KR20160021058A - Use of LYPD1 for cancer diagnostic or therapeutic marker - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising LY6/PLAUR domain containing 1 (LYPD1) gene expression inhibitor, an LYPD1 protein activity inhibitor, or a mixture thereof as an active component; a composition for diagnosing cancer comprising a preparation for measuring the level of LYPD1 gene mRNA or a protein thereof; a kit for diagnosing cancer comprising the diagnostic composition; a method for providing information for diagnosing cancer by measuring the level of LYPD1 gene mRNA or a protein thereof; or a method for screening a cancer treating agent.

Description

Use of LYPD1 as a cancer diagnostic or therapeutic target [

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an inhibitor of expression of LYPD1 (LY6 / PLAUR domain containing 1) gene, an inhibitor of LYPD1 protein activity or a mixture thereof as an active ingredient, a level of LYPD1 gene mRNA or its protein A cancer diagnosis kit comprising the diagnostic composition, and a method of providing information for cancer diagnosis by measuring the level of mRNA or a protein of the LYPD1 gene or a method of screening a cancer therapeutic agent .

Cancer is a disease in which cell cycle is not controlled and cell division continues, such as lung cancer, esophageal cancer, uterine cancer, breast cancer, colon cancer and prostate cancer. Among them, colorectal cancer is most commonly adenocarcinoma, and it is divided into colon cancer and rectal cancer. The incidence of each site is highest in the lower colon, or rectum, about 50%. Colon cancer is the second leading cause of cancer-related deaths in the United States and Europe (American Cancer Society statics for 2009). The treatment of colorectal cancer is based on surgical resection, and chemotherapy and radiotherapy are combined. Despite advances such as surgical treatment, chemotherapy and radiotherapy, it is frequently diagnosed after metastasis to other organs due to the characteristics of colorectal cancer progressing without specific symptoms, . Therefore, effective diagnosis and treatment targets for colorectal cancer are required for this reason.

In addition, prostate cancer is a malignant tumor originating from the prostate gland. In the western world, it is the most common cancer among men. However, since prostate cancer is uncommon, it is difficult to detect it early. Therefore, it is often difficult to find a hospital after the cancer tissue is enlarged or the prostate cancer is transferred to other organs such as bone or the like. Therefore, it is required to develop an effective diagnosis and treatment target of prostate cancer for this reason.

On the other hand, LYPD1 (LY6 / PLAUR domain containing 1) gene is found in chimpanzee, dog, cattle, mouse and chicken, and human LYPD1 gene has 81 organisms and orthologs. Although there is no published report on LYPD1, it has been reported that Hela cells have a tumor suppressor role (Exp Cell Res, 2006, 312 (6): 865-76) There is no known function.

Under these circumstances, the present inventors have made intensive efforts to develop a marker for diagnosis and treatment of cancer. As a result, LYPD1 can be effectively used as a cancer diagnostic marker. In addition, suppression of the expression of LYPD1 gene leads to invasion of cancer cells, Cell growth is inhibited, while the expression of LYPD1 gene is enhanced, leading to an increase in major cell signal transduction associated with cancer cell infiltration and carcinogenesis. Therefore, when the expression or activity of the LYPD1 gene is inhibited, it is effective in prevention or treatment of cancer, .

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of LYPD1 gene expression, an inhibitor of LYPD1 protein activity, or a mixture thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or ameliorating cancer comprising an inhibitor of LYPD1 gene expression, an inhibitor of LYPD1 protein activity, or a mixture thereof as an active ingredient.

It is still another object of the present invention to provide a cancer diagnostic composition comprising an agent for measuring the level of LYPD1 gene mRNA or a protein thereof.

Yet another object of the present invention is to provide a cancer diagnostic kit comprising the composition.

Yet another object of the present invention is to provide a method for detecting cancer, comprising: (a) measuring the level of the mRNA of LYPD1 gene or a protein thereof from a separate sample of a subject suspected of having cancer; And (b) comparing the level of the mRNA of the gene or the protein thereof to a level of the mRNA of the gene of the normal control sample or a level of the mRNA of the gene of the normal control sample, or a method of diagnosing cancer .

It is still another object of the present invention to provide a method for treating cancer, which comprises the steps of (a) treating a candidate substance for cancer treatment to a separate sample expressing the LYPD1 gene, and then measuring the level of the mRNA of the LYPD1 gene or a protein thereof; And (b) when the level of the mRNA of the gene or the protein thereof is lower than that of the control sample not treated with the sample, a method for screening a cancer therapeutic agent.

One aspect of the present invention for solving the above problems is a pharmaceutical composition for preventing or treating cancer comprising an inhibitor of expression of LYPD1 (LY6 / PLAUR domain containing 1) gene, an inhibitor of LYPD1 protein activity or a mixture thereof as an active ingredient Lt; / RTI >

According to the present invention, when the expression or activity of LYPD1 gene is inhibited in cancer cells, the infiltration of cancer cells, cell mobility and cell growth are reduced. Thus, prevention or treatment of cancer can be achieved by inhibiting the expression of LYPD1 gene or the activity of the protein.

As used herein, the term "LYPD1" is an abbreviation of "LY6 / PLAUR domain containing 1 ", and LYPD1 has been reported to inhibit the tumor in Hela cells (Yu DH, et al., Exp Cell Res, 2006), and the function of LYPD1 in relation to other cancers is unknown.

Rather, in the present invention, LYPD1 has an activity of promoting invasion of cancer cells, cell mobility and cell growth, and inhibiting the expression thereof inhibits invasion of cancer cells, cell mobility and cell growth, so that it is used as a cancer diagnostic marker or cancer therapeutic agent .

The gene and protein information of LYPD1 can be easily confirmed through a known database such as NCBI gene bank, for example, but not limited to, LYPD1 which is Gene ID: 116372 of NCBI gene bank. The LYPD1 may have the amino acid sequence shown in SEQ ID NO: 7, and may have the nucleotide sequence shown in SEQ ID NO: 1, though it is not particularly limited thereto.

