KR101007576B1 - Colon cancer diagnostic markers using up-regulated genes - Google Patents

Abstract

The present invention relates to diagnostic markers specific for colorectal cancer. The present invention also relates to compositions, kits, and methods for diagnosing colorectal cancer using the formulations comprising agents for measuring the presence of the marker.

Description

Colon cancer diagnostic markers using up-regulated genes

The present invention relates to a composition, a kit including an agent for selecting a diagnostic marker specific for colon cancer and measuring the presence of the marker, and a method for diagnosing colon cancer using the same.

The large intestine is a place where food ingested by mouth is digested and absorbed, and is also a place where excess food stays. In addition, it absorbs moisture and makes feces, and it is also a place where many types of bacteria live. In humans, the length of the large intestine is about 2m and consists of a colon, rectum, and anus. Wherever there is the colonic mucosa, cancer can occur, but the areas prone to cancer are the S colon and rectum.

Currently, the incidence of colorectal cancer in Korea is increasing remarkably. The deaths from colon cancer are the fourth in men after gastric cancer, lung cancer, and liver cancer, and are similar in women. The incidence of colorectal cancer is being investigated for men to be more frequent than women. By age, 50s are the most, followed by 60s. Compared to Europe and the United States, Korea tends to be about 10 years younger than the age of onset. It occurs even in young people in their 30s with a frequency of 5%-10%, and colorectal cancer, which appears in younger people, tends to occur frequently among families. It is believed that environmental factors affect the incidence of colon cancer more than genetic factors, and it is recognized that the rapid westernization of dietary life, especially excessive intake of animal fats and proteins, is the cause. However, it is known that colon cancer around 5% is caused by a genetic predisposition. Taken together, risk factors prone to colorectal cancer include: 1) experience of colon polyps, 2) family history, 3) long-term ulcerative colitis, 4) difficult to cure fistula, etc. There is this.

The Dukes classification method and the stage classification method of UICC are typical for colorectal cancer. The standard is not based on the size of the cancer, but the degree of progression is defined according to the depth of the cancer entering the colon wall and the presence or absence of distant metastasis. Tables 1 and 2 below describe the classification criteria of the Dukes classification method and the stage classification method, respectively.

Features of the Dukes classification method designation 5-year survival rate after surgery Pathological aspect Dukes A over 90 Cancer staying within the walls of the large intestine Dukes B 60~80% Cancer has penetrated the colon wall, but lymph node metastasis has not occurred. Dukes C 20-50% Lymph node metastasis has occurred Dukes D Less than 20% Distant metastasis to the peritoneum, liver, lungs, etc.

Characteristics of stage classification designation Pathological aspect Period 0 Cancer staying in the mucous membrane 1st Cancer staying in the wall of the large intestine 2nd The cancer has crossed the wall of the large intestine but has not reached the adjacent organs. Stage 3 Cancer infiltrating adjacent organs or lymph node metastasis 4th Distant metastasis to the peritoneum, liver, lungs, etc.

Because there is only a very small difference between the two classification methods, it is currently accepted that Dukes A corresponds to the 0 stage, 1 stage, Dukes B stage 2, Dukes C stage 3, and Dukes D stage 4, especially. The Dukes classification is an internationally widely used method.

Colon cancer can be completely cured by appropriate endoscopy or surgical therapy at early detection, and even if it has progressed and metastasized to the liver or lungs (remote metastasis), if the operation is possible, a cure by surgical therapy can be expected. In other words, among the current treatments, surgical treatment is the most effective. However, if the detection is delayed, since the metastasis to the lungs, liver, lymph nodes, or peritoneum, which is difficult to be resected, cannot be used as in the above case, surgery cannot be used. Ultimately, early diagnosis and treatment are required for effective treatment of colon cancer. I would say it is essential.

As a surgical treatment, recurrence often occurs after surgery. After surgery, regular examinations should be taken at intervals of 3-4 months to check for recurrence. Among the organs in the body, the liver, lungs, and peritoneum are organs that are prone to recurrence, and may recur locally at the resected site. Compared to other cancers, colorectal cancer recurs quickly and can be completely cured by resecting the recurrent lesion. Since more than 80% of recurrences are detected within 3 years after surgery, it is the standard of cure that does not recur within 5 years after surgery.

