KR100892588B1 - Diagnosis Kit and Chip For Gastric Cancer Using Gastric Cancer Specific Methylation Marker Gene - Google Patents

Abstract

The present invention relates to a method for screening gastric cancer specific marker genes, a kit for detecting gastric cancer using the screened gene and a nucleic acid chip, and more particularly, to a gastric cancer specific marker gene in which a promoter region is specifically methylated in gastric cancer transformed cells. The present invention relates to a screening method, a kit for detecting gastric cancer and gastric cancer progression stage, and a nucleic acid chip capable of confirming promoter methylation of the marker gene.

By using the diagnostic kit and the diagnostic nucleic acid chip of the present invention, gastric cancer can be diagnosed at an early transformation stage, so that early diagnosis is possible, and the advanced stage of gastric cancer and gastric cancer can be diagnosed more accurately and quickly than conventional methods.

Gastric cancer, Methylation, Methylation marker, Gastric cancer diagnosis, Early diagnosis

Description

Diagnosis Kit and Chip For Gastric Cancer Using Gastric Cancer Specific Methylation Marker Gene}

1 is a schematic diagram showing a method for selecting a gastric cancer marker gene.

2 is a schematic diagram illustrating a method of confirming methylation by restriction enzyme cleavage and gene amplification to confirm that the marker candidate gene is actually methylated.

3 is a flowchart illustrating a method of selecting a gastric cancer marker gene.

Figure 4 shows the promoter methylation status of 23 identified methylated genes in cancer cell lines AGS, MKN1, MKN28 and SNU484 cells.

5 shows the promoter methylation status of 23 identified methylated genes in tumors and non-tumor gastric tissue in the vicinity of the tumor.

6 shows the reactivation of 23 gastric cancer markers after treatment with the dimethylating agent.

FIG. 7A shows the methylation frequency of 23 gastric cancer markers in normal tissues (3 samples), tumor tissues (12 samples) and consequent tumor tissues (10 samples) taken from normal subjects.

Figure 7b shows that 23 marker genes are highly methylated in tumor tissue as well as tumor tissue.

Figure 8 shows the methylation frequency of 23 confirmed genes in advanced gastric cancer tissue.

Field of invention

The present invention relates to a method for screening gastric cancer specific marker genes, a kit for detecting gastric cancer using the screened gene and a nucleic acid chip, and more particularly, to a gastric cancer specific marker gene in which a promoter region is specifically methylated in gastric cancer transformed cells. The present invention relates to a screening method, a kit for detecting gastric cancer and gastric cancer progression stage, and a nucleic acid chip capable of confirming promoter methylation of the marker gene.

Background of the Invention

Despite advances in modern medicine, solid cancers, the majority of cancers (non-blood cancers), have a five-year survival rate of less than 50%. About two-thirds of all cancer patients are found with advanced cancer, most of whom die within two years of being diagnosed. This result is not only a problem of cancer treatment methods, but also because it is not easy to diagnose cancer at an early stage, to accurately diagnose advanced cancer, or to observe the prognosis for newly developed therapeutics.

Recent diagnosis of cancer in the clinic is typically confirmed by performing a tissue biopsy after medical history examination, physical examination and clinical evaluation. However, clinical diagnosis of cancer is possible only when the number of cancer cells is more than 1 billion and the diameter of the cancer is more than 1 cm. In this case, the cancer cells already have metastatic capacity and at least half of them have already metastasized. On the other hand, although tumor markers for monitoring substances produced directly or indirectly from cancer are used for cancer screening, more than half of them appear normal even in the presence of cancer, and often positive in the absence of cancer. There is a limit that causes confusion. In addition, there is a problem that the anticancer substance mainly used for treating cancer is effective only when the tumor is small in size.

Diagnosis and treatment of cancer is difficult because cancer cells are very complex and diverse. Cancer cells grow excessively continuously, penetrate into surrounding tissues, metastasize to terminal organs, and eventually lead to death. Despite the attack of the immune system or chemotherapy, cancer cells survive, continue to evolve, and selectively spread to the cell groups that are best suited to survive. Cancer cells are living organisms with a high degree of vitality that are caused by mutations in numerous genes. In order for a cell to be transformed into a cancer cell and develop into a malignant cancer mass diagnosed in a hospital, numerous genetic variations must occur. Therefore, a genetic level approach is needed to diagnose and treat cancer at the source.

Recently, genetic analysis has been actively attempted to diagnose cancer. The simplest and typical method is to use PCR to detect the presence of the ABL: BCR fusion gene (a genetic feature of leukemia) in the blood. The method is more than 95% accurate and uses this simple and easy genetic analysis to treat chronic myeloid leukemia and to be used for outcome assessment and subsequent studies. However, there is a drawback that the method applies only to a few blood cancers.

Recently, genetic testing using DNA in serum or plasma has been actively attempted. The method is to detect cancer-related genes that are isolated from cancer cells and secreted into the blood and present in the form of free DNA in serum.

The concentration of DNA present in the serum was found to be 5 to 10 times higher in actual cancer patients than normal individuals, and this increased DNA is mostly derived from cancer cells. Cancer can be diagnosed by analyzing cancer-specific gene abnormalities such as mutations, deletions or functional loss of oncogenes and tumor-suppressor genes, such as DNA isolated from cancer cells.

Active attempts to diagnose lung cancer, head and neck cancer, breast cancer, colon cancer and liver cancer by examining the promoter methylation of the mutated K-Ras oncogenes, p53 tumor-suppressor genes and p-16 genes, and the labeling and instability of microsatellites (Chen, X. et al ., Clin. Cancer Res ., 5: 2297, 1999; Esteller, M. et al ., Cancer Res ., 59:67, 1999; Sanchez-Cespedes. M. et al ., Cancer Res ., 60: 892, 2000; Sozzi, G. et al ., Clin. Cancer Res ., 5: 2689, 1999).

DNA from cancer cells can also be detected in samples other than blood. Attempts have been made to detect the presence of cancer cells using gene or antibody tests in sputum or bronchoalveolar lavage fluid of lung cancer patients (Palmisano, W. et al ., Cancer Res ., 60: 5954, 2000; Sueoka, E. et. al ., Cancer Res ., 59: 1404, 1999). In addition, methods for detecting the presence of oncogenes in the colon and rectal excretion of cancer patients (ahlquist, D. et al ., Gastroenterol ., 119: 1219, 2000) and methods for detecting abnormality of promoter methylation in urine and prostate fluid ( Goessl, C. et al ., Cancer Res ., 60L: 5941, 2000). However, in order to precisely diagnose cancers that cause numerous gene abnormalities and show various mutation characteristics of each gene, a method of simultaneously analyzing a large number of genes in an accurate and automated manner is required. It is not established.

Therefore, we propose a method for diagnosing cancer by measuring DNA methylation. When the promoter CpG island of a particular gene is hyper-methylated, the gene is inhibited in expression. Even if there are no mutations in the protein-coding sequence of a gene in vivo, the function of the gene is lost and the main mechanism is disturbed. In addition, the loss of function of numerous tumor suppressor genes in human cancers is also analyzed as a factor. Therefore, detecting methylation of the promoter CpG island of tumor suppressor genes is urgently needed in cancer research. Recently, methods for determining promoter methylation by methods such as methylation specific PCR (hereinafter referred to as MSP) or automatic DNA sequencing for cancer diagnosis and screening have been actively attempted.

In genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base called 5-methylcytosine with a methyl group attached to the fifth carbon of the cytosine ring (5-mC). . 5-mC is attached only to C of CG dinucleotide (5'-mCG-3 '), called CpG. C in CpG is mostly methylated by the contact of methyl groups. Methylation of CpG inhibits the expression of alu or transposons and repeats of the genome. In addition, the CpG is a site where most epigenetic changes occur frequently in mammalian cells. The 5-mC of the CpG naturally deaminoates to become T, and the CpG of the mammalian genome appears with only 1% probability, which is much lower than the normal probability (1/4 ㅧ 1/4 = 6.25%). .

The region where CpG is exceptionally collected is called CpG island. CpG islands are sites of 0.2-3kb in length with a C + G content of 50% or more and a CpG ratio of 3.75% or more. There are about 45,000 CpG islands in the human genome, most of which are found at promoter sites that regulate gene expression. Indeed, the CpG islands are found in promoters of housekeeping genes, about 50% of human genes (Cross, S. and Bird, A., Curr. Opin. Gene Develop ., 5: 309, 1995). .

In somatic cells of normal individuals, CpG islands of the housekeeping gene promoter region are unmethylated and genes that are not expressed during development, such as imprinted genes and genes on inactive X chromosome, are methylated.

During the cancer-causing process, methylation is found on the promoter CpG islands and expression of the corresponding genes is inhibited. In particular, the methylation of the promoter CpG island of the tumor suppressor gene, which regulates cell cycle or apoptosis, repairs DNA, is involved in cell adhesion and intercellular interactions, and inhibits cell invasion and metastasis, is methylated. Inhibits the expression and function of the gene in the same manner as mutation of the coding sequence, thereby promoting the development and progression of cancer. In addition, aging also causes partial methylation of the CpG islands.

It is interesting to note that mutations in genes affect the development of innate cancers, and in acquired cancers, for genes that do not have mutations, methylation of the promoter CpG island occurs instead of mutations. Typical examples include acquired kidney cancer VHL (von Hippel Lindau), breast cancer BRCA1, colorectal cancer MLH1 and gastric cancer E-CAD. In addition, promoter methylation of p16 or mutation of Rb occurs in about half of all cancers, and mutation of p53 or promoter methylation of p73, p14, etc. occurs in the remaining cancers.

An important fact is that epigenetic changes caused by promoter methylation cause genetic changes (eg, mutations in coding sequences), and cancer development proceeds by a combination of such genetic and epigenetic changes. For example, in the case of the MLH1 gene, the function of one allele of the MLH1 gene in colon cancer is lost by mutation or deletion, and the other allele is incapable of functioning by promoter methylation. In addition, if MLH1, a DNA repair gene, fails to function by promoter methylation, mutations may occur in other major genes to further promote cancer development.

Most cancers exhibit three characteristics of CpG: hypermethylation of the promoter CpG island of the tumor suppressor gene, hypermethylation of the remaining CpG base sites, and increased activity of a methylation enzyme called DNA cytosine methyltransferase (DNMT) (Singal, R. and Ginder, G., Blood, 93: 4059, 1999; Robertson, K. and Jones, P., Carcinogensis, 21:. 461, 2000; Malik, K. and Brown, K., Brit J. Cancer, 83: 1583 , 2000).

It is not yet fully understood why the expression of the corresponding gene is inhibited when the promoter CpG island is methylated, but methyl CpG-binding protein (MECP) or methyl CpG-binding domain protein (MBD) and histone deacetylases. It is hypothesized that is due to the attachment of methylated cytosine, thereby changing the chromatin structure of the chromosome, thereby changing the histone protein.

Promoter methylation can be used to accurately analyze the methylation of all cytosine bases on the promoter CpG islands in order to maximize the accuracy of cancer diagnosis, to analyze cancer development at each stage, and to distinguish between cancer and aging changes. I need a way.

Authentic methods use amplification of a gene region containing CpG islands by methylation specific PCR (MSP) and a sequencing method (bisulfite genome sequencing method). However, there is no way to accurately and quickly and automatically analyze various changes in promoter methylation of many genes that can be used to evaluate each stage of various cancers in diagnostic, early diagnosis or clinical trials.

Much research has been done in the field of screening new tumor suppressor genes associated with methylation. Until now, screening methods for tumor suppressor genes known to be methylated in various cancers have been mainly used in reference to existing literature. Recently, a method has been developed for screening genes with increased expression by dimethylation in cell lines treated with a dimethylating agent, compared to cell lines that have not been treated with a dimethylating agent by treating the cancer cell lines (Suzuki et al ., 2002., Nature Genetics, 31: 141-149). However, this method is inefficient in detecting methylation-related genes with high accuracy because screening for methylation-related tumor genes using only dimethylation phenomena in cancer cell lines is performed without performing gene expression analysis using tissue-derived clinical samples. Therefore, in order to screen tumor-specific methylated genes with high sensitivity and specificity in clinical applications, it is necessary to develop a systematic method for screening tissue-specific and cancer-specific methylated tumor suppressor genes.

In this regard, the present inventors performed gene expression analysis in transformed cells of gastric tissue clinical samples in order to effectively screen methylation-related tumor suppressor genes that are methylated specifically in gastric cancer tissues. From this, a list of genes with reduced expression in tumor tissue cells compared to normal cells was prepared. In addition, a list of genes that were expressed more in cells treated with the dimethylating agent was compared to tumor tissues (transformed) cells not treated with the dimethylating agent. By comparing the two gene lists, a common gene list was selected as a methylation related gene promoter marker. By using the screened genetic markers, the degree of methylation was measured using actual clinical samples diagnosed with dysplasia, which is a stage of cancer and early cancer progression, and these gene markers differentially diagnose tissues undergoing transformation into gastric cancer and cancer cells. The present invention was confirmed after confirming that it can be done.

