[go: up one dir, main page]

WO2023144708A1 - Nouveau biomarqueur pour le diagnostic du cancer de l'ovaire - Google Patents

Nouveau biomarqueur pour le diagnostic du cancer de l'ovaire Download PDF

Info

Publication number
WO2023144708A1
WO2023144708A1 PCT/IB2023/050614 IB2023050614W WO2023144708A1 WO 2023144708 A1 WO2023144708 A1 WO 2023144708A1 IB 2023050614 W IB2023050614 W IB 2023050614W WO 2023144708 A1 WO2023144708 A1 WO 2023144708A1
Authority
WO
WIPO (PCT)
Prior art keywords
ovarian cancer
protein
expression level
gene
present
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2023/050614
Other languages
English (en)
Korean (ko)
Inventor
한승만
노동영
유명희
강운범
허성현
정현훈
김세익
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bertis Inc
Original Assignee
Bertis Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bertis Inc filed Critical Bertis Inc
Publication of WO2023144708A1 publication Critical patent/WO2023144708A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • 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
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for preventing or treating ovarian cancer by measuring the expression level of a factor specifically regulated in ovarian cancer to predict onset or regulating the expression of the factor.
  • Ovarian cancer is the most lethal gynecological cancer and accounts for 5% of all cancer-related deaths in women worldwide. Because ovarian cancer has no symptoms specific to ovarian cancer and no effective screening tools are available, most OC patients are diagnosed at a fairly advanced stage with a 5-year survival rate of less than 30%. However, if ovarian cancer can be diagnosed at an early stage, this 5-year survival rate can be improved by up to 90%.
  • High-grade serous ovarian cancer is the most common histologic type, accounting for 70% of all OC cases.
  • HGSOC is the most aggressive type of ovarian cancer, and is often diagnosed at a fairly advanced stage due to the rapid growth of the tumor.
  • the overall survival rate of HGSOC patients has hardly improved for decades, and accordingly, there is a great clinical demand for early diagnosis of HGSOC with high accuracy.
  • a highly reliable biomarker that can be used to accurately diagnose HGSOC with high sensitivity and specificity has not yet been discovered.
  • MS Mass spectrometry
  • DIA Data Independent Acquisition
  • DIA is based on the fragmentation of all precursor ions in a large isolation window over the entire range of mass-to-charge values (m/z), which is a prior art data-dependent acquisition (DDA) method.
  • DDA data-dependent acquisition
  • the present inventors analyzed the serum proteome using DIA-based mass spectrometry to discover an efficient diagnostic biomarker for ovarian cancer, through which ovarian cancer-specific blood in the blood that can diagnose ovarian cancer with high accuracy
  • This study aimed to discover potential biomarkers and confirm their effects.
  • the present inventors discovered an efficient diagnostic biomarker for ovarian cancer, which has the poorest prognosis among gynecological cancers but has a low survival rate due to the absence of early diagnosis methods, and ultimately, the mortality rate due to ovarian cancer, especially high-grade serous ovarian cancer.
  • an efficient diagnostic biomarker for ovarian cancer which has the poorest prognosis among gynecological cancers but has a low survival rate due to the absence of early diagnosis methods, and ultimately, the mortality rate due to ovarian cancer, especially high-grade serous ovarian cancer.
  • proteins that are specifically up- or down-regulated in ovarian cancer patients or genes encoding them were discovered, and by measuring the expression levels of these markers, ovarian cancer can be diagnosed at an early stage with high accuracy, as well as using blood
  • the present invention was completed by discovering that it is a highly reliable marker that can also be applied to a mass spectrometry diagnosis method.
  • an object of the present invention is to provide a composition for diagnosing ovarian cancer.
  • Another object of the present invention is to provide a method for providing information necessary for the diagnosis of ovarian cancer.
  • Another object of the present invention is to provide a composition for preventing or treating ovarian cancer.
  • Another object of the present invention is to provide a screening method for a composition for preventing or treating ovarian cancer.
  • the present invention is an ovarian cancer comprising, as an active ingredient, an agent for measuring the expression level of one or more genes selected from the group consisting of FGA , VWF , ARHGDIB and SERPINF2 or proteins encoded by them. ) Provides a composition for diagnosis.
  • the present inventors discovered an efficient diagnostic biomarker for ovarian cancer, which has the poorest prognosis among gynecological cancers but has a low survival rate due to the absence of early diagnosis methods, and ultimately, the mortality rate due to ovarian cancer, especially high-grade serous ovarian cancer.
  • an efficient diagnostic biomarker for ovarian cancer which has the poorest prognosis among gynecological cancers but has a low survival rate due to the absence of early diagnosis methods, and ultimately, the mortality rate due to ovarian cancer, especially high-grade serous ovarian cancer.
  • proteins that are specifically up- or down-regulated in ovarian cancer patients or genes encoding them were discovered, and by measuring the expression levels of these markers, ovarian cancer can be diagnosed at an early stage with high accuracy, as well as using blood
  • the present invention was completed by discovering that it is a highly reliable marker that can also be applied to a mass spectrometry diagnosis method.
  • FGA Fiinogen alpha chain protein
  • VWF is the name of a gene encoding the von Willebrand Factor protein in humans.
  • the aforementioned von Willebrand factor protein may be referred to as VWF, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the corresponding protein.
  • ARHGDIB is the name of a gene encoding Rho GDP-dissociation inhibitor 2 protein in humans.
  • Rho GDP-dissociation inhibitor 2 protein described above may be referred to as RhoGD12, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the protein.
  • SERPINF2 is the name of a gene encoding the alpha 2-antiplasmin protein in humans.
  • the above-mentioned alpha 2-antiplasmin protein may be referred to as A2AP, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • genes used herein can be used as biomarkers for diagnosis of ovarian cancer independently or in combination of two or more, and when used in combination, a set of corresponding genes can be a “biomarker panel”.
  • biomarker panel may also be referred to as “biomarker detection panel”, and detection; Diagnosis; judgment of prognosis; diagnosis of stage; or a set of two or more biomarkers that can be used to monitor a disease or condition.
  • the biomarker components of this biomarker set may be packaged together or physically linked by reversibly or irreversibly binding to a solid support.
  • the biomarker panel of the present invention may be provided through a separate tube sold or shipped together as part of a kit;
  • diagnosis includes determination of a subject's susceptibility to a particular disease, determination of whether a subject currently has a particular disease, and determination of the prognosis of a subject suffering from a particular disease. do.
  • the term “diagnostic composition” refers to FGA , VWF , ARHGDIB , and SERPINF2 in order to determine whether or not to develop ovarian cancer in a subject or predict the possibility of onset. It means an integrated mixture or device including a means for measuring the expression level of one or more genes selected from the group consisting of genes or proteins they encode, and thus may be expressed as a “diagnostic kit”.
  • the agent for measuring the expression level of a gene used as a marker in the present invention is a primer or probe that specifically binds to the nucleic acid molecule of the gene.
  • nucleic acid molecule has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugars or bases that are modified. (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).
