WO2010095879A2 - Sirna to be bound to transcripts of apoptosis-related genes, and compositions for treating cancer using same - Google Patents

Abstract

The present invention relates to siRNA to be bound to transcripts of apoptosis-related genes, and to compositions for treating cancer using same. More particularly, the present invention relates to siRNA which is bound to the mRNA of TRAF7 (tumor necrosis factor receptor associated factor) genes or TAB2 (TAK1 binding protein 2) genes to inhibit the expression of said genes, thus leading to cancer cell-specific apoptosis and to compositions for treating cancer containing said siRNA as an active ingredient. The siRNA and the compositions containing same according to the present invention induce cancer cell-specific apoptosis, and therefore, can be used as effective anticancer therapeutic agents which cause no damage to normal cells.

Description

SiRNA, which binds to a transcript of apoptosis-related genes, and a composition for treating cancer using the same

The present invention relates to siRNA that binds to a transcript of apoptosis-related genes and a composition for treating cancer using the same, and more specifically, mRNA of a tumor necrosis factor receptor associated factor 7 (TAF7) or TAB2 (TAK1 binding protein 2) gene. By binding to and inhibiting the expression of the gene, the present invention relates to siRNA that leads to cancer cell specific cell death and an anticancer agent comprising the siRNA as an active ingredient.

RNA interference is a mechanism that regulates gene expression after nucleotide sequence specificity by double-stranded RNA (dsRNA) in the expression process of genes, such as C. elegans was first discovered plants, fruit flies, mammals Common to animals (Fire et al ., Nature, 391: 806-811, 1998; Novina & Sharp, Nature, 430: 161-164, 2004). RNA interference occurs when 19-25 bp dsRNA enters the cell and binds to the RNA-induced silencing complex (RISC) complex, and only the antisense (guide) strand is complementary to the mRNA and is present in the RISC complex. It is known to degrade target mRNAs by endonuclease domains (Rana, TM, Nat. Rev. Mol. Cell Biol., 8: 23-36, 2007; Tomari, Y. and Zamore, PD, Genes Dev. , 19: 517-529, 2005).

When the dsRNA is delivered into the cell, the dsRNA specifically binds to the target mRNA sequence and degrades the mRNA, and thus it is considered as a new tool that can control the expression of the gene using the dsRNA. However, in humans, the introduction of dsRNA into cells caused antiviral interferon pathways, making it difficult to see RNA interference effects.In 2001, Elbashir and Tuschl et al. When introduced into the cell, it was found that the interferon pathway was not induced and specifically degraded the target mRNA (Elbashir, SM, Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T., Nature, 411, 494-498, 2001; Elbashir, SM, Lendeckel, W., Tuschl, T., Genes & Dev., 15, 188-200, 2001; Elbashir, SM, Martinez, J., Patkaniowska , A., Lendeckel, W., Tuschl, T., EMBO J., 20, 6877-6888, 2001). Since then, 21 nt dsRNA has been spotlighted as a tool of new functional genomics under the name of siRNA (small interfering RNA).

RNA interference has several advantages over conventional antisense RNA technology as a means of functional genetics and therapy. First, in antisense RNA, a large amount of antisense RNA has to be synthesized and experimented in order to find an efficient target sequence, whereas siRNA can be predicted to some extent through some algorithms. Only a few experiments can find high efficiency siRNA.

Second, siRNA (RNAi) is known to be able to efficiently inhibit gene expression at lower concentrations than antisense RNA. This means that smaller amounts can be used when used for research and can be very effective, especially when used as a therapeutic.

Third, the inhibition of gene expression by RNAi is a mechanism that occurs naturally in vivo and its action is very specific.

Due to the above advantages, siRNAs have recently been found to have an excellent effect on inhibiting the expression of specific genes in animal cells, and have been spotlighted as gene therapeutics. Due to their high activity and precise gene selectivity, siRNA has been studied for the past 20 years. It is expected to replace antisense oligonucleotide (ODN) as a therapeutic agent. Recently developed by OPKO as a drug for the treatment of wet age-related Macular disease, 'Bevasiranib' is an siRNA that inhibits the expression of VEGF by selectively acting on vascular endotherial growth factor (VEGF) that causes angiogenesis. As part of clinical trials (Dejneka NS et al. , Mol Vis., 28 (14): 997-1005, 2008). In addition, disease therapies including siRNAs targeting various genes are currently under development (Ryan P. Million, Nature Reviews Drug Discovery 7: 115-116, 2008).

Cancer has characteristics that are not regulated in cell growth compared to normal cells, invading into adjacent tissues, and sometimes metastatic. Cancer is a disease primarily related to tissue growth control. When normal cells are transformed into cancer cells, the expression of genes that regulate cell growth and division changes (Croce CM., The New England journal of medicine, 358 (5): 502-511, 2008). Genes known as tumor suppressor genes inhibit genes associated with cell division and survival and other cancer cell characteristics (Carlos G. Ferreira et al., Clinical Cancer Research, 8: 2024-2034, 2002) They are transcriptional regulators that are activated by intracellular stress or DNA damage. These genes are involved in DNA repair and prevent mutations from transferring to daughter cells during division.

Since RNA interference is known to inhibit gene expression with high specificity and efficiency, siRNAs targeting various genes have been studied for cancer treatment. The siRNAs include cancer cells such as oncogenes, anti-apoptotic molecules, telomerases, growth factor receptor genes, signaling molecules, and the like. Inhibit the expression of genes necessary for survival or induce apoptosis (Knudson AG, Nature reviews. Cancer 1 (2): 157-162, 2001). Since no cell death is a major cause of cancer development and growth, chemical treatments such as irradiation cause apoptosis and induce cancer cell death (Carlos G. Ferreira et al ., Clinical Cancer Research, 8: 2024-2034, 2002).

