JP2012090555A - Carcinogenesis risk estimation using measurement of quantitative methylation of rasgrf1 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for extensively analyzing genes receiving epigenetic silencing in a tissue other than cancer in an organism where the cancer is generated, identifying a gene related to carcinogenesis risks, finding roles of the gene in cancer by and obtaining indicators for determining a new carcinogenesis risk related to the gene; and to provide a therapeutic agent for cancer.SOLUTION: A method detects a carcinogenesis risk in a subject, and the method includes detection of methylated CpG of a CpG island in a promoter area of a RASGRF1 (Ras Protein Specific Guanine Nucleotide-Releasing Factor 1) gene in a biological sample obtained from the subject. The invention also provides a therapeutic agent for cancer which contains a nucleic acid encoding the RASGRF1 gene or RASGRF1.

Description

  The present invention provides a method for obtaining an index for detecting cancer, a method for obtaining an index for determining cancer risk, a kit for detecting cancer, a kit for detecting cancer risk, a prophylactic agent for cancer, a therapeutic agent for cancer, The present invention relates to a method for searching for a preventive agent for cancer and a method for searching for a therapeutic agent for cancer.

  Cancer is one of the top causes of death in developed countries. For example, 31% of Japanese people died of cancer in 2003, and it has been the top cause of death in Japan for the past 20 years.

  When looking at the cancer that caused the death by location, gastric cancer has consistently ranked first or second in the last 20 years, but there is a clear change over time in the breakdown of cases with gastric cancer, In the past, Stage III and IV, which are represented by advanced gastric cancer, occupied the main patient group, but now more than 50% are occupied by Stage I. StageI consists of StageIa and Ib, both of which are characterized by cancer remaining in the submucosa and are classified as so-called early cancers.

  Recently, it has been recognized that gastritis caused by Helicobacter pylori (H. pylori) infection causes gastric cancer, but the infection rate of H. pylori is much higher than the incidence of gastric cancer. Among them, setting a high risk group for gastric cancer is extremely important for early detection and prevention of gastric cancer.

  In such early detection of cancer, currently, preoperative evaluation by macroscopic diagnosis does not have 100% diagnostic ability.

In general, about 90% of gastric cancers are differentiated. Differentiated gastric cancer is easy to diagnose early because it is known that cancer is more likely to become cancerous as strong atrophy or intestinal metaplasia is seen in endoscopy. In addition, the serum pepsinogen method, which is an index of atrophy, is considered useful for identifying high-risk groups of differentiated gastric cancer, and has recently been applied in health examinations (Fig. 1). Such atrophy and intestinal metaplasia are found in the background mucosa of differentiated gastric cancer. It is known that abnormal methylation of CpG islands of tumor suppressor genes such as P16 and MLH1 is accumulated in adenomas and intestinal metaplasias that are precancerous lesions of differentiated gastric cancer (Kang GH, Shim YH, Jung HY, et al. CpG island methylation in premalignant stages of gastric carcinoma.Cancer Res 2001; 61: 2847-51. Lee JH, Park SJ, Abraham SC, et al. Frequent CpG island methylation in precursor lesions and early gastric adenocarcinomas. Oncogene 2004; 23: 4646-54.).
However, none of the markers for judging the carcinogenic risk of differentiated gastric cancer could be used to judge the risk of undifferentiated carcinogenesis.
Furthermore, the relationship between DNA methylation of CpG islands of genes that promote oncogene function and gastric cancer is not known. In addition, there is no known relationship between DNA methylation of CpG islands of genes that promote the function of oncogenes and methods for obtaining an index for judging the risk of carcinogenesis in gastric cancer.

  On the other hand, undifferentiated gastric cancer, which accounts for about 10% of gastric cancer, is known to occur without atrophy and intestinal metaplasia, and is negative even in the serum pepsinogen method, making it difficult to identify high-risk groups. Finding early lesions of undifferentiated gastric cancer by endoscopy is difficult compared to differentiated cancers, progresses faster, and leads to early lymph node metastasis (Figure 2). However, at present, there is no known method for obtaining an index for judging the carcinogenic risk of undifferentiated gastric cancer. Therefore, establishment of a molecular marker for predicting the carcinogenic risk of such undifferentiated gastric cancer is required.

  The occurrence of undifferentiated cancer is considered to be caused by a mechanism different from that of differentiated cancer, and it seems necessary to identify a new gene that can detect the risk of occurrence of undifferentiated cancer. In addition, when detecting the risk of developing an undifferentiated cancer, it is considered necessary to be able to detect the risk of developing an undifferentiated cancer using a tissue that has not yet become cancerous. Therefore, it is considered necessary to identify a novel gene that can detect the risk of developing undifferentiated gastric cancer using tissue that has not yet become cancerous.

  H. pylori infection causes both differentiated and undifferentiated gastric cancer (Uemura N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 784-9.).

  Under these circumstances, regarding the early detection of cancer, it has recently been found that gene expression suppression by abnormal methylation plays an important role in various cancer cell lines, and cancer using abnormal methylation as an index. The detection method of this is proposed. For example, it has been reported that suppression of gene expression by abnormal methylation of interferon regulatory factor 4 gene, interferon regulatory factor 5 gene, and interferon regulatory factor 8 gene plays an important role in various cancer cell lines (non-patented) Reference 1: Yamashita M, et al, Cancer Science 2010, 101 (7), 1708-1716). It has also been clarified that H.pylori infection causes abnormal methylation of various genes such as LOX, HAND1, THBD, p41ARC, and FLNc in the gastric mucosa, increasing the risk of differentiated cancer (Non-Patent Document). 2: Maekita T. et al., Clinical Cancer Research, 2006, 12 (3), 989-995; Non-patent document 3: Nakajima T., Cancer Epidemiology, Biomarkers and Prevention, 2006, 15 (11), 2317-2321 ).

  As a mechanism for inactivating a tumor suppressor gene, epigenetic gene expression suppression (silencing) is widely known. In particular, cytosine methylation (DNA methylation) of CpG islands in the transcription initiation region of tumor suppressor genes is a phenomenon seen in almost all cancers, and many known tumor suppressor genes are silenced by DNA methylation. It has been reported. In recent years, genes with specific DNA methylation in cancer have been discovered one after another, and DNA methylation in cancer is a mutation and deletion as a mechanism of genetic abnormalities that are associated with an increased risk of cancer and the development and progression of cancer. (Jones PA and Baylin SB. The epigenomics of cancer. Cell 2007; 128: 683-92.).

  However, so far, the relationship between undifferentiated cancer and DNA methylation of specific genes is not known.

  On the other hand, as one of the factors of increased carcinogenic risk and the occurrence / progress of cancer, inactivation of a tumor suppressor gene, activation by mutation of an oncogene, and the like can be mentioned.

  On the other hand, the most well known oncogene is the ras gene. The ras gene is an oncogene with the most frequent abnormality in human cancers, and ras gene abnormality is often reported as one of the causes of human carcinogenesis (for example, Ellis RW et al. Mol. Cell. Biol. 2: 1339-1345, 1982.).

  Three types of human ras genes, H-ras, K-ras, and N-ras, have been found and are known to exist on human chromosomes 11, 12, and 1. (See, for example, Hall A et al. Nature 303: 396-400, 1983).

  The ras gene is a proto-oncogene that has no oncogenic activity as it is, and becomes a ras protein that is constantly activated by mutations that cause amino acid substitutions at the 12th, 13th, 59th, and 61st amino acids of the ras gene. Acquired carcinogenic activity (see, eg, Storer RD et al. Cancer Res. 46: 1458-1464, 1986; Adams, JM & Cory, S., Science 254, 1161-1167 (1991)). .)

  Approximately 30% of all human tumors have a ras gene mutation (Adjei, AA, Journal of National Cancer Institute, 93 (14): 1062-1074 (July 18,2001); Almoguera, C. et al., Cell ( 1988) 53: 549-54; Smit, VTet al., Nucleic Acids Res (1988) 16,7773-82; Bos, JL, Cancer Research, 49: 4682-4689 (1989)), three tumorigenic ras Mutations leading to gene, H-ras, K-ras, and N-ras activation are 90% of pancreatic cancer, 50% of colorectal cancer, 50% of lung cancer, 50% of thyroid cancer, 30 of liver cancer. %, Skin cancer, 25%, and 30% of myeloid leukemia (Clark, GJ & Der, CJ, Cellular Cancer Markers (eds Garrett, CT & Sell, S. ) 17-52 (Humana Press, Totowa, New Jersey (1995), Bos, JL, Cancer Res. 49, 4682-4689 (1989); Rodenhuis, S. et al., Cancer Res. (1988) 48: 5738- 41; Adjei, AA, Journal of National Cancer Institute, 93 (14): 1062-1074 (July 18,2001); Almoguera, C. et al., Cell (1988) 53: 549-54; Migley, RS Kerr, D.J., Crit Rev Oncol Hematol. (2002) 44: 109-20).

  The ras gene encodes a GTP-binding protein with a molecular weight of 21 kDa. When a ligand binds to an extracellular receptor that is coupled to the GTP-binding protein, GDP is released and GTP binds instead. (Hall A, Annu. Rev Cell Biol 1994, 10: 31-54) and acts as a molecular switch that transmits signals into cells via proteins such as Raf, phosphoinositide 3-kinase, RalGDS (Campbell SL et al , Oncogene 1998, 17: 1395-413; Quillam LA et al., Bioessays 1995, 17: 395-404).

  Conversion from the inactive GDP-bound ras protein to the active GTP-bound ras protein is performed by the GDP / GTP exchange factor (GEF) protein, and the active GTP-bound to inactive form Conversion to the GDP-linked form is performed by GTPase-promoting protein (GAP).

  The number of GEFs acting on ras protein is limited, for example, the RASGRF1 gene is known (Schweighoffer et al., Oncogene 8: 1477-1485, 1993), but the DNA methylation of the RASGRF1 gene and the risk of cancer or cancer There is no known relationship with the occurrence or development of

Yamashita M, Toyota M, Suzuki H, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae. Cancer Sci 2010 Maekita T, Nakazawa K, Mihara M, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk.Clin Cancer Res 2006; 12: 989-95. Nakajima T, Maekita T, Oda I, et al. Higher methylation levels in gastric mucosae significantly correlate with higher risk of gastric cancers.Cancer Epidemiol Biomarkers Prev 2006; 15: 2317-21.

  Therefore, the object of the present invention is to identify a gene related to cancer by conducting a comprehensive analysis of a gene that undergoes epigenetic silencing by the occurrence of cancer, elucidate its role in cancer, Related methods for obtaining an indicator for detecting cancer, methods for obtaining an indicator for determining cancer risk, kits for detecting cancer, kits for detecting cancer risk, preventive agents for cancer, therapeutic agents for cancer, cancer It is an object of the present invention to provide a method for searching for a preventive agent for cancer and a method for searching for a therapeutic agent for cancer.

  In addition, the subject of the present invention is to identify genes related to cancer by conducting a comprehensive analysis of genes that undergo epigenetic silencing due to the occurrence of cancer, particularly for undifferentiated cancers. Elucidating the role and obtaining an index for detecting undifferentiated cancer related to the gene, a method for obtaining an index for determining the carcinogenic risk of undifferentiated cancer, an undifferentiated cancer Kit for detection, kit for detection of carcinogenic risk of undifferentiated cancer, preventive agent for undifferentiated cancer, therapeutic agent for undifferentiated cancer, method for searching for preventive agent for undifferentiated cancer, and undifferentiated The object is to provide a method for searching for a therapeutic agent for a type of cancer.

  Furthermore, the subject of the present invention is to perform a comprehensive analysis of genes that undergo epigenetic silencing in a state in which the risk of cancer development is increased, using a background tissue among biopsy tissues obtained from a subject. , Identify genes related to carcinogenic risk, elucidate their role in cancer-prone states in tissues that have not yet become cancerous, and obtain indicators for detecting cancer using background tissues in relation to the genes Method, method for obtaining index for determining cancer risk using background tissue, kit for detecting cancer using background tissue, kit for detecting cancer risk using background tissue, cancer preventive agent, cancer It is to provide a method for searching for a therapeutic agent for cancer, a method for searching for a preventive agent for cancer, and a method for searching for a therapeutic agent for cancer.

  Furthermore, the object of the present invention is to comprehensively analyze genes that undergo epigenetic silencing due to the occurrence of gastric cancer, to identify genes related to gastric cancer and to elucidate their roles in gastric cancer. Of obtaining an index for detecting gastric cancer, a method for obtaining an index for determining the risk of developing cancer of the stomach, a kit for detecting gastric cancer, a kit for detecting the risk of developing cancer of the stomach, a prophylactic agent for gastric cancer, and gastric cancer It is intended to provide a method for searching for a therapeutic agent for gastric cancer, a method for searching for a preventive agent for gastric cancer, and a method for searching for a therapeutic agent for gastric cancer.

  Therefore, the present inventors have intensively studied to solve the above-mentioned problems, and genes whose expression is decreased under the control of specific epigenetic silencing in cancer tissues of organs where cancer has occurred RAGRF1 was identified for the first time, and DNA methylation of CpG islands in the promoter region of the gene was rarely observed in non-cancerous tissues, whereas it was observed at high levels in cancer tissues of organs where cancer occurred. From this, a method for obtaining an index for detecting cancer in a subject by detecting DNA methylation of a CpG island in the promoter region of the RASGRF1 gene in a biological sample obtained from the subject was found, and the present invention was completed. It was. The present invention is excellent in that it can be applied not only to differentiated cancers but also to undifferentiated cancers.

