WO2012091220A1 - Composition pour la prévention ou le traitement de maladies néoplasiques, comprenant un miarn en tant que substance active - Google Patents

Composition pour la prévention ou le traitement de maladies néoplasiques, comprenant un miarn en tant que substance active Download PDF

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WO2012091220A1
WO2012091220A1 PCT/KR2011/001688 KR2011001688W WO2012091220A1 WO 2012091220 A1 WO2012091220 A1 WO 2012091220A1 KR 2011001688 W KR2011001688 W KR 2011001688W WO 2012091220 A1 WO2012091220 A1 WO 2012091220A1
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kit
mir
disease
sequence
expression
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김호근
김원규
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • composition for the prevention or treatment of tumor diseases comprising m i RNA as an active ingredient
  • the present invention relates to miR-494 and its use as negative regulators of KIT in GIST.
  • GISTs gastrointestinal stromal tumors
  • KIT oncogenes of the receptor tyrosine kinase family
  • PDGFRA v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
  • KIT oncogenes of the receptor tyrosine kinase family
  • PDGFRA v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
  • KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
  • PDGFRA v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
  • Mutations in these genes lead to their continued activation, resulting in constant stimulation of downstream signaling pathways of KIT and PDGFRA [4, 5].
  • KIT activation is important for the development and progression of GIST [3].
  • the downstream molecular pathways involved in GIST tumorigenesis following KIT mutations are PI3-kinase, Src family kinase and Ras-E. And JAK-STAT, etc. [6] ⁇ The activation of the molecular pathways described above after KIT activation results in a GIST tumor formation process through cell proliferation activation and apoptotic signal inhibition [4, 6, 7].
  • imatinib (STI571), which competitively binds to the ATP binding pocket.
  • Treatment of imatinib inhibits KIT activation and molecularly blocks downstream MAP kinase and PI3-kinase -AKT pathways [7, 8].
  • GIST patients who become imatinib-resistant during imatinib treatment are resistant to the inhibitory effects of imatinib.
  • KIT Overexpression of KIT has been reported as a typical feature of GIST, and the presence of KIT mutations always leads to potent KIT expression [12, 13]. Although KIT mutations are present in about 70% of GIST patients, overexpression of KIT is found in over 90% of GIST patients, indicating that another complementary mechanism is present in KIT overexpression [2, 14]. Because microRNAs (miRNAs) play an important role in regulating gene expression in cancer, they are mechanisms that can regulate their aberrations [15, 16]. Currently, miRNAs known to target KIT are miR-221 and miR-222 [17]. Previously, we have identified five candidate miRNAs showing relatively inverse expression to KIT expression by comparing KIT expression and miRNA expression profiles in GIST patients [13].
  • the inventors have sought to develop new targets for the treatment of gastrointestinal stromal tumors (GISTs).
  • GISTs gastrointestinal stromal tumors
  • the inventors directly bind miR-494 to two different seed match sites present in KIT mRNA to down-regulate KIT expression, and downstream molecules of the KIT signaling transition pathway (eg, The present invention was completed by discovering that the expression of phospho-AKT and phospho-STAT3) is inhibited to inhibit the growth and proliferation of GIST cell lines (eg, GIST882 cell line) with KIT-activating mutations.
  • Kit-v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog KIT-mediated disease, disorder or condition.
  • a pharmaceutical composition for preventing or treating diseases, disorders or conditions is provided.
  • Another object of the present invention to provide a nucleotide sequence for the prevention or treatment of the disease, disorder or condition.
  • Another object of the present invention to provide a method for treating the disease, disorder or condition.
  • the present invention provides microRNA-494 (miR-494). Or v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) comprising an agent that induces overexpression of miR-494-a mediated disease, disease or condition (tyrosine kinase—mediated diseases, A pharmaceutical composition for preventing or treating disorders or conditions) is provided.
  • miR-494 miR-494
  • KIT Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
  • the present invention provides a sequence of 20-100 for the treatment of tumor diseases comprising an antisense oligonucleotide sequence having a sequence complementary to the 8th to 15th nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2 sequence Provide the nucleotide sequence.
  • the present invention provides a continuous sequence of 20-100 for the treatment of tumor diseases comprising an antisense oligonucleotide sequence having a sequence complementary to the 8th to 15th nucleotide sequence of the nucleotide sequence of SEQ ID NO: 3 sequence Provide the nucleotide sequence.
  • the invention provides a method of treating a KIT-mediated disease, disorder or condition comprising the step of treating an agent that induces micro RNA-494 (miR-494) or miR-494 overexpression. to provide.
  • the invention processes 20-100 consecutive nucleotide sequences comprising an antisense oligonucleotide sequence having a sequence complementary to the 8th to 15th nucleotide sequences of the nucleotide sequence of SEQ ID NO: 2. It provides a method of treating a KIT-mediated disease, disorder or condition comprising the steps of:
  • the invention processes 20-100 consecutive nucleotide sequences comprising an antisense oligonucleotide sequence having a complementary sequence to the 8th to 15th nucleotide sequences of the nucleotide sequence of SEQ ID NO: 3 It provides a method of treating a KIT-mediated disease, disorder or condition comprising the steps of:
  • the inventors have sought to develop new targets for the treatment of gastrointestinal stromal tumors (GISTs).
  • KIT kinase is one of the receptor tyrosine kinases.
  • KIT kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase.
  • SCF stem cell factor
  • GIST gastrointestinal stromal tumors
  • SCLC small cell lung cancer
  • AML acute myelogenous leukemia
  • Mast cell leukemia Mast cell leukemia
  • GIST Gastrointestinal stromal tumors
  • the present invention is the first invention to discover the function of miR-494 as a negative regulator of KIT kinase that causes gastrointestinal stromal tumors.
  • miR-494 of the present invention directly binds to 3′-UTR (untranslated region) of KIT mRNA and down-regulates expression of KIT.
