# Patent

## Title
Composition for promoting apoptosis or inhibiting cell growth, comprising epstein-barr virus microrna

## Abstract

Provided is a use of an miRNA of an Epstein-Barr virus (EBV), more specifically, EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, miR-BART2-3p, and mimics thereof for promoting apoptosis or inhibiting cell growth.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the national stage of International Application No. PCT/KR2012/004149, filed May 24, 2012, entitled, “Composition For Promoting Apoptosis Or Inhibiting Cell Growth, Comprising Epstein-Barr Virus Microrna,” which claims the benefit of priority of Korean Patent Application No. 10-2011-0049812, filed May 25, 2011, the contents of both of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
Disclosed is use of an miRNA of an Epstein-Barr virus (EBV), more specifically, miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, miR-BART2-3p, and a mimic thereof for promoting apoptosis or inhibiting cell growth.
A study for the present invention was supported by grants from the Gyeonggi Regional Research Centre (GRRC) of the Catholic University of Korea [(GRRC Catholic 2010-A01). RNA-based development of biopharmaceutical lead molecules] and from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (0920210).
BACKGROUND ART
Apoptosis is an important process in normal development and functioning of multicellular organisms. Physiological apoptosis plays an important role in various normal processes, however, abnormal apoptosis causes various diseases (Science. 267, 1456-1462 (1995); Biochem Biophys Res Commun. 266 (3), 699-717 (1999)).
For example, inhibited apoptosis may cause a cancer, an autoimmune disease, an inflammatory disease, and a virus infection. On the other hand, excessive apoptosis may cause a degenerative neurologic disease or a cardiac disease. Thus, as regulation of apoptosis in a concerned tissue or cell is very valuable, a substance promoting apoptosis is useful for preventing and treating an autoimmune disease, a lymphoproliferative disease, an inflammatory disease, and a virus infection and a substance inhibiting apoptosis is useful for preventing and treating a degenerative neurologic disease or a cardiac disease.
MicroRNAs (miRNAs) are non-coding RNAs formed when a long hairpin type transcript produced in a cell is cleaved by enzymes named as Drosha and Dicer into 19 to 25 nt. miRNAs have a base sequence which is incompletely complementary to a 3′ untranslated region of a target gene mRNA, and thus inhibit translation. However, miRNAs have a fully complementary base sequence, and thus induce mRNA degradation.
In 2004, a first case of a virus that EBV expresses its own viral miRNA was reported. Afterwards, 25 pre-miRNAs were discovered. Among the pre-miRNAs, 22 pre-miRNAs produced from a BART transcript were largely divided into Cluster 1 and Cluster 2, and were expressed in most of the EBV-related tumor or cell strain.
miR-BART5-5p, which is expressed in a EBV-infected cell, was reported to inhibit expression of PUMA, which is a pro-apoptotic protein, to increase a rate of cell survival rate (J Exp Med. 205(11), 2551-2560 (2008)). On the other hand, it was reported that, while LMP1, which is an oncogenic EBV protein, induces cell growth and transformation at a low LMP1 expression level, LMP1 inhibits cell growth at a high LMP1 expression level, and miR-BART1-5p, 16-5p, and 17-5p inhibit an LMP1 expression to decrease apoptosis (Proc Natl Acad Sci USA. 104(41), 16164-16169 (2007)). It was reported that miR-BART22-3p inhibits expression of LMP2A, which is known to be necessary to maintain EBV latency, and, although miR-BART22-3p does not affect cell growth or apoptosis, miR-BART22-3p may promote immune evasion (Neoplasia. 11(11), 1174-1184 (2009)). It was reported that miR-BART2-5p targets BALF5, which is an EBV DNA polymerase, to contribute to maintenance of EBV latency and, at the same time, targets MICB, which is a ligand of a natural killer cell, to evade an immune response by the natural killer cell (Nucleic Acids Res. 36(2), 666-675 (2008); Cell Host Microbe. 5(4), 376-385 (2009)). It was reported that miR-BART6-5p targets Dicer, which is related with miRNA biogenesis, and contributes to maintenance of EBV latency (J Biol. Chem. 285(43), 33358-33370 (2010)). Recently, it was proved through a luciferase reporter assay that miRNAs of BART Cluster 1 and Cluster 2 inhibit expression of Bim, which is a pro-apoptotic gene, and reduce apoptosis, but the specific BART miRNA having such functions was not identified (Virology. 412(2), 392-400 (2011)). As described above, EBV BART miRNAs are known to promote cell growth and inhibit apoptosis.
DETAILED DESCRIPTION OF THE INVENTION 
Technical Problem
The present invention provides finding a substance promoting apoptosis and inhibiting cell growth and a method of using the substance.
Technical Solution
According to an aspect of the present invention, there is provided a use of EBV miRNA, more specifically, miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, miR-BART2-3p, and mimics thereof for promoting apoptosis or inhibiting cell growth.
The inventors of the present invention synthesized and prepared mimics of BART miRNAs in a form of a double stranded RNA and investigated the effects by transfecting the mimics of BART miRNAs to AGS, which is a tumor cell strain not infected with EBV. As a result, it was unexpectedly shown that the mimics of miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p inhibit cell growth and promote apoptosis. In addition, the apoptosis promoting effect of the BART miRNA mimics showed a synergic effect when 5-FU, which is an anticancer agent, was simultaneously administered.
In addition, in order to investigate an apoptosis mechanism of miR-BART15-3p, which is a representative example of the BART miRNAs and mimics thereof having an effect of promoting apoptosis, the inventors of the present invention selected an expected target gene of miR-BART15-3p and tested the expected target gene. A decrease of BIRC6 protein level and a decrease of mRNA level of TAX1BP1 by miR-BART15-3p were verified by Western blotting and QRT-PCR, respectively, indicating that BIRC6 and TAX1BP were the target genes.
The mimics of EBV BART miRNA used in the experiments are shown below.









TABLE 1





miRNA Name

Duplex Sequence







ebv-miR-
sense
5′ACCUAGUGUUAGUGUUGUGCU 3′


BART3-5p

(SEQ NO: 1)


 


ebv-miR-
antisense
5′AGCACAACACUAACACUAGGU 3′


BART3-5p

(SEQ NO: 2)


 


ebv-miR-
sense
5′CGCACCACUAGUCACCAGGUGU 3′


BART3-3p

(SEQ NO: 3)


 


ebv-miR-
antisense
5′ACACCUGGUGACUAGUGGUGCG 3′


BART3-3p

(SEQ NO: 4)


 


ebv-miR-
sense
5′GACCUGAUGCUGCUGGUGUGCU 3′


BART4-5p

(SEQ NO: 5)


 


ebv-miR-
antisense
5′AGCACACCAGCAGCAUCAGGUC 3′


BART4-5p

(SEQ NO: 6)


 


ebv-miR-
sense
5′CACAUCACGUAGGCACCAGGUGU 3′


BART4-3p

(SEQ NO: 7)


 


ebv-miR-
antisense
5′ACACCUGGUGCCUACGUGAUGUG 3′


BART4-3p

(SEQ NO: 8)


 


ebv-miR-
sense
5′UCUUAGUGGAAGUGACGUGCUGUG 3′


BART1-5p

(SEQ NO: 9)


 


ebv-miR-
antisense
5′CACAGCACGUCACUUCCACUAAGA3′


BART1-5p

(SEQ NO: 10)


 


ebv-miR-
sense
5′UAGCACCGCUAUCCACUAUGUC 3′


BART1-3p

(SEQ NO: 11)


 


ebv-miR-
antisense
5′GACAUAGUGGAUAGCGGUGCUA 3′


BART1-3p

(SEQ NO: 12)


 


ebv-miR-
sense
5′AGGGAAACAUGACCACCUGAAGUC3′


BART15-5p

(SEQ NO: 13)


 


ebv-miR-
antisense
5GACUUCAGGUGGUCAUGUUUCCCU 3′


BART15-5p

(SEQ NO: 14)


 


ebv-miR-
sense
5′GUCAGUGGUUUUGUUUCCUUGA 3′


BART15-3p

(SEQ NO: 15)


 


ebv-miR-
antisense
5′UCAAGGAAACAAAACCACUGAC 3′


BART15-3p

(SEQ NO: 16)


 


ebv-miR-
sense
5′CAAGGUGAAUAUAGCUGCCCAUCG 3′


BART5-5p

(SEQ NO: 17)


 


ebv-miR-
antisense
5′CGAUGGGCAGCUAUAUUCACCUUG 3′


BART5-5p

(SEQ NO: 18)


 


ebv-miR-
sense
5′GUGGGCCGCUGUUCACCU3′


BART5-3p

(SEQ NO: 19)


 


ebv-miR-
antisense
5′AGGUGAACAGCGGCCCAC 3′


BART5-3p

(SEQ NO: 20)


 


ebv-miR-
sense
5′UUAGAUAGAGUGGGUGUGUGCUCU 3′


BART16-5p

(SEQ NO: 21)


 


ebv-miR-
antisense
5′AGAGCACACACCCACUCUAUCUAA 3′


BART16-5p

(SEQ NO: 22)


