KR101808658B1 - Diagnostic Kit for Cancer and Pharmaceutical Composition for Prevention and Treatment of Cancer - Google Patents

Abstract

The present invention relates to a cancer diagnostic kit selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, a method of detecting cancer diagnostic markers, a pharmaceutical composition for preventing or treating cancer, and a screening method of cancer therapeutic agent .
The present invention provides an accurate cancer diagnosis by providing a target gene specific for gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer, and provides a cancer-specific therapeutic agent through the target gene, Development.

Description

[0001] The present invention relates to a cancer diagnostic kit and a pharmaceutical composition for preventing or treating cancer,

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

More particularly, the present invention relates to a cancer diagnostic kit using a C10orf81 gene and a protein expressed therefrom, a method for detecting a cancer diagnostic marker, a pharmaceutical composition for preventing or treating cancer, and a cancer screening method.

Human Genome Project Research shows that there is a new era of bio-biotechnology that aims to understand human diseases at the molecular level, to discover new disease-related targets, and to develop new drugs using them. As a result, the era of personalized medicine that diagnoses, treats, and predicts various diseases according to individual patients with different genetic environments is coming to the reality. Customized medicine includes biomarkers for genome diagnosis, targeting treatment technology, discovery of disease-specific drug action points, new drugs for target treatment, accurate clinical information, genome information of patients, genome dynamics results, Matrix and others should be complemented by the development of drug genome technology. In particular, in order to be competitive in customized medicine, it is important to develop a drug prediction system for diseases and individuals, to discover diagnostic biomarkers, to develop new target genes and proteins, and to develop therapeutic agents using them.

Thus, since the formation of cancer is progressed by a complex combination of various genes and the expression and regulation mechanisms of these genes, recently, the expression of cancer-related genes using oligo chips using a large number of genes has been compared with a new diagnosis of cancer Studies are being conducted to identify therapeutic markers. The genes whose expression is increased or decreased in cancer cells are involved in various parts such as cell division, cell signaling, cytoskeleton, cell movement, cell defense, gene and protein expression, and intracellular metabolism. There are a number of genes that show changes but others that show different expression changes. Because each of these patients is likely to be due to their specificity, the exact pathological characterization and classification of the patient's tissues should be followed, and more accurate identification and identification of new genes is required for accurate diagnosis. By examining the frequency of expression of a specific gene in a specific cell, a cancer-related gene can be uncovered, thereby making it possible to understand the molecular mechanism of cancer progression, and to use it as a cancer diagnosis and therapeutic target.

In recent years, there has been a fierce competition for exploring therapeutic targets and studying the functions of genes involved in the generation and treatment of cancer, which is a worldwide incurable disease, in order to use them for diagnosis and therapeutic drug development. With the activation of the genome research, studies on human gene DNA chip or proteome analysis have been actively conducted, and a large number of genes related to cancer have been discovered. Therefore, a list of many genes and related databases have been established, but most of these genes Biological function and cancer relevance have not yet been studied or are uncertain, and there are considerable difficulties in actual cancer-related or diagnostic and target genes.

In addition, as the need for research on the function of numerous human genes is highlighted, the utilization and interest of model organisms that can perform simple functions and morphological studies are increasing. In order to understand basic and evolutionally well-preserved mechanisms occurring within the cell, studies are underway to utilize the model organism, which has been used in various fields for a long time, in cancer diagnosis or therapeutic target discovery.

On the other hand, the use of RNA interference (RNAi) technique for large-scale gene expression in cancer cells can be used to confirm the possibility of use as a therapeutic cancer target. RNAi is a technique for inducing the degradation of mRNA of the corresponding nucleotide sequence in cells by using various types of oligosubstituted ribonucleic acid (miRNA, expressed dsRNA, synthetic siRNA) to prevent the function of the gene from appearing. By observing the phenomenon of expression of cells in which gene expression has been inhibited, new gene development studies and signal pathway analysis can be performed, and new drug development studies such as target validation are being conducted. Among the oligo-ribonucleic acids that can be used for the RNAi technique, a technique using a small interfering RNA (siRNA), which is currently in the spotlight, is a breakthrough method of selecting "breakthrough of the year" in the 2002 Science Journal. siRNA is a short 19-23 double ribonucleic acid chain that is introduced into a cell to inhibit the expression of a specific gene without non-specific inhibition. Therefore, the siRNA promotes the production of cancer, siRNA inhibits the action of the corresponding gene in the cell and kills cancer cells, which can be used to verify the therapeutic target gene. Therefore, recent research on siRNAs for target gene discovery, target gene verification, and therapeutic agent development is very active.

Currently, C10orf81 (chromosome 10 open reading frame 81, synonyms: FLJ23537, HEL185 (Epididymis luminal protein 185)) gene is a PH domain-containing protein and its function is not known at all.

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

The present inventors have made intensive researches for discovering genes that can be used as targets for cancer therapy and drug screening, and as a result, the expression of C10orf81 gene in various cancer cells is remarkably increased, and when they are inhibited, , Thereby completing the present invention.

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

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

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

According to one aspect of the present invention, the present invention provides a method for producing a nucleotide sequence comprising the steps of (i) a nucleotide sequence of C10orf81 (Chromosome 10 open reading frame 81) gene, (ii) a fragment of the nucleotide, (iii) a sequence complementary to the nucleotide sequence or fragment thereof, (iv) a protein expressed from a C10orf81 gene, or (v) a binding agent specific to the protein; and (c) a kit for the diagnosis of cancer selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer .

According to another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the step of measuring the expression level of the C10orf81 gene in a biological sample of a human to provide information necessary for diagnosis of cancer, which comprises gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer A method for detecting a cancer diagnostic marker selected from the group.

According to another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the steps of: (a) detecting a level of a protein expressed from a C10orf81 gene in a biological sample of a human, A method of detecting a cancer diagnostic marker selected from the group consisting of prostate cancer.

The present inventors have made intensive researches for discovering genes that can be used as targets for cancer therapy and drug screening, and as a result, the expression of C10orf81 gene in various cancer cells is remarkably increased, and when they are inhibited, , Thereby completing the present invention.

The C10orf81 gene of the present invention is a gene also called FLJ23537 or HEL185 (Epididymis luminal protein 185), which encodes a PH domain-containing protein. The mRNA permutation of C10orf81 is as shown in SEQ ID NO: 1 and its ORF sequence is shown in SEQ ID NO: 2. The third sequence shows the amino acid sequence of the protein expressed from the C10orf81 gene. In this specification, the term 'C10orf81 gene' refers to the DNA or mRNA of these genes, and includes all or a part of DNA or mRNA.

