KR101492024B1 - Markers for diagnosis of metastatic brain tumor - Google Patents

Abstract

The present invention relates to a marker capable of diagnosing metastatic brain tumors, a diagnostic composition using the same, a method of diagnosing or predicting metastatic brain tumors, and a method of selecting drugs capable of inhibiting the expression or activity thereof.
Specifically, there is provided a method for diagnosing or predicting metastatic brain tumors, a method for diagnosing or diagnosing metastatic brain tumors, a method for diagnosing or diagnosing metastatic brain tumors, at least one metastatic brain tumor marker selected from the group consisting of MELK gene, protein encoded from MELK gene, Vimentin gene and Viemntin gene, And a method for screening a drug capable of inhibiting the expression or activity thereof.

Description

[0002] Markers for diagnosis of metastatic brain tumors [

The present invention relates to a marker capable of diagnosing metastatic brain tumors, a diagnostic composition using the same, a method of diagnosing or predicting metastatic brain tumors, and a method of selecting drugs capable of inhibiting the expression or activity thereof.

Brain tumors are tumors that occur abnormally within the palatine bone, and are largely divided into those caused by other causes, and primary or metastatic (secondary) brain tumors, depending on the cause. Primary brain tumors originate from cells in the brain and surrounding structures, whereas metastatic (secondary) brain tumors occur when cancer cells spread from the other parts of the body (for example, lungs, kidneys, breast, skin) to the brain. Primary brain tumors have many forms and can divide the shape of the tumor depending on the cells that have been started. For example, astrocytomas originate from astrocytes, and astrocytes, the most common cell types in the brain, support neurons (nerve cells). Primary brain tumors rarely spread out of the central nervous system.

Metastatic brain tumors that metastasize from the outside of the brain, such as the lung, breast, or melanoma, are systemic and account for more than half of all brain tumors and are one of the most common tumors of the intracranial tumors. In the case of metastatic brain lesions, histologic findings of the tumor represent the features of the primary site, but symptoms such as headache, nausea, muscle weakness and cognitive functioning may manifest themselves as in primary brain tumors. Therefore, the treatment depends on the clinical characteristics of the patient. However, first the radiation therapy to prevent the increase of the pressure of the brain and to inhibit the growth of the tumor is preceded and the chemotherapy of chemotherapy is performed according to the cancer of the primary lesion after the metastasis .

One third of patients with metastatic brain tumors do not have a history of cancer (National Cancer Center, treatment of metastatic brain tumors). Confirmation of metastatic brain tumors is possible only if the origin cancer has been confirmed histologically or after biopsy do. In 16-35% of patients with metastatic brain tumors, there is a statistic that brain lesions are found first without systemic cancer. Most of the patients with metastatic brain tumors are systemically unable to control because of the large spread of cancer, which may limit their lifespan. Unfortunately, most metastatic brain tumors do not respond well to radiation and chemotherapy, and the degree of metastasis of primary cancer is crucial in determining treatment options.

Metastatic brain tumors may develop months or even years after the treatment of primary cancer, so the diagnosis rate for treatment is very low. Therefore, it is urgent to develop a new method for diagnosing or predicting the metastasis of brain tumors in order to increase the survival rate and accelerate the treatment period in the absence of such primary cancer or in the case of the prognosis of primary cancer.

Recently, a diagnostic method using a tumor marker for early diagnosis of cancer has been developed. In the case of a tumor marker, it is often distinguished from a specific cancer cell in the body. Therefore, when the tumor marker is abnormally overexpressed, . However, tumor markers vary in their types depending on the location of cancer, and there are no markers that can identify all types of cancer.

Blot J. et al., Dev. Biol. 2002, 241 (2): 327-338 Davezac N. et al., Oncogene, 2002, 21: 7630-7641 Dmitry V. et al., Nat. Cell Biol., 2003,5: 545-551 Hyun-A Seoung et al., J. Biol. Chem., 2003, 278: 9655-9662 Ichiro Nakano et al., J. Neurosci. Res., 2008, 86: 48-60; Haiyoung Jung et al., J. Biol. Chem., 2008, 283: 34541-34553

The present invention aims at finding new markers capable of accurately and promptly diagnosing metastatic brain tumors or predicting whether or not they metastasize. The present invention aims to use this marker for a new diagnostic method for metastatic brain tumors and further to select drugs that inhibit the expression or activity of these markers to be used for research and development of target cancer drugs.

The present invention relates to a marker for diagnosing at least one metastatic brain tumor selected from the group consisting of a MELK gene, a protein encoded from a MELK gene, a Vimentin gene, and a protein coated from a Viemntin gene. Preferably, MELK has the amino acid sequence of SEQ ID NO: 1 and Vimentin has the amino acid sequence of SEQ ID NO: 2.

The present invention also relates to a composition for the diagnosis of metastatic brain tumor comprising an agent for measuring the level of one or more genes or proteins from the group consisting of MELK gene, protein encoded from MELK gene, Vimentin gene and protein coated from Vimentin gene. Preferably, the substance measuring the level of the MELK or Vimentin gene may be a primer or a probe capable of amplifying the gene, and the substance measuring the level of the MELK or Vimentin protein may be an antibody that specifically recognizes the protein .

