KR101779147B1 - Compositions for Diagnosing or Treating Pancreatic Cancer Using KIAA1199 - Google Patents

Abstract

The present invention provides a method for screening a substance for preventing or treating pancreatic cancer using a novel molecular marker for pancreatic cancer stem cells and pancreatic cancer. The present invention relates to a method for screening a substance for preventing or treating pancreatic cancer using a novel molecular marker for pancreatic cancer stem cell and pancreatic cancer, a kit for detecting pancreatic cancer stem cell, a kit for analyzing pancreatic cancer diagnosis or prognosis, a composition for inhibiting pancreatic cancer stem cell formation, There is provided a pharmaceutical composition for treating or inhibiting metastasis. The target for treatment of pancreatic cancer of the present invention is very useful for screening candidate therapeutic agents that specifically act on pancreatic cancer stem cells. In addition, the present invention can be used to accurately analyze the diagnosis and prognosis of pancreatic cancer.

Description

Compositions for Diagnosing or Treating Pancreatic Cancer Using KIAA1199 [0002]

The present invention relates to a composition for diagnosis or treatment of pancreatic cancer using KIAA1199.

It has been suggested that cancer stem cells or tumor initiating cells that maintain and regenerate cancer tissues, such as general organs, may be involved in the initial onset of cancer, as well as cancer cells that are reduced after cancer treatment (BJ Zhou et al., Nature Reviews Drug Discovery , 8: 806-823 (2009)). In addition, it has been reported that cancer is associated with recurrence and metastasis and cancer induction resistance. Therefore, in order to fundamentally diagnose and treat cancer, it is necessary to focus on cancer stem cells, which occupy only a small part of cancer tissues and play a key role in the development, maintenance and recurrence of cancer. Further understanding of cancer stem cells It is also helpful to develop diagnostic markers for

Pancreatic cancer is the fatal cancer with a 5-year survival rate of 1-4% and a median survival time of 5 months. It has the worst prognosis in cancer of the human body. In the 80-90% of patients, the diagnosis is made in a state in which a radical resection is not possible, which is expected to be cured. Therefore, the prognosis is poor and the treatment depends on chemotherapy.

The therapeutic effects of some anticancer drugs including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective against pancreatic cancer, are extremely disappointing, and the response rate to chemotherapy is only about 15% This suggests that there is an urgent need to develop more effective early diagnosis and treatment methods to improve the prognosis of pancreatic cancer patients. Cancer cells refer to cancer cells that have the ability to self-renew and have recently emerged as a major target of cancer treatment conception. It is also known that cancers arising in organs of the body have molecular patterns that are differentiated according to the organ of origin. In the same vein, research has been reported that the cancer stem cells from each organ also have molecular markers that differentiate them from other organs.

Therefore, the inventors of the present invention reported and characterized the molecular markers of new pancreatic cancer stem cells in pancreatic carcinoma without definite molecular markers.

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 sought to find novel biomarkers for therapeutic targets of pancreatic cancer based on the characteristics of pancreatic cancer stem cells and pancreatic cancer stem cells. As a result, the present inventors have found that KIAA1199 specifically expressed in pancreatic cancer stem cells can be a molecular target for treating pancreatic cancer. In addition, the present inventors have found that biomarkers discovered can be used as markers capable of diagnosing pancreatic cancer, particularly early diagnosis, prognosis and future therapeutic targets based on cancer stem cell biological characteristics, Thereby completing the invention.

Accordingly, it is an object of the present invention to provide a method for screening a substance for preventing or treating pancreatic cancer.

It is another object of the present invention to provide a kit for detecting pancreatic cancer stem cells.

It is still another object of the present invention to provide a kit for pancreatic cancer diagnosis or prognosis analysis.

It is another object of the present invention to provide a composition for inhibiting pancreatic cancer stem cell formation.

It is another object of the present invention to provide a pharmaceutical composition for treating or preventing pancreatic cancer.

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

According to one aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating pancreatic cancer comprising the steps of:

(a) contacting a cell or tissue comprising the polynucleotide of the first sequence or the polynucleotide of the second sequence of the sequence listing with a sample to be analyzed; And

(b) measuring the expression level of the protein or polynucleotide in step (a), wherein when the sample reduces the expression of the protein or polynucleotide, the sample is a substance for preventing or treating pancreatic cancer .

The present inventors have made extensive efforts to find novel biomarkers for cancer therapeutic targets and cancer stem cells based on the characteristics of pancreatic cancer stem cells. As a result, the present inventors have found that KIAA1199 specifically expressed in pancreatic cancer stem cells can be a molecular target for treating pancreatic cancer. In addition, the present inventors have found that the biomarkers discovered are markers capable of diagnosing pancreatic cancer based on cancer stem cell biological characteristics, particularly early diagnosis, prognostic markers, and future therapeutic targets.

The present invention can be used as a therapeutic target of pancreatic cancer, which is specifically expressed in pancreatic cancer stem cells isolated from pancreatic cancer cell line, and the diagnosis and prognosis of pancreatic cancer can be accurately diagnosed at an early stage.

The marker of the present invention is specifically expressed in pancreatic cancer stem cells. Furthermore, the marker is a marker showing an expression pattern that is highly expressed in pancreatic cancer stem cells compared with pancreatic cancer cells, that is, the ability to distinguish pancreatic cancer cells from pancreatic cancer stem cells.

The expression " cell or tissue " used in the present invention for screening a substance for the prevention or treatment of pancreatic cancer is preferably pancreatic cancer stem cell or pancreatic cancer tissue.

The term " pancreatic cancer " as used herein means cancer originating from pancreatic cells. There are several types of pancreatic cancer, including pancreatic adenocarcinoma, which accounts for about 90% of cases. Generally, pancreatic cancer means pancreatic adenocarcinoma. In addition, there are cystic adenocarcinoma (adenocarcinoma) and endocrine tumor. About 5% to 10% of pancreatic cancer patients have a genetic predisposition. About 7.8% of pancreatic cancer patients have a family history of pancreatic cancer, compared with 0.6% of pancreatic cancer patients in the general population. Pancreatic cancer is a very poor prognosis with a 5 - year survival rate of less than 5%. The reason for this is that most of the cases are found after cancer has progressed, so surgical resection is possible within 20% of cases, and even if completely resected by visual examination, survival rate is not improved by micro metastasis and response to chemotherapy and radiotherapy It is low. Therefore, the most important method for improving survival rate is early detection and operation when symptomless or nonspecific.

The present inventors found that the expression of the KIAA1199 protein or the polynucleotide encoding the KIAA1199 protein is significantly increased in pancreatic cancer stem cells compared with pancreatic cancer cells. Unlike pancreatic cancer cells, the specific increase in the expression of the target protein or the polynucleotide encoding the target protein only in the pancreatic cancer stem cell indicates that it is an essential factor in the survival of the pancreatic cancer stem cell, and the substance that blocks the expression thereof is pancreatic cancer Stem cell growth inhibition and death by inducing the fundamental treatment of pancreatic cancer is helpful in the treatment of substances, pancreatic cancer treatment is determined.

The therapeutic agent for pancreatic cancer identified by the screening method of the present invention does not only target general pancreatic cancer cells which occupy the majority of cancer tissues but also treats pancreatic cancer stem cells that are a key part of the onset and maintenance of pancreatic cancer, Because it targets the cells, it enables the fundamental treatment of pancreatic cancer.

The sample used in step (a) is a single compound or a mixture of compounds (e.g., a natural extract or a cell or tissue culture). The test substance can be obtained from a library of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA).

Samples can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, , The " 1-bead 1-compound " library method, and the synthetic library method using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, and the like.

According to another aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating pancreatic cancer comprising the steps of:

(a) preparing a protein of the first sequence;

(b) contacting the test substance with the protein; And

(c) analyzing whether the test substance binds to the protein or whether the test substance inhibits the function of the protein; When the test substance binds to the protein or inhibits the function of the protein, it is judged to be a substance for preventing or treating pancreatic cancer.

According to the screening method of the present invention, the test substance is contacted with the protein of the first sequence listing.

The protein used in the present invention may be in the form displayed on a cell surface, in an isolated form or in a purified form displayed on the surface of a virus (for example, a bacteriophage).

When proteins displayed on the cell surface or virus surface are used, it is preferable that the cells or viruses are immobilized on a solid substrate in order to speed up or automate screening. It is also desirable to immobilize the isolated or purified form of the protein on a solid substrate. Available as substrates are any of those conventionally used in the art, including, but not limited to, hydrocarbon polymers such as polystyrene and polypropylene, glass, metals and gels. Solid phase substrates can be provided in the form of dip sticks, microtiter plates, particles (e.g., beads), affinity columns and immunoblot membranes (e.g., polyvinylidene fluoride membranes) (see U.S. Patent No. 5,143,825 No. 5,374,530, 4,908,305, and 5,498,551). Most preferably, the solid substrate is a microtiter plate.

The screening method of the present invention can be carried out in various ways, and can be carried out in a high throughput manner according to various binding assays known in the art.

In the screening method of the present invention, the test substance or the protein may be labeled with a detectable label. For example, the detectable label may be a chemical label (e.g., biotin), an enzyme label (e.g., horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, Let dehydratase and β- glucosidase), a radiolabel (e.g., ¹⁴ C, ¹²⁵ I, ³² P and ³⁵ S), fluorescent labels [e.g., coumarin, fluorescein, FITC (fluoresein Isothiocyanate), rhodamine 6G (rhodamine 6G), rhodamine B, TAMRA (6-carboxytetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino- , Dabsyl and FAM], luminescent markers, chemiluminescent markers, FRET (fluorescence resonance energy transfer) markers or metal markers (e.g., gold and silver).

If a detectable label is labeled or a test substance is used, the occurrence of a bond between the protein and the test substance can be detected by analyzing the signal from the label. For example, when alkaline phosphatase is used as a label, it is possible to use bromochloroindoleyl phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate ) And enhanced chemifluorescence (ECF). When horseradish peroxidase is used as a label, it is preferable to use chlorinaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorpine benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazone), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), and naphthol / pyronin.

