WO2017146530A1 - Composition de diagnostic du carcinome à cellules rénales et procédé de détection d'un marqueur diagnostique - Google Patents

Composition de diagnostic du carcinome à cellules rénales et procédé de détection d'un marqueur diagnostique Download PDF

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WO2017146530A1
WO2017146530A1 PCT/KR2017/002082 KR2017002082W WO2017146530A1 WO 2017146530 A1 WO2017146530 A1 WO 2017146530A1 KR 2017002082 W KR2017002082 W KR 2017002082W WO 2017146530 A1 WO2017146530 A1 WO 2017146530A1
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protein
expression level
grs
mrna
hrs
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PCT/KR2017/002082
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Korean (ko)
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김성훈
박민철
찰스 고흐너 피터
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재단법인 의약바이오컨버젼스연구단
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Publication of WO2017146530A1 publication Critical patent/WO2017146530A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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

Definitions

  • Kidney cancer diagnostic composition and diagnostic marker detection method Kidney cancer diagnostic composition and diagnostic marker detection method
  • GRS glycyl-tRNA synthetase
  • AIMPl aminoacyl-tRNA synthetase.
  • a composition for diagnosing kidney cancer comprising an agent for measuring the expression level of one or more mRNAs selected from the group consisting of, obtained from a subject to provide information necessary for diagnosing kidney cancer
  • GRS glycyl-tRNA synthetase
  • HRS hi st idyl-tRNA synthetase
  • aminoacyl-tRNA synthetase complex ⁇
  • AIMPl aminoacyl-tRNA synthetase complex
  • the number of cancer (malignant neoplasm) deaths in Korea was 62,887, which was 25.5% (29.6% of male deaths and 20.5% of female deaths) out of 246,515 deaths in Korea (512 deaths per 100,000 population). Cancer deaths (130.7 deaths per 100,000 population) are the leading cause of death.
  • the cancer mortality order is lung cancer, stomach cancer, liver cancer, colon cancer, and pancreatic cancer. The deaths of these five cancers account for about 70% of all cancer deaths.
  • the main cause of cancer deaths in men is lung cancer, stomach cancer, liver cancer and colorectal cancer.
  • the number of deaths from these four cancers (28, 147) accounts for 70% of the total number of deaths from men (40, 177).
  • the main cause of cancer deaths in women is gastric cancer, lung cancer, liver cancer, colon cancer and pancreatic cancer.
  • the number of deaths from these five cancers (13, 630) is the total number of female cancer deaths (22, 710). Accounted for 60% of the total.
  • Kidney cancer rarely causes symptoms when the tumor is small, The symptoms do not appear until they are big enough to push the organs out. Therefore, the diagnosis is often delayed, and when the first diagnosis is made, about 30% of the patients are already metastasized.
  • the most common symptom is hematur ia, but this only occurs in 60% of patients. Rather, according to the transition area shortness of breath, cough i, because this transition is shown symptoms such as headache symptoms are diagnosed with kidney cancer case amounts to 30% of patients.
  • Kidney cancers can cause high blood pressure, hypercalemia, and liver dysfunction, especially because of certain hormones produced by cancer cells. Recently, however, many cases have been discovered by imaging during accidental medical examination without any symptoms. These cases are mainly found early, so the treatment results are relatively good. Kidney cancer accounts for about 3% of adult cancers in the United States, with approximately 32,000 new cases per year. In addition, about 12, 000 people are estimated to die from kidney cancer, and the frequency is increasing every year worldwide. In Korea, the incidence rate is lower than 1, and according to 2002 Central Cancer Registration Data, 578 patients were newly registered. It accounts for 1.6% of cancer cases. In particular, men are twice as likely as women, and in 2002, 1,104 patients occurred in men, indicating the long-term enrollment rate of men's cancer 2.03 ⁇ 4>.
