KR101413581B1 - Compositon for preventing or treating cancer comprising miR-186, miR-216b, miR-337-3p and miR-760 - Google Patents

Abstract

The present invention relates to a composition for preventing and treating cancer comprising as an active ingredient a nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760. The miR-186, miR-216b, miR-337-3p and miR-760 according to the present invention combine with each other in cancer cells to inhibit the expression of CKII alpha gene, thereby inducing the production of ROS and the expression of p53 and p21 Induces cell senescence in cancer cells and thus has an excellent effect of inhibiting the proliferation of cancer cells. Therefore, the present invention can be usefully used as a preventive or therapeutic agent for cancer.

Description

The present invention relates to a composition for preventing or treating cancer comprising as an active ingredient miR-186, miR-216b, miR-337-3p and miR- 3p and miR-760}

The present invention relates to a composition for preventing or treating cancer comprising, as an active ingredient, a nucleic acid molecule composed of miR-186, miR-216b, miR-337-3p and miR-760.

Protein kinase CKII (hereinafter referred to as CKII) is a universal serine / threonine kinase that catalyzes the phosphorylation of a number of cellular and nuclear proteins. CKII consisted of two active subunits (alpha and / or alpha) and two regulatory subunits beta and beta. The regulatory subunits β and β 'induce catalytic activity of α and α` subunits by inducing tetramer formation and thus substrate recognition. Overexpression of the CKII [alpha] subunit causes tumors to form in the over-expressing mouse Myc gene. In addition, it has recently been shown that CKII plays an important role in anti-apoptotic activities as well as cell growth and proliferation. In normal early cells, the cell division cycle is stopped after a limited number of cleavage, and these cells become irreversible cellular senescence. Cell senescence can be caused by oxidative stress and tumorigenic activity. Aging can be defined by molecules and phenotypes such as large flat cell morphology and aging-associated [beta] -galactosidase activity (SA-beta-gal). CKII activity is down-regulated in old human lung fibroblasts (IMR-90) and aged rat tissues, and inhibits CKII activity at the time of human fibroblast (IMR-90) and human colorectal cancer cells (HCT116) Early aging was found to be induced. The p53-p21Cip1 / WAF1-RB pathway is essential in aging induced by inhibition of CKII. The uptake of p53 by acetylation of the SIRT1 (silent information regulator 2; analog of Sir2) and the production of superoxide anion by the activity of NADPH oxidase inhibited the stabilization of p53 in senescent cells by inhibition of CKII It functions as an upstream activator. DNA methylation is involved in the silencing of CKII alpha and CKII alpha genes during cell senescence. However, the precise mechanism described above is not clearly understood.

Small RNA (hereinafter referred to as "sRNA") refers to a ribonucleic acid having a length of about 17 to 25 nucleotides (hereinafter referred to as "nt") which functions to regulate gene expression in vivo. Is classified into microRNA (hereinafter referred to as "miRNA") and small interfering RNA (hereinafter referred to as "siRNA") depending on the manner in which it is produced. MiRNA is a hairpin RNA (hairpin RNA), and the siRNA is derived from a double stranded RNA (hereinafter referred to as "dsRNA"). In general, sRNA that plays an important role in various regulation processes in vivo is miRNA, and sRNA which is used to control the expression of a specific gene is classified as siRNA.

