WO2012011777A2 - Kit for diagnosing colorectal cancer and pharmaceutical composition for prevention or treatment of colorectal cancer - Google Patents

Abstract

The present invention relates to: a composition for diagnosing colorectal cancer containing a formulation for measuring the expression level of a chromosome 1 open reading frame 135 (C1orf135) gene mRNA or the protein thereof; a kit for diagnosing colorectal cancer containing the composition; a method for providing information on diagnosis of colorectal cancer, the method comprising a step for measuring the expression level of a C1orf135 gene mRNA or the expression level of the protein coded by the gene; a pharmaceutical composition for the prevention or the treatment of colorectal cancer, the composition comprising an expression inhibitor for a C1orf135 gene or an activity inhibitor for the protein thereof; and a method for screening a therapeutic agent for colorectal cancer. The present invention enables the accurate diagnosis of colorectal cancer by providing a gene specific to colorectal cancer, and a diagnostic marker for determining the metastasis and prognosis of colorectal cancer, and can be useful in the diagnosis and prognosis management of colorectal cancer. Also, the present invention enables the development of new bio-medicines having fewer side effects and customized anticancer drugs by providing a colorectal cancer-specific therapeutic agent capable of inhibiting the gene.

Description

Colorectal cancer diagnostic kit and pharmaceutical composition for preventing or treating colorectal cancer

The present invention provides a composition for diagnosing colorectal cancer comprising an agent for measuring the expression level of mRNA of C1orf135 (Chromosome 1 open reading frame 135) gene or a protein thereof, a kit for diagnosing colorectal cancer comprising the composition, mRNA expression level of C1orf135 gene or A method for providing information for diagnosing colorectal cancer comprising measuring the expression level of a protein encoded by the gene, a pharmaceutical composition for preventing or treating colorectal cancer comprising an inhibitor of C1orf135 gene or an activity inhibitor of a protein thereof And a method for screening a colorectal cancer therapeutic agent.

Colorectal cancer is generally recognized as an 'advanced cancer' because of its high incidence in high-income groups. In the West, cancer ranks second in cancer death, accounting for about 15% of all cancers. have. In the United States, approximately 56,000 people die of colorectal cancer and 130,000 new cases of colorectal cancer occur annually.In Korea, the proportion of total cancer in mortality is ranked fourth after gastric cancer, liver cancer and lung cancer. It is cancer that occupies. 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 examining the frequency of expression of specific genes in specific cells, it is possible to discover genes related to colorectal cancer, thereby understanding the molecular mechanism of colorectal cancer progression, and furthermore, to be used as targets for colon cancer diagnosis and treatment. will be.

Meanwhile, the C1orf135 (Chromosome 1 open reading frame 135, FLJ14264, AIBP, MGC2603) gene is known as a gene encoding the Aurora A-binding protein and is a substrate of ATM or ATR1 in response to DNA damage. It is known to play a role, but the specific research on the association with colorectal cancer is not known.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. 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 for the discovery of genes that are used for colorectal cancer diagnosis and drug screening and may be targets for colorectal cancer treatment. As a result, the expression of C1orf135 gene in the colorectal cancer cells is significantly increased. By finding out that there existed, this invention was completed.

An object of the present invention is to provide a composition for diagnosing colorectal cancer comprising an agent for measuring the expression level of the mRNA or protein of C1orf135 (Chromosome 1 open reading frame 135) gene.

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

Another object of the present invention is to measure the mRNA expression level of the C1orf135 gene or the protein encoded by the gene from a biological sample of suspected colorectal cancer; And (b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample. It is to provide a method of providing.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating colorectal cancer, comprising an inhibitor of the C1orf135 gene expression or an inhibitor of a protein thereof.

Another object of the present invention is to perform the following steps: (a) analyzing the expression of the C1orf135 gene or the activity of a protein thereof after treating the test substance; And (b) judging the expression of the C1orf135 gene or the activity of the protein thereof after the test substance is inhibited compared to the expression of the C1orf135 gene or the activity of the protein thereof without treatment of the test substance. It provides a screening method for treating colorectal cancer.

