KR101899057B1 - Pharmaceutical composition for treatment of cancer comprising rna oligonucleotide - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer comprising an RNA oligonucleotide having a specific sequence and structure. More particularly, the present invention relates to a pharmaceutical composition for treating cancer comprising an RNA oligonucleotide having a specific sequence and a spiral bend structure according to the present invention, And induces apoptosis of cancer cells. Therefore, a composition comprising the RNA oligonucleotide can be usefully used for cancer treatment.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR TREATMENT OF CANCER COMPRISING RNA OLIGONUCLEOTIDE [0002]

The present invention relates to a pharmaceutical composition for treating cancer comprising an RNA oligonucleotide having a specific sequence and structure.

Cells divide and grow by the regulatory function of the cell itself, and die by themselves when the lifespan is complete or damaged, thereby maintaining an overall number balance. However, if there is a problem in the regulation function of these cells due to various reasons, the abnormal cells to be killed are infinitely proliferated and invade into the surrounding normal tissues or organs to form lumps and destroy or deform the existing structures. The condition is called cancer. There are surgery, radiation, and chemotherapy for cancer, but there are problems such as low cure rate and many side effects. Therefore, it is required to develop new anticancer drugs that can reduce the side effects and improve the treatment rate.

The incidence of pancreatic cancer is lower than that of other cancers, but it is known that cancer mortality rate is the highest among cancer patients. Pancreatic cancer is a cancer that is difficult to diagnose early, and is characterized by its ability to easily migrate into surrounding organs or lymph nodes, and the mortality rate from it is continuously increasing.

Chemotherapy and radiation therapy are used to treat pancreatic cancer. Gemcitabine, the most commonly used anticancer drug for pancreatic cancer, is used in combination with other drugs such as oxalate and 5-fluorouracil (5-FU), but it has a significant impact on the significant survival rate of patients with pancreatic cancer I'm not getting crazy.

On the other hand, researches are currently under way to use RNA oligonucleotides for the treatment of various diseases. U.S. Patent Application Publication No. 2012/0288476 discloses that an oligonucleotide in which a phosphate group is bonded at the 5'-end and a cap-free oligonucleotide expresses 1-type interferon, interleukin-18, interleukin- Can be increased.

In addition, U.S. Patent Publication No. 2012/0121551 discloses that an RNA composed of four nucleotides induces the activity of interferon-alpha to promote an immune response.

All of the published RNAs have a common feature that they contain triphosphate at the 5 ' -end. Thus, it is known that RNA does not have a cap at its 5'-end and that it contains triphosphate, which binds to retinoic acid-inducible gene I (RIG-I) protein in cells to activate the expression of interferon.

Interferon is a glycoprotein derived from most cells in which nuclei exist. Since interferon- alpha and interferon-beta genes of type 1 interferon gene clusters have been reported to be deleted in tumor cells, interferon has been known to be an important factor in the growth of tumors. Recombinant interferon- (Jun Yoshida et al ., Cancer Sci ., 2004, Vol. 95, No. 11, 858-865).

U.S. Patent Application Publication No. 2012/0288476 U.S. Patent Application Publication No. 2012/0121551

 Jun Yoshida et al., Cancer Sci., 2004, Vol. 95, No. 11, 858-865

The inventors of the present invention have found that, while researching a substance usable for the treatment of cancer, RNA oligonucleotides having a specific sequence and structure without intervening triphosphate at the 5'-terminus have been found to be interferon- 56 (interferon stimulated gene 56; ISG56) and induce apoptosis of cancer cells. The present invention has been completed based on this finding.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating cancer comprising an RNA oligonucleotide having a specific sequence and structure.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating cancer comprising an RNA oligonucleotide as an active ingredient, wherein the RNA oligonucleotide comprises a nucleotide sequence represented by SEQ ID NO: 1 (5'-N ₁ GUAGAN ₂ N ₃ - 3 ') and the nucleotide sequence represented by SEQ ID NO: 2 (5'-N ₄ N ₅ UUUGCN ₆ -3') complementarily bind to each other to form a double strand, wherein the double strand is a helical bend structure Wherein the 3'-terminal of the nucleotide sequence of SEQ ID NO: 1 and the 5'-terminal of the nucleotide sequence of SEQ ID NO: 2 are connected by a loop to form a hairpin structure, 1 has a hydroxyl group (OH) at the 5'-terminal thereof. The present invention also provides a pharmaceutical composition for treating cancer.

