Double-helix oligonucleotide construct comprising double-stranded miRNA and use thereof

Abstract

The present invention relates to a double-helix oligonucleotide construct comprising a double-stranded miRNA and a composition for preventing or treating cancer comprising the same. More particularly, the present invention relates to a double-helix oligonucleotide construct comprising miR-544a characterized by a method that effectively inhibits the proliferation of cancer cells or induces a voluntary death of cancer cells, and an anticancer composition comprising the construct.

Claims

1. A method for treatment of lung cancer by inhibiting EGF signaling comprising administering a double-stranded oligonucleotide structure comprising a structure of the following structural formula (1):
A-X—R—Y—B  (1) wherein A represents a hydrophilic compound; B represents a hydrophobic compound; X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond; and R represents miR-544a.

2. The method of claim 1, wherein the hydrophilic compound A is represented by (P).sub.n, (P.sub.m-J) or (J-P.sub.m).sub.n, wherein P is a hydrophilic monomer, n is 1 to 200, m is 1 to 15, and J is a linker that connects between m hydrophilic monomers or between m hydrophilic monomers and the oligonucleotide.

3. The method of claim 2, wherein the hydrophilic compound has a molecular weight of 200 to 10,000.

4. The method of claim 2, wherein the hydrophilic monomer (P) has a structure of the following compound (1): ##STR00010## wherein G is selected from the group consisting of CH.sub.2, O, S and NH.

5. The method of claim 2, wherein the linker (J) is selected from the group consisting of PO.sub.3.sup.−, SO.sub.3 and CO.sub.2.

6. The method of claim 2, wherein the hydrophobic compound has a molecular weight of 250 to 1,000.

7. The method of claim 6, wherein the hydrophobic compound is selected from the group consisting of a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, a C.sub.12-C.sub.50 unsaturated or saturated hydrocarbon, diacylphosphatidylcholine, fatty acid, phospholipid, and lipopolyamine.

8. The method of claim 7, wherein the steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine.

9. The method of claim 7, wherein the glyceride derivative is selected from among mono-, di-, and tri-glycerides.

10. The method of claim 1, wherein the covalent bond represented by each of X or Y is a non-degradable bond or a degradable bond.

11. The method of claim 10, wherein the non-degradable bond is an amide bond or a phosphate bond.

12. The method of claim 10, wherein the degradable bond is a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-degradable bond.

13. The method of claim 1, wherein the miR-544a comprises, as an active ingredient, a double strand composed of a double-stranded RNA consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

14. The method of claim 1, wherein the oligonucleotide treats lung cancer by inducing lung cancer cell apoptosis.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1 to 4 show the results of miRNA library screening obtained by transfecting eight kinds of lung cancer cell lines with a miRNA library and measuring the inhibition of proliferation of the cells.

(2) FIG. 5 shows the apoptosis-inducing effect of miR-544a on the PC9 and PC9/ER cell lines.

(3) FIG. 6 shows the results of Western blot analysis performed to measure the effect of miR-544a on the regulation of proteins involved in the EGFR signaling pathway.

(4) FIG. 7 shows the results of RT-qPCR assay performed to analyze the effect of miR-544a on the inhibition of EGFR mRNA in the PC9, PC9/ER, H1975 and H596 cell lines.

(5) FIG. 8 shows the results of verifying a target sequence of miR-544a by luciferase assay and indicates that, when the target sequence is removed from the EGFR 3′UTR sequence, the inhibition of luciferase activity by miR-544a disappears.

(6) FIG. 9 shows the cell viability of lung cancer cell lines having EGFR mutation after treatment with erlotinib and miR544a.

(7) FIG. 10 shows the effect of a miRNA, prepared as an oligonucleotide structure, on the apoptosis of lung cancer cell lines.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION

(8) Unless otherwise defined, all the technical and scientific terms used in the present specification have the same meanings as commonly understood by those skilled in the art to which the present disclosure pertains. In general, the nomenclature used in the present specification is well known and commonly used in the art.

(9) In the present invention, a miRNA, which exhibits an excellent effect by inhibiting the EGFR signaling pathway in lung cancer cells having EGFR mutation and inhibiting the proliferation of the cells, was discovered and the anticancer effect of the miRNA was evaluated.

