WO2009131366A2 - Cdca5 en tant que marqueur diagnostique et agent thérapeutique pour le cancer gastrique ou le cancer colorectal - Google Patents

Cdca5 en tant que marqueur diagnostique et agent thérapeutique pour le cancer gastrique ou le cancer colorectal Download PDF

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WO2009131366A2
WO2009131366A2 PCT/KR2009/002085 KR2009002085W WO2009131366A2 WO 2009131366 A2 WO2009131366 A2 WO 2009131366A2 KR 2009002085 W KR2009002085 W KR 2009002085W WO 2009131366 A2 WO2009131366 A2 WO 2009131366A2
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cancer
cdca5
gastric
colorectal cancer
gene
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PCT/KR2009/002085
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WO2009131366A3 (fr
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염영일
이희구
윤효란
김재화
송은영
김종태
김영호
전호경
정경숙
원미선
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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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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 relates to a biomarker CDCA5 (cell division cycle associated 5) specific for gastric cancer or colorectal cancer, and more particularly, to a composition for diagnosing gastric cancer or colorectal cancer comprising an agent for measuring the expression level of CDCA5.
  • a kit comprising a composition, a method for detecting the marker, and a method for screening a gastric or colorectal cancer therapeutic agent using the marker.
  • the present invention also relates to a pharmaceutical composition for the treatment and prevention of cancer comprising a substance that inhibits the expression or activity of CDCA5.
  • the third cause is the presence of Helicobacter pylori (H.P) infection in the development of gastric cancer.
  • H.P Helicobacter pylori
  • the causal relationship between Helicobacter pylori infection and gastric cancer may not be decisive, it may be premature to conclude that the infection of the Helicobacter pylori is detected in 40-60% of Korean gastrointestinal disease and gastric cancer patients.
  • the relative risk of stomach cancer is relatively high compared to Therefore, elimination of Helicobacter pylori has emerged as a method of preventing gastritis and gastric cancer.
  • cancer cells are produced. However, it is a general view that cancer is caused by the generation of cells that are not controlled by the alteration of genes that function to control the growth of normal cells. All. Therefore, in the early stages of gastric cancer, cancer cells are classified as early gastric cancer, and the prognosis of treatment for patients found in early gastric cancer was relatively good. Therefore, early diagnosis and treatment of gastric cancer is expected to contribute to lowering the mortality rate of gastric cancer and lowering the cost of treating cancer.
  • Stomach cancer symptoms range from no symptoms to severe pain.
  • the symptoms of gastric cancer rather than having a certain characteristic is a general digestive symptoms.
  • the early stages of gastric cancer asymptomatic cases, even if the slightest indigestion or upper abdominal discomfort, so most people overlook it, causing the death rate of stomach cancer.
  • gastrointestinal X-ray imaging which includes dual imaging, compression, and mucosal imaging.
  • gastroscopy allows direct visual observation of the stomach gland, and even the smallest lesions that do not appear on X-rays. Not only can it be done, but biopsy can be performed directly in places where stomach cancer is suspected, increasing the diagnosis rate.
  • this method has the disadvantages of hygiene problems and patient suffering during the examination.
  • the best treatment of gastric cancer that has been advanced so far is to remove the lesion by surgery, and thus, the only treatment for the cure is surgical resection.
  • Surgical excision can be done in various ways.
  • the extent of resection may be determined in consideration of the sequelae after extensive resection.
  • gastric cancer has metastasized to other organs, radical surgery is impossible. Therefore, other methods such as chemotherapy are taken.
  • anti-cancer drugs that are on the market can be used to relieve temporary symptoms or to suppress recurrence and prolong survival. Although it has a temporary effect, the patient may suffer from double pain due to side effects and economic burdens caused by chemotherapy.
  • biomarkers for the development of a diagnostic agent for gastric cancer and the development of a therapeutic agent that complements the shortcomings of surgical resections or anticancer agents, and the development of agents that measure the diagnostic markers are intractable disease. It is one of the leading tasks to conquer stomach cancer, which is one of the goals of this study.
  • colorectal cancer is adenocarcinoma (adenocarcinoma) in pathology, and largely divided into colon cancer and rectal cancer.
  • adenocarcinoma adenocarcinoma
  • the incidence of site-specific incidence occurs most often in the lower colon, or rectum, at about 50%.
