KR20120013693A - Markers for diagnosing angiogenesis-related diseases and use thereof - Google Patents

Abstract

PURPOSE: A novel marker for diagnosing angiogenesis-associated diseases is provided to effectively diagnose and predict angiogenesis-associated diseases. CONSTITUTION: A marker for diagnosing angiogenesis-associated diseases contains one or more genes selected among 91 genes or proteins expressed from the genes. The diseases are caused by abnormal NFAT5 expression. The marker for diagnosing the diseases caused by abnormality of cell cycle or proliferation contains the genes or proteins. A marker for diagnosing the diseases caused by abnormal apoptotic function contains the genes or proteins.

Description

Markers for diagnosing angiogenesis-related diseases and use thereof

The present invention relates to a novel diagnostic marker for angiogenesis-related diseases, diagnostic kits, microarrays, and diagnostic methods for angiogenesis-related diseases using the diagnostic markers.

Angiogenesis is the process by which new blood vessels are formed from existing blood vessels and plays an important role in normal human defense and physiological phenomena and early development, such as wound healing and inflammatory reactions. The angiogenesis is a step in which new capillaries are generated by the reconstitution of blood vessels by the breakdown of the basal membrane by proteolytic enzymes, the movement of vascular endothelial cells forming the vascular wall, the proliferation, and the formation of tubes (tubes) by vascular endothelial cell differentiation. It takes place through a series of sequential steps, including.

Also the process of blood vessel is new is there strictly is regulated by a variety of negative and positive regulatory factor (Folkman and Cotran., Int. Rev. Exp. Patho., 16, 207 ~ 248, 1976), these angiogenesis is normally controlled Failure to do so can lead to a number of conditions, including cancer, rheumatoid arthritis, and diabetic retinopathy.

Therefore, the study of the mechanism of angiogenesis and the development of a material that can suppress it has been the focus of attention in the prevention and treatment of various diseases including cancer, and recently in animal cancer models and human clinical trials Investigation of the development of angiogenesis inhibitors is being actively conducted as it has been shown that inhibition of tumor angiogenesis can effectively inhibit tumor growth and development and prolong patient life.

Rheumatoid arthritis (RA), a disease associated with angiogenesis, is a chronic inflammatory disease that progressively destroys joints, proliferating fibroblast-like synoviocytes (FSLs), and angiogenesis. , And invasive pannus formation. In joint synovial (joint synovium), FLS is actively involved in the production of inflammatory RA (Firestein, GS Invasive fibroblast- like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors? Arthritis Rheum 39 , 1781-1790 (1996)). These cells are resistant to apoptosis, proliferate abnormally, pro-inflammatory cytokines such as IL-1 and IL-6, and VEGF (vascular), as well as several matrix metalloproteinases (MMPs). produce angiogenic factors such as endothelial growth factors (Bucala, R., Ritchlin, C., Winchester, R. & Cerami, A. Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts. J Exp Med 173 , 569-574 (1991), Fava, RA , et al . Vascular permeability factor / endothelial growth factor (VPF / VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med 180 , 341-346 (1994). Angiogenesis is also very active in RA, particularly in the early invention of the disease (Koch, AE Review: angiogenesis: implications for rheumatoid arthritis. Arthritis Rheum 41 , 951-962 (1998). Newly formed blood vessels not only supply nutrients and oxygen to the pannus, but can also maintain a chronic inflammatory state by moving the inflammatory cells to the site of synovitis (Firestein, GS Starving the synovium: angiogenesis and inflammation in rheumatoid) arthritis. J Clin Invest 103 , 3-4 (1999)).

NFAT5 is a new member of the NFAT family transcription factors based on the homology of DNA-binding domains. Initially, NFAT5 was identified as tonicity enhancer binding protein (TonebP) that binds tonicity response elements in upstream regulatory regions of genes induced by hypertonic stress (Miyakawa, H., Woo, SK, Dahl, SC). , Handler, JS & Kwon, HM Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 96 , 2538-2542 (1999). However, recent reports show that NFAT5 plays an additional role in other organizations. For example, NFAT5 plays a role in the proliferation and survival of lymphocytes (Go, WY, Liu, X., Roti, MA, Liu, F. & Ho, SN NFAT5 / TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proc Natl Acad Sci USA 101 , 10673-10678 (2004), Trama, J., Lu, Q., Hawley, RG & Ho, SN The NFAT-related protein NFATL1 (TonEBP / NFAT5) is induced upon T cell activation in a calcineurin-dependent manner. J Immunol 165 , 4884-4894 (2000)), mediate myoblast migration during skeletal muscle myogenesis via the CYR61 dependent pathway (O'Connor, RS, Mills, ST, Jones, KA, Ho, SN & Pavlath, GK A combinatorial role for NFAT5 in both myoblast migration and differentiation during skeletal muscle myogenesis. J Cell Sci 120 , 149-159 (2007)). In breast cancer, NFAT5 is induced by α ₆ β ₄ clustering, which results in increased cell migration (Jauliac, S. , et. al . The role of NFAT transcription factors in integrin-mediated carcinoma invasion. Nat Cell Biol 4 , 540-544 (2002)). In addition, NFAT5-VEGFC signaling in mononuclear phagocytic cells is a major determinant of extracellular volume and blood pressure homeostasis (Machnik, A. , et. al . Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism. Nat Med 15 , 545-552 (2009)). Despite an improved understanding of the functional role of NFAT5, little is known about its role in the development of inflammatory diseases.

On the other hand, the inventors have confirmed that NFAT5 is highly expressed in RA synoviiums, which is strongly induced by inflammatory stimuli. In particular, heterozygous deficiency of the NFAT5 gene in mice has been shown to inhibit the development of arthritis, and by NFAT5 siRNA-treated RA FLS and endothelial cells mRNA profiling, NFAT5 is associated with RA onset. As major cellular processes-1) cell cycle and survival, 2) neovascularization, 3) cell migration was regulated. Based on these data, we used cultured FLS and endothelial cells to confirm the proliferation, angiogenesis, and migration function of NFAT5, and cell migration and pro-inflammatory cytokine production was also significant in macrophages of NFAT5 deficient mice. Decreased. Therefore, NFAT5 is an important regulator of rheumatoid inflammation, and furthermore, it can be a potential target of angiogenesis-related diseases by playing an important role in the mechanism of angiogenesis-related diseases.

Accordingly, the present inventors completed the present invention by identifying subgenes having different expressions according to the expression of NFAT5 based on the results of the study on the function of NFAT5, and confirming their association with angiogenesis-related diseases.

Accordingly, an object of the present invention is to provide a novel diagnostic marker for angiogenesis-related diseases that can predict or diagnose angiogenesis-related diseases.

In addition, another object of the present invention is a kit for diagnosing angiogenesis-related diseases comprising a material for measuring the expression level of a gene that can be used as a diagnostic marker for angiogenesis-related diseases according to the present invention or the amount of protein expressed from the gene. To provide.

In addition, another object of the present invention is to provide a microarray for diagnosing angiogenesis-related diseases including a diagnostic marker for angiogenesis-related diseases according to the present invention.

Another object of the present invention is to provide a method for predicting and diagnosing angiogenesis-related diseases using the diagnostic marker for angiogenesis-related diseases according to the present invention.

In order to achieve the object of the present invention as described above, the present invention provides a diagnostic marker for angiogenesis-related diseases comprising at least one gene selected from 91 genes listed in Table 1 or a protein expressed from the gene. .

In one embodiment of the present invention, the angiogenesis-related disease is characterized by the abnormal expression of NFAT5 transcription factor, the gene of NFAT5 may have a nucleotide sequence represented by SEQ ID NO: 1. Although not limited thereto, preferably, the disease may be rheumatoid arthritis.

In one embodiment of the present invention, the marker for diagnosing angiogenesis-related diseases according to the present invention includes one or more genes selected from genes 1 to 51 of Table 1 or a protein expressed from the genes. It is a diagnostic marker for angiogenesis-related diseases caused by abnormal cell cycle and / or proliferation.

In one embodiment of the present invention, the marker for diagnosing angiogenesis-related diseases according to the present invention includes one or more genes selected from genes 52 to 71 of Table 1 or a protein expressed from the genes. In addition, it is a diagnostic marker for angiogenesis-related diseases caused by apoptosis dysfunction.

In one embodiment of the present invention, the diagnostic marker for the angiogenesis-related disease according to the present invention includes one or more genes selected from the genes of serial numbers 72 to 77 of Table 1 or a protein expressed from the genes In this regard, it is a diagnostic marker for angiogenesis-related diseases caused by angiogenesis dysfunction.

In one embodiment of the present invention, the marker for diagnosing angiogenesis-related diseases according to the present invention includes one or more genes selected from the genes from serial numbers 78 to 84 of Table 1 or a protein expressed from the genes. In addition, it is a diagnostic marker for angiogenesis-related diseases caused by abnormal cell adhesion (adhesion) function.

In one embodiment of the present invention, the marker for diagnosing angiogenesis-related diseases according to the present invention includes one or more genes selected from the genes of serial numbers 85 to 91 of Table 1 or a protein expressed from the genes. In this regard, it is a diagnostic marker for angiogenesis-related diseases caused by abnormal cell migration.

In another aspect, the present invention provides a kit for diagnosing angiogenesis-related diseases comprising a substance for measuring the expression level of one or more genes selected from the 91 genes listed in Table 1 or the amount of protein expressed from the genes.

In one embodiment of the present invention, the substance may be a primer capable of amplifying any one of the 91 genes or an antibody against a protein expressed from the gene.

The present invention also provides a microarray for diagnosing angiogenesis-related diseases comprising the marker according to the present invention.

In addition, the present invention comprises the steps of (a) measuring the expression level of the one or more genes selected from the 91 genes listed in Table 1 or the amount of the expressed protein from a biological sample of suspected angiogenesis-related disease; And (b) measuring the expression level of the gene or the amount of the expressed protein thereof from the normal control sample and comparing the result with the measurement result of step (a).

In one embodiment of the invention, the measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot, ELISA (enzyme linked) immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.

Since the marker gene according to the present invention is found to be reduced or increased in the tissues or cells of angiogenesis-related disease animals compared to normal tissues or cells, the marker genes can be rapidly used when used as a marker for diagnosing angiogenesis-related diseases. It is effective in diagnosing and predicting angiogenesis-related diseases. In particular, it is effective in diagnosing and predicting angiogenesis-related diseases that may be caused by various functional abnormalities of NFAT5 transcription factors, including rheumatoid arthritis.

