KR101601941B1 - MTA3, a marker for predicting the response to anti-cancer drug in a patient with ovarian cancer - Google Patents

Abstract

The present invention relates to a composition, a kit, and a method for predicting the response of an ovarian cancer patient to chemotherapeutic treatment by detecting the methylation level or MTA3 expression level of the CpG region of MTA3 (Metastasis Associated Family, Member 3) gene.

Description

MTA3, a marker for predicting the response to anticancer drugs in a patient with ovarian cancer,

The present invention relates to a composition, a kit, and a method for predicting the response to chemotherapeutic drugs in ovarian cancer patients by detecting the methylation level or MTA3 expression level of the CpG region of MTA3 (Metastasis Associated Family, Member 3) gene from a sample of ovarian cancer patients .

According to the National Cancer Registry annual report (2010 cancer registration statistics), ovarian cancer is the tenth most common cancer in women in Korea, and the number of generators is increasing at an annual rate of 1.6%. In addition, the five-year relative survival rate of cancer patients increased from 63.9% to 73.3%, while the 5-year survival rate of ovarian cancer patients decreased from 61.3% to 60.4% , Are suffering from the treatment of ovarian cancer.

Ovarian cancer is found in only 15% of all patients in the early stages, and more than 65% of all patients are found to have metastasized to other tissues. Currently, the treatment of ovarian cancer is usually performed in combination with platinum-based chemotherapy such as cisplatin and surgical operation to remove metastatic lesion and metastatic lesion such as uterus, ovary, lymph node and the like. However, because the ovaries of both sides are removed during the first operation, the prognosis is not good when recurred after metastatic recurrence. Also, patients who have recurred after treatment often have resistance to chemotherapy, and subsequent treatment is very difficult.

Furthermore, among patients with similar clinical characteristics, the response to chemotherapy varies widely, and even patients with the same stage have significant differences in patient survival. Therefore, numerous studies have been conducted to find molecular biomarkers that better predict the patient's clinical outcome and select patients that are effective in specific therapies.

In the case of patients resistant to anticancer drugs, it is possible that the factors of welfare genetics may play a major role because they are affected by the expression of genes due to other environmental reasons rather than the change of the base sequence, thereby obtaining tolerance. Representative research areas of epigenetic genomics include DNA methylation, methylation of cytosines in DNA sequences, chemical modification of histones, and regulation of expression by small RNAs. However, there have been few developments in biomarkers that can predict the response and survival of cancer chemotherapy by analyzing the linkage between welfare genetic factors and anticancer drug response.

Korean Patent Publication No. KR 2010-0075857 United States patent publication US 2013-0177912 United States patent publication US 2012-0302572 US patent registration US 8323941

Thus, in the present invention, gene expression patterns between cells showing sensitivity to anticancer drugs and cells showing anticancer drug resistance were compared, and genes showing specific expression changes in cells showing anticancer drug resistance were selected. Furthermore, among the genes showing the change in the gene expression, the genes whose methylation changes in the CpG region were confirmed to have an influence on the gene expression were finally selected to identify specific CpG sites that affected the gene expression, It was confirmed that the reactivity of cancer patients with ovarian cancer can be predicted by measuring the degree of methylation occurring at a specific CpG position of the gene, thereby completing the present invention.

Accordingly, one object of the present invention is to provide a method of selecting a member selected from the group consisting of MTA3 (Metastasis Associated Family, Member 3), DLX5 (Distal Less Homeobox 5), TET1 (Tet Methylcytosine Dioxygenase 1) and UCHL1 (Ubiquitin Carboxyl-Terminal Esterase L1) Or an agent for measuring the level of methylation of the CpG region of one or more genes selected from the group consisting of MTA3, DLX5, TET1, and UCHL1. And to provide a composition for predicting reactivity.

It is another object of the present invention to provide a kit for predicting the reactivity of an ovarian cancer patient to an anticancer agent, which comprises the above composition.

It is another object of the present invention to provide a method for detecting the reactivity of an ovarian cancer patient against an anticancer agent comprising a primer, a probe or an antisense oligonucleotide that specifically binds to at least one gene selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 And to provide a microarray for prediction.

It is another object of the present invention to provide a method for predicting the response of an ovarian cancer patient to an anticancer agent, which comprises administering to a patient a sample comprising the CpG region of one or more genes selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 And to provide a method for detecting methylation.

It is another object of the present invention to provide a method for detecting at least one selected from the group consisting of MTA3, DLX5, TET1, and UCHL1 from a sample of a patient in order to provide information necessary for prediction of reactivity to an anticancer agent in ovarian cancer patients .

The present invention is based on the discovery that specific CpG sites of the MTA3, DLX5, TET1 and UCHL1 genes are specifically hypermethylated in anticancer drug resistant cells, whereby the expression of the gene is specifically reduced, and that the CpG site The degree of methylation or the degree of expression of the gene is used as a biomarker to predict the reactivity of an ovarian cancer patient to an anticancer agent.

The CpGs of the MTA3, DLX5, TET1 and UCHL1 genes are specifically hypermethylated in the anticancer drug resistant cells, respectively. Thus, the expression of the gene is specifically reduced, and therefore, each of these genes is used for predicting patient response to anticancer agents Or may be used as a single biomarker, or two or more of them may be used as a complex biomarker.

Thus, in one embodiment, the present invention provides a method for predicting the responsiveness of an ovarian cancer patient to an anticancer agent, comprising the step of determining the level of methylation of the CpG region of one or more genes selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 And a kit comprising the same.

In a preferred embodiment, the present invention relates to a composition for predicting the responsiveness of an ovarian cancer patient to an anticancer agent and a kit comprising the same, which comprises an agent for measuring the methylation level of the CpG region of the MTA3 gene.

In this case, more preferably, the composition and kit may further comprise an agent for measuring the methylation level of the CpG region of one or more genes selected from the group consisting of DLX5, TET1 and UCHL1.

In another preferred embodiment, the present invention relates to a composition for predicting the reactivity of an ovarian cancer patient to an anticancer agent and a kit comprising the same, which comprises an agent for measuring the methylation level of the CpG region of the DLX5 gene.

In this case, more preferably, the composition and kit may further comprise an agent for measuring the methylation level of the CpG region of one or more genes selected from the group consisting of MTA3, TET1, and UCHL1.

In another preferred embodiment, the present invention relates to a composition for predicting the responsiveness of an ovarian cancer patient to an anticancer agent and a kit comprising the same, which comprises an agent for measuring the methylation level of the CpG region of the TET1 gene.

In this case, more preferably, the composition and kit may further comprise an agent for measuring the methylation level of the CpG region of one or more genes selected from the group consisting of MTA3, DLX5 and UCHL1.

