하기 위한 프라이머, 프로브 또는 선택적으로 마커를 인지하는 항체뿐만 아니라 분석방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치가 포함될 수 있다.The kit of the present invention can detect a marker by confirming the expression level of mRNA or a protein thereof in the expression level of the cancer diagnostic marker C10orf81. Kits of the present invention may include primers, probes or optionally antibodies that recognize the expression level of a cancer diagnostic marker, as well as one or more other component compositions, solutions or devices suitable for the assay.
상기 키트는 C10orf81 유전자와 대상체로부터 얻은 시료 간의 반응을 확인하여 암을 진단하는 원리를 이용할 수 있다. 상기 C10orf81 유전자와 대상체로부터 얻은 시료 간의 반응의 확인은 DNA-DNA, DNA-RNA, DNA-단백질 간의 반응 여부를 확인하는데 사용되는 통상적인 방법들, 예컨대 DNA 칩, 단백질 칩, 중합효소 연쇄반응(PCR), 노던 블롯팅, 서던 블롯팅, ELISA(Enzyme Linked Immunosorbent assay), 효모 이중 혼성법(yeast two-hybrid), 2-D 겔 분석 및 시험관 내 결합 에세이(in vitro binding assay) 등을 이용할 수 있다. 즉, 상기 유전자의 전부 또는 일부를 프로브로 사용하여 대상자의 체액으로부터 분리한 핵산과 하이브리드화한 후 당 분야에 공지된 다양한 방법, 예컨대 역전사 중합효소 연쇄반응(reverse transcription polymerase chain reaction), 서던 블로팅, 노던 블롯팅 등으로 이를 검출함으로써 대상자에서 상기 유전자가 고발현된 상태인지 또는 저발현된 상태인지 조사하면 암의 발생 여부를 판단할 수 있다.The kit may use the principle of diagnosing cancer by confirming a reaction between the C10orf81 gene and a sample obtained from a subject. Confirmation of the reaction between the C10orf81 gene and the sample obtained from the subject is a common method used to confirm the reaction between DNA-DNA, DNA-RNA, DNA-protein, such as DNA chip, protein chip, polymerase chain reaction (PCR). ), Northern blotting, Southern blotting, Enzyme Linked Immunosorbent assay (ELISA), yeast two-hybrid, 2-D gel analysis and in vitro binding assays can be used. . That is, all or part of the gene is used as a probe to hybridize with nucleic acid isolated from the body fluid of the subject, and then various methods known in the art, such as reverse transcription polymerase chain reaction and southern blotting. By detecting this by Northern blotting, the subject can determine whether the cancer has occurred by investigating whether the gene is in a high or low expression state.
또한, 상기 키트는 C10orf81 유전자로부터 발현된 단백질과 대상체로부터 얻은 시료 간의 반응을 확인하여 암을 진단하는 원리를 이용할 수도 있다. 앞서 언급한 바와 같이, 상기 C10orf81 유전자는 위암, 간암, 대장암, 폐암 또는 전립선암 세포에서 특이적으로 발현이 증가하므로 상기 유전자의 과발현 여부를 조사하는 것뿐만 아니라, 상기 유전자로부터 발현된 단백질의 과발현 여부를 조사하면 암을 진단할 수 있다. 본 발명의 암 진단용 키트에서 상기 단백질을 포함하는 조성물과 시료 간의 반응의 확인은 DNA-단백질, RNA-단백질, 단백질-단백질 간의 반응 여부를 확인하는데 사용되는 통상적인 방법들, 예컨대 DNA 칩, 단백질 칩, 중합효소 연쇄반응 (PCR), 노던 블롯팅, 서던 블롯팅, 웨스턴 블롯팅, ELISA(Enzyme Linked Immunosorbent assay), 특이적 면역염색(histoimmunostaining), 효모 이중 혼성법(yeast two-hybrid), 2-D 겔 분석 및 시험관 내 결합 에세이 (in vitro binding assay) 등을 이용할 수 있다. 예컨대 상기 유전자들로부터 발현된 단백질의 전부 또는 일부를 프로브로 사용하여 대상자의 체액으로부터 분리한 핵산 또는 단백질과 하이브리드화한 후 당분야에 공지된 다양한 방법, 예컨대 역전사 중합효소 연쇄반응(reverse transcription polymerases chain reaction), 웨스턴 블로팅(western blotting) 등으로 이를 검출함으로써 대상자에서 상기 유전자가 고발현된 상태인지 조사하면 암의 발생 여부를 판단할 수 있다.In addition, the kit may use the principle of diagnosing cancer by confirming a reaction between a protein expressed from the C10orf81 gene and a sample obtained from a subject. As mentioned above, the C10orf81 gene is specifically increased in gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer cells, and thus not only to investigate whether the gene is overexpressed, but also to overexpress the protein expressed from the gene. Investigate whether the cancer can be diagnosed. Confirmation of the reaction between the composition and the sample containing the protein in the cancer diagnostic kit of the present invention is a common method used to confirm the reaction between the DNA-protein, RNA-protein, protein-protein, such as DNA chips, protein chips Polymerase chain reaction (PCR), Northern blotting, Southern blotting, Western blotting, Enzyme Linked Immunosorbent assay (ELISA), specific immunostaining, yeast two-hybrid, 2- D gel analysis and in vitro binding assays can be used. For example, all or part of a protein expressed from the genes as a probe may be hybridized with a nucleic acid or protein isolated from a subject's body fluid, and then various methods known in the art, such as reverse transcription polymerases chain. By detecting this by reaction, western blotting, or the like, it is possible to determine whether the cancer has occurred by examining whether the gene is highly expressed in the subject.
본 발명의 키트는 이에 제한되지는 않으나, 바람직하게는 마이크로어레이, 유전자 증폭 키트 또는 면역분석(immunoassay)용 키트일 수 있다.The kit of the present invention is not limited thereto, but may preferably be a microarray, a gene amplification kit or an immunoassay kit.
본 발명의 키트가 마이크로어레이인 경우, 상기 마이크로어레이의 고상표면에 프로브가 고정화 되어 있다. 본 발명의 마이크로어레이에 있어서, 상기한 프로브는 혼성화 어레이 요소(hybridizable array element)로서 이용되며, 기체(substrate) 상에 고정화된다. 바람직한 기체는 적합한 견고성 또는 반-견고성 지지체로서, 예컨대, 막, 필터, 칩, 슬라이드, 웨이퍼, 파이버, 자기성 비드 또는 비자기성 비드, 겔, 튜빙, 플레이트, 고분자, 미소입자 및 모세관을 포함한다. 상기한 혼성화 어레이 요소는 상기의 기체 상에 배열되고 고정화된다. 이와 같은 고정화는 화학적 결합 방법 또는 UV와 같은 공유 결합적 방법에 의해 실시된다. 예를 들어, 상기 혼성화 어레이 요소는 에폭시 화합물 또는 알데히드기를 포함하도록 변형된 글래스 표면에 결합될 수 있고, 또한 폴리라이신 코팅 표면에서 UV에 의해 결합될 수 있다. 또한, 상기 혼성화 어레이 요소는 링커(예: 에틸렌 글리콜 올리고머 및 디아민)를 통해 기체에 결합될 수 있다.When the kit of the present invention is a microarray, a probe is immobilized on the solid surface of the microarray. In the microarray of the present invention, the probe is used as a hybridizable array element and is immobilized on a substrate. Preferred gases include suitable rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. Said hybridization array element is arranged and immobilized on said gas. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (eg, ethylene glycol oligomer and diamine).
한편, 본 발명의 마이크로어레이에 적용되는 시료 DNA 또는 RNA는 표지(labeling)될 수 있고, 마이크로어레이상의 어레이 요소와 혼성화된다. 혼성화 조건은 다양하게 할 수 있으며, 혼성화 정도의 검출 및 분석은 표지 물질에 따라 다양하게 실시될 수 있다.On the other hand, the sample DNA or RNA applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may be carried out in various ways depending on the labeling substance.
본 발명의 암 진단용 키트는 혼성화에 기초하여 실시할 수 있다. 이 경우, 상술한 본 발명의 마커의 뉴클레오타이드 서열에 대하여 상보적인 서열을 가지는 프로브가 이용된다.The cancer diagnostic kit of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.
프로브의 표지는 혼성화 여부를 검출케 하는 시그널을 제공할 수 있으며, 이는 올리고뉴클레오타이드에 연결될 수 있다. 적합한 표지는 형광단(예컨대, 플루오리신(fluorescein), 피코에리트린(phycoerythrin), 로다민, 리사민(lissamine), 그리고 Cy3와 Cy5(Pharmacia)), 발색단, 화학발광단, 자기입자, 방사능동위원소(P32 및 S35), 매스 표지, 전자밀집입자, 효소(알칼린 포스파타아제 또는 호스래디쉬 퍼옥시다아제), 조인자, 효소에 대한 기질, 중금속(예컨대, 금) 그리고 항체, 스트렙타비딘, 바이오틴, 디곡시게닌과 킬레이팅기와 같은 특정 결합 파트너를 갖는 햅텐을 포함하나, 이에 한정되는 것은 아니다. 표지는 당업계에서 통상적으로 실시되는 다양한 방법, 예컨대, 닉 트랜스레이션 (nick translation) 방법, 무작위 프라이밍 방법 (Multiprime DNA labelling systems booklet, "Amersham"(1989)) 및 카이네이션 방법 (Maxam & Gilbert, Methods in Enzymology, 65:499(1986))을 통해 실시될 수 있다. 상기 표지는 형광, 방사능, 발색 측정, 중량 측정, X-선 회절 또는 흡수, 자기, 효소적 활성, 매스 분석, 결합 친화도, 혼성화 고주파, 나노크리스탈에 의하여 검출할 수 있는 시그널을 제공한다.The label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin And hapten with specific binding partners such as digoxigenin and chelating groups. Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology , 65: 499 (1986)). The label provides a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.
본 발명에서 적합한 혼성화 조건은 최적화 절차에 의하여 일련의 과정으로 결정될 수 있다. 이런 절차는 연구실에서 사용을 위한 프로토콜을 수립하기 위하여 당업자에 의하여 일련의 과정으로 실시된다. 예를 들어, 온도, 성분의 농도, 혼성화 및 세척 시간, 완충액 성분 및 이들의 pH 및 이온세기 등의 조건은 프로브의 길이 및 GC 양 및 타겟 뉴클레오타이드 서열 등의 다양한 인자에 의존한다. 혼성화를 위한 상세한 조건은 Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); 및 M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999)에서 확인할 수 있다. 예를 들어, 상기 엄격조건 중에서 고 엄격조건은 0.5 M NaHPO4, 7% SDS(sodium dodecyl sulfate), 1 mM EDTA에서 65℃ 조건으로 혼성화하고, 0.1 x SSC(standard saline citrate)/0.1% SDS에서 68℃ 조건으로 세척하는 것을 의미한다. 또는, 고 엄격조건은 6 x SSC/0.05% 소듐 파이로포스페이트에서 48℃ 조건으로 세척하는 것을 의미한다. 저 엄격조건은 예를 들어, 0.2 x SSC/0.1% SDS에서 42℃ 조건으로 세척하는 것을 의미한다.Suitable hybridization conditions in the present invention can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). For example, among the stringent conditions, the higher stringency conditions were hybridized to 65 ° C. in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and at 0.1 × standard saline citrate / 0.1% SDS. It means to wash at 68 ℃ conditions. Alternatively, high stringency conditions mean washing at 48 ° C. in 6 × SSC / 0.05% sodium pyrophosphate. Low stringency means washing at 42 ° C. conditions, for example, at 0.2 × SSC / 0.1% SDS.
