KR101588018B1 - Biomarker Indicative of Cancer and Diagnosis Method Using the Same - Google Patents

Abstract

The present invention relates to a kit for cancer diagnosis or prognosis, which comprises (a) an oligonucleotide that specifically binds to a nucleotide sequence encoding embigin, or (b) a binding agent that specifically binds to an embryonic protein, and And a screening method of a therapeutic agent for pancreatic cancer. According to the present invention, the amount of mRNA or protein expressed by the biomarker of the present invention is specifically increased in cancer tissues than in normal tissues. Therefore, by using embycin, which is a marker of the present invention, cancer can be diagnosed clearly by distinguishing between normal tissue and cancer.

Description

Technical Field [0001] The present invention relates to a biomarker for cancer,

The present invention relates to cancer-specific biomarkers and uses thereof.

Pancreatic cancer is one of the most frequent cancers in Korea with colorectal cancer, breast cancer, and thyroid cancer. It is the ninth most common cause of cancer and the fifth most common cause of cancer deaths. These pancreatic cancers are the most deadly cancer in the human body, with a 5-year survival rate of less than 5% and an average survival of only 5 months. At the time of diagnosis, 80-90% of the patients were found to be unable to undergo surgical resection. Therefore, they are totally dependent on chemotherapy and radiotherapy. However, in spite of anticancer drugs or radiation therapy, the response to therapy is very low. Most patients are resistant to anticancer drugs within 2-4 months of treatment, and most of them die from metastatic disease such as liver metastasis . Therefore, if you are diagnosed with pancreatic cancer earlier and are looking for factors that regulate cancer growth and metastasis, they are of great use as new diagnostic markers or anticancer drugs.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to find a novel biomarker capable of rapidly and accurately molecular diagnosis of cancer. As a result, the present invention was completed by confirming that embigin can diagnose cancer early and is a marker for determining prognosis.

Accordingly, an object of the present invention is to provide a kit for cancer diagnosis or prognosis analysis.

It is another object of the present invention to provide a method for detecting a cancer marker in order to provide information necessary for diagnosis or prognosis of cancer.

It is still another object of the present invention to provide a screening method of a cancer therapeutic agent.

It is another object of the present invention to provide a pharmaceutical composition for preventing, treating or inhibiting cancer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a kit for cancer diagnosis or prognosis, comprising a binding agent that specifically binds to an oligonucleotide or embryonic protein that specifically binds to a nucleotide encoding embigin to provide.

According to another aspect of the present invention, there is provided a method for diagnosing cancer or diagnosing cancer through the method of detecting the expression of nucleotide sequence of embjin protein or embjin in a biological sample of a human in order to provide information necessary for cancer diagnosis or prognosis analysis. A method for detecting a marker is provided.

The present inventors have tried to find a novel molecular marker capable of rapidly and accurately molecular diagnosis of cancer. As a result, it was confirmed that the above-mentioned molecular marker can diagnose cancer early and is a marker capable of determining prognosis.

The molecular marker of the present invention can be an index for the generation and development of cancer and can be used for diagnosis of cancer development and development.

As used herein, the term " diagnosis " is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of an object (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining the stage of a cancer, or determining the response of a cancer to treatment), or determining therametrics And monitoring the status of the object to provide information about the object.

In the kit for cancer diagnosis or prognosis analysis according to the present invention, the cancer is preferably selected from the group consisting of pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, Pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, liver cancer, stomach cancer, colon cancer, colon cancer, colon cancer, cervix cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, Pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, liver cancer, stomach cancer or colon cancer, and most preferably pancreatic cancer.

According to the embodiment of the present invention, it can be seen that the embjin is highly specifically expressed in pancreatic cancer tissue than normal pancreatic tissue (see Fig. 1). By using embycin, a marker of the present invention, pancreatic cancer can be diagnosed by distinguishing between normal pancreas and pancreatic cancer.

According to a preferred embodiment of the present invention, the kit of the present invention can selectively diagnose and analyze only pancreatic cancer samples from a normal pancreas sample and a pancreatic cancer sample. Preferably, the sample is human tissue, cell, blood, serum or plasma.

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

According to a preferred embodiment of the present invention, the nucleotide encoding embycin of the present invention has the nucleotide sequence of the first sequence of the sequence listing.

According to a preferred embodiment of the present invention, the embedding protein of the present invention has the amino acid sequence of the second sequence of the sequence listing.

According to a preferred embodiment of the present invention, the present invention can be carried out by an immunoassay method, that is, an antigen-antibody reaction method. In this case, an antibody or an aptamer that specifically binds to the above-described pancreatic cancer marker of the present invention is used.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies can be prepared by methods routinely practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the method of the present invention is carried out using an antibody or an aptamer, the present invention can be carried out according to a conventional immunoassay method to diagnose pancreatic cancer.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotope is an antibody labeled (e.g., C _14, I _125, P ₃₂ and S ₃₅₎ of detecting the marker molecules of the present invention .

When the method of the present invention is carried out by an ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating the surface of a solid substrate with an unknown cell sample lysate to be analyzed; (Ii) reacting the cell lysate with an antibody to a marker as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P450. In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable that the substrate is selected from the group consisting of bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (N-methylpyrrolidone), tetramethylbenzidine, ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronin, glucose oxidase and nitroblue tetrazolium a substrate such as phenzaine methosulfate may be used The.

