KR101853731B1 - Composition for inducing cell senescence comprising Linc-ASEN gene expression inhibitor - Google Patents

Abstract

The present invention relates to a composition for inducing cell senescence comprising a Linc-ASEN expression inhibitor as an active ingredient, a composition for preventing or treating cancer, and a chemotherapeutic adjuvant. The composition according to the present invention can inhibit the expression of Linc-ASEN (Long intergenic noncoding RNA-Associated with Senescence) gene in cells to induce aging of cells, particularly aging in cancer cells, And can be used as anticancer drugs, anticancer drugs, and the like.

Description

A composition for inducing cell senescence comprising a Linc-ASEN expression inhibitor as an active ingredient {Composition for inducing cell senescence-containing Linc-ASEN gene expression inhibitor}

The present invention relates to a composition for inducing cell senescence comprising a Linc-ASEN expression inhibitor as an active ingredient, a composition for preventing or treating cancer, and a chemotherapeutic adjuvant.

The human genome consists of 3 billion nucleotides and only 2% of the genomes produce protein. Previous researchers focused on genes that produce proteins. It was later found that RNA (non-coding RNA), which does not produce proteins, also plays an important biological role through the ENCODE (Encyclopedia of DNA Elements) project. Noncoding RNAs It is classified into RNA (small non-coding RNA) and long non-coding RNA (lncRNA).

Small non-coding RNAs are known to be highly conserved in living organisms. Small non-coding RNAs are known as microRNAs, short interfering RNAs (siRNAs) ), Piwi-interfering RNA (piRNAs), among which the most well-known microRNAs bind to the 3 'untranslated region (3' UTR) of the target messenger RNA and inhibit translation .

Unlike small non-coding RNAs, long intergenic RNAs (long intergenic RNAs) are known to have low nucleotide sequence conservation among species, It is known to be involved in biological, developmental and pathological mechanisms. For example, MEG3 lncRNA binds to p53, a tumor suppressor protein, which inhibits tumors by activating genes regulated by p53. HOTAIR lncRNA binds to histone degeneration complex PRC2 (Polycomb-repressive complex 2) to reduce the expression of cancer-suppressing genes, and overexpressed HOTAIR lncRNA promotes metastasis of breast and liver cancer.

On the other hand, premature aging of tumor cells is also called stress-induced premature aging, which means aging that occurs in tumor cells by various stimuli. Unlike normal cells, which divide and aging only a certain number of times, tumor cells are cells that change indefinitely due to the change of the characteristics of replication aging in the process of carcinogenesis. Therefore, the concept that tumor cells do not progress in cell aging process was common. Recently, however, it has become known that cell aging is rapidly induced by various stimuli in tumor cells. Genetic toxic chemicals, radiation, and ultraviolet rays have been reported as stressors that can induce aging of tumor cells.

Recently, it has been suggested that inhibition of tumor cell proliferation through aging of tumor cells using stress induction premature aging has been suggested as an effective mechanism for the treatment of cancer patients, and it is essential that the efficiency of cancer treatment is increased through research on the mechanism of cell senescence induction. In addition, it has been reported that tumor cell senescence is efficiently induced, and that tumor suppressor protein p53 acts to efficiently remove tumor tissue through cell senescence. Proven. This suggests that cell senescence may be useful for tumor therapy. Actually, the activation of aging mechanism of tumor cells enables to reduce the side effects of cancer treatment by enabling the administration of lower dose of anti-cancer agent or radiation than the activation of cell death mechanism, and overcoming resistance to cancer treatment of tumor cells that have acquired resistance to apoptosis Can be used.

The present inventors have been studying cell senescence using LncRNA to identify a novel LncRNA Linc-ASEN and to analyze the correlation between Linc-ASEN and cell senescence and to inhibit it and induce cancer cell senescence As a result, it has been confirmed that Linc-ASEN expression inhibitor can be used as a cancer treatment agent and anticancer adjuvant through ultimately these characteristics and the present invention has been completed.

KR Patent Registration No. 10-1466372 KR Patent Registration No. 10-1497930

It is an object of the present invention to provide a composition for inducing cell senescence, a composition for preventing or treating cancer, and a chemotherapeutic adjuvant comprising Linc-ASEN (long intergenic noncoding RNA-Associated with Senescence) expression inhibitor as an active ingredient.

In order to achieve the above object, the present invention provides a composition for inducing cell senescence comprising Linc-ASEN expression inhibitor as an active ingredient.

