KR20130010831A - Novel biomarkers for biliary tract cancer based on biliary tract cancer stem cell characteristics and uses thereof - Google Patents

Abstract

PURPOSE: A method for screening a material which prevents or treats biliary tract cancer using a novel molecular marker is provided to screen a therapeutic agent candidate which is specific to biliary tract cancer stem cells or cancer stem cells. CONSTITUTION: A method for screening a material which prevents or treats biliary cancer comprises: a step of contacting a sample with a cell or a tissue containing a protein of sequence number 1 or 3 or a polynucleotide of sequence number 2 or 4; and a step of measuring expression level of the protein or polynucleotide. The protein exists on the cell surface, viral surface, or in an isolated form. A kit for detecting biliary cancer stem cells contains a primer or a probe which specifically binds to the protein or polynucleotide. [Reference numerals] (AA) Expressing TAGLN2 at normal biliary epithalial tissue and biliary cancer tissue

Description

Novel Biomarkers for Biliary tract Cancer Based on Biliary tract Cancer Stem Cell Characteristics and Uses Technical}

The present invention relates to novel biomarkers and their use for biliary tract cancer based on biliary tract cancer stem cell properties.

Gastrointestinal cancers account for more than 55% of cancers in the human body, and gastric cancer, liver cancer, and colorectal cancer are mostly cancers. However, biliary tract cancer is an increasing number of patients worldwide. In the US, 5,000 new cases of biliary tract cancer occur annually. Gallbladder and biliary tract cancer account for 3.6% of all cancers in Korea.

The prognosis of biliary tract cancer is so bad that the prevalence and mortality rate is similar, and there is no treatment yet established. Curative resection is the only treatment that can be expected to be cured. Because metastasis is common, 5-year survival is less than 5%. In addition, when it is found that there is no special early diagnosis, the disease is already advanced and treatment is often delayed.

Cancer cells also contain cancer stem cells that maintain and regenerate cancerous tissues like normal organs, and these cells form spheres in suspension culture in vitro , and the cells obtained by spear cultures are more potent than the original cell lines. It has been reported to have the ability to form (A. Dubrovska et al. Proc Natl Acad Sci USA .: 106, 268-273 (2009)). In addition, cancer stem cells are involved not only in the development of cancer but also in the regeneration of cancer cells reduced after the conventional cancer treatment is applied, thereby inducing cancer recurrence or metastasis as well as resistance to anticancer drugs. (BB Zhou et al. Nature Reviews Drug Discovery : 8, 806-823 (2009).

At present, in advanced countries, breast cancer (M. Al-Hajj et al., Proc Natl Acad Sci USA USA : 100 (7), 3983-3988 (2003)), liver cancer (ZF Yang et al., Cancer cell ; 13, 153-166 (2008)), colorectal cancer (P. Dalerba et al., Proc Natl Acad Sci . USA : 104 (24), 10158-63 (2007)), pancreatic cancer (C. Li et al. Cancer Res : 67 (3) 1030-1037 (2007)), since some information on cancer stem cells is already secured, cancer stem cells of organs and tissues can be differentiated from those of advanced countries. There is a need for research.

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 invention has made extensive research efforts to discover novel biomarkers and targets for the treatment of biliary tract cancer based on the characteristics of biliary tract cancer stem cells. As a result, the present inventors found that proteins specifically expressed in biliary tract cancer stem cells may be molecular targets for biliary tract cancer treatment. In addition, the present inventors have found that the biomarkers that have been discovered can be diagnosed, especially early diagnosis of biliary tract cancer based on the biological characteristics of biliary cancer cancer stem cells, markers for determining the prognosis, and markers for future treatment targets. Thus, the present invention has been completed.

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

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

Still another object of the present invention is to provide a kit for detecting bile cancer cancer stem cell specific biomarkers.

Another object of the present invention to provide a kit for detecting cancer stem cell specific biomarkers.

Another object of the present invention to provide a kit for diagnosing or prognostic analysis of biliary tract cancer.

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

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

According to one aspect of the present invention, the present invention provides a method for screening a material for preventing or treating biliary tract cancer, comprising the following steps:

(a) a protein selected from the group consisting of the protein of the first sequence and the protein of the third sequence, or a polynucleotide of the second sequence and a polynucleotide of the fourth sequence Contacting a cell or tissue comprising the nucleotide 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 bile duct cancer Is determined.

The present inventors made extensive efforts to discover novel biomarkers for biliary cancer and therapeutic targets for biliary tract cancer based on the characteristics of biliary cancer cancer stem cells, and as a result, proteins specifically expressed in biliary cancer cancer stem cells were biliary tract. It has been found to be a molecular target for cancer treatment. In addition, the inventors have identified that the biomarkers that have been excavated are markers capable of diagnosing biliary tract cancer, especially early diagnosis and determining prognosis.

The present invention identifies proteins expressing specifically from biliary cancer cancer stem cells isolated from biliary cancer cell lines, and they can be targets for biliary tract cancer treatment. Has

The two markers of the present invention are specifically expressed in biliary cancer cancer stem cells. Moreover, the two markers are markers for the ability to distinguish between biliary cancer cells and biliary cancer stem cells, that is, expression patterns that are highly expressed in biliary cancer cancer stem cells as compared to biliary cancer cells.

In the present invention, the expression "cell or tissue" used for screening a substance for preventing or treating biliary tract cancer is a cancer stem cell or a cancer tissue, and preferably a biliary cancer cancer stem cell or a biliary tract cancer tissue.

The present inventors have found that, in the case of proteins selected from the group consisting of the above proteins or respective polynucleotides encoding them, their expression is significantly increased in biliary tract cancer stem cells, unlike normal biliary tract cancer cells. Unlike general bile duct cancer cells, the specific remarkable increase in the expression of target proteins or polynucleotides encoding them only in biliary cancer cancer stem cells indicates that it is an essential factor for the survival of cancer stem cells and blocks their expression. Substances to be induced growth inhibition and death of cancer stem cells, it is determined to be a substance that helps in the treatment of biliary tract cancer, that is, biliary tract cancer.

The sample used in step (a) is a single compound or a mixture of compounds (eg a natural extract or a cell or tissue culture). Test substances can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc . Natl . Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci . USA 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, there is provided a method for screening a substance for preventing or treating biliary tract cancer, comprising the following steps:

(a) preparing a protein selected from the group consisting of the protein of the first sequence and the protein of the third sequence of the sequence listing;

(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 determined as a substance for preventing or treating biliary tract cancer.

According to the screening method of the present invention, the test substance is brought into contact with a protein selected from the group consisting of the protein of the first sequence of the sequence listing and the protein of the third sequence of the sequence listing.

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

In the case of using a protein exhibited on the cell surface or on the virus surface, it is preferable to immobilize the cell or virus on a solid substrate in order to expedite or automate screening. It is also desirable to immobilize the isolated or purified form of the protein onto a solid substrate. Available as substrates are any conventionally used in the art, including but not limited to hydrocarbon polymers such as polystyrene and polypropylene, glass, metals and gels. Solid phase substrates may be provided in the form of dipsticks, microtiter plates, particles (eg beads), affinity columns and immunoblot membranes (eg polyvinylidene fluoride membranes). See US Pat. No. 5,143,825. 5,374,530, 4,908,305 and 5,498,551). Most preferably, the solid substrate is a microtiter plate.

The screening methods of the present invention can be carried out in a variety of ways, in particular 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 proteins 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 S ³⁵ ), fluorescent labels such as coumarin, fluorescein isothiocyanate, rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Cy 3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl and FAM], luminescent, chemiluminescent, FRET transfer labels or metal labels (e.g., gold and silver).

In the case of using a protein or test substance labeled with a detectable label, the binding between the protein and the test substance can be analyzed by detecting a signal from the label. For example, when alkaline phosphatase is used as a label, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS-B1-phosphate) Signal is detected using a chromogenic reaction substrate such as) and enhanced chemifluorescence (ECF). When hose radish peroxidase is used as a label, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and substrates such as naphthol / pyronin to detect the signal.

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 the 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. The change in acidification rate can be used as an indicator for binding between test substance and QP-C (McConnell et al., Science 257: 19061912 (1992)).

The ability of the test substance to bind to proteins can be analyzed using real-time bimolecular interaction analysis (BIA) (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 analysis or a three-hybrid analysis method (US Pat. No. 5,283,317; Zervos et al., Cell 72, 223232, 1993; Madura et al., J. Biol . Chem . 268, 1204612054, 1993; 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, it is possible to screen substances, particularly proteins, which bind to the proteins. 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 selected from the group consisting of the polynucleotide of the second sequence of the sequence listing and the polynucleotide of the fourth sequence of the sequence listing may be introduced into the DNA binding domain of a known transcription factor (e.g., GAL-4) -Coding polynucleotide. ≪ / RTI > 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 in in vivo to form a complex, the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of the reporter gene (eg, LacZ). Expression of the reporter gene can be detected, which indicates that the protein of analysis can bind to the protein, and consequently, it can be used as a material for preventing or treating biliary tract cancer.

The sample used in the present invention is a peptide, antibody, peptide aptamer, AdNectin, affibody (US Pat. No. 5,831,012), Avimer (Avimer, Silverman, J. et al, Nature Biotechnology 23 (12): 1556 (2005)) or 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 invention, the present invention provides a method of screening for a substance for the prophylaxis or treatment of cancer comprising the steps of:

(a) a protein selected from the group consisting of the protein of the first sequence and the protein of the third sequence, or a polynucleotide of the second sequence and a polynucleotide of the fourth sequence Contacting a cell or tissue comprising the nucleotide with a sample to be analyzed; And

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

In the present invention, the expression cells or tissues used for the screening of a substance for preventing or treating cancer are cancer stem cells or cancer tissues, and preferably, biliary tract cancer stem cells or biliary cancer tissues.