Colorectal cancer is cured almost 100% if it is early, but it is very difficult to detect when asymptomatic because there are generally no subjective symptoms, so periodic examination should be preceded. The representative screening test for colorectal cancer is the occult blood test, but just because a positive test for this test does not mean that you have colorectal cancer, and conversely, a negative response does not mean that you do not have colorectal cancer. It is unreasonable to be used for accurate diagnosis. Therefore, the colorectal cancer test method that guarantees effective results currently in use is shown in Table 3 below.

Colon cancer test Inspection name Inspection method and features Colonoscopy After eating restriction, the organ is sufficiently emptied, and barium and air are injected from the anus to take an X-ray picture to examine the shape of the intestine. Colonoscopy There are short endoscopes to observe the S-colon, and long endoscopes to observe all the large intestines down to the appendix.
The polyp may be removed immediately at the same time as the test Tumor marker How to use blood tests to diagnose cancer that is hiding somewhere in the body
However, to date, there are no tumor markers that can detect colon cancer early.
A marker called CEA is common, but only about half of them are positive in the presence of colon cancer.
Used as an index to determine the progression and treatment effect of colorectal cancer Radiation diagnosis
(CT, MRI,
Ultrasound, etc.) Used to determine the degree of progression of the primary lesion and whether it has distant metastases to the liver.

To date, a tumor marker peculiar to colon cancer that can detect colon cancer at an early stage has not been identified. Of course, there is a marker called CEA, but as mentioned in Table 3 above, only about half of the colon cancer is positive, so it is mainly used as an index to determine the progression and treatment effect of colon cancer. It is somewhat unreasonable to be used as a marker for diagnosis.

Accordingly, the present inventors produced DNA chips for genes expected to be related to colon cancer, and compared the expression levels in gastric cancer, breast cancer, prostate cancer, and liver cancer as well as colon cancer. As a result, it was possible to select genes that are specifically overexpressed only in colorectal cancer tissues, and finally select genes that are possible as diagnostic markers for colorectal cancer, and through these, it was confirmed that colorectal cancer can be diagnosed early and accurately, and the present invention Was completed.

One object of the present invention is CHEK1 (CHK1 checkpoint homolog (S. pombe), GenBank No. NM_001114121), MAD2L1 (MAD2 mitotic arrest deficient-like 1 (yeast), GenBank No. NM_002358), KRT23 (keratin 23 (histone deacetylase) inducible), GenBank No. NM_015515), PROX1 (prospero homeobox 1, GenBank No. NM_002763), SLC7A11 (solute carrier family 7, (cationic amino acid transporter, y+ system) member 11, GenBank No. NM_014331), ASB9 (ankyrin repeat and SOCS box-containing 9, GenBank No. NM_001031739), ENC1 (ectodermal-neural cortex (with BTB-like domain), GenBank No. NM_003633), PLAU (plasminogen activator, urokinase, GenBank No. NM_001145031), RNF183 (ring finger protein 183, GenBank No. NM_145051), CSEIL (CSE1 chromosome segregation 1-like (yeast), GenBank No. NM_001316), ATAD2 (ATPase family, AAA domain containing 2, GenBank No. NM_014109), RAD51AP1 (RAD51 associated protein 1, GenBank No. NM_001130862), C60RF173 (chromosome 6 open reading frame 173, GenBank No. NM_001012507), and CCNB1 (cyclin B1, GenBank No. NM_031966) selected from One or more colon cancer diagnostic markers are provided.

Another object of the present invention is to measure the mRNA or protein level thereof of one or more genes selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1. It is to provide a composition for diagnosing colorectal cancer comprising an agent.

Another object of the present invention is to provide a colon cancer diagnostic kit comprising the composition for diagnosing colon cancer.

Another object of the present invention is to provide a method for diagnosing colon cancer using the composition or kit for diagnosing colon cancer.

In one embodiment, the present invention relates to at least one colon cancer diagnostic marker selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1.

In the present invention, the term "diagnosis" means to confirm the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether the onset of colon cancer.

In the present invention, the term "colon cancer" refers to cancer arising from the mucous membrane, which is the innermost surface of the large intestine, and collectively refers to rectal cancer, colon cancer, and anal cancer.

In the present invention, the term “diagnosis marker, diagnostic marker, or diagnostic marker” refers to a substance capable of diagnosing colorectal cancer cells by distinguishing them from normal cells, and is an increase in colorectal cancer cells compared to normal cells. And organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.). For the purposes of the present invention, colon cancer diagnostic markers are CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1, which are genes whose expression increases in colon cancer cells. .