1 is a photograph showing the expression level of the NFAT5 expressed in the cells of the synovial cells of rheumatoid arthritis and osteoarthritis of the angiogenesis-related diseases through immunochemical staining, Figures 1a to 1e is the synovial membrane of rheumatoid arthritis 1F to 1H are observed in synovial cells of osteoarthritis, and FIG. 1I is a diagram illustrating the expression of NFAT5 over time after inducing osmotic stress by treating 100 mM NaCl in synovial cells. 1J and 1K show that the expression level of NFAT5 and the movement in the cells according to the cytokine treatment were confirmed.
Figure 2 shows the results of examining the effect on the symptoms of angiogenesis-related diseases when inhibiting the expression of NFAT5, Figure 2a is a collagen in the control mice and the mice lacking the expression of the NFAT5 gene NFAT5 gene expression After inducing arthritis as a graph showing the degree of pain over time, Figure 2b is a graph showing the measure of the swelling of the mouse foot, Figure 2c is a portion of the joint tissue of the mouse hematoxylene and eosin It is a photograph observing the degree of inflammation, the proliferation of synovial cells and the degree of joint destruction by staining with, and FIG. One average value is plotted.
Figure 3 shows the results of using NFAT5 siRNA to investigate the effect of NFAT5 on the survival and proliferation of synovial cells, Figure 3a is a CCK cell survival rate and morphology of the scrambled siRNA (control) and the introduction of NFAT5 siRNA CCK -8 shows the results confirmed by microscopic observation, 3b shows the result confirmed by the TUNEL analysis of the degree of apoptosis, 3c is treated with scrambled siRNA (control) and NFAT5 siRNA to the cells and induces cell proliferation, respectively After treatment with cytokines TNF-a and TGF-β, respectively, a graph showing the degree of cell proliferation, 3d is a graph showing the change of cell number by NFAT5 siRNA, 3e is a cell growth factor by NFAT5 siRNA The change in expression of incyclin D and E was confirmed by Western blot.
Figure 4 shows the results of the changes in the proliferation and migration of vascular endothelial cells according to the inhibition of NFAT5 expression, Figure 4a after treatment with NFAT5 siRNA in vascular endothelial cells, the cell survival rate over time analyzed by MTT assay One result. Figure 4b shows the result of examining the effect of the endothelial growth factor VEGF on the degree of proliferation of vascular endothelial cells treated with NFAT5 siRNA, Figure 4c shows the degree of inhibition of tube formation by endothelial cells according to NFAT5 siRNA under a microscope 4D and 4E show that NFAT5 siRNA inhibits the migration and chemotaxis of vascular endothelial cells.
FIG. 5 shows the results of investigating the effects of NFAT5 on macrophage migration and cytokine production. FIG. 5A shows 3% thioglycol in control mice expressing NFAT5 gene and mice lacking expression of NFAT5 gene. After intraperitoneal injection of rate, macrophages isolated from the mice were treated with LPS and IL-1β, and then the degree of migration of macrophages was shown in a graph. FIG. 5B shows control mice and NFAT5 genes expressing NFAT5 gene The graph shows the result of confirming the expression level of TNF-a in macrophages of mice lacking expression by flow cytometry. FIGS. 5C and 5D show control mice expressing NFAT5 gene and mice lacking expression of NFAT5 gene. After stimulating macrophages of LPS or IL-1β, the amount of TNF-a and IL-12 produced was measured by ELISA. Shows.
FIG. 6 shows the results of confirming molecular signatures regulated by NFAT5 in FLS and HUVEC, where 6a introduces NFAF5 siRNA into cells and expresses NFAT5 mRNA and protein expression levels in RA FLS by RT-PCR (upper). panel) and Western blot analysis (lower panel), 6b shows that performed on HUVEC cells, 6c is the expression pattern of 1373 DEGs in RA FLS or HUVEC 48 hours NFAF5 siRNA transfection Is a heatmap, 6d is a venn diagram of DEGs, showing overlap between RA FLS and HUVEC microarray data sets, and 6e is interrupted by NFAT5 siRNA in RA FLS and HUVEC. The functional analysis of biological processes and cell type-dependent DEGs is shown using DAVID software, 6f is NFAT5 in RA FLS and HUVEC. Receiving modulated by NFAT5, it shows a quantitative real-time PCR results of measurement of DEGs subset including CCNB2, CYR61, and RHOB.
FIG. 7 shows the results of real-time PCR analysis of genes regulated by NFAT5 in RA FLS and HUVEC, showing three major cellular processes (cell cycle and survival, neovascularization, in RA FLS (a) and HUVEC (b)). And mRNA expression of DEGs involved in cell migration).

The present invention is characterized by providing a marker for diagnosing angiogenesis-related diseases for diagnosing and predicting angiogenesis-related diseases, and more particularly, providing a marker for diagnosing and predicting diseases related to NFAT5 transcription factors.

In the present invention, the focus of the NFAT5 gene as a target of a novel inhibitor that inhibits angiogenesis, the transcription factor NFAT5 (nuclear factor of activated T cells 5) is a type of NFAT family, NFAT protein is IL-2 promoter It has been shown to act to promote transcription by binding to, and five types have been identified to date. In other words, the NFAT family consists of NFAT1 (NFATp or NFATc2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3), and NFAT5. It is known to modulate the immune response. In addition, NFAT protein in T cells is involved in the development and activity of thymocytes, differentiation of T cells, self-tolerance, etc., and by the interaction of NFAT family calcium signal with other signals NFAT5 was initially identified as a tonity enhancer binding protein (TonEBP) in the NFAT family, and binds to tonicity response elements present in the upregulatory region of genes induced by osmotic stress. Recently, NFAT5 plays an important role in the proliferation and survival of lymphocytes, and CYR61-dependent pathways have been reported to mediate myoblast migration during muscle differentiation.

On the other hand, in the present invention, in the case of a disease caused by excessive angiogenesis, it was confirmed that the NFAT5 gene is overexpressed in the cells of the disease, and furthermore, when the expression of the NFAT5 gene was suppressed, the severity of the disease was confirmed to be reduced. As a result, it was found that the inhibitor of NFAT5 can be used as a therapeutic agent for preventing and treating angiogenesis-related diseases.

That is, according to one embodiment of the present invention, NFAT5 protein expressed in the cells of the synovial cells obtained from patients with rheumatoid arthritis and osteoarthritis, which are diseases caused by excessive angiogenesis, through immunochemical staining, It was confirmed that NFAT5 was overexpressed in synovial cells of rheumatoid arthritis and osteoarthritis patients compared to normal synovial cells (see Example 1).

Therefore, the present inventors found that NFAT5 is overexpressed when angiogenesis is not normally regulated.

In general, angiogenesis, or neovascularization, refers to the process by which new capillaries are formed from existing vasculature in tissues, and pathological angiogenesis is almost consistently associated with some degree of inflammation, and thus the substances that cause inflammation. Blood vessels can be formed or neoplastic.

The present inventors confirmed that the expression of NFAT5 gene overexpressed in angiogenic diseases is regulated by inflammatory cytokines and osmotic stress. According to one embodiment of the present invention, synovial cells obtained from patients with rheumatoid arthritis and osteoarthritis After treatment with inflammatory cytokines TNF-a and IL-1β and NaCl to induce osmotic stress, the expression and migration of NFAT5 in cells were analyzed by Western blot and confocal microscopy. It was found that stress further increased the expression of NFAT5 in angiogenic diseases, and it was found that the NFAT5 protein expressed by this treatment was transferred to the nucleus in the cell (see Example 2).

Furthermore, the present inventors anticipated that the inhibition of NFAT5 expression or activity could prevent or treat angiogenesis-related diseases. In order to elucidate such an expectation, in one embodiment of the present invention, mice lacking the NFAT5 gene were identified. Phosphorus NFAT5 ^+/- and NFAT5 ^{+ / +} mice as controls, intravenous anti-type II collagen antibodies were used to induce arthritis, an angiogenesis-related disease. NFAT5 ^+/- mice showed significantly decreased disease severity compared to NFAT5 ^{+ / +} mice, and the degree of inflammation, synovial proliferation, and joint destruction were significantly reduced compared to controls. See Example 3).

Therefore, the results indicate that only a partial defect of the NFAT5 gene in angiogenesis-related disease cells can reduce the severity of the disease.

In addition, according to another embodiment of the present invention, after transducing NFAT5 siRNA to synovial cells of rheumatoid arthritis and osteoarthritis to inhibit the expression of the NFAT5 gene, cell survival rate and cell proliferation of synovial cells according to NFAT5 siRNA As a result, in the synovial cells into which NFAT5 siRNA was introduced, apoptosis was increased compared to the control group, and cell survival rate was decreased, whereas cell death was increased. Factors affecting cell proliferation, cyclin D and The expression of E was also shown to be reduced (see Example 4). Therefore, inhibiting the expression or activity of NFAT5 inhibited the cell proliferation of pathological arthritis synovial cells, while inducing apoptosis was found to have a therapeutic effect.

Furthermore, the inhibition or expression of NFAT5 according to the present invention is characterized by inhibiting the proliferation and migration of vascular endothelial cells.

On the other hand, in the case of cancer caused by angiogenesis abnormality, the supply of oxygen and nutrients is required in order to grow cancer tissue, and angiogenesis is involved to make such a supply passage. In the angiogenesis process in cancer tissues, cancer cells secrete angiogenesis inducers, and these inducers bind to receptors present in endothelial cells to form new blood vessels. Stimulated endothelial cells begin to grow and secrete proteolytic enzymes, which cause degradation of the base membrane, which causes endothelial cells to move toward the angiogenesis inducer. It is called cell migration. The endothelial cells thus migrated between the blood vessels (cell attachment), then moved to the place to penetrate, then penetrated the basement membrane (cell infiltration) and connected to each other to form a tube, and finally the ends of the hollow sprouts. This combination creates a loop where blood enters. In other words, when any one of these processes is blocked, angiogenesis may be reduced, and thus an effect of treating diseases induced by angiogenesis including cancer may be expected.

The present inventors investigated whether the inhibition or expression of NFAT5 can inhibit the proliferation and migration of endothelial cells. According to one embodiment of the present invention, after introducing NFAT5 siRNA into HUVEC cells, which are vascular endothelial cells, As a result, endothelial cell apoptosis and cell proliferation were confirmed. As a result, in the endothelial cells transduced with NFAT5 siRNA, cell viability and cell proliferation were reduced compared to the control group into which scrambled siRNA was introduced. Cell chemotaxis was shown to decrease with migration and tube formation (see Example 5).

