KR20130013675A - A pharmaceutical composition for inhibiting metastasis comprising sirna of bc200 rna - Google Patents

Abstract

PURPOSE: An siRNA which suppresses BC200 RNA activation and a pharmaceutical composition containing the same for suppressing tumor metastasis are provided to inhibit cancer cell migration and metastasis and to effectively treat cancer. CONSTITUTION: An siRNA which suppresses BC200 RNA(sequence number 1) activation has a base sequence of 5'-GUAACUUCCCUCAAAGCAA-3'(sequence number 2) or 5'- UUGCUUUGAGGGAAGUUAC-3'(sequence number 3). The siRNA additionally contains a dTdT base sequence at the 3'-end of the siRNA. A vector contains a polynucleotide which enables transcription of shRNA. The vector transcribes shRNA in a cell and the transcribed shRNA is converted into siRNA by cleavage using RNase III. A pharmaceutical composition for suppressing tumor metastasis contains the vector as an active ingredient. The composition additionally contains a pharmaceutically acceptable carrier. [Reference numerals] (AA) Negative control group siRNA

Description

A pharmaceutical composition for inhibiting metastasis comprising siRNA of BC200 RNA}

The present invention relates to a pharmaceutical composition for inhibiting tumor metastasis comprising siRNA against BC200 RNA, and more particularly, the present invention relates to siRNA capable of inhibiting the activity of BC200 RNA, shRNA that can be transformed intracellularly into the siRNA. It relates to a vector and a pharmaceutical composition for inhibiting tumor metastasis comprising the siRNA or the vector.

If ncRNAs that do not encode proteins have various cellular functions, ncRNAs can be expected to play an important role in cancer metabolism. However, only ncRNA expression in cancer cells has been reported, and biosynthesis and its mechanisms have been reported. There is not much research on this.

BC200 (Brain Cytoplasmic 200) RNA was originally discovered as a neuron-specific ncRNA (non-coding RNA) expressed in the primate brain (Tiedge, H., et. al ., J Neurosci, 1993 and Watson, JB and Sutcliffe, JG, Mol Cell Biol, 1987), are thought to be factors that regulate local protein synthesis in dendritic processes of neurons (Lin, D., et. al ., Mol Cell Biol, 2008). It has also been reported that BC200 RNA is expressed in tumor tissues of various origins in addition to neural tissues, especially in breast cancer, which is higher in metastatic breast cancer than in benign tumors (Chen, W., et. al ., J Pathol, 1997 and Iacoangeli, A., et al ., Carcinogenesis, 2004). In addition, many BC200 RNAs are known to be found in neurological diseases such as Alzheimer's disease (Lukiw, WJ, et al ., Neurochem Res, 1992 and Mus, E., et. al ., Proc Natl Acad Sci USA, 2007).

BC200 RNA is produced by transcription by RNA polymerase III in cells (Martignetti, JA and Brosius, J., Proc Natl Acad Sci USA, 1993). Within neurons, Poly (A) -binding proteins (Muddashetty, R., et al ., J Mol Biol, 2002), Fragile X mental retardation (FMRP) proteins (Zalfa, F., et al ., J Biol Chem, 2005), SRP9 / 14 protein (Kremerskothen, J., et al ., Neurosci Lett, 1998), SYNCRIP protein (Duning, K., et al ., J Neurochem, 2008).

On the other hand, in relation to the method of inhibiting genes, siRNA (small interfering RNA) is a double stranded RNA (dsRNA) consisting of 12 to 21 nucleotide sequences and has a characteristic of inhibiting gene expression specifically. Originally found in nematodes, fruit flies, and natural mechanisms that regulate genes in plants (Fire, A., et. al . Nature, 1998), and recent studies have shown that mammalian cells may be designed to interfere with expression by blocking the synthesis of proteins by inducing degradation of specific genes using sequence-specific siRNAs. Accordingly, the use of siRNA has suggested the possibility of treating the disease by suppressing the expression of exogenous and endogenous genes in vitro or in vivo (Xia, H., et. al . Nat Biotechnol, 2002).

After the fact that BC200 RNA has been linked to cancer cells, efforts have been made to develop methods for inhibiting BC200 RNA. For example, International Patent Application Publication No. WO04058939 discloses a method for inhibiting BC200 RNA using an antisense single strand, but the prior art focuses on using an antisense single strand, and moreover, expresses a gene using an antisense single strand. When inhibiting, there is a problem about specificity and stability of the target, and thus it cannot be regarded as an effective expression inhibition method. A method for suppressing the expression of BC200 RNA using siRNA that supplements the above problem has not been developed.

