WO2018004240A1 - Use of nupr1 in diagnosis and treatment of brain tumor - Google Patents
Use of nupr1 in diagnosis and treatment of brain tumor Download PDFInfo
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- WO2018004240A1 WO2018004240A1 PCT/KR2017/006783 KR2017006783W WO2018004240A1 WO 2018004240 A1 WO2018004240 A1 WO 2018004240A1 KR 2017006783 W KR2017006783 W KR 2017006783W WO 2018004240 A1 WO2018004240 A1 WO 2018004240A1
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- nupr1
- isoform
- expression
- brain
- brain tumor
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Definitions
- the present invention relates to the use of NUPR1 in the diagnosis and treatment of brain tumors. More specifically, the present invention relates to the prevention of brain tumors comprising a biomarker composition for diagnosing or prognostic brain tumors comprising NUPR1 (NUPR1) and a NUPR1 inhibitor or A therapeutic composition.
- NUPR1 NUPR1
- NUPR1 inhibitor or A therapeutic composition NUPR1 inhibitor or A therapeutic composition.
- Brain cancer is a generic term for primary brain cancers that occur in the brain tissue and the brain envelope that surrounds the brain, and secondary brain cancers that metastasize from cancers that occur in the skull or other parts of the body. Such brain cancers are often distinguished from cancers occurring in other organs.
- the lung, stomach, breast and other cancers are limited to one or two types of organs, and their properties are the same or similar.
- the brain develops a variety of cancers, including glioblastoma multiforme, malignant glioma, lymphadenomas, germ cell tumors, and metastatic tumors.
- the glioma is the most frequent tumor of primary brain.
- Meningioma is the most frequent of the brain's benign tumors with pituitary adenoma, accounting for about 15-20% of primary brain tumors. Pituitary adenoma accounts for about 10-15% of all brain tumors. Depending on the size of the tumor, it may be divided into microadenoma less than 10mm in diameter and giant adenoma greater than 10mm. Metastatic tumors account for 30 to 40% of all brain tumors, about 60% of which are observed as multiple masses, but about 40% to single masses. Therefore, it is difficult to differentiate between primary malignancies, especially glioblastoma, when they appear as a single mass. In Korea, brain cancer caused by lung cancer is the most common.
- the 2000 HHO taxonomy includes glioblastomas, including giant cell glioblastoma and gliosarcoma.
- the neuroeducoma (gliosarcoma) is a rare variant that contains 1.8-2.4% of glioblastoma and is clinically similar to glioblastoma. Because of the sarcomatous nature of neuroeducoma tumor cells, neuroedema is more aggressive and invasive because of its greater propensity to metastasize extracranial in neuroedema patients.
- NUPR1 particularly NUPR1 isoform A
- the inventors have found that NUPR1, particularly NUPR1 isoform A, is significantly increased in malignant brain tumor patients with poor prognosis among brain tumor patients, and has completed the present invention by confirming that the brain tumor can be treated by inhibiting it.
- An object of the present invention is to provide a biomarker composition for brain tumor diagnosis or prognosis prediction comprising NUPR1 (Nuclear protein 1).
- Another object of the present invention is a composition for diagnosing or prognosticing a brain tumor comprising a detection agent of NUPR1 (Nuclear protein 1), a kit for diagnosing or prognosticing a brain tumor comprising the composition, and for diagnosing or diagnosing a brain tumor using the composition It is to provide a method of providing information.
- NUPR1 Nuclear protein 1
- Still another object of the present invention is to provide a composition for preventing or treating brain tumors, including a NUPR1 (Nuclear protein 1) expression or activity inhibitor.
- NUPR1 Nuclear protein 1
- Still another object of the present invention is to provide a method for screening a brain tumor therapeutic agent using NUPR1 (Nuclear protein 1).
- the present invention provides a biomarker composition for brain tumor diagnosis or prognostic prediction comprising NUPR1 (Nuclear protein 1).
- the present invention provides a composition for diagnosing or predicting a brain tumor comprising a preparation for measuring the expression or activity level of NUPR1 (Nuclear protein 1).
- the present invention provides a kit for diagnosing or predicting a brain tumor comprising the composition.
- the present invention provides a method for providing information for diagnosing or predicting a brain tumor comprising a; measuring the mRNA expression of the NUPR1 gene or its protein activity level from a biological sample.
- the present invention provides a pharmaceutical composition for preventing or treating brain tumors, including an expression or activity inhibitor of NUPR1 (Nuclear protein 1).
- the present invention provides a food composition for preventing or improving brain tumors, including the expression or activity inhibitor of NUPR1 (Nuclear protein 1).
- the present invention also provides a method for preventing or treating brain tumors, comprising administering an expression or activity inhibitor of NUPR1 (Nuclear protein 1) to a subject in need thereof.
- NUPR1 Nuclear protein 1
- the present invention comprises the steps of (a) treating a brain tumor treatment candidate material to the isolated cells; And (b) measuring the mRNA expression of the NUPR1 gene or its protein activity level in the cell.
- NUPR1, in particular NUPR1 isoform A is significantly increased in brain tumor patients compared to healthy normal people, especially in patients with malignant brain tumors with a poor prognosis, which can be used as a biomarker for diagnosing brain tumors or predicting prognosis. have.
- it is possible to inhibit the growth, metastasis and invasion of brain tumors through the inhibition of NUPR1, in particular NUPR1 isoform A according to the present invention it can be used as a target for the treatment of brain tumors, it can be used in the screening of future brain tumor therapeutics. .
- 1 is a diagram confirming the protein expression of NUPR1 isoform A in normal and brain tumor-derived cells.
- Figure 2 is a diagram showing the brain MRI pictures of patients before obtaining the GBM-28 and GBM-37 samples (from left to T1, T2, T1-enhanced MRI).
- Figure 3 is a diagram showing the results of immunohistochemistry (H & E, GFAP, Vimentin) in brain tumor tissue obtained from GBM-28 (left) and GBM-37 (right) samples (X50).
- FIG. 5 shows the results of performing cEA DNA array by extracting total RNA from GBM-28 and GBM-37 samples, and performing GSEA (Gene Set Enrichment Analysis) using the transcripts of each sample. It is a figure confirming whether the type is a proneural subtype or a mesenchymal subtype.
- GSEA Gene Set Enrichment Analysis
- Figure 6 shows the result of performing the GSEA (Gene Set Enrichment Analysis) by using the total RNA extracted from the GBM-28 and GBM-37 samples obtained cDNA microarray data, using the transcript and specific functions of each sample Figure shows the results of analyzing the correlation between the set of genes representing.
- GSEA Gene Set Enrichment Analysis
- FIG. 7 shows MTS proliferation assay results of primary cultured cells obtained from GBM-28 and GBM-37 samples.
- FIG. 8 is a diagram showing the results of categorizing various disease and biological functions through IPA after extracting total RNA from GBM-28 and GBM-37 samples to obtain cDNA microarray data.
- Figure 9 shows the extraction of total RNA from GBM-28 and GBM-37 samples, followed by RT-PCR for NUPR1 total, NUPR1 isoform A (varient 1, v.1) and NUPR1 isoform B (varient 2, v.2) mRNAs.
- Figure 9 shows the results of analyzing the expression level.
- FIG. 10 is a diagram showing the results of analysis of the expression of the total NUPR1 and NUPR1 isoform A (varient 1, v.1) expression by Western blot after extracting the whole protein from the GBM-28 and GBM-37 samples.
- FIG. 11 is a diagram illustrating the expression level of NUPR1 isoform A in each group after dividing the primary glioblastoma patient group registered into TCGA (The Cancer Genome Atlas) into five subtypes.
- FIG. 12 is a diagram showing the result of analyzing the mRNA expression of NUPR1 isoform A through RT-PCR after extracting the whole RNA from cells derived from various brain tumors.
- FIG. 13 is a diagram showing the results of survival analysis after dividing the primary glioblastoma patient group registered in TCGA (The Cancer Genome Atlas) into high and low expression according to the degree of NUPR1 isoform A expression.
- FIG. 14 is a first screening of MGMP methylated patients among primary glioblastoma patient groups registered in The Cancer Genome Atlas (TCGA), and then divided into high and low expression according to NUPR1 isoform A expression. This figure shows the results of the survival analysis.
- TCGA Cancer Genome Atlas
- FIG. 15 is a primary screening of MGMP unmethylated patients among primary glioblastoma patients registered with The Cancer Genome Atlas (TCGA), and divided into high and low expression according to the degree of NUPR1 isoform A expression. It is a figure which shows the result of performing a survival analysis.
- TCGA Cancer Genome Atlas
- FIG. 16 shows clinical information of 79 primary glioblastoma patients who visited Seoul National University Hospital.
- FIG. 17 stains tissues of 79 primary glioblastoma patients who visited Seoul National University Hospital using antibodies against NUPR1 isoform A, and then divides the expression into high and low expression according to the expression level of NUPR1 isoform A. Representative tumor tissue from each group of patients is shown.
- FIG. 18 is a diagram showing the results of a survival analysis after dividing 79 primary glioblastoma patients at Seoul National University Hospital into high and low expression according to the degree of NUPR1 isoform A expression.
- NUPR1 isoform A isoform A
- NUPR1 isoform B is a diagram confirming the effects on the mRNA expression of NUPR1 isoform A and NUPR1 isoform B after transducing five siRNAs (# 1 to # 5) for NUPR1 isoform A into GBM-37 cells, respectively.
- NUPR1 isoform A protein after transfection into GBM-37 cells with five siRNAs (# 1 to # 5) for NUPR1 isoform A, respectively.
- siRNA-NUPR1 ⁇ isoform A into GBM-37 cells.
- siRNA-NUPR1 ⁇ isoform A into GBM-37 cells.
- Fig. 3 shows the transcript subtype changes before and after siRNA transfection.
- Figure 24 is transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a) and extracted the entire RNA to obtain cDNA microarray data, using the GSEA (Gene Set Enrichment Analysis) results
- GSEA Gene Set Enrichment Analysis
- FIG. 25 is transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a) and then extracted the entire RNA to obtain cDNA microarray data, using the GSEA (Gene Set Enrichment Analysis) results
- Figure 3 shows the expression changes of related gene sets such as Inflammatory Response and Reactive Oxygen Species Pathway before and after siRNA transfection.
- FIG. 26 transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a), extracted the entire RNA to obtain cDNA microarray data, and then used GSEA (Gene Set Enrichment Analysis) The results of the experiments were shown to confirm the expression changes of related gene sets such as Chemokine Signaling Pathway and IL6-JAK-STAT3 before and after siRNA transfection.
- GBM-37_siNUPR1a GBM-37 cells
- FIG. 27 is a diagram showing the results of H & E staining of brain tumor tissues generated after injection of GBM-37 cells transformed with siRNA (siRNA NUPR1v.1) for scRNA or NUPR1 isoform A into mice.
- siRNA siRNA NUPR1v.1
- FIG. 28 is a view showing the enlarged observation result of the brain tumor tissue of FIG. 27.
- FIG. 29 shows the injection of GBM-37 cells transformed with siRNA (siRNA-NUPR1 ⁇ ) for scRNA (CTL) or NUPR1 isoform A into mice, followed by extraction of whole protein from the resulting brain tumor tissues, and NUPR1 via Western blot. It is a figure which shows the result of analyzing the degree of homozygous A expression.
- the present invention provides a biomarker composition for diagnosing or predicting a brain tumor comprising NUPR1 (Nuclear protein 1).
- NUPR1 Nuclear protein 1
- NUPR1 Nuclear protein 1
- the NUPR1 (Nuclear protein 1) includes NUPR1 isoform A and NUPR1 isoform B, preferably NUPR1 isoform A.
- prognosis refers to prediction of progression and recovery of a condition and refers to prospective or preliminary evaluation.
- the prognosis means prediction of treatment success, survival, recurrence, metastasis, etc. in the subject after treatment of brain tumor patients, and preferably means prognosis of survival.
- a “biomarker” is a substance capable of predicting a brain tumor diagnosis or prognosis, and can be diagnosed by discriminating whether a brain tumor occurs in a biological sample, and used as a prognostic factor of a brain tumor individual having a poor prognosis. It can be used in organic biomolecules such as polypeptides, nucleic acids (e.g. mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) with significant differences between normal and brain tumor individuals. And the like.
- the biomarker is NUPR1, which is increased in brain tumor individuals as compared to normal individuals, especially in malignant brain tumor patients with poor prognosis, and the lower the expression level, the better the prognosis factor.
- brain tumor is a tumor occurring in the brain and the central nervous system and can be used in combination with “brain cancer", the most representative disease of the brain tumor and mixed with a high frequency "glioma” (glioma) Can be used.
- the brain tumors collectively refer to primary brain tumors that arise from brain tissue or the brain envelope that envelops the brain, and secondary brain tumors that originate in the skull or surrounding structures, or that metastasize to the brain from other cancers of the body. Brain tumors may also be classified by cell type, morphology, cytogenetics, molecular genetics, immunologic markers, or a combination thereof, and may be classified by the World Health Organization for brain tumors. Classification classifies central nervous system tumors on a scale of malignancy based on the histological characteristics of the tumor.
- brain tumors include glioblastomas, glioblastoma, anaplastic astrocytomas, oligodendrogliomas, ependymomas, and low-grade astrocytomas.
- brain tumors can be divided into four subtypes in molecular aspect, and more specifically, proneural based on clinical values such as DNA methylation pattern, degree of signal transduction activation, survival and therapeutic response.
- subtype mesenchymal subtype, classic subtype, or neural subtype.
- the proneural subtype includes the G-CIMP subtype, and recently, there are also cases of dividing it into five subtypes.
- the prognosis of the proneural subtype is known to be the best, and the prognosis of the mesenchymal subtype is known to be the worst, and the NUPR1, in particular, the NUPR1 isoform according to the present invention.
- NUPR1 in particular NUPR1 isoform A
- A is characterized by a higher expression pattern in the mesenchymal subtype than the systemic subtype. That is, NUPR1, in particular NUPR1 isoform A, according to the present invention is significantly increased in brain tumor individuals with poor prognosis among brain tumor individuals suffering from the same disease, and can not only diagnose brain tumors but also distinguish subtypes. Prognosis can be used.
- the present invention provides a composition for diagnosing brain tumor or predicting prognosis comprising an agent for measuring the expression or activity level of Nuclear protein 1 (NUPR1).
- NUPR1 Nuclear protein 1
- NUPR1 particularly NUPR1 isoform A
- Diagnosis or prognosis of brain tumors is possible by measuring mRNA expression of isoform A gene or its protein activity level.
- "measurement of mRNA expression level of NUPR1 gene” is a process of confirming the presence and expression level of a biomarker gene in a biological sample for diagnosis or prognosis of brain tumor.
- the amount of mRNA is measured using the agent used in the measurement method.
- the agent for measuring the mRNA level of the NUPR1 gene in the present invention is preferably an antisense oligonucleotide, primer pair or probe, since the base sequence of the gene encoding the NUPR1 is registered in the gene bank, those skilled in the art Based on this, antisense oligonucleotides, primer pairs or probes can be designed that specifically amplify specific regions of these genes.
- a "primer” is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and starts for copying the template. By a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
- the primer means all primers that can be used for amplification of the NUPR1 gene, and the sequence of the primer is not limited as long as it can bind and amplify complementarily with the gene.
- the term "probe” refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which is capable of specific binding with mRNA. You can check the presence.
- the probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, and the like, as long as the probe may complementarily bind to the gene.
- the sequence of is not limited.
- Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidates, carbamates, etc.) or charged linkages (eg, phosphorothioates, phosphorodithioates, etc.).
- NUPR1 protein activity level measurement is a process of confirming the presence and expression level of the marker protein in a biological sample for the diagnosis or prognosis of brain tumor, preferably binding specifically to the NUPR1 protein Antibodies can be used to determine the amount of protein.
- an antibody refers to a specific protein molecule directed to an antigenic site as it is known in the art.
- an antibody refers to an antibody that specifically binds to NUPR1, which antibody is cloned into an expression vector according to a conventional method to obtain a protein encoded by the gene, and from the protein obtained It can be prepared by the phosphorus method.
- partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids.
- the form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
- Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
- the functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.
- the present invention provides a kit for diagnosing or predicting a brain tumor comprising the composition.
- the kit for measuring the mRNA expression level of the NUPR1 gene in the present invention may be a kit containing the necessary elements necessary to perform RT-PCR.
- the RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water and the like.
- the kit of the present invention may be a kit including essential elements necessary for performing the microarray chip.
- the microarray chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached by a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof, using the marker of the present invention. It can be easily prepared by the manufacturing method commonly used in the art.
- the substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto.
- the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
- the kit for measuring the NUPR1 protein activity level in the present invention may include a substrate, a suitable buffer, a secondary antibody labeled with a coloring enzyme or a fluorescent substance, and a coloring substrate for immunological detection of the antibody.
- the substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like.
- the chromophore may be a peroxidase or an alkaline force.
- Fatase alkaline phosphatase
- the fluorescent material may be used FITC, RITC, etc.
- the color substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) ))
- OPD o-phenylenediamine
- TMB tetramethyl benzidine
- the present invention provides a method for providing information for diagnosing or predicting a brain tumor, including measuring mRNA expression of a NUPR1 gene or a protein activity level thereof from a biological sample.
- the expression of mRNA or protein activity of NUPR1, particularly NUPR1 isoform A gene is increased in tissues in brain tumor patients, especially in patients with malignant brain tumors having a short survival time due to poor prognosis. MRNA expression of the NUPR1 gene or its protein activity was found to be significantly higher. Thus, expression analysis of NUPR1 in biological samples can provide information for diagnosing brain tumors or predicting prognosis.
- the "information providing method for diagnosis or prognosis prediction” is a preliminary step for diagnosis or prediction of prognosis and provides objective basic information necessary for the diagnosis or prognosis of brain tumor and the clinical judgment or findings of a doctor. Is excluded.
- biological sample means a direct subject separated from an individual and measuring the expression level of a gene or protein of interest, and a sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine. And the like.
- a sample of the present invention may be used to predict clinical outcome after treatment, i.e., recurrence and survival prognosis, from a sample for diagnosing the onset of a brain tumor in a suspicious brain tumor or from a subject undergoing surgical or chemical treatment for a brain tumor.
- the sample preferably brain tissue or cells isolated therefrom are not limited thereto.
- the "analytical method for measuring mRNA expression level” includes a polymerase reaction (PCR), a reverse transcriptase polymerase reaction (RT-PCR), a competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), a real time reverse transcriptase polymerase.
- PCR polymerase reaction
- RT-PCR reverse transcriptase polymerase reaction
- Competitive RT-PCR competitive reverse transcriptase polymerase reaction
- Realtime RT-PCR RNase protection assay (RPA), northern blotting, or DNA microarray analysis.
- the "analytical method for measuring protein activity level” includes western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, and ouktero. Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis, but is not limited thereto.
- the method of the present invention may comprise comparing the measured mRNA expression or protein activity level of the NUPR1 gene with the level measured in the control.
- the control group includes a negative control group (normal group) or a positive control group (brain tumor patient group).
- the positive control group may be an individual known to have a poor prognosis, for example, an individual having a history of metastasis or death after the onset of a brain tumor.
- the positive control group may be an individual capable of confirming survival information through a known database, and preferably may be a brain tumor patient whose survival value is below average.
- the mRNA expression of the NUPR1 gene or its protein activity level is higher than that of the negative control group (normal group)
- the prognosis when the mRNA expression of the NUPR1 gene or its protein activity level is higher than that of the positive control group (brain tumor patient group), it may be determined that the prognosis is relatively poor and lower than that of the positive control group (brain tumor patient group). In this case, the prognosis is relatively good.
- the present invention provides a pharmaceutical composition for preventing or treating brain tumors, including an inhibitor of NUPR1 (Nuclear protein 1) expression or activity.
- the present invention provides a food composition for preventing or improving brain tumors, including the expression or activity inhibitor of NUPR1 (Nuclear protein 1).
- the present invention also provides a method for preventing or treating brain tumors, comprising administering an expression or activity inhibitor of NUPR1 (Nuclear protein 1) to a subject in need thereof.
- NUPR1 Nuclear protein 1
- expression or activity inhibitor of NUPR1 (Nuclear protein 1) is used as a generic term for a substance that reduces the expression or activity of NUPR1, and more specifically, directly acts on NUPR1 or indirectly to its ligand. It may include any substance that reduces the expression or activity of NUPR1 by reducing the expression of NUPR1 at the transcription level or by inhibiting its activity, such as by acting.
- the substance that inhibits the expression of NUPR1 can be used without limitation in the form of a compound, a nucleic acid, a peptide, a virus or a vector containing the nucleic acid that can target NUPR1 and inhibit the expression or activity of NUPR1.
- Examples of substances that inhibit NUPR1 expression include siRNAs, shRNAs, miRNAs, antisense oligonucleotides, ribozymes, DNAzyme, peptide nucleic acids (PNAs) that specifically bind to NUPR1 mRNA and thereby inhibit the expression of NUPR1 mRNA.