In another preferred embodiment, the present invention relates to a composition for predicting the responsiveness of an ovarian cancer patient to an anticancer agent and a kit comprising the same, which comprises an agent for measuring the methylation level of the CpG region of the UCHL1 gene.

In this case, more preferably, the composition and the kit may further comprise an agent for measuring the methylation level of the CpG region of one or more genes selected from the group consisting of MTA3, DLX5 and TET1.

In the present invention, the mRNAs of the MTA3, DLX5, TET1 and UCHL1 genes can be confirmed by their sequence information in a known gene database. For example, the nucleic acid sequence of human MTA3 gene can be found in Genbank registration number NM_001282756.1, the nucleic acid sequence of human DLX5 gene can be confirmed in Genbank registration number NM_005221.5, and the nucleic acid sequence of human TET1 gene can be found in Genbank registration number The nucleic acid sequence of the human UCHL1 gene can be found in Genbank registration number NM_004181.4.

In the present invention, the term "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, the methylation in the present invention means whether methylation occurs in the cytosine of a specific CpG site of a specific gene. When methylation occurs, the binding of the transcription factor is hindered and the expression of the specific gene is inhibited. On the other hand, when the methylation or the low methylation occurs, the expression of the specific gene is increased.

Genomic DNA of mammalian cells contains 5th methyl-cytosine (5-methylcytosine, 5-mC) base with a methyl group attached to the fifth carbon of the cytosine ring in addition to A, C, G and T do. Methylation of 5-methylcytosine occurs only in C of a CG dinucleotide (5'-mCG-3 ') called CpG, and methylation of CpG inhibits expression of repeated sequences of alu or transposon and genome. Further, since 5-mC of the CpG is naturally deaminated and is likely to become a thymine (T), CpG is a site where most of the epigenetic changes occur frequently in mammalian cells.

The term "measurement of methylation level" in the present invention means the measurement of the methylation level of the CpG region of the MTA3, DLX5, TET1 and / or UCHL1 gene and includes methylation-specific PCR such as methylation- chain reaction, MSP), real-time methylation-specific polymerase chain reaction (PCR), PCR using methylated DNA-specific binding protein, or quantitative PCR. Or by automated base analysis such as pyrosequencing and bisulfite sequencing, but are not limited thereto.

Preferably, the CpG site of the MTA3, DLX5, TET1 and / or UCHL1 gene refers to a CpG site present on the DNA of the gene. The DNA of the gene includes a promoter region, an enhancer region, a protein coding region (open reading region), and a promoter region, which are necessary to express the gene and include a series of constituent units operatively linked to each other. frame, ORF) and a terminator area. Thus, the CpG site of the MTA3, DLX5, TET1 and / or UCHL1 gene may be present in the promoter region, enhancer region, protein coding region (ORF) or terminator region of the gene.

Preferably, in the present invention, measuring the methylation level of the CpG region of the MTA3 gene may mean measuring the methylation level of the enhancer region of the MTA3 gene, and determining the methylation level of the MTA3 gene at 42794620 to 42795547 (928 bp) regions of chromosome 2 RTI ID = 0.0 > cytosine < / RTI > In the present invention, the 42794620 to 42795547 base of the above chromosome 2 is represented by SEQ ID NO: 1.

Also, preferably, in the present invention, measuring the methylation level of the CpG region of the DLX5 gene may mean measuring the methylation level of the enhancer region of the DLX5 gene, and 96651477 to 96652214 (738 bp) of chromosome 7 (Chr7) RTI ID = 0.0 > cytosine < / RTI > In the present invention, the 96651477 to 96652214th base of the above chromosome 7 is represented by SEQ ID NO: 2.

Further, preferably, in the present invention, the methylation level of the CpG region of the TET1 gene may be measured by measuring the methylation level of the promoter region of the TET1 gene, and the cytotoxicity of the 70321183 to 70322935 regions of chromosome 10 (Chr10) This may mean measuring the level of methylation of the god. In the present invention, nucleotides 70321183 to 70322935 of the chromosome 10 are shown in SEQ ID NO: 3.

Also, preferably, in the present invention, measuring the methylation level of the CpG region of the UCHL1 gene may mean measuring the methylation level of the promoter region of the UCHL1 gene, and measuring the methylation level of the 41258314 to 41259927 regions of chromosome 4 (Chr4) This may mean measuring the level of methylation of the god. In the present invention, nucleotides 41258314 to 41259927 of the chromosome 4 are represented by SEQ ID NO: 4.

In the present invention, the nucleotide sequence of the human genome chromosome region is expressed according to the latest version of The February 2009 Human reference sequence (GRCh 37) The specific sequence of the human genome chromosome region may be changed somewhat as the result of the genome sequence study is updated, and the expression of the human genome chromosome region of the present invention may be different according to the modification. Accordingly, the human genome chromosome region expressed in accordance with The February 2009 Human reference sequence (GRCh 37) of the present invention has been updated since the filing date of the present invention so that the expression of the human genome chromosome region is now It is obvious that the scope of the present invention extends to the modified human genome chromosome region. Such modifications are readily apparent to those skilled in the art to which the present invention pertains.

The present inventors compared gene expression patterns between cells showing sensitivity to anticancer drugs and cells showing anticancer drug resistance and selected genes showing specific expression changes in cells showing resistance to anticancer drugs. In addition, among the genes showing changes in the gene expression, the genes whose methylation changes in the CpG region were confirmed to have an influence on the gene expression were finally selected to identify specific CpG sites that affected the gene expression, It has been confirmed that the reactivity of cancer patients with ovarian cancer can be predicted by measuring the degree of methylation occurring at a specific CpG position of the gene.

Thus, hypermethylation of DNA methylation at the CpG positions of MTA3, DLX5, TET1, and / or UCHL1 can be used as a biomarker to predict the response of cancer patients to ovarian cancer.

In the present invention, the patient refers to a patient who has developed ovarian cancer. A gene sample is obtained from tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine obtained from these ovarian cancer patients. The gene sample includes cDNA synthesized from DNA, mRNA or mRNA.

As used herein, the term "prediction of responsiveness to an anticancer agent" means predicting whether a patient will preferentially or non-preferentially respond to the anticancer agent treatment, or predicting the risk of the anticancer agent resistance. The predictive methods of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment regimen for patients with ovarian cancer. Preferably, the anticancer agent may be a platinum-based anticancer agent, for example, cisplatin.