According to the present invention, by providing a gene specific for colorectal cancer and providing a diagnostic marker for judging metastasis and prognosis of colorectal cancer, it is possible to accurately diagnose colorectal cancer and to be useful for the treatment and prognosis of colorectal cancer. Can be. In addition, by providing a colorectal cancer-specific therapeutic agent that inhibits the gene, it is possible to enable the development of biopharmaceuticals and customized anticancer agents with fewer side effects.

1 is a graph and data analysis results showing that the expression of the C1orf135 gene in 66 colorectal cancer clinical tissues through a microarray experiment using the 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 through RT-PCR. GAPDH was used as a control gene.

Figure 3 is a result of quantitative analysis of changes 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.

Figure 5a is a result of verifying the effect of C1orf135 siRNA by selecting the colorectal cancer cell line KM12C as a representative cell line. Treatment of the siRNA of C1orf135 shows that the amount of actual C1orf135 mRNA is reduced, resulting in reduced protein.

5B shows growth inhibition of KM12C cells when treated 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.

As one aspect for achieving the above object, the present invention provides a composition for diagnosing colorectal cancer comprising an agent for measuring the expression level of the mRNA of the C1orf135 (Chromosome 1 open reading frame 135) gene or protein thereof.

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

In the present invention, the term "diagnosis" refers to confirming the presence or characteristics of a pathological state, and for the purpose of the present invention, the diagnosis is to confirm the development of colorectal cancer.

As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is a substance capable of diagnosing colon cancer cells from normal cells, and increases or decreases in cells with colon cancer compared to normal cells. Organic biomolecules such as polypeptides or nucleic acids (eg, mRNAs), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, and the like) that exhibit a decrease. For the purposes of the present invention, the colorectal cancer diagnostic markers of the present invention are the C1orf135 gene and proteins encoded by it, which show a particularly high level of expression in colorectal cancer cells as compared to cells of normal colon tissue.

Gene expression levels in biological samples can be confirmed by identifying the amount of mRNA or protein.

In the present invention, mRNA expression level measurement is a process of confirming the presence and expression of mRNA of colorectal cancer marker genes in a biological sample to diagnose colorectal cancer, and can be known by measuring the amount of mRNA. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting or DNA chips, but are not limited thereto.

The agent for measuring the expression level of mRNA of the gene means a molecule that can be used for detection of the marker by checking the expression level of C1orf135, a marker that increases expression in colorectal cancer cells, but is not limited thereto. It may be to include a primer or probe that specifically binds to the gene. Based on the mRNA sequence of C1orf135 (SEQ ID NO: 1) and its ORF sequence (SEQ ID NO: 2), one skilled in the art can design primers or probes that specifically amplify specific regions of these genes.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, colon cancer can be diagnosed through the generation of a desired product by PCR amplification using sense and antisense primers of C1orf135 polynucleotide. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which is capable of specific binding with mRNA, and is labeled to indicate the presence or absence of a specific mRNA. You can check it. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, hybridization may be performed using a probe complementary to C1orf135 polynucleotide, and colon cancer may be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, "capsulation", substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, Phosphoramidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

In the present invention, the protein expression level measurement is a process of confirming the presence and expression level of the protein expressed from the colorectal cancer marker gene in a biological sample to diagnose colorectal cancer, preferably specifically binding to the protein of the gene The amount of protein can be confirmed using an antibody. Analysis methods for this include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoelectrophoresis. , Tissue immunity staining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chips (protein chip) and the like, but is not limited thereto.

The agent for measuring the expression level of the protein means a molecule that can be used for detection of the marker by confirming the expression level of the protein expressed from the C1orf135 gene, which is a marker for increasing expression in colorectal cancer cells, but is not limited thereto. For example, it may include an antibody specific for the protein. Based on the amino acid sequence of the Aurora A-binding protein (SEQ ID NO: 3), which is a protein expressed from the C1orf135 gene, a person skilled in the art can design an antibody specific for the protein.