  • Recent studies have shown that changes in dietary habits have significantly increased the incidence and mortality of colorectal cancer in Korea, and the incidence of colorectal cancer increased 420% from 1995 to 2002, making it the number one in cancer. (The 2003 Health Insurance Statistics Yearbook, published by the National Health Insurance Service).
  • the cause of colorectal cancer is still unknown, but genetic factors, dietary habits associated with eating high-fat and low-fiber foods, and inflammatory bowel disease are being considered.
  • Colorectal cancer can occur in all age groups, but as the age increases, the incidence increases and occurs frequently in the 50s and 60s. The incidence of men and women is higher in colon cancer in women and rectal cancer in men. Treatment of colorectal cancer is performed by chemotherapy and radiotherapy based on surgical resection. Despite advances in surgical therapies, chemotherapy and radiotherapy, mortality rates are very high when people miss the point of surgery because they are metastasized to other organs due to the characteristics of colorectal cancer that progress without specific symptoms. The 5-year average survival rate is at least 90% in stage 1, at least 70% in stage 2, at least 50% in stage 3 and below 5% in stage 4.However, the earlier the early detection and treatment of colorectal cancer, Significantly increased (2004 Cancer Information, published by the National Cancer Center). Therefore, the development of a method for effectively and early diagnosis of colorectal cancer is urgently required.
  • cancer-labeled antigens Human tumors express and secrete a variety of molecules called cancer-labeled antigens.
  • antigens that can be used for the diagnosis and treatment of cancer onset and metastasis have been suggested by analyzing the serum of various cancer patients.
  • NSE lung cancer
  • C15-3 breast cancer
  • CA19-9 colon cancer, pancreatic cancer
  • no diagnostic markers or anticancer drugs have been developed that have an excellent effect on the diagnosis and treatment of gastric or colorectal cancer.
  • the present inventors prepared DNA chips for genes predicted to be related to gastric cancer or colorectal cancer, and compared expression levels in gastric cancer as well as colorectal cancer, breast cancer, and pancreatic cancer.
  • genes that were specifically overexpressed only in gastric or colorectal cancer tissues, and to finally select potential CDCA5 genes as a diagnostic marker for gastric or colorectal cancer, thereby enabling early and accurate diagnosis of gastric or colorectal cancer.
  • the present invention was completed by revealing that a substance that inhibits the production or secretion of the gene can be used for the treatment of gastric or colorectal cancer.
  • One object of the present invention to provide a composition for diagnosing gastric cancer or colorectal cancer comprising an agent for measuring the level of mRNA of the CDCA5 gene or protein thereof.
  • Another object of the present invention is to provide a gastric or colorectal cancer diagnostic kit comprising the composition for diagnosing gastric or colorectal cancer.
  • Another object of the present invention is to provide a method for detecting gastric or colorectal cancer marker CDCA5.
  • the present invention is expected to find an effect that can be used as an index of data useful for the treatment and prognosis of gastric cancer or colorectal cancer by discovering and providing a diagnostic marker that can determine the metastasis and prognosis of gastric cancer or colorectal cancer.
  • Using the gastric cancer or colorectal cancer marker CDCA5 according to the present invention can accurately and easily determine the presence or absence of gastric cancer or colorectal cancer, can be used as a specific target in the development of gastric cancer or colorectal cancer-specific anticancer drugs, furthermore, It can be used for the study of tumor formation of cancer and is expected to contribute to the early diagnosis of liver cancer.
  • the composition comprising a substance that inhibits the expression or activity of CDCA5 according to the present invention can be usefully used for the prevention and treatment of cancer by inhibiting the expression or activity of CDCA5.
  • 1 is a result of comparing gene expression in normal gastric tissue and gastric cancer or colorectal cancer tissue using a 48K human micro array chip (Illumina).
  • FIG. 2 is a diagram of genes specifically overexpressed in (a) gastric cancer or (b) colorectal cancer cells based on 48K human micro array chip (Illumina) analysis results.
  • FIG. 5 shows DNA chip results of the same genes in gastric cancer, colorectal cancer, breast cancer, prostate cancer and liver cancer, and compares their expressions by data mining.
  • FIG. 6 is an electrophoretic photograph confirming the expression level of CDCA5 in normal tissue and gastric cancer tissue using reverse transcriptase polymerase reaction of CDCA5 gene.
