Composition for diagnosing bladder cancer using changes in CpG methylation of specific genes and use thereof

Abstract

The present invention relates to a composition, kit, nucleic acid chip, and method for diagnosing bladder cancer by detecting the methylation level of CpG sites in one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL. Hypermethylation of CpG sites in the genes of the present invention is specifically observed in bladder cancer, and therefore the composition, kit, chip, or method according to the present invention can be used not only to accurately and quickly diagnose bladder cancer, but also for early diagnosis and recurrence monitoring.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0010053, filed on January 28, 2020, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a composition, a kit, a nucleic acid chip, and a method for diagnosing bladder cancer by detecting the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL. [Background technology]

[0003] Bladder cancer is cancer that develops in the bladder mucosa and is the most common type of urological cancer. The most common symptom of bladder cancer is hematuria, and in approximately 25% of cases in elderly people aged 65 or older, bladder cancer is the cause of hematuria. Generally, if hematuria or bladder irritation symptoms persist, the possibility of bladder cancer is suspected and tests to diagnose bladder cancer are performed.

[0004] The most basic tests for bladder cancer are urine tests and urine cytology. Urine tests check for the presence of red blood cells and inflammatory cells in the urine, while urine cytology checks for the presence of cancer cells in the urine. If the test comes back positive, there is a high probability of urinary system cancer, including bladder cancer. It is also a non-invasive test, which has the advantage of being inexpensive, but the disadvantage is that it has reduced accuracy.

[0005] Cystoscopy is performed when bladder cancer is suspected through a urine test or urine cytology test and hematuria is visible to the naked eye. This test involves inserting an endoscope into the urethra under local anesthesia to directly observe the inside of the bladder to determine the presence, location, shape, number, and size of tumors within the bladder. However, this method has drawbacks: it is expensive, invasive, and difficult to detect early-stage bladder cancer.

[0006] Early detection of bladder cancer is associated with a good prognosis and is directly linked to quality of life. Furthermore, bladder cancer frequently recurs, making periodic follow-up examinations essential. Therefore, there is a need to develop a non-invasive test that overcomes the shortcomings of various conventional bladder cancer diagnostic methods and enables efficient early diagnosis and recurrence monitoring.

[0007] On the other hand, epigenetics is a field that studies the regulation of gene expression that occurs without changes in DNA base sequence. Epigenetics studies the regulation of gene expression by epigenetic alterations such as DNA methylation, miRNA or histone acetylation, methylation, phosphorylation, and ubiquitination.

[0008] Double DNA methylation is the most widely studied epigenetic mutation. Epigenetic mutations can lead to alterations in gene function and tumor cells. Therefore, DNA methylation is associated with the expression (or suppression and induction) of disease-regulating genes within cells, and cancer diagnostic methods based on DNA methylation measurement have recently been proposed. In particular, because cancer-specific methylation can occur in advance in tissues at the precancerous stage, the detection of cancer-specific methylation has great potential for use in cancer diagnosis.

[0009] Therefore, it is necessary to develop effective bladder cancer-specific methylation markers that can predict the risk of bladder cancer. Summary of the Invention [Problem to be solved by the invention]

[0010] Here, the present inventors discovered that specific gene CpG sites in bladder cancer are hypermethylated, and developed a composition, kit, nucleic acid chip and method that can diagnose bladder cancer by detecting the methylation level, thereby completing the present invention.

[0011] Therefore, an object of the present invention is to provide a composition for diagnosing bladder cancer, which comprises a preparation for measuring the methylation level of CpG sites in specific genes.

[0012] Another object of the present invention is to provide a composition for diagnosing bladder cancer, which comprises a preparation for measuring the methylation level of CpG sites in specific genes.

[0013] Another object of the present invention is to provide a composition for diagnosing bladder cancer that is essential for a preparation that measures the methylation level of CpG sites in specific genes.

[0014] Another object of the present invention is to provide a bladder cancer diagnostic kit containing a PCR primer pair for amplifying a fragment containing a CpG site of a specific gene and a sequencing primer for pyrosequencing the PCR product amplified by the primer pair.

[0015] Yet another object of the present invention is to provide a nucleic acid chip for diagnosing bladder cancer, on which a fragment containing a CpG site of a specific gene and a probe capable of hybridizing under strict conditions are immobilized.

