METHOD FOR INCREASING CYTOTOXICITY OF NATURAL KILLER CELL BY REGULATING MIR-150 EXPRESSION
Film capable of suppressing the occurrence of of the present invention refers to miR-150 substituted method for increasing the cell cytotoxicity using the same for preventing or treating cancer is directed to pharmaceutical composition. Immune component in particular during cells, NK cells (natural killer cells, hereinafter NK by cells) the non-specifically cancer cells killing ability to found been proceeds the protein complex from a while, a mammary carcinoma deficiency activity of the differentiation and NK ( NK into a host in which cells infected for counteract or cancer pathogenic congenital immune response through the secretion of cytokines (Innate immune response) and immune response calix arene (Adaptive immune response) to make an important role ( Micro RNA (MicroRNA) Dicer1 and nucleus, thus in a cytoplasmic has two types of called Drosha in ribonuclease (ribonuclease) (primary) miRNA primary produced thereby RNA (19-22 nt) untranslated small as is. In animal cells maturation of miRNA has specific target mRNA 3' presenting duty on RNA-induced silencing complexes (UTR) to the target mRNA or translation of target protein (translational suppression) inhibitors due to. reduces the expression of gene ( Hematopoietic immune derived from the stem cells in cell differentiation reactions and innate immunity Argonaute relationships may with miRNA ( The, the present inventor are NK cell cytotoxicity of modulating an adder adds the contact corresponding to a target molecule new effort results, microRNA-150 (miR-150) of mouse and human, and is and to (cytotoxicity) cell cytotoxicity NK Prf1 of 3' UTR bio-sequence listing specifically bind to reduce the cell growth rate by enhancing expression of a target protein, substantially cancer artificially implantation of the NK miR-150 from mouse model of cells which lack is the size of the cancer and is attached on the perfect and transition by verify, NK through inhibitors miR-150 portion of the compound that activates the cells, of treating cancer by using the same may be found by the present invention, is completed. The present purpose of the invention the film capable of suppressing the occurrence of of miR-150 substituted method for increasing the cell cytotoxicity using the same for preventing or treating cancer is directed to pharmaceutical composition. Said end of the, Film capable of suppressing the occurrence of of the present invention refers to miR-150 substituted method for increasing the cell cytotoxicity provides. Furthermore, the present invention refers to miR-150 substituted cells which lack diseases which contain as the active ingredient for treatment or prophylaxis of cancer provides pharmaceutical compositions. Furthermore, the present invention refers to miR-150 substituted cells which lack of cancer and as an active ingredient provides pharmaceutical compositions for inhibiting the. Furthermore, the present invention refers to miR-150 complementary to nucleotide antisense bind, small interfering RNA (short interfering RNA) or a short hair pin RNA (Short hairpin RNA) selected from the group consisting of inhibitors of miR-150 sound absorbing member is partly substituted diseases which contain as the active ingredient provides a kit for cell activation. Furthermore, the present invention refers to 1) miR-150 a cell that expresses a step of treating the compound to; 2) said step 1) in cells of processed a step of measuring a expression dose miR-150; and 3) the control group compared to said step 2) in a subject to reduce expression of miR-150 of selecting compounds including step of screening anticancer provides method. In the present invention miR-150 inhibit expression of induced inhibiting or through (Knock-out, KO) of the mouse and the substituted Prf1 to increase the feeling expression of proteins in cells in vitro as well as in vivo is increased activity of living cells substituted even, is miR-150 prf1 mRNA of 3' UTR Prf1 the specifically bind to portion can increase expression of the, substituted lack miR-150 growth of cell constitution: metastasis, or, lack said miR-150 cytotoxicity cells substituted treatment of cancer to increase the feeling and is thus useful for the inhibiting the, and cancer can be. Mouse miR-150 KO and (wild type) a wild-type such cell which also 1a NK IL-15 (25ng/ml) mouse after the separation of the cells include media activated protein expression amount GzmB and Prf1 then immune blot (immunoblotting) through a diagram in which film is very (upper), semi-qPCR ptk7 MRI GzmB and Prf1 to (mRNA) level of confirming the receivable channel degrees and is about (lower), also a be schematized to door has 1b 1a Prf1 is indicated by graph filars ratio of protein expression, expression of miR-150 between multiple layer PCR TaqMan embodiment against a wild-type expression of KO door is represented by graph filars, also 1c has human umbilical cord blood isolated from primary NK IL-15 cells (30 ng/ml) for treating with, immune blot 888000108 8888 Prf1 and GzmB protein expression amount (upper) and diagram in through, between and Prf1 to qPCR embodiment GzmB mRNA level of confirming the receivable channel degrees and is about (lower), 1d also be schematized to 1c door has a Prf1 protein expression ratio (lower) is indicated by graph filars, PCR TaqMan embodiment miR-150 between expression of multiple layer against a wild-type expression of filars KO door is represented by graph (upper). Mouse miR-150 KO and from the wild-type has 2a also NK IL-15 (25ng/ml) mouse after the separation of the cells include media activated cell growth rate by enhancing expression of protein Prf1 then immune blot (immunoblotting) (upper) and diagram in through, between embodiment to qPCR Prf1 mRNA level of confirming the receivable channel degrees and is about (lower), and also from the wild-type has 2b NK mouse miR-150 KO IL-15 (25ng/ml) mouse after the separation of the cells with the supernatant may be processed to recover cells culture nor post-incubation 2 improves NK cytotoxic through Cr release assay measures the saturated in range where R has considerable line folded door is, and also from the wild-type has 2c NK mouse miR-150 KO IL-15 (25ng/ml) mouse after the