The present invention also provides a method of diagnosing metastatic brain tumor comprising the steps of: a) measuring the expression level or amount of at least one of MELK gene, Vimentin gene, MELK protein and Vimentin protein present in a biological sample; and b ) Comparing the measurement result in the step (a) with the expression amount of the MELK or Vimentin gene of the normal control sample or the amount of the protein, to a method for diagnosing or predicting metastatic brain tumors. The biological sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine, and the measurement may be performed using a reverse transcriptase-polymerase chain reaction, a radioimmunoassay (RIA), a radioimmunodiffusion, and an immunoprecipitation assay (ELISA), a real time polymerase chain reaction (PCR), Western blot, northern blot, enzyme linked immunosorbent assay Can be selected. Preferably, in the method of diagnosing, it may be determined that the metastatic brain tumor is diagnosed when the expression amount of MELK or Vimentin gene present in the biological sample or the amount of protein is increased as compared with the control.

Also, the present invention provides a method for preparing a cell line, comprising the steps of: a) treating a test substance with an MELK or Vimentin expressing cell line; b) measuring the expression level of MELK or Vimentin in the cell line; And c) selecting a substance for preventing or treating metastatic brain tumor that inhibits the expression of MELK or Vimentin, wherein the degree of expression of MELK or Vimentin is lower than that of a control without treatment of the test substance ≪ / RTI > Preferably, the degree of expression of step b) can be measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), Western blot and RT-PCR.

The present invention also relates to a method for the preparation of a pharmaceutical composition comprising the steps of: a) treating a test substance with MELK or Vimentin; b) measuring the activity of the MELK or Vimentin; And c) selecting a substance for preventing or treating metastatic brain tumor that inhibits the activity of MELK or Vimentin, comprising the step of selecting the test substance whose activity level of MELK or Vimentin is decreased compared to the control group not treated with the test substance ≪ / RTI > Preferably, the degree of activity of step b) can be measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, protein chip, enzyme substrate staining and antigen-antibody aggregation .

The present invention also relates to a method for the diagnosis of metastatic brain tumor comprising a composition for diagnosing metastatic brain tumor comprising a substance measuring at least one level from the group consisting of MELK gene, protein encoded from MELK gene, protein encoded by Vimentin gene and protein from Vimentin gene Or a prediction kit. Preferably these kits can be RT-PCR kits, DNA microarray chip kits or protein chip kits.

Maternal embryonic leucine zipper kinase (MELK) is a type of serine / threonine kinase that is activated by adenosine monophosphate and plays an important role in various cancers. MELK was first found in embryos of African clawed frogs (Xenopus) and is known to maximize its amount or activation in the mitotic phase of the cell cycle. This phenomenon has also been confirmed in vertebrate cells (Blot J. et al., Dev. Biol. 2002, 241 (2): 327-338; Davezac N. et al., Oncogene , 2002, 21: 7630-7641). As a protein that is phosphorylated by MELK, CDC25 is known to be involved in the cell cycle (Dmitry V. et al., Nat. Cell Biol., 2003, 5: 545-551), which phosphorylates the transcription protein ZPR9 (Hyun-A Seoung et al., J. Biol. Chem., 2003, 278: 9655-9662). Recently, it has been shown that the expression of MELK is increased in the tissues of patients with breast cancer or adventitia cancer, and inhibiting the expression of MELK alone has been shown to inhibit the growth of cancer cells (Ichiro Nakano et al., J. Neurosci. Res , 2008, 86: 48-60; Haiyoung Jung et al., J. Biol. Chem., 2008, 283: 34541-34553).

Viemntin is a type III intermediate fiber protein expressed in mesenchymal cells and is a major component of cytoskeleton of mesenchymal cells. Vimentin plays an important role in supporting and fixing the location of cytoplasmic organelles. Vimentin is attached to the sides or ends of nuclei, vesicles and mitochondria. The dynamic nature of vimentin plays an important role in the flexibility of cells, and recent scientists have found that under vivo mechanical stress, they provide elasticity in cells in the absence of microtubule or actin filament networks. Therefore, Vimentin is considered to be an important cytoskeletal component in maintaining cell integrity. (Goldman RD, Khuon S., Chou Y., Opal P., Steinert P. 1996. "The function of intermediate filaments in cell shape and cytoskeletal integrity" J Cell Biol 134 (4): pp. 97183.)

In studies of transgenic mice lacking vimentin, these mice were functionally normal. This suggests that the micropipe network can compensate for the absence of the intermediate network, suggesting that the microtubules and Vimentin interact closely. Vimentin plays an important role in stabilizing cell morphology, cytoplasmic integrity and cytoskeletal interactions. It has also been shown that vimentin regulates the transport of low density lipoproteins (LDL) (Sarria AJ, Panini SR, Evans RM (1992). "A functional role for vimentin in the metabolism of lipoprotein-derived cholesterol in human SW-13 cells ". J Biol Chem 267 (27): pp. 1945563) Blocking cholesterol-derived LDL transport in cells resulted in lower amounts of lipoproteins than normal cells with Vimentin.