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

(a) preparing a protein selected from the group consisting of the protein of the first sequence and the protein of the third sequence of the sequence listing;

(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 determined as a substance for preventing or treating cancer.

The cancer treatment agent identified by the screening method of the present invention does not target only general cancer cells, which occupy most of the cancer tissues, but occupies only a small portion of the cancer tissues, but plays a key role in the development, maintenance, and recurrence of cancer. Stem cells are targeted, allowing for the fundamental treatment of cancer.

According to another aspect of the present invention, the present invention provides a binding agent or a polynucleotide of SEQ ID NO: 2 sequence specifically binding to a protein selected from the group consisting of the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 3 and Provided is a kit for detecting a biliary tract cancer stem cell comprising a primer or a probe specifically binding to a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NO: 4.

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

According to another aspect of the present invention, the present invention provides a binding agent or a polynucleotide of SEQ ID NO: 2 sequence specifically binding to a protein selected from the group consisting of the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 3 and Provided is a kit for diagnosing or prognostic biliary cancer comprising a primer or a probe specifically binding to a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NO: 4.

In particular, the kit for diagnosing or prognostic biliary cancer of the present invention is a kit for diagnosing or prognostic biliary cancer derived from biliary cancer cancer stem cells.

The expression “kites for diagnosing or prognostic analysis of bile duct cancer” means a kit including a composition for diagnosing or prognostic analysis of biliary tract cancer. Therefore, the expression "kites for diagnosing or prognostic analysis of biliary tract cancer" may be used interchangeably or mixed with "compositions for diagnosing or prognostic analysis of biliary tract cancer".

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or as long as a person has a particular disease or condition. Determining the prognosis of the subject (eg, identifying a metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (eg, for treatment efficacy Monitoring the state of an object to provide information).

In the present invention, the term "biliary duct cancer" refers to a mass composed of cancer cells generated in the biliary tract. Biliary cancer is less common than other cancers, but it is difficult to diagnose early and has a good prognosis because of its spread to surrounding organs and lymph nodes. Biliary cancer accounts for 3% of all gastrointestinal cancers, and is rare in the West, but East Asia, including Korea, is a popular area of biliary tract cancer. In terms of types of biliary tract cancers, extrahepatic biliary tract cancers are declining worldwide, but hepatic biliary tract cancers are increasing. Biliary cancer develops in 50-70 years of age and is more common in men. In addition, most tumors occurring in the biliary tract are malignant and are often diagnosed due to jaundice due to biliary obstruction. The cause of the disease is still unclear, but it is reported that carcinogenic substances in bile are caused by stimulation of the biliary tract.

The present inventors confirmed that using the marker of the present invention, a highly sensitive and reliable result for the development of biliary tract cancer from an individual can be obtained.

The expression "protein expression analysis" used in diagnosing biliary tract cancer in the present invention is a process of confirming the presence and degree of expression of a protein expressed from the genes in a biological sample, preferably, specific for the protein. By means of the antibody to bind to means that the amount of protein. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket. Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc. The method of analysis of the invention is not limited.

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

"Antibody" in the present invention means a specific protein molecule directed against an antigenic site. For the 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 new biliary tract cancer marker proteins have been identified as described above, the production of antibodies using them can be readily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the biliary cancer marker protein antigens described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. 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 are well known in the art by the hybridoma method (Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature , 352: 624-628, 1991; Marks et al, J. Mol . Biol . , 222: 58, 1-597, 1991) It can be prepared using. Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

The dimmer aepta oligo nucleic acid or peptide molecules that bind to the active form of the enzyme in the present invention, general information of the aptamer is 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 provides oligopeptides, monoclonal antibodies, which specifically bind to at least one protein selected from the above protein group for diagnosing biliary tract cancer and / or biliary tract cancer stem cells. Polyclonal antibodies, chimeric antibodies, ligands, PNA (Peptide nucleic acid) or aptamers, more preferably oligopeptides, monoclonal antibodies, polyclonal antibodies or chimeric antibodies Still more preferably monoclonal antibodies or polyclonal antibodies, most preferably monoclonal antibodies.

In the present invention, the antibody is preferably a conjugated antibody (micro particle) (conjugated antibody). In addition, the microparticles 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 20 markers described above, but preferably, the kit is immunoassay. ), Most preferably the kit is a Luminex assay kit, a protein microarray kit or an ELISA kit.

The LUMINEX assay kit, protein microarray kit, and ELISA kit are polyclonal and monoclonal antibodies against any one or more proteins selected from the above protein group, and the polyclonal antibody and monoclonal antibody to which a label is bound. Secondary antibodies against.

Examples of the types of kits in the present invention include immunochromatography strip kits, luminex assay kits, protein microarray kits, eliza kits, or immunological dot kits. Kind is not limited.

The kit may further include the necessary elements necessary to perform the ELISA. ELISA kits contain antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. 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 the necessary elements necessary for performing protein microarrays to simultaneously analyze the complex markers. The microarray kit includes antibodies specific for the marker protein bound to the solid phase. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The protein microarray kit can also include antibodies specific for the control protein. Other protein microarray kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (such as fused with antibodies), and substrates thereof or other materials that can bind to antibodies. And the like. In a method of analyzing a sample using a protein microarray, the protein is separated from the sample, and the separated protein is hybridized with a protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein. In addition, it can provide information necessary for diagnosing biliary tract cancer.

Luminex Assay is a high-throughput quantitative method that can simultaneously measure up to 100 different analytes without pretreatment of small (10-20 μl) patient samples It is very sensitive (pg unit) and can be quantified in a short time (3-4 hours), and it is an analysis method that can replace the existing ELISA or ELISPOT. The Luminex Assay is a multiplexed fluorescent microplate assay that can simultaneously analyze more than 100 biological samples from each well in a 96-well plate. By using the laser detector of the real-time signal transmission to distinguish the polystyrene bead (polystyrene bead) of more than 100 different color groups. The 100 beads are configured to be distinguished in the following manner. On the one hand, the red fluorescence bead is divided into ten or more steps, and on the other, the orange fluorescence bead is divided into ten steps, showing the difference in intensity, and the beads therebetween. The red and orange ratios are mixed in different proportions, making up a total of 100 color-coded bead sets. In addition, each bead is attached to the antibody of the protein to be analyzed, it is possible to quantify the protein by an immune antibody reaction using the same. The sample is analyzed using two lasers, one of which detects the beads to determine the bead identification number, and the other laser reacts with the antibody attached to the beads. The protein in the sample is detected. Thus, 100 in vivo proteins can be analyzed simultaneously in one well. This analysis has the advantage of being able to detect samples as small as 15 μl.

Luminex kits capable of performing the Luminex assay of the present invention include antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Luminex kits can also include antibodies specific for control proteins. 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.

In the kit for diagnosing or prognostic analysis of the biliary tract cancer of the present invention, the biliary tract cancer diagnostic kit including the immunochromatography strip for diagnosing the biliary tract cancer is necessary for performing a rapid test capable of knowing the analysis result within 5 minutes. It may be a diagnostic kit characterized by including an element. The immunochromatography strip may include (a) a sample pad into which a sample is absorbed; (b) a conjugate pad that binds to a protein of any one or more genes selected from the 20 gene groups in the sample; (c) a test membrane in which a test line and a control line including monoclonal antibodies against proteins of any one or more genes selected from the 20 gene groups are treated; (d) an absorption pad on which the remaining sample is absorbed; And (e) a support. Rapid test kits, which also include immunochromatographic strips, include antibodies specific for the marker protein. The antibody is an antibody having high specificity and affinity for each marker protein and having little cross-reactivity to other proteins. The antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. Rapid test kits may also include antibodies specific for the control protein. Other rapid test kits include reagents that can detect bound antibodies, such as nitrocellulose membranes to which specific and secondary antibodies are immobilized, membranes bound to beads to which antibodies are bound, absorbent pads and sample pads, and the like. Other substances necessary for diagnosis, and the like.

Determination of protein expression levels by immunological dot assay in the present invention comprises the steps of (a) dotting a biological sample on the membrane; (b) reacting an antibody specific for a protein of any one or more genes selected from the group consisting of the 20 genes with the instilled membrane; And (c) adding and developing a secondary antibody conjugated with a marker to the reacted membrane, wherein the ELISA assay comprises (a) a gene group having base sequences for the 20 markers. Adsorbing the antibody 1 specific to the protein of any one or more genes selected from the solid phase; (b) contacting the antibody 1 adsorbed to the solid body with a biological sample of a suspected biliary cancer patient to form an antigen-antibody complex; (c) binding the complex to the complex by treating antibody 2 specific for a protein encoded by at least one gene selected from the group of genes having base sequences for the 20 markers to which the label is bound; And (d) is preferably a sandwich ELISA assay comprising the step of detecting the concentration of the protein by detecting the label, the protein microarray assay (a) is selected from the gene group consisting of the 20 markers Immobilizing polyclonal antibodies specific to proteins of one or more genes on a chip; (b) contacting the immobilized polyclonal antibody 1 with a biological sample of a suspected biliary cancer patient to form an antigen-antibody complex; (c) binding to the complex by treating a monoclonal antibody specific for a protein encoded by any one or more genes selected from the group of genes having base sequences for the 20 markers to which the label is bound; And (d) detecting the label and measuring the concentration of the protein.

