KR20130007974A - Novel therapeutic target for gastric cancer based on gastric cancer stem cell - 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 curative resection, but in gastric cancer, 20% of patients were diagnosed as advanced gastric cancer that was not curable at the time of diagnosis, including those who recurred after resection and postoperative adjuvant chemotherapy after radical resection. 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), 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.

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.

Still another object of the present invention is to provide a kit for diagnosing or prognosticing 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 apparent from the following detailed description and claims.

According to one aspect of the present invention, the present invention provides a method for screening a 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 (eg a natural extract or a cell or tissue culture). Test substances can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc . Natl . Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci . USA 91, 11422, 1994; Zuckermann et al., J. Med. Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int. Ed . Engl . 33, 2059, 1994; Carell et al., Angew . Chem . Int . Ed . Engl . 33, 2061; Gallop et al., J. Med . Chem . 37, 1233, 1994 and the like.

According to another aspect of the present invention, there is provided a method for screening a substance for preventing or treating 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 a form displayed on a cell surface, a form displayed on a virus (eg, bacteriophage) surface, an isolated form, or a purified form.

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

The screening 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).

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

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

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

In addition, the screening method of the present invention can be carried out according to a two-hybrid analysis or a three-hybrid analysis method (US Pat. No. 5,283,317; Zervos et al., Cell 72, 223232, 1993; Madura et al., J. Biol . Chem . 268, 1204612054, 1993; Bartel et al., BioTechniques 14, 920924,1993; Iwabuchi et al., Oncogene 8, 16931696, 1993; And WO 94/10300). In this case, the protein may be used as a bait protein. According to this method, it is possible to screen substances, particularly proteins, which bind to the proteins. To-hybrid system is based on the modular nature of the transcription factor consisting of segmentable DNA-binding and activation domains. Briefly, this analysis method uses two DNA constructs. For example, in one construct, 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 in in vivo to form a complex, the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of the reporter gene (eg, LacZ). Expression of the reporter gene can be detected, which indicates that the protein of analysis can bind to the protein, and consequently, it can be used as a material for preventing or treating gastric cancer.

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

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

(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 material for preventing or treating cancer, comprising the following steps:

(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 determined as a substance for preventing or treating cancer.

The cancer treatment agent identified by the screening method of the present invention does not target only general cancer cells, which occupy most of the cancer tissues, but occupies only a small portion of the cancer tissues, but plays a key role in the development, maintenance, and recurrence of cancer. 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, a binding agent that specifically binds to a protein may be, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a PNA (Peptide nucleic acid) or an aptamer. to be.

According to another aspect of the 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” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or having a particular disease or condition. Determining the prognosis of a subject (eg, identifying a metastatic or metastatic cancer state, determining the stage of the cancer or responsiveness of the cancer to treatment), or therametrics (eg, treating efficacy Monitoring the status of an object to provide information about it).

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. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket. Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc. The method of analysis of the invention is not limited.

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

As used herein, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

Since new 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 are well known in the art by the hybridoma method (Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature , 352: 624-628, 1991; Marks et al, J. Mol . Biol . , 222: 58, 1-597, 1991) It can be prepared using. Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

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

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

According to a preferred embodiment of the present invention, the present invention provides oligopeptides, monoclonal antibodies, 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 (micro particle) (conjugated antibody). In addition, the microparticles are preferably colored latex or colloidal gold particles. In the present invention, the antibody may be any antibody capable of measuring the expression level of a protein encoded by a known mRNA gene for the 20 markers described above, but preferably, the kit is immunoassay. ), Most preferably the kit is a Luminex assay kit, a protein microarray kit or an ELISA kit.

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

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

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

In addition, the kit may additionally include the necessary elements necessary for performing protein microarrays to simultaneously analyze the complex markers. The microarray kit includes antibodies specific for the marker protein bound to the solid phase. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The protein microarray kit can also include antibodies specific for the control protein. Other protein microarray kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (such as fused with antibodies), and substrates thereof or other materials that can bind to antibodies. And the like. In a method of analyzing a sample using a protein microarray, the protein is separated from the sample, and the separated protein is hybridized with a protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein. 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 that can simultaneously analyze more than 100 biological samples in each well of a 96-well plate. By using the laser detector of the real-time signal transmission to distinguish the polystyrene bead (polystyrene bead) of more than 100 different color groups. The 100 beads are configured to be distinguished in the following manner. On the one hand, the red fluorescence bead is divided into ten or more steps, and on the other, the orange fluorescence bead is divided into ten steps, showing the difference in intensity, and the beads therebetween. The red and orange ratios are mixed in different proportions, making up a total of 100 color-coded bead sets. In addition, each bead is attached to the antibody of the protein to be analyzed, it is possible to quantify the protein by an immune antibody reaction using the same. The sample is analyzed using two lasers, one of which detects the beads to determine the bead identification number, and the other laser reacts with the antibody attached to the beads. The protein in the sample is detected. Thus, 100 in vivo proteins can be analyzed simultaneously in one well. This analysis has the advantage of being able to detect samples as small as 15 μl.

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

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

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

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

Protein expression level measurement is preferably by using an ELISA method. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. More preferably, the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. By confirming the degree of complex formation of the 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 sliced to a thickness of several micrometers and attached to glass slides, and then reacted with one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

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, cells, blood, serum, plasma, saliva, cerebrospinal fluid or urine, preferably blood or serum, 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. . Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, and the like.

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

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

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

By “primer” of the present invention is meant a nucleic acid sequence having a short free 3-terminal hydroxyl group which can form complementary templates and base pairs and which serves as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroami Date, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like. Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention may also be modified using a label capable of directly or indirectly providing a detectable signal. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

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

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

According to another aspect of the present invention, a 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 an adherent state by attaching the same culture to a culture dish (Nalgene Nunc Int., Rochester, NY) were used as controls. 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 for one hour at 42 ° C. was reverse transcribed through the reaction of Superscript II (Gibco BRL, Grand Island, NY). Expression of cancer stem cell related genes in spear cells was analyzed by primers of cancer stem cell related genes and PCR with Taq polymerase, and the expression of 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 ℃ / 30 Notch3 ATGGTGGGAACTAAACACAGCT ATGACCCTGGAGGAAGCACA 55 ℃ / 35 Hes1 GTGCTGTCTGGATGCGGAGT GAACACTCACACTCAAAGCCC 55 ℃ / 35 Ihh CCTGAACTCGCTGGCTATCT AATACACCCAGTCAAAGCCG 56 ℃ / 35 Smo GAATCGCTACCCTGCTGTTA TGAGCAGGTGGAAGTAGGAG 59 ℃ / 30 Gli1 AGAGTCCAGGGGGTTACATA AGAGTCCAGGGGGTTACATA 57 ℃ / 35 FZD7 CCAACGGCCTGATGTACTTT GCCATGCCGAAGAAGTAGAG 61 ℃ / 30 β-catenin GTATGAGTGGGAACAGGGATTT CCTGGTCCTCGTCATTTAGC 52 ℃ / 25 Nanog ACTGTCTCTCCTCTTCCTTCCT AGAGTAAAGGCTGGGGTAGGTA 61 ℃ / 30 Oct4 GTGGAGGAAGCTGACAACAA AGCAGCCTCAAAATCCTCTC 62 ℃ / 27 PTEN GGACGAACTGGTGTAATGAT CAGACCACAAACTGAGGATT 56 ℃ / 30 β -actin GGCATCCTCACCCTGAAGTA GGGGTGTTGAAGGTCTCAAA 55 ℃ / 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 for one hour at 42 ° C. was reverse transcribed 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 Cancer  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 for one hour at 42 ° C. was reverse transcribed 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 Blot

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 Cultivation

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 Analysis of Cancer Stem Cell Related Genes 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 Cancer  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 Cancer  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.

