WO2023144708A1 - Novel biomarker for diagnosis of ovarian cancer - Google Patents

Abstract

The present invention relates to a method for diagnosing ovarian cancer by measuring an expression level of a gene or protein involved in the onset of ovarian cancer and a method for preventing or treating ovarian cancer by regulating the expression thereof. Designed to provide a biomarker useful for ovarian cancer, especially high-grade serous ovarian cancer (HGSOC), the present invention provides a method for predicting the onset of ovarian cancer at an early stage with high reliance while suppressing or activating the expression of corresponding factors, whereby the present invention can be advantageously used for effectively preventing or treating ovarian cancer.

Description

Novel biomarkers for diagnosis of ovarian cancer

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 (OC) 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 (HGSOC) 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. In particular, 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. However, a highly reliable biomarker that can be used to accurately diagnose HGSOC with high sensitivity and specificity has not yet been discovered.

Meanwhile, blood has been the focus of extensive cancer biomarker research due to the minimally invasive nature of liquid biopsy. Previous studies have identified potential blood biomarkers for OC ranging from microRNAs to exosomes to proteins. Measurement of CA-125, a cancer antigen, and human epididymis protein-4 (HE4) in the blood are currently used as gold standards for OC detection (diagnosis) and monitoring. However, although these biomarkers are currently widely used in clinical environments, they have limitations in reducing the mortality of OC patients due to low sensitivity and low specificity. Due to this point, since the options of diagnostic methods for OC patients are significantly limited, additional discovery of new biomarkers for diagnosing OC is urgently needed.

Proteome-wide analysis is a useful method for discovering and prioritizing new biomarkers that have not been previously identified. Mass spectrometry (MS) is effectively used for comprehensive studies in proteomics because it can provide unbiased profiling of human proteomes. Recently, MS analysis based on Data Independent Acquisition (DIA) is emerging as an analysis methodology for next-generation proteomics. 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. ) has advantages such as improved sensitivity, reproducibility, and minimization of fractionation requirements.

Accordingly, 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.

A number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.

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. In order to develop a new diagnostic method that can significantly lower the As a result, 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.

Accordingly, 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.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, 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. In order to develop a new diagnostic method that can significantly lower the As a result, 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.

As used herein, the term “ FGA ” is the name of a gene encoding the fibrinogen alpha chain protein in humans. The fibrinogen alpha chain protein described above may be referred to as FIBA, but is not limited thereto and may be referred to as all aliases or synonyms commonly used as the name of the protein.

As used herein, the term “ 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.

As used herein, the term “ ARHGDIB ” is the name of a gene encoding Rho GDP-dissociation inhibitor 2 protein in humans. The 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.

As used herein, the term “ 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.

All 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”.

In this specification, the term “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. For example, the biomarker panel of the present invention may be provided through a separate tube sold or shipped together as part of a kit; The surface of a chip, membrane, strip, filter, bead, particle, filament, fiber, gel or matrix; It may be provided inside, coupled to the wells of multi-well plates or other supports, but is not limited to the above example, and all kinds of biomarker panels that can be used for diagnosis of diseases as a combination of biomarkers include

As used herein, the term "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.

As used herein, 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”.

According to a specific embodiment of the present invention, 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.

In this specification, the term “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)).

As used herein, the term “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. As used herein, the term "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).

As used herein, 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. As 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.

Conditions suitable for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington , can be determined by referring to the matters disclosed in DC (1985).

According to a specific embodiment of the present invention, 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.

According to the present invention, 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. Such an immunoassay can be performed according to various immunoassay or immunostaining protocols previously developed.

For example, when the method of the present invention is performed according to the radioimmunoassay method, antibodies labeled with radioactive isotopes (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) 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. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984.

By analyzing the strength of the final signal by the above-described immunoassay process, it is possible to predict whether or not to develop ovarian cancer. That is, when the signal for the above-mentioned protein is stronger than in the normal sample in the sample of the object, the object has a high possibility that ovarian cancer has developed or will develop in the future. It is judged to be

As used herein, 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.

As used herein, 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

In the present invention, an aptamer that specifically binds to the above-described protein may be used instead of an antibody. As used herein, 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. For a general discussion of aptamers see Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

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.

According to a specific embodiment of the present invention, 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.

