KR102283910B1 - Novel use of βPIX having cellular senescence modulating activity - Google Patents

Abstract

The present invention relates to a novel use of βPIX having the ability to control aging. Specifically, according to the present invention, an object of the present invention is to prevent or treat cellular senescence-related diseases comprising βPIX or an activator of βPIX as an active ingredient, a composition for inhibiting cellular senescence, a composition for regulating cellular senescence, and measurement of cellular senescence It relates to a biomarker composition for use, a method for screening an inhibitor of cellular senescence, and a method for inhibiting cellular senescence. In the present invention, when βPIX is overexpressed, it is possible to reduce the level of cellular senescence through inhibition of SA-β-galactosidase activity, inhibition of senescence-associated secretion phenotype (SASP), and inhibition of expression of senescence markers such as p16INK and pγH2AX. It was confirmed that there is, and also, when suppressing the expression of βPIX, it was confirmed that it can promote the level of cellular senescence. Therefore, since aging can be controlled by regulating the expression level of βPIX, there is an effect that βPIX can be used as a new aging index factor that can measure the level of aging. There is an effect that can be used as a new treatment.

Description

Novel use of βPIX having cellular senescence modulating activity

The present invention relates to a novel use of βPIX having the ability to control aging.

Cellular senescence is a major cause of aging in individuals and is caused by numerous factors such as DNA damage, telomere erosion, accumulation of cellular stress, and loss of cellular replication ability. As an index defining cellular senescence, aged cells exhibit morphological features such as enlarged size, flattened shape, increased heterochromatin in the nucleus, and increased vacuole in the cytoplasm. In addition, representative biochemical indicators defining aging include increased SA-β-gal (senescence-associated β-galatosidase) activity, increased expression of molecules that inhibit cell growth such as p53, p16/INK4 (p16), and p21, and insulin Inflammation-related such as insulin-like growth factor binding proteins (IGFBPs), interleukin-6, transforming growth factor-β (TGF-β), and interferon There is an increase in secretion of proteins.

Cellular aging not only contributes to the aging of an individual or tissue, but also plays an important role in the pathogenesis of various diseases. Senescent cells are frequently observed in inflammatory lesion tissues such as rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissues, and arteriosclerotic vascular tissues, and cellular aging is also observed in prostatic hyperplasia, hepatitis, and liver cancer.

When senescent cells accumulate, senescent cells do not divide well, and damaged tissues cannot properly repair. In addition, it promotes the secretion of enzymes or inflammatory cytokines that decompose surrounding tissues, thereby accelerating tissue damage and contributing to the onset of diseases related to aging. Therefore, since the aging of cells is closely related to the aging of the individual, studies are being actively conducted to control the aging of the individual by delaying the senescent aging.

On the other hand, βPIX (PAK-interacting exchange factor beta, ARHGEF 7) protein is a guanine nucleotide exchange factor and acts as an enzyme that converts inactive Rac and Cdc42 into active form, and acts as an enzyme for cell adhesion (focal adhesion). It is known as one of the major proteins that regulate cell adhesion and migration in However, it has not yet been reported that it can act as a major factor regulating the aging of cells to be demonstrated in the present invention.

Republic of Korea Patent Registration 10-0573880

Accordingly, the present inventors studied whether βPIX is related to aging. As a result, βPIX expression was reduced as the individual or cells aged, and when βPIX expression was suppressed at the gene level, it was found that cellular senescence and tissue senescence were induced. In tissues or cells with reduced βPIX expression, it was observed that the expression of p16/INK4 (p16) and phospho-γH2AX (pγH2AX) proteins, which are representative aging indicators, and Senescence associated-β-galactosidase (SA-β-gal) activity increased. did. In addition, it was confirmed that aging was delayed (suppressed) through changes in aging indicators when βPIX was overexpressed in aged mice. Therefore, through these results, the present invention by confirming that the βPIX protein of the present invention can be used as a new regulatory factor and aging indicator that can regulate aging, as well as delay aging through increased expression of βPIX. completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of aging or aging-related diseases, comprising βPIX or a component that induces an increase in expression of βPIX as an active ingredient.

Another object of the present invention is to provide a composition for inhibiting cellular senescence, comprising the βPIX protein or the βPIX gene encoding the same as an active ingredient.

Another object of the present invention is to provide a composition for regulating cellular senescence, comprising a substance regulating the expression of the βPIX gene or the activity of the βPIX protein.

Another object of the present invention is to provide a biomarker composition for measuring cellular aging, including the βPIX protein or the βPIX gene.

Another object of the present invention is to provide a method for screening an inhibitor of cellular senescence.

Another object of the present invention is to provide a method for regulating cellular senescence, comprising treating the isolated cells with an expression vector containing βPIX protein or βPIX gene.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating aging or aging-related diseases, comprising βPIX or an activator of βPIX as an active ingredient.

In one embodiment of the present invention, the βPIX is composed of the amino acid sequence of SEQ ID NO: 1, and the βPIX activator may be a protein, compound or nucleic acid capable of enhancing the expression or activity of βPIX.

In one embodiment of the present invention, the βPIX may be one in which the βPIX gene consisting of the nucleotide sequence of SEQ ID NO: 2 is inserted into an expression vector.

In one embodiment of the present invention, the composition inhibits SA-β-galactosidase activity; inhibition of senescence-associated secretory phenotype (SASP); and inhibition of expression of p16/INK4 and pγH2AX; It may have activity.

In addition, the present invention provides a composition for inhibiting cellular aging comprising the βPIX protein or the βPIX gene encoding the same as an active ingredient.