The miRNA is an endogenous hairpin-shaped transcript (Bartel, DP, Cell 116: 281-9) as a 19-25 nt long single stranded RNA (ssRNA) MiRNAs complementarily bind to the 3 'untranslated regions (UTRs) of the target mRNA, resulting in post-transcriptional gene suppression (post-transcriptional translation) -transcriptional gene suppressor, which suppresses target genes by inducing translational inhibition and mRNA destabilization. miRNA plays an important role in various processes such as development, differentiation, proliferation, apoptosis and metabolism These processes are often disturbed during tumor formation and miRNAs appear to play a role in tumorigenesis, as many miRNAs remain low in human cancers. A strong association between miRNAs and cancer has recently been demonstrated, New research in MiRNA expression is dramatically altered during development and cell differentiation, and miRNAs are expressed in miRNAs (miRNAs) (Lu, J. et al., Nature 435: 834-838, 2005). Previous research into the regulatory network for miRNA function has also shown that the above- (Kim, VN Nat, Rev. Mol. Cell. Biol. 6: 376-385, 2005) miRNA biosynthesis is initiated via transcription by RNA polymerase II (Cai. X., et al., RNA 10: 1957-1966, 2004; Kim, VN Nat. Rev. Mol. Cell. Biol. 6: 376-385, 2005; Lee, Y., et al., EMBO J 21: 4663-4670, 2002; Lee, Y., et al., EMBO J 23: 4051-4060, 2004). Primary miRNAs (hereinafter referred to as "pri-miRNAs") usually exceed the length of a few Kb and include 5 'cap and poly A teil. pri-miRNA has been shown to be a key component of ribonuclease III, Drosha (Lee, Y., et al., Nature 425: 415-419, 2003) and its subgenus DGCR8 (Gregory, RI et al., Nature 432: 235-240 A microprocessor consisting of a microprocessor, a microprocessor, a microprocessor, and a microprocessor, ) And cleaved into a hairpin-structure precursor (pre-miRNA) of about 65 nt. The recombinant DGCR8 or Drosha protein alone has no cleavage activity in the pri-miRNA process and shows cleavage activity when the two proteins are bound, indicating that the two proteins play the most important role in the pre-miRNA process (Gregory, RI et al., Nature 432: 235-240, 2004; Han, J., et al., Genes Dev. 18: 3016-3027, 2004). The pre-miRNA process is the central step in miRNA biosynthesis and is defined as the process of generating one end of a molecule containing an mRNA sequence in a long pre-miRNA. The pre-miRNA generated after the initiation process is transported to the cytoplasm by the nuclear transport element Exp5 (exportin-5) (Bohnsack, MT, et al., RNA 10: 185-191, 2004; Lund, E., et al., Science 303: 95-98, 2004; Yi, R., et al., Genes Dev 17: 3011-3016, 2003). In the cytoplasm, a cytoplasmic RNase Ⅲ type protein, Dicer, cleaves the transferred pre-miRNA to produce a 22-nt miRNA double strand. One strand of the dicer product is a mature miRNA that is present in the cytoplasm and is assembled with an effector complex called miRNP (microribonucleoprotein) or miRISC (miRNA-induced silencing complex) (Khvorova, A., et al. , Cell 115: 209-216, 2003; Schwarz, DS, et al., Cell 115: 199-208, 2003).

The RNAi phenomenon, a gene silencing mechanism by sRNA, has been used as an effective genetic research tool in mammalian systems. An effective and stable gene knockdown is expressed as small hairpin RNA (shRNA) and processed as small interfering RNA (siRNA). Recent breakthroughs of RNAi technology have been made by producing shRNA expression cassettes mimicking natural miRNA genes (Dickins, RA, et al., Nat. Genet. 37: 1289-1295, 2005; Silva, JM et al., Nat Genet., 37: 1281-1288, 2005: Zeng, Y., et al., Mol Cell 9: 1327-1333, 2002). MiRNA-based shRNAs regulated by the RNA polymerase II promoter induce efficient and stable gene silencing modulation in cultured cells such as animal models.

In addition, bioinformatics analysis predicts that miRNAs can regulate 30% of human genes, and recently miRNAs have been found to be involved in many cellular mechanisms such as aging, proliferation, apoptosis, and differentiation of cells .

Accordingly, the inventors of the present invention confirmed that the miRNA of the present invention inhibits the expression of CKII alpha in cancer cells and induces the production of ROS, thereby increasing the expression of p53 and p21 and inducing cell senescence of colon cancer cells. .

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a nucleic acid molecule composed of miR-186, miR-216b, miR-337-3p and miR-760.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760.

The miR-186, miR-216b, miR-337-3p and miR-760 of the present invention combine with each other in cancer cells to inhibit the expression of CKII alpha gene, thereby inducing the production of ROS and inducing the expression of p53 and p21 , Which is effective for inhibiting the proliferation of cancer cells by inducing cell senescence in cancer cells.