  • the expression level of each gene in normal tissues Represents the expression level of each gene.
  • Hs.683 shows the expression level of the gastric cancer diagnostic marker of the present invention through reverse transcriptase.
  • FIG. 8 is an electrophoretic photograph confirming the expression level of CDCA5 in normal tissue and colorectal cancer tissue using reverse transcriptase polymerase reaction of CDCA5 gene, in which odd lanes represent expression levels of each gene in normal tissues and cancer tissues in even lanes. The expression level of each gene in is shown.
  • Figure 9 is a graph comparing the expression level of CDCA5 in normal tissues (a) gastric cancer tissue and (b) colorectal cancer tissue using real-time polymerase reaction of the CDCA5 gene.
  • FIG. 12 shows that CDCA5 is specifically expressed in colorectal cancer tissues using immunohistostaining in colorectal cancer tissues and normal tissues using CDCA5 specific antibodies.
  • the present invention relates to a composition for diagnosing gastric cancer or colorectal cancer comprising an agent for measuring the expression level of the cell division cycle associated 5 (CDCA5).
  • diagnosis means identifying the presence or characteristic of a pathological condition.
  • diagnosis is to determine whether the cancer of the stomach or colorectal cancer.
  • diagnostic marker is a substance that can distinguish gastric cancer or colorectal cancer cells from normal cells, and has gastric cancer or colorectal cancer as compared to normal cells.
  • Polypeptides or nucleic acids eg, mRNA, etc.
  • organic biomolecules such as lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.
  • the gastric cancer or colorectal cancer diagnostic markers of the present invention have a CDCA5 (cell division cycle associated 5) gene which exhibits a particularly high level of expression in gastric or colorectal cancer cells compared to cells of normal gastric or colorectal tissue. And proteins encoded thereby.
  • CDCA5 cell division cycle associated 5
  • CDCA5 (cell division cycle associated 5) is a factor that regulates chromosome adhesion and S or G2 phase, and its gene and protein information is registered in the National Center for Biotechnology Information (NCBI) (NM_080668, NP_542399). CDCA5 is known to be involved in chromosomal adhesions, but the function of CDCA5 and its association with gastric or colorectal cancer are not known at all.
  • CDCA5 can be used as a marker for diagnosing gastric or colorectal cancer through the following verification.
  • 1,601 genes specifically overexpressed in gastric cancer cells were first extracted from DNA samples using DNA chips, and then divided into two clusters using clustering analysis.
  • FIG. 1 a gene expressing a doubling expression difference in more than 60% of patients
  • FIG. 2 which is a gene that is specifically expressed only in gastric cancer or colon cancer as compared with colorectal cancer, breast cancer and pancreatic cancer CDCA5 was finally screened ( Figures 3-5).
  • overexpression of CDCA5 was confirmed in gastric cancer tissue or colorectal cancer tissue obtained from colorectal cancer patients by immunohistochemistry (FIG. 12).
  • the term "agent measuring the expression level of cell division cycle associated 5 (CDCA5)” refers to the expression level of CDCA5, which is a marker for increasing expression in gastric or colorectal cancer cells, as described above. It refers to a molecule capable of, preferably refers to an antibody, primer or probe specific to the marker.
  • the expression level of CDCA5 can be known by identifying the expression level of the mRNA of the CDCA5 gene or the protein encoded by the gene.
  • "mRNA expression level measurement” is a process of confirming the presence and expression level of mRNA of gastric cancer or colorectal cancer marker gene in a biological sample to diagnose gastric cancer or colorectal cancer, and can be known by measuring the amount of mRNA. Analytical methods for this include RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting (Northern) blotting) and DNA chips, but are not limited thereto.
  • Agents for measuring mRNA levels of genes are preferably primer pairs or probes, and since the nucleic acid sequence of the CDCA5 gene is identified in NM_080668 (NCBI), those skilled in the art will appreciate primers that specifically amplify specific regions of these genes based on the sequence.
  • the probe can be designed.
  • 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.
  • PCR amplification using sense and antisense primers of CDCA5 polynucleotide can be used to diagnose gastric cancer or colorectal cancer through the generation of desired products. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.
  • the term "probe” refers to nucleic acid fragments such as RNA or DNA, which are short to several bases to hundreds of bases, which are capable of specific binding with mRNA, and are labeled to identify the presence of a specific mRNA.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.
