KR20120090110A - Compositions for preventing or treating neoplastic disorders comprising mirna as an active ingredient - Google Patents

Abstract

PURPOSE: A composition containing an agent which induces miR-494 or miR-494 overexpression is reduce the suppress growth of tumor cells. CONSTITUTION: A pharmaceutical composition for preventing or treating KIT(vkit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)-mediated diseases or KIT-mediated diseases contains an agent which induces miRNA-494 or miR-494 overexpression. 20-100 serial nucleotides for preventing or treating KIT-mediated diseases or disorder contain an antisense oligonucleotide having a sequence which is complementary to 8th to 15th nucleotide sequences of sequence number 2.

Description

Compositions for preventing or treating neoplastic diseases comprising miRNA as an active ingredient {Compositions for Preventing or Treating Neoplastic Disorders Comprising miRNA as an Active Ingredient}

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

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. function 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 them, in particular, activation of KIT is important for the development and progression of GIST [3]. Downstream molecular pathways involved in GIST tumorigenesis following KIT mutations include PI3-kinase, Src family kinase, Ras-E and JAK-STAT [6]. The activation of the aforementioned molecular pathways after KIT activation results in GIST tumorigenic processes 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 effect of imatinib. Thus, more diverse approaches are needed for GIST patients in which KIT activation is important [9-11], which can inhibit KIT activated by both posttranscriptional and posttranslational regulation [9-11].

Overexpression of KIT has been reported as a typical feature of GIST, and the presence of KIT mutations always leads to strong 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]. Since microRNAs (miRNAs) play an important role in regulating gene expression in cancer, they are a mechanism that can regulate their aberrations [15, 16]. Currently, miRNAs known to target KIT are miR-221 and miR-222 [17]. Previously, we compared five KIT expression and miRNA expression profiles in GIST patients to identify five candidate miRNAs that exhibited expression inversely relative to KIT expression [13].

Numerous papers and patent documents are referenced and cited throughout this specification. 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.

The inventors have sought to develop new targets for the treatment of gastrointestinal stromal tumors (GISTs). As a result, we 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) inhibits 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 pharmaceutical composition for the prevention or treatment of KIT (mediated diseases, disorders or conditions) -mediated v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog.

It is another object of the present invention to provide a nucleotide sequence for preventing or treating the above diseases, diseases or conditions.

Another object of the present invention is to provide a method for screening a therapeutic agent for neoplastic disorders.

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

According to an aspect of the present invention, the present invention provides a microorganism-494 (miR-494) or miR-494 over-expression (agents) including KIT (v-kit Hardy-Zuckerman 4 feline sarcoma) as an active ingredient It provides a pharmaceutical composition for the prevention or treatment of viral oncogene homolog-mediated diseases, diseases or conditions (tyrosine kinase-mediated diseases, disorders or conditions).

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 Provide the nucleotide sequence.

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: 3 Provide the nucleotide sequence.

The inventors have sought to develop new targets for the treatment of gastrointestinal stromal tumors (GISTs). As a result, we 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, It was found that the expression of phospho-AKT and phospho-STAT3) was 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 and binds to a specific ligand, stem cell factor (SCF). The binding results in dimerization of the kinase and then activation of the kinase. As a result, many substrates of KIT kinase are phosphorylated (Blume-Jensen P. et al., EMBO J. 10, 4121-4128, 1991; and Lev S. et al., EMBO 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 the 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 refers to 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 50%, and has a very high malignancy. In GIST patients the mutation was found 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 “microRNA (miRNA)” refers to 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 (RNase III type enzyme) into precursor miRNAs (pre-miRNAs) of stem-loop structure, migrated 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 miRNAs prepared as described above are involved in development, cell proliferation and death, fat metabolism, tumor formation, etc. by regulating 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 at 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, siRNAs (small interference RNA) and natural product 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. The 2nd to 8th nucleotide sequence in SEQ ID NO: 1 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. Therefore, the antisense oligonucleotide of the present invention is a sequence capable of binding to a sequence present in the target (preferably KIT) of miR-494, and preferably to bind to SEQ ID NO: 2 and SEQ ID NO: 3 20-100 contiguous nucleotide sequences or SEQ ID NO: 3, more preferably, 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 20-100 contiguous nucleotide sequences comprising an antisense oligonucleotide sequence having a sequence complementary to the 8th to 15th nucleotide sequence of the nucleotide sequence of the sequence, and most preferably the sequence complementary to the nucleotide sequence of SEQ ID NO: 2 Antisense oligonucleotides 20-100 contiguous nucleotide sequences comprising a nucleotide sequence or 20-100 contiguous nucleotide sequences comprising an antisense oligonucleotide sequence having a sequence complementary to the nucleotide sequence of SEQ ID NO: 3.