Through the above assay methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected biliary cancer patients can be compared, and the significant increase in the expression level of the biliary cancer marker gene to the protein can be compared. By determining whether or not, the actual suspected onset of biliary tract cancer can be diagnosed.

In the present invention, the term antigen-antibody complex means a combination of a biliary 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 a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is 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. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2+, [RU (bpy) 3] 2+, [MO (CN) 8] 4- and the like. Radioisotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like.

Protein expression level measurement is preferably by using an ELISA method. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. More preferably, the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. By confirming the degree of complex formation of the biliary tract marker protein and the antibody, it is possible to confirm the onset of biliary tract cancer.

Also preferably, Western blot using at least one antibody against the biliary tract cancer marker. 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. By checking the amount of the generated antigen-antibody complexes using labeled antibodies, the amount of protein produced by the expression of genes can be checked to determine whether biliary tract cancer develops. The detection method consists of a method of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the cells of biliary tract cancer. mRNA or protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.

Also, preferably, immunohistostaining is performed using at least one antibody against the biliary tract cancer marker. After collecting and fixing normal biliary tissue and suspected biliary tract cancer, paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several micrometers and attached to glass slides, and then reacted with one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

Also, preferably, at least one antibody against the biliary tract cancer marker is arranged at a predetermined position on the substrate to be immobilized at a high density. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read and confirmed the presence or expression of the protein and the biliary tract. You can check for cancer.

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

Expression polynucleotide measurement used for diagnosing biliary tract cancer in the present invention refers to measuring the amount of polynucleotide by confirming the presence and degree of expression of the polynucleotide encoding the protein marker of the present invention in a biological sample. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, and the like.

The base sequences encoding respective protein markers selected from the above protein group used as markers in the present invention may include base sequences having homology with these sequences.

Preferably, 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 the polynucleotide sequence and the polynucleotide sequence encoding the respective proteins.

"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 copying. 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. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers 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 methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroami Date, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like. Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, 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 radioisotopes, fluorescent molecules, biotin, and the like.

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

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

According to another aspect of the present invention, a binding agent or a polynucleotide and sequence listing agent of SEQ ID NO: 2, which specifically binds to a protein selected from the group consisting of the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 3 Provided are a kit for detecting cancer stem cells, comprising a primer or probe specifically binding to a polynucleotide selected from the group consisting of four sequences of polynucleotides.

The cancer may include, for example, biliary tract cancer, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, and head and neck cancer. , Skin cancer, thyroid cancer, parathyroid cancer and ureter cancer.

According to another aspect of the present invention, a binding agent or a polynucleotide and sequence listing agent of SEQ ID NO: 2, which specifically binds to a protein selected from the group consisting of the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 3 Provided is a kit for diagnosing or prognostic bile duct cancer comprising a primer or a probe specifically binding to a polynucleotide selected from the group consisting of four sequences of polynucleotides.

The expression “kites for diagnosing or prognostic analysis of bile duct cancer” means a kit including a composition for diagnosing or prognostic analysis of biliary tract cancer. Therefore, the expression "kites for diagnosing or prognostic analysis of biliary tract cancer" may be used interchangeably or mixed with "compositions for diagnosing or prognostic analysis of biliary tract cancer".

According to another aspect of the present invention, a binding agent or a polynucleotide and sequence listing agent of SEQ ID NO: 2, which specifically binds to a protein selected from the group consisting of the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 3 It provides a kit for cancer diagnosis or prognosis analysis comprising a primer or probe that specifically binds to a polynucleotide selected from the group consisting of four sequences of polynucleotides.

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

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

(Iii) The present invention provides a method for screening a substance for preventing or treating biliary tract cancer by using a novel molecular marker for biliary tract cancer stem cells and biliary tract cancer.

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

(Iii) A biliary cancer or cancer therapeutic target of the present invention is very useful for screening therapeutic agent candidates specifically acting on biliary cancer cancer stem cells or cancer stem cells.

(Iii) In addition, the present invention can accurately analyze the diagnosis and prognosis of biliary tract cancer or cancer.

1 is a diagram showing the difference in expression of cancer stem cell-related conventional markers in biliary tract cancer and adjacent normal tissues through immunohistochemistry.
a) anti-Notch-1 antibody, b) anti-sonic hedgehog antibody, c) anti-Gli-1 antibody
2 is a diagram showing the expression of CD24, CD44, ESA and CD133, known as cancer stem cell markers in human biliary tract cancer cell lines SNU245 and SNU1196, through flow cytometry.
Figure 3 is a diagram showing the spear cells having the characteristics of cancer stem cells cultured from human biliary tract cancer cell line.
Figure 4 is a diagram showing the results confirmed by the RT-PCR expression of cancer stem cell-related genes in human biliary tract cancer spheres.
5 is a diagram showing the characteristics of the spear cells cultured in human biliary duct cancer cell lines SNU245 and SNU1196 using a colonogenic assay.
Figure 6 shows the adhesion of SNU1196 cells and histologic morphology through tumor size and H & E staining 12 weeks after subcutaneous injection of spear cells into nude mice.
Figure 7 shows the results of expression analysis of new marker TAGLN2 related to biliary cancer cancer stem cells in human biliary tract cancer tissue.
a) difference in TAGLN2 expression in normal and cancerous tissues (10 × 10 magnification), b) tissue microarray (TMA) 1, c) tissue microarray 2, de) difference in TAGLN2 expression in normal and cancerous tissues (40 X 10 magnification)
8 is a diagram showing the results of expression analysis of new marker TMEM165 related to biliary cancer cancer stem cells in human biliary cancer tissue.
a) Difference in TMEM165 expression in normal and cancerous tissues (10 × 10 magnification), b) Tissue microarray (TMA) 1, c) Tissue microarray 2, de) Difference in TMEM165 expression in normal and cancerous tissues ( 40 x 10 magnification)
9 is a result of confirming the expression of TAGLN2 through RT-PCR after extracting mRNA from six human cholangiocarcinoma cell lines of SNU245, SNU308, SBU478, SNU869, SNU1079 and SNU1196. RT-PCR data was normalized to the mRNA level of the Beta-actin gene.
10 shows the results of transfecting TAGLN2 siRNA into human biliary tract cancer cell line SNU1196 and then confirming the decrease in TAGLN2 expression with RT-PCR and Western blot. RT-PCR data was matched to the mRNA level of the Beta-actin gene, and Western blot data was normalized to the expression level of the GAPDH protein.
(a) RT-PCR, (b) Western Blot
Figure 11 shows the results of cancer cell metastasis assay (Migration assay) according to the inhibition of TAGLN2 expression.
12 is a graph analyzing the sensitivity to the anticancer drug gemcitabine when inhibiting TAGLN2 expression.
Figure 13 shows the results of Western blot changes in various signaling systems in accordance with the inhibition of TAGLN2 expression. Western blot data was normalized to the expression level of GAPDH protein.
(a) cancer stem cell markers and cancer stem cell-related Wnt signaling systems, (b) PI3K signaling, MAPK signaling, and apoptosis-related markers
14 is a result of confirming the expression of TMEM165 through RT-PCR after extracting mRNA from six human cholangiocarcinoma cell lines of SNU245, SNU308, SBU478, SNU869, SNU1079 and SNU1196. RT-PCR data was normalized to the mRNA level of the Beta-actin gene.
Figure 15 shows the results of transfection of the TMEM165 shRNA plasmid into the biliary duct cancer cell line SNU1196, and the decrease in expression of TMEM165 by Western blot. Western blot data was normalized to the expression level of GAPDH protein.
FIG. 16 shows the results of analyzing changes in various characteristics of cancer cells according to TMEM165 knock-down (k / d).
(a) Proliferation assay, (b) Sphere formation assay, (c) Cancer metastasis assay, (d) Cancer cell invasion assay, (e) Anticancer susceptibility assay
17 shows the results of Western blot changes in various signaling systems according to inhibition of TMEM165 expression. Western blot data was normalized to the expression level of GAPDH protein.
(a) cancer stem cell related markers, (b) PI3K signaling system and MAPK signaling system

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 Methods

Expression Analysis of Cancer Stem Cell Signaling System in Human Bile Cancer Cells by Immunohistochemical Staining

Human normal and biliary cancer tissue slides were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide containing methanol solution at room temperature for 20 minutes. Microwave antigen search was performed for 5 minutes in citrate buffer (0.01 M, pH 6.0). The slides were then incubated for 1 hour with 10% normal donkey serum solution to reduce non-specific background staining. The primary antibody reacted by diluting at 1: 200 was rabbit polyclonal Notch3 (santa cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal Shh (santa cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal Gli1 (santa cruz biotechnology Inc, santa cruz, CA). Blocked sections were incubated overnight at 4 ° C. with the primary antibody. Subsequent reactions were performed according to standard protocols included in the Envision kit (DakoCytomation, Carpineteria, Calif., USA). Finally, the slides were incubated with 3,3'-diaminobenzidine (DakoCytomation, Carpinteria, CA, USA) and transformed with Harris hematoxylin (Sigma-Aldrich, Inc., St. Louis, Mo.).

Expression of cancer stem cell markers in biliary tract cancer cell lines was confirmed by flow cytometry

Human biliary tract cancer cell lines SNU-245 and SNU-1196 cells were treated with accutase (Accutase; Sigma-Aldrich, St. Louis, MO) to prepare the same number of single cell suspensions. It was then stained with antibodies known as cancer stem cell markers, and the antibodies were PE-CD24 (BD Biosciences PharMingen, San Diego, Calif.), APC-CD44 (BD Biosciences PharMingen, San Diego, Calif.), FITC-ESA ( BD Biosciences PharMingen, San Diego, CA) and PE-CD133 (BD Biosciences PharMingen, San Diego, CA). Flow cytometry was performed using BD LSR II (BD Biosciences, Franklin Lakes, NJ) and analyzed using BD FACSDiva software.

