KR100861278B1 - Ribonucleic acid-based pharmaceutical composition for treating cancer - Google Patents

Abstract

An siRNA complementary to a sequence of GASC1 mRNA is provided to apoptosize cancer cells by inhibiting expression of GASC1, which is commonly expressed in the cancer cells through RNA-mediated interference(RNAi), thereby being usefully used as an excellent anti-cancer agent. A ribonucleic acid-based pharmaceutical composition for treating cancer comprises at least one siRNA which is complementarily bound to a GASC1 mRNA transcript sequence of SEQ ID : NO. 6, 9, 12 or 14 to inhibit expression of the GASC1 in cells. Further, the siRNA is a form transformed chemically by Phosphorothioate formula or boranophosphate formula. The composition additionally comprises at least one siRNA capable of inhibiting expression of Wnt-1, Hec1, Survivin, Livin, Bcl-2, XIAP, Mdm2, EGF, EGFR, VEGF, VEGFR, Mcl-1, IGF1R, Akt1, Grp78, STAT3, STAT5a, beta-catenin, WISP1, or c-myc.

Description

Ribonucleic acid-based pharmaceutical composition for treating cancer

The present invention relates to a siRNA (small interfering RNA, siRNA) that binds to the GASC1 transcript (mRNA) nucleotide sequence complementarily to inhibit the expression of GASC1 in a cell, and a ribonucleic acid pharmaceutical composition for cancer treatment.

In eukaryotic cells, proteins are synthesized in ribosomes and undergo various modifications. Post-translational histone modifications include phosphorylation, acetylation, and methylation. Formulas except methyl have been known to be reversible. The discovery of lysine-specific demethylase 1 (LSD1), an enzyme specific for lysine, revealed that the methyl group modification was also reversible (Shi Y. et al., Cell, 119: 941, 2004). The recently discovered enzyme, JHDM1 (jumonji C (JmjC) -domain-containing histone demethylase 1), preferentially demethylates H3K36me2 through oxidation using Fe (II) and α-ketoglutatate. (Tsukada Y et al., Nature, 439: 811, 2006).

In the case of LSD1 and JHDM1, mono or dimethylation was known to act only on lysine residues, and in 2006, Y. Shi and P.Cloos reversible trimethylation. JMJD2 group was found (Cloos PA et al., Nature, 442: 307, 2006, Whetstine JR et al., Cell, 125: 467, 2006). JMJD2C demethylates H3K9me3 and H3K9me3 in the form of a single methyl group on nucleosomes (Adam G. West et al., EMBO reports, 7: 1206, 2006). Also called gene amplified in squamous cell carcinorma (GASC1) and esophageal cancer, It is a putative oncogene that is highly regulated in small and breast cancers. Overexpression of GASC1 reduces heterochromatin formation by eliminating heterochromatin protein-1 (HP1). Reducing GASC1 inhibits cell proliferation. It is not yet known exactly why the demethylation function of GASC1 is required for cell proliferation and how it contributes to cancer formation. (Arianne H., Nat. Rev. Mol. Bio., 7: 1, 2006)

Ribonucleic acid-mediated interference (RNAi) is a siRNA with 21-25 nucleotide-sized double-helix structures that specifically binds to a transcript that has a complementary sequence (mRNA transcript) to break down the transcript to express specific proteins. This phenomenon is suppressed. In recent years, studies on the use of ribonucleic acid-mediated interference phenomena in the development of chemical synthetic medicines have been conducted to selectively inhibit the expression of specific proteins at the transcript level and to use them in the development of various therapeutic drugs, especially tumor therapies. It is becoming. Small molecule chemical drugs require long development time and development costs to be optimized for specific protein targets, while the biggest advantage of siRNA medicine using ribonucleic acid mediated interference is incapable of medicinal The development of lead compounds optimized for all protein targets, including non-druggable target materials, can be accelerated. While protein and antibody drugs have difficulty in production due to complex manufacturing process, siRNA has the advantage of being relatively easy to mass-produce due to the ease of synthesis and separation purification, and has higher storage stability than protein medicine due to the characteristics of nucleic acid material. . It is also emerging as a new drug candidate based on several advantages, such as being able to antagonize specific molecular targets unlike conventional drugs (David et al., Nature Chemical Biology, 2: 711-719, 2006). .

The first consideration in treatment with siRNA is to select the optimal siRNA sequence with the highest activity in the target sequence. The efficiency of ribonucleic acid mediated interference is known to have a significant effect on the specific binding site to the target transcript. Based on the database of the past few years, algorithms have been developed and provided to the users to design the sequence position of siRNA that does not only bind to the transcript but actually inhibits the expression of the target ribonucleic acid. However, it cannot be said that all siRNAs determined by in silico method using computer algorithm can effectively inhibit target ribonucleic acid in real cells and in vivo. Although siRNAs may meet the requirements of complementary binding to target transcripts, several factors have yet to be identified, including the stability and intracellular location of ribonucleic acids and proteins, and the status of proteins involved in ribonucleic acid-mediated interference. Are known to be involved in determining the efficiency of ribonucleic acid mediated interference phenomena. Therefore, it is necessary to carry out a technique for selecting siRNA by selecting several target sequence positions per transcript of a gene and finding an optimal sequence having excellent expression suppression effect among these candidate groups (Derek et al., Annu. Rev. Biomed. Eng., 2006.8: 377-402).

An object of the present invention is to provide a ribonucleic acid pharmaceutical composition for cancer treatment comprising at least one siRNA that binds to the GASC1 transcript (mRNA) sequence and inhibits the expression of GASC1 in cells.

The present invention provides a cancer treatment comprising one or more siRNAs that complementarily bind to the GASC1 transcript (mRNA) sequences of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 14 to inhibit the expression of GASC1 in cells. Provided is a ribonucleic acid pharmaceutical composition.

The ribonucleic acid pharmaceutical composition for cancer treatment containing siRNA that inhibits the expression of GASC1 of the present invention may include only one siRNA so as to selectively bind to only one sequence position of the transcript (mRNA) of GASC1, or more than one sequence It may also include two or more siRNAs to enable targeting at the location.

In the present invention, the term “siRNA” includes siRNAs that have been chemically modified to prevent rapid degradation by nucleases in vivo. One skilled in the art can synthesize and modify the siRNA in a desired manner using methods known in the art. Andreas Henschel, Frank Buchholz 1 and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs.Nucleic Acids Research 32 (Web Server Issue): W113-W120. Since siRNA has a double helix structure, it is relatively more stable than single-stranded ribonucleic acid or antisense oligonucleotide, but since it is rapidly decomposed by nucleic acid degrading enzymes in vivo, it can be reduced by chemical modification. have. Methods of chemically modifying siRNAs to make them stable and resistant so that they are not readily degraded are well known to those skilled in the art. The most commonly used method of chemical modification of siRNA is phosphorothioate or boranophosphate modification. These substances stably form the linkage between the nucleosides of the siRNA, consequently conferring resistance to nucleic acid degradation. Ribonucleic acid modified with boranophosphate is characterized by poor nucleic acid degradation, but is not synthesized by chemical reactions, and is synthesized only by boranophosphate entering ribonucleic acid by in vitro transcription. Boranophosphate formula belongs to a relatively easy method, but has a disadvantage that it is difficult to modify in a specific position. On the other hand, phosphorothioate modification method has the advantage of introducing elemental sulfur to the desired part, but severe phosphothioation reduces the efficacy, toxicity, nonspecific RNA-induced silencing complex (RISC) Problems such as formation may appear. Therefore, in recent years, in addition to the above two methods, a method of chemically modifying only the end position of ribonucleic acid (above the 3 'end) to give resistance to nuclease is more preferred. In addition, chemical modification of the ribose ring is known to increase resistance to nucleases. Particularly, the variation in the ribose 2'-position originally present in the cell stabilizes the double helix ribonucleic acid. However, when the methyl group is precisely entered at this position, the stability is increased, and too many methyl groups have the problem that the ribonucleic acid mediated interference phenomenon is lost. The reason for such chemical modification is also to increase the efficacy and increase the pharmacokinetic residence time in vivo (Mark et. Al., Molecular Therapy, 13: 644-670, 2006).