  • miR-494 of the present invention reduced phosphorylation (eg, phospho-AKT and phospho-STAT3) of downstream molecules of the KIT signaling transition pathway (see FIG. 5).
  • miRNA miRNA microRNA
  • miRNA miRNA
  • Drosha RNasem type enzymes
  • pre-miRNAs precursor miRNAs of stem-loop structure, which are transported into the cytoplasm and cleaved by Dicer into mature miRNAs [Kim VN et al. . , Nat Rev Mol Cell Biol. 2005 May; 6 (5): 376-385.
  • the miRNA prepared as described above is involved in development, cell proliferation and death, fat metabolism, tumor formation, etc. by controlling the expression of target proteins [Wienholds E et al. , Science, 309 (5732): 310-311 (2005); Nelson P et al., Trends Biochem Sci. , 28: 534-540 (2003); Lee RC et al. , Cell, 75: 843-854 (1993); and Esquela—Kerscher A et al., Nat Rev Cancer. 6: 259-269 (2006).
  • the number of the miRNA is a number assigned according to the found order of small RNA (small RNA)
  • miR-494 means the miRNA found in the 494th, which is obvious to those skilled in the art (http: //www.mirbase.org).
  • miR-494 overexpression agents encompasses all agents that induce overexpression of miR-494 of the present invention and include chemicals, nucleotides, antisense-RNAs, siRNACsmall interference RNAs. ) And natural extracts, but is not limited thereto.
  • miR-494 is located at No. 14 of the human chromosome and is described in SEQ ID NO: 1.
  • the base sequence is a mature form of miRNA-494, which is derived from the precursor miRNA (precusor microRNA) of the miR-494 hairpin structure. From 2nd in Sequence 1
  • the nucleotide sequence up to the eighth is the key sequence of miR-494.
  • the key sequence of miRNA is a very important sequence for target recognition (Krenz, M. et al., J. Am. Coll. Cardiol. 44: 2390-2397 (2004); H. Kiriazis, et. al., Annu. Rev. Physiol. 62: 321 (2000)), conserved for various species.
  • the antisense oligonucleotides of the present invention are sequences capable of binding to sequences present in the target (preferably KIT) of miR-494, and are preferably capable of binding to the sequence 2nd sequence and the sequence 3rd sequence.
  • nucleotide sequence is the second sequence Having a sequence complementary to T sense oligonucleotide is 20 to 100 contiguous nucleotide sequence or the antisense oligonucleotide SEQ ID NO. 2 coming from 100 consecutive nucleotide sequence containing a nucleotide sequence having a sequence complementary to the nucleotide sequence of the third sequence comprising a nucleotide sequence.
  • antisense oligonucleotide herein includes a nucleic acid-based molecule that has a complementary sequence to a miRNA, particularly the key sequence of the miRNA, and that can then hybridize to the target sequence of the miRNA.
  • antisense oligonucleotide can be described herein as a "complementary nucleic acid-based inhibitor.”
  • the term "complementary" as used to refer to an antisense oligonucleotide is sufficient to allow the antisense oligonucleotide to selectively localize to a miR-494 target under certain localization or annealing conditions, preferably physiological conditions.
  • Complementary means having one or more mismatch sequences, meaning substantially encompassing both substantially com lementary and perfectly complementary, preferably Means completely complementary.
  • the key match sites / sequences of the miR-494 of the invention are described in SEQ ID NO: 2 and SEQ ID NO: 3. Most preferably, the position / sequence is at 3′-UTR of KIT mRNA.
  • Antisense oligonucleotides in the present invention include various molecules. Antisense oligonucleotides are DNA or RNA molecules, more preferably RNA molecules. Optionally, the antisense oligonucleotides used in the present invention include ribonucleotides (RNA), deoxyribonucleotides (DNA), 2'-0-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, peptide nucleic acid (PNA) or LNAGocked. nucleic acid).
  • RNA ribonucleotides
  • DNA deoxyribonucleotides
  • PNA phosphorothioate-backbone deoxyribonucleotides
  • PNA peptide nucleic acid
  • LNAGocked. nucleic acid LNAGocked. nucleic acid
  • the 2'-0-modified oligonucleotide is preferably a 2'-0-alkyl oligonucleotide, more preferably a 2'-0-d-3 alkyl oligonucleotide, even more preferably a 2'-0- d-3 methyl oligonucleotide.
  • antisense oligonucleotides in the present invention have a broad meaning including nucleic acid-based inhibitors having sequences complementary to the target sequence of miR-494. Included in the antisense oligonucleotides of the present invention include, for example, antisense oligonucleotides in a narrow sense. Inhibition of function on the miR-494 target of the invention can be achieved by administering a typical antisense oligonucleotide. Antisense oligonucleotides are ribonucleotides
  • Antisense oligonucleotides may comprise one or more locked nucleic acids (LNAs).
  • LNA is a modified ribonucleotide that has a locked form, including an additional bridge between 2 'and 4' carbons of the ribose sugar moiety, so that oligonucleotides with LNA have improved thermal stability [J Weiler, J. Hunziker and J Hall Gene Therapy (2006) 13, 496.502].
  • antisense oligonucleotides may include peptide nucleic acids (PNAs), which include peptide-based backbones instead of sugar-phosphate backbones.
  • PNAs peptide nucleic acids
  • antisense oligonucleotides may include include 2'-0-alkyl (eg, 2'-0_methyl, 2'-0-methoxyethyl), 2'-fluoro and 4'-thio modifications.
  • Sugar modifications such as;
  • Backbone modifications such as phosphorothioate, morpholino or phosphonocarboxylate linkages (eg, US Pat. Nos. 6,693,187 and 7,067,641).
  • a suitable antisense oligonucleotide is 2'-0_methoxyethyl "gapmer” 'which includes 2'-0-methoxyethyl-modified ribonucleotides at the 5'-terminus and 3'-terminus Has at least 10 deoxyribonucleotides in.