Therefore, suppressing the expression or activity of NFAT5 has the effect of reducing the proliferation, migration and tube formation of endothelial cells has the effect of treating diseases caused by angiogenesis, including cancer.

 Therefore, the present invention provides a marker for diagnosing angiogenesis-related diseases including a gene expressed differently according to the expression level of NFAT5 or a protein expressed from the gene.

In the present invention, the term diagnosis refers to confirming a pathological condition. For the purpose of the present invention, the diagnosis is performed by confirming the presence or absence of the expression of a diagnostic marker of an angiogenesis-related disease. In addition, the diagnosis in the present invention includes determining whether an angiogenesis related disease is diagnosed, whether development or reduction of angiogenesis related disease is confirmed by checking the presence or absence of an angiogenesis related disease diagnostic marker.

In addition, the diagnostic marker (diagnosis marker) refers to a substance capable of diagnosing the cells of angiogenesis-related diseases from normal cells, polypeptides showing an increase or decrease in cells of angiogenesis-related diseases compared to normal cells or Organic biomolecules such as nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.

The marker for diagnosing angiogenesis-related diseases provided by the present invention may be a gene expressed in the following table or a protein expressed from the gene in which the amount of expression is increased or decreased in cells of angiogenesis-related diseases compared to normal cells.

In another aspect, the present invention provides a kit for diagnosing angiogenesis-related diseases comprising a substance for measuring the expression level of one or more genes selected from the 91 genes listed in the above table or the amount of protein expressed from the genes.

In the present invention, the level of the gene, preferably means the mRNA level, that is, the amount of mRNA expressed in the gene, the material that can measure the level may include a primer or probe specific for the gene. have. In the present invention, the primer or probe specific to the gene may be a primer or probe capable of specifically amplifying the entire gene or a specific region of the gene, and the primer or probe may be designed through a method known in the art. have.

The term primer in the present invention refers to a single-strand that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers. Oligonucleotides. Suitable lengths of primers can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can hybridize with the template to perform the primer-specific action. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the gene, which is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence to act as a primer.

The amplification reaction refers to an amplification reaction of a nucleic acid molecule, and the amplification reactions of such genes are well known in the art, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), and Liga. Aze chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), and the like.

In the present invention, the term probra refers to a linear oligomer of natural or modified monomers or linkages, and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, and is present naturally. Or artificially synthesized. The probe according to the invention may be single chain, preferably oligodioxyribonucleotides. Probes of the invention can include natural dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probe of the present invention may also include a ribonucleotide. For example, the probes of the present invention can be used in combination with a framework-modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexy Tolyl DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having C-7 substituents (the substituents being fluoro, bromo, chloro, bromo, chloro, , Iodo-, me -, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine.

In addition, the substance capable of measuring the level of the protein in the present invention may include antibodies such as polyclonal antibodies, monoclonal antibodies and recombinant antibodies that can specifically bind to the protein.

In addition, in the present invention, since the proteins have been identified as a marker protein for diagnosing angiogenesis-related diseases as described above, a method for generating an antibody using the protein is known to those skilled in the art. It can be manufactured easily using a technique. For example, in the case of polyclonal antibodies, the protein antigen can be produced by a method well known in the art for injecting the protein antigen into an animal and collecting blood from the animal to obtain a serum containing the antibody. It can be produced from any animal species host such as sheep, monkeys, horses, pigs, cattle, dogs and the like. In the case of monoclonal antibodies, the hybridoma method (Kohler et al., European Jounral of Immunology , 6, 511-519, 1976) or phage antibody libraries (Clackson et al, Nature , 352, 624-628, 1991, Marks et al, J. Mol . Biol ., 222: 58, 1-597, 1991). In addition, the antibodies according to the invention may comprise 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.

If the kit for diagnosing angiogenesis-related diseases of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus). thermophilus (Tth), Thermus filiformis, Thermis flavus, thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs, the kit for diagnosing angiogenesis-related diseases of the present invention When is applied to an immunoassay, the kits of the invention may optionally comprise a secondary antibody and a substrate of the label. Furthermore, the kits according to the invention can be produced in a number of separate packaging or compartments comprising the reagent components described above.

The present invention also provides a microarray for diagnosing angiogenesis-related diseases, including a composition for diagnosing angiogenesis-related diseases capable of measuring the expression level of the protein or a gene encoding the same.

In the microarray of the present invention, primers, probes or antibodies capable of measuring the expression level of the protein or gene encoding the same are used as a hybridizable array element and immobilized on a substrate. Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. The hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by a chemical bonding method or a covalent binding method such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine).

On the other hand, when the sample to be applied to the microarray of the present invention is a nucleic acid can be labeled (labeled), it can be hybridized with the array element on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

In addition, the present invention can provide a method for diagnosing angiogenesis-related diseases by measuring the expression level or the protein level of the genes described in the table according to the present invention, the method (a) suspected angiogenesis-related diseases Measuring the expression level or amount of the expressed protein of at least one gene selected from the 91 genes listed in the table of claim 1 from a biological sample of; And (b) measuring the expression level of the gene or the amount of the expressed protein thereof from a normal control sample and comparing the result with the measurement result of step (a). The method of measuring the expression level of the gene or the expression protein in the above may be carried out including a known process for separating mRNA or protein from a biological sample using a known technique.

In the present invention, the "biological sample" refers to a sample collected from a living body, wherein the expression level of the gene or the expression protein level is different from the normal control group according to the development or progression of angiogenesis-related disease. Examples include, but are not limited to, tissues, cells, blood, serum, plasma, saliva, urine, and the like.

The expression level of the gene is preferably to measure the level of mRNA, the method for measuring the level of mRNA reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern Blots and DNA chips, etc., but is not limited thereto.

The protein level can be measured using an antibody, in which case the marker protein in the biological sample and the antibody specific thereto form a conjugate, i.e., an antigen-antibody complex, and the amount of antigen-antibody complex formed is determined by the detection label ( Quantitative measurements can be made through the magnitude of the signal in the detection label. Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement fixation assays, FACS, protein chips, and the like.

Therefore, the present invention can determine the amount of mRNA expression or protein of the control group and the amount of mRNA expression or protein in patients with angiogenic diseases or patients suspected of angiogenesis-related diseases through the detection methods as described above. By comparing the degree of with a control group, it is possible to predict and diagnose the onset, progression or prognosis of angiogenesis-related diseases.

On the other hand, but not limited thereto, the angiogenesis-related diseases of the present invention may include rheumatoid arthritis.

Rheumatoid arthritis is an autoimmune disease characterized by abnormal proliferation due to inflammation of intraarticular synovial cells, infiltration of inflammatory cells, and excess of neovascularization. Although the cause of rheumatoid arthritis has not yet been precisely identified, immune response mediators, such as cytokines produced as a result of immune responses to certain antigenic substances, cause abnormal increases in inflammatory cells, abnormal increases in synovial cells due to synovitis, and It is known to produce and sustain an inflammatory response, which can lead to cartilage damage and bone damage, resulting in improper joint function. In particular, the formation of neovascularization plays an essential role in the breakdown of the joint by the proliferation of synovial cells and proliferation of inflammatory cells and the progression of arthritis. However, it is not known how the interaction between lymphocytes, which are immune cells, and synovial cells with inflammatory reactions, is responsible for the mechanism of neovascularization in rheumatoid arthritis. When the CD40 ligand on the surface of lymphocytes, which are immune cells, and CD40 on the surface of the synovial cells, the inventors found that the secretion of vascular endothelial growth factor (VEGF), which is known as a potent neovascular promoter, increased by four times. Academia was the first to discover the involvement of a carrier (Journal of Immunology, 164 (10): 5055-5061).

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

In cells of angiogenesis-related diseases NFAT5 Expression Analysis of

The present inventors obtained fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis and osteoarthritis, respectively, to investigate the expression of NFAT5 gene in cells of diseases caused by excessive angiogenesis (Yoo, SA et al. , Calcineurin is expressed and plays acritical role in inflammatory arthritis. JImmunol 177,2681-2690, 2006), fetal calf serum (FCS) or insulin-transferrin-selenium (ITSA) added to DMEM medium. In addition, HUVEC cells, vascular endothelial cells, were isolated from normal cord blood veins and cultured in M199 medium containing 20% FCS. Thereafter, the amount of NFAT5 expressed in the cells was confirmed by immunochemical staining, that is, the synovial membrane obtained from the patients with rheumatoid arthritis and osteoarthritis was cut into 5 μm, treated with pepsin for 30 minutes, and at room temperature for 30 minutes. Blocking with bovine serum albumin (BSA). The antibody against NFAT5 (purchased from University of Maryland) was then reacted overnight at 4 ° C., using IgG of nonspecific mice purchased from Sigma as a control. The slides containing the sections were then washed three times with TBS buffer and reacted with biotin-bound anti-mouse IgG in a humid chamber. NFAT5 positive cells were then detected using peroxidase-linked streptavidin with 33-diamino-bendidine tetrahydrochloride (DAB) and the slides were stained with hematoxylene.

In addition, the present inventors reported that NFAT5 is regulated by surrounding osmoticity. After treating NaCl with FLS cells, the present inventors analyzed the expression of NFAT5 through Western blot, wherein the Western blot was After dissolving FLS cells treated with NaCl at a concentration of 100 mM using a conventional lysis buffer known in the art, insoluble materials were removed by centrifugation at 12000 g for 20 minutes at 4 ° C. The concentration of the final protein obtained was then quantified by Bradford analysis, followed by electrophoresis on SDS-PAGE and electrophoresis onto nitrocellulose membranes, followed by reaction with an antibody to NFAT5 (purchased from University of Maryland) and ECL solution. Was visualized on the film.

As a result, as shown in Fig. 1, NFAT5 was found to be expressed in the synovial tissue of patients with rheumatoid arthritis and osteoarthritis very much, and especially in the synovial tissue of rheumatoid arthritis, more NFAT5 is expressed than osteoarthritis. (See FIGS. 1A-1H).

In addition, as a result of measuring the expression level of NFAT5 after treating NaCl at each concentration of the cells, the expression of NFAT5 was increased in NaCl concentration-dependently in both synovial cells of patients with rheumatoid arthritis and osteoarthritis (see FIG. 1I).

Therefore, the present inventors found that the NFAT5 gene was overexpressed in the angiogenic disease, and furthermore, when the surrounding osmotic environment was established, the expression amount was increased.

