KR20100115157A - Composition for preventing or treating aging or age-related diseases comprising aimp3 si-rna or anti-aimp3 antibody - Google Patents

Abstract

PURPOSE: A composition containing AIMP3 inhibitor for preventing or treating aging and aging-associated diseases is provided to promote ubiquitin of raminin A. CONSTITUTION: A composition for preventing or treating aging or aging-associated diseases contains AIMP3 small interfering RNA(AIMP3 siRNA) or anti-AIMP3 antibody. The aging-associated diseases are alopecia, wrinkle, or osteoporosis. The AIMP3 small interfering RNA(AIMP3 siRNA) is sequence number 1 and 2, 3 and 4, or 5 and 6.

Description

Composition for preventing or treating aging-related diseases containing AI-RNA or antibody of AIMP3 as an active ingredient {Composition for preventing or treating aging or age-related diseases comprising AIMP3 si-RNA or anti-AIMP3 antibody}

The present invention relates to a substance having inhibitory activity of AMP3 (ARS-interacting multifunctional protein 3) and a composition for preventing or treating aging and aging-related diseases, including the same, and more specifically, ubiquitin of laminin A It relates to a pharmaceutical composition for the prevention or treatment of aging or age-related diseases, including AIMP3 si-RNA or anti-AIMP3 that inhibits the activity of AIMP3.

Aging is the collective term for all physiological changes in the body that occur over time and is a life phenomenon that is caused by a number of factors that vary from person to person, including cancer, cardiovascular failure, obesity, and non-alcoholic fatty liver. The state is associated not only with the degeneration of cells, tissues and organs, but also with the decay of most physiological performance limits. Aging varies in shape and speed from individual to individual, and even in each individual tissue, the pattern of aging is difficult to study at the individual level. On the other hand, if you look specifically at the aging phenomenon, changes in the function of each component organ and tissue occurs, which is due to the change in the function of the cells as a structural unit. In other words, the aging of the individual may be caused by the loss of nerve cells in the brain, the loss of cognitive function, the loss of elasticity of the skin due to the loss of subcutaneous fat cells, or the whitening of hair root melanocytes due to loss of melanin pigment production. This is due to the aging of the cells that make up the individual.

On the other hand, in order to understand the aging of the individual it is necessary to identify the molecular mechanism through the study of aging at the cellular level, that is, cellular senescence (cellular senescence). According to the results of cell aging, senescent cells cease cell division at G1 phase and can no longer enter S cell phase by physiological growth factors. It also shows several functional declines and the appearance of final differentiated cells (Goldstein, Science, 249: 1129-1133 (1990); Campisi J., Cell, 84: 497-500 (1996)). Many studies of cellular aging have been done using human fibroblasts, which have been reported to reflect the aging phenomena of individuals (Campisi J., Cell, 84: 497-500 (1996)).

Aminoacyl-tRNA synthetases (ARSs) in higher eukaryotic systems consist of a unique polymer complex consisting of nine different ARSs and three cofactors called ARS-interacting multi-functional proteins (AIMPs). AIMP3 / p18 is the smallest component that hides in the complex and plays an important role in the stability of the entire complex. Although hidden small in the complex, it is activated in the presence of DNA damage or carcinogenic stimuli and transferred to the nucleus to activate ATM or ATR, the upstream kinases of p53.

Point mutations or allele deletions have been observed in cancer patients that inactivate or reduce the ability of AIMP3 to activate p53. AIMP3 homozygous mice die in the early embryonic state, while heterozygous mice are born live but develop various cancers. AIMP3 heterozygous cells transfected with oncogenes such as Ras and Myc show severe nuclear fragmentation and chromosomal instability.

The present inventors have revealed that AIMP3 has tumor suppression ability, but the anti-aging activity of the si-RNA or anti-AIMP3 antibody of AIMP3 has not been revealed and there is no research on this.

The present inventors have discovered that overexpression of this protein induces cellular aging in studies related to AIMP3 through a decrease in cell differentiation, an increase in aging markers, and nuclear modification by reduction of laminin A, a nuclear membrane protein. Animal studies have revealed that aging leads to aging of individuals. In particular, promoting the degradation of laminin A is due to the mechanism of inducing the binding of laminin A to Siah-1 and ubiquitin, and that AIMP3 small interfering RNA (si-AIMP3) or anti-AIMP3 antibody inhibits this mechanism. It was found that the present invention was completed.

Therefore, an object of the present invention is to provide a composition for the prevention or treatment of aging or aging-related diseases comprising AIMP3 si-RNA (si-AIMP3) or anti-AIMP3 antibody.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of aging or aging-related diseases comprising AIMP3 si-RNA (si-AIMP3) or anti-AIMP3 antibody.

Hereinafter, the present invention will be described in detail.

The present invention provides AIMP3 small interfering RNA (si-AIMP3) or anti-AIMP3 antibody comprising a composition for the prevention or treatment of aging phenomena or aging-related diseases.

si-RNA refers to double-chain RNA that can induce RNA interference through the cleavage of specific mRNA. Si-AIMP3 of the present invention refers to a short double-chain RNA, characterized in that it inhibits the expression of genes by interfering with the mRNA making AIMP3. The si-RNA of the present invention can inhibit the cellular expression of AIMP3 to prevent aging of cells by AIMP3, and in particular, to prevent degradation of the nuclear membrane by preventing degradation of the nuclear membrane protein laminin A by AIMP3. In one embodiment of the present invention, as the concentration of si-AIMP3 administered increases, the ubiquitin of laminin A decreases (see FIG. 3c) and the amount of laminin A increases in whole cell lysate and the amount of AIMP3 increases. It was confirmed to shrink (see FIG. 3F).

The si-AIMP3 of the present invention is not limited to the paired double-chain RNA portion paired with RNA, but may include a portion not paired by a mismatch, bulge and the like. The terminal structure of si-AIMP3 can be either a blunt end or a protruding end as long as the expression of the target gene can be suppressed by the RNAi structure. si-AIMP3 may include low-molecular RNA (eg, tRNA, rRNA, viral RNA) at one protrusion in a range capable of maintaining the expression inhibitory effect of the AIMP3 gene.

The method for preparing si-RNA is a method of directly synthesizing si-RNA in vitro, then transfecting into cells and si-RNA expression vector or PCR-derived si prepared to express si-RNA in cells. There is a method of transforming or infecting an RNA expression cassette into a cell. The determination of how to prepare an si-RNA and introduce it into a cell or animal may depend on the purpose of the experiment and the cellular biological function of the target gene product.

The si-RNA of the present invention specifically reacts with AIMP3 and is preferably si-RNA consisting of a pair of SEQ ID NOs: 1 and 2, a pair of SEQ ID NOs: 3 and 4, or a pair of SEQ ID NOs: 5 and 6, respectively.