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

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> PN110290D <130> PN110290D <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 1642 <212> PRT <213> ABCA5 protein <400> 1 Met Ser Thr Ala Ile Arg Glu Val Gly Val Trp Arg Gln Thr Arg Thr   1 5 10 15 Leu Leu Leu Lys Asn Tyr Leu Ile Lys Cys Arg Thr Lys Lys Ser Ser              20 25 30 Val Gln Glu Ile Leu Phe Pro Leu Phe Phe Leu Phe Trp Leu Ile Leu          35 40 45 Ile Ser Met Met His Pro Asn Lys Lys Tyr Glu Glu Val Pro Asn Ile      50 55 60 Glu Leu Asn Pro Met Asp Lys Phe Thr Leu Ser Asn Leu Ile Leu Gly  65 70 75 80 Tyr Thr Pro Val Thr Asn Ile Thr Ser Ser Ile Met Gln Lys Val Ser                  85 90 95 Thr Asp His Leu Pro Asp Val Ile Thr Glu Glu Tyr Thr Asn Glu             100 105 110 Lys Glu Met Leu Thr Ser Ser Leu Ser Lys Pro Ser Asn Phe Val Gly         115 120 125 Val Val Phe Lys Asp Ser Met Ser Tyr Glu Leu Arg Phe Phe Pro Asp     130 135 140 Met Ile Pro Val Ser Ser Ile Tyr Met Asp Ser Arg Ala Gly Cys Ser 145 150 155 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Leu Leu Leu Gln Asn Ser Ala Asp Ser Asp Ile Ser Asp Leu         915 920 925 Ile Ser Phe Phe Thr Ser Gln Asn Ile Met Val Thr Met Ile Asn Asp     930 935 940 Ser Asp Tyr Val Ser Val Ala Pro His Ser Ala Ala Leu Asn Val Met 945 950 955 960 His Ser Glu Lys Asp Tyr Val Phe Ala Ala Val Phe Asn Ser Thr Met                 965 970 975 Val Tyr Ser Leu Pro Ile Leu Val Asn Ile Ile Ser Asn Tyr Tyr Leu             980 985 990 Tyr His Leu Asn Val Thr Glu Thr Ile Gln Ile Trp Ser Thr Pro Phe         995 1000 1005 Phe Gln Glu Ile Thr Asp Ile Val Phe Lys Ile Glu Leu Tyr Phe Gln    1010 1015 1020 Ala Ala Leu Leu Gly Ile Ile Val Thr Ala Met Pro Pro Tyr Phe Ala 1025 1030 1035 1040 Met Glu Asn Ala Glu Asn His Lys Ile Lys Ala Tyr Thr Gln Leu Lys                1045 1050 1055 Leu Ser Gly Leu Leu Pro Ser Ala Tyr Trp Ile Gly Gln Ala Val Val            1060 1065 1070 Asp Ile Pro Leu Phe Phe Ile Ile Leu Ile Leu Met Leu Gly Ser Leu        1075 1080 1085 Leu Ala Phe His Tyr Gly Leu Tyr Phe Tyr Thr Val Lys Phe Leu Ala    1090 1095 1100 Val Val Phe Cys Leu Ile Gly Tyr Val Pro Ser Val Ile Leu Phe Thr 1105 1110 1115 1120 Tyr Ile Ala Ser Phe Thr Phe Lys Lys Ile Leu Asn Thr Lys Glu Phe                1125 1130 1135 Trp Ser Phe Ile Tyr Ser Val Ala Ala Leu Ala Cys Ile Ala Ile Thr            1140 1145 1150 Glu Ile Thr Phe Phe Met Gly Tyr Thr Ile Ala Thr Ile Leu His Tyr        1155 1160 1165 Ala Phe Cys Ile Ile Ile Pro Ile Tyr Pro Leu Leu Gly Cys Leu Ile    1170 1175 1180 Ser Phe Ile Lys Ile Ser Trp Lys Asn Val Arg Lys Asn Val Asp Thr 1185 1190 1195 1200 Tyr Asn Pro Trp Asp Arg Leu Ser Val Ala Val Ile Ser Pro Tyr Leu                1205 1210 1215 Gln Cys Val Leu Trp Ile Phe Leu Leu Gln Tyr Tyr Glu Lys Lys Tyr            1220 1225 1230 Gly Gly Arg Ser Ile Arg Lys Asp Pro Phe Phe Arg Asn Leu Ser Thr        1235 1240 1245 Lys Ser Lys Asn Arg Lys Leu Pro Glu Pro Pro Asp Asn Glu Asp Glu    1250 1255 1260 Asp Glu Asp Val Lys Ala Glu Arg Leu Lys Val Lys Glu Leu Met Gly 1265 1270 1275 1280 Cys Gln Cys Cys Glu Glu Lys Pro Ser Ile Met Val Ser Asn Leu His                1285 1290 1295 Lys Glu Tyr Asp Asp Lys Lys Asp Phe Leu Leu Ser Arg Lys Val Lys            1300 1305 1310 Lys Val Ala Thr Lys Tyr Ile Ser Phe Cys Val Lys Lys Gly Glu Ile        1315 1320 1325 Leu Gly Leu Leu Gly Pro Asn Gly Ala Gly Lys Ser Thr Ile Ile Asn    1330 1335 1340 Ile Leu Val Gly Asp Ile Glu Pro Thr Ser Gly Gln Val Phe Leu Gly 1345 1350 1355 1360 Asp Tyr Ser Ser Glu Thr Ser Glu Asp Asp Asp Ser Leu Lys Cys Met                1365 1370 1375 Gly Tyr Cys Pro Gln Ile Asn Pro Leu Trp Pro Asp Thr Thr Leu Gln            1380 1385 1390 Glu His Phe Glu Ile Tyr Gly Ala Val Lys Gly Met Ser Ala Ser Asp        1395 1400 1405 Met Lys Glu Val Ile Ser Arg Ile Thr His Ala Leu Asp Leu Lys Glu    1410 1415 1420 His Leu Gln Lys Thr Val Lys Lys Leu Pro Ala Gly Ile Lys Arg Lys 1425 1430 1435 1440 Leu Cys Phe Ala Leu Ser Met Leu Gly Asn Pro Gln Ile Thr Leu Leu                1445 1450 1455 Asp Glu Pro Ser Thr Gly Met Asp Pro Lys Ala Lys Gln His Met Trp            1460 1465 1470 Arg Ala Ile Arg Thr Ala Phe Lys Asn Arg Lys Arg Ala Ala Ile Leu        1475 1480 1485 Thr Thr His Tyr Met Glu Glu Ala Glu Ala Val Cys Asp Arg Val Ala    1490 1495 1500 Ile Met Val Ser Gly Gln Leu Arg Cys Ile Gly Thr Val Gln His Leu 1505 1510 1515 1520 Lys Ser Lys Phe Gly Lys Gly Tyr Phe Leu Glu Ile Lys Leu Lys Asp                1525 1530 1535 Trp Ile Glu Asn Leu Glu Val Asp Arg Leu Gln Arg Glu Ile Gln Tyr            1540 1545 1550 Ile Phe Pro Asn Ala Ser Arg Gln Glu Ser Phe Ser Ser Ile Leu Ala        1555 1560 1565 Tyr Lys Ile Pro Lys Glu Asp Val Gln Ser Leu Ser Gln Ser Phe Phe    1570 1575 1580 Lys Leu Glu Glu Ala Lys His Ala Phe Ala Ile Glu Glu Tyr Ser Phe 1585 1590 1595 1600 Ser Gln Ala Thr Leu Glu Gln Val Phe Val Glu Leu Thr Lys Glu Gln                1605 1610 1615 Glu Glu Glu Asp Asn Ser Cys Gly Thr Leu Asn Ser Thr Leu Trp Trp            1620 1625 1630 Glu Arg Thr Gln Glu Asp Arg Val Val Phe        1635 1640 <210> 2 <211> 8177 <212> DNA <213> ABCA5 nucleotide <220> <221> gene (222) (1) .. (8177) <220> <221> CDS (147) .. (5072) <400> 2 agctctgcct gcgccaggag cgcgcgggtg cgcgcggccg cgccctcgca cagatcccag 60 ctgggtcacc cgcactgagt caacagactg agcgcgtcca ggcctgacag ctctgcggct 120 cgggccctga ggtttattca gaaaac atg tcc act gca att agg gag gta 170                                  Met Ser Thr Ala Ile Arg Glu Val                                    1 5 gga gtt tgg aga cag acc aga aca ctt cta ctg aag aat tac tta att 218 Gly Val Trp Arg Gln Thr Arg Thr Leu Leu Leu Lys Asn Tyr Leu Ile      10 15 20 aaa tgc aga acc aaa aag agt agt gtt cag gaa att ctt ttt cca cta 266 Lys Cys Arg Thr Lys Lys Ser Ser Val Gln Glu Ile Leu Phe Pro Leu  25 30 35 40 ttt ttt tta ttt tgg tta ata tta att agc atg atg cat cca aat aag 314 Phe Phe Leu Phe Trp Leu Ile Leu Ile Ser Met Met His Pro Asn Lys                  45 50 55 aaa tat gaa gaa gtg cct aat ata gaa ctc aat cct atg gac aag ttt 362 Lys Tyr Glu Glu Val Pro Asn Ile Glu Leu Asn Pro Met Asp Lys Phe              60 65 70 act ctt tct aat cta att ctt gga tat act cca gtg act aat att aca 410 Thr Leu Ser Asn Leu Ile Leu Gly Tyr Thr Pro Val Thr Asn Ile Thr          75 80 85 agc agc atc atg cag aaa gtg tct act gat cat cta cct gat gtc ata 458 Ser Ser Ile Met Gln Lys Val Ser Thr Asp His Leu Pro Asp Val Ile      90 95 100 att act gaa gaa tat aca aat gaa aaa gaa atg tta aca tcc agt ctc 506 Ile Thr Glu Glu Tyr Thr Asn Glu Lys Glu Met Leu Thr Ser Ser Leu 105 110 115 120 tct aag ccg agc aac ttt gta ggt gtg gtt ttc aaa gac tcc atg tcc 554 Ser Lys Pro Ser Asn Phe Val Gly Val Val Phe Lys Asp Ser Met Ser                 125 130 135 tat gaa ctt cgt ttt ttt cct gat atg att cca gta tct tct att tat 602 Tyr Glu Leu Arg Phe Phe Pro Asp Met Ile Pro Val Ser Ser Ile Tyr             140 145 150 atg gat tca aga gct ggc tgt tca aaa tca tgt gag gct gct cag tac 650 Met Asp Ser Arg Ala Gly Cys Ser Lys Ser Cys Glu Ala Ala Gln Tyr         155 160 165 tgg tcc tca ggt ttc aca gtt tta caa gca tcc ata gat gct gcc att 698 Trp Ser Ser Gly Phe Thr Val Leu Gln Ala Ser Ile Asp Ala Ala Ile     170 175 180 ata cag ttg aag acc aat gtt tct ctt tgg aag gag ctg gag tca act 746 Ile Gln Leu Lys Thr Asn Val Ser Leu Trp Lys Glu Leu Glu Ser Thr 185 190 195 200 aaa gct gtt att atg gga gaa act gct gtt gta gaa ata gat acc ttt 794 Lys Ala Val Ile Met Gly Glu Thr Ala Val Val Glu Ile Asp Thr Phe                 205 210 215 ccc cga gga gta att tta ata tac cta gtt ata gca ttt tca cct ttt 842 Pro Arg Gly Val Ile Leu Ile Tyr Leu Val Ile Ala Phe Ser Pro Phe             220 225 230 gga tac ttt ttg gca att cat atc gta gca gaa aaa gaa aaa aaa ata 890 Gly Tyr Phe Leu Ala Ile His Ile Val Ala Glu Lys Glu Lys Lys Ile         235 240 245 aaa gaa ttt tta aag ata atg gga ctt cat gat act gcc ttt tgg ctt 938 Lys Glu Phe Leu Lys Ile Met Gly Leu His Asp Thr Ala Phe Trp Leu     250 255 260 tcc tgg gtt ctt cta tat aca agt tta att ttt ctt atg tcc ctt ctt 986 Ser Trp Val Leu Leu Tyr Thr Ser Leu Ile Phe Leu Met Ser Leu Leu 265 270 275 280 atg gca gtc att gcg aca gct tct ttg tta ttt cct caa agt agc agc 1034 Met Ala Val Ile Ala Thr Ala Ser Leu Leu Phe Pro Gln Ser Ser Ser                 285 290 295 att gtg ata ttt ctg ctt ttt ttc ctt tat gga tta tca tct gta ttt 1082 Ile Val Ile Phe Leu Leu Phe Phe Leu Tyr Gly Leu Ser Ser Val Phe             300 305 310 ttt gct tta atg ctg aca cct ctt ttt aaa aaa tca aaa cat gtg gga 1130 Phe Ala Leu Met Leu Thr Pro Leu Phe Lys Lys Ser Lys His Val Gly         315 320 325 ata gtt gaa ttt ttt gtt act gtg gct ttt gga ttt att ggc ctt atg 1178 Ile Val Glu Phe Phe Val Thr Val Ala Phe Gly Phe Ile Gly Leu Met     330 335 340 ata atc ctc ata gaa agt ttt ccc aaa tcg tta gtg tgg ctt ttc agt 1226 Ile Ile Leu Ile Glu Ser Phe Pro Lys Ser Leu Val Trp Leu Phe Ser 345 350 355 360 cct ttc tgt cac tgt act ttt gtg att ggt att gca cag gtc atg cat 1274 Pro Phe Cys His Cys Thr Phe Val Ile Gly Ile Ala Gln Val Met His                 365 370 375 tta gaa gat ttt aat gaa ggt gct tca ttt tca aat ttg act gca ggc 1322 Leu Glu Asp Phe Asn Glu Gly Ala Ser Phe Ser Asn Leu Thr Ala Gly             380 385 390 cca tat cct cta att att aca att atc atg ctc aca ctt aat agt ata 1370 Pro Tyr Pro Leu Ile Ile Thr Ile Met Leu Thr Leu Asn Ser Ile         395 400 405 ttc tat gtc ctc ttg gct gtc tat ctt gat caa gtc att cca ggg gaa 1418 Phe Tyr Val Leu Leu Ala Val Tyr Leu Asp Gln Val Ile Pro Gly Glu     410 415 420 ttt ggc tta cgg aga tca tct tta tat ttt ctg aag cct tca tat tgg 1466 Phe Gly Leu Arg Arg Ser Ser Leu Tyr Phe Leu Lys Pro Ser Tyr Trp 425 430 435 440 tca aag agc aaa aga aat tat gag gag tta tca gag ggc aat gtt aat 1514 Ser Lys Ser Lys Arg Asn Tyr Glu Glu Leu Ser Glu Gly Asn Val Asn                 445 450 455 gga aat att agt ttt agt gaa att att gag cca gtt tct tca gaa ttt 1562 Gly Asn Ile Ser Phe Ser Glu Ile Ile Glu Pro Val Ser Ser Glu Phe             460 465 470 gta gga aaa gaa gcc ata aga att agt ggt att cag aag aca tac aga 1610 Val Gly Lys Glu Ala Ile Arg Ile Ser Gly Ile Gln Lys Thr Tyr Arg         475 480 485 aag aag ggt gaa aat gtg gag gct ttg aga aat ttg