WO2010140824A2 - Composition for diagnosis and determination of prognosis of hepatocellular carcinoma - Google Patents

Composition for diagnosis and determination of prognosis of hepatocellular carcinoma Download PDF

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WO2010140824A2
WO2010140824A2 PCT/KR2010/003513 KR2010003513W WO2010140824A2 WO 2010140824 A2 WO2010140824 A2 WO 2010140824A2 KR 2010003513 W KR2010003513 W KR 2010003513W WO 2010140824 A2 WO2010140824 A2 WO 2010140824A2
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efna1
hepatocellular carcinoma
epha2
gene
composition
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Korean (ko)
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WO2010140824A3 (en
WO2010140824A9 (en
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김대곤
유경란
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전북대학교산학협력단
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/207—Modifications characterised by siRNA, miRNA
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the present invention relates to a composition for diagnosing and prognosticting hepatocellular carcinoma, and more particularly, to a composition for diagnosing hepatocellular carcinoma and a receptor for EFNA1, comprising an Ephrine A1 (EFNA1) gene or a protein expressed from the EFNA1 gene. It relates to a composition for determining hepatocellular carcinoma comprising an EPHA2 gene or a protein expressed from the EPHA2 gene.
  • EFNA1 Ephrine A1
  • Tumor markers vary depending on where the cancer occurs, and studies using monoclonal antibodies, laser beams, radioisotopes, chemiluminescence, and automated assays have been conducted to make better tumor marker diagnostics.
  • monoclonal antibodies, laser beams, radioisotopes, chemiluminescence, and automated assays have been conducted to make better tumor marker diagnostics.
  • tumor markers only certain cancer cells in the body can make them, and when the abnormalities are identified, certain organ cancers can be diagnosed. This relationship is called "long-term specificity.”
  • Hepatocellular carcinoma means primary liver cancer that occurs in the liver. Cancer that develops at another site and spread to the liver is called metastatic liver cancer. Hepatocellular carcinoma accounts for over 90% of all liver cancers. Even if this cancer is treated, 40 to 80% of patients relapse. Most relapses in the liver, but may also occur in the inner walls and bones surrounding the lungs, lymph nodes and abdominal cavity. The ratio of male to female patients is around 4 to 1, most often occurring after middle age. The causes are hepatitis B virus and hepatitis C virus, alcoholic epilepsy, metabolic chronic epilepsy, and toxic substances. In particular, 65-80% of patients with this disease are known as carriers of hepatitis B antigen.
  • Symptoms usually include fatigue, abdominal pain or sometimes bloating and loss of appetite.
  • a characteristic symptom is the development of a core that can be felt in the middle. If hepatocellular carcinoma burst in the abdominal cavity, sudden abdominal pain and bloating, hypotension or shock may occur.
  • liver cancer Recently, mutations in tumor suppressor genes or oncogenes such as p53, ⁇ -catenin, and Axin1 have been found in tumor tissues, and these genes have been reported to be involved in the development of liver cancer. The frequency of mutations is so low that such genetic modifications are insufficient to explain the causal relationship between liver cancer development or onset. Thus, the molecular and biological mechanisms of the cause and progression of liver cancer remain challenges.
  • AFP a representative molecular marker of hepatocellular carcinoma
  • primary liver cancer hepatitis, cirrhosis and fetal cancer.
  • fetal liver or yolk sac peaked at 13 weeks, then sharply lowered in concentration, and is present at very low levels (normally below 7-10 ng / mL in normal adults), but within the blood AFP
  • Increasing levels may indicate a serious increase in tumor cells, especially in 50-70% or more of tumor patients associated with liver cells, indicating increased levels of AFP, typically 7-15 ng AFP per ml of blood It is determined that the following is negative.
  • tumor marker AFP is also produced in hepatitis and cirrhosis, which frequently coincide with liver cancer, and the amount is known to rise up to about 500 ng / mL. It is also used as an indicator of congenital abnormalities such as rupture and hydrocephalus. AFP shows low and low specificity, especially in early liver cancer, and increases with cirrhosis and aggravation of chronic hepatitis, respectively. Therefore, several new biomarkers are currently premature for des-gamma carboxyprothrombin (DCP), prothrombin induced by vitamin K absence-II (PIVKA-II), lens cularis agglutinin-reactive (AFP-L3), and gPC3 (glypican-3). There is a study on whether liver cancer can be diagnosed, and there is a need for additional markers that can effectively increase the diagnosis rate of early stage cancer.
  • DCP des-gamma carboxyprothrombin
  • PIVKA-II prothrombin induced by vitamin K absence-I
  • An object of the present invention is to provide a marker for diagnosis and prognosis of hepatocellular carcinoma to search for hepatocellular carcinoma more quickly and efficiently, and also to develop a therapeutic method that contributes to national health by using the same.
  • the present invention provides a composition for diagnosing hepatocellular carcinoma comprising a protein expressed from the Ephrine A1 (EFNA1) gene or the EFNA1 gene.
  • EFNA1 Ephrine A1
  • the present invention provides a composition for hepatocellular carcinoma prognosis, characterized in that it comprises an EPHA2 gene or a protein expressed from the EPHA2 gene of the receptor of EFNA1.
  • the present invention is a hepatocellular carcinoma diagnostic marker using Ephrine A1 (EFNA1) and its receptor, EPHA2, and can be used as a biomarker useful for diagnosing and prognostic hepatocellular carcinoma, providing EFNA1 and EPHA2 genes and expressing a protein that is an EFNA1 gene product. It can be effectively detected in body fluids for early diagnosis of hepatocellular carcinoma, rapid and sensitive quantification of EPHA2 gene expression in patient tissues as tumor markers, and can be used to determine the prognosis of hepatocellular carcinoma, ie invasive and metastatic.
  • EFNA1 Ephrine A1
  • EPHA2 Ephrine A1
  • the present invention is a hepatocellular carcinoma diagnostic marker using Ephrine A1 (EFNA1) and its receptor, EPHA2, and can be used as a biomarker useful for diagnosing and prognostic hepatocellular carcinoma, providing EFNA1 and EPHA2 genes and expressing a protein that is an EFNA1 gene product
  • FIG. 1 shows the results of Northern blot analysis of mRNA expression of Ephrine A1 (EFNA1) in 40 hepatocellular carcinoma tissues (N) and hepatocellular carcinoma tissues (T). Show the histogram (right).
  • EFNA1 Ephrine A1
  • Figure 2 is an electrophoresis picture of the results of RT-PCR using primers of SEQ ID NO: 1 and SEQ ID NO: 2 in 40 non-hepatic cancer tissue (N) and hepatocellular carcinoma tissue (T). RT-PCR results of RT-PCR mRNA expression of EPHA2 (left) and a histogram (right) showing the ratio amplified compared to the non-hepatic cancer tissue were shown.
  • Figure 3 shows the results of EFNA1 Western blot using sc-911 polyclonal antibody after the introduction of GFP and c-myc labeled EFNA1 expression vector in 293 T cells.
  • Figure 4 shows the results of Western blot after introducing GFP and c-myc labeled EPHA2 expression vector in 293 T cells using sc-924 polyclonal antibody.
  • Figure 5 shows the results of analysis of the intracellular location and expression of EFNA1 by immunofluorescence method.
  • Figure 6 shows the results of immunofluorescence analysis of the intracellular location and expression of EPHA2.
  • Figure 7 shows the immunochemical staining of hepatocellular carcinoma and non-cancerous tissues by sc-911 polyclonal antibody with EFNA1 expression, and the immunochemical staining of hepatocellular carcinoma and non-cancerous tissues by sc-924 polyclonal antibody with EPHA2 expression.
  • A is normal liver
  • B is highly differentiated hepatocellular carcinoma tissue selectively stained with EFNA1 antibody
  • C is neutralized hepatocellular carcinoma tissue selectively stained with EFNA1 antibody
  • D is lowly differentiated hepatocellular carcinoma tissue selectively stained with EFNA1 antibody
  • E is normal liver
  • F is highly differentiated hepatocellular carcinoma tissue selectively stained with EPHA2 antibody
  • G is moderately differentiated hepatocellular carcinoma tissue selectively stained with EPHA2 antibody
  • H is low-differentiated hepatocellular carcinoma tissue selectively stained with EFNA1 antibody Indicates.
  • FIG. 8 shows Northern blots (top) and quantitative histographs (bottom) for comparative analysis of EFNA1, EPHA2 and AFP expression in 10 hepatocellular carcinoma cell lines.
  • FIG. 9 shows a northern blot (top) and a quantitative histograph (bottom) of comparative analysis of EFNA1, EPHA2, and AFP expression in surrounding non-hepatic cancer tissues and liver cancer tissues in 12 patients with liver cancer.
  • FIG. 10A shows the results of Western blot comparing and analyzing EFNA1 secretion protein in culture supernatant from 10 hepatocellular carcinoma cell lines, and B is secreted EFNA1 from normal liver, hepatocellular carcinoma of less than 4 cm in diameter and hepatocellular carcinoma of more than 4 cm Western blot analysis of the protein is shown.
  • Figure 11 shows the survival graph of EPHA2 expression state, that is, high or low expression in 68 hepatocellular carcinoma tissues.
  • the present invention provides a composition for diagnosing hepatocellular carcinoma comprising an Ephrine A1 (EFNA1) gene or a protein expressed from the EFNA1 gene.
  • EFNA1 Ephrine A1
  • the present invention also provides a composition for the prognosis of hepatocellular carcinoma characterized in that it comprises an EPHA2 gene or a protein expressed from the EPHA2 gene, which is the receptor for EFNA1.
  • the composition may include a sense or antisense primer complementary to the mRNA of the EFNA1 gene.
  • the composition may include a sense or antisense primer complementary to the mRNA of the EPHA2 gene.
  • the composition may include an antibody that specifically recognizes a protein expressed from the EFNA1 gene.
  • the composition may include an antibody that specifically recognizes a protein expressed from the EPHA2 gene.
  • the composition may include siRNA that inhibits the expression of a protein expressed from the EFNA1 gene.
  • the composition may include an siRNA that inhibits the expression of a protein expressed from the EPHA2 gene.
  • the present invention provides a hepatocyte cancer diagnostic kit comprising the composition.
  • EFNA1 Ephrine A1
  • EPHA2 Ephrine A1
  • expression protein thereof an expression protein thereof, and the like of the present invention
  • the present invention relates to novel hepatocellular carcinoma diagnosis and prognostic markers including mRNA or protein measurements of EFNA1 and EPHA2.
  • diagnostic marker means a substance capable of diagnosing hepatocellular carcinoma cells from normal cells, and is increased in cells with hepatocellular carcinoma compared to normal cells.
  • organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) exhibiting a decrease.
  • Hepatocellular carcinoma diagnostic markers provided by the present invention are EFNA1 genes and proteins that increase in cells with hepatocellular carcinoma compared to normal cells.
  • a “prognosis marker, a prognosis marker” is a marker associated with a stage of hypodifferentiation or metastasis invasion in hepatocellular carcinoma cells, and increases or decreases when hepatocellular carcinoma becomes poorly differentiated or as the disease progresses.
  • Polypeptides or nucleic acids eg, mRNA, etc.
  • lipids eg, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.
  • Hepatocellular carcinoma prognostic markers provided herein are EPHA2 genes and proteins that increase in hepatocellular carcinoma cells or tissues.
  • EFNA1 is a typical ligand that binds to the EPHA receptor tyrosine kinase and is present in the cell membrane by linkage to glycosy-phosphatidylinositol (GPI).
  • the ligand EFNA1 binds to EPHA2 receptor tyrosine kinase to promote autophosphorylation of EPHA2 and down-regulate signaling to regulate cell growth and migration.
  • Expression of EFNA1 in tumor endothelial cells has been observed to promote tumor endothelial migration.
  • Blocking EFNA1 and soluble EPHA2-Fc receptors inhibited EPHA2 deep delivery and reduced tumor-associated angiogenesis. Eventually, tumor progression was reduced.
  • HCC is a typical angioplasty tumor.
  • EFNA1 Arterial hyperplasia on radiation is the HCC diagnostic standard.
  • EFNA1 is a family of proteoglycans, heparin sulfate, on the surface of cells anchored to glycosyl phatidylinositol.
  • EFNA1 expression in HCC is 88% vs. 55% for larger HCCs and 77% vs. 43% for smaller HCCs (based on 3 cm diameter) compared to AFP.
  • EFNA1 was mostly overexpressed in HCC and specifically increased in serum of HCC patients.
  • the present invention provides a composition for diagnosing hepatocellular carcinoma comprising a nucleic acid material capable of measuring the expression level of EFNA1 and EPHA2 proteins or genes encoding the same.
  • the diagnostic composition means a substance capable of measuring the expression level of the EFNA1 and EPHA2 proteins or genes encoding the same, preferably the mRNA levels of the EFNA1 and EPHA2 genes, which are specific to the EFNA1 and EPHA2 genes.
  • Primer pairs or probes are specific for the EFNA1 and EPHA2 genes.
  • Those skilled in the art can design primers or probes that specifically amplify known sequences, particularly for the EFNA1 and EPHA2 genes, by NM_004428 and NM_004431 (NCBI).
  • a "primer” is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and functions as a starting point for copying template strands. Means a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) at appropriate buffers and temperatures.
  • reagents for polymerization DNA polymerase or reverse transcriptase
  • dNTPs deoxynucleoside triphospates
  • the primers of the present invention are primers specific for each of the markers EFNA1 and EPHA2 genes, and may preferably consist of sense (forward) and antisense (forward) nucleic acids having 7 to 50 nucleotide sequences. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.
  • the primer nucleic acid sequences of the present invention may, if necessary, include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
  • labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg 32 P), fluorescent molecules, chemical groups (eg biotin), and the like.
  • enzymes eg horseradish peroxidase, alkaline phosphatase
  • radioisotopes eg 32 P
  • fluorescent molecules eg biotin
  • primer pairs in the present invention include primer pairs of all combinations of forward and reverse primers that recognize the target gene sequence, but are preferably primer pairs that provide assay results with specificity and sensitivity.
  • a hepatocellular carcinoma diagnostic kit comprising a composition for diagnosing hepatocellular carcinoma capable of measuring expression levels of EFNA1 and EPHA2 proteins or genes encoding the same.
  • the diagnostic kit may be a diagnostic kit containing the necessary elements necessary to perform the ELISA.
  • ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromopores, enzymes (eg, conjugated with antibodies) and substrates thereof. It may also include antibodies specific for quantitative control proteins.
  • Another embodiment of the present invention provides a method for diagnosing hepatocellular carcinoma by measuring the expression level of EFNA1 and EPHA2 gene or EFNA1 and EPHA2 protein level, and a method for measuring the progression or prognosis of hepatocellular carcinoma by the above method. More specifically, the expression level of a gene can be detected at the mRNA level or at the protein level, which can be performed including known processes for separating mRNA or protein from biological samples using known techniques.
  • biological sample includes, but is not limited to, samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in EFNA1 and EPHA2 gene expression levels by the development of hepatocellular carcinoma. Do not.
  • Analytical methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting, DNA chips, and the like.
  • the EFNA1 and EPHA2 marker proteins in the biological sample and the antibodies specific thereto form a conjugate, which is expressed as an "antigen-antibody complex" in the present invention.
  • the amount of antigen-antibody complex formed can be measured quantitatively through the magnitude of the signal of the detection label.
  • a detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto.
  • Assays 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 Northern blot auto-radiogram was scanned using the LAS3000 system (Fuji photo-film, Japan) to measure the signal intensity.
  • Expression level of hepatocellular carcinoma tissues compared to non-hepatic cancer tissues was calculated by standardizing the expression level of 18S.
  • EFNA1 and EPHA2 measurements between groups were examined by the unpair-t test or Mann-Whitney test. Significance criteria were determined when P ⁇ 0.05.
