KR20120009896A - Diagnostic Kit for Colon Cancer and Pharmaceutical Composition for Prevention and Treatment of Colon Cancer - Google Patents

Abstract

PURPOSE: A kit for diagnosing colon cancer and a pharmaceutical composition are provided to ensure accurate diagnosis and to develop customized anti-cancer drug. CONSTITUTION: A kit for diagnosing colon cancer contains a nucleotide sequence of C1orf135(chromosome 1 open reading frame 135) gene, a fragment of the nucleotide, a complementary sequence of the nucleotide sequence or fragment, a protein expressed from C1orf135 gene, or a specific binder to the protein. The kit is a microarray for amplifying genes. A pharmaceutical composition for preventing or treating colon cancer contains an inhibitor of C1orf135 gene expression and a pharmaceutically acceptable carrier.

Description

Diagnostic Kit for Colon Cancer and Pharmaceutical Composition for Prevention and Treatment of Colon Cancer}

The present invention relates to a colorectal cancer diagnostic kit and a pharmaceutical composition for preventing or treating colorectal cancer.

More specifically, the present invention relates to a colorectal cancer diagnostic kit using the C1orf135 gene and a protein expressed therefrom, a method for detecting a colorectal cancer diagnostic marker, a pharmaceutical composition for preventing or treating colorectal cancer, and a method for screening a colorectal cancer therapeutic agent.

Colorectal cancer is generally recognized as a 'developed cancer' due to its high incidence in high income groups. In the West, cancer is the second largest cancer death, accounting for about 15% of all cancers. . In the U.S., approximately 56,000 people die of colorectal cancer and 130,000 new cases of colorectal cancer occur annually.In Korea, color cancer accounts for the fourth largest cancer mortality rate after gastric cancer, liver cancer and lung cancer. to be. 5.8% of all cancers in 1980 were 6.1% in 1985, 8.2% in 1995, and 11.2% in 2002. Compared with 1991 data, stomach, liver and cervical cancers Mortality from colorectal cancer has more than doubled from 4.7 to 11.4 per 100,000 population. If this trend continues, the incidence of colorectal cancer in Korea will reach the western level by 2010, and the incidence of colorectal cancer in 2006 is second.

Although many efforts have been made to increase the survival rate of colorectal cancer patients, it is difficult to improve the survival rate through existing treatments. In order to increase the survival rate of colorectal cancer patients, it is urgently needed to find early diagnosis and new targets for colorectal cancer. Most colorectal cancers occur in adenomas, and it takes about 10 years to change to cancer, so there are many opportunities for early diagnosis to remove adenocarcinomas or early cancers. Current screening tests include 1) fecal occult blood test, 2) S colonoscopy, 3) colonoscopy, and 4) colonography. The occult blood test is less useful for early diagnosis, and colonography is less accurate. It is 20-50% below 1 cm, 10-30% above 1 cm, and 15-45% for early colorectal cancer. Therefore, a more accurate and early diagnosis system is needed, which is the part that needs to find colon cancer-related marker genes. In addition, colorectal cancer markers can be used to predict prognosis and establish appropriate treatment policy when colon cancer develops.

The development of colorectal cancer is known to be involved in various genetic changes, such as various types of cancer genes, tumor suppressor genes. In fact, colorectal cancer is one of the most identified genetic changes in the carcinogenesis process. Colorectal cancer is not a single cancer gene or a tumor suppressor gene change that can cause cancer alone, and many colon cancers have undergone several years of development in order for normal colon mucosa to progress to colorectal cancer. Changes in related genes need to accumulate, which is called multistage changes in colorectal cancer. What is important here is not the order of gene change at each step, but the sum of the changes in the genes that ultimately accumulate. Genetic changes involved in the development of colorectal cancer include the K-ras, APC, MCC gene, DCC gene on chromosome 18, p53 gene on chromosome 17, and DNA methylation abnormalities. HMSH2, hMSH1, hPMS1 And mutations such as hPMS2.

As such, the formation of cancer proceeds by a combination of various genes and the expression and regulation mechanisms of these genes. In recent years, the expression rate of cancer-related genes using oligo chips using a large amount of genes is compared to a new diagnosis of cancer. Research is being done to uncover markers of treatment. Genes whose expression increases or decreases in cancer cells are involved in many parts, including cell division, cell signaling, cytoskeleton, cell movement, cell defense, expression of genes and proteins, and intracellular metabolism. While there are genes that change, many genes show other changes in expression. This is likely due to the specificity of each patient, so the exact pathologic findings and classification of the patient's tissues under study should be followed.

By investigating the frequency of expression of specific genes in specific cells, it is possible to discover genes related to colon cancer, thereby understanding the molecular mechanism of colon cancer progression, and furthermore, to be used as targets for colon cancer diagnosis and treatment. will be.

The C1orf135 (Chromosome 1 open reading frame 135, FLJ14264, AIBP, MGC2603) gene is now known as the gene encoding the Aurora A-binding protein and acts as a substrate for ATM or ATR1 in response to DNA damage. It is known that the results of the study of the association with colorectal cancer are not well known.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made intensive studies to find genes that can be used for diagnosing colorectal cancer and screening drugs and can be targets for treating colorectal cancer. As a result, the expression of C1orf135 gene in colon cancer cells is significantly increased, and the colorectal cancer treatment is inhibited. By finding out that there is efficacy, the present invention has been completed.

Accordingly, an object of the present invention is to provide a diagnostic kit for colorectal cancer and a pharmaceutical composition for preventing or treating colorectal cancer.

Another object of the present invention is to provide a method for detecting a colorectal cancer diagnostic marker and a method for screening a colorectal cancer therapeutic agent.

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

According to one aspect of the present invention, the present invention provides a kit comprising: (i) a nucleotide sequence of a C1orf135 (Chromosome 1 open reading frame 135) gene, (ii) a fragment of the nucleotide, (iii) a sequence complementary to the nucleotide sequence or a fragment thereof, It provides a kit for diagnosing colorectal cancer comprising (iv) a protein expressed from the C1orf135 gene, or (v) a binder specific for the protein.

According to another aspect of the present invention, the present invention provides a method for detecting colorectal cancer diagnostic markers by measuring the expression level of C1orf135 gene in a biological sample of human in order to provide information necessary for diagnosing colorectal cancer.