(10) In the present invention, lung cancer cell lines having EGFR mutation were treated with a screening library of about 1,700 miRNAs and the ability to inhibit cancer cell growth was measured. As a result, miR-544a having the nucleotide sequence described below was discovered (FIGS. 1 to 4), and it was found that miR-544a had excellent anticancer efficacy (FIGS. 5, 6 and 9).

(11) Therefore, in one aspect, the present invention is directed to a double-stranded oligonucleotide structure, which comprises miR-544a and comprises a structure of the following structural formula (1):
A-X—R—Y—B  (1)

(12) In the structural formula (1), A represents a hydrophilic compound; B represents a hydrophobic compound; X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond; and R represents an miR-544a sequence.

(13) In the present invention, the miR-544a may be a double-stranded RNA, DNA or RNA-DNA hybrid consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2.

(14) TABLE-US-00001 miR-544a (SEQ ID NO: 1) 5'-AUUCUGCAUUUUUAGCAAGUUC-3' (SEQ ID NO: 2) 5'-ACUUGCUAAAAAUGCAGAAUUU-3'

(15) As described in the above “Background Art” section, the seed sequence corresponding to 8-9.sup.th nucleotides counting from the second nucleotide of the active sequence of miRNA is the major factor of activity. A long double-stranded sequence comprising the seed region may be used in production of the double-stranded oligonucleotide.

(16) In the present invention, the miRNA discovered by library screening exhibited anticancer efficacy by inhibiting the EGFR signaling pathway in lung cancer cell lines having EGFR mutation.

(17) This miRNA may comprise a duplex or single-stranded molecule polynucleotide, and may be an antisense oligonucleotide or microRNA (miRNA), but is not limited thereto.

(18) In the case of an oligo conjugate in which a hydrophilic compound and a hydrophobic compound are bound to an RNA or DNA oligonucleotide as described in the present invention, the oligonucleotide can be efficiently delivered in vivo and the stability thereof can also be improved, through the conjugate in which the hydrophilic compound and the hydrophobic compound are conjugated to both ends of the RNA or DNA oligonucleotide.

(19) Self-assembled nanoparticles are formed by the hydrophobic interaction of the hydrophobic compound moieties. These nanoparticles have advantages in that they have excellent in vivo delivery efficiency and in vivo stability, and have a very uniform particle size by improving the structure thereof, which makes quality control (QC) easy, and a process of preparing the same as drugs is simple.

(20) In one embodiment, A representing the hydrophilic compound in the double-stranded oligonucleotide structure comprising miRNA according to the present invention is represented by (P).sub.n, (P.sub.mJ).sub.n or (J-P.sub.m).sub.n, wherein P may be a hydrophilic monomer; n may be 1 to 200; m may be 1 to 15; and J may be a linker that connects between m hydrophilic monomers or between m hydrophilic monomers and the oligonucleotide.

(21) When the hydrophilic material is A, the double-stranded oligonucleotide structure according to the present invention has the following structural formula (1′):

(22) ##STR00001##

(23) In the structural formula (1′) A, B, X and Y are as defined in structural formula (1) above, S represents the sense strand of the miRNA, and AS represents the antisense strand of the miRNA.

(24) In one embodiment, the double-stranded oligonucleotide structure comprising miRNA according to the present invention may be a double-stranded oligonucleotide structure comprising a structure of the following structural formula (2):
A-X-5′R3′Y—B  (2)

(25) In the structural formula (2), A, B, X, Y and R are as defined in structural formula (1) above.

(26) More preferably, the double-stranded oligonucleotide structure has a structure of the following structural formula (2′):

(27) ##STR00002##

(28) In one embodiment, the hydrophilic compound may be a cationic or nonionic polymer compound having a molecular weight of 200 to 10,000, preferably a nonionic polymer compound having a molecular weight of 1,000 to 2,000. As the hydrophilic compound, a nonionic hydrophilic polymer compound, for example, polyethylene glycol, polyvinyl pyrrolidone or polyoxazoline, is preferably used, without being limited thereto.