Alternatively, the binding of the test substance to the protein may be assayed without labeling of the interactants.

For example, a microphysiometer can be used to analyze whether a test substance binds to QP-C. The microphysiometer is an analytical tool that uses a light-addressable potentiometric sensor (LAPS) to measure the rate at which a cell acidifies its environment. Changes in the rate of acidification can be used as an indicator of the binding between the test substance and QP-C (McConnell et al., Science 257: 19061912 (1992)).

The ability of the test substances to bind to proteins can be analyzed using real-time bimolecular interaction analysis (Sjolander & Urbaniczky, Anal. Chem. 63: 23382345 (1991), and Szabo et al., Curr Opin. Struct. Biol. 5: 699705 (1995)). BIA is a technique for analyzing specific interactions in real time, and can be performed without labeling of interactants (e.g., BIAcore ™). Changes in surface plasmon resonance (SPR) can be used as indicators for real-time reactions between molecules.

In addition, the screening method of the present invention can be carried out according to a two-hybrid assay or a three-hybrid assay (US Pat. No. 5,283,317; Zervos et al., Cell 72, 223232, 1993; Madura et al., J Bartel et al., BioTechniques 14, 920924, 1993; Iwabuchi et al., Oncogene 8, 16931696, 1993; and WO 94/10300). In this case, the protein may be used as a bait protein. According to this method, substances binding to the protein, particularly proteins, can be screened. To-hybrid system is based on the modular nature of the transcription factor consisting of segmentable DNA-binding and activation domains. Briefly, this analysis method uses two DNA constructs. For example, in one construct, a polynucleotide of the second sequence of the sequence listing is fused to a DNA binding domain-coding polynucleotide of a known transcription factor (e.g., GAL-4). In another construct, a DNA sequence encoding a protein of interest ("prey" or "sample") is fused to a polynucleotide encoding the activation domain of the known transcription factor. If bait and prey interact to form a complex in vivo, the DNA-binding and activation domains of the transcription factor become adjacent, which triggers transcription of the reporter gene (e.g., LacZ). The expression of the reporter gene can be detected. This indicates that the protein to be analyzed can bind to the protein, and consequently it can be used as a substance for preventing or treating cancer.

Examples of the sample used in the present invention include peptides, antibodies, peptide aptamers, AdNectin, affibody (US Pat. No. 5,831,012), Avimer, Silverman, (Kunitz domain, Arnoux B et al., Acta Crystallogr. D Biol. Crystallogr. 58 (Pt 7): 12524 (2002)), , And Nixon, AE, Current opinion in drug discovery & development 9 (2): 2618 (2006)).

According to another aspect of the present invention, there is provided a kit for detecting pancreatic cancer stem cells comprising a binding agent that specifically binds to the protein of the first sequence of the sequence listing or a primer or probe that binds to the polynucleotide of the second sequence of the sequence listing Lt; / RTI >

In the present invention, the binding agent that specifically binds to a protein includes, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA) to be.

The kit for detecting pancreatic cancer stem cells according to the present invention is characterized by measuring the expression level of a polynucleotide encoding a marker protein or a protein thereof from a human pancreatic cell sample and comparing the measured expression level with a protein or nucleotide expression level of a normal control sample And the like.

The normal control sample is a sample that has already been confirmed as not containing pancreatic cells, normal pancreatic cells or cancer stem cells that have not been taken from a human, and that does not form spheres under non-adherent culture conditions, for example, , Or a cell sample other than cancer of a pancreatic cancer patient identified to be non-malignant, but are not necessarily limited thereto. The expression level of the protein or the polynucleotide encoding the same in the normal control sample can also be measured using the same method as described above.

Through the above detection methods, it is possible to compare the amount of expression of the protein or the polynucleotide encoding the protein or the polynucleotide encoding the protein or the polynucleotide in the pancreatic cancer patient, which is the target of detection of cancer stem cells, in the normal control group, The presence or absence of cancer stem cells in the pancreatic cancer patient sample can be diagnosed.

Specifically, it is determined that the expression level of the protein or each of the polynucleotides encoding the protein in the patient sample is at least 150% of the level of expression of the protein or the polynucleotide encoding the same in the normal control sample.

According to another aspect of the present invention, the present invention provides a method for diagnosing or prognosing pancreatic cancer comprising a binding agent that specifically binds to the protein of the first sequence of the sequence listing or a primer or a probe that binds to the polynucleotide of the second sequence of the sequence listing Provide a kit.

In particular, the kit for diagnosing or prognosing pancreatic cancer according to the present invention is a kit derived from pancreatic cancer stem cells.

As used herein, the expression " kit for pancreatic cancer diagnosis or prognosis " means a kit containing a composition for the diagnosis or prognosis of pancreatic cancer. Therefore, the above expression " kit for diagnosis or prognosis analysis of pancreatic cancer " can be used interchangeably or in combination with " composition for diagnosis or prognosis analysis of pancreatic cancer ".

The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of an object (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining a stage of a cancer, or determining a cancer's response to treatment), or determining therametrics (e.g., And monitoring the status of the object to provide information).

Expression protein expression analysis used in the diagnosis of pancreatic cancer in the present invention is a process for confirming the presence and the expression level of a protein expressed from the gene in a biological sample. Preferably, the antibody expresses an antibody that specifically binds to the protein To identify the amount of protein. Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip are examples of immunoprecipitation, protein immunoassay, immunohistochemical staining, The method of analysis of the invention is not limited.

In the present invention, the binding agent that specifically binds to a protein includes, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA) to be.

&Quot; Antibody " in the present invention means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since the novel pancreatic cancer marker protein has been identified as described above, the production of an antibody using the pancreatic cancer marker protein can be easily performed using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injection of the above-described pancreatic cancer marker protein antigen into an animal and blood sampling from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies can be prepared using hybridoma methods well known in the art (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 1992, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).

The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

In addition, the antibody of the present invention can be used in a complete form having two full-length light chains and two full-length heavy chains,

Functional fragments of antibody molecules. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In the present invention, the aptamer binding to the active form of the enzyme is an oligonucleotide or a peptide molecule. The general contents of the aptamer are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

According to a preferred embodiment of the present invention, the present invention relates to an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a chimeric antibody that specifically binds to the protein to diagnose pancreatic cancer and / Antibody, ligand, peptide nucleic acid (PNA) or aptamer, more preferably an oligopeptide, a monoclonal antibody, a polyclonal antibody or a chimeric antibody, more preferably a monoclonal antibody, Antibody or polyclonal antibody, most preferably a monoclonal antibody.

In the present invention, the antibody is preferably a conjugated antibody conjugated with microparticles.

The fine particles are preferably colored latex or colloidal gold particles. In the present invention, the antibody may be any antibody capable of measuring the expression level of a protein encoded by a known mRNA gene for the marker described above. Preferably, the kit is used for immunoassay Kit, and most preferably the kit is a Luminex assay kit, a protein microarray kit or an ELISA kit.

The Luminex assay, protein microarray kit and ELISA kit comprise a polyclonal antibody and a monoclonal antibody to the protein, and a polyclonal antibody to which the labeling substance is bound and a secondary antibody to the monoclonal antibody .

Examples of the kit in the present invention include an immunochromatography strip kit, a lumenex assay kit, a protein microarray kit, an ELISA kit, or an immunological dot kit. The kind is not limited.

The kit may additionally include essential elements necessary for performing ELISA. ELISA kits contain antibodies specific for the marker protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

In addition, the kit may additionally include essential elements necessary for performing a protein microarray to simultaneously analyze complex markers. Microarray kits contain antibodies specific for the marker protein bound to the solid phase. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The protein microarray kit may also include antibodies specific for the control protein. Other protein microarray kits can be used to detect a bound antibody, such as a labeled secondary antibody, a chromophore, an enzyme (e.g., fused with an antibody), and a substrate or other substance capable of binding to the antibody And the like. A method for analyzing a sample using a protein microarray is a method for separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, and detecting the presence or the expression level of the protein, Can provide the necessary information.

Luminex Assay is a high-throughput quantitative assay that can simultaneously measure up to 100 different analyte without pretreatment small (10-20 μl) patient samples Is an analytical method capable of replacing existing ELISA or ELISPOT with high sensitivity (in pg) and rapid quantification (3-4 hours). The Luminex Assay is a multiplexed fluorescent microplate assay capable of analyzing more than 100 biological samples simultaneously in each well in a 96-well plate. And more than 100 different color groups of polystyrene beads are discriminated and quantified by carrying out signal transmission in real time using a laser detector of the present invention.

The 100 beads are configured to be distinguished in the following manner. On one side, the red fluorescence bead is divided into more than ten stages. On the other side, the orange fluorescence bead is divided into ten stages to show the intensity difference and the bead between them. Are mixed in different ratios of red and orange to form a total of 100 color-coded bead sets. In addition, each bead has an antibody of the protein to be analyzed, so that it is possible to quantify the protein by the immunoassay using the antibody. The sample is analyzed using two lasers, one laser detects the bead's unique number and the other laser reacts with the antibody attached to the bead The protein in the sample is detected. Thus, 100 in vivo protein analysis is possible simultaneously in one well. This assay has the advantage of being detectable with as little as 15 μl of sample.

Luminex kits capable of carrying out the Luminex assays of the present invention include antibodies specific for marker proteins. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The lumenex kit may also include antibodies specific for the control protein. Other luminex kits may also include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies) and other substrates capable of binding to the substrate or antibody . ≪ / RTI > The antibody may be a conjugated antibody conjugated with microparticles, and the microparticles may be colored latex or colloidal gold particles.

