KR101360409B1 - Therapeutic Targets of Gastric Cancer Based on the Characteristics of Gastric Cancer Stem Cells - Google Patents

Abstract

The present invention provides a method for screening a substance for preventing or treating gastric cancer using gastric cancer stem cells and novel molecular markers for gastric cancer. The present invention provides a method for screening a substance for preventing or treating gastric cancer by using a novel molecular marker for cancer stem cells and cancer. The gastric cancer or cancer therapeutic target of the present invention is very useful for screening gastric cancer stem cells or therapeutic agent candidates that specifically act on cancer stem cells. In addition, the present invention can accurately analyze the diagnosis and prognosis of gastric cancer or cancer.

Description

Novel Therapeutic Target for Gastric Cancer Based on Gastric Cancer Stem Cell}

The present invention relates to novel biomarkers for gastric cancer stem cells and targets for the treatment of gastric cancer based on gastric cancer stem cell properties.

There are cancer stem cells or tumor initiating cells in cancer tissues that maintain and regenerate cancer tissues like normal organs, and these cancer stem cells are believed to be involved in the cancer process as the first cancers. It has also been reported to be involved in angiogenesis and neovascularization in cancer tissues . In addition, it has been reported to have a profound effect on cancer recurrence, metastasis and induction of chemo resistance by accelerating cancer cell invasion and regeneration of cancer cells reduced after cancer treatment (Y.-F. Ping and X.-W). Bian, Current Molecular Medicine , 11: 69-75 (2011), BB Zhou et al. Nature Reviews Drug Discovery , 8: 806-823 (2009)). Therefore, in order to fundamentally diagnose and treat cancer, it is necessary to focus on cancer stem cells, which occupy only a small part of cancer tissues and play a key role in the development and maintenance of cancer, resistance to anticancer drugs, and recurrence. It will help develop diagnostic and therapeutic markers for early diagnosis.

Cancer is the world's leading mortality rate of 1-2, and gastrointestinal cancer accounts for 45% of all cancers in Korea. Gastric cancer is the leading cause of death from cancer in Korea. Gastrointestinal cancer can be cured only by radical resection, but gastric cancer includes 20% of patients diagnosed as advanced gastric cancer that cannot be cured at the time of diagnosis. More than 84% of patients with gastric cancer are treated with chemotherapy. However, the reason why the existing drugs do not make a significant contribution to the patient's life extension even if the response rate is high is that most of the cancer cells constituting the tumor have an anticancer effect and contribute to reducing the size of the tumor, but ultimately such anticancer Cancer stem cells that survive even treatment and cancer stem cell-derived anticancer drug-resistant cancer cells are thought to be due to failure to target. These cancer stem cells or cancer initiating cells have characteristics of stem cells such as self renewal, differentiation, and proliferation, and the presence of cancer stem cells is known as breast cancer (M. Al). Hajj et al.,Proc Natl Acad Sci 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). These cancer stem cells are known to have a profound effect on cancer recurrence, metastasis, and induction of chemotherapy resistance by surviving cancer cells after their initial onset and after cancer treatment (BBB Zhou et al., 8). , 806-823, 2009). Spheres form spheres during incubation in vitro, and cells obtained from spear cultures have been reported to have stronger cancer-forming ability than the original cell lines (A. Dubrovska et al. ,Proc Natl Acad Sci USA. 106, 268-273, 2009). Therefore, in order to fundamentally treat gastric cancer, an antibiotic-resistant tumor, in addition to conventional cancer treatment, the focus is on cancer stem cells, which play a key role in the development and maintenance of cancer, neovascularization, chemotherapy resistance and recurrence, while taking up only a part of cancer tissue. It must be correct.

In the present invention, by culturing the spear cells having cancer stem cell characteristics to analyze the characteristics of cancer stem cells in gastric cancer, by identifying the specific markers of gastric cancer stem cell through the cDNA array in the spear cells early diagnosis markers, new anticancer drugs or existing anticancer drugs To develop a new anticancer therapy for cancer stem cells to treat stomach cancer that is resistant to cancer.

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 efforts to discover novel biomarkers for gastric cancer stem cells and targets for the treatment of gastric cancer based on gastric cancer stem cell characteristics. As a result, the inventors have found that proteins specifically expressed in gastric cancer stem cells may be molecular targets for gastric cancer treatment. In addition, the present inventors have identified the present invention by identifying the biomarkers discovered as markers for diagnosis based on biological characteristics of gastric cancer stem cell biological characteristics, in particular, early diagnosis, markers for prognosis, and markers for future therapeutic targets. It was completed.

Accordingly, it is an object of the present invention to provide a method for screening a substance for preventing or treating gastric 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 gastric cancer stem cell specific biomarkers.

It is still another object of the present invention to provide a kit for detecting cancer stem cell-specific biomarkers.

Still another object of the present invention is to provide a kit for diagnosing or prognosticting gastric cancer.

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

Still another object of the present invention is to provide a target of a therapeutic material for the development of anticancer drugs for gastric cancer.

Still another object of the present invention is to provide a target of a therapeutic material for developing cancer anticancer drugs.

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

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

(a) a protein selected from the group consisting of a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7 and a protein of SEQ ID NO: 9, or A polynucleotide of SEQ ID NO: 2, a polynucleotide of SEQ ID NO: 4, a polynucleotide of SEQ ID NO: 6, a polynucleotide of SEQ ID NO: 8, and a polynucleotide of SEQ ID NO: 10 Contacting a sample to be analyzed with a cell or tissue comprising a polynucleotide; 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 gastric cancer. Is determined.

The present inventors made an intensive study to discover new biomarkers for gastric cancer stem cells and targets for the treatment of gastric cancer based on the characteristics of gastric cancer stem cells, and as a result, proteins specifically expressed in gastric cancer stem cells were used to treat gastric cancer. It has been found that it can be a molecular target. In addition, the inventors have found that the biomarkers that have been excavated are markers for diagnosing gastric cancer stem cells, particularly for early diagnosis of gastric cancer, and for determining the prognosis.

The present invention has the characteristics of identifying proteins expressing specifically from gastric cancer stem cells isolated from gastric cancer cell lines, and they can be a target for gastric cancer treatment, and have a very accurate diagnosis and prognosis of gastric cancer early.

The five markers of the present invention are specifically expressed in gastric cancer stem cells. Furthermore, the five markers are markers for the ability to distinguish gastric cancer cells from gastric cancer stem cells, that is, expression patterns that are highly expressed in gastric cancer stem cells as compared to gastric cancer cells.

In the present invention, the expression "cell or tissue" used for screening a substance for preventing or treating gastric cancer is cancer stem cells or cancer tissues, preferably gastric cancer stem cells or gastric cancer tissues.

The inventors have found that the expression of a protein selected from the group consisting of the above proteins or each polynucleotide encoding them significantly increases in gastric cancer stem cells, unlike ordinary gastric cancer cells. Unlike general gastric cancer cells, the specific remarkably increased expression of a target protein or polynucleotide encoding the same in gastric cancer stem cells indicates that it is an essential factor for the survival of cancer stem cells, and blocks the expression thereof. Is determined to be a substance that helps in the fundamental treatment of gastric cancer by inducing growth inhibition and death of cancer stem cells, that is, a gastric cancer therapeutic agent.

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

(a) a protein selected from the group consisting of a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7, and a protein of SEQ ID NO: 9 Preparing;

(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 gastric cancer.

According to the screening method of the present invention, the group consisting of the protein of SEQ ID NO: 1, the protein of SEQ ID NO: 3, the protein of SEQ ID NO: 5, the protein of SEQ ID NO: 7 and the protein of SEQ ID NO: 9 The test material is contacted with a protein selected from.

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

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

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

In the screening method of the present invention, the test substance or the 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).