In general, ovarian cancer is treated with chemotherapy, especially platinum-based chemotherapy combined with cisplatin. However, among patients with similar clinical features or similar stages, chemotherapy There is a wide range of responses and significant differences in survival. By using the method of the present invention, the responsiveness to chemotherapy can be easily predicted, the survival prognosis can be determined, and it is possible to decide whether or not to use additional treatment methods. This increases the survival rate after the onset of ovarian cancer.

In addition, since the abnormal methylation change of cancer-resistant cancer cells exhibits significant similarity to the methylation change of genomic DNA obtained from a biological sample such as tissue, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, When using the marker, it is possible to easily diagnose the reactivity of the patient to the anticancer drug through blood or body fluids.

In the present invention, the agent for measuring the methylation level of the CpG region may be a compound that modifies an unmethylated cytosine base or a primer specific for a methylated sensitive restriction enzyme, a methylated allele sequence of MTA3, DLX5, TET1 and / or UCHL1 gene, And primers specific for unmethylated allele sequences.

The compound capable of modifying the unmethylated cytosine base may be bisulfite or a salt thereof, but is not limited thereto, preferably sodium bisulfite. Methods for detecting the methylation of a CpG site by modifying an unmethylated cytosine residue using such a bisulfite are well known in the art (WO01 / 26536; US2003 / 0148326A1).

In addition, the methylation sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting the methylation of the CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like. The methylation or non-methylation of the restriction enzyme recognition site at C may or may not be cleaved by restriction enzymes and may be detected by PCR or Southern blot analysis. Other methylation sensitive restriction enzymes than the above restriction enzymes are well known in the art.

Representative methods for determining the level of methylation at the CpG site of the patient's MTA3, DLX5, TET1, and / or UCHL1 genes include obtaining genomic DNA from a biological sample of a patient and modifying cytosine bases that are not methylated in the resulting DNA A compound or a methylation-sensitive restriction enzyme, amplifying the treated DNA by PCR using a primer, and confirming presence or absence of the amplified product.

Thus, the formulations of the present invention may include primers specific for the methylated allele sequence of the MTA3, DLX5, TET1, and / or UCHL1 genes, and primers specific for unmethylated allele sequences. In the present invention, the term "primer" means a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free 3-terminal hydroxyl group and serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. Also, primers can incorporate additional features that do not alter the primer properties of primers that serve as a starting point for DNA synthesis, as sense and antisense nucleic acids with 7 to 50 nucleotide sequences. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary that it can hybridize with the template. The position of the primer or the primer binding site refers to a target DNA fragment to which the primer hybridizes.

The primer of the present invention can be preferably designed according to the sequence of a specific CpG site to be subjected to methylation analysis and includes a pair of primers capable of specifically amplifying cytosine that has been methylated and not modified by bisulfite, And a primer pair that is not methylated and can specifically amplify cytosine modified by bisulfite.

In addition to the above preparation, the composition and kit may further contain a polymerase agarose, a buffer solution necessary for electrophoresis, and the like.

In another aspect, the present invention provides a method for predicting the response of an ovarian cancer patient to an anticancer agent, comprising the steps of: (a) detecting a CpG site of one or more genes selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 Lt; RTI ID = 0.0 > methylation. ≪ / RTI >

For example, the present invention provides a method for diagnosing ovarian cancer, comprising the steps of: measuring the methylation level of the CpG region of the MTA3 gene from a sample of an ovarian cancer patient; And comparing the level of methylation with the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises the steps of: measuring the methylation level of the CpG region of the at least one gene selected from the group consisting of DLX5, TET1 and UCHL1 from the patient sample; And comparing the methylation level to the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample.

In another aspect, the present invention provides a method for diagnosing ovarian cancer, comprising the steps of: measuring the methylation level of the CpG region of the DLX5 gene from a sample of an ovarian cancer patient; And comparing the level of methylation with the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises: measuring the methylation level of the CpG region of the at least one gene selected from the group consisting of MTA3, TET1 and UCHL1 from the patient sample; And comparing the methylation level to the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample.

In another aspect, the present invention provides a method for diagnosing ovarian cancer, comprising the steps of: measuring the methylation level of CpG region of TET1 gene from a sample of ovarian cancer patient; And comparing the level of methylation with the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises: measuring the methylation level of the CpG region of the at least one gene selected from the group consisting of MTA3, DLX5 and UCHL1 from the patient sample; And comparing the methylation level to the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample.

In another aspect, the present invention provides a method for detecting the level of methylation of a CpG region of a UCHL1 gene from a sample of an ovarian cancer patient; And comparing the level of methylation with the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises the steps of: measuring the methylation level of the CpG region of the at least one gene selected from the group consisting of MTA3, DLX5 and TETl from the patient sample; And comparing the methylation level to the methylation level of CpG of the corresponding gene of the anti-cancer agent sensitive control sample.

The term "patient sample" in the present invention refers to a sample such as a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine which have different levels of methylation of MTA3, DLX5, TET1 and / or UCHL1 gene due to anticancer drug resistance But are not limited thereto.

First, to obtain genomic DNA from a patient and measure the methylation level, the genomic DNA can be obtained by the phenol / chloroform extraction method, SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), Cetyl Trimethyl Ammonium Bromide (Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

The step of measuring the methylation level of the CpG region of the gene comprises, more particularly, (a) treating the genomic DNA in the obtained sample with a compound that modifies the unmethylated cytosine base or a methylation-sensitive restriction enzyme; And (b) amplifying the treated DNA by PCR using a primer capable of amplifying a specific CpG region of the gene.

The compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite. Methods for detecting the methylation of a particular CpG site by modifying an unmethylated cytosine residue using such a bisulfite are well known in the art.

In addition, as described above, the methylation-sensitive restriction enzyme in step (a) may be a restriction enzyme capable of specifically detecting methylation of a specific CpG site and may be a restriction enzyme containing CG as a recognition site of a restriction enzyme For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like.

In the step (b), the amplification may be performed by a conventional PCR method. As described above, the primers used herein can be desirably designed according to the sequence of a specific CpG site to be subjected to methylation analysis, and can be specifically amplified by specifically amplifying cytosine which has been methylated and not modified by bisulfite And a primer pair capable of specifically amplifying cytosine modified by bisulfite without being methylated.

The step of measuring the methylation level of the CpG region of the gene may further include the step of (c) confirming presence or absence of the amplified product in the step (b). The presence or absence of the amplified product in the step (c) may be performed according to a method known in the art, for example, electrophoresis to detect a band at a desired position. For example, in the case of using a compound for modifying an unmethylated cytosine residue, two kinds of primer pairs used in the step (a), that is, a primer capable of specifically amplifying cytosine that has been methylated and not modified by bisulfite The degree of methylation can be determined according to the presence or absence of the PCR product amplified by the pair of primers that can specifically amplify cytosine modified by bisulfite and methylation, respectively. Preferably, the methylation can be determined by treating the sample genomic DNA with bisulfite, amplifying the CpG region of the gene by PCR, and analyzing the base sequence of the amplified region using a bisulfite genomic sequencing method .