혼성화 반응 이후에, 혼성화 반응을 통하여 나오는 혼성화 시그널을 검출한다. 혼성화 시그널은 예컨대, 프로브에 결합된 표지의 종류에 따라 다양한 방법으로 실시할 수 있다. 예를 들어, 프로브가 효소에 의해 표지된 경우, 이 효소의 기질을 혼성화 반응 결과물과 반응시켜 혼성화 여부를 확인할 수 있다. 이용될 수 있는 효소/기질의 조합은, 퍼옥시다아제(예컨대, 호스래디쉬 퍼옥시다아제)와 클로로나프톨, 아미노에틸카바졸, 디아미노벤지딘, D-루시페린, 루시게닌(비스-N-메틸아크리디늄 니트레이트), 레소루핀 벤질 에테르, 루미놀, 암플렉스 레드 시약(10-아세틸-3,7-디하이드록시페녹사진), HYR(p-phenylenediamine-HCl and pyrocatechol), TMB(tetramethylbenzidine), ABTS(2,2-Azine-di[3-ethylbenzthiazoline sulfonate]), o-페닐렌디아민(OPD) 및 나프톨/파이로닌; 알칼린 포스파타아제와 브로모클로로인돌일 포스페이트(BCIP), 니트로 블루 테트라졸리움(NBT), 나프톨-AS-B1-포스페이트(naphthol-AS-B1-phosphate) 및 ECF 기질; 글루코스 옥시다아제와 t-NBT(nitroblue tetrazolium) 및 m-PMS(phenzaine methosulfate) 등이다. After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. The hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe. For example, if the probe is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate).
본 발명의 키트는 바람직하게는 유전자 증폭 키트일 수 있다.The kit of the present invention may preferably be a gene amplification kit.
본 발명에서 용어, "증폭"은 핵산 분자를 증폭하는 반응을 의미한다. 다양한 증폭 반응들이 당업계에 보고되어 있으며, 이는 중합효소 연쇄반응(PCR)(미국 특허 제4,683,195, 4,683,202, 및 4,800,159호), 역전사-중합효소 연쇄반응(RT-PCR)(Sambrook 등, Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press(2001)), Miller, H. I.(WO 89/06700) 및 Davey, C. 등(EP 329,822)의 방법, 리가아제 연쇄 반응(ligase chain reaction; LCR), Gap-LCR(WO 90/01069), 복구 연쇄 반응(repair chain reaction; EP 439,182), 전사-매개 증폭(transcription-mediated amplification; TMA, WO 88/10315), 자가 유지 염기서열 복제(self sustained sequence replication, WO 90/06995), 타깃 폴리뉴클레오티드 염기서열의 선택적 증폭(selective amplification of target polynucleotide sequences, 미국 특허 제6,410,276호), 컨센서스 서열 프라이밍 중합효소 연쇄 반응(consensus sequence primed polymerase chain reaction(CP-PCR), 미국 특허 제4,437,975호), 임의적 프라이밍 중합효소 연쇄 반응(arbitrarily primed polymerase chain reaction(AP-PCR), 미국 특허 제5,413,909호 및 제5,861,245호), 핵산 염기서열 기반 증폭(nucleic acid sequence based amplification(NASBA), 미국 특허 제5,130,238호, 제5,409,818호, 제5,554,517호, 및 제6,063,603호), 가닥 치환 증폭(strand displacement amplification) 및 고리-중재 항온성 증폭(loop-mediated isothermal amplification; LAMP)을 포함하나, 이에 제한되지는 않는다. 사용 가능한 다른 증폭 방법들은 미국특허 제5,242,794, 5,494,810, 4,988,617호 및 미국 특허 제09/854,317호에 기술되어 있다. As used herein, the term "amplification" means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329, 822), ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self sustained sequence replication , WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR), U.S. Patent 4,437,975), optional priming Arbitrarily primed polymerase chain reaction (AP-PCR), US Pat. Nos. 5,413,909 and 5,861,245, Nucleic acid sequence based amplification (NASBA), US Pat. Nos. 5,130,238, 5,409,818 Nos. 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP), but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.
PCR은 가장 잘 알려진 핵산 증폭 방법으로, 그의 많은 변형과 응용들이 개발되어 있다. 예를 들어, PCR의 특이성 또는 민감성을 증진시키기 위해 전통적인 PCR 절차를 변형시켜 터치다운(touchdown) PCR, 핫 스타트(hot start) PCR, 네스티드(nested) PCR 및 부스터(booster) PCR이 개발되었다. 또한, 실시간(real-time) PCR, 분별 디스플레이 PCR(differential display PCR: DD-PCR), cDNA 말단의 신속 증폭(rapid amplification of cDNA ends: RACE), 멀티플렉스 PCR, 인버스 중합효소 연쇄반응(inverse polymerase chain reaction: IPCR), 벡토레트(vectorette) PCR 및 TAIL-PCR(thermal asymmetric interlaced PCR)이 특정한 응용을 위해 개발되었다. PCR에 대한 자세한 내용은 McPherson, M.J., 및 Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000)에 기재되어 있으며, 그의 교시사항은 본 명세서에 참조로 삽입된다.PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.
중합 반응을 실시할 때, 반응 용기에 반응에 필요한 성분들을 과량으로 제공하는 것이 바람직하다. 증폭 반응에 필요한 성분들의 과량은, 증폭반응이 성분의 농도에 실질적으로 제한되지 않는 정도의 양을 의미한다. Mg2+와 같은 조인자, dATP, dCTP, dGTP 및 dTTP를 원하는 증폭 정도가 달성될 수 있을 정도로 반응 혼합물에 제공하는 것이 요구된다. 증폭 반응에 이용되는 모든 효소들은 동일한 반응 조건에서 활성 상태일 수 있다. 완충액은 모든 효소들이 최적의 반응 조건에 근접하도록 한다. 따라서 본 발명의 증폭 과정은 반응물의 첨가와 같은 조건의 변화 없이 단일 반응물에서 실시될 수 있다.When carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel. Excess of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is desired to provide cofactors such as Mg 2+ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. The buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.
본 발명의 키트는 면역분석용 키트일 수 있으며, 본 발명의 암 마커에 특이적으로 결합하는 항체 또는 앱타머를 이용하여 실시될 수 있다.The kit of the present invention may be a kit for immunoassay, and may be performed using an antibody or aptamer specifically binding to the cancer marker of the present invention.
본 발명에서 이용되는 항체는 폴리클로날 또는 모노클로날 항체이며, 바람직하게는 모노클로날 항체이다. 항체는 시판되는 것을 사용할 수도 있고 당업계에서 통상적으로 실시되는 방법들, 예를 들어, 융합 방법(Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)), 재조합 DNA 방법(미국 특허 제4,816,56호) 또는 파아지 항체 라이브러리 방법(Clackson et al, Nature, 352:624-628(1991) 및 Marks et al, J. Mol. Biol., 222:58, 1-597(1991))에 의해 제조될 수도 있다. 항체 제조에 대한 일반적인 과정은 Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; 및 Coligan , CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991에 상세하게 기재되어 있으며, 상기 문헌들은 본 명세서에 참조로서 삽입된다. 예를 들어, 단일클론 항체를 생산하는 하이브리도마 세포의 제조는 불사멸화 세포주를 항체-생산 림프구와 융합시켜 이루어지며, 이 과정에 필요한 기술은 당업자에게 잘 알려져 있으며 용이하게 실시할 수 있다. 모노클로날 항체는 일반적으로 알칼라인 포스파타아제(alkaline phosphatase, AP) 또는 호올스래디쉬 퍼록시다제(horseradish peroxidase, HRP) 등의 효소가 컨쥬게이션된 2차 항체 및 이들의 기질을 사용하여 발색반응시킴으로써 정량분석할 수도 있고, 아니면 직접 상기 단백질에 대한 모노클로날 항체에 AP 또는 HRP 효소 등이 컨쥬게이션된 것을 사용하여 정량분석할 수도 있다.The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies may be commercially available and methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US patents) 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol. Biol. , 222: 58, 1-597 (1991)). It may be prepared by. General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Monoclonal antibodies are generally developed by using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a substrate thereof. It may be quantitatively analyzed, or may be directly quantitated using a conjugate of AP or HRP enzyme or the like to a monoclonal antibody directed against the protein.
폴리클로날 항체는 단백질 항원을 적합한 동물에게 주사하고, 이 동물로부터 항혈청을 수집한 다음, 공지의 친화성(affinity) 기술을 이용하여 항혈청으로부터 항체를 분리하여 얻을 수 있다.Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.
또한, 항원결합성을 갖는 것이면 모노클로날 항체 또는 폴리클로날 항체의 일부도 본 발명의 항체에 포함되고, 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수항체도 포함된다.In addition, a monoclonal antibody or a part of polyclonal antibody is also included in the antibody of the present invention as long as it has antigen binding, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
또 다른 하나의 양태로서, 본 발명은 (a) 암 의심 환자의 생물학적 시료로부터 C10orf81 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 측정하는 단계; 및 (b) 상기 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 정상 대조구 시료의 해당 유전자의 mRNA 발현수준 또는 단백질 발현수준과 비교하는 단계를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단을 위한 정보의 제공 방법을 제공한다.In another aspect, the present invention provides a method for treating cancer, comprising the steps of: (a) measuring the mRNA expression level of the C10orf81 gene or the expression level of a protein encoded by a biological sample of a suspected cancer patient; And (b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample, gastric cancer, liver cancer, colorectal cancer, Provided is a method of providing information for diagnosing cancer selected from the group consisting of lung cancer and prostate cancer.
본 발명에서 용어, "생물학적 시료"는 위암, 간암, 대장암, 폐암 또는 전립선암 발병에 의해 암 마커의 유전자 발현수준이 차이나는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨와 같은 시료 등을 포함하나, 이에 제한되지는 않는다. As used herein, the term "biological sample" refers to tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in gene expression levels of cancer markers due to the development of gastric cancer, liver cancer, colon cancer, lung cancer, or prostate cancer. Samples such as, but not limited to.
상기 생물학적 시료에서 mRNA 또는 이의 단백질을 분리하는 과정은 공지의 공정을 이용하여 수행할 수 있으며, 상기 mRNA 또는 이의 단백질의 발현수준은 다양한 방법으로 측정할 수 있다.Separation of mRNA or protein thereof from the biological sample may be performed using a known process, and the expression level of the mRNA or protein thereof may be measured by various methods.
상기 mRNA 발현수준의 측정은 이에 제한되지는 않으나, 바람직하게는 역전사효소 중합효소반응, 경쟁적 역전사효소 중합효소반응, 실시간 역전사효소 중합효소반응, RNase 보호 분석법, 노던 블랏팅 또는 DNA 칩에 의한 것일 수 있으며, 보다 바람직하게는 역전사효소 중합효소반응 또는 DNA 칩에 의한 것일 수 있다.The mRNA expression level is not limited thereto, but may be preferably by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. It may be more preferably by reverse transcriptase polymerase reaction or DNA chip.
상기의 역전사효소 중합효소반응은 반응 후 전기영동하여 밴드 패턴과 밴드의 두께를 확인함으로써 암 진단 마커로 사용되는 유전자의 mRNA 발현 여부와 정도를 확인 가능하고 이를 대조군과 비교함으로써, 암 발생 여부를 간편하게 진단할 수 있다. 또한, 상기 DNA 칩은 상기 암 마커 유전자 또는 그 단편에 해당하는 핵산이 유리 같은 기판에 고밀도로 부착되어 있는 DNA 칩을 이용하는 것으로서, 시료에서 mRNA를 분리하고, 그 말단 또는 내부를 형광 물질로 표지된 cDNA 프로브를 조제하여, DNA 칩에 혼성화시킨 다음 암의 발병 여부를 판독할 수 있다.The reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band by checking the mRNA expression and degree of the gene used as a cancer diagnostic marker and compare it with the control group, it is easy to determine whether cancer Diagnosis can be made. In addition, the DNA chip is a DNA chip in which the nucleic acid corresponding to the cancer marker gene or a fragment thereof is attached to a glass-like substrate at a high density. The DNA chip is isolated from a sample, and the terminal or the inside thereof is labeled with a fluorescent material. cDNA probes can be prepared, hybridized to DNA chips, and read for cancer.
