KR101178510B1 - Genetic Markers for Human Mucinous Colon Carcinoma - Google Patents

Abstract

The present invention relates to a kit for diagnosing mucous bowel cancer in humans comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide. According to the present invention, genes representing differences between mucous bowel cancer and non-mucus bowel cancer can be selected according to over- or low-expression and can be used to diagnose human mucinous bowel cancer more simply and accurately. The present invention also provides a method for screening a mucus enteric cancer-specific anticancer agent using a gene that is overexpressed in mucus enteric cancer. Therefore, the present invention can diagnose and treat mucus intestinal cancer more quickly and efficiently at low cost, compared to the conventional method for identifying mucus intestinal cancer tissues or biochemical mucus intestinal cancer requiring a long time.

Mucous Bowel Cancer, Diagnostic Kits, Markers, cDNA Microarray, PAM

Description

Genetic Markers for Human Mucinous Colon Carcinoma

The present invention relates to a kit for diagnosing human mucous bowel cancer, a method for detecting human bowel cancer markers, and a method for screening a substance for preventing or treating human mucous bowel cancer.

Mucinous adenocarcinoma is a part of adenocarcinoma when the distribution of extracellular mucin is more than 50% of adenocarcinoma. Mucus intestinal cancer is known to show many differences from non-mucinous intestinal cancers in clinicopathologic and molecular genetics. Clinically, it is known that about 10% of intestinal cancers occur in Europe and the United States, but about 2.9% to 7.4% in Japan. Compared with non-mucosal bowel cancer, mucous bowel cancer shows a high incidence in patients with bowel cancer under 50 years of age, right colon. Mucous bowel cancer is known to show more metastasis to peripheral organs and lymph nodes than non-mucosa, especially because it is resistant to drugs based on fluorouracil, which has a better prognosis. Not so, the 5-year survival rate is lower. Molecular genetics, several recent studies have been conducted on the association between genes and the genetic variation of microsatellite instability (MSI), CpG methylation phenotype, MUC2 and MUC5AC. However, the link between the biological characteristics and the genetic characteristics of mucous bowel cancer, such as tumor formation ability, has not been clearly explained.

To this end, it is important to select a target gene that can distinguish the characteristics of mucosal bowel cancer. In other words, it is important to identify the characteristics of each gene by selecting genes that show differences in the expression form and amount of genes according to mucosal bowel cancer and non-mucus bowel cancer.

The microarray technique is an experimental technique that can distinguish genes showing differences according to the expression levels of genes according to biological characteristics through a single experiment, and is a method useful for screening mucus intestinal cancer-specific genes desired in the present invention.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors studied a method for quickly and accurately distinguishing the difference between mucous bowel cancer and non-mucus bowel cancer. As a result, the microarray analysis was performed to identify genes that are specifically overexpressed or underexpressed in mucous bowel cancer tissues. The present invention has been completed by finding a method of identification.

Therefore, it is an object of the present invention to provide a kit for diagnosing mucous bowel cancer in humans.

Another object of the present invention is to provide a method for detecting a mucus intestinal cancer marker in humans.

It is another object of the present invention to provide a method for screening a substance for preventing or treating human mucous bowel cancer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 3, the sequence complementary to the nucleotide sequence or a fragment of the nucleotide for human diagnostic mucosal bowel cancer Provide the kit.

The present inventors studied a method for quickly and accurately distinguishing the difference between mucous bowel cancer and non-mucus bowel cancer. A method of identification was found.

Intestinal cancer includes duodenal cancer, jejunal cancer, ileal cancer and colorectal cancer (rectal and colon cancer). Most bowel cancers occurring in humans are occupied by colon cancer and rectal cancer. The cause of intestinal cancer is not yet clear, but it is well known for familial polyposis, idiopathic non-specific ulcerative colitis, polyps in the large intestine and rectum, and especially in the case of chorionic adenomas. . Mucinous bowel cancer refers to the case where mucin accounts for more than 50% of the tumor site through histological staining. In addition, mucosal bowel cancer is considered essentially the same as colloidal carcinoma. However, it is distinguished from signal-ring cells in which large amounts of mucin are produced but not secreted. Most bowel cancers produce very few mucins, and it is not clear whether their distribution (10-50%) affects signs of the disease. Compared to non-mucosal bowel cancer, the incidence of mucous bowel cancer is about 6-19%. These mucosal bowel cancers are known to not only facilitate cancer metastasis as compared to non-mucosal bowel cancers, but also have lower 5-year survival rates due to increased drug resistance.