It is an object of the present invention to provide a kit for diagnosing gastric cancer comprising a primer for amplifying a fragment comprising a CpG island of a gastric cancer marker gene in which a promoter region is specifically methylated in a gastric cancer transformed cell and a methylation-sensitive restriction endonuclease. have.

Another object of the present invention is to provide a nucleic acid chip for diagnosis of gastric cancer and gastric cancer progression, comprising a fragment comprising a CpG island of the gastric cancer specific marker gene and a probe capable of hybridizing under strict conditions.

In order to achieve the above object, the present invention comprises the steps of (a) comparing the gene expression list of the transformed cells and non-transformed cells, identifying a gene whose expression is reduced in the transformed cells; (b) treating the transformed cells with a dimethylating agent, comparing the gene expression list of the transformed cells with the gene expression list of the transformed cells not treated with the dimethylating agent, thereby expressing more in the cells treated with the dimethylating agent. Identifying a gene; (c) reviewing the sequences of genes identified in common in steps (a) and (b) to exclude genes without CpG islands in the promoter sequence; And (d) provides a screening method of the methylation marker gene that can determine whether the cell transformation from normal cells to gastric cancer cells comprising the step of selecting the remaining gene as the methylation marker gene except for the above.

In the present invention, the dimethylating agent may be characterized in that the 5 aza 2'-deoxysidine (DAC).

In the present invention, by analyzing the methylation of the gene commonly identified in cells transformed with cancer, the step of identifying a gene identified as methylated marker gene, the promoter region of the commonly identified gene It can be characterized.

In the present invention, methylation analysis of the identified gene comprises the steps of: (a) preparing a primer comprising a methylation site in the nucleic acid site to be amplified; (b) treating the genome of the transformed cell with a methylation specific restriction endonuclease; And (c) amplifying the nucleic acid by contacting the primers with the treated genomic nucleic acid, but if no amplification product is produced, the identified gene is considered unmethylated, and if the amplification product is produced, the identification Characterized in that it is carried out by the step of selecting the gene as methylated.

In the present invention, the genome treated with isochizomer of methylation-sensitive restriction endonuclease in which the transformed cell genome cleaves both methylated and unmethylated CpG sites is used as a control. can do.

In the present invention, the amplification products may be generated by hybridization using a probe.

In the present invention, the probe may be characterized in that it is immobilized on a solid substrate.

In the present invention, amplification may be performed by a method selected from the group consisting of PCR, real-time PCR, amplification, and linear amplification using isothermal enzymes.

The present invention also relates to MTCBP-1 (NT_022270)-Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_007933) -Mitotrophin; MTSS1 (NT_008046) —Metastasis suppressor 1; PELI2 (NT_026437)-Pellino homolog 2, (Drosophila) {Pellino homolog 2, (Drosophila)}; PLEKHF2 (NT008046) —Pleckstrin homology domain containing, family F (with FYVE domain) member 2], including flextrin homology domains; RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory agent (RAS-like, estrogen-regulated, growth inhibitor); THBD (NT_011387) Thrombomodulin; TP53INP1 (NT_008046)-Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_016354)-hypothetical protein MGC11324; ZFHX1B (NT_005058)-Zinc finger homeobox 1b; ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface (Adrenergic, beta-2-, receptor surface); AR (NT_011669) -Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and soft muscle atrophy; kidney disease) {Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; BLVRB (NT_011109)-Bliiverdin Reductase B (flavin reductase B) (NADPH); CALCR (NT_007933) —Calcitonin receptor; CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; CKAP4 (NT_019546) -Cytoskeleton-associated protein 4; CYBRD1 (NT_005403)-Cytochrome b reductase 1; GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_023935)-Golgi phosphoprotein 2; HHEX (NT_030059) —Hematopoietically expressed homeobox; LAMA2 (NT_025741)-laminin, alpha 2 (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; CPEB3 (NT_030059) —cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_007299)-5-hydroxytrytamine (serotonin) receptor 1B {5-hyroxytryptamine (serotonin) receptor 1B} gene and amplification fragments containing CpG islands of gastric cancer marker genes selected from the group consisting of combinations thereof Provided are a kit for the diagnosis of gastric cancer and gastric cancer progression, comprising a primer and a methylation sensitive restriction endonuclease.

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The present invention also relates to MTCBP-1 (NT_022270)-Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_007933) -Mitotrophin; MTSS1 (NT_008046) —Metastasis suppressor 1; PELI2 (NT_026437)-Pellino homolog 2, (Drosophila) {Pellino homolog 2, (Drosophila)}; PLEKHF2 (NT_008046) -Plexckstrin homology domain containing, family F (with FYVE domain) member 2], containing flextrin homology domains; RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory agent (RAS-like, estrogen-regulated, growth inhibitor); THBD (NT_011387) Thrombomodulin; TP53INP1 (NT_008046)-Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_016354)-hypothetical protein MGC11324; ZFHX1B (NT_005058)-Zinc finger homeobox 1b; ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface (Adrenergic, beta-2-, receptor surface); Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; BLVRB (NT_011109)-Bliiverdin Reductase B (flavin reductase B) (NADPH); CALCR (NT_007933) —Calcitonin receptor; CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; CKAP4 (NT_019546) -Cytoskeleton-associated protein 4; CYBRD1 (NT_005403)-Cytochrome b reductase 1; GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_023935)-Golgi phosphoprotein 2; HHEX (NT_030059) —Hematopoietically expressed homeobox; LAMA2 (NT_025741)-laminin, alpha 2 (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; CPEB3 (NT_030059) —cytoplasmic polyadenylation element binding protein 3; Fragments containing CpG islands of gastric cancer marker genes selected from the group consisting of the HTR1B (NT_007299) -5-hydroxytryptamine (serotonin) receptor 1B gene and combinations thereof It provides a nucleic acid chip for diagnosis of gastric cancer and gastric cancer progression step comprising a probe capable of hybridization.

One aspect of the present invention relates to a method for screening a methylation marker gene capable of confirming cell transformation from normal cells to gastric cancer cells.

In the present invention, marker genes that regulate the process of transforming normal cells into gastric cancer cells in gastric tissue by methylation were selected. The methylation marker gene may comprise (1) comparing the gene expression list of the transformed cell or cell line and the non-transformed cell or cell line, and sorting the list of genes that are more expressed in the non-transformed cell or cell line; (2) treating the transformed cell or cell line with a methylation inhibitor and sorting the list of genes that are more expressed in the transformed cell or cell line treated with the methylation inhibitor as compared to the untreated transformed cell or cell line; (3) Excluding the genes without CpG islands by comparing the gene lists obtained in steps (1) and (2) and examining the sequence of genes present in both lists to see if there is a CpG island in the promoter sequence. ; And (4) considering the remaining genes excluded as above as marker gene candidates regulated by methylation.

As an additional method of identification, it may be confirmed by biochemical analysis whether the gene is substantially methylated.

The screening method can find genes that are methylated in various stages of dysplasia in gastric cancer as well as gastric cancer, and the selected genes can be screened for gastric cancer, risk assessment, prediction, disease identification, stage diagnosis and treatment of disease. Can also be used for selection of.

Identifying genes that are methylated in gastric cancer and at various stages of abnormality enables early and accurate diagnosis of gastric cancer, and can identify new targets for establishing and treating methylation lists using multiple genes. In addition, methylation data according to the present invention will be able to establish a more accurate gastric cancer diagnosis system in conjunction with other non-methylated associated biomarker detection methods.

With the methods of the present invention, the progression of gastric cancer at various stages or stages can be diagnosed, including determining the methylation stage of one or more nucleic acid biomarkers obtained from a sample. Comparing the methylation step of the nucleic acid isolated from the sample of each stage of gastric cancer with the methylation step of one or more nucleic acids obtained from a sample having no cell proliferative abnormality of the gastric tissue, the specific stage of gastric cancer of the sample can be identified. The step may be hypermethylation.

In one example of the invention, the nucleic acid may be methylated at the regulatory site of a gene. As another example, since methylation starts from the outside of the regulatory region of the gene and proceeds to the inside, detection of methylation at the outside of the regulatory region enables early diagnosis of genes involved in cellular transformation.

As another example, in the present invention, a cell growth abnormality (dysplasia) of gastric tissue present in a specimen may be diagnosed by detecting a methylation state of one or more nucleic acids of the following examples using a kit or a nucleic acid chip: MTCBP-1 (NT_022270)-Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_007933) -Myotrophin; MTSS1 (NT_008046) —Metastasis suppressor 1; PELI2 (NT_026437)-Pellino homolog 2, (Drosophila) {Pellino homolog 2, (Drosophila)}; PLEKHF2 (NT_008046) -Plexckstrin homology domain containing, family F (with FYVE domain) member 2], containing flextrin homology domains; RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory agent (RAS-like, estrogen-regulated, growth inhibitor); THBD (NT_011387) Thrombomodulin; TP53INP1 (NT_008046)-Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_016354)-hypothetical protein MGC11324; ZFHX1B (NT_005058)-Zinc finger homeobox 1b; ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface (Adrenergic, beta-2-, receptor surface); AR (NT_011669) -Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and soft muscle atrophy; kidney disease) {Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; BLVRB (NT_011109)-Bliiverdin Reductase B (flavin reductase B) (NADPH); CALCR (NT_007933) —Calcitonin receptor; CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; CKAP4 (NT_019546) -Cytoskeleton-associated protein 4; CYBRD1 (NT_005403)-Cytochrome b reductase 1; GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_023935)-Golgi phosphoprotein 2; HHEX (NT_030059) —Hematopoietically expressed homeobox; LAMA2 (NT_025741)-laminin, alpha 2 (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; CPEB3 (NT_030059) —cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_007299)-5-hydroxytryptamine (serotonin) receptor 1B {5-hyroxytryptamine (serotonin) receptor 1B} gene and combinations thereof.

By using the diagnostic kit or nucleic acid chip of the present invention, it is possible to determine the propensity of cell growth abnormality (dysplasia progression) of the gastric tissue of the specimen. The method includes determining the methylation status of one or more nucleic acids isolated from the sample, wherein the methylation step of the one or more nucleic acids is compared to the methylation step of the nucleic acid isolated from the sample without cell growth abnormality (dysplasia) of the tissue. It can be characterized by.

Exemplary nucleic acids include MTCBP-1 (NT_022270) —Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_007933) -Mitotrophin; MTSS1 (NT_008046) —Metastasis suppressor 1; PELI2 (NT_026437)-Pellino homolog 2, (Drosophila) {Pellino homolog 2, (Drosophila)}; PLEKHF2 (NT_008046) -Plexckstrin homology domain containing, family F (with FYVE domain) member 2], containing flextrin homology domains; RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory agent (RAS-like, estrogen-regulated, growth inhibitor); THBD (NT_011387) Thrombomodulin; TP53INP1 (NT_008046)-Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_016354)-hypothetical protein MGC11324; ZFHX1B (NT_005058)-Zinc finger homeobox 1b; ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface (Adrenergic, beta-2-, receptor surface); Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; BLVRB (NT_011109)-Bliiverdin Reductase B (flavin reductase B) (NADPH); CALCR (NT_007933) —Calcitonin receptor; CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; CKAP4 (NT_019546) -Cytoskeleton-associated protein 4; CYBRD1 (NT_005403)-Cytochrome b reductase 1; GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_023935)-Golgi phosphoprotein 2; HHEX (NT_030059) —Hematopoietically expressed homeobox; LAMA2 (NT_025741)-laminin, alpha 2 (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; CPEB3 (NT_030059) —cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_007299)-5-hydroxytryptamine (serotonin) receptor 1B {5-hyroxytryptamine (serotonin) receptor 1B} gene and combinations thereof.

In another embodiment of the present invention, the methylation gene marker can be used for early diagnosis of tissues that are likely to form gastric cancer. If a gene identified to be methylated in cancer tissue is methylated in a tissue that appears clinically or morphologically normal, the tissue that appears to be normal is in progress of cancer. Therefore, gastric cancer specific genes in seemingly tissues can confirm the methylation, thereby allowing early diagnosis of gastric cancer.

By using the methylation marker gene of the present invention, it is possible to detect cell growth abnormality (heterogeneous progression) of gastric tissue in a sample. The method includes contacting a sample comprising one or more nucleic acids isolated from a sample with an agent capable of determining one or more methylation states. The method comprises identifying the methylation status of one or more sites in one or more nucleic acids, wherein the methylation status of the nucleic acid is determined by the methylation status of the same site in the nucleic acid of the sample that does not have cell growth abnormalities (progressive dysplasia) of the tissue. It may be characterized by a difference.

