JP2014161308A - Method for obtaining the index of detection of gastric cancer or risk assessment of gastric cancer onset - Google Patents

Abstract

Provided is a method for obtaining an index for detecting gastric cancer or for evaluating the risk of developing gastric cancer.
At least one of the expression level of BARHL2 gene and the frequency of cytosine methylation is used as an index for detection of gastric cancer or risk assessment of gastric cancer development. Preferably, in addition, at least one of the expression level and cytosine methylation frequency of at least one of the SOX17 gene and the MINT25 gene is used as an index for detection of gastric cancer or risk assessment of gastric cancer development.
[Selection figure] None

Description

  The present invention relates to a method for obtaining an index for detecting gastric cancer or for evaluating the risk of developing gastric cancer.

  Conventionally, as a method for diagnosing cancer, somatic cells are removed by biopsy, immunostaining is performed using a cancer-specific antigen on the cell surface, and a pathologist makes a definitive diagnosis.

  Although genetic abnormalities such as single nucleotide polymorphisms (SNPs) are known as risk factors for cancer, it has been known that cancer also develops due to epigenetic abnormalities that are acquired by lifestyle habits and the like. . Epigenetic abnormalities are due to methylation of the 5th carbon of cytosine (C) of DNA 5′-CG-3 ′ (CpG sequence), and histones undergo modifications such as acetylation, methylation and phosphorylation This is mainly due to changes in chromatin structure. In particular, DNA methylation is considered to contribute to carcinogenesis by suppressing cytosine (C) methylation without changing the DNA base sequence in a gene promoter region having a high density of CpG sequences, thereby suppressing gene expression. (Non-Patent Document 1-2). It is known that methylation is caused by gastritis, Helicobacter pylori, chemical substances, and the like (Non-patent Documents 3-5).

  Cancer markers such as CEA, CA19-9, PSA, CA125, SCC antigen, AFP, PIVKA-II, and pepsinogen have been used so far for cancer testing. However, since the marker does not rise significantly unless cancer has occurred, it has not led to early cancer detection even if it is useful in advanced cancer. In addition to conventional cancer markers, in recent years, cancer diagnosis methods targeting DNA methylation have been proposed (Patent Document 1). In gastric cancer, gastric cancer markers of MINT25 gene and Sox17 gene targeting DNA methylation have been proposed (Patent Documents 2-3). However, when these gastric cancer markers are used, a significant difference is observed in gastric cancer patients with H. pylori infection, but there is a problem that no significant difference is observed in gastric cancer patients not infected with H. pylori. In recent years, the incidence of gastric cancer in non-infected persons with H. pylori has increased mainly among young people, and therefore, an index for detection of early gastric cancer or evaluation of the risk of developing gastric cancer regardless of the presence or absence of H. pylori infection is desired.

JP 2008-283947 A Japanese Patent No. 4426549 International Publication No. 2009/136501

Cell, 128: 683-692, 2007, J Natl Cancer Inst. 85 (15): 1235-1240, 1993. Clin Cancer Res, 12 (3): 989-995, 2006. Cancer Epidemiol Biomarkers Prev. , 17 (10): 2555-2564, 2008 Cancer Res. , 70 (4): 1430-440, 2010

  An object of the present invention is to provide a method for obtaining an index for detecting early gastric cancer or for evaluating the risk of developing gastric cancer.

  As a result of intensive studies, the present inventors have found that at least one of the expression level of the BARHL2 gene and the frequency of cytosine methylation can be used as a gastric cancer marker, and have completed the present invention.

  That is, the present invention provides a method for obtaining an index for detecting gastric cancer or evaluating the risk of developing gastric cancer, comprising the step of measuring at least one of the expression level of BARHL2 gene and the frequency of cytosine methylation in a sample collected from a subject. A method of using at least one of the expression level of the BARHL2 gene and the frequency of cytosine methylation as an index for detection of gastric cancer or risk assessment of gastric cancer development.