상기 단백질 발현수준의 측정은 이에 제한되지는 않으나, 바람직하게는 해당 단백질에 특이적인 항체를 이용하는 것일 수 있다. 상기 항체를 이용하여 항원-항체 복합체의 형성량을 비교함으로써, 암의 발병 여부를 진단할 수 있으며, 상기 항체에 대해서는 상기에서 설명한 바와 같다.The measurement of the protein expression level is not limited thereto, but preferably may be to use an antibody specific for the protein. By comparing the amounts of antigen-antibody complexes formed using the antibodies, it is possible to diagnose whether cancer has occurred, and the antibodies have been described above.
상기 단백질 발현수준의 측정은 이에 제한되지는 않으나, 바람직하게는 웨스턴 블랏, ELISA, 방사선면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 면역침전 분석법, 보체 고정 분석법, FACS 또는 단백질 칩을 이용하는 것일 수 있다.The protein expression level is not limited thereto, but preferably, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement It may be using a fixed assay, FACS or protein chip.
ELISA법을 이용하는 경우, ELISA는 고체 지지체에 부착된 항원을 인지하는 표지된 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 2차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함할 수 있다. 보다 바람직하게는, 고체 지지체에 항체를 부착시키고 시료를 반응시킨 후 항원-항체 복합체의 항원을 인지하는 표지된 항체를 부착시켜 효소적으로 발색시키거나 항원-항체 복합체의 항원을 인지하는 항체에 대해 표지된 2차 항체를 부착시켜 효소적으로 발색시키는 샌드위치 ELISA 방법에 의해서 검출할 수 있다.When using the ELISA method, ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support. Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antibody attached to the solid support, followed by reaction with another antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support. Various ELISA methods can be included, such as indirect sandwich ELISA using labeled secondary antibodies that recognize the antibody. More preferably, the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It can be detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops.
암 마커에 대한 하나 이상의 항체를 이용한 웨스턴 블랏을 이용하는 경우, 시료에서 전체 단백질을 분리하고, 이를 전기영동하여 단백질을 크기에 따라 분리한 다음, 니트로셀루로즈 막으로 이동시켜 항체와 반응시킨다. 이때 생성된 항원-항체 복합체의 양을 표지된 항체를 이용하여 확인하는 방법으로 유전자의 발현에 의해 생성된 단백질의 양을 확인하여, 암 발병 여부를 확인할 수 있다. 상기 검출 방법은 대조군에서의 마커의 발현량과 암이 발병한 세포에서의 마커의 발현량을 조사하는 방법으로 이루어진다. 상기 발현수준은 마커 단백질의 절대적(예: ㎍/㎖) 또는 상대적(예: 시그널의 상대 강도) 차이로 나타낼 수 있다.When using Western blots with one or more antibodies against cancer markers, the entire protein is isolated from the sample, electrophoresed to separate the proteins according to size, and then transferred to a nitrocellulose membrane to react with the antibody. In this case, the amount of the generated antigen-antibody complex can be confirmed by using a labeled antibody to check the amount of the protein produced by the expression of a gene, thereby determining whether the cancer has developed. The detection method comprises a method of examining the expression level of the marker in the control group and the expression level of the marker in the cancer-causing cells. The expression level can be expressed as an absolute (eg μg / ml) or relative (eg relative intensity of signal) difference of the marker protein.
암 마커에 대한 하나 이상의 항체를 이용한 면역조직 염색을 이용하는 경우, 정상 대장 상피 조직 및 암으로 의심되는 조직을 채취 및 고정한 후, 당업계에서 널리 공지된 방법으로 파라핀 포매 블록을 제조한다. 이들을 수 ㎛ 두께의 절편으로 만들어 유리 슬라이드에 붙인 후, 이와 상기의 항체를 공지의 방법에 의하여 반응시킨다. 이후, 반응하지 못한 항체는 세척하고, 검출라벨로 표지하여 현미경 상에서 항체의 표지 여부를 판독한다.When using immunohistostaining with one or more antibodies to cancer markers, normal colon epithelial tissue and tissue suspected of cancer are collected and fixed, followed by preparation of paraffin embedding blocks by methods well known in the art. These are sliced to a thickness of several 탆 and attached to a glass slide, and then the antibodies are reacted by a known method. The unreacted antibody is then washed and labeled with a detection label to read whether the antibody is labeled on the microscope.
암 마커에 대한 하나 이상의 항체가 기판 위의 정해진 위치에 배열되어 고밀도로 고정화되어 있는 단백질 칩을 이용하는 경우, 시료에서 단백질을 분리하고, 분리한 단백질을 단백질 칩과 혼성화시켜서 항원-항체 복합체를 형성시키고 이를 판독하여, 단백질의 존재 또는 발현 정도를 확인하여, 암 발병 여부를 확인할 수 있다.When using a protein chip in which one or more antibodies against a cancer marker are arranged at a predetermined position on a substrate and immobilized at high density, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex. By reading this, the presence or expression of the protein can be checked to determine whether the cancer develops.
상기 방법들을 통하여, 정상 대조군에서의 mRNA 또는 이의 단백질의 발현수준과 암 의심환자에서의 mRNA 또는 이의 단백질의 발현수준을 비교할 수 있고, 암 마커 유전자에서 mRNA 또는 이의 단백질로의 유의한 발현량의 증가여부를 판단하여, 암 의심 환자의 실제 암 발병 여부를 진단할 수 있다.Through the above methods, it is possible to compare the expression level of mRNA or protein thereof in a normal control group with the expression level of mRNA or protein thereof in cancer suspected patients, and increase the significant expression level of the cancer marker gene to mRNA or protein thereof. By determining whether or not the cancer suspected patient can actually diagnose the onset of cancer.
또 다른 하나의 양태로서, 본 발명은 C10orf81 유전자의 발현 억제제 또는 이의 단백질의 활성 억제제를 포함하는 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암의 예방 또는 치료용 약제학적 조성물을 제공한다.As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer comprising an inhibitor of the expression of the C10orf81 gene or a protein inhibitor thereof. to provide.
본 발명의 약제학적 조성물에서 유효성분으로 포함되는 C10orf81 유전자의 발현 억제제는 이에 제한되지는 않으나, 바람직하게는 C10orf81 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 microRNA일 수 있다.The inhibitor of expression of the C10orf81 gene included as an active ingredient in the pharmaceutical composition of the present invention is not limited thereto, but may preferably be an antisense oligonucleotide, siRNA, shRNA or microRNA specific to the C10orf81 gene.
본 발명에서 상기 안티센스 올리고뉴클레오타이드는 특정 DNA 또는 RNA 타겟에 대해 안티센스(또는 상보)인 짧은 길이의 DNA 합성 가닥(또는 DNA 아날로그)으로서, 생체 내 뿐만 아니라 생체 외에서도 유전자-특이적 억제를 달성하기 위해 성공적으로 사용되어 왔다. 안티센스 올리고뉴클레오타이드는 타겟에 결합하고 전사, 번역 또는 스플라이싱의 단계에서 발현을 멈추게 함으로써 DNA 또는 RNA 타겟에 의해 인코드된 단백질의 발현을 막기 위해 제안되었다. 안티센스 올리고뉴클레오타이드는 세포 배양 및 질병의 동물 모델에서도 성공적으로 이용되어 왔다. 올리고뉴클레오타이드가 분해되지 않도록 더욱 안정하고 저항적이 되게 하기 위한 안티센스 올리고뉴클레오타이드의 또 다른 변형이 당업자에게 알려져 있고 이해된다. 여기서 사용된 안티센스 올리고뉴클레오타이드는 이중나선 또는 단일나선 DNA, 이중나선 또는 단일나선 RNA, DNA/RNA 하이브리드, DNA 및 RNA 아날로그 및 염기, 당 또는 백본 변형을 지닌 올리고뉴클레오타이드를 포함한다. 올리고뉴클레오타이드는 안정성을 증가시키고, 뉴클레아제 분해에 대한 저항성을 증가시키기 위해 당분야에 알려진 방법에 의해 변형된다. 이들 변형은 당분야에 알려져 있는 올리고뉴클레오타이드 백본의 변형, 당 모이어티의 변형 또는 염기의 변형을 포함하나 이에 제한되지는 않는다.In the present invention, the antisense oligonucleotide is a short length DNA synthesis strand (or DNA analog) that is antisense (or complementary) to a specific DNA or RNA target, to achieve gene-specific inhibition in vivo as well as in vitro. It has been used successfully. Antisense oligonucleotides have been proposed to prevent the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides have been successfully used in cell culture and animal models of disease. Other modifications of antisense oligonucleotides to make them more stable and resistant to degradation of oligonucleotides are known and understood by those skilled in the art. Antisense oligonucleotides as used herein include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and to increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications to oligonucleotide backbones, modifications of sugar moieties or bases known in the art.
본 발명에서 상기 siRNA(small interfering RNA)는 RNA 방해 또는 유전자 사일런싱(silencing)을 매개할 수 있는 핵산 분자로서, 표적 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 녹다운(knockdown) 방법 또는 유전자치료 방법으로 사용된다. 본 발명에서 siRNA 분자가 이용되는 경우, 센스 가닥(C10orf81 mRNA 서열(서열번호 1)에 상응하는 서열)과 안티센스 가닥(C10orf81 mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조 또는 자기-상보성(self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다. siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 불일치(mismatch)(대응하는 염기가 상보적이지 않음), 팽창/돌출(bulge)(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. siRNA 말단 구조는 C10orf81 유전자의 발현을 RNA 간섭(RNA interference, RNAi) 효과에 의하여 억제할 수 있는 것이면 평활(blunt) 말단 혹은 점착(cohesive) 말단 모두 가능하다. 점착 말단 구조는 3'-말단 돌출 구조와 5'-말단 돌출 구조 모두 가능하다. 상기 siRNA 분자는 이에 제한되지는 않으나, 전체 길이가 15 내지 30 염기, 바람직하게는 19 내지 21 염기일 수 있다.In the present invention, the siRNA (small interfering RNA) is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and because it can suppress expression of a target gene, it is an efficient gene knockdown method or gene therapy method. Used as When the siRNA molecule is used in the present invention, a double stranded structure is formed in which the sense strand (the sequence corresponding to the C10orf81 mRNA sequence (SEQ ID NO: 1)) and the antisense strand (the sequence complementary to the C10orf81 mRNA sequence) are positioned opposite to each other. It may have a single chain structure with self-complementary sense and antisense strands. siRNAs are not limited to the complete pairing of double-stranded RNA pairs that pair with RNA, but include mismatches (corresponding bases are not complementary), expansion / bulge (bases corresponding to one chain). None) and the like may be included. The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the C10orf81 gene can be inhibited by RNA interference (RNAi) effects. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures. The siRNA molecule is not limited thereto, but may have a total length of 15 to 30 bases, preferably 19 to 21 bases.