  • hepatocellular carcinoma is a real cancer patient by comparing gene expression levels in a normal control group and suspected hepatocellular carcinoma, and to predict the progression or prognosis of hepatocellular carcinoma.
  • the EFNA1 and EPHA2 overexpressing cell lines by introducing a recombinant expression vector and the cells treated with EFNA1 and EPHA2 expression inhibitory candidates to compare the mRNA or protein levels of the control and control cells compared to EFNA1 and EPHA2 Methods of screening expression inhibitors are provided.
  • Screening method of the EFNA1 and EPHA2 expression inhibitor according to a specific embodiment of the present invention, after culturing the cells expressing the EFNA1 and EPHA2 protein treated with the candidate cells for inhibiting EFNA1 and EPHA2 expression, and then do not process the inhibitor candidate Comparing the expression level of hepatocellular carcinoma diagnostic marker (protein, mRNA) in the non-control cells.
  • the present invention enables the search for anticancer substances that inhibit EFNA1 and EPHA2 expression and can effectively treat liver cancer cells.
  • a method of measuring the expression level of the EFNA1 and EPHA2 genes, preferably the mRNA level using a primer or a probe can be preferably used.
  • RT-PCR is preferable.
  • Inhibitors that reduce the expression of diagnostic markers can be selected and used to treat hepatocellular carcinoma in comparison to the control.
  • a composition for preventing or treating hepatocellular carcinoma comprising an EFNA1 and EPHA2 expression inhibitor.
  • the EFNA1 and EPHA2 expression inhibitors include antisense RNA and complementary sense RNA strands complementary to the mRNA of the EFNA1 and EPHA2 gene, induces EFNA1 and EPHA2 gene specific RNA interference (RNA interference).
  • RNA interference RNA interference
  • It relates to a composition for preventing or treating hepatocellular carcinoma comprising an siRNA and a composition for preventing or treating hepatocellular carcinoma comprising an antibody specific for EFNA1 and EPHA2 proteins.
  • siRNA refers to a double-chain RNA capable of inducing RNA interference (RNAi) through cleavage of mRNA of a target gene, and refers to a sequence homologous to mRNA of a target gene.
  • the branch consists of a sense RNA strand and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit expression of a target gene, it is provided by an efficient gene knockdown method or gene therapy method.
  • siRNAs are not limited to the complete pairing of double-stranded RNA portions paired with RNA, but mismatches (the corresponding bases are not complementary), bulges (the bases that do not correspond to one chain), and the like. It may include parts that are not paired by.
  • the total length is 10 to 80 bases, preferably 15 to 60 bases, more preferably 20 to 40 bases.
  • siRNA terminal structures can be either blunt or cohesive.
  • the cohesive end structure can be either a 3 'end protruding structure or a 5' end protruding structure, and the number of protruding bases is not limited.
  • the number of bases may be 1 to 8 bases, preferably 2 to 6 bases.
  • siRNA is a low-molecular RNA (e.g., a natural RNA molecule such as tRNA, rRNA, viral RNA or artificial RNA) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene Molecules).
  • the siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA.
  • the length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.
  • the method of preparing siRNA is a method of directly synthesizing siRNA in vitro, introducing the cell into a cell through a gene transfection process, and an siRNA expression vector or a PCR-derived siRNA expression cassette prepared to express siRNA in a cell. There is a method of gene transfer or transduction into cells.
  • the term “specific” or “specific” refers to the ability to inhibit only the target gene without affecting other genes in the cell, and is specific for EFNA1 and EPHA2 in the present invention.
  • the siRNA of the present invention can specifically reduce the mRNA of EFNA1 and EPHA2, and the sequence and length are not particularly limited.
  • an antibody specific for the EFNA1 and EPHA2 proteins when used as a therapeutic antibody, the antibody may be coupled (eg, covalently) to an existing therapeutic agent directly or indirectly through a linker or the like.
  • Therapeutic agents that can be combined with antibodies include, but are not limited to, radionuclides, drugs, lymphokines, toxins, heterozygous antibodies, and the like.
  • the antibody can be administered by itself or in a composition comprising the antibody.
  • they are prepared in appropriate formulations, including an acceptable carrier depending on the mode of administration.
  • Formulations suitable for the mode of administration are known and typically comprise a surfactant which facilitates migration across the membrane.
  • surfactants may be steroid derived or N- [1- (2,3 diole oil (propyl) Cationic lipids such as) -N, N, N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidyl glycerol.
  • compositions comprising the antibodies of the invention can be administered in pharmaceutically effective amounts to treat cancer cells or their metastases.
  • the pharmaceutical composition can be administered single or multiple.
  • Compositions comprising the antibody are administered by suitable methods including subcutaneous, intraperitoneal, pulmonary and nasal, and lesion re-administration if necessary for local immunosuppressive therapy.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like.
  • the pH of the formulation can be designed to balance antibody stability (chemical and physical stability) and to employ other techniques for administration, as appropriate, to formulate suitable formulations. Typical dosage levels can be optimized using standard impressive techniques.
  • the antibodies of the invention can be administered in the form of nucleic acids encoding the antibodies to allow the antibodies to be produced intracellularly.
  • RNA of the hepatocellular carcinoma tissue and the non-hepatic cancer tissue extracted in Example 1 was first prepared using fluorescent cDNA microarray hybridization (cDNA microarray hybridization) containing approximately 3000 genes, and then unsupervised hierarchical clustering analysis. Divided into two main groups. A statistical program was used to identify genes that can distinguish hepatocellular carcinoma from non-hepatic cancer at 1% or less of FDR.
  • fluorescent cDNA microarray hybridization cDNA microarray hybridization
  • Labeled probes were dissolved in hybridization buffer after precipitation with isopropanol. The dissolved probe was placed on a slide, covered with a hybrid slip, and placed in a hybridization chamber to react overnight at 65 ° C. After hybridization, slides were washed for 10 minutes each with 1 ⁇ SSC / 0.1% SDS, 0.1 ⁇ SSC / 0.1% SDS (50 ° C.), 0.1 ⁇ SSC buffer. Reaction slides were scanned using Quantray program and analyzed using ImaGene 4.2 (Biodiscovery) program.
  • the fluorescence intensities of Cy5 and Cy3 were normalized using the whole spot after local background correction using the two-margin program.
  • Qualitative analysis of the spot used the following criteria. Among the spots with a signal mean-background mean / background value of SD> 2.0 or more, the spots where the average signal passes a standard of 1.5 times or more of the local background were used for the analysis. This criterion applied equally to the analysis of all array slides.
  • Carcinoma classification was analyzed using the "Cluster” and "TreeView” available on the website.Only array elements that can be measured in at least 80% of the samples used were analyzed. Before and after the fluorescence ratio of each spot was subjected to log-transformation, median centering was performed to remove experimental biases. By scoring significant genes.
  • Genes expressed in at least 80% of hepatocellular carcinoma and non-hepatic carcinoma tissues were selected, and a noncensored systematic clustering analysis was attempted for all tissues based on similarity with the expression patterns of all genes.
  • the gene expression patterns were observed by cDNA microarry analysis in primary HCC tumor samples from 40 patients and corresponding non-tumor liver tissue samples. If genes were expressed at least 80% and were selected based on SD> 0.5, noncensored hierarchical clustering analysis was performed on all tissues based on similarity with the overall gene expression pattern.
  • genes that are specifically expressed in precancerous cirrhosis and early or full differentiation stages of hepatocellular carcinoma can be used as early screening markers for the development of hepatocellular carcinoma or as molecular targets for hepatocellular carcinoma evolution.
  • Group 5 genes are selected from genes encoded by the secreted proteins or from membrane proteins expressed in cirrhosis and differentiation stages in I / II hepatocellular carcinoma.
  • the 13 genes in Group III chose to downregulate hepatocellular carcinoma in the III / IV differentiation stage.
  • EFNA1 a gene encoding a secreted protein in Table 1
  • hepatocytes by measuring EFNA1 RNA and protein expression in biological samples including tissues and body fluids. The purpose of this study was to confirm that cancer can be used as a diagnostic and prognostic indicator.
  • a sample containing 20 ⁇ g of nontumor and tumor total RNA was fractionated to 1% Agarose containing 2.2% formaldehyde and 50 mM 3- (N-morpholono) propanesulfonic acid (MOPS) and transferred to nylon membrane.
  • the nylon membrane was cross-linked by UV cross linker, labeled with a random primer in a 32 P dCTP-labeled EFNA1 cDNA probe, hybridized for one day, washed and exposed to X-ray film at -70 °C.
  • the amount of mRNA loaded in each lane was used as a control by hybridizing 18s cDNA after stripping the membrane.
  • RNA was added to 10 ⁇ g total RNA, 50M decamer RT-PCR superscriptII (200unit, invitrogen) to complete the reaction after incubation for 50 minutes at 37 °C.
  • PCR amplification was performed by adding 10 ⁇ m, 2.5 mM dNTP, 2 ⁇ l, and tag polymerase to 100 ⁇ l of each of EPHA2 to 2 ⁇ l of cDNA. PCR was performed at 94 ° C for 1 min. 35 cycles were reacted at 65 ° C. for 1 min and at 72 ° C. for 1 min. Internal control was performed using the Quantum RNATM 18S internal standard kit.
  • the primer sequences of EPHA2 were 5'-ATGGAGCTCCAGG CAGC CCGCGC-3 '(sense, SEQ ID NO: 1) and 5'-AGGGAAGCTGTTGCAGTCA-3' (antisense, SEQ ID NO: 2).
  • PCR products were isolated by electrophoresis on 2% agarose gel and confirmed by UV after staining with Ethidium bromide. 18s was used as endogenous control. Among them, it was observed that EFNA1 mRNA was selectively overexpressed in 36 of 40 (90%) HCC tissues (see FIG. 1).
  • Expression of EFNA1 and EPHA2 was not associated with Edmondson differentiation of TMN tumors.
  • Immunohistochemical staining was used to fix tissues fixed in 10% forrmalin and buried in paraffin with 4 ⁇ m thickness. After rehydration, microwave epitope retrieval was used to remove paraffin (in 15 mmol at 750 W for 15 min in a citrate buffer of pH 6.0). Before incorporation of primary antibody, intrinsic peroxidase was inhibited with 3% hydrogen peroxide and bovine albumin blocking. biotin was attached.
  • the primary mouse monoclonal antibody of EFNA1 and EPHA2 was attached to rabbit polyclonal antibody (PMA06) as a primary antibody, along with the secondary antibody, biotin attached antibody and streptavidine-horseradishperoxidase (DAKO, Glostrup, Denmark). Color development.
  • EFNA1 or EPHA2 antibody In order to determine the specificity of the immunoreactivity of EFNA1 or EPHA2 antibody, GFP-labeled EFNA1 or GFP-labeled EPHA2 or Myc-labeled EFNA1 or Myc-labeled EPHA2 expression vector was introduced and immunoblotting was performed to immunoreactive RTMW2E7 mAb or PMA06B. Investigate. Since the introduced EFNA1 cDNA is labeled with GFP or Myc at the COOH-terminus, immunoblot was performed using anti-GFP rabbit polyclonal antibody (santacruz) or anti-C-Myc Mouse monoclonal antibody (9E10, Santa Cruz). EFNA1 antibody or EPHA2 antibody was confirmed to specifically detect the corresponding band (see FIGS. 3 and 4).
  • GFP-labeled EFNA1 or EPHA2 expression vector and control vector were introduced into Hep3B and HepG2 cell lines to confirm the immunoreactivity of RTMWZED mAb or DMAO6B Ab by immunofluorescence. Comparing fluorescence and transmission images, GFP-labeled EFNA1 exogenous expression was expressed in the major nucleus or cytoplasm, consistent with previous reports. Green fluorescence was completely consistent with the immunoreactivity of EFNA1, and the expression of endogenous EFNA1 was mainly expressed in the cytoplasm of cells by RTMWZE7 mAb. In contrast, endogenous EPHA2 and GFP-labeled EPHA2 expression were mainly identified in cell node membranes (see FIGS. 5 and 6).
  • Immunohistochemical staining showed that the immunoreactivity of EFNA1 was negative in hepatocytes, blood vessels, bile ducts, and was found in many hepatic cytoplasms.
  • Normal livers include hepatocytes, fibroblasts and invasive blood cells, EPHA2 is expressed in the cytoplasm and cell membrane of malignant liver cells, and weakly expressed in normal liver and non-hepatic cancer tissues (see FIG. 7).
  • HCC validated pathologically histologically obtained liver tissue specimens and applied to all groups (see FIG. 10B).
  • the secretion of EFNA1 from serum supernatants of HCC patients and normal control groups was compared.
  • EFNA1 protein was expressed higher than in normal control serum.
  • the secretion of EFNA1 in patients with larger diameters (> 4 cm in diameter) and patients with smaller diameters ( ⁇ 4 cm in diameter) was not significantly different.
  • Proteins were separated from cell lysate, loaded with 30 ⁇ g SDS PAGE, and transferred to nitrocellulose membrane.
  • the membrane was blocked with 5% skin milk and treated with primary EFNA1 and EPHA2 antibodies (1: 1000 dilution) at room temperature for 4 hours. After treatment, the cells were washed three times with TBS containing 0.1% Tween 20.
  • EFNA1 and EPHA2 were identified using alkaline phosphatase-conjugated goat anti-rabbit IgG or chemiluminescence.
  • Opti-MEM without FBS for one day and only media supernatants were collected. The media were concentrated with a centricon YM10 filter and immunoblotting 20 ⁇ g of potein.
  • 1% BSA and serum were mixed 1: 1 and transferred to nitrocellulose membrane after reaching SDS-PAGE.
  • EFNA1 and EPHA2 were then analyzed in comparison to the expression of AFP in various hepatocellular carcinoma cells (see FIG. 8). Immunoblot analyses showed higher expression of EFNA1 in SK-Hep1, SH-J1, HLK-3, HKK2, and HLK-5 cells in Hep G2, Hep 3B, PLC / PRF / 5, and Huh7 cells. However, the majority of EPHA2 was overexpressed in Huh7, SK-Hep1 and SH-J1 cells. In particular, EPHA2 was overexpressed in SK-Hep1 and SH-1 cells, but EFNA1 and AFP were weakly expressed. Partial expression of this EPHA2 in liver cancer is in contrast to the expression of AFP and that of ENA1.
  • HCC cells We expressed p53 expression in HCC cells because it regulated EPHA2 by p53-family proteins. Expression of EFNA1 and EPHA2 was seen in eleven HCC patient samples. Most HCC tissues (91%, 10 out of 11) showed higher expression of EFNA1 than in non-hepatic cancer patients. In some HCC patient tissues (2 out 11), EPHA2 was expressed higher than in non-hepatic cancers. AFP was overexpressed in 45% (5 out of 11) HCC patient tissues. These data indicate that EFNA1 is more meaningful as a biochemical marker than AFP.
  • the autoradiograph of Northern hybridization was scanned using LAS3000system (Fuji) and analyzed for densitometric data.
  • the expression levels of each cancerous and noncancerous tissue were compared with 18s expression and averaged for analysis. Differences in the expression of EFNA1 between groups were confirmed by Mann-whitney test or unpaired-test, and the significance level was set to P value less than 0.05.
  • HepG2 a liver cancer cell line purchased from ATCC, was incubated at 37 ° C. under 5% CO 2 , and the medium was used by adding essential amino acid and pyruvate to MEM medium containing 10% FBS.
  • Myc labeled EPHA2 was prepared using a pCDNA3.1 / Myc vector (Invitrogen, USA) as a plasmid, respectively, and introduced into cells, and only a blank vector (pCDNA3.1 / Myc-HisA vector) was introduced as a control experiment.