(29) In other embodiments, when the hydrophilic compound is (P.sub.m-J).sub.n or (J-P.sub.m).sub.n, the double-stranded oligonucleotide structure according to the present invention has a structure of the following structural formula (3) or (4):
(P.sub.m-J).sub.n-X—R—Y—B  (3)
(J-P.sub.m).sub.n—X—R—Y—B  (4)

(30) In the structural formula (3) and (4), P may be a hydrophilic monomer; n may be 1 to 200; m may be 1 to 15; J may be a linker that connects between m hydrophilic monomers or between m hydrophilic monomers and the oligonucleotide; X and Y may be each independently a simple covalent bond or a linker-mediated covalent bond; and R may be the specific miRNA of the present invention. More preferably, the double-stranded oligonucleotide comprising miRNA according to the present invention has a structure of the following structural formula (3′):

(31) ##STR00003##

(32) In the structural formula (3′), P, B, J, m, n, X and Y are as defined in structural formula (3) above, S represents the sense strand of the miRNA, and AS represents the antisense strand of the miRNA.

(33) More preferably, the double-stranded oligonucleotide structure comprising miRNA according to the present invention has a structure of the following structural formula (4′):

(34) ##STR00004##

(35) In the structural formula (4′), P, B, J, m, n, X and Y are as defined in structural formula (4) above, S represents the sense strand of the miRNA, and AS represents the antisense strand of the miRNA.

(36) As the hydrophilic monomer (P) in structural formula (3) and structural formula (4) above, any one selected from among nonionic hydrophilic monomers may be used without limitation as long as it satisfies the purpose of the present invention. Preferably, it is possible to use a monomer selected from among compounds (1) to (3) shown in Table 1 below, more preferably a monomer of compound (1). G in compound (1) may preferably be selected from among CH.sub.2, O, S and NH.

(37) In particular, among hydrophilic monomers, the monomer represented by compound (1) has advantages in that it may have excellent biocompatibility such as being introduced with various functional groups, having excellent bioaffinity, inducing less immune response, and can increase the in vivo stability and delivery efficiency of the oligonucleotide contained in the structure according to structural formula (3) or structural formula (4). Due to these advantages, the monomer is very suitable for production of the structure according to the present invention.

(38) TABLE-US-00002 TABLE 1 Preferred hydrophilic monomer structures in the present invention Compound (1) Compound (2) Compound (3) G is CH.sub.2, O, S or NH.

(39) The total molecular weight of the hydrophilic compound in structural formula (3) or structural formula (4) is preferably in the range of 1,000 to 2,000. Thus, for example, when the hexa(ethylene glycol) of compound (1) is used, that is, when a compound, in which G in structural formula (3) or structural formula (4) is O and m is 6, is used, the repeat number (n) is preferably 3 to because the hexa(ethylene glycol) spacer has a molecular weight of 344.

(40) The present invention is characterized in that a suitable number (represented by n) of repeat units of the hydrophilic group (hydrophilic blocks) represented by (P.sub.m-J) or (J-P.sub.m) in structural formula (3) or structural formula (4) may be used as required. The hydrophilic monomer P and linker J included in each hydrophilic block may be the same or different between the hydrophilic blocks. In other words, when 3 hydrophilic blocks are used (n=3), the hydrophilic monomer of compound (1), the hydrophilic monomer of compound (2) and the hydrophilic monomer of compound (3) may be used in the first, second and third blocks, respectively, suggesting that different monomers may be used in all hydrophilic blocks. Alternatively, any one selected from the hydrophilic monomers of compounds (1) to (3) may also be used in all the hydrophilic blocks. Similarly, as the linker that mediates bonding of the hydrophilic monomer, the same linker may be used in all hydrophilic blocks, or different linkers may be used in the hydrophilic blocks. In addition, m, which is the number of the hydrophilic monomers, may also be the same or different between the hydrophilic blocks. In other words, in the first hydrophilic block, three hydrophilic monomers are connected (m=3), and in the second hydrophilic block, five hydrophilic monomers are connected (m=5), and in the third hydrophilic block, four hydrophilic monomers are connected (m=4), suggesting that different numbers of the hydrophilic monomers may be used in the hydrophilic blocks. Alternatively, the same number of the hydrophilic monomers may also be used in all the hydrophilic blocks.

(41) In addition, in the present invention, the linker (J) is preferably selected from the group consisting of PO.sub.3.sup.−, SO.sub.3 and CO.sub.2, but is not limited thereto. It will be obvious to those skilled in the art that any linker selected depending on the hydrophilic monomer used may be used, as long as it satisfies the purpose of the present invention.

(42) All or part of the hydrophilic material monomer may be modified to have a functional group necessary for binding to other materials, such as a target specific ligand, as necessary.

(43) In some cases, one to three phosphate groups may be bound to the 5′ end of the antisense strand of the double-stranded oligonucleotide structure comprising the gene-specific miRNA.