In the present specification, the term “ 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.

As used herein, the term “ 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.

As used herein, the term “ 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.

In the present specification, the term “ MPO” is the name of a gene encoding a myeloperoxidase protein in humans. 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.

In the present specification, the term “ 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.

As used herein, the term “ 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.

As used herein, the term “ 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.

In this specification, the term “ 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.

As used herein, the term “ 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.

As used herein, the term “ 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.

In the present specification, the term “ 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.

As used herein, the term “ PPBP” is the name of a gene encoding 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.

As used herein, the term “ 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.

In this specification, the term “ 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.

As used herein, the term “ 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.

In the present specification, the term “ 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.

In the present specification, the term “ 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.

According to a specific embodiment of the present invention, 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.

Among the components of the present invention, 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.

Among the components of the present invention, 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.

According to a specific embodiment of the present invention, the S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA, LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP or CD14 genes or those encoding in an individual with ovarian cancer 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.

Since the genes used in the present invention, the proteins they encode, and the terms "increase in expression level" and "decrease in expression level" of the present invention have already been described in detail, their descriptions are omitted to prevent excessive redundancy.

According to a specific embodiment of the present invention, the ovarian cancer that can be diagnosed with the composition of the present invention is high-grade serous ovarian cancer (HGSOC).

As used herein, the term "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.

According to another aspect of the present invention, 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

We found that FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP and CD14 in patients with ovarian cancer, especially high-grade serous ovarian cancer. For the first time, the expression levels of proteins or genes encoding them have a positive correlation with the likelihood of developing ovarian cancer, and the expression levels of APOA1, APOA4 and SERPINF2 proteins or genes encoding them have a negative correlation with the likelihood of developing ovarian cancer. identified. Thus, 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; When 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.

As used herein, 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.

As used herein, 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. A decrease of 10% or more, a decrease of about 20% or more, a decrease of about 30% or more, a decrease of about 40% or more, a decrease of about 50% or more, or a decrease of about 60% or more, but not excluding the range outside this range.

As used herein, 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.

According to a specific embodiment of the present invention, measuring the expression level is performed by mass spectrometry (MS).

In this specification, the term “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. In general, 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.

According to a specific embodiment of the present invention, 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: 1 (FGA): HPDEAAFFDTASTGK

SEQ ID NO: 2 (VWF): HIVTFDGQNFK

SEQ ID NO: 3 (S100A9): NIETIINTFHQYSVK

SEQ ID NO: 4 (VCL): LLAVAATAPPDAPNR

SEQ ID NO: 5 (THBS1): TIVTTLQDSIR

SEQ ID NO: 6 (MPO): VVLEGGIDPILR

SEQ ID NO: 7 (IGFBP2): LIQGAPTIR

SEQ ID NO: 8 (SRGN): IQDLNR

SEQ ID NO: 9 (PF4): TTSQVRPR

SEQ ID NO: 10 (GP1BA): LTSLPLGALR

SEQ ID NO: 11 (LRG1): DLLLPQPDLR

SEQ ID NO: 12 (ARHGDIB): TLLGDGPVVTDPK

SEQ ID NO: 13 (PRG4): DQYYNIDVPSR

SEQ ID NO: 14 (LBP): ITLPDFTGDLR

SEQ ID NO: 15 (PPBP): NIQSLEVIGK

SEQ ID NO: 16 (C3): TGLQEVEVK

SEQ ID NO: 17 (FCGBP): GNPAVSYVR

SEQ ID NO: 18 (CD14): FPAIQNLALR

SEQ ID NO: 19 (APOA1): VQPYLDDFQK

SEQ ID NO: 20 (APOA4): QLTPYAQR

SEQ ID NO: 21 (SERPINF2): EDFLEQSEQLFGAK.

As used herein, the term “multiple reaction monitoring” is an analytical technique capable of selectively separating, detecting, and quantifying a specific analyte and monitoring its concentration change. In a method capable of accurately performing multi-measurement, parent ions among the ion fragments generated in the ionization source are selectively transferred to the collision tube using the first mass filter (Q1). Subsequently, 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. In this way, it is an analysis method with high selectivity and sensitivity that can detect only the information of the target component. 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. In addition, because of its excellent sensitivity and selectivity, 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).