Figure 1 shows the sequence of the miRNA of the present invention and its target sequence CKII < RTI ID = 0.0 >3'UTR< / RTI >
Figure 2 shows the levels of protein expression of CKII a when miR-186, miR-216b, miR-337-3p and miR-760 of the invention were transfected into HCT116 human colon cancer cells (hereinafter HCT116 cells) .
Figure 3 is a graph showing protein expression levels of CKII alpha when HCT116 cells are transfected with three miRNA combinations selected from miR-186, miR-216b, miR-337-3p and miR-760 of the present invention.
Figure 4 is a graph showing protein expression levels of CKII alpha when co-transfected with HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760 of the present invention.
5 is a miR-186, miR-216b, miR-337-3p , and miR-760 with the ball in HCT116 cells is a diagram showing the protein expression levels in the case of transfection, CKII α` CKII and β.
Figure 6 shows the level of mRNA expression of CKII a using RT-PCR when co-transfected with HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760.
Figure 7 shows the results of luciferase assay when co-transfected into HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760.
Figure 8 shows activity levels of CKII protein when co-transfected into HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760.
Figure 9 shows SA-beta-gal activity levels when co-transfected into HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760.
FIG. 10 is a graph showing protein expression levels of p53 and p21 when co-transfected into HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760.
Figure 11 is a graph showing levels of production of reactive oxygen species (ROS) when co-transfected into HCT116 cells together with miR-186, miR-216b, miR-337-3p and miR-760.
12 is miR-186, miR-216b, miR-337-3p , and miR-760 in a ball HCT116 cells transfected if infection, overexpression of the α CKII, SA-β-gal activity (A), p53 and p21 (B) and the production level (C) of active oxygen (ROS).

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760.

Hereinafter, the present invention will be described in detail.

The miR-186, miR-216b, miR-337-3p and miR-760 of the present invention combine with each other in cancer cells to inhibit the expression of the CKII alpha gene, thereby inducing the production of ROS and inducing the expression of p53 and p21 Thereby inducing cell senescence in cancer cells and inhibiting the proliferation of cancer cells. Therefore, the nucleic acid molecule composed of miR-186, miR-216b, miR-337-3p and miR-760 of the present invention can be usefully used as a preventive or therapeutic agent for cancer.

Said arm comprises a cancer caused by the over-expression of CKII α gene, cancer caused by the over-expression of the CKII α gene is the various cancer of the human body, gynecological tumors, endocrine cancer, central nervous system cancer, ureter cancer, etc. The present invention relates to a method for screening for a cancer in a patient suffering from a cancer selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, Renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, pancreatic cancer, vulvar carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, Carcinoma, primary central nervous system lymphoma, cholesteatoma tumor, brainstem glioma, or pituitary adenoma.

MicroRNAs (hereinafter referred to as miRNAs) that can be used in the present invention include animals including humans, such as monkeys, pigs, horses, cows, sheeps, dogs (miR-186, miR-216b, miR-337-3p and miR-760 derived from human beings), preferably from dogs, cats, mice, rabbits, More preferably miR-186, miR-216b, miR-337-3p and miR-760 of SEQ ID NOS: 1-4. MiRNA nucleic acid molecules of the present invention may have a length of 18-100 nt nucleotides and preferably 19-25 nt long as mature miR-186, miR-216b, miR-337-3p and miR-760, Has a length of 21, 22 or 23 nt. In addition, the miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules of the present invention are precursors miR-186, miR-216b, miR-337 -3p and miR-760 nucleic acid molecules.

Also, miR-186, miR-216b, miR-337-3p and miR-760 used in the present invention are functional equivalents of the nucleic acid molecule constituting them, for example miR-186, miR- Although miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules were modified by deletion, substitution or insertion of some nucleotide sequences of miR-3p and miR- It is a concept that includes variants that can function in the same way as a molecule. In the present invention, the mutant is also referred to as mimics.

In addition, miR-186, miR-216b, miR-337-3p and miR-760 of the present invention may exist in a single-stranded or double-stranded form. The mature miRNA molecules are predominantly single stranded, but the precursor miRNA molecules are at least partially self-complementary (e.g., stem- and loop-structure), which can form mainly double-stranded portions. In addition, the nucleic acid molecule of the present invention can be configured in the form of RNA, DNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA).