  The method includes the step of measuring at least one of the expression level and cytosine methylation frequency of at least one of the SOX17 gene and the MINT25 gene in a sample collected from a subject, and comprising at least one of the expression level of the BARHL2 gene and the cytosine methylation frequency It is preferable that at least one of the expression level of at least one of the SOX17 gene and the MINT25 gene and the frequency of cytosine methylation be used as an index for detection of gastric cancer or risk assessment of gastric cancer development. By adding at least one of the SOX17 gene and the MINT25 gene already used as a gastric cancer marker as an index, a more accurate diagnosis can be performed.

  In the above method, gastric mucosa washing liquid, biopsy, gastric juice, blood or feces can be used as the specimen, and gastric mucosa washing liquid is particularly preferable. The gastric mucosa washing solution has an advantage that information on the entire stomach can be obtained and an advantage that the DNA strands in the specimen are not easily broken.

  The present invention also provides a kit for measuring the cytosine methylation frequency of the BARHL2 gene, comprising a nucleic acid sequence of SEQ ID NO: 1, comprising an oligonucleotide having a length of 40 bases or less and a nucleic acid sequence of SEQ ID NO: 2, There is provided a kit comprising a primer set comprising an oligonucleotide having a length of 40 bases or less and a probe comprising an oligonucleotide having a nucleic acid sequence of SEQ ID NO: 3 and a length of 30 bases or less. The above method can be carried out using such a kit.

  In the kit, the primer set is preferably composed of an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 4 and an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 5, and the probe is preferably composed of an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 6. .

  According to the method of the present invention, it is possible to detect early gastric cancer or to evaluate the risk of developing gastric cancer. Furthermore, according to the method of the present invention, it is possible to detect early gastric cancer or evaluate the risk of developing gastric cancer regardless of the presence or absence of Helicobacter pylori infection.

It is a schematic diagram which shows the CpG island of a BARHL2 gene. The bar represents the position where CpG is present. It is a figure which shows the change of methylation frequency before and behind ESD. (A) represents all genes on the array, and (b) represents genes whose methyl frequency has decreased after ESD. Numbers 1-6 represent patients. It is a figure showing the mRNA expression level and methylation frequency of BARHL2 gene in a gastric cancer cell. It is a figure showing the influence which a demethylating agent has on the mRNA expression level of BARHL2 gene of a gastric cancer cell. It is a figure showing the staining result of the gastric mucosa tissue fragment of gastritis and gastric adenoma. It is a figure showing the dyeing | staining result of the gastric mucosa tissue fragment | piece of gastric cancer. It is a figure which shows the change of methylation frequency before and behind ESD. (A) MINT25 gene, (b) BARHL2 gene, (c) SOX17 gene.

(Gene and CpG island)
The BARHL2 gene (Gene Map Locus: 1p22.2, Sequence: NC — 000001.10 (91177579..91182794)) is a gene consisting of 5216 base pairs and expressing the BarH-like homeobox 2 protein, and is expressed during development of the nervous system It is a gene of the nerve axon system. The DNA sequence of the BARHL2 gene is shown in SEQ ID NO: 13.

  The SOX17 gene (Gene Map Locus: 8q11.23, Sequence: NC — 000008.10 (55370495..553373456)) is composed of 2962 base pairs and is an SRY (sex determining region Y) -box 17 gene. The DNA sequence of the SOX17 gene is shown in SEQ ID NO: 14.

  The MINT25 (Methylation in tumor 25) gene is a gene fragment of the CABIN1 gene (Gene Map Locus: 22p11.23, Sequence: NC — 000022.10 (24407765.24574596). The DNA sequence of the MINT25 gene is shown in SEQ ID NO: 15.