In this specification, the term “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.

In the present invention, mass spectrometry for proteins used as biomarkers of ovarian cancer can be performed through Data-Independent Acquisition (DIA). In this specification, the term "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.

According to a specific embodiment of the present invention, 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.

According to a specific embodiment of the present invention, measuring the expression level is performed by mass spectrometry (MS).

According to a specific embodiment of the present invention, 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.

According to a specific embodiment of the present invention, the biological sample is blood.

In this specification, the term “blood” means “whole blood”, and such blood includes plasma and serum.

As used herein, the term “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%.

As used herein, the term “plasma” refers to the liquid component of blood and functions as a transport medium in supplying nutrients to cells and organs of the body.

As used herein, 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.

According to the present invention, 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. Compared to the conventional tissue biopsy procedure, it is non-invasive and has the advantage of minimizing the pain of the patient and obtaining information about cancer more quickly.

According to a specific embodiment of the present invention, the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

Since the meaning of ovarian cancer or high-grade serous ovarian cancer in the present invention has already been described above, description thereof is omitted to prevent excessive duplication.

According to another aspect of the present invention, 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; Alternatively, 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.

Since the genes used in the present invention and the proteins they encode have already been described above, their descriptions are omitted to prevent excessive redundancy.

According to the present invention, 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.

As used herein, 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.

In the present specification, the term "small hairpin RNA (shRNA)" 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. Usually, 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.

As used herein, the term "siRNA" 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.

As used herein, the term “miRNA (microRNA)” 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.

In the present specification, the term “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.

In the present specification, the term “peptide nucleic acid (PNA)” refers to a molecule that has properties of both nucleic acid and protein and can complementarily bind to DNA or RNA. PNA is not found in nature, but is artificially synthesized by chemical methods, and forms double strands through hybridization with natural nucleic acids having complementary nucleotide sequences to regulate the expression of target genes.

As used herein, the term “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. A nucleic acid molecule that inhibits an essential activity for overall biological function. 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.

As used herein, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cut the recognized locus. A typical example of such a gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.

According to the present invention, 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.

Since the antibodies or aptamers that can be used in the present invention have already been described above, their descriptions are omitted to prevent excessive duplication.

As used herein, 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”.

As used herein, the term “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.

As used herein, the term “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. Therefore, the 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. Accordingly, the term "treatment" or "therapeutic agent" in the present specification includes the meaning of "therapeutic aid" or "therapeutic aid".

According to a specific embodiment of the present invention, the ovarian cancer that can be prevented or treated with the composition of the present invention is high-grade serous ovarian cancer (HGSOC).

Since the meaning of ovarian cancer or high-grade serous ovarian cancer in the present invention has already been described above, description thereof is omitted to prevent excessive duplication.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating ovarian cancer comprising the following steps:

(a) 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;

(b) measuring the expression level of the protein or gene in the biological sample;

Reduction in activity or expression of the FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP or CD14 protein or gene in the biological sample Or, if the activity or expression level of the protein or gene of APOA1, APOA4, or SERPINF2 is increased, it is determined as a composition for preventing or treating ovarian cancer.

ovarian cancer pathogenesis-related genes used as markers in the present invention or proteins encoded by them; And ovarian cancer that can be prevented or treated through regulation of their expression has already been described above, so description thereof is omitted to avoid excessive redundancy.

In the present invention, the term “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.

The term "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.

According to a specific embodiment of the present invention, the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

Since the meaning of ovarian cancer or high-grade serous ovarian cancer in the present invention has already been described above, description thereof is omitted to prevent excessive duplication.

According to another aspect of the present invention, the present invention is an ovarian cancer (Ovarian Cancer) to a subject.

According to a specific embodiment of the present invention, in the diagnostic method of the present invention, 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.

According to another aspect of the present invention, 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 features and advantages of the present invention are summarized as follows:

(a) 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.

(b) 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.

FIG. 4A shows treatment with a control siRNA (siControl) or the indicated siRNAs against FGA, VWF and ARHGDIB, together with DMSO; Or, after overexpression of SERPINF2 (n=3 per condition), it is a graph showing the results of measuring the viability value of SK-OV-3 cells through proliferation analysis.