The nucleic acid molecule of the present invention can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available. In addition, the composition of the present invention may include not only the miRNA nucleic acid molecule itself, but also other substances capable of increasing the expression rate of the miRNA of the present invention in a cell, for example, a compound, a natural product, a novel protein and the like.

Meanwhile, the miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules in the composition of the present invention can be provided as a vector for intracellular expression.

The miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules of the present invention can be used in a variety of transformation techniques such as DNA and DEAE-dextran complexes, DNA and nuclear protein complexes, DNA and lipid complexes The miR-186, miR-216b, miR-337-3p, and miR-760 nucleic acid molecules may be contained in a carrier that enables efficient introduction into the cell . The carrier is preferably a vector, and both viral vectors and non-viral vectors are usable. As the viral vector, for example, lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector can be used, Preferably a lentiviral vector, but is not limited thereto. Lentiviruses are a type of retrovirus that is not only a mitotic cell but also a mitotic cell due to the nucleophilicity of a nucleopore or a pre-integrated complex (a virus "shell") that allows active incorporation into a complete nuclear membrane There is a feature that can be made.

It is also preferred that the vector comprising miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules further comprises a selectable marker. The term "selection marker" in the present invention is intended to facilitate selection of transformed cells with the introduction of miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules. The selection marker which can be used in the vector of the present invention is not particularly limited as long as it is a gene capable of easily detecting or measuring the introduction of a vector, but it is typically a gene having resistance to drug resistance, nutritional requirement, cytotoxic agent, Such as GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol Histidinol dehydrogenase gene hisD) and guanine phosphosribosyltransferase (Gpt). Preferably, GFP (green fluorescent protein) and puromycin marker can be used.

Meanwhile, the miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules in the anticancer composition of the present invention can be provided in a form introduced into a cell.

The term "introduction" in the present invention means introduction of foreign DNA into a cell by transfection or transduction. The transfection can be carried out in the presence or absence of a sugar such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion Can be carried out by various methods known in the art. Transfection also refers to the transfer of genes into cells using virus or viral vector particles as a means of infection.

In this way, cells into which miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules were introduced exhibited high levels of miR-186, miR-216b, miR-337-3p and miR- By transplanting these cells into cancer tissues, the proliferation of cancer can be suppressed. Therefore, the composition of the present invention comprising cells into which miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules have been introduced can be used as a cell therapy agent for cancer treatment.

On the other hand, the composition comprising the nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760 of the present invention may further comprise a pharmaceutically acceptable carrier, . As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition of the present invention can be applied to any formulation containing nucleic acid molecules composed of miR-186, miR-216b, miR-337-3p and miR-760 as an active ingredient and can be manufactured into oral or parenteral formulations have. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.

Formulations for oral administration of the present invention may be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. A binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, Calcium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned materials, a liquid carrier such as a fatty oil may be further contained.

The formulation for parenteral administration of the present invention may be formulated for use such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection system, or aerosol formulation for allowing inhalation through a respiratory apparatus. For formulation into injectable formulations, the compositions of the present invention may be formulated as solutions or suspensions in water with stabilizers or buffers in water, and formulated for unitary administration of ampoules or vials. For injection into suppositories, it may be formulated into rectal compositions such as suppositories or enema preservatives, including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying, such as an aerosol formulation, a propellant or the like may be formulated with the additive such that the dispersed concentrate or wet powder is dispersed.

The present invention also provides a method of preventing or treating cancer, comprising the step of administering to a mammal a nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760.

The term "administering" as used herein means introducing the miRNA nucleic acid molecule of the present invention to a mammal in any suitable manner, and the expression of miR-186, miR-216b, miR-337-3p and miR- Or non-viral techniques, or transplantation of cells expressing miR-186, miR-216b, miR-337-3p and miR-760 nucleic acid molecules.

The method of treatment of the present invention comprises administering a therapeutically effective amount of the anti-cancer composition of the present invention. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by treatment within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, the drugs used or co-used with the specific composition, and the like, well known in the medical arts. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned factors. The daily dose of the composition of the present invention is preferably 0.1 to 10 mg / kg, and is preferably administered once to several times a day.