  The CpG island is a region in which the GC content, which is a ratio of guanine (G) and cytosine (C), is 50% or more, and the ratio of CpG (CG sequence) is 60% or more of the amount expected from the GC content. It is. This methylation of CpG island is an addition reaction of a methyl group to the carbon atom at the 5-position of the pyrimidine ring of cytosine. When this methylation occurs, gene expression is suppressed. It is known that if the frequency of methylation increases, necessary gene expression cannot be obtained, which is one of the causes of cancer.

(Method for obtaining an index for detecting gastric cancer or for assessing the risk of developing gastric cancer)
In one embodiment of the present invention, at least one of the expression level of BARHL2 gene and the frequency of cytosine methylation is used as an index for detection of gastric cancer or risk assessment of gastric cancer development. Therefore, at least one of the expression level of the BARHL2 gene and the cytosine methylation frequency in a sample collected from the subject is measured.

  In another embodiment of the present invention, detection of gastric cancer or at least one of the expression level and cytosine methylation frequency of BARHL2 gene and at least one of the expression level and cytosine methylation frequency of at least one of SOX17 gene and MINT25 gene is performed. It is used as an index for risk assessment of gastric cancer. Therefore, at least one of the expression level and cytosine methylation frequency of the BARHL2 gene in the sample collected from the subject is measured, and the expression level and cytosine methylation frequency of at least one of the SOX17 gene and the MINT25 gene in the sample collected from the subject are measured. At least one is measured.

  Subjects include all patients with Stage I to IV gastric cancer, as well as patients such as adenoma who are not diagnosed with Stage I and cannot be judged as malignant or benign. Furthermore, the risk of developing gastric cancer can be evaluated using healthy subjects as subjects. In particular, StageI is preferable for early gastric cancer detection.

  Samples are not limited to gastric-derived samples such as gastric mucosal lavage fluid, biopsy and gastric juice, but also include oral wipes, blood, plasma, serum, lymph, saliva, ascites, urine, feces and the like. The specimen is preferably a gastric mucosa washing solution, biopsy, gastric juice, blood or stool from the viewpoint of ease of handling. Among them, a gastric mucosa washing solution is more preferable as a sample from the point that information from the whole stomach is included and the fact that DNA strands in the sample are less cleaved.

  As a method of isolating cell components from the obtained specimen, centrifugal isolation is performed in the case of components mixed in a liquid such as gastric mucosa washing liquid and gastric juice, and in the case of a tissue piece such as a biopsy, it is used as it is. The isolated cells extract DNA, RNA and protein as necessary. Any of these can be performed by methods well known to those skilled in the art, and various commercially available kits can be used. As a DNA extraction method, for example, a phenol / chloroform extraction method can be used. As an RNA extraction method, for example, the AGPC method can be used. As a protein extraction method, for example, a method using a RIPA buffer or the like can be used.

  The gene expression level may be a transcription product, ie, mRNA, or a translation product, ie, protein. The obtained RNA is synthesized with cDNA by reverse transcriptase and used for measuring the gene expression level. The gene expression level may be corrected by the amount of housekeeping genes such as GAPDH and β-actin. As a method for quantifying mRNA, for example, a real-time RT-PCR method can be used. You may correct | amend the obtained protein with the protein amount of housekeeping genes, such as GAPDH and (beta) -actin. As a protein quantification method, for example, an ELISA method can be used.

  Methods for measuring cytosine methylation frequency of genes include Southern blotting using restriction enzymes, PCR using restriction enzymes, bisulfite reaction and sequence analysis, and Restriction Mapping using bisulfite PCR. Examples thereof include a method and a method using real-time PCR. For the sequence analysis method, real-time PCR, pyrosequencing method or the like is used.

  As a typical example of measuring the methylation frequency of CpG islands of BARHL2, SOX17, and MINT25 genes, a method of measuring a nucleotide sequence by pyrosequencing after bisulfite treatment will be described. .