According to another aspect of the present invention, the present invention provides a method for detecting a colorectal cancer diagnostic marker through a method of measuring the level of protein expressed from the C1orf135 gene in a biological sample of human to provide information necessary for the diagnosis of colorectal cancer To provide.

The present inventors have made intensive studies to find genes that can be used for diagnosing colorectal cancer and screening drugs and can be targets for treating colorectal cancer. As a result, the expression of C1orf135 gene in colon cancer cells is significantly increased, and the colorectal cancer treatment is inhibited. By finding out that there is efficacy, the present invention has been completed.

The C1orf135 (Chromosome 1 open reading frame 135) gene of the present invention is also called AIPB and encodes an Aurora A-binding protein. The mRNA sequence of C1orf135 is as shown in SEQ ID NO: 1, and the ORF sequence is shown in SEQ ID NO: 2. SEQ ID NO: 3 shows the amino acid sequence of the Aurora A-binding protein, a protein expressed from the C1orf135 gene. As used herein, the term “C1orf135 gene” refers to DNA or mRNA of these genes, and is a concept that includes all or part of DNA or mRNA.

In one embodiment of the present invention, the method for detecting a colorectal cancer diagnostic kit or colorectal cancer diagnostic marker is a method for diagnosing colorectal cancer by confirming a reaction between a C1orf135 gene or a fragment thereof or a complementary sequence thereof and a sample obtained from a subject. I use it. Confirmation of the reaction between the C1orf135 gene and the sample obtained from the subject is a common method used to confirm the reaction between DNA-DNA, DNA-RNA, DNA-protein, such as DNA chip, protein chip, polymerase chain reaction (PCR). ), Northern blotting, Southern blotting, Enzyme Linked Immunosorbent assay (ELISA), yeast two-hybrid, 2-D gel analysis and in vitro binding assays ( in in vitro binding assay) and the like. That is, by using all or part of the gene as a probe to hybridize with the nucleic acid isolated from the body fluids of the subject, various methods known in the art, such as reverse transcription polymerase chain reaction, Southern blotting ( By detecting this by Southern blotting, Northern blotting, etc., it is possible to determine whether colorectal cancer is generated by investigating whether the gene is high or low in the subject.

In addition, the method for detecting a colorectal cancer diagnostic kit or colorectal cancer diagnostic marker of the present invention may use the principle of diagnosing colorectal cancer by confirming a reaction between a protein expressed from the C1orf135 gene and a sample obtained from a subject. As mentioned above, the C1orf135 gene is specifically expressed in colorectal cancer cells, so it is possible to diagnose colon cancer by not only examining whether the gene is overexpressed but also by overexpressing the protein expressed from the gene. have. In the colorectal cancer diagnostic kit of the present invention, confirmation of the reaction between the composition and the sample containing the protein may be performed using conventional methods, such as DNA chips, proteins, etc. Chip, polymerase chain reaction (PCR), Northern blotting, Southern blotting, Western blotting, Enzyme Linked Immunosorbent assay (ELISA), specific immunostaining, yeast two-hybrid, 2 -D gel analysis and in vitro binding assay (in in vitro binding assay) and the like. For example, all or part of a protein expressed from the genes as a probe may be hybridized with a nucleic acid or protein isolated from a subject's body fluid, and then various methods known in the art, such as reverse transcription polymerases chain. By detecting this by reaction, western blotting, or the like, it is possible to determine whether colorectal cancer occurs by examining whether the gene is highly expressed in the subject.

According to a preferred embodiment of the present invention, the kit of the present invention is characterized in that the microarray. The probe is immobilized on the solid surface of the microarray. 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 binding 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 or RNA applied to the microarray of the present invention can be labeled and hybridized with the 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 colorectal cancer of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

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 (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin And hapten with specific binding partners such as digoxigenin and chelating groups. Labeling is carried out in a variety of ways conventionally practiced in the art, such as the nick translation method, the random priming method (Multiprime DNA labeling systems booklet, "Amersham" (1989)), and the chination method (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

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 time, 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 by 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).

According to another preferred embodiment of the present invention, the colorectal cancer diagnostic kit of the present invention is 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 may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations 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, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

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. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. 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.

According to another preferred embodiment of the present invention, the colorectal cancer diagnostic kit of the present invention is an immunoassay kit, characterized in that the antibody or aptamer specifically binding to the colorectal cancer markers of the present invention. It can be carried out using.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The antibody may be commercially available and methods conventionally practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. . Mol Biol, 222:.. 58, may be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Monoclonal antibodies are generally developed by using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a substrate thereof. It may be quantitatively analyzed, or may be directly quantitated using a conjugate of AP or HRP enzyme or the like to a monoclonal antibody directed against the protein.

Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

In addition, a monoclonal antibody or a part of polyclonal antibody is also included in the antibody of the present invention as long as it has antigen binding, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) an inhibitor of expression of the C1orf135 gene; And (b) provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising a pharmaceutically acceptable carrier.

In the present invention, the expression inhibitor of the C1orf135 gene may be an antisense oligonucleotide to the mRNA of the gene.

Antisense oligonucleotides have been successfully used to achieve gene-specific inhibition in vivo as well as in vitro. Antisense nucleotides are short length DNA synthesis strands (or DNA analogs) that are antisense (or complementary) to a particular DNA or RNA target. Antisense oligonucleotides have been proposed to prevent the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides have been successfully used in cell culture and animal models of disease (Hogrefe, 1999). Other modifications of antisense oligonucleotides to make them more stable and resistant to degradation of oligonucleotides are known and understood by those skilled in the art. Antisense oligonucleotides as used herein include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and to increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications to oligonucleotide backbones, modifications of sugar moieties or bases known in the art.

In addition, the inhibitor for the C1orf135 gene may be a siRNA (Small Interfering RNA) of the gene and the base sequence may be 19-23 consecutive sequential sequences in each sequence of the base sequence of the C1orf135 gene (mRNA) have.