separation of the cells include media activated then cell assay fluorescence Prf1 protein expression amount (flow cytometry) confirming through degrees and is about 8 880000135888 (lower), a bar graph is door represented by mean fluorescence intensity (MFI) (upper). Also from the wild-type has 3a NK mouse miR-150 KO and after the separation of the cells including mouse IL-15 48h improves then activated fluorescence level of expression of the cd52 cell membrane receptor (flow cytometry) cell assay indicating a degrees and is about, also from the wild-type has 3b NK mouse miR-150 KO and after the separation of the cells including mouse IL-15 48h improves NK-treating with before antibodies anti-NKp46 cells marker secretion Prf1 CD107a level of fluorescence is in (flow cytometry) cell assay, also from the wild-type has 3c NK mouse miR-150 KO and after the separation of the cells including mouse IL-15 improves apoptosis of then activated 48h FasL ligands, TRAIL cell assay fluorescence level of a degrees and is about (flow cytometry), also from the wild-type has 3d NK mouse miR-150 KO and after the separation of the cells including mouse improves IL15 48h are then expressed active marker secretion Prf1 CD107a cell assay fluorescence level is in (flow cytometry). Also displayed in a diagram in the 4a (reporter vector) reporter vectors pcAANAT a degrees and is about, the 4b also miR-150 Prf1 of target protein 3 'UTR miR-150 bio-sequence listing of a bonded to the portion of the mutant and a (mutant) is in of mimetic for bio-sequence listing, the 4c also miR-150 Prf1 and GzmB of human in which the target proteins are of 3' UTR miR-150 a bonded to the portion of the of a (mutant) mutant and bio-sequence listing bio-sequence listing for mimetic the degrees and is about, s to 4d also the HEK293T cells (reporter vector) reporter vectors pcAANAT Prf1 and GzmB of mouse coupled to 3 '-UTR portion separably coupling the RFID circuit site miR-150 stable (wild-type) and a (transfection) injection transgenic mutant miR-150 miR-150 and then simultaneously, reporter gene (reporter gene) in a viewpoint from which to view the amount of expression of AANAT immune blot (Immunoblotting) AANAT and a actin protein expression amount through a diagram in which film is very (upper), embodiment AANAT mRNA expression amount between qPCR through door is represented by histograms (lower), also s pcAANAT to 4e the HEK293T cells (reporter vector) reporter vectors of GzmB and Prf1 human coupled to 3' -UTR portion separably coupling the RFID circuit stable miR-150 (wild-type) and a site miR-150 and then a transgenic mutant miR-150 simultaneously, reporter gene (reporter gene) in a viewpoint from which to view the amount of expression of AANAT immune blot (Immunoblotting) AANAT and a actin protein expression amount through a diagram in which film is very (upper), embodiment AANAT mRNA expression amount between qPCR through door is represented by histograms (lower). The 5a also miR-150 NK lentivirus for overexpressing the cells into MICE and 48 (immunoblotting) blot system with an immune process has been completed a gene targeted Prf1 being impeded as much as possible in expression of which film is very a shown that, also the 5b 5a mouse NK in cells and also said toxicity is door main appear as Cr release assay, in vitro human has 5c also differentiated mNK miR-150 NK92MI a and cell lines cells in the comparison of the expression of a degrees and is about between qPCR embodiment, also the 5d are expressed from one network to another with reduced miR-150 NK92MI to a human cell line by introducing lentivirus for overexpressing miR-150 miR-150 embodiment for increases expression of qPCR is door main appear as between, the said also 5d 5e also, such as is performed in the condition that the a the target for miR-150 Prf1 (immunoblotting) blot immune protein expression of a degrees and is about showing, also the 888000060488 8 miR-150 to 5e also said lentivirus such as a which has been that overexpress HSP NK cell toxicity door is represented by Cr release assay. Also 6a has immune cells which lack Rag2-/-rC-/-mouse, capable of producing wild-type/miR-150 KO NK IL-15 a (100 ng/ml) after activated after injection of to intravenous tail, the growth by blood shipper mouth B16/F10 melanoma is measurement of the degrees and is about, which lack immune cells has 6b also Rag2-/-rC-/-mouse, capable of producing wild-type/miR-150 KO NK IL-15 a (100 ng/ml) after activated, and injection into a intravenous tail after injection of intravenous melanoma B16/F10, pulmonary growth metastasis of cancer is formed of cancer is in measuring colony number. Which lack immune cells has 6c also Rag2-/-rC-/-mouse, capable of producing wild-type/miR-150 KO NK IL-15 a (100 ng/ml) after activated, and injection into a intravenous tail B16/F10 after injection of intravenous melanoma, cancer formed pulmonary door is indicative of the state of. Hereinafter, the present invention described a detail the. In the present invention refers to separated substituted, including inhibiting expression of miR-150 substituted method provides increased cell cytotoxicity. Terms used in the present invention "miR-150" mammalian, including human as one found in family precursor microRNA, miR-150-aca (Accession No. MI0018761), miR-150-bta (Accession No. MI0005058), miR-150-cfa (Accession No. MiR-150-MI0007998) dre (Accession No. MI0002016), miR-150-eca (Accession No. MI0012762), miR-150-ggo (Accession No. MI0020764), miR-150-hsa (Accession No. MI0000479), miR-150-ipu (Accession No. MI0024515), miR-150-mdo (Accession No. MI0012504), miR-150-mml (Accession No. MI0007641), mmu-miR-150 (Accession No. MI0000172), miR-150-oan (Accession No. MI0006840), miR-150-oar (Accession No. MI0025255), miR-150-ppy (Accession No. MI0014840), miR-150-ptr (Accession No. MI0008550), miR-150-rno (Accession No. MI0000920), sha-miR-150 (Accession No. MI0019633), miR-150-1-ssc (Accession No. MI0022133), miR-150-2-ssc (Accession No. MI0022134), miR-150-xtr (Accession No. MI0004846), the logical source including the device one, are not limited to, Prf1 mRNA and a complementary capable of binding to a similar sequence and may include both whether until, the logical source including the device, two 20-25 a nucleotides in length, and which is, prf1 