WO2005001126A1

WO2005001126A1 - Detection kit for gastric cancer and metastatic gastric cancer

Info

Publication number: WO2005001126A1
Application number: PCT/KR2004/000677
Authority: WO
Inventors: Nam-Soon Kim; Yong Sung Kim; Ju-Yeon Lee; Jung-Hwa Oh; Hong-Seog Park; Hee-Young Ahn; Sun-Young Yoon; Yoonsoo Han; Sangsoo Kim; Jeong-Min Kim; Sang-Soon Byun; Seung-Moo Noh; Kyu-Sang Song; Hyang Sook Yoo
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2003-06-12
Filing date: 2004-03-25
Publication date: 2005-01-06
Also published as: JP2006526998A

Abstract

Disclosed is a kit for diagnosing gastric cancer and metastatic gastric cancer, more particularly to the diagnostic kit for detecting gastric cancer, which has been designed developed for detecting the expression levels of the up-regulated and the down-regulated gene in gastric cancer to determine whether tested gastric sample is a gastric cancer or not; and to the diagnostic kit for detecting the metastatic gastric cancer, designed and developed for detecting the expression levels of the up-regulated and down-regulated gene in metastatic gastric cancer to diagnose whether tested gastric cancer sample is metastatic cancer or not.

Description

Detection Kit for Gastric Cancer and Metastatic Gastric Cancer

Field of the Invention The present invention relates to a detection kit for diagnosing gastric cancer and metastatic gastric cancer, more particularly to the diagnostic kit for detecting gastric cancer, which has been designed and developed for detecting the expression levels of the up-regulated and the down-regulated genes in gastric cancer to determine whether or not a given gastric sample is cancer; and to the diagnostic kit for detecting the metastatic gastric cancer, designed and developed for detecting the expression levels of the up-regulated and the down-regulated genes in the metastatic gastric cancer to diagnose whether the gastric cancer sample is metastatic cancer or not. Gastric cancer is one of the leading causes of cancer death in East Asia, especially in Korea and Japan and the mortality due to the cancer has been ranked in the top among various diseases [Parkin et al, Int. J. Cancer 80: 827-841, 1999; Neugut et al., Semin. Oncol. 23: 281-291, 1996]. However, many of the genes associated with gastric cancer have not been known yet. Earlier studies have shown that development of primary and metastatic gastric cancer are not due to actions of specific genes alone, but result from rather complex interactions among many genes involved in various signal transduction pathways and control mechanisms in cells of malignant cancer [Fang, Chin. Med. J., 81: 193-194, 2001]. Therefore, an approach to identify new genes related to the development of primary and metastatic gastric cancer via comparisons and analyses of the levels of expression for numerous genes between the normal gastric tissues and the gastric cancer cell lines/ tissues or between the primary gastric cancer and the metastatic gastric cancer cell lines would be more useful than merely concentrating on identifying the mechanisms of development of primary and metastatic gastric cancer for only a few selected genes. As mentioned above, primary and metastatic cancers appear to be progressed by involving multiple steps related to a given cancer and requiring a combination of altered gene expression. However, exact mechanism for development of primary and metastatic gastric cancer has not been fully understood until now. According to recent molecular analyses, it has been reported that many genetic alterations occur in genes such as ρ53 [Yokozaki et al, Int. Rev. Cyfol. 204: 49- 95, 2001], β-catenin [Park et al., Cancer Res. 59: 4257-4260, 1999], E-cadherin [Berx et al., Hum. Mutat. 12: 226-237, 1998], trefoil factor 1 [Park et al., Gastroenterology 119: 691-698, 2000], and c-met [Lee et al., Oncogene 19: 4947-4953, 2000] in gastric cancer. In addition, it has been known that some genes such as CA11 [Yoshikawa et al., Jpn. /. Cancer Res. 91: 459-463, 2000; Shiozaki et al., Int. J. Oncol. 19: 701-707, 2001], and the trefoil factor genes such as TFF1 and TFF2 [Shiozaki et al., Int. J. Oncol. 19: 701-707, 2001; Kirikoshi and KatohKirikoshi, Int. J. Oncol. 21: 655-659, 2002], synthesized in the mucous membrane of stomach, are down-regulated in gastric cancer. Further, Hasegawa et al. has shown that the expressions of RPL10, HSPCB, LOC56287, IGHM, PG REGIA, RNASE1, TFF1, TFF2 genes are presumed to be involved in the metastasis of gastric cancer, when using a cDNA microarray consisting of 23,040 genes which are expressed in gastrointestinal cancer [Hasegawa et al., Cancer Res. 62: 7012-7017, 2002]. However, it is not yet sufficient to diagnose gastric cancer only based on these genes. On the other hand, until recently, approximately 90% of cancer patients die as s result of metastasis of the primary tumor rather than due to the primary tumor itself. However, studies on prognosis of the metastatic gastric cancer, which is progressed(developed) as far as into the gastric serosa, is still not very active [Yamazaki et al., Cancer 63: 613-617, 1989; Shimada et al., Cancer 1: 1657-1668, 1999]. There have been only a few attempts trying to discovery genes involved in a serial course of metastasis from the primary gastric cancer to the malignant tumor, for which the expression ratio of genes related to the metastasis of cancer were compared by using the gene chips or cDNA microarray containing a plenty of genes, in order to find useful markers for diagnosis or treatment of the metastatic gastric cancer [Sakakura et al., Br. J. Cancer 87: 1153-1161, 2002; Wang et al, J. Cancer Res. Clin. Oncol. 128: 547-553, 2002]. Earlier studies have also shown that the reduced α-katenin and E-cadherin receptor as the adhesion receptor carrying out the migration, invasion and metastasis of cancer cell play a role in dedifferentiation of gastric cancer cells and the metastasis of these cells to lymph nodes [Streit at al., J. Mol. Med. 74: 253-268, 1996], and the over-expression of CD44-6v mutant among various mutants of CD44 receptor is playing an important role in the metastasis from gastric cancer cell to lymph nodes [Streit et al., Recent Results Cancer Res. 142: 19-50, 1996]. From the pathological point of view, it has been suggested that over- expression of epidermal growth factor (EGF), a well-known cell growth factor, EGF- receptor (EGFR) and c-erbB-2 are involved in invasion and metastasis of gastric cancer to lymph nodes [Tokunaga et al., Cancer 75: 1418-1425, 1995]. Further, Sakakura et al. has reported that as a result of comparison and analysis of differential expression between the primary gastric cancer cells and the metastatic cancer cells derived from malignant ascites thereof, the altered expressions of CD44, keratin 7, keratin 8, keratin 14, aldehyde dehydrogenase, CD9, IP3 receptor type 3, IL-2 receptor, IL-4 Stat, p27 and integrin (3 4 genes are believed to be involved in the process of metastatic gastric cancer [Sakakura et al., Br. J. Cancer 87: 1153-1161, 2002]. Most of the previous studies were focused on identifying the functions of only a single gene or several genes related to the metastasis, however, it has been reported recently that attempts were made for comparison and analyses of numerous genes involved in the intracellular processes of signal transduction, apoptosis, cell adhesion, etc., which are known closely related to the process of metastatic cancers [Sakakura et al., Br. J. Cancer 87: 1153-1161, 2002; Wang et al., J. Cancer Res. Clin. Oncol. 128: 547-553, 2002; Weiss et al., Oncogene 22: 1872-1879, 2003]. Therefore, it is essential to perform studies for identifying new target genes that are involved in metastasis for establishing more accurate standards for diagnosis of metastatic cancer. The gastric cancer cell lines utilized in the present invention comprise 11 kinds of gastric cancer cell lines (SNU-1, SNU-5, SNU- 6, SNU-216, SNU-484, SNU- 520, SNU-601, SNU-620, SNU-638, SNU-668, SNU-719) kindly established by Park et al., and one cell line.(KMS5) established by Korean Research Institute of Bioscience and Biotechnology, in which the primary gastric cancer cell lines derived from primary tumor were SNU-1, SNU-484, SNU-520 and SNU-719, and the metastatic gastric cancer cell lines derived from malignant ascites after gastric cancer became malignant were SNU-5, SNU-16, SNU-601, SNU-620, SNU-638, SNU-668 [Park et al., Cancer Res. 50: 2773-2780, 1990; Park et al., Int. J. Cancer 70: 43-449, 1997] . Further, the clinical tissues of gastric cancer used in the present invention are 30 gastric tissues (4 normal gastric tissues, 1 gastric cancer tissue, 4 pairs of normal/ gastric cancer tissues, 10 primary gastric cancer tissues and 7 metastatic gastric cancer tissues), which were provided by the College of Medicine, Chungnam National University (Korea). To date, studies to identify a few of genes showing specific expressions among SNU gastric cancer cell lines have been reported. These studies have revealed that c-met is amplified in SNU-16 cells, and the TGF-type II receptor, CEA, CA19-9 and c-erbB2 genes are over-expressed in SNU-1, SNU-5 and SNU-16 cells [Hara et al., Lab. Invest. 78: 1143-1153, 1998; Park et al., Proc. Natl. Acad. Sci. USA 91: 8772-8776, 1994; Bae et al., J Korean Med. Sci. 8: 153-159, 1993]. However, no gene has been found to show common change in gene expression in aforementioned cell lines. Therefore, the inventors of the present invention selected out those genes which show similar changes of gene expression in the primary gastric cancer cell Tines, the metastatic cancer cell lines and the normal gastric tissues, respectively, and tested the for their potentials as markers for diagnosis of gastric cancer or metastatic gastric cancer.