The pancreatic cancer diagnostic kit comprising the immunochromatographic strip for diagnosis of pancreatic cancer in the kit for pancreatic cancer diagnosis or prognosis according to the present invention is characterized in that it comprises essential elements necessary for performing a rapid test in which the analysis result can be obtained within 5 minutes And the like. The immunochromatographic strip comprises (a) a sample pad on which a sample is absorbed; (b) a conjugate pad that binds to the protein of the gene in the sample; (c) a test membrane in which a test line and a control line containing a monoclonal antibody against the protein of the gene are treated; (d) an absorption pad on which a residual amount of the sample is absorbed; And (e) a support. Also, a rapid test kit containing an immunochromatographic strip contains antibodies specific for the marker protein. The antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The rapid test kit may also include an antibody specific for the control protein. Other rapid test kits include reagents capable of detecting conjugated antibodies, for example, a nitrocellulose membrane with a specific antibody and a secondary antibody immobilized thereon, a membrane bound to an antibody-bound bead, an absorbent pad and a sample pad Other substances necessary for diagnosis, and the like.

The measurement of the level of protein expression by the immunodet assay in the present invention includes (a) dotting a biological sample on the membrane; (b) reacting an antibody specific for the protein of the gene to the dormant membrane; And (c) a step of adding a secondary antibody conjugated with a label to the membrane to be subjected to the reaction, followed by color development, wherein the ELISA assay comprises the steps of: (a) contacting the protein of the gene having the nucleotide sequence with the marker Adsorbing a specific antibody 1 on a solid body; (b) contacting an antibody 1 adsorbed on the solid body with a biological sample of a suspected cancer patient to form an antigen-antibody complex; (c) treating antibody 2 specific for the protein encoded by the gene having the nucleotide sequence for the marker to which the marker substance is bound, to bind to the complex; And (d) measuring the concentration of the protein by detecting the labeling substance, wherein the protein microarray assay comprises (a) a polyclonal antibody specific to the protein of the marker gene, Immobilizing the antibody on the chip; (b) contacting the immobilized polyclonal antibody 1 with a biological sample of a patient suspected of having cancer to form an antigen-antibody complex; (c) treating the monoclonal antibody specific to the protein encoded by the gene having the nucleotide sequence for the marker to which the marker substance is bound, to bind to the complex; And (d) measuring the concentration of the protein by detecting the labeled substance.

Through the above analysis methods, it is possible to compare the amount of the antigen-antibody complex formed in the normal control group with the amount of the antigen-antibody complex formed in the suspected patient of pancreatic cancer, and judge whether the expression level of the pancreatic cancer marker gene is significantly increased Thus, it is possible to diagnose the actual pancreatic cancer in patients suspected of having pancreatic cancer.

In the present invention, the term antigen-antibody complex refers to a combination of a pancreatic cancer marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label.

Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructoketase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like.

The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like.

Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radioisotope includes include ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re are not limited to .

Measurement of protein expression levels is preferably performed using an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. The pancreatic cancer marker protein and antibody complex formation level can be confirmed to confirm the onset of pancreatic cancer.

In addition, preferably, Western blotting using one or more antibodies against the pancreatic cancer marker is used. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the protein produced by the expression of the gene can be confirmed by confirming the amount of the generated antigen-antibody complex using the labeled antibody, thereby confirming whether or not the pancreatic cancer has occurred. The detection method comprises a method of examining the expression level of a marker gene in a control group and the expression level of a marker gene in a cancer-causing cell. The level of mRNA or protein may be expressed as the absolute (e.g., [mu] g / ml) or relative (e.g., the relative intensity of the signal) difference of the marker protein.

Preferably, immunostaining is performed using at least one antibody against the pancreatic cancer marker. Normal tissues and suspected pancreatic cancer tissues are collected and fixed, and paraffin-embedded blocks are prepared by methods well known in the art. These are cut into pieces of several μm thick and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.

Preferably, the protein chip is used in which at least one antibody against the pancreatic cancer marker is arranged at a predetermined position on the substrate and immobilized at a high density. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It is possible to confirm whether or not the disease has occurred.

The biological sample in the present invention means a tissue, a cell, a blood, a serum, a plasma, a saliva, a cerebrospinal fluid or urine, preferably a tissue or a cell, and most preferably a cell.

The expression " polynucleotide measurement " used to diagnose pancreatic cancer in the present invention means measuring the amount of polynucleotide in the biological sample by confirming the presence and the degree of expression of the polynucleotide encoding the protein marker of the present invention . RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

The nucleotide sequence encoding the protein used as a marker in the present invention may include a nucleotide sequence having homology with this nucleotide sequence.

Preferably, the polynucleotide in the present invention means a fragment of DNA or mRNA.

The probe or primer used in the diagnostic kit of the present invention has a sequence complementary to a polynucleotide sequence and a polynucleotide sequence encoding the protein.

The term " primer " of the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand replication.

The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences as a marker primer specific primer.

Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art.

Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., dodecanedioic acid, dodecanedioic acid, dodecanedioic acid, tartaric acid, tartaric acid, tartaric acid, The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention may also be modified using a label capable of directly or indirectly providing a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

As used herein, the term " probe " refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

When a probe is used, the probe is hybridized with the cDNA molecule. 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. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); And M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; N.Y. (1999). For example, high stringency conditions were hybridized under conditions of 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS) and 1 mM EDTA at 65 ° C, followed by hybridization in 0.1 x SSC (standard saline citrate) /0.1% SDS at 68 Lt; 0 > C. 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.

According to another aspect of the present invention, the present invention provides a pancreatic cancer stem cell comprising an antibody that specifically binds to the protein of the first sequence of the sequence listing or a nucleic acid molecule for suppressing the expression of the polynucleotide of the second sequence of the sequence listing as an active ingredient The present invention provides a composition for inhibiting the formation of a cell.

According to another aspect of the present invention, the present invention provides a method for treating pancreatic cancer, comprising an antibody that specifically binds to the protein of the first sequence of the sequence listing or a nucleic acid molecule for suppressing the expression of the polynucleotide of the second sequence of the sequence, Or a pharmaceutical composition for inhibiting metastasis.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, percutaneous administration, mucosal administration, or topical administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the present invention are within the range of 0.0001-10000 / kg on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

As used herein, an antibody against KIAA1199 protein or a nucleic acid molecule for inhibiting the expression of KIAA1199 reduces sphere and colony formation, decreases the proliferation and migration ability of cancer stem cells, and consequently reduces cancer stem cells It is possible to inhibit proliferation.

The KIAA1199 expression-suppressing nucleic acid molecule used in the present invention is an antisense oligonucleotide or siRNA oligonucleotide or an shRNA oligonucleotide that specifically binds to the KIAA1199 gene.

The term " antisense oligonucleotide " as used herein refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into a protein . The antisense sequence refers to a DNA or RNA sequence complementary to KIAA1199 mRNA and capable of binding to KIAA1199 mRNA and is capable of translating KIAA1199 mRNA, translocating it into the cytoplasm, maturation or any other essential biological function . ≪ / RTI > The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases.

The antisense nucleic acid can be modified at one or more base, sugar or backbone locations to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5 (3): 343-55 (1995) ). The nucleic acid backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic biantennary bond, and the like. In addition, the antisense nucleic acid may comprise one or more substituted sugar moieties. The antisense nucleic acid may comprise a modified base. Modified bases include, but are not limited to, hypoxanthin, 6-methyladenine, 5-me pyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine . In addition, the antisense nucleic acid of the present invention may be chemically combined with one or more moieties or conjugates that enhance the activity and cytotoxicity of the antisense nucleic acid. A cholesterol moiety, a cholesteryl moiety, a cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, octadecylamine, hexylamino- And liposoluble moieties such as Cole sterol moieties. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase the stability to nuclease and increase the binding affinity of the antisense nucleic acid with the target mRNA.

In the case of antisense oligonucleotides, they can be synthesized in vitro in a conventional manner and administered in vivo or in vivo to synthesize antisense oligonucleotides. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

The design of the antisense oligonucleotides that can be used in the present invention can be readily made by methods known in the art with reference to the nucleotide sequence of SEQ ID No. 2 (Weiss, B. (ed.): Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, FL, 1997; Weiss, B., et al., Antisense RNA gene therapy for studying and modulating biological processes. 358 (1999).

According to a specific embodiment of the present invention, the nucleic acid molecule for inhibiting the expression of KIAA1199 is an siRNA or shRNA comprising a sequence complementary to the KIAA1199 gene.

The term "siRNA " in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 And WO 00/44914.) Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.

The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit expression of the gene by the RNAi effect. The sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure.

SiRNA molecules of the invention may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between self-complementary sense and antisense strands, in which case the siRNA molecule is formed by the expression of a nucleotide sequence The siRNA molecules form a hairpin structure by intramolecular hybridization and form a stem-and-loop structure as a whole. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

According to a specific embodiment of the present invention, the siRNA is a sequence complementary to nucleotides 2137 to 2161 of the KIAA1199 gene of SEQ ID NO: 2, more specifically siRNA described in SEQ ID NO: 3, The sequence complementary to nucleotides 3664 to 3688 of the KIAA1199 gene of the sequence, more specifically, the siRNA described in SEQ ID NO: 4.

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

(a) The present invention provides a method for screening a substance for preventing or treating pancreatic cancer using a novel molecular marker for pancreatic cancer stem cell and pancreatic cancer.

(Ii) The present invention relates to a method for screening a substance for preventing or treating pancreatic cancer using a novel molecular marker for pancreatic cancer stem cells and pancreatic cancer, a kit for detecting pancreatic cancer stem cells, a kit for analyzing pancreatic cancer diagnosis or prognosis, Compositions and pharmaceutical compositions for treating or preventing pancreatic cancer.

(Iii) The target for treating pancreatic cancer of the present invention is very useful for screening candidate therapeutic agents that specifically function on pancreatic cancer stem cells.

(Iv) Using the present invention, the diagnosis and prognosis of pancreatic cancer can be accurately analyzed.