  • Human full-length EPHA2 gene was obtained from 21C Frontier Human Genome Bank of Korea Research Institute of Bioscience and Biotechnology, and used as a template, a forward primer having a HindIII restriction enzyme site (SEQ ID NO: 3'5'-CCCAAGCTTCGCGGCATGGCGCTCCAGGCAGCCCG-3 ') and XhoI restriction enzyme A reverse primer (SEQ ID NO: 4: 5'-CCGCTCGAGGATGGGGATCCCCACAGTGTTCACCT-3 ') having a site was prepared, and cDNA encoding the EPHA2 protein amplified by PCR was cleaved with restriction enzymes HindIII and Xho1 and electrophoresed to the gel and banded.
  • the insert was inserted into the vector frame, cloned, introduced into E. coli, amplified, and the plasmid was purified.
  • genes were introduced using lipofectin (Gibco, invitrogen). After 48 hours, the cells were incubated for 2-3 weeks in a selection medium containing G418, and each colony formed was transferred to a 6-well plate, transferred to a T25 flask, expanded, and observed for morphology.
  • the biomarkers of the present invention are expected to contribute to the prevention of diagnosis and diagnosis of hepatocellular carcinoma and to the prevention and diagnosis of hepatocellular carcinoma, and to contribute to the national health of life by contributing to the treatment and prevention of cancer in Korea.

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Abstract

The present invention relates to a composition for the diagnosis and determination of the prognosis of hepatocellular carcinoma, and more particularly to a composition for the diagnosis of hepatocellular carcinoma comprising the Ephrine A1 (EFNA1) gene or a protein expressed from the EFNA1 gene and a composition for the determination of the prognosis of hepatocellular carcinoma comprising the EPHA2 gene which is a receptor for EFNA1 or a protein expressed from the EPHA2 gene. The present invention is a marker for diagnosing hepatocellular carcinoma using Ephrine A1 (EFNA1) and its receptor, EPHA2, and can be useful for a biomarker for the diagnosis and determination of the prognosis of hepatocellular carcinoma. The present invention can diagnose hepatocellular carcinoma early by providing the EFNA1 gene and the EPHA2 gene and effectively detecting the expression of a protein which is the EFNA1 gene product in a body fluid. The present invention can quantify the EPHA2 gene expression as a tumor marker in patients' tissues quickly and sensitively and be useful for determining the prognosis of hepatocellular carcinoma progress, that is, the invasive and metastatic potential.

Description

간세포암의 진단 및 예후결정용 조성물Composition for Diagnosis and Prognosis of Hepatocellular Carcinoma
본 발명은 간세포암의 진단 및 예후결정용 조성물에 관한 것으로, 보다 구체적으로는 Ephrine A1(EFNA1) 유전자 또는 상기 EFNA1 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 진단용 조성물 및 EFNA1의 수용체인 EPHA2 유전자 또는 상기 EPHA2 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물에 관한 것이다. The present invention relates to a composition for diagnosing and prognosticting hepatocellular carcinoma, and more particularly, to a composition for diagnosing hepatocellular carcinoma and a receptor for EFNA1, comprising an Ephrine A1 (EFNA1) gene or a protein expressed from the EFNA1 gene. It relates to a composition for determining hepatocellular carcinoma comprising an EPHA2 gene or a protein expressed from the EPHA2 gene.
종양 마커를 이용한 암 진단 검사 및 암 예후 검사에는 여러 가지가 있으나 대부분 항체를 이용한 혈액 검사에 의하며, 일부에서는 조직 추출액, 소변, 대변 등을 이용하여 검사하는 방법도 있다. 종양 마커는 암이 발생하는 장소에 따라 다양하여 보다 나은 종양 표지자 진단법을 만들기 위해 단클론 항체, 레이저광선, 방사성동위원소, 화학발광, 자동분석장치 등을 사용한 연구가 진행되고 있다. 종양 마커 중에는 체내의 특정 암 세포만이 만들 수 있어 그 이상치가 확인되면 바로 특정 장기의 암을 진단할 수 있는 것이 있다. 이런 관계를 「장기특이성이 높다」라고 한다.There are many types of cancer diagnostic tests and cancer prognosis tests using tumor markers, but most of them are blood tests using antibodies, and in some cases, tests using tissue extracts, urine, and feces are also available. Tumor markers vary depending on where the cancer occurs, and studies using monoclonal antibodies, laser beams, radioisotopes, chemiluminescence, and automated assays have been conducted to make better tumor marker diagnostics. Among tumor markers, only certain cancer cells in the body can make them, and when the abnormalities are identified, certain organ cancers can be diagnosed. This relationship is called "long-term specificity."
간세포암은 간에서 발생하는 원발성 간암을 의미한다. 다른 부위에서 생겨서 간으로 전이된 암은 전이성 간암이라고 한다. 간세포암은 전체 간암의 90％ 이상을 차지한다. 이 암은 치료를 하더라도 환자의 40∼80％는 재발한다. 대부분 다시 간에 재발하지만, 폐와 림프절, 복강을 둘러싸고 있는 안쪽 벽과 골에서 나타날 수도 있다. 남성과 여성 환자의 비율은 4 대 1 정도이고, 대부분 중년 이후에 발생한다. 원인은 B형 간염바이러스와 C형 간염바이러스, 알코올성 간질병, 대사성 만성 간질병, 독성물질 등이다. 특히 국내에서 이 질병 환자의 65∼80％는 B형 간염 항원 보균자로 알려져 있다. 초기에는 증세가 나타나지 않는데, 만약 증세가 나타났다면 이미 많이 진행된 것이다. 증세는 주로 피로감, 복부통증이나 때로 팽만감과 식욕부진이 나타난다. 특징적인 증세는 명치에서 응어리가 느껴지는 것으로 상당히 진행된 상태에서 나타난다. 만약 복강 안에서 간세포암이 터진 경우에는 갑작스러운 복부통증 및 팽만, 저혈압이나 쇼크 등이 일어난다.Hepatocellular carcinoma means primary liver cancer that occurs in the liver. Cancer that develops at another site and spread to the liver is called metastatic liver cancer. Hepatocellular carcinoma accounts for over 90% of all liver cancers. Even if this cancer is treated, 40 to 80% of patients relapse. Most relapses in the liver, but may also occur in the inner walls and bones surrounding the lungs, lymph nodes and abdominal cavity. The ratio of male to female patients is around 4 to 1, most often occurring after middle age. The causes are hepatitis B virus and hepatitis C virus, alcoholic epilepsy, metabolic chronic epilepsy, and toxic substances. In particular, 65-80% of patients with this disease are known as carriers of hepatitis B antigen. Initially, symptoms do not appear, but if they do, there is a lot going on. Symptoms usually include fatigue, abdominal pain or sometimes bloating and loss of appetite. A characteristic symptom is the development of a core that can be felt in the middle. If hepatocellular carcinoma burst in the abdominal cavity, sudden abdominal pain and bloating, hypotension or shock may occur.
이 질병 환자는 간세포암이 진행하여 사망하기도 하지만 동반하고 있는 간경화증 때문에 사망하는 경우도 있으므로 간세포암의 진행을 막는 동시에 간경화증의 진행을 방지하는 치료를 시행하여야 한다. 조기에 암을 발견하여 수술을 시행하면 완전히 치료할 수 있다. 그러나 암이 많이 진행한 경우, 간기능 상태가 나쁜 경우, 신체 다른 부위로 이미 전이한 경우에는 수술을 할 수 없어 경피적 에탄올주입법과 경동맥화학색전술, 고주파치료 등을 시행한다. 예후가 나쁜 질병이기 때문에 예방과 조기발견이 중요하다. 이를 위해서는 B형 간염 백신을 접종하고, 만성간염 또는 간경화증을 가진 환자의 경우에는 3∼6개월에 1번씩 검진을 한다.Patients with this disease may die of hepatocellular carcinoma, but they may die due to accompanying cirrhosis. Therefore, treatment should be performed to prevent the progression of hepatocellular carcinoma and prevent the progression of cirrhosis. Early detection of cancer and surgery can be completely cured. However, if the cancer progresses a lot, if the liver function is bad, or if it has already metastasized to another part of the body, surgery cannot be performed. Therefore, percutaneous ethanol injection, carotid artery embolization, and radiofrequency therapy are performed. Because prognosis is a bad disease, prevention and early detection are important. To do this, hepatitis B vaccine is given, and patients with chronic hepatitis or cirrhosis are examined once every 3 to 6 months.
최근에 p53, β-catenin, Axin1과 같은 종양억제 유전자(tumor suppressor gene) 또는 종양성 유전자(oncogene)의 변이(mutation)가 종양 조직에서 발견되었고 이들이 간암발생에 관여할 것이라는 보고가 있지만, 이들 유전자 변이 빈도가 매우 낮아 이러한 유전자 변형으로 간암 발생 또는 발병의 인과관계를 설명하기는 부족하다. 따라서 간암의 원인과 진행에 대한 분자 생물학적 기전은 아직도 연구해야 할 과제로 남아있다.Recently, mutations in tumor suppressor genes or oncogenes such as p53, β-catenin, and Axin1 have been found in tumor tissues, and these genes have been reported to be involved in the development of liver cancer. The frequency of mutations is so low that such genetic modifications are insufficient to explain the causal relationship between liver cancer development or onset. Thus, the molecular and biological mechanisms of the cause and progression of liver cancer remain challenges.
간세포암의 대표적인 분자마커인 AFP는 간세포가 형질전환되어 암세포로 탈분화 되었을때 비정상적으로 생성되는 당단백으로, 원발성 간암, 간염, 간경변과 태아성암 등의 발견과 치료경과 관찰에 이용된다. 대개 태아의 간이나 난황막에서 주로 만들어져 13주째에 최고치를 보이다가 이후에는 급격히 농도가 낮아져 정상의 성인에게서는 극히 낮은 농도(정상의 경우 7-10 ng/mL 이하)로만 존재하나, 혈액 내부의 AFP 수준의 증가는 심각한 종양세포의 증가를 의미할 수 있으며, 특히 간(liver)세포와 관련된 종양 환자의 50-70% 이상에서 AFP의 수치 증가가 확인되어, 일반적으로 혈액 1ml당 AFP 7~15 ng 이하를 음성이라고 판정한다.AFP, a representative molecular marker of hepatocellular carcinoma, is a glycoprotein that is abnormally produced when hepatocytes are transformed into cancer cells and is used for the detection and treatment of primary liver cancer, hepatitis, cirrhosis and fetal cancer. Usually made in fetal liver or yolk sac, peaked at 13 weeks, then sharply lowered in concentration, and is present at very low levels (normally below 7-10 ng / mL in normal adults), but within the blood AFP Increasing levels may indicate a serious increase in tumor cells, especially in 50-70% or more of tumor patients associated with liver cells, indicating increased levels of AFP, typically 7-15 ng AFP per ml of blood It is determined that the following is negative.
간암의 경우, 간암과 동시에 합병하는 일이 많은 간염, 간경변에서도 종양 마커 AFP가 만들어지기도 하며 양은 500 ng/mL 정도까지는 상승한다고 알려져 있고, 산부인과 질환에서 임신부의 혈중 및 양수 중 AFP 증가는 무뇌아, 척추파열, 수두증 등 선천성 이상의 지표로 활용되기도 한다. AFP는 특히 조기 간암에서 저감도와 저 특이성을 보이며 간경변이나 만성간염의 악화시에도 각각 증가한다. 따라서 몇 가지의 새로운 바이오 마커 DCP(Des-gamma carboxyprothrombin), PIVKA-II(prothrombin induced by vitamin K absence-II), AFP-L3(lens cularis agglutinin-reactive), GPC3(glypican-3) 등에 대하여 현재 조기간암을 진단할 수 있는지에 관한 연구가 이루어지고 있으며, 조기간암의 진단율을 효과적으로 증가시킬 수 있는 추가적 마커가 필요한 실정이다.In the case of liver cancer, tumor marker AFP is also produced in hepatitis and cirrhosis, which frequently coincide with liver cancer, and the amount is known to rise up to about 500 ng / mL. It is also used as an indicator of congenital abnormalities such as rupture and hydrocephalus. AFP shows low and low specificity, especially in early liver cancer, and increases with cirrhosis and aggravation of chronic hepatitis, respectively. Therefore, several new biomarkers are currently premature for des-gamma carboxyprothrombin (DCP), prothrombin induced by vitamin K absence-II (PIVKA-II), lens cularis agglutinin-reactive (AFP-L3), and gPC3 (glypican-3). There is a study on whether liver cancer can be diagnosed, and there is a need for additional markers that can effectively increase the diagnosis rate of early stage cancer.
본 발명의 목적은 간세포암을 보다 신속하고 효율적으로 검색하기 위한 간세포암 진단 및 예후결정용 마커를 제공하고자 함이며, 또한 이를 이용하여 국민생활건강에 이바지하는 치료법을 개발하고자 함이다.An object of the present invention is to provide a marker for diagnosis and prognosis of hepatocellular carcinoma to search for hepatocellular carcinoma more quickly and efficiently, and also to develop a therapeutic method that contributes to national health by using the same.
상기의 과제를 해결하고자, 본 발명은 Ephrine A1(EFNA1) 유전자 또는 상기 EFNA1 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 진단용 조성물을 제공한다. 또한 본 발명은 EFNA1의 수용체인 EPHA2 유전자 또는 상기 EPHA2 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물을 제공한다.In order to solve the above problems, the present invention provides a composition for diagnosing hepatocellular carcinoma comprising a protein expressed from the Ephrine A1 (EFNA1) gene or the EFNA1 gene. In another aspect, the present invention provides a composition for hepatocellular carcinoma prognosis, characterized in that it comprises an EPHA2 gene or a protein expressed from the EPHA2 gene of the receptor of EFNA1.
본 발명은 Ephrine A1(EFNA1)와 그 수용체인 EPHA2을 이용한 간세포암 진단마커로서 간세포암 진단 및 예후 판정에 유용한 바이오마커로써 이용이 가능하며, EFNA1와 EPHA2 유전자를 제공하고 EFNA1 유전자 산물인 단백질의 발현을 체액에서 효과적으로 검출하여 간세포암을 조기진단하고 EPHA2 유전자 발현을 종양 마커로 환자조직에 신속하고 민감하게 정량하며 간세포암의 진행성, 즉 침습성 및 전이성의 예후를 판단하는 데 사용될 수 있다.The present invention is a hepatocellular carcinoma diagnostic marker using Ephrine A1 (EFNA1) and its receptor, EPHA2, and can be used as a biomarker useful for diagnosing and prognostic hepatocellular carcinoma, providing EFNA1 and EPHA2 genes and expressing a protein that is an EFNA1 gene product. It can be effectively detected in body fluids for early diagnosis of hepatocellular carcinoma, rapid and sensitive quantification of EPHA2 gene expression in patient tissues as tumor markers, and can be used to determine the prognosis of hepatocellular carcinoma, ie invasive and metastatic.
도 1은 40예의 비간암 조직(N)과 간세포암 조직(T)에서 Ephrine A1(EFNA1)의 mRNA 발현을 노던블룻 분석한 결과(왼쪽)와, 정량화하여 비간암조직에 비하여 증폭된 비를 그린 히스토그램(오른쪽)을 나타낸다.FIG. 1 shows the results of Northern blot analysis of mRNA expression of Ephrine A1 (EFNA1) in 40 hepatocellular carcinoma tissues (N) and hepatocellular carcinoma tissues (T). Show the histogram (right).
도 2는 40예의 비간암 조직(N)와 간세포암 조직(T)에서 서열번호 1과 서열번호 2의 프라이머를 이용한 RT-PCR 결과의 전기영동사진이다. EPHA2의 mRNA 발현을 RT-PCR한 결과(왼쪽)와, 정량화하여 비간암조직에 비하여 증폭된 비를 그린 히스토그램(오른쪽)을 나타낸다.Figure 2 is an electrophoresis picture of the results of RT-PCR using primers of SEQ ID NO: 1 and SEQ ID NO: 2 in 40 non-hepatic cancer tissue (N) and hepatocellular carcinoma tissue (T). RT-PCR results of RT-PCR mRNA expression of EPHA2 (left) and a histogram (right) showing the ratio amplified compared to the non-hepatic cancer tissue were shown.
