KR20070051033A - Method for diagnosing hyperplastic polyp of intestine - Google Patents

Abstract

The present invention relates to a method for diagnosing a new hyperplastic polyp, and more particularly, a diagnostic kit using a specific gene group for colonic proliferative polyps, specific genes for colonic proliferative polyps used therein, and a colon using the same. The present invention relates to a diagnostic method for proliferating polyps, and the diagnostic kit can be effectively used for diagnosis of all tumors and cancers related to proliferating polyps including colorectal cancer.

Colorectal cancer diagnosis method, colon proliferation polyps, specific genes for proliferation polyps, kit for colon cancer diagnosis

Description

Method for diagnosing colonic proliferative polyps {Method for diagnosing hyperplastic polyp of intestine}

1 is an overall image photograph showing the expression of specific genes in the proliferating polyp tissue and normal mucosa of the present invention.

The present invention relates to a method for diagnosing a new hyperplastic polyp, and more particularly, a diagnostic kit using a specific gene group for colonic proliferative polyps, specific genes for colonic proliferative polyps used therein, and a colon using the same. A method for diagnosing proliferating polyps.

In recent years, the analysis of functional genomes has been highlighted as a new field of study with the development of bioinformatics due to the advance of computer science. It has advanced to the point where it is possible to analyze tens of thousands of samples in half a day, so that many genetic information can be easily analyzed to predict the course of disease for the target disease, and the diagnosis and treatment are based on the genetic predisposition of each patient. Foreshadowing the arrival of "tailored medicine."

The expression of a particular gene can be known indirectly by measuring the mRNA amount of that gene, which is expected to be accompanied by changes in the expression of this gene in almost all diseases. To date, molecular biology techniques have only been able to measure the expression level of one or several genes for a disease or stimulus. Recently, microarray methods have been introduced and started to be distributed, which allows analysis of hundreds to thousands of genes simultaneously, and the increase or decrease of gene expression in a single response. . As described above, the microarray method is quantitative and quantitative in terms of genetic analysis compared to the conventional methods.

In 1995, Pat Brown and David Bostine of Stanford University and their team first introduced biochips, a quantitative measure of gene expression, a kind of microarray method. Since then, tumor research using this method has continued, but as the human genomic project is completed, the need for studying gene expression in large quantities at the genome level has increased.

Specifically, microarrays, like Southern blot or Northern blot analysis, are based on the principle of array based hybridization, where hundreds to thousands of genes are densely aligned onto a substrate such as slide glass rather than nitrocellulose membranes. It is immobilized. It consists of hybridizing cDNA synthesized by reverse transcription from single-stranded DNA immobilized on actual slide glass and mRNA of cells or tissues to be interpreted, and then measuring signal density of spots changed by binding between them. It uses the principle of indirectly measuring the amount of mRNA, a prior step in the synthesis. The advantage of this microarray analysis is that one experiment can detect changes in expression patterns for a large number of genes simultaneously. It is also a new level of high-throughput screening (HTS system) that can be widely applied to gene diagnosis, mutation investigation, drug efficacy and differential diagnosis of diseases.

However, even if the cDNA gene is analyzed using a microarray method, cancer cell-related genes are unstable and the gene expression pattern of cancer cells may change over time. Therefore, questions remain about how to interpret the results of such microarrays. In the future, it is important to select only a few specific genes that can be applied for diagnosis and treatment among various gene groups whose expression is increased or decreased in a specific disease.

Most colorectal cancers arise from adenomatous polyps that already exist, and it is reported that about 10% of patients with these polyps progress slowly into carcinoma polyps throughout their lives. Indeed, most polyps found in the large intestine are non-tumour and appear in the form of hyperplastic polyps that protrude by simple proliferation. So far it has been called hyperplastic polyp, but it is recommended to express it as "proliferative polyp" in recent years in view of the need to improve the term. It is accepted that the proliferating polyps are non-neoplastic polyps and therefore not clinically important because they have no malignant potential, but recently, studies have reported that the proliferating polyps of the lower colon are markers of adenomatous polyps of the upper colon. Attracting attention. Some studies have shared genetic and epidemiological as well as anatomical distribution characteristics with colon adenomas, reporting progenitor disease before adenomas polyps.