1 shows a microarray list for selecting protein candidate groups. Capan-1 and HPAC cell lines were used to make adherent and prototype cells. Differences in gene patterns between the two were confirmed by microarray, and some of the results of this microarray were listed.
Figure 2a shows the number of adherent cells and circular ellipsoids in AsPC-1, Capan-1, HPAC and Miapaca-2
0.0 > mRNA < / RTI > level in KIAA1199 cells.
FIG. 2B is a graph showing mRNA levels of KIAA1199 in adherent cells and circular ellipsoid cells of Capan-1, HPAC.
Figure 2c is a graph showing the protein levels of KIAA1199 in adherent cells and tumor progenitor cells of Capan-1, HPAC.
FIG. 2d shows the results of immunohistochemical staining of cancer tissue, normal tissue, and KIAA1199 in pancreatic cancer patients.
FIG. 3A shows Western blotting results of KIAA1199 cDNA vectors (K8, K9 and K10) and empty vectors (E6 and E9) tagged with Flag in Capan-1 cell line.
FIG. 3B is a photograph showing a cell shape of E6 cell line and K10 cell line observed with a light microscope.
3c is a view showing a mobile assay and an invasion assay using E6 and K10 cell lines.
FIG. 3D is a diagram showing recovery patterns of E6 and K10 cells. FIG.
3E is a diagram showing the difference in the molecular pattern of E6 and K10 cells.
FIG. 4A is a graph showing the expression pattern of KIAA1199 after treatment with K10 cell line (overexpressing cell line of KIAA1199) of siRNA variants # 1 and # 2 specific to KIAA1199.
FIG. 4B shows KIAA1199 expression levels after treatment of naive Capan1 cells, samples treated with scrambled oligos on E6, K10 and K10 cells, and siRNAs specific for K10 cells.
Figure 5a shows the expression patterns of each molecule in E6, K10 and K10 + scrambled oligos and K10 + siRNA samples by Western blotting.
FIG. 5B shows mRNA expression patterns of KIAA1199, beta -catenin, Dvl2, Wnt3, Wnt3a and Wnt7b by qRT-PCR.
FIG. 5C is a graph showing the results of experiments in which qRT-PCR was performed on cells treated with scrambling and siRNA in AsPC-1 cell line, respectively.
FIG. 5d shows the results of the ELISA using an FFP plasmid tagged with luciferase-tagged ORF with a TOP plasmid tagged with an open reading frame (cf / Lef promoter sequence) and a mutated Tcf / Lef promoter sequence Fig. 5 shows experimental results of a luciferase assay. Fig.
FIG. 5E is a graph showing mRNA levels of OCT4, Nanog, and Sox-2 in the four scrambled and siRNA-treated samples of the E6, K10 and AsPC-1 cell lines of the Capan1 cell line.
FIG. 5f is a graph showing Western blotting results of KIAA1199 protein expression patterns in cytoplasm, membrane, nucleus and cytoskeletal fractions by performing subcellular fractionation of K10 cells.
FIG. 5g shows a result of a ChIP assay experiment to determine whether KIAA1199 acts as a transcription factor in the nucleus.
5H is a graph showing the results of western blotting experiments of secretory Wnt3.
6A is a diagram showing the interaction between KIAA1199 and OCT4.
FIG. 6B is a graph showing the results of an experiment in which a sumoylation assay was performed after immunoprecipitation with OCT4 and SUMO-1, respectively.
FIG. 7 is a graph showing the results of an experiment for verifying the tumor forming ability of KIAA1199 in vivo . FIG.
8A is a graph showing the results of measurement of mRNA levels of Oct4, Nanog, Dvl2, Wnt3 and Wnt3a in adherent cells and protoplast cells of Capan-1 and HPAC cell lines.
FIG. 8B is a graph showing the results of measurement of protein expression levels of β-catenin and Dvl2 in adherent cells and protoplast cells of Capan-1 and HPAC cell lines.
FIG. 8C is a graph showing the results of measurement of the interaction between β-catenin and KIAA1199 in the protoplast cells of Capan-1 and HPAC cell lines by immunoprecipitation using a β-catenin antibody.
FIG. 8D is a graph showing the results of measurement of the interaction between β-catenin and KIAA1199 in the adherent cells and protoplast cells of Capan-1 and HPAC cell lines by immunoassay using KIAA1199 antibody.
FIG. 8E is a graph showing the results of measurement of the β-catenin and KIAA1199 as a transcription factor for the OCT4 and Nanog promoters through a chromatin immunoprecipitation assay.
FIG. 9 is a graph showing the results of IHC using KIAA1199 for TMA tissue of pancreatic cancer patients and the average survival rate of patients. FIG.
10a-f show the IC50 values of Irinotecan, Carboplatin, 5-FU, Gemcitabine, Etoposide, and Paclitaxel in E6 and K10 cells, respectively And Fig.
FIG. 10g shows the results of measuring end points of survival rates for gemcitabine after treating scaffold siRNA or siKIAA1199 in E6 and K10 cells, respectively.

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

Example

Materials and Experiments

1. Tumor culture (sphere culture)

The cells were cultured for 5 or 7 days to maintain a concentration of 1,000 cells / mL in a culture medium containing 0.5% BSA, 0.5% FBS, 1xITS, bFGF 10 ng / mL and EGF 10 ng / mL.

2. RT-PCR and qRT-PCR

RNA was extracted from pancreatic cancer cells or pancreatic cancer cells derived from tumor protoplast cells using an RNeasy mini kit (Quiagen). CDNA was synthesized from the obtained RNA using Super Script II System (Invitrogen). RT-PCR and qRT-PCR were performed with target-specific primers using Taq polymerase (Takara) and SYBR mastmix (ABI), respectively.

3. Cell lines, antibodies and siRNA

Human pancreatic cancer cell lines AsPC-1, Capan-1 and HPAC were used in this experiment. (CST), pKT (CST), pMTOR (CST), pGSK3? (CST), pancreatic islets (CST) CST), cadherin, N-cadherin,? -Catenin (Santa cruz), IGFR1? (Santa cruz), IGFR1? (Santa cruz), SUMO-1 (Santa cruz) and GAPDH specific antibodies were used. SiRNA specific for the KIAA1199 gene was purchased from Invitrogen (Stealth).

4. Construction of plasmids and stable cell lines

The human KIAA1199 ORF (NM_018689) was purchased from GenScript and cloned into the pcDNA3.1 (+) plasmid tagged with Flag at the c-terminus. The pcDNA3.1 (+) plasmid and the pcDNA3.1 plasmid were transfected into Capan-1 cell line using GENEIN transfection reagent (Gliostem). A stabilized cell line E6 with a control vector and a stabilized cell line K10 with overexpressed KIAA1199 were constructed.

5. Migration, invasiveness and wound healing assays

Cells of 2x10 ⁵ or 3x10 ⁵ cells / mL were dispensed into Transwell (3422, corning). After 3 days, the cells were fixed with 5% glutaraldehyde for 10 minutes and then washed three times with distilled water. Cells were stained for 20-30 minutes using crystal violet. The cells were washed several times with distilled water, dried and mounted on slides.

For invasion assays, the top surface was coated with agarose and then the same number of cells were dispensed into the transwell inserts. The same steps as the move assay were then performed.

Culture plates of E6 and K10 cells having a density of 90% or more for scratching healing were scratched by a tip and cultured in a growth medium for 24 hours.

6. Sumoylation Assay

(Naive) samples using IP buffer containing 50 mM Tris-HCl (pH 6.7) buffer, 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 0.5% NP40 and 1x protease inhibitor cocktail, Cells, E6 cells, K10 cells and cells treated with siRNA). The lysate was reacted with the appropriate antibody overnight at 4 ° C and treated with ultra biolink support resin for 2 hours to aid binding. The resin was collected by centrifugation and washed three times with wash buffer containing 20 mM Tris-HCL, 500 mM NaCl, 1 mM EDTA and 0.5% NP40. SDS-PAGE was performed using the sample and Western blotting analysis was performed.

7. Self-renewal and colony formation assays

Pancreatic cancer cells, naïve cells, cells transfected with scrable siRNA, and cells specific for KIAA1199 Tumor protoplasts of siRNA-transfected cells were isolated into single cells. Cells were cultured in stem cell medium and 2nd and 3rd generation tumor rounds were obtained. The numbers of floating tumor progenitors and total cells were counted under an optical microscope. For colony formation, isolated single cells were seeded in 24 well low attached dishes (Corning) at a density of 100 cells per well in 0.3% soft agar containing growth medium. The plates were then incubated for 14 days at 37 ° C in 5% CO ² until colonies were visible. The colonies were stained with 0.01% crystal violet, and the number of colonies was counted under an inverted microscope.

8. Chip (chromatin immunoprecipitation) assay

Cells transfected with E6, K10, AsPC-1 scrambled cells, cells transfected with AsPC-1 siKIAA1199, capan-1-derived adherent and protoplast cells, HPAC-derived adhesion and protoplast cells were used for ChIP assays. This experiment was performed according to the EZ ChIP kit (Up state) protocol. The antibody used for IP is the same as that used for Western blotting.

9. In vivo  Xenotransplantation

Five Balb / C nude mice were used in each experimental group. E6 and < RTI ID = 0.0 > K10 &⁵ Cells / mouse, and mice were sacrificed 7 days later.

10. TMA (Tissue Microarray) and Immunohistochemical staining (IHC)

Immunohistochemical staining (IHC) was performed on TMA containing cancer tissues and adjacent normal tissues of pancreatic cancer patients using KIAA1199 antibody (Sigma-Aldrich). Immunohistochemical staining was performed in the following manner: The tissues stained with the slides were incubated at 60 ° C. for 30 minutes, and then immersed in diluted alcohol such as 70%, 50%, and 30% starting from 100% ethanol The paraffin was removed. Subsequently, the plate was washed three times with PBS, immersed in a peroxidase quenching solution (100% methanol and 30% hydrogen peroxide mixture) for 20 minutes, washed three times with PBS, and then incubated with 10 mM sodium citrate solution (pH 6.0) For 3 minutes. The tap water was poured on the slide for 10 minutes, washed three times with PBS, and 10% normal donkey serum was treated at room temperature for 1 hour. After washing three times with PBS, the cells were reacted with an antibody specific for KIAA1199 protein (Sigma-Aldrich) at 4 ° C for 24 hours and washed with PBS. The Envision kit solution from DAKO was processed and then allowed to stand in the chamber for 20 minutes. Washed three times each with PBS and distilled water, treated with hematoxylin for 1 min, washed three times with distilled water, dried, and then mounted on a cover slide and covered with a cover slide and observed under a microscope. Immunoreactivity of cancer tissues was classified as not expressed, weakly expressed, moderately expressed, and strongly expressed depending on the intensity of the staining. To score for each, more than 50% of the cells under the low-power field (100x) should exhibit a corresponding degree of immunoreactivity. The OS is defined as the interval between the date of diagnosis and the date of death. Recurrence-free survival (RFS) was defined as the interval between the date of death and the date of relapse or last visit.