  • primer refers to conditions in which synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of nucleotides and a polymerizer such as DNA polymerase, synthesis under conditions of suitable temperature and pH. refers to an oligonucleotide that serves as the starting point of Specifically, the primer is a single chain deoxyribonucleotide.
  • Primers used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides, and may also include ribonucleotides.
  • the primer of the present invention may be an extension primer that anneals to a target nucleic acid to form a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which is extended to a position where the immobilized probe is annealed, so that the probe becomes occupies the annealed area.
  • the extension primer used in the present invention includes a hybrid nucleotide sequence complementary to a target nucleic acid, for example, a specific nucleotide sequence of genes used as markers in the present invention.
  • the term "complementary” means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, substantially complementary and perfectly complementary. ), and specifically means completely complementary cases.
  • substantially complementary sequence is intended to include not only completely identical sequences, but also sequences that are partially inconsistent with the sequence to be compared, within the range of annealing to a specific sequence and acting as a primer.
  • the primer must be long enough to prime the synthesis of the extension product in the presence of the polymerization agent.
  • the suitable length of a primer depends on a number of factors, such as temperature, pH and the source of the primer, but is typically 15-30 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.
  • the design of such primers can be easily performed by those skilled in the art by referring to the target nucleotide sequence, and can be performed using, for example, a primer design program (eg, PRIMER 3 program).
  • the term “probe” refers to a natural or modified monomer including deoxyribonucleotide and ribonucleotide capable of hybridizing to a specific nucleotide sequence, or a linear oligomer having a linkage. Specifically, the probe is single-stranded for maximum efficiency in hybridization, more specifically a deoxyribonucleotide.
  • a probe used in the present invention a sequence perfectly complementary to a specific nucleotide sequence of genes used as markers in the present invention may be used, but substantially within a range that does not interfere with specific hybridization Complementary sequences may also be used. In general, since the stability of a duplex formed by hybridization tends to be determined by the matching of the terminal sequence, it is preferable to use a probe complementary to the 3'-end or 5'-end of the target sequence. do.
  • an agent for measuring a protein encoded by genes used as markers in the present invention includes an antibody or an antigen-binding fragment thereof that specifically binds to the protein; or an aptamer that specifically binds to the corresponding protein.
  • proteins encoded by genes used as markers in the present invention can be detected according to an immunoassay method using an antigen-antibody reaction and used to analyze whether or not ovarian cancer has occurred.
  • an immunoassay can be performed according to various immunoassay or immunostaining protocols previously developed.
  • antibodies labeled with radioactive isotopes may be used.
  • Antibodies that specifically recognize proteins used as markers in the present invention are polyclonal or monoclonal antibodies, preferably monoclonal antibodies.
  • Antibodies of the present invention can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,567 ) or phage antibody library methods (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). . General procedures for antibody preparation are described in Harlow, E.
  • the term “antigen binding fragment” refers to a part of a polypeptide capable of binding to an antigen in the overall immunoglobulin structure, and includes, for example, F(ab')2, Fab', Fab, Fv and scFvs, but are not limited thereto.
  • the term “specifically binding” has the same meaning as “specifically recognizing”, and refers to a specific interaction between an antigen and an antibody (or a fragment thereof) through an immunological reaction. means that
  • an aptamer that specifically binds to the above-described protein may be used instead of an antibody.
  • the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity.
  • RNA or DNA nucleic acid
  • peptide molecule that binds to a specific target substance with high affinity and specificity.
  • the markers used in the diagnosis of ovarian cancer in the present invention are proteins having the entire sequence and can be targets for diagnosis of ovarian cancer, as well as some fragments of each protein described in Table 2 or SEQ ID NOs: 1 to 21 of the present specification. It can be a target for cancer diagnosis, and in this case, in addition to mass spectrometry, all types of protein or peptide fragment detection methods including the above-described protein measurement method can be used.
  • the above diagnostic composition is a group consisting of S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA , LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP , CD14 , APOA1 and APOA4 It further comprises an agent for measuring the expression level of one or more genes selected from or the protein they encode as an active ingredient.
  • S100A9 is the name of a gene encoding calcium-binding protein A9 protein in humans.
  • the aforementioned calcium-binding protein A9 protein may be referred to as S100A9, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as a name for the protein.
  • VCL is the name of a gene encoding the Vinculin protein in humans.
  • the above-described vinculin protein may be referred to as VCL, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • THBS1 is the name of a gene encoding the Thrombospondin 1 protein in humans.
  • the aforementioned thrombospondin 1 protein may be referred to as TSP1, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the protein.
  • MPO myeloperoxidase protein
  • the aforementioned myeloid cell peroxidase protein may be referred to as MPO, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • IGFBP2 is the name of a gene encoding insulin-like growth factor-binding protein 2 in humans.
  • the above-described insulin-like growth factor-binding protein 2 may be referred to as IBP2, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • SRGN is the name of a gene encoding Serglycin protein in humans.
  • the aforementioned serglycin protein may be referred to as SRGN, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • PF4 is the name of a gene encoding the platelet factor 4 protein in humans.
  • the aforementioned platelet factor 4 protein may be referred to as PF4, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the protein.
  • GP1BA is the name of a gene encoding platelet glycoprotein Ib alpha chain protein in humans.
  • the aforementioned platelet glycoprotein Ib alpha chain protein may be referred to as GP1BA, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • LRG1 is the name of a gene encoding the leucine-rich alpha-2-glycoprotein 1 protein in humans.
  • the aforementioned leucine-rich alpha-2-glycoprotein 1 protein may be referred to as LRG1, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the protein.
  • PRG4 is the name of a gene encoding the protein Proteoglycan 4 in humans.
  • the aforementioned proteoglycan 4 protein may be referred to as PRG4, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • LBP is the name of a gene encoding a lipopolysaccharide binding protein protein in humans.
  • the aforementioned lipopolysaccharide-binding protein may be referred to as LBP, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • PPBP Pro-Platelet basic protein in humans.
  • the aforementioned all-platelet basic protein may be referred to as PPBP, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • C3 is the name of a gene encoding the complement component 3 protein in humans.
  • the aforementioned complement component 3 protein may be referred to as C3, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the protein.