(44) For example, the double-stranded oligonucleotide structure comprising the miRNA may have a structure of the following structural formula (3′) or structural formula (4′):

(45) ##STR00008##

(46) The hydrophobic compound (B) serves to form nanoparticles consisting of the oligonucleotide of structural formula (1) by hydrophobic interaction.

(47) The hydrophobic compound preferably has a molecular weight of 250 to 1,000, and may be selected from among a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, a C.sub.12 to C.sub.50 unsaturated or saturated hydrocarbon, diacylphosphatidylcholine, fatty acid, phospholipid, lipopolyamine or the like, but is not limited thereto. It will be obvious to those skilled in the art that any hydrophobic compound may be used as long as it satisfies the purpose of the present invention.

(48) The steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine, and the glyceride derivative may be selected from among mono-, di-, and tri-glycerides. Here, fatty acid of the glyceride is preferably C.sub.12-C.sub.50 unsaturated or saturated fatty acid.

(49) In particular, among the hydrophobic compounds, the saturated or unsaturated hydrocarbon or cholesterol is preferably used, because it has an advantage of that it can be easily bound in a process of synthesizing the oligonucleotide structure according to the present invention.

(50) The hydrophobic compound may be bound to the opposite end to the hydrophilic compound, and may be bound to any position of the sense or antisense strand of the miRNA.

(51) In the present invention, the hydrophilic compound, the hydrophilic compound block or the hydrophobic compound is bound to the oligonucleotide by a single covalent bond or a linker-mediated covalent (X or Y). The covalent bond may be any one of a non-degradable bond and a degradable bond. Here, examples of the non-degradable bond include, but are not limited to, an amide bond and a phosphate bond, and examples of the degradable bond include, but are not limited to, a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, and an enzyme-degradable bond.

(52) In other examples of the present invention, the miRNA oligonucleotide structure according to the present invention was produced, lung cancer cell lines were treated with the produced oligonucleotide structure, and the cell lines were stained with Annexin V and analyzed by flow cytometry. As a result, as shown in FIG. 9, it could be confirmed that, when nanoparticles consisting of the miRNA structure were used to increase stability in vivo, apoptosis of the cell lines could be induced dependently in a concentration-dependent manner.

(53) Therefore, in another aspect, the present invention is directed to a composition for cancer prevention or treatment. The present invention is also directed to a method for cancer prevention or treatment comprising a step of administering the oligonucleotide structure. The present invention also provides the oligonucleotide structure for use in cancer prevention or treatment. The present invention also provides the use of the oligonucleotide structure in the manufacture of a medicament for cancer prevention or treatment.

(54) In the present invention, the cancer may be one or more cancers selected from the group consisting of a primary cancer such as lung cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, gallbladder and bile duct cancer, breast cancer, leukemia, esophageal cancer, non-Hodgkin's lymphoma, thyroid cancer, cervical cancer, or skin cancer; a metastatic carcinoma arising from metastasis to other organs from the primary cancer site of origin; and a neoplastic cell disease caused by the promotion of abnormally excessive cell division, but is not limited thereto.

(55) The sequence of miRNA that may be used as an active ingredient of the pharmaceutical composition for cancer treatment according to the present invention is a sequence derived from the human genome, but may be a miRNA sequence obtained from other animal genomes without limiting the miRNA-derived genome to the human genome.

(56) The miRNA may be used as various miRNA mimics, which generate biologically equivalent effect. For example, modified miRNA comprising a miRNA sequence containing the same seed region may be used. Here, the length of SEQ ID NO: 1 or SEQ ID NO: 2 may be reduced, and a short-length miRNA mimic consisting of 15 nucleotides may also be used.

(57) miRNA mimics for the miRNA may partially comprise a phosphorothioate structure in which an RNA phosphate backbone structure is substituted with another element such as sulfur. Moreover, those obtained by wholly or partially substituting RNA with a DNA, PNA (peptide nucleic acid) or LNA (locked nucleic acid) molecule may also be used. In addition, those obtained by substituting the 2′ hydroxyl group of RNA sugar with various functional structures including methylation, methoxylation, fluorination or the like may also be used, but is not limited to.