Therefore, the present inventors directly introduce siRNA or shRNA capable of complementary binding to BC200 RNA specifically overexpressed in a cancer cell line, or transform the animal cell into a vector capable of transcripting the siRNA or shRNA, thereby transforming BC200. When inhibiting RNA, not only the migration and invasive ability of cancer cells are lowered, but also the expression of tumor metastasis markers is reduced, thereby confirming that tumor metastasis is inhibited by the siRNA, thus completing the present invention.

One object of the present invention is to provide an siRNA capable of inhibiting the activity of BC200 RNA.

Another object of the present invention is to provide a vector expressing shRNA that can be transformed intracellularly into the siRNA.

Another object of the present invention to provide a pharmaceutical composition for inhibiting tumor metastasis comprising the siRNA or vector as an active ingredient.

According to one embodiment for achieving the above object, the present invention provides an siRNA capable of inhibiting the activity of BC200 RNA (SEQ ID NO: 1). At this time, the base sequence of the siRNA is not particularly limited, but it is preferable to use 5'-GUAACUUCCCUCAAAGCAA-3 '(SEQ ID NO: 2), 5'- UUGCUUUGAGGGAAGUUAC-3' (SEQ ID NO: 3), and the like. The 3'-terminus may further include the base sequence of dTdT.

As used herein, the term 'siRNA (short interfering RNA)' refers to double-stranded RNA capable of inducing RNAi that inhibits gene activity. In the present invention, siRNA means siRNA capable of inhibiting the activity of BC200 RNA, which may be in any form as long as it induces RNAi, for example, by chemical synthesis or biochemical synthesis or in vivo synthesis. The obtained siRNA, or double-stranded RNA of at least 10 bases in which the double-stranded RNA of at least about 40 bases is degraded in the body, and the like can be used.

The siRNA is at least about 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, in particular to a portion of the nucleic acid sequence of BC200 RNA Preferably at least 95%, most preferably at least 100% of the sequence having a homology, it is also possible to use RNA or modified variants thereof comprising a double stranded portion. The sequence portion having homology is usually at least 15 nucleotides or more, preferably about 19 nucleotides or more, more preferably at least 20 nucleotides, and still more preferably 21 nucleotides or more. In addition, the siRNA is preferably composed of a base sequence capable of complementarily binding to the region with the lowest homology with other RNA in the base sequence of BC200 RNA. For example, the siRNA may be composed of a base sequence that does not include a common base sequence between BC200 RNA and 7SL RNA having high homology thereto.

In another embodiment of the invention, the invention provides a vector comprising a polynucleotide capable of transcribing shRNA that can be converted into said siRNA in a cell.

The term 'shRNA (short hairpin RNA)' of the present invention includes a partial sequence of palindromic sequences in single-stranded RNA, thereby forming a hairpin-like structure having a double-stranded structure in the 3 'region. , Refers to RNA which can be transformed into siRNA by being cleaved by dicer, which is a kind of RNase present in the cell after being expressed in the cell. The length of the double-stranded structure is not particularly limited, but is preferably 10 nucleotides or more. More preferably 20 nucleotides or more. In the present invention, the shRNA may be included in a vector. When the vector containing the shRNA is introduced into a cell, the shRNA is expressed in the cell, and the expressed shRNA is a kind of RNase III present in the cell. It is cleaved by and converted to siRNA, the converted siRNA can induce RNAi to inhibit the activity of BC200 RNA.

The vector is not particularly limited, but viral vectors such as retroviruses, vaccinia viruses, adenoviruses, and sinrinseliki viruses can be used.

The method of introducing the vector into the cell is not particularly limited, and may be performed by a calcium phosphate method, a liposome, or a method using various transformation reagents (for example, oligofectamine, lipofectamine, lipofection, etc.).

As another embodiment of the present invention, the present invention provides a pharmaceutical composition for inhibiting tumor metastasis comprising the siRNA or vector as an active ingredient.

Tumors that can be prevented or treated by the composition of the present invention are not particularly limited, but target cancers in which BC200 RNA is expressed in cells, specifically, breast cancer, cervical cancer, esophageal cancer, liver cancer, parotid cancer, and tongue cancer. And so on.