  • FCGBP is the name of a gene encoding an IgGFc-binding protein in humans.
  • the aforementioned IgGFc-binding protein may be referred to as FCGBP, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • CD14 is the name of a gene encoding Cluster of differentiation 14 protein in humans.
  • the aforementioned differentiation cluster 14 protein may be referred to as CD14, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as names for the protein.
  • APOA1 is the name of a gene encoding Apolipoprotein A1 protein in humans.
  • the apolipoprotein A1 protein described above may be referred to as APOA1, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • APOA4 is the name of a gene encoding Apolipoprotein A4 (Apolipoprotein A4) protein in humans.
  • the apolipoprotein A4 protein described above may be referred to as APOA4, but is not limited thereto and may be referred to by all aliases or synonyms commonly used as the name of the protein.
  • the expression level of the FGA , VWF or ARHGDIB gene or the protein they encode is increased in an individual with ovarian cancer, and the expression level of the SERPINF2 gene or the protein they encode is decreased.
  • the term “increased expression level” used while referring to the “composition for diagnosing ovarian cancer” refers to a case in which the expression level of a corresponding gene or a protein encoded by the corresponding gene is significantly higher than that of a control group or a normal group. Specifically, the expression level is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, or about 60% or more compared to the control or normal group. It means an increased case, but does not exclude a range outside of this.
  • the term “decreased expression level” used while referring to the “diagnostic composition for ovarian cancer” refers to a case in which the expression level of a corresponding gene or a protein encoded by the corresponding gene is significantly lower than that of a control group or a normal group. Specifically, the expression level is reduced by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, or about 60% or more compared to the control or normal group. It means a reduced case, but does not exclude a range outside of this.
  • the expression level of the protein is increased, and the expression level of the APOA1 or APOA4 gene or the protein they encode is decreased.
  • the ovarian cancer that can be diagnosed with the composition of the present invention is high-grade serous ovarian cancer (HGSOC).
  • HGSOC high-grade serous ovarian cancer
  • HGSOC is one of several subtypes of ovarian cancer, and includes clear cell and endometrioid subtypes. Together they constitute the three major subtypes of ovarian cancer.
  • HGSOC is the most malignant form of ovarian cancer and is a subtype that accounts for up to 70% of all ovarian cancer cases. It is known to have a poor prognosis.
  • the present invention provides information necessary for diagnosis of ovarian cancer, including the step of measuring the expression levels of FGA , VWF , ARHGDIB and SERPINF2 genes or proteins encoded by them in a biological sample isolated from an individual. Provides a way to provide
  • proteins or genes encoding them having the above positive correlation in an individual or in a biological sample isolated from the individual are highly expressed;
  • proteins having the aforementioned negative correlation or genes encoding them are underexpressed, the subject is determined to have ovarian cancer or is likely to develop in the future.
  • the term “high expression” refers to a case in which the expression level of a corresponding gene or a protein encoded by the corresponding gene is significantly higher than that of the control group or normal group, and specifically, the expression level is about approx. An increase of 10% or more, an increase of about 20% or more, an increase of about 30% or more, an increase of about 40% or more, an increase of about 50% or more, or an increase of about 60% or more.
  • the term “low expression” refers to a case in which the expression level of a corresponding gene or a protein encoded by the corresponding gene is significantly lower than that of the control group or normal group, and specifically, the expression level is about approx.
  • the term “individual” refers to an individual to whom a sample is provided to measure the expression level of the gene or the protein encoded by the gene of the present invention, and is ultimately analyzed for the onset of ovarian cancer.
  • Subjects include, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys, specifically humans. Since the composition of the present invention also provides information for predicting the genetic risk of developing ovarian cancer in the future as well as the current onset of ovarian cancer, the subject of the present invention may be an ovarian cancer patient and has not yet developed ovarian cancer. It may be an unhealthy individual.
  • measuring the expression level is performed by mass spectrometry (MS).
  • mass spectrometry may also be referred to as “mass spectrometry” and means a method of analyzing unknown compounds by their mass.
  • Mass spectrometry is performed by filtering, detecting, and measuring ions using the m/z value, which is the mass-to-charge ratio.
  • mass spectrometry (1) ionizes a compound to charge it.
  • Step (2) measuring the molecular weight of the charged compound and calculating the m/z value.
  • the calculated m/z value is used as a reference to identify and quantify the target compound in complex mixtures.
  • the mass spectrometry of the present specification can be performed through all types of mass spectrometry using the above principle.
  • the mass spectrometry measures the expression level of one or more peptides selected from the group consisting of SEQ ID NOs: 1, 2, 12 and 21.
  • Mass spectrometry for proteins used as biomarkers of ovarian cancer in the present invention is specifically performed by multiple reaction monitoring (MRM) or parallel reaction monitoring (Parallel reaction monitoring) through peptide fragments represented by SEQ ID NOs: 1 to 21 below. monitoring, PRM), but is not limited to:
  • SEQ ID NO: 2 HIVTFDGQNFK
  • SEQ ID NO: 3 S100A9: NIETIINTFHQYSVK
  • SEQ ID NO: 4 (VCL): LLAVAATAPPDAPNR
  • SEQ ID NO: 5 (THBS1): TIVTTLQDSIR
  • SEQ ID NO: 7 (IGFBP2): LIQGAPTIR
  • SEQ ID NO: 8 (SRGN): IQDLNR
  • SEQ ID NO: 10 (GP1BA): LTSLPLGALR
  • SEQ ID NO: 12 (ARHGDIB): TLLGDGPVVTDPK
  • SEQ ID NO: 15 (PPBP): NIQSLEVIGK
  • SEQ ID NO: 18 (CD14): FPAIQNLALR
  • SEQ ID NO: 21 (SERPINF2): EDFLEQSEQLFGAK.
  • the term “multiple reaction monitoring” is an analytical technique capable of selectively separating, detecting, and quantifying a specific analyte and monitoring its concentration change.
  • parent ions among the ion fragments generated in the ionization source are selectively transferred to the collision tube using the first mass filter (Q1).
  • the mother ions that have reached the collider tube collide with the internal collider gas are split to generate daughter ions, and are sent to the second mass filter (Q2), where only characteristic ions are delivered to the detector.
  • Q2 the second mass filter
  • MRM is used for quantitative analysis of small molecules and is used to diagnose specific genetic diseases.
  • the MRM method is advantageous in that it is easy to simultaneously measure a plurality of peptides, and it is possible to confirm the relative concentration difference of protein diagnostic marker candidates between a normal person and a cancer patient without an antibody.
  • MRM analysis is being introduced for the analysis of complex proteins and peptides in blood, especially in proteome analysis using mass spectrometry (Anderson L. et al., Mol CellProteomics, 5: 375-88 , 2006; DeSouza, L. V. et al., Anal. Chem., 81: 3462-70, 2009).
  • parallel reaction monitoring is a parallel application of MRM. Unlike MRM, which analyzes a pair of parent/daughter ions at once, all daughter ions generated from a selected parent ion are simultaneously monitored. way to analyze it.
  • mass spectrometry for proteins used as biomarkers of ovarian cancer can be performed through Data-Independent Acquisition (DIA).
  • DIA Data-Independent Acquisition
  • data-independent acquisition analysis method is a method of analyzing all ions belonging to the m/z value of a selected range without selecting a specific parent ion.
