KR20120078269A - Use of anxa10 as a diagnostic marker for gastric cancer - Google Patents

Abstract

PURPOSE: A marker for diagnosing gastric cancer, ANXA 10, is provided to quickly and accurately diagnose and predict gastric cancer and to develop a therapeutic agent for treating gastric cancer. CONSTITUTION: A marker for diagnosing gastric contains ANXA10(annexin A10) or ANXA10 encoded from the gene. The ANXA10 gene has a base sequence of sequence number 1. A composition for diagnosing gastric cancer contains a material for measuring expression level of ANXA10 gene or ANXA10 protein. A method for diagnosing gastric cancer comprises: a step of measuring expression level of ANXA10 gene or ANXA10 protein in a biological sample; and a step of comparing the gene or protein expression of the sample with the expression level of a control group. The biological sample is a tissue, cells, blood, serum, plasma, spittle or urine. A composition for preventing or treating gastric cancer contains the material as an active ingredient.

Description

Use of ANXA10 as a diagnostic marker for gastric cancer

The present invention relates to a novel gastric cancer diagnostic marker, gastric cancer diagnostic composition, gastric cancer diagnostic kit, microarray, a method for diagnosing gastric cancer using the gastric cancer diagnostic marker, and gastric cancer therapeutic agent capable of effectively diagnosing and predicting gastric cancer early.

Gastric cancer is the most common malignancy in South Korea and parts of Southeast Asia, and is the second leading cause of cancer deaths worldwide (Parkin DM, Pisani P, Ferlay J: Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 80 : 827-841, 1999.). However, the mechanisms for the development and progression of gastric cancer are not yet clear. Carcinogenesis is a multistep process involving changes in intracellular oncogenes and tumor suppressor genes required for tumor metastasis (Parkin DM, Pisani P, Ferlay J: Global cancer statistics.CA Cancer J Clin 49: 33-64, 31, 1999., Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis.Cell 61: 759-767, 1990., Kinzler KW, Vogelstein B: Life (and death) in a malignant tumour.Nature 379: 19-20, 1996.). In the development of gastric cancer, various genetic changes are responsible for the development and development of gastric cancer, causing changes in specific genes that play an important role in various cellular activities such as cell adhesion, signaling, differentiation, development and DNA repair. Thus, genes that perform these functions have been identified (Park WS, Lee JH, Shin MS , et. al . Inactivating mutations of the caspase-10 gene in gastric cancer. Oncogene 21: 2919-2925, 2002., Park WS, Oh RR, Park JY , et al. Somatic mutations of the trefoil factor family 1 gene in gastric cancer. Gastroenterology 119: 691-698, 2000., Werner M, Becker KF, Keller G, Hofler H: Gastric adenocarcinoma: Pathomorphology and molecular pathology. J Cancer Res Clin Oncol 127: 207-216, 2001., Fuchs CS, Mayer RJ: Gastric carcinoma. N Engl J Med 333: 32-41, 1995.), there is a need to study more of these potential oncogenes or tumor suppressors as targets for future gastric cancer treatment. Recent studies have identified chromosomal aberrations in gastric cancer and include multiple loci on chromosomes such as 1p, 2p, 3p, 4p, 5q, 6q, 7q, 8q, 12q, 13p, 14q, 17p, 17q and 18q in gastric cancer. The losses of locus) were found. However, little is known about the mechanism of loss of these loci or specific genes. It is also unclear how these genetic changes are associated with the clinical characteristics of individual gastric cancer patients. Therefore, it is necessary to further study various gastric carcinogenesis mechanisms to improve the diagnosis, defense and treatment strategy.

Annexins (ANXs) are composed of regulated membrane domains and / or membrane cytoskeletal linkages, certain exocytic and endocytic transport steps, and ion transport across the membrane ( It is a family of cytosolic calcium-regulated proteins and membrane-binding proteins that are involved in a variety of biological processes that are linked to many membrane-related activities such as regulation of ion fluxes (Gerke V, Creutz CE, Moss SE: Annexins: Linking ca2 + signaling to membrane dynamics.Nat Rev Mol Cell Biol 6: 449-461, 2005.). Activity of these proteins is mediated by Ca ^{2 +} dependent signaling pathways, cell cycle, is associated with growth, vesicle movement (vesicle trafficking), and regulation of apoptosis. In addition, recent studies have reported that ANXs are involved in complex mechanisms and that abnormal expression of ANX protein levels is associated with cancer progression. In various cancers, it is believed that certain ANXs are involved in carcinogenic mechanisms. In certain types of cancer, expression of ANXA1, 2, 7, 8, and 11 was up- or down-regulated, ANXA3, 4 and 6 were up-regulated and ANXA10 were down-regulated (Mussunoor S, Murray GI: The role of annexins in tumour development and progression.J Pathol 216: 131-140, 2008). Downregulation of ANXA10 protein was first found in higher vertebrates and was associated with the presence of p53 mutations and poor prognosis in liver cancer patients (21). Recently, expression of ANXA10 has been reported to decrease in mucinous adenocarcinoma, and low levels of ANXA10 expression have also been associated with poor prognosis (Kim J, Kim MA, Jee CD, Jung EJ, Kim WH: Reduced expression and homozygous deletion of annexin a10 in gastric carcinoma.Int J Cancer 125: 1842-1850, 2009.). These results show, at least in part, that ANXA10 is involved in the stage of tumor progression.