도 3은 293 T세포에서 GFP와 c-myc 표지 EFNA1 발현 벡타를 도입시킨 후 sc-911 다클론항체를 이용한 EFNA1 웨스턴블롯 결과의 나타낸다.Figure 3 shows the results of EFNA1 Western blot using sc-911 polyclonal antibody after the introduction of GFP and c-myc labeled EFNA1 expression vector in 293 T cells.
도 4는 sc-924 다단클론항체를 이용하여 293 T세포에서 GFP와 c-myc 표지 EPHA2 발현 벡타를 도입시킨 후 웨스턴블롯 결과의 나타낸다.Figure 4 shows the results of Western blot after introducing GFP and c-myc labeled EPHA2 expression vector in 293 T cells using sc-924 polyclonal antibody.
도 5는 EFNA1의 세포내 위치와 발현양태를 면역형광법으로 분석한 결과를 나타낸다.Figure 5 shows the results of analysis of the intracellular location and expression of EFNA1 by immunofluorescence method.
도 6은 EPHA2의 세포내 위치와 발현양태를 면역형광법으로 분석한 결과를 나타낸다.Figure 6 shows the results of immunofluorescence analysis of the intracellular location and expression of EPHA2.
도 7은 EFNA1 발현을 sc-911 다클론항체의한 간세포암과 비암조직의 면역화학염색 결과와 EPHA2 발현을 sc-924 다클론항체의한 간세포암과 비암조직의 면역화학염색 결과를 나타낸다.(A는 정상간, B는 EFNA1 항체에 선택적으로 염색되는 고분화 간세포암조직, C는 EFNA1 항체에 선택적으로 염색되는 중분화 간세포암조직, D는 EFNA1 항체에 선택적으로 염색되는 저분화 간세포암조직, E는 정상간, F는 EPHA2 항체에 선택적으로 염색되는 고분화 간세포암조직, G는 EPHA2 항체에 선택적으로 염색되는 중분화 간세포암조직, H는 EFNA1 항체에 선택적으로 염색되는 저분화 간세포암조직을 나타낸다.)Figure 7 shows the immunochemical staining of hepatocellular carcinoma and non-cancerous tissues by sc-911 polyclonal antibody with EFNA1 expression, and the immunochemical staining of hepatocellular carcinoma and non-cancerous tissues by sc-924 polyclonal antibody with EPHA2 expression. A is normal liver, B is highly differentiated hepatocellular carcinoma tissue selectively stained with EFNA1 antibody, C is neutralized hepatocellular carcinoma tissue selectively stained with EFNA1 antibody, D is lowly differentiated hepatocellular carcinoma tissue selectively stained with EFNA1 antibody, E is normal liver, F is highly differentiated hepatocellular carcinoma tissue selectively stained with EPHA2 antibody, G is moderately differentiated hepatocellular carcinoma tissue selectively stained with EPHA2 antibody, H is low-differentiated hepatocellular carcinoma tissue selectively stained with EFNA1 antibody Indicates.)
도 8은 10종의 간세포암 세포주에서 EFNA1과 EPHA2 및 AFP 발현을 비교 분석한 노던불롯(상)과 정량적 히스토그래프(하)를 나타낸다.8 shows Northern blots (top) and quantitative histographs (bottom) for comparative analysis of EFNA1, EPHA2 and AFP expression in 10 hepatocellular carcinoma cell lines.
도 9는 간암환자 12예에서 주위비간암조직과 간암조직에서 EFNA1과 EPHA2 및 AFP 발현을 비교 분석한 노던불롯(상)과 정량적 히스토그래프(하)를 나타낸다.FIG. 9 shows a northern blot (top) and a quantitative histograph (bottom) of comparative analysis of EFNA1, EPHA2, and AFP expression in surrounding non-hepatic cancer tissues and liver cancer tissues in 12 patients with liver cancer.
도 10의 A는 10종의 간세포암 세포주에서 배양상청액에서 EFNA1 분비단백을 비교 분석한 웨스턴블롯의 결과를 나타내며, B는 정상간, 직경 4cm 미만 간세암조직과 4cm이상 간세포암 환자 혈청에서 EFNA1 분비단백의 웨스턴블롯 분석을 나타낸다. FIG. 10A shows the results of Western blot comparing and analyzing EFNA1 secretion protein in culture supernatant from 10 hepatocellular carcinoma cell lines, and B is secreted EFNA1 from normal liver, hepatocellular carcinoma of less than 4 cm in diameter and hepatocellular carcinoma of more than 4 cm Western blot analysis of the protein is shown.
도 11은 68명의 간세포암 조직에서 EPHA2 발현상태, 즉 고발현 또는 저발현에 따른 생존그래프를 나타낸다.Figure 11 shows the survival graph of EPHA2 expression state, that is, high or low expression in 68 hepatocellular carcinoma tissues.
본 발명은 Ephrine A1(EFNA1) 유전자 또는 상기 EFNA1 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 진단용 조성물을 제공한다.The present invention provides a composition for diagnosing hepatocellular carcinoma comprising an Ephrine A1 (EFNA1) gene or a protein expressed from the EFNA1 gene.
본 발명은 또한 EFNA1의 수용체인 EPHA2 유전자 또는 상기 EPHA2 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물을 제공한다.The present invention also provides a composition for the prognosis of hepatocellular carcinoma characterized in that it comprises an EPHA2 gene or a protein expressed from the EPHA2 gene, which is the receptor for EFNA1.
본 발명에서 상기 조성물은 EFNA1 유전자의 mRNA에 상보적인 센스 또는 안티센스 프라이머를 포함할 수 있다.In the present invention, the composition may include a sense or antisense primer complementary to the mRNA of the EFNA1 gene.
또한 본 발명에서 상기 조성물은 EPHA2 유전자의 mRNA에 상보적인 센스 또는 안티센스 프라이머를 포함할 수 있다.In the present invention, the composition may include a sense or antisense primer complementary to the mRNA of the EPHA2 gene.
또한 본 발명에서 상기 조성물은 EFNA1 유전자로부터 발현되는 단백질을 특이적으로 인식하는 항체를 포함할 수 있다.In the present invention, the composition may include an antibody that specifically recognizes a protein expressed from the EFNA1 gene.
또한 본 발명에서 상기 조성물은 EPHA2 유전자로부터 발현되는 단백질을 특이적으로 인식하는 항체를 포함할 수 있다.In the present invention, the composition may include an antibody that specifically recognizes a protein expressed from the EPHA2 gene.
또한 본 발명에서 상기 조성물은 EFNA1 유전자로부터 발현되는 단백질의 발현을 억제하는 siRNA를 포함할 수 있다.In addition, in the present invention, the composition may include siRNA that inhibits the expression of a protein expressed from the EFNA1 gene.
또한 본 발명에서 상기 조성물은 EPHA2 유전자로부터 발현되는 단백질의 발현을 억제하는 siRNA를 포함할 수 있다.In addition, in the present invention, the composition may include an siRNA that inhibits the expression of a protein expressed from the EPHA2 gene.
본 발명은 상기의 조성물을 포함하는 간세포암 진단키트를 제공한다.The present invention provides a hepatocyte cancer diagnostic kit comprising the composition.
이하 본 발명의 Ephrine A1(EFNA1), EPHA2 유전자, 그 발현 단백질 등을 포함한 간세포암 진단 또는 예후결정용 마커에 대하여 보다 상세하게 설명한다.Hereinafter, a marker for diagnosing or prognostic hepatocellular carcinoma including Ephrine A1 (EFNA1), an EPHA2 gene, an expression protein thereof, and the like of the present invention will be described in more detail.
본 발명은 EFNA1와 EPHA2의 mRNA 또는 단백질 측정을 포함하는 새로운 간세포암 진단 및 예후 마커에 관한 것이다.The present invention relates to novel hepatocellular carcinoma diagnosis and prognostic markers including mRNA or protein measurements of EFNA1 and EPHA2.
본 발명에서,“진단용 마커, 진단하기 위한 마커 또는 진단 마커(diagnosis marker)”란 간세포암 세포를 정상 세포와 구분하여 진단할 수 있는 물질을 의미하고, 정상 세포에 비하여 간세포암을 가진 세포에서 증가 또는 감소를 보이는 폴리펩타이드 또는 핵산(예: mRNA 등), 지질 , 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다. 본 발명에서 제공하는 간세포암 진단 마커는 정상 세포에 비해 간세포암을 가진 세포에서 증가하는 EFNA1 유전자와 단백질이다.In the present invention, "diagnostic marker, diagnostic marker, or diagnostic marker" means a substance capable of diagnosing hepatocellular carcinoma cells from normal cells, and is increased in cells with hepatocellular carcinoma compared to normal cells. Or organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) exhibiting a decrease. Hepatocellular carcinoma diagnostic markers provided by the present invention are EFNA1 genes and proteins that increase in cells with hepatocellular carcinoma compared to normal cells.
본 발명에서,“예후 마커, 예후 결정을 위한 마커(prognosis marker)”란 간세포암 세포에서 저분화 또는 전이 침습의 병기와 관련된 마커로서 간세포암이 분화가 나빠질 때 또는 병기의 진해에 따라 증가 또는 감소를 보이는 폴리펩타이드 또는 핵산(예: mRNA 등), 지질 , 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다. 본 발명에서 제공하는 간세포암 예후 마커는 간세포암 세포 또는 조직에서 증가하는 EPHA2 유전자와 단백질이다.In the present invention, a “prognosis marker, a prognosis marker” is a marker associated with a stage of hypodifferentiation or metastasis invasion in hepatocellular carcinoma cells, and increases or decreases when hepatocellular carcinoma becomes poorly differentiated or as the disease progresses. Polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. Hepatocellular carcinoma prognostic markers provided herein are EPHA2 genes and proteins that increase in hepatocellular carcinoma cells or tissues.
EFNA1은 전형적인 리간드로 EPHA receptor tyrosine kinase와 결합하여 glycosy-phosphatidylinositol(GPI)에 linkage에 의해 세포막에서 존재한다. 리간드 EFNA1은 EPHA2 receptor tyrosine kinase 와 결합하여 EPHA2의 자가인산화를 촉진하고 신호전달을 하향 조절하여 세포성장과 이동을 조절한다. 종양내피세포에서 EFNA1의 발현은 종양내피 이동을 촉진하는 것으로 관찰되었다. EFNA1와 가용성 EPHA2-Fc receptor을 차단하면 EPHA2 심호전달을 억제하고 종양관련 신생혈관생성을 감소시켰다. 결국 종양의 진행을 감소시켰다. HCC는 전형적인 혈관과형성종양으로서 방사선상에서 동맥과형성은 HCC진단 표준으로 된다. EFNA1은 glycosyl phatidylinositol에 앵커된 세포표면에 있는 헤파린설페이트(heparin sulfate)인 프로테오그리칸(proteoglycan) 의 계통의 일종이다. EFNA1이 HCC에서 발현을 AFP와 비교해 볼때 큰 사이즈 HCC에서 88%대 55%,작은 사이즈 HCC에서는 77%대 43%(3cm직경을 기준으로)이다. 흥미로운 것은 EFNA1은 단백발현이 대부분 HCC에서 과발현되었고, HCC 환자 혈청에서 특이하게 증가되었다.EFNA1 is a typical ligand that binds to the EPHA receptor tyrosine kinase and is present in the cell membrane by linkage to glycosy-phosphatidylinositol (GPI). The ligand EFNA1 binds to EPHA2 receptor tyrosine kinase to promote autophosphorylation of EPHA2 and down-regulate signaling to regulate cell growth and migration. Expression of EFNA1 in tumor endothelial cells has been observed to promote tumor endothelial migration. Blocking EFNA1 and soluble EPHA2-Fc receptors inhibited EPHA2 deep delivery and reduced tumor-associated angiogenesis. Eventually, tumor progression was reduced. HCC is a typical angioplasty tumor. Arterial hyperplasia on radiation is the HCC diagnostic standard. EFNA1 is a family of proteoglycans, heparin sulfate, on the surface of cells anchored to glycosyl phatidylinositol. EFNA1 expression in HCC is 88% vs. 55% for larger HCCs and 77% vs. 43% for smaller HCCs (based on 3 cm diameter) compared to AFP. Interestingly, EFNA1 was mostly overexpressed in HCC and specifically increased in serum of HCC patients.
따라서, 본 발명은 EFNA1 및 EPHA2 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정할 수 있는 핵산물질을 포함하는 간세포암 진단용 조성물을 제공한다.Accordingly, the present invention provides a composition for diagnosing hepatocellular carcinoma comprising a nucleic acid material capable of measuring the expression level of EFNA1 and EPHA2 proteins or genes encoding the same.
본 발명에서, 진단용 조성물은 상기 EFNA1 및 EPHA2 단백질 또는 이를 암호화하는 유전자의 발현 수준, 바람직하게는 EFNA1 및 EPHA2 유전자의 mRNA 수준을 측정할 수 있는 물질을 의미하며, 이러한 물질은 EFNA1 및 EPHA2 유전자에 특이적인 프라이머 쌍 또는 프로브를 포함한다. EFNA1 및 EPHA2 유전자에 특이적인 프라이머 쌍 또는 프로브를 이용하여 EFNA1 및 EPHA2의 발현을 mRNA수준에서 효과적으로 검출할 수 있다. 당업자는 알려진 서열을, 특히 EFNA1 및 EPHA2 유전자의 경우 NM_004428 및 NM_004431 (NCBI)에 의해 이들 유전자의 특정 영역을 특이적으로 증폭하는 프라이머 또는 프로브를 디자인할 수 있다.In the present invention, the diagnostic composition means a substance capable of measuring the expression level of the EFNA1 and EPHA2 proteins or genes encoding the same, preferably the mRNA levels of the EFNA1 and EPHA2 genes, which are specific to the EFNA1 and EPHA2 genes. Primer pairs or probes. Expression of EFNA1 and EPHA2 can be effectively detected at the mRNA level using primer pairs or probes specific for the EFNA1 and EPHA2 genes. Those skilled in the art can design primers or probes that specifically amplify known sequences, particularly for the EFNA1 and EPHA2 genes, by NM_004428 and NM_004431 (NCBI).
본 발명에서 "프라이머"란 짧은 자유 3말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(DNA 중합효소 또는 역전사효소) 및 상이한 4가지 dNTP(deoxynucleoside triphospate)의 존재하에서 DNA합성을 개시할 수 있다. In the present invention, a "primer" is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and functions as a starting point for copying template strands. Means a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) at appropriate buffers and temperatures.
본 발명의 프라이머는, 각 마커 EFNA1 및 EPHA2 유전자에 특이적인 프라이머로, 바람직하게 7개 내지 50개의 뉴클레오타이드 서열을 가진 센스(전향) 및 안티센스(후향) 핵산으로 구성될 수 있다. 프라이머는 DNA 합성의 개시점으로 작용하는 프라이머의 기본 성질을 변화시키지 않는 추가의 특징을 혼입할 수 있다. 또한, 본 발명의 프라이머 핵산 서열은 필요한 경우, 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 포함할 수 있다. The primers of the present invention are primers specific for each of the markers EFNA1 and EPHA2 genes, and may preferably consist of sense (forward) and antisense (forward) nucleic acids having 7 to 50 nucleotide sequences. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. In addition, the primer nucleic acid sequences of the present invention may, if necessary, include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
표지의 예로는, 효소(예를 들어, 호스래디쉬 퍼옥시다제, 알칼린 포스파타아제), 방사성 동위원소(예를 들어, 32P), 형광성 분자, 화학그룹(예를 들어, 바이오틴) 등이 있다. 본 발명에서 프라이머 쌍은 표적 유전자 서열을 인지하는 정방향 및 역방향의 프라이머로 이루어진 모든 조합의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다.Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg 32 P), fluorescent molecules, chemical groups (eg biotin), and the like. There is this. Primer pairs in the present invention include primer pairs of all combinations of forward and reverse primers that recognize the target gene sequence, but are preferably primer pairs that provide assay results with specificity and sensitivity.