Figure 4b treated SK-OV-3 cells with the indicated siRNAs; Or, after overexpressing SERPINF2 (n=3 per condition), representative images (left) and quantification results (right) of the wound area measured at 0 or 12 hours are shown.

4C shows representative images (left) and resultant graphs (right) of colony formation results of SK-OV-3 cells when treated with the indicated siRNAs or when SERPINF2 was overexpressed (n=3 per condition).

4D shows treatment with control siRNA (siControl) or indicated siRNAs against FGA, VWF and ARHGDIB, together with DMSO; Alternatively, after overexpression of SERPINF2 (n=3 per condition), the figure shows the result of measuring the expression level by Western blotting.

Figure 4e shows the results of measuring the viability values of SK-OV-3 cells through a proliferation assay after treatment with siControl as a control or siRNAs indicated for FGA, VWF and ARHGDIB (n = 3 per condition). This is the graph shown.

Figure 4f shows representative images (left) and quantification results (right) of the wound area measured at 0 or 12 hours after SK-OV-3 cells were treated with the indicated siRNAs (n=3 per condition).

Figure 4g shows representative images (left) and result graphs (right) of colony formation results of SK-OV-3 cells when treated with the indicated siRNAs (n=3 per condition).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method and analysis method

study population

In this study, serum samples from high-grade serous ovarian cancer (HGSOC) patients and controls (HCs) were used. For the population group studied, patients who met the following criteria were identified: (1) patients diagnosed with HGSOC; (2) Those who underwent primary surgical treatment between August 2012 and December 2020 (3) Those who donated a blood sample taken one day before surgery for scientific purposes after providing written informed consent. Meanwhile, patients with malignancies other than HGSOC were excluded.

We collected baseline clinicopathologic characteristics of the patients, such as age at diagnosis, International Federation of Obstetricians and Gynecologists (FIGO) stage, and initial serum CA-125 level. In terms of survival outcome, progression-free survival (PFS) was defined as the time interval from the date of diagnosis to the date of disease progression. Serum samples of the test subjects were frozen and kept fresh, and were recovered from the Human Biobank of Seoul National University Hospital. The control group was healthy people who underwent regular health checkups at Seoul National University Hospital Health Care System Gangnam Center.

reagent

multiple affinity elimination columns (Agilent Technology, Palo Alto, Calif., USA); S-Trap (Protifi, Huntington, NY, USA); triethylammonium bicarbonate buffer (TEAB; Sigma); acetonitrile (ACN; Macron, Avantor Performance Materials, Center Valley, PA); DL-dithiothreitol (DTT; Sigma); iodoacetamide (IAA; Sigma); trifluoroacetic acid (TFA; Pierce, Rockford, IL); Formic acid (FA; Sigma); Dimethyl sulfoxide (DMSO; Sigma) was used.

Serum sample preparation

The 14 most abundant proteins in plasma (albumin, IgA, IgG, IgM, α1-antitrypsin, α1-acid glycoprotein, apolipoprotein A1, apolipoprotein A2, complement C3, transferrin) using a multiple affinity removal column (MARS14) , α2-macroglobulin, transthyretin, haptoglobin and fibrinogen) were removed. An aliquot of 40 μL 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 μL 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 μL of 50 mM TEAB solution and sonicated for 10 minutes. 100 μg 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. Briefly, after loading, 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.

Comprehensive Serum Proteome Profiling Experiments for Serum Spectral Library Generation

For serum proteome profiling experiments, a total of 157 serum peptide samples were combined to create 6 pooled sample groups (Benign, Clear Cell, Endometriod, High-Grade Serous, Mucinous and Healthy).

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 In summary, the online NCFC device consisted of a mid-pH RP column (150 μm × 50 cm) and two NCFC valves (25-port, 1-channel, C5M-66024D, VICI) interconnected by 24 fractional loops (200 μm). For the dual online liquid chromatography system, two SPE columns (150 μm × 3 cm) and two analytical columns (75 μm × 150 cm) were used. All columns were built in-house, packed with Jupiter C18 material (3 μm, 300 Å, Phenomenex), and heated at 60 °C.

