WO2019112242A1 - Composition comprising sirna for prevention or treatment of neurofibrosarcoma - Google Patents

Abstract

The present invention relates to a composition comprising TNS3 gene expression-suppressing short interfering RNA (siRNA) as an effective ingredient for preventing, alleviating, or treating malignant tumors of neurofibromatosis. According to the present invention, the siRNA was found to have good effects on the proliferation suppression and treatment of malignant tumors by suppressing the expression of TNS3 gene, which is overexpressed in tumors of neurofibromatosis type 1 patients, as the TNS3 gene expression-suppressing siRNA exhibited good effects in cell and animal tests. In addition, the TNS3 siRNA composition particularly has an excellent effect on the proliferation suppression and treatment of malignant tumors of neurofibromatosis type 1 when used in combination with the anticancer agents ICE (ifosfamide, carboplatin, etoposide).

Description

composition for preventing or treating nerve fibrosis including siRNA

The present invention relates to an siRNA that inhibits the expression of the TNS3 gene and a composition for treating neurofibromatosis malignancies comprising the same as an effective ingredient.

Neurofibromatosis Type 1 (NF1) was first described by Fredrich von Recklin-ghausen in 1882 and is an autosomal dominant genetic disorder that forms tumors along the peripheral nerves. It occurs in about 1 in 3,500 patients. The tumor is called neurofibromatosis because it can occur in any part of the nervous body, and the number is more than one. The main symptoms are neurofibroma and cafe-au-lait spots, Lisch nodules, optic pathway glioma, scoliosis, Tibial dysplasia, plexiform neurofibroma, pontine glioma, etc. (see Fig. 1 of the present invention).

The most common type 1 neurofibromatosis patient is a neurofibroma, a benign tumor that occurs in the skin. About 30-40% of patients with type 1 neurofibromatosis have gunshot nerve fibromatosis, a benign tumor that occurs under the skin or in the deep part of the body. In particular, gunshot neurofibromas show a massive lump (lump) shape that grows sideways and spreads sideways in various body parts (see FIG. 2 of the present invention), and a large number of multiple gunshot neurofibromas develop in the deep part 3 of the present invention). Although plexiform neurofibroma (PN) is a benign tumor, it develops very seriously in patients, and in about 10% of patients with type 1 neurofibromatosis, neurofibromatosis is a malignant neurofibroma neurofibrosarcoma, malignant peripheral nerve sheath tumor (MPNST) (see FIG. 4 of the present invention). In patients with type I neurofibromatosis, the malignant progression of benign tumors develops rapidly, resulting in a very high mortality rate.

(PN) stimulates the growth of bone and surrounding tissues and grows to a large size, but it may also appear to be a skewed appearance, and about 8 to 13% of such patients are malignant with neurofibromatosis (MPNSTs) 5). MPNST is a highly malignant metastatic cancer that is known to have a high mortality rate and low response to chemotherapy and radiation.

Type 1 neurofibromatosis is caused by a mutation in the NF1 gene, and about 50% of the patients are caused by naturally occurring mutations that are not hereditary. The NF1 gene is located on chromosome 17q11.2, and consists of 350 kb genomic DNA and 57 exons. The mRNA is about 11 to 13 kb. The tumor suppressor gene NF1 encodes a neurofibromin protein in the cytoplasm of ~ 320 kDa that encodes a GAP-related protein that negatively regulates the Ras signal at amino acid 1125-1537 Domain (GAP-related domain). GTPase-activating protein (GAP) modulates the activity of Ras by stimulating intrinsic GTPase of Ras and converting it into an inactive form of Ras-GDP in the activated form, Ras-GTP. Oncogene The Ras protein, which is encoded by Ras, is expressed in both neuronal and endothelial cells, and the signal is transmitted downstream (downstream). Increased GTP-Ras induces signal transduction through a representative downstream pathway, Raf kinase, and Raf induces signaling through kinase kinase including Erk1 and Erk2 isoforms of MEK kinase and mitogen-activated protein (MAP) kinase (See FIG. 6 of the present invention) by activating the cell.

There are three types of Ras genes: K-Ras, H-Ras, and N-Ras. Mutations in the Ras gene are found in about 50% of human cancers. Mutation of the Ras gene and excessive activity of the Ras protein are involved in the carcinogenesis of the cell, and thus research on the cancer treatment through suppression of the intracellular expression of the Ras protein has been actively carried out. It is known that all three Ras proteins, K-Ras, H-Ras and N-Ras, are overactivated by the mutation of the NF1 gene. However, recent studies have shown that astrocytes in patients with type I neurofibromatosis have H- Ras and N-Ras are not activated, only K-Ras is specifically activated, and hematopoietic cells are activated only by H-Ras.

