KR20230069414A - Composition for diagnosing oral cavity cancer comprising an agent for determining level of methylation of specific gene and a method for diagnosing oral cavity cancer the same - Google Patents

Abstract

The present invention relates to a composition for diagnosing oral cavity cancer comprising an agent for determining the level of methylation of a specific gene and a method for diagnosing oral cavity cancer using the same. In the present invention, it was confirmed that DNA methylation was specifically detected in a CpG island region present in a promoter region of five genes (GPX3, ANG, GPRC5B, CTGF, BAMBI) in oral cancer patients, and thus, the methylation of the genes can be useful for diagnosis or prognosis prediction of the oral cancer.

Description

Composition for diagnosing oral cavity cancer comprising an agent for determining level of methylation of specific gene and a method for diagnosing oral cavity cancer the same}

The present invention relates to a composition for diagnosing oral cancer, including an agent for measuring the methylation level of a specific gene, and a method for diagnosing oral cancer using the same.

Head and Neck Cancer (HNC), which includes cancer of the oral cavity, pharynx, and larynx, is the sixth most common cancer worldwide, with a high morbidity rate and a 5-year survival rate of only 50%. . Among head and neck cancers, more than 90% of oral cancers are oral squamous cell carcinoma (OSCC), which arises from squamous epithelium, and usually develops between the ages of 50 and 60. Oral squamous cell carcinoma arises through a series of molecular mutations that lead to uncontrolled cell growth from hyperplasia to dysplasia, carcinoma in situ, and invasive carcinoma.

Although these oral squamous cell carcinomas occur in the most accessible area on visual examination, there are no clear early risk signs, which affects staging and tumor progression. Accordingly, most cases are diagnosed at an advanced stage of the disease, and the survival rate is low. Until now, surgery and radiotherapy have been the main treatments, but oral cancers such as oral squamous cell carcinoma are usually located in the head and neck, resulting in postoperative defects and functional disorders in patients. Therefore, it is necessary to develop a diagnostic marker that can diagnose oral cancer early and determine its prognosis.

Although the cause of oral cancer has not been clearly identified, it is known to be caused by genetic predisposition and environmental factors. Although environmental factors are also important factors in the pathogenesis of oral cancer, the results of genetic studies are helpful in understanding the susceptibility of oral cancer. Regarding the correlation between genetic and environmental factors, epigenetic mechanisms are predicted to contribute to the pathogenesis of oral cancer.

Epigenetics is extensively studied in the field of tumor biology. It deals in particular with the regulation of gene expression mainly through DNA methylation, miRNA or histone mutations such as acetylation, methylation, phosphorylation or ubiquitination. Among these, DNA methylation is the most studied epigenetic mutation in mammals. Dysregulation of epigenetic processes can lead to mutations in gene function and changes to tumor cells. DNA methylation is known to be associated with the suppression of transcriptional gene expression in human diseases, including a well-established role in tumors, and recently, various methods for diagnosing various diseases including cancer through DNA methylation measurement have been proposed.

DNA methylation mainly occurs at cytosine of the CpG island of the promoter region of a specific gene, and as a result, the binding of transcription factors is hindered, and the expression of a specific gene is blocked (gene silencing), coding It is the main mechanism by which the function of the gene is lost even without a mutation in the coding sequence. Abnormal methylation/demethylation in CpG islands has been reported in various diseases including cancer, and active attempts are being made to examine promoter methylation of disease-related genes and use it for diagnosis of various diseases.

Although DNA methylation has been known to be related to tumor development and heteromorphism by many studies, among the studies to identify the relationship between oral cancer incidence and clinical aspects, studies on DNA methylation mutations in whole genes of oral cancer patients have been identified. no bar

Republic of Korea Patent Publication No. 10-2021-0029823 (published on 21.03.16)

An object of the present invention is to provide a composition for diagnosing oral cancer.

Another object of the present invention is to provide a kit for diagnosing oral cancer.

Another object of the present invention is to provide a method for providing information necessary for diagnosing oral cancer.