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

Alternatively, the binding of the test substance to the protein may be assayed without labeling of the interactants. For example, a microphysiometer can be used to analyze whether a test substance binds to QP-C. The microphysiometer is an analytical tool that uses a light-addressable potentiometric sensor (LAPS) to measure the rate at which a cell acidifies its environment. Changes in the rate of acidification can be used as an indicator of the binding between the test substance and QP-C (McConnell et al., Science 257: 19061912 (1992)).

Binding capacity of the protein and the test substance can be analyzed with the 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.

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, substances binding to the protein, particularly proteins, can be screened. To-hybrid system is based on the modular nature of the transcription factor consisting of segmentable DNA-binding and activation domains. Briefly, this analysis method uses two DNA constructs. For example, in one construct, the polynucleotide of SEQ ID NO: 2, the polynucleotide of SEQ ID NO: 4, the polynucleotide of SEQ ID NO: 6, the polynucleotide of SEQ ID NO: 8, and the sequence 10 A polynucleotide selected from the group consisting of polynucleotides of is fused to a DNA binding domain-encoding polynucleotide of a known transcription factor (eg, GAL-4). In another construct, a DNA sequence encoding a protein of interest ("prey" or "sample") is fused to a polynucleotide encoding the activation domain of the known transcription factor. If bait and prey interact to form a complex in vivo, the DNA-binding and activation domains of the transcription factor become adjacent, which triggers transcription of the reporter gene (e.g., LacZ). 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 gastric cancer.

Examples of the sample used in the present invention include peptides, antibodies, peptide aptamers, AdNectin, affibody (US Pat. No. 5,831,012), Avimer, Silverman, J. et al, Nature Biotechnology 23 (12): 1556 (2005)) or the Kunitz domain (Arnoux B et al., Acta Crystallogr . D Biol . Crystallogr . 58 (Pt 7): 12524 (2002)) and Nixon, AE, Current opinion in drug discovery & development 9 (2): 2618 (2006)).

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

(a) contacting a sample to be analyzed with a cell or tissue comprising a protein of SEQ ID NO: 3 or a polynucleotide of SEQ ID NO: 4; 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 .

The expression "cell or tissue" used for screening a substance for preventing or treating cancer in the present invention is cancer stem cells or cancer tissue, preferably gastric cancer stem cells or gastric cancer tissue.

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

(a) preparing a protein of SEQ ID NO: 3;

(b) contacting the test substance to be analyzed 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 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. By targeting stem cells, it enables cancer or the fundamental treatment of cancer.

According to another aspect of the present invention, the present invention provides a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7 and a protein of SEQ ID NO: 9 Binder or polynucleotide of SEQ ID NO: 2, polynucleotide of SEQ ID NO: 4, polynucleotide of SEQ ID NO: 6, polynucleotide of SEQ ID NO: 8, specifically binding to a protein selected from the group consisting of And a primer or probe specifically binding to a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NO: 10 sequence.

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

According to another aspect of the invention, the group consisting of the protein of SEQ ID NO: 1, the protein of SEQ ID NO: 3, the protein of SEQ ID NO: 5, the protein of SEQ ID NO: 7 and the protein of SEQ ID NO: 9 Binder or polynucleotide of SEQ ID NO: 2, polynucleotide of SEQ ID NO: 4, polynucleotide of SEQ ID NO: 6, polynucleotide of SEQ ID NO: 8, SEQ ID NO: 8 It provides a kit for gastric cancer diagnosis or prognostic analysis comprising a primer or probe specifically binding to a polynucleotide selected from the group consisting of the polynucleotide of the tenth sequence.

In particular, the kit for gastric cancer diagnosis or prognosis analysis of the present invention is a kit for gastric cancer diagnosis or prognosis analysis derived from gastric cancer stem cells.

As used herein, the expression “kit for diagnosis or prognosis of gastric cancer” means a kit including a composition for diagnosis or prognosis of gastric cancer. Therefore, the expression "kit for diagnosis or prognosis of gastric cancer" can be used interchangeably or mixed with "composition for diagnosis or prognosis of gastric cancer".

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

In the present invention, the term "gastric cancer" generally refers to a cancer that originates in gastric cells. Stomach cancer is secreted by less gastric acid, the bactericidal power is reduced and the intestinal bacteria are increased to produce a lot of nitroso compounds.The main cause of the stomach is the transition from the bony part of the stomach to the duodenum. Can be. Cancer cells in the gastric mucosa of the stomach penetrates deeply along the mucosa, submucosa, muscle layer and the mucous membrane, and severely penetrates the stomach wall and invades other organs around it. The exact cause of gastric cancer has not yet been identified, as in other cancers, but foods of risk factors that increase gastric cancer include salted or smoked foods, nitric acid, nitrite processed foods, or vegetables or drinking water with high content. Salty foods and the like are also known as a risk factor for gastric cancer. In particular, gastric cancer has no symptoms in the early stages, so it is difficult to detect early, and the symptoms are sudden, chronic gastritis, duodenum, stomach ulcer, etc. Similar to the symptoms of common diseases, early detection is more difficult.

The present inventors confirmed that using the marker of the present invention, a highly sensitive and reliable result for gastric cancer was obtained from an individual.

The expression "measurement of protein expression level" used in diagnosing gastric 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. Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip are examples of immunoprecipitation, protein immunoassay, immunohistochemical staining, The method of analysis of the invention is not limited.

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

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

Since new gastric 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 gastric 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 can be prepared using the hybridoma method (Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or a phage antibody library (Clackson et al, Nature , 352: 624-628, 1991; Marks et al, J. Mol . Biol . , 222: 58, . ≪ / RTI > The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

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.

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

According to a preferred embodiment of the present invention, the present invention provides oligopeptides, monoclonal antibodies, polyclones that specifically bind to at least one protein selected from the above protein group for diagnosing gastric cancer stem cells and / or gastric cancer. Raw antibody, chimeric antibody, ligand, PNA (Peptide nucleic acid) or aptamer, more preferably oligopeptide, monoclonal antibody, polyclonal antibody or chimeric antibody, Even more preferred are monoclonal antibodies or polyclonal antibodies, most preferably monoclonal antibodies.

In the present invention, the antibody is preferably a conjugated antibody conjugated with microparticles. The fine particles are preferably colored latex or colloidal gold particles. In the present invention, the antibody may be any antibody capable of measuring the level of expression of a protein encoded by a known mRNA gene for the 20 markers described above. Preferably, the kit is an immunoassay ), And most preferably the kit is a luminex assay kit, a protein microarray kit or an ELISA kit.

The lumenex assay, the protein microarray kit and the ELISA kit may be prepared by mixing polyclonal antibody and monoclonal antibody against any one or more proteins selected from the above protein group, and the polyclonal antibody and monoclonal antibody Lt; RTI ID = 0.0 > secondary < / RTI >

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

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

In addition, the kit may additionally include essential elements necessary for performing a protein microarray to simultaneously analyze complex markers. Microarray kits contain antibodies specific for the marker protein bound to the solid phase. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The protein microarray kit may also include antibodies specific for the control protein. Other protein microarray kits 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. It can also provide the information needed to diagnose gastric 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 As a result, it has good sensitivity (pg unit) and can be quantified in a short time (3-4 hours), and it can replace ELISA or ELISPOT. The Luminex Assay is a multiplexed fluorescent microplate assay capable of analyzing more than 100 biological samples simultaneously in each well in a 96-well plate. And more than 100 different color groups of polystyrene beads are discriminated and quantified by carrying out signal transmission in real time using a laser detector of the present invention. The 100 beads are configured to be distinguished in the following manner. On one side, the red fluorescence bead is divided into more than ten stages. On the other side, the orange fluorescence bead is divided into ten stages to show the intensity difference and the bead between them. Are mixed in different ratios of red and orange to form a total of 100 color-coded bead sets. In addition, each bead has an antibody of the protein to be analyzed, so that it is possible to quantify the protein by the immunoassay using the antibody. The sample is analyzed using two lasers, one laser detects the bead's unique number and the other laser reacts with the antibody attached to the bead The protein in the sample is detected. Thus, 100 in vivo protein analysis is possible simultaneously in one well. This assay has the advantage of being detectable with as little as 15 μl of sample.