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. A significant diagnostic marker refers to a marker with high reliability so that the result obtained by diagnosis is accurate and thus has high validity and consistent results even when measured repeatedly. The colon cancer diagnostic marker of the present invention is genes whose expression is always increased due to direct or indirect factors along with the onset of colon cancer, and shows the same result even in repeated experiments, and the difference in expression level is very large compared to the control group, resulting in an incorrect result. These are highly reliable markers with little probability of dropping. Therefore, the diagnosis result based on the result obtained by measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

In this case, except for genes expressed in almost the same amount in normal colon epithelial cells and colon cancer cells, for example, the expression in colon cancer tissues is 2-9 times higher than that of genes expressed in normal tissues used as a control. Genes to be increased were selected.

In another aspect, the present invention is the expression level or protein level of one or more genes selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1. It relates to a composition for diagnosing colorectal cancer comprising an agent to be measured.

The level of gene expression in a biological sample can be ascertained by checking the amount of mRNA or protein.

In the present invention, “measurement of mRNA expression level” is a process of determining the presence and expression level of mRNA of colon cancer marker genes in a biological sample in order to diagnose colorectal cancer, and measures the amount of mRNA. Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), and RNase protection assay (RPA; RNase protection). assay), Northern blotting, DNA chip, etc., but are not limited thereto. In the present invention, “protein expression level measurement” is a process of determining the presence and expression level of a protein expressed from a colon cancer marker gene in a biological sample in order to diagnose colorectal cancer. The amount of protein can be confirmed by using an antibody that binds to it. Analysis methods for this include western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket. Immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited thereto. .

Accordingly, in a specific aspect of the present invention, a primer sequence specific to one or more genes selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1 is included. It provides a colorectal cancer diagnostic marker composition.

The term "primer" of the present invention refers to a short nucleic acid sequence having a short free three-terminal hydroxyl group, capable of forming a base pair with a complementary template, and serving as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers serving as the starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, “encapsulation”, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (eg, methyl phosphonate, phosphotriester, phos Poroamidate, carbamate, etc.) or to a charged linker (eg phosphorothioate, phosphorodithioate, etc.). Nucleic acids may be one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.). ), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequence of the present invention can also be modified using a label that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, and biotin.

According to a specific embodiment of the present invention, the composition for detecting a diagnostic marker for colon cancer is 1 selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1. Each primer pair specific for the above genes is included. Specifically, these are the primers represented by SEQ ID NOs: 1 to 28 in the nucleotide sequence list.

In another aspect, the present invention is a colorectal cancer comprising an antibody specific for one or more proteins selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1. A diagnostic marker composition is provided.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since the colon cancer marker protein has been identified as described above, generating an antibody using it can be easily prepared using techniques well known in the art.

The polyclonal antibody can be produced by a method well known in the art by injecting the above-described colon cancer marker protein antigen into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow and dog.

Monoclonal antibodies are hybridoma methods well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6:511-519), or phage antibody library (Clackson et al, Nature, 352:624 -628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

In addition, the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. A functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv. In another aspect of the present invention, it provides a colon cancer diagnostic kit comprising the composition for diagnosing colon cancer according to the present invention. Preferably, the colorectal cancer diagnostic kit may be configured to further include one kind or more other component compositions, solutions, or devices suitable for the analysis method.

As a specific aspect, the diagnostic kit may be a diagnostic kit comprising essential elements necessary to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. In addition, a primer specific to the nucleic acid sequence of the control gene may be included. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitors DEPC. -May include DEPC-water, sterilized water, etc.

In another aspect, it may be a diagnostic kit characterized in that it preferably contains essential elements necessary to perform a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, and the like for preparing a fluorescently labeled probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

Also preferably, it may be a diagnostic kit characterized in that it contains the essential elements necessary to perform the ELISA. The ELISA kit contains antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity with other proteins, and are monoclonal, polyclonal, or recombinant antibodies. In addition, the ELISA kit may contain an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or antibodies capable of binding. Other materials may be included.

In another aspect, the present invention provides a method for diagnosing colorectal cancer using the composition for diagnosing colorectal cancer or the colorectal cancer diagnosis kit.

As a specific aspect, the present invention uses a primer specific for one or more genes selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1. Measuring mRNA from a biological sample of a patient suspected of cancer; And comparing the increase in the mRNA level with the mRNA level of a normal control sample.