Another object of the present invention is to provide a composition for predicting oral cancer prognosis.

Another object of the present invention is to provide a kit for predicting oral cancer prognosis.

In order to achieve the above object, the present invention provides a composition for diagnosing oral cancer comprising an agent for measuring the methylation level of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes.

In addition, the present invention provides a kit for diagnosing oral cancer comprising the composition.

In addition, the present invention provides (a) measuring the methylation level of the CpG island region of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes from a biological sample isolated from the subject; and (b) comparing the measured methylation level with that of a normal control sample.

In addition, the present invention provides a composition for predicting prognosis of oral cancer comprising an agent for measuring the methylation level of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes.

In addition, the present invention provides a kit for predicting oral cancer prognosis comprising the composition.

In the present invention, it was confirmed that DNA methylation was specifically detected in CpG island regions present in the promoter regions of five genes (GPX3, ANG, GPRC5B, CTGF, and BAMBI) in patients with oral cancer. Accordingly, a composition including an agent for measuring the methylation level of the gene can be usefully used for oral cancer diagnosis or prognosis prediction.

1 is a comparison result of transcriptional expression changes of genes according to the present invention (GPX3, ANG, GPRC5B, CTGF, BAMBI) before and after treatment with DNA methylation inhibitors in oral squamous cell carcinoma cell lines.
Figure 2 schematically shows the methylation analysis region within the CpG island present in the promoter for methylation analysis of the genes (GPX3, ANG, GPRC5B, CTGF, BAMBI) according to the present invention.
Figure 3 is a result of comparing each methylation degree (%) of the genes (GPX3, ANG, GPRC5B, CTGF, BAMBI) according to the present invention in the oral cancer group and the normal group.
Figure 4 is a result of confirming the overall survival rate by TCGA cohort analysis of the genes (GPX3, ANG, GPRC5B, CTGF, BAMBI) according to the present invention in the oral cancer group.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "methylation" occurs at cytosine of a CpG island in the promoter region of a specific gene, thereby interfering with the binding of transcription factors, thereby blocking the expression of a specific gene. For purposes of this purpose, methylation means methylation in the CpG island of the promoter region of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes.

In the present invention, the term "measurement of methylation level" refers to measuring the methylation level of the promoter CpG island of the gene, such as methylation-specific PCR (methylation-specific polymerase chain reaction, hereinafter also referred to as MSP) or automatic sequencing. method can be checked.

In the present invention, the term "CpG island" refers to a genomic region in which CpG is gathered at an exceptionally high frequency, with a C+G content of 50% or more and a CpG ratio of 3.75% or more, 0.2 to 3 kb in length. say the part In CpG, C represents cytosine and G represents guanine, and p represents a phosphodiester bond between cytosine and guanine. There are about 45,000 CpG islands in the human genome, most of which are found in promoter regions that control gene expression. In fact, these CpG islands are found in the promoters of housekeeping genes in about 50% of human genes (Cross, S. and Bird, A., Curr. Opin. Gene Develop., 5:309, 1995). . In normal human somatic cells, the CpG island of the housekeeping gene promoter region is unmethylated, and genes that are not expressed during development, such as imprinted genes and inactive genes on the X chromosome, are methylated.

In the genomic DNA of mammalian cells, in addition to A, C, G and T, there is a 5th base called 5-methylcytosine with a methyl group attached to the 5th carbon of the cytosine ring (5-mC) do. 5-mC is attached only to the C of the CG dinucleotide (5'-mCG-3'), called CpG. C in CpG is mostly methylated by contact with methyl groups. Methylation of CpG inhibits the expression of transposons and genomic repeats. In addition, the CpG is a site where most epigenetic changes frequently occur in mammalian cells. This is because 5-mC of the CpG is easily deaminated to become thymine (T).

As used herein, the term "diagnosis" means confirming the presence or characteristics of a pathological condition. For purposes of the present invention, diagnosis is to determine whether oral cancer has occurred.

