WO2012091220A1 - Composition for the prevention or treatment of neoplastic diseases, comprising mirna as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of KIT(v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue)-mediated diseases, disorders or conditions, which comprises microRNA-494 (miR-494) or an agent (agents) that induces (induce) miR-494 overexpression. The present invention also relates to a screening method using the pharmaceutical composition. The miR-494 of the present invention binds directly to two different core sequence sites (seed match sites/sequences) in KIT 3'-UTR and so effects top-down control of KIT, and reduces the expression of downstream molecules (e.g. phospho-AKT and phospho-STAT3) in the KIT signalling transition pathway, and the miR-494 overexpression induction of the present invention suppresses the growth of neoplastic cells and preferably GIST cell lines. Consequently, when preventing or treating KIT-mediated diseases, disorders or conditions, it is advantageous to use the composition of the present invention which comprises miR-494 and a substance that promotes expression thereof or a substance that suppresses KIT expression of the present invention as an active ingredient.

Description

 【Specification】

 [Name of invention]

 composition for the prevention or treatment of tumor diseases comprising m i RNA as an active ingredient

[Technical field]

 The present invention relates to miR-494 and its use as negative regulators of KIT in GIST.

[Background technology]

 Molecular genetics of gastrointestinal stromal tumors (GISTs) is one of the best understood tumors of human tumors [1]. Two of the oncogenes of the receptor tyrosine kinase family, the v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and PDGFRA, are gain-of-gain mutations in about 70% and 15% of GIST, respectively. f unction mutations [2, 3]. Mutations in these genes lead to their continued activation, resulting in constant stimulation of downstream signaling pathways of KIT and PDGFRA [4, 5]. Among these, 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].

Progression of GIST is successfully blocked by 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]. However, GIST patients who become imatinib-resistant during imatinib treatment are resistant to the inhibitory effects of imatinib. Thus, it is possible to inhibit activated KIT by both posttranscriptional and posttranslational regulation. More diverse approaches are needed for GIST patients where KIT activation is important [9–11].

 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]. Is referenced and its citation is indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained. [Detailed Description of the Invention]

 The inventors have sought to develop new targets for the treatment of gastrointestinal stromal tumors (GISTs). As a result, 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.

It is an object of the present invention to provide a kit-v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT-mediated disease, disorder or condition). Provided is a pharmaceutical composition for preventing or treating diseases, disorders or conditions).

 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.

 It is another object of the present invention to provide a method for treating a KIT-mediated disease, disease or condition comprising the step of processing the nucleotide sequence.

 It is another object of the present invention to provide a method for screening an agent for treating neoplastic disorders. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings. According to one aspect of the present invention, 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. According to another aspect of the present invention, 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.

 According to another aspect of the present invention, 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.

According to another aspect of the invention, 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. According to another aspect of the invention, 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:

 According to another aspect of the invention, 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). As a result, we directly bind to two different seed match sites where miR-494 is present in KIT mRNA, down-regulate KIT expression, and downstream molecules of the KIT signaling transition pathway (eg, It has been found 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 kinase is one of the receptor tyrosine kinases.

Combines with stem cell factor (SCF). The binding results in dimerization of the kinase and subsequently activation of the kinase. As a result, many substrates of KIT kinase are phosphorylated (Blume-Jensen P. et al., EMB0 J. 10, 4121-4128, 1991; and Lev S. et al., EMB0 J., 10, 647-654, 1991). Abnormal activation of KIT kinase generates proliferative signals in certain types of cancer cells, which are thought to be the cause of cancer development or malignant transformation. Representative diseases caused by abnormal activation of KIT kinase include gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), acute myelogenous leukemia (AML), adipocyte leukemia ( Mast cell leukemia) and cancer. "Gastrointestinal stromal tumors (GIST)" of the present invention means an epileptic tumor that occurs in the gastrointestinal tract expressing KIT kinase, and the mutation of KIT kinase is activated at a frequency of about 5 OT, and GIST with very high malignancy Patients were found to have the mutation at a higher frequency, suggesting that the possibility of the mutation may be a diagnostic factor (Lasota J. et al., Am. J. Pathol., 157, 1091-1095, 2000; and Taniguchi M et al., Cancer Res., 59, 4297-4300, 1999). However, no correlation has been reported on the correlation between KIT expression and miRNA.