본 발명의 또 다른 양태에서는, EFNA1 및 EPHA2 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정할 수 있는 간세포암 진단용 조성물을 포함하는 간세포암 진단키트를 제공한다. In still another aspect of the present invention, there is provided a hepatocellular carcinoma diagnostic kit comprising a composition for diagnosing hepatocellular carcinoma capable of measuring expression levels of EFNA1 and EPHA2 proteins or genes encoding the same.
또한, 본 발명에서 단백질 수준을 측정하는 제재가 바람직하게 항체인 경우, 상기 진단키트는 ELISA를 수행하기 위해 필요한 필수 요소를 포함하는 진단키트일 수 있다. 이러한 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면 표지된 2차 항체, 발색단(chromopores), 효소(예: 항체와 접합) 및 그의 기질을 포함할 수 있다. 또한, 정량 대조구 단백질에 특이적인 항체를 포함할 수 있다.In addition, when the agent for measuring the protein level in the present invention is preferably an antibody, the diagnostic kit may be a diagnostic kit containing the necessary elements necessary to perform the ELISA. Such ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromopores, enzymes (eg, conjugated with antibodies) and substrates thereof. It may also include antibodies specific for quantitative control proteins.
본 발명의 또 다른 양태에서는 EFNA1 및 EPHA2 유전자의 발현수준 또는 EFNA1 및 EPHA2 단백질 수준을 측정하여 간세포암을 진단하는 방법 및 상기의 방법으로 간세포암의 진행단계 또는 예후를 측정하는 방법을 제공한다. 보다 구체적으로는, 유전자의 발현 수준을 mRNA 수준 또는 단백질 수준에서 검출할 수 있고, 이는 공지의 기술을 이용하여 생물학적 시료로부터 mRNA 또는 단백질을 분리하는 공지의 공정을 포함하여 수행될 수 있다.Another embodiment of the present invention provides a method for diagnosing hepatocellular carcinoma by measuring the expression level of EFNA1 and EPHA2 gene or EFNA1 and EPHA2 protein level, and a method for measuring the progression or prognosis of hepatocellular carcinoma by the above method. More specifically, the expression level of a gene can be detected at the mRNA level or at the protein level, which can be performed including known processes for separating mRNA or protein from biological samples using known techniques.
본 발명에서 "생물학적 시료"란 간세포암 발생에 의해 EFNA1 및 EPHA2 유전자 발현 수준이 차이나는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨와 같은 시료를 포함하나, 이에 제한되지는 않는다. As used herein, the term "biological sample" includes, but is not limited to, samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in EFNA1 and EPHA2 gene expression levels by the development of hepatocellular carcinoma. Do not.
mRNA 수준을 측정하기 위한 분석 방법으로는 RT-PCR, 경쟁적 RT-PCR , 실시간 RT-PCR, RNase 보호 분석법, 노던 블랏팅, DNA 칩 등이 있으나 이로 제한되는 것은 아니다. 상기 검출 방법들을 통하여, 대조구에서의 mRNA 발현량과 간세포암 환자 또는 간세포암 의심환자에서의 mRNA 발현량을 확인할 수 있고, mRNA로의 유의한 발현량 정도를 대조구와 비교함으로써 간세포암의 이환여부를 예측할 수 있다.Analytical methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting, DNA chips, and the like. Through the above detection methods, it is possible to confirm the mRNA expression level in the control group and the mRNA expression level in hepatocellular carcinoma patients or suspected hepatocellular carcinoma patients, and to predict whether the hepatocellular carcinoma is affected by comparing the significant expression level with mRNA with the control group. have.
본 발명에서는 EFNA1 및 EPHA2 단백질 수준을 측정하기 위해 항체를 이용하는 경우, 생물학적 시료 내의 EFNA1 및 EPHA2 마커 단백질과 이에 특이적인 항체는 결합물을 형성하며, 이를 본 발명에서는“항원-항체 복합체”로 표현한다. 항원-항체 복합체의 형성량은 검출 라벨(detection label)의 시그널의 크기를 통해서 정량적으로 측정 가능하다. 이러한 검출 라벨은 효소, 형광물, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹 중에서 선택할 수 있으며, 반드시 이로 제한되는 것은 아니다. 단백질 수준을 측정하기 위한 분석방법으로는 웨스턴 블랏, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 면역침전 분석법, 보체 고정분석법, FACS, 단백질 칩 등이 있으나, 이로 제한되는 것은 아니다.In the present invention, when the antibody is used to measure the EFNA1 and EPHA2 protein levels, the EFNA1 and EPHA2 marker proteins in the biological sample and the antibodies specific thereto form a conjugate, which is expressed as an "antigen-antibody complex" in the present invention. . The amount of antigen-antibody complex formed can be measured quantitatively through the magnitude of the signal of the detection label. Such a detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. Assays 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.
정량 및 통계분석을 위하여는 노던블롯의 오토-라디오그램(auto-radiogram)을 LAS3000 시스템(Fuji photo-film, 일본)을 이용하여 스캔하여 신호강도를 측정하였다. 비간암조직에 비해 간세포암조직의 발현 수준은 18S의 발현수준에 대해 표준화하여 계산하였다. 각 군간의 EFNA1 및 EPHA2 측정치는 언페어-티 테스트(unpair-t test)나 맨-휘트니 테스트(Mann-Whitney test)에 의해 검사하였다. P < 0.05일 때 유의기준을 정하였다.For quantitative and statistical analysis, the Northern blot auto-radiogram was scanned using the LAS3000 system (Fuji photo-film, Japan) to measure the signal intensity. Expression level of hepatocellular carcinoma tissues compared to non-hepatic cancer tissues was calculated by standardizing the expression level of 18S. EFNA1 and EPHA2 measurements between groups were examined by the unpair-t test or Mann-Whitney test. Significance criteria were determined when P <0.05.
상기 검출 방법들을 통하여, 정상 대조군과 간세포암 의심환자에서의 유전자 발현 수준을 비교함으로써 간세포암 실제 암 환자여부를 진단할 수 있고, 간세포암의 진행단계 또는 예후를 예측할 수 있다.Through the detection methods, it is possible to diagnose whether hepatocellular carcinoma is a real cancer patient by comparing gene expression levels in a normal control group and suspected hepatocellular carcinoma, and to predict the progression or prognosis of hepatocellular carcinoma.
본 발명의 또 다른 양태에서는, 재조합 발현벡터의 도입에 의한 EFNA1 및 EPHA2 과발현 세포주와 상기 세포주에 EFNA1 및 EPHA2 발현 억제 후보물질을 처리하여 처리된 세포와 대조구의 mRNA 또는 단백질 수준을 비교하여 EFNA1 및 EPHA2 발현 억제제를 스크리닝하는 방법을 제공한다.In another embodiment of the present invention, the EFNA1 and EPHA2 overexpressing cell lines by introducing a recombinant expression vector and the cells treated with EFNA1 and EPHA2 expression inhibitory candidates to compare the mRNA or protein levels of the control and control cells compared to EFNA1 and EPHA2 Methods of screening expression inhibitors are provided.
본 발명의 구체적 양태에 따른 EFNA1 및 EPHA2 발현 억제제의 스크리닝 방법은, EFNA1 및 EPHA2 단백질이 발현되는 세포를 배양하여 상기 세포에 EFNA1 및 EPHA2 발현 억제 후보 물질을 처리한 뒤, 이를 억제 후보 물질을 처리하지 않은 대조구 세포에서의 간세포암 진단 마커(단백질, mRNA)의 발현 정도와 비교하는 단계를 포함한다. 이와 같은 스크리닝 방법을 이용하여, 본 발명은 EFNA1 및 EPHA2 발현을 억제하며 간암세포를 효과적으로 치료할 수 있는 항암물질을 검색할 수 있게 한다.Screening method of the EFNA1 and EPHA2 expression inhibitor according to a specific embodiment of the present invention, after culturing the cells expressing the EFNA1 and EPHA2 protein treated with the candidate cells for inhibiting EFNA1 and EPHA2 expression, and then do not process the inhibitor candidate Comparing the expression level of hepatocellular carcinoma diagnostic marker (protein, mRNA) in the non-control cells. Using this screening method, the present invention enables the search for anticancer substances that inhibit EFNA1 and EPHA2 expression and can effectively treat liver cancer cells.
스크리닝 방법으로는, 바람직하게 EFNA1 및 EPHA2 유전자의 발현 수준, 바람직하게 mRNA 수준을 프라이머 또는 프로브를 이용하는 측정하는 방법이 이용될 수 있으며, 그 중에서도 RT-PCR이 바람직하다. 대조구와 비교하여 진단마커의 발현을 감소시키는 억제제를 선별하여 간세포암 치료에 사용할 수 있다.As the screening method, a method of measuring the expression level of the EFNA1 and EPHA2 genes, preferably the mRNA level using a primer or a probe, can be preferably used. Among them, RT-PCR is preferable. Inhibitors that reduce the expression of diagnostic markers can be selected and used to treat hepatocellular carcinoma in comparison to the control.
본 발명의 또 다른 양태에서는, EFNA1 및 EPHA2 발현 억제물질을 포함하는 간세포암 예방 또는 치료용 조성물을 제공한다. 구체적인 양태로서 본 발명은, 상기 EFNA1 및 EPHA2 발현 억제물질은 EFNA1 및 EPHA2 유전자의 mRNA에 상보적인 안티센스 RNA와 상보적인 센스 RNA 가닥을 포함하는, EFNA1 및 EPHA2 유전자 특이적 RNA간섭(RNA interference)을 유도하는 siRNA를 포함하는 간세포암 예방 또는 치료용 조성물 및 EFNA1 및 EPHA2 단백질에 특이적인 항체를 포함하는 간세포암 예방 또는 치료용 조성물에 관한 것이다.In still another aspect of the present invention, there is provided a composition for preventing or treating hepatocellular carcinoma comprising an EFNA1 and EPHA2 expression inhibitor. In a specific embodiment, the present invention, the EFNA1 and EPHA2 expression inhibitors include antisense RNA and complementary sense RNA strands complementary to the mRNA of the EFNA1 and EPHA2 gene, induces EFNA1 and EPHA2 gene specific RNA interference (RNA interference). It relates to a composition for preventing or treating hepatocellular carcinoma comprising an siRNA and a composition for preventing or treating hepatocellular carcinoma comprising an antibody specific for EFNA1 and EPHA2 proteins.
본 발명에서 용어,“siRNA”는 표적유전자의 mRNA의 절단(cleavage)을 통하여 RNA 간섭(RNAi: RNA interference) 현상을 유도할 수 있는 이중사슬 RNA를 의미하고, 표적유전자의 mRNA와 상동인 서열을 가지는 센스 RNA 가닥과 이와 상보적인 서열을 가지는 안티센스 RNA 가닥으로 구성된다. siRNA는 표적유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 넉다운 방법 또는 유전자치료(gene therapy)의 방법으로 제공된다.As used herein, the term “siRNA” refers to a double-chain RNA capable of inducing RNA interference (RNAi) through cleavage of mRNA of a target gene, and refers to a sequence homologous to mRNA of a target gene. The branch consists of a sense RNA strand and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit expression of a target gene, it is provided by an efficient gene knockdown method or gene therapy method.
siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 미스매치(mismatch, 대응하는 염기가 상보적이지 않음), 벌지(bulge, 일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. 전체 길이는 10 내지 80 염기, 바람직하게는 15 내지 60 염기, 더욱 바람직하게는 20 내지 40 염기이다. siRNA 말단 구조는 평활(blunt)말단 혹은 점착(cohesive) 말단 모두 가능하다. 점착 말단 구조는 3' 말단 돌출한 구조와 5' 말단 쪽이 돌출한 구조 모두 가능하고 돌출하는 염기 수는 한정되지 않는다. siRNAs are not limited to the complete pairing of double-stranded RNA portions paired with RNA, but mismatches (the corresponding bases are not complementary), bulges (the bases that do not correspond to one chain), and the like. It may include parts that are not paired by. The total length is 10 to 80 bases, preferably 15 to 60 bases, more preferably 20 to 40 bases. siRNA terminal structures can be either blunt or cohesive. The cohesive end structure can be either a 3 'end protruding structure or a 5' end protruding structure, and the number of protruding bases is not limited.
예를 들어, 염기 수로는 1 내지 8 염기, 바람직하게는 2 내지 6 염기로 할 수 있다. 또한, siRNA는 표적유전자의 발현억제 효과를 유지할 수 있는 범위에서 예를 들어, 한 쪽 말단의 돌출 부분에 저분자 RNA(예를 들어, tRNA, rRNA, 바이러스 RNA와 같은 천연의 RNA분자 또는 인공의 RNA분자)를 포함할 수 있다. siRNA 말단구조는 양측 모두 절단 구조를 가질 필요는 없고, 이중사슬 RNA의 일방의 말단 부위가 링커 RNA에 의하여 접속된 스템 루프형 구조일 수도 있다. 링커의 길이는 스템 부분의 쌍을 이루는 데 지장이 없는 길이면 특별히 한정되지 않는다. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. In addition, siRNA is a low-molecular RNA (e.g., a natural RNA molecule such as tRNA, rRNA, viral RNA or artificial RNA) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene Molecules). The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.
siRNA를 제조하는 방법은 시험관에서 siRNA를 직접 합성한 뒤, 유전자 전달감염(transfection) 과정을 거쳐 세포 안으로 도입시키는 방법과 siRNA가 세포 안에서 발현되도록 제조된 siRNA 발현 벡터 또는 PCR-derived siRNA 발현 카세트 등을 세포안으로 유전자 전달 또는 형질도입(transduction) 시키는 방법이 있다.The method of preparing siRNA is a method of directly synthesizing siRNA in vitro, introducing the cell into a cell through a gene transfection process, and an siRNA expression vector or a PCR-derived siRNA expression cassette prepared to express siRNA in a cell. There is a method of gene transfer or transduction into cells.
본 발명에서, 용어“특이적”또는“특이적인”은 세포내에서 다른 유전자에 영향을 미치지 않고 표적 유전자만 억제하는 능력을 의미하고, 본 발명에서는 EFNA1 및 EPHA2 에 특이적이다.In the present invention, the term "specific" or "specific" refers to the ability to inhibit only the target gene without affecting other genes in the cell, and is specific for EFNA1 and EPHA2 in the present invention.
본 발명의 siRNA는 EFNA1 및 EPHA2의 mRNA를 특이적으로 감소시킬 수 있으며 서열과 길이는 특별히 제한되지 않는다.The siRNA of the present invention can specifically reduce the mRNA of EFNA1 and EPHA2, and the sequence and length are not particularly limited.
EFNA1 및 EPHA2 단백질에 특이적인 항체가 치료용 항체로 사용될 경우, 항체는 기존의 치료제와 직접 또는 링커 등을 통하여 간접적으로 커플링(예를 들어, 공유결합)시킬 수 있다. 항체와 결합될 수 있는 치료제에는 방사성핵종(radionuclide), 약제, 림포카인, 독소, 이형가능성 항체 등이 있으나 이로 제한되지는 않는다. When an antibody specific for the EFNA1 and EPHA2 proteins is used as a therapeutic antibody, the antibody may be coupled (eg, covalently) to an existing therapeutic agent directly or indirectly through a linker or the like. Therapeutic agents that can be combined with antibodies include, but are not limited to, radionuclides, drugs, lymphokines, toxins, heterozygous antibodies, and the like.