The siRNA sequence shows a forward strand (sense strand) for convenience, and constitutes a double ribonucleic acid chain together with a reverse sequence (antisense strand) indicating the actual gene suppression effect.

siRNA has the potential to be a very strong drug against inhibition of expression of specific genes in vivo in terms of cell culture and long lasting effects, ability to transfect cells in vivo and resistance to serum degradation. (Bertrand et al., 2002). Delivery of siRNAs and expression constructs / vectors including siRNAs are known to those skilled in the art. For example, US applications 2004/106567 and 2004/0086884 describe many expression constructs as well as delivery mechanisms including viral vectors, nonviral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates. Provides a vector / vector.

siRNAs have been proposed as alternatives to antisense oligonucleotides because they can achieve an effect equal to or higher than that obtained by antisense oligonucleotides even at relatively low concentrations (Thompson, 2002). The use of siRNAs has shown popularity in inhibiting gene expression in animal models of disease. One skilled in the art can synthesize and modify the antisense oligonucleotides and siRNAs in any desired manner using methods known in the art (eg, Andreas Henschel, Frank Buchholz1 and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs.Nucleic Acids Research 32 (Web Server Issue): W113-W120. Reference). Those skilled in the art also understand well regulatory sequences (eg, constitutive promoters, inducible promoters, tissue-specific promoters or combinations thereof) useful for expression constructs / vectors with antisense oligonucleotides or siRNAs.

In addition, the inhibitor for the gene may be a short hairpin RNA (shRNA) of the gene. shRNA is a single-stranded RNA with a length of 45 to 70 nucleotides. The shRNA synthesizes oligo DNA linking 3-10 base linkers between the sense of target gene siRNA and complementary nonsense, and then clones into a plasmid vector or shRNA. When inserted into the retroviruses lentivirus (adetivirus) and adenovirus (adenovirus) to express a loop of the hairpin structure of the shRNA is produced and the intracellular dicer is converted to siRNA by the RNAi effect. It shows a relatively long RNAi effect compared to the shRNA.

In addition, the inhibitor for the C1orf135 gene may be micro RNA of the gene. Micro RNA (micro RNA or miRNA) regulates a variety of biological processes, including development, differentiation, proliferation, conservation and apoptosis. MicroRNAs generally regulate the expression of the gene encoding the target mRNA by destabilizing the target mRNA or disrupting translation.

In addition, regulatory sequences useful in expression constructs / vectors with antisense oligonucleotides, siRNAs, shRNAs or microRNAs (eg, constitutive promoters, inducible promoters, tissue specific promoters or combinations thereof) are also known in the art. We can choose appropriately from contents.

The inhibitor of the gene may be any substance that inhibits the expression of the gene in addition to the antisense oligonucleotide, siRNA, shRNA or microRNA. Thus, compounds known in the art as inhibitors of such genes are also available.

Antisense oligonucleotides, siRNAs, shRNAs or microRNAs of the invention used for the treatment of colorectal cancer may be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide fast release, or sustained or delayed release of the active ingredient after administration.

According to another aspect of the invention, the invention is a protein inhibitor expressed from (a) C1orf135 gene; And (b) provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising a pharmaceutically acceptable carrier.

When the gene of the present invention is inhibited, the expression of the protein is suppressed and colon cancer is inhibited. Therefore, when the protein of the gene is inhibited, the colon cancer can be suppressed.

Accordingly, the present invention provides a method for treating colorectal cancer, comprising the use of an inhibitor for the protein, and administering an effective amount of the inhibitor of the protein to a patient. Treatment of colorectal cancer in the present invention includes the prevention and inhibition of colorectal cancer.

The inhibitor for the protein may be, but is not necessarily limited to, an antibody or aptamer for a protein expressed from a gene of the present invention. The monoclonal antibody to the protein may be prepared and used through a monoclonal antibody manufacturing method customary in the art, or may be used commercially. In addition, a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through an antiserum production method conventional in the art.

The composition for treating colorectal cancer of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. If the inhibitor for a protein of the invention is an antibody, the pharmaceutically acceptable carrier may consist of a minimum amount of auxiliary material, such as a wetting or emulsifying agent, preservative or buffer, which increases the shelf life or effectiveness of the binding protein.

In addition, the composition for treating colorectal cancer of the present invention may be used together with one or more colorectal cancer therapeutic agents. Compositions for treating colorectal cancer of the present invention are, for example, chemotherapeutic agents well known to those skilled in the art, such as cyclophosphamide, aziridine, alkylalconsulfonate, nitrosourea, dacarbazine, carboplatin , Alkylating agents such as cisplatin, antibiotics such as mitomycin C, anthracycline, doxorubicin (adriamycin), antimetabolitic agents such as methotrexate, 5-fluorouracin, cytarabine, vinca alkaloids, etc. Plant-derived drugs and hormones may be further included.

The composition for treating colorectal cancer of the present invention may include a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and such adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Lubricants, solubilizers and the like.

In addition, the composition of the present invention may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration.

Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical formulation forms of the compositions for treating colorectal cancer of the present invention are granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds. And so on.

The composition for treating colorectal cancer of the present invention may be administered through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It may be administered in a conventional manner.

In the method of treatment of the present invention, "effective amount" means the amount required to achieve the effect of inhibiting colorectal cancer. Thus, the "effective amount" of the active ingredient of the present invention may include the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, body weight, general state of health, sex and It can be adjusted according to various factors including the diet, the time of administration, the route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently. In adults, when the inhibitor of the gene or protein is administered once or several times a day, 0.01ng / kg-10mg / kg for siRNA, 0.01ng / kg-10 for antisense oligonucleotides to mRNA of the gene Mg / kg, 0.1ng / kg-10mg / kg for compounds, and 0.1ng / kg-10mg / kg for monoclonal antibodies to the protein.

According to another aspect of the invention, the present invention comprises the steps of (a) contacting a test substance to a cell expressing the nucleotide represented by SEQ ID NO: 1; And (b) analyzing the effect of the test substance on the expression of the nucleotide, and when the test substance inhibits the expression of the nucleotide, the method for screening a colorectal cancer therapeutic agent is determined as a colorectal cancer therapeutic agent.

In the screening method of the present invention, confirming the reaction between the composition containing the gene and the test substance may use conventional methods used to confirm the reaction between DNA-DNA, DNA-RNA, DNA-protein, and DNA-compound. have. For example, outside of a living body ( in In vitro ) hybridization test to confirm the binding between the gene and the test substance, a method of measuring the expression rate of the gene by Northern analysis, quantitative PCR, quantitative real-time PCR after reacting the mammalian cells and the test substance, or the The reporter gene may be linked to a gene, introduced into a cell, reacted with a test substance, and a method of measuring the expression rate of the reporter protein may be used. In such a case, the composition of the present invention may include distilled water or a buffer solution to keep the structure of the nucleic acid stable, in addition to the gene.