On the other hand, diagnostic methods of bowel cancer known to date include occult blood test, tumor marker test, intestinal angiography, endoscopy, abdominal ultrasonography or abdominal computed tomography and tumor marker test, but these methods are expensive and time-consuming. There is a disadvantage that it is inadequate for patients who do not have external symptoms.

Therefore, in order to overcome these drawbacks, the present inventors have developed a kit for diagnosing mucous bowel cancer in humans, which can quickly and accurately determine whether mucous bowel cancer exists.

As used herein, the term “intestinal cancer” refers to cancers occurring in the intestine, including precursors of rectal cancer, colorectal cancer and intestinal cancer (eg, adenomatous polyps).

According to a preferred embodiment of the present invention, the human bowel cancer diagnostic kit of the present invention may be a microarray.

The probe or primer used in the diagnostic kit of the present invention has a sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 3 sequences. As used herein, the term “complementary” refers to the degree of complementarity that can be selectively hybridized to a nucleotide sequence selected from the group consisting of SEQ ID NOs. 1 to 3 under certain specific hybridization or annealing conditions. It means to have. Thus, the term “complementary” has a different meaning from the term perfectly complementary, and the primer or probe of the present invention selectively hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 If enough, one or more mismatch sequences may be present.

As used herein, the term “primer” may serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. By single-stranded oligonucleotide. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 as a template, and may hybridize to this gene sequence to act as a primer. It is sufficient to have sufficient complementarity in the range. The design of such primers can be easily carried out by those skilled in the art by referring to nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 3, for example, using a primer design program (eg, PRIMER 3 program). You can do it.

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally Present or artificially synthesized. Probes of the invention are preferably single chain and oligodioxyribonucleotides. Probes of the invention can include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probes of the present invention may also comprise ribonucleotides. For example, the probes of the present invention may be backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al. , Nature , 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA , Phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2 '-Fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethyl -, Propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro-, Iodo-, methyl- , Ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-, inosine and diaminopurine.

In the microarray of the present invention, the probe is used as a hybridizable array element and is immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. This immobilization is carried out by chemical bonding methods or by covalent bonding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, the sample DNA applied to the microarray of the present invention can be labeled and hybridized with array elements on the microarray. Hybridization conditions can vary. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The kit for diagnosing mucous bowel cancer of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 is used.

Intestinal cancer may be determined by hybridization-based analysis using a probe hybridized to a nucleotide sequence selected from the group consisting of SEQ ID NOs. 1 to 3 sequences. In addition, the human cancerous cancer diagnostic kit of the present invention can be produced according to GenBank Accession No. AA040387; AA042990; AA054073; AA181085; AA278698; AA279145; AA400973; AA412284; AA452933; AA453310; AA459012; AA465495; AA481464; AA488868; AA490263; AA490497; AA598652; AA865729; AA872095; AA873578; AA922903; AA939100; AA971714; AA976544; AA995282; AI094854; AI147237; AI203404; AI269774; AI336626; AI341793; AI346446; AI349090; AI359768; AI369785; AI675714; AI688155; AI700308; AI990099; AI990501; AW009769; AW072118; AW073291; AW075441; AW082097; H04789; H10045; H13623; N30553; N35888; N62394; N63845; N63943; N74131; N74236; R09561; R73909; R99562; T70057; W47576; And at least one nucleotide sequence selected from the group consisting of AA931716 and AA933744, a sequence complementary to said nucleotide sequence, or a fragment of said nucleotide.

The label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin Hapten with specific binding partners, such as, but not limited to, biotin, digoxigenin and chelating groups. Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology , 65: 499 (1986)). Labels provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biosamples. The raw sample is preferably enteric cancer cells, most preferably colon cancer or rectal cancer cells. The hybridization reaction-based assay may be performed by labeling the cDNA to be analyzed instead of the probe.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc. NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. The hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe. For example, if the probe is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver dyeing using silver nitrate. Therefore, when the mucosal bowel cancer marker of the present invention is carried out on the basis of hybridization, specifically, (i) a sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 Hybridizing the branched probe to the nucleic acid sample; (ii) detecting whether the hybridization reaction occurs. By analyzing the intensity of the hybridization signal by the hybridization process, it is possible to determine whether mucous bowel cancer. That is, the hybridization signal for the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and the second sequence in the sample is stronger than the normal sample (normal cells) or to the nucleotide sequence selected from the group consisting of SEQ ID NO: 3 If the hybridization signal is weaker than a normal sample (normal cell), it is diagnosed as mucinous bowel cancer.