Clinically, not all of the small polyps commonly seen through colonoscopy go through adenoma or cancer. The present inventors anticipate that the information on gene expression patterns will differ in cases where tumors do not become tumors and remain as proliferative polyps and progress to adenomas and eventually into cancerous processes. I had done it. In Korea, Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR), RNA dehydrogenase protection assay, and nuclear run-on assay, etc. Some studies have been conducted to analyze the genes related to the polyp using. However, the results of microarray studies of genes related to colonic proliferation polyps have not been reported to date.

Accordingly, the present inventors have continued to apply new molecular biological techniques to clinical diagnosis and treatment. As a result, the microarray method is useful for diagnosing the proliferating polyps of the colon, and the specific genes for the colon proliferating polyps are identified. The present invention has been successfully completed by developing and providing a diagnostic method and a diagnostic kit for colonic proliferation polyps applying the microarray method for screening and measuring the expression of the specific genes.

The present invention aims to provide a diagnostic method for tumors and cancers including colon cancer, which is a new clinical diagnostic method applying a novel molecular biological technique, using expression patterns of specific genes for colonic proliferation polyps.

In order to achieve the above object, the present invention provides a diagnostic kit using a specific gene group for colonic proliferation polyps, specific genes for colonic proliferation polyps used therein, and a method for diagnosing colonic proliferation polyps using the same.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for diagnosing colorectal cancer using a gene specific for a hyperplastic polyp.

Preferably, the present invention relates to the Ras homolog enriched in brain (RHEB) gene, WAAS protein family, member 2 (WASF2), tyrosinase-related protein Tyrosinase-related protein 1 (TYRP1), a visual system. Homeobox-like gene VSX1 (Visual system homeobox 1 homolog), Sacoma virus oncogene-like gene ROS1 (V-ros UR2 sarcoma virus oncogene homolog 1), WEE1 (WEE1 homolog), protein tyrosine kinase TEC (Tec protein tyrosine kinase) And tumor necrosis factor receptor superfamily TNFRSF10A (Tumor necrosis factor receptor superfamily, member 10a).

In addition, the present invention, in addition to the above-described specific genes such as cyaryl transferase SIAT7D (Sialyltransferase 7D), dopamine receptor DRD1 (Dopamine receptor D1), Saaryltransferase SIAT1 (Sialyltransferase 1), intersectin ITSN1 (Intersectin 1), tumor A method for diagnosing colorectal cancer using any one or more selected from the group consisting of a necrosis factor superfamily TNFSF 12 ( Tumor necrosis factor superfamily, member 12 ) and a checkpoint suppressor CHES1 (Checkpoint suppressor 1) is provided.

The diagnostic method of the present invention comprises the steps of: (1) separating the total RNA of the colon polyp patient; (2) preparing cDNA therefrom; (3) performing a hybridization reaction using a slide glass for microarray; (4) measuring the data with the scanner; And (5) analyzing the image with various analysis programs; And so on.

In the above process, genes including GAPDH, β-actin gene and the like are used to normalize signal data, and the basal signal intensity is determined using yeast DNA. At this time, if it is at least two times higher than the basic signal strength, it is determined as a meaningful value.

The present invention also provides a diagnostic kit for detecting a variety of tumors and cancers, including colon cancer and the like associated with colonic proliferation polyps using a specific gene for the hyperplastic polyp.

The diagnostic kit of the present invention includes the Ras homolog enriched in brain (RHEB) gene, WAAS protein family, member 2 (WASF2), tyrosinase-related protein tyrosinase-related protein 1 (TYRP1), Visual system homeobox-like gene VSX1 (Visual system homeobox 1 homolog), Sacoma virus oncogene-like gene ROS1 (V-ros UR2 sarcoma virus oncogene homolog 1), WEE1 (WEE1 homolog), protein tyrosine kinase Tec protein tyrosine kinase) and tumor necrosis factor receptor superfamily TNFRSF10A (Tumor necrosis factor receptor superfamily, member 10a) is characterized in that the detection of any one or more selected from the group is overexpressed and to diagnose colorectal cancer.