Spear Isolation and Culture of Human Stem Cells from Human Biliary Cancer Cell Lines

Six strains of human cholangiocarcinoma cell line (SNU245, SNU478, SNU869, SNU1196, SNU308, SNU1079) were purchased from the Korean Cell Line Bank (KCLB, Korean Cell Line Bank, Seoul, Korea). Cell lines were cultured in RPMI1640 (Invitrogen Gibco, Grand Island, NY) containing growth medium ie 10% fetal bovine serum (FBS; Hyclone, Logan, UT) by the protocol presented by the Korean Cell Line Bank. Biliary cancer cell lines were cultivated with EGF (10 ng / ml, R & D Systems Inc., Minneapolis, MN), bFGF (10 ng / ml, R & D Systems Inc.), 1ITS (insulin transferring selenium, Gibco) and Serum free RPMI medium supplemented with 0.5% BSA (Invitrogen Gibco, Grand Island, NY) at a rate of 1000 cells / ml for 6-10 days after inoculation into 6-well ultra low cluster plates (Corning Inc., Corning NY) (D. Ponti et. Al, Cancer Res : 65 (13), 5506-5511 (2005)). Cells grown in an adherent state by attaching the same culture to a culture dish (Nalgene Nunc Int., Rochester, NY) were used as controls.

Measurement of Cancer Stem Cell-Related Polynucleotides in Spear Cells

Total RNA was extracted from spear cells and adhesion of SNU245 and SNU1196 cells using RNAeasy Mini kit (QIAGEN, Valencia, Calif.). 2 μg of each RNA extracted for one hour at 42 ° C. was reverse transcribed through the reaction of Superscript II (Gibco BRL, Grand Island, NY). Expression of cancer stem cell related genes in spear cells was analyzed by primers of cancer stem cell related genes and PCR with Taq polymerase, and the expression of betaactin gene was used as a control. Primer base sequence, PCR reaction temperature and cycle number of cancer stem cell related genes are shown in Table 1 below.

gene Forward primer sequence (5'-3 ') Reverse primer sequence (5'-3 ') Tm / cycle Notch3 ATGGTGGGAACTAAACACAGCT ATGACCCTGGAGGAAGCACA 55 ℃ / 35 Hes1 GTGCTGTCTGGATGCGGAGT GAACACTCACACTCAAAGCCC 55 ℃ / 35 Ihh CCTGAACTCGCTGGCTATCT AATACACCCAGTCAAAGCCG 56 ℃ / 35 Gli1 AGAGTCCAGGGGGTTACATA AGAGTCCAGGGGGTTACATA 57 ℃ / 35 Nanog ACTGTCTCTCCTCTTCCTTCCT AGAGTAAAGGCTGGGGTAGGTA 61 ℃ / 30 Oct4 GTGGAGGAAGCTGACAACAA AGCAGCCTCAAAATCCTCTC 62 ℃ / 27 PTEN GGACGAACTGGTGTAATGAT CAGACCACAAACTGAGGATT 56 ℃ / 30 FZD7 CCAACGGCCTGATGTACTTT GCCATGCCGAAGAAGTAGAG 61 ℃ / 30 β-catenin GTATGAGTGGGAACAGGGATTT CCTGGTCCTCGTCATTTAGC 52 ℃ / 25 C-met CAATGTGAGATGTCTCCAGC CCTTGTAGATTGCAGGCAGA 56 ℃ / 28 Snail AAGCTTCCATGGCGCGCTCTTTCCTCGTCAGGAAGCCC GGATCCTCAGCGGGGACATCCTGAGCAGCCGGACTCTTG 61 ℃ / 30 Vimentin GAGAACTTTGCCGTTGAAGC GCTTCCTGTAGGTGGCAATC 55 ℃ / 27 CD90 GACCCGTGAGACAAAGAAGC ACTGTGACGTTCTGGGAGGA 61 ℃ / 35 CEACAM1 ATACCTGCCACGCCAATAAC TTATGCTGAGGGTGGTGTTG 61 ℃ / 35 β -actin GGCATCCTCACCCTGAAGTA GGGGTGTTGAAGGTCTCAAA 55 ℃ / 25

Spear  Cell self-renewal and colonization

The soft agar colony forming assay was performed by modifying standard protocols (MJ Son et al., Cell stem cells : 4, 440-452 (2009)). Difco agar noble (Becton, Dickinson company, Sparks, MD) was used, and 0.6% of the bottom agar (0.6% bottom agar) in a ratio of 1: 1 with 2X spheres was added to a 24-well plate and solidified at room temperature. Adhesion and spear cells cultured from human biliary tract cancer cell lines SNU245 and SNU1196 were treated with 0.25% trypsin EDTA (Gibco BRL, Grand Island, NY) to prepare the same number of single cell suspensions. Adhesion and spear cells and agar suspended in the spear culture were mixed to make a top agar of 0.3% and dispensed on the lower agar. Spear cultures containing growth factors were replaced every 2-3 days. 37 ℃ CO _{2 for} 2-3 weeks After culturing in an incubator, the cells were stained with crystal violet solution, and the cell populations were counted for comparative analysis of autologous regeneration and anchorage independent growth.

Biliary system Cancer cell line SNU1196 Spear  Cell Cancer  Confirm

Adhesion and spear cells cultured from human biliary tract cancer cell line SNU1196 were treated with 0.25% trypsin EDTA (Gibco BRL, Grand Island, NY) to prepare a single cell suspension of the same number (1000 cells). Then, subcutaneous injection was performed on both sides of nude mice (Orientbio, Seoul). After observing for 12 weeks and then dissected to confirm the cancer ability of the spear cells.

cDNA Microarray

Total RNA was extracted using the RNAeasy Mini kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Total RNA was quantitated using an ND-1000 nanodrop spectrometer (NanoDrop Technologies, Inc., DE) and an RNA 6000 nanochip (Agilent Technologies) by an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clare, Calif.) Qualitative analysis. Microarray experimental procedures were performed according to the manufacturer's protocol. CDNA microarray analysis was performed in Digital Genomics (Seoul, Korea). Briefly, the double yarn cDNA was prepared using 6 μg of each total RNA, and amplified by biotin-labeling (IVT labeling kit; Affymetrix). The labeled cDNA was fragmented and hybridized to Affymetrix GeneChip human genome U133 plus 2.0 high concentration oligonucleotide arrays (Affymatrix, Santa Clara, Calif.). Microarrays were then washed with Genechip Fluidics Station 450 (Affymetrix) and scanned using Genechip Array Scanner 3000 7G (Affymetrix). The Affymetrix Expression Console software version 1.1 was used to generate expression data and standardize the MAS5 algorithm. Expression intensity data in raw .cel files were normalized with the MAS5 algorithm to reduce noise. In over 50% of samples in at least one sample group, probe sets other than the presence call were filtered out by MAS5 detection call. Paired t-tests were performed on MASS expression values to determine genes that expressed differently between the two groups and considered genes with more than 1.2-fold differences. All individual chip analyzes were performed twice for independent total RNA samples. Thereafter, the inventors derived more refined candidate groups through integrated analysis with cDNA chip data of human biliary tract cancer tissues.

Human body through immunohistochemical staining Biliary tract  In the organization Biliary tract  Cancer Stem Cell Related Proteins TAGLN2  And expression analysis of TMEM165

Human biliary tract slides (normal and biliary total slides and TMA tissue) were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide containing methanol solution at room temperature for 20 minutes. Microwave antigen search was performed for 5 minutes in citrate buffer (0.01 M, pH 6.0). The slides were then incubated for 1 hour with 10% normal donkey serum solution to reduce non-specific background staining. Primary antibodies are rabbit polyclonal TAGLN2 (Sigma-Aldrich, Inc., St. Louis, MO), rabbit polyclonal TMEM165 (Sigma-Aldrich, Inc., St. Louis, MO). Dilution concentrations are 1: 500 and 1: 1000, respectively. Blocked sections were incubated overnight at 4 ° C. with the primary antibody. Subsequent reactions were performed using the recommended procedures included in the Envision kit (DakoCytomation, Carpineteria, CA, USA). Finally, slides were incubated with 3,3'-diaminobenzidine (DkoCytomation, Carpinteria, Calif., USA), and modified Harris hematoxylin (Sigma-Aldrich, Inc., St. Louis, MO) solution.

Biliary tract  In cell lines TAGLN2  And TMEM165 mRNA  Confirmation of expression

Total RNA was extracted from a total of six human cholangiocarcinoma cell lines (SNU245, SNU308, SNU478, SNU869, SNU1079 and SNU1196) using the RNAeasy Mini kit (QIAGEN, Valencia, CA). 2 μg of each RNA extracted for one hour at 42 ° C. was reverse transcribed through the reaction of Superscript II (Gibco BRL, Grand Island, NY). TAGLN2 or TMEM165 expression was analyzed in biliary tract cancer cell lines by PCR with TAGLN2 or TMEM165 gene primers and Taq polymerase, and the beta-actin gene was used as a control. Primer nucleotide sequences of the TAGLN2 and TMEM165 genes are shown in Table 2.

gene Forward primer sequence (5'-3 ') Reverse primer sequence (5'-3 ') TAGLN2 TAT GGC ATT AAC ACC ACT GA GGA TTC TCC TTG GAT TTC TT TMEM165 CTG ACC CCT TTT TAA GGA AT CTT GTT GTG CCA TGT TAT TG

TAGLN2 siRNA Transfection of transfection )

SiRNA for TAGLN2 was used by Santa cruz sc-106633 (santa cruz biotechnology Inc, santa cruz, CA). The siRNA 30 nM per 9 x 10 ⁴ bile duct cancer cell line SNU1196 was detected using lipofectamine RNAiMAX (Invitrogen) to determine the expression of TAGLN2 at 30, 48, 72 and 96 hours after transfection. Cells into which a control siRNA (santa cruz, sc-37007) was introduced were used as a control.