Treatment of a cell line with an RNA oligonucleotide having a specific sequence and a spiral bending structure according to the present invention increases the expression of ISG56 and induces apoptosis of cancer cells. Therefore, the composition comprising the RNA oligonucleotide is useful for treating cancer .

1 is a diagram showing the sequence and structure of RNA oligonucleotides prepared according to an embodiment of the present invention.
FIG. 2 is a view showing the structure of 5'-OH-iav or 5'-PPP-iav which is an RNA oligonucleotide prepared according to an embodiment of the present invention.
FIG. 3 is a graph showing an increase in the expression of ISG56 by 5'-OH-Bend-GC-8bp-PS, an RNA oligonucleotide prepared according to an embodiment of the present invention.
4 is a graph showing an increase in expression of ISG56 by 5'-OH-Long_Bend, an RNA oligonucleotide prepared according to an embodiment of the present invention.
5 shows the effect of 5'-OH-Bend-GC-8bp-PS and 5'-OH-Long_Bend, which are RNA oligonucleotides prepared according to an embodiment of the present invention, on the apoptosis inducing effect of pancreatic cancer cell line (Panc02) As shown in FIG.
FIG. 6 shows the effect of 5'-OH-Bend-GC-8bp-PS and 5'-OH-Long_Bend on the apoptosis induction effect of the normal cell line (HEK293), which is an RNA oligonucleotide prepared according to an embodiment of the present invention, As shown in FIG.
FIG. 7 is a graph showing FACS-induced effect of 5'-OH-Bend-GC-8bp-PS on the hepatoma cell line (SNU886) induced by the RNA oligonucleotide prepared according to an embodiment of the present invention.
8a and 8b are graphs showing the effect of 5'-OH-Bend-GC-8bp-PS on RNAi oligonucleotides prepared according to an embodiment of the present invention on the apoptosis inducing effect of the gastric cancer cell lines SNU216 and MKN74 using FACS This is the graph I checked.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for treating cancer comprising an RNA oligonucleotide as an active ingredient, wherein the RNA oligonucleotide comprises a nucleotide sequence (5'-N ₁ GUAGAN ₂ N ₃ -3 ') represented by SEQ ID NO: 1 and SEQ ID NO: (5'-N ₄ N ₅ UUUGCN ₆ -3 ') represented by SEQ ID NO: 1 are complementarily bonded to each other to form a double strand, the double strand has a helical bend structure, Terminal of the nucleotide sequence shown in SEQ ID NO: 1 is connected to the 5'-terminal of the nucleotide sequence shown in SEQ ID NO: 2 by a loop to have a hairpin structure, and the nucleotide sequence shown in SEQ ID NO: And a hydroxyl group (OH) at the 5'-terminal thereof.

The RNA oligonucleotides according to the present invention may comprise 8 to 100, 8 to 50, 8 to 30, 8 to 20, 10 to 100, 10 to 50, 10 to 30, 20 to 500, 10 to 200, 10 to 100, 20 to 100, 20 to 90 or 20 to 50 bases.

In the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 wherein the oligonucleotide RNA constituting the nucleotides N ₁ to N ₆ may be any one selected from the group consisting of A, G, C and U, specifically, G or C Lt; / RTI > In one embodiment according to the present invention, shown in the nucleotide sequence shown in the SEQ ID NO: 1 to N ₁ is G, N ₂ is C, and N ₃ may be a G (SEQ ID NO: 3 corresponds to a), SEQ ID NO: 2 in the nucleotide sequence of N ₄ may be a C, ₅ N is G, and N ₆ is a C (corresponding to SEQ ID NO: 4).

In addition, the RNA oligonucleotide according to the present invention has a helical bend structure between the fourth base (A) of the nucleotide sequence of SEQ ID NO: 1 and the fifth base (U) of the nucleotide sequence of SEQ ID NO: 2 Is formed.

