KR101890392B1 - A composition, and marker for diagnosing ovarian cancer comprising WNT3A - Google Patents

Abstract

A composition, kit and method for predicting the prognosis of ovarian cancer or recurrence of ovarian cancer, and a composition for preventing ovarian cancer recurrence or a composition for preventing recurrence of ovarian cancer, and a method for screening a substance therefor, It is possible to efficiently diagnose recurrence, and to efficiently select candidates that can treat ovarian cancer or prevent recurrence of ovarian cancer.

Description

[A composition, and marker for diagnosing ovarian cancer comprising WNT3A]

A composition, kit, and method for predicting the prognosis of ovarian cancer or the risk of recurrence of ovarian cancer, and a composition for treating ovarian cancer or preventing recurrence of ovarian cancer, and a method for screening a substance therefor.

Ovarian carcinoma is a cancer with the highest mortality rate among female cancers. This high mortality rate is due to the chemoresistance and frequent recurrence of ovarian carcinoma. Several agents for preventing or treating ovarian carcinoma have been developed, but ovarian carcinoma still has a high recurrence rate and mortality rate.

Recently, interest in cancer stem cells (CSC), which exist in a small number in tumors but have high cancer-causing ability, is increasing. CSC was first isolated from ovarian serous carcinoma (OSC) by spheroid formation assay, and it is speculated that these are one of the major causes of cancer recurrence and chemical resistance. However, an effective strategy for reducing ovarian CSC and reducing the high recurrence rate and mortality rate of ovarian cancer has not been developed yet.

One aspect provides a composition for diagnosing the prognosis of ovarian cancer or the risk of ovarian cancer recurrence in a subject.

Another aspect provides a kit for diagnosing the prognosis of ovarian cancer in a subject or the risk of ovarian cancer recurrence.

Another aspect provides a method for diagnosing the prognosis of ovarian cancer or the risk of ovarian cancer recurrence in a subject.

Another aspect provides a composition for treating ovarian cancer or preventing recurrence of ovarian cancer.

Another aspect provides a method of screening an agent for treating ovarian cancer or preventing recurrence of ovarian cancer.

One aspect includes an agent for measuring the expression level of one or more genes selected from the group consisting of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A and PDK4. It provides a composition for predicting the prognosis of ovarian cancer or the risk of ovarian cancer recurrence in an individual.

The gusecoid homeobox (GSC) gene may have an mRNA sequence of NCBI Accession No: NM_173849.2. The VAV3 guanine nucleotide exchange factor (VAV3) gene may have an mRNA sequence of NCBI Accession No: NM_006113.4 or NM_001079874.1. The forkhead box A2 (FOXA2) gene may have an mRNA sequence of NCBI Accession No: NM_021784.4 or NM_153675.2. The lymphoid enhancer-binding factor 1 (LEF1) gene may have an mRNA sequence of NCBI Accession No: NM_016269.4, NM_001166119.1, NM_001130714.2 or NM_001130713.2. The cartilage oligomeric matrix protein (COMP) gene may have an mRNA sequence of NCBI Accession No: NM_000095.2. The azurocidin 1 (azurocidin 1: AZU1) gene may have an mRNA sequence of NCBI Accession No: NM_001700.4. The glutamate receptor, ionotropic, N-methyl D-aspartate 2A (GRIN2A) gene is of NCBI Accession No: NM_001134408.2, NM_000833.4 or NM_001134407.2. It may have an mRNA sequence. The interleukin 17F (IL17F) gene may have an mRNA sequence of NCBI Accession No: NM_052872.3. The CD86 molecule (CD86) gene may have an mRNA sequence of NCBI Accession No: NM_001206925.1, NM_001206924.1, NM_176892.1, NM_175862.4 or NM_006889.4. The peptide YY (peptide YY: PYY) gene may have an mRNA sequence of NCBI Accession No: NM_004160.4. The c-fos-induced growth factor (vascular endothelial growth factor D) (FIGF) gene may have an mRNA sequence of NCBI Accession No: NM_004469.4. The NK3 homeobox 2 (NK3 homeobox 2: NKX3-2) gene may have an mRNA sequence of NCBI Accession No: NM_001189.3. Wingless-type MMRV integration site family member 3A (wingless-type MMTV integration site family member 3A: WNT3A) gene may have an mRNA sequence of NCBI Accession No: NM_033131.3. Pyruvate dehydrogenase kinase, isozyme 4 (pyruvate dehydrogenase kinase, isozyme 4: PDK4) gene may have an mRNA sequence of NCBI Accession No: NM_002612.3.

An increase or decrease in the expression of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 genes is associated with the prognosis of ovarian cancer or recurrence of ovarian cancer. It was discovered by the inventors. Therefore, the expression levels of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 genes can be used to diagnose ovarian cancer prognosis or risk of ovarian cancer recurrence. I can.

In the present specification, GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 proteins are naturally present wild-type GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 and functional variants thereof. In addition, in the present specification, GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 genes are naturally present wild-type GSC, VAV3, FOXA2, It is interpreted to include a gene encoding a LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein and a gene encoding a functional variant thereof.

The expression level may include any expression level of a gene encoding GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein. The expression level may be the level of expression at the level of mRNA or protein. Therefore, the composition is to measure the amount of mRNA, amount of protein, or combination thereof of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene. It may include a formulation for. The agent may be a substance that specifically binds to the transcript of the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene. The agent is a primer, a probe that specifically binds to the mRNA of a gene of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 or a complementary sequence thereof, Or antisense sequences thereof; Or a combination thereof. The agent is a primer, probe, or a primer that specifically binds to the gene encoding the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein. Antisense sequence; Or a combination thereof.