TMEM165  Expression inhibition (knock-down, k / d) cell line construction

TMEM165 shRNA plasmids and control shRNA plasmids (SureSiliencing shRNA plasmids) were purchased from QIAGEN (QIAGEN, Valencia, Calif.) And permanently injected into the SNU1196 cell line to identify various characteristic changes due to inhibition of expression of TMEM165. k / d) cell lines were established.

The sequence of shRNA-1 for TMEM165 used in the present invention is GGA AGA AGT TCA AGC TGA ATT, the sequence of shRNA-4 is TCC TGC TAC CCA AAC TAA TTT, and the sequence of control shRNA is GGA ATC TCA TTC GAT GCA TAC. As a selection marker, 2.0 µg / ml of puromycin (Sigma-Aldrich, Inc., St. Louis, Mo.) was used. Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) Was used to transfect cells with TMEM165 shRNA plasmid and control shRNA plasmid. After 24 hours, the culture medium was changed to a culture medium containing 2 μg / ml puromycin, followed by continuous culture for at least 3 days. Cells surviving in puromycin cultures were cultured by attaching a small number of 20-30 to 100 mm plates, followed by selection of single colonies using cloning cylinders (Sigma-Aldrich, Inc., St. Louis, MO). TMEM165 knock-down was then confirmed via Western blot.

Protein expression analysis using Western blot

After acquitase treatment (Accutase; Sigma-Aldrich, St. Louis, MO), the cells were washed with PBS and the proteins were extracted, and the proteins were extracted, loaded on SDS-polyacrylamide gel and electrophoresed. . Proteins fractionated by size by electrophoresis were transferred to PVDF membranes (Millipore corporation, Billerica, Mass., USA), and then TBS- with 5% non-fat milk added to reduce nonspecific reactions. Blocking in T (Tris-buffered saline / 0.05% Tween-20) for 1 hour. Each membrane was then reacted with the primary antibody overnight at 4 ° C. Primary antibodies were rabbit polyclonal TAGLN2 (Sigma-Aldrich, Inc., St. Louis, MO), rabbit polyclonal TMEM165 (Sigma-Aldrich, Inc., St. Louis, MO), rabbit polyclonal c- MET (Cell Signaling Technology, Inc., Danver, MA), rabbit polyclonal phospho-GSK3 (Cell Signaling Technology, Inc., Danver, MA), mouse monoclonal b-catenin (santa cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal Ihh (santa cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal phospho-AKT (Cell Signaling Technology, Inc., Danver, MA), rabbit polyclonal AKT (santa cruz biotechnology Inc , santa cruz, CA), mouse monoclonal p21RAS (BD transduction laboratories, San Diego, CA), rabbit polyclonal phospho-MEK (Cell Signaling Technology, Inc., Danver, MA), rabbit polyclonal MEK (santa) cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal phospho-ERK (Cell Signaling Technology, Inc., Danver, MA), goth polyclonal ERK (santa cruz biotechnology Inc, santa cr) uz, CA), rabbit polyclonal BAX (santa cruz biotechnology Inc, santa cruz, CA), mouse monoclonal BCLxL (santa cruz biotechnology Inc, santa cruz, CA) and mouse monoclonal GAPDH (santa cruz biotechnology Inc, santa cruz, CA). Each membrane reacted with the primary antibody was washed in TBS-T buffer, and then reacted with a horseradish peroxidase (HRP) conjugated secondary antibody (santa cruz biotechnology Inc, santa cruz, CA) for 1 hour. Each protein was detected using an enhanced chemiluminescence system (PIERCE, Rockford, IL).

Cell proliferation rate assay ( Proliferation assay )

Each cell was attached to a 24-well-plate of 1.1 × 10 ⁴ , and then, every 8 days, cells were separated with 0.25% trypsin EDTA at a predetermined time and the number of cells was measured on a hematocytometer.

Sphere forming assay

For spear culture, TMEM165 knock-down (k / d) cell lines and control cell lines were treated with EGF (10 ng / ml, R & D Systems Inc., Minneapolis, MN), bFGF (10 ng / ml, R & D Systems Inc.), 1ITS. 6-well ultra-low cluster plates (Corning Inc., Corning NY) at a rate of 1000 cells / ml in serum-free RPMI medium supplemented with (insulin transferring selenium, Gibco) and 0.5% BSA (Invitrogen Gibco, Grand Island, NY) After incubation for 7-10 days after inoculation (D. Ponti et. Al, Cancer Res : 65 (13), 5506-5511 (2005)) the difference in sphere formation was observed under a microscope.

Cancer Cell Metastasis Analysis migration assay ) And cancer cell invasion assay invasion assay )

Cancer cell metastasis and infiltration assays were performed in Transwell Inc., Tewksbury MA. In the case of cancer cell invasion assay, Matrigel (BD Biosciences PharMingen, San Diego, Calif.), Diluted 1/4 in serum-free media, was added to the upper well by 85 μl and solidified at 37 ° C. Dispense 1.0 × 10 ⁴ cells suspended in serum-free medium in the upper wells and 500 μl of NIH3T3 fibroblasts in the lower wells for 24 hours and 72 hours for infiltration analysis. After passing through the filter, the cells attached to the bottom of the filter were fixed and stained, and the number of metastases and infiltrated cells was measured through microscopic observation.

Anticancer Susceptibility Analysis

Each cell was attached to 96 well-plates in 10 ³ cells, and the following day, gemcitabine (Gemcitabine, Lilly france SAS) was treated at concentrations of 0, 20, 200, 2000 nM and incubated for 72 hours. MTT solution (Sigma-Aldrich, Inc., St. Louis, MO) was added and reacted for 4 hours, and then treated with DMSO (sigma-Aldrich) to measure absorbance at 570 nm to analyze sensitivity.

Experiment result

Various existing cancer stem cells in biliary tract cancer and adjacent normal tissues Marker  Expression analysis through immunochemical staining

Immunohistochemical staining was performed to determine whether Notch and Hedgehog, which are signals related to stem cells, were expressed in human cholangiocarcinoma tissues. Expression of stem cell-related proteins such as notch-1, sonic hedgehog, and Gli-1 is significantly increased in biliary tract cancer tissues compared to gastrointestinal cancer tissues (FIGS. 1a-c). It is confirmed that they are very involved in.

Flow cell  Cancer Stem Cells Through Analysis Markers  Confirmation of expression

Flow cytometry was performed to confirm the expression of CD24, CD44, ESA and CD133 as cancer stem cell markers in human biliary tract cancer cell lines SNU245 and SNU1196. The expression of each marker was higher in SNU1196 cells than in SNU245 cells. CD44 and ESA expressed 50-90% higher in SNU245 and SNU1196, whereas CD133 expressed only 0.5% in SNU1196. In the case of CD24, expression of 2.3% in SNU245 and 96% in SNU1196 was confirmed, indicating that the expression difference between cell lines is large in the biliary tract cancer cell line (FIG. 2).

anatomy Biliary system  Characteristics of cancer stem cells from cell lines Spear  Isolation and Cultivation

Spear formation indicating the characteristics of cancer stem cells was confirmed, and SNU245 and SNU1196 were selected from a total of six biliary tract cancer cell lines, and subsequent experiments were performed (FIG. 3).

Biliary tract Spear  Cancer stem cell association in cell Polynucleotides  analysis

Spears were isolated and cultured by selecting SNU245 and SNU1196 among biliary cancer cell lines in which spear formation was confirmed, and expression of various cancer stem cell-related genes was confirmed through RT-PCR. It was confirmed that stem cell-related signaling systems such as Notch, Hedgehog, and Wnt were activated in spear cells compared to the adhesion of biliary cancer cell lines. In addition, the expression of c-met, snail and vimentin, markers of epithelial to mesenchymal transition, which is a characteristic of cancer stem cells, and CD90, which was recently reported as a liver cancer stem cell marker, was also increased (FIG. 4). This suggests that a large amount of cancer stem cells are contained in the spear cells, and the biliary cancer stem cell specific markers can be predicted by examining gene and protein profiles specifically expressed in spear cells.

Biliary tract Spear  In a cell Colony formation Clonogenic assay  Characterization of cancer stem cells through

Colony formation assays were performed to examine the characteristics of spear cells cultured in human biliary tract cancer cell lines SNU245 and SNU1196. Compared to the adherent cells, the spear cells form more colonies, indicating that the self-renewal and non-adhesion proliferation ability, which is the most important characteristic of the stem cells, is high (FIG. 5).