  • Kidney cancer is most common in people in their 40s and 60s, and the most common is in their 60s (479, 30.2%), and in their 50s (412 people, 26.0>). (268 people, 16.9%) in that order. Therefore, it can be said that the development of marker for early diagnosis of kidney cancer is very important.
  • the present inventors have made efforts to develop a biomarker that can effectively diagnose kidney cancer. As a result, the present inventors have a simple and rapid detection in the serum of kidney cancer patients. A biomarker capable of high ability, high sensitivity and specificity was discovered and the present invention was completed.
  • an object of the present invention is one or more mRNAs selected from the group consisting of GRS (glycyl-tRNA synthetase), HRSChi st idyl-tRNA synthetase (HRSC), and AIMPKaminoacyl-tRNA synthetase ramp lex® inter acting mult i funct ional Protein 1)
  • GRS glycyl-tRNA synthetase
  • Another object of the invention is the eu air and selected from the group consisting of the GRS, HRS and AIMP1 - Do - or _ more mRNA is eu protein-of-for a _ first yo claim for measuring the expression ⁇ level renal cancer To provide a diagnostic kit. Another object of the present invention to provide information necessary for diagnosing kidney cancer,
  • Another object of the present invention is to prepare a glycyl-tRNA synthetase (GRS), hi st idyl-tRNA synthetase (HRS) and aminoacyl-tRNA synthetase com lex-interacting mul ti funct ional Protein 1) To provide a use of the agent for measuring the expression level of one or more mRNAs or proteins thereof selected from the group consisting of. .
  • the present invention is GRS (glycyl-tRNA synthetase), HRS (hi st idyl-tRNA synthetase) and AIMPl (aminoacyl-tRNA synthetase complex-inter acting mul ti funct ional Protein 1)
  • GRS glycyl-tRNA synthetase
  • HRS hi st idyl-tRNA synthetase
  • AIMPl aminoacyl-tRNA synthetase complex-inter acting mul ti funct ional Protein 1
  • the present invention provides a kit for diagnosing kidney cancer comprising an agent for measuring the expression level of at least one mRNA or protein selected from the group consisting of GRS, HRS and AIMP1.
  • the present invention provides information necessary for diagnosing kidney cancer,
  • GRS glycosyl-tRNA synthetase
  • HRS HRSChi st idyl-tRNA synthetase
  • AIMPKarainoacyl-tRNA synthetase comp 1 ex-interacting mul ti funct ional Protein for the preparation of a diagnostic agent for kidney cancer It is to provide a use of the agent for measuring the expression level of one or more mRNAs or proteins thereof selected from the group consisting of 1).
  • HRS HRSChi st idyl-tRNA synthetase
  • AIMPKarainoacyl-tRNA synthetase comp 1 ex-interacting mul ti funct ional Protein for the preparation of a diagnostic agent for kidney cancer It is to provide a use of the agent for measuring the expression level of one or more mRNAs or proteins thereof selected from the group consisting of 1).
  • the present invention will be described in detail.
  • the present invention measures the expression level of one or more mRNAs or proteins thereof selected from the group consisting of GRS (glycyl-tRNA synthetase), HRSChi st idyl-tRNA synthetase (HRS), and AIMPKaminoacyl-tRNA synthetase complex-interacting mul ti funct ional Protein 1) It provides a composition for diagnosing kidney cancer comprising a formulation.
  • GRS glycyl-tRNA synthetase
  • GRS glycyl-tRNA synthetase
  • HRSChi st idyl-tRNA synthetase HRSChi st idyl-tRNA synthetase
  • AIMPl aminoacyl eu tRNA synthetase complex-interact ing mul ti funct ional Protein 1
  • GRS glycosyl-tRNA synthetase
  • HRS hi st idyl-tRNA synthetase
  • AIMPl aminoacyl-tRNA synthetase complex-interacting multifunctional Protein 1
  • the present invention relates to the expression levels of GRS (glycyl-tRNA synthetase), HRS (hi st idyl-tRNA synthetase) and AIMPl (aminoacyl-tRNA synthetase com lex-interacting multifunctional Protein 1), ie GRS (glycyl) -tRNA synthetase), HRS (histidyl- tRNA synthetase) and AIMPl (aminoacyl-tRNA synthetase eomp 1 ex- i-nt erae ti ng mul-t-ifunet onal Protein- 1) protein 'quality - the mRNA level i ⁇ - or —Provide a composition for diagnosing kidney cancer comprising the agent to be measured.