The primer may be one that provides a polymerization initiation point in a polymerization reaction by a polymerase. The primer may be one used in a nucleic acid amplification reaction. The term “amplification” refers to increasing the copy number of a target sequence or its complementary sequence. The nucleic acid amplification reaction may be performed by any method known in the art. Amplification of nucleic acids includes methods that require multiple cycles during amplification or methods performed at a single temperature. Examples of cycling techniques include those requiring thermal cycling. Methods requiring thermal cycling include polymerase chain reaction (PCR). PCR is known in the art. PCR usually involves denaturing double-stranded DNA into single-stranded DNA by heat denaturation, and annealing a primer to the single-stranded DNA; And synthesizing a complementary strand from the primer. The isothermal amplification method is a method in which a single temperature or the main aspect of the amplification process is performed at a single temperature. In contrast to PCR, which heats the reaction product to allow additional primers to bind to separate the double strands, the isothermal method relies on a strand displacing polymerase to separate the double strands and recopy the template. do. The isothermal method can be divided into a method that relies on substitution of a primer to initiate repetitive template copying and a method that relies on continuous reuse or novel synthesis of a single primer molecule. Methods that depend on the substitution of primers include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA), and loop-mediated amplification. It includes a method selected from the group consisting of (loop mediated amplification: LAMP). Methods that rely on continuous reuse or novel synthesis of single primer molecules include methods selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA). The primers may be included in one or two or more sets depending on the selected amplification method. The primer may be a primer for PCR.

The measurement of mRNA expression level was performed in a biological sample to diagnose the prognosis of ovarian cancer or the risk of ovarian cancer recurrence. And as a process of checking the presence and expression of the mRNA of the PDK4 gene, the amount of mRNA may be measured. This can be measured by directly separating the mRNA or using a primer or probe for the mRNA. Analysis methods for this are RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting. ), a nucleic acid microarray including DNA, or a combination thereof. RT-PCR is a method of analyzing RNA. It is a method of amplifying and analyzing cDNA obtained by reverse transcription of mRNA by PCR. In the amplification step of the RT-PCR, a primer pair specifically prepared for the gene is used, and by checking the band pattern and the thickness of the band by electrophoresis after RT-PCR, it is possible to check the mRNA expression and expression level of the gene. And by comparing this with the control group, it is possible to easily determine the prognosis or risk of recurrence of ovarian cancer in an individual. The control may be a normal control or a negative control obtained from a sample of an individual who does not suffer from ovarian cancer or has been cured. The control may also be a positive control obtained from a sample of an individual suffering from ovarian cancer or recurring ovarian cancer.

In the present specification, the term "primer" is a nucleic acid sequence having a free 3-terminal hydroxyl group, which can form a base pair with a template that is complementary to a specific base sequence, and the template strand is copied. It refers to a nucleic acid sequence that acts as a starting point for Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. For example, as a specific primer for the mRNA of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene, having 7 to 50 nucleotide sequences. PCR amplification using sense and antisense primers can be performed to determine the prognosis of ovarian cancer or the risk of ovarian cancer recurrence by measuring the amount of production of a desired product. PCR conditions, the length of the sense and antisense primers can be appropriately selected according to techniques known in the art. The primers are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80 , 20 to 50, or may be one having 20 to 30 nt.

In the present specification, the term "probe" refers to a nucleic acid fragment such as RNA or DNA capable of specifically binding to a target nucleic acid, for example, mRNA, and the presence, content, and expression level of a specific mRNA are confirmed. It may be labeled so that it can be used. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. For example, hybridization was performed using a probe having a nucleic acid sequence complementary to the mRNA of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene. , By measuring the expression level of mRNA through the degree of hybridization, the prognosis of ovarian cancer or the risk of ovarian cancer recurrence of an individual can be confirmed. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art. The probe is 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80 , 20 to 50, or may be one having 20 to 30 nt.

In addition, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known method. In addition, such nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications are methylation, encapsulation, substitution of one or more homologs of natural nucleotides, or modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoroamidate, carbamate. Etc.) or to a charged linker (eg, phosphorothioate, phosphorodithioate, etc.). In addition, the primer or probe may be modified using a label that can directly or indirectly provide a detectable signal. Examples of the label may include radioactive isotopes, fluorescent molecules, or biotin.

The agent may be a peptide or protein that specifically binds to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein or mRNA encoding the same. have. The peptide or protein may be an antibody, a ligand, a receptor, an agonist, an antagonist, or a fragment thereof; Or a combination thereof.

Measurement of protein expression level is performed in biological samples to diagnose the prognosis of ovarian cancer or the risk of ovarian cancer recurrence. It may be a process of confirming the presence and degree of expression of the protein expressed in the WNT3A or PDK4 gene. It is for example directly isolated GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein, or GSC, VAV3, FOXA2, LEF1, COMP, AZU1 , GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein specific binding antibody or fragment thereof using the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, It may be to identify CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 proteins. Analysis methods for this include western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, rocket immunoelectrophoresis. , Tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS, mass spectrometry, magnetic bead-antibody immunoprecipitation, protein chip, or a combination thereof. . For example, the ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, or after reacting with another antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support. An indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody may be included. In addition, after attaching the antibody to the solid support and reacting the sample, a labeled antibody that recognizes the antigen of the antigen-antibody complex is attached to develop enzymatically, or the secondary labeled for the antibody that recognizes the antigen of the antigen-antibody complex. It can be detected by a sandwich ELISA method in which an antibody is attached and colored enzymatically, and the prognosis of ovarian cancer or the risk of recurrence of ovarian cancer can be confirmed by confirming the degree of formation of a complex between the protein and the antibody.

In addition, for example, Western blot analysis using one or more antibodies against the protein may be used. In the Western blot analysis, the whole protein is separated from the sample, electrophoresis is performed, the protein is separated according to size, and then transferred to a nitrocellulose membrane to react with the antibody. Thereafter, by confirming the amount of the protein by a method of confirming the amount of the generated antigen-antibody complex using a labeled antibody, the prognosis of ovarian cancer or the risk of ovarian cancer recurrence of the individual can be confirmed.

The detection method may consist of a method of examining the expression level of the protein in a normal control group that did not have or is cured of ovarian cancer, and did not apply other manipulations, and the level of the protein in a biological sample. Can be expressed as an absolute (eg, μg/ml) or relative (eg, relative intensity of a signal) difference between the above-described proteins.

The term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody can specifically bind to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein or fragment thereof. Protein fragments of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 may be, for example, immunogenic fragments. The fragment has one or more epitopes that can be recognized by antibodies against GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 proteins. Refers to a protein fragment that is present. GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 genes were cloned into an expression vector and GSC, VAV3, FOXA2, LEF1, encoded by the genes, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 proteins are obtained, and antibodies can be prepared from the obtained proteins according to conventional methods in the art.