In addition, the anticancer effect can be enhanced by administering a known anticancer agent in combination with the composition of the present invention.

The method of treatment of cancer of the present invention is applicable to any mammal which has developed cancer induced by overexpression of the CKII alpha gene and mammals such as cows, pigs, sheep, horses, dogs and cats Of livestock.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

[ Example  1] Using bioinformatics, CKII alpha mRNA For miRNA  Screening

MiRNAs that could be targets of CKII α mRNA were selected from MicroRNA, TargetScan, miRBase, Mami, Segal lab, psRNATarget, and RNA hybrid. As a result, miR-186, miR-216b, miR-337-3p and miR-760 were selected as targets of 3'UTR of CKII alpha mRNA. This is shown in FIG. In addition, the mimic sequences of the miRNAs are shown in SEQ ID NOS: 1 to 4.

[ Example  2] miR -186, miR -216b, miR -337-3p and miR -760 Imitative CKII Confirmation of regulation of α

1. Cell culture and miR -186, miR -216b, miR -337-3p and miR -760 Imitation  HCT116 transfected human colon cancer cells

HCT116 human colon cancer cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (fetal bovine serum) at 5% CO ₂ at 37 ° C. miR-186, miR-216b, miR-337-3p and miR-760 mimic and control miRNA were purchased from Genolution Inc (Seoul, Korea) and used. The RNAs were transfected into HCT116 cells at a final concentration of 40 nM using lipofectamine RNAi max (Invitrogen, Carlsbad, Calif.). The transfected cells were analyzed by the conventional method FEBS Lett. P53 deacetylation by SIRT1 during protein kinase CKII downregulation-mediated cellular senescence). Forty-eight hours after transfection, cells were harvested.

2. HCT116  In human colon cancer cells Western Brotting  Used miR -186, miR -216b, miR -337-3p and miR -760 On the imitation  by CKII α suppression

At the protein level Western blotting was performed to confirm that the expression of CKII alpha was inhibited by miR-186, miR-216b, miR-337-3p and miR-760.

More specifically, the cells transfected in the dish were washed with cold PBS, scraped with a rubber policeman to collect the cells and washed with 100 μl of cold radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris- Cells were cultured in HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, 1 μg / ml aprotinin, 1 μg / ml leupeptin, 1 μg / ml pepstatin . The protein concentration of the supernatant was determined using a Bradford protein dye reagent (Bio-Rad, Hercules, Calif.). The supernatant was adjusted to an equivalent protein concentration and immunoblotting was performed by known methods. At this time, antibodies specific for CKIIα, CKIIβ, p53, p21Cip1 / WAF1, and β-actin purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.) Were used and anti-HA antibodies purchased from Roche (Basel, Switzerland) .

The protein expression levels of CKII [alpha] in the cells transfected with miR-186, miR-216b, miR-337-3p and miR-760 are shown in Fig. 2 and the expression levels of miR-186, miR-216b, miR- The level of protein expression of CKII [alpha] in the co-transfected cells of the combination of three miRNAs selected from the 3p and miR-760 mimetics is shown in Fig. 3 and miR-186, miR-216b, miR-337-3p and miR The expression levels of CKII [alpha] protein in all-co-transfected co-transfected cells are shown in FIG. In addition, miR-186, miR-216b , miR-337-3p , and miR-760 both mimetics co-transfected CKII in infected cells and α` CKII beta protein expression level.

As shown in Fig. 2, the expression levels of CKII a protein in the cells transfected with miR-186, miR-216b, mir-337-3p and miR-760 mimetics were not significantly different from the control group As shown in FIG. 3, the protein expression levels of the co-transfected cells of the combination of three miRNAs selected from the miR-186, miR-216b, miR-337-3p and miR-760 mimetics were significantly higher than those of the control CKII a protein expression level was not significantly different.

In addition, as shown in FIG. 4, in the cells co-transfected with miR-186, miR-216b, miR-337-3p and miR-760 mimetics, the protein expression level of CKII a was 50% , And the expression level of the protein was substantially reduced compared with the cells transfected with siRNA against CKII a positive control group.