< Example  2>

Caused by inflammatory cytokines NFAT5 Expression Analysis of

To investigate whether the expression patterns of NFAT5 are altered by inflammatory cytokines, the amount of pro-inflammatory cytokines TNF-a and IL-1β, respectively, was 10ng / ml in synovial cells obtained from patients with rheumatoid arthritis and osteoarthritis. And then incubated for 12 hours, Western blot was performed by the same method as described in Example 1 using an antibody against NFAT5. In addition, the present inventors treated TNF-a and IL-1β at 10 ng / ml, respectively, in order to observe intracellular migration of NFAT5 protein following treatment with TNF-a, IL-1β and NaCl, at a concentration of 100 mM. Cells treated with NaCl were each dispensed into glass chamber slides, fixed with cold methanol, treated with PBS solution containing 0.25% Triton X-100, and then reacted at 4 ° C. with an antibody against NFAT5. The Alexa 488-bound anti-rabbit IgG was then reacted for 30 minutes and the location of NFAT5 protein in the cells was confirmed by confocal microscopy, where the nucleus contained 4,6-diamino-2-phenylridol (DAPI). And stained.

As a result, as shown in Figure 1j, when treated with inflammatory cytokines TNF-a and IL-1β, the expression of NFAT5 was significantly increased compared to the untreated control group, the degree of expression is increased osteoarthritis Rheumatoid arthritis was found to be significantly increased. In addition, when the position of the NFAT5 protein was confirmed by confocal microscopy, it was confirmed that the NFAT5 protein was moved to the nucleus when treated with TNF-a, IL-1β and NaCl (see FIG. 1K).

Therefore, the above results showed that the present inventors found that inflammatory cytokines and osmotic environments increase the expression of NFAT5 in angiogenic diseases, and that the expressed NFAT5 has been transferred to the nucleus within the cell. Could.

< Example  3>

NFAT5  Severity alleviation of angiogenic diseases due to gene deficiency

The above examples confirmed that NFAT5 is overexpressed in the case of angiogenic diseases, and thus, whether the NFAT5 gene is deficient and the NFAT5 protein is not expressed, the severity of the angiogenic disease may be reduced. To this end, 8-week-old heterozygous mice NFAT5 ^+/- deficient in the NFAT5 gene obtained from the University of Maryland University School of Medicine and anti-type II collagen antibodies (5 mg) inducing arthritis in NFAT5 ^{+ / +} mice as controls. ) 3 days after intravenous injection, 50 ug of LPS was administered intraperitoneally. Afterwards, the mice were observed daily, and the degree of pain was observed by the limbs of the mice to be given 0 to 4 points according to the degree of pain, and the maximum score for each mouse was 16 points. In addition, the degree of pain was analyzed by measuring the thickness of the foot of the mouse, that is, the forefoot and hind feet were measured using a microcaliper daily, and at a specific time the diameter of the ankle divided by the thickness measurement on day 0 after the antibody injection The value was used as the thickness index and expressed in%. Severity scores were measured from 0 to 3, with 0 being no pain, 1 being mild, 2 being moderate, and 3 being severe.

In addition, the present inventors observed the degree of inflammation, proliferation of the synovial membrane and the degree of joint destruction of the tissues of the joints in the NFAT5 ^+/- and NFAT5 ^{+ / +} mice using hematoxylene and eosin staining methods known in the art.

As a result, as shown in Figure 2, the symptoms of arthritis induced by collagen in the case of NFAT5 ^+/- NFAT5 compared to ^{+ / +} mice that normally NFAT5 gene expression, was found to be a moderate decrease (Fig. 2a), measure the thickness of the mouse to analysis of the degree of abnormality of the joints, the thickness of the ankle joint also compared to NFAT5 ^{+ / +} mice showed that the NFAT5 ^+/- remarkably thin (see Fig. 2b), via immunological staining NFAT5 ^In the case of ^+/- , the degree of inflammation, synovial proliferation, and joint destruction were significantly reduced compared to NFAT5 ^{+ / +} mice.

Therefore, the present inventors have found that only a partial defect of the NFAT5 gene can significantly reduce the symptoms of angiogenic diseases including arthritis.

< Example  4>

NFAT5 of Synovial cells  Role analysis

The results confirm that the defect of the NFAT5 gene can reduce the symptoms of angiogenic diseases, the present inventors investigated how the NFAT5 gene actually plays a role in the survival and proliferation of cells in synovial cells. To this end, NFAT5 siRNA having a nucleotide sequence shown in Table 1 as a siRNA capable of inhibiting the expression of NFAT5 and a control NFAT5 siRNA for use as a control were prepared, respectively. Subsequently, NFAT5 siRNA (SEQ ID NOs: 2-7 and SEQ ID NOs: 12-17) were injected into synovial cells of rheumatoid arthritis or osteoarthritis, and then cultured for 72 hours, and then the survival rate and morphology of the cells were observed. In this case, the control siRNA (SEQ ID NOS: 8-11) described in Table 1 was used as a control group, respectively. The control siRNAs for the NFAT5 siRNAs of SEQ ID NOS: 2-5 and SEQ ID NOs: 12-17 were SEQ ID NO: And siRNAs of 9 were used, and siRNAs of SEQ ID NOs: 10 and 11 were used as controls for the NFAT5 siRNAs of SEQ ID NOs: 6 and 7. Cell morphology was observed under a microscope, cell viability was performed using MTT assay methods or cell counting kit-8 (cck-8) known in the art, and apoptosis was performed via TUNEL analysis. TUNEL analysis is performed using Apotag peroxidase, in which synovial cells of arthritis are cultured in four chamber slides, NFAT5 siRNA is transduced into the cells and washed with PBS 24 hours later, followed by 4% PFA. Cells were fixed for 10 minutes. The immobilized cells were then incubated with digoxigenin-bound dUTP at 37 ° C. for 1 hour to react with TdT catalase. The reaction stop solution was then treated at room temperature, reacted with an anti-digoxigenin antibody bound to peroxidase for 30 minutes, and then DNA fragments cleaved by apoptosis were measured by staining with DAB, a substrate of peroxidase. .

NFAT5 siRNA sequence NFAT5 siRNA Types Sequence SEQ ID NO: 297R NFAT5 siRNA (S)  5-augcccucggacuucaucucauu-3 ' 2 297R NFAT5 siRNA (AS)  5'-ugagaugaaguccgagggcauuu-3 ' 3 569R NFAT5 siRNA (S)  5'-augggcggugcuugcagcuccuu-3 ' 4 569R NFAT5 siRNA (AS)  5'-ggagcugcaagcaccgcccauuu-3 ' 5 Control inv569R (S)  5'-ccucgacguucguggcggguauu-3 ' 8 Control inv569R (AS)  5'-uacccgccacgaacgucgagguu-3 ' 9 NFAT5 siRNA (S)  5′-cccucucagcaaggguuau (dtdt) -3 ′ 6 NFAT5 siRNA (AS)  5′-auaacccuugcugagaggg (dtdt) -3 ′ 7 Control siRNA (S)  5′-uucuccgaacgugucacgu (tt) -3 ′ 10 Control siRNA (AS)  5′-acgugacacguucggagaa (tt) -3 ′ 11 A_NFAT5 siRNA (S)  5′-GCAAAGAAGUGGACAUUGA (tt) -3 ′ 12 A_NFAT5 siRNA (AS)  5'-UCAAUGUCCACUUCUUUGC (tt) -3 ' 13 B_NFAT5 siRNA (S)  5′-GCAUAGCUGUUCAGUGAAA (tt) -3 ′ 14 B_NFAT5 siRN (AS)  5'-UUUCACUGAACAGCUAUGC (tt) -3 ' 15 C_NFAT5 siRNA (S)  5′-GCAAGUAACUCUCUUCUUA (tt) -3 ′ 16 C_NFAT5 siRNA (AS)  5′-UAAGAAGAGAGUUACUUGC (tt) -3 ′ 17

In addition, the degree of cell proliferation was transduced with NFAT5 siRNA in the cells, and then treated with 10 ng / ml of TNF-a or TNF-β, respectively, followed by [ ³ H] thymidine incorporation. Measured using, the extent of proliferation was expressed in cpm, as a control was used for cells treated nothing, the assay was calculated by the average value obtained by performing the same method three times. In addition, the expression level of cyclin D, E, which is a factor involved in cell proliferation, was also confirmed by Western blot, and Western blot was used in the same manner as described in the above example except that antibodies of cyclin D and E were used. .

As a result, when the NFAT5 siRNAs of SEQ ID NO: 2-7 and SEQ ID NO: 12-17 were transduced into the synovial cells of arthritis, the cell viability of the synovial cells was reduced compared to the control group (see FIG. 3A). , TUNEL analysis showed that the synovial cells treated with NFAT5 siRNA significantly increased apoptosis compared to the cells treated with nothing and the group treated with scrapable siRNA (FIG. 3B). Therefore, the results showed that the present inventors found that NFAT5 plays a role in regulating apoptosis of synovial cells. In particular, when the expression of NFAT5 gene is inhibited, apoptosis is induced in synovial cells of arthritis disease. there was.

Furthermore, synovial cells treated with NFAT5 siRNA were shown to inhibit cell proliferation of synovial cells despite treatment with inflammatory cytokines (see FIGS. 3C and 3D), and expression of cyclins D and E, factors affecting cell proliferation. It was also shown to be reduced in synovial cells treated with NFAT5 siRNA (see FIG. 3E).

< Example  5>

NFAT5 Of the effects on endothelial cell proliferation and migration

In order to investigate whether NFAT5 affects the proliferation and migration of endothelial cells that play an important role in angiogenesis in addition to synovial cells, the present inventors transduced the NFAT5 siRNA used in Example 4 on HUVEC cells, which are vascular endothelial cells. The degree of apoptosis and cell proliferation was examined at each time, and cell death and cell proliferation were performed in the same manner as described in Example 4 above. In addition, after introducing NFAT5 siRNA into HUVEC cells, the migration and tube formation of HUVEC cells were examined. First, cell migration measurements were performed on HUVEC cells in which NFAT5 siRNA was introduced into a 60 mm cell culture dish and cells not treated with siRNA. After dispensing and incubating the dish to full, the dish was scraped using pipette tips, and then treated with VEGF ₁₆₂ (20ng / ml) in M199 medium containing 1 mM thymidine and 1% FBS and incubated for 12 hours. Then, the number of cells was counted to measure the degree of movement of the cells. In addition, tube formation was analyzed by dispensing HUVEC cells into polymerized Matrigel (BD Bioscience, CA) added with VEGF ₁₆₂ (20 ng / ml), incubating for 12 hours, and then measuring the length of the formed tubes. Observed using -Pro Plus v4.5.