The present invention, in one aspect, for the diagnosis of high abnormalities (high-risk group dysplasia) of gastric dysplasia containing a target-specific primer for amplifying a gene identified as the gastric cancer specific methylation marker gene and an agent for sensitively cutting non-methylated nucleic acid Provide the kit.

In a further aspect of the invention the kit comprises a first container containing a carrier means for partitioning a sample contained therein and an agent that sensitively cleaves unmethylated nucleic acids and a second containing a target-specific primer for amplification of the biomarker It may be characterized by including one or more containers including the container.

In another embodiment of the present invention, screening the marker gene, (1) comparing the gene expression list of the transformed cells and non-transformed cells, identifying a gene whose expression is reduced in the transformed cells; (2) Treating the transformed cells with a dimethylating agent, comparing the gene expression list of the transformed cells with the gene expression list of the transformed cells not treated with the dimethylating agent, thereby expressing more genes in the cells treated with the dimethylating agent. Confirming; (3) identifying the sequence of genes identified in common in steps (1) and (2) and excluding genes without CpG islands in the sequences of promoter and exon 1 among the screened genes; And (4) a method of screening a methylation marker gene capable of confirming the transformation of gastric cancer cells from normal cells, including selecting the remaining gene as the methylation marker gene.

In the above method, the comparison may be performed by direct comparison or indirect comparison. The dimethylating agent may be 5 aza 3'-dioxycytidine (DAC). The method for identifying a methylation marker gene in the industry includes analyzing the methylation of the commonly identified gene in transformed cells, wherein the methylation is present at the promoter region of the commonly identified gene, wherein the gene Confirm marker gene.

 In another embodiment, the analysis of methylation of the commonly identified genes according to the method comprises the steps of: (i) identifying a primer comprising a methylation site within the amplified nucleic acid site, (ii) transforming with a methylation specific restriction endonuclease Processing the genome of the cell, and (iii) confirming that the gene was methylated that the amplification product produced by contacting the primers with the genomic nucleic acid was successful, and the amplification product was not generated. By confirming that the gene is not methylated.

As a control, the transformed cell genome can be treated with isochiomers of methylation sensitive restriction endonucleases that cleave both methylated and unmethylated CpG sites.

In another aspect, the present invention provides a nucleic acid chip for gastric cancer and gastric cancer advanced stage diagnosis comprising a fragment capable of hybridizing with a fragment comprising a CpG island of a marker gene which detects the presence of the amplified nucleic acid by hybridization. It is about.

Additionally, the amplification can be performed by PCR or real-time PCR, amplification using isothermal enzymes or linear amplification. Cell transformation can be detected early by detecting methylation outside the promoter.

In an embodiment of the present invention, transformed gastric cancer cells can be identified by examining methylation of the marker gene using the kit or nucleic acid chip of the present invention.

In another embodiment of the present invention, the progression of gastric cancer or gastric cancer can be diagnosed by examining the methylation of the marker gene using the kit or the nucleic acid chip of the present invention.

In another embodiment of the present invention, the possibility of progressing to gastric cancer can be diagnosed by examining methylation of the marker gene using a kit or nucleic acid chip of the present invention using a sample showing a normal phenotype. The sample may use solid or liquid tissue, serum or plasma.

In another embodiment of the present invention, by examining the methylation frequency of genes specifically methylated in gastric cancer, and determining the methylation frequency of tissues having the potential for gastric cancer progression, the possibility of tissue development into gastric cancer can be evaluated.

In the present invention, "dimethylation agents" include, but are not limited to, chemical agents or enzymes that remove methyl groups from nucleic acids or inhibit methylation from occurring.

Examples of such dimethylated agents include 5-azacytidine, 5-acza 2-deoxycytidine (DAC), arabinofuranosyl-5-azacytosine, 5-fluoro- 5-fluoro-2'-deoxycytidine, pyrimidone, trifluoromethyldeoxycytidine, pseudoisocytidine, dihydro-5-azasai As competitive inhibitors such as thydine, AdoHcy, AdoMet / AdoHcy analogs, sinefungin and analogs, SIBA (5'-deoxy-5'-S-isobutyladenosine), MTA (5'-methylthio-5 ' Drugs that affect levels of AdoMet, such as deoxyadenosine and ethionine analogs, methionine, L-cis-AMB, cycloleucine, antifolates, and methotre Low levels of AdoHcy hydrolase such as methotrexate, drugs that affect levels of AdoHcy, and dc-AdoMet and MTA Third, 3-deaza-adenosine, neplanocin A, 3-dezaneplanocin, 4'-thioadenosine, 3-deza-aristeromycin 3-deza-aristeromycin, ornithine synthetase inhibitors, inhibitors of DFMO (α-difluoromethylornithine), spermine and spermidine syntheses, MTA (S -Methyl-5'-methylthioadenosine), L-cis-AMB, AdoDATO, MGBG, methylthioadenosine phosphorylase inhibitor, DFMTA (difluoromethylthioadenosine), metanin, spermine / spermidine , Sodium butyrate, procainamide, hydralazine, dimethylsulfoxide, free radical DNA adduct, UV, 8-hydroxy guanine, N-methyl-N-nitrosourea (N-methyl N-nitrosourea, nobiobiocine, phenolbarnital, benzo [a] pyrene, ethylmethanesulfonate, ethylnitrosourea (eth ylnitrosourea), N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, nitrogen mustard, N-methyl-N-nitro-N-nitroso Guanidine, diethylnitrosamine, chlordene, N-acetoxy-N-2-acethylaminofluorene, aflatoxin B1, nalidixic acid ), N-2-fluorenylacetamine, 3-methyl-4 '-(dimethylamino) azobenzene, 1,3-bis (2-chloroethyl) -1-nitrosourea, Cyclophosphamide, 6-mercaptopurine, 4-nitroquinoline-1-oxide, N-nitrosodiethylamine, hexamethylenebisacetamide, rethonic acid, cAMP and retionic acid, Antisense mRNAs for aromatic hydrocarbon carcinogens, dibutyryl cAMP or methyltransferases (Zingg et al. ., Carcinogenesis , 18: 869, 1997).

As used herein, "cell transformation" is characterized from one form to another, such as from normal to abnormal, from non-tumor to tumorous, from undifferentiated to differentiation, from stem cells to non-stem cells. It means to change. Additionally, the transformation can be recognized by the morphology, phenotype, biochemical properties, etc. of the cell. There are many examples of the transformation of such cells, but the present invention relates to the presence of abnormal cells and cancerous cells in the stomach, and the marker for transformation of the tissue is a marker for conversion to gastric cancer cells.

In the present invention, the term "direct comparison" is used to competitively probe probes between differently labeled nucleic acids separated from one or more sources in order to confirm the correspondence between one type of differently labeled nucleic acid and another type of nucleic acid. To combine.

In the present invention, the possibility of cancer is discovered before the "early confirmation" of cancer, preferably, before morphological changes are observed in the sample tissue or cells. In addition, "early identification" of cell transformation refers to the possibility of transformation occurring at an early stage before the cell is transformed.

"Hypermethylation" in the present invention means methylation of CpG islands.

In the present invention, "indirect comparison" evaluates the level of nucleic acid isolated from the first source and the same allele level in the nucleic acid isolated from the second source using a reference probe hybridized to the nucleic acid isolated from the first source and the second source, respectively. And comparing the results to determine the relative amount of nucleic acid present in the sample without competing direct binding to the reference probe.

In the present invention, "sample" or "sample sample" means a wide range of samples including all biological samples obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of assay being performed. As indicated above, biological samples include semen, lymph, body fluids, blood plasma, and the like. Methods of obtaining bodily fluids and tissue biopsies from mammals are commonly known. Preferred source is gastric biopsy.

As used herein, "near tumor tissue" or "paired tumor tissue" refers to tissue that exhibits clinical and morphologically normal phenotypes present in the vicinity of cancerous tissue sites.

Screening of Methylation Regulated Biomarkers

In the present invention, a biomarker gene that is methylated is screened when the cell or tissue is transformed or the cell is changed to another form. Here, "transformed" cells means that the normal form is abnormal, non-neoplastic is neoplastic, undifferentiated form is differentiated into different forms (Fig. 1). ).

Therefore, in the present invention, the methylation regulatory marker gene in the transformation into gastric cancer cells was identified by a systematic method. In one aspect of the method, (1) comparing the gene expression list of the transformed gastric cancer cells and non-transformed cells or cell lines, and sorting the list of genes that are more present in the non-transformed cells or cell lines; (2) Genes present in more gastric cancer cells or cell lines treated with methylation inhibitors as compared to untreated transformed gastric cancer cells or cell lines treated with methylated inhibitors and transformed gastric cancer cells or cell lines. Sorting the list; (3) comparing the gene lists obtained in steps (1) and (2) to examine the sequence of genes present in both lists to exclude genes that do not have a CpG island in the promoter sequence; And (d) selecting the remaining gene except for the methylation marker gene.

In addition to the above, a nucleic acid methylation detection assay is performed to further refine the biomarker candidate list and confirm that the biomarker candidate is indeed methylated under conversion conditions. Numerous methods are available for detecting the methylation in DNA fragments. As one non-limiting example, the following method can be used. Genomic DNA is treated with methylation sensitivity restriction enzymes and the methylation site is contacted with a probe having a gene sequence specific for the marker. If a site to which an uncleaved probe is bound is detected, it means that methylation has occurred at the site to which the probe is bound.

The method of practicing the present invention using microarray technology is described below.

(1) The transformed cell expression library and the non-converted cell expression library are fluorescently labeled differently, preferably with greenish Cy3 and redish Cy5. The labeled library is competitively coupled to a microarray to which a probe set of known genes is immobilized. Identify genes that are more expressed in nontransformed cells. Optionally, an indirect comparison method is performed.

(2) The transformed cell line is treated with a dimethylation agent and the expression library is labeled with a fluorescent label. In addition, an expression library labeled differently is obtained from a conversion cell line not treated with a dimethylating agent. The two libraries bind competitively to a microarray substrate to which a set of known gene probes is immobilized. Genes that are more expressed in transformed cells treated with dimethylation agents are identified. The genes will be reactivated in dimethylation conditions. Optionally, an indirect comparison method is performed.

(3) Compare genes identified from the two methods and select genes that are common to both lists.

In addition, comparison of gene expression between transformed and non-transformed cells and between cells treated with a dimethylating agent and untreated cells can be performed by direct competition of the probe set. Optionally, the comparison may be an indirect comparison. For example, the expressed gene can be independently associated with a probe set of known standard comparative RNAs. Therefore, the relative amounts of genes expressed from different cells can be indirectly compared. The probe set of the comparative RNA is optimized because it contains a complete set of expressed genes (FIG. 1).

(4) Check whether the CpG island is present in the nucleic acid sequence of the promoter region or exon 1 region of the gene. Genes with exons 1 or promoters without CpG islands are deleted from the list. It measures the methylation levels of the genes in the list, which can be used in various ways. In one embodiment of the invention, the genome of the transformed cells is treated with methylation sensitive restriction endonucleases and nucleic acid amplification is performed using several primers with methylation sites located in the region to be amplified. If the amplification was successful at the end of the nucleic acid amplification step, methylation occurred because the gene was not cleaved by methylation sensitive restriction endonucleases. Without the amplification product, methylation did not occur because the gene was cleaved by methylation sensitive endonucleases.

Biomarkers for Gastric Cancer

The present invention provides a biomarker for diagnosing gastric cancer.

Use of Cancer Cells for Comparison with Biomarker-Normal Cells for Gastric Cancer

In this example, "normal" cells refers to cells that do not exhibit abnormal cell morphology or changes in cytological properties. A "tumor" cell refers to a cancer cell, and a "non-tumor" cell refers to a cell that is part of the diseased tissue but is not considered to be a tumor site.

The gene expression list of gastric tumor cells was indirectly compared with the gene expression lists of non-tumor cells and tumor cells in the form of competitive hybridization of microarrays. The common list of reference genes was compared with the list of genes of non-tumor cells, such as tissues near tumors, and the common list of reference genes was compared with the list of tumor cell genes. Compared with non-tumor cells, genes that are inhibited in tumor cells were found and recorded as tumor suppressor genes.

Alternatively, the list of gene expressions obtained from tumor cells can be compared directly with non-tumor and / or normal cells in the form of microarray hybridization to obtain tumor suppressor genes. In addition, both the direct and indirect comparison methods can be used to find a fungal inhibitor gene.