The pharmaceutical composition for inhibiting tumor metastasis of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions according to conventional methods. Can be used. In addition, the composition may further comprise a suitable carrier commonly used in the manufacture of pharmaceutical compositions. The carrier is not particularly limited, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undetermined Vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, in the red bean extract and fractions thereof. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In another embodiment of the invention, the invention provides a method of inhibiting metastasis of a tumor comprising administering the siRNA or vector to a patient in need of tumor treatment.

The term 'patient' of the present invention refers to a patient having a tumor expressing BC200 RNA, but is not particularly limited thereto, and may be a breast cancer patient, a cervical cancer patient, an esophageal cancer patient, a liver cancer patient, a parotid cancer patient, a tongue cancer patient, or the like. Can be.

The dosage of the pharmaceutical composition for inhibiting tumor metastasis of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient. For example, a composition of the present invention comprising siRNA is preferably administered at about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg, per adult, and comprises the vector The composition of the present invention is preferably administered at about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, more preferably 0.01 to 1 mg / kg per adult, for the prevention or treatment of cancer of the present invention The frequency of administration of the composition is not particularly limited, but may be administered once a day or several times in divided doses.

The siRNA against BC200 RNA of the present invention may inhibit the activity of BC200 RNA, thereby inhibiting tumor cell migration and subsequent tumor cell metastasis, and thus may be widely used for more effective cancer treatment.

1 is an RT-PCR photograph showing the regulation of BC200 RNA in cervical cancer cell lines transformed with siRNA.
2A is a micrograph showing the migration capacity of cells treated with BC200 RNA siRNA.
2B is a bar graph showing the migration ability index numerically.
3A is a micrograph of the invasion ability of cells treated with BC200 RNA siRNA.
3B is a bar graph showing the above-mentioned invasion capacity numerically.
4 is a cleavage map of the expression vector p-1010BCT capable of overexpressing BC200 RNA.
Figure 5 is a Western blot analysis picture showing the expression level of PRL-3 protein tumor metastasis marker according to the expression level of BC200 RNA.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: BC200 RNA Complementary to siRNA Transformants comprising

Example  1-1: Cell Culture

Human-derived cervical cancer cell line (HeLa) was cultured with DMEM (Dulbecco's modified Eagle's minimal essential medium, DMEM) containing 10% (v / v) fetal calf serum (FBS) and 1% (v / v) antibiotic solution. Was incubated in an incubator at 37% carbon dioxide concentration of 5% (v / v).

Example  1-2: siRNA  Ready

A 19 nucleotide siRNA capable of binding to cancer cell specific BC200 RNA (SEQ ID NO: 1) was prepared. The prepared siRNA was prepared by oligonucleotide synthesis so as to have a double stranded form consisting of a sense sequence (5'-GUAACUUCCCUCAAAGCAA-3 ', SEQ ID NO: 2) and an antisense sequence (5'- UUGCUUUGAGGGAAGUUAC-3', SEQ ID NO: 3). It was.

On the other hand, siRNA used as a control is oligonucleotide synthesis to have a double strand consisting of a sense sequence (5'-CCUACGCCACCAAUUUCGU-3 ', SEQ ID NO: 4) and an antisense sequence (5'-ACGAAAUUGGUGGCGUAGG-3', SEQ ID NO: 5) It was prepared through.

Example  1-3: Transformation

In siRNA transfection targeting BC200 RNA, lipofectamine 2000 (Invitrogen) was used. Typically, 200 pmol of siRNA was injected into a 50% confluent cell in a medium without antibiotics in a 6 cm culture dish. Thereafter, the cells were incubated for 6 hours, replaced with medium containing 1% (v / v) antibiotic solution, and incubated for 42 hours. The cell proliferation rate was examined by performing a proliferation assay by microscopic observation.

Example  2: of the cells to which the transformants were administered RT -PCR ( Reverse transcription  중합체 chain reaction ) analysis

After removing the medium from the culture dish, it was washed twice with PBS. Then, using the Easy blue solution (Intron Biotechnology) to extract the total RNA (total RNA), using the total RNA as a template for reverse transcription using a random hexamer (random hexaer), followed by PCR reaction ONE-STEP RT-PCR PreMix Kit (Intron Biotechnology) was used. For BC200 RNA, sense primer 5'-GCCTGTAATCCCAGCTCTCA-3 '(SEQ ID NO: 6) and antisense primer 5'-GTTGCTTTGAGGGAAGTTACGCT-3' (SEQ ID NO: 7); for GAPDH mRNA, sense primer 5'-CGCGGATCCCCGGCCGGGCGCGGTG-3 ' (SEQ ID NO: 8) and antisense primer 5'-CCCAAGCTTAAAAAGGGGGGGGGGGGTTG-3 '(SEQ ID NO: 9). GAPDH mRNA was used as a positive control for quantification of RNA, and only siRNA was used as a negative control (FIG. 1).