Based on the sequence of LYPD1, an inhibitor of LYPD1 expression or an activity inhibitor can be designed, and the sequence can be modified to some extent in this design. Those skilled in the art will appreciate that sequences that retain more than 80%, in particular more than 90%, more specifically more than 95%, and more particularly 98% Do.

Specifically, in one embodiment of the present invention, it was confirmed that when the expression of LYPD1 is inhibited by siRNA in SW480sub cell line, which is a colorectal cancer cell, cell infiltration, mobility and cell growth are reduced (Example 8) It was confirmed that when the expression is enhanced in the cancer cells, the infiltration ability of the cells is increased (FIG. 5).

In another embodiment of the present invention, SW480 and HEK293E cells were transfected and then Western blotted, commonly used for the phosphorylation of JNK and ATF-2, a component of transcription factor AP-1 (activator protein 1) Phosphorylation was increased (FIG. 6). As a result, in SW480, the expression of vimentin, vimentin, which is a mesenchymal cell marker, and the expression of cyclin D1 and cyclin A were increased. In addition to ATF-2 in HEK293E, phosphorylation of c-Jun, And cyclin A, cyclin A were increased in the control group.

In addition, in another embodiment of the present invention, the activity of AP-1 was confirmed by the luciferase assay in the HEK293E cell line, and the activity of AP-1 was increased by the expression of LYPD1 (FIG. 7) . Thus, AP-1 activity is closely related to cancer cell activity (growth and invasion), indicating that AP-1 activity regulation by LYPD1 expression contributes to invasion / cell migration and cell growth regulation.

In conclusion, when the expression of LYPD1 of the present invention is decreased in various cancer cells including colon cancer cells, AP-1 activity is decreased, and the infiltration / cell mobility and cell growth of cancer cells are decreased. That is, the LYPD1 of the present invention can diagnose cancer by measuring the expression of LYPD1 as a diagnostic or therapeutic target of cancer associated with invasion / cell mobility and cell growth of various cancer cells, and can also treat cancer by suppressing its expression or activity .

In another embodiment of the present invention, cell growth and LYPD1 expression are decreased when the expression of LYPD1 is inhibited by siRNA in the prostate cancer cell line PC3 (FIGS. 9 and 10), and cell mobility is decreased (Fig. 11). This suggests that inhibiting the expression of LYPD1 of the present invention has a remarkable effect on cancer treatment.

As used herein, the term "inhibitor of LYPD1 gene expression" is used collectively to refer to a substance that decreases the expression or activity of LYPD1. More specifically, the expression of LYPD1 is decreased at the transcription level or protein level Which may include all materials which make it possible. The substance that inhibits the expression of LYPD1 may be used in any form such as a compound, a nucleic acid, a peptide, a virus, or a vector containing the nucleic acid, which can target LYPD1 and inhibit the expression or activity of LYPD1.

Specifically, the LYPD1 gene expression inhibitor may be at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the LYPD1 gene.

Specifically, the LYPD1 gene expression inhibitor may bind to one or more sequences selected from the group consisting of SEQ ID NOS: 2 to 5. The nucleotide sequence of SEQ ID NO: 2 to 5 is a nucleotide sequence part capable of binding to the inhibitor in the LYPD1 gene.

Such an expression inhibitor can be easily designed so that a person skilled in the art can come to restrain the expression of LYPD1 according to techniques commonly known in the art.

In addition, the activity inhibitor of the LYPD1 protein may be an antibody or an aptamer that specifically binds to a protein expressed from the LYPD1 gene, but is not limited thereto.

Such an antibody may be a polyclonal antibody, a monoclonal antibody or fragments of the antibody as long as the antibody has antigen binding ability. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody, and a human antibody. In addition to the novel antibody, antibodies that are already known in the art may also be included. The antibody comprises a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains, so long as the antibody has the property of binding specifically recognizing the protein expressed from the LYPD1 gene. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes, but is not limited to, Fab, F (ab ') 2, F (ab') ₂ and Fv.

In addition, the composition of the present invention may be used alone, but it may be used alone or in combination with radiation therapy or chemotherapy (including cell growth arrest or cytotoxic substance, antibiotic substance, alkylating agent, antimetabolite, hormone, An anti-EGFR agent, an anti-angiogenic agent, a paroxetine transfer agent, an anti-angiogenic agent, an anti-angiogenesis inhibitor, a metallo-matrix protease inhibitor, a telomerase inhibitor, a tyrosine kinase inhibitor, Such as, but not limited to, inhibitors, ras-raf signaling pathway pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin binders, topoisomerase I inhibitors, topoisomerase II inhibitors, It is not limited.

 For the purpose of the present invention, the cancer is not limited as long as it is a cancer that can be prevented or treated by inhibiting the expression of the LYPD1 gene or the activity of the protein. Specifically, the cancer is not limited to colon cancer, prostate cancer, lung cancer, , Pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, endometrial carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, Hodgkin's disease, Esophageal cancer, Small intestine cancer, Endocrine cancer, Thyroid cancer, Parathyroid cancer, Adenocarcinoma, Soft tissue sarcoma, Urethral cancer, Penile cancer, Chronic or acute leukemia, Lymphocytic lymphoma, Bladder cancer, Cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, and more specifically Colon cancer, or prostate cancer.

The colon cancer is a cancer arising from the mucosa, which is the innermost surface of the large intestine, and refers to rectal cancer, colon cancer and anal cancer.

The prostate cancer refers to a cancer that occurs in the prostate gland.

As used herein, the term "prevention" refers to any act that inhibits or delays the onset of cancer by administering the composition to an individual. The term "treatment" as used in the present invention means all the actions that cause the cancer symptom to be improved or benefited by administering the composition to a cancerous subject.

The composition of the present invention may be administered together with a pharmaceutically acceptable carrier. In oral administration, a conjugate, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, In the case of injections, a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer and the like may be mixed. In the case of topical administration, a base, excipient, lubricant and preservative may be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, unit dosage ampoules or multiple dose forms may be prepared.