Background of Invention "Expressed Sequence Tag (EST) collection" is a human genome research project for collecting certain genes expressed in specific tissues or cells by constructing cDNA libraries using specific tissues or cells and analyzing the nucleotide sequences of cDNA clones randomly selected from those cDNA libraries. The analysis oi ESTs generated by cDNA libraries has been shown to provide efficient selection of new target genes [Adams et al., Science 252: 1651-1656, 1991; Adams et al., Nature 377:(SuppI.) 3-174, 1995; Hillier et al., Genome Res. 6: 807-828, 1996; Marra et al., Nat. Genet. 21:191-194,1999], and an extensive and quantitative measure of the transcriptional activity of expressed genes [Okubo et al., Nat. Genet. 2: 173-179, 1992; Adams et al., Science 252: 1651-1656, 1991; Adams et al., Nature 377:(Suppl.) 3-174, 1995; Liew et al., Proc. Natl. Acad. Sci. USA 91: 10645-10649, 1994; Mao et al., Proc. Natl. Acad. Sci. USA 95: 8175-8180, 1998; Ryo et al., Nucleic Acids Res. 26: 2586-2592, 1998; Sterky et al., Proc. Natl. Acad. Sci. USA 95: 13330-13335, 1998]. Accordingly, analysis of a specific gene frequency in a specific cell line may be able to reveal the presence of certain genes related to gastric cancer, and thus selected genes then may help to elucidate the underlying molecular mechanisms on development of gastric cancer, and also enable accurate diagnosis of gastric cancer. In the present invention, the inventors have identified 26 genes which are up-regulated in gastric cancer and 6 genes which are down-regulated in gastric cancer, all of which have never been reported to be related to gastric cancer. To date, the cellular functions for those genes have been reported as ollows. TUBA6 (Tubulin-alpha 6 chain) is a crucial element of microtubule. FKBPl A (FK506 binding protein 1A) is a constituent of myoplasmic reticulum of skeletal muscle involved in calcium secretion pathway which serves to control ryanodine receptor isoform-1. As for RPL4 (ribosomal protein L4) its function has not been yet elucidated. ARF1 (ADP-ribosylation factor 1), known as a GTP-hinding protein, effects as a heterotropic allosteric kinase of chlorella toxin catalytic subunits. FTH1 (ferritin, heavy polypeptide 1) is an intracellular molecule which stores iron. SH3GLB2 (SH3- domain GRB2-like endophilin B2) is similar to RING-3 protein; and HSPCA (heat shock 90kDa protein 1, alpha) has ATPase activity. TMSB4X (thymosin, beta 4, X chromosome) plays an important role in constituting cytoskeleton, and PYCR1 (pyrroline-5-carboxylate reductase 1) is involved in activating a catalyst and related to prostatic cancer [Ernst et al., Am. }. Pαthol. 160: 2169-2180, 2002]. Further, it has been known that the activating transcription factor 4 (ATF4) is an enhancer element of tax reaction, and surfeit 4 (SURF4) is a membrane protein. Further, ACTB (actin, beta), K-ALPHA-1 and keratin 8 (KRT8) participate in the formation of cell structure; LDHA (lactate dehydrogenase A), GAPD (glyceraldehyde-3-phosphate dehydrogenase), PKM2 (pyruvate kinase, muscle) and PGK1 (phosphoglycerate kinase 1) participate in glycolysis pathway; HMGIY (high mobility group protein isoforms I and Y), JUN (v-jun sarcoma virus 17 oncogene homolog (avian)) and CD44 (CD44 antigen) participate in signal transduction pathway; HSPA8 (heat shock 70kDa protein 8), HSPCB (heat shock 90kDa protein 1, beta) and HSPB1 (heat shock 27kDa protein 1) are related with heat shock mechanism; and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1) is a cof actor for protein synthesis in eukaryotes. Syndecan 1 (SDC1) is known to be a heparan sulfate-bearing protoglycan and is related to human malignant tumors [Mastumoto et al., Int. J. Cancer 74: 482-91, 1997] including liver cancer [Mastumoto et al., Int. }. Cancer 74: 482-91, 1997], head and neck cancer [Inki et al., Br. J. Cancer 70: 319-323, 1994], colon cancer [Day et al., Virchows Arch. 434: 121-125, 1999], etc. Nevertheless, little has been known on genes such as CD74 (invariant polypeptide of major histocompatibility complex), LOC131177 (FAM3D), AGR2 (anterior gradient 2 homolog, Xenopus laevis), IMAGE:4296901 (pepsinA), SNC73 and IGKC (immunoglobulin kappa constant) which are highly expressed in normal gastric tissues than gastric cancer cell lines. Additionally, in the present invention, 9 genes which are up-regulated in metastatic gastric cancer and 9 genes which are down-regulated in metastatic gastric cancer were newly identified. Among those genes, over-expression of CD44 has been already known to be closely related with the metastasis of gastric cancer into lymph nodes and also involved in its development into malignancy [Streit et al., J. Mol. Med. 74: 253-268, 1996; Joo et al., Anticancer Res. 23: 1581-1588, 2003]. In the present invention, keratin-8, a known cell adhesion molecule which is related to cell infiltration, was shown that its expression is decreased and thus classified as such, and this is contrary to the result of Sakakura et al who classified the gene as the one whose expression is increased in metastatic gastric cancer [Sakakura et al., Br. J. Cancer 87: 1153-1161, 2002]. The above contrary results may be due to the diverse nature having each of the primary gastric cancer cell lines. However, the inventors of the present invention also observed high expression of keratin 8 in SNU-16 cell line in comparison with other primary gastric cancer cell lines as shown in the study of Sakakura et al. The relevance of other genes to the metastasis of gastric cancer has been rarely reported until now and the followings are major information that has been achieved so far in this regard. Among the genes whose expression increase in the metastatic gastric cancer cell lines, GADD45b (growth arrest and DNA-damage- inducible gene 45beta) is a component involved in signal transduction which is over- expressed upon death of cells. This gene was recently reported that its expression is significantly reduced in hepatoma cell lines as compared to when it is in the normal hepatocyte, thus suggesting that GADD45b gene would be involved in mechanism of tumor suppression [Abdollahi et al., Oncogene 6: 165-167, 991; Qiu et al., Am. J. Pathol. 162:1961-1974, 2003]. JUN (v-jun sarcoma virus 17 oncogene homolog (avian)) is a proto-oncogene, an intracellular substance for signal transduction, and is involved in cell proliferation by controlling AP-1, a transcription factor [Shaulian et al., Nat. Cell. Biol. 4: E131-136, 2002]. It has been reported that the expression of the gene is increased in various cancer cells, and is especially related to the malignant tumors such as breast cancer, ovarian cancer, hematologic malignancy, osteosarcoma, etc. [Selvamurugan et al., Mol. Cell. Biol. Res. Commun. 3: 218-223, 2000; Volm et al., Clin. Exp. Metastasis 14: 209-214, 1996; Rossi et al., Int. J. Cancer 57: 86-89, 1994; Honoki et al., Mol. Carcinog. 7: 111-115, 1993]. However, it has not been reported that the expression of the gene is involved in the metastasis of gastric cancer. HMGIY (high mobility group protein isoforms I and Y) is a transcription factor involved in DNA binding, and abnormalities have been reported in the region of chromosome 6p21.3, at which this gene is located, in the case of benign mesenchymal tumors [Kazmierczak et al., Genes Chromosomes Cancer 23: 279-285, 1998]. However, it has not been known whether the expression of HMGIY genes is related to other tumors or malignant degeneration of tumors. GSTP1 (Glutathione S-transferase PI) is known as one of GST (Glutathione S-transferase) enzymes involved in detoxification of various carcinogens, and its level of expression is known to increase in various cancers such as lung cancer, gastric cancer, breast cancer, etc. Further, recent reports showed that the methylation of this gene would be deeply involved in carcinogenesis of gastric cancer [Howie et al., Carcinogenesis 11: 451-458, 1990; Kang et al., Lab. Investigation 83: 635-641, 2003]. LMNA (Lamin A/C) gene is one of nuclear membrane proteins, and very little is known about its function in carcinogenesis [Wydner et al., Genomics 32: 474-478, 1996]. ESRRA (Estrogen-related receptor alpha) gene encodes a protein for the membrane receptor of estrogen, an important hormone for human reproduction and osteogenesis, and the protein is known essential in estrogen signal transduction [Giguere, Trends Endocrinol. Metab. 13: 220-225, 2002]. There is also a report on the possibility of ESRRA as a target substance for treating patients suffering from malignant breast cancer [Ariazi et al., Cancer Res. 62: 6510-6518, 2002]. PLK (Polo-like kinase) is a serine-threordne phosphorylating enzyme and is essential for accurate regulation oi mitosis. It has been reported that the expression of the gene and protein is increased in patients having lung cancer, head and neck cancers, and colon cancer