In the screening method of the present invention, the test subject may be individual nucleic acids, proteins, other extracts or natural products, or the like, which are estimated to have a potential as a colorectal cancer metastasis inhibitor according to a conventional selection method or randomly selected. .

Inhibition of the expression of the test substance and the activity of the colorectal cancer high expression gene obtained by the screening method of the present invention to enhance the expression of the high-expression gene or enhance the function of the protein, and conversely inhibits the function of the protein In the case of the former, the test substance exhibiting activity may be a candidate for colorectal cancer treatment by developing an inhibitor for the test substance, and in the latter case, it may be a candidate for colorectal cancer treatment. Such a candidate drug for colorectal cancer treatment acts as a leading compound in the development process of colorectal cancer treatment in the future, and modifies its structure so that the leading substance can exhibit a function inhibitory effect of the gene or protein expressed therefrom. By optimizing, new colon cancer therapies can be developed.

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

(Iii) The present invention provides a kit for diagnosing colorectal cancer, a method for detecting a colorectal cancer diagnostic marker, a pharmaceutical composition for preventing or treating colorectal cancer, and a screening method for treating colorectal cancer.

(Ii) The present invention provides a colorectal cancer specific target gene to enable accurate colorectal cancer diagnosis, and provides a new colorectal cancer specific therapeutic agent through the target gene, thereby enabling the development of bio-drugs and customized anticancer drugs with fewer side effects. do.

1 is a graph and data analysis results showing that the expression of the C1orf135 gene was increased in 66 colorectal cancer clinical tissues through microarray experiment using Illumina 48K chip.
Figure 2 shows the results of analyzing the change in the expression level of the C1orf135 gene in 16 colon cancer patients tissues by RT-PCR. GAPDH is a control gene.
Figure 3 is a result of quantitative analysis of the change in the expression level of the C1orf135 gene in colorectal cancer cell lines and normal cells using Realtime-PCR. GAPDH was used as a control gene.
4A shows the cloning of the C1orf135 gene.
Figure 4b is a graph showing the change in cell growth rate due to the overexpression of C1orf135 gene and SRB staining to investigate the growth of cells.
5a shows the effect of C1orf135 siRNA by selecting colorectal cancer cell line KM12C as a representative cell line. Treatment of C1orf135 siRNA shows that the amount of actual C1orf135 mRNA is reduced and protein is reduced.
5B shows the inhibition of growth of KM12C cells following treatment with siRNA of C1orf135.
Figure 6 is a graph showing the effect of inhibiting cell growth rate by the treatment of siRNA in normal cells and colorectal cancer cell lines.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Example 1: Confirmation of the effect of increasing the expression level of C1orf135 in clinical tissue of colorectal cancer patients by microarray experiment

(1) Preparation of colorectal cancer clinical tissue

Colorectal cancer and normal tissue from 66 patients with colorectal cancer were obtained from Samsung Medical Center (Seoul, South Korea). Each tissue was surgically removed from the patient and stored in liquid nitrogen until analysis.

(2) total RNA isolation

Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantified using Experion RNA StdSens (Bio-Rad) chip. First, the colorectal cancer clinical tissues and normal tissues were cut to an appropriate size, and then dissolved in 15 ml of a digestion buffer in a kit to which 150 μl of beta mercapto ethanol was added. 15 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After washing twice, total RNA was isolated by adding 1.2 ml of RNase-free water.

(3) Microarray

The extracted total RNA was hybridized using an Illumina TotalPrep RNA Amplification Kit (Ambion). CDNA was synthesized using a T7 Oligo (dT) primer, and in vitro transcription was performed using biotin-UTP to prepare a biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. CRNAs prepared from normal colon epithelial cells and colon cancer cells were hybridized to a Human-6 V2 (Illumina) chip. In order to remove non-specific hybridization after hybridization, DNA chips were washed using Illumina Gene Expression System wash (Illumina) and the washed DNA chips were labeled with streptavidin-Cy3 (Amersham) fluorescent dye. Fluorescently labeled DNA chips were scanned using a confocal laser scanner (Illumina, Inc.) to obtain fluorescence data present in each spot and stored as image files in TIFF form. TIFF image files were quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence values of each spot. Quantified results were corrected using the 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.

The results are shown in FIG. 1 is a C1orf135 gene expression profile measured in cancerous tissues and normal tissues of colorectal cancer patients, it is shown in red when high expression, green when low expression. The C1orf135 gene had an average six-fold increase in expression in colorectal cancer tissues. These results indicate that the C1orf135 gene shows a large expression difference compared to normal colon epithelial cells, and thus, can be used as a diagnosis of colorectal cancer, drug screening, or therapeutic target. In addition, FIG. 1 shows that C1orf135 gene expression was increased by an average of 4 times or more in 64 colorectal cancer tissues out of 66 colorectal cancer tissues compared to normal tissues.

Example  2: Confirmation of Gene Expression for Colorectal Cancer in Individual Clinical Samples

17 pairs of tissue tissues (T1-T66) and normal tissues (N1-N66) from 17 pairs of colorectal cancer patients (66 cancer patients of Example 1) (T1-T66) The expression levels of the C1orf135 gene identified in Example 1 using T17 and N1-N17) were analyzed by RT-PCR method. Total RNA was isolated via the method of Example 1.