In addition, the diagnostic kit of the present invention is a cyaryl transferase SIAT7D (Sialyltransferase 7D), dopamine receptor DRD1 (Dopamine receptor D1), a aryltransferase SIAT1 (Sialyltransferase 1), intersectin ITSN1 (Intersectin 1), tumors Necrosis factor superfamily TNFSF 12 ( Tumor necrosis factor superfamily, member 12 ) and the checkpoint suppressor CHES1 (Checkpoint suppressor 1) by detecting the decrease in expression of any one or more of the group characterized in that it is characterized by diagnosing colorectal cancer do.

The present invention (1) cDNA preparation reagents; (2) gene marker reaction reagents; (3) gene integration reactor; And / or (4) genetic analysis instrument; It provides a kit for diagnosing colorectal cancer for detecting the specific gene for colonic proliferation polyps by a microarray method.

Specifically, the present invention is to prepare a cDNA by separating the total RNA of the colon polyp patient; Attach a genetic label; The hybridization reaction proceeds with a slide glass for microarray; Signal data is measured with a scanner and image analysis is performed with various analysis programs to diagnose proliferative polyp-related diseases including colon cancer. At this time, the signal data is normalized using genes including GAPDH, β-actin gene, and the like, and the basal signal intensity is determined by using yeast DNA.

In addition, the present invention, in addition to the microarray method, Northern blot, polymerase chain reaction, reverse transcriptase-polymerase chain reaction, RT-PCR, RNAase protection assay In addition, a restriction enzyme fragment polymorphism analysis (RFLP) and / or a nuclear run (nuclear run-on assay) may be performed to provide a kit for diagnosing colorectal cancer for detecting the specific gene for colonic proliferation polyps. As described above, all diagnostic kits for detecting various tumors and cancers including colorectal cancer by detecting the specific genes and their expression patterns of the colon proliferation polyps are included in the scope of the present invention.

In addition, the (1) cDNA preparation reagent; (2) gene marker reaction reagents; (3) gene integration reactor; And / or (4) the genetic analysis instrument employs all reagents, reactors, and instruments that are naturally known to those skilled in the art, including conventionally used and reported commercially available reagents, reactors, and instruments, and includes genes specific for said propagation polyps. Diagnostic kits are included within the scope of this invention.

In addition, the present invention is the specific gene, that is, Ras-like gene Ras homolog enriched in brain (RHEB), wass protein family (WASF2, member 2), tyrosinase-related protein Tyrosinase-related protein 1 (TYRP1), visual system call Meobox-like gene VSX1 (Visual system homeobox 1 homolog), Sacoma virus oncogene-like gene ROS1 (V-ros UR2 sarcoma virus oncogene homolog 1), WEE1 (WEE1 homolog), protein tyrosine kinase TEC (Tec protein tyrosine kinase) and / Or Tumor necrosis factor receptor superfamily TNFRSF10A (Tumor necrosis factor receptor superfamily, member 10a) alone or in combination to provide for use in the diagnosis of various tumors and cancers, including proliferative polyps and colorectal cancer.

In addition, the present invention is the specific gene, that is, cyclyl transferase SIAT7D (Sialyltransferase 7D), dopamine receptor DRD1 (Dopamine receptor D1), aryl transferase SIAT1 (Sialyltransferase 1), intersectin ITSN1 (Intersectin 1), tumor necrosis factor Use for diagnosis of various tumors and cancers, including proliferative polyps and colorectal cancer, alone or in combination with superfamily TNFSF 12 ( Tumor necrosis factor superfamily, member 12 ) and / or checkpoint suppressor 1 (CHES1). To provide.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Diagnostic Samples for Colon Proliferation Polyps

In the present invention, first, the patients who have undergone colonoscopy in the last few months were screened for colonic polyps of 1 cm or less and determined to conduct genetic screening. In addition, the pathologically histologically confirmed hyperplastic polyp among 36 samples, which were judged to be capable of further experiments due to good RNA status in RNA quality control, that is, 18 pairs of normal mucosa and polyp tissues. Five patients were selected. As a result, the mean age of 5 patients was 65.4 years on average and the sex ratio of male and female was 4: 1 (see Table 1). Histopathologically, the polyps were all measured to be 1 cm or less in diameter and the average size was 0.6 (0.5-0.9) cm in diameter.

