JP6103866B2 - Colorectal cancer detection method, diagnostic kit and DNA chip - Google Patents

Description

  The present invention relates to a colorectal cancer detection or test method using a diagnostic (test or detection) polynucleotide useful for the test or detection of colorectal cancer, and a colorectal cancer diagnosis or detection kit using the polynucleotide.

  The large intestine begins with the cecum and consists of the ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. The large intestine is an important digestive organ that stores the remaining intestinal contents that have been digested and absorbed, making it stool while absorbing water. It also protects by excreting a large amount of intestinal bacteria.

  The death rate from cancer in Japan in 2009 was 273.5 out of 100,000 people, and the death rate of colorectal cancer tends to increase with the westernization of dietary habits. The death rate is 3rd for men and 1st for women after lung cancer and stomach cancer.

  Cancers that develop in the large intestine are broadly divided into adenocarcinoma, squamous cell carcinoma, and adenosquamous carcinoma, and most are adenocarcinoma, which originates from a colon polyp (polyp). Stem polyps grow into mushroom-like shapes, usually benign cancers, some of which progress to adenocarcinoma. In addition, it is now clear that flat and non-polyp lesions can lead to advanced colorectal cancer. Genetic factors are also known as risk factors for colorectal cancer, but generally include smoking, obesity, and alcohol consumption.

  Standard treatment methods for colorectal cancer are described in (Colon Cancer Study Group / Edition Colorectal Cancer Treatment Guidelines 2009, Kanehara Publishing Co., Ltd.). Endoscopic treatment, surgery, chemotherapy, radiation therapy, etc. It is given in consideration of the stage, the size and depth of cancer, and the degree of metastasis. In the case of early colorectal cancer, it can be completely removed by endoscopic resection or surgery, and the recurrence rate is very low. In advanced colorectal cancer, metastases that are difficult to remove may occur in the lungs, liver, lymph nodes, peritoneum, etc., and local recurrence may occur in the removed site. At these times, in addition to surgery, radiation therapy and chemotherapy (anticancer drug treatment) are performed.

  Early colorectal cancer (advanced cancer) and advanced colorectal cancer (advanced cancer) are defined by the depth of wall penetration. Early colorectal cancer refers to cancer in which the advanced part of the cancer is confined to the mucosa or submucosa of the colon wall and does not exceed this, and advanced colorectal cancer refers to cancer that extends beyond the submucosa and reaches deeper than the intrinsic muscle layer. These are defined in the 7th edition of the rules for handling colorectal cancer (Edited by Colorectal Cancer Study Group, Kanehara Publishing, 2006). Colorectal cancer can be classified in detail by Duke's stage. Duke's stage A is a state where cancer invasion is confined to the submucosa layer of the intestinal wall, Duke's stage B is a state where cancer invasion reaches the intrinsic muscle layer of the intestinal wall, Duke's stage C Is a state in which cancer invasion reaches the intrinsic muscle layer of the intestinal wall and further metastasis to the lymph node is observed, and Duke's stage D indicates a state in which the cancer has spread beyond the lymph node to other organs.

  When colorectal cancer is detected at a relatively early stage, the prognosis is relatively good, and the 5-year survival rate is 94.3% for Duke's stage A and 84.4% for stage B (Non-patent Document 1). ). However, treatment results for large cancers and cancers with metastases are inferior, and the importance of early detection is recognized.

  However, in most cases, it is asymptomatic at the stage of early colorectal cancer, and it is difficult to detect colorectal cancer at an early stage by subjective symptoms. Symptoms after cancer progresses include bloody stools, stool thinning (small stool column), residual stool, abdominal pain, symptoms related to defecation such as repeated diarrhea and constipation, anemia, and vomiting. The most common subjective symptom is bloody stool, but it is often confused with noncancerous diseases such as hemorrhoids. In addition, the ease of appearance of subjective symptoms of cancer varies depending on the site of cancer occurrence, the left colorectal cancer is a cancer existing in the descending colon, sigmoid colon, and rectum, the left colon has a thin lumen, and stool becomes solid As a result, the subjective symptoms of passing obstacles appear relatively early. On the other hand, right-sided colorectal cancer that occurs in the cecum, ascending colon, and transverse colon has a wide lumen and liquid stool that passes through, so it is difficult to detect early symptoms until cancer progresses and becomes large.

  Examples of colorectal cancer examination methods include digital rectal examination, fecal occult blood examination, ultrasound examination, CT examination, colonoscopy, and enema contrast examination. A typical test method performed in a large-scale mass screening is a fecal occult blood test (Non-patent Document 1). The fecal occult blood test is used to screen subjects who have a more accurate test, and colon cancer is a definitive diagnosis method for colorectal cancer performed on subjects who test positive. There are methods such as barium enema and image diagnosis, but all methods require the excretion of all stool, and in some cases, the examination is painful, and the physical and mental burden on the patient is great, and detailed examination There is a need to reduce false negatives and false positives in the previous screening stage.

  Therefore, in addition to or instead of the fecal occult blood test, it is strongly desired to discover a specific and highly sensitive cancer marker and perform a test using it. Marker detection methods for detecting colorectal cancer include, among colon cancer cells, the amount of gene expression, protein expression, cell metabolites, etc. in colon cancer cells derived from patients and non-cancer cells derived from patients at the time of surgery. By some means, or by measuring the amount of genes, proteins, metabolites, etc. contained in the body fluid of colorectal cancer patients and non-cancer patients, or from the large intestine contained in the stool of colorectal cancer patients and non-cancer patients A method for measuring the amount of genes, proteins, metabolites, etc. contained in the cells.

  So far, CEA (Carcinoembryonic antigen) and CA19-9 shown in Non-Patent Document 2 have been clinically applied as blood markers for colorectal cancer.

  Moreover, although it is in a research stage, the possibility of various markers is examined as shown in Patent Documents 1 to 8 and Non-Patent Documents 1 to 3, and stool contains colon cells detached from the intestine. There are also reports on the detection of a colon cancer cell-derived gene in stool as a colon cancer marker. In Patent Document 1, RNA is extracted from colon cancer tissue and colon cancer cells in stool, and the expression levels of REG1A gene (regenerating islet-derived 3 alpha), COL1A1 gene (collagen type 1 alpha 1), etc. are RT-PCR (reversed). It has been shown that it can be used as a marker using the expression of these genes as an index, measured with a photo-polymerase chain reaction) or a DNA chip.

  On the other hand, in Non-Patent Document 3, colon cancer cells are selected from antibodies from frozen stool, RNA is extracted, and the expression levels of SEPP1 gene (selenoprotein P, plasma, 1), RPL27A gene (ribosomal protein L27a) and the like are expressed. It has been shown to measure by RT-PCR.

  In Patent Document 2, RNA is extracted from frozen stool, and the expression levels of COX-2 gene (cyclooxygenase 2), SNAIL gene and MMP-7 gene (matrix metallopeptidase 7) are expressed by RT-PCR (reverse transcription-polymerase). It is shown to be measured by a chain reaction).

  Patent Document 3 discloses a plurality of genes such as a JUND gene (jun-D proto-oncogene) and a TPT1 gene (tumor protein, translationally-controlled 1) that are isolated from cells that survive from stool and are expressed in the cells. A method for detecting colorectal cancer cells by detecting colorectal cancer cells using as a guide the detected amount of is shown.

  Patent Document 4 discloses a method for detecting a colorectal cancer patient by combining a number of gene expression levels such as CEBPB gene (CCAAT / enhancer binding protein, beta) in blood.

  Patent Document 5 shows that the expression level of the FCGR3A gene (Fc fragment of IgG, low affinity IIIa, receptor) is increased in colon cancer cells than in normal colon epithelial cells.

  Patent Document 6 discloses a method for detecting a colorectal cancer patient by combining a number of gene expression levels such as a PFKFB3 gene (6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 3) in blood. ing.

  Patent Document 7 discloses a method for detecting colorectal cancer, lung cancer, and the like based on gene expression levels such as IL8 gene (interleukin 8) in blood and stool.

  Patent Document 8 reports the SOD2 gene (superoxide dismutase 2, mitochondrial, nuclear gene encoding mitochondrial protein) whose expression level increases in the cancerous part.

JP 2007-159491 A Japanese Patent No. 4798514 JP 2008-306976 A US Patent Application Publication No. 2010/0196889 Special table 2012-506253 gazette International Publication No. 2011/144718 Special table 2008-514234 gazette US Patent Application Publication No. 2008/0026385

Morikawa T et al., 2005, Gastroenterology, 129, p422-8. Locker GY et al., 2006, JOURNAL OF CLINICAL ONCOLOGY 33, p5313-27 Yajima S et al., 2007, International journal of oncology, 31, p1029-37.

  As described above, various novel colon cancer marker candidates have been discovered, but none of them has sufficient diagnostic performance and is actually widely clinically applied.

  The sensitivity of the fecal occult blood test shown in Non-Patent Document 1 is 52.8% for Duke's stage A, 70% for stage B, and 78.3% for stage C and D. There is a problem that false negative is high in colorectal cancer that is not accompanied. For example, stool is liquid in the right colorectal cancer, so it is difficult to bleed from the cancer area, or the antigenicity of hemoglobin decreases due to the long time it exists in the intestine until the stool with blood attached is discharged Can cause false negatives.

  CEA and CA19-9 shown in Non-Patent Document 2 cannot detect colon cancer specifically and early.

  In the method described in Patent Document 1, it is necessary to collect a large intestine tissue in order to measure the expression level of the REG1A gene, the COL1A1 gene, and the like, and there is a problem that the invasiveness is high.

  In the method described in Non-Patent Document 3, in order to measure the expression level of the SEPP1 gene, the RPL27A gene, etc., it is necessary to select colon cancer cells using antibodies from frozen stool. There are concerns about RNA degradation during operation due to RNase derived from intestinal bacteria in stool, their dead bodies, and digestive fluid, and fluctuations in gene expression levels due to complicated operations.

  In the method described in Patent Document 2, in order to measure the expression level of COX-2 gene, SNAIL gene and MMP-7 gene, stool is quickly frozen after RNA collection so that RNA degradation does not occur, and RNA extraction It is difficult to transport to the facility in a frozen state.

  In the method described in Patent Document 3, it is necessary to select colon cancer cells from the stool with an antibody in order to isolate the surviving cells from the stool and measure the expression level of JUN gene, TPT1 gene, etc. expressed in the cells. And Therefore, there are concerns about RNA degradation during operation by RNase derived from intestinal bacteria in the stool, their dead bodies, and digestive fluid, and fluctuations in gene expression levels due to complicated operations.

  In Patent Document 4, although many genes including CEBPB gene in blood-derived RNA are listed as measurement targets, there is no description of whether the gene is expressed in colon cancer cells in stool, and stool was used. No specific method for diagnosing colorectal cancer is described. In addition, a simpler and less invasive method of collecting a specimen than blood collection is desirable.

  Patent Document 5 does not describe whether the FCGR3A gene is expressed in colon cancer cells in stool, and does not describe a specific method for diagnosing colorectal cancer using stool. In addition, a simple and non-invasive method of collecting a sample is desirable rather than collecting large intestine tissue.

  In Patent Document 6, although many genes including PFKFB3 gene in blood-derived RNA are listed as measurement targets, there is no description of whether the gene is expressed in colon cancer cells in stool, and stool is used. No specific method for diagnosing colorectal cancer has been described. In addition, a simpler and less invasive method of collecting a specimen than blood collection is desirable.

  Patent Document 7 mentions stool as a sample for extracting RNA for measuring the expression level of IL8 gene or the like, but does not describe a specific method for diagnosing colorectal cancer using stool.

  Patent Document 8 describes that the expression level of the SOD2 gene increases in the cancerous part, but there is no description of whether the gene is expressed in colon cancer cells in the stool, and a specific example using stool is described. No method for diagnosing colorectal cancer is described.

  As described above, the above existing markers are poor in sensitivity and / or specificity, and a wasteful additional examination by misidentifying a non-colon cancer patient as a colorectal cancer patient or a treatment opportunity by overlooking a colorectal cancer patient May be lost. In addition, a method for efficiently detecting the RNA quality in the frozen stool has not been established, and the detection may be inaccurate. Moreover, it is not preferable to give pain by invading a patient at the time of collecting a large intestine tissue. Therefore, detection from stool that can be collected without causing pain to the patient is possible, and it is possible to correctly distinguish colorectal cancer patients from colorectal cancer patients and non-colorectal cancer patients from non-colorectal cancer patients. Desired. In particular, if colorectal cancer can be detected early, it can be completely removed by endoscopic resection or surgery, and the recurrence rate can be kept low, so a highly sensitive colorectal cancer marker is eagerly desired. .