The composition of the present invention may use si-AIMP3 alone or may include additional substances known to be effective in preventing aging.

The present invention provides antibodies specific for AIMP3. "Antibody" refers to specific protein molecules directed against antigenic sites. Antibodies of the invention include both polyclonal and monoclonal antibodies. The preparation of antibodies can be readily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods of injecting AIMP3 protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like.

Monoclonal antibodies can be prepared using fusion methods, synthetic DNA methods or phage antibody library techniques well known in the art.

The present invention provides a method for screening an anti-aging substance, wherein the candidate substance inhibits the activity of AIMP3 that promotes the ubiquitin of laminin A. To this end, methods for detecting protein-protein interactions known in the art can be used, for example, in vitro protein-protein binding assays (in vitro pull-down assays), electrophoretic mobility shift assays (EMSA), Immunoassays, functional assays (such as phosphorylation assays) for protein binding, non-immunoprecipitation assays, immunoprecipitation western blot assays, immuno-co-localization assays, cross-linking ( cross-linking, affinity chromatography, immunoprecipitation (IP), yeast two-hybrid (Y2H), fluorescence resonance energy transfer (FRET), split protein Recombination (bimolecular fluorescence complementation, Bi-FC) can be performed by a variety of methods known in the art.

When the composition of the present invention is administered clinically, the composition of the present invention may be formulated in a unit dosage form suitable for oral or parenteral administration. When formulated in the form of a general medicine, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations include at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, ointments, creams. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

In addition, the composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into a parenteral formulation, the compounds of formulas 1 to 6 of the present invention are mixed in water with stabilizers or buffers to prepare solutions or suspensions, which are formulated in unit dosage forms of ampoules or vials. Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method and excretion, drug combination and severity of the disease.

These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

The drawings described in the present invention will be described as follows.

Figure 1 relates to the contents that AIMP3 inhibits cell proliferation and induces cell aging accompanied with modification of the nucleus. In FIG. 1A, changes of 293 cells according to culture time after transfection of GFP and GFP-AIMP3 were confirmed by light microscopy and fluorescence microscopy. (Bar = 100 μm) In FIG. 1B, growth curves of 293 cells expressing GFP and GFP-AIMP3 were confirmed. In FIG. 1C, a list of cell cycle changes of 293 cells expressing GFP-AIMP3 was confirmed. After expressing GFP-AIMP3, the nuclei were separated and stained with propidium iodide at the indicated time and analyzed using flow cytometry. The vertical axis represents the ratio of the G0-G1, S and G2-M groups. 20,000 analyzes per sample were analyzed. In FIG. 1D, the effect of AIMP3 (+ means 6 μg / ml plasmid DNA) on aging-related β-galactosidase activity in p53 ^{+ / +} and p53 ^{− / −} HCT116 cells is indicated by X-gal staining. One photo was confirmed. In FIG. 1E, Western blotting results of DcR2 were performed to investigate the effect of p53 on PCc and HCT116 cells on DcR2 amount of AIMP3. In FIG. 1F, HeLa cell lines were transfected with empty vector (EV) or AIMP3 and stained with laminin A using an anti-laminin A antibody to confirm immunofluorescence comparison of nuclear structures. In FIG. 1G, the distribution of laminin A was observed by transfection of GFP-laminin A with EV or AIMP3. In FIG. 1H, the photos observed by β-galactosidase staining of AIMP3 WT and TG MEF cells after passage 8 were confirmed. In FIG. 1i, the immunoblot analysis of AIMP3 and senescence markers DcR2 and p16 in the heart cells of AIMP3 WT and TG mice was confirmed. In FIG. 1J, the amount of intracellular laminin A and AIMP3 measured by western blotting using anti-laminin A and AIMP3 in AIMP3 WT and TG MEFs was confirmed.

2 shows that AIMP3 increases ubiquitin-mediated degradation of laminin A. In FIG. 2A, p53 ^{+ / +} and p53 ^{− / −} HCT116 cells were injected with AIMP3 at different concentrations (µg / ml) and the amount of laminin A was measured by western blotting. In FIG. 2B, Western blotting test results of the effects of the inhibition of AIMP3 on the amount of laminin A using specific siRNA in 293 and Hela cells were confirmed. GFP-laminin A was transfected into both cells and the amount was measured by western blotting using anti-GFP antibody. In Figure 2c, the results of the test of the interaction of endogenous AIMP3 and laminin A through co-immunoprecipitation was confirmed. Protein extracts from 293 cells were immunoprecipitated with anti-laminin A antibodies, and AIMP3 precipitated together was confirmed by western blotting. In FIG. 2D, GFP-Laminin A and Myc-AIMP3 were transfected into 293 cells, and then immunoprecipitated Myc-AIMP3 with an anti-Myc antibody, and together precipitated GFP-Laminin A by western blotting using an anti-GFP antibody. Confirmed. In Figure 2E, 293 cells were transfected with GFP-laminin A. GST-AIMP3 was immunoprecipitated with glutathione-Sepharose beads and coprecipitated GFP-laminin A was confirmed by western blotting using anti-GFP antibody. In Figure 2f, it was confirmed that AIMP3 performs laminin A reduction function depending on proteasome. p53 ^-/- HCT116 cells were transfected with plasmids expressing Myc-AIMP3 and GFP-laminin A. After 6 hours treatment with MG132, cell lysates were analyzed by western blotting using anti-Myc and -GFP antibodies. In Figure 2g, p53 ^-/- HCT116 cells were transfected with EV or Myc-AIMP3 and subjected to immunoprecipitation using anti-laminin A antibody after MG132 treatment. The precipitate was separated by SDS-PAGE and the ubiquitin of laminin A was observed by western blotting using anti-ubiquitin antibody. In FIG. 2H, Ap53 ^{− / −} HCT116 cells expressing HA-ubiquitin, Myc-AIMP3 and GFP-laminin were cultured with and without MG132. Cell lysates were immunoprecipitated with anti-GFP antibody and precipitates were western blotting with anti-HA antibody.