The information of the genes is registered in Genebank and is MELK's Acc. No. NM_014791 and is specified by the amino acid sequence of SEQ ID NO: 1. The registration number of Vimentin is NM_003380, which is specified by the amino acid sequence of SEQ ID NO: 2.

The amino acid sequence of NELK (NP 055606) is as shown in Fig. 6 (a).

The amino acid sequence of Vimentin (NP 003371) is as shown in Figure 6 (b).

The inventors of the present invention constructed a cell in which MELK was selectively overexpressed and introduced a protein expression vector that reacted with doxycycline and introduced a vector system in which the amount of MELK expression was regulated by the doxycycline protein (Tet-MELK-C4) was constructed to induce MELK expression in the presence of Doxycycline. In the present invention, it was confirmed that the protein expression of MELK was increased after 6 hours after treatment with 200 ng / ml Doxycycline, and it was confirmed that the increased MELK protein was retained for 48 hours. After treatment of 200 ng / ml Doxycycline for 24 h with tet-MELK-C4 cells, proteins were separated and subjected to two-dimensional protein analysis to analyze proteins increased by MELK as compared to tetoxelecine-untreated tet-MELK-C4 cells .

As a result, Vimentin protein was increased in MELK - enhanced cells and this result was consistent with Western blotting results.

The inventors analyzed the genes specifically expressed in cells extracted from patients with metastatic brain tumors and found that MELK and Vimentin were specifically expressed, leading to the present invention.

As can be seen in FIG. 1, the expression of MELK and Vimentin is specifically elevated after 24 hours in comparison with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), which is a control gene in the present invention.

The above results show that the genes of MELK or Vimentin or the proteins of these genes can be markers specific for the diagnosis of metastatic brain tumors. Little is known about this effect. Therefore, the present inventors have developed at least one metastatic brain tumor diagnostic marker selected from the group consisting of MELK gene, protein encoded from MELK gene, Vimentin gene and Viemntin gene, and the marker is one embodiment of the present invention to provide. Preferably, MELK has the amino acid sequence of SEQ ID NO: 1 and Vimentin has the amino acid sequence of SEQ ID NO: 2.

Another aspect of the present invention relates to a metastatic brain tumor diagnostic composition comprising an agent for measuring the level of at least one gene or protein selected from the group consisting of MELK gene, protein encoded from MELK gene, protein Vimentin gene and protein coated from Vimentin gene .

Specifically, the expression level of the gene can be detected at the mRNA level or the protein level, and the separation of the mRNA or protein in the biological sample can be performed using a known process.

A primer or a probe can be used to measure the mRNA level of a gene, and a primer or a probe that specifically amplifies a specific region can be designed by those skilled in the art using the sequence of gene information (Genebank).

The primers of the present invention can form a base pair with a complementary template with a nucleic acid sequence having a short free 3 'hydroxyl group and function as a starting point for template strand copying Quot; means a short nucleic acid sequence. In the present invention, the degree of expression can be measured at the mRNA level of the sample using a sense or antisense primer of MELK or Vimentin polynucleotide.

In the present invention, a probe utilizes a substance that specifically binds to a complementary base sequence of a nucleic acid, such as an oligonucleotide, a DNA or RNA probe, or the like. In the present invention, the level of expression can be measured using a probe complementary to polynucleotide of MELK or Vimentin.

The production or selection of the primer or probe can be easily performed by those skilled in the art and can be modified using known conditions.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods, and such nucleic acid sequences can also be modified using many means known in the art . Examples include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides into homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, Mate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.).

In the present invention, the measurement of the protein expression level is a process of confirming the presence and the degree of expression of a protein expressed in a marker gene, and it is an antigen-antibody binding reaction or a protein-ligand binding reaction that specifically binds to the protein of the gene (ELISA), radioimmunoassay (RIA), mass spectrometry, protein chip, fluorescence immunoassay, enzyme substrate colorimetry, and the like. Method, antigen-antibody aggregation method, and the like, and the method is not limited thereto.

In the present invention, an antibody refers to a specific protein molecule which is known in the art and directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to MELK or Vimentin, which is a marker of the present invention. These antibodies can be obtained by cloning each gene into an expression vector according to a conventional method, To obtain a protein to be coded, and can be produced from the obtained protein by a conventional method.

An antibody of the invention comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

Through these detection methods, it is possible to diagnose the actual cancer incidence in patients with suspected metastatic brain tumor by comparing the level of gene expression in the normal control group with the gene expression level in the suspected patients with metastatic brain tumor. That is, the expression levels of the marker of the present invention are measured from cells presumed to be metastatic brain tumors, the expression level of the marker of the present invention is measured from normal cells, and the expression level of the marker of the present invention is compared with that of normal cells And more likely to be expressed in metastatic brain tumors than in those that are presumed to be metastatic brain tumors.

Accordingly, another aspect of the present invention is a method for diagnosing metastatic brain tumor comprising the steps of: a) measuring the expression level or amount of at least one of MELK gene, Vimentin gene, MELK protein and Vimentin protein present in a biological sample , And b) comparing the measurement result in step a) with an expression amount or protein amount of the MELK or Vimentin gene of a normal control sample, to diagnose or predict a metastatic brain tumor.