As used herein, the term “antisense oligonucleotide” includes nucleic acid-based molecules that have complementary sequences to miRNAs, in particular the key sequences of miRNAs, which can hybridize to the target sequences of miRNAs. Thus, the term "antisense oligonucleotide" can be described herein as a "complementary nucleic acid-based inhibitor."

The term “complementary” as used herein referring to antisense oligonucleotides means that the antisense oligonucleotides are sufficiently complementary to selectively hybridize to miR-494 targets under certain hybridization or annealing conditions, preferably physiological conditions. To have one or more mismatch sequences, meaning substantially encompassing both substantially complementary and perfectly complementary, preferably completely complementary it means.

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 LNA. (locked nucleic acid). The 2'-0-modified oligonucleotide is preferably 2'-0-alkyl oligonucleotide, more preferably 2'-OC _1-3 alkyl oligonucleotide, still more preferably 2'-OC _1-3 Methyl oligonucleotides.

As mentioned above, the 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 or deoxyribonucleotides. Preferably, the antisense oligonucleotides comprise at least one chemical modification. Antisense oligonucleotides may comprise one or more locked nucleic acids (LNAs). LNAs are modified ribonucleotides that have a locked form, including additional bridges between the 2 'and 4' carbons of the ribose sugar moiety, such 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 comprise 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 a 2'-0-methoxyethyl "gapmer" which comprises a 2'-0-methoxyethyl-modified ribonucleotide at the 5'-end and a 3'-end and is centrally located. At least 10 deoxyribonucleotides. This "gapmer" can trigger the RNase I-dependent disruption mechanism of the RNA target. The antisense oligonucleotides are 7-50 nucleotides in length, preferably 10-40 nucleotides, more preferably 15-30 nucleotides, and most preferably 20-25 nucleotides.

According to a preferred embodiment of the invention, agents that induce 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, inducing overexpression of miR-494 of the present invention inhibited GIST cell growth (consistent with changes in G ₁ and S phases of the cell cycle: see, Figure 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, 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 (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914) siRNAs are provided as an efficient gene knockdown method or as a gene therapy method because they can inhibit the expression of target genes siRNA was first discovered in plants, worms, fruit flies and parasites. siRNA was developed / used and applied to mammalian cell research (Degot S, et al. 2002; Degot S, et al. 2004; Ballut L, et al. 2005).

On the other hand, a disease that can be treated by the pharmaceutical composition of the present invention is a KIT-mediated disease, disease or condition, and includes a disease, disease or condition induced by an 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, neurodegenerative diseases, mast-cell associated disease, pain, substance-abuse disease, prion disease, heart disease, fibrotic disease, idiopathic pulmonary hypertension (IPAH), primary lung Hypertension (PPH), glioma or cardiovascular disease, even more preferably neoplastic disease.

According to a preferred embodiment of the present invention, the tumorous disease of the present invention 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, colorectal 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 compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. 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 present invention may be oral or parenteral (eg, intravenous, subcutaneous or topical).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . 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.

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. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the invention, the invention comprises the steps of (a) treating a test substance to a cell comprising a microRNA-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 determining that the test substance is an agent for treating tumorous disease. 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 which 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 method 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, small interference RNAs (siRNAs), 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, and as a result of the measurement, when the expression of the nucleotide sequence of the marker of the present invention is increased or the expression of its target KIT 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 (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 102-108, CRC press), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning.A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al. , Molecular Cloning.A Laboratory Manual , 3rd ed.Cold Spring Harbor Press (2001)).