The tumor necrosis factor receptor associated factor (TRAF) is a family that accepts signaling from the TNF receptor family and toll / interleukin 1 (TIR 1) receptor members. Recent studies have shown that TRAF6,7, in particular, toll-like receptor 2 (TLR2). It is known that tumor suppressor cylindromatosis (CYLD) and negative cross talking (negative cross talking), which perform negative regulation during signaling, are known (Hiroki Yoshida, THE JOURNAL OF BIOLOGICAL CHEMISTRY, 280). 49): 41111-41121, 2005). The TRAF7 gene is also known to be particularly involved in MEKK3 and MEKK3-mediated AP1 and CHOP activation and apoptosis (Liang-Guo Xu et al. , THE JOURNAL OF BIOLOGICAL CHEMISTRY, 279 (17): 17278-17282, 2004).

TAB2 [TAK (Transforming Growth Factor-β-activated Kinase) Binding Protein 2] is a protein that expresses JNK (c-Jun N-terminal kinase) and NF-kB (nuclear transcription factor kB) during IL-1 (interleukin 1) signaling. Adapter proteins for activating TAK1 and TRAF6 are known to link the two (Giichi Takaesu et al ., Molecular Cell, 5: 649-658, 2000). Recently, embryos lacking the TAB2 gene resulted in lethal death and induction of cell death, similar to those in mice lacking NF-kB, p65, IKK, and NEMO / IKK (Beg, AA et al. , Nature 376: 167-170, 1995; Li, Q., D. Van Antwerp et al. , Science, 284: 321-325, 1999; Li, ZW et al. , Exp. Med. 189: 1839-1845, 1999; Makris, C. et al. , Mol. Cell 5: 969-979., 2000; Rudolph, D. et al. , Genes Dev. 14: 854-862, 2000). Thus, TAB2 appears to play a major role in preventing liver cell death in embryonic development (Hideki Sanjo et al ., MOLECULAR AND CELLULAR BIOLOGY, 23 (4): 1231-1238, 2003).

Recent studies have shown that TRAF7 and TAB2 proteins are involved in apoptosis processes, and studies are underway to induce or delay cell death by regulating this protein (Liang-Guo Xu et al. , THE JOURNAL OF BIOLOGICAL CHEMISTRY, 279 (17): 17278-17282, 2004, Hideki Sanjo et al ., MOLECULAR AND CELLULAR BIOLOGY, 23 (4): 1231-1238, 2003). For example, US Patent Publication No. 2007-0161565 discloses a study for controlling the inflammatory response by inhibiting IL-1 signaling through TAB2 using a dominant negative mutation of TAB2, but a method using dominant negative mutation is disclosed. Inhibiting the role of the protein, it is difficult to induce mutations using gene therapy methods, and even if the mutations are temporarily induced, the efficiency is difficult to apply to the actual tumor treatment (Spitzweg C and Morris JC, Thyroid. 14 (6): 424-34, 2004). In addition, the existing siRNA treatment technology has a low protein expression inhibitory effect, there was a problem that the manufacturing process is more complicated by cloning the expression vector.

Accordingly, the present inventors can effectively treat tumor cells by inducing tumor cell-specific apoptosis by directly introducing tumor-specific siRNA capable of inactivating the mRNA by binding to mRNA transcribed from TRAF7 and TAB2 genes related to cell death. Confirmed that the present invention was completed.

It is an object of the present invention to provide a composition for treating cancer and a method of inhibiting cancer using siRNA that induces cancer cell-specific apoptosis, which has fewer side effects and higher effects than conventional cancer therapeutic agents.

In order to achieve the above object,

[1] The present invention provides an siRNA having one nucleotide sequence selected from the group consisting of nucleotide sequences shown in SEQ ID NOs: 1 to 12 and having mRNA binding activity.

[2] The present invention provides an siRNA wherein the mRNA is transcribed from a tumor necrosis factor receptor associated factor (TRAF7) gene or a TAB1 binding protein 2 (TAB2) gene.

[3] The present invention provides an siRNA that induces cancer cell specific apoptosis.

[4] The present invention provides a composition for treating cancer comprising at least one of siRNA of [1] as an active ingredient and a carrier.

[5] The present invention provides a composition for treating cancer, wherein the cancer of [4] is cancer associated with uterine cancer, lung cancer, prostate cancer, breast cancer, oral cancer, TRAF7 or TAB2 gene.

[6] The present invention provides a method for inhibiting cancer cells, comprising administering to a subject one or more siRNAs selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 1-12.

[7] The present invention provides a method for inhibiting tumor cells, comprising treating a cell with a solution containing at least one siRNA selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 1-12.

[8] The present invention provides a method for inhibiting tumor cells in which the siRNA concentration is 5 nM or more in the solution of [7].

Hereinafter, the present invention will be described in more detail.

The present invention has one nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NOs: 1 to 12, and mRNA transcribed from a tumor necrosis factor receptor associated factor (TRAF7) gene or a TAB1 binding protein 2 (TAB2) gene. It provides siRNA having binding activity.

The present inventors conducted a study to develop a method for inhibiting the expression of TRAF7 or TAB2, and when using siRNA designed to complementarily bind to the mRNA sequence encoding the gene, the expression of the gene is effectively It was found that it can be suppressed. The siRNA developed by the present inventors consists of a double-stranded RNA having a sense sequence in which a base T is converted to U in a DNA sequence of a target gene (see sequence below) and an antisense sequence complementary thereto. The double stranded RNA may also include an overhang at one or both strands' 3'-terminal sites, meaning a structure having one or more unpaired nucleotides at the double stranded RNA 3'-end. do.