Figure 3 shows that AIMP3 promotes Siah1-dependent ubiquitin of laminin A. In FIG. 3A, Flag-Siah1 was transfected into Myc-p53 ^{− / −} HCT116 cells with or without Myc-AIMP3. After 24 hours, the amount of laminin A was measured by western blotting. The amounts of laminin A, Siah1 and AIMP3 were measured with anti-laminin A, -Flag, and -Myc antibodies. Tubulin was included as a loading control. In Figure 3b, 293 cells were transfected with a combination of HA-ubiquitin, si-Siah1 and Myc-AIMP3 shown in the figure. Thereafter, the cells were incubated with or without MG132 for 10 hours. Cell lysates were immunoprecipitated with anti-laminin A antibodies and precipitates were immunoblotting with anti-HA antibodies. The expression levels of Myc-AIMP3, Siah1 and tubulin in whole cell lysate (WCL) were measured using anti-Myc, -Siah1 and tubulin antibodies. In FIG. 3C, p53 ^{− / −} HCT116 cells were transfected with a combination of Flag-Siah1, HA-ubiquitin and si-AIMP3 at various concentrations (0, 5 and 10 uM) shown in the figure. After immunoprecipitation of laminin A, ubiquitinated laminin A was confirmed by immunoblotting using anti-HA antibody. In FIG. 3D, p53 ^{− / −} HCT116 was transfected with AIMP3 and Flag-Siah1 at various concentrations (0, 2, 4 μg / ml). Cell lysates were immunoprecipitated with anti-Myc antibody, and the precipitated Siah1 was confirmed by immunoboltting using anti-Flag antibody. In Figure 3e, ³⁵ S-labelled full-length Siah1 was prepared by in vitro translation and mixed with GST or GST-AIMP3. GST protein was precipitated by glutathione S-Sepharose, and the precipitated Siah1 was confirmed by radiographic imaging. In FIG. 3F, p53 ^{− / −} HCT116 cells were transfected with Flag-Siah1 and with different amounts of si-AIMP3. After 48 hours of incubation, cells were harvested and immunoprecipitated using anti-Flag antibody. The co-precipitated laminin A was confirmed by western blotting using anti-laminin A antibody.

4 relates to the premature aging phenotype of AIMP3 transfected mice. In FIG. 4A, the growth status of AIMP3 transfected mice (TG, n = 70) was wild-type (WT, n = 70) and heterozygous mice (HE, n = 70) by measuring body weight every two weeks for 24 months. Compared to. The number of AIMP3 transfected mice gradually decreased over time. In FIG. 4B, survival numbers of AIMP3 transfected mice, WT, and HE were measured and recorded monthly. In Figure 4c, the image of the 10-month-old AIMP3 TG and WT mice were confirmed by photographs. Transfected mice show alopecia. In Figure 4d, the number of mice showing the symptoms of hair loss by age was confirmed by a graph measuring. In FIG. 4E, skin tissues of AIMP3 WT and TG mice were compared by photomicrograph by hematoxylin and eosin staining. E, D and A mean epidermis, dermis and subcutaneous fat. The skin of TG mice was clearly wrinkled and the subcutaneous fat layer was reduced. In FIG. 4F, whole body radiographs of 18 months old WT and TG mice were confirmed. AIMP3 TG mice show a marked multiplication. In Figure 4g, bone density (gcm ^-2 ) of WT and TG mice was compared graphically (n = 8 in each group). In Fig. 4h, bone sections of 18-month-old TG and WT mice were compared by staining with hematoxylin and eosin. The thickness of the cortical bones of the tibia is greatly reduced in AIMP3 TG mice.

Figure 5 relates to the confirmation that the mouse aIMP3 cDNA entered into the chromosome of the mouse. In FIG. 5A, 234 amino acid long full-length mouse AIMP3 cDNAs were subcloned to the Eco RI site of the pCAGGS expression vector to make AIMP3 transfected mice. Expression of the entered AIMP3 cDNA is achieved by human cytomegalovirus immediate-early enhancers linked to the chicken-β-actin promoter. The primers to be used to specify that the mouse AIMP3 cDNA was entered was indicated. In FIG. 5B, genomic DNA was isolated from the tails of AIMP3 WT, HE and TG mice. PCR analysis was performed using A5 'and A3' primers. NC stands for negative control. In FIG. 5C, gemonic DNA was isolated from six mice and PCR was performed using B5 ′ and B3 ′ primers. Two of them produced PCR products of the desired size. In FIG. 5D, proteins were isolated from MEFs of 6 mice to measure the amount of AIMP3 expression. The measurement was performed by western blotting using anti-AIMP3 antibody. In FIG. 5E, RNA was isolated from MEF cells, and the expression level of AIMP3 was measured by RT-PCR using B5 'and B3' primers. In Figure 5f was confirmed the nucleotide sequence of the primer used to identify the AIMP3 cDNA in the chromosome.

Figure 6 relates to the degree of cellular senescence of AIMP3 WT, HE mice determined by β-galactosidase activity. In FIG. 6A, Senescence-Associated (SA) β-galactosidase activity was measured using X-gal after AIMP3 WT and HE MEF cells were cultured in media causing cell senescence. In FIG. 6B, the percentage of cells showing positive β-galactosidase reaction in total cells was measured and displayed graphically. In Figure 6c, AIMP3 WT and HE skin cells were subjected to SA-β-galactosidase staining. In FIG. 6D, control and si-AIMP3 RNAs were added to AIMP3 WT MEFs cells and subjected to SA-β-galactosidase staining.

7 shows that AIMP3 induces modification of the nucleus. Immunohistochemistry of laminin A was performed on the skin of AIMP3 WT and TG mice. In the bottom line, you can see the blue form and laminin A stained (brown). In AIMP3 TG cells, structural changes in the nucleus and reduction of laminin A can be clearly confirmed.

8 relates to that AIMP3 selectively increases the ubiquitin of laminin A. In FIG. 8A, 293 cells were transfected with EV and AIMP3, respectively, and treated with cycloheximide to prevent synthesis of new proteins. Cleavage of caspase 6 produced fragments of laminin A (70 kDa) at 40-45 and 28 kDa, and laminin A / C antibodies acted on total laminin A and 28kDa laminin A. In FIG. 8B, GFP-Laminin A, Myc-AIMP3, and Myc-AIMP2 were transfected into 293 cells in the combinations shown in the figure and the expression of this protein was confirmed by western blotting. In FIG. 8C, 293 cells were transfected with GFP-laminin A and Myc-Aimp3, and the expression of each protein shown in the figure was confirmed by western blotting.

9 relates to the interaction of GST-AIMP3 with Siah1. Various amounts of Flag-Siah1 were injected into HCT116p53 ^{− / −} cells. GST-AIMP3 was precipitated with glutathione-Sepharose and the precipitated Siah1 was measured by western blotting.