The process of separating mRNA from a biological sample can be performed using a known process, and the level of mRNA can be measured by various methods.

In the present invention, the term "biological sample" includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine whose gene expression levels of colon cancer markers differ due to colon cancer, but this Not limited.

Analysis methods for measuring mRNA levels include reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, DNA chip, etc., but are not limited thereto.

Through the above detection methods, it is possible to compare the mRNA expression level in the normal control and the mRNA expression level in the suspected colorectal cancer patient, and determine whether a significant increase in the amount of expression from the colorectal cancer marker gene to the mRNA is determined to You can diagnose whether you develop colorectal cancer.

The mRNA expression level is preferably measured using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for a gene used as a colon cancer marker.

In the reverse transcriptase polymerase reaction, by checking the band pattern and the thickness of the band by electrophoresis after the reaction, it is possible to check the mRNA expression and degree of the gene used as a diagnostic marker for colorectal cancer. Can be easily diagnosed (Figs. 8 to 11). Meanwhile, the DNA chip uses a DNA chip in which a nucleic acid corresponding to the colon cancer marker gene or its fragment is attached at high density to a glass-like substrate, and the mRNA is separated from the sample, and the end or the inside thereof is labeled with a fluorescent substance. A cDNA probe is prepared, hybridized to a DNA chip, and then the onset of colon cancer can be read.

In another specific embodiment, the present invention uses an antibody specific for one or more proteins selected from CHEK1, MAD2L1, KRT23, PROX1, SLC7A11, ASB9, ENC1, PLAU, RNF183, CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1 for colon cancer. It provides a method for diagnosing colorectal cancer comprising the steps of confirming the protein level by contacting a biological sample of a suspected patient to form an antigen-antibody complex, and comparing the increase in the amount of protein formation with the protein level of a normal control sample.

The process of separating a protein from a biological sample can be performed using a known process, and the protein level can be measured by various methods.

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, Okteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but are not limited thereto.

Through the above analysis methods, it is possible to compare the amount of formation of the antigen-antibody complex in the normal control and the amount of the antigen-antibody complex in the suspected colorectal cancer patient, and a significant increase in the amount of expression from the colon cancer marker gene to the protein. By judging whether or not, it is possible to diagnose whether a patient suspected of colon cancer actually develops colorectal cancer.

In the present invention, the term “antigen-antibody complex” refers to a combination of a colon cancer marker protein and an antibody specific thereto, and the amount of formation of the antigen-antibody complex is quantitatively measured through the size of the signal of the detection label. It is possible.

The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but is not limited thereto. When an enzyme is used as a detection label, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese. Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phospholpyruvate deca But not limited to, but not limited to, boxylase, β-latamase, and the like. Fluorescein includes fluorescein, isothiocyanate, rhodamine, phycoerytherin, phycocyanine, allophycocyanin, o-phthaldehyde, and fluorescarmine, but is not limited thereto. Ligands include biotin derivatives, but are not limited thereto. The luminescent material includes, but is not limited to, acridinium ester, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, but are not limited thereto. Redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W(CN) ₈ , [Os(bpy) ₃ ] ²⁺ ,[RU(bpy) ₃ ] ²⁺ , [MO(CN) ₈ ] ^4-, etc. are included, but are not limited thereto. Radioisotopes include, but are not limited to, ^{3 H, 14} C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, etc. .

The protein expression level is preferably measured using an ELISA method. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes the capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and a solid support. Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen, and a labeled that recognizes this antibody after reacting with another antibody that recognizes the antigen in a complex of an antibody attached to a solid support and an antigen. It includes various ELISA methods such as indirect sandwich ELISA using secondary antibodies. More preferably, after attaching the antibody to a solid support and reacting the sample, a labeled antibody that recognizes the antigen of the antigen-antibody complex is attached to develop enzymatically or against the antibody that recognizes the antigen of the antigen-antibody complex. It is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and colored enzymatically. By checking the degree of formation of a complex between the colon cancer marker protein and the antibody, it is possible to determine whether or not colon cancer has occurred.

In addition, preferably, Western blot using at least one antibody against the colon cancer marker is used. The whole protein is separated from the sample, electrophoresed to separate the protein according to its size, and then transferred to a nitrocellulose membrane to react with the antibody. By confirming the amount of the generated antigen-antibody complex using a labeled antibody, the amount of protein generated by the expression of the gene can be checked to confirm whether or not colon cancer has occurred. The detection method comprises a method of examining the expression level of the marker gene in a control group and the expression level of the marker gene in cells with colon cancer. The mRNA or protein level can be expressed as an absolute (eg, μg/ml) or relative (eg, relative intensity of a signal) difference between the above-described marker protein.