상기 siRNA는 세포 배양 및 생체 내에서 오래 지속되는 효과, 생체 내에서 세포를 트랜스펙션시키는 능력 및 혈청 내 분해에 대한 저항력의 측면에서 생체 내에서 특정한 유전자의 발현의 저해에 대해 매우 강한 약물이 되는 잠재력을 지닌다. siRNA의 전달 및 siRNA를 포함한 발현 컨스트럭트/벡터는 당업자에게 알려져 있다. 예를 들어, 미국 출원 제2004/106567 및 2004/0086884호에서는 바이러스성 벡터, 비바이러스성 벡터, 리포솜 전달 운반체, 플라스미드 주입 시스템, 인공 바이러스 엔벨로프 및 폴리라이신 컨쥬게이트를 포함한 전달 메커니즘뿐만 아니라 많은 발현 컨스트럭트/벡터를 제공하고 있다.The siRNA is a very strong drug against the inhibition of expression of specific genes in vivo in terms of long lasting effects in cell culture and in vivo, the ability to transfect cells in vivo and resistance to serum degradation. Has the potential. Delivery of siRNAs and expression constructs / vectors including siRNAs are known to those skilled in the art. For example, US applications 2004/106567 and 2004/0086884 disclose many expression consensus as well as delivery mechanisms including viral vectors, nonviral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates. Provide truck / vector.
본 발명에서 상기 shRNA(short hairpin RNA)은 45 내지 70 뉴클레오타이드의 길이를 가지는 단일가닥의 RNA로서, 타겟유전자 siRNA 염기서열의 센스가닥과 상보적인 안티센스가닥 사이에 3-10개의 염기 링커를 연결하는 올리고 DNA를 합성한 후, 플라스미드 벡터에 클로닝하거나 또는 shRNA를 레트로바이러스인 렌티바이러스(lentivirus) 및 아데노 바이러스(adenovirus)에 삽입하여 발현시키면 루프(loop)가 있는 헤어핀 구조의 shRNA가 만들어지고 세포내의 다이서(dicer)에 의해 siRNA로 전환되어 RNAi 효과를 나타낸다.In the present invention, the shRNA (short hairpin RNA) is a single-stranded RNA having a length of 45 to 70 nucleotides, an oligo linking 3-10 base linkers between the sense strand of the target gene siRNA sequence and the complementary antisense strand. After synthesizing DNA, cloning into plasmid vectors or inserting and expressing shRNAs into retroviruses lentivirus and adenovirus results in loops of hairpin structured shRNAs and intracellular dicers. It is converted into siRNA by (dicer) to show RNAi effect.
본 발명에서 상기 마이크로 RNA(microRNA)는 발생, 분화, 증식, 보존 및 아폽토시스 등 다양한 생물학적 과정을 조절한다. 마이크로 RNA는 일반적으로 타겟 mRNA를 불안정하게 하거나, 번역을 방해함으로써 타겟 mRNA를 코딩하는 유전자의 발현을 조절한다.In the present invention, the microRNA (microRNA) regulates various biological processes such as development, differentiation, proliferation, conservation and apoptosis. MicroRNAs generally regulate the expression of the gene encoding the target mRNA by destabilizing the target mRNA or disrupting translation.
상기 안티센스 올리고 뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA를 지닌 발현 컨스트럭트/벡터에 유용한 조절 서열(예를 들어, 구성적 프로모터, 유도성 프로모터, 조직 특이적 프로모터 또는 그의 결합) 역시 당분야에서 공지된 내용으로부터 적절히 선택할 수 있으며, 상기 유전자의 발현 억제제는 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA 외에도 상기 유전자의 발현을 억제하는 물질이면 어떤 것이든 가능하다.Regulatory sequences useful in expression constructs / vectors with such antisense oligonucleotides, siRNAs, shRNAs or microRNAs (eg, constitutive promoters, inducible promoters, tissue specific promoters or combinations thereof) are also known in the art. The expression inhibitor of the gene may be any material that inhibits the expression of the gene in addition to the antisense oligonucleotide, siRNA, shRNA or microRNA.
본 발명의 약제학적 조성물에서 유효성분으로 포함되는 C10orf81 유전자로부터 발현된 단백질의 활성 억제제는 이에 제한되지는 않으나, 바람직하게는 C10orf81 단백질에 특이적인 항체일 수 있다.The inhibitor of activity of the protein expressed from the C10orf81 gene included as an active ingredient in the pharmaceutical composition of the present invention is not limited thereto, but may preferably be an antibody specific for the C10orf81 protein.
상기 항체에 대하여는 상기에서 설명한 바와 같으며, 상기 단백질에 대한 모노클로날 항체는 당업계에 통상적인 모노클로날 항체 제작 방법을 통해 제작되어 사용될 수도 있고, 시판되는 것을 사용할 수 있다. 또한, 모노클로날 항체 대신에 상기 단백질을 인식하는 폴리클로날 항체를 사용할 수도 있고, 이는 당업계에 통상적인 항혈청 제작 방법을 통해 제작되어 사용될 수도 있다.As described above with respect to the antibody, the monoclonal antibody to the protein may be produced and used through a monoclonal antibody production method common in the art, or may be commercially available. In addition, a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through an antiserum production method conventional in the art.
본 발명의 약제학적 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가적으로 약제학적으로 허용되는 담체를 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다. 적당한 약제학적으로 허용되는 담체는 예를 들어 물, 식염수, 인산 완충 식염수, 덱스트린, 글리세롤, 에탄올뿐만 아니라 이들의 조합을 포함할 수 있다. 또한, 단백질의 활성 억제제가 항체일 경우 약제학적으로 허용되는 담체는 결합 단백질의 저장 수명 또는 유효성을 증가시키는 습윤제 또는 유화제, 방부제 또는 완충액과 같은 최소량의 보조 물질로 구성될 수 있다.The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including an additional pharmaceutically acceptable carrier in addition to the active ingredient described above for administration. Suitable pharmaceutically acceptable carriers can include, for example, water, saline, phosphate buffered saline, dextrin, glycerol, ethanol, as well as combinations thereof. In addition, when the activity inhibitor of the protein is an antibody, a pharmaceutically acceptable carrier may consist of a minimum amount of auxiliary material, such as a wetting or emulsifying agent, preservative or buffer, which increases the shelf life or effectiveness of the binding protein.
상기 담체는 투여 방법에 따라 다르게 포함할 수 있으며, 경구 투여시에는 결합체, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소 투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭시르, 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조될 수 있다.The carrier may be included differently depending on the method of administration, and oral administration may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like. For example, buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used. For topical administration, bases, excipients, lubricants, preservatives and the like can be used. Formulations of the compositions of the present invention can be prepared in a variety of mixtures with the pharmaceutically acceptable carriers described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampoules or in multiple dosage forms.
또 다른 하나의 양태로서, 본 발명은 상기 약제학적 조성물을 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 의심개체에 투여하는 단계를 포함하는 암의 치료 방법을 제공한다.In another aspect, the present invention provides a method for treating cancer, comprising administering the pharmaceutical composition to a cancer suspect selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer, and prostate cancer.
본 발명에서 상기 암 의심 개체는 암이 발병하였거나 발병할 수 있는 인간을 포함한 모든 동물을 의미하며, 본 발명의 C10orf81 유전자의 발현 억제제 또는 이의 단백질의 활성 억제제를 포함하는 약제학적 조성물을 암 의심 개체에 투여함으로써, 개체를 효율적으로 치료할 수 있다.In the present invention, the cancer suspected subject refers to all animals including humans having or may develop cancer, and the pharmaceutical composition comprising an inhibitor of expression of the C10orf81 gene of the present invention or an activity inhibitor of a protein thereof is included in the cancer suspected subject. By administering, the individual can be treated efficiently.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 경구 투여, 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 비내 투여, 폐내 투여, 직장내 투여, 강내 투여, 복강 내 투여, 경막 내 투여가 이루어질 수 있으나, 이에 제한되지는 않는다.As used herein, the term "administration" means introducing a composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the desired tissue. have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but are not limited thereto. Do not.
본 발명의 치료 방법은 상기 약제학적 조성물을 약제학적 유효량으로 투여하는 것을 포함할 수 있다. 본 발명에서 유효량은 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 성인의 경우, 상기 유전자의 발현 억제제 또는 단백질의 활성 억제제를 1일 1회 내지 수회 투여시, siRNA일 경우 0.01ng/kg-10㎎/kg, 상기 유전자의 mRNA에 대한 안티센스 올리고뉴클레오타이드인 경우 0.01ng/kg-10㎎/kg, 화합물일 경우 0.1ng/kg-10㎎/kg, 상기 단백질에 대한 모노클로날 항체일 경우 0.1ng/kg-10㎎/kg의 용량으로 투여될 수 있다.The method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount. In the present invention, the effective amount is defined as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently. In adults, when the expression inhibitor of the gene or the activity of the protein is administered once or several times, 0.01ng / kg-10mg / kg for siRNA, 0.01ng for the antisense oligonucleotide for mRNA of the gene / kg-10mg / kg, 0.1ng / kg-10mg / kg for the compound, 0.1ng / kg-10mg / kg for the monoclonal antibody to the protein can be administered.
또 다른 하나의 양태로서, 본 발명은 (a) 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성을 분석하는 단계; 및 (b) 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성이 시험물질을 처리하지 않은 C10orf81 유전자의 발현 또는 이의 단백질의 활성에 비하여 억제되면 암 치료제로 판단하는 단계를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 치료제의 스크리닝 방법을 제공한다.In another embodiment, the present invention provides a method for treating a C10orf81 gene or analyzing a protein thereof after treating a test substance; And (b) judging the expression of the C10orf81 gene or the activity of the protein thereof after the test substance is inhibited compared to the expression of the C10orf81 gene or the activity of the protein thereof without treatment of the test substance, the cancer treatment comprising: The present invention provides a method for screening a cancer therapeutic agent selected from the group consisting of liver cancer, colorectal cancer, lung cancer and prostate cancer.
본 발명에서 상기 (a) 단계는 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성을 분석하는 단계로서, 상기 분석은 세포 내에서 또는 시험관 내에서 분석할 수 있으며, 이에 제한되지는 않으나 예컨대 RT-PCR, 노던 블로팅, cDNA 마이크로어레이 혼성화 반응, 인 시투(in situ) 혼성화 반응, 방사능면역분석, 면역침전, ELISA, 웨스턴 블로팅 등을 이용하여 분석할 수 있다.In the present invention, the step (a) is a step of analyzing the expression of the C10orf81 gene or the activity of the protein after the test material, the assay can be analyzed in cells or in vitro, but is not limited thereto. For example, it can be analyzed using RT-PCR, northern blotting, cDNA microarray hybridization reaction, in situ hybridization reaction, radioimmunoassay, immunoprecipitation, ELISA, western blotting and the like.
본 발명에서 용어, "시험물질"은 C10orf81 유전자의 발현 또는 이의 단백질의 활성에 영향을 미치는지 여부를 검사하기 위하여 스크리닝에서 이용되는 미지의 물질을 의미한다. 상기 시험물질은 통상적인 선정방식에 따라 암 전이 억제제로서의 가능성을 지닌 것으로 추정되거나 또는 무작위적으로 선정된 개별 물질이 될 수 있으며, 이에 제한되는 것은 아니나 핵산, 단백질, 화학물질 및 천연 추출물을 포함할 수 있다. 본 발명의 스크리닝 방법에 의해 분석되는 시험물질은 단일 화합물 또는 화합물들의 혼합물일 수 있으며, 합성 또는 천연 화합물의 라이브러리로부터 얻을 수 있다.As used herein, the term "test substance" refers to an unknown substance used in screening to test whether the expression of the C10orf81 gene or the activity of a protein thereof is affected. The test substance may be an individual substance that is estimated or randomly selected as a cancer metastasis inhibitor according to a conventional selection method, and may include, but is not limited to, nucleic acids, proteins, chemicals, and natural extracts. Can be. The test substance analyzed by the screening method of the present invention may be a single compound or a mixture of compounds, and may be obtained from a library of synthetic or natural compounds.