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

The primers of the present invention can be preferably designed according to the sequence of the CpG island to be analyzed for methylation, and a pair of primers and methylation that can specifically amplify cytosine that is methylated and not modified by bisulfite, respectively. It may be a primer pair capable of specifically amplifying cytosine modified by bisulfite.

The present invention provides a composition for diagnosing oral cancer comprising an agent for measuring the methylation level of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes.

The methylation level may be to detect the methylation level of a CpG island region of a gene.

The agent is a primer pair capable of amplifying a fragment containing a methylated CpG island region, a primer pair capable of amplifying a fragment containing an unmethylated CpG island region, a compound modifying an unmethylated cytosine base or methylation sensitivity It may be any one or more selected from the group consisting of restriction enzymes, but it is specified that it is not limited thereto.

Primer pairs capable of amplifying the fragment containing the methylated CpG island region include a primer pair composed of SEQ ID NOs: 13 and 14, a primer pair composed of SEQ ID NOs: 17 and 18, and SEQ ID NOs: 21 and 18. It may be any one or more selected from the group consisting of a primer pair consisting of SEQ ID NO: 22, a primer pair consisting of SEQ ID NOS: 25 and 26, and a primer pair consisting of SEQ ID NOS: 29 and 30.

Primer pairs capable of amplifying the fragment containing the unmethylated CpG island region include a primer pair consisting of SEQ ID NOs: 11 and 12, a primer pair consisting of SEQ ID NOs: 15 and 16, and SEQ ID NO: 19 And it may be any one or more selected from the group consisting of a primer pair consisting of SEQ ID NO: 20, a primer pair consisting of SEQ ID NOS: 23 and 24, and a primer pair consisting of SEQ ID NOS: 27 and 28.

The compound that modifies the unmethylated cytosine base may be at least one selected from bisulfite, hydrogen sulfite, disulfite, and salts thereof. A method of detecting methylation of a promoter by modifying unmethylated cytosine residues using such bisulfite is well known in the art (Herman JG et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 9821 - 9826; WO01/26536; US2003/0148326A1).

The methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of a CpG island, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, and NotI. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, cleavage by the restriction enzyme varies, and this can be detected through PCR or Southern blot analysis. Other methylation sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

The composition for diagnosing oral cancer may further include polymerase agarose, a buffer solution required for electrophoresis, and the like, in addition to the above agents.

In addition, the present invention provides a kit for diagnosing oral cancer comprising the composition.

The kit may be any one selected from the group consisting of a RT-PCR kit, a microarray chip kit, a DNA kit, and a protein chip kit, but is not limited thereto.

In addition, the present invention provides (a) measuring the methylation level of the CpG island region of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes from a biological sample isolated from the subject; and (b) comparing the measured methylation level with that of a normal control sample.

In the step (b), when the methylation level of the CpG island region of the gene measured from the biological sample isolated from the individual is increased compared to the normal control group, oral cancer may be determined.

The methylation level is measured by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pyrosequencing and bisulfite It may be performed by a method selected from the group consisting of sequencing, but it is specified that it is not limited thereto.

The biological sample may be selected from the group consisting of cells, tissues, blood, saliva, and urine isolated from an individual suspected of having oral cancer, but is not limited thereto.

In addition, the present invention provides a composition for predicting prognosis of oral cancer comprising an agent for measuring the methylation level of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes.

The methylation level may be to detect the methylation level of a CpG island region of a gene.

The agent is a primer pair capable of amplifying a fragment containing a methylated CpG island region, a primer pair capable of amplifying a fragment containing an unmethylated CpG island region, a compound modifying an unmethylated cytosine base or methylation sensitivity It may be any one or more selected from the group consisting of restriction enzymes.

In addition, the present invention provides a kit for predicting oral cancer prognosis comprising the composition.

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

Example 1: Oral cancer-specific methylation gene screening

(1) Whole transcriptome change (RNA-seq) analysis

In order to identify the transcriptome, which is one of the ways to identify transcriptional expression changes of genes, oral squamous carcinoma cell (OSCC) 5 types (Ca9-22, HSC3, SAS, OSC20, YD10B) Transcriptional changes of genes before and after treatment with DNA methylation inhibitor (DNMT inhibitor) in the cell line of were analyzed by RNA-sequencing method.