In one embodiment of the present invention, a method for detecting a cancer diagnostic kit or a cancer diagnostic marker selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer, and prostate cancer includes detecting a C10orf81 gene or a fragment thereof or a complementary sequence thereof And the principle of diagnosing cancer is used. Confirmation of the reaction between the C10orf81 gene and the sample obtained from a subject can be carried out by a conventional method used for confirming the reaction between DNA-DNA, DNA-RNA and DNA-protein such as a DNA chip, a protein chip, a polymerase chain reaction ), Northern blotting, Southern blotting, ELISA (Enzyme Linked Immunosorbent assay) , yeast double hybrid method (yeast two-hybrid), 2 -D gel analysis, and in vitro binding assay (in vitro binding assay) can be used. That is, all or a part of the gene may be used as a probe and hybridized with a nucleic acid separated from a body fluid of a subject. Thereafter, various methods known in the art such as reverse transcription polymerase chain reaction, Southern blotting Southern blotting, northern blotting, or the like to detect whether the gene is highly expressed or underexpressed in the subject.

The method for detecting a cancer diagnostic kit or cancer diagnostic marker selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer according to the present invention is characterized in that the reaction between a protein expressed from C10orf81 gene and a sample obtained from a subject is confirmed, May be used. As described above, since the expression of C10orf81 specifically increases in gastric cancer, liver cancer and colon cancer cells, not only the overexpression of the gene but also the overexpression of the protein expressed from the gene can be investigated, Liver cancer, colon cancer, lung cancer and prostate cancer. In the cancer diagnosis kit selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, lung cancer and prostate cancer of the present invention, confirmation of the reaction between the composition containing the protein and the sample can be carried out by using DNA- (DNA), protein chips, PCR, Northern blotting, Southern blotting, Western blotting, ELISA (enzyme linked immunosorbent assay), specific immuno staining histoimmunostaining, yeast two-hybrid, 2-D gel analysis and in vitro binding assay ( in vitro binding assay) can be used. For example, all or a part of proteins expressed from the genes may be used as a probe to be hybridized with a nucleic acid or a protein separated from a body fluid of a subject, and then subjected to various methods known in the art, for example, reverse transcription polymerases chain reaction, western blotting or the like to detect whether the gene is highly expressed in the subject, thereby determining whether or not cancer has occurred.

According to a preferred embodiment of the present invention, the kit of the present invention is a microarray. A probe is immobilized on the solid-phase surface of the microarray. In the microarray of the present invention, the probe is used as a hybridizable array element and 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. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a 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).

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

The kit for the diagnosis of gastric cancer, liver cancer, colon cancer, lung cancer or prostate 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 may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (Such as P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin, biotin , Hapten having specific binding partners such as digoxigenin and chelating groups. Markers can be obtained by a variety of methods routinely practiced in the art, such as the nick translation method, the Multiprime DNA labeling systems booklet ("Amersham" (1989)) and the kaination method (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, color measurement, 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 by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. The detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization. Combinations of enzymes / substrates that may be used include, but are not limited to, peroxidases (such as horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2) , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate).

According to another preferred embodiment of the present invention, the kit for the diagnosis of gastric cancer, liver cancer, colorectal cancer, lung cancer or prostate cancer of the present invention is a gene amplification kit.

The term " amplification " as used herein refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315) (SEQ ID NO: 1), self-sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction CP-PCR), U.S. Patent No. 4,437,975) (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA), U.S. Patent 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 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, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

According to another preferred embodiment of the present invention, the kit for the diagnosis of gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer according to the present invention is an immunoassay kit, For example, an antibody or an aptamer that specifically binds to colon, lung, or prostate cancer markers.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The antibody may be commercially available or may be prepared by methods conventionally practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods . 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, the disclosures of 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. The monoclonal antibody is generally prepared by performing a color reaction using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and substrates thereof Or quantitatively analyzed using a monoclonal antibody against the protein directly conjugated with an AP or HRP enzyme or the like.

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

Further, monoclonal antibodies or polyclonal antibodies are included in the antibody of the present invention, and all immunoglobulin antibodies are included, if they have antigen binding properties. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a C10orf81 gene expression inhibitor; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating cancer is selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer.

In the present invention, the expression inhibitor of C10orf81 gene may be an antisense oligonucleotide for the mRNA of the gene.

Antisense oligonucleotides have been used successfully to achieve gene-specific inhibition both 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 inhibit the expression of proteins encoded by DNA or RNA targets by binding to the target and stopping expression at the transcription, translation or splicing step. Antisense oligonucleotides have also been successfully used in animal models of cell culture and disease (Hogrefe, 1999). Other variations of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art. Antisense oligonucleotides used herein include double stranded or single stranded DNA, double stranded or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and oligonucleotides with base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications of the oligonucleotide backbone known in the art, modifications of the sugar moiety, or modifications of the base.

In addition, the inhibitor for the C10orf81 gene may be siRNA (Small Interfering RNA) of the gene, and the base sequence thereof may be 19-23 consecutive nucleotide sequences in each of the nucleotide sequences of the C10orf81 gene (mRNA) have.

The sequence of the siRNA represents a sense strand for convenience, and constitutes a double ribonucleic acid chain together with an antisense strand indicating an actual gene suppression effect.

siRNA has the potential to become a very potent drug against the inhibition of the 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 (Bertrand et al., 2002). Expression constructs / vectors including siRNA delivery and siRNA are known to those skilled in the art. For example, U.S. Patent Application Nos. 2004/106567 and 2004/0086884 disclose delivery mechanisms including viral vectors, non-viral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates, / Vector.

siRNAs have been proposed as an alternative to antisense oligonucleotides because they can achieve equivalent or even higher effects than those obtained by antisense oligonucleotides at relatively low concentrations (Thompson, 2002). The use of siRNAs has been shown to inhibit gene expression in animal models of disease. Those skilled in the art can synthesize and modify the antisense oligonucleotides and siRNAs in any desired manner using methods known in the art (see, for example, Andreas Henschel, Frank Buchholzl and Bianca Habermann (2004) DEQOR: a web-based Nucleic Acids Research 32 (Web Server Issue): W113-W120. Reference). Also, those skilled in the art are well aware of regulatory sequences (e.g., constitutive promoters, inducible promoters, tissue-specific promoters, or combinations thereof) useful in expression constructs / vectors with antisense oligonucleotides or siRNA.