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

In the kit for diagnosing gastric cancer or prognostic analysis of the present invention, the gastric cancer diagnostic kit including the immunochromatography strip for diagnosing gastric cancer includes essential elements necessary for performing a rapid test in which a result of analysis can be known within 5 minutes. It may be a diagnostic kit characterized in that. The immunochromatographic strip comprises (a) a sample pad on which a sample is absorbed; (b) a conjugate pad which binds to a protein of any one or more genes selected from the above 20 genes in the sample; (c) a test membrane on which a test line and a control line containing a monoclonal antibody against the protein of one or more genes selected from the 20 gene groups are treated; (d) an absorption pad on which a residual amount of the sample is absorbed; And (e) a support. Also, a rapid test kit containing an immunochromatographic strip contains antibodies specific for the marker protein. The antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for each marker protein and little cross-reactivity to other proteins. The rapid test kit may also include an antibody specific for the control protein. Other rapid test kits include reagents 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.

The measurement of the level of protein expression by the immunodet assay in the present invention includes (a) dotting a biological sample on the membrane; (b) reacting an antibody specific to the protein of one or more genes selected from the group consisting of the 20 genes to the dormant membrane; And (c) a step of adding a secondary antibody conjugated with a label to the membrane to be subjected to the reaction and coloring, and wherein the ELISA assay comprises (a) a gene group having a nucleotide sequence for the 20 markers The method comprising the steps of: (a) adsorbing antibody 1 specific to a protein of any one or more genes selected from the group consisting of: (b) contacting the antibody 1 adsorbed to the solid body with a biological sample of a suspected gastric cancer patient to form an antigen-antibody complex; (c) treating antibody 2 specific to a protein encoded by at least one gene selected from the group of genes having the nucleotide sequence of the 20 markers to which the labeling substance is bound, to bind with the complex; And (d) measuring the concentration of the protein by detecting the labeling substance, wherein the protein microarray assay comprises the steps of: (a) selecting a gene group selected from the group consisting of the 20 markers; Immobilizing a polyclonal antibody specific to the protein of one or more genes on the chip; (b) contacting the immobilized polyclonal antibody 1 with a biological sample of a suspected gastric cancer to form an antigen-antibody complex; (c) treating a monoclonal antibody specific to a protein encoded by any one or more genes selected from the group consisting of the genes having the nucleotide sequences of the 20 markers to which the labeling substance is bound, to bind with the complex; And (d) measuring the concentration of the protein by detecting the labeled substance.

Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected gastric cancer patients can be compared, and whether significant increase in the expression level of gastric cancer marker gene to protein can be compared. By judging, it is possible to diagnose whether the gastric cancer suspect patient actually develops gastric cancer.

As used herein, the term “antigen-antibody complex” means a combination of a gastric cancer marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be measured quantitatively through the size of a signal of a detection label. Do.

Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructoketase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re.

Protein expression level measurement is preferably by using an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is 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 gastric cancer marker protein and antibody, it is possible to determine whether the gastric cancer.

In addition, preferably, Western blot using at least one antibody against the gastric 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 confirming the amount of the generated antigen-antibody complexes using a labeled antibody, the amount of protein produced by the expression of a gene can be confirmed, thereby confirming whether gastric cancer develops. The detection method consists of a method for examining the expression level of the marker gene in the control group and the expression level of the marker gene in cells with gastric 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 gastric cancer marker. After collecting and fixing normal gastric tissue and tissue suspected of gastric cancer, paraffin embedding blocks are prepared by methods well known in the art. These are cut into pieces of several μm thick and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.

In addition, preferably, one or more antibodies against the gastric cancer marker are arranged at a predetermined position on the substrate to use a protein chip 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 to confirm stomach cancer. You can check whether you have the disease.

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

The expression “polynucleotide measurement” used for diagnosing gastric 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. . RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

The nucleotide sequence encoding each of the protein markers selected from the protein group used as a marker in the present invention may include a nucleotide sequence having homology with these nucleotide sequences.

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

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

The term " primer " of the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences for each marker gene-specific primer. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include 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. The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention may also be modified using a label capable of directly or indirectly providing a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

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

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

According to another aspect of the present invention, a cancer stem cell detection kit comprising a binding agent that specifically binds to a protein of SEQ ID NO: 3 or a primer or probe that specifically binds to a polynucleotide of SEQ ID NO: 4 to provide.

The cancer may include, for example, 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, head and neck cancer, skin cancer, But not limited to thyroid cancer, parathyroid cancer and ureter cancer.

According to another aspect of the invention, the group consisting of the protein of SEQ ID NO: 1, the protein of SEQ ID NO: 3, the protein of SEQ ID NO: 5, the protein of SEQ ID NO: 7 and the protein of SEQ ID NO: 9 Binder or polynucleotide of SEQ ID NO: 2, polynucleotide of SEQ ID NO: 4, polynucleotide of SEQ ID NO: 6, polynucleotide of SEQ ID NO: 8, SEQ ID NO: 8 It provides a kit for gastric cancer diagnosis or prognostic analysis comprising a primer or probe specifically binding to a polynucleotide selected from the group consisting of the polynucleotide of the tenth sequence.

As used herein, the expression “kit for diagnosis or prognosis of gastric cancer” means a kit including a composition for diagnosis or prognosis of gastric cancer. Therefore, the expression "kit for diagnosis or prognosis of gastric cancer" can be used interchangeably or mixed with "composition for diagnosis or prognosis of gastric cancer".

According to another aspect of the present invention, a kit for diagnosing cancer or prognostic analysis comprising a binder that specifically binds to a protein of SEQ ID NO: 3 or a primer or probe that specifically binds to a polynucleotide of SEQ ID NO: 4 To provide.

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 gastric cancer using gastric cancer stem cells and novel molecular markers for gastric cancer.

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

(Iii) The gastric cancer or cancer therapeutic target of the present invention is very useful for screening gastric cancer stem cells or therapeutic agent candidates that specifically act on cancer stem cells.