In addition, the present inventors investigated cell chemotaxis by NFAT5 in HUVEC cells, which were analyzed using a transwell chamber equipped with a 6.5 mm diameter polycarbonate filter (8 μm pore size). That is, VEGF (50 ng / ml) prepared in DMEM medium containing 1% FBS was added to the lower wells, and 5 x 10 ⁴ cells / ml of cells suspended in DMEM containing 1% FBS were added to the upper wells. After that, the cells were incubated at 37 ° C. for 12 hours. Cells that did not migrate on the filter top surface were removed using a cotton swab, and the migrated cells were fixed and stained using the Diff-Quik kit. Chemotaxis assays were performed by counting the number of cells migrated at x 200 magnification using a microscope.

As a result, as shown in Figure 4, when the NFAT5 siRNA of SEQ ID NO: 2 to 7 and SEQ ID NO: 12 to 17 transduced into HUVEC cells, vascular endothelial cells, cell survival rate and the degree of cell proliferation was reduced compared to the control group Cell proliferation analysis showed that cell proliferation was reduced by treatment with NFAT5 siRNA, regardless of the presence of growth factor VEGF (see FIGS. 4A and 4B). In addition, the tube formation of vascular endothelial cells also showed a decrease in the degree of tube formation in the group treated with the NFAT5 siRNA of SEQ ID NOS: 2 to 7 and SEQ ID NOs: 12 to 17 (see FIG. 4C). Analysis of cell migration also showed that when NFAT5 siRNA according to the present invention was treated, cell migration by VEGF was inhibited (see FIG. 4D). As a result of cell chemotaxis assay, when the NFAT5 siRNA was transfected into HUVEC cells, chemotaxis of vascular endothelial cells by VEGF ₁₆₂ was also significantly reduced (see FIG. 4E).

Therefore, the present inventors showed that when NFAT5 siRNA, an inhibitor of NFAT5, according to the present invention was treated to vascular endothelial cells, cell proliferation, tube formation, cell migration and cell chemotaxis of vascular endothelial cells were inhibited. , Cell death was shown to increase.

Therefore, the inventors have found that the inhibitor of NFAT5, in particular NFAT5 siRNA, according to the present invention can inhibit the proliferation of vascular endothelial cells and can prevent or treat diseases that may be caused by neovascularization or angiogenesis. .

< Example  6>

NFAT5 end Macrophage  Analysis of effects on migration and cytokine production

In addition, the present inventors injected 3% of thioglycolate into NFAT5 ^+/- and NFAT5 ^{+ / +} mice intraperitoneally to confirm that NFAT5 also affects macrophage migration and cytokine production. I was separated from the peritoneal macrophages (Kreckler, LM, Wan, TC , Ge, ZD & Auchampach, JA Adenosine inhibits tumor necrosis factor-alpha release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor. JPharmacolExpTher 317, 172 -18, LPS (1ug / ml) or IL-1β (10ng / ml) to the macrophages for 12 hours, and counted the number of cells to determine the degree of macrophage migration Investigate.

In addition, the production of cytokines was treated with LPS (1ug / ml) or IL-1β (10ng / ml) for 12 hours after intraperitoneal macrophages (3X10 ⁴ cells / ml) isolated by the above method, and then the supernatant of the medium. After collection, the production of LPS or IL-12 was measured using ELISA. In addition, the cultured cells were reacted with FITC-labeled anti-TNF-a antibody for 30 minutes, diluted with PBS buffer containing 0.1% FBS and 0.01% NaN ₃ , and then FACS caliber (Becton dickinson, mountain view, The amount of TNF-a produced was measured using a CA) instrument.

As a result, as shown in Figure 6, in the case of mice lacking the NFAT5 gene in the macrophage migration involved in angiogenesis and vascular inflammation, the NFAT5 gene is normally expressed in the macrophage migration by treatment with inflammatory cytokines. It was shown to be reduced compared to the mouse group (see FIG. 5A).

In addition, as a result of comparative analysis of the amount of cytokines produced, the production of IL-12 and TNF-a was found to be significantly reduced in mice lacking the NFAT5 gene compared to mice in which the NFAT5 gene is present (FIG. 5B). To FIG. 5D).

Therefore, the above results suggest that the present inventors inhibited the production of cytokines that stimulate angiogenesis and inhibits the migration of macrophages involved in angiogenesis when cells, especially those with vascular overdose, inhibit the expression of the NFAT5 gene. Inhibition revealed that angiogenesis could be suppressed, and ultimately, inhibitors of NFAT5 could be used as therapeutic agents for preventing or treating angiogenic diseases.

< Example  7>

NFAT5 Down adjustment of downregulation Genes differentially expressed by DEG : differentially expressed  identification of genes)

(One) DEGs Pity

Profiling RA FLS (n = 3) and HUVEC (n = 4) mRNA transfected with dummy siRNA or NFAT5 siRNA using Illumina HumanRef-8 (for RA FLS) and HumanHT-12 (for HUVEC) It was. Untransfected cells were used as controls. Intensities from the arrays were normalized using quantile normalization methods (Bolstad, BM, Irizarry, RA, Astrand, M. & Speed, TP A comparison of normalization methods for high density oligonucleotide arrays data based on variance and bias. Bioinformatics 19, 185-193 (2003)). Using standardized intensity, DEGs between control siRNA and NFAT5 siRNA-transfected samples were determined. The decision was made using the integrated statistical hypothesis testing as follows: 1) Two independent tests, T-test and log2 median ratio test, were performed; 2) The P values of each test were calculated using an empirical distribution of null hypotheses that the mean of the genes did not differ, which was obtained from the random permutation of the samples; 3) The Stouffer method was used to combine the individual P values to calculate the FDR (Hwang, D. , et. al . A data integration methodology for systems biology. Proc Natl Acad Sci USA 102 , 17296-17301 (2005)), 4) DEGs were selected with genes with FDR <0.01 and fold change of 1.5 cutoff or more. Finally, functional enrichment analysis of DEGs was performed using DAVID software to identify cellular processes overrepresented by DEGs governed by NFAT5.

(2) real time ( real - time ) PCR

CDNA was synthesized from total RNA using the High Capacity cDNA RT Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time PCR was performed using Thermal cycler dice real-time systems (Takara) using SYBR Premix (Takara) according to the manufacturer's instructions. β-actin (ACTB) was used as an internal control of PCR amplification. The level of transcript was measured relative to the control and expressed in 2 ^{-ΔΔ CT} (Yuan, JS, Reed, A., Chen, F. & Stewart, CN, Jr. Statistical analysis of real-time PCR data BMC Bioinformatics 7 , 85 (2006)). The data are presented as mean and standard deviation of three repetitions. Gene-specific primers are as follows.

Primers for real time PCR analysis gene Forward direction primer SEQ ID NO: Reverse primer SEQ ID NO: ACTB 5'-CAACTGGGACGATATGGAGAAG-3 ' 18 5'-TCTCCTTCTGCATCCTGTCAG-3 ' 19 AURKA 5'-CATTCCTTTGCAAGCACAAA-3 ' 20 5'-TTTTGATGCCAGTTCCTCCT-3 ' 21 AURKB 5'-TGCATTGGAGTGCTTTGCTA-3 ' 22 5'-TTATGCCTGAGCAGTTTGGA-3 ' 23 BMP4 5'-ACGGTGGGAAACTTTTGATG-3 ' 24 5'-TTGAGGTAACGATCGGCTAA-3 ' 25 CASP4 5'-AAGGACAAACCCAAGGTCAT-3 ' 26 5'-CGTTGAAGAGCAGAAAGCAA-3 ' 27 CCL2 5'-GCTGAGACTAACCCAGAAACATC-3 ' 28 5'-GGAATGAAGGTGGCTGCTAT-3 ' 29 CCNB2 5'-TTCTTAAGGCGAGCATCAAA-3 ' 30 5'-GCTGCTTTAAGTTCCATTTTCC-3 ' 31 CCNE2 5'-CACTGAAAAACCACCAGGAAA-3 ' 32 5'-TAGGGCAATCAATCACAGCA-3 ' 33 CDH11 5'-TGTCCGTGAGAACATCATTACTT-3 ' 34 5'-TGTCTTTGCGGGGGATAAAT-3 ' 35 CIDEA 5'-AGAAAAGGAAAGGGCTTGGT-3 ' 36 5'-GTCATTCTTGCGATGGGTTT-3 ' 37 CTGF 5'-TGACAGTCCGTCAAAACAGA-3 ' 38 5'-CAAATGCTTCCAGGTGAAAAA-3 ' 39 CYR61 5'-TTTGGAGCTTGTGGAGTTGA-3 ' 40 5'-TGCCCTCCCATTTACTTTTG-3 ' 41 HRK 5'-TTCGAGAAGGAAGTGGAGAGTAAAG-3 ' 42 5'-TCTGTTTCTGCAGCTGGATTT-3 ' 43 HSPA2 5'-GCGTAAACCTCTTTGCCTTTC-3 ' 44 5'-GCACCTCTCCTTTCATTTGC-3 ' 45 NRP1 5'-TAACAGCACAGGGAAGCAAA-3 ' 46 5'-GCAAATGTGACTCCCAACAA-3 ' 47 RHOB 5'-TGCCATAAGCGAACTTTGTG-3 ' 48 5'-TCGTATGCAAATGACGAGTG-3 ' 49 TGFB2 5'-TGTTCATGAATGGCTTCACC-3 ' 50 5'-GTGCCATCAATACCTGCAAA-3 ' 51 TNFRSF1B 5'-AGTTGGACTGATTGTGGGTGT-3 ' 52 5'-TTATCGGCAGGCAAGTGA-3 ' 53 TNFRSF6B 5'-TTCTCACAGACGTGCACAGA-3 ' 54 5'-ACGAGATCTTGCTCTGGGTCTT-3 ' 55

(3) results

To systematically investigate possible mechanism (s) for arthritis inhibition in mice lacking NFAT5, mRNA profiling of RA FLS and human umbilical vascular endothelial cells (HUVEC) was performed. These are the two cellular constructs of invasive pannus. Cells were treated with NFAT5 siRNA, and then incubated for 48 hours in a complete medium. We found that NFAT5 siRNA in RA FLS reduced the level and protein expression of NFAT5 mRNA (FIG. 6A) and decreased the level of protein in HUVEC (FIG. 6B). Then, using quantile normalization methods (Bolstad, BM, Irizarry, RA, Astrand, M. & Speed, TP A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics 19 , 185-193 (2003)), the intensity of all probes was normalized for each of the RA FLS and HUVEC datasets. Then, differently expressed genes (DEGs) between samples treated with control siRNA and NFAT5 siRNA in each cell type were identified using integrative statistical testing. The result was 682 (310 upregulated and 372 downregulated genes in NFAT5 siRNA treated samples) and 910 (457 upregulated and 453 downregulated genes) DEGs in the RA FLS and HUVEC datasets, respectively. Were identified (false discovery rate (FDR) <0.01 and fold change> 1.5) (FIG. 6C) (Table 1).