  In recent years, expression gene amount analysis using a DNA array has been widely used as such a marker search technique. Probes using base sequences corresponding to hundreds to tens of thousands of genes are fixed to the DNA array. By adding the test sample to the DNA array, the gene in the sample binds to the probe, and the amount of gene in the test sample can be known by measuring the amount of binding by some means. The gene corresponding to the probe to be immobilized on the DNA array can be freely selected, and the amount of gene expressed in the sample using colon cancer cells and non-cancer cells from colon cancer patients at the time of surgery or endoscopy It is possible to estimate a gene group that can serve as a colon cancer marker. However, invasive collection of tissue at the time of surgery or endoscopy is disadvantageous for early diagnosis of cancer, and a simpler and noninvasive diagnostic method is required.

  The present invention uses an expression level of RNA directly extracted from stool that can be collected non-invasively as an index, and detects or tests colorectal cancer using a diagnostic (test or detection) polynucleotide useful for colorectal cancer testing or detection. It is an object of the present invention to provide a method and a kit for diagnosis or detection of colorectal cancer using the polynucleotide.

  In order to solve the above problems, the present inventors refrigerated the collected stool in a reagent that suppresses RNA degradation, and then extracted RNA from the stool to express gene expression in the stool of colorectal cancer patients. The gene expression in the stool of non-colon cancer patients is analyzed using a DNA chip, genes that can be used as detection markers for colon cancer are found, and the expression levels of those genes are compared with those of samples derived from non-colon cancer patients. As a result, it was found that the number of specimens derived from colon cancer patients decreased, decreased, increased or increased, and the present invention was completed.

1. SUMMARY OF THE INVENTION The present invention has the following features.
In the first aspect, the present invention provides in vitro expression levels of all five genes CEBPB, FCGR3A, PFKFB3, IL8, and SOD2, which are genes expressed in the stool of colorectal cancer patients, in the stool from the subject. A method for detecting colorectal cancer comprising measuring.

  In another embodiment, there is provided a colorectal cancer detection method as described above, which is performed using a nucleic acid probe for specifically detecting the expression of the gene.

  In another embodiment, the method for detecting colorectal cancer as described above, wherein the polynucleotides for detecting all expression levels of CEBPB, FCGR3A, PFKFB3, IL8 and SOD2 are the following (1) to (5 The detection method of colon cancer which is 5 types of polynucleotides.

(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences, A variant thereof, a derivative thereof, or a polynucleotide fragment thereof comprising 18 or more consecutive bases, (b) a nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which t is u in the nucleotide sequence, or these nucleotide sequences A polynucleotide comprising a complementary base sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) above under stringent conditions or a polynucleotide comprising 18 or more consecutive bases thereof Nucleotide fragment (2) (a) a nucleotide sequence represented by SEQ ID NO: 2 or a salt thereof for detecting the FCGR3A gene A polynucleotide comprising a nucleotide sequence wherein t is u in the sequence or a nucleotide sequence complementary to these nucleotide sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases, (b) SEQ ID NO: Or a polynucleotide comprising a base sequence represented by 2 or a base sequence in which t is u in the base sequence or a base sequence complementary to these base sequences, or (c) any one of (a) or (b) above (3) a nucleotide sequence represented by SEQ ID NO: 3 for detecting a PFKFB3 gene, (3) a polynucleotide fragment comprising a polynucleotide that hybridizes with a polynucleotide under stringent conditions, or a polynucleotide fragment thereof comprising 18 or more consecutive bases; A polynucleotide comprising a nucleotide sequence in which t is u in the nucleotide sequence or a nucleotide sequence complementary to these nucleotide sequences Otide, a variant thereof, a derivative thereof, or a polynucleotide fragment containing 18 or more consecutive bases thereof, (b) a base sequence represented by SEQ ID NO: 3, or a base sequence in which t is u in the base sequence, or these bases A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof (4) for detecting the IL8 gene, (a) from the base sequence represented by SEQ ID NO: 4 or the base sequence having t in the base sequence or a base sequence complementary to these base sequences A polynucleotide, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases thereof ( b) a nucleotide sequence represented by SEQ ID NO: 4, a nucleotide sequence in which t is u in the nucleotide sequence, or a polynucleotide comprising a nucleotide sequence complementary to these nucleotide sequences, or (c) (a) or (b) above (5) A polynucleotide fragment containing at least 18 consecutive bases or a polynucleotide fragment that hybridizes with any one of the polynucleotides under stringent conditions (5) (a) represented by SEQ ID NO: 5 Or a polynucleotide comprising a base sequence having t in the base sequence or a base sequence complementary to these base sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising 18 or more consecutive bases (B) the nucleotide sequence represented by SEQ ID NO: 5, or the nucleotide sequence in which t is u in the nucleotide sequence or a salt thereof A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof Polynucleotide fragment containing

  In another embodiment, the method for detecting colorectal cancer as described above, wherein the nucleic acid probe is five kinds of polynucleotides comprising the nucleotide sequences represented by SEQ ID NOs: 6 to 10. I will provide a.

  In another embodiment, the colorectal cancer detection method described above, wherein the CEBPB gene, the FCGR3A gene, the PFKFB3 gene, the IL8 gene, and the SOD2 gene each have a base sequence represented by SEQ ID NOs: 1 to 5. A method for detecting colon cancer is provided.

  In another embodiment, the method for detecting colorectal cancer as described above, wherein a sample sample derived from a colorectal cancer patient is obtained using a nucleic acid probe comprising the following five types of polynucleotides (1) to (5): A first step of measuring in vitro the expression level of a target gene in a plurality of stool known to be a specimen sample derived from a non-colon cancer patient, the target obtained in the first step A second step of creating a discriminant equation (SVM) using the measured value of the gene expression level as a teacher, and a step of measuring the expression level of the target gene in the stool derived from the subject in vitro as in the first step Substituting the measured value of the expression level of the target gene obtained in the third step into the discriminant obtained in the third step and the second step, and based on the result obtained from the discriminant, The target gene from colon cancer exists in the stool Or a fourth step of determining or evaluating whether or not a target gene derived from colorectal cancer is present.

(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences, A variant thereof, a derivative thereof, or a polynucleotide fragment thereof comprising 18 or more consecutive bases, (b) a nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which t is u in the nucleotide sequence, or these nucleotide sequences A polynucleotide comprising a complementary base sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) above under stringent conditions or a polynucleotide comprising 18 or more consecutive bases thereof Nucleotide fragment (2) (a) a nucleotide sequence represented by SEQ ID NO: 2 or a salt thereof for detecting the FCGR3A gene A polynucleotide comprising a nucleotide sequence wherein t is u in the sequence or a nucleotide sequence complementary to these nucleotide sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases, (b) SEQ ID NO: Or a polynucleotide comprising a base sequence represented by 2 or a base sequence in which t is u in the base sequence or a base sequence complementary to these base sequences, or (c) any one of (a) or (b) above (3) a nucleotide sequence represented by SEQ ID NO: 3 for detecting a PFKFB3 gene, (3) a polynucleotide fragment comprising a polynucleotide that hybridizes with a polynucleotide under stringent conditions, or a polynucleotide fragment thereof comprising 18 or more consecutive bases; A polynucleotide comprising a nucleotide sequence in which t is u in the nucleotide sequence or a nucleotide sequence complementary to these nucleotide sequences Otide, a variant thereof, a derivative thereof, or a polynucleotide fragment containing 18 or more consecutive bases thereof, (b) a base sequence represented by SEQ ID NO: 3, or a base sequence in which t is u in the base sequence, or these bases A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof (4) for detecting the IL8 gene, (a) from the base sequence represented by SEQ ID NO: 4 or the base sequence having t in the base sequence or a base sequence complementary to these base sequences A polynucleotide, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases thereof ( b) a nucleotide sequence represented by SEQ ID NO: 4, a nucleotide sequence in which t is u in the nucleotide sequence, or a polynucleotide comprising a nucleotide sequence complementary to these nucleotide sequences, or (c) (a) or (b) above (5) A polynucleotide fragment containing at least 18 consecutive bases or a polynucleotide fragment that hybridizes with any one of the polynucleotides under stringent conditions (5) (a) represented by SEQ ID NO: 5 Or a polynucleotide comprising a base sequence having t in the base sequence or a base sequence complementary to these base sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising 18 or more consecutive bases (B) the nucleotide sequence represented by SEQ ID NO: 5, or the nucleotide sequence in which t is u in the nucleotide sequence or a salt thereof A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof Polynucleotide fragment containing

  In addition, in an embodiment of the present invention, there is provided a method for detecting colorectal cancer, wherein the nucleic acid probe is five types of polynucleotides consisting of the base sequences represented by SEQ ID NOs: 6 to 10.

  In the second aspect, the present invention provides a kit for diagnosing colorectal cancer comprising a nucleic acid probe containing the following five polynucleotides (1) to (5).

(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences, A variant thereof, a derivative thereof, or a polynucleotide fragment thereof comprising 18 or more consecutive bases, (b) a nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which t is u in the nucleotide sequence, or these nucleotide sequences A polynucleotide comprising a complementary base sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) above under stringent conditions or a polynucleotide comprising 18 or more consecutive bases thereof Nucleotide fragment (2) (a) a nucleotide sequence represented by SEQ ID NO: 2 or a salt thereof for detecting the FCGR3A gene A polynucleotide comprising a nucleotide sequence wherein t is u in the sequence or a nucleotide sequence complementary to these nucleotide sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases, (b) SEQ ID NO: Or a polynucleotide comprising a base sequence represented by 2 or a base sequence in which t is u in the base sequence or a base sequence complementary to these base sequences, or (c) any one of (a) or (b) above (3) a nucleotide sequence represented by SEQ ID NO: 3 for detecting a PFKFB3 gene, (3) a polynucleotide fragment comprising a polynucleotide that hybridizes with a polynucleotide under stringent conditions, or a polynucleotide fragment thereof comprising 18 or more consecutive bases; A polynucleotide comprising a nucleotide sequence in which t is u in the nucleotide sequence or a nucleotide sequence complementary to these nucleotide sequences Otide, a variant thereof, a derivative thereof, or a polynucleotide fragment containing 18 or more consecutive bases thereof, (b) a base sequence represented by SEQ ID NO: 3, or a base sequence in which t is u in the base sequence, or these bases A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof (4) for detecting the IL8 gene, (a) from the base sequence represented by SEQ ID NO: 4 or the base sequence having t in the base sequence or a base sequence complementary to these base sequences A polynucleotide, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases thereof ( b) a nucleotide sequence represented by SEQ ID NO: 4, a nucleotide sequence in which t is u in the nucleotide sequence, or a polynucleotide comprising a nucleotide sequence complementary to these nucleotide sequences, or (c) (a) or (b) above (5) A polynucleotide fragment containing at least 18 consecutive bases or a polynucleotide fragment that hybridizes with any one of the polynucleotides under stringent conditions (5) (a) represented by SEQ ID NO: 5 Or a polynucleotide comprising a base sequence having t in the base sequence or a base sequence complementary to these base sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising 18 or more consecutive bases (B) the nucleotide sequence represented by SEQ ID NO: 5, or the nucleotide sequence in which t is u in the nucleotide sequence or a salt thereof A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof Polynucleotide fragment containing

  Moreover, in this embodiment of the present invention, a kit for diagnosing colorectal cancer is provided, wherein the nucleic acid probe is five types of polynucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 6 to 10.

  In the third aspect, the present invention provides a DNA chip for colorectal cancer diagnosis comprising a nucleic acid probe comprising the following five types of polynucleotides (1) to (5).