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

1 is a diagram showing a cell line forming a sphere among 11 human gastric cancer cell lines.
Figure 2 is a diagram showing the stem cell-related gene expression changes in adhesion cells and spear cells of gastric cancer cell line N87. RT-PCR data was normalized to the mRNA level of the Beta-actin gene.
Figure 3 is a diagram showing the change in gene expression of gastric cancer stem cell-related novel markers in spear cells cultured from N87 and SNU484 cells. RT-PCR data was normalized to the mRNA level of the Beta-actin gene.
Figure 4 is a diagram showing the change in expression of gastric cancer stem cell-related new markers in tissue TMA of human gastric cancer patients through immunohistochemistry (10 x 10 magnification).
(a) anti-ABCA5 antibody, (b) anti-LINGO2 antibody, (c) anti-LGR4 antibody, (d) anti-LRRN3 antibody, (e) anti-Ero1L antibody
5 is a diagram showing the expression changes of gastric cancer stem cell-related new markers in tissue TMA of human gastric cancer patients through immunohistochemistry (40 × 10 magnification).
(a) anti-ABCA5 antibody, (b) anti-LINGO2 antibody, (c) anti-LGR4 antibody, (d) anti-LRRN3 antibody, (e) anti-Ero1L antibody
6 is a diagram showing cell sorting after indirect FACS staining of human gastric cancer cell lines N87 and SNU484 cells cultured in normal medium using ABCA5, LINGO2, LGR4 and LRRN3 antibodies.
(a) cells sorted using anti-ABCA5 antibody, (b) cells sorted using anti-LINGO2 antibody, (c) cells sorted using anti-LGR4 antibody, and (d) anti-LRRN3 antibodies. Cell sorted using
Figure 7 is a diagram showing the cancer stem cell characteristics of the cells separated by each marker antibody via Western blot. Data was normalized to the expression level of GAPDH protein.
8 is a diagram showing the cancer stem cell characteristics of the cells separated by each marker antibody through a colonogenic assay.
(a) cells sorted using anti-ABCA5 antibody, (b) cells sorted using anti-LINGO2 antibody, (c) cells sorted using anti-LGR4 antibody
9 is a subcutaneous injection of LGR4 expressing cells into immune control mice (NOD-SCID), observed for 10 weeks, and dissected and immunostained.
(a) Tumor size comparison, (b) H & E staining to confirm histopathologic morphology
FIG. 10 is a diagram showing LINGO2 expressing cells subcutaneously in the flanks of immune control mice (NOD-SCID) and observed for 15 weeks, followed by dissection to perform immunostaining.
(a) Tumor size change over time after subcutaneous injection, (b) Tumor size difference according to the number of subcutaneous injection cells, (c) Immunostaining using anti-CD34 antibody or anti-pVEGFR2 antibody.
11 is a result of confirming the expression of LINGO2 through RT-PCR after extracting mRNA from nine gastric cancer cell lines of AGS, N87, SNU1, SNU5, SNU16, SNU484, SNU601, SNU638, and SNU668. RT-PCR data was normalized to the mRNA level of the Beta-actin gene.
12 shows the results of transfection of LINGO2 shRNA plasmid into gastric cancer cell line SNU484, followed by Western blot and flow cytometry for decreased expression of LINGO2. Western blot data was normalized to the expression level of GAPDH protein.
(a) Western blot, (b) Flow cytometry
Figure 13 is the result of analyzing the change of various characteristics of cancer cells according to LINGO2 knock-down (k / d).
(a) cell morphology, (b) cell proliferation assay, (c) cell healing assay, (d) cancer cell metastasis assay, (e) cancer cell Invasion assay
Figure 14 shows the results confirmed by Western blot changes in various signaling systems in accordance with the inhibition of LINGO2 expression. Western blot data was normalized to the expression level of GAPDH protein. [Epithelial to Mesenchymal transition related proteins, PI3K signaling system and MAPK signaling system]
FIG. 15 shows the results of Western blot and zymography confirming changes in expression and activity of MMP proteins in LINGO2 knock-down (k / d) cell line cultures.

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

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

Of the 11 human gastric cancer cell lines, N87, AGS, SNU1, SNU5, SNU16, and NIH3T3, a mouse fibroblast, were purchased from the American Type Culture Collection (ATCC), and human gastric cancer cell lines SNU216, SNU484, SNU601, SNU638, and SNU668. And SNU719 were purchased from Korean Cell Line Bank (KCLB, Korean Cell Line Bank, Seoul, Korea). Cell lines were cultured in RPMI1640 (Invitrogen Gibco, Grand Island, NY) growth medium containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT) by the protocol presented by ATCC and the Korean Cell Line Bank. Gastric cancer cell lines were cultured with EGF (10 ng / ml, R & D Systems Inc., Minneapolis, MN), bFGF (10 ng / ml, R & D Systems Inc.), insulin transfer selenium (GIbco) and 0.5 for sphere culture. Incubate in 6-well ultra-low cluster plates (Corning Inc., Corning NY) at a rate of 1000 cells / ml in serum-free RPMI medium supplemented with% BSA (Invitrogen Gibco, Grand Island, NY) for 7-10 days (D. Ponti et al., Cancer Res. 65 (13), 5506-5511 (2005)). Cells grown in the adherent state were attached to a culture dish (Nalgene Nunc Int., Rochester, NY) with the same culture medium as a control. Culture supernatants of NIH3T3 cells were used for cell metastasis and infiltration assays.

Expression Analysis of Cancer Stem Cell Related Genes in Spear Cells

Total RNA was extracted from adherent and spear cells of N87 and SNU484 using RNAeasy Mini kit (QIAGEN, Valencia, Calif.). 2 μg of each RNA extracted at 42 ° C for one hour was subjected to reverse transcription reaction 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 beta-actin 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 Jagged 2 GTGGATGTCGACCTTTGTGA GGCAGTCGTCAATGTTCTCA 61 ° C / 30 Notch3 ATGGTGGGAACTAAACACAGCT ATGACCCTGGAGGAAGCACA 55 ° C / 35 Hes1 GTGCTGTCTGGATGCGGAGT GAACACTCACACTCAAAGCCC 55 ° C / 35 Ihh CCTGAACTCGCTGGCTATCT AATACACCCAGTCAAAGCCG 56 ° C / 35 Smo GAATCGCTACCCTGCTGTTA TGAGCAGGTGGAAGTAGGAG 59 ° C / 30 Gli1 AGAGTCCAGGGGGTTACATA AGAGTCCAGGGGGTTACATA 57 ℃ / 35 FZD7 CCAACGGCCTGATGTACTTT GCCATGCCGAAGAAGTAGAG 61 ° C / 30 β-catenin GTATGAGTGGGAACAGGGATTT CCTGGTCCTCGTCATTTAGC 52 ℃ / 25 Nanog ACTGTCTCTCCTCTTCCTTCCT AGAGTAAAGGCTGGGGTAGGTA 61 ° C / 30 Oct4 GTGGAGGAAGCTGACAACAA AGCAGCCTCAAAATCCTCTC 62 ° C / 27 PTEN GGACGAACTGGTGTAATGAT CAGACCACAAACTGAGGATT 56 ° C / 30 β -actin GGCATCCTCACCCTGAAGTA GGGGTGTTGAAGGTCTCAAA 55 ° C / 25

cDNA Microarray

Total RNA was extracted from adherent and spear cells of N87 and SNU484 using the RNAeasy Mini kit (QIAGEN, Valencia, Calif.) 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). Dual yarn cDNAs were 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). Expression data was generated using Affymetrix Expression Console software version 1.1 and standardized with 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 1.5-fold differences. All individual chip analyzes were performed twice for independent total RNA samples.

Expression of gastric cancer stem cell-related targets derived from cDNA array

1) Semi-quantitative RT-PCR

Total RNA was extracted from adherent and spear cells of N87 and SNU484 using RNAeasy Mini kit (QIAGEN, Valencia, Calif.). 2 μg of each RNA extracted at 42 ° C for one hour was subjected to reverse transcription reaction through the reaction of Superscript II (Gibco BRL, Grand Island, NY). Expression of spear cells was analyzed by PCR with Taq polymerase and primers of gastric cancer stem cell-related target genes derived from cDNA array analysis, and the expression of betaactin gene was used as a control.

2) Gastric cancer stem cells in human gastric cancer tissues through immunohistochemical staining Of targets  Expression analysis

Human gastric 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 mouse monoclonal LINGO2 (Lifespan biosciences, seattle, WA), goth polyclonal ABCA5 (Abnova corporation, Taipei), rabbit polyclonal LGR4 (Sigma, St. Louis, MO), rabbit polyclonal LRRN3 (Abcam, Cambridge, UK), mouse monoclonal Ero1L (santa cruz biotechnology Inc, santa cruz, CA). Blocked sections were incubated overnight at 4 ° C. with the primary antibody. Subsequent reactions were carried out using the recommended procedures included in the Envision kit (DakoCytomation, Carpineteria, Calif., USA) or the Goat HRP-Polymer kit (Biocare medical, Pike Lane Concord, CA). Finally, slides were incubated with 3,3'-diaminobenzidine (DkoCytomation, Carpinteria, Calif., USA), and modified Harris hematoxylin (Sigma-Aldrich, Inc., St. Louis, MO) solution.