As used herein, the term “antibody” refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed from the C1orf135 gene, which is a marker of the present invention. Such an antibody may be cloned into an expression vector according to a conventional method for the marker gene. The protein encoded by is obtained and can be prepared by conventional methods from the obtained protein. Also included are partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody. Antibodies used in the detection of colorectal cancer diagnostic markers of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.

As another aspect, the present invention provides a kit for diagnosing colorectal cancer comprising the composition.

The kit of the present invention can detect the marker by checking the expression level of the mRNA or protein thereof expression level of C1orf135, a colorectal cancer diagnostic marker. Kits of the invention may include primers, probes or optionally antibodies that recognize the expression level of the colorectal cancer diagnostic marker, as well as one or more other component compositions, solutions or devices suitable for the assay. .

The kit may use the principle of diagnosing colon cancer by confirming a reaction between the C1orf135 gene and a sample obtained from a subject. 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 can be used. . That is, all or part of the gene is used as a probe to hybridize with nucleic acid isolated from the body fluid of the subject, and then various methods known in the art, such as reverse transcription polymerase chain reaction and southern blotting. By detecting this by Northern blotting or the like, it is possible to determine whether colorectal cancer is generated by examining whether the gene is in a high or low expression state.

In addition, the kit may use the principle of diagnosing colon 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., for determining the reaction between DNA-proteins, RNA-proteins, and protein-proteins. Chip, polymerase chain reaction (PCR), Northern blotting, Southern blotting, Western blotting, Enzyme Linked Immunosorbent assay (ELISA), specific immunostaining, yeast two-hybrid, 2 -D gel assays and in vitro binding assays can be used. 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.

The kit of the present invention is not limited thereto, but may preferably be a microarray, a gene amplification kit or an immunoassay kit.

When the kit of the present invention is a microarray, a 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 suitable rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. Said hybridization array element is arranged and immobilized on said gas. 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 (eg, 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 may vary, and detection and analysis of the degree of hybridization may be carried out in various ways depending on 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 can be performed 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)). The label provides a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.

Suitable hybridization conditions in the present invention 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, among the stringent conditions, the high stringency conditions are hybridized at 65 ° C. in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and at 0.1 × standard saline citrate / 0.1% SDS. It means to wash at 68 ℃ conditions. Alternatively, high stringency conditions mean washing at 48 ° C. in 6 × SSC / 0.05% sodium pyrophosphate. Low stringency means washing at 42 ° C. conditions, for example, at 0.2 × SSC / 0.1% SDS.

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).

The kit of the present invention may preferably be a gene amplification kit.

As used herein, the term "amplification" means 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), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), 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), U.S. Patent 4,437,975), optional priming 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. Nos. 5,130,238, 5,409,818 Nos. 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP), 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, 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 carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel. Excess of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. 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. 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.

The kit of the present invention may be a kit for immunoassay, and may be performed using an antibody or aptamer specifically binding to the colorectal cancer marker of the present invention.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies may be commercially available and methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US patents) 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, 1-597 (1991)). It may also be prepared by. 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 immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. 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 to 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.

In another aspect, the present invention provides a method for treating cancer, comprising the steps of: (a) measuring the mRNA expression level of the C1orf135 gene or the expression level of a protein encoded by a biological sample of suspected colorectal cancer; And (b) comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample. Provide the method of providing.

As used herein, the term "biological sample" includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in the level of gene expression of colorectal cancer markers due to colorectal cancer. It is not limited to this.

Separation of mRNA or protein thereof from the biological sample may be performed using a known process, and the expression level of the mRNA or protein thereof may be measured by various methods.

The mRNA expression level is not limited thereto, but may be preferably by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. It may be more preferably by reverse transcriptase polymerase reaction or DNA chip.