(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences, A variant thereof, a derivative thereof, or a polynucleotide fragment thereof comprising 18 or more consecutive bases, (b) a nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which t is u in the nucleotide sequence, or these nucleotide sequences A polynucleotide comprising a complementary base sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) above under stringent conditions or a polynucleotide comprising 18 or more consecutive bases thereof Nucleotide fragment (2) (a) a nucleotide sequence represented by SEQ ID NO: 2 or a salt thereof for detecting the FCGR3A gene A polynucleotide comprising a nucleotide sequence wherein t is u in the sequence or a nucleotide sequence complementary to these nucleotide sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases, (b) SEQ ID NO: Or a polynucleotide comprising a base sequence represented by 2 or a base sequence in which t is u in the base sequence or a base sequence complementary to these base sequences, or (c) any one of (a) or (b) above (3) a nucleotide sequence represented by SEQ ID NO: 3 for detecting a PFKFB3 gene, (3) a polynucleotide fragment comprising a polynucleotide that hybridizes with a polynucleotide under stringent conditions, or a polynucleotide fragment thereof comprising 18 or more consecutive bases; A polynucleotide comprising a nucleotide sequence in which t is u in the nucleotide sequence or a nucleotide sequence complementary to these nucleotide sequences Otide, a variant thereof, a derivative thereof, or a polynucleotide fragment containing 18 or more consecutive bases thereof, (b) a base sequence represented by SEQ ID NO: 3, or a base sequence in which t is u in the base sequence, or these bases A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof (4) for detecting the IL8 gene, (a) from the base sequence represented by SEQ ID NO: 4 or the base sequence having t in the base sequence or a base sequence complementary to these base sequences A polynucleotide, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising the 18 or more consecutive bases thereof ( b) a nucleotide sequence represented by SEQ ID NO: 4, a nucleotide sequence in which t is u in the nucleotide sequence, or a polynucleotide comprising a nucleotide sequence complementary to these nucleotide sequences, or (c) (a) or (b) above (5) A polynucleotide fragment containing at least 18 consecutive bases or a polynucleotide fragment that hybridizes with any one of the polynucleotides under stringent conditions (5) (a) represented by SEQ ID NO: 5 Or a polynucleotide comprising a base sequence having t in the base sequence or a base sequence complementary to these base sequences, a variant thereof, a derivative thereof, or a polynucleotide fragment comprising 18 or more consecutive bases (B) the nucleotide sequence represented by SEQ ID NO: 5, or the nucleotide sequence in which t is u in the nucleotide sequence or a salt thereof A polynucleotide comprising a base sequence complementary to the sequence, or (c) a polynucleotide that hybridizes with the polynucleotide of any one of (a) or (b) under stringent conditions or 18 or more consecutive bases thereof Polynucleotide fragment containing

  In addition, in an embodiment of the present invention, there is provided a DNA chip for colorectal cancer diagnosis, wherein the nucleic acid probe is 5 types of polynucleotides consisting of the base sequences represented by SEQ ID NOs: 6 to 10.

2. Definitions Terms used herein have the following definitions.
Indications using abbreviations such as nucleotides, polynucleotides, DNA, RNA, etc. shall be in accordance with “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office) and customary in this technical field. .

  In the present specification, “polynucleotide” is used for nucleic acids including both RNA and DNA. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, aRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. In the present specification, the polynucleotide is used interchangeably with the nucleic acid.

  In this specification, “gene” includes not only RNA and double-stranded DNA, but also each single-stranded DNA such as positive strand (or sense strand) or complementary strand (or antisense strand) constituting the same. It is intended to be used. The length is not particularly limited.

  Accordingly, in the present specification, unless otherwise specified, “gene” refers to double-stranded DNA containing human genomic DNA, single-stranded DNA containing cDNA (positive strand), and single-stranded DNA having a sequence complementary to the positive strand. All of DNA (complementary strand) and fragments thereof, and human genome are included. The “gene” is not limited to a “gene” represented by a specific base sequence (or SEQ ID NO), but is a protein having a biological function equivalent to a protein encoded by these, for example, a homolog (ie, homolog). , Variants, such as splice variants, and “genes” encoding derivatives are included. Specifically, the “gene” encoding the homologue, variant or derivative is, for example, any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 5 under the stringent conditions described later, Examples thereof include “genes” having a base sequence that hybridizes with a complementary sequence of a base sequence in which t is u in the base sequence. For example, as a homologue of a human-derived protein (that is, a homolog) or a gene encoding the same, a protein or gene of another species corresponding to the protein or a human gene encoding the protein can be exemplified, and these proteins or genes Homologs can be identified by HomoLoGene (http://www.ncbi.nlm.nih.gov/homoloGene/). Specifically, a specific human amino acid or nucleotide sequence can be determined using the BLAST program (Stephen F et al., Nucleic Acids Res., 1997, 17, p3389-3402, http://blast.ncbi.nlm.nih.gov/Blast. cgi), the registration number (accession number) of the sequence can be obtained (Score is the highest, E-value is 0, and Identify is 100%). Known BLAST programs include BLASTN (gene) and BLASTX (protein). For example, in the case of gene search, the registration number from the BLAST search is input to HomoLomoGene. It is possible to select a gene of another species as a gene homolog corresponding to the human gene indicated by a specific base sequence from the list showing the correlation of the gene homologue between the other species gene and the human gene obtained as a result. it can. In this method, a FASTA program (http://www.genome.jp/tools/fasta/) may be used instead of the BLAST program. The “gene” does not ask whether the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron. In addition, the “gene” may be contained in the cell, may be released outside the cell and may exist alone, or may be encapsulated in a vesicle encapsulated in a lipid bilayer called an exosome. There may be.

  Exosomes are small vesicles secreted from cells. Exosomes are derived from multivesicular endosomes, and are fused with cell membranes to release internal vesicles to the extracellular environment. At that time, vesicles may contain “genes” such as RNA and DNA. It is known that exosomes are contained in body fluids such as blood, serum, plasma, stool and lymph.

  In the present specification, the “transcript” refers to RNA synthesized using a DNA sequence of a gene as a template. RNA is synthesized in such a manner that RNA polymerase binds to a site called a promoter located upstream of the gene and ribonucleotides are bound to the 3 'end so as to be complementary to the DNA base sequence. This RNA includes not only the gene itself but also the entire sequence from the transcription start point to the end of the poly A sequence, including the expression control region, coding region, exon or intron.

  In the present specification, the “probe” includes a polynucleotide used for specifically detecting RNA produced by gene expression or a polynucleotide derived therefrom and / or a polynucleotide complementary thereto.

  In the present specification, the “primer” includes a continuous polynucleotide and / or a complementary polynucleotide that specifically recognizes and amplifies RNA generated by gene expression or a polynucleotide derived therefrom.

  Here, a complementary polynucleotide (complementary strand, reverse strand) is a full-length sequence of a polynucleotide comprising a base sequence defined by a sequence number, or a base sequence in which t is u in the base sequence, or a portion thereof It means a polynucleotide having a base-complementary relationship based on a base pair relationship such as A: T (U), G: C with respect to a sequence (for convenience sake, this is referred to as a positive strand). However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows hybridization with the target normal strand under stringent conditions. There may be.

  As used herein, “stringent conditions” refers to conditions under which a probe hybridizes to its target sequence to a detectably greater extent (eg, at least twice background) than to other sequences. Say. Stringent conditions are sequence-dependent and depend on the environment in which hybridization is performed. By controlling the stringency of the hybridization and / or wash conditions, target sequences that are 100% complementary to the probe can be identified. Examples of stringent hybridization conditions are not limited to the following conditions as described later, but include, for example, 30 ° C. to 60 ° C., preferably 3-4 × SSC, 0.1-0.5% SDS. Hybridization in solution, for example, a solution containing 0.5 × SSC and 0.1% SDS at 30 ° C., and a solution containing 0.2 × SSC and 0.1% SDS at 30 ° C., and 30 Conditions such as continuous washing with 0.05 × SSC solution at 0 ° C. can be mentioned. Here, 1 × SSC is an aqueous solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate, and the surfactant contains SDS, Triton, Tween, or the like.

  In the present specification, the term “variant” refers to a natural variant caused by polymorphism, mutation, or the like, or the nucleotide sequence represented by SEQ ID NOs: 1 to 5, or t in the nucleotide sequence. a variant comprising a deletion, substitution, addition or insertion of 1, 2 or 3 or more, preferably 1 or 2 bases in the base sequence which is u, or a partial sequence thereof, or SEQ ID NOs: 1 to 5 (however, , T is u.) In the base sequence of the precursor RNA of mRNA, or the base sequence in which u is t in the base sequence, or a partial sequence thereof, 1 or 2 or more, preferably 1 or several bases About 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about about 80% or more, about 85% or more, about 90% or more, about 95% or more, about a variant containing deletion, substitution, addition or insertion; 98% or more, approx. Mutant shows a more than 9% percent identity, or refers to a polynucleotide or a nucleic acid which hybridises under stringent conditions of oligonucleotides as previously defined comprising a nucleotide sequence or subsequence thereof.

  As used herein, “several” means an integer of about 10, 9, 8, 7, 6, 5, 4, 3, or 2.

  In the present specification, a mutant can be produced using a well-known technique such as site-directed mutagenesis or PCR-based mutagenesis.

  As used herein, “% identity” can be determined using a BLAST or FASTA protein or gene search system with or without a gap (Zheng Zhang et al., 2000). J Comput Biol, 7, p203-214; Altschul, SF et al., 1990, Journal of Molecular Biology, 215, p403-410; Pearson, WR et al., 1988, Proceedings of the. National Academic Sciences USA, Vol. 85, p2444-2, 448).

  As used herein, the term “derivative” refers to a modified nucleic acid, a non-limiting group such as a labeled derivative such as a fluorophore, a modified nucleotide (for example, a halogen, an alkyl such as methyl, an alkoxy such as methoxy, a group such as thio, carboxymethyl, etc. A derivative containing PNA (peptide nucleotide acid; Nielsen, PE, etc.). 1991, Science, 254, p1497-500), LNA (locked nucleic acid; Obika, S. et al., 1998, Tetrahedron Lett., 39, p5401-5404) and the like.

  In the present specification, “diagnostic (or detection or test) polynucleotide” refers to the presence or absence of colorectal cancer, the degree of morbidity, the presence or absence of improvement, and the degree of improvement, and prevention and improvement of colorectal cancer. Or what is used directly or indirectly in order to screen the candidate substance useful for a treatment. This includes nucleotides, oligonucleotides and polynucleotides that can specifically recognize and bind to genes whose expression varies in vivo in relation to the onset of colorectal cancer, particularly in the stool of colorectal tissues and colon cancer patients. Is included. These nucleotides, oligonucleotides and polynucleotides are used as probes for detecting the gene expressed in vivo, in tissues or cells based on the above properties, and for amplifying the gene expressed in vivo. It can be effectively used as a primer.

  In the present specification, the “specimen sample” to be detected and diagnosed refers to a biological tissue in which the gene of the present invention changes in expression with the occurrence of colorectal cancer, a substance derived from the biological tissue, a substance that comes into contact with the biological tissue, Specifically, it refers to colon tissue, colon epithelial cells, stool, and the like.

  As used herein, “stool occult blood test” means a method for colorectal cancer diagnosis in which hemoglobin in stool is detected and bleeding from the luminal surface of the large intestine is evaluated. Specifically, it is an immunological method using an antibody that specifically detects human hemoglobin in stool.

In this specification, “colon endoscopy” refers to the number, size, location and type of cancer in the large intestine (surface characteristics, characteristics of the cancer base). Means a method to evaluate and make a definitive diagnosis of colorectal cancer.
In this specification, “accuracy” is the sum of “the number of times that a colorectal cancer patient is correctly identified as a colorectal cancer patient” and “the number of times that a non-colorectal cancer patient is correctly determined as a non-colorectal cancer patient” / (total number of cases) Means. The accuracy indicates the rate at which the discrimination results for all the samples are correct, and is a first index for evaluating the diagnostic performance.

  In this specification, “sensitivity” means (the number of times that a colorectal cancer patient is correctly distinguished from a colorectal cancer patient) / (“number of times that a colorectal cancer patient is correctly distinguished from a colorectal cancer patient”) and “the number of colorectal cancer patients as a non-colorectal cancer patient. This is the sum of the number of times of erroneous determination. High sensitivity makes it possible to detect colorectal cancer at an early stage, leading to complete removal of the cancerous part and a reduction in the recurrence rate.

  In the present specification, “specificity” is (number of times a non-colon cancer patient is correctly identified as a non-colon cancer patient) / (“number of times a non-colon cancer patient is mistakenly identified as a colorectal cancer patient”) and “non-colon cancer patient. Is the sum of the number of times correctly identified as a non-colon cancer patient. If the specificity is high, it is possible to prevent a wasteful additional examination by misidentifying a non-colon cancer patient as a colorectal cancer patient, thereby reducing the burden on the patient and medical costs.

  As used herein, the term “CEBPB gene” or “CEBPB” means a CCAAT / enhancer-binding protein beta gene (DNA) represented by a specific nucleotide sequence (SEQ ID NO: 1), C, unless specified by a SEQ ID NO. / EBP-beta, CRP2, IL6DBP, LAP, NF-IL6, TCF5, homologues, variants and derivatives thereof (DNA) are included. Specifically, the CEBPB gene (GenBank Accession No. NM_005194) described in SEQ ID NO: 1 and other species homologues are included. The CEBPB gene is described in Tsushima, H et al., 2012, Ann. Rheum. Dis. 71, p99-107.