According to a preferred embodiment of the present invention, the human bowel cancer diagnostic kit of the present invention may be a gene amplification kit.

As used herein, the term "amplification" refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual , 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR) ( 17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self-maintaining sequence replication (self sustained sequence replication, WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction ( CP-PCR), US Pat. No. 4,437,975), optional print Arbitrarily primed polymerase chain reaction (AP-PCR), US Pat. Nos. 5,413,909 and 5,861,245, Nucleic acid sequence based amplification (NASBA), US Pat. No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.

PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

When the diagnostic kit of the present invention is carried out using a primer, a gene amplification reaction is carried out to investigate the expression level of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3. Since the present invention is to analyze the expression level of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 3, the sequence from the sample of analysis (e.g., enteric cancer cells) to SEQ ID NO 1 to 3 The amount of mRNA of the nucleotide sequence selected from the group consisting of the group is examined to determine the expression level of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 3 sequences.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be carried out according to conventional methods known in the art. See Sambrook, J. et al., Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001); Tesniere. , C. et al., Plant Mol. Biol. Rep. , 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); and Chomczynski, P. et al. , Anal.Biochem. 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from human samples, the end of the mRNA has a poly-A tail, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptases using these sequence characteristics. PNAS USA , 85: 8998 (1988); Libert F, et al., Science , 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual , 3rd ed.Cold Spring Harbor Press (2001). Next, the synthesized cDNA is amplified through gene amplification reaction.

Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention and include “Clenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu). Include.

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desired to provide cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.

CDNA of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 sequences thus amplified is analyzed by a suitable method to investigate the expression level of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 do. For example, the degree of expression of the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 3 is examined by gel electrophoresis of the amplification reaction product described above, and by observing and analyzing the resulting band. Through this amplification reaction, the expression of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and 2 in biosamples is higher than that of a normal sample (normal cell) or selected from SEQ ID NO: 3 Mucosal bowel cancer is diagnosed when the expression of the nucleotide sequence is lower than that of normal samples (normal cells).

Therefore, when the method for detecting a cancerous cancer marker of the present invention is carried out based on an amplification reaction using cDNA, specifically, (i) a primer annealed to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 Amplification reaction using the amplification reaction; And (ii) analyzing the product of the amplification reaction to determine the expression level of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus ). (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

According to a preferred embodiment of the present invention, the nucleotide sequence selected from SEQ ID NO: 1 and 2 in the kit for diagnosing mucous bowel cancer of human is highly expressed in mucous bowel cancer tissue, selected from SEQ ID NO: 3 The resulting nucleotide sequence is low expressed in mucosal bowel cancer tissue.

The term “high expression” as used herein referring to a nucleotide sequence refers to gene expression analysis methods commonly used in the art, such as real-time RT-PCR methods (Sambrook, J. et al., Molecular Cloning. A Laboratory). Manual , 3rd Ed., Cold Spring Harbor Press (2001)). For example, the first and second sequences of the present invention are 1.7-2.5 times higher in mucinous bowel cancer samples than in non-mucus bowel cancer samples. The term “low expression” as used herein to refer to a nucleotide sequence refers to gene expression analysis methods commonly used in the art, such as real-time RT-PCR methods (Sambrook, J. et al., Molecular Cloning. A Laboratory). Manual , 3rd Ed., Cold Spring Harbor Press (2001)). For example, in the case of the third sequence of the present invention, the mucinous bowel cancer sample is expressed 2.5-3 times higher than the non-mucus bowel cancer sample. Human bowel cancer diagnostic kit of the present invention mucinous bowel cancer can be diagnosed by analyzing whether the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 is high or low.

According to another aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating human intestinal cancer, comprising the steps of: (a) selecting from the group consisting of SEQ ID NOs: 1 to 3 Contacting a sample to be analyzed with a cell comprising the nucleotide sequence of interest; And (b) measuring the expression level of the nucleotide sequence, wherein the high expression of the nucleotide sequence selected from SEQ ID NO: 1 and 2 in the nucleotide sequence is suppressed or of a nucleotide sequence selected from SEQ ID NO: 3 When the low expression is suppressed, the sample is determined to be a substance for the prevention or treatment of human mucinous bowel cancer.