Figure 10 relates to AIMP3 specifically ubiquitin laminin A but not progerin. In Figure 10a, the difference in exon structure of laminin A and progerin. Laminin A cDNA consists of 12 exons. progerin is produced by a miscution between the double-endon 11 and the N-terminus of 12. In FIG. 10B, 293 cells were transfected with HA-ubiquitin and Flag-Siah1 and GFP-progerin and GFP laminin A in the combination shown in the figure. AIMP3 and laminin A were immunoprecipitated and the precipitate was separated by SDS-PAGE. The ubiquitin of laminin A and progerin was confirmed by immunoblotting using anti-HA antibody. In FIG. 10c, the direct interaction of laminin A with progerin on AIMP3 was measured by an in vitro pull-down assay. Radioactive AIMP3 was prepared by in vitro translation and mixed with GST-laminin A (exon 11-12 pieces) or GST-progerin (Δexon 11-12). GST protein was precipitated using glutathione-Sepharose, and the precipitated AIMP3 was confirmed by radiograph. 10d and 10e, the effect of AIMP2 on the intracellular rotation of laminin A (10d) and progerin (10e) was confirmed by pulse-chase experiments. 293 cells were transfected with Myc-AIMP3 and GFP-laminin A or GFP-progerin and incubated for 3 hours in radioactive methonine. Intracellular protein production was inhibited by cycloheximide, and GFP-laminin A or GFP-progerin was immunoprecipitated by anti-GFP matrix and confirmed by radiograph.

11 shows that AIMP3 increases with a decrease in laminin A in some aged tissue. The immunoblotting test of AIMP3, laminin A / C, and senescence markers DcR2 was performed on tissue cells of 2 months (Y) and 28 months (O) mice. The upper band of the two bands of laminin A / C is the band of laminin A. Five samples were tested and one of them was displayed. Increased expression of AIMP3 was likewise seen in other samples.

As described above, the present inventors have found that AIMP3 induces cell aging and in particular promotes the ubiquitin of laminin A to promote aging through modification of the nucleus, and that a substance that inhibits this mechanism inhibits aging by AIMP3. It was clarified. In particular, it was confirmed that the si-RNA of AIMP3 was excellent in the ubiquitin inhibitory activity of laminin A of AIMP3. Accordingly, the composition of the present invention provides a new method for the treatment of aging and related diseases.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Example 1

Materials and Experiment Methods

Material

Mammalian a-type laminin and progerin expression vectors were provided by Tom Misteli (NIH / NCI). Lipofectamine was used for transfection according to the provider's method. Laminin A / C and p16 antibodies were purchased from Cell Signaling and DcR2 and GFP were purchased from Santa Cruz Biotechnology. The anti-AIMP3 antibody (polyclonal rabbit anti-AIMP3 antibody) was prepared according to Kim et al. (Ki, T. et al., J. Biol. Chem., 275: 21768-21772, 2000). Western blotting ECL solution (WEST-one) was purchased from Intron biotechnology.

2. Construction of AIMP3 Transfected Mice.

To prepare AIMP3 transfected mice, a 234 amino acid-length full-length mouse AIMP3 cDNA was obtained by RT-PCR using mouse transcripts as a template, and included a human cytomegalovirus immediate-early enhancer linked to the chicken-β-actin promoter. Subcloning was performed at the Eco RI site of the pCAGGS expression vector. The fragments were cleaved with Sal I and Hind III of the vector and purified DNA was injected into fertility embryos of fertilized eggs of FVB / N mice and transplanted into fertile pregnant female FVB / N mice. Founder genotypes were determined by PCR using primers specific for DNA sequences and nucleotide sequences isolated from the tails of mice. Three founders were made. Mice were reared in cages with a 12 h light / dark cycle. The first generation of heterozygous transfected offspring was made by backcrossing the founder with C57Bl / 6J. AIMP3 expression in transfected mice was confirmed by immunoblotting and RT-PCR of AIM3. All experiments were conducted at a facility accredited by the Laboratory Animal Care Assessment Certification Association in accordance with Sungkyunkwan Medical School's accredited animal policy.

3. Phenotypic analysis.

Mice are subjected to systemic X-ray analysis for 20 seconds at 20KV under anesthesia. Tibia is measured for bone thickness and osteoitis based on tibia. Dorsal skin fragments were fixed, embedded with paraffin and stained with haematoxylin and eosin. The thickness of the dermis and subcutaneous fat layer was measured by taking four skin samples from each age group of each genotype.

4. β-galactosidase staining.

Cells are washed with PBS and fixed with 0.5% glutaraldehyde. After washing with PBS containing 1 mM MgCl ₂ and stained overnight at 37 ° C with X-gal solution (Senescence β-galactocidase staining kit, Cell signaling).

5. Fluorescent staining.

Cells are planted in cover glass and transfected with GFP-Laminin A. Blocking buffer (PBS containing 0.01% bovine serum) albumin and 0.05% Triton X-100) for 1 hour. After washing with PBST, the primary antibody (1: 200) is treated followed by the Alexa488-conjugated secondary antibody (1: 300). The cells are then stained with DAPI. After mounting, observe laminin A with a fluorescent confocal microscope (, Bio-Rad).

6. Western blotting and co-immunoprecipitation.

Lysate the cells in RIPA buffer containing protease inhibitor and centrifuge the lysate at 14,000 rpm for 30 minutes. The extracted 20μg of protein is separated by SDS-PAGE. To test the interaction of AIMP3 and laminin A, protein extracts were reacted with normal IgG and protein A / G agrose for 2 hours and centrifuged to remove IgG-specific proteins. Take the supernatant, add the antibody to laminin A or GFP, and react with stirring at 4 ° C. for 2 hours, and then add protein A / G agarose. After washing twice with cold PBS and washing once with RIPA, the precipitate is placed in SDS buffer and separated by SDS-PAGE. The protein was transferred to PVDF membrane and AIMP3 reacted with anti-AIMP3 monoclonal antibody and horseradish peroxidase-conjugated secondary antibody.

7. In vitro  pull-down.

For in vitro pull down assay, GST-laminin A or GST was first mixed with glutathione-Sepharose for 2 hours in 4 ° C PBS buffer containing 1% Triton X-100 and 0.5% N-laurylsarcosine. TMP Quick coupled Transcription / Translation system (Promega) is used to prepare AIMP3 by in vitro translation in the presence of [ ³⁵ S] methionine. The prepared peptide was added to the GST protein mixture and reacted with stirring in PBS buffer containing 1% TritonX-100, 0.5% N-laurylsarcosine, 1 mM DTT, 2 mM EDTA and 300 μm phenylmethysulfonyl fluoride for 4 hours at 4 ° C. Let's do it. Then wash 6 times in the same buffer containing 0.5% Triton X-100. Isolate proteins bound to Sepharose bead with SDS buffer and separate them with SDS-PAGE to make radiographs.

8. RT-PCR.

Sol the cells. After dissolving with D solution (4 M guanidine thiocyanate, 1% laurosarcosine, 25 mM sodium citrate and 0.1% β-mercaptoenthanol), it is reacted with chloroform containing acidic phenol and 4% isoamylalcohol. After shaking, the mixture was centrifuged at 14,000 rpm, the supernatant was taken and isopropanol was added to precipitate RNA. After washing with 100% ethanol, 1 μg of RNA is dissolved in sterile water and used with a specific primer as a template for RT-PCR.