(1) c-DNA synthesis and concentration correction of template

2 μg of total RNA of each sample, 1 μl of 50 μM oligo (dT) primer and 2.5 μl of 10 mM dNTP were added and sterile water containing RNase inhibitor DEPC (diethyl pyrocarbonate) was added to make 25 μl total RNA / primer. A mixed solution was made. After reacting at 65 ° C. for 5 minutes, the mixture was transferred to 55 ° C. and stored. Next, add 5 µl of 10X RT buffer, 10 µl of 25 mM MgCl ₂ , 5 µl of 0.1M DTT, 1 µl of RNase inhibitor, and 1 µl of SuperScript III RT enzyme to make 25 µl of the total. After mixing with, it was reacted at 55 ℃ for 50 minutes. Thereafter, the reaction was carried out at 85 ° C. for 5 minutes to inactivate the RT enzyme, followed by termination on the reaction. GAPDH was used as a standard gene for quantifying marker genes. RT-PCR reaction was performed using primers of the standard gene, and the concentration of cDNA was corrected so that the expression level of the standard gene GAPDH was the same. First, each cDNA was diluted 20-fold, and then PCR reaction was performed using 2 μl of the diluted sample. PCR was performed using 15 µl of 2X PCR premix (Hot start), 2 µl of GAPDH 5 'primer, 2 µl of 3' primer, and 11 µl of distilled water, and 20, 23 and 25 cycles were performed. At this time, RT-PCR reaction conditions were performed at 94 ℃ 30 seconds, 50 ℃ 30 seconds and 72 ℃ 1 minutes, the product size was 457 bp. The GAPDH primers used were N-terminal 5'-TCATGACCACAGTCCATGCC-3 '(SEQ ID NO: 6) and C-terminal' -TCCACCACCCTGTTGCTGTA-3 '(SEQ ID NO: 7). Each product was loaded into a 2% agarose gel, electrophoresed, electrophoresed, and gel images were taken. The images were quantified using the TotalLab v1.0 program (Nonlinear Dynamix), and then corrected for PCR. The concentration of was equally corrected.

(2) Expression analysis using RT-PCR

CDNA diluted to the same amount was PCR using the sense and antisense primers of the C1orf135 gene. cDNA was mixed with 3 μl, 10 μl of 2X premix, 2 μl each of primer (20 pmole) and 2 μl of distilled water to make 20 μl of total solution. The PCR reactions were 94 degrees 1 minute, 54 degrees 30 seconds and 72 degrees 1 minute. The cycle was performed. In order to confirm the PCR product, it was electrophoresed using 2% agarose gel and analyzed using an imaging apparatus. Realtime RT-PCR was used with DNASYBRI reagent from Qiagen (CA, USA) and LightCycler (Roche). Melt Curve analysis was used to assess the quality of PCR products and gene expression was analyzed using LightCycler version 3.5 software (Roche).

C1orf135 specific primers used in the RT-PCR are as follows. N-terminal primer 5'-AGAAAAAGGGGATTCTGCCA-3 '(SEQ ID NO: 4), C-terminal primer 5'-CTACGGCTTTGTTTCACCGA-3' (SEQ ID NO: 5). The results are shown in Figure 2, in the picture, N means normal tissue (Non-tumor tissue), T means the corresponding colorectal cancer tissue. As a result, it was confirmed that the C1orf135 gene is highly expressed in colorectal cancer samples, and thus, the C1orf135 gene may be used as a colorectal cancer marker or therapeutic target for diagnosis or drug screening of colorectal cancer.

Example 3. Confirmation of the increased expression level of the C1orf135 gene in colorectal cancer cell line

A simple way of estimating the cancer's relevance to a gene is to compare changes in the expression level of that gene in cancer tissues or cancer cell lines. The relationship between the expression level according to the cancer production and progression should be investigated by comparing whether the cancer cell line is expressed or reduced in a large amount compared to normal cells. To do this, extract the total RNA from the colorectal cancer cell line, and then perform RT-PCR and Realtime-PCR using the oligonucleotides of the gene to compare the amount of each gene with the normal cell line. In comparison, the expression level of the gene in the cell is confirmed. In the present invention, the expression level of the C1orf135 gene was analyzed for mRNA levels in colorectal cancer cell lines and normal cells.

(1) Preparation of colorectal cancer cell line

DMEM or RPMI1640 (GIBCO) cultures containing 10% fetal bovine serum (Fetal Bovine Serum, GIBCO), penicillin (10000 U / mL) and streptomycin (10 mg / mL) were used. Colorectal cancer cell lines DLD-1 (Korea Cell Line Bank), Colo205 (Korea Cell Line Bank), SW480 (ATCC), SW620 (Korea Cell Line Bank), SNU-C1 (Korea Cell Line Bank), SNU-C2A (Korea Cell Line Bank), Inoculate KM12C (Korea Cell Line Bank), KM12SM (Korea Cell Line Bank), HT29 (ATCC) and Hct116 (Korea Cell Line Bank, KCLB) to have a cell count of 1 × 10 ⁶ per dish and incubate it at 37 ° C with 5% CO _2. Incubated at. IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as a normal cell line. Colon cancer cell lines were inoculated with a cell number of 1 × 10 ⁶ and cultured in an incubator at 37 ° C. with 5% CO ₂ .

(2) total RNA isolation

Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantified using Experion RNA StdSens (Bio-Rad) chip. Cells were recovered by centrifugation and lysed by adding 10 μl of beta mercapto ethanol to 1 ml of digestion buffer RLN (50 mM TrisCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl 2 and 0.5% NP-40) in the kit. 1 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After two washes, total RNA was isolated by adding 100 μl of RNase-free water.

(3) Analysis of expression level using c-DNA synthesis and Realtime PCR

c-DNA was synthesized by the same method as in Example 2, and the concentration correction process of the template was performed in the same manner as in Example 2. CDNA diluted to the same amount was subjected to Realtime-PCR using the sense and antisense primers of the C1orf135 gene. Realtime RT-PCR was performed using DNASYBRGreen I reagent and LightCycler (Roche) from Qiagen (CA, USA). Melt Curve analysis was used to assess the quality of PCR products and gene expression was analyzed using LightCycler version 3.5 software (Roche). The C1orf135 specific primer used was the same as the primer used in Example 2, except that GAPDH used as a control was a Qiagen primer (QT00079247). As a result of performing Realtime-PCR, it was confirmed that the mRNA expression of C1orf135 was significantly increased in many colorectal cancer cell lines compared to normal cell lines (FIG. 3). These results indicate that increased expression level of the C1orf135 gene may be an index and treatment target for colorectal cancer diagnosis.