In addition, as shown in FIG. 5, miR-186, miR- 216b, miR-337-3p , and miR-760 mimic the co-transfection in the infected cells and CKII α` CKII β was not significantly different from that of the control group.

These results indicate that miR-186, miR-216b, miR-337-3p and miR-760 mimetics of the present invention do not affect protein expression of CKII alpha in human colon cancer cells, , It was confirmed that the expression of CKII [alpha] subunit was markedly reduced, thereby effectively aging colon cancer cells and inhibiting colorectal cancer.

3. HCT116  In human colon cancer cells RT - PCR Using miR -186, miR -216b, miR -337-3p and miR -760 copy By sieve CKII α suppression

RNA was extracted from the HCT116 cells using TRIzol reagent (Invitrogen, Carlsbad, Calif.). RNA reverse transcriptase and the gene-PCR was performed using a specific reverse primer (gene-specific reverse primer) a was carried out with reverse transcription by using, as a result, the primer of the CKII α genes of Table 1 to the obtained cDNA. In order to standardize the amount of RNA in each sample, a selective primer was used for β-actin RNA, and the PCR product was subjected to electrophoresis on 1.5% agarose gel.

primer order SEQ ID NO: CKII α forward primer 5`-GACAAGCTTATGTCGGGACCC-3` SEQ ID NO: 5 CKII α reverse direction primer 5`-GACAAGCTTTTACTGCTGAGC-3` SEQ ID NO: 6

The degree of expression of CKII [alpha] mRNA in the co-transformed cells of both miR-186, miR-216b, miR-337-3p and miR-760 mimetics into HCT116 cells is shown in Fig.

As shown in Fig. 6, the amount of CKII < alpha > mRNA in the co-transformed cells of all of the miR-186, miR-216b, miR-337-3p and miR-760 mimetics in HCT116 cells Respectively.

4. Luciferase Assay  Used miR -186, miR -216b, miR -337-3p and miR -760 by imitation CKII α suppression

CKII [alpha] 3 ' UTR receptor was generated by inserting human CKII [alpha] mRNA into XhoI / NotI restriction enzyme cleavage site of siCHECK-2 vector (Promega), and the restriction enzyme cleavage site was Renilla Lucifer It is located downstream of the Lase gene. More specifically, PCR was performed using the primers shown in Table 2 below to amplify 3 ' UTR of CKII a, which was then transfected into siCHECK-2 vector (Promega). CKII α 3`UTR reporter transfected cells to 1 x 10 ⁵ miR-186, miR-216b, miR-337-3p , and miR-760 mimic the ball-to transfection, in the manufacturer's protocol And then placed on a 24-well plate using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, half-litter and renilla luciferase activity were measured using dual luciferase promega. The results are shown in Fig.

primer order SEQ ID NO: CKII α 3` UTR  Forward primer 5`-AATCTCGAGCGGCCCTATCTGTCTCCTGAT-3` SEQ ID NO: 7 CKII α 3` UTR  Reverse primer 5`-TCGCGGCCGCCACCAAAGAATTCCAACACTGGATC-3` SEQ ID NO: 8

As shown in Fig. 7, in HCT116 cells co-transfected with miR-186, miR-216b, miR-337-3p and miR-760 mimetics, luciferase activity Was reduced by 55%. In addition, it was confirmed that only the microRNAs containing all of these microRNAs inhibited the activity of luciferase.

By by the result, miR-186, miR-216b , miR-337-3p , and miR-760 are in a mutually cooperative, by targeting a region of the CKII 3`UTR α specifically to decrease the stability of the CKII α mRNA, And suppressed its expression.

[ Example  3] HCT116  In human colon cancer cells miR -186, miR -216b, miR -337-3p and miR -760 Imitative  Aging Inducibility  Confirm

One. miR -186, miR -216b, miR -337-3p and miR -760 Imitative CKII  Measurement of protein inhibitory activity

To confirm the role of miR-186, miR-216b, miR-337-3p and miR-760 in cell senescence, [γ-32P] ATP and 1 mM of synthetic CKII peptide substrate (peptide sequence: RRREEETEEE) The phosphotransferase activity of the CKII protein was measured. The results are shown in Fig.