20% of NF1 patients with overactive and accumulating RAS due to neurofibromin loss or dysfunction cause severe life-threatening complications and about 8 to 13% And the clinical symptoms are severe, but inherited, so it is impossible to treat it. In the case of malignant neurofibrosarcoma, only mass therapy such as resection of the tumor is the only treatment, or surgery is impossible according to the location of the tumor, and it is directly related to the life of the patient regardless of the timing of the cancer. The exact mechanism of the variability and malignancy of the clinical phenotype of type 1 neurofibromatosis remains unclear. Traditional therapies include radiotherapy, surgical resection, and cytotoxic drugs, but no long-term effects were found.

It is an object of the present invention to provide short interfering RNA capable of inhibiting TNS3 gene expression.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating nerve fibrosis, which is a malignant tumor of type 1 neurofibromatosis.

In order to achieve the above object, the present invention provides short interfering RNA (siRNA) comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 4.

In order to accomplish the above object, the present invention provides a neurofibrosarcoma (malignant) tumor comprising short interfering RNA (siRNA) comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 4 as an active ingredient. peripheral nerve sheath tumor, MPNST).

The present invention relates to a composition comprising an siRNA which effectively inhibits TNS3 gene expression as an active ingredient. In the treatment of siRNA, the survival rate of cancer cells in malignant tumors is remarkably decreased, and isopamide, Etoposide ) And carboplatin (Carboplatin). The siRNA-containing composition can effectively prevent, ameliorate, or treat malignant tumors of type 1 neurofibromatosis, A pharmaceutical composition for prevention or treatment of sarcoma, a health functional food composition for preventing or ameliorating nerve fibrosis, and the like.

Figure 1 shows representative symptoms of type 1 neurofibromatosis (A) milk-cough-au-lait spots, (B) neurofibroma, (C) Lisch nodules, (D) optic pathway glioma, (E) scoliosis, (F) tibial dysplasia, (G) plexiform neurofibroma, (H) glioma).

Figure 2 shows gunshot neurofibroma occurring in various body parts.

FIG. 3 shows an example of a gunshot neurofibroma occurring in the deep part.

Fig. 4 shows an example of nerve fibrosarcoma developed malignantly in benign bullet neurofibroma.

Figure 5 shows the process of tumor formation and malignancy of type 1 neurofibromatosis.

Fig. 6 schematically shows the role of neurofibromin and the mechanism of Ras activation.

FIG. 7 shows the results of comparing the amount of Tensin 3 protein and TNS3 mRNA expression in normal Schwann cells (HSC) and NF1 malignant tumor Schwann cell lines (FIG. 7a: increased expression of TNS3 gene in malignant tumor cells (S462) , Fig. 7b: expression of TNS3 gene is increased in malignant tumor (MPNST) sNF02.2, sNF96.2, S462 cell line).

FIG. 8 shows ERK1 / 2 activation (phospho-ERK1 / 2) and Bcl-2 overexpression (FIG. 8A) upon inhibition of TNS3 expression using siRNA # 1 and # 2 in type 1 neurofibromatosis malignant tumor Schwann cell line (S462) RAS activation (Fig. 8B).

9 shows the results of confirming the cell survival rate of siRNA (# 2) alone or in combination with an anticancer drug against the TNS3 gene.

FIG. 10 shows the in vivo efficacy of TNS3 gene inhibition inhibition. FIG. 10 shows the effect of SNS2, a type 1 neurofibromatous malignant tumor cell, on the size, volume, and volume of malignant tumor tissue of a xenograft mouse, (Observation of the anticancer effect of ICE and TNS3 gene siRNA (# 2) alone and in combination treatment).

Fig. 11 shows the result of comparing the size of the tumor and the size of the tumor in vivo by inhibition of TNS3 gene expression.

Neurofibromatosis Type 1 (NF1) was first described by Fredrich von Recklin-ghausen in 1882 and is an autosomal dominant genetic disorder that forms tumors along the peripheral nerves. It occurs in about 1 in 3,500 cases. Malignant tumors present in about 8 to 13% of patients are unpredictable, have severe clinical symptoms and high mortality rates, but are difficult to treat due to the inability to perform surgery depending on the location of the tumor. The efficacy of anticancer drugs is low and recurrence rates are high.