5'-GUCUCUGCGUCUACUCCAU-3 '(SEQ ID NO: 1),

5'-CAAGUGCAAAGGGACCUUU-3 '(SEQ ID NO: 2),

5'-GGUUGAAAUAAAUGCUCCA-3 '(SEQ ID NO: 3),

5'-CCAAUCAGUUCACCUACUA-3 '(SEQ ID NO: 4),

5'-UCAAUAGCCAGACCUUAAA-3 '(SEQ ID NO: 5),

5'-GCACAUGUGGAUAGAAUAA-3 '(SEQ ID NO: 6),

When siRNAs of SEQ ID NOs: 1 to 3 (sample names TRAF7-1301, TRAF7-1257, and TRAF7-2449, respectively) relating to TRAF7 are introduced into tumor cells, the level of mRNA encoding TRAF7 in the cells is reduced. It was confirmed that the proliferation of tumor cells was suppressed. In particular, it was confirmed that the siRNA of SEQ ID NO: 3 not only effectively inhibits TRAF7 mRNA expression, but also induces cell death in cancer cells and effectively inhibits cancer cell proliferation.

Meanwhile, as a result of confirming that the variants prepared by partially modifying the siRNA nucleotide sequence of SEQ ID NO: 3 showed a similar effect to the siRNA of SEQ ID NO: 3, siRNAs having the modified nucleotide sequence as follows (SEQ ID NOS: 7 to 9) were also identified. In addition, it was confirmed that it can still suppress the expression of TRAF7 mRNA. The siRNA variants of SEQ ID NOS: 7-9 (sample names TRAF7-2449-M1, TRAF7-2449-M2, TRAF7-2449-M3, respectively) all replaced some bases in SEQ ID NO: 3 siRNA, as shown below.

5'-GGUUGAAAU G AAUGCUCCA-3 '(SEQ ID NO: 7),

5'-GGUUGAAAU GC AUGCUCCA-3 '(SEQ ID NO: 8),

5'-GGUUGAAAUAAAUGCU GU A-3 '(SEQ ID NO: 9),

In addition, even when introducing SEQ ID Nos. 4 to 6 (sample names TAB2-1031, TAB2-1239, and TAB2-1709, respectively), which are siRNAs for TAB2, into the tumor cells, the level of mRNA encoding TAB2 in the cells was increased. As well as lowering, it was confirmed that the proliferation of tumor cells was suppressed. In particular, it was confirmed that the siRNA of SEQ ID NO: 4 not only effectively inhibits TAB2 mRNA expression, but also induces apoptosis in cancer cells and effectively inhibits proliferation of cancer cells.

As a result of confirming that the variants prepared by partially modifying the siRNA nucleotide sequence of SEQ ID NO: 4 show a similar effect to the siRNA of SEQ ID NO: 4, siRNA having the nucleotide sequence modified as follows (SEQ ID NOs: 10 to 12) is also included. In addition, it was confirmed that still can suppress the expression of TAB2 mRNA. The siRNA variants of SEQ ID NOs: 10-12 (sample names TAB2-1031-M1, TAB2-1031-M2, TAB2-1031-M3, respectively) replaced some bases in SEQ ID NO: 4 siRNAs with the following underlined portions.

5'-CCAAUCAGU G CACCUACUA-3 '(SEQ ID NO: 10),

5'-CCAAUCAGU GU ACCUACUA-3 '(SEQ ID NO: 11),

5'-CCAAUCAGUUCACCUA UG A-3 '(SEQ ID NO: 12)

In addition, the present invention is a method for inhibiting cancer cells comprising the step of administering at least one siRNA of the siRNA as an active ingredient, a composition for treating cancer comprising a pharmaceutically acceptable carrier and at least one siRNA of the siRNA to the individual To provide.

When the siRNA is introduced in the tumor-induced animal model, it was confirmed that the proliferation of the tumor induced in the animal model is suppressed and the induced tumor is killed. In addition, it was confirmed that the siRNA of the present invention can be used as a cancer-specific anticancer agent because it did not induce apoptosis in normal cells but not tumor cells.

The composition for treating cancer of the present invention is mixed with an injection composition of siRNA capable of complementarily binding to mRNA encoding TRAF7 or TAB2 in the form of injection to the site where the tumor occurred, or gel molding or transdermal absorption adhesive composition It is preferred to mix with or directly apply to affected areas. At this time, the injectable composition is preferably an isotonic aqueous solution or suspension, and the composition is sterile and / or adjuvants (e.g. preservatives, stabilizers, wetting or emulsifier solution promoters, salts for controlling osmotic pressure, buffers and / or liposomes). Gel composition, such as carboxymethyl cellulose, methyl cellulose, acrylic acid polymer, carbopol, and a pharmaceutically acceptable carrier and / or liposome preparation. The active ingredient layer includes an adhesive layer, an adsorption layer for sebum absorption, and a therapeutic drug layer, and the therapeutic drug layer contains a pharmaceutically acceptable carrier and / or liposome preparation.

The dosage of siRNA capable of complementarily binding to the mRNA encoding TRAF7 or TAB2 of the present invention is parenteral administration of 0.05 to 0.1 μg / kg body weight, depending on the patient's age, sex, symptoms, method of administration or prophylactic purpose. can do. The level of dosage for a patient with specific symptoms can vary by the person skilled in the art depending on the weight, age, sex, health condition, diet, time of administration, method of administration, and the like of the patient.