9. Construction of si-RNA for AIMP3

In order to suppress the expression of AIMP3 si-RNA for AIMP3 was constructed.

The mRNA sequence of human-derived AIMP3 protein (GenBank accession No. AB011079) was searched for in vitro-generated si-RNA sequences that specifically act on AIMP3.

si-RNA sequences SEQ ID NO: name order Number One AB011079_Stealth_115_sence cca agu cua aca gga uug acu acu a 25 2 AB011079_Stealth_115_antisence uag uag uca auc cug uua gac uug g 25 3 AB011079_Stealth_107_sence aca aug guc caa gyc yaa cag gau u 25 4 AB011079_Stealth_107_antisence aau ccu guu rgr cuu gga cca uug u 25 5 AB011079_Stealth_154_sence guc aag caa gcc aac aaa gaa uau u 25 6 AB011079_Stealth_154_antisence aau auu cuu ugu ugg cuu gcu uga c 25

* Degenerate sequence: Y means (C or T), R means (A or G).

In order to experience the function of the prepared si-RNA, control and the prepared si-AIMP3 RNA were added to the HEK293 cell line, respectively, and 48 hours later, the amount of intracellular AIMP3 was measured using anti-AIMP3 antibody. As a result, as shown in Figure 12 it was confirmed that the amount of AIMP3 specifically reduced only when the si-RNA of the present invention is administered.

≪ Example 1 >

Inhibition of Cell Proliferation of AIMP3 and Induction of Cell Aging with Nuclear Modification

To determine whether AIMP3 affects cellular senescence, we transfected AIMP3 and GFP into human embryo kidney cell line 293 and observed how AIMP3 affects cell growth.

As shown in the GFP production of the transfectants, it was confirmed that the AIMP3 transfectants clearly lag behind the growth of the control cell lines (FIGS. 1a and b).

AIMP3 transfectants showed an increase in G0 / G1 phase with a decrease in S phase, suggesting the possibility of AIMP3 cell proliferation inhibitory activity (FIG. 1C). Because AIMP3 activates p53 via ATM and ATR in response to DNA damage, and activated p53 can cause cell death as well as cell death, leading to the conclusion that AIMP3 will induce cell aging through p53. To verify this possibility, we examined whether ß-galactosidase activity associated with aging was determined by transfecting AIMP3 into p53 ^{+ / +} and p53 ^-/- HCT116 cells to determine whether AIMP3 could induce cellular senescence.

Although, as expected, ß-galactosidase activity was increased in p53 ^{+ / +} HCT116 cell line by AIMP3 transfection, similar increase in ß-galactosidase activity was observed in p53 ^{− / −} HCT116 cell line (FIG. 1D). This suggests that AIMP3 promotes cell aging independent of p53.

To further demonstrate this, the degree of aging was observed by treating AIPM3 to two different cell lines, p53-inactive PC3 and HCT116, and measuring the amount of DcR2, another marker of aging. Treatment with AIMP3 increased the amount of DcR2 in both cell lines (FIG. 1E), further demonstrating that AIMP3 can augment cell aging independent of p53. We also stained the nuclear membrane protein laminin A by immunofluorescence to observe aging of the cells. The nuclear membrane structure was modified by transfection of AIMP3 (FIG. 1F). To demonstrate this observation, we treated GFP-fused laminin A with AIMP3 to determine the response of laminin A to AIMP3. In addition, the appearance of normal nuclei was altered by the imported AIMP3, and foci formation of GFP-laminin A was observed (FIG. 1G). All these results show that AIMP3 can induce cell senescence regardless of p53 activation.

To investigate whether AIMP3-induced cellular senescence occurs in vivo, we continuously constructed AIMP3 transfected mice expressing more AIMP3 than wild type. Transgenic mice were prepared according to the method described in the method by adding AIMP3 cDNA of the mouse to the vector with the ß-actin promoter of the chicken (Fig. 5a). The incorporation of AIMP3 cDNA into the FVB / N mouse chromosome was demonstrated using PCR using different primer pairs (Figure 5b, c and f). Increased expression of AIMP3 in transfected mice was confirmed by western blotting and RT-PCR (FIG. 5D and e). Comparison of ß-galactosidase activity of AIMP3 transfectant (TG) mouse embryonic fibroblasts (MEFs) with wild-type (WT) resulted in more intense X-gal staining in AIMP3 TG cells (FIG. 1H). We also isolated heart cells and compared DcR2 and p16, known markers of aging, by Western blotting. Again, AIMP3 TG cells showed higher amounts of DCR2 and p16 compared to wild type (FIG. 1I). In contrast, we compared the activity of ß-galacosidase in heterozygous AIMP3 TG, wild-type MEFs and skin tissue. It was observed that ß-galacosidase activity was markedly reduced in AIMP3 heterozygote cells compared to wild type (Fig. 6a, b and c). The activity of ß-galactosidase is also markedly degraded when AIMP3 transcription is interrupted by specific siRNAs. Since transfection of AIMP3 induces nuclear transformation, we examined whether Western blotting of laminin A levels in AIMP3 transfected MEF cells.

As expected, the amount of laminin A was reduced in AIMP3 transfected cells compared to wild-type cells. Laminin A analysis by immunohistochemistry on mouse skin tissue confirmed that the amount of laminin A in AIMP3 transfected mice was reduced compared to wild type. AIMP3 transfected skin cells often showed modified nuclei with reduced laminin A staining by AIMP3 transfection (Supplementary Figure 7 right arrows).

<Example 2>

Promoting ubiquitin-mediated Laminin A Degradation of AIMP3

To determine whether AIMP3 actually plays a role in reducing the amount of laminin A, we examined the change in the amount of laminin A by transfecting AIMP3 into p53 ^{+ / +} and p53 ^-/- HCT116 cells. The amount of laminin A decreased as the amount of AIMP3 in both cells increased (FIG. 2A). In any case, the transfectants of laminin A did not affect the amount of AIMP3 in the cells (results not shown), so it could be expected that AIMP3 is a higher regulator of laminin A. In contrast, we observed two different cell lines to determine how inactivating AIMP3 affects the amount of laminin A. To more accurately measure the change in laminin A amount, we transfected GFP-laminin A into 293 and HeLa cells and inhibited the expression of AIMP3 using specific siRNA. The amount of GFP-laminin A increased with inhibition of AIMP3 in both cell lines (FIG. 2B).

Since there was no change in laminin A transcription by transfection of AIMP3 (results not shown), the reduction of AIMP3-dependent laminin A does not appear to be involved in the regulation of laminin A. Therefore, we assumed that AIMP3 influences the protein stability of laminin A in some way. To find out, we first tested whether AIMP3 temporarily interacts with laminin A by co-immunoprecipitation. In 293 cell lines, when endogenous AIMP3 was immunoprecipitated using antibody, laminin A was also precipitated together (FIG. 2C).