In order to investigate the biological role of ANXA10 in gastric cancer, the present inventors compared the expression of ANXA10 in gastric cancer with that in the corresponding normal gastric tissue and confirmed it in gastric cancer cell lines. In addition, recombinant ANXA10 was constructed and in vitro ( in In vitro ) tumor growth and apoptosis assay was performed to complete the present invention.

Accordingly, an object of the present invention is to provide a marker for diagnosing gastric cancer, which comprises the ANXA10 gene or a protein encoded from the gene.

In addition, another object of the present invention to provide a composition for diagnosing gastric cancer comprising a substance for measuring the expression level of ANXA10 gene or ANXA10 protein.

In addition, another object of the present invention to provide a gastric cancer diagnostic kit and gastric cancer diagnostic microarray comprising the gastric cancer diagnostic composition of the present invention.

In addition, another object of the present invention is to provide a method for diagnosing gastric cancer, which comprises measuring the expression amount or protein amount of ANXA10 gene.

Another object of the present invention is to provide a method for screening a substance capable of preventing or treating gastric cancer.

Furthermore, another object of the present invention is to provide a composition for preventing or treating gastric cancer, comprising a substance that increases the expression of ANXA10 gene.

In order to achieve the object of the present invention as described above, the present invention is ANXA10 It provides a marker for diagnosing gastric cancer comprising a gene or ANXA10 protein encoded from the gene.

In one embodiment of the present invention, the ANXA10 gene may be one having a nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention provides a composition for diagnosing gastric cancer, comprising a substance for measuring the expression level of ANXA10 gene or the level of ANXA10 protein.

In one embodiment of the present invention, a substance for measuring the level of expression of the gene ANXA10 it may be a primer or a probe to amplify the ANXA10 gene or mRNA.

In one embodiment of the present invention, the substance for measuring the level of ANXA10 protein may be an antibody that specifically recognizes ANXA10 protein.

The present invention also provides a kit for diagnosing gastric cancer comprising the composition for diagnosing gastric cancer of the present invention.

In addition, the present invention provides a gastric cancer diagnostic microarray comprising the composition for diagnosing gastric cancer of the present invention.

Further, according to the present invention,

(a) measuring the amount of expression or the amount of protein of the ANXA10 gene present in the biological sample; And (b) it provides a method for diagnosing gastric cancer comprising the step of comparing the results of the step (a) with the amount of expression or protein expression of the ANXA10 gene of the control sample.

In one embodiment of the invention, the biological sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine.

In one embodiment of the present invention, the method for measuring the expression amount or protein amount of the ANXA10 gene is reverse transcriptase-polymerase chain reaction, real time polymerase chain reaction (real time-polymerase chain reaction) ), Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation assay, and immunohistochemical analysis It may be selected from the group.

Further, according to the present invention,

(a) contacting a sample to be analyzed with a cell comprising the ANXA10 gene or ANXA10 protein; (b) measuring the expression amount of the ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein; And (c) determining that the sample is a material for preventing or treating gastric cancer when the amount of expression of ANXA10 gene, the amount of ANXA10 protein, or the activity of ANXA10 protein is increased as a result of the measurement in step (b). It provides a method for screening a substance for preventing or treating gastric cancer.

In one embodiment of the present invention, the method for measuring the expression amount of ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein is reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction ( real time-polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation assay and immunohistochemical analysis It may be selected from the crowd consisting of (immunohistochemical analysis).

In addition, the present invention provides a composition for preventing or treating gastric cancer, comprising as an active ingredient a substance for increasing the expression amount of ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein as a result of the screening.

Since the expression level of the ANXA10 gene according to the present invention is reduced in tissues or cells of gastric cancer compared to non-gastric cancer tissues or cells, when the ANXA10 gene is used as a diagnostic marker for gastric cancer, gastric cancer can be diagnosed and predicted early and quickly and accurately. It is effective, and furthermore, it can be used as a target for the development of gastric cancer treatments.