When the method of the present invention is carried out in a Capture-ELISA system, a specific embodiment of the present invention comprises the steps of (i) coating an antibody against a marker of the present invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the sample; (Iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating label and specifically reacts with an embedding protein; And (iv) measuring a signal originating from said label.

The detection antibody has a label that generates a detectable signal. The label may be a chemical (e.g., biotin), an enzyme (alkaline phosphatase,? -Galactosidase, horseradish peroxidase and cytochrome P450), a radioactive material (such as C14, I125, P32, and S35 But are not limited to, fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane , ≪ / RTI > Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. This detection of the signal enables a qualitative or quantitative analysis of the marker of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

According to another variant of the invention, an aptamer which specifically binds to the marker of the invention can be used in place of the antibody. Aptamers are oligonucleic acid or peptide molecules, and the general contents of aptamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).

By analyzing the intensity of the final signal by the above-described immunoassay, pancreatic cancer can be diagnosed. That is, when the signal for the marker of the present invention is stronger than the normal sample in the sample, it is diagnosed as pancreatic cancer.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally comprise reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

According to a preferred embodiment of the present invention, the kit of the present invention may be a microarray.

According to a preferred embodiment of the present invention, the kit of the present invention is a gene amplification kit.

When the kit of the present invention is a microarray, the probe is immobilized on the solid-phase surface of the microarray. When the kit of the present invention is a gene amplification kit, it includes a primer.

The probe or primer used in the diagnostic kit of the present invention has a sequence complementary to the embinyin nucleotide sequence. The term " complementary " as used herein means having complementarity enough to selectively hybridize to the nucleotide sequences described above under any particular hybridization or annealing conditions. Thus, the term " complementary " has a different meaning from the term complementary, and the primers or probes of the present invention may include one or more mismatches mismatch < / RTI > sequence.

As used herein, the term " primer " refers to a primer that, under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) in a suitable buffer at a suitable temperature, - means strand oligonucleotide. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the above-mentioned nucleotide sequence which is a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the gene sequence and acting as a primer. The design of such a primer can be easily carried out by a person skilled in the art with reference to the above-mentioned nucleotide sequence, for example, by using a program for primer design (for example, PRIMER 3 program).

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

The nucleotide sequence of the marker of the present invention to be referred to in the preparation of the primer or the probe can be found in Sequence Listing 1, and a primer or a probe can be designed with reference to this sequence.

In the microarray of the present invention, the probe is used as a hybridizable array element and immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, the sample DNA to be applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions can be varied. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The pancreatic cancer diagnostic kit of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

Hybridization-based analysis can be performed using a probe hybridized to the nucleotide sequence of the marker of the present invention to determine the presence or absence of pancreatic cancer.

The label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin , Haptens with specific binding partners such as biotin, digoxigenin and chelating groups. Markers can be obtained by a variety of methods routinely practiced in the art, such as the nick translation method, the Multiprime DNA labeling systems booklet ("Amersham" (1989)) and the kaination method (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, color measurement, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biosamples. The raw sample is preferably a pancreas tissue cell. Instead of the probe, the cDNA to be analyzed may be labeled and subjected to a hybridization reaction-based analysis.

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

After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization. Combinations of enzymes / substrates that may be used include, but are not limited to, peroxidases (such as horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2) , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by a silver staining method using silver nitrate. Therefore, when the method for detecting pancreatic cancer markers of the present invention is carried out based on hybridization, specifically (i) hybridizing a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention to a nucleic acid sample; (ii) detecting whether the hybridization reaction has occurred. By analyzing the intensity of the hybridization signal by the hybridization process, it is possible to judge whether or not the cancer is pancreatic cancer. That is, the sample is diagnosed as pancreatic cancer when the hybridization signal for the nucleotide sequence of the marker of the present invention is stronger than a normal sample (for example, normal pancreatic tissue cells).

According to a preferred embodiment of the present invention, the human pancreatic cancer diagnostic kit of the present invention may be a gene amplification kit.

The term " amplification " as used herein refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315) (SEQ ID NO: 1), self-sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction CP-PCR), U.S. Patent No. 4,437,975) (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA), U.S. Patent No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21,22), and loop-mediated isothermal amplification. (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the diagnostic kit of the present invention is carried out using a primer, the gene amplification reaction is carried out to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. Since the present invention analyzes the degree of expression of the nucleotide sequence of the marker of the present invention, the amount of mRNA of the nucleotide sequence of the marker of the present invention is examined in a sample (for example, pancreatic tissue, blood or urine) The degree of expression of the nucleotide sequence of the marker is determined.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere , C. et al., Plant Mol . Biol . Rep . , ≪ / RTI > 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); And Chomczynski, P. et al., Anal . Biochem . 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of the mRNA, and the cDNA can be easily synthesized using the oligo dT primer and the reverse transcriptase using such a sequence characteristic ( See, for example, PNAS USA, 85: 8998 (1988); Libert F, et al., Science , 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning . A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the " Clenow " fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The cDNA of the nucleotide sequence of the thus amplified marker of the present invention is analyzed by a suitable method to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. For example, the result of amplification reaction described above is subjected to gel electrophoresis, and the resulting band is observed and analyzed to examine the expression level of the nucleotide sequence of the marker of the present invention. Through this amplification reaction, when the expression of the nucleotide sequence of the marker of the present invention in the raw sample is higher than that of the normal sample (normal cells), it is diagnosed as pancreatic cancer.