본 발명에서 상기 (b) 단계는 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성이 시험물질을 처리하지 않은 C10orf81 유전자의 발현 또는 이의 단백질의 활성에 비하여 억제되면 암 치료제로 판단하는 단계로서, 상기 시험물질에 의하여 C10orf81 유전자의 발현 또는 C10orf81 단백질의 활성이 하향 조절(down-regulation)되는 것을 측정하여 암 치료제로 판정할 수 있다.In the present invention, the step (b) is a step of judging the expression of C10orf81 gene or the activity of the protein thereof after treatment with the test substance is inhibited compared to the expression of the C10orf81 gene or the activity of the protein thereof without treatment with the test substance. As a test agent, the expression of the C10orf81 gene or the activity of the C10orf81 protein is down-regulated and measured as a cancer drug.
본 발명의 스크리닝 방법을 통해 얻은 암 고발현 유전자의 발현을 증진시키거나 단백질의 활성을 증진시키는 기능을 나타내는 시험물질 및 반대로 암 고발현 유전자의 발현을 억제시키거나 단백질의 활성을 억제시키는 기능을 나타내는 시험물질은, 전자의 경우, 시험물질에 대한 억제제를 개발함으로써 암 치료제 후보물질이 될 수 있고, 후자의 경우는 암 치료제 후보물질이 될 수 있다. 이와 같은 암 치료제 후보물질은 이후의 암 치료제 개발과정에서 선도물질(leading compound)로서 작용하게 되며, 선도물질이 상기 유전자 또는 그로부터 발현되는 단백질의 기능 억제효과를 나타낼 수 있도록 그 구조를 변형시키고 최적화함으로써, 새로운 암 치료제를 개발할 수 있다.A test substance that has a function of enhancing the expression of a cancer high expression gene or a protein activity obtained through the screening method of the present invention, and vice versa, a function of inhibiting the expression of a cancer high expression gene or inhibiting the activity of a protein. The test substance, in the former case, may be a cancer drug candidate by developing an inhibitor for the test substance, and in the latter case, it may be a cancer drug candidate. Such candidates for cancer treatment will act as leading compounds in the development of cancer treatments in the future, and by modifying and optimizing the structure so that the leading substances can exhibit the inhibitory effect of the gene or protein expressed therefrom. Can develop new cancer therapies.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 단, 하기 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.
실시예 1: C10orf81 유전자의 암 세포주에서의 발현양 증가 효과 확인Example 1: Confirmation of the effect of increasing the amount of C10orf81 gene in cancer cell line
유전자의 암 관련성을 추정하는 간단한 방법은 암조직 또는 암세포주에서의 해당 유전자의 발현양의 변화를 비교하는 것이다. 정상 세포에 비해 암세포주에서 다량 발현되어 있는지 또는 감소되어 있는지 비교하여 암 생성 및 진행에 따른 발현량과의 관계를 조사하여야 한다. 이를 위해서는 암세포주에서 실제 해당 유전자의 발현이 어떻게 나타나고 있는지 암 세포주에서 총 RNA를 추출한 후 해당 유전자의 올리고뉴클레오타이드를 사용하여 RT-PCR 및 Realtime-PCR을 수행하여 각 유전자의 양을 정상 세포주와 비교하여 유전자의 세포 내 발현양을 확인하게 된다. 이에 본 발명에서는 C10orf81 유전자의 발현 정도를 인간 위암, 간암, 대장암 세포주 및 정상 세포에서 mRNA 레벨을 분석하였다. A simple way of estimating the cancer's relevance to a gene is to compare changes in the amount of expression of that gene in cancer tissue or cancer cell lines. The relationship between the expression level according to the cancer production and progression should be investigated by comparing whether the cancer cell line is expressed or reduced in a large amount compared to normal cells. To do this, extract the total RNA from the cancer cell line to see how the expression of the gene is actually expressed in the cancer cell line, perform RT-PCR and Realtime-PCR using the oligonucleotide of the gene, and compare the amount of each gene with the normal cell line. The expression level of the gene in the cell is confirmed. In the present invention, the expression level of the C10orf81 gene was analyzed for mRNA levels in human gastric cancer, liver cancer, colorectal cancer cell lines and normal cells.
1-1. 암 세포주의 준비1-1. Preparation of Cancer Cell Lines
10% 우태아혈청(Fetal Bovine Serum, GIBCO사), 페니실린(10000 U/㎖)과 스트렙토마이신(10 mg/㎖)을 첨가한 RPMI1640(GIBCO) 배양액을 사용하였다. 위암 세포주로서 SNU620, SNU484 및 SNU638(모두 서울대학교 세포주 은행), 간암세포주로서 Huh7(ATCC), Chang(ATCC) 및 SNU354(서울대학교 세포주 은행), 대장암세포주로서는 KM12C(서울대학교 세포주 은행), Colo205(서울대학교 세포주 은행), SW480(ATCC) 및 HT29(ATCC)를 이용하였다. 정상 세포주로는 IMR90(human fetal lung fibroblasts, ATCC#CCL-186)을 이용하였다. 암 세포주를 세포수가 1X106이 되도록 접종하고 이를 37℃, 5% CO2 가 존재하는 배양기에서 배양하였다.RPMI1640 (GIBCO) cultures containing 10% fetal bovine serum (Fetal Bovine Serum, GIBCO), penicillin (10000 U / mL) and streptomycin (10 mg / mL) were used. SNU620, SNU484 and SNU638 (all of Seoul National University Cell Line Bank) as gastric cancer cell lines, Huh7 (ATCC), Chang (ATCC) and SNU354 (Seoul National University Cell Line Bank) as liver cancer cell lines, KM12C (Seoul National University Cell Line Bank), Colo205 as colon cancer cell lines (Seoul National University Cell Line Bank), SW480 (ATCC) and HT29 (ATCC) were used. IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as a normal cell line. Cancer cell lines were inoculated with a cell number of 1 × 10 6 and cultured in an incubator at 37 ° C. with 5% CO 2 .
1-2. 총 RNA 분리1-2. Total RNA Isolation
총 RNA는 QIAGEN 킷트(RNeasy Maxi kit: cat #75162)를 사용하여 분리하였고, Experion RNA StdSens(Bio-Rad사) 칩을 이용하여 정량하였다. 세포를 원심분리하여 회수한 후 키트 내 분해 완충액 RLN(50 mM TrisCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40) 1 ㎖에 베타 멀캅토 에탄올 10 ㎕를 첨가하여 용해시켰다. 여기에 1 ㎖의 70% 에탄올을 넣어 잘 섞은 후, 3000 g에서 5분간 원심분리하여 총 RNA를 막에 부착시켰다. 두 차례의 세척을 한 후 100 ㎕의 RNase가 없는 물을 첨가하여 총 RNA를 분리하였다.Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantified using Experion RNA StdSens (Bio-Rad) chip. The cells were collected by centrifugation and lysed by adding 10 µl of beta mercapto ethanol to 1 ml of digestion buffer RLN (50 mM TrisCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP-40) in the kit. . 1 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After two washes, total RNA was isolated by adding 100 μl of RNase-free water.
1-3. c-DNA 합성과 주형의 농도 보정1-3. c-DNA synthesis and concentration correction of template
시료 각각의 총 RNA 2 ㎍, 프라이머인 50 μM 올리고(dT) 1 ㎕와 10 mM dNTP 2.5 ㎕를 넣고 RNase 저해제인 DEPC(diethyl pyrocarbonate)가 들어 있는 멸균수로 전체가 25 ㎕가 되도록 하여 RNA/프라이머 혼합용액을 만들었다. 65℃에서 5분간 반응시킨 후 55℃로 옮겨 보관하였다. 다음 10X RT 완충액 5 ㎕, 25mM MgCl2 10 ㎕, 0.1M DTT 5 ㎕, RNase 억제제 1 ㎕, SuperScriptIII RT 효소를 1 ㎕ 넣고 전체가 25 ㎕가 되도록 한 후 55℃에서 보관 중인 RNA/프라이머 혼합용액과 섞어준 후, 55℃에서 50분간 반응시켰다. 그 후 85℃에서 5분간 반응시켜 RT 효소를 불활성화 한 후 얼음에 넣어 반응을 종결시켰다. 마커유전자를 정량하기 위한 표준 유전자로서 GAPDH를 사용하였다. 표준 유전자의 프라이머를 이용하여 PCR 반응을 수행하고 표준 유전자 GAPDH의 발현량이 동일해지도록 cDNA의 농도를 보정하였다. 우선 각각의 cDNA를 20배 희석한 후, 희석된 샘플 2 ㎕를 이용하여 PCR 반응을 수행하였다. PCR은 2X PCR premix(Hot start) 15 ㎕, 2 ㎕의 GAPDH 5' 프라이머, 2 ㎕의 3' 프라이머 및 11 ㎕의 증류수를 넣어 사용하였고, 20 사이클, 23 사이클 및 25 사이클을 수행하였다. 이 때 PCR 반응 조건은 94℃ 30초, 50℃ 30초 및 72℃ 1분으로 수행하였으며, 산물의 크기는 457 bp이었다. PCR 산물을 2% 아가로스 젤에 로딩하여 전기영동한 후 젤 사진을 찍고, 이미지를 TotalLab v1.0 프로그램(Nonlinear Dynamix사) 으로 정량한 후, 다시 보정하여 PCR을 수행하여 정량하는 방식으로 각 시료의 농도를 동일하게 보정하였다.2 μg of total RNA of each sample, 1 μl of 50 μM oligo (dT) primer and 2.5 μl of 10 mM dNTP were added and sterile water containing RNase inhibitor DEPC (diethyl pyrocarbonate) was added to make 25 μl total RNA / primer. A mixed solution was made. After reacting at 65 ° C. for 5 minutes, the mixture was transferred to 55 ° C. and stored. Next, add 5 µl of 10X RT buffer, 10 µl of 25mM MgCl 2 , 5 µl of 0.1M DTT, 1 µl of RNase inhibitor, 1 µl of SuperScript III RT enzyme, and add 25 µl of the total amount of the RNA / primer mixture. After mixing, the mixture was reacted at 55 ° C. for 50 minutes. Thereafter, the reaction was carried out at 85 ° C. for 5 minutes to inactivate the RT enzyme, followed by termination on the reaction. GAPDH was used as a standard gene for quantifying marker genes. PCR reaction was performed using primers of the standard gene, and the concentration of cDNA was corrected so that the expression level of the standard gene GAPDH was the same. First, each cDNA was diluted 20-fold, and then PCR reaction was performed using 2 μl of the diluted sample. PCR was performed using 15 µl of 2X PCR premix (Hot start), 2 µl of GAPDH 5 'primer, 2 µl of 3' primer, and 11 µl of distilled water, and 20, 23 and 25 cycles were performed. At this time, PCR reaction conditions were performed at 94 ℃ 30 seconds, 50 ℃ 30 seconds and 72 ℃ 1 minute, the product size was 457 bp. Each product was loaded into a 2% agarose gel, electrophoresed, electrophoresed, and gel images were taken. The images were quantified using the TotalLab v1.0 program (Nonlinear Dynamix), and then corrected again to perform PCR for quantification. The concentration of was equally corrected.