  In addition, the inventors of the present invention are diligently studying to solve the above-mentioned problems, and the genes whose expression is decreased under the control of epigenetic silencing specifically in the background tissue of the organ in which cancer has occurred RASGRF1 was identified for the first time, and DNA methylation of CpG islands in the promoter region of the gene was rarely observed in tissues that were not cancerous, but was found at a high level in the background tissues of organs where cancer occurred. The present inventors have found a method for obtaining an index for judging cancer risk in a subject by detecting DNA methylation of CpG island in the promoter region of the RASGRF1 gene in a biological sample obtained from the subject, and completed the present invention I let you. The present invention is excellent in that it can be applied not only to differentiated cancers but also to undifferentiated cancers.

  The present invention also relates to a method for obtaining an index for detecting cancer in a subject or a method for obtaining an index for determining carcinogenic risk, in a promoter region of the RASGRF1 gene in a biological sample obtained from the subject. Detecting said DNA methylation of CpG islands.

  The present invention also relates to a method for obtaining an index for detecting undifferentiated cancer in a subject or a method for obtaining an index for determining carcinogenic risk, wherein the RASGRF1 gene in a biological sample obtained from the subject Detecting DNA methylation of CpG islands in the promoter region of

  The present invention also relates to a method for obtaining an index for detecting cancer in a subject who is negative for serum pepsinogen or a method for obtaining an index for determining carcinogenic risk, wherein the method comprises the steps of: Detecting DNA methylation of CpG islands in the promoter region of RASGRF1 gene.

  The present invention also provides a method for obtaining an index for detecting cancer in a subject or a method for obtaining an index for determining carcinogenic risk, the method comprising: obtaining a CpG island in the promoter region of the RASGRF1 gene of a background tissue obtained from a subject. It relates to said method comprising detecting DNA methylation.

  Furthermore, the present invention provides the method as described above, wherein the methylated region of the CpG island in the promoter region of the RASGRF1 gene is a DNA region having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 7. About.

  In addition, the present invention was methylated using one or more methods selected from the group consisting of methylation-specific PCR, bisulfite sequencing, pyrosequencing, cobra and methylate methods. It relates to said method for detecting CpG.

  Further, the present invention provides that the DNA methylation of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is the same as the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject. The present invention relates to the above-mentioned method, wherein an index for determining the presence or absence of cancer or the risk of carcinogenesis is obtained on the basis that it is contained in CpG in an amount exceeding 8%.

  Further, the present invention provides that the DNA methylation of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is the same as the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject. The present invention relates to the above-mentioned method, wherein an index for determining the presence or absence of cancer or the risk of carcinogenesis is obtained on the basis that CpG contains at least 10%.

  Further, the present invention provides that the DNA methylation of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is the same as the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject. The present invention relates to the above-mentioned method, wherein an index for determining the presence or absence of cancer or the risk of carcinogenesis is obtained on the basis that CpG contains at least 15%.

  Further, the present invention provides that the DNA methylation of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is the same as the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject. The present invention relates to the above method, wherein an index for judging the presence or absence of cancer or the risk of carcinogenesis is obtained on the basis that it is contained in CpG in an amount exceeding 20%.

  Further, the present invention provides that the DNA methylation of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is the same as the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject. The present invention relates to the above-mentioned method, wherein an index for judging the presence or absence of cancer or the risk of carcinogenesis is obtained based on the fact that CpG contains at least 30%.

  Further, the present invention provides that the DNA methylation of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is the same as the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject. The present invention relates to the above-mentioned method, wherein an index for determining the presence or absence of cancer or the risk of carcinogenesis is obtained on the basis that CpG contains at least 50%.

  The present invention also provides a forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in SEQ ID NO: 7, and a base sequence shown in SEQ ID NO: 7. By amplifying a CpG island in the promoter region of the RASGRF1 gene using a reverse primer comprising 15 to 40 consecutive bases in a base sequence complementary to the same or substantially the same base sequence, methylated CpG To the above method.

  The present invention also includes a forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as that represented by SEQ ID NO: 11, and a base sequence represented by SEQ ID NO: 11. A CpG island in the promoter region of the RASGRF1 gene in bisulfite-treated genomic DNA is amplified using a reverse primer containing 15 to 40 consecutive nucleotides in a nucleotide sequence complementary to the same or substantially the same nucleotide sequence. Thus, it relates to said method, wherein unmethylated CpG is detected.

  The present invention also includes a forward primer comprising 15 to 40 bases in the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 15, and a base sequence represented by SEQ ID NO: 15. A CpG island in the promoter region of the RASGRF1 gene in bisulfite-treated genomic DNA is amplified using a reverse primer containing 15 to 40 consecutive nucleotides in a nucleotide sequence complementary to the same or substantially the same nucleotide sequence. In particular, the present invention relates to a method as described above, wherein methylated CpG is detected.

  The present invention further detects methylated CpG by amplifying a CpG island in the promoter region of the RASGRF1 gene using the oligonucleotide having the base sequence set forth in any of SEQ ID NOs: 19 to 32. Concerning the method.

  The present invention also includes SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29, Oligo having a base sequence that is substantially the same as the combination of oligonucleotides having the base sequences represented by SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, or the combination of oligonucleotides having these base sequences The present invention relates to the above method, wherein DNA methylation of CpG island in the promoter region of RASGRF1 gene is detected using any one or more oligonucleotide combinations among the nucleotide combinations.

  Furthermore, the present invention also provides that the biological sample is one or two selected from the group consisting of a biopsy sample of stomach, duodenum, colon, pancreas or lung, serum, stool, colon lavage fluid, gastric lavage fluid, and pancreatic juice. It relates to said method, which is more than a species.

  The present invention also relates to the above-described method, wherein the index is obtained using a biopsy sample of the gastric vestibular part which is free or mild in stomach atrophy.

  The present invention also relates to the above method, wherein the index is obtained using a biopsy sample of the vestibular part of the stomach where no intestinal metaplasia is observed.

  The present invention also relates to the method described above, wherein the specimen obtained from the subject is a gastric lavage fluid or a gastric biopsy sample.

  The present invention also relates to the above method, wherein the specimen obtained from the subject is a biopsy sample of the vestibular part of the stomach.

  The present invention also relates to the above method, wherein the specimen obtained from the subject is a duodenal wash or a duodenal biopsy sample.

  The present invention also relates to the above method, wherein the specimen obtained from the subject is a large intestine washing liquid or a large intestine biopsy sample.

  The present invention also relates to the above method, wherein the specimen obtained from the subject is pancreatic juice or a biopsy sample of pancreas.

  The present invention also relates to the above method, wherein the specimen obtained from the subject is a sputum or lung biopsy sample.

  The present invention also relates to the above method, wherein the specimen obtained from the subject is blood, serum, ascites, urine, or stool.

  The present invention also relates to the method as described above, wherein the cancer is gastric cancer, duodenal cancer, colon cancer, pancreatic cancer or lung cancer.

  The present invention also relates to the above method, wherein an index is obtained by measuring the expression level of the RASGRF1 gene in a sample obtained from a subject.

  The present invention also relates to a PCR primer set comprising a forward primer and a reverse primer for amplifying a CpG island in the transcription start region of the RASGRF1 gene.

  Further, the present invention is the same as the forward primer containing 15 to 40 bases in the same or substantially the same base sequence as shown in SEQ ID NO: 7, and the base sequence shown in SEQ ID NO: 7. Alternatively, the present invention relates to a PCR primer set for detecting DNA methylation of a CpG island in the promoter region of the RASGRF1 gene, comprising a reverse primer comprising 15 to 40 consecutive nucleotides in a nucleotide sequence complementary to a substantially identical nucleotide sequence.

  In addition, the present invention is the same as the forward primer containing 15 to 40 bases in the same or substantially the same base sequence as shown in SEQ ID NO: 11, and the base sequence shown in SEQ ID NO: 11. Alternatively, the present invention relates to a PCR primer set for detecting DNA methylation of a CpG island in the promoter region of the RASGRF1 gene, comprising a reverse primer comprising 15 to 40 consecutive nucleotides in a nucleotide sequence complementary to a substantially identical nucleotide sequence.

  Further, the present invention is the same as the forward primer containing 15 to 40 bases in the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 15, and the base sequence represented by SEQ ID NO: 15. Alternatively, the present invention relates to a PCR primer set for detecting DNA methylation of a CpG island in the promoter region of the RASGRF1 gene, comprising a reverse primer comprising 15 to 40 consecutive nucleotides in a nucleotide sequence complementary to a substantially identical nucleotide sequence.

  The present invention also includes SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29, Oligo having a base sequence that is substantially the same as the combination of oligonucleotides having the base sequences represented by SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, or the combination of oligonucleotides having these base sequences The present invention relates to a PCR primer set for detecting DNA methylation of CpG islands in the promoter region of the RASGRF1 gene, which comprises any one or more oligonucleotide combinations among nucleotide combinations.

  The present invention also includes any one of the above PCR primer sets, and one or more selected from sodium hydroxide, hydroquinone, bisulfite, deoxyribonucleotide triphosphate, mineral oil, or a buffer. And a kit for obtaining an index for detecting cancer or an index for determining cancer risk.

  The present invention also relates to a preventive or therapeutic agent for cancer comprising the RASGRF1 protein or the RASGRF1 gene.

  The present invention also relates to a preventive or therapeutic agent for cancer comprising an agent that suppresses DNA methylation of CpG islands in the promoter region of the RASGRF1 gene.

  In addition, the present invention is a method for searching for a prophylactic or therapeutic agent for cancer, wherein animal cells are cultured in the presence and absence of a test substance, and CpG islands in the promoter region of the RASGRF1 gene in each cell are cultured. The present invention relates to the above method comprising measuring the frequency of DNA methylation and selecting a test substance having an effect of suppressing the methylation as a candidate substance for a prophylactic or therapeutic agent for cancer.

  The present invention is also a method for searching for a prophylactic or therapeutic agent for cancer, comprising culturing animal cells in the presence and absence of a test substance, measuring the expression level of the RASGRF1 gene in each cell, and And the method comprising selecting a test substance having an effect of promoting the expression of the RASGRF1 gene as a candidate substance for a prophylactic or therapeutic agent for cancer.

  A method for obtaining an index for detecting cancer according to the present invention and a method for obtaining an index for determining cancer risk are obtained by methylating CpG islands in the promoter region of the RASGRF1 gene in a biological sample obtained from a subject. It is based on detecting CpG. Since the RASGRF1 gene is highly expressed in normal tissues and specifically decreased in cancer tissues and tissues with a high risk of carcinogenesis, methylation of CpG islands in the promoter region directly controlling the expression According to the method of the present invention for detecting a CpG that has been detected, it is possible to detect cancer and the risk of carcinogenesis very accurately. Further, according to the method of the present invention, the biological sample used for detection is a stomach tissue or stomach cancer tissue, colon tissue or colon cancer tissue, duodenal tissue or duodenal cancer tissue, pancreatic tissue or pancreatic cancer tissue obtained from a subject, lung In addition to tissues or cancer tissues such as tissues or lung cancer tissues, relatively easily available materials such as serum, stool, gastric lavage fluid, colon lavage fluid and pancreatic juice can be used, so that cancer can be easily detected. it can. In addition, according to the method of the present invention, it is possible to detect a state in which cancer has not yet occurred but the risk of cancer is increased. Furthermore, the method according to the present invention can be performed using a methylation-specific PCR method, a bisulfite sequencing method, a pyrosequencing method, a cobra method, a methylite method, and the like, so that it is highly possible to use a very small amount of biological sample. With detection sensitivity, accurate detection results can be obtained in a short time without burdening the subject, early detection of cancer, prediction of cancer progress, prediction of cancer recurrence risk, It also contributes to the prediction of cancer risk. Furthermore, the method according to the present invention is an epoch-making method in that it can detect not only differentiated cancer but also carcinogenic risk of undifferentiated cancer.

  Similarly, in the cancer detection kit and the cancer risk detection kit according to the present invention, the methylated CpG of the CpG island in the promoter region of the RASGRF1 gene, which is the target of detection, is specifically increased in cancer tissues. On the other hand, since it is rare in tissues that are not cancerous, it enables simple and easy detection of cancer with good accuracy, detection of cancer at the very early stage, monitoring of cancer recurrence, carcinogenesis It is suitable for risk detection and cancer progression prediction. In addition, the method and kit of the present invention are useful in combination with other inspection methods such as endoscopy, and can obtain more precise inspection results.