Therefore, when the method for detecting a pancreatic cancer marker of the present invention is carried out based on an amplification reaction using cDNA, specifically (i) performing an amplification reaction using a primer annealed to the nucleotide sequence of the marker of the present invention; And (ii) analyzing the product of the amplification reaction to determine the degree of expression of the nucleotide sequence of the marker of the present invention.

The marker of the present invention is a biomolecule which is highly expressed in pancreatic cancer. High expression of these markers can be measured at the mRNA or protein level. As used herein, the term " high expression " means that the degree of expression of a nucleotide sequence to be a target in a sample to be investigated is higher than that of a normal sample. For example, expression analysis methods conventionally used in the art, such as RT-PCR or ELISA methods (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001) In the case of expression analysis, it means that the expression is analyzed to be high. For example, when the marker according to the present invention is highly expressed about 1.5-10 times as much as a normal cell, it is determined to be " high expression " in the present invention and determined to be pancreatic cancer.

According to another aspect of the present invention, the present invention provides a screening method for a cancer therapeutic agent comprising the steps of: (a) treating a test substance with a cell comprising an embjin-encoding nucleotide sequence; And (b) analyzing the expression of the nucleotide sequence or the expression of the embedding protein. When the test substance suppresses the expression of the nucleotide sequence or the expression of the embedding protein, It is judged as a cure.

In the screening method for a cancer therapeutic agent of the present invention, the cancer is preferably selected from the group consisting of pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, Pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, liver cancer, stomach cancer, colon cancer, colon cancer, prostate cancer, urinary bladder cancer, pancreatic cancer, cancer of the uterine cervix, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, Pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, liver cancer, stomach cancer or colon cancer, and most preferably pancreatic cancer. The term " cancer "

In the screening method of the present invention, the test substance or the Ebbiegin-encoding nucleotide sequence or protein may be labeled with a detectable label. For example, the detectable label may be a chemical label (e.g., biotin), an enzyme label (such as horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase,? -Galactosidase and? - glucosidase), radioactive labels (e.g., C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent labels such as coumarin, fluorescein isothiocyanate, rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Cy 3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl and FAM], luminescent, chemiluminescent, FRET transfer labels or metal labels (e.g., gold and silver).

According to the method of the present invention, first, the test substance is contacted with cells containing the embjin-encoding nucleotide sequence of the present invention. The term " test substance " used in referring to the screening method of the present invention means an unknown substance used in screening to check whether or not it affects the expression level of the embjin-encoding nucleotide sequence of the present invention. Such test substances include, but are not limited to, chemical, protein or natural product extracts.

Next, the expression level of the embryogenic-encoding nucleotide sequence of the present invention is measured in the cells treated with the test substance. Measurement of the expression level can be carried out as described below. When the result of the measurement shows that expression of the embzin-encoding nucleotide sequence of the present invention or expression of embryonic protein is inhibited, it can be determined as a cancer treatment agent.

The measurement of the change in the expression level of the above-described nucleotide sequence can be carried out through various methods known in the art. For example, such as RT-PCR (Sambrook, Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 102-108, CRC press), hybridization reaction using a cDNA microarray (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or in situ (in situ hybridization reaction (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)).

When carrying out according to the RT-PCR protocol, first, total RNA is isolated from cells treated with the test substance, and first strand cDNA is prepared using oligo dT primer and reverse transcriptase. Next, PCR is carried out using the first strand cDNA as a template and the target substance-coding nucleotide-specific primer set. The above-mentioned primer set is a sequence contained in an embjin-encoding nucleotide sequence to be used. Then, the PCR amplification product is electrophoresed, and the formed band is analyzed to measure changes in the expression amount of the nucleotide sequence.

In addition, changes in the amount of embryonic cells can be performed through various immunoassay methods known in the art. For example, changes in the amount of embryonic cells may be detected by immunodiffusion, radioimmunoassay, radioimmunoprecipitation, Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay, capture-ELISA, inhibition or competitive assay, But are not limited to,

Methods of immunoassay or immunostaining include, but are not limited to, Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme - linked immunosorbent assay ( ELISA ), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e.g., ¹⁴ C, ¹²⁵ I, ³² P And S ³⁵ ) can be used to detect the marker molecules of the present invention.

When the method of the present invention is carried out by an ELISA method, the present invention relates to a method for screening a solid substrate, comprising the steps of: (i) coating the surface of a solid substrate with an analyte of an unknown cell sample to be analyzed; (Ii) reacting said cell lysate with an antibody to a target as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable that the substrate is selected from the group consisting of bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'- Azine-di [3-ethylbenzthiazoline sulfonate]), o - phenylenediamine (OPD) and naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. The detection of such signals enables a qualitative or quantitative analysis of the embodies of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing, treating or inhibiting cancer, which comprises an embryonic expression inhibitor or an activity inhibitor as an active ingredient.

According to a preferred embodiment of the present invention, the embryonic expression inhibitor or activity inhibitor of the present invention may be used as siRNA, shRNA, miRNA, ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, peptides, More preferably siRNA, shRNA, miRNA, antisense oligonucleotide, peptide or antibody, even more preferably siRNA, shRNA, miRNA or antisense oligonucleotide, most preferably siRNA.

The term "siRNA " in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914) siRNA is provided as an efficient gene knockdown method or as a gene therapy method because it can inhibit the expression of a target gene. SiRNA was first discovered in plants, insects, fruit flies and parasites, Recently, siRNA has been developed and used in mammalian cell research (Degot S, et al., 2002; Degot S, et al., 2004; Ballut L, et al.