[0016] It is yet another object of the present invention to provide a method for providing information for diagnosing bladder cancer, which comprises measuring and comparing the methylation levels of CpG sites of specific genes from different samples.

[0017] Yet another object of the present invention is to provide a method for measuring the methylation level of CpG sites of specific genes in a biological sample isolated from an individual to provide information necessary for diagnosing the recurrence or progression of bladder cancer.

[0018] Yet another object of the present invention is to provide use of a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL for the manufacture of a preparation for diagnosing bladder cancer.

[0019] Further, still another object of the present invention is to a) obtaining a sample from an individual; b) measuring the methylation level of CpG sites of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL from the sample; and c) comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample. [Means for solving the problem]

[0020] To achieve the above object, the present invention provides a composition for diagnosing bladder cancer, comprising a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL.

[0021] The present invention also provides a composition for diagnosing bladder cancer, comprising a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL.

[0022] The present invention also provides a composition for diagnosing bladder cancer, which essentially consists of a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL.

[0023] To achieve another object of the present invention, the present invention provides a kit for diagnosing bladder cancer, comprising a primer pair for amplifying a fragment containing a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL.

[0024] In addition, to achieve yet another object of the present invention, the present invention provides a nucleic acid chip for diagnosing bladder cancer, on which a probe capable of hybridizing with a fragment containing a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL is immobilized.

[0025] In order to achieve still another object of the present invention, the present invention provides a method for treating bladder cancer, comprising the steps of: measuring the methylation level of CpG sites of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL from a sample of a patient suspected of having bladder cancer;

[0026] comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample.

[0027] In order to achieve yet another object of the present invention, the present invention provides a method for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL in a biological sample isolated from an individual, in order to provide information necessary for diagnosing the recurrence or progression of bladder cancer.

[0028] In order to achieve yet another object of the present invention, the present invention provides use of a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL for the manufacture of a bladder cancer diagnostic preparation. In order to achieve still another object of the present invention, the present invention provides a) obtaining a sample from an individual; b) measuring the methylation level of CpG sites of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL from the sample; and c) comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide one of skill with general definitions of terms used in the present specification: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); and Hale & Marsh, J.D. ham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

[0030] The present invention will be described in detail below.

[0031] The present invention provides a composition for diagnosing bladder cancer, comprising a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL.

[0032] In the present invention, the term "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, the presence or absence of methylation in the present invention refers to the presence or absence of methylation occurring at a cytosine at a specific CpG site of a specific gene. When methylation occurs, it prevents the binding of transcription factors, thereby suppressing the expression of the specific gene. Conversely, when non-methylation or hypomethylation occurs, the expression of the specific gene increases.

[0033] In addition to A, C, G, and T, mammalian genomic DNA contains a fifth base, 5-methylcytosine (5-mC), in which a methyl group is attached to the fifth carbon of the cytosine ring. Methylation of 5-methylcytosine occurs only at the C of CpG, a CG dinucleotide (5'-mCG-3'). CpG methylation suppresses the expression of alu or transposons and genomic repeat sequences. Furthermore, because the 5-mC of CpG is prone to spontaneous deamination to thymine (T), CpG is the site where most epigenetic changes occur frequently in mammalian cells.

[0034] The term "measuring methylation levels" as used herein refers to measuring the methylation level of CpG sites in one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL. This measurement can be performed using a bisulfite treatment or a bisulfite-independent detection method. Methylation levels can be measured using methylation-specific PCR, such as methylation-specific polymerase chain reaction (MSP), real-time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding proteins, or quantitative PCR. Alternatively, the measurement can be performed using automated base analysis, such as pyrosequencing and bisulfite sequencing. Furthermore, bisulfite-independent detection methods can be performed using a detection method using the ten-eleven translocation protein (TET) (see Nature Biotechnology, volume 37, pages 424-429 (2019)). The TET protein is an enzyme that acts on DNA and is involved in the chemical conversion of bases. When bisulfite is treated, all Cs except methylated Cs are converted to T bases, whereas Tet protein converts only methylated Cs to Ts, allowing for more efficient detection.

[0035] Preferably, the CpG site of one or more genes selected from the group consisting of IFF01, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL refers to a CpG site present in the DNA of the gene. The DNA of the gene is a concept that includes all of the series of structural units necessary for the expression of the gene that are operably linked to each other, such as a promoter region, a protein-coding region (open reading frame, ORF), and a terminator region. Therefore, the CpG site of one or more genes selected from the group consisting of IFF01, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL may be present in the promoter region, protein-coding region (ORF), or terminator region of the gene.