[Wolf et al., Oncogene 14: 543-549, 1997; Knecht et al., Cancer Res. 59: 2794-2797, 1999; Takahashi et al., Cancer Sci. 94: 148-152, 2003]. IGFBP3 (insulin-like growth factor binding protein 3) is a protein which is important or IGF (Insulin-like growth factor) signal transduction pathway regulating the mitosis and cell death, and the increase of IGFBP3 protein in the patients suffering from various cancers provides a key index for diagnosis of cancer [Deal et al., J. Clin. Endoclinol. Metab. 86: 1274- 1280, 2001; Furstenberger et al., Lancet Oncol.3: 298-302, 002]. The aforementioned genes are those whose level of expression increases in cell lines derived from the malignant ascites where the metastasis of gastric cancer was progressed than in the primary gastric cancer cell lines. Numerous studies have been conducted to prove the relevance of these genes to cancers, however, these studies have been rarely focused on identifying their functional relationship to gastric cancer and its metastasis. Besides, the inventors of the present invention also selected those genes whose level of expression is reduced in the metastatic gastric cancer progresses in order to use them as markers for diagnosis of metastasis of gastric cancer. The genes are down-regulated in metastatic gastric cancer compared to the primary gastric cancer cell lines/ tissues. Of these genes as such, FKBPIA (FK506 binding protein 1A) and TMSB4X (thymosin, beta 4, X chromosome) play an important role in constructing cytoskeleton, and PKM2 (pyruvate kinase, muscle) and GAPD (glyceraldehyde-3-phosphate dehydrogenase) are known as enzymes involved in glycolytic pathway, and the expression of these genes is decreased as gastric cancer becomes malignant. KRT8 (Keratin 8) is known as a binding protein which is related to cell migration and invasion [Martens et al., Cancer 87: 87-92, 1999; Sakakura et al., Bri. J. Cancer 87: 1153-1161, 2002]. However, its gene was identified as a low level expression gene in metastatic gastric cancer until now. PTMA (prothymosin-alpha) is a nuclear protein involved in mitosis whose expression is regulated by c-myc transcriptional factor but there is no relation between its gene and gastric cancer or metastatic gastric cancer [Szabo et al, Hum. Genet. 90: 629-634, 1993; Haggerty et al., Proc. Natl. Acad. Sci. USA 100: 5313-5318, 2003]. ATP5A1 (ATP synthase alpha subunit) is an enzyme located in mitochondria which involved in energy metabolism, and CALM2 (calmodulin 2) is a protein playing a key role in calcium signal transduction, but there is no report on whether it is related to a specific cancer. NET1 (neuroepithelial cell transforming gene 1) is a proto- oncogene, which was first identified in 1996 by Chan et al., little has been known on the functional role of its gene. However, there is one report about that the expression of NETI gene relates to mitosis of cervical cancer [Chan et al., Oncogene 12: 1259-1266, 1996; Wollscheid et al, Int. J. Cancer 99: 771-775, 2002], Under these circumstances, the present inventors completed the present invention by sequencing the nucleotide sequences of genes in the cDNA libraries constructed from the primary gastric cancer cell lines, the normal tissues, a gastric cancer tissue and the metastatic gastric cancer cell lines; analyzing the up-regulated or down-regulated genes in gastric cancer or metastatic gastric cancer based on counting the obtained frequency of these genes; confirming if these genes expressed at high or low level from gastric cancer via real-time RT-PCR and competitive RT- PCR; and discovering new gene markers for detecting gastric cancer or metastatic gastric cancer. Accordingly, it is an object of the present invention to provide a method and a kit for diagnosis of gastric cancer based on the predetermined expression level of specific genes expressed in gastric cancer. Further, it is another object of the present invention to provide a method and a kit for diagnosis of metastatic gastric cancer based on the predetermined expression level of specific genes expressed in metastatic gastric cancer.

The aforementioned and other objects of the present invention will be accomplished by the present invention as described hereinafter.

Summary of Invention According to one aspect of the present invention, the invention provides a method and a kit for diagnosis of gastric cancer based on the predetermined expression level of at least one gastric cancer-related gene selected from a group consisting of EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARF1, SURF4, KRT8, GAPD, HSPCB, PGK1, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl, IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2 and IMAGE:4296901 (pepsinA). According to another aspect of the present invention, the invention also provides a method and a kit ior diagnosis of the metastatic gastric cancer based on the predetermined expression level of at least one metastatic gastric cancer- related gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTP1, LMNA, ESRRA, PLK, CD44, IGFBP3, PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1.

According to still another aspect of the present invention, the kit for diagnosis of gastric cancer according to the present invention comprises: a sense primer and an anti-sense primer of at least one gene selected from a group consisting of EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARF1, SURF4, KRT8, GAPD, HSPCB, PGK1, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein the above genes are up-regulated in gastric cancers compared to the normal gastric tissues; and a sense primer and an anti-sense primer of at least one gene selected irom a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA) and a mixture thereof, wherein the above genes are down-regulated in gastric cancers compared to the normal gastric tissues. According to a further aspect of the present invention, the kit for diagnosis oi gastric cancer according to the present invention comprises: a probe corresponding to at least one gene selected from a group consisting of EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARF1, SURF4, KRT8, GAPD, HSPCB, PGK1, HMGIY, K- ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein the above genes are up-regulated in gastric cancers compared to the normal gastric tissues; and a probe corresponding to at least one gene selected from a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA) and a mixture thereof, wherein the above genes are down-regulated in gastric cancers compared to the normal gastric tissues.

According to a further aspect of the present invention, the kit for diagnosis of gastric cancer according to the present invention comprises: an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARF1, SURF4, KRT8, GAPD, HSPCB, PGK1, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB,

HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein the above genes are up-regulated in gastric cancers compared to the normal gastric tissues; and an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA) and a mixture thereof, wherein the above genes are down-regulated in gastric cancers compared to the normal gastric tissues. According to a further aspect of the present invention, the kit for diagnosis of metastatic gastric cancer according to the present invention comprises: a sense primer and an anti-sense primer of at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTP1, LMNA, ESRRA, PLK, CD44 and IGFBP3 and a mixture thereof, wherein the above genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and a sense primer and an anti-sense primer of at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1 and a mixture thereof, wherein the above genes are down-regulated in metastatic gastric cancers compared to the primary gastric cancers. According to a further aspect of the present invention, the kit for diagnosis of gastric cancer according to the present invention comprises: a probe corresponding to at least one gene selected irom. a group consisting of GADD45B, JUN, HMGIY, GSTP1, LMNA, ESRRA, PLK, CD44 and IGFBP3 and a mixture thereof, wherein the above genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and a probe corresponding to at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1 and a mixture thereof, wherein the above are down-regulated in metastatic gastric cancers compared to the primary gastric cancers. According to a further aspect of the present invention, the kit for diagnosis of metastatic gastric cancer according to the present invention comprises: an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTP1, LMNA, ESRRA, PLK, CD44 and IGFBP3, wherein the above genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1, wherein the above genes are down-regulated in metastatic gastric cancers compared to the primary gastric cancers.

Hereinafter, the present invention is described in detail.

The present invention provides 32 marker genes for diagnosing gastric cancer, consisting of 26 up-regulated genes in gastric cancers, and 6 down-regulated genes in gastric cancers. The present invention also provides 18 marker genes for diagnosing metastatic gastric cancer, consisting of 9 up-regulated genes in metastatic gastric cancers, and 9 down-regulated genes in metastatic gastric cancers. The marker genes for gastric cancer according to the present invention comprise full-length and/ or fragments of up-regulated or down-regulated genes in gastric cancers. The marker genes for metastatic gastric cancer according to the present invention comprise full-length and/ or fragments of up-regulated or down-regulated genes in metastatic gastric cancers. The information for nucleotide sequence of gastric cancer marker genes is provided in the ioϊlowing Table 1, and the nucleotide sequence information of marker genes for metastatic gastric cancer is provided in the following Table 2.

Table 1. Sequence Information of Gastric Cancer Marker Genes

Table 2. Sequence Information of Metastatic Gastric Cancer Marker Genes

According to the present invention, 19 cDNA libraries were constructed from 14 gastric cancer cell lines (SNU5, SNU668, SNU16, SNU484, SNU1[3 strains], SNU620, SNU719, SNU638, SNU601, SNU216, SNU520, KMS5), 1 gastric cancer tissue (T665307) and 4 normal gastric tissues (K402, N258215, JM669761, N665307); nucleotide sequences of about 65,209 EST from these cDNA libraries were determined, and EST frequencies for the specific genes from a gastric cancer pool (gastric cancer cell lines + gastric cancer tissues) and a normal gastric pool (normal gastric tissues) were analyzed; and 26 genes (EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARF1, SURF4, KRT8, GAPD, HSPCB, PGK1, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl) which are up-regulated in the gastric cancer pool, i.e. the candidate genes that may be involved with cancer progression, and 6 strains of genes (IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA)) which are down-regulated in the cancer pool, i.e. the candidate genes that may be associated with suppression of gastric cancer, were finally selected as marker genes for gastric cancer. Further, in the present invention, of the above 19 cDNA libraries, 12 cDNA libraries constructed from 6 primary gastric cancer cell lines (SNU1[3 strains], SNU484, SNU719, SNU520) and 6 metastatic gastric cancer cell lines derived from malignant ascites (SNU5, SNU668, SNU16, SNU620, SNU638, SNU601) were selected; about 39,315 ESTs were sequenced from the selected libraries; the EST frequency for the specific genes from primary gastric cancer cell lines (primary pool) and the metastatic cancer cell lines derived from malignant ascites (ascites pool) were analyzed; and 9 genes (GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44, IGFBP3) which are up-regulated in the ascites pool, and 9 genes (PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1) which are down- regulated in the ascites pool, finally were selected as marker genes for metastatic gastric cancer. When the expression levels of 26 candidate genes selected by EST frequency were examined in gastric cancers and the normal gastric tissues using quantitative RT-PCR, it was observed that the candidate genes showing high frequency in gastric cancers were up-regulated in gastric cancers compared to the normal gastric tissues while the candidate genes having low frequency in gastric cancer were down-regulated in gastric cancers, thus showing that transcriptional levels of marker genes for gastric cancer selected by the EST frequency are closely concurred with data obtained from the quantitative RT-PCR.