After treatment with a DNA methylation inhibitor (DNMT inhibitor), genes whose gene expression increased 1.5 times compared to the normal group were distinguished, and in detail, the final genes were selected based on the following criteria. 1) Among the genes, genes were selected by classifying the presence or absence of CpG islands in the promoter region, and 2) genes whose expression increased after treatment with methylation inhibitors in oral squamous cell carcinoma cell lines were selected.

(2) Verification of gene expression changes using quantitative real-time RT-PCR

For transcriptional expression analysis of genes, transcript sequences were obtained from the Ensembl Genome Browser (https://asia.ensembl.org), which holds information on Exon and Intron regions of genes, and Primer3 web tool (http://frodo. wi.mit.edu/primer3) was used to construct primers. Primers used are shown in Table 1 below.

Genes Primers Sequences (5' - 3') sequence number GPX3 GPX3-F ACTGCCATGGTGGCATAAGT One GPX3-R CCAGAATGACCAGACCGAAT 2 ANG ANG-F AGAAGCGGGTGAGAAACAAA 3 ANG-R AGTGCTGGGTCAGGAAGTGT 4 GPRC5B GPRC5B-F TTTTCCAAGCCCATTGTTTC 5 GPRC5B-R AGAAGGACCCACCCTCATTT 6 CTGF CTGF-F CCTGGTCCAGACCACAGAGT 7 CTGF-R TGGAGATTTTGGGAGTACGG 8 BAMBI BAMBI-F TGCTGGACAGGAGCACTTTA 9 BAMBI-R TGCAAATAACCCTGGTGACA 10

For quantitative real-time RT-PCR analysis, total RNA was isolated from 10 oral squamous cell carcinoma cell lines, and cDNA was synthesized using a CFX96 ^TM real-time system (Biorad) using SYBR green master mix (Themo Scientific). . The expression level of the target gene was normalized to the beta actin level value, and the relative quantification of gene expression was performed using the ΔΔCt method.

Referring to FIG. 1 , it was confirmed that the expression of five genes (GPX3, ANG, GPRC5B, CTGF, and BAMBI) was significantly increased after treatment with the DNMT inhibitor than before treatment in the oral squamous cell carcinoma cell line. Based on the above information, 5 genes showing significant differences were finally selected. In addition, the above results prove that the transcriptional expression of these genes is regulated by DNA methylation. Therefore, we sought to identify and validate their promoter methylation.

(3) DNA methylation analysis

DNA was extracted from 10 oral squamous cell carcinoma cell lines using the phenol/chloroform method. Primer pairs used for methylation analysis were constructed within the CpG island of the gene of interest. Primer locations for CpG island analysis and methylation-specific PCR of genes were obtained from the California State University Santa Cruz Genome Browser (http://www.genome.ucsc.edu) and MethPrimer (http://www.urogene.org/cgi- bin/methprimer/methprimer.cgi) to predict the CpG island of the gene of interest and conduct the experiment. The positions of the primers subjected to methylation analysis in the promoter regions of the genes are shown in FIG. 2 . Primers used in the methylation-specific PCR are shown in Table 2 below.

Genes Primers Sequences (5' - 3') sequence number GPX3


GPX3-U-F AGTGTTGGATATTTTAGATGGATGG 11 GPX3-U-R AAAACAAAAAAAACAAACAAAACAC 12 GPX3-M-F CGTCGGATATTTTAGACGGAC 13 GPX3-M-R AAAAACAAAAAAAACAAACAAAAACG 14 ANG


ANG-U-F TTTTGATAGTTTAGTTGGTAGAGTGG 15 ANG-U-R AAACAAAAACAAATTTAATCACATC 16 ANG-M-F TTTTCGATAGTTTAGTTGGTAGAGC 17 ANG-M-R AAACAAAAACAAATTTAATCACGTC 18 GPRC5B