  Furthermore, the cancer preventive agent and cancer therapeutic agent of the present invention comprise a drug that suppresses DNA methylation of CpG islands in the promoter region of the RASGRF1 protein, the nucleic acid encoding the RASGRF1 gene, or the RASGRF1 gene. . Accordingly, the therapeutic agent of the present invention can introduce RASGRF1 protein into cancer cells, expression of RASGRF1 gene in cancer cells, or introduction of drugs that suppress CpG island DNA methylation in the promoter region of RASGRF1 gene into cancer cells. Thus, the growth of cancer cells can be suppressed, and an excellent therapeutic effect can be obtained. In addition, since the expression of the RASGRF1 protein or the RASGRF1 gene contained in the therapeutic agent of the present invention is suppressed in cancer tissues and tissues with a high risk of carcinogenesis, high expression is observed in normal tissues. The therapeutic agent of the present invention makes it possible to specifically restore in cancer cells the expression of a gene or its gene product that has been inactivated by an epigenetic abnormality, and is toxic to normal tissues. It is useful for the development of more effective and less effective side-effect therapy that can exert its effect specifically in target tissues that need prevention or treatment It is.

  Furthermore, the method for searching for a cancer preventive agent and the method for searching for a cancer therapeutic agent according to the present invention comprises culturing mammalian cells in the presence and absence of a test substance, and CpG in the promoter region of the RASGRF1 gene in each cell. Measure the frequency of DNA methylation in the island or the expression level of the RASGRF1 gene, and use a test substance that has the effect of suppressing the methylation or a test substance that increases the expression level of the RASGRF1 gene as a prophylactic or therapeutic agent for cancer. The method comprising selecting as a candidate substance. Therefore, the search method of the present invention is a search method that can be efficiently performed using cells, and can search for a prophylactic or therapeutic agent for cancer having a new mechanism of action against a wide range of test substances. Is.

It is a schematic diagram which shows the carcinogenic process from H.pylori infection, and the raise of the carcinogenic risk. H. pylori infection is thought to play an important role in the path of development of differentiated gastric cancer through chronic gastritis to atrophic gastritis and intestinal metaplasia. Atrophic gastritis and intestinal metaplasia are a high risk of gastric cancer. Well known as a group. Serum pepsinogen is useful as an index representing the degree of atrophy. In addition, H. pylori antibody becomes negative as intestinal metaplasia progresses. H. pylori infection is also known to induce abnormal DNA methylation in the gastric epithelium, suggesting that abnormal methylation accumulates in the intestinal metaplasia and is involved in carcinogenesis. On the other hand, the path of undifferentiated gastric cancer is still unclear. One of the characteristics of undifferentiated gastric cancer that is different from differentiated gastric cancer is that there is a pathway that occurs without atrophic gastritis or intestinal metaplasia.

It is the table | surface which showed the frequency of the lymph node metastasis of undifferentiated gastric cancer for every depth of penetration. The number in% is the percentage of cases with lymph node metastasis. In intramucosal cancer, it is considered that differentiated gastric cancer does not involve lymph node metastasis in the absence of ulceration, regardless of the size, and even in the case of ulceration, if it is 3 cm or less. On the other hand, it has been shown that undifferentiated gastric cancer is 2 cm or more without ulceration, and if it is accompanied by ulceration, it may have caused lymph node metastasis regardless of the size. Is considered to cause lymph node metastasis earlier than differentiated gastric cancer. (Gotoda T, et al: Incidence of lymph node metastasis from early gastric cancer.The estimation using a large number of cases in two large centers.Gastric Cancer 3: 219-225, 2000)

It is a figure which shows the ratio of DNA methylation of the RASGRF1 gene in various gastric cancer cell lines. The vertical axis represents the percentage of DNA methylation of the RASGRF1 gene in%, and each bar represents a gastric cancer cell line. For gastric cancer cell lines, DNA methylation of RASGRF1 gene was measured for MKN1, MKN7, MKN74, SH101, SNU1, SNU638, JRST, KatoIII, AZ521, MKN28, MKN45, NUGC3, NUGC4, AGS, and NCI-N87. did. The method for measuring the DNA methylation of RASGRF1 gene is described in Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae., Cancer Sci 2010, vol. 101, No. 7, 1708-1716. Based on the method, it was carried out by the pyrosequencing method. RASGRF1 gene shows 90% or more DNA methylation for MKN7, SH101, SNU638, JRST, KatoIII, MKN28, AGS, NCI-N87 gastric cancer cell lines, and 70% or more DNA methylation for MKN74 and SNU1 AZ521 showed about 55% DNA methylation, NUGC4 about 45% DNA methylation, and MKN45 about 20% DNA methylation. Thus, the RASGRF1 gene was frequently methylated in gastric cancer cell lines.

It is a figure which shows that the expression of a RASGRF1 gene is lose | disappearing in the gastric cancer cell line in which the RASGRF1 gene is DNA methylated. The vertical axis shows the expression level of the RASGRF1 gene as a relative amount (RQ: Relative Quantitation), and each bar shows a gastric cancer cell line. For gastric cancer cell lines, the expression level of the RASGRF1 gene was measured for each gastric cancer cell line of MKN1, MKN7, MKN74, SH101, SNU1, SNU638, JRST, KatoIII, AZ521, MKN28, MKN45, NUGC3, NUGC4, AGS, and NCI-N87. . The method for measuring the expression level of RASGRF1 gene is described in Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae., Cancer Sci 2010, vol. 101, No. 7, 1708-1716. Based on the above, the sequence of 5 ′-[TTCGGACAACACAAAATGGCAAACC] -3 ′ (SEQ ID NO: 34) is used as a forward primer, and 5 ′-[TCTTCTCTGTCTCCACGATCTGCA] -3 ′ (SEQ ID NO: 35) or 5 ′-[CCATTCGTCACAATCTTTTGCGTC] is used as a reverse primer. It was carried out by TaqMan RT-PCR method using the sequence of -3 ′ (SEQ ID NO: 36). The expression of RASGRF1 gene is SH101, SNU638, JRST, MKN28, AGS, NCI-N87 gastric cancer cell lines that showed 90% or more DNA methylation of the same gene, NUGC4 that also showed about 45% DNA methylation, It was almost lost in MKN45, which also showed about 20% DNA methylation. On the other hand, the expression level of the NUGC3 gene, in which DNA methylation of the RASGRF1 gene hardly occurred, was high. Thus, the expression of the RASGRF1 gene disappeared in cells in which methylation of the RASGRF1 gene was observed.

It is a figure which shows that the expression of a RASGRF1 gene will be recovered if the gastric cancer cell line in which the RASGRF1 gene is DNA methylated is treated with 5-aza-2'deoxycytidine (5-aza-dc). The vertical axis shows the expression level of the RASGRF1 gene as a relative amount (RQ: Relative Quantitation), and each bar shows a gastric cancer cell line. Gastric cancer cell lines are MKN1, MKN7, MKN74, SH101, SNU1, SNU638, JRST, KatoIII, AZ521, MKN28, MKN45, NUGC3, NUGC4, AGS, NCI-N87, and 5-aza-dc The expression level of the RASGRF1 gene was measured for each of the untreated cell line and the 5-aza-dc treated cell line. Each bar shows the case where it is not treated with 5-aza-dc and the case where it is treated with 5-aza-dc. The bar labeled “AZA” indicates the case where it is processed with 5-aza-dc, and the bar not labeled “AZA” indicates the case where it is not processed with 5-aza-dc. For example, “MKN1” indicates an MKN1 gastric cancer cell line not treated with 5-aza-dc, and “MKN1 AZA” indicates an MKN1 gastric cancer cell line treated with 5-aza-dc. The method for measuring the expression level of RASGRF1 gene is described in Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae., Cancer Sci 2010, vol. 101, No. 7, 1708-1716. Based on the above, the sequence of 5 ′-[TTCGGACAACACAAAATGGCAAACC] -3 ′ (SEQ ID NO: 34) is used as a forward primer, and 5 ′-[TCTTCTCTGTCTCCACGATCTGCA] -3 ′ (SEQ ID NO: 35) or 5 ′-[CCATTCGTCACAATCTTTTGCGTC] is used as a reverse primer. It was carried out by TaqMan RT-PCR method using the sequence of -3 ′ (SEQ ID NO: 36). In cell lines except MKN74 and AZ521, it was confirmed that the expression of the RASGRF1 gene was restored by 5-aza-dc treatment.

It is a figure which shows having identified RASGRF1 as a gene which recognizes abnormal methylation in the background mucosa of undifferentiated gastric cancer by MCAM method (Methylated CpG Island Amplification Microarray method). It is a figure which shows the result of the methylation analysis using the pyrosequencing method of the RASGRF1 gene in the gastric mucosa biopsy specimen in the stomach mucosa of the stomach of a gastric cancer case, and the gastric mucosa of an H.pylori gastric inflammation case. Each row corresponds to a target, and a filled cell indicates that the methylation frequency is 15% or more. Abnormal methylation is frequently observed in the background mucosa of cancer cases, but few cases show abnormal methylation in H. pylori gastric inflammation cases.

It is a figure which shows the result of the quantitative methylation analysis by the pyrosequencing method of the RASGRF1 gene in a gastric mucosa biopsy specimen. Each point represents each case. The left figure shows the methylation frequency of the RASGRF1 gene in the biopsy sample obtained from the gastric vestibule, and the right figure shows the methylation frequency of the RASGRF1 gene in the biopsy sample obtained from the body part of the stomach. “Undifferentiated” refers to cases of gastric cancer that were determined to be undifferentiated in histomorphology by hematoxylin and eosin-stained specimens. “Differentiated” refers to cases of gastric cancer that were determined to be differentiated by the same method. "Indicates a case of H. pylori infection but not suffering from gastric cancer. The horizontal bar represents the average methylation frequency of each group, and the P value represents a significant difference by one-way analysis of variance. The methylation rate of the background mucosa in the gastric vestibule of patients with undifferentiated gastric cancer showed significantly higher methylation than the mucosa in the gastric vestibule of noncancerous cases.

Pyrosequencing of the RASGRF1 gene in gastric mucosal biopsy specimens of the gastric vestibule diagnosed as having no atrophy, mild atrophy, or no intestinal metaplasia based on an updated Sydney System histopathological evaluation It is a figure which shows the result of the quantitative methylation analysis by a singing method. Each point represents each case. The figure on the left shows the methylation frequency of the RASGRF1 gene in a biopsy sample obtained from a subject with no atrophy or a mild degree of atrophy, and the figure on the right shows a case without intestinal metaplasia The methylation frequency of the RASGRF1 gene in the obtained biopsy sample is shown. `` Undifferentiated type '' refers to a case of gastric cancer that was determined to be undifferentiated in histomorphology by hematoxylin and eosin stained specimens, `` differentiated type '' refers to a case of gastric cancer that was determined to be differentiated by the same method, “Non-cancer” refers to cases that are infected with H. pylori but do not suffer from gastric cancer. The horizontal bar represents the average methylation frequency of each group, and the P value represents a significant difference by one-way analysis of variance. The methylation frequency of the RASGRF1 gene in the background mucosa of undifferentiated cancer cases diagnosed with no atrophy, mild degree of atrophy, or no intestinal metaplasia The frequency was significantly higher than the methylation frequency.

It is a figure which shows the result of the quantitative methylation analysis by the pyrosequencing method of RASGRF1 gene in the gastric mucosa biopsy sample of a serum pepsinogen negative case. Each point represents each case. The figure on the left shows the methylation frequency of the RASGRF1 gene in a biopsy sample obtained from a case with negative serum pepsinogen, and the figure on the right shows the RASGRF1 gene in a biopsy sample obtained from a case with positive serum pepsinogen. Shows methylation frequency. `` Cancer background vestibule '' is the background mucosa of the gastric vestibule in cases suffering from stomach cancer, `` Non-cancer vestibule '' is the stomach vestibule of cases that are infected with H. pylori but do not suffer from gastric cancer Showing the mucous membranes. The horizontal bar indicates the average value of methylation frequency of each group, and the P value indicates a significant difference by t-test. Regardless of whether serum pepsinogen was positive or negative, the methylation frequency of the RASGRF1 gene in the cancerous vestibule was significantly higher than the methylation frequency of the RASGRF1 gene in the noncancerous vestibule.

  Hereinafter, the present invention will be described in detail.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In general, the terms and techniques used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genes and proteins and nucleic acid chemistry described herein are well known in the art. It shall be normally used. In addition, unless otherwise indicated, the methods and techniques of the present invention are subject to various general and more specialized, cited and discussed herein, according to conventional methods well known in the art. As described in the public references. Such references include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring. Harbor Press (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992 and 2000 supplement); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology -4 ^th Ed., Wiley & Sons (1999); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1990); and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1999) Etc.

  The terms used herein for analytical chemistry, synthetic organic chemistry and medicinal chemistry and medicinal chemistry, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques shall be used for chemical synthesis, chemical analysis, drug production, formulation and delivery, and treatment of subjects.

  The term “subject” in the present invention means any living individual, preferably a vertebrate, more preferably a mammal, and still more preferably a human individual. In the present invention, the subject may be healthy or afflicted with some disease, but when treatment for cancer is intended, the subject afflicted with the disease or experimentally afflicted Subjects such as rodents such as mice, rats, gerbils, guinea pigs, felines such as cats, pumas and tigers, deer animals such as deer and elk, rabbits, dogs, minks, sheep and goats Cattle, horses, monkeys, humans and the like are preferable.

  The term “case” in the present invention refers to a subject that is a human patient.