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. According to the present invention _: miR-494 of the present invention directly binds to 3′-UTR (untranslated region) of KIT mRNA and down-regulates expression of KIT. In addition, 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).

 As used herein, the term “micro RNA microRNA (miRNA)” is a 21-25 nt single-stranded RNA molecule that binds to the 3′-UTR of mRNA (messengerRNA) to control gene expression in eukaryotes [Bartel DP et. al. , Cell. 2004 Jan 23; 116 (2): 281-297]. The production of miRNAs is made by Drosha (RNasem type enzymes) into precursor miRNAs (pre-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).

In the composition of the present invention, 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). As used herein, the term “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.

 Preferably, 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. In general, 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. Thus, 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. 20-100 contiguous nucleotide sequences or SEQ ID NO: 3, more preferably comprising an antisense oligonucleotide sequence having a sequence complementary to the eighth to fifteenth nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2 and of the nucleotide sequence of SEQ ID from the 8th 15th nucleotide sequence complementary to 20 to 100 consecutive nucleoside ^another, id sequence antisense oligonucleotide having a sequence containing a nucleotide sequence in, most preferably in the sequence Listing, the 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.

The term “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. Thus, the term "antisense oligonucleotide" can be described herein as a "complementary nucleic acid-based inhibitor."

 As used herein, 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.

 According to a preferred embodiment of the invention, 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). 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.

As described above, 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

Deoxyribonucleotides. Preferably, the itisense oligonucleotide comprises at least one chemical modification. 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]. Alternatively, antisense oligonucleotides may include peptide nucleic acids (PNAs), which include peptide-based backbones instead of sugar-phosphate backbones. Other chemical modifications that 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). In another embodiment, 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.

According to a preferred embodiment of the invention, 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). In addition, of the present invention Inducing overexpression of miR-494 inhibited GIST cell growth (consistent with changes in cell cycle phase and S phase: see FIG. 6).

 Alternatively, another approach similar to miR-494 of the present invention is to administer an inhibitory RNA molecule, wherein the inhibitory RNA molecule comprises a sequence complementary to the target sequence of miR-494. Such 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).

 As used herein, the term "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).

 On the other hand, 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.

According to a preferred embodiment, 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.

 According to a preferred embodiment of the present invention, 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. In addition to the above components, 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. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

 The pharmaceutical 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 ᅳ 100 mg / kg (body weight) per day.

The pharmaceutical 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. Wherein 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. According to another aspect of the invention, 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, the method comprising: increasing the expression of miR-494 and treating it as a tumor drug do.

 According to the method of the present invention, first, 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. Most preferably, the cell comprising the nucleotide sequence of the present invention is a human gastrointestinal stromal tumor line. As used to refer to the screening methods of the present invention, the term "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.

 Then, 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. When the result of the measurement indicates that the expression of the nucleotide sequence of the marker of the present invention is increased or the expression of KIT, which is a target thereof, is suppressed, 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. aufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press), and localization using cDNA microarrays (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)) or in situ) oscillation reaction (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)) It can be carried out.

 In case of carrying out the RT-PCR protocol, first, total 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.

 According to a preferred embodiment of the invention, 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. Compared to conventional PCR methods, 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.

When the amount of PCR amplification products reaches a detectable amount by fluorescence, an amplification curve begins to occur, and the signal rises exponentially and reaches a steady state. The larger the initial DNA amount, the less the number of cycles the amplification product reaches to detectable amount, so the amplification curve appears faster. Therefore, real-time PCR reaction using a standard sample diluted in stages yields amplification curves arranged at equal intervals in the order of increasing initial DNA content. Setting a threshold at an appropriate point here yields the point C _r at which the threshold intersects the amplification curve.

 In real-time PCR, 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.

