KR20100064516A - Anticancer agent comprising microrna as an active ingredient and the preparation method thereof - Google Patents

Abstract

PURPOSE: An anticancer agent containing miRNA is provided to induce apoptosis of cancer cells and to treat cancer. CONSTITUTION: An anticancer agent contains miR-29 as an active ingredient. The miR-29 is miR-29a, miR-29b, or miR-29c miRNA. The miR-29a, miR-29b, and miR-29c have a nucleotide of sequence number 149, 150, and 152, respectively. The anticancer agent is manufactured by mixing miR-29 and pharmaceutically acceptable carrier or media.

Description

Anticancer agent comprising micro-RNA as an active ingredient and a method for producing the same {Anticancer agent comprising microRNA as an active ingredient and the preparation method

The present invention relates to an anticancer agent comprising a microRNA (hereinafter, referred to as "miRNA") as an active ingredient and a method for producing the same.

Small RNA refers to ribonucleic acid having a length of about 17 to 25 nucleotides (hereinafter referred to as “nt”) that plays a role in regulating gene expression in vivo. sRNAs are largely classified into microRNAs (hereinafter referred to as "miRNA") and small interfering RNAs (hereinafter referred to as "siRNA") depending on the manner in which they are generated. miRNAs are partially produced from double-stranded hairpin RNAs, and siRNAs are derived from long double stranded RNAs (hereinafter referred to as “dsRNAs”). The sRNA is miRNA, and the sRNA used to experimentally control the expression of a specific gene is classified as siRNA.

miRNAs are 19-25 nt long single stranded RNA (hereinafter referred to as 'ssRNA') molecules and are endogenous hairpin-shaped transcripts (Bartel, DP, Cell 116: 281-). 297, 2004; Kim, VN, Mol . Cells . 19: 1-15, 2005. miRNAs complementarily bind to the 3 'untranslated regions (UTRs) of the target mRNA and post-transcriptional gene suppressors (posts). acts as a transcriptional gene suppressor and suppresses target genes by inducing translation inhibition and mRNA destabilization miRNAs play an important role in a variety of processes, such as development, differentiation, proliferation, apoptosis and metabolism These processes are sometimes disturbed during tumorigenicity, and given that many miRNAs remain low in human cancer, miRNAs appear to play a role in tumorigenicity. New research from Has emerged as a field (Esquela-kerscher, A. and Slack , FJ, Nat Rev Cancer 6 (4):... 259-269, 2006), the expression of miRNA is dramatically changing the process development and cell differentiation, miRNA Profiling has shown reliable results at the developmental line and disease stage (Lu, J. et al ., Nature 435: 834-838, 2005.) and also through previous analyzes of regulatory networks for miRNA function. I could understand how molecules are generated and controlled (Kim, VN Nat Rev Mol Cell Biol 6:..... 376-385, 2005).

miRNA biosynthesis is initiated via transcription by RNA polymerase II (Cai. X., et al., RNA 10: 1957-1966, 2004; Kim, VN Nat . Rev. Mol . Cell . Biol . 6: 376-385, 2005; Lee, Y., et al ., EMBO J 21: 4663-4670, 2002; Lee, Y., et al ., EMBO J 23: 4051-4060, 2004). Primary miRNAs (hereinafter referred to as "pri-miRNAs") are usually over several Kb in length and include a 5 'cap and a poly A tail. pri-miRNA has been described in ribocuclease III, Drosha (Lee, Y., et. al ., Nature 425: 415-419, 2003) and its subelements DGCR8 (Gregory, RI et. al ., Nature 432: 235-240, 2004; Han, J., et al ., Genes Dev . 18: 3016-3027, 2004; Landthaler, M., et al ., Curr . Biol . 14: 2162-2167, 2004), which is first cleaved by a complex called a microprocessor and separated into about 65 nt of hairpin-structure precursor (pre-miRNA). Recombinant DGCR8 or Drosha protein singles do not have cleavage activity in the pri-miRNA process, and cleavage activity when the two proteins combine, indicating that these two proteins act as the most important role of the pre-miRNA process. Gregory, RI et al ., Nature 432: 235-240, 2004; Han, J., et al ., Genes Dev . 18: 3016-3027, 2004). The pre-miRNA process is a central step in miRNA biosynthesis and is defined as the process of generating one end of a molecule containing an mRNA sequence in a long pre-miRNA. The pre-miRNA generated after the initiation process is transported to the cytoplasm by nuclear transport element Exp5 (exportin-5) (Bohnsack, MT, et. al ., RNA 10: 185-191, 2004; Lund, E., et al ., Science 303: 95-98, 2004; Yi, R., et al ., Genes Dev . 17: 3011-3016, 2003). In the cytoplasm, the pre-miRNA delivered by Dicer, a RNase III type protein in the cytoplasm, is cut to generate a miRNA double strand of about 22 nt. One strand of the Dicer product is present in the cytoplasm as a mature miRNA and assembled with an effector complex called a microribonucleoprotein (miRNP) or a miRNA-induced silencing complex (miRISC) (Khvorova, A., et. al ., Cell 115: 209-216, 2003; Schwarz, DS, et al ., Cell 115: 199-208, 2003).

RNAi, a gene silecing mechanism by sRNA, has been used as an effective gene research tool in mammalian systems. Effective and stable gene knockdown occurs by expressing small hairpin RNA (hereinafter referred to as “shRNA”) and processing it into small interfering RNA (siRNA). Recent breakthroughs in RNAi technology have been made by making shRNA expression cassettes that mimic natural miRNA genes (Dickins, RA, et. al ., Nat . Genet . 37: 1289-1295, 2005; Silva, JM et al ., Nat . Genet. 37: 1281-1288, 2005: Zeng, Y., et al ., Mol . Cell 9: 1327-1333. 2002). MiRNA-based shRNAs regulated by RNA polymerase II promoters effectively and stably induce gene silencing regulation in cultured cells such as animal models.

The p53 gene is a tumor suppressor gene, and the activity of the p53 gene in approximately 50-60% of cancer patients is mutated or deleted, indicating that the p53 pathway is inactivated in most human cancers. This shows the critical role of p53. Recent studies have shown that the recovery of p53 expression itself is effective in inducing strong inhibition of cancer in mice. Therefore, expression of the p53 gene in cancer cells with p53 mutation or increased activity of p53 in cancer cells may induce the death of cancer cells.

This means that cancer progression strongly requires p53's loss of function, and the recovery of p53 could be a new and effective approach to cancer therapy.

As a transcription factor, p53 protein regulates many genes that cause anti-proliferative responses such as cell cycle arrest, DNA repair, cell death and cellular senescence. Intracellular levels of p53 are tightly controlled by the ubiquitin-proteasome system, allowing p53 to respond sensitively to a variety of signals. While cell survival signals accelerate p53 disruption, intracellular stressors, such as DNA damage, stimulate specific sets of kinases to stabilize p53 proteins. To date, several protein factors have been known to regulate p53 levels. However, it is unknown whether regulatory RNA is also involved in the process.

Although many studies have been conducted on protein factors that regulate p53, a tumor suppressor, the role of microRNAs in controlling p53 is unknown. In the screening of miRNAs that regulate p53 activity, we found that those belonging to the same family as miR-29 (family members) increased p53 levels by inhibiting Cdc42 and p53α that negatively regulates p53. .

It is an object of the present invention to provide an anticancer agent comprising miRNA as an active ingredient.

In order to achieve the above object, the present invention provides an anticancer agent comprising miRNA that increases the activity of p53, mi85 that inhibits the activity of p85α or Cdc42 as an active ingredient.

The anticancer agent has a therapeutic effect of cancer by inducing cell death in cancer cells whose cell cycle progresses abnormally due to inhibition of p53 activity.

Hereinafter, the present invention will be described in detail.

The inventors screened miRNAs that activate p53 and treated them with cells, followed by Western blot, MTT, and FACS analysis. As a result, miR29 family, miR-29a, miR-29b and miR-29c were selected as candidate miRNAs that activate p53 and induce cell death. The miRNAs induce cell death through p53 activation through p53 protein stabilization.

The inventors have found that the miRNAs do not directly target p53, but activate p53 by inhibiting the activity of p85α and Cdc42, and found a site in the proteins that reacts with the miRNAs. It was also confirmed that knockdown of p85α and Cdc42 actually increased the activity of p53 protein.

The present invention provides an anticancer agent comprising any one miRNA selected from the group consisting of miR-29a, miR-29b and miR-29c as an active ingredient.

The therapeutic agent for a disease of the present invention may be formulated to be suitable for the route to be administered. Examples of routes of administration include parenteral or oral administration, such as intravenous, blood, subcutaneous, inhalation, transdermal (local), mucosal and rectal. Solutions or suspensions used for oral administration such as blood or subcutaneous administration include solvents for injection, saline solutions, fixed oils, aseptic diluents such as polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, such as benzine alcohol or methyl parabens. Bacterial agents; Antioxidants such as ascorbic acid or sodium sulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and extender modifiers such as sodium chloride or dextrose may be included. Suitable carriers for the preparation of suppositories are natural and hardened oils, waxes, fats, semi-liquid polyols and the like. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be sealed with ampoules, disposable syringes, multiple dose vials made of glass or plastic.

Suitable disease therapeutic agents for injection include sterile aqueous solutions or dispersions and sterile powders for the instant preparation of sterile injections or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, CremophorEL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the disease treatment agent must be sterile and must be fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or spray medium containing water, ethanol, polyols (glycerol, propylene glycol and liquid polyreylene glycol, etc.), and suitable mixtures thereof. By using a coating such as for example lecithin, proper fluidity can be maintained by maintaining the required particle size in the case of by-products and by using surfactants. Microbial action can be prevented by various antibacterial and antibacterial agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. It is preferable that sugars, polyhydric alcohols such as mannitol, sorbitol, and isotonic agents such as sodium chloride are included in the disease treatment agent. Prolonged absorption of the injectable composition can be achieved by, for example, incorporating delayed absorption agents such as aluminum monostearate and gelatin in the treatment of disease.

Sterile injectable solutions can be prepared by incorporating the required active ingredient in one or a combination of the ingredients described above in a suitable solvent and, if necessary, by filter sterilization. Generally, dispersions can be prepared by incorporating the active ingredient in a sterile vehicle which may contain a basic dispersion medium and the required other ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, by which methods powders of the desired components can be obtained from the active ingredient and the previously sterilized filtration solution. Examples of suitable carriers for the preparation of injectable solutions include water, alcohols, polyols, glycerin, vegetable oils and the like.