In addition, the inhibitor for the gene may be shRNA (short hairpin RNA) of the gene. shRNA is a single-stranded RNA having a length of 45 to 70 nucleotides. Oligo DNA is synthesized by ligating 3-10 base linkers between the sense of the target gene siRNA sequence and the complementary nonsense, followed by cloning into a plasmid vector or shRNA Is inserted into retroviruses such as lentivirus and adenovirus to express a hairpin shRNA with a loop, which is transformed into siRNA by intracellular dicer to produce an RNAi effect. And exhibit a relatively long-term RNAi effect as compared to the shRNA.

In addition, the inhibitor for the C10orf81 gene may be a microRNA of the above gene. MicroRNAs (microRNAs or miRNAs) regulate a variety of biological processes, including development, differentiation, proliferation, retention, and apoptosis. MicroRNAs generally regulate the expression of a gene encoding a target mRNA by destabilizing or mobilizing the target mRNA.

In addition, regulatory sequences (e.g., constitutive promoters, inducible promoters, tissue-specific promoters or combinations thereof) useful in expression constructs / vectors with antisense oligonucleotides, siRNAs, shRNAs or microRNAs are also known in the art The contents can be selected appropriately.

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. Therefore, compounds known as inhibitors of the genes in the art are also available.

An antisense oligonucleotide, siRNA, shRNA or microRNA of the present invention used for the treatment of gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer may be administered in the form of a composition additionally containing a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide a rapid release, sustained or delayed release of the active ingredient after administration.

(A) a protein inhibitor expressed from the C10orf81 gene; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating cancer is selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer.

Inhibition of the gene of the present invention inhibits the expression of the protein, thereby inhibiting cancer. Thus, inhibition of the protein of the gene can inhibit gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer.

Accordingly, the present invention provides a method for treating gastric cancer, liver cancer, colon cancer, lung cancer, or prostate cancer, comprising administering to a patient an inhibitor of said protein and an effective amount of an inhibitor of said protein. In the present invention, cancer treatment includes prevention and inhibition of cancer.

The inhibitor for the protein may be an antibody or an aptamer to a protein expressed from the gene of the present invention, but is not necessarily limited thereto. The monoclonal antibody to the protein may be prepared by using a monoclonal antibody production method customary in the art or may be commercially available. In addition, a polyclonal antibody recognizing the protein may be used in place of the monoclonal antibody, or it may be prepared and used through an antiserum preparation method customary in the art.

The composition for treating cancer of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. When the inhibitor for the protein of the present invention is an antibody, the pharmaceutically acceptable carrier may be composed of a minimum amount of auxiliary substance such as a wetting agent or an emulsifying agent, an antiseptic or a buffer to increase the shelf life or effectiveness of the binding protein.

Also, the composition for treating gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer of the present invention can be used together with one or more cancer therapeutic agents. The composition for treating cancer of the present invention may be administered orally or parenterally, for example, using chemotherapeutic agents well known to those skilled in the art, such as cyclophosphamide, aziridine, alkylalkanesulfonate, nitroso urea, Antibiotics such as alkylating agents such as cisplatin, mitomycin C, anthracycline and doxorubicin (adriamycin), antimetabolitic agents such as methotrexate, 5-fluorouracil and cytarabine, plants such as vinca alkaloids Derived medicines, hormones, and the like.

The composition for treating cancer of the present invention may include pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective ingredients. Examples of such adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Or solubilizing agents.

In addition, the composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients 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 preparation form of the cancer therapeutic composition of the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drips or injectable solutions, .

The composition for treating cancer of the present invention may be administered orally, intravenously, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, intranasally, inhalation, topically, rectally, Lt; / RTI >

In the method of treatment of the present invention, the "effective amount" means an amount required for achieving the effect of suppressing gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer. Thus, the "effective amount" of the active ingredient of the present invention will vary depending upon factors such 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 of formulation and the age, weight, The dosage, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used concurrently, and the like. In the case of an adult, 0.01 ng / kg-10 mg / kg in the case of siRNA and 0.01 ng / kg-10 in the case of the antisense oligonucleotide against the mRNA of the gene when the gene or protein inhibitor is administered once to several times a day Kg of the compound, 0.1 ng / kg-10 mg / kg of the compound, and 0.1 ng / kg-10 mg / kg of the monoclonal antibody to the protein.

According to another aspect of the present invention, the present invention provides a method for producing a nucleic acid comprising the steps of: (a) contacting a test substance to a cell expressing a nucleotide represented by the first sequence of the sequence listing; 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, wherein the test substance is selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer And a method for screening a cancer treating agent selected from the group consisting of

In the screening method of the present invention, the reaction between the composition containing the gene and the test substance can be confirmed by conventional methods used for confirming whether DNA-DNA, DNA-RNA, DNA-protein or DNA- have. For example, in the living body outside (in vitro) hybridization test, after reacting the mammalian cells with the test substance, Northern analysis, quantitative PCR, quantitative real-time PCR, such as expression Determination of the gene through to ensure the combination whether between the gene and the test substance, or the A method in which a reporter gene is ligated into a gene, introduced into a cell, reacted with the substance to be tested, and the expression ratio of the reporter protein is measured. In this case, in addition to the gene, the composition of the present invention may contain distilled water or a buffer to stably maintain the structure of the nucleic acid.

In the screening method of the present invention, the substance to be tested may be an individual nucleic acid, protein, other extract or natural product, compound, etc., which are presumed to have potential as inhibitors of large-bowel cancer by a conventional selection method or randomly selected .

The test substance obtained through the screening method of the present invention exhibiting the activity of enhancing the expression of the cancer-expressing gene or enhancing the function of the protein and the activity of suppressing the expression of the cancer-expressing gene or suppressing the function of the protein Can be a cancer candidate candidate by developing an inhibitor against the test substance in the case of the former, and can be a cancer candidate candidate in the latter case. Such cancer candidate drug candidate acts as a leading compound in the development of a therapeutic agent for colorectal cancer in the future, and its structure is modified and optimized so that the leading substance can exhibit the function suppression effect of the gene or the protein expressed therefrom , A new cancer treatment agent can be developed.

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

(I) The present invention provides a kit for the diagnosis of gastric cancer, liver cancer or colorectal cancer, a method of detecting a cancer diagnosis marker, a pharmaceutical composition for the prevention or treatment of gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer, do.

(Ii) The present invention provides a precise cancer diagnosis by providing a target gene specific for gastric cancer, liver cancer, colon cancer, lung cancer or prostate cancer, and is capable of diagnosing cancer precisely, and is capable of diagnosing gastric cancer, liver cancer, colon cancer, lung cancer, It is possible to develop biopharmaceuticals and customized anticancer drugs with fewer side effects by providing therapeutic agents.