The reverse transcriptase polymerase reaction can be confirmed by the electrophoresis after the reaction by confirming the band pattern and the thickness of the gene mRNA expression and degree of genes used as diagnostic markers for colorectal cancer and by comparing it with the control group, colon cancer occurrence It is easy to diagnose. In addition, the DNA chip uses a DNA chip in which nucleic acid corresponding to the colorectal cancer marker gene or fragment thereof is attached to a glass-like substrate at a high density, to isolate mRNA from a sample, and to label the terminal or the inside with a fluorescent substance. CDNA probes can be prepared, hybridized to DNA chips, and then read for the development of colorectal cancer.

The measurement of the protein expression level is not limited thereto, but preferably may be to use an antibody specific for the protein. By comparing the amount of antigen-antibody complex formed using the antibody, it is possible to diagnose the onset of colorectal cancer, and the antibody is as described above.

In addition, the measurement of the protein expression level is not limited thereto, but preferably, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay , Complement fixation assays, FACS or protein chips.

When using the ELISA method, ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support. Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antibody attached to the solid support, followed by reaction with another antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support. Various ELISA methods can be included, such as indirect sandwich ELISA using labeled secondary antibodies that recognize the antibody. More preferably, the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It can be detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops.

In the case of using Western blots with one or more antibodies against colorectal cancer markers, the entire protein is isolated from the sample, electrophoresed to separate proteins according to size, and then transferred to a nitrocellulose membrane to react with the antibody. In this case, by checking the amount of the generated antigen-antibody complex using a labeled antibody, the amount of the protein produced by the expression of the gene may be confirmed to determine whether colorectal cancer is developed. The detection method comprises a method of examining the expression level of the marker in the control group and the expression level of the marker in the cells in which the colorectal cancer develops. The expression level can be expressed as an absolute (eg μg / ml) or relative (eg relative intensity of signal) difference of the marker protein.

When using immunohistostaining with one or more antibodies to colon cancer markers, normal colon epithelial tissue and tissue suspected of colon cancer are collected and fixed, followed by preparation of paraffin embedding blocks by methods well known in the art. These are sliced to a thickness of several 탆 and attached to a glass slide, and then the antibodies are reacted by a known method. The unreacted antibody is then washed and labeled with a detection label to read whether the antibody is labeled on the microscope.

When using a protein chip in which one or more antibodies against the colorectal cancer marker are arranged at a predetermined position on a substrate and immobilized at high density, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex. By reading this and confirming the presence or expression level of the protein, it is possible to determine the onset of colorectal cancer.

Through the above methods, it is possible to compare the expression level of mRNA or protein thereof in a normal control group with the expression level of mRNA or protein thereof in suspected colorectal cancer patients, and the significant expression level of the colorectal cancer marker gene to mRNA or protein thereof. By determining whether the increase of, the suspected colorectal cancer can diagnose whether the actual colorectal cancer.

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising an inhibitor of the C1orf135 gene expression or inhibitory activity of the protein thereof.

The inhibitor of expression of the C1orf135 gene included as an active ingredient in the pharmaceutical composition of the present invention is not limited thereto, but may preferably be an antisense oligonucleotide, siRNA, shRNA or microRNA specific to the C1orf135 gene.

In the present invention, the antisense oligonucleotide is a short length DNA synthesis strand (or DNA analog) that is antisense (or complementary) to a specific DNA or RNA target, to achieve gene-specific inhibition in vivo as well as in vitro. It has been used successfully. 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. 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 the present invention, the siRNA (small interfering RNA) is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and because it can suppress expression of a target gene, it is an efficient gene knockdown method or gene therapy method. Used as When the siRNA molecule is used in the present invention, a double stranded structure is formed in which the sense strand (the sequence corresponding to the C1orf135 mRNA sequence (SEQ ID NO: 1)) and the antisense strand (the sequence complementary to the C1orf135 mRNA sequence) are positioned opposite to each other. It may have a single chain structure with self-complementary sense and antisense strands. siRNAs are not limited to the complete pairing of double-stranded RNA pairs that pair with RNA, but include mismatches (corresponding bases are not complementary), expansion / bulge (bases corresponding to one chain). None) and the like may be included. The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the C1orf135 gene can be suppressed by RNA interference (RNAi) effects. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures. The siRNA molecule is not limited thereto, but may have a total length of 15 to 30 bases, preferably 19 to 21 bases.