Serum peptides (25 μg, 40 μg or 50 μg) 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 μL/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. During mRP gradient elution, two NCFC valves were rotated simultaneously at 1-minute intervals for 5 cycles to include 5 discontinuously connected 1-minute eluates in each of the 24 fractionation loops. For two-dimensional separation, 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). Using low-pH solvent A (water solvent, 0.1% formic acid) and 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 μL/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 Exploris™ 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. For each CV, full MS scans (300–1400 Th) were obtained at a resolution of 60,000 and the full MS AGC target was 500 with an IT of 20 ms. An isolation window of 1.2 Th and a normalized collision energy (NCE) of 28 were used for high-energy collision dissociation (HCD). 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. For protein inference, bipartite graph analysis was used. A protein with the most sibling peptides was selected as a representative protein in each protein group. When more than one protein has the same number of sibling peptides, the protein with the highest sequence coverage was selected as the representative protein. In addition, proteins with unique peptides were regarded as separate representative proteins.

A spectral library database was constructed using representative MS/MS spectra of the final peptides. In summary, the MS2 spectrum with the highest search score (i.e., -log(SpecE-value)) for each peptide ion was selected as a representative MS/MS spectrum. 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. Finally, 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.

Data-independent acquisition methods and data processing

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 Acclaim™ Pepmap RSLC C18 column (150 mm × 150 μm i.d., 2 μm, 100 Å) equipped with a C18 Pepmap trap column (20 mm × 100 μm i.d., 5 μm, 100 Å; Thermo Scientific). Separation was performed over 60 minutes (1 μL/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 Exploris™ 480 mass spectrometer with an EASY-SPRAY™ source (Thermo Fisher Scientific). Mass spectrometry runs were run in DIA mode. For the DIA experiment, the overall MS resolution was set to 60,000, the overall MS AGC target was 300% with an IT of 25 ms, and 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. The default settings were used except that no gas flow was applied and the spray voltage was set to 2.5 kV for FAIMS with dual CVs of -35V and -45V. 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.

differential Expression analysis

An integrative statistical method was applied to define differentially expressed peptides and proteins (DEPeptides and DEProteins, respectively). Briefly, for each peptide or protein, 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. Finally, we identified DEPeptides with an overall p-value <0.05 for each comparison and an absolute log2-median-ratio greater than the mean of the 10th and 90th percentiles of the empirical distribution. We found that the t-test p-value was <0.05, the absolute log2-median-ratios value was greater than the mean of the 10th and 90th percentiles of the empirical distribution for log2-median-ratios, and the number of consistent DEPeptides (the DEProteins were defined as number of consistent DEPeptides)≥1. In each comparison, only peptides and proteins expressed in more than half of the total samples of the two test groups were used for hypothesis testing.

path analysis

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-β 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.

Multiple and parallel reaction monitoring assays

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 μm i.d., 3.5 μm, 100 Å; Agilent) using a 5-40% acetonitrile gradient in 0.1% formic acid over 15 min (20 μL/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 Exploris™ 480 mass spectrometer. Trypsinized peptides were prepared on an Acclaim™ Pepmap RSLC C18 column (150 mm × 150 μm i.d., 2 μm, 100 Å) equipped with a C18 Pepmap trap column (20 mm × 100 μm i.d., 5 μm, 100 Å, Thermo Scientific) at 50 °C in 0.1% formic acid and Separation was carried out over 60 min (1.5 μL/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. For the PRM experiment, the overall MS resolution was set to 60,000, the overall MS AGC target was set to 300 with an IT of 25 ms, and the m/z range was set to 280-1,200. MS/MS resolution was set to 15,000, IT was set to 22 ms, and 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.

In vitro functional analysis

cell culture

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.

Plasmid and siRNA transfection

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):

Negative Control 5'-CCUCGUGCCGUUCCAUCAGGUAGUU-3' (SEQ ID NO: 22),

siFGA-1 5'-CUCUGUAUCUGGUAGUACUUU-3' (SEQ ID NO: 23),

siFGA-2 5'-GGAUUUAGGCACAUUGUCUUU-3' (SEQ ID NO: 24),

siVWF-1 5'-CCUUCUGAGCCCACAAUAAUU-3' (SEQ ID NO: 25),

siVWF-2 5'-GCUGUAAGUCUGAAGUAGAUU-3' (SEQ ID NO: 26),

siARHGDIB-1 5'-GGAAAUGGACAAAGAUGAUUU-3' (SEQ ID NO: 27) and

siARHGDIB-2 5'-AAACCAUUGUGUUAAAGGAUU-3' (SEQ ID NO: 28).