As used herein, the term administration refers to the introduction of a composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any conventional route so long as it can reach the target tissue have. Intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intramuscular, subcutaneous, intradermal, But is not limited thereto. The effective dose range of the composition of the present invention can be varied depending on the sex, body surface area, kind and severity of disease, age, sensitivity to the drug, administration route and discharge rate, administration time, treatment period, target cell, And may be readily determined by one of ordinary skill in the art.

Another aspect of the present invention provides a health functional food for preventing or ameliorating cancer comprising an inhibitor of LYPD1 gene expression, an inhibitor of LYPD1 protein activity, or a mixture thereof as an active ingredient.

The kind of the food is not particularly limited, and includes food in a conventional sense. Non-limiting examples of foods to which the material can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice creams, , A drink, an alcoholic beverage, and a vitamin complex.

When the health functional food of the present invention is a beverage composition, it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Non-limiting examples of such natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweetening agents such as dextrin, cyclodextrin; Synthetic sweetening agents such as saccharin and aspartame, and the like. The proportion of the additional component added may be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit drink or vegetable drink. These components may be used independently or in combination of two or more. The ratios of these additives can also be suitably selected by those skilled in the art.

 Another aspect of the present invention provides a cancer diagnostic composition comprising an agent for measuring the level of LYPD1 (LY6 / PLAUR domain containing 1) gene mRNA or a protein thereof.

The LYPD1 is as described above.

As described above, the gene and protein information of LYPD1 can be easily confirmed through a known database such as NCBI gene bank, for example, LYPD1 which is Gene ID: 116372 of NCBI gene bank, but is not limited thereto . The LYPD1 may have the amino acid sequence shown in SEQ ID NO: 7, and may have the nucleotide sequence shown in SEQ ID NO: 1, though it is not particularly limited thereto.

Based on the sequence of LYPD1, the expression level of LYPD1 can be measured to diagnose cancer progression or onset. The sequence may be modified to some extent in diagnosing cancer progression or onset. Those skilled in the art will appreciate that sequences which retain more than 80%, in particular more than 90%, more specifically more than 95%, more specifically more than 98% homology by such an artificial modification, It will be easily understood that the present invention can be used as a cancer diagnostic marker and is equivalent to the above sequence of the present invention as long as the difference between the normal individuals and the suspected cancer can be significantly compared.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming whether or not the cancer has progressed or developed.

As used herein, the term "marker or diagnostic marker" refers to a substance capable of diagnosing a cancer cell or an individual having cancer by distinguishing it from a normal cell or a normal cell. The cell or object Such as polypeptides, proteins or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that exhibit increased or decreased activity. For the purpose of the present invention, the cancer diagnostic marker of the present invention is LYPD1, which is a gene whose expression is increased in cancer cells.

The term "agent for measuring the level of LYPD1 gene mRNA" used in the present invention means a preparation used for confirming the expression of LYPD1 contained in a sample, preferably RT-PCR, competitive RT-PCR A target gene used in methods such as competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, But it is not particularly limited thereto.

The term "primer" as used herein refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primers 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.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides, which can specifically bind to a gene or mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection, but the present invention is not limited thereto.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

 As used herein, the term "measurement of protein level" refers to a process for determining the presence and expression level of a protein expressed from a cancer marker gene in a biological sample in order to diagnose cancer. Specifically, The amount of the protein can be confirmed by using a specifically binding antibody.

In the present invention, "antibody" means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the cancer marker protein has been identified as described above, the production of the antibody using the cancer marker protein can be easily performed using techniques well known in the art.

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

Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 1992, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991). The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with 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 an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

Methods for determining the amount of the target protein bound to the antibody using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Fluorescence Activated Cell Sorter Chip, and the like, but are not limited thereto.

Another aspect of the present invention provides a cancer diagnostic kit comprising the cancer diagnostic composition.

Since the level of the mRNA or the protein of the LYPD1 gene of the present invention is higher than that of the normal cells, the level of the mRNA or the protein of the LYPD1 gene of the normal cell is compared with that of the normal cell. If the level is high, cancer cells can be diagnosed.

The kit can be used to diagnose cancer by measuring the expression level of LYPD1 from a sample of a subject suspected of having cancer, including, but not limited to, a primer or a probe for measuring the expression level of LYPD1, One or more other component compositions, solutions or devices suitable for use in the present invention may be included.

Specifically, the diagnostic kit for measuring the expression level of LYPD1 of the present invention may be a kit containing essential elements necessary for conducting RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

In particular, the kit of the present invention may contain essential elements necessary for performing gene chip analysis. The kit for gene chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

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

For the purpose of the present invention, the cancer is not limited as long as the level of the mRNA or the protein of the LYPD1 gene is higher than that of a normal cell, but specifically, the cancer is not limited to colon cancer, prostate cancer, lung cancer, non- Ovarian cancer, rectal cancer, stomach cancer, anorectal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, ovarian cancer, ovarian cancer, ovarian cancer, Cancer of the kidney, cancer of the urethra, cancer of the urethra, cancer of the urethra, cancer of the urethra, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cancer, endometrioid cancer, , Renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, and more specifically, Amil it is, but is not limited thereto.

Another aspect of the present invention provides a method for providing information for cancer diagnosis using the diagnostic composition or kit. Specifically, there is provided a method for diagnosing cancer, comprising the steps of: (a) isolating a mRNA of LYPD1 gene Or measuring the level of the protein thereof; And (b) comparing the level of the mRNA of the gene or the protein thereof to a level of the mRNA of the gene of the normal control sample or a protein thereof. In addition, the method of providing information for cancer diagnosis may be a cancer diagnosis method.

The method may further comprise the step of (c) determining that the cancer is onset if the level of the mRNA of the gene or the protein thereof is higher than the mRNA of the gene of the normal control sample or the protein thereof.