  • the method for providing information necessary for the diagnosis of ovarian cancer described above includes S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA , LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP , CD14 , APOA1 and APOA4 , and one or more genes selected from the group consisting of, or the step of measuring the expression level of the protein they encode additionally comprises.
  • measuring the expression level is performed by mass spectrometry (MS).
  • the mass spectrometry is composed of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19 and 20
  • the expression level of one or more peptides selected from the group is measured.
  • the biological sample is blood.
  • blood means “whole blood”, and such blood includes plasma and serum.
  • whole blood refers to blood that is generally composed of uncoagulated plasma and cellular components.
  • Plasma accounts for about 50-60% of the total blood volume, and cellular components (eg red blood cells, white blood cells or platelets) may account for about 40-50%.
  • plasma refers to the liquid component of blood and functions as a transport medium in supplying nutrients to cells and organs of the body.
  • the term “serum” is a word meaning a pale yellow liquid collected from blood. Specifically, when blood is left unattended after being collected, red clots are formed as the fluidity of blood decreases. It refers to the light yellow body fluid component that remains when the coagulum is removed.
  • diagnosis of ovarian cancer is possible using the biomarkers of the present invention, and the diagnosis can be made through liquid biopsy using a patient-derived bodily fluid such as blood as a biological sample.
  • a patient-derived bodily fluid such as blood as a biological sample.
  • the ovarian cancer is high-grade serous ovarian cancer (HGSOC).
  • the present invention relates to the group consisting of FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP and CD14 Inhibitors for one or more selected from;
  • a composition for preventing or treating ovarian cancer comprising, as an active ingredient, an activator for at least one selected from the group consisting of APOA1, APOA4, and SERPINF2 is provided.
  • the present inventors found that the occurrence of ovarian cancer can be suppressed when FGA, VWF and ARHGDIB proteins or genes encoding them are inhibited, or when SERPINF2 protein or genes encoding them are overexpressed or activated in ovarian cancer patients. proved experimentally.
  • the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of target genes, specifically FGA , VWF and ARHGDIB genes, whereby the activity or expression of the target gene becomes undetectable or to an insignificant level. Not only when present, but also means a substance that lowers the activity or expression to the extent that the biological function of the target gene can be significantly lowered.
  • Inhibitors of target genes are, for example, shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) antisense oligonucleotides or targets that inhibit the expression of the gene at the gene level, the sequence of which is already known in the art All known in the art including, but not limited to, CRISPR systems containing guide RNAs recognizing genes, antibodies or aptamers that inhibit at the protein level, as well as compounds, peptides and natural products that inhibit their activity Means of inhibition at the gene and protein level can be used.
  • small hairpin RNA is a single strand consisting of 50-70 nucleotides forming a stem-loop structure in vivo , which is used to suppress the expression of a target gene through RNA interference. It refers to the RNA sequence that creates a tight hairpin structure.
  • long RNAs of 19-29 nucleotides complementary to both sides of the loop region of 5-10 nucleotides form a double-stranded stem, which is introduced into the cell through a vector containing a U6 promoter so that it is always expressed. It is transduced and is usually passed on to daughter cells, allowing inheritance of suppression of the target gene.
  • RNA refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, the blunt end or cohesive All ends are possible. As for the sticky end structure, both a structure with 3 ends protruding and a structure with 5 ends protruding are possible.
  • miRNA refers to a single-stranded RNA molecule that inhibits target gene expression through complementary binding with mRNA of a target gene while having a short stem-loop structure as an oligonucleotide that is not expressed in cells. do.
  • ribozyme is a type of RNA and refers to an RNA molecule having a function such as an enzyme that recognizes a specific RNA base sequence and cuts it itself.
  • a ribozyme is composed of a region that binds with specificity to a complementary nucleotide sequence of a target mRNA strand and a region that cleaves a target RNA.
  • PNA peptide nucleic acid
  • antisense oligonucleotide refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and performs translation into a protein, translocation into the cytoplasm, maturation, or all other functions.
  • Antisense oligonucleotides can be modified at one or more bases, sugars or backbone positions to enhance potency (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). .
  • the oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomic, heterocyclic sugarsulfones, and the like.
  • gRNA guideRNA
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the expression inhibitor of the present invention may be a specific antibody that inhibits the activity of the protein encoded by the genes.
  • An antibody that specifically recognizes a target protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
  • the term "activator” refers to an active ingredient that enhances the expression level or activity of a target gene, specifically, the SERPINF2 protein, for example, the expression of SERPINF2, a protein whose sequence and structure are already known in the art It includes, but is not limited to, nucleic acid molecules, peptides, proteins, compounds, and natural products that enhance at the gene or protein level or enhance intrinsic biological activity. Accordingly, “SERPINF2 protein activator” is used as the same meaning as “SERPINF2 protein agonist”.
  • prevention refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed with the disease or disease, but is likely to suffer from the disease or disease.
  • treatment refers to (a) inhibition of the development of a disease, condition or condition; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition.
  • Administration of the composition of the present invention to a subject inhibits the expression of FGA, VWF and ARHGDIB proteins or genes encoding them; As the expression of the SERPINF2 protein or the gene encoding it is activated, the generation of ovarian cancer cells is inhibited, thereby suppressing the development of symptoms due to ovarian cancer, removing them, or alleviating them.
  • composition of the present invention itself may be a composition for treating these diseases, or may be administered together with other pharmacological ingredients to be applied as a treatment adjuvant for the above diseases.
  • treatment or “therapeutic agent” in the present specification includes the meaning of "therapeutic aid” or “therapeutic aid”.
  • the ovarian cancer that can be prevented or treated with the composition of the present invention is high-grade serous ovarian cancer (HGSOC).
  • the present invention provides a method for screening a composition for preventing or treating ovarian cancer comprising the following steps:
  • test substance selected from the group consisting of FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP, CD14, APOA1, APOA4, and SERPINF2 contacting a test substance with a biological sample comprising at least one protein, genes encoding them, or cells expressing them;
  • biological sample refers to any sample obtained from mammals, including humans, containing cells expressing the above-described genes or proteins produced by the expression of the above-described genes, including tissues, organs, cells, or cell cultures. Including, but not limited to. More specifically, the biological sample may be cancer tissue, cancer cells, a culture thereof, or blood.