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.

western blotting

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. 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). For Western blotting, the following antibodies were used: FGA (Mα 1:500; ab92572, Abcam), VWF (Rα 1:500; ab189500, Abcam), ARHGDIB (D4GDI 1:1000; ab181252, Abcam), α-Tubulin DM1A Ma 1:1000; Cell Signaling Technology Inc.), FLAG (GT231 Mα 1:1000; GTX629631, GeneTex Inc), anti-mouse IgG antibody HRP (1:3000; 170-6516, BIORAD) and anti-rabbit IgG antibody HRP (1:3000; 170-6515, BIORAD).

cell proliferation assay

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 μl 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.

Wound Healing Assay

Cell migration was assessed by scratching SK-OV-3 cells in 12-well plates cultured at a density of 500 cells per well. Wound healing image snaps were acquired at 0 and 12 h time points using a LEICA DMi1 microscope system with a 10×/0.22 PH1 lens and an MC170 HD camera. Experiments were performed in triplicate through three wells. The average width of the wound closure at 4 different locations in each experiment was imaged using ImageJ software.

Colony formation assay

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.

Statistical analysis of in vitro functional analysis

Two-tailed t-tests were used to estimate the statistical significance of all functional test results. A p-value of less than 0.05 was considered significant and each experiment was performed in three biological replicates.

Experiment result

Characterization of the study population

For the discovery of potential diagnostic biomarkers for HGSOC, 54 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. During the interim observation period of 25 months, 42.3% of HGSOC patients in the discovery set and 57.1% in the validation set experienced recurrence after primary treatment surgery.

Comprehensive OC serum spectral library

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 ). A total of 216 LC-FAIMS-MS/MS global proteome datasets were obtained, and 137,086 peptides and 12,066 protein groups were identified through MS-GF+ search. The identified serum proteome was estimated to cover approximately 8 orders of dynamic ranges (Ceruloplasmin at 440 μg/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. Among tandem spectra of identified serum peptides, 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.

Discovery of potential diagnostic biomarkers for HGSOC

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). For downstream analysis, 14,755 peptides and 951 proteins quantified in more than half of the total samples in both HGSOC and HC groups were used. Differential expression analysis was then performed to identify potential diagnostic biomarkers for HGSOC. As a result, compared to HC, 100 up-regulated and 54 down-regulated proteins were identified in the serum of HGSOC patients (Fig. 2c).

Pathway analysis revealed that up-regulated proteins were involved in complement and coagulation cascades, platelet activation and aggregation, toll-like receptor 4 (TLR4) signaling, neutrophil extracellular trap (NET) formation, as well as insulin-like growth factor (IGF) and It has been confirmed that it is involved in immune processes including growth factor β (TGF-β) signaling and extracellular matrix (ECM) organization. On the other hand, proteins involved in Fc gamma receptor (FCγR) activation and plasma lipoprotein assembly were significantly down-regulated (FIG. 2d).

Next, we constructed a network model describing the interactions between proteins involved in the process overrepresented in HGSOC serum (Fig. 2e). 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. showed

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 FCγR-mediated phagocytosis (IGLC2/3/HG2/HG3/KC/KV1-5/KV1D-33/KV3-20/KV3D-20/KV4-1) were co-regulated. In addition, it was confirmed that upregulation of FCGBP together with activation of TLR4 signaling can cause macrophage recruitment to the tumor microenvironment (TME). In addition, 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. .

Finally, the network model including IGF, TGF-β 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-β signaling (LRG1, LTBP1, THBS1, PRG4 and SERPINE1) collectively contribute to cancer cell proliferation, migration and invasion in HGSOC.

In the network model, 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.

coagulation and complement cascade (VWF, SERPINF2, MBL2, C3 and C4B); NET formation (MPO); 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); and TGF-β signaling (LRG1, LTBP1, THBS1 and PRG4) (shown in red in Fig. 2e).

Validation of potential diagnostic biomarkers for HGSOC

Targeted MS analysis (MRM or PRM) 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.