항체는 그 자체 또는 항체를 포함하는 조성물로 투여될 수 있다. 치료용 조성물의 경우 투여 방식에 따라 허용가능한 담체를 포함하여 적절한 제제로 제조된다. 투여방식에 적합한 제제는 공지되어 있고, 전형적으로 막을 통과한 이동을 용이하게 하는 계면활성제를 포함한다, 이러한 계면활성제는 스테로이드에서 유도된 것이거나 N-[1-(2,3 디올레 오일(프로필)-N,N,N-트리메틸암모늄클로라이드(DOTMA) 등의 양이온성 지질, 또는 콜레스테롤 헤미숙시네이트, 포스파티딜 글리세롤 등의 각종 화합물 등이 있다.The antibody can be administered by itself or in a composition comprising the antibody. For therapeutic compositions, they are prepared in appropriate formulations, including an acceptable carrier depending on the mode of administration. Formulations suitable for the mode of administration are known and typically comprise a surfactant which facilitates migration across the membrane. Such surfactants may be steroid derived or N- [1- (2,3 diole oil (propyl) Cationic lipids such as) -N, N, N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidyl glycerol.
본 발명의 항체를 포함하는 조성물은 암세포 또는 그들의 전이를 치료하기 위하여 약학적으로 효과적인 양으로 투여될 수 있다. 약학적 조성물은 단일 또는 다중 투여될 수 있다. 항체를 포함하는 조성물은 피하, 복강 내, 폐 내 및 비강 내, 및 국부적 면역억제치료를 위해 필요하다면 병변 재투여를 포함하는 적합한 방법에 의해 투여된다. 비경구 주입에는 근육내, 정맥내, 동맥내, 복강내 또는 피하투여가 포함된다. 바람직한 투여방식 및 제제는 정맥 주사제, 피하 주사제, 피내 주사제, 근육 주사제, 점적 주사제 등이다. 제제의 pH는 항체 안정성(화학적 및 물리적 안정성)의 균형을 맞추고 투여를 위한 다른 기술들을 적절하게 변형시켜 이용하여, 적당한 제제를 고안화할 수 있다. 전형적인 투여량 수준은 표준 인상적 기술을 사용하여 최적화 할 수 있다. 또한, 본 발명의 항체는 항체를 암호화하는 핵산의 형태로 투여되어 세포내에서 항체가 생성되도록 할 수 있다.Compositions comprising the antibodies of the invention can be administered in pharmaceutically effective amounts to treat cancer cells or their metastases. The pharmaceutical composition can be administered single or multiple. Compositions comprising the antibody are administered by suitable methods including subcutaneous, intraperitoneal, pulmonary and nasal, and lesion re-administration if necessary for local immunosuppressive therapy. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. The pH of the formulation can be designed to balance antibody stability (chemical and physical stability) and to employ other techniques for administration, as appropriate, to formulate suitable formulations. Typical dosage levels can be optimized using standard impressive techniques. In addition, the antibodies of the invention can be administered in the form of nucleic acids encoding the antibodies to allow the antibodies to be produced intracellularly.
이하, 본 발명의 실시예에 대하여 보다 구체적으로 설명한다. 다만, 이들 실시예는 본 발명을 예시하기 위한 것이로, 본 발명의 권리범위가 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, embodiments of the present invention will be described in more detail. However, these examples are for illustrating the present invention, the scope of the present invention is not limited by these examples.
<실시예 1> 간세포암조직과 비간암조직으로 부터의 총 RNA의 분리Example 1 Isolation of Total RNA from Hepatocellular Carcinoma and Non-Liver Cancer
40명의 간세포암 환자의 간세포암 조직과 주위 비간암 조직을 수술적으로 절제하여 연구를 실행했으며 본 연구는 전북대학교 병원 연구윤리위원회의 승인을 받았다. 40명의 간세포암 환자는 근치적 치료를 위해 수술을 받았으며 암조직과 주위 간경변성 조직은 이 절제된 간 조직에서 얻었다. 서류에 의한 동의를 받았으며 간세포암과 비간암조직은 병리학자에 의해 조직학적으로 확인되었다. 절제 후 이 조직들은 멸균된 인산 완충액으로 씻고 총 RNA분리를 위해 질소탱크 속에 보관하였다. 총RNA는 트리졸 키트(Trizol Kit, Invitrogen사, 미국)에 의해 추출하고 전기영동에 의하여 RNA의 질을 확인하였고, 일부 절제된 조직은 10% 포르말린 완충액에 고정하고 파라핀 매몰로 표준 조직병리학적 분석을 시행하였다. 이 프로트콜은 기관윤리위원회의 윤리지침에 의거하여 수행하였고, 간세포암 및 주위 비간암조직으로부터 총RNA를 각각 트리졸(Trisol) 용액을 이용하여 추출하였다.The study was performed by surgically resecting the hepatocellular carcinoma and surrounding non-hepatic carcinoma of 40 hepatocellular carcinoma patients. This study was approved by the Research Ethics Committee of Chonbuk National University Hospital. Forty hepatocellular carcinoma patients underwent surgery for curative treatment and cancerous and surrounding cirrhosis tissues were obtained from this resected liver tissue. Hepatocellular and non-hepatic cancer tissues were histologically confirmed by a pathologist. After excision, the tissues were washed with sterile phosphate buffer and stored in a nitrogen tank for total RNA isolation. Total RNA was extracted by Trizol Kit (Invitrogen, USA) and confirmed the quality of RNA by electrophoresis, some excised tissues were fixed in 10% formalin buffer and paraffin embedded for standard histopathological analysis. Was implemented. This protocol was performed according to the ethical guidelines of the Institutional Ethics Committee. Total RNA was extracted from hepatocellular carcinoma and surrounding non-hepatic cancer tissues using Trizol solution, respectively.
<실시예 2> 간세포암 관련 유전자 프로필 작성Example 2 Gene Preparation for Hepatocellular Carcinoma
실시예 1에서 추출한 간세포암 조직과 비간암조직의 총 RNA를 먼저 약 3000개의 유전자가 탑재된 cDNA 마이크로어레이 혼성화(hybridization)를 하여 형광 표지 cDNA를 제작하고, 비검열 계통적 분석(unsupervised hierarchical clustering analysis)에 의해 2군의 주그룹으로 나누었다. 통계 프로그램에 의하여 FDR(위발견율) 1% 이하에서 비간암 조직과 간세포암을 구별할 수 있는 유전자를 동정하였다. Total RNA of the hepatocellular carcinoma tissue and the non-hepatic cancer tissue extracted in Example 1 was first prepared using fluorescent cDNA microarray hybridization (cDNA microarray hybridization) containing approximately 3000 genes, and then unsupervised hierarchical clustering analysis. Divided into two main groups. A statistical program was used to identify genes that can distinguish hepatocellular carcinoma from non-hepatic cancer at 1% or less of FDR.
간세포암 및 주위 비간암조직으로부터 총 RNA를 각각 트리졸(Trisol)용액을 이용하여 추출하여, 형광 표지 cDNA 제작하고 마이크로어레이 혼성화(hybridization)를 하였다. 분리한 총RNA 100μg을 14㎕에 용해하여 수퍼스크립트(SuperSript) II 역전사 효소와 0.5mM dATP, dGTP, dCTP 및 0.2mM dTTP를 추가하고 각각 0.1mM Cy5-dUTP 또는 Cy3-dUTP를 넣어 40㎕ 최종 부피에서 표지는 42℃에서 2시간 실시하였으며, 500 mM EDTA 5㎕를 가하여 종료시키고 표지하지 않은 RNA는 1N NaOH 10㎕를 가하여 65℃에서 30분 간 가열 가수분해하고 25㎕ 트리스(Tris) HCL(pH 7.5)를 가하여 중화한 후 바이오스핀(Biospin)6 컬럼을 이용하여 표지되지 않은 핵산염기와 염을 제거하였다. Total RNA was extracted from hepatocellular carcinoma and surrounding non-liver carcinoma tissue using Trizol solution, respectively, to prepare fluorescent labeled cDNA, and to perform microarray hybridization. Dissolve 100 μg of total RNA in 14 μl, add SuperSript II reverse transcriptase, 0.5 mM dATP, dGTP, dCTP, and 0.2 mM dTTP, add 0.1 mM Cy5-dUTP or Cy3-dUTP, respectively, and 40 μl final volume. Labeling was carried out at 42 ° C. for 2 hours, and 5 μl of 500 mM EDTA was terminated. Unlabeled RNA was heat hydrolyzed at 65 ° C. for 30 minutes by adding 10 μl of 1N NaOH and 25 μl Tris HCL (pH). 7.5) was added for neutralization and then the unlabeled nucleic acid bases and salts were removed using a Biospin 6 column.
표지된 프로브는 이소프로판올로 침전 후 혼성화(hybridization) 완충액에 녹였다. 녹인 프로브는 슬라이드에 놓고 하이브리드 슬립(hybri-slip)을 덮어주고, 혼성화(hybridization) 챔버에 넣어 65℃에서 하룻밤 반응시켰다. 혼성화 후 슬라이드들은 1× SSC/0.1% SDS, 0.1× SSC/0.1% SDS(50℃), 0.1× SSC 완충액으로 각 10분간 세척하였다. 반응이 끝난 슬라이드들은 콴트어레이(Quantarray) 프로그램를 사용하여 스캔한 후 이마진 4.2(ImaGene 4.2, Biodiscovery)프로그램을 사용하여 분석하였다.Labeled probes were dissolved in hybridization buffer after precipitation with isopropanol. The dissolved probe was placed on a slide, covered with a hybrid slip, and placed in a hybridization chamber to react overnight at 65 ° C. After hybridization, slides were washed for 10 minutes each with 1 × SSC / 0.1% SDS, 0.1 × SSC / 0.1% SDS (50 ° C.), 0.1 × SSC buffer. Reaction slides were scanned using Quantray program and analyzed using ImaGene 4.2 (Biodiscovery) program.
Cy5 및 Cy3의 형광강도는 이마진 프로그램을 이용하여 국소 배경교정(local background correction)을 거친 후 전체 스팟을 이용하여 표준화(normalization)하였다. 스팟의 정성분석은 다음의 기준을 사용하였다. 신호 평균-배경 평균(Signal mean-Background mean)/배경(Background) 값이 SD>2.0 이상인 스팟 중에서 평균 신호가 국소 배경(local background)의 1.5배 이상의 기준을 통과하는 스팟을 분석에 사용하였다. 이 기준은 모든 어레이 슬라이드의 분석에 동일하게 적용시켰다.The fluorescence intensities of Cy5 and Cy3 were normalized using the whole spot after local background correction using the two-margin program. Qualitative analysis of the spot used the following criteria. Among the spots with a signal mean-background mean / background value of SD> 2.0 or more, the spots where the average signal passes a standard of 1.5 times or more of the local background were used for the analysis. This criterion applied equally to the analysis of all array slides.
암종 분류는 웹사이트에서 이용할 수 있는 "클러스터(Cluster)"와“트리뷰(TreeView)"를 이용하여 분석하였다. 사용한 샘플 중 적어도 80% 이상에서 측정될 수 있는 어레이 요소만을 분석대상으로 하였다. 분석 전, 각 스팟의 형광비는 로그 전환(log-transformation)을 거친 후 실험적 편차(experimental biases)을 제거하기 위하여 중간값 센터링(median centering)을 하였다. 통계학적 처리는 SAM(Significance analysis of microarray)프로그램에 의하여 유의한 유전자를 점수화 하였다.Carcinoma classification was analyzed using the "Cluster" and "TreeView" available on the website.Only array elements that can be measured in at least 80% of the samples used were analyzed. Before and after the fluorescence ratio of each spot was subjected to log-transformation, median centering was performed to remove experimental biases. By scoring significant genes.
간세포암과 비간암조직에서 적어도 80%에서 발현되는 유전자를 선택하였으며, 전체 유전자 발현 양상과의 유사성을 근거로 모든조직에 대해 비검열 계통적 클러스터링(hierachical clustering) 분석을 시도하였다.Genes expressed in at least 80% of hepatocellular carcinoma and non-hepatic carcinoma tissues were selected, and a noncensored systematic clustering analysis was attempted for all tissues based on similarity with the expression patterns of all genes.
40명의 환자로부터 얻은 primary HCC tumor 표본과 이에 상응하는 non-tumor 간 조직 표본에서 cDNA microarry 분석을 통하여 유전자 발현 양상을 관찰하였다. 유전자는 최소 80%에서 발현되었고 SD>0.5인 것을 기반으로 선택하였으면 전체 유전자 발현 양상과의 유사성을 근거로 모든 조직에 대해 비검열 계층적 클러스터링 분석을 하였다. The gene expression patterns were observed by cDNA microarry analysis in primary HCC tumor samples from 40 patients and corresponding non-tumor liver tissue samples. If genes were expressed at least 80% and were selected based on SD> 0.5, noncensored hierarchical clustering analysis was performed on all tissues based on similarity with the overall gene expression pattern.
Group I에서 선택된 10개의 분비단백질을 코딩하는 유전자 혹은 막 단백질 중 오직 한 개만이 간세포암 클러스터에 포함되는 것을 제외하고 대부분 비간세포암 조직을 나타내었다. 때문에 암전 간경변, 간세포암의 초기 혹은 완전 분화 단계에서 특이하게 발현되는 유전자는 간세포암 발병의 조기검진 마커 혹은 간세포암 진화를 나타내는 분자적 표적물로 사용 가능하다. Group II의 5 유전자는 분비단백질이 코딩하는 유전자 혹은 막 단백질이 간경변과 분화단계가 I/II 간세포암에서 발현되는 유전자에서 선택한 것이다. Group III의 13 유전자는 III/IV 분화단계의 간세포암에서 하향조절하는 것을 선택한 것이다.Most of the non- hepatocellular carcinoma tissues were represented except that only one of the genes or membrane proteins encoding the 10 secretory proteins selected from Group I was included in the hepatocellular carcinoma cluster. Therefore, genes that are specifically expressed in precancerous cirrhosis and early or full differentiation stages of hepatocellular carcinoma can be used as early screening markers for the development of hepatocellular carcinoma or as molecular targets for hepatocellular carcinoma evolution. Group 5 genes are selected from genes encoded by the secreted proteins or from membrane proteins expressed in cirrhosis and differentiation stages in I / II hepatocellular carcinoma. The 13 genes in Group III chose to downregulate hepatocellular carcinoma in the III / IV differentiation stage.
[표 1] 간세포암에서 차별 발현되는 분비 또는 막단백질을 코딩하는 유전자 목록(List of genes which encode secretory or membrane proteins preferentially expressed in HCC)Table of List of genes which encode secretory or membrane proteins preferentially expressed in HCC
Figure PCTKR2010003513-appb-I000001
Figure PCTKR2010003513-appb-I000001
상기 표 1중 분비 단백질을 코딩하는 유전자인 EFNA1이 간세포암조직에서 선택적으로 상향 발현되는 점은 중요한 진단적 가치를 가지고 있어, 조직 및 체액을 포함하는 생물학적 시료에서 EFNA1 RNA 및 단백질 발현을 측정하여 간세포암의 진단 및 예후지표로서 사용할 수 있음을 확인하고자 하였다.Selective upward expression of EFNA1, a gene encoding a secreted protein in Table 1, has important diagnostic value, and thus, hepatocytes by measuring EFNA1 RNA and protein expression in biological samples including tissues and body fluids. The purpose of this study was to confirm that cancer can be used as a diagnostic and prognostic indicator.