In case of carrying out the RT-PCR protocol, first, total RNA is isolated from cells treated with the test substance, and then the 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, PCR amplification products are electrophoresed and the formed bands are analyzed to measure changes 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 real-time increases in PCR amplification products (Levak KJ, et al. , PCR Methods Appl. , 4 (6): 357-62 (1995)). The PCR reaction can be monitored by recording the amount of fluorescence emission in each cycle during the exponential phase, where 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 _T (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 at 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) detection of amplified DNA without separation via electrophoresis is possible; (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 fewer cycles the amount of amplification products can detect, resulting in faster amplification curves. Therefore, real-time PCR reaction using a standard sample diluted in steps 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 _T at the intersection of the threshold and the amplification curve.

In real-time PCR, PCR amplification products are detected via 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 is a method using a double stranded DNA binding die, which includes non-specific amplification and primer-dimer complexes using a non-sequence specific fluorescent 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 and 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 amplification products 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 green is used for singleplex reactions, but it 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).

Value C _T (threshold cycle) refers to the fluorescence is the number of cycles exceeds the threshold value (threshold) occurs in the reaction, which is inversely proportional to the logarithm of initial number of copies. Therefore, the C _T value assigned to a particular well reflects the number of cycles in which a sufficient number of amplicons have accumulated in the reaction. The C _T value is the cycle in which the increase in ΔRn was first detected. Rn refers to the magnitude of the fluorescence signal generated during PCR at each time point, and ΔRn refers to the fluorescence emission intensity (standardized reporter signal) of the reporter die divided by the fluorescence emission intensity of the reference die. The C _T value is also called Cp (crossing point) in LightCycler. The C _T 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 the reaction. The slope of the log-linear phase represents the amplification efficiency (Eff) ( http://www.appliedbiosystems.co.kr/ ).

TaqMan probes, on the other hand, typically contain primers (e.g., 20-30 nucleotides) that include a fluorophore at the 5'-end and a quencher at the 3'-end (e.g., TAMRA or non-fluorescent quencher (NFQ)). Longer oligonucleotides. The excited fluorescent material transfers energy to a nearby quencher rather than to fluoresce (FRET = For fluorescence resonance energy transfer; Chen, X., et al. , Proc Natl Acad Sci USA , 94 (20): 10756-61 ( 1997)). Therefore, if the probe is normal, no fluorescence is generated. 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 SEQ ID NO.

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 the 5'to 3'nuclease activity degrades the Taqman probe hybridized to the template and releases the fluorescent dye from the probe, thereby releasing the inhibition by the quencher, thereby showing fluorescence. 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 quencher molecule bound to the Taqman probe are fluorescent materials. Fluorescent reporter molecules and quencher 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), YO-PRO ™ -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), TO-PRO ™ -1 (533), TOTO1 (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY558 / 568 (568 ), BODIPY564 / 570 (570), Cy3 ™ (570), Alexa ™ 546 (570), TRITC (572), Magnesium Orange ™ (575), Phycoerythrin R & B (575), Rhodamine Phalloidin (575), Calcium Orange ™ ( 576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ™ (590), Cy3.5 ™ (596), ROX (608), Calcium Crimson ™ (615), Alexa ™ 594 (615), Texas Red (615), Nile Red (628), YO-PRO ™ -3 (631), YOYO ™ -3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-PRO ™ -3 660), TOTO3 (660) , DiD DilC (5) (665), Cy5 ™ (670), Thiadicarbocyanine (671) and Cy5.5 (694).

Suitable reporter-quencher 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, 1970); 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); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, RP, HANDBOOK OF FLUORESCENT PROBES 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 and includes all DNA (gDNA 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 may be carried out by hybridization methods known in the art. In the present invention, suitable hybridization 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, hybridization and reaction time, 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 hybridization can be found in Joseph Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; NY (1999).

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 obtainable from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , Pyrococcus furiosus ( Pfu), Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species spr. , DNA polymerase of Thermotoga neapolitana and Thermosipho africanus .

“Template-dependent extension reaction” catalyzed by a template-dependent nucleic acid polymerase refers to 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 (ELISA), capture-ELISA, inhibition or competition assay, and Sandwich analysis includes, but is not limited to.

The immunoassay or method of immunostaining is described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotope is an antibody labeled (e.g., C ^14, I ^125, P ³² and S ³⁵⁾ of detecting the marker molecules of the present invention .