11. MTT assay

E6 and K10 cells were seeded in 96 well plates at 2,500 cells / well. After 24 hours, each plate was treated with concentrations indicating 5-FU, Gemcitabine, Irinotecan, Etoposide, Carboplatin and Paclitaxel. After 72 hours of incubation, the cells were washed with PBS and treated with MTT solution (Amresco) diluted in growth medium for 3 hours. Fluorescence was then measured at a wavelength of 570 nm using an ELISA instrument.

12. Statistical analysis

All statistical analyzes were performed using the SPSS version of Windows version 18.0 (SPSS, Inc., Chicago, IL, USA). Survival curves were generated using the Kaplan-Meier method and the differences between the curves were evaluated using the log-rank test. The P-value <0.05 was considered statistically significant.

Experiment result

1. Selection of protein candidates through microarray

Tumor cells of each cell line were cultured in human pancreatic cancer cell lines CAPAN-1 and HPAC via sphere culture, which is a culture method of cancer stem cells, and adherent cells were used as a control. Chip analysis was performed. The results are shown in Fig.

Figure 1 shows fold change values (FC_HS and FC_CS) in tumor progenitor cells versus adherent cells. Among the candidates in the list shown in Fig. 1, KIAA1199 was selected as a research target.

2. Verification of expression level of KIAA1199 protein in cell line

Tumor protoplasts were cultured in human pancreatic cancer cell lines AsPC-1, Capan-1, HPAC and Miapaca-2, and qRT-PCR was performed to confirm the mRNA levels of KIAA1199 using adherent cells Respectively. As a result, it was confirmed that the mRNA of KIAA1199 was increased in the tumor cell form of each cell line as compared with the adherent cell as shown in FIG.

In addition, RT-PCR was performed to confirm the protein level of KIAA1199 in adherent cells and tumor protoplast cells of human pancreatic cancer cell line Capan-1, HPAC. As a result, the mRNA of KIAA1199 was increased in tumor progenitor cells (Fig. 2B).

Western blotting was performed to confirm the protein level of KIAA1199 in adherent cells and tumor progenitor cells of the human pancreatic cancer cell line Capan-1, HPAC. As a result, the protein of KIAA1199 was increased in tumor progenitor cells than in the adherent cells of both cell lines (Fig. 2C).

Immunohistochemical staining using cancer, normal tissue and antibody specific to KIAA1199 protein in pancreatic cancer patients showed that KIAA1199 protein was increased in cancer tissues than normal tissues in pancreatic cancer patients (FIG. 2d).

3. Flag- KIAA1199  Establishment and characterization of overexpressed stable cell line

The KIAA1199-cDNA vector (K8, K9 and K10 cells) and the co-vector (E6 and E9 cells), in which Flag was tagged, were selected for Capan-1 cell line and then selected as the selective drug G418. The expression pattern of the Flag-KIAA1199 protein in each clone was verified by Western blotting using an antibody specific for Flag. The results are shown in Fig.

E6 cell line and K10 cell line were observed with a fluorescence microscope. As a result, it was confirmed that the shape of the K10 cell line changed into a cell-shaped form with a branch, unlike E6 (FIG. 3B).

Migration and invasion assays of the E6 and K10 cell lines resulted in a large number of cells migrating or invasive in the K10 cell line (FIG. 3c).

In addition, E6 and K10 cells were dispensed and maintained in a 24-well plate dish, followed by scratching with a tip. After 24 hours, recovery was observed in the K10 cell line (FIG. 3D).

On the other hand, Western blotting revealed differences in the molecular patterns of E6 and K10 cells, indicating that EMT-related molecules, IGF-1 signaling-related molecules, OCT4, which is known to regulate self-renewal, Beta-catenin expression was altered (Fig. 3E).

4. Functional verification of KIAA1199

In order to examine the function of KIAA1199, siRNA variants # 1 and # 2 specific to KIAA1199 were treated with K10 cell line (overexpressing KIAA1199 cell line) and their expression pattern was confirmed. As shown in FIG. 4A, it was confirmed that the expression of KIAA1199 was more efficiently reduced than that of variant # 1. Also, it was confirmed that molecules such as OCT4 and IGF1R, which were increased in K10, decrease together.

Naive Capan1 cells, E6, K10 cells; K10 cells treated with scrambled oligos; And KIAA1199 expression level in the group treated with siRNA specific for K10 cells (Fig. 4B).

E6, K10 cells, K10 cells treated with scrambled oligos, and K10A1199-specific siRNA-treated K10 cells showed that tumor cells were formed and colony formation was observed in cells treated with scramble oligos in K10 and K10 cells compared to E6 The number of tumor protoplasts and colonies was increased, and the cells treated with KIAA1199-specific siRNA in K10 were found to be inferior in tumor protoplasm and colony forming ability (FIG. 4C).

In addition, AsPC-1 cells were treated with scrambling or KIAA1199 specific siRNA, and Western blotting was performed. As a result, a molecular change pattern similar to that of K10 cell line produced in Capan-1 cells was confirmed (FIG.

5. Verification of the relationship between KIAA1199 and Wnt signaling

Beta - catenin was expressed by KIAA1199 expression and its relationship with Wnt signaling was verified.

Molecular expression patterns of E6, K10 and K10 + scrambled oligos and K10 + siRNA samples were measured by western blotting and mRNA expression patterns of KIAA1199, beta -catenin, Dvl2, Wnt3, Wnt3a and Wnt7b were measured by qRT-PCR. As a result, it was confirmed that mRNA levels of beta -catenin, Dvl2, Wnt3, and Wnt3a, which are typical molecules involved in Wnt signal transduction, correspond to the level of KIAA1199, as shown in Figs. 5A and 5B. However, it was confirmed that the level of Wnt7b mRNA, an atypical Wnt signaling-related molecule, appears to be opposite to that of KIAA1199.

QRT-PCR was performed on cells treated with scrambling and siRNA in AsPC-1 cell line, respectively. As a result, it was confirmed that mRNA levels of Dvl2, Wnt3, and Wnt3a, which are involved in typical Wnt signaling, change according to mRNA levels of KIAA1199 (Fig. 5C).

In order to confirm that KIAA1199 acts on the typical Wnt signal transduction in which beta-catenin acts as a main molecule, beta-catenin is transferred to the nucleus and then luciferase is added to an ORF (open reading frame) with a binding Tcf / Lef promoter sequence. Luciferase assay was carried out using an FOP plasmid tagged with luciferase in an ORF having a mutated TOP plasmid and a mutated Tcf / Lef promoter sequence. As a result, the TOP / FOP ratio was increased in K10A1199 overexpressed K10 cells, and the TOP / FOP ratio was decreased in the other cell lines in which KIAA1199 expression was reduced by siRNA (Fig. 5d).

The mRNA levels of OCT4, Nanog and Sox-2, known as targets of typical Wnt signaling in the four samples scrambled and siRNA treated with E6, K10 and AsPC-1 cell lines of Capanl cell line, (Fig. 5 (e)).

K10 cells were subjected to subcellular fractionation to obtain fractions of cytoplasm, membrane, nucleus and cytoskeleton, and the expression pattern of KIAA1199 in each fraction was confirmed by Western blotting. As a result, it was confirmed that KIAA1199 was expressed in the nucleus (Fig. 5F).

In order to confirm whether KIAA1199 acts as a transcription factor in the nucleus, ChIP assay showed that the binding of beta-catenin to the target promoter was increased as KIAA1199 protein expression level was increased (panel a of FIG. 5g) As the level decreased, binding with the target promoter decreased (panel e in Figure 5g). In addition, it was confirmed that KIAA1199 binds to the OCT4, Nanog promoter (panels b, c, and d in FIG. 5g).

The medium of E6 and K10 cells was discarded and the secretion Wnt3 was observed by western blotting after sedimentation with acetone. As a result, secretory Wnt3 was increased in K10 cells (FIG. 5h).

6. Verification of relevance to OCT4

As the KIAA1199 increased, the intensity of the OCT4 modified band was found to be stronger. Therefore, the relationship between KIAA1199 and OCT4 was investigated. The results are shown in Figs. 6A and 6B.

As shown in Fig. 6A, it was confirmed that the KIAA1199 protein of 110 kDa was associated with OCT4.

In addition, a sumoylation assay was performed to identify the modified band of OCT4. The capillary K10 cell line and the AsPC-1 cell line were treated with scrambling and KIAA1199-specific siRNA, followed by immunoprecipitation with OCT4 and SUMO-1, respectively, followed by humilation. As a result, (Fig. 6 (b)).

7. in vivo  Verification of tumor formation ability of KIAA1199

5x10 ⁵ E6 and K10 cells were injected into subcutaneous fat on the right leg of each of five nude Balb / C mice, and mice were sacrificed 7 weeks later. As a result, as shown in Fig. 7, it was confirmed that no mice in which tumors occurred in 5 mice injected with E6 were observed. However, it was confirmed that tumors occurred in 4 out of 5 mice injected with K10 cells.