  In the present specification, unless otherwise specified, the name of a gene is indicated by adding “gene” to an alphabet (or alphabet and numeral), and the protein encoded by the gene is the above-mentioned alphabet (or alphabet and (Protein) is added to what is indicated by numbers). When only indicated by alphabets (or alphabets and numbers), it means a gene or protein. The above-mentioned “gene” means any one of a genomic gene, messenger RNA, and cDNA, and when any one is specified, it is specified and described. Therefore, unless otherwise specified, for example, simply “RASGRF1” means a gene of RASGRF1 (Ras Protein Specific Guanine Nucleotide-Releasing Factor 1) or a protein encoded by the RASGRF1 gene, and “RASGRF1 protein” The notation means a protein encoded by the RASGRF1 gene, and particularly the notation “RASGRF1 genomic gene” means a genomic gene encoding RASGRF1.

  In the present invention, “background tissue” refers to a tissue obtained from a portion other than cancer in an organ having cancer of a subject affected with cancer. In the present invention, “background mucosa” refers to a background tissue that is a mucosal tissue. In the present invention, “cancer background” refers to the background tissue of a case suffering from cancer. Thus, for example, the expression “cancer background gastric vestibule” means the background tissue of the gastric vestibule of a case suffering from gastric cancer.

  In the present invention, “cancer tissue” or “cancerous part” refers to a tissue obtained from a part containing cancer in an organ having cancer of a subject affected with cancer.

  In the present specification, “epigenetic” has a normal meaning in the technical field to which the present invention belongs, and for example, gene expression is controlled by an acquired modification to chromatin without any change in DNA sequence. Can be illustrated. Examples of the acquired modification to chromatin include chemical modification such as methylation of a histone, acetylation, phosphorylation, etc. in addition to methylation of a DNA base.

  In the present specification, “silencing” has a normal meaning in the technical field to which the present invention belongs, and means that the amount of messenger RNA is reduced by a mechanism other than gene recombination. “Silencing” includes transcriptional gene silencing and post-transcriptional gene silencing. Examples of transcriptional gene silencing include epigenetic silencing, genome imprinting, paramutation, transposon suppression, transgene suppression, and placement effects. Post-transcriptional gene silencing includes RNA interference and nonsense. Mediated decay can be illustrated.

  The RASGRF1 protein used in the present invention is a protein derived from RNA transcribed from the RASGRF1 genomic gene located on chromosome 15q24 in humans. Type 1 isoform (NP_002882) consisting of 1273 amino acids, type 2 isoform consisting of 489 amino acids. Three types of isoforms are known: a form (NP_722522) and a type 3 isoform consisting of 1257 amino acids (NP_001139120). Each isoform has the mRNA sequence registered with accession number NM_002891 for the type 1 isoform, the mRNA sequence registered with accession number NM_153815 for the type 2 isoform, and the accession number NM_001145648 for the type 3 isoform. Each is expressed as a registered mRNA sequence. Therefore, the RASGRF1 protein includes a protein consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. Examples of the mRNA of the RASGRF1 gene include nucleic acids having the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

  Here, the nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5 is approximately 70% or more, preferably approximately the same as the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5. Examples thereof include base sequences having homology of 80% or more, more preferably about 90% or more, still more preferably about 95% or more, and most preferably about 98% or more. Furthermore, the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5 is the same as or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. Examples of the nucleotide sequence of a nucleic acid that encodes a protein consisting of the amino acid sequence of the nucleic acid that encodes a protein having the function of the RASGRF1 protein can be given. The function of RASGRF1 protein is the function of RASGRF1 protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, or 6, or the function of promoting the release of GDP bound to ras protein and converting it to GTP-binding type. Can be illustrated.

  Similarly, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 is about 70% or more of the amino acid sequence represented by SEQ ID NO: 2, 4, or 6. An amino acid sequence having a homology of preferably about 80% or more, more preferably about 90% or more, still more preferably about 95% or more, and most preferably about 98% or more can be exemplified. Further, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 is preferably 1 to 10 in the amino acid sequence represented by SEQ ID NO: 2, 4, or 6. A sequence in which any amino acid is deleted, substituted or added, more preferably, a sequence in which any one to 5 amino acids are deleted, substituted or added, and more preferably any one to 3 amino acids is deleted. , Substituted and added sequences can be exemplified.

  In addition, the RASGRF1 gene used in the present invention includes, for example, a nucleic acid containing a base sequence represented by SEQ ID NO: 1, 3, or 5, or a base sequence represented by SEQ ID NO: 1, 3, or 5 and a string. A protein containing a nucleotide sequence that specifically hybridizes under a gentle condition and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 above Nucleic acid to be included. Here, examples of the nucleic acid containing a base sequence that specifically hybridizes with the base sequence represented by SEQ ID NO: 1, 3, or 5 under stringent conditions include, for example, SEQ ID NO: 1, 3, or 5 About 60% or more, preferably about 70% or more, more preferably about 80% or more, still more preferably about 90% or more, particularly preferably about 95% or more, with a base sequence complementary to the represented base sequence, Most preferably, a nucleic acid containing a base sequence having a homology of about 98% or more can be used.

  In the present specification, “stringent conditions” are usually conditions of 42 ° C., 2 × SSC and 0.1% SDS, preferably 65 ° C., 0.1 × SSC and 0.1% SDS. Is the condition.

  It should be noted that there may be differences in the base sequence of the RASGRF1 gene due to polymorphisms, isoforms, etc., as well as between different species, as well as between different species, but even if the base sequence is different, the RASGRF1 protein As long as it is encoded, it is included in the RASGRF1 gene.

  Examples of the RASGRF1 genomic gene used in the present invention include the RASGRF1 genomic gene located in human chromosome 15q24, and the genomic gene homologous to the RASGRF1 gene in mammals.

  In the present invention, the promoter region of the RASGRF1 gene refers to a specific region on DNA that is involved in the initiation of transcription of the RASGRF1 gene on the chromosome, and is a region where RNA polymerase binds to this and transcription is initiated. In general, the promoter sequence is located at −20 to −110 bp from the transcription start position, and often has a consensus sequence such as TATA box, CAAT box or GC box. However, even if it is not located at -20 to -110 bp from the transcription start position or does not have these consensus sequences, it is located upstream of the transcription start position and is involved in the initiation of transcription of the RASGRF1 gene If it is a specific region on the genomic DNA, it is the promoter region of the RASGRF1 gene. In the present invention, it means a region having a total length of 2000 bp from the transcription start position or 5 'end of the start codon to 2000 bp upstream, and is preferably a region represented by SEQ ID NO: 7.

  In the present invention, the CpG island is a region rich in CpG (ie, cytosine on the 5 ′ side and guanine adjacent to each other on the 5 ′ side and bonded to each other by a phosphodiester bond), and has a GC content of 50%. This means a DNA region having a ratio of measured frequency to predicted frequency of CpG dinucleotide of 0.6 or more.

  In the present invention, “DNA methylation” means that cytosine in the CpG island of the promoter region of any genomic gene is methylated.

  In the present invention, “methylation of CpG island” refers to DNA methylation in a CpG island present in the promoter region of an arbitrary genomic gene.

  In the present invention, “abnormal methylation” refers to a state in which DNA methylation in a CpG island present in the promoter region of an arbitrary genomic gene is higher than that in a normal tissue.

  The CpG island in the promoter region of the RASGRF1 gene can be used for detection of cancer by the method of the present invention without limitation as long as it is a region corresponding to the CpG island among the promoter regions described above.

  Among the CpG islands in the promoter region of the human RASGRF1 gene, in the method for detecting cancer according to the present invention, for example, the total length of 405 bp from the 267th to the 671st bases of the sequence represented by SEQ ID NO: 7 is preferable. Region (region consisting of the base sequence represented by SEQ ID NO: 8) or a region having substantially the same base sequence, more preferably the 296th to 610th sequences of the sequence represented by SEQ ID NO: 7. A region having a total length of 315 bp up to the 1 st base (region consisting of the base sequence represented by SEQ ID NO: 9) or a region having substantially the same base sequence, and most preferred is the sequence represented by SEQ ID NO: 7 It is a region having a total length of 138 bp from the 387th to the 524th (region consisting of the base sequence represented by SEQ ID NO: 10) Properly is a region having substantially the same nucleotide sequence therewith.

  Here, as the base sequence substantially the same as the base sequence represented by SEQ ID NO: 7, 8, 9 or 10, the base sequence represented by SEQ ID NO: 7, 8, 9 or 10 is about 70% or more, Preferred examples include base sequences having homology of preferably about 80% or more, more preferably about 90% or more, still more preferably about 95% or more, and most preferably about 98% or more.

In the method of the present invention, “detecting methylated CpG” means detecting methylated cytosine (ie, DNA methylation) in a DNA base sequence.
As long as the method of the present invention includes "detecting methylated CpG of a CpG island in the promoter region of the RASGRF1 gene in a biological sample obtained from a subject", the subject is judged to have a high risk of carcinogenesis. Criteria to be performed can be carried out without any particular limitation, and the risk of carcinogenesis may be detected by adopting criteria appropriately selected according to individual circumstances, cancer types, etc. Since the proportion of methylated CpG in islands is low and the proportion of methylated CpG in CpG islands in cancer tissues tends to be high, methylation of the CpG islands in biological samples obtained from subjects Compared to the methylated CpG of a CpG island in a biological sample obtained from a healthy subject of the same species as the subject, or a pair obtained from the same individual as the subject. Compared to methylated CpG of CpG islands in biological samples that are normal tissues to be used as reference, it is preferable to obtain a detection result that the risk of carcinogenesis is high in the subject on the basis of quantitatively higher .

  In the present invention, a method for detecting cancer in a subject, the method comprising detecting methylated CpG of a CpG island in the promoter region of the RASGRF1 gene in a biological sample obtained from the subject. Provided.

  Furthermore, according to the study by the present inventors, the methylated CpG of the CpG island in the promoter region of the RASGRF1 gene indicates that if the biological sample obtained from the subject is a normal tissue, the total CpG in the sample The ratio of the total amount of CpG in the sample is much higher when the biological sample obtained from the subject is cancer tissue or background tissue. It has been found that in many cases it exceeds 50%. However, when a biopsy tissue or the like is used as a biological sample, cancer tissue and normal tissue are often mixed, so normal tissue even if only the portion of cancer tissue or background tissue is high. The ratio of methylated CpG to the total CpG in the sample, which is averaged by the low value of the part of, tends to appear as a low value even when cancer is actually present. Therefore, in a more preferred embodiment, the method of the present invention is such that the methylated CpG of the CpG island in the promoter region of the RASGRF1 gene in the biological sample obtained from the subject is obtained from the subject. In all CpGs of CpG islands in said region (ie in all alleles in said sample), at least 8%, preferably at least 10%, more preferably at least 15%, even more preferably at least 20%, even more Preferably, cancer is detected based on the inclusion of at least 30%, and most preferably at least 50%, and the detection result that cancer is present can be obtained.

  In the method of the present invention, detection of methylated CpG is not particularly limited, and any method may be used. However, bisulfite sequencing, pyrosequencing, cobra ( It is preferable to use known methods for detecting DNA methylation (methylated cytosine), such as a cobra) method, a methylation-specific PCR (MSP) method, and a methylight method. Any one of the methods may be used alone, or two or more methods may be used in combination. Among the above-mentioned methods, the methylite method is particularly preferably used because methylation can be detected with a high sensitivity from a very small amount of sample, and the pyrosequencing method is used for the purpose of quantitative methylation detection. Particularly preferably used.

  The DNA methylation analysis by the bisulfite sequence method was performed by simultaneously amplifying a methylated allele and an unmethylated allele using DNA treated with bisulfite (bisulfite), and the resulting PCR product was TOPO-cloning vector. After cloning into (Invitorogen) etc., the presence or absence of methylation is detected by sequencing reaction. Although it has the advantage of knowing the methylation status of all CpG sequences in the amplified region, it is not suitable for the analysis of a large number of specimens because of the labor required for the analysis (Methods. 2002 Jun) 27 (2): 101-7 and Taku Suzuki, Minoru Toyoda, Kozo Imai: bisulfite PCR, new genetic engineering handbook revised 4th edition (Masamatsu Muramatsu and Masaru Yamamoto), Yodosha, pp99-106, 2003 ).

Specifically, in the DNA methylation analysis by the bisulfite sequencing method, bisulfite treatment was performed using EpiTect Bisulfite Kits (Qiagen), and then the target region was amplified by PCR with a pair of bisulfite sequencing primers. The amplified PCR product can be cloned with TOPO TA Cloning Kit (Invitrogen) and analyzed by analyzing the nucleotide sequence with ABI3130x sequencer (Applied Biosystems). Primers can be designed to specifically amplify specific regions in the CpG island.

  In the pyrosequencing method, methylated alleles and unmethylated alleles are simultaneously amplified using bisulfite-treated DNA, and the obtained PCR products are analyzed by the pyrosequencing method. The ratio of DNA methylation is detected as a nucleotide polymorphism of cytosine and thymine, and is calculated as a value obtained by dividing the fluorescence intensity of cytosine by the sum of the fluorescence intensity of cytosine and the fluorescence intensity of thymine. The pyrosequencing method is useful as a quantitative and high-throughput methylation analysis method (see Nat Protoc. 2007; 2 (9): 2265-75).

  Specifically, for DNA methylation analysis by the pyrosequencing method, bisulfite treatment using EpiTect Bisulfite Kits (Qiagen), the target region was amplified by PCR with a set of pyrosequencing primers, and Pyro It can be performed by performing a sequence analysis using Gold Regent (Biotage) and sequencing primers. These primers can be designed to specifically amplify specific regions in the CpG island.