<실시예3> 간세포암군과 상응하는 비간암군에서 EFNA1 및 EPHA2 발현 분석Example 3 Analysis of EFNA1 and EPHA2 Expression in Hepatocellular Carcinoma and Corresponding Non-Liver Cancer
1. 노던블롯 분석(Northern blot analysis)과 RT-PCRNorthern blot analysis and RT-PCR
Nontumor와 tumor의 total RNA 20㎍을 함유한 sample을 2.2％formaldehyde와 50mM 3-(N-morpholono)propanesulfonic acid (MOPS)를 함유하는 1％ Agarose에 분획하여 nylon membrane에 전이했다. Nylon membrane은 UV cross linker로 처리하여 cross link 하였고 32P dCTP로 표지된 EFNA1 cDNA 프로브(probe)에서 random primer 로 labeling 한 후 하루동안 hybridization 한 후 washing 후 -70℃에서 X-ray film 에 노출시켰다. 각 lane 에 loading한 mRNA의 양은 membrane을 stripping 한후 18s cDNA를 hybridization함으로써 대조군으로 이용하였다.A sample containing 20 µg of nontumor and tumor total RNA was fractionated to 1% Agarose containing 2.2% formaldehyde and 50 mM 3- (N-morpholono) propanesulfonic acid (MOPS) and transferred to nylon membrane. The nylon membrane was cross-linked by UV cross linker, labeled with a random primer in a 32 P dCTP-labeled EFNA1 cDNA probe, hybridized for one day, washed and exposed to X-ray film at -70 ℃. The amount of mRNA loaded in each lane was used as a control by hybridizing 18s cDNA after stripping the membrane.
선택적으로 reverse transcription(RT)은 10㎍ total RNA, 50M decamer RT-PCR superscriptII(200unit,invitrogen)을 가하여 37℃에서 50min동안 incubation 후 반응을 완성하였다. 이 cDNA를 이용하여 cDNA 2㎕에 EPHA2 각각의 100 pmol의 primer를 10㎛, 2.5mM dNTP 2㎕, Tag polymerase를 가하여 PCR 증폭을 하였다. PCR은 94℃에서 1min. 65℃에서 1min, 72℃에서 1min씩 35cycle을 반응을 시켰다. Quantum RNATM 18S internal Standard kit를 이용하여 internal control을 잡았다. EPHA2의 primer sequence는 5'-ATGGAGCTCCAGG CAGC CCGCGC-3' (센스(sense), 서열번호 1)와 5'-AGGGAAGCTGTTGCAGTCA-3' (안티센스(antisense), 서열번호 2)였다. Selectively reverse transcription (RT) was added to 10㎍ total RNA, 50M decamer RT-PCR superscriptII (200unit, invitrogen) to complete the reaction after incubation for 50 minutes at 37 ℃. Using the cDNA, PCR amplification was performed by adding 10 μm, 2.5 mM dNTP, 2 μl, and tag polymerase to 100 μl of each of EPHA2 to 2 μl of cDNA. PCR was performed at 94 ° C for 1 min. 35 cycles were reacted at 65 ° C. for 1 min and at 72 ° C. for 1 min. Internal control was performed using the Quantum RNATM 18S internal standard kit. The primer sequences of EPHA2 were 5'-ATGGAGCTCCAGG CAGC CCGCGC-3 '(sense, SEQ ID NO: 1) and 5'-AGGGAAGCTGTTGCAGTCA-3' (antisense, SEQ ID NO: 2).
PCR산물은 2％ agarose gel에서 electrophoresis에 의해 분리하였고 Ethidium bromide 염색 후 UV를 이용하여 확인하였다. 내인적 control로 18s를 이용하였다. 그 중에서 EFNA1 mRNA 가 40예 중 36예(90%) HCC 조직에서 선택적으로 과발현된다는 것을 관찰하였다(도 1 참조). PCR products were isolated by electrophoresis on 2% agarose gel and confirmed by UV after staining with Ethidium bromide. 18s was used as endogenous control. Among them, it was observed that EFNA1 mRNA was selectively overexpressed in 36 of 40 (90%) HCC tissues (see FIG. 1).
그 발현 수는 간세포암 조직에서 3.94(95% CI, 4.76에서 1.97), 상응하는 비간세포암의 조직에서는 1.4(95%CI, 2.57에서 0.96)이었다(P=0.0006). 또한, EPHA2는 간세포암에서 불균형적으로 발현되었다. 간세포암 조직에서는 1.49(95% CI, 2.54에서 1.04), 비간세포암 조직에서는 1.83(95% CI, 2.95에서 0.93)으로 각자 발현이 특이하지 않았다(P=0.5966)(도 2 참조). 결정적으로 EFNA1과 EPHA2의 발현은 TMN 종양의 Edmondson 분화와 관련되지 않는다는 것을 알 수 있었다.The expression number was 3.94 (95% CI, 4.76 to 1.97) in hepatocellular carcinoma tissue and 1.4 (95% CI, 0.97 to 2.57) in tissue of corresponding non-hepatic cell carcinoma (P = 0.0006). EPHA2 was also disproportionately expressed in hepatocellular carcinoma. 1.49 (95% CI, 2.54 to 1.04) in hepatocellular carcinoma tissues and 1.83 (95% CI, 2.95 to 0.93) in hepatocellular carcinoma tissues did not have specific expression (P = 0.5966) (see FIG. 2). Crucially, expression of EFNA1 and EPHA2 was not associated with Edmondson differentiation of TMN tumors.
2. 면역블롯 면역형광 염색 및 면역화학염색2. Immunoblot Immunofluorescence Staining and Immunochemical Staining
면역조직화학염색은 10% forrmalin에 고정하고 paraffin에 매몰시킨 조직을 4㎛두께로 자른 것을 가지고 이용하였다. Rehydration 후에 파라핀제거를 위해microwave epitope retrieval 을 이용하였다.(750W에서 15min 동안 10mmol, PH 6.0의 citrate buffer에 담아) 일차항체를 붙이기 전에 3% hydrogen peroxide와 함께 내재적인 peroxidase는 저해하였고 bovine albumin blocking 과정에서 biotin을 부착하였다. EFNA1과 EPHA2의 일차적인 mouse단일 항체(RJMWZE7, Alexis biochemical, Lausen, Switzerland) rabbit 다클론항체(PMA06)을 일차항체로 붙이고 이차항체인 바이오틴부착항체와 streptavidine-horseradishperoxidase (DAKO,Glostrup, Denmark) 와 함께 발색시켰다.Immunohistochemical staining was used to fix tissues fixed in 10% forrmalin and buried in paraffin with 4 ㎛ thickness. After rehydration, microwave epitope retrieval was used to remove paraffin (in 15 mmol at 750 W for 15 min in a citrate buffer of pH 6.0). Before incorporation of primary antibody, intrinsic peroxidase was inhibited with 3% hydrogen peroxide and bovine albumin blocking. biotin was attached. The primary mouse monoclonal antibody of EFNA1 and EPHA2 (RJMWZE7, Alexis biochemical, Lausen, Switzerland) was attached to rabbit polyclonal antibody (PMA06) as a primary antibody, along with the secondary antibody, biotin attached antibody and streptavidine-horseradishperoxidase (DAKO, Glostrup, Denmark). Color development.
Meyer’s hematoxylin을 이용하여 counter-staining 을 하였다. 음성대조 염색은 생리적 식염수나 mouse IgG 면역단백질을 일차항체로 사용하였다. 면역형광법을 위해 세포를 glasscoverslips위에 키운 후 , GFP-targged EFNA1와 EPHA2 발현 vecter나 대조군으로서 공벡터를 도입후 4% paroformaldehyde로 고정시킨 후 0.2% triton-X 를 유하는 phosphate buffered saline (PBS)로 투과성을 유도하여 1% bovine serum albumin (BSA)로 블로킹하였다. 세포는 mouse 단클론항체인 RJMW2E로 4℃에서 밤새 배양 후 적형광 TRITC부착된 anti-mouse immunoglobulin을 이차적으로 붙였다. 최종 세척 후 세포는 1㎍/ml Hoechst 33258 에 15분간 핵을 염색하고 50% glycerol에 마운팅하고 레이저 스캔현미경(LCM510,Carcl Zeiss, Germany)으로 관찰하였다.Counter-staining was performed using Meyer ’s hematoxylin. Negative control staining was performed using physiological saline or mouse IgG immunoprotein as the primary antibody. Cells were grown on glasscoverslips for immunofluorescence, permeabilized with phosphate buffered saline (PBS) containing 0.2% triton-X and then fixed with 4% paroformaldehyde after introduction of a GFP-targged EFNA1 and EPHA2 expression vecter or control vector. Induction was blocked with 1% bovine serum albumin (BSA). Cells were incubated overnight at 4 ° C. with RJMW2E, a mouse monoclonal antibody, followed by secondary attachment of red fluorescent TRITC-attached anti-mouse immunoglobulin. After the final wash, the cells were stained for 15 minutes in 1 μg / ml Hoechst 33258, mounted on 50% glycerol, and observed by laser scanning microscope (LCM510, Carcl Zeiss, Germany).
EFNA1이나 EPHA2항체의 면역반응성의 특이성을 결정하기 위하여 293T 세포주에 GFP표지 EFNA1 혹은 GFP표지 EPHA2나 Myc 표지 EFNA1 혹은 Myc표지 EPHA2 발현 벡터를 도입하고 면역블로팅을 실행하여 RTMW2E7 mAb나 PMA06B의 면역반응성을 조사하였다. 도입된 EFNA1 cDNA는 COOH-말단부위에 GFP나 Myc이 표지되어 있기 때문에 anti-GFP rabbit polyclonal antibody(santacruz)나 anti-C-Myc Mouse monoclonal antibody(9E10,Santacruz)를 이용하여 면역블랏을 실행하였다. EFNA1 항체 혹은 EPHA2 항체는 상응한 밴드를 특이적으로 검출하는 것을 확인하였다(도 3 및 4 참조). In order to determine the specificity of the immunoreactivity of EFNA1 or EPHA2 antibody, GFP-labeled EFNA1 or GFP-labeled EPHA2 or Myc-labeled EFNA1 or Myc-labeled EPHA2 expression vector was introduced and immunoblotting was performed to immunoreactive RTMW2E7 mAb or PMA06B. Investigate. Since the introduced EFNA1 cDNA is labeled with GFP or Myc at the COOH-terminus, immunoblot was performed using anti-GFP rabbit polyclonal antibody (santacruz) or anti-C-Myc Mouse monoclonal antibody (9E10, Santa Cruz). EFNA1 antibody or EPHA2 antibody was confirmed to specifically detect the corresponding band (see FIGS. 3 and 4).
다음 방법으로 Hep3B와 HepG2 세포주에 GFP표지 EFNA1 혹은 EPHA2 발현벡터와 대조 공벡터를 도입하여 면역형광법으로 RTMWZED mAb 나 DMAO6B Ab의 면역반응성을 확인하였다. 형광과 투과이미지를 비교하여보면 GFP표지 EFNA1외인적 발현은 주요 핵이나 세포질에서 발현되었으며 이는 이전의 보고와 일치하였다. 그린 형광은 EFNA1의 면역반응성과 완전하게 일치하였으며 내재적 EFNA1의 발현은 RTMWZE7 mAb 에 의해 주로 세포의 세포질에서 발현됨을 확인하였다. 이와 대조적으로, 내인적 EPHA2 와 GFP표지 EPHA2발현은 주요하게 세포절 막에서 확인되었다(도 5 및 6 참조). In the following method, GFP-labeled EFNA1 or EPHA2 expression vector and control vector were introduced into Hep3B and HepG2 cell lines to confirm the immunoreactivity of RTMWZED mAb or DMAO6B Ab by immunofluorescence. Comparing fluorescence and transmission images, GFP-labeled EFNA1 exogenous expression was expressed in the major nucleus or cytoplasm, consistent with previous reports. Green fluorescence was completely consistent with the immunoreactivity of EFNA1, and the expression of endogenous EFNA1 was mainly expressed in the cytoplasm of cells by RTMWZE7 mAb. In contrast, endogenous EPHA2 and GFP-labeled EPHA2 expression were mainly identified in cell node membranes (see FIGS. 5 and 6).
면역조직화학염색의 결과로부터 EFNA1의 면역반응성이 간세포, 혈관, 담관 등에서 소극적이고 악성 간 세포질에서 많이 위치하여 있다는 것을 알 수 있었다. 정상적인 간에는 간세포, 섬유성 낭포와 침투성 혈액 세포등이 포함되고, EPHA2는 악성 간 세포의 세포질 및 세포막에 발현되고, 정상적인 간과 비간암 조직에서 미약하게 발현되었다(도 7 참조).Immunohistochemical staining showed that the immunoreactivity of EFNA1 was negative in hepatocytes, blood vessels, bile ducts, and was found in many hepatic cytoplasms. Normal livers include hepatocytes, fibroblasts and invasive blood cells, EPHA2 is expressed in the cytoplasm and cell membrane of malignant liver cells, and weakly expressed in normal liver and non-hepatic cancer tissues (see FIG. 7).
<실시예4> 세포배양 상청액 및 환자 혈청 실험Example 4 Cell Culture Supernatant and Patient Serum Experiment
1. Chang liver 세포와 HepG2, Hep3B, PLC/PRF/5, Huh7, SK-HEP-1, SH-J1, HKL3, HKK2, HKL5 등 간암세포의 배양 상층액에서 EFNA1이 분비된다는 것을 분석하였다. 대다수 EFNA1은 HepG2 and Huh7 세포에서 Chang liver 세포에서보다 명확히 분비된다는 것을 관찰하였다(도 10A 참조). 하지만 SK-HEP-1와 SH-J1 세포에서는 EFNA1 단백질이 미소하게 분비되었다. 또한 APF 단백질이 EFNA1과 마찬가지로 Hep G2 , Hep 3B, PLC/PRF/5 세포에서 많이 분비된다는 분석을 진행하였다. 흥미로운 것은 Huh7세포에서 AFP 단백질이 미소하게 분비되지만 EFNA1 단백질은 같은 조건하의 상층액에서 풍부하게 분비되었다. 1. We analyzed the secretion of EFNA1 from cultured supernatants of Chang liver cells and liver cancer cells such as HepG2, Hep3B, PLC / PRF / 5, Huh7, SK-HEP-1, SH-J1, HKL3, HKK2, HKL5. It was observed that the majority of EFNA1 is secreted more clearly in HepG2 and Huh7 cells than in Chang liver cells (see FIG. 10A). However, EFNA1 protein was secreted slightly in SK-HEP-1 and SH-J1 cells. In addition, as in EFNA1, APF protein was secreted in Hep G2, Hep 3B, PLC / PRF / 5 cells. Interestingly, AFP protein was secreted slightly in Huh7 cells, but EFNA1 protein was abundantly secreted in the supernatant under the same conditions.
다음 인구통계학적인 임상정보는 각 환자에서 수집하였다. 세 그룹은 등록되어 있는 tumor사이즈에 따라 일관되었다. 그룹(Group)1 NL(G1,n=3)은 건강한 간, 과거에 간질환이 없는 정상간 생화화적 소견을 보이는 정상군이다. 그룹(Group)2 (G2,n=3)은 조직학적으로 확인된 간암 조직(HCC)으로 이루어졌고 tumor 사이즈는 4cm 이하이다. 그룹(Group)3 (G3,n=3)은 조직학적으로 확인된 사이즈가 4cm 이상의 간세포암환자군(HCC)이다. 모든 혈청은 수집 즉시 초속원심분리기에 의해 분리하였고 -20°C에서 냉동보관하였다. The following demographic clinical information was collected from each patient. The three groups were consistent with the tumor size registered. Group 1 NL (G1, n = 3) is a normal group with healthy liver and normal liver biochemical findings without past liver disease. Group 2 (G2, n = 3) consisted of histologically confirmed liver cancer tissue (HCC) with a tumor size of 4 cm or less. Group 3 (G3, n = 3) is a hepatocellular carcinoma patient (HCC) group whose size is more than 4 cm. All serum was isolated by ultracentrifuge immediately after collection and frozen at -20 ° C.