mRNA and a synergetically combines the method and apparatus for controlling communication in any if you could only can be includes whether. Said substituted substituted human mouse or cells manner cells in a logical source, the logical source, limited to not. Said miR-150 miR-150 step for inhibiting the expression of the antisense bind complementary to, small interfering RNA (short interfering RNA) and short hair pin RNA (Short hairpin RNA) selected from the group consisting of substituted is safe to skin without any side transforming cells can be degraded and performed method, limited to not. Inhibiting the expression of said miR-150 miR-150 of the step function by untransformed method inhibiting can be degraded and performed, limited to not. In said method, expression of proteins in cells substituted Prf1 of: confirming whether is increased additionally comprises the step can be. Whether or not a is increased expression of proteins said Prf1 immune blot, such as immune blot method but can be identified by their thermally, are not limited to all known art publicly known method of. can be accomplished through. In said method, said substituted for glyphosate having increased activity, cells a step for identifying a whether may be added but, limited to not. For glyphosate having increased activity, cells substituted said devices and confirming whether the substituted IL-15 cells when stimulated, the control group compared to experimental groups γ-IFN method a is used to check whether the non-viscous coating layer is increased, or the control group compared to experimental groups target cytotoxicity increased capacity is used to check whether a method but may include, limited to not. Terms used in the present invention "increased cell cytotoxicity as an end point, substituted" or "substituted cells increase activity" the substituted or to promote activity of living cells, or to promote cell cytotoxicity substituted, substituted expression of Prf1 of cells is enhanced or otherwise, expression of GzmB cell substituted is enhanced or otherwise, is enhanced or otherwise expression of γ-IFN, miR-150 inhibited expression of the means, limited to not. In specific embodiment of the present invention, the present inventor are miR-150 NK Prf1 in cells on the opposite side of the pattern, where the expression of the show to make sure that that wild-type mouse NK mouse miR-150 KO IL-15 herein obtained cells NK processes a portion of the compound that activates the cells is related to the amount of mRNA of GrzmB miR-150 Prf1 and amount of the n bit parallel data inputted as measured by PCR-between embodiment, expression of proteins GzmB and Prf1 have been measuring blot immune amount, mouse Prf1 expression of proteins GzmB and it is confirmed that abruptly increases (also upper 1a) has been, increased expression of mouse GzmB mRNA Prf1 mRNA but with being burned is scarcely changed expression of (lower 1a also), expression of a mouse miR-150 Prf1 Prf1 with low level of opposite pattern is enhanced when, when abruptly increases in Prf1 a, so as to reduce abrasion of it has been confirmed (also 1b). Furthermore, the present inventor are human substituted active IL-15 the cells are separated Prf1 and then immune blot as measured by an amount of protein result, the level of a low cost remain almost unchanged Prf1 mRNA Prf1 the n bit parallel data inputted confirm that the user abruptly increases in protein (also 1c), the patterns of expression of protein Prf1 miR-150 expression of opposite pattern he is showing identifying improving (also 1d). Furthermore, the present inventor are NK miR-150 Prf1 cells lack expression of cells and, at the same time, is increased verify that the cytotoxicity is augmented immune blot and fluorescent cells by and determining the overexpression of Prf1,51 Cr-release analysis method targeted cytotoxicity (cytotoxicity) cytotoxic prodrugs to a method which directly measures the ability is measurement of the result, compared to wild-type mouse NK cells lack miR-150 NK Prf1 protein expression amount increased cells (also 2a, 2c), cytotoxicity (also 2b) 80 M. has been confirmed. Furthermore, the present inventor are NK Prf1 is high activity in the cells due to increased expression of proteins to make sure that that, wild-type NK NK miR-150 cells lack expression of receptor and ligand of cells (effector) effector cell assay fluorescence amount secretion protein as measured by an result, wild-type and lack miR-150 NK emulating cell-to-cell the cell membrane of receptor expression dose a large thin film transistors are electrically connected to the n bit parallel data inputted identifying improving (also 3a), marker secretion of levels Prf1 CD107a thin film transistors are electrically connected to confirm it can be obtained (also 3b, 3d), substantially levels of cell death receptor (death receptor) of thin film transistors are electrically connected (also 3c) by identifying improving, NK high mouse lack miR-150 Prf1 active cells caused with an increased expression of proteins that it has been confirmed. Furthermore, the present inventor are miR-150 is Prf1 mRNA of 3 'UTR portion specifically bind to the achieved through strong and long-lasting expression of proteins prf1 to reporter system (reporter system) (also 4a) to target protein miR-150 Prf1 of 3' UTR portion transforming (also 4b, 4c) the n bit parallel data inputted, miR-150 mutant soldiers contrast result when the low pass filter that overexpress HSP inhibit expression of reporter gene by identifying no inclusion of (also 4d, e), is miR-150 Prf1 mRNA 3' UTR specifically bind to the it has been confirmed that inhibit expression. Furthermore, the present inventor are substituted use of cells of miR-150 with revelation system Prf1 protein expression and cell killing and wounding decline supervis ion portion confirms a inhibitors miR-150 to a lentivirus NK cells that overexpress HSP immune blot (immunoblotting) in the n bit parallel data inputted is provided, which expresses Prf1, infected NK NK Prf1 of cells that overexpress HSP miR-150 cells by and reduced protein expression of (also 5a, 5e) is conform to cytotoxic ° apart and can be used as (also 5b, 5f). Thus miR-150 Prf1 NK and expression of proteins is a leakage of oil to the cell cytotoxicity, the expression