  • This "gapmer” can trigger an RNase I-dependent disruption mechanism of an RNA target.
  • the antisense oligonucleotides are 7-50 nucleotides in length, preferably 10-40 nucleotides, more preferably 15-30 nucleotides, and most preferably 2-25 nucleotides.
  • the agents inducing miR-494 or miR-494 overexpression of the invention inhibit the expression of KIT or inhibit the phosphorylation of AKT or STAT3.
  • MiR-494 of the present invention inhibited the expression of the KIT at the mRNA or protein level, and phosphorylation of the AKT or STAT3 was inhibited (see FIGS. 4B and 5).
  • Inducing overexpression of miR-494 inhibited GIST cell growth (consistent with changes in cell cycle phase and S phase: see FIG. 6).
  • inhibitory RNA molecules include, but are not limited to, small interference RNA (siRNA), short hairpin RNA (shRNA), ribozyme (r ibozyme), DNAzyme or peptide nucleic acids (PNA).
  • siRNA small interference RNA
  • shRNA short hairpin RNA
  • r ibozyme ribozyme
  • PNA peptide nucleic acids
  • siRNA refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (W0 00/44895, W0 01/36646, W0 99/32619, W0 01/29058, W0 99 / 07409 and W0 00/44914).
  • siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of the target gene.
  • siRNA was first discovered in plants, worms, fruit flies and parasites, but has recently been used in mammalian cell research by developing / using siRNA (Degot S, et al. 2002; Degot S, et al. 2004; Ballut L, et al. 2005).
  • diseases that can be treated by the pharmaceutical composition of the present invention include KIT-mediated diseases, diseases or conditions, diseases, diseases or conditions induced by the overexpressed or mutated form of the KIT gene or protein More preferably, neoplastic disorder, inflammatory disease, autoimmune disease, cancer, allergic disease, irritable bowel syndrome (IBS), graft versus host disease (GVHD), metabolic syndrome, CNS (central nervous system) )-Related diseases Degenerative neuropathy, mast-cell associated disease, pain, substance-abuse disease, prion disease, heart disease, fibrotic disease, idiopathic pulmonary hypertension (IPAH), primary pulmonary hypertension (PPH), glioma or cardiovascular disease, even more preferably neoplastic disease.
  • neoplastic disorder preferably, inflammatory disease, autoimmune disease, cancer, allergic disease, irritable bowel syndrome (IBS), graft versus host disease (GVHD), metabolic syndrome, CNS (central nervous system)
  • IBS irritable bowel syndrome
  • the neoplastic disorder of the present invention is gastrointestinal stromal tumor (gatrointe 'stinal stromal tumors, GISTs), small cell lung cancer (small cell lung cancer), non-small cell lung cancer, acute myeloid leukemia (acute myelocytic leukemia, acute lymphocytic leukemia, myelodyplastic syndrome, chronic myeloid leukemia, large intestine Carcinoma, gastric carcinoma, testicular cancer, glioblastoma astrocytoma or mastocytosis, most preferably gastrointestinal stromal tumor.
  • GISTs gastrointestinal stromal tumor
  • small cell lung cancer small cell lung cancer
  • non-small cell lung cancer non-small cell lung cancer
  • acute myeloid leukemia acute myelocytic leukemia, acute lymphocytic leukemia, myelodyplastic syndrome, chronic myeloid leukemia, large intestine Carcinoma, gastric carcinoma, testicular cancer, glioblastoma astro
  • the carcinoma of the present invention is testicular cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, brain cancer, colon cancer, neuroendocrine cancer, gastric cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, Adrenal cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer or ureter cancer.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in the preparation, lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, phosphate chalc, alginate, gelatin, calcium silicate, microcrystalline saelreul Ross, polyvinyl an pyrrolidone, selreul Ross, water, syrup, methyl selreul Ross, methyl hydroxybenzoate, propyl hydroxybenzoate,, talc, magnesium stearate, and mineral oils such as Including, but not limited to.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
  • compositions of the invention may be oral or parenteral (eg, intravenous, subcutaneous or topical).
  • Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction in response to the patient. It may be prescribed. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.0001 ⁇ 100 mg / kg (body weight) per day.
  • compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container.
  • the formulation is a solution suspension or emulsion in an oil or aqueous medium It may also be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.
  • the invention comprises the steps of (a) treating a test substance to a cell comprising a micro RNA-494 (miR-494) -encoding nucleotide sequence; And (b) analyzing the expression of miR-494 in the cells.
  • a method for screening a therapeutic agent for neoplastic disorders comprising: increasing the expression of miR-494 and treating it as a tumor drug do.
  • a sample to be analyzed is contacted with a cell comprising the nucleotide sequence of the target of the present invention (preferably miR-494-encoding nucleotide sequence).
  • Cells comprising the nucleotide sequence of the target of the present invention are not particularly limited, preferably cells that overexpress KIT or contain KIT-activating mutations, and more preferably are gastrointestinal stromal cell lines.
  • the cell is preferably a primary cultured cell, an established cell line or a tumor cell.
  • the cell comprising the nucleotide sequence of the present invention is a human gastrointestinal stromal tumor line.
  • test material refers to an unknown material used in screening to examine whether it affects the expression level of the marker of the present invention.
  • the test substance includes, but is not limited to, chemicals, nucleotides, antisense -RNAs, siRNAC small interference RNAs) and natural extracts.
  • the expression level of the target of the present invention is measured in the cells treated with the test substance.
  • the amount of expression can be measured as described below.
  • the test substance is used for tumorous disease. It can be determined as a prophylactic or therapeutic substance.
  • Measurement of the expression level change of miR-494 and KIT of the present invention can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (200D), Northern blotting (Peter B.