In one embodiment according to the present invention, the helical bending structure is a structure in which the third base (U) and the fifth base (G) of the nucleotide sequence shown in SEQ ID NO: 1 are the sixth bases of the nucleotide sequences shown in SEQ ID NO: 2 (A) of the nucleotide sequence shown in SEQ ID NO: 1 and the fifth nucleotide (U) of the nucleotide sequence shown in SEQ ID NO: 2 when the nucleotide sequence is a wobble base pair with the fourth nucleotide (G) and the fourth nucleotide (U) .

The helical bending structure has a shape bent by 10 to 90 degrees, specifically, 30 to 70 degrees, more specifically 40 to 50 degrees, based on the plane formed by the double stranded RNA.

The loops in the hairpin RNA structure may be composed of at least four bases, for example, 4 to 80, 4 to 75, 4 to 70, 4 to 65, 4 to 60, 4 to 55, 4 to 50, 4 to 45, 4 to 40, 4 to 35, 4 to 30, 4 to 25, 4 to 20, 4 to 15 or 4 to 10 bases. In one embodiment of the present invention, the loop may comprise four or 73 bases. In one embodiment according to the present invention, the four nucleotide sequences constituting the loop are UUCG.

In addition, if some of the nucleotide sequences constituting the loop are complementary to each other, a Watson-Crick base pair may be formed to have a stem structure. The stem structure may comprise an AU motif with a Watson-Crick base pair formed between A and U.

The AU motif may comprise 10 to 50, 15 to 40, 20 to 35, and 25 to 30 AU base pairs. In one embodiment according to the present invention, the AU motif may consist of 26 consecutive AU base pairs.

In one embodiment of the present invention, the oligonucleotide having the hairpin RNA structure may be the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8.

The RNA oligonucleotides according to the present invention are useful for inhibiting degradation by endonuclease and for enhancing in vivo stability, at least one of phosphodiester bonds forming the RNA oligonucleotide is expressed by a phosphor A phosphorothioate bond, a boranophosphate bond, and a methylphosphonate bond. The term " bond " In a specific embodiment according to the present invention, said modification is at least one phosphorothioate bond.

The present inventors prepared RNA oligonucleotides having a hairpin RNA structure (FIG. 1), wherein 5'-OH-iav or 5'-PPP-iav had a helical bending structure (FIG. 2) (Fig. 3 and Fig. 4).

In addition, it was confirmed that the RNA oligonucleotide having the helical bending structure induces apoptosis of pancreatic cancer, liver cancer and gastric cancer cell lines without inducing apoptosis of normal cells (FIGS. 5 to 8B).

Accordingly, the RNA oligonucleotide having the helical bending structure according to the present invention increases the expression of ISG56 and induces apoptosis of cancer cells, and thus the composition comprising the RNA oligonucleotide can be usefully used for cancer treatment.

The pharmaceutical composition for the treatment of cancer of the present invention can be used for the treatment of cancer such as pancreatic cancer, liver cancer, stomach cancer, lung cancer, colon cancer, rectal cancer, thyroid cancer, esophageal cancer, kidney cancer, bladder cancer, prostate cancer, cervical cancer, breast cancer, Or ovarian cancer. ≪ / RTI >

The pharmaceutical composition for treating cancer according to the present invention may contain 10 to 95% by weight of the RNA oligonucleotide according to the present invention, which is an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further contain one or more kinds of active ingredients showing the same or similar functions in addition to the above-mentioned effective ingredients.

The pharmaceutical composition of the present invention may contain, in addition to the above-described effective ingredients for administration, one or more pharmaceutically acceptable additives such as excipients, carriers, diluents, and other adjuvants.

The dosage of the pharmaceutical composition according to the present invention may vary depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of the formulation and the age, body weight, The time of administration, the route of administration, the duration of treatment, and concurrent medications. However, for the desired effect, the effective amount of the RNA oligonucleotide contained in the pharmaceutical composition according to the present invention is such that the intracellular concentration is 1 to 1,000 nM, specifically 100 to 500 nM. The administration may be carried out once a day or divided into several doses.