(58) The miRNA is not limited to the mature miRNA and the double-stranded RNA of the miRNA mimic derived therefrom, but may be used in the form of a miRNA precursor. The miRNA precursor may also be obtained by substitution of the above-described RNA phosphate backbone structure, whole or partial substitution of RNA nucleic acid with DNA, PNA or LNA, or modification of the 2′ hydroxyl group of RNA sugar.

(59) The miRNA may be used in the form of a miRNA precursor or primary miRNA (pri-miRNA), and can be synthesized by a chemical method or delivered to cells in the form of a plasmid so as to be expressed.

(60) Examples of a method of delivering the miRNA to cells cultured in a culture dish, which may be used in the present invention, include, but are not limited to, a method of using a mixture of miRNA and a cationic lipid, a method of delivering the miRNA to cells by electrical stimulus, and a method of using a virus.

(61) The composition for cancer treatment comprising the miRNA as an active ingredient may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, and may be formulated together with the carrier.

(62) As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not impair the biological activity and characteristics of an administered compound without irritating an organism. As a pharmaceutically acceptable carrier in a composition that is formulated as a liquid solution, as a sterile and biocompatible carrier, physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more thereof may be used. In addition, the composition of the present invention may, if necessary, comprise other conventional additives, including antioxidants, buffers, and bacteriostatic agents. Furthermore, the composition of the present invention may be formulated as injectable forms such as aqueous solutions, suspensions or emulsions, pills, capsules, granules, or tablets with the aid of diluents, dispersants, surfactants, binders and lubricants.

(63) The composition for cancer prevention or treatment, which comprises the miRNA and a pharmaceutically acceptable carrier, can be applied as any formulation comprising the same as an active ingredient and may be prepared as an oral or parenteral formulation. Pharmaceutical formulations of the present invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or a form suitable for administration by inhalation or insufflation.

(64) Examples of oral formulations comprising the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or emulsified suspensions, powders, granules, emulsions, hard or soft capsules, syrups, or elixirs. Formulations such as tablets or capsules may comprise a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, and a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsule formulations may comprise, in addition to the above-mentioned substances, a liquid carrier such as fatty oil.

(65) Parenteral formulations comprising the composition of the present invention as an active ingredient include injectable forms for subcutaneous, intravenous or intramuscular injection, suppositories, or sprays inhalable via the respiratory organ, such as aerosols. Injectable formulations may be prepared by mixing the composition of the present invention with a stabilizer or a buffer in water to prepare a solution or a suspension, and loading the solution or suspension into ampules or vials to prepare unit dosage forms. Suppository formulations include suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides. For spray formulations, such as aerosols, a propellant for spraying a water-dispersed concentrate or wet powder may be used in combination with an additive.

EXAMPLES

(66) Hereinafter, the present invention will be described in more detail with reference to examples. It will be obvious to skilled in the art that these examples are merely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

Example 1: miRNA Screening Using miRNA Library

(67) Using the same miRNA library used in Korean Patent Application No. 10-2016-0022462, an experiment was performed to screen miRNAs that induce apoptosis of lung cancer cell lines. The lung cancer cell lines used in the experiment are as follows: H2009, H596, H1650, PC9, PC9/GR, H1975, HCC827, and A549 (purchased from the ATCC or the Korean Cell Line Bank). Each of the cell lines was seeded into a 96-well plate and treated with 40 nM of each miRNA together with the transfection reagent RNAiMax (Invitrogen), and the miRNA was delivered into the cells. After 96 hours of additional culture, the relative growth of the cells was measured using CellTiter-Glo reagent (Promega) (FIGS. 1 to 4). Among the miRNAs, miR-544a was selected as a miRNA having an excellent effect against the cell lines used.

Example 2: Analysis of Apoptotic Effect of miRNA

(68) The experiment performed in Example 1 was performed by a method of measuring the inhibitory effect of miRNA on cell proliferation. Methods capable of inhibiting cell proliferation can be broadly divided into two: one is a method of preventing the transition from a specific stage to the next stage in the cell cycle, and the other is a method for inducing apoptosis.

(69) In order to examine how the miRNA selected in Example 1 exhibits a cell proliferation inhibitory effect, an experiment was performed on the PC9 and PC9/ER cell lines. Each of the cell lines was seeded and cultured in a 6-well plate, and then each of a miRNA control and miR-544a was delivered into cells using the transfection reagent RNAiMax (Invitrogen) to reach a concentration of nM. After 48 hours of additional culture, the cells were treated with FITC fluorescent dye-labeled annexin V and propidium iodide (PI) and analyzed by flow cytometry (FACS). As a result, it was confirmed that the number of dead cells in the cell line treated with miR-544a was significantly higher than that in the cell line treated with the miRNA control. This suggests that miR-544a exhibits a cell proliferation inhibitory effect by inducing apoptosis (FIG. 5).