  • hybridization may be performed using a probe complementary to a CDCA5 polynucleotide, and gastric cancer or colorectal cancer may 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 composition for detecting gastric or colorectal cancer diagnostic markers includes respective primer pairs specific for the CDCA5 gene.
  • measurement of protein expression level refers to a process of confirming the presence and degree of expression of a protein expressed in a gastric or colorectal marker gene in a biological sample to diagnose gastric or colorectal cancer.
  • Specific amounts of the antibodies are used to determine the amount of protein.
  • 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), FACS and protein chips (protein chip) and the like, but are not limited thereto.
  • Agents for measuring protein levels are preferably antibodies.
  • an antibody refers to a specific protein molecule directed to an antigenic site as it is known in the art.
  • an antibody means an antibody that specifically binds to CDCA5, a marker of the present invention, which is encoded by the marker gene by cloning each gene into an expression vector according to a conventional method.
  • Proteins can be obtained and prepared from conventional proteins by conventional methods.
  • 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 gastric or colorectal 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 relates to a kit for diagnosing gastric or colorectal cancer comprising the composition for diagnosing gastric or colorectal cancer.
  • the kit of the present invention can detect markers by confirming the expression level of mRNA or protein with the expression level of CDCA5, which is a gastric or colorectal cancer diagnostic marker.
  • the marker detection kit of the present invention includes one or more other component compositions, solutions or solutions suitable for analytical methods as well as primers, probes or optionally antibodies that detect the expression level of a gastric or colorectal diagnostic marker. Device may be included.
  • the kit for measuring the mRNA expression level of CDCA5 in the present invention may be a kit containing the necessary elements necessary to perform RT-PCR.
  • RT-PCR kits in addition to each primer pair specific for the marker gene, RT-PCR kits can be used in test tubes or other appropriate containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymers. Enzymes such as lyase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as the quantitative control.
  • the kit of the present invention may be a diagnostic kit including essential elements necessary for performing a DNA chip.
  • the DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, an agent, an enzyme, or the like for preparing a fluorescent probe.
  • the substrate may comprise cDNA corresponding to the quantitative control gene or fragment thereof.
  • the kit for measuring the protein expression level of CDCA5 in the present invention includes a substrate, a suitable buffer, a secondary antibody labeled with a chromophore or a fluorescent substance, a chromogenic substrate, and the like for immunological detection of the antibody. can do.
  • the substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like.
  • the chromophore may be a peroxidase or an alkaline force.
  • Fatase Alkaline Phosphatase
  • the fluorescent material can be used FITC, RITC, etc.
  • the color substrate substrate is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ) Or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.
  • the present invention relates to a method for detecting gastric cancer or colorectal cancer marker CDCA5 by comparing the expression level of CDCA5 from a sample of a patient with the expression level of normal cells to provide information necessary for diagnosing gastric or colorectal cancer. will be.
  • the expression level of the gene can be detected at the mRNA level or the protein level, and separation of the mRNA or protein from the biological sample can be performed using known processes.
  • patient's sample includes but is not limited to tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, which differ in expression level of CDCA5, which is a gastric or colon cancer marker gene. Do not.
  • the above detection methods it is possible to diagnose whether a cancer patient is suspected of cancer or colorectal cancer by comparing the gene expression level in a normal control group with the gene expression level in a gastric cancer or colorectal cancer suspected patient. That is, after measuring the expression level of the marker of the present invention from cells suspected of gastric cancer or colorectal cancer, and comparing the two by measuring the expression level of the marker of the present invention from normal cells, the expression level of the marker of the present invention is normal. If more expression is derived from a cell suspected of gastric or colorectal cancer than that of a cell, a cell suspected of gastric or colorectal cancer can be predicted as gastric cancer or colorectal cancer.
  • Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, and DNA chip.
  • mRNA expression level measurement is preferably by using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for the gene used as a gastric or colon cancer marker.
  • the reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band to determine the mRNA expression and degree of genes used as diagnostic markers for gastric or colorectal cancer, and compared with the control, stomach cancer or Colon cancer can be diagnosed easily.