According to the method of the present invention, first, a sample to be analyzed is contacted with a cell comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 sequences. Preferably, the cell comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 3 is a human intestinal cancer cell. The term “sample” used while referring to the screening method of the present invention is unknown used in screening to examine whether it affects the expression level of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3 Means the substance. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

Then, the expression level of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 3 in the cells treated with the sample is measured. The measurement of the expression level is as described above, and as a result of the measurement, the high expression of the nucleotide sequence selected from SEQ ID NO: 1 and 2 in the nucleotide sequence is suppressed or of the nucleotide sequence selected from SEQ ID NO: 3 When the low expression is suppressed, the sample may be determined as a substance for the prevention or treatment of human mucinous bowel cancer.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a kit for diagnosing mucous bowel cancer in humans.

(Ii) According to the present invention, genes showing differences between mucosal bowel cancer and non-mucus bowel cancer can be selected according to over- or low-expression and can be used to diagnose human mucinous bowel cancer more simply and accurately.

(Iii) The present invention also provides a method for screening a mucus enteric cancer-specific anticancer agent using a gene that is overexpressed in mucus enteric cancer.

(Iii) Therefore, the present invention can diagnose and treat mucus intestinal cancer more efficiently and quickly and at low cost, compared to conventional mucus intestinal cancer tissue identification or biochemical mucus intestinal cancer determination method requiring a long time.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Subject Patients and Specimen Collection

From 2003 to 2006, 39 patients with mucinous bowel cancer containing more than 50% of mucin were found in clinical pathology among patients diagnosed with colorectal cancer at Shinchon Severance Hospital and Yonsei Cancer Center, Yonsei University College of Medicine. Tissues and 71 non-mucus intestinal cancer tissues containing up to 10% mucin were used. The collected tissue was immediately frozen with liquid nitrogen and collected at -80 ° C until the experiment. All studies were conducted on patients who voluntarily agreed to an agreement approved by the Institutional Review Board of Yonsei University Severance Hospital.

Example 2 RNA Extraction and Amplification

About 100 mg of tissue stored was homogenized using a mortar and pestle in liquid nitrogen, and then dissolved using 1 mL of trizol (Invitrogen, Carlsbad, CA, USA). After 200 μl of chloroform was reacted at 4 ° C. for 10 minutes, the mixture was centrifuged at 14,000 rpm for 25 minutes at 4 ° C. to separate only the supernatant. After the separated supernatant and isopropanol were reacted for 30 minutes at -20 ° C, RNA was precipitated by centrifugation at 14,000 rpm for 25 minutes at 4 ° C. Precipitated RNA was washed with 70% ethyl alcohol, the alcohol remaining at room temperature was dried and dissolved in ultra-pure water. The amount and quality of total RNA was confirmed using nanodrop (Nanodrop, Nanodrop technology, Rockland, USA), 1% agarose gel electrophoresis. Yonsei reference RNA (Yonsei University Cancer Metastasis Research Center, Seoul, Korea) to be used as a control is YCC-3, YCC-B1, HCT-116, SK-Hep-1, A549, HL-60, MOLT-4, HeLa, HT The same amount of total RNA extracted from 11 human tumor cell lines of -1080, Caki-2, and T98G was prepared by mixing.

RNA amplification was performed according to the standard protocol of Yonsei University Cancer Metastasis Research Center. First, primary amplification was performed using 4 μl of total RNA as a template. 2 μg of oligo-dT / T7 primer (5'-GGCCAGTAATTGTAATACGACTCACTATAGGGAGGCGG-3 ', Genotech, Daejeon, Korea) was added to the RNA template and reacted for 9 minutes at 65 ° C. for 9 minutes of RNase-deficient water. . After cooling on ice for 2 minutes, 4 μl of 5 x first strand buffer (Invitrogen, Carlsbad, CA, USA), 2 μl of 100 mM DTT (Invitrogen, Carlsbad, CA, USA), 2 μl of 10 mM dNTP mix (Invitrogen, Carlsbad, CA, USA), 2 μl of RNAsin (Promega, Madison, WI, USA), 2 μl of SuperScript II (Invitrogen, Carlsbad, CA, USA) and reverse transcription reaction at 42 ° C. for 1 hour. I was.