The process of isolating mRNA from an individual suspected of having cancer can be performed using a known process. The term "sample of an individual" used in the present invention refers to a gene having a different level of the mRNA of LYPD1 gene or its protein But are not limited to, tissue, cells, blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

The mRNA level can be measured by various methods. Specifically, a primer or a probe that specifically binds to the LYPD1 gene can be used.

Specifically, the mRNA level may be measured by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay But are not limited to, those determined by RNase protection method, Northern blotting or gene chips.

Methods for measuring the LYPD1 protein level include Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc. But is not limited thereto.

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

The term "antigen-antibody complex" in the present invention means a combination of a cancer marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through a signal label of a detection label Do.

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

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

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

More specifically, immunostaining is performed using one or more antibodies against the cancer marker. Normal colon epithelial tissues and suspected cancerous tissues are collected and fixed, and paraffin-embedded blocks are prepared by methods well known in the art. They are made into sections with a thickness of several um and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.

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

For the purpose of the present invention, the cancer is not limited as long as the level of the mRNA or the protein of the LYPD1 gene is higher than that of a normal cell, but specifically, the cancer is not limited to colon cancer, prostate cancer, lung cancer, non- Ovarian cancer, rectal cancer, stomach cancer, anorectal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, ovarian cancer, ovarian cancer, ovarian cancer, Cancer of the kidney, cancer of the urethra, cancer of the urethra, cancer of the urethra, cancer of the urethra, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cancer, endometrioid cancer, , Renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, and more specifically, Amil it is, but is not limited thereto.

Another aspect of the present invention is a method for treating cancer, comprising the steps of: (a) treating a candidate substance for cancer treatment to a separate sample expressing the LYPD1 gene, and then measuring the level of the mRNA of the LYPD1 gene or a protein thereof; And (b) when the level of the mRNA or the protein of the gene is lower than that of the control sample not treated with the sample, a method for screening a cancer therapeutic agent.

The level of LYPD1 of the present invention is measured in a cell expressing the LYPD1 gene in the absence of a candidate substance capable of preventing or treating cancer and the level of the LYPD1 of the present invention is measured in the presence of the candidate substance, , A substance which reduces the level of expression of the LYPD1 of the present invention in the absence of the candidate substance in the absence of the candidate substance can be predicted by an agent for the prophylaxis or treatment of cancer.

Specifically, the cell expressing the LYPD1 gene may be a colon, prostate, lung, non-small cell lung, colon, bone, pancreatic, skin, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary adenocarcinoma, adenocarcinoma, adenocarcinoma of the uterine cervix, adenocarcinoma, vulvar carcinoma, vulvar carcinoma, (CNS) tumors, primary central nervous system (CNS) neoplasms, renal cell carcinomas, renal pelvic carcinomas, central nervous system (CNS) tumors, Lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma cell. More specifically, it may be a colon cancer or a prostate cancer cell. Accordingly, the present invention provides a method for treating colon cancer or prostate cancer But the present invention is not limited thereto.

The present inventors have confirmed that a LYPD1 (LY6 / PLAUR domain containing 1) gene, whose function is not well known until now, regulates the infiltration of cancer cells, cell mobility and cell growth and regulates cell signal transduction. A cancer diagnosis kit comprising the composition, a method of providing information for cancer diagnosis, a screening method of cancer treatment agent, And a pharmaceutical composition for preventing or treating cancer and the like.

FIG. 1 shows that the inhibition of LYPD1 expression by siRNA in colorectal cancer cell lines leads to a decrease in cancer cell infiltration and cell mobility. a is a photograph showing the result of staining a colon cancer cell which migrated from an upper chamber to a lower chamber with a crystal violet solution. and b is a graph showing the relative proportion of colon cancer cells transferred from the upper chamber to the lower chamber.
FIG. 2 is a graph showing that the growth of cancer cells is reduced as a result of suppression of expression of LYPD1 by siRNA in colon cancer cell lines.
FIG. 3 is a graph showing that LYPD1 expression is reduced as a result of suppression of expression of LYPD1 by siRNA for 48 hours (a) and 72 hours (b) in a colon cancer cell line.
FIG. 4 is a graph showing the degree of LYPD1 expression by transforming LYPD1 expression vectors (p3XFlagCMV14-LYPD1 and pFlagCMV1-LYPD1) into normal cells.
FIG. 5 shows (a) the increase in the expression of LYPD1 in the colon cancer cell line and (b) the increase in the infiltration ability. FIG.
FIG. 6 is a graph showing the activation of cell signaling when the expression of LYPD1 is increased in the normal cell (a) and the colon cancer cell line (b), respectively.
FIG. 7 shows the increase in AP-1 reporter activity according to the expression of LYPD1 in normal cells.
Fig. 8 is a graph showing the results of RT-qPCR expression of LYPD1 expression in 6 colon cancer cell lines and 2 prostate cancer cell lines.
FIG. 9 shows that the growth of cancer cells is reduced as a result of inhibiting the expression of LYPD1 in prostate cancer cell lines with siRNA for 48 hours (a) and 72 hours (b).
FIG. 10 is a graph showing that LYPD1 expression is reduced as a result of inhibition of LYPD1 expression by siRNA in a prostate cancer cell line. FIG.
11 is a graph showing that the mobility of cancer cells is reduced as a result of suppression of expression of LYPD1 by siRNA in the prostate cancer cell line. a is a photograph showing the results of staining of prostate cancer cells that have moved from the upper chamber to the lower chamber with a crystal violet solution. and b is a graph showing the relative proportion of prostate cancer cells transferred from the upper chamber to the lower chamber.

Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example  1. Cell culture

All human cell lines were obtained from the American Type Culture Collection (USA). The colon cancer cell lines SW480, HCT116, HT29, HCT15, Lovo, Colo205 and prostate cancer cell lines DU145 and PC3 cell lines were cultured in RPMI1640 (GIBCO, USA) containing 10% FBS, penicillin-streptomycin, L-glutamine and sodium pyruvate ) Medium, HEK293E cells were cultured in DMEM medium at 37 ° C and 5% CO ₂ . SW480sub was a subpopulation obtained from SW480.