  The Cobra method (COBRA method: Combined Bisulfite Restriction Analysis) is a method in which a methylated allele and an unmethylated allele are simultaneously amplified using bisulfite-treated DNA, and the resulting PCR product is cleaved with a restriction enzyme, and then agarose gel is used. Electrophoresis, staining with ethidium bromide, and determining the presence or absence of methylation by the presence or absence of a band cleaved by a restriction enzyme (Methods Mol Biol. 2002; 200: 71-85, Taku Suzuki, Minoru Toyota) , Kozo Imai: bisulfite PCR, 4th revised genetic engineering handbook (Masamatsu Muramatsu, Masaru Yamamoto), Yodosha, pp99-106, 2003 and Taku Suzuki Minoru COBRA method Epigenetics experimental protocol (Toshikazu Ushijima (See Yoichi Kaikai) Yodosha p.69).

  Specifically, the DNA methylation analysis by the Cobra method was performed using an EZ DNA methylation kit (Zymo Research, CA, USA), and genomic DNA derived from a gastric mucosa biopsy specimen in a sodium bisulfite solution. The treatment can be carried out overnight at 0 ° C., PCR is performed using primers designed to amplify the target region, and the resulting PCR product is digested with an appropriate restriction enzyme. By appropriately selecting a restriction enzyme, if the target genomic DNA amplified is not methylated, the sequence generated by cytosine modification with sodium hydrogen sulfite is not digested, while the amplified target DNA is If the genomic DNA is methylated, the experiment can be designed so that cytosine is not modified with sodium bisulfite, so that it can be digested by restriction enzymes, and the resulting PCR fragment is electrophoresed and then methylated. The concentration ratio of the fragment band to the non-methylated fragment band can be measured by densitometry.

  Methylation-specific PCR is a primer specific to methylated alleles and unmethylated alleles, taking advantage of the difference in sensitivity to conversion from cytosine to uracil depending on the presence or absence of methylation during the bisulfite reaction. The PCR product obtained can be amplified and detected by PCR, and the obtained PCR product can be electrophoresed on an agarose gel and stained with ethidium bromide to determine the presence or absence of methylation based on the presence or absence of a band. Yes (Methods Mol Med. 2005; 113: 279-91 and Taku Suzuki, Minoru Toyoda, Kozo Imai: Bisulfite PCR New Genetic Engineering Handbook Revised 4th Edition (Masamatsu Muramatsu and Masaru Yamamoto), Yodosha, pp99-106 , 2003).

  Specifically, in the methylation-specific PCR method, bisulfite treatment is performed using EpiTect Bisulfite Kits (Qiagen), methylation is detected with a set of methylation-specific primers, and a set of non-specific PCR methods. Unmethylation can be detected with methylation specific primers. These primers can be designed to specifically amplify specific sites in the CpG island.

  The methylite method is a kind of methylation-specific PCR method, in which methylation is detected by fluorescence using a real-time PCR method in combination with a primer that recognizes a methylation-specific sequence and TaqMan Probe. By using the methylite method, highly sensitive methylation detection is possible, and it is useful when performing methylation detection from a very small amount of sample (see Methods. 2001 Dec; 25 (4): 456-62).

  Specifically, DNA methylation analysis by the methylite method involves modifying the DNA by bisulfite treatment and using a position-specific PCR primer adjacent to the oligonucleotide probe along with a 5 ′ fluorescent reporter dye and a 3 ′ quenching dye. Amplify by fluorescence-based real-time quantitative PCR. During the reaction, the fluorescent reporter dye is released by the enzyme. Since the fluorescent reporter dye released is proportional to the amount of PCR product, fluorescence proportional to the degree of methylation can be continuously detected in an automated nucleotide sequencer device (Eads CA, et al., Cancer Res. 1999 May 15; 59 (10): 2302-6). DNA methylation analysis by the methylite method can be performed using, for example, EpiTect MethyLight PCR Kit (Qiagen).

  When using bisulfite sequencing, pyrosequencing, cobra, methylation-specific PCR, methylate, etc., bisulfite treatment specifically converts unmethylated cytosine to uracil, resulting in methylation. Cytosine is not converted to uracil.

  Therefore, when the cytosine of the CpG island in the promoter region of the RASGRF1 genomic gene is not methylated, the region represented by SEQ ID NO: 7, 8, 9 or 10 is subjected to bisulfite treatment, respectively. , 13 or 14 is converted. On the other hand, when the cytosine of the CpG island in the promoter region of the RASGRF1 genomic gene is methylated, the region represented by SEQ ID NO: 7, 8, 9 or 10 is subjected to bisulfite treatment, respectively. , 17 or 18 is converted into an array.

  Therefore, in a preferred embodiment, the detection of CpG according to the present invention is the same as a pair of primers specific to the base sequence of CpG island in the promoter region of the RASGRF1 gene, for example, the base sequence represented by SEQ ID NO: 7 or A forward primer containing 15 to 40 bases in a substantially identical base sequence, and a base sequence complementary to a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 7 A reverse primer comprising 15 to 40 bases, more preferably a forward primer comprising 15 to 40 bases in the same or substantially the same base sequence as that represented by SEQ ID NO: 8, and a sequence A continuous sequence in a base sequence complementary to the base sequence identical or substantially identical to the base sequence represented by No. 8 A reverse primer containing 15 to 40 bases, more preferably a forward primer containing 15 to 40 bases in the same or substantially the same base sequence as shown in SEQ ID NO: 9, and A reverse primer comprising 15 to 40 consecutive nucleotides in a nucleotide sequence complementary to the nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 9 is particularly preferably used. Complementary to a forward primer containing 15 to 40 bases in the same or substantially the same base sequence as the base sequence to be detected, and the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 10 This is performed using a reverse primer containing 15 to 40 bases in a continuous base sequence.

  In the method of the present invention, when the sample is subjected to bisulfite treatment, the CpG island in the promoter region of the RASGRF1 gene is methylated in the DNA region having the base sequence represented by SEQ ID NO: 7 contained in the sample. If the CpG island in the promoter region of the RASGRF1 gene is methylated, the region having the base sequence shown in SEQ ID NO: 11 is converted to the region having the base sequence shown in SEQ ID NO: 15. .

  Therefore, the detection of the above-mentioned non-methylated CpG according to the present invention includes, for example, a forward primer containing 15 to 40 bases in the same or substantially the same base sequence as shown in SEQ ID NO: 11. And a reverse primer comprising 15 to 40 consecutive bases in a base sequence complementary to the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 11, more preferably SEQ ID NO: 12 A forward primer containing 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in (1), and a base sequence that is the same or substantially the same as the base sequence shown in SEQ ID NO: 12 More preferably, a reverse primer containing 15 to 40 bases in a complementary base sequence is used, and more preferably represented by SEQ ID NO: 13. A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as the base sequence, and a base complementary to the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 13 Using a reverse primer containing 15 to 40 bases in a sequence, particularly preferably, containing 15 to 40 bases in a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 14 This can be carried out using a forward primer and a reverse primer comprising 15 to 40 consecutive bases in a base sequence complementary to the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 14.

  Similarly, the detection of the above-mentioned methylated CpG according to the present invention includes, for example, a forward sequence containing 15 to 40 bases in the same or substantially the same base sequence as shown in SEQ ID NO: 15. More preferably, using a primer and a reverse primer containing 15 to 40 consecutive bases in a base sequence complementary to the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 15, A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in FIG. 16, and a base sequence that is the same as or substantially the same as the base sequence shown in SEQ ID NO: 16 More preferably, a salt represented by SEQ ID NO: 17 using a reverse primer containing 15 to 40 consecutive bases in a base sequence complementary to A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as the sequence, and a base sequence complementary to the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 17 In particular, a reverse primer containing 15 to 40 bases in sequence, particularly preferably a forward sequence containing 15 to 40 bases in the same or substantially the same base sequence as shown in SEQ ID NO: 18 It can be carried out using a primer and a reverse primer containing 15 to 40 bases in a base sequence complementary to the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 18.

  Here, the base sequence substantially the same as the base sequence represented by SEQ ID NOs: 7 to 18 is about 70% or more, preferably about 80% or more from the base sequence represented by SEQ ID NOs: 7 to 18, more Preferred examples include base sequences having homology of preferably about 90% or more, more preferably about 95% or more, and most preferably about 98% or more. The base length of the primer is, for example, 15 to 40 bases, preferably 17 to 35 bases.

  Therefore, for example, when the method of the present invention is carried out by the bisulfite sequence method, the pyrosequence method, or the cobra method using the region described in SEQ ID NO: 8 as a CpG island in the promoter region of the RASGRF1 gene, By bisulfite treatment, when the CpG island in the sequence represented by SEQ ID NO: 8 in the sample is not methylated, it is converted to the sequence represented by SEQ ID NO: 12, while If the CpG island in the sequence represented is methylated, it is converted to the sequence represented by SEQ ID NO: 16. The sequence represented by SEQ ID NO: 12 and the sequence represented by SEQ ID NO: 16 are both forward primer: 5 '-[AGTGTGGGGTGTGTGTGTGT] -3' (SEQ ID NO: 19) and reverse primer: 5 '-[ACTAAAAATTTTCTACAAAT] -3 '(SEQ ID NO: 20) can be amplified by PCR, and methylated CpG can be detected by sequencing the amplified sequence.

  For example, when the method of the present invention is carried out by the bisulfite sequencing method, the pyrosequencing method, or the cobra method using the region described in SEQ ID NO: 9 as the CpG island in the promoter region of the RASGRF1 gene, By bisulfite treatment, when the CpG island in the sequence represented by SEQ ID NO: 9 in the sample is not methylated, it is converted to the sequence represented by SEQ ID NO: 13, while When the CpG island in the sequence represented is methylated, it is converted to the sequence represented by SEQ ID NO: 17. The sequence represented by SEQ ID NO: 13 and the sequence represented by SEQ ID NO: 17 are both forward primer: 5 ′-[AAGATGGTGTTTTTTTTTTATAGGGTTG] -3 ′ (sequence number 21) and reverse primer: 5 ′-[AAAACCATAAAAACCTTCAAATAACTAC] -3 It can be amplified by PCR using '(SEQ ID NO: 22), and methylated CpG can be detected by sequencing the amplified sequence. Similarly, in the case where the CpG island is not methylated, the 1204th to 1504th base sequences in the base sequence represented by SEQ ID NO: 7 in the sample are not methylated. Even so, both should be amplified by PCR using forward primer: 5 '-[GGAGGAGGTATTAAGTTGTAGTTTG] -3' (SEQ ID NO: 23) and reverse primer: 5'- [CCAAATATCTACACTCCAAAATCTAAC] -3 '(SEQ ID NO: 24) Methylated CpG can be detected by sequencing the amplified sequence.

  Further, for example, when the method of the present invention is performed by a methylation-specific PCR method or a methylite method, a region of 138 bp in total length from the 387th to 524th positions of the sequence represented by SEQ ID NO: 7 (SEQ ID NO: 10 Can be used). In this case, by bisulfite treatment of the sample, when the CpG island in the sequence represented by SEQ ID NO: 10 in the sample is not methylated, the sample is converted to the sequence represented by SEQ ID NO: 14, and the sequence in the sample When the CpG island in the sequence represented by No. 10 is methylated, it is converted to the sequence represented by SEQ ID No. 18.

  Therefore, when the method of the present invention is carried out by methylation-specific PCR method or methylite method, forward primer: 5 ′-[AGTTTGTTGTTTTTTGTGGTAGATAG] -3 ′ (SEQ ID NO: 25) and reverse primer: 5 ′-[CCTACACAATTACACAACATTTATCC] -3 ′ (SEQ ID NO: 26) can be used to perform a PCR reaction to specifically amplify unmethylated CpG islands. Similarly, PCR was carried out using forward primer: 5 ′-[AGTTCGTTGTTTTTTGCGGTAGATAG] -3 ′ (SEQ ID NO: 27) and reverse primer: 5 ′-[CCTACACAATTACACGACATTTATCC] -3 ′ (SEQ ID NO: 28), and methylated. CpG islands can be specifically amplified. Then, methylated CpG can be detected based on the presence or absence of a methylation specific band and an unmethylation specific band.

  When the method of the present invention is carried out by methylation-specific PCR method or methylite method, forward primer: 5 ′-[GTTGTTTTTTGTGGTAGATAGT] -3 ′ (SEQ ID NO: 29) and reverse primer: 5 ′-[ACAACATTTATCCAAAAACA] It is also possible to specifically amplify a non-methylated CpG island by performing a PCR reaction using -3 ′ (SEQ ID NO: 30). Similarly, PCR was carried out using forward primer: 5 ′-[GTTGTTTTTTGCGGTAGATAGC] -3 ′ (SEQ ID NO: 31) and reverse primer: 5 ′-[ACGACATTTATCCGAAAACG] -3 ′ (SEQ ID NO: 32), and methylated. It is also possible to specifically amplify the CpG island. Then, methylated CpG can be detected based on the presence or absence of a methylation specific band and an unmethylation specific band.