When the method of the present invention is carried out by 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; (Ii) reacting said cell lysate with an antibody to a target as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) 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, fluorescence, luminescence 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 (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as (phenzaine methosulfate) may be used. All.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise (i) 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 result of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with granulation factor proteins; And (iv) measuring a signal originating from said label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-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 luciferase 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) Accordingly, the composition 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 is effective for preventing or treating KIT-mediated diseases, diseases or conditions. It can be usefully applied.

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. NC represents negative control.
2 shows the results of investigating KIT expression inhibited by miR-494. GIST882 cells were transfected with 25 nM of nontargeting miRNA, miR-221, miR-494 or miR-494 inhibitors. 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 decreased in cells transfected with miR-494 and miR-221 (FIG. 2B). NC represents negative control.
3 shows KIT protein (3a), miR-494 levels (3b) and graphs (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 (GTTTCCGG) 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. O vectors were used to confirm that all constructs function properly using miR-221 and miR-222 (GTAGCAGA), previously reported to target the 3'-UTR of KIT. 4B transfected a He construct with M constructs containing miR-494 or O constructs containing miR-221. Two days after transfection, cells were harvested and subjected to dual luciferase assays. 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 additionally mutated at positions 1760-1763 and miR-494 was transfected, the luciferase activity was fully restored relative to the sample to which the M construct was transfected ( 4b, bottom panel).
FIG. 4C is the result of a Taqman-miRNA assay 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 containing a 50 nM miR-494 inhibitor into the cell line showed markedly 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 showed little difference between the samples treated with the control and the samples treated with the miR-494 inhibitor. NC represents negative control.
6 shows the results showing that GIST cell proliferation is inhibited by miR-494. 6A shows the GIST882 morphology 12 days after transfection. Cells were incubated 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). 6B shows GIST882 cells were cultured in 60-mm dishes with 2 × 10 ⁶ cells and dispensed into each dish. Three, six and twelve days after transfection, cells were counted by hand twice and the counted average was used. Cells were transfected twice on days 0 and 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). 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 GIST882 cells transfected with non-targeting miRNA (top panel) or miR-494 (bottom panel) on day 4, and cells were obtained and stained with propidium iodide. Both samples were analyzed by flow cytometry. miR-494 treatment showed a 5.6% increase in G ₀ -G ₁ phase cells and a 5.7% decrease in S phase cells compared to cells transfected with non-targeting miRNAs. NC represents negative control.

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

Example

Materials and Experiments

Cell line and cell culture

GIST882 cell lines with activated KIT mutations (Exon 13, K642E7) were kindly presented by Dr. Jonathan Fletcher at 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 (Olympus, Tokyo, Japan).

Patient and Tissue Samples

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).

Clinicopathological and genetic status of 31 GISTs. case Age / gender Mutation status tumor cell
shape Immunohistochemistry KIT PDGFR Size (cm) location Mitosis (/ 50HPF) Risk factor KIT CD34 One 76 / F V560 del Wild type 8 top 4 middle Fusiform + ¹⁾ + 2 56 / F V559A Wild type 5.5 top One middle Fusiform + + 3 64 / F D579 del Wild type 17 top 20 height Fusiform + + 4 45 / M T574_R586 ins Wild type 10 top One middle Fusiform + + 5 68 / F V559D Wild type 8 top 6 height Fusiform + + 6 58 / M Y553_Q556 del Wild type 6 top 14 height Fusiform + + 7 58 / M V559D Wild type 16 top 2 height Mixed + + 8 74 / F D579 del Wild type 3 top 4 lowness Fusiform + + 9 51 / M W557R Wild type 3.5 top One lowness Fusiform - + 10 67 / F K550_Q556 del Wild type 9 top 14 height Mixed + + 11 43 / F W557R Wild type 9.8 top 5 middle Fusiform + + 12 42 / F L556_D569 ins Wild type 33 Stomach and spleen 4 height Fusiform + + 13 65 / M A504_Y505 ins Wild type 6.5 Abdominal cavity 23 height Mixed + + 14 41 / F V559D Wild type 3.5 top 5 lowness Fusiform + + 15 49 / F V555_W557 del Wild type 13 Intestine 15 height Fusiform + - 16 64 / F W557_K558 del Wild type 7.5 top 10 height Fusiform + + 17 50 / M W557_K558 del Wild type 8 top 23 height Mixed + + 18 39 / F V559D Wild type 6.3 top 8 height Fusiform + + 19 69 / F V559D Wild type 7 top 2 middle Fusiform + + 20 74 / M Q556_V559 del Wild type 6 top 40 height Mixed + + 21 46 / F M552_N572 del Wild type 5 Intestine 74 height Mixed + + 22 61 / F F509_F511 ins Wild type 5 Intestine 0 lowness Mixed + - 23 79 / F W557_K558 del Wild type 6.5 Intestine 28 height Fusiform + + 24 64 / M A504_Y505 ins Wild type 9.5 Intestine 0 height Fusiform + + 25 47 / M A504_Y505 ins Wild type 9 Intestine 20 height Fusiform + - 26 57 / F Wild type Wild type 15 top One height Fusiform + + 27 78 / M Wild type Wild type 17 top 49 height Epithelium + + 28 66 / M Wild type D842V 3 top 0 lowness Mixed - - 29 44 / F Wild type Wild type 1.3 top 0 Very low Fusiform + + 30 38 / F Wild type Wild type 5.5 Intestine 0 height Fusiform + + 31 26 / M Wild type Wild type 5.5 top 4 middle Fusiform - +