  • GRS glycyl-tRNA synthetase
  • HRS hi st idyl-tRNA synthe
  • Aminoacyl-tRNA synthetase is an enzyme that attaches a specific amino acid to its corresponding tRNA.
  • Higher organisms consist of 23 enzymes, including 3 types of enzymes involved in the formation of multi-synthetase complexes, including AIMPl (p43), (AIMP2) p38, and (AIMP3) pl8.
  • AIMPl p43
  • AIMP2 p38
  • AIMP3 AIMP3
  • GRS has recently been reported to promote the apoptosis of certain cancer cells by secreting from macrophages (.MC Park, et al. (2012) Secreted human glycyl-tRNA synthetase implicated in defense against ERK-act ivated tumor i genesis.PNAS109 (ll): E640-7)
  • Fas ligand secreted by Fas ligand in the presence of cancer cells was found to induce cell death by inhibiting CDH6 and ERK activity in certain cancer cells.
  • autoimmune antibodies against GRS exist in autoimmune diseases, breast cancer cells (Mun J, et al.
  • GRS can be used as a marker for diagnosing renal cancer because the protein level is significantly higher than that in the serum of renal cancer patients.
  • HRS has been reported to be the most common autoantigen in autoimmune diseases including myositis and promotes the migration of lymphocytes (CD4 + and CD8 + lymphocyte) monocytes (IL-2 activated monocytes) and immature dendritic eel Is. Is a protein that has been reported to exhibit chemoaxis activity (Howard,
  • AIMPKARS-interacting multi-functional protein 1 previously known as the p43 protein, is a protein recently renamed AIMP1 (Sang Gyu Park, et al., Trends in Biochemical Sciences, 30: 569-574, 2005).
  • AIMPl is a protein consisting of 312 amino acids, which binds to a multiple t-RNA synthetase complex (Deutscher, MP, Method Enzymol, 29, 577-583, 1974; Dang CV et al., Int. J. Biochem. 14, 539—543, 1982; Mirande, M. et al., EMBO J.
  • AIMP1 is a protein that enhances the catalytic activity of the multiple -t.RNA synthetase (Park SG et al., J. Biol. Chem. 274, 16673-16676, 1999) .
  • Secreted AIMPl is known to act on a variety of target cells such as monocytes / macrophages, endothelial cells and fibroblasts.
  • target cells such as monocytes / macrophages, endothelial cells and fibroblasts.
  • AIMP1 can be used as a marker for diagnosing renal cancer because the protein level is significantly higher than that in the serum of renal cancer patients, which is normal, and is disclosed for the first time in the present invention.
  • diagnostic marker is a material that can diagnose a kidney cancer patient from a normal control group, and increases or decreases a patient with kidney cancer compared to a normal control group.
  • Organic biomolecules such as reduced polypeptides or nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.).
  • the kidney cancer diagnostic markers of the present invention are GRS (glycyl-tRNA synthetase), HRS (histidyl-tRNA synthetase), and HRS, which show high levels of expression in cancer cells compared to cells of normal gastric tissue.
  • GRS glycyl-tRNA synthetase
  • HRS histidyl-tRNA synthetase
  • HRS which show high levels of expression in cancer cells compared to cells of normal gastric tissue.