In addition, preferably, immunohistochemistry using one or more antibodies against the colon cancer marker is performed. After collecting and fixing normal colon epithelial tissue and tissue suspected of colon cancer, a paraffin-embedded block is prepared by a method well known in the art. These are made into slices several um thick and attached to a glass slide, and then reacted with one selected from the above antibodies by a known method. Thereafter, the unreacted antibody is washed, labeled with one of the above-mentioned detection labels, and whether or not the antibody is labeled is read under a microscope.

In addition, preferably, at least one antibody against the colon cancer marker is arranged at a predetermined position on a substrate to use a protein chip that is immobilized at high density. The method of analyzing a sample using a protein chip involves separating a protein from the sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading this, and confirming the presence or expression of the protein, You can check whether you have colon cancer.

The present invention provides useful data for the treatment and prognosis of colon cancer by providing diagnostic markers that can determine the metastasis and prognosis of colon cancer. In addition, the colon cancer diagnostic markers may be used for research on the development of an anticancer drug specific for colon cancer.

1 is a result of comparing the expression of 2,230 genes in normal colon tissue and colon cancer tissue using a 48K human microarray chip (Illumina) (p value <0.05). DF at the top of the figure means Disease-free and RC means Recurrent.
2 is a diagram of genes specifically overexpressed in colon cancer tissues based on the analysis results of a 48K human microarray chip (Illumina).
3 to 6 are examples of comparative analysis (data mining) of various microarray data published based on the microarray results of a colorectal cancer-specific gene.FIG. 3 is a CHEK1 gene, FIG. 4 is a MAD2L1 gene, and FIG. 5 is KRT23 gene, and Figure 6 is a comparative analysis result of the PROX1 gene.
7 is a result of comparing gene overexpression using a DNA chip targeting the same genes in colorectal cancer, gastric cancer, breast cancer, prostate cancer, and liver cancer.
8 is an electrophoresis picture confirming the expression levels of CHEK1, MAD2L1, KRT23, PROX1 and SLC7A11 diagnostic markers in normal tissues and colon cancer tissues using reverse transcription polymerase reaction (odd lanes are normal tissues, even lanes are cancer tissues) .
9 is an electrophoresis picture confirming the expression levels of ASB9, ENC1, PLAU and RNF183 diagnostic markers in normal tissues and colon cancer tissues using reverse transcription polymerase reaction (odd lanes are normal tissues, even lanes are cancer tissues).
Figure 10 is an electrophoresis picture confirming the expression levels of CSEIL, ATAD2, RAD51AP1, C60RF173 and CCNB1 diagnostic markers in normal tissues and colon cancer tissues using reverse transcription polymerase reaction (odd lanes are normal tissues, even lanes are cancer tissues) .
11 shows 10 colon cancer cell lines (Lane 1, DLD-1; Lane 2, HT29; Lane 3, HCT116; Lane 4, colo205; Lane 5, SW480;
Lane 6, SW620; Lane 7, SNU C1; Lane 8, SNU C2A; Lane 9, KM 12C; Lane 10, KM 12SM).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example One. DNA Discovery of overexpressing genes for colon cancer using chips

Compared to normal colon epithelial cells, in order to primarily extract genes that are specifically overexpressed only in colon cancer cells, the expression level of 2,230 genes was investigated using a DNA chip (48K human microarray, Illumina).

First, total RNA was extracted from normal colon epithelial cells and colon cancer cells. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN), and quantified using an Experion RNA StdSens (Bio-Rad) chip. For hybridization, the extracted total RNA was used as an Illumina TotalPrep RNA Amplification Kit (Ambion). CDNA was synthesized using T7 Oligo (dT) primer, and in vitro transcription was performed using biotin-UTP to prepare biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. The cRNA prepared from normal colon epithelial cells and colon cancer cells was hybridized to a Human-6 V2 (Illumina) chip. After hybridization, in order to remove non-specific hybridization, the DNA chip was washed using Illumina Gene Expression System Wash Buffer (Illumina), and the washed DNA chip was labeled with streptavidin-Cy3 (Amersham) fluorescent dye. The fluorescence-labeled DNA chip was scanned using a confocal laser scanner (Illumina) to obtain fluorescence data present in each spot, and stored as a TIFF image file. The TIFF image file was quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence value of each spot. The quantified result was corrected using the'quantile' function with the Avadis Prophetic version 3.3 (Strand Genomics) program.