The composition for treating cancer of the present invention can be used to treat uterine cancer, lung cancer, prostate cancer, breast cancer, oral cancer and cancer associated with TRAF7 or TAB2 gene.

In addition, the present invention provides a method for inhibiting tumor cells comprising the step of treating the cells with the solution containing the siRNA. The concentration of siRNA to be treated at this time is 5 nM or more.

A siRNA that inhibits the expression of TRAF7 or TAB2 of the present invention and a composition for treating cancer using the siRNA as an active ingredient can effectively inhibit tumor cell proliferation by inducing apoptosis of tumor cells and thus anticancer therapy for various uses. It can be widely used in.

1 is a graph showing mRNA levels of TRAF7 expressed in cell lines 24 hours after transfection with three sequences of siRNA for TRAF7 of the present invention, wherein NC is transfected with negative control siRNA (negative control siRNA) Means control group.

Figure 2 is a graph showing the mRNA level of TRAF7 expressed over time after transfection with the SEQ ID NO: 2,3 siRNA of the present invention.

3 is a graph showing the mRNA levels of TAB2 expressed in cell lines transfected with three sequences of siRNA for TAB2 of the present invention.

Figure 4 is a graph showing the mRNA level of TAB2 expressed over time after transfection with the SEQ ID NO: 4, 5 siRNA of the present invention.

5 is a graph showing the mRNA level of TAB2 expressed after transfection with siRNA of SEQ ID NO: 4 and the prior art siRNA of the present invention.

Ref: siRNA described in prior art (SEQ ID NO: 13)

TAB2-1301: siRNA of the present invention (SEQ ID NO: 4)

Figure 6 is a photograph showing whether apoptosis is induced in human prostate cancer cell line (PC3) and human lung cancer cell line (A549) transfected with the SEQ ID NO: 3, 4 siRNA of the present invention.

7 is a photograph showing whether apoptosis is induced in human breast cancer cell lines (MCF7) and human cervical cancer cell lines (HeLa) transfected with SEQ ID NO: 3, 4 siRNA.

FIG. 8 is a diagram illustrating lung cancer cell line (A549) and human articular synovial cell (synoviocyte), which are normal cells, were transfected with siRNA to compare whether siRNA SEQ IDs 3 and 4 induce apoptosis. It is a photograph observing whether death was induced.

9 is transfected with a siRNA of various concentrations SEQID 3 siRNA for TRAF7, and analyzed the degree of apoptosis in accordance with the concentration.

FIG. 10 is a photograph of transfection of SEQ ID No. 4 siRNA for TAB2 with siRNAs of various concentrations, followed by induction of apoptosis according to concentration.

FIG. 11 is a graph showing mRNA levels of TRAF7 expressed in cell lines after transfection with three variant siRNAs (TRAF7 2449-M1, M2, M3) for SEQ ID NO: 3 siRNA (TRAF7) and SEQ ID NO: 3 of the present invention. to be.

12 is a photograph showing the induction of apoptosis in a cell line transfected with three variant siRNAs to the SEQ ID NO: 3 siRNA of the present invention. * Indicates a substituted portion of the sequence.

FIG. 13 is a graph showing mRNA levels of TAB2 expressed in cell lines transfected with three variant siRNAs against the SEQ ID NO: 4 siRNA of the present invention.

Figure 14 is a photograph showing the induction of apoptosis in cell lines transfected with siRNA of SEQ ID NO: 4 and three variant siRNAs (TAB2-M1, M2, M3) for SEQ ID NO: 4 of the present invention. * Indicates a substituted portion of the sequence.

15 is a graph analyzing how the SEQID 3 siRNA of the present invention affects the growth of tumors.

TRAF7-1 to TRAF7-6: 6 rats administered human oral cancer cells treated with SEQID 3 siRNA at a concentration of 100 nM

NC-1 to NC-3: 3 mice administered oral cancer cells (KB) not transfected with siRNA

FIG. 16 compares tumor formation patterns of rats and controls treated with human oral cancer cell lines (KB) transfected with SEQID 3 siRNA after 14, 20, 30, and 43 days of cancer cell line administration. One picture.

TRAF7-1, TRAF7-2: Two rats treated with human oral cancer cell line (KB) in which SEQID 3 siRNA was treated at a concentration of 100 nM

NC-1, NC-2: Two mice administered human oral cancer cell line (KB) not transfected with siRNA

FIG. 17 is a graph illustrating how SEQID 4 siRNA of the present invention affects tumor growth after 14, 20, 30, and 43 days of administration of cancer cell lines.

TAB2-1 to TAB2-6: 6 rats administered human oral cancer cells treated with SEQID 4 siRNA at a concentration of 100 nM

NC-1 to NC-3: 3 mice administered human oral cancer cells (KB) not transfected with siRNA

Hereinafter, an Example demonstrates this invention. However, the following examples are only for illustrating the present invention and do not limit the content of the present invention.

Example 1 Identification of Target Sequence and Preparation of siRNA

First, the target base to which siRNA can bind in TRAF7 mRNA sequence (NCBI accession number: NM_032271.2, NM_206835.1) and TAB2 mRNA sequence (NM_015093.2) using the Turbo si-Designer program (Bionia, Korea). The sequence was found.