In addition, the present invention provides a composition for preventing or treating cancer comprising a Linc-ASEN expression inhibitor.

The present invention also provides an anticancer adjuvant comprising a Linc-ASEN expression inhibitor.

The composition according to the present invention can inhibit the expression of Linc-ASEN (Long intergenic noncoding RNA-Associated with Senescence) gene in cells to induce aging of cells, particularly aging in cancer cells, And can be used as anticancer drugs, anticancer drugs, and the like.

FIG. 1 is a graph showing the growth rate of cultured cells analyzed by trypan blue staining in order to confirm the effect of inhibiting Linc-ASEN RNA by small interference RNAs on cell senescence.
FIG. 2 is an optical microscope photograph showing the effect of inhibiting Linc-ASEN RNA by small interference RNAs on cell senescence.
FIG. 3 is a graph showing the cell aging-induced effects of Linc-ASEN RNA inhibition by small interfering RNAs. The cells were stained with the aging-related beta-galactosidase staining method and the ratio of beta-galactosidase- Fig.
FIG. 4 is a graph showing the results of Western blot analysis of the expression levels of (Phospho) -pRb, p53 and p21, which are aging-specific expression proteins, in order to confirm the cell aging induction effect by Linc-ASEN RNA inhibition by small interference RNA Fig.

The present invention provides a composition for inducing cell senescence comprising Linc-ASEN (long intergenic noncoding RNA-Associated with Senescence) expression inhibitor.

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

In the present invention, Linc-ASEN is a kind of LncRNA (long intergenic RNA) and has a total of 794 base pairs located at 59491502 to 59492295 on human chromosome 17.

In the present invention, Linc-ASEN is characterized in that it is transcribed from the DNA sequence shown in SEQ ID NO: 1. It is to be understood by those skilled in the art that the above-mentioned SEQ ID NO: 1 is merely illustrative. Sequences having substantial sequence identity or substantial sequence homology to the sequences are also within the scope of the present invention. As used herein, the term "substantial sequence identity" or "substantial sequence identity" is used to denote that the sequence represents substantial structural or functional identity with another sequence. These differences are due, for example, to inherent variations in the codon usage between different species. If there is a significant amount of sequence redundancy or similarity between two or more different sequences, the structural differences will be negligible if they have similar physical properties even if the length or structure of these sequences is different. The present invention can include a sequence having a sequence homology of 80% or more with the above-described sequence of Linc-ASEN, preferably 90%, more preferably 95% or more sequence homology .

The term "expression inhibitor " as used herein means that the expression level of RNA as a target gene is eliminated or decreased, preferably RNA interference caused by RNA cleavage, It does not.

The Linc-ASEN expression inhibitor according to the present invention is useful as an antisense nucleotide complementary to Linc-ASEN RNA, a small hairpin RNA, shRNA, small interfering RNA, siRNA and ribozyme. , But is not limited thereto.

The small interfering RNA is composed of a sense sequence of 15 to 30 mers selected in the nucleotide sequence of Linc-ASEN RNA and an antisense sequence complementarily binding to the sense sequence, And is not particularly limited.

The small interfering RNA according to the present invention may be, for example, a sense RNA represented by SEQ ID NO: 2 and an antisense RNA represented by SEQ ID NO: 3, or a sense RNA represented by SEQ ID NO: 4 and an antisense RNA represented by SEQ ID NO: 5 However, it is not limited to the RNA, which can be selected based on the nucleotide sequence of Linc-ASEN RNA.

Small interfering RNAs represented by SEQ ID NOS: 2 to 5 include variants having one or more substitutions, insertions, deletions, and combinations thereof that do not degrade their activity. Such modifications may have a sequence homology of 80% or more, preferably 90%, more preferably 95% or more, with the sequence of the above siRNA.

The method of producing the small interference RNA is not particularly limited. The small interfering RNA may be synthesized directly (Sui G et al., (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells, Proc Natl Acad Sci USA 99: 5515-5520) (Brummelkamp TR et al., (2002) A system for stable expression of short interfering RNAs in mammalian cells, Science 296: 550-553) using a transcription transcription method .

The composition of the present invention may contain at least one known active ingredient having a Linc-ASEN expression inhibiting effect or a known active ingredient having a cell aging-inducing effect together with the Linc-ASEN expression inhibitor.

In the present invention, the cell may preferably be a cancer cell.