Polyps found from the patient were resected in principle noose, and small polyps up to 5 mm in diameter were excised to a size of at least 75% using biopsy forceps. The size of the polyps was measured based on the width of the biopsy forceps. Five pairs of samples extracted from the final screened patients were prepared as the diagnostic sample of the present invention as follows. Half of the polyp tissue is RNA later ^® to prevent damage to RNA; was transported to the dipping (Am by-products on - Austin), a cold storage for one day in less than 4 ℃, then quick-frozen in liquid nitrogen and the laboratory vessel.

[Table 1] Characteristics of Patient Polyps

Example 2 Fluorescent Labeling and Hybridization of Specific Genes for Colon Proliferation Polyps

In the present invention, in order to develop a method for diagnosing colonic proliferation polyps using a specific gene, a microarray analysis using a GenoCheck Platinum Biochip Human cancer 3.0K oligochip is performed as follows. It was performed together.

Total RNA was extracted from 5 cases of histopathologically confirmed proliferating polyp specimens using the Trizol reagent (manufactured by Zipco BRL, USA), ie, samples of patients selected in Example 1 above. 400 units of Superscript RNase H-reverse transcriptase (from Zipco RBL; USA), oligo (dT) _18, 0.5 mM dATP and dTTP, 0.2 mM dCTP and dGTP, 0.1 mM Cy3 or Cy5, labeled with 30 μg total RNA A total of 30 μl of fluorescently labeled cDNA probe was synthesized by addition of a reverse transcription mixture consisting of (NEN Life Sciences; USA). After the reverse transcription reaction, 5 μl of reaction stopper (0.5 M NaOH / 50 mM EDTA) was added thereto and reacted at 65 ° C. for 10 minutes. The Cy3 or Cy5 fluorescently labeled cDNA was precipitated using ethanol and concentrated, followed by 12 μl of hybridization buffer (2 × Denhardt's solution, 4.5% SDS, 1 × SSC, 1 mM EDTA, 0.25 M Na ₂ HPO ₄ , 0.05 mg / ml yeast tRNA), respectively. Then, the two cDNAs synthesized above were mixed in the same amount, denatured at 95 ° C. for 2 minutes, and reacted at 45 ° C. for 20 minutes.

The cDNA mixture was placed on a slide glass (Geneno Co., Ltd., Korea) in which DNAs of 3,000 genes were accumulated, covered with a cover slip (22 mm × 22 mm), and then the mixture was well distributed on the slide glass. Treated. Then, the slide was placed in a reaction chamber, and the hybridization reaction was performed at 62 ° C. for 14 hours. After the reaction, the mixture was first washed at room temperature using 2 × SSC solution and 0.1% SDS solution for 2 minutes, and then again at 1 × SSC. Washed for 3 minutes in solution and 2 minutes in 0.2 × SSC solution.

Example 3 Analysis of Diagnostic Results Using Specific Genes for Colon Proliferation Polyps

In the present invention, the result of hybridization using a specific gene in the above to diagnose colon proliferation polyps was scanned and analyzed as follows.

Specifically, the hybridized slide glass was scanned using a GenePix 4000B scanner (Axon, California, USA). The scanned results were then image analyzed using analytical programs of GenePix Pro 3.0 (Axon, California, USA) and GeneSpring (Silicon Genetics, CA, USA). At this time, the analysis of individual spots was performed using the algorithm built into GenePix, and the normalization of the data was performed using the signals of GAPDH and β-actin genes, which are housekeeping genes. The background signal intensity was determined using yeast DNA accumulated on each slide, and it was determined that the values that are at least two times higher than the basic signal intensity were meaningful. Also, in order to increase the accuracy of the data, the value that the signal / noise ratio is less than 3 is excluded. The experimental results obtained were interpreted through the correlation analysis of genes using the Tree View microarray software program.

In addition, to confirm the accuracy of the analysis, statistical analysis was performed as follows. Statistical differences between the experimental and control groups were compared using the t-test, and the significance level for multiple comparisons was determined according to Bonferroni adjustment.