Example  2. siRNA  Or transfection of the plasmid

SW480 and PC3 cells were transfected using the Microporation technique (Neon Transfection system, Invitrogen) as described. After harvesting the cells, washed with PBS, after which 3 x10 ⁵ material in a concentration of cell / 12㎕ suspended in buffer R, the addition of 40 μM siRNA 2㎕ or 1 μg plasmid and transfected using Microporator device. Forty-eight hours later, cells were harvested and infiltration experiments and cell lysis were performed to Western blot. The LYPD1 specific siRNA used was purchased from Dharmacon (four mixtures). The four target sequences are shown in Table 1 (5 'to 3' direction).

SEQ ID NO: The target sequence (5 'to 3' direction) 2 GCA CAC UGC UGA ACU GAA G 3 GAA CGU UCA AGA CAU GUGI 4 GGA GCA AAG UGC CGG GAU C 5 CAA GUC CUG UGC AUC AUCA

The integrin alpha5-specific siRNA sequence was 5'-GGACCAAGGCAGAAGGCAG-3 '(SEQ ID NO: 6) and TT was added at the 3' end for stability.

In addition, plasmid was introduced into HEK293E cells using PEI (polyethyleneimine) (using 2 μg of 6-well plasmid + 4 μg of PEI).

Example  3. LYPD1  Expression Construct  making

The region coding for LYPD1 (the gene coding for amino acids 1 to 141 of the LYPD1 amino acid sequence (SEQ ID NO: 7, NP_653187.3)) was obtained by PCR and subcloned into HindIII-BamHI of p3XFlag-CMV14 (Sigma). The primer set used was 5'-aacccaagcttgccgccatgtgggtcctaggcatcg-3 '(SEQ ID NO: 8) as the forward primer and 5'-aacccggatccgca gtg tgc cga gaa gag g-3' (SEQ ID NO: 9) as the reverse primer.

A part of the region encoding LYPD1 (the gene coding for amino acid sequence 23 to 141 of SEQ ID NO: 7 except for the signal peptide) was subcloned into HindIII-BamHI of pFlag-CMV1 (Sigma) by PCR. The primer set used 5'-aagcccaagcttatccagtgctaccagtgtgaag-3 '(SEQ ID NO: 10) as the forward primer and 5'-aacccggatccctatca gca gtg tgc cga gaa g-3' (SEQ ID NO: 11) as the reverse primer.

At this time, the substrate used in the PCR (plasmid in which the region encoding LYPD1 was subcloned) was purchased from the Korean Human Gene Bank (genbank.kribb.re.kr), and the final plasmid was confirmed by sequencing.

Example 4. Invasion assay and cell migration assay.

In order to analyze the change of the cancer cell infiltration ability, the control siRNA was synthesized from the sense strand 5'-CUU ACG CUG AGU ACU UCG A-3 '(SEQ ID NO: 12), the antisense strand 5'-UCG AAG UAC UCA GCG UAA G-3' SEQ ID NO: 13), and the control siRNA or LYPD1-specific siRNA was transformed into the SW480sub cell line. In addition, SW480 cell line was transformed with LYPD1 expression vector vs empty vector. Infiltration analysis was performed 48 hours later. Specifically, the porous membrane of a 24-well transfer plate (8 pore size; Costar, USA) was coated with 100 μl of a matrigel (BD Biosciences, USA) at a concentration of 250 μg / ml diluted in serum-free medium, ≪ / RTI > for a period of time. The lower layer of the Transwell plate was coated with 100 [mu] l of collagen type I (Sigma) at a concentration of 5 [mu] g / ml as a chemoattractant.

3 × 10 ⁴ cells resuspended in serum-free medium were dispensed into the upper chambers and allowed to migrate from the upper chamber to the lower chamber while incubating for 48 h at 37 ° C and 5% CO ₂ . Unmoved cells were removed from the surface of the upper chamber. Cells transformed into the lower chamber were fixed with 3.7% paraformaldehyde dissolved in PBS and stained with 2% crystal violet solution. The extra crystal violet solution was washed with distilled water, and a selected area (× 100) was photographed. The number of migrated cells was counted in five selected areas. The average value was calculated, and the relative percentage was calculated.

To analyze the changes in cell migration ability, 1.5 x 10 ⁴ cells were dispensed into the upper chamber without matrigel coating.

Example 5. Reverse transcription Polymerase chain reaction (reverse transcription-polymerase chain reaction)

The entire RNA was extracted using TRIzol (Invitrogen), and complementary DNA was synthesized using reverse transcriptase (Bioneer, Daejeon, Korea). LYPDl specific primers (5'-CCCGAGTTCATTGTGAATTG-3 '(SEQ ID NO: 14) and 5'-ACAGGACTTGCGGTACATGA-3' (SEQ ID NO: 15)); And SYBR Green (PKT, Seoul, Korea) with the glyceraldehyde 3-phosphate dehydrogenase specific primer (5'-CATGACCACAGTCCATGCCAT-3 '(SEQ ID NO: 16) and 5'- AAGGCCATGCCAGTGAGCTTC- ) Was used to perform real-time quantitative PCR (annealing temperature 58 ° C).

Example  6. Western Blat  Western blot analysis

Cells were lysed in RIPA buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, complete protease inhibitor) . Cell lysates were quantitated by the modified Bradford assay (Bio-Rad Laboratories, Hercules, CA). 30 μg of cell lysate was mixed with SDS sample buffer, heated, and subjected to 8-15% SDS-PAGE gels. The separated proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk. After that, anti-cyclin A, anti-cyclin A (1: 1000 dilution; Santa Cruz), anti-phospho-ERK1 / 2, anti-ERK1 / 2, anti-phospho-JNK, anti-JNK , anti-phospho-ATF2, anti-ATF2, anti-c-Jun, anti-c-Jun, anti-cyclin D1 (1: ) And reacted with a secondary antibody conjugated with horseradish peroxidase. The ECL kit (ECL Plus, Amersham, USA) was then processed according to the manufacturer's instructions.