  • RNA is isolated from cells treated with the test substance, and then, first-chain cDNA is prepared using oligo dT primer and reverse transcriptase. Subsequently, the first chain cDNA is used as a template, and a PCR reaction is performed using a miR-494- or KIT-specific primer set. Then, the PCR amplification product is electrophoresed, and the formed band is analyzed to measure the change in the expression level of miR-494 or KIT.
  • the amplification of the invention is carried out according to real-time PCR.
  • Real-time PCR is a technique for monitoring and analyzing the increase in PCR amplification products in real time (Levak KJ, et al., PCR Methods Appl., 4 (6): 357-62 (1995)).
  • PCR reactions can be monitored by recording the fluorescence emission in each cycle during an exponential phase in which the increase in PCR product is proportional to the initial amount of the target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the C r value (threshold cycle).
  • a marked increase in fluorescence above the reference value measured between 3-15 cycles means detection of accumulated PCR product.
  • real-time PCR has the following advantages: (a) Conventional PCR is measured in the plateau, while real-time PCR is used during the exponential growth phase. Data can be obtained; (b) the increase in the reporter fluorescence signal is directly proportional to the number of amplicons generated; (c) the cleaved probe provides permanent record amplification of the amplicon; (d) increase the detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) electrophoresis Detection of amplified DNA is possible without separation through; (g) small amplicon sizes may be used to achieve increased amplification efficiency; And (h) the risk of contamination is low.
  • PCR amplification products are detected by fluorescence.
  • Detection methods are largely an interchelating method (SYBR Green I method), a method using a fluorescent labeled probe (TaqMan probe method), and the like.
  • the interchelating method uses a double-stranded DNA binding die, which includes non-specific amplification and primer-dimer complexes using a non-sequence specific fluorescence interchelating reagent (SYBR Green I or ethidium bromide).
  • SYBR Green I is a fluorescent die that binds to the minor groove of double-stranded DNA. It is a reagent that shows little fluorescence in solution but shows strong fluorescence when bound to double-stranded DNA (Morrison TB, Biotechniques., 24 (6): 954-8, 960, 962 (1998).
  • SYBR Green Real-Time PCR is accompanied by optimization procedures such as melting point or dissociation curve analysis for amplicon identification. Normally, SYBR greens are used for singleplex reactions, but they can be used for multiplex reactions when accompanied by melting curve analysis (Siraj AK, et al., Clin Cancer Res., 8 ( 12): 3832-40 (2002); and Vrettou C., et al., Hum Mutat., Vol 23 (5): 513-521 (2004)).
  • the threshold cycle (C T ) value is the number of cycles in which the fluorescence generated in the reaction exceeds the threshold, which is the initial copy number. Inversely proportional to algebra Therefore, the Cr value assigned to a particular well reflects the number of cycles in which the stratified number of amplicons accumulated in the reaction.
  • the C r value is the cycle in which an increase in ARn was first detected. Rn means the magnitude of the fluorescence signal generated during PCR at each time point, and ARn means the fluorescence emission intensity (standardized reporter signal) of the reporter die divided by the fluorescence emission intensity of the reference die.
  • the Cr value is also named CpCcrossing point in LightCycler.
  • the Cr value represents the point in time when the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in a log-linear phase. This period provides the most useful information about reaction.
  • the slope of the log-linear step represents the amplification efficiency (Eff) (hUp: //w.appl iedbiosystems.co.kr/).
  • TaqMan probes typically contain primers (eg, 20-30 nucleotides) comprising a fluorescent at the 5'-end and a quencher at the 3'-end (eg, TAM A or non-fluorescent something (NFQ)). It is longer than ligonucleotides.
  • Taqman probes are designed to anneal to internal sites of PCR products. Preferably, the Taqman probe may be designed as an internal sequence of the first sequence of Sequence Listing.
  • Taqman probes specifically hybridize to template DNA in the annealing step, but fluorescence is suppressed by the quencher on the probe.
  • the Taq DNA polymerase activity possessed by Taq DNA polymerase decomposes the Taqman probe, which is localized in the template, and the fluorescent dye is released from the probe.
  • the 5'-end of the Taqman probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by the template-dependent nucleic acid polymerase, the 5'-end of the Taqman probe is cleaved by the 5 'to 3' nuclease activity of the polymerase to The fluorescent signal is generated. .
  • Fluorescent reporter molecules and those molecules that can be used in the present invention can be any known in the art, for example: (The number in parentheses is the maximum emission wavelength in nanometers): Cy2 TM (506), Y0-PR0 TM -1 (509), YOYO TM -1 (509), Calcein (517), FITC (518), FluorX TM (519), Alexa TM (520), Rhodamine 110 (520) , 5-FAM (522), Oregon Green TM 500 (522), Oregon Green TM 488 (524), RiboGreen TM (525), Rhodamine Green TM (527), Rhodamine 123 (529), Magnesium Green TM (531), Calcium Green TM (533), T0-PR0 TM -1 (533), T0T01 (533), JOE (548), B0DIPY530 / 550 (550), Di 1 (565), B0DIPY TMR (568), B0
  • DNACgDNA or cDNA DNACgDNA or cDNA
  • RNA molecules more preferably RNA molecules.
  • the method of annealing or hybridizing the target nucleic acid to the extension primers and probes can be carried out by the method of known in the art.
  • suitable isomerization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory.
  • conditions such as temperature, concentration of components, time to shake and reaction, buffer components and their pH and ionic strength depend on various factors such as the length and GC amount of the oligonucleotide and the target nucleotide sequence.
  • Detailed conditions for the shake are described by Joseph Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer I Verlag New York Inc. N.Y. (1999).
  • the template-dependent nucleic acid polymerase used in the present invention is an enzyme having 5 'to 3' nuclease activity.
  • the template-dependent nucleic acid polymerase used in the present invention is preferably a DNA polymerase.