In the case where restriction enzymes are used, it is judged that the CpG region has been methylated in the case where there is a PCR result in DNAs treated with restriction enzymes in a state known in the art, for example, mock DNA, If there is no PCR result in the DNA treated with the restriction enzyme, the methylation can be judged by judging that the CpG site is unmethylated, which is obvious to a person skilled in the art. In the above, mock DNA refers to a sample DNA that has been separated from the sample and has not undergone any treatment.

According to this method, when the specific CpG site of the MTA3, DLX5, TET1 and / or UCHL1 gene in the patient sample is measured in the hypermethylated state, it can be predicted that the patient will show reactivity to the anticancer drug.

Therefore, according to the present invention, the methylation of CpG of MTA3, DLX5, TET1, and / or UCHL1 gene can be effectively checked to easily predict the chemotactic response of ovarian cancer patients.

In the present invention, specific CpG sites of MTA3, DLX5, TET1 and UCHL1 genes are specifically hypermethylated in anticancer drug resistant cells, thereby confirming that the expression of the gene is specifically reduced.

Accordingly, in one aspect, the present invention provides a composition for predicting the response to an anticancer agent in an ovarian cancer patient, comprising the agent for measuring an expression level of at least one selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 .

In another aspect, the present invention provides a method of screening for an anticancer agent for ovarian cancer patients, which comprises a primer, a probe, or an antisense oligonucleotide that specifically binds to at least one gene selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 To a microarray for predicting reactivity.

The term " agent that measures the level of expression of at least one selected from the group consisting of MTA3, DLX5, TET1 and UCHL1 "of the present invention refers to an agent that measures the expression level of one or more genes selected from the group consisting of MTA3, Quot; means an agent that directly or indirectly measures the expression of a protein to be coded and confirms the degree of expression. For example, the agent may include a primer, a probe, or an antisense oligonucleotide capable of specifically binding to at least one gene selected from the group consisting of MTA3, DLX5, TET1, and UCHL1. In addition, the preparation may include an antibody that specifically binds to at least one protein selected from the group consisting of MTA3, DLX5, TET1, and UCHL1.

In the present invention, the expression levels of MTA3, DLX5, TET1 and / or UCHL1 can be determined by confirming the expression level of the mRNA of the gene or the expression level of the protein encoded by the gene. In the present invention, "measurement of mRNA expression level" is a process of confirming the presence or absence of mRNA and the expression level of a marker gene in a sample of a patient, and can be determined by measuring the amount of mRNA. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), and reverse transcriptase-polymerase chain reaction RNase protection assay, Northern blotting, DNA chip, and the like. In the present invention, "measurement of protein expression level" is a process of confirming the presence and the degree of expression of a protein encoded by a marker gene in a sample of a patient. Using the antibody specifically binding to the protein of the gene, You can check the amount. Examples of the assay method include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis , Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.

The term "primer" of the present invention is as described above.

The term "probe" in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to mRNA, and is labeled, Can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, hybridization is performed using a probe complementary to the marker polynucleotide of the present invention, and anticancer reactivity can be predicted through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The antisense oligonucleotides, primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The term "antibody" as used herein in the present invention means a specific protein molecule indicated for an antigenic site as a term known in the art. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the marker of the present invention, and the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, Part thereof is included in the antibody of the present invention and includes all the immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The kit of the present invention can detect markers by identifying mRNA expression levels or protein expression levels of MTA3, DLX5, TET1 and / or UCHL1. In the present invention, the kit may comprise a primer, a probe, or an antibody that selectively recognizes a marker for measuring the expression level of MTA3, DLX5, TET1, and / or UCHL1 that is specifically reduced in anticancer drug resistant cells, One or more other component compositions, solutions or devices may be included.

As a specific example, in the present invention, the kit for measuring the mRNA expression level of the MTA3, DLX5, TET1 and / or UCHL1 gene may be a kit containing essential elements for performing RT-PCR. The RT-PCR kit can be used to detect the presence of enzymes such as test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNases, RNase inhibitors , DEPC-water (DEPC-water), sterilized water, and the like.

In addition, the kit of the present invention may be a gene detection kit including essential elements necessary for performing a microarray. A substrate on which a cDNA corresponding to a microarray, a gene, or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. More specifically, it may be a DNA microarray in which oligonucleotides or complementary strand molecules corresponding to 10 or more genes selected in the microarray of the present invention, or fragments thereof, are integrated. The oligonucleotide or its complementary strand molecule comprises 18 to 30 nucleotides of the gene, preferably 20 to 25 nucleotides. The microarray of the present invention can be easily prepared by a method commonly used in the art using the anticancer drug resistance-specific gene of the present invention. In order to fabricate a microarray chip, a micropipetting method using a piezo electric method or a pin micropipetting method for immobilizing the above-mentioned detected marker gene as a probe DNA molecule on a substrate of a DNA chip, A method using a spotter of the above-mentioned type is preferably used, but the present invention is not limited thereto. The substrate of the microarray chip is preferably coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto . In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

In addition, the kit for measuring the level of protein expression in the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The chromogenic enzyme may be peroxidase, Alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline- ), Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

In another aspect, the present invention provides a method for detecting at least one selected from the group consisting of MTA3, DLX5, TET1, and UCHL1 from a sample of a patient in order to provide information necessary for predicting reactivity of an ovarian cancer patient to an anticancer agent .

For example, the invention provides a method comprising: measuring the level of expression of MTA3 from a sample of a patient; And comparing the expression level of the MTA3 with an expression level of MTA3 in an anticancer drug sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises measuring at least one expression level selected from the group consisting of DLX5, TETl and UCHLl from a sample of the patient; And comparing the expression level to at least one expression level selected from the group consisting of DLX5, TET1 and UCHL1 of the anti-cancer drug sensitive control sample.

For example, the invention provides a method comprising: measuring the level of expression of DLX5 from a sample of a patient; And comparing the expression level of DLX5 with an expression level of DLX5 in an anticancer sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises measuring at least one expression level selected from the group consisting of MTA3, TET1 and UCHL1 from a sample of the patient; And comparing the expression level to one or more levels of expression selected from the group consisting of MTA3, TET1 and UCHL1 of the anti-cancer drug sensitive control sample.

For example, the invention provides a method comprising: measuring the level of expression of TET1 from a sample of a patient; And comparing the expression level of TET1 with the expression level of TET1 in an anticancer drug sensitive control sample, to a method for providing information for predicting responsiveness to an anticancer agent in an ovarian cancer patient.