The form of the antibody includes a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody includes special antibodies such as humanized antibodies. Polyclonal antibodies can be prepared by injecting a biomarker protein or fragment thereof, which is an immunogen, into an external host according to conventional methods known to those skilled in the art. As the external host, mammals such as mice, rats, sheep, and rabbits can be used. When the immunogen is injected by intramuscular, intraperitoneal or subcutaneous injection, it can be administered with an adjuvant to increase antigenicity in general. Thereafter, blood may be regularly collected from an external host to collect serum showing improved titer and specificity for an antigen, or an antibody may be isolated and purified therefrom.

Monoclonal antibodies can be prepared by techniques for generating immortalized cell lines by fusion known to those skilled in the art. Briefly explaining the production method of monoclonal antibody, the protein is purified and an appropriate amount of about 10 μg is immunized to Balb/C mice, or a polypeptide fragment of the protein is synthesized and bound to bovine serum albumin to immunize mice. Then, the antigen-producing lymphocytes isolated from the mouse are fused with human or mouse myeloma to generate immortalized hybridomas, and only hybridoma cells that produce the desired monoclonal antibody are selected and proliferated using an ELISA method. After that, the monoclonal antibody can be isolated and purified from the culture. In addition, monoclonal antibodies can be used by obtaining commercially available antibodies against GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 proteins.

Using these antibodies, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting or immunoblotting on polyacrylic gels known in the art It can be confirmed whether the protein was expressed in a biological sample by a method such as.

The term “fragment” means, for example, a polypeptide that does not have the structure of an intact antibody, peptide or protein, but has a specific antigen-binding site or binding domain directed against an antigenic site. Such fragments include functional fragments of antibody molecules that are not in full form antibodies having two light chains and two heavy chains. The functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') ₂ or Fv. The binding fragment may comprise at least 7 amino acids, for example 9 or more amino acids, 12 or more amino acids.

The expression level may be an expression level in a sample isolated from an individual. The sample may be isolated from the ovary. The sample may be blood, plasma, serum, urine, feces, saliva, tears, cerebrospinal fluid, cells, tissues, or a combination thereof isolated from an individual. The individual may include mammals including humans, primates, dogs, cats, cows, horses, pigs, rabbits, mice, and the like.

Another aspect includes an agent for measuring the expression level of one or more genes selected from the group consisting of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A and PDK4. It provides a kit for diagnosing the prognosis of ovarian cancer in an individual or the risk of ovarian cancer recurrence.

The kit can measure the mRNA expression level of, for example, GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein or a gene encoding it. , May be a microarray. The microarray is easy for those skilled in the art according to a method known in the art using the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 genes. Can be manufactured. The microarray is the mRNA of the gene encoding the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein, or cDNA of the sequence corresponding to the fragment thereof. It may be attached to the substrate as a probe.

When the kit is for measuring the mRNA expression level of, for example, GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene, RT-PCR It may be a kit containing the essential elements necessary to perform. The RT-PCR kit includes test tubes or other suitable containers, reaction buffers, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase, in addition to each primer specific for the mRNA of the marker gene. Inhibitors, DEPC-water, or sterile water. In addition, a primer pair specific to a gene used as a quantitative control may be included.

In addition, the kit is an antibody that specifically binds to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein, for immunological detection of antibodies. It may comprise a substrate, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, or a chromogenic substrate. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, or a glass slide glass. As the color developing enzyme, peroxidase or alkaline phosphatase may be used. FITC or RITC may be used as the fluorescent material. As the chromogenic substrate, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethyl benzidine) may be used.

It is understood that any of the terms or elements mentioned in the kit, such as those mentioned in the description of the composition, are as mentioned in the description of the composition above.

Another aspect is a sample isolated from an individual and GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, and a sample isolated from an individual in order to provide information necessary to diagnose the prognosis of ovarian cancer or the risk of ovarian cancer recurrence Forming a complex thereof by contacting a substance that specifically binds to one or more selected from PYY, FIGF, NKX3-2, WNT3A, or PDK4 protein or mRNA encoding the same; Measuring the level of the complex to measure the expression level of one or more selected from the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 genes; Comparing the measured expression level with the expression level measured in a control; And determining that the prognosis of ovarian cancer of the individual is poor or that the risk of ovarian cancer recurrence is high when the expression level is changed compared to the expression level of the control group. Provides a way to diagnose the risk of

The method specifically binds a sample isolated from an individual to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein or mRNA encoding the same. Contacting the material to form a composite thereof.

The expression level may be an expression level at the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 protein level. Therefore, the measurement is to measure the amount of mRNA, the amount of protein, or a combination thereof of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene. You can do it.

The method also includes measuring the level of the complex to measure the expression level of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene in the sample. Includes.

Measuring the level of the complex may be performed by detecting a signal from a detectable label attached to the protein or a substance that specifically binds to the mRNA encoding the protein. Measuring the level of the complex comprises separating the complex again to measure the level of the protein or mRNA encoding the protein, or measuring the level of a substance specifically binding to the protein or mRNA encoding the protein or the It may be to measure the level of the complex without separating it. The measurement was RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, Nucleic acid microarray including DNA, western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, rocket immunity Electrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS, mass spectrometry, magnetic bead-antibody immunoprecipitation, protein chip, or a combination thereof. I can.

The method includes the measured expression level of the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene with the expression level of the same gene measured in the control group. And comparing.

The method also includes the step of determining, when the expression level of the measured gene is higher than the expression level of the same gene measured in the normal control or negative control, the individual has a poor prognosis of ovarian cancer or a risk of ovarian cancer recurrence. It may include determining that it is high. In addition, the determining step is that when the expression level of the measured gene is equal to or lower than the expression level of the same gene measured in a normal control or a negative control, the individual does not have a poor prognosis of ovarian cancer or recurrence of ovarian cancer. It may be to include the step of determining that the risk of is low.