Example  4. C1orf135  Gene Cloning

The C1orf135 gene was amplified using PCR technique from Invitrogen's human brain c-DNA library. The forward primer 5'-TGC GGATCC ATGAGGCGGACAGGCCCCGAG-3 ' (BamHI enzyme cleavage site comprises - underline, SEQ ID No. 8, SEQ) and the reverse primer 5'-ATG GATATC TTAGAATTGGTGTCTGATAAC-3' (EcoRV enzyme cleavage site comprises - underline, SEQ ID NO. Gene was amplified by PCR using pfu premix (iNtRON, Inc.) at 55 ° C for 35 cycles. The DNA band of the C1orf135 gene was purified, cut with BamHI / EcoRV restriction enzyme, and cloned into pENTR3C vector. DNA sequencing confirmed the nucleotide sequence (1071 bp) of the C1orf135 gene and cloned it according to the manufacturer's manual using the Gateway system (Invitrogen Corp. www.invitrogen.com). To clone C1orf135, pENTR3C-C1orf135 was cloned by LR reaction with pDEST-Flag (Invitrogen Corp. www.invitrogen.com). The plasmid pENTR3C-C1orf135 was mixed with the vector pDEST-Flag DNA, and then LR clonase II enzyme mixture was added and incubated for 1 hour at room temperature. After culturing, proteinase K was added and incubated at 37 ° C. for 10 minutes to complete the LR reaction, and then transformed into E. coli DH5α competent cells to obtain a pDEST-Flag-C1orf135 clone.

Example  5. C1orf135 Effect of cell growth rate on cell overexpression

DMEM medium containing a mixture of 5% bovine calf serum and penicillin (10,000 units / ml) and streptomycin (10 mg / ml) in a normal cell line, NIH3T3 fibroblasts (ATCC), diluted 100-fold. (GIBCO) was inoculated with 5 ml in a 60 mm dish at a concentration of 1.4 × 10 ⁵ cells / ml and then incubated at 37 ° C. for 24 hours in a 5% CO ₂ incubator. C1orf135 plasmid DNA was transfected into the cultured cells. After 6 hours, all of the medium was removed, washed three times with PBS (Phosphate Buffered Saline), and then changed to a medium containing 5% serum. 24 hours after transfection, the cells were treated with 0.5% EDTA-trypsin, collected and counted by a hemocytometer, and seeded in a 24-well plate at a concentration of 1 × 10 ⁵ cells / ml. ) Was incubated in a 5% CO2 incubator at 37 ° C. until SRB (Sulforhodamine B) analysis. One plate was taken out every 12 hours after incubation, and cell growth rate was measured by SRB analysis. For comparison, the cells were transfected with pDEST-flag vector DNA and treated in the same manner to the NIH3T3 cell line, which is a normal cell line, and used as a control. The results are shown in FIG. 4, the horizontal axis represents time, and the vertical axis represents cell growth rate. PDEST-Flag refers to a cell transfected with a vector without the C1orf135 gene, and pDEST-Flag-C1orf135 is a vector cloned with the C1orf135 gene. Refers to cells overexpressing the C1orf135 protein.

As a result, the growth rate was increased by about 30% in cells overexpressing the C1orf135 gene, indicating that the C1orf135 gene is associated with increasing the growth rate of cells characteristic of tumor cells (FIG. 4). These results indicate that increased expression level of the C1orf135 gene may be an indicator of tumor cell diagnosis.

Example  6. Colorectal cancer cell line siC1orf135 Effect of Growth Inhibition on Colorectal Cancer Cell Lines

In the case of genes expressed in large amounts in colorectal cancer cell tissues or colorectal cancer cell lines, siRNA techniques can be used to observe whether there is a change in cell proliferation rate. In the present invention, each siRNA for C1orf135 was designed and introduced into the colorectal cancer cell line, and then the growth rate of the colorectal cancer cell line was observed according to the decrease in the gene mRNA expression. Eight colorectal cancer cell lines, DLD-1 (Korea Cell Line Bank), Colo205 (Korea Cell Line Bank), SW480 (ATCC), SW620 (Korea Cell Line Bank), SNU-C1 (Korea Cell Line Bank), SNU-C2A (Korea Cell Line Bank) ), KM12C (Korea Cell Line Bank) and KM12SM (Korea Cell Line Bank, KCLB) and normal cell line IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) were used.

(1) Confirmation of mRNA inhibitory effect of C1orf135 by siC1orf135 using KM12C cell line

First, C1orf135 and siRNA to be used as a control were designed and synthesized (Samchully Pharmaceutical, South Korea). The synthesized siRNA consists of a structure in which two bases of a single chain are suspended in a double ribonucleic acid chain of 19 oligonucleotides designed based on gene sequences. A control siRNA (siControl) was used as a negative control of siRNA experiments that had no effect on cell proliferation.

After incubating the colon cancer cell line KM12C with 70% confluency, siC1orf135 (sense sequence, SEQ ID NO: 10) and siControl (sense sequence, SEQ ID NO: 11) were introduced into the cells by the hyperfect method (Qiagen Co.). Cultured for 72 hours and transformed. RNA was extracted from each cell and RT-PCR was performed as described in detail above to confirm that the gene mRNA was reduced, and protein expression was analyzed by Western blot to confirm that gene expression was inhibited by siRNA ( 5). siControl represents the negative control. siControl shows no siRNA targets for sequences that are not homologous to human genes. Each cell was stained with Sulfur rhodamine B (Sigma Co.) and microscopic observation of the SRB stained cells of the siControl-treated samples showed no significant changes in cell growth. It is shown that it happened (Fig. 5B).

(2) Verification of growth inhibition effect by siC1orf135 in various colorectal cancer cell lines

To investigate the extent of cell growth degradation by siRNA in a more diverse cell line, 0.5 ml seeding in a 24 well plate at a concentration of 1.0 × 10 ⁵ cells / ml was followed by 24 hours at 37 ° C. in a 5% CO ₂ incubator. Incubated. The control siRNA (sense sequence, SEQ ID NO: 11) and siC1orf135 (sense sequence, SEQ ID NO: 10) were transfected into the cell according to the above method and cultured for 72 hours, and the cells were cultured by siRNA. The degree of growth inhibition was also investigated. Each cell was stained with SRB (Sulfur rhodamine, Sigma Co.) and compared with the control cell line treated with siControl, and the growth inhibition of the cells was compared and the results are shown in FIG. 6. In FIG. 6, the X axis represents each cell line, and the Y axis represents relative cell growth. As a result, inhibition of gene expression of C1orf135 in most colorectal cancer cell lines showed cell growth inhibition. According to the above experimental results, the siRNA of the C1orf135 gene can induce growth inhibition of the colorectal cancer cell line, which means that the C1orf135 gene can be used as a target for treating colorectal cancer.