In this case, more preferably, the method further comprises: measuring at least one expression level selected from the group consisting of MTA3, DLX5 and UCHL1 from a sample of the patient; And comparing the expression level to at least one expression level selected from the group consisting of MTA3, DLX5 and UCHL1 of the anti-cancer drug sensitive control sample.

For example, the present invention provides a method comprising: measuring the level of expression of UCHL1 from a sample of a patient; And comparing the expression level of the UCHL1 with the expression level of UCHL1 in an anticancer sensitive control sample. The present invention also relates to a method for providing information for predicting reactivity of an ovarian cancer patient to an anticancer agent.

In this case, more preferably, the method further comprises: measuring at least one level of expression selected from the group consisting of MTA3, DLX5 and TET1 from a sample of the patient; And comparing the expression level to at least one expression level selected from the group consisting of MTA3, DLX5 and TETl of the anti-cancer drug sensitive control sample.

The biomarker identified in the present invention can be usefully used to select patients who have a therapeutic effect on an anticancer drug or to determine a therapeutic regimen of a patient by predicting the response of the patient to chemotherapy and the risk of resistance to the anticancer drug.

FIG. 1 shows the results of the MTA3 gene expression difference between the A2780 cell line and the A2780cis cell line resistant to the anticancer drug, and the MTA3 gene expression difference between the anticancer drug sensitive cell line and the anticancer drug resistant cell line through the mRNA microarray, respectively.
FIG. 2 is a graph showing the difference in DNA methylation between the A2780 cell line and the A2780cis cell line resistant to the anticancer drug, and the DNA methylation level of the MTA3 gene between the anticancer drug-sensitive cell line and the anticancer drug resistant cell line (U: unmethylated, M: methylated).
FIG. 3 shows the difference in expression of DLX5 gene between the A2780 cell line and the A2780cis cell line resistant to the anticancer drug, and the difference in DLX5 gene expression between the anticancer drug sensitive cell line and the anticancer drug resistant cell line through the mRNA microarray, respectively.
FIG. 4 is a graph showing the difference in DNA methylation level of the DLX5 gene between the A2780 cell line and the A2780cis cell line resistant to the anticancer drug, and the DNA methylation degree of the DLX5 gene between the anticancer drug sensitive cell line and the anticancer drug resistant cell line (U: unmethylated, M: methylated).
FIG. 5 shows the results of confirming the difference in expression of TET1 gene between an anticancer drug sensitive cell line and an anticancer drug resistant cell line through an mRNA microarray.
FIG. 6 shows the results of qRT-PCR for the difference in TET1 gene expression between an anticancer sensitive cell line and an anticancer drug resistant cell line.
FIG. 7 shows the results of DNA methylation microarray analysis of differences in TET1 gene methylation between the anticancer drug-sensitive cell line and the anticancer drug resistant cell line (U: unmethylated, M: methylated).
FIG. 8 shows the results of the survival analysis according to the expression abnormality of TET1.
FIG. 9 shows the results of confirming the difference in expression of UCHL1 gene between an anticancer drug sensitive cell line and an anticancer drug resistant cell line through an mRNA microarray.
FIG. 10 shows the results of confirming the difference in UCHL1 gene expression between the A2780 cell line and the A2780cis cell line resistant to the anticancer drug through the mRNA microarray.
11 shows the difference in DNA methylation level of UCHL1 gene between A2780 cell line and A2780cis cell line resistant to anticancer drug and DNA methylation degree of UCHL1 gene between anticancer drug sensitive cell line and anticancer drug resistant cell line by DNA methylation microarray (U: unmethylated, M: methylated).
Figure 12 shows DNA methylation massARRAY results of the UCHL1 gene.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Cell line and Cisplatin  Cancer Resistant Cell Model

A total of 10 cell lines of PA-1, TOV-21G, TOV-112D, CAOV3, A2780, MDAH2774, ES2, OVCAR3, OV-90 and SKOV3, ATLAS), 100 units penicillin (Gibco), and 100 μg / ml streptomycin (Gibco) under the following conditions. In addition, 1 μM / ml of cisplatin was added to the A2780 cell line under subculture to intentionally obtain a cell model that was resistant to the anticancer drug, which was named "A2780cis".

Cat. (cell) Cell line Cell-derived / related diseases morphology badge Availability HTB-77 SKOV-3 Derived from human / adenocarcinoma / metastatic state epithelial McCoy's 5a + 10% FBS + 1% P / S (REF) 16600-082 (Gibco) CRL-1978 ES-2 Human / carcinoma fibroblast McCoy's 5a + 10% FBS + 1% P / S (REF) 16600-082 (Gibco) CRL-1572 PA-1 Human / teratocarcinoma epithelial MEM alpha + 10% FBS + 1% P / S 41061-029 (Gibco) HTB-75 Caov-3 Human / adenocarcinoma epithelial DMEM (1.5 g / L sodium bicarbonate) + 10% FBS + 1% P / S LM001-51 (welgene) CRL-11730 TOV-21G Human / adenocarcinoma epithelial MCDB 105 (1.5 g / L sodium bicarbonate) & Medium 199 (2.2 g / L sodium bicarbonate) 1: 1 mix 11150-059 (Gibco, Medium 199) CRL-11731 TOV-112D Human / adenocarcinoma epithelial MCDB 105 (1.5 g / L sodium bicarbonate) & Medium 199 (2.2 g / L sodium bicarbonate) 1: 1 mix 11150-059 (Gibco, Medium 199) CRL-11732 OV-90 Human / plural epithelial MCDB 105 (1.5 g / L sodium bicarbonate) & Medium 199 (2.2 g / L sodium bicarbonate) 1: 1 mix 11150-059 (Gibco, Medium 199) HTB-161 OVCAR-3 Human / adenocarcinoma epithelial RPMI 1640 (25 mM HEPES) + 10% FBS + 1% P / S LM011-03 (welgene) CRL-10303 MDAH 2774 Human / adenocarcinoma mucinous RPMI 1640 (25 mM HEPES) + 10% FBS + 1% P / S LM011-03 (welgene) 93112519 A2780 (parent) Human / carcinoma epithelial RPMI 1640 (25 mM HEPES) + 10% FBS + 1% P / S LM011-03 (welgene) 93112517 A2780cis Human / carcinoma epithelial RPMI 1640 (25 mM HEPES) + 10% FBS + 1% P / S LM011-03 (welgene)

Example  2. Total RNA  extraction

In each cell line, total RNA was extracted with 700 μl of Trizol (RNA-Bee), and the extracted total RNA was electrophoresed in 1.5% agarose gel to confirm the extent of degradation and then quantified using Nanodrop Lite (Thermo) Respectively.

