KR101985067B1 - Composition for preventing or treating cancer comprising miR-BART1-3p inhibitor as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cancer comprising an miR-BART1-3p inhibitor as an active ingredient, and more particularly to a composition for preventing or treating cancer comprising an antisense oligonucleotide complementary to miR-BART1-3p inhibitor miR- May be useful for treating gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, or nasopharyngeal carcinoma.

Description

A composition for preventing or treating cancer comprising an miR-BART1-3p inhibitor as an active ingredient (Composition for preventing or treating cancer comprising miR-BART1-3p inhibitor as active ingredient)

The present invention relates to a composition for preventing or treating cancer comprising an miR-BART1-3p inhibitor as an active ingredient.

miRNAs (microRNAs) are short nucleotides of 22 bp or less, produced by cutting a long transcription product in the form of a hairpin, produced in the cell, by a Drosha and Dicer enzyme as a single-stranded, non-coding RNA molecule (Nature. 2003 Sep 25 ; 425 (6956): 415-9). Most miRNAs bind to the 3 'untranslated region (UTR) of the target gene mRNA to inhibit translation and display various functions within the cell. For example, it is associated with various diseases including cancer, as well as intracellular functions such as cell growth, cell differentiation and apoptosis. In particular, more than 50% of miRNAs in relation to cancer are found in the gene region associated with cancer (Proc Natl Acad Sci U S A. 2004 Mar 2; 101 (9): 2999-3004). miRNA modulation disorders are associated with stomach cancer, lung cancer, breast cancer, and colorectal cancer.

It was reported in 2004 that EBV (Epstein Barr Virus) was the first virus to express miRNA. Thereafter, 25 pre-miRNAs and 44 mature miRNAs were found in EBV. EBV miRNAs are known to be divided into four BHRF1 miRNAs made in BHRF1 transcripts and 40 BART miRNAs made in BART transcripts. In addition, BHRF1 miRNA is expressed only in latency III, but BART miRNA is reported to be expressed in the majority of EBV-associated tumors and cell lines.

We found that miR-BART1-3p, among the EBV BART miRNAs, regulates Cyclin D1, which plays an important role in cell growth. EBV was infected and miR-BART1-3p inhibitor was transferred to AGS-EBV expressing miR-BART1-3. As a result, it was confirmed that cell growth of AGS-EBV was reduced.

Our results suggest that miR-BART1-3p inhibitors may be delivered to EBV-associated gastric cancer and may be used for treatment or inhibition of EBV-associated gastric cancer.

1. Korean Patent No. 10-1308941 2. Korean Patent No. 10-1465319

It is an object of the present invention to provide a composition for preventing or treating cancer comprising an miR-BART1-3p inhibitor as an active ingredient.

It is still another object of the present invention to provide a diagnostic composition for cancer comprising miR-BART1-3p mimetic as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing or treating cancer comprising an miR-BART1-3p inhibitor as an active ingredient.

In one embodiment of the invention, the miR-BART1-3p inhibitor is selected from the group consisting of an antisense oligonucleotide complementary to miR-BART1-3p, a small interfering RNA (siRNA), a short hairpin RNA (shRNA) And the like.

In one embodiment of the present invention, the miR-BART1-3p inhibitor may be an antisense oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 4.

In one embodiment of the present invention, the antisense oligonucleotide is a ribonucleotide, a deoxyribonucleotide, a 2'-O-modified oligonucleotide, a phosphorothioate-backbone deoxyribonucleotide, a PNA (peptide nucleic acid) ).

In one embodiment of the present invention, the cancer may be a cancer formed by infection with EBV (Epstein-Barr virus).

In one embodiment of the present invention, the cancer is any one selected from the group consisting of EBV-related gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngeal carcinoma Lt; / RTI >

The present invention also provides a diagnostic composition for cancer comprising miR-BART1-3p mimetic as an active ingredient.

In one embodiment of the present invention, the miR-BART1-3p may be SEQ ID NO: 1.