3) Separation of Cells by Gastric Cancer Stem Cell-Related Marker Antibodies and Characterization of Cancer Stem Cells of Isolated Cells

Gastric cancer cell lines N87 or SNU484 cells cultured in normal culture were treated with accutase (Accutase; Sigma-Aldrich, St. Louis, MO) to prepare the same number of single cell suspensions. Then, in direct staining was performed with the antibodies of gastric cancer stem cell-related targets derived from the present invention. Incubated for 20 minutes with primary antibody or isotype control corresponding to each antibody, and then incubated for 20 minutes on ice with a fluorescent antibody conjugated secondary antibody. Primary antibodies were mouse monoclonal LINGO2 (Lifespan biosciences, seattle, WA), goth polyclonal ABCA5 (Abnova corporation, Taipei), rabbit polyclonal LGR4 (Sigma, St. Louis, MO), rabbit polyclonal LRRN3 (Abcam, Cambridge, UK). Isotype control and fluorescent secondary antibodies were purified mouse IgG2a, k isotype control, PE goth anti-mouse IgG (BD Biosciences PharMingen, San Diego, Calif.), Normal rabbit IgG, goth anti-rabbit IgG-PE (santa cruz). biotechnology Inc, santa cruz, CA), normal goth IgG and donkey anti-goth IgG-PE (santa cruz biotechnology Inc, santa cruz, CA). Cell sorting was performed using FACSAria II (BD Immunocytochemistry System, Franklin Lakes, NJ).

4) Gastric cancer stem cell related target  Expression Analysis of Cancer Stem Cell-Related Proteins in Cells Isolated by Antibodies

Cells classified by antibodies of gastric cancer stem cell-related targets derived from the present invention were cultured in the same number and cultured for 7-10 days, followed by protein extraction, and electrophoresis after loading on SDS-polyacrylamide gel. It was. 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 overnight at 4 ° C. with a primary antibody corresponding to well known cancer stem cell related proteins. Primary antibodies include rabbit polyclonal OCT4 (Cell Signaling Technology, Inc., Danver, MA), rabbit polyclonal Sox2 (Cell Signaling Technology, Inc., Danver, MA), mouse monoclonal PTEN (santa cruz biotechnology Inc , santa cruz, CA), rabbit polyclonal GLI1 (santa cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal HEY1 (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).

5) Gastric cancer stem cell related target  Confirmation of colonization of cells separated by antibody

Modifications were made to standard protocols of the soft agar colony forming assay (MJ Son. Et al., Cell stem cells . 4, 440-452, 2009). Difco agar noble (Becton, Dickinson company, Sparks, MD) was used, and 0.6% bottom agar was added to a 24 well plate at a ratio of 1: 1 with 2X sphere culture and allowed to solidify at room temperature. The sorted cells suspended in the spear culture and agar were mixed to make 0.3% top agar, which was dispensed on the lower agar. Spear culture medium containing the growth factor (Growth factor) was changed 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 cell colonies were counted to compare self renewal and anchorage independent growth.

6) Gastric cancer stem cell related target  Of cells isolated by antibodies Darkening ability  Confirm

Cells sorted via FACS were washed with serum-free HBSS (Gibco BRL, Grand Island, NY), followed by serum-free RPMI (Gibco BRL, Grand Island, NY) / Matrigel (BD Biosciences PharMingen, San Diego, CA) (1: 1 volume) (C. Li. Et al., Cancer Res . 67 (3) 1030-1037, 2007). Subsequently, subcutaneous injection was performed on both sides of nude mice (Orientbio, Seoul) or NOD-SCID mice (FOX Chase Cancer Center, Philadelphia, PA). After 14-16 weeks of observation and dissection.

7) CD34  & pVEGFR2  Immunohistochemical staining

Tissue slides of mouse cancer obtained by subcutaneous injection of cells sorted with LINGO2 antibody 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. To confirm angiogenesis, rat monoclonal CD34 (Abcam, Cambridge, UK) and rabbit polyclonal phospho-VEGFR2 (Cell signaling technology, Inc., Danver, MA) antibodies were diluted 1:50 to overnight at 4 ° C. Incubated. The secondary antibody against CD34 was diluted 1: 200 anti-rat IgG-HRP (santa cruz biotechnology Inc, santa cruz, CA) and incubated for 1 hour at room temperature. Responses to pVEGFR2 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.

Gastric cancer cell line LINGO2 mRNA  Confirmation of expression

Total RNA was extracted from a total of nine gastric cancer cell lines (AGS, N87, SNU1, SNU5, SNU16, SNU484, SNU601, SNU638 and SNU668) using the RNAeasy Mini kit (QIAGEN, Valencia, CA). 2 μg of each RNA extracted at 42 ° C for one hour was subjected to reverse transcription reaction through the reaction of Superscript II (Gibco BRL, Grand Island, NY). LINGO2 expression in gastric cancer cell lines was analyzed by PCR with the LINGO2 gene primer and Taq polymerase, and the beta-actin gene was used as a control.

LINGO2  Expression inhibition (knock-down, k / d) cell line construction

LINGO2 shRNA plasmids and control shRNA plasmids (SureSiliencing shRNA plasmids) were purchased from QIAGEN (QIAGEN, Valencia, CA), and transfected into the SNU484 cell line to identify various characteristic changes due to inhibition of LINGO2 expression. k / d) cell lines were established.

The sequence of shRNA for LINGO2 used in the present invention is AGA CTT GAG TGA CAA CAT CAT, the sequence of control shRNA is GGA ATC TCA TTC GAT GCA TAC, and as a selection marker, 2.0 μg / ml of puromycin (Sigma-Aldrich, Inc., St. Louis, Mo.). Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) Was used to transfect cells with LINGO2 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). Later, Western blot and flow cytometry confirmed LINGO2 knock-down.

LINGO2  Knock- Down (k / d) cell line  Check for various characteristic changes

1) rate of cell proliferation ( Proliferation assay )

Each cell was attached to a 24 well-plate of 1.1 × 10 ⁴ , and then, every 2 days for 8 days, the cells were separated by 0.25% trypsin 37 EDTA and the number of cells was measured on a hematocytometer.

2) Cell proliferation assay wound healing assay )

After cell culture, the number of cells was almost 100% filled and the scratches were given using a 200 μl tip, and the scratch recovery ability over time was observed under a microscope.

3) 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.

4) Western Blat

The primary antibodies used in Western blot to examine various signaling systems by inhibition of LINGO2 expression were mouse monoclonal LINGO2 (Lifespan biosciences, seattle, WA), rabbit polyclonal E-cadherin (santa cruz biotechnology Inc, santa cruz). , CA), mouse monoclonal N-cadherin (Abcam plc., Cambridge, MA), rabbit polyclonal phospho-GSK3 (Cell Signaling Technology, Inc., Danver, MA), rabbit polyclonal phospho-AKT (Cell Signaling Technology, Inc., Danver, MA), rabbit polyclonal AKT (santa cruz biotechnology Inc, santa cruz, CA), rabbit polyclonal phospho-MEK (Cell Signaling Technology, Inc., Danver, MA), rabbit poly Clonal 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 cruz, CA) ), Mouse monoclonal MMP-1 (santa cruz biotechnology Inc, santa cruz, CA), rabbit monoclonal MMP-9 (Abcam pl c., Cambridge, MA) and mouse monoclonal GAPDH (santa cruz biotechnology Inc, santa cruz, CA).