The present invention relates to a method for detecting MELK or Vimentin by comparing the expression level of MELK or Vimentin with the expression level of normal cells from a patient's sample in order to provide information necessary for diagnosis of metastatic brain tumor. In the present invention, a sample of a patient includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, etc. in which the expression level of marker gene MELK or Vimentin is different.

As described above, a method for confirming the expression level of MELK or Vimentin is to use an antibody, a primer or a probe specific for a protein encoded by mRNA or gene of MELK or Vimentin gene. Methods for analysis include reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, Western blot, northern blot, enzyme linked immunosorbent assay (ELISA) But are not limited to, radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.

The metastatic brain tumor marker of the present invention can be a target of a new drug and can be utilized for various drug development. Accordingly, another aspect of the present invention is a method of treating a test substance comprising: a) treating a test substance with an expression cell line of MELK or Vimentin; b) measuring the expression level of MELK or Vimentin in the cell line; And c) selecting a substance for preventing or treating metastatic brain tumor that inhibits the expression of MELK or Vimentin, wherein the degree of expression of MELK or Vimentin is lower than that of a control without treatment of the test substance Method. The degree of expression of the step b) can be measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), Western blot and RT-PCR.

Specifically, the method can be useful for screening a therapeutic agent for metastatic brain tumors by comparing the increase or decrease in the expression of MELK or Vimentin in the presence or absence of a candidate for metastatic brain tumor treatment. Materials that indirectly or directly reduce the concentration of MELK or Vimentin may be selected as therapeutic agents for metastatic brain tumors. That is, the expression level of the marker MELK or Vimentin of the present invention in metastatic brain tumor cells is measured in the absence of a candidate for metastatic brain tumor treatment and the expression level of the marker MELK or Vimentin of the present invention is measured in the presence of a metastatic brain tumor candidate And then compare the quantities to predict the substance as a therapeutic agent for metastatic brain tumors in which the level of expression of the marker of the present invention in the presence of metastatic brain tumor treatment candidate is lower than the level of marker expression in the absence of metastatic brain tumor treatment candidate.

Further, another aspect of the present invention is a method for preparing a test substance, comprising the steps of: a) treating a test substance with MELK or Vimentin; b) measuring the activity of the MELK or Vimentin; And c) selecting a substance for preventing or treating metastatic brain tumor that inhibits the activity of MELK or Vimentin, comprising the step of selecting the test substance whose activity level of MELK or Vimentin is decreased compared to the control group not treated with the test substance Method. Preferably, the activity level of step b) can be measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, protein chip, enzyme substrate staining and antigen-antibody aggregation.

Specifically, the present invention relates to a screening method for a therapeutic agent for metastatic brain tumors by selecting a substance that inhibits its activity at the level of a protein encoded by MELK or Vimentin gene after administration of a substance expected to treat metastatic brain tumor.

In another aspect, the present invention relates to a kit for diagnosing or predicting metastatic brain tumors comprising the composition for diagnosing metastatic brain tumors.

The kit of the present invention can detect the marker by confirming the expression level of MELK or Vimentin, which is a metastatic brain tumor diagnostic marker, or the expression level of mRNA or protein. The kit of the present invention may include one or more other component compositions, solutions or devices suitable for the assay, as well as antibodies recognizing primers, probes or optionally markers for measuring the level of expression of metastatic brain tumor diagnostic markers .

As a specific example, the kit for measuring the mRNA expression level of MELK in the present invention may be a kit containing essential elements necessary for performing RT-PCR. When a PCR amplification reaction is applied, the reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus Pfu), DNA polymerase joins, and dNTPs.

Also preferably, the kit of the present invention can be a diagnostic kit containing essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

Further, in a metastatic brain tumor diagnostic kit comprising an antibody specific for MELK or Vimentin of the present invention, the anti-MELK or Vimentin antibody may be biotin, biotin derivative having substantially the same binding action to biotin against avidin or streptavidin And a visualization conjugate conjugated with a chromophore, a coloring material or a fluorescent molecule bound to the ligand-specific binding molecule is bound to the ligand, but not limited thereto. Do not.

Methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase, competitive reverse transcriptase polymerase, real-time reverse transcriptase polymerase, RNase protection assay, northern blotting, and DNA chip. Through these detection methods, it is possible to compare the mRNA expression amount in the normal control group and the mRNA expression amount in the suspected patients with metastatic brain tumor, and judge whether the expression level of the marker gene to mRNA is increased or not, Can be diagnosed.

The measurement of the mRNA expression level is preferably performed using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific to a gene used as a metastatic brain tumor marker. The above reverse transcriptase polymerase reaction can be confirmed by the electrophoresis after the reaction and the band pattern and the thickness of the band can be confirmed by comparing the mRNA expression level and the degree of the gene used as a diagnostic marker for metastatic brain tumor and comparing with the control group, Can be diagnosed easily.