Separately, gastric cancer cell lines MKN1, MKN28 and SNU484 were treated with DAC, a dimethylating agent, and the reactivation of genes normally inhibited in tumor cells was analyzed. Genes that overlap (overlap) in the tumor suppressor gene set and the dimethylated reactivation gene set were considered candidates for gastric cancer biomarker genes, and 61 overlapping genes were found. It was confirmed by in silico analysis that the genes have essential CpG islands. Twenty-one genes without CpG islands were found and excluded from the list. In addition, a biochemical test is needed to confirm that the 40 remaining genes are actually methylated when isolated from tumor cells.

Methylation sensitive enzyme / nucleic acid sequence based amplification (NASBA) analysis, such as HpaII / MsPI enzyme cleavage / PCR (or post enzyme digestion-PCR), excluded 17 genes that were not methylated in gastric cancer cell lines. In order to further confirm by biochemical methods whether the candidate gene is indeed methylated in tumor cells, bisulfite sequencing was performed and all 23 genes were identified as methylated.

A gene expression list of the 23 genes was prepared. Expression levels of the 23 genes were measured in tumors and non-tumor tissues near tumors (FIG. 5). The degree of methylation of the gene was also determined using methylation sensitive enzyme / nucleic acid sequence based amplification (NASBA) analysis, such as HpaII / MsPI enzyme cleavage / PCR (or post-enzyme digestion-PCR) in clinical samples. Results of analysis in gastric tissue scraps are shown in FIG. 5. The identified genes were not methylated in normal cells. However, they were methylated in tumor cells as well as near tumor tumors. 7 and 8 further show that tumor cells are methylated more frequently than near tumor tissue.

 One aspect of the invention is based on the discovery of a relationship between gastric cancer and promoter hypermethylation of the 23 genes described below: MTCBP-1 (NT_022270) -membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein -1 (Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1); MTPN (NT_007933) -Mitotrophin; MTSS1 (NT_008046) —Metastasis suppressor 1; PELI2 (NT_026437) -Pellino homolog 2, (chopari) {Pellino homolog 2, (Drosophila)}; PLEKHF2 (NT_008046) -Plexckstrin homology domain containing, family F (with FYVE domain) member 2], containing flextrin homology domains; RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory agent (RAS-like, estrogen-regulated, growth inhibitor); THBD (NT_011387) Thrombomodulin; TP53INP1 (NT_008046)-Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_016354)-hypothetical protein MGC11324; ZFHX1B (NT_005058)-Zinc finger homeobox 1b; ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface (Adrenergic, beta-2-, receptor surface); AR (NT_011669) -Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and soft muscle atrophy; kidney disease) {Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; BLVRB (NT_011109)-Bliiverdin Reductase B (flavin reductase B) (NADPH); CALCR (NT_007933) —Calcitonin receptor; CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; CKAP4 (NT_019546) -Cytoskeleton-associated protein 4; CYBRD1 (NT_005403)-Cytochrome b reductase 1; GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_023935)-Golgi phosphoprotein 2; HHEX (NT_030059) —Hematopoietically expressed homeobox; LAMA2 (NT_025741)-laminin, alpha 2 (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; CPEB3 (NT_030059) —cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_007299)-5-hydroxytryptamine (serotonin) receptor 1B {5-hyroxytryptamine (serotonin) receptor 1B} gene.

For other uses of the diagnostic kits and nucleic acid chips of the present invention, the methylation stage of one or more nucleic acids isolated from a sample can be determined to prematurely diagnose cell growth abnormalities of the sample's stomach tissue. The methylation step of the at least one nucleic acid may be compared with the methylation status of at least one nucleic acid isolated from a sample having no cell growth potential of the tissue. The nucleic acid is preferably a CpG-containing nucleic acid such as a CpG island.

For other uses of the diagnostic kits and nucleic acid chips of the present invention, one can diagnose abnormalities in cell growth of the stomach tissue of a sample comprising determining methylation of one or more nucleic acids isolated from the sample. The nucleic acid is MTCBP-1 (NT_022270) -membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_007933) -Mitotrophin; MTSS1 (NT_008046) —Metastasis suppressor 1; PELI2 (NT_026437)-Pellino homolog 2, (Drosophila) {Pellino homolog 2, (Drosophila)}; PLEKHF2 (NT_008046) -Plexckstrin homology domain containing, family F (with FYVE domain) member 2], containing flextrin homology domains; RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory agent (RAS-like, estrogen-regulated, growth inhibitor); THBD (NT_011387) Thrombomodulin; TP53INP1 (NT_008046)-Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_016354)-hypothetical protein MGC11324; ZFHX1B (NT_005058)-Zinc finger homeobox 1b; ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface (Adrenergic, beta-2-, receptor surface); AR (NT_011669) -Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and soft muscle atrophy; kidney disease) {Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; BLVRB (NT_011109)-Bliiverdin Reductase B (flavin reductase B) (NADPH); CALCR (NT_007933) —Calcitonin receptor; CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; CKAP4 (NT_019546) -Cytoskeleton-associated protein 4; CYBRD1 (NT_005403)-Cytochrome b reductase 1; GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_023935)-Golgi phosphoprotein 2; HHEX (NT_030059) —Hematopoietically expressed homeobox; LAMA2 (NT_025741)-laminin, alpha 2 (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; CPEB3 (NT_030059) —cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_007299)-5-hydroxytryptamine (serotonin) receptor 1B {5-hyroxytryptamine (serotonin) receptor 1B} gene and combinations thereof, characterized in that the methylation step of the at least one nucleic acid is gastric tissue It may be characterized by comparison with the methylation status of one or more nucleic acids isolated from a sample that does not have a predisposition to abnormal cell growth.

The term "prescription" means a property prone to abnormal cell growth. A cultivated sample does not yet have cell growth abnormalities, but refers to a sample in which cell growth abnormalities exist or have increased.

Another aspect of the invention involves contacting a sample comprising a nucleic acid of a sample with an agent capable of determining the methylation status of the sample and confirming methylation of one or more sites of the one or more nucleic acids. It provides a way to diagnose. Here, the methylation of one or more sites of one or more nucleic acids can be characterized as different from the methylation step of the same site of the same nucleic acid of a sample having no cell growth potential.

The methods of the present invention comprise determining methylation of one or more sites of one or more nucleic acids isolated from a sample. Here, "nucleic acid" or "nucleic acid sequence" means oligonucleotides, nucleotides, polynucleotides, or genomic origins of DNA or RNA, sense or antisense strands of genomic origin or synthetic origin of fragments, single strands or double strands thereof, or Refers to DNA or RNA of synthetic origin, peptide nucleic acid (PNA) or DNA amount or RNA quantity material of natural or synthetic origin. If the nucleic acid is RNA, it is apparent to those skilled in the art that in place of deoxynucleotides A, G, C, and T, each is replaced by ribonucleotides A, G, C, and U.

Any nucleic acid capable of detecting the presence of differently methylated CpG islands can be used. The CpG island is a CpG rich site in the nucleic acid sequence. The nucleic acid includes, for example, a sequence encoding the following gene (denoted GenBank Acession Number):

1. MTCBP-1 (NT_022270); Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1

Amplicon Size: 231 bp

cagg acagggtctg ggcttgaatg cctttgcttc cgaaaacagc ggagccgtgg ggcctccccc gcgccggccc tgcctccaac cagccgccca atc ccgg ctc ccatgcgcgt ccacgcctcc ctataagaca aagcgcggcc gacgggctcc ggcgcggcc

MTCBP-1-F: 5'-caggacagggtctgggcttg-3 '(SEQ ID NO: 2)

MTCBP-1-R: 5'-cggggcgtcgtccatatacc-3 '(SEQ ID NO: 3)

2. MTPN (NT_007933); Myotrophin

Amplicon Size: 249 bp

tggtggtgca gaagcgtcct aatcccatcg aaagtactct cactcttcct ccattcgctg tcttgacctc cc ccgg gcct ccttcattct gcctcccagt cccgcccact tgtcg ccgg c tcctaccctc cactctagcc ttcccggcag cctgtaccg gcgcatgcg

MTPN-F: 5'-tggtggtgcagaagcgtcct-3 '(SEQ ID NO: 5)

MTPN-R: 5'-ggaccctcccccaacttcct-3 '(SEQ ID NO: 6)

3. MTSS1 (NT_008046); Metastasis suppressor 1

Amplicon Size: 236 bp

ta caagcgggct ctgggctagg cgcgccaccc gtgcaagtcc ccgg ggagcg gcggtgcacc ctccgtcccg cgcgctcgca gccattgtag gggtgggcgc tcgccaggca gggtgccgac acgccctctc cgcgctgcga cgggcgg ccg g gggaggaga gggtgcgctg tgcgca ccgg agggagaggc t ccgg cccag cgccgcctgc ccgccagcag accagcaggc tcct (SEQ ID NO: 7)

MTSS1-F: 5'-tacaagcgggctctgggcta-3 '(SEQ ID NO: 8)

MTSS1-R: 5'-aggagcctgctggtctgctg-3 '(SEQ ID NO: 9)

4. PELI2 (NT_026437); Pellino homolog 2 (Drosophila)

Amplicon Size: 216 bp

gcgccttcg aaacgtcctc tacgccagca ccaactggca aaaccttcta attttctaga cgcctttctg cttggttttg gaaggggagg cacccaagtg ggtgtgtgcg acacctctag ttgtaag ccg g gacacagtg acgtcgagag agcgctattc tactcggaga ggaagaaacc

PELI2-F: 5'-gcgccttcgaaacgtcctct-3 '(SEQ ID NO.:11)

PELI2-R: 5'-tcccaagcccacgttttcct-3 '(SEQ ID NO: 12)

5. PLEKHF2 (NT_008046); Pleckstrin homology domain containing, family F (with FYVE domain) member 2

Amplicon Size: 203 bp

aaggg ctggtcggag tcaggaaagt caggtaaggc gcctcacgtg cacctcaacg cgtcgcggga gcgcgtcccg acctcacaca tggacaagct ccgcccgcgg ccgg cctgag tgggtgtggc ctccgccaaa ggccccgccc ctagagcg tcgcg aggggg

PLEKHF2-F: 5'-aagggctggtcggagtcagg-3 '(SEQ ID NO.:14)

PLEKHF2-R: 5'-cgccctttcttgccagttcc-3 '(SEQ ID NO.:15)

6. RERG (NT_009714); RAS-like, estrogen-regulated, growth inhibitor

Amplicon Size: 226 bp

gg agcctggagg cttggaaata accagtgaaa aagggaagcc cgtcttgggt gcagcacgtt aaagacccaa gctcgcaagc ctgggaggca gcgcggcggg aggagcctgc ccctgccccc agtagggggc gccgaagcgc cgcactgcag catcctggcc gctgagcgca gcggccttgg ccgg gctcag ctcgcgtcct gccgcagtcc ctccgccgct agtc (SEQ ID NO: 16)

RERG-F: 5'-ggagcctggaggcttggaaa-3 '(SEQ ID NO.:17)

RERG-R: 5'-gactagcggcggagggactg-3 '(SEQ ID NO.:18)

7. THBD (NT_011387); Thrombomodulin

Amplicon Size: 182 bp

cgccag ggcagggttt actcatc ccg g cgaggtgat cccatgcgcg agggcgggcg caagggcggc cagagaaccc agcaatccga gtatgcggca tcagcccttc ccaccaggca cttccttcct tttcccgaac gtccagggag ggaggg ccgg gcacttataa actcgg

THBD-F: 5'-cgccagggcagggtttactc-3 '(SEQ ID NO.:20)

THBD-R: 5'-cggccagggctcgagtttat-3 '(SEQ ID NO: 21)

8. TP53INP1 (NT_008046); Tumor protein p53 inducible nuclear protein 1

Amplicon Size: 229 bp

ctccca gcaccctggc tacacgtcta accctaggct gaccaggtgg ggctctcgga ggcgttagcc ccagccctcc caggagtctt aatgttcctc tcacaggaaa aaacgttctg cgccctttgt gccccaaacc ctcgaccctt cactcggcga gaggggtcgc tgagggagag atccacctct gcccttcccg ttgcccg ccg g ttcttcctc gcccgcctct tac (SEQ ID NO: 22)

TP53INP1-F: 5'-ctcccagcaccctggctaca-3 '(SEQ ID NO: 23)

TP53INP1-R: 5'-gtaagaggcgggcgaggaag-3 '(SEQ ID NO.:24)

9. MGC11324 (NT_016354); Hypothetical protein MGC11324

Amplicon Size: 236 bp

ggtggcttc agcccagacc tgggcagcca gcggagaaag agttaactgg caggggcgag gaggagccca gggaggaagg aaggatattg ccgtaattct gaaagttttt ttccttcctc tcttcccttc gcagaggtga gtg ccgg gct cggcgctct ccc tgg