dose miR-150 NK cell cytotoxicity have the ability to modulate the activity.. Furthermore, the present invention refers to said miR-150 substituted cells lack diseases which contain as the active ingredient for treatment or prophylaxis of cancer provides pharmaceutical compositions. Terms used in the present invention "treatment" has cancer cell is killing reduction of cancer growth as well as, reduction of recurrence, cancer cells an immune system to a anticancer active memory or to the individual having reduced toxicity such as anticancer activity of cancer cells, cancer cells all without deteriorating longer causes the mixture by the addition of an initiator effect, limited to not. Lack of the present invention miR-150 substituted cells (NK cells) or inhibited expression of the miR miR (knock-out) pleasure out is a substituted cells, or said miR-150 stages for inhibiting the expression of the substituted, including, but cells, are not limited to. Furthermore, lack of the present invention miR-150 substituted miR-150 cells isolated from individual it became pleasure out is substituted can be cells, not limited to. Furthermore, the present invention refers to miR-150 for cells into which the substituted lack miR-150 and work is conducted without being affected which is effective to suppress the transformed cells substituted.. "Vector" terms used in the present invention a linear DNA, plasmid DNA, linear RNA, RNA plasmid, or recombinant may include viral vector, limited to not. A lung tumor cancer as defined above, hepatocellular carcinoma, stomach, colon cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid, may be and haematological malignancies melanoma, are not limited to. Furthermore, the present invention refers to said miR-150 substituted cells lack diseases which contain as the active ingredient tumor metastasis suppressor provides pharmaceutical compositions for. Terms used in the present invention "transition" determines which one organs, blood vessel or lymphatic cancer cell generated and the other tissue along the clutch gone out meaning as well as method/system for monitoring remote base also includes the otherwise. Furthermore, diseases which contain as the active ingredient inhibitors of the present invention refers to miR-150 substituted provides a kit for cell activation. The present invention according to said miR-150 synergetically combines inhibitors of antisense nucleotide, small interfering RNA (short interfering RNA) or a short hair pin RNA (Short hairpin RNA) at least one selected from the group consisting of can be, are not limited to. In addition the present invention refers to, 1) miR-150 a cell that expresses a step of treating the compound to; 2) said step 1) in cells of processed a step of measuring a expression dose miR-150; and 3) the control group compared to said step 2) in a subject to reduce expression of miR-150 of selecting compounds including step of screening anticancer provides method. Said natural compounds compounds are subject, compound, RNA, DNA, polypeptide, enzyme, protein, ligands, antibodies, antigen, bacteria or of fungi on metabolites selected from the group consisting of bioactive molecules and be decreased which may be, limited to not. the step of measuring the expression dose said miR-150, RT-PCR, quantitatively or semi-quantitatively stoichiometric RT-PCR (quantitative or semi-quantitative RT-PCR), quantitatively or semi-quantitatively PCR (quantitative or semi-quantitative real-time PCR) between stoichiometric embodiment, of southern blot (northern blot) and DNA or RNA chip (chip) selected from the group consisting of of either the method includes identifying a can be degraded and, limited to not. In specific embodiment of the present invention, the present inventor are actually cells lack miR-150 NK mouse growth and metastasis, or verify that the, immune cells lack the requisite a mouth that activation of injecting cells into a 150 KO NK-miR cancer again in the state of growth for the injection of an and transfer monitors a result, cancer cells NK mouse lack miR-150 and vastly reduces the growth of the n bit parallel data inputted the output insurance document (also 6a), mouse lack miR-150 NK and vastly reduces the transition of cancer cells in addition been has been confirmed (also 6b, 6c). In in vivo results such as said, a and is free of miR-150 NK cell cytotoxicity is relative to the wild-type exhibits that excellent. Cytotoxicity cells NK miR-150 thus lack an electrode the pharmaceutical composition for preventing or treating cancer can be used, and is useful. Of the present invention composition is an oral or parenteral a variety of formulations can be. When the formulated composition said common filling number, number extender, coupled number, wetting, disintegrating, or of a thinning agent surfactant is formulated excipient. Solid medicinal preparation is finished, the perfume ingredient for oral administration, bolus, masked powders that are to be, granules number, capsule number including an, such solid formulation contains at least one compound at least one or more excipients number for example, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose), is prepared of gelatin. Furthermore, simple number magnesium stearic acid in addition to excipients, such as talc is used lubricants provide. Liquid formulations for parenteral administration include suspension number, content liquid, emulsion or syrup number which, including, the additive and water as the diluent a simple commonly used, in addition to paraffin a bioliquid number various excipient, for example wetting, sweetener, fragrance or a and preservatives may include. Agents are for parenteral administration in the sterilized aqueous solution, non-aqueous solvent, solvent suspension, emulsion, comprises such as a suppository or freeze-dried preparation. Non-aqueous solvent and a suspension solvent include propylene glycol (propylene glycol), polyethylene glycol, vegetable oil olive, such as an oil, ethyl [...] the scanning electrode and the ester can be or the like is used as an. [...] include base left proposal (witepsol), macrogol glyceride, twin (tween) 61, carcass five fingers, does comprising lauric acid, glycerol, gelatin can be or the like is used as an. Of the present invention