FIG. 1A shows the results of RT-PCR analysis of changes in the expression level of C10orf81 gene in human gastric cancer cell line, liver cancer cell line, colon cancer cell line and normal cells. GAPDH is a control gene.
FIG. 1B shows the results of quantitative analysis of changes in the expression level of C10orf81 gene in human gastric cancer cell line, liver cancer cell line, colon cancer cell line and normal cell using Realtime-PCR. GAPDH was used as a control gene.
FIG. 2 is a diagram and data analysis showing that expression of C10orf81 gene was increased in 66 colon cancer clinical tissues through microarray experiment using Illumina 48K chip.
FIG. 3 shows the results of RT-PCR analysis of changes in the expression level of C10orf81 gene in 10 colorectal cancer patients, 5 liver cancer patient tissues and gastric cancer patient tissues. GAPDH is a control gene.
FIG. 4 is a graph showing the expression of C10orf81 protein in colon cancer tissue using C10orf81 antibody. FIG. Normal tissues were used as a comparison group.
FIG. 5 is a graph showing changes in cell growth rate due to overexpression of C10orf81 gene, and SRB staining for the promotion of cell growth.
FIG. 6A shows the results of a test of the effect of C10orf81 siRNA by selecting a colon cancer cell line KM12C as a representative cell line. Treatment of C10orf81 siRNA shows that the amount of actual C10orf81 mRNA is reduced and protein is reduced.
Figure 6b shows the inhibition of cell growth by treating C10orf81 siRNA.
FIG. 7 is a graph showing inhibitory effect on cell growth rate by treatment of siRNA in normal cells, stomach cancer, liver cancer, colon cancer, lung cancer and prostate cancer cell line.

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 Increase of Expression Level of C10orf81 Gene in Cancer Cell Lines

A simple way of estimating the cancer association of a gene is to compare changes in the amount of expression of that gene in cancer tissues or cancer cell lines. The relationship between the expression level of cancer cells and the progression of cancer should be investigated by comparing the expression level of cancer cells with that of normal cells. To do this, we extracted the total RNA from the cancer cell line and analyzed RT-PCR and Realtime-PCR using the oligonucleotide of the gene, comparing the amount of each gene with the normal cell line Thereby confirming the expression amount of the gene in the cell. In the present invention, the expression level of C10orf81 gene was analyzed for mRNA levels in human gastric cancer, liver cancer, colon cancer cell line and normal cells.

(1) Preparation of Cancer Cell Lines

(GIBCO) supplemented with 10% Fetal Bovine Serum (GIBCO), penicillin (10000 U / ml) and streptomycin (10 mg / ml) was used. (ATCC), Chang (ATCC) and SNU354 (Seoul National University cell line bank) as colon cancer cell lines, KM12C (Seoul National University cell line bank) as colonic cancer cell line, Colo205 (Seoul National University cell line bank), SW480 (ATCC) and HT29 (ATCC) were used. IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as the normal cell line. The cancer cell line was inoculated to a cell number of 1 × 10 ⁶ and cultured in an incubator in the presence of 5% CO ₂ at 37 ° C.

(2) Total RNA Isolation

Total RNA was isolated using a QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantitated using an Experion RNA StdSens (Bio-Rad) chip. Cells were recovered by centrifugation, and then 10 μl of beta-mercaptoethanol was added to 1 ml of RLN (50 mM TrisCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl ₂ , 0.5% NP-40) . 1 ml of 70% ethanol was added thereto, followed by centrifugation at 3000 g for 5 minutes to adhere the total RNA to the membrane. After two washes, 100 μl of RNase-free water was added to separate the total RNA.

(3) Synthesis of c-DNA and calibration of template concentration

2 μg of total RNA of each sample, 1 μl of 50 μM oligo (dT) as a primer and 2.5 μl of 10 mM dNTP were added and sterilized water containing DEPC (diethyl pyrocarbonate) as an RNase inhibitor was added to make 25 μl as a whole. Mixed solution. The reaction was carried out at 65 ° C for 5 minutes, then transferred to 55 ° C and stored. Next, add 5 μl of 10 × 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 the total volume 25 μl, and then store the RNA / The mixture was allowed to react at 55 캜 for 50 minutes. The reaction was then terminated at 85 ° C for 5 min to inactivate the RT enzyme and place in ice. GAPDH was used as a standard gene for quantifying marker genes. PCR was performed using a primer of a standard gene and the concentration of cDNA was corrected so that the expression level of the standard gene GAPDH became equal. First, each cDNA was diluted 20-fold and then 2 μl of the diluted sample was used for PCR reaction. The PCR was carried out by adding 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, followed by 20 cycles, 23 cycles and 25 cycles. The PCR reaction conditions were 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute, and the size of the product was 457 bp. The PCR product was loaded on 2% agarose gel, electrophoresed, and then the gel was photographed. The image was quantified with TotalLab v1.0 program (Nonlinear Dynamix) and then corrected again to perform PCR. Was corrected to be the same.

(4) Expression analysis using RT-PCR and Realtime PCR

The cDNA diluted to the same amount was subjected to PCR using the sense and antisense primers of the C10orf81 gene. The PCR reaction was performed at 94 ° C for 1 minute, 54 ° C for 30 seconds, and 72 ° C for 1 minute. The PCR reaction was carried out at 3 ° C for 3 minutes, 2x premix (10 μl), 2 μl of each primer (20 pmoles) and distilled water Cycle. PCR products were analyzed by electrophoresis using 2% agarose gel and image analysis. Realtime RT-PCR was performed with DNASYBRI reagent from Qiagen (CA, USA) and LightCycler (Roche). Melt curve analysis was used to evaluate the quality of PCR products and gene expression was analyzed using LightCycler version 3.5 software (Roche).

The C10orf81-specific primers used in the RT-PCR were as follows. The N-terminal primer 5'-AAGAGGAACCCCAGACCCTT-3 '(SEQ ID No. 4), the C-terminal primer 5'-TCTCCACTTGGCTTTCATCG-3' (SEQ ID No. 5). The GAPDH primer for RT-PCR is an N-terminal 5'-TCATGACCACAGTCCATGCC-3 '(SEQ ID No. 6), a C-terminal 5'-TCCACCACCCTGTTGCTGTA-3' (SEQ ID No. 7) The GAPDH used was a Qiagen primer (QT00079247). RT-PCR and Realtime-PCR showed that the expression level of C10orf81 was significantly increased in many cancer cell lines as compared with the normal cell line (FIG. 1). These results indicate that the increased expression level of the C10orf81 gene can be a diagnostic marker for cancer.