Accordingly, the inventors of the present invention have found that the suppression of the overexpression of the TNS3 gene (tensin 3 protein) in malignant tumors of patients with type 1 neurofibromatosis inhibits the proliferation and treatment of malignant tumors. Thereby completing the invention.

Accordingly, the present invention provides a short interfering RNA (siRNA) comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 4.

In one embodiment of the present invention, the siRNA can inhibit TNS3 gene expression and thus can be used to prevent, ameliorate, or treat neurofibromatosis by inhibiting TNS3.

Accordingly, the present invention provides neurofibrosarcoma (MPNST) comprising short interfering RNA (siRNA) consisting of any one of the nucleotide sequences of SEQ ID NOS: 1 to 4, ) ≪ / RTI >

The neurofilament sarcoma may be caused by malignant transformation of the bulbar neurofibroma. That is, the present invention can treat neurofibrosarcoma corresponding to a malignant tumor. It is known that the neurofilament sarcoma is caused by malignant development in about 8-13% of patients with gunshot neurofibroma.

At this time, the siRNA can prevent or treat nerve fibrosis by inhibiting the expression of the TNS3 gene.

In addition, the siRNA can prevent or treat nerve fibrosis by inhibiting the activation of ERK1 / 2 and RAS or the expression of Bcl-2.

The composition may be administered in combination with, but not limited to, any one or more anticancer agents selected from the group consisting of isopamide, carboplatin, and etoposide. That is, the composition may further comprise at least one anticancer agent selected from the group consisting of isoparamide, carboplatin and etoposide. In this case, the composition exhibits the best synergy and increases the killing effect of malignant tumor cells .

When the composition of the present invention is a pharmaceutical composition, for administration, it may contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned effective ingredient. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to conventional methods . In detail, when formulating, it can be prepared using diluents or excipients such as fillers, weights, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid form preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules and the like. Such a solid preparation may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and tasks. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, it is possible to use witepsol, macrosole, tween 61, cacao paper, laurin, glycerogelatin and the like.

The appropriate dose of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, and the time, but can be appropriately selected by the person skilled in the art. 0.001 mg / kg to 50 mg / kg, and may be administered once to several times per day as needed.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. It is to be understood that both the foregoing description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. no.

Example 1: Cell culture

Human cells (HSC) (ScienCell Research Laboratories) and sNF96.2, sNF02.2, S462 (American Type Culture Collection, USA), which are neurofibrosarcoma (MPNST) Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (FBS, Hyclone Laboratories, Logan, USA), penicillin (100 U / ml) and streptomycin (100 μg / ml) The cells were cultured in a CO ₂ incubator at 37 ° C using a Bio-Rad Laboratories, Logan, USA.

Example 2: Western blot analysis

The cultured cells were lysed with 50 μg / ml phenylmethylsulfonyl fluoride (PMSF) and lysis buffer (150 mM NaCl, 1% Nonidet P-40, pH 7.4) supplemented with protease inhibitor cocktail , 0.5% sodium deoxycholate, and 0.1% SDS). Proteins were quantitated using the Lowry protein assay reagent kit (Bio-Rad Laboratories, USA).

The same amount of protein was taken for each condition, mixed with SDS-PAGE sample loading buffer (5X), heated at 100 ° C for 10 minutes, loaded and loaded on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) Respectively. The proteins separated by electrophoresis were immobilized on an immobilon-P transfer membrane (0.45) at 350 mA for 120 minutes using a transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine) (PVDF transfer membrane, Millipore Corporation, USA). Protein-transferred membranes were blocked with 3% non-fat dry milk (skim milk solution), reacted with primary antibody for 24 h at 4 ° C, and incubated with TBST (tris-buffered saline and tween 20 ) ≪ / RTI > Thereafter, Western blot analysis was performed with each specific antibody as follows.

The primary antibodies used were TNS3 (Tensin 3) 1: 3000 (Sigma Aldrich, USA), ERK, phospo-ERK, cleaved caspase 3, Bcl- , Neurofibromin, β-actin 1: 3000 (Santa Cruz Biotechnology, INC, USA), the second antibody was peroxidase-conjugated chlorine The cells were reacted with a goat anti-rabbit antibody, a goat anti-mouse antibody 1: 5000 (Santa Cruz Biotechnology, INC, USA), followed by WEST-ZOL Plus (iNtRON Biotechnology , KOREA) were processed and developed on the film.