 First, 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). To quantify amplicon production. The reagent does not bind to ssDNA. 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). Therefore, since the fluorescence is emitted through the binding between SYBR Green I and the double-stranded DNA, the amount of amplified product can be measured. 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, on the other hand, 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. The excited fluorescent material transfers energy to something nearby rather than to fluoresce (FRET = Forster or fluorescence resonance energy transfer; Chen, X., et al., Proc Natl Acad Sci USA, 94 (20): 10756-61 (1997)). Therefore, no fluorescence is generated when the probe is normal. 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. During the extension reaction, 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. At this time, 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. . Both the reporter molecule and the molecule somewhere bound to the Taqman probe are fluorescent materials. 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 ™ (506), Y0-PR0 ™ -1 (509), YOYO ™ -1 (509), Calcein (517), FITC (518), FluorX ™ (519), Alexa ™ (520), Rhodamine 110 (520) , 5-FAM (522), Oregon Green ™ 500 (522), Oregon Green ™ 488 (524), RiboGreen ™ (525), Rhodamine Green ™ (527), Rhodamine 123 (529), Magnesium Green ™ (531), Calcium Green ™ (533), T0-PR0 ™ -1 (533), T0T01 (533), JOE (548), B0DIPY530 / 550 (550), Di 1 (565), B0DIPY TMR (568), B0DIPY558 / 568 ( 568), B0DIPY564 / 570 (570), Cy3 ™ (570), Alexa ™ 546 (570), TRITC (572), Magnesium Orange ™ (575), Phycoerythr in R & B (575), Rhodamine Phalloidin (575), Calcium 0range ™ (576), Pyronin Y (580), Rhodamine B (580), TAMRAC582), Rhodamine Red ™ (590), Cy3.5 ™ (596), ROX (608), Calcium Crimson ™ (615), Alexa ™ 594 (615 ), Texas Red (615), Nile Red (628), Y0-PR0 ™ -3 (631), Y0Y0 ™ -3 (631), R-phycocyanin (642), C- Phycocyanin (648), T0-PR0 ™ -3 660), T0T03 (660), DiD Di 1C (5) (665), Cy5 ™ (670), Thiadicarbocyanine (671) and Cy5.5 (694).

Suitable reporter-something pairs are disclosed in many literatures: Pesce et al., Editors, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2nd EDITION (Academic Press, New York, 1971); Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pingsheim, FLUORESCENCE AND PHOSPHORESCENCE (Inter science Publishers, New York, 1949); Haugland, RP, HANDBOOK B FLUORESCENT AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Oreg. , 1996; US Pat. Nos. 3,996,345 and 4,351,760. The target nucleic acid used in the present invention is not particularly limited,

DNACgDNA or cDNA) or 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. In the present invention, 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. For example, 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. Preferably, 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. By “template-dependent extension reaction” catalyzed by a template-dependent nucleic acid polymerase is meant a reaction that synthesizes a nucleotide sequence complementary to the sequence of the template.

 According to a preferred embodiment of the present invention, the real-time PCR of the present invention is carried out by Taqman probe method.

 In addition, the change in the amount of KIT protein can be carried out through various immunoassay methods known in the art. For example, 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-Ά Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out according to a radioimmunoassay, an antibody labeled with a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) detects a marker molecule of the present invention. It can be used to.

 When the method of the present invention is carried out by ELISA, the present invention comprises the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (Π) 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, β_galacto Oxidase, horse radish peroxidase and cytochrome p ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (ΝΒΤ), 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 (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazol ine sulfonate]), phenylenediamine (0PD) and naph / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m— PMS (phenzai substrates such as ne methosulfate may be used.

 When the method of the invention is carried out in a capture—ELISA mode, 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 포스 β-galactosidase, horse radish peroxidase and cytochrome Ρ ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , Ρ ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemi luminescent, and fluorescence resonance energy transfer (FRET). Method: Ed Harlow and David Lane, Using Ant i bodies ： A The Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

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

 (a) The present invention relates to a novel function of miR-494 as a negative regulator of KIT in GIST.

 (b) 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).

 (c) In addition, the induction of miR-494 overexpression of the present invention inhibited the growth of tumor cells, preferably GIST cell lines.

 (d) Therefore, the 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.

[Brief Description of Drawings]

 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.

 2 shows the results of investigating KIT expression inhibited by miR-494.

25 nM of nontargeting miRNA, miR-221, miR-494 or miR-494 in GIST882 cells Inhibitors were transfected. After 3 days, cells were obtained and analyzed by Western blot (FIG. 2A). KIT expression in cells transfected with miR-494 was reduced by about 57% compared to expression in cells transfected with non-targeted miRNAs. As a result of qRT-PCR analysis, KIT mRNA levels were decreased in cells transfected with miR-494 and miR-221 (FIG. 2B). N. Indicates 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. The reverse correlation between KIT protein and miR-494 expression was clear (r; -0.490, P = .005).

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). When the Mb construct comprising the nucleotide sequence and miR-494 additionally mutated at positions 1760-1763 was transfected, the luciferase activity was completely restored relative to the sample to which the M construct was transfected ( 4b, bottom panel).