  • DNA polymerases typically have 5 'to 3' nuclease activity.
  • Template-dependent nucleic acid polymerases used in the present invention include E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase.
  • the template-dependent nucleic acid polymerase is a thermostable DNA polymerase obtained from various bacterial species, which is Thermus aguatjeusi Taq), Thermus , Thermus filiformis, Therm is flavus, Thermococcus literal is, Pyrococcus fur / osusiPfu), Thermus antranikiani i, Thermus caldophi lus, Thermus chl iarophi lus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus rubenoshier, Thermus rubenoshier DNA polymerases of scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophi lus, Ther otoga maritima, Thermotoga neapol itana and Thermos ipho africanus.
  • template-dependent extension reaction catalyze
  • the real-time PCR of the present invention is carried out by Taqman probe method.
  • changes in the amount of KIT protein can be carried out through various immunoassay methods known in the art.
  • changes in the amount of granulation factor protein may include immunostaining, radioimmunoassay, radioimmunoprecipitation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay, capture-ELISA, inhibition or competition assay, and Including but not limited to sandwich analysis.
  • the immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed. , CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-1 inked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed. , Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Ant i bodies- ⁇ Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.
  • an antibody labeled with a radioisotope detects a marker molecule of the present invention. It can be used to.
  • the present invention comprises the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; ( ⁇ ) reacting the cell lysate with the antibody to the target as a primary antibody; (iii) reacting the resultant of step (ii) with the secondary antibody to which the enzyme is bound; And (iv) measuring the activity of the enzyme.
  • Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.
  • Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescent reaction, luminescence reaction or infrared reaction, for example, luciferase, alkaline phosphatase, ⁇ _galacto Oxidase, horse radish peroxidase and cytochrome p 450 .
  • alkaline phosphatase When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium ( ⁇ ), and naph are -AS-B1-phosphate (naphthol— When chromogenic reaction substrates such as AS-Bl-phosphate (ECF) and enhanced chemi fluorescence (ECF) are used, and when horse radish peroxidase is used, chloronaph, aminoethylcarbazole, diaminobenzidine, D—luciferin, lucifer Genine (bis-N-methylacridinium nitrate) Resorphin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (-pheny 1 ened i am i ne ⁇ HC 1 and pyrocatechol), TMB (tetra
  • certain embodiments of the invention comprise the steps of (coating an antibody against a target of the invention as a capturing antibody on the surface of a solid substrate; (ii) Reacting the capture antibody with the cell sample (iii) reacting the product of step (ii) with a detecting antibody that has a label that generates a signal and specifically reacts with the granulation factor protein And (iv) measuring the signal resulting from the label.
  • the detection antibody carries a label which generates a detectable signal.
  • the label may include chemicals (eg biotin), enzymes (alkaline phosphatase ⁇ ⁇ -galactosidase, horse radish peroxidase and cytochrome ⁇ 450 ), radioactive substances (eg C 14 , I 125 , ⁇ 32 and S 35 ), fluorescent materials (eg, fluorescein), luminescent materials, chemi luminescent, and fluorescence resonance energy transfer (FRET).
  • chemicals eg biotin
  • enzymes alkaline phosphatase ⁇ ⁇ -galactosidase, horse radish peroxidase and cytochrome ⁇ 450
  • radioactive substances eg C 14 , I 125 , ⁇ 32 and S 35
  • fluorescent materials eg, fluorescein
  • luminescent materials eg, chemi luminescent, and fluorescence resonance energy transfer (FRET).
  • the ELISA Methods and Capture The measurement of the final enzyme activity or signal in the ELISA method can be carried out according to various methods known in the art. Detection of such signals allows for qualitative or quantitative analysis of the targets of the present invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.
  • the present invention relates to a novel function of miR-494 as a negative regulator of KIT in GIST.
  • miR-494 of the present invention binds directly to two other key match sites / sequences within the KIT 3′-UTR to down-regulate KIT and downstream molecules of the KIT signaling transition pathway (Eg, phospho-AKT and phospho-STAT3).
  • compositions and methods of the present invention comprising a miR-494 of the present invention and a substance that promotes its expression or a substance that inhibits KIT expression as an active ingredient can be used for the prevention or prevention of KIT-mediated diseases, diseases or conditions. It can be usefully applied to treatment.
  • FIG. 1 is a Western blot result investigating the effects of five candidate miRNAs on KIT protein expression.
  • Five candidate miRNAs miR-9, miR-142-5p, miR-370, miR-494 and miR-510) in total 25 nM were transfected into KIT-overexpressing GIST882 cell lines, respectively.
  • GAPDH was used as loading control.
  • N.C. represents a negative control.
  • FIG. 3 shows KIT protein (3a), miR-494 levels (3b) and graph (3c) simultaneously expressed in 31 GIST patients.
  • GIST patient tissues from Cases 1 to 25 had KIT mutations, and GIST patient tissues from Cases 26 to 31 did not contain KIT mutations.
  • Each case extracted both RNA and protein from the same tissue.
  • KIT expression levels were quantified from Western blot results.
  • the amount of mature miR-494 was measured using a Taqman miRNA assay.
  • Expression levels of miR-494 and KIT were normalized to U6 RNA and GAPDH, respectively and normalized values of miR-494 and KIT expression levels were divided by the mean value of miR-494 and KIT for comparison.
  • 4 is a reporter assay result for determining miR-494 binding sites present within the KIT 3′-UTR site.
  • 4A is a diagrammatic representation of vector constructs used in a reporter assay.
  • the original vector (N vector) comprises the total sequence of the Renilla luciferase-encoding site and the 3′-UTR site of KIT obtained from GIST882 cDNA.
  • the M construct comprises four mutated nucleotide sequences (GTTKXGG) from the binding seed sequence of miR-494 predicted according to target scan 3.0.