  • test substance used while referring to the screening method of the present invention is added to a sample containing cells expressing the gene of the present invention and used in screening to examine whether or not it affects the activity or expression level of these genes. means an unknown substance.
  • the test substance includes, but is not limited to, compounds, nucleotides, peptides and natural extracts.
  • the step of measuring the expression level or activity of the gene in the biological sample treated with the test substance may be performed by various methods for measuring the expression level and activity known in the art.
  • the ovarian cancer is high-grade serous ovarian cancer (HGSOC).
  • the present invention is an ovarian cancer (Ovarian Cancer) to a subject.
  • S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA , LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP , CD14 , APOA1 and APOA4 consisting of Provided is a method for diagnosing ovarian cancer, which further comprises administering to a subject a composition characterized in that it further comprises, as an active ingredient, an agent for measuring the expression level of at least one gene selected from the group or a protein encoded by the gene selected from the group.
  • the present invention relates to the group consisting of FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP and CD14 Inhibitors for one or more selected from; Or a method for preventing or treating ovarian cancer comprising administering to a subject a composition for preventing or treating ovarian cancer comprising, as an active ingredient, an activator for at least one selected from the group consisting of APOA1, APOA4, and SERPINF2. to provide.
  • Genes that can be used for diagnosis, prevention or treatment of ovarian cancer in the present invention proteins encoded by the genes; Since the expression inhibitors or activators of the genes and proteins have already been described above, they are omitted to avoid excessive redundancy.
  • the present invention provides a method for diagnosing ovarian cancer by measuring the expression level of genes or proteins involved in the onset of ovarian cancer, and a method for preventing or treating ovarian cancer by regulating their expression.
  • the present invention provides an effective biomarker for ovarian cancer, particularly high-grade serous ovarian cancer, thereby providing a method for predicting the onset of ovarian cancer early and with high reliability, while suppressing the expression of the relevant factors. Or by activating it, it can be usefully used for efficient prevention or treatment of ovarian cancer.
  • 1A is a schematic illustration of the experimental procedure for targeted MS analysis followed by comprehensive serum proteome profiling experiments.
  • Figure 1b is a graph showing the protein concentration dynamic range of serum proteins identified by comprehensive serum proteome profiling for spectral library generation, and protein concentration estimates were obtained from the Human Protein Atlas (blood proteins).
  • Figure 1c is a picture showing the MS/MS spectrum annotated with the peptide (QCYLQQVK) of RNF213.
  • Figure 2a is a graph showing the number of peptides (top) and proteins (bottom) identified by LC-DIA-MS / MS in HGSOC and HC, total and average number of identified peptides and proteins (Average number) is indicated.
  • Figure 2b is a picture showing the results of PCA analysis of the HGSOC and HC serum proteomes, and the deviations explained by each principal component are indicated.
  • Figure 2c is a heatmap of the expression of DEProteins in HGSOC and HC, showing the number of up- and down-regulated proteins.
  • Figure 2d is a picture showing the pathway enriched by DEProteins, and the dot plot shows the importance of the pathway enriched by DEProteins as log10 (p-value).
  • Figure 2e is a diagram showing a network model showing interactions between DEProteins involved in the enriched pathway of Figure 2d.
  • 3A is a box plot showing normalized peak areas (light/heavy) of 21 potential HGSOC diagnostic biomarkers in a validation cohort using targeted MS experiments.
  • 3B is a box plot showing normalized protein abundance of 21 biomarker candidates in a discovery cohort using LC-DIA-MS/MS.
  • HGSOC high-grade serous ovarian cancer
  • HCs controls
  • PFS progression-free survival
  • the 14 most abundant proteins in plasma (albumin, IgA, IgG, IgM, ⁇ 1-antitrypsin, ⁇ 1-acid glycoprotein, apolipoprotein A1, apolipoprotein A2, complement C3, transferrin) using a multiple affinity removal column (MARS14) , ⁇ 2-macroglobulin, transthyretin, haptoglobin and fibrinogen) were removed.
  • MARS14 multiple affinity removal column
  • An aliquot of 40 ⁇ L of serum diluted 4-fold with proprietary “Buffer A” was injected onto the MARS14 depletion column of a binary HPLC system (20A Prominence, Shimadzu, Tokyo, Japan).
  • the unbound fraction was collected in a collection tube containing 100 ⁇ L of 5% SDS in 100 mM TEAB solution and then completely dried in a speed-vac concentrator (Thermo Fisher Scientific, Waltham, MA, USA). The dried samples were resuspended in 100 ⁇ L of 50 mM TEAB solution and sonicated for 10 minutes. 100 ⁇ g of protein was reduced (DTT, 10 mM, 56° C., 30 minutes) and alkylated (IAA, 20 mM, room temperature in the dark, 30 minutes). Samples were then prepared by suspension trapping (S-Trap) based tryptic digestion according to the manufacturer's instructions with slight modifications.
  • S-Trap suspension trapping
  • samples were washed with 90:10% methanol/50 mM ammonium bicarbonate. Samples were then digested with 50 mM ammonium bicarbonate and trypsin (1:25 trypsin/protein) was added to this fraction followed by overnight incubation at 37°C. Digested peptides were eluted by centrifugation at 1,000 g for 60 seconds. Two additional elutions were performed using 0.2% formic acid and 0.2% formic acid in 50% acetonitrile. The three eluents were pooled together, dried by vacuum centrifugation and stored at -80°C until use.
  • a previously developed dual online noncontiguous fractionating and concatenating reverse phase/reverse phase liquid chromatography (DO-NCFC-RP/RPLC) system was used to obtain 24 NCFC fractions. modified to create
  • the online NCFC device consisted of a mid-pH RP column (150 ⁇ m ⁇ 50 cm) and two NCFC valves (25-port, 1-channel, C5M-66024D, VICI) interconnected by 24 fractional loops (200 ⁇ m).
  • two SPE columns 150 ⁇ m ⁇ 3 cm
  • two analytical columns 75 ⁇ m ⁇ 150 cm
  • Serum peptides (25 ⁇ g, 40 ⁇ g or 50 ⁇ g) from each pooled sample group were injected and separated on a mid-pH column for online 1st dimensional separation.
  • Gradient (1-50% solvent B at a flow rate of 1 ⁇ L/min) using mid-pH solvent A (10 mM TEAB, pH 7.5 in water solvent) and mid-pH solvent B (10 mM TEAB, pH 7.5 in 99% ACN solvent). 120 min) was created.
  • mid-pH solvent A (10 mM TEAB, pH 7.5 in water solvent
  • mid-pH solvent B (10 mM TEAB, pH 7.5 in 99% ACN solvent
  • each of the 24 online NCFC fractions was transferred to a SPE column while diluting 10-fold with an acidification and dilution buffer (0.2% TFA in water).
  • an acidification and dilution buffer (0.2% TFA in water).
  • low-pH solvent A water solvent, 0.1% formic acid
  • low-pH solvent B ACN solvent, 0.1% formic acid
  • a gradient (10-37.5% Sol B, 37.5% Sol B over 160 min at a flow rate of 0.3 ⁇ L/min) -80% Sol B for 5 minutes, 80% Sol B for 13 minutes, 10% Sol B for 2 minutes).
  • the DO-NCFC-RP/RPLC is an Orbitrap ExplorisTM 480 Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a high field asymmetric waveform ion mobility spectrometry (FAIMS ProTM, Thermo Fisher Scientific, Bremen, Germany). , Germany) online.
  • Peptides were ionized using our company's nanoelectrospray source with 3 kV spray voltage, and the temperature of the desolvation capillary was set at 250 °C.
  • Three compensation voltages (CVs) of -35V, -50V and -60V were used with a cycle time of 1 second. Temperatures of the inner and outer electrodes were both set to 100°C.
  • MS/MS spectra were obtained at a resolution of 15,000, an AGC target of 1000, and an IT of 32 ms.
  • MS/MS data were subjected to post-experimental monoisotopic mass refinement (mPE-MMR), and the resulting MS/MS data (e.g.