1 shows expression of ANXA10 in gastric cancer tissues and cell lines. Human gastric tissue (patients are represented by numbers. N = corresponding normal tissue, T = tumor tissue) and nine gastric cancer cell lines were collected from five gastric cancer patients to extract proteins or RNA. (A) Western blot analysis showing the expression of ANXA10 protein in gastric cancer tissue. GAPDH is the internal control (bottom). The data represents at least two independent experiments. (B) RT-PCR and Western blot analysis confirmed the expression of ANXA10 mRNA and protein in gastric cancer cell lines, respectively. The data represents at least two independent experiments.
2 shows immunohistochemical images of ANXA10 expression in human gastric cancer and adjacent normal tissues. ANXA10 antibody was used to immunostain gastric cancer and adjacent normal tissues (1:50 dilution). (A) Representative diagram of gastric cancer and adjacent normal tissues at magnification x100. Adjacent normal tissues at (B) magnification x200 and gastric cancer at (C) magnification x200 were developed with DAB chromogen and counterstained with hematoxylin.
3 shows the results that the introduction of ANXA10 expressing plasmids into MKN-1 cells inhibits their proliferation. (A) Western blot analysis showing that ANXA10 protein levels were overexpressed in MKN-1 cells transfected with the indicated concentrations of ANXA10 expressing recombinant plasmid, but not in the control plasmid (Mock). The cleavage form of PARP, a sign of apoptosis, was determined by western blotting using an antibody against PARP. (B) Cell growth rate of MKN-1 cells transfected with pcDNA3.1 / HisC_Mock, pcDNA3.1 / HisC_ANXA10-clone1 and clone3. The cells were seeded at the same density into a 24-well plate and incubated for a designated time before the cells were counted. Data is expressed as mean ± standard error for 3 experiments (unpaired student t -test, * p <0.05 vs. control).
4 shows an increase in the percentage of apoptosis MKN-1 cells overexpressing MKN-1. MKN-1 cells were transiently transfected with pcDNA3.1 / HisC_Mock or pcDNA3.1 / HisC_ANXA10-clone1 or clone3, stained with annexin V and propidium iodide and quantified by fluorescence-activated cell sorter (FACS) analysis. . The population shown in the figure is the total distribution of the cells. Significant induction of apoptosis region (M2) was observed in MKN-1 cells overexpressing ANXA10 in both clones. Data was obtained from three independent experiments.

ANXs are a multigene family of closely related calcium binding proteins that do not have charged phospholipids and the helix-loop-helix calcium-binding motif (EF-hands) of membrane binding proteins that exhibit type specific expression (Smith PD, Moss SE: Structural evolnution of the annexin supergene family.Trends Genet 10: 241-246, 1994.). There are at least 12 human ANX members, who have been named A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, and A13 (Mussunoor S, Murray GI: The role of annexins in tumour development and progression.J Pathol 216: 131-140, 2008.). ANXs share a similar structure characterized by the presence of 70-amino acid motifs and 4 or 8 repeats of a wide variety of amino termini (Smith PD, Moss SE: Structural evolution of the annexin supergene family. Trends Genet 10: 241-246 , 1994., Dubois T, Oudinet JP, Mira JP, Russo-Marie F: Annexins and protein kinases c. Biochim Biophys Acta 1313: 290-294, 1996., Benz J, Hofmann A: Annexins: From structure to function. Chem 378: 177-183, 1997.). Among 13 or more ANX family members, other ANXs have a distinctive tissue and cell distribution in vertebrates. ANXA2, -A5, -A6, and -A11 are very common, while ANXA3, -A8, and -A13 have more restrictive tissue distribution (Smith PD, Moss SE: Structural evolution of the annexin supergene family. Trends Genet 10 241-246, 1994., Raynal P, Pollard HB: Annexins: The problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins.Biochim Biophys Acta 1197: 63-93, 1994., Morgan RO, Fernandez MP: Annexin gene structures and molecular evolutionary genetics. Cell Mol Life Sci 53: 508-515, 1997.). Some ANXs are overexpressed in many types of cancers, while others do not show expression. There is also increasing evidence showing that changes in ANX expression and / or subcellular localization contribute to cancer development and progression. Several ANX genes are located in chromosomal regions that show a high frequency of loss in certain types of cancer, indicating that these ANXs may be tumor suppressor genes (Mussunoor S, Murray GI: The role of annexins in tumour development and progression. J Pathol 216: 131-140, 2008., Srivastava M, Bubendorf L, Srikantan V , et al . : Anx7, a candidate tumor suppressor gene for prostate cancer.Proc Natl Acad Sci USA 98: 4575-4580, 2001., Garcia Pedrero JM, Fernandez MP, Morgan RO , et al . : Annexin a1 down-regulation in head and neck cancer is associated with epithelial differentiation status.Am J Pathol 164: 73-79, 2004.). ANXA10, a member of the ANX family, has several distinctive features, including rare expression, codon deletions in conservative repeat 3, and abnormal ablation of type II calcium binding sites in tetrad core repeats. Have ANXA10 has recently attracted attention as a potential gene therapy target for liver cancer because of its decreased expression associated with vascular invasion, premature relapse, and poor prognosis associated with p53 mutations. Located in the q33 band of Chromosome (Deloukas P, Schuler GD, Gyapay G , et al . A physical map of 30,000 human genes. Science 282: 744-746, 1998.). Chromosome 4q is one of the most common areas associated with high frequency locus loss in liver cancer. However, despite the accumulated evidence supporting aberrant expression of ANXA10 and the high frequency of allele loss of the ANXA10 gene, information regarding its biological activity in various types of cancer, in particular gastric cancer, in certain types of cancer none. Previous studies have shown that ANXA10 expression is reduced in gastric cancer. In addition, expression profiling indicates that there are large-scale molecular changes in colorectal cancer (Koh KH, Rhee H, Kang HJ , et. al . : Differential gene expression profiles of metastases in paired primary and metastatic colorectal carcinomas. Oncology 75: 92-101, 2008). The results of the embodiments of the present invention showed down-regulation of ANXA10 in gastric cancer; In addition, recombinant plasmid-mediated ANXA10 protein overexpression inhibited the growth properties of MKN-1 cancer cells. Endogenous expression analysis of ANXA10 in gastric cancer cell lines showed no detectable levels of mRNA, except for SNU-638 and -719 (FIG. 1B).