Biliary tract Spear  Cell Cancer  Verification

In order to confirm the cancerous ability of the human biliary duct cancer cell line SNU1196 spear cells, 1000 control cells and 1000 spear cells were subcutaneously injected to both sides of nude mice and observed for 12 weeks. The group injecting the spear cells with the characteristics of cancer stem cells had higher cancer formation ability than the group injected with the adhesive cells, resulting in faster growth of the spear cells and histologically more severe internal degeneration. It was confirmed that it is positive (Fig. 6). Spear cells formed from biliary tract cancer cell lines express genes and markers related to cancer stem cells, and by examining biomarkers specifically expressed in spheres based on the results of increased cancerous ability (FIGS. 1-6). The biliary cancer stem cell specific markers could be predicted.

cDNA  Through the array Biliary tract  Specifically expressed in stem cells Biomarker  excavation

CDNA microarrays using Affymetrix GeneChip HG U133 were performed to analyze genes expressed differently in spheres and adherent cells of SNU245 and SNU1196 cells and to identify target markers related to biliary tract cancer cells. Then, by integrating and analyzing the cDNA microarray data of human cholangiocarcinoma tissues of the present inventors, we found TAGLN2 and TMEM165, which are increased in both spear cells and human cholangiocarcinoma tissues (Table 3).

Gene
symbol Gene name RefSeq
Transcript ID Sphere_Fold change Chip data_Fold change of existing biliary tract cancer tissues TAGLN2 Transgelin 2 NM_003564 SNU245 1.7 6.6 SNU1196 1.5 TMEM165 Transmembrane165 NM_018475 SNU245 1.4 3.3 SNU1196 1.2

anatomy Biliary tract  Novel Markers Associated with Biliary Cancer Cancer Stem Cells in Tissues TAGLN2 Expression analysis

Expression of TAGLN2, a novel marker related to biliary tract cancer stem cells derived from the present invention, was confirmed in human biliary tract cancer tissues. As a result of confirming the expression of TAGLN2 in human biliary tract epithelial tissue and biliary tract cancer tissue, it was confirmed that the expression in the normal tissue was significantly lower than that in the biliary tract cancer tissue (FIG. 7A). In addition, as a result of confirming the expression of TAGLN2 in the elevated TMA of 33 biliary tract cancer tissues, it was confirmed that 96.5% of the biliary tract cancer tissues were expressed in the tissues (Fig. 7B-E).

anatomy Biliary tract  Novel Markers Associated with Biliary Cancer Cancer Stem Cells in Tissues TMEM165 Expression analysis

Expression of TMEM165, a novel marker related to biliary tract cancer stem cells derived from the present invention, was confirmed in human biliary tract cancer tissues. As a result of confirming the expression of TMEM165 in normal biliary tract epithelial tissue and biliary tract cancer tissue, it was verified that the expression in the normal tissue was significantly lower than that in the biliary tract cancer tissue (FIG. 8A). In addition, as a result of confirming the expression of TMEM165 in the elevated TMA of 33 biliary cancer patient tissues, it was confirmed that the expression of 88.9% of the biliary cancer patient tissues (Fig. 8b-e).

TAGLN2  Analysis of various characteristics according to inhibition of expression

RT-PCR confirmed TAGLN2 expression in human biliary tract cancer cell lines prior to inhibition of TAGLN2 expression by siRNA, indicating that all six biliary duct cancer cell lines express TAGLN2 strongly (FIG. 9). When TAGLN2 siRNA was transfected into the SNU1196 cell line, it was confirmed that the expression of TAGLN2 was significantly reduced from 48 hours to 96 hours (FIGS. 10A-B).

At 48 hours after TAGLN2 siRNA transfection, cancer cell metastasis and anticancer susceptibility were analyzed, and at 72 hours, expression changes of various proteins were confirmed by Western blot.

11 and 12 show that as a result of cancer cell metastasis capacity and anticancer drug sensitivity analysis, when the expression of TAGLN2 is inhibited, cancer cell metastasis capacity is significantly reduced and sensitivity to anticancer drugs is also increased. In addition, the expression of c-met, a pancreatic cancer stem cell marker and known to be related to various characteristics of cancer stem cells, was decreased with inhibition of TAGLN2 expression and b-catenin in cancer stem cell-related Wnt signaling system was also reduced. Figure 13a). In addition, it was confirmed that the expression of phosphorylated AKT and ERK, the increase of BAX, a pro-apoptotic marker, and the expression of BCLxL, an anti-apoptotic marker, were associated with apoptosis (FIG. 13B).

TMEM165  Expression inhibition (knock-down, k / d) cell line construction and characterization

RT-PCR confirmed the expression of TMEM165 in human cholangiocarcinoma cell lines before constructing the TMEM165 expression inhibitory cell line, and it was found that all 6 biliary cell carcinoma cell lines express TMEM165 strongly (FIG. 14). In order to analyze the various properties that appear in suppressing the expression of TMEM165, a TMEM165 knock-down (k / d) cell line was established by permanently injecting shRNA plasmid against TMEM165 and control shRNA plasmid into SNU1196 cells. The established TMEM165 knock-down (k / d) cell line was found to have reduced expression of TMEM165 protein compared to the control (FIG. 15).

When the expression of TMEM165 was suppressed, the cell proliferation rate was significantly lower than that of the control group (Fig. 16a), and the formation of spear cells was also inhibited, indicating that the self-renewal capacity of cancer stem cells was reduced (Fig. 16b). . In addition, cancer cell metastasis capacity (FIG. 16c) and cancer cell invasion capacity (FIG. 16d) were significantly reduced compared to the control. In addition, when the expression of TMEM165 was suppressed, the sensitivity to the anticancer gemcitabine was shown to increase (Fig. 16e).

17 shows the results of Western blot changes of various proteins according to inhibition of TMEM165 expression. When TMEM165 expression was inhibited, the expression of c-met, which has recently emerged as a cancer stem cell, also decreased, and the expression of b-catenin and Ihh of the Hedgehog signaling system of stem cell-related Wnt signaling system was also reduced. Once again it could be confirmed (Fig. 17a). In addition, the expression of pGSK3-beta and p-MEK was significantly reduced, and it was found that PI3K and MAPK signaling could be regulated together when artificially inhibiting the expression of TMEM165 (FIG. 17B).

Review

The prognosis for biliary tract cancer is very poor, with similar rates of morbidity and mortality, and no treatment has yet been established. Since recurrence or metastasis is common, 5-year survival is less than 5%. In addition, when it is found that there is no special early diagnosis, the disease is already advanced and treatment is often delayed.

In the present invention, a new diagnosis targeting cancer stem cells in biliary tract cancers, focusing on cancer stem cells or tumor initiating cells, which are considered to be the first initiation of cancer and are known to cause recurrence, metastasis and chemotherapy resistance. And markers related to biliary tract cancer stem cells to develop treatments.

Spear cells with cancer stem cell characteristics were cultured from human biliary tract cancer cell lines, and it was verified that spear cells have cancer stem cell characteristics. Well-known stem cell signaling systems (T. Reya et al., Nature , 414: 105-111,2001), such as Notch, Hedgehog, and Wnt signals, which are involved in cancer development and stem cell characteristics, were activated in these spear cells. , C-met (Li C. et al., Gastroenterology , 141 (6): 2218-2227,2011), also reported as pancreatic cancer stem cells, and the mesenchymal cell marker vimentin were increased.

In addition, the self-renewal ability, which is the most important characteristic of cancer stem cells, was higher in the spear cells of the biliary cancer cell line.In vivo experiments showed that the spear cell cancer ability was higher than that of the control cells. Consistent with previous reports of stem cell inclusions (J. Zhou et al., Proc Natl Acad Sci USA , 104: 16158-16163 (2007).

Based on these results, TAGLN2 and TMEM165, which are related to biliary tract cancer stem cell markers, were derived from cDNA arrays in spheres formed from human biliary tract cancer cell lines. Both TAGLN2 and TMEM165 were strongly expressed in all 6 biliary duct cancer cell lines, but not in normal biliary tract tissues, whereas they were strongly expressed in biliary duct cancer tissues, indicating the potential as a diagnostic marker for biliary tract cancers.

In addition, when the expression of these markers is artificially inhibited, cancer cell metastasis, decreased invasive capacity, and anticancer drug sensitivity are shown to be therapeutic targets, and c-met, b-catenin, PI3K and MAPK signaling system activity are also decreased. Showed results. It has been known that PI3K or MAPK signaling is activated through the activation of c-met in various cancer cells (Joseph Paul Eder et al., Clin) . Caner Res ., 15 (7) 2207-2214, (2009)). In addition, c-met is an important protein for epithelial mesenchymal cell migration and invasion growth related to cancer stem cells, and has recently been reported as a pancreatic cancer stem cell marker and therapeutic target (Li C. et al., Gastroenterology , 141 ( 6): 2218-2227 (2011)).

Based on the above results, it was confirmed that TAGLN2 and TMEM165 could be a diagnostic marker and therapeutic target for biliary tract cancer based on cancer stem cells.

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.

references

1. A. Dubrovska et al. Proc Natl Acad Sci USA .: 106, 268-273, 2009.

2. BB Zhou et al. Nature Reviews Drug Discovery : 8,806-823,2009)

3.M. Al-Hajj et al., Proc Natl Acad Sci USA .: 100 (7), 3983-3988, 2003.

4. ZF Yang et al., Cancer cell ; 13,153-166, 2008.

5. P. Dalerba et al., Proc Natl Acad Sci USA .: 104 (24), 10158-63, 2007.

6. C. Li et al. Cancer Res : 67 (3) 1030-1037, 2007.

7.MJ Son et al., Cellstem cells : 4,440-452,2009.