  The DNA extracted from the specimen is subjected to bisulfite treatment using sodium bisulfite and then subjected to desulfonic acid treatment. Thereby, unmethylated C is converted to uracil (U), but methylated C remains C without being converted. Therefore, methylation frequency can be measured by examining the ratio of U and C at the same location in DNA. Although the methylation frequency of CpG at an arbitrary position in the CpG island may be measured, it is preferable to measure the methylation frequency of CpG in the range of 2 Kb upstream or downstream of the gene transcription start point. It is also possible to analyze the sequence of the DNA as it is.

  Usually, a sequence analysis is facilitated by amplifying a specific site by PCR or the like using a primer capable of amplifying only the specific site. As a method for amplifying DNA, PCR method (Polymerase Chain Reaction), LCR method (Ligase Chain Reaction), SDA method (Strand Displacement Amplificon) and ICAN method (Isothermal and Chimeric Primer Amplification Primer) Can do. The PCR method is preferred because it is widely used.

  FIG. 1 will be specifically described with reference to the BARHL2 gene as an example. Genes can be amplified using forward and reverse primers, and amplification products can be detected using probes.

  As a primer set for measuring the frequency of cytosine methylation of the BARHL2 gene, (i) an oligonucleotide having a nucleic acid sequence of SEQ ID NO: 1 as a forward primer and having a length of 40 bases or less, and (ii) a sequence as a reverse primer Examples include combinations of oligonucleotides containing the nucleic acid sequence of No. 2 and having a length of 40 bases or less. The length of such oligonucleotide is preferably 35 bases or less, more preferably 30 bases or less, and even more preferably 25 bases or less. A specific example of the forward primer is an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 4. A specific example of the reverse primer is an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 5.

  As a probe for detecting the amplification product amplified by the above primer set, an oligonucleotide containing the nucleic acid sequence of SEQ ID NO: 3 and having a length of 30 bases or less can be mentioned. The length of the oligonucleotide is preferably 25 bases or less, and more preferably 20 bases or less. A specific example of the probe is an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 6.

  As a primer set for measuring the frequency of cytosine methylation of the SOX17 gene, a combination of an oligonucleotide consisting of a nucleic acid sequence of SEQ ID NO: 7 (forward primer) and an oligonucleotide consisting of a nucleic acid sequence of SEQ ID NO: 8 (reverse primer) is mentioned. Examples of the probe include an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 9.

  As a primer set for measuring the frequency of cytosine methylation of the MINT25 gene, a combination of an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 10 (forward primer) and an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 11 (reverse primer) is mentioned. Examples of the probe include an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 12.

  Hereinafter, the present invention will be described based on examples. However, the present invention is not limited to the following examples.

Example 1
Endoscopic mucosal resection (EMR) and endoscopic mucosal dissection (ESD) for patients with early gastric cancer using an Olympus endoscope (EVIS LUCERA series) and early gastric cancer was treated endoscopically. Before and after treatment, the patient's gastric mucosa lavage fluid was collected and used as a sample.

  Prior to endoscopic observation, a mucus dissolution remover (pronase (registered trademark) MS 20,000 units, sodium bicarbonate 1 g, and gascon (registered trademark) drop 4 mL (containing dimethicone 80 mg) was added to normal water 100 mL or more). By endoscopic observation, mucus dissolved in the mucus dissolution remover was sucked and removed by an endoscope. Thereafter, a gastric mucosa washing liquid (described in International Publication No. 2007/132844) obtained by washing the gastric mucosa with physiological saline (Otsuka Pharmaceutical) is used as an endoscope bottle (Forte Glow Medical Co., Ltd., medical device). (Notification number 09B1X0000400138). This gastric mucosa washing solution was divided into 50 mL centrifuge tubes, centrifuged at 1500 G for 10 minutes with a centrifuge, and after removing the supernatant, gastric mucosa cells contained in the gastric mucosa washing solution were obtained.