composition is an oral or parenterally can be administered, , either intraperitoneally or external the skin upon parenteral administration, rectal, intravenous, muscle, avoid, intrauterine epidural or cerebrovascular injection it is preferable that the scheme corresponds to, most preferably employs skin external. Effective compositions comprise pharmaceutical of the present invention the administered in an amount. In the present invention, "in a pharmaceutically effective amount and a" applicable to medical treatment through a rational will benefit/risk ratio a unit, device and method to a sufficient amount meaning, effective capacity type of diseases, a patient level, in the severity, drug action, sensitivity to drugs, administration time, route, and discharge ratio, therapy period, the inhaler includes a mechanism to open signal by using an elements and other medical well-known to the field can be determined according to factors. Useful when administered therapeutic agent individual composition of the present invention or with additional therapeutic agents administered of the existing method can be sequential or from therapeutic agent can be administered simultaneously, can be single or multiple administration. Considers both the components least side effect without a starting switch enables the maximum amount of administration of a amount are important, by the one skilled in the art can be the storing apparatus. Dosage is the body mass of the subject of the present invention composition, age, sex, health status, dietary, administration time, administration method, the flexible bumper structure for severe and/or prevention of diseases and rate disposed, saccharin natrium, the range, daily dose constitutes a saururus extract based on the amount of polyvinylarene, and 0.01 to 1000 mg/kg, preferably 30 to 500 mg/kg and, more preferably is 50 to 300 mg/kg, 1-6 day can be administered once. However route, , the severity of obesity, sex, weight, age and the like increase/decrease can be said even any method with a dosage of the present invention. are not limited to range. Of the present invention composition can be applied alone, or surgical, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers are using conjunction with method, use can be made of,. Hereinafter, the present invention to. as further described and/or at least two different embodiment. Stage, embodiment examples of the present invention relate to a is, embodiment a contents of the present invention and/or at least two different limited not. < 실시예 1>NK Prf1 of and mouse human in identification of relationship expression miR-150 and The present inventor are Prf1 of and mouse human in NK miR-150 and a [...] pattern, where the expression of the show to make sure that that for is experimentally is performed for all the. <1-1>Mouse NK IL-15 mouse cells for processing when GzmB and Prf1 identification of amount mRNA and protein expression The present inventor are that is controlled by the mouse Prf1 miR-150 is to make sure that, first activated NK mouse obtained cells prf1 protein, protein GzmB and mouse prf1 mRNA (seq ID no:1), mouse GzmB mRNA (seq ID no:2) expression it is found out that amount. Specifically, the 8-12 gene (Genotyping)- week zerofall miR-150 Knock out (KO) from mouse male wild-type mouse, and then an aqueous solution of a (spleen), the red blood cells using buffer Ack removing and complete fall obtained a (total splenocyte) cells. At B cells a then rinsed with a liquid to remove (scrubbed) nylon (nylon) fiber (fiber) to every after, mature mouse NK cells separated the in NK separation kit (isolation kit) II (MACS). These through mouse IL-15 RPMI 1640 (10% fetal bovine serum) with active per time period on, the third to eo and then. Wild type mouse miR-150 KO NK IL-15 (25ng/ml) mouse after the separation of the cells include media activated protein expression GzmB and Prf1 then it ladles, the gun phosphorus anti-amount (abcam) and anti- will be and it will growthe LAN pretension B (polyclonal anti-granzymeB) [...] immune using a metal thin film substrate, wherein the n bit parallel data inputted, it is found out that to qPCR semi-amount mRNA expression. Specifically, immune blot (Immunoblotting) (anti-perforin) (abcam) it ladles, the gun phosphorus anti-antibodies difference 1 analysis, will be and it will grow anti- the LAN pretension B and a A (B and a polyclonal anti-granzymeA) (SantaCruz Biotechnology), magnetron will be and it will grow AANAT anti-(polyclonal) (gift from Dr. Klein DC), monoclonal anti-Erk (monoclonal), anti-phospho Erk (Signaling cells), monoclonal anti-a standing (monoclonal) 1 (Dicer1) (abcam), monoclonal anti-GAPDH (Ab FRONTIER) (monoclonal) antibodies difference. 2 the ground terminal of time displayed on time using Immobilon Western kit (MILLIPORE) formed by implanting, semi-qPCR the Trizol reagent (Invitrogen, Carlsbad, CA) (Reagent) using total RNA 1 μ g to separate microneedle manage with mouse style leukemia virus (Moloney murine leukemia virus, M-MLV) reverse transcriptase (reverse transcriptase) (Roche Diagnostics, Basel, Switzerland) and a oligo-dT 42 °C according to manual using 1 in reacting time have been synthesised a cDNA. Synthesized cDNA using semi-quantitative (semi-quantitative) PCR in the 95 °C 30 seconds, in 55 °C 30 seconds, 30 seconds 72 °C in corresponding advertisement based on the shown list performing cycle 22 about an article which he/she, added for extending in 5 ingredient is performed for all the 72 °C conditions. PCR product is a electrophoresis EtBr (ethidium bromide) dyeing is confirmed. Synthesized cDNA using primer pairs for PCR the Prf1 between embodiment (seq ID no 3, 4) or GzmB to a primer pair (seq ID no 5, 6) using SYBR premix (Premix) Ex Taq (Takara Bio, Tokyo, Japan) using in performed for all the (Thermal Cycler) (TakaraBio) amplifier Dice TP800 gene. The level mRNA showed to a value which is relative to GAPDH mRNA. Quantitative relations of miRNA between the PCR TaqMan MicroRNA assay kit embodiment according to performed for all the/m (Assay kit) (Applied Biosysems). small nuclearribonucleic acid miRNA expression is U6 (small nuclear RNA, snRNA) showed in a relative value of a. As a result, expression of mouse Prf1 GzmB and abruptly increases to day 3 can be it is confirmed that (also