8. D. Ponti et. al, Cancer Res : 65 (13), 5506-5511,2005.

9.T. Reya et al., Nature , 414: 105-111,2001

10. Li C. et al., Gastroenterology , 141 (6): 2218-2227,2011

11.J. Zhou et al., Proc Natl Acad Sci USA , 104: 16158-16163,2007

12.Joseph Paul Eder et al., Clin Caner Res ., 15 (7) 2207-2214, 2009

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. 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> PN110407 <130> PN110407 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 199 <212> PRT <213> TAGLN2 human protein <400> 1 Met Ala Asn Arg Gly Pro Ala Tyr Gly Leu Ser Arg Glu Val Gln Gln   1 5 10 15 Lys Ile Glu Lys Gln Tyr Asp Ala Asp Leu Glu Gln Ile Leu Ile Gln              20 25 30 Trp Ile Thr Thr Gln Cys Arg Lys Asp Val Gly Arg Pro Gln Pro Gly          35 40 45 Arg Glu Asn Phe Gln Asn Trp Leu Lys Asp Gly Thr Val Leu Cys Glu      50 55 60 Leu Ile Asn Ala Leu Tyr Pro Glu Gly Gln Ala Pro Val Lys Lys Ile  65 70 75 80 Gln Ala Ser Thr Met Ala Phe Lys Gln Met Glu Gln Ile Ser Gln Phe                  85 90 95 Leu Gln Ala Ala Glu Arg Tyr Gly Ile Asn Thr Thr Asp Ile Phe Gln             100 105 110 Thr Val Asp Leu Trp Glu Gly Lys Asn Met Ala Cys Val Gln Arg Thr         115 120 125 Leu Met Asn Leu Gly Gly Leu Ala Val Ala Arg Asp Asp Gly Leu Phe     130 135 140 Ser Gly Asp Pro Asn Trp Phe Pro Lys Lys Ser Lys Glu Asn Pro Arg 145 150 155 160 Asn Phe Ser Asp Asn Gln Leu Gln Glu Gly Lys Asn Val Ile Gly Leu                 165 170 175 Gln Met Gly Thr Asn Arg Gly Ala Ser Gln Ala Gly Met Thr Gly Tyr             180 185 190 Gly Met Pro Arg Gln Ile Leu         195 <210> 2 <211> 1360 <212> DNA <213> TAGLN2 human nucleotide <220> <221> gene (222) (1) .. (1360) <220> <221> CDS <222> (74) .. (670) <220> <221> polyA_signal (222) (1339) .. (1344) <220> <221> polyA_site <222> (1360) <400> 2 gcccttgcct tgagtcagtg cgctgctctc cagcccgctt gaacgctccc cgcagccacc 60 gccacccatt gga atg gcc aac agg gga cct gca tat ggc ctg agc 106                       Met Ala Asn Arg Gly Pro Ala Tyr Gly Leu Ser                         1 5 10 cgg gag gtg cag cag aag att gag aaa caa tat gat gca gat ctg gag 154 Arg Glu Val Gln Gln Lys Ile Glu Lys Gln Tyr Asp Ala Asp Leu Glu              15 20 25 cag atc ctg atc cag tgg atc acc acc cag tgc cga aag gat gtg ggc 202 Gln Ile Leu Ile Gln Trp Ile Thr Thr Gln Cys Arg Lys Asp Val Gly          30 35 40 cgg ccc cag cct gga cgc gag aac ttc cag aac tgg ctc aag gat ggc 250 Arg Pro Gln Pro Gly Arg Glu Asn Phe Gln Asn Trp Leu Lys Asp Gly      45 50 55 acg gtg cta tgt gag ctc att aat gca ctg tac ccc gag ggg cag gcc 298 Thr Val Leu Cys Glu Leu Ile Asn Ala Leu Tyr Pro Glu Gly Gln Ala  60 65 70 75 cca gta aag aag atc cag gcc tcc acc atg gcc ttc aag cag atg gag 346 Pro Val Lys Lys Ile Gln Ala Ser Thr Met Ala Phe Lys Gln Met Glu                  80 85 90 cag atc tct cag ttc ctg caa gca gct gag cgc tat ggc att aac acc 394 Gln Ile Ser Gln Phe Leu Gln Ala Ala Glu Arg Tyr Gly Ile Asn Thr              95 100 105 act gac atc ttc caa act gtg gac ctc tgg gaa gga aag aac atg gcc 442 Thr Asp Ile Phe Gln Thr Val Asp Leu Trp Glu Gly Lys Asn Met Ala         110 115 120 tgt gtg cag cgg acg ctg atg aat ctg ggt ggg ctg gca gta gcc cga 490 Cys Val Gln Arg Thr Leu Met Asn Leu Gly Gly Leu Ala Val Ala Arg     125 130 135 gat gat ggg ctc ttc tct ggg gat ccc aac tgg ttc cct aag aaa tcc 538 Asp Asp Gly Leu Phe Ser Gly Asp Pro Asn Trp Phe Pro Lys Lys Ser 140 145 150 155 aag gag aat cct cgg aac ttc tca gat aac cag ctg caa gag ggc aag 586 Lys Glu Asn Pro Arg Asn Phe Ser Asp Asn Gln Leu Gln Glu Gly Lys                 160 165 170 aac gtg atc ggg tta cag atg ggc acc aac cgc ggg gcg tct cag gca 634 Asn Val Ile Gly Leu Gln Met Gly Thr Asn Arg Gly Ala Ser Gln Ala             175 180 185 ggc atg act ggc tac ggg atg cca cgc cag atc ctc tgatcccacc 680 Gly Met Thr Gly Tyr Gly Met Pro Arg Gln Ile Leu         190 195 ccaggccttg cccctgccct cccacgaatg gttaatatat atgtagatat atattttagc 740 agtgacattc ccagagagcc ccagagctct caagctcctt tctgtcaggg tggggggttc 800 agcctgtcct gtcacctctg aggtgcctgc tggcatcctc tcccccatgc ttactaatac 860 attcccttcc ccatagccat caaaactgga ccaactggcc tcttcctttc ccctgggacc 920 aaaatttagg ggcctcagtc cctcaccgcc atgccctggc ctattctgtc tctccttctt 980 ccccctggcc tgttctgtct ctgagctctg tgtcctccgt tcattccatg gctgggagtc 1040 actgatgctg cctctgcctt ctgatgctgg actggccttg cttctacaag tatgcttctc 1100 ccacagctgt ggctgcagga acttaattta tagggaggag cctgtggcag ctgctgcccc 1160 agccacagct gcactgactg tgctcaccac acatctgggg cagccttccc tggcaggggc 1220 cctcgtggct tctcattttc cattcccttc actgtggcta aggggtgggg tgaggggatg 1280 gagagggagg gctgcctacc atggtctggg gcttgaggaa gatgagtttg ttgatttaaa 1340 taaagaattt gtcatttttg 1360 <210> 3 <211> 324 <212> PRT <213> TMEM165 human protein <400> 3 Met Ala Ala Ala Ala Pro Gly Asn Gly Arg Ala Ser Ala Pro Arg Leu   1 5 10 15 Leu Leu Leu Phe Leu Val Pro Leu Leu Trp Ala Pro Ala Ala Val Arg              20 25 30 Ala Gly Pro Asp Glu Asp Leu Ser His Arg Asn Lys Glu Pro Pro Ala          35 40 45 Pro Ala Gln Gln Leu Gln Pro Gln Pro Val Ala Val Gln Gly Pro Glu      50 55 60 Pro Ala Arg Val Glu Lys Ile Phe Thr Pro Ala Ala Pro Val His Thr  65 70 75 80 Asn Lys Glu Asp Pro Ala Thr Gln Thr Asn Leu Gly Phe Ile His Ala                  85 90 95 Phe Val Ala Ala Ile Ser Val Ile Val Ser Glu Leu Gly Asp Lys             100 105 110 Thr Phe Phe Ile Ala Ala Ile Met Ala Met Arg Tyr Asn Arg Leu Thr         115 120 125 Val Leu Ala Gly Ala Met Leu Ala Leu Gly Leu Met Thr Cys Leu Ser     130 135 140 Val Leu Phe Gly Tyr Ala Thr Thr Val Ile Pro Arg Val Tyr Thr Tyr 145 150 155 160 Tyr Val Ser Thr Val Leu Phe Ala Ile Phe Gly Ile Arg Met Leu Arg                 165 170 175 Glu Gly Leu Lys Met Ser Pro Asp Glu Gly Gln Glu Glu Leu Glu Glu             180 185 190 Val Gln Ala Glu Leu Lys Lys Lys Asp Glu Glu Phe Gln Arg Thr Lys         195 200 205 Leu Leu Asn Gly Pro Gly Asp Val Glu Thr Gly Thr Ser Ile Thr Val     210 215 220 Pro Gln Lys Lys Trp Leu His Phe Ile Ser Pro Ile Phe Val Gln Ala 225 230 235 240 Leu Thr Leu Thr Phe Leu Ala Glu Trp Gly Asp Arg Ser Gln Leu Thr                 245 250 255 Thr Ile Val Leu Ala Ala Arg Glu Asp Pro Tyr Gly Val Ala Val Gly             260 265 270 Gly Thr Val Gly His Cys Leu Cys Thr Gly Leu Ala Val Ile Gly Gly         275 280 285 Arg Met Ile Ala Gln Lys Ile Ser Val Arg Thr Val Thr Ile Ily Gly     290 295 300 Gly Ile Val Phe Leu Ala Phe Ala Phe Ser Ala Leu Phe Ile Ser Pro 305 310 315 320 Asp Ser Gly Phe                 <210> 4 <211> 1983 <212> DNA <213> TMEM165 human nucleotide <220> <221> gene (222) (1) .. (1983) <220> <221> CDS 222 (268) .. (1239) <400> 4 gagtcggggc tgggccgcgc cgaggcagct ggctgactcc agtttagccg ccgccggaga 60 ggacgggcgc cgagccgggg ctgcggactt cggcctgccc ctcacctcac tcccgctgct 120 tgcacctccc ggatggtgct gactgctccc taagcggcgg cggcggcgag tcgtgaggac 180 gcgccgcgga ggctgttcgg ggtcgaggct tcccgtcgcc ggcacttcct cttgcggcgc 240 ccgtgcgcgg ccggcccggc aggcggg atg gcg gcc gcg gct cca ggg aac 291                                  Met Ala Ala Ala Ala Pro Gly Asn                                    1 5 ggc cgc gca tcg gcg ccc cgg ctg ctt ctg ctc ttt ctg gtt ccg ctg 339 Gly Arg Ala Ser Ala Pro Arg Leu Leu Leu Leu Phe Leu Val Pro Leu      10 15 20 ctg tgg gcc ccg gct gcg gtc cgg gcc ggc cca gat gaa gac ctt agc 387 Leu Trp Ala Pro Ala Ala Val Arg Ala Gly Pro Asp Glu Asp Leu Ser  25 30 35 40 cac cgg aac aaa gaa ccg ccg gcg ccg gcc cag cag ctg cag ccg cag 435 His Arg Asn Lys Glu Pro Pro Ala Pro Ala Gln Gln Leu Gln Pro Gln                  45 50 55 cct gtg gct gtg cag ggc ccc gag ccg gcc cgg gtc gag aaa ata ttt 483 Pro Val Ala Val Gln Gly Pro Glu Pro Ala Arg Val Glu Lys Ile Phe              60 65 70 aca cca gca gct cca gtt cat acc aat aaa gaa gat cct gct acc caa 531 Thr Pro Ala Ala Pro Val His Thr Asn Lys Glu Asp Pro Ala Thr Gln          75 80 85 act aat ttg gga ttt atc cat gca ttt gtc gct gcc ata tca gtt att 579 Thr Asn Leu Gly Phe Ile His Ala Phe Val Ala Ala Ile Ser Val Ile      90 95 100 att gta tct gaa ttg ggt gat aag aca ttt ttt ata gca gcc atc atg 627 Ile Val Ser Glu Leu Gly Asp Lys Thr Phe Phe Ile Ala Ala Ile Met 105 110 115 120 gca atg cgc tat aac cgc ctg acc gtg ctg gct ggt gca atg ctt gcc 675 Ala Met Arg Tyr Asn Arg Leu Thr Val Leu Ala Gly Ala Met Leu Ala                 125 130 135 ttg gga cta atg aca tgc ttg tca gtt ttg ttt ggc tat gcc acc aca 723 Leu Gly Leu Met Thr Cys Leu Ser Val Leu Phe Gly Tyr Ala Thr Thr             140 145 150 gtc atc ccc agg gtc tat aca tac tat gtt tca act gta tta ttt gcc 771 Val Ile Pro Arg Val Tyr Thr Tyr Tyr Val Ser Thr Val Leu Phe Ala         155 160 165 att ttt ggc att aga atg ctt cgg gaa ggc tta aag atg agc cct gat 819 Ile Phe Gly Ile Arg Met Leu Arg Glu Gly Leu Lys Met Ser Pro Asp     170 175 180 gag ggt caa gag gaa ctg gaa gaa gtt caa gct gaa tta aag aag aaa 867 Glu Gly Gln Glu Glu Leu Glu Glu Val Gln Ala Glu Leu Lys Lys Lys 185 190 195 200 gat gaa gaa ttt caa cga acc aaa ctt tta aat gga ccg gga gat gtt 915 Asp Glu Glu Phe Gln Arg Thr Lys Leu Leu Asn Gly Pro Gly Asp Val                 205 210 215 gaa acg ggt aca agc ata aca gta cct cag aaa aag tgg ttg cat ttt 963 Glu Thr Gly Thr Ser Ile Thr Val Pro Gln Lys Lys Trp Leu His Phe             220 225 230 att tca ccc att ttt gtt caa gct ctt aca tta aca ttc tta gca gaa 1011 Ile Ser Pro Ile Phe Val Gln Ala Leu Thr Leu Thr Phe Leu Ala Glu         235 240 245 tgg ggt gat cgc tct caa cta act aca att gta ttg gca gct aga gag 1059 Trp Gly Asp Arg Ser Gln Leu Thr Thr Ile Val Leu Ala Ala Arg Glu     250 255 260 gac ccc tat ggt gta gcc gtg ggt gga act gtg ggg cac tgc ctg tgc 1107 Asp Pro Tyr Gly Val Ala Val Gly Gly Thr Val Gly His Cys Leu Cys 265 270 275 280 acg gga ttg gca gta att gga gga aga atg ata gca cag aaa atc tct 1155 Thr Gly Leu Ala Val Ile Gly Gly Arg Met Ile Ala Gln Lys Ile Ser                 285 290 295 gtc aga act gtg aca atc ata gga ggc atc gtt ttt ttg gcg ttt gca 1203 Val Arg Thr Val Thr Ile Ile Gly Gly Ile Val Phe Leu Ala Phe Ala             300 305 310 ttt tct gca cta ttt ata agc cct gat tct ggt ttt t aacaagctgt 1250 Phe Ser Ala Leu Phe Ile Ser Pro Asp Ser Gly Phe         315 320 ttgttcatct atatttagtt taaaataggt agtattatct ttctgtacat agtgtacatt 1310 acaactaaaa gtgatggaaa aatactgtat tttgtagcac tgattttgtg agtttgaccc 1370 attattatgt ctgagatata atcattgatt ctatttgtaa caaggagttt taaaagaaac 1430 ctgacttcta agtgtgggtt tttcttctct ccaacataat tatgttaata tggtcctcat 1490 ttttcttttg gtgcagaacc gttgtgcagt ggggtctacc atgcaatttt ctttcagcac 1550 tgaccccttt ttaaggaata caaattttct ccttcatcac ttaggtgttt taagatgttt 1610 accttaaagt ttttcttggg gaaagaatga attaatttct atttcttaaa acatttccct 1670 gagccagtaa acagtagttt aatcattggt cttttcaaaa ctaggtgttt aaaaaaagag 1730 acatatatga tattgctgtt atatcaataa catggcacaa caagaactgt ctgccaggtc 1790 attcttcctc tttttttttt aattgggtag gacacccaat ataaaaacag tcaatatttg 1850 acaatgtgga attaccaaat taaaagagaa tactatgaat gtattcatat tttttctata 1910 ttgaataaac aatgtaacat agataacaat ataaataaaa gtggtatgac cagtgaaaaa 1970 aaaaaaaaaa aaa 1983