Example 2: The effect of increasing the expression level of C10orf81 in clinical tissues of colon cancer patients by microarray experiment

(1) Preparation of Cancer Clinical Tissue

From the Samsung Medical Center (Seoul, Korea), 66 colon cancer patients and normal tissues were obtained, respectively. Each tissue was surgically removed from the patient and stored in liquid nitrogen until analysis.

(2) Total RNA Isolation

Total RNA was isolated using a QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantitated using an Experion RNA StdSens (Bio-Rad) chip. First, the cancer clinical tissue and the normal tissue were cut into an appropriate size, and then dissolved in a 15 ml kit of digestion buffer supplemented with 150 μl of beta mercaptoethanol. 15 ml of 70% ethanol was added thereto, followed by centrifugation at 3000 g for 5 minutes to adhere the total RNA to the membrane. After two rounds of washing, 1.2 ml of distilled water without RNase was added to the total RNA.

(3) Microarray implementation

The extracted total RNA was hybridized using an Illumina TotalPrep RNA amplification kit (Ambion). CDNA was synthesized using T7 Oligo (dT) primer, and biotin-labeled cRNA was prepared by in vitro transcription using biotin-UTP. The prepared cRNA was quantified using NanoDrop. CRNA produced in normal colon epithelial cells and colon cancer cells was hybridized with human-6 V2 (Illumina) chip. To remove non-specific hybridization after hybridization, the DNA chip was washed with Illumina Gene Expression System washing solution (Illumina), and the washed DNA chip was labeled with streptavidin-Cy3 (Amersham) fluorescent dye. The fluorescence-labeled DNA chip was scanned using a confocal laser scanner (Illumina) to obtain fluorescence data existing in each spot and stored as a TIFF image file. The TIFF image file was quantified with BeadStudio version 3 (Illumina), and fluorescence values of each spot were quantified. The quantified results were calibrated using the 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics).

The results are shown in Fig. FIG. 2 shows C10orf81 gene expression profiles measured in cancer tissues and normal tissues of patients with colorectal cancer, and when expressed high, it was red, while when low expression was expressed as green. The expression level of C10orf81 gene in cancer tissue was increased by an average of 6 times. These results indicate that the C10orf81 gene exhibits a large difference in expression compared to normal colon epithelial cells, and thus can be used as a cancer diagnosis, drug screening, or therapeutic target.

Example 3. Confirmation of gene expression for cancer diagnosis in individual clinical samples

Ten pairs of colon cancer patient samples (10 pairs of cancer tissues (T1-T66) and normal tissues (N1-N66) collected from 66 pairs of cancer patients in Example 2 , N1-N10), and the amount of expression of C10orf81 gene identified in Example 1 was analyzed by RT-PCR using four pairs of gastric cancer samples (Samsung Medical Center) and five liver cancer clinical samples (Samsung Medical Center) Respectively. Total RNA was isolated by the method of Example 2 and cDNA was synthesized by the same method as in Example 1. The template calibration procedure and RT-PCR were also carried out in the same manner as in Example 1. The results are shown in FIG. 3. In the photograph, N means normal tissue (non-tumor tissue), and T means cancer tissue corresponding thereto. As a result, the C10orf81 gene has been confirmed to be highly expressed in cancer samples. Therefore, the C10orf81 gene can be used as a cancer marker or a therapeutic target for cancer diagnosis or drug screening.

Example  4. Comparison of protein expression in tissues using immunostaining

Immunostaining was performed on tissue slides in order to determine the presence and location of protein in normal colon epithelium and colon cancer tissues. First, colonoscopic epithelial cells and colon cancer cells were excised from colon cancer patients through surgical resection to make paraffin embedded blocks. This was cut into a thickness of 5 ㎛ using a microtome and attached to a glass slide to form a tissue slide. They were stained with a known immuno-staining method, and the existence and position of proteins in tissues were confirmed by a microscope. Antibodies used for immunization were the anti-C10orf81 antibody (Novus, 1: 1000). As a result, in immunohistochemical staining, C10orf81 protein was expressed in cell membrane and cytoplasm, and was expressed more strongly than normal mucosa (Fig. 4).

Example  5. C10orf81  Gene Cloning

The C10orf81 gene was amplified using Invitrogen's human brain c-DNA library using PCR. The forward primer 5'-TGC GGATCC ATGGAACCCAAACCTCAGAAGA-3 ' (BamHI enzyme cleavage site comprises - underline, SEQ ID No. 8, SEQ) and the forward primer 5'-ATG GATATC TCATATCCGTCCTGTGGCTTC-3' (EcoRV enzyme cleavage site comprises - underline, SEQ ID NO. Ninth sequence) amplified by PCR using pfu premix (iNtRON, Inc.) at 37 ° C at 37 ° C. The DNA band of the C10orf81 gene was purified and then cut with BamHI / EcoRV restriction enzyme and cloned into the pENTR3C vector. The DNA sequence (1089 bp) of the C10orf81 gene was confirmed by sequencing and cloned according to the manufacturer's manual using the GATE Way system (Invitrogen Corp. www.invitrogen.com). To clone C10orf81, pENTR3C-C10orf81 was cloned by LR reaction with pDEST (Invitrogen Corp. www.invitrogen.com). The plasmid pENTR3C-C10orf81 was mixed with the vector pDEST DNA, and the LR clonase II enzyme mixture was added thereto, followed by incubation at room temperature for 1 hour. After the incubation, Proteinase K was added and cultured at 37 ° C for 10 minutes to complete the LR reaction. Then, E. coli DH5α competent cells were transformed into pDEST-C10orf81 clones.

Example  6. C10orf81 Of the cell growth rate

The normal cell line, NIH3T3 fibroblast (ATCC), was inoculated into a DMEM medium (GIBCO, 15140) diluted 100 times with a mixture of 5% bovine calf serum and penicillin (10,000 units / ml) -streptomycin (10 mg / (GIBCO) at a concentration of 1.4 × 10 ⁵ cells / ml in a 60 mM dish and incubated at 37 ° C. for 24 hours in a 5% CO 2 incubator. The C10orf81 plasmid DNA was transfected into the cultured cells. After 6 hours, the medium was removed, washed three times with PBS (phosphate buffered saline), and the medium was replaced with 5% serum. Twenty-four hours after the transfection, the cells were treated with 0.5% EDTA-trypsin and the cells were counted using a heamacytometer. The cells were seeded in a 24-well plate at a concentration of 1 × 10 ⁵ cells / ) And incubated at 37 ° C in a 5% CO2 incubator until SRB (Sulforhodamine B) analysis. Each plate was taken out every 12 hours after the incubation and the cell growth rate was measured by SRB assay. For comparison, pDEST vector DNA was transfected and treated in the same manner as NIH3T3 cells, and used as a control. The results are shown in Fig. 5, the horizontal axis represents time, the vertical axis represents cell growth rate, pDEST represents a cell transfected with a vector lacking the C10orf81 gene, and pDEST-C10orf81 represents a cell in which a C10orf81 gene is cloned. ≪ RTI ID = 0.0 > C10orf81 < / RTI > protein.