Example  7. Promoter reporter assay

Cells were transformed with Lipofectamine 200 (Invitrogen). For transfection, 2 × 10 ⁵ cells after the seeded in 6 well plates cultivation for 24 hours, 2 ㎍ reporter plasmid (AP-1 reporter DNA; AP-1 cis-Reporting system, pAP-1-Luc; Stratagene) , and 1.8 < RTI ID = 0.0 > pg < / RTI > LYPD1 expression vector. The activity of firefly luciferase was measured 48 hours or 72 hours after the transfection by Dual-luciferase reporter assay system (Promega). The transformation efficiency was analyzed by measuring the Renilla luciferase activity, which was coded by the 2 [mu] g Renilla luciferase vector pRL-TK (Promega) transformed together.

Example  8. In colorectal cancer cell lines LYPD1 Expression siRNA Suppressed by

Example  8-1. Infiltration of cancer cells and decrease of cell mobility

Forty - eight hours after the introduction of LYPD1 - specific siRNA into the SW480sub cell line by microporation technique, the cells were harvested and analyzed for invasion and cell migration through Transwell.

As a result, the infiltration was reduced by about 34% and the cell mobility by about 45%. In the case of using intergreen alpha 5 -specific siRNA as a positive control, infiltration was reduced by about 29% and cell migration by 32% (FIG.

This suggests that suppression of LYPD1 expression inhibits the invasion and cell mobility of colon cancer cells more than suppression of intergreen alpha 5 expression, Suggesting that there is a significant effect on

Example  8-2. Reduced cell growth of cancer cells

Cells were harvested 48 hours after introducing LYPD1-specific siRNA into SW480sub cell line by microporation technique. Cells were plated on 96-well plates at 3000 cells / 100 μl / well (medium containing 10% FBS) Growth was measured by colorimetric techniques. As a result of measuring the OD450nm-OD650nm value after 2 hours from adding 10 CC of CCK-8 reagent, it was confirmed that the cell growth was reduced by 34% and 46% by LYPD1-siRNA (siLYPD1) (Fig. 2).

In addition, real-time quantitative PCR confirmed the inhibition of LYPD1 expression by siRNA, indicating that LYPD1 expression was reduced by 51% (FIG. 3).

As a result, it was found that the inhibition of the expression of LYPD1 reduced the growth of cancer cells. Thus, it was confirmed that inhibiting the expression of LYPD1 of the present invention has a remarkable effect on the cancer treatment.

Example  9. LYPD1 And the expression of

Example  9-1. In normal cells LYPD1 The expression level of

In order to confirm the level of LYPD1 expression of the LYPD1 expression vectors (p3XFlagCMV14-LYPD1 and pFlagCMV1-LYPD1) prepared in Example 3, the vector was transformed into HEK293E cells as normal cells and after 48 hours, the whole lysate was obtained Or changed into serum-free medium, and cultured for 48 hours to obtain a conditioned medium. The lysates and conditioned media were analyzed by Western blot with anti-flag (Fig. 4).

As a result, it was confirmed that LYPD1 was clearly expressed by p3XFlagCMV14-LYPD1. Also, it was confirmed that the signal was detected in the conditioned medium, indicating that all or part of LYPD1 was secreted or shedded outside the cell.

Example  9-2. Increased invasive capacity of cancer cells

In order to analyze the change of invasion capacity of cancer cells according to the increase of LYPD1 expression, SW480 cells were transiently transfected with LYPD1 expression vector (p3XFLAG-CMV14-LYPD1), and after 48 hours, cells were harvested and infiltrated with Transwell As a result, infiltration increased by 26% (FIG. 5).

Example  9-3. Activation of key cell signaling

In addition, SW480 and HEK293E cells were transformed and the cells were lysed at 48 hours and 72 hours. Western blotting revealed that the phosphorylation of JNK and the constitutive component of transcription factor AP-1 (activator protein 1) It was confirmed that the phosphorylation reaction of ATF-2 was increased (FIG. 6).

As a result, expression of cyclin D1 and cyclin A was increased in SW480, and expression of cyclin D1 and cyclin A was increased. In addition to ATF-2 in HEK293E, phosphorylation of c-Jun, a component of AP-1 And the expression of cyclin E and cyclin A was increased.

This suggests a cell signaling pathway associated with pathways regulating LYPD1 cell growth and cell migration.

Example  10. LYPD1 Regulation of AP-1 reporter activity

After 48 hours of introduction of the LYPD1 expression vector or empty vector (p3XFLAG-CMV14) into the HEK293E cell line using LF2000, the activity of the transcription factor AP-1 was confirmed through the luciferase assay (Fig. 7).

As a result, it was confirmed that the activity of AP-1 was increased by LYPD1 expression (2-fold increase at 48 hours and 45% increase at 72 hours). These results are consistent with the increase of phospho-c-Jun by LYPD1 expression in Western blot analysis.

As a result, AP-1 activity is closely related to cancer cell activity (growth and invasion), so that the regulation of AP-1 activity by LYPD1 expression contributes to invasion / cell migration and cell growth regulation.

In conclusion, when the expression of LYPD1 of the present invention is decreased in various cancer cells including colon cancer cells, AP-1 activity is decreased, and the infiltration / cell mobility and cell growth of cancer cells are decreased. That is, LYPD1 of the present invention is a cancer diagnostic marker related to invasion / cell mobility and cell growth of various cancer cells, and it was found that inhibiting the expression of LYPD1 has a remarkable effect on cancer treatment.

Example  11. Prostate cancer cell lines LYPD1 Expression siRNA Suppressed by

Example  11-1. In colorectal cancer cell lines and prostate cancer cell lines LYPD1 Expression analysis

In order to confirm the expression of LYPD1 gene in various cancer cell lines, total RNA was isolated from various cell lines cultured under the conditions of Example 1 using Trizol (Invitrogen), and cDNA was synthesized using this. Also, the real time quantitative PCR of Example 5 was performed.