[0036] Preferably, measuring the methylation level of CpG sites of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL in the present invention also means measuring the methylation level of cytosine at CpG sites of the genes listed in Table 1 below.

[0037] [Table 1]

[0038] In the present invention, the CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL is characterized by being located within + / - 3000 bases (3 kb) from the transcription start site (TSS) of the gene.

[0039] In the present invention, the base sequences of the human genome chromosomal regions are expressed according to the February 2009 Human Reference Sequence (GRCh37). However, the specific sequences of the human genome chromosomal regions may be slightly modified as the results of genome sequence research are updated, and such modifications may result in the expressions of the human genome chromosomal regions of the present invention being different from the current expressions. Therefore, even if the human reference sequence is updated after the filing date of the present invention, and the expressions of the human genome chromosomal regions expressed according to the February 2009 Human Reference Sequence (GRCh37) of the present invention are modified to be different from the current expressions, it is deemed that the scope of the present invention clearly includes the modified human genome chromosomal regions. The details of such modifications are readily apparent to anyone skilled in the art to which the present invention pertains.

[0040] In the present invention, the preparation for measuring the methylation level of the CpG site may comprise a compound that modifies a cytosine base or a methylation-sensitive restriction enzyme, a primer specific to a methylated allele sequence of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL, and a primer specific to an unmethylated allele sequence.

[0041] The compound that modifies the cytosine base may be a compound that modifies unmethylated or methylated cytosine, and may be, but is not limited to, bisulfite or its salt, preferably sodium sulfite, which modifies unmethylated cytosine, or TET protein, which modifies methylated cytosine. Methods for modifying cytosine bases to detect the presence or absence of methyl at CpG sites are known in the art (WO01 / 26536; US2003 / 0148326A1).

[0042] The methylation-sensitive restriction enzyme may be a restriction enzyme that contains a CG as its recognition site and is capable of specifically detecting methylation at CpG sites. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, and NotI. Methylation or non-methylation at the C in the restriction enzyme recognition site determines whether or not the restriction enzyme cleaves, which can be detected by PCR or Southern blot analysis. Methylation-sensitive restriction enzymes other than the above restriction enzymes are known in the art.

[0043] The primers may include a primer specific to a methylated allele sequence and a primer specific to an unmethylated allele sequence of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL.

[0044] In the present invention, the term "primer" refers to a short nucleic acid sequence with a short free 3-terminal hydroxyl group that can form base pairs with a complementary template and serve as a starting point for template strand copying. A primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature. Primers are sense and antisense nucleic acids with a sequence of 7 to 50 nucleotides and may incorporate additional features that do not alter the basic property of a primer acting as a starting point for DNA synthesis.

[0045] The primers of the present invention can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and more preferably, can be any one or more selected from the group consisting of a primer pair capable of specifically amplifying cytosin that has been methylated and not modified by bisulfite, a primer pair capable of specifically amplifying cytosin that has been unmethylated and modified by bisulfite, a primer pair capable of specifically amplifying cytosin that has been methylated and modified by a Tet-series protein, and a primer pair capable of specifically amplifying cytosin that has not been unmethylated and not modified by a Tet-series protein.

[0046] Therefore, the present invention provides a kit for diagnosing bladder cancer, comprising a primer pair for amplifying a fragment containing a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL.

[0047] The composition and kit may further contain, in addition to the preparation, a polymerizing enzyme agarose, a buffer solution necessary for electrophoresis, and the like.

[0048] The present invention also provides a nucleic acid chip for diagnosing bladder cancer, on which a probe capable of hybridizing with a fragment containing a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL is immobilized.

[0049] In the present invention, the term "nucleic acid" refers to an oligonucleotide, nucleotide, polynucleotide, or fragment thereof, single-stranded or double-stranded DNA or RNA of genomic or synthetic origin, sense or antisense strand DNA or RNA of genomic or synthetic origin, PNA (peptide nucleic acid), or any other DNA or RNA substance of natural or synthetic origin. It will be apparent to those skilled in the art that when the nucleic acid is RNA, the deoxynucleotides A, G, C, and T are substituted with ribonucleotides A, G, C, and U, respectively.