  • Ma, Mb and Mc constructs were prepared by replacing the possible miR-494 binding sites found by the inventors with four nucleotide sequences (GTTTCCGG) in the M construct.
  • the 0 vector was used to confirm that all constructs function properly using miR-221 and miR-222 (GTAGCAGA), previously reported to target the 3'-UTR of the KIT.
  • 4B transfected the He constructs containing M constructs containing miR-494 or zero constructs containing miR-221. Transfection After 2 days, cells were obtained and subjected to a dual luciferase assay. When the predicted miR-221 and miR-222 binding sites were mutated, luciferase activity was restored. Transfection with miR-494 and M mutant vectors resulted in only slight recovery (FIG. 4B, top panel).
  • FIG. 4C is the result of Tapan-miRNA assay (top panel) examining the expression level of miR-494 in different cancer cell lines (top panel) from which HeLa, SNU216 and GIST882 cell lines were selected for future study (bottom panel).
  • HeLa and SNU216 cells exhibited high expression levels of miR-494.
  • Transfection of the N vector with a 50 nM miR-494 inhibitor in the cell line showed significantly increased luciferase activity compared to cells transfected with non-targeting miRNA (negative control).
  • the GIST882 cell line showed low expression levels of miR-494 and did not show any significant change in luciferase activity compared to cells transfected with non-targeted miRNA (negative control) (bottom panel).
  • the above results indicate that endogenous miR-494 directly down-regulates KIT.
  • NC represents negative control.
  • FIG. 5 is a Western blot result of measuring the expression of p-AKT and p_STAT in cell lines transfected with miR-494.
  • Transfection of miR-494 into the GIST882 cell line caused significant changes in the AKT and STAT signaling pathways.
  • GIST882 cell lines were transfected with 50 nM of untargeted miRNA, miR-221, miR-494 or miR-494 inhibitors.
  • Western blots for KIT, phospho-KIT, STAT3, phospho-STAT3, AKT phospho-AKT, ERK, phospho-ERK and GAPDH were repeated in the same blot for each antibody.
  • Expression levels of KIT and phospho-KIT were markedly reduced by transfection of GIST882 with miR-494. Both downstream molecules of the KIT signaling pathway, phospho-AKT and phospho-STAT3, were reduced. Other results indicate that the control group There was little difference between the samples and the samples treated with the miR-494 inhibitor. NC
  • FIG. 6 shows that GIST cell proliferation is inhibited by miR-494.
  • FIG. 6A shows the GIST882 morphology 12 days after transfection. Cells were cultured in 60-mm cell culture dishes and pictures were taken before cell counting. Poor cell clusters and irregular cell morphology appeared on day 12 in GIST882 cell line transfected with miR-494 (right panel).
  • FIG. 6B shows GIST882 cells were cultured in 60-mm dish with 2 ⁇ 10 6 cells and dispensed into each dish. Cells were counted twice manually at 3, 6 and 12 days after transfection and counted average values were used. Cells were transfected twice on day 0 and day 6.
  • FIG. 6C shows the results of transfecting GIST882 cells with non-targeted miRNA (top panel) or miR-494 (bottom panel) on day 4, and then staining with propidium iodide. Both samples were analyzed by flow cytometry.
  • MiR-494 treatment showed a 5.6% increase in stage III cells and a 5.7% decrease in S phase cells compared to cells transfected with non-targeted miRNAs. NC represents negative control.
  • GIST882 cell lines with activated KIT mutations were kindly presented by Dr. Jonathan Fletcher of Harvard University (Cambridge, Mass.).
  • SNU216, SNU638, SNU1, NCI-N-87 and HeLa cell lines were purchased from Korean Cell Line Bank; Cancer Research Institute, Seoul Korea.
  • Cell culture images were obtained using IX71 (01ympus, Tokyo, Japan). Patient and tissue feed
  • GIST patients were divided into four groups based on tumor risk according to Fletcher et al. [33].
  • qRT-PCR Quantitative RT-PCR
  • KITCPrimerBank ID; 4557695a2) and the qRT-PCR primer sequences for GAPDH were obtained from Primerbank database (ht tp: / /pga.mgh.harvard.edu/pr i merbank /).
  • the reaction was carried out at a total dose of 20 ⁇ containing Premix Ex Taq (TAKARA, Tokyo, Japan) according to the manufacturer's instructions. All reactions were run in three pairs in an ABI Prism 7300 real-time PCR system. miRNA mimetics (mimics) and transformation
  • Total lysates from the samples were obtained with passive lysis buffer (Pr omega, Madison, Wis., USA).
  • Primary antibodies used in the present invention are GAPDHCTrevigen, Gaitherburg, MD, USA), c-KIT, STAT3, ERK, phospho-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho c—KIT (Invitrogen) , AKT, phospho-AKT and phospho STAT3 (Cel 1 Signaling, Danvers, MA USA).
  • Western blot images were analyzed using the LAS-4000 Mini (Fuj i f i lm, Tokyo, Japan). Luciferase Reporter Assay
  • N vectors were prepared. Total 3′-URT sequences obtained from GIST882 via PCR amplification were cloned downstream of the SV40 enhancer and early promoter-regulated Renilla luciferase cassette in pRL3 vector (Promega). The N vector was then used to prepare five mutation constructs to replace mutations in the key sequences complementary to the miR-494, miR-221 or miR-222 binding sites. The PGL3 luciferase reporter vector was used as a control vector for the dual luciferase assay (Promega). Oligonucleotide sequences used for the vector constructs are listed in Table 2. Table 2
  • Dual luciferase assays were performed each time by co-transfecting the control vector with N, M, 0, Ma, Mb or Mc vectors. All miRNA mimetics were transfected with 5 nM. After 2 days of transfection, luciferase activity was measured according to the manufacturer's instructions.