8. Reaffirm the relevance of basic WNT signaling and KIAA1199 to adherent cells and tumor protoplasts

Capan-1 and HPAC cell lines were used to make adherent cells and protoplast cells, and the mRNA levels of Oct4, Nanog, Dvl2, Wnt3 and Wnt3a were confirmed. The primer sequences used for mRNA level identification were as follows: Wnt3 forward, 5'-GCG TGT TAG TGT CCA GGG AGT T-3 '; Wnt3 reverse direction, 5'-TGA GGT GCA TGT GGT CCA GGA T-3 '; Wnt3a forward, 5'-ATG AAC CGC CAC AAC AAC GAG G-3 '; Wnt3a forward, 5'-GTC CTT GAG GAA GTC ACC GAT G-3 '; Oct4 forward, 5'-CCT GAA GCA GAA GAG GAT CAC C-3 ', Oct4 reverse, 5'-AAA GCG GCA GAT GGT CGT TTG G-3'; Nanog Forward, 5'-CTC CAA CAT CCT GAA CCT CAG C-3 '; Nanog reverse, 5'-CGT CAC ACC ATT GCT ATT CTT CG-3 '; Dvl2 forward, 5'-TCC ATA CGG ACA TGG CAT CGG T-3 '; Dvl2 reverse, 5'-CGT GAT GGT AGA GCC AGT CAA C-3 '. Protein levels were confirmed using β-catenin and an antibody against Dvl2 (Santa Cruz) in both cell lines. As a result, the mRNA level and protein level of each cell line increased in the protoplast cells (Fig. 8A-b).

The interaction between β-catenin and KIAA1199 was confirmed by immunoprecipitation in the protoplast cells of both cell lines, and the transcriptional activity of β-catenin and KIAA1199 on OCT4 and Nanog genes was confirmed by chromatin immunoprecipitation assay 8c-d). In addition, using Upstate's EZ Chip Kit, it was confirmed that KIAA1199 also acts as a transcription factor for OCT4 and Nanog promoters, as well as β-catenin, which is known as OCT4 and Nanog transcription factor (FIG. These results suggest that KIAA1199 acts as a transcription factor for OCT4 and Nanog promoter in cell protoplasts compared to adherent cells.

9. Analysis of survival rate of KIAA1199 and pancreatic cancer patients using TMA (Tissue microarray) analysis

IHC was performed on 30 pancreatic cancer TMA tissues using an antibody specific for KIAA1199 (Sigma-Aldrich). As a result, 40% of cancer tissues were scarcely stained or weakly stained, and 60% of cancer tissues showed moderate or strong staining (Fig. 9). The average total survival time of patients with almost unstained or weakly stained tissues was 1,847 days, while the average survival time of patients with intermediate or strong staining was 226 days. This analysis was performed in the SPSS program on Windows version 18.0 and the survival curves were obtained by the Kaplan-Meier method and the p-value was 0.018. The results showed that the stronger the expression of KIAA1199, the lower the average survival time of patients.

10. Sensitivity test for anti-cancer drugs

MTT assays were performed by diluting each anticancer drug at one-tenth of the maximum concentration in the graph x axis, and the end point of survival rate was measured after 3 days of culture. Gemcitabine, Irinotecan, Etoposide, Carboplatin, which are used as pancreatic cancer chemotherapeutic agents in K10 cells expressing KIAA1199 more than in E6 cells expressing less KIAA1199, Lt; RTI ID = 0.0 &gt; IC50 &lt; / RTI &gt; On the other hand, the opposite result was obtained for Paclitaxel (Fig. 10f). In addition, the survival rate end point of gemcitabine was measured after treatment of E6 and K10 cells with scrambled siRNA or siKIAA1199, respectively, and the IC50 value of the KIAA1199 knockdown sample was decreased (FIG. 10g).