The method also includes a change in the expression level of the GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A or PDK4 gene of the individual compared to the expression level of the control group. And determining that the subject has a poor prognosis of ovarian cancer or a high risk of recurrence of ovarian cancer. The change in the expression level is that the expression level of the individual is similar to that of the normal control or negative control, or 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%. , 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more increase, or compared to the positive control Similar level or decreasing by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more May include.

The method may also include evaluating the clinical pathologic parameters of the patient, including chemical resistance of cancer cells, lymph node metastasis, distant metastasis or patient survival.

Among the terms or elements mentioned in the method of diagnosing, as mentioned in the description of the composition or kit, it is understood to be as mentioned in the description of the composition or kit above.

Another aspect provides a composition for treating ovarian cancer or preventing recurrence of ovarian cancer, comprising an agent for inhibiting the expression level of the VAV3 gene.

The agent can directly or indirectly treat ovarian cancer or prevent recurrence of ovarian cancer by providing an effect of inhibiting the spheroid formation ability of cancer cells, inhibiting the formation of cancer cell colonies, inhibiting the proliferation or growth of cancer cells, or improving drug sensitivity to other anticancer agents. have. The cancer cells may be CSC.

The agent may specifically bind to a protein of the VAV3 gene or an mRNA sequence encoding it. The agent may inhibit the expression of the VAV3 gene by binding to the protein of the VAV3 gene or an mRNA sequence encoding the same.

The agent may be a nucleotide complementary to the sequence of the VAV3 gene. The nucleotides are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80 , May have a length of 90 or 100 nt. The nucleotide may be a sequence having a sequence homology of 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or more with the sequence of the VAV3 gene. , Includes all sequences capable of complementary binding to the VAV3 gene sequence. The nucleotide may be siRNA. The nucleotide may include the sequence of SEQ ID NO: 1 or 2.

The agent may be an antibody or fragment thereof that specifically binds to the VAV3 protein.

Among the terms or elements mentioned in the composition for treating ovarian cancer or preventing recurrence of ovarian cancer, the same as those mentioned in the description of the diagnostic composition, kit, or method, described above for the diagnostic composition, kit, or method. It is understood to be as mentioned in.

Another aspect includes administering a candidate substance to the subject; Forming a complex thereof by contacting the sample isolated from the individual with a substance that specifically binds to the VAV3 protein or the mRNA encoding the VAV3 protein; Measuring the level of the complex to measure the expression level of the VAV3 gene in the sample; Comparing the measured expression level with the expression level of the VAV3 gene measured in a control; And determining that the candidate substance is effective for treating ovarian cancer or preventing recurrence of ovarian cancer when the expression level changes compared to the expression level of the control group. It provides a method for screening.

The method includes administering a candidate substance to the subject. The candidate substance may be any substance expected to be effective in treating ovarian cancer or preventing recurrence of ovarian cancer. The route for administering the candidate substance may be appropriately selected according to the substance to be selected. The route of administration may be by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The substance can be administered systemically or locally, for example to the ovary.

The method includes determining that the candidate substance is effective in treating ovarian cancer or preventing recurrence of ovarian cancer when the expression level of the VAV3 gene of the individual is changed compared to the expression level of the control group. The change in the expression level is that the expression level of the individual is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% compared to the control, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000% or more, or 1%, 2%, 3%, 4% , 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% or more.

Among the terms or elements mentioned in the above method of screening, such as those mentioned in the description of the composition or method of diagnosing, it is understood that they are as mentioned above in the description of the composition or method of diagnosing.

The composition according to an aspect may be effectively used to diagnose the prognosis of ovarian cancer or the risk of ovarian cancer recurrence in an individual.

The kit according to another aspect can be effectively used to diagnose the prognosis of ovarian cancer or the risk of ovarian cancer recurrence in an individual.

The method according to another aspect can be effectively used to diagnose the prognosis of ovarian cancer or the risk of ovarian cancer recurrence in an individual.

The composition according to another aspect may be effectively used to treat ovarian cancer or prevent recurrence of ovarian cancer.

According to the method of screening a substance for treating ovarian cancer or preventing recurrence of ovarian cancer according to another aspect, it is possible to efficiently select a candidate substance capable of treating ovarian cancer or preventing recurrence of ovarian cancer.