The siRNA molecule of the present invention may have a structure in which the sense strand (the corresponding sequence corresponding to the p21 mRNA sequence) and the antisense strand (the sequence complementary to the p21 mRNA sequence) are located on opposite sides to form a double strand. Also, according to another embodiment, the siRNA molecules of the invention may have a single stranded structure with self-complementary sense and antisense strands. The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of mucin gene (preferably MUC5AC) by the RNAi effect. The sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure.

SiRNA molecules of the present invention may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and antisense strands, wherein the siRNA molecule formed by the expression of the nucleotide sequence is a molecule The hairpin structure is formed by hybridization, and the stem-and-loop structure as a whole is formed. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The term "antisense oligonucleotide " in the present invention means DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into a protein The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to a mucin gene (preferably, MUC5AC) and capable of binding to mucin gene mRNA, and includes a translation of p21 mRNA, translocation ), Maturation, or any other overall biological function. The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 40 bases in 10 to be.

As used herein, the term " miRNA (microRNA) " is a single stranded RNA molecule of 21-25 nucleotides that is a regulatory element that controls gene expression in a eukaryotic organism through inhibition in the step of disrupting or detoxifying the target mRNA. These miRNAs are made up of two stages of processing. The first miRNA transcript is made in the nucleus by the RNase III type enzyme called Drosha, which is made into a stem-loop structure of about 70-90 bases, that is, a pre-miRNA, which is then transferred to the cytoplasm and transferred to an enzyme called Dicer To produce a mature miRNA of 21-25 bases. These miRNAs complementarily bind to the target mRNA and act as post-transcriptional gene suppressors, leading to translation inhibition and mRNA destabilization. miRNAs are involved in a variety of physiological phenomena and diseases.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a pharmaceutically effective amount of microparticles secreted from cancer cells or cancerous tissues; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing and treating ischemic diseases.

According to the present invention, the microparticles of the present invention induce angiogenesis, and can be applied to ischemic diseases to exhibit preventive or therapeutic effects.

According to a preferred embodiment of the present invention, the microparticles of the present invention are secreted from cancer cells or cancer tissues, more preferably under hypoxic conditions, from cancer cells or cancer tissues.

According to a preferred embodiment of the present invention, the ischemic diseases to which the composition of the present invention is applied are ischemic heart diseases, ischemic myocardial infarction, ischemic heart failure, ischemic enteric disease, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, Renal failure, ischemic baldness, ischemic stroke, and ischemic leg ischemic disease.

According to a preferred embodiment of the present invention, the microparticles of the present invention are secreted from cancer cells or cancer tissues, and the cancer is preferably selected from the group consisting of pancreatic cancer, biliary cancer, neuroendocrine tumors, lung cancer, breast cancer, ovarian cancer, liver cancer, Cancer of the thyroid, pituitary cancer, or ureter, more preferably pancreatic cancer, biliary cancer, neuroendocrine carcinoma, pancreatic cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, Pancreatic cancer, biliary cancer, neuroendocrine tumor, lung cancer, liver cancer, stomach cancer, or colon cancer, and more preferably a pancreatic cancer, a breast cancer, a lung cancer, a liver cancer, a stomach cancer, a colon cancer, a colon cancer, a prostate cancer, a bone cancer, .

As used herein, the term " pharmaceutically effective amount " means an amount necessary to induce angiogenesis properly and to demonstrate the pharmacological effect of the treatment of the ischemic disease.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides novel molecular markers for cancer

(Ii) The amount of mRNA or protein expressed by the biomarker of the present invention is specifically increased in cancer tissues than in normal tissues.

(Iii) Furthermore, by using embin, which is a marker of the present invention, early diagnosis and prognosis of cancer can be determined by distinguishing between normal tissues and cancer tissues.

Figure 1 shows the results of RT-PCR analysis of changes in embryonic expression in cancer tissue (CA) versus normal tissue (NL).
FIG. 2 shows the results of confirming embryonic expression changes in cancer tissues (a, b) and normal tissues (c) by tissue immunostaining.
FIG. 3 shows the result of western blot analysis of intracellular signal changes after embedding of siRNA.
Fig. 4 is a photograph showing morphological changes of cells after embedding siRNA treatment.
FIG. 5 shows the result of measurement of cell proliferation after treatment with embryonic siRNA using WST-8.
FIG. 6 shows the results of measurement of changes in cell migration and invasion ability according to embedding of siRNA.
FIG. 7 shows the results of measuring the change in wound healing ability according to the embedding of siRNA.
FIG. 8 shows the results of measuring the expression and activity of MMR-2/9 by carrying out an ego-migration using gelatin.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

In colorectal cancer tissue Embjin ( embigin ) Expression change

In pancreatic cancer surgical tissues, we resected the cancer tissues and parts of the normal tissues that are far away from the cancer tissues. For this, the tissue was detached and immersed in RNAlater RNA stabilization reagent (Qiagen, USA), and total RNA was obtained using RNeasy Protect Mini Kit (Qiagen, USA). Superscript II (Invitrogen, USA), RNaseOut (Invitrogen, USA), oligo dT primer (Invitrogen, USA), dNTP (Invitrogen, USA) were used to reverse mRNA in total RNA. The mRNA obtained from the total RNA was subjected to PCR using primers of the emboxin (forward direction -5'-TACAAGTCCACCTCTCAGAGAAG-3 ', reverse direction -5'-CCCAGATGTTGTGAACTGGCATG-3') and beta-actin primer, Ex-Taq polymerase Respectively.