MGC11324-F: 5'-ggtggcttcagcccagacct-3 '(SEQ ID NO.:26)

MGC11324-R: 5'-tcaggagaggtccgcagtgg-3 '(SEQ ID NO: 27)

10. ZFHX1B (NT_005058); Zinc finger homeobox 1b

Amplicon Size: 215 bp

cctgcctccc gacactcttg gcgaggtttt tgtacagttt gct ccgg gag ctgtttcttc gcttccacct ttttctcccc cacacttcgc ggcttcttca tgctttttct tctcaccatt tctggccaaa actacaaaca

agacttcgca ggtaggtttt ttttcctccc cttttctctc tttttatccc tttttggtgt gctcgtcctc catcc (SEQ ID NO: 28)

ZFHX1B-F: 5'-cctgcctcccgacactcttg-3 '(SEQ ID NO .: 29)

ZFHX1B-R: 5'-ggatggaggacgagcacacc-3 '(SEQ ID NO: 30)

11.ADRB2 (NT_029289); Adrenergic, beta-2-, receptor, surface

  Amplicon size; 261 bp

gagctgggag ggtgtgtctc agtgtctatg gctgtggttc ggtataagtc tgagcatgtc tgccagggtg tatttgtgcc tgtatgtgcg tgcctcggtg ggcactctcg tttccttccg aatgtggggc agtg ccgg tg tgctgccctc tgccttgaga cctcaagccg cgcaggcgcc cagggcaggc aggtagcggc cacagaagag ccaaaagctc ccgg gttggc tggtaaggac accacctcca gctttagccc t (SEQ ID NO: 31)

Forward; 5'-gagctgggagggtgtgtctcag-3 '(SEQ ID NO: 32)

Reverse; 5'-agggctaaagctggaggtggtg-3 '(SEQ ID NO: 33)

12. AR (NT_011669); Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)

Amplicon Size: 195 bp

ggacccga ctcgcaaact gttgcatttg ctctccacct cccagcgccc cctccgagat c ccgg ggagc cagcttgctg ggagagcggg acggtccgga gcaagcccac aggcagagga ggcgacagag ggaaaaagg

Forward; 5'- cag gca acc cag acg tcc aga g -3 '(SEQ ID NO: 35)

Reverse; 5'- gct ggc gtg gtg cgt ccc t-3 '(SEQ ID NO: 36)

13. BLVRB (NT_011109); Biliverdin reductase B (flavin reductase (NADPH)

Amplicon Size: 256 bp

tttctcccct tgctggttct gggaggccct ggggcttaga gtgcgcccca gatccgctcc aggctccggg agagggggcg tgagctacga gaggccgtgg gtgaggctcg cctggccccgc ccctct ccgg gaggtggagc gcggaggcag ggcgctgagt gacaagttat ctcggctccg tccactctat ttgttgctat gtggaggcgt ggccttctgt ggccccgcct tccagtctaa gtggcgcgca gagag (SEQ ID NO: 37)

Forward; 5'-tttctccccttgctggttctgg-3 '(SEQ ID NO: 38)

Reverse; 5'-ctctctgcgcgccacttagact-3 '(SEQ ID NO: 39)

14. CALCR (NT_007933); Calcitonin receptor

Amplicon Size: 229 bp

cct gtgtttacgc ggcgctttag tctcccggac tcgcagggtg agccccagcc ctgactggag cgagacagca gccgcgagcg cagccccact cgcggg ccgg ggcgactggg gctggcgcga ggctcacgga gctcaccagc tcgcccctcc ctctcctggg acaggagggg gctgactggg gtggcggggt ccgg gaaggg gggctggctc tcatcaattc tgctgc (SEQ ID NO: 40)

Forward; 5'- cct gtg ttt acg cgg cgc ttt-3 '(SEQ ID NO: 41)

Reverse; 5'- gca gca gaa ttg atg aga gcc a-3 '(SEQ ID NO: 42)

15. CDH2 (NT_010966); Cadherin 2, type 1, N-cadherin (neuronal)

Amplicon Size: 203 bp

tct aaactcccag ggggacaaaa aaaacaaaac agaacacaaa acttgggctg tccagtacat ctcaagggtg gagctgaag tgcgagctcc gagaggagcc cggcctccg cctcccccgc cgcaggtggc tc ccgg cgag gcctcagaca caatagctag atgagct

Forward; 5'-tctcaaactcccagaggggaca-3 '(SEQ ID NO: 44)

Reverse; 5'-aaactaaggacgcaggccaagc-3 '(SEQ ID NO: 45)

16. CKAP4 (NT_019546); Cytoskeleton-associated protein 4

Amplicon Size: 214 bp

ggctggattt tggacagcct tttttgtctc cgccttcaaa cccaaggcaa aggacaaggc ccaaccccat ggcgggctggg agagtgaaag cagggggagt cccccaattc ccagcggaaa ggaagggcgat ctgttcccac ccgctgactc ccactc ccgg gtt

Forward; 5'-ggctggattttggacagccttt-3 '(SEQ ID NO: 47)

Reverse; 5'-gacctgtgcggagaggagagaa-3 '(SEQ ID NO: 48)

17. CYBRD1 (NT_005403); Cytochrome b reductase 1

Amplicon Size: 221 bp

ggagacagcc ccaagaagtc gacgcc ccgg tcccgccgc c cgg ccactac ccagagggct gccgccgcct ctccaagttc ttgtggcccc cgcggtgcgg agtatggggc gctgatggcc atggagggct actggcgctt cctggcgctg ctggggt cgt cgt

Forward; 5'-ggagacagccccaagaagtcg-3 '(SEQ ID NO: 50)

Reverse; 5'-tctcggtagtggaggacccagac-3 '(SEQ ID NO: 51)

18. GFPT1 (NT_022184); Glutamine-fructose-6-phosphate transaminase 1

Amplicon Size: 200 bp

tcctcctctt tcctcgcttcg tgcagtgctgg cgcctggggc ctggggctgc acggggcacg cac gct ccgg attgctttgc taacaattcc atccttctct ttccgtcatt ccctgccagg tgctgagttt ctccctccct ctcggctcgg tccctcccgc ggctcgcc cc gg ggcgggcg tctccaggaa ctccaggc (SEQ ID NO: 52)

Forward; 5'-tcctcctctttcctcgcttcgt-3 '(SEQ ID NO: 53)

Reverse; 5'-gcctggagttcctggagacg-3 '(SEQ ID NO: 54)

19. GOLPH2 (NT_023935); Golgi phosphoprotein 2

Amplicon Size: 208 bp

acggcctcat agggcttctc aaacatcagtg cgcctgagcg tcatctgagg cgctgtttca aatgcagctg c ccgg gcta taagatcaca cccgaaggcg t ccgg gaatc ttcacttttt ccgttgctag cagtggaagg gtcacagacc aaacactaag gcctgagcga tgagga

Forward; 5'- acggcctcatagggcttctcaa-3 '(SEQ ID NO: 56)

Reverse; 5'- ggcattccctgttgaccatcat-3 '(SEQ ID NO: 57)

20. HHEX (NT_030059); Hematopoietically expressed homeobox

Amplicon Size: 209 bp

gcagggaagt ctttccattt ccatgattaa tggaggacct tagcagaaac ggctcaccgc gaggtgtgac gaccgcagga gggcgtcaat caagaaaatg cccccttgcc ccgg aggcga gtggagccgc acagagccga ggccatacgg gccagcgga cggtagaaa

Forward; 5'- gcagggaagtctttccatttcca-3 '(SEQ ID NO: 59)

Reverse; 5'- cgcagtctggctctctggggtttt-3 '(SEQ ID NO: 60)

21. LAMA2 (NT_025741); laminin, alpha 2 (merosin, congenital muscular dystrophy)

Amplicon Size: 378 bp

cctagctgt ttgcatttcc cagaaataat gttttccatg tgtagggatt ttacagattt caaagtgctt tcatgtcaat tactttcttt aatttaaaag aagttcagat accaggtcaa gctaggaatg atccggtctc agagggaagg agcgctctag gaaaggagga tcctttaata gagggccgtc ctggggccgc gtgcccatgg aaggcgagag tggaggagtg tcctctttct cccccaccct caggcggcgg c ccgg ccaaa gccagagggg gctgtctcct cctcttcccc agcagctgct gctcgctcag ctcacaagcc aaggccaggg gacagggcgg cagcgactcc tctggctccc gagaagtgg (SEQ ID NO: 61)

Forward: 5'-cct agc tgt ttg cat ttc cca g-3 '(SEQ ID NO: 62)

Reverse: 5'-cca ctt ctc ggg agc cag ag-3 '(SEQ ID NO: 63)

22. CPEB3 (NT_030059); cytoplasmic polyadenylation element binding protein 3

Amplicon Size: 216 bp

taagggggtc attcccctga aagataac cc gg ttcttgcc agtgacatcg ctgacaaaca cttcacaagt tggggagggg gaagggggag gggaagaggg taaggaaaac taattttgtg gattaacagg agtccctctc aggtcaaaac ggggttccaa ggaaaccaga cgccaccg

Forward; 5'-taagggggtcattcccctgaaa-3 '(SEQ ID NO: 65)

Reverse; 5'-cggcctgggtcgtagactttg-3 '(SEQ ID NO: 66)

23. HTR1B (NT_007299); 5-hyroxytryptamine (serotonin) receptor 1B gene

Amplicon Size: 434 bp

gggagcttcc ttggccagga aaggaacagg gcggggggaa aagagggagg gagagagaaa aagggaaggt gatggggagc gggcgtccgc acccgccgcg ccgcacgagt tgcactgctc tggcgga ccg g acctggact ctatataaag agcccatctg ctccgtagct cgcacgcttc tc ccgg gctg gtgcacgccg cgtccctcca gctcccccag acacctgccc cttcccagtg tgccgcgcca ggtcctccag acccgcgcac ccagtggcat ggctccgagt ggctcccgtg ggaccagggt ggcggtggcg gcggcgcggc ccgagcagcc gcaactccag cccccgtgtc ccccctttta tggctccgtc tccgcggggc agctcgtccg agtggccaga gagtgaaaag agagggaggg cagag (SEQ ID NO: Number: 67)

Forward: 5'-gggagcttccttggccagga-3 '(SEQ ID NO: 68)

Reverse: 5'-ctctgccctccctctcttttc-3 '(SEQ ID NO: 69)

"Ccgg" denoted in bold above means a methylated site and is also a site recognized by methylation sensitive restriction enzyme HpaII.

Methylaton

Any nucleic acid in purified or unpurified form may be used in the present invention, and any nucleic acid containing or suspected of containing a nucleic acid sequence containing a target site (eg, CpG-containing nucleic acid) may be used. . A nucleic acid site that can be differentially methylated is a CpG island, which is a nucleic acid sequence having a high CpG density compared to other dinucleotide CpG nucleic acid sites. Doublet CpG is only 20% probable in vertebrate DNA when predicted by the ratio of G * C base pairs. At certain sites, the density of double CpG is ten times higher than at other sites in the genome. CpG islands have an average G * C ratio of about 60%, with the average DNA G * C ratio of 40%. CpG islands are typically about 1 to 2 kb in length and there are about 45,000 CpG islands in the human genome.

In many genes, CpG islands start upstream of the promoter and extend to the transcriptional site downstream. Methylation of CpG islands in promoters usually inhibits expression of genes. CpG islands may also surround the 3 'region of the gene coding region, as well as the 5' region of the gene coding region. Therefore, CpG islands are found at several sites, including upstream of a coding sequence of a regulatory region comprising a promoter region, a coding region (eg exon region), downstream of a coding region, eg, an enhancer region and an intron. .

Typically, the CpG-containing nucleic acid is DNA. However, the method of the present invention may apply for example a sample containing DNA or RNA containing DNA and mRNA, wherein the DNA or RNA may be single stranded or double stranded, or DNA-RNA hybrids. It may be characterized by the contained sample.

Nucleic acid mixtures may also be used. The specific nucleic acid sequence to be detected may be a fraction of a large molecule, and from the beginning the specific sequence may exist in the form of isolated molecules that make up the entire nucleic acid sequence. The nucleic acid sequence need not be a nucleic acid present in pure form, and the nucleic acid may be a small fraction in a complex mixture, such as containing whole human DNA. Nucleic acids included in the sample used to measure the degree of methylation of nucleic acids contained in the sample or used to detect methylated CpG islands are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY., 1989). It can be extracted by various methods described in.