Oral disease therapeutic agents generally include an inert diluent or an edible carrier. For oral therapeutic administration, the active ingredient is incorporated into excipients and used in capsules in the form of tablets, troches, or gelatin. Oral disease therapies can also be prepared using fluid carriers for use as mouthwashes. Examples of suitable carriers for soft gelatin capsules are vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Examples of suitable carriers for the preparation of solutions and syrups are water, polyols, saccharose, invert sugar, glucose and the like.

Pharmaceutically suitable binders and / or adjuvant materials may be included as part of the disease treatment. The pharmaceutical preparations may also contain preservatives, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for controlling osmotic pressure, buffers, coatings or antioxidants. Tablets, pills, capsules, trucks and the like may contain any or a mixture of similar properties of the following components: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel ^™ or corn starch; Lubricants such as magnesium stearate or Sterotes ^™ ; Gildants, such as silicon dioxide in the colloidal state; Sweetening agents such as sucrose or saccharin; Or flavoring agents such as peppermint, methyl salicylate, or orange flavor. Oral or parenteral disease therapeutic agents are advantageously formulated in dosage unit form to facilitate uniformity under dose administration. The miRNAs may be formulated as a medicament in a conventional manner using one or more pharmaceutically acceptable carriers. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used, for example, as carriers for tablets and hard gelatin capsules.

The dosage for the treatment of cancer can vary widely and will, of course, be adjusted to the individual requirements in each particular case.

EXAMPLE

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are intended to specifically illustrate the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Screening of miRNAs to Activate p53

We selected 91 miRNAs known to remain high or low in human cancers. Selected miRNAs were chemically synthesized in the form of double siRNA (siRNA duplex) (Table 1).