On the other hand, expression was confirmed in a paraffin block made of the patient's tissue to confirm the location of the expression. Paraffin was removed from the paraffin tissues using xylene and dehydration was performed using ethanol at different concentrations. After incubation with anti-HRP, Dako RealEnvision (Dako) was treated with donkey serum (Hyclone) to block the antibody, followed by incubation with substrate-chromogen Dako) to induce color development.

Analysis of genetic effects through gene expression changes

(# 1 target sequence 5'-GCCGUGCACUAUUCCAAUU-3 '# 2 target sequence 5'-GCAAACAAAUGGGAAGUUA-3') and scrambled siRNA (5'-CCUACGCCACCAAUUUCGU-3 ') in pancreatic cancer cell line BxPC- Were each mixed with lipofectamine RNAi MAX (Invitrogen, USA) and then treated with 20 nM of the cells. The siRNA was transfected three times at 72-hour intervals, and the amount of gene expression was confirmed by real-time PCR and the amount of protein expression was confirmed by Western blotting.

Real-time PCR was performed using an Applied Biosystems machine and confirmed by X2 SyberGreen (Applied Biosystems, USA). Experimental results were normalized to the expression level of beta-actin. Western blotting was performed by scraping cell lines treated with siRNA through a cell scraper, mixing with a protein lysis buffer to induce lysis, measuring the concentration of the protein, electrophoresis on 10% SDS-PAGE, and transferring the cells to nitrocellulose paper (Milipore, USA ), Blocked with 5% skim milk, and then treated with anti-Abbeam (USA) antibody, anti-GAPDH (SantaCruz, USA) antibody. Secondary antibodies were anti-rabbit (SantaCruz, USA) and anti-mouse (SantaCruz, USA) antibodies, respectively. Antibodies were developed using HRP. (Cell Signaling, USA), PI3K-110α (SantaCruz, USA), and snail / slug (Abcam, USA) in order to examine the expression changes in the BxPC- To perform Western blotting.

Observation of cell shape change through gene expression change

Cells were treated with siRNA and examined using a microscope (IX50, Olympus) for changes in cell shape.

Changes in growth rate of cells due to changes in gene expression

Cells were treated with siRNA and the following day, WST-8 (EZ-Cytox, Daeil Lab Service) was added to the cell culture medium for 2 hours at 37 ° C and then absorbance (450 nm) was measured using an ELISA reader . The growth rate of the cells was measured in consideration of the change in the color of the culture medium of the cells and the number of existing cells.

Cell migration due to gene expression changes migration )/invasion( invasion ) Ability measurement

In order to examine the migration of cells, 2x10 ⁴ cells treated with siRNA in Transwell (Costar, USA) were each suspended in a medium containing 1% FBS and dispensed. A 20% FBS cell culture medium was laid on the bottom as an attractant to attract the cells. The cells were incubated in a 37 ° C cell incubator for 12 hours, and the cells were fixed and stained with crystal violet.

To examine the ability of the cells to pass through the matrix, metrizel (Invitrogen, USA) was coated on transwells diluted in serum-free medium at a concentration of 1: 5 and 2 x 10 ⁴ cells treated with siRNA were each treated with 1% FBS Lt; RTI ID = 0.0 > ml < / RTI > A 20% FBS cell culture medium was laid on the bottom as an attractant to attract the cells. The cells were incubated in a 37 ° C cell incubator for 12 hours, and the cells were fixed and stained with crystal violet.

Measurement of the wound healing ability of cells according to the change of gene expression

Using a tip, we scraped the bottom of the culture dish with the cells to examine the process of artificially wounding the cells treated with the siRNA and healing the wound.

Cells following changes in gene expression MMP -2/9 Measurement of expression change

To investigate the expression and function of MMP-2/9, we performed zymography. Cells treated with siRNA were grown in serum-free medium for 2 days. The medium was collected, lyophilized, and the same amount of protein was electrophoresed on SDS-PAGE gel containing 10% gelatin (Sigma, USA) Deg.] C to confirm the degree of degradation of the gelatin contained in the gel.

Experiment result

Normal tissues of pancreatic cancer patients In cancer tissue Embjin  Expression

mRNA expression was found to be increased in pancreatic cancer tissues compared with the normal tissues of patients. Immunoblotting with paraffin tissue of the patient revealed that embryogen expressed in pancreatic islets of the normal pancreas was expressed in the pancreatic cancer tissues Metaplastic duct and cancer tissue (Fig. 1-2).

Embjin siRNA  After processing Intracellular  Signal change

It was confirmed that the expression of phosphorylated-GSK-3ß, PIdK-110α, and Snail / Slug was inhibited when the expression of embjin was inhibited similarly to the change according to the previously reported expression of Basigin through Western blotting 3).

Observation of cell shape change through gene expression change

Microscopic observation revealed that embryogenic siRNA-treated cells were generally rounded compared to conventional BxPC-3, and most of the apical cells were not visible (FIG. 4).

Changes in growth rate, migration / invasion capacity of cells with changes in gene expression

Inhibition of Embjin expression by embryonic siRNA treatment was observed to slow the cell growth by about 13% compared to the control group (Fig. 5). Cells treated with embryonic siRNA showed decreased cell migration and penetration ability by inhibition of embryonic expression (Fig. 6).

Measurement of the wound healing ability of cells according to the change of gene expression

The wound was induced in the cells, and the cell state after 21 hours was observed. As a result, it was confirmed that the wound-healing ability was reduced in cells treated with embedding siRNA (FIG. 7).