Example 3: Analysis of Mechanism by which miR-544a Induces Apoptosis

(70) In order to examine the mechanism by which miR-544a identified in Examples 1 and 2 induces apoptosis of the lung cancer cell lines, the effects of miR-544a on the signaling pathway in the cell lines used were measured. It is known that the EGFR signaling pathway in the lung cancer cell lines used in the Examples is activated due to the presence of mutation in the EGFR protein and activation of the EGFR signaling pathway plays an important role in the survival of the cell lines.

(71) Thus, since the mechanism by which miR-544a induces apoptosis of the cell lines was predicted to regulate the EGFR signaling pathway, the expression levels of the proteins involved in the EGFR signaling pathway were measured by Western blot analysis. The results of the measurement are shown in FIG. 6. It can be seen that the expression level of the EGFR protein in the sample treated with miR-544a significantly decreased compared to that in the control group, and the expression level of phosphorylated EGFR, indicative of the activity of the EGFR protein, also decreased compared to that in the control group.

(72) The ERK signaling pathway is located downstream of the EGFR signaling pathway, and it could be confirmed that, because the EGFR signaling pathway was inhibited by miR-544a, the amount of ERK, an activated form of the ERK protein, decreased (FIG. 6).

Example 4: Confirmation that miR-544a Inhibits Expression of EGFR mRNA

(73) It is known that, when the expression of protein is decreased by miRNA as indicated by the data shown in FIG. 6, the expression of mRNA is also generally inhibited by miRNA. To confirm this fact, the lung cancer cell lines PC9, PC9/ER, H1975 and H596 were treated with miR-544a or a negative control, and then the relative expression level of EGFR mRNA was analyzed.

(74) As a result, it could be confirmed that the expression of EGFR mRNA decreased in all the cell lines used in the experiment (FIG. 7).

Example 5: Identification of Direct Target mRNA of miR-544a by Luciferase Assay

(75) Since miRNA inhibits protein production from target mRNA by binding to the 3′ UTR (untranslated region) of the target mRNA, luciferase assay is generally used as a method of directly measuring the relationship between miRNA and the target mRNA. Using the TargetScan software, a 3′ UTR sequence comprising a miRNA binding sequence was predicted. The predicted 3′ UTR sequence was cloned into the 3′ UTR of firefly luciferase by a gene cloning technique. The constructed vector and the miRNA of interest were co-transfected into human embryonic kidney (HEK) cells, and the luciferase expression level of the vector was measured. To examine whether EGFR mRNA becomes a target of miR-544a, the 3′ UTR of EGFR mRNA was divided into a and b and inserted into the firefly luciferase vector. At this time, in order to correct the transfection efficiency, Renilla luciferase was also transfected to correct the measurement value. After co-transfection of the miRNA, firefly luciferase and Renilla luciferase, the cells were cultured for 48 hours, and then the luciferase activity was measured using a luminometer.

(76) As a result, as shown in FIG. 8, it could be confirmed that each target mRNA was controlled directly by the miRNA. In addition, it was confirmed that, when the sequences of the EGFR 3′UTR, on which the miRNA acts, were predicted and mutations (EGFR 3′UTR 250-256, 2288-2295, and 3476-3482 nt) in the corresponding regions were induced, the expression inhibition phenomenon by the miRNA disappeared. Thus, it was confirmed that the miRNA controls the target mRNA by direct binding to the regions on which it acts.