miRNA 5 'to 3' sequence nt let-7a guide UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 20) 22 passenger CUAUACAACCUACUACCUCAUU (SEQ ID NO: 21) 22 let-7d guide AGAGGUAGUAGGUUGCAUAGU (SEQ ID NO: 22) 21 passenger UAUGCAACCUACUACCUUUUU (SEQ ID NO: 23) 21 miR-1 guide UGGAAUGUAAAGAAGUAUGUA (SEQ ID NO. 24) 21 passenger CAUACUUCUUUACAUUCCAUU (SEQ ID NO: 25) 21 miR-100 guide AACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 26) 22 passenger CAAGUUCGGAUCUACGGGUUUU (SEQ ID NO: 27) 22 miR-101 guide UACAGUACUGUGAUAACUGAAG (SEQ ID NO 28) 22 passenger UCAGUUAUCACAGUACUGUAUU (SEQ ID NO: 29) 22 miR-106a guide AAAAGUGCUUACAGUGCAGGUAGC (SEQ ID NO: 30) 24 passenger UACCUGCACUGUAAGCACUUUUUU (SEQ ID NO: 31) 24 miR-106b guide UAAAGUGCUGACAGUGCAGAU (SEQ ID NO: 32) 21 passenger CUGCACUGUCAGCACUUUAUG (SEQ ID NO: 33) 21 miR-107 guide AGCAGCAUUGUACAGGGCUAUCA (SEQ ID NO: 34) 23 passenger AUAGCCCUGUACAAUGCUGUUUU (SEQ ID NO 35) 23 miR-124a guide UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 36) 22 passenger GCAUUCACCGCGUGCCUUAAUU (SEQ ID NO: 37) 22 miR-125a guide UCCCUGAGACCCUUUAACCUGUG (SEQ ID NO: 38) 23 passenger CAGGUUAAAGGGUCUCAGGGAUU (SEQ ID NO: 39) 23 miR-125b guide UCCCUGAGACCCUAACUUGUGA (SEQ ID NO: 40) 22 passenger ACAAGUUAGGGUCUCAGGAAUU (SEQ ID NO: 41) 22 miR-128b guide UCACAGUGAACCGGUCUCUUUC (SEQ ID NO: 42) 22 passenger AAGAGACCGGUUCACUGUCAUU (SEQ ID NO: 43) 22 miR-130a guide CAGUGCAAUGUUAAAAGGGCAU (SEQ ID NO: 44) 22 passenger GCCCUUUUAACAUUGCACUGUU (SEQ ID NO 45) 22 miR-133a guide UUGGUCCCCUUCAACCAGCUGU (SEQ ID NO: 46) 22 passenger AGCUGGUUGAAGGGGACCAAUU (SEQ ID NO: 47) 22 miR-135a guide UAUGGCUUUUUAUUCCUAUGUGA (SEQ ID NO 48) 23 passenger ACAUAGGAAUAAAAAGCCAUAUU (SEQ ID NO: 49) 23 miR-138 guide AGCUGGUGUUGUGAAUC (SEQ ID NO: 50) 17 passenger UUCACAACACCAGUUUU (SEQ ID NO: 51) 17 miR-141 guide UAACACUGUCUGGUAAAGAUGG (SEQ ID NO: 52) 22 passenger AUCUUUACCAGACAGUGUUAUU (SEQ ID NO: 53) 22 miR-145 guide GUCCAGUUUUCCCAGGAAUCCCUU (SEQ ID NO: 54) 24 passenger GGGAUUCCUGGGAAAACUGGAUUU (SEQ ID NO: 55) 24 miR-146 guide UGAGAACUGAAUUCCAUGGGUU (SEQ ID NO 56) 22 passenger CCCAUGGAAUUCAGUUCUCAUU (SEQ ID NO: 57) 22 miR-148a guide UCAGUGCACUACAGAACUUUGU (SEQ ID NO: 58) 22 passenger AAAGUUCUGUAGUGCACUCAUU (SEQ ID NO: 59) 22 miR-148b guide UCAGUGCAUCACAGAACUUUGU (SEQ ID NO: 60) 22 passenger AAAGUUCUGUGAUGCACUAAUU (SEQ ID NO: 61) 22 miR-149 guide UCUGGCUCCGUGUCUUCACUCC (SEQ ID NO: 62) 22 passenger AGUGAAGACACGGAGCUAGUUU (SEQ ID NO: 63) 22 miR-151 guide ACUAGACUGAAGCUCCUUGAGG (SEQ ID NO: 64) 22 passenger UCAAGGAGCUUCAGUCUAAUUU (SEQ ID NO: 65) 22 miR-152 guide UCAGUGCAUGACAGAACUUGGG (SEQ ID NO: 66) 22 passenger CAAGUUCUGUCAUGCACUGAUU (SEQ ID NO: 67) 22 miR-153 guide UUGCAUAGUCACAAAAGUGA (SEQ ID NO: 68) 20 passenger ACUUUUGUGACUAUGCAAUU (SEQ ID NO: 69) 20 miR-155 guide UUAAUGCUAAUCGUGAUAGGGG (SEQ ID NO: 70) 22 passenger CCUAUCACGAUUAGCAUUAAUU (SEQ ID NO: 71) 22 miR-15a guide UAGCAGCACAUAAUGGUUUGUG (SEQ ID NO: 72) 22 passenger CAAACCAUUAUGUGCUGCUAUU (SEQ ID NO: 73) 22 miR-16 guide UAGCAGCACGUAAAUAUUGGCG (SEQ ID NO: 74) 22 passenger CCAAUAUUUACGUGCUGCUGUU (SEQ ID NO: 75) 22 miR-17-5p guide CAAAGUGCUUACAGUGCAGGUAGU (SEQ ID NO: 76) 24 passenger UACCUGCACUGUAAGCACUUUAUU (SEQ ID NO: 77) 24 miR-181b guide AACAUUCAUUGCUGUCGGUGGG (SEQ ID NO: 78) 22 passenger CACCGACAGCAAUGAAUGUUUU (SEQ ID NO: 79) 22 miR-182 guide UGGUUCUAGACUUGCCAACUA (SEQ ID NO: 80) 21 passenger GUUGGCAAGUCUAGAACCAUU (SEQ ID NO: 81) 21 miR-183 guide UAUGGCACUGGUAGAAUUCACUG (SEQ ID NO: 82) 23 passenger GUGAAUUCUACCAGUGCCAUAUU (SEQ ID NO: 83) 23 miR-184 guide UGGACGGAGAACUGAUAAGGGU (SEQ ID NO: 84) 22 passenger CCUUAUCAGUUCUCCGUCCAUU (SEQ ID NO: 85) 22 miR-186 guide CAAAGAAUUCUCCUUUUGGGCUU (SEQ ID NO: 86) 23 passenger GCCCAAAAGGAGAAUUCUUUGUU (SEQ ID NO: 87) 23 miR-187 guide UCGUGUCUUGUGUUGCAGCCG (SEQ ID NO: 88) 21 passenger GCUGCAACACAAGACACGAUU (SEQ ID NO: 89) 21 miR-18a guide UAAGGUGCAUCUAGUGCAGAUA (SEQ ID NO: 90) 22 passenger UCUGCACUAGAUGCACCUUAUU (SEQ ID NO: 91) 22 miR-191 guide CAACGGAAUCCCAAAAGCAGCU (SEQ ID NO: 92) 22 passenger CUGCUUUUGGGAUUCCGUUGUU (SEQ ID NO: 93) 22 miR-194 guide UGUAACAGCAACUCCAUGUGGA (SEQ ID NO: 94) 22 passenger CACAUGGAGUUGCUGUUACAUU (SEQ ID NO: 95) 22 miR-196a guide UAGGUAGUUUCAUGUUGUUGG (SEQ ID NO: 96) 21 passenger AACAACAUGAAACUACUUAUU (SEQ ID NO: 97) 21 miR-197 guide UUCACCACCUUCUCCACCCAGC (SEQ ID NO: 98) 22 passenger UGGGUGGAGAAGGUGGUGAAUU (SEQ ID NO: 99) 22 miR-198 guide GGUCCAGAGGGGAGAUAGG (SEQ ID NO: 100) 19 passenger UAUCUCCCCUCUGGAUUUU (SEQ ID NO: 101) 19 miR-199a guide CCCAGUGUUCAGACUACCUGUUC (SEQ ID NO: 102) 23 passenger ACAGGUAGUCUGAACACUGGUUU (SEQ ID NO: 103) 23 miR-19a guide UGUGCAAAUCUAUGCAAAACUGA (SEQ ID NO: 104) 23 passenger AGUUUUGCAUAGAUUUGCACAUU (SEQ ID NO: 105) 23 miR-19b guide UGUGCAAAUCCAUGCAAAACUGA (SEQ ID NO: 106) 23 passenger AGUUUUGCAUGGAUUUGCAUAUU (SEQ ID NO: 107) 23 miR-200b guide UAAUACUGCCUGGUAAUGAUGAC (SEQ ID NO: 108) 23 passenger CAUCAUUACCAGGCAGUAUUAUU (SEQ ID NO: 109) 23 miR-203 guide GUGAAAUGUUUAGGACCACUAG (SEQ ID NO: 110) 22 passenger AGUGGUCCUAAACAUUUCAUUU (SEQ ID NO: 111) 22 miR-205 guide UCCUUCAUUCCACCGGAGUCUG (SEQ ID NO: 112) 22 passenger GACUCCGGUGGAAUGAAGGAUU (SEQ ID NO: 113) 22 miR-206 guide UGGAAUGUAAGGAAGUGUGUGG (SEQ ID NO: 114) 22 passenger ACACACUUCCUUACAUUCUAUU (SEQ ID NO: 115) 22 miR-20a guide UAAAGUGCUUAUAGUGCAGGUAG (SEQ ID NO: 116) 23 passenger ACCUGCACUAUAAGCACUUUAUU (SEQ ID NO: 117) 23 miR-21 guide UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 118) 22 passenger AACAUCAGUCUGAUAAGUUAUU (SEQ ID NO: 119) 22 miR-212 guide UAACAGUCUCCAGUCACGGCC (SEQ ID NO: 120) 21 passenger CCGUGACUGGAGACUGUUAUU (SEQ ID NO: 121) 21 miR-214 guide ACAGCAGGCACAGACAGGCAG (SEQ ID NO: 122) 21 passenger GCCUGUCUGUGCCUGCUGUUU (SEQ ID NO: 123) 21 miR-215 guide AUGACCUAUGAAUUGACAGAC (SEQ ID NO: 124) 21 passenger CUGUCAAUUCAUAGGUCAUUU (SEQ ID NO: 125) 21 miR-218 guide UUGUGCUUGAUCUAACCAUGU (SEQ ID NO: 126) 21 passenger AUGGUUAGAUCAAGCAUAAUU (SEQ ID NO: 127) 21 miR-219 guide UGAUUGUCCAAACGCAAUUCU (SEQ ID NO: 128) 21 passenger AAUUGCGUUUGGACAAUUAUU (SEQ ID NO: 129) 21 miR-221 guide AGCUACAUUGUCUGCUGGGUUUC (SEQ ID NO: 130) 23 passenger AACCCAGCAGACAAUGUAGUUGU (SEQ ID NO: 131) 23 miR-222 guide AGCUACAUCUGGCUACUGGGUCUC (SEQ ID NO: 132) 24 passenger GACCCAGUAGCCAGAUGUAGCUCC (SEQ ID NO: 133) 24 miR-223 guide UGUCAGUUUGUCAAAUACCCC (SEQ ID NO: 134) 21 passenger GGUAUUUGACAAACUGACAUU (SEQ ID NO: 135) 21 miR-23b guide AUCACAUUGCCAGGGAUUACC (SEQ ID NO: 136) 21 passenger UAAUCCCUGGCAAUGUGCUUU (SEQ ID NO: 137) 21 miR-24 guide UGGCUCAGUUCAGCAGGAACAG (SEQ ID NO: 138) 22 passenger GUUCCUGCUGAACUGAGCCAUU (SEQ ID NO: 139) 22 miR-25 guide CAUUGCACUUGUCUCGGUCUGA (SEQ ID NO: 140) 22 passenger AGACCGAGACAAGUGCAAUUCU (SEQ ID NO: 141) 22 miR-26a guide UUCAAGUAAUCCAGGAUAGGC (SEQ ID NO: 142) 21 passenger CUAUCCUGGAUUACUUGAAUU (SEQ ID NO: 143) 21 miR-27a guide UUCACAGUGGCUAAGUUCCGC (SEQ ID NO: 144) 21 passenger GGAACUUAGCCACUGUGAAUU (SEQ ID NO: 145) 21 miR-296 guide AGGGCCCCCCCUCAAUCCUGU (SEQ ID NO: 146) 21 passenger AGGAUUGAGGGGGGGCUUUUU (SEQ ID NO: 147) 21 miR-29a guide UAGCACCAUCUGAAAUCGGUUA (SEQ ID NO: 148) 22 passenger ACCGAUUUCAGAUGGUGCUAUU (SEQ ID NO: 149) 22 miR-29b guide UAGCACCAUUUGAAAUCAGUGUU (SEQ ID NO: 150) 23 passenger CACUGAUUUCAAAUGGUGCUAUU (SEQ ID NO: 151) 23 miR-29c guide UAGCACCAUUUGAAAUCGGUUA (SEQ ID NO: 152) 22 passenger ACCGAUUUCAAAUGGUGCUAUU (SEQ ID NO: 153) 22 miR-301 guide CAGUGCAAUAGUAUUGUCAAAGC (SEQ ID NO: 154) 23 passenger UUUGACAAUACUAUUGCAUUAUU (SEQ ID NO: 155) 23 miR-302b guide UAAGUGCUUCCAUGUUUUAGUAG (SEQ ID NO: 156) 23 passenger ACUAAAACAUGGAAGCACUUAUU (SEQ ID NO: 157) 23 miR-30a guide UGUAAACAUCCUCGACUGGAAG (SEQ ID NO: 158) 22 passenger UCCAGUCGAGGAUGUUUAUAUU (SEQ ID NO: 159) 22 miR-30c guide UGUAAACAUCCUACACUCUCAGC (SEQ ID NO: 160) 23 passenger UGAGAGUGUAGGAUGUUUAUAUU (SEQ ID NO: 161) 23 miR-31 guide GGCAAGAUGCUGGCAUAGCUG (SEQ ID NO: 162) 21 passenger GCUAUGCCAGCAUCUUGUUUU (SEQ ID NO: 163) 21 miR-32 guide UAUUGCACAUUACUAAGUUGC (SEQ ID NO: 164) 21 passenger AACUUAGUAAUGUGCAAUAUU (SEQ ID NO: 165) 21 miR-320 guide AAAAGCUGGGUUGAGAGGGCGAA (SEQ ID NO: 166) 23 passenger CGCCCUCUCAACCCAGCUUUUUU (SEQ ID NO: 167) 23 miR-324-3p guide CCACUGCCCCAGGUGCUGCUGG (SEQ ID NO: 168) 22 passenger AGCAGCACCUGGGGCAGUAGUU (SEQ ID NO: 169) 22 miR-338 guide UCCAGCAUCAGUGAUUUUGUUGA (SEQ ID NO: 170) 23 passenger AACAAAAUCACUGAUGCUGCAUU (SEQ ID NO: 171) 23 miR-339 guide UCCCUGUCCUCCAGGAGCUCA (SEQ ID NO: 172) 21 passenger AGCUCCUGGAGGACAGGAAUU (SEQ ID NO: 173) 21 miR-345 guide UGCUGACUCCUAGUCCAGGGC (SEQ ID NO: 174) 21 passenger CCUGGACUAGGAGUCAGCAUU (SEQ ID NO: 175) 21 miR-34a guide UGGCAGUGUCUUAGCUGGUUGUU (SEQ ID NO: 176) 23 passenger CAACCAGCUAAGACACUGCCAUU (SEQ ID NO: 177) 23 miR-367 guide AAUUGCACUUUAGCAAUGGUGA (SEQ ID NO: 178) 22 passenger ACCAUUGCUAAAGUGCAAUUUU (SEQ ID NO: 179) 22 miR-372 guide AAAGUGCUGCGACAUUUGAGCGU (SEQ ID NO: 180) 23 passenger GCUCAAAUGUCGCAGCACUUUUU (SEQ ID NO: 181) 23 miR-373 guide GAAGUGCUUCGAUUUUGGGGUGU (SEQ ID NO: 182) 23 passenger ACCCCAAAAUCGAAGCACUUUUU (SEQ ID NO: 183) 23 miR-383 guide AGAUCAGAAGGUGAUUGUGGCU (SEQ ID NO: 184) 22 passenger CCACAAUCACCUUCUGAUCUUU (SEQ ID NO: 185) 22 miR-429 guide UAAUACUGUCUGGUAAAACCGU (SEQ ID NO: 186) 22 passenger GGUUUUACCAGACAGUAUUAUU (SEQ ID NO: 187) 22 miR-451 guide AAACCGUUACCAUUACUGAGUUU (SEQ ID NO: 188) 23 passenger ACUCAGUAAUGGUAACGGUUUUU (SEQ ID NO: 189) 23 miR-452 guide UGUUUGCAGAGGAAACUGAGAC (SEQ ID NO: 190) 22 passenger CUCAGUUUCCUCUGCAAACAUU (SEQ ID NO: 191) 22 miR-488 guide CCCAGAUAAUGGCACUCUCAA (SEQ ID NO: 192) 21 passenger GAGAGUGCCAUUAUCUGUGUU (SEQ ID NO: 193) 21 miR-7 guide UGGAAGACUAGUGAUUUUGUUG (SEQ ID NO: 194) 22 passenger ACAAAAUCACUAGUCUUCUAUU (SEQ ID NO: 195) 22 miR-9 guide UCUUUGGUUAUCUAGCUGUAUGA (SEQ ID NO: 196) 23 passenger AUACAGCUAGAUAACCAAAGGUU (SEQ ID NO: 197) 23 miR-92 guide UAUUGCACUUGUCCCGGCCUG (SEQ ID NO: 198) 21 passenger GGCCGGGACAAGUGCAAUAUU (SEQ ID NO: 199) 21 miR-93 guide AAAGUGCUGUUCGUGCAGGUAG (SEQ ID NO: 200) 22 passenger ACCUGCACGAACAGCACUUUCC (SEQ ID NO: 201) 22 miR-95 guide UUCAACGGGUAUUUAUUGAGCA (SEQ ID NO: 202) 22 passenger CUCAAUAAAUACCCGUUGAAUU (SEQ ID NO: 203) 22 miR-99b guide CACCCGUAGAACCGACCUUGCG (SEQ ID NO: 204) 22 passenger CAAGGUCGGUUCUACGGGUCUU (SEQ ID NO: 205) 22

 The miRNA may be synthesized by single-stranded RNA (shRNA, short hairpin RNA) or double stranded RNA (siRNA, small intefering RNA) composed of loops and stems by chemical synthesis, and synthesizes a DNA sequence encoding the miRNA. Recombinant vector prepared by inserting into a virus or plasmid vector for siRNA expression may be provided in the form of a plasmid vector. The expression vector comprises a promoter efficient for siRNA expression, the promoter by RNA polymerase II or RNA polymerase III It is preferred to be recognized, in particular to be recognized by RNA polymerase III, most preferably the U6 promoter or the H1 promoter. Such siRNA expression vectors include pSilencer (Ambion, Inc.), pSiEx (Novagen, Inc.), siXpress (Takara Bio, Inc.) and pBLOCK-iT ™ (Invitrogen, Inc.) and SilenCircle ™ (Allele). Can be used, but is not limited thereto.