91 μl of RNase-deficient water, 30 μl of 5 × second strand buffer (Invitrogen, Carlsbad, CA, USA), 3 μl of 10 mM dNTP mix, 10 U DNA ligase, 4 U DNA polymerase I, and 2 U RNase H were added and reacted at 16 ° C. for 2 hours. After the reaction, 20 U of T4 DNA polymerase (Invitrogen, Carlsbad, CA, USA) was added and reacted at 16 ° C. for 5 minutes, and 10 μl of 1 M NaOH and 0.5 M EDTA were added for 10 minutes at 65 ° C. The reaction was deactivated. Then neutralize with 25 μl Tris-HCl, add 97 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1), mix well, and place in Phase Lock GelTM (Eppendorf, Westbury, NY, USA). Only cDNA was purified by centrifugation at 13,000 rpm for 2 minutes. Separate the supernatant containing cDNA separately, add 0.5 times 7.5M ammonium acetate and 2.5 times 95% EtOH of the supernatant volume, and centrifuge at 13,000 rpm or more at 4 ° C for 30 minutes to remove cDNA deposits. It was obtained, washed with 80% EtOH and dried at room temperature. The dried cDNA deposit was dissolved by adding 8 μl of RNase-deficient water.

In vitro transcription was performed using the T7 MEGAscript kit (Ambion, Austin, TX, USA) using two strands of cDNA synthesized. Briefly, 2 μl of 75 mM rATP, rUTP, rCTP, rGTP, enzyme mixture, and 10 × reaction buffer were added to 8 μl of the cDNA solution prepared above, and reacted at 37 ° C. for 5 hours. The amplified mRNA was obtained by purification using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.The amount and quality of the amplified mRNA was determined using nanodrops and 1% agarose gel electrophoresis. Evaluated.

Example 3 cDNA Microarray Hybridization

The cDNA microarray used a human 17K cDNA chip (CMRC-GT, Seoul, Korea), which contains 17,104 genes containing known genes and EST. The experiment used an indirect method to compare the experimental group and the reference RNA. . At this time, the experimental group was stained with Cy5-dUTP and the reference RNA was stained with Cy3-dUTP.

This procedure was performed according to the CMRC standard protocol. Briefly, 6 μg of random primers (Invitrogen, Carlsbad, Calif., USA) were added to 2 μg of amplified mRNA so that the total amount was 21 μl with RNase-deficient water, followed by reaction at 65 ° C. for 10 minutes. 8 μl 5 x first strand buffer, 4 μl 100 mM DTT, 2 μl SuperScript II RT, 2 μl 20 x low dT / dNTP mix, 1 μl RNAsin, in amplified mRNA and random primer mixed solution. 1 μl of Cy3 or Cy5-dUTP (Genomic Tree, Daejeon, Korea) was added and reacted at 42 ° C. for 1 hour 30 minutes. 15 μl of 0.1 M NaOH was added to decompose the RNA template and incubate at 65 ° C. for 30 minutes to inactivate the reaction. After neutralization with 0.1 M NaOH and the same amount of 0.1 M HCl, using the QIA quick PCR purification kit (Qiagen, Valencia, CA, USA), only purified Cy3 or Cy5 stained probes were dissolved twice with 200 μl ultrapure water. Paid. In each of the solutions thus obtained, 20 μg of human COT-1 DNA (Invitrogen, Carlsbad, CA, USA), yeast tRNA (Invitrogen, Carlsbad, CA, USA), poly (A) RNA (Sigma, Saint Louis, MO, USA) After adding the Condensate using a Microcon YM-30 column (Millipore, Bedford, MA, USA) to a final amount of 40 μl was used after denatured at 100 ℃.

The cDNA microarray chip was pre-treated at 42 ° C. in a solution of 3.5 x Sodium chloride / Sodium Citrate buffer (SSC), 0.1% Sodium Dodesyl Sulfate (SDS), 10 mg / ml Bovine Serum Albumin (BSA) for 1 hour before use. After hybridization (pre-hybridization) and washed twice with tertiary distilled water and completely dried using isopropanol was used. The concentrated probe was finally placed in a glass of cDNA microarray chip prepared at 80 μl of 25% formamide, 5 × SSC, 0.1% SDS, and injected into the gap, followed by light at 42 ° C. for 16 hours. The reaction was blocked. After completion of the reaction, the microarrays were washed with 2 x SSC and 0.1% SDS solution, 1 x SSC and 0.1% SDS solution, 0.2 x SSC solution, and 0.05 x SSC solution for 2 minutes each, and dried using a rotary dryer.

The hybridized microarrays were detected using a GenePix 4000B scanner (Axon Instruments, Foster City, CA, USA) to detect fluorescence signals and imaged using GenePix pro 4.0 software (Axon Instruments, Foster City, CA, USA). At this time, in order to minimize the interference caused by the non-specific fluorescence signal, the background signal was removed according to a local background subtraction method and the numerical results were obtained.