Table 1 shows an example of siRNA for the C1orf135 gene.

siRNA position within mRNA order GC content Identity 794 CGUAGUCCAGUUUCUGUGUTT 0.47 19 850 GAGUCAACUUUUCACUGAATT 0.37 19 807 CUGUGUUUUCCUGGGACAATT 0.47 19 896 CACAACACUAGAGCUCCUUTT 0.47 19 261 CAGAAAGUCAGAUCAACAATT 0.37 19 274 CAACAAAGAGUCCAAGAAATT 0.37 19 668 CAGCCCUUAGGCAAGACUATT 0.53 19 911 CCUUUUCAAGAUGUAACCATT 0.37 19

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. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Korea Research Institute of Bioscience & Biotechnology <120> Diagnostic Kit for Colon Cancer and Pharmaceutical Composition          for Prevention and Treatment of Colon Cancer <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 2062 <212> DNA <213> Homo sapiens, C1orf135 mRNA <400> 1 atgaggcgga caggccccga ggaggaggcc tgcggcgtgt ggctggacgc ggcggcgctg 60 aagaggcgga aagtgcagac acatttaatc aaaccaggca ccaaaatgct aacactcctt 120 cctggagaaa gaaaggctaa tatttatttt actcaaagaa gagctccatc tacaggcatt 180 caccagagaa gcattgcttc cttcttcacc ttgcagccag gaaagacaaa tggcagtgac 240 cagaagagtg tttcatctca tacagaaagt cagatcaaca aagagtccaa gaaaaatgcg 300 acccagctag accatttgat cccaggctta gcacacgatt gcatggcatc ccctttagcc 360 acttcaacca ctgcggacat ccaggaagct ggactctctc ctcagtccct ccagacttct 420 ggccaccaca gaatgaaaac cccattttca actgagctat ctttgctcca gcctgatact 480 ccagactgtg ctggagatag tcatacccca ctggcttttt ccttcaccga ggacttggaa 540 agttcttgtt tgctagaccg aaaggaagaa aaaggggatt ctgccaggaa atgggaatgg 600 cttcatgagt ctaagaagaa ctatcagagt atggagaaac acaccaaact acctggggac 660 aaatgctgtc agcccttagg caagactaaa ttggaaagaa aggtgtctgc caaagaaaac 720 aggcaggccc ctgtcctcct tcaaacatac agggaatcct ggaatggaga aaacatagaa 780 tcggtgaaac aaagccgtag tccagtttct gtgttttcct gggacaatga aaagaatgac 840 aaggactcct ggagtcaact tttcactgaa gattctcaag gccagcgggt cattgcccac 900 aacactagag ctccttttca agatgtaacc aataactgga attgggactt agggccgttt 960 cctaacagtc cttgggctca gtgccaggag gatgggccaa ctcaaaatct gaagcctgat 1020 ttgctcttta cccaggactc tgaaggtaat caagttatca gacaccaatt ctaaatgttt 1080 gaagctttgt ttctaaaagt accttgaaat gatagagatg taggaaaata tagttgtggg 1140 tggagagagg agtgagtttg tttaggtggg aaggtggcat gggatgaagt tgtcattact 1200 gagcatcttc tctgtgtaaa taaagggcag taccattgtt aagacagtgg gattggcatc 1260 atggctttcc ctcaggaagg tggtggctgg taaattccct gaatgagtct atgatgaaca 1320 ctgaggcagc acagtgggta tttatctcta tgaaagtgcc ttttactcag cctgcacaga 1380 gccatctctt tgcccttcca gatgtctgac tgggaccttg cttatggatg tgtttttttt 1440 tttttttttt tgagatggag tctcgctctg tcgccaggct ggagtgcagt ggtgcgacct 1500 cagctcactg caccctctgt gtcccggatt caagcgattc tcctgcctca gcctcccgaa 1560 tagcagggac tacaggcatg cgccaccacg cccagctaat tttttttgga tttttagtag 1620 agacgaggtt tcaccatatt agccaggatg gtctccatct cctgacctcc tgatccgccc 1680 acctcagcct cccaaagtgc tgagattaca ggcataagcc accgcgccca gccagatgtg 1740 tgagctttta atctctggct gatcttaacc cacatcagcc taagcttggg atgattactc 1800 ttgacccttt tttttcagtg attagcaaat ctccccacaa cccaggtgtg gagagaagag 1860 aggtagaatg gtgctagttt cctattttat ttttgtggta actgtacagc actttaaagt 1920 tatatactct atgtttaaat atctccctta aaaagcctga gctgtacaac aatctggatg 1980 tgactctgtt acccttttcc cacaagatag gagggaatcc cctttgtaaa actatgaatc 2040 caaataaatg tttacaaagt gt 2062 <210> 2 <211> 1074 <212> DNA <213> Homo sapiens, C1orf135 ORF <400> 2 atgaggcgga caggccccga ggaggaggcc tgcggcgtgt ggctggacgc ggcggcgctg 60 aagaggcgga aagtgcagac acatttaatc aaaccaggca ccaaaatgct aacactcctt 120 cctggagaaa gaaaggctaa tatttatttt actcaaagaa gagctccatc tacaggcatt 180 caccagagaa gcattgcttc cttcttcacc ttgcagccag gaaagacaaa tggcagtgac 240 cagaagagtg tttcatctca tacagaaagt cagatcaaca aagagtccaa gaaaaatgcg 300 acccagctag accatttgat cccaggctta gcacacgatt gcatggcatc ccctttagcc 360 acttcaacca ctgcggacat ccaggaagct ggactctctc ctcagtccct ccagacttct 420 ggccaccaca gaatgaaaac cccattttca actgagctat ctttgctcca gcctgatact 480 ccagactgtg ctggagatag tcatacccca ctggcttttt ccttcaccga ggacttggaa 540 agttcttgtt tgctagaccg aaaggaagaa aaaggggatt ctgccaggaa atgggaatgg 600 cttcatgagt ctaagaagaa ctatcagagt atggagaaac acaccaaact acctggggac 660 aaatgctgtc agcccttagg caagactaaa ttggaaagaa aggtgtctgc caaagaaaac 720 aggcaggccc ctgtcctcct tcaaacatac agggaatcct ggaatggaga aaacatagaa 780 tcggtgaaac aaagccgtag tccagtttct gtgttttcct gggacaatga aaagaatgac 840 aaggactcct ggagtcaact tttcactgaa gattctcaag gccagcgggt cattgcccac 900 aacactagag ctccttttca agatgtaacc aataactgga attgggactt agggccgttt 960 cctaacagtc cttgggctca gtgccaggag gatgggccaa ctcaaaatct gaagcctgat 1020 ttgctcttta cccaggactc tgaaggtaat caagttatca gacaccaatt ctaa 1074 <210> 3 <211> 357 <212> PRT <213> Homo sapiens, aurora A-binding protein <400> 3 Met Arg Arg Thr Gly Pro Glu Glu Glu Ala Cys Gly Val Trp Leu Asp   1 5 10 15 Ala Ala Ala Leu Lys Arg Arg Lys Val Gln Thr His Leu Ile Lys Pro              20 25 30 Gly Thr Lys Met Leu Thr Leu Leu Pro Gly Glu Arg Lys Ala Asn Ile          35 40 45 Tyr Phe Thr Gln Arg Arg Ala Pro Ser Thr Gly Ile His Gln Arg Ser      50 55 60 Ile Ala Ser Phe Phe Thr Leu Gln Pro Gly Lys Thr Asn Gly Ser Asp  65 70 75 80 Gln Lys Ser Val Ser Ser His Thr Glu Ser Gln Ile Asn Lys Glu Ser                  85 90 95 Lys Lys Asn Ala Thr Gln Leu Asp His Leu Ile Pro Gly Leu Ala His             100 105 110 Asp Cys Met Ala Ser Pro Leu Ala Thr Ser Thr Thr Ala Asp Ile Gln         115 120 125 Glu Ala Gly Leu Ser Pro Gln Ser Leu Gln Thr Ser Gly His His Arg     130 135 140 Met Lys Thr Pro Phe Ser Thr Glu Leu Ser Leu Leu Gln Pro Asp Thr 145 150 155 160 Pro Asp Cys Ala Gly Asp Ser His Thr Pro Leu Ala Phe Ser Phe Thr                 165 170 175 Glu Asp Leu Glu Ser Ser Cys Leu Leu Asp Arg Lys Glu Glu Lys Gly             180 185 190 Asp Ser Ala Arg Lys Trp Glu Trp Leu His Glu Ser Lys Lys Asn Tyr         195 200 205 Gln Ser Met Glu Lys His Thr Lys Leu Pro Gly Asp Lys Cys Cys Gln     210 215 220 Pro Leu Gly Lys Thr Lys Leu Glu Arg Lys Val Ser Ala Lys Glu Asn 225 230 235 240 Arg Gln Ala Pro Val Leu Leu Gln Thr Tyr Arg Glu Ser Trp Asn Gly                 245 250 255 Glu Asn Ile Glu Ser Val Lys Gln Ser Arg Ser Pro Val Ser Val Phe             260 265 270 Ser Trp Asp Asn Glu Lys Asn Asp Lys Asp Ser Trp Ser Gln Leu Phe         275 280 285 Thr Glu Asp Ser Gln Gly Gln Arg Val Ile Ala His Asn Thr Arg Ala     290 295 300 Pro Phe Gln Asp Val Thr Asn Asn Trp Asn Trp Asp Leu Gly Pro Phe 305 310 315 320 Pro Asn Ser Pro Trp Ala Gln Cys Gln Glu Asp Gly Pro Thr Gln Asn                 325 330 335 Leu Lys Pro Asp Leu Leu Phe Thr Gln Asp Ser Glu Gly Asn Gln Val             340 345 350 Ile Arg His Gln Phe         355 <210> 4 <211> 20 <212> DNA <213> Homo sapiens, C1orf135 specific primer (N-teminal) <400> 4 agaaaaaggg gattctgcca 20 <210> 5 <211> 20 <212> DNA <213> Homo sapiens, C1orf135 specific primer (C-teminal) <400> 5 ctacggcttt gtttcaccga 20 <210> 6 <211> 20 <212> DNA <213> Homo sapiens, GAPDH specific primer (N-teminal) <400> 6 tcatgaccac agtccatgcc 20 <210> 7 <211> 20 <212> DNA <213> Homo sapiens, GAPDH specific primer (C-teminal) <400> 7 tccaccaccc tgttgctgta 20 <210> 8 <211> 30 <212> DNA <213> Homo sapiens, C1orf135 specific primer (forward) <400> 8 tgcggatcca tgaggcggac aggccccgag 30 <210> 9 <211> 30 <212> DNA <213> Homo sapiens, C1orf135 specific primer (reverse) <400> 9 atggatatct tagaattggt gtctgataac 30 <210> 10 <211> 21 <212> RNA <213> Homo sapiens, siC1orf135 (sense sequence) <400> 10 cguaguccag uuucuguguu u 21 <210> 11 <211> 21 <212> RNA Homo sapiens, siControl (sense sequence) <400> 11 ccuacgccac caauuucguu u 21