Further, when the expression levels of 18 metastatic candidate genes selected by EST frequency were examined in primary gastric cancers and the metastatic gastric cancers using quantitative RT-PCR, it was observed that the candidate genes showing high frequency in metastatic gastric cancers were up-regulated in metastatic gastric cancer cell lines compared to the primary gastric cancer cell lines while the candidate genes having low frequency in metastatic gastric cancer were down-regulated in metastatic gastric cancer cell lines, thus showing that transcriptional levels of marker genes for metastatic gastric cancer selected by the EST frequency are closely concurred with data obtained from the quantitative RT- PCR.

The above results therefore suggest that gastric cancer can be diagnosed based on the expression level of gastric cancer-related genes such as EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl, IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA), and also metastatic gastric cancer can be diagnosed by measuring the expression level of metastatic gastric cancer-related genes such as GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44, IGFBP3, PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1.

Further, the present invention also provides a method for diagnosing gastric cancer by measuring the expression levels of the gastric cancer-related genes, which comprises the steps of: (a) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in gastric cancers consisting of EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and/ or their mixture in tested gastric cancer tissues. (b) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in gastric cancers consisting of EEFA1A, TUBA6, FKBPIA, PKM2, LDHA, RPU, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and/ or their mixture in tested normal tissues; (c) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in gastric cancers consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsin A) and/ or their mixture in tested gastric cancer tissues; (d) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in gastric cancers consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE.-4296901 (pepsin A) and/ or their mixture in tested normal gastric tissues; and (e) comparing the amount obtained in step (a) with that obtained in step (b), and comparing the amount measured by the step (c) with the amount measured by the step (d), thereby, determining whether the tested gastric samples are gastric cancer or not. The present invention also provides a method for diagnosing metastatic gastric cancer by measuring the expression levels of the metastatic gastric cancer- related genes, which comprises the steps of : (a) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in metastatic gastric cancers consisting of

GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 , IGFBP3 and/ or their mixture in tested metastatic gastric cancer tissues; (b) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in metastatic gastric cancers consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44, IGFBP3 and/ or their mixture in tested primary gastric cancer tissues; (c) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in metastatic gastric cancers consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1 and/ or their mixture in tested metastatic gastric cancer tissues; (d) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in metastatic gastric cancers consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1 and/ or their mixture in tested primary gastric cancer tissues; and (e) comparing the amount measured by the step (a) with the amount measured by the step (b), and comparing the amount measured by the step (c) with the amount measured by the step (d), thereby, determining whether the tested metastatic gastric samples are metastatic gastric cancer or not. The expression levels of the marker genes of gastric cancer and metastatic gastric cancer can be measured by the known method using a sense primer and an anti-sense primer having nucleotide sequences complementary to the marker genes or the fragments thereof. For example, the expressed amounts of the genes are measured by real-time RT-PCR and competitive RT-PCR. Such primers should include a partial sequence of DNA region encoding the protein from the marker genes, and the primer is a DNA fragment with a size greater than 15 bp in length. For example, the primers listed in Table 5 to Table 7 can be used in the present invention. The term "complementary" is not to be limited to the case having the complete complement in the successive 15 bp long sequence, but it can be applied to cases having 70% or more, preferably 80% or more of homology in the nucleotide sequence. Moreover, the expression level of gastric cancer and metastatic gastric cancer marker genes according to the present invention can be measured by the known hybridization methods using the fragments of marker genes as probes. For example, Northern hybridization ["Molecular Cloning - A Laboratory Manual "Cold Spring Habor Laboratory, NY, Maniatis, T. at al., 1982, section 7.37-7.52], in situ hybridization [Jacquemier et al., Bull Cancer 90: 31-8, 2003] or microarray [Macgregor, Expert Rev Mol Diagn 3: 185-200, 2003] may be used for the measurement. The probes has 200~1000bp containing nucleotide sequences of selected 32 marker genes in general, preferably 400~800bp. The nucleotide sequences may only have the homology of at least 70% to the nucleotide sequences of gastric cancer and metastatic gastric cancer marker genes. The probes of gastric cancer and metastatic gastric cancer marker genes according to the present invention can be produced by the common process, such as PCR method using the sense primer and anti-sense primer of gastric cancer marker genes and metastatic gastric cancer marker genes. Further, the expression level of gastric cancer and metastatic gastric cancer marker genes according to the present invention can be determined by measuring the amount of protein encoded by each gene. In the above process, the protein can be quantified by the common processes, such as ELISA and immunoprecipitation method, using an antibody which specifically binds to the protein oi gastric cancer and metastatic gastric cancer marker genes. The antibodies corresponding to the up-regulated genes and the down- regulated genes in gastric cancers or metastatic gastric cancers can be produced by cloning the genes into the expression vector using the common processes, harvesting the proteins encoded by the marker gene, and obtaining the antibodies from those proteins using the common method. The present invention includes the peptide fragments derived from the above proteins, and the peptide fragments of the invention comprise at least 7 amino acids, preferably at least 9 amino acids, more preferably at least 12 amino acids. The antibody of the invention is not limited to specific antibody type and may include the polyclonal antibodies [Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. Jhon Wiley & Sons Section 11.12-11.13], monoclonal antibodies [Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. Jhon Wiley & Sons Section 11.4-11.11], their parts having antigen-binding capacity, or substantially all immunoglobulin antibodies against the protein encoded by marker genes. In addition, the antibody according to the present invention also includes special antibody such as a humanized antibody [Methods in Enzymology 203, 99-121 (1991)], etc. Thirty-two gastric cancer marker genes or their proteins may be used alone or in combination for diagnosing gastric cancer, and 18 of metastatic gastric cancer marker genes or the proteins may also be used alone or in combination for diagnosing metastatic gastric cancer. Further, the kit for . diagnosing gastric cancer or metastatic gastric cancer includes the reagents for isolating RNA or poly(A)+RNA, in addition to the sense and anti-sense primers or probes of marker genes specific to the gastric cancer and metastatic gastric cancer, and may also include a solid supporter for the above marker genes when the expression level is measured by microarray. Further, since 32 gastric cancer marker genes may possibly be the activating genes or suppressing genes of gastric cancer, the compounds having low molecular weight which binds to the proteins encoded by the above marker genes may be the candidate compounds inhibiting or promoting the function of target proteins encoded by these marker genes, and may also be used as a drug, such as anticancer drugs, therapeutic agents, etc. Further, since 18 metastatic gastric cancer marker genes may possibly be the activating genes or suppressing genes for metastasis of gastric cancer, the compounds having low molecular weight which binds to the proteins encoded by the above marker genes may be the candidate compounds inhibiting or promoting the function of target proteins encoded by these marker genes, and may also be used as a drug, such as antimetastatic cancer drugs, therapeutic agents, etc.

The methods to screen these compounds include various known methods, such as a method of fixing each protein encoded by 32 gastric cancer marker genes or 18 metastatic gastric cancer marker genes onto the affinity column and purifying the proteins by contacting with the samples for screening [Pandya et al., Virus Res 87: 135-143, 2002], a method using two-hybrid system [Fields, S and Song, O., Nature 340: 245-246, 1989], Western blotting analysis ["Molecular Cloning - A Laboratory Manual" Cold Spring Harbor Laboratory, NY, Maniatis, T. et al. (1982) section 18.30- 18.74], high throughput screening [Aviezer et al., / Biomol Screen 6: 171-7, 2001], etc. The tested samples may include, but not limited to cell extracts, expression products from gene libraries, synthesized compounds with low molecular weight, synthesized peptides, natural compounds, etc.

Hereinafter, the invention will be described in detail by following Examples, however, it is provided only for better understanding of the invention and not intended to limit the scope of invention by the examples.