The siRNA is a very strong drug against the inhibition of expression of specific genes in vivo in terms of long lasting effects in cell culture and in vivo, the ability to transfect cells in vivo and resistance to serum degradation. Has the potential. 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 disclose many expression consensus as well as delivery mechanisms including viral vectors, nonviral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates. Provide truck / vector.

In the present invention, the shRNA (short hairpin RNA) is a single-stranded RNA having a length of 45 to 70 nucleotides, an oligo linking 3-10 base linkers between the sense strand of the target gene siRNA sequence and the complementary antisense strand. After synthesizing DNA, cloning into plasmid vectors or inserting and expressing shRNAs into retroviruses lentivirus and adenovirus results in loops of hairpin structured shRNAs and intracellular dicers. It is converted into siRNA by (dicer) to show RNAi effect.

In the present invention, the microRNA (microRNA) regulates various biological processes such as 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.

Regulatory sequences useful in expression constructs / vectors with such antisense oligonucleotides, siRNAs, shRNAs or microRNAs (eg, constitutive promoters, inducible promoters, tissue specific promoters or combinations thereof) are also known in the art. The expression inhibitor of the gene may be any material that inhibits the expression of the gene in addition to the antisense oligonucleotide, siRNA, shRNA or microRNA.

The inhibitor of activity of the protein expressed from the C1orf135 gene included as an active ingredient in the pharmaceutical composition of the present invention is not limited thereto, but may preferably be an antibody specific for the C1orf135 protein.

As described above with respect to the antibody, the monoclonal antibody to the protein may be produced and used through a monoclonal antibody production method common in the art, or may be commercially available. 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 pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including an additional pharmaceutically acceptable carrier in addition to the active ingredient described above for administration. Suitable pharmaceutically acceptable carriers can include, for example, water, saline, phosphate buffered saline, dextrin, glycerol, ethanol, as well as combinations thereof. In addition, when the activity inhibitor of the protein is an antibody, a 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.

The carrier may be included differently depending on the method of administration, and oral administration may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like. For example, buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used. For topical administration, bases, excipients, lubricants, preservatives and the like can be used. Formulations of the compositions of the present invention can be prepared in a variety of mixtures with the pharmaceutically acceptable carriers described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampoules or in multiple dosage forms.

As another aspect, the present invention provides a method of treating colorectal cancer comprising administering the pharmaceutical composition to a suspected colorectal cancer subject.

In the present invention, the suspected colorectal cancer means all animals including humans having or may develop colorectal cancer, and the pharmaceutical composition comprising an inhibitor of expression of the C1orf135 gene of the present invention or an activity inhibitor of a protein thereof is colorectal cancer. By administering to a suspect subject, the subject can be treated efficiently.

As used herein, the term "administration" means introducing a composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the desired tissue. have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but are not limited thereto. Do not.

The method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount. In the present invention, the effective amount is defined as 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 and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently. In adults, when the expression inhibitor of the gene or activity of the protein thereof is administered once or several times a day, 0.01ng / kg-10mg / kg for siRNA, 0.01 for an antisense oligonucleotide for mRNA of the gene ng / kg-10 mg / kg, the compound may be administered at a dose of 0.1 ng / kg-10 mg / kg and the monoclonal antibody to the protein at a dose of 0.1 ng / kg-10 mg / kg.

In another aspect, the present invention provides a method of treating a test substance, comprising: (a) analyzing the expression of the C1orf135 gene or activity of a protein thereof after treating a test substance; And (b) judging the expression of the C1orf135 gene or the activity of the protein thereof after the test substance is inhibited compared to the expression of the C1orf135 gene or the activity of the protein thereof without treatment of the test substance. Provided are methods for screening a colorectal cancer treatment agent.