  As used herein, the term “FCGR3A gene” or “FCGR3A” refers to the Low affinity immunoglobulin gamma Fc region receptor III-A Precursor gene represented by a specific base sequence (SEQ ID NO: 2) unless otherwise specified by a SEQ ID NO: DNA), CD16, CD16A, FCG3, FCGR3, FCGRIII, FCR-10, FCRIII, FCRIIIA, IGFR3, homologues, variants and derivatives thereof, and the like (DNA). Specifically, the FCGR3A gene (GenBank Accession No. NM_000569) described in SEQ ID NO: 2 and other species homologues are included. The FCGR3A gene has been described by Nambodiri, A .; M.M. Et al., 2011, Clin. Exp. Immunol. 166, p361-365.

  As used herein, the term “PFKFB3 gene” or “PFKFB3” is a 6-phosphofructo-2-kinase / fructose-2,6 represented by a specific base sequence (SEQ ID NO: 3) unless otherwise specified by a SEQ ID NO. -The gene (DNA) which codes biphosphatase 3 gene (DNA), IPFK2, PFK2, its homologue, a variant, a derivative, etc. is included. Specifically, the PFKFB3 gene (GenBank Accession No. NM — 004566) described in SEQ ID NO: 3 and other species homologues are included. Columbo, S .; L et al., 2011, Proc. Natl. Acad. Sci. 108, p21069-21074.

  As used herein, the term “IL8 gene” or “IL8” means the IL8 gene (DNA), Interleukin-8 Precursor gene (DNA) represented by the specific base sequence (SEQ ID NO: 4), unless otherwise specified by the SEQ ID NO: ), CXCL8, GCP-1, GCP1, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF, NAP-1, NAP1, homologues, mutants and derivatives thereof (DNA). Specifically, the IL8 gene (GenBank Accession No. NM_000584) described in SEQ ID NO: 4 and other species homologues are included. Santos, J.M. C. Et al., 2012, Clin. Exp. Immunol. 167, p129-136.

  As used herein, the term “SOD2 gene” or “SOD2” means a superoxide dismutase [Mn], a mitochondrial precursor gene (DNA) represented by a specific base sequence (SEQ ID NO: 5), unless specified by a SEQ ID NO: It includes genes (DNA) encoding IPOB, MNSOD, MVCD6, homologues, mutants and derivatives thereof. Specifically, the SOD2 gene (GenBank Accession No. NM_001024466, NM_001024465, NM_000636) described in SEQ ID NO: 5 and other species homologues are included. Black, J. et al. L. Et al., 2012, 36, p59-66.

  As used herein, “P” or “P value” indicates the probability that a statistical quantity assumes a value that is contrary to the assumption in the distribution assumed by the statistical test. Therefore, it is assumed that the smaller the “P” or “P value”, the closer the assumption is to true.

  The present invention uses, as an index, the expression level of RNA directly extracted from stool that can be collected non-invasively, and a polynucleotide for disease diagnosis useful for diagnosis or treatment of colorectal cancer, and high accuracy of colorectal cancer using the polynucleotide. In addition, a highly sensitive detection or inspection method is provided, and the present invention enables quick and simple diagnosis or inspection. In addition, the present invention provides the advantage that early cancer of Duke's stage A can be detected with high sensitivity and high accuracy, and right colorectal cancer can be detected with high sensitivity compared to fecal occult blood test. To do.

The flow of analysis for determining the genes described in Table 1 is shown.

The present invention will be described more specifically below. Target gene for colorectal cancer Target gene as a colorectal cancer marker for determining or evaluating the presence and / or absence of colorectal cancer or colorectal cancer cells using the polynucleotide and kit for colorectal cancer diagnosis as defined above of the present invention Includes, for example, human genes containing the nucleotide sequences represented by SEQ ID NOs: 1 to 5 (ie, CEBPB, FCGR3A, PFKFB3, IL8, SOD2, respectively), homologues thereof, transcripts thereof, or mutations thereof Body or derivative. Here, genes, homologues, transcripts, mutants and derivatives are as defined above. A preferred target gene is a human gene containing the nucleotide sequence represented by SEQ ID NOs: 1 to 5, a transcription product thereof, more preferably the transcription product, ie, mRNA, its precursor mRNA.

  In the present invention, any of the above genes serving as indicators of colorectal cancer has an increased expression level in subjects suffering from colorectal cancer as compared to healthy subjects (see Table 1 in Examples below).

  The first target gene is a CEBPB gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. There has been known a report that changes in the expression of the CEBPB gene or its transcription product can serve as a marker for colorectal cancer (Patent Document 3 above).

  The second target gene is the FCGR3A gene, their homologs, their transcripts, or their mutants or derivatives. It has been known that the gene expression level of the FCGR3A gene or a transcription product thereof is increased in a colon cancer tissue or a colon cancer cell line (Patent Document 4 above), but the FCGR3A8 gene or its transcription product in stool is increased. There are no known reports that increased expression can be a marker for colon cancer.

  The third target gene is the PFKFB3 gene, their homologs, their transcripts, or their mutants or derivatives. So far, it has been reported that the expression level of PFKFB3 gene or its transcription product in blood can be a marker for colorectal cancer (Patent Document 5 above), but the expression of PFKFB3 gene or its transcription product in stool is known. There are no known reports that an increase in can be a marker for colorectal cancer.

  The fourth target gene is the IL8 gene, their homologs, their transcripts, or their variants or derivatives. Until now, it has been known that the expression level of IL8 gene or its transcript in the stool is measured and used as a marker for colorectal cancer (Patent Document 6 above).

  The fifth target gene is the SOD2 gene, their homologs, their transcripts, or their mutants or derivatives. So far, it has been reported that the expression level of SOD2 gene or its transcription product is increased in the cancerous part (Patent Document 7 above), but the increase in the expression of SOD2 gene or its transcription product in stool is a colon cancer. There is no known report that it can be a marker for.

2. Polynucleotide for Diagnosing Colorectal Cancer 2.1 Nucleic Acid In the present invention, a polynucleotide that can be used for detecting or examining colorectal cancer or for diagnosing colorectal cancer is derived from a human as a target gene for colorectal cancer. Allows qualitative and / or quantitative determination of the presence, expression level or abundance of CEBPB, FCGR3A, PFKFB3, IL8, SOD2, their homologues, their transcripts or their variants or derivatives .

  The expression level of the target gene is increased in subjects suffering from colon cancer compared to healthy subjects. Therefore, the diagnostic polynucleotide of the present invention can be used effectively for measuring the expression level of a target gene in colorectal cancer tissue and normal colorectal tissue and comparing them.

  A diagnostic polynucleotide that can be used in the present invention includes a nucleotide sequence represented by SEQ ID NOs: 1 to 5 in a living tissue of a patient suffering from colorectal cancer, or a nucleotide sequence in which t is u in the nucleotide sequence. A nucleotide group and a complementary polynucleotide group thereof, a polynucleotide group that hybridizes with DNA comprising a base sequence complementary to the base sequence under stringent conditions, a complementary polynucleotide group thereof, and a polynucleotide group thereof. A combination of one or more polynucleotides selected from a group of polynucleotides containing 18 or more, 20 or more, 25 or more, preferably 30 or more, 40 or more, 50 or more, more preferably 60 or more consecutive bases in the base sequence Including. These polynucleotides can be used as probes and primers for detecting the colon cancer marker, which is a target gene.

Specifically, the diagnostic polynucleotide of the present invention can include the following one or more polynucleotides, variants thereof, derivatives thereof, or fragments thereof.
(1) A polynucleotide group consisting of the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences, variants thereof, derivatives thereof, or Those fragments containing 18 or more consecutive bases.
(2) A polynucleotide group comprising the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences.
(3) a polynucleotide group consisting of the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences, variants thereof, or 18 or more consecutive sequences Those fragments containing the bases.
(4) A polynucleotide group comprising the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences.
(5) A polynucleotide group consisting of a base sequence complementary to the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences, and variants thereof , Derivatives thereof, or fragments thereof containing 18 or more consecutive bases.
(6) A polynucleotide group comprising a base sequence complementary to the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences.
(7) A polynucleotide group that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to each of the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or 18 or more consecutive groups Those fragments containing bases.
(8) A polynucleotide group that hybridizes under stringent conditions with a DNA comprising each of the base sequences in which t is u in the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or 18 or more Those fragments containing consecutive bases.

  The polynucleotide fragments of the above (1) to (8) are, for example, in the base sequence of each polynucleotide, for example, the total number of bases in the range of 18 to 18 bases, 18 to 30 bases, 18 to 60 bases, etc. However, it is not limited to these.

  Any of the polynucleotides or fragments thereof used in the present invention may be DNA or RNA.

  The diagnostic polynucleotide of the present invention can be prepared using general techniques such as a DNA recombination technique, a PCR method, a site-directed mutagenesis method, and a method using a DNA / RNA automatic synthesizer.

  Techniques such as DNA recombination techniques, site-directed mutagenesis, and PCR techniques are described in, for example, Ausubel et al., Current Protocols in Molecular Biology, John Willy & Sons, US (1993); Sambrook et al. Techniques such as those described in Spring Harbor Laboratory Press, US (1989) can be used.

  Human-derived CEBPB, FCGR3A, PFKFB3, IL8, and SOD2 are known, and the method for obtaining them is also known as described above. Therefore, the diagnostic polynucleotide of the present invention can be produced by cloning these genes.

  The diagnostic polynucleotide of the present invention can be chemically synthesized using an automatic DNA synthesizer. In general, the phosphoramidite method is used for this synthesis, and single-stranded DNA of up to about 100 bases can be automatically synthesized by this method. Automatic DNA synthesizers are commercially available from, for example, Polygen, ABI, and Applied BioSystems.

  Alternatively, the polynucleotide of the present invention can be prepared by a cDNA cloning method. For example, microRNA Cloning Kit Wako can be used as the cDNA cloning technique.

3. Colon cancer diagnosis kit (nucleic acid)
The present invention also provides a colorectal cancer diagnosis (detection) kit comprising one or more of the diagnostic polynucleotide of the present invention, a variant thereof, a derivative thereof, and / or a fragment thereof. Here, variants and derivatives include those defined above.

  The kit of the present invention preferably contains one or a plurality of polynucleotides selected from the polynucleotides described in 2.1 above or a fragment thereof.

  The kit of the present invention comprises a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1 to 5, or a nucleotide sequence wherein t is u in the nucleotide sequence, a polynucleotide comprising the complementary sequence thereof, the polynucleotide and One or more, preferably a plurality, of polynucleotides that hybridize under stringent conditions, or fragments of the polynucleotides, variants, or derivatives thereof can be included.

The polynucleotide fragment that can be included in the kit of the present invention is, for example, one or more DNAs selected from the group consisting of the following (1) to (3):
(1) A DNA comprising 18 or more consecutive bases in a base sequence represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10 wherein t is u or a complementary sequence thereof.
(2) A DNA comprising 60 or more consecutive bases in a base sequence represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10 wherein t is u or a complementary sequence thereof.
(3) DNA containing 60 or more consecutive bases in each of the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10 in which t is u or a complementary sequence thereof.

  In a preferred embodiment, the polynucleotide comprises a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a nucleotide sequence in which t is u in the nucleotide sequence, Polynucleotides consisting of complementary sequences, polynucleotides that hybridize with these polynucleotides under stringent conditions, or those 18 bases to the total number of bases or 20 bases to the total number of bases, preferably 25 to 1000 Examples having a base length of 25 to 100 bases, such as 30 to 100 bases, 40 to 100 bases, 50 to 100 bases, more preferably 60 to 100 bases, are not limited to this range. .

  In a preferred embodiment, the fragment is a polynucleotide comprising 18 or more, 20 or more, 25 or more, preferably 30 or more, 40 or more, 50 or more, more preferably 60 or more, such as 60 to 100 consecutive bases. be able to.

  In another preferred embodiment, the fragment is a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 6 to 10.

  In another preferred embodiment, the fragment is a polynucleotide comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 6 to 10.

  In another preferred embodiment, the fragment is a polynucleotide consisting of the base sequence represented by any of SEQ ID NOs: 6 to 10.

  Examples of the above combinations are the nucleotide sequences shown in SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or the nucleotide sequences in which t is u in the nucleotide sequences, or polynucleotides comprising the complementary sequences thereof, their polynucleotides And polynucleotides that hybridize under stringent conditions. When the detection accuracy (%) of colorectal cancer when 5 kinds of genes are combined in this manner is plotted against the number of genes necessary for colorectal cancer detection, the probability is, for example, CEBPB (SEQ ID NO: 1), FCGR3A The combination of (SEQ ID NO: 2) and PFKFB3 (SEQ ID NO: 3) is 68.4%, and when all five genes of IL8 (SEQ ID NO: 4) and SOD2 (SEQ ID NO: 5) are combined, the accuracy is 84. 2% (Table 2).