  • AIMPl aminoacyl-tRNA synthetase complex-interacting multifunctional Protein 1
  • Expression in the present invention 'expression' means that the protein or nucleic acid is produced in the cell. Proteins are used interchangeably with polypeptides or peptide ides and refer to polymers of amino acid residues, for example as commonly found in proteins in nature.
  • Polynucleotide or nucleic acid refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-strands. Unless otherwise limited, known analogues of natural nucleotides that are localized to nucleic acids in a manner similar to naturally occurring nucleotides are also included. 'MRNA' is RNA that transfers genetic information (gene-specific nucleotide sequences) to ribosomes that specify amino acid sequences from specific genes during protein synthesis. "Diagnosis means to determine the presence or characteristics of the pathology.
  • Diagnosis in the present invention is to determine the presence or pathology of kidney cancer by measuring the expression level of any one or more selected from the group consisting of GRS, HRS and AIMP1, that is, the protein or mRNA of one or more of the markers will be.
  • the agent for measuring mRNA expression level is a probe or primer specifically binding to any one or more mRNA selected from the group consisting of GRS, HRS and AIMP1 May be a set.
  • the GRS, HRS and AIMP1 mRNA may be derived from a mammal including a human, preferably, the mRNA of GRS is SEQ ID NO 1, the HRS mRNA is SEQ ID NO 2 and the AIMP1 mRNA is represented by SEQ ID NO 3 It may include a base sequence.
  • the diagnostic composition of the present invention comprising at least one mRNA specific probe or primer set selected from the group consisting of GRS, HRS and AIMP1 as an agent for measuring the expression level of any one or more selected from the group consisting of GRS, HRS and AIMP1. May further comprise agents required for methods of detecting known RNA.
  • the present composition can be used to measure the level of mRNA of the markers in a subject using a known method for detecting RA.
  • a primer is a single strand ol igonucleotide that acts as a starting point for DNA synthesis.
  • the primer specifically binds to a polynucleotide that is a template at appropriate buffer and temperature conditions, and the DNA polymerase is linked to the primer by adding nucleoside triphosphate having a base complementary to the template DNA. DNA is synthesized by this.
  • the primer is generally composed of 15 to 30 base sequences, and the melting temperature (TJ) varies depending on the base composition and length of the template strand.
  • the sequence of the primer does not need to have a sequence that is completely complementary to some nucleotide sequences of the template, and is sufficient as long as it has sufficient complementarity within a range that can be hybridized with the template to perform a primer-specific function. Therefore, in the present invention, the primers for measuring the mRNA expression level of the respective markers do not need to have a sequence completely complementary to each gene sequence, and amplify a specific section of the mRNA or cDNA through DNA synthesis. If there is a length and complementarity suitable for the purpose of measuring the quantity, it is divided into layers.
  • the primer for amplification reaction consists of a set (pair) of complementary binding to the template (or sense) and the opposite (antisense, ant isense) at each end of the specific section of the mRNA to be amplified.
  • Primers can be easily designed by those skilled in the art by referring to mRNA or cDNA sequences of GRS, HRS and AIMP1.
  • the primer of the present invention is preferably one set specifically binding to the GRS mRNA sequence represented by SEQ ID NO: 1, the HRS mRNA sequence represented by SEQ ID NO: 2, the AIMP1 mRNA sequence represented by SEQ ID NO: 3, It may be a pair or a combination thereof, most preferably in the forward primer selected from the group consisting of SEQ ID NO: 7, 8 and 9 and the reverse primer selected from the group consisting of SEQ ID NO: 10, 11 and 12, respectively One or more may be selected, but is not limited thereto.
  • SEQ ID NOs: 7 and 10 are primers specific for the GRS mRNA sequence
  • SEQ ID NOs: 8 and 11 are primers specific for the HRS mRNA sequence
  • SEQ ID NOs: 9 and 12 are primers specific for the AIMP1 mRNA sequence .