As a result, in the cells overexpressing the C10orf81 gene, the growth rate was increased by about 35%, indicating that the C10orf81 gene is involved in increasing the growth rate of the cells characteristic of the tumor cells (FIG. 5). These results indicate that the expression level of the C10orf81 gene can be an indicator of tumor cell cytology.

Example  7. Cancer cell siC10orf81 Of Cancer Cell Growth by Introducing

In the case of genes that are expressed in large amounts in cancer cell lines or cancer cells, it is possible to observe whether there is a change in cell proliferation rate by inhibiting gene expression through siRNA technique. In the present invention, each siRNA for C10orf81 was designed and introduced into a cancer cell line, and the change in the growth rate of the cancer cell following the decrease of the mRNA expression of the gene was observed. SNU484 (Seoul National University Cell Line Bank), and SNU638 (Seoul National University Cell Line Bank), Liver Cancer Cell Chang (ATCC) and SNU354 (Seoul National University Cell Line Bank), Colon Cancer Cell Line KM12C (Seoul National University Cell Line Bank) and Colo205 , Lung cancer cell line A549 (ATCC), and prostate cancer cell line PC3 (ATCC). IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as the normal cell line.

(1) Confirmation of mRNA inhibition effect of C10orf81 by siC10orf81 using KM12C cell line

First, C10orf81 and siRNA to be used as a control group were designed and synthesized (Samchullyi Pharmaceutical, Korea). The synthesized siRNA consists of a double ribonucleotide chain of 19 oligonucleotides designed on the basis of the gene base sequence and a structure in which a short base of two nucleotides is suspended. Control siRNA was used as a negative control for siRNA experiments that had no effect on cell proliferation.

After culturing the cancer cell line KM12C with 70% confluency, siC10orf81 (sense sequence, SEQ ID NO: 10 sequence) and siControl (sense sequence, SEQ ID NO: 11 sequence) were introduced into the cells by the high-throughput 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 mRNA of the gene was reduced, and analysis of protein expression through Western blot analysis revealed that gene expression was suppressed by siRNA 6A). siControl represents a negative control. siControl shows that there are no siRNA targets for sequences that are not homologous to human genes. Each cell was stained with SRB (Sulfur rhodamine B, Sigma Co.) and observed by photomicrographs of SRB-stained cells in a sample treated with siControl which showed no significant change in cell growth. Thus, siC10orf81- (Fig. 6B).

(2) Inhibition of growth inhibition by siC10orf81 in various cancer cell lines

To investigate the degree of cell growth depression by siRNA in a wider variety of cell lines, 0.5 ml of seed was seeded in a 24-well plate at a concentration of 1.0 x 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours Lt; / RTI > The control siRNA (control siRNA) (sense sequence, SEQ ID NO: 11) and siC10orf81 (sense sequence, SEQ ID NO: 10 sequence) were transfected into the cells in the same manner as described above and cultured for 72 hours. Growth inhibition was also investigated. Each cell was stained with SRB (Sulfur rhodamine, Sigma Co.), and the inhibition degree of cell growth was compared based on the cell line treated with siControl, which is a control, and the results are shown in FIG. The X axis in FIG. 7 represents each cell line, and the Y axis represents relative cell growth. As a result, inhibition of gene expression of C10orf81 showed cell growth inhibition in most cancer cell lines. These results suggest that C10orf81 gene siRNA can be used as a cancer treatment target because siRNA of C10orf81 gene can inhibit growth of colon cancer cell line, lung cancer, liver cancer, stomach cancer and prostate cancer cell line.

Table 1 shows an example of siRNA against the C10orf81 gene.