BRIEF DESCRIPTION OF DRAWINGS Fig.l depicts a result oi analyzing the EST frequency for selecting gastric cancer-related genes, according to the present invention. Fig. 2 depicts a result of analyzing the EST frequency for selecting metastatic gastric cancer-related genes, according to the present invention. Fig. 3 shows the expression levels of the up-regulated genes and down- regulated genes in gastric cancers by using real time RT-PCR in various gastric samples, [from (a) to (n) represent the up-regulated genes; and from (o) to (q) represent down-regulated genes. X-axis represents various samples, in which S14, S17, S18, S19 indicate the normal gastric tissues; SI, S2, S3, S5, S6, S7, S8, S9, S10, S12, S13, S21 indicate gastric cancer cell lines; and S20 indicates a gastric cancer tissue. Y-axis represents the expression level of a target gene in various samples, in which the expression levels of the up-regulated genes were indicated as a rate against the average expression levels in normal gastric tissues; and the expression levels of the down-regulated genes were indicated as a rate against the average of expression levels in gastric cancer cell lines and gastric cancer tissues. Expression level of each gene was calculated relative to the amount of B2M expressed in each sample]. Fig. 4 depicts the expression levels of the up-regulated genes and down- regulated genes in gastric cancers by using competitive RT-PCR in various gastric samples, [from (a) to (1) represent the up-regulated genes; and from (m) to (o) represent the down-regulated genes. X-axis represents the various samples, in which S14, S18, S19 indicate the normal gastric tissues; SI, S2, S3, S5, S6, S7, S8, S9, S10, S12, S13, S21 indicate gastric cancer cell lines; and S20 indicates a gastric cancer tissue. Y-axis represents the expression level of a target gene in various samples, in which the expression levels of the up-regulated genes were indicated as a rate against the average of expression levels in the normal gastric tissues; and the expression levels of the down-regulated genes were indicated as a rate against the average oi expression levels in gastric cancer cell lines and gastric cancer tissues. Expression level of each gene was calculated relative to the amount of B2M expressed in each sample]. Fig. 5 depicts the expression levels of the up-regulated genes and down-regulated genes in the metastatic gastric cancers by using competitive RT-PCR in various gastric samples, [from (a) to (i) represent the up-regulated genes; and from (j) to (r) represent the down-regulated genes. X-axis represents the various samples, in which S10, S5, S7, S21 indicate the primary gastric cancer cell lines; SI, S2, S3, S6, S8, S9 indicate the metastatic gastric cancer cell lines. Y-axis represents the expression level of a target gene in various samples, in which the expression levels of up- regulated genes were indicated as a rate against the average of expression levels in the primary gastric cancer cell lines; and the expression levels of down-regulated genes were indicated as a rate against the average of expression levels in the metastatic gastric cancer cell lines. Expression level of each gene was calculated relative to the amount of B2M expressed in each sample].

Fig. 6 depicts the expression levels of the up-regulated genes and down-regulated genes in gastric cancers by using competitive RT-PCR in patient samples with gastric cancer, [from (a) to (d) represent the up-regulated genes; and from (e) to (h) represent the down-regulated genes. X-axis represents the tested samples taken from 4 patients suffering gastric cancer, in which Nl, N2, N3, N4 indicate the normal gastric tissues; Tl, T2, T3, T4 indicate gastric cancer tissues. Y-axis represents the expression level of a target gene in patient tissues, which expression level of each gene was calculated relative to the amount of B2M expressed in each sample]. Fig. 7 depicts the expression levels of the up-regulated genes and down-regulated genes in metastatic gastric cancers by using competitive RT-PCR in patient samples with metastatic gastric cancer, [from (a) to (d) represent the up-regulated genes; and from (e) to (h) represent the down-regulated genes. X-axis represents the patient samples taken from 10 patients suffering the primary gastric cancer and 7 patients suffering the metastatic gastric cancer, in which El, E2, E3, E4, E5, E6, E7, E8, E9, E10 indicate the primary gastric cancer tissues; and Al, A2, A3, A4, A5, A6, A7 indicate the metastatic gastric cancer tissues. Y-axis represents the expression level of a target gene in the patient samples, which expression level of each gene was calculated relative to the amount of B2M expressed in each sample].

BEST MODE FOR EMBODIMENT OF INVENTION

Example 1: Isolation of total RNA from gastric cancer cell lines, gastric cancer tissues and normal gastric tissues Gastric cancer cell lines, SNU5, SNU668, SNU16, SNU1, SNU484, SNU620, SNU719, SNU638, SNU601, SNU216, SNU520 (Korean Cell Line Bank, http://cellbank.snu.ac.kr) and KMS5 (the Korea Research Institute of Bioscience and Biotechnology (KRIBB)) were cultured in RPMI culture media containing 10% FBS; and 1 sample of gastric cancer tissue (T665307) and 4 samples of normal gastric tissues, K402, N258215, N669761, N665307 (The College of Medicine, Chungnam National University, Korea) were obtained from the tissues removed by surgery and stored in liquid nitrogen until it is used for analysis. Total RNAs were isolated from cells and tissues by using QIAGEN kit (RNeasy Maxi kit: cat#75162). First, the adherent cells were recovered using trypsin-EDTA solution and then dissolved in 15ml of RLT buffer in the kit where 150μi of β-Mercaptoethanol was added. Meanwhile, about lg of tissue was taken, dissolved in 15ml of RLT buffer solution in the kit where 150μl of β- Mercaptoethanol was added, and crushed using a homogenizer. The sample solution was centrifuged at 3000g for 10 min to separate the supernatant, to which 15 ml of 70% EtOH was added and homogeneously mixed, and was centrifuged at 3000g for 5 min to attach total RNAs to the membrane. After washing twice, 1.2ml oi RNase-f ree water was added, and total RNAs were eluted.

Example 2: Screening of the up-regulated genes and the down-regulated genes in gastric cancer and metastatic gastric cancer by analysis of EST frequency In order to analyze the genes expressed in gastric cancer and malignant metastatic gastric cancer, the present inventors constructed various cDNA libraries from gastric cancer cell lines including the primary gastric cancer cell lines and the metastatic gastric cancer cell lines, a gastric cancer tissue and normal gastric tissues, randomly selected the cDNA clones from these cDNA libraries, sequenced these cDNAs and selected the up-regulated genes and the down-regulated genes in gastric cancer or metastatic gastric cancer by analysis of EST frequency.

(1) Construction of cDNA library Each l O^g of total RNAs from each sample obtained in Example 1 was treated with 3U of BAP (Bacterial alkaline Phosphatase, TakaRa) enzyme in the BAP enzyme reaction solution (lOOmM Tris-HCl (pH 7.0), 2mM DTT, 80U Rnasin (promega)), and then was reacted with 1000U of TAP (tobacco acid pyrophosphatase) enzyme in the TAP (Waco) enzyme reaction solution (50mM sodium acetate (pH 5.5), ImM EDTA, 2mM DTT, 80U Rnasin (promega)). Then, a reaction between 40 pmole of oligonucleotides (SEQ No. 1) and 250U RNA ligase

(TakaRa) was carried out in the presence of 50mM Tris-HCl (pH 7.5), 5mM MgCra, 2mM DTT, 0.5mM ATP, 26% PEG and 100U Rnasin, and thus synthesized oligomer was added only to intacted mRNA having a phosphate group. From total RNAs treated with the aforementioned reaction, mRNA was isolated by using oligotex mRNA purification kit (QIAGEN), and 1st cDNA was synthesized by using oligomer (SEQ No. 2) containing dTi7 as a primer. Thus synthesized cDNA was amplified in PCR reaction containing 5'-primer (SEQ No. 3) and 3'-ρrimer (SEQ No. 4) by using XL PCR kit (PerkinEImer). PCR products were treated with an enzyme Sfil and were isolated by agarose gel electrophoresis to produce 1.3 kb or more of cDNA fragments. Thus isolated cDNA fragments were linked to pCNS-D2 vector treated with Dralll enzyme by using TaKaRa linking kit, and then transformed into ToplOF' strain of E. coli (Invitrogen) by electroporation to construct cDNA library. According to the method of the invention, 14 cDNA libraries of gastric cancer cell lines, one cDNA library of gastric cancer tissue and 4 cDNA libraries of normal gastric tissues were constructed as shown in Table 3.

Table 3. Construction of various cDNA libraries and Analysis of EST UniGene #151^a Source Library Clone Clusters

Cancer Cell Line SNU5 ^' S1SNU5 1876 805 SNU668 S2SNU668 1696 684 SNU16 S3SNU16 2674 964 SNU1 S4SNU1 1800 736 S10SNU1 1973 1007 S11SNU1 6089 2417 SNU484 S5SNU484 2351 934 SNU620 S6SNU620 2778 1051 SNU719 S7SNU719 2781 853 SNU638 S8SNU638 2483 487 SNU601 S9SNU601 6883 2902 SNU216 S12SNU216 7634 1780 SNU520 S21SNU520 5931 2402 KMS5 S13KMS5 5444 2025 Normal Tissue K402 S14K402 5800 2026 N258215 S17N258215 527 263 N669761 S18N669761 1808 521 N665307 S19N665307 1310 469

Tumor Tissue T665307 S20T665307 3371 1274 Total 65209 19762 ^aNumber of clones and clusters in NCBI UniGene build #151 contributed by our EST sequence. Among these libraries constructed according to the method of invention, 6 cDNA libraries of primary gastric cancer cell lines and 6 cDNA libraries of metastatic gastric cancer cell lines are as seen from the following Table 4.