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 Preparation and Taqman miRNA Assays

Total RNA was extracted from frozen tissue using TRIZOL (Life Technology, Rockville, MD). Expression levels of miRNA were quantified by a Taqman miRNA assay (Applied Biosystems) and analyzed using the Applied Biosystems 7300 real-time PCR system (Applied Biosystems). All assays were performed in triplicates.

Quantitative RT-PCR (qRT-PCR)

The qRT-PCR primer sequences for KIT (PrimerBank ID; 4557695a2) and GAPDH (PrimerBank ID; 2282013a2) were obtained from the Primerbank database ( http://pga.mgh.harvard.edu/primerbank/ ). The reaction was carried out at a total volume of 20 μl 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 mimics and transformations

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 (Promega, Madison, Wis., USA). Primary antibodies used in the present invention are GAPDH (Trevigen, 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 (Cell Signaling, Danvers, MA USA). Western blot images were analyzed using the LAS-4000 Mini (Fujifilm, Tokyo, Japan).

Luciferase Reporter Assay

To produce another construct, an N vector was first prepared. The total 3′-URT sequence obtained from GIST882 via PCR amplification was cloned downstream of the SV40 enhancer and early promoter-regulated Renilla Luciferase cassette in the 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.

Construct order N-construct F ¹⁾ 5’-GCAGAATCAGTGTTTGGGTCA-3 ’ N-construct R ²⁾ 5’-CAGGAATAAAACACGTGGAACA-3 ’ M-Construction F 5'-TAATTTTTTCTTACATCAGATCCGGCTTTGCAGTGGCTTAATGT-3 ' M-Construction R 5'-ACATTAAGCCACTGCAAAGCCGGATCTGATGTAAGAAAAAATTA-3 ' O-construct F 5'-TGTAAATATTGAAATCAGACAATAATGTCTTTTGAATATT-3 ' O-construct R 5'-AATATTCAAAAGACATTATTGTCTGATTTCAATATTTACA-3 ' Ma-Construction F 5’-TCATTATTAGTACTGCTCTTCCGGCTTTTCACATAGCTGTCTAGA-3 ’ Ma-Construction R 5’-TCTAGACAGCTATGTGAAAAGCCGGAAGAGCAGTACTAATAATGA-3 ’ Mb-construct F 5'-TGTCTGAAAAATTCCTTTGTCCGGCTATTGACTTCAATGATAGTA-3 ' Mb Construct R 5’-TACTATCATTGAAGTCAATAGCCGGACAAAGGAATTTTTCAGACA-3 ’ Mc-Construct F 5’-CCGGCTTTGCAGTGGCTTAATCCGGGAAATTATTTTGTGGCTTTT-3 ’ Mc-Construct R 5'-AAAGCCACAAAATAATTTCCCGGATTAAGCCACTGCAAAGCCGG-3 '

Dual luciferase assays were performed each time by co-transfecting control vectors with N, M, O, Ma, Mb or Mc vectors. All miRNA mimetics were transfected at 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 numbers 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 a 60-mm dish. On day 4 of transfection, cells from each sample were stained with a solution consisting of PBS, propidium iodide (Abcam, Cambridge, MA, USA) and RNase A. All samples were analyzed by FACS Calibur (BD Biosciences, San Jose, CA, USA).