1 shows CSC cells isolated from primary cells cultured from 17 OSCs by spheroid formation assay. 1A is a phase difference image of the primary culture of OSC cells, (a) is a low view, (b) is a high view, and (c) is a spheroid morphology of the power view. FIG. 1B shows that the spheroid-forming ability of primary cancer cells is constant between the first and second generation at 2.17%-2.37%, indicating that the spheroid-forming cells have self-renewing properties. Figure 1C shows that the blood-collapsed expression of stem cell markers was detected in SFC cells (*P<0.05). Markers of stem cells known in the art were used, indicating that all markers except CD117 are significantly overexpressed in SFC. This indicates that SFCs are abundant in CSCs compared to cultured primary cells.
2 shows cDNA microarray analysis of OSC SFCs and corresponding primary cancer cells. FIG. 2A shows hierarchical clustering of genes having a difference in expression level of 15 times or more between SFC and primary cancer cells. Samples on the X-axis and genes on the Y-axis are listed, respectively, and clustered expression data are shown on the heat map. The relative gene expression levels of the SFC sample compared to the corresponding primary cancer cells are shown in grayscale from light (down) to dark (up). 28 genes were highly expressed in all 4 SFC samples compared to control cells, and 34 genes were expressed lower in all 4 SFC samples compared to control cells. The pie chart of FIG. 2B shows the gene ontology in which the expression was changed more than twice in SFC (P<0.05). Genes related to apoptosis and proliferation account for 13%, genes related to cell cycle 10%, genes related to signal transduction and CSC account for 8%, and genes related to drug response account for 5%. Figure 2C shows the confirmation by qRT-PCR of a gene whose expression is remarkably increased in a cDNA microarray. qRT-PCR was performed on 17 pairs of SFC and its paternal cancer cells, GSC (4.26 times), VAV3 (7.05 times), FOXA2 (12.06 times), LEF1 (17.26 times), COMP (21.33 times), GRIN2A (9.36 times) ), CD86 (23.14 fold), PYY (4.18 fold), NKX3-2 (10.35 fold), and PDK4 (74.26 fold) were significantly increased in SFC compared to their paternal cancer cells (*P<0.05).
3A shows the mRNA expression levels of genes selected by qRT-PCR. It indicates that the mRNA expression level of 13 genes was significantly increased in SFC compared to primary cancer cells. 11 genes were significantly overexpressed in OSC compared to the control fallopian tubes (*P<0.05). 3B shows that the LEF1, PYY, NKX3-2 and WNT3A genes significantly increased the relative mRNA levels in chemo-resistant cancer cells compared to chemo-sensitive cancer cells. Figure 3C shows the correlation between the clinical pathological parameters and the mRNA expression level confirmed by qRT-PCR. (a) shows that the increased expression level of PYY (more than 2 times) was significantly associated with lymph node metastasis (*P<0.05). (b) shows that an increase in the expression level of VAV3 (more than 2 times) is associated with distant metastasis (*P<0.05). (c) shows that the increase in the expression level of PYY (more than 2 times), FOXA2 (more than 6 times), and NKX3-2 (more than 10 times) is associated with an increase in chemical resistance of OSC, respectively (*P<0.05) .
4A shows immunohistochemical staining of OSC and control tissues. Figures (a,b) indicate that FOXA2 was positive in the nucleus, but negative in the fallopian tube in OSC staining for FOXA2. (c,d) LEF1 shows positive in the nucleus in OSC staining, but negative in the fallopian tube. (e,f) Shows positive in nuclear and cytoplasmic reactivity in OSC to VAV3, but negative in fallopian tube. (g,h) NKX3-2 staining was observed in the nucleus in the OSC, but not in the fallopian tube. (i,j) Membrane staining for Wnt3A was observed in the OSC, but only lumen staining was observed in the fallopian tube. (k,l) PYY-positive staining in OSC was observed, indicating negative in the fallopian tube. Figure 4B shows the survival curve according to the immunohistochemistry of VAV3 and FOXA2. It is shown that patients with high levels of VAV3(a) or FOXA2(b) expression have lower survival rates than those with low levels of VAV3 or FOXA2 expression (P<0.05).
5 shows the effect of VAV3 knockdown on PTX-resistant ovarian cancer cells. 5A shows the results of analysis of spheroid formation. After VAV3 siRNA addition, the number of spheroid formations was reduced by 30% compared to the negative siRNA control. The size of spheroids was also significantly reduced in SKpac cells to which VAV3 siRNA was added compared to the negative siRNA control. The graph represents the mean ± standard error. (*P<0.001). 5B shows the colony formation assay. VAV3 knockdown cells were dispensed at 300 cells per well and cultured for 14 days. The number of colonies of SKpac cells to which VAV3 siRNA was applied was reduced by 41% compared to control cells to which negative siRNA was applied. The graph represents the mean ± standard error of 3 replicate experiments (*P<0.001). Figure 5C shows the MTT analysis. Cell viability was evaluated after addition of VAV3 siRNA. Cell viability decreased by 25% and 18%, respectively, after 24 and 48 hours compared to the negative siRNA control (*P<0.05). After addition of VAV3 siRNA and 40 nM PTX, the number of SKpac cells decreased by 27% and 16% at 24 and 48 hours, respectively, compared to the control group with PTX and negative siRNA (*P<0.05). The graph represents the mean±standard error of 3 replicate experiments.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Selection of markers indicating poor prognosis of ovarian cancer

1. Materials and Experiment Methods

(1) Patient and tissue samples

For the isolation and evaluation of CSCs, tumor cells from 17 highly advanced OSC ovarian cancer patients were acquired and cultured during oophorectomy. Analysis of spheroid formation was performed using these cultured primary cancer cells.

Thirty-six fresh tissue samples were obtained from patients with highly OSC, and qRT-PCR was performed to confirm the expression of mRNAs differently expressed in cDNA microarrays using this. Table 1 below shows the clinical pathological characteristics of the patients.

A fallopian tube from a patient undergoing hysterectomy with tubal resection due to benign uterine fibroids was used as a health control.

For immunohistochemical analysis, formalin-fixed paraffin-embedded tissues from 74 highly OSC patients treated at the Bundang Cha Medical Center were used. The clinical pathological characteristics of the patients are shown in Table 1 below.

Histological diagnosis and clinical stage were classified according to the World Health Organization (WHO) classification. The histological stage followed a two-stage grading system, and the tumor stage followed the tumor-lymph node-metastasis (TNM) stage method.

The samples were divided into chemosensitive and chemoresistant groups according to their responsiveness to the postoperative primary chemotherapy regimen (taxol/platinum-based combination therapy).

This study was approved by the ethics council of Cha Hospital Bundang, and was conducted with prior consent from all patients.

[Table 1] Clinical pathologic characteristics of patients used in quantitative real-time PCR analysis and immunochemical tissue staining

IHC: immunochemical tissue; qRT-PCR: quantitative real-time PCR

(2) Primary cell culture and separation of spheroid-forming cells

Primary tumor cells were obtained from 17 patients with advanced OSC during oophorectomy. The tumor mass was cut into small pieces and enzymatically digested with a single-cell suspension. Then, it was cultured in a ^{Ca 2+} /Mg ²⁺ phosphate buffered saline solution containing 50 U/mL collagenase A (Roche, Pleasanton, CA). Cells were cultured with Ber-EP4-coated magnetic Dynabead (Life Technologies, Grand Island, NY) to select epithelial cells, followed by 10% fetal bovine serum, 1% penicillin-streptomycin and 20 ng/mL epidermal growth. It was cultured in RPMI medium (Gibcoo/Life Technologies, Grand Island, NY) containing factor (Life Technologies).

For spheroid formation assay, single cells were harvested with 20 ng/mL epidermal growth factor (Life Technologies), 10 ng/mL basic fibroblast growth factor (Sigma-Aldrich, St Louis, MO), 0.4% fetal bovine albumin (Sigma- Aldrich) and 5 μg/mL insulin (Sigma-Aldrich) in serum-free Dulbecco's modified Eagles's medium/F12 medium (Life Technologies) at ^{a density of 1 ×10 3} cells/cm 2 ultra-low-adhesion 6-well culture plate (Corning) , Acton, MA). The spheroid formation of 50-100 cells was evaluated on the 7th day after dispensing. The efficiency of spheroid formation is defined as cells dispensed per colony/well.