In addition, the pharmaceutical composition of the present invention may be administered to a subject by various methods known in the art. The administration route can be appropriately selected by a person skilled in the art in consideration of the administration method, the volume of the body fluid, the viscosity, and the like.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1. Preparation of RNA oligonucleotides

RNA oligonucleotides capable of increasing the expression of interferon-beta or ISG56 were prepared.

First, an RNA oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 and having a triphosphate at the 5'-terminal was prepared using a technique known in the art. On the other hand, an RNA oligonucleotide composed of the nucleotide sequence shown in SEQ ID NOS: 5 to 8 and having a hydroxy group at the 5'-terminal or a phosphodiester bond is substituted with a phosphorothioate bond is referred to as an integrated DNA technology Integrated DNA Technologies) or Dharmacon.

Thus, as shown in Fig. 1, 5'-OH-iav and 5'-PPP-iav RNA oligonucleotides composed of the nucleotide sequence shown in SEQ ID NO: 5 and having a hydroxyl group or triphosphate at the 5'- Nucleotides were prepared. Further, a 5'-OH-Cont-GC-8bp RNA oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6 and having a hydroxy group at the 5'-terminal was prepared. Further, 5'-OH-Bend consisting of the nucleotide sequence shown in SEQ ID NO: 7 and having a hydroxy group at the 5'-terminal and having a phosphodiester bond substituted with a phosphorothioate bond to form an RNA oligonucleotide -GC-8bp-PS RNA oligonucleotide. Further, a 5'-OH-Long_Bend RNA oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 and having a hydroxy group at its 5'-terminal was prepared.

Example 2. Identification of RNA oligonucleotide structure

The following experiment was conducted to confirm the structure of the 5'-OH-iav and 5'-PPP-iav RNA oligonucleotides prepared in Example 1 above.

First, the RNA oligonucleotide prepared in Example 1 was dissolved in a buffer solution containing 10 mM sodium phosphate (pH 6.5), 0.01 mM EDTA, and 10 (v / v)% D ₂ O to prepare a sample. Various spectroscopic experiments were conducted by a known method. At this time, the two-dimensional NOE spectroscopic experiment (NOESY) was performed with a nuclear magnetic resonance (NMR) spectrometer (Bruker, USA) of 400, 600 and 800 MHz at a mixing time of 100 and 200 ms. In addition, experiments were carried out with a ¹ H- ¹⁵ H HSQC (heteronuclear single quantum coherence) spectroscopy at a temperature of 278 K, double quantum filtered correlation (DQF-COOSY) and homogeneous total correlation (TOCSY) spectroscopy at 125 ms mixing time, 30 ms mixing time , ¹ H- ³¹ P HETCOR (heteronuclear correlation) and ¹ H- ³¹ P Hetero-TOCSY spectroscopy, and NOESY spectroscopy at 80, 150 and 250 ms mixing times. In addition, ¹ H- ¹³ C CT-HSQC, HCCH-COZY, 2D HCCH-relayed COZY, 2D HCCH-TOCSY and 3D HCCH-TOCSY spectroscopic experiments were performed.

NMR spectroscopy results were used to determine NMR peaks of base hydrogens of the RNA oligonucleotide and H1 ', H2', H3 ', H4', and H5 '/ H5''. Approximately 563 NOE distance constraints were obtained from the NOESY spectroscopy experiment and the distance specification values were varied from 3 to 4 groups (e.g., 1.8 to 3.4 A, 1.8 to 5.0 A and 3.8 to 7.0 A, Or 1.8 to 3.4 A, 2.5 to 4.5 A, 3.5 to 6.0 A and 4.0 to 7.0 A). But did not have hydrogen bonding limitations for non-Watson-Crick bonds. The dihedral angle was obtained from ³ J _{H1 ', H2'} obtained from DQF-COZY, and the angle was fixed at -158 ± 15 degrees for all χ. Other surface angles (eg, α, β, γ, ε, ζ) were restricted to the A-type helical structure of RNA. The bulge portion, except for some β and ε, does not restrict the other back angles. Residual dipolar coupling values were measured by HSQC experiments with increased sensitivity to ± 1 Hz accuracy. In addition, an alignment tensor was analyzed by singular value decomposition to obtain an anisotropy value of -8.0 Hz and a rhombicity value of 0.32. Calculation of all structures was done with X-PLOR 3.1 and CNS. 100 structures were generated according to the distance specification, simulated annealing at 3,000 K for 10 seconds, and simulated annealing for cooling at 300 K for 50 seconds. The distance force constant was maintained at 50 ㎉ / mol / Å, and the backside constant was changed from 20 ㎉ / mol / 400 to 400 ㎉ / mol /.. The structures with the lowest energy states were refined to 300 20 to 20,, and the last 5 rest were subjected to restrained energy minimization. A total of 220 structures obtained from these were purified by adding 22 residual bipolar coupling specification values, and the force constant value of the residual bipolar coupling was maintained at 3.0 ㎉ / mol. Finally, 32 structures were obtained and analyzed with Insight II (Biosym Technologies, USA) and CURVES 5.2 software.