Cells following changes in gene expression MMP -2/9 Measurement of expression change

It was confirmed that a relatively small amount of MMP-2/9 was expressed in embryogenic siRNA-treated cells through the euormagy (Fig. 8). MMP is a zinc-dependent endopeptidase that selectively degrades substances in the extracellular matrix and is involved in normal cell remodeling and tumor angiogenesis and metastasis. Among the MMPs, MMP-9 and MMP-2 are involved in tumor invasion and metastasis. As is evident from the above experimental results, it is considered that the cell migration / invasion ability and the wound restoration ability decrease due to the inhibition of embjin expression is due to a decrease in the expression of MMP-2/9.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> Embigin <130> PN120606 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 4309 <212> RNA <213> Homo sapiens embigin mRNA <220> <221> gene &Lt; 222 > (1) .. (4309) <220> <221> CDS (250). (1230) <400> 1 acatccggga acgccagcag ccggctgagg gctgcataac tgatggaggg ccgggcgcgg 60 taagagcgtc tcgggggagt ggggcaaggc ggccgggccc ctcccattcc gccttttctt 120 cagcgtccta cccgcggcac tggctgcgag cgccgggcca cctgcgagtg tgcgcaggga 180 ctctggacac ccgcggcggc gagctgaggg agcagtctcc acgaggaccc aggcggaccc 240 tctggcgcc atg cgc gcc ctc ccc ggc ctg ctg gag gcc agg gcg cgt acg 291            Met Arg Ala Leu Pro Gly Leu Leu Glu Ala Arg Ala Arg Thr              1 5 10 ccc cgg ctg ctc ctc ctc cag tgc ctt ctc gct gcc gcg cgc cca agc 339 Pro Arg Leu Leu Leu Leu Gln Cys Leu Leu Ala Ala Ala Arg Pro Ser  15 20 25 30 tcg gcg gac ggc agt gcc cca gat tcg cct ttt aca agt cca cct ctc 387 Ser Ala Asp Gly Ser Ala Pro Asp Ser Pro Phe Thr Ser Pro Pro Leu                  35 40 45 aga gaa gaa ata gg aaat aac ttt tcc ttg gag agt cat aac ata 435 Arg Glu Glu Ile Met Ala Asn Asn Phe Ser Leu Glu Ser His Asn Ile              50 55 60 tca ctg act gaa cat tct agt atg cca gta gaa aaa aat atc act tta 483 Ser Leu Thr Glu His Ser Ser Met Pro Val Glu Lys Asn Ile Thr Leu          65 70 75 gaa agg cct tct aat gta aat ctc aca tgc cag ttc aca aca tct ggg 531 Glu Arg Pro Ser Asn Val Asn Leu Thr Cys Gln Phe Thr Thr Ser Gly      80 85 90 gat ttg aat gca gta aat gtg act tgg aaa aaa gat ggt gaa caa ctt 579 Asp Leu Asn Ala Val Asn Val Thr Trp Lys Lys Asp Gly Glu Gln Leu  95 100 105 110 gag aat aat tat ctt gtc agt gca aca gga agc acc ttg tat acc caa 627 Glu Asn Asn Tyr Leu Val Ser Ala Thr Gly Ser Thr Leu Tyr Thr Gln                 115 120 125 tac agg ttc acc atc att aat agc aaa caa atg gga agt tat tct tgt 675 Tyr Arg Phe Thr Ile Ile Asn Ser Lys Gln Met Gly Ser Ser Ser Cys             130 135 140 ttc ttt cga gag gaa aag gaa caa agg gga aca ttt aat ttc aaa gtc 723 Phe Phe Arg Glu Glu Lys Glu Gln Arg Gly Thr Phe Asn Phe Lys Val         145 150 155 cct gaa ctt cat ggg aaa aac aag cca ttg atc tct tac gta ggg gat 771 Pro Glu Leu His Gly Lys Asn Lys Pro Leu Ile Ser Tyr Val Gly Asp     160 165 170 tct act gtc ttg aca tgt aaa tgt caa aat tgt ttt cct tta aat tgg 819 Ser Thr Val Leu Thr Cys Lys Cys Gln Asn Cys Phe Pro Leu Asn Trp 175 180 185 190 acc tgg tac agt agt aat ggg agt gta aag gtt cct gtt ggt gtt caa 867 Thr Trp Ser Ser Asn Gly Ser Val Lys Val Pro Val Gly Val Gln                 195 200 205 atg aat aaa tat gtg atc aat gga aca tat gct aac gaa aca aag ctg 915 Met Asn Lys Tyr Val Ile Asn Gly Thr Tyr Ala Asn Glu Thr Lys Leu             210 215 220 aag ata aca caa ctt ttg gag gaa gat ggg gaa tct tac tgg tgc cgt 963 Lys Ile Thr Gln Leu Leu Glu Glu Asp Gly Glu Ser Tyr Trp Cys Arg         225 230 235 gca cta ttc caa tta ggc gag agt gaa gaa cac att gag ctt gtg gtg 1011 Ala Leu Phe Gln Leu Gly Glu Ser Glu Glu His Ile Glu Leu Val Val     240 245 250 ctg agc tat ttg gtg ccc ctc aaa cca ttt ctt gta ata gtg gct gag 1059 Leu Ser Tyr Leu Val Le Le Lys Pro Le Leu Val Ile Val Ala Glu 255 260 265 270 gtg att ctt tta gtg gcc acc ctg ctt tgt gaa aag tac aca caa 1107 Val Ile Leu Leu Val Ala Thr Ile Leu Leu Cys Glu Lys Tyr Thr Gln                 275 280 285 aag aaa aag cag tac gat gag ggg aaa gaa ttt gag cag att gaa 1155 Lys Lys Lys Lys His Ser Asp Glu Gly Lys Glu Phe Glu Gln Ile Glu             290 295 300 cag ctg aaa tca gat gat agc aat ggt ata gaa aat aat gtc ccc agg 1203 Gln Leu Lys Ser Asp Asp Ser Asn Gly Ile Glu Asn Asn Val Pro Arg         305 310 315 cat aga aaa aat gag tct ctg ggc cag tgaatacaaa acatcatgtc 1250 His Arg Lys Asn Glu Ser Leu Gly Gln     320 325 gagaatcatt ggaagatata cagagttcgt atttcagctt tgtttatcct tcctgttaag 1310 agcctctgag tttttagttt taaaaggatg aaaagcttat gcaacatgct cagcaggagc 1370 ttcatcaacg atatatgtca gatctaaagg tatattttca ttctgtaatt atgttacata 1430 aaagcaatgt aaatcagaat aaatatgtta gaccagaata aaattaatta tattctggtc 1490 ttcaaaggac acacagaaca gatatcagca gaatcactta atacttcata gaacaaaaat 1550 cactcaaaac ctgtttataa ccaaagaatt catgaaaaag aaagcctttg ccatttgtct 1610 tagaaagtta ttttttaaaa aaaaatcata cttactatta gtatctatgg aagtatatgt 