  • RNA or DNA fragments of polynucleotides, such as RNA or DNA, ranging from short to several hundred bases in length that can specifically bind mRNA or cDNA (complementary DNA) of specific genes. And it's labeled The presence or absence of mRNA or cDNA to bind to, and the amount of expression can be confirmed.
  • a probe complementary to GRS, HRS or AIMP1 mRNA is used to perform a hybridization reaction with a sample of a subject to measure the expression level of GRS, HRS or AIMP1 mRNA to diagnose kidney cancer. It is available. The choice and probe conditions of the probe can be appropriately selected according to techniques known in the art.
  • primers or probes of the present invention can be synthesized chemically using phosphoramidite solid support synthesis or other well known methods.
  • primers or probes can be variously modified according to methods known in the art within a range that does not interfere with the hybridization with GRS, HRS or AIMP1 mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides and modifications between nucleotides, for example, uncharged linkages (e.g., methyl phosphonates, phosphoesters, phosphoramidates, carbamates, etc.).
  • the agent for measuring the protein expression level may be an antibody that specifically binds to GRS, HRS or AIMP1 protein, respectively.
  • the GRS, HRS and AIMP1 proteins may be derived from mammals including humans.
  • the GRS protein includes SEQ ID NO. 4
  • the HRS protein includes SEQ ID NO. 5
  • the AIMP1 protein includes SEQ ID NO. It may be.
  • Antibody antibody refers to an immunoglobulin (i ⁇ unoglobul in) that specifically binds to the antigenic site.
  • the antibody in the present invention is an antibody that specifically binds only to GRS, HRS or AIMP1 protein, without reacting to other proteins including other types of aminoacyl thiA-N synthase other than GRS, HRS or AIMP1.
  • GRS, HRS or AIMP1 antibodies can be prepared by cloning each gene into an expression vector to obtain a protein that is encoded by the gene, and from the obtained protein according to conventional methods in the art.
  • Each protein specific antibody may also be prepared using fragments of GRS, HRS or AIMP1 proteins comprising GRS, HRS or AIMP1 antigenic sites. have.
  • the form of the antibody of the present invention is not particularly limited and includes a polyclonal ant ibody or a monoclonal antibody.
  • a part of the whole antibody is included in the antibody of the present invention as long as it has antigen-antibody binding, and includes all kinds of immunoglobulin antibodies that specifically bind to GRS, HRS or AIMP1.
  • the antibodies of the present invention also include special antibodies and recombinant antibodies such as humanized antibodies and chimeric antibodies as long as they can specifically bind to GRS, HRS or AIMP1 protein.
  • the diagnostic composition of the present invention comprising each of the marker protein specific antibodies as an agent for measuring the expression level of GRS, HRS or AIMP1 may further include an agent necessary for a method for detecting a known protein, and using the present composition By using a method of detecting a known protein without limitation, the level of one or more proteins selected from the group consisting of GRS, HRS or AIMP1 in the subject can be measured.
  • the present invention provides a kit for diagnosing kidney cancer comprising an agent for measuring the expression level of any one or more mRNA or protein selected from the group consisting of GRS, HRS and AIMP1.
  • the diagnostic kit of the present invention includes a primer for recognizing one or more mRNAs selected from the group consisting of GRS, HRS and AIMP1 as a marker or at least one mRNA selected from the group consisting of GRS, HRS and AIMP1 as a marker, Probes as well as one or more other component compositions, solutions or devices suitable for analytical methods can be included.
  • the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing reverse transcription polymerase reaction.
  • the reverse transcriptase polymerase kit includes each primer pair specific for the marker gene.
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific for the nucleic acid sequence of the control gene.
  • Extra-transcriptase polymerase reaction kits can be used in test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (which may include DEPOwater sterilized water, etc.)
  • the kit may be a diagnostic kit characterized by including the necessary elements necessary to carry out the DNA chip. Substrates to which the corresponding cDNA or oligonucleotide is attached, and reagents, preparations, enzymes, etc., for the manufacture of fluorescence-labeled probes.