- examples of a substance that inhibits NUPR1 activity include an antibody or an antigen-binding fragment thereof, an aptamer, a compound thereof, or the like that specifically binds to the protein of NUPR1 to inhibit the activity of the NUPR1 protein, and preferably may be an antibody. This is not restrictive.
- siRNA small interference RNA
- siRNA is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and refers to a small RNA fragment of 21 to 25 nucleotides in size.
- the siRNA of the present invention may have a double-stranded structure in which the sense strand (corresponding sequence) and the antisense strand (sequence complementary to the mRNA sequence) are positioned opposite to each other and form a double-stranded structure. -complementary) can have a single chain structure with sense and antisense strands.
- the siRNA of the present invention is not limited to the complete pairing of double-stranded RNA portions paired with RNA, but by mismatching (corresponding base is not complementary), bulge (no base corresponding to one chain), or the like. Unpaired parts may be included.
- siRNA consisting of SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, or SEQ ID NO: 11 and 12, the siRNA of the NUPR1 mRNA If the expression can be inhibited, the sequence is not particularly limited.
- shRNA short hairpin RNA
- Viruses, lentiviruses, and plasmid expression vector systems can be used to introduce and express them into cells, and these shRNAs are converted into siRNAs with the correct structure by siRNA processing enzymes (Dicer or Rnase III) present in the cells. It is well known to induce silencing of.
- miRNA is a single-stranded RNA molecule of 21-25 nucleotides, and is a regulator that controls gene expression in eukaryotes through inhibition of the disruption or translation of target mRNAs.
- This miRNA consists of two stages of processing.
- the first miRNA transcript (primary miRNA) is made into a 70-90 base stem-loop structure, or pre-miRNA, by an RNaseIII type enzyme called Drosha in the nucleus, which then migrates into the cytoplasm and is called Dicer. Is cleaved into a mature miRNA of 21-25 bases.
- the miRNA thus produced complementarily binds to the target mRNA and acts as a post-transcriptional gene suppressor, inducing translation inhibition and mRNA destabilization. miRNAs are involved in a variety of physiological phenomena and diseases.
- antisense oligonucleotide refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a particular mRNA, binding to the complementary sequence in the mRNA to translate the mRNA into a protein It shows an inhibitory effect.
- aptamer means a nucleic acid molecule having binding activity to a given target molecule.
- the aptamers may be RNA, DNA, modified nucleic acids, or mixtures thereof, and may be in linear or cyclic form, using an oligonucleotide library called systemic evolution of ligands by exponential enrichment (SELEX). It is a substance obtained by isolating oligomers which have high affinity and selectivity for binding to specific chemical or biological molecules by evolutionary methods.
- the aptamer can specifically bind to the target and modulate the target's activity, for example, by blocking the ability of the target to function through binding.
- prevention means any action that inhibits or delays the onset of brain tumors by administration of the composition.
- treatment prevents the occurrence, metastasis or recurrence of brain tumors and alleviates the symptoms by administering the composition, lowers all direct or indirect pathological consequences of the disease, reduces the rate of disease progression and These include alleviating or temporarily mitigating a disease state, driving off or improving the prognosis.
- improvement means any action that at least reduces the parameters associated with the condition being treated, for example the extent of symptoms.
- NUPR1 inhibitor by inhibiting the expression of NUPR1, in particular NUPR1 isoform A (varient 1, v.1), not only can the growth, metastasis and invasion of brain tumors be suppressed, but also the effect of converting into a prognosis subtype is It has been confirmed that the NUPR1 inhibitor can be used as a medicine and dietary supplement for the prevention, improvement, or treatment of brain tumors.
- composition of the present invention may further contain one or more known active ingredients having a therapeutic effect on brain tumors, together with the expression or activity inhibitor of NUPR1.
- compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
- Carriers, excipients and diluents which may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
- the pharmaceutical composition of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions according to conventional methods. .
- Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA.
- when formulating the composition can be prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants.
- solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and prepared.
- excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and prepared.
- lubricants such as magnesium stearate and talc are also used.
- Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .
- Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
- non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used.
- base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
- administration means providing a subject with a composition of the present invention in any suitable manner.
- Preferred dosages of the pharmaceutical compositions of the present invention depend on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. The dosage does not limit the scope of the invention in any aspect.
- the pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intracranial injection. However, since oral administration may denature the composition by gastric acid, oral compositions may be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.
- the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of brain tumors.
- the expression or activity inhibitor of NUPR1 of the present invention can be added to food compositions, preferably functional foods for the purpose of preventing or improving brain tumors.
- health functional food refers to a food having a bioregulatory function, such as prevention or improvement of disease, biological defense, immunity, recovery of symptoms, inhibition of aging, and should be harmless to the human body when taken in the long term.
- the composition When the food composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.
- the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment), and there is no particular limitation on the type of food.
- the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents used in beverages and the like.
- the present invention comprises the steps of (a) treating a brain tumor treatment candidate to isolated brain tumor cells; And (b) measuring the mRNA expression of the NUPR1 gene or its protein activity level in the brain tumor cells.
- the candidate of step (a) is a substance that is expected to be able to treat brain tumors or an unknown substance that is expected to improve the prognosis, and includes a compound, protein or natural product extract, but is not limited thereto. It is not.
- the method of the present invention (c) when the mRNA expression or protein activity level of the NUPR1 gene measured in step (b) shows a lower level than the brain tumor cells not treated with the candidate material, the candidate material as a brain tumor treatment agent Determining that it can be used.
- the method of measuring the mRNA or protein level of the gene is the same as described above.
- NUPR1 isoform A (v.1) protein.
- SVG p12 used as a control means normal cells as human derived astroglia.
- a primary antibody specific for NUPR1 isoform A a polyclonal antibody prepared in Example 2-4 described below was used.
- ⁇ -actin was used as a housekeeping protein for quantification, and brain tumor patient information is shown in Table 1 below. The results are shown in FIG.
- GBM-12 Male 46 Glioblastoma (grade IV) GBM-14 Male 52 Anaplastic Astrocytoma (grade III) GBM-15 Female 37 Glioblastoma (grade IV) GBM-30 Female 46 Glioblastoma (grade IV)
- NUPR1 isoform A protein is hardly expressed in the SVG p12 sample which is normal cells, whereas NUPR1 isoform A protein is expressed in the GBM-12, GBM-14, GBM-15 and GBM-30 samples of brain tumor patients. It was confirmed that this is markedly high.
- NUPR1 isoform A (varient 1, v.1) is expressed higher in the brain tumor patient group than healthy normal group, it was confirmed that it can be used as a diagnostic marker for brain tumors.
- Brain tumor tissue samples were obtained from primary glioblastoma patients (Glioblastoma, GBM-28) and secondary neuroeducoma patients (Gliosarcoma, GBM-37), followed by primary culture.
- GBM-28 and GBM-37 are samples derived from the same patient, and GBM-37 is a sample at the time of recurrence after 3 months after tumor removal surgery and concurrent chemoradiotherapy after obtaining the GBM-28 sample. .
- Brain MRI photographs of patients before sample acquisition are shown in FIG. 2 (T1, T2, T1-enhanced MRI from left).
- FBS fetal bovine serum, Gibco Corp., 16000, Grand Island, NY, USA
- DMEM medium Dulbecco's modified Eagle medium, WelGENE, LM001-05, Korea), including Island, NY, USA
- H & E GFAP, Vimentin, X50
- morphology observations were performed on cells obtained from patients with primary glioblastoma (GBM-28) and patients with secondary neuroedema (GBM-37). 3 and 4 are shown.
- GBM-28 cells isolated from primary glioblastoma patients show typical characteristics of glioblastoma
- GBM-37 cells isolated from secondary neuroedema patients The right side of each figure was confirmed that the GFAP staining and distinguished from the normal glioblastoma, such as showing a specific form (fascicular arranged spindle cells).
- cDNA microarray was performed. Transcript analysis was performed by oligonucleotide microarray analysis using MA-human Agilent 44k (Agilent Technologies), and one-channel microarray data was analyzed by global median normalization method using GeneSpring GX 7.3 (EBIOGEN, Seoul, Korea). Using the analyzed cDNA microarray data, Gene Set Enrichment Analysis, Broad Institute, USA (GSEA) was performed to select a gene set specifically correlated in the transcripts of cells derived from each brain tumor patient.
- GSEA Gene Set Enrichment Analysis, Broad Institute, USA
- GSEA was run through the javaGSEA desktop application (GSEA v2.1.) And the gene set was downloaded from a database called MSigDb (http://www.broadinstitute.org/gsea/msigdb/). GSEA produces a enrichment score, a normalized enrichment score, a nominal p-value, and a false discovery rate (q-value). Only gene sets with p-values less than 0.05 were selected to compare GBM-28 transcripts and GBM-37 transcripts. The results are shown in FIGS. 5 and 6.
- GBM-28 shows a proneural subtype with a prognosis.
- GBM-37 confirms that the prognosis represents a very poor Mesenchymal subtype.
- MTS cell proliferation assays were performed on cells isolated from primary glioblastoma patients (GBM-28) and secondary neuroeducoma patients (GBM-37). . First, they seeded the cells in each 96-well culture plate with 1.0 ⁇ 10 4 cells / well ratios. After cell culture for a period of time, CellTiter 96® AQueous One Solution cell proliferation assay (Promega, 3580) solution was added to each well. The absorbance at 490 nm was then measured using Infinite M200 Pro (Tecan). The results are shown in FIG.
- GBM-37 cells isolated from secondary neuroeducoma patients were found to have higher cell proliferation than GBM-28 cells isolated from primary glioblastoma patients.
- Example 2-1 a bioinformatics program called IPA was used.
- the cDNA microarray data obtained in Example 2-1 was analyzed using a tool called Core analysis of IPA, and transcriptome analysis was performed using a detailed program technique called “Disease and bio functions” provided by Core analysis. .
- the results are shown in FIG.
- GBM-37 As shown in FIG. 8, as a result of classifying the transcripts isolated from primary glioblastoma patients (GBM-28) and secondary neuroeducoma patients (GBM-37), GBM-37 was more malignant than GBM-28. It was again confirmed that the island had high properties. Through the above experimental results, it can be seen that the subjects corresponding to GBM-37 recurred as a more malignant brain tumor, that is, a secondary neuroedema with a poor prognosis after chemotherapy.
- NUPR1 isoform A (varient 1, v.1) was found to be expressed at a significantly high level in GBM-37.
- NUPR1 isoform A (varient 1, v.1) is found to be higher in malignant tumor patients with a poorer prognosis than among brain tumors.
- total protein was obtained using RIPA buffer according to a known method in brain tumor patient derived cells obtained from primary glioblastoma patient (GBM-28) and secondary neuroeducoma patient (GBM-37).
- the protein samples were subjected to Western blot using antibodies against NUPR1 as a whole and NUPR1 isoform A (varient 1, v.1).
- ⁇ -actin was used as a housekeeping protein for quantification.
- the antibody against NUPR1 isoform A (varient 1, v.1) was prepared by the following procedure. First, cysteine was added to the N-terminus of the NUPR1a specific peptide (18 amino acids 38 to 55 positions from the N-terminus of NUPR1a (NP_001035948.1)), and then conjugated to KLH. The fusion peptide was immunized subcutaneously with 0.5 mg each of two rabbits using PBS and Freund's adjuvant. The same amount of fusion peptide was immunized subcutaneously three times at two week intervals after primary immunization. One week after the final immunization, blood was collected and polyclonal antibodies were obtained using Protein A resin. The polyclonal antibody against NUPR1 isoform A (variant 1, v.1) obtained through the above procedure was used for experiments after confirming specificity through assays with antibodies produced by expressing recombinant proteins of NUPR1a and NUPR1b.
- the total NUPR1 protein expression in the GBM-28 and GBM-37 samples did not show a specific difference, but the NUPR1 isoform A (varient 1, v.1) protein was compared with the GBM-28 sample. It was found to be significantly higher in 37 samples. In other words, NUPR1 isoform A (v.1) protein was expressed higher in malignant tumor patients with poorer prognosis than among brain tumors as in mRNA expression analysis.
- the primary glioblastoma patient group enrolled in the Cancer Genome Atlas was divided into five subtypes (Proneural, Classical, Neural, Mesenchymal, G-CIMP), followed by NUPR1 isoform A (ID: ENST00000395641).
- the expression level was analyzed.
- the G-CIMP subtype belongs to the Proneural subtype and is a subtype known to have a good prognosis. This subtype is known to be closely associated with the prognostic factor of a patient called the IDH1 / 2 mutation. The results are shown in FIG.
- NUPR1 isoform A (varient 1, v.1) is expressed at a higher level in malignant brain tumors classified as mesenchymal subtypes with the poorest prognosis among brain tumors. It was confirmed that it can be used not only for the diagnosis of brain tumors but also for the diagnosis of subtypes of brain tumors.
- RNA was isolated from brain tumor patient derived cells obtained from brain tumor patients with different diagnosis names and survival rates. RT-PCR was performed in the same manner as in 2-3. Specific patient information is shown in Table 2, and the experimental results are shown in FIG. U87 is a human primary glioblastoma cell line sold by the American Type Culture Collection (ATCC).
- ATCC American Type Culture Collection
- the mRNA expression level of NUPR1 isoform A (varient 1, v.1) was significantly higher in the GBM-37 sample derived from the patient with extremely poor prognosis due to only 7 months of survival compared to the sample derived from other brain tumor patients. It was confirmed.
- the primary glioblastoma patient group registered in The Cancer Genome Atlas (TCGA) was divided into high and low expression according to the degree of NUPR1 isoform A expression, and then survival analysis was performed. More specifically, an RNA-sequencing dataset was used, and the ID of NUPR1 isoform A was identified as ENST00000395641 (uc002dqd.2). Using the ID, the expression value of NUPR1 isoform A was confirmed for each patient, and then classified into high and low expression according to the expression level of NUPR1 isoform A and survival analysis was performed.
- MGMT methylation which is used as a prognostic factor for glioblastoma patients, was classified primarily in the same patient group, and then expression level and survival analysis of NUPR1 isoform A were performed. The results are shown in FIGS. 13 to 15.
- NUPR1 isoform A (varient 1, v.1) shows a significant correlation with the survival rate, which can be used as a marker for predicting the prognosis of brain tumor patients. Confirmed.
- siRNAs capable of specifically inhibiting the NUPR1 isoform A were prepared (Genolution). After culturing GBM-37 cells for 24 hours, siRNA was transfected into cells using LipofectamineTM RNAiMAX (Invitrogen). 24 hours after transfection, total RNA and protein were isolated from each cell and RT-PCR and Western blots were performed according to known methods. The siRNA sequences are shown in Table 3, with RT-PCR results shown in FIG. 19 and Western blot results shown in FIG. 20.
- NUPR1 ⁇ As shown in FIG. 19, it was confirmed that all five siRNAs for NUPR1 isoform A did not affect NUPR1 isoform B (NUPR1 ⁇ ), but specifically downregulated mRNA expression of NUPR1 isoform A (NUPR1 ⁇ ).
- NUPR1 ⁇ protein expression of NUPR1 isoform A (NUPR1 ⁇ ) is also down-regulated by the transfection of siRNA against NUPR1 isoform A.
- GBM-37 cells isolated from malignant brain tumor patients were transfected with siRNA (NUPR1 V1 # 2) prepared in Example 4-1, followed by MTS cell proliferation assay.
- siRNA NUPR1 V1 # 2
- MTS cell proliferation assay MTS cell proliferation assay.
- scRNA scRNA
- each cell was seeded in a 96-well culture plate at a rate of 1.0 ⁇ 10 4 cells / well. After cell culture for a period of time, CellTiter 96® AQueous One Solution cell proliferation assay (Promega, 3580) solution was added to each well. The absorbance at 490 nm was then measured using Infinite M200 Pro (Tecan). The results are shown in FIG.
- GBM-37 cells isolated from malignant brain tumor patients were transfected with siRNA (NUPR1 V1 # 2) prepared in Example 4-1, followed by a transwell migration assay.
- siRNA NUPR1 V1 # 2
- scRNA transfected with scRNA were used.
- each cell was released in serum-free medium, and the cells were incubated for 24 hours in transwell inserts of 24-well plates (BD Biosciences, # 3422). Then medium with 10% FBS (fetal bovine serum) was added to the lower chamber of the plate as a chemoattractant. After incubating the cells for 24 hours, non-migrated cells were removed using a cotton swab. The remaining metastatic cells were stained using crystal violet (0.5% in 20% methanol) and then harvested.
- Invasion assays were performed under the same conditions as the transwell transfer assay, and growth factor-reduced matrigel-coated insert wells (BD Biosciences, # 356234) were used. Cell metastasis and invasion values were calculated by dividing the microscopic field into quarters and calculating the average number of cells. The results are shown in FIG. 22.
- siRNA against NUPR1 isoform A significantly inhibited metastasis and invasion of GBM-37 cells.
- GBM-37 before transfection showed a Mesenchymal subtype with poor prognosis (see FIG. 5), but it was possible to inhibit the expression of NUPR1 isoform A using siRNA against NUPR1 isoform A.
- the prognosis showed good proneural subtype. This is a result showing that the properties of cancer cells have been changed to a proneural subtype with a better prognosis by siRNA for NUPR1 isoform A.
- FIGS. 24 to 26 The results are shown in FIGS. 24 to 26.
- the left side is the GBM-37 sample and the right side is the GBM-37 sample after transformation with siRNA for NUPR1 isoform A.
- Metastasis related gene set was down-regulated in the GBM-37 sample (GBM-37_siNUPR1a) after transformation with siRNA for NUPR1 isoform A compared to wild type.
- NUPR1 V1 # 2 siRNA (NUPR1 V1 # 2) or scRNA-transformed GBM-37 cells (2 ⁇ 10 6 cells) for NUPR1 isoform A were each examined for 5 weeks of athymic mice (BALB / c nu / nu) (Central Laboratory Animal). , Inc, Korea). Intracellular injection was performed in the left forehead and the coordinates were set 2 mm laterally and 0.5 mm forward from bregma. Tumors formed 7 days after cell injection were extracted and immediately stored in Bouin's fixative for 2-3 hours. Deparaffinized tumor tissue was H & E stained. Images of the stained tissue were obtained through Olympus whole-slide scanner with OlyVIA software (Olympus Life Science, Center Valley, PA, USA). The results are shown in FIGS. 27 and 28.
- tumor size was decreased in the group to which GBM-37 cells transfected with siRNA for NUPR1 isoform A (siRNA NUPRv.1).
- NUPR1, in particular NUPR1 isoform A is significantly increased in brain tumor patients compared to healthy normal persons, especially in patients with malignant brain tumors with a poor prognosis, which is a biomarker for diagnosing brain tumors or predicting prognosis. Can be utilized as In addition, it is possible to inhibit the growth, metastasis and invasion of brain tumors through the inhibition of NUPR1, in particular NUPR1 isoform A according to the present invention, it can be used as a target for the treatment of brain tumors, it can be used in the screening of future brain tumor therapeutics. .
Abstract
The present invention relates to use of nuclear protein 1 (NUPR1) in diagnosing and treating brain tumors and, more particularly, to a biomarker composition comprising NUPR1 for diagnosing brain tumors or for predicting prognosis and a composition comprising an NUPR1 inhibitor for preventing or treating brain tumors. The level of NUPR1, particularly NUPR1 isoform A, according to the present invention is significantly increased in patients with brain tumors and further remarkably in patients with malignant brain tumors of poor prognosis, compared to healthy normal persons, so that NUPR1 can be used as a biomarker for diagnosis of brain tumors or for prediction of prognosis. In addition, NUPR1, particularly NUPR1 isoform A according to the present invention can be used as a target for brain tumor treatment and also, in future, in screening a therapeutic agent for brain tumors as the inhibition thereof can suppress the growth, metastasis and invasion of brain tumors.
Description
본 발명은 뇌종양의 진단 및 치료에서의 NUPR1의 용도에 관한 것으로, 보다 구체적으로 본 발명은 NUPR1(Nuclear protein 1)을 포함하는 뇌종양 진단 또는 예후 예측용 바이오 마커 조성물 및 NUPR1 억제제를 포함하는 뇌종양 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to the use of NUPR1 in the diagnosis and treatment of brain tumors. More specifically, the present invention relates to the prevention of brain tumors comprising a biomarker composition for diagnosing or prognostic brain tumors comprising NUPR1 (NUPR1) and a NUPR1 inhibitor or A therapeutic composition.