In addition to chemical modification methods, a safe and efficient delivery system is required to increase the intracellular delivery efficiency of siRNA. To this end, the siRNA of the present invention may be included in a pharmaceutical composition for treating cancer in the form of a complex with a nucleic acid delivery system.

Nucleic acid carriers for delivering nucleic acid materials into cells can be largely divided into viral and non-viral vectors. The most widely used is a viral vector because of its high delivery efficiency and long duration. Among various viral vectors, retroviral vectors, adenovirus vectors, and adeno-associated viral vectors are mainly used. Such a viral vector is efficient in intracellular delivery of ribonucleic acid, but has various problems in terms of safety, such as recombination into a virus having in vivo activity, inducing an immune response, and random insertion into a host chromosome. In contrast, nonviral vectors have advantages over viral vectors, which have a low toxicity and immune response, can be repeatedly administered, easily form complexes with ribonucleic acids, and facilitate mass production. Further, ligands specific to diseased cells or tissues are conjugated to non-viral vectors to enable long-term cell-selective nucleic acid delivery. As the non-viral vector, various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles and the like can be used. Nucleic acid carriers can significantly enhance the transport efficiency of a desired nucleic acid in an animal cell and can be delivered to any animal cell depending on the purpose of use of the nucleic acid to be delivered.

In one embodiment of the invention, the nucleic acid carrier may be a cationic liposome.

The cationic liposomes include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, EDOPC), 1- Palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, EPOPC), 1,2-dimyristoyl-sn- Glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1, 2-distearoyl-sn- glycero-3-ethylphosphocholine (SPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, EDPPC ), 1,2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3- trimethylammonium-propane, DOTAP), N- [1- (2,3-dioleoyloxy) propyl] -N , N, N-trimethylammonium chloride (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride, DOTMA) and 3ß- [N- (N '(N', N'-dimethylamino) Ethane) -camamoyl] call And one or more cationic lipids selected from the group consisting of resterol (3ß- [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol, DC-Cholesterol).

In addition, the cationic liposomes include, but are not limited to, 1,2-diacyl-sn-glycero-3-phosphoethanolamine (1,2-diacyl-sn-glycero-3-phosphoethanolamine, DOPE), 1 , 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, DPhPE), 1,2-dioleoyl-sn-glycero-3 -Phosphocholine (1,2-dioleoyl-sn-glycero-3 -phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3- [phospho-L-serine] (1,2- dioleoyl-sn-glycero-3- [phospho-L-serine], DOPS), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3 -ethylphosphocholine, DO-Ethyl-PC), cholesterol and the like may further comprise one or more auxiliary lipids.

In another embodiment of the invention, the nucleic acid carrier may be a cationic polymer.

The cationic polymer is, but is not limited to, poly-L-lysine (PLL), poly-L-ornithine, poly-L-histidine (poly-L -histidine), polytetrahydrofuran, bis (3-aminopropyl) terminated (polytetrahydrofuran, bis (3-aminopropyl) terminated, PTHF), polyacrylamide (PA), poly (α- [4- Aminobutyl] -L-glycolic acid (poly (α- [4-aminobutyl] -L-glycolic acid, PAGA), poly (2-aminoethyl propylene phosphate) (poly (2-aminoethyl propylene phosphate), PPE-EA) Cationic derivatives of cyclodextrin, poly (2- (dimethylamino) ethyl methacrylate, pDMAEMA), poly (4-vinylpyridine) ( poly (4-vinylpyridine), P4VP), O, O'-bis (2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol (O, O'-Bis (2-aminopropyl) p olypropylene glycol-block-polyethylene glycol-block-polypropylene glycol), poly-N-ethyl-4-vinylpyridinium tribromide (PVP), chitosan, polyamidoamine, PAMAM), fractured PAMAM, and polyethyleneimine (PEI).

Since the nucleic acid carrier of the present invention having a formulation of a cationic liposome, a cationic polymer, or the like has a positive charge, the nucleic acid carrier and the nucleic acid are electrostatically bonded by a simple mix by the positive charge of the nucleic acid carrier and the anionic charge of the nucleic acid. Can form complexes of the liver.

In addition, the composition for treating cancer of the present invention can be expected to have a combined effect by additionally including a conventionally known anticancer chemotherapeutic agent in addition to siRNA that inhibits the expression of GASC1. Anticancer chemotherapeutic agents that can be coadministered with siRNAs that inhibit the expression of GASC1 of the present invention include, for example, cisplatin, carboplatin, oxaliplatin, doxorubicin, and daunorubicin ( daunorubicin, epirubicin, epirubicin, idarubicin, mitoxantrone, valubicin, curcumin, gefitinib, erlotinib, erlotinib, irinotecan (irinotecan), topotecan (topotecan), vinblastine (vinblastine), vincristine (vincristine), docetaxel (docetaxel), paclitaxel (paclitaxel) and the like can be used.

The present invention may further comprise a pharmaceutically acceptable carrier in addition to siRNA or a complex of siRNA with a nucleic acid carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide fast release, or sustained or delayed release of the active ingredient after administration.

The siRNA of the present invention or a complex of siRNA with a nucleic acid carrier can be introduced into cells for the treatment of cancer. As can be seen in the following examples, the introduction of the siRNA or the complex of the siRNA and nucleic acid carriers of the present invention into the cell is suppressed the expression of GASC1 involved in the generation of cancer to kill the cancer cells.

Introduction of the desired nucleic acid into cells in vivo or ex vivo through the ribonucleic acid pharmaceutical composition for cancer treatment of the present invention selectively reduces the expression of GASC1, a target protein, or modifies mutations in the target gene. Thus, cancer caused by overexpression of GASC1 can be treated.

In the present invention, the therapeutically effective amount of the siRNA or the complex of siRNA and nucleic acid carrier means the amount required for administration in order to anticipate a cancer therapeutic effect. Therefore, the type of disease, the severity of the disease, the type of nucleic acid to be administered, the type of formulation, the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and the duration of treatment, the chemotherapy drug used simultaneously, etc. It can be adjusted according to various factors including a drug. It is preferable to administer the cancer treatment composition to an adult at a dose of 0.001 mg / kg to 100 mg / kg once daily.

In the following example, siRNAs were synthesized by selecting candidate sequence groups suitable for siRNA composition effective for suppressing expression in the entire transcript sequence of GASC1, and then treated with tumor cell lines using carriers such as liposomes and cationic polymers to mediate ribonucleic acid. Expression interference by was confirmed by reverse transcriptase reaction at the transcript stage. In addition, to evaluate the effect of GASC1 inhibition on tumor cell growth, tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl was treated in small-interfering ribonucleic acid treated groups against GASC1. Staining method using tetrazolium bromide, lactate dehydrogenase (LDH) measurement, Annexin V-FITC / PI staining and cell staining were used.