1-4. RT-PCR 및 Realtime PCR 을 이용한 발현량 분석1-4. Expression Analysis Using RT-PCR and Realtime PCR
동일한 양이 되도록 희석한 cDNA를 C10orf81 유전자의 센스 및 안티센스 프라이머를 이용하여 PCR 하였다. cDNA 는 3 ㎕, 2X premix 10 ㎕, 프라이머 각 2 ㎕(20 pmole) 및 증류수 2 ㎕를 섞어 총 용액 20 ㎕로 만들고 PCR 반응은 94℃ 1분, 54℃ 30초 및 72℃ 1분으로 하였으며 35 사이클을 수행하였다. PCR 산물을 확인하기 위하여 2% 아가로스 젤을 이용하여 전기영동하고 이미지 장비를 이용하여 분석하였다. Realtime RT-PCR은 Qiagen사(CA, USA)의 DNASYBRI 시약과 LightCycler(Roche)를 이용하였다. Melt Curve 분석을 이용하여 PCR 산물의 질을 평가하였고 유전자 발현량은 LightCycler 버전 3.5 소프트웨어(Roche)를 사용하여 분석하였다.CDNA diluted to the same amount was PCR using the sense and antisense primers of the C10orf81 gene. cDNA was mixed with 3 μl, 10 μl of 2X premix, 2 μl of primer each (20 pmole) and 2 μl of distilled water to make 20 μl of total solution. The PCR reaction was performed at 94 ° C for 1 minute, 54 ° C for 30 seconds and 72 ° C for 1 minute. The cycle was performed. In order to confirm the PCR product, it was electrophoresed using 2% agarose gel and analyzed using an imaging apparatus. Realtime RT-PCR was used with DNASYBRI reagent from Qiagen (CA, USA) and LightCycler (Roche). Melt Curve analysis was used to assess the quality of PCR products and gene expression was analyzed using LightCycler version 3.5 software (Roche).
상기 RT-PCR에서 사용한 C10orf81 특이적 프라이머는 다음과 같다. N-말단 프라이머 5'-AAGAGGAACCCCAGACCCTT-3'(서열번호 4), C-말단 프라이머 5'-TCTCCACTTGGCTTTCATCG-3'(서열번호 5). RT-PCR 용 GAPDH 프라이머는 N-말단 5'-TCATGACCACAGTCCATGCC-3'(서열번호 6), C-말단 5'-TCCACCACCCTGTTGCTGTA-3'(서열번호 7)이며, Realtim-PCR의 콘트롤로 사용한 GAPDH는 Qiagen 프라이머(QT00079247)를 사용하였다. RT-PCR 및 Realtime-PCR을 수행한 결과, C10orf81은 정상 세포주와 비교하여 많은 암세포 주에서 그 mRNA 발현 정도가 확실히 증가됨을 확인할 수 있었다(도 1a 및 1b). 이러한 결과는 C10orf81 유전자의 발현량 증가가 암진단 지표가 될 수 있음을 의미한다.The C10orf81 specific primers used in the RT-PCR are as follows. N-terminal primer 5'-AAGAGGAACCCCAGACCCTT-3 '(SEQ ID NO: 4), C-terminal primer 5'-TCTCCACTTGGCTTTCATCG-3' (SEQ ID NO: 5). GAPDH primers for RT-PCR are N-terminal 5'-TCATGACCACAGTCCATGCC-3 '(SEQ ID NO: 6), C-terminal 5'-TCCACCACCCTGTTGCTGTA-3' (SEQ ID NO: 7), GAPDH used as control of Realtim-PCR is Qiagen Primer (QT00079247) was used. As a result of performing RT-PCR and Realtime-PCR, it was confirmed that the expression level of C10orf81 was significantly increased in many cancer cell lines compared to normal cell lines (FIGS. 1A and 1B). These results indicate that increased expression of the C10orf81 gene may be an indicator of cancer diagnosis.
실시예 2: 마이크로 어레이 실험에 의한 대장암 환자 임상조직에서의 C10orf81의 발현양 증가 효과 확인Example 2 Confirmation of Increased Expression of C10orf81 in Clinical Tissues of Colorectal Cancer Patients by Microarray Experiment
2-1. 암 임상조직의 준비2-1. Preparation of Cancer Clinical Tissue
삼성의료원(서울, 대한민국)으로부터 66명의 대장암 환자의 대장암 조직과 정상 조직을 각각 입수하였다. 각각의 조직은 환자로부터 외과적으로 제거된 후, 분석 때 까지 이를 액체질소에 보관하였다.Colorectal cancer and normal tissue from 66 patients with colorectal cancer were obtained from Samsung Medical Center (Seoul, South Korea). Each tissue was surgically removed from the patient and stored in liquid nitrogen until analysis.
2-2. 총 RNA 분리2-2. Total RNA Isolation
총 RNA는 QIAGEN 킷트(RNeasy Maxi kit: cat #75162)를 사용하여 분리하였고, Experion RNA StdSens(Bio-Rad사) 칩을 이용하여 정량하였다. 우선 상기 암 임상조직과 정상조직을 적절한 크기로 자른 후, 150 ㎕의 베타 멀캅토 에탄올을 첨가한 15 ㎖의 키트 내 분해 완충액에 용해시켰다. 여기에 15 ㎖의 70% 에탄올을 넣어 잘 섞은 후, 3000 g에서 5분간 원심분리하여 총 RNA를 막에 부착시켰다. 두 차례의 세척을 한 후 1.2 ㎖의 RNase가 없는 증류수를 첨가하여 총 RNA를 분리하였다.Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantified using Experion RNA StdSens (Bio-Rad) chip. First, the cancer clinical tissues and normal tissues were cut to an appropriate size, and then dissolved in 15 ml of a dissolution buffer in a kit containing 150 μl of beta mercapto ethanol. 15 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After washing twice, total RNA was isolated by adding 1.2 ml of RNase-free distilled water.
2-3. 마이크로어레이 실시2-3. Microarray
상기 추출된 총 RNA를 Illumina TotalPrep RNA 증폭 키트(Ambion사)를 이용하여 하이브리드화 하였다. T7 Oligo(dT) 프라이머를 이용하여 cDNA를 합성하고, 바이오틴-UTP를 이용하여 인 비트로 전사를 실시하여 바이오틴-표지 cRNA를 제조하였다. 제조된 cRNA는 NanoDrop을 이용하여 정량하였다. 정상 대장 상피 세포 및 대장암 세포에서 제조된 cRNA를 인간-6 V2(Illumina사) 칩에 하이브리드화 하였다. 하이브리드화 후 비 특이적 하이브리드화를 제거하기 위하여 Illumina Gene Expression System 세척액(Illumina사)을 이용하여 DNA 칩을 세척하였고 세척된 DNA 칩은 스트렙트아비딘-Cy3(Amersham사) 형광 염색약으로 표지하였다. 형광 표지된 DNA 칩은 공초점(confocal) 레이저 스캐너(Illumina사)를 이용하여 스캐닝하여 각 스팟에 존재하는 형광의 데이터를 얻어서 TIFF 형태의 이미지 파일로 저장하였다. TIFF 이미지 파일을 BeadStudio 버전 3(Illumina 사)으로 정량하여 각 스팟의 형광값을 정량하였다. 정량된 결과는 Avadis Prophetic 버전 3.3(Strand Genomics사) 프로그램으로 'quantile' 기능을 이용하여 보정하였다.The extracted total RNA was hybridized using Illumina TotalPrep RNA Amplification Kit (Ambion). CDNA was synthesized using T7 Oligo (dT) primers, and in vitro transcription was performed using biotin-UTP to prepare biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. CRNA prepared from normal colon epithelial cells and colon cancer cells was hybridized to a human-6 V2 (Illumina) chip. In order to remove non-specific hybridization after hybridization, DNA chips were washed using Illumina Gene Expression System wash (Illumina) and the washed DNA chips were labeled with streptavidin-Cy3 (Amersham) fluorescent dye. Fluorescently labeled DNA chips were scanned using a confocal laser scanner (Illumina, Inc.) to obtain fluorescence data present in each spot and stored as image files in TIFF form. TIFF image files were quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence values of each spot. Quantified results were corrected using the 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.
그 결과를 도 2에 나타내었다. 도 2는 대장암 환자의 암조직과 정상조직에서 측정된 C10orf81 유전자 발현 프로파일로, 고발현 되는 경우 붉은색으로, 저발현되는 경우 녹색으로 나타내었다. C10orf81 유전자는 암 조직에서 발현양이 평균 6배 증가되었다. 이러한 결과는 C10orf81 유전자는 정상 대장 상피세포와 비교하여 발현차이를 크게 나타내므로, 암 진단, 약물 스크리닝 또는 치료타겟 등으로 사용할 수 있음을 알 수 있다.The results are shown in FIG. FIG. 2 is a C10orf81 gene expression profile measured in cancerous and normal tissues of colorectal cancer patients, and is shown in red when high expressed and green when low expressed. The C10orf81 gene increased the mean amount of expression 6 times in cancer tissues. These results indicate that the C10orf81 gene shows a large expression difference compared to normal colon epithelial cells, and thus can be used as a cancer diagnosis, drug screening, or therapeutic target.
실시예 3: 개별 임상시료에서 암 진단용 유전자 발현 확인Example 3: Confirmation of Gene Expression for Cancer Diagnosis in Individual Clinical Samples
10쌍의 대장암 임상환자 샘플(실시예 2에서 66쌍의 암 환자로부터 채취한 암조직(T1-T66)과 정상조직(N1-N66) 중 임상 2기에 해당하는 환자조직 10쌍(T1-T10, N1-N10))을 이용하였으며, 4쌍의 위암 임상 샘플(삼성의료원), 5개의 간암 임상 샘플(삼성의료원)을 이용하여 실시예 1에서 확인된 C10orf81 유전자의 발현량을 RT-PCR 방법을 통하여 분석하였다. 실시예 2의 방법을 통하여 총 RNA를 분리하고 실시예 1에서의 방법과 동일한 방법을 통하여 cDNA를 합성하였으며 주형의 농도 보정 과정 및 RT-PCR도 실시예 1과 동일하게 수행하였다. 그 결과를 도 3에 나타내었으며, 사진에서 N은 정상조직(non tumor 조직)을 의미하며, T는 그에 해당하는 암 조직을 의미한다. 그 결과, C10orf81 유전자는 확연히 암 시료에서 고발현되는 것을 확인함으로써, C10orf81 유전자는 암의 진단 또는 약물스크리닝 등을 위한 암 마커 또는 치료 타겟 등으로 사용할 수 있음을 확인할 수 있었다.Ten pairs of colorectal cancer patient samples (cancer tissue (T1-T66) and normal tissues (N1-N66) taken from 66 pairs of cancer patients in Example 2) , N1-N10)), four pairs of gastric cancer clinical samples (Samsung Medical Center), five liver cancer clinical samples (Samsung Medical Center) using the RT-PCR method to determine the expression level of the C10orf81 gene identified in Example 1 Analyzed through. Total RNA was isolated by the method of Example 2 and cDNA was synthesized by the same method as in Example 1, and the concentration correction process and RT-PCR of the template was performed in the same manner as in Example 1. The results are shown in Figure 3, in the picture, N means normal tissue (non tumor tissue), T means the corresponding cancer tissue. As a result, by clearly confirming that the C10orf81 gene is highly expressed in cancer samples, it was confirmed that the C10orf81 gene can be used as a cancer marker or therapeutic target for diagnosis or drug screening of cancer.
실시예 4: 면역염색을 이용한 조직 내 단백질 발현량 비교Example 4 Comparison of Protein Expression in Tissues Using Immunostaining
정상 대장 상피 조직과 대장암 조직에서의 단백질 존재여부 및 발현 위치를 확인하고자 조직 슬라이드를 대상으로 면역 염색을 실시하였다. 우선, 외과적 절제술을 통하여 대장암 환자들로부터 대장 정상 상피세포 조직과 대장암 세포조직을 적출하여 파라핀 포매 블록을 만들었다. 이를 마이크로톰을 이용하여 5 ㎛의 두께로 잘라 유리슬라이드에 붙여 조직 슬라이드를 만들었다. 이들을 공지의 면역염색법으로 염색하여 현미경으로 조직 내 단백질의 존재 여부 및 위치를 확인하였다. 면역에 사용된 항체는 항 C10orf81 항체(Novus, 1:1000)이다. 그 결과, 면역조직화학 염색에서 C10orf81 단백질은 세포막과 세포질에서 발현되었고, 정상 점막보다 강하게 발현되었다(도 4).Tissue slides were immunostained to confirm the presence and expression of proteins in normal colon epithelial and colon cancer tissues. First, paraffin embedding blocks were made by removing colonic epithelial cells and colon cancer cells from colon cancer patients through surgical resection. This was cut to a thickness of 5 μm using a microtome and attached to a glass slide to form a tissue slide. These were stained by a known immunostaining method to confirm the presence and location of proteins in the tissue under a microscope. The antibody used for immunization is an anti C10orf81 antibody (Novus, 1: 1000). As a result, in immunohistochemical staining, C10orf81 protein was expressed in cell membrane and cytoplasm, and was stronger than normal mucosa (FIG. 4).