In the present invention, the cancer cells may be selected from the group consisting of liver cancer cells, stomach cancer cells, lung cancer cells, blood cancer, breast cancer cells, skin cancer cells, cervical cancer cells, brain tumor cells, endometrial cancer cells, thyroid cancer cells, colorectal cancer cells, Cancer cells, pancreatic cancer cells, or rectal cancer cells.

The composition for inducing cell senescence of the present invention may be a pharmaceutical composition, and the aged cells have a general characteristic that the size of cells becomes larger and flattened and exhibits beta-galactosidase activity at a specific pH.

Although induction of aging may be detrimental to life (for example, tissue aging) because it is lethal to regeneration and neoplasia of tissue, it may cause permanent tumor suppressor in tumor cells with strong cell division And thus can have beneficial effects as anticancer or anticancer adjuvants.

The composition for inducing cell senescence of the present invention may comprise a carrier, diluent, excipient or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, and may be a compound, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, And if necessary, other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, the pharmaceutical composition can be suitably formulated according to each disease or ingredient, using known methods in the art.

The Linc-ASEN expression inhibitor has an effect of increasing p21.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising Linc-ASEN (long intergenic noncoding RNA-Associated with Senescence) expression inhibitor.

In the present invention, the term "prevention" means any action that delays cancer by administration of the composition.

In the present invention, the term "treatment" means any action that improves or alleviates the symptoms of cancer by administration of the composition.

In the present invention, the cancer may be liver cancer, stomach cancer, lung cancer, blood cancer, breast cancer, skin cancer, cervical cancer, endometrial cancer, thyroid cancer, colon cancer, bladder cancer, ovarian cancer, prostate cancer, pancreatic cancer or rectal cancer Do not.

The composition of the present invention, together with the Linc-ASEN expression inhibitor, may contain at least one known active ingredient having a preventive or therapeutic effect on cancer.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, calcium stearate, White sugar, etc. may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but not limited thereto.

The composition of the present invention can be administered orally or parenterally in various clinical formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, And suitable formulations known in the art are preferably those described in Remington ' s Pharmaceutical Science (Mack Publishing Company, Easton PA, recently). Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, oligosaccharide, 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, mineral oil and the like.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like May be included.

The preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used. The parenteral administration can be carried out using an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection or intra-thoracic injection.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will depend on the species and severity, age, sex, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the treatment of cancer.

The present invention also provides an anticancer adjuvant comprising Linc-ASEN (long intergenic noncoding RNA-Associated with Senescence) expression inhibitor.

The anti-cancer adjuvant according to the present invention may be in the form of a pharmaceutical composition or a food composition, more specifically, an anti-cancer pharmaceutical adjuvant or an anti-cancer food adjuvant.

The anti-cancer adjuvant means a material that can be used as an adjunct to enhance the effect of the cancer therapeutic agent generally used in the art, and the effect of the cancer therapeutic agent can be improved by using the adjuvant according to the present invention.

The term "food" is a concept that includes all the foods that people consume on a daily basis, and includes health functional foods.

The Linc-ASEN expression inhibitor of the present invention may be added to a food composition as an anti-cancer adjuvant. When the Linc-ASEN expression inhibitor of the present invention is used as a food additive, the effective ingredient may be added intact or used together with other food or food ingredients And can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

The kind of the anticancer agent that can be used together with the anticancer adjuvant of the present invention is not particularly limited. The anticancer agent can be selected under the general principle considered when selecting an anticancer agent, such as the type of cancer cells, the rate of absorption of the anticancer drug (treatment period and route of administration of the anticancer drug), the location of the tumor, and the size of the tumor.

According to one embodiment of the present invention, the Linc-ASEN expression inhibitor has an excellent cancer prevention or therapeutic effect by inducing aging of cancer cells. Therefore, the Linc-ASEN expression inhibitor of the present invention can be usefully used for anti-cancer adjuvant drugs or health functional foods for enhancing the anticancer effect of cancer therapeutic agents.

The above description of the composition of the present invention is omitted in order to avoid excessive complexity due to duplicated description.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited thereto.