Regarding the biological activity of ANXA10 during the course of tumor progression, there is evidence that ANXA10 acts as a proliferative and anti-prosurvival factor in several types of cancer cells, including liver and gastric cancer cells.

The results of the embodiments of the present invention show the dysregulation of ANXA10 in gastric cancer. To determine the biological role of ANXA10 in gastric cancer, recombinant plasmid-mediated gene overexpression was used. In MKN-1 cells, overexpression of endogenous ANXA10 resulted in decreased cell growth (FIG. 3B) and increased apoptotic cells (FIG. 4). According to a recent study, silencing of endogenous ANXA10 stimulated the migration of cells, increased the number of colonies of MKN-1 cells, and then decreased when transfected with the expression plasmid cDNA (Kim). J, Kim MA, Jee CD, Jung EJ, Kim WH: Reduced expression and homozygous deletion of annexin a10 in gastric carcinoma.Int J Cancer 125: 1842-1850, 2009.). The results of the present invention are consistent with previous findings that ANXA10 can act as a tumor suppressor in gastric cancer cells by inhibiting cell growth and inducing basic cell death.

To determine the clinical significance of ANXA10 expression, gastric cancer samples were analyzed using immunostaining methods; As a result, the expression of ANXA10 protein was slightly reduced (25.4%) in 31 of the 122 gastric cancers tested and showed negative or weak positive in ANXA10 antibody staining. However, 20 of 122 (16.4%) normal gastric cancer tissues showed negative or weak positive staining (FIG. 2 and Table 1). ANXA10 expression has been shown to decrease in mucinous adenocarcinoma, meaning that ANXA10 loss may reflect a loss of exocytotic function in the dedifferentiation process of gastric cancer development. Low ANXA10 expression was associated with poor prognosis in patients with HCC and gastric cancer (Kim J, Kim MA, Jee CD, Jung EJ, Kim WH: Reduced expression and homozygous deletion of annexin a10 in gastric carcinoma.Int J Cancer 125: 1842- 1850, 2009., Liu SH, Lin CY, Peng SY , et al . : Down-regulation of annexin a10 in hepatocellular carcinoma is associated with vascular invasion, early recurrence, and poor prognosis in synergy with p53 mutation. Am J Pathol 160: 1831-1837, 2002.).

In short, the results of the embodiments according to the present invention showed reduced expression of ANXA10 in both cancer cell lines and human gastric cancer. In addition, overexpression of ANXA10 in gastric cancer cell lines inhibited tumor cell growth and induced apoptosis. These results indicate that loss of ANXA10 expression may contribute to tumor cell growth and survival.

Accordingly, the present invention provides a novel gastric cancer diagnostic marker comprising an ANXA10 gene or an ANXA10 protein encoded from the gene. The base sequence of the ANXA10 gene is shown in SEQ ID NO: 1.

In the present invention, the term diagnosis refers to confirming a pathological condition. For the purpose of the present invention, the diagnosis means confirming the expression of gastric cancer diagnostic markers and confirming the onset of gastric cancer. Diagnosis in the present invention includes determining whether the gastric cancer diagnostic markers are expressed and the degree of expression, and determining whether the gastric cancer is developed, developed, and reduced.

In addition, the diagnostic marker (diagnosis marker) means a substance capable of diagnosing the cells of gastric cancer from normal cells, polypeptides or nucleic acids (eg, mRNA, etc.) showing an increase or decrease in the cells of gastric cancer compared to normal cells ), Lipids, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, and the like) and the like. The gastric cancer diagnostic marker provided by the present invention may be an ANXA10 gene or a protein whose expression amount is reduced in cells of gastric cancer compared to normal cells, and preferably, the ANXA10 gene has a nucleotide sequence as set forth in SEQ ID NO: 1.

In another aspect, the present invention provides a composition for diagnosing gastric cancer comprising a substance for measuring the level of ANXA10 gene or the level of ANXA10 protein.

In the present invention, the level of the ANXA10 gene, preferably means the mRNA level, that is, the amount of mRNA, the ANXA10 gene is expressed, and the substance capable of measuring the level may include a primer or probe specific for the ANXA10 gene. Can be. Primers specific to the ANXA10 gene in the present invention or the probe may be designed to amplify a specific region of the entire or gene ANXA10 gene-specific primers or probes shown in SEQ ID NO: 1, the primer or probe is known in the art You can design it through the method.