  • the DNA chip is a DNA chip in which the nucleic acid corresponding to the gastric cancer or colorectal cancer marker gene or fragment thereof is attached to a glass-like substrate with high density, and isolates the mRNA from the sample, and the end or the inside of the DNA chip as a fluorescent material Labeled cDNA probes can be prepared, hybridized to DNA chips and read for the development of gastric or colorectal cancer.
  • Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto.
  • the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected gastric cancer or colorectal cancer patients can be compared, and the significance of gastric cancer or colorectal cancer marker gene to protein By determining whether the amount of expression is increased, it is possible to diagnose whether the actual gastric or colorectal cancer of the patient suspected of stomach or colorectal cancer.
  • antigen-antibody complex means a combination of gastric cancer or colorectal cancer marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formation is quantitatively measured through the magnitude of a signal of a detection label. It is possible.
  • Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto.
  • enzymes include ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, ⁇ -latamase, and the like.
  • Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like.
  • Ligands include, but are not limited to, biotin derivatives.
  • Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like.
  • Microparticles include, but are not limited to, colloidal gold, colored latex, and the like.
  • Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K 4 W (CN) 8 , [Os (bpy) 3 ] 2+ , [RU (bpy) 3 ] 2+ , [MO (CN) 8 ] 4-, and the like.
  • Radioisotopes include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, and the like. .
  • Protein expression level measurement is preferably by using an 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, attached to a solid support
  • Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support
  • Various ELISA methods include indirect sandwich ELISA using secondary antibodies.
  • 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 is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. The degree of complex formation of gastric or colorectal marker protein and antibody can be checked to determine whether gastric or colorectal cancer develops.
  • Western blot using at least one antibody against the gastric or colorectal cancer marker is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody.
  • the detection method consists of a method for examining the expression level of the marker gene in the control group and the expression level of the marker gene in cells with gastric or colorectal cancer.
  • mRNA or protein levels can be expressed as absolute (eg ⁇ g / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.
  • immunohistostaining is performed using one or more antibodies against the gastric or colorectal cancer markers.
  • paraffin embedding blocks are prepared by methods well known in the art. These are cut into slices of several ⁇ m thickness, adhered to glass slides, and reacted with a selected one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.
  • one or more antibodies against the gastric or colorectal cancer markers are arranged at a predetermined position on the substrate to be immobilized at a 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, which is read to confirm the presence or expression level of the protein, You can check whether you have gastric or colorectal cancer.
  • the present invention comprises measuring the level of expression of the CDCA5 gene or the level of protein encoded by administration of a substance expected to be able to treat gastric or colorectal cancer. It relates to a screening method of.
  • CDCA5 can be usefully used for screening gastric cancer or colorectal cancer therapeutic agents by a method of comparing the increase or decrease in the expression of CDCA5 in the presence and absence of a candidate for treating gastric or colorectal cancer.
  • Substances that directly or indirectly reduce the concentration of CDCA5 can be selected as therapeutic agents for gastric or colorectal cancer.
  • the expression level of the marker CDCA5 of the present invention in gastric cancer or colorectal cancer cells in the absence of a candidate for gastric or colorectal cancer treatment is measured, and also the expression level of the marker CDCA5 of the present invention in the presence of a candidate for gastric or colorectal cancer treatment
  • the substance which reduces the expression level of the marker of the present invention in the presence of a candidate for treating gastric or colorectal cancer is lower than the marker expression level in the absence of the candidate for treating gastric or colorectal cancer. It can be predicted as a therapeutic agent.
  • the present invention relates to a pharmaceutical composition for treating and preventing cancer comprising oligonucleotides that inhibit the expression of CDCA5.
  • the cancer is preferably colon cancer, lung cancer, liver cancer or stomach cancer.
  • the oligonucleotide is an antisense oligonucleotide sequence complementary to siRNA, shRNA or CDCA5 mRNA for CDCA5 mRNA.
  • siRNA refers to a short double-chain RNA capable of inducing RNA interference (RNAi) through the cleavage of a particular mRNA. It consists of a sense RNA strand having a sequence homologous to mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. siRNAs can be provided as an efficient gene knockdown method or as a method of gene therapy because they can inhibit the expression of a target gene.
  • RNAi RNA interference
  • siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included.
  • the total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.
  • the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the target gene can be inhibited by the RNAi effect.
  • the cohesive end structure can be either a 3 'end projecting structure or a 5' end projecting structure.
  • the number of protruding bases is not limited.