 


ebv-miR-
sense
5′AUCACCACCCUCUAUCCAUAU 3′


BART16-3p

(SEQ NO: 23)


 


ebv-miR-
antisense
5′AUAUGGAUAGAGGGUGGUGAU 3′


BART16-3p

(SEQ NO: 24)


 


ebv-miR-
sense
5′UAAGAGGACGCAGGCAUACAAG 3′


BART17-5p

(SEQ NO: 25)


 


ebv-miR-
antisense
5′CUUGUAUGCCUGCGUCCUCUUA 3′


BART17-5p

(SEQ NO: 26)


 


ebv-miR-
sense
5′UGUAUGCCUGGUGUCCCCUUAGU 3′


BART17-3p

(SEQ NO: 27)


 


ebv-miR-
antisense
5′ACUAAGGGGACACCAGGCAUACA 3′


BART17-3p

(SEQ NO: 28)


 


ebv-miR-
sense
5′UAAGGUUGGUCCAAUCCAUAGG 3


BART6-5p

(SEQ NO: 29)


 


ebv-miR-
antisense
5′CCUAUGGAUUGGACCAACCUUA 3′


BART6-5p

(SEQ NO: 30)


 


ebv-miR-
sense
5′CGGGGAUCGGACUAGCCUUAGA 3′


BART6-3p

(SEQ NO: 31)


 


ebv-miR-
antisense
5′UCUAAGGCUAGUCCGAUCCCCG 3′


BART6-3p

(SEQ NO: 32)









Sense and Antisense Sequences of Cluster 1 BART miRNA Mimics









TABLE 2







ebv-miR-
Sense
5′UCACUAGUGAAGGCAACUAAC3′


BART21-5p

(SEQ NO: 33)


 



Antisense
5′GUUAGUUGCCUUCACUAGUGA3′




(SEQ NO: 34)


 


ebv-miR-
Sense
5′CUAGUUGUGCCCACUGGUGUUU3′


BART21-3p

(SEQ NO: 35)


 



Antisense
5′AAACACCAGUGGGCACAACUAG3′




(SEQ NO: 36)


 


ebv-miR-
Sense
5′UCAAGUUCGCACUUCCUAUACA3′


BART18-5p

(SEQ NO: 37)


 



Antisense
5′UGUAUAGGAAGUGCGAACUUGA3′




(SEQ NO: 38)


 


ebv-miR-
Sense
5′UAUCGGAAGUUUGGGCUUCGUC3′


BART18-3p

(SEQ NO: 39)


 



Antisense
5′GACGAAGCCCAAACUUCCGAUA3′




(SEQ NO: 40)


 


ebv-miR-
Sense
5′CCUGGACCUUGACUAUGAAACA3′


BART7-5p

(SEQ NO: 41)


 



Antisense
5′UGUUUCAUAGUCAAGGUCCAGG3′




(SEQ NO: 42)


 


ebv-miR-
Sense
5′CAUCAUAGUCCAGUGUCCAGGG3′


BART7-3p

(SEQ NO: 43)


 



Antisense
5′CCCUGGACACUGGACUAUGAUG3′




(SEQ NO: 44)


 


ebv-miR-
Sense
5′UACGGUUUCCUAGAUUGUACAG3′


BART8-5p

(SEQ NO: 45)


 



Antisense
5′CUGUACAAUCUAGGAAACCGUA3′




(SEQ NO: 46)


 


ebv-miR-
Sense
5′GUCACAAUCUAUGGGGUCGUAGA3′


BART8-3p

(SEQ NO: 47)


 



Antisense
5′UCUACGACCCCAUAGAUUGUGAC3′




(SEQ NO: 48)


 


ebv-miR-
Sense
5′UACUGGACCCUGAAUUGGAAAC3′


BART9-5p

(SEQ NO: 49)


 



Antisense
5′GUUUCCAAUUCAGGGUCCAGUA3′




(SEQ NO: 50)


 


ebv-miR-
Sense
5′UAACACUUCAUGGGUCCCGUAGU3′


BART9-3p

(SEQ NO: 51)


 



Antisense
5′ACUACGGGACCCAUGAAGUGUUA3′




(SEQ NO: 52)


 


ebv-miR-
Sense
5′UGCUAGACCCUGGAGUUGAACC3′


BART22-5p

(SEQ NO: 53)


 



Antisense
5′GGUUCAACUCCAGGGUCUAGCA3′




(SEQ NO: 54)


 


ebv-miR-
Sense
5′UUACAAAGUCAUGGUCUAGUAGU3′


BART22-3p

(SEQ NO: 55)


 



Antisense
5′ACUACUAGACCAUGACUUUGUAA3′




(SEQ NO: 56)


 


ebv-miR-
Sense
5′GCCACCUCUUUGGUUCUGUACA3′


BART10-5p

(SEQ NO: 57)


 



Antisense
5′UGUACAGAACCAAAGAGGUGGC3′




(SEQ NO: 58)


 


ebv-miR-
Sense
5′UACAUAACCAUGGAGUUGGCUGU3′


BART10-3p

(SEQ NO: 59)


 



Antisense
5′ACAGCCAACUCCAUGGUUAUGUA3′




(SEQ NO: 60)


 


ebv-miR-
Sense
5′UCAGACAGUUUGGUGCGCUAGUUG3′


BART11-5p

(SEQ NO: 61)


 



Antisense
5′CAACUAGCGCACCAAACUGUCUGA3′




(SEQ NO: 62)


 


ebv-miR-
Sense
5′ACGCACACCAGGCUGACUGCC3′


BART11-5p

(SEQ NO: 63)


 



Antisense
5′GGCAGUCAGCCUGGUGUGCGU3′




(SEQ NO: 64)


 


ebv-miR-
Sense
5′ACCCGCCCAUCACCACCGGAC3′


BART12-5p

(SEQ NO: 65)


 



Antisense
5′GUCCGGUGGUGAUGGGCGGGU3′




(SEQ NO: 66)


 


ebv-miR-
Sense
5′UCCUGUGGUGUUUGGUGUGGUU3′


BART12-3p

(SEQ NO: 67)


 



Antisense
5′AACCACACCAAACACCACAGGA3′




(SEQ NO: 68)


 


ebv-miR-
Sense
5′ACAUUCCCCGCAAACAUGACAUG3′


BART19-5p

(SEQ NO: 69)


 



Antisense
5′CAUGUCAUGUUUGCGGGGAAUGU3′




(SEQ NO: 70)


 


ebv-miR-
Sense
5′UUUUGUUUGCUUGGGAAUGCU3′


BART19-3p

(SEQ NO: 71)


 



Antisense
5′AGCAUUCCCAAGCAAACAAAA3′




(SEQ NO: 72)


 


ebv-miR-
Sense
5′UAGCAGGCAUGUCUUCAUUCC3′


BART20-5p

(SEQ NO: 73)


 



Antisense
5′GGAAUGAAGACAUGCCUGCUA3′




(SEQ NO: 74)


 


ebv-miR-
Sense
5′CAUGAAGGCACAGCCUGUUACC3′


BART20-3p

(SEQ NO: 75)


 



Antisense
5′GGUAACAGGCUGUGCCUUCAUG3′




(SEQ NO: 76)


 


ebv-miR-
Sense
5′AACCGGCUCGUGGCUCGUACAG3′


BART13-5p

(SEQ NO: 77)


 



Antisense
5′CUGUACGAGCCACGAGCCGGUU3




(SEQ NO: 78)


 


ebv-miR-
Sense
5′UGUAACUUGCCAGGGACGGCUGA3′


BART13-3p

(SEQ NO: 79)


 



Antisense
5′UCAGCCGUCCCUGGCAAGUUACA3′




(SEQ NO: 80)


 


ebv-miR-
Sense
5′UACCCUACGCUGCCGAUUUACA3′


BART14-5p

(SEQ NO: 81)


 



Antisense
5′UGUAAAUCGGCAGCGUAGGGUA3′




(SEQ NO: 82)


 


ebv-miR-
Sense
5′UAAAUGCUGCAGUAGUAGGGAU3′


BART14-3p

(SEQ NO: 83)


 



Antisense
5′AUCCCUACUACUGCAGCAUUUA3′




(SEQ NO: 84)


 


ebv-miR-
Sense
5′UAUUUUCUGCAUUCGCCCUUGC3′


BART2-5p

(SEQ NO: 85)


 



Antisense
5′GCAAGGGCGAAUGCAGAAAAUA3′




(SEQ NO: 86)


 


ebv-miR-
Sense
5′AAGGAGCGAUUUGGAGAAAAUAAA3′


BART2-3p

(SEQ NO: 87)


 



Antisense
5′UUUAUUUUCUCCAAAUCGCUCCUU3′




(SEQ NO: 88)