  • Cell proliferation assay
  • GIST882 cells were aliquoted into 60-mm dishes with 2 ⁇ 10 6 cells and then transfected with 50 nM of untargeted miRNA or miR-494. Cell counts were manually counted on days 3, 6 and 12. Another transfection was performed on day 6 for the samples to be counted on day 12. Cell morphology was checked daily. Samples consisted of two pairs and mean values were used for further analysis. Each sample was obtained and analyzed by Western blot to confirm sustained inhibition on KIT expression. Cell cycle analysis GIST882 cells were transfected with 50 nM of untargeted miRNA or miR-494 in 60-dish.
  • miRNAs (miR-9, miR-142-5p, miR-370, miR-494 and miR-510) were transfected into KIT-overexpressing GIST882 cell lines, respectively.
  • Western blot analysis of the transfected samples revealed that only miR-494 consistently reduced KIT protein expression (FIG. 1). The inventors then conducted further studies on miR-494.
  • m iR-494 The ability of m iR-494 to induce KIT down-regulation was confirmed by transfecting 25 nM of nontargeting miRNA miR-494, miR-221 or miR-494 inhibitors to the GIST882 cell line. Untargeted miRNA was a negative control compared to miR-494, and miR-221 was a positive control compared to miR-494. miR-221 and miR-222 are both known to directly target the 3'-UTR of KIT mRNA [17]. Three days after transfection, cells were obtained and subjected to quantitative reverse transcription polymerase chain react ion (qRT-PCR) and western blotting.
  • qRT-PCR quantitative reverse transcription polymerase chain react ion
  • the inventors performed miRNA qRT-PCR to analyze the expression level of miR-494 and western blot to analyze the expression level of KIT protein, thereby inversely correlating KIT expression and miR-494 expression in GIST. correlat ion).
  • the assay used 31 freshly-frozen samples of GIST patients consisting of 25 GIST patients with KIT mutations and 6 GIST patients without KIT mutations (Table 1).
  • the miR-494 levels were determined using a Taqman miRNA assay and analyzed by ABI real-time PCR 7300 using mean values obtained from three independent experiments in each case.
  • the levels of KIT and miR-494 from each case were presented as a relative fold change over the mean value of KIT and miR-494 compared to the respective expression levels (FIGS.
  • KIT mRNA is a direct target of miR-494 by identifying the binding position of miR-494 to 3′-UTR of KIT mRNA. 1897-1903 via a target scan 3.0 database (http: // www, target scan, org /) to detect miR-494's algorithm-based binding site for the 3'-UTR of KIT mRNA One binding site located at was expected (FIG. 4A).
  • N vector a vector named N (N vector) comprising the encoding sequence for the total 3′-UTR sequence of Renilla luciferase and KIT mRNA obtained from cDNA of GIST883 cell line. Devised.
  • the KIT 3′-UTR position was amplified by PCR and cloned to the right after the Renilla luciferase encoding sequence in the pRL3 vector.
  • N vectors (5 nM) comprising non-targeting miRNA, miR-494, miR-221 or miR-222 were tested.
  • HeLa cell line was transfected. Two days after transfection, cells were harvested and subjected to dual luciferase assay using a dual luciferase assay (DLR) assay kit.
  • DLR dual luciferase assay
  • Non-targeting miRNA, miR-494, miR-221 or miR-222 was transfected into an N, M or 0 vector, and a reporter assay was performed. Luciferase activity in cells transfected with 0 vectors containing miR-221 or miR-222 was fully restored compared to the negative control (cells transfected with N vectors containing untargeted miRNAs). miR-221 and miR-222 directly target KIT mRNA. However, samples transfected with M vector and miR-494 showed very slightly restored luciferase activity compared to the negative control (FIG. 4B, top panel). Above The results show that miR-494 can directly target the 3'-UTR of KIT mRNA or there may be additional binding sites.
  • KIT expression was reduced in cells transfected with m iR-221, but less than miR-494.
  • miR-494 inhibitors did not affect KIT expression, probably because the expression level of miR-494 in the GIST882 cell line is very low.
  • the expression of p-KIT (pY703) was also tested with phospho-specific antibodies, which significantly reduced expression levels.
  • the activation forms of the three downstream molecules of the KIT signaling pathway (AKT, ERK and STAT3) were also analyzed. As measured with phospho-AKT specific antibodies, phospho-AKT levels decreased after miR-221 and miR-494 treatment (FIG. 5), and were more phospho after miR-494 treatment than miR-221 treatment. Down-regulation of AKT occurred.
  • the effect of miR-494 on proliferation of GIST882 cells was confirmed by a cell proliferation assay in which GIST882 cells were cultured by dispensing GIST882 cells into 60-mm dishes with 2 ⁇ 10 6 cells. Normal cell morphology was checked regularly, and transfection was performed in a total of 12 dishes on the third day of subculture. Cells were transfected with 50 nM of untargeted miRNA or miR-494 on the first and sixth days, respectively, and counted the cell number on days 3, 6 and 12 after initial transfection. Each experiment was conducted in two pairs and used for the data analysis. After 3 days no distinct change occurred.
  • miRNAs can play a major regulatory role in signal transduction and tumorigenesis [15, 16]. Mutations and overexpression of KIT are internally associated with KIT-induced tumorigenic processes, and dysregulated miRNAs may explain KIT overexpression and subsequent tumorigenic processes [12, 13]. miR-221 and miR-222 have been reported as negative regulators of erythroid cell proliferation by targeting KIT [17]. However, miRNAs involved in GIST tumor formation by targeting KIT have not been reported yet. In the present invention, we propose that miR-494 directly targets KIT and that inhibition of miR-494 induces KIT overexpression by demonstrating that endogenous miR-494 induces KIT down-regulation.