As shown in Fig. 8, the activity of CKII protein in the extracts of cells transfected with four miRNAs was found to be lowered to 40% as compared with that of the control without miRNA.

2. miR -186, miR -216b, miR -337-3p and miR -760 Imitative SA -β- gal  Verify Active

In order to confirm that miR-186, miR-216b, miR-337-3p and miR-760 mimic induce senescence in colon cancer cells, miR-186, miR-216b, miR-337-3p and miR- After 2 days of transfection, transfection, SA-β-gal activity was measured. The measurement method is described in FEBS Lett. 584 (2010) 3137-3142 (SM Jeon, SJ Lee, TK Kwon, et al., NADPH oxidase is involved in protein kinase CKII downregulation-mediated senescence through elevation of the levels of reactive oxygen species in human colon cancer cells) . The result of measurement of the SA-beta-gal activity is shown in FIG.

As shown in FIG. 9, in the HCT116 cells co-transfected with the miR-186, miR-216b, miR-337-3p and miR-760 mimetics, SA-β-gal inhibited the miRNAs with non- The cells were stained significantly better than the cells. The above results indicate that the high activity of SA-β-gal can be used as an indicator of cell senescence, so that miR-186, miR-216b, miR-337-3p and miR-760 mimetics can inhibit cell senescence in colorectal cancer cells .

3. miR -186, miR -216b, miR -337-3p and miR -760 Imitative p53  And p21 ^{Cip1 / WAF1} Confirmation of expression enhancement effect

Western blotting was carried out in the same manner as in Example 2 to confirm that miR-186, miR-216b, miR-337-3p and miR-760 mimetics cause senescence in colon cancer cells. At this time, p53 and p21 ^{Cip1 / WAF1} , And Western blot results are shown in FIG.

As shown in Figure 10, p53 and p21 ^{Cip1 / WAF1} protein expression in co-transfected HCT116 cells with miR-186, miR-216b, miR-337-3p and miR-760 mimetics, Compared to the control group. The results show that miR-186, miR-216b, miR-337-3p and miR-760 mimetics cooperate to form CKII It is possible to inhibit the expression of the gene and increase the expression of p53 and p21 to cause cell senescence.

4. miR -186, miR -216b, miR -337-3p and miR -760 Imitative ROS (Active oxygen) Generation  Confirm

 In cells with suppressed CKII and aged cells, ROS production is known to be an up-regulator of p53 stabilization. Thus, in order to determine whether miR-186, miR-216b, miR-337-3p and miR-760 mimics induce ROS production, HCT116 cells were cultured in CM-H2DCFDA or DHE to produce dichlorofluorescein diacetate (DCF) ROS production was confirmed by ETH fluorescent image. The results are shown in Fig.

As shown in FIG. 11, in HCT116 cells co-transfected with the miR-186, miR-216b, miR-337-3p and miR-760 mimetics or both miRNAs were treated with hydrogen peroxide and excess oxidation anion the level of superoxide anion was significantly increased compared to the control group in which the miRNA was not co-transformed or the miRNA-treated control group Respectively.

These results show that miR-186, miR-216b, miR-337-3p, and miR-760 mimics induce the production of ROS by inhibiting CKII.

5. miR -186, miR -216b, miR -337-3p and miR -760 Imitative CK Through inhibition of Ⅱα Aging Inducibility  reconfirm

miR-186, miR-216b, miR-337-3p and miR-760 mimics were co-transfected into HCT116 cells co-transfected The methods of Examples 3-1 to 3-4 above were carried out without transfecting or transfecting pcDNA-HA-CKIIa. The results are shown in Fig.

As shown in Figure 12A, when co-transfected with miR-186, miR-216b, miR-337-3p and miR-760 mimetics, SA- gal activity was significantly increased and then SA-β-gal activity was restored to its original level when pcDNA-HA-CKIIα was transfected and CKIIα was overexpressed.