실시예 5: C10orf81 유전자의 클로닝Example 5: Cloning of the C10orf81 Gene
Invitrogen의 인간 두뇌 c-DNA 라이브러리에서 PCR 기법을 이용하여 C10orf81 유전자를 증폭하였다. 이때 정방향 프라이머 5'-TGCGGATCCATGGAACCCAAACCTCAGAAGA-3'(BamHI 효소 절단 부위 포함-밑줄, 서열번호 8) 및 정방향 프라이머 5'-ATGGATATCTCATATCCGTCCTGTGGCTTC-3'(EcoRV 효소 절단 부위 포함-밑줄, 서열번호 9)을 이용해 55℃에서 37 사이클로 pfu premix(iNtRON, Inc.)를 사용하여 PCR로 유전자를 증폭하였다. C10orf81 유전자의 DNA 밴드를 정제한 후 BamHI/EcoRV 제한효소로 자르고, pENTR3C 벡터에 클로닝(cloning)하였다. DNA 시퀀싱(sequencing)으로 C10orf81 유전자의 염기서열(1089 bp)을 확인하고, GATE Way 시스템(Invitrogen Corp. www.invitrogen.com)을 이용한 제조회사의 매뉴얼에 따라 클로닝을 하였다. C10orf81을 클론하기 위해 pENTR3C-C10orf81을 pDEST(Invitrogen Corp. www.invitrogen.com)와 LR 반응시켜 클로닝하였다. 플라스미드 pENTR3C-C10orf81을 벡터 pDEST DNA와 함께 섞은 후 LR clonase Ⅱ 효소 혼합물을 각각 첨가하고 상온에서 1 시간 동안 배양하였다. 배양이 끝나고, 프로테나제 K를 첨가하고 37℃에서 10분간 배양하여 LR 반응을 끝낸 후, E. coli DH5α 컴피턴트 세포에 형질전환하여 pDEST-C10orf81 클론을 얻었다.The C10orf81 gene was amplified using PCR technique from Invitrogen's human brain c-DNA library. The forward primer 5'-TGC GGATCC ATGGAACCCAAACCTCAGAAGA-3 ' (BamHI enzyme cleavage site comprises - underline, SEQ ID NO: 8) and reverse primer 5'-ATG GATATC TCATATCCGTCCTGTGGCTTC-3' (EcoRV cleavage site include an enzyme-underline, SEQ ID NO: 9) Gene was amplified by PCR using pfu premix (iNtRON, Inc.) at 37 cycles at 55 ℃. The DNA band of the C10orf81 gene was purified, cut with BamHI / EcoRV restriction enzyme, and cloned into the pENTR3C vector. DNA sequencing confirmed the nucleotide sequence (1089 bp) of the C10orf81 gene, and was cloned according to the manufacturer's manual using the GATE Way system (Invitrogen Corp. www.invitrogen.com). To clone C10orf81, pENTR3C-C10orf81 was cloned by LR reaction with pDEST (Invitrogen Corp. www.invitrogen.com). The plasmid pENTR3C-C10orf81 was mixed with the vector pDEST DNA, and then the LR clonase II enzyme mixture was added and incubated for 1 hour at room temperature. After culturing, proteinase K was added and incubated at 37 ° C. for 10 minutes to complete the LR reaction. E. coli DH5α competent cells were transformed to obtain a pDEST-C10orf81 clone.
실시예 6: C10orf81의 과발현에 의한 세포 성장 속도 증가 효과 확인Example 6: Confirmation of the effect of increasing the cell growth rate by overexpression of C10orf81
정상세포주인 NIH3T3 섬유아세포(ATCC)를 5% 송아지 혈청(bovine calf serum)과 페니실린(10,000 단위/㎖)-스트렙토마이신(10 ㎎/㎖)의 혼합액(GIBCO, 15140)을 100배 희석한 DMEM 배지(GIBCO)에 1.4 X 105 세포/㎖의 농도로 60 mm 디쉬에 5 ㎖ 접종한 후 5% CO2 배양기에서 37℃로 24시간 동안 배양하였다. C10orf81 플라스미드 DNA를 배양된 상기 세포에 트랜스펙션(transfection)하였다. 6시간 후 배지를 모두 제거하고 PBS(Phosphate Buffered Saline)로 3회 씻어준 후, 5% 혈청이 든 배지로 갈아주었다. 트랜스펙션 후 24시간이 경과하면 0.5% EDTA-트립신을 처리하여 세포를 모아 혈구계수기(heamacytometer)로 세포수를 측정한 다음, 24 웰 플레이트에 1 X 105 세포/㎖의 농도로 접종(seeding)하여 5% CO2 배양기에서 37℃로 SRB(Sulforhodamine B) 분석을 할때까지 배양하였다. 배양 후 12시간 마다 플레이트를 하나씩 꺼내어 SRB 분석 방법으로 세포성장율(cell growth rate)을 측정하였다. 비교를 위해 pDEST 벡터 DNA로 트랜스펙션하고 상기 NIH3T3 세포와 동일하게 처리하여 대조군으로 사용하였다. 그 결과를 도 5에 나타내었다. 도 5의 가로축은 시간을 세로축은 세포성장율을 나타내며, pDEST는 C10orf81 유전자가 없는 벡터를 트랜스펙션한 세포를 의미하고, pDEST-C10orf81은 C10orf81 유전자가 클론된 벡터를 트랜스펙션하여 C10orf81 단백질이 과발현된 세포를 의미한다.DMEM medium containing a mixture of 5% bovine calf serum and penicillin (10,000 units / ml) and streptomycin (10 mg / ml) in a normal cell line, NIH3T3 fibroblasts (ATCC), diluted 100-fold. (GIBCO) was inoculated with 5 ml in a 60 mm dish at a concentration of 1.4 × 10 5 cells / ml and then incubated for 24 hours at 37 ° C. in a 5% CO 2 incubator. C10orf81 plasmid DNA was transfected into the cultured cells. After 6 hours, all of the medium was removed, washed three times with PBS (Phosphate Buffered Saline), and then changed to a medium containing 5% serum. 24 hours after transfection, the cells were treated with 0.5% EDTA-trypsin, collected and counted by a hemocytometer, and seeded in a 24-well plate at a concentration of 1 × 10 5 cells / ml. ) Was cultured in a 5% CO 2 incubator at 37 ° C. until SRB (Sulforhodamine B) analysis. One plate was taken out every 12 hours after incubation, and cell growth rate was measured by SRB analysis. For comparison, the cells were transfected with pDEST vector DNA and treated the same as the NIH3T3 cells to be used as a control. The results are shown in FIG. 5, the horizontal axis represents time, and the vertical axis represents cell growth rate. PDEST represents a cell transfected with the vector without the C10orf81 gene, and pDEST-C10orf81 transfects the vector cloned with the C10orf81 gene to overexpress the C10orf81 protein. Means cells.
그 결과, C10orf81 유전자가 과량 발현된 세포에서는 성장 속도가 약 35% 정도 증가하였는데, 이것은 C10orf81 유전자는 종양세포의 특징인 세포의 성장 속도를 증가시키는 것과 관련되어 있음을 나타낸다(도 5). 이러한 결과는 C10orf81 유전자의 발현량 증가가 종양세포 진단 지표가 될 수 있음을 의미한다.As a result, the growth rate was increased by about 35% in cells overexpressing the C10orf81 gene, indicating that the C10orf81 gene is associated with increasing the growth rate of cells characteristic of tumor cells (FIG. 5). These results indicate that increased expression level of the C10orf81 gene may be a diagnostic marker for tumor cells.
실시예 7: 암세포주에 siC10orf81의 도입에 따른 암세포주의 성장 억제 효과 확인Example 7: Confirmation of the growth inhibitory effect of the cancer cell line according to the introduction of siC10orf81 in the cancer cell line
암세포 조직 또는 암세포주에서 다량 발현되는 유전자의 경우 siRNA 기법을 통해 유전자 발현을 억제하여 세포의 증식 속도에 변화가 있는지를 관찰할 수 있다. 이에 본 발명에서는 C10orf81에 대한 각각의 siRNA를 디자인하여 암세포주에 도입한 후 해당유전자 mRNA 발현 저하에 따른 암세포주의 성장 속도의 변화를 관찰하였다. 위암 세포주 SNU484(서울대학교 세포주 은행), 및 SNU638(서울대학교 세포주 은행), 간암세포주 Chang(ATCC) 및 SNU354(서울대학교 세포주 은행), 대장암세포주 KM12C(서울대학교 세포주 은행) 및 Colo205(서울대학교 세포주 은행), 폐암 세포주 A549(ATCC), 전립선암 세포주 PC3(ATCC)를 이용하였다. 정상 세포주로는 IMR90(human fetal lung fibroblasts, ATCC#CCL-186)을 이용하였다.In the case of genes that are expressed in large amounts in cancer cell tissues or cancer cell lines, siRNA techniques can be used to observe whether there is a change in cell proliferation rate by inhibiting gene expression. In the present invention, each siRNA for C10orf81 was designed and introduced into the cancer cell line, and then the growth rate of the cancer cell line was observed according to the decrease in the gene mRNA expression. Gastric cancer cell lines SNU484 (Seoul National University Cell Line Bank), SNU638 (Seoul National University Cell Line Bank), Liver Cancer Cell Chang (ATCC) and SNU354 (Seoul National University Cell Line Bank), Colon Cancer Cell Lines KM12C (Seoul National University Cell Line Bank) and Colo205 (Seoul National University Cell Line) Bank), lung cancer cell line A549 (ATCC), prostate cancer cell line PC3 (ATCC). IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as a normal cell line.
7-1. KM12C 세포주를 이용한 siC10orf81에 의한 C10orf81의 mRNA 저해효과 확인7-1. Confirmation of mRNA Inhibitory Effect of C10orf81 by siC10orf81 Using KM12C Cell Line
먼저 C10orf81 및 대조군으로 사용할 siRNA를 디자인하여 합성하였다(삼천리 제약, 대한민국). 합성된 siRNA는 유전자 염기서열을 바탕으로 디자인된 19개 올리고뉴클레오타이드의 이중 리보핵산쇄에 단쇄의 2 염기가 매달린 구조로 이루어져 있다. 대조군 siRNA는 세포의 증식에 아무 영향을 미치지 않는 siRNA 실험의 음성대조군으로 사용되었다.First, C10orf81 and siRNA to be used as a control were designed and synthesized (Samchully Pharmaceutical, Korea). The synthesized siRNA consists of a structure in which two bases of a single chain are suspended in a double ribonucleic acid chain of 19 oligonucleotides designed based on gene sequences. Control siRNA was used as a negative control of siRNA experiments that had no effect on cell proliferation.