On the other hand, a DNA chip uses a DNA chip in which a nucleic acid corresponding to the metastatic brain tumor marker gene or a fragment thereof is attached to a glass-like substrate at a high density. The DNA chip separates the mRNA from the sample, A cDNA probe can be prepared, hybridized to a DNA chip, and then detected for the occurrence of a metastatic brain tumor.

Analysis methods for measuring protein levels include Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips, and the like. Through these analytical methods, it is possible to compare the formation amount of the antigen-antibody complex in the normal control group with the amount of the antigen-antibody complex formed in the suspected patient of metastatic brain tumor, and the increase in the expression amount of the protein from the metastatic brain tumor marker gene , It is possible to diagnose the onset of metastatic brain tumor in patients with suspected metastatic brain tumor.

The antigen-antibody complex of the present invention refers to a combination of a metastatic brain tumor marker protein and an antibody specific thereto, and the formation amount of an antigen-antibody complex can be quantitatively measured through a signal label of a detection label.

Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re.

Measurement of protein expression levels is preferably performed using an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. The extent of complex formation of the metastatic brain tumor marker protein and antibody can be confirmed to identify the onset of metastatic brain tumors.

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

In addition, preferably, immunostaining is performed using one or more antibodies against the metastatic brain tumor marker. Normal colon epithelial tissues and tissues suspected to be metastatic brain tumors are harvested and fixed, and paraffin-embedded blocks are prepared by methods well known in the art. They are made into sections with a thickness of several micrometers and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.

Preferably, a protein chip is used in which one or more antibodies against the metastatic brain tumor marker are arranged at predetermined positions on the substrate and immobilized at a high density. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It is possible to confirm the onset of metastatic brain tumor.

The present invention can be usefully used for the treatment and prognosis of metastatic brain tumors by providing diagnostic markers of metastatic brain tumors which are difficult to predict or diagnose. The simple and rapid diagnosis of the metastatic brain tumor according to the present invention enables accurate and easy measurement of the presence or absence of the tumor to advance the treatment period and lower the current high mortality rate.

In addition, these markers can be used as targets for the research and development of target specific anticancer drugs and can be used for screening of various drugs.

Figure 1 compares intracellular expression of MELK and Vimentin with control.
MELK (maternal embryonic leucine zipper kinase); Vim (Vimentin); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)
FIG. 2 shows the result of investigation of migration assay.
FIG. 3 shows the result of measuring invasion assay.
Fig. 4 shows the results of the investigation of migration ability of U87-melk cells.
FIG. 5 shows the result of investigation of cell migration ability by scratch wound assay.
(a) Not treated with doxycycline (DOX)
(b) Treatment with 200 ng / ml of doxycycline (DOX)
6 shows the respective amino acid sequences of MELK and Viemntin.
(a) MELK, (b) Vimentin

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are illustrative only and are not intended to be limiting.

Example 1 Production of MELK Induced Expression Cell Lines

MELK expression vector was introduced into U87MG using Clontech's Tet-On 3G system. G418 and Hygromycine were each treated at a concentration of 50 ug / ml to stably maintain the desired vector in the cells. In the case of MELK-inducing cell lines, the amount of MELK protein can be increased by treating 200 ng / ml of Doxycyline. The increase of MELK protein after 6 hours after docycline treatment was observed and it was confirmed that it was maintained for more than 48 hours.

Example 2: Protein analysis by secondary electrophoresis

DMSO was treated as a control for the MELK inducible cell line, and Doxycycline 200 ng / ml for the MELK expression group. After 24 hours, the cells were washed with cold phosphate buffer saline (PBS) and treated with 10% TCA (Trichloroacetic acid) Next, the supernatant was discarded, the cell mass was suspended in acetone, subjected to ultrasonic disruption, and centrifuged. The separated proteins were dissolved in a cell lysis solution containing urea (8M Uream, 4% CHAPS, 40 mM Tris-HCl) and quantified. Quantified protein pellets were mixed with rehydration buffer (48% Urea, 2% CHPAS, 0.5% IPG) and separated between pH 3 and 10 on an IPG strip. The protein separated by pH was separated from the acrylamide gel, and the pH axis of the horizontal axis was separated by the protein size, and the protein with the change in the comparison with the control group was selected and separated from the acrylamide gel. The isolated proteins were treated with trypsin to identify proteins in MALD-TOF TOF instruments.

Example 3: Cell culture

(GIBCO BRL., Gaithersburg, MD, USA), 50 ug / ml and 1% penicillin / streptomycin (GIBCO BRL. (GIBCO BRL., Gaithersburg, MD, USA) containing penicillin / streptomycine (GIBCO BRL., Gaithersburg, MD, USA)