  • mgf files were submitted to the UniProt human reference database (released August 2020, containing 97.093 entries) and general The protein database consisting of contaminants (179 items) was searched using the MS-GF+ search engine (v9949, http://proteomics.ucsd.edu/software-tools/ms-gf/).
  • the search parameters were semi-tryptic cleavage and a precursor mass tolerance of 10 ppm. Carbamidomethylation of cysteine was used for static modification, and oxidation of methionine and deamidation of asparagine and glutamine were used for variable modifications.
  • the search results were filtered by target-decoy analysis with a False Discovery Rate (FDR) of 1% at the PSM (Peptide Spectrum Match) level.
  • FDR False Discovery Rate
  • a spectral library database was constructed using representative MS/MS spectra of the final peptides.
  • the MS2 spectrum with the highest search score i.e., -log(SpecE-value)
  • the fragment peaks of the representative MS/MS spectra for each peptide matched the theoretical fragments (type b and y) within 0.01 Da.
  • Annotated fragments were collected, and other unannotated fragments were removed.
  • representative MS/MS spectra with at least 5 annotated fragments were included in the spectral library, along with experimental mass information and retention time information. Experimental retention times were converted to normalized elution times using an iRT peptide (Ki-3002-2, Biognosys) spiked into each NCFC fraction.
  • Digested peptides were isolated using a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). Trypsinized peptides were reconstituted in 0.1% TFA and run on an AcclaimTM Pepmap RSLC C18 column (150 mm ⁇ 150 ⁇ m i.d., 2 ⁇ m, 100 ⁇ ) equipped with a C18 Pepmap trap column (20 mm ⁇ 100 ⁇ m i.d., 5 ⁇ m, 100 ⁇ ; Thermo Scientific). Separation was performed over 60 minutes (1 ⁇ L/min) using a 5-40% acetonitrile gradient in a 50° C. environment and 0.1% formic acid and 5% DMSO.
  • the LC was connected to an Orbitrap ExplorisTM 480 mass spectrometer with an EASY-SPRAYTM source (Thermo Fisher Scientific). Mass spectrometry runs were run in DIA mode.
  • the overall MS resolution was set to 60,000
  • the overall MS AGC target was 300% with an IT of 25 ms
  • the m/z range was set to 300-1,400.
  • the AGC target value for the MS2 spectrum was set at 1000%, and 44 windows of 24 Da with an overlap of 1 Da were used. Resolution was set to 15,000 and IT was set to 22 ms. NCE was set at 27.
  • LC-DIA-MS/MS data were processed using DIA-NN17 (version 1.7.10) with its own serum spectral library. Spectra were retrieved with default settings except that the m/z range was 300-1,300 for precursors and 200-1,300 for fragment ions. Identification results were filtered with an FDR of 1% at the precursor level. Quantities of peptides and proteins were obtained using the MaxLFQ algorithm, and the corresponding quantity data were quantile normalized. All LC-MS/MS data have been deposited with the ProteomeXchange consortium through the PRIDE partner repository under the dataset identifier PXD033169.
  • test statistics were calculated using Students' t-test, Wilcoxon-Ranksum test and log2-median-ratio at each comparison. Then, all samples were randomly permuted 10,000 times to estimate the test statistics and empirical distributions of log2-median-ratio for the null hypothesis. Calculate the observed test statistic and adjusted p-value for the log2-median-ratio for each peptide or protein using the estimated empirical distribution, then combine these p-values using Stouffer's method to calculate the overall p-value.
  • Pathway over-representation analysis was performed using ConsensusPathDB.
  • the pathway represented by DEProteins was identified as a pathway with a q-value of less than 0.05 and a number of molecules involved in the pathway of 3 or more.
  • the entire set of genes identified in the present study was used as a background gene list.
  • the identified pathways were classified into 'immune system', 'IGF signaling', 'TGF- ⁇ signaling', 'ECM organization' or 'lipoprotein assembly' according to their functional relevance.
  • a network model was constructed for the DEProteins associated with the overrepresented pathways.
  • the network was constructed by setting nodes to proteins and adding protein-protein interactions and activation/inhibition relationships. Nodes were aligned based on localization and activation/inhibition relationships obtained from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database and literature search. Networks were drawn using Cytoscape.
  • MRM-MS Multiple Reaction Monitoring-Mass Spectrometry analysis was applied with Qtrap5500 plus (Sciex). Trypsinized peptides were sampled on a ZORBAX 300SB-C18 reversed-phase column (0.5 mm ⁇ 150 ⁇ m i.d., 3.5 ⁇ m, 100 ⁇ ; Agilent) using a 5-40% acetonitrile gradient in 0.1% formic acid over 15 min (20 ⁇ L/min). separated. Collision energy (CE) values for each ionized peptide were determined using SKYLINE, which provides values. Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) analysis was applied with an Orbitrap ExplorisTM 480 mass spectrometer.
  • PRM-MS Parallel Reaction Monitoring-Mass Spectrometry
  • Trypsinized peptides were prepared on an AcclaimTM Pepmap RSLC C18 column (150 mm ⁇ 150 ⁇ m i.d., 2 ⁇ m, 100 ⁇ ) equipped with a C18 Pepmap trap column (20 mm ⁇ 100 ⁇ m i.d., 5 ⁇ m, 100 ⁇ , Thermo Scientific) at 50 °C in 0.1% formic acid and Separation was carried out over 60 min (1.5 ⁇ L/min) using a 5-40% acetonitrile gradient in 5% DMSO. Mass spectrometry was performed in targeted mass mode. Precursor target mass, m/z value and charge state were listed through data-dependent acquisition analysis using synthetic peptides.
  • the overall MS resolution was set to 60,000
  • the overall MS AGC target was set to 300 with an IT of 25 ms
  • the m/z range was set to 280-1,200.
  • MS/MS resolution was set to 15,000
  • IT was set to 22 ms
  • NCE was set to 27.
  • the minute voltage was set to 2.5 kV, and the peaks of the PRM-MS spectrum were selected and calculated by SKYLINE.
  • Human HGSOC cancer cell line SK-OV-3 was obtained from Seoul National University College of Medicine (Korea). SK-OV-3 cells were cultured at 37°C in 5 medium containing RPMI 1640 (Corning; 10-041-CV), 10% fetal bovine serum (FBS; Invitrogen), and 1% Penicillin/streptomycin (WelGENE Inc.). Incubated with % CO2. Cells were maintained with fresh nutrients for 3 days.
  • Plasmids tagged with Myc-DDK were purchased from OriGene Technologies, Inc. (Rockville, MD). A Myc-DDK tagged SERPINF2-human serpin peptidase inhibitor (RC206435) plasmid was used for transfection. Plasmids were transfected into SK-OV-3 cells using Lipofectamine 3000 reagent according to the manufacturer's protocol from Invitrogen (#L3000008). For knockdown experiments, the following siRNAs were purchased from Genolution, Inc. (Korea):
  • siVWF-1 5'-CCUUCUGAGCCCACAAUAAUU-3' SEQ ID NO: 25
  • siVWF-2 5'-GCUGUAAGUCUGAAGUAGAUU-3' SEQ ID NO: 26
  • siRNA was transfected into SK-OV-3 cells using Lipofectamine RNAiMAX transfection reagent according to the manufacturer's protocol from Invitrogen (#13778075). SK-OV-3 cells were exposed to siRNA reagent for 72 hours and harvested with PBS (WelGENE Inc.). In addition, DNA transfections were incubated for 28 hours in Opti-MEM diluted with Lipofectamine and harvested for Western blotting. For proliferation assays and cell migration, cells were transfected at 48 hours with siRNA reagents and at 24 hours with DNA transfected reagents.
  • Cells were lysed in RIPA lysis buffer (cell biolabs, Inc.) containing a phosphatase inhibitor (100X cocktail GenDEPOT). Lysates were boiled in SDS sample buffer (Alfa Aesar Thermo) at 95° C. for 5 minutes. Proteins were separated on a 4-20% precast SDS-PAGE gel (BIORAD) and transferred to a FL-PVDF membrane (MERCK) in a 15 min ATTO EZ Fast protocol. Membrane blocking was performed by overnight incubation at 4° C. with primary antibodies in 5% bovine albumin serum (BSA; EcoCell) in 1X TBST.
  • BSA bovine albumin serum
  • Membranes were first reduced, washed three times with 1X TBST, and incubated with secondary antibodies for 30 minutes at room temperature. Then, the detected proteins were used for western blotting reagent solution (Roche) and ECL (Electrochemiluminescence) of ODYSSEY Fc System (Li-Cor Bioscience).
  • FGA M ⁇ 1:500; ab92572, Abcam