As a result, as shown in Fig. 2, the 5'-PPP-iav and 5'-OH-iav RNA oligonucleotides were forming a helical bending structure. Such a helical bending structure has two single strands composed of sequences of 5'-GUAGA-3 'and 5'-UUUGC-3' among the sequences constituting 5'-PPP-iav and 5'- It was confirmed that the structure was formed by forming double strands through Watson-Creek bond. Thus, the 5'-OH-Bend-GC-8bp-PS and 5'-OH-Long_Bend RNAs of the present invention, which contain the sequence of 5'-GUAGA-3 'and 5'-UUUGC-3' It can be seen that the oligonucleotide also forms a helical bending structure.

Experimental Example 1. Confirmation of increase of ISG56 expression by 5'-OH-Bend-GC-8bp-PS

The RNA oligonucleotide (5'-OH-Bend-GC-8bp-PS) linked by a phosphorothioate bond among the RNA oligonucleotides having the bending structure prepared in the present invention was confirmed to have an interferon- , The expression of ISG56 induced by the expression of interferon-beta was confirmed.

1.1. Preparation of cell line

First, 3 × 10 ⁶ HEK293T cells (ATCC, USA) were plated on a 100 mm tissue culture plate in 7 ml of DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum (FBS, Gibco, USA) And the cells were cultured at 37 ° C and 5% CO ₂ for 24 hours to prepare cell lines.

1.2. Treatment of RNA oligonucleotides

The 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide was treated with the cell line prepared in Experimental Example 1.1.

First, the cultured cells were treated with trypsin-EDTA (Gibco, USA) to remove the cells, and the cells were counted and dispensed into a 6-well plate at 1 × 10 ³ cells. Thereafter, it was cultured at 37 DEG C and 5% CO ₂ for 42 hours, and after removing the medium, 400 μL of OPTI-MEM (Gibco, USA) and RNA oligonucleotides without FBS were treated.

The treatment of the RNA oligonucleotides was carried out by adding 4 μl of Lipofectamine LTX (Invitrogen, USA) and 1 μM of 5'-OH-Cont-GC-8bp and 5'-OH-Bend- Mu] l of plus-reagent (Invitrogen, USA) were mixed and treated with 200 [mu] l of cells. The cells were then cultured for another 4 hours at 37 ° C and 5% CO _2. At this time, poly (I: C) [poly (I: C)], also known as RIG-I ligand, , And negative control group. After 4 hours, the medium was removed, and 2 ml of DMEM medium containing 10% FBS was added thereto, followed by further incubation at 37 ° C and 5% CO ₂ for 2 hours.

1.3. Expression of ISG56

In order to confirm the expression of ISG56 in the cells treated with the RNA oligonucleotide as described above, RNA was isolated by the following method.

First, the medium was removed and the cells were recovered with 500 μl of TRI-reagent (Ambion, USA), and the collected RNA was separated by adding chloroform to the collected cells. Isopropanol was added thereto to make a pellet. The pellet was washed with 75% ethanol, dried and dissolved in sterilized distilled water. DNase (Promega, USA) was added to the separated RNA and treated at room temperature for 30 minutes to remove contaminating DNA and inactivated with a stop solution. Thereafter, cDNA was synthesized from RNA by reaction with superscript III reverse transcriptase (Invitrogen, USA) at 50 ° C for 1 hour.