In vitro functional testing of serum biomarkers on HGSOC

Three strongly up-regulated biomarkers (FGA, VWF and ARHGDIB) and one strongly down-regulated biomarker, under the assumption that the HGSOC serum biomarkers may play a functional role rather than a mere consequence of biological processes regulated in HGSOC. Functional analysis was performed for a marker (SERPINF2). First, the cell survival, migration and colony formation abilities of SK-OV-3 human HGSOC cells expressing control siRNA and siRNAs for each of the three upregulated biomarkers were compared (FIG. 4d). In SK-OV-3 cells, it was confirmed that cell viability, migration, and colony formation ability were significantly reduced by knockdown of FGA, VWF, and ARHGDIB (P <0.05) (FIG. 4a to c and e to g). On the other hand, overexpression of SERPINF2 in SK-OV-3 cells (Fig. 4d) significantly (P <0.05) reduced their viability, migration and colony forming ability (Fig. 4a to c and e to g). Through the above data, it was found that HGSOC serum biomarkers such as FGA, VWF, ARHGDIB and SERPINF2 can play an important regulatory role in cancer cell proliferation and migration in HGSOC.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (17)

1. A composition for diagnosis of ovarian cancer comprising, as an active ingredient, an agent for measuring the expression level of at least one gene selected from the group consisting of FGA , VWF , ARHGDIB and SERPINF2 or a protein encoded by them.
2. According to claim 1, S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA , LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP , CD14 , APOA1 and one or more genes selected from the group consisting of APOA4 or these A composition characterized in that it further comprises an agent for measuring the expression level of the protein encoded as an active ingredient.
3. The composition according to claim 1, wherein the expression level of the FGA , VWF or ARHGDIB gene or the protein they encode is increased, and the expression level of the SERPINF2 gene or the protein they encode is decreased in the subject with ovarian cancer. .
4. The method of claim 2, wherein the expression of the S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA , LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP or CD14 genes or the proteins they encode in an individual with ovarian cancer A composition characterized in that the amount is increased and the expression level of the APOA1 or APOA4 gene or the protein they encode is reduced.
5. The composition according to claim 1, wherein the ovarian cancer is high-grade serous ovarian cancer (HGSOC).
6. A method for providing information necessary for diagnosing ovarian cancer, comprising measuring the expression levels of FGA , VWF , ARHGDIB and SERPINF2 genes or proteins encoded by them in a biological sample isolated from a subject.
7. The method according to claim 6, wherein the step of measuring the expression level is performed by mass spectrometry (MS).
8. 8. The method of claim 7, wherein 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.
9. According to claim 6, S100A9 , VCL , THBS1 , MPO , IGFBP2 , SRGN , PF4 , GP1BA , LRG1 , PRG4 , LBP , PPBP , C3 , FCGBP , CD14 , One or more genes selected from the group consisting of APOA1 and APOA4 or these A method characterized in that it further comprises the step of measuring the expression level of the protein encoded.
10. 10. The method of claim 9, wherein the step of measuring the expression level is performed by mass spectrometry (MS).
11. 11. The method of claim 10, wherein the mass spectrometry is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19 and 20 A method comprising measuring the expression level of one or more peptides.
12. 7. The method of claim 6, wherein the biological sample is blood.
13. 7. The method of claim 6, wherein the ovarian cancer is high-grade serous ovarian cancer (HGSOC).
14. an inhibitor for one or more selected from the group consisting of FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP and CD14; Or 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.
15. 15. The composition according to claim 14, wherein the ovarian cancer is high-grade serous ovarian cancer (HGSOC).
16. A method for screening a composition for preventing or treating ovarian cancer comprising the following steps:

    (a) 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;

    (b) measuring the expression level of the protein or gene in the biological sample;

    Reduction in activity or expression of the FGA, VWF, S100A9, VCL, THBS1, MPO, IGFBP2, SRGN, PF4, GP1BA, LRG1, ARHGDIB, PRG4, LBP, PPBP, C3, FCGBP or CD14 protein or gene in the biological sample Or, if the activity or expression level of the protein or gene of APOA1, APOA4, or SERPINF2 is increased, it is determined as a composition for preventing or treating ovarian cancer.
17. 17. The method of claim 16, wherein the ovarian cancer is High-grade serous ovarian cancer (HGSOC).

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