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 ANXA10 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. In addition, it is preferable that the primer according to the present invention can be used for gene amplification reactions.

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 that can measure the level of the ANXA10 protein in the present invention may include antibodies such as polyclonal antibodies, monoclonal antibodies and recombinant antibodies that can specifically bind to ANXA10 protein.

In addition, in the present invention, since ANXA10 protein has been identified as a marker protein for diagnosing gastric cancer as described above, a method for producing an antibody using the protein uses techniques known to those skilled in the art. It can be manufactured easily. For example, in the case of polyclonal antibodies, ANXA10 antigen can be produced by methods well known in the art, which are injected into animals and collected from animals to obtain serum comprising the antibodies, which polyclonal antibodies are goats, rabbits, sheep. Can be produced from any animal species host such as, monkey, horse, pig, cow, dog, 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.

The present invention also provides a kit for diagnosing gastric cancer, comprising a composition for diagnosing gastric cancer which can measure the expression level of ANXA10 protein or a gene encoding the same.

Gastric cancer diagnostic composition included in the kit for diagnosing gastric cancer of the present invention may include a primer, a probe or an antibody capable of measuring the expression level of ANXA10 protein or a gene encoding the same, and the definition thereof is as described above.

When the kit for diagnosing gastric cancer of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include 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, wherein the kit for diagnosing gastric cancer of the present invention is applied to immunoassay. In the case, the kit of the present invention may optionally comprise a substrate of a secondary antibody and a 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.

In another aspect, the present invention provides a gastric cancer diagnostic microarray comprising a composition for diagnosing gastric cancer that can measure the expression level of ANXA10 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 ANXA10 protein or gene encoding it 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 gastric cancer by measuring the expression level or ANXA10 protein level of the ANXA10 gene, the method comprising the steps of (a) measuring the expression amount or protein amount of the ANXA10 gene present in the biological sample Doing; And (b) comparing the measurement result of step (a) with the expression amount or protein amount of ANXA10 gene of the control sample. The method for measuring the expression level or ANXA10 protein level of the ANXA10 gene in the above may be carried out including a known process for separating mRNA or protein from biological samples using known techniques.

In the present invention, the "biological sample" refers to a sample collected from a living body in which the expression level or protein level of the ANXA10 gene is different from a normal control group according to the incidence or progression of gastric cancer. But not limited to, tissue, cells, blood, serum, plasma, saliva, urine, and the like.

The expression level of the ANXA10 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, and the like, but is not limited thereto.

The measurement of the ANXA10 protein level may use an antibody, in which case the ANXA10 marker protein in the biological sample and the antibody specific thereto form a binding, ie, an antigen-antibody complex, and the amount of antigen-antibody complex formation is detected. Quantitative measurements can be made through the magnitude of the signal on the 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 ANXA10 mRNA expression or protein in the control group and the amount of ANXA10 mRNA expression or protein in gastric cancer patients or suspected gastric cancer patients through the detection method as described above, and the degree of the expression amount is By comparing with, it is possible to predict and diagnose the onset, progression or prognosis of gastric cancer.

The present invention further comprises the steps of (a) contacting a sample to be analyzed with a cell comprising the ANXA10 gene or ANXA10 protein; (b) measuring the expression amount of the ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein; And (c) determining that the sample is a material for preventing or treating gastric cancer when the amount of expression of ANXA10 gene, the amount of ANXA10 protein, or the activity of ANXA10 protein is increased as a result of the measurement in step (b). It provides a method for screening a substance for preventing or treating gastric cancer.

According to the method of the present invention, first, a sample to be analyzed may be contacted with a cell containing the ANXA10 gene or protein. Herein, the sample refers to an unknown substance used in screening to examine whether the amount of expression of ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein is affected. The sample may include, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts. Thereafter, the expression level of the ANXA10 gene, the amount of ANXA10 protein or the activity of the ANXA10 protein can be measured in the cells treated with the sample, and as a result, the expression amount of the ANXA10 gene, the amount of the ANXA10 protein or the activity of the ANXA10 protein is increased. If the sample is measured, the sample can be determined as a substance capable of treating or preventing gastric cancer.

The method of measuring the expression amount of the ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein may be performed through various methods known in the art, for example, but not limited thereto, reverse transcriptase polymerase chain. Reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoassay radioimmunodiffusion, immunoprecipitation assay, and immunohistochemical analysis.

The present invention also provides a pharmaceutical composition for preventing or treating gastric cancer, comprising as an active ingredient a substance that increases the expression of ANXA10 gene or increases the expression and activity of ANXA10 protein. The pharmaceutical composition according to the present invention may include a chemical, nucleotide or natural extract as an active ingredient.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as an injectable formulation as a typical parenteral dosage form. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in an effective amount via a variety of routes including oral, transdermal, subcutaneous, intravenous or intramuscular, which administration is intended to introduce any substance into the patient in any suitable manner. Means and route of administration of the substance can be administered via any general route as long as it can reach the target tissue.