Real-time PCR was performed on the synthesized cDNA as a template. Specifically, real-time PCR was performed by using a hybridization method using h-tag DNA polymerase (solgent, Korea), dNTP, tetraethylammonium chloride, evagreen dye (Biotium, USA) Primer for GAPDH gene. Real-time PCR was carried out at 95 ° C for 15 minutes, followed by 40 cycles of 95 ° C for 20 seconds, 60 ° C for 40 seconds, and 72 ° C for 20 seconds. Primers targeting ISG56 and GAPDH are shown in Table 1 below.

SEQ ID NO: name order SEQ ID NO: 9 ISG56 forward 5'-gcctccttgggttcgtctacaa-3 ' SEQ ID NO: 10 ISG56 reverse 5'-tcaaagtcagcagccagtctca-3 ' SEQ ID NO: 11 GAPDH forward 5'-gcattgccctcaacgaccac-3 ' SEQ ID NO: 12 GAPDH reverse 5'-gaggccatgtgggccatgag-3 '

As a result, the change in expression of ISG56 is shown graphically in Fig.

As shown in FIG. 3, it was confirmed that the 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide having a 5'-terminal hydroxyl group and a spiral bend structure increased the expression of ISG56 to a significant level.

Experimental Example 2. Expression of ISG56 by 5'-OH-Long_Bend

In order to confirm whether or not the long hairpin RNA oligonucleotide (5'-OH-Long_Bend) among the RNA oligonucleotides having the bending structure prepared in the present invention had interferon-β expression increasing activity, the expression of ISG56 was confirmed Respectively.

All experiments were carried out in the same manner as in Experimental Example 1 above. However, the negative control group was treated with bait, and the positive control group was treated with 5'-PPP-iav. For the experimental group, 5'-OH-Long_Bend RNA oligonucleotides were treated.

As a result, the change in expression of ISG56 is shown graphically in Fig.

As shown in FIG. 4, it was confirmed that the 5'-OH-Long_Bend RNA oligonucleotide having a spiral bend structure having a hydroxy group at the 5'-terminal and having a long hairpin increased the expression of ISG56.

Experimental Example 3. Confirmation of the induction effect of 5'-OH-Bend-GC-8bp-PS and 5'-OH-Long_Bend RNA oligonucleotides on pancreatic cancer cell death

3.1. Preparation of cell line and RNA oligonucleotide treatment

First, 0.5 × 10 ⁵ Panc02 cells (Prof. Dr. med. Christiane Bruns, Universittsklinikum Magdeburg) and 2 × vitamin (Gibco, USA) diluted 50 × and 2 × NEAA (Non-essential) were added to each well of a 24- (Dulbecco's modified Eagle's medium; WELGENE, USA) containing Amino Acid solution (Sigma, USA).

On the other hand, a 1 μg / mL RNA oligonucleotide (5'-OH-Bend-GC-8bp-PS or 5'-OH-Long_Bend) and a poly (I: C) ) Were mixed with 40 [mu] l of each OPTI-MEM (Gibco, USA). Then, 1 μl of Lipofectamine LTX (Invitrogen, USA) and 0.6 μl of a positive reagent (Invitrogen, USA) were mixed and placed in 40 μl of OPTI-MEM (Gibco, USA) . Then, 40 μl of the mixture of the prepared RNA oligonucleotide or poly (I: C) [poly (I: C)] and 40 μl of the mixture of lipofectamine-plus reagents were mixed and incubated at room temperature for 20 minutes to obtain RNA oligonucleotides Or Poly I: C and lipofectamine were able to bind to each other.

Then, the oligonucleotide of 80 nucleotides ㎕ RNA-lipoic specifications were administered the vitamin conjugate to the wells containing the Panc02 cells, and incubated the cells 37 ℃ again, under the conditions of 5% CO ₂ for 24 hours. At this time, poly (I: C) [poly (I: C)], also known as RIG-I ligand, was treated as a positive control and 5'-OH-Cont-GC-8bp as a negative control.