miRNAs were co-transfected with HeLa cells along with the reporter plasmid. HeLa cells were cultured in DMEM medium with 10% fetal bovine serum. MCF-7, MDA-MB-231, SW480 and SNU-638 cells were cultured in RPMI1640 medium to which 10% fetal bovine serum was added. Plasmids and RNA oligonucleotides were transformed into mammalian cells using Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol. siRNA oligonucleotides were purchased from Samchully Pharmaceutical.

sip85α 5'-UAUUGAAGCUGUAGGGAAA-3 '(SEQ ID NO: 1);

siCdc42 5'-CUGCAGGGCAAGAGGAUUA-3 '(SEQ ID NO: 2);

sip53 5'-AACUACUUCCUGAAAACAA-3 '(SEQ ID NO: 3);

si luciferase 5 ′ CUUACGCUGAGUACUUCGATT-3 ′ (SEQ ID NO: 4); and

siGFP 5’-UGAAUUAGAUGGCGAUGUU-3 ＇(SEQ ID NO: 5)

Since the reporter plasmid pGL3-luc has 13 p53 binding sites upstream of the luciferase gene, quantitative measurement of p53 activity is possible. 10 miRNAs that most potentiate p53 (miR-186, miR-187, miR-95, miR-191, miR-181b, miR-155, miR-29a, miR-183, miR-125a) in the pG13-luc measurement , miR-302b), and miR-451 and miR-212 were selected as controls, which do not seem to affect p53 activity. The results of the screening show that miRNAs have different effects on p53 activity (FIG. 1A).

Example 2 p53 Activity Promotion and Cell Death Induction of miR-29

Cells treated with the miRNAs were subjected to Western blot (FIG. 1B), MTT measurement (FIG. 1C) and FACS analysis (FIG. 1D). MTT measurements were inoculated HeLa cells into 96-well plates, transformed with each of the miRNAs, and after 3 days, 50 μl of MTT solution (2 mg / ml) was added to each well and 2 hours at 37 ° C. Incubated for And 150 μl of DMSO was added to each well, and was performed by measuring the absorbance at 590 nm using a microplate reader. FACS analysis was performed two days after transformation, cells were fixed with 70% ethanol, treated with RNase (5 μg / ml) and stained with propidium iodide (50 μg / ml), and Cell Quest The program was performed by analyzing the DNA content of the stained cells in FACSCalibur (Becton Dickinson). As shown in FIG. 1A, miR-29a is the only miRNA that showed positive results in all three tests, suggesting the possibility that miR-29a activates p53 and induces cell death.

miR-29a and two analogs, miR-29b and miR-29c (FIG. 2), respectively or in mixtures, increase p53 protein levels in transformed HeLa cells (FIG. 8B). It was confirmed by Annexin V staining that miR-29 miRNAs (miR-29) induce cell death (FIG. 3A). Annexin V staining was performed one day after transformation, HeLa cells were washed with PBS, fixed with 4% paraformaldehyde for 1 hour, cells washed twice and bound buffered with FITC-bound Annexin V. (binding buffer, 10mM Hepes, pH 7.4, 140mM NaCl, 25mM CaCl2) was performed by staining for 2 hours. Stained cells were visualized by fluorescence microscopy (40X).

This also coincides with the number of HeLa cells in culture also reduced with miR-29 treatment (FIG. 3B). However, the mutant miR-29 (miR-29Mut, 4nt replacement, Fig. 2) did not induce p53 (Fig. 8b). Mutations were derived using the Stratagene's Quick-Change Site-Directed Mutagenesis kit.

Primer set for p85α 3'UTR mutation

5'-CAAAGAGGCCTTTAACCATGGGTAGTGTTAATGCTTTCTGAAGC-3 '(forward: SEQ ID NO: 6); and

5'-GCTTCAGAAAGCATTAACACTACCCATGGTTAAAGGCCTCTTTG-3 '(reverse: SEQ ID NO: 7).

The primers used for the upstream target region of Cdc42 3 'UTR (positions 577-599)

5'-CATGTTCCCCATCTGGGTAGCTTAGGAAGGAGTATAG-3 '(forward: SEQ ID NO: 8); and

5'-CTATACTCCTTCCTAAGCTACCCAGATGGGGAACATG-3 '(reverse: SEQ ID NO: 9).

Primers used downstream of the Cdc42 UTR (positions 997-1019)

5'-GAGGAAATACGAGGGGTGGGTAGAGAAGACAGACATCTGTGG-3 '(SEQ ID NO: 10); and

5'-CCACAGATGTCTGTCTTCTCTACCCACCCCTCGTATTTCCTC-3 '(SEQ ID NO: 11).

Cell death induced by miR-29 occurs only in cells expressing wild type p53 (eg HeLa and MCF-7) (FIG. 8C). MDA-MB-231 and SW480 cells that inactivate the p53 mutation in miR-29 treatment did not induce cell death (Fig. 8c), indicating that wild type p53 is required for miR-29-induced cell death. In agreement, miR-29c is much less effective at inducing cell death when MCF-7 cells are reduced in p53 by RNAi (FIG. 8D). Therefore, this means that miR-29s induce cell death through p53 activation.

We measured the half-life of p53 after treatment with cycloheximide, which inhibits protein synthesis (FIG. 8E). The turnover rate of p53 is usually very high in HeLa cells, and when miR-29s are present, p53 is fairly stable. RT-PCR analysis showed that treatment of miR-29s did not affect p53 mRNA levels. It was found that (Fig. 4), which means that p53 accumulates through protein stabilization. When performing RT-PCR, the whole RNA was prepared using Trizol of Invitrogen, treated with DNase I of Takara, and reverse transcription was performed according to the manufacturer's protocol with M-MuLV reverse transcriptase (Fermentas). Since p53 mRNA does not have a miR-29 binding site, miR29 does not seem to target p53 directly.

Example 3 Inhibition of p85α Activity of miR-29

The inventors have found that, using TargetScan (TargetScan, www.targetscan.org), p85α and Cdc42 (Cell division cycle 42) have regions estimated to be one and two target regions in their 3'UTR, respectively. (FIGS. 5A and 5B). p85α protein is a regulatory unit of phosphatidylinositol 3 kinase (PI3K), which plays an important role in balancing cell survival and cell death, and Cdc42 protein is a cell type, cell migration And Rho family of GTPases that regulate cell cycle progression. P85α 3'UTR was amplified using the following primers.

5'-CACACGTTCCTAAGCTGGAGTG-3 '(SEQ ID NO: 12); And

5'-CAGTCCAGAGCAGTGACAGTATGAC-3 '(SEQ ID NO: 13)

The amplification products were transformed downstream of the luciferase gene in the pGL3_CMV vector (modified form of pGL3 of PRomega).

As measured by Western blot, a decrease in p85α protein level was observed in the presence of miR-29s (FIG. 9A). For the performance of Western blot, 2 days after the transformation of miRNA and siRNA, the whole cell lysate was transferred to RIPA buffer (25 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). It was prepared using, 30 ~ 50 ㎍ each protein sample was separated by SDS-PAGE 10 ~ 12.5%. Proteins were transferred to PVDF membrane using Amersham semi-dry transfer system to perform Western blot. At this time, p85α monoclonal antibody was purchased from Cell Signaling, p53, Cdc42 and GAPDH monoclonal antibodies were purchased from SantaCruz.

To confirm whether miR-29 directly targets p85α, a reporter with p85α 3′UTR was generated and tested. For luciferase analysis, HeLa or SNU-638 cells were seeded in 24-well plates one day before transformation. For cotransformation of siRNA, 180 ng of the firefly luciferase reporter plasmid and 20 ng reporter plasmid of renilla luciferase were rapidly transformed into HeLa cells along with duplex siRNA 30 nM.

For cotransformation of 2'-O-Me miRNA inhibitors, 90 ng of firefly luciferase reporter plasmid and 10 ng reporter plasmid of renilla luciferase with SNU-638 with 2'-O-Me miRNA inhibitor 200 nM The cells were transiently transformed transiently.

For measuring p53 activity, 500 ng of pGL3-Luc reporter and 20 mg of Renilla Luciferase reporter plasmid were cotransformed with miRNA 30 nM.

For p85α and Cdc42 overexpression, 200 ng of protein expression plasmid co-transformed with 400 ng of pGL3-Luc and 20 ng of the Renilla luciferase reporter plasmid.

After 48 hours, luciferase activity was measured using Promega's Dual-luciferase assay kit according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity, and all experiments were performed three times.

 When transformed into HeLa cells with miR-29, it can be seen that the luciferase activity is reduced by 20-30% (Fig. 9b. black bars). In contrast, reporters carrying mutations where they were predicted to miR-29 target sites (Fig. 9B. White bars) were not inhibited by miR-29s. In addition, inhibition of endogenous miR-29s with antisense oligonucleotides with 2'-0-methyl modification increased luciferase expression in SNU-638 cells with high levels of miR-29s (FIG. 6.).

The expression level of miRNA was measured by Northern blotting. The entire RNA was dissolved in 15% urea-acrylamide gel and then transferred to Zetaprobe-GT-membrane (Bio-Rad), and oligonucleotides complementary to each miRNA Probes were end-labeled using T4 polynucleotide activase (Takara).

2′-O-Me modified with antisense oligonucleotide was purchased from SAmchully Pharmaceutical and used for specific inhibition of the miR-29 family member.

Anti-miR-29a 5'-UAACCGAUUUCAGAUGGUGCUA-3 '(SEQ ID NO: 14);

Anti-miR-29b 5'-AACACUGAUUUCAAAUGGUGCUA-3 '(SEQ ID NO: 15);

Anti-miR-29c 5'-UAACCGAUUUCAAAUGGUGCUA-3 '(SEQ ID NO: 16); and

Anti-siLuc 5'-AACUUACGCUGAGUACUUCGA-3 '(SEQ ID NO: 17)

Example 4 Inhibition of Cdc42 Activity of miR-29

Another potential target of miR-29s is Cdc42 mRNA with two target regions for miR-29s (FIG. 5B.). Cdc42 3'UTR primers

5'-CTCTCCAGAGCCCTTTCTGC-3 '(SEQ ID NO: 18); and

5'-CAAAGAATTGAGACATGAGAAAGC-3 '(SEQ ID NO: 19).