Claims (11)

Screening methods for the prevention or treatment of biliary tract cancer, comprising the following steps:
(a) a protein selected from the group consisting of the proteins of the first sequence and the proteins of the third sequence, or a polynucleotide of the second sequence and a polynucleotide of the fourth sequence Contacting a cell or tissue comprising the polynucleotide to be analyzed 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 bile duct cancer Is determined.
Screening methods for the prevention or treatment of biliary tract cancer, comprising the following steps:
(a) preparing a protein selected from the group consisting of the protein of the first sequence and the protein of the third sequence of the sequence listing;
(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 determined as a substance for preventing or treating biliary tract cancer.
A screening method of a substance for preventing or treating cancer, comprising the steps of:
(a) a polynucleotide selected from the group consisting of a protein of SEQ ID NO: 1 and a protein of SEQ ID NO: 3 or a polynucleotide of SEQ ID NO: 2 and polynucleotide of SEQ ID NO: 4 Contacting a sample to be analyzed with a cell or tissue comprising a nucleotide; 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 cancer. It is determined.
A screening method of a substance for preventing or treating cancer, comprising the steps of:
(a) preparing a protein selected from the group consisting of the protein of the first sequence and the protein of the third sequence of the sequence listing;
(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 cancer.
The method of claim 2, wherein the protein is present in the cell surface, in the viral surface, or in isolated form.
A binder that specifically binds to a protein selected from the group consisting of a protein of SEQ ID NO: 1 and a protein of SEQ ID NO: 3 or a polynucleotide of SEQ ID NO: 2 and a group of polynucleotides of SEQ ID NO: 4 Kit for detecting biliary tract cancer stem cell comprising a primer or probe specifically binding to a polynucleotide selected from.
A binding agent that specifically binds to a protein selected from the group consisting of the proteins of the first sequence and a protein of the third sequence, or a polynucleotide of the second sequence and a polynucleotide of the fourth sequence Wherein the primer or probe specifically binds to a polynucleotide selected from the group consisting of:
A binder that specifically binds to a protein selected from the group consisting of a protein of SEQ ID NO: 1 and a protein of SEQ ID NO: 3 or a polynucleotide of SEQ ID NO: 2 and a group of polynucleotides of SEQ ID NO: 4 Kit for diagnosing or prognostic biliary cancer comprising a primer or probe that specifically binds to a polynucleotide selected from.
A binding agent that specifically binds to a protein selected from the group consisting of the proteins of the first sequence and a protein of the third sequence, or a polynucleotide of the second sequence and a polynucleotide of the fourth sequence And a primer or a probe that specifically binds to a polynucleotide selected from the group consisting of:
10. The kit according to any one of claims 6 to 9, wherein the kit is an immunoassay kit.
The kit according to any one of claims 6 to 9, wherein the kit is a kit for gene amplification or microarray.

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