[0050] Methylation begins at the outer edge of a gene's regulatory site and progresses inward. Therefore, detecting methylation at the outer edge of a regulatory site can be used to diagnose genes involved in cell transformation early and monitor progression or recurrence after treatment.

[0051] Therefore, the methylation gene markers enable early diagnosis of cells that may develop bladder cancer. If genes confirmed to be methylated in cancer cells are also methylated in clinically or morphologically normal-appearing cells, the normal-appearing cells are undergoing cancer progression. Therefore, by confirming the methylation of bladder cancer-specific genes in normal-appearing cells, early diagnosis of bladder cancer and monitoring of progression or recurrence after treatment are possible.

[0052] The present invention also provides a method for detecting bladder cancer in a patient sample suspected of having bladder cancer, comprising the steps of: measuring the methylation level of a CpG site in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL; and comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample.

[0053] The method for measuring the methylation level may be selected from the group consisting of, but not limited to, PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR using a methylated DNA-specific binding protein, measurement of the presence or absence of methylation using a methylation-sensitive restriction enzyme, quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing.

[0054] Specifically, methylation-specific PCR involves treating sample DNA with bisulfite, then designing and using different primers to perform PCR depending on whether the CpG dinucleotide is methylated. If the primer binding site is methylated, PCR proceeds with the methylated primer; if it is not, PCR proceeds with the normal primer. In other words, the method involves treating sample DNA with bisulfite, then performing PCR using two primers simultaneously, and then comparing the results.

[0055] Real-time methylation-specific PCR is a real-time measurement method based on methylation-specific PCR. Genomic DNA is treated with bisulfite, and PCR primers corresponding to methylated DNA are designed and used for real-time PCR. There are two methods for detection: using a TanMan probe complementary to the amplified base sequence, or using Sybergreen. Therefore, real-time methylation-specific PCR can selectively quantify only methylated DNA. In this method, a standard curve is created using in vitro methylated DNA samples, and a negative control group containing genes lacking the 5'-CpG-3' sequence is amplified for standardization purposes to quantitatively analyze the degree of methylation.

[0056] In a method for measuring the presence or absence of methylation using a methylation-sensitive restriction enzyme, the methylation-sensitive restriction enzyme acts on CpG dinucleotides, and if this site is methylated, it cannot act as an enzyme. Therefore, if sample DNA is treated with the methylation-sensitive restriction enzyme and then amplified by PCR to include the enzyme's target site, if the DNA is methylated, the restriction enzyme will not act and PCR will be amplified, but if the unmethylated site is cleaved by the restriction enzyme, PCR will not be amplified, allowing the presence or absence of methylation at a specific DNA site to be measured.

[0057] PCR or DNA chip methods using methylated DNA-specific binding proteins allow selective isolation of methylated DNA by mixing DNA with a protein that specifically binds only to methylated DNA. Genomic DNA is mixed with the methylated DNA-specific binding protein, and then the methylated DNA is selectively isolated. The isolated DNA is amplified using PCR primers corresponding to intron regions, and the presence or absence of methylation is measured by agarose gel electrophoresis. Quantitative PCR can also be used to measure the presence or absence of methylation. The methylated DNA separated with the methylated DNA-specific binding protein can be labeled with a fluorescent dye and hybridized to a DNA chip containing complementary probes. The methylated DNA-specific binding protein is not limited to MBD2bt.

[0058] Furthermore, pyrosequencing of bisulfite-treated DNA is based on the following principle: When methylation occurs at a CpG dinucleotide site, 5-methylcytosine (5-mC) is formed, and this modified base is converted to uracil upon bisulfite treatment. If CpG dinucleotides are methylated during bisulfite treatment of DNA extracted from a sample, they are preserved as cytosines, and the remaining unmethylated cytosines are converted to uracil. Sequence analysis of bisulfite-treated DNA can be preferably performed using pyrosequencing. Detailed descriptions of pyrosequencing are known in the art.

[0059] Alternatively, a bisulfite-independent detection method using Tet protein can be used to convert only methylated C to T, allowing detection of methylated bases (see LIU, Yibin, et al., Nature Biotechnology volume 37, pages 424-429 (2019)).