  • ELISA kits include specific antibodies to marker proteins, which are antibodies with high specificity and affinity for each marker protein and little cross-reactivity to other proteins.
  • the ELISA kit may comprise an antibody specific for the control protein, and the ELISA kit may contain a reagent capable of detecting the bound antibody, for example, labeled 2.
  • the kits of the present invention may also include enzymes and substrates to develop reactions.
  • unbound proteins, etc. can include washes or eluents that can retain only bound protein markers.
  • biological samples capable of identifying infectious inflammatory disease specific proteins that can be distinguished from normal conditions of blood, serum, urine, lacrimal fluid and saliva, preferably biological liquid samples, such as blood, serum, plasma Can be measured from Samples may be prepared to increase the detection sensitivity of protein markers, for example serum samples obtained from patients may be anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous It may be pretreated using methods such as sequential ial extract ion or gel electrophoresis. To provide you with the information you need to diagnose kidney cancer,
  • Step (c) comparing the expression level of each gene mRNA or the expression level of the protein with the expression level or protein expression level of the corresponding gene mRNA in the normal control sample to determine that the subject with increased expression level has kidney cancer; It provides a method for detecting a marker of renal cancer comprising.
  • GRS, HR-S, and MMP1- could be key as a standard marker of cabin cancer, and we measured the expression levels of each of these markers to help diagnose kidney cancer. It provides a way to provide information.
  • Step (a) of the method of the present invention is a step of providing a sample of a subject.
  • the sample may be used without limitation as long as it is collected from a subject to diagnose kidney cancer, for example, a cell or tissue obtained by a biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various secretions, urine, Feces and the like.
  • Step (b) of the method of the present invention is the step of measuring at least one expression level selected from the group consisting of GRS, HRS and AIMP1 in the sample provided in step (a).
  • the expression level may be the expression level of one or more mRNAs or proteins selected from the group consisting of GRS, HRS and AIMP1.
  • the expression level of each protein can be detected or measured using an antibody that specifically binds to each protein. Protein specific antibodies are as described in the diagnostic composition of the present invention.
  • Methods for measuring the expression level of the protein can be used without limitation methods known in the art, such as Western blotting blotting, dot blotting, enzyme immunosorbent assay (EL ISA), radioimmunoassay (RIA), radioimmunoassay, ukuteroni immunodiffusion, rocket immunoelectrophoresis, immune tissue Chemical staining, immunoprecipitation, complement fixation assay, flow cytometry (FACS) or protein chip method, etc., but are not limited to these.
  • an ELISA method can be used.
  • the mRNA level of each marker was determined by amplifying the mRNA or cDNA of each marker from the subject's sample using a primer set or probe that specifically binds to the mRNA, or by using probes and hybridization. The presence and expression level of mRNA of each marker can be measured. Primers and probes are as described in the diagnostic composition of the present invention.
  • Determination of mRNA expression level can be used without limitation the expression level determination method conventional in the art, and examples of analytical methods are reverse transcription polymerase chain react ion (RT-PCR), competitive RT_PCR (competitive RT) -PCR), real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA microarray chip, RNA sequencing ( RNA sequencing, nanostringing method, and in situ hybridization method (//? Situ hybridization) of tissue sections, but are not limited to these.
  • RT-PCR reverse transcription polymerase chain react ion
  • competitive RT_PCR competitive RT
  • RPA RNase protection assay
  • Northern blotting DNA microarray chip
  • RNA sequencing RNA sequencing, nanostringing method, and in situ hybridization method (//? Situ hybridization) of tissue sections, but are not limited to these.
  • Step (c) of the method of the present invention compares the level of one or more mRNAs or proteins selected from the group consisting of GRS, HRS and AIMP1 of the subject sample measured in step (b) with a normal person and its expression level compared to a normal person. It is a step which determines that this increased test subject has kidney cancer.