Table 4. Construction of various cDNA libraries and Analysis of EST Unigene #151^a Source Library Clones Clusters Primary Cancer Cell Line

Metastatic Cancer Cell Line r SNU5 S1SNU5 1876 805 SNU668 S2SNU668 1696 684 SNU16 S3SNU16 2674 964

^aNumber of clones and clusters in NCBI UniGene build #151 contributed by our EST sequence

(2) cDNA Sequencing and Data Analysis cDNA clones were picked from constructed cDNA libraries and cultured on LB agar medium which contains ampicillin (100_#g/ml). The plasmids DNA of cultured clones were isolated by using a MWG 96 well plasmid prep-system (MWG Biotech), and sequencing reactions of the cDNAs were performed using automatic sequencer ABI 3700 (PE Applied Biosy stems). The individual ESTs were searched against the human mRNA subset extracted from the GenBank database and then against the UniGene database (Hs.seq.all, build#151) for similarity comparisons using BLASTN. As shown in Table 3, 19,762 human UniGene clusters were collected by sequencing 65,209 of ESTs from 19 gastric cDNA libraries and analyzing sequence data of these ESTs against the UniGene data base. The number of determined EST per each library and the cell types of each library are also shown in Table 3. As shown in Table 4, 15,242 human UniGene clusters were collected by sequencing 39,315 of ESTs from 12 cDNA libraries constructed from the primary gastric cancer cell lines and the metastatic gastric cancer cell lines, and analyzing sequence data of these ESTs against the UniGene data base. The number of determined EST per each library and the cell types of each library are also shown in Table 4.

(3) Analysis for EST frequency Among the constructed cDNA libraries, 14 cDNA libraries for gastric cancer cell lines and one cDNA library for gastric cancer tissue were classified into the cancer pool, and 4 cDNA libraries for normal gastric tissues were classified into the normal pool. In addition, 6 cDNA libraries for the primary gastric cancer cell lines were classified into the primary pool, and 6 cDNA libraries for the metastatic gastric cancer cell lines were classified into the ascites pool. The frequencies for each gene were indicated as the ratio of total number of the specific genes in each sample against the number oi total clones. Further, the frequencies of each gene in each library were calculated by the ratio of total number of the specific genes in each library against total number of genes, and displayed by the various colors. According to the analysis, 26 up-regulated genes and 6 down-regulated genes in gastric cancer were selected as gastric cancer marker genes (Fig. 1). Further, 9 up-regulated genes, and 9 down-regulated genes in metastatic gastric cancer were selected as metastatic gastric cancer marker genes (Fig. 2).

Example 3: Analysis of expression level of selected marker genes by RT-PCR Each expression level of gastric cancer-related genes and metastatic gastric cancer-related genes were quantitatively analyzed by RT-PCR.

(1) Reaction of Reverse Transcriptase Each 5jxg of 17 total RNAs isolated in Example 1 was reacted for 60 min in the reaction buffer for reverse transcription at 42 °C to obtain 17 kinds of 1st cDNA.

The cDNA synthesis was completed by reacting on the 70 ^°C heating block for 15 min.

poly dT(12-18) primer (OAμg/ μi) ltd

5X first-strand buffer 4μi lOmM dNTP mixture Iμi

0.1M DTT 2μi

RNaseOUT (40U//_i) μi

Reverse Transcriptase (200U/ μi) Iμi

Total RNA (5/«g) X d Distilled water (10-X)

Total 20 i

(2) Quantitation of Expression level of Gastric Cancer-related Marker genes using by real-time PCR (a) Amplification of marker genes by real-time PCR A real-time PCR was performed by using FastStart-DNA Master SYBR Green I kit (Roche, Switzerland), with total reaction amount of 20^. That is, to the following real-time PCR solution containing sense- and antisense-primers of each gene, 2μi each of cDNA synthesized was added after it was diluted 1/500. As listed in Table 5, primers for 14 of the up-regulated genes and 3 of the down- regulated genes, were designed in the protein encoding region, the length of each primer is 17~20bρ, and GC content is around 50~70% (GenoTech, Korea). PCR was conducted for 45 cycles under the condition of 95 ^°C (15sec), 55 °C (5sec), 72 ^°C (30sec).

25mM MgCl₂ 0.8/d sense primer (5pmole/ μi) 2μi antisense primer (5pmoie/ l) ltd SYBR Green I polymerase mixture 2μi distilled water 11.2μi Total 20μi

Table 5. Primers for Gastric Cancer-Related Marker Genes used for Real-Time PCR

Gene Sense-primer Antisense-primer

EEFA1A SEQ No.: 5 SEQ No.: 6

ACTB SEQ No.: 7 SEQ No.: 8

K-ALPHA-1 SEQ No.: 9 SEQ No.: 10

FTH1 SEQ No.: 11 SEQ No.: 12

KRT8 SEQ No.: 13 SEQ No.: 14

FKBPIA SEQ No.: 15 SEQ No.: 16 GAPD SEQ No.: 17 SEQ No.: 18

LDHA SEQ No.: 19 SEQ No. 20

PKM2 SEQ No.: 21 SEQ No.: 22

PGKl SEQ No.: 23 SEQ No.: 24

HSPCA SEQ No.: 25 SEQ No.: 26

HSPA8 SEQ No.: 27 SEQ No.: 28

HSPCB SEQ No.: 29 SEQ No.: 30

HSPBl SEQ No.: 31 SEQ No.: 32

SNC73 SEQ No.: 33 SEQ No.: 34

Pepsin A SEQ No.: 35 SEQ No.: 36

IGKC SEQ No.: 37 SEQ No.: 38

β-2-microglobulin (B2M) was used as a standard gene for the quantitative analysis of marker genes. That is, 1.2μg of B2M DNA, was diluted to 1/10, 1/100, 1/1000 and 1/10000, respectively, and then 2μi of each diluted solutions was used as templates of the real-time PCR as aforementioned. The standard graph for the amount of B2M was calculated from the amplified B2M products. Based on the standard data, the amount of marker genes in gastric samples was quantitatively analyzed by the PCR products of marker genes. In PCR reaction of B2M, 5'-ρrimer (SEQ No. 39) and 3'-ρrimer (SEQ No. 40) were used as primers, respectively. These primers were designed based on the nucleotide sequence in the protein coding region of the gene.

(b) Identification of Expression level of marker genes by real-time PCR The amount of PCR products of the marker genes amplified by real-time RT- PCR was divided by the amount of PCR products of the standard genes (the amount of B2M is average amount obtained from 3 experiments). Then, the expression level oi the up-regulated genes was represented by the ratio of the target genes expressed in the tissue/ cell lines of gastric cancer against the average amount of expression of target genes expressed in the normal gastric tissues, and the expression level of the down-regulated genes was represented by the ratio of the target genes expressed in the normal gastric tissues against the average amount of expression of target genes expressed in the gastric cancer samples, as shown in Fig. 3 (S14, S17, S18, S19: normal gastric tissues; SI, S2, S3, S5, S6, S7, S8, S9, S10, S12, S13, S21: gastric cancer cell lines; S20: gastric cancer tissue). As a result, the amount of real-time RT-PCR products from the up-regulated genes (data given in Example 2-(3)) in gastric cancers, which are selected by EST high frequency, were high in gastric cancer samples compared to normal gastric tissues, and the amount of real-time RT-PCR products from the down-regulated genes, which are selected by EST low frequency, were low in gastric cancer samples. That is, the genes EEFA1A, FKBPIA, PKM2, LDHA, KRT8, GAPD, HSPCB, PGKl, K-ALPHA-1, FTH1, HSPA8, ACTB, HSPCA, HSPBl were shown to be potential and efficient carcinogenic markers of gastric cancer. The genes of IGKC, SNC73, IMAGE:4296901 (pepsinA) were also shown to be potential and efficient suppressing markers of gastric cancer.

(3) Quantitation of Expression level of Gastric Cancer-related Marker genes using Competitive RT-PCR (a) Normalization of Sample Concentration for Competitive RT-PCR In the present experiment, B2M gene was used as a standard gene for the quantitative analysis of the marker genes. For normalization of sample concentration, a competitor DNA for B2M, which have same priming parts with the B2M of standard genes but have different size of PCR products, are used in competitive RT- PCR. B2M competitor DNA was prepared by performing PCR in 50 i of reaction buffer containing 3_/d oi pCNS vector, DNA (2ng), 10_/ of 5x PCR premix (Bioneer), Iμi of 5'-primer (20pmole: SEQ No. 41), Iμi oi 3'-ρrimer (20ρmole: SEQ No. 42) and ?>5βi oi distilled water. PCR reaction was performed for 30 cycles under the condition of 94 °C 30 (sec), 50 °C 30 (sec), 72 °C 30 (sec), and the PCR products for B2M competitor DNA was found to be 322bp in size. Thus produced B2M competitor DNA was diluted to 7/108, 1/107, 3/107, 7/107, 1/106 and 3/106 via 6 steps, each 2μi oi dilutions were mixed with 1^st cDNA (an amount corresponding to lOng of RNA used for reverse transcription) of Example 3-(l), and a competitive RT-PCR for 6 samples was performed in 20μi oi reaction solutions containing μi of 5X Taq DNA polymerase, 2μi of B2M primers (5ρmole/_/κ ) (SEQ No. 39 and 40), Sμi of distilled water. The condition for PCR was 94 ^°C (40sec), 55 ^°C (lmin), 72 °C (lmin) and conducted for 25 cycles. 3μl of PCR products were loaded on 3% agarose gel, were separated at 100 V for 30min., and then were taken image files by using FrogTM apparatus (Gel Image

Analysis System) [Core Bio]. By using TotalLab vl.O program (NonLinear Dynamix

Ltd.), from the finally obtained gel band image files (.tif), the concentration having the similar sensitivity between two bands (322bp in B2M competitor DNA, 390bρ in the original B2M genes) was selected, and quantitatively analyzed to correct the concentrations oi each sample.