  • miR-494 Down-regulation of KIT occurs more strongly at int by miR-494 than miR-221 and miR-222 previously reported with KIT-targeting miRNAs. Furthermore, expression levels of miR-221 and miR-222 were not associated with KIT expression in GIST [13]. Thus, we could conclude that miR-494 is the major miRNA that regulates KIT expression in GIST. To date, reports on the function of miR-494 have shown that miR-494 induces down-regulation of PTEN in chemically transformed cell lines and miR-494 expression is reduced in lymphoma and head and neck squamous cell carcinoma according to miRNA profile studies. There were no reports but [19-21]. However, no direct target has been reported on miR-494's direct targets. This is the first study to demonstrate that miR-494 is a direct target of KIT in GIST.
  • KIT proteins are high oncogenic tyrosine kinases belonging to the RTK family and are included in the major signal transduction pathways of PI3-kinase, Six family kinases and Ras-Erk, and the minor signal transduction pathways of JAK-STAT [6]. Mutations in transmembrane domains continue to activate downstream signals by activating dimerization of the KIT receptor [22] .Acquired mutations are often found in exons 9, 11, 13 and 17 of the KIT gene, which is a GIST tumor formation process. Contribute to [3, 23]. This study shows that down-regulation of KIT induced by miR-494 It has been demonstrated that regulation affects the levels of p-AKT and p-STAT3.
  • Tumor proteins can be inhibited in post-translational fashion or in post-transcriptional fashion [27].
  • Inhibition of KIT by imatinib is an example of post-translational inhibition via competitive binding of KIT to the ATP binding forge.
  • inhibition of KIT by imatinib is incomplete because resistance through potential mutational changes of amino acid residues that may occur during processing may occur.
  • imatinib resistance occurs by inhibiting the binding of imatinib to ATP binding forks of KIT [11].
  • Such possible mutation-derived resistance is urgently needed for more powerful therapeutic means including miR-494.
  • miR-494 therapeutic use of miR-494 to inactivate KIT would have to be supplemented because conventional RNA-based therapies are paradoxically unstable in the blood with the enormous molecular size of small interfering RNAs.
  • novel delivery systems such as 3 ⁇ 4 timer, nanoparticles and liposomes [28-30].
  • miR-494 could be used as a novel therapeutic means for treating GIST.
  • miR-494 is a potent regulator of KIT in GIST, and introducing miR-494 into GIST patients may be a novel way to suppress tumor progression.
  • Kang HJ Nam SW, Kim H, et al. Correlation of KIT and platelet-derived growth factor receptor al ha mutat ions with gene activation and expression profiles in gastrointestinal stromal tumors. Oncogene 2005; 24: 1066-74.

Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies, troubles ou affections médiés par KIT (homologue de l'oncogène viral du sarcome félin Hardy-Zuckerman 4 v-kit), qui comprend microARN-494 (miR-494) ou un agent (agents) qui induit (induisent) la surexpression de miR-494. La présente invention concerne en outre un procédé de criblage utilisant la composition pharmaceutique. Le miR-494 de la présente invention se lie directement à deux sites de séquence centrale différents (sites/séquences de correspondance d'amorce) dans 3'-UTR de KIT et effectue donc le contrôle descendant de KIT, et réduit l'expression de molécules en aval (par exemple phospho-AKT et phospho-STAT3) dans la voie de transition de signalisation KIT, et l'induction de la surexpression de miR-494 de la présente invention supprime la croissance de cellules néoplasiques et de préférence des lignées cellulaires GIST. Par conséquent, lors de la prévention ou du traitement de maladies, troubles ou affections médiés par KIT, il est avantageux d'utiliser la composition de la présente invention qui comprend miR-494 et une substance qui stimule l'expression de celui-ci ou une substance qui supprime l'expression de KIT de la présente invention en tant que substance active.
PCT/KR2011/001688 2010-12-30 2011-03-10 Composition pour la prévention ou le traitement de maladies néoplasiques, comprenant un miarn en tant que substance active WO2012091220A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112210554A (zh) * 2019-07-11 2021-01-12 东北林业大学 源于鹿茸由CNBP介导的microRNA及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101668518B1 (ko) * 2014-05-23 2016-10-24 강원대학교산학협력단 혈관신생을 촉진하는 마이크로rna-101

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090186348A1 (en) * 2007-09-14 2009-07-23 Asuragen, Inc. Micrornas differentially expressed in cervical cancer and uses thereof
US20100144850A1 (en) * 2007-04-30 2010-06-10 The Ohio State University Research Foundation Methods for Differentiating Pancreatic Cancer from Normal Pancreatic Function and/or Chronic Pancreatitis
KR20100064516A (ko) * 2008-12-05 2010-06-15 서울대학교산학협력단 마이크로ｒｎａ를 유효성분으로 포함하는 항암제 및 그 제조방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100144850A1 (en) * 2007-04-30 2010-06-10 The Ohio State University Research Foundation Methods for Differentiating Pancreatic Cancer from Normal Pancreatic Function and/or Chronic Pancreatitis
US20090186348A1 (en) * 2007-09-14 2009-07-23 Asuragen, Inc. Micrornas differentially expressed in cervical cancer and uses thereof
KR20100064516A (ko) * 2008-12-05 2010-06-15 서울대학교산학협력단 마이크로ｒｎａ를 유효성분으로 포함하는 항암제 및 그 제조방법

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHOI, HJ. ET AL.: "MicroRNA expression profile of gastrointestinal stromal tumors is distinguished by 14q loss and anatomic site.", INT J CANCER, vol. 126, no. 7, 1 April 2010 (2010-04-01), pages 1640 - 1650 *
WANG, X. ET AL.: "MicroRNA-494 Targeting Both Proapoptotic and Antiapoptotic Proteins Protects Against Ischemia/Reperfusion-Induced Cardiac Injury", CIRCULATION, 28 September 2010 (2010-09-28) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112210554A (zh) * 2019-07-11 2021-01-12 东北林业大学 源于鹿茸由CNBP介导的microRNA及其应用

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