Of the total 1373 DEGs, 463 and 691 genes were expressed differently in RA FLS and HUVEC, respectively, and 219 DEGs were shared by RA FLS and HUVEC ( P = 0.001001) (FIGS. 6C and 6D). The shared or DAVID software for DEGs dependent on cell type (da Huang, W., Sherman, BT & Lempicki, RA Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat As a result of functional enrichment analysis using Protoc 4 , 44-57 (2009)), DEGs are mainly related to three cellular processes that are closely related to the onset of RA: 1) cell cycle and survival, 2) angiogenesis, and 3) cell migration (FIG. 6E). For example, 219 of 41 was 41 DEGs is DEGs is related to cell cycle (P = 2.2010 ^-14), were down-regulated of which 38 DEGs (downregulated) was only three are up-regulated (upregulated), which NFAT5 Has shown that it may play an important role in promoting the proliferation of RA FLS and endothelial cells. Interestingly, 39 of the 463 DEGs predominant in RA FLS are involved in the regulation of cell death, while 25 and 21 of 691 DEGs predominant in HUVEC are involved in cell migration and angiogenesis, respectively. (FIG. 6E), suggesting that they represent cellular processes specific to the cell-type. Quantitative real time PCR analysis of 19 DEGs associated with cell cycle, apoptosis, angiogenesis, and migration confirmed changes in their expression levels in independent RA FLS and HUVEC samples (FIGS. 6F and 7). ).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Markers for diagnosing angiogenesis-related diseases and use          the <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 14174 <212> DNA <213> Artificial Sequence <220> NFAT5 gene sequence (transcript varient 6) <400> 1 tgcaacggaa acttttggct ccacgaacag aaaagcaaga gcaaaaactc agttaaatgc 60 gttcttcctc catctttgcc tagtccaaaa ggaaaaaaaa agaaacagaa aaaagaaaaa 120 ctgtttggaa gagtagcgtt gaggtttgct ttttacctgt ttttcctcta gaacagccag 180 gtggattcac atgatccaat tttttaattg tcttctatgt cccactccga ataacccgga 240 tgccccagca caatcagaga gatcgttttt tttaaaaaaa atttcagcct ccaaaggtag 300 gaggggagtg ttaggggaga aagtacttta attaaaaatc aataactcga ggtttttggg 360 atacggtttg ccatttccta attaagaaat gggtttgaca gtccctttgt acacactgct 420 atgcaaaccc caaagggttg gcggctgtcc gggcgatgac actccggtcc cctgcgagac 480 cccgggccag ccaggcccgt ccgccgccgg cctctggggt ccgtccccgg ctcgcgcaga 540 cctctcgctt ctctcggctc tgtctcctgc gctcagctct gctcggggcc ggccgcctca 600 ggctcgccgc caccaggtcg ttgcaaatac cttttccctc cccggggccc cagcgcgcgg 660 ccacctccca gcctcccccc ctcccaccct ggcagcgggg ccctttcccg gctcaggaac 720 agcagcagcc cgggccgcgc cggcaggaag cgaggcccat gttgctgctg ttccctggcg 780 cgcctccccg ccctccgggg gccgccacgg ctcttccgcg ctcccgggca cccccctccg 840 cgcctgcgct gtgccccacg ggggcggggc tcagattcct gtcagcggcg gcggcggtgg 900 cggcgaccgt cagttttcgc tgaggagaaa cacgaaacgg accctttggc tctccccctt 960 ccccttcccc gtcctgaacc cctctcctgg tcaccgagaa tcagtccccg tggagttccc 1020 cctccacctc gccatcgttt cctcggtcct cggcccagtg gaagtcacta ccctcgagga 1080 ggaggcagcg gcagccgccc tcgcgtcgcc gcccccggtt cggtgcccgc ggtcccggag 1140 aggaggtgcc gccgccaccg ccgctccccc cctcccgctg ccctcgggcc gggctgggtc 1200 gagctgcgat gccctcggac ttcatctcat tgctcagcgc ggacctagac ctggaatcgc 1260 ccaagtccct ctactcgcga gattctctga agttacaccc atcacagaat tttcatagag 1320 ctggactatt ggaagaatct gtctatgatc ttctcccaaa ggagttacag ttacctccat 1380 ctagagaaac atctgtagca tcaatgagtc agacaagcgg tggtgaggca ggctcgcctc 1440 ctccagctgt tgttgctgct gatgcttctt cagctccctc ctcttcctcc atgggcggtg 1500 cttgcagctc ctttaccacc tcttccagcc ctaccattta ttctacctca gtcaccgaca 1560 gcaaggctat gcaagtggag agctgctcct cagccgtggg ggtaagtaac agaggggtaa 1620 gtgaaaagca gttaaccagt aacacagttc agcagcatcc atcaacaccg aagaggcaca 1680 cagtcttgta catctcacca ccacctgagg acttgctgga taacagtcgg atgtcctgcc 1740 aggatgaggg gtgtggattg gaatctgagc agagctgcag tatgtggatg gaggattccc 1800 cctccaactt cagtaacatg agcaccagtt cctacaatga taacactgag gtacctcgta 1860 aatcacgaaa acgaaatcca aagcagaggc cgggggtcaa acgacgagat tgtgaagaat 1920 ctaatatgga tatatttgat gccgacagtg ccaaagcacc tcactatgtg ctttctcagc 1980 ttaccacgga caacaaaggc aactcaaaag cgggaaatgg aacattggaa aaccaaaaag 2040 gaactggagt aaagaagagc cctatgttgt gtggacaata tcctgttaaa agtgagggaa 2100 aggagctgaa gatagttgta caacctgaga cacagcaccg agctcggtac ctgactgagg 2160 gcagccgtgg ctcagtgaaa gatagaacac agcaaggctt tcctacagta aagctggaag 2220 gccataatga acctgtagtg ttgcaagtgt ttgtgggcaa cgactctgga cgagtgaaac 2280 cacatggatt ttatcaggcc tgcagagtaa ctggacgaaa tacaactcct tgcaaagaag 2340 tggacattga aggcactact gttatagaag tcggccttga tcctagcaac aacatgacac 2400 tggcggtgga ctgcgtaggg atattgaaat tgaggaatgc tgatgtcgaa gccagaatag 2460 gaattgctgg ttccaagaag aaaagcactc gtgccagatt ggtttttcga gttaatatca 2520 tgaggaaaga tggctccact ttgacactgc aaacaccctc ttctccaatt ttgtgtactc 2580 agccagcagg agtgccagaa atcttaaaga aaagcttgca tagctgttca gtgaaaggag 2640 aagaagaagt gtttttaatc ggcaagaact ttctgaaagg aactaaagtt attttccaag 2700 aaaatgtttc tgatgaaaac tcttggaagt cagaagctga aattgatatg gaactatttc 2760 atcagaatca tcttattgtg aaggttcctc cctatcatga ccaacatata actttgcctg 2820 tgtcagtggg aatatatgta gtgacaaatg ctggaagatc tcatgatgtt caaccattca 2880 cttacactcc agacccagca gctggtgctt tgaatgtaaa tgtgaagaag gaaatatcta 2940 gtccagcaag accttgctct tttgaagagg ccatgaaagc aatgaaaact actggatgta 3000 atttagataa ggtaaatatt atccctaatg ccctgatgac tccactcata ccaagcagta 3060 tgattaagag tgaagatgtt actccaatgg aagtaacagc agaaaaaaga tcttccacta 3120 tttttaagac tacaaagtct gttggatcaa ctcagcaaac attagaaaac atctcaaaca 3180 tagcaggaaa tggctctttt tcatcaccat catcttccca cctaccttct gaaaatgaaa 3240 aacagcagca gattcagccc aaggcataca acccagagac cctgacaact attcaaaccc 3300 aggacatctc acagcctggt acttttccag cagtttctgc ttctagtcag ctgcccaaca 3360 gcgatgcact attgcagcag gctacacagt ttcagacaag agaaactcag tctagagaga 3420 tattacagtc agatggtaca gtggttaatt tgtcacaact gactgaggca tcacaacaac 3480 agcagcagtc accactacaa gaacaagcac agactttaca gcagcagatt tcatcaaata 3540 tttttccatc accaaatagt gtgagtcagc ttcagaatac tattcagcag ctgcaagcag 3600 ggagtttcac aggcagtact gctagtggca gcagtggaag tgttgacttg gtccaacaag 3660 ttttagaggc acagcagcag ttatcttcag ttttattttc tgctccagat ggtaatgaga 3720 atgttcaaga gcagcttagt gcagatattt ttcaacaagt cagtcaaatt cagagtggtg 3780 taagccctgg aatgttttcc tcaacagagc caacagtcca taccagacca gataatttat 3840 tacctggaag agctgaaagt gttcatccac agtctgaaaa cacgttatct aatcaacagc 3900 agcagcagca gcagcaacag caagtgatgg aatcttcagc cgcaatggtg atggagatgc 3960 aacagagtat ctgccaggca gctgcccaga ttcagtcaga gttattccct tcaactgctt 4020 cagcaaatgg aaaccttcag caatcgccag tttaccagca gacttctcac atgatgagtg 4080 cattgtctac caatgaggat atgcaaatgc agtgtgaatt gttttcttct cctcctgcag 4140 tttctggaaa tgaaacttct acaactacca cacagcaggt tgcaacccct ggcactacca 4200 tgtttcagac atcaagttca ggagatggag aagaaactgg aacacaagca aaacagattc 4260 agaacagtgt ctttcagacc atggtccaaa tgcaacatag tggggacaat caacctcaag 4320 ttaacctttt ttcatccaca aaaagtatga tgagtgttca gaatagtggt acccaacaac 4380 aaggtaatgg tttattccag caagggaatg agatgatgtc acttcaatct ggaaattttt 4440 tgcagcagtc ttctcattca caggcccaac tttttcatcc tcaaaatcct attgccgatg 4500 ctcagaacct ttcccaggaa actcaaggtt ctctctttca tagtccaaat cctattgtcc 4560 acagtcagac ttctacaacc tcctctgaac aaatgcagcc tccaatgttt cactctcaaa 4620 gtaccattgc tgtgttacag ggctcttcag ttcctcaaga ccagcagtca accaacatat 4680 ttctttccca gagtcccatg aataatcttc agactaacac agtagcccaa gaagcatttt 4740 ttgcagcacc gaactcaatt tctccacttc agtcaacatc aaacagtgaa caacaagctg 4800 ctttccaaca gcaagctcca atatcacaca tccagactcc tatgctttcc caagaacagg 4860 cacaaccccc gcagcagggt ttatttcagc ctcaggtggc cctgggctcc cttccaccta 4920 atccaatgcc tcaaagccaa caaggaacca tgttccagtc acagcactca atagttgcca 4980 tgcagagtaa ctctccatcc caggaacagc agcagcagca gcaacagcag cagcaacagc 5040 agcagcaaca acaacagagc attttattca gtaatcagaa taccatggct acaatggcgt 5100 ctccaaagca accaccacca aacatgatat tcaacccaaa tcaaaatcca atggctaatc 5160 aggagcaaca gaaccagtca atttttcacc aacaaagtaa catggcccca atgaatcaag 5220 agcaacagcc catgcaattt cagagtcagt ccacagtttc ctcacttcag aacccaggtc 5280 ctacccagtc ggaatcatca cagaccccct tgttccatag ctctcctcag attcagttgg 5340 tacaagggtc acctagttct caagagcagc aagtaactct cttcttatct ccagcatcca 5400 tgtctgcctt gcagaccagt ataaatcaac aagatatgca acagtctcct ctttattccc 5460 ctcagaacaa catgcctgga attcaaggag ccacatcttc gcctcaacca caggctactt 5520 tatttcacaa cacagcagga ggcacaatga accaactgca gaattctcct ggctcatctc 5580 agcagacatc aggaatgttc ttatttggca ttcaaaataa ctgtagtcag cttttaacct 5640 ctggaccagc tacattgcct gatcagttga tggccataag tcagccaggc caaccacaaa 5700 acgagggcca gccacctgtg acaacacttc tttctcagca aatgccagag aattctccac 5760 tggcatcctc tataaacacc aaccagaaca tcgaaaagat tgatttgctt gtttcattgc 5820 aaaaccaagg gaacaacttg actggctcct tttaactgga tataaattcc acgaagaaaa 5880 tcctgattcc aagatgtcct gagatcttgt ggttccatga gaattattac tttaaaaaca 5940 aaacaaaata taaaaaactg tgtttgagta aactgataga ttttactctg actgcaaaag 6000 agcacaccta tgctgcttgt tgcagtaact aaccaccaat gttaacatct tcatatttta 6060 tattcctaat aacagtgatg actgagaatc tatttgagtt tccagctggc agaattaatt 6120 gttattattt tcctaggcgc aatttcctta aacgtacagt ttaaattcaa ggctggacca 6180 ctcagttatt attgctatta gaaaataata tatcatgttt acttttgttc ttcattattt 6240 tctttcctgc attgttttag tcaagtaatg gcttttgaaa aagtaaagtt caataataac 6300 taaggctgtg atttttttca atataaaagg cacagctgtt ggccaaagtg aaggaatctt 6360 ttttcagttt tattggagaa actgaagggt aacattctaa caagtaaact gtatgtgcag 6420 ataaaagtac tcttgattta acacaaaggc agatgataca cttataaaac tgggaacagc 6480 tggaatgctt cttgatttta ttttttcaga gagttgttag ttctctgggt ttctactaag 6540 gggtttagcc ataactgtgc atagaaaaat aattatctgt aaaaaatgaa ggggataata 6600 tatgataaat tatgttctga tatcctccta cagtagttta aattgacaga aaaatttgaa 6660 tgttttcttc ttaacccagt cttaggctgg tattcccttt ttatatatat ctatattact 6720 tttcacctct ttttcacttt actttagaga actattaata tactactggc ttcatgaccc 6780 tgtagcatct ttggccactt taatctaggg tgacctagca atcctgcagc acagggcaga 6840 gagtactgtc ttaggaatta ttaggagttg attcctgaga aacaacacat ttttccccat 6900 gaacggtgct gttctgaagt cttcaaattt ttccctctaa taggaaacag tataaatttt 6960 aattaaaaaa aaaaggcaaa ctaaaatttc ttgaaatatc acttctccct gatctgcagt 7020 gagtataaat tcacttgtca cctcagtgct ttacagtttg aagtggtcac ttacctgatg 7080 gttcccacaa gccttaggct ttacagggtt gtatcattga cttaaaatga agaattaact 7140 tgtgttacat