In addition, the effective amount in the above means an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the disease type and severity of the patient, age, sex, weight, sensitivity to the drug, type of current treatment, administration method, target cell, and the like. It can be easily determined by experts. In addition, the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be single or multiple administrations. Preferably all of the above factors can be administered in an amount that can achieve the maximum effect in a minimum amount without side effects, more preferably 1 to 10000 ㎍ / weight kg / day, even more preferably 10 to 1000 mg It may be administered repeatedly several times a day at an effective dose of / kg body weight / day.

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>

<1-1> Tissue Samples and Organizations Microarray

Five frozen human gastric cancers and their corresponding normal tissues were obtained from Jilin University in China and randomly selected for the experiment. There was no family history in any of the patients. In tissue microarray (TMA), the intestinal-type was 40, the diffuse-type was 62, and the mixed-type of gastric cancer was 20. The average tumor size was 6.37 cm and 122 tumors were located in the upper or middle section. For TMA production, a total of 122 formalin-fixed, paraffin-embedded gastric cancer specimens were obtained in the Department of Pathology, Catholic University College of Medicine. Two pathologists screened histological sections and selected representative tumor cell regions. Using commercially available microarray devices (Beecher Instruments, Micro-Array Technologies, Silver Spring, MD, USA), two and one tissue centric samples were obtained in each tumor and normal tissue, according to known methods, and It was placed in a recipient paraffin block (Kononen J, Bubendorf L, Kallioniemi A , et al . : Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4: 844-847, 1998.).

<1-2> Cell Culture and Sample

MKN-1, MKN-28, MKN-74, SNU-216, SNU-484, SNU-638, SNU-668 and SNU-719 cells were purchased from Korea Cell Line Bank (KCLB; Seoul, Korea) and American Type Culture AGS cells were purchased from Collection (ATCC; Manassas, VA, USA). Each cell line was cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (Sigma, St Louis, MO, USA). After trypsinized the MKN-1 cells, inoculated in a 60 mm dish at a concentration of 2 × 10 ⁵ cells / plate, 5% CO ₂ It was grown to overnight at 37 ° C. in a wet incubator. 24 hours after inoculation, ANXA10-specific siRNAs were transfected into the cells in Opti-MEM (Life Technologies, Gaithersburg, MD, USA). Transfection was performed using oligofectamine reagent (Invitrogen, Carlsbad, Calif., USA) according to the instructions.

<1-3> RT - PCR  ( Reverse transcriptase - 중합체 chain reaction )

Total RNA was isolated from MKN-1 cells using Trizol reagent (Invitrogen) according to the producer's instructions. The concentration of the RNA was measured by NanoDrop (Thermo Fisher Scientific Inc., Waltham, Mass., USA). Thereafter, 1 μg of total RNA was reverse transcribed using 2.5 uM oligo (d) T (Amersham biosciences, Piscataway, NJ, USA) and Superscript II enzyme (Gibco-BRL, Gaithersburg, MD, USA). The reaction mixture was incubated at 42 ° C. for 60 minutes and then incubated at 72 ° C. for 15 minutes. In order to normalize the deviation of the total cDNA amount added to each reaction, GAPDH (glyceraldehydes-3-phosphate dehydrogenase) gene expression was used as an endogenous control. Cycling conditions were as follows: 40 cycles of 95 ° C 15 seconds, 58 ° C 5 seconds, 72 ° C 5 seconds after initial denaturation at 95 ° C for 10 minutes. Agarose electrophoresis was used to identify ANXA10 and GAPDH transcripts.

<1-4> Weston Blot  analysis

Anti-ANXA10, anti-GAPDH, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PARP (Cell Signaling Technology, Danvers, MA, USA), anti-mouse IgG (Amershambiosciences), anti rabbit IgG (Bio-rad Laboratories, Hercules, Calif., USA) antibodies were used and bound antibodies were detected using an ECL plus Western blotting detection system (Amersham biosciences). The intensity of the western blot band was quantified using LAS 3000 (Fuji Photo Film Co., Tokyo, Japan).

<1-5> battlefield ( full - length ) Person Annexin  A10 ( ANXA10 Production

Full length cDNA encoding ANXA10 of SNU-620 cells was amplified by PCR. ANXA10 primers used were as follows (EcoR I and Xho I restriction enzyme sites are inserted into the primers, which correspond to the underlined portions in the following primer sequences.

Primer sequence

ANXA10 Forward Primer:

5'-TGGTG GAATTC ATGTTTTGTGGAGACTATGAGCA-3 '(SEQ ID NO: 2)

ANXA10 Reverse Primer:

5'-GGCCG CTCGAG TTAGTA GTCCTCAGCATCACCAG-3 '(SEQ ID NO: 3).