In one embodiment of the present invention, the cancer may be a cancer formed by infection with EBV (Epstein-Barr virus).

In one embodiment of the present invention, the cancer is any one selected from the group consisting of EBV-related gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngeal carcinoma Lt; / RTI >

The miR-BART1-3p inhibitor according to the present invention is effective for the treatment of cancer, specifically for the treatment of EBV-related cancer, more specifically for the treatment of EBV-related gastric cancer, and thus can be useful for the treatment of gastric cancer.

Figure 1 shows the results of a change in the expression level of Cyclin Dl by overexpression of miR-BART1-3p (scrambled control: control group; and miR-BART1-3p: miR-BART1-3p-transformed group). (A) is the result of qRT-PCR analysis of cyclin D1 mRNA expression, (B) is the result of western blot analysis of the amount of cyclin D1 protein expression, and (C) This is a result.
Figure 2 shows the results of confirming the change in the expression level of cyclin D1 by inhibiting or inhibiting the expression of miR-BART1-3p (control LNA: control group; and LNA-miR-BART1-3p (i): miR-BART1-3p inhibitor Transformed group). (A) is the result of qRT-PCR analysis of cyclin D1 mRNA expression, (B) is the result of western blot analysis of the amount of cyclin D1 protein expression, and (C) This is a result.
Figure 3 shows the cell growth curve of EBV-reperfusion cell lines inhibited or inhibited by miR-BART1-3p expression (negative control: control; and miR-BART1-3pi: miR-BART1-3p inhibitor ).

In the present invention, " miRNA inhibitor " may be any one selected from the group consisting of antisense oligonucleotides complementary to miRNA, siRNA (small interfering RNA), shRNA (short hairpin RNA) and ribozyme , But is not limited thereto.

The term " antisense oligonucleotide " of the present invention encompasses a nucleic acid-based molecule capable of forming a duplex with a miRNA, in particular, a complementary sequence to the leader sequence of the miRNA, to form a miRNA. Thus, the term " antisense oligonucleotide " herein can be described as a " complementary nucleic acid-based inhibitor ".

The term " complementary " of the present invention means that the antisense oligonucleotide is sufficiently complementary to selectively hybridize to a miR-BART1-3p target under any hybridization or annealing conditions, preferably physiological conditions, Substantially complementary " and " perfectly complementary ", and preferably means completely complementary.

In the present invention, the antisense oligonucleotides include various molecules. The antisense oligonucleotide is a DNA or RNA molecule, more preferably an RNA molecule. Alternatively, the antisense oligonucleotides used in the present invention include ribonucleotides (RNA), deoxyribonucleotides (DNA), 2'-O-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, PNA lt; / RTI > The 2'-O-modified oligonucleotide is preferably a 2'-O-alkyl oligonucleotide, more preferably a 2'-O-C1-3 alkyl oligonucleotide, most preferably a 2'- 3 methyl oligonucleotide.

Inhibition of miR-BART1-3p in the present invention can be achieved by administering a typical antisense oligonucleotide. Antisense oligonucleotides are ribonucleotides or deoxyribonucleotides. Preferably, the antisense oligonucleotide comprises at least one chemical modification. Antisense oligonucleotides may comprise one or more LNAs (Locked nucleic acids). The LNA is a modified ribonucleotide that has an additional bridge between the 2 'to 4' carbon of the ribose sugar site to have a locked form so that the oligonucleotide with the LNA has improved thermal stability. Alternatively, the antisense oligonucleotides may comprise PNAs (peptide nucleic acids), which include a peptide-based backbone instead of a sugar-phosphate backbone. Other chemical modifications that the antisense oligonucleotides may include are 2'-O-alkyl (eg, 2'-O-methyl, 2'-O-methoxyethyl), 2'-fluoro and 4'- Sugar modifications such as; Backbones such as phosphorothioates, morpholino or phosphonocarboxylate linkages (e.g., U.S. Patent Nos. 6,693,187 and 7,067,641). In another embodiment, a suitable antisense oligonucleotide is a 2'-O-methoxyethyl " gapmer " which comprises a 2'-O-methoxyethyl-modified ribonucleotide at the 5'- and 3'- At least 10 deoxyribonucleotides. This "gapmer" can trigger the RNase I-dependent breakdown mechanism of the RNA target. The length of the antisense oligonucleotide is 7-50 nucleotides, preferably 10-40 nucleotides, more preferably 15-30 nucleotides, and most preferably 20-25 nucleotides.