Example 4: Western blotting < RTI ID = 0.0 >

U87MG cells were removed from the incubator at 37 ° C, and the medium was removed and washed twice with PBS solution. U87MG cells were collected with a scrapper and centrifuged at 2500 rpm for 5 minutes. After the supernatant was removed, the pro-prep solution was mixed with the pellet in a ratio of 1: 1, mixed well, and left on ice for 10 minutes. After centrifugation at 13000 rpm for 10 minutes, protein was quantified using supernatant. 30 μg of protein, sample buffer, reducing agent, and pro-prep solution were mixed in a new container, left at 70 ° C for 10 minutes, and then cooled on ice. 200 ml of Mops SDS buffer and 500 μl of antioxidant were placed inside the dams of the electrophoresis tank, and 600 ml of Mops SDS buffer was filled in the outside of the dam. 10 μl of the mixed solution was added to each well of the gel and electrophoresed at 100-150 V. The electrophoresed gel was transferred to a PVDF membrane, and the obtained membrane was placed in a blocking buffer and left at 80 rpm for about 1 hour. The primary antibody was diluted 1: 1000 in the blocking buffer and left at 4 캜 for about 24 hours. The PVDF membrane was washed 6 times with 0.1% TBST at 120 rpm for 10 minutes. HRT fluorescently labeled secondary antibodies were diluted 1: 2000 in 0.1% TBST and left at 80 rpm for 1 hour. The PVDF membrane was washed 6 times with 0.1% TBST at 120 rpm for 10 minutes. The ECL solution (Amersham) was uniformly applied to the PVDF film and developed on the film in the dark room.

Example 5: Migration Assay (Cell Biolabs Inc, San Diego, CA, USA)

A 96-well mobile plate was left at room temperature for 10 minutes. U87MG cell suspension was prepared at a concentration of ³ × 10 ⁴ cells / ml in serum-free medium. The compounds were treated with the cell suspension to the desired concentration. A plate cover and a membrane chamber were separated from the 96-well mobile plate. In each well of the feeder tray, 150 μl of DMEM medium containing 10% fetal bovine serum was added. I put the membrane chamber back onto the feeder tray. 100 μl of the pre-prepared U87MG cell suspension was placed in the membrane chamber. The plate cover was covered and incubated at 37 ° C for 4 hours. Before the end of incubation, 150 μl of cell detachment solution was added to a 96-well cell harvesting tray. The 96 well mobile plate was removed from the incubator, the plate cover was opened, and the membrane chamber was carefully removed from the feeder tray. The membrane chamber was turned over and dipped in a towel to remove medium containing U87MG cells. The separated membrane chamber was carefully placed in a cell collection tray containing 150 μl of cell separation solution and incubated at 37 ° C for 30 minutes. The plate cover was covered with a cell collection tray with a membrane chamber, and lightly shaken and shaken to move the cells completely from the membrane interface to the cell separation solution. 4X dissolution buffer (Lysis buffer) and CyQuant GR dye were prepared at a ratio of 75: 1. 4X dissolution buffer and CyQuant GR staining solution was added to a 96-well plate containing 150 μl of cell separation solution in an amount of 50 μl per well, followed by incubation at room temperature for 20 minutes. 150 μl per well of the mixed solution was transferred to a 96-well plate (Griner) suitable for fluorescence measurement, and then measured at 480/520 nm (FIG. 2).

As shown in FIG. 2 showing the above results, it was confirmed that when Doxycycline was treated, the migration ability was remarkably increased.

Example 6 Invasion Assay (Cell Biolabs Inc, San Diego, CA, USA)

The 96-well invasion plate on sterile conditions was left at room temperature for 10 minutes. 100 μl of warm serum-free medium was rehydrated in the base membrane layer of the membrane chamber for 1 hour. U87MG cell suspension was prepared at a concentration of 1 × 10 ⁶ cells / ml in serum-free medium. The compounds were treated with the cell suspension to the desired concentration. And then shaken off with a hydrated paper towel. Separate the cover of the 96-well infiltration plate and the membrane chamber and add 150 μl of DMEM medium containing 10% fetal bovine serum to each well of the feeder tray. I put the membrane chamber back onto the feeder tray. 100 μl of the pre-prepared U87MG cell suspension was placed in the membrane chamber. The plate cover was covered and cultured at 37 ° C for 24 hours. Before the end of incubation, 150 μl of cell detachment solution was added to a 96-well cell harvesting tray. The 96 well mobile plate was removed from the incubator, the plate cover was opened, and the membrane chamber was carefully removed from the feeder tray. The membrane chamber was turned over and dipped in a towel to remove medium containing U87MG cells. The separated membrane chamber was carefully placed in a cell collection tray containing 150 μl of cell separation solution and incubated at 37 ° C for 30 minutes. The plate cover was covered with a cell collection tray with a membrane chamber, and lightly shaken and shaken to move the cells completely from the membrane interface to the cell separation solution. 4X dissolution buffer (Lysis buffer) and CyQuant GR dye were prepared at a ratio of 75: 1. 4X dissolution buffer and CyQuant GR staining solution was added to a 96-well plate containing 150 μl of cell separation solution in an amount of 50 μl per well, followed by incubation at room temperature for 20 minutes. 150 [mu] l per well of the mixed solution was transferred to a 96-well plate (Griner) suitable for fluorescence measurement, and then measured at 480/520 nm (Fig. 3).

The results are shown in Fig. 3, and the treatment with doxycycline showed a significantly increased infiltration ability of the cells.