Cellular interactions between the two proteins were also confirmed by co-immunoprecipitation of added GFP-lamia A and Myc-AMIP3 (FIG. 2D.). We performed an in vitro pull-dawn assay using GFP-laminin A and GST-AIMP3 to confirm direct interaction between the two proteins (Fig. 2e.).

The amount of intracellular laminin A can be regulated post transcription by several different mechanisms. First we tested whether AIMP3 induces caspase6 dependent laminin A degradation. Although control group 293 cells were treated with cycloheximide, a novel protein production inhibitor, a hydrolysis product of laminin A was found (Fig. 8a left), but AIMP3 was reduced even though the amount of laminin A decreased more rapidly when AIMP3 was added. When transfected it was not observed (Fig. 8a right).

These results indicate that the regulation of AIPM3-dependent laminin A inhibition occurs through different pathways. We also tested the possibility of degradation of laminin A by lysosomes. When cells were treated with NH ₄ Cl, which inhibits the activity of lysosomes, this did not affect the degradation of AIMP3-dependent laminin A (results not shown).

Therefore, it was found that laminin A was not degraded by lysosomes.

Laminin A is known to interact with UBC9 (ubiquitin conjugating enzyme E21), so the stability of laminin A could be controlled by the ubiquitin system. We tested whether the reduction of AIMP3-dependent laminin A was associated with ubiquitin. When AIMP3 was added to HCT116 p53 ^{− / −} cells, the amount of intracellular GFP-laminin A decreased significantly as expected (FIG. 2F left). However, cells were not affected by AIMP3 when treated with MG132, which blocks the activity of the proteasome (FIG. 2F right). These results indicate that AIMP3 may induce ubiquitin-dependent laminin A degradation. We tested whether AIMP3 actually increased the ubiquitin of laminin A. The ubiquitin of endogenous laminin A was increased when p53 ^-/- HCT116 cells were treated with MG132 and AIMP3 was added (Figure 2g). We also found that p53 ^-/- HCT116 transfected with GFP-laminin A and HA-ubiquitin. After the cells were treated with MG132, the effect of AIMP3 on the ubiquitin of GFP-laminin A was observed. As a result, it was confirmed that the ubiquitin of GFP-laminin A by HA-ubiquitin was increased by the addition of AIMP3 (FIG. 2H). We also tested whether AIMP3 specifically requires AIMP3 in the ubiquitin of laminin A. To solve this problem, we transfected 293 cells with AIMP2, another cofactor associated with the multi-tRNA synthetase complex, and compared the effect on laminin A with that of AIMP3. As a result, unlike AIMP3, AIMP2 did not reduce the amount of laminin A (FIG. 8B). We also tested if AIMP3 could reduce the intracellular levels of another laminin protein, B1, and β-catenin, another substrate of ubiquitin. As a result, AIMP3 reduced not only the endogenous laminin A but also the amount of externally injected GFP-laminin A, but did not affect the amount of the other two proteins (Fig. 8c). All these results indicated that AIMP3 selectively regulates laminin A through the ubiquitin system.

Example 3

Siah1-dependent Laminin A Ubiquitin Promotion of AIMP3 and AIMP3 Inhibitory Activity by si-RNA on AIMP3

Siah1, an E3 ubiquitin ligase, is known to catalyze the ubiquitin of laminin A. We tested whether Siah 1 is involved in the ubiquitin of laminin A induced by AIMP3. First we checked whether the amount of laminin A was affected by the combination of Siah1 and AIMP3. As expected, the amount of laminin A was decreased when Siah1 was added, and the effect of Siah1 was further increased when AIMP3 was further added thereto (FIG. 3A). This suggests that both proteins have a cooperative effect on the inhibition of laminin A. When Siah1 is inhibited by siRNA selective to Siah1, the degree of AIMP3-dependent ubiquitin of laminin A decreases (FIG. 3B).

When the expression of AIMP3 is inhibited by si-RNA (si-AIMP3) to AIMP3, the degree of Siah1-dependent ubiquitin of laminin A decreases depending on the concentration of si-AIMP3 (FIG. 3C).

We investigated the interaction of Siah1 with AIMP3 by co-immunoprecipitation. Increasing expression of Siah1 also increased its interaction with AIMP3 (FIG. 3D). Interaction between the two proteins could also be observed between GST-AIMP3 and Siah1 (FIG. 9).

Direct interaction of Siah1 and AIMP3 was confirmed by an in vitro pull-down assay using Siah1 and AIMP3 prepared using isotopes (FIG. 3E).

We then tested whether the binding of Siah1 to laminin A was affected by AIMP3 by co-immunoprecipitation. As the expression of AIMP3 was inhibited by si-RNA against AIMP3 (si-AIMP3), the amount of laminin A immunoprecipitated with Siah1 was also reduced (FIG. 3F). This means that AIMP3 binds to both Siah1 and laminin A, and in some way mediates the interaction between the enzyme and the subject, and that the action of AIMP3 is inhibited by si-RNA (si-AIMP3) on AIMP3.

It is known that laminin A gene can make a deletion variant progerin (Δ50) (Fig. 10a). The amount of this variant increases as the amount of laminin A decreases in older cells. Transfection of AIMP3 increased ubiquitin of laminin A but not ubiquitin of progerin (FIG. 10B). To determine whether AIMP3 binds to progerin as well as laminin A, we prepared a GST-progerin fragment comprising a portion of GST-laminin A and exon11-12 spanning exon 11-12 (FIG. 10A). The interaction of this fusion protein with AIMP3 was measured by an in vitro pull-down assay. AIMP3 co-precipitated with GST-laminin A but not with GST-progerin (FIG. 10C). We also compared the effect of AIMP3 on the intracellular turnover rates of laminin A and progerin by pulse-chase experiments. The amount of laminin A was sharply decreased compared to the control group by AIMP3 transfection, but the amount of progerin was not significantly affected by the injection of AIMP3 (FIGS. 10D and e).

Example 4

Premature Aging Phenotype in Mice Transfected with AIMP3

We then tested in AIMP3 transfected mice whether the senescence of cells induced by increased expression of AIMP3 is also associated with organizational phenotypes. AIMP3 transfected mice were born alive, but the body growth of the mice stopped much longer than in the same embryo heterozygous or wild type (FIG. 4A). In addition, transfected mice showed a higher mortality rate compared to one-fold heterozygotes (HE) or wild-type (WT) (FIG. 4B). Transfected mice showed alopecia (Fig. 4c. And d), reduced fat cells with wrinkled skin (Fig. 4e), with lordokyphosis (Fig. 4f), and reduction of bone minerals in females. (FIG. 4E) and a decrease in bone thickness (FIG. 4H). Thus, overexpression of AIMP3 resulted in a form of aging of the cells as well as over-mature aging at the biological level.