In the present invention, the step (a) is a step of analyzing the expression of the C1orf135 gene or the activity of the protein after processing the test material, the assay may be analyzed in cells or in vitro, but is not limited thereto. For example, it can be analyzed using RT-PCR, northern blotting, cDNA microarray hybridization reaction, in situ hybridization reaction, radioimmunoassay, immunoprecipitation, ELISA, western blotting and the like.

As used herein, the term "test substance" refers to an unknown substance used in screening to test whether the expression of the C1orf135 gene or the activity of a protein thereof is affected. The test substance may be an individual substance estimated to have a potential as a colorectal cancer metastasis inhibitor or randomly selected according to a conventional selection method, including but not limited to nucleic acids, proteins, chemicals, and natural extracts. can do. The test substance analyzed by the screening method of the present invention may be a single compound or a mixture of compounds, and may be obtained from a library of synthetic or natural compounds.

In the present invention, the step (b) is the treatment of the test substance after the expression of the C1orf135 gene or the activity of the protein is suppressed compared to the expression of the C1orf135 gene or the activity of the protein that does not process the test substance to determine the treatment for colorectal cancer As a step, the expression of the C1orf135 gene or the activity of the C1orf135 protein is down-regulated by the test substance, and thus, it may be determined as a therapeutic agent for colorectal cancer.

Test substances exhibiting a function of enhancing the expression of a high colorectal cancer high expression gene or a protein activity obtained through the screening method of the present invention and vice versa a function of inhibiting the expression of a high colorectal cancer high expression gene or inhibiting the activity of a protein In the former case, the test substance may be a candidate for colorectal cancer treatment by developing an inhibitor for the test substance, and the latter 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, and the structure is modified so that the leading material can exhibit the function inhibitory effect of the gene or the protein expressed therefrom. By optimizing, new colorectal cancer therapies can be developed.

Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

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

1-1. 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 then stored in liquid nitrogen until analysis.

1-2. 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 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.

1-3. 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 nonspecific 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 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 diagnostic for colon cancer, drug screening, or therapeutic target. In addition, FIG. 1 shows that the C1orf135 gene expression is 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 Diagnosis in Individual Clinical Samples

17 pairs of tissue tissues (T1-T66) and normal tissues (N1-N66) from 66 pairs of colorectal cancer clinical patients samples (cancer tissues (T1-T66) and normal tissues (N1-N66) obtained from 66 pairs of cancer patients of Example 1) 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.

2-1. 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. Then 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 SuperScriptIII RT enzyme to a total of 25 μl, followed by RNA / primer mixing at 55 ° C. After mixing with the solution, it was reacted for 50 minutes at 55 ℃. 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.

2-2. 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 of primer each (20 pmole) and 2 μl of distilled water to make 20 μl of total solution. PCR reaction was performed at 94 ℃ for 1 minute, 54 ℃ for 30 seconds and 72 ℃ for 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 the colorectal cancer sample, and thus, the C1orf135 gene could be used as a colorectal cancer marker or therapeutic target for diagnosis or drug screening of colorectal cancer.

Example 3: Confirmation of the effect of increasing the 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.

3-1. Preparation of Colon Cancer Cell Lines

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 .

3-2. 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.

3-3. Expression Analysis 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 of the C1orf135 gene may be a diagnostic indicator and therapeutic target for colorectal cancer.

Example 4: Cloning of the C1orf135 Gene

The C1orf135 gene was amplified using PCR technique from Invitrogen's human brain c-DNA library. At this time, the forward primer 5'-TGC GGATCC ATGAGGCGGACAGGCCCCGAG-3 ' (BamHI enzyme cleavage site comprises - underline, SEQ ID NO: 8) and reverse primer 5'-ATG GATATC TTAGAATTGGTGTCTGATAAC-3' (EcoRV cleavage site include an enzyme-underline, SEQ ID NO: 9 Gene was amplified by PCR using pfu premix (iNtRON, Inc.) at 55 cycles at 55 ° C. 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 mixtures were added and incubated at room temperature for 1 hour. After incubation, proteinase K was added and incubated at 37 ° C. for 10 minutes to complete the LR reaction. E. coli DH5α competent cells were transformed to obtain a pDEST-Flag-C1orf135 clone.