tca ttt gac ata 1658 Lys Lys Gly Glu Asn Val Glu Ala Leu Arg Asn Leu Ser Phe Asp Ile     490 495 500 tat gag ggt cag att act gcc tta ctt ggc cac agt gga aca gga aag 1706 Tyr Glu Gly Gln Ile Thr Ala Leu Leu Gly His Ser Gly Thr Gly Lys 505 510 515 520 agt aca ttg atg aat att ctt tgt gga ctc tgc cca cct tct gat ggg 1754 Ser Thr Leu Met Asn Ile Leu Cys Gly Leu Cys Pro Pro Ser Asp Gly                 525 530 535 ttt gca tct ata tat gga cac aga gtc tca gaa ata gat gaa atg ttt 1802 Phe Ala Ser Ile Tyr Gly His Arg Val Ser Glu Ile Asp Glu Met Phe             540 545 550 gaa gca aga aaa atg att ggc att tgt cca cag tta gat ata cac ttt 1850 Glu Ala Arg Lys Met Ile Gly Ile Cys Pro Gln Leu Asp Ile His Phe         555 560 565 gat gtt ttg aca gta gaa gaa aat tta tca att ttg gct tca atc aaa 1898 Asp Val Leu Thr Val Glu Glu Asn Leu Ser Ile Leu Ala Ser Ile Lys     570 575 580 ggg ata cca gcc aac aat ata ata caa gaa gtg cag aag gtt tta cta 1946 Gly Ile Pro Ala Asn Asn Ile Ile Gln Glu Val Gln Lys Val Leu Leu 585 590 595 600 gat tta gac atg cag act atc aaa gat aac caa gct aaa aaa tta agt 1994 Asp Leu Asp Met Gln Thr Ile Lys Asp Asn Gln Ala Lys Lys Leu Ser                 605 610 615 ggt ggt caa aaa aga aag ctg tca tta gga att gct gtt ctt ggg aac 2042 Gly Gly Gln Lys Arg Lys Leu Ser Leu Gly Ile Ala Val Leu Gly Asn             620 625 630 cca aag ata ctg ctg cta gat gaa cca aca gct gga atg gac ccc tgt 2090 Pro Lys Ile Leu Leu Leu Asp Glu Pro Thr Ala Gly Met Asp Pro Cys         635 640 645 tct cga cat att gta tgg aat ctt tta aaa tac aga aaa gcc aat cgg 2138 Ser Arg His Ile Val Trp Asn Leu Leu Lys Tyr Arg Lys Ala Asn Arg     650 655 660 gtg aca gtg ttc agt act cat ttc atg gat gaa gct gac att ctt gca 2186 Val Thr Val Phe Ser Thr His Phe Met Asp Glu Ala Asp Ile Leu Ala 665 670 675 680 gat agg aaa gct gtg ata tca caa gga atg ctg aaa tgt gtt ggt tct 2234 Asp Arg Lys Ala Val Ile Ser Gln Gly Met Leu Lys Cys Val Gly Ser                 685 690 695 tca atg ttc ctc aaa agt aaa tgg ggg atc ggc tac cgc ctg agc atg 2282 Ser Met Phe Leu Lys Ser Lys Trp Gly Ile Gly Tyr Arg Leu Ser Met             700 705 710 tac ata gac aaa tat tgt gcc aca gaa tct ctt tct tca ctg gtt aaa 2330 Tyr Ile Asp Lys Tyr Cys Ala Thr Glu Ser Leu Ser Ser Leu Val Lys         715 720 725 caa cat ata cct gga gct act tta tta caa cag aat gac caa caa ctt 2378 Gln His Ile Pro Gly Ala Thr Leu Leu Gln Gln Asn Asp Gln Gln Leu     730 735 740 gtg tat agc ttg cct ttc aag gac atg gac aaa ttt tca ggt ttg ttt 2426 Val Tyr Ser Leu Pro Phe Lys Asp Met Asp Lys Phe Ser Gly Leu Phe 745 750 755 760 tct gcc cta gac agt cat tca aat ttg ggt gtc att tct tat ggt gtt 2474 Ser Ala Leu Asp Ser His Ser Asn Leu Gly Val Ile Ser Tyr Gly Val                 765 770 775 tcc atg acg act ttg gaa gac gta ttt tta aag cta gaa gtt gaa gca 2522 Ser Met Thr Thr Leu Glu Asp Val Phe Leu Lys Leu Glu Val Glu Ala             780 785 790 gaa att gac caa gca gat tat agt gta ttt act cag cag cca ctg gag 2570 Glu Ile Asp Gln Ala Asp Tyr Ser Val Phe Thr Gln Gln Pro Leu Glu         795 800 805 gaa gaa atg gat tca aaa tct ttt gat gaa atg gaa cag agc tta ctt 2618 Glu Glu Met Asp Ser Lys Ser Phe Asp Glu Met Glu Gln Ser Leu Leu     810 815 820 att ctt tct gaa acc aag gct gct cta gtg agc acc atg agc ctt tgg 2666 Ile Leu Ser Glu Thr Lys Ala Ala Leu Val Ser Thr Met Ser Leu Trp 825 830 835 840 aaa caa cag atg tat aca ata gca aag ttt cat ttc ttt acc ttg aaa 2714 Lys Gln Gln Met Tyr Thr Ile Ala Lys Phe His Phe Phe Thr Leu Lys                 845 850 855 cgt gaa agt aaa tca gtg aga tca gtg ttg ctt ctg ctt tta att ttt 2762 Arg Glu Ser Lys Ser Val Arg Ser Val Leu Leu Leu Leu Leu Ile Phe             860 865 870 ttc aca gtt cag att ttt atg ttt ttg gtt cat cac tct ttt aaa aat 2810 Phe Thr Val Gln Ile Phe Met Phe Leu Val His His Ser Phe Lys Asn         875 880 885 gct gtg gtt ccc atc aaa ctt gtt cca gac tta tat ttt cta aaa cct 2858 Ala Val Val Pro Ile Lys Leu Val Pro Asp Leu Tyr Phe Leu Lys Pro     890 895 900 gga gac aaa cca cat aaa tac aaa aca agt ctg ctt ctt caa aat tct 2906 Gly Asp Lys Pro His Lys Tyr Lys Thr Ser Leu Leu Leu Gln Asn Ser 905 910 915 920 gct gac tca gat atc agt gat ctt att agc ttt ttc aca agc cag aac 2954 Ala Asp Ser Asp Ile Ser Asp Leu Ile Ser Phe Phe Thr Ser Gln Asn                 925 930 935 ata atg gtg acg atg att aat gac agt gac tat gta tcc gtg gct ccc 3002 Ile Met Val Thr Met Ile Asn Asp Ser Asp Tyr Val Ser Val Ala Pro             940 945 950 cat agt gcg gct tta aat gtg atg cat tca gaa aag gac tat gtt ttt 3050 His Ser Ala Ala Leu Asn Val Met His Ser Glu Lys Asp Tyr Val Phe         955 960 965 gca gct gtt ttc aac agt act atg gtt tat tct tta cct ata tta gtg 3098 Ala Ala Val Phe Asn Ser Thr Met Val Tyr Ser Leu Pro Ile Leu Val     970 975 980 aat atc att agt aac tac tat ctt tat cat tta aat gtg act gaa acc 3146 Asn Ile Ile Ser Asn Tyr Tyr Leu Tyr His Leu Asn Val Thr Glu Thr 985 990 995 1000 atc cag atc tgg agt acc cca ttc ttt caa gaa att act gat ata gtt 3194 Ile Gln Ile Trp Ser Thr Pro Phe Phe Gln Glu Ile Thr Asp Ile Val                1005 1010 1015 ttt aaa att gag ctg tat ttt caa gca gct ttg ctt gga atc att gtt 3242 Phe Lys Ile Glu Leu Tyr Phe Gln Ala Ala Leu Leu Gly Ile Ile Val            1020 1025 1030 act gca atg cca cct tac ttt gcc atg gaa aat gca gag aat cat aag 3290 Thr Ala Met Pro Pro Tyr Phe Ala Met Glu Asn Ala Glu Asn His Lys        1035 1040 1045 atc aaa gct tat act caa ctt aaa ctt tca ggt ctt ttg cca tct gca 3338 Ile Lys Ala Tyr Thr Gln Leu Lys Leu Ser Gly Leu Leu Pro Ser Ala    1050 1055 1060 tat tgg att gga caa gct gtt gtt gat atc ccc tta ttt ttt atc att 3386 Tyr Trp Ile Gly Gln Ala Val Val Asp Ile Pro Leu Phe Phe Ile Ile 1065 1070 1075 1080 ctt att ttg atg cta gga agc tta ttg gca ttt cat tat gga tta tat 3434 Leu Ile Leu Met Leu Gly Ser Leu Leu Ala Phe His Tyr Gly Leu Tyr                1085 1090 1095 ttt tat act gta aag ttc ctt gct gtg gtt ttt tgc ctt att ggt tat 3482 Phe Tyr Thr Val Lys Phe Leu Ala Val Val Phe Cys Leu Ile Gly Tyr            1100 1105 1110 gtt cca tca gtt att ctg ttc act tat att gct tct ttc acc ttt aag 3530 Val Pro Ser Val Ile Leu Phe Thr Tyr Ile Ala Ser Phe Thr Phe Lys        1115 1120 1125 aaa att tta aat acc aaa gaa ttt tgg tca ttt atc tat tct gtg gca 3578 Lys Ile Leu Asn Thr Lys Glu Phe Trp Ser Phe Ile Tyr Ser Val Ala    1130 1135 1140 gcg ttg gct tgt att gca atc act gaa ata act ttc ttt atg gga tac 3626 Ala Leu Ala Cys Ile Ala Ile Thr Glu Ile Thr Phe Phe Met Gly Tyr 1145 1150 1155 1160 aca att gca act att ctt cat tat gcc ttt tgt atc atc att cca atc 3674 Thr Ile Ala Thr Ile Leu His Tyr Ala Phe Cys Ile Ile Ile Pro Ile                1165 1170 1175 tat cca ctt cta ggt tgc ctg att tct ttc ata aag att tct tgg aag 3722 Tyr Pro Leu Leu Gly Cys Leu Ile Ser Phe Ile Lys Ile Ser Trp Lys            1180 1185 1190 aat gta cga aaa aat gtg gac acc tat aat cca tgg gat agg ctt tca 3770 Asn Val Arg Lys Asn Val Asp Thr Tyr Asn Pro Trp Asp Arg Leu Ser        1195 1200 1205 gta gct gtt ata tcg cct tac ctg cag tgt gta ctg tgg att ttc ctc 3818 Val Ala Val Ile Ser Pro Tyr Leu Gln Cys Val Leu Trp Ile Phe Leu    1210 1215 1220 tta caa tac tat gag aaa aaa tat gga ggc aga tca ata aga aaa gat 3866 Leu Gln Tyr Tyr Glu Lys Lys Tyr Gly Gly Arg Ser Ile Arg Lys Asp 1225 1230 1235 1240 ccc ttt ttc aga aac ctt tca acg aag tct aaa aat agg aag ctt cca 3914 Pro Phe Phe Arg Asn Leu Ser Thr Lys Ser Lys Asn Arg Lys Leu Pro                1245 1250 1255 gaa cca cca gac aat gag gat gaa gat gaa gat gtc aaa gct gaa aga 3962 Glu Pro Pro Asp Asn Glu Asp Glu Asp Glu Asp Val Lys Ala Glu Arg            1260 1265 1270 cta aag gtc aaa gag ctg atg ggt tgc cag tgt tgt gag gag aaa cca 4010 Leu Lys Val Lys Glu Leu Met Gly Cys Gln Cys Cys Glu Glu Lys Pro        1275 1280 1285 tcc att atg gtc agc aat ttg cat aaa gaa tat gat gac aag aaa gat 4058 Ser Ile Met Val Ser Asn Leu His Lys Glu Tyr Asp Asp Lys Lys Asp    1290 1295 1300 ttt ctt ctt tca aga aaa gta aag aaa gtg gca act aaa tac atc tct 4106 Phe Leu Leu Ser Arg Lys Val Lys Lys Val Ala Thr Lys Tyr Ile Ser 1305 1310 1315 1320 ttc tgt gtg aaa aaa gga gag atc tta gga cta ttg ggt cca aat ggt 4154 Phe Cys Val Lys Lys Gly Glu Ile Leu Gly Leu Leu Gly Pro Asn Gly                1325 1330 1335 gct ggc aaa agc aca att att aat att ctg gtt ggt gat att gaa cca 4202 Ala Gly Lys Ser Thr Ile Ile Asn Ile Leu Val Gly Asp Ile Glu Pro            1340 1345 1350 act tca ggc cag gta ttt tta gga gat tat tct tca gag aca agt gaa 4250 Thr Ser Gly Gln Val Phe Leu Gly Asp Tyr Ser Ser Glu Thr Ser Glu        1355 1360 1365 gat gat gat tca ctg aag tgt atg ggt tac tgt cct cag ata aac cct 4298 Asp Asp Asp Ser Leu Lys Cys Met Gly Tyr Cys Pro Gln Ile Asn Pro    1370 1375 1380 ttg tgg cca gat act aca ttg cag gaa cat ttt gaa att tat gga gct 4346 Leu Trp Pro Asp Thr Thr Leu Gln Glu His Phe Glu Ile Tyr Gly Ala 1385 1390 1395 1400 gtc aaa gga atg agt gca agt gac atg aaa gaa gtc ata agt cga ata 4394 Val Lys Gly Met Ser Ala Ser Asp Met Lys Glu Val Ile Ser Arg Ile                1405 1410 1415 aca cat gca ctt gat tta aaa gaa cat ctt cag aag act gta aag aaa 4442 Thr His Ala Leu Asp Leu Lys Glu His Leu Gln Lys Thr Val Lys Lys            1420 1425 1430 cta cct gca gga atc aaa cga aag ttg tgt ttt gct cta agt atg cta 4490 Leu Pro Ala Gly Ile Lys Arg Lys Leu Cys Phe Ala Leu Ser Met Leu        1435 1440 1445 ggg aat cct cag att act ttg cta gat gaa cca tct aca ggt atg gat 4538 Gly Asn Pro Gln Ile Thr Leu Leu Asp Glu Pro Ser Thr Gly Met Asp    1450 1455 1460 ccc aaa gcc aaa cag cac atg tgg cga gca att cga act gca ttt aaa 4586 Pro Lys Ala Lys Gln His Met Trp Arg Ala Ile Arg Thr Ala Phe Lys 1465 1470 1475 1480 aac aga aag cgg gct gct att ctg acc act cac tat atg gag gag gca 4634 Asn Arg Lys Arg Ala Ala Ile Leu Thr Thr His Tyr Met Glu Glu Ala                1485 1490 1495 gag gct gtc tgt gat cga gta gct atc atg gtg tct ggg cag tta aga 4682 Glu Ala Val Cys Asp Arg Val Ala Ile Met Val Ser Gly Gln Leu Arg            1500 1505 1510 tgt atc gga aca gta caa cat cta aag agt aaa ttt gga aaa ggc tac 4730 Cys Ile Gly Thr Val Gln His Leu Lys Ser Lys Phe Gly Lys Gly Tyr        1515 1520 1525 ttt ttg gaa att aaa ttg aag gac tgg ata gaa aac cta gaa gta gac 4778 Phe Leu Glu Ile Lys Leu Lys Asp