암 세포주 KM12C를 70% 컨플루언시로 배양 후 하이퍼펙트 방법(Qiagen Co.)으로 siC10orf81(센스서열, 서열번호 10), siControl(센스서열, 서열번호 11)를 세포 내에 도입하여 72시간 동안 배양하며 형질 전환하였다. 각 세포에서 RNA를 추출하여 상기에서 상세히 기술한 바와 같이 RT-PCR을 수행하여 해당 유전자 mRNA가 감소되었음을 확인하고 웨스턴 블롯을 통한 단백질 발현을 분석하여 siRNA에 의한 유전자 발현 억제가 이루어졌음을 확인하였다(도 6a). siControl은 음성대조군을 나타낸다. siControl은 인간의 유전자와 상동성이 없는 시퀀스에 대한 siRNA로 타겟하는 부분이 없는 것을 보여준다. 각 세포를 SRB(Sulfur rhodamine B, Sigma Co.)로 염색하고 현미경을 통해 세포 성장에 큰 변화가 없는 siControl를 처리한 시료의 SRB 염색한 세포의 사진을 관찰함으로써 siC10orf81을 처리한 세포에서 성장 억제가 일어났음을 보여주고 있다(도 6b).After culturing cancer cell line KM12C with 70% confluency, siC10orf81 (sense sequence, SEQ ID NO: 10) and siControl (sense sequence, SEQ ID NO: 11) were introduced into cells by hyperfect method (Qiagen Co.) for 72 hours. And transformed. As described in detail above, RNA was extracted from each cell to confirm that gene mRNA was reduced by RT-PCR, and protein expression was analyzed by Western blot to confirm that gene expression was inhibited by siRNA ( 6a). siControl represents the negative control. siControl shows no siRNA targets for sequences that are not homologous to human genes. Each cell was stained with SRB (Sulfur rhodamine B, Sigma Co.), and microscopic observation of the SRB-stained cells of the siControl-treated samples showed no significant changes in cell growth. It is shown that it happened (Fig. 6b).
7-2. 다양한 암 세포주에서의 siC10orf81 에 의한 성장 저해 효과 검증7-2. Validation of growth inhibition by siC10orf81 in various cancer cell lines
좀 더 다양한 세포주에서 siRNA에 의한 세포 성장 저하 정도를 조사하기 위해 1.0 x 105 세포/㎖의 농도로 24 웰 플레이트에 0.5 ㎖ 접종(seeding)한 후 5% CO2 배양기에서 37℃로 24시간 동안 배양하였다. 대조군 siRNA(Control siRNA)(센스서열, 서열번호 11)와 siC10orf81(센스서열, 서열번호 10)을 위의 방법대로 세포 내로 트랜스펙션한 후 72시간 동안 배양하며 siRNA에 의한 세포의 성장 억제정도도 조사하였다. 각 세포를 SRB(Sulfur rhodamine, Sigma Co.)로 염색하고 대조군(control)인 siControl을 처리한 세포주를 기준으로, 세포의 성장 억제 정도를 비교하여 그 결과를 도 7에 나타내었다. 도 7의 X축은 각 세포주를 나타내며, Y축은 상대세포성장비(Relative cell growth)를 나타낸다. 그 결과 대부분의 암 세포주에서 C10orf81의 유전자 발현 억제가 세포 성장 억제를 나타내었다. 이러한 결과는, C10orf81 유전자의 siRNA는 대장암 세포주 및 폐암, 간암, 위암 및 전립선암 세포주의 성장 억제를 유발할 수 있으므로 C10orf81 유전자가 암 치료 타겟으로 활용 가능함을 의미한다. To investigate the extent of cell growth degradation by siRNA in more diverse cell lines, 0.5 ml seeding in 24 well plates at a concentration of 1.0 x 10 5 cells / ml was followed by 24 hours at 37 ° C. in a 5% CO 2 incubator. Incubated. Control siRNA (sense sequence, SEQ ID NO: 11) and siC10orf81 (sense sequence, SEQ ID NO: 10) were transfected into the cells according to the above method and cultured for 72 hours. Investigate. Each cell was stained with SRB (Sulfur rhodamine, Sigma Co.) and the control group treated with siControl, and compared with the degree of growth inhibition of the cells, the results are shown in FIG. The X axis of FIG. 7 represents each cell line, and the Y axis represents Relative cell growth. As a result, the inhibition of gene expression of C10orf81 in most cancer cell lines showed cell growth inhibition. These results indicate that siRNA of the C10orf81 gene can induce growth inhibition of colorectal cancer cell lines and lung cancer, liver cancer, gastric cancer and prostate cancer cell lines, so that the C10orf81 gene can be used as a cancer therapeutic target.
표 1은 C10orf81 유전자에 대한 siRNA 의 예를 보여준다.Table 1 shows an example of siRNA for the C10orf81 gene.
표 1
mRNA 내 siRNA 위치 서열 GC 함량 Identity
1531 CUGAACAGAGCUCCCAAGATT 0.53 19
3066 CGUUCAGUUGCAGAACCAATT 0.47 19
74 GUAUGAAGGCCCAGAACUUTT 0.47 19
163 ACUUCCUGGUGGUUCAGAUTT 0.47 19
1116 AGACAUCCCAUGAGUCUGUTT 0.47 19
1847 AGACACAGAUUGGGUCAGUTT 0.47 19
978 CUCGAAGUGUUCUUUUAGATT 0.37 19
2961 CAAAAACAGGCUUCAGAAATT 0.37 19
234 GCAGGAGUGAGUUCAGGAATT 0.53 19
1516 CUGGAUAAGCCUAGACUGATT 0.47 19
Table 1
siRNA position within mRNA order GC content Identity
1531 CUGAACAGAGCUCCCAAGATT 0.53 19
3066 CGUUCAGUUGCAGAACCAATT 0.47 19
74 GUAUGAAGGCCCAGAACUUTT 0.47 19
163 ACUUCCUGGUGGUUCAGAUTT 0.47 19
1116 AGACAUCCCAUGAGUCUGUTT 0.47 19
1847 AGACACAGAUUGGGUCAGUTT 0.47 19
978 CUCGAAGUGUUCUUUUAGATT 0.37 19
2961 CAAAAACAGGCUUCAGAAATT 0.37 19
234 GCAGGAGUGAGUUCAGGAATT 0.53 19
1516 CUGGAUAAGCCUAGACUGATT 0.47 19
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
본 발명에 따라 위암, 간암, 대장암, 폐암 또는 전립선암에 특이적인 유전자를 제공하고 상기 암의 전이 및 예후를 판단할 수 있는 진단 마커를 제공함으로써, 정확한 암 진단을 가능하게 하며, 암의 치료 및 예후관리에 유용하게 사용할 수 있다. 또한, 상기 유전자를 억제하는 암 특이적인 치료제를 제공함으로써, 부작용이 적은 바이오 신약 및 맞춤형 항암제의 개발을 가능하게 할 수 있다.According to the present invention, by providing genes specific for gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer and providing diagnostic markers for determining the metastasis and prognosis of the cancer, accurate cancer diagnosis is possible and treatment of cancer And it can be useful for prognosis management. In addition, by providing a cancer-specific therapeutic agent that inhibits the gene, it is possible to enable the development of bio-new drugs and customized anti-cancer agents with fewer side effects.

Claims (13)

  1. C10orf81(Chromosome 10 open reading frame 81) 유전자의 mRNA 또는 이의 단백질의 발현수준을 측정하는 제제를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단용 조성물.A cancer diagnostic composition selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer, comprising an agent for measuring the expression level of mRNA of C10orf81 (Chromosome 10 open reading frame 81) gene or protein thereof.
  2. 제1항에 있어서, 상기 유전자의 mRNA의 발현수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머 또는 프로브를 포함하는 것인 조성물.The composition of claim 1, wherein the agent for measuring the expression level of mRNA of the gene comprises a primer or a probe that specifically binds to the gene.
  3. 제1항에 있어서, 상기 단백질의 발현수준을 측정하는 제제는 상기 단백질에 특이적인 항체를 포함하는 것인 조성물.The composition of claim 1, wherein the agent for measuring the expression level of the protein comprises an antibody specific for the protein.
  4. 제1항 내지 제3항 중 어느 한 항의 조성물을 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단용 키트.A cancer diagnostic kit selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, comprising the composition of any one of claims 1 to 3.
  5. 제4항에 있어서, 상기 키트는 마이크로어레이, 유전자 증폭 키트 또는 면역분석(immunoassay)용 키트로 구성된 군으로부터 선택된 것인 키트.The kit of claim 4, wherein the kit is selected from the group consisting of a microarray, a gene amplification kit, or an immunoassay kit.
  6. (a) 암 의심 환자의 생물학적 시료로부터 C10orf81 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 측정하는 단계; 및(a) measuring the mRNA expression level of the C10orf81 gene or the protein encoded by the gene from a biological sample of a suspected cancer patient; And
    (b) 상기 유전자의 mRNA 발현수준 또는 상기 유전자가 코딩하는 단백질의 발현수준을 정상 대조구 시료의 해당 유전자의 mRNA 발현수준 또는 단백질 발현수준과 비교하는 단계를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 진단을 위한 정보의 제공 방법.(b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample; gastric cancer, liver cancer, colon cancer, lung cancer And providing information for diagnosing cancer selected from the group consisting of prostate cancer.
  7. 제6항에 있어서, 상기 mRNA 발현수준의 측정은 역전사효소 중합효소반응, 경쟁적 역전사효소 중합효소반응, 실시간 역전사효소 중합효소반응, RNase 보호 분석법, 노던 블랏팅 또는 DNA 칩에 의한 것인 방법.The method of claim 6, wherein the measurement of mRNA expression level is by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting or DNA chip.
  8. 제6항에 있어서, 상기 단백질 발현수준의 측정은 해당 단백질에 특이적인 항체를 이용하는 것인 방법.The method of claim 6, wherein the measuring of the protein expression level uses an antibody specific for the protein.
  9. 제6항에 있어서, 상기 단백질 발현수준의 측정은 웨스턴 블랏, ELISA, 방사선면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 면역침전 분석법, 보체 고정 분석법, FACS 또는 단백질 칩을 이용하는 것인 방법.The method of claim 6, wherein the protein expression level is measured by Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunity staining, immunoprecipitation assay, complement fixation assay, Using FACS or protein chips.
  10. C10orf81 유전자의 발현 억제제 또는 이의 단백질의 활성 억제제를 포함하는 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암의 예방 또는 치료용 약제학적 조성물.A pharmaceutical composition for preventing or treating cancer selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer comprising an inhibitor of the C10orf81 gene or an activity inhibitor of a protein thereof.
  11. 제10항에 있어서, 상기 유전자의 발현 억제제는 C10orf81 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 microRNA로 구성된 군으로부터 선택된 것인 조성물.The composition of claim 10, wherein the expression inhibitor of the gene is selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the C10orf81 gene.
  12. 제10항에 있어서, 상기 단백질의 활성 억제제는 C10orf81 단백질에 특이적인 항체인 것인 조성물.The composition of claim 10, wherein the activity inhibitor of the protein is an antibody specific for the C10orf81 protein.
  13. (a) 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성을 분석하는 단계; 및(a) analyzing the expression of the C10orf81 gene or the activity of a protein thereof after treating the test substance; And
    (b) 시험물질을 처리한 후 C10orf81 유전자의 발현 또는 이의 단백질의 활성이 시험물질을 처리하지 않은 C10orf81 유전자의 발현 또는 이의 단백질의 활성에 비하여 억제되면 암 치료제로 판단하는 단계를 포함하는, 위암, 간암, 대장암, 폐암 및 전립선암으로 구성된 군으로부터 선택된 암 치료제의 스크리닝 방법.(b) after treating the test substance, if the expression of the C10orf81 gene or the activity of the protein thereof is inhibited compared to the expression of the C10orf81 gene or the activity of the protein which did not process the test substance, gastric cancer, comprising: A method for screening a cancer therapeutic agent selected from the group consisting of liver cancer, colon cancer, lung cancer and prostate cancer.
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