On the other hand, by using a method of linking the phosphodiester bonds that form the DNA skeleton structure using β-cyanoethyl phosphoramidite (β-cyanoethyl phosphoramidite), siRNA capable of binding to the designed target sequence Synthesized (Sinha et al., Nucleic Acids Research, 12: 4539-4557, 1984). Specifically, using an RNA synthesizer (384 Synthesizer, BIONEER, Korea), a series of processes consisting of deblocking, coupling, oxidation, and capping on a nucleotide-attached solid support Was repeated to obtain a reaction containing RNA of the desired length. Subsequently, RNA was separated from the reaction by HPLC (LC-20A Prominence, SHIMADZU, Japan), and the molecular weight was measured using a MALDI-TOF mass absorption spectrometer (MALDI TOF-MS, SHIMADZU, Japan) to prepare a sequence to be synthesized. Check if it is in conformity with. Thereafter, the same amount of sense and antisense RNA strands were mixed and placed in 1X annealing buffer (30mM HEPES, 100mM Potassium acetate, 2mM Magnesium acetate, pH 7.0 ~ 7.5), reacted for 3 minutes in a 90 ° C constant temperature water bath, and then again at 37 ° C. To prepare the desired double-stranded siRNA (SEQ ID NO: 1 to SEQ ID NO: 12), respectively. As a control siRNA, a commercially available and untargeted Accutarget negative control siRNA (NC-siRNA, BIONEER, Korea) was used.

Example 2 Inhibition of Target Gene Expression Using siRNA in Tumor Cell Line

Using each siRNA prepared in Example 1, human uterine cancer cell line (HeLa), human lung cancer cell line (A549), human prostate cancer cell line (PC3), human breast cancer cell line (MCF7) and normal cell line Joint synovial cell lines (RA Synoviocytes) were transfected, and the expression patterns of TAB2 and TRAF7 were measured and the effects of inducing apoptosis were analyzed.

Example 2-1 Tumor Cell Line Cultures

Human uterine cancer cells (HeLa), human prostate cancer cell lines (PC3) and human oral cancer cell lines (KB) obtained from the American Type Culture Collection (ATCC) were RPMI 1640 culture medium (GIBCO / Invitorgen, USA), human lung cancer. Cell line (A549) and human synovial cell line (RA synoviocyte) were cultured in DMEM medium (GIBCO / Invitorgen, USA) and human breast cancer cell line (MCF7) in EMEM medium (GIBCO / Invitorgen, USA). At this time, 10% (v / v) fetal calf serum, penicillin 100 units / ml, streptomycin 100 μg / ml were added to the medium, and cultured under 37 ° C. and 5% (v / v) CO ₂ .

Example 2-2 Inhibition Experiment of Tumor Cell Line Using siRNA

Each tumor cell line 3.3 × 10 ⁵ cultured in Example 2-1 was incubated in Opti-MEM (GIBCO, Invitorgen, USA) for 24 hours in a 6-well plate under the conditions of Example 2-1. After removal, 800 μl of Opti-MEM medium was dispensed for each well.

Meanwhile, 3 μl of Lipofectamine ^™ RNAiMAX (Invitrogen, USA) and 195 μl of Opti-MEM medium were mixed and allowed to react at room temperature for 5 minutes, followed by addition of 2 μl of each siRNA prepared in Example 1 above (50 pmole / μl). (Final 100 nM treatment), and reacted again at room temperature for 20 minutes to prepare a solution.

Thereafter, 200 μl of the transforming solution was dispensed into each well of the tumor cell line to which Opti-MEM was dispensed and incubated for 6 hours, and then the Opti-MEM medium was removed. Depending on the cell line, RPMI1640 culture medium or DMEM culture medium or 2.5 ml of EMEM was dispensed and then cultured at 37 ° C. and 5% (v / v) CO ₂ for 24 and 72 hours, respectively.

Example 2-3 mRNA Quantitative Analysis of TRAF7 and TAB2 Genes

After total RNA was extracted from the cell line transfected in Example 2-2, cDNA was synthesized, and mRNA amounts of TAB2 and TRAF7 genes were quantified by real-time PCR.

Example 2-3-1 RNA Isolation and cDNA Synthesis from Transfected Cells

RNA Extraction Kit (AccuPrep  Cell total RNA extraction kit, BIONEER, Korea), extracting the total RNA from the cell line transfected in Example 2-2, the extracted RNA is RNA reverse transcriptase (AccuPower  Using CycleScript RT Premix / dT _20, Bioneer, Korea), cDNA was synthesized as follows.

Specifically, AccuPower contained in 0.25 ml Eppendorf tubes  1 µg of extracted RNA per tube was added to CycleScript RT Premix / dT ₂₀ (Bioneer, Korea) and distilled water treated with diethyl pyrocarbonate (DEPC) was added so that the total volume was 20 µl. This is a gene amplifier (MyGenie ^™ 96 Gradient Thermal Block, BIONEER, Korea), which hybridizes RNA and primer for 1 minute at 30 ° C, synthesizes cDNA for 6 minutes at 52 ° C, and repeats the two processes 95 times. The amplification reaction was terminated by deactivating the enzyme for 5 minutes at < RTI ID = 0.0 >

Example 2-3-2 mRNA Quantitative Analysis of TRAF7

Using the cDNA synthesized in Example 2-3-1 as a template, the relative amount of TRAF7 mRNA was quantified by real-time PCR in the following manner.