  In addition, methylated CpG in the sequence amplified by the combination of each primer of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, and SEQ ID NO: 31 and SEQ ID NO: 32 Can also be detected by the pyrosequencing method, in which case the methylated CpG can be detected using the sequence primer: 5 ′-[GTTTTTTGYGGTAGATAG] -3 ′ (SEQ ID NO: 33).

In the present invention, the base represented by “Y” in the sequence of the primer used for the detection of methylated DNA is thymine (T) when cytosine is detected in an unmethylated CpG island. It means, and when detecting methylated cytosine in a methylated CpG island, it means cytosine (C).
In the present invention, the base represented by “R” in the sequence of the primer used for detection of methylated DNA means adenine (A) or guanine (G).

  Bisulfite treatment converts double-stranded DNA into single strand and then converts cytosine to uracil, so that the treated DNA loses complementarity and is amplified by PCR based on the sense strand after treatment. The sequence of the PCR product is partially different between PCR amplification based on the antisense strand. This is a phenomenon based on the chemical nature of the bisulfite treatment, and the same analysis result can be theoretically obtained regardless of which strand is used for PCR. Normally, PCR primers are designed based on the base sequence of the sense strand of the gene, but if PCR amplification or sequencing reaction is difficult due to a large number of repeated sequences, the primer is based on the base sequence of the antisense strand. In some cases, it is possible to solve such technical difficulties.

  Still further, according to the present invention, as a reagent for detecting or quantifying the above methylated CpG in a biological sample obtained from a subject, it is specific to the base sequence of the CpG island in the promoter region of the RASGRF1 gene. A kit for detecting cancer or a kit for detecting cancer risk, comprising an oligonucleotide as a pair of primers is provided.

  As a primer used for detecting or quantifying CpG of the CpG island in the promoter region of the RASGRF1 gene, which is included in the above-described cancer detection kit or carcinogenic risk detection kit, for example, SEQ ID NO: 7, 8, 9 or 10 A forward primer comprising a part of the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 8, and a base identical or substantially identical to the base sequence represented by SEQ ID NO: 7, 8, 9 or 10, respectively. The reverse primer containing a part of the base sequence complementary to the sequence can be used. In addition, when CpG islands in the promoter region of the RASGRF1 gene are detected specifically in an unmethylated CpG, the genomic DNA after bisulfite treatment is treated with the base represented by SEQ ID NO: 11, 12, 13 or 14. A forward primer containing a part of the same or substantially the same base sequence as that of the sequence, and complementary to the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 11, 12, 13 or 14, respectively. The reverse primer containing a part of the base sequence can be appropriately designed and used. Furthermore, when CpG islands in the promoter region of the RASGRF1 gene are specifically detected for CpG that is methylated, the genomic DNA after bisulfite treatment is represented by SEQ ID NO: 15, 16, 17 or 18. A forward primer containing a part of the same or substantially the same base sequence as the base sequence, and complementary to the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 15, 16, 17, or 18, respectively. A reverse primer containing a part of a simple base sequence can be used by appropriately designing and using each.

  As an example of a particularly preferred combination of oligonucleotides for PCR primer set contained in the kit of the present invention, an oligonucleotide having a base sequence represented by SEQ ID NO: 19 and an oligonucleotide having a base sequence represented by SEQ ID NO: 20 A combination, a combination of an oligonucleotide having the base sequence represented by SEQ ID NO: 21 and an oligonucleotide having the base sequence represented by SEQ ID NO: 22, an oligonucleotide having the base sequence represented by SEQ ID NO: 23 and the sequence represented by SEQ ID NO: 24 A combination of an oligonucleotide having a base sequence, a combination of an oligonucleotide having a base sequence represented by SEQ ID NO: 25 and an oligonucleotide having a base sequence represented by SEQ ID NO: 26, represented by SEQ ID NO: 27 Combination of oligonucleotide having base sequence and oligonucleotide having base sequence represented by SEQ ID NO: 28, combination of oligonucleotide having base sequence represented by SEQ ID NO: 29 and oligonucleotide having base sequence represented by SEQ ID NO: 30 Or a combination of an oligonucleotide having the base sequence represented by SEQ ID NO: 31 and an oligonucleotide having the base sequence represented by SEQ ID NO: 32, or a combination of oligonucleotides having these base sequences. Examples include a combination of oligonucleotides having a certain base sequence.

  Here, the combination of oligonucleotides consisting of base sequences substantially the same as the combination of oligonucleotides for PCR primer set described above is the base sequence of SEQ ID NO: 19 to SEQ ID NO: 32 constituting these PCR primer sets An oligonucleotide having a base sequence having a deletion, substitution or addition of 1 or 2 bases, preferably a deletion or substitution of 1 base in the base sequences of SEQ ID NO: 19 to SEQ ID NO: 32, An oligonucleotide consisting of a base sequence having an addition, more preferably an oligonucleotide consisting of a base sequence having one base substitution in the base sequences of SEQ ID NO: 19 to SEQ ID NO: 32.

  As described above, the oligonucleotides represented by SEQ ID NOs: 19 to 24 can be used when the method of the present invention is performed by the bisulfite sequencing method, the pyro sequencing method, or the cobra method, and are represented by SEQ ID NOs: 25 to 32. These oligonucleotides can be used when the method of the present invention is carried out by a methylation-specific PCR method or a methylite method.

  Since the cancer detection kit of the present invention is suitable for detecting methylated CpG of the CpG island in the promoter region of the RASGRF1 gene, an index for detecting cancer according to the present invention using the kit Or a method for obtaining an index for determining a cancer risk.

The cancer detection kit of the present invention may be in any form as long as it contains any of the above-described oligonucleotides or primers, and can appropriately include reagents, instruments, instruction manuals, etc., and organisms such as tissues and cells. Solutions (eg, buffer for tissue or cell lysis, phenol / chloroform, ethanol, etc.) and instruments used for extraction of DNA from biological samples, reagents necessary for PCR (eg, H ₂ O, buffer, MgCl ₂ , dNTP) 1 type or 2 types or more, such as a reagent required for fixed_quantity | quantitative_assay of PCR amplification fragment (for example, RI, fluorescent dye, etc.).

In the present invention, cancer refers to a malignant tumor, and generally includes those included in the concept of cancer.
In addition, gastric cancer generally includes those included in the concept of gastric cancer, including malignant tumors that develop from the gastric epithelial mucosa, and duodenal cancer generally includes those included in the concept of duodenal cancer. Including, cancer that has developed directly from the duodenal mucosa, cancer of the duodenal adenoma or duodenal polyp, cancer of the pancreatic tissue that has entered the duodenum, and cancer of the Brunel gland hamartoma. Colorectal cancer generally includes those included in the concept of colorectal cancer, and includes malignant tumors arising from the colorectal epithelial mucosa, colorectal adenomas, and the like.

  And pancreatic cancer includes what is generally included in the concept of pancreatic cancer, and includes invasive pancreatic duct cancer, pancreatic endocrine tumor, intraductal papillary mucinous tumor, mucinous cystic tumor, and acinar cell carcinoma. Lung cancer includes those generally included in the concept of lung cancer, and includes small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma, lung large cell carcinoma, carcinoid, columnar tumor, and mucoepidermoid carcinoma.

  The “biological sample obtained from the subject” in the present invention is not particularly limited as long as cancer can be detected. For example, blood, serum, ascites, urine, stool, gastric lavage fluid, pancreatic juice Alternatively, biopsy samples of gastrointestinal tissues (stomach, duodenum, small intestine, large intestine, pancreas, etc.), lung biopsy samples, sputum and the like can be used. Since the expression level of RASGRF1 gene is generally high in any normal tissue and tends to be significantly reduced in cancer tissue, the biological sample obtained from the subject can be appropriately selected according to the purpose. For example, in the case of detecting colon cancer, it is preferably serum, feces, colon washing fluid, colon tissue, etc., and in the case of detecting duodenal cancer, a biopsy sample of duodenal tissue In the case of aiming at detection of gastric cancer, it is preferably serum, gastric lavage fluid, stomach tissue and the like. In the case of aiming at detection of pancreatic cancer, serum, pancreatic juice, pancreatic tissue and the like. Preferably, for the purpose of detecting lung cancer, it is preferably a lung biopsy sample, sputum.

  In addition, any biological sample collected by this method can be used in the method of the present invention. For example, the biological sample may be collected by a surgical operation and collected by a puncture needle for tissue collection. In addition to pancreatic juice, it may be collected as a gastric lavage fluid, a secretion secreted from the mammary gland, or a lavage fluid secreted from the mammary gland (ductal fluid).

  In carrying out the method of the present invention, it is also possible to directly detect the above methylated CpG without performing pretreatment such as DNA extraction on a biological sample obtained from the above-mentioned subject. However, after extracting DNA from a biological sample, methylated CpG can be detected by the above-mentioned methods such as the bisulfite sequencing method, pyrosequencing method, cobra method, methylation-specific PCR method, methylite method, etc. Preferably it is done. Methods for extracting DNA from biological samples are well known in the art, and can be performed using, for example, phenol / chloroform, ethanol, or a commercially available DNA extraction reagent.

  Furthermore, the present inventors have found that the expression level of RASGRF1 is remarkably suppressed in cancer cells while high expression level is observed in normal cells. Accordingly, since RASGRF1 is considered to be effective in suppressing cancer cell proliferation and metastasis, the present invention also provides a cancer therapeutic agent comprising the RASGRF1 protein, and further a cancer therapeutic agent comprising the RASGRF1 gene. To do.

  Here, as described above, the RASGRF1 protein specifically includes a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6.

  The RASGRF1 gene includes, for example, a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5 or a stringent condition with the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5. And a nucleic acid encoding a protein having a nucleotide sequence that specifically hybridizes with and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 2, 4, or 6. included. Here, examples of the nucleic acid containing a base sequence that specifically hybridizes with the base sequence represented by SEQ ID NO: 1, 3, or 5 under stringent conditions include, for example, SEQ ID NO: 1, 3, or 5 About 60% or more, preferably about 70% or more, more preferably about 80% or more, still more preferably about 90% or more, particularly preferably about 95% or more, with a base sequence complementary to the represented base sequence, Most preferably, a nucleic acid containing a base sequence having a homology of about 98% or more can be used.

  Any of the above cancer therapeutic agents according to the present invention can be used for any cancer, but is particularly preferably used for gastric cancer, duodenal cancer, colon cancer, pancreatic cancer, and lung cancer as described above. .

  The therapeutic agent for cancer of the present invention is a pharmaceutically acceptable carrier (eg, excipient, binder, disintegrant, lubricant, stabilizer, preservative, pH adjuster, flavoring agent, diluent, injection). In combination with a solvent for preparation, etc.) and can be provided as a therapeutic agent for cancer. In addition, the therapeutic agent of the present invention may contain a label or nanocapsule that can be specifically delivered to a target tissue. The therapeutic agent of the present invention may contain either the RASGRF1 protein or the nucleic acid encoding RASGRF1 alone, or may contain two or more in combination.

  Furthermore, the therapeutic agent of the present invention may further contain other medicinal ingredients such as known anticancer agents effective for cancer treatment, and can also be used in combination with these active ingredients. Here, as a known anticancer agent, for example, fluorouracil, tamoxifen, anastrozole, aclarubicin, doxorubicin, tegafur, cyclophosphamide, irinotecan, cytarabine, paclitaxel, docetaxel, epirubicin, carboplatin, cisplatin, thiotepa, or these pharmaceuticals Examples of the acceptable salt can be exemplified.

  Examples of the administration route of the therapeutic agent of the present invention include intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, parenteral administration such as local administration, and oral administration. Examples thereof include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, suspensions and the like. Specifically, in the case of local administration, the affected area is exposed by surgery, and the therapeutic agent of the present invention can be directly administered to the cancer tissue by means of a syringe or the like. This can be done by intravascular administration. A preferred route of administration is topical administration.

  In particular, the cancer therapeutic agent of the present invention containing a nucleic acid encoding the RASGRF1 gene can be used as a gene therapeutic agent for the purpose of introducing and expressing the RASGRF1 gene in cancer cells.

  The cancer therapeutic agent of the present invention used as a gene therapeutic agent is an HVJ liposome method, a method in which the base sequence of the gene is directly administered by injection, a calcium phosphate method, a DEAE-dextran method, an electroporation method, a method using a gene gun, It can be administered to a subject by a parenteral administration route by a method of administration by a lipofection method, a method using an appropriate expression vector, or the like. Examples of suitable expression vectors include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, and the like.

  The dose and frequency of administration of the cancer therapeutic agent of the present invention vary depending on the intended effect, administration method, treatment period, subject age, body weight, sex, etc., but the dose when using the RASGRF1 protein is subject When human is a human, it can be appropriately selected from the range of usually 100 μg to 100 mg, preferably 1 to 20 mg per day for an adult, and when using a nucleic acid encoding the RASGRF1 gene or an expression vector containing these, In the case of a human being, it can be appropriately selected from the range of usually 0.01 mg / kg to 1 g / kg, preferably 0.1 mg / kg to 500 mg / kg, more preferably 1 mg / kg to 50 mg / kg per adult day, The frequency of administration can be appropriately selected from a range of once to several times a day. However, the dosage is not limited to the above range because it may vary depending on various conditions.

  The invention also provides the use of a nucleic acid encoding the RASGRF1 protein, in particular in the production of a therapeutic agent according to the invention and as a therapeutic agent according to the invention.