Statistical analysis

The correlation between miR-494 expression level and KIT expression level was determined by analyzing the Spearman correlation 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 because of their statistically significant correlation with KIT expression in GIST [13]. 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. 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 miR-494 to induce KIT down-regulation was confirmed by transfecting 25 nM of nontargeting miRNA, miR-494, miR-221 or miR-494 inhibitors into the GIST882 cell line. Non-targeting miRNA was a negative control compared to miR-494, miR-221 was a positive control compared to miR-494. Both miR-221 and miR-222 are 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 reaction (qRT-PCR) and western blotting. When cells were transfected with miR-494 or miR-221 (miR-494 showed a better effect than miR-221), Western blotting analysis confirmed 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 miR-494 inhibitors 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 somewhat increased levels of KIT mRNA in cells transfected with miR-494 inhibitors (FIG. 2B). The above results indicate that KIT expression is regulated by miR-494.

Inverse correlation between miR-494 expression 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 to inverse correlation between KIT expression and miR-494 expression in GIST. ) Was confirmed. 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 fold change relative to 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. See 1897-1903 via the Target scan 3.0 database ( http://www.targetscan.org/ ) to find the algorithm-based binding site of miR-494 for the 3'-UTR of KIT mRNA. One binding site located 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, an N vector (5 nM) comprising a nontargeting miRNA, miR-494, miR-221 or miR-222 was used. HeLa cell line was transfected. Two days after transfection, cells were obtained and subjected to dual luciferase assays using a dual luciferase assay (DLR) assay kit. Samples treated with N vectors and miR-494 were only about half the luciferase activity of samples transfected with N vectors and untargeted miRNAs, and more pronounced than samples treated with miR-221 and miR-222. A decrease was shown (FIG. 4B, top panel). Site-directed mutagenesis of 3′-UTR of KIT mRNA of N vector, M vector for mutant 1899-1902 (GTTTCCGG) miR-494 and nucleotide of 1961-1964 O vectors for miR-221 / miR-222 with altered sequence (GTAGCAGA) were constructed. N, M or O vectors were transfected with non-targeting miRNA, miR-494, miR-221 or miR-222, and a reporter assay was performed. Luciferase activity in cells transfected with O vectors comprising miR-221 or miR-222 was fully restored compared to the negative control (cells transfected with N vectors containing nontargeted miRNAs). miR-221 and miR-222 directly target KIT mRNA. However, samples transfected with the M vector and miR-494 showed very slightly restored luciferase activity compared to the negative control (FIG. 4B, top panel). The above 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 powerful 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 containing 6-7 seed match sequences for miR-494. The potential miR-494 binding sites retrieved were: 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 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

In addition, the inventors 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, whereas GIST882 cell lines had little detectable miR-494 expression (FIG. 4C, top panel). The three cell lines described above were each transfected with N vectors containing nontargeting miRNAs or N vectors containing miR-494 inhibitors as negative controls. Cells were harvested two days later 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 the 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

KIT 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 important 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 nontargeting miRNA, 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 miR-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. Expression of p-KIT (pY703) was also tested with phospho-specific antibodies, with a marked decrease in 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 by 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. Taken together, the above results indicate that miR-494 treatment induces KIT down-regulation and affects downstream molecules in the same way as KIT-inhibiting molecules such as imatinib or KIT shRNA.

Inhibition of GIST Cell Proliferation by Induced miR-494 Overexpression

The effect of miR-494 on the 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. Normally confirmed normal cell morphology, transfection was carried out in a total of 12 dishes on the third day of passage culture. 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 clear 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 further amplified on day 12 of assay. For GIST882 cells, the normal cell morphology is fusiform with pin-point terminal structures, but miR-494 treated GIST882 cells showed a changed cell morphology on day 12 (FIG. 6A), and the number of cells was also almost as compared to the control cell number. Decreased by 37% (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. Cell lines treated with miR-494 had a 6% increase in G ₀ -G ₁ phase cells and a 5% decrease in S phase cells compared to cells transfected with non-targeting miRNAs. The above findings indicate that miR-494 inhibits GIST882 cell proliferation by regulating the cell cycle.