Through the analysis of the expression level of 1,601 genes obtained by the above process, the gene expression patterns of normal colon epithelial cells and colon cancer cells were compared and analyzed. At this time, a hierarchical clustering analysis was used, and as a result, it was found that normal colon epithelial cells and colon cancer cells are largely divided into two clusters (FIG. 1).

In addition, compared to normal and colonic epithelial cells, genes showing a difference in expression of more than twofold in more than 60% of patients were found (Table 4), of which 281 and 605 overexpression and underexpression were respectively, gastric cancer and breast cancer. , As a result of comparing the expression of the same genes in pancreatic cancer (Fig. 7), genes that are specifically expressed only in colon cancer were finally selected (Figs. 3 to 6 and Figs. 8 to 11).

Genes overexpressed in colorectal cancer Gene symbol Unigene Cluster Log2( FC ) P value MMP7 Hs .2256 9.268 7.74E-08 CST1 Hs .123114 7.686 6.19E-04 CDH3 Hs .461074 5.874 0.025 CLDN1 Hs .439060 5.748 2.29E-05 ESM1 Hs .129944 5.701 0.022 DPEP1 Hs .109 5.379 0.002 CLDN2 Hs .522746 5.262 2.14E-06 HS6ST2 Hs .385956 4.201 1.47E-06 ABHD7 Hs .201555 3.743 4.05E-08 SLC35D3 Hs .369703 3.692 0.004 CXCL1 Hs .789 3.624 3.84E-09 CDCA1 - 1.76 5.37e-06 CA9 Hs .63287 3.47 0.001 VSNL1 Hs .444212 3.159 3.28E-04 MMP1 Hs .83169 3.116 0.032 ANLN Hs .62180 3.091 0.021 LCN2 Hs .204238 3.059 0.001 TPX2 Hs .244580 3.029 0.035 EGFL6 Hs .12844 2.974 0.009 LGR5 Hs .658889 2.899 6.75E-07 KLK6 Hs .79361 2.842 1.68E-05 COMP Hs .1584 2.818 2.98E-06 NLF1 Hs .202656 2.799 3.92E-06 MAD2L1 Hs .591697 2.787 0.002 PROX1 Hs .585369 2.731 1.46E-09 CKS2 Hs .83758 2.522 0.001 MELK Hs .184339 2.517 0.003 HCAP -G - 2.509 0.045 OLR1 Hs .412484 2.503 0.002 ATAD2 Hs .370834 2.467 0.005 PROSAPIP1 Hs .90232 2.35 1.53E-07 SOX9 Hs .694731 2.318 8.75E-08 MMP12 Hs .1695 2.306 0.022 CCNB1 Hs .23960 2.293 0.046 KRT23 Hs .9029 2.241 1.14E-04 ASB9 Hs .19404 2.209 2.44E-05 CHEK1 Hs .24529 2.201 2.94E-09 CSE1L Hs .90073 2.185 6.10E-06 SLC7A11 Hs .390594 2.105 3.05E-05 ENC1 Hs .104925 2.059 0.004 PLAU Hs .77274 2.001 0.003 HIG2 Hs .433213 1.995 2.96E-04 RNF183 Hs .211374 1.97 7.35E-07 RAD51AP1 Hs .591046 1.932 0.021 RRM2 Hs .226390 1.818 0.044 UBE2S - 1.818 3.66E-09 PCSK9 Hs .18844 1.77 9.31E-07 C6ORF173 Hs .486401 1.734 5.45E-09 SLC12A2 - 1.728 0.003 TSTA3 Hs .404119 1.716 0.006 TGFBI Hs .369397 1.711 1.02E-04 PRDX4 Hs .83383 1.281 0.038

Example 2. In tissues and cells mRNA Separation

For reverse transcription polymerase reaction, normal colonic epithelial tissue and colon cancer tissue were extracted from 20 colon cancer patients, and mRNA was isolated from a total of 40 tissues. First, tissues removed by surgical resection were removed from sterilized phosphate-buffered saline immediately after removal, and then frozen with liquid nitrogen. Thereafter, total RNA was isolated by the Guanidinium Method, and single-step RNA isolation was performed. Total RNA isolated as described above was quantified using a spectrophotometer, and then stored in a -70°C freezer until use.