According to the following examples, the present invention inhibits the expression of GASC1 in cells by complementarily binding to GASC1 transcript (mRNA) sequences of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 14 Among siRNAs, siRNA having a sense sequence of SEQ ID NO: 26 and an antisense sequence of SEQ ID NO: 27, a siRNA having a sense sequence of SEQ ID NO: 32, and an antisense sequence of SEQ ID NO: 33, a sense sequence of SEQ ID NO: 38, and an antisense sequence of SEQ ID NO: 39 SiRNA having a siRNA having a sense sequence of SEQ ID NO: 42 and an antisense sequence of SEQ ID NO: 43 was found to effectively inhibit the expression of GASC1 in particular.

Therefore, the siRNA included in the ribonucleic acid pharmaceutical composition of the present invention is preferably the sense sequence of SEQ ID NO: 26 and the antisense sequence of SEQ ID NO: 27.

Therefore, the siRNA included in the ribonucleic acid pharmaceutical composition of the present invention is preferably the sense sequence of SEQ ID NO: 32 and the antisense sequence of SEQ ID NO: 33.

Therefore, the siRNA included in the ribonucleic acid pharmaceutical composition of the present invention is preferably the sense sequence of SEQ ID NO: 38 and the antisense sequence of SEQ ID NO: 39.

Therefore, the siRNA included in the ribonucleic acid pharmaceutical composition of the present invention is preferably the sense sequence of SEQ ID NO: 42 and the antisense sequence of SEQ ID NO: 43.

The present invention therefore also provides an siRNA that inhibits the expression of GASC1 and has a sense sequence of SEQ ID NO: 26 and an antisense sequence of SEQ ID NO: 27.

The present invention therefore also provides an siRNA that inhibits the expression of GASC1 and has a sense sequence of SEQ ID NO: 32 and an antisense sequence of SEQ ID NO: 33.

The present invention therefore also provides an siRNA that inhibits the expression of GASC1 and has a sense sequence of SEQ ID NO: 38 and an antisense sequence of SEQ ID NO: 39.

The present invention therefore also provides an siRNA that inhibits expression of GASC1 and has a sense sequence of SEQ ID NO: 42 and an antisense sequence of SEQ ID NO: 43.

The ribonucleic acid pharmaceutical composition of the present invention may further include an siRNA that inhibits the expression of cancer-related genes other than GASC1.

The siRNA further included in the ribonucleic acid pharmaceutical composition of the present invention may be any siRNA for a gene causing cancer by overexpression. For example, siRNAs that inhibit the expression of genes other than GASC1 include Wnt-1, Hec1, Survivin, Livin, Bcl-2, XIAP, Mdm2, EGF, EGFR, VEGF, VEGFR, Mcl-1, IGF1R, Akt1, Grp78 , STAT3, STAT5a, β-catenin, WISP1, c-myc may be selected from the group consisting of siRNA that inhibits the expression.

The composition of the present invention further comprises an siRNA that inhibits the expression of cancer-related genes other than GASC1, compared to the case of processing siRNA against a single gene, siRNAs for cancer-related genes whose cancer is caused by overexpression of the gene. In the case of combined production and treatment of each can more effectively cure cancer.

The invention also provides the use of siRNA for the manufacture of an anticancer agent comprising siRNA included in the composition of the invention described above.

The invention also provides a method of treating cancer comprising introducing the composition of the invention described above into cells of a subject.

Cancer treatment in the present invention includes the prevention and suppression of cancer.

The siRNA complementary to the nucleotide sequence of the GASC1 transcript of the present invention kills cancer cells by inhibiting the expression of GASC1 commonly expressed in cancer cells by ribonucleic acid mediated interference (RNAi). The composition of can be used as an excellent anticancer agent.

Figure 1 shows the results of the GASC1 transcript expression inhibitory effect mediated by GASC1 expression inhibition siRNA in human liver cancer cell line Hep3B using reverse transcriptase polymerase chain reaction.

Figure 2 shows the effect of tumor cell death mediated by GASC1 inhibitory siRNA in human liver cancer cell line using MTT (tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) staining. The results confirmed by Hep3B are shown.

Figure 3 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in human liver cancer cell line Hep3B using lactate dehydrogenase (LDH) assay.

4 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in Hep3B, a human liver cancer cell line, using annexin V-FITC / PI staining.

Figure 5 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in human liver cancer cell line Hep3B using a cell staining method using a crystal violet (crystal violet) dye.

FIG. 6 shows MTT (tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2, mediated by apoptosis of GASC1 expression inhibitory siRNA and sicl for suppressing Mcl-1 and Wnt-1 expression; Using the 5-diphenyl tetrazolium bromide staining method, Hep3B is a human liver cancer cell line.

Figure 7 shows the results of confirming the apoptosis effect of chemically modified GASC1 expression inhibitory siRNA in human liver cancer cell line Hep3B using lactate dehydrogenase (LDH) assay.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Example ]

Example  One. GASC1  For suppressing expression siRNA Can combine target  Design of Sequence Candidates

A target sequence candidate group to which siRNA can bind in the transcript of GASC1 was designed.

First, a siRNA design program for a desired nucleotide sequence was used to design a target nucleotide sequence to which siRNA can bind in the GASC1 mRNA sequence (NM_015061).

Table 1 shows the siRNA design program and URL provided on the Internet, Table 2 is a candidate group of the target sequence of siRNA by the in silico method finally selected in the present invention using the programs of Table 1. The number in the open reading frame (ORF) sequence of Table 2 describes the sequence sequence using “A” of ATG as the start codon.

Table 1 . siRNA design program

Design program name URL siRNA Sequence Designer (Clontech) http://bioinfo.clontech.com/rnaidesigner/sirnaSequenceDesign.do GenScript siRNA Target Finder http://www.genscript.com/ssl-bin/app/rnai siRNA Design Tool (Qiagen) http://www1.qiagen.com/Products/GeneSilencing/CustomSiRna/SiRnaDesigner.aspx Find siRNA sequences (Invivogen) http://www.sirnawizard.com/design.php siRNA Selection Program (WI) http://jura.wi.mit.edu/bioc/siRNAext/ siRNA Target Finder (Ambion) http://ambion.com/techlib/misc/siRNA_finder.html

Table 2 . Target sequence candidate group to which siRNA can bind in GASC1 mRNA (NM_015061)

SEQ ID NO: ORF sequence number Sequence One 33 CCCCAGCUGUAAGAUAAUG 2 117 AGGAGCCCAUCGUGCGGGU 3 218 UUCAGCAGAUGGUCACAGG 4 352 GAUUUGGAGCGCAAGUACU 5 521 AUACCCCAUAUCUCUAUUU 6 729 GAUGACAUUGAUUUCUCCA 7 846 UCAUGGUUUCAACUGUGCA 8 1005 ACAAGGAAAGGAUAUAUAC 9 1206 CCCCGACUCAGUCACAGAU 10 1427 GUGUACCUUCUAUAUCCAG 11 1778 GUGAUGAAGAAUUGCCUGA 12 1926 GCCACACUGUGCCAUCUGC 13 2112 UGCCUUCCUUGAAGAGGAU 14 2607 CGUGAAGUCCAAGGCUUGC 15 3062 AGAGACAAAGAGUGCUGAG

Example  2. GASC1  For suppressing expression siRNA  Production of candidates

Fifteen small interfering ribonucleic acids that can bind to the target nucleotide sequence designed in Example 1 were prepared by Ambion (Ambion Inc., Texas, USA), a Silencer siRNA Construction kit was synthesized according to the method described in the instructions. The sequences of 15 siRNA candidate groups are shown in Table 3.