The amplification products were transformed downstream of the luciferase gene in the pGL3_CMV vector (modified form of pGL3 of PRomega). As measured by Western blot, Cdc42 protein levels in HeLa cells transformed with miR-29s were reduced. It was confirmed that the decrease (Fig. 9c.). In addition, when cells are transformed with miR-29s, luciferase reporters with 3'UTR of Cdc42 mRNA are inhibited by 40% (Fig. 9d, black bars), but reporters with mutated target regions It was not inhibited by miR-29s (FIG. 9d. white bars). This means that the two regions mediate Cdc42 regulation of miR-29. 2′-O-methyl inhibitors against miR-29s increased the expression of reporter in SNU-638 cells (FIG. 6).

<Example 5> Confirmation of p53 negative regulation of Cdc42 and p85α using knockdown of Cdc42 and p85α

To test whether p85α and Cdc42 are required for miR-29-mediated p53 activation, we first constructed RNAi for these genes and measured p53 activity. Knockdown of p85α and Cdc42 increased p53 protein activity in the pGL3-luc reporter assay (FIG. 7A) and increased p53 protein levels (FIG. 7B). In this context, FACS analysis showed that knockdown of Cdc42 and p85α increased the number of apoptotic cells (FIG. 7C). These data indicate that Cdc42 and p85α negatively regulate p53.

Example 6 Confirmation whether Cdc42 and p85α Inhibition by miR-29 Promotes the Activity of p53

If the inhibition of Cdc42 and p85α by miR-29s is really critical for the induction of p53, overexpression of these proteins should restore the effects of miR-29 on p53. To test this, plasmids expressing Cdc42 or p85α were transformed into HeLa cells with miR-29 mix. Overexpression of Cdc42, p85α, or both reverses p53 induction (FIG. 9E, white bar), which occurs only in the wild type miR-29 and expression of Cdc42 or p85α in the presence of the miR-29 mutation. It was confirmed that little effect on the activity (Fig. 9e, gray bar).

We tested whether overexpression of Cdc42 and p85α could protect cells from miR-29 induced cell death. FACS analysis shows that when Cdc42 and p85α are expressed with miR-29s, the number of cell death cells is indeed reduced (Fig. 9f), which miR-29 activates p53 through inhibition of Cdc42 and p85α. Means that.

The methods described herein will be useful for the treatment and alleviation of various cancers, including colorectal cancer, cervical cancer, breast cancer. In addition, the data reported herein provide clues to the comprehensive understanding of cancer, to facilitate the development of new diagnostic strategies, and to identify molecular targets for making therapeutics and preventive substances. This information will help to better understand tumorigenesis and will be an indicator in developing new strategies for the diagnosis, treatment and ultimately prevention of cancer. Although the present invention has been described in detail and with reference to specific embodiments, the description is intended to be exemplary or explanatory, and is intended to illustrate the invention and its suitable examples. Through a formal description, those skilled in the art will readily realize that various changes and modifications can be made without departing from the scope and spirit of the invention. Accordingly, the present invention is not intended to be limited to the above description, but is intended to define the scope of the present invention in the following claims and equivalent ranges.

1 shows functional screening of miRNAs that regulate p53.

(a) shows the distinct effects of miRNA transformation on p53 activity.

Using the luciferase reporter gene (pGL3-Luc) comprising 13 p53 binding regions, p53 activity was measured after 91 miRNAs were transformed into HeLa cells. Synthetic miRNA (30 nM), pGL3-Luc (0.5 μg) and Renilla luciferase plasmid (0.02 μg) were co-transformed into HeLa cells in 24 well plates and luciferase activity measured 24 hours after transformation It was. Reporter activity values were normalized to the group mean and listed in decreasing order (n = 2).

(b) Western blotting for measurement of p53 protein levels 2 days after MiRNA transformation in HeLa cells

(c) MTT assay (n = 3, mean ± SD) for viability measurement after 3 days of miRNA-transformed HeLa cells

(d) FACS analysis of HeLa cells transformed with miRNAs. As shown in Fig. 1C (bottom right, n = 3, mean ± SD), the number of dead cells is quantified.

Figure 2 shows the results of analyzing the sequence and genome of miR-29 family members.

3 shows induction of cell death by miR-29;

(a) miR-29 induces cell death in HeLa cells. Two days after miRNA transformation, HeLa cells were fixed with 4% paraformaldehyde and then stained with Annexin V-FITC. Cells were visualized with fluorescence microscopy (400 ×).

(b) miR-29 inhibits HeLa cell growth. Cell proliferation rates are estimated daily using a hematocytometer.

(c) miR-29 increases cell death in HdLA cells and MCF-7 cells, but not in MDA-MB-231 and SW480 cells. The sub diploid G1 peak from FACS analysis is used to estimate the number of apoptosis cells.

4 shows that miR-29 does not affect p53 mRNA levels.

RT-PCR of p53 mRNA of HeLa cells transformed with miR-29 complex or miR-29 mutant, and the product of cells transformed with lipofectamine (Mock) is amplified as a control.

FIG. 5 shows the sequence of miRNA target regions of p85α and Cdc42 3′UTRs.

From 326 nucleotides of 3′UTR of p85α to 348 nucleotides are 5′- AGAGGCCUUUAACCAUGGUGCUU-3 ′ and from 326 nucleotides of 326 nucleotides of 3′UTR of mutant p85α are 5′-AGAGGCCUUUAACCAUGGGUAGU-3 ′. 577 nucleotides from 577 nucleotides of 3′UTR of Cdc42 are 5′-AACAUGUUCCCCAUCUGGUGCUC-3 ′ and 577 nucleotides to 599 nucleotides of 3′UTR of mutant Cdc42 are 5′-AACAUGUUCCCCAUCUGGGUAGC-3 ′. 997 nucleotides of 3'UTR of Cdc42 to 1019 nucleotides are 5'-AGGAAAUACGAGGGGUGGUGCUA-3 'and 997 nucleotides to 1019 nucleotides of 3'UTR of mutant Cdc42 are 5'-AGGAAAUACGAGGGGUGGGUAGA-3'.

FIG. 6 shows that miR-29 targets p85α and Cdc42. FIG.

(a) Northern blotting comparing the expression levels of miR-29a, miR-29b and miR-29c in HeLa cells and SNU-638 cells to analyze synthetic miR-29 family members (0.05 ng) for comparison. It was. HeLa cells express much lower levels of miR-29 compared to SNU-638 cells.

(b) Antagonism of miR-29 increases p85α expression. p85α 3'UTR plasmids were transformed into SNU-638 cells with miRNA inhibitors specific for miR-29 family members. Since the cells do not express siLuc, antisense oligo of siLuc (anti-siLuc) was used as a control (m = 3, mean ± SD).

(c) Antagonism of miR-29 increases Cdc42 expression. The same test as in (b) was conducted except for Cdc42 3'UTR.

(d) RT-PCR to measure the levels of p85α and Cdc42 mRNA after transducing miRNA to HeLa cells.

FIG. 7 shows that inhibition of Cdc42 and p85α activates p53.

(a) Knockdown of Cdc42 and p85α activates p53. p53 activity was measured using pGL3-Luc reporter plasmid in HeLa cells transformed with miRNA or siRNA.

(b) Knockdown of Cdc42 and p85α increases p53 protein levels. Two days after transfection of HeLa cells, p53 protein levels were analyzed using Western blot.

(c) Knockdown of Cdc42 and p85α induces cell death. Cell death was estimated as shown in Figure 1c.

8 shows that miR-29 family members positively relgulate p53.

(a) A summary of the screening of miRNAs that regulate p53. increased the p53 protein level (> 2 fold) and reduced cell viability (30%) compared to the negative controls (miR-451 and miR-212) of the 10 miRNAs with the highest activity in the pGL3-luc reporter assay ), The ability to induce cell death (> 2 fold) was tested.

(b) miR-29 and its members increase p53 protein levels. HeLa cells transformed with each miRNA were analyzed by Western blot.

(c) miR-29 increased cell death in HeLa and MCF-7 cells, but not in MDA-MB-231 and SW480 cells. The sub diploid G1 peak from the FACS analysis is used to estimate the number of apoptosis cells (n = 3, mean ± 3), see FIG. 4C for a representative plot.

(d) Knockdown of p53 inhibits cell death in MCF-7 cells transformed with miR-29c. The number of cell deaths is estimated as in (c).

(e) MiR-29 enhances the stability of p53 protein. Unlike cells treated with miR-29Mut (left panel, right), HeLa cells transformed with miR-29 mix and treated with 50 μg / ml cyclohexide (CHX) for the indicated time (Outer panel, left) maintain high p53 levels. Note that the blot representing the miR-29 Mut treated cells was exposed longer to zero point equilibrium than the cells treated with miR-29mix. Half of the protein samples at time 0 were loaded for comparison (0.5 ×). The intensity of the p53 protein band is quantified and normalized to GAPDH intensity (right panel, n = 3, mean ± SD).

9 shows that miR-29 targets p85α and Cdc42 directly. .

(a) Western blot showed that miR-29 mix reduced p85α protein levels in HeLa cells.

(b) miR-29α targets p85α 3′UTR. HeLa cells are transformed with a reporter prepared by fusing the luciferase genes downstream of wild type or mutant p85α of 3′UTR. Concurrent transformation of miR-29 reduces the reporter activity of wild type 3′UTR, but miR-29Mut does not. The results were unrelated siRNA; Normalized to siGFP (n = 3, mean ± SD).

(c) Western blot shows that miR-29 reduces CDC42 protein levels in HeLa cells.

(d) miR-29 targets Cdc 42 3'UTR. Reporter analysis in (b) was used except for the Cdc42 3'UTR.

(e) pGL3-luc reporter assay (n = 3, mean ± SD), overexpression of CDC42 and p85α in HeLa cells inhibited the increased p53 activity induced by miR-29.

(f) Overexpression of CDC42 and p85α in HeLa cells offsets cell death induced by miR-29. Dead cells are quantified as shown in FIG. 8C.