Another way of inhibiting miR-BART1-3p is to administer an inhibitory RNA molecule, which contains a sequence complementary to the maturation sequence of miR-BART1-3p. Such inhibitory RNA molecules include double interfering RNA (siRNA), short hairpin RNA (shRNA) and ribozymes.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those conventionally used in the field of the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous infusion, local injection, intracerebral infusion, spinal cord infusion, subcutaneous infusion, intraperitoneal injection, .

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Materials and Methods

1.1. miRNA Imitation (mimic) and miRNA  Manufacture of inhibitors

The sequence of miR-BART1-3p (5'-UAGCACCGCUAUCCACUAUGUC-3 ': SEQ ID NO: 1), mutated miR-BART1-3p (SEQ ID NO: 5'-UAGGTGCGCUAUCCACUAUGUC-3 ': SEQ ID NO: 2), scrambled control (5'-ACGUGACACGUUCGGAGAAUU-3': SEQ ID NO: 3). miR-BART1-3p inhibitor (5'-ACAUAGUGGAUAGCGGUGCU-3 ': SEQ ID NO: 4), control inhibitor (5'-GTGTAACACGTCTATACGCCCA-3 ': SEQ ID NO: 5).

1.2. Cell culture and transformation

AGS cell line, a gastric cancer cell line, was purchased from ATCC (American Type Culture Collection, US) and cultured in RPMI1640 (Gibco) medium supplemented with 10% FBS (fetal bovine serum). AGS-EBV was an EBV (Epstein-Barr virus) -inactivated AGS cell line, which was cultured in the same culture conditions as AGS, supplemented with 400 μg / ml of G418 (geneticin). miRNA mimics and miRNA inhibitors were transferred to AGS and AGS-EBV cell lines according to the manufacturer's protocol using Lipofectamine® 2000 (Invitrogen).

1.3. CCK -8 Assay

Cell viability was measured with a CCK-8 assay (Dojindo Molecular Technologies, Inc.). The EBV-infected AGS-EBV cell line was seeded at 1 × 10 ³ cells / well in a 96-well plate and the miRNA inhibitor was transformed with Lipofectamine® 2000 (Invitrogen). After transfection, CCK-8 was added to each well at a rate of 10 μl per well. Cells were incubated in a 37 ° C incubator for 2 hours, and the absorbance at 450 nm was measured with an ELISA meter.

1.4. qRT -PCT (Quantitative Real-time Polymerase Chain Reaction) method

AGS cells (1 × 10 ⁶ cells) were cultured in a 100 mm dish, transformed into 20 nM scrambled control and miR-BART1-3p mimic using Lipofectamine® 2000 (Invitrogen), respectively, and cells were harvested 48 hours later.

AGS-EBV cells (1x10 ⁶ cells) were cultured in 100 mm dishes and then treated with 50 nM of each control inhibitor and antisense LNA (TM) for miR-BART1-3p using Lipofectamine®2000 (Thermo Fisher Scientific) acid), and cells were collected after 48 hours.