Table 3 . Base sequence candidate group of siRNA for GASC1 expression inhibition

Example SEQ ID NO: Sense base sequence (5'-3 ') ORF target sequence number Antisense Sequence (5'-3 ') 2-1 16 CCCCAGCUGUAAGAUAAUGTT 33 17 CAUUAUCUUACAGCUGGGGTT 2-2 18 AGGAGCCCAUCGUGCGGGUTT 117 19 ACCCGCACGAUGGGCUCCUTT 2-3 20 UUCAGCAGAUGGUCACAGGTT 218 21 CCUGUGACCAUCUGCUGAATT 2-4 22 GAUUUGGAGCGCAAGUACUTT 352 23 AGUACUUGCGCUCCAAAUCTT 2-5 24 AUACCCCAUAUCUCUAUUUTT 521 25 AAAUAGAGAUAUGGGGUAUTT 2-6 26 GAUGACAUUGAUUUCUCCATT 729 27 UGGAGAAAUCAAUGUCAUCTT 2-7 28 UCAUGGUUUCAACUGUGCATT 846 29 UGCACAGUUGAAACCAUGATT 2-8 30 ACAAGGAAAGGAUAUAUACTT 1005 31 GUAUAUAUCCUUUCCUUGUTT 2-9 32 CCCCGACUCAGUCACAGAUTT 1206 33 AUCUGUGACUGAGUCGGGGTT 2-10 34 GUGUACCUUCUAUAUCCAGTT 1427 35 CUGGAUAUAGAAGGUACACTT 2-11 36 GUGAUGAAGAAUUGCCUGATT 1778 37 UCAGGCAAUUCUUCAUCACTT 2-12 38 GCCACACUGUGCCAUCUGCTT 1926 39 GCAGAUGGCACAGUGUGGCTT 2-13 40 UGCCUUCCUUGAAGAGGAUTT 2112 41 AUCCUCUUCAAGGAAGGCATT 2-14 42 CGUGAAGUCCAAGGCUUGCTT 2607 43 GCAAGCCUUGGACUUCACGTT 2-15 44 AGAGACAAAGAGUGCUGAGTT 3062 45 CUCAGCACUCUUUGUCUCUTT

Example  3. GASC1  For suppressing expression siRNA Wow Cationic With liposomes  Preparation of the complex

Fifteen kinds of siRNA for inhibiting expression of GASC1 prepared synthetically in Example 2 and a complex with a cationic liposome delivering the same were prepared.

First, 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE), a cell fusion phospholipid, cholesterol, and cationic phospholipid 1 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) (Avanti Polar Lipid Inc., USA) molar ratio 1: 1: 1 The mixture was taken in a glass vial and mixed, and then rotary evaporated at low speed until all chloroform was evaporated in a nitrogen environment to prepare a lipid thin film. To prepare a lipid multilamellar vesicle, 1 ml of a phosphate buffer solution was added to the thin film, and the vial was sealed at 37 ° C., and then stirred (vortexing) for 3 minutes. Cationic liposomes were prepared by passing a 0.2 μm polycarbonate membrane three times using a particle homogenization maker (extruder, Northern Lipid Inc., Canada) to make a uniform size. The obtained cationic liposomes were mixed with each of 15 kinds of siSCs for inhibiting GASC1 expression of Example 2 and maintained at room temperature for 20 minutes to prepare a complex with siRNA and cationic liposomes. Table 4 summarizes the composition of the siRNA and the cationic liposomes prepared in Example 3.

Table 4 . Composition of the complex of siRNA prepared with Example 3 with a cationic liposome

Example GASC1 siRNA Cationic liposomes 3-1 Example 2-1 DOPE + Cholesterol + EDOPC 3-2 Example 2-2 3-3 Example 2-3 3-4 Example 2-4 3-5 Example 2-5 3-6 Example 2-6 3-7 Example 2-7 3-8 Example 2-8 3-9 Example 2-9 3-10 Example 2-10 3-11 Example 2-11 3-12 Example 2-12 3-13 Example 2-13 3-14 Example 2-14 3-15 Example 2-15

Example  4. GASC1  For suppressing expression siRNA Wow Cationic  Preparation of Complexes with Polymers

Three complexes were prepared by mixing GASC1 expression inhibitory siRNA synthesized in Example 2 with polyethyleneimine (PEI), a cationic polymer. Molecular weight 25kD polyethyleneimine (PEI) (Sigma-Aldrich, USA) was dissolved in water, adjusted to a molar concentration of 1 mM, taken in a Pyrex 100 ml glass round bottom flask and dissolved until adjusted to pH 5 with 1N hydrochloric acid. . In order to remove impurities, a cationic polymer was prepared by passing a 0.2 μm membrane through a syringe filter. Two kinds of siRNA for inhibiting GASC1 expression prepared in Example 2 were selected as shown in Table 4, mixed with the cationic polymer prepared above, and left at room temperature for 20 minutes to prepare a complex.

Table 5 . Composition of the complex of siRNA prepared by Example 4 with a cationic polymer

Example GASC1 siRNA Cationic polymer 4-1 Example 2-6 + Example 2-9 Polyethyleneimine 4-2 Examples 2-12 + Examples 2-14

Comparative example  One. Luciferase GL2 ( luciferase GL2 ) Expression suppression siRNA Wow Cationic  Preparation of Complexes with Liposomes

As a negative control for comparing the cytotoxicity of siRNA itself, the siRNA that inhibits the expression of the commercially available luciferase GL2 was used as Samchully Pharmaceuticals, Seoul, Korea). The base sequence of the siRNA for inhibiting luciferase GL2 expression is 5'-CGUACGCGGAAUACUUCGATT-3 'in the forward direction and 5'-UCGAAGUAUUCCGCGUACGTT-3' in the reverse direction. A siRNA for inhibiting luciferase GL2 expression was mixed with the cationic liposomes prepared in Example 3 and maintained at room temperature for 20 minutes to prepare a complex with siRNA and cationic liposomes.

< Liver cancer cell line Hep3B Cultivation of>

Hep3B cell line, a liver cancer cell, was purchased from ATCC (American Type Culture Collection, USA). Hep3B cell lines were cultured in DMEM (Dulbecco's modified eagles medium, Gibco, USA) containing 10% fetal calf serum w / v (HyClone laboratories Inc, USA) and 100 unit / ml penicillin or 100 μg / ml streptomycin.

Example  5. Reverse transcription  Using polymerase chain reaction GASC1  Suppress expression siRNA GASC1 on Transcript  Expression inhibition efficacy evaluation

In order to evaluate the effect of the siRNA-containing composition on cancer cell death on GASC1 experiments were performed using the reverse transcriptase polymerase chain reaction in the following process.

Hep3B cell lines were seeded 8 × 10 μs of cells per well in 24-well plates the day before the experiment. When the cells of each plate grew uniformly by 50-70%, the medium was removed and fresh medium was added at 250 μL per well.

50 μl of serum-free medium was added to the Eppendorf tube, and the composite composition of the luciferase GL2 expression inhibitory siRNA of Comparative Example 1 and the cationic liposome, and 15 kinds of GASC1 expression inhibitory to Examples 3-1 to 3-15 The composite composition of the siRNA and the cationic liposome was added, respectively. The final concentration of siRNA contained in the culture medium was adjusted to 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a CO 2 cell incubator at 37 ° C. for 24 hours.