PCR products were cut with EcoRI and XhoI, purified and inserted into pcDNA 3.1 / HisC vector. Sequencing confirmed the recombinant plasmid (pcDNA 3.1 / HisC_ANXA10-1, -3).

<1-6> Cell counts for measuring cell growth counts )

The effect of cell growth was measured by counting the number of cells at the indicated times using a hemocytometer. Briefly, 12-well flat-bottomed microtiter plates were inoculated with MKN-1 cells at a concentration of 2 × 10 ⁴ cells per well. The cells were transfected with recombinant plasmid or Mock vector containing ANXA10 and allowed to grow for a designated time. The cells were trypsinized and then counted using trypan blue (0.8 mM in PBS) staining. All measurements were performed in three replicates and each measurement repeated at least three times.

<1-7> apoptosis ( Apoptosis ) analysis

Apoptosis levels in samples were quantified using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, Calif., USA). Briefly, after transfection, the cells were trypsinized, washed twice with cold PBS, suspended in 1 × binding buffer at a concentration of 1 × 10 ⁶ cells / ml, and 100 μl of solution. (1 × 10 ⁵ cells) was transferred to a 5 ml culture tube and then 5 μl of Annexin V-FITC solution was added; 10 μl of PI was placed in each tube and the cells were analyzed by flow cytometry.

<1-8> Statistics Processing

Each experiment was performed at least three times. Data is expressed as mean ± standard error over the number of experiments. Statistical significance was defined as the result of the unpaired student's t- test at P <0.05 and the chi square test for immunohistochemistry.

< Example  2>

In stomach cancer ANXA10 Expression reduction

ANXA10 is present in the q33 band on chromosome 4 (Raynal P, Pollard HB: Annexins: The problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins.Biochim Biophys Acta 1197: 63-93, 1994.); Chromosome 4q is one of the most common regions of the high frequency of allele loss in HCC. Recent studies have reported that reduced expression of ANXA10 is associated with gastric cancer. However, its role in gastric cancer is not yet clear. Therefore, to investigate the abnormal expression of ANXA10 in gastric cancer, the expression of ANXA10 in both gastric cancer cell lines and human gastric cancer tissues was measured and compared. As a result of measuring the expression of ANXA10 by RT-PCR and Western blot analysis, in gastric cancer cell lines, most cell lines derived from gastric carcinoma showed decreased ANXA10 expression. In gastric cancer cells, ANXA10 could not be detected by RT-PCR and Western blot analysis; The exceptions are the SNU-620 and SNU-719 cell lines (FIG. 1B). In order to confirm the abnormal expression of ANXA10 in gastric cancer, differential expression of ANXA10 was measured in five selected human gastric cancer tissues. As shown in FIG. 1A, the expression level of ANXA10 protein was shown to decrease in cancerous tissues as compared to the corresponding normal tissues. In addition, protein expression of ANXA10 was verified by immunostaining of gastric cancer specimens using ANXA10 antibody. The results are summarized in FIG. 2 and Table 1.

Of the 122 gastric cancers tested, 31 (25.4%) showed negative or weak positive for staining with ANXA10 antibody, and 102 of 122 (83.6%) normal gastric cancer tissues were moderate in normal adjacent tissues. Or strong ANXA10 expression. 2 shows representative images of normal and gastric cancer tissues (FIG. 2A), enlarged normal gastric tissues (FIG. 2B) and gastric cancer tissues (FIG. 2C).

<Example 3>

Inhibition of Cell Growth and Induction of Apoptosis of MKN-1 Cells by Overexpression of ANXA10

In order to examine the biological results of ANXA10 expression regulation abnormality, we examined whether ANXA10 acts as an inhibitor of growth in gastric cancer cell lines. MKN-1 cells that did not express ANXA10 were transiently transfected with a control plasmid (pcDNA3.1 / HisC) or ANXA10 plasmid (pcDNA3.1 / HisC_ANXA10-clone1, -clone3).

As a result, as shown in FIG. 3A, introducing ANXA10 expression vector into MKN-1 cells is associated with effective expression of ANXA10 in MKN-1 cells as compared to non-expression (Mock) vectors. To determine the biological effect of ANXA10 overexpression, the growth rate of MKN-1 cells was measured by cell counting assay. As in FIG. 3B, overexpression of ANXA10 significantly reduced cell growth compared to cells treated with the pcDNA3.1 / HisC_Mock vector. Anti-mitogen effects on MKN-1 cells can be explained, in part, by increased cell senescence or apoptosis. Indeed, when ANXA 10 was transfected, cleavage of poly ADP-ribose polymerase (PARP), indicating apoptosis, was observed (FIG. 3A).