<110> seoul <120> microRNA <130> 8p-07-06 <160> 205 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> RNA <213> Homo sapiens <400> 1 uauugaagcu guagggaaa 19 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <400> 2 cugcagggca agaggauua 19 <210> 3 <211> 19 <212> RNA <213> Homo sapiens <400> 3 aacuacuucc ugaaaacaa 19 <210> 4 <211> 21 <212> RNA <213> Homo sapiens <220> <221> variation (222) (20) .. (21) N is thymine. <400> 4 cuuacgcuga guacuucgan n 21 <210> 5 <211> 19 <212> RNA <213> Homo sapiens <400> 5 ugaauuagau ggcgauguu 19 <210> 6 <211> 44 <212> DNA <213> Homo sapiens <400> 6 caaagaggcc tttaaccatg ggtagtgtta atgctttctg aagc 44 <210> 7 <211> 44 <212> DNA <213> Homo sapiens <400> 7 gcttcagaaa gcattaacac tacccatggt taaaggcctc tttg 44 <210> 8 <211> 37 <212> DNA <213> Homo sapiens <400> 8 catgttcccc atctgggtag cttaggaagg agtatag 37 <210> 9 <211> 37 <212> DNA <213> Homo sapiens <400> 9 ctatactcct tcctaagcta cccagatggg gaacatg 37 <210> 10 <211> 42 <212> DNA <213> Homo sapiens <400> 10 gaggaaatac gaggggtggg tagagaagac agacatctgt gg 42 <210> 11 <211> 42 <212> DNA <213> Homo sapiens <400> 11 ccacagatgt ctgtcttctc tacccacccc tcgtatttcc tc 42 <210> 12 <211> 22 <212> DNA <213> Homo sapiens <400> 12 cacacgttcc taagctggag tg 22 <210> 13 <211> 25 <212> DNA <213> Homo sapiens <400> 13 cagtccagag cagtgacagt atgac 25 <210> 14 <211> 22 <212> RNA <213> Homo sapiens <400> 14 uaaccgauuu cagauggugc ua 22 <210> 15 <211> 23 <212> RNA <213> Homo sapiens <400> 15 aacacugauu ucaaauggug cua 23 <210> 16 <211> 22 <212> RNA <213> Homo sapiens <400> 16 uaaccgauuu caaauggugc ua 22 <210> 17 <211> 21 <212> RNA <213> Homo sapiens <400> 17 aacuuacgcu gaguacuucg a 21 <210> 18 <211> 20 <212> DNA <213> Homo sapiens <400> 18 ctctccagag ccctttctgc 20 <210> 19 <211> 24 <212> DNA <213> Homo sapiens <400> 19 caaagaattg agacatgaga aagc 24 <210> 20 <211> 22 <212> RNA <213> Homo sapiens <400> 20 ugagguagua gguuguauag uu 22 <210> 21 <211> 22 <212> RNA <213> Homo sapiens <400> 21 ugagguagua gguuguauag uu 22 <210> 22 <211> 22 <212> RNA <213> Homo sapiens <400> 22 cuauacaacc uacuaccuca uu 22 <210> 23 <211> 21 <212> RNA <213> Homo sapiens <400> 23 uaugcaaccu acuaccuuuu u 21 <210> 24 <211> 21 <212> RNA <213> Homo sapiens <400> 24 uggaauguaa agaaguaugu a 21 <210> 25 <211> 21 <212> RNA <213> Homo sapiens <400> 25 cauacuucuu uacauuccau u 21 <210> 26 <211> 22 <212> RNA <213> Homo sapiens <400> 26 aacccguaga uccgaacuug ug 22 <210> 27 <211> 22 <212> RNA <213> Homo sapiens <400> 27 caaguucgga ucuacggguu uu 22 <210> 28 <211> 22 <212> RNA <213> Homo sapiens <400> 28 uacaguacug ugauaacuga ag 22 <210> 29 <211> 22 <212> RNA <213> Homo sapiens <400> 29 ucaguuauca caguacugua uu 22 <210> 30 <211> 24 <212> RNA <213> Homo sapiens <400> 30 aaaagugcuu acagugcagg uagc 24 <210> 31 <211> 24 <212> RNA <213> Homo sapiens <400> 31 uaccugcacu guaagcacuu uuuu 24 <210> 32 <211> 21 <212> RNA <213> Homo sapiens <400> 32 uaaagugcug acagugcaga u 21 <210> 33 <211> 21 <212> RNA <213> Homo sapiens <400> 33 cugcacuguc agcacuuuau g 21 <210> 34 <211> 23 <212> RNA <213> Homo sapiens <400> 34 agcagcauug uacagggcua uca 23 <210> 35 <211> 23 <212> RNA <213> Homo sapiens <400> 35 auagcccugu acaaugcugu uuu 23 <210> 36 <211> 22 <212> RNA <213> Homo sapiens <400> 36 uuaaggcacg cggugaaugc ca 22 <210> 37 <211> 22 <212> RNA <213> Homo sapiens <400> 37 gcauucaccg cgugccuuaa uu 22 <210> 38 <211> 23 <212> RNA <213> Homo sapiens <400> 38 ucccugagac ccuuuaaccu gug 23 <210> 39 <211> 23 <212> RNA <213> Homo sapiens <400> 39 cagguuaaag ggucucaggg auu 23 <210> 40 <211> 22 <212> RNA <213> Homo sapiens <400> 40 ucccugagac ccuaacuugu ga 22 <210> 41 <211> 22 <212> RNA <213> Homo sapiens <400> 41 acaaguuagg gucucaggaa uu 22 <210> 42 <211> 22 <212> RNA <213> Homo sapiens <400> 42 ucacagugaa ccggucucuu uc 22 <210> 43 <211> 22 <212> RNA <213> Homo sapiens <400> 43 aagagaccgg uucacuguca uu 22 <210> 44 <211> 22 <212> RNA <213> Homo sapiens <400> 44 cagugcaaug uuaaaagggc au 22 <210> 45 <211> 22 <212> RNA <213> Homo sapiens <400> 45 gcccuuuuaa cauugcacug uu 22 <210> 46 <211> 22 <212> RNA <213> Homo sapiens <400> 46 uugguccccu ucaaccagcu gu 22 <210> 47 <211> 22 <212> RNA <213> Homo sapiens <400> 47 agcugguuga aggggaccaa uu 22 <210> 48 <211> 23 <212> RNA <213> Homo sapiens <400> 48 uauggcuuuu uauuccuaug uga 23 <210> 49 <211> 23 <212> RNA <213> Homo sapiens <400> 49 acauaggaau aaaaagccau auu 23 <210> 50 <211> 17 <212> RNA <213> Homo sapiens <400> 50 agcugguguu gugaauc 17 <210> 51 <211> 17 <212> RNA <213> Homo sapiens <400> 51 uucacaacac caguuuu 17 <210> 52 <211> 22 <212> RNA <213> Homo sapiens <400> 52 uaacacuguc ugguaaagau gg 22 <210> 53 <211> 22 <212> RNA <213> Homo sapiens <400> 53 aucuuuacca gacaguguua uu 22 <210> 54 <211> 24 <212> RNA <213> Homo sapiens <400> 54 guccaguuuu cccaggaauc ccuu 24 <210> 55 <211> 24 <212> RNA <213> Homo sapiens <400> 55 gggauuccug ggaaaacugg auuu 24 <210> 56 <211> 22 <212> RNA <213> Homo sapiens <400> 56 ugagaacuga auuccauggg uu 22 <210> 57 <211> 22 <212> RNA <213> Homo sapiens <400> 57 cccauggaau ucaguucuca uu 22 <210> 58 <211> 22 <212> RNA <213> Homo sapiens <400> 58 ucagugcacu acagaacuuu gu 22 <210> 59 <211> 22 <212> RNA <213> Homo sapiens <400> 59 aaaguucugu agugcacuca uu 22 <210> 60 <211> 22 <212> RNA <213> Homo sapiens <400> 60 ucagugcauc acagaacuuu gu 22 <210> 61 <211> 22 <212> RNA <213> Homo sapiens <400> 61 aaaguucugu gaugcacuaa uu 22 <210> 62 <211> 22 <212> RNA <213> Homo sapiens <400> 62 ucuggcuccg ugucuucacu cc 22 <210> 63 <211> 22 <212> RNA <213> Homo sapiens <400> 63 agugaagaca cggagcuagu uu 22 <210> 64 <211> 22 <212> RNA <213> Homo sapiens <400> 64 acuagacuga agcuccuuga gg 22 <210> 65 <211> 22 <212> RNA <213> Homo sapiens <400> 65 ucaaggagcu ucagucuaau uu 22 <210> 66 <211> 22 <212> RNA <213> Homo sapiens <400> 66 ucagugcaug acagaacuug gg 22 <210> 67 <211> 22 <212> RNA <213> Homo sapiens <400> 67 caaguucugu caugcacuga uu 22 <210> 68 <211> 20 <212> RNA <213> Homo sapiens <400> 68 uugcauaguc acaaaaguga 20 <210> 69 <211> 20 <212> RNA <213> Homo sapiens <400> 69 acuuuuguga cuaugcaauu 20 <210> 70 <211> 22 <212> RNA <213> Homo sapiens <400> 70 uuaaugcuaa ucgugauagg gg 22 <210> 71 <211> 22 <212> RNA <213> Homo sapiens <400> 71 ccuaucacga uuagcauuaa uu 22 <210> 72 <211> 22 <212> RNA <213> Homo sapiens <400> 72 uagcagcaca uaaugguuug ug 22 <210> 73 <211> 22 <212> RNA <213> Homo sapiens <400> 73 caaaccauua ugugcugcua uu 22 <210> 74 <211> 22 <212> RNA <213> Homo sapiens <400> 74 uagcagcacg uaaauauugg cg 22 <210> 75 <211> 22 <212> RNA <213> Homo sapiens <400> 75 ccaauauuua cgugcugcug uu 22 <210> 76 <211> 24 <212> RNA <213> Homo sapiens <400> 76 caaagugcuu acagugcagg uagu 24 <210> 77 <211> 24 <212> RNA <213> Homo sapiens <400> 77 uaccugcacu guaagcacuu uauu 24 <210> 78 <211> 22 <212> RNA <213> Homo sapiens <400> 78 aacauucauu gcugucggug gg 22 <210> 79 <211> 22 <212> RNA <213> Homo sapiens <400> 79 caccgacagc aaugaauguu uu 22 <210> 80 <211> 21 <212> RNA <213> Homo sapiens <400> 80 ugguucuaga cuugccaacu a 21 <210> 81 <211> 21 <212> RNA <213> Homo sapiens <400> 81 guuggcaagu cuagaaccau u 21 <210> 82 <211> 23 <212> RNA <213> Homo sapiens <400> 82 uauggcacug guagaauuca cug 23 <210> 83 <211> 23 <212> RNA <213> Homo sapiens <400> 83 gugaauucua ccagugccau auu 23 <210> 84 <211> 22 <212> RNA <213> Homo sapiens <400> 84 uggacggaga acugauaagg gu 22 <210> 85 <211> 22 <212> RNA <213> Homo sapiens <400> 85 ccuuaucagu ucuccgucca uu 22 <210> 86 <211> 23 <212> RNA <213> Homo sapiens <400> 86 caaagaauuc uccuuuuggg cuu 23 <210> 87 <211> 23 <212> RNA <213> Homo sapiens <400> 87 gcccaaaagg agaauucuuu guu 23 <210> 88 <211> 21 <212> RNA <213> Homo sapiens <400> 88 ucgugucuug uguugcagcc g 21 <210> 89 <211> 21 <212> RNA <213> Homo sapiens <400> 89 gcugcaacac aagacacgau u 21 <210> 90 <211> 22 <212> RNA <213> Homo sapiens <400> 90 uaaggugcau cuagugcaga ua 22 <210> 91 <211> 22 <212> RNA <213> Homo sapiens <400> 91 ucugcacuag augcaccuua uu 22 <210> 92 <211> 22 <212> RNA <213> Homo sapiens <400> 92 caacggaauc ccaaaagcag cu 22 <210> 93 <211> 22 <212> RNA <213> Homo sapiens <400> 93 cugcuuuugg gauuccguug uu 22 <210> 94 <211> 22 <212> RNA <213> Homo sapiens <400> 94 uguaacagca acuccaugug ga 22 <210> 95 <211> 22 <212> RNA <213> Homo sapiens <400> 95 cacauggagu ugcuguuaca uu 22 <210> 96 <211> 21 <212> RNA <213> Homo sapiens <400> 96 uagguaguuu cauguuguug g 21 <210> 97 <211> 21 <212> RNA <213> Homo sapiens <400> 97 aacaacauga aacuacuuau u 21 <210> 98 <211> 22 <212> RNA <213> Homo sapiens <400> 98 uucaccaccu ucuccaccca gc 22 <210> 99 <211> 22 <212> RNA <213> Homo sapiens <400> 99 uggguggaga agguggugaa uu 22 <210> 100 <211> 19 <212> RNA <213> Homo sapiens <400> 100 gguccagagg ggagauagg 19 <210> 101 <211> 19 <212> RNA <213> Homo sapiens <400> 101 uaucuccccu cuggauuuu 19 <210> 102 <211> 23 <212> RNA <213> Homo sapiens <400> 102 cccaguguuc agacuaccug uuc 23 <210> 103 <211> 23 <212> RNA <213> Homo sapiens <400> 103 acagguaguc ugaacacugg uuu 23 <210> 104 <211> 23 <212> RNA <213> Homo sapiens <400> 104 ugugcaaauc uaugcaaaac uga 23 <210> 105 <211> 23 <212> RNA <213> Homo sapiens <400> 105 aguuuugcau agauuugcac auu 23 <210> 106 <211> 23 <212> RNA <213> Homo sapiens <400> 106 ugugcaaauc caugcaaaac uga 23 <210> 107 <211> 23 <212> RNA <213> Homo sapiens <400> 107 aguuuugcau ggauuugcau auu 23 <210> 108 <211> 23 <212> RNA <213> Homo sapiens <400> 108 uaauacugcc ugguaaugau gac 23 <210> 109 <211> 23 <212> RNA <213> Homo sapiens <400> 109 caucauuacc aggcaguauu auu 23 <210> 110 <211> 22 <212> RNA <213> Homo sapiens <400> 110 gugaaauguu uaggaccacu ag 22 <210> 111 <211> 22 <212> RNA <213> Homo sapiens <400> 111 agugguccua aacauuucau uu 22 <210> 112 <211> 22 <212> RNA <213> Homo sapiens <400> 112 uccuucauuc caccggaguc ug 22 <210> 113 <211> 22 <212> RNA <213> Homo sapiens <400> 113 gacuccggug gaaugaagga uu 22 <210> 114 <211> 22 <212> RNA <213> Homo sapiens <400> 114 uggaauguaa ggaagugugu gg 22 <210> 115 <211> 22 <212> RNA <213> Homo sapiens <400> 115 acacacuucc uuacauucua uu 22 <210> 116 <211> 23 <212> RNA <213> Homo sapiens <400> 116 uaaagugcuu auagugcagg uag 23 <210> 117 <211> 23 <212> RNA <213> Homo sapiens <400> 117 accugcacua uaagcacuuu auu 23 <210> 118 <211> 22 <212> RNA <213> Homo sapiens <400> 118 uagcuuauca gacugauguu ga 22 <210> 119 <211> 22 <212> RNA <213> Homo sapiens <400> 119 aacaucaguc ugauaaguua uu 22 <210> 120 <211> 21 <212> RNA <213> Homo sapiens <400> 120 uaacagucuc cagucacggc c 21 <210> 121 <211> 21 <212> RNA <213> Homo sapiens <400> 121 ccgugacugg agacuguuau u 21 <210> 122 <211> 21 <212> RNA <213> Homo sapiens <400> 122 acagcaggca cagacaggca g 21 <210> 123 <211> 21 <212> RNA <213> Homo sapiens <400> 123 gccugucugu gccugcuguu u 21 <210> 124 <211> 21 <212> RNA <213> Homo sapiens <400> 124 augaccuaug aauugacaga c 21 <210> 125 <211> 21 <212> RNA <213> Homo sapiens <400> 125 cugucaauuc auaggucauu u 21 <210> 126 <211> 21 <212> RNA <213> Homo sapiens <400> 126 uugugcuuga ucuaaccaug u 21 <210> 127 <211> 21 <212> RNA <213> Homo sapiens <400> 127 augguuagau caagcauaau u 21 <210> 128 <211> 21 <212> RNA <213> Homo sapiens <400> 128 ugauugucca aacgcaauuc u 21 <210> 129 <211> 21 <212> RNA <213> Homo sapiens <400> 129 aauugcguuu ggacaauuau u 21 <210> 130 <211> 23 <212> RNA <213> Homo sapiens <400> 130 agcuacauug ucugcugggu uuc 23 <210> 131 <211> 23 <212> RNA <213> Homo sapiens <400> 131 aacccagcag acaauguagu ugu 23 <210> 132 <211> 24 <212> RNA <213> Homo sapiens <400> 132 agcuacaucu ggcuacuggg ucuc 24 <210> 133 <211> 24 <212> RNA <213> Homo sapiens <133> 133 gacccaguag ccagauguag cucc 24 <210> 134 <211> 21 <212> RNA <213> Homo sapiens <400> 134 ugucaguuug ucaaauaccc c 21 <210> 135 <211> 21 <212> RNA <213> Homo sapiens <400> 135 gguauuugac aaacugacau u 21 <210> 136 <211> 21 <212> RNA <213> Homo sapiens <400> 136 aucacauugc cagggauuac c 21 <210> 137 <211> 21 <212> RNA <213> Homo sapiens <400> 137 uaaucccugg caaugugcuu u 21 <210> 138 <211> 22 <212> RNA <213> Homo sapiens <400> 138 uggcucaguu cagcaggaac ag 22 <139> <211> 22 <212> RNA <213> Homo sapiens <400> 139 guuccugcug aacugagcca uu 22 <210> 140 <211> 22 <212> RNA <213> Homo sapiens <400> 140 cauugcacuu gucucggucu ga 22 <210> 141 <211> 22 <212> RNA <213> Homo sapiens <400> 141 agaccgagac aagugcaauu cu 22 <210> 142 <211> 21 <212> RNA <213> Homo sapiens <400> 142 uucaaguaau ccaggauagg c 21 <210> 143 <211> 21 <212> RNA <213> Homo sapiens <400> 143 cuauccugga uuacuugaau u 21 <210> 144 <211> 21 <212> RNA <213> Homo sapiens <400> 144 uucacagugg cuaaguuccg c 21 <210> 145 <211> 21 <212> RNA <213> Homo sapiens <400> 145 ggaacuuagc cacugugaau u 21 <210> 146 <211> 21 <212> RNA <213> Homo sapiens <400> 146 agggcccccc cucaauccug u 21 <210> 147 <211> 21 <212> RNA <213> Homo sapiens <400> 147 aggauugagg gggggcuuuu u 21 <210> 148 <211> 22 <212> RNA <213> Homo sapiens <400> 148 uagcaccauc ugaaaucggu ua 22 <210> 149 <211> 22 <212> RNA <213> Homo sapiens <400> 149 accgauuuca gauggugcua uu 22 <210> 150 <211> 23 <212> RNA <213> Homo sapiens <400> 150 uagcaccauu ugaaaucagu guu 23 <210> 151 <211> 23 <212> RNA <213> Homo sapiens <400> 151 cacugauuuc aaauggugcu auu 23 <210> 152 <211> 22 <212> RNA <213> Homo sapiens <400> 152 uagcaccauu ugaaaucggu ua 22 <210> 153 <211> 22 <212> RNA <213> Homo sapiens <400> 153 accgauuuca aauggugcua uu 22 <210> 154 <211> 23 <212> RNA <213> Homo sapiens <400> 154 cagugcaaua guauugucaa agc 23 <210> 155 <211> 23 <212> RNA <213> Homo sapiens <400> 155 uuugacaaua cuauugcauu auu 23 <210> 156 <211> 23 <212> RNA <213> Homo sapiens <400> 156 uaagugcuuc cauguuuuag uag 23 <210> 157 <211> 23 <212> RNA <213> Homo sapiens <400> 157 acuaaaacau ggaagcacuu auu 23 <210> 158 <211> 22 <212> RNA <213> Homo sapiens <400> 158 uguaaacauc cucgacugga ag 22 <210> 159 <211> 22 <212> RNA <213> Homo sapiens <400> 159 uccagucgag gauguuuaua uu 22 <210> 160 <211> 23 <212> RNA <213> Homo sapiens <400> 160 uguaaacauc cuacacucuc agc 23 <210> 161 <211> 23 <212> RNA <213> Homo sapiens <400> 161 ugagagugua ggauguuuau auu 23 <210> 162 <211> 21 <212> RNA <213> Homo sapiens <400> 162 ggcaagaugc uggcauagcu g 21 <210> 163 <211> 21 <212> RNA <213> Homo sapiens <400> 163 gcuaugccag caucuuguuu u 21 <210> 164 <211> 21 <212> RNA <213> Homo sapiens <400> 164 uauugcacau uacuaaguug c 21 <210> 165 <211> 21 <212> RNA <213> Homo sapiens <400> 165 aacuuaguaa ugugcaauau u 21 <210> 166 <211> 23 <212> RNA <213> Homo sapiens <400> 166 aaaagcuggg uugagagggc gaa 23 <210> 167 <211> 23 <212> RNA <213> Homo sapiens <400> 167 cgcccucuca acccagcuuu uuu 23 <210> 168 <211> 22 <212> RNA <213> Homo sapiens <400> 168 ccacugcccc aggugcugcu gg 22 <210> 169 <211> 22 <212> RNA <213> Homo sapiens <400> 169 agcagcaccu ggggcaguag uu 22 <210> 170 <211> 23 <212> RNA <213> Homo sapiens <400> 170 uccagcauca gugauuuugu uga 23 <210> 171 <211> 23 <212> RNA <213> Homo sapiens <400> 171 aacaaaauca cugaugcugc auu 23 <210> 172 <211> 21 <212> RNA <213> Homo sapiens <400> 172 ucccuguccu ccaggagcuc a 21 <210> 173 <211> 21 <212> RNA <213> Homo sapiens <400> 173 agcuccugga ggacaggaau u 21 <210> 174 <211> 21 <212> RNA <213> Homo sapiens <400> 174 ugcugacucc uaguccaggg c 21 <175> 175 <211> 21 <212> RNA <213> Homo sapiens <400> 175 ccuggacuag gagucagcau u 21 <210> 176 <211> 23 <212> RNA <213> Homo sapiens <400> 176 uggcaguguc uuagcugguu guu 23 <210> 177 <211> 23 <212> RNA <213> Homo sapiens <400> 177 caaccagcua agacacugcc auu 23 <210> 178 <211> 22 <212> RNA <213> Homo sapiens <400> 178 aauugcacuu uagcaauggu ga 22 <210> 179 <211> 22 <212> RNA <213> Homo sapiens <400> 179 accauugcua aagugcaauu uu 22 <210> 180 <211> 23 <212> RNA <213> Homo sapiens <400> 180 aaagugcugc gacauuugag cgu 23 <210> 181 <211> 23 <212> RNA <213> Homo sapiens <400> 181 gcucaaaugu cgcagcacuu uuu 23 <210> 182 <211> 23 <212> RNA <213> Homo sapiens <400> 182 gaagugcuuc gauuuugggg ugu 23 <210> 183 <211> 23 <212> RNA <213> Homo sapiens <400> 183 accccaaaau cgaagcacuu uuu 23 <210> 184 <211> 22 <212> RNA <213> Homo sapiens <400> 184 agaucagaag gugauugugg cu 22 <210> 185 <211> 22 <212> RNA <213> Homo sapiens <400> 185 ccacaaucac cuucugaucu uu 22 <210> 186 <211> 22 <212> RNA <213> Homo sapiens <400> 186 uaauacuguc ugguaaaacc gu 22 <210> 187 <211> 22 <212> RNA <213> Homo sapiens <400> 187 gguuuuacca gacaguauua uu 22 <210> 188 <211> 23 <212> RNA <213> Homo sapiens <400> 188 aaaccguuac cauuacugag uuu 23 <210> 189 <211> 23 <212> RNA <213> Homo sapiens <400> 189 acucaguaau gguaacgguu uuu 23 <210> 190 <211> 22 <212> RNA <213> Homo sapiens <400> 190 uguuugcaga ggaaacugag ac 22 <210> 191 <211> 22 <212> RNA <213> Homo sapiens <400> 191 cucaguuucc ucugcaaaca uu 22 <210> 192 <211> 21 <212> RNA <213> Homo sapiens <400> 192 cccagauaau ggcacucuca a 21 <210> 193 <211> 21 <212> RNA <213> Homo sapiens <400> 193 gagagugcca uuaucugugu u 21 <210> 194 <211> 22 <212> RNA <213> Homo sapiens <400> 194 uggaagacua gugauuuugu ug 22 <210> 195 <211> 22 <212> RNA <213> Homo sapiens <400> 195 acaaaaucac uagucuucua uu 22 <210> 196 <211> 23 <212> RNA <213> Homo sapiens <400> 196 ucuuugguua ucuagcugua uga 23 <210> 197 <211> 23 <212> RNA <213> Homo sapiens <400> 197 auacagcuag auaaccaaag guu 23 <210> 198 <211> 21 <212> RNA <213> Homo sapiens <400> 198 uauugcacuu gucccggccu g 21 <210> 199 <211> 21 <212> RNA <213> Homo sapiens <400> 199 ggccgggaca agugcaauau u 21 <210> 200 <211> 22 <212> RNA <213> Homo sapiens <400> 200 aaagugcugu ucgugcaggu ag 22 <210> 201 <211> 22 <212> RNA <213> Homo sapiens <400> 201 accugcacga acagcacuuu cc 22 <210> 202 <211> 22 <212> RNA <213> Homo sapiens <400> 202 uucaacgggu auuuauugag ca 22 <210> 203 <211> 22 <212> RNA <213> Homo sapiens <400> 203 cucaauaaau acccguugaa uu 22 <210> 204 <211> 22 <212> RNA <213> Homo sapiens <400> 204 cacccguaga accgaccuug cg 22 <210> 205 <211> 22 <212> RNA <213> Homo sapiens <400> 205 caaggucggu ucuacggguc uu 22  