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. miRNA ^* -transfected miRNA; Vector-Transfected vector used in the reporter assay.

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 represents negative control.

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. The difference in cell numbers between GIST882 cells treated with non-targeting miRNA (negative control) and GIST882 cells treated with miR-494 was small at 3 days, larger at 6 days, and nearly tripled at 12 days. (Top panel) 웨스턴 Western blotting of all samples in the proliferation assay showed that GIST882 cells treated with miR-494 showed reduced levels of KIT (bottom panel). 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.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident. Example Experimental Materials and Methods

Cell line and cell culture

 GIST882 cell lines with activated KIT mutations (Axon 13, K642E7) 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

 The 31 GIST patients included in this study were identified from 1997 to July 2006 in the Department of Pathology, Yonsei University College of Medicine. Approval for use of these organizations for research purposes was obtained from the Ethics Review Board of Yonsei University College of Medicine. Some fresh samples were obtained from the Liver Cancer Specimen Bank of the National Research Resource Bank Program of the Korea Science and Engineering Foundation of the Ministry of Science and Technology. Seventeen of the samples from the GIST patients were previously used for miRNA profiles and proteome analysis [12 ′ 13, 31, 32]. Conventional pathological parameters such as anatomical location, risk and tumor size were examined without confirmation of molecular data (Table 1).

Table 1

Clinicopathological and genetic status of 31 GISTs.

del

 1) +, tumor cells expressing KIT; Tumor cells that do not express KIT.

GIST patients were divided into four groups based on tumor risk according to Fletcher et al. [33].

RNA Manufacturing and Taqman miRNA Assays

 Total RNA was extracted from frozen tissue using TRIZ0L (Life Technology, Rockville, MD). Taqman miRNA assay (Applied

Expression levels of miRNA were analyzed and analyzed using Applied Biosystems 7300 real-time PCR system (Applied Biosystems). All assays were performed in triplicates. Quantitative RT-PCR (qRT-PCR)

KITCPrimerBank ID; 4557695a2) and the qRT-PCR primer sequences for GAPDH (PrimerBank ID; 2282013a2) 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

 All mature forms of the miRNA mimetics (nontargeting miRNA, miR-221, miR-222 miR-494 and miR-494 inhibitors) used in this study are miRIDIAN miRNA mimetics (Thermo Scientific, Waltham, MA, USA). All transformation experiments were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Three days after transformation, all cells were obtained and used for subsequent Western blot analysis. Western blot

 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

To create another construct, first 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. On day 4 of transfection, cells of each sample were stained with a solution consisting of PBS propidium iodide (Abeam, Cambridge, MA, USA) and RNase A. All samples were analyzed by FACS Calibur (BD Biosciences, San Jose, CA, USA). Statistical analysis

 Correlation between miR-494 expression level and KIT expression level was determined by analyzing the Spearman correlat ion by examining the miR-494 qRT-PCR value and the band intensity of the KIT Western blot. P values (<0.05) were judged to be significant. Statistical analysis was performed using SPSS software version 10.0 (SPSS, Inc., Chicago, IL, USA). Experiment result

Identification of miR-494 as voice regulator of KIT

Our previous microarray data were used to evaluate the effect on KIT expression of five candidate miRNAs selected [13] because of the statistically significant correlation with KIT expression in GIST [13]. 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. 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. If cells have transfected miR-494 or miR— 221 (miR- 494 shows a better effect than miR-221), Western blotting analysis confirmed a significant down-regulation of KIT expression. The effect of miR-221 or miR-222 on KIT down-regulation did not show any significant difference (results not shown). Cell lines transfected with the miR-494 inhibitor showed the same KIT expression as the negative control sample, probably because miR-494 had very low expression levels in the GIST882 cell line (FIG. 2A). The same pattern was confirmed in qRT-PCR analysis as in Western blot analysis, except that it showed a somewhat increased level of KIT mRNA in cells transfected with miR-49.4 billion agents (FIG. 2B). The above results indicate that KIT expression is regulated by miR-494.

Inverse correlation between miR-494 and KIT expression in GIST

 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. 3A-3C). The analysis showed an inverse correlation between the expression of KIT and miR-494 (r = -0.490 and P = .005). As expected, cases with KIT mutations showed higher KIT expression than those without KIT mutations. The above findings indicate that although KIT overexpression is closely associated with KIT mutation status, KIT expression is inversely associated with miR-494 expression in GIST.