Example  3. mRNA Microarray ( Microarray ) analysis

The RNA was extracted from each cell line, the background was corrected using Affymetrix Human Gene 1.0 ST, and the degree of expression of each gene was analyzed using RMA standardization procedure and log2 transformed expression level. Ten cell lines were sequenced according to half-maximal inhibitory concentration (IC ₅₀ ) in response to cisplatin chemotherapy. The results were divided into two groups: sensitive and resistant (anti-cancer) 0.3 or more were analyzed.

Example  4. Quantitative real time ( Quantitative real - time ) PCR  ( qRT - PCR )

Reverse transcriptase (Thermo) was used to synthesize RNA with complement DNA. 2 μl of DNAseI buffer (Thermo) was added to 1 μg of RNA, and 1 μl of DNase I (Thermo) enzyme was added to remove the DNA. The DNA was then reacted at 37 ° C. for 30 minutes so that the enzyme could work, and then 50 mM EDTA And the enzyme was denatured by reacting at 65 ° C for 10 minutes. Then, 2 μl of 10 mM dNTP and 1 μl of 50 uM oligodT were added and reacted at 65 ° C. for 5 minutes. After that, 4 μl of 5X RT buffer and 20 μl of reverse transcriptase (100 μl) were reacted at 42 ° C for 60 minutes and 70 ° C for 10 minutes to synthesize cDNA, which was diluted 1: 4 and 2 μl And used as a template for qRT-PCR. For qRT-PCR, 10 μl of cDNA (2 μl), SYBER green master mix (Qiagen) and 5 μl of complementary primer (10 pmole) of each gene were reacted at 95 ° C for 5 minutes, followed by 40 cycles (95 ° C for 5 seconds, Sec). MRNA expression of TBP and GAPDH was used as an internal control. Expression of each gene was corrected by the expression level of internal control Ct = 2- (interesting gene ct - internal gene ct). The sequence of the oligonucleotide primer used is as follows.

Base sequence Human TET1 F (forward) 5'-cagccatcagatctgtaagaaaag-3 '(SEQ ID NO: 5) R (reverse) 5'-gcctcttgttttcctttataacctc-3 '(SEQ ID NO: 6) Human GAPDH F (forward) 5'-gaaggtgaaggtcggagtca-3 '(SEQ ID NO: 7) R (reverse) 5'-catgggtggaatcatattgga-3 '(SEQ ID NO: 8)

Example  5. DNA Methylation Microarray  analysis

DNA methylation microarrays were analyzed using the Infinium (®) Human Methylation 450K BeadChip to measure the degree of methylation in DNA. The degree of methylation is expressed as a value having a value of 0 to 1. When the value of 0 is 0, it means that no methylation occurs in the CpG site having the corresponding probe. Conversely, when 1 Lt; RTI ID = 0.0 > cytosine < / RTI > around the probe. To compare the degree of methylation between the two groups with sensitivity to chemotherapy, a CpG site with a p-value of less than 0.05 and a degree of methylation greater than 0.2 was selected using t-test Respectively.

Example  6. DNA  Extraction Bisulfite ( Bisulfite ) process

The bisulfite treatment is maintained when the methyl group is substituted for the cytosine in the DNA base sequence, but when the methyl group is not present, it is converted to uracil and the degree of methylation can be measured. DNA was extracted from each cell line using DNeasy blood & tissue kit (Qiagen) according to the manufacturer's manual, and the extracted DNA was used as a template for bisulfite treatment. 500 ng of DNA was bisulfite treated according to the manual of Zymo EZ DNA methylation kit (zymo).

Example  7. MassARRAY Analysis of the degree of methylation using

The bisulfite-treated DNA was quantitatively analyzed by MassARRAY analysis using BT-specific primers. The treated DNA was analyzed by dephosphorylation using the reagents provided by Sequenom, the transcription with RNA, and the degree of methylation by classifying cytosine and thiamine into masses using a MALDI-TOF mass spectrometer. I used the manual provided by the manufacturer Sequenom.

Base sequence Human UCHL1_BT F (forward) 5'-gagattgtaaggtttgggggtt-3 '(SEQ ID NO: 9) R (reverse) 5'-ccactcactttattcaacatctaaaaa-3 '(SEQ ID NO: 10)

Experiment result

1. Anticancer drug resistant cells MTA3  And DLX5 Enhancer  Site CpG Methylation  And methylation and decrease in gene expression

In A2780cis cell line resistant to cisplatin anticancer drug, the genes with probes with 0.3 or more difference in gene expression and DNA methylation compared to the existing cell line (A2780) were selected. Based on the microarray results, we have identified genes that show the same tendency in the resistant cell line (A2780cis) and in the resistant cell line. As a result, the expression level of MTA3 and DLX5 genes was decreased and hypermethylation was observed (FIGS. 1 to 4). In addition, the location of the hypermethylated probe with increased DNA methylation was confirmed using the H3K4Me1, H3K27Ac (enhancer marker), and H3K4Me3 (promoter marker) provided in the ENCODE data. As a result, And methylation of the gene was observed. Specifically, the position of the hypermethylated probe of MTA3 was found in the region of 42794620-42795547 (928bp) of chromosome 2 (Chr2), and the position of hypermethylated probe of DLX5 was located at 96651477-96652214 (Chr7) of chromosome 7 738bp).

The enhancer site enhances the transcription efficiency by facilitating the binding of the transcription factor, and it is shown that the transcription factor is unable to bind properly due to hypermethylation of this part and the expression amount decreases.

2. Anticancer drug-resistant cells TET1  Gene Promoter promoter Hypermethylation of CpG and  Confirm reduction of gene expression

TET1 was selected as a candidate gene for tolerance-specific molecular target by commonly searching for genes whose mRNA expression level varies in DNA methylation in cancer-resistant and resistant cell models through integration analysis. In the TET1 promoter region, probes showing a significant difference (p-value of 0.05) between the anticancer-resistant cell line and the sensitive cell line were selected, and the cells were hypermethylated and down-expressed in the anticancer drug resistant cell line. In addition, the methylation level of 5 'UTR, which is the promoter region of TET1 gene, tended to be very high when resistance to anticancer drugs was present. In the hypermethylation of the promoter region, the expression level of TET1 was decreased. This tendency was confirmed by microarray data and qRT-PCR (Figs. 5 to 7).

The location of the hypermethylated probe of TET1 was found in the region 70321183 to 70322935 of chromosome 10 (Chr10).