  The cells were resuspended in 4.5 mL SDS / EDTA / Tris solution. To this, 0.5 mL of 10% SDS and 50 mL of 20 mg / mL proteinase K (Takara Bio Inc., Code No. 9033) were added and incubated at 55 ° C. for 1 hour. 5 mL of phenol (UltraPure Buffer-Saturated Phenol, Invitrogen Life Technologies) was added, mixed by inversion, centrifuged at 2700 rpm, 4 ° C. for 15 minutes, and the supernatant was transferred to a new tube. This operation was further repeated once or twice, and the solvent was changed to the same amount of chloroform (Wako Pure Chemical Industries, Ltd.), and further repeated once or twice. Glycogen 5 mL (Ambion, Cat # 9510) and 100% ethanol 9 mL were added, and the mixture was inverted and incubated at 4 ° C. for 12 hours. Thereafter, the specimen is centrifuged at 2700 rpm for 15 minutes at 4 ° C. and the supernatant is discarded. The pellet is suspended in 10 mL of 70% ethanol, and then centrifuged at 2700 rpm and 4 ° C. for 15 minutes. It melt | dissolved in 200 mL and obtained the DNA analysis sample.

  Using 6 cases of the test set, candidate genes effective for early gastric cancer diagnosis were selected by the MCAM (Methylated CpG Island Amplification Microarray) method for the similarly processed DNA analysis samples. Thereafter, candidate gene methylation abnormalities were compared before and after ESD using the validation set 64 cases.

  The MCAM method enables selective collection of methylated DNA fragments from sample DNA in a gene-wide manner by performing treatment with a methylated cytosine sensitive restriction enzyme. By applying microarray technology to the amplified DNA, comprehensive methylation is enabled (Genome Res. 2007 Oct; 17 (10): 1529-36. Epub 2007 Sep 4, CSH Protoc. 2008). Mar 1; 2008: pdb.prot 4974. doi: 10.1101 / pdb.prot 4974.).

  The result is shown in FIG. FIG. 2 shows a value obtained by dividing the methylation frequency before ESD by the methylation frequency after ESD. Red indicates that the frequency of methylation is high before ESD surgery, and the frequency of methylation is reduced after gastric cancer resection by ESD surgery. (A) shows the results of all genes on the array, and (b) shows the results of selecting genes whose methylation frequency decreased after ESD. BARHL2, SOX17, PTPRN2, SLC2A9, PACAP, IFNGR2, CYP26C1, LRPC4, SOX9, has-mir-365-1 and has-nir-193b, showing high values, so gastric cancer is removed by ESD surgery This shows that the frequency of methylation of these genes has decreased. Among these genes, the BARHL2 gene was found to decrease the frequency of methylation without distinction between H. pylori-infected persons and H. pylori-infected persons.

(Example 2)
After bisulfite treatment was performed on the sample DNA, PCR was performed with a biotinylated primer targeting a portion of the CpG sequence present in the BARHL2 gene promoter region. After confirming the obtained PCR product by electrophoresis using an agarose gel, methylation frequency analysis was performed using a pyrosequencer. Measure the methylation frequency of the BARHL2 gene in various types of gastric cancer cells, synthesize the cDNA by reverse transcriptase using the mRNA of the BARHL2 gene, measure the amount of RNA, and specify the mRNA expression level of the GAPDH gene Turned into. As gastric cancer cells, MKN1, MKN7, MKN45, MKN74, NUGC3 and KatoIII were used. The results are shown in FIG. In cells with high methylation frequency (MKN7 and MKN45), it was found that the mRNA expression level of the BARHL2 gene was low.

Cells with high methylation frequency are cultured in a CO ₂ incubator, and the DNA demethylating agent 5-Aza-dC (5-aza-2′-deoxycytidine; 1 mM or 5 mM) is 24 hours later and 48 hours later. And 72 hours later, after 96 hours, the cells were recovered and RNA was extracted. After synthesizing cDNA, the mRNA expression level of the BARHL2 gene depending on the presence or absence of addition was examined. The results are shown in FIG. It was found that the mRNA expression level of the BARHL2 gene increases with the addition of the demethylating agent. From this, it was found that the expression of the BARHL2 gene was suppressed by methylation.

