WO2012011778A2 - Trousse de diagnostic du cancer et composition pharmaceutique pour la prévention ou le traitement du cancer - Google Patents

Trousse de diagnostic du cancer et composition pharmaceutique pour la prévention ou le traitement du 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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WO2012011778A3 (fr
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정경숙
원미선
안지원
김영주
송은영
염영일
이희구
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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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
    • G01N33/575—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.

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Abstract

L'invention concerne une composition de diagnostic du cancer contenant une formulation pour mesurer le niveau d'expression d'un ARNm du gène 81 à cadre ouvert de lecture, situé sur le chromosome 10 (C10orf81), ou le niveau d'expression de la protéine dudit gène; une trousse de diagnostic du cancer contenant la composition; une méthode d'apport d'informations sur le diagnostic du cancer comprenant une étape de mesure du niveau d'expression de l'ARNm du gène C10orf81 ou du niveau d'expression de la protéine codée par ledit gène; et une méthode de criblage de compositions pharmaceutiques destinées à la prévention ou au traitement du cancer, dont un inhibiteur d'expression du gène C10orf81 ou un inhibiteur d'activité de la protéine dudit gène et des agents thérapeutiques contre le cancer. L'invention permet de poser un diagnostic précis du cancer au moyen d'un gène spécifique du cancer de l'estomac, du cancer du foie, du cancer colorectal, du cancer du poumon, ou du cancer de la prostate, et au moyen de marqueurs de diagnostic permettant de déterminer la métastase et le pronostic du cancer. L'invention peut être utile dans la gestion du diagnostic et du pronostic du cancer. L'invention permet en outre de mettre au point de nouveaux biomédicaments présentant moins d'effets secondaires et des médicaments anticancéreux personnalisés, par la mise en oeuvre d'un agent thérapeutique spécifique du cancer colorectal pouvant inhiber le gène.
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