  • siRNA is a low-molecular RNA (for example, natural RNA molecules such as tRNA, rRNA, viral RNA or artificial RNA molecules) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene It may include.
  • the siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a step loop structure in which the terminal portion of the double chain RNA general is connected by linker RNA.
  • the siRNA used in the present invention is itself a complete form with polynucleotide pairing, that is, a form introduced into a cell through two transformation processes in which the siRNA is directly synthesized in vitro, or one form to have such a form after administration in vivo.
  • Single-chain oligonucleotide fragments and their reverse complements may be derived from single-chain polynucleotides separated by spacers, for example siRNA expression vectors or PCR-derived siRNAs prepared to be expressed in cells
  • the expression cassette may be in a form introduced into a cell through a transformation or infection process. Determination of how to prepare an siRNA and introduce it into a cell or animal may depend on the purpose and cellular biological function of the target gene product. In one embodiment, 5′-GAC AUG ACU CUC CCU GGA ATT-3 ′ or 5′-GCA GGG AGC UUA CUA AGG ATT-3 ′ was used as siRNA in the present invention.
  • shRNA is intended to overcome the disadvantages of the high cost of biosynthesis of siRNA, the short-term maintenance of RNA interference effect due to low cell transfection efficiency, and the adenovirus, lenti from the promoter of RNA polymerase III.
  • Viral and plasmid expression vector systems can be used to introduce them into cells for expression, and these shRNAs are converted into siRNAs with the correct structure by siRNA processing enzymes (Dicer or Rnase III) present in the cells to silence the target genes. It is well known to induce.
  • antisense oligonucleotide means a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to the complementary sequence in the mRNA to a protein There is a feature that inhibits translation.
  • Antisense sequences of the present invention refer to DNA or RNA sequences that are complementary to CDCA5 mRNA and capable of binding to CDCA5 mRNA, and are responsible for translation, translocation, maturation, or any other overall biological function of CDCA5 mRNA. May inhibit essential activity.
  • the length of the antisense oligonucleotide is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases.
  • Antisense RNA can be synthesized in vitro and administered in vivo by conventional methods, or the antisense RNA can be synthesized in vivo.
  • One example of synthesizing antisense RNA in vitro is using RNA polymerase I.
  • One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin of the recognition site (MCS) is in the opposite direction.
  • MCS origin of the recognition site
  • composition of the present invention may comprise an additional substance that inhibits the proliferation of cancer cells of a patient, in addition to including at least one of siRNA or CDCA5 antisense oligonucleotides of CDCA5, or may be used to prevent influx of siRNA or antisense oligonucleotide molecules.
  • Promoting agents for example liposomes (US Pat. Nos. 4,897,355, 4,394,448, 4,23,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270).
  • lipophilic carriers of one of many sterols including cholesterol, cholate and deoxycholic acid.
  • Antisense oligonucleotides can also be conjugated to peptides that are taken up by the cell. Examples of useful peptides include peptide hormones, antigens or antibodies and peptide toxins.
  • the growth of the colorectal cancer cell lines Colo205, HCT116, lung cancer cell line A549, liver cancer cell lines Huh7, Snu387, gastric cancer cell line Snu638 as a result of analyzing the cell growth inhibition level by transfecting various cancer cells siRNA against CDCA5
  • the CDCA5 gene can be used as a cancer treatment target because it may cause inhibition (FIG. 10).
  • the present invention relates to a pharmaceutical composition for treating and preventing cancer, comprising an antibody or antigen-binding fragment thereof specific for CDCA5 protein that inhibits CDCA5 activity.
  • the cancer is preferably colon cancer, lung cancer, liver cancer or stomach cancer.
  • Such antibodies include polyclonal antibodies, monoclonal antibodies or antigen binding, some of which are included in the antibodies of the invention and all immunoglobulin antibodies.
  • the antibodies of the present invention also include special antibodies such as humanized antibodies, and may include antibodies already known in the art in addition to novel antibodies.
  • Such antibodies include functional fragments of antibody molecules, as well as complete forms having the full length of two heavy and two light chains, as long as they have the property of binding specifically to recognize CDCA5.
  • the functional fragment of the molecule of an antibody means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 , Fv, etc.