Example  1. New Lnc  RNA (long intergenic  RNA) and Small interference  RNA (small interfering RNA, siRNA )

1-1. new Lnc  RNA identification

Human breast cancer cells with 10% fetal bovine serum (FBS, TBC) and 1% penicillin streptomycin (WelGENE) was maintained in DMEM (WelGENE) containing a, 37 ℃ and 5% CO ₂ air is maintained every two days in an incubator that ≪ / RTI > The human breast cancer cells were treated with 50ng / ml doxorubicin or 6Gy irradiation to induce senescence. After this, total RNA was extracted and it was confirmed that the expression of a specific gene (hereinafter referred to as "Linc-ASEN") in aged human breast cancer cells was changed based on the result of RNA sequencing. Using the UCSC genome browser, it was confirmed that Linc-ASEN, whose expression is changed in the aged cancer cells, is a novel Lnc RNA which has not been known so far.

The RACE method (Rapid Amplification of CDNA Ends) was used to identify and determine the entire nucleotide sequence of the gene. RACE was performed using the 5 'RACE System for Rapid Amplification of cDNA Ends kits (Invitrogen, Carlsbad, Calif.) And the 3' RACE System for Rapid Amplification of cDNA Ends kit (Invitrogen, Carlsbad, Calif.). As a result, it was confirmed that the nucleotide sequence of Linc-ASEN is a nucleotide sequence that is transcribed from the DNA of SEQ ID NO: 1 and is located at 59491502 to 59492295 (total 794 base pairs) of human chromosome 17.

1-2. Linc - ASEN  Manufacture of small interfering RNA

In order to produce Linc-ASEN small interfering RNA that inhibits the expression of Linc-ASEN identified in Example 1-1, BLOCK-iT ™ RNAi Designer (Invitrogen, https://rnaidesigner.thermofisher.com/rnaiexpress/) was used Respectively. Control small interfering RNAs used as controls used small interfering RNAs previously used in the art (Kim et al. 2012).

The nucleotide sequence of the control small-interfering RNA and Linc-ASEN small interfering RNA object SEQ ID NO: Base sequence Control small interfering RNA - 5'-CCUACGCCACCAAUUUCGU (dTdT) -3 ' Linc-ASEN Small interfering RNA 1 (sense) 2 5'-GCUCAUGUUUGUCUCAAU (dTdT) -3 ' Linc-ASEN Small interfering RNA 1 (antisense) 3 5'-AUUGAGAGCAAACAUGAGC (dTdT) -3 ' Linc-ASEN Small interfering RNA 2 (sense) 4 5'-GGAAACUUUCGCUGAUGAA (dTdT) -3 ' Linc-ASEN Small interfering RNA 2 (antisense) 5 5'-UUCAUCAGCGAAAGUUUCC (dTdT) -3 '

Example  2. Trip plate blue  Through staining Linc - ASEN  Identify the effect of small interfering RNAs on cancer cell growth

In order to confirm the induction effect of Linc-ASEN small interfering RNA on cancer cell senescence, the change of cultured cell proliferation rate was observed and trippine blue staining was performed. First, Linc-ASEN small interfering RNAs (SEQ ID NOS: 2 and 3) were treated with human breast cancer cells (MCF7) using Lipofectamine RNAiMAX (Invitrogen Corp.). Thereafter, the number of human breast cancer cells (MCF7) living on days 1, 2, 3, and 4, respectively, on the day of treatment with Linc-ASEN small interfering RNA was measured using trypan blue staining known in the art Respectively. After confirming the number of cells in each day, relative cell proliferation was shown based on day 0 as 1, and control small interfering RNAs were simultaneously treated and compared. The results are shown in Fig.

As shown in Fig. 1, the cells treated with the control small-interfering RNA were confirmed to normally proliferate, whereas the cells proliferated by the Linc-ASEN small interfering RNA were significantly reduced. This suggests that Linc-ASEN small interfering RNA inhibits the proliferation of cancer cells through the inhibition of Linc-ASEN expression, that is, induces senescence.

Example  3. Observation of cell appearance Linc - ASEN  Small interfering RNA of  Determination of aging induction effect of cancer cells

In order to confirm the induction effect of Linc-ASEN small interfering RNA on cancer cell aging, cell appearance was examined using an optical microscope. First, human breast cancer cells (MCF7) were treated with small interfering RNAs and Linc-ASEN small interfering RNAs (SEQ ID NOS: 2 and 3) in the same manner as in Example 2 above. Each of the cells was subcultured at an interval of 2 days in an incubator maintained at 37 ° C and 5% CO ₂ air. 2 days and 4 days, the cell appearance was observed using an optical microscope, and the results are shown in FIG.