뇌암은 뇌조직과 뇌를 싸고 있는 뇌막에서 발생 되는 원발성 뇌암과 두개골이나 신체의 다른 부위에서 발생한 암으로부터 전이된 이차성 뇌암을 통칭하는 것이다. 이와 같은 뇌암은 다른 장기에서 발생하는 암과 구분되는 점이 많다. 우선 폐, 위, 유방 등에 생기는 암은 장기별로 한두 종류에 국한되고, 그 성질이 동일하거나 유사한 편이다. 그러나 뇌에는 다형성 교모세포종, 악성신경교종, 임파선종, 배아세포종, 전이성 종양 등 다양한 암이 발생한다. 상기 교종(glioma)은 뇌의 원발성 종양 중 가장 빈도가 높은 종양이다. 교종은 일반적으로 종양의 악성도가 높을수록 종양의 CT 음영이나 MRI 신호강도가 일정하지 않고 주위의 부종이 심하며 종양 내부에 괴사가 나타난다. 수막종(meningioma)은 뇌의 양성 종양 중에서 뇌하수체선종과 함께 가장 빈도가 높으며 원발성 뇌종양의 약 15~20%를 차지한다. 뇌하수체선종(pituitary adenoma)은 전체 뇌종양의 약 10~15%를 차지한다. 종양의 크기에 따라 직경 10mm 이하의 미세선종과 10mm 이상의 거대선종으로 나누기도 한다. 전이암(metastatic tumor)은 전체 뇌종양의 30~40%에 해당하며, 약 60%에서는 다발성 종괴로 관찰되나 약 40%에서는 단일 종괴로 나타난다. 따라서 단일 종괴로 나타날 때는 원발성 악성 종양, 특히 교모세모종과의 감별진단이 어렵다. 우리나라에서는 폐암에 의한 뇌 전이암이 가장 많다.Brain cancer is a generic term for primary brain cancers that occur in the brain tissue and the brain envelope that surrounds the brain, and secondary brain cancers that metastasize from cancers that occur in the skull or other parts of the body. Such brain cancers are often distinguished from cancers occurring in other organs. First of all, the lung, stomach, breast and other cancers are limited to one or two types of organs, and their properties are the same or similar. However, the brain develops a variety of cancers, including glioblastoma multiforme, malignant glioma, lymphadenomas, germ cell tumors, and metastatic tumors. The glioma is the most frequent tumor of primary brain. In general, the higher the malignancy of the tumor, the CT shadow or MRI signal intensity of the tumor is not constant, the surrounding swelling is severe, and necrosis appears inside the tumor. Meningioma is the most frequent of the brain's benign tumors with pituitary adenoma, accounting for about 15-20% of primary brain tumors. Pituitary adenoma accounts for about 10-15% of all brain tumors. Depending on the size of the tumor, it may be divided into microadenoma less than 10mm in diameter and giant adenoma greater than 10mm. Metastatic tumors account for 30 to 40% of all brain tumors, about 60% of which are observed as multiple masses, but about 40% to single masses. Therefore, it is difficult to differentiate between primary malignancies, especially glioblastoma, when they appear as a single mass. In Korea, brain cancer caused by lung cancer is the most common.
그 중, 교모세포종(glioblastoma)은 뇌-척수 조직이나 이를 싸고 있는 막으로부터 발생하는 원발성 종양으로, 정상적으로 뇌조직에 풍부하게 존재하고 있는 신경교세포(Neuroglia Cell)에서 시작된 종양이다. 교모세포종은 성상세포종 중에서 가장 악성이며 조직학적으로는 역형성 성상세포종에 괴사 소견이 추가된 것이다. 이 종양은 가장 흔한 원발성 뇌종양으로 신경교종의 1/2, 소아 신경교종의 15%를 차지하며 소뇌에 발생하는 경우는 흔치 않은 것으로 보고되고 있다. 2000년 개정된 WHO의 분류표를 보면 교모세포종에는 거대세포 교모세포종(Giant cell glioblastoma)과 신경교육종(gliosarcoma)등도 포함된다. 상기 신경교육종(gliosarcoma)은 교모세포종의 1.8~2.4%를 포함하는 희귀한 변종이고 교모세포종과 임상적으로 유사하다. 신경교육종 종양 세포의 육종적인(sarcomatous) 특징 때문에, 신경교육종 환자에서 두개강 외 전이되는 경향이 크므로, 신경교육종은 더 공격적이고 침윤성인 특징이 있다. Among them, glioblastoma is a primary tumor originating from the brain-spinal tissue or the membrane surrounding the glioblastoma. The glioblastoma is a tumor originating from glioma cells (Neuroglia Cells) normally abundantly present in brain tissue. Glioblastoma is the most malignant of astrocytoma and histologically, necrosis is added to anaplastic astrocytoma. This tumor is the most common primary brain tumor, accounting for 1/2 of glioma and 15% of pediatric glioma, and is rarely reported in the cerebellum. The 2000 HHO taxonomy includes glioblastomas, including giant cell glioblastoma and gliosarcoma. The neuroeducoma (gliosarcoma) is a rare variant that contains 1.8-2.4% of glioblastoma and is clinically similar to glioblastoma. Because of the sarcomatous nature of neuroeducoma tumor cells, neuroedema is more aggressive and invasive because of its greater propensity to metastasize extracranial in neuroedema patients.
기존의 뇌종양 환자들의 예후를 판별하는 방법으로는 임상치료의 경과, 방사선 요법 및 화학요법의 경과들을 종합하고, 뇌종양의 악성도와 위치, 연령 등을 고려하여 재발 가능성을 비롯한 생존 예후를 결정하는 것이 일반적인 방법이었으나, 이는 환자의 유형마다 다양한 차이가 존재하며, 임상적 치료에 대한 결과도 달라지므로 정확한 예후 예측방법이라고 할 수 없다. 따라서 악성 뇌종양을 진단하고, 예후를 예측할 수 있는 새로운 진단 및 치료 표적으로서의 바이오 마커 개발의 필요성이 대두되고 있다. In order to determine the prognosis of existing patients with brain tumors, it is common to synthesize the course of clinical treatment, radiotherapy and chemotherapy, and to determine the survival prognosis including the possibility of recurrence by considering the malignancy, location, and age of brain tumors. This method, however, has various differences according to the types of patients, and the result of clinical treatment is also different, and thus it is not an accurate prognostic method. Therefore, there is a need to develop a biomarker as a new diagnostic and therapeutic target for diagnosing malignant brain tumors and predicting prognosis.
이러한 배경하에서 본 발명자들은 NUPR1, 특히 NUPR1 동형 A가 뇌종양 환자 중에서도 예후가 나쁜 악성 뇌종양 환자에서 현저하게 증가함을 확인하였으며, 이를 억제함으로써 뇌종양을 치료할 수 있음을 확인함으로써 본 발명을 완성하였다. Under these backgrounds, the inventors have found that NUPR1, particularly NUPR1 isoform A, is significantly increased in malignant brain tumor patients with poor prognosis among brain tumor patients, and has completed the present invention by confirming that the brain tumor can be treated by inhibiting it.
본 발명의 목적은 NUPR1(Nuclear protein 1)을 포함하는 뇌종양 진단 또는 예후 예측용 바이오 마커 조성물을 제공하는 것이다.An object of the present invention is to provide a biomarker composition for brain tumor diagnosis or prognosis prediction comprising NUPR1 (Nuclear protein 1).
본 발명의 다른 목적은 NUPR1(Nuclear protein 1)의 검출 제제를 포함하는 뇌종양 진단 또는 예후 예측용 조성물, 상기 조성물을 포함하는 뇌종양 진단 또는 예후 예측용 키트 및 상기 조성물을 이용한 뇌종양 진단 또는 예후 예측을 위한 정보제공방법을 제공하는 것이다.Another object of the present invention is a composition for diagnosing or prognosticing a brain tumor comprising a detection agent of NUPR1 (Nuclear protein 1), a kit for diagnosing or prognosticing a brain tumor comprising the composition, and for diagnosing or diagnosing a brain tumor using the composition It is to provide a method of providing information.
본 발명의 또 다른 목적은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 치료용 조성물을 제공하는 것이다.Still another object of the present invention is to provide a composition for preventing or treating brain tumors, including a NUPR1 (Nuclear protein 1) expression or activity inhibitor.
본 발명의 또 다른 목적은 NUPR1(Nuclear protein 1)을 이용한 뇌종양 치료제의 스크리닝 방법을 제공하는 것이다. Still another object of the present invention is to provide a method for screening a brain tumor therapeutic agent using NUPR1 (Nuclear protein 1).
상기와 같은 과제를 해결하기 위해, 본 발명은 NUPR1(Nuclear protein 1)을 포함하는 뇌종양 진단 또는 예후 예측용 바이오 마커 조성물을 제공한다.In order to solve the above problems, the present invention provides a biomarker composition for brain tumor diagnosis or prognostic prediction comprising NUPR1 (Nuclear protein 1).
또한, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성 수준을 측정하는 제제를 포함하는 뇌종양 진단 또는 예후 예측용 조성물을 제공한다. In addition, the present invention provides a composition for diagnosing or predicting a brain tumor comprising a preparation for measuring the expression or activity level of NUPR1 (Nuclear protein 1).
또한 본 발명은 상기 조성물을 포함하는 뇌종양 진단 또는 예후 예측용 키트를 제공한다. In another aspect, the present invention provides a kit for diagnosing or predicting a brain tumor comprising the composition.
또한 본 발명은 생물학적 시료로부터 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계;를 포함하는 뇌종양 진단 또는 예후 예측을 위한 정보제공방법을 제공한다.In another aspect, the present invention provides a method for providing information for diagnosing or predicting a brain tumor comprising a; measuring the mRNA expression of the NUPR1 gene or its protein activity level from a biological sample.
또한, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating brain tumors, including an expression or activity inhibitor of NUPR1 (Nuclear protein 1).
또한, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 개선용 식품 조성물을 제공한다.In another aspect, the present invention provides a food composition for preventing or improving brain tumors, including the expression or activity inhibitor of NUPR1 (Nuclear protein 1).
또한, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 이를 필요로하는 개체에 투여하는 단계를 포함하는, 뇌종양의 예방 또는 치료 방법을 제공한다.The present invention also provides a method for preventing or treating brain tumors, comprising administering an expression or activity inhibitor of NUPR1 (Nuclear protein 1) to a subject in need thereof.
또한, 본 발명은 (a) 분리된 세포에 뇌종양 치료 후보물질을 처리하는 단계; 및 (b) 상기 세포에서 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계를 포함하는 뇌종양 치료제의 스크리닝 방법을 제공한다. In addition, the present invention comprises the steps of (a) treating a brain tumor treatment candidate material to the isolated cells; And (b) measuring the mRNA expression of the NUPR1 gene or its protein activity level in the cell.
본 발명에 따른 NUPR1, 특히 NUPR1 동형 A는 건강한 정상인에 비해 뇌종양 환자에서 유의하게 증가하며, 특히 예후가 나쁜 악성 뇌종양 환자에서 더욱 현저하게 증가하는바, 이를 뇌종양 진단 또는 예후 예측용 바이오 마커로 활용할 수 있다. 또한, 본 발명에 따른 NUPR1, 특히 NUPR1 동형 A의 억제를 통해 뇌종양의 성장, 전이 및 침입을 억제할 수 있는바, 이를 뇌종양 치료를 위한 타겟으로 활용할 수 있고, 향후 뇌종양 치료제의 스크리닝에도 이용할 수 있다. NUPR1, in particular NUPR1 isoform A, according to the present invention is significantly increased in brain tumor patients compared to healthy normal people, especially in patients with malignant brain tumors with a poor prognosis, which can be used as a biomarker for diagnosing brain tumors or predicting prognosis. have. In addition, it is possible to inhibit the growth, metastasis and invasion of brain tumors through the inhibition of NUPR1, in particular NUPR1 isoform A according to the present invention, it can be used as a target for the treatment of brain tumors, it can be used in the screening of future brain tumor therapeutics. .
도 1은 정상 및 뇌종양 환자 유래 세포에서 NUPR1 동형 A의 단백질 발현을 확인한 도이다. 1 is a diagram confirming the protein expression of NUPR1 isoform A in normal and brain tumor-derived cells.
도 2는 GBM-28 및 GBM-37 샘플 수득 전 환자의 뇌 MRI 사진을 나타낸 도이다 (왼쪽부터 T1, T2, T1-enhanced MRI). Figure 2 is a diagram showing the brain MRI pictures of patients before obtaining the GBM-28 and GBM-37 samples (from left to T1, T2, T1-enhanced MRI).
도 3은 GBM-28(왼쪽) 및 GBM-37(오른쪽) 샘플로부터 수득한 뇌종양 조직에서 면역조직화학 분석(H&E, GFAP, Vimentin) 결과를 나타낸 도이다 (X50). Figure 3 is a diagram showing the results of immunohistochemistry (H & E, GFAP, Vimentin) in brain tumor tissue obtained from GBM-28 (left) and GBM-37 (right) samples (X50).
도 4는 GBM-28(왼쪽) 및 GBM-37(오른쪽) 샘플로부터 수득한 1차 배양 세포의 형태를 현미경으로 관찰한 결과를 나타낸 도이다.4 shows the results of microscopic observation of the morphology of primary cultured cells obtained from GBM-28 (left) and GBM-37 (right) samples.
도 5는 GBM-28 및 GBM-37 샘플에서 전체 RNA를 추출하여 cDNA 마이크로어레이 데이터를 얻은 후, 이를 활용해 GSEA(Gene Set Enrichment Analysis)를 실시한 결과를 나타낸 것으로, 각 샘플의 전사체가 신경교종 서브타입 중 전신경(Proneural) 아류형인지 간엽성(Mesenchymal) 아류형인지를 확인한 도이다. FIG. 5 shows the results of performing cEA DNA array by extracting total RNA from GBM-28 and GBM-37 samples, and performing GSEA (Gene Set Enrichment Analysis) using the transcripts of each sample. It is a figure confirming whether the type is a proneural subtype or a mesenchymal subtype.
도 6은 GBM-28 및 GBM-37 샘플에서 전체 RNA를 추출하여 cDNA 마이크로어레이 데이터를 얻은 후, 이를 활용해 GSEA(Gene Set Enrichment Analysis)를 실시한 결과를 나타낸 것으로, 각 샘플의 전사체와 특정 기능을 대표하는 유전자 세트 간의 상관성을 분석한 결과를 나타낸 도이다. Figure 6 shows the result of performing the GSEA (Gene Set Enrichment Analysis) by using the total RNA extracted from the GBM-28 and GBM-37 samples obtained cDNA microarray data, using the transcript and specific functions of each sample Figure shows the results of analyzing the correlation between the set of genes representing.
도 7은 GBM-28 및 GBM-37 샘플로부터 수득한 1차 배양 세포의 MTS 증식 어쎄이 결과를 나타낸 도이다. FIG. 7 shows MTS proliferation assay results of primary cultured cells obtained from GBM-28 and GBM-37 samples.
도 8은 GBM-28 및 GBM-37 샘플에서 전체 RNA를 추출하여 cDNA 마이크로어레이 데이터를 얻은 후, IPA를 통해 여러가지의 Disease and Biological Functions들을 카테고리화 한 결과를 나타낸 도이다.8 is a diagram showing the results of categorizing various disease and biological functions through IPA after extracting total RNA from GBM-28 and GBM-37 samples to obtain cDNA microarray data.
도 9는 GBM-28 및 GBM-37 샘플에서 전체 RNA를 추출한 후, RT-PCR을 통해 NUPR1 전체, NUPR1 동형 A(varient 1, v.1) 및 NUPR1 동형 B(varient 2, v.2) mRNA 발현 정도를 분석한 결과를 나타낸 도이다. Figure 9 shows the extraction of total RNA from GBM-28 and GBM-37 samples, followed by RT-PCR for NUPR1 total, NUPR1 isoform A (varient 1, v.1) and NUPR1 isoform B (varient 2, v.2) mRNAs. Figure showing the results of analyzing the expression level.
도 10은 GBM-28 및 GBM-37 샘플에서 전체 단백질을 추출한 후, 웨스턴 블랏을 통해 NUPR1 전체 및 NUPR1 동형 A(varient 1, v.1) 발현 정도를 분석한 결과를 나타낸 도이다. 10 is a diagram showing the results of analysis of the expression of the total NUPR1 and NUPR1 isoform A (varient 1, v.1) expression by Western blot after extracting the whole protein from the GBM-28 and GBM-37 samples.
도 11은 TCGA (The Cancer Genome Atlas)에 등록되어 있는 원발성 교모세포종 (primary glioblastoma) 환자군을 다섯 가지 서브타입으로 나눈 후, 각 군에서 NUPR1 동형 A의 발현 정도를 확인한 도이다. FIG. 11 is a diagram illustrating the expression level of NUPR1 isoform A in each group after dividing the primary glioblastoma patient group registered into TCGA (The Cancer Genome Atlas) into five subtypes.
도 12는 여러 뇌종양 환자 유래 세포에서 전체 RNA를 추출한 후, RT-PCR을 통해 NUPR1 동형 A의 mRNA 발현 정도를 분석한 결과를 나타낸 도이다. 12 is a diagram showing the result of analyzing the mRNA expression of NUPR1 isoform A through RT-PCR after extracting the whole RNA from cells derived from various brain tumors.
도 13은 TCGA (The Cancer Genome Atlas)에 등록되어 있는 원발성 교모세포종 (primary glioblastoma) 환자군을 NUPR1 동형 A 발현 정도에 따라 고발현 및 저발현으로 나눈 후, 생존 분석을 수행한 결과를 나타낸 도이다. 13 is a diagram showing the results of survival analysis after dividing the primary glioblastoma patient group registered in TCGA (The Cancer Genome Atlas) into high and low expression according to the degree of NUPR1 isoform A expression.
도 14는 TCGA (The Cancer Genome Atlas)에 등록되어 있는 원발성 교모세포종 (primary glioblastoma) 환자군 중 MGMP 메틸화된 환자를 1차 선별하고, 이를 다시 NUPR1 동형 A 발현 정도에 따라 고발현 및 저발현으로 나눈 후, 생존 분석을 수행한 결과를 나타낸 도이다.FIG. 14 is a first screening of MGMP methylated patients among primary glioblastoma patient groups registered in The Cancer Genome Atlas (TCGA), and then divided into high and low expression according to NUPR1 isoform A expression. This figure shows the results of the survival analysis.
도 15는 TCGA (The Cancer Genome Atlas)에 등록되어 있는 원발성 교모세포종 (primary glioblastoma) 환자군 중 MGMP 비메틸화된 환자를 1차 선별하고, 이를 다시 NUPR1 동형 A 발현 정도에 따라 고발현 및 저발현으로 나눈 후, 생존 분석을 수행한 결과를 나타낸 도이다.FIG. 15 is a primary screening of MGMP unmethylated patients among primary glioblastoma patients registered with The Cancer Genome Atlas (TCGA), and divided into high and low expression according to the degree of NUPR1 isoform A expression. It is a figure which shows the result of performing a survival analysis.
도 16은 서울대학교 병원에 내원한 79명의 원발성 교모세포종 (primary glioblastoma) 환자의 임상 정보를 나타낸 도이다. FIG. 16 shows clinical information of 79 primary glioblastoma patients who visited Seoul National University Hospital.
도 17은 서울대학교 병원에 내원한 79명의 원발성 교모세포종 (primary glioblastoma) 환자의 조직을 NUPR1 동형 A에 대한 항체를 사용하여 염색한 후 NUPR1 동형 A 발현 정도에 따라 고발현 및 저발현으로 나눈 다음, 각 그룹 환자 유래 대표적인 종양 조직을 나타낸 도이다. FIG. 17 stains tissues of 79 primary glioblastoma patients who visited Seoul National University Hospital using antibodies against NUPR1 isoform A, and then divides the expression into high and low expression according to the expression level of NUPR1 isoform A. Representative tumor tissue from each group of patients is shown.
도 18은 서울대학교 병원에 내원한 79명의 원발성 교모세포종 (primary glioblastoma) 환자를 NUPR1 동형 A 발현 정도에 따라 고발현 및 저발현으로 나눈 후, 생존 분석을 수행한 결과를 나타낸 도이다.FIG. 18 is a diagram showing the results of a survival analysis after dividing 79 primary glioblastoma patients at Seoul National University Hospital into high and low expression according to the degree of NUPR1 isoform A expression.
도 19는 NUPR1 동형 A에 대한 5가지 siRNA(#1~#5)를 각각 GBM-37 세포에 형질 주입한 후, NUPR1 동형 A 및 NUPR1 동형 B의 mRNA 발현에 미치는 영향을 확인한 도이다. 19 is a diagram confirming the effects on the mRNA expression of NUPR1 isoform A and NUPR1 isoform B after transducing five siRNAs (# 1 to # 5) for NUPR1 isoform A into GBM-37 cells, respectively.
도 20은 NUPR1 동형 A에 대한 5가지 siRNA(#1~#5)를 각각 GBM-37 세포에 형질 주입한 후, NUPR1 동형 A 단백질 발현에 미치는 영향을 확인한 도이다. 20 is a diagram confirming the effect on the expression of NUPR1 isoform A protein after transfection into GBM-37 cells with five siRNAs (# 1 to # 5) for NUPR1 isoform A, respectively.