  Furthermore, according to the present invention, there is provided a method for treating cancer, comprising administering a nucleic acid encoding the RASGRF1 gene. In this case, the effective dose, administration method, administration form, and the like can be based on the above.

  The method for searching for a prophylactic or therapeutic agent for cancer according to the present invention can be carried out using cells or animals derived from animals. The cells may be primary cultured cells, established cells, or genetically modified cells. The animal may be a wild type or a genetically modified animal as long as it is a non-human animal. Cells derived from animals used in the method for searching for a prophylactic or therapeutic agent for cancer according to the present invention are preferably derived from mammals. When animals are used, mammals other than humans can be used.

  In the search method of the present invention, animal cells are cultured in the presence and absence of a test substance, the frequency of DNA methylation of CpG islands in the promoter region of the RASGRF1 gene in each cell is measured, and the methylation is performed. The present invention relates to the above-mentioned method, comprising selecting a test substance having an effect of suppressing or a test substance having an effect of reducing the frequency of methylation as a candidate substance for a prophylactic or therapeutic agent for cancer.

  The search method of the present invention has an effect of culturing animal cells in the presence and absence of a test substance, measuring the expression level of the RASGRF1 gene in each cell, and promoting the expression of the RASGRF1 gene. The method includes selecting a test substance as a candidate substance for a prophylactic or therapeutic agent for cancer.

  The search method of the present invention measures the frequency of DNA methylation of CpG islands in the promoter region of the RASGRF1 gene in a specific tissue after administering a test substance to an animal, and compared with the case where no test substance is administered. And selecting a test substance having an effect of suppressing the methylation or a test substance having an effect of reducing the frequency of the methylation as a candidate substance for a prophylactic or therapeutic agent for cancer.

  In addition, the search method of the present invention measures the expression level of the RASGRF1 gene in a specific tissue after administering a test substance to an animal, and promotes the expression of the RASGRF1 gene compared to when no test substance is administered. And selecting a test substance having an effect as a candidate substance for a prophylactic or therapeutic agent for cancer.

  The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited to these examples.

<Example 1> Collection of gastric mucosa biopsy specimen At the time of upper gastrointestinal endoscopy, with informed consent to the patient and the approval of the ethics committee of the facility, for diagnosis of H. pylori-infected gastritis DNA was extracted with phenol / chloroform from some of the collected non-cancerous gastric mucosa biopsy specimens and analyzed for methylation. 142 cases and 284 specimens (32 undifferentiated cancer cases (DGC), 59 differentiated cancer cases (IGC), 51 non-cancer cases (NGC)) were included. Diagnosis of H.pylori infection was determined by serum H.pylori antibody, microscopy, rapid urease method, or breath test.

<Example 2> Comprehensive screening of genes that are methylated in the undifferentiated cancer background mucosa 3 specimens of cancer of undifferentiated gastric cancer, 6 specimens of non-cancerous mucosa of stomach vestibule and stomach body, differentiated gastric cancer Methylation by MCAM (Methylated CpG Island Amplification Microarray) using 3 specimens of cancer, 4 specimens of stomach vestibule and stomach part, and 6 specimens of stomach vestibule and stomach part of non-cancer cases The genome gene search that has been done was performed. The MCAM method is based on Estecio MRH, et al., High-throughput methylation profiling by MCA coupled to CpG island microarray., Genome Research, 2007; 17: 1529-1536 or Gao W, et al., Variable DNA methylation patterns associated with progression. Carcinogenesis 2008; 29: 1901-10 was performed according to the method described in the disease in hepatocellular carcinomas.
As a result, abnormal methylation of tumor suppressor genes such as microRNA34b / c, IRF4, SFRP2, and DKK2 reported so far was examined in the background mucosa of undifferentiated gastric cancer, and undifferentiated among these tumor suppressor genes. No genes with abnormal methylation accumulated in the background mucosa of gastric cancer.
On the other hand, the cluster analysis extracted RASGRF1 as a gene that recognized methylation in some undifferentiated cancer tissues and the gastric vestibular background mucosa, but did not recognize methylation in the background of differentiated cancer tissues and differentiated cancer. Cluster analysis was performed using genespring ver. 11 (Agilent Technology).

<Example 3> Methylation of RASGRF1 gene in gastric cancer cell line DNA methylation of RASGRF1 gene was measured using various gastric cancer cell lines. For gastric cancer cell lines, DNA methylation of RASGRF1 gene was measured for MKN1, MKN7, MKN74, SH101, SNU1, SNU638, JRST, KatoIII, AZ521, MKN28, MKN45, NUGC3, NUGC4, AGS, and NCI-N87. did. The method for measuring the DNA methylation of RASGRF1 gene is described in Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae., Cancer Sci 2010, vol. 101, No. 7, 1708-1716. Based on the method, it was carried out by the pyrosequencing method. As shown in FIG. 3, the RASGRF1 gene shows 90% or more DNA methylation for each gastric cancer cell line of MKN7, SH101, SNU638, JRST, KatoIII, MKN28, AGS, NCI-N87, and 70% for MKN74 and SNU1. The above DNA methylation was shown, about 55% DNA methylation for AZ521, about 45% DNA methylation for NUGC4, and about 20% DNA methylation for MKN45. Thus, the RASGRF1 gene was frequently methylated in gastric cancer cell lines.

<Example 4> Loss of expression of RASGRF1 gene in gastric cancer cell lines The expression level of RASGRF1 gene was measured using various gastric cancer cell lines. For gastric cancer cell lines, the expression level of the RASGRF1 gene was measured for each gastric cancer cell line of MKN1, MKN7, MKN74, SH101, SNU1, SNU638, JRST, KatoIII, AZ521, MKN28, MKN45, NUGC3, NUGC4, AGS, and NCI-N87. . The method for measuring the expression level of RASGRF1 gene is described in Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae., Cancer Sci 2010, vol. 101, No. 7, 1708-1716. Based on the above, the sequence of 5 ′-[TTCGGACAACACAAAATGGCAAACC] -3 ′ (SEQ ID NO: 34) is used as a forward primer, and 5 ′-[TCTTCTCTGTCTCCACGATCTGCA] -3 ′ (SEQ ID NO: 35) or 5 ′-[CCATTCGTCACAATCTTTTGCGTC] is used as a reverse primer. This was performed by TaqMan RT-PCR using the sequence of -3 ′ (SEQ ID NO: 36). As shown in FIG. 4, the expression of the RASGRF1 gene is about 45% of SH101, SNU638, JRST, MKN28, AGS, and NCI-N87 gastric cancer cell lines that showed 90% or more DNA methylation of the same gene. NUGC4, which showed DNA methylation, was almost lost in MKN45, which also showed about 20% DNA methylation. On the other hand, the expression level of the NUGC3 gene, in which DNA methylation of the RASGRF1 gene hardly occurred, was high. Thus, the expression of the RASGRF1 gene disappeared in cells in which methylation of the RASGRF1 gene was observed.

<Example 5> Recovery of expression of RASGRF1 gene in gastric cancer cell line by treatment with 5-aza-2'-deoxycytidine (5-aza-dc) The same gastric cancer cell line used in Example 3 and Example 4 was used. After treatment with 5-aza-2'-deoxycytidine, the expression level of the RASGRF1 gene was measured. 5-aza-dc treatment was performed by culturing each gastric cancer cell line for 72 hours in the presence of 2 μM of 5-aza-dc. The method for measuring the expression level of the RASGRF1 gene is the same as in Example 4. The treatment with 5-aza-2'-deoxycytidine is performed by Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae. , Cancer Sci 2010, vol. 101, No. 7, 1708-1716. The treatment of 5-aza-2'-deoxycytidine, a methylase inhibitor, restored the expression of the RASGRF1 gene in cell lines except MKN74 and AZ521.

<Example 6> Methylation analysis of RASGRF1 gene in gastric mucosa biopsy specimen by MCAM method Next, methylation analysis of CpG island in the promoter region of RASGRF1 gene by clinical specimen was performed. 58 cases (28 undifferentiated cancers, 25 differentiated cancers) and 22 cases of gastric inflammation infected with H.pylori were collected by MCAM (Methylated CpG Island Amplification Microarray). Genome genome search was conducted. The MCAM method is based on Estecio MRH, et al., High-throughput methylation profiling by MCA coupled to CpG island microarray., Genome Research, 2007; 17: 1529-1536 or Gao W, et al., Variable DNA methylation patterns associated with progression. Carcinogenesis 2008; 29: 1901-10 is performed according to the method described in the above, and when the ratio of methylated CpG of the CpG island in the promoter region of the RASGRF1 gene is 15% or more, the result was positive. (Figure 6).
Methylation was observed in 14 cases (50%) of undifferentiated gastric cancer, 14 cases of background mucosa (50%), 13 cases of cancer of differentiated gastric cancer (52%), and 15 cases of background mucosa (60%) . Only 2 cases (9%) of gastric inflammation with H.pylori showed methylation. When methylation of the RASGRF1 gene was observed in the non-cancerous gastric mucosa, the odds ratio at which gastric cancer was present was 12.1 times.

<Example 7> Quantitative methylation analysis of RASGRF1 gene in gastric mucosa biopsy specimen by pyrosequencing method Next, methylation analysis of CpG island in the promoter region of RASGRF1 gene was quantitatively performed for all specimens. RASGRF1 gene methylation analysis method is described in Yamashita M, et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae., Cancer Sci 2010, vol. 101, No. 7, 1708-1716. On the basis of the pyrosequencing method. As a result, the average ratio of methylated CpG in the background mucosa of the gastric vestibule was 17.67% in undifferentiated cancer cases, 19.07% in differentiated cancer cases, and 10.10% in H.pylori-infected gastric inflammation cases. It was. The proportion of methylated CpG was significantly higher (p <0.05, p <0.001) in undifferentiated cancer cases and differentiated cancer cases than in H. pylori-infected gastric inflammation cases. Therefore, it was shown that methylation analysis of CpG islands in the promoter region of the RASGRF1 gene in the background mucosa of the gastric vestibule is useful for predicting cancer risk (FIG. 7). The ratio of methylated CpG in the background mucosa of the gastric body was not significantly different among undifferentiated cancer cases, differentiated cancer cases, and H. pylori-infected gastric inflammation cases.

<Example 8> Histopathological evaluation of gastric mucosal biopsy specimen by Updated Sydney System Diagnosis of gastritis was performed by biopsy from vaginal vestibule and gastrointestinal vaginal cavity, and updated Sydney System was used for activity, inflammation and atrophy. The degree of intestinal metaplasia was evaluated. Updated Sydney System was based on Dixon MF, et al. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol 1996; 20: 1161-81. .

Example 9 Quantitative methylation analysis by pyrosequencing of RASGRF1 gene in gastric mucosal biopsy specimen of gastric vestibule with no or slight histopathology
Example 3 and CpG island methylation analysis in the promoter region of the RASGRF1 gene in 30 cases of which histopathological evaluation was obtained by the Updated Sydney System and which were judged as having little or no atrophy It carried out by the same method as Example 7 (FIG. 8). As a result, the average ratio of methylated CpG in the background mucosa of the gastric vestibule was 17.58% in undifferentiated cancer cases, 13.2% in differentiated cancer cases, and 7.29% in H. pylori-infected gastric inflammation cases. It was. The proportion of methylated CpG in undifferentiated cancer cases was significantly higher (p <0.05) than the proportion of methylated CpG in H. pylori-infected gastric inflammation cases.

<Example 10> Quantitative methylation analysis of RASGRF1 gene by pyrosequencing method in gastric mucosa biopsy specimens in which intestinal metaplasia is not observed histopathologically
Methylation analysis of CpG islands in the promoter region of the RASGRF1 gene was performed in the same manner as in Example 3 and Example 7 in 33 cases in which intestinal metaplasia was not observed among cases that were evaluated by the Updated Sydney System. (Fig. 8). As a result, the average ratio of methylated CpG in the background mucosa of the gastric vestibule was 10.65% in undifferentiated cancer cases, 7.45% in differentiated cancer cases, and 4.15% in H. pylori-infected gastric inflammation cases. It was. The proportion of methylated CpG in undifferentiated cancer cases was significantly higher (p <0.05) than the proportion of methylated CpG in H. pylori-infected gastric inflammation cases.

<Example 11> Measurement of serum pepsinogen Pepsinogens I and II were measured with the serum collected during the serum H.pylori antibody test. If the serum concentration of pepsinogen I exceeds 70 ng / ml or the serum concentration of pepsinogen I exceeds 3 times the serum concentration of pepsinogen II, serum pepsinogen is negative and stomach atrophy It was determined that no stagnation was observed.

<Example 12> Quantitative methylation analysis of RASGRF1 gene by pyrosequencing method in gastric mucosal biopsy specimen of serum pepsinogen negative case About 29 cases of gastric vestibular mucosa negative in serum pepsinogen in the promoter region of RASGRF1 gene The methylation analysis of CpG islands was performed in the same manner as in Example 3 and Example 7. As a result, the average ratio of methylated CpG in the background mucosa of the gastric vestibule was 7.1% in H. pylori-infected gastric inflammation cases (non-cancerous vestibule), whereas gastric cancer-affected cases (cancer background) In the vestibule), it was 15.78%, indicating significantly (p <0.05) high methylation (FIG. 9). In pepsinogen-negative cases, the probability of suffering from gastric cancer when the proportion of methylated CpG in the CpG island in the promoter region of the RASGRF1 gene is 10% or more is higher than the odds of not suffering from gastric cancer. The ratio was 9.3.