Sense and Antisense Sequences of Cluster 2 BART miRNA Mimics
Hereinafter, the present invention is described in more detail.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
An embodiment of the present invention is a pharmaceutical composition, which is for preventing or treating a disease related to decreased apoptosis or abnormal cell growth, comprising at least one selected from the group consisting of miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p of EBV and a pharmaceutically acceptable carriers. Another embodiment of the present invention is a pharmaceutical composition, which is for preventing or treating a disease related to decreased apoptosis or abnormal cell growth, comprising at least one selected from the group consisting of an miR-BART4-5p mimic, miR-BART4-3p mimic, miR-BART1-5p mimic, miR-BART15-3p mimic, miR-BART5-5p mimic, miR-BART5-3p mimic, miR-BART16-5p mimic, miR-BART16-3p mimic, miR-BART17-3p mimic, miR-BART21-3p mimic, miR-BART18-5p mimic, miR-BART7-5p mimic, miR-BART9-5p mimic, miR-BART22-5p mimic, miR-BART20-3p mimic, miR-BART13-5p mimic, miR-BART13-3p mimic, and miR-BART2-3p mimic of EBV and a pharmaceutically acceptable carriers.
The structure of BART miRNAs or miR-BART mimics used in various embodiments of the present invention may have either a blunt end or a cohesive end as long as the end allows for inhibiting expression of a target gene by an effect of RNAi. The cohesive end structure may have a 3′-end overhang structure or a 5′-end overhang structure. The number of overhang nucleotides is not limited but, it may be, for example, one to eight, or appropriately, one to four. The BART miRNAs or miR-BART mimics used in various embodiments of the present invention may be chemically modified by a general method known in this art to prevent rapid degradation by an in vivo nuclease and increase in vivo stability. For example, a hydroxyl group at a 2′-position of a ribose ring may be modified with H, OR, R, R′OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, or I (Herein, R may be an alkyl or aryl, or appropriately, a C1 to C6 alkyl group and R′ may be an alkylene, or appropriately, a C1 to C6 alkylene.), or an phosphate backbone may be modified with phosphorothioate, phosphorodithioate, alkyl phosphonate, phosphoramidate, or boranophosphate. In addition, BART miRNAs or mimics of miRNAs may be used by substituting at least one site of the sequences of the BART miRNAs or mimics of miRNAs with a locked nucleic acid (LNA), a peptide nucleic acid (PNA) or a morpholino, which is a nucleic acid analog, to prevent a rapid in vivo degradation and increase stability of the BART miRNAs or mimics of miRNAs. The BART miRNAs or miR-BART mimics used in various embodiments of the present invention include a variant, which is a functional equivalent including a change which does not decrease activity of BART miRNAs or miR-BART mimics, having at least one of substitution, addition, deletion and their combination of the BART miRNAs or miR-BART mimics.
The BART miRNAs or miR-BART mimics used in various embodiments of the present invention may have a complete type in which two RNA strands as a pair form a double-strand RNA, which is a type formed as an miRNA is directly synthesized in vitro and then introduced to a cell through transfection, or a type which is formed by a modification to a structure having a short hairpin which may be used for transfection by a plasmid pre-miRNA vector and a PCR-induced miRNA expression cassette.
The BART miRNAs or miR-BART mimics used in various embodiments of the present invention may be prepared by various synthesis methods known in this art including a direct chemical synthesis method (Sui G et al., Proc Natl Acad Sci USA 99:5515-5520, 2002), a synthesis method using in vitro transcription (Brummelkamp T R et al., Science. 296:550-553, 2002), and a method of cleaving a long double-strand RNA, which is synthesized by in vitro transcription, with an RNase III family enzyme (Paul C P et al., Nature Biotechnology 20:505-508, 2002).
The BART miRNAs or miR-BART mimics used in various embodiments of the present invention may be included as composites with various nucleic acid vectors (viral or non-viral vectors) known in this art in order to increase intracellular transfer efficiency. For example, the BART miRNAs or miR-BART mimics may be included as a recombinant plasmid or virus vector expressing the BART miRNAs or miR-BART mimics. Plasmids which may be used for this purpose are pSilencer (Ambion), pSiEx (Novagen), siXpress (Takara Bio), pBLOCK-iTTM (Invitrogen), pcDNA3.1 (Invitrogen), pCEP4 (Invitrogen), and SilenCircleTM (Allele), but are not limited thereto. Viral vectors which may be used to include the BART miRNAs or miR-BART mimics are a retroviral vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a lentivirus vector, a herpes virus vector, an alpha-viral vector, an EB virus vector, a papilloma virus vector, and a foamy virus vector, but are not limited thereto. In addition, nonviral vectors which may be used to include the BART miRNAs or miR-BART mimics are, as a carrier reagent, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, G-fectin, a cationic phospholipid nanoparticle, a cationic polymer, a cationic micelle, a cationic emulsion or liposome, a ligand-DNA complex, and a gene gun, but are not limited thereto. A nonviral vector as a liposome is an amphipathic agent, for example, a lipid existing as a micelle, an insoluble single layer, or a lamella layer in liquid crystal or an aqueous solution. Lipids for preparing a liposome include a monoglyceride, a diglyceride, a sulfatide, a lysolecithin, a lechitin phospholipod, aponin, bile acid, and lipofectin, but are not limited thereto.
In addition, to increase in vivo stability of the BART miRNAs or miR-BART mimics, the BART miRNAs or miR-BART mimics may be prepared as a pharmaceutical preparation by using general intracellular RNA transfer techniques known in this art such as a method of increasing intracellular absorption by coupling a biocompatible polymer such as polyethylene glycol. The intracellular RNA transfer techniques include a method of making a cell suspension by electroporation in a solution including an miRNA or a mimic thereof to introduce the miRNA or the mimic thereof to the cell by applying a high voltage DC pulse to the solution. By an in vivo method, a solution including an miRNA or a mimic thereof may be injected into a part of a body and a DC voltage may be applied as a pulse through an electrode to introduce the miRNA or the mimic thereof to a cell.
As the mimics used in various embodiments of the present invention, an miR-BART4-5p mimic to which an RNA having a sequence of SEQ NO:5 and an RNA having a sequence of SEQ NO:6 are hybridized, an miR-BART4-3p to which an RNA having a sequence of SEQ NO:7 and an RNA having a sequence of SEQ NO:8 are hybridized, an miR-BART1-5p to which an RNA having a sequence of SEQ NO:9 and an RNA having a sequence of SEQ NO:10 are hybridized, an miR-BART15-3p mimic to which an RNA having a sequence of SEQ NO:15 and an RNA having a sequence of SEQ NO:16 are hybridized, an miR-BART5-5p mimic to which an RNA having a sequence of SEQ NO:17 and an RNA having a sequence of SEQ NO:18 are hybridized, an miR-BART5-3p mimic to which an RNA having a sequence of SEQ NO:19 and an RNA having a sequence of SEQ NO:20 are hybridized, an miR-BART16-5p mimic to which an RNA having a sequence of SEQ NO:21 and an RNA having a sequence of SEQ NO:22 are hybridized, an miR-BART16-3p mimic to which an RNA having a sequence of SEQ NO:23 and an RNA having a sequence of SEQ NO:24 are hybridized, an miR-BART17-3p mimic to which an RNA having a sequence of SEQ NO:27 and an RNA having a sequence of SEQ NO:28 are hybridized, an miR-BART21-3p mimic to which an RNA having a sequence of SEQ NO:35 and an RNA having a sequence of SEQ NO:36 are hybridized, an miR-BART18-5p mimic to which an RNA having a sequence of SEQ NO:37 and an RNA having a sequence of SEQ NO:38 are hybridized, an miR-BART7-5p mimic to which an RNA having a sequence of SEQ NO:41 and an RNA having a sequence of SEQ NO:42 are hybridized, an miR-BART9-5p mimic to which an RNA having a sequence of SEQ NO:49 and an RNA having a sequence of SEQ NO:50 are hybridized, an miR-BART22-5p mimic to which an RNA having a sequence of SEQ NO:53 and an RNA having a sequence of SEQ NO:54 are hybridized, an miR-BART20-3p mimic to which an RNA having a sequence of SEQ NO:75 and an RNA having a sequence of SEQ NO:76 are hybridized, an miR-BART13-5p mimic to which an RNA having a sequence of SEQ NO:77 and an RNA having a sequence of SEQ NO:78 are hybridized, an miR-BART13-3p mimic to which an RNA having a sequence of SEQ NO:79 and an RNA having a sequence of SEQ NO:80 are hybridized, and an miR-BART2-3p to which an RNA having a sequence of SEQ NO:87 and an RNA having a sequence of SEQ NO:88 are hybridized may be used.
Another embodiment of the present invention is a method of preventing or treating a disease related to decreased apoptosis or abnormal cell growth by using at least one selected from the group consisting of EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p.
Another embodiment of the present invention is a method of preventing or treating a disease related to decreased apoptosis or abnormal cell growth by using at least one selected from the group consisting of an miR-BART4-5p mimic, an miR-BART4-3p mimic, an miR-BART1-5p mimic, an miR-BART15-3p mimic, an miR-BART5-5p mimic, an miR-BART5-3p mimic, an miR-BART16-5p mimic, an miR-BART16-3p mimic, an miR-BART17-3p mimic, an miR-BART21-3p mimic, an miR-BART18-5p mimic, an miR-BART7-5p mimic, an miR-BART9-5p mimic, an miR-BART22-5p mimic, an miR-BART20-3p mimic, an miR-BART13-5p mimic, an miR-BART13-3p mimic, and an miR-BART2-3p of EBV. As the mimics used in various embodiments of the present invention, an miR-BART4-5p mimic to which an RNA having a sequence of SEQ NO:5 and an RNA having a sequence of SEQ NO:6 are hybridized, an miR-BART4-3p to which an RNA having a sequence of SEQ NO:7 and an RNA having a sequence of SEQ NO:8 are hybridized, an miR-BART1-5p to which an RNA having a sequence of SEQ NO:9 and an RNA having a sequence of SEQ NO:10 are hybridized, an miR-BART15-3p mimic to which an RNA having a sequence of SEQ NO:15 and an RNA having a sequence of SEQ NO:16 are hybridized, an miR-BART5-5p mimic to which an RNA having a sequence of SEQ NO:17 and an RNA having a sequence of SEQ NO:18 are hybridized, an miR-BART5-3p mimic to which an RNA having a sequence of SEQ NO:19 and an RNA having a sequence of SEQ NO:20 are hybridized, an miR-BART16-5p mimic to which an RNA having a sequence of SEQ NO:21 and an RNA having a sequence of SEQ NO:22 are hybridized, an miR-BART16-3p mimic to which an RNA having a sequence of SEQ NO:23 and an RNA having a sequence of SEQ NO:24 are hybridized, an miR-BART17-3p mimic to which an RNA having a sequence of SEQ NO:27 and an RNA having a sequence of SEQ NO:28 are hybridized, an miR-BART21-3p mimic to which an RNA having a sequence of SEQ NO:35 and an RNA having a sequence of SEQ NO:36 are hybridized, an miR-BART18-5p mimic to which an RNA having a sequence of SEQ NO:37 and an RNA having a sequence of SEQ NO:38 are hybridized, an miR-BART7-5p mimic to which an RNA having a sequence of SEQ NO:41 and an RNA having a sequence of SEQ NO:42 are hybridized, an miR-BART9-5p mimic to which an RNA having a sequence of SEQ NO:49 and an RNA having a sequence of SEQ NO:50 are hybridized, an miR-BART22-5p mimic to which an RNA having a sequence of SEQ NO:53 and an RNA having a sequence of SEQ NO:54 are hybridized, an miR-BART20-3p mimic to which an RNA having a sequence of SEQ NO:75 and an RNA having a sequence of SEQ NO:76 are hybridized, an miR-BART13-5p mimic to which an RNA having a sequence of SEQ NO:77 and an RNA having a sequence of SEQ NO:78 are hybridized, an miR-BART13-3p mimic to which an RNA having a sequence of SEQ NO:79 and an RNA having a sequence of SEQ NO:80 are hybridized, and an miR-BART2-3p to which an RNA having a sequence of SEQ NO:87 and an RNA having a sequence of SEQ NO:88 are hybridized may be used.
The diseases related to decreased apoptosis or abnormal cell growth are, for example, tumors, autoimmune diseases, lymphoproliferative diseases, inflammatory diseases, viral infections, restenosis of blood vessels by angioplasty, and fibrosis by overgrowth of fibroblasts, but are not limited thereto. The tumors include stomach cancer, bladder cancer, a brain tumor, breast cancer, bone cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, T-cell or B-cell originated lymphoid malignancies, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, spleen cancer, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioblastoma, synovioma, mesothelioma, an Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, a colon tumor, colorectal cancer, pancreatic cancer, uterine cancer, head and neck cancer, skin cancer, a squamous cell tumor, a sebaceous gland tumor, a papillary tumor, papillary adenoma, cystadenocarcinoma, a medullary tumor, a bronchogenic tumor, a renal cell tumor, liver cancer, a bile gland tumor, choriocarcinoma, seminoma, fetal servant, a Wilm's tumor, testicular cancer, a lung tumor, a small cell lung tumor, a non-small cell lung tumor, a bladder tumor, epithelioma, neuroglioma, astrocytoma, medulloblastoma, craniopharyngioma, cerebral ventricle ependymoma, pinealocytoma, angioblastoma, a acoustic nerve tumor, oligodendrocyte, meningioma, neuroblastoma, retinoblastoma, leukemia, lymphoma, Kaposi sarcoma, and nasopharyngeal epithelial carcinoma, but are not limited thereto.