As a result, a total of 67 genes were analyzed to show differences in gene expression in the proliferating polyps. In particular, 34 of these genes were overexpressed and 33 were underexpressed. Specifically, Ras homolog enriched in brain (RHEB), WAS protein family, member 2 (WASF2), tyrosinase-related protein TYRP1 (TYRP1), visual system homeobox-like gene VSX1 (Visual system homeobox 1 homolog), Sacoma virus oncogene-like gene ROS1 (V-ros UR2 sarcoma virus oncogene homolog 1), WEE1 (WEE1 homolog), protein tyrosine kinase TEC (Tec protein tyrosine kinase) and tumor necrosis factor receptor super Eight gene groups, including the family TNFRSF10A (Tumor necrosis factor receptor superfamily, member 10a), were significantly overexpressed by more than 10-fold. In addition, cyaryl transferase SIAT7D (Sialyltransferase 7D), dopamine receptor DRD1 (Dopamine receptor D1), tetraaryl transferase SIAT1 (Sialyltransferase 1), intersectin ITSN1 (Intersectin 1), tumor necrosis factor superfamily TNFSF 12 ( Tumor necrosis factor) superfamily, member 12 ), and six gene groups, including the checkpoint suppressor 1 (CHES1), were significantly underexpressed by 10-fold or less.

Figure 1 shows the overall image of the expression of specific genes in the proliferating polyp tissue and normal mucosa of the present invention. In Figure 1, the red line shows the degree of overexpression and the blue line shows the degree of underexpression, respectively.

As described above, the diagnostic kit using the specific gene group for the new colon proliferation polyps of the present invention, the specific genes for the colon proliferation polyps used therein, and the method for diagnosing the colon proliferation polyps using the same are conventional microarray methods. By effectively analyzing the expression patterns of the specific genes, it can be widely used in the diagnosis of various tumors and cancers, including colorectal cancer associated with colonic proliferation polyps.

Claims (7)

A kit for diagnosing colorectal cancer using a specific gene for a hyperplastic polyp. The method of claim 1, The specific genes include Ras homolog enriched in brain (RHEB), WAS protein family, member 2 (WASF2), tyrosinase-related protein Tyrosinase-related protein (TYRP1), and visual system homeobox-like gene. Visual system homeobox 1 homolog (VSX1), V-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), WEE1 homolog (WEE1), protein tyrosine kinase TEC (Tec protein tyrosine kinase) and tumor necrosis factor receptor The superfamily TNFRSF10A (Tumor necrosis factor receptor superfamily, member 10a) of any one or more selected from the group consisting of colon cancer diagnostic kit, characterized in that the overexpression. The method according to claim 1 or 2, The specific genes are siaryl transferase SIAT7D (Sialyltransferase 7D), dopamine receptor DRD1 (Dopamine receptor D1), tetraaryl transferase SIAT1 (Sialyltransferase 1), intersectin ITSN1 (Intersectin 1), tumor necrosis factor superfamily TNFSF 12 ( Tumor) Necrosis factor superfamily, member 12 ) and the checkpoint suppressor CHES1 (Checkpoint suppressor 1) A diagnostic kit for colorectal cancer, characterized in that any one or more selected from the group consisting of. The method according to claim 1, wherein (1) cDNA preparation reagents; (2) gene marker reaction reagents; (3) gene integration reactor; And / or (4) a genetic analysis instrument. Colorectal cancer diagnostic method for detecting the expression pattern of a specific gene for colonic proliferation polyps. The method of claim 5, The specific genes include Ras homolog enriched in brain (RHEB), WAS protein family, member 2 (WASF2), tyrosinase-related protein TYRP1, and visual system homeobox-like gene VSX1. (Visual system homeobox 1 homolog), Sacoma virus oncogene-like gene ROS1 (V-ros UR2 sarcoma virus oncogene homolog 1), WEE1 (WEE1 homolog), protein tyrosine kinase TEC (Tec protein tyrosine kinase) and tumor necrosis factor receptor super Colorectal cancer diagnostic method, characterized in that at least one selected from the group consisting of a family TNFRSF10A (Tumor necrosis factor receptor superfamily, member 10a). The method of claim 5, The specific genes are siaryl transferase SIAT7D (Sialyltransferase 7D), dopamine receptor DRD1 (Dopamine receptor D1), tetraaryl transferase SIAT1 (Sialyltransferase 1), intersectin ITSN1 (Intersectin 1), tumor necrosis factor superfamily TNFSF 12 ( Tumor) necrosis factor superfamily, member 12 ) and the checkpoint suppressor CHES1 (Checkpoint suppressor 1), characterized in that any one or more selected from the group consisting of colon cancer.

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