도 21은 NUPR1 동형 A에 대한 siRNA(siRNA-NUPR1α)를 GBM-37 세포에 형질 주입한 후, MTS 증식 어쎄이 결과를 나타낸 도이다. 21 is a diagram showing the results of MTS proliferation assay after transducing siRNA (siRNA-NUPR1α) for NUPR1 isoform A into GBM-37 cells.
도 22는 NUPR1 동형 A에 대한 siRNA(siRNA-NUPR1α)를 GBM-37 세포에 형질 주입한 후, 트랜스웰 전이 어쎄이 및 침입 어쎄이를 수행한 결과를 나타낸 도이다. 22 is a diagram showing the results of transwell transfer assay and invasion assay after transducing siRNA (siRNA-NUPR1α) for NUPR1 isoform A into GBM-37 cells.
도 23은 NUPR1 동형 A에 대한 siRNA를 GBM-37 세포에 형질 주입(GBM-37_siNUPR1a)한 후 전체 RNA를 추출하여 cDNA 마이크로어레이 데이터를 얻은 다음, 이를 활용해 GSEA(Gene Set Enrichment Analysis)를 실시한 결과를 나타낸 것으로, siRNA 형질 주입 전후의 전사체 서브타입 변화를 확인한 도이다. 23 is transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a) and extracted the entire RNA to obtain cDNA microarray data, using the GSEA (Gene Set Enrichment Analysis) results Fig. 3 shows the transcript subtype changes before and after siRNA transfection.
도 24는 NUPR1 동형 A에 대한 siRNA를 GBM-37 세포에 형질 주입(GBM-37_siNUPR1a)한 후 전체 RNA를 추출하여 cDNA 마이크로어레이 데이터를 얻은 다음, 이를 활용해 GSEA(Gene Set Enrichment Analysis)를 실시한 결과를 나타낸 것으로, siRNA 형질 주입 전후의 Epithelial-Mesenchymal Transition, Metastasis 등 관련 유전자 세트의 발현 변화를 확인한 도이다. Figure 24 is transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a) and extracted the entire RNA to obtain cDNA microarray data, using the GSEA (Gene Set Enrichment Analysis) results This figure shows the expression changes of related gene sets such as Epithelial-Mesenchymal Transition and Metastasis before and after siRNA transfection.
도 25는 NUPR1 동형 A에 대한 siRNA를 GBM-37 세포에 형질 주입(GBM-37_siNUPR1a)한 후 전체 RNA를 추출하여 cDNA 마이크로어레이 데이터를 얻은 다음, 이를 활용해 GSEA(Gene Set Enrichment Analysis)를 실시한 결과를 나타낸 것으로, siRNA 형질 주입 전후의 Inflammatory Response, Reactive Oxygen Species Pathway 등 관련 유전자 세트의 발현 변화를 확인한 도이다. 25 is transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a) and then extracted the entire RNA to obtain cDNA microarray data, using the GSEA (Gene Set Enrichment Analysis) results Figure 3 shows the expression changes of related gene sets such as Inflammatory Response and Reactive Oxygen Species Pathway before and after siRNA transfection.
도 26은 NUPR1 동형 A에 대한 siRNA를 GBM-37 세포에 형질 주입(GBM-37_siNUPR1a)한 후, 전체 RNA를 추출하여, cDNA 마이크로어레이 데이터를 얻은 후, 이를 활용해 GSEA(Gene Set Enrichment Analysis)를 실시한 결과를 나타낸 것으로, siRNA 형질 주입 전후의 Chemokine Signaling Pathway, IL6-JAK-STAT3 등 관련 유전자 세트의 발현 변화를 확인한 도이다. Figure 26 transfected siRNA for NUPR1 isoform A into GBM-37 cells (GBM-37_siNUPR1a), extracted the entire RNA to obtain cDNA microarray data, and then used GSEA (Gene Set Enrichment Analysis) The results of the experiments were shown to confirm the expression changes of related gene sets such as Chemokine Signaling Pathway and IL6-JAK-STAT3 before and after siRNA transfection.
도 27은 scRNA 또는 NUPR1 동형 A에 대한 siRNA(siRNA NUPR1v.1)로 형질전환 시킨 GBM-37 세포를 마우스에 주입한 후, 생성된 뇌종양 조직을 H&E 염색하여 관찰한 결과를 나타낸 도이다. 27 is a diagram showing the results of H & E staining of brain tumor tissues generated after injection of GBM-37 cells transformed with siRNA (siRNA NUPR1v.1) for scRNA or NUPR1 isoform A into mice.
도 28은 도 27의 뇌종양 조직을 확대하여 관찰한 결과를 나타낸 도이다. FIG. 28 is a view showing the enlarged observation result of the brain tumor tissue of FIG. 27.
도 29는 scRNA(CTL) 또는 NUPR1 동형 A에 대한 siRNA(siRNA-NUPR1α)로 형질전환시킨 GBM-37 세포를 마우스에 주입한 후, 생성된 뇌종양 조직에서 전체 단백질을 추출하고, 웨스턴 블랏을 통해 NUPR1 동형 A 발현 정도를 분석한 결과를 나타낸 도이다. FIG. 29 shows the injection of GBM-37 cells transformed with siRNA (siRNA-NUPR1α) for scRNA (CTL) or NUPR1 isoform A into mice, followed by extraction of whole protein from the resulting brain tumor tissues, and NUPR1 via Western blot. It is a figure which shows the result of analyzing the degree of homozygous A expression.
이하, 본 발명에 대하여 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
일 양태로, 본 발명은 NUPR1(Nuclear protein 1)을 포함하는 뇌종양 진단 또는 예후 예측용 바이오 마커 조성물을 제공한다.In one aspect, the present invention provides a biomarker composition for diagnosing or predicting a brain tumor comprising NUPR1 (Nuclear protein 1).
본 발명에 있어서, "NUPR1(Nuclear protein 1)"는 작은 크로마틴 단백질로, 다양한 스트레스 자극에 반응하는 것으로 알려져 있다. In the present invention, "NUPR1 (Nuclear protein 1)" is a small chromatin protein and is known to respond to various stress stimuli.
상기 NUPR1(Nuclear protein 1)은 NUPR1 동형(isoform) A 및 NUPR1 동형(isoform) B를 포함하는 것으로, 바람직하게는 NUPR1 동형(isoform) A이다. The NUPR1 (Nuclear protein 1) includes NUPR1 isoform A and NUPR1 isoform B, preferably NUPR1 isoform A.
본 발명에 있어서, “NUPR1 동형(isoform) A”는 “NUPR1 변이체 1(varient 1, v.1.)”또는 "NUPR1α"라고도 지칭하며, 서열번호 1로 표시되는 100개의 아미노산 서열로 이루어져 있고, 구체적인 정보는 NCBI Reference Sequence ID인 NP_001035948.1 및 NM_001042483.1에서 확인할 수 있다. In the present invention, “NUPR1 isoform A” is also referred to as “NUPR1 variant 1 (v.1.)” Or “NUPR1α” and consists of 100 amino acid sequences represented by SEQ ID NO: 1. Detailed information can be found in the NCBI Reference Sequence IDs NP_001035948.1 and NM_001042483.1.
본 발명에 있어서, “NUPR1 동형(isoform) B”는 “NUPR1 변이체 2(varient 2, v.2.)”또는 "NUPR1β"라고도 지칭하며, 서열번호 2로 표시되는 82개의 아미노산 서열로 이루어져 있고, 구체적인 정보는 NCBI Reference Sequence ID인 NP_036517.1 및 NM_012385.2에서 확인할 수 있다. In the present invention, “NUPR1 isoform B” is also referred to as “NUPR1 variant 2 (v.2.)” Or “NUPR1β” and consists of 82 amino acid sequences represented by SEQ ID NO: 2, Detailed information can be found in the NCBI Reference Sequence IDs NP_036517.1 and NM_012385.2.
본 발명에 있어서, "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단이란 뇌종양의 발병 여부, 바람직하게는 악성 뇌종양의 발병 여부를 확인하는 것이다. In the present invention, "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether a brain tumor develops, preferably whether malignant brain tumor develops.
본 발명에 있어서, "예후"란 병세의 진행, 회복에 관한 예측을 의미하는 것으로, 전망 내지는 예비적 평가를 말한다. 본 발명의 목적상, 예후란 뇌종양 환자의 치료 후 해당 개체에서 치료 성공 여부, 생존, 재발, 전이 등에 대한 예측을 의미하며, 바람직하게는 생존 예후를 의미한다. In the present invention, "prognosis" refers to prediction of progression and recovery of a condition and refers to prospective or preliminary evaluation. For the purposes of the present invention, the prognosis means prediction of treatment success, survival, recurrence, metastasis, etc. in the subject after treatment of brain tumor patients, and preferably means prognosis of survival.
본 발명에 있어서, "바이오 마커"란 뇌종양 진단 또는 예후를 예측할 수 있는 물질로, 생물학적 시료에서 뇌종양 발생 여부를 구분하여 진단할 수 있고, 예후가 좋지 않은 뇌종양 개체의 예후 인자(prognostic factor)로 사용할 수 있는 물질로, 정상 개체와 뇌종양 개체간에서 유의적인 차이를 보이는 폴리펩타이드, 핵산 (예: mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다. 본 발명의 목적상, 바이오 마커는 NUPR1으로 정상 개체에 비해 뇌종양 개체에서 증가하며, 특히 예후가 나쁜 악성 뇌종양 환자에서 더욱 증가하는바, 발현 정도가 낮을수록 예후가 좋은 인자이다. In the present invention, a "biomarker" is a substance capable of predicting a brain tumor diagnosis or prognosis, and can be diagnosed by discriminating whether a brain tumor occurs in a biological sample, and used as a prognostic factor of a brain tumor individual having a poor prognosis. It can be used in organic biomolecules such as polypeptides, nucleic acids (e.g. mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) with significant differences between normal and brain tumor individuals. And the like. For the purposes of the present invention, the biomarker is NUPR1, which is increased in brain tumor individuals as compared to normal individuals, especially in malignant brain tumor patients with poor prognosis, and the lower the expression level, the better the prognosis factor.
본 발명에 있어서, “뇌종양”은 뇌 및 중추신경계에서 발생되는 종양으로서 "뇌암(brain cancer)"과 혼용하여 사용될 수 있으며, 뇌종양의 가장 대표적인 질환이며 빈도가 높은 “신경교종(glioma)”과 혼용하여 사용될 수 있다. 상기 뇌종양은 뇌조직이나 뇌를 싸고 있는 뇌막으로부터 발생되는 원발성 뇌종양과, 두개골이나 주변구조물에서 발생하거나 신체의 다른 암으로부터 뇌로 전이된 이차성 뇌종양을 총칭한다. 또한, 뇌종양은 세포타입, 형태학(morphology), 세포유전학(cytogenetics), 분자유전학(molecular genetics), 면역표지자(immunologic markers), 또는 이들의 조합에 의해 분류될 수 있으며, 세계보건기구에 의한 뇌종양의 분류는 종양의 조직학적 특징에 근거한 악성의 스케일에 따라 중추신경계 종양을 분류한다.In the present invention, "brain tumor" is a tumor occurring in the brain and the central nervous system and can be used in combination with "brain cancer", the most representative disease of the brain tumor and mixed with a high frequency "glioma" (glioma) Can be used. The brain tumors collectively refer to primary brain tumors that arise from brain tissue or the brain envelope that envelops the brain, and secondary brain tumors that originate in the skull or surrounding structures, or that metastasize to the brain from other cancers of the body. Brain tumors may also be classified by cell type, morphology, cytogenetics, molecular genetics, immunologic markers, or a combination thereof, and may be classified by the World Health Organization for brain tumors. Classification classifies central nervous system tumors on a scale of malignancy based on the histological characteristics of the tumor.
예를 들어, 뇌종양에는 교모세포종(glioblastomas), 신경교육종(gliosarcoma), 역형성 성상세포종(anaplastic astrocytomas), 희소돌기아교세포종(oligodendrogliomas), 상의세포종(ependymomas), 저등급 성상세포종(low-grade astrocytomas), 수모세포종(medulloblastomas), 성상세포종 종양(astrocytic tumors), 모양세포성 성상세포종 (Pilocytic astrocytoma), 미만성 성상세포종(diffuse astrocytomas), 다형성 황색성상세포종(pleomorphic xanthoastrocytomas), 상의하 거대세포 성상세포종(subependymal giant cell astrocytomas), 역형성 희소돌기아교세포종(anaplastic oligodendrogliomas), 희소돌기성상세포종(oligoastrocytomas), 역형성 희소돌기성상세포종(anaplastic oligoastrocytomas), 점액성 유두상 상의세포종(myxopapillary ependymomas), 상의하세포종(subependymomas), 뇌실상의세포종(ependymomas), 역형성 뇌실상의세포종(anaplastic ependymomas), 아스트로블라스토마(astroblastomas), 제3뇌실의 척삭모양 신경아교종(chordoid gliomas of the third ventricle), 대뇌 신경교종증(gliomatosis cerebris), 글랜글리오사이토마스(glangliocytomas), 결합조직생성 유아 성상세포종(desmoplastic infantile astrocytomas), 결합조직생성 유아신경절교종(desmoplastic infantile gangliogliomas), 태생기발육부전 신경상피종(dysembryoplastic neuroepithelial tumors), 상의모세포종(ependymoblastomas), 천막위 원시신경외배엽성 종양(supratentorial primitive neuroectodermal tumors), 맥락막총 유두종(choroids plexus papilloma), 중등도 분화형의 솔방울샘 실질 종양(pineal parenchymal tumors of intermediate differentiation), 혈관주위세포종(hemangiopericytomas), 안장영역의 종양(tumors of the sellar region), 두개인두종(craniopharyngioma), 혈관모세포종(capillary hemangioblastoma), 또는 일차성 중추신경계 임파종(primary CNS lymphoma)이 포함되며, 이에 제한되지 않는다. For example, brain tumors include glioblastomas, glioblastoma, anaplastic astrocytomas, oligodendrogliomas, ependymomas, and low-grade astrocytomas. astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, pleomorphic xanthoastrocytomas, epidermoid astrocytomas (subependymal giant cell astrocytomas), anaplastic oligodendrogliomas, oligoastrocytomas, anaplastic oligoastrocytomas, myxopapillary ependymomas Subependymomas, ependymomas, anaplastic ependymomas, astroblas Astroblastomas, chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, degenerated astrocytomas (desmoplastic infantile astrocytomas) ), Connective tissue-forming infantile gangliogliomas, dysembryoplastic neuroepithelial tumors, epidemic moblastomas, supratentorial primitive neuroectodermal tumors, choroid plexus (choroids plexus papilloma), pineal parenchymal tumors of intermediate differentiation, hemangiopericytomas, tumors of the sellar region, craniopharyngioma, hemangioblastoma (capillary hemangioblastoma), or primary CNS lymphoma. , But it is not limited thereto.
또한, 뇌종양은 분자적 측면에서 4가지 아형으로 구분될 수 있으며, 보다 구체적으로는, DNA 메틸화 양상, 신호 전달 과정 활성화 정도, 생존 및 치료 반응 등의 임상적 수치를 기준으로 전신경 아류형(proneural subtype), 간엽성 아류형(mesenchymal subtype), 고전적 아류형(classical subtype) 또는 신경형 아류형(neural subtype)으로 구분될 수 있다. 상기 전신경 아류형(proneural subtype)에는 G-CIMP 아류형이 포함되며, 최근에는 이를 분리 구분하여 5가지 아형으로 구분하는 사례도 존재한다. 상기 4가지 아형 중 전신경 아류형(proneural subtype)의 예후가 가장 좋은 것으로 알려져있으며, 간엽성 아류형(mesenchymal subtype)의 예후가 가장 나쁜 것으로 알려져있고, 본 발명에 따른 NUPR1, 특히 NUPR1 동형(isoform) A는 전신경 아류형에 비해 간엽성 아류형에서 더 높은 발현 양상을 나타내는 특징을 보인다. 즉, 본 발명에 따른 NUPR1, 특히 NUPR1 동형 A는 동일한 질환을 앓고 있는 뇌종양 개체 중에서도 예후가 나쁜 뇌종양 개체에서 현저하게 증가하는바, 단순하게 뇌종양 발병 여부를 진단하는 것뿐만 아니라 아형을 구분할 수 있어 이를 이용한 예후 예측이 가능하다. In addition, brain tumors can be divided into four subtypes in molecular aspect, and more specifically, proneural based on clinical values such as DNA methylation pattern, degree of signal transduction activation, survival and therapeutic response. subtype, mesenchymal subtype, classic subtype, or neural subtype. The proneural subtype includes the G-CIMP subtype, and recently, there are also cases of dividing it into five subtypes. Among the four subtypes, the prognosis of the proneural subtype is known to be the best, and the prognosis of the mesenchymal subtype is known to be the worst, and the NUPR1, in particular, the NUPR1 isoform according to the present invention. ) A is characterized by a higher expression pattern in the mesenchymal subtype than the systemic subtype. That is, NUPR1, in particular NUPR1 isoform A, according to the present invention is significantly increased in brain tumor individuals with poor prognosis among brain tumor individuals suffering from the same disease, and can not only diagnose brain tumors but also distinguish subtypes. Prognosis can be used.
다른 양태로, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성 수준을 측정하는 제제를 포함하는 뇌종양 진단 또는 예후 예측용 조성물을 제공한다. In another aspect, the present invention provides a composition for diagnosing brain tumor or predicting prognosis comprising an agent for measuring the expression or activity level of Nuclear protein 1 (NUPR1).
본 발명의 일 실시예에 따르면, NUPR1, 특히 NUPR1 동형 A가 건강한 정상인에 비해 뇌종양 환자에서 유의하게 증가하며, 특히 예후가 나쁜 악성 뇌종양 환자에서 더욱 현저하게 증가함을 확인하였는바, NUPR1, 특히 NUPR1 동형 A 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정함으로써 뇌종양 진단 또는 예후 예측이 가능하다. According to one embodiment of the present invention, NUPR1, particularly NUPR1 isoform A, was significantly increased in brain tumor patients compared to healthy normal subjects, particularly in patients with malignant brain tumors with poor prognosis, NUPR1, in particular NUPR1 Diagnosis or prognosis of brain tumors is possible by measuring mRNA expression of isoform A gene or its protein activity level.
본 발명에 있어서, "NUPR1 유전자의 mRNA 발현 수준 측정"은 뇌종양의 진단 또는 예후 예측을 위하여 생물학적 시료에서 바이오 마커 유전자의 존재 여부와 발현 정도를 확인하는 과정으로, 표적 유전자로부터 전사된 mRNA의 수준을 측정하는 방법에 사용되는 제제를 이용해 mRNA의 양을 측정한다. 구체적으로, 본 발명에서 NUPR1 유전자의 mRNA 수준을 측정하는 제제는 바람직하게는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이며, 상기 NUPR1을 암호화하는 유전자의 염기 서열이 유전자 은행에 등록되어 있으므로 당업자는 상기 서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 디자인할 수 있다.In the present invention, "measurement of mRNA expression level of NUPR1 gene" is a process of confirming the presence and expression level of a biomarker gene in a biological sample for diagnosis or prognosis of brain tumor. The amount of mRNA is measured using the agent used in the measurement method. Specifically, the agent for measuring the mRNA level of the NUPR1 gene in the present invention is preferably an antisense oligonucleotide, primer pair or probe, since the base sequence of the gene encoding the NUPR1 is registered in the gene bank, those skilled in the art Based on this, antisense oligonucleotides, primer pairs or probes can be designed that specifically amplify specific regions of these genes.
본 발명에 있어서, "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어 표적 서열 내에서 전형적으로 mRNA와 헤테로이중체를 형성할 수 있는 뉴클레오티드 염기서열 및 서브유닛간 백본을 갖는 올리고머를 지칭한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 유사 상보성을 가질 수 있다. In the present invention, an "antisense" is a backbone between subunits and nucleotide sequences where antisense oligomers can hybridize with target sequences in RNA by Watson-Crick base pairing to form heterodimers with mRNA typically within the target sequence. Refers to an oligomer having The oligomer may have precise sequence complementarity or similar complementarity to the target sequence.
본 발명에 있어서, "프라이머"는 짧은 자유 3` 말단 수산화기(free 3` hydroxyl group)를 가지는 핵산 서열로 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고 주형의 복사를 위한 시작지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 중합효소 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다. 본 발명에서 프라이머는 NUPR1 유전자의 증폭에 사용될 수 있는 모든 프라이머를 의미하며, 상기 유전자와 상보적으로 결합하여 증폭시킬 수 있는 한, 프라이머의 서열은 제한되지 않는다. In the present invention, a "primer" is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and starts for copying the template. By a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, the primer means all primers that can be used for amplification of the NUPR1 gene, and the sequence of the primer is not limited as long as it can bind and amplify complementarily with the gene.
본 발명에 있어서, "프로브"는 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며, 표지(Labelling)되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 상기 프로브는 올리고 뉴클레오티드 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있으며, 상기 유전자와 상보적으로 결합할 수 있는 한, 프로브의 서열은 제한되지 않는다. In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which is capable of specific binding with mRNA. You can check the presence. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, and the like, as long as the probe may complementarily bind to the gene. The sequence of is not limited.