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> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> Compositions for Diagnosing or Treating Pancreatic Cancer Using          KIAA1199 <130> PN140319P <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1361 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE &Lt; 222 > (1) .. (1361) <223> KIAA1199 protein sequence <400> 1 Met Gly Ala Ala Gly Arg Gln Asp Phe Leu Phe Lys Ala Met Leu Thr   1 5 10 15 Ile Ser Trp Leu Thr Leu Thr Cys Phe Pro Gly Ala Thr Ser Thr Val              20 25 30 Ala Ala Gly Cys Pro Asp Gln Ser Pro Glu Leu Gln Pro Trp Asn Pro          35 40 45 Gly His Asp Gln Asp His His Val His Ile Gly Gln Gly Lys Thr Leu      50 55 60 Leu Leu Thr Ser Ser Ala Thr Val Ser Ser Ile Ser Ser Glu Gly  65 70 75 80 Gly Lys Leu Val Ile Lys Asp His Asp Glu Pro Ile Val Leu Arg Thr                  85 90 95 Arg His Ile Leu Ile Asp Asn Gly Gly Glu Leu His Ala Gly Ser Ala             100 105 110 Leu Cys Pro Phe Gln Gly Asn Phe Thr Ile Ile Leu Tyr Gly Arg Ala         115 120 125 Asp Glu Gly Ile Gln Pro Asp Pro Tyr Tyr Gly Leu Lys Tyr Ile Gly     130 135 140 Val Gly Lys Gly Gly Ala Leu Glu Leu His Gly Gln Lys Lys Leu Ser 145 150 155 160 Trp Thr Phe Leu Asn Lys Thr Leu His Pro Gly Gly Met Ala Glu Gly                 165 170 175 Gly Tyr Phe Phe Glu Arg Ser Trp Gly His Arg Gly Val Ile Val His             180 185 190 Val Ile Asp Pro Lys Ser Gly Thr Val Ile His Ser Asp Arg Phe Asp         195 200 205 Thr Tyr Arg Ser Lys Lys Glu Ser Glu Arg Leu Val Gln Tyr Leu Asn     210 215 220 Ala Val Pro Asp Gly Arg Ile Leu Ser Val Ala Val Asn Asp Glu Gly 225 230 235 240 Ser Arg Asn Leu Asp Asp Met Ala Arg Lys Ala Met Thr Lys Leu Gly                 245 250 255 Ser Lys His Phe Leu His Leu Gly Phe Arg His Pro Trp Ser Phe Leu             260 265 270 Thr Val Lys Gly Asn Pro Ser Ser Val Glu Asp His Ile Glu Tyr         275 280 285 His Gly His Arg Gly Ser Ala Ala Arg Val Phe Lys Leu Phe Gln     290 295 300 Thr Glu His Gly Glu Tyr Phe Asn Val Ser Leu Ser Ser Glu Trp Val 305 310 315 320 Gln Asp Val Glu Trp Thr Glu Trp Phe Asp His Asp Lys Val Ser Gln                 325 330 335 Thr Lys Gly Gly Glu Lys Ile Ser Asp Leu Trp Lys Ala His Pro Gly             340 345 350 Lys Ile Cys Asn Arg Pro Ile Asp Ile Gln Ala Thr Thr Met Asp Gly         355 360 365 Val Asn Leu Ser Thr Glu Val Val Tyr Lys Lys Gly Gln Asp Tyr Arg     370 375 380 Phe Ala Cys Tyr Asp Arg Gly Arg Ala Cys Arg Ser Tyr Arg Val Arg 385 390 395 400 Phe Leu Cys Gly Lys Pro Val Arg Pro Lys Leu Thr Val Thr Ile Asp                 405 410 415 Thr Asn Val Asn Ser Thr Ile Leu Asn Leu Glu Asp Asn Val Gln Ser             420 425 430 Trp Lys Pro Gly Asp Thr Leu Val Ile Ala Ser Thr Asp Tyr Ser Met         435 440 445 Tyr Gln Ala Glu Glu Phe Gln Val Leu Pro Cys Arg Ser Cys Ala Pro     450 455 460 Asn Gln Val Lys Val Ala Gly Lys Pro Met Tyr Leu His Ile Gly Glu 465 470 475 480 Glu Ile Asp Gly Val Asp Met Arg Ala Glu Val Gly Leu Leu Ser Arg                 485 490 495 Asn Ile Val Met Gly Glu Met Glu Asp Lys Cys Tyr Pro Tyr Arg             500 505 510 Asn His Ile Cys Asn Phe Phe Asp Phe Asp Thr Phe Gly Gly His Ile         515 520 525 Lys Phe Ala Leu Gly Phe Lys Ala Ala His Leu Glu Gly Thr Glu Leu     530 535 540 Lys His Met Gly Gln Gln Leu Val Gly Gln Tyr Pro Ile His Phe His 545 550 555 560 Leu Ala Gly Asp Val Asp Glu Arg Gly Gly Tyr Asp Pro Pro Thr Tyr                 565 570 575 Ile Arg Asp Leu Ser Ile His His Thr Phe Ser Arg Cys Val Thr Val             580 585 590 His Gly Ser Asn Gly Leu Leu Ile Lys Asp Val Val Gly Tyr Asn Ser         595 600 605 Leu Gly His Cys Phe Phe Thr Glu Asp Gly Pro Glu Glu Arg Asn Thr     610 615 620 Phe Asp His Cys Leu Gly Leu Leu Val Lys Ser Gly Thr Leu Leu Pro 625 630 635 640 Ser Asp Arg Asp Ser Lys Met Cys Lys Met Ile Thr Glu Asp Ser Tyr                 645 650 655 Pro Gly Tyr Ile Pro Lys Pro Arg Gln Asp Cys Asn Ala Val Ser Thr             660 665 670 Phe Trp Met Ala Asn Pro Asn Asn Asn Leu Ile Asn Cys Ala Ala Ala         675 680 685 Gly Ser Glu Glu Thr Gly Phe Trp Phe Ile Phe His His Val Thr     690 695 700 Gly Pro Ser Val Gly Met Tyr Ser Pro Gly Tyr Ser Glu His Ile Pro 705 710 715 720 Leu Gly Lys Phe Tyr Asn Asn Arg Ala His Ser Asn Tyr Arg Ala Gly                 725 730 735 Met Ile Ile Asp Asn Gly Val Lys Thr Thr Glu Ala Ser Ala Lys Asp             740 745 750 Lys Arg Pro Phe Leu Ser Ile Ile Ser Ala Arg Tyr Ser Pro His Gln         755 760 765 Asp Ala Asp Pro Leu Lys Pro Arg Glu Pro Ala Ile Ile Arg His Phe     770 775 780 Ile Ala Tyr Lys Asn Gln Asp His Gly Ala Trp Leu Arg Gly Gly Asp 785 790 795 800 Val Trp Leu Asp Ser Cys Arg Phe Ala Asp Asn Gly Ile Gly Leu Thr                 805 810 815 Leu Ala Ser Gly Gly Thr Phe Pro Tyr Asp Asp Gly Ser Lys Gln Glu             820 825 830 Ile Lys Asn Ser Leu Phe Val Gly Glu Ser Gly Asn Val Gly Thr Glu         835 840 845 Met Met Asp Asn Arg Ile Trp Gly Pro Gly Gly Leu Asp His Ser Gly     850 855 860 Arg Thr Leu Pro Ile Gly Gln Asn Phe Pro Ile Arg Gly Ile Gln Leu 865 870 875 880 Tyr Asp Gly Pro Ile Asn Ile Gln Asn Cys Thr Phe Arg Lys Phe Val                 885 890 895 Ala Leu Glu Gly Arg His Thr Ser Ala Leu Ala Phe Arg Leu Asn Asn             900 905 910 Ala Trp Gln Ser Cys Pro His Asn Asn Val Thr Gly Ile Ala Phe Glu         915 920 925 Asp Val Pro Ile Thr Ser Arg Val Phe Phe Gly Glu Pro Gly Pro Trp     930 935 940 Phe Asn Gln Leu Asp Met Asp Gly Asp Lys Thr Ser Val Phe His Asp 945 950 955 960 Val Asp Gly Ser Val Ser Glu Tyr Pro Gly Ser Tyr Leu Thr Lys Asn                 965 970 975 Asp Asn Trp Leu Val Arg His Pro Asp Cys Ile Asn Val Pro Asp Trp             980 985 990 Arg Gly Ala Ile Cys Ser Gly Cys Tyr Ala Gln Met Tyr Ile Gln Ala         995 1000 1005 Tyr Lys Thr Ser Asn Leu Arg Met Lys Ile Ile Lys Asn Asp Phe Pro    1010 1015 1020 Ser His Pro Leu Tyr Leu Glu Gly Ala Leu Thr Arg Ser Thr His Tyr 1025 1030 1035 1040 Gln Gln Tyr Gln Pro Val Val Thr Leu Gln Lys Gly Tyr Thr Ile His                1045 1050 1055 Trp Asp Gln Thr Ala Pro Ala Glu Leu Ala Ile Trp Leu Ile Asn Phe            1060 1065 1070 Asn Lys Gly Asp Trp Ile Arg Val Gly Leu Cys Tyr Pro Arg Gly Thr        1075 1080 1085 Thr Phe Ser Ile Leu Ser Asp Val His Asn Arg Leu Leu Lys Gln Thr    1090 1095 1100 Ser Lys Thr Gly Val Phe Val Arg Thr Leu Gln Met Asp Lys Val Glu 1105 1110 1115 1120 Gln Ser Tyr Pro Gly Arg Ser His Tyr Tyr Trp Asp Glu Asp Ser Gly                1125 1130 1135 Leu Leu Phe Leu Lys Leu Lys Ala Gln Asn Glu Arg Glu Lys Phe Ala            1140 1145 1150 Phe Cys Ser Met Lys Gly Cys Glu Arg Ile Lys Ile Lys Ala Leu Ile        1155 1160 1165 Pro Lys Asn Ala Gly Val Ser Asp Cys Thr Ala Thr Ala Tyr Pro Lys    1170 1175 1180 Phe Thr Glu Arg Ala Val Val Asp Val Pro Met Pro Lys Lys Leu Phe 1185 1190 1195 1200 Gly Ser Gln Leu Lys Thr Lys Asp His Phe Leu Glu Val Lys Met Glu                1205 1210 1215 Ser Ser Lys Gln His Phe Phe His Leu Trp Asn Asp Phe Ala Tyr Ile            1220 1225 1230 Glu Val Asp Gly Lys Lys Tyr Pro Ser Ser Glu Asp Gly Ile Gln Val        1235 1240 1245 Val Val Ile Asp Gly Asn Gln Gly Arg Val Val Ser His Thr Ser Phe    1250 1255 1260 Arg Asn Ser Ile Leu Gln Gly Ile Pro Trp Gln Leu Phe Asn Tyr Val 1265 1270 1275 1280 Ala Thr Ile Pro Asp Asn Ser Ile Val Leu Met Ala Ser Lys Gly Arg                1285 1290 1295 Tyr Val Ser Arg Gly Pro Trp Thr Arg Val Leu Glu Lys Leu Gly Ala            1300 1305 1310 Asp Arg Gly Leu Lys Leu Lys Glu Gln Met Ala Phe Val Gly Phe Lys        1315 1320 1325 Gly Ser Phe Arg Pro Ile Trp Val Thr Leu Asp Thr Glu Asp His Lys    1330 1335 1340 Ala Lys Ile Phe Gln Val Val Pro Ile Pro Val Val Lys Lys Lys Lys 1345 1350 1355 1360 Leu     <210> 2 <211> 7080 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1). (7080) <223> KIAA1199 DNA sequence <400> 2 gagctagcgc tcaagcagag cccagcgcgg tgctatcgga cagagcctgg cgagcgcaag 60 cggcgcgggg agccagcggg gctgagcgcg gccagggtct gaacccagat ttcccagact 120 agctaccact ccgcttgccc acgccccggg agctcgcggc gcctggcggt cagcgaccag 180 acgtccgggg ccgctgcgct cctggcccgc gaggcgtgac actgtctcgg ctacagaccc 240 agagggagca cactgccagg atgggagctg ctgggaggca ggacttcctc ttcaaggcca 300 tgctgaccat cagctggctc actctgacct gcttccctgg ggccacatcc acagtggctg 360 ctgggtgccc tgaccagagc cctgagttgc aaccctggaa ccctggccat gaccaagacc 420 accatgtgca tatcggccag ggcaagacac tgctgctcac ctcttctgcc acggtctatt 480 ccatccacat ctcagaggga ggcaagctgg tcattaaaga ccacgacgag ccgattgttt 540 tgcgaacccg gcacatcctg attgacaacg gaggagagct gcatgctggg agtgccctct 600 gccctttcca gggcaatttc accatcattt tgtatggaag ggctgatgaa ggtattcagc 660 cggatcctta ctatggtctg aagtacattg gggttggtaa aggaggcgct cttgagttgc 720 atggacagaa aaagctctcc tggacatttc tgaacaagac ccttcaccca ggtggcatgg 780 cagaaggagg ctattttttt gaaaggagct ggggccaccg tggagttatt gttcatgtca 840 tcgaccccaa atcaggcaca gtcatccatt ctgaccggtt tgacacctat agatccaaga 900 aagagagtga acgtctggtc cagtatttga acgcggtgcc cgatggcagg atcctttctg 960 ttgcagtgaa tgatgaaggt tctcgaaatc tggatgacat ggccaggaag gcgatgacca 1020 aattgggaag caaacacttc ctgcaccttg gatttagaca cccttggagt tttctaactg 1080 tgaaaggaaa tccatcatct tcagtggaag accatattga atatcatgga catcgaggct 1140 ctgctgctgc ccgggtattc aaattgttcc agacagagca tggcgaatat ttcaatgttt 1200 ctttgtccag tgagtgggtt caagacgtgg agtggacgga gtggttcgat catgataaag 1260 tatctcagac taaaggtggg gagaaaattt cagacctctg gaaagctcac ccaggaaaaa 1320 tatgcaatcg tcccattgat atacaggcca ctacaatgga tggagttaac ctcagcaccg 1380 aggttgtcta caaaaaaggc caggattata ggtttgcttg ctacgaccgg ggcagagcct 1440 gccggagcta ccgtgtacgg ttcctctgtg ggaagcctgt gaggcccaaa ctcacagtca 1500 ccattgacac caatgtgaac agcaccattc tgaacttgga ggataatgta cagtcatgga 1560 aacctggaga taccctggtc attgccagta ctgattactc catgtaccag gcagaagagt 1620 tccaggtgct tccctgcaga tcctgcgccc ccaaccaggt caaagtggca gggaaaccaa 1680 tgtacctgca catcggggag gagatagacg gcgtggacat gcgggcggag gttgggcttc 1740 tgagccggaa catcatagtg atgggggaga tggaggacaa atgctacccc tacagaaacc 1800 acatctgcaa tttctttgac ttcgatacct ttgggggcca catcaagttt gctctgggat 1860 ttaaggcagc acacttggag ggcacggagc tgaagcatat gggacagcag ctggtgggtc 1920 agtacccgat tcacttccac ctggccggtg atgtagacga aaggggaggt tatgacccac 1980 ccacatacat cagggacctc tccatccatc atacattctc tcgctgcgtc acagtccatg 2040 gctccaatgg cttgttgatc aaggacgttg tgggctataa ctctttgggc cactgcttct 2100 tcacggaaga tgggccggag gaacgcaaca cttttgacca ctgtcttggc ctccttgtca 2160 agtctggaac cctcctcccc tcggaccgtg acagcaagat gtgcaagatg atcacagagg 2220 actcctaccc ggggtacatc cccaagccca ggcaagactg caatgctgtg tccaccttct 2280 ggatggccaa tcccaacaac aacctcatca actgtgccgc tgcaggatct gaggaaactg 2340 gattttggtt tatttttcac cacgtaccaa cgggcccctc cgtgggaatg tactccccag 2400 gttattcaga gcacattcca ctgggaaaat tctataacaa ccgagcacat tccaactacc 2460 gggctggcat gatcatagac aacggagtca aaaccaccga ggcctctgcc aaggacaagc 2520 ggccgttcct ctcaatcatc tctgccagat acagccctca ccaggacgcc gacccgctga 2580 agccccggga gccggccatc atcagacact tcattgccta caagaaccag gaccacgggg 2640 cctggctgcg cggcggggat gtgtggctgg acagctgccg gtttgctgac aatggcattg 2700 gcctgaccct ggccagtggt ggaaccttcc cgtatgacga cggctccaag caagagataa 2760 agaacagctt gtttgttggc gagagtggca acgtggggac ggaaatgatg gacaatagga 2820 tctggggccc tggcggcttg gaccatagcg gaaggaccct ccctataggc cagaattttc 2880 caattagagg aattcagtta tatgatggcc ccatcaacat ccaaaactgc actttccgaa 2940 agtttgtggc cctggagggc cggcacacca gcgccctggc cttccgcctg aataatgcct 3000 ggcagagctg cccccataac aacgtgaccg gcattgcctt tgaggacgtt ccgattactt 3060 ccagagtgtt cttcggagag cctgggccct ggttcaacca gctggacatg gatggggata 3120 agacatctgt gttccatgac gtcgacggct ccgtgtccga gtaccctggc tcctacctca 3180 cgaagaatga caactggctg gtccggcacc cagactgcat caatgttccc gactggagag 3240 gggccatttg cagtgggtgc tatgcacaga tgtacattca agcctacaag accagtaacc 3300 tgcgaatgaa gatcatcaag aatgacttcc ccagccaccc tctttacctg gagggggcgc 3360 tcaccaggag cacccattac cagcaatacc aaccggttgt caccctgcag aagggctaca 3420 ccatccactg ggaccagacg gcccccgccg aactcgccat ctggctcatc aacttcaaca 3480 agggcgactg gatccgagtg gggctctgct acccgcgagg caccacattc tccatcctct 3540 cggatgttca caatcgcctg ctgaagcaaa cgtccaagac gggcgtcttc gtgaggacct 3600 tgcagatgga caaagtggag cagagctacc ctggcaggag ccactactac tgggacgagg 3660 actcagggct gttgttcctg aagctgaaag ctcagaacga gagagagaag tttgctttct 3720 gctccatgaa aggctgtgag aggataaaga ttaaagctct gattccaaag aacgcaggcg 3780 tcagtgactg cacagccaca gcttacccca agttcaccga gagggctgtc gtagacgtgc 3840 cgatgcccaa gaagctcttt ggttctcagc tgaaaacaaa ggaccatttc ttggaggtga 3900 agatggagag ttccaagcag cacttcttcc acctctggaa cgacttcgct tacattgaag 3960 tggatgggaa gaagtacccc agttcggagg atggcatcca ggtggtggtg attgacggga 4020 accaagggcg cgtggtgagc cacacgagct tcaggaactc cattctgcaa ggcataccat 4080 ggcagctttt caactatgtg gcgaccatcc ctgacaattc catagtgctt atggcatcaa 4140 agggaagata cgtctccaga ggcccatgga ccagagtgct ggaaaagctt ggggcagaca 4200 ggggtctcaa gttgaaagag caaatggcat tcgttggctt caaaggcagc ttccggccca 4260 tctgggtgac actggacact gaggatcaca aagccaaaat cttccaagtt gtgcccatcc 4320 ctgtggtgaa gaagaagaag ttgtgaggac agctgccgcc cggtgccacc tcgtggtaga 4380 ctatgacggt gactcttggc agcagaccag tgggggatgg ctgggtcccc cagcccctgc 4440 cagcagctgc ctgggaaggc cgtgtttcag ccctgatggg ccaagggaag gctatcagag 4500 accctggtgc tgccacctgc ccctactcaa gtgtctacct ggagcccctg gggcggtgct 4560 ggccaatgct ggaaacattc actttcctgc agcctcttgg gtgcttctct cctatctgtg 4620 cctcttcagt gggggtttgg ggaccatatc aggagacctg ggttgtgctg acagcaaaga 4680 tccactttgg caggagccct gacccagcta ggaggtagtc tggagggctg gtcattcaca 4740 gatccccatg gtcttcagca gacaagtgag ggtggtaaat gtaggagaaa gagccttggc 4800 cttaaggaaa tctttactcc tgtaagcaag agccaacctc acaggattag gagctggggt 4860 agaactggct atccttgggg aagaggcaag ccctgcctct ggccgtgtcc acctttcagg 4920 agactttgag tggcaggttt ggacttggac tagatgactc tcaaaggccc ttttagttct 4980 gagattccag aaatctgctg catttcacat ggtacctgga acccaacagt tcatggatat 5040 ccactgatat ccatgatgct gggtgcccca gcgcacacgg gatggagagg tgagaactaa 5100 tgcctagctt gaggggtctg cagtccagta gggcaggcag tcaggtccat gtgcactgca 5160 atgccaggtg gagaaatcac agagaggtaa aatggaggcc agtgccattt cagaggggag 5220 gctcaggaag gcttcttgct tacaggaatg aaggctgggg gcattttgct ggggggagat 5280 gaggcagcct ctggaatggc tcagggattc agccctccct gccgctgcct gctgaagctg 5340 gtgactacgg ggtcgccctt tgctcacgtc tctctggccc actcatgatg gagaagtgtg 5400 gtcagagggg agcaatgggc tttgctgctt atgagcacag aggaattcag tccccaggca 5460 gccctgcctc tgactccaag agggtgaagt ccacagaagt gagctcctgc cttagggcct 5520 catttgctct tcatccaggg aactgagcac agggggcctc caggagaccc tagatgtgct 5580 cgtactccct cggcctggga tttcagagct ggaaatatag aaaatatcta gcccaaagcc 5640 ttcattttaa cagatgggga aagtgagccc ccaagatggg aaagaaccac acagctaagg 5700 gagggcctgg ggagccccac cctagccctt gctgccacac cacattgcct caacaaccgg 5760 ccccagagtg cccaggcact cctgaggtag cttctggaaa tggggacaag tcccctcgaa 5820 ggaaaggaaa tgactagagt agaatgacag ctagcagatc tcttccctcc tgctcccagc 5880 gcacacaaac ccgccctccc cttggtgttg gcggtccctg tggccttcac tttgttcact 5940 acctgtcagc ccagcctggg tgcacagtag ctgcaactcc ccattggtgc tacctggctc 6000 tcctgtctct gcagctctac aggtgaggcc cagcagaggg agtagggctc gccatgtttc 6060 tggtgagcca atttggctga tcttgggtgt ctgaacagct attgggtcca ccccagtccc 6120 tttcagctgc tgcttaatgc cctgctctct ccctggccca ccttatagag agcccaaaga 6180 gctcctgtaa gagggagaac tctatctgtg gtttataatc ttgcacgagg caccagagtc 6240 tccctgggtc ttgtgatgaa ctacatttat cccctttcct gccccaacca caaactcttt 6300 ccttcaaaga gggcctgcct ggctccctcc acccaactgc acccatgaga ctcggtccaa 6360 gagtccattc cccaggtggg agccaactgt cagggaggtc tttcccacca aacatctttc 6420 agctgctggg aggtgaccat agggctctgc ttttaaagat atggctgctt caaaggccag 6480 agtcacagga aggacttctt ccagggagat tagtggtgat ggagaggaga gttaaaatga 6540 cctcatgtcc ttcttgtcca cggttttgtt gagttttcac tcttctaatg caagggtctc 6600 acactgtgaa ccacttagga tgtgatcact ttcaggtggc caggaatgtt gaatgtcttt 6660 ggctcagttc atttaaaaaa gatatctatt tgaaagttct cagagttgta catatgtttc 6720 acagtacagg atctgtacat aaaagtttct ttcctaaacc attcaccaag agccaatatc 6780 taggcatttt cttggtagca caaattttct tattgcttag aaaattgtcc tccttgttat 6840 ttctgtttgt aagacttaag tgagttaggt ctttaaggaa agcaacgctc ctctgaaatg 6900 cttgtctttt ttctgttgcc gaaatagctg gtcctttttc gggagttaga tgtatagagt 6960 gtttgtatgt aaacatttct tgtaggcatc accatgaaca aagatatatt ttctatttat 7020 ttattatatg tgcacttcaa gaagtcactg tcagagaaat aaagaattgt cttaaatgtc 7080                                                                         7080 <210> 3 <211> 25 <212> RNA <213> Homo sapiens <220> <221> misc_RNA <222> (1) <223> KIAA1199 siRNA # 1 <400> 3 accacugucu uggccuccuu gucaa 25 <210> 4 <211> 25 <212> RNA <213> Homo sapiens <220> <221> misc_RNA <222> (1) <223> KIAA1199 siRNA # 2 <400> 4 cagggctgtt gttcctgaag ctgaa 25