After 24 hours, the whole ribonucleic acid (RNA) is isolated from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and this ribonucleic acid is complementary deoxyribonucleic acid using AccuPower RT PreMix (Bioneer, Daejeon, Korea). reverse transcription with (cDNA). The primer sequences specific for GASC1 used for polymerase chain reaction were 5'-CACCTGCTGAGGGAGAAGTC-3 '(left) and 5'-TCCCCTTGG ATAATGTCTGC-3' (right), and the size of the polymerase chain reaction product was 250 base pairs. . The expression level of the GASC1 transcript was measured quantitatively by correcting the band density of the GASC1-specific chain reaction product by amplifying the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript.

Figure 1 shows the results of the GASC1 transcript expression inhibitory effect mediated by GASC1 expression inhibition siRNA in human liver cancer cell line Hep3B using reverse transcriptase polymerase chain reaction. Figure 1A is a quantification of the relative expression of the transcript of GASC1, Figure 1B is a representative electrophoresis picture showing the expression level of the transcript of GASC1. In Figure 1, "C" is a control group, "NC" is a comparative example 1 treatment group, 3-1 to 3-15 shows a complex treatment group from Examples 3-1 to 3-15. In the control group (C), the expression of GASC1 transcript was observed in the untreated group, and Comparative Example 1 treatment group (NC) was the luciferase GL2 ribonucleic acid treated group, and there was no change in the expression of the GASC1 transcript as compared to the control group, Example 3 The complex treated groups of -1 to 3-15 showed various expression inhibitory effects compared to the control group. The expression of GASC1 transcripts was significantly reduced in the complex treatment groups of Examples 3-6, 3-9, 3-12, and 3-14. Therefore, it can be seen from FIG. 1 that siRNAs prepared in Examples 2-6, 2-9, 2-12, and 2-14 are delivered into Hep3B cells to selectively inhibit the expression of GASC1.

Example  6. MTT  Method GASC1  Suppress expression siRNA Of antitumor efficacy

In order to evaluate the effects of siRNA-containing compositions on GASC1 on cancer cell death, MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) method was used as follows. The experiment was carried out by the process.

Hep3B cell lines were seeded 8 × 10 μs of cells per well in 24-well plates the day before the experiment. When the cells of each plate grew uniformly by 50-70%, the medium in the plate was removed, and fresh medium without serum was added at 250 μL per well.

50 μl of serum-free medium was added to the Eppendorf tube, and the composite composition of the luciferase GL2 expression inhibitory siRNA and the cationic liposome of Comparative Example 1, and 15 GASC1 expression inhibitory siRNAs of Examples 3-1 to 3-15 And a composite composition with cationic liposomes were added, respectively. The final concentration of siRNA contained in the culture medium was 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a CO 2 cell incubator at 37 ° C. for 24 hours.

After 48 hours of treatment with the complex, MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) solution was added to 10% of the medium and incubated for another 4 hours. The supernatant was then removed and 0.06 N hydrochloric acid isopropanol solution was added and its absorbance was measured at 570 nm using an ELISA reader. As a control, untreated cells were used.

Figure 2 shows the effect of tumor cell death mediated by GASC1 inhibitory siRNA in human liver cancer cell line using MTT (tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) staining. The results confirmed by Hep3B are shown. In Figure 2, "C" is a control group, "NC1" is a nucleic acid carrier cationic liposome single treatment group, "NC2" is Comparative Example 1 treatment group, 3-1 to 3-15 are Examples 3-1 to 3- The complex treatment group of the siRNA up to 15 and cationic liposomes is shown. The control group “C” was untreated group, and the tumor cell survival rate was estimated to 100%. The carrier liposome-treated group “NC1” did not contain siRNA, so there was no significant change in cell viability compared to the control group. "Luciferase GL2 inhibitory siRNA had no effect on tumor cell viability, and the complex treatment group of the siRNAs of Examples 3-1 to 3-15 with cationic liposomes was compared with the control group, Example 3-6 , 3-9, 3-12, 3-14 group treated with reduced tumor cell viability. Therefore, it can be seen from FIG. 2 that siRNAs prepared in Examples 2-6, 2-9, 2-12, and 2-14 are delivered into Hep3B cells to selectively inhibit the expression of GASC1 and thereby exhibit antitumor efficacy. have.

Example  7. Lactic Acid Dehydrogenase  ( LDH ) Method GASC1  Suppress expression siRNA Of antitumor efficacy

LDH Cytotoxicity Detection Kit (TAKARA Bio Inc.), which highly sensitively detects lactate dehydrogenase (LDH) released from cells due to tumor cell damage, to evaluate the extent of siRNA-containing compositions that inhibit the expression of GASC1. , Otsu Shiga, Japan) was used to perform the experiment as follows.

Hep3B cell lines were seeded 8 × 10 μs of cells per well in 24-well plates the day before the experiment. When the cells of each plate grew uniformly by 50-70%, the medium was removed, and 250 μl of wells were added per well.

50 μl each of serum-free medium was added to the Eppendorf tube, and the composite composition of the luciferase GL2 expression inhibitory siRNA and the cationic liposome of Comparative Example 1, the siRNA and cation for inhibiting GASC1 expression of Examples 3-1 to 3-15 Each of the complex compositions with sex liposomes was added. The final concentration of siRNA included in the media was set to 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a 37 ° C. CO 2 cell incubator. At this time, Triton X-100 was treated to 3% to prepare for measurement of the maximum LDH activity was also incubated in 37 ℃ CO₂ cell culture. 48 hours after the complex was treated, the tissue culture plate was centrifuged at 250 × g for 10 minutes, and then 100 μl of the supernatant was transferred to a separate transparent 96-well plate, and the reaction mixture was prepared according to the manufacturer's protocol. Added. After the plate was shielded and allowed to stand at room temperature for 30 minutes, its absorbance was measured at 492 nm using an ELISA reader. A culture medium containing nothing was used as a negative control and cells treated with 3% Triton X-100 were used as a positive control. Tumor cell damage rate was calculated using the formula [(absorbance of experimental group-absorbance of negative control group) / (absorbance of positive control group-absorbance of negative control group) x 100].

Figure 3 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in human liver cancer cell line Hep3B using lactate dehydrogenase (LDH) assay. In Figure 3, "C" is a control group, "NC1" is a nucleic acid carrier cationic liposome single treatment group, "NC2" is Comparative Example 1 treatment group, 3-1 to 3-15 are Examples 3-1 to 3- The complex treatment group of 15 is shown. The control group “C” was untreated group, and there was no change in tumor cell damage rate. The cationic liposome-treated group “NC1” did not contain siRNA, so there was no significant change in tumor cell damage rate compared to the control group. The soldier "NC2" did not cause significant changes in tumor cell damage rate because luciferase GL2 inhibitory siRNA did not cause ribonucleic acid mediated interference, and the treatment groups of Examples 3-1 to 3-15 were compared to the control group, Example 3 Tumor cell damage rate was increased in the complex treatment group of -6, 3-9, 3-12, 3-14 siRNA and cationic liposomes. Accordingly, it can be seen from FIG. 3 that siRNAs prepared in Examples 2-6, 2-9, 2-12, and 2-14 are delivered into Hep3B cells to selectively inhibit expression of GASC1 and have antitumor efficacy as a result. have.

Example  8. Fluorescence Flow cell  Through analysis GASC1  Suppress expression siRNA Of cancer cell death

In order to evaluate the effect of the siRNA-containing composition that inhibits the expression of GASC1 on cancer cell death, experiments were performed by the following procedure using fluorescence flow cytometry.