(3) cDNA microarray analysis

The cDNA microarray was performed on four spheroid-forming cell (SFC) samples and corresponding primary cancer cells.

CSC was obtained using the iScript cDNA synthesis kit (Bio-Rad, Reymond, WA). Synthesis and hybridization of the target cDNA probe was performed using Agilent's Low RNA Input Linear Amplification kit (Agilent Technology, Santa Clara, CA). The amplified and labeled cRNA was purified on the cRNA Cleanup Module (Agilent Technology). The fragmented cRNA was pipetted onto directly assembled Human Oligo Microarrays (60K) (Aligent Technology).

Hybridization images were scanned using a DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology). Data normalization and selection of significantly changed genes were performed with GeneSpring GX 7.3 (Agilent Technology). The average of the normalized ratio was calculated by dividing the average normalized signal channel intensity by the average normalized control channel intensity. Functional annotation of the gene was performed according to the Gene Ontology Consortium (www.geneontology.org/index.shtml) by GeneSpring GX 7.3. Gene classification was performed based on DAVID (http://david.abcc.ncifcrf.gov/) and Medline database (www.ncbi.nlm.nih.gov/).

(4) Quantitative real-time PCR

Total RNA was isolated from tissues or primary cells using TRIzol reagent (Life Technologies). First-strand cDNA synthesis was performed using Superscript III kit (Life Technologies). Real-time PCR was performed three times using the CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). 20 μL of final reaction contained 0.5 μL cDNA template, 10 μL TaqMAn Master Mix (Applied Biosystems, Paisley, United Kindom), and 1 μL of primer and probe mixture. Transcription levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Gene expression was calculated using the ^{formula 2 -ΔΔCt.}

(5) Tissue microarray and IHC staining

For IHC analysis, 74 OSC samples were used to construct a tissue microarray. In each sample, two centers of tissue of 3 mm diameter were pierced from each tissue mass, and they were aligned on a paraffin block using a ^{manual microarray device (UNITMA; Quick-RAY TM UNITech Science, Seoul, Korea).} The tissue microarray paraffin compartment was deparaffinized in xylene. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. For antigen recovery, the compartments were microwaved for 15 minutes in 0.1 M citrate buffer (pH 6.0).

Slides were incubated overnight at 4° C. with the following primary antibodies and dilutions: FOXA2 (1:200; Abcam, Cambridge, United Kingdom), LEF1 (1:250; Abcam), PYY (1:50; Abcam), VAV3 (1:50; Novus Biologicals, Littleton, CO), NKX3-2 (1:75; Novus Biologicals), and Wnt3A (1:750; Novus Biologicals). The slides were then incubated for 15-30 minutes at room temperature with a secondary antibody using an HRP polymer Ultravision LP Detection System (Thermo Scientific, Waltham, MA). The compartments were then colored with diaminobenzidine and counter-stained with hematoxylin. IHC staining was interpreted by two independent pathologists.

Protein expression was determined by immunoreactivity score. The percentage of positive cells is 0 (negative), 1 (less than 10% positive cells), 2 (10-50% positive cells), 3 (51-75% positive cells), or 4 (>75% positive cells) Scored as. The staining intensity was divided by 0 (negative), 1 (weak), 2 (medium), or 3 (strong). The final immunoreactivity score was calculated by multiplying the above two types of scores.

(6) Functional analysis for VAV3

Transfection of VAV3 siRNA

In the VAV3 functional analysis, PTX-resistant SKpac cells resulting from continuous exposure of SKOV3 (ATCC, Manassas, VA) to paclitaxel (PTX) at a stepwise increasing concentration for more than 8 months were used. VAV3 siRNA and negative siRNA (normal control) were synthesized (Invitrogen, Carlsbad, CA). The day before transfection, 1×10 ⁵ cells were dispensed into each of the 6-well plates. The next day, the cells were transfected with siRNA oligonucleotides annealed according to the manufacturer's instructions using Lipofectamine 2000 (Invitrogen). After 24 hours and 48 hours of transfection, cells were harvested and the efficiency of VAV3 knockdown after siRNA transfection was confirmed by qRT-PCR. The identified cells are used in subsequent MTT, colony formation, and spheroid-forming assays.

(7) MTT analysis for cell viability

Cells (1×10 ⁴ ) were dispensed into a 96-well culture plate, and then 10 pmol VAV3 siRNA was added to the culture medium for 24 hours and 48 hours. For control cells, 10 pmol of negative siRNA was added into the culture medium. After 24 and 48 hours, the cells were incubated with MTT reagent (0.5 mg/mL) at 37° C. for 4 hours. The resulting formazan crystal was dissolved by adding 100 μL of DMSO to each well.

(8) colony formation analysis

Cells were dispensed at a density of ^{1×10 5} cells per well of each of the 6 well plates. The next day, cells were transfected with siRNA and cultured for 48 hours. The transfected cells were then placed again in a gelatin-coated 6 well culture dish at a density of 300 cells per well. After 14 days, colonies were fixed with 4% paraformaldehyde for 10 minutes and stained with hematoxylin. Groups exceeding 50 cells were counted as colonies.

(9) statistical analysis

Statistical analysis was performed with the SPSS statistics software package (IBM SPSS Statistics Data Editor 20). The relationship between mRNA and protein expression levels and clinical pathologic factors in each tumor group were evaluated by χ ² analysis, Fisher's exact test, or Mann-Whitney test. For survival analysis, Kaplan-Meier method and Cox proportional hazard model were used. The results of each functional analysis were expressed as mean±standard error. Student's t-test was performed and P<0.05 was considered valid.