Example 7: Scratch Assay

Cell mobility was determined using an Incucyte 96-well WoundMaker system (Essen Bioscience, Ann Arbor, MI, USA). (Figs. Methods and results were analyzed using Essen Bioscience. Imaging equipment was operated in a cell incubator and real-time measurements were performed.

The results are shown in Figs. 4 and 5, and it can be confirmed that the mobility of the cells is remarkably increased as a result of treatment with doxycyclin.

The foregoing description is merely illustrative of the technical idea of the present invention and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, and thus the scope of the technical idea of the present invention is not limited to this embodiment. The scope of protection of the present invention should be construed in accordance with the following claims and all technical ideas within the technical scope of the claims should be construed as falling within the scope of the present invention.

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Markers for diagonosis of metastatic brain tumor <130> P12111311498 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 651 <212> PRT <213> Homo sapiens <400> 1 Met Lys Asp Tyr Asp Glu Leu Leu Lys Tyr Tyr Glu Leu His Glu Thr   1 5 10 15 Ile Gly Thr Gly Gly Phe Ala Lys Val Lys Leu Ala Cys His Ile Leu              20 25 30 Thr Gly Glu Met Val Ala Ile Lys Ile Met Asp Lys Asn Thr Leu Gly          35 40 45 Ser Asp Leu Pro Arg Ile Lys Thr Glu Ile Glu Ala Leu Lys Asn Leu      50 55 60 Arg His Gln His Ile Cys Gln Leu Tyr His Val Leu Glu Thr Ala Asn  65 70 75 80 Lys Ile Phe Met Val Leu Glu Tyr Cys Pro Gly Gly Glu Leu Phe Asp                  85 90 95 Tyr Ile Ile Ser Gln Asp Arg Leu Ser Glu Glu Glu Thr Arg Val Val             100 105 110 Phe Arg Gln Ile Val Ser Ala Val Ala Tyr Val His Ser Gln Gly Tyr         115 120 125 Ala His Arg Asp Leu Lys Pro Glu Asn Leu Leu Phe Asp Glu Tyr His     130 135 140 Lys Leu Lys Leu Ile Asp Phe Gly Leu Cys Ala Lys Pro Lys Gly Asn 145 150 155 160 Lys Asp Tyr His Leu Gln Thr Cys Cys Gly Ser Leu Ala Tyr Ala Ala                 165 170 175 Pro Glu Leu Ile Gln Gly Lys Ser Ser Tyr Leu Gly Ser Glu Ala Asp Val             180 185 190 Trp Ser Met Gly Ile Leu Leu Tyr Val Leu Met Cys Gly Phe Leu Pro         195 200 205 Phe Asp Asp Asp Asn Val Met Ala Leu Tyr Lys Lys Ile Met Arg Gly     210 215 220 Lys Tyr Asp Val Pro Lys Trp Leu Ser Pro Ser Ser Leu Leu Leu 225 230 235 240 Gln Gln Met Leu Gln Val Asp Pro Lys Lys Arg Ile Ser Met Lys Asn                 245 250 255 Leu Leu Asn His Pro Trp Ile Met Gln Asp Tyr Asn Tyr Pro Val Glu             260 265 270 Trp Gln Ser Lys Asn Pro Phe Ile His Leu Asp Asp Asp Cys Val Thr         275 280 285 Glu Leu Ser Val His His Arg Asn Asn Arg Gln Thr Met Glu Asp Leu     290 295 300 Ile Ser Leu Trp Gln Tyr Asp His Leu Thr Ala Thr Tyr Leu Leu Leu 305 310 315 320 Leu Ala Lys Lys Ala Arg Gly Lys Pro Val Arg Leu Arg Leu Ser Ser                 325 330 335 Phe Ser Cys Gly Gln Ala Ser Ala Thr Pro Phe Thr Asp Ile Lys Ser             340 345 350 Asn Asn Trp Ser Leu Glu Asp Val Thr Ala Ser Asp Lys Asn Tyr Val         355 360 365 Ala Gly Leu Ile Asp Tyr Asp Trp Cys Glu Asp Asp Leu Ser Thr Gly     370 375 380 Ala Ala Thr Pro Arg Thr Ser Gln Phe Thr Lys Tyr Trp Thr Glu Ser 385 390 395 400 Asn Gly Val Glu Ser Lys Ser Leu Thr Pro Ala Leu Cys Arg Thr Pro                 405 410 415 Ala Asn Lys Leu Lys Asn Lys Glu Asn Val Tyr Thr Pro Lys Ser Ala             420 425 430 Val Lys Asn Glu Glu Tyr Phe Met Phe Pro Glu Pro Lys Thr Pro Val         435 440 445 Asn Lys Asn Gln His Lys Arg Glu Ile Leu Thr Thr Pro Asn Arg Tyr     450 455 460 Thr Thr Pro Ser Lys Ala Arg Asn Gln Cys Leu Lys Glu Thr Pro Ile 465 470 475 480 Lys Ile Pro Val Asn Ser Thr Gly Thr Asp Lys Leu Met Thr Gly Val                 485 490 495 Ile Ser Pro Glu Arg Arg Cys Arg Ser Val Glu Leu Asp Leu Asn Gln             500 505 510 Ala His Met Glu Glu Thr Pro Lys Arg Lys Gly Ala Lys Val Phe Gly         515 520 525 Ser Leu Glu Arg Gly Leu Asp Lys Val Ile Thr Val Leu Thr Arg Ser     530 535 540 Lys Arg Lys Gly Ser Ala Arg Asp Gly Pro Arg Arg Leu Lys Leu His 545 550 555 560 Tyr Asn Val Thr Thr Thr Arg Leu Val Asn Pro Asp Gln Leu Leu Asn                 565 570 575 Glu Ile Met Ser Ile Leu Pro Lys Lys His Val Asp Phe Val Gln Lys             580 585 590 Gly Tyr Thr Leu Lys Cys Gln Thr Gln Ser Asp Phe Gly Lys Val Thr         595 600 605 Met Gln Phe Glu Leu Glu Val Cys Gln Leu Gln Lys Pro Asp Val Val     610 615 620 Gly Ile Arg Arg Gln Arg Leu Lys Gly Asp Ala Trp Val Tyr Lys Arg 625 630 635 640 Leu Val Glu Asp Ile Leu Ser Ser Cys Lys Val                 645 650 <210> 2 <211> 466 <212> PRT <213> Homo sapiens <400> 2 Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly   1 5 10 15 Gly Pro Gly Thr Ala Ser Arg Ser Ser Ser Ser Ser Tyr Val Thr              20 25 30 Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr          35 40 45 Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg      50 55 60 Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu  65 70 75 80 Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe                  85 90 95 Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp             100 105 110 Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn         115 120 125 Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser     130 135 140 Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln 145 150 155 160 Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp                 165 170 175 Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu             180 185 190 Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln         195 200 205 Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val     210 215 220 Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu 225 230 235 240 Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile                 245 250 255 Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val             260 265 270 Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu         275 280 285 Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg     290 295 300 Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg 305 310 315 320 Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr                 325 330 335 Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala             340 345 350 Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu         355 360 365 Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln     370 375 380 Asp Leu Asu Val Lys Met Aslan Leu Asp Ile Glu Ile Ala Thr Tyr 385 390 395 400 Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro                 405 410 415 Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro             420 425 430 Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu         435 440 445 Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp     450 455 460 Leu Glu 465