3. In the blood of patients with ovarian cancer TET1 More than the expression level of Between disease free survival rates  Closely related

(C-bioportal) analysis of blood samples from 316 patients with ovarian cancer (Ovarian Serous Cystadenocarcinoma, TCGA, Nature 2011) (Cerami et al. Cancer Discov. 2012 & Gao et al. As a result, the duration of disease free survival was shorter in 17 patients who had problems with TET1 gene expression (p value 0.039). On the other hand, there was no significant effect on overall survival (p value 0.09).

In addition, when the expression level of TET1 promoter is changed by hypermethylaion, the disease-free survival rate is shortened rather than the survival rate of the patient. If the expression of TET1 is decreased, the cancer is resistant to the anticancer drug, (Fig. 8). In addition, the present invention can be used as a diagnostic biomarker capable of measuring the risk of recurrence.

4. Anticancer drug resistant cells UCHL1  Gene promoter Hypermethylation of CpG and  Confirm reduction of gene expression

The methylation of UCHL1 gene, which is different from the DNA methylation in A2780cis, which is an anticancer drug resistant cell model, and the degree of methylation of the UCHL1 gene in the candidate gene group whose mRNA expression level is changed, was hypermethylated at the promoter region of UCHL1, The results were low in the anticancer drug-resistant cell model (A2780cis) and in the resistant cell line, and the promoter region was hypermethylated (Fig. 9 to Fig. 12).

The location of the hypermethylated probe of UCHL1 was located at 41258314 to 41259927 sites on chromosome 4 (Chr4).