  • the expression level of each marker of the subject measured by the method of step (b) described above is compared with the marker level of a normal person measured by the same method. Subjects with increased levels of expression of each marker compared to healthy subjects are determined to have kidney cancer.
  • the invention also
  • the present invention may be useful for screening kidney cancer therapeutic agents by comparing the increase or decrease of expression of one or more mRNAs or proteins selected from the group consisting of GRS, KRS, and AIMP1 in the presence and absence of a candidate candidate for treating cancer.
  • a substance that indirectly or directly reduces the expression level of one or more mRNAs or proteins selected from the group consisting of GRS, HRS and AIMP1 may be selected as a therapeutic agent for kidney cancer.
  • a substance which reduces the expression level of the marker of the present invention when the complement substance is present after the renal cancer treatment is lower than the marker expression level under the absence of the candidate substance for renal cancer treatment may be selected as a therapeutic agent for renal cancer.
  • the 'anticancer activity' of the present invention means inhibiting abnormal increase in cell division, conversion from normal cells to cancer cells, cell division and proliferation of cancer cells, tumor growth and growth.
  • the 'cell or animal' of the present invention is a cell or animal of a cancer or tumor model, and is commonly used in the art, and may be a cell, tissue, organ or the like derived from a mammal, including a human.
  • the invention is selected from the group consisting of GRS (glycyl-tRNA synthetase), HRS (hist idyl-tR A synthetase) and AIMPKaminoacyl-tRNA synthetase complex—interacting mult i funct ional Protein 1) for the preparation of a diagnostic agent for kidney cancer
  • GRS glycyl-tRNA synthetase
  • HRS hist idyl-tR A synthetase
  • an agent to measure the expression level of one or more mRNAs or proteins thereof is a probe or primer set that specifically
  • the agent for measuring the expression level of the protein of the present invention may be an antibody specific for one or more proteins selected from the group consisting of GRS, HRS and AIMP1, as described above.
  • the mRNA of the GRS of the present invention is SEQ ID NO: 1
  • the mRNA of HRS may be a nucleotide sequence represented by SEQ ID NO: 2 and AIMP1 of SEQ ID NO: 3
  • the GRS protein of the present invention is SEQ ID NO: 4
  • the HRS protein may be one comprising the amino acid sequence represented by SEQ ID NO: 5 and the AIMP1 protein represented by SEQ ID NO: 6, as described above.
  • the present invention can provide the use of an agent for measuring the expression level of one or more mRNA or protein selected from the group consisting of GRS, HRS and AIMP1 for preparing a kit for diagnosing kidney cancer.
  • the kit of the present invention may be an RT-PCR kit, a DNA chip kit or a protein chip kit, but is not limited thereto.
  • Renal cancer diagnostic markers of the present invention consisting of GRS, H S and AIMP1 have increased expression levels in serum of kidney cancer patients compared to normal controls.
  • GRS GRS
  • HRS HRS
  • AIMP1 AIMP1
  • FIG. 1 is a dot blot showing the serum protein levels of the normal and renal cancer patients (A: GRS, B: RS, C: AIMPl, D: HRS, E: WRS, F: TNF- ⁇ , G: IL-10)
  • Figure 2 is the result of confirming the serum GRS protein level according to gender.
  • 3 shows the R0C curve of serum protein levels.
  • Serum from kidney cancer patients was obtained from Samsung Medical Center (Seoul, Korea) according to the regulations of the Institutional Review Board. 32 samples of normal patients and 100 samples of kidney cancer patients were analyzed.