1 is an RT-PCR photograph showing the regulation of BC200 RNA in cervical cancer cell lines transformed with siRNA. As shown in Figure 1, it can be seen that the amount of the BC200 RNA siRNA is reduced compared to the control.

Example  3: Analysis of the migration capacity of cells to which the transformants were administered

To analyze the migration ability, the transformed cells were incubated for 42 hours in a petri dish and the cells were removed from the petri dish with trypsin, washed and diluted in DMEM medium containing 0.1% BSA. Incubate at 2 x 10 ⁵ cells per well of 24-well transwell (8um pore, Costar Corning) coated at a concentration of / ml, incubate in serum-free medium for 6 hours, and then transwell Stained with violet dye, a microscope photograph was taken at 200 times magnification and read to observe the shape of the cells (FIG. 2a), and the density of the black part was measured after converting the micrograph into a black and white image, measured in a control group. Based on the calculated values, the miRNA ability index was converted into a graph by converting the siRNA-treated value of the present invention (FIG. 2B).

Figure 2a is a micrograph showing the migration ability of the cells treated with BC200 RNA siRNA, Figure 2b is a bar graph showing the quantification of the migration ability index. As shown in Figure 2a and 2b, it was confirmed that the migration ability of the cells treated with BC200 RNA siRNA is reduced compared to the negative control.

Therefore, it was found that the migration ability of the cervical cancer cell line transformed with siRNA that complementarily binds to BC200 RNA decreases.

Example  4: Analysis of invasive capacity of cells to which the transformants were administered

Also, to analyze the invasive ability, the type I collagen was coated with a Matrigel at a concentration of 250 ug / ml on the top of a 24-well transwell coated with a concentration of 5 ug / ml. Transwell was placed in a culture dish filled with DMEM medium containing 0.5% FBS, 2 x 10 ⁵ cells were placed on the top, and 100% of 0.1% BSA / DMEM was added to CO ₂ at 37 ° C for 24 to 48 hours. Incubated with injection. After that, the cells on the upper side of the transwell were removed and the cells on the lower side were fixed with 4% formaldehyde for 30 minutes. The shape of the cells was observed through the microscope in the same manner as described above (FIG. 3a) and the graph of the invasiveness index was quantified (FIG. 3b).

Figure 3a is a micrograph showing the invasion capacity of the cells treated with BC200 RNA siRNA, Figure 3b is a bar graph showing the invasion capacity numerically. As shown in Figure 3a and 3b, it was confirmed that the invasion ability of the cells treated with BC200 RNA siRNA is reduced compared to the negative control.

Therefore, it was found that the invasive ability of the cervical cancer cell line transformed with siRNA complementary to BC200 RNA was reduced.

Example  5: BC200 RNA For siRNA of Tumor metastasis  Inhibitory effect

In order to confirm the relationship between the overexpression or expression suppression of BC200 RNA and tumor metastasis, the relationship was confirmed using PRL (Phosphatase Regenerating of Liver) -3 known as a conventional tumor metastasis marker.

Example  5-1: BC200 RNA  Preparation of Overexpression Vectors

The nucleotide sequence corresponding to the BC200 RNA gene subregion 100nt to the BC200 RNA gene subregion 100nt was cloned into a T-Blunt vector (Solgent) to prepare an expression vector p-1010BCT capable of overexpressing BC200 RNA in the cell. (FIG. 4). 4 is a cleavage map of the expression vector p-1010BCT capable of overexpressing BC200 RNA.