Example 4 Results Analysis Using Bioinformatics Techniques

1 is a general schematic diagram of the present invention and shows an assay including microarray experiments and gene selection, cross validation and prediction. Raw data normalization, gene filtering, classification gene screening, cross-checking of training sets, testing, and class prediction in an independent set were performed using GeneSpring 7.0 software (Silicon Genetics, Redwood city, CA) and Prediction Analysis of Microarray (PAM). , EXCEL, http://www-stat.stanford.edu/~tibs/PAM/ ). The analysis content of the entire experiment is shown in FIG. 1.

To select mucus intestinal cancer related genes, each group selected 25 mucus intestinal cancer samples and 27 non-mucus intestinal cancer samples as training groups. Welch's t-test method was used to select genes with significantly different FDR (false discovery rate) of 0.05 or less in the training group. Selected genes were predicted using five mucus intestinal cancer samples and four non-mucus intestinal cancer samples using predictive analysis (PAM). Selected genes were obtained genetic information using the SOURCE ( http://source.stanford.edu ) database. Among the selected genes, LOOCV (leave-one-out cross validation, R package, http://www.r-project.org/ ) method was used to select mucosal bowel cancer-related genes for more efficient prediction. . Selected tumorigenic genes were compared with microarray experiments using RT-PCR method.

Example 5: Cross-validation in class prediction genes and training sets and prediction of tissue types in test sets

As described in Example 4, 62 genes were selected from 11,621 genes through gene screening between normal or tumor tissues of the training set, and based on the difference in relative expression change of these genes, the principal component analysis method (Principal Component) 50 genes were selected through Analysis, GeneSpring 7.3, Silicon Genetics, Redwood city, CA). The selected genes were 100% predicted between the two groups at 1.519 and 1.688. When the predicted intensity was 1.688, 50 genes selected by the stratification analysis were used. As a result, it was confirmed that the mucous bowel cancer group and the non-mucus bowel cancer group were clearly distinguished.

Table 1 shows 62 genes representing the differences between the mucus and non-mucosa tissues of the training set, and the average expression ratio of the mucosal and non-mucosa cancer tissues was The average of the Cy5 / Cy3 ratios of each mucous bowel and non-mucus bowel tissue in the set is shown.

GenBank Access Number Gene name Viscous / Non-Viscous Average Expression Ratio One AA017125 SORT1 0.34 2 AA040387 XPNPEP2 -0.46 3 AA042990 SEMA3C -0.82 4 AA127096 FUS -0.49 5 AA155640 TCN1 1.46 6 AA181085 KDELR3 0.58 7 AA278698 HDHD1A -0.61 8 AA279145 CAB39L -0.98 9 AA282208 -0.56 10 AA282253 HERC3 -0.47 11 AA283090 CD44 0.61 12 AA412284 PVR -0.81 13 AA446884 TRIM13 -0.32 14 AA453310 AMACR -0.66 15 AA458870 CDC37 0.3 16 AA459012 LMBRD1 -0.56 17 AA465396 SLC25A37 0.51 18 AA481464 PPIB 0.62 19 AA488433 SLMO2 -0.71 20 AA488868 AYTL2 0.74 21 AA490263 NEK3 -0.85 22 AA490497 UBL3 -0.57 23 AA598652 -1.06 24 AA633997 YWHAQ -0.34 25 AA669068 STAU1 -0.48 26 AA872095 YWHAB -0.49 27 AA918102 -0.46 28 AA931716 TRIM7 0.91 29 AA933744 L1TD1 1.56 30 AA976544 MLPH 0.73 31 AA995282 FHL2 0.53 32 AI051281 C13orf23 -0.53 33 AI203404 0.73 34 AI269774 PHYH -0.51 35 AI298104 AGL -0.61 36 AI339538 SLC26A3 -2.47 37 AI349090 0.64 38 AI350851 0.72 39 AI359768 RHOU -0.64 40 AI360206 C20orf112 -0.55 41 AI369785 NPDC1 0.76 42 AI393075 CYP4F3 -0.5 43 AI632018 TINAG -0.35 44 AI654481 CLCN2 -0.36 45 AI688155 SEPHS2 -0.58 46 AI700308 PPP1R3D -0.62 47 AI769855 DEFB1 -0.54 48 AI989542 CEBPA -0.42 49 AI990104 MAPRE1 -0.37 50 AI990501 STMN2 -0.54 51 AW009769 TFF1 2.01 52 AW072118 FSCN1 1.15 53 AW073291 AGR2 1.71 54 AW082097 PI3 1.73 55 H04789 GYG2 -0.67 56 H10045 VAV3 -1.27 57 H14359 ELF4 -0.46 58 H73234 CDC42EP1 0.44 59 H87106 -0.8 60 N74236 MPP1 -0.86 61 R09561 CD55 0.82 62 W47576 ASAHL -0.66