As described above, the present inventors have found that AIMP3 induces cell aging and in particular promotes ubiquitin of laminin A to promote aging through modification of the nucleus. It was found. In particular, it was confirmed that the si-RNA of AIMP3 was excellent in the ubiquitin inhibitory activity of laminin A of AIMP3. Therefore, the composition of the present invention is highly industrially available by providing a new method for the treatment of aging and related diseases.

1 relates to the inhibition of cell proliferation of AIMP3 and induction of cell aging accompanied with modification of the nucleus. (a) Optical and fluorescence micrographs of 293 cells according to culture time after transfection of GFP and GFP-AIMP3. (Bar = 100 μm) (b) Growth curves of 293 cells expressing GFP and GFP-AIMP3. (c) List of cell cycle changes of 293 cells expressing GFP-AIMP3. The vertical axis represents the ratio of the G0-G1, S and G2-M groups. (20,000 assays per sample) (d) Effect of AIMP3 on β-galactosidase activity associated with aging in p53 ^{+ / +} and p53 ^{− / −} HCT116 cells (+ means 6 μg / ml of plasmid DNA) Photographs with X-gal staining. (e) Western blotting of DcR2 was performed to investigate the effect of AIMP3 on the amount of DcR2 in PC53 and HCT116 cells in which p53 was inactivated. (f) Immunofluorescence comparison of nuclei by transfecting HeLa cell lines with empty vector (EV) or AIMP3 and staining laminin A with anti-laminin A antibodies. (g) Photograph of the distribution of laminin A by transfection of GFP-laminin A with EV or AIMP3. (h) Photographed by β-galactosidase staining of AIMP3 WT and TG MEF cells after passage 8 culture. (i) Picture of immunoblot analysis of AIMP3 and aging markers DcR2 and p16 in heart cells of AIMP3 WT and TG mice. (j) Western blotting results for the measurement of intracellular laminin A and AIMP3 levels using anti-laminin A and AIMP3 in AIMP3 WT and TG MEFs

2 relates to an increase in ubiquitin-mediated degradation of laminin A of AIMP3. (a) After injection of AIMP3 into p53 ^{+ / +} and p53 ^-/- HCT116 cells by concentration (㎍ / ml), the amount of laminin A was measured by western blotting. (b) Western blotting of 923 and Hela cells for the effect of the inhibition of AIMP3 using specific siRNA on the amount of laminin A. GFP-laminin A was transfected into both cells and the amount was measured by western blotting using anti-GFP antibody. (c) Co-immunoprecipitation of endogenous AIMP3 and laminin A interactions. Protein extracts from 293 cells were immunoprecipitated with anti-laminin A antibodies, and AIMP3 precipitated together was confirmed by western blotting. (d) GFP-Laminin A and Myc-AIMP3 were transfected into 293 cells, Myc-AIMP3 was immunoprecipitated with anti-Myc antibody and co-precipitated GFP-Laminin A was confirmed by western blotting with anti-GFP antibody. . (e) GFP-Laminin A was transfected into 293 cells. GST-AIMP3 was immunoprecipitated with glutathione-Sepharose beads and coprecipitated GFP-laminin A was confirmed by western blotting using anti-GFP antibody. (f) AIMP3 performs laminin A reduction function proteasome-dependently. p53 ^-/- HCT116 cells were transfected with plasmids expressing Myc-AIMP3 and GFP-laminin A. After 6 hours treatment with MG132, cell lysates were analyzed by western blotting using anti-Myc and -GFP antibodies. (g) p53 ^-/- HCT116 cells were transfected with EV or Myc-AIMP3 and immunoprecipitated with anti-laminin A antibody after MG132 treatment. (h) Ap53 ^-/- HCT116 cells expressing HA-ubiquitin, Myc-AIMP3 and GFP-laminin were cultured with and without MG132. Cell lysates were immunoprecipitated with anti-GFP antibody and precipitates were western blotting with anti-HA antibody.

Figure 3 relates to the Siah1-dependent ubiquitin promotion of laminin A of AIMP3. (a) Flag-Siah1 was transfected into Myc-p53 ^-/- HCT116 cells with or without Myc-AIMP3. After 24 hours, the amount of laminin A was measured by western blotting. Laminin A, Siah1 and AIMP3 levels were measured with anti-laminin A, -Flag, and -Myc antibodies. Tubulin was included as a loading control. (b) 293 cells were transfected with a combination of HA-ubiquitin, si-Siah1 and Myc-AIMP3 as shown in the figure. Thereafter, the cells were incubated with or without MG132 for 10 hours. Cell lysates were immunoprecipitated with anti-laminin A antibodies and precipitates were immunoblotting with anti-HA antibodies. The expression levels of Myc-AIMP3, Siah1 and tubulin in whole cell lysate (WCL) were measured using anti-Myc, -Siah1 and tubulin antibodies. (c) p53 ^{− / −} HCT116 cells were transfected with a combination of Flag-Siah1, HA-ubiquitin and si-AIMP3 (0, 5 and 10 uM) shown in the figure. After immunoprecipitation of laminin A, the results of immunoblotting of ubiquitinated laminin A using anti-HA antibody. (d) p53 ^{− / −} HCT116 was transfected with AIMP3 and Flag-Siah1 (0, 2, 4 μg / ml) by concentration. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoboltting of Siah1 precipitated together with anti-Flag antibody. (e) ³⁵ S-labelled full-length Siah1 was prepared by in vitro translation and mixed with GST or GST-AIMP3. GST protein was precipitated by glutathione S-Sepharose, and the precipitated Siah1 was confirmed by radiographic imaging. (f) p53 ^{− / −} HCT116 cells were transfected with Flag-Siah1 and with different amounts of si-AIMP3. After 48 hours of incubation, cells were harvested and immunoprecipitated using anti-Flag antibody. The co-precipitated laminin A was confirmed by western blotting using anti-laminin A antibody.

4 relates to premature aging of AIMP3 transfected mice. (a) Body weight was measured every two weeks for 24 months, and the growth status of AIMP3 transfected mice (TG, n = 70) was compared with wild type (WT, n = 70) and heterozygous mice (HE, n = 70). Graph compared. (B) Monthly survival of AIMP3 transfected mice, WT and HE. (c) Photographs of 10 month old AIMP3 TG and WT mice. Transfected mice show alopecia. (D) A graph measuring the number of mice showing signs of hair loss by age. (e) Photographs of skin tissue of AIMP3 WT and TG mice by hematoxylin and eosin staining. E, D and A mean epidermis, dermis and subcutaneous fat. The skin of TG mice is clearly wrinkled and the subcutaneous fat layer is reduced. (f) Whole body radiographs of 18 months old WT and TG mice. AIMP3 TG mice show a marked multiplication. (g) Comparison of bone density (gcm ^-2 ) between WT and TG mice (n = 8 in each group). (h) Comparison of hematoxylin and eosin stained bone cross-sections of 18-month-old TG and WT mice.