  In the present invention, the size of the polynucleotide fragment is, for example, the total number of bases in 18 to 18 bases, 18 to 30 bases or more, 18 to 40 bases or more, 18 to 18 in the base sequence of each polynucleotide. 60 bases or more, 18 to 80 bases or more, 18 to 100 bases or more, 20 to 20 bases or more, 20 to 30 bases or more, 20 to 40 bases or more, 20 to 60 bases Or more, 20 to 80 bases or more, 20 to 100 bases or more, 25 to the total number of bases in the sequence, 25 to 30 bases or more, 25 to 40 bases or more, 25 to 60 bases or more Above, ranges of 25-80 bases or more, 25-100 bases or more, etc. Is a number of bases, and shall not be limited thereto.

  The above combinations constituting the kit of the present invention are merely examples, and all other various possible combinations are included in the present invention.

  In addition to the above-described polynucleotide, variant or fragment thereof of the present invention, the kit of the present invention can also include a known or future-found polynucleotide that enables colorectal cancer diagnosis.

  The polynucleotides, variants or fragments thereof contained in the kit of the present invention are packaged individually or in any combination in different containers.

4). DNA chip The present invention is further described in the same polynucleotide as that contained in the diagnostic polynucleotide and / or kit of the present invention (or the diagnostic polynucleotide described in Section 2.1 and / or the kit described in Section 3 above). A DNA chip for diagnosing colorectal cancer comprising a polynucleotide), a variant, a derivative, a fragment, and a combination thereof.

  The substrate of the DNA chip is not particularly limited as long as DNA can be solid-phased, and examples thereof include a slide glass, a silicon chip, a polymer chip, and a nylon membrane. These substrates may be subjected to surface treatment such as poly L-lysine coating, introduction of functional groups such as amino groups and carboxyl groups.

  The solid phase immobilization method is not particularly limited as long as it is a commonly used method, such as a method of spotting DNA using a high-density dispenser called a spotter or an arrayer, a fine droplet from a nozzle, a piezoelectric element, etc. Examples include a method of spraying DNA onto a substrate using an apparatus (inkjet) ejected by the above, or a method of sequentially synthesizing nucleotides on a substrate. When using a high-density dispenser, for example, put different gene solutions in each well of a plate with a large number of wells, pick up this solution with a pin (needle), and spot it on the substrate in order. by. In the ink jet method, genes are ejected from a nozzle, and the genes are arranged and arranged at high speed on a substrate. For DNA synthesis on a substrate, the base bonded to the substrate is protected with a functional group that can be removed by light or heat, and the functional group is removed by applying light or heat only to the base at a specific site by using a mask. Let Thereafter, the step of adding a base to the reaction solution and coupling with the base on the substrate is repeated.

  The polynucleotide to be immobilized is all the polynucleotides of the present invention described above.

For example, such polynucleotides can include one or more of the following polynucleotides or fragments thereof.
(1) a polynucleotide group consisting of the nucleotide sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a nucleotide sequence in which t is u in the nucleotide sequences, variants thereof, derivatives thereof, or Those fragments containing 18 or more consecutive bases.
(2) A polynucleotide group comprising the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences.
(3) a polynucleotide group comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a nucleotide sequence in which t is u in the nucleotide sequences, variants thereof, derivatives thereof, or Those fragments containing 18 or more consecutive bases.
(4) a polynucleotide group comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a nucleotide sequence in which t is u in the nucleotide sequences, variants thereof, derivatives thereof, or Those fragments containing 18 or more consecutive bases.
(5) A polynucleotide group consisting of a base sequence complementary to the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences, and variants thereof , Derivatives thereof, or fragments thereof containing 18 or more consecutive bases.
(6) A polynucleotide group comprising a base sequence complementary to the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or a base sequence in which t is u in the base sequences.
(7) A polynucleotide that hybridizes under stringent conditions with DNA comprising a base sequence complementary to each of the base sequences in which t is u in the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10 Groups or fragments thereof containing 18 or more consecutive bases.
(8) A polynucleotide group that hybridizes under stringent conditions with a DNA comprising each of the base sequences in which t is u in the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or 18 or more Those fragments containing consecutive bases.
(9) In each of the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or the base sequence in which t is u in the base sequence, or a complementary sequence thereof, 18 or more consecutive bases A polynucleotide comprising.
(10) In each of the base sequence represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, or the base sequence in which t is u in the base sequence, or a complementary sequence thereof, 60 or more consecutive bases A polynucleotide comprising.

  In the present invention, the size of the polynucleotide fragment is, for example, the total number of bases in 18 to 18 bases, 18 to 30 bases or more, 18 to 40 bases or more, 18 to 18 in the base sequence of each polynucleotide. 60 bases or more, 18 to 80 bases or more, 18 to 100 bases or more, 20 to the total number of bases or more, 20 to 30 bases or more, 20 to 40 bases or more, 20 ~ 60 bases or more, 20 to 80 bases or more, 20 to 100 bases or more, 25 to the total number of bases in the sequence, 25 to 30 bases or more, 25 to 40 bases or more, 25 to 60 Base or more 25 to 80 base or more 25 to 100 base or more On a base number in the range such as, but not intended to be limited thereto.

  According to a preferred embodiment, the DNA chip of the present invention is a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1 to 5, the nucleotide sequence in which t is u, or a complementary sequence thereof. All of five or more of the five genes selected from above can be included.

  In the present invention, the polynucleotide to be immobilized may be any of genomic DNA, cDNA, RNA, synthetic DNA, and synthetic RNA, or may be single-stranded or double-stranded. Here, the synthetic DNA and the synthetic RNA include a modified nucleic acid as described in the definition of “derivative” above.

  Examples of DNA chips that can detect and measure the expression level of a target gene, RNA or cDNA include 3D-Gene (registered trademark) Human Oligo chip 25k ver 2.1 manufactured by Toray Industries, Inc. and SurePrint G3 Human manufactured by Agilent. GE microarray kit 8x60K, GeneChip Human Gene 1.0 ST Array of Affymetrix, etc. can be mentioned.

  For production of a DNA chip, for example, a method of immobilizing a probe prepared in advance on a solid surface can be used. In the method of immobilizing a polynucleotide probe prepared in advance on a solid phase surface, a polynucleotide having a functional group introduced therein is synthesized, and an oligonucleotide or polynucleotide is spotted on the surface of the surface-treated solid support and covalently bonded ( For example, J. B. Lamture et al., Nucleic. Acids. Research, 1994, Vol. 22, p. 2121-2125, Z. Guo et al., Nucleic. Acids. Research, 1994, Vol. 5465). In general, a polynucleotide is covalently bound to a surface-treated solid support via a spacer or a crosslinker. A method is also known in which polyacrylamide gel fragments are aligned on a glass surface and a synthetic polynucleotide is covalently bound thereto (G. Yershov et al., Proceedings of the National Academic Sciences USA, 1996). 94, p. 4913). In addition, an array of microelectrodes was prepared on a silica microarray, an agarose permeation layer containing streptavidin was provided on the electrode as a reaction site, and this site was positively charged to immobilize the biotinylated polynucleotide. There is also known a method that enables precise hybridization at high speed by controlling the charge of the site (RG Sonoski et al., Proceedings of the National Academic Sciences USA, 1997, No. 1). 94, pp. 1119-1123).

5. Detection method of colorectal cancer The present invention detects in vitro whether or not a colorectal cancer-derived gene is contained in a specimen sample using the diagnostic polynucleotide, kit, DNA chip, or a combination thereof of the present invention, A method for testing or evaluating, wherein a sample sample such as stool collected from a colon cancer patient and a non-colon cancer patient is used to compare the expression gene amount in the sample, and the expression level of the target gene in the sample sample Increase / decrease includes determining or evaluating the presence of a colorectal cancer-derived gene in the specimen sample, wherein the target gene is included in the diagnostic polynucleotide, kit or DNA chip Methods are provided that are detectable by a polynucleotide, variant thereof or fragment thereof.

  By detecting, examining or diagnosing genes derived from colorectal cancer from a sample sample such as stool by the above method of the present invention, it is possible only to enable early diagnosis of cancer noninvasively with high accuracy and sensitivity. Provide methods that provide early treatment and improved prognosis, and further enable monitoring of disease exacerbations and the effectiveness of surgical, radiotherapeutic, and chemotherapeutic treatments.

  According to a preferred embodiment, the method for preserving a specimen sample such as stool according to the present invention is to preserve the stool with a reagent that suppresses RNA degradation in the specimen sample, such as RNAlater (registered trademark) Solutions (Life technologies). May be. When RNAlater (registered trademark) Solutions is used, the specimen sample is stored for 0 days to 5 years, preferably 0 days to 1 year, more preferably 0 to 1 month. The storage temperature is -80 to 40 ° C, preferably 0 to 25 ° C, more preferably 4 ° C. In another preferred embodiment, a reagent containing a guanidine salt or an acidic phenol that inactivates RNase may be added to the specimen sample. In addition, refrigerated storage or frozen storage in which no reagent is added to the sample can be used, but the storage method is not limited to these methods.

  According to a preferred embodiment, a method for extracting a gene derived from colorectal cancer from a specimen sample such as stool according to the present invention adds a reagent for RNA extraction containing a guanidine salt to a specimen sample such as stool. In order to reduce the risk of infection and reagent exposure, vortex in a sealed tube to inactivate RNase to prevent RNA degradation and lyse cells in stool, and then RNeasy Total RNA is prepared according to a conventional method such as mini kit (QIAGEN). Further, an RNA extraction reagent containing an acidic phenol such as Trizol (Life technologies) or Isogen (Nippon Gene), or a homogenizer may be used, but the method is not limited thereto.

  In the above-described method of the present invention, the diagnostic polynucleotide, kit or DNA chip contains the polynucleotide of the present invention, a variant thereof or a fragment thereof, as described above, singly or in any possible combination. used.

  In the detection, test or (gene) diagnosis of colorectal cancer of the present invention, the polynucleotide, variant or fragment thereof contained in the diagnostic polynucleotide, kit or DNA chip of the present invention may be used as a primer or a probe. it can. When used as a primer, TaqMan (registered trademark) MicroRNA Assays from Applied Biosystems can be used, but is not limited to this method.

  Polynucleotides, variants thereof or fragments thereof contained in the diagnostic polynucleotide or kit of the present invention are identified as Northern blot, Southern blot, RT-PCR, in situ hybridization, Southern hybridization, etc. In a known method for specifically detecting a gene, it can be used as a primer or a probe according to a conventional method. Depending on the type of detection method used, a sample of the cancerous lesion of the large intestine and part or all of the normal tissue may be collected with a biopsy or collected from a living tissue removed by surgery. Or collect a biological tissue such as stool from the subject. Furthermore, total RNA prepared therefrom according to a conventional method may be used, or various polynucleotides containing cDNA or aRNA amplified therefrom prepared based on the RNA may be used.

  Alternatively, the expression level of a nucleic acid such as the gene, RNA, and cDNA of the present invention in a living tissue can be detected or quantified using a DNA chip (including a DNA macroarray). In this case, the diagnostic polynucleotide or kit of the present invention can be used as a probe for a DNA array. Such a DNA array is hybridized with labeled DNA or RNA prepared based on RNA collected from a living tissue, and a complex of the probe and labeled DNA or RNA formed by the hybridization is labeled with the labeled DNA or RNA. By detecting RNA labeling as an index, the presence or absence or expression level (expression level) of the colon cancer-related gene of the present invention in living tissue can be evaluated. In the method of the present invention, a DNA chip can be preferably used, which can evaluate the presence / absence or expression level of a plurality of genes for one biological sample at the same time.

  The diagnostic polynucleotide, kit or DNA chip of the present invention is useful for diagnosis, examination or detection of colon cancer (diagnosis of presence / absence or degree of morbidity). Specifically, the diagnosis of colorectal cancer using the diagnostic polynucleotide, kit or DNA chip is carried out by using colorectal cancer cells and non-colorectal cancer cells collected at the time of surgery or endoscopy from a colorectal cancer patient, and / or Or, by using stool from colon cancer patients and non-colon cancer patients, the amount of expressed genes in the sample is compared, and the difference in the expression level of the gene detected by the diagnostic polynucleotide is determined or evaluated. be able to. In this case, the difference in gene expression level is not only the presence or absence of expression, but also compared the expression level between both living tissues derived from colon cancer patients and living tissues derived from non-colon cancer patients. This includes cases where there is a difference in time. For example, CEBPB gene is induced / decreased in expression in colorectal cancer, so it is expressed / decreased in the body tissue of the subject. If there is a difference between the expression level and the expression level in normal tissue, the sample is derived from colon cancer. It is possible to determine or evaluate that the gene is not included and / or the gene derived from colon cancer is included.