[0060] When methylation occurs at a CpG dinucleotide site, forming 5-methylcytosine (5-mC), the CpG dinucleotide is converted to uracil when treated with the Tet (ten-eleven translocation) protein, while the unmethylated cytosine is preserved. Sequence analysis of Tet-treated DNA is not limited to pyrosequencing alone; it can also be analyzed using methods such as methylation-sensitive PCR (MSP), microarrays, and next-generation sequencing (NGS).

[0061] Preferably, the method of the present invention for providing information for diagnosing bladder cancer may be performed by a method comprising the steps of: a) obtaining a sample from an individual; b) obtaining genomic DNA from the sample; c) treating the obtained genomic DNA with a compound that modifies unmethylated cytosine bases; d) amplifying the treated DNA by PCR using pyrosequencing primers capable of amplifying the promoter of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL to obtain a PCR product; and e) measuring the degree of methylation by pyrosequencing the PCR product using sequencing primers.

[0062] The genomic DNA in step b) can be obtained by phenol / chloroform extraction, SDS extraction, or CTAB separation, which are commonly used in the art, or by using a commercially available DNA extraction kit.

[0063] In the present invention, the term "sample" refers to a broad range of biological fluids, including all biological fluids obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of analysis to be performed. Methods for obtaining biopsies of body fluids and tissues from mammals are generally well known, and in the present invention, the sample may be selected from the group consisting of human-derived materials, including tissues, cells, blood, plasma, serum, feces, and urine. Since abnormal methylation changes in cancer tissues show considerable similarity to methylation changes in genomic DNA obtained from biological samples such as cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, the use of the markers of the present invention has the advantage of enabling simple diagnosis of bladder cancer using blood or body fluids.

[0064] Meanwhile, the present invention provides a method for measuring the methylation level of CpG sites of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL in a biological sample isolated from an individual to provide information necessary for diagnosing the recurrence or progression of bladder cancer.

[0065] The "individual" in this method may be an animal, preferably a mammal, particularly an animal including a human, or a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect, preferably a patient suspected of having, diagnosed with, or treated for bladder cancer.

[0066] Furthermore, the "sample" is as described above, but preferably the sample for diagnosing recurrence or progression may be urine collected from an individual or patient.

[0067] In the present invention, the diagnosis of bladder cancer recurrence or progression may include bladder cancer prognosis, where "prognosis" refers to the progression of the disease during or after treatment for bladder cancer, preferably the progression of the disease after treatment, and includes, but is not limited to, overall survival, disease-free survival, and distant metastasis-free survival. Furthermore, the progression of bladder cancer refers to a concept including complete recovery, recurrence, metastasis, or metastatic recurrence of bladder cancer, more preferably, metastatic recurrence, but is not limited to this.

[0068] The present invention provides use of a preparation for measuring the methylation level of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL for the manufacture of a preparation for diagnosing bladder cancer.

[0069] The present invention a) obtaining a sample from an individual; b) measuring the methylation level of CpG sites of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL from the sample; and c) comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample.

[0070] In one embodiment, the present invention provides a method of diagnosing and treating bladder cancer in an individual, comprising the steps of: i) obtaining a sample from an individual; ii) measuring the methylation level of a CpG site in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL from the sample; iii) comparing the measured methylation level with the CpG region of the same gene in a normal control sample; comparing the methylation level of the target gene with that of the target gene; and iv) administering to the determined individual a therapeutic agent for treating bladder cancer or treating the neurodegenerative disease through surgery.

[0071] The methods including steps i) to iv) can be understood based on the method including steps a) to c) above.

[0072] The step iv) is a step of treating the disease in the individual diagnosed with the disease in the step iii) by means of administering a therapeutic drug or performing surgery.

[0073] The term "treatment" in the present invention refers comprehensively to improving the symptoms of bladder cancer or the disease, which may include curing, substantially preventing, or improving the condition of the disease, including, but not limited to, alleviating, curing, or preventing one or most symptoms, including bladder cancer.

[0074] The "therapeutic agent" is not particularly limited as long as it is a type of drug commonly used in the treatment of bladder cancer. The therapeutic agent is administered to an individual in a "therapeutically effective amount," which can be determined by those skilled in the art, taking into account various factors, such as the specific properties of the drug, the route of administration, the number of treatments, as well as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate. The route of administration of the therapeutic agent is not particularly limited and may be oral or parenteral, and includes both local and systemic administration. Examples of parenteral administration include, but are not limited to, intranasal drug application, subcutaneous injection, and the like, as well as intramuscular and intravenous injection.