Complex composition of luciferase GL2 expression inhibitory siRNA of Comparative Example 1 and cationic liposomes in liver cancer cell line (Hep3B), GASC1 expression inhibitory siRNA of Example 3-3, 3-6, and 3-12 with cationic liposomes The composite composition, the composite composition of two siRNA for inhibiting GASC1 expression of Example 4-1, 4-2 and a polyethyleneimine cationic polymer were treated and evaluated for cell death. As an evaluation method, an Annexin V-FITC Apoptosis Detection kit purchased from BD Biosciences, USA was used.

Hep3B cell lines were seeded 2 × 10 ⁵ cells per well in 6-well plates the day before the experiment. When the cells of each plate grew uniformly by 40-50%, the medium in the plate was removed, and fresh medium containing no serum was added at 1400 μl per well.

50 μl each of serum-free medium was added to the eppendorf tube, and the luciferase GL2 expression inhibitory siRNA of Comparative Example 1 and a complex of liposomes, and the GASC1 expression inhibitory siRNAs of Examples 3-3, 3-6, and 3-12 A composite composition with a cationic liposome, and a composite composition with two siRNAs for inhibiting GASC1 expression in Examples 4-1 and 4-2 and a polyethyleneimine cationic polymer were added, respectively. The final concentration of siRNA contained in the culture medium was adjusted to 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a 37 ° C. CO 2 cell incubator. 92 hours after the complex was treated, the cultured cells were collected, washed twice with phosphate buffer solution, and stained for 30 minutes with light shielding using annexin V-FITC and propium iodide (PI) provided by the kit. . Subsequently, apoptosis efficiency by shifting the fluorescence intensity peak was analyzed using a fluorescence flow cytometer, BD FACS CALIBUR (BD Bioscience, USA). Ribonucleic acid untreated cell group, annexin V-FITC and PI alone treated cells were used to correct the shift of fluorescence intensity peak, respectively.

4 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in Hep3B, a human liver cancer cell line, using annexin V-FITC / PI staining. In Figure 4, (A) is a complex of Comparative Example 1, (B) is a complex composition of Example 3-3, which had no GASC1 transcript inhibitory effect, (C) is a complex composition of Example 3-6, (D) Is the composite composition of Example 3-12, (E) is the composite composition of Example 4-1, (F) represents the composite composition treatment group of Example 4-2. In the composite composition treatment group of Comparative Example 1, the ratio of annexin V positive cells, that is, cells in which apoptosis occurred was 10% (see FIG. 4A). In Example 5, there was no GASC1 transcript inhibitory effect. In the group treated with the composite composition of Example 3-3, which showed no antitumor efficacy in 6 and 7, the annexin-positive cells were 15%, similar to those of the comparative example 1 treatment group (FIG. 4B). On the other hand, the siRNA compositions of Examples 3-6 and 3-12 showed a significant increase in apoptosis-prone cells as the percentage of annexin positive cells was 80% and 84%, respectively (FIGS. 4C and 4D). . Complex composition of Example 4-1 (siRNA of Example 2-6 + siRNA of Example 2-9) and complex composition of Example 4-2 comprising two GASC1 expression inhibiting siRNAs (Example 2- In the group treated with siRNA of 12 + siRNA of Examples 2-14, the percentage of annexin-positive cells undergoing apoptosis was increased to 91% and 90%, respectively (FIGS. 4E and 4F). Therefore, it can be seen from FIG. 4 that the siRNA compositions prepared in Examples 3-6 and 3-12 selectively inhibit the expression of GASC1 and have antitumor efficacy as a result, and also Example 4-1 and Example 4- It can be seen that the use of two effective siRNAs together as observed in the complex composition treatment group 2 also has significant anti-tumor efficacy.

Example  9. Through remaining cell staining method GASC1 siRNA Of cancer cell death

In order to evaluate the effect of the GASC1 siRNA-containing composition on cancer cell death, experiments were performed using the remaining cell staining method as follows.

A complex of luciferase GL2 expression inhibitory siRNA of Comparative Example 1 and a cationic liposome in a liver cancer cell line (Hep3B), and a complex of a cationic liposome with a siRNA for inhibiting GASC1 expression of Examples 3-1, 3-9, and 3-14 Compositions, composite compositions of two siRNAs for inhibiting GASC1 expression of Example 4-1 and 4-2 and a polyethyleneimine cationic polymer were treated and evaluated for cell death. As an evaluation method, a cell staining method using a crystal violet dye (dye) was used.

Hep3B cell lines were seeded by 2 × 10 ⁵ cells per well in 6-well plates the day before the experiment. When the cells of each plate grew uniformly by 40-50%, the medium in the plate was removed, and fresh medium containing no serum was added at 1400 μl per well.

50 μl each of serum-free medium was added to the Eppendorf tube, and the complex composition of luciferase GL2 expression inhibitory siRNA and liposome of Comparative Example 1, siRNA for inhibitory GASC1 expression of Examples 3-1, 3-9, and 3-14 And a composite composition of liposomes, and a composite composition of two siRNAs for inhibiting GASC1 expression in Examples 4-1 and 4-2 and a polyethyleneimine cationic polymer. The final concentration of siRNA contained in the culture medium was adjusted to 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the composite composition thus prepared was added to a well plate and cultured in a CO 2 cell incubator at 37 ° C. After 92 hours of treatment with the composite composition, the solution was washed with phosphate buffer solution, treated with 500 µl of a solution containing 0.5% crystal violet and 20% methanol, stained for 1 minute, and then the solution was removed. Was observed. Figure 5 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in human liver cancer cell line Hep3B using a cell staining method using a crystal violet (crystal violet) dye. In Figure 5, (A) is a complex of Comparative Example 1, (B) is a complex composition of Example 3-1, which did not have GASC1 transcript inhibitory efficacy, (C) is a complex composition of Example 3-9, (D) The composite composition of Example 3-14, (E) shows the composite composition of Example 4-1, (F) shows the composite composition treatment group of Example 4-2. The ribonucleic acid and liposome composition treatment group (Example 5-1) of Example 3-1, which does not have GASC1 expression inhibitory ability, as compared to the complex treatment group of Comparative Example 1 (FIG. 5A), was apoptosis due to the large amount of residual cells attached to the well plates. Did not show a significant difference, whereas in Example 5, the composition of Examples 3-9, the compositions of Examples 3-14, and the compositions of Examples 4-1 and 4-2, in which GASC1 expression was suppressed (FIG. 5C, 5D, 5E, and 5F) showed that the damaged tumor cells fell off the well plate and the percentage of stained remaining cells was reduced. Therefore, it can be seen from FIG. 5 that the siRNA and liposome compositions prepared in Examples 3-9 and 3-14 selectively inhibit the expression of GASC1 and as a result have antitumor efficacy, and also Examples 4-1 and 4- It can be seen that the use of two effective siRNAs together as observed in the composition treatment group of 2 also has significant antitumor efficacy.

Example  10. GASC1  Suppress expression siRNA And other gene expression inhibition siRNA Of antitumor efficacy after combination treatment

In addition to the siRNA against GASC1, we tried to evaluate the effect of the siRNA that inhibits the expression of two or more genes in combination with another siRNA that induces cancer cell death. To this end, the experiment was performed using the MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) method as described below.

Hep3B cell lines were seeded 8 × 10 μs of cells per well in 24-well plates the day before the experiment. When the cells of each plate grew uniformly by 50-70%, the medium in the plate was removed, and fresh medium without serum was added at 250 μL per well.