As a result, it was confirmed that the expression level of LYPD1 was varied in the colon cancer cell line and the prostate cancer cell line (FIG. 8).

Example  11-2. Reduced cell growth of cancer cells

Cells were harvested 48 hours after introducing LYPD1-specific siRNA into the PC3 cell line by microporation technique. Cells were plated in 96-well plates at 3000 cells / 100 μl / well (medium containing 10% FBS) Growth was measured by colorimetric techniques. As a result of measuring the OD450 nm-OD650 nm value after 2 hours from adding 10 μl of CCK-8 reagent, it was confirmed that cell growth was reduced by 21% and 27% by LYPD1-siRNA (siLYPD1), respectively (FIG.

In addition, the inhibition of LYPD1 expression by siRNA was confirmed by real time quantitative PCR, and LYPD1 expression was reduced by 70% (FIG. 10).

As a result, it was found that the inhibition of LYPD1 expression decreased the growth of prostate cancer cells. Thus, it was confirmed that the inhibition of LYPD1 expression of the present invention had a remarkable effect on cancer treatment.

Example  11-3. Reduced mobility of cancer cells

Cells were harvested 48 hours after introduction of LYPD1 specific siRNA into PC3 cell line by Microporation technique and cell mobility was analyzed through Transwell.

As a result, cell mobility decreased by about 21% (Fig. 11).

Thus, it can be seen that when the expression of LYPD1 is inhibited, the mobility of prostate cancer cells is highly suppressed, suggesting that inhibiting the expression of LYPD1 of the present invention has a remarkable effect on cancer treatment.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, as well as all changes or modifications derived from the meaning and scope of the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Use of LYPD1 for cancer diagnostic or therapeutic marker <130> KPA140674-KR-P1 <150> KR 10-2014-0105989 <151> 2014-08-14 <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 2683 <212> DNA <213> Homo sapiens LYPD1 <400> 1 agggcggtgt caatgcaccc tccagcggtg cgcgcaggcg ggagaaggga gggcggcccg 60 ggcaagtgag acagttaagg cagtgtcccc accacacccc cacccagatt ggccacgccg 120 agctggttct tgacagaagg ccttcgcgga ggaagagggg gcacagctgc acaggacacc 180 ctacggagcc tgcgggcgtg gaactttgcc aggcgcacgg gaacgcgcgc ccttcctgtc 240 agcctcccgg ggcgccaggc tcccgcggcc cgcagcggga cagcctcagt tgtgtgggct 300 ggacccagtc gctggggtac cgaccagtcc tggaaggcgc agaggacgtg gagtggggag 360 gctgccttcc tatgtgcgaa gggccagccg ggcacgcagt cctcagaccc tagtccgcac 420 ccggcaggtc cccacggcac ctgctgcgcc ctcctcgccg ctcccccaac ctccccatct 480 cagaaaacta ccagttctct cccgcccccc ggcgcccctt tcccaggaac gtgcggaggc 540 gggagaagag gaagacagga agggggtggg gatgtgaagc gaccgtccca gccttccccg 600 cccgccaccc ccaccccaac tcggcagccg tcacgtgatg cctggagtgg gaggtgggga 660 gaaaaggcga gacttttgtg ggtgctcccg atcgccagta gttccttcag tctcagccgc 720 caactccgga ggcgcggtgc tcggcccggg agcgcgagcg ggaggagcag agacccgcag 780 ccgggagccc gagcgcgggc gatgcaggct ccgcgagcgg cacctgcggc tcctctaagc 840 tacgaccgtc gtctccgcgg cagcagcgcg ggccccagca gcctcggcag ccacagccgc 900 tgcagccggg gcagcctccg ctgctgtcgc ctcctctgat gcgcttgccc tctcccggcc 960 ccgggactcc gggagaatgt gggtcctagg catcgcggca actttttgcg gattgttctt 1020 gcttccaggc tttgcgctgc aaatccagtg ctaccagtgt gaagaattcc agctgaacaa 1080 cgactgctcc tcccccgagt tcattgtgaa ttgcacggtg aacgttcaag acatgtgtca 1140 gaaagaagtg atggagcaaa gtgccgggat catgtaccgc aagtcctgtg catcatcagc 1200 ggcctgtctc atcgcctctg ccgggtacca gtccttctgc tccccaggga aactgaactc 1260 agtttgcatc agctgctgca acacccctct ttgtaacggg ccaaggccca agaaaagggg 1320 aagttctgcc tcggccctca ggccagggct ccgcaccacc atcctgttcc tcaaattagc 1380 cctcttctcg gcacactgct gaagctgaag gagatgccac cccctcctgc attgttcttc 1440 cagccctcgc ccccaacccc ccacctccct gagtgagttt cttctgggtg tccttttatt 1500 ctgggtaggg agcgggagtc cgtgttctct tttgttcctg tgcaaataat gaaagagctc 1560 ggtaaagcat tctgaataaa ttcagcctga ctgaattttc agtatgtact tgaaggaagg 1620 aggtggagtg aaagttcacc cccatgtctg tgtaaccgga gtcaaggcca ggctggcaga 1680 gtcagtcctt agaagtcact gaggtgggca tctgcctttt gtaaagcctc cagtgtccat 1740 tccatccctg atgggggcat agtttgagac tgcagagtga gagtgacgtt ttcttagggc 1800 tggagggcca gttcccactc aaggctccct cgcttgacat tcaaacttca tgctcctgaa 1860 aaccattctc tgcagcagaa ttggctggtt tcgcgcctga gttgggctct agtgactcga 1920 gactcaatga ctgggactta gactggggct cggcctcgct ctgaaaagtg cttaagaaaa 1980 tcttctcagt tctccttgca gaggactggc gccgggacgc gaagagcaac gggcgctgca 2040 caaagcgggc gctgtcggtg gtggagtgcg catgtacgcg caggcgcttc tcgtggttgg 2100 cgtgctgcag cgacaggcgg cagcacagca cctgcacgaa cacccgccga aactgctgcg 2160 aggacaccgt gtacaggagc gggttgatga ccgagctgag gtagaaaaac gtctccgaga 2220 aggggaggag gatcatgtac gcccggaagt aggacctcgt ccagtcgtgc ttgggtttgg 2280 ccgcagccat gatcctccga atctggttgg gcatccagca tacggccaat gtcacaacaa 2340 tcagccctgg gcagacacga gcaggaggga gagacagaga aaagaaaaac acagcatgag 2400 aacacagtaa atgaataaaa ccataaaata tttagcccct ctgttctgtg cttactggcc 2460 aggaaatggt accaattttt cagtgttgga cttgacagct tcttttgcca caagcaagag 2520 agaatttaac actgtttcaa acccggggga gttggctgtg ttaaagaaag accattaaat 2580 gctttagaca gtgtatttat accagttgat gtctgttaat tttaaaaaaa tgttttcatt 2640 ggtgtttgtt tgcgtatcca gaaagcagtt catgttatcc ata 2683 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <400> 2 gcacacugcu gaacugaag 19 <210> 3 <211> 19 <212> RNA <213> Homo sapiens <400> 3 gaacguucaa gacaugugu 19 <210> 4 <211> 19 <212> RNA <213> Homo sapiens <400> 4 ggagcaaagu gccgggauc 19 <210> 5 <211> 19 <212> RNA <213> Homo sapiens <400> 5 caaguccugu gcaucauca 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> integrin alpha5-specific siRNA <400> 6 ggaccaaggc agaaggcag 19 <210> 7 <211> 141 <212> PRT <213> Homo sapiens LYPD1 <400> 7 Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu Leu   1 5 10 15 Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu Phe Gln              20 25 30 Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn Cys Thr Val          35 40 45 Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu Gln Ser Ala Gly      50 55 60 Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala Ala Cys Leu Ile Ala  65 70 75 80 Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro Gly Lys Leu Asn Ser Val                  85 90 95 Cys Ile Ser Cys Cys Asn Thr Pro Leu Cys Asn Gly Pro Arg Pro Lys             100 105 110 Lys Arg Gly Ser Ser Ala Ser Ala Leu Arg Pro Gly Leu Arg Thr Thr         115 120 125 Ile Leu Phe Leu Lys Leu Ala Leu Phe Ser Ala His Cys     130 135 140 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 forward primer <400> 8 aacccaagct tgccgccatg tgggtcctag gcatcg 36 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 reverse primer <400> 9 aacccggatc cgcagtgtgc cgagaagagg 30 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 except signal peptide forward primer <400> 10 aagcccaagc ttatccagtg ctaccagtgt gaag 34 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 except signal peptide_reverse primer <400> 11 aacccggatc cctatcagca gtgtgccgag aag 33 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siGL3 sense strand <400> 12 cuuacgcuga guacuucga 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siGL3 anti-sense strand <400> 13 ucgaaguacu cagcguaag 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LYPD1-specific primer <400> 14 cccgagttca ttgtgaattg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LYPD1-specific primer <400> 15 acaggacttg cggtacatga 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> glyceraldehyde 3-phosphate dehydrogenase-specific primer <400> 16 catgaccaca gtccatgcca t 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> glyceraldehyde 3-phosphate dehydrogenase-specific primer <400> 17 aaggccatgc cagtgagctt c 21