Identification of two key match sites in KIT's 3'-UTR We have identified that 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). We prepared a reporter assay by preparing 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. In a pilot experiment to confirm that our N vector constructs function properly, 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. Samples treated with N vector and miR-494 were only about half the luciferase activity of samples transfected with N vector and non-targeted miRNA, more prominent than samples treated with miR—221 and miR-222. A decrease was shown (FIG. 4B, top panel). M vector for 1899-1902 nucleotide sequence changed (GTTTCCGG) miR-494 and nucleotide of 1961-1964 via site-directed mutagenesis of 3'-UTR of N vector KIT mRNA A zero vector was constructed for sequence changed (GTAGCAGA) miR-221 / miR-222. 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.

 We manually examined the more potent miR-494 binding sites using Vector NTI software (Invitrogen, Carlsbad, Calif., USA) (FIG. 4A). As a result, the 3'-UTR of KIT mRNA had three potential miR-494 binding sites, including 6-7 seed match sequences for miR-494. The potential miR-494 binding sites detected were as follows: 5'-TGTTTCT-3 '(position, 1222-1228), 5' -GTGTTTCT-3 '(position 1758-1765) and 5' -ATGTTT-3 '( Location, 1918-1923). Through the above findings, we constructed three vectors (Ma, Mb and Mc vectors) derived from the M vector by an additional mutation of the potential miR-494 binding sites (GTTTCCGG). The reporter assay was performed by transfecting the HeLa cell lines with M, Ma, Mb and Mc vectors containing miR-494, respectively. Only cells transfected with Mb and miR-494 showed fully restored luciferase activity compared to the negative control (FIG. 4B, bottom panel). Since the G base of the next 5'-GTGTTTCT-3 '(position, 1753-1760) can hydrogen bond with the U base by a wobble match, the position is unique as suggested by the target scan 3.0 It functions almost the same as the binding position. Taken together, we can conclude that two different positions are important for binding miR-494 to the 3'-UTR site of KIT mRNA. Recovery of KIT Expression After Inhibition of miR-494

We also confirmed the ability of endogenous miR-494 to bind to the 3′-UTR site of KIT mRNA through a reporter assay using N vector and miR-494 inhibitors. Prior to the inhibition assay, we measured the expression level of miR-494 by Taqman miRNA assay in six cell lines and then selected three cell lines: HeLa, GIST882 and SNU216. HeLa and SNU216 cell lines had relatively high expression levels of miR-494, while GIST882 cell lines had little detectable miR-494 expression (FIG. 4C, top panel). Each of the three cell lines described above is either a N vector containing non-targeting miRNA or an N vector containing miR-494 inhibitors as a negative control. Transfection. The cells were later harvested and subjected to a DLR assay. Luciferase activity of SNU216 and HeLa cells transfected with miR-494 inhibitors increased more than twice the activity of negative control, but the activity of GIST882 cells remained almost unchanged (FIG. 4C, bottom panel). The above results indicate that KIT expression is directly regulated by endogenous miR-494 even in cell lines that do not express KIT such as HeLa and SNU216. Disturbed Signaling After miR-494 Treatment

IT downstream molecular pathways are known to be involved in cell proliferation, differentiation and apoptosis. As suggested by many studies [6], regulation of KIT expression affects critical pathways including the AKT, ERK and JACK-STAT pathways. The effect of miR-494 on these pathways was verified by analyzing the status of p-AKT, p-ERK, p-KIT and p-STAT3 after miR-494 transfection. GIST882 cell lines were transfected with 50 nM of untargeted miR A, miR-494, miR-221 or miR-494 inhibitors. KIT expression was significantly reduced in cells transfected with miR-494. In addition, KIT expression was reduced in cells transfected with _m iR-221, but less than miR-494. However, 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. In addition, 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. Treatment of the miR-494 inhibitor did not result in measurable changes. The pattern of change in P-STAT3 was similar to phospho-AKT in that the level of p-STAT3 decreased after miR-221 and miR-494 treatment and the decrease was greater after miR-494 treatment. No change in p-ERK levels was observed. The above findings are consistent with previous reports [4, 8, 9, 18] that expression of p-AKT and p-STAT3 is affected when KIT is inactivated by imatinib or KIT shRNA that inhibits KIT activation. In summary, The above results indicate that miR-494 treatment induces KIT down-regulation and affects downstream molecules in the same manner as KIT-inhibitory molecules such as imatinib or KIT shRNA. Inhibition of GIST Cell Proliferation by Induced MiR-494 Overexpression