The nucleic acid may comprise a regulatory site, which is a DNA site that encodes information or regulates transcription of the nucleic acid. The regulatory site comprises at least one promoter. A "promoter" is the minimum sequence necessary to direct transcription and can regulate inducibility by cell type specific, tissue specific or external signal or agent in a promoter-dependent gene. The promoter is located at the 5 'or 3' site of the gene. Nucleic acid numbers of all or part of a promoter site can be applied to measure methylation of CpG island sites. Methylation of the target gene promoter proceeds from outside to inside. Therefore, the early stages of cell turnover can be detected by analyzing methylation outside of the promoter region.

The nucleic acid isolated from the sample is obtained by biological sample of the sample. If you want to diagnose gastric cancer or the progression of gastric cancer, the nucleic acid should be separated from the tissue by scrap or biopsy. Such samples may be obtained by various medical procedures known in the art.

In one embodiment of the invention, the degree of methylation of the nucleic acid of the sample obtained from the sample is measured in comparison to the same nucleic acid portion of the sample that is free of cell growth abnormalities of the gastric tissue. Hypermethylation refers to the presence of methylated alleles in one or more nucleic acids. Samples without cell growth abnormalities of the above tissues do not show methylation alleles when the same nucleic acid is tested.

Sample

The present invention describes the early identification of gastric cancer, and uses gastric cancer specific gene methylation. Methylation of gastric cancer specific genes also occurred in tissues near the tumor site. Therefore, the early detection method of gastric cancer can confirm the presence or absence of methylation of gastric cancer-specific genes in all samples including liquid or solid tissue. The sample includes, but is not limited to, serum or plasma.

Individual genes and panels

The present invention can be used individually as a diagnostic or predictive marker, or a combination of several marker genes in the form of a panel display, and several marker genes can be used to improve reliability and efficiency through an overall pattern or a list of methylated genes. It can confirm that it improves. The genes identified in the present invention can be used individually or as a gene set in which the genes mentioned in this example are combined. For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or gastric The methylation of the gene specific for the gene indicates the possibility of developing cancer. Alternatively, genes can be ranked, weighted, and selected for the level of likelihood of developing cancer according to the number and importance of the genes methylated together. Such algorithms belong to the present invention.

Methylation Detection Method

Detection of Differential Methylation—Methylation Sensitivity Restriction Endonuclease

Detection of differential methylation can be accomplished by contacting the nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites.

In a separate reaction, the sample was contacted with an isochizomer of a methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG sites, thereby cleaving the methylated nucleic acid.

Specific primers were added to the nucleic acid sample and the nucleic acid was amplified by conventional methods. If there is an amplification product in the sample treated with methylation sensitive restriction endonuclease, and there is no amplification product in the isomerized sample of methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG sites , Methylation occurred in the analyzed nucleic acid site. However, no amplification products were present in the samples treated with methylation sensitive restriction endonucleases, and the amplification products were also found in the samples treated with isocymers of methylation sensitive restriction endonucleases that cleave both methylated and unmethylated CpG sites. Existence means that no methylation occurs in the analyzed nucleic acid site.

Here, "methylation sensitivity restriction endonuclease" is a restriction enzyme that contains CG at the recognition site and has activity when C is methylated compared to when C is not methylated (eg, SmaI). Non-limiting examples of methylation sensitivity limiting endonucleases include SmaI, BssHII or HpaII, BSTUI and NotI. The enzymes may be used alone or in combination. Other methylation sensitivity limiting endonucleotides include, but are not limited to, for example, SacII and EagI.

 Isocymers of methylation sensitive restriction endonucleases are restriction endonucleases that have the same recognition site as methylation sensitive restriction endonucleases, but cleave both methylated and unmethylated CGs, for example MspI. Can be mentioned.

Primers of the invention are constructed to have "alternatively" complementarity with each strand of the locus to be amplified and, as described above, contain suitable G or C nucleotides. This means that the primers have sufficient complementarity to hybridize with the corresponding nucleic acid strands under the conditions for carrying out the polymerization. The primer of the present invention is used in the amplification process, which is an enzymatic continuous reaction in which the target locus, such as PCR, increases to an exponential number through many reaction steps. Typically, one primer (antisense primer) has homology to the negative (-) strand of the locus, and the other primer (sense primer) has homology to the positive (+) strand. When primers are annealed to the denatured nucleic acid, the chain is stretched by enzymes and reactants such as DNA polymerase I (Klenow) and nucleotides, resulting in newly synthesized + and-strands containing the target locus sequence. The newly synthesized target locus is also used as a template, and exponential synthesis of the target locus sequence proceeds when the cycle of denaturation, primer annealing and chain extension is repeated. The product of the continuous reaction is an independent double stranded nucleic acid having an end corresponding to the end of the specific primer used in the reaction.

The amplification reaction is preferably a PCR that is commonly used in the art. However, alternative methods such as real-time PCR or linear amplification with isothermal enzymes can also be used, and multiplex amplification reactions can also be used.

Detection of Differential Methylation-Bisulfite Sequencing Method

Other methods of detecting nucleic acids containing methylated CpG include contacting a sample containing nucleic acid with an agent that modifies unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using CpG-specific oligonucleotide primers. It includes. Here, the oligonucleotide primer may be characterized by detecting the methylated nucleic acid by distinguishing the modified methylated and unmethylated nucleic acid. The amplification step is optional and desirable but not necessary. The method relies on a PCR reaction that distinguishes between modified (eg, chemically modified) methylated and unmethylated DNA. Such methods are disclosed in US Pat. No. 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

temperament

Once the target nucleic acid site is amplified, the nucleic acid amplification product can be hybridized with a known gene probe immobilized on a solid support (substrate) to detect the presence of the nucleic acid sequence.

Here, a "substrate" is a mixture means comprising a substance, structure, surface or material, abiotic, synthetic, inanimate, planar, spherical or specific binding, flat surface material, hybridization or enzyme recognition site Or many other recognition sites beyond many other recognition sites or many other molecular species composed of surfaces, structures or materials. Such substrates include, for example, semiconductors, (organic) synthetic metals, synthetic semiconductors, insulators and dopants; Metals, alloys, elements, compounds and minerals; Synthesized, degraded, etched, lithographic, printed and microfabricated slides, devices, structures and surfaces; Industrial, polymers, plastics, membranes, silicones, silicates, glass, metals and ceramics; Wood, paper, cardboard, cotton, wool, cloth, woven and non-woven fibers, materials and fabrics, but are not limited to these.

Some types of membranes are known in the art to have adhesion to nucleic acid sequences. Specific and non-limiting examples of such membranes are membranes for gene expression detection such as nitrocellulose or polyvinylchloride, diaotized paper and commercially available membranes such as GENESCREEN ^™ , ZETAPROBE ^™ (Biorad) and NYTRAN ^™ . Can be mentioned. Beads, glass, wafers and metal substrates are also included. Methods of attaching nucleic acids to such objects are well known in the art. Alternatively, screening can also be performed in the liquid phase.

Hybridization conditions

In nucleic acid hybridization reactions, the conditions used to achieve stringent specific levels vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, degree of homology, nucleotide sequence composition (eg, GC / AT composition ratio), and nucleic acid type (eg, RNA, DNA) select hybridization conditions. Is considered. An additional consideration is whether the nucleic acid is immobilized, for example in a filter or the like.

Examples of very stringent conditions are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2XSSC / 0.1% SDS at room temperature (low stringency conditions); 0.2XSSC / 0.1% SDS at 42 ° C. (conditions with moderate stringency); 0.1X SSC at 68 ° C. conditions with high stringency. The washing process can be carried out using one of these conditions, for example, conditions having a high stringency, or each of the above conditions can be used, and each of the conditions described above in each of 10 to 15 minutes in the order described above. Or some iteration However, as described above, the optimum conditions vary with the particular hybridization reaction involved and can be determined experimentally. In general, conditions of high stringency are used for hybridization of critical probes.

Label

Important probes are labeled so that they can be detected, for example, with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. Proper labeling of such probes is a technique well known in the art and can be carried out by conventional methods.

Kit

According to one aspect of the present invention, there is provided a kit useful for detecting a cell growth abnormality of a specimen. Kits of the invention include a compartmentalized carrier means for holding a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, a second container containing a primer for amplifying a CpG containing nucleic acid, and a cleaved or uncleaved nucleic acid. One or more containers including a third container containing means for detecting the presence. Primers used in accordance with the present invention include the sequences set forth in SEQ ID NOS: 1-69, functional combinations, and fragments thereof. Functional combinations or fragments are used as primers to detect whether methylation has occurred on the genome.

The carrier means is suitable for containing one or more containers, such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in the container. For example, a vessel containing methylation sensitive restriction endonucleases can be configured as one vessel. One or more containers may contain primers that have homology with important nucleic acid locus. In addition, one or more of the containers may contain isoxisomers of methylation sensitive restriction enzymes.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example

Example 1 Identification of Genes Inhibited in Gastric Cancer

To identify genes that are inhibited in gastric cancer, microarray hybridization was performed. Microarray hybridization was performed using standard protocols (Schena et al ., Science , 270: 467, 1995). Tissues near tumors (4 samples) and tumor tissues (4 samples) were isolated from paired gastric cancer patients (Chungnam University Hospital Surgery) and total RNA was isolated therefrom. In order to indirectly compare the relative difference in gene expression levels of the paired near tumor tissue and tumor tissue, standard comparison RNA (indirect comparison) was constructed. In order to prepare standard RNA, lung cancer cell line A549 (Korea Cell Line Bank (KCLB) 10185), gastric cancer cell line AGS (KCLB 21739), kidney cancer cell line Caki-2 (KCLB 30047), colon cancer cell line HCT116 (KCLB 10247), cervical cancer Cell line Hela (KCLB 10002), blood cancer cell line HK-60 (KCLB 10240), HT1080 (KCLB 10121), breast cancer cell line MDA-MB2 (KCLB 30026), liver cancer cell line SK-hep1 (KCLB 30052) and T cell derived cell line Molt- Total RNA was isolated from 11 human cancer cell lines of 4 (KCLB 21582), brain cancer cell line U-87MG (KCLB 30014). Total RNA of the cell line and gastric tissue was isolated using Tri Reagent (Sigma, USA). To prepare standard comparative RNAs, total RNA isolated from 11 cell lines were mixed in equal amounts. The standard comparative RNA was used as an internal control. In order to compare relative gene expression levels of the paired near-tumor and tumor tissues, RNAs isolated from non-tumor and tumor tissues were compared indirectly with standard RNA. 100 μg of total RNA was labeled with Cy3-dUTP or Cy5-dUTP. The standard comparative RNA was labeled with Cy3, RNA isolated from the tissue was labeled with Cy5. The two cDNAs labeled with Cy3- and Cy5- were purified using a PCR purification kit (Qiagen, Germany). Purified cDNA was mixed and concentrated to a final volume of 27 μl using Microcon YM-30 (Millipore Corp., USA).

A total of 80 μl of the hybridization reaction solution included: 27 μl of labeled cDNA target, 20 μl of 20X SSC, 8 μl of 1% SDS, 24 μl of formamide (Sigma, USA) and 20 μg of human Cot1 DNA (Invitrogen Corp. , USA). The hybridization reaction solution was heated at 100 ° C. for 2 minutes and immediately hybridized to a human 22K oligonucleotide (GenomicTree, Inc., Korea) microarray. The hybridization was performed for 12-16 hours at 42 ° C. in HybChamber X (GenomicTree, Inc., Korea) with humidity control. After hybridization, the microarray slides were read using Axon 4000B (Axon Instrument Inc., USA). Signal and background fluorescence intensities were measured for each probe by averaging the intensity of all the pixels inside the target site using GenePix Pro 4.0 software (Axon Instrument Inc., USA). Spots showing obvious abnormalities were excluded from the analysis. All data were subjected to normalization, statistical analysis and cluster analysis using GeneSpring 7.2 (Agilent, USA).

To determine the relative differences in gene expression levels of non-tumor and tumor tissues, statistical analysis for indirect comparison (ANOVA, p <0.05) was performed. From the results of the statistical analysis, it was confirmed that all of the 818 genes were down regulated in the tumor tissues compared to the tissues near the paired tumors (FIG. 1).

Example 2. Confirmation of Methylation Regulator Gene Expression

Dimethylating agent, 5-aza-2-, in gastric cancer cell lines MKN1 (KCLB 80101), MKN28 (KCLB 80102) and SNU484 (KCLB 484) to confirm that expression of the gene identified in Example 1 is regulated by promoter methylation. Deoxycytidine (DAC, Sigma, USA) was treated for 3 days at 200 nM concentration. Untreated cell lines and treated cell lines were treated with Tri reagent to separate total RNA. To determine gene expression changes by DAC treatment, the level of transcription was directly compared between untreated and treated cell lines. As a result, 3,036 genes with increased expression were identified when the DAC was treated, compared to the control without the DAC. Comparing the list of 818 tumor suppressor genes obtained in Example 1 and the genes re-expressed by 3,036 dimethylation, 61 common genes were found (FIG. 1).