2. Selection of SFC-specific biomarkers to evaluate the pathological state of ovarian cancer

(1) Confirmation of the presence of a large amount of cancer stem cells in the SFC isolated from the primary cell culture of human ovarian carcinoma

In the spheroid-forming assay, non-adherent spheroid bundles of 50-100 cells were observed 1 week after dispensing (FIG. 1A). After collecting the SFCs, the ability to form spheroids was evaluated. The efficiency of spheroid formation of the inoculated cells was 2.37%±0.4% in the first generation. The above floating spheres were enzymatically digested and single cells were harvested and used to form secondary spheroids under the same culture conditions. The second generation spheroid formation ability (2.17%±0.3%) was similar to that of the first generation (Fig. 1B and Table 2).

[Table 2] OCS's 1st and 2nd generation spheroid formation ability

SCN: serum carcinoma; SD: standard deviation

In order to confirm whether CSC is abundant in SFC, mRNA expression in SFC of well-known stem cell markers was analyzed by qRT-PCR and compared with paternal primary cancer cells. Stem cell markers evaluated were ALDH1, CD24, CD44, CD133, NOTCH3, SOX2, and CD117 . Except for CD117 , all stem cell markers were highly expressed in SFCs than in primary cancer cells (P<0.05). Therefore, it was confirmed that CSCs are abundant in SFCs (Fig. 1C), and these SFCs can be called CSC-like cells.

(2) Confirmation of gene expression profile of CSC-like cells by cDNA microarray

A cDNA microarray was performed to evaluate the gene expression profile of spheroid-forming CSC-like cells. Gene expression was compared between four CSC-like cell samples and their corresponding paternal primary cancer cells. As a result, the expression of 619 genes in CSC-like cells was significantly increased or decreased by at least 5 times or more compared to the control cancer cells (P<0.05). Of these, 381 genes increased and 238 genes decreased. In particular, the hierarchical clustering of 62 genes with a 15-fold or more change is shown in FIG. 2A. This microarray data was provided in accordance with the recommendations of minimum information about a microarray experiment (MIAME), and can be viewed through the Gene Expression Omnibus (GEO) series registration number GSE60765 (www.ncbi. nlm.nih.gov/geo).

Genes that increased or decreased by at least 2 times or more (P<0.05) in CSC-like cells were classified according to biological process gene ontology. 2B is a circular diagram showing the proportion of genes representing each process. The largest number of genes are associated with apoptosis and proliferation (13% each); Cell cycle (10%); CSC (8%) and signaling (8%); Transforming growth factor-β (TGF-β) (6%); Mitogen-activated protein kinase (MAPK) (6%); Tyrosine kinase (6%) and Notch signal (6%); Drug responsiveness (5%); And Wnt signaling (5%); Hedgehog signal (4%); Epithelial-mesenchymal mutation (3%) and Toll-like signal (3%); And genes of the insulin receptor pathway (2%).

Among the CSC markers, CD24 (13.85 fold), ALDH1A1 (6.80 fold) and SOX2 (5.36 fold) were significantly upregulated in CSC-like cells forming spheroids. Cancer-related genes whose expression is increased by at least 5 times compared to the control in CSC-like cells are summarized in Table 3 along with their related functions.

[Table 3] Genes that are known to be related to tumor development and are upregulated by at least 5 times in CSC compared to primary cancer cells

APO: apoptosis; CSC: cancer stem cell marker; CYC: cell cycle; DRU: drug reaction; EMT: epithelial mesenchymal transformation; HED: Hedgehog path; INS: insulin receptor; NOT: Notch signal path; PRO: proliferation; SCT: stem cell transcription; SIG: signaling; TGF: transforming growth factor-β pathway; TOL: Toll-like receptor signaling pathway; TYR: tyrosine kinase signal; WNT: Wnt path.

(3) Selection of 14 genes as biomarker candidate groups and confirmation by qRT-PCR

To identify biomarker candidates for predicting poor prognosis and chemical resistance, 14 genes were selected based on cDNA microarrays and gene ontology. Specifically, cDNA microarrays were used to identify genes whose gene expression was increased by 5 times or more in CSC-like cells, and the most highly expressed oncogenic genes were selected. The relative expression levels of the 14 genes are as follows: GSC (8.39 times), VAV3 (11.75 times), FOXA2 (15.11 times), LEF1 (28.76 times), COMP (15.34 times), AZU1 (13.36 times), GRIN2A (7.34 times), IL17F (17.36 times), CD86 (8.67 times), PYY (11.98 times), FIGF (14.01 times), NKX3-2 (9.43 times), WNT3A (6.19 times), and PDK4 (11.83 times).

In addition, the levels of mRNA expression in SFCs of the 14 genes and their paternal cancer cells were confirmed by qRT-PCR. As a result, GSC (4.26 times; P <0.001), VAV3 (7.05 times; P <0.001), FOXA2 (12.06 times; P <0.05), LEF1 (17.26 times; P <0.05), COMP (21.33 times; P < 0.001), GRIN2A (9.36 times; P <0.001), CD86 (23.14 times; P <0.001), PYY (4.18 times; P <0.001), NKX3-2 (10.35 times; P <0.001), and PDK4 (74.26 times) ; It was confirmed that P <0.001) significantly increased the expression level in the SFC compared to the paternal cancer cells (FIG. 2C).

(4) In chemically resistant cancer cells LEF1, PYY, NKX3-2 And WNT3A Confirmation of an increase in expression level of

To measure the mRNA expression of the candidate genes, qRT-PCR was performed on 36 fresh human OSC tissues. The levels of mRNA expression in OSC tissues were compared with normal fallopian tube control tissues.

As a result, in human OSC, the following 11 genes were significantly overexpressed: VAV3 (7.92 times), FOXA2 (7.32 times), LEF1 (9.64 times), COMP (25.01 times), GRIN2A (12.51 times), IL17F( 2.50 fold), CD86 (6.35 fold), PYY (13.50 fold), NKX3-2 (10.79 fold), WNT3A (15.67 fold), and PDK4 (10.19 fold) (all P<0.05).

Figure 3A shows the relative expression levels in the OSC of 13 candidate gene compared to the control group. There was a significant difference in the expression levels of LEF1, PYY, NKX3-2, and WNT3A (P <0.05). Specifically, in OSC, which is a chemically resistant cancer cell, LEF1 is 2.85 times as compared to that of the chemically sensitive control cells; PYY is 20.03 times; NKX3-2 is 3.02 times; And WNT3A increased the expression level 3.91 times (Fig. 3B).