upper 1a). Day 3 in addition GzmB mRNA abruptly increases in to the level of the protein show pattern similar and is formed integrally with the screw and, as there is no change the level Prf mRNA could confirm it. (Lower 1a also). <1-2>Mouse NK IL-15 mouse cells for processing identification of amount miR-150 when The present inventor are activated NK of cells in which the amount of an expression GzmB and Prf1 miR-150 high amount to confirm the expression of, said in the embodiment <1-1>Between miR-150 to PCR TaqMan embodiment such as expression of an experiment using a computer to is performed for all the confirming amount. As a result, expression of mouse Prf1 Prf1 Day 1 with low level of opposite pattern and increased, the processing unit vibrates the Prf1 Day 2, 3 (also 1b) abruptly reduced. <1-3>When human NK cells for processing IL-15 Prf1 miR-150 level identification of mRNA expression and the expression of a protein and The present inventor are ointment, drink or injection occurs phenomenon and said human supervis ion portion confirms a NK primary the mesenchymal stem to the cells are separated human and then active IL-15 human protein and Prf1 Prf1 mRNA (seq ID no 7) expression amount was miR-150 expression for determining the level. Specifically, human primary (primary) NK HISTOPAQUE-1077 (sigma-aldrich)-ACCUSPIN System cells obtained from UCB (full name) using. Kit cell separation MACS NK NK cells according to manual (isolation KIT) been isolated by negative selection (negative selection). The, the cell population NK CD56+/CD3- cells of at least 93%, CD3+ 5% was less than. As a result, the level of mRNA is formed integrally with the screw and with a view to presenting a clear a big difference in protein abruptly increases in by Day 2, 3 (also 1c). The patterns of expression of protein in addition Prf1 miR-150 expression of opposite pattern he is showing identifying improving (also 1d). Said s and result such as mouse Prf1 miR-150 commonly by the protein expression after transfer by exhibits that adjustment. < 실시예 2>Mouse lack miR-150 NK Prf1 of cells confirming increase cytotoxic and protein expression The present inventor are miR-150 of Prf1 due to lack of protein expression is increased in, yet NK cell cytotoxicity is augmented verify that the for is experimentally is performed for all the. <2-1>Mouse lack miR-150 NK Prf1 of cells increased protein expression The present inventor are wild type mouse miR-150 KO NK miR-150 IL-15 cells after activated expression of said in the embodiment <1-1>Such as immune blot (immunobloting) and of the following fluorescent cell assay human power by operating all systems by through (flow cytometry). Specifically in the case of fluorescent nanoparticles (flow cytometry) cell assay, fluorescent cell assay for all antibodies Becton, Dickinson and Company and a BD Pharmingen from drew his. Dyeing buffer NK cells (staining buffer, phosphate-buffered saline[PBS] containing 1% fetal bovine serum[FBS] and a 0.01% NaN3) to 20 minutes in 4 °C been for dying antibodies designated. As a result, cells which lack miR-150 NK mouse IL-15 in prior to and after is activated by increased relative to the wild-type level of protein Prf1 or show, volume a large level of mRNA Prf1 show differences could confirm it not. (Also 2a). In addition even fluorescent cell assay (flow cytometry) mouse which lack miR-150 NK increased relative to the wild-type cells Prf1 protein containing cytoplasm could confirm it is (also 2c). <2-2>Mouse lack miR-150 NK confirming increased cell cytotoxicity The present inventor are NK mouse lack miR-150 to make sure that cell cytotoxicity, NK isolated from the cytotoxicity assay (assay) is performed for all the cells. Specifically, the (cytotoxicity) cell cytotoxicity as an end point, NK51 Cr-release in performed for all the analysis method. Mouse NK cells corresponding advertisement based on the shown list used YAC1 a of target cells, K562 human NK cells of target cells include the first voice portion out of an. tax paid by the sweat cord and separately or preadipocytes differentiated NK NK implanted cells and that were cells. Living NK cells petal blue tree water exclusion dye been computed using a method (trypan blue dye exclusion). Living NK using the same number of cells51 Cr release analysis reactor for a been used cells (effector). YAC1, K562 target cells of 1.551 Cr 37 °C to reacted time 1 in independently, making various experimental conditions. Labeled YAC1, K562 target cells which washes the a second, 96-well plate (plate) to 10000 per well was frequency divides the so that canine cells. Reactor proportion of cells (effector) and a target (target) (E:T) is 1.5:1 to 5:1 in until then divides the 37 °C, 5% CO2 is supplied in independently, making various experimental conditions of the environment shown in the reacted time 4. When reaction is completed, is recovered and allowed to supernatant increased radioactive (radioactivity) for [...] (scintillation counter, RACTOBETA; LKB Instruments) is measured with. As a result, the present invention according to miR-150 NK than in a wild-type such cells lack a binary 2 identifying 80 M. cytotoxicity (also 2b) can be. Said result such as a high level that the presence of the Prf1 mRNA and inhibited by miR-150, miR-150 Prf1 surface for the key devoid of expression of cytotoxic such as his ID using the light the light source provides 2000. < 실시예 3>Wild-type mouse, and lack miR-150 NK effector expression of receptor and ligand of cells confirming protein secretion The present inventor are NK Prf1 is high activity in the cells due to increased expression of proteins to make sure that that, wild-type NK NK miR-150 cells lack expression of receptor and ligand of cells (effector) effector such as said in the embodiment 2 amount of secretion protein fluorescent cell assay is confirmed. Specifically, wild type mouse lack miR-150 NK IL-15 mouse after the separation of the cells including time 48 improves then activated fluorescence level of expression of the cd52 cell membrane receptor analyzes cell assay, wild type mouse lack miR-150 NK IL-15 mouse after the separation of the cells including 48 time active improves anti-then treating with before antibodies NKp46 Prf1 secretion level of marker fluorescence CD107a analyzes cell