5) Zymography ( Zymography )

In order to determine the gelatinase activity of MMP2 and MMP9 in the culture of control (mock) and LINGO2 knock-down (k / d) cells, egoography was performed. The same number of cells of 5 × 10 ⁵ were dispensed into 100 mm dishes, and 10 ml of serum-free RPMI medium (Invitrogen, Carlsbad, CA) was added thereto and cultured for 48 hours. The culture supernatant was then obtained via centrifugation and concentrated using an Amicon Ultra Centrifugal Filter Device (Milipore ireland Ltd., Tullagreen) with a 3 kDa molecular-mass cut off (MWCO). The concentrates were mixed at a ratio of 1: 1 with the sample buffer at 20 μl each, and then loaded on 10% Zymogram gel (Bio-rad, Hercules CA) and electrophoresed at 100 V for 90 minutes. After immersing the gel in a buffer containing 2.5% Triton X100, and then regenerated at room temperature for 30 minutes, it was changed to a development solution (Bio-rad, Hercules CA) and reacted at 37 ° C. overnight. After staining with Coomassie blue (Bio-rad, Hercules CA), it was discolored until a clear band showing the gelatinase activity of MMP2 and MMP9 was clearly identified. The gel was scanned after drying.

 Experiment result

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

As a result of confirming whether or not the spear formation showing the characteristics of the cancer stem cells, it was confirmed that spear is formed in the N87 and SNU484 cell line of a total of 11 human gastric cancer cell lines (Fig. 1).

Spear  Expression of Cancer Stem Cell Related Gene in Cells

Semi-quantitative RT-PCR confirmed that the stem cell-related signaling systems such as Notch, Hedgehog, and Wnt were activated in spear cells compared to the adhesion cells of gastric cancer cell line N87. In addition, oct4, nanog, which indicates the multipotent of stem cells, and PTEN involved in the maintenance of stem cells have been increased, and it was confirmed that a large amount of cancer stem cells were contained in the spear cells (FIG. 2).

Development of Cancer Stem Cell Gene Database to Identify Gastric Cancer Stem Cell-related Markers

In order to analyze differently expressed genes in the spheres and adherent cells of N87 and SNU484 cells and to identify target markers related to gastric cancer cancer stem cells, cDNA microarray was performed using Affymetrix GeneChip HG U133. Exposed markers of gastric cancer stem cells are shown in Table 2.

Spear Genetic symbol Gene name Ratio change of expression level RefSeq
Transcript ID N87 SNU484 ABCA5 ATP-binding cassette, sub-family A (ABC1), member 5 1.631


2.306
NM_018672 N87 ERO1L ERO1-like (S. cerevisiae) 2.245 NM_014584 SNU484 LGR4 Leucine-rich repeat-containing G protein-coupled receptor 4 2.684 NM_018490 SNU484 LINGO2 Leucine rich repeat and Ig domain containing 2 2.881 NM_152570 SNU484 LRRN3 Leucine rich repeat neuronal 3 6.658 NM_001099658

Gastric cancer stem cell-related new Markers mRNA  Confirmation of expression

RT-PCR was performed on spear cells cultured from N87 and SNU484 cells to verify mRNA expression of new markers related to gastric cancer stem cells, and the primer sequences for the genes are shown in Table 3.

gene Forward primer sequence (5'-3 ') Reverse primer sequence (5'-3 ') ABCA5 CATGCTTTTGCCATTGAAGA GCGTTCTTGGTTCTCCAGTT Ero1L CACCTCAGCCTCCCAAGTAG GAGGGCACGATTTTGAGGTA LGR4 TGTGGCTTCTGTTCATTTCG TGGCCTTACAAAATTACAACACA LINGO2 TTGCAAATATTGGCGTTCTG TGATGCAAGGCTTTAACAAATG LRRN3 AGGCCAATTCAGTTTTGGTG GTGCAAACCTTTGGTGGTG

RT-PCR showed that the mRNA expression of ABCA5 and LINGO2 was increased in both the spheres of N87 and SNU484, and the expression of Ero1L, LGR4 and LRRN3 was increased in the N87 or SNU484 spheres (Fig. 3). Like the genes that previously showed the characteristics of cancer stem cells shown in FIG. 2, the increase in the spear cells found in this study indicates the potential as a novel marker for gastric cancer stem cells.

New Gastric Cancer Stem Cells in Human Gastric Cancer Tissues On the markers  Expression analysis

Expression of new markers related to gastric cancer stem cells was verified by immunohistochemistry in tissue TMA of human gastric cancer patients (FIGS. 4A-E and 5). Five new cancer-related markers of gastric cancer stem cells, including ABCA5, do not express in adjacent normal cells, but are expressed in more than 50% of adenomatous polyp and gastric cancer tissue known as precancerous lesions. It can be seen that it is involved in the early cancer process. Figure 5 shows the expression of each marker in the gastric cancer tissue in a large magnification photograph.

Cell Separation by Gastric Cancer Cancer Stem Cell-Related Marker Antibodies and Identification of Cancer Stem Cell Characteristics of Isolated Cells

In order to verify the cancer stem cell association of the cells isolated by the gastric cancer stem cell-associated marker antibody derived from the present invention, human gastric cancer cell lines N87 and SNU484 cells cultured in normal medium using ABCA5, LINGO2, LGR4 and LRRN3 antibodies. By indirect FACS staining and cell sorting was confirmed.

The expression of ABCA5 in gastric cancer cell lines N87 and SNU484 cells was 5.7% and 18.1% (Figure 6a), LINGO2 expression was 68.8% and 81.5% (Figure 6b), LGR4 expression was 38.2% and 87.7% (Figure 6c) and LRRN3 Expression of 27.5% and 9.2% (Fig. 6D), respectively. The expression difference was different according to each marker and cell line. This difference is thought to be greater in patients with gastric cancer, and the expression of CD44, CD24 and ESA markers, already known as various cancer stem cell markers, is also shown in C. Li. Back 2007 Cancer It can also be confirmed by the results reported in Res . (C. Li. Et al., Cancer Res . 67 (3) 1030-1037 (2007). It was confirmed that cell separation by novel markers ABCA5, LINGO2, LGR4 and LRRN3 antibodies (FIGS. 6A-6D), and Western blot and colony assays to determine the cancer stem cell characteristics of the cells separated by each marker. clonogenic assay) was performed (FIGS. 7 and 8A-C).

Among the cells isolated by the novel marker ABCA5 or LGR4 antibody related to gastric cancer stem cells, it was confirmed that the expression of OCT4 and SOX2, which indicates the multipotent of stem cells, was increased in cells expressing each marker (Fig. 7 ( B) and (C)). In the case of cells isolated by the LINGO2 antibody (Fig. 7 (A)), OCT4, PTEN involved in stem cell maintenance, and Hedgehog pathway, which are signals activated by cancer stem cells, are also expressed in cells expressing LINGO2. The transcription factors GLI1 and HEY1, the transcription factors of the Notch pathway, have been increased, confirming that these new markers are closely related to cancer stem cells. In addition, the results of colony formation analysis for determining the self-renewal and non-adherent proliferation, which are the most important characteristics of cancer stem cells, are shown in FIGS. 8A-C.

Gastric cancer stem cell-related markers LINGO2, ABCA5, LRRN3 and LGR4 cells expressing more colony results, showing the self-renewal ability and non-adherent proliferation characteristics of cancer stem cells .

Gastric cancer stem cell novel marker LGR4 Separated by LGR4 - high  Cell Darkening ability  Verification

In order to confirm the tumorigenic potential of LGR4 expressing cells (LGR4-high cells) which have been confirmed to have stem cell characteristics through the above experiment, cells expressing LGR4 low on both sides of NOD-SCID mice (LGR4). -low cells) and LGR4-high cells were injected subcutaneously. Observation and dissection for 10 weeks is shown in FIG. 9. Subcutaneous injection was divided into 500 and 1000 cell groups through limited dilution assay to confirm tumor initiation. As shown in FIG. 9A, when 500 cells are injected subcutaneously, cells expressing LGR4 low do not form tumors, whereas cells expressing LGR4 high form tumors, and LGR4 is a marker for gastric cancer stem cells and gastric cancer. It was confirmed that it has the potential as a therapeutic target.