  A method for detecting the absence of a colon cancer-derived gene / or the presence of a colon cancer-derived gene in a specimen sample using the diagnostic polynucleotide, kit or DNA chip of the present invention is as follows. A part or the whole is collected by biopsy or the like, collected from a living tissue removed by surgery, or stool of a subject is collected, and five genes contained therein are selected from the polynucleotide group of the present invention. Inspecting, diagnosing, or evaluating the presence or absence of colorectal cancer by detecting using one or a plurality of polynucleotides, mutants or fragments thereof, and measuring the gene expression level . The colorectal cancer detection method of the present invention can also examine, diagnose, or evaluate the presence or absence or extent of improvement of the disease when a therapeutic agent is administered to improve the disease, for example, in a colon cancer patient. .

The method of the present invention includes, for example, the following steps (a), (b) and (c):
(A) contacting a specimen sample derived from a subject with the polynucleotide of the diagnostic polynucleotide, kit or DNA chip of the present invention,
(B) a step of measuring the expression level of the target gene in the biological sample using the polynucleotide as a probe;
(C) A step of determining or evaluating the presence or absence of colorectal cancer (cells) in the specimen sample based on the result of (b),
Can be included.

  Examples of the specimen sample used in the method of the present invention include a sample prepared from a biological tissue of a subject, for example, a large intestine tissue and its surrounding tissues, a tissue suspected to be a colon cancer, stool, and the like. Specifically, an RNA-containing sample prepared from the tissue, or a sample containing a polynucleotide further prepared therefrom, is obtained by collecting a part or all of a subject's biological tissue with a biopsy or the like, or removing it by surgery Or collected from the stool and prepared according to a conventional method.

  The term “subject” as used herein refers to mammals such as, but not limited to, humans, monkeys, mice, rats, and the like, preferably humans.

  The method of this invention can change a process according to the kind of biological sample used as a measuring object.

When RNA is used as a measurement object, colon cancer (cells) is detected, for example, by the following steps (a), (b) and (c):
(A) RNA prepared from a biological sample of a subject or complementary polynucleotide (cDNA) transcribed therefrom or RNA (aRNA) amplified therefrom is used as the polynucleotide of the diagnostic polynucleotide, kit or DNA chip of the present invention Combining with,
(B) a step of measuring RNA derived from a biological sample bound to the polynucleotide or a complementary polynucleotide transcribed from the RNA using the polynucleotide as a probe;
(C) determining or evaluating the presence or absence of colorectal cancer (derived gene) based on the measurement result of (b) above,
Can be included.

  In order to detect, test or diagnose colorectal cancer (derived gene) according to the present invention, for example, various hybridization methods can be used. As such hybridization method, for example, Northern blot method, Southern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization method, Southern hybridization method and the like can be used.

When using the Northern blot method, the presence or absence of each gene expression in RNA and its expression level can be detected and measured by using the diagnostic polynucleotide of the present invention as a probe. Specifically, labeled diagnostic polynucleotide of the invention (complementary strand) radioactive isotopes ^{(32 P,} 33 ^P, etc. 35 ^S), fluorescent substance (cyan, rhodamine, fluorescamine-based, etc.) or the like, After hybridizing it with RNA derived from the biological tissue of the subject transferred to a nylon membrane or the like according to a conventional method, the diagnostic polynucleotide (DNA) and the double strand of RNA formed are diagnostic polynucleotides A signal derived from the label (radioisotope or fluorescent substance) of a radiation detector (BAS-1800II, Fuji Photo Film Co., Ltd. can be exemplified) or a fluorescence detector (STORM860, GE Healthcare, etc.) The method of detecting and measuring can be illustrated.

  When the quantitative RT-PCR method is used, the presence or absence of gene expression and its expression level in RNA can be detected and measured by using the diagnostic polynucleotide of the present invention as a primer. Specifically, cDNA is prepared from RNA derived from the biological tissue of a subject according to a conventional method, and prepared from the diagnostic polynucleotide of the present invention so that each target gene region can be amplified using this as a template. A method of detecting the resulting double-stranded DNA by hybridizing the pair of primers (consisting of a positive strand and a reverse strand that binds to the cDNA) to cDNA, performing PCR by a conventional method, and it can. In addition, as a method for detecting double-stranded DNA, the above PCR is performed using a primer previously labeled with a radioisotope or a fluorescent substance, the PCR product is electrophoresed on an agarose gel, and then is detected with ethidium bromide or the like. A method of detecting by staining the double-stranded DNA and a method of detecting by detecting the produced double-stranded DNA by transferring it to a nylon membrane or the like according to a conventional method and hybridizing it with a probe as a probe be able to.

  When DNA array analysis is used, a DNA chip in which the diagnostic polynucleotide of the present invention is attached to a substrate as a DNA probe (single strand or double strand) is used. Those obtained by immobilizing a gene group on a substrate generally have names of DNA chip and DNA array, and the DNA array includes a DNA macroarray and a DNA microarray. DNA array is included.

  Although hybridization conditions are not limited, For example, it shall be 30 to 60 degreeC, and shall be the conditions for 1 to 2 to 4 hours in the solution containing SSC and surfactant. Here, 1 × SSC is an aqueous solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate, and the surfactant contains SDS, Triton, Tween, or the like. More preferably, the hybridization conditions include 3 to 4 × SSC and 0.1 to 0.5% SDS. Washing conditions after hybridization include, for example, a solution containing 0.5 × SSC and 0.1% SDS at 30 ° C., a solution containing 0.2 × SSC and 0.1% SDS at 30 ° C., and 30 Conditions such as continuous washing with 0.05 × SSC solution at 0 ° C. can be mentioned. It is desirable that the complementary strand maintain a hybridized state with the target positive strand even when washed under such conditions. Specifically, as such a complementary strand, a strand composed of a base sequence that is completely complementary to the base sequence of the target positive strand, and a strand composed of a base sequence having at least 80% homology with the strand Can be illustrated.

Examples of stringent hybridization conditions when PCR is performed using the diagnostic polynucleotide of the present invention or the polynucleotide fragment of the kit as a primer include, for example, 10 mM Tris-HCL (pH 8.3), 50 mM KCL, 1-2 mM MgCl _2. And the like, using a PCR buffer having the composition as described above, and treating for about 15 seconds to 1 minute at Tm + 5 to 10 ° C. calculated from the primer sequence. Examples of such a calculation method of Tm include Tm = 2 × (number of adenine residues + number of thymine residues) + 4 × (number of guanine residues + number of cytosine residues).

  For other examples of “stringent conditions” in these hybridizations, see, for example, Sambrook, J. et al. & Russel, D.C. Written by Molecular Cloning, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, published on January 15, 2001, Volumes 7.42-7.45, Volumes 8.9-8.17, etc. And can be used in the present invention.

  The present invention also uses the diagnostic polynucleotide, kit, DNA chip, or combination thereof of the present invention to measure the expression level of a target gene or gene in a specimen sample derived from a subject, and Determine whether the specimen sample contains and / or does not contain a colon cancer-derived gene, using a support vector machine (SVM) with the gene expression level of the sample and a biological sample derived from a non-colon cancer patient as a teacher sample Provide a way to evaluate.

  That is, the present invention further uses the diagnostic polynucleotide of the present invention, kit, DNA chip, or a combination thereof, and the specimen sample contains a colon cancer-derived gene / or does not contain a colon cancer-derived gene. A first step of measuring the expression level of a target gene in a plurality of biological samples known to be determined or evaluated in vitro, and a measurement value of the expression level of the target gene obtained in the first step. A second step of creating a discriminant using SVM as a teacher sample, a third step of measuring the expression level of the target gene in a specimen sample derived from a subject in vitro as in the first step, the second step Substituting the measured value of the expression level of the target gene obtained in the third step into the discriminant obtained in step 3, and based on the result obtained from the discriminant, the specimen sample is a gene derived from colon cancer And / or a fourth step of determining or evaluating the absence of a gene derived from colon cancer, wherein the target gene is contained in the polynucleotide, kit or DNA chip, or a variant thereof Or a method thereof, which is detectable by a fragment thereof.

Alternatively, the method of the present invention includes, for example, the following steps (a), (b) and (c):
(A) The expression level of a target gene in a biological sample known to be a tissue containing a colon cancer-derived gene derived from a colon cancer patient and / or a tissue not containing a colon cancer-derived gene derived from a non-colon cancer patient A step of measuring using a polynucleotide (kit) or DNA chip for diagnosis (detection) according to the invention,
(B) substituting the measured value of the expression level measured in (a) into the following formulas 1 to 3 to create a discriminant called SVM;
(C) The expression level of the target gene in the specimen sample derived from the subject is measured using the diagnostic (detection) polynucleotide, kit or DNA chip according to the present invention, and the discriminant created in (b) Substituting and determining or evaluating that the specimen sample contains and / or does not contain a colon cancer-derived gene based on the obtained results;
Can be included.

  What is SVM? It is a discriminant analysis method devised by Vapnik (The Nature of Statistical Learning Theory, Springer, 1995). A boundary surface called a hyperplane for correctly classifying the data set into a known grouping, with a specific data item of the data set with a known grouping as an explanatory variable and a grouping as a target variable. And a discriminant for classifying the data using the boundary surface. In the discriminant, grouping can be discriminated by substituting the measured value of the newly given data set as an explanatory variable into the discriminant. Further, the discrimination result at this time may be a group to be classified, may be a probability of being classified into a group to be classified, or may be a distance from a hyperplane. In SVM, as a method for dealing with a non-linear problem, a method is known in which a feature vector is non-linearly transformed into a higher dimension and linear identification is performed in the space. An expression in which the inner product of two elements in a non-linearly mapped space is expressed only by the input in the original space is called a kernel. As an example of the kernel, a linear kernel, RBF (Radial Basis Function) Kernel and Gaussian kernel. It is possible to construct an optimum discriminant function, that is, a discriminant, only by calculating the kernel while actually calculating the feature in the mapped space while mapping to a higher dimension by the kernel. (For example, Hideki Aso et al., Frontier 6 of Statistical Science, “New Concepts and Methods of Pattern Recognition and Learning, Iwanami Shoten (2004), Nero Cristianini et al., SVM Introduction, Kyoritsu Publishing (2008)).

  The one-class SVM method, which is a kind of SVM method, creates a hyperplane by learning with only one group of explanatory variables, and an unknown data set is included in the known data set or becomes an outlier. (Pai-Hsuen Chen et al., 2005, Applied Stochastic Models in Business and Industry, Vol. 21, p111-136). Outliers tend to be located near the origin of a two-dimensional plane when mapped from a high-dimensional plane to a two-dimensional plane using a kernel function.

The explanatory variable of the discriminant by the one-class SVM method of the present invention includes a value obtained by measuring a polynucleotide selected from the polynucleotides described in Section 2 above or a fragment thereof. The explanatory variable for discriminating between the colon cancer patient and the non-colon cancer patient of the invention is, for example, a gene expression level selected from the group consisting of the following (1) to (2):
(1) A colon cancer patient or non-colon cancer measured by any one of DNAs containing 18 or more consecutive bases in the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10 or their complementary sequences Gene expression level in the patient's stool.
(2) In the base sequences represented by SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10 or their complementary sequences, 60 or more contiguous sequences in the DNA base sequence or its complementary sequence containing 18 or more consecutive bases, respectively Gene expression level in stool of colorectal cancer patients or non-colorectal cancer patients as measured by any of the DNAs containing the selected bases.

An example of calculating a discriminant that can be used in the method of the present invention is shown below.
First, all subjects are divided into two groups: colon cancer patients and non-colon cancer patients. As a reference for determining that the subject is a colon cancer patient or a non-colon cancer patient, for example, a close examination such as a colonoscopy can be used.

  Next, a data set (hereinafter referred to as a learning set) consisting of comprehensive gene expression levels of the two groups of biological samples derived from stool is prepared, and there is a clear difference in gene expression levels between the two groups. A discriminant is determined by the one-class SVM method using the gene as an explanatory variable and the grouping as an objective variable (for example, 0 and 1) (Equations 1 and 2). At this time, the discriminant has a kernel function defined by Equation 3. For example, the discriminant is created by learning the gene expression level of a non-colon cancer patient in the learning set by the one-class SVM method.

（数１はＬａｇｕｒａｎｇｅの未定定数法を用いることで帰着するＬａｇｕｒａｎｇｅ定数αを用いた最適化問題を示す。νは学習セットの誤判別率を調整するパラメータを表す。）

(Equation 1 represents an optimization problem using the Lagrangian constant α that results from using Lagrange's undetermined constant method. Ν represents a parameter for adjusting the misclassification rate of the learning set.)