50 μl of serum-free medium was added to the eppendorf tube, and the composition of the luciferase GL2 expression inhibitory siRNA of Comparative Example 1 and the cationic liposome complex composition, and the GASC1 expression inhibitory of Example 3-12, which had the best antitumor activity in Example 6 Complex composition of a siRNA with a cationic liposome, a siRNA for inhibiting GASC1 expression of Example 2-12 and a cationic liposome of a siRNA for suppressing Mcl-1 expression, a siRNA for inhibiting GASC1 expression of Example 2-12 And a complex composition with cationic liposomes of siRNA for Wnt-1 expression inhibition were added, respectively. Mcl-1 (Catalog No .: M-004501-04) and Wnt-1 (Catalog No .: M-003937-00) siRNAs for suppressing expression were purchased from DHARMACON (Lafayette, CO, USA).

The final concentration of each siRNA in the culture medium was 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a CO 2 cell incubator at 37 ° C. for 24 hours.

After 48 hours of treatment with the complex, MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) solution was added to 10% of the medium and incubated for another 4 hours. The supernatant was then removed and 0.06 N hydrochloric acid isopropanol solution was added and its absorbance was measured at 570 nm using an ELISA reader. As a control, untreated cells were used.

Figure 6 shows the effect of tumor cell death mediated by a combination of GASC1 inhibitory siRNA and Mcl-1 and Wnt-1 siRNA, MTT (tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl Using tetrazolium bromide staining method, Hep3B is a human liver cancer cell line. In Figure 6, "C" is a control group, "NC1" is a nucleic acid carrier cationic liposomes only group, "NC2" is Comparative Example 1 treatment group, "siGASC1" is the siRNA and cationic liposomes of Examples 3-12 Complex treatment group, “siGASC1 + siMcl-1”, “siGASC1 + siWnt-1”, is a combination of a siRNA for inhibiting GASC1 expression with a siRNA for suppressing expression of Mcl-1 or Wnt-1 and a cationic liposome of Examples 2-12. The composite treatment group is shown. The control group “C” was untreated group, and the tumor cell survival rate was estimated to 100%. The carrier liposome-treated group “NC1” did not contain siRNA, so there was no significant change in cell viability compared to the control group. "Luciferase GL2 inhibitory siRNA had no effect on tumor cell viability, and Mcl-1 or" siGASC1 + siMcl-1 "and" siGASC1 + siWnt-1 "in the GASC1 expression inhibitory siRNAs of Examples 2-12 Treatment of the composition further comprising a siRNA for Wnt-1 expression suppression resulted in a reduction in tumor cell viability compared to treatment with the siRNA of Example 3-12 expressed as "siGASC1" and only the complex treatment group with the cationic liposome. Therefore, from FIG. 6, a composition additionally containing siRNAs that induce cancer cell death, such as Mcl-1 and Wnt-1 expression inhibitory siRNAs, along with GASC1 expression inhibitory siRNAs, is delivered into Hep3B cells to determine the genes targeted by each siRNA. It can be seen that the selective inhibition of expression and as a result shows a higher anti-tumor efficacy than when the siRNA for inhibiting GASC1 expression alone.

Example  11. Chemically modified GASC1  For suppressing expression siRNA Wow Cationic With liposomes  Preparation of the complex

A complex with a cationic liposome that modified and delivered phosphorothioate at the 3 ′ end of the siRNA for inhibiting the expression of 15 GASC1 siRNAs of Example 2 was prepared.

Chemical modification of siRNA for inhibiting GASC1 expression was performed by Samchully Pharmaceuticals, Seoul, Korea) to order and use.

For the preparation of cationic liposomes, 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol, cell fusion phospholipids 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) (Avanti Polar Lipid Inc., USA) The mixture was taken in a molar ratio of 1: 1: 1, mixed in a glass vial, and then evaporated at low speed until all chloroform was evaporated in a nitrogen environment to prepare a lipid thin film. To prepare a lipid multilamellar vesicle, 1 ml of a phosphate buffer solution was added to the thin film, and the vial was sealed at 37 ° C., and then stirred (vortexing) for 3 minutes. Cationic liposomes were prepared by passing a 0.2 μm polycarbonate membrane three times using a particle homogenization maker (extruder, Northern Lipid Inc., Canada) to make a uniform size.

The resulting cationic liposomes were mixed with 15 kinds of GASC1 expression inhibitory siRNAs modified with phosphorothioate at the 3 ′ end of the siRNA, respectively, and maintained at room temperature for 20 minutes to prepare a complex with siRNA and cationic liposomes.

Table 6 summarizes the composition of the conjugated siRNA modified with the phosphorothioate prepared through Example 11 and the cationic liposome.

Table 6 . Composition of the complex of the phosphorothioate-modified siRNA prepared with Example 11 with a cationic liposome

Example GASC1 siRNA Cationic Liposomes 11-1 SiRNA of Example 2-1 modified with phosphorothioate DOPE + Cholesterol + EDOPC 11-2 SiRNA of Example 2-2 modified with phosphorothioate 11-3 SiRNA of Example 2-3 modified with phosphorothioate 11-4 SiRNA of Examples 2-4 modified with phosphorothioate 11-5 SiRNA of Example 2-5 modified with phosphorothioate 11-6 SiRNA of Examples 2-6 modified with phosphorothioate 11-7 SiRNA of Examples 2-7 modified with phosphorothioate 11-8 SiRNA of Example 2-8 modified with phosphorothioate 11-9 SiRNA of Examples 2-9 modified with phosphorothioate 11-10 SiRNA of Examples 2-10 modified with phosphorothioate 11-11 SiRNA of Examples 2-11 modified with phosphorothioate 11-12 SiRNA of Examples 2-12 modified with phosphorothioate 11-13 SiRNA of Example 2-13 modified with phosphorothioate 11-14 SiRNA of Examples 2-14 modified with phosphorothioate 11-15 SiRNA of Examples 2-15 modified with phosphorothioate

Example 12 Evaluation of Antitumor Efficacy of Chemically Modified GASC1 Inhibitory siRNA Using Lactic Acid Dehydrogenase (LDH) Method

LDH Cytotoxicity Detection Kit for high sensitivity detection of lactate dehydrogenase (LDH) released from cells due to tumor cell damage in order to assess the extent of siRNA-containing compositions that inhibit the expression of chemically modified GASC1. (TAKARA Bio Inc., Otsu Shiga, Japan) was used to perform the experiment in the following process.

Hep3B cell lines were seeded 8 × 10 μs of cells per well in 24-well plates the day before the experiment. When the cells of each plate grew uniformly by 50-70%, the medium was removed, and 250 μl of wells were added per well.

50 μl each of serum-free medium was added to the Eppendorf tube, and the composite composition of the luciferase GL2 expression inhibitory siRNA and the cationic liposome of Comparative Example 1, and the 3 ′ terminal excess portion of the siRNA of Examples 11-1 to 11-15 To the above, 15 complexes of GASC1 expression inhibitory siRNA and cationic liposomes chemically modified with phosphorothioate were added. The final concentration of siRNA included in the media was set to 50 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a 37 ° C. CO 2 cell incubator. At this time, Triton X-100 was treated to 3% to prepare for measurement of the maximum LDH activity was also incubated in 37 ℃ CO₂ cell culture. 48 hours after the complex was treated, the tissue culture plate was centrifuged at 250 × g for 10 minutes, and then 100 μl of the supernatant was transferred to a separate transparent 96-well plate, and the reaction mixture was prepared according to the manufacturer's protocol. Added. After the plate was shielded and allowed to stand at room temperature for 30 minutes, its absorbance was measured at 492 nm using an ELISA reader. A culture medium containing nothing was used as a negative control and cells treated with 3% Triton X-100 were used as a positive control. Tumor cell damage rate was calculated using the formula [(absorbance of experimental group-absorbance of negative control group) / (absorbance of positive control group-absorbance of negative control group) x 100].