  • VWF R ⁇ 1:500; ab189500, Abcam
  • ARHGDIB D4GDI 1:1000; ab181252, Abcam
  • FLAG GT231 M ⁇ 1:1000; GTX629631, GeneTex Inc
  • anti-mouse IgG antibody HRP 1:3000; 170-6516, BIORAD
  • anti-rabbit IgG antibody HRP 1:3000; 170-6515, BIORAD.
  • the WST-1 assay kit was used for cell proliferation assay according to TaKara's manufacturer's protocol (MK400; Premix WST-1 Cell Proliferation Assay System). Transfected cells were plated in 96-well plates at a density of 1000 cells per well in 100 ⁇ l medium and incubated for 5 days. Then, the cells were treated with 10ul WST-1 reagent, and after 4 hours, the absorbance of the formazan dye was measured at 450 nm to analyze cell proliferation.
  • MK400 Premix WST-1 Cell Proliferation Assay System
  • Transfected cells were plated in 6-well plates at a density of 500 cells per well. After 10 days, the colonies were washed once with PBS at room temperature, stained with 0.5% crystal violet (V5265, Sigma) for 1 hour, and washed three times to remove residual dye. Colonies were then dried overnight and analyzed using ImageJ software.
  • HGSOC patient cases and 48 healthy control (HC) subjects were prepared: (1) a discovery set for proteomics profiling consisting of 26 HGSOC and 24 HC ( discovery set) and (2) a validation set for targeted MS experiments consisting of 28 HGSOCs and 24 HCs (Table 1).
  • the mean age of HGSOC patients was 59.5 and 56 years in the discovery and validation sets, respectively, and 46 and 48.5 years in the discovery and validation sets, respectively, for HC.
  • the number of FIGO stage I, II, and III HGSOC patients was 3, 3, and 20 in the discovery set and 3, 10, and 15 in the validation set, respectively.
  • Dual online non-contiguous fractionating and concatenating reverse-phase/reverse phase liquid chromatography combined with a FAIMS-MS/MS instrument (DO-NCFC-RP/RPLC-FAIMS-MS) -phase liquid chromatography coupled to a FAIMS-MS/MS instrument (DO-NCFC-RP/RPLC-FAIMS-MS/MS) was used to perform comprehensive global proteome profiling experiments of serum samples.
  • a total of 9 sets of DO-NCFC-RP/RPLC-FAIMS-MS/MS experiments were performed on 6 groups of depleted serum samples, each group included 24 online NCFC fractions and 3 CVs (Fig. 1a ).
  • the identified serum proteome was estimated to cover approximately 8 orders of dynamic ranges (Ceruloplasmin at 440 ⁇ g/mL to Ring finger protein 213 at 3.5 pg/mL). 213), Figs. 1b and 1c).
  • the serum proteome of the present invention provides a high coverage serum proteome and can be used to construct an OC serum spectral library.
  • a spectral library was constructed using the tandem spectrum with the highest search score for the peptide and five or more annotated fragment ion peaks.
  • the spectral library contained 127,444 spectra of 94,212 non-overlapping peptides covering 8,458 protein groups.
  • LC-DIA-MS/MS analysis was performed on 24 HCs and 26 HGSOCs in the discovery cohort for HGSOC diagnostic biomarker discovery.
  • a DIA-NN search using the OC serum spectral library identified 29,721 peptides and 2,439 proteins in all samples (Fig. 2a), which is the largest quantification of the serum proteome of OC to date.
  • the number of peptides identified across samples varied from 13,818-15,987 with an average of 14,797 and proteins varied from 1,012-1,240 with an average of 1,104.
  • Principal component analysis (PCA) confirmed that the corresponding serum proteome clearly separated HGSOC from HC (Fig. 2b).
  • TLR4 toll-like receptor 4
  • NET neutrophil extracellular trap
  • IGF insulin-like growth factor
  • FC ⁇ R Fc gamma receptor
  • a network model involving the coagulation and complement cascade is characterized by (1) coagulation action, including upregulation of procoagulants (F11, VWF and FGA/B), downregulation of anticoagulant factors (SERPINA5/F2 and A2M); and (2) commonly upregulated complement processes (MBL2, C3/9/C4A/B/BPA/BPB and CFHR2/5) that cross talk with the coagulation cascade to promote coagulation or carry out the reverse reaction.
  • procoagulants F11, VWF and FGA/B
  • SERPINA5/F2 and A2M downregulation of anticoagulant factors
  • MBL2, C3/9/C4A/B/BPA/BPB and CFHR2/5 commonly upregulated complement processes
  • Networks involving neutrophils and macrophages can (1) form NETs (LBP, ACTB and MPO); (2) TLR4 signaling, including upregulation of TLR4 signaling activating proteins (LBP, FGA/B, CD14, S100A8) and downregulation of TLR4 signaling inhibitory proteins (APOA1/4/C3); and (3) downregulation of FC ⁇ R-mediated phagocytosis (IGLC2/3/HG2/HG3/KC/KV1-5/KV1D-33/KV3-20/KV3D-20/KV4-1) were co-regulated.
  • upregulation of FCGBP together with activation of TLR4 signaling can cause macrophage recruitment to the tumor microenvironment (TME).
  • networks comprising platelets showed coordinated upregulation of platelet activation and aggregation (e.g., VCL, GP1BA, PF4, PPBP, SRGN, VWF, and FGA) that can be promoted by NET formation and lead to the coagulation process.
  • platelet activation and aggregation e.g., VCL, GP1BA, PF4, PPBP, SRGN, VWF, and FGA
  • the network model including IGF, TGF- ⁇ signaling and ECM organization was (1) upstream of ECM organization (EFEMP1, SERPINE1, PCOLCE, SPARC, TIMP1, FN1, NID1, ENO1, LCP1, ARHGDIB and FGA/B). control; (2) IGF signaling (IGFBP2 and APP); and (3) TGF- ⁇ signaling (LRG1, LTBP1, THBS1, PRG4 and SERPINE1) collectively contribute to cancer cell proliferation, migration and invasion in HGSOC.
  • EFEMP1, SERPINE1, PCOLCE, SPARC, TIMP1, FN1, NID1, ENO1, LCP1, ARHGDIB and FGA/B IGF signaling
  • IGFBP2 and APP IGF signaling
  • TGF- ⁇ signaling LRG1, LTBP1, THBS1, PRG4 and SERPINE1
  • potential HGSOC-related diagnostic biomarkers for targeted MS experiments were selected according to the following criteria: fold change, expression consistency across samples, and comparison with previously reported OC pathophysiology. correlation.
  • VWF coagulation and complement cascade
  • MPO NET formation
  • TLR4 signaling LBP, CD14 and S100A8/9
  • lipoprotein assembly APOA1/4
  • macrophage recruitment to the TME FCGBP
  • platelet activation and aggregation VCL, PFN1, FLNA, GP1BA, VWF, FN1, FGA, PF4, PPBP, SRGN and CALU
  • ECM organization SPARC, TIMP1, FN1, ENO1, LCP1 and ARHGDIB
  • IGF signaling IGFBP2
  • TGF- ⁇ signaling LRG1, LTBP1, THBS1 and PRG4
  • Targeted MS analysis was performed in an independent cohort of 25 HC and 28 HGSOC patients using stable isotope labeled (SIL) peptides of 33 biomarker candidates to validate their potential diagnostic value. Seven candidates (FLNA, FN1, LTBP1, PFN1, S100A8, TIMP1, and CALU) did not show detectable transition signals in MRM or PRM experiments, so they were excluded from the final biomarker candidate set. Expression patterns of the final set of 26 diagnostic biomarker candidates were compared with each other with target MS (FIG. 3A) and LC-DIA-MS/MS analysis (FIG. 3B) results. Targeted MS analysis showed that the expression of 21 of 26 candidates showed a statistically significant (P ⁇ 0.05) change between HGSOC and HC (Table 2), which was consistent with the LC-DIA-MS/MS analysis.
  • SIL stable isotope labeled
  • HGSOC serum biomarkers such as FGA, VWF, ARHGDIB and SERPINF2 can play an important regulatory role in cancer cell proliferation and migration in HGSOC.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Reproductive Health (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de diagnostic du cancer de l'ovaire par mesure d'un niveau d'expression d'un gène ou d'une protéine impliqué dans l'apparition du cancer de l'ovaire et un procédé de prévention ou de traitement du cancer de l'ovaire par régulation de l'expression de celui-ci. Conçue pour fournir un biomarqueur utile pour le cancer de l'ovaire, en particulier le cancer de l'ovaire grave (HGSOC), la présente invention fournit un procédé permettant de prédire l'apparition du cancer de l'ovaire à un stade précoce avec une fiabilité élevée tout en supprimant ou en activant l'expression des facteurs correspondants, ce qui permet d'utiliser avantageusement la présente invention pour prévenir ou traiter efficacement le cancer de l'ovaire.
PCT/IB2023/050614 2022-01-25 2023-01-25 Nouveau biomarqueur pour le diagnostic du cancer de l'ovaire Ceased WO2023144708A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2022-0010746 2022-01-25
KR20220010746 2022-01-25