(Example 3)
Using gastric mucosal tissue fragments of gastritis, gastric adenoma and gastric cancer, nuclear staining and BARHL2 antibody staining were performed for comparison. DAPI staining (Boehringer Mannheim GmbH (Boehringer Mannheim Cat. No. 236-276) (blue), BARHL2 antibody (SIGMA-ALDRICH Anti-BARHL2 AV31981) staining (green), and an image (Merge) in which both are superimposed (Merge). Fig. 6 shows the results of gastritis and gastric adenoma, and Fig. 6 shows the results of gastric cancer, and Fig. 6 also shows the results of hematoxylin and eosin staining (HE). Thus, BARHL2 protein is present in gastric mucosa tissue fragments of gastritis and gastric adenoma, however, as is apparent from Fig. 6, BARHL2 protein is low in gastric cancer gastric mucosa tissue fragments. Therefore, in gastric cancer cells, the frequency of BARHL2 methylation No longer, thereby BARHL2 gene expression (transcription and translation) was found to be suppressed.

Example 4
For patients with early gastric cancer, the methylation frequency of each gene was measured before and after ESD treatment. The DNA analysis sample was subjected to bisulfite treatment, PCR (biotinylation) was performed using primers specific to the promoter region of the MINT25 gene, BRAHL2 gene or SOX17 gene, and the amplified biotinylated fragment was used for methylation analysis. By using a pyrosequencing method, a specific analysis program was set up in the portion of the CG sequence, and the frequency of methylation was measured (Qiagen, PyroMark Q24). The results are shown in FIG. (A) shows the results of the MINT gene, (b) shows the results of the BARHL2 gene, and (c) shows the results of the SOX17 gene. For all genes, the frequency of methylation after ESD surgery after gastric cancer resection was significantly reduced. From this, it is considered that a risk test for gastric cancer becomes possible by measuring the frequency of methylation of the BARHL2 gene. Furthermore, by considering the frequency of methylation of at least one of the SOX17 gene and the gene MINT25 gene in addition to the frequency of methylation of the BARHL2 gene, more accurate detection of early gastric cancer and risk assessment of gastric cancer development can be achieved. Conceivable.

Claims (6)

A method for obtaining an index for detecting gastric cancer or assessing the risk of developing gastric cancer,
Measuring at least one of the expression level of the BARHL2 gene and the cytosine methylation frequency in a sample collected from the subject,
Using at least one of the expression level of the BARHL2 gene and the frequency of cytosine methylation as an index for detection of gastric cancer or risk assessment of gastric cancer development,
Method. Measuring at least one of the expression level and cytosine methylation frequency of at least one of the SOX17 gene and the MINT25 gene in a sample collected from the subject,
At least one of the expression level and cytosine methylation frequency of the BARHL2 gene and at least one of the expression level and cytosine methylation frequency of at least one of the SOX17 gene and the MINT25 gene is used as an index for detection of gastric cancer or risk evaluation of gastric cancer development. ,
The method of claim 1.   The method according to claim 1 or 2, wherein the specimen is gastric mucosa lavage fluid, biopsy, gastric juice, blood or feces.   The method according to claim 3, wherein the specimen is a gastric mucosa washing solution. A kit for measuring the cytosine methylation frequency of the BARHL2 gene,
A primer set comprising a nucleic acid sequence of SEQ ID NO: 1 and having a length of 40 bases or less and a oligonucleotide set comprising a nucleic acid sequence of SEQ ID NO: 2 and having a length of 40 bases or less;
A probe comprising an oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 3 and having a length of 30 bases or less. The primer set consists of an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 4 and an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 5,
The probe consists of an oligonucleotide consisting of the nucleic acid sequence of SEQ ID NO: 6;
The kit according to claim 5.