ctataaagag caaaataaca cactccagaa cttggcagtt gtagcattag 7200 ttatacagtt ttgggtgttc ttgccacccg tgggatgcct gcttctcact accacctgtg 7260 tctggacaca tgcttatgtc tcattttcct tttggcatgt ggaaagctgt caatgcagtg 7320 taaggccaac gtgtgtgtgg cttctatgtg ttgagataat gttttggtat ccttgtccgt 7380 ttcatttatt ttttaagtgt acaaaaaata acctgttaat tgttgaaggc tacttttctg 7440 ttcttttttt tttttttttt ctatcctgta catttagttg aactgtgcgg aattgtggtg 7500 ttggttttgt ttacacagcc agatttttcc ttctttttgt tttgtgatga tcttcctttg 7560 ttctttgaat gtgctctttt gtctttttct cttttttctc atgttttctt ccctccacct 7620 ccaccccttt ctttctttct ctctctgatt gagaggcatt gaattacgtt ttcagtagta 7680 caggcttctt gccgatatga agggaacttt tcagaaagag acctactctg ggtcatttaa 7740 ttttgaatac agttttcaat cgttcaagtt ttggatggtt tatatctaat gtgtgtttca 7800 tttttttgga aagctatatt ttgtatttag gaaatggtat actattttgc tatttgtact 7860 gagtgagtac attggcataa atatagaaat ttatatatat acatatatat aaactattct 7920 tttttgccac acatttttgt ggtaaatttg tgagtttgtc tgatgttcta ccacaacgtg 7980 gcgtctgata acagtgaggg ggggtggggt ttgttatgtc tttattgagt atttaagtat 8040 cttttgaaac aaatgacctg ttcatctgtg gccattccat caggcagtta gttccttgat 8100 gtcagtagtg ggctaaaggc agcttactgt gtgtttgctg gagctttcac tcagccaagt 8160 gttagagtca ggaaacccat tgaggcaatg gcgtcaaatg gtgtttcaca agaatgagcc 8220 attcagtctt tgctcactat atatttaata ttttattatt gttgttattg ttattattaa 8280 ttggctttct gtattctatg ccttttattt ataaagacac taagaaaacc catgtttgta 8340 attttaataa catttttccc atcttgtaat atccagagct actttataaa ttctctgaac 8400 caaaagtatt ttcctcagtg tatctcttct cccccagccc ctattgggaa aaattaccca 8460 gtatagttca ggttatgagg aggatcagcc acacaatcca gtgcttcagt ttgaaaatgt 8520 aaaattctaa ccctaaagta gggttggttg aaatttcaga caaagcaaac ccagcaggta 8580 taaaaagtag tataaataca aatctgtaag ttatttttga attttctgaa cttttttcta 8640 agagattaca taggagacta aagaaatcta tctgttcaag ttctaattag gatgattgtt 8700 aatactgcac tgtggatgaa gtggcgactg gcttgtgtgc tgacttctgt ggtttagcaa 8760 gaggtttatt gttatcaaat gctaattggc aatgccaagt cactgggacc aattttctgt 8820 tttataatat ctaagtttag aacagaatat atacctgaac tgtagtggtt tgatcggatg 8880 gagacagaaa acccgatttt tattctcata aattttgtgg ttatttatac aagggctgtg 8940 ctatgctacc atattcttgt tcaataataa taggtttgtt gtttttttta cattgttaaa 9000 tgttccttac ccctaaaggt caatgttaag tacaacattc tgaaaataca atttggctac 9060 gaagagtatt catcttcttt gaagctcagt ggttgatatt tgtgctaata atgcaatttc 9120 ctgattactg ttacaagtta tagctacata tgggagagac tcagtgagcc agcaaaggcc 9180 atagaaacaa caatttatta aatgtattta tggcagaagg acctaaataa actgtgagcc 9240 accttttctt ctttatattg ttacatttaa gtgttcttgc tttcagcaac tcacattaat 9300 gcttggagct tatctctttc tctctctctc tctctctctc tgtgtgtgtg tgtgtatgtg 9360 tgtgtgtgtg tgtgtgtgtt tccttattgt cattccatta tatatccaca ccaacatggg 9420 tgacgataat tcaaagtcat attttgcctc taagcttgat catgttacct ttatgattaa 9480 agtatcatgt tatttagcca atgcaaatct gttttaaaac aaatagttta aaaaaagaac 9540 aagtttttaa gggctttatt atagaagaag tattaatgaa ggactttcct tcctccctcc 9600 ctttcctccc ctccctgcct cccttcttcc cttccatctc cccctcctcc ctgccttctt 9660 tgtttctcct tcccttattc ctccctccct cctttctccc ttccttcctt tcttccattc 9720 atccttcctt gccttttatt tttatttttt gtaatatcac atgtgctgta gtttggaatt 9780 ttattctagt gcatttcttg ctcatcagaa cctcagctaa tctacctagg aaaaatagta 9840 tcaaaggaaa tgagaaagtt gtatctgagt ccctccagaa ctaagataat tctttttgac 9900 catttaagcc tttataaatg cgttttgacc atttaagcct ttataaatgc ttgttttagg 9960 aaagtgaatc tgttagatgc atcaacaaat aatgaccagg acaaaacgat ttaataatta 10020 aagtctcaaa tcaccatggt tatacatttt caccagaaat agtaatctta caatttttca 10080 tttttctgat gaagatttct gttccaatat ctgtttccta atagattttt taaattaatt 10140 agctttcctc tgctttatga ccacaggttt tatccctaac cgagacagct gtcttatatc 10200 tgcatgcctt agactgtgtg gagggactcc atgaagaaag accataggtt agaaaaataa 10260 ctcatagtat ataccctagt aagtgggtta gtagaatctc ataacatgta ttaaaaagag 10320 gttttcttct ctgcttgttt gtgtcactag agcaaaattg tagagataat gctcataatg 10380 cagtaaatat cagaataata tctacaatat catttgtgga tggtcccagg tcccagtgct 10440 ctagttactt tacttctttt tttttttttg agatggagtc ttgctctgtc tctcaggcta 10500 gagcagtgtg cgatctcagc tcactgcagc ctccacctcc caggttcaag cgattctcct 10560 gcctcagcct cccaagtagc caggattaca ggcaccctcc actaggcccg gctaattttt 10620 tttgtatttt tttagtagag atggggtttt gccatgttgg ccaggctggt ttcgaactcc 10680 taacctccag tgatccacct gcctcggcgt cccaaagtgc taggattaca ggcatgagcc 10740 accacatccg gcctaattac ttctttaatc cccatttatt tttatgccat tctagcctca 10800 tttattaata aaattatgtt tttactttct ctttcaggaa attttttaaa ttaatatttt 10860 atatctagat ctaatgctat ggaaaagtgc ctttttatca tttataattt catttttcac 10920 tatttccaaa aacacataaa caaatagttt cagtaggtcc cagcttttac tttttccatt 10980 taaaccttct tttctccatt tcttcccttt ggcttaagaa taaaagaaaa ggtacattgc 11040 tagaattgtt tctttgggag agggtaaaag attacagaat tagactgttc agcctttata 11100 taaactaaat ttgtcttcat ctcaaccagc taatggtagg tcttatctga atactcatga 11160 gaattttagc atctgtgaaa ctccatgcac cagatgtgtg taaatttcag gaagaaagtg 11220 ttgaaagcat tttctctgat gttaattaga tggaaataaa tcactaaaac atagtttagg 11280 taaagcctga ttatgccact tttttttaac tagacagggc aaagttgttt atgttagtgt 11340 acttcttgtc tatcctcagt taatttacct agacaaaaag tgtcaaagga aatgagaaaa 11400 aggttatatc tgactccctc cagacctaag ataattcctt ttgatcagat acagtcagat 11460 ggagtgcctt ggtttttgtt aattttgcct ctattccagc tccttaccac agcggtggtg 11520 cttaaagaaa ggatcatcag caacaggtca ggatagttct acctttggga tagggctgct 11580 ttccccgtgc tagtatttct gtgactgtta gtggcactga ggactgcaaa cttttatgca 11640 atattcttaa taccctattg atattatgca ctttaatcat tccaaagaag ccaagaatgc 11700 tgtatagtga tgattccttc ctaatgaatt catcttaact atttagaatg ttatgtccct 11760 tttcttttgg atagccaact tggtataaat gttatatgga tttttctaaa atgactatat 11820 aggacttaag actttgaaat gtaatttact tataagggga aataattatg ctttagcaca 11880 tcattttaga aacgtcacat tttagaaaca ttcagcttgc taacctacat gtttgggaat 11940 tcattaaaac cagttgtcta tatattttgt gccatgtata taagaacatt acaatatatc 12000 tttttctaca tatgtagtat gtgcaaccag tggttctcag agtatggttc tcagcccacc 12060 agctagtatc agtatcacct gggaactagt tagaaatgta aattctttgg ccccatccca 12120 gacatactga gtcagaaact ctggaatagg gcccccgcaa tctgttttca caagccctcc 12180 aggtgattct gatgcacact ttaaagttta ggaaccactg ggctaagact ctgttgagat 12240 atagagtttt tcttccactc agactgatat agttatacat tgttcttcat gtaaattcag 12300 cttaacctgg ttatctataa tcttttattg gcaaaagtta attctcagta ctgcctatag 12360 agatacagtg tattttatgt acatacacaa ttagtctaat tcttgataat tcagttaatt 12420 tagtttggca ttttcctacc acttactaaa aggtttacat taaatgactg atttaaatat 12480 ataggtgcaa tgttctatgt ttattttaat tgttatgaca tttaagtagc taatataatt 12540 gaccggtgct aaagtctcct gtttatccat aaaatgggta cattatgggc agtgtaatac 12600 aagctttctt ttcattgcct agtactttac cagcagacca cagttttgcc ctggctagac 12660 caaccctcag aacaaaatca tcattccttg tatttatatt tgtatctgag atagtaaaca 12720 agatggctgg ccaggtcaac atggcacctt aacttatttt tttaataggt aaaacttctt 12780 caaaagtagc ttgctttgta taagaactaa gctatcagta tagatatagc tatccttgga 12840 gcttatgttt cagacaagaa ttatttacta aaataaataa taaacaagat aatgcattat 12900 acaatttggg catttctcgt ttctcaagtg tatgcatcat ggtaaatata aactaaccac 12960 aagataggta gattgattca tttcatttta atctccttgt gtaattcagt acctccataa 13020 ttgttctaat cttcttccca ctgtttacaa attaccagtt aattaactcg tgaaagaaaa 13080 attcacatat cagaataaaa ataaatgtat actcacttta taaaaatcac cactgctgtc 13140 tttccttaat actagcagtg gaaatgtaag tggcttactc tacaaatttt ggtgctggca 13200 aatacatagg caaactgttg ggagctgctc tagttacatt cctcccttct tattcccttt 13260 ttctcttcct cactttattg cataacatat tcctgtaccc aaagcattct accacagttc 13320 tatttgactc ccacttgtaa taactccttt aaaaaattcc atgtttaacc atatgaccct 13380 gcttgcttac tcatattctc cctccctctc cccttccttt ctctctcttc cagaagtcat 13440 ttgcctggtt tgaaatattt tgtagggatt gcttattata ttattttagc tgatgaacct 13500 caggacaacg tctacacaca cacacataca tacacgcaca caaaatctca gctgttgaag 13560 agtgggcttg gaatcagact tctgtgtcca gtaaaaaact cctgcactga agtcattgtg 13620 acttgagtag ttacagactg attccagtga acttgatcta atttcttttg atctaatgaa 13680 tgtgtctgct taccttgttt ccttttaatt gataagctcc aagtagttgc taattttttg 13740 acaactttaa atgagtttca ttcacttctt ttacttaatg ttttaagtat agtaccaata 13800 atttcattaa cctgttctca agtggtttag ctaccattct gccattttta atttttattt 13860 aattttattt gcttgagcac actgatcaac cactgaactg ccttcttcca ttgtcctgca 13920 atgatataag ggttacattt ttgtgtatat ggctttcata gttgggattt cagagcactg 13980 ataccagata ttttcagttt gttctctggg ggaatttcat ttgcatctat gtttttagct 14040 atctgtgata acttgttaaa tattaaaaag atattttgct tctattggaa catttgtata 14100 ctcgcaacta tatttctgta aacagctgca gtcaaaaata aaacactgaa agttaaaaaa 14160 aaaaaaaaaa aaaa 14174 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> 297R NFAT5 siRNA (S) <400> 2 augcccucgg acuucaucuc auu 23 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> 297R NFAT5 siRNA (AS) <400> 3 ugagaugaag uccgagggca uuu 23 <210> 4 <211> 23 <212> RNA <213> Artificial Sequence <220> 569R NFAT5 siRNA (S) <400> 4 augggcggug cuugcagcuc cuu 23 <210> 5 <211> 23 <212> RNA <213> Artificial Sequence <220> 569R NFAT5 siRNA (AS) <400> 5 ggagcugcaa gcaccgccca uuu 23 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NFAT5 siRNA (S) <400> 6 cccucucagc aaggguuau 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> NFAT5 siRNA (AS) <400> 7 auaacccuug cugagaggg 19 <210> 8 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> inv569R (S) <400> 8 ccucgacguu cguggcgggu auu 23 <210> 9 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> inv569R (AS) <400> 9 uacccgccac gaacgucgag guu 23 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> Control siRNA (S) <400> 10 uucuccgaac gugucacgu 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> Control siRNA (AS) <400> 11 acgugacacg uucggagaa 19 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A_NFAT5 siRNA (S) <400> 12 gcaaagaagu ggacauuga 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A_NFAT5 siRNA (AS) <400> 13 ucaaugucca cuucuuugc 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> B_NFAT5 siRNA (S) <400> 14 gcauagcugu ucagugaaa 19 <210> 15 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> B_NFAT5 siRN (AS) <400> 15 uuucacugaa cagcuaugc 19 <210> 16 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> C_NFAT5 siRNA (S) <400> 16 gcaaguaacu cucuucuua 19 <210> 17 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> C_NFAT5 siRNA (AS) <400> 17 uaagaagaga guuacuugc 19