본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미 데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidates, carbamates, etc.) or charged linkages (eg, phosphorothioates, phosphorodithioates, etc.).
본 발명에 있어서, "NUPR1 단백질 활성 수준 측정"은 뇌종양의 진단 또는 예후 예측을 위하여 생물학적 시료에서 마커 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 바람직하게는 NUPR1 단백질에 대하여 특이적으로 결합하는 항체를 이용해 단백질의 양을 확인할 수 있다. In the present invention, "NUPR1 protein activity level measurement" is a process of confirming the presence and expression level of the marker protein in a biological sample for the diagnosis or prognosis of brain tumor, preferably binding specifically to the NUPR1 protein Antibodies can be used to determine the amount of protein.
본 발명에 있어서, "항체"란 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 NUPR1에 특이적으로 결합하는 항체를 의미하며, 이러한 항체는 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함되며, 본 발명의 부분 펩티드로는, 최소한 7개 아미노산, 바람직하게는 9개 아미노산, 더욱 바람직하게는 12개 이상의 아미노산을 포함한다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수항체도 포함된다. 본 발명에 사용되는 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합기능을 보유하고 있는 단편을 뜻하며 Fab, F(ab'), F(ab') 2 및 Fv 등이 있다.In the present invention, "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to NUPR1, which antibody is cloned into an expression vector according to a conventional method to obtain a protein encoded by the gene, and from the protein obtained It can be prepared by the phosphorus method. Also included are partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.
다른 양태로, 본 발명은 상기 조성물을 포함하는 뇌종양 진단 또는 예후 예측용 키트를 제공한다.In another aspect, the present invention provides a kit for diagnosing or predicting a brain tumor comprising the composition.
본 발명의 키트에는 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 제제 외에, 분석방법에 적합한 한 종류 이상의 다른 구성성분 조성물, 용액 또는 장치가 포함될 수 있으며, 어떠한 형태로든 본 발명의 범위를 제한하지 않는다. Kits of the present invention may include one or more other component compositions, solutions or devices suitable for analytical methods, in addition to agents for measuring mRNA expression or protein activity levels of the NUPR1 gene, which in any form limit the scope of the present invention. I never do that.
구체적인 일례로서, 본 발명에서 상기 NUPR1 유전자의 mRNA 발현 수준을 측정하기 위한 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. 상기 RT-PCR 키트는 마커 유전자에 대한 특이적인 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소 및 역전사효소와 같은 효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다.As a specific example, the kit for measuring the mRNA expression level of the NUPR1 gene in the present invention may be a kit containing the necessary elements necessary to perform RT-PCR. The RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water and the like.
또한, 본 발명의 키트는 마이크로어레이 칩을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. 상기 마이크로어레이 칩 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조구 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있으며, 본 발명의 마커를 이용하여 당업계에서 통상적으로 사용되는 제조 방법에 의하여 용이하게 제조될 수 있다. 마이크로어레이 칩을 제작하기 위해서, 상기 탐색된 마커를 탐침 DNA 분자로 이용하여 DNA 칩의 기판상에 고정화시키기 위해 파이조일렉트릭(piezoelectric) 방식을 이용한 마이크로피펫팅(micropipetting)법 또는 핀(pin) 형태의 스폿터(spotter)를 이용한 방법 등을 사용하는 것이 바람직하나 이에 한정되는 것은 아니다. 상기 마이크로어레이 칩의 기판은 아미노-실란(amino-silane), 폴리-L-라이신(poly-Llysine) 및 알데히드(aldehyde)로 이루어진 군에서 선택되는 활성기가 코팅된 것이 바람직하나, 이에 한정되는 것은 아니다. 또한, 상기 기판은 슬라이드 글래스, 플라스틱, 금속, 실리콘, 나일론 막 및 니트로셀룰로스 막(nitrocellulose membrane)으로 이루어진 군에서 선택되는 것이 바람직하나 이에 한정되는 것은 아니다.In addition, the kit of the present invention may be a kit including essential elements necessary for performing the microarray chip. The microarray chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached by a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof, using the marker of the present invention. It can be easily prepared by the manufacturing method commonly used in the art. In order to fabricate a microarray chip, a micropipetting method or a pin type using a piezoelectric method for immobilizing the searched marker as a probe DNA molecule on a substrate of a DNA chip. It is preferable to use a method using a spotter such as but not limited thereto. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto. . In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
또한, 본 발명에서 상기 NUPR1 단백질 활성 수준을 측정하기 위한 키트는 항체의 면역학적 검출을 위하여 기질, 적당한 완충용액, 발색 효소 또는 형광물질로 표지된 2차 항체, 및 발색 기질 등을 포함할 수 있다. 상기에서 기질은 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스틸렌 수지로 합성된 96 웰 플레이트 및 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등이 사용될 수 있고, 형광물질은 FITC, RITC등이 사용될 수 있고, 발색기질은 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)) 또는 OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘)가 사용될 수 있다.In addition, the kit for measuring the NUPR1 protein activity level in the present invention may include a substrate, a suitable buffer, a secondary antibody labeled with a coloring enzyme or a fluorescent substance, and a coloring substrate for immunological detection of the antibody. . The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase or an alkaline force. Fatase (alkaline phosphatase) and the like can be used, the fluorescent material may be used FITC, RITC, etc., the color substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) )) Or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.
다른 양태로, 본 발명은 생물학적 시료로부터 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계;를 포함하는 뇌종양 진단 또는 예후 예측을 위한 정보제공방법을 제공한다. In another aspect, the present invention provides a method for providing information for diagnosing or predicting a brain tumor, including measuring mRNA expression of a NUPR1 gene or a protein activity level thereof from a biological sample.
본 발명의 일 실시예에 따르면, 음성대조군에 비해 뇌종양 환자에서 조직 내 NUPR1, 특히 NUPR1 동형 A 유전자의 mRNA 발현 또는 이의 단백질 활성 정도가 증가하고, 특히 예후가 나빠 생존 시간이 짧은 악성 뇌종양 환자에서 조직 내 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성이 현저하게 높게 나타남을 확인하였다. 따라서 생물학적 시료에서 NUPR1의 발현 분석을 통해 뇌종양 진단 또는 예후 예측을 위한 정보를 제공할 수 있다.According to one embodiment of the present invention, compared to the negative control group, the expression of mRNA or protein activity of NUPR1, particularly NUPR1 isoform A gene, is increased in tissues in brain tumor patients, especially in patients with malignant brain tumors having a short survival time due to poor prognosis. MRNA expression of the NUPR1 gene or its protein activity was found to be significantly higher. Thus, expression analysis of NUPR1 in biological samples can provide information for diagnosing brain tumors or predicting prognosis.
본 발명에 있어서, “진단 또는 예후 예측을 위한 정보제공방법”은 진단 또는 예후 예측을 위한 예비적 단계로써 뇌종양의 진단 또는 예후 예측을 위하여 필요한 객관적인 기초정보를 제공하는 것이며 의사의 임상학적 판단 또는 소견은 제외된다. In the present invention, the "information providing method for diagnosis or prognosis prediction" is a preliminary step for diagnosis or prediction of prognosis and provides objective basic information necessary for the diagnosis or prognosis of brain tumor and the clinical judgment or findings of a doctor. Is excluded.
본 발명에 있어서, "생물학적 시료"란 개체로부터 분리되어 목적 유전자 또는 단백질의 발현 수준을 측정하는 직접적인 대상을 의미하고, 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨와 같은 시료 등을 포함한다. 예를 들어, 본 발명의 시료란 뇌종양 의심 개체에서 뇌종양 발병 여부를 진단하기 위한 시료 또는 뇌종양에 대한 수술적 또는 화학적 치료를 받은 개체로부터 치료 후의 임상 결과, 즉 재발 가능성 및 생존 예후를 예측하는데 사용되는 시료로서, 바람직하게는 뇌 조직 또는 이로부터 분리한 세포이나 이에 제한되지 않는다.In the present invention, "biological sample" means a direct subject separated from an individual and measuring the expression level of a gene or protein of interest, and a sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine. And the like. For example, a sample of the present invention may be used to predict clinical outcome after treatment, i.e., recurrence and survival prognosis, from a sample for diagnosing the onset of a brain tumor in a suspicious brain tumor or from a subject undergoing surgical or chemical treatment for a brain tumor. As the sample, preferably brain tissue or cells isolated therefrom are not limited thereto.
본 발명에 있어서, "mRNA 발현 수준을 측정하기 위한 분석 방법"에는 중합효소반응(PCR), 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Realtime RT-PCR), RNase 보호분석법(RPA; RNase protection assay), 노던 블랏팅(northern blotting), 또는 DNA 마이크로어레이 분석법 등이 있으나, 이에 제한되지 않는다.In the present invention, the "analytical method for measuring mRNA expression level" includes a polymerase reaction (PCR), a reverse transcriptase polymerase reaction (RT-PCR), a competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), a real time reverse transcriptase polymerase. Realtime RT-PCR, RNase protection assay (RPA), northern blotting, or DNA microarray analysis.
본 발명에 있어서, "단백질 활성 수준을 측정하기 위한 분석 방법"에는 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(Radioimmunoassay), 방사면역 확산법(Radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(Rocket) 면역전기영동, 조직면역염색, 면역 침전분석법(immunoprecipitation assay), 보체 고정 분석법(complete fixation assay), 유세포분석법(Fluorescence Activated Cell Sorter, FACS) 또는 단백질 칩(protein chip) 분석법 등이 있으나, 이에 제한되지 않는다.In the present invention, the "analytical method for measuring protein activity level" includes western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, and ouktero. Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis, but is not limited thereto.
본 발명의 방법은 상기 측정된 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 대조군에서 측정된 수준과 비교하는 단계를 포함할 수 있다. The method of the present invention may comprise comparing the measured mRNA expression or protein activity level of the NUPR1 gene with the level measured in the control.
상기 대조군은 음성대조군(정상군) 또는 양성대조군(뇌종양 환자군)을 포함한다. The control group includes a negative control group (normal group) or a positive control group (brain tumor patient group).
상기 양성대조군은 예후가 나쁜 것으로 알려진 개체일 수 있으며, 예를 들어 뇌종양의 발병 후 전이, 사망 등의 이력이 발생한 개체를 의미한다. 또한, 상기 양성대조군은 공지된 데이터베이스를 통해 생존 정보를 확인할 수 있는 개체일 수 있으며, 바람직하게는 생존값이 평균 이하인 뇌종양 환자일 수 있다. The positive control group may be an individual known to have a poor prognosis, for example, an individual having a history of metastasis or death after the onset of a brain tumor. In addition, the positive control group may be an individual capable of confirming survival information through a known database, and preferably may be a brain tumor patient whose survival value is below average.
본 발명의 방법에 있어서, NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준 수준이 음성대조군(정상군)보다 높을 경우, 뇌종양, 특히 예후가 나쁜 악성 뇌종양이 이미 발병하였거나 또는 발병할 위험성이 높은 것으로 판단할 수 있다. In the method of the present invention, when the mRNA expression of the NUPR1 gene or its protein activity level is higher than that of the negative control group (normal group), it is determined that a brain tumor, particularly a malignant brain tumor with a poor prognosis, is already at risk or high risk of developing. Can be.
또한, 본 발명의 방법에 있어서, NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준 수준이 양성대조군(뇌종양 환자군)보다 높을 경우, 상대적으로 예후가 나쁠 것으로 판단할 수 있으며, 양성대조군(뇌종양 환자군)보다 낮을 경우, 상대적으로 예후가 좋을 것으로 판단할 수 있다. In addition, in the method of the present invention, when the mRNA expression of the NUPR1 gene or its protein activity level is higher than that of the positive control group (brain tumor patient group), it may be determined that the prognosis is relatively poor and lower than that of the positive control group (brain tumor patient group). In this case, the prognosis is relatively good.
또 다른 양태로, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 치료용 약학적 조성물을 제공한다. 또한, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 개선용 식품 조성물을 제공한다. In another aspect, the present invention provides a pharmaceutical composition for preventing or treating brain tumors, including an inhibitor of NUPR1 (Nuclear protein 1) expression or activity. In another aspect, the present invention provides a food composition for preventing or improving brain tumors, including the expression or activity inhibitor of NUPR1 (Nuclear protein 1).
또한, 본 발명은 NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 이를 필요로하는 개체에 투여하는 단계를 포함하는, 뇌종양의 예방 또는 치료 방법을 제공한다. The present invention also provides a method for preventing or treating brain tumors, comprising administering an expression or activity inhibitor of NUPR1 (Nuclear protein 1) to a subject in need thereof.
본 발명에 있어서, "NUPR1(Nuclear protein 1)의 발현 또는 활성억제제"는 NUPR1의 발현 또는 활성을 감소시키는 물질을 통칭하는 의미로 사용되며, 보다 구체적으로는 NUPR1에 직접적으로 작용하거나 그의 리간드에 간접적으로 작용하는 등의 방식을 통해 NUPR1의 발현을 전사 수준에서 감소시키거나 그 활성을 방해함으로써 NUPR1의 발현 또는 활성을 감소시키는 모든 물질을 포함할 수 있다. 상기 NUPR1 발현을 저해하는 물질은 NUPR1을 표적으로 하여 NUPR1의 발현 또는 활성을 억제할 수 있는 화합물, 핵산, 펩타이드, 바이러스 또는 상기 핵산을 포함하는 벡터 등 그 형태에 제한없이 사용 가능하다. NUPR1 발현을 저해하는 물질의 예로, NUPR1의 mRNA에 특이적으로 결합하여 NUPR1 mRNA의 발현을 저해하는 siRNA, shRNA, miRNA, 안티센스 올리고뉴클레오티드, 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids) 등이 포함되며, 바람직하게는 siRNA, shRNA, miRNA, 안티센스 올리고뉴클레오티드일 수 있고, 이에 제한되지 않는다. 또한, NUPR1 활성을 저해하는 물질의 예로, NUPR1의 단백질에 특이적으로 결합하여 NUPR1 단백질의 활성을 억제하는 항체 또는 그의 항원 결합 단편, 앱타머, 화합물 등이 포함되며, 바람직하게는 항체일 수 있고, 이에 제한되지 않는다. In the present invention, "expression or activity inhibitor of NUPR1 (Nuclear protein 1)" is used as a generic term for a substance that reduces the expression or activity of NUPR1, and more specifically, directly acts on NUPR1 or indirectly to its ligand. It may include any substance that reduces the expression or activity of NUPR1 by reducing the expression of NUPR1 at the transcription level or by inhibiting its activity, such as by acting. The substance that inhibits the expression of NUPR1 can be used without limitation in the form of a compound, a nucleic acid, a peptide, a virus or a vector containing the nucleic acid that can target NUPR1 and inhibit the expression or activity of NUPR1. Examples of substances that inhibit NUPR1 expression include siRNAs, shRNAs, miRNAs, antisense oligonucleotides, ribozymes, DNAzyme, peptide nucleic acids (PNAs) that specifically bind to NUPR1 mRNA and thereby inhibit the expression of NUPR1 mRNA. This includes, but is not limited to, siRNA, shRNA, miRNA, antisense oligonucleotides. In addition, examples of a substance that inhibits NUPR1 activity include an antibody or an antigen-binding fragment thereof, an aptamer, a compound thereof, or the like that specifically binds to the protein of NUPR1 to inhibit the activity of the NUPR1 protein, and preferably may be an antibody. This is not restrictive.
본 발명에 있어서, "siRNA(small interference RNA)"는 RNA 방해 또는 유전자 사일런싱을 매개할 수 있는 핵산 분자로서, 21 내지 25 뉴클레오티드 크기의 작은 RNA 조각을 의미한다. 본 발명의 siRNA는 센스 가닥(mRNA 서열에 상응하는(corresponding) 서열)과 안티센스 가닥(mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조를 가질 수 있으며, 자기-상보성(self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다. 본 발명의 siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 미스매치(대응하는 염기가 상보적이지 않음), 벌지(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. 본 발명의 일 실시예에서는 서열번호 3 및 4, 서열번호 5 및 6, 서열번호 7 및 8, 서열번호 9 및 10, 또는 서열번호 11 및 12로 이루어진 siRNA를 이용하였으나, 상기 siRNA가 NUPR1 mRNA의 발현을 저해할 수 있다면 그 서열은 특별히 제한되지 않는다. In the present invention, "small interference RNA" (siRNA) is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and refers to a small RNA fragment of 21 to 25 nucleotides in size. The siRNA of the present invention may have a double-stranded structure in which the sense strand (corresponding sequence) and the antisense strand (sequence complementary to the mRNA sequence) are positioned opposite to each other and form a double-stranded structure. -complementary) can have a single chain structure with sense and antisense strands. The siRNA of the present invention is not limited to the complete pairing of double-stranded RNA portions paired with RNA, but by mismatching (corresponding base is not complementary), bulge (no base corresponding to one chain), or the like. Unpaired parts may be included. In one embodiment of the present invention was used siRNA consisting of SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, or SEQ ID NO: 11 and 12, the siRNA of the NUPR1 mRNA If the expression can be inhibited, the sequence is not particularly limited.
본 발명에 있어서, "shRNA(short hairpin RNA)"는 siRNA의 고가의 생합성 비용, 낮은 세포 형질감염 효율로 인한 RNA 간섭 효과의 단시간 유지 등의 단점을 극복하기 위한 것으로 RNA 중합효소 Ⅲ의 프로모터로부터 아데노바이러스, 렌티 바이러스 및 플라스미드 발현 벡터 시스템을 이용하여 이를 세포 내로 도입하여 발현시킬 수 있으며, 이러한 shRNA는 세포 내에 존재하는 siRNA 프로세싱 효소(Dicer or Rnase Ⅲ)에 의해 정확한 구조를 갖는 siRNA로 전환되어 목적 유전자의 사일런싱을 유도함이 널리 알려져 있다.In the present invention, "shRNA (short hairpin RNA)" is to overcome the disadvantages such as the high cost of biosynthesis of siRNA, short-term maintenance of RNA interference effect due to low cell transfection efficiency, and adeno from the promoter of RNA polymerase III. Viruses, lentiviruses, and plasmid expression vector systems can be used to introduce and express them into cells, and these shRNAs are converted into siRNAs with the correct structure by siRNA processing enzymes (Dicer or Rnase III) present in the cells. It is well known to induce silencing of.
본 발명에 있어서, "miRNA(microRNA)"는 21-25 뉴클레오타이드의 단일 가닥 RNA 분자로써, 타겟 mRNA의 파쇄 또는 해독단계에서의 억제를 통하여 진핵생물의 유전자 발현을 제어하는 조절물질이다. 이러한 miRNA는 두 단계의 프로세싱으로 이루어진다. 최초의 miRNA 전사체(primary miRNA)가 핵 안에서 Drosha라는 RNaseⅢ 타입효소에 의해 70-90 염기 정도의 스템-루프 구조, 즉 pre-miRNA로 만들어지고, 이후 세포질로 이동하여 다이서(Dicer)라는 효소에 의해 절단되어 21-25 염기의 성숙한 miRNA로 만들어진다. 이렇게 생성된 miRNA는 표적 mRNA에 상보적으로 결합하여 전사 후 유전자 억압자(post-transcriptional gene suppressor)로써 작용하며, 번역 억제와 mRNA 불안정화를 유도한다. miRNAs는 다양한 생리학적 현상 및 질환에 관여한다.In the present invention, "miRNA (microRNA)" is a single-stranded RNA molecule of 21-25 nucleotides, and is a regulator that controls gene expression in eukaryotes through inhibition of the disruption or translation of target mRNAs. This miRNA consists of two stages of processing. The first miRNA transcript (primary miRNA) is made into a 70-90 base stem-loop structure, or pre-miRNA, by an RNaseIII type enzyme called Drosha in the nucleus, which then migrates into the cytoplasm and is called Dicer. Is cleaved into a mature miRNA of 21-25 bases. The miRNA thus produced complementarily binds to the target mRNA and acts as a post-transcriptional gene suppressor, inducing translation inhibition and mRNA destabilization. miRNAs are involved in a variety of physiological phenomena and diseases.
본 발명에 있어서, "안티센스 올리고뉴클레오티드"는 특정 mRNA의 서열에 상보적인 핵산 서열을 함유하고 있는 DNA 또는 RNA 또는 이들의 유도체를 의미하는데, mRNA 내의 상보적인 서열에 결합하여 mRNA의 단백질로의 번역을 저해하는 효과를 나타낸다. In the present invention, "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a particular mRNA, binding to the complementary sequence in the mRNA to translate the mRNA into a protein It shows an inhibitory effect.