[0075] The "sample" of the present invention refers to a sample isolated and obtained from an individual suspected of having a disease, and may be selected from the group consisting of, but not limited to, cells, tissues, blood, serum, plasma, saliva, sputum, mucosal fluid, and urine. The "individual" may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the therapeutic effect.

[0076] As used herein, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components or method steps not specifically recited in a composition or method according to the present invention. Similarly, the term "consisting of" means excluding additional elements, steps, or ingredients not specifically recited. The term "essentially consisting of" means that a composition or method may include, in addition to the recited substances or steps, substances or steps that do not substantially affect the basic characteristics of the composition or method. [Effects of the Invention]

[0077] As described above, hypermethylation of CpG sites in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL is specifically observed in bladder cancer. Therefore, the composition, kit, chip, or method according to the present invention can be used to accurately and quickly diagnose bladder cancer, and further enables early diagnosis and subsequent monitoring of recurrence after treatment. [Brief explanation of the drawings]

[0078] [Figure 1] FIG. 1 shows the results of examining the differences in methylation between tumor tissues and non-tumor tissues for a total of six genes selected according to the present invention. [Figure 2] FIG. 2 shows the results of confirming the methylation information of a total of six genes selected according to the present invention in a group of tumor tissue cell lines. [Figure 3] FIG. 3 shows the results of confirming the methylation information of a total of six genes selected by the present invention in peripheral blood mononuclear cells. [Figure 4] FIG. 4 shows the results of confirming the methylation information of a total of six genes selected according to the present invention for each cell type of peripheral blood mononuclear cells. [Figure 5] FIG. 5 shows the results of confirming the diagnostic accuracy of bladder cancer for a total of six genes selected according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0079] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. [Example]

[0080] Example 1: Selection of bladder cancer-specific methylated genes To identify methylated genes specifically found in bladder cancer, we conducted a large-scale comparative study of methylation in tumor tissues and normal tissues obtained from bladder cancer patients after surgery using large-scale methylation microarray chip data. In this study, tumor tissue refers to bladder cancer tissue, and non-tumor tissue refers to tissues other than cancer, including normal tissue. A total of 412 bladder cancer tumor tissues and 21 non-tumor tissues were used for the analysis.

[0081] To select bladder cancer-specific methylated genes, DNA was extracted from each tissue and the methylation level of gene sites was confirmed using Infinium Human Methylation 450 Beadchip microarray.

[0082] DNA extracted from each tissue was converted by bisulfite treatment, which altered cytosine bases depending on whether the DNA site was methylated. The probes used in the microarray experiment were designed to be specific for methylation and non-methylation to identify whether cytosine bases at the methylated site of the gene were altered.

[0083] The microarray experiment measured the degree of gene methylation through approximately 450,000 (450k) probes, each representing a methylation site in the gene, and the results of each probe obtained through the test were expressed as a β value. The β value ranges from 0 to 1, and the closer to 1, the higher the degree of methylation of the corresponding genetic site.

[0084] To identify differentially methylated regions (DMRs) between the tumor and non-tumor groups, we used the Limma (Linear Models for Microarray Data) method, an empirical Bayes t-test, to identify gene sites showing statistically significant methylation differences between the groups.

[0085] The Limma method is known to be the least affected by outliers among multiple statistical methylation analysis methods used to confirm differences between groups. Therefore, it is a suitable method for finding cancer-specific markers because it is less affected by abnormal measurements in some samples. In this experiment, the smaller the adjusted p-value derived by the Limma method, the more significant the difference in methylation between the two groups.

[0086] In particular, to search for tumor-specific methylation sites, we identified gene sites with significant differences in beta values ​​between tumor and non-tumor groups, and identified sites with higher methylation levels in tumor tissues than in non-tumor tissues as cancer-specific methylation sites. It was selected as a candidate for O-Mark.

[0087] As a result of the Limma analysis of the dataset, gene sites with the lowest p-values ​​among 450,000 probes when comparing the tumor group to the non-tumor group and with a large difference of β-values ​​of 0.3 or more between the groups were selected as tumor-specific hypermethylated regions. As a result, 352 gene sites that showed tumor-specific hypermethylation in the dataset out of approximately 450,000 gene sites were selected as biomarker candidates.