As shown in FIG. 2, unlike the cells treated with the control small interfering RNA, it was confirmed that the cells treated with the Linc-ASEN small interfering RNA showed a wide and flat-shaped, aging-cell-specific form. This suggests that the Linc-ASEN inhibitor, Linc-ASEN small interfering RNA, has the effect of inducing senescence in cancer cells.

Example  4. Beta- Galactosidase (β- 가alat 시드세제 ) Through staining Linc - ASEN  Confirming the induction effect of small interference RNA on human breast cancer cell aging

Beta-galactosidase staining was performed to confirm the aging induction effect of Linc-ASEN small interfering RNA based on the appearance of a modified pattern of β-galactosidase expression in aged cells. First, a human breast cancer cell line MCF7 was cultured in a 60 mm culture dish in the same manner as in Example 2, and the cells were treated with a small interference RNA treatment group, a control group, and a LincASEN small interference RNA (SEQ ID NOs: 2 and 3) .

The cells were washed with PBS (phosphate-buffered saline) and the cells were fixed with 3.7% formaldehyde for 5 minutes. (5-bromo-4-chloro-3-indole? -D-galactoside), 40 mM citric acid / sodium phosphate (pH 5.7), 5 mM The cells were stained with a solution containing potassium ferrocyanide, 150 mM sodium chloride and 2 mM magnesium chloride. The staining was carried out in a 37 ° C incubator for 12 to 16 hours. The proportion of positive cells, which are about the level of beta-galactosidase activity, was observed using an optical microscope, and the results are shown in FIG.

As shown in Fig. 3, it was confirmed that the proportion of cells showing aging-related beta-galactosidase-positive cells in the experimental group treated with Linc-ASEN small interfering RNA was increased as compared with the control group. This indicates that the Linc-ASEN inhibitor Linc-ASEN small interfering RNA has the effect of inducing senescence in human breast cancer cells.

Example  5. Western Blotting  through Linc - ASEN  Confirming the induction effect of small interference RNA on human breast cancer cell aging

Western blotting was performed to determine whether aging induction effects of Linc-ASEN small interfering RNA were due to changes in protein level expression. First, in the human breast cancer cell line MCF7 cultured in the same manner as in Example 2, a control group, which is a small interference RNA treatment group, and an experimental group, which is a treatment group of LincASEN small interference RNA (SEQ ID NOs: 2 and 3), were prepared.

After washing the cell culture medium with PBS (phosphate buffered saline), the cells were collected with a scraper. The collected cells were treated with RIPA buffer (5 M sodium chloride, 10% Triton X-100, Deoxycholic Acid, 10% SDS, 1.5 M Tris-HCl pH 8.0, 0.5 M EDTA, distilled water) And the protein was extracted. The extracted proteins were quantified using a BCA protein assay kit (Thermo Scientific Pierce) and separated by SDS-PAGE gel.

The separated proteins were transferred to a nitrocellulose membrane, and primary antibodies and secondary antibodies specific to each protein were sequentially treated as shown in [Table 2] and [Table 3] below. The nitrocellulose membrane was washed with PBST (phosphate buffered saline with tween 20) and treated with ECL reagent (Thermo Scientific) to observe changes in protein expression. The results are shown in Fig. Actin was used as a control.

Primary antibodies specific for proteins Antibody host manufacturer In Ser807 / 811 the anti-in-pRb Rb Cell signaling Inc. (Danvers, MA) Anti-p53 Mo Novocastra Inc. (Newcastle, UK) Anti-p21 Rb Santa Cruz Biotechnology (Santa Cruz, CA) Anti-beta actin Mo Applied Biological material Inc. (Richmond BC, Canada)

Secondary antibody specific for protein Antibody host manufacturer HRP-conjugated anti-mouse Mo Santa Cruz Biotechnology (Santa Cruz, CA) HRP-conjugated anti-rabbit Rb Santa Cruz Biotechnology (Santa Cruz, CA)

As shown in FIG. 4, the expression of p21, which is an index of cell senescence, was specifically increased and phosphorylation of Rb was reduced in the experimental group treated with Linc-ASEN small interfering RNA as compared with the control group. This indicates that Linc-ASEN small interfering RNA has the effect of regulating expression at the protein level and inducing cancer cell senescence.