（数２は最終的に得られた識別関数を示す）
カーネルとしては以下のＲＢＦカーネルを用いることができる。

(Equation 2 shows the discriminant function finally obtained)
The following RBF kernel can be used as the kernel.

（ここで、γは超平面の複雑さを調整するカーネルパラメータを表す。）

(Where γ represents a kernel parameter that adjusts the complexity of the hyperplane.)

  For an unknown colorectal cancer patient or non-colorectal cancer patient, the expression level of the gene used in the discriminant in the stool of the colorectal cancer patient or the colon cancer patient is measured, and these are expressed as x in the discriminant. By substituting, it can be determined whether the patient belongs to a colorectal cancer patient or a non-colorectal cancer patient.

  As described above, it is determined or evaluated whether a subject unknown whether it is a colorectal cancer patient or a non-colorectal cancer patient belongs to a group of colorectal cancer patients or non-colorectal cancer patients The discriminant created from the learning set is necessary to create the discriminant to be used. In order to increase the discriminant accuracy of the discriminant, a gene having a clear difference between the two groups in the learning set is used as the discriminant. It is necessary to use it.

  Moreover, it is preferable to determine the gene used for the explanatory variable of the discriminant as follows. First, the p-value of the t-test, which is a parametric analysis, is a data set of the stool-derived comprehensive gene expression level of the colon cancer patient group and the non-colorectal cancer patient group stool-derived comprehensive gene expression level. Using the P value of the Mann-Whitney U test or the P value of the Wilcoxon test, which is nonparametric analysis, the difference in the expression level of each gene between the two groups is determined.

  It can be regarded as statistically significant when the risk factor (significance level) of the P value obtained by the test is smaller than 5%, 1% or 0.01%, for example.

  In order to correct the increase in the probability of the first type of error due to repeated testing, it can be corrected by a known method, such as Bonferroni, Holm, etc. Basic multiple comparison method ", Scientist (2007)) Bonferroni correction, for example, the P value obtained by the test is multiplied by the number of test iterations, that is, the number of genes used in the analysis, and the desired significance level The probability of causing the first type of error in the entire test can be suppressed by comparing with.

  Next, a discriminant using an arbitrary number of genes with a large difference in the gene expression level obtained here is created, and this discriminant is derived from the stool of another independent colon cancer patient or non-colon cancer patient. By substituting the gene expression level into an explanatory variable, the discrimination result of the group to which this independent colon cancer patient or non-colon cancer patient belongs is calculated. In order to construct a discriminant that obtains the maximum discriminating accuracy, the discriminant is created and the discriminant accuracy is calculated repeatedly by increasing the genes one by one in descending order of gene expression difference (Furey TS et al. 2000, Bioinformatics., Vol. 16, p906-14).

  In addition, it is preferable to use the Split-sample method for determining the gene used in the discriminant and calculating the discrimination accuracy (FIG. 1). In other words, the data set is divided into a learning set and a test set, and a test and discriminant are created with the learning set, and the test group is discriminated with the discriminant and the true group to which the test set belongs is used for accuracy and sensitivity.・ Calculate specificity and evaluate discrimination performance.

  The present invention relates to a polynucleotide for disease diagnosis useful for diagnosis and treatment of colorectal cancer, a method for detecting (or examining) colorectal cancer using the polynucleotide, and diagnosis (or detection or detection) of colorectal cancer using the polynucleotide. Test) Kit is provided. In particular, in the method of the present invention, for example, in the fecal occult blood test, it was determined to be negative in order to carry out selection of a diagnostic gene exhibiting an accuracy exceeding the existing colorectal cancer diagnostic method from stool and creation of a diagnostic algorithm. Regardless, expression genes derived from stool specimens derived from patients whose colon cancer was finally revealed by detailed examinations such as colonoscopy and stool specimens derived from patients without colon cancer By comparing the expressed genes, it is possible to construct a diagnostic gene set and a diagnostic algorithm that show an accuracy exceeding the fecal occult blood test.

  For example, one or more of the above polynucleotides for each gene based on the polynucleotide sequence of SEQ ID NO: 1-5 or SEQ ID NO: 6-10 for the above five genes as described above, and / or as described above Using the polynucleotides of SEQ ID NOs: 1 to 5 and SEQ ID NOs: 6 to 10, the expression levels of the above five target genes are different between the sample samples derived from colorectal cancer patients and the sample samples derived from non-colon cancer patients. It is highly likely that the specimen sample contains a colon cancer-derived gene or does not contain a colon cancer-derived gene, using as an index the difference between them or the increase / decrease in expression in a specimen sample from a colon cancer patient Can be distinguished by accuracy. In particular, in any stage from Duke's stage A to C, colorectal cancer can be detected with high sensitivity and high accuracy. However, it provides a special effect of higher sensitivity and accuracy.

  The present invention is more specifically described by the following examples. However, the scope of the present invention shall not be limited by this example.

<Example 1>
1. Sample collection Intestinal endoscopy with informed consent was diagnosed by Duke's stage A in 15 cases, stage B in 18 cases, stage C in 15 cases, stage D in 5 cases, and a total of 53 cases of large intestine Stool 0.5 g was obtained from a total of 114 subjects including cancer patients and 61 non-colon cancer patients. The obtained stool was immediately added with 10 ml of RNAlater (registered trademark) Solutions (Life technologies) and stored at 4 ° C. to suppress RNA degradation.

2. Extraction of total RNA RNeasy mini kit (QIAGEN) for stool of 53 colorectal cancer patients and 61 non-colorectal cancer patients preserved in RNAlater (registered trademark) Solutions obtained in “1” above as a sample Was used to obtain total RNA. The operation basically followed the procedure specified by the company. Specifically, 2 ml of Buffer RLT and 20 μl of β-mercaptoethanol were mixed, homogenized by vortex in addition to the stool extracted from RNAlater (registered trademark) Solutions, and then the kit. The total RNA was purified by the column attached to 1.

3. Measurement of gene expression level Using the target RNA obtained in the above “2”, TargetAmp (registered trademark) 1-Round Aminoallyl-aRNA Amplification Kit 101 (Epicentre (registered trademark) Biotechnologies) was used in accordance with the procedure prescribed by the company. Sense RNA was obtained.

  As oligo DNA microarray, 3D-Gene (registered trademark) Human Oligo chip 25k ver 2.1 manufactured by Toray Industries, Inc. was used to narrow down the genes based on the procedure defined by the company. The hybridized DNA chip was scanned using a DNA array scanner (3D-Gene (registered trademark) Scanner 3000, Toray Industries, Inc.) to acquire an image and Extraction 2.0.0.4 (Toray Industries, Inc.) The fluorescence intensity was digitized. Statistical processing is described in Speed T. et al. "Statistical analysis of gene expression microarray data" by Helen Causton et al., "Microarray Gene Expression Data Analysis: A Beginner's Guide" The gene expression level deficit value was replaced with a value obtained by subtracting 0.1 from the logarithmic value of the minimum value of the gene expression level in each DNA chip. As a result, nucleic acid sequences detected by hybridization of each probe of Human Oligo chip 25k ver 2.1 for 53 colon cancer patients or 61 non-colon cancer patients, that is, comprehensive gene expression levels of mRNA were obtained.

4). Discrimination scoring system The gene expression level of mRNA detected from stool-derived total RNA of a total of 114 specimens of 53 colon cancer patients obtained in “3” above or 61 specimens of non-colorectal cancer patients, And non-colon cancer patient groups, a colon cancer discrimination gene is determined, and the discrimination result of the colon cancer patient or non-colon cancer patient when the gene is used is expressed as R language 2.13.2 (R Development Core). Team (2011) R: A language and environmental for statistical computing. R Foundation for Statistical Computing, URL http://www.R-project.org/. (Evgenia Dimitriadou, Kurt Hornik, Friedrich Leisch, David Meyer and Andreas Weingessel (2011)., TU Wien. R package version 1.6.) Was calculated using the. That is, according to the Split-sample method shown in FIG. 1, first, 53 colorectal cancer patient groups and 61 non-colorectal cancer patient groups obtained in “3” above are classified into 41 colorectal cancer patient samples (Duke's stage A). 11 cases, stage B 14 cases, stage C 11 cases, stage D 5 cases) and 54 non-colon cancer patients 54 learning sets, colon cancer patients 12 samples (Duke's stage A 4 cases, stage B was 4 cases and stage C was 4 cases) and 7 non-colon cancer patients. The learning set was normalized with quantile normalization (Bolstad, B. M., et al., 2003, Bioinformatics, Vol. 19, p185-193), and using the gene average expression level corresponding to the rank in the gene expression level of the gene at that time Quantile normalization of the test set was performed. In 20% of the samples (19 samples) in the learning set, only genes having a gene expression level of 2 6 or more were selected, and only genes corresponding to the genes selected in the learning set were selected from the test set. . In the learning set, between the gene expression level of the colorectal cancer patient group and the gene expression level of the non-colon cancer patient group, the difference between the two groups is determined for each gene expression level by the Wilcoxon test to calculate the P value and correct Bonferroni The rank indicating the high performance of discriminating between the colon cancer patient group and the non-colon cancer patient group of each gene in the learning set was calculated (Table 1). In the learning set, the gene expression level of the gene with the smallest P value obtained from the Wilcoxon test is sequentially input to the SVM (Formula 1 to Formula 3) to create a discriminant, and this discriminant is used to discriminate the test set. went. SVM is in accordance with A Practical Guide to Support Vector Classification (Chih-Wei Hsu et al., Http://www.csie.ntu.edu.tw/˜cjlin/paper-guide/guide/guide. Was used to determine the test set by optimizing the parameters ν and γ in the learning set, and the accuracy, sensitivity and specificity of the result were calculated.

5. Discrimination result 1
As a result of the discriminant scoring system, CEBPB, FCGR3A, PFKFB3, IL8 and SOD2 genes represented by SEQ ID NOs: 1 to 5 were found as the first to fifth genes in the ranking. Furthermore, when all the gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 which are partial sequences of the polynucleotide sequences of SEQ ID NOs: 1 to 5 are used as probes corresponding to these genes, Accuracy and sensitivity are 83.2%, sensitivity is 78.0%, and specificity is 87.0% for the learning set consisting of stool from 41 cancer patients and 54 non-colon cancer patients. It was found that the minimal gene set becomes “SEQ ID NO: 1 + 2 + 3 + 4 + 5” in Table 2.

  Furthermore, in order to confirm the more general performance of the discriminant formula created by the SVM, the stool of 12 colon cancer patients (Duke's stage A 4 cases, stage B 4 cases, stage C 4 cases) For a test set consisting of stool from 7 non-colon cancer patients, the gene expression level was measured in the same manner as described above, and determined using the above SVM discriminant. CEBPB and FCGR3A represented by SEQ ID NOs: 1 to 5 When all the gene expression levels measured with the polynucleotides of SEQ ID NOs: 6 to 10 are used as probes corresponding to the PFKFB3, IL8 and SOD2 genes, the accuracy is 84.2 when all the above five genes are combined. %, Sensitivity is 83.3%, specificity is 85.7%, the smallest gene set with the highest accuracy and sensitivity ( 3 'SEQ ID NO: 1 + 2 + 3 + 4 + 5 "), and the training set results were supported. Further, Duke's stage A, which has a low degree of progression of colorectal cancer, was correctly identified as cancer in 4 out of 4 samples of Duke's stage A and 3 out of 4 samples of Duke's stage B.

  As described above, since the method of the present invention can detect colon cancer early and with high sensitivity, it is possible to make an early judgment of performing cancer resection by endoscope or surgery, and as a result, the recurrence rate can be reduced. Become.

<Example 2>
SVM constructed above for the test set consisting of 12 colon cancer patients (Duke's stage A 4 cases, stage B 4 cases, stage C 4 cases) and 7 non-colon cancer patients The accuracy, sensitivity, and specificity of the discriminant result and fecal occult blood test were compared. The diagnostic result of the fecal occult blood test has an accuracy of 76.2%, a sensitivity of 64.3%, and a specificity of 100%, and the colorectal cancer detection method of the present invention (SEQ ID NO: 1 + 2 + 3 + 4 + 5 “DNA chip”) It was found that the accuracy was about 10% and the sensitivity was about 20% higher than the fecal occult blood test.

  In the fecal occult blood test, Duke's stage A was correctly determined as cancer in 4 out of 4 specimens and Duke's stage B in 2 out of 4 specimens (Table 4), and the colorectal cancer detection method of the present invention was more effective. Duke's stage B with the second lowest progression of colorectal cancer could be detected one sample more.