Example 3. Methylation Structure of Identified Genes

(1) In silico analysis of promoter site CpG islands

MethPrimer (http://itsa.ucsf.edu/~urolab/methprimer/index1.html) was used to find the presence of CpG islands at the promoter regions of the 61 genes. 21 of the 61 genes did not have CpG islands and the 21 genes were excluded from the list of common genes.

(2) Biochemical Analysis of Methylation

To biochemically confirm the methylation status of the remaining 24 genes, the restriction enzymes HpaII (methylation-sensitive) and MspI (methylation-insensitive) were treated, followed by PCR to detect the methylation status of each promoter (Fig. 2).

The two enzymes identically recognize the 5'-CCGG-3 'sequence. HpaII does not show activity when the internal cytosine residues are methylated, whereas MspI is not affected by methylation of the internal cytosine residues. If the cytosine residues of the CpG site are not methylated, both types of enzymes can cleave the target sequence. To determine the methylation status of a particular gene, a PCR target was selected that has one or more HpaII sites on the CpG island of the promoter region. 100 ng of genomic DNA isolated from gastric cancer cell lines AGS, MKN1, MKN28 and SNU484 were treated with 5 U of HpaII and 10 U of MspI, respectively, and purified by Quiagen PCR purification kit. Specific primers were used to amplify important sites. The purified genomic DNA 5 ng was amplified by PCR using a gene-specific primer set. The PCR reaction was carried out for 30 cycles by treating one minute at 94 ° C., one minute at 66 ° C., and one minute at 72 ° C., and finally extended at 72 ° C. for 10 minutes. Each amplification product was run on a 2% agarose gel containing ethidium bromide. If the band density of the HpaII treated product was greater than 1.5 times greater than the MspII treated product, the target site was considered methylated, and if the band density of the HpaII treated product was less than 1.5 times untreated. As a result, 17 of the 40 genes were not methylated, down-regulated in tumor tissues, up-regulated in dimethylation conditions, containing CpG islands in the promoter, and in cancer cell lines. Only 23 candidate genes were left that met the conditions that were substantially methylated (FIGS. 3 and 4).

(3) bisulfite sequencing of methylated promoters

In order to further confirm the methylation status of the 23 genes, bisulfite sequencing of each promoter was performed. Treatment of DNA with bisulfite leaves unmethylated cytosine modified with uracil and methylated cytosine remains unchanged. The bisulfite modification was performed by the method of Sato et al . (Sato et al ., Cancer Research , 63: 3735, 2003). 1 μg of genomic DNA of gastric cancer cell lines AGS, MKN1, MKN28 and SNU484 was treated with bisulfite using a methylation-specific PCR (MSP) bisulfite modification kit (In2Gen, Inc., Korea). The bisulfite treated AGS, MKN1, MKN28 and SNU484 genomic DNA were amplified by PCR and then sequenced for PCR products. As a result, it was confirmed that all of the above genes were methylated.

Example 4 Gene Expression List of Identified Genes

A gene expression list of 23 genes identified is shown in FIG. 5. As shown in FIG. 5, gene expression was inhibited in corresponding tumor tissues, as compared to tissues near tumors.

Example 5. Reactivation of 23 Identified Genes by Dimethylating Agent Treatment

6 shows the reactivation of 23 identified genes. As shown in FIG. 6, gene expression was reactivated as compared to gastric cancer cells which were not treated in gastric cancer cells treated with a dimethylation preparation (DAC).

Example 6 Promoter Methylation Assay in Clinical Samples

To confirm the clinical applicability of the methylated promoters of the 23 genes selected in the present invention, methylation assays were performed using clinical, non-patient, normal tumor and early gastric cancer tissue samples. Methylation assays were performed by the methods described in the examples above, using restriction enzyme / PCR methods.

Figure 7 shows the consolidation of the methylation assay for early gastric cancer, as shown in Figure 7, no gene methylation occurred in normal tissues obtained from normal samples (Biochain). However, methylation occurred not only in gastric cancer tissues but also in all genes in the tissues near the tumors that are associated with them. As expected, all genes were methylated in cancer samples, but not methylated in normal cells. In addition, as shown in FIG. 7, since 23 identified genes were methylated in the tissues near the paired tumors, it was confirmed that the 23 identified genes were useful for early detection of gastric cancer. FIG. 7B shows the frequency of methylation of identified marker genes in tumor tissues and tissues adjacent to tumors paired with them.

The methylation frequency of the marker in tumor tissue was obtained by dividing the number of tumor tissue samples to which the marker was methylated by the total number of samples including the tumor tissue and the tissues near the tumor paired thereto, and the frequency of methylation of the markers in the adjacent tumor tissue. Was obtained by dividing the number of methylated markers of tissue samples near paired tumors by the total number of samples, expressed as a percentage.

Example 7 Promoter Methylation Analysis in Clinical Samples of Advanced Gastric Cancer

In order to analyze the adequacy of the clinical application of the methylated promoter of 23 genes selected in the present invention, methylation analysis was performed using clinical samples of advanced gastric cancer tissues, and compared with normal samples. Methylation analysis was performed by supra method using restriction enzyme / PCR. 8 shows the methylation frequency (%) of 23 identified genes in advanced gastric cancer. As shown in FIG. 8, all genes were highly methylated in advanced gastric cancer tissues. As expected, all genes were methylated in gastric cancer samples, and methylation did not occur in normal cells.

The methylation frequency of the identified gene markers in tumor tissue was obtained by dividing the number of methylated markers in tumor tissue samples by dividing the total number of samples including tumor tissue and normal tissue, and expressed as a percentage.

The present invention provides a method for screening gastric cancer specific marker genes, a kit for diagnosing gastric cancer and gastric cancer progression stage diagnostic kit and a nucleic acid chip for gastric cancer and gastric cancer progression stage diagnosis that can diagnose methylation of CpG island of the screened gastric cancer specific marker genes. There is. By using the diagnostic kit and the diagnostic nucleic acid chip of the present invention, gastric cancer can be diagnosed at an early transformation stage, so that early diagnosis is possible, and the stage of gastric cancer and gastric cancer can be diagnosed more accurately and quickly than conventional methods. .