Example 5 Confirmation of Increasing Cell Growth Rate by Overexpression of C1orf135

DMEM medium containing 100% dilution of a mixture of 5% bovine calf serum and penicillin (10,000 units / ml) and streptomycin (10 mg / ml) of NIH3T3 fibroblasts (ATCC), a normal cell line (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 Figure 4b. 4B, 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. 4B). These results indicate that increased expression level of the C1orf135 gene may be an indicator of tumor cell diagnosis.

Example 6: Confirmation of the growth inhibitory effect of the colon cancer cell line according to the introduction of siC1orf135 in the colon cancer cell line

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.

6-1. 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 double-stranded double-stranded structure of double ribonucleic acid chains 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 culturing 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 cell by hyperfect method (Qiagen Co.) 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 ( 5a). 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).

6-2. Validation of growth inhibition 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. Control siRNA (sense sequence, SEQ ID NO: 11) and siC1orf135 (sense sequence, SEQ ID NO: 10) were transfected into the cells according to the above method and cultured for 72 hours, and the growth inhibition of the cells by siRNA was examined. Investigate. 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. These results indicate that siRNA of the C1orf135 gene can induce growth inhibition of the colorectal cancer cell line, and thus the C1orf135 gene can be used as a target for treating colorectal cancer.

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

Table 1

siRNA position within mRNA	order	GC content	Indentity
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

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

According to the present invention, by providing a gene specific for colorectal cancer and providing a diagnostic marker for judging metastasis and prognosis of colorectal cancer, it is possible to accurately diagnose colorectal cancer and to be useful for the treatment and prognosis of colorectal cancer. Can be. In addition, by providing a colorectal cancer-specific therapeutic agent that inhibits the gene, it is possible to enable the development of biopharmaceuticals and customized anticancer agents with fewer side effects.

Claims (13)

1. C1orf135 (Chromosome 1 open reading frame 135) A composition for diagnosing colorectal cancer comprising an agent for measuring the expression level of mRNA of the gene or protein thereof.
2. The composition of claim 1, wherein the agent for measuring the expression level of mRNA of the gene comprises a primer or a probe that specifically binds to the gene.
3. The composition of claim 1, wherein the agent for measuring the expression level of the protein comprises an antibody specific for the protein.
4. A kit for diagnosing colorectal cancer, comprising the composition of claim 1.
5. The kit of claim 4, wherein the kit is selected from the group consisting of a microarray, a gene amplification kit, and an immunoassay kit.
6. (a) measuring the mRNA expression level of the C1orf135 gene or the protein encoded by the gene from a biological sample of suspected colorectal cancer; And

   (b) providing information for diagnosing colorectal cancer, comprising comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level or protein expression level of the gene of the normal control sample. Way.
7. The method of claim 6, wherein the measurement of mRNA expression level is by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting or DNA chip.
8. The method of claim 6, wherein the measuring of the protein expression level uses an antibody specific for the protein.
9. The method of claim 6, wherein the protein expression level is measured by Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunity staining, immunoprecipitation assay, complement fixation assay, Using FACS or protein chips.
10. A pharmaceutical composition for preventing or treating colorectal cancer, comprising the inhibitor of the C1orf135 gene or an activity inhibitor of a protein thereof.
11. The composition of claim 10, wherein the expression inhibitor of the gene is selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the C1orf135 gene.
12. The composition of claim 10, wherein the activity inhibitor of the protein is an antibody specific for C1orf135 protein.
13. (a) analyzing the expression of the C1orf135 gene or the activity of a protein thereof after treating the test substance; And

    (b) judging the expression of the C1orf135 gene or the activity of the protein thereof after treatment with the test substance is inhibited compared to the expression of the C1orf135 gene or the activity of the protein thereof without treatment of the test substance; Screening method for cancer treatment.

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