GPRC5B-U-F TTTAGTGTGTGTAAATGGATAATGA 19 GPRC5B-U-R AATCCAAAACACAAAAAAAATCAAT 20 GPRC5B-M-F TTTTAGTGTGCGTAAACGGATAAC 21 GPRC5B-M-R CAAAACGCAAAAAAAATCGAT 22 CTGF


CTGF-U-F GAGTGTTAAGGGGTTAGGATTAATTT 23 CTGF-U-R ACACTAACTATCTCCTCTCAACAAA 24 CTGF-M-F AGTGTTAAGGGGTTAGGATTAATTC 25 CTGF-M-R GCACTAACTATCTCCTCTCAACGA 26 BAMBI


BAMBI-U-F GGGAGAATTTAGTGGTTTTTAATG 27 BAMBI-U-R CAATACCCCAACCTATACAAATAACA 28 BAMBI-M-F GGGAGAATTTAGCGGTTTTTAAC 29 BAMBI-M-R ATACCCCGACCTATACAAATAACG 30

(4) Bisulfite treatment and methylation specific PCR

Bisulfite modification (methylated CG site remains unchanged, unmethylated cytosine (C) is changed to thymine (T) for 2 μg of genomic DNA using EZ DNA methylation kit (Zymo Research) process) was performed. For positive and negative controls, ex vivo methylated DNA (IVD) and water were treated, respectively. For methylation analysis of target genes. Quantitative MSP amplification was performed on the samples treated with bisulfite, and PCR products were confirmed using an agarose gel. Methylation-specific PCR Methylation analysis was performed with reference to the method detailed in Kim et al 2014.

Example 2: Methylation trend analysis

(1) Acquisition of oral mucosal tissue samples

In order to screen for oral cancer-specific methylation genes, tissue samples were obtained from patients diagnosed with oral cancer (OSCC-T) and normal subjects (Normal). For samples, FFPE (Formalin-Fixed paraffin-embedded) tissues owned by the Department of Oral Pathology, Pusan National University School of Dentistry were used.

(2) Candidate gene methylation trends in normal group and oral cancer patient group

In order to identify novel genetic markers that can effectively diagnose oral cancer, five candidate genes (GPX3, GPX3, ANG, GPRC5B, CTGF, BAMBI) methylation trends were analyzed using a methylation-specific PCR method.

Referring to FIG. 3, the GPX3, ANG, GPRC5B, CTGF, and BAMBI showed high methylation levels, such as 83%, 83%, 50%, 62%, and 31%, respectively, in the oral cancer patient group sample (n = 103). , It was confirmed that the normal group samples (n = 47) showed low methylation levels of 15%, 11%, 21%, 23% and 11%, respectively (FIG. 3). The above result means that the expression of the 5 genes selected in this experiment is affected by promoter DNA methylation specifically in oral cancer.

Taken together, the methylation and expression level results indicate that the hypermethylation of the 5 genes is specifically associated with the inhibition of transcriptional expression in oral cancer patient samples, and the 5 genes identified by the present invention are effective in diagnosing oral cancer. means it can be used.

Example 3: Overall survival rate analysis

The five genes were divided into oral cancer patients (n = 344) in the TCGA cohort (head and neck squamous cell carcinoma; HNSCC), and the overall survival rate of oral cancer patients was analyzed by Kaplan-Meier survival curves. The TCGA cohort has methylation pattern analysis data through methylation analysis in patient samples.

In conclusion, referring to FIG. 4 , it can be seen that the prognosis of patients in which each of the five genes is methylated is worse than that of patients who are not methylated. Five genes are methylated at a high rate in oral cancer patient samples, and these results prove that the prognosis of methylated patients is poor when examined statistically using cohorts rather than clinical information of actual patients.