본 발명에 있어서, "앱타머"는 소정의 표적 분자에 대한 결합 활성을 갖는 핵산 분자를 의미한다. 상기 앱타머는 RNA, DNA, 수식(modified) 핵산 또는 이들의 혼합물일 수 있으며, 직쇄상 또는 환상의 형태일 수 있는데, SELEX(systematic evolution of ligands by exponential enrichment)라 불리는 올리고뉴클레오타이드(oligonucleotide) 라이브러리를 이용한 진화적인 방법에 의해 특정 화학 분자나 생물학적 분자에 높은 친화력과 선별력을 갖고 결합하는 올리고머를 분리하여 수득되는 물질이다. 상기 앱타머는 표적에 특이적으로 결합하고 표적의 활성을 조정할 수 있는데, 예컨대, 결합을 통하여 표적이 기능한 능력을 차단할 수 있다.In the present invention, "aptamer" means a nucleic acid molecule having binding activity to a given target molecule. The aptamers may be RNA, DNA, modified nucleic acids, or mixtures thereof, and may be in linear or cyclic form, using an oligonucleotide library called systemic evolution of ligands by exponential enrichment (SELEX). It is a substance obtained by isolating oligomers which have high affinity and selectivity for binding to specific chemical or biological molecules by evolutionary methods. The aptamer can specifically bind to the target and modulate the target's activity, for example, by blocking the ability of the target to function through binding.
본 발명에 있어서, "예방"은 조성물의 투여에 의해 뇌종양의 발병을 억제시키거나 또는 지연시키는 모든 행위를 의미한다.In the present invention, "prevention" means any action that inhibits or delays the onset of brain tumors by administration of the composition.
본 발명에 있어서, "치료"는 조성물의 투여에 의해 뇌종양의 발생, 전이 또는 재발을 예방하고 증상을 완화시키며, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키고, 질병 진행 속도를 감소시키며, 질병 상태를 경감 또는 일시적 완화시키고, 차도시키거나 예후를 개선시키는 것이 모두 포함된다.In the present invention, “treatment” prevents the occurrence, metastasis or recurrence of brain tumors and alleviates the symptoms by administering the composition, lowers all direct or indirect pathological consequences of the disease, reduces the rate of disease progression and These include alleviating or temporarily mitigating a disease state, driving off or improving the prognosis.
본 발명에 있어서, "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.In the present invention, "improvement" means any action that at least reduces the parameters associated with the condition being treated, for example the extent of symptoms.
본 발명의 일 실시예에서는 NUPR1, 특히 NUPR1 동형 A(varient 1, v.1)의 발현을 억제함으로써 뇌종양의 성장, 전이 및 침입을 억제할 수 있을 뿐만 아니라 예후가 좋은 서브타입으로 전환시키는 효과가 나타남을 확인하였는바, NUPR1 억제제는 뇌종양 예방, 개선, 또는 치료를 위한 의약품 및 건강기능식품으로 활용될 수 있다. In one embodiment of the present invention, by inhibiting the expression of NUPR1, in particular NUPR1 isoform A (varient 1, v.1), not only can the growth, metastasis and invasion of brain tumors be suppressed, but also the effect of converting into a prognosis subtype is It has been confirmed that the NUPR1 inhibitor can be used as a medicine and dietary supplement for the prevention, improvement, or treatment of brain tumors.
본 발명의 조성물은 NUPR1의 발현 또는 활성억제제와 함께 뇌종양에 대한 치료 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다. The composition of the present invention may further contain one or more known active ingredients having a therapeutic effect on brain tumors, together with the expression or activity inhibitor of NUPR1.
본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 본 발명의 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Carriers, excipients and diluents which may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학적 조성물은 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 당해 기술 분야에 알려진 적합한 제제는 문헌 (Remington's Pharmaceutical Science, 최근, Mack Publishing Company, Easton PA)에 개시되어 있는 것을 사용하는 것이 바람직하다. 구체적으로, 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있다. 예를 들어, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로오스, 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제에는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 상기 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. The pharmaceutical composition of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions according to conventional methods. . Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Specifically, when formulating the composition can be prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants. For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명에 있어서, "투여"는 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다. 본 발명의 약학적 조성물의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르며, 당업자에 의해 적절하게 선택될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.In the present invention, "administration" means providing a subject with a composition of the present invention in any suitable manner. Preferred dosages of the pharmaceutical compositions of the present invention depend on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. The dosage does not limit the scope of the invention in any aspect.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 뇌 내 주사에 의해 투여될 수 있다. 다만, 경구 투여 시에는 위산에 의하여 상기 조성물이 변성될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 될 수 있다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intracranial injection. However, since oral administration may denature the composition by gastric acid, oral compositions may be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.
본 발명의 약학적 조성물은 뇌종양의 예방 또는 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of brain tumors.
또한, 본 발명의 NUPR1의 발현 또는 활성억제제는 뇌종양의 예방 또는 개선을 목적으로 식품 조성물, 바람직하게는 건강기능식품에 첨가될 수 있다. In addition, the expression or activity inhibitor of NUPR1 of the present invention can be added to food compositions, preferably functional foods for the purpose of preventing or improving brain tumors.
본 발명에 있어서, ‘건강기능식품’이란 질병의 예방 또는 개선, 생체방어, 면역, 병후의 회복, 노화 억제 등 생체조절기능을 가지는 식품을 말하는 것으로, 장기적으로 복용하였을 때 인체에 무해하여야 한다. In the present invention, "health functional food" refers to a food having a bioregulatory function, such as prevention or improvement of disease, biological defense, immunity, recovery of symptoms, inhibition of aging, and should be harmless to the human body when taken in the long term.
본 발명의 식품 조성물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있으며, 상기 식품의 종류에는 특별한 제한은 없다. When the food composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment), and there is no particular limitation on the type of food.
본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다. The food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents used in beverages and the like.
또 다른 양태로, 본 발명은 (a) 분리된 뇌종양 세포에 뇌종양 치료 후보물질을 처리하는 단계; 및 (b) 상기 뇌종양 세포에서 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계를 포함하는 뇌종양 치료제의 스크리닝 방법을 제공한다. In another aspect, the present invention comprises the steps of (a) treating a brain tumor treatment candidate to isolated brain tumor cells; And (b) measuring the mRNA expression of the NUPR1 gene or its protein activity level in the brain tumor cells.
본 발명에서 상기 (a) 단계의 뇌종양 세포는 NUPR1을 발현하는 세포이다. In the present invention, the brain tumor cells of step (a) are cells expressing NUPR1.
본 발명에서 상기 (a) 단계의 후보물질은 뇌종양을 치료할 수 있을 것으로 예상되는 물질 또는 그 예후를 개선시킬 수 있을 것으로 기대되는 미지의 물질로서, 화합물, 단백질 또는 천연물 추출물을 포함하나, 이에 한정되는 것은 아니다.In the present invention, the candidate of step (a) is a substance that is expected to be able to treat brain tumors or an unknown substance that is expected to improve the prognosis, and includes a compound, protein or natural product extract, but is not limited thereto. It is not.
본 발명의 방법은 (c) 상기 (b) 단계에서 측정된 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준이 후보물질을 처리하지 않은 뇌종양 세포에 비해 낮은 수준을 나타내는 경우, 상기 후보물질을 뇌종양 치료제로 사용할 수 있을 것으로 판단하는 단계를 포함한다. The method of the present invention (c) when the mRNA expression or protein activity level of the NUPR1 gene measured in step (b) shows a lower level than the brain tumor cells not treated with the candidate material, the candidate material as a brain tumor treatment agent Determining that it can be used.
한편, 상기 유전자의 mRNA 또는 이의 단백질 수준을 측정하는 방법은 상술한 바와 동일하다.On the other hand, the method of measuring the mRNA or protein level of the gene is the same as described above.
이하, 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.
실시예 1. 정상군 및 뇌종양 환자군에서 NUPR1 동형(isoform) A의 발현 분석Example 1 Analysis of Expression of NUPR1 Isoform A in Normal and Brain Tumor Patients
다양한 진단명을 가진 다수의 환자로부터 뇌 조직 샘플을 분리한 후, 웨스턴 블랏을 통해 NUPR1 동형 A(varient 1, v.1) 단백질 발현 정도를 분석하였다. 대조군으로 이용한 SVG p12는 인간 유래 성상세포(astroglia)로서 정상 세포를 의미한다. NUPR1 동형 A에 특이적인 1차 항체는 후술하는 실시예 2-4에서 제조한 다클론항체를 이용하였다. 정량을 위한 하우스킵핑 단백질로 β-액틴을 이용하였으며, 뇌종양 환자 정보는 하기 표 1에 나타내었다. 그 결과를 도 1에 나타내었다. Brain tissue samples were isolated from multiple patients with various diagnostic names and analyzed by Western blot for the expression of NUPR1 isoform A (v.1) protein. SVG p12 used as a control means normal cells as human derived astroglia. As a primary antibody specific for NUPR1 isoform A, a polyclonal antibody prepared in Example 2-4 described below was used. Β-actin was used as a housekeeping protein for quantification, and brain tumor patient information is shown in Table 1 below. The results are shown in FIG.
| IDID | 성별gender | 나이age | 진단명Diagnosis Name |
| GBM-12GBM-12 | MaleMale | 4646 | Glioblastoma (grade IV)Glioblastoma (grade IV) |
| GBM-14GBM-14 | MaleMale | 5252 | Anaplastic Astrocytoma (grade III)Anaplastic Astrocytoma (grade III) |
| GBM-15GBM-15 |
Female |
3737 | Glioblastoma (grade IV)Glioblastoma (grade IV) |
| GBM-30GBM-30 | FemaleFemale | 4646 | Glioblastoma (grade IV)Glioblastoma (grade IV) |
도 1에 나타낸 바와 같이, 정상 세포인 SVG p12 샘플에서 NUPR1 동형 A 단백질이 거의 발현되지 않는데 반해, 뇌종양 환자인 GBM-12, GBM-14, GBM-15 및 GBM-30 샘플에서는 NUPR1 동형 A 단백질 발현이 현저하게 높게 나타남을 확인하였다. As shown in FIG. 1, NUPR1 isoform A protein is hardly expressed in the SVG p12 sample which is normal cells, whereas NUPR1 isoform A protein is expressed in the GBM-12, GBM-14, GBM-15 and GBM-30 samples of brain tumor patients. It was confirmed that this is markedly high.
이상에 개시한 실시예 1의 실험을 통하여, NUPR1 동형 A(varient 1, v.1)는 건강한 정상군에 비해 뇌종양 환자군에서 더 높게 발현되는바, 이를 뇌종양 진단 마커로 활용할 수 있음을 확인하였다. Through experiments of Example 1 described above, NUPR1 isoform A (varient 1, v.1) is expressed higher in the brain tumor patient group than healthy normal group, it was confirmed that it can be used as a diagnostic marker for brain tumors.
실시예 2. 뇌종양 환자군에서 NUPR1 동형(isoform) A의 발현 분석Example 2 Expression Analysis of NUPR1 Isoform A in Brain Tumor Patients
2-1. 교모세포종 및 신경교육종 환자의 세포 특성 및 전사체 서브타입 분석2-1. Analysis of Cell Characteristics and Transcript Subtypes in Glioblastoma and Neuroeducoma Patients
원발성 교모세포종 환자(Glioblastoma, GBM-28) 및 이차 신경교육종 환자(Gliosarcoma, GBM-37)로부터 뇌종양 조직 샘플을 수득한 후, 1차 배양(Primary Culture)을 수행하였다. 상기 GBM-28 및 GBM-37은 동일 환자로부터 유래된 샘플이며, GBM-37은 GBM-28 샘플 수득 후 종양의 제거 수술과 동시화학방사선요법을 받고 난 후 3개월 만에 재발했을 당시의 샘플이다. 샘플 수득 전 환자의 뇌 MRI 사진을 도 2에 나타내었다(왼쪽부터 T1, T2, T1-enhanced MRI).Brain tumor tissue samples were obtained from primary glioblastoma patients (Glioblastoma, GBM-28) and secondary neuroeducoma patients (Gliosarcoma, GBM-37), followed by primary culture. GBM-28 and GBM-37 are samples derived from the same patient, and GBM-37 is a sample at the time of recurrence after 3 months after tumor removal surgery and concurrent chemoradiotherapy after obtaining the GBM-28 sample. . Brain MRI photographs of patients before sample acquisition are shown in FIG. 2 (T1, T2, T1-enhanced MRI from left).
다음으로, 뇌 세포 배양에는 10% FBS(fetal bovine serum, Gibco Corp., 16000, Grand Island, NY, USA) 및 항생제인 1% 100U/ml 페니실린/스트렙토마이신(Gibco Corp., 15140-122, Grand Island, NY, USA)을 포함하는 DMEM 배지(Dulbecco’s modified Eagle medium, WelGENE, LM001-05, Korea)를 이용하였으며, 5% CO2, 37℃가 유지되는 인큐베이터 안에서 배양하였다. 원발성 교모세포종 환자(GBM-28) 및 이차 신경교육종 환자(GBM-37)로부터 수득한 세포를 대상으로 면역조직화학 분석(H&E, GFAP, Vimentin, X50) 및 형태 관찰을 수행하였으며, 그 결과를 도 3 및 도 4에 나타내었다. Next, 10% FBS (fetal bovine serum, Gibco Corp., 16000, Grand Island, NY, USA) and 1% 100 U / ml penicillin / streptomycin (Gibco Corp., 15140-122, Grand) DMEM medium (Dulbecco's modified Eagle medium, WelGENE, LM001-05, Korea), including Island, NY, USA), was used and incubated in an incubator maintained at 5% CO 2, 37 ° C. Immunohistochemical analysis (H & E, GFAP, Vimentin, X50) and morphology observations were performed on cells obtained from patients with primary glioblastoma (GBM-28) and patients with secondary neuroedema (GBM-37). 3 and 4 are shown.
도 3 및 도 4에 나타낸 바와 같이, 원발성 교모세포종 환자로부터 분리한 GBM-28 세포(각 도면 왼쪽)는 교모세포종의 전형적인 특성을 나타내고 있는데 반해, 이차 신경교육종 환자로부터 분리한 GBM-37 세포(각 도면 오른쪽)는 GFAP 염색이 되지 않고 특이적인 형태(fascicular arranged spindle cells)를 나타내는 등 일반 교모세포종과는 구분됨을 확인하였다. As shown in FIGS. 3 and 4, GBM-28 cells isolated from primary glioblastoma patients (left side of each figure) show typical characteristics of glioblastoma, whereas GBM-37 cells isolated from secondary neuroedema patients ( The right side of each figure) was confirmed that the GFAP staining and distinguished from the normal glioblastoma, such as showing a specific form (fascicular arranged spindle cells).
다음으로, 상기 과정을 통해 수득한 뇌종양 환자 유래 세포(<passage 10)에서 TRIzol reagent를 이용하여 전체 RNA를 추출한 후, cDNA 마이크로어레이를 수행하였다. 전사체 분석은 MA-human Agilent 44k (Agilent Technologies)를 사용한 oligonucleotide microarray analysis를 통해 이루어졌으며, one-channel microarray data는 GeneSpring GX 7.3 (EBIOGEN, Seoul, Korea)를 사용한 global median normalization method를 통해 분석하였다. 분석된 cDNA 마이크로어레이 데이터를 활용하여 각 뇌종양 환자 유래 세포의 전사체에서 특이적으로 상관성(correlation)이 있는 유전자 세트를 선별하기 위하여 GSEA(Gene Set Enrichment Analysis, Broad Institute, USA)를 실시하였다. GSEA는 javaGSEA desktop application(GSEA v2.1.)을 통해 실행되었고, 유전자 세트는 MSigDb (http://www.broadinstitute.org/gsea/msigdb/)라는 데이터베이스에서 다운로드하였다. GSEA는 enrichment score, normalized enrichment score, nominal p-value, 그리고 false discovery rate (q-value)를 생산한다. GBM-28의 전사체와 GBM-37의 전사체를 비교하기 위해 0.05 미만의 p-value를 갖는 유전자 세트만을 선별하였다. 그 결과를 도 5 및 도 6에 나타내었다. Next, after extracting the total RNA using the TRIzol reagent from the brain tumor patient-derived cells (<passage 10) obtained through the above process, cDNA microarray was performed. Transcript analysis was performed by oligonucleotide microarray analysis using MA-human Agilent 44k (Agilent Technologies), and one-channel microarray data was analyzed by global median normalization method using GeneSpring GX 7.3 (EBIOGEN, Seoul, Korea). Using the analyzed cDNA microarray data, Gene Set Enrichment Analysis, Broad Institute, USA (GSEA) was performed to select a gene set specifically correlated in the transcripts of cells derived from each brain tumor patient. GSEA was run through the javaGSEA desktop application (GSEA v2.1.) And the gene set was downloaded from a database called MSigDb (http://www.broadinstitute.org/gsea/msigdb/). GSEA produces a enrichment score, a normalized enrichment score, a nominal p-value, and a false discovery rate (q-value). Only gene sets with p-values less than 0.05 were selected to compare GBM-28 transcripts and GBM-37 transcripts. The results are shown in FIGS. 5 and 6.
도 5에 나타낸 바와 같이, 신경교종에 대한 4가지의 전사체 서브타입(Proneural, Neural, Classical 및 Mesenchymal subtype) 중에서 GBM-28은 예후가 좋은 편에 속하는 전신경(Proneural) 서브타입을 보이는데 반해, GBM-37은 예후가 매우 나쁜 간엽성(Mesenchymal) 서브타입을 나타냄을 확인하였다. As shown in FIG. 5, of the four transcript subtypes for glioma (Proneural, Neural, Classical and Mesenchymal subtypes), GBM-28 shows a proneural subtype with a prognosis. GBM-37 confirms that the prognosis represents a very poor Mesenchymal subtype.
또한, 도 6에 나타낸 바와 같이, Epithelial-Mesenchymal transition, Leukocyte Migration, Hypoxia 유전자 세트 등 암의 악성도에 기여한다고 알려진 유전자들이 GBM-28 보다 GBM-37과 상관성(correlation)이 높음을 확인하였다. In addition, as shown in Figure 6, it was confirmed that genes known to contribute to cancer malignancy such as Epithelial-Mesenchymal transition, Leukocyte Migration, Hypoxia gene set has a higher correlation with GBM-37 than GBM-28.
2-2. 교모세포종 및 신경교육종 환자의 악성도 분석2-2. Malignant analysis of glioblastoma and neuroeducoma patients
뇌종양 환자 유래 세포의 악성도를 분석하기 위하여, 원발성 교모세포종 환자(GBM-28) 및 이차 신경교육종 환자(GBM-37)로부터 분리한 세포를 대상으로 MTS 증식 어쎄이(cell proliferation assay)를 수행하였다. 먼저, 각 세포를 1.0×104 cells/well 비율로 96-웰 배양 플레이트에 시딩하였다. 일정 시간 세포 배양 후, CellTiter 96® AQueous One Solution cell proliferation assay (Promega, 3580) 용액을 각 웰에 첨가하였다. 그리고 나서 Infinite M200 Pro (Tecan)를 사용하여 490 nm에서의 흡광도를 측정하였다. 그 결과를 도 7에 나타내었다. To analyze the malignancy of brain tumor-derived cells, MTS cell proliferation assays were performed on cells isolated from primary glioblastoma patients (GBM-28) and secondary neuroeducoma patients (GBM-37). . First, they seeded the cells in each 96-well culture plate with 1.0 × 10 4 cells / well ratios. After cell culture for a period of time, CellTiter 96® AQueous One Solution cell proliferation assay (Promega, 3580) solution was added to each well. The absorbance at 490 nm was then measured using Infinite M200 Pro (Tecan). The results are shown in FIG.
도 7에 나타낸 바와 같이, 이차 신경교육종 환자로부터 분리한 GBM-37 세포는 원발성 교모세포종 환자로부터 분리한 GBM-28 세포에 비해 세포 증식 정도가 높음을 확인하였다. As shown in FIG. 7, GBM-37 cells isolated from secondary neuroeducoma patients were found to have higher cell proliferation than GBM-28 cells isolated from primary glioblastoma patients.
다음으로, 상기 실시예 2-1의 결과를 더 심층적으로 분석하기 위하여 IPA라는 생물정보학 프로그램을 사용하였다. 상기 실시예 2-1에서 수득한 cDNA 마이크로어레이 데이터를 IPA의 Core analysis 라는 툴을 사용하여 분석하였으며, Core analysis에서 제공하는 “Disease and bio functions”라는 세부 프로그램 기법을 사용하여 전사체 분석을 수행하였다. 그 결과를 도 8에 나타내었다. Next, in order to further analyze the results of Example 2-1, a bioinformatics program called IPA was used. The cDNA microarray data obtained in Example 2-1 was analyzed using a tool called Core analysis of IPA, and transcriptome analysis was performed using a detailed program technique called “Disease and bio functions” provided by Core analysis. . The results are shown in FIG.