<110> Ewha University - Industry Collaboration Foundation <120> MTA3, a marker for predicting the response to anti-cancer drug in          a patient with ovarian cancer <130> DPP20140605KR <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 928 <212> DNA <213> Homo sapiens <220> <221> enhancer &Lt; 222 > (1) .. (928) <223> Chr2: 42794620-42795547 <400> 1 agcacacagc tgtgtatcac cacacattcc aaacaagcca ccacaccaag aaatcttctt 60 cgtgtttatt atgcagcaat gtctgggagt actttggaat ggactggctt ctctaaagaa 120 agttatttgt gttcatttgg gggatcgcca gctaagattc aacgttgttt tttttttttt 180 gtatctgtga gagtaggaag gccgtgaaaa cagaggttgt ttagagaatt ggcctgggac 240 aataaccatt tagaagcttt aggcacgtaa ccagttaagt gtccatcgac atgaaactga 300 ttgaatacaa tctcctctgt ttgggggtgg tggtgattgt gaactgtagg gccctgttcc 360 cccatggaca aaggaagccc ctcactccta aaggtccctt ttcccagcag aacaggccct 420 gcatatacag tgcccgtcta gagccagggc ctccatctgg gtgttggaca gttggttctg 480 ctcagcccag ttttggggta aacgatgacg cagttagggt ggcctaggaa gggagttttt 540 ctacctctgt ccccagctgg gtctctggcg aggcgggaag cacccggaat cttcctggcc 600 ctagagcctg caggctccag gccggcccct tgaatctcac cgcgaggaag gcaccctgct 660 gcctgcactt atttgcatcc aagagtttgc attgagactg gcgcttgcct actagggcag 720 ccacaggggg gttccccagg gacagaggta gggaaggagg acctgtcttg gggtggacag 780 gtgacggggt accctgggtg ggggaagagg tgctgggag aggctaagcc ccagccccca 840 ctctaattct ctaacagccc ctggtccgca tttgagtgca ggcctctgcc tcagccagga 900 ccctgacccc agggccctcc acttccag 928 <210> 2 <211> 738 <212> DNA <213> Homo sapiens <220> <221> enhancer <222> (1). (738) <223> Chr7: 96651477-96652214 <400> 2 catggcagcg ccgtatttac ctgtgtttgt gtcaatccca gcgaggcggc cagctcggcg 60 cgttccggca aggcgaggta ctgagtcttc tgaaaccttc tctgtaatgc ggccagctga 120 aagctggaat aaatagtcct gggtttacga actttctttg gtttgccatt caccattctc 180 acctcgggct cggtcacttc tttctctaaa taatcaaaac agactcagtt aaagcttgtc 240 accagacaat gccccttttg cggaagggcc tcaaatagag tcctagagtt tccttaccact 300 cagtcttcat tctttgccca tatcaccacc acctttgctt ttcaggaccg cgataaatat 360 gcacctaccc caaatatgga aaagcgggat agcctctgat taattcctac tctgttcatt 420 cagatagttt gtgcaaagcg cttcctacaa cactgctttc tccttggcat ctgggattca 480 gctcttgcgg tcgttgcttc ttctcccgga cttgttttta cgcccaccct gtgctcctcc 540 gcgtttccgc ccctccccag gcttcaagaa caccagcccc tccccaggct tcaaggaggt 600 ggccctgggc cgtgacccta cttctgtcgg cgtctgttcg ggcgcccgca gaagccagac 660 tcttgggcag ggaagacgct aaccgaggtc tccaaagttc aaagacccac aagcaaactg 720 ctccttcact tcagcagc 738 <210> 3 <211> 1753 <212> DNA <213> Homo sapiens <220> <221> promoter &Lt; 222 > (1) .. (1753) <223> chr10: 70321183-70322935 <400> 3 gagtagagtg tgttctgtca aaatggcatt taagaaactc atttatgagc accactgggt 60 cgctggacgc gctggccttg cgatcccatc agtccccgtc tggcgggatc ccggagcggg 120 ccccgagggt gggaggtgcg cggagctggg tgctggggct ggtcgtgcag cacgtgaggg 180 gcctggtcct cgctctcggg cgaggaggcg gccgcacaac ctaccgcccc cagagaaaaa 240 atgctctcct ctctctctgt gcttccctcc ctccctccct ctcccccgct ccggtccccg 300 cctctctcgc cccaggctct tgaatatata tatatatata ttctacaaga agtggtctgg 360 aagggactgg ccgacgctgg taatctttcc acgtgaacga gaaacccacg ctgggcattt 420 ctgatccact aaaaactaaa gtgtttccaa aagaacactt aaagcttcag cctcggagtc 480 tttcccgcgc ccccaactcg tccctctccc aaattaacca acctttctgc atacgtctct 540 tagggtgcct cttcccctcc gtcccggttc cactcctggg cactaaacga gctgcacaat 600 atgtcctagt aatgactgct ttttgaagaa atgtttgaaa agtgctgcaa gaccttggag 660 cggaacagg attaggtaca cagccctggg ccggcggggt gagccgcgcg cgccactgga 720 agagcggcgc tgtatagcag cagccccagg tctcccctgg acctgagtgt gctcggcgtc 780 cccaccccca atataaagta aaataaaagc aatggtttaa ttaggcctca ggatacgaaa 840 aatatggaaa aggcgcttct taaccagtaa gggcaacagt tatttggagc acactaggaa 900 tttatgacta caattaaacc aggggcaggg gtacacgtag ataggtacgg aaataaggag 960 attggtgtga gttaataatc gaaagactta ggtgaatcaa accaagaatc aaaagactta 1020 ggtggctttt ggtgtgtttc cgattttatc ggtattgatc atacgggtcc cccttttccc 1080 caattaaggg atttttaatt agtttgtcag tctttaaagc taagttgtgg tttcaattaa 1140 atttcagaat caagatatac gggaaatggc agacagtgtc cttttctctt tgcaaagatg 1200 aggagtccag gcttacaact ctgggactgc cactttccat tttaataaga gttaactatc 1260 gctctccggg ctggtctccc agaatagatg ggtgggaggg ttgggtctcc actgtccctg 1320 ccctttgttt ctcttccttc tcccatctga tttgtctccc ctcccccacc ttctgagcgt 1380 ccgtatcttc cctgcacact tgatttcccc tatccccttg gtcttttagt tttcggcctt 1440 ttgatcccag ttgccttttt atgttctagc tttattacta tagatgtcga accccttcct 1500 cagtttttac tataagtaat ttttgccctt cctcagtttt ttactataca tttcttcccc 1560 ttctttcgtt tttggccttt tttgttttct ttttgggcat tttcttcctt ctcttgcttt 1620 ttcttctact tcctcctgtt gtgtgttatg tctttcttgt cttcggaagg acagttctat 1680 gtccattcta acgtctccct tcccttttct tggccctttt cccccctttt ttcccccttc 1740 ctctctgcag ggc 1753 <210> 4 <211> 1614 <212> DNA <213> Homo sapiens <220> <221> promoter &Lt; 222 > (1) .. (1614) <223> chr4: 41258314-41259927 <400> 4 tgtattaccc tcacatcccc cagctttcta ctgctctccc aggaccaacc atttcttccg 60 cgggagtcac attacatcag cattcctaat gcagtatctg ttatctacca gattctgttt 120 tattctaggt agtcacttaa aaacgaacct cggtactggt ctgacttaac atggaggagg 180 aattgtctaa ggttaaacgc aaactgctga gagatttggg gcggggggca cacatttaca 240 ttcattcgta ttaaatatat acctgttgaa tttgtgcttt ttctcaaatg cttcagagac 300 tcgagcttta gagtaattgg gatggtgaaa ggatgggttt ccagaaactt cgcccaaaat 360 taaagactcc atcaaaagga ctgctccata cactcaagga acacccacca acaaatcccg 420 tctccacaac caccagatta tctcaccggc gagtgagact gcaaggtttg ggggcccggc 480 cgtaccactc cgcgctgcgc acggggggtt cgtacccatc tggccgcgac cgtccgtttc 540 cccctcgctt ggttctgccc ctgctccccc tgcacaggcc tcacagtgcg tctggccggc 600 gctttatagc tgcagcctgg gcggctccgc tagctgtttt tcgtcttccc taggctattt 660 ctgccgggcg ctccgcgaag atgcagctca agccgatgga gatcaacccc gaggtgagcg 720 ccaggtgcac cgctacccgg agagcgcgag gccgagggag ggggagccga gtcgctgatc 780 ggttcggttt tgcctttttc tttgcatttg cctttcagat gctgaacaaa gtgagtggcg 840 tctcgcgccg tctctggccc cctcccccgc gagcgccgag gcgggggcgc ccaccggttc 900 cggctgctgg cagggaccaa gccgcccgct gcgagcaccg gagacggccg ggctggggcg 960 tgggctgggc gcctttcctg ggcccctgca tttagcgggt gactctacga aaccggtcac 1020 ggggagacgg agggggctgc gccccgtggc gagcgagcgc caggcggagc tcccgagggc 1080 gtggcgcggg cagcacagac tcggctgcac gggcttcgcg ggcgccacgt gtgggccgcg 1140 ctttgtgctg tgtcattgcg ccggcccggg tgggggtggc agggcgggac tggggctcct 1200 cccaggctcg ggtgcgggcg cggagggcgc gcgcctcctg gccccgcccc ctggcaggtg 1260 cccgcgaccc gcgtgtcccc gtgcgcctgg ccgccttgtc tcctctccgc aggtgctgtc 1320 ccggctgggg gtcgccggcc agtggcgctt cgtggacgtg ctggggctgg aagaggagtc 1380 tctgggctcg gtgccagcgc ctgcctgcgc gctgctgctg ctgtttcccc tcacggccca 1440 ggtagggcgt ggggcccagg atgcgccggc cgccggcagt gcacgccgct ccccagcttg 1500 agtcctcggg ggctccccgc cccgccccct cccctgtagg tgatgcgggg cgcgcctaca 1560 aggaagggag gagcctgcat tttcgtggta cctactccct gggctcctgt tcca 1614 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Human TET1 <400> 5 cagccatcag atctgtaaga aaag 24 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Human TET1 <400> 6 gcctcttgtt ttcctttata acctc 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Human GAPDH <400> 7 gaaggtgaag gtcggagtca 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Human GAPDH <400> 8 catgggtgga atcatattgg a 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Human UCHL1_BT <400> 9 gagattgtaa ggtttggggg tt 22 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Human UCHL1_BT <400> 10 ccactcactt tattcaacat ctaaaaa 27

Claims (10)

1. A composition comprising an agent that measures the level of methylation of the CpG region of the MTA3 (Metastasis Associated Family, Member 3)
Wherein the CpG region of the gene comprises CpG represented in the nucleotide sequence of SEQ ID NO:
Compositions for predicting responsiveness to anticancer agents in ovarian cancer patients.
2. The method according to claim 1, wherein the agent for measuring the methylation level of the CpG region of the gene is
Compounds or methylation sensitive restriction enzymes that modify unmethylated cytosine bases;
A primer specific for the methylated sequence of the CpG region of the gene; And
Wherein the primer comprises a primer specific for the unmethylated sequence.
3. The composition of claim 2, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.
3. The composition of claim 2, wherein the methylation sensitive restriction enzyme is Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI or Not I.
delete delete delete A composition comprising a composition according to any one of claims 1 to 4,
Kits for the prediction of response to anticancer drugs in ovarian cancer patients.
A method for detecting methylation of a CpG region of an MTA3 gene comprising CpG in a nucleotide sequence of SEQ ID NO: 1 from a sample of a patient, in order to provide information necessary for predicting reactivity of an ovarian cancer patient to an anticancer agent.
delete

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