Trp Ile Glu Asn Leu Glu Val Asp    1530 1535 1540 cgc ctt caa aga gaa att cag tat att ttc cca aat gca agc cgt cag 4826 Arg Leu Gln Arg Glu Ile Gln Tyr Ile Phe Pro Asn Ala Ser Arg Gln 1545 1550 1555 1560 gaa agt ttt tct tct att ttg gct tat aaa att cct aag gaa gat gtt 4874 Glu Ser Phe Ser Ser Ile Leu Ala Tyr Lys Ile Pro Lys Glu Asp Val                1565 1570 1575 cag tcc ctt tca caa tct ttt ttt aag ctg gaa gaa gct aaa cat gct 4922 Gln Ser Leu Ser Gln Ser Phe Phe Lys Leu Glu Glu Ala Lys His Ala            1580 1585 1590 ttt gcc att gaa gaa tat agc ttt tct caa gca aca ttg gaa cag gtt 4970 Phe Ala Ile Glu Glu Tyr Ser Phe Ser Gln Ala Thr Leu Glu Gln Val        1595 1600 1605 ttt gta gaa ctc act aaa gaa caa gag gag gaa gat aat agt tgt gga 5018 Phe Val Glu Leu Thr Lys Glu Gln Glu Glu Glu Asp Asn Ser Cys Gly    1610 1615 1620 act tta aac agc aca ctt tgg tgg gaa cga aca caa gaa gat aga gta 5066 Thr Leu Asn Ser Thr Leu Trp Trp Glu Arg Thr Gln Glu Asp Arg Val 1625 1630 1635 1640 gta ttt tgaatttg tattgttcgg tctgcttact gggacttctt tctttttcac 5120 Val Phe ttaattttaa ctttggttta aaaagttttt tattggaatg gtaactggag aaccaagaac 5180 gcacttgaaa tttttctaag ctccttaatt gaaatgctgt ggttgtgtgt tttgcttttc 5240 tttaaataaa acgtatgtat aattaagtga agctgcatgt ttgtattgaa gtatattgaa 5300 ctatatagtt tgtatgtcat ctttttcacc attcagaaac agtgcttctg aatttgtgat 5360 ttaaaggaat tgtaatagaa tagttttatt tttaagttat ctttaagttt atgccatctt 5420 cttaaataag tacgtaatgt tccaatctaa ataaaaaact aattcataac taatgcatag 5480 aaaagataca taaagcaatg tgaaagtttc ttgcttctcc tttttaattt ctaaaaaagc 5540 cactttgaat ggaagttgtc atccgtaaaa gctgaagtgt aagcactagg aaatctcaat 5600 atagagattt gaggaaagtt atatccacta ggtggcagtc attgatcata ataagtgaaa 5660 tgagcccttg ttctagtaca tgattttagg cttaggtaat taggtatgtg aaattacatt 5720 tctttaattt aaagtaaaat tcagaaggtt ttagttatta taattaaagg aagactgtgt 5780 gtagaatctt acgtaatagt ctgattcttt gactctgtgg ctagaatgac agttatctat 5840 ggaggtggta gaattaagcc ataccttttc cttcatcact cttggaacat ataaattttt 5900 tgtcatcttc cttgcaaagg gcacatttaa ttttgttctt gaataaaata ttttattggg 5960 tatttgtttt tattggtaaa ctttagtgaa tctcttctat aaaattgtaa gtagatcagt 6020 gtgtagattt atttagtaac ttacctcatt gactttggaa catggtagga tatgaataaa 6080 ctctttcaag ggttaaatta agaattgtaa gtgcagtgcg cctatttctt tttaataaga 6140 accagaattt catttttgat agagttaaag gcatggctaa tattttctta gaaatacttc 6200 cttgtacaac ccatgtattg gctcaaggta taaaatagtg taaataatgc tagttgacat 6260 tacatttcaa ccaaatgaaa gtattaactt cataaaacaa atatattgag agtgtgatta 6320 ttgtattaga caacaacaac agcaatgaca gaaggatttg gtactgaatt actttaaata 6380 agcaaaacct ttcagtggca ttattcattc tttaatgtag gcaaaattta atgaactttt 6440 agctttattg tgtaataaga catctgtgat tttaggctct agcagtggat ttaaaagtgt 6500 tctttctcgg tacaaagtac gtggctgata ttttccccca ttgctggggg cagttgctat 6560 acactttcct aaagtatata taatgttcac ataaataccc agggacctct tttttgggat 6620 gtgcttttgt aattatgtgt aaccttgaat acttaagttt agtaaaagca acaaaattat 6680 atctatagtt tactaaggca gtcacgaaaa agcatagttt tttttagaac ctgaccatcc 6740 taagaaagtt atgtcatctg aagatagagc atgactaatg aagaaaatta cctgcttttg 6800 cacgtagatt tggtaacttt cactttacat ttgtgaatgt gtaattggtg caaagtaaat 6860 tttttttcct gcaaagtgaa agttgagtag tcacaatcat gttatttatt ttgttcattt 6920 agttactttg cttttaatgt atatgaatac atgctcttgt aaacaatttt aaaagacatt 6980 caaatttttt tttagttttt atatactcag aatatatttt ggttgttaaa ttaaattatg 7040 ttcccttttt cacagtaaca aaatctgact caggagaaat ataacttaat tcttaggaaa 7100 acaagggtaa gtgagctgga aagcaaagga ggcaaaaagt gaaatatatt tatttacagt 7160 gtacagttca tgtttatgaa catactaatt ttaaaatgtc ttaaattact gatctcaaaa 7220 cttttttcta tgtcagtaca gataaataaa taaatataca tattaggcaa aagcaacatc 7280 accaacctta tatttcccat tcatggtcaa attttcaaag gaaagtaaaa gctgagcttt 7340 actgagtgag tgtttactga ctaataaggc aagaaaaaac atgatgagtg tactttacat 7400 cttacttttg tgggctctta atttttttct ctagtttgtt ttcagtaaag acagtgggtt 7460 ttctttaaaa taattttttt cttttaaatt tgcacaaatt atcttaggtc agtattttgg 7520 ttttaatcat taaaacattt ttagagtaat gtaatttaac aaatatttta agtgattatg 7580 tttccagaaa tggattcttt ggagagaact cggatgacta tcagttttgg ccctgtaaga 7640 tcgtggagat ctgtaagata gatacttata taacttacat aaaatatata taatatatta 7700 cttatataac ttatataact agctattatt ttatataact aactataata tgacaaatcc 7760 tttgctagta gtactaacaa cgttttatag gagcacaatt aattttactt aggataagtg 7820 ttgttattat tgtttttatt gttgttctgt tagttactca aaacttcatt ctaattgtgc 7880 cctgagtttg ttaaaatacc atactgtatt tttgtgtaac atgtaaatag gcattaattt 7940 ttgagaaata gaaatgttta tccttaatgt atttttaatt tgctaacatt gattttttat 8000 tttctttcct gaaatagctt atttcctaaa atgaaagaat ttattctcag atgaataatt 8060 tttatatcag ctattcttat caggtaaaag tttaattatt ttttatttat aatatatgta 8120 agcaattctc aatgctgctc tttaagaaaa ataaatatga ttatattaat aataagc 8177 <210> 3 <211> 606 <212> PRT <213> LINGO2 protein <400> 3 Met Leu His Thr Ala Ile Ser Cys Trp Gln Pro Phe Leu Gly Leu Ala   1 5 10 15 Val Val Leu Ile Phe Met Gly Ser Thr Ile Gly Cys Pro Ala Arg Cys              20 25 30 Glu Cys Ser Ala Gln Asn Lys Ser Val Ser Cys His Arg Arg Arg Leu          35 40 45 Ile Ala Ile Pro Glu Gly Ile Pro Ile Glu Thr Lys Ile Leu Asp Leu      50 55 60 Ser Lys Asn Arg Leu Lys Ser Val Asn Pro Glu Glu Phe Ile Ser Tyr  65 70 75 80 Pro Leu Leu Glu Glu Ile Asp Leu Ser Asp Asn Ile Ile Ala Asn Val                  85 90 95 Glu Pro Gly Ala Phe Asn Asn Leu Phe Asn Leu Arg Ser Leu Arg Leu             100 105 110 Lys Gly Asn Arg Leu Lys Leu Val Pro Leu Gly Val Phe Thr Gly Leu         115 120 125 Ser Asn Leu Thr Lys Leu Asp Ile Ser Glu Asn Lys Ile Val Ile Leu     130 135 140 Leu Asp Tyr Met Phe Gln Asp Leu His Asn Leu Lys Ser Leu Glu Val 145 150 155 160 Gly Asp Asn Asp Leu Val Tyr Ile Ser His Arg Ala Phe Ser Gly Leu                 165 170 175 Leu Ser Leu Glu Gln Leu Thr Leu Glu Lys Cys Asn Leu Thr Ala Val             180 185 190 Pro Thr Glu Ala Leu Ser His Leu Arg Ser Leu Ile Ser Leu His Leu         195 200 205 Lys His Leu Asn Ile Asn Asn Met Pro Val Tyr Ala Phe Lys Arg Leu     210 215 220 Phe His Leu Lys His Leu Glu Ile Asp Tyr Trp Pro Leu Leu Asp Met 225 230 235 240 Met Pro Ala Asn Ser Leu Tyr Gly Leu Asn Leu Thr Ser Leu Ser Val                 245 250 255 Thr Asn Thr Asn Leu Ser Thr Val Pro Phe Leu Ala Phe Lys His Leu             260 265 270 Val Tyr Leu Thr His Leu Asn Leu Ser Tyr Asn Pro Ile Ser Thr Ile         275 280 285 Glu Ala Gly Met Phe Ser Asp Leu Ile Arg Leu Gln Glu Leu His Ile     290 295 300 Val Gly Ala Gln Pro Arg Thr Ile Glu Pro His Ser Phe Gln Gly Leu 305 310 315 320 Arg Phe Leu Arg Val Leu Asn Val Ser Gln Asn Leu Leu Glu Thr Leu                 325 330 335 Glu Glu Asn Val Phe Ser Ser Pro Arg Ala Leu Glu Val Leu Ser Ile             340 345 350 Asn Asn Asn Pro Leu Ala Cys Asp Cys Arg Leu Leu Trp Ile Leu Gln         355 360 365 Arg Gln Pro Thr Leu Gln Phe Gly Gly Gln Gln Pro Met Cys Ala Gly     370 375 380 Pro Asp Thr Ile Arg Glu Arg Ser Phe Lys Asp Phe His Ser Thr Ala 385 390 395 400 Leu Ser Phe Tyr Phe Thr Cys Lys Lys Pro Lys Ile Arg Glu Lys Lys                 405 410 415 Leu Gln His Leu Leu Val Asp Glu Gly Gln Thr Val Gln Leu Glu Cys             420 425 430 Ser Ala Asp Gly Asp Pro Gln Pro Val Ile Ser Trp Val Thr Pro Arg         435 440 445 Arg Arg Phe Ile Thr Thr Lys Ser Asn Gly Arg Ala Thr Val Leu Gly     450 455 460 Asp Gly Thr Leu Glu Ile Arg Phe Ala Gln Asp Gln Asp Ser Gly Met 465 470 475 480 Tyr Val Cys Ile Ala Ser Asn Ala Ala Gly Asn Asp Thr Phe Thr Ala                 485 490 495 Ser Leu Thr Val Lys Gly Phe Ala Ser Asp Arg Phe Leu Tyr Ala Asn             500 505 510 Arg Thr Pro Met Tyr Met Thr Asp Ser Asn Asp Thr Ile Ser Asn Gly         515 520 525 Thr Asn Ala Asn Thr Phe Ser Leu Asp Leu Lys Thr Ile Leu Val Ser     530 535 540 Thr Ala Met Gly Cys Phe Thr Phe Leu Gly Val Val Leu Phe Cys Phe 545 550 555 560 Leu Leu Leu Phe Val Trp Ser Arg Gly Lys Gly Lys His Lys Asn Ser                 565 570 575 Ile Asp Leu Glu Tyr Val Pro Arg Lys Asn Asn Gly Ala Val Val Glu             580 585 590 Gly Glu Val Ala Gly Pro Arg Arg Phe Asn Met Lys Met Ile         595 600 605 <210> 4 <211> 2620 <212> DNA <213> LINGO2 nucleotide <220> <221> gene (222) (1) .. (2620) <220> <221> CDS (511) .. (2328) <400> 4 agctcagagc agagcaccga aagtggccac taccagcatg aagagcccaa caattcaaac 60 tggtgaagtg agaaaaacag aatgcagctt tcaaggttcg tttcaagcag ttggcttgtg 120 ggactctgag agatgctgct gcccatgaca tgcgggaatt atcatgatca actacccagc 180 ttggatttca cccagtggcc aagagctttg tgtgggagac ggcaagggtt ggatttttca 240 aaagagtaaa ccaggtggat acatttgcaa agtggaatcc tccaggtgcc tttaaggagt 300 ttacagtcta gtaggtatga atccagccat cctcagcagt ggcaccgata tcttcaaaag 360 tgtcgtttgt ctggctctct gccttctgga tgggaagcct accctgatgt ctctcttcaa 420 atctgataaa tcatgaggaa cctataaccc ttttggccac atgcaaaaaa gcaagacccg 480 tgaccaaggt gtagactaag aagtggagtc atg ctt cac acg gcc ata tca tgc 534                                  Met Leu His Thr Ala Ile Ser Cys                                    1 5 tgg cag cca ttc ctg ggt ctg gct gtg gtg tta atc ttc atg gga tcc 582 Trp Gln Pro Phe Leu Gly Leu Ala Val Val Leu Ile Phe Met Gly Ser      10 15 20 acc att ggc tgc ccc gct cgc tgt gag tgc tct gcc cag aac aaa tct 630 Thr Ile Gly Cys Pro Ala Arg Cys Glu Cys Ser Ala Gln Asn Lys Ser  25 30 35 40 gtt agc tgt cac aga agg cga ttg atc gcc atc cca gag ggc att ccc 678 Val Ser Cys His Arg Arg Arg Leu Ile Ala Ile Pro Glu Gly Ile Pro                  45 50 55 atc gaa acc aaa atc ttg gac ctc agt aaa aac agg cta aaa agc gtc 726 Ile Glu Thr Lys Ile Leu Asp Leu Ser Lys Asn Arg Leu Lys Ser Val              60 65 70 aac cct gaa gaa ttc ata tca tat cct ctg ctg gaa gag ata gac ttg 774 Asn Pro Glu Glu Phe Ile Ser Tyr Pro Leu Leu Glu Glu Ile Asp Leu          75 80 85 agt gac aac atc att gcc aat gtg gaa cca gga gca ttc aac aat ctg 822 Ser Asp Asn Ile Ile Ala Asn Val Glu Pro Gly Ala Phe Asn Asn Leu      90 95 100 ttt aac ctg cgt tcc ctc cgc cta aaa ggc aat cgt cta aag ctg gtc 870 Phe Asn Leu Arg Ser Leu Arg Leu Lys Gly Asn Arg Leu Lys Leu Val 105 110 115 120 cct ttg gga gta ttc acg ggg ctg tcc aat ctc act aag ctt gac att 918 Pro Leu Gly Val Phe Thr Gly Leu Ser Asn Leu Thr Lys Leu Asp Ile                 125 130 135 agt gag aat aag att gtc att tta cta gac tac atg ttc caa gat cta 966 Ser Glu Asn Lys Ile Val Ile Leu Leu Asp Tyr Met Phe Gln Asp