Example  5-2: BC200 RNA According to the expression level of Tumor metastasis Marker  Change of expression

T-Blunt vector used as a control, the expression vector p-1010BCT capable of overexpressing the BC200 RNA prepared in Example 5-1, the control siRNA prepared in Example 1-2 and siRNA for the BC200 RNA of the present invention Each transformant was prepared by introducing into each cervical cancer cell line, and the level of PRL-3 protein expressed from each transformant was analyzed by Western blot. At this time, siRNA for the T-Blunt vector, p-1010BCT, control siRNA and BC200 RNA were introduced into cervical cancer cell lines using lipofectamine 2000 (Invitrogen), and the transformants were cultured for 48 hours, followed by anti- Western blot using PRL-3-antibody and a secondary antibody recognizing the antibody (FIG. 5). In this case, α-tubulin was used as the internal control group.

Figure 5 is a Western blot analysis picture showing the expression level of the tumor metastasis marker PRL-3 protein according to the expression level of BC200 RNA, lane 1 is the PRL-3 protein expressed in the transformant to which the T-Blunt vector is introduced Lane 2 represents PRL-3 protein expressed in the transformant to which p-1010BCT was introduced, lane 3 represents the PRL-3 protein expressed in the transformant to which the control siRNA was introduced, and Lanes represent PRL-3 protein expressed in a transformant into which siRNA against BC200 RNA was introduced.

As shown in FIG. 5, the PRL-3 protein levels expressed in the transformants to which the T-Blunt vector (lane 1) and the control siRNA (lane 3) were introduced showed similar levels to each other, whereas the expressed PRL- Compared to the level of 3 protein, the level of PRL-3 protein expressed in the transformants into which p-1010BCT (lane 2) was introduced was increased significantly, and in transformants into which siRNA (lane 4) for BC200 RNA was introduced. The expressed PRL-3 protein level was found to be greatly reduced.

From the above results, when the expression level of BC200 RNA is increased, the expression of tumor metastasis marker protein is increased to promote tumor metastasis, whereas when the expression level of BC200 RNA is decreased, the expression of tumor metastasis marker protein is decreased. It was confirmed that tumor metastasis was suppressed.

Incorporating the results of Examples 2 to 5, it can be seen that BC200 RNA siRNA can reduce the amount of BC200 RNA in cervical cancer cell lines, and has the effect of reducing the migration and invasion ability related to the metastasis of cancer cells.

<110> Korea Advanced Institute of Science and Technology <120> A pharmaceutical composition for inhibiting metastasis comprising          siRNA of BC200 RNA <130> PA110532 / KR <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 200 <212> RNA <213> BC200 RNA <400> 1 ggccgggcgc gguggcucac gccuguaauc ccagcucuca gggaggcuaa gaggcgggag 60 gauagcuuga gcccaggagu ucgagaccug ccugggcaau auagcgagac cccguucucc 120 agaaaaagga aaaaaaaaaa caaaagacaa aaaaaaaaua accguaacuu cccucaaagc 180 aacaaccccc cccccccuuu 200 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 2 guaacuuccc ucaaagcaa 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 3 uugcuuugag ggaaguuac 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 4 ccuacgccac caauuucgu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 5 acgaaauugg uggcguagg 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcctgtaatc ccagctctca 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gttgctttga gggaagttac gct 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgcggatccc cggccgggcg cggtg 25 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cccaagctta aaaagggggg ggggggttg 29

Claims (9)

SiRNA capable of inhibiting the activity of BC200 RNA (SEQ ID NO: 1).
The method of claim 1,
SiRNA that inhibits the activity of BC200 RNA.
The method of claim 1,
The siRNA is siRNA having a nucleotide sequence of 5'-GUAACUUCCCUCAAAGCAA-3 '(SEQ ID NO: 2) or 5'- UUGCUUUGAGGGAAGUUAC-3' (SEQ ID NO: 3).
The method of claim 3,
SiRNA further comprising a base sequence of dTdT at the 3'-end of the siRNA.
A vector comprising a polynucleotide capable of transcribing shRNA which can be transformed into the siRNA of any one of claims 1 to 4 in a cell.
The method of claim 5,
Said vector transcribes shRNA intracellularly, and said transcribed shRNA is cleaved by RNase III and converted into siRNA.
A pharmaceutical composition for inhibiting tumor metastasis comprising as an active ingredient a vector comprising a siRNA of any one of claims 1 to 4 or a polynucleotide capable of transcribing an shRNA that can be converted into the siRNA.
The method of claim 7, wherein
The composition further comprises a pharmaceutically acceptable carrier.
The method of claim 7, wherein
Wherein the tumor is selected from the group consisting of breast cancer, cervical cancer, esophageal cancer, liver cancer, ovarian cancer, parotid cancer and tongue cancer.

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