Gastric cancer stem cell novel marker LINGO2 Separated by LINGO2 - high  Cell Darkening ability  Association of neovascularization and neovascularization

Expression of mRNA in spear cells and cell surface was increased and involved in the early cancerous process of gastric cancer, and a small group of LINGO2-high cells (LINGO2-high cells), which have been shown to have various cancer stem cell characteristics, in vivo Preliminary experiments were performed by first subcutaneously injecting LINGO2-high cells and LINGO2-highly expressed cells (LINGO2-low cells) sorted through FACS to nude mice to confirm whether they had tumorigenic potential in the stomach. Is shown in Figure 10a. Cells were isolated using LINGO2 antibody, and the isolated LINGO2-low cells and LINGO2-high cells were subcutaneously injected into the flanks of nude mice, respectively, 15 weeks and dissected 15 weeks later.

When 500 cells injected by LINGO2 marker antibody were injected, the size and weight of tumors formed in mice injected with LINGO2-high cells after 15 weeks was 1.5 times higher than that of mice injected with LINGO2-low cells. It was. In addition, tumors formed from LINGO2-high cells showed much more blood vessel formation than tumors of LINGO2-low cells, indicating that LINGO2 markers are associated with neovascularization. For more accurate verification, the results obtained after observing the cells separated by the LINGO2 marker in the immune control mice (NOD-SCID) for 15 weeks by subcutaneous injection of 250 and 1000 cells, respectively, are shown in FIGS. 10B and 10C.

As with the results in nude mice, the tumor was formed larger when LINGO2-high cells were injected, and the neovascularization was confirmed again. In addition, tumors obtained by injecting LINGO2-high cells into mice and tumors obtained by injecting LINGO2-low cells into mice were immunized with angiogenesis markers CD34 and phospho VEGFR2 to verify the association between LINGO2 and angiogenesis. As a result of staining, it was confirmed that the expression of CD34 and phospho VEGFR2 was stronger in LINGO2-high tumors.

LINGO2  Expression inhibition (knock-down, k / d) cell line construction and characterization

RT-PCR confirmed whether LINGO2 expression in gastric cancer cell lines before constructing LINGO2 expression inhibition cell line, it was found that all nine gastric cancer cell lines express LINGO2 (FIG. 11).

In order to analyze the various properties that appear upon inhibition of LINGO2 expression, the LRNAO knock-down (k / d) cell line was established by permanent injection of shRNA plasmid against LINGO2 and control shRNA plasmid into SNU484 cells. The established LINGO2 knock-down (k / d) cell line was found to have reduced expression of LINGO2 protein compared to the control (FIGS. 12A-B). Inhibition of LINGO2 expression did not show a significant difference in cell proliferation rate (FIG. 13B), but the rate of cell proliferation movement (FIG. 13C), cancer cell metastasis capacity (FIG. 13D) and cancer cell infiltration capacity (FIG. 13E) were significantly lower than those of the control group. Indicated. In addition, when the expression of LINGO2 was suppressed, it was confirmed that the expression of N-cadherin, which is a marker of mesenchymal cells, and the phosphorylation activity of AKT and ERK were greatly reduced (FIG. 14).

FIG. 15 shows the results of confirming changes in expression and activity of MMP proteins in culture of control cells and LINGO2 knock-down (k / d) cells. MMP proteins play an important role in neovascularization. MMP1 and MMP9 secretion is significantly lowered in LINGO2-inhibited cells compared to control cells. Could know.

Epithelial mesenchymal cell migration has resulted in cancerous processes, recurrence and metastasis, and cell survival and drug resistance have been shown through AKT and ERK phosphorylation in cancer cells of various organs. In addition, activation of signaling systems by phosphorylation of these proteins is known to promote the expression and activation of MMP2 and MMP9, which play a large role in angiogenesis (Lionel Larue and Alfonso Bellacosa, Oncogene , 24: 7443-7454 (2005). )). In conclusion, this series of processes is initiated and accelerated by cancer stem cells, and based on the above results, LINGO2 plays an important role in the microenvironment of cancer stem cells and may be a potential target for neovascularization therapy. Can be predicted.

Review

In the present invention, cancer is considered to be the first initiation of cancer, focusing on cancer stem cells or tumor initiating cells known to cause neovascularization, recurrence or metastasis, and chemotherapy resistance. To develop new therapies targeting stem cells, markers related to gastric cancer stem cells were derived. Spear cells with cancer stem cell characteristics were cultured from gastric cancer cell lines, and the spheres were verified to have cancer stem cell characteristics. Well-known stem cell-related 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, are activated in these sphere cells. In addition, it was found that the expression of oct4 and nanog genes, which are known to increase in the early stage of development and indicate the multipotent ability of stem cells, was increased. In addition, the expression of PTEN, which plays an important role in the survival and maintenance of cancer stem cells, has also been increased, consistent with previous reports on stem cells (J. Zhou et al., Proc) . Natl Acad Sci USA , 104: 16158-16163, (2007)).

Based on these results, gastric cancer stem cell-related markers (ABCA5, Ero1L, LGR4, LINGO2, LRRN3) were selected through cDNA array in spheres formed from gastric cancer cell lines. Five new markers were increased in spheres formed from gastric cancer cell lines, and expression in human gastric cancer tissues was confirmed to be involved in the early cancer process. When the cells were separated by each marker antibody, signaling indicating the characteristics of cancer stem cells was activated in the cells expressing the new markers, and the stem cells had self-renewal characteristics. there was.

ABCA5 is known to be a member of the ABC (ATP-binding cassette) transporter superfamily and has been reported to be elevated in malignant melanoma (I Vet al., Actas Dermosifiliogr . 101 (4): 341-8 (2010)). In addition, human mesothelioma cells have been reported to inhibit the expression of ABC genes including ABCA5 and susceptibility to doxorubicin by inhibiting ERK1 and ERK2, which play an important role in multidrug resistance and cancer cell survival. (A. Shukla et al., Mol Cancer , 15: 314, (2010)) can be expected to be potential targets for the treatment of anticancer drug resistant tumors.

In 2005 Oncogene, D. May et al. Reported that Ero1L is induced by the hypoxia environment (Hypoxia) in vitro and co-induced with VEGF in the hypoxic tumor microenvironment (D. May etl al., Oncogne) . , 24 (6): 1011-20 (2005)). The hypoxic environment is a common environment for fast-growing tumor cells. Considering that the secretion of VEGF induced by hypoxic conditions plays an important role in the neovascularization of cancer tissues, Ero1L is a potential anti-angiogenic target. It can be inferred that

Although LRRN3 (Neuronal leucine rich repeat protein 3) has been reported to belong to the LRR superfamily, few associations have been reported with cancer. In 2002, the JBC rat NLRR- isolated from c-Ha-ras transgenic rat tumors. 3 expression is mainly regulated through Ras-MAPK signaling and reported to activate it more (K. Fukamachi et al., J. Biol . Chem . 277 (46): 43549-52 (2002)).

LGR4, also known as GPCR48 (G protein-coupled receptor 48), is a member of the LGR family and is known for its diverse physiological functions. In 2006, cancer research reported that LGR4 plays an important role in invasion and metastasis of colorectal cancer cell lines (Y. Gao et al., Cancer Res , 66 (24): 11623-31 (2006)), the most well known LGR5 in the LGR family, has recently been reported as a colorectal cancer stem cell marker (H. Takahashi et al., Ann Surg Oncol ., 18 (4): 1166-74 (2011)). The 2011 EMBO report reports that LGR4 is also a permissive factor for wnt signaling in the gut and a potential target for the treatment of bowel cancer (RC Mustata et al., EMBO) . Rep ., 2011). The LGR4 derived from the present invention has characteristics of stem cells and has been shown to be involved in the early cancerous process of gastric cancer. In addition, NOD-SCID transplantation confirmed that LGR4 highly expressing cells have a possibility of tumor formation, and LGR4 may be a target for the treatment of gastric cancer as a gastric cancer stem cell-related marker.