Total RNA was extracted from each cell collected in the above manner. After cDNA was synthesized, mRNA expression was observed using a qRT-PCR system (Applied Biosystems). Primer information for the gene is shown in Table 1 below.

gene direction Sequence information Cyclin D1 Forward 5'-CAA TGA CCC CGCG ATT TC-3 '(SEQ ID NO: 6) Reverse 5'-CAT GGA GGG CGG ATT GGA A-3 '(SEQ ID NO: 7) GAPDH Forward 5'-CAT GAG AAG TAT GAC AAC AGC CT-3 '(SEQ ID NO: 8) Reverse 5'-AGT CCT TCC ACG ATA CCA AAG T-3 '(SEQ ID NO: 9)

1.5. Western Blotting  Way

After obtaining the cells in 1.4, the proteins in the obtained cells were obtained using RIPA buffer. The obtained protein was separated through a polyacrylamide gel and then transferred to a PVDF membrane. The expression levels of cyclin D1 and beta-actin were confirmed using anti-cyclin D1 and anti-beta-actin antibodies and secondary antibodies.

Example  2. miR - BART1 Effect of -3p on cell proliferation

In order to observe the effect of miR-BART1-3p on Cyclin D1, miR-BART1-3p mimetic was transferred to AGS cells, and the expression level of cyclin D1 mRNA and protein was measured by qRT-PCR and Western blotting Respectively.

As a result, it was confirmed that the amount of cyclin D1 mRNA expression and protein expression was increased in AGS cells transfected with miR-BART1-3p compared to cells transfected with scrambled control (FIG. 1).

Example  3. miR - BART1 Cells by the -3p inhibitor

To investigate the effect of intrinsic miR-BART1-3p, miR-BART1-3p inhibitor (LNA ™) was transferred to AGS-EBV to investigate the amount of cyclin D1 mRNA expression and protein expression.

As a result, it was confirmed that the amount of cyclin D1 mRNA expression and protein expression was reduced in the AGS-EBV cells transfected with the miR-BART1-3p inhibitor, compared to cells transfected with control LNA (FIG. 2).

In addition, when miR-BART1-3p inhibitor was delivered to AGS-EBV cells expressing miR-BART1-3p, cell growth was reduced compared to AGS-EBV cells to which control LNA was transferred (Fig. 3).

The present inventors found that miR-BART1-3p increased the expression of Cyclin D1, which plays an important role in cell growth, and that miR-BART1-3p, which inhibits the expression of miR-BART1-3p in EBV-infected gastric cancer cell line AGS- 3p inhibitor inhibited cell growth compared with AGS-EBV cells delivered with negative control inhibitors.

Thus, miR-BART1-3p-specific inhibition of miR-BART1-3p inhibitors by miR-BART1-3p inhibitors has been shown to be able to treat EBV-associated gastric cancer.

<110> Industry-Academic Cooperation Foundation of Catholic University &Lt; 120 > Composition for;          miR-BART1-3p inhibitor as active ingredient <130> PN1610-351 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-BART1-3p <400> 1 uagcaccgcu auccacuaug uc 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> mutated miR-BART1-3p <400> 2 uaggtgcgcu auccacuaug uc 22 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> scrambled control <400> 3 acgugacacg uucggagaau u 21 <210> 4 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> miR-BART1-3p inhibitor <400> 4 acauagugga uagcggugcu 20 <210> 5 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> control inhibitor <400> 5 gtgtaacacg tctatacgcc ca 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cyclin D1 forward <400> 6 caatgacccc gcacgatttc 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cyclin D1 reverse <400> 7 catggagggc ggattggaa 19 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward <400> 8 catgagaagt atgacaacag cct 23 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse <400> 9 agtccttcca cgataccaaa gt 22

Claims (10)

CLAIMS 1. A method for inhibiting EBV-infected AGS gastric cancer cell growth, comprising the step of treating miR-BART1-3p inhibitor with EBV (Epstein-Barr virus) -infected AGS gastric cancer cells in vitro ,
The miR-BART1-3p inhibitor is a LNA (locked nucleic acid) comprising the nucleotide sequence of SEQ ID NO: 4,
Wherein said miR-BART1-3p inhibitor inhibits EBV-infected AGS cancer cell growth by reducing the expression level of mRNA and protein of Cyclin D1, and inhibits EBV-infected AGS cancer cell growth.
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