  Furthermore, regarding right-sided colon cancer, all 5 out of 5 samples could be diagnosed as cancer using the DNA chip according to the present invention (Table 4). Since the colorectal cancer detection method of the present invention detects the expression levels of the above five genes derived from colorectal cancer cells detached from the large intestine in the stool, even if there is a cancer part in the right side colon where the stool is liquid, the large intestine It is thought that the cancer can be detected. On the other hand, the fecal occult blood test was able to diagnose only 2 out of 5 samples for right colorectal cancer (Table 4). In the right colorectal cancer, stool is liquid, so it is difficult to bleed from the cancer part, and because the long time it exists in the intestine until stool with blood attached is discharged, the antigenicity of hemoglobin decreases, etc. It is thought that it was difficult to detect in the fecal occult blood test.

  From the comparison of the results of Example 1 and Example 2, the colorectal cancer detection method of the present invention is about 10% more accurate and about 20% more sensitive than the fecal occult blood test, and the degree of progression of colorectal cancer is relatively high. It was found that the Duke's stage B specimen having a low A can be detected as cancer, and the right colorectal cancer can be detected with high sensitivity.

  As described above, since the method of the present invention can detect colorectal cancer earlier than fecal occult blood test, it is possible to make an early judgment on the resection of the cancer part by endoscope or surgery, and as a result, the recurrence rate. Can be lowered.

<Example 3>
As a probe corresponding to the genes represented by SEQ ID NOs: 1 to 5, discrimination results when using all five gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10, and 1 to 4 gene expression The discrimination results combining the amounts were compared, and the superiority of using the gene expression levels of all five genes (“SEQ ID NO: 1 + 2 + 3 + 4 + 5”) was evaluated.

  As a comparison, any one of the gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 was selected, and the same test set as in Example 2 was determined by the above-described determination method using SVM. The accuracy and sensitivity were not exceeded when using all five gene expression levels measured with 10 polynucleotides (Table 5).

  For comparison, two types of gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 were selected and determined in the same manner, and all five gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 were determined. The accuracy and sensitivity when using was not exceeded (Table 6).

  For comparison, three gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 were selected and discriminated in the same manner. As a result, all five gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 were determined. The accuracy and sensitivity when using was not exceeded (Table 7).

  For comparison, four gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 were selected and determined in the same manner. As a result, all five gene expression levels measured by the polynucleotides of SEQ ID NOs: 6 to 10 were determined. The accuracy and sensitivity when using was not exceeded (Table 8).

  From the comparison of the results of Example 1 and Example 3, the accuracy was 84.2% when discriminating by the expression level of all 5 types of genes instead of 1 to 4 or less combinations of the polynucleotides of SEQ ID NOs: 6 to 10. It was found that the sensitivity was 83.3%, the accuracy of the fecal occult blood test was 76.2%, and the highest accuracy and sensitivity exceeded the sensitivity of 64.3%.

  Thus, the use of all five genes as genetic markers is optimal for the diagnosis of colorectal cancer superior to the existing fecal occult blood test.

  According to the present invention, since it is possible to provide a colorectal cancer detection or examination method excellent in accuracy and sensitivity, at least to discriminate a colorectal cancer-derived gene contained in stool collected from a colorectal cancer patient before endoscopic examination Very useful.

Claims (11)

A method for assisting in the detection of colorectal cancer, comprising measuring in vitro expression levels of CEBPB gene, FCGR3A gene, PFKFB3 gene, IL8 gene and SOD2 gene in stool derived from a subject. The method for assisting detection of colorectal cancer according to claim 1, which is performed using a nucleic acid probe for specifically detecting the expression of the gene. The method for assisting detection of colorectal cancer according to claim 2, wherein the nucleic acid probe is the following five types of polynucleotides (1) to (5).
(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences , or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 1 complementary base polynucleotide, or (c) comprising said sequence (a) or any polynucleotide that hybridizes to the polynucleotide under stringent conditions (b) (2) FCGR3A gene for the detection of, (a) The base sequence represented by SEQ ID NO: 2, the base sequence in which t is u in the base sequence, or the base sequence complementary to these base sequences Ranaru polynucleotide, or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 2 a polynucleotide comprising a nucleotide sequence complementary, or (c) (a) or (b) polynucleotide (3) that hybridizes under any of the polynucleotides under stringent conditions of PFKFB3 to detect gene of, (a) sequence t is in the nucleotide sequence or the base sequence represented by numbers 3 consisting of a nucleotide sequence complementary to the nucleotide sequence or those nucleotide sequences which are u polynucleotide or sequence of its 18 or more 100 or less (B) a nucleotide sequence represented by SEQ ID NO: 3 or t in the nucleotide sequence polynucleotide comprising a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is u, or (c) (a) or (b) hybridizes to the polynucleotide in any of the polynucleotides under stringent conditions (4) (a) a polynucleotide comprising a base sequence represented by SEQ ID NO: 4, a base sequence in which t is u in the base sequence, or a base sequence complementary to these base sequences, for detecting the IL8 gene ; or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 4 complementary base A polynucleotide containing a sequence, or (c) a stringent condition with a polynucleotide of either (a) or (b) above. Iburidaizu to polynucleotide (5) SOD2 gene for detecting a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 5 (a) polynucleotide consisting, or polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 5 polynucleotide, or (c) (a) or (b) any of the polynucleotides that hybridize to polynucleotide under stringent conditions, including a nucleotide sequence complementary to the The method for assisting the detection of colorectal cancer according to claim 2, wherein the nucleic acid probe is five kinds of polynucleotides comprising the nucleotide sequences represented by SEQ ID NOs: 6 to 10. The CEBPB gene, the FCGR3A gene, the PFKFB3 gene, the IL8 gene, and the SOD2 gene each have a base sequence represented by SEQ ID NOs: 1 to 5, and assist the detection of colorectal cancer according to any one of claims 1 to 4. how to. Using a nucleic acid probe containing the following five types of polynucleotides (1) to (5), a plurality of known specimen samples derived from colon cancer patients or specimen samples derived from non-colon cancer patients A first step of measuring the expression level of the target gene in the stool in vitro, and a discriminant formula (SVM) using the measurement value of the expression level of the target gene obtained in the first step as a teacher Step 3, the third step of measuring the expression level of the target gene in the stool derived from the subject in vitro as in the first step, the third step in the discriminant obtained in the second step Substituting the measured value of the expression level of the target gene obtained in step 1, and based on the result obtained from the discriminant, the presence of a target gene derived from colorectal cancer in the stool derived from the subject, or That no cancer-derived target gene exists. Teimata includes a fourth step of evaluating, the detection method of colon cancer.
(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences , or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 1 complementary base polynucleotide, or (c) comprising said sequence (a) or any polynucleotide that hybridizes to the polynucleotide under stringent conditions (b) (2) FCGR3A gene for the detection of, (a) The base sequence represented by SEQ ID NO: 2, the base sequence in which t is u in the base sequence, or the base sequence complementary to these base sequences Ranaru polynucleotide, or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 2 a polynucleotide comprising a nucleotide sequence complementary, or (c) (a) or (b) polynucleotide (3) that hybridizes under any of the polynucleotides under stringent conditions of PFKFB3 to detect gene of, (a) sequence t is in the nucleotide sequence or the base sequence represented by numbers 3 consisting of a nucleotide sequence complementary to the nucleotide sequence or those nucleotide sequences which are u polynucleotide or sequence of its 18 or more 100 or less (B) a nucleotide sequence represented by SEQ ID NO: 3 or t in the nucleotide sequence polynucleotide comprising a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is u, or (c) (a) or (b) hybridizes to the polynucleotide in any of the polynucleotides under stringent conditions (4) (a) a polynucleotide comprising a base sequence represented by SEQ ID NO: 4, a base sequence in which t is u in the base sequence, or a base sequence complementary to these base sequences, for detecting the IL8 gene ; or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 4 complementary base A polynucleotide containing a sequence, or (c) a stringent condition with a polynucleotide of either (a) or (b) above. Iburidaizu to polynucleotide (5) SOD2 gene for detecting a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 5 (a) polynucleotide consisting, or polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 5 polynucleotide, or (c) (a) or (b) any of the polynucleotides that hybridize to polynucleotide under stringent conditions, including a nucleotide sequence complementary to the   The method for detecting colorectal cancer according to claim 6, wherein the nucleic acid probes are five types of polynucleotides comprising the nucleotide sequences represented by SEQ ID NOs: 6 to 10. A kit for diagnosing colorectal cancer using stool as a sample sample , comprising a nucleic acid probe comprising the following five types of polynucleotides (1) to (5).
(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences , or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 1 complementary base polynucleotide, or (c) comprising said sequence (a) or any polynucleotide that hybridizes to the polynucleotide under stringent conditions (b) (2) FCGR3A gene for the detection of, (a) The base sequence represented by SEQ ID NO: 2, the base sequence in which t is u in the base sequence, or the base sequence complementary to these base sequences Ranaru polynucleotide, or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 2 a polynucleotide comprising a nucleotide sequence complementary, or (c) (a) or (b) polynucleotide (3) that hybridizes under any of the polynucleotides under stringent conditions of PFKFB3 to detect gene of, (a) sequence t is in the nucleotide sequence or the base sequence represented by numbers 3 consisting of a nucleotide sequence complementary to the nucleotide sequence or those nucleotide sequences which are u polynucleotide or sequence of its 18 or more 100 or less (B) a nucleotide sequence represented by SEQ ID NO: 3 or t in the nucleotide sequence polynucleotide comprising a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is u, or (c) (a) or (b) hybridizes to the polynucleotide in any of the polynucleotides under stringent conditions (4) (a) a polynucleotide comprising a base sequence represented by SEQ ID NO: 4, a base sequence in which t is u in the base sequence, or a base sequence complementary to these base sequences, for detecting the IL8 gene ; or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 4 complementary base A polynucleotide containing a sequence, or (c) a stringent condition with a polynucleotide of either (a) or (b) above. Iburidaizu to polynucleotide (5) SOD2 gene for detecting a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 5 (a) polynucleotide consisting, or polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 5 polynucleotide, or (c) (a) or (b) any of the polynucleotides that hybridize to polynucleotide under stringent conditions, including a nucleotide sequence complementary to the   The kit for colorectal cancer diagnosis according to claim 8, wherein the nucleic acid probe is 5 types of polynucleotides comprising the nucleotide sequences represented by SEQ ID NOs: 6 to 10. A DNA chip for colorectal cancer diagnosis using stool as a sample sample , comprising a nucleic acid probe comprising the following five types of polynucleotides (1) to (5).
(1) For detecting the CEBPB gene, (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base sequence having t in the base sequence, or a base sequence complementary to these base sequences , or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 1 complementary base polynucleotide, or (c) comprising said sequence (a) or any polynucleotide that hybridizes to the polynucleotide under stringent conditions (b) (2) FCGR3A gene for the detection of, (a) The base sequence represented by SEQ ID NO: 2, the base sequence in which t is u in the base sequence, or the base sequence complementary to these base sequences Ranaru polynucleotide, or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 2 a polynucleotide comprising a nucleotide sequence complementary, or (c) (a) or (b) polynucleotide (3) that hybridizes under any of the polynucleotides under stringent conditions of PFKFB3 to detect gene of, (a) sequence t is in the nucleotide sequence or the base sequence represented by numbers 3 consisting of a nucleotide sequence complementary to the nucleotide sequence or those nucleotide sequences which are u polynucleotide or sequence of its 18 or more 100 or less (B) a nucleotide sequence represented by SEQ ID NO: 3 or t in the nucleotide sequence polynucleotide comprising a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is u, or (c) (a) or (b) hybridizes to the polynucleotide in any of the polynucleotides under stringent conditions (4) (a) a polynucleotide comprising a base sequence represented by SEQ ID NO: 4, a base sequence in which t is u in the base sequence, or a base sequence complementary to these base sequences, for detecting the IL8 gene ; or a polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, (b) a nucleotide sequence or those nucleotide sequences t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 4 complementary base A polynucleotide containing a sequence, or (c) a stringent condition with a polynucleotide of either (a) or (b) above. Iburidaizu to polynucleotide (5) SOD2 gene for detecting a nucleotide sequence or complementary nucleotide sequence thereto nucleotide sequence is t is u in the nucleotide sequence or the base sequence represented by SEQ ID NO: 5 (a) polynucleotide consisting, or polynucleotide fragment comprising the 18 or more 100 or less contiguous bases, nucleotide sequence, or these nucleotide sequences is t is u in the nucleotide sequence or the base sequence represented by (b) SEQ ID NO: 5 polynucleotide, or (c) (a) or (b) any of the polynucleotides that hybridize to polynucleotide under stringent conditions, including a nucleotide sequence complementary to the   The DNA chip for colorectal cancer diagnosis according to claim 10, wherein the nucleic acid probe is five kinds of polynucleotides consisting of base sequences represented by SEQ ID NOs: 6 to 10.

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