assay, wild type mouse lack miR-150 NK IL-15 mouse after the separation of the cells including 48 improves of then active time fasL it is an extinction ligand cells, fluorescence level of TRAIL analyzes cell assay, wild type mouse lack miR-150 NK IL-15 mouse after the separation of the cells including then active time 48 improves marker secretion Prf1 CD107a cell assay the level of fluorescence. As a result, lack wild-type and miR-150 NK emulating cell-to-cell the cell membrane of receptor expression dose a large thin film transistors are electrically connected to the n bit parallel data inputted identifying improving (also 3a), marker secretion Prf1 CD107a even level of thin film transistors are electrically connected to confirm it could (also 3b, 3d). In addition substantially levels of cell death receptor (death receptor) thin film transistors are electrically connected to confirm it could (also 3c). Results such as said mouse high NK miR-150 Prf1 active cells lack expression of proteins with an increased exhibits that caused. < 실시예 4>And mouse of human miR-150 Prf1 mRNA of inhibitors to confirming The present inventor are prf1 mRNA of human miR-150 and mouse of 3' UTR portion specifically bind to the achieved through strong and long-lasting expression of proteins prf1 to suppressed using reporter system (reporter system) is arranged underneath an experiment using a computer to is performed for all the. Specifically, reporter system (reporter system) (also 4a) in pcAANAT to human, in which the target proteins are of miR-150 GzmB and a Prf1 of 3' UTR and a crossing portion that couples the adaptation and cloned (also 4b, c). Plasmid pcAANAT manufacturing, or by a sand blast, an PCR amplifies the portion was a desired gene. CDNA amplification is performed (SolGent) polymerase Pfu sequence analysis technique bio-sequence listing has been confirmed. In addition using specific primer Prf1 GzmB and 3 of ' UTRs pcAANAT a plasmid binding of proteins reporter site and constructed with a. Next HEK293T cells (reporter vector) and said pcAANAT reporter vectors, in conjunction with a human and mouse Prf1 and GzmB of 3'-UTR portion in stable association wild-type miR-150 or mutant miR-150 then a transgenic simultaneously, for viewing amount expression of AANAT reporter gene (reporter gene) in immune blot (immunoblotting) protein expression (actin) actin and a AANAT through it is found out that amount. HEK293T DNA reporter cells (reporter DNA) and a microRNA (mimics) (ThermoFisherScientific) mimic the Metafectene (Biontex) (transfection) injection transgenic according to performed for all the/m. < 실시예 1-1>The same said protein purification and immune blot (immunoblotting) current method, the solubilization cytoplasm of cells NK 1% NP-40 and the perfect protease inhibitors including number (complete proteinase inhibitor) (Roche) PBS (phosphate-buffer saline) was made. As a result, the expression of mouse and human, and GzmB miR-150 are involved but that cannot support the high-speed, and mouse of human Prf1 3' UTR corresponding advertisement based on the shown list inhibitors abruptly protein expression of the reporter gene, when the low pass filter that overexpress HSP reporter gene mutant miR-150 regulate the expression of a sense that it does not it has been confirmed (also 4d, e). Results such as said and mouse of human miR-150 Prf1 mRNA 3' UTR specifically bind to the exhibits that inhibit expression. < 실시예 5>MiR-150 Prf1 overexpression of confirming relationship and cytotoxic expression of proteins <5-1>MiR-150 Prf1 mouse overexpression of confirming relationship and cytotoxic expression of proteins The present inventor are lack miR-150 NK made of a cytotoxicity is augmented in cells and based on, with revelation system of miR-150 supervis ion portion confirms a appears opposite effect use of paradigm was developed for. Specifically the control group (pMIRNA1-GFP control, SBI System Biosciences) that overexpress HSP miR-150 and NK lentivirus pMIR-150 (SBI System Biosciences) into MICE and said in the embodiment 1-1 the cells 48 hours the same method (immunoblotting) blot immunizing a Prf1 performing a target gene expression of has been confirmed. As a result, reduced protein expression of abruptly Prf1 and (also 5a) is conform to cytotoxic ° apart and can be used as (also 5b). <5-2>MiR-150 Prf1 human overexpression of confirming relationship and cytotoxic expression of proteins The present inventor are mouse NK cells lack miR-150 Prf1 protein cytotoxic and Prf1 mRNA 3 and increasing the ' UTR directly target is absent in the data processing system based on a viewing end and an output end, human mature NK cells even serve, such as within constitution: the mouse miR-150 supervis ion portion confirms a method to < 실시예 1-1>the same said immunizing expression of the [...] Prf1 it is found out that amount. Specifically, in in vitro CD34+ mature from cells and along specific cell and tissue lineages of a human being for the differentiation (mature) NK (mNK) a cord blood derived from (umbilical cord blood, UCB) using centrifugal separating Ficoll-Hypaque from using monocytes cells (mononuclear cells, MNCs) been is extracted. CD56+, CD3+, CD14+, CD19+, CD2+, CD11b+, CD15+, CD123+ using lineage cell depletion kit (MiltenyiBiotec) cells having the second interlayer from MNC, CD34+ lineage negative CD34 cell isolation kit (MiltenyiBiotec) cells using been isolated from cells. Separated CD34+ cells as measured by the purity of the (purity) become FACS 97% or more. CD34+ mNK cells of differentiating into the step is briefly described, separated CD34+ cells 106 M a melt to HC (Sigma), SCF (30ng/ml), FL (50ng/ml), IL-7 (5 ng/mL) in MyeloCult H5100 (Stem Cell Technologies) including 37 °C, 5% CO2 is supplied in independently, making various experimental conditions of the environment shown in the good during 14. Complete mNK order to of differentiating into IL-15 derived from human (30 ng/ml, R & DSystems) by adding further good daytime 2. CD56+/CD3- through automatic analysis FACS a purity of cells become 90% or more. A relatively NK92-MI cell line in vitro differentiation thus prevents generation of skin wound when mature NK cells influence miR-150 level compared to the n bit parallel data inputted observation provided an opening (also 5c), method