Claims (9)

A method of screening for a substance for the prevention or treatment of pancreatic cancer comprising the steps of:
(a) contacting a cell or tissue comprising the polynucleotide of the first sequence or the polynucleotide of the second sequence of the sequence listing with a sample to be analyzed; And
(b) measuring the expression level of the protein or polynucleotide in step (a), wherein when the sample reduces the expression of the protein or polynucleotide, the sample is a substance for preventing or treating pancreatic cancer .
A method of screening for a substance for the prevention or treatment of pancreatic cancer comprising the steps of:
(a) preparing a protein of the first sequence;
(b) contacting the test substance with the protein; And
(c) analyzing whether the test substance binds to the protein or whether the test substance inhibits the function of the protein; When the test substance binds to the protein or inhibits the function of the protein, it is judged to be a substance for preventing or treating pancreatic cancer.
3. The method of claim 2, wherein the protein is present on the cell surface or on the virus surface, or in isolated form.
A kit for detecting pancreatic cancer stem cells comprising a binding agent that specifically binds to a protein of the first sequence or a primer or probe that binds to a polynucleotide of the second sequence.
delete The kit according to claim 4, wherein the kit is a kit for immunoassay, gene amplification or microarray.
delete A composition for inhibiting the formation of pancreatic cancer stem cells comprising an antibody specifically binding to the protein of the first sequence of the sequence listing or a nucleic acid molecule for suppressing the expression of the polynucleotide of the second sequence as an active ingredient.
An antibody that specifically binds to the protein of the first sequence of the sequence listing, or a nucleic acid molecule for suppressing the expression of the polynucleotide of the second sequence, as an active ingredient, for the treatment or prevention of metastasis of pancreatic cancer.

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