  • Glycyl-tRNA synthetase secreted into the serum of normal or kidney cancer patients Lysyl-tRNA synthetase (KRS), hist idyl-tRNA synthetase (HRS), tryptophanyl-tRNA synthetase (WRS), AIMP ⁇ l (aminoacyl ⁇ tRNA synthetase complex-interacting multifunctional Protein 1), TNF— ⁇ , IL-10, and CA Levels of -19-9 were analyzed using the enzyme immunoassay kit according to the manufacturer's instructions. Protein secretion was measured using a microplate reader (TECAN). Kits for measuring the respective protein levels were purchased from the following manufacturers:
  • TNF- a TNF- a
  • IL-10 BD science, USA
  • the P value between blood serum secreted proteins in normal and renal cancer patients was analyzed by XLASTAT software using the Mann-Whitney test / Two-tailed test. Dotblot plots, ROC curves, AUC, and standard deviations were analyzed using Graphpad Prism 6 software.
  • Table 2 and FIG. 1-as shown in Figure 1 loading-in the intestinal cancer patients and serum-GRS (glycyl-tRNA synthetase), HRS (histidyl-tRNA synthetase) and AIMPl (aminoacyl-tRNA synthetase com
  • serum-GRS glycyl-tRNA synthetase
  • HRS histidyl-tRNA synthetase
  • AIMPl aminoacyl-tRNA synthetase com
  • Example 1 the GRS protein levels, which showed the largest difference in serum levels in the normal group and the kidney cancer group, were analyzed according to gender. The results are shown in Table 3 and FIG. 2.
  • the AUC of R0C of GRS, AIMP1, and HRS is greater than 0.6, and the p value is less than 0.01, which is statistically significant since the value in serum of kidney cancer patients is significantly larger than that of normal persons. It was found that the biomarker. In addition, GRS was very good as a biomarker for male kidney cancer.
  • Renal cancer diagnostic marker of the present invention consisting of GRS, HRS and AIMP1 is increased in the expression level in the serum of kidney cancer patients compared to the normal control. Therefore, by measuring the expression level of one or more markers selected from the group consisting of GRS, HRS and AIMP1 can accurately and quickly determine the presence of kidney cancer, the industrial availability is very excellent.

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Abstract

La présente invention concerne une composition de diagnostic du carcinome à cellules rénales et un procédé de détection d'un marqueur diagnostique, plus spécifiquement une composition de diagnostic du carcinome à cellules rénales comprenant un ou plusieurs ARNm choisis dans le groupe constitué par la glysyl-ARNt synthétase (GRS), l'histidyl-ARNt synthétase (HRS) et une protéine multifonctionnelle 1 interagissant avec un complexe aminoacyl-ARNt synthétase (AIMP1) ou une préparation permettant de mesurer les niveaux d'expression protéiques de celles-ci, et un procédé de détection d'un marqueur à partir d'un échantillon provenant d'un sujet testé pour obtenir les informations nécessaires au diagnostic du carcinome à cellules rénales. Le marqueur diagnostique du carcinome à cellules rénales comprenant GRS, HRS et AIMP1 selon la présente invention a des niveaux d'expression en hausse dans le sérum d'un patient atteint d'un carcinome à cellules rénales comparativement à un groupe comparatif normal. Par conséquent, la présence du carcinome à cellules rénales peut être déterminée précisément et rapidement par mesure des niveaux d'expression d'un ou de plusieurs marqueurs choisis dans le groupe constitué par GRS, HRS et AIMP1.
PCT/KR2017/002082 2016-02-25 2017-02-24 Composition de diagnostic du carcinome à cellules rénales et procédé de détection d'un marqueur diagnostique WO2017146530A1 (fr)

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KR101968046B1 (ko) * 2018-07-19 2019-04-11 (주) 바이오인프라생명과학 암의 조기 진단을 위한 복합 바이오마커
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KR20150077749A (ko) * 2013-12-30 2015-07-08 재단법인 의약바이오컨버젼스연구단 항 grs 모노클로날 항체 및 이의 용도
KR20150078472A (ko) * 2013-12-30 2015-07-08 재단법인 의약바이오컨버젼스연구단 항 AIMP1/p43 모노클로날 항체 및 이의 용도
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