[0088] Among these genetic sites, if they were not pseudogenes, but were located in a CpG island region, within + / - 3000 bases (3 kb) of the gene's transcription start site (TSS), and present on an autosome, they were selected as bladder cancer-specific hypermethylated genes. As a result, a total of six genes were selected (see Figure 1), as shown in Table 2 below.

[0089] [Table 2]

[0090] Example 2: Confirmation of bladder cancer specificity of bladder cancer diagnostic genes in cell lines To confirm whether the high methylation of the six selected genes is due to bladder cancer cells, we utilized public databases and analyzed the methylation patterns in 20 cancer cell lines derived from bladder cancer. The data were analyzed using the Infinium Human Methylation 450 B (Infinium Human Methylation 450 B) assay according to the manufacturer's standardized methylation analysis testing procedure for DNA extracted from each cell line. A headchip microarray experiment was performed.

[0091] The results of the experiment were as follows: the degree of methylation of genes was measured using approximately 450,000 probes, and the methylation value of each probe was expressed as a β value, which ranges from 0 to 1, with the β value being closer to 1, indicating a higher degree of methylation of the corresponding genetic site.

[0092] The 20 bladder cancer cell lines are as follows: 5637 (comic ID: 687452), 639-V (comic ID: 906798), 647-V (comic ID: 906797), BFTC-905 (comic ID: 910926), CAL-29 (comic ID: 1290730), DSH1 (comic ID: 753552), HT-1197 (comic ID: 907065), HT-1376 (comic ID: 907066), J82 (comic ID: 753566), KU-19-19 (comic ID: 907312), LB831-BLC (comic ID: 753584), RT-112 (comic ID: 909704), RT4 (comic ID: 909705), and RT4 (comic ID: 909706). ID:687455), SCABER(comic ID:1299051), SW1710 (comic ID: 909749), SW780(comic ID:687457), T-24(comic ID:724812), TCCSUP(comic ID:687459), UM-UC-3(comic ID:724838), VM-CUB-1(comic ID:909780)

[0093] The methylation levels of the six selected genes were confirmed in bladder cancer cell lines. The median methylation value for each gene was 0.93 or higher, indicating a high level of methylation, consistent with the high methylation levels of the selected genes in tumor tissues (see Figure 2). Therefore, it was confirmed that the selected genes are specifically methylated in bladder cancer.

[0094] Example 3: Confirmation of hypomethylation levels of bladder cancer diagnostic genes in peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) are peripheral blood cells with spherical nuclei, including immune-related cells such as T cells, B cells, macrophages, dendritic cells, and natural killer cells (NK cells).

[0095] To confirm the methylation levels of the six selected genes, a cancer sample must be obtained. This refers to a wide range of body fluids, including all cancer-related biological fluids obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of analysis being performed. During the sample collection process, contamination with peripheral blood mononuclear cells is the most frequent factor that can interfere with methylation measurement, and the gene methylation levels of the cancer sample are affected by the methylation of the genes to be measured in peripheral blood mononuclear cells.

[0096] To determine whether the six selected genes were affected by contamination with peripheral blood mononuclear cells, the methylation patterns of peripheral blood mononuclear cells from a total of 110 individuals were analyzed. DNA extracted from peripheral blood mononuclear cells was subjected to an Infinium Human Methylation 450 Beadchip microarray experiment using the manufacturer's standardized methylation analysis testing procedure, as in Examples 1 and 2. The results of the experiment were obtained by measuring the degree of gene methylation using approximately 450,000 probes, and the methylation value of each probe was expressed as a β value. β values ​​range from 0 to 1, with the closer the value is to 1, the higher the degree of methylation of the corresponding genetic site.

[0097] The methylation levels of the six selected genes were confirmed in peripheral blood mononuclear cells. The median methylation level of each gene was less than 0.10, which indicated a low level of methylation, unlike the high methylation levels of the selected genes in tumor tissues (see Figure 3). Therefore, it was confirmed that the selected genes are specifically methylated in bladder cancer.