Location of siRNA in mRNA order GC content Identity 1531 CUGAACAGAGCUCCCAAGATT 0.53 19 3066 CGUUCAGUUGCAGAACCAATT 0.47 19 74 GUAUGAAGGCCCAGAACUUTT 0.47 19 163 ACUUCCUGGUGGUUCAGAUTT 0.47 19 1116 AGACAUCCCAUGAGUCUGUTT 0.47 19 1847 AGACACAGAUUGGGUCAGUTT 0.47 19 978 CUCGAAGUGUUCUUUUAGATT 0.37 19 2961 CAAAAACAGGCUUCAGAAATT 0.37 19 234 GCAGGAGUGAGUUCAGGAATT 0.53 19 1516 CUGGAUAAGC CUAGACUGATT 0.47 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. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience & Biotechnology <120> Diagnostic Kit for Cancer and Pharmaceutical Composition for          Prevention and Treatment of Cancer <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 3250 <212> DNA <213> Homo sapiens, C10orf81 cDNA <400> 1 attattaatg tgtttgcttt catttttggt gctaggcagc tgggacacca aggagagttc 60 tgtggcaagc cacaagtatg aaggcccaga acttcccctg agggcatagc ttccatttca 120 gtttggggca agaataaaat ctgctcaagg atattagttt gggaacttcc tggtggttca 180 gatttcaagc agagtttgtg cttaatcctc acccaggcac caaggctcag agtcagcagg 240 agtgagttca ggaatcctcg ggacaaggca ctttcctgag cactggacca gcgacctctt 300 ggcttccagg gaggacacac agccatcatg gaacccaaac ctcagaagag tccaggcaaa 360 caatttacat tttcttatga aaatgaagtc tgcaaacaag attactttat taaatcacca 420 ccttctcagc tgttctcctc tgtgacctct tggaaaaagc ggtttttcat cctgtcaaag 480 gctggggaaa agagctttag tctttcctat tataaagacc atcatcaccg aggttccatt 540 gaaattgatc aaaattccag tgtagaagtt ggcataagta gccaggaaaa gatgcaatct 600 gtgcagaaga tgtttaaatg ccaccctgat gaggtcatgt ccatcagaac cactaacagg 660 gaatacttcc tcattggcca cgacagggag aagattaaag actgggtctc cttcatgtca 720 tcatttcgcc aggatataaa agcaacacag cagaacacag aggaggaact ctcattgggt 780 aataaaagaa ccctcttcta ctccagccct ctccttggcc cttccagcac atcagaggct 840 gttggctcca gctcaccaag aaatggtctc caagacaagc atttaatgga acaaagttct 900 ccaggattta ggcaaactca cctacaagat ttatcagaag ccactcaaga tgtgaaggaa 960 gagaatcatt atcttactcc tcgaagtgtt cttttagagt tggataatat cattgcttcc 1020 agtgattctg gtgaatccat tgaaactgat ggtccagacc aggtctctgg aagaattgag 1080 tgtcattatg agccaatgga atcctatttt ttcaaagaga catcccatga gtctgtggat 1140 agcagcaaag aggaacccca gacccttcca gagacccagg atggggacct ccacctgcaa 1200 gaacaaggct caggaattga ttggtgtctt tcccctgccg atgtggaagc acagaccaca 1260 aatgaccaaa aggggtcggc ctcactaact gttgtgcaat tgtctatatt aatcaataat 1320 atccccgatg aaagccaagt ggagaaactg aacgttttcc tttctcctcc tgatgtcatc 1380 aactatcttg ctctcacaga agccacagga cggatatgaa attaaagctt accatcggca 1440 ggatcccaaa ttcagagaca ttccatgctg catcctatat gtgtccctca aaatgccaaa 1500 gtgctgcacc ttctcagctg gataagccta gactgaacag agctcccaag aggagtccgg 1560 ccattaaaaa gagccagcag aaaggagcca gggagtaacg caccccagac ccatggcagc 1620 agaaccagga tggagctggg actgtccagc tctgccccct gctgctgcca tgtgatagga 1680 gacagtcggc acccccctct gaatttctgc atctgcatct taacaatggg gatgactatc 1740 ccctctctgg ttattgtatc agagatgcta agagggtcat gtggcatgat tggagaacct 1800 gggggaattg gaaggcctta ttatctcagc tattgtccca aacaccacag acacagattg 1860 ggtcagtcct tcatgtaata catgctgtgt tctgtgagga tgtggtccac acaattcctt 1920 ctttgttaag ggacatacag ttgcaaatac tcactgcatg aaggcaagat tcccaaggga 1980 gatgtgatag ctgatcaggc ttcccagaca cctccttccc aaacacctcc ttcccaacac 2040 ctccttcccc aacatccttc ccaacacctc cttcccaaca cctccttccc aaacacctcc 2100 ttcccaaaca cctccttccc aacacctcct tccccaacac ctccttccca aacacctctt 2160 tcccaaacac ctccttccca gacacctcct tcccaacacc tccttcccaa cacctccttc 2220 ccaaacccct ctttcccaaa cacctccttc cccaacacct ccttcccaac acctccttcc 2280 cccttcccca acacctcctt ccccaacacc tccttcccaa cacctccttc ccaaacccct 2340 ccttccccaa catccttccc aacacctcct tcccaacacc tccttcccaa acacctcctt 2400 cccaaacacc tccttcccaa cacctccttc cccaacacct ccttcccaaa cacctctttc 2460 ccaaacacct ccttcccaga cacctccttc ccaacaccgc cttcccaaca cctccttccc 2520 aaacccctct ttcccaaaca cctccttccc caacacctcc ttccccaaca cctccttccc 2580 aacacctcct tcccccttcc ccaacaactc cttccccaac acctccttcc caaacatccc 2640 cttcccaaac acctgcctct cttcaacccc acaggccaga gtgctgagac agagtggcct 2700 tttggattca ataagtatct tgttctctta aagactcagc aacgatttta gaagtcgcag 2760 cagttttaca tcacatgcag ccaagatcag cttgctctac aagcaataac agaactactt 2820 agcacttcaa ggttgaaagt tcttcactaa tggatccatt gactaattga tcctggaagg 2880 ccaaaggaat aaaattcttt tatataaata ggaaaacaaa ggcagagagc taaagcacta 2940 atcaaatcgg ggggtgttag agcaaaaaca ggcttcagaa agagtatttt accatgcttc 3000 acatggaaaa aatcgagccc cggagcgatg aaaggcatat tttctttgtt tctccaagtt 3060 tcataaccgt tcagttgcag aaccaagaat ctaaaaccag ctctgggaaa caaatgtcca 3120 gatgccagcc tcatagttga acttggattt gaaaatacct tcagcactta gaagagacat 3180 tcaaatacat ttcatttcct gttacccaga ttgttcggaa agtattaaaa atttttcatt 3240 tacatgctgt 3250 <210> 2 <211> 1092 <212> DNA <213> Homo sapiens, C10orf81 ORF <400> 2 atggaaccca aacctcagaa gagtccaggc aaacaattta cattttctta tgaaaatgaa 60 gtctgcaaac aagattactt tattaaatca ccaccttctc agctgttctc ctctgtgacc 120 tcttggaaaa agcggttttt catcctgtca aaggctgggg aaaagagctt tagtctttcc 180 tattataaag accatcatca ccgaggttcc attgaaattg atcaaaattc cagtgtagaa 240 gtggcataa gtagccagga aaagatgcaa tctgtgcaga agatgtttaa atgccaccct 300 gatgaggtca tgtccatcag aaccactaac agggaatact tcctcattgg ccacgacagg 360 gagaagatta aagactgggt ctccttcatg tcatcatttc gccaggatat aaaagcaaca 420 cagcagaaca cagaggagga actctcattg ggtaataaaa gaaccctctt ctactccagc 480 cctctccttg gcccttccag cacatcagag gctgttggct ccagctcacc aagaaatggt 540 ctccaagaca agcatttaat ggaacaaagt tctccaggat ttaggcaaac tcacctacaa 600 gatttatcag aagccactca agatgtgaag gaagagaatc attatcttac tcctcgaagt 660 gttcttttag agttggataa tatcattgct tccagtgatt ctggtgaatc cattgaaact 720 gatggtccag accaggtctc tggaagaatt gagtgtcatt atgagccaat ggaatcctat 780 tttttcaaag agacatccca tgagtctgtg gatagcagca aagaggaacc ccagaccctt 840 ccagagaccc aggatgggga cctccacctg caagaacaag gctcaggaat tgattggtgt 900 ctttcccctg ccgatgtgga agcacagacc acaaatgacc aaaaggggtc ggcctcacta 960 actgttgtgc aattgtctat attaatcaat aatatccccg atgaaagcca agtggagaaa 1020 ctgaacgttt tcctttctcc tcctgatgtc atcaactatc ttgctctcac agaagccaca 1080 ggacggatat ga 1092 <210> 3 <211> 462 <212> PRT <213> Homo sapiens, PH domain-containing protein <400> 3 Met Ala Gly Gly Lys Gln Phe Thr Phe Ser Tyr Glu Asn Glu Val Cys   1 5 10 15 Lys Gln Asp Tyr Phe Ile Lys Ser Pro Pro Ser Gln Leu Phe Ser Ser              20 25 30 Val Thr Ser Trp Lys Lys Arg Phe Phe Ile Leu Ser Lys Ala Gly Glu          35 40 45 Lys Ser Phe Ser Leu Ser Tyr Tyr Lys Asp His His His Arg Gly Ser      50 55 60 Ile Glu Ile Asp Gln Asn Ser Ser Val Glu Val Gly Ile Ser Ser Gln  65 70 75 80 Glu Lys Met Gln Ser Val Gln Lys Met Phe Lys Cys His Pro Asp Glu                  85 90 95 Val Met Ser Ile Arg Thr Thr Asn Arg Glu Tyr Phe Leu Ile Gly His             100 105 110 Asp Arg Glu Lys Ile Lys Asp Trp Val Ser Phe Met Ser Ser Phe Arg         115 120 125 Gln Asp Ile Lys Ala Thr Gln Gln Asn Thr Glu Glu Glu Leu Ser Leu     130 135 140 Gly Asn Lys Arg Thr Leu Phe Tyr Ser Ser Pro Leu Leu Gly Pro Ser 145 150 155 160 Ser Thr Ser Glu Ala Val Gly Ser Ser Ser Pro Arg Asn Gly Leu Gln                 165 170 175 Asp Lys His Leu Met Glu Gln Ser Ser Pro Gly Phe Arg Gln Thr His             180 185 190 Leu Gln Asp Leu Ser Glu Ala Thr Gln Asp Val Lys Glu Glu Asn His         195 200 205 Tyr Leu Thr Pro Arg Ser Val Leu Leu Glu Leu Asp Asn Ile Ile Ala     210 215 220 Ser Ser Asp Ser Gly Glu Ser Ile Glu Thr Asp Gly Pro Asp Gln Val 225 230 235 240 Ser Gly Arg Ile Glu Cys His Tyr Glu Pro Met Glu Ser Tyr Phe Phe                 245 250 255 Lys Glu Thr Ser His Glu Ser Val Asp Ser Ser Lys Glu Glu Pro Gln             260 265 270 Thr Leu Pro Glu Thr Gln Asp Gly Asp Leu His Leu Gln Glu Gln Gly         275 280 285 Ser Gly Ile Asp Trp Cys Leu Ser Pro Ala Asp Val Glu Ala Gln Thr     290 295 300 Thr Asn Gln Lys Gly Ser Ala Ser Leu Thr Val Val Gln Leu Ser 305 310 315 320 Ile Leu Ile Asn Asn Ile Pro Asp Glu Ser Gln Val Glu Lys Leu Asn                 325 330 335 Val Phe Leu Ser Pro Pro Asp Val Ile Asn Tyr Leu Ala Leu Thr Glu             340 345 350 Ala Thr Gly Arg Ile Cys Val Ser Gln Trp Glu Gly Pro Pro Arg Leu         355 360 365 Gly Cys Ile Phe Cys His Gly Asp His Leu Leu Ala Val Asn Asp Leu     370 375 380 Lys Pro Gln Ser Leu Glu Glu Val Ser Leu Phe Leu Thr Arg Ser Ile 385 390 395 400 Gln Lys Glu Lys Leu Lys Leu Thr Ile Gly Arg Ile Pro Asn Ser Glu                 405 410 415 Thr Phe His Ala Ala Ser Cys Met Cys Pro Ser Lys Cys Gln Ser Ala             420 425 430 Ala Pro Ser Gln Leu Asp Lys Pro Arg Leu Asn Arg Ala Pro Lys Arg         435 440 445 Ser Pro Ala Ile Lys Lys Ser Gln Gln Lys Gly Ala Arg Glu     450 455 460 <210> 4 <211> 20 <212> DNA <213> Homo sapiens, C10orf81 specific primer (N-teminal) <400> 4 aagaggaacc ccagaccctt 20 <210> 5 <211> 20 <212> DNA <213> Homo sapiens, C10orf81 specific primer (C-terminal) <400> 5 tctccacttg gctttcatcg 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-terminal) <400> 7 tccaccaccc tgttgctgta 20 <210> 8 <211> 31 <212> DNA <213> Homo sapiens, C10orf81 specific primer (forward) <400> 8 tgcggatcca tggaacccaa acctcagaag a 31 <210> 9 <211> 30 <212> DNA <213> Homo sapiens, C10orf81 specific primer (reverse) <400> 9 atggatatct catatccgtc ctgtggcttc 30 <210> 10 <211> 21 <212> RNA <213> Homo sapiens, siC10orf81 (sense sequence) <400> 10 agacauccca ugagucuguu u 21 <210> 11 <211> 21 <212> RNA <213> Homo sapiens, siControl (sense sequence) <400> 11 ccuacgccac caauuucguu u 21