3.2. Cell death using FACS (Fluorecence-activated Cell Sorting)

Cell lines treated with RNA oligonucleotides or poly (I: C) [poly (I: C)] as in Example 3.1 were seeded in 5 mL round-bottom tubes using 0.25% trypsin- , And the supernatant was removed by spin-down at 1500 rpm at 4 ^° C for 5 minutes. Cells were then incubated with 100 μl of 1x binding buffer (10 mM HEPES (Sigma)) containing FITC conjugated Annexin V (Biolegend, USA) and 7-aminoactinomycin D , 140 mM NaCl, 2.5 mM CaCl ₂ ) for 15 minutes in a dark room. After that, stained cells were measured by LSRFortessa flow cytometry (BD Bioscience, USA) and analyzed by FlowJo software (Treestar, USA).

As a result, the proportion of cells killed by apoptosis is shown in Fig.

As shown in FIG. 5, the 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide having a 5'-terminal hydroxyl group and a bend structure has a significant level of 5'-OH- Long_Bend RNA oligonucleotide, And the death of the cell line was induced.

Experimental Example 4. Confirmation of normal cell death inducing effect of 5'-OH-Bend-GC-8bp-PS and 5'-OH-Long_Bend RNA oligonucleotides

The normal cell line (HEK293; ATCC, USA) was divided into DMEM medium (WELGENE, USA) as in Experimental Example 3.1, and the RNA oligonucleotide was treated as described in Experimental Example 3.1. Thereafter, the cell death effect was confirmed using FACS as in Experimental Example 3.2.

As a result, the percentage of cells killed by apoptosis is shown in Fig. As shown in FIG. 6, the poly-I: C induces the death of the HEK293 cell line while the 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide having a hydroxy group at the 5'- The 5'-OH-Long_Bend RNA oligonucleotides did not induce the death of HEK293 cell line at significant levels.

As a result of synthesis of Experimental Examples 3 and 4, the 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide and the 5'-OH-Long_Bend RNA oligonucleotide were synthesized between normal cells (HEK293) and cancer cells (pancreatic cancer cells; Panc02) The cancer cells were selectively killed.

EXPERIMENTAL EXAMPLE 5. Confirmation of 5'-OH-Bend-GC-8bp-PS Oligonucleotide Inducing Effect on Hepatocellular Carcinoma Cells

The liver cancer cell line SNU886 (Korean Cell Line Bank, Republic of Korea) was divided into DMEM medium (WELGENE, USA) as in Experimental Example 3.1 and the 5'-OH-Bend- GC-8bp-PS oligonucleotide 3.1. Thereafter, the cell death effect was confirmed using FACS as in Experimental Example 3.2.

As a result, the percentage of cells killed by apoptosis is shown in Fig. As shown in FIG. 7, the 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide having a 5'-terminal hydroxyl group and a bending structure induced death of the liver cancer cell line at a significant level.

Experimental Example 6. Confirmation of 5'-OH-Bend-GC-8bp-PS oligonucleotides inducing gastric cancer cell death

(SNU216 and MKN74; Korean Cell Line Bank, Republic of Korea) were divided into DMEM medium (WELGENE, USA) as in Experimental Example 3.1, and 5'-OH-Bend-GC-8bp-PS oligonucleotides Treated in the same manner as in Experimental Example 3.1. Thereafter, the cell death effect was confirmed using FACS as in Experimental Example 3.2.

As a result, the percentage of cells killed by apoptosis is shown in FIGS. 8A and 8B, respectively. As shown in FIGS. 8A and 8B, the 5'-OH-Bend-GC-8bp-PS RNA oligonucleotide having a 5'-terminal hydroxy group and a bending structure induced the death of gastric cancer cell lines at a significant level.