  • compositions of the present invention can be used alone, but radiation therapy or chemotherapy (cell growth arrest or cytotoxic substances, antibiotic-like substances, alkylating agents, anti-metabolic substances, hormonal agents, immunizing agents, interferon type) to increase the treatment efficiency Substances, cyclooxygenase inhibitors, metallomatrix protease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor substances, anti-HER substances, anti-EGFR substances, anti-angiogenic substances, farnesyl transferase inhibitors , ras-raf signal conduction pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin conjugates, topoisomerase I inhibitors, topoisomerase II inhibitors, etc.).
  • composition of the present invention may be administered with a pharmaceutically acceptable carrier, and when used orally, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like may be used.
  • 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.
  • administration refers to introducing a composition of the invention to a patient in any suitable manner, and the route of administration of the composition of the invention may be administered via any general route as long as it can reach the desired tissue.
  • the range of effective dosages of the compositions of the present invention may range from sex, body surface area, type and severity of disease, age, sensitivity to drugs, route of administration and rate of release, time of administration, duration of treatment, target cells, expression levels, etc. It may vary according to various well-known factors, and can be easily determined by those skilled in the art.
  • the DNA chip 48K human microarray sold by Illumina was used for 2,230 genes to extract genes specifically overexpressed only in gastric or colorectal cancer cells. Expression level was investigated.
  • CRNAs prepared from normal gastric epithelial and gastric cancer cells were hybridized to a Human-6 V2 (Illumina) chip.
  • DNA chips were washed using Illumina Gene Expression System Wash Buffer (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) to obtain data of fluorescence present in each spot and stored as image files in TIFF format. 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 the Avadis Prophetic version 3.3 (Strand Genomics) program.
  • genes showing more than two-fold differences in expression were found in more than 60% of patients compared with normal and gastric epithelial cells and colorectal cancer epithelial cells (FIG. 2), with 281 and 605 double overexpression and low expression, respectively.
  • FIG. 2 shows the results of comparing data mining data sets with microarray results.
  • gastric normal epithelial tissue and gastric or colorectal cancer tissue were extracted from 5 gastric cancer or 20 colorectal cancer patients, and mRNA was isolated from 10 tissues and 40 colorectal cancer tissues.
  • the tissues removed by surgical excision removed blood from sterile phosphate-buffered saline immediately after extraction and were frozen with liquid nitrogen. Thereafter, total RNA was isolated and single-step RNA isolation was performed by the Guaniidinium Method. Total RNA isolated as described above was quantified using a spectrophotometer, and stored in a -70 °C freezer until use.
  • Example 1 As a result of Example 1, one of the selected gastric or colorectal specific overexpression genes was selected (data mining) and subjected to a polymerase reaction.
  • each gene was obtained from NCBI's CoreNucleotide (http://www.ncbi.nlm.nih.gov/), and primer sequences of these genes were designed through the Primer3 program. Using the primers designed as described above, the degree of expression of each gene was confirmed by performing a polymerase reaction (FIGS. 6 to 9 and 11). Each primer sequence is shown in Table 2.
  • cDNA prepared by reverse transcriptase was prepared from mRNA extracted from the tissue and cell line of Example 2. cDNA production was carried out using AccuAcript High Fielity 1st Stand cDNA Synthesis Kit (STRATAGENE), and the reverse transcriptase reaction and real-time polymerase reaction were performed using the cDNA obtained from the above reaction and the primers of Table 2.
  • STRATAGENE AccuAcript High Fielity 1st Stand cDNA Synthesis Kit
  • siRNA techniques can be used to observe whether there is a change in cell proliferation rate by inhibiting gene expression.
  • the siRNA for CDCA5 was designed and introduced into various cancer cells, and the growth rate of the cancer cell line was observed according to the decrease in the expression of the corresponding gene mRNA.
  • siRNAs of CDCA5 and scrambled were designed and synthesized in Samchully Pharmaceutical (1: GAC AUG ACU CUC CCU GGA ATT, 2: GCA GGG AGC UUA CUA AGG ATT).
  • 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. Scrambled siRNA was used as a negative control for siRNA experiments that had no effect on cell proliferation.
  • Various cancer cells were cultured in 70% confluency and then transformed by incorporating the siRNA and the control scrambled siRNA into the cells by incubation for 72 hours by K-Max method.
  • RT-PCR was performed by RNA extraction from each cell to confirm that the gene mRNA was reduced, and that the gene expression was suppressed by siRNA.