Claims (10)

(i) a nucleotide sequence of C10orf81 (Chromosome 10 open reading frame 81), (ii) a fragment of the nucleotide, (iii) a sequence complementary to the nucleotide sequence or a fragment thereof, (iv) (v) a cancer-diagnostic kit selected from the group consisting of gastric cancer, hepatic cancer and colorectal cancer, characterized in that it comprises a binding agent specific to the protein, said kit comprising a C10orf81 gene or a protein Wherein cancer is judged to be cancer when the expression level is highly expressed relative to normal cells or tissues.
The cancer diagnostic kit according to claim 1, wherein the kit is a microarray.
The cancer diagnostic kit according to claim 1, wherein the kit is a gene amplification kit.
The cancer diagnostic kit according to claim 1, wherein the kit is an immunoassay kit.
Measuring the expression level of C10orf81 (Chromosome 10 open reading frame 81) gene from a cell or tissue isolated from the subject; And determining the cancer as a cancer when the level of expression of the C10orf81 gene is highly expressed as compared to normal cells or tissues, wherein the cancer is cancerous, comprising gastric cancer, liver cancer and colon cancer The method comprising the steps of:
Measuring the expression level of C10orf81 (Chromosome 10 open reading frame 81) protein from a cell or tissue isolated from the subject; And determining the cancer as a cancer when the measured expression level of the C10orf81 protein is high compared to normal cells or tissues, wherein the cancer is a cancer comprising gastric cancer, liver cancer and colon cancer A method for providing information necessary for cancer diagnosis, which is selected from the group
(a) C10orf81 gene expression inhibitor; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating cancer is selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, 10. The pharmaceutical composition for preventing or treating cancer according to claim 1,
delete delete A screening method for cancer treatment selected from the group consisting of gastric cancer, liver cancer, colon cancer, lung cancer and prostate cancer, comprising the steps of:
(a) contacting a test substance to 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, the cancer therapeutic agent is determined.

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