In other words, in each well of a 96-well plate, dilute the cDNA synthesized in Example 2-3-1 by 1/5, and analyze 3 μl of the diluted cDNA and 2 × GreenStar ^™ PCR master to analyze the expression level of TRAF7. 10 μl of mix (BIONEER, Korea), 6 μl of distilled water, and 1 μl of TRAF7 qPCR primer (10 pmole / μl respectively, BIONEER, Korea) were prepared. In order to normalize the expression level of mRNA, 3 μl of the same cDNA and 2 × GreenStar ^™ PCR master mix (BIONEER, Korea) diluted in each well of 96-well plate using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a standard gene 10 μl, distilled water 6 μl, GAPDH qPCR primer (10 pmole / μL each, BIONEER, Korea) were added to make a GAPDH real-time PCR mixture. The 96-well plate containing the mixed solution was subjected to the following reaction using the Exicycler ^™ 96 Real-Time Quantitative Thermal Block (BIONEER, Korea): 15 minutes at 95 ° C to remove enzyme activation and cDNA secondary structure. Then, 42 cycles of four steps of 30 seconds denaturing at 94 ° C., 30 seconds annealing at 58 ° C., 30 seconds extension at 72 ° C., and SYBR green scan were performed. After performing the final extension for 3 minutes at 72 ° C, the temperature was maintained at 55 ° C for 1 minute, and the melting curve was analyzed from 55 ° C to 95 ° C. After the PCR was completed, the Ct (threshold cycle) value of TRAF7 and the Ct value of GAPDH were measured, and the ΔCt value, which is the difference between the two values, was obtained, followed by negative control siRNA (NC-siRNA, BIONEER, Korea). The ΔΔCt value, which is the difference of the ΔCt value from the transfected control, was obtained. The percent reduction in mRNA expression of TRAF7 was compared using the ΔΔCt value and Formula 2 ^(−ΔΔCt) × 100.

As a result, the levels of mRNA of TRAF7 present in the cell lines transfected with three siRNAs were 8.5% (SEQ ID NO: 1), 6.7% (SEQ ID NO: 2) of the control, and 3.9% (SEQ ID NO: 3) of the control, respectively. Was confirmed. In particular, the siRNA of SEQ ID NO: 3 shows 3.9% of the control group, and it can be seen that the expression of TRAF7 can be most effectively suppressed (FIG. 1). In addition, in order to confirm the effect of siRNA over time, comparing the mRNA amount of 24 hours and 72 hours after transfection, it was found that even after 72 hours, the expression of TRAF7 is effectively inhibited by siRNA (Fig. 2).

Example 2-3-3 mRNA Quantitative Analysis of TAB2

Using the cDNA synthesized in Example 2-3-1 as a template, the relative amount of TAB2 mRNA was quantified by real-time PCR in the following manner.

Specifically, dilute the cDNA synthesized in Example 2-3-1 to 1/5 in each well of a 96-well plate, and 3 μl of diluted cDNA and 2 × GreenStar ^™ PCR to analyze the expression level of TAB2. 10 μl of master mix (BIONEER, Korea), 6 μl of distilled water, and 1 μl of TAB2 qPCR primer (10 pmole / μl, respectively, BIONEER, Korea) were prepared. In addition, to normalize mRNA expression, 10 μl of the same cDNA and 2 × GreenStar ^™ PCR master mix (BIONEER, Korea) diluted in each well of a 96-well plate using GAPDH as a standard gene, 6 μl of distilled water, and GAPDH 1 μl of qPCR primer (10 pmole / μL each, BIONEER, Korea) was added to make a real-time PCR mixture. The 96-well plate containing the mixed solution was subjected to the following reaction using an Exicycler ^™ 96 Real-Time Quantitative Thermal Block (BIONEER, Korea): 15 minutes at 95 ° C to remove enzyme activation and cDNA secondary structure. Then, repeat the four steps of 30 seconds denaturation at 94 ° C., 30 seconds annealing at 58 ° C., 30 seconds extension at 72 ° C., SYBR green scan, and repeat the final extension for 3 minutes at 72 ° C., followed by 55 ° C. The temperature was maintained for 1 minute at and the melting curve was analyzed from 55 ° C to 95 ° C. After the PCR was completed, the Ct value of the obtained TAB2 and the Ct value of GAPDH were measured, and the ΔCt value, which is the difference between the two values, was obtained and then transfected with negative control siRNA (NC-siRNA, BIONEER, Korea). The ΔΔCt value, which is the difference between the ΔCt values and the control group, was obtained. The reduction rate of mRNA expression of TAB2 was compared using the ΔΔCt value and the formula 2 ^(−ΔΔCt) × 100.

As a result, the level of mRNA of TAB2 present in the cell lines transfected with three siRNA was 5.5% of the control group (SEQ ID NO: 4), 12.1% of the control group (SEQ ID NO: 5), 14.3% of the control group (SEQ ID NO: 6) I could confirm that. In particular, the siRNA of SEQ ID NO: 4 showed 5.5% of the control group, and it was found that the expression of TAB2 was most effectively suppressed (FIG. 3). In addition, in order to confirm the effect of siRNA of SEQ ID NO: 4,5 over time, mRNA amount was compared 72 hours after transfection. As a result, it was found that even after 72 hours, expression of TAB2 was effectively inhibited by siRNA (FIG. 4).

Example 2-3-4 Comparative Experiments on Inhibitory Effects of siRNA and Gene Expressions of the Prior Art

According to a recently published paper, embryonic cells that inhibited the expression of TAB2 did not occur anymore, and these observations have led to the study of inducing cell death by inhibiting the action of TAB2 through antibodies. MOLECULAR AND CELLULAR BIOLOGY, 2003, 23 (4): 1231-1238). In addition, studies have been conducted to induce apoptosis of cancer cells by inhibiting the action of growth factor receptors through siRNA against TAB2 (Cancer Res 2007; 67 (13): 6253-6262). Thus, the experiment was performed to compare the effect of the siRNA and the siRNA of the present invention.