Example 6: Evaluation of Whether miR-544a Overcomes Drug Resistance

(77) The efficacy of erlotinib, which is clinically used as a therapeutic agent for lung cancer having EGFR mutation, was evaluated comparatively with that of miR-544a. The cell lines having EGFR mutation, PC9, PC9/GR, PC9/ER, H1975, H596 and H1650, were seeded and cultured in 96-well plates, and then treated with erlotinib at the concentrations shown in FIG. 9. After 96 hours of additional culture, the relative viability of the cells was measured. These cell lines were used because they have genetic characteristics and drug resistance as follows. PC9 contains delE764-A750 mutation and has excellent sensitivity to erlotinib, and thus when PC9 is treated with erlotinib, it can be effectively killed. On the other hand, PC9/GR and PC9/ER contain delE764-A750 mutation, like PC9, and have T790M mutation. For this reason, they are cell lines having resistance to erlotinib. H1975 has L858R and T790M mutations, H596 has overexpressed EGFR, and H1650 has delE764-A750 mutation. These cell lines are all cell lines having resistance to erlotinib. The cell viability was measured using the CellTiter-Glo used in Example 1. Similarly, the cell lines were treated with each of the miR control and miR-544a at the concentrations shown in FIG. 9 together with the RNAiMax transfection reagent, and the relative viability of the cells was measured under the same conditions as those for the erlotinib-treated group.

(78) As a result, as shown in FIG. 9, it was confirmed that the PC9 cell line known to be highly sensitive to erlotinib could be killed by erlotinib at a concentration of 0.1 μM, but when the other cell lines having resistance to erlotinib due to mutations such as EGFR T790M were treated with erlotinib at a concentration of 10 μM which is 100-fold higher than that for the PC9 cell line, apoptosis of the cells could be induced. On the other hand, it was confirmed that miR-544a could induce apoptosis of the cells at a treatment concentration of 0.001 to 0.01 μM regardless of the presence of EGFR T790M mutation.

(79) This suggests that the miRNA is effective at a lower concentration than erlotinib and can effectively act regardless of erlotinib resistance caused by EGFR T790M mutation. In addition, the same results could also be confirmed from the Western blot results shown in FIG. 6. In the PC9 cell line, miR-544a and erlotinib inhibit activated EGFR (pEGFR) and inhibit phosphorylation of the downstream signaling factor ERK. As a result, they induce apoptosis as can be seen from PARP. However, in the other cell lines having erlotinib resistance due to EGFR T790M mutation, only miR-544a exhibits this effect.

Example 7: Synthesis of RNA Oligonucleotide Structure

(80) The double-stranded oligonucleotide structure produced in the present invention has a structure represented by the following structural formula (5):

(81) ##STR00009##

(82) In structural formula (5) above, S represents the sense strand of the miRNA; AS represents the antisense strand of the miRNA; PO.sub.4 is a phosphate group; the ethylene glycol is a hydrophilic monomer, and the hexa(ethylene glycol) is bonded via the linker (J) phosphate group (PO.sub.3.sup.−); C.sub.24 is tetradocosane which is a hydrophobic compound containing a disulfide bond; and 5′ and 3′ refer to the directions of the ends of the double-stranded oligo RNA.

(83) The sense strand of the miRNA in structural formula (5) above was synthesized as follows. Using DMT-hexa(ethylene glycol)-CPG as a support and β-cyanoethyl phosphoramidite, an oligonucleotide-hydrophilic compound structure comprising a sense strand having hexa(ethylene glycol) bonded to the 3′ end was synthesized by a method of connecting a phosphodiester bond forming an oligonucleotide framework structure. Then, tetradocosane containing a disulfide bond was bonded to the 5′ end, thus preparing the sense strand of the desired oligonucleotide-polymer structure. In the case of the antisense strand to be annealed to the sense strand, the antisense strand having a sequence complementary to the sense strand was prepared through the above-mentioned reaction.

Example 8: Induction of Apoptosis by Oligonucleotide Structure Comprising miRNA Sequence

(84) To ensure the in vivo stability of the miRNA screened through the above Examples, an oligonucleotide structure was produced according to the method of Example 7. In order to evaluate whether the produced nanoparticles also induce apoptosis of lung cancer cell lines, the lung cancer cell lines A549 and H1650 were seeded and cultured in 96-well plates, and the nanoparticles were added to media at a concentration of 1000 nM. The cells were cultured in the media containing the nanoparticles, and then the relative growth of the cells was measured using CellTiter-Glo reagent (Promega).

(85) As a result, it was confirmed that apoptosis was induced by the miRNA prepared as the oligonucleotide structure (FIG. 10).

(86) Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

(87) The double-stranded oligonucleotide structure according to the present invention and a composition for cancer treatment comprising the same comprise miR-544a which exhibits an improved anticancer effect compared to the drug erlotinib that is clinically used for lung cancer having EGFR mutation. Thus, the double-stranded oligonucleotide structure and the composition may be widely used as an anticancer therapeutic agent.

SEQUENCE LIST FREE TEXT

(88) Electronic file is attached.