5 relates to the demonstration of the fact that the mouse aIMP3 cDNA has entered the chromosome of the mouse. (a) Schematic diagram of full-length mouse AIMP3 cDNA, 234 amino acids long, subclone to the Eco RI site of the pCAGGS expression vector to make AIMP3 transfected mice. (b) PCR analysis of genomic DNA isolated from tails of AIMP3 WT, HE and TG mice. NC stands for negative control. (c) Genomic DNA was isolated from 6 mice and PCR was performed using B5 'and B3' primers. Two of them produced PCR products of the desired size. (d) Proteins were isolated from MEFs of six mice, and the expression levels of AIMP3 were measured. The measurement was performed by western blotting using anti-AIMP3 antibody. (e) RNA was isolated from MEF cells, and the expression level of AIMP3 was measured by RT-PCR using B5 'and B3' primers. (f) Nucleotide sequence of the primer used to identify AIMP3 cDNA in the chromosome.

Figure 6 is a measure of the degree of cell aging of AIMP3 WT, HE mice by β-galactosidase activity. (a) Photographs of Senescence-Associated (SA) β-galactosidase activity measured using X-gal after AIMP3 WT and HE MEF cells were cultured in media causing cell aging. (b) A graph measuring the proportion of cells that showed β-galactosidase positive reaction in total cells. (c) Photos of SA-β-galactosidase staining on AIMP3 WT and HE skin cells. (d) Pictures of control and si-AIMP3 RNAs injected into AIMP3 WT MEFs cells and subjected to SA-β-galactosidase staining.

7 is a photograph showing that AIMP3 induces nuclear deformation. In the bottom line, you can see the blue form and laminin A stained (brown). In AIMP3 TG cells, structural changes in the nucleus and reduction of laminin A can be clearly confirmed.

8 relates to AIMP3 selectively increasing the ubiquitin of laminin A. FIG. (a) 293 cells were transfected with EV and AIMP3, respectively, and treated with cycloheximide to prevent the synthesis of new proteins. Cleavage of caspase 6 produced fragments of laminin A (70 kDa) at 40-45 and 28 kDa, and laminin A / C antibodies acted on total laminin A and 28kDa laminin A. (b) GFP-Laminin A, Myc-AIMP3, and Myc-AIMP2 were transfected into 293 cells by the combinations shown in the figure, and the expression of this protein was confirmed by western blotting. (c) 293 cells were transfected with GFP-laminin A and Myc-Aimp3 and Western blotting confirmed the expression of each protein shown in the figure.

9 relates to the interaction of GST-AIMP3 with Siah1. Various amounts of Flag-Siah1 were injected into HCT116p53 ^{− / −} cells. GST-AIMP3 was precipitated with glutathione-Sepharose and the precipitated Siah1 was measured by western blotting.

Figure 10 is to demonstrate that AIMP3 specifically ubiquitin laminin A but not progerin. (a) Differences in exon structure of laminin A and progerin. Laminin A cDNA consists of 12 exons. progerin is produced by a miscution between the double-endon 11 and the N-terminus of 12. (b) 293 cells were transfected with HA-ubiquitin and Flag-Siah1 and GFP-progerin and GFP laminin A in the combination shown in the figure. AIMP3 and laminin A were immunoprecipitated and the precipitate was separated by SDS-PAGE. Laminin A and progerin ubiquitin were identified by immunoblotting using anti-HA antibody. (c) The direct interaction of laminin A with progerin on AIMP3 was determined by an in vitro pull-down assay. Radioactive AIMP3 was prepared by in vitro translation and mixed with GST-laminin A (exon 11-12 pieces) or GST-progerin (△ exon 11-12). GST protein was precipitated using glutathione-Sepharose, and the precipitated AIMP3 was confirmed by radiograph. (d) and (e) The effect of AIMP2 on intracellular rotation of laminin A (d) and progerin (e) was confirmed by pulse-chase experiments. 293 cells were transfected with Myc-AIMP3 and GFP-laminin A or GFP-progerin and incubated for 3 hours in radioactive methonine. Intracellular protein production was inhibited by cycloheximide, and GFP-laminin A or GFP-progerin was immunoprecipitated by anti-GFP matrix and confirmed by radiograph.

Figure 11 relates to AIMP3 increased with a decrease in laminin A in some aged tissue. The immunoblotting test of AIMP3, laminin A / C, and senescence markers DcR2 was performed on tissue cells of 2 months (Y) and 28 months (O) mice. The upper band of the two bands of laminin A / C is the band of laminin A. Five samples were tested and one of them was displayed. Increased expression of AIMP3 was likewise seen in other samples.

12 shows western blotting results of testing the effect of si-RNA specifically reacting with AIMP3.

<110> SNU R & DB FOUNDATION <120> Composition for preventing or treating aging or age-related          diseases comprising AIMP3 si-RNA or anti-AIMP3 antibody <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> AB011079_Stealth_115_sence: sence siRNA specific to AB011079          gene <400> 1 ccaagucuaa caggauugac uacua 25 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> AB011079_Stealth_115_antisence: antisence siRNA specific to          AB011079 gene <400> 2 uaguagucaa uccuguuaga cuugg 25 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> AB011079_Stealth_107_sence: sence siRNA specific to AB011079          gene <400> 3 acaauggucc aagycyaaca ggauu 25 <210> 4 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> AB011079_Stealth_107_antisence: antisence siRNA specific to          AB011079 gene <400> 4 aauccuguur grcuuggacc auugu 25 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> AB011079_Stealth_154_sence: sence siRNA specific to AB011079          gene <400> 5 gucaagcaag ccaacaaaga auauu 25 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> AB011079_Stealth_154_antisence: antisence siRNA specific to          AB011079 gene <400> 6 aauauucuuu guuggcuugc uugac 25  

Claims (3)

AIMP3 small interfering RNA (si-AIMP3) or anti-AIMP3 antibody comprising a composition for the prevention or treatment of aging or aging-related diseases comprising a. The composition of claim 1, wherein the aging-related disease is selected from alopecia, wrinkles, mulch, and osteoporosis. The composition of claim 1, wherein the AIMP3 small interfering RNA (si-AIMP3) is selected from a pair of SEQ ID NOs: 1 and 2, a pair of SEQ ID NOs: 3 and 4, and a pair of SEQ ID NOs: 5 and 6.

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