Specifically, the TAB2 siRNA (Ref, 5'-GGAACGACUUCAAAGAGAATT-3 ', SEQ ID NO: 13) described in the related art (Cancer Res 2007; 67 (13): 6253-6262) and the siRNA of the present invention (SEQ ID NO: 4). ) Was transfected with 20 nM of HeLa cell line, and mRNA amount was analyzed by real-time PCR in the same manner as in Example 2-3-3 after 24 hours. As a result, when transfected with the siRNA of SEQ ID NO: 4 of the present invention, the amount of mRNA expression of TAB2 represented 8.4% of the control group, thus confirming that the target mRNA expression inhibition was higher than that of 21.1% of the siRNA described above. (FIG. 5).

Example 2-4 Apoptosis Induction Experiments on Cancer Cell Lines (Annexin V Staining)

Annexin V staining (Annexin-V-Alexa 568, Roche Applied Science, Germany) was performed to determine the extent of apoptosis in the transfected cell line in the same manner as in Example 2-2.

Specifically, 72 hours after transfection of the experimental cell lines (HeLa, PC-3, MCF-7, A549), the culture medium was removed and Annexin V-Alexa 568 Labeling solution (Roche Applied Science, Germany) was used. 100 μl / well (based on a 24-well plate) was added, followed by reaction at room temperature for 15 minutes, followed by microscopic observation (FIGS. 6 and 7). Annexin V is a protein expressed on the surface of cells in which apoptosis occurs, and it can be observed by adding a specific coloring reagent.

As a result, it was confirmed that abundant annexin V was stained in human prostate cancer cell line (PC3) and human lung cancer cell line (A549) as compared to the control group which was not transfected with siRNA of the present invention. (FIG. 6). In human breast cancer cells (MCF7) and human cervical cancer cells (HeLa), it was confirmed that apoptosis was induced by a large amount of annexin V staining compared to the control group not transfected with the siRNA of the present invention (FIG. 7). ).

Example 2-5 Apoptosis Induction Experiment on Cancer Cell Line (Plate Adhesion)

The degree of apoptosis induction in the transfected cell line in the same manner as in Example 2-2 was analyzed through a microscope (FIG. 8).

Specifically, 72 hours after transfection of the lung cancer cell line (A549) and the human articular synovial cell line (RA synoviocyte), the degree of apoptosis was confirmed under a microscope. Wherein the human joint synovial cell line is a normal cell line selected to compare whether the siRNAs of SEQ ID NOs: 3, 4 induce apoptosis specifically to cancer cells. Human lung cancer cell lines and human joint synovial cell lines generally grow on a support such as a wall and grow. When the cells die, the attached protein is degraded and detached from the plate.

As a result, the human lung cancer cell line, which is a cancer cell line, was found to have more apoptosis in the siRNA-transfected cell line compared to the control group which was not transfected with the siRNA of the present invention. The cell death was less than that of the cells. Therefore, it was confirmed that the siRNA of the present invention induces apoptosis specifically to cancer cells.

Example 2-6 Analysis of Apoptosis Inducing Effect According to SiRNA Concentration

Except for treating the siRNA at various concentrations, human uterine cancer cell line (HeLa) was transfected using the same method as in Example 2-2 to analyze the effect of apoptosis according to the concentration.

Specifically, the human uterine cancer cell line (HeLa) was transfected with various concentrations of siRNA (SEQ ID NO: 3 or 4, 100nM, 20nM, 5nM, 1nM, 0.5nM), and culture medium was removed after 72 hours. Annexin V-Alexa 568 Labeling solution was added to the cell line by 100 μl / well (24-well plate), followed by reaction at room temperature for 15 minutes, followed by observation under a microscope (FIGS. 9 and 10).

As a result, the siRNA of the present invention until treatment with siRNA at a concentration of 5 nM or more in human cervical cancer cell lines transfected with siRNA for TRAF7 (SEQ ID NO: 3, FIG. 9) or siRNA for TAB2 (SEQ ID NO: 4, FIG. 10). Compared to the control group without treatment, it was confirmed that a large amount of annexin V was stained, that is, apoptosis was induced.

Example 2-7 Analysis of TRAF7 Expression Inhibitory Effect of Modified TRAF7 siRNA

Modified TRAF7 2449-M1 (SEQ ID NO: 7), 2449-M2 (SEQ ID NO: 8), 2449-M3 (SEQ ID NO: 3) partially modified with the nucleotide sequence of siRNA (SEQ ID NO: 3), which showed excellent effects in Example 2-3. 9) were produced respectively.

Subsequently, the transformed lung cancer cell line (A549) was obtained using the same method as in Example 2-2. RNA was obtained from the obtained cancer cell line in the same manner as in Example 2-3-1, and cDNA was synthesized using this as a template. The amount of TRAF7 mRNA was analyzed for the synthesized cDNA in the same manner as in Example 2-3-2. In other words, after PCR, the Ct values of TRAF7 and GAPDH were measured, the ΔCt value, which is the difference between the two values, was obtained, and then the ΔCt with the control transfected with a negative control siRNA (NC-siRNA, BIONEER, Korea). The value of ΔΔCt, which is the difference between the values, was obtained. Reduction rate of mRNA expression of TRAF7 was compared using the above values and Formula 2 ^(−ΔΔCt) × 100.

As a result, the cell lines transfected with the three variant siRNAs had less content of TRAF7 mRNA than the cell lines transfected with the control siRNA, and this effect was effective even 72 hours after transfection with siRNA. Could be (FIG. 11).

However, in order to determine the degree of cell death induction in the cell line as in Example 2-4, as a result of analysis by Annexin V staining, the cell line transfected with the variant siRNA was transfected with the siRNA of SEQ ID NO: 3 Compared with the cell line, a small amount of annexin V was stained, indicating that apoptosis was induced less (FIG. 12).