A total of 10 colon cancer cell lines were selected (DLD-1, HT29. HCT116, colo205, SW480, SW620, SNU C1, SNU C2A, KM 12C, KM 12SM), and the Korea Cell Line Bank at 28 Yeongeon-dong, Jongno-gu, Seoul, Korea (Korean Cell Line Bank, KCLB). 10% fetal bovine serum (Fetal Bovine Serum, FBS, Hyclon) and penicillin/streptomycin (1mg/ml Penicillin/ Streptomycin, Sigma) in each of the optimal culture media, DMEM (Invitrogen) or RPMI1640 (Invitrogen) medium. ) Was added and cultured for 5-6 days, and then total RNA was isolated by the Guanidinium Method in the same manner as in the above tissues, and single-step RNA separation was performed. The isolated RNA was quantified using a spectrophotometer as described above, and stored in a -70°C freezer until use.

Example 3. Reverse transcription Comparison of gene expression using polymerase reaction

Among the selected colon cancer-specific overexpression genes as a result of Example 1, 14 were selected (data mining) and subjected to a polymerase reaction.

For the preparation of primers, the entire DNA nucleotide sequence of each gene was obtained from NCBI's CoreNucleotide (http://www.ncbi.nlm.nih.gov/), and primer sequences of these genes were designed through the Primer3 program. Using the primers designed as described above, a polymerase reaction was performed to confirm the expression level of each gene (FIGS. 8-10). Each primer sequence is shown in Table 5.

Primer sequence Oligo name Product size Seqences One CHEK1 (L) 301 bp AGC GGT TGG TCA AAA GAA TG (SEQ ID NO: 1) CHEK1 (R) ACC TTC TGG CTG CTC ACA AT (SEQ ID NO: 2) 2 MAD2L1 (L) 326 bp GTG GTG AGG TCC TGG AAA GA (SEQ ID NO: 3) MAD2L1 (R) AAT TTT GTA GGC CAC CAT GC (SEQ ID NO: 4) 3 KRT23 (L) 309 bp GTC TCG GTA CTC CTG CAA GC (SEQ ID NO: 5) KRT23 (R) GCG TGC TTT TGG ATT TCA TT (SEQ ID NO: 6) 4 PROX1 (L) 355 bp CAG CCC GAA AAG AAC AGA AG (SEQ ID NO: 7) PROX1 (R) TCC GGT TGT AAG GAG TTT GG (SEQ ID NO: 8) 5 SLC7A11 (L) 304 bp GGC AGT GAC CTT TTC TGA GC (SEQ ID NO: 9) SLC7A11 (R) AAC TGC CAG CCC AAT AAA AA (SEQ ID NO: 10) 6 ASB9 (L) 303 bp CAT CAT GGA TGG CAA ACA AG (SEQ ID NO: 11) ASB9 (R) GCT GTC ACA CCA TTC ACC TG (SEQ ID NO: 12) 7 ENC1 (L) 302 bp ATG CAT GTG CAG AGT TCC TG (SEQ ID NO: 13) ENC1 (R) CTG CAA CAG TTC TGG GAG GT (SEQ ID NO: 14) 8 PLAU (L) 301 bp GTG GCC AAA AGA CTC TGA GG (SEQ ID NO: 15) PLAU (R) TCA GCG CTG TAG TCC TTG TG (SEQ ID NO: 16) 9 RNF183 (L) 307 bp ACC TCA GCC TTG TGA CTC CA (SEQ ID NO: 17) RNF183 (R) ATG AGG ATG GGC GTA GAC AC (SEQ ID NO: 18) 10 CSE1L (L) 300 bp CTG CAG TAC CCA TGC AAA TG (SEQ ID NO: 19) CSE1L (R) ACA AAA CGA GGC AGG TAT CG (SEQ ID NO: 20) 11 ATAD2 (L) 302 bp TAT GGA TGG ATT GGA CAG CA (SEQ ID NO: 21) ATAD2 (R) AGA TCT GTG GGT AGC GTC GT (SEQ ID NO: 22) 12 RAD51AP1 (L) 304 bp GGT GGT GTT CAA GGG AAA AG (SEQ ID NO: 23) RAD51AP1 (R) GAG CAG AGT CCA CCG AAG TC (SEQ ID NO: 24) 13 C6ORF173 (L) 318 bp TCG GAC TGA GGT TTT TCT GC (SEQ ID NO: 25) C6ORF173 (R) GCG TTT GTC CTG GAC TCT TC (SEQ ID NO: 26) 14 CCNB1 (L) 306 bp TAC CTA TGC TGG TGC CAG TG (SEQ ID NO: 27) CCNB1 (R) TGA CTG CTT GCT CTT CCT CA (SEQ ID NO: 28)