  • NT represents the amount of gene expression in cells not treated with siRNA, and scrambled shows no target portion.
  • Colorectal cancer cell lines Colo205 (Seoul National University Cell Line Bank), HCT116 (Seoul National University Cell Line Bank), lung cancer cell line A549, liver cancer cell line Huh7 (pre-distribution from Chonnam National University), Snu387 (Seoul National University Cell Line Bank), gastric cancer cell line Snu638, cervical cancer Hela 70% After incubation with confluency, the siRNA and the control scrambled siRNA were introduced into the cells by the K-Max method, cultured for 72 hours, and the degree of growth inhibition of the cells by the siRNA was also investigated.
  • CDCA5 gene can be used as a cancer treatment target because it can induce growth inhibition of colorectal cancer cell line, lung cancer and liver cancer cell line through siRNA treatment of CDCA5 gene (FIG. 10).

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Abstract

L'invention concerne le biomarqueur CDCA5 (protéine associée au cycle de division cellulaire 5) qui est spécifique du cancer gastrique ou du cancer colorectal. L'invention concerne plus particulièrement une composition servant à diagnostiquer un cancer gastrique ou un cancer colorectal et contenant un agent mesurant le niveau d'expression de CDCA5, un nécessaire contenant cette composition, un procédé de détermination du marqueur, ainsi qu'un procédé de criblage d'un agent thérapeutique pour le cancer gastrique ou colorectal au moyen de ce marqueur. L'invention concerne en outre une composition pharmaceutique servant à prévenir et à traiter le cancer et contenant une matière inhibant l'expression ou l'activation de CDCA5.
PCT/KR2009/002085 2008-04-21 2009-04-21 Cdca5 en tant que marqueur diagnostique et agent thérapeutique pour le cancer gastrique ou le cancer colorectal WO2009131366A2 (fr)

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EP2362905A1 (fr) * 2008-10-24 2011-09-07 Oncotherapy Science, Inc. Procédé d'analyse de composés contre le cancer du poumon ou de l' sophage
CN107619835A (zh) * 2017-05-11 2018-01-23 广东医科大学 Cdca5在胃癌中的表达载体及其构建方法及cdca5在胃癌中特异干扰片段
CN109652548A (zh) * 2019-01-21 2019-04-19 首都医科大学附属北京朝阳医院 circ-CCNB1作为胃癌和结直肠癌诊断生物标志物和治疗靶点的应用

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WO2007013671A2 (fr) * 2005-07-27 2007-02-01 Oncotherapy Science, Inc. Procédé de diagnostic du cancer de l'oesophage
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WO2006002378A2 (fr) * 2004-06-23 2006-01-05 Avalon Pharmaceuticals Determination de genes associes au cancer et de cibles therapeutiques utilisant des methodes de cytogenetique moleculaire
US20060074565A1 (en) * 2004-10-06 2006-04-06 Miller Lance D Methods, systems, and compositions for classification, prognosis, and diagnosis of cancers
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WO2007013671A2 (fr) * 2005-07-27 2007-02-01 Oncotherapy Science, Inc. Procédé de diagnostic du cancer de l'oesophage
US20080057514A1 (en) * 2006-09-06 2008-03-06 Vanderbilt University Methods of screening for gastrointestinal cancer

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2362905A1 (fr) * 2008-10-24 2011-09-07 Oncotherapy Science, Inc. Procédé d'analyse de composés contre le cancer du poumon ou de l' sophage
EP2362905A4 (fr) * 2008-10-24 2012-07-04 Oncotherapy Science Inc Procédé d'analyse de composés contre le cancer du poumon ou de l' sophage
CN107619835A (zh) * 2017-05-11 2018-01-23 广东医科大学 Cdca5在胃癌中的表达载体及其构建方法及cdca5在胃癌中特异干扰片段
CN109652548A (zh) * 2019-01-21 2019-04-19 首都医科大学附属北京朝阳医院 circ-CCNB1作为胃癌和结直肠癌诊断生物标志物和治疗靶点的应用
CN109652548B (zh) * 2019-01-21 2022-08-05 首都医科大学附属北京朝阳医院 circ-CCNB1作为胃癌和结直肠癌诊断生物标志物和治疗靶点的应用

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