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. Cells transfected with miR-494 significantly reduced the number of cells and cell clusters 6 days after initial transfection, and this change was aggravated on day 12 of assay. In the case of GIST882 cells, the normal cell morphology was pin-point and spindle-shaped, but miR-494 treated GIST882 cells showed a changed cell morphology on day 12 (FIG. 6A), and the number of cells was also higher than that of control cells. It decreased to nearly 37¾ (FIG. 6B, top panel). Sustained down-regulation of KIT was confirmed by western blotting of the same sample used in the proliferation assay (FIG. 6B, bottom panel).

Through flow cytometry, changes in cell cycle after miR-494 transfection were investigated. GIST882 cells were transfected with 50 nM of nontargeting miRNA or miR-494, respectively. Samples were obtained on day 4 of transfection and stained with propidium iodide for flow cytometry. the cell line treated with miR-494 to G ₀ are compared to the transfected cells in a non-targeting miRNA-increased cell 6> group Gi, S phase cells was reduced 5). The above findings indicate that miR-494 inhibits GIST882 cell proliferation by regulating the cell cycle. Additional discussion MicroRNAs can directly and indirectly influence signal transduct ion by regulating target genes and components of target gene regulatory pathways in a post-transcription manner. These findings indicate that 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. 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. The findings indicate that when GIST882 cells with activated KIT are treated with imatinib or KIT shRNA, p— AKT and p-STAT3 expression decreases, thereby blocking cell proliferation and ultimately cell growth following increased apoptosis. Consistent with previous reports showing suppression [4, 8, 9, 18]. Imatinib inhibits KIT through the ATP binding pocket, which affects KIT phosphorylation, activating downstream molecules [24-26]. Our study shows that miR-494 treatment disrupts signaling pathways and inhibits cell proliferation. It is shown that it affects the GIST882 cell line in a similar manner to imatinib. The above findings mean that miR-494 may be another option for treating GIST. Our identification of two different key match sites of miR-494 at 3'-UTR of KIT further supports the concept of KIT mRNA as a direct target of miR-494, and KIT activation Deriving more specific treatment possibilities for GIST patients with

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. However, inhibition of KIT by imatinib is incomplete because resistance through potential mutational changes of amino acid residues that may occur during processing may occur. As a result, 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. 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. However, many of the problems described above are solved by novel delivery systems such as ¾ timer, nanoparticles and liposomes [28-30]. By applying the novel delivery system, miR-494 could be used as a novel therapeutic means for treating GIST. In conclusion, 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. The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that these specific technologies are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. references

 Fletcher CD, Berman J J, Cor less C, et al. Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol 2002; 33: 459-65.

2. Miett inen M, Lasota J. Gastrointestinal stromal tumors ᅳ definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Vir chows Arch 2001: 438 1-12.

 3. Miett inen M, Lasota J. Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med 2006; 130: 1466-78.

 4. Duensing A, Medeiros F, McConarty B, et al. Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs). Oncogene 2004; 23: 3999-4006.

 5. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299: 708—10.

6. Lennartsson J, Ronnstrand L. The stem cell factor receptor / c-Kit as a drug target in cancer. Curr Cancer Drug Targets 2006; 6: 65 ᅳ 75.

7. Tuveson DA, Willis NA, Jacks T, et al. STI571 inactivation of the gastrointestinal stromal tumor c_KIT oncoprotein: biological and clinical implications. Oncogene 2001; 20: 5054—8. 8. Gordon PM, Fisher DE. Role for the proapoptotic factor BIM in mediating imat inib-induced apoptosis in a c-KIT-dependent gastrointestinal stromal tumor cell line. J Biol Chem 2010; 285: 14109-14.

9. Bauer S, Duensing A, Demet r i GD, et al. KIT oncogenic signaling mechanisms in imat inib— resistant gastrointestinal stromal tumor: PI3-kinase / AKT is a crucial survival pathway. Oncogene 2007; 26: 7560-8.

 10. Ma Y, Zeng S, Metcalfe DD, et al. The c-KIT mutat ion causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wi ld-type kinases and those with regulatory- type mutations. Blood 2002; 99: 1741-4.

 11. McLean SR, Gana-Weisz M, Hartzoulakis B, et al. Imatinib binding and cKIT inhibition is abrogated by the cKIT kinase domain I missense mutation Val654Ala. Mol Cancer Ther 2005; 4: 2008-15.