Claims (22)

An anticancer agent comprising miR-29 as an active ingredient. The anticancer agent according to claim 1, wherein the miR-29 is any one miRNA selected from the group consisting of miR-29a, miR-29b, and miR-29c. The anticancer agent according to claim 2, wherein the miR-29a has a sequence of SEQ ID NO: 148. The anticancer agent according to claim 2, wherein the miR-29b has a sequence of SEQ ID NO: 150.  The anticancer agent according to claim 2, wherein the miR-29c has a sequence of SEQ ID NO: 152. The method according to any one of claims 1 to 5, wherein the anticancer agent is chronic lymphocytic leukemia, breast cancer, cervical cancer, fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma. , Lymphangiosarcoma, lymphangiopiapia, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon tumor, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer , Skin cancer, brain cancer, impression cell tumor, sebaceous gland tumor, papillary tumor, papilloma adenocarcinoma, cystic adenocarcinoma, medulla tumor, organ supportive tumor, renal cell tumor, liver cancer, biliary gland tumor, chorionic cancer, normal carcinoma, fetal tumor, Wilms (Wilm's) tumors, testicular cancer, lung tumors, small cell lung tumors, non-small cell lung tumors, bladder tumors, epithelial tumors, glioma, astrocytoma, medulloblastoma, craniocytoma, cerebral hepatocellular tumor, pineal carcinoma, hemangioblastoma, hearing neuroma, Spinal gliomas, subarachnoid species, melanoma, An anticancer agent for the purpose of treating and alleviating any one cancer selected from the group consisting of neuroblastoma, retinoblastoma, leukemia, lymphoma and Kaposi sarcoma.  The anticancer agent according to any one of claims 1 to 5, wherein the anticancer agent inhibits the activity of p85α. The anticancer agent according to any one of claims 1 to 5, wherein the anticancer agent inhibits the activity of Cdc42.  The anticancer agent according to any one of claims 1 to 5, wherein the anticancer agent promotes the activity of p53. A method for producing an anticancer agent comprising the step of mixing miR-29 with a pharmaceutically acceptable carrier or medium. The method of claim 10, wherein the miR-29 is any one miRNA selected from the group consisting of miR-29a, miR-29b, and miR-29c. 12. The method of claim 11, wherein the miR-29a has a sequence of SEQ ID NO: 148. 12. The method of claim 11, wherein the miR-29b has a sequence of SEQ ID NO: 150. 12. The method of claim 11, wherein the miR-29c has a sequence of SEQ ID NO: 152. 15. The method according to any one of claims 10 to 14, wherein the anticancer agent is chronic lymphocytic leukemia, breast cancer, cervical cancer, fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma. , Lymphangiosarcoma, lymphangiopiapia, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon tumor, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer , Skin cancer, brain cancer, impression cell tumor, sebaceous gland tumor, papillary tumor, papilloma adenocarcinoma, cystic adenocarcinoma, medulla tumor, organ supportive tumor, renal cell tumor, liver cancer, biliary gland tumor, chorionic cancer, normal carcinoma, fetal tumor, Wilms (Wilm's) tumors, testicular cancer, lung tumors, small cell lung tumors, non-small cell lung tumors, bladder tumors, epithelial tumors, glioma, astrocytoma, medulloblastoma, craniocytoma, cerebral hepatocellular tumor, pineal carcinoma, hemangioblastoma, hearing neuroma, Spinolioma, Suakjong, Black , Neuroblastoma, retinoblastoma, leukemia, lymphoma, and Kaposi (Kaposi) method for producing an anticancer agent, characterized in that for the purpose of the treatment and alleviation of any selected one of the arms from the group consisting of sarcoma. A method of treating cancer comprising administering to a subject a pharmaceutically effective amount of one or more miR-29. The method of claim 16, wherein the miR-29 is any one miRNA selected from the group consisting of miR-29a, miR-29b, and miR-29c. 18. The method of claim 17, wherein miR-29a has the sequence of SEQ ID NO: 148. 18. The method of claim 17, wherein the miR-29b has a sequence of SEQ ID NO: 150. The method of claim 17, wherein the miR-29c has a sequence of SEQ ID NO: 152. 18. 21. The method of any one of claims 16-20, wherein the treatment method is chronic lymphocytic leukemia, breast cancer, cervical cancer, fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial. Sarcoma, Lymphangiosarcoma, Lymphangiopiaoma, Synovial Sarcoma, Mesothelioma, Ewing Tumor, Leiomyosarcoma, Rhabdomyosarcoma, Gastric Cancer, Esophageal Cancer, Colon Tumor, Rectal Cancer, Pancreatic Cancer, Breast Cancer, Ovarian Cancer, Prostate Cancer, Uterine Cancer, Head and Neck Cancer, skin cancer, brain cancer, impression cell tumor, sebaceous gland tumor, papillary tumor, papilloma adenocarcinoma, cystic adenocarcinoma, medulla tumor, tracheal support tumor, renal cell tumor, liver cancer, cholestatic tumor, chorionic cancer, normal carcinoma, fetal tumor, bill Wilm's tumor, testicular cancer, lung tumor, small cell lung tumor, non-small cell lung tumor, bladder tumor, epithelial tumor, glioma, astrocytoma, medulloblastoma, craniocytoma, ventricular hematoma, pineal carcinoma, hemangioblastoma, auditory neuroma , Spinnerets, Suakjong, black Species, neuroblastoma, retinoblastoma, leukemia, lymphoma, and Kaposi (Kaposi) A method for treating cancer, characterized in that for the purpose of the treatment and alleviation of any selected one of the arms from the group consisting of sarcoma. 21. The method of any one of claims 16 to 20, wherein the method of treatment comprises a human or a mammal other than a human.

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