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not intended to limit the scope of the present invention. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Genomic Tree, Inc. <120> Diagnosis Kit and Chip For Gastric Cancer Using Gastric Cancer          Specific Methylation Marker Gene <130> PI07-B022 <150> US60746358 <151> 2006-05-03 <160> 69 <170> KopatentIn 1.71 <210> 1 <211> 231 <212> DNA <213> Homo sapiens <400> 1 caggacaggg tctgggcttg aatgcctttg cttccgaaaa cagcggagcc gtggggcctc 60 ccccgcgccg gccctgcctc caaccagccg cccaatcccg gctcccatgc gcgtccacgc 120 ctccctataa gacaaagcgc ggccgacggg ctccgagcgc ggcccctggg ttcgaacacg 180 gcacccgcac tgcgcgtcat ggtgcaggcc tggtatatgg acgacgcccc g 231 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caggacaggg tctgggcttg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggggcgtcg tccatatacc 20 <210> 4 <211> 249 <212> DNA <213> Homo sapiens <400> 4 tggtggtgca gaagcgtcct aatcccatcg aaagtactct cactcttcct ccattcgctg 60 tcttgacctc ccccgggcct ccttcattct gcctcccagt cccgcccact tgtcgccggc 120 tcctaccctc cactctagcc ttcccggcag cctgtacatt gcgcatgcgc actagtggcg 180 ttcgcgccgc ggacccagag agaggcgttc cgcggaggag aagaggaaga ggaagttggg 240 ggagggtcc 249 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tggtggtgca gaagcgtcct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggaccctccc ccaacttcct 20 <210> 7 <211> 236 <212> DNA <213> Homo sapiens <400> 7 tacaagcggg ctctgggcta ggcgcgccac ccgtgcaagt ccccggggag cggcggtgca 60 ccctccgtcc cgcgcgctcg cagccattgt aggggtgggc gctcgccagg cagggtgccg 120 acacgccctc tccgcgctgc gacgggcggc cgggggagga gagggtgcgc tgtgcgcacc 180 ggagggagag gctccggccc agcgccgcct gcccgccagc agaccagcag gctcct 236 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tacaagcggg ctctgggcta 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aggagcctgc tggtctgctg 20 <210> 10 <211> 216 <212> DNA <213> Homo sapiens <400> 10 gcgccttcga aacgtcctct acgccagcac caactggcaa aaccttctaa ttttctagac 60 gcctttctgc ttggttttgg aaggggaggc acccaagtgg gtgtgtgcga cacctctagt 120 tgtaagccgg gacacagtga cgtcgagaga gcgctattct actcggagag gaagttaatc 180 ccatcgaact ccagccagga aaacgtgggc ttggga 216 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gcgccttcga aacgtcctct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tcccaagccc acgttttcct 20 <210> 13 <211> 203 <212> DNA <213> Homo sapiens <400> 13 aagggctggt cggagtcagg aaagtcaggt aaggcgcctc acgtgcacct caacgcgtcg 60 cgggagcgcg tcccgacctc acacatggac aagctccgcc cgcggccggc ctgagtgggt 120 gtggcctccg ccaaaggccc cgcccctaga gcgcgtcgcg aggggcgcga ggggcggggc 180 gagggaactg gcaagaaagg gcg 203 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aagggctggt cggagtcagg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgccctttct tgccagttcc 20 <210> 16 <211> 226 <212> DNA <213> Homo sapiens <400> 16 ggagcctgga ggcttggaaa taaccagtga aaaagggaag cccgtcttgg gtgcagcacg 60 ttaaagaccc aagctcgcaa gcctgggagg cagcgcggcg ggaggagcct gcccctgccc 120 ccagtagggg gcgccgaagc gccgcactgc agcatcctgg ccgctgagcg cagcggcctt 180 ggccgggctc agctcgcgtc ctgccgcagt ccctccgccg ctagtc 226 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggagcctgga ggcttggaaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gactagcggc ggagggactg 20 <210> 19 <211> 182 <212> DNA <213> Homo sapiens <400> 19 cgccagggca gggtttactc atcccggcga ggtgatccca tgcgcgaggg cgggcgcaag 60 ggcggccaga gaacccagca atccgagtat gcggcatcag cccttcccac caggcacttc 120 cttccttttc ccgaacgtcc agggagggag ggccgggcac ttataaactc gagccctggc 180 cg 182 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cgccagggca gggtttactc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cggccagggc tcgagtttat 20 <210> 22 <211> 229 <212> DNA <213> Homo sapiens <400> 22 ctcccagcac cctggctaca cgtctaaccc taggctgacc aggtggggct ctcggaggcg 60 ttagccccag ccctcccagg agtcttaatg ttcctctcac aggaaaaaac gttctgcgcc 120 ctttgtgccc caaaccctcg acccttcact cggcgagagg ggtcgctgag ggagagatcc 180 acctctgccc ttcccgttgc ccgccggttc ttcctcgccc gcctcttac 229 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ctcccagcac cctggctaca 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gtaagaggcg ggcgaggaag 20 <210> 25 <211> 236 <212> DNA <213> Homo sapiens <400> 25 ggtggcttca gcccagacct gggcagccag cggagaaaga gttaactggc aggggcgagg 60 aggagcccag ggaggaagga aggatattgc cgtaattctg aaagtttttt tccttcctct 120 cttcccttcg cagaggtgag tgccgggctc ggcgctctgc tcctggagct cccgcgggac 180 tgcctgggga cagggactgc tgtggcgctc ggccctccac tgcggacctc tcctga 236 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ggtggcttca gcccagacct 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tcaggagagg tccgcagtgg 20 <210> 28 <211> 215 <212> DNA <213> Homo sapiens <400> 28 cctgcctccc gacactcttg gcgaggtttt tgtacagttt gctccgggag ctgtttcttc 60 gcttccacct ttttctcccc cacacttcgc ggcttcttca tgctttttct tctcaccatt 120 tctggccaaa actacaaaca agacttcgca ggtaggtttt ttttcctccc cttttctctc 180 tttttatccc tttttggtgt gctcgtcctc catcc 215 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 cctgcctccc gacactcttg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 ggatggagga cgagcacacc 20 <210> 31 <211> 261 <212> DNA <213> Homo sapiens <400> 31 gagctgggag ggtgtgtctc agtgtctatg gctgtggttc ggtataagtc tgagcatgtc 60 tgccagggtg tatttgtgcc tgtatgtgcg tgcctcggtg ggcactctcg tttccttccg 120 aatgtggggc agtgccggtg tgctgccctc tgccttgaga cctcaagccg cgcaggcgcc 180 cagggcaggc aggtagcggc cacagaagag ccaaaagctc ccgggttggc tggtaaggac 240 accacctcca gctttagccc t 261 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gagctgggag ggtgtgtctc ag 22 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 agggctaaag ctggaggtgg tg 22 <210> 34 <211> 191 <212> DNA <213> Homo sapiens <400> 34 ggacccgact cgcaaactgt tgcatttgct ctccacctcc cagcgccccc tccgagatcc 60 cggggagcca gcttgctggg agagcgggac ggtccggagc aagcccacag gcagaggagg 120 cgacagaggg aaaaagggcc gagctagccg ctccagtgct gtacaggagc cgaagggacg 180 caccacgcca g 191 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 caggcaaccc agacgtccag ag 22 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 gctggcgtgg tgcgtccct 19 <210> 37 <211> 256 <212> DNA <213> Homo sapiens <400> 37 tttctcccct tgctggttct gggaggccct ggggcttaga gtgcgcccca gatccgctcc 60 aggctccggg agagggggcg tgagctacga gaggccgtgg gtgaggctcg cctggccccg 120 cccctctccg ggaggtggag cgcggaggca gggcgctgag tgacaagtta tctcggctcc 180 gtccactcta tttgttgcta tgtggaggcg tggccttctg tggccccgcc ttccagtcta 240 agtggcgcgc agagag 256 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 tttctcccct tgctggttct gg 22 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 ctctctgcgc gccacttaga ct 22 <210> 40 <211> 229 <212> DNA <213> Homo sapiens <400> 40 cctgtgttta cgcggcgctt tagtctcccg gactcgcagg gtgagcccca gccctgactg 60 gagcgagaca gcagccgcga gcgcagcccc actcgcgggc cggggcgact ggggctggcg 120 cgaggctcac ggagctcacc agctcgcccc tccctctcct gggacaggag ggggctgact 180 ggggtggcgg ggtccgggaa ggggggctgg ctctcatcaa ttctgctgc 229 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 cctgtgttta cgcggcgctt t 21 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 gcagcagaat tgatgagagc ca 22 <210> 43 <211> 185 <212> DNA <213> Homo sapiens <400> 43 tctaaactcc cagggggaca aaaaaaacaa aacagaacac aaaacttggg ctgtccagta 60 catctcaagg gtggagctga agtgcgagct ccgagaggag cccggcctcc gcctcccccg 120 ccgcaggtgg ctcccggcga ggcctcagac acaatagcta gatgagcttg gctgcgtcct 180 agttt 185 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 tctcaaactc ccagagggga ca 22 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 aaactaagga cgcaggccaa gc 22 <210> 46 <211> 214 <212> DNA <213> Homo sapiens <400> 46 ggctggattt tggacagcct tttttgtctc cgccttcaaa cccaaggcaa aggacaaggc 60 ccaaccccat ggcgggctgg gagagtgaaa gcagggggag tcccccaatt cccagcggaa 120 aggaagggcg atctgttccc acccgctgac tcccactccc ggggccaggg ctccttgggc 180 gccccccttc atttctctcc tctccgcaca ggtc 214 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 ggctggattt tggacagcct tt 22 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 gacctgtgcg gagaggagag aa 22 <210> 49 <211> 221 <212> DNA <213> Homo sapiens <400> 49 ggagacagcc ccaagaagtc gacgccccgg tcccgccgcc cggccactac ccagagggct 60 gccgccgcct ctccaagttc ttgtggcccc cgcggtgcgg agtatggggc gctgatggcc 120 atggagggct actggcgctt cctggcgctg ctggggtcgg cactgctcgt cggcttcctg 180 tcggtgatct tcgccctcgt ctgggtcctc cactaccgag a 221 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 ggagacagcc ccaagaagtc g 21 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 tctcggtagt ggaggaccca gac 23 <210> 52 <211> 200 <212> DNA <213> Homo sapiens <400> 52 tcctcctctt tcctcgcttc gtgcagtgct ggcgcctggg gcctggggct gcacggggca 60 cgcacccggg ctattgcttt gctaacaatt ccatccttct ctttccgtca ttccctgcca 120 ggtgctgagt ttctccctcc ctctcggctc ggtccctccc gcggctcgcc ccggggcggg 180 cgtctccagg aactccaggc 200 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 tcctcctctt tcctcgcttc gt 22 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 gcctggagtt cctggagacg 20 <210> 55 <211> 208 <212> DNA <213> Homo sapiens <400> 55 acggcctcat agggcttctc aaacatcagt gcgcctgagc gtcatctgag gcgctgtttc 60 aaatgcagct gcccgggcta taagatcaca cccgaaggcg tccgggaatc ttcacttttt 120 ccgttgctag cagtggaagg gtcacagacc aaacactaag gcctgagcgg tgacaaccga 180 ggcgagatga tggtcaacag ggaatgcc 208 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 acggcctcat agggcttctc aa 22 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 57 ggcattccct gttgaccatc at 22 <210> 58 <211> 209 <212> DNA <213> Homo sapiens <400> 58 gcagggaagt ctttccattt ccatgattaa tggaggacct tagcagaaac ggctcaccgc 60 gaggtgtgac gaccgcagga gggcgtcaat caagaaaatg cccccttgcc ccggaggcga 120 gtggagccgc acagagccga ggccatacgg gccagcagta cggactgctt caatagaaaa 180 cacgtgcaaa accagagagc cagactgcg 209 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 gcagggaagt ctttccattt cca 23 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 cgcagtctgg ctctctggtt tt 22 <210> 61 <211> 378 <212> DNA <213> Homo sapiens <400> 61 cctagctgtt tgcatttccc agaaataatg ttttccatgt gtagggattt tacagatttc 60 aaagtgcttt catgtcaatt actttcttta atttaaaaga agttcagata ccaggtcaag 120 ctaggaatga tccggtctca gagggaagga gcgctctagg aaaggaggat cctttaatag 180 agggccgtcc tggggccgcg tgcccatgga aggcgagagt ggaggagtgt cctctttctc 240 ccccaccctc aggcggcggc ccggccaaag ccagaggggg ctgtctcctc ctcttcccca 300 gcagctgctg ctcgctcagc tcacaagcca aggccagggg acagggcggc agcgactcct 360 ctggctcccg agaagtgg 378 <210> 62 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 cctagctgtt tgcatttccc ag 22 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 ccacttctcg ggagccagag 20 <210> 64 <211> 216 <212> DNA <213> Homo sapiens <400> 64 taagggggtc attcccctga aagataaccc ggttcttgcc agtgacatcg ctgacaaaca 60 cttcacaagt tggggagggg gaagggggag gggaagaggg taaggaaaac taattttgtg 120 gattaacagg agtccctctc aggtcaaaac ggggttccaa ggaaaccaga cgccactcct 180 tagctaagtt tcacccaaag tctacgaccc aggccg 216 <210> 65 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 taagggggtc attcccctga aa 22 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 cggcctgggt cgtagacttt g 21 <210> 67 <211> 435 <212> DNA <213> Homo sapiens <400> 67 gggagcttcc ttggccagga aaggaacagg gcggggggaa aagagggagg gagagagaaa 60 aagggaaggt gatggggagc gggcgtccgc acccgccgcg ccgcacgagt tgcactgctc 120 tggcggaccg gacctggact ctatataaag agcccatctg ctccgtagct cgcacgcttc 180 tcccgggctg gtgcacgccg cgtccctcca gctcccccag acacctgccc cttcccagtg 240 tgccgcgcca ggtcctccag acccgcgcac ccagtggcat ggctccgagt ggctcccgtg 300 ggaccagggt ggcggtggcg gcggcgcggc ccgagcagcc gcaactccag cccccgtgtc 360 ccccctttta tggctccgtc tccgcggggc agctcgtccg agtggccaga gagtgaaaag 420 agagggaggg cagag 435 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 gggagcttcc ttggccagga 20 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 ctctgccctc cctctctttt c 21  

Claims (14)

delete delete delete delete delete delete delete delete delete delete delete delete A kit for the diagnosis of gastric cancer or gastric cancer progression, comprising a primer for amplifying a fragment comprising a CpG island of a gastric cancer marker gene promoter selected from the group consisting of and a methylation sensitive restriction endonuclease: (1) MTCBP-1 (NT_022270)-membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1 gene; (2) MTPN (NT_007933) -Mitotrophin gene (Myotrophin); (3) MTSS1 (NT_008046)-Cancer Metastasis Suppressor 1; (4) PELI2 (NT_026437)-pelino homolog 2 gene, (Drosophila) {Pellino homolog 2, (Drosophila)}; (5) PLEKHF2 (NT_008046)-Plextrin homology domain containing, family F (with FYVE domain) member 2; (6) RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory gene (RAS-like, estrogen-regulated, growth inhibitor); (7) MGC11324 (NT_016354)-Hypothetical protein MGC11324; (8) ZFHX1B (NT_005058)-Zinc finger homeobox 1b gene; (9) ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface genes (Adrenergic, beta-2-, receptor surface); (10) AR (NT_011669) -androgen receptor gene (dihydrotestosterone receptor; testicular feminization; spinal and soft muscle atrophy; nephropathy); Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; (11) BLVRB (NT_011109) -blubberdin reductase B gene (flavin reductase: NADPH) {Biliverdin reductase B (flavin reductase: NADPH)}; (12) CALCR (NT_007933)-calcitonin receptor gene; (13) CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin gene (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; (14) CKAP4 (NT_019546) —Cytoskeleton-associated protein 4; (15) CYBRD1 (NT_005403)-Cytochrome b reductase 1; (16) GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; (17) GOLPH2 (NT_023935) -Golgi phosphoprotein 2; (18) HHEX (NT_030059) —Hematopoietically expressed homeobox that is expressed upon hematopoiesis; (19) LAMA2 (NT_025741)-laminin, alpha 2 gene (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; (20) CPEB3 (NT_030059)-cytoplasmic polyadenylation element binding protein 3; And (21) HTR1B (NT_007299)-5-hydroxytryptamine (serotonin) receptor 1B gene {5-hyroxytryptamine (serotonin) receptor 1B}. Nucleic acid chip for the diagnosis of gastric cancer or gastric cancer progression comprising a fragment comprising a promoter CpG island of the gastric cancer marker gene selected from the group consisting of a probe capable of hybridizing with: (1) MTCBP-1 (NT_022270)-membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1 gene; (2) MTPN (NT_007933) -Mitotrophin gene (Myotrophin); (3) MTSS1 (NT_008046)-Cancer Metastasis Suppressor 1; (4) PELI2 (NT_026437)-pelino homolog 2 gene, (Drosophila) {Pellino homolog 2, (Drosophila)}; (5) PLEKHF2 (NT_008046)-Plextrin homology domain containing, family F (with FYVE domain) member 2; (6) RERG (NT_009714)-RAS amount, estrogen-regulated growth inhibitory gene (RAS-like, estrogen-regulated, growth inhibitor); (7) MGC11324 (NT_016354)-hypothetical protein MGC11324 gene (Hypothetical protein MGC11324); (8) ZFHX1B (NT_005058)-Zinc finger homeobox 1b gene; (9) ADRB2 (NT — 029289) —adrenergic, beta-2-, receptor-surface genes (Adrenergic, beta-2-, receptor surface); (10) AR (NT_011669) -androgen receptor gene (dihydrotestosterone receptor; testicular feminization; spinal and soft muscle atrophy; nephropathy); Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)}; (11) BLVRB (NT_011109) -blubberdin reductase B gene (flavin reductase: NADPH) {Biliverdin reductase B (flavin reductase: NADPH)}; (12) CALCR (NT_007933)-calcitonin receptor gene (Calcitonin receptor); (13) CDH2 (NT_010966) -cadherin 2, type 1, N-cadherin gene (neuron) {Cadherin 2, type 1, N-cadherin (neuronal)}; (14) CKAP4 (NT_019546) —Cytoskeleton-associated protein 4; (15) CYBRD1 (NT_005403)-Cytochrome b reductase 1; (16) GFPT1 (NT_022184)-Glutamine-fructose-6-phosphate transaminase 1; (17) GOLPH2 (NT_023935) -Golgi phosphoprotein 2; (18) HHEX (NT_030059) —Hematopoietically expressed homeobox that is expressed upon hematopoiesis; (19) LAMA2 (NT_025741)-laminin, alpha 2 gene (merosin, congenital muscular dystrophy) {laminin, alpha 2 (merosin, congenital muscular dystrophy)}; (20) CPEB3 (NT_030059)-cytoplasmic polyadenylation element binding protein 3; And (21) HTR1B (NT_007299)-5-hydroxytryptamine (serotonin) receptor 1B gene {5-hyroxytryptamine (serotonin) receptor 1B}.

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