 12. 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.

13. Choi HJ, Lee H, Kim H, et al. MicroRNA expression profile of gastrointestinal stromal tumors is distinguished by 14q loss and anatomic site. Int J Cancer 2009; 126: 1640-50.

 14. Quek R, George S. Gastrointestinal stromal tumor: a clinical overview. Hematol Oncol Clin North ½ 2009; 23: 69-78, viii.

15. Schickel R, Boyerinas B, Park SM, et al. MicroRNAs: key players in the immune system, differentiation, tumori genes is and cell death. Oncogene 2008; 27: 5959-74.

16. Cal in GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: 857-66. 17. Felli N, Font ana L, Pelosi E, et al. MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. Proc Natl Acad Sci USA 2005; 102: 18081-6.

 18. Ou WB, Zhu MJ, Demet r i GD, et al. Protein kinase C-theta regulates KIT expression and proliferation in gastrointestinal stromal tumors.

Oncogene 2008; 27: 5624-34.

 19. Liu L, Jiang Y, Zhang H, et al. Overexpressed miR-494 down-regulates PTEN gene expression in eel Is transformed by anti ᅳ benzo (a) yr ene-1 r ans-7, 8-d i hydr od i o 1 -9, 10-epox i de. Life Sci 2009; 86: 192-8.

 20. Chang SS, Jiang WW, Smith I, et al. MicroRNA alterations in head and neck squamous cell carcinoma. Int J Cancer 2008; 123: 2791—7.

 21. Robert us JL, Kluiver J, Weggemans C, et al. MiRNA profiling in B non-Hodgkin lymphoma: a MYC 一 related miRNA profile characterizes Burkitt lymphoma. Br J Haematol; 149: 896-9.

 22. Lux ML, Rubin BP, Biase TL, et al. KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors. Am J Pathol 2000; 156: 791-5.

 23. Nakahara M, Isozaki K, Hirota S, et al. A novel gain—of ᅳ funct ion mutat ion of c ᅳ kit gene in gastrointestinal stromal tumors.

Gastroenterology 1998; 115: 1090-5.

 24. Heinrich MC, Griffith DJ, Druker BJ, et al. Inhibition of c-kit receptor tyrosine kinase activity by ST I 571 ， a selective tyrosine kinase inhibitor. Blood 2000; 96: 925-32.

25. Joensuu H, Roberts PJ, Sar lomo-Rikala M, et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001; 344: 1052-6.

26. Demet ri GD. Structural reengineer ing of imat inib to decrease cardiac risk in cancer therapy. J CI in Invest 2007; 117: 365 3. O Ji

pgpeot.t rr ⁱ n ^I n J C ^li n Exato _; . Ph ^lc no367241 _:

pTO asw2sresn? A ⁱ w ^l ha ⁱ ztre½ ₁ 2 ⁱ nh r _f a stadac _i ednr _i z -.-.-.

Claims

[Range of request]

 [Claim 1]

 V_kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) containing micro RNA-494 (miR-494) or miR-494 overexpression as an active ingredient—mediated disease, disease or condition ( A pharmaceutical composition for preventing or treating KIT-mediated diseases, disorders or conditions.

[Claim 2]

 The composition of claim 1, wherein the agent inducing miR-494 or miR-494 overexpression inhibits KIT expression at the mRNA or protein level.

[Claim 3]

 The composition of claim 1, wherein miR-494 inhibits phosphorylation of AKT or STAT3.

[Claim 4]

 The composition of claim 1, wherein the KIT-mediated disease, disease or condition is a disease, disease or condition induced by a form in which the KIT gene or protein is overexpressed or mutated.

[Claim 5]

The method of claim 1, wherein the KIT-mediated disease, disease or condition is neoplastic disorders, inflammatory disease, autoimmune disease, cancer, allergic disease, irritable bowel syndrome (IBS), graft versus host disease (GVHD). ), Metabolic syndrome, central nervous system (CNS) -related diseases, neurodegenerative diseases, mast cell-related diseases, pain, substance-abuse disease, fryer disease, heart disease, fibrotic disease, A composition characterized in that it is idiopathic pulmonary hypertension (ΙΡΑΗ), primary pulmonary hypertension (ΡΡΗ), glioma or cardiovascular disease. [Claim 6]

 6. The composition of claim 5, wherein said KIT-mediated disease, disease or condition is a disease.

[Claim 7]

 The method of claim 6, wherein the tumorous disease is gastrointestinal stromal tumors (GISTs), small cell lung cancer, non-small cell lung cancer, acute myelocytic leukemia, acute lymphocytic leukemia , Myelodyplastic syndrome, chronic myeloid leukemia, colon carcinoma, gastric carcinoma, testicular cancer, glioblastoma, astrocytoma or mastocytosis.