Claims (12)

A diagnostic marker for angiogenesis-related diseases comprising at least one gene selected from 91 genes listed in the following table or a protein expressed from the gene.

The method of claim 1,
The angiogenesis related disease marker for diagnosing angiogenesis related disease, characterized in that caused by abnormal expression of NFAT5. The method according to claim 1 or 2,
The angiogenesis related disease is a marker for diagnosing angiogenesis related disease, characterized in that rheumatoid arthritis. The method of claim 1,
Marker for diagnosing angiogenesis-related diseases caused by cell cycle or cell proliferation abnormality, characterized in that it comprises one or more genes selected from genes 1 to 51 of the table or a protein expressed from the genes. The method of claim 1,
Marker for diagnosing angiogenesis-related diseases caused by apoptosis function characterized in that it comprises one or more genes selected from the genes 52 to 71 of the table or a protein expressed from the genes. The method of claim 1,
Marker for diagnosing angiogenesis-related diseases caused by angiogenesis dysfunction, characterized in that it comprises one or more genes selected from the genes of SEQ ID NOs: 72 to 77 in the table or a protein expressed from the genes. The method of claim 1,
Marker for diagnosing angiogenesis-related diseases caused by adhesion dysfunction, characterized in that it comprises one or more genes selected from the genes 78 to 84 of the above table or a protein expressed from the genes. The method of claim 1,
Marker for diagnosis of angiogenesis-related diseases caused by a cell migration (migration) function characterized in that it comprises one or more genes selected from the genes of the numbers 85 to 91 in the table or a protein expressed from the genes. Kit for diagnosing angiogenesis-related diseases comprising a substance measuring the expression level of one or more genes selected from the 91 genes listed in the table of claim 1 or the amount of protein expressed from the genes. A microarray for diagnosing angiogenesis-related diseases comprising the marker of claim 1. (a) measuring the expression level of the one or more genes selected from the 91 genes listed in the table of claim 1 or the amount of the expressed protein from a biological sample of suspected angiogenesis related disease; And
(b) a method for predicting and diagnosing angiogenesis-related diseases comprising measuring the expression level of the gene or the amount of the expressed protein thereof from a normal control sample and comparing the result with the measurement result of step (a). The method of claim 11,
The measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA). : A method for predicting and diagnosing angiogenesis-related diseases, characterized in that it is selected from the group consisting of radioimmunoassay, radioimmunodiffusion, and immunoprecipitation assay.

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