Publications (1)

Publication Number Publication Date
WO2023144708A1 true WO2023144708A1 (fr) 2023-08-03

Family

ID=87470867

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2023/050614 Ceased WO2023144708A1 (fr) 2022-01-25 2023-01-25 Nouveau biomarqueur pour le diagnostic du cancer de l'ovaire

Country Status (2)

Country Link
KR (2) KR102573076B1 (fr)
WO (1) WO2023144708A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102799841B1 (ko) * 2024-06-13 2025-04-28 주식회사 엔도믹스 난소암 진단을 위한 메틸화 마커 유전자 및 이의 용도

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009009890A1 (fr) * 2007-07-16 2009-01-22 University Health Network Biomarqueurs du cancer de l'ovaire
KR20200057652A (ko) * 2018-11-16 2020-05-26 가톨릭대학교 산학협력단 유전성 난소암 발병 예측용 바이오마커 및 이의 용도
WO2021146659A1 (fr) * 2020-01-17 2021-07-22 Mercy Bioanalytics, Inc. Compositions et méthodes pour la détection du cancer de l'ovaire
CN113391072A (zh) * 2021-05-30 2021-09-14 浙江省肿瘤医院 卵巢癌尿液标志物组合及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009009890A1 (fr) * 2007-07-16 2009-01-22 University Health Network Biomarqueurs du cancer de l'ovaire
KR20200057652A (ko) * 2018-11-16 2020-05-26 가톨릭대학교 산학협력단 유전성 난소암 발병 예측용 바이오마커 및 이의 용도
WO2021146659A1 (fr) * 2020-01-17 2021-07-22 Mercy Bioanalytics, Inc. Compositions et méthodes pour la détection du cancer de l'ovaire
CN113391072A (zh) * 2021-05-30 2021-09-14 浙江省肿瘤医院 卵巢癌尿液标志物组合及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUH SUNGHYUN, KANG CHAEWON, PARK JI EUN, NAM DOWOON, KIM SE IK, SEOL AERAN, CHOI KYERIM, HWANG DAEHEE, YU MYEONG-HEE, CHUNG HYUN H: "Novel Diagnostic Biomarkers for High-Grade Serous Ovarian Cancer Uncovered by Data-Independent Acquisition Mass Spectrometry", JOURNAL OF PROTEOME RESEARCH, AMERICAN CHEMICAL SOCIETY, vol. 21, no. 9, 2 September 2022 (2022-09-02), pages 2146 - 2159, XP093082170, ISSN: 1535-3893, DOI: 10.1021/acs.jproteome.2c00218 *
XIU, L. ET AL.: "Identification of serum peptide biomarker for ovarian cancer diagnosis by Clin-TOF-II-MS combined with magnetic beads technology", ZHONGHUA ZHONGLIU ZAZHI (CHINESE JOURNAL OF ONCOLOGY), ZHONGGUO KEXUE YIXUE KEXUEYUAN, CN, vol. 43, no. 11, 23 November 2021 (2021-11-23), CN , pages 1188 - 1195, XP009547807, ISSN: 0253-3766 *

Also Published As

Publication number Publication date
KR102573076B1 (ko) 2023-09-01
KR20230114677A (ko) 2023-08-01
KR20230115268A (ko) 2023-08-02

Similar Documents

Publication Publication Date Title
WO2018208091A2 (fr) Biomarqueur permettant de surveiller ou de diagnostiquer l'apparition d'un cancer du foie dans un groupe fortement exposé à un cancer du foie et son utilisation
Roberts et al. Comparison of human and murine enteroendocrine cells by transcriptomic and peptidomic profiling
EP3444267B1 (fr) Marqueur différentiel du cancer du poumon
WO2009113814A2 (fr) Marqueur protéique utilisé pour le diagnostic précoce du cancer du foie
WO2017043679A1 (fr) Panel de biomarqueurs protéiques pour dépister le cancer du poumon non à petites cellules et méthode diagnostique l'utilisant
Yoo et al. Discovery of gastric cancer specific biomarkers by the application of serum proteomics
WO2019182371A1 (fr) Composition pour le diagnostic de maladies neurologiques dégénératives
WO2023144708A1 (fr) Nouveau biomarqueur pour le diagnostic du cancer de l'ovaire
WO2021177691A1 (fr) Composition pour le diagnostic ou le traitement de la résistance aux médicaments anticancéreux
WO2023191543A1 (fr) Composition pour le diagnostic du cancer
US20100285971A1 (en) Traceability of Cellular Cycle Anomalies Targeting Oncology and Neurodegeneration
Khan et al. Protein interactome of the cancerous inhibitor of protein phosphatase 2A (CIP2A) in Th17 cells
WO2022265392A1 (fr) Biomarqueurs multiples pour le diagnostic du cancer des ovaires et leur utilisation
WO2015130115A1 (fr) Nouvel anticorps spécifique de la tspan8 et ses utilisations
WO2019225923A1 (fr) Composition pour diagnostiquer des maladies
Tian et al. Interleukin‐4‒induced posttranscriptional gene regulation of CCL26 by the RNA‐binding protein HuR in primary human nasal polyp‒derived epithelial cells
WO2020166992A1 (fr) Composition pour un diagnostic du cancer
WO2023090901A1 (fr) Procédé de sélection d'un marqueur de sénescence cellulaire utilisant un apprentissage automatique, biomarqueur pour la sénescence cellulaire, et procédé de criblage d'agent senolytique l'utilisant
WO2018088826A2 (fr) Composition de diagnostic de métastases du cancer colorectal ou de prédiction de pronostic et son utilisation
WO2024063523A1 (fr) Biomarqueur pour le diagnostic de la résistance à un médicament anticancéreux dans le cadre du cancer de la vessie, et son utilisation
WO2023008858A1 (fr) Nouvel anticorps pour prévenir ou traiter une maladie fibrotique
WO2025244187A1 (fr) Nouveau biomarqueur pour le diagnostic du cancer du pancréas
WO2021167177A1 (fr) Composition de biomarqueur de diagnostic compagnon et kit de diagnostic compagnon la contenant
WO2024219820A1 (fr) Peptide dérivé de fcn1 pour le diagnostic du glaucome et son utilisation
WO2024219823A1 (fr) Peptide dérivé de trfe pour le diagnostic du glaucome et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23746557

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 11202403853Q

Country of ref document: SG

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23746557

Country of ref document: EP

Kind code of ref document: A1