1670 aacaattttt atgtaaaggt catctttctg tgatagtgaa aaaatatgtc tttactaagt 1730 tgaaatgaat actttctgcc tttgctaatg atagttattc tacaatctcc acaagaaaaa 1790 tatacctttt atccggaaat attggtttaa ggcaaataaa taaaactgtg cttgctctaa 1850 agctctgcac tacaaaagca aattttccag tctttattcc actcataacc actgctttcc 1910 ccaccaggct aaagtgcttc ttagtcttaa atagggtttt taacttcata aggtacacca 1970 ttgtttatgt tttagggtgc aggtccatat agtcgtaata cttaaaacac tgctacactg 2030 atgcttttgt ggctcatggt ttcctggttc tttgatttaa cacctggctt cctggtatct 2090 atggctggtt aaatatctaa acctgcatta ttagtgcttt acaataata ttcccttgac 2150 tgaagtccag aaatactaaa gcacttcttt tttattgctg tgaatgcctt agacttatct 2210 tccaaaatag gatacttcgt ttctattgca tatataactg aaatatttta taaactcact 2270 gtaatcaaat cttataaaat ctgaatcaac atgaaaagtt gtcaatgctt gaatgaatac 2330 taaaatcata ccacaagata ctagataaca taaatgacca tgaagtgtcc cactgctcaa 2390 agggcaacaa ctttaagaac agaagcaaag agtggtggtg tggaacccaa gattcttggc 2450 gactagggaa tttaaaggtt ggggaggagg aattcaatga tttgacaaga aatactttat 2510 aaaaatttgc tccatgtatt ggctaagaaa aatataataa aattttggta gtgaaaattg 2570 tatcactaat gtttaccaat attactagta tgaataatac acataaggta gtgacttgga 2630 agctgctcaa tactttgttg ctataacaaa atgcaatatt ttgttgtaaa gcatcttaac 2690 tacacatata agtttgctga acttgaatac atcccggtaa tggcaaactt cattttattc 2750 ttggttttca ttttattctg gtctcagggc catacattta ttttagttta attaattatt 2810 attttgacaa cattacttgg gtaatatcta tataatggta gataaccaat cagttagctt 2870 aattaatcta agctatttga attcagggaa atattagtca aatctgctgt ttgactaaat 2930 acaattcaaa gcagtgtatt tgtgacttgg aatatagaac acaaacaaat gtgtaaccca 2990 tctgcagtat gcattcttcc attttaccac cactaaccat tgaggctcat acaattttca 3050 agcaacaagc attgcttccc aaaggtaaaa taatcaggaa tatttctata gtaaatggag 3110 tttttaattg gcaatactgt ttattttttt ctagcaaaga caatttatct cagtaacctt 3170 aaataggaaa agtactcaaa aattaaagtt gacttttggc tttttgtagt ctctctggag 3230 gacttccaga aaattaccac tgttggattt taatctgctt ccattaacca atcttacaga 3290 gatgataaat gattaacta atattgacca aatataaatt gactgcaaat aataagccgc 3350 ataaaagttg aagtaggaaa actactccaa gtttatatat taggcacctt ggggaattct 3410 cttagtcttt ccagctaaca aggattttca gcataataag tagatttaat gatggcataa 3470 aattcccaga tactttcata tacatttttt gccaaagtgt aaatcttctc cttttgcagt 3530 ctttttgtta ttatcccaac atctagctgc tcaggcccta atttagaact cattctgata 3590 ttttcttctt ctgtattaat taatacttat gggtcatgaa tccctgctcc tttgaccttt 3650 ttaatagctc atgaatccat cccattgcct ccccaactct gcccacacct gctgcaacac 3710 taatgccttg attttcctac ctcagtctcc ttttctatta atccctcttt tgcgtggctg 3770 cctgagtcca ctttctcaaa cacaaacctc actatgcaac tttggaatct ctgtaggctc 3830 acctttgcca tttcttaata ttgcattctc caaattgcta atctctatcc agtctcttga 3890 actagacttg gcacatggag attccgctca gaaagtgctc ttcacacttc tacctgcttg 3950 actaacccca gattatcttt caagacttta atctgatctt gtgtcttaga gaagccccca 4010 tacctggtag agcatgtacc atcttacatg cttaaataac tccacattta tttgtgttta 4070 ttactctgtg ttataaatat acatttgttg gtctctctct tggattattt tgtttctttg 4130 tcctgtaact accactgaaa gggtgcaata cagctttctt gaaatgtgta ttgaacggat 4190 gaatgtataa ataaaaatta aattttgtaa atttctgctt attcttagaa aaagaatcta 4250 aattgtgaca aatcagaatt gaaaaaagta ttctaataaa gaaaaacaag cttttataa 4309 <210> 2 <211> 327 <212> PRT <213> Homo sapiens embigin amino acids <400> 2 Met Arg Ala Leu Pro Gly Leu Leu Glu Ala Arg Ala Arg Thr Pro Arg   1 5 10 15 Leu Leu Leu Leu Gln Cys Leu Leu Ala Ala Ala Arg Pro Ser Ser Ala              20 25 30 Asp Gly Ser Ala Pro Asp Ser Pro Phe Thr Ser Pro Pro Leu Arg Glu          35 40 45 Glu Ile Met Ala Asn Asn Phe Ser Leu Glu Ser His Asn Ile Ser Leu      50 55 60 Thr Glu His Ser Ser Met Pro Val Glu Lys Asn Ile Thr Leu Glu Arg  65 70 75 80 Pro Ser Asn Val Asn Leu Thr Cys Gln Phe Thr Thr Ser Gly Asp Leu                  85 90 95 Asn Ala Val Asn Val Thr Trp Lys Lys Asp Gly Glu Gln Leu Glu Asn             100 105 110 Asn Tyr Leu Val Ser Ala Thr Gly Ser Thr Leu Tyr Thr Gln Tyr Arg         115 120 125 Phe Thr Ile Ile Asn Ser Lys Gln Met Gly Ser Ser Ser Cys Phe Phe     130 135 140 Arg Glu Glu Lys Glu Gln Arg Gly Thr Phe Asn Phe Lys Val Pro Glu 145 150 155 160 Leu His Gly Lys Asn Lys Pro Leu Ile Ser Tyr Val Gly Asp Ser Thr                 165 170 175 Val Leu Thr Cys Lys Cys Gln Asn Cys Phe Pro Leu Asn Trp Thr Trp             180 185 190 Tyr Ser Ser Asn Gly Ser Val Lys Val Pro Val Gly Val Gln Met Asn         195 200 205 Lys Tyr Val Ile Asn Gly Thr Tyr Ala Asn Glu Thr Lys Leu Lys Ile     210 215 220 Thr Gln Leu Leu Glu Glu Asp Gly Glu Ser Tyr Trp Cys Arg Ala Leu 225 230 235 240 Phe Gln Leu Gly Glu Ser Glu Glu His Ile Glu Leu Val Val Leu Ser                 245 250 255 Tyr Leu Val Pro Leu Lys Pro Phe Leu Val Ile Val Ala Glu Val Ile             260 265 270 Leu Leu Val Ala Thr Ile Leu Leu Cys Glu Lys Tyr Thr Gln Lys Lys         275 280 285 Lys Lys His Ser Asp Glu Gly Lys Glu Phe Glu Gln Ile Glu Gln Leu     290 295 300 Lys Ser Asp Asp Ser Asn Gly Ile Glu Asn Asn Val Pro Arg His Arg 305 310 315 320 Lys Asn Glu Ser Leu Gly Gln                 325