Figure 7 shows the results of confirming the apoptosis effect of GASC1 expression inhibitory siRNA in human liver cancer cell line Hep3B using lactate dehydrogenase (LDH) assay. In Figure 7, "C" is a control group, "NC1" is a nucleic acid carrier cationic liposome only treatment group, "NC2" is Comparative Example 1 treatment group, 11-1 to 11-15 are Examples 11-1 to 11- The complex treatment group of 15 is shown. The control group “C” was untreated group, and there was no change in tumor cell damage rate. The cationic liposome-treated group “NC1” did not contain siRNA, so there was no significant change in tumor cell damage rate compared to the control group. The soldier "NC2" did not cause significant change in tumor cell damage rate because luciferase GL2 inhibitory siRNA did not cause ribonucleic acid mediated interference, and the treatment groups of Examples 11-1 to 11-15 were compared with the control group, Example 11 Tumor cell damage rate was increased in the complex treatment group of -6, 11-9, 11-12, 11-14 with siRNA and cationic liposomes. Thus, siRNA for inhibiting GASC1 expression of chemically modified GASC1 of Examples 11-6, 11-9, 11-12, and 11-14 from FIG. 7 was transferred into Hep3B cells to selectively inhibit the expression of GASC1 and consequently antitumor. It can be seen that it has efficacy.

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<220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 19 acccgcacga ugggcuccut t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 20 uucagcagau ggucacaggt t 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 21 ccugugacca ucugcugaat t 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 22 gauuuggagc gcaaguacut t 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 23 aguacuugcg cuccaaauct t 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 24 auaccccaua ucucuauuut t 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 25 aaauagagau augggguaut t 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 26 gaugacauug auuucuccat t 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 27 uggagaaauc aaugucauct t 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 28 ucaugguuuc aacugugcat t 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 29 ugcacaguug aaaccaugat t 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 30 acaaggaaag gauauauact t 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 31 guauauaucc uuuccuugut t 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 32 ccccgacuca gucacagaut t 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 33 aucugugacu gagucggggt t 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 34 guguaccuuc uauauccagt t 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 35 cuggauauag aagguacact t 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 36 gugaugaaga auugccugat t 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 37 ucaggcaauu cuucaucact t 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 38 gccacacugu gccaucugct t 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 39 gcagauggca caguguggct t 21 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 40 ugccuuccuu gaagaggaut t 21 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 41 auccucuuca aggaaggcat t 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 42 cgugaagucc aaggcuugct t 21 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 43 gcaagccuug gacuucacgt t 21 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, sense sequence <220> <223> siRNA against GASC1, sense sequence <400> 44 agagacaaag agugcugagt t 21 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA against GASC1, antisense sequence <220> <223> siRNA against GASC1, antisense sequence <400> 45 cucagcacuc uuugucucut t 21

Claims (19)

Ribo for cancer treatment comprising one or more siRNAs that complementarily bind to the GASC1 transcript nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 14 to inhibit expression of GASC1 in cells Nucleic acid pharmaceutical composition. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 1, wherein the siRNA is chemically modified by phosphorothioate modification or boranophosphate modification. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 1, wherein the siRNA is contained in a complex form with a nucleic acid carrier. 4. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 3, wherein the nucleic acid carrier is a cationic liposome. The method of claim 4, wherein the cationic liposome is 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, EDOPC), 1- Palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, EPOPC), 1,2-dimyristoyl-sn- Glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1, 2-distearoyl-sn-glycero-3-ethylphosphocholine (SPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, EDPPC ), 1,2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), N- [1- (2,3-dioleoyloxy) propyl] -N , N, N-trimethylammonium chloride (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride, DOTMA) and 3ß- [N- (N '(N', N'-dimethylamino) Ethane) -carbamoyl] cholesterol (3ß- A ribonucleic acid pharmaceutical composition for cancer treatment comprising at least one cationic lipid selected from the group consisting of [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol, DC-Cholesterol). The method of claim 5, wherein the cationic liposome is 1,2-diacyl-sn-glycero-3-phosphoethanolamine (1,2-diacyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2 -Diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, DPhPE), 1,2-dioleoyl-sn-glycero-3-force Focoline (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3- [phospho-L-serine] (1,2-dioleoyl- sn-glycero-3-phospho-L-serine], DOPS), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, DO-Ethyl-PC) and cholesterol, ribonucleic acid pharmaceutical composition for cancer treatment further comprising one or more auxiliary lipids selected from the group consisting of. 4. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 3, wherein the nucleic acid carrier is a cationic polymer. The method according to claim 7, wherein the cationic polymer is poly-L-lysine, poly-L-ornithine, poly-L-histidine , Poly-L-arginine, bis (3-aminopropyl) terminated polytetrahydrofuran (bis (3-aminopropyl) terminated polytetrahydrofuran), polyacrylamide (PA), poly (α- [4-aminobutyl] -L-glycolic acid (poly (α- [4-aminobutyl] -L-glycolic acid, PAGA), poly (2-aminoethyl propylene phosphate) (poly (2-aminoethyl propylene phosphate) ), PPE-EA), cationic derivatives of cyclodextrin, poly (2- (dimethylamino) ethyl methacrylate), pDMAEMA), poly ( 4-vinylpyridine) (poly (4-vinylpyridine), P4VP), O, O'-bis (2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol (O, O'-bis ( 2-a minopropyl) polypropylene glycol-block-polyethylene glycol-blockpolypropyleneglycol), chitosan, chitosan derivatives, polyethylenimine (PEI), polyethyleneimine derivatives, polyamidoamine (polyamidoamine, PAMAM), fractured PAMAM and poly-N- A ribonucleic acid pharmaceutical composition for cancer treatment selected from the group consisting of ethyl-4-vinylpyridinium tribromide (poly-N-ethyl-4-vinylpyridinium tribromide). The ribonucleic acid pharmaceutical composition according to claim 1, further comprising an anticancer chemotherapeutic agent. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 1, wherein the siRNA has a sense sequence of SEQ ID NO: 26 and an antisense sequence of SEQ ID NO: 27. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 1, wherein the siRNA has a sense sequence of SEQ ID NO: 32 and an antisense sequence of SEQ ID NO: 33. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 1, wherein the siRNA has a sense sequence of SEQ ID NO: 38 and an antisense sequence of SEQ ID NO: 39. The ribonucleic acid pharmaceutical composition for treating cancer according to claim 1, wherein the siRNA has a sense sequence of SEQ ID NO: 42 and an antisense sequence of SEQ ID NO: 43. An siRNA that inhibits expression of GASC1 and has a sense sequence of SEQ ID NO: 26 and an antisense sequence of SEQ ID NO: 27; An siRNA that inhibits expression of GASC1 and has a sense sequence of SEQ ID NO: 32 and an antisense sequence of SEQ ID NO: 33; An siRNA that inhibits expression of GASC1 and has a sense sequence of SEQ ID NO: 38 and an antisense sequence of SEQ ID NO: 39; An siRNA that inhibits expression of GASC1 and has a sense sequence of SEQ ID NO: 42 and an antisense sequence of SEQ ID NO: 43; The method of claim 1, wherein Wnt-1, Hec1, Survivin, Livin, Bcl-2, XIAP, Mdm2, EGF, EGFR, VEGF, VEGFR, Mcl-1, IGF1R, Akt1, Grp78, STAT3, STAT5a, β-catenin, A ribonucleic acid pharmaceutical composition for treating cancer further comprising at least one siRNA selected from the group consisting of WISP1 or siRNA that inhibits the expression of c-myc. delete