도 8에 나타낸 바와 같이, 원발성 교모세포종 환자(GBM-28) 및 이차 신경교육종 환자(GBM-37)로부터 분리한 전사체가 나타내는 특성을 분류한 결과, GBM-37이 GBM-28에 비해 더 악성도가 높은 특성을 가지고 있음을 재확인하였다. 이상의 실험 결과를 통하여, GBM-37에 해당하는 대상 개체는 항암 치료 후 더 악성인 뇌종양, 즉 예후가 나쁜 이차 신경교육종으로 재발하였음을 알 수 있다. As shown in FIG. 8, as a result of classifying the transcripts isolated from primary glioblastoma patients (GBM-28) and secondary neuroeducoma patients (GBM-37), GBM-37 was more malignant than GBM-28. It was again confirmed that the island had high properties. Through the above experimental results, it can be seen that the subjects corresponding to GBM-37 recurred as a more malignant brain tumor, that is, a secondary neuroedema with a poor prognosis after chemotherapy.
2-3. 교모세포종 및 2-3. Glioblastoma and
신경교육종Neuroeducation
환자에서 In the patient
NUPR1NUPR1
동형( Isomorphism
isoformisoform
) A 유전자 발현 분석A gene expression analysis
먼저, 원발성 교모세포종 환자(GBM-28) 및 이차 신경교육종 환자(GBM-37)로부터 수득한 뇌종양 환자 유래 세포에서 공지된 방법에 따라 TRIzol reagent를 이용하여 전체 RNA를 분리하였다. 상기 전체 RNA로부터 cDNA를 생성한 후, NUPR1 전체, NUPR1 동형 A(varient 1, v.1) 및 NUPR1 동형 B(varient 2, v.2)에 대한 프라이머를 이용하여 RT-PCR을 수행하였다. 정량을 위한 하우스킵핑 유전자로 GAPDH를 이용하였다. 그 결과를 도 9에 나타내었다. First, total RNA was isolated from a brain tumor patient derived cells obtained from primary glioblastoma patient (GBM-28) and secondary neuroeducoma patient (GBM-37) using TRIzol reagent according to a known method. After generating cDNA from the total RNA, RT-PCR was performed using primers for NUPR1 whole, NUPR1 isoform A (varient 1, v.1) and NUPR1 isoform B (varient 2, v.2). GAPDH was used as a housekeeping gene for quantification. The results are shown in FIG.
도 9에 나타낸 바와 같이, GBM-28 및 GBM-37 샘플에서 NUPR1 전체 및 NUPR1 동형 B(varient 2, v.2)에 대한 mRNA 발현량은 특이적인 차이가 나타나지 않았으나, NUPR1 동형 A(varient 1, v.1)은 GBM-37에서 현저하게 높은 수치로 발현됨을 확인하였다. 즉, NUPR1 동형 A(varient 1, v.1)는 뇌종양 중에서도 보다 더 예후가 나쁜 악성 종양 환자에서 더 높게 발현됨을 알 수 있다. As shown in FIG. 9, mRNA expression levels of the whole NUPR1 and NUPR1 isoform B (varient 2, v.2) did not show specific differences in the GBM-28 and GBM-37 samples, but the NUPR1 isoform A (varient 1, v.1) was found to be expressed at a significantly high level in GBM-37. In other words, NUPR1 isoform A (varient 1, v.1) is found to be higher in malignant tumor patients with a poorer prognosis than among brain tumors.
2-4. 교모세포종 및 2-4. Glioblastoma and
신경교육종Neuroeducation
환자에서 In the patient
NUPR1NUPR1
동형( Isomorphism
isoformisoform
) A 단백질 발현 분석A protein expression analysis
먼저, 원발성 교모세포종 환자(GBM-28) 및 이차 신경교육종 환자(GBM-37)로부터 수득한 뇌종양 환자 유래 세포에서 공지된 방법에 따라 RIPA 버퍼를 이용하여 전체 단백질을 수득하였다. 상기 단백질 샘플을 대상으로 NUPR1 전체 및 NUPR1 동형 A(varient 1, v.1)에 대한 항체를 이용하여 웨스턴 블랏을 수행하였다. 정량을 위한 하우스킵핑 단백질로 β-액틴을 이용하였다. First, total protein was obtained using RIPA buffer according to a known method in brain tumor patient derived cells obtained from primary glioblastoma patient (GBM-28) and secondary neuroeducoma patient (GBM-37). The protein samples were subjected to Western blot using antibodies against NUPR1 as a whole and NUPR1 isoform A (varient 1, v.1). Β-actin was used as a housekeeping protein for quantification.
이때 NUPR1 동형 A(varient 1, v.1)에 대한 항체는 다음 과정을 통해 제조하였다. 먼저, NUPR1a 특이 펩타이드(NUPR1a(NP_001035948.1) N-말단으로부터 38~55 위치의 18 개 아미노산)의 N-말단에 시스테인(Cysteine)을 추가한 후, KLH에 접합하였다. PBS와 Freund’s adjuvant를 이용하여 상기 융합 펩타이드를 토끼 2마리에 각 0.5mg씩 피하에 면역하였다. 1차 면역 후 2주 간격으로 3회 동일량의 융합 펩타이드를 피하에 면역하였다. 최종 면역 후 1주 뒤에 전 채혈을 진행한 뒤 Protein A resin을 이용하여 다클론 항체를 확보하였다. 상기 과정을 통해 수득한 NUPR1 동형 A(varient 1, v.1)에 대한 다클론 항체는 NUPR1a와 NUPR1b의 재조합 단백질을 발현시켜서 생산한 항체와의 검정을 통해 특이성을 확인한 후 실험에 이용하였다. The antibody against NUPR1 isoform A (varient 1, v.1) was prepared by the following procedure. First, cysteine was added to the N-terminus of the NUPR1a specific peptide (18 amino acids 38 to 55 positions from the N-terminus of NUPR1a (NP_001035948.1)), and then conjugated to KLH. The fusion peptide was immunized subcutaneously with 0.5 mg each of two rabbits using PBS and Freund's adjuvant. The same amount of fusion peptide was immunized subcutaneously three times at two week intervals after primary immunization. One week after the final immunization, blood was collected and polyclonal antibodies were obtained using Protein A resin. The polyclonal antibody against NUPR1 isoform A (variant 1, v.1) obtained through the above procedure was used for experiments after confirming specificity through assays with antibodies produced by expressing recombinant proteins of NUPR1a and NUPR1b.
상기 과정을 통해 제조한 NUPR1 동형 A(varient 1, v.1)에 대한 다클론 항체를 이용하여 웨스턴 블랏을 수행한 결과를 도 10에 나타내었다. 10 shows the results of Western blot using the polyclonal antibody against NUPR1 isoform A (varient 1, v.1) prepared by the above procedure.
도 10에 나타낸 바와 같이, GBM-28 및 GBM-37 샘플에서 NUPR1 전체 단백질 발현량은 특이적인 차이가 나타나지 않았으나, NUPR1 동형 A(varient 1, v.1) 단백질은 GBM-28 샘플에 비해 GBM-37 샘플에서 현저하게 높게 발현됨을 확인하였다. 즉, NUPR1 동형 A(varient 1, v.1) 단백질은 mRNA 발현 분석 결과와 마찬가지로 뇌종양 중에서도 보다 더 예후가 나쁜 악성 종양 환자에서 더 높게 발현됨을 알 수 있다. As shown in FIG. 10, the total NUPR1 protein expression in the GBM-28 and GBM-37 samples did not show a specific difference, but the NUPR1 isoform A (varient 1, v.1) protein was compared with the GBM-28 sample. It was found to be significantly higher in 37 samples. In other words, NUPR1 isoform A (v.1) protein was expressed higher in malignant tumor patients with poorer prognosis than among brain tumors as in mRNA expression analysis.
2-5. 교모세포종 환자에서 2-5. In patients with glioblastoma
전사체Transcript
서브타입에 따른 By subtype
NUPR1NUPR1
동형( Isomorphism
isoformisoform
) A 단백질 발현 분석A protein expression analysis
TCGA (The Cancer Genome Atlas)에 등록되어 있는 원발성 교모세포종 (primary glioblastoma) 환자군을 다섯 가지 서브타입(Proneural, Classical, Neural, Mesenchymal, G-CIMP)으로 나눈 후, NUPR1 동형 A(ID: ENST00000395641)의 발현 정도를 분석하였다. G-CIMP 서브타입은 Proneural 서브타입에 속하는 것으로 예후가 좋은 것으로 알려진 서브타입이다. 이 서브타입은 IDH1/2 mutation이라는 환자의 예후 마커(prognostic factor)와 밀접한 관련이 있다고 알려져있다. 그 결과를 도 11에 나타내었다. The primary glioblastoma patient group enrolled in the Cancer Genome Atlas (TCGA) was divided into five subtypes (Proneural, Classical, Neural, Mesenchymal, G-CIMP), followed by NUPR1 isoform A (ID: ENST00000395641). The expression level was analyzed. The G-CIMP subtype belongs to the Proneural subtype and is a subtype known to have a good prognosis. This subtype is known to be closely associated with the prognostic factor of a patient called the IDH1 / 2 mutation. The results are shown in FIG.
도 11에 나타낸 바와 같이, 다섯 가지의 서브타입 중에서 예후가 가장 나쁘다고 알려진 간엽성 (Mesenchymal) 서브타입을 가지고 있는 환자 군에서 NUPR1 동형 A의 발현이 가장 높게 나타남을 확인하였다. As shown in FIG. 11, it was confirmed that the expression of NUPR1 isoform A was highest in the patient group having the mesenchymal subtype, which is known to have the worst prognosis among the five subtypes.
이상에 개시한 실시예 2의 실험을 통하여, NUPR1 동형 A(varient 1, v.1)는 뇌종양 중에서도 예후가 가장 나쁜 간엽성 아류형(mesenchymal subtype)으로 분류되는 악성 뇌종양에서 더 높은 수준으로 발현되는바, 단순한 뇌종양 진단 뿐만 아니라 뇌종양의 서브타입 진단에까지 이용될 수 있음을 확인하였다. Through experiments of Example 2 described above, NUPR1 isoform A (varient 1, v.1) is expressed at a higher level in malignant brain tumors classified as mesenchymal subtypes with the poorest prognosis among brain tumors. It was confirmed that it can be used not only for the diagnosis of brain tumors but also for the diagnosis of subtypes of brain tumors.
실시예Example
3. 뇌종양 환자군에서 3. In patients with brain tumors
NUPR1NUPR1
동형( Isomorphism
isoformisoform
) A의 발현 정도와 예후와의 상관관계 분석Correlation analysis between A expression level and prognosis
3-1. 다수의 뇌종양 환자에서 NUPR1 동형 A의 발현 및 생존률 분석3-1. Expression and Survival Analysis of NUPR1 Isoform A in Multiple Brain Tumors
NUPR1 동형 A(varient 1, v.1)의 악성 뇌종양 진단 가능성을 추가 검증하기 위하여, 서로 다른 진단명 및 생존률을 보인 뇌종양 환자로부터 수득한 뇌종양 환자 유래 세포에서 전체 RNA를 분리한 후 대상으로 상기 실시예 2-3과 동일한 방식으로 RT-PCR을 수행하였다. 구체적인 환자 정보는 표 2에 나타내었으며, 실험 결과는 도 12에 나타내었다. U87은 ATCC(American Type Culture Collection)에서 판매중인 human primary glioblastoma cell line이다. In order to further verify the possibility of diagnosing malignant brain tumor of NUPR1 isoform A (varient 1, v.1), total RNA was isolated from brain tumor patient derived cells obtained from brain tumor patients with different diagnosis names and survival rates. RT-PCR was performed in the same manner as in 2-3. Specific patient information is shown in Table 2, and the experimental results are shown in FIG. U87 is a human primary glioblastoma cell line sold by the American Type Culture Collection (ATCC).
| IDID | 성별gender | 나이age | 진단명Diagnosis Name | 생존율Survival rate |
| GBM-12GBM-12 | MaleMale | 4646 | Glioblastoma (grade IV)Glioblastoma (grade IV) | 36 months36 months |
| GBM-15GBM-15 |
Female |
3737 | Glioblastoma (grade IV)Glioblastoma (grade IV) | 18 months18 months |
| GBM-37 GBM-37 |
Female |
4040 | Gliosarcoma (grade IV)Gliosarcoma (grade IV) | 7 months7 months |
도 12에 나타낸 바와 같이, 생존률이 7개월에 불과하여 예후가 극히 나쁜 환자 유래 GBM-37 샘플에서 다른 뇌종양 환자 유래 샘플에 비해 NUPR1 동형 A(varient 1, v.1)의 mRNA 발현량이 현저하게 높은 것을 확인하였다. As shown in FIG. 12, the mRNA expression level of NUPR1 isoform A (varient 1, v.1) was significantly higher in the GBM-37 sample derived from the patient with extremely poor prognosis due to only 7 months of survival compared to the sample derived from other brain tumor patients. It was confirmed.
3-2. 원발성 교모세포종 환자군에서 3-2. In patients with primary glioblastoma
NUPR1NUPR1
동형 A의 발현 수준에 따른 According to the expression level of isoform A
생존률Survival rate
분석 analysis
TCGA (The Cancer Genome Atlas)에 등록되어 있는 원발성 교모세포종 (primary glioblastoma) 환자군을 NUPR1 동형 A 발현 정도에 따라 고발현 및 저발현으로 나눈 후, 생존 분석을 수행하였다. 보다 구체적으로, RNA-sequencing dataset을 사용하였으며, NUPR1 동형 A의 ID는 ENST00000395641 (uc002dqd.2)로 확인되었다. 상기 ID를 사용해 NUPR1 동형 A의 발현 값을 환자별로 확인한 후, NUPR1 동형 A의 발현 정도에 따라 고발현(High) 및 저발현(Low)으로 분류하고 생존 분석을 수행하였다. 또한, 동일한 환자군을 대상으로 교모세포종 환자의 예후 마커(prognostic factor)로 사용되고 있는 MGMT 메틸화 유무를 1차 적으로 분류한 후, NUPR1 동형 A의 발현 정도와 생존 분석을 수행하였다. 그 결과를 도 13 내지 15에 나타내었다. The primary glioblastoma patient group registered in The Cancer Genome Atlas (TCGA) was divided into high and low expression according to the degree of NUPR1 isoform A expression, and then survival analysis was performed. More specifically, an RNA-sequencing dataset was used, and the ID of NUPR1 isoform A was identified as ENST00000395641 (uc002dqd.2). Using the ID, the expression value of NUPR1 isoform A was confirmed for each patient, and then classified into high and low expression according to the expression level of NUPR1 isoform A and survival analysis was performed. In addition, MGMT methylation, which is used as a prognostic factor for glioblastoma patients, was classified primarily in the same patient group, and then expression level and survival analysis of NUPR1 isoform A were performed. The results are shown in FIGS. 13 to 15.
도 13에 나타낸 바와 같이, NUPR1 동형 A의 발현 정도가 높은 군(High)에서 생존률이 더 낮은 것을 확인하였으며, 이는 통계적 유의성을 가지는 결과임을 확인하였다. As shown in Figure 13, it was confirmed that the survival rate is lower in the group (High) where the expression level of NUPR1 isoform A is high, which is a result having statistical significance.
또한, 도 14 및 도 15에 나타낸 바와 같이, 교모세포종 환자의 예후 마커인 MGMT 메틸화 유무와 관계없이 NUPR1 동형 A의 발현 정도에 따라 환자들의 생존률에 유의한 차이가 나타남을 확인하였다. In addition, as shown in Figures 14 and 15, regardless of the presence or absence of MGMT methylation, a prognostic marker of glioblastoma patients, it was confirmed that there is a significant difference in the survival rate of patients according to the expression level of NUPR1 isoform A.
3-3. 신경교종 환자군에서 NUPR1 동형 A의 발현 수준에 따른 생존률 분석3-3. Survival analysis according to the expression level of NUPR1 isoform A in glioma group
서울대학교 병원에 내원한 79명의 원발성 교모세포종 (primary glioblastoma) 환자들을 NUPR1 동형 A의 발현 정도에 따라 고발현(High) 및 저발현(Low)으로 나눈 후, 생존 분석을 수행하였다. 환자들의 임상 정보를 도 16에, 각 그룹의 대표 환자의 종양 조직을 NUPR1 동형 A에 대한 항체를 사용하여 염색한 결과를 도 17에, 생존 분석 결과를 도 18에 나타내었다. 79 primary glioblastoma patients who visited Seoul National University Hospital were divided into high and low expression according to the expression level of NUPR1 isoform A, and then survival analysis was performed. The clinical information of the patients is shown in FIG. 16, the tumor tissues of the representative patients of each group were stained with an antibody against NUPR1 isoform A in FIG. 17, and the results of survival analysis are shown in FIG. 18.
도 18에 나타낸 바와 같이, NUPR1 동형 A 단백질에 대한 항체를 이용하여 발현 정도에 따라 나눈 두 그룹 중, NUPR1 동형 A의 발현 정도가 낮은 군(Low_Score)의 생존률이 더 좋은 편임을 확인하였다. As shown in FIG. 18, it was confirmed that the survival rate of the group having a low expression level of NUPR1 isoform A (Low_Score) was better among the two groups divided by the expression level using the antibody against the NUPR1 isoform A protein.
이상에 개시한 실시예 3의 실험을 통하여, NUPR1 동형 A(varient 1, v.1)의 발현 정도는 생존률과 유의미한 상관관계를 나타내는바, 이를 뇌종양 환자의 예후를 예측하는 마커로 활용할 수 있음을 확인하였다. Through the experiment of Example 3 described above, the expression level of NUPR1 isoform A (varient 1, v.1) shows a significant correlation with the survival rate, which can be used as a marker for predicting the prognosis of brain tumor patients. Confirmed.
실시예Example
4. 뇌종양 환자군에서 4. In patients with brain tumors
NUPR1NUPR1
동형 A의 발현 억제에 따른 항암 효과 분석 Analysis of anticancer effects by suppressing the expression of isoform A
4-1. 4-1.
NUPR1NUPR1
동형 A에 대한 For isoform A
siRNA의siRNA
제조 Produce
공지된 서열번호 2의 NUPR1 동형 A에 대한 염기서열을 바탕으로, NUPR1 동형 A를 특이적으로 억제할 수 있는 5개의 siRNA를 제조하였다(Genolution). GBM-37 세포를 24시간동안 배양한 후, LipofectamineTM RNAiMAX (Invitrogen)를 사용하여 siRNA를 세포 내로 형질 주입(Transfection)하였다. 형질 주입 24시간 후, 각 세포로부터 전체 RNA 및 단백질을 분리하였으며, 공지된 방법에 따라 RT-PCR 및 웨스턴 블랏을 수행하였다. siRNA 서열은 표 3에 나타내었으며, RT-PCR 결과를 도 19에, 웨스턴 블랏 결과를 도 20에 나타내었다. Based on the nucleotide sequence for the known NUPR1 isoform A of SEQ ID NO: 2, five siRNAs capable of specifically inhibiting the NUPR1 isoform A were prepared (Genolution). After culturing GBM-37 cells for 24 hours, siRNA was transfected into cells using LipofectamineTM RNAiMAX (Invitrogen). 24 hours after transfection, total RNA and protein were isolated from each cell and RT-PCR and Western blots were performed according to known methods. The siRNA sequences are shown in Table 3, with RT-PCR results shown in FIG. 19 and Western blot results shown in FIG. 20.
도 19에 나타낸 바와 같이, NUPR1 동형 A에 대한 5개의 siRNA는 모두 NUPR1 동형 B(NUPR1β)에는 영향을 주지 않고, NUPR1 동형 A(NUPR1α)의 mRNA 발현만을 특이적으로 하향 조절함을 확인하였다. As shown in FIG. 19, it was confirmed that all five siRNAs for NUPR1 isoform A did not affect NUPR1 isoform B (NUPR1β), but specifically downregulated mRNA expression of NUPR1 isoform A (NUPR1α).
또한, 도 20에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA의 형질 주입에 의하여 NUPR1 동형 A(NUPR1α)의 단백질 발현 역시 하향 조절됨을 확인하였다. In addition, as shown in Figure 20, it was confirmed that the protein expression of NUPR1 isoform A (NUPR1α) is also down-regulated by the transfection of siRNA against NUPR1 isoform A.
이하 실험에서는 NUPR1 동형 B에 가장 영향을 덜 주면서도 NUPR1 동형 A에 대한 억제효과가 우수한 #2 siRNA (서열번호 5 및 6)를 이용하여 실험하였다. In the following experiments, experiments were performed using # 2 siRNA (SEQ ID NOs: 5 and 6), which had the least effect on NUPR1 isoform B, but had an excellent inhibitory effect on NUPR1 isoform A.
4-2. 4-2.