<110> Korea Advanced Institute of Science and Technology <120> PHARMACEUTICAL COMPOSITION FOR TREATMENT OF CANCER COMPRISING RNA          OLIGONUCLEOTIDE <130> FPD201607-0081 <150> KR 2015-0144306 <151> 2015-10-15 <150> KR 2016-0085582 <151> 2016-07-06 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> RNA oligonucleotide <400> 1 nguagann 8 <210> 2 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> RNA oligonucleotide <400> 2 nnuuugcn 8 <210> 3 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> RNA oligonucleotide <400> 3 gguagacg 8 <210> 4 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> RNA oligonucleotide <400> 4 cguuugcc 8 <210> 5 <211> 33 <212> RNA <213> Artificial Sequence <220> <223> iav RNA oligonucleotide <400> 5 gaguagaaac aaggcuucgg ccugcuuuug cuc 33 <210> 6 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Cont-GC-8bp RNA oligonucleotide <400> 6 ggcagacguu cgcgucugcc 20 <210> 7 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Bend-GC-8bp-PS RNA oligonucleotide <400> 7 gguagacguu cgcguuugcc 20 <210> 8 <211> 89 <212> RNA <213> Artificial Sequence <220> <223> Long_Bend RNA oligonucleotide <400> 8 gguagacgaa accagauaaa aaaaaaaaaa aaaaaaaaaa aaauaauuuu uuuuuuuuuu 60 uuuuuuuuuu uuaucugguu ucguuugcc 89 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ISG56 forward primer <400> 9 gcctccttgg gttcgtctac aa 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ISG56 reverse primer <400> 10 tcaaagtcag cagccagtct ca 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 11 gcattgccct caacgaccac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 12 gaggccatgt gggccatgag 20

Claims (11)

(5'-N ₁ GUAGAN ₂ N ₃ -3 ') represented by SEQ ID NO: 1 and an RNA oligonucleotide having an RNA oligonucleotide as an active ingredient, wherein the RNA oligonucleotide comprises a nucleotide sequence represented by SEQ ID NO: (5'-N ₄ N ₅ UUUGCN ₆ -3 ') complementarily bind to each other to form double strands, wherein N ₁ to N ₆ are each selected from the group consisting of A, G, C and U Wherein either N ₁ and N ₆ form a Watson-Creek base pair, N ₂ and N ₅ form a Watson-Creek base pair, N ₃ and N ₄ form a Watson-Creek base pair), the double strand The 3'-terminal of the nucleotide sequence shown in SEQ ID NO: 1 and the 5'-terminal of the nucleotide sequence shown in SEQ ID NO: 2 are connected by a loop to form a hairpin ), And the nucleotide sequence shown in SEQ ID NO: 1 has a hydroxyl group (OH) at its 5'- A pharmaceutical composition for, the treatment of cancer.
The method according to claim 1,
The SEQ ID NO: 1 or from the nucleotide sequence shown in SEQ ID NO: 2 N ₁ to N ₆ is a pharmaceutical composition for, the treatment of cancer, characterized in that G or C.
3. The method of claim 2,
In the nucleotide sequence shown in the SEQ ID NO: 1 N ₁ is G, N ₂ is C, N ₃ is G is (corresponding to SEQ ID NO: 3), in the nucleotide sequence shown in the SEQ ID NO: 2 N ₄ is C, N ₅ is G, N, C _6, characterized in that (SEQ ID NO: 4 correspond to) a pharmaceutical composition for the treatment of cancer.
The method according to claim 1,
Wherein the loop comprises at least four bases.
5. The method of claim 4,
RTI ID = 0.0 &gt; UUCG &lt; / RTI &gt; base.
The method according to claim 1,
Wherein the loop is composed of 4 to 80 bases.
The method according to claim 6,
Characterized in that the loop forms a Watson-Crick base pair and has a stem structure, wherein the stem structure comprises an AU motif consisting of an AU base pair.
8. The method of claim 7,
Wherein the AU motif is composed of 10 to 50 AU base pairs.
The method according to claim 1,
Characterized in that the RNA oligonucleotide is a nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8.
10. The method according to any one of claims 1 to 9,
Wherein at least one of the phosphodiester linkages in which the RNA oligonucleotide forms is modified with at least one bond selected from the group consisting of a phosphorothioate bond, a boranophosphate bond and a methylphosphonate bond. A pharmaceutical composition for treating cancer.
The method according to claim 1,
Wherein said cancer is selected from the group consisting of pancreatic cancer, liver cancer, stomach cancer, lung cancer, colon cancer, rectal cancer, thyroid cancer, esophageal cancer, kidney cancer, bladder cancer, prostate cancer, cervical cancer, breast cancer, blood cancer, skin cancer, &Lt; / RTI &gt;

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