HCC는 외과수술에서 얻은 간 조직 표본을 병리조직학적 진단으로 유효하게 검증하였고 모든 그룹에 적용하였다(도 10 B 참조). HCC 환자와 정상적인 비교군의 혈청 상층액에서 EFNA1이 분비되는 것을 비교하였다. HCC 환자에서 정상적인 비교군의 혈청에서보다 EFNA1 단백질이 높게 발현되었다. 하지만 HCC 종양의 크기에 따라, 즉 직경이 큰 종양(> 4 cm in diameter)환자와 직경이 작은 종양(< 4 cm in diameter)환자에서 EFNA1의 분비되는 것은 크게 구별되지 않았다. HCC validated pathologically histologically obtained liver tissue specimens and applied to all groups (see FIG. 10B). The secretion of EFNA1 from serum supernatants of HCC patients and normal control groups was compared. In HCC patients, EFNA1 protein was expressed higher than in normal control serum. However, according to the size of HCC tumors, that is, the secretion of EFNA1 in patients with larger diameters (> 4 cm in diameter) and patients with smaller diameters (<4 cm in diameter) was not significantly different.
2. 면역블로팅2. Immunoblotting
Cell lysate로부터 단백질을 분리하여 SDS PAGE 30㎍을 loading하여 분리한후 nitrocellulose membrane에 옮겼다. membrane은 5% skin milk로 블록킹 한 후 primary EFNA1과 EPHA2 antibody를 (1:1000 dilution) 실온에서 4시간 동안 처리하였다. 처리 후 0.1% Tween 20이 포함된 TBS로 세번 세척하였다. EFNA1과 EPHA2는 alkaline phosphatase-conjugated goat anti-rabbit IgG나 화학발광방법을 이용하여 확인하였다. 분비단백질을 확인하기 위해 cell은 FBS가 없는 Opti-MEM으로 하루동안 배양후 Media supernatant만 수거하였다. Media는 centricon YM10 filter로 concentration 하여 20㎍의 potein을 immunoblotting하였다. 혈청 면역반응을 위하여 1%BSA와 혈청을 1:1로 mix후 10㎍만을 SDS-PAGE에 도달후 nitrocellulose membrane에 이동시켰다.Proteins were separated from cell lysate, loaded with 30µg SDS PAGE, and transferred to nitrocellulose membrane. The membrane was blocked with 5% skin milk and treated with primary EFNA1 and EPHA2 antibodies (1: 1000 dilution) at room temperature for 4 hours. After treatment, the cells were washed three times with TBS containing 0.1% Tween 20. EFNA1 and EPHA2 were identified using alkaline phosphatase-conjugated goat anti-rabbit IgG or chemiluminescence. To confirm the secreted protein, cells were cultured with Opti-MEM without FBS for one day and only media supernatants were collected. The media were concentrated with a centricon YM10 filter and immunoblotting 20 μg of potein. For serum immune response, 1% BSA and serum were mixed 1: 1 and transferred to nitrocellulose membrane after reaching SDS-PAGE.
다음 EFNA1과 EPHA2은 각종 간세포암 세포에서 AFP의 발현과 비교하여 분석하였다.(도 8 참조). Immunoblot analyses에서 EFNA1이 Hep G2, Hep 3B, PLC/PRF/5, Huh7 세포에서 SK-Hep1, SH-J1, HLK-3, HKK2, HLK-5 세포에서 보다 높게 발현되었다. 하지만 Huh7, SK-Hep1, SH-J1 세포에서는 대다수의 EPHA2가 과발현되었다. 특히, SK-Hep1와 SH-1 세포에서 EPHA2가 과발현되지만 EFNA1과 AFP은 약하게 발현되었다. 간암에서 이런 EPHA2의 부분적인 발현은 AFP 발현과 ENA1의 발현과 상반된다. p53-family proteins에 의하여 EPHA2를 조절한다고 하기 때문에 HCC 세포에서 p53 의 발현을 보았다. 11개 HCC 환자 샘플에서 EFNA1과 EPHA2의 발현을 보았다. 대부분의 HCC환자 조직(91%, 10 out of 11)에서 비 간암환자에서보다 EFNA1이 높게 발현되었다. 일부분의 HCC환자 조직(2 out 11)에서 비간암에서보다 EPHA2가 높게 발현되었다. AFP는 45%(5 out of 11) HCC환자 조직에서 과발현되었다. 이러한 데이터는 EFNA1이 AFP보다 생화학적 마커로 더욱 의미있다는 것을 알려준다. EFNA1 and EPHA2 were then analyzed in comparison to the expression of AFP in various hepatocellular carcinoma cells (see FIG. 8). Immunoblot analyses showed higher expression of EFNA1 in SK-Hep1, SH-J1, HLK-3, HKK2, and HLK-5 cells in Hep G2, Hep 3B, PLC / PRF / 5, and Huh7 cells. However, the majority of EPHA2 was overexpressed in Huh7, SK-Hep1 and SH-J1 cells. In particular, EPHA2 was overexpressed in SK-Hep1 and SH-1 cells, but EFNA1 and AFP were weakly expressed. Partial expression of this EPHA2 in liver cancer is in contrast to the expression of AFP and that of ENA1. We expressed p53 expression in HCC cells because it regulated EPHA2 by p53-family proteins. Expression of EFNA1 and EPHA2 was seen in eleven HCC patient samples. Most HCC tissues (91%, 10 out of 11) showed higher expression of EFNA1 than in non-hepatic cancer patients. In some HCC patient tissues (2 out 11), EPHA2 was expressed higher than in non-hepatic cancers. AFP was overexpressed in 45% (5 out of 11) HCC patient tissues. These data indicate that EFNA1 is more meaningful as a biochemical marker than AFP.
3. 정량화 및 통계학적 분석3. Quantification and Statistical Analysis
Northern hybridization의 autoradiograph는 LAS3000system(Fuji)을 이용하여 스캔하였고 densitometric data를 분석하였다. 각각의 암 조직과 비암 조직의 발현수준을 18s 발현과 대조하여 평균화 시킨 후 분석하였다. 그룹간의 EFNA1의 발현의 차이는 Mann-whitney test나 unpaired-test를 이용하여 확인했고 significance 의 수준은 P값이 0.05이하로 하였다.The autoradiograph of Northern hybridization was scanned using LAS3000system (Fuji) and analyzed for densitometric data. The expression levels of each cancerous and noncancerous tissue were compared with 18s expression and averaged for analysis. Differences in the expression of EFNA1 between groups were confirmed by Mann-whitney test or unpaired-test, and the significance level was set to P value less than 0.05.
<실시예5> EPHA2 재조합 유전자 제작과 EPHA2 재조합 유전자 과발현 세포주 확립Example 5 EPHA2 Recombinant Gene Preparation and EPHA2 Recombinant Gene Overexpression Cell Line Establishment
미국 ATCC사에서 구입한 간암세포주 HepG2를 37℃에서 5% CO2 상태하에 배양하였고, 배지는 10% FBS가 포함된 MEM 배지에 필수아미노산과 피루베이트(Pyruvate)를 가하여 사용하였다. Myc 표지 EPHA2를 각각 플라스미드로 pCDNA3.1/Myc 벡터 (Invitrogen, 미국)를 이용하여 제조하여 세포에 도입하였고, 대조실험으로는 공벡터(pCDNA3.1/Myc-HisA 벡터)만을 도입하였다. HepG2, a liver cancer cell line purchased from ATCC, was incubated at 37 ° C. under 5% CO 2 , and the medium was used by adding essential amino acid and pyruvate to MEM medium containing 10% FBS. Myc labeled EPHA2 was prepared using a pCDNA3.1 / Myc vector (Invitrogen, USA) as a plasmid, respectively, and introduced into cells, and only a blank vector (pCDNA3.1 / Myc-HisA vector) was introduced as a control experiment.
인간의 전장 EPHA2 유전자를 한국생명공학연구원 21C 프론티어 인간유전체은행에서 분양받아 이 유전자를 템플리트로 사용하여 HindIII 제한효소부위를 갖는 정방향 프라이머(서열번호 3: 5'-CCCAAGCTTCGCGGCATGGCGCTCCAGGCAGCCCG-3')과 XhoI 제한효소부위를 갖는 역방향 프라이머(서열번호 4: 5'-CCGCTCGAGGATGGGGATCCCCACAGTGTTCACCT-3')를 제조하고, 이를 이용하여 PCR로 증폭된 EPHA2 단백질을 코딩하는 cDNA를 제한효소 HindIII 및 Xho1으로 절단하여 젤에 전기영동하고 밴드를 절취하여 유전자 추출 키트(Accuprep gel purification kit; Bioneer, Daejeon, Korea)를 이용하여 용출시켜서 인서트로 사용하였다. 벡터 pcDNA3.1/Myc-HisA를 HindIII 및 SalI으로 절단하여 같은 방법으로 젤에 전기영동한 후 밴드를 절취하여 유전자 추출 키트(Accuprep gel purification kit)를 이용하여 용출시킨 다음 벡터로 사용하였다. Human full-length EPHA2 gene was obtained from 21C Frontier Human Genome Bank of Korea Research Institute of Bioscience and Biotechnology, and used as a template, a forward primer having a HindIII restriction enzyme site (SEQ ID NO: 3'5'-CCCAAGCTTCGCGGCATGGCGCTCCAGGCAGCCCG-3 ') and XhoI restriction enzyme A reverse primer (SEQ ID NO: 4: 5'-CCGCTCGAGGATGGGGATCCCCACAGTGTTCACCT-3 ') having a site was prepared, and cDNA encoding the EPHA2 protein amplified by PCR was cleaved with restriction enzymes HindIII and Xho1 and electrophoresed to the gel and banded. Was eluted using a gene extraction kit (Accuprep gel purification kit; Bioneer, Daejeon, Korea) was used as an insert. Vector pcDNA3.1 / Myc-HisA was cleaved with HindIII and SalI, electrophoresed in gel in the same manner, and the bands were cut and eluted using an Accuprep gel purification kit, which was then used as a vector.
라이게이션 키트을 이용하여 인서트를 벡터 프레임에 삽입하여 클로닝하고 대장균에 도입시켜 증폭시킨 후 플라스미드를 정제하였다. HepG2 간암세포가 6cm 디쉬에 70% 정도 찼을때 리포펙틴(Gibco, invitrogen사)을 이용하여 유전자를 도입하였다. 48시간 후에 G418을 함유한 선택 배지에서 2-3주간 배양하도록 하고 형성된 각 콜로니를 6웰 플레트에 옮기고 T25 플라스크에 옮겨 확장시킨 후 형태를 관찰하였다. Using the ligation kit, the insert was inserted into the vector frame, cloned, introduced into E. coli, amplified, and the plasmid was purified. When HepG2 liver cancer cells were about 70% filled in 6 cm dishes, genes were introduced using lipofectin (Gibco, invitrogen). After 48 hours, the cells were incubated for 2-3 weeks in a selection medium containing G418, and each colony formed was transferred to a 6-well plate, transferred to a T25 flask, expanded, and observed for morphology.
2주의 대조 세포주와 4주의 EPHA2 과발현 세포주를 분리하였는데 이들은 EPHA2 면역블롯에 의하여 EPHA2 단백 과발현이 확인되었다(도 9 참조). 공벡터(pCDNA3.1/Myc-HisA 벡터) 발현세포주 VC2 및 VC4 대조세포주에 비해, pCDNA3.1/Myc-HisA-EPHA2 벡터 도입 후 EPHA2 과발현이 관찰된 세포주 EPHA2 #7 및 #12를 확립하였다. 외형적으로는 변화가 없는 본 세포주는 장차 EPHA2 단백 발현이 간암세포주에 미치는 영향을 연구할 수 있는 재료가 되며, 장차 간암진단 및 치료의 분자생물학적 표적연구에 이용될 수 있을 것이다.Two control cell lines and four EPHA2 overexpressing cell lines were isolated, and EPHA2 protein overexpression was confirmed by EPHA2 immunoblot (see FIG. 9). Compared to the empty vector (pCDNA3.1 / Myc-HisA vector) expressing cell lines VC2 and VC4 control cell lines, cell lines EPHA2 # 7 and # 12 were observed after the introduction of the pCDNA3.1 / Myc-HisA-EPHA2 vector. The cell line, which is not changed in appearance, is a material for studying the effect of EPHA2 protein expression on hepatocellular carcinoma cell lines in the future, and may be used for molecular biological targets of liver cancer diagnosis and treatment in the future.
본 발명의 바이오 마커는 간세포암을 조기에 보다 효율적으로 검색할 수 있게 함으로써, 간암의 예방 및 진단에 도움을 주고 국내의 암 치료 및 예방에 기여하여 국민생활건강에 이바지 할 것으로 기대된다. The biomarkers of the present invention are expected to contribute to the prevention of diagnosis and diagnosis of hepatocellular carcinoma and to the prevention and diagnosis of hepatocellular carcinoma, and to contribute to the national health of life by contributing to the treatment and prevention of cancer in Korea.

Claims (9)

  1. Ephrine A1(EFNA1) 유전자 또는 상기 EFNA1 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 진단용 조성물.Ephrine A1 (EFNA1) gene or hepatocellular carcinoma diagnostic composition comprising a protein expressed from the EFNA1 gene.
  2. EFNA1의 수용체인 EPHA2 유전자 또는 상기 EPHA2 유전자로부터 발현되는 단백질을 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물. Hepatocellular carcinoma prognosis composition, characterized in that it comprises an EPHA2 gene or a protein expressed from the EPHA2 gene of the receptor of EFNA1.
  3. 제1항에 있어서, 상기 조성물은 EFNA1 유전자의 mRNA에 상보적인 센스 또는 안티센스 프라이머를 포함하는 것을 특징으로 하는 간세포암 진단용 조성물.The method of claim 1, wherein the composition is a composition for diagnosing hepatocellular carcinoma, characterized in that it comprises a sense or antisense primer complementary to the mRNA of the EFNA1 gene.
  4. 제2항에 있어서, 상기 조성물은 EPHA2 유전자의 mRNA에 상보적인 센스 또는 안티센스 프라이머를 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물.According to claim 2, wherein the composition is a hepatocellular carcinoma prognosis composition, characterized in that it comprises a sense or antisense primer complementary to the mRNA of the EPHA2 gene.
  5. 제1항에 있어서, 상기 조성물은 EFNA1 유전자로부터 발현되는 단백질을 특이적으로 인식하는 항체를 포함하는 것을 특징으로 하는 간세포암 진단용 조성물.The composition of claim 1, wherein the composition comprises an antibody that specifically recognizes a protein expressed from the EFNA1 gene.
  6. 제2항에 있어서, 상기 조성물은 EPHA2 유전자로부터 발현되는 단백질을 특이적으로 인식하는 항체를 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물.The composition of claim 2, wherein the composition comprises an antibody that specifically recognizes a protein expressed from an EPHA2 gene.
  7. 제1항에 있어서, 상기 조성물은 EFNA1 유전자로부터 발현되는 단백질의 발현을 억제하는 siRNA를 포함하는 것을 특징으로 하는 간세포암 진단용 조성물.The composition for diagnosing hepatocellular carcinoma of claim 1, wherein the composition comprises siRNA that inhibits expression of a protein expressed from the EFNA1 gene.
  8. 제2항에 있어서, 상기 조성물은 EPHA2 유전자로부터 발현되는 단백질의 발현을 억제하는 siRNA를 포함하는 것을 특징으로 하는 간세포암 예후결정용 조성물.According to claim 2, wherein the composition is a composition for hepatocellular carcinoma prognosis characterized in that it comprises siRNA that inhibits the expression of the protein expressed from the EPHA2 gene.
  9. 제1항 내지 제8항 중에서 선택된 어느 하나의 조성물을 포함하는 간세포암 진단키트.Hepatocyte cancer diagnostic kit comprising any one of the compositions selected from claim 1 to claim 9.
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