Another embodiment of the present invention is a composition, which is for promoting apoptosis or inhibiting cell growth, comprising at least one selected from the group consisting of EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p.
Another embodiment of the present invention is a pharmaceutical composition, which is for promoting apoptosis or inhibiting cell growth, including at least one selected from the group consisting of an miR-BART4-5p mimic, an miR-BART4-3p mimic, an miR-BART1-5p mimic, an miR-BART15-3p mimic, an miR-BART5-5p mimic, an miR-BART5-3p mimic, an miR-BART16-5p mimic, an miR-BART16-3p mimic, an miR-BART17-3p mimic, an miR-BART21-3p mimic, an miR-BART18-5p mimic, an miR-BART7-5p mimic, an miR-BART9-5p mimic, an miR-BART22-5p mimic, an miR-BART20-3p mimic, an miR-BART13-5p mimic, an miR-BART13-3p mimic, and an miR-BART2-3p of EBV.
An Example of the present invention includes a composition, which is for promoting apoptosis or inhibiting cell growth, comprising at least one selected from the group consisting of an miR-BART4-5p mimic to which an RNA having a sequence of SEQ NO:5 and an RNA having a sequence of SEQ NO:6 are hybridized, an miR-BART4-3p to which an RNA having a sequence of SEQ NO:7 and an RNA having a sequence of SEQ NO:8 are hybridized, an miR-BART1-5p to which an RNA having a sequence of SEQ NO:9 and an RNA having a sequence of SEQ NO:10 are hybridized, an miR-BART15-3p mimic to which an RNA having a sequence of SEQ NO:15 and an RNA having a sequence of SEQ NO:16 are hybridized, an miR-BART5-5p mimic to which an RNA having a sequence of SEQ NO:17 and an RNA having a sequence of SEQ NO:18 are hybridized, an miR-BART5-3p mimic to which an RNA having a sequence of SEQ NO:19 and an RNA having a sequence of SEQ NO:20 are hybridized, an miR-BART16-5p mimic to which an RNA having a sequence of SEQ NO:21 and an RNA having a sequence of SEQ NO:22 are hybridized, an miR-BART16-3p mimic to which an RNA having a sequence of SEQ NO:23 and an RNA having a sequence of SEQ NO:24 are hybridized, an miR-BART17-3p mimic to which an RNA having a sequence of SEQ NO:27 and an RNA having a sequence of SEQ NO:28 are hybridized, an miR-BART21-3p mimic to which an RNA having a sequence of SEQ NO:35 and an RNA having a sequence of SEQ NO:36 are hybridized, an miR-BART18-5p mimic to which an RNA having a sequence of SEQ NO:37 and an RNA having a sequence of SEQ NO:38 are hybridized, an miR-BART7-5p mimic to which an RNA having a sequence of SEQ NO:41 and an RNA having a sequence of SEQ NO:42 are hybridized, an miR-BART9-5p mimic to which an RNA having a sequence of SEQ NO:49 and an RNA having a sequence of SEQ NO:50 are hybridized, an miR-BART22-5p mimic to which an RNA having a sequence of SEQ NO:53 and an RNA having a sequence of SEQ NO:54 are hybridized, an miR-BART20-3p mimic to which an RNA having a sequence of SEQ NO:75 and an RNA having a sequence of SEQ NO:76 are hybridized, an miR-BART13-5p mimic to which an RNA having a sequence of SEQ NO:77 and an RNA having a sequence of SEQ NO:78 are hybridized, an miR-BART13-3p mimic to which an RNA having a sequence of SEQ NO:79 and an RNA having a sequence of SEQ NO:80 are hybridized, and an miR-BART2-3p to which an RNA having a sequence of SEQ NO:87 and an RNA having a sequence of SEQ NO:88 are hybridized. The compositions of the present invention may be used in vivo or in vitro.
Another embodiment of the present invention is a method of promoting apoptosis or inhibiting cell growth by using at least one selected from the group consisting of EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p.
Another embodiment of the present invention is a method of promoting apoptosis or inhibiting cell growth by using at least one selected from the group consisting of an miR-BART4-5p mimic, an miR-BART4-3p mimic, an miR-BART1-5p mimic, an miR-BART15-3p mimic, an miR-BART5-5p mimic, an miR-BART5-3p mimic, an miR-BART16-5p mimic, an miR-BART16-3p mimic, an miR-BART17-3p mimic, an miR-BART21-3p mimic, an miR-BART18-5p mimic, an miR-BART7-5p mimic, an miR-BART9-5p mimic, an miR-BART22-5p mimic, an miR-BART20-3p mimic, an miR-BART13-5p mimic, an miR-BART13-3p mimic, and an miR-BART2-3p of EBV. As the mimics, an miR-BART4-5p mimic to which an RNA having a sequence of SEQ NO:5 and an RNA having a sequence of SEQ NO:6 are hybridized, an miR-BART4-3p to which an RNA having a sequence of SEQ NO:7 and an RNA having a sequence of SEQ NO:8 are hybridized, an miR-BART1-5p to which an RNA having a sequence of SEQ NO:9 and an RNA having a sequence of SEQ NO:10 are hybridized, an miR-BART15-3p mimic to which an RNA having a sequence of SEQ NO:15 and an RNA having a sequence of SEQ NO:16 are hybridized, an miR-BART5-5p mimic to which an RNA having a sequence of SEQ NO:17 and an RNA having a sequence of SEQ NO:18 are hybridized, an miR-BART5-3p mimic to which an RNA having a sequence of SEQ NO:19 and an RNA having a sequence of SEQ NO:20 are hybridized, an miR-BART16-5p mimic to which an RNA having a sequence of SEQ NO:21 and an RNA having a sequence of SEQ NO:22 are hybridized, an miR-BART16-3p mimic to which an RNA having a sequence of SEQ NO:23 and an RNA having a sequence of SEQ NO:24 are hybridized, an miR-BART17-3p mimic to which an RNA having a sequence of SEQ NO:27 and an RNA having a sequence of SEQ NO:28 are hybridized, an miR-BART21-3p mimic to which an RNA having a sequence of SEQ NO:35 and an RNA having a sequence of SEQ NO:36 are hybridized, an miR-BART18-5p mimic to which an RNA having a sequence of SEQ NO:37 and an RNA having a sequence of SEQ NO:38 are hybridized, an miR-BART7-5p mimic to which an RNA having a sequence of SEQ NO:41 and an RNA having a sequence of SEQ NO:42 are hybridized, an miR-BART9-5p mimic to which an RNA having a sequence of SEQ NO:49 and an RNA having a sequence of SEQ NO:50 are hybridized, an miR-BART22-5p mimic to which an RNA having a sequence of SEQ NO:53 and an RNA having a sequence of SEQ NO:54 are hybridized, an miR-BART20-3p mimic to which an RNA having a sequence of SEQ NO:75 and an RNA having a sequence of SEQ NO:76 are hybridized, an miR-BART13-5p mimic to which an RNA having a sequence of SEQ NO:77 and an RNA having a sequence of SEQ NO:78 are hybridized, an miR-BART13-3p mimic to which an RNA having a sequence of SEQ NO:79 and an RNA having a sequence of SEQ NO:80 are hybridized, and an miR-BART2-3p to which an RNA having a sequence of SEQ NO:87 and an RNA having a sequence of SEQ NO:88 are hybridized may be used.
Another embodiment of the present invention is a kit, which is for promoting apoptosis or inhibiting cell growth, comprising at least one selected from the group consisting of EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p.
Another embodiment of the present invention is a kit, which is for promoting apoptosis or inhibiting cell growth, comprising at least one selected from the group consisting of an miR-BART4-5p mimic, an miR-BART4-3p mimic, an miR-BART1-5p mimic, an miR-BART15-3p mimic, an miR-BART5-5p mimic, an miR-BART5-3p mimic, an miR-BART16-5p mimic, an miR-BART16-3p mimic, an miR-BART17-3p mimic, an miR-BART21-3p mimic, an miR-BART18-5p mimic, an miR-BART7-5p mimic, an miR-BART9-5p mimic, an miR-BART22-5p mimic, an miR-BART20-3p mimic, an miR-BART13-5p mimic, an miR-BART13-3p mimic, and an miR-BART2-3p of EBV. As the mimics, an miR-BART4-5p mimic to which an RNA having a sequence of SEQ NO:5 and an RNA having a sequence of SEQ NO:6 are hybridized, an miR-BART4-3p to which an RNA having a sequence of SEQ NO:7 and an RNA having a sequence of SEQ NO:8 are hybridized, an miR-BART1-5p to which an RNA having a sequence of SEQ NO:9 and an RNA having a sequence of SEQ NO:10 are hybridized, an miR-BART15-3p mimic to which an RNA having a sequence of SEQ NO:15 and an RNA having a sequence of SEQ NO:16 are hybridized, an miR-BART5-5p mimic to which an RNA having a sequence of SEQ NO:17 and an RNA having a sequence of SEQ NO:18 are hybridized, an miR-BART5-3p mimic to which an RNA having a sequence of SEQ NO:19 and an RNA having a sequence of SEQ NO:20 are hybridized, an miR-BART16-5p mimic to which an RNA having a sequence of SEQ NO:21 and an RNA having a sequence of SEQ NO:22 are hybridized, an miR-BART16-3p mimic to which an RNA having a sequence of SEQ NO:23 and an RNA having a sequence of SEQ NO:24 are hybridized, an miR-BART17-3p mimic to which an RNA having a sequence of SEQ NO:27 and an RNA having a sequence of SEQ NO:28 are hybridized, an miR-BART21-3p mimic to which an RNA having a sequence of SEQ NO:35 and an RNA having a sequence of SEQ NO:36 are hybridized, an miR-BART18-5p mimic to which an RNA having a sequence of SEQ NO:37 and an RNA having a sequence of SEQ NO:38 are hybridized, an miR-BART7-5p mimic to which an RNA having a sequence of SEQ NO:41 and an RNA having a sequence of SEQ NO:42 are hybridized, an miR-BART9-5p mimic to which an RNA having a sequence of SEQ NO:49 and an RNA having a sequence of SEQ NO:50 are hybridized, an miR-BART22-5p mimic to which an RNA having a sequence of SEQ NO:53 and an RNA having a sequence of SEQ NO:54 are hybridized, an miR-BART20-3p mimic to which an RNA having a sequence of SEQ NO:75 and an RNA having a sequence of SEQ NO:76 are hybridized, an miR-BART13-5p mimic to which an RNA having a sequence of SEQ NO:77 and an RNA having a sequence of SEQ NO:78 are hybridized, an miR-BART13-3p mimic to which an RNA having a sequence of SEQ NO:79 and an RNA having a sequence of SEQ NO:80 are hybridized, and an miR-BART2-3p to which an RNA having a sequence of SEQ NO:87 and an RNA having a sequence of SEQ NO:88 are hybridized may be used.
The kits may be used in vivo or in vitro to fulfill a purpose.
Another embodiment of the present invention may include at least one additional effective component in each of the embodiments of the present invention besides at least one selected from the group consisting of EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p or at least one selected from the group consisting of an miR-BART4-5p mimic, an miR-BART4-3p mimic, an miR-BART1-5p mimic, an miR-BART15-3p mimic, an miR-BART5-5p mimic, an miR-BART5-3p mimic, an miR-BART16-5p mimic, an miR-BART16-3p mimic, an miR-BART17-3p mimic, an miR-BART21-3p mimic, an miR-BART18-5p mimic, an miR-BART7-5p mimic, an miR-BART9-5p mimic, an miR-BART22-5p mimic, an miR-BART20-3p mimic, an miR-BART13-5p mimic, an miR-BART13-3p mimic, and an miR-BART2-3p of EBV. The additional effective component may be administered in combination with another therapeutic mode which includes an anticancer agent, a chemotherapeutic agent, an immunotherapeutic agent, an antimicrobial agent, and an antivirus agent, but is not limited thereto, or may be administered in combination with radiation therapy or photodynamic therapy. The chemotherapeutic agent includes an antimetabolite agent, a DNA damaging agent, a microtube instabilizing agent, a microtube stabilizing agent, an actin depolymerizing agent, a growth inhibitor, a topoisomerase inhibitor, an HMG-CoA inhibitor, a purine inhibitor, a pyrimidine inhibitor, a metalloproteinase inhibitor, a CDK inhibitor, an angiogenesis inhibitor, a differentiation promoter, and an immunotherapy agent, but is not limited thereto.
Each of the embodiments of the present invention as described above may be applied to a human or an animal. A carrier included in the pharmaceutical composition of an embodiment of the present invention may be appropriately selected according to the administration route. In addition to the carrier, a diluent, a filler, a salt, a buffer, a stabilizer, a solubilizer or another substance known in this art may be used for the preparation of in the pharmaceutical composition.
An administration route of the pharmaceutical composition of an embodiment of the present invention includes parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhaled, intranasal, aerosol, intraocular, intravascular, endotracheal, intramuscular, intraperitoneal, intrarectal, intravaginal, a gene gun, a skin patch, an eye drop or mouthwash, and an injection, but is not limited thereto.
The quantity of miRNA or a mimic thereof included in the pharmaceutical composition of an embodiment of the present invention may be adjusted according to the characteristics of the disease to treat and severity thereof and characteristics of previous treatments given to a patient. An amount of from about 10 μg to about 20 mg per 1 kg of body weight or organ weight may be included in the pharmaceutical composition. In addition, the administration amount of the pharmaceutical composition is dependent upon an expression vector and a subject to be administered. For example, in a case of a viral vector, the amount of a recombinant virus including a viral vector is in the range from about 103 to about 1012 pfu/kg.
Advantageous Effects
EBV miR-BART4-5p, miR-BART4-3p, miR-BART1-5p, miR-BART15-3p, miR-BART5-5p, miR-BART5-3p, miR-BART16-5p, miR-BART16-3p, miR-BART17-3p, miR-BART21-3p, miR-BART18-5p, miR-BART7-5p, miR-BART9-5p, miR-BART22-5p, miR-BART20-3p, miR-BART13-5p, miR-BART13-3p, and miR-BART2-3p or mimics thereof of the present invention have an effect of promoting apoptosis and inhibiting cell growth, and thus may be used as an active ingredient in a pharmaceutical composition for promoting apoptosis and inhibiting cell growth, a method of preventing or treating a disease related to decreased apoptosis or abnormal cell growth, and a pharmaceutical composition or a kit used in the method.



DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effect of individual EBV BART miRNA mimics on cell growth of an AGS cell strain. n=9 and * denotes P<0.05 and † denotes P<0.01.
FIG. 2 shows the effect of miR-BART5-5p, miR-BART15-3p mimic on cell growth of an AGS cell strain depending on the treatment concentration. n=3, †:P<0.01.
FIG. 3 shows the effect of miR-BART5-5p, miR-BART15-3p or miR-BART20-3p mimic on cell growth of an AGS cell strain depending on the treatment time. n=6, †:P<0.01.
FIGS. 4 and 5 shows the effect on cell cycle of an AGS cell strain observed by PI staining when only an miR-BART5-5p mimic or an miR-BART15-3p mimic was treated and when miR-BART5-5p and miR-BART15-3p mimic were treated with 5-FU simultaneously. n=3, *:P<0.05, †:P<0.01.
FIG. 6 shows the effect on cell cycle of an AGS cell strain observed by PI staining when a BART miRNA mimic was treated with 5-FU simultaneously. n=6, *:P<0.05, †, P<0.01.
FIG. 7 shows the apoptosis effect investigated by Annexin V staining when only an miR-BART5-5p mimic or an miR-BART15-3p mimic was treated and when an miR-BART5-5p or an miR-BART15-3p mimic was treated with 5-FU simultaneously.
FIG. 8 shows the apoptosis effect on an AGS cell strain investigated by Annexin V staining when individual BART miRNA mimics were treated with 5-FU simultaneously. n=9, *:P<0.05, †: P<0.01.
FIGS. 9 and 10 show the results of verifying whether a gene which might be targeted by miR-BART15-3p was actually targeted. The top shows a schematic diagram of psiCHECK which is specially designed for an miRNA targeting test. The bottom shows the result of testing a gene which might be targeted by miR-BART15-3p performed by a luciferase reporter assay. n=3; †: P<0.01.
FIG. 11 shows the result of verifying whether miR-BART15-3p direct targets BIRC6 3′UTR. A shows the expected binding sites of miR-BART15-3p included in psiC-BIRC6, BIRC6m1, BIRC6m2, and BIRC6m1m2. B shows the result of a luciferase reporter assay performed after delivering each vector and miR-BART15-3p together into an HEK293T cell.
FIG. 12 shows the effect of miR-BART15-3p on mRNA and protein levels of genes which were identified as a target. A. BIRC6 mRNA level verified by QRT-PCR in an AGS to which miR-BART15-3p was delivered. B. BIRC6 protein level verified by Western blotting in an AGS to which miR-BART15-3p was delivered. C. BIRC6 mRNA level verified by QRT-PCR in an AGS-EBV to which an miR-BART15-3p inhibitor was delivered. D. BIRC6 protein level verified by Western blotting in an AGS-EBV to which an miR-BART15-3p inhibitor was delivered. E. TAX1BP1 mRNA level verified by QRT-PCR in an AGS to which miR-BART15-3p was delivered.