From these experiments, Linc-ASEN small interfering RNA, a Linc-ASEN expression inhibitor, has been shown to reduce cancer proliferation and to induce cancer cell senescence. Therefore, the Linc-ASEN small interfering RNA of the present invention is effective in inhibiting cancer cell proliferation through induction of cancer cell senescence, and thus can be used in various fields such as a composition for inducing cell senescence, an anticancer agent, and a cancer adjuvant.

Hereinafter, examples of pharmaceutical compositions for preventing or treating cancer, including the Linc-ASEN expression inhibitor of the present invention, will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1. Preparation of pharmaceutical preparations

1. Manufacturing of powder

Linc-ASEN expression inhibitor 20 mg

Lactose 100 mg

Talc 10 mg

The above components can be mixed and filled in an airtight container to prepare powders.

2. Preparation of tablets

Linc-ASEN expression inhibitor 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets may be prepared by tableting according to a conventional method for producing tablets.

3. Preparation of capsules

Linc-ASEN expression inhibitor 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components may be mixed and filled in a gelatin capsule according to a conventional method for preparing a capsule, thereby preparing a capsule.

4. Preparation of injections

Linc-ASEN expression inhibitor 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

5. Manufacture of liquids

Linc-ASEN expression inhibitor 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component is added to and dissolved in purified water according to a conventional liquid preparation method, and the lemon flavor is added in an appropriate amount, then the above components are mixed, and then purified water is added. The total amount of the liquid agent added to the purified water is adjusted to 100 ml, and the liquid agent can be finally prepared by filling it in a brown bottle and sterilizing it.

<110> Inha University Research and Business Foundation <120> Composition for inducing cell senescence comprising Linc-ASEN          gene expression inhibitor <130> 1-203P <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 794 <212> DNA <213> Homo sapiens <400> 1 ggtgactttg gtgcccagca ctacaatttg ttgactgtgt gtgacttctc tccaaagaga 60 gaccccaaag agagaaagcg ataccaatga aggccattca ggcacccatg tttaaggggg 120 tcccccaggc taatttctta cttgcgagga agccttcatg gcagtcaatg cgctttcaca 180 tgtggcctct ccccgaccct cactggccgg gtggagacta tgagtcctcg tttcacagac 240 atgacattgg ggttcagaga aatgaagtga ggtgcccaag gccatccagt aaataaagga 300 cggagctctt tcacaatccc ttgggcctgg cccttggaaa ctttcgctga tgaagtgatg 360 atgggtgaga gtcaggcttg cagcaggaaa agggcaaacg gggcagggga gggagtgccc 420 ggatgtcctg gccaggaggt gtgtccgtgg gagaagcagc ctggctcaga gcccaagtcc 480 agcaggcccg ggggagggtg gcctcatggc tccagtcagc ccagctgggc ctgcaaggac 540 ccctggtcgg gcaggggctt ctcagtgtaa aaccctaaca ggggcgttga gaggattacc 600 cgagaggggg ctgcatcctg tgcttagctc agggcatggc tcatgtttgc tctcaatatt 660 atttatttct ctcccaaaga gaacaagcag gatgtcctct ggctccatgg tgcctgctcc 720 tcagcccgac aggagcagct gtggaacagc cactgtgtgc ttaataaaca tgcagacatt 780 tgattgaatt ctca 794 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Linc-ASEN siRNA 1-SENSE <400> 2 gcucauguuu gcucucaau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Linc-ASEN siRNA 1-ANTISENSE <400> 3 auugagagca aacaugagc 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Linc-ASEN siRNA 2-SENSE <400> 4 ggaaacuuuc gcugaugaa 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Linc-ASEN siRNA 2-ANTISENSE <400> 5 uucaucagcg aaaguuucc 19

Claims (9)

(RNA), small interfering RNA (siRNA), and ribozyme, which bind complementarily to Linc-ASEN (long intergenic noncoding RNA-Associated with Senescence) Wherein the pharmaceutical composition comprises at least one inhibitor of Linc-ASEN expression.
The method according to claim 1,
Wherein said Linc-ASEN is RNA transcribed from the DNA of SEQ ID NO: 1.
delete The method according to claim 1,
Wherein the small interfering RNA is at least one selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 2-5.
delete delete delete delete (RNA), small interfering RNA (siRNA), and ribozyme, which bind complementarily to Linc-ASEN (long intergenic noncoding RNA-Associated with Senescence) Cancer-preventing or treating cancer comprising at least one Linc-ASEN expression inhibitor selected from the group consisting of

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