Example 2-8 Inhibitory Effect of Modified TAB2 siRNA on TAB2 Expression

Partial modification of the nucleotide sequence of the siRNA (SEQ ID NO: 4) showing the excellent effect in Example 2-3, variants TAB2 1031-M1 (SEQ ID NO: 10), 1031-M2 (SEQ ID NO: 11), 1031-M1 (SEQ ID NO: 12) was produced.

Subsequently, the transformed lung cancer cell line (A549) was obtained using the same method as in Example 2-2. RNA was obtained from the obtained cancer cell line in the same manner as in Example 2-3-1, and cDNA was synthesized using this as a template. The amount of TAB2 mRNA was analyzed in the same manner as in Example 2-3-2 on the synthesized cDNA. That is, after completion of PCR, the Ct values of the obtained TAB2 and GAPDH were measured, respectively, and the ΔCt value, which is the difference between the two values, was obtained, and then the control group transfected with negative control siRNA (NC-siRNA, BIONEER, Korea) The value of ΔΔCt which is the difference between the values of ΔCt and ΔCt was determined. From this value, the value of 2 ^(-ΔΔCt) X 100 was obtained, and the reduction rate of mRNA expression of TAB2 was compared.

As a result, the cell lines transfected with the three variant siRNAs contained less TAB2 mRNA compared to the cell lines transfected with the control siRNA (NC-siRNA, BIONEER, Korea), and the effect was 72 hours after transfection. It can be seen that after this last time (Fig. 13).

However, in order to determine the degree of apoptosis induction in the cell line as in Example 2-4, as a result of analysis by Annexin V staining, the cell line transfected with the variant siRNA was transfected with the siRNA of SEQ ID NO: 4 Compared with the cell line, a small amount of annexin V was stained, indicating that apoptosis was induced less (FIG. 14).

Example 3 Tumor Proliferation Inhibition Experiment of TRAF7 siRNA (in vivo)

Tumor cells were injected into nude mice to induce tumor formation and confirmed the tumor growth inhibition effect of TRAF7 siRNA in vivo.

Example 3-1 Tumor Cell Line Culture and Transfection

Human oral cancer cell lines (KB) were cultured as in Example 2-1, and then transfected with siRNA of SEQ ID NO: 3 as in Example 2-2.

Example 3-2 Tumor Cell Administration and Tumor Size Measurement in Nude Mice

Tumor cells transfected in Example 3-1 were injected subcutaneously with 3 × 10 ⁶ cells per head in Balb / c nude mice (5 weeks old, CB, Korea), and then the size of the formed tumor was fixed. It was analyzed whether the siRNA of SEQ ID NO: 3 affects tumor formation inhibition.

Specifically, the transfected human oral cancer cell line (KB) in Example 3-1 was treated with trypsin and isolated, and then only cells were collected by centrifugation and suspended in PBS. The suspended cell line was counted with a hematocytometer, and 3 × 10 ⁶ cells per nude mouse were subcutaneously injected into the nude mouse once. In addition, as a control, the cell lines not transfected with siRNA of the present invention were injected in equal amounts into separate nude mice to analyze how TRAF7 siRNA affects tumor growth.

As a result, when the cell line transfected with the TRAF7 siRNA (SEQ ID NO: 3) of the present invention was administered as compared to the control group, it was confirmed that the growth rate of the tumor was low and the size was also small (FIGS. 15 and 16).

Example 4 Tumor Proliferation Inhibition Experiment of TAB2 siRNA (in vivo)

Example 4-1 Tumor Cell Line Culture and Transfection

Human oral cancer cell lines (KB) were cultured as in Example 2-1, and then transfected with siRNA of SEQ ID NO: 4 as in Example 2-2.

Example 4-2 Tumor Cell Administration and Tumor Size Measurement in Nude Mice

Tumor cells transfected with siRNA in Example 4-1 were injected subcutaneously with 3 × 10 ⁶ cells per head in a Balb / c nude mouse, and the size of the formed tumor was measured on a fixed date to determine the siRNA of SEQ ID NO. Was analyzed to affect tumor formation inhibition.

Specifically, the transfected human oral cancer cell line (KB) in Example 3-1 was treated with trypsin and isolated, and then only cells were collected by centrifugation and suspended in PBS. The suspended cell line was counted with a hematocytometer, and 3 × 10 ⁶ cells per nude mouse were subcutaneously injected into the nude mouse once. In addition, as a control, a similar injection of a cell line not transfected with the siRNA of the present invention into separate nude mice was analyzed to determine the effect of TAB2 siRNA on tumor growth.

As a result, when the cell line transfected with TAB2 siRNA (SEQ ID NO: 4) was administered as compared to the control group, it was confirmed that the tumor growth rate was low and the size was also small (FIG. 17).

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Claims (8)

SiRNA having one or more nucleotide sequences selected from the group consisting of nucleotide sequences set forth in SEQ ID NOs: 1 to 12, and having mRNA binding activity. The method according to claim 1, The mRNA is transcribed from a tumor necrosis factor receptor associated factor (TRAF7) gene or a TAB1 (TAK1 binding protein 2) gene. The method according to claim 1 or 2, SiRNA inducing cancer cell specific apoptosis. A composition for treating cancer, comprising at least one siRNA of claim 1 as an active ingredient and a carrier. The method according to claim 4, The cancer is uterine cancer, lung cancer, prostate cancer, breast cancer, oral cancer, cancer treatment composition for cancer associated with the TRAF7 or TAB2 gene. A method of inhibiting cancer cells, comprising administering to a subject one or more siRNAs selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1-12. A method of inhibiting tumor cells, comprising treating a cell with a solution comprising at least one siRNA selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1-12. The method according to claim 7, The method of inhibiting tumor cells in which the concentration of the siRNA is 5nM or more in the solution.

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