Claims (16)

At least one marker selected from the group consisting of a doxycyclin-treated MELK gene and a protein encoded from the treated MELK gene; And
At least one marker selected from the group consisting of a protein encoded by a Vimentin gene and a Vimentin gene;
Wherein the markers are selected from the group consisting of: &lt; RTI ID = 0.0 &gt;
2. The marker composition according to claim 1, wherein the MELK has the amino acid sequence of SEQ ID NO: 1.
2. The marker composition of claim 1, wherein the Vimentin has the amino acid sequence of SEQ ID NO: 2.
9. A metastatic brain tumor diagnostic composition comprising a substance that measures the level of the marker composition of claim 1.
5. The composition according to claim 4, wherein the substance measuring the level of MELK or Vimentin gene is a primer or a probe capable of amplifying a gene.
6. The composition of claim 5, wherein the substance that measures the level of the MELK or Vimentin protein is an antibody that specifically recognizes the protein.
A method for diagnosing metastatic brain tumor comprising the steps of: a) measuring the expression level or amount of a protein of any one of claims 1 to 3 present in a biological sample; and b) Comparing the expression level of the MELK or Vimentin gene of the normal control sample with the amount of the protein or the amount of the MELK or Vimentin gene in the normal control sample.
8. The method of claim 7, wherein the biological sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine.
8. The method of claim 7, wherein the measurement is performed using a reverse transcriptase-polymerase chain reaction, a real time-polymerase chain reaction, a Western blot, northern blot, an enzyme linked immunosorbent assay (ELISA) , Radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay. The method of claim 1,
8. The method according to claim 7, comprising the step of determining that metastatic brain tumor has occurred when the amount of expression of MELK or Vimentin gene or amount of protein present in said biological sample is increased as compared with the control.
a) treating the test substance with an expression cell line of the marker composition of claim 1; b) measuring the expression level of the marker composition in the cell line; And c) selecting a test substance whose degree of expression of the marker composition is lower than that of a control group not treated with the test substance, and a method for screening a substance for preventing or treating metastatic brain tumor suppressing the expression of MELK or Vimentin .
The method according to claim 11, wherein the degree of expression of step b) is measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), Western blot and RT-PCR Way.
a) treating the test substance with the marker composition according to any one of claims 1 to 3; b) measuring the activity of the marker composition; And c) selecting a test substance whose activity level of the marker composition is lower than that of a control group in which the test substance is not treated. A method for screening a substance for preventing or treating metastatic brain tumor suppressing the expression of MELK or Vimentin .
The method according to claim 13, wherein the activity level of step b) is measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, protein chip, enzyme substrate staining and antigen- . &Lt; / RTI &gt;
A kit for the diagnosis or prediction of a metastatic brain tumor comprising the composition of claim 4.
16. The kit according to claim 15, wherein the kit is an RT-PCR kit, a DNA microarray chip kit or a protein chip kit.

Images