Claims (15)

(a) an oligonucleotide that specifically binds to a nucleotide encoding embigin, or (b) a binding agent that specifically binds to an embryonic protein.
2. The kit for diagnosing or prognosticating pancreatic cancer according to claim 1, wherein the nucleotide has a nucleotide sequence of SEQ ID NO: 1.
The kit for diagnosing or prognosticating pancreatic cancer according to claim 1, wherein the embjin protein has an amino acid sequence of SEQ ID NO: 2.
delete The method of claim 1, wherein the binding agent is an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer. Or a prognostic assay kit.
The kit for diagnosing or prognosticating pancreatic cancer according to claim 1, wherein the kit is an immunoassay kit.
The kit for diagnosing or prognosticating pancreatic cancer according to claim 1, wherein the kit is a microarray.
The kit for diagnosing or prognosticating pancreatic cancer according to claim 1, wherein the kit is a gene amplification kit.
Characterized in that the expression of the nucleotide sequence of embjin protein or embycin in a biological sample isolated from a human is detected by a method for providing information necessary for pancreatic cancer diagnosis or prognosis analysis.
delete 10. The method of claim 9, wherein the biological sample is human tissue, cell, blood, serum or plasma.
(a) treating the test substance with a cell comprising an embjin-encoding nucleotide sequence; And (b) analyzing the expression of the nucleotide sequence or the expression of the embedding protein. When the test substance inhibits the expression of the nucleotide sequence or the expression of the embedding protein, It is judged as a cure.
delete SiRNA, shRNA, miRNA, ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, peptides, antibodies or aptamers targeting embjin as an inhibitor or inhibitor of embigin expression. A pharmaceutical composition for the prevention, treatment or inhibition of metastasis of pancreatic cancer comprising as an active ingredient. delete

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