(b) Amplification of marker genes using competitive RT-PCR The marker genes were amplified by PCR reaction containing 1st cDNA of the sample corrected according to (a) as template. The sense- and antisense-primers for gastric cancer-related marker genes were showed in Table 6, and the sense- and antisense-primers for metastatic gastric cancer related marker genes were showed in Table 7. The PCR condition was 94 ^°C (lmin), 55 °C (30sec), 72 "C (lmin) and conducted for 25 cycles. The primers for the genes in Table 6 and Table 7 were designed in the protein encoding region, and the size of each primer was 17 ~ 20bp and GC contents were about 50 ~ 70% (CoreBiosystem, Korea). The composition oi PCR reaction mixture is as follows. cDNA 5μi

5X PCR mix (Bioneer, Korea) 3μi sense primer (1 Opmole/ i) Iμi anti sense primer (1 Opmole/ μi) Iμi distilled water 5jd Total 15μi

Table 6. Primers for Gastric Cancer-Related Marker Genes used for Competitive RT-PCR Gene Sense-primer Antisense-primer

TMSB4X SEQ No.: 43 SEQ No.: 44

RPL4 SEQ No.: 45 SEQ No.:46

TUBA6 SEQ No.: 47 SEQ No.:48

HMGIY SEQ No.: 49 SEQ No.: 50

ATF4 SEQ No.: 51 SEQ No.: 52

JUN SEQ No.: 53 SEQ No.: 54

SDCl SEQ No.: 55 SEQ No.: 56

ARFl SEQ No.: 57 SEQ No.: 58

PYCRl SEQ No.: 59 SEQ No.: 60

SURF4 SEQ No.: 61 SEQ No.: 62

SH3GLB2 SEQ No.: 63 SEQ No.: 64

CD44 SEQ No.: 65 SEQ No.: 66

CD74 SEQ No.: 67 SEQ No.: 68

AGR2 SEQ No.: 69 SEQ No.: 70

FAM3D SEQ No.: 71 SEQ No.: 72

Table 7. Primers for Metastatic Gastric Cancer-Related Marker Genes used for Competitive RT-PCR

Gene Sense-primer Antisense-primer

GADD45B SEQ No.: 73 SEQ No.: 74

JUN SEQ No.: 53 SEQ No.: 54

HNGIY SEQ No.: 49 SEQ No.: 50

GSTPl SEQ No.: 75 SEQ No.: 76

LMNA SEQ No.: 77 SEQ No.: 78

ESRRA SEQ No.: 79 SEQ No.: 80

PLK SEQ No.: 81 SEQ No.: 82

CD44 SEQ No.: 65 SEQ No.: 66

IGFBP3 SEQ No.: 83 SEQ No.: 84

PKM2 SEQ No.: 21 SEQ No.: 22

FKBPIA SEQ No.: 15 SEQ No.: 16 KRT8 SEQ No.: 85 SEQ No.: 86

TMSB4X SEQ No.: 43 SEQ No.: 44

GAPD SEQ No.: 17 SEQ No.: 18

ATP5A1 SEQ No.: 87 SEQ No.: 88

PTMA SEQ No.: 89 SEQ No.: 90

CALM2 SEQ No.: 91 SEQ No.: 92

NET1 SEQ No.: 93 SEQ No.: 94

(c) Identification of Expression Level of Marker Genes by Competitive RT-PCR In order to identify the products amplified by PCR, the products were separated on 2% agarose gel at 100V for 30 min, and were quantitatively analyzed using TotalLab vl.O program (NonLinear Dynamix Ltd.) as described in (3)-(a) of Example 3. As a result, as shown in Fig. 4, the amount of competitive RT-PCR products of the up-regulated genes in gastric cancer (Example 2-(3)) having high EST frequency was relatively high in the gastric cancer samples, while the amount of competitive RT-PCR products of the down-regulated genes in gastric cancer having low EST frequency was low in the gastric cancer samples. That is, 12 up-regulated genes such as TMSB4X, RPL4, TUBA6, HMGIY, ATF4, JUN, SDCl, ARFl, PYCRl, SURF4, SH3GLB2, CD44 are shown to be potential and efficient carcinogenic markers of gastric cancer, and the 3 genes such as CD74, AGR2, LOC131177 (FAM3D) are shown to be potential and efficient suppressing markers of gastric cancer. Further, as shown in Fig. 5, the amount of competitive RT-PCR products of the up-regulated genes in metastatic gastric cancer (Example 2-(3)) having high EST frequency was relatively high in the metastatic gastric cancer samples, while the amount of competitive RT-PCR products of the down-regulated genes in metastatic gastric cancer having low EST frequency was low in the metastatic gastric cancer samples. That is, 9 up-regulated genes in the metastatic gastric cancer, such as GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44, IGFBP3 and 9 down- regulated genes in the metastatic gastric cancer, such as PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1 are shown to be able to be useful markers of the metastatic gastric cancer.

Example 4: Diagnosis for Gastric Cancer or Metastatic Gastric Cancer by Quantitative analysis of up- or down-regulated genes in Gastric Cancer or by Quantitative analysis of up- or down-regulated genes in Metastatic Gastric Cancer in Patient Samples In this experiment, the expression levels for gastric cancer-related marker genes were identified in the 4 pairs of patient samples (376454, 663593, 668217, 670500) which consist of test tissues/ normal tissues (Tl ~ T4 and Nl ~ N4, respectively) taken from 4 patients with gastric cancer from the College of Medicine, Chungnam National University (Korea). After random selection oi FKBPIA, LDHA, HSPCB, TUBA6 genes among up-regulated genes in gastric cancer, and of SNC73, IGKC, LOC131177 (FAM3D), CD74 among down-regulated genes in gastric cancer, competitive RT-PCR was performed to determine the amount of these gene expression. In addition, the expression levels of the genes which comprise JUN, GSTPl, LMNA, CD44 genes randomly selected by up-regulated genes in metastatic gastric cancer and PKM2, FKBPIA, GAPD, ATP5A1 genes randomly selected by down- regulated genes in metastatic gastric cancer, were identified in the patient samples, which were directly taken from 10 patients (E1HE10) suffering the primary gastric cancer and 7 patients (A1~A7) suffering the metastatic gastric cancer from the College of Medicine, Chungnam National University (Korea).

(1) Isolation of total RNAs and Reaction of Reverse Transcriptase Total RNAs were isolated by the same method as Example 1, and a reaction of reverse transcription was performed using 5μg oi total RNA at 42 "C for 60 min to produce 8 kinds of 1st cDNA from the patient samples of gastric cancer and 17 of 1st cDNA from the patient samples of the metastatic gastric cancer. Then, cDNA construction was completed by the reaction on the heating block at 70 ^°C for 15min.

(2) Quantitation of Expression level of Gastric Cancer-related Genes by Competitive RT-PCR As described in (3)-(a) of Example 3, the concentrations of samples were corrected to adjust each concentration of templates for PCR to become equal. That is, with respect to the quantified Tl, N4 and T4 at the lowest concentrations, the concentrations of the remaining 5 samples were corrected by the dilution using the distilled water. The 1^st cDNA of the corrected sample was used by lOμi as template, and PCR was performed in 15 μi of PCR solution containing sense- and antisense-primer. The PCR condition was 94 °C (lmin), 55"C (30sec), 72 ^°C (lmin) and was conducted for 30 cycles. The used primers are as seen from Table 5 and Table 6. In order to identify the PCR products, an electrophoresis was performed, for all PCR solution, on 2% agarose gel at 100 V for 30 min, and PCR products were quantitatively analyzed using TotalLab vl.O program (NonLinear Dynamix Ltd.) as described in 3)-a) of Example 3. As a result, as seen from Fig. 6, the amount of competitive RT-PCR products of up-regulated genes, such as FKBPIA, LDHA, HSPCB, and TUBA6, in gastric cancer was higher in most tested cancer tissues than those of normal tissues collected from the same patient, and the amount of competitive RT-PCR products of down- regulated genes, such as SNC73, IGKC, LOC131177 (FAM3D), and CD74, in gastric cancer was low in most tested cancer tissues than those of normal tissues. Four tested tissues (T1XT4) were diagnosed to be gastric cancer, and such result meets the result of clinical test for gastric cancer according to prior art (Tl; gastric cancer stage IV; T2: gastric cancer stage IDA; T3 and T4: gastric cancer stage II).