BEST MODE
Hereinafter, the embodiments of the present invention are described in detail with reference to Examples, but the embodiments of the present invention are not limited thereto.
EXAMPLE 1
Preparation of BART miRNA Mimics
Mature type BART miRNA mimics were synthesized by Genolution Pharmaceuticals (Seoul, Korea) according to our request and each sequence of the mimic is shown in Table 1 and Table 2.
EXAMPLE 2
Cell Culture and Transformation
An AGS cell strain (Korean Cell Line Bank), which is a stomach cancer cell strain, was cultured in an RPMI1640 (Gibco) culture medium including 10% fetal bovine serum. Mature type synthesized BART miRNAs were transfected to the AGS cell strain by using G-fectin (Genolution) according to a protocol of the manufacture.
EXAMPLE 3
Cell Growth Test (CCK-8 Assay)
To investigate the effect of individual EBV BART miRNAs on the AGS cell strain, the synthesized miRNAs were transfected to the AGS cell strain and, after 72 hours, cells were counted by CCK-8 assay. CCK-8 assay is an analytical method of counting cells by measuring colored formazans which are formed as tetrazolium salts are easily reduced by enzymes such as dehydrogenase included in a mitochondria.
Immediately after the AGS cell strain, which is an EBV-negative cell strain, was divided into 96-well plate having 1×103 cells/well, a synthesized miRNA (10 nM) was transfected by using G-fectin. After the transfection, according to the experimental objective, the cells were cultured at 37° C. in an incubator, and then CCK-8 was divided among the wells by allocating 10 μl of CCK-8 to each well and the resulting mixture was cultured in an incubator at 37° C. for two hours. Then, the light absorbance was measured with an ELISA measuring instrument.
Astonishingly, it was showing that mimics of miR-BART15-3p, miR-BART5-5p, miR-BART16-5p, miR-BART17-3p, and miR-BART20-3p inhibited cell growth (Table 3 and FIG. 1).