Additional discussion

MicroRNAs can affect signal transduction directly and indirectly by controlling the target genes and components of the target gene regulatory pathway in a post-transcriptional manner. The findings indicate that miRNAs can play key regulatory functions in signal transduction and tumorigenesis [15, 16]. Mutations and overexpression of KITs 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 in vitro 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. So far, 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 protein is a high oncogenic tyrosine kinase belonging to the RTK family and is included in the major signal transduction pathway of PI3-kinase, Src family kinase and Ras-Erk and of JAK-STAT [6]. Mutation of the transmembrane domain activates 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 and contribute to the GIST tumorigenic process [3, 23]. This study demonstrated that down-regulation of KIT induced by miR-494 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 to activate downstream molecules [24-26]. Our study shows that miR-494 treatment affects the GIST882 cell line in a manner similar to imatinib, which disrupts signaling pathways and inhibits cell proliferation. The findings described above mean that miR-494 may be another option for treating GIST. Our identification of two different key match sites of miR-494 in the 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 or post-transcriptional fashion [27]. Inhibition of KIT by imatinib is an example of post-translational inhibition through competitive binding of KIT to the ATP binding forge. However, inhibition of KIT by imatinib is incomplete because resistance can occur through potential mutational changes of amino acid residues that may occur during processing. 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. The therapeutic use of miR-494 to inactivate KIT would have to be complemented because conventional RNA-based therapies are paradoxically unstable in the blood with the huge molecular size of siRNAs (small interfering RNAs). However, many of the problems described above are solved by novel delivery systems such as aptamers, 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 inhibit tumor progression.

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

references

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<110> Industry-Academic Cooperation Foundation, Younsei University <120> Compositions for Preventing or Treating Neoplastic Disorders          Comprising miRNA as an Active Ingredient <130> PN100650 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> RNA <213> Homo sapiens <400> 1 ugaaacauac acgggaaacc uc 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> A site in KIT 3'-UTR <400> 2 acaucagwws www swuugca gu 22 <210> 3 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> B site in KIT 3'-UTR <400> 3 uuccuuusws wwwswauuga cu 22

Claims (15)

V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) -mediated diseases, disorders or agents comprising microRNA-494 (miR-494) or miR-494 overexpression as an active ingredient Pharmaceutical compositions for the prevention or treatment of KIT-mediated diseases, disorders or conditions.
The composition of claim 1, wherein the agent inducing miR-494 or miR-494 overexpression inhibits expression of KIT.
The composition of claim 1, wherein miR-494 inhibits phosphorylation of AKT or STAT3.
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.
The method of claim 1, wherein the KIT-mediated disease, disease or condition is neoplastic disorder, 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 associated disease, pain, substance-abuse disease, prion disease, heart disease, fibrotic disease , Idiopathic pulmonary hypertension (IPAH), primary pulmonary hypertension (PPH), glioma or cardiovascular disease.
6. The composition of claim 5, wherein said KIT-mediated disease, disease or condition is a neoplastic disease.
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 myelogenous leukemia, colorectal carcinoma, gastric carcinoma, testicular cancer, glioblastoma, astrocytoma or mastocytoma.
20-100 contiguous nucleotide sequences for the prevention or treatment of a KIT-mediated disease, disease or condition comprising an antisense oligonucleotide sequence having a complementary sequence to the 8th to 15th nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2.
20-100 contiguous nucleotide sequences for the prevention or treatment of a KIT-mediated disease, disease or condition comprising an antisense oligonucleotide sequence having a complementary sequence to the 8th to 15th nucleotide sequence of the nucleotide sequence of SEQ ID NO: 3.
10. The sequence of claim 8 or 9, wherein the antisense oligonucleotide sequence comprises a sequence complementary to the key match sequences of miR-494.
The sequence of claim 10, wherein the key binding sequence of miR-494 is in a 3'-UTR (untranslated region) of KIT.
10. The sequence of claim 8 or 9, wherein the antisense oligonucleotide is a DNA or RNA molecule.
(a) treating a test substance to a cell comprising a microRNA-494 (miR-494) -encoding nucleotide sequence; And (b) analyzing the expression of miR-494 in the cells. do.
The method of claim 13, wherein miR-494 inhibits expression of KIT or inhibits phosphorylation of AKT or STAT3.
The method of claim 13, 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, neurology Glioma, astrocytoma or mast cell tumor.

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