Claims (16)

A pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of expression of LYPD1 (LY6 / PLAUR domain containing 1) gene, an inhibitor of activity of LYPD1 protein, or a mixture thereof as an active ingredient.
The composition according to claim 1, wherein the LYPD1 gene expression inhibitor is selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the LYPD1 gene.
3. The composition according to claim 2, wherein the LYPD1 gene expression inhibitor binds to at least one sequence selected from the group consisting of SEQ ID NOS: 2-5.
The composition of claim 1, wherein the activity inhibitor is an antibody that specifically binds to a LYPD1 protein.
A health functional food for preventing or ameliorating cancer, which comprises an inhibitor of expression of LYPD1 gene, an inhibitor of activity of LYPD1 protein, or a mixture thereof as an active ingredient.
A composition for diagnosing cancer, comprising an agent for measuring the level of LYPD1 gene mRNA or a protein thereof.
7. The composition for cancer diagnosis according to claim 6, wherein the agent for measuring the level of the gene mRNA comprises a primer or a probe specifically binding to the gene.
7. The composition according to claim 6, wherein the agent for measuring the level of the protein comprises an antibody specific to the protein.
7. The composition according to claim 6, wherein the cancer is colon cancer or prostate cancer.
A cancer diagnostic kit comprising the composition of claim 6.
[Claim 11] The cancer diagnostic kit according to claim 10, wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
(a) measuring the level of mRNA or a protein thereof of the LYPD1 gene from a separate sample of a subject suspected of having cancer; And
(b) comparing the level of the mRNA of the gene or the protein thereof to a level of the mRNA of the gene of the normal control sample or a protein thereof.
The method according to claim 12, wherein the step of measuring the mRNA level of the LYPD1 gene comprises the steps of: RT-PCR; competitive RT-PCR; real-time reverse transcriptase polymerase wherein the cancer is measured by quantitative RT-PCR, RNase protection method, Northern blotting or a gene chip.
13. The method according to claim 12, wherein the step of measuring the level of the mRNA of the LYPD1 gene uses a primer or a probe specifically binding to the LYPD1 gene.
13. The method according to claim 12, wherein the step of measuring the level of the protein of the LYPD1 gene uses an antibody that specifically binds to the protein.
(a) treating a candidate substance for cancer treatment to a separate sample expressing the LYPD1 gene, and then measuring the level of the mRNA or the protein thereof of the LYPD1 gene; And
(b) selecting a cancer therapeutic agent when the level of the mRNA or the protein of the gene is lower than that of the control sample not treated with the sample.

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