[Claim 8]

 20-100 contiguous nucleotide sequences for the prophylaxis or treatment of a KIT-mediated disease, disease or condition comprising an antisense oligonucleotide sequence having a sequence complementary to the eighth to fifteenth nucleotide sequence of the nucleotide sequence of the second sequence.

[Claim 9]

 20-100 contiguous nucleotide sequences for the prophylaxis or treatment of a KIT-mediated disease, disease or condition comprising an antisense oligonucleotide sequence having a sequence complementary to the eighth to fifteenth nucleotide sequence of the nucleotide sequence of the third sequence.

[Claim 10]

Claim 8 or claim 9 wherein the anti hitting the sensor nucleotide sequence is SEQ ID NO: characterized in that it comprises a sequence complementary to the key combination of the miR-494 sequence _(see d mat sequences). [Claim 111]

 The sequence of claim 10, wherein the key binding sequence of miR-494 is in a 3′-untranslated region (UTR) of KIT.

[Claim 12]

 10. The sequence of claim 8 or 9, wherein the antisense oligonucleotide is a DNA or RNA molecule. [Claim 13]

 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. [Claim 14]

 The method of claim 13, wherein said inducing miR-494 or miR-494 overexpression; The agent is characterized in that the expression of KIT is inhibited at the mRNA or protein level.

 The method of claim 13, wherein the miR-494 inhibits phosphorylation of AKT or STAT3.

[Claim 16]

 The method of claim 13, wherein the KIT-mediated disease, disease or condition is a disease, disease or condition induced by a form in which the KIT gene or protein is overexpressed or mutated.

[Claim 17]

The method according to claim 13, wherein the KIT-mediated disease, disease or condition is neoplastic disease, inflammatory disease, autoimmune disease, cancer, allergic disease, irritable bowel syndrome, Graft versus host disease, metabolic syndrome, CNS-related disease, neurodegenerative disease, mast cell-related disease, pain, substance-abuse disease, prion disease, heart disease, fibrotic disease, idiopathic pulmonary hypertension, primary pulmonary hypertension , Glioma or cardiovascular disease.

[Claim 18]

 18. The method of claim 17, wherein the KIT-mediated disease, disease or condition is a neoplastic disease. [Claim 19]

 19. The method of claim 18, wherein the neoplastic disease is gastrointestinal stromal tumor, small cell lung cancer, non-small cell lung cancer, acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, colorectal carcinoma, gastric carcinoma, testicular cancer, nerve A glioma, an astrocytoma or mast cell tumor.

[Claim 20]

 KIT-mediated disease, disease comprising processing 20-100 contiguous nucleotide sequences comprising an antisense oligonucleotide sequence having a complementary sequence to the eighth to fifteenth nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2 Or method of treating the condition.

[Claim 21]

KIT—mediated disease, disease comprising processing 20-100 contiguous nucleotide sequences comprising an antisense oligonucleotide sequence having a complementary sequence to the eighth to fifteenth nucleotide sequence of the nucleotide sequence of SEQ ID NO: 3 Or method of treating the condition. [Claim 22] 22. The method of claim 20 or 21, wherein said antisense oligonucleotide sequence comprises a sequence complementary to the core binding sequence of miR-494. [Claim 23]

 The method of claim 22, wherein the key binding sequence of miR-494 is in the 3′-UTR of KIT.

[Claim 24]

 22. The method of claim 20 or 21, wherein said antisense oligonucleotide is a DNA or RNA molecule.

[Claim 25]

 (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. do. [Claim 26]

 The method of claim 25, wherein the miR-494 inhibits expression of KIT or inhibits phosphorylation of AKT or STAT3.

[Claim 27]

 26. The method of claim 25, wherein the tumorous disease is gastrointestinal stromal tumor, small cell lung cancer, non-small cell lung cancer, acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, colorectal carcinoma, gastric carcinoma, testicular cancer, nerve Glioma, astrocytoma or mast cell tumor.