Example 3: Ras activity assay

In order to measure Ras activity, the amount of GTP-Ras was analyzed by affinity precipitation method using Ras activation assay kit (Upstate Biotech. Lake Placid, NY). The cultured S462 cells were dissolved in lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl ₂ , 1 mM EDTA and 2% glycerol) ) Was reacted with agarose beads binding to Raf-1 RBD fusion protein at 4 ° C for 1 hour. The agarose beads were washed three times with 1 ml of dissolution buffer, mixed with a loading buffer (5X), heated at 100 ° C for 10 minutes, and subjected to 12-15% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The agarose beads were collected by centrifugation and then washed with a dissolution buffer. Finally, a sample buffer was added and heated at 95 ° C for 5 minutes. Then, western blotting was performed using Ras specific antibody to analyze the amount of GTP-Ras.

Example 4: Design and production of short interfering RNA (siRNA) for inhibiting TNS3 gene expression

To suppress the expression of the TNS3 gene, siRNAs complementary to the TNS3 gene mRNA were designed and synthesized (Cosmogenetech, Korea). A complementary two-stranded RNA-DNA hybrid oligomer (Sense and Antisense) was prepared by synthesizing each of the preceding 19 sequences with RNA and the last 2 TTs with DNA, (Double strand RNA) was used as the siRNA. The nucleotide sequences of the sense and antisense siRNAs represented by SEQ ID NOS: 1 and 2 (# 1) and SEQ ID NOS: 3 and 4 (# 2) prepared at this time are shown in Table 1 below.

siRNA	Kinds	Base sequence	SEQ ID NO:
siTNS3 # 1	Sense	5'-CCC AGC AAA GCG TTC AAA CTT-3	One
	Antisense	5'-GUU UGA ACG CUU UGC UGG GTT-3 '	2
siTNS3 # 2	Sense	5'-GGU CCG AAC ACU UGU ACA ATT-3 ''	3
	Antisense	5'-UUG UAC AAG UGU UCG GAC CTT-3 '	4

Example 5: Total RNA Extraction, cDNA Synthesis and Quantitative PCR

To confirm the expression level of TNS3, RNA was extracted from the cells cultured in Example 1 using Trizol (TM) reagent (Invitrogen Co., USA), and DNase I (Invitrogen Co., USA) was added thereto using 3 μg of RNA. , USA). After removing the remaining gDNA, cDNA was synthesized and amplified by PCR using the synthesized cDNA. Primers used (Table 2) were constructed using the property that the RNA can not be a template for PCR and the intron region exists only in gDNA. CDNA was synthesized using TransScript II Reverse Transcriptase PCR Kit (Qiagen, USA). For tumors isolated from xenotransplantation mice, tumors were pulverized using a homogenizer, and RNA was extracted using Trizol ^™ reagent (Invitrogen Co., USA), and cDNA was synthesized as described above.

Total RNA was extracted from the cultured cells and amplified by real-time quantitative real-time PCR using the synthesized cDNA as a template using a gene-specific primer. STBR Green (TaKaRa, Shiga, Japan), which binds to double-stranded DNA and exhibits fluorescence, was added to cDNA (150 ng) and the fluorescence intensity was measured using an ABI Prism 7000 Sequence Detection System (Applied Biosystems; Foster City, CA, U, S , A). The primer sequences used were as shown in Table 2 below.

gene	Kinds	Base sequence	SEQ ID NO:
Human	Forward	5'-CGT TCT TTG GGC TCA GTC TC-3 '	5
	Reverse	5'-CTG AAG CCT TGG AAA AGT CG-3 '	6
Mouse	Forward	5'-GAG GGG TGG TAA AGG ACG C-3 '	7
	Reverse	5'-GGA GGG CTC CAT TAA TGC TGAA-3 '	8

Example 6: Measurement of cell viability

Cell viability was analyzed using the D-Plus CCK cell viability assay kit (Donglin LS, Korea). Cells were seeded at a cell density of 7 x 10 < 3 > cells per well in 96 well plates and cultured for 24 hours after siRNA (see Table 1 above) and / or anticancer treatment. Subsequently, the culture medium was replaced with 100 μl of a culture medium supplemented with 10 μl of CCK reagent and further cultured for 2 hours. Cell viability was determined by measuring the absorbance at 450 nm using an ELISA plate reader (Bio-Rad Model 680).