As shown in Figure 12B, when co-transfected with the miR-186, miR-216b, miR-337-3p and miR-760 mimetics, p53 and p21 ^{Cip1 / WAF1} expression was increased. When pcDNA-HA-CKIIα was transfected and CKIIα was overexpressed, expression of p53 and p21 ^{Cip1 / WAF1} was restored to its original level.

As shown in Figure 12C, when co-transfected with miR-186, miR-216b, miR-337-3p and miR-760 mimetics, ROS production was increased , And then the ROS level was restored to its original level when pcDNA-HA-CKIIα was transfected and CKIIα was overexpressed.

These results indicate that miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively inhibit the expression of CKIIa, inducing the production of ROS and inducing cell senescence.

<110> KYUNGBUK COLLEGE INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Compositon for preventing or treating cancer comprising miR-186,          miR-216b, miR-337-3p and miR-760 <130> P-128 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> it is miR-186 sequence <400> 1 ucggguuuuc cucuuaagaa ac 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> it is miR-216b sequence <400> 2 aguguaaacg gacgucucua aa 22 <210> 3 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> it is miR-337-3p sequence <400> 3 cuucuuuccg uaguauaucc uc 22 <210> 4 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> it is miR-760 sequence <400> 4 aggggugucu gggucucggc 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer sequence for CK2 alpha gene <400> 5 gacaagctta tgtcgggacc c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer sequence for CK2 alpha gene <400> 6 gacaagcttt tactgctgag c 21 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > it is forward primer sequence for CK2 alpha 3 &apos; UTR <400> 7 aatctcgagc ggccctatct gtctcctgat 30 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> &Lt; 223 &gt; it is reverse primer sequence for CK2 alpha 3 &apos; UTR <400> 8 tcgcggccgc caccaaagaa ttccaacact ggatc 35

Claims (17)

A pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the miR-186 comprises the nucleotide sequence of SEQ ID NO: 1. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the miR-216b is a nucleotide sequence of SEQ ID NO: 2. 2. The pharmaceutical composition according to claim 1, wherein the miR-337-3p comprises the nucleotide sequence of SEQ ID NO: 3. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the miR-760 comprises the nucleotide sequence of SEQ ID NO: 4. 2. The method according to claim 1, wherein the nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760 inhibits the expression of CKII alpha, a protein kinase. A pharmaceutical composition. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the nucleic acid molecule composed of miR-186, miR-216b, miR-337-3p and miR-760 increases the production of active oxygen. The method according to claim 1, wherein the nucleic acid molecule comprising miR-186, miR-216b, miR-337-3p and miR-760 induces cell senescence by increasing the expression of p53 and p21. Or &lt; / RTI &gt; The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is cancer caused by overexpression of the CKII alpha gene. 10. The method of claim 9, wherein the cancer induced by overexpression of the CKII alpha gene is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, Papillary carcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, childhood solid tumor, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Wherein the cancer is at least one selected from the group consisting of benign lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, first central nervous system lymphoma, cholesteatoma tumor, brainstem glioma and pituitary adenoma. &Lt; / RTI &gt; A method of preventing or treating cancer, comprising administering to a mammal, excluding a human, a therapeutically effective amount of a nucleic acid molecule consisting of miR-186, miR-216b, miR-337-3p and miR-760. 12. The method for preventing or treating cancer according to claim 11, wherein the miR-186 comprises the nucleotide sequence of SEQ ID NO: 1. 12. The method for preventing or treating cancer according to claim 11, wherein the miR-216b comprises the nucleotide sequence of SEQ ID NO: 2. 12. The method for preventing or treating cancer according to claim 11, wherein the miR-337-3p comprises the nucleotide sequence of SEQ ID NO: 3. 12. The method for preventing or treating cancer according to claim 11, wherein the miR-760 comprises the nucleotide sequence of SEQ ID NO: 4. 12. The method of claim 11, wherein the nucleic acid molecule comprising the miR-186, miR-216b, miR-337-3p, and miR-760 inhibits the expression of CKII to induce the production of active oxygen and increase the expression of p53 and p21 To induce senescence of the cells. 12. The method for preventing or treating cancer according to claim 11, wherein the mammal is at least one selected from the group consisting of cattle, pigs, sheep, horses, dogs, and cats.

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