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

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

<110> Inje University Industry-Academic Cooperation Foundation Pusan National University Industry-University Cooperation Foundation <120> Composition for diagnosing oral cavity cancer comprising an agent for determining level of methylation of specific gene and a method for diagnosing oral cavity cancer the same <130> ADP-2021-0662 <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> GPX3-F <400> 1 actgccatgg tggcataagt 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> GPX3-R <400> 2 ccagaatgac cagaccgaat 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> ANG-F <400> 3 agaagcgggt gagaaacaaa 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> ANG-R <400> 4 agtgctgggt caggaagtgt 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> GPRC5B-F <400> 5 ttttccaagc ccattgtttc 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> GPRC5B-R <400> 6 agaaggaccc accctcattt 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> CTGF-F <400> 7 cctggtccag accacagagt 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> CTGF-R <400> 8 tggagattt gggagtacgg 20 <210> 9 <211> 20 <212> DNA <213> artificial sequence <220> <223> BAMBI-F <400> 9 tgctggacag gagcacttta 20 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> BAMBI-R <400> 10 tgcaaataac cctggtgaca 20 <210> 11 <211> 25 <212> DNA <213> artificial sequence <220> <223> GPX3-U-F <400> 11 agtgttggat attttagatg gatgg 25 <210> 12 <211> 24 <212> DNA <213> artificial sequence <220> <223> GPX3-U-R <400> 12 aaacaaaaaa aacaaacaaa acac 24 <210> 13 <211> 21 <212> DNA <213> artificial sequence <220> <223> GPX3-M-F <400> 13 cgtcggatat tttagacgga c 21 <210> 14 <211> 24 <212> DNA <213> artificial sequence <220> <223> GPX3-M-R <400> 14 aaaacaaaaa aaacaaacaa aacg 24 <210> 15 <211> 25 <212> DNA <213> artificial sequence <220> <223> ANG-U-F <400> 15 tttgatagtt tagttggtag agtgg 25 <210> 16 <211> 25 <212> DNA <213> artificial sequence <220> <223> ANG-U-R <400> 16 aaacaaaaac aaatttaatc acatc 25 <210> 17 <211> 25 <212> DNA <213> artificial sequence <220> <223> ANG-M-F <400> 17 ttttcgatag tttagttggt agagc 25 <210> 18 <211> 25 <212> DNA <213> artificial sequence <220> <223> ANG-M-R <400> 18 aaacaaaaac aaatttaatc acgtc 25 <210> 19 <211> 25 <212> DNA <213> artificial sequence <220> <223> GPRC5B-U-F <400> 19 tttagtgtgt gtaaatggat aatga 25 <210> 20 <211> 25 <212> DNA <213> artificial sequence <220> <223> GPRC5B-U-R <400> 20 aatccaaaac acaaaaaaaa tcaat 25 <210> 21 <211> 24 <212> DNA <213> artificial sequence <220> <223> GPRC5B-M-F <400> 21 ttttagtgtg cgtaaacgga taac 24 <210> 22 <211> 21 <212> DNA <213> artificial sequence <220> <223> GPRC5B-M-R <400> 22 caaaacgcaa aaaaaatcga t 21 <210> 23 <211> 26 <212> DNA <213> artificial sequence <220> <223> CTGF-U-F <400> 23 gagtgttaag gggttaggat taattt 26 <210> 24 <211> 25 <212> DNA <213> artificial sequence <220> <223> CTGF-U-R <400> 24 acactaacta tctcctctca acaaa 25 <210> 25 <211> 25 <212> DNA <213> artificial sequence <220> <223> CTGF-M-F <400> 25 agtgttaagg ggttaggatt aattc 25 <210> 26 <211> 24 <212> DNA <213> artificial sequence <220> <223> CTGF-M-R <400> 26 gcactaacta tctcctctca acga 24 <210> 27 <211> 24 <212> DNA <213> artificial sequence <220> <223> BAMBI-U-F <400> 27 gggagaattt agtggttttt aatg 24 <210> 28 <211> 25 <212> DNA <213> artificial sequence <220> <223> BAMBI-U-R <400> 28 aataccccaa cctatacaaa taaca 25 <210> 29 <211> 23 <212> DNA <213> artificial sequence <220> <223> BAMBI-M-F <400> 29 gggagaattt agcggttttt aac 23 <210> 30 <211> 24 <212> DNA <213> artificial sequence <220> <223> BAMBI-M-R <400> 30 ataccccgac ctatacaaat aacg 24