Example 7: Formation of type 1 neurofibromatosis malignant xenograft mouse model and administration of short interfering RNA (siRNA)

7-1. Generation of type 1 neurofibromatosis malignant xenograft mouse model

Nerve fiber sarcoma S462 cells were centrifuged at 1,000 rpm for 5 minutes to remove the supernatant, followed by 1 × 10 ⁷ cells / ml in phosphate-buffered saline (PBS), followed by subcutaneous injection of 0.1 ml Respectively. Tumor volume was measured and individuals reaching about 80 to 120 mm ³ were selected and grouped to equalize on the basis of tumor volume and body weight.

7-2. Administration of anticancer agents and / or siRNA

SiTNS3 (# 2) prepared in Example 4 was mixed with siTNS3 (10 μg) and in vivo-JETPEI (Polyplus, France) in a 5% glucose solution for 15 minutes and then local injection was performed on the tumor tissue . siTNS3 was directly administered to the tumor, and siTNS3 10 μg / 100 μl was administered three times weekly (Monday, Wednesday, and Friday) for 3 weeks.

In addition, ifosfamide, carboplatin, and etoposide were prepared by intraperitoneal injection with physiological saline according to the concentration of the drug, kg) and etoposide (3.3 mg / kg) were administered intraperitoneally for 5 days (Mon. through Fri) for the first week, and intraperitoneal administration of carboplatin (13.3 mg / Respectively.

7-3. Tumor volume measurement, tumor excision and weight analysis

The maximum length (L) and the perpendicular width (W) of the tumor were measured using a caliper twice a week twice during the observation period, and the tumor volume (TV) Respectively.

[Equation 1]

TV (mm ³ ) = L (mm) x W ² (mm ² ) x 1/2

In addition, the mice were euthanized with CO ₂ gas, and tumors were harvested and weighed. Tumor growth inhibition rate (IR) was calculated by substituting the extracted tumor into the following equation (2).

[Equation 2]

IR (%) = (1-T / C) 100

T: Mean tumor weight in experimental and control groups

C: Tumor weight of negative control

Example 8: Statistical analysis

Student's t-test was used to verify the significance of the in vitro graph. Body weights, tumor volume, and tumor weights obtained in the in vivo experiment were assayed using SAS (Version 9.3, SAS Institute Inc., USA). Body weights, tumor volume and tumor weights were tested by Bartlett test (significance level: <0.05). In the case of equal distribution, one-way analysis of variance (ANOVA) was performed (significance level: <0.05). When significance was observed, a multiple test of Dunnett's t-test was performed to confirm the significance of each test group for negative control (Significance level: one side <0.05 and <0.01). When the equal distribution was rejected, the Kruskal-Wallis test was performed (significance level: <0.05). When significance was observed, a multiple test of the steel's test was performed to confirm the significance of each test group for the negative control group.

Experimental Example 1: Determination of overexpression of TNS3

To confirm the expression level of TNS3, Western blotting and quantitative real-time PCR were performed. As a result, malignant tumor cell lines derived from patients with type 1 neurofibromatosis contained TNS3 (tensin-like SH2 domain containing 1, and Tensin 3) overexpression. That is, the expression levels of Tensin 3 protein and TNS3 mRNA in normal schwann cells (HSC) and NF1 malignant tumor Schwann cell line (S462) were increased, and as shown in FIG. 7A, expression of TNS3 gene was increased in malignant tumors Respectively. Tensin 3 has been shown to be involved in Rho GTPase signaling and cell adhesion regulation, and is known to be phosphorylated (activated) by Src and is involved in malignant tumors.

In addition, as shown in FIG. 7B, it was confirmed that the expression of TNS3 gene was also increased in other types of malignant tumors (MPNST) sNF02.2 and sNF96.2 cells. Therefore, it was hypothesized that inhibition of TNS3 (Tensin 3) expression would have a beneficial effect in the treatment of type 1 neurofibromatosis malignancy.