Claims (10)

(i) the nucleotide sequence of the Chromosome 1 open reading frame 135 (C1orf135) gene, (ii) a fragment of the nucleotide, (iii) a sequence complementary to the nucleotide sequence or a fragment thereof, (iv) a protein expressed from the C1orf135 gene, or (v) A kit for diagnosing colorectal cancer, comprising a binder specific for the protein.
The kit for diagnosing colorectal cancer according to claim 1, wherein the kit is a microarray.
The kit for diagnosing colorectal cancer according to claim 1, wherein the kit is a gene amplification kit.
The kit for diagnosing colorectal cancer according to claim 1, wherein the kit is a kit for immunoassay.
A method for detecting colorectal cancer diagnostic markers by measuring the expression level of the C1orf135 gene in a human biological sample to provide information necessary for diagnosing colorectal cancer.
A method for detecting a colorectal cancer diagnostic marker by measuring the level of a protein expressed from the C1orf135 gene in a human biological sample to provide information necessary for diagnosing colorectal cancer.
(a) inhibitors of expression of the C1orf135 gene; And (b) a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising a pharmaceutically acceptable carrier.
The pharmaceutical composition for preventing or treating colorectal cancer according to claim 7, wherein the inhibitor is selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs of the C1orf135 gene.
(a) a protein inhibitor expressed from the C1orf135 gene; And (b) a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising a pharmaceutically acceptable carrier.
Screening methods for treating colorectal cancer, comprising the following steps:
(a) contacting a test substance with a cell expressing a nucleotide represented by SEQ ID NO: 2; And
(b) analyzing the effect of the test substance on the expression of the nucleotide, wherein the test substance inhibits the expression of the nucleotide, and is determined as a therapeutic agent for colorectal cancer.

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