<Example 13> Quantitative methylation analysis of RASGRF1 gene by pyrosequencing method in gastric mucosa biopsy specimens of serum pepsinogen positive cases About 30 cases of gastric vestibular mucosa positive in serum pepsinogen in the promoter region of RASGRF1 gene The methylation analysis of CpG islands was performed in the same manner as in Example 3 and Example 7. As a result, the average ratio of methylated CpG in the background mucosa of the gastric vestibule was 11.27% in H. pylori-infected gastric inflammation cases (non-cancerous vestibule), whereas gastric cancer affected cases (cancer background) In the vestibule part, it was 20.4%, showing significantly (p <0.05) high methylation (FIG. 9).
In pepsinogen positive cases, the probability of suffering from gastric cancer when the percentage of CpG island methylated in the promoter region of the RASGRF1 gene is 10% or more is odd compared to the probability of not suffering from gastric cancer. The ratio was 3.0.

<Example 14> Statistical analysis All statistical analyzes were performed using PRISM version 5 for windows, and statistical tests using one-way ANOVA (one-way analysis of variance) and Student's t-test (student's t-test) were performed.

  From the above results, DNA methylation of CpG islands in the promoter region of the RASGRF1 gene was detected at a high level in the non-cancerous part (background mucosa) of gastric mucosal tissue in cases affected by gastric cancer, and H. pylori As the risk of carcinogenesis of gastric cancer is higher, CpG islands in the promoter region of the RASGRF1 gene are methylated at a specifically higher rate. It was revealed that the RASGRF1 gene was silenced specifically for gastric cancer. In addition to gastric cancer, gastrointestinal cancers such as duodenal cancer, colon cancer, and cancers in which ras genes such as pancreatic cancer or lung cancer are involved in carcinogenesis, the CpG island in the promoter region of the RASGRF1 gene is methylated and the RASGRF1 gene Was considered to be silenced.

  RASGRF1 is a GTP exchange promoting factor of ras, an oncogene, and suppression of gene expression of RASGRF1 should be supposed to contribute to suppression of cancer. However, the inventor of the present application, contrary to expectation, found that the expression of the RASGRF1 gene was suppressed by high-frequency methylation in the non-cancerous part of the gastric mucosa tissue in cases suffering from gastric cancer. Is.

  As described above, the present inventors have found that CpG of the CpG island in the promoter region of the RASGRF1 gene is methylated at a high ratio in the background mucosa of undifferentiated gastric cancer. The RASGRF1 gene was methylated in about 50% of gastric cancer (FIG. 6), and was frequently methylated even in the non-cancerous part of the gastric mucosal tissue in cases affected with gastric cancer. It was also revealed that methylation was not observed only by suffering from H. pylori gastritis, and the ratio of methylated CpG in the CpG island in the promoter region of the RASGRF1 gene is an indicator of detection of gastric cancer and the risk of carcinogenesis of gastric cancer. It was thought that it was useful as an index to judge. In addition, the CpG island in the promoter region of the RASGRF1 gene is the gastric mucosa affected by H. pylori gastritis in the non-cancerous part (background mucosa) of the gastric mucosa tissue in cases suffering from undifferentiated gastric cancer and differentiated gastric cancer. It was methylated more significantly and frequently. Surprisingly, even in cancer cases without pathological atrophy and intestinal metaplasia, or in serum pepsinogen negative cases, the RASGRF1 gene was detected in the non-cancerous part of the gastric mucosal tissue in gastric cancer cases. It was revealed that CpG islands in the promoter region were methylated at an abnormally high rate. Based on these results, methylation of CpG islands in the promoter region of the RASGRF1 gene can be measured using gastric cancer that does not undergo atrophy and intestinal metaplasia, especially the diagnosis of the presence of undifferentiated cancer, and the carcinogenesis of undifferentiated cancer. It proved useful for risk prediction.

From these results, by using the biological sample obtained from the subject by the method of the present invention or by using the kit of the present invention, cancer in the subject, particularly stomach cancer, duodenal cancer, colon cancer, pancreatic cancer, It has been shown that lung cancer can be detected easily and accurately. In addition, the method of the present invention or the kit of the present invention was considered to be extremely useful not only for cancer detection but also for cancer progress prediction, cancer risk prediction, and recurrence risk prediction. As described above, the CpG island directly regulates the expression of the RASGRF1 gene, which is specifically expressed in cancer background tissues, in cancer cells, so that cancer is detected based on methylation in this region. The method of the present invention is extremely accurate.
The cancer preventive or therapeutic agent of the present invention increases the expression of RASGRF1 that functions as a tumor suppressor gene product in cancers such as gastric cancer, duodenal cancer, colon cancer, pancreatic cancer, and lung cancer. It was shown that the cancer cell growth can be effectively suppressed.
Furthermore, the method for searching for a preventive or therapeutic agent for cancer of the present invention is a search method that can be efficiently performed using cells, and is useful for the prevention or treatment of undifferentiated gastric cancer based on a new mechanism of action. It was shown that it was possible to search for new substances.

The RASGRF1 gene is specifically suppressed in cancers such as gastric cancer, duodenal cancer, colon cancer, pancreatic cancer, lung cancer and the like. According to the present invention, the RASGRF1 gene promoter directly controls the suppression of the expression of the RASGRF1 gene. A method for obtaining an index for determining cancer risk including detecting DNA methylation in a CpG island of a region, and a kit for obtaining an index of cancer risk based on such a method can be provided. Therefore, the present invention greatly contributes to the treatment and research in addition to the diagnosis of cancers such as gastric cancer, duodenal cancer, colon cancer, pancreatic cancer and lung cancer.
In addition, RASGRF1 contains RAGSRF1 or a nucleic acid that encodes the RASGRF1 gene because it is specifically decreased in the background tissue of cancer and cancer cells and has an action of suppressing the expression of oncogenes. A method for searching for a therapeutic agent for cancer can be provided, and such a method for searching for a therapeutic agent for cancer is expected to be useful for the development of a drug that can provide a high therapeutic effect.

Claims (33)

  A method for obtaining an index for detecting cancer or an index for determining cancer risk in a subject by detecting DNA methylation of a CpG island in the promoter region of the RASGRF1 gene in a biological sample obtained from the subject .   2. The method according to claim 1, wherein an index for detecting a cancer of undifferentiated cancer or determining a cancer risk is obtained.   The method according to claim 1 or 2, wherein an index for detecting cancer or an index for determining cancer risk is obtained in a subject who is negative for serum pepsinogen.   The method according to any one of claims 1 to 3, wherein the biological sample is background tissue.   The method according to any one of claims 1 to 4, wherein the CpG island in the promoter region of the RASGRF1 gene is a DNA region having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 7.   One or two methods for measuring methylation of CpG islands in the promoter region of RASGRF1 gene are selected from the group consisting of methylation-specific PCR, bisulfite sequencing, pyrosequencing, cobra and methylate The method as described in any one of Claims 1-5 which is the above method.   The method according to any one of claims 1 to 6, wherein the cancer risk is determined to be high when CpG island methylation in the promoter region of the RASGRF1 gene exceeds at least 8%.   The method according to any one of claims 1 to 7, wherein it is determined that the cancer risk is high when methylation of a CpG island in the promoter region of the RASGRF1 gene exceeds at least 10%.   The method according to any one of claims 1 to 8, wherein it is determined that the cancer risk is high when methylation of a CpG island in the promoter region of the RASGRF1 gene exceeds at least 15%.   The method according to any one of claims 1 to 9, wherein it is determined that the cancer risk is high when methylation of CpG islands in the promoter region of the RASGRF1 gene exceeds at least 20%.   The method according to any one of claims 1 to 10, wherein the cancer risk is determined to be high when methylation of CpG islands in the promoter region of the RASGRF1 gene exceeds at least 30%.   The method according to any one of claims 1 to 11, wherein the cancer risk is determined to be high when methylation of CpG islands in the promoter region of the RASGRF1 gene exceeds at least 50%.   A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence represented by SEQ ID NO: 7, and the same or substantially the same as the base sequence represented by SEQ ID NO: 7 By amplifying a CpG island in the promoter region of the RASGRF1 gene using a reverse primer containing 15 to 40 consecutive bases in the base sequence complementary to the base sequence, the methylated CpG of the CpG island is detected. The method according to any one of claims 1 to 12.   A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in SEQ ID NO: 11, and the same or substantially the same as the base sequence shown in SEQ ID NO: 11 By amplifying the CpG island in the promoter region of the RASGRF1 gene in the bisulfite-treated genomic DNA using a reverse primer containing 15 to 40 consecutive bases in the base sequence complementary to the base sequence, the CpG island 13. The method according to any one of claims 1 to 12, comprising detecting unmethylated CpG.   A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in SEQ ID NO: 15, and the same or substantially the same as the base sequence shown in SEQ ID NO: 15 By amplifying the CpG island in the promoter region of the RASGRF1 gene in the bisulfite-treated genomic DNA using a reverse primer containing 15 to 40 consecutive bases in the base sequence complementary to the base sequence, the CpG island 13. The method according to any one of claims 1 to 12, comprising detecting the methylated CpG.   Sequence number 19 and sequence number 20, sequence number 21 and sequence number 22, sequence number 23 and sequence number 24, sequence number 25 and sequence number 26, sequence number 27 and sequence number 28, sequence number 29 and sequence number 30, sequence number Of the combination of oligonucleotides having the base sequence represented by SEQ ID NO: 32 and SEQ ID NO: 32, or the combination of oligonucleotides having the base sequence substantially the same as the combination of oligonucleotides having these base sequences, 16. The method according to any one of claims 1 to 15, comprising detecting DNA methylation of a CpG island in the promoter region of the RASGRF1 gene using a combination of any one or more oligonucleotides.   The method according to any one of claims 1 to 16, wherein the index is obtained by using a biopsy sample of the vestibular portion of the stomach which has no or slight stomach atrophy.   The method according to any one of claims 1 to 17, wherein an index is obtained using a biopsy sample of the vestibular part of the stomach where no intestinal metaplasia is observed.   The method according to claim 1, wherein the specimen obtained from the subject is a gastric lavage fluid or a gastric biopsy sample.   20. The method of any one of claims 1-19, wherein the specimen obtained from the subject is a biopsy sample of the gastric vestibule.   21. The method according to any one of claims 1 to 20, wherein the specimen obtained from the subject is a duodenal lavage fluid or a duodenal biopsy sample.   The method according to any one of claims 1 to 21, wherein the specimen obtained from the subject is a large intestine washing liquid or a large intestine biopsy sample.   The method according to any one of claims 1 to 22, wherein the specimen obtained from the subject is pancreatic juice or a biopsy sample of pancreas.   24. The method according to any one of claims 1 to 23, wherein the specimen obtained from the subject is a sputum or lung biopsy sample.   The method according to any one of claims 1 to 24, wherein the specimen obtained from the subject is blood, serum, ascites, urine, or feces.   26. The method according to any one of claims 1 to 25, wherein the cancer is gastric cancer, duodenal cancer, colon cancer, pancreatic cancer or lung cancer.   A method of obtaining an index for detecting cancer or an index for determining cancer risk in a subject by measuring the expression level of the RASGRF1 gene in a biological sample obtained from the subject.   A PCR primer set consisting of a forward primer and a reverse primer for amplifying a CpG island in the promoter region of the RASGRF1 gene.   A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence represented by SEQ ID NO: 7, and the same or substantially the same as the base sequence represented by SEQ ID NO: 7 The PCR primer set according to claim 28, comprising a reverse primer comprising 15 to 40 consecutive bases in a base sequence complementary to the base sequence.   A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in SEQ ID NO: 11, and the same or substantially the same as the base sequence shown in SEQ ID NO: 11 The PCR primer set according to claim 28, comprising a reverse primer comprising 15 to 40 consecutive bases in a base sequence complementary to the base sequence.   A forward primer comprising 15 to 40 consecutive bases in the same or substantially the same base sequence as shown in SEQ ID NO: 15, and the same or substantially the same as the base sequence shown in SEQ ID NO: 15 The PCR primer set according to claim 28, comprising a reverse primer comprising 15 to 40 consecutive bases in a base sequence complementary to the base sequence.   Sequence number 19 and sequence number 20, sequence number 21 and sequence number 22, sequence number 23 and sequence number 24, sequence number 25 and sequence number 26, sequence number 27 and sequence number 28, sequence number 29 and sequence number 30, sequence number Of the combination of oligonucleotides having the base sequence represented by SEQ ID NO: 32 and SEQ ID NO: 32, or the combination of oligonucleotides having the base sequence substantially the same as the combination of oligonucleotides having these base sequences, 29. The PCR primer set of claim 28, comprising a combination of any one or more oligonucleotides.   One or more selected from sodium hydroxide, hydroquinone, bisulfite, deoxyribonucleotide triphosphate, mineral oil or buffer, comprising the PCR primer set according to any one of claims 28 to 32. A kit for obtaining an index for detecting cancer or an index for determining the risk of carcinogenesis.