To confirm the apoptosis capacity of ANXA10, cells transfected with ANXA10 were stained with Annexin V / propidium iodide (PI) and compared with the control by flow cytometry. As shown in FIG. 4, flow cytometry using PI and Annexin V staining for dead or apoptotic sandpaper increased by insertion of ANXA10 into MKN-1 cells in both early and late stages of apoptosis (M2). Shows that Significant induction of apoptosis cells was observed in clones with ANXA10 expression vector compared to cells treated with pcDNA3.1 / HisC_Mock vector (non-treated or Mock vector treated). Apoptosis cells (dots in FIG. 4) in the ANXA10 expression vector groups pcDNA3.1 / HisC_ANXA10-clone1 and pcDNA3.1 / HisC_ANXA10-clone3 compared to the untreated (17.97%) or Mock vector (17.56%) treated group Right in the plot graph increased from 40.67% to 50.69%.

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> Use of ANXA10 as a diagnostic marker for gastric cancer <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 975 <212> DNA <213> Artificial Sequence <220> <223> ANXA10 cDNA sequence <400> 1 atgttttgtg gagactatgt gcaaggaacc atcttcccag ctcccaattt caatcccata 60 atggatgccc aaatgctagg aggagcactc caaggatttg actgtgacaa agacatgctg 120 atcaacattc tgactcagcg ctgcaatgca caaaggatga tgattgcaga ggcataccag 180 agcatgtatg gccgggacct gattggggat atgagggagc agctttcgga tcacttcaaa 240 gatgtgatgg ctggcctcat gtacccacca ccactgtatg atgctcatga gctctggcat 300 gccatgaagg gagtaggcac tgatgagaat tgcctcattg aaatactagc ttcaagaaca 360 aatggagaaa ttttccagat gcgagaagcc tactgcttgc aatacagcaa taacctccaa 420 gaggacattt attcagagac ctcaggacac ttcagagata ctctcatgaa cttggtccag 480 gggaccagag aggaaggata tacagaccct gcgatggctg ctcaggatgc aatggtccta 540 tgggaagcct gtcagcagaa gacgggggag cacaaaacca tgctgcaaat gatcctgtgc 600 aacaagagct accagcagct gcggctggtt ttccaggaat ttcaaaatat ttctgggcaa 660 gatatggtag atgccattaa tgaatgttat gatggatact ttcaggagct gctggttgca 720 attgttctct gtgttcgaga caaaccagcc tattttgctt atagattata tagtgcaatt 780 catgactttg gtttccataa taaaactgta atcaggattc tcattgccag aagtgaaata 840 gacctgctga ccataaggaa acgatacaaa gagcgatatg gaaaatccct atttcatgat 900 atcagaaatt ttgcttcagg gcattataag aaagcactgc ttgccatctg tgctggtgat 960 gctgaggact actaa 975 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> ANXA10 F-primer <400> 2 tggtggaatt catgttttgt ggagactatg agca 34 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> ANXA10 R-primer <400> 3 ggccgctcga gttagtagtc ctcagcatca ccag 34

Claims (11)

Gastric cancer diagnostic markers comprising ANXA10 (Annexin A10) gene or ANXA10 protein encoded from the gene. According to claim 1, wherein the ANXA10 gene gastric cancer diagnostic marker, characterized in that it has a nucleotide sequence of SEQ ID NO: 1. Gastric cancer diagnostic composition comprising a material for measuring the expression level of the ANXA10 gene or ANXA10 protein. The method of claim 3,
The substance for measuring the level of the ANXA10 gene is a gastric cancer diagnostic composition, characterized in that the primer or probe that can amplify the ANXA10 gene or its mRNA. The method of claim 3,
The substance for measuring the level of the ANXA10 protein is a gastric cancer diagnostic composition, characterized in that the antibody that specifically recognizes ANXA10 protein. (a) measuring the amount of ANXA10 gene expression or the amount of ANXA10 protein present in the biological sample; And
(B) gastric cancer diagnostic method comprising the step of comparing the measurement results of step (a) with the amount of ANXA10 gene expression or the amount of ANXA10 protein of the control sample. The method of claim 6,
Wherein said biological sample is selected from the group consisting of tissues, cells, blood, serum, plasma, saliva, and urine. The method of claim 6,
The measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA). : Method for predicting and diagnosing gastric cancer, characterized in that it is selected from the group consisting of radioimmunoassay, radioimmunodiffusion, immunoprecipitation assay, and immunohistochemical analysis. (a) contacting a sample to be analyzed with a cell comprising the ANXA10 gene or ANXA10 protein;
(b) measuring the expression amount of the ANXA10 gene, the amount of ANXA10 protein or the activity of ANXA10 protein; And
(c) when the measurement result of step (b) indicates that the expression amount of ANXA10 gene, the amount of ANXA10 protein, or the activity of ANXA10 protein is increased, the sample includes the step of determining the substance for preventing or treating gastric cancer, A method of screening for the prevention or treatment of gastric cancer. 10. The method of claim 9,
The measurement of step (b) includes reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation assay, and immunohistochemical analysis are selected from the group consisting of a substance for the prevention or treatment of gastric cancer. How to screen. A composition for preventing or treating gastric cancer, comprising as an active ingredient an amount of expression of ANXA10 gene, an amount of ANXA10 protein, or a substance that increases the activity of ANXA10 protein.

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