(3) Quantitation of Expression level of Metastatic Gastric Cancer-related Marker Genes by Competitive RT-PCR As described in (3)-(a) of Example 3, the concentrations of samples were corrected to adjust each concentration of templates for PCR to be same. That is, with respect to the sample having the lowest concentration, the concentrations of the remaining 13 samples were diluted. Although the concentrations of samples used as templates were dependent on the kinds of genes, at least 1/50 diluted concentration of 1st cDNA was used. 1st cDNA oi the corrected sample was used in the volume of 2μi as template, and PCR was performed in 15μi of PCR solution containing sense- and antisense-primer. The PCR condition was 94 °C (30sec), 55 ^°C (lmin), 72 °C (lmin) and was conducted for 25 cycles. The used primers are as seen from Table 7. In order to identify the PCR products, an electrophoresis was performed, for all PCR solution, on 2% agarose gel at 100V for 30 min, and PCR products were quantitatively analyzed using TotalLab vl.O program (NonLinear Dynamix Ltd.) as described in 3)-a) of Example 3. As a result, as seen from Fig. 7, the amount oi competitive RT-PCR products of up-regulated genes comprised of JUN, GSTPl, LMNA and CD44 in metastatic gastric cancer was higher in most tested metastatic cancer tissues than those of primary cancer tissues, and the amount of competitive RT-PCR products of down- regulated genes comprised of PKM2, FKBPIA, GAPD, ATP5A1 in metastatic gastric cancer was lower in most tested tissues than those oi primary cancer tissues. All 17 patient tissues (E1~E10 and A1~A7) were diagnosed to be gastric cancer; 10 patient samples of primary gastric cancer were diagnosed to be the stage I A of gastric cancer; and 7 tissues of metastatic gastric cancer were diagnosed to be the stage IV of gastric cancer.

INDUSTRIAL APPLICABILITY As described above, the present invention provides 6 down-regulated genes and 26 up-regulated genes in gastric cancer compared to the normal gastric tissues, as the marker of tumor very available for diagnosis of gastric cancer, which marker can perform the quantification sensitively and quickly in the tissues of patients. Further, the present invention provides 9 low-regulated genes and 9 high- regulated genes in the metastatic gastric cancer compared to the primary gastric cancers, as the marker very available for diagnosis for the metastasis of malignant gastric cancer, which marker can perform the quantification sensitively and quickly in the tissues of patients to diagnose metastatic gastric cancer.

Claims

What is claimed is:

1. A method for diagnosing gastric cancer, which comprises the steps of: (a) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in gastric cancers consisting of EEFAIA, TUBA6, FKBPIA, PKM2, LDHA, RPU, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and/ or their mixture in tested gastric cancer tissue samples. (b) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in gastric cancers consisting of EEFAIA, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and/ or their mixture in tested normal tissue samples; (c) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in gastric cancers consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE.-4296901 (pepsin A) and/or their mixture in tested gastric cancer tissue samples; (d) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in gastric cancers consisting of IGKC, SNC73,

CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsin A) and/or their mixture in tested normal gastric tissue samples; and (e) comparing the amount obtained in step (a) with that obtained in step (b), and comparing the amount measured by the step (c) with the amount measured by the step (d), thereby, determining whether the tested gastric samples is a gastric cancer or not.

2. The method for diagnosing gastric cancer according to Claim 1, wherein the amount of expression of said genes are measured by real-time RT-PCR and competitive RT-PCR.

3. A method for diagnosing metastatic gastric cancer, which comprises the steps of: (a) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in metastatic gastric cancers consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 , IGFBP3 and/ or their mixture in tested metastatic gastric cancer tissue samples; (b) measuring the expression levels of RNA or protein of at least one gene selected from up-regulated genes in metastatic gastric cancers consisting of

GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44, IGFBP3 and/or their mixture in tested primary gastric cancer tissue samples; (c) measuring the expression levels of RNA or protein oi at least one gene selected from down-regulated genes in metastatic gastric cancers consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1 and/ or their mixture in tested metastatic gastric cancer tissue samples; (d) measuring the expression levels of RNA or protein of at least one gene selected from down-regulated genes in metastatic gastric cancers consisting oi PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2, NET1 and/ or their mixture in tested primary gastric cancer tissue samples; and (e) comparing the amount measured by the step (a) with the amount measured by the step (b), and comparing the amount measured by the step (c) with the amount measured by the step (d), thereby determining whether the tested gastric sample is metastatic gastric cancer or not.

4. The method for diagnosing gastric cancer according to Claim 3, wherein the amount of expression of said genes are measured by competitive RT-PCR.

5. A kit for diagnosing gastric cancer, which comprises: (a) a sense primer and an anti-sense primer of at least one gene selected from a group consisting of EEFAIA, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTH1, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein said genes are up-regulated in gastric cancers compared to the normal gastric tissues; and (b) a sense primer and an anti-sense primer oi at least one gene selected from a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE-.4296901 (pepsinA) and a mixture thereof, wherein said genes are down- regulated in gastric cancers compared to the normal gastric tissues.

6. The kit for diagnosing gastric cancer according to Claim 5, wherein said primer is a DNA fragment with a size greater than 15 bp in length.

7. A kit for diagnosing gastric cancer, which comprises: a probe corresponding to at least one gene selected from a group consisting of EEFA A, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTHl, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein said genes are up-regulated in gastric cancers compared to the normal gastric tissues; and a probe corresponding to at least one gene selected from a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA) and a mixture thereof, wherein said genes are down-regulated in gastric cancers compared to the normal gastric tissues.

8. The kit for diagnosing gastric cancer according to Claim 7, wherein the amount of expression of said genes are measured via hybridization using said probes.

9. The kit for diagnosing gastric cancer according to Claim 7 or 8, wherein said probes are 200-1000 bp of nucleotides in length.

10. A kit for diagnosing gastric cancer, which comprises: an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of

EEFAIA, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTHl, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein said genes are up-regulated in gastric cancers compared to the normal gastric tissues; and an antibody that recognizes the proteins encoded by at least one gene selected from a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA) and a mixture thereof, wherein said genes are down-regulated in gastric cancers compared to the normal gastric tissues.

11. The kit for diagnosing gastric cancer according to Claim 10, wherein the expression levels of said proteins are measured by antigen-antibody reaction.

12. A kit for diagnosing metastatic gastric cancer, which comprises: a sense primer and an anti-sense primer of at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 and IGFBP3 and a mixture thereof, wherein said genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and a sense primer and an anti-sense primer of at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1 and a mixture thereof, wherein said genes are down-regulated in metastatic gastric cancers compared to the primary gastric cancers.

13. The kit for diagnosing metastatic gastric cancer according to Claim 12, wherein said primer is a DNA fragment with a size greater than 15 bp of nucleotides in length.

14. A kit for diagnosing metastatic gastric cancer, which comprises: a complimentary probe corresponding to at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 and IGFBP3 and a mixture thereof, wherein said genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and a complimentary probe corresponding to at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1 and a mixture thereof, wherein said genes are down-regulated in metastatic gastric cancers compared to the primary gastric cancers.

15. The kit for diagnosing metastatic gastric cancer according to Claim 14, wherein the amount of expression of said genes are measured via hybridization using said probes.

16. The kit for diagnosing metastatic gastric cancer according to Claim 14 or 15, wherein said probes are 200-1000 bp in length.

17. A kit for diagnosing metastatic gastric cancer, which comprises: an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 and IGFBP3, said genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and an antibody that recognizes the protein encoded by at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1, wherein said genes are down-regulated in metastatic gastric cancers compared to the primary gastric cancers.

18. The kit for diagnosing metastatic gastric cancer according to Claim 17, wherein the expression levels of said proteins are measured by antigen-antibody reaction.

19. A kit for diagnosing metastatic gastric cancer, which comprises: an antibody that recognizes the proteins encoded by at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 and IGFBP3, wherein said genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and an antibody that recognizes the proteins encoded by at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1, wherein said genes are down- regulated in metastatic gastric cancers compared to the primary gastric cancers.

20. A method for screening a suppressor of gastric cancer, which comprises the steps of: (a) binding a test compound to (i) the proteins encoded by at least one gene selected from a group consisting of EEFAIA, TUBA6, FKBPIA, PKM2, LDHA, RPL4, ARFl, SURF4, KRT8, GAPD, HSPCB, PGKl, HMGIY, K-ALPHA-1, FTHl, HSPA8, SH3GLB2, ACTB, HSPCA, TMSB4X, SDCl, PYCRl, ATF4, JUN, CD44, HSPBl and a mixture thereof, wherein said genes are up-regulated in gastric cancers compared to the normal gastric tissues; and (ii) the proteins encoded by at least one gene selected from a group consisting of IGKC, SNC73, CD74, LOC131177 (FAM3D), AGR2, IMAGE:4296901 (pepsinA) and a mixture thereof, wherein said genes are down-regulated in gastric cancers compared to the normal gastric tissues; and (b) determining whether said test compound promotes or suppresses the reaction of proteins.

21. A method for screening a suppressor of metastatic gastric cancer, which comprises the steps of: (a) binding a test compound to (i) the proteins encoded by at least one gene selected from a group consisting of GADD45B, JUN, HMGIY, GSTPl, LMNA, ESRRA, PLK, CD44 and IGFBP3, wherein said genes are up-regulated in metastatic gastric cancers compared to the primary gastric cancers; and (ii) the proteins encoded by at least one gene selected from a group consisting of PKM2, FKBPIA, KRT8, TMSB4X, GAPD, ATP5A1, PTMA, CALM2 and NET1, wherein said genes are down-regulated in metastatic gastric cancers compared to the primary gastric cancers; and (b) determining whether said test compound promotes or suppresses the reaction of proteins.