Experimental Example 2 Inhibition of ERK1 / 2 and RAS Activation and Bcl-2 Expression of siRNA in NF1 Malignant Tumor Model

Based on the results obtained in Experimental Example 1, siTNS3 # 1 and # 2 prepared in Example 4 were cultured in the presence of type 1 neurofibromatous malignant tumor Schwann cell line (S462) using RNAi MAX (Invitrogen) ) To inhibit TNS3 expression. Western blot analysis revealed the presence of ERK1 / 2 activation (phospho-ERK1 / 2, p-ERK1 / 2) and Bcl-2 expression level. In addition, the degree of RAS activation (GTP-RAS) was examined.

As a result, as shown in FIG. 8, the ERK1 / 2 activation (p-ERK1 / 2) and Bcl-2 expression levels were remarkably decreased in the neurofilament sarcoma cell line (S462) when siTNS3 # Respectively. In addition, it was confirmed that RTP activation (GTP-RAS) was significantly reduced during siTNS3 # 2 treatment. These results suggest that the expression level of TNS3 is closely related to the type 1 neurofibromatosis and that inhibition of TNS3 expression by siTNS3 # 1 and # 2 of the present invention will be effective for the treatment of neurofibromatosis malignancy.

Experimental Example 3: Effect of siRNA on tumor cell death in NF1 malignant tumor Schwann cells

First, as described in Experimental Example 2, the siTNS3 # 2 thus prepared was treated to inhibit the expression of TNS3, and then the change of NF1 malignant tumor Schwann cell line (S462) was observed. At this time, siTNS3 # 2 was treated alone or in combination with ICE (Ifosfamide 1.8 mM, Carboplatin 90 μM, and Etoposide 30 μM), which are typical anticancer drugs used, by 10 pmole / ml, respectively, and cell viability was analyzed.

As a result, as shown in FIG. 9, it was confirmed that the cell survival rate was significantly decreased upon treatment with TNS3 gene siRNA (siTNS3 # 2) in S462 malignant cells, and the cell survival rate was significantly decreased in combination with ICO (Ifosfamide, Carboplatin, Etoposide) It is confirmed that there is synergy effect in city. In HSC cells, TNS3 gene siRNA (siTNS3 # 2) treatment did not show a synergistic effect with the combination of ICE (Ifosfamide, Carboplatin, Etoposide) and cell viability.

Experimental Example 4: Malignant tumor suppression effect of siRNA in mouse model of type 1 neurofibromatosis malignant xenograft

In order to confirm the inhibitory effect of TNS gene expression in vivo, as shown in Example 7-1, NF1 malignant tumor Schwannoma cell line (S462) was inoculated into nude mice to induce carcinogenesis of type 1 neurofibromatosis malignant xenograft Mouse, siTNS3 # 2 alone, chemotherapeutic agent ICE alone, siTNS3 # 2 and anti-cancer agent ICE (3 times a week, local injection, anticancer agent) were used as complementary binding to TNS3 gene as shown in Example 7-2 The first dose was administered to the abdomen for 21 days, and the size of the tumor was observed for 43 days.

As a result, as shown in Fig. 10, excellent anticancer activity (tumor cell proliferation inhibition) was observed even with the TNS3 gene siTNS3 # 2 alone, and synergistic effect was further improved in combination with ICE, which is an anticancer agent. In addition, when the mean tumor volume and weight of each group were compared at 43 days, a significant tumor suppression effect was observed in the TNS3 gene siTNS3 # 2 ICE combination treatment group. Furthermore, as shown in Figs. 11A and 11B, even when the size of the tumor and the size of the tumor removed were compared, there was an excellent malignant tumor suppression effect in siTNS3 # 2 and ICE combination treatment group.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

1. A prophylactic or therapeutic agent for neurofibrosarcoma (MPNST) containing short interfering RNA (siRNA) consisting of any one of the nucleotide sequences of SEQ ID NOS: 1 to 4 as an active ingredient A pharmaceutical composition.
2. The pharmaceutical composition according to claim 1, wherein the neurofilament sarcoma is caused by malignant transformation of neurofibromatosis.
3. The pharmaceutical composition according to claim 1, wherein the siRNA inhibits the expression of TNS3 gene to prevent or treat neurofilament sarcoma.
4. The pharmaceutical composition for preventing or treating neurofibromatosis according to claim 1, wherein the siRNA inhibits or treats neurofilament sarcoma by inhibiting ERK1 / 2 and RAS activation or Bcl-2 expression.
5. The method according to claim 1, wherein the composition is administered in combination with any one or more anticancer agents selected from the group consisting of isopamide, carboplatin, and etoposide. Or &lt; / RTI &gt;

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