For reverse transcription enzyme reaction, cDNA made through reverse transcription reaction was prepared from mRNA extracted from the tissues and cell lines of Example 2 above. cDNA production was performed using the AccuAcript High Fielity 1st Stand cDNA Synthesis Kit (STRATAGENE), and a reverse transcriptase reaction was used using the cDNA obtained as a result of the above reaction and the primers in Table 5. As a result, it was possible to confirm the difference in expression between normal colon cells and colon cancer cells as shown in FIGS. 8 to 10, and expression in colon cancer cell lines as shown in FIG.

Attach electronic file of sequence list

Claims (9)

MAD2L1 (MAD2 mitotic arrest deficient-like 1 (yeast), NCBI GenBank Accession No. NM_002358) A composition for diagnosing colorectal cancer comprising an agent measuring the level of a gene mRNA or a protein thereof. According to claim 1, wherein the composition is CHEK1 (CHK1 checkpoint homolog (S. pombe), NCBI GenBank Accession No. NM_001114121), ENC1 (ectodermal-neural cortex (with BTB-like domain), NCBI GenBank Accession No. NM_003633), RAD51AP1 (RAD51 associated protein 1, NCBI GenBank Accession No. NM_001130862), CCNB1 (cyclin B1, NCBI GenBank Accession No. NM_031966), CSEIL (CSE1 chromosome segregation 1-like (yeast), NCBI GenBank Accession No. NM_001316), KRT23 (KRT23 keratin 23 (histone deacetylase inducible), NCBI GenBank Acession No. NM_015515) PLAU (plasminogen activator, urokinase, NCBI GenBank Acession No. NM_001145031) , SLC7A11 (solute carrier family 7, (cationic amino acid transporter, y + system) member 11, GenBank No. NM_014331) and C60RF173 (chromosome 6 open reading frame 173, NCBI GenBank Accession No. NM_001012507) A composition for diagnosing colorectal cancer comprising an agent for measuring the level of one or more gene mRNAs or proteins thereof. According to claim 1 or claim 2, wherein the agent for measuring the level of the gene mRNA composition for diagnosing colorectal cancer comprising a primer that specifically binds to the gene. 4. The primer of claim 3, wherein the primer for MAD2L1 among the primers specifically binding to the gene is a primer pair of SEQ ID NOs: 3 and 4, the primer pair for CCNB1 is a primer pair of SEQ ID NOs: 27 and 28, and the primer for CSEIL is a sequence. Primer pairs of SEQ ID NOs: 19 and 20, primer pairs of SEQ ID NOs: 1 and 2, primer pairs of SEQ ID NOs: 1 and 2, primer pairs of SEQ ID NOs: 13 and 14, primer pairs of RAD51AP1, primer pairs of SEQ ID NOs: 23, 24, Primer for KRT23 is a primer pair of SEQ ID NOs: 5 and 6, primer pairs of SEQ ID NOs: 15 and 16 for PLAU, primer pairs of SEQ ID NOs: 9 and 10 for SLC7A11, and primer number for C60RF173 A composition for diagnosing colorectal cancer which is a pair of primers 25 and 26. The composition for diagnosing colorectal cancer according to claim 1 or 2, wherein the agent for measuring the level of the protein comprises an antibody specific for the protein. A kit for diagnosing colorectal cancer, comprising the composition of claim 1. The kit for diagnosing colorectal cancer according to claim 6, which is an RT-PCR kit, a DNA chip kit or a protein chip kit. Measuring mRNA from a biological sample of a suspected colorectal cancer patient using a primer specifically binding to MAD2L1 (MAD2 mitotic arrest deficient-like 1 (yeast), GenBank No. NM_002358) gene; And comparing the increase of the mRNA level with the mRNA level of a normal control sample. The method of claim 8, further comprising measuring and comparing mRNA levels of at least one gene selected from CHEK1, ENC1, RAD51AP1, CCNB1, CSEIL, KRT23, PLAU, SLC7A11, and C60RF173. How to provide.

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