Additionally, the relationship between mRNA expression level and clinical pathological parameters was analyzed. The increase in the expression level of VAV3 (twice compared to the normal control group) was associated with distant metastasis (P<0.05). Elevated expression of PYY (more than 2 times) was associated with lymph node metastasis (P<0.05) and chemical resistance (P<0.05). The increase in the expression level of FoxA2 (6 times or more) and the increase in the expression level of NKX3-2 (10 times or more) were associated with chemical resistance (P<0.05) (Fig. 3C).

(5) within OSC VAV3, NKX3-2 And LEF1 The association between overexpression and poor prognosis

IHC studies were performed on 74 OSC tissue sample microarrays to confirm the protein expression of candidate genes and their relationship with clinical stage, lymph node and distant metastasis, and clinical pathological parameters such as chemical resistance and survival. Six proteins of LEF1, PYY, NKX3-2, WNT3A6, VAV3 and FOXA2 were selected for the study. LEF1, NKX3-2, PYY and Wnt3A had an immunoreactivity score of 4 or higher; FOXA2 has an immune response score of 2 or higher; And VAV3 was considered to be overexpressed if the immune response score was 6 or higher.

As a result, local or diffuse staining was observed in the cytoplasm or nucleus of the OSC tissue (Fig. 4A). Specifically, FOXA2, LEF1 and NKX3-2 were positive in the nucleus, PYY was positive in the cytoplasm, and VAV3 was positive in both the nucleus and cytoplasm.

Benign serous cystadenoma and epithelial and stromal cells from normal fallopian tubes showed negative or weakly positive (less than 5% positive cells) for FOXA2, LEF1, NKX3-2 and VAV3.

Near-terminal staining for Wnt3A was found in epithelial cells of normal fallopian tubes and benign serous cystic adenoma. On the other hand, membrane staining was observed in OSC tissues.

Most OSCs overexpressed these proteins. FOXA2 in 66.2% (49/74) cases, and LEF1 in 59.5% (44/74) cases, and PYY, NKX3-2 and Wnt3A in 58.1% (43/74) and 58.1% (43/74), respectively. ), and 63.5% (47/74) of cases. The relationship between IHC staining and clinical pathological parameters is shown in Table 4 below. Distant metastasis was significantly associated with the expression of LEF1, VAV3 and NKX3-2 (P<0.05). Chemical resistance was significantly associated with overexpression of NKX3-2 (P<0.05).

[Table 4] Relationship between immunohistochemical expression patterns of OSC and clinical pathological parameters (N=74)

^a : p<0.05. An immunoreactivity score of 2 or greater for FOXA2, 4 or greater for LEF1, PYY, NKX3-2 and Wnt3A, and 6 or greater for VAV3 is considered overexpression.

Example 2. Confirmation of the effect of VAV3 knockdown on CSC using VAV3 siRNA

(1) Confirmation of association between high VAV3 immunoreactivity and poor prognosis of OSC

74 OSC patients were analyzed for survival according to their protein expression. Experimental materials and methods are the same as those described in Example 1 above. Protein expression was analyzed by immunohistochemistry. The mean follow-up period was 31 months (1-112 months). During this observation period, 21 patients (28.4%) died from disease. Kaplan-Meier analysis revealed that VAV3 overexpression (immune response score of 6 or higher) and FOXA2 overexpression (immune response score of 2 or higher) were significantly associated with short survival (OS) (VAV3, OS: Overexpression group 64.2% vs. control 94.4%, P <0.05; FOXA2, OS: overexpression group 63.3% vs. control 88%, P <0.05). The expression of other proteins was not significantly associated with patient survival.

A multivariate Cox regression analysis was performed for clinical pathological parameters and VAV3 and FOXA2 immune responsiveness. VAV3 overexpression was confirmed to be an independent marker for poor prognosis in OSC patients (Table 5; risk rate 15.27, P<0.05). However, FOXA2 overexpression was not significantly associated with OS in this analysis. Chemical resistance was also associated with a low survival rate (hazard rate=17.74, P<0.001).

[Table 5] Multivariate Cox regression analysis on clinical pathological parameters and immunohistochemical expression levels of VAV3 and FOXA2

^a : P<0.05.

(2) VAV3 knockdown inhibits spheroid formation and reduces cancer cell survival and proliferation.

1) Analysis of spheroid formation

When VAV3 siRNA (SEQ ID NO: 1 or 2) was added to cancer cells, in order to confirm the effect of VAV3 siRNA on CSC activity, the number and size of spheroids of VAV3 knockdown cells were compared with a normal control.

As a result, VAV3 siRNA treatment significantly reduced the number of spheroid formations in CSCs (30% reduction compared to the negative siRNA control) (P<0.001), and the size of the spheroids was also significantly reduced (FIG. 5A). That is, it was confirmed that VAV3 knockdown inhibited CSC activity.

2) Colony formation analysis

Colony formation analysis confirmed that the clonal proliferation of SKpac cells was significantly inhibited by VAV3 siRNA. Specifically, the number of colonies of SKpac cells was reduced by 41% compared to the control to which the negative siRNA was added by VAV3 siRNA (P<0.001) (FIG. 5B).

3) PTX drug sensitivity analysis

The effect of VAV3 inhibition on the sensitivity of PTX-resistant SKpac cells to PTX was evaluated. Specifically, cell viability was tested using MTT assay.

As a result, when VAV3 siRNA + PTX was added, the number of SKpac cells was reduced by 27% and 16%, respectively, compared to the control group with PTX + negative siRNA added 24 hours and 48 hours after addition (P<0.05) (Fig. 5C).

<110> SUNGKWANG MEDICAL FOUNDATION <120> A composition, and marker for diagnosing ovarian cancer comprising WNT3A <130> PN122416 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> VAV3 siRNA <400> 1 caggaccaaa gagucaggag aauau 25 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> VAV3 siRNA <400> 2 auauucuccu gacucuuugg uccug 25

Claims (1)

A composition for predicting the prognosis of an individual ovarian cancer or the risk of ovarian cancer recurrence, comprising an agent for measuring expression levels of WNT3A, LEF1, VAV3, AZU1, and GSC genes.

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