Claims (16)

A composition for diagnosing oral cancer comprising an agent for measuring the methylation level of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes. The composition for diagnosing oral cancer according to claim 1, wherein the methylation level is detected by detecting the methylation level of the CpG island region of the gene. The method of claim 1, wherein the preparation comprises a primer pair capable of amplifying a fragment comprising a methylated CpG island region, a primer pair capable of amplifying a fragment comprising an unmethylated CpG island region, and an unmethylated cytosine base. A composition for diagnosing oral cancer, characterized in that it is at least one selected from the group consisting of a modifying compound or a methylation sensitive restriction enzyme. The method of claim 3, wherein the primer pair capable of amplifying the fragment containing the methylated CpG island region is a primer pair composed of SEQ ID NOs: 13 and 14, and a primer composed of SEQ ID NOs: 17 and 18 Any one selected from the group consisting of a pair, a primer pair consisting of SEQ ID NOs: 21 and 22, a primer pair consisting of SEQ ID NOs: 25 and 26, and a primer pair consisting of SEQ ID NOs: 29 and 30 A composition for diagnosing oral cancer, characterized in that the above. The method of claim 3, wherein the primer pair capable of amplifying the fragment containing the unmethylated CpG island region consists of a primer pair consisting of SEQ ID NOs: 11 and 12 and SEQ ID NOs: 15 and 16 Any one selected from the group consisting of a primer pair, a primer pair consisting of SEQ ID NOs: 19 and 20, a primer pair consisting of SEQ ID NOs: 23 and 24, and a primer pair consisting of SEQ ID NOs: 27 and 28 A composition for diagnosing oral cancer, characterized in that one or more. The composition for diagnosing oral cancer according to claim 3, wherein the compound that modifies the unmethylated cytosine base is at least one selected from bisulfite, hydrogen sulfite, disulfite, and salts thereof. A kit for diagnosing oral cancer comprising the composition of any one of claims 1 to 6. The kit for diagnosing oral cancer according to claim 7, wherein the kit is any one selected from the group consisting of a RT-PCR kit, a microarray chip kit, a DNA kit, and a protein chip kit. (a) measuring the methylation level of the CpG island region of one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes from a biological sample isolated from the subject; and
(b) a method for providing information necessary for oral cancer diagnosis comprising the step of comparing the measured methylation level with that of a normal control sample. 10. The method of claim 9, wherein step (b) further comprises determining oral cancer if the methylation level of the CpG island region of the gene measured from the biological sample isolated from the subject is increased compared to the normal control group. A method for providing information necessary for diagnosis of oral cancer. The method of claim 9, wherein the methylation level is determined by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pi A method for providing information necessary for diagnosing oral cancer, characterized in that it is performed by a method selected from the group consisting of sequencing and bisulfite sequencing. 10. The method of claim 9, wherein the biological sample is selected from the group consisting of cells, tissues, blood, saliva, and urine isolated from an individual suspected of having oral cancer. A composition for predicting prognosis of oral cancer comprising an agent for measuring the methylation level of any one or more genes selected from the group consisting of GPX3, ANG, GPRC5B, CTGF and BAMBI genes. The composition for predicting prognosis of oral cancer according to claim 13, wherein the methylation level is detected by detecting the methylation level of the CpG island region of the gene. The method of claim 13, wherein the preparation comprises a primer pair capable of amplifying a fragment comprising a methylated CpG island region, a primer pair capable of amplifying a fragment comprising an unmethylated CpG island region, and an unmethylated cytosine base. A composition for predicting the prognosis of oral cancer, characterized in that at least one selected from the group consisting of a compound or a methylation-sensitive restriction enzyme that transforms. A kit for predicting the prognosis of oral cancer comprising the composition of any one of claims 13 to 15.

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