Leu             140 145 150 cat aac ctg aag tct cta gaa gtg ggg gac aat gat ttg gtt tat ata 1014 His Asn Leu Lys Ser Leu Glu Val Gly Asp Asn Asp Leu Val Tyr Ile         155 160 165 tca cac agg gca ttc agt ggg ctt ctt agc ttg gag cag ctc acc ctg 1062 Ser His Arg Ala Phe Ser Gly Leu Leu Ser Leu Glu Gln Leu Thr Leu     170 175 180 gag aaa tgc aac tta aca gca gta cca aca gaa gcc ctc tcc cac ctc 1110 Glu Lys Cys Asn Leu Thr Ala Val Pro Thr Glu Ala Leu Ser His Leu 185 190 195 200 cgc agc ctc atc agc ctg cat ctg aag cat ctc aat atc aac aat atg 1158 Arg Ser Leu Ile Ser Leu His Leu Lys His Leu Asn Ile Asn Asn Met                 205 210 215 cct gtg tat gcc ttt aaa aga ttg ttc cac ctg aaa cac cta gag att 1206 Pro Val Tyr Ala Phe Lys Arg Leu Phe His Leu Lys His Leu Glu Ile             220 225 230 gac tat tgg cct tta ctg gat atg atg cct gcc aat agc ctc tac ggt 1254 Asp Tyr Trp Pro Leu Leu Asp Met Met Pro Ala Asn Ser Leu Tyr Gly         235 240 245 ctc aac ctc aca tcc ctt tca gtc acc aac acc aat ctg tct act gta 1302 Leu Asn Leu Thr Ser Leu Ser Val Thr Asn Thr Asn Leu Ser Thr Val     250 255 260 ccc ttc ctt gcc ttt aaa cac ctg gta tac ctg act cac ctt aac ctc 1350 Pro Phe Leu Ala Phe Lys His Leu Val Tyr Leu Thr His Leu Asn Leu 265 270 275 280 tcc tac aat ccc atc agc act att gaa gca ggc atg ttc tct gac ctg 1398 Ser Tyr Asn Pro Ile Ser Thr Ile Glu Ala Gly Met Phe Ser Asp Leu                 285 290 295 atc cgc ctt cag gag ctt cat ata gtg ggg gcc cag cct cgc acc att 1446 Ile Arg Leu Gln Glu Leu His Ile Val Gly Ala Gln Pro Arg Thr Ile             300 305 310 gag cct cac tcc ttc caa ggg ctc cgc ttc cta cgc gtg ctc aat gtg 1494 Glu Pro His Ser Phe Gln Gly Leu Arg Phe Leu Arg Val Leu Asn Val         315 320 325 tct cag aac ctg ctg gaa act ttg gaa gag aat gtc ttc tcc tcc cct 1542 Ser Gln Asn Leu Leu Glu Thr Leu Glu Glu Asn Val Phe Ser Ser Pro     330 335 340 agg gct ctg gag gtc ttg agc att aac aac aac cct ctg gcc tgt gac 1590 Arg Ala Leu Glu Val Leu Ser Ile Asn Asn Asn Pro Leu Ala Cys Asp 345 350 355 360 tgc cgc ctt ctc tgg atc ttg cag cga cag ccc acc ctg cag ttt ggt 1638 Cys Arg Leu Leu Trp Ile Leu Gln Arg Gln Pro Thr Leu Gln Phe Gly                 365 370 375 ggc cag caa cct atg tgt gct ggc cca gac acc atc cgt gag agg tct 1686 Gly Gln Gln Pro Met Cys Ala Gly Pro Asp Thr Ile Arg Glu Arg Ser             380 385 390 ttc aag gat ttc cat agc act gcc ctt tct ttt tac ttt acc tgc aaa 1734 Phe Lys Asp Phe His Ser Thr Ala Leu Ser Phe Tyr Phe Thr Cys Lys         395 400 405 aaa ccc aaa atc cgt gaa aag aag ttg cag cat ctg cta gta gat gaa 1782 Lys Pro Lys Ile Arg Glu Lys Lys Leu Gln His Leu Leu Val Asp Glu     410 415 420 ggg cag aca gtc cag cta gaa tgc agt gca gat gga gac ccg cag cct 1830 Gly Gln Thr Val Gln Leu Glu Cys Ser Ala Asp Gly Asp Pro Gln Pro 425 430 435 440 gtg att tcc tgg gtg aca ccc cga agg cgt ttc atc acc acc aag tcc 1878 Val Ile Ser Trp Val Thr Pro Arg Arg Arg Phe Ile Thr Thr Lys Ser                 445 450 455 aat gga aga gcc acc gtg ttg ggt gat ggc acc ttg gaa atc cgc ttt 1926 Asn Gly Arg Ala Thr Val Leu Gly Asp Gly Thr Leu Glu Ile Arg Phe             460 465 470 gcc cag gat caa gac agc ggg atg tat gtt tgc atc gct agc aat gct 1974 Ala Gln Asp Gln Asp Ser Gly Met Tyr Val Cys Ile Ala Ser Asn Ala         475 480 485 gct ggg aat gat acc ttc aca gcc tcc tta act gtg aaa gga ttc gct 2022 Ala Gly Asn Asp Thr Phe Thr Ala Ser Leu Thr Val Lys Gly Phe Ala     490 495 500 tca gat cgt ttt ctt tat gcg aac agg acc cct atg tac atg acc gac 2070 Ser Asp Arg Phe Leu Tyr Ala Asn Arg Thr Pro Met Tyr Met Thr Asp 505 510 515 520 tcc aat gac acc att tcc aat ggc acc aat gcc aat act ttt tcc ctg 2118 Ser Asn Asp Thr Ile Ser Asn Gly Thr Asn Ala Asn Thr Phe Ser Leu                 525 530 535 gac ctt aaa aca ata ctg gtg tct aca gct atg ggc tgc ttc aca ttc 2166 Asp Leu Lys Thr Ile Leu Val Ser Thr Ala Met Gly Cys Phe Thr Phe             540 545 550 ctg gga gtg gtt tta ttt tgt ttt ctt ctc ctt ttt gtg tgg agc cga 2214 Leu Gly Val Val Leu Phe Cys Phe Leu Leu Leu Phe Val Trp Ser Arg         555 560 565 ggg aaa ggc aag cac aaa aac agc att gac ctt gag tat gtg ccc aga 2262 Gly Lys Gly Lys His Lys Asn Ser Ile Asp Leu Glu Tyr Val Pro Arg     570 575 580 aaa aac aat ggt gct gtt gtg gaa ggg gag gta gct gga ccc agg agg 2310 Lys Asn Asn Gly Ala Val Val Glu Gly Glu Val Ala Gly Pro Arg Arg 585 590 595 600 ttc aac atg aaa atg att tg aaggcccacc cctcacatta ctgtctcttt 2360 Phe Asn Met Lys Met Ile                 605 gtcaatgtgg gtaatcagta agacagtatg gcacagtaaa ttactagatt aagaggcagc 2420 catgtgcagc tgcccctgta tcaaaagcag ggtctatgga agcaggagga cttccaatgg 2480 agactctcca tcgaaaggca ggcaggcagg catgtgtcag agcccttcac acagtgggat 2540 actaagtgtt tgcgttgcaa atattggcgt tctggggatc tcagtaatga acctgaatat 2600 ttggctcaca ctcacggaca 2620 <210> 5 <211> 927 <212> PRT <213> LGR4 protein <400> 5 Met Pro Gly Pro Leu Gly Leu Leu Cys Phe Leu Ala Leu Gly Leu Leu   1 5 10 15 Gly Ser Ala Gly Pro Gly Gly Ala Ala Pro Pro Leu Cys Ala Ala Pro              20 25 30 Cys Ser Cys Asp Gly Asp Arg Arg Val Asp Cys Ser Gly Lys Gly Leu          35 40 45 Thr Ala Val Pro Glu Gly Leu Ser Ala Phe Thr Gln Ala Leu Gln Leu      50 55 60 Ala Gly Asn Asp Leu Ser Phe Ile His Pro Lys Ala Leu Ser Gly Leu  65 70 75 80 Lys Glu Leu Lys Val Leu Thr Leu Gln Asn Asn Gln Leu Lys Thr Val                  85 90 95 Pro Ser Glu Ala Ile Arg Gly Leu Ser Ala Leu Gln Ser Leu Arg Leu             100 105 110 Asp Ala Asn His Ile Thr Ser Val Pro Glu Asp Ser Phe Glu Gly Leu         115 120 125 Val Gln Leu Arg His Leu Trp Leu Asp Asp Asn Ser Leu Thr Glu Val     130 135 140 Pro Val His His Leu Ser Asn Leu Pro Thr Leu Gln Ala Leu Thr Leu 145 150 155 160 Ala Leu Asn Lys Ile Ser Ser Ile Pro Asp Phe Ala Phe Thr Asn Leu                 165 170 175 Ser Ser Leu Val Val Leu His Leu His Asn Asn Lys Ile Arg Ser Leu             180 185 190 Ser Gln His Cys Phe Asp Gly Leu Asp Asn Leu Glu Thr Leu Asp Leu         195 200 205 Asn Tyr Asn Asn Leu Gly Glu Phe Pro Gln Ala Ile Lys Ala Leu Pro     210 215 220 Ser Leu Lys Glu Leu Gly Phe His Ser Asn Ser Ile Ser Val Ile Pro 225 230 235 240 Asp Gly Ala Phe Asp Gly Asn Pro Leu Leu Arg Thr Ile His Leu Tyr                 245 250 255 Asp Asn Pro Leu Ser Phe Val Gly Asn Ser Ala Phe His Asn Leu Ser             260 265 270 Asp Leu His Ser Leu Val Ile Arg Gly Ala Ser Met Val Gln Gln Phe         275 280 285 Pro Asn Leu Thr Gly Thr Val His Leu Glu Ser Leu Thr Leu Thr Gly     290 295 300 Thr Lys Ile Ser Ser Ile Pro Asn Asn Leu Cys Gln Glu Gln Lys Met 305 310 315 320 Leu Arg Thr Leu Asp Leu Ser Tyr Asn Asn Ile Arg Asp Leu Pro Ser                 325 330 335 Phe Asn Gly Cys His Ala Leu Glu Glu Ile Ser Leu Gln Arg Asn Gln             340 345 350 Ile Tyr Gln Ile Lys Glu Gly Thr Phe Gln Gly Leu Ile Ser Leu Arg         355 360 365 Ile Leu Asp Leu Ser Arg Asn Leu Ile His Glu Ile His Ser Arg Ala     370 375 380 Phe Ala Thr Leu Gly Pro Ile Thr Asn Leu Asp Val Ser Phe Asn Glu 385 390 395 400 Leu Thr Ser Phe Pro Thr Glu Gly Leu Asn Gly Leu Asn Gln Leu Lys                 405 410 415 Leu Val Gly Asn Phe Lys Leu Lys Glu Ala Leu Ala Ala Lys Asp Phe             420 425 430 Val Asn Leu Arg Ser Leu Ser Val Pro Tyr Ala Tyr Gln Cys Cys Ala         435 440 445 Phe Trp Gly Cys Asp Ser Tyr Ala Asn Leu Asn Thr Glu Asp Asn Ser     450 455 460 Leu Gln Asp His Ser Val Ala Gln Glu Lys Gly Thr Ala Asp Ala Ala 465 470 475 480 Asn Val Thr Ser Thr Leu Glu Asn Glu Glu His Ser Gln Ile Ile Ile                 485 490 495 His Cys Thr Pro Ser Thr Gly Ala Phe Lys Pro Cys Glu Tyr Leu Leu             500 505 510 Gly Ser Trp Met Ile Arg Leu Thr Val Trp Phe Ile Phe Leu Val Ala         515 520 525 Leu Phe Phe Asn Leu Leu Val Ile Leu Thr Thr Phe Ala Ser Cys Thr     530 535 540 Ser Leu Pro Ser Ser Lys Leu Phe Ile Gly Leu Ile Ser Val Ser Asn 545 550 555 560 Leu Phe Met Gly Ile Tyr Thr Gly Ile Leu Thr Phe Leu Asp Ala Val                 565 570 575 Ser Trp Gly Arg Phe Ala Glu Phe Gly Ile Trp Trp Glu Thr Gly Ser             580 585 590 Gly Cys Lys Val Ala Gly Phe Leu Ala Val Phe Ser Ser Glu Ser Ala         595 600 605 Ile Phe Leu Leu Met Leu Ala Thr Val Glu Arg Ser Leu Ser Ala Lys     610 615 620 Asp Ile Met Lys Asn Gly Lys Ser Asn His Leu Lys Gln Phe Arg Val 625 630 635 640 Ala Ala Leu Leu Ala Phe Leu Gly Ala Thr Val Ala Gly Cys Phe Pro                 645 650 655 Ile Phe His Arg Gly Glu Tyr Ser Ala Ser Pro Leu Cys Leu Pro Phe             660 665 670 Pro Thr Gly Glu Thr Pro Ser Leu Gly Phe Thr Val Thr Leu Val Leu         675 680 685 Leu Asn Ser Leu Ala Phe Leu Le Au Met Ala Val Ile Tyr Thr Lys Leu     690 695 700 Tyr Cys Asn Leu Glu Lys Glu Asp Leu Ser Glu Asn Ser Gln Ser Ser 705 710 715 720 Met Ile Lys His Val Ala Trp Leu Ile Phe Thr Asn Cys Ile Phe Phe                 725 730 735 Cys Pro Val Ala Phe Phe Ser Phe Ala Pro Leu Ile Thr Ala Ile Ser             740 745 750 Ile Ser Pro Glu Ile Met Lys Ser Val Thr Leu Ile Phe Phe Pro Leu         755 760 765 Pro Ala Cys Leu Asn Pro Val Leu Tyr Val Phe Phe Asn Pro Lys Phe     770 775 780 Lys Glu Asp Trp Lys Leu Leu Lys Arg Arg Val Thr Lys Lys Ser Gly 785 790 795 800 Ser Val Ser Val Ser Ile Ser Ser Gln Gly Gly Cys Leu Glu Gln Asp                 805 810 815 Phe Tyr Tyr Asp Cys Gly Met Tyr Ser His Leu Gln Gly Asn Leu Thr             820 825 830 Val Cys Asp Cys Cys Glu Ser Phe Leu Leu Thr Lys Pro Val Ser Cys         835 840 845 Lys His Leu Ile Lys Ser His Ser Cys Pro Ala Leu Ala Val Ala Ser     850 855 860 Cys Gln Arg Pro Glu Gly Tyr Trp Ser Asp Cys Gly Thr Gln Ser Ala 865 870 875 880 His Ser Asp Tyr Ala Asp Glu Glu Asp Ser Phe Val Ser Asp Ser Ser                 885 890 895 Asp Gln Val Gln Ala Cys Gly Arg Ala Cys Phe Tyr Gln Ser Arg Gly             900 905 910 Phe Pro Leu Val Arg Tyr Ala Tyr Asn Leu Pro Arg Val Lys Asp         915 920 925 <210> 6 <211> 5041 <212> DNA <213> LGR4 nucleotide <220> <221> gene (222) (1) .. (5041) <220> <221> CDS (474) (474) (3254) <400> 6 agaagggaaa aggacgggaa gagattgagc cgcggctggg agacagcgag ccagagtctg 60 ggtgtttgtg cgagagccac ggcgggggct ggggcgagtg gccggcatgg ctgaaggctg 120 cgctctgcaa ccttgaagag ccgctgcatt gagaggccag ggacagggag accggtgcga 180 tggcagagcg cggcccccgc cgctgcgccg ggccggcccg gctggcctga gccgccggag 240 gagcggggct gcctctgcgc