[0098] In addition, we performed methylation analysis on each cell group that constitutes peripheral blood mononuclear cells (PBMCs) to analyze whether specific cell groups affect the methylation level measurement within the sample. We analyzed not only whole blood and peripheral blood mononuclear cells (PBMCs) from blood samples of a total of six normal subjects, but also granulocytes, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and C Cell populations were classified into D19+ B cells, CD14+ monocytes, neutrophils, and eosinophils, and DNA methylation levels were analyzed using the Infinium Human Methylation 450 Beadchip microarray experiment, as described in Examples 1 and 2.

[0099] The levels of the six selected genes were examined in peripheral blood mononuclear cells by cell group. The median methylation level of each gene was found to be less than 0.16 at maximum. The selected genes also showed low levels of methylation in peripheral blood mononuclear cells (see Figure 4).

[0100] This result indicates that the selected genes show low methylation levels in peripheral blood mononuclear cells, and that the influence of contamination with peripheral blood mononuclear cells on the measurement of the methylation of the genes from the sample is very limited.

[0101] Example 4: Evaluation of diagnostic performance of bladder cancer diagnostic genes To confirm the usefulness of the selected genes as diagnostic markers for bladder cancer, the accuracy of bladder cancer diagnosis based on methylation degree was evaluated.

[0102] Sensitivity and specificity are used to evaluate diagnostic accuracy. By calculating sensitivity and specificity values ​​for possible cutoff values ​​of continuous diagnostic test measurements, a receiver operating characteristic (ROC) curve can be drawn, showing the change in sensitivity and specificity depending on the cutoff value. Diagnostic accuracy can be measured by the area under the ROC curve (AUC). AUC values ​​range from 0.5 to 1, with higher values ​​indicating higher diagnostic accuracy. An AUC value of 1 indicates a perfectly accurate diagnostic result, while an AUC value of 0.5 is considered to be the same as a random result.

[0103] The cancer classification accuracy according to the degree of methylation between non-tumor and tumor tissues using the selected genes was analyzed using a collected methylation dataset. As shown in Figure 5, all selected genes had an AUC value of 0.906 or higher, indicating high diagnostic accuracy, confirming that the selected genes are useful for diagnosing bladder cancer (see Figure 5). [Industrial Applicability]

[0104] As described above, hypermethylation of CpG sites in one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL is specifically observed in bladder cancer. Therefore, the composition, kit, chip, or method according to the present invention can be used not only to accurately and quickly diagnose bladder cancer, but also for early diagnosis and recurrence monitoring.

Claims

1. A composition for diagnosing bladder cancer, comprising a preparation for measuring the methylation level of a CpG site of NR2E1 and a preparation for measuring the methylation level of a CpG site of KCNA3, or comprising a preparation for measuring the methylation level of a CpG site of NR2E1, a preparation for measuring the methylation level of a CpG site of KCNA3, and a preparation for measuring the methylation level of a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2 and BOLL.

2. 2. The composition of claim 1, wherein the CpG site is located between + / - 3000 bases (3 kb) from the transcription start site of the gene.

3. The preparation for measuring the methylation level of the CpG site of the gene comprises: Compounds that modify unmethylated cytosine or methylated cytosine bases; a primer specific to a methylated sequence of a CpG site of NR2E1 and KCNA3, or a primer specific to a methylated sequence between a CpG site of NR2E1 and KCNA3 and a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2, and BOLL; and The composition of claim 1, comprising a primer specific for an unmethylated sequence.

4. The composition of claim 3, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof, and the compound that modifies the methylated cytosine base is Tet protein.

5. A platform for amplifying a fragment containing the CpG sites of NR2E1 and KCNA3, or a fragment containing the CpG sites of NR2E1 and KCNA3 and the CpG sites of one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2 and BOLL. A kit for diagnosing bladder cancer comprising a marker.

6. A nucleic acid chip for diagnosing bladder cancer on which a probe is fixed that can hybridize with a fragment containing the CpG sites of NR2E1 and KCNA3, or a fragment containing the CpG sites of NR2E1 and KCNA3 and a CpG site of any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2 and BOLL.

7. For producing a bladder cancer diagnostic preparation, Use of a preparation for measuring the methylation level of a CpG site in NR2E1 together with a preparation for measuring the methylation level of a CpG site in KCNA3, or use of a preparation for measuring the methylation level of a CpG site in NR2E1 together with a preparation for measuring the methylation level of a CpG site in KCNA3 together with a preparation for measuring the methylation level of a CpG site in any one or more genes selected from the group consisting of IFFO1, MARCH11, BARHL2 and BOLL.

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