TABLE 3







Relative





Cell
Standard



Growth
Deviation
p value





















Scrambled
1
±0.105353




control



BART3-5p
1.272325
±0.217357
0.00544481



BART3-3p
1.134944
±0.151438
0.04557112



BART4-5p
0.934781
±0.177295
0.36007269



BART4-3p
1.064824
±0.240507
0.47442005



BART1-5p
1.141337
±0.213522
0.10024868



BART1-3p
1.201069
±0.192482
0.01763492



BART15-5p
0.954474
±0.112565
0.38881645



BART15-3p
0.567640
±0.115939
1.0446E−08



BART5-5p
0.523116
±0.125143
5.8566E−08



BART5-3p
0.958293
±0.185292
0.56725316



BART16-5p
0.704990
±0.097954
1.3912E−05



BART16-3p
1.158869
±0.216577
0.07123824



BART17-5p
0.955098
±0.121512
0.41459821



BART17-3p
0.786497
±0.200822
0.01533499



BART6-5p
0.880951
±0.150107
0.07181715



BART6-3p
0.910063
±0.190801
0.23941914



BART21-5p
1.303997
±0.204928
0.00190053



BART21-3p
1.214906
±0.230037
0.02708391



BART18-5p
1.394629
±0.167148
4.5047E−05



BART18-3p
1.392545
±0.199891
0.00021762



BART7-5p
1.538728
±0.185522
4.0421E−06



BART7-3p
1.156463
±0.162895
0.02973117



BART8-5p
1.447761
±0.383293
0.00813662



BART8-3p
1.395663
±0.210488
0.00028794



BART9-5p
1.481661
±0.197746
3.1646E−05



BART9-3p
1.569261
±0.412069
0.00303969



BART22-5p
1.529268
±0.173392
2.8427E−06



BART22-3p
1.495125
±0.233896
0.000121



BART10-5p
1.646048
±0.208024
2.5367E−06



BART10-3p
1.591101
±0.414520
0.00097895



BART11-5p
1.079796
±0.086483
0.09942768



BART11-3p
0.945864
±0.134622
0.35714111



BART12-5p
1.158610
±0.214861
0.07006222



BART12-3p
1.102165
±0.143919
0.10628436



BART19-5p
1.151466
±0.136228
0.01861102



BART19-3p
1.236815
±0.195064
0.00756775



BART20-5p
1.245400
±0.324239
0.0561646



BART20-3p
0.857667
±0.125343
0.01903579



BART13-5p
1.325692
±0.235629
0.0030181



BART13-3p
1.254611
±0.227980
0.01121857



BART14-5p
1.408238
±0.271466
0.00181178



BART14-3p
1.446585
±0.336094
0.00346446



BART2-5p
1.084926
±0.222797
0.3234295



BART2-3p
1.432948
±0.382729
0.00965267










EXAMPLE 4
Effect on Cell Growth Depending on Treatment Time
By the method described in Example 3, miR-BART5-5p mimic or miR-BART15-3p mimic (10 nM) were transfected to the AGS cell strain, and the effect thereof on cell growth was investigated over time. Both of the EBV BART miRNAs greatly decreased cell growth and, particularly, the miR-BART15-3p mimic inhibited cell growth almost entirely (FIG. 2).
EXAMPLE 5
Effect on Cell Growth Depending on Mimic Concentration 
miR-BART5-5p, miR-BART15-3p, and miR-BART20-3p were transfected to the AGS cell strain (1×103 cells/well) in a concentration of 1, 3, 10, and 30 nM, and the effect on cell growth was investigated. Immediately after the AGS cell strain was divided into a 96-well plate such that 1×103 AGS cells were allocated to each well, a synthesized miRNA was transfected by using G-fectin. Seventy two hours after the transfection, CCK-8 was divided among the wells by allocating 10 μl of CCK-8 to each well, and the resulting mixture was cultured at 37° C. in an incubator for two hours. Then, the light absorbance was measured with an ELISA measuring instrument. miR-BART15-3p showed cell growth which was 50% lower than that of a scrambled control at 3 nM and almost no cell growth at 10 nM and 30 nM. miR-BART5-5p showed a tendency to inhibit cell growth beginning at 10 nM. miR-BART20-3p showed decreased cell growth, beginning at 10 nm, and almost no cell growth at 30 nm (FIG. 3).
EXAMPLE 6
Effect on Cell Cycle (Propidium Iodide Staining)
Effect of individual EBV BART miRNAs on cell cycle of the AGS cell strain was observed. The effect on cell cycle of an AGS cell strain was observed by PI staining when an miR-BART5-5p mimic or an miR-BART15-3p mimic was treated alone or treated in combination with 5-FU.
The AGS cells were divided into a 96-well plate such that 2×105 AGS cells were allocated to each well, and synthesized miRNAs (10 nM) were transfected and cultured at 37° C. in an incubator. Twenty four hours after the transfection, 10 uM of 5-FU, which is an anti-cancer agent, was treated in one case or not treated in another case, and then the resulting mixture was cultured in an incubator at 37° C. for 48 hours. The cells were detached by using Trypsin-EDTA and washed with PBS two times. Afterwards, the ratio of cells that had changed cell cycles to cells that did not have changed cell cycles was measured by FACS using propidium iodide staining (FIG. 4 and FIG. 5).
In the case of the AGS cells to which an miR-BAR15-3p mimic was transfected alone, the percentage of sub G1 was significantly higher than that of the scrambled control, indicating that apoptosis was promoted. In addition, in both of the miR-BART5-5p and miR-BART15-3p mimic, the ratio of sub G1, which is corresponding to an apoptosis cell, was greatly increased after the 5-FU treatment.
EXAMPLE 7
Effect of Combination Treatment with Anti-Cancer Agent on Cell Cycle
Immediately after the AGS cell strains, which are EBV-negative cell strains, were divided into a 6-well plate such that 2×105 AGS cells were allocated to each well, a synthesized miRNA (10 nM) was transfected by using G-fectin and cultured at 37° C. in an incubator. Twenty four hours after the transfection, 10 uM of 5-FU was treated and the resulting mixture was cultured at 37° C. in an incubator for 48 hours. The cells were detached by using Trypsin-EDTA and washed with PBS two times. Afterwards, DNA was stained by propidium iodide staining and the ratio of cells where cell cycle was changed was investigated by FACS.
The FACS measurement results showed that, when a mimic of miR-BART4-5p, 5-5p, 6-3p, 15-3p, 16-5p, 17-3p, 12-5p, 12-3p, 19-5p, 19-3p, 20-5p, 20-3p, 13-5p was treated, the ratio of sub G1 was increased, indicating that the apoptosis was increased (Table 4 and FIG. 6).











TABLE 4







Relative





Sub G1
Stand. Dev.
p value





















Scrambled
1
±0.031059




control



BART3-5p
0.822417
±0.067281
0.049080409



BART3-3p
0.964756
±0.047266
0.326058798



BART4-5p
1.235970
±0.118464
0.076996648



BART4-3p
0.754954
±0.049416
0.004307754



BART1-5p
0.802753
±0.046515
0.006954738



BART1-3p
0.794689
±0.070658
0.000329204



BART15-5p
0.819543
±0.131573
0.143851905



BART15-3p
2.507941
±0.074841
0.000890606



BART5-5p
2.111330
±0.385397
0.037943576



BART5-3p
0.948571
±0.075790
0.37614083



BART16-5p
1.181969
±0.053320
0.012037789



BART16-3p
0.985176
±0.019290
0.413525999



BART17-5p
1.098926
±0.033329
0.01271764



BART17-3p
1.067312
±0.086918
0.323110984



BART6-5p
1.022841
±0.042981
0.472476656



BART6-3p
1.239752
±0.103959
0.059670258



BART21-5p
1.086786
±0.090225
0.067468002



BART21-3p
0.964995
±0.143659
0.584959365



BART18-5p
0.982845
±0.079117
0.636157842



BART18-3p
0.996285
±0.119193
0.943515502



BART7-5p
0.991846
±0.075438
0.813623836



BART7-3p
0.939265
±0.094216
0.184372669



BART8-5p
1.012969
±0.113624
0.79643115



BART8-3p
0.961632
±0.091309
0.36746866



BART9-5p
1.005308
±0.138199
0.92984722



BART9-3p
0.866864
±0.113797
0.03265356



BART22-5p
0.965417
±0.051049
0.1940472



BART22-3p
1.053684
±0.103996
0.27121849



BART10-5p
0.833050
±0.118185
0.01548568



BART10-3p
0.839359
±0.092229
0.00677711



BART11-5p
1.081256
±0.064301
0.02701403



BART11-3p
1.127625
±0.175298
0.13865741



BART12-5p
1.134805
±0.103104
0.02204065



BART12-3p
1.316871
±0.233791
0.0216891



BART19-5p
1.348848
±0.126873
0.00060989



BART19-3p
1.243943
±0.119867
0.00292325



BART20-5p
1.378976
±0.042194
2.6358E−08



BART20-3p
1.618129
±0.09943
6.6505E−06



BART13-5p
1.178035
±0.055734
0.00013301



BART13-3p
0.981342
±0.246613
0.86134039



BART14-5p
0.907649
±0.168803
0.24466241



BART14-3p
0.875700
±0.061192
0.00301909



BART2-5p
1.092187
±0.086943
0.05007166



BART2-3p
1.178367
±0.170677
0.05327269










EXAMPLE 8
Measurement of Apoptosis (Annexin V Staining)
In the cases when an miR-BART5-5p mimic or an miR-BART15-3p mimic was treated alone or treated in combination with 5-FU, the effect thereof on apoptosis of the AGS cell strain was observed.
Immediately after the AGS cell strain, which is an EBV-negative cell strain, was divided was divided into a 6-well plate such that 2×105 AGS cells were allocated to each well, a synthesized miRNA (10 nM) was transfected by using G-fectin and the resulting mixture was cultured at 37 t in an incubatorTwenty four hours after the transfection, 10 uM of 5-FU was treated and the resulting mixture was cultured in an incubator at 37° C. for 48 hours. The cells were detached by using Trypsin-EDTA and washed with PBS two times. Afterwards, the cells were treated with a PE Annexin V Apoptosis Detection Kit and the changed ratio of apoptotic cell was investigated by FACS (FIG. 7).
In both of the cases when an miR-BART5-5p mimic or an miR-BART15-3p mimic was treated alone and when miR-BART5-5p and miR-BART15-3p mimic were treated in combination with 5-FU, the ratio of the lower right in FIG. 7, which corresponds to early apoptotic cell, and the ratio of the upper right in FIG. 7, which corresponds to late apoptotic cell, were increased. Treatment of both of the two miR-BART5-5p and miR-BART15-3p mimics in combination with 5-FU showed a synergetic effect in view that the degree of apoptosis by the combination treatment was significantly greater than the sum of the degree of the apoptosis by the 5-FU treatment alone and the degree of the apoptosis by the apoptosis transfection of each miRNA mimic alone.
EXAMPLE 9
Effect of Combination Treatment with Anti-Cancer Agent on Apoptosis Inducement (Annexin V Staining)
Effect of individual EBV BART miRNAs on apoptosis of the AGS cell strain was observed by an Annexin V staining method.
Immediately after the AGS cell strain, which is an EBV-negative cell strain, is divided into a 6-well plate such that 2×105 AGS cells were allocated to each well, a synthesized miRNA (10 nM) was transfected by using G-fectin and the resulting mixture was cultured at 37° C. Twenty four hours after the transfection, 10 uM of 5-FU, which is an anti-cancer agent, was treated and the resulting mixture was cultured in an incubator at 37° C. for 48 hours. The cells were detached by using Trypsin-EDTA and washed with PBS two times. Afterwards, the cells were treated with a PE Annexin V Apoptosis Detection Kit and the ratio of cells where apoptosis took place was measured by FACS.
Staining with Annexin V, which is a fluorescent pigment which may stain an external surface of a cell in which apoptosis is induced, was significantly higher in the cells to which synthesized miR-BART4-5p, 4-3p, 1-5p, 15-3p, 5-5p, 16-5p, 16-3p, 17-3p, 21-3p, 18-5p, 7-5p, 9-5p, 22-5p, 20-3p, 13-5p, 13-3p, and 2-3p mimics were transfected than that of the cell to which a scrambled control was transfected (Table 5 and FIG. 8).