Example 6 real-time RT-PCR

In order to validate the expression ratio obtained in the microarray experiment, real-time RT-PCR was performed with the same mRNA to confirm the gene expression pattern (Kim TM et al. Clin Cancer Res 11:79 ( 2005)). The reaction solution was a total volume of 20 μl containing 10 μl of QuantiTect SYBR Green PCR mixture (Qiagen, CA, USA) consisting of 2.5 mM MgCl ₂ (Qiagen, CA), 2 μl cDNA, 20 pmol oligonucleotide primers. In the Roter Gene 2072D real-time PCR machine (Corbett Research, Sydney, Austrialia), PCR was performed at 95 ° C. for 15 minutes (activating HotstarTaq ^™ DNA polymerase (Qiagen, CA, USA)) and at 30 cycles for 20 seconds at 95 ° C. Amplification), 30 seconds at 50 ℃, 45 seconds at 72 ℃.

The fluorescent signal amplified in each sample was measured at the late extension phase of each cycle. To determine the amount of signal from each gene, 10-fold serially diluted human genomic DNA was used as a control. The standard curve was drawn by plotting the threshold frequency measured for arbitrary numbers of RT-PCR products in response to gene expression of beta-actin in diluted genomic DNA. The threshold period (Ct) value is determined by the number of cycles when the fluorescence exceeds the threshold value. In the negative control, no fluorescence signal was detected to increase the number of cycles to 35.

Among the selected genes, TFF1 (Trefoil factor 1, GeneBank Accession No .: AW009769, SEQ ID NO: 1), AGR2 (Anterior gradient 2 homolog, GeneBank Accession No .: AW073291, SEQ ID NO: 2), which shows increased expression in mucosal bowel cancer SLC26A3 (Solute carrier family 26 member 3, GeneBank accession number: AI339538, 3rd sequence) and the mucin-associated gene, MUC2 gene, which express the expression pattern, were selected and used for this purpose.

The sequences of the primers used are as follows: TFF1 forward, 5'-TTGTGGTTTTCCTGGTGTCA-3 ', TFF1 reverse, 5'-CCGAGCTCTGGGACTAATCA-3'; AGR2 forward, 5'-TCCCTTCCTTGAGCATTTTG-3 ', AGR2 reverse, 5'-GGCCTTGAGACTTGAAACCA-3'; SLC26A3 forward, 5'-TGGCGCCACTATACTGCTAA-3 ', SLC26A3 reverse, 5'-TTCAAACTTTGGAACAAGATGG-3'; MUC2 forward, 5′-TGGAAAGCAAGGACTGAACA-3 ′, MUC2 reverse, 5′-TACACCCACATCGAGAGCTG-3 ′.

Relative expression levels of 15 non-mucosal bowel cancer tissues and 11 mucous bowel cancer tissues were correlated with the rate of gene expression by microarray analysis and real-time RT-PCR. Correlation coefficients were determined by comparing the expression values of genes with data measured by microarray and RT-PCR. Expression of three genes by microarray and expression patterns of one known gene were completely identical by RT-PCR (FIG. 4).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Figure 1 is a schematic diagram showing the microarray experiment and analysis process according to the invention as a whole.

2 is a graph showing the predictive strength of the training group and the test group using 62 genes. The x-axis shows the predicted intensity for each gene in the group, and the y-axis shows the predicted accuracy.

FIG. 3 is a dendogram showing the supervised hierarchical clustering of 50 genes and 52 cancer tissue samples selected through principal component analysis (Principal Component Analysis, GeneSpring 7.3, Agilent technologies, USA). to be. The horizontal axis represents the sample, the vertical axis represents the gene, and the upper and left phylogeny trees are grouped into groups of similar expression levels. NMC and MC refer to non-mucoid and mucinous bowel cancer, respectively, and green (2), yellow (3) and purple (4) represent the cancer's progression stages (tumors / nodes / metastases).

Figure 4a is a graph confirming the significance through the RT-PCR experiment through random selection among the genes selected by microarray analysis. FIG. 4B is a histogram showing the correlation between the data of four genes using microarray and RT-PCR at each gene level ( ^* P <0.05, ^** P <0.005).