gtccatggag cagcgggaag ggcgaaactc cggagcgccg 300 cgtccctgcg ccgctgcggc ggactgctga aggggccgag cccgcgcgga ccgccgagga 360 agagaccccc gctccagccc gcaggccggc tgcccggggg cggcggggga catcggaggg 420 cagcggagcg agcagcgccg cgggagaggc cggcgcggga ggcggccgca gca 473 atg ccg ggc ccg cta ggg ctg ctc tgc ttc ctc gcc ctg ggg ctg ctc 521 Met Pro Gly Pro Leu Gly Leu Leu Cys Phe Leu Ala Leu Gly Leu Leu   1 5 10 15 ggc tcg gcc ggg ccc ggc ggc gcg gcg ccg cct ctc tgc gcg gcg ccc 569 Gly Ser Ala Gly Pro Gly Gly Ala Ala Pro Pro Leu Cys Ala Ala Pro              20 25 30 tgc agc tgc gac ggc gac cgt cgg gtg gac tgc tcc ggg aag ggg ctg 617 Cys Ser Cys Asp Gly Asp Arg Arg Val Asp Cys Ser Gly Lys Gly Leu          35 40 45 acg gcc gtg ccc gag ggg ctc agc gcc ttc acc caa gcg cta caa ttg 665 Thr Ala Val Pro Glu Gly Leu Ser Ala Phe Thr Gln Ala Leu Gln Leu      50 55 60 gcg ggc aac gac ctt tct ttt atc cac cca aag gcc ttg tct ggg ttg 713 Ala Gly Asn Asp Leu Ser Phe Ile His Pro Lys Ala Leu Ser Gly Leu  65 70 75 80 aaa gaa ctc aaa gtt cta acg ctc cag aat aat cag ttg aaa aca gta 761 Lys Glu Leu Lys Val Leu Thr Leu Gln Asn Asn Gln Leu Lys Thr Val                  85 90 95 ccc agt gaa gcc att cga ggg ctg agt gct ttg cag tct ttg cgt tta 809 Pro Ser Glu Ala Ile Arg Gly Leu Ser Ala Leu Gln Ser Leu Arg Leu             100 105 110 gat gcc aac cat att acc tca gtc ccc gag gac agt ttt gaa gga ctt 857 Asp Ala Asn His Ile Thr Ser Val Pro Glu Asp Ser Phe Glu Gly Leu         115 120 125 gtt cag tta cgg cat ctg tgg ctg gat gac aac agc ttg acg gag gtg 905 Val Gln Leu Arg His Leu Trp Leu Asp Asp Asn Ser Leu Thr Glu Val     130 135 140 cct gtg cac cac ctc agc aat ctg ccc acc cta cag gcg ctg acc ctg 953 Pro Val His His Leu Ser Asn Leu Pro Thr Leu Gln Ala Leu Thr Leu 145 150 155 160 gct ctc aac aag atc tca agc atc cct gac ttt gca ttt acc aac ctt 1001 Ala Leu Asn Lys Ile Ser Ser Ile Pro Asp Phe Ala Phe Thr Asn Leu                 165 170 175 tca agc ctg gta gtt ctg cat ctt cat aac aat aaa att aga agc ctg 1049 Ser Ser Leu Val Val Leu His Leu His Asn Asn Lys Ile Arg Ser Leu             180 185 190 agt caa cac tgt ttt gat gga cta gat aac ctg gag acc tta gac ttg 1097 Ser Gln His Cys Phe Asp Gly Leu Asp Asn Leu Glu Thr Leu Asp Leu         195 200 205 aat tat aat aac ttg ggg gaa ttt cct cag gct att aaa gcc ctt cct 1145 Asn Tyr Asn Asn Leu Gly Glu Phe Pro Gln Ala Ile Lys Ala Leu Pro     210 215 220 agc ctt aaa gag cta gga ttt cat agt aat tct att tct gtt atc cct 1193 Ser Leu Lys Glu Leu Gly Phe His Ser Asn Ser Ile Ser Val Ile Pro 225 230 235 240 gat gga gca ttt gat ggt aat cca ctc tta aga act ata cat ttg tat 1241 Asp Gly Ala Phe Asp Gly Asn Pro Leu Leu Arg Thr Ile His Leu Tyr                 245 250 255 gat aat cct ctg tct ttt gtg ggg aac tca gca ttt cac aat tta tct 1289 Asp Asn Pro Leu Ser Phe Val Gly Asn Ser Ala Phe His Asn Leu Ser             260 265 270 gat ctt cat tcc cta gtc att cgt ggt gca agc atg gtg cag cag ttc 1337 Asp Leu His Ser Leu Val Ile Arg Gly Ala Ser Met Val Gln Gln Phe         275 280 285 ccc aat ctt aca gga act gtc cac ctg gaa agt ctg act ttg aca ggt 1385 Pro Asn Leu Thr Gly Thr Val His Leu Glu Ser Leu Thr Leu Thr Gly     290 295 300 aca aag ata agc agc ata cct aat aat ttg tgt caa gaa caa aag atg 1433 Thr Lys Ile Ser Ser Ile Pro Asn Asn Leu Cys Gln Glu Gln Lys Met 305 310 315 320 ctt agg act ttg gac ttg tct tac aat aat ata aga gac ctt cca agt 1481 Leu Arg Thr Leu Asp Leu Ser Tyr Asn Asn Ile Arg Asp Leu Pro Ser                 325 330 335 ttt aat ggt tgc cat gct ctg gaa gaa att tct tta cag cgt aat caa 1529 Phe Asn Gly Cys His Ala Leu Glu Glu Ile Ser Leu Gln Arg Asn Gln             340 345 350 atc tac caa ata aag gaa ggc acc ttt caa ggc ctg ata tct cta agg 1577 Ile Tyr Gln Ile Lys Glu Gly Thr Phe Gln Gly Leu Ile Ser Leu Arg         355 360 365 att cta gat ctg agt aga aac ctg ata cat gaa att cac agt aga gct 1625 Ile Leu Asp Leu Ser Arg Asn Leu Ile His Glu Ile His Ser Arg Ala     370 375 380 ttt gcc aca ctt ggg cca ata act aac cta gat gta agt ttc aat gaa 1673 Phe Ala Thr Leu Gly Pro Ile Thr Asn Leu Asp Val Ser Phe Asn Glu 385 390 395 400 tta act tcc ttt cct acg gaa ggc ctg aat ggg cta aat caa ctg aaa 1721 Leu Thr Ser Phe Pro Thr Glu Gly Leu Asn Gly Leu Asn Gln Leu Lys                 405 410 415 ctt gtg ggc aac ttc aag ctg aaa gaa gcc tta gca gca aaa gac ttt 1769 Leu Val Gly Asn Phe Lys Leu Lys Glu Ala Leu Ala Ala Lys Asp Phe             420 425 430 gtt aac ctc agg tct tta tca gta cca tat gct tat cag tgc tgt gca 1817 Val Asn Leu Arg Ser Leu Ser Val Pro Tyr Ala Tyr Gln Cys Cys Ala         435 440 445 ttt tgg ggt tgt gac tct tat gca aat tta aac aca gaa gat aac agc 1865 Phe Trp Gly Cys Asp Ser Tyr Ala Asn Leu Asn Thr Glu Asp Asn Ser     450 455 460 ctc cag gac cac agt gtg gca cag gag aaa ggt act gct gat gca gca 1913 Leu Gln Asp His Ser Val Ala Gln Glu Lys Gly Thr Ala Asp Ala Ala 465 470 475 480 aat gtc aca agc act ctt gaa aat gaa gaa cat agt caa ata att atc 1961 Asn Val Thr Ser Thr Leu Glu Asn Glu Glu His Ser Gln Ile Ile Ile                 485 490 495 cat tgt aca cct tca aca ggt gct ttt aag ccc tgt gaa tat tta ctg 2009 His Cys Thr Pro Ser Thr Gly Ala Phe Lys Pro Cys Glu Tyr Leu Leu             500 505 510 gga agc tgg atg att cgt ctt act gtg tgg ttc att ttc ttg gtt gca 2057 Gly Ser Trp Met Ile Arg Leu Thr Val Trp Phe Ile Phe Leu Val Ala         515 520 525 tta ttt ttc aac ctg ctt gtt att tta aca aca ttt gca tct tgt aca 2105 Leu Phe Phe Asn Leu Leu Val Ile Leu Thr Thr Phe Ala Ser Cys Thr     530 535 540 tca ctg cct tcg tcc aaa ttg ttt ata ggc ttg att tct gtg tct aac 2153 Ser Leu Pro Ser Ser Lys Leu Phe Ile Gly Leu Ile Ser Val Ser Asn 545 550 555 560 tta ttc atg gga atc tat act ggc atc cta act ttt ctt gat gct gtg 2201 Leu Phe Met Gly Ile Tyr Thr Gly Ile Leu Thr Phe Leu Asp Ala Val                 565 570 575 tcc tgg ggc aga ttc gct gaa ttt ggc att tgg tgg gaa act ggc agt 2249 Ser Trp Gly Arg Phe Ala Glu Phe Gly Ile Trp Trp Glu Thr Gly Ser             580 585 590 ggc tgc aaa gta gct ggg ttt ctt gca gtt ttc tcc tca gaa agt gcc 2297 Gly Cys Lys Val Ala Gly Phe Leu Ala Val Phe Ser Ser Glu Ser Ala         595 600 605 ata ttt tta tta atg cta gca act gtc gaa aga agc tta tct gca aaa 2345 Ile Phe Leu Leu Met Leu Ala Thr Val Glu Arg Ser Leu Ser Ala Lys     610 615 620 gat ata atg aaa aat ggg aag agc aat cat ctc aaa cag ttc cgg gtt 2393 Asp Ile Met Lys Asn Gly Lys Ser Asn His Leu Lys Gln Phe Arg Val 625 630 635 640 gct gcc ctt ttg gct ttc cta ggt gct aca gta gca ggc tgt ttt ccc 2441 Ala Ala Leu Leu Ala Phe Leu Gly Ala Thr Val Ala Gly Cys Phe Pro                 645 650 655 att ttc cat aga ggg gaa tat tct gca tca ccc ctt tgt ttg cca ttt 2489 Ile Phe His Arg Gly Glu Tyr Ser Ala Ser Pro Leu Cys Leu Pro Phe             660 665 670 cct aca ggt gaa acg cca tca tta gga ttc act gta acg tta gtg cta 2537 Pro Thr Gly Glu Thr Pro Ser Leu Gly Phe Thr Val Thr Leu Val Leu         675 680 685 tta aac tca cta gca ttt tta tta atg gcc gtt atc tac act aag cta 2585 Leu Asn Ser Leu Ala Phe Leu Le Au Met Ala Val Ile Tyr Thr Lys Leu     690 695 700 tac tgc aac ttg gaa aaa gag gac ctc tca gaa aac tca caa tct agc 2633 Tyr Cys Asn Leu Glu Lys Glu Asp Leu Ser Glu Asn Ser Gln Ser Ser 705 710 715 720 atg att aag cat gtc gct tgg cta atc ttc acc aat tgc atc ttt ttc 2681 Met Ile Lys His Val Ala Trp Leu Ile Phe Thr Asn Cys Ile Phe Phe                 725 730 735 tgc cct gtg gcg ttt ttt tca ttt gca cca ttg atc act gca atc tct 2729 Cys Pro Val Ala Phe Phe Ser Phe Ala Pro Leu Ile Thr Ala Ile Ser             740 745 750 atc agc ccc gaa ata atg aag tct gtt act ctg ata ttt ttt cca ttg 2777 Ile Ser Pro Glu Ile Met Lys Ser Val Thr Leu Ile Phe Phe Pro Leu         755 760 765 cct gct tgc ctg aat cca gtc ctg tat gtt ttc ttc aac cca aag ttt 2825 Pro Ala Cys Leu Asn Pro Val Leu Tyr Val Phe Phe Asn Pro Lys Phe     770 775 780 aaa gaa gac tgg aag tta ctg aag cga cgt gtt acc aag aaa agt gga 2873 Lys Glu Asp Trp Lys Leu Leu Lys Arg Arg Val Thr Lys Lys Ser Gly 785 790 795 800 tca gtt tca gtt tcc atc agt agc caa ggt ggt tgt ctg gaa cag gat 2921 Ser Val Ser Val Ser Ile Ser Ser Gln Gly Gly Cys Leu Glu Gln Asp                 805 810 815 ttc tac tac gac tgt ggc atg tac tca cat ttg cag ggc aac ctg act 2969 Phe Tyr Tyr Asp Cys Gly Met Tyr Ser His Leu Gln Gly Asn Leu Thr             820 825 830 gtt tgc gac tgc tgc gaa tcg ttt ctt tta aca aag cca gta tca tgc 3017 Val Cys Asp Cys Cys Glu Ser Phe Leu Leu Thr Lys Pro Val Ser Cys         835 840 845 aaa cac ttg ata aaa tca cac agc tgt cct gca ttg gca gtg gct tct 3065 Lys His Leu Ile Lys Ser His Ser Cys Pro Ala Leu Ala Val Ala Ser     850 855 860 tgc caa aga cct gag ggc tac tgg tcc gac tgt ggc aca cag tcg gcc 3113 Cys Gln Arg Pro Glu Gly Tyr Trp Ser Asp Cys Gly Thr Gln Ser Ala 865 870 875 880 cac tct gat tat gca gat gaa gaa gat tcc ttt gtc tca gac agt tct 3161 His Ser Asp Tyr Ala Asp Glu Glu Asp Ser Phe Val Ser Asp Ser Ser                 885 890 895 gac cag gtg cag gcc tgt gga cga gcc tgc ttc tac cag agt aga gga 3209 Asp Gln Val Gln Ala Cys Gly Arg Ala Cys Phe Tyr Gln Ser Arg Gly             900 905 910 ttc cct ttg gtg cgc tat gct tac aat cta cca aga gtt aaa gac 3254 Phe Pro Leu Val Arg Tyr Ala Tyr Asn Leu Pro Arg Val Lys Asp         915 920 925     tgaact actgagtgtg taaccgtttc ccccgtcaac caaaatcagt gtttatagag 3310 tgaaccctat tctcatcttt catctgggaa gcacttctgt aatcactgcc tggtgtcact 3370 tagaagaagg agaggtggca gtttatttct caaaccagtc attttcaaag aacaggtgct 3430 taaattataa attggtgaaa aatgcaatgt ccaagcaatg tatgatctgt ttgaaacaaa 3490 tatatgactt gaaaaggatc ttaggtgtag tagagcaata taatgttagt tttttctgat 3550 ccataagaag caaatttata cctatttgtg tattaagcac aagataaaga acagctgtta 3610 atatttttta aaaatctatt ttaaaatgtg attttctata actgaagaaa atatcttgct 3670 aattttacct aatgtttcat ccttaatctc aggacaactt actgcagggc caaaaaaggg 3730 actgtcccag ctagaactgt gagagtatac ataggcatta ctttattatg ttttcacttg 3790 ccatccttga cataagagaa ctataaattt tgtttaagca atttataaat ctaaaacctg 3850 aagatgtttt taaaacaata ttaacagctg ttaggttaaa aaaatagctg gacatttgtt 3910 ttcagtcatt atacattgct ttggtccaat cagtaatttt ttcttaagtg ttttgtgatt 3970 acactactag aaaaaaagta aaaggctaat tgctgtgtgg gtttagtcga tttggctaaa 4030 ctactaacta atgtgggggt ttaatagtat ctgagggatt tggtggcttc atgtaatgtt 4090 