TABLE 5







Relative





Apoptotic
Standard



cell
Deviation
p value





















Scrambled
1
±0.045449




control



BART3-5p
1.015518
±0.057258
0.533833207



BART3-3p
1.111229
±0.179385
0.104858241



BART4-5p
1.203786
±0.098988
0.0001573



BART4-3p
1.132958
±0.112067
0.007098806



BART1-5p
1.080029
±0.076102
0.017899831



BART1-3p
0.923128
±0.05671
0.006299649



BART15-5p
1.022471
±0.067605
0.42180601



BART15-3p
1.664526
±0.262502
7.03897E−05



BART5-5p
1.325288
±0.131761
3.70881E−05



BART5-3p
1.170317
±0.118036
0.002362445



BART16-5p
1.311740
±0.095923
2.57932E−06



BART16-3p
1.369744
±0.531194
0.071050536



BART17-5p
1.003707
±0.045914
0.865469677



BART17-3p
1.087938
±0.038403
0.000417



BART6-5p
1.116871
±0.553642
0.545540951



BART6-3p
1.018077
±0.126517
0.695135495



BART21-5p
0.920701
±0.110456
0.071812901



BART21-3p
1.155482
±0.199968
0.048993185



BART18-5p
1.201565
±0.139068
0.002034155



BART18-3p
0.870541
±0.072638
0.000562715



BART7-5p
1.096568
±0.072284
0.00480732



BART7-3p
1.106590
±0.223946
0.195211986



BART8-5p
0.975743
±0.05407
0.318217



BART8-3p
1.072157
±0.238263
0.395391



BART9-5p
1.249817
±0.252558
0.017017



BART9-3p
0.794846
±0.11368
0.000516



BART22-5p
1.196878
±0.120093
0.00098



BART22-3p
1.105684
±0.247556
0.239469



BART10-5p
0.837272
±0.066876
3.05E−05



BART10-3p
0.897144
±0.108305
0.023527



BART11-5p
1.005647
±0.085212
0.863686



BART11-3p
1.058308
±0.148333
0.288678



BART12-5p
0.945826
±0.106574
0.188281



BART12-3p
1.084376
±0.129566
0.095047



BART19-5p
1.071132
±0.109755
0.099917



BART19-3p
0.974170
±0.127526
0.5797



BART20-5p
1.074948
±0.102514
0.07019



BART20-3p
1.415718
±0.298863
0.003319



BART13-5p
1.077471
±0.092429
0.043486



BART13-3p
1.186893
±0.15828
0.007815



BART14-5p
1.053543
±0.062082
0.054294



BART14-3p
0.942639
±0.062966
0.042576



BART2-5p
1.081581
±0.108739
0.062056



BART2-3p
1.061974
±0.070423
0.043585










Although an antisense sequence of a synthesized double stranded RNA is known to be almost degraded, a remaining antisense sequence of miR-BART4-5p, miR-BART15-3p, miR-BART5-5p, miR-BART16-5p, miR-BART17-3p, miR-BART9-5p, and miR-BART20-3p which is not degraded may function as siRNA. Thus, a siDirect software program analyzing an off-target effect of a double stranded RNA was used to investigate the possibility of off-target effect. The result showed that the antisense sequences included at least two mismatches, and thus antisense sequences are deemed to have no possibility to act as a siRNA.
EXAMPLE 10
Selection of Expected Target Genes of miR-BART15-3p
A software program of scanning expected target genes of an miRNA, such as Targetscan (http://www.targetscan.org/), Reptar (http://bioinformatics.ekmd.huji.ac.il/reptar/), or DIANA-microT (http://diana.cslab.ece.ntua.gr/microT/) was used to make a list of expected target genes, investigate functions of individual expected target genes, and genes related to apoptosis were separated. Then, a degree of hybridization between target sites of the expected target genes and miR-BART15-3p was investigated with a RNA hybrid software program (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). The result of the RNA hybridization of the finally selected genes is shown in Table 6.









TABLE 6





Expected




Target

Schematic Diagram of Hybridization


Gene
Function
between Target Site and miRNA







BCL2
anti-
target 5′ A CU   UGU  CU         G 3′



apoptotic
         C    GGA   CA  GGCCACUGA



gene
         G    CCU   GU  UUGGUGACU




miRNA  3′ A UU   UU   U          G 5′


 


BCL2L2
BCL2 type
target 5′ A CU   UGU  CU         G 3′



anti-apoptotic
           C  GGA   CA  GGCCACUGA



gene
           G  CCU   GU  UUGGUGACU




miRNA  3′ A UU   UU   U          G 5′


 


BIRC6
protecting cell
target 5′ U    UU  UG            U 3′



under apoptosis
           UCAA   G  AGAACCACUGAU




           AGUU   U  UUUUGGUGACUG




miRNA  3′      CCU UG              5′


 




target 5′ A    UC     CUUU       A 3′




            AAG  AGCAA    UCACUGA




            UUC  UUGUU    GGUGACU




miRNA  3′ AG   CU     UU         G 5′


 


TAX1BP1
inhibiting
target 5′      A               G 3′



TNF-induced
                AAAU AGACCACUGA



apoptosis
                UUUG UUUGGUGACU




miRNA  3′ AGUUCC    U          G 5′


 


DDX42
increasing cell
target 5′ U      UUGGCUUCU     UUCAAU       G 3′



survival by
             AGGAG        GCAAA      CCAUUGA



interacting with
             UCCUU        UGUUU      GGUGACU



TP53BP2
miRNA  3′ AGU                  U            G 5′









EXAMPLE 11
Test of Expected Target Genes of miR-BART15-3p
For an miRNA target test, to psiCHECK, which is a luciferase vector (FIG. 9) specially designed for an miRNA target test including a firefly gene and a renilla gene in the same vector, a 3′UTR of the five genes shown in Table 5, which might be targeted by miR-BART15-3p, was individually cloned to prepare a vector for a target test for each of the genes.
HEK293T was divided into a 96-well plate such that 5×103 cells were allocated to each well, and miR-BART15-3p and a vector for a target test were transfected by using lipofectamine2000. Forty eight hours after the transfection, a dual luciferase reporter assay (Virology. 412(2), 392-400 (2011)) was performed to normalize the results with reference to firefly luciferase and observe a variation of the renilla luciferase gene expression. Among the five candidate genes of the experiment, only the psiCHECK vector to which 3′UTR of BIRC6 and TAX1BP1 was introduced showed a decrease of the renilla value by miR-BART15-3p. The renilla value was not decreased by miR-BART15-3 μm which was formed by mutating a seed part of miR-BART15-3p (FIG. 10). This result indicates that miR-BART15-3p targeted 3′UTR of BIRC6 and TAX1BP1 to decrease expression of renilla luciferase gene.
BIRC6 3′UTR included two target sites which might be hybridized with miR-BART15-3p. To verify whether these two sites are directly targeted by miR-BART15-3p, a point mutation was performed at a psiC-BIRC6 site which was seed-matched with miR-BART15-3p to prepare psiC-BIRC6m1 and psiC-BIRC6m2. Both of the sites were mutated to prepare psiC-BIRC6m1m2 (FIG. 11A).
HEK293T was divided into a 96-well plate such that 5×103 cells were allocated to each well, and miR-BART15-3p and a vector for a target test were transfected by using lipofectamine-2000. Forty eight hours after the transfection, a dual luciferase reporter assay (Virology. 412(2), 392-400 (2011)) was performed to normalize the results with reference to firefly luciferase and observe a variation of the renilla luciferase gene expression. The renilla value was not decreased in psiC-BIRC6m1 and psiC-BIRC6m1m2 by, while the renilla value was decreased in psiC-BIRC6m2 as in the case of psiC-BIRC6. The result indicates that miR-BART15-3p directly targets only a mutation site in psiC-BIRC6m1 among the two expected binding sites in BIRC6 (FIG. 11B).
EXAMPLE 12
Effect of miR-BART15-3p on Expression of Target Gene
It was investigated whether the mRNA level and the protein level of BIRC6, which was considered as a direct target of miR-BART15-3p, were changed by miR-BART15-3p.
miR-BART15-3p was transfected to AGS by using lipofectamine-2000. Forty eight hours after the transfection, the cells were harvested and a QRT-PCR and a Western blotting were performed (FIGS. 12A and 12B). The BIRC6 mRNA level was not changed in the AGS to which miR-BART15-3p was transfected, while the protein level was decreased.
An miR-BART15-3p inhibitor was transfected to AGS-EBV by using lipofectamine-2000. Forty eight hours after the transfection, the cells were harvested and a QRT-PCR and a Western blotting were performed (FIGS. 12C and 12D). The BIRC6 mRNA level was not changed in the AGS-EBV to which an miR-BART15-3p inhibitor was transfected, while the protein level was increased.
Different from BIRC6, the mRNA level of TAX1BP1 was decreased in the AGS to which miR-BART15-3p was transfected (FIG. 12E).

## Claims

1. A method for promoting apoptosis or inhibiting cell growth, the method comprising administering to a subject in need thereof an effective amount of miR-BART15-3p.

2. The method for promoting apoptosis or inhibiting cell growth of claim 1, wherein the mimics is an miR-BART15-3p mimic to which an RNA having a sequence of SEQ NO:15 and an RNA having a sequence of SEQ NO:16 are hybridized.

3. The method of claim 1, wherein the miR-BART15-3P is included in a recombinant plasmid or viral vector expressing miR-BART15-3P included in a nonviral vector.