<110> Yonsei University <120> Genetic Markers for Human Mucinous Colon Carcinoma <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 417 <212> DNA <213> Homo sapiens <400> 1 tgctggccct cggcaccctg gccaaggccc agacagagac gtgtacagtg gccccccgtg 60 aaagacagaa ttgtggtttt cctggtgtca cgccctccca gtgtgcaaat aagggctgct 120 gtttcgacga ccccgttcgt ggggtcccct ggtgcttcta tcctaatacc atcgacgtcc 180 ctccaaaaga ggagtgtgaa ttttagacac ttctgcaggg atctgcctgc atcctgacgc 240 ggtgccgtcc ccagcacggt gattagtccc agagctcggc tgccccctcc cccggacacc 300 tcagacacgc ttctgcagct gtgcctcggc tcacaacaca gattgactgc tctgactttg 360 actactcaaa attggcctaa aaattaaaag agatcgatat taaaaaaaaa aaaaaaa 417 <210> 2 <211> 507 <212> DNA <213> Homo sapiens <400> 2 tggagaaaac agaacacccc caaaacattt attttttttt ttagaaaatc atggctcact 60 atggtagtat acaatattgt tttcacacat gtacacttga aaccaaattt ctaaaacttg 120 tttttcttaa aaaatagttg ttgtaacatt aaaccataac ctaatcagtg tgttcactat 180 gcttccacac tagccagtct tctcacactt cttctggttt caagtctcaa ggcctgacag 240 acagaagggc ttggagattt tttttcttta caattcagtc ttcagcaact tgagagcttt 300 cttcatgttg tcaagcaaca gagctgtatc tgcaggttcg taagcataga aacggtttga 360 atatcttcca gtgatatcgg ctctaactgt cagagatggg tcaacaaaca taatcctggg 420 gacatactgg ccatcaggag aaaggtgttt gtcagttggt tcataaacca nattgaggag 480 gacaaactgc tctgccaatt tctggat 507 <210> 3 <211> 491 <212> DNA <213> Homo sapiens <400> 3 cactgtacaa ctgatttttt aatggaaata tattaatatg acctgtataa attatgagat 60 tcaaaacagt ggcgccacta tactgctaaa cctatgcatg aaggtagtga ctaggatgga 120 aatctgtcag tgctacaaaa atatgtatga acaaaataat tttcaccctt tgataaagct 180 acaagatata aaatttagaa tacttatata atttcatact agatatgtga aaaatatgcc 240 atgctagaac catcttgttc caaagtttga aacatattct gtcaaaaata ctcttcgtac 300 aatgtatgaa cttatcaata actttctggg tataaagttg tttttatgtc atagtcagat 360 gaagatcctt ctgaattata tgttgattag aattttgttt caactggcac ctcatatacc 420 cgattacgta atcctccatt tgtatttatg gtaaaatcaa tttttccatc tttttcctga 480 ctgggattaa a 491  

Claims (12)

Human mucus intestinal cancer kit comprising a primer consisting of a nucleotide sequence of SEQ ID NO: 4 and a primer consisting of a nucleotide sequence of SEQ ID NO: 5. delete The kit for diagnosing mucus intestinal cancer according to claim 1, wherein the kit is a gene amplification kit. delete delete In order to provide information necessary for the diagnosis of human intestinal cancer, expression of a nucleotide sequence consisting of SEQ ID NO: 1 in a biological sample of humans may be performed using a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 4 and a nucleotide sequence of SEQ ID NO: 5 A method for detecting a mucus intestinal cancer marker in humans by a method using a primer. delete 7. The method of claim 6, wherein the method is performed by gene amplification. 7. The method of claim 6, wherein the nucleotide sequence consisting of SEQ ID NO: 1 is highly expressed in mucosal bowel cancer tissues. delete The method of claim 6, wherein the biological sample is enteric cancer cells. A method for screening a substance for preventing or treating human mucous bowel cancer, comprising the following steps: (a) contacting a sample to be analyzed with a cell comprising a nucleotide sequence consisting of SEQ ID NO: 1; And (b) measuring the expression level of the nucleotide sequence using a primer consisting of a nucleotide sequence of SEQ ID NO: 4 and a primer consisting of a nucleotide sequence of SEQ ID NO: 5, wherein the nucleotide sequence consisting of SEQ ID NO: 1 When high expression of is suppressed, the sample is determined to be a substance for the prevention or treatment of mucus intestinal cancer in humans.

Images