ctcattaatg aatacttcct aatatcgttg gctctactaa tattttccaa tttgctggga 4150 tgtcacctag caatagtttg gattatatag aaagtaaact gtggtcaata cttgcattta 4210 attagacgaa acggggagta attatgacac gaagtactta tgtttatttc ttagtgagct 4270 ggattatctt gaacctgtgc tattaaatgg aaatttccat acatcttccc catactattt 4330 tttataaaag agcctattca atagctcaga ggttgaactc tggttaaaca agataatatg 4390 ttattaataa agatagaaga agaaagaata aagcttagtc ctgtgtcttt aaaaattaaa 4450 aattttactt gattcccatc tatgggcttt agacctatta ctgggtggag tcttaaagtt 4510 ataattgttc aatatgtttt ttgaacagtg tgctaaatca atagcaaacc cactgccata 4570 ttagttattc tgaatatact aaaaaaatcc agctagattg cagtttaata attaaactgt 4630 acatactgtg catataatga atttttatct tatgtaaatt atttttagaa cacaagttgg 4690 gaaatgtggc ttctgttcat ttcgtttaat taaagctacc tcctaaacta tagtggctgc 4750 cagtagcaga ctgttaaatt gtggtttata tactttttgc attgtaaata gtctttgttg 4810 tacattgtca gtgtaataaa aacagaatct ttgtatatca aaatcatgta gtttgtataa 4870 aatgtgggaa ggatttattt acagtgtgtt gtaattttgt aaggccaact atttacaagt 4930 tttaaaaatt gctatcatgt atatttacac atctgataaa tattaaatca taacttggta 4990 agaaactcct aattaaaagg ttttttccaa aatccaaaaa aaaaaaaaaa a 5041 <210> 7 <211> 708 <212> PRT <213> LRRN3 protein <400> 7 Met Lys Asp Met Pro Leu Arg Ile His Val Leu Leu Gly Leu Ala Ile   1 5 10 15 Thr Thr Leu Val Gln Ala Val Asp Lys Lys Val Asp Cys Pro Arg Leu              20 25 30 Cys Thr Cys Glu Ile Arg Pro Trp Phe Thr Pro Arg Ser Ile Tyr Met          35 40 45 Glu Ala Ser Thr Val Asp Cys Asn Asp Leu Gly Leu Leu Thr Phe Pro      50 55 60 Ala Arg Leu Pro Ala Asn Thr Gln Ile Leu Leu Leu Gln Thr Asn Asn  65 70 75 80 Ile Ala Lys Ile Glu Tyr Ser Thr Asp Phe Pro Val Asn Leu Thr Gly                  85 90 95 Leu Asp Leu Ser Gln Asn Asn Leu Ser Ser Val Thr Asn Ile Asn Val             100 105 110 Lys Lys Met Pro Gln Leu Leu Ser Val Tyr Leu Glu Glu Asn Lys Leu         115 120 125 Thr Glu Leu Pro Glu Lys Cys Leu Ser Glu Leu Ser Asn Leu Gln Glu     130 135 140 Leu Tyr Ile Asn His Asn Leu Leu Ser Thr Ile Ser Pro Gly Ala Phe 145 150 155 160 Ile Gly Leu His Asn Leu Leu Arg Leu His Leu Asn Ser Asn Arg Leu                 165 170 175 Gln Met Ile Asn Ser Lys Trp Phe Asp Ala Leu Pro Asn Leu Glu Ile             180 185 190 Leu Met Ile Gly Glu Asn Pro Ile Ile Arg Ile Lys Asp Met Asn Phe         195 200 205 Lys Pro Leu Ile Asn Leu Arg Ser Leu Val Ile Ala Gly Ile Asn Leu     210 215 220 Thr Glu Ile Pro Asp Asn Ala Leu Val Gly Leu Glu Asn Leu Glu Ser 225 230 235 240 Ile Ser Phe Tyr Asp Asn Arg Leu Ile Lys Val Pro His Val Ala Leu                 245 250 255 Gln Lys Val Val Asn Leu Lys Phe Leu Asp Leu Asn Lys Asn Pro Ile             260 265 270 Asn Arg Ile Arg Arg Gly Asp Phe Ser Asn Met Leu His Leu Lys Glu         275 280 285 Leu Gly Ile Asn Asn Met Pro Glu Leu Ile Ser Ile Asp Ser Leu Ala     290 295 300 Val Asp Asn Leu Pro Asp Leu Arg Lys Ile Glu Ala Thr Asn Asn Pro 305 310 315 320 Arg Leu Ser Tyr Ile His Pro Asn Ala Phe Phe Arg Leu Pro Lys Leu                 325 330 335 Glu Ser Leu Met Leu Asn Ser Asn Ala Leu Ser Ala Leu Tyr His Gly             340 345 350 Thr Ile Glu Ser Leu Pro Asn Leu Lys Glu Ile Ser Ile His Ser Asn         355 360 365 Pro Ile Arg Cys Asp Cys Val Ile Arg Trp Met Asn Met Asn Lys Thr     370 375 380 Asn Ile Arg Phe Met Glu Pro Asp Ser Leu Phe Cys Val Asp Pro Pro 385 390 395 400 Glu Phe Gln Gly Gln Asn Val Arg Gln Val His Phe Arg Asp Met Met                 405 410 415 Glu Ile Cys Leu Pro Leu Ile Ala Pro Glu Ser Phe Pro Ser Asn Leu             420 425 430 Asn Val Glu Ala Gly Ser Tyr Val Ser Phe His Cys Arg Ala Thr Ala         435 440 445 Glu Pro Gln Pro Glu Ile Tyr Trp Ile Thr Pro Ser Gly Gln Lys Leu     450 455 460 Leu Pro Asn Thr Leu Thr Asp Lys Phe Tyr Val His Ser Glu Gly Thr 465 470 475 480 Leu Asp Ile Asn Gly Val Thr Pro Lys Glu Gly Gly Leu Tyr Thr Cys                 485 490 495 Ile Ala Thr Asn Leu Val Gly Ala Asp Leu Lys Ser Val Met Ile Lys             500 505 510 Val Asp Gly Ser Phe Pro Gln Asp Asn Asn Gly Ser Leu Asn Ile Lys         515 520 525 Ile Arg Asp Ile Gln Ala Asn Ser Val Leu Val Ser Arg Lys Ala Ser     530 535 540 Ser Lys Ile Leu Lys Ser Ser Val Lys Trp Thr Ala Phe Val Lys Thr 545 550 555 560 Glu Asn Ser His Ala Ala Gln Ser Ala Arg Ile Pro Ser Asp Val Lys                 565 570 575 Val Tyr Asn Leu Thr His Leu Asn Pro Ser Thr Glu Tyr Lys Ile Cys             580 585 590 Ile Asp Ile Pro Thr Ile Tyr Gln Lys Asn Arg Lys Lys Cys Val Asn         595 600 605 Val Thr Thr Lys Gly Leu His Pro Asp Gln Lys Glu Tyr Glu Lys Asn     610 615 620 Asn Thr Thr Thr Leu Met Ala Cys Leu Gly Gly Leu Leu Gly Ile Ile 625 630 635 640 Gly Val Ile Cys Leu Ile Ser Cys Leu Ser Pro Glu Met Asn Cys Asp                 645 650 655 Gly Gly His Ser Tyr Val Arg Asn Tyr Leu Gln Lys Pro Thr Phe Ala             660 665 670 Leu Gly Glu Leu Tyr Pro Pro Leu Ile Asn Leu Trp Glu Ala Gly Lys         675 680 685 Glu Lys Ser Thr Ser Le Le Lys Val Lys Ala Thr Val Ile Gly Leu Pro     690 695 700 Thr Asn Met Ser 705 <210> 8 <211> 2127 <212> DNA <213> LRRN3 nucleotide <220> <221> gene (222) (1) .. (2127) <220> <221> CDS (222) (1) .. (2124) <400> 8 atg aag gac atg cca ctc cga att cat gtg cta ctt ggc cta gct atc 48 Met Lys Asp Met Pro Leu Arg Ile His Val Leu Leu Gly Leu Ala Ile   1 5 10 15 act aca cta gta caa gct gta gat aaa aaa gtg gat tgt cca cgg tta 96 Thr Thr Leu Val Gln Ala Val Asp Lys Lys Val Asp Cys Pro Arg Leu              20 25 30 tgt acg tgt gaa atc agg cct tgg ttt aca ccc aga tcc att tat atg 144 Cys Thr Cys Glu Ile Arg Pro Trp Phe Thr Pro Arg Ser Ile Tyr Met          35 40 45 gaa gca tct aca gtg gat tgt aat gat tta ggt ctt tta act ttc cca 192 Glu Ala Ser Thr Val Asp Cys Asn Asp Leu Gly Leu Leu Thr Phe Pro      50 55 60 gcc aga ttg cca gct aac aca cag att ctt ctc cta cag act aac aat 240 Ala Arg Leu Pro Ala Asn Thr Gln Ile Leu Leu Leu Gln Thr Asn Asn  65 70 75 80 att gca aaa att gaa tac tcc aca gac ttt cca gta aac ctt act ggc 288 Ile Ala Lys Ile Glu Tyr Ser Thr Asp Phe Pro Val Asn Leu Thr Gly                  85 90 95 ctg gat tta tct caa aac aat tta tct tca gtc acc aat att aat gta 336 Leu Asp Leu Ser Gln Asn Asn Leu Ser Ser Val Thr Asn Ile Asn Val             100 105 110 aaa aag atg cct cag ctc ctt tct gtg tac cta gag gaa aac aaa ctt 384 Lys Lys Met Pro Gln Leu Leu Ser Val Tyr Leu Glu Glu Asn Lys Leu         115 120 125 act gaa ctg cct gaa aaa tgt ctg tcc gaa ctg agc aac tta caa gaa 432 Thr Glu Leu Pro Glu Lys Cys Leu Ser Glu Leu Ser Asn Leu Gln Glu     130 135 140 ctc tat att aat cac aac ttg ctt tct aca att tca cct gga gcc ttt 480 Leu Tyr Ile Asn His Asn Leu Leu Ser Thr Ile Ser Pro Gly Ala Phe 145 150 155 160 att ggc cta cat aat ctt ctt cga ctt cat ctc aat tca aat aga ttg 528 Ile Gly Leu His Asn Leu Leu Arg Leu His Leu Asn Ser Asn Arg Leu                 165 170 175 cag atg atc aac agt aag tgg ttt gat gct ctt cca aat cta gag att 576 Gln Met Ile Asn Ser Lys Trp Phe Asp Ala Leu Pro Asn Leu Glu Ile             180 185 190 ctg atg att ggg gaa aat cca att atc aga atc aaa gac atg aac ttt 624 Leu Met Ile Gly Glu Asn Pro Ile Ile Arg Ile Lys Asp Met Asn Phe         195 200 205 aag cct ctt atc aat ctt cgc agc ctg gtt ata gct ggt ata aac ctc 672 Lys Pro Leu Ile Asn Leu Arg Ser Leu Val Ile Ala Gly Ile Asn Leu     210 215 220 aca gaa ata cca gat aac gcc ttg gtt gga ctg gaa aac tta gaa agc 720 Thr Glu Ile Pro Asp Asn Ala Leu Val Gly Leu Glu Asn Leu Glu Ser 225 230 235 240 atc tct ttt tac gat aac agg ctt att aaa gta ccc cat gtt gct ctt 768 Ile Ser Phe Tyr Asp Asn Arg Leu Ile Lys Val Pro His Val Ala Leu                 245 250 255 caa aaa gtt gta aat ctc aaa ttt ttg gat cta aat aaa aat cct att 816 Gln Lys Val Val Asn Leu Lys Phe Leu Asp Leu Asn Lys Asn Pro Ile             260 265 270 aat aga ata cga agg ggt gat ttt agc aat atg cta cac tta aaa gag 864 Asn Arg Ile Arg Arg Gly Asp Phe Ser Asn Met Leu His Leu Lys Glu         275 280 285 ttg ggg ata aat aat atg cct gag ctg att tcc atc gat agt ctt gct 912 Leu Gly Ile Asn Asn Met Pro Glu Leu Ile Ser Ile Asp Ser Leu Ala     290 295 300 gtg gat aac ctg cca gat tta aga aaa ata gaa gct act aac aac cct 960 Val Asp Asn Leu Pro Asp Leu Arg Lys Ile Glu Ala Thr Asn Asn Pro 305 310 315 320 aga ttg tct tac att cac ccc aat gca ttt ttc aga ctc ccc aag ctg 1008 Arg Leu Ser Tyr Ile His Pro Asn Ala Phe Phe Arg Leu Pro Lys Leu                 325 330 335 gaa tca ctc atg ctg aac agc aat gct ctc agt gcc ctg tac cat ggt 1056 Glu Ser Leu Met Leu Asn Ser Asn Ala Leu Ser Ala Leu Tyr His Gly             340 345 350 acc att gag tct ctg cca aac ctc aag gaa atc agc ata cac agt aac 1104 Thr Ile Glu Ser Leu Pro Asn Leu Lys Glu Ile Ser Ile His Ser Asn         355 360 365 ccc atc agg tgt gac tgt gtc atc cgt tgg atg aac atg aac aaa acc 1152 Pro Ile Arg Cys Asp Cys Val Ile Arg Trp Met Asn Met Asn Lys Thr     370 375 380 aac att cga ttc atg gag cca gat tca ctg ttt tgc gtg gac cca cct 1200 Asn Ile Arg Phe Met Glu Pro Asp Ser Leu Phe Cys Val Asp Pro Pro 385 390 395 400 gaa ttc caa ggt cag aat gtt cgg caa gtg cat ttc agg gac atg atg 1248 Glu Phe Gln Gly Gln Asn Val Arg Gln Val His Phe Arg Asp Met Met                 405 410 415 gaa att tgt ctc cct ctt ata gct cct gag agc ttt cct tct aat cta 1296 Glu Ile Cys Leu Pro Leu Ile Ala Pro Glu Ser Phe Pro Ser Asn Leu             420 425 430 aat gta gaa gct ggg agc tat gtt tcc ttt cac tgt aga gct act gca 1344 Asn Val Glu Ala Gly Ser Tyr Val Ser Phe His Cys Arg Ala Thr Ala         435 440 445 gaa cca cag cct gaa atc tac tgg ata aca cct tct ggt caa aaa ctc 1392 Glu Pro Gln Pro Glu Ile Tyr Trp Ile Thr Pro Ser Gly Gln Lys Leu     450 455 460 ttg cct aat acc ctg aca gac aag ttc tat gtc cat tct gag gga aca 1440 Leu Pro Asn Thr Leu Thr Asp Lys Phe Tyr Val His Ser Glu Gly Thr 465 470 475 480 cta gat ata aat ggc gta act ccc aaa gaa ggg ggt tta tat act tgt 1488 Leu Asp Ile Asn Gly Val Thr Pro Lys Glu Gly Gly Leu Tyr Thr Cys                 485 490 495 ata gca act aac cta gtt ggc gct gac ttg aag tct gtt atg atc aaa 1536 Ile Ala Thr Asn Leu Val Gly Ala Asp Leu Lys Ser Val Met Ile Lys             500 505 510 gtg gat gga tct ttt cca caa gat aac aat ggc tct ttg aat att aaa 1584 Val Asp Gly Ser Phe Pro Gln Asp Asn Asn Gly Ser Leu Asn Ile Lys         515 520 525 ata aga gat att cag gcc aat tca gtt ttg gtg