NUPR1NUPR1
동형 A에 대한 For isoform A
siRNA가siRNA
신경교육종Neuroeducation
세포의 증식에 미치는 영향 분석 Analysis of the effect on cell proliferation
악성 뇌종양 환자로부터 분리한 GBM-37 세포에 상기 실시예４-1에서 제조한 siRNA(NUPR1 V1 #2)를 형질주입한 후, MTS 증식 어쎄이(cell proliferation assay)를 수행하였다. 대조군으로는 scRNA(표 3의 Negative contro)를 형질주입한 GBM-37 세포를 이용하였다. 먼저, 각 세포를 1.0×104 cells/well 비율로 96-웰 배양 플레이트에 시딩하였다. 일정 시간 세포 배양 후, CellTiter 96® AQueous One Solution cell proliferation assay (Promega, 3580) 용액을 각 웰에 첨가하였다. 그리고 나서 Infinite M200 Pro (Tecan)를 사용하여 490 nm에서의 흡광도를 측정하였다. 그 결과를 도 21에 나타내었다. GBM-37 cells isolated from malignant brain tumor patients were transfected with siRNA (NUPR1 V1 # 2) prepared in Example 4-1, followed by MTS cell proliferation assay. As a control, GBM-37 cells transfected with scRNA (Negative contro of Table 3) were used. First, each cell was seeded in a 96-well culture plate at a rate of 1.0 × 10 4 cells / well. After cell culture for a period of time, CellTiter 96® AQueous One Solution cell proliferation assay (Promega, 3580) solution was added to each well. The absorbance at 490 nm was then measured using Infinite M200 Pro (Tecan). The results are shown in FIG.
도 21에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA에 의해 GBM-37 세포의 증식이 현저하게 억제됨을 확인하였다. As shown in FIG. 21, it was confirmed that the proliferation of GBM-37 cells was significantly inhibited by siRNA against NUPR1 isoform A.
4-3. 4-3.
NUPR1NUPR1
동형 A에 대한 For isoform A
siRNA가siRNA
신경교육종Neuroeducation
세포의 전이 및 침입에 미치는 영향 분석 Analysis of the effect on cell metastasis and invasion
악성 뇌종양 환자로부터 분리한 GBM-37 세포에 상기 실시예４-1에서 제조한 siRNA(NUPR1 V1 #2)를 형질주입한 후, 트랜스웰 전이 어쎄이(Trans-well migration assay)를 수행하였다. 대조군으로는 scRNA를 형질주입한 GBM-37 세포를 이용하였다. 먼저, 혈청-프리 배지에 각 세포를 풀어준 후, 24-웰 플레이트(BD Biosciences, #3422)의 트랜스웰 인서트(transwell inserts)에서 24시간동안 세포를 배양시켰다. 그 다음에 10% FBS(fetal bovine serum)를 첨가한 배지를 화학유인물질(chemoattractant)로써 플레이트의 하부 챔버에 첨가시켰다. 상기 세포를 24시간동안 배양한 후, 비-전이(non-migrated) 세포를 면봉을 사용하여 제거하였다. 남아있는 전이 세포를 크리스탈 바이올렛(0.5% in 20% methanol)을 사용하여 염색한 후 개수하였다. 한편, 침입 어쎄이(Invasion assay)는 상기 트랜스웰 전이 어쎄이와 동일한 조건에서 진행되었으며, growth factor-reduced matrigel-coated insert wells (BD Biosciences, #356234)을 사용하였다. 세포 전이와 침입 수치는 현미경 필드를 4등분 하여 세포들의 개수의 평균치를 계산하였다. 그 결과를 도 22에 나타내었다. GBM-37 cells isolated from malignant brain tumor patients were transfected with siRNA (NUPR1 V1 # 2) prepared in Example 4-1, followed by a transwell migration assay. As a control, GBM-37 cells transfected with scRNA were used. First, each cell was released in serum-free medium, and the cells were incubated for 24 hours in transwell inserts of 24-well plates (BD Biosciences, # 3422). Then medium with 10% FBS (fetal bovine serum) was added to the lower chamber of the plate as a chemoattractant. After incubating the cells for 24 hours, non-migrated cells were removed using a cotton swab. The remaining metastatic cells were stained using crystal violet (0.5% in 20% methanol) and then harvested. Invasion assays were performed under the same conditions as the transwell transfer assay, and growth factor-reduced matrigel-coated insert wells (BD Biosciences, # 356234) were used. Cell metastasis and invasion values were calculated by dividing the microscopic field into quarters and calculating the average number of cells. The results are shown in FIG. 22.
도 22에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA에 의해 GBM-37 세포의 전이 및 침입이 현저하게 억제됨을 확인하였다. As shown in FIG. 22, it was confirmed that siRNA against NUPR1 isoform A significantly inhibited metastasis and invasion of GBM-37 cells.
4-4. 4-4.
NUPR1NUPR1
동형 A에 대한 For isoform A
siRNA가siRNA
신경교육종Neuroeducation
세포의 Cellular
전사체Transcript
서브타입에 미치는 영향 분석 Impact Analysis on Subtypes
악성 뇌종양 환자로부터 분리한 GBM-37 세포에 상기 실시예４-1에서 제조한 siRNA(NUPR1 V1 #2)를 형질주입하고 48시간 후, 상기 실시예 2-1과 동일한 방식으로 GSEA(Gene Set Enrichment Analysis, Broad Institute, USA)를 실시하였다. 그 결과를 도 23에 나타내었다. 48 hours after transfection of siRNA (NUPR1 V1 # 2) prepared in Example 4-1 to GBM-37 cells isolated from malignant brain tumor patients, GSEA (Gene Set Enrichment) was performed in the same manner as in Example 2-1. Analysis, Broad Institute, USA). The results are shown in FIG.
도 23에 나타낸 바와 같이, 형질주입 전 GBM-37은 예후가 나쁜 간엽성(Mesenchymal) 서브타입을 나타내었으나 (도 5 참조), NUPR1 동형 A에 대한 siRNA를 이용하여 NUPR1 동형 A의 발현을 억제할 경우 다시 재발 전(GBM-28)과 동일하게 예후가 좋은 전신경(Proneural) 서브타입을 나타냄을 확인하였다. 이는 NUPR1 동형 A에 대한 siRNA에 의해 보다 좋은 예후를 보이는 전신경(Proneural) 서브타입으로 암세포의 성질이 바뀌었음을 보여주는 결과이다. As shown in FIG. 23, GBM-37 before transfection showed a Mesenchymal subtype with poor prognosis (see FIG. 5), but it was possible to inhibit the expression of NUPR1 isoform A using siRNA against NUPR1 isoform A. In case of recurrence (GBM-28), the prognosis showed good proneural subtype. This is a result showing that the properties of cancer cells have been changed to a proneural subtype with a better prognosis by siRNA for NUPR1 isoform A.
4-5. 4-5.
NUPR1NUPR1
동형 A에 대한 For isoform A
siRNA가siRNA
신경교육종Neuroeducation
세포에서 유전자 발현에 미치는 영향 분석 Analysis of the effect on gene expression in cells
상기 실시예 4-4와 동일한 전사체 샘플에서 침입(Invasion), 염증(Inflammation) 및 신호 전달(signaling)-관련(related) 유전자 발현 변화를 분석하였다. 그 결과를 도 24 내지 도 26에 나타내었다. 각 도에서 왼쪽이 GBM-37 샘플이고, 오른쪽이 NUPR1 동형 A에 대한 siRNA로 형질전환 시킨 후의 GBM-37 샘플이다. Invasion, Inflammation, and signaling-related gene expression changes in the same transcript samples as in Example 4-4 were analyzed. The results are shown in FIGS. 24 to 26. In each figure, the left side is the GBM-37 sample and the right side is the GBM-37 sample after transformation with siRNA for NUPR1 isoform A.
도 24에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA로 형질전환 시킨 후의 GBM-37 샘플(GBM-37_siNUPR1a)에서 야생형에 비하여 Epithelial-Mesenchymal Transition, Metastasis 관련 유전자 세트의 발현이 하향 조절됨을 확인하였다. As shown in FIG. 24, it was confirmed that the expression of Epithelial-Mesenchymal Transition, Metastasis related gene set was down-regulated in the GBM-37 sample (GBM-37_siNUPR1a) after transformation with siRNA for NUPR1 isoform A compared to wild type.
또한, 도 25에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA로 형질전환 시킨 후의 GBM-37 샘플(GBM-37_siNUPR1a)에서 야생형에 비하여 Inflammatory Response, Reactive Oxygen Species Pathway에 관련된 유전자 세트의 발현이 하향 조절됨을 확인하였다. In addition, in the GBM-37 sample (GBM-37_siNUPR1a) after transformation with siRNA for NUPR1 isoform A, expression of gene sets related to Inflammatory Response and Reactive Oxygen Species Pathway was down-regulated as shown in FIG. 25. Confirmed.
또한, 도 26에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA로 형질전환 시킨 후의 GBM-37 샘플(GBM-37_siNUPR1a)에서 야생형에 비하여 Chemokine Signaling Pathway, IL6-JAK-STAT3에 관련된 유전자 세트의 발현이 하향 조절됨을 확인하였다. In addition, in the GBM-37 sample (GBM-37_siNUPR1a) after transformation with siRNA for NUPR1 isoform A, the expression of the gene set related to Chemokine Signaling Pathway, IL6-JAK-STAT3 was lowered as shown in FIG. 26. It was confirmed to be controlled.
4-5. 4-5.
NUPR1NUPR1
동형 Isomorphism
A에 대한 억제가 뇌종양Inhibition of A Brain Tumor
질환 동물 모델의 생존에 미치는 영향 분석 Analysis of the impact on survival of disease animal models
먼저, NUPR1 동형 A에 대한 siRNA(NUPR1 V1 #2) 또는 scRNA를 형질전환 시킨 GBM-37 세포 (2×106 cells)를 각각 5주령의 athymic mice (BALB/c nu/nu) (Central Laboratory Animal, Inc, Korea)의 뇌에 주입하였다. 두 개 내의 세포 주입은 좌측 이마엽에 수행되었으며, 좌표는 bregma로부터 옆으로 2 mm, 앞쪽으로 0.5 mm로 설정하였다. 세포 주입 7일 후 형성된 종양을 추출하고, 바로 Bouin’s fixative에 2~3시간동안 보관하였다. Deparaffinized된 종양 조직을 H&E 염색하였다. 상기 염색된 조직의 이미지는 Olympus whole-slide scanner with OlyVIA software (Olympus Life Science, Center Valley, PA, USA)을 통해 수득하였다. 그 결과를 도 27 및 도 28에 나타내었다.First, siRNA (NUPR1 V1 # 2) or scRNA-transformed GBM-37 cells (2 × 10 6 cells) for NUPR1 isoform A were each examined for 5 weeks of athymic mice (BALB / c nu / nu) (Central Laboratory Animal). , Inc, Korea). Intracellular injection was performed in the left forehead and the coordinates were set 2 mm laterally and 0.5 mm forward from bregma. Tumors formed 7 days after cell injection were extracted and immediately stored in Bouin's fixative for 2-3 hours. Deparaffinized tumor tissue was H & E stained. Images of the stained tissue were obtained through Olympus whole-slide scanner with OlyVIA software (Olympus Life Science, Center Valley, PA, USA). The results are shown in FIGS. 27 and 28.
도 27 및 도 28에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA로 형질주입시킨 GBM-37 세포를 투여한 군(siRNA NUPRv.1)에서 종양의 크기가 감소함을 확인하였다. As shown in FIG. 27 and FIG. 28, it was confirmed that tumor size was decreased in the group to which GBM-37 cells transfected with siRNA for NUPR1 isoform A (siRNA NUPRv.1).
다음으로, 각 마우스의 종양 조직으로부터 단백질을 추출하여 NUPR1 동형 A의 발현 정도를 분석하였다. 그 결과를 도 29에 나타내었다. Next, the protein was extracted from the tumor tissue of each mouse and analyzed for the expression level of NUPR1 isoform A. The results are shown in FIG. 29.
도 29에 나타낸 바와 같이, NUPR1 동형 A에 대한 siRNA로 형질주입시킨 GBM-37 세포를 투여한 동물모델의 종양 조직에서 NUPR1 동형 A의 단백질 발현이 현저하게 감소함을 확인하였다. As shown in FIG. 29, it was confirmed that protein expression of NUPR1 isoform A was significantly decreased in tumor tissues of animal models to which GBM-37 cells transfected with siRNA against NUPR1 isoform A were administered.
이상에 개시한 실시예 4의 실험을 통하여, NUPR1 동형 A의 발현을 억제함으로써 뇌종양의 성장, 전이 및 침입을 억제할 수 있을 뿐만 아니라 예후가 좋은 서브타입으로 바뀌는바, NUPR1 동형 A의 특이적인 억제제는 뇌종양 치료제로 활용할 수 있음을 확인하였다. Through the experiments of Example 4 described above, by inhibiting the expression of NUPR1 isoform A, not only the growth, metastasis, and invasion of brain tumors can be suppressed, but also the prognosis is changed into a subtype. Confirmed that it can be used as a treatment for brain tumors.
종합적으로, 본 발명에 따른 NUPR1, 특히 NUPR1 동형 A는 건강한 정상인에 비해 뇌종양 환자에서 유의하게 증가하며, 특히 예후가 나쁜 악성 뇌종양 환자에서 더욱 현저하게 증가하는바, 이를 뇌종양 진단 또는 예후 예측용 바이오 마커로 활용할 수 있다. 또한, 본 발명에 따른 NUPR1, 특히 NUPR1 동형 A의 억제를 통해 뇌종양의 성장, 전이 및 침입을 억제할 수 있는바, 이를 뇌종양 치료를 위한 타겟으로 활용할 수 있고, 향후 뇌종양 치료제의 스크리닝에도 이용할 수 있다. Overall, NUPR1, in particular NUPR1 isoform A, according to the present invention is significantly increased in brain tumor patients compared to healthy normal persons, especially in patients with malignant brain tumors with a poor prognosis, which is a biomarker for diagnosing brain tumors or predicting prognosis. Can be utilized as In addition, it is possible to inhibit the growth, metastasis and invasion of brain tumors through the inhibition of NUPR1, in particular NUPR1 isoform A according to the present invention, it can be used as a target for the treatment of brain tumors, it can be used in the screening of future brain tumor therapeutics. .
Claims (15)
- NUPR1(Nuclear protein 1)을 포함하는 뇌종양 진단 또는 예후 예측용 바이오 마커 조성물. Biomarker composition for brain tumor diagnosis or prognosis prediction comprising NUPR1 (Nuclear protein 1).
- 제1항에 있어서, 상기 NUPR1은 NUPR1 동형(isoform) A인 것을 특징으로 하는, 바이오 마커 조성물. The biomarker composition of claim 1, wherein the NUPR1 is NUPR1 isoform A.
- 제2항에 있어서, 상기 NUPR1 동형(isoform) A는 서열번호 1로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는, 바이오 마커 조성물. The biomarker composition of claim 2, wherein the NUPR1 isoform A consists of an amino acid sequence represented by SEQ ID NO: 1.
- 제1항에 있어서, 상기 뇌종양은 교모세포종(glioblastomas), 신경교육종(gliosarcoma), 역형성 성상세포종(anaplastic astrocytomas), 희소돌기아교세포종(oligodendrogliomas), 상의세포종(ependymomas), 저등급 성상세포종(low-grade astrocytomas), 수모세포종(medulloblastomas), 성상세포종 종양(astrocytic tumors), 모양세포성 성상세포종 (Pilocytic astrocytoma), 미만성 성상세포종(diffuse astrocytomas), 다형성 황색성상세포종(pleomorphic xanthoastrocytomas), 상의하 거대세포 성상세포종(subependymal giant cell astrocytomas), 역형성 희소돌기아교세포종(anaplastic oligodendrogliomas), 희소돌기성상세포종(oligoastrocytomas), 역형성 희소돌기성상세포종(anaplastic oligoastrocytomas), 점액성 유두상 상의세포종(myxopapillary ependymomas), 상의하세포종(subependymomas), 뇌실상의세포종(ependymomas), 역형성 뇌실상의세포종(anaplastic ependymomas), 아스트로블라스토마(astroblastomas), 제3뇌실의 척삭모양 신경아교종(chordoid gliomas of the third ventricle), 대뇌 신경교종증(gliomatosis cerebris), 글랜글리오사이토마스(glangliocytomas), 결합조직생성 유아 성상세포종(desmoplastic infantile astrocytomas), 결합조직생성 유아신경절교종(desmoplastic infantile gangliogliomas), 태생기발육부전 신경상피종(dysembryoplastic neuroepithelial tumors), 상의모세포종(ependymoblastomas), 천막위 원시신경외배엽성 종양(supratentorial primitive neuroectodermal tumors), 맥락막총 유두종(choroids plexus papilloma), 중등도 분화형의 솔방울샘 실질 종양(pineal parenchymal tumors of intermediate differentiation), 혈관주위세포종(hemangiopericytomas), 안장영역의 종양(tumors of the sellar region), 두개인두종(craniopharyngioma), 혈관모세포종(capillary hemangioblastoma), 및 일차성 중추신경계 임파종(primary CNS lymphoma)으로 이루어진 군으로부터 선택된 것을 특징으로 하는, 바이오 마커 조성물. The method of claim 1, wherein the brain tumor is glioblastomas, glioblastoma, anaplastic astrocytomas, oligodendrogliomas, ependymomas, low grade astrocytomas low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, polymorphic xanthoastrocytomas Subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, anaplastic oligoastrocytomas, myxoidal papillary ymomas , Suprapendymomas, ependymomas, anaplastic ependymomas, as Astroblastomas, chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, connective tissue-forming infantile astrocytoma astrocytomas, connective tissue-forming infantile gangliogliomas, dysembryoplastic neuroepithelial tumors, ependymoblastomas, supratentorial primitive neuroectodermal tumors Choroids plexus papilloma, moderately differentiated pineal parenchymal tumors of intermediate differentiation, hemangiopericytomas, tumors of the sellar region, craniopharyngioma, blood vessels To capillary hemangioblastoma, and primary CNS lymphoma Biomarker composition, characterized in that selected from the group consisting of.
- NUPR1(Nuclear protein 1)의 발현 또는 활성 수준을 측정하는 제제를 포함하는 뇌종양 진단 또는 예후 예측용 조성물.A composition for diagnosing or predicting a brain tumor comprising a preparation for measuring the expression or activity level of NUPR1 (Nuclear protein 1).
- 제5항에 있어서, 상기 제제는 NUPR1 유전자에 특이적으로 결합하는 프라이머 쌍 또는 프로브인 것을 특징으로 하는, 뇌종양 진단 또는 예후 예측용 조성물.According to claim 5, The agent is characterized in that the primer pair or probe specifically binding to the NUPR1 gene, brain tumor diagnosis or prognostic composition.
- 제5항에 있어서, 상기 제제는 NUPR1 단백질에 특이적으로 결합하는 항체인 것을 특징으로 하는, 뇌종양 진단 또는 예후 예측용 조성물.According to claim 5, The agent is characterized in that the antibody that specifically binds to NUPR1 protein, brain tumor diagnosis or prognostic composition.
- 제5항 내지 제7항 중 어느 한 항의 조성물을 포함하는 뇌종양 진단 또는 예후 예측용 키트.A kit for diagnosing or prognosticing a brain tumor comprising the composition of claim 5.
- 생물학적 시료로부터 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계;를 포함하는 뇌종양 진단 또는 예후 예측을 위한 정보제공방법.Measuring the mRNA expression of the NUPR1 gene or its protein activity level from a biological sample; Information providing method for diagnosing or predicting brain tumors comprising.
- NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating brain tumors, including an expression or activity inhibitor of NUPR1 (Nuclear protein 1).
- 제10항에 있어서, 상기 발현억제제는 NUPR1의 mRNA에 특이적으로 결합하는 miRNA, siRNA, shRNA 및 안티센스 올리코뉴클레오티드로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 뇌종양 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating brain tumors according to claim 10, wherein the expression inhibitor is at least one selected from the group consisting of miRNA, siRNA, shRNA and antisense oligonucleotide that specifically binds to mRNA of NUPR1. .
- 제10항에 있어서, 상기 활성억제제는 NUPR1의 단백질에 특이적으로 결합하는 항체인 것을 특징으로 하는, 뇌종양 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating brain tumors of claim 10, wherein the activity inhibitor is an antibody that specifically binds to a protein of NUPR1.
- NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 포함하는 뇌종양 예방 또는 개선용 식품 조성물.Food composition for the prevention or improvement of brain tumors, including NUPR1 (Nuclear protein 1) expression or activity inhibitor.
- NUPR1(Nuclear protein 1)의 발현 또는 활성억제제를 이를 필요로하는 개체에 투여하는 단계를 포함하는, 뇌종양의 예방 또는 치료 방법.A method of preventing or treating brain tumors, comprising administering an expression or activity inhibitor of NUPR1 (Nuclear protein 1) to a subject in need thereof.
- (a) 분리된 뇌종양 세포에 뇌종양 치료 후보물질을 처리하는 단계; 및 (a) treating the brain tumor treatment candidate with the isolated brain tumor cells; And(b) 상기 뇌종양 세포에서 NUPR1 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계를 포함하는 뇌종양 치료제의 스크리닝 방법.(b) measuring the mRNA expression of the NUPR1 gene or its protein activity level in the brain tumor cells.
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