of the cell lines is miR-150 miR-150 in role of has been confirmed. Specifically, the cell lines NK92-MI 10% FBS, 1% penicillin / (streptomycin) include Streptomyces erythromycin (penicillin) alpha (alpha)-MEM was in the. As a result, a lentivirus miR-150 for processing simultaneously abruptly increases in level 4 to influence protein Prf1 falling cytotoxic and is and, at the same time, it has been confirmed (also 5d, 5e, 5f). Results such as said and mouse human miR-150 Prf1 common inhibiting the expression of a general inhibition of the expression of factor the.. < 실시예 6>Mouse lack miR-150 NK cells confirming effect metastasis, or growth of cancer, The present inventor are actually cells lack miR-150 NK of mouse (Jackson Laboratory) growth and metastasis, or verify that the immune cells lack the requisite a mouth that activation after of injecting cells into a 150 KO NK-miR, again the n bit parallel data inputted injection cancer cells has been observed on transition growth of cancer cells. Specifically miR-150 NK is controlled by the any effects's growth cancer activity of living cells to see of the immune cell (B, T, NK) a lack the requisite Rag2-/-rC-/-mouth (Jackson Laboratory) to wild-type/miR-150 KO NK IL-15 a (100 ng/ml) intravenous tail and activated after injection of to, B16F10 melanoma blood shipper mouth and the growth has been observed on. Third step cancer transitions in affecting any miR-150 to supervis ion portion confirms a Rag2-/-rC-/-mouse, capable of producing wild-type/miR-150 KO NK IL-15 a (100 ng/ml) intravenous tail and activated after injection of to, intravenous melanoma B16F10 tumor transfer of a pulmonary it was determined that the colony number. As a result, cancer cells NK mouse lack miR-150 and vastly reduces the growth of the n bit parallel data inputted the output insurance document (also 6a), mouse lack miR-150 NK and vastly reduces the transition of cancer cells in addition been has been confirmed (also 6b, 6c). The present invention relates to a method for increasing cytotoxicity of natural killer cells including a step for inhibiting miR-150 expression or a pharmaceutical composition for preventing or treating cancer through miR-150-deficient natural killer cells and, specifically, to a method for increasing cytotoxicity of natural killer cells including a step for inhibiting miR-150 expression, which inhibits Prf1 protein expression of natural killer cells by using miR-150 which is complementarily bonded to 3′UTR of Prf1 mRNA, and to a pharmaceutical composition for preventing or treating cancer using miR-150-deficient natural killer cells. COPYRIGHT KIPO 2015 Separated substituted cell, including inhibiting expression of miR-150 substituted increased cell cytotoxicity method. According to Claim 1, said substituted substituted human mouse or cells to cells in characterized by method. According to Claim 1, said substituted Prf1 (Perforin1) expression of proteins in cells is increased whether to including ascertaining a characterized by method. According to Claim 1, said miR-150 miR-150 a inhibiting expression of lectin-like complementary to nucleotide antisense bind, small interfering RNA (short interfering RNA) and short hair pin RNA (Short hairpin RNA) selected from the group consisting of substituted is safe to skin without any side characterized by method the transformed cells. According to Claim 1, said cells substituted for glyphosate having increased activity, whether to including ascertaining a characterized by method. According to Claim 5, said substituted i whether or not a is increased activity of living cells) stimulated IL-15 cells when substituted, the control group compared to experimental groups γ-IFN method a is used to check whether the non-viscous coating layer is increased; or, ii) experimental compared to the control group increased capacity cytotoxicity target groups is used to check whether a method characterized by method identifying a through. Substituted cells which lack miR-150 diseases which contain as the active ingredient pharmaceutical compositions for the treatment of or prophylaxis of cancer. According to Claim 7, said a lung tumor cancer, hepatocellular carcinoma, stomach, colon cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid, melanoma and haematological malignancies selected from the group consisting of arm either characterized by a pharmaceutical composition. Substituted which lack according to Claim 7 said miR-150 Prf1 cells characterized by for increased expression of pharmaceutical compositions for the treatment of or prophylaxis of cancer. According to Claim 7, which lack said miR-150 human or mouse, substituted characterized in derived pharmaceutical compositions for the treatment of or prophylaxis of cancer. MiR-150 substituted cells which lack of cancer and as an active ingredient pharmaceutical composition for inhibiting the. MiR-150 complementary to nucleotide antisense bind, small interfering RNA (short interfering RNA) or a short hair pin RNA (Short hairpin RNA) selected from the group consisting of inhibitors of miR-150 sound absorbing member is partly substituted diseases which contain as the active ingredient kit for cell activation. 1) miR-150 a cell that expresses a step of treating the compound to; 2) said step 1) in cells of processed a step of measuring a expression dose miR-150; and 3) the control group compared to said step 2) in a subject to reduce expression of miR-150 of selecting compounds including the screening of the method. According to Claim 13, said natural compounds compounds are subject, compound, RNA, DNA, polypeptide, enzyme, protein, ligands, antibodies, antigen, bacteria or of fungi on metabolites and selected from the group consisting of acid molecules sound absorbing member is partly characterized by anticancer screening method. According to Claim 13, the expression dose said miR-150 RT-PCR, quantitatively or semi-quantitatively stoichiometric RT-PCR (quantitative or semi-quantitative RT-PCR), quantitatively or semi-quantitatively PCR (quantitative or semi-quantitative real-time PCR) between stoichiometric embodiment, of southern blot (northern blot) and DNA or RNA chip (chip) selected from the group consisting of of either the method characterized by ascertaining the screening of the method.