tcc cgg aaa gca agt 1632 Ile Arg Asp Ile Gln Ala Asn Ser Val Leu Val Ser Arg Lys Ala Ser     530 535 540 tct aaa att ctc aaa tct agt gtt aaa tgg aca gcc ttt gtc aag act 1680 Ser Lys Ile Leu Lys Ser Ser Val Lys Trp Thr Ala Phe Val Lys Thr 545 550 555 560 gaa aat tct cat gct gcg caa agt gct cga ata cca tct gat gtc aag 1728 Glu Asn Ser His Ala Ala Gln Ser Ala Arg Ile Pro Ser Asp Val Lys                 565 570 575 gta tat aat ctt act cat ctg aat cca tca act gag tat aaa att tgt 1776 Val Tyr Asn Leu Thr His Leu Asn Pro Ser Thr Glu Tyr Lys Ile Cys             580 585 590 att gat att ccc acc atc tat cag aaa aac aga aaa aaa tgt gta aat 1824 Ile Asp Ile Pro Thr Ile Tyr Gln Lys Asn Arg Lys Lys Cys Val Asn         595 600 605 gtc acc acc aaa ggt ttg cac cct gat caa aaa gag tat gaa aag aat 1872 Val Thr Thr Lys Gly Leu His Pro Asp Gln Lys Glu Tyr Glu Lys Asn     610 615 620 aat acc aca aca ctt atg gcc tgt ctt gga ggc ctt ctg ggg att att 1920 Asn Thr Thr Thr Leu Met Ala Cys Leu Gly Gly Leu Leu Gly Ile Ile 625 630 635 640 ggt gtg ata tgt ctt atc agc tgc ctc tct cca gaa atg aac tgt gat 1968 Gly Val Ile Cys Leu Ile Ser Cys Leu Ser Pro Glu Met Asn Cys Asp                 645 650 655 ggt gga cac agc tat gtg agg aat tac tta cag aaa cca acc ttt gca 2016 Gly Gly His Ser Tyr Val Arg Asn Tyr Leu Gln Lys Pro Thr Phe Ala             660 665 670 tta ggt gag ctt tat cct cct ctg ata aat ctc tgg gaa gca gga aaa 2064 Leu Gly Glu Leu Tyr Pro Pro Leu Ile Asn Leu Trp Glu Ala Gly Lys         675 680 685 gaa aaa agt aca tca ctg aaa gta aaa gca act gtt ata ggt tta cca 2112 Glu Lys Ser Thr Ser Le Le Lys Val Lys Ala Thr Val Ile Gly Leu Pro     690 695 700 aca aat atg tct taa 2127 Thr Asn Met Ser 705 <210> 9 <211> 468 <212> PRT <213> Ero1L protein <400> 9 Met Gly Arg Gly Trp Gly Phe Leu Phe Gly Leu Leu Gly Ala Val Trp   1 5 10 15 Leu Leu Ser Ser Gly His Gly Glu Glu Gln Pro Pro Glu Thr Ala Ala              20 25 30 Gln Arg Cys Phe Cys Gln Val Ser Gly Tyr Leu Asp Asp Cys Thr Cys          35 40 45 Asp Val Glu Thr Ile Asp Arg Phe Asn Asn Tyr Arg Leu Phe Pro Arg      50 55 60 Leu Gln Lys Leu Leu Glu Ser Asp Tyr Phe Arg Tyr Tyr Lys Val Asn  65 70 75 80 Leu Lys Arg Pro Cys Pro Phe Trp Asn Asp Ile Ser Gln Cys Gly Arg                  85 90 95 Arg Asp Cys Ala Val Lys Pro Cys Gln Ser Asp Glu Val Pro Asp Gly             100 105 110 Ile Lys Ser Ala Ser Tyr Lys Tyr Ser Glu Glu Ala Asn Asn Leu Ile         115 120 125 Glu Glu Cys Glu Gln Ala Glu Arg Leu Gly Ala Val Asp Glu Ser Leu     130 135 140 Ser Glu Glu Thr Gln Lys Ala Val Leu Gln Trp Thr Lys His Asp Asp 145 150 155 160 Ser Ser Asp Asn Phe Cys Glu Ala Asp Asp Ile Gln Ser Pro Glu Ala                 165 170 175 Glu Tyr Val Asp Leu Leu Leu Asn Pro Glu Arg Tyr Thr Gly Tyr Lys             180 185 190 Gly Pro Asp Ala Trp Lys Ile Trp Asn Val Ile Tyr Glu Glu Asn Cys         195 200 205 Phe Lys Pro Gln Thr Ile Lys Arg Pro Leu Asn Pro Leu Ala Ser Gly     210 215 220 Gln Gly Thr Ser Glu Glu Asn Thr Phe Tyr Ser Trp Leu Glu Gly Leu 225 230 235 240 Cys Val Glu Lys Arg Ala Phe Tyr Arg Leu Ile Ser Gly Leu His Ala                 245 250 255 Ser Ile Asn Val His Leu Ser Ala Arg Tyr Leu Leu Gln Glu Thr Trp             260 265 270 Leu Glu Lys Lys Trp Gly His Asn Ile Thr Glu Phe Gln Gln Arg Phe         275 280 285 Asp Gly Ile Leu Thr Glu Gly Glu Gly Pro Arg Arg Leu Lys Asn Leu     290 295 300 Tyr Phe Leu Tyr Leu Ile Glu Leu Arg Ala Leu Ser Lys Val Leu Pro 305 310 315 320 Phe Phe Glu Arg Pro Asp Phe Gln Leu Phe Thr Gly Asn Lys Ile Gln                 325 330 335 Asp Glu Glu Asn Lys Met Leu Leu Leu Glu Ile Leu His Glu Ile Lys             340 345 350 Ser Phe Pro Leu His Phe Asp Glu Asn Ser Phe Phe Ala Gly Asp Lys         355 360 365 Lys Glu Ala His Lys Leu Lys Glu Asp Phe Arg Leu His Phe Arg Asn     370 375 380 Ile Ser Arg Ile Met Asp Cys Val Gly Cys Phe Lys Cys Arg Leu Trp 385 390 395 400 Gly Lys Leu Gln Thr Gln Gly Leu Gly Thr Ala Leu Lys Ile Leu Phe                 405 410 415 Ser Glu Lys Leu Ile Ala Asn Met Pro Glu Ser Gly Pro Ser Tyr Glu             420 425 430 Phe His Leu Thr Arg Gln Glu Ile Val Ser Leu Phe Asn Ala Phe Gly         435 440 445 Arg Ile Ser Thr Ser Val Lys Glu Leu Glu Asn Phe Arg Asn Leu Leu     450 455 460 Gln Asn Ile His 465 <210> 10 <211> 1885 <212> DNA <213> Ero1L nucleotide <220> <221> gene (222) (1) .. (1885) <220> <221> CDS (222) (173) .. (1576) <400> 10 gcgaggtggc gatcgctgag aggcaggagg gccgaggcgg gcctgggagg cggcccggag 60 gtggggcgcc gctggggccg gcccgcacgg gcttcatctg agggcgcacg gcccgcgacc 120 gagcgtgcgg actggcctcc caagcgtggg gcgacaagct gccggagctg ca 172 atg ggc cgc ggc tgg gga ttc ttg ttt ggc ctc ctg ggc gcc gtg tgg 220 Met Gly Arg Gly Trp Gly Phe Leu Phe Gly Leu Leu Gly Ala Val Trp   1 5 10 15 ctg ctc agc tcg ggc cac gga gag gag cag ccc ccg gag aca gcg gca 268 Leu Leu Ser Ser Gly His Gly Glu Glu Gln Pro Pro Glu Thr Ala Ala              20 25 30 cag agg tgc ttc tgc cag gtt agt ggt tac ttg gat gat tgt acc tgt 316 Gln Arg Cys Phe Cys Gln Val Ser Gly Tyr Leu Asp Asp Cys Thr Cys          35 40 45 gat gtt gaa acc att gat aga ttt aat aac tac agg ctt ttc cca aga 364 Asp Val Glu Thr Ile Asp Arg Phe Asn Asn Tyr Arg Leu Phe Pro Arg      50 55 60 cta caa aaa ctt ctt gaa agt gac tac ttt agg tat tac aag gta aac 412 Leu Gln Lys Leu Leu Glu Ser Asp Tyr Phe Arg Tyr Tyr Lys Val Asn  65 70 75 80 ctg aag agg ccg tgt cct ttc tgg aat gac atc agc cag tgt gga aga 460 Leu Lys Arg Pro Cys Pro Phe Trp Asn Asp Ile Ser Gln Cys Gly Arg                  85 90 95 agg gac tgt gct gtc aaa cca tgt caa tct gat gaa gtt cct gat gga 508 Arg Asp Cys Ala Val Lys Pro Cys Gln Ser Asp Glu Val Pro Asp Gly             100 105 110 att aaa tct gcg agc tac aag tat tct gaa gaa gcc aat aat ctc att 556 Ile Lys Ser Ala Ser Tyr Lys Tyr Ser Glu Glu Ala Asn Asn Leu Ile         115 120 125 gaa gaa tgt gaa caa gct gaa cga ctt gga gca gtg gat gaa tct ctg 604 Glu Glu Cys Glu Gln Ala Glu Arg Leu Gly Ala Val Asp Glu Ser Leu     130 135 140 agt gag gaa aca cag aag gct gtt ctt cag tgg acc aag cat gat gat 652 Ser Glu Glu Thr Gln Lys Ala Val Leu Gln Trp Thr Lys His Asp Asp 145 150 155 160 tct tca gat aac ttc tgt gaa gct gat gac att cag tcc cct gaa gct 700 Ser Ser Asp Asn Phe Cys Glu Ala Asp Asp Ile Gln Ser Pro Glu Ala                 165 170 175 gaa tat gta gat ttg ctt ctt aat cct gag cgc tac act ggt tac aag 748 Glu Tyr Val Asp Leu Leu Leu Asn Pro Glu Arg Tyr Thr Gly Tyr Lys             180 185 190 gga cca gat gct tgg aaa ata tgg aat gtc atc tac gaa gaa aac tgt 796 Gly Pro Asp Ala Trp Lys Ile Trp Asn Val Ile Tyr Glu Glu Asn Cys         195 200 205 ttt aag cca cag aca att aaa aga cct tta aat cct ttg gct tct ggt 844 Phe Lys Pro Gln Thr Ile Lys Arg Pro Leu Asn Pro Leu Ala Ser Gly     210 215 220 caa ggg aca agt gaa gag aac act ttt tac agt tgg cta gaa ggt ctc 892 Gln Gly Thr Ser Glu Glu Asn Thr Phe Tyr Ser Trp Leu Glu Gly Leu 225 230 235 240 tgt gta gaa aaa aga gca ttc tac aga ctt ata tct ggc cta cat gca 940 Cys Val Glu Lys Arg Ala Phe Tyr Arg Leu Ile Ser Gly Leu His Ala                 245 250 255 agc att aat gtg cat ttg agt gca aga tat ctt tta caa gag acc tgg 988 Ser Ile Asn Val His Leu Ser Ala Arg Tyr Leu Leu Gln Glu Thr Trp             260 265 270 tta gaa aag aaa tgg gga cac aac att aca gaa ttt caa cag cga ttt 1036 Leu Glu Lys Lys Trp Gly His Asn Ile Thr Glu Phe Gln Gln Arg Phe         275 280 285 gat gga att ttg act gaa gga gaa ggt cca aga agg ctt aag aac ttg 1084 Asp Gly Ile Leu Thr Glu Gly Glu Gly Pro Arg Arg Leu Lys Asn Leu     290 295 300 tat ttt ctc tac tta ata gaa cta agg gct tta tcc aaa gtg tta cca 1132 Tyr Phe Leu Tyr Leu Ile Glu Leu Arg Ala Leu Ser Lys Val Leu Pro 305 310 315 320 ttc ttc gag cgc cca gat ttt caa ctc ttt act gga aat aaa att cag 1180 Phe Phe Glu Arg Pro Asp Phe Gln Leu Phe Thr Gly Asn Lys Ile Gln                 325 330 335 gat gag gaa aac aaa atg tta ctt ctg gaa ata ctt cat gaa atc aag 1228 Asp Glu Glu Asn Lys Met Leu Leu Leu Glu Ile Leu His Glu Ile Lys             340 345 350 tca ttt cct ttg cat ttt gat gag aat tca ttt ttt gct ggg gat aaa 1276 Ser Phe Pro Leu His Phe Asp Glu Asn Ser Phe Phe Ala Gly Asp Lys         355 360 365 aaa gaa gca cac aaa cta aag gag gac ttt cga ctg cat ttt aga aat 1324 Lys Glu Ala His Lys Leu Lys Glu Asp Phe Arg Leu His Phe Arg Asn     370 375 380 att tca aga att atg gat tgt gtt ggt tgt ttt aaa tgt cgt ctg tgg 1372 Ile Ser Arg Ile Met Asp Cys Val Gly Cys Phe Lys Cys Arg Leu Trp 385 390 395 400 gga aag ctt cag act cag ggt ttg ggc act gct ctg aag atc tta ttt 1420 Gly Lys Leu Gln Thr Gln Gly Leu Gly Thr Ala Leu Lys Ile Leu Phe                 405 410 415 tct gag aaa ttg ata gca aat atg cca gaa agt gga cct agt tat gaa 1468 Ser Glu Lys Leu Ile Ala Asn Met Pro Glu Ser Gly Pro Ser Tyr Glu             420 425 430 ttc cat cta acc aga caa gaa ata gta tca tta ttc aac gca ttt gga 1516 Phe His Leu Thr Arg Gln Glu Ile Val Ser Leu Phe Asn Ala Phe Gly         435 440 445 aga att tct aca agt gtg aaa gaa tta gaa aac ttc agg aac ttg tta 1564 Arg Ile Ser Thr Ser Val Lys Glu Leu Glu Asn Phe Arg Asn Leu Leu     450 455 460 cag aat att cat taaa gaaaacaagc tgatatgtgc ctgtttctgg acaatggagg 1620 Gln Asn Ile His 465 cgaaagagtg gaatttcatt caaaggcata atagcaatga cagtcttaag ccaaacattt 1680 tatataaagt tgcttttgta aaggagaatt atattgtttt aagtaaacac atttttaaaa 1740 attgtgttaa gtctatgtat aatactactg tgagtaaaag taatacttta ataatgtggt 1800 acaaatttta aagtttaata ttgaataaaa ggaggattat caaattaaaa aaaaaaaaaa 1860 aaaaaaaaaa aaaaaaaaaa aaaaa 1885

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 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.
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 step (a), wherein when the sample reduces the expression of the protein or polynucleotide, the sample is a substance for preventing or treating cancer. It is determined.
A screening method of a substance for preventing or treating cancer, comprising the steps of:
(a) preparing a protein of SEQ ID NO: 3;
(b) contacting the sample 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.
4. The method of claim 3, wherein the protein is present in the cell surface, in the viral surface or in 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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