LINGO2 is a member of the LRR gene family and is known to be expressed in early mouse embryos during mouse embryogenesis.In 2009, the LINGO family performed various signal transduction processes including RAS / MAPK signaling and PI3K signaling in the Gene Expression Pattern. It has been reported to be found in the domain structure of the activating Trk receptor (S. Homma et al., Gene Expr Patterns , 9 (1): 1-26 (2009)). And Parkinson's disease has been reported (YW Wu et al., Hum Genet (2011)), have not been reported for cancer. However, in the results of the present invention, LINGO2 has been shown to be associated with tumorigenicity and neovascularization, thus predicting the potential target of anti-angiogenesis.

Currently, research on gastric cancer stem cells is at an early stage in the world. In 2011, ME Han et al. Used CD44 and EpCAM to isolate cells from cancers such as breast cancer and pancreatic cancer. Gastric cancer stem cells were collected in the CD44 + / EpCAM + subpopulation, which showed higher resistance to anticancer drugs such as 5-FU and doxorubicin. Cancer spheres have also been reported as an ideal model system for cancer stem cell research (ME Han et al., Cell . Mol . Life Sci ., (2011)).

Thus, to date, all of the well-known cancer stem cell markers are reported, and based on the above reports and various experiments of the present invention, five new markers are used for diagnosis of gastric cancer using gastric cancer stem cell characteristics and anticancer drug resistance. It may be a useful target for gastric cancer treatment.

references

1. Y.-F. Ping and X.-W. Bian, Cancer stem cells switch on tumor neovascularization. Current Molecular Medicine, 11: 69-75, 2011

2. BB Zhou et al. Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nature Reviews Drug Discovery: 8, 806-823, 2009.

3. M. Al-Hajj et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A .: 100 (7), 3983-3988, 2003.

4. ZF Yang et al. Significance of CD90 + cancer stem cells in human liver cancer. Cancer cell; 13, 153-166, 2008.

5. P. Dalerba et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A .: 104 (24), 10158-63, 2007.

6. C. Li et al. Identification of pancreatic cancer stem cells. Cancer Res: 67 (3) 1030-1037, 2007.

7. A. Dubrovska et al. The role of PTEN / Akt / PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proc Natl Acad Sci U S A .: 106, 268-273, 2009.

8. MJ Son et al. SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell stem cells: 4, 440-452, 2009.

9.Lionel Larue and Alfonso Bellacosa, Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3 'kinase / AKT pathways. Oncogene, 24: 7443-7454, 2005

10. T. Reya et al. Stem cells, cancer, and cancer stem cells. Nature, 414: 105-111, 2001.

11. J. Zhou et al. Activation of the PTEN / mTOR / STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance.Proc Natl Acad Sci USA, 104: 16158-16163, 2007.

12. I V et al. ATP-binding cassette transporter ABCB5 gene is expressed with variability in malignant melanoma. Actas Dermosifiliogr. 101 (4): 341-8, 2010.

13. A. Shukla et al. Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin. Mol Cancer, 15: 314, 2010.

14. D. May et al. Ero1-L alpha plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia: implication for cancer.Oncogne, 24 (6): 1011-20, 2005.

15. K. Fukamachi et al. Neuronal leucine-rich repeat protein-3 amplifies MAPK activation by epidermal growth factor through a carboxyl-terminal region containing endocytosis motifs. J. Biol. Chem. 277 (46): 43549-52, 2002.

16. Y. Gao et al. Up-regulation of GPR48 induced by down-regulation of p27Kip1 enhances carcinoma cell invasiveness and metastasis. Cancer Res, 66 (24): 11623-31, 2006.

17. H. Takahashi et al. Significance of Lgr 5 (+ ve) cancer stem cells in the colon and rectum. Ann Surg Oncol., 18 (4): 1166-74, 2011.

18. RC Mustata et al. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.EMBO Rep., 2011.

19. S. Homma et al. Expression pattern of LRR and Ig domain-containing protein (LRRIG protein) in the early mouse embryo. Gene Expr Patterns, 9 (1): 1-26, 2009.

20. YW Wu et al. Lingo2 variants associated with essential tremor and Parkinson's disease. Hum Genet, 2011.

21. ME Han et al. Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research. Cell. Mol. Life Sci., 2011.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (11)

Screening methods for preventing or treating gastric cancer, comprising the following steps:
(a) a protein selected from the group consisting of a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7 and a protein of SEQ ID NO: 9, or The polynucleotide of SEQ ID NO: 2, the polynucleotide of SEQ ID NO: 4, the polynucleotide of SEQ ID NO: 6, the polynucleotide of SEQ ID NO: 8, and the polynucleotide of SEQ ID NO: 10 Contacting a sample to be analyzed with a cell or tissue comprising a polynucleotide; 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 gastric cancer. It is determined.
Screening methods for preventing or treating gastric cancer, comprising the following steps:
(a) a protein selected from the group consisting of a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7, and a protein of SEQ ID NO: 9 Preparing;
(b) contacting the sample to be analyzed with the protein; And
(c) analyzing whether the sample binds to the protein or whether the sample inhibits the function of the protein; When the sample binds to the protein or inhibits the function of the protein, it is determined as a substance for preventing or treating gastric cancer.
A screening method of a substance for preventing or treating cancer, comprising the steps of:
(a) contacting a sample to be analyzed with a cell or tissue comprising a protein of SEQ ID NO: 3 or a polynucleotide of SEQ ID NO: 4; 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 .
A screening method of a substance for preventing or treating cancer, comprising the steps of:
(a) preparing a protein of SEQ ID NO: 3;
(b) contacting the sample to be analyzed with the protein; And
(c) analyzing whether the sample binds to the protein or whether the sample inhibits the function of the protein; When the sample binds to the protein or inhibits the function of the protein, it is determined as a substance for preventing or treating cancer.
4. The method of claim 3, wherein the protein is present on a cell surface, on a virus surface or in an isolated form.
Specifically, a protein selected from the group consisting of a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7, and a protein of SEQ ID NO: 9 A binding agent or polynucleotide of SEQ ID NO: 2, polynucleotide of SEQ ID NO: 4, polynucleotide of SEQ ID NO: 6, polynucleotide of SEQ ID NO: 8, and polynucleotide of SEQ ID NO: 10 Gastric cancer stem cell detection kit comprising a primer or probe specifically binding to a polynucleotide selected from the group.
A kit for cancer stem cell detection comprising a primer or probe specifically binding to a polynucleotide of SEQ ID NO: 4 or a binding agent that specifically binds to a protein of SEQ ID NO: 3.
Specifically, a protein selected from the group consisting of a protein of SEQ ID NO: 1, a protein of SEQ ID NO: 3, a protein of SEQ ID NO: 5, a protein of SEQ ID NO: 7, and a protein of SEQ ID NO: 9 A binding agent or polynucleotide of SEQ ID NO: 2, polynucleotide of SEQ ID NO: 4, polynucleotide of SEQ ID NO: 6, polynucleotide of SEQ ID NO: 8, and polynucleotide of SEQ ID NO: 10 Gastric cancer diagnostic or prognostic kit comprising a primer or probe specifically binding to a polynucleotide selected from the group.
A kit for cancer diagnosis or prognosis analysis comprising a binding agent that specifically binds to a protein of SEQ ID NO: 3 or a primer or probe that specifically binds to a polynucleotide of SEQ ID NO: 4.
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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