In one embodiment of the present invention, the βPIX protein may consist of the amino acid sequence of SEQ ID NO: 1, and the βPIX gene may consist of the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention provides a composition for regulating cell aging, comprising a substance that modulates the expression of the βPIX gene or the activity of the βPIX protein.

In one embodiment of the present invention, the substance regulating the expression of the βPIX gene may be a substance inhibiting the expression of the βPIX gene or a substance overexpressing the βPIX gene.

In one embodiment of the present invention, the substance inhibiting the expression of the βPIX gene is small interfering RNA (siRNA) for βPIX, small hairpin RNA (shRNA), micro RNA (microRNA: miRNA) ) or an antisense oligonucleotide.

In one embodiment of the present invention, the substance overexpressing the βPIX gene may be an expression vector containing the βPIX gene.

In one embodiment of the present invention, the substance inhibiting the expression of the βPIX gene, SA-β- galactosidase activity promotion; induction of senescence-associated secretory phenotype (SASP); delay of cell cycle and cell proliferation; And it may be to increase the level of senescence of cells through the enhancement of the expression of p16 / INK4 and pγH2AX.

In one embodiment of the present invention, the substance overexpressing the βPIX gene, SA-β-galactosidase activity inhibition; inhibition of senescence-associated secretory phenotype (SASP); And it may be to reduce the level of cellular senescence by suppressing the expression of p16 and pγH2AX.

In addition, the present invention provides a biomarker composition for measuring cellular aging, including the βPIX protein or the βPIX gene.

In one embodiment of the present invention, the βPIX protein may consist of the amino acid sequence of SEQ ID NO: 1, and the βPIX gene may consist of the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention comprises the steps of treating any candidate material in a biological sample; detecting the expression level of βPIX protein or βPIX gene from the biological sample; And it provides a screening method for cellular senescence inhibitors, comprising the step of comparing the expression level with the expression level of the same gene or the same protein in a control not treated with any candidate substance.

In one embodiment of the present invention, when the expression level of the βPIX protein or βPIX gene is increased in the group treated with the candidate substance compared to the control group, the step of determining the candidate substance as an inhibitor of cellular senescence may be further included. .

The present invention also provides a method for inhibiting cellular senescence, comprising treating the isolated cells with an expression vector containing βPIX protein or βPIX gene.

In one embodiment of the present invention, the βPIX protein may consist of the amino acid sequence of SEQ ID NO: 1, and the βPIX gene may consist of the nucleotide sequence of SEQ ID NO: 2.

In the present invention, when the expression of βPIX is overexpressed, the level of senescence of cells through inhibition of SA-β-galactosidase activity, inhibition of senescence-associated secretion phenotype (SASP), and inhibition of expression of aging markers such as p16/INK4 and pγH2AX It was confirmed that it is possible to reduce In addition, it was confirmed that when the expression of βPIX is suppressed, it can promote the level of cellular senescence. Therefore, since aging can be controlled by regulating the expression level of βPIX, there is an effect that βPIX can be used as a new aging index factor that can measure the level of aging. There is an effect that can be used as a new treatment.

1 is a result of analyzing the change in the expression level of βPIX according to aging, 1A is the expression of βPIX in tissues (lung, kidney, spleen, heart and skin) obtained from mice aged 3 months, 15 months, and 24 months; It is the result of confirming the level through immunoblotting. 1B is the result of analyzing the change in the expression level of βPIX according to aging in mouse lung tissue, 2A shows SA-β-Gal, βPIX and SA-β-Gal, βPIX and The results of staining each of the aging indicators (p16, pγH2AX) are shown, and 1C is a bar graph showing the expression levels of SA-β-Gal, βPIX and aging indicators (p16, pγH2AX).
2 is an observation of changes in βPIX expression according to aging in human lung tissue and human-derived fibroblasts (human diploid fibroblast, HDF). Figure 2A shows the results of confirming the expression levels of βPIX and p16 by immunohistochemistry in lung tissues obtained from young people and elderly people, and each expression level is shown as a bar graph, respectively, and 1C is HDF cells with different subcultures. βPIX and p16 expression levels were analyzed by immunoblotting, 1D shows the results of SA-β-Gal activity assay, and 1E shows SA-β-Gal positive through SA-β-Gal staining. A photograph of cells observed under a microscope is shown.
3 is a result of confirming the induction of cellular senescence by inhibition of βPIX expression. 3A is SA-β-Gal after treatment with siRNA (siPIX1, siPIX2) and control siRNA (siCtrl) for βPIX targeting HDF cell lines, respectively. (blue) and pγH2AX (brown) are shown. 3B shows the result of measuring the number of SA-β-Gal positive cells through SA-β-Gal staining in 3A experimental group, 3C is 3A experimental group Shows the results of confirming the protein levels of βPIX and p16 by immunoblotting. 3D is a schematic diagram of the experimental procedure of 3E and 3F, and 3E is siRNA against βPIX (siPIXm1, siPIXm2) and control siRNA (siCtrl) into the bronchioles tissues of 3-month-old mice, respectively, and after 4 weeks, SA- Shows the results of confirming the expression levels of β-Gal, βPIX, p16 and pγH2AX through immunohistochemistry. 3F is a bar graph showing the quantification of the above results.
Figure 4 confirms the cell cycle and cell proliferation delay induction results according to the inhibition of βPIX expression, 4A is a HDF cell line after treatment with siRNA (siPIX1, siPIX2) and control siRNA (siCtrl) for βPIX, respectively, After 3 days, the expression level of the cell cycle-related factors was confirmed through immunoblotting, 4B is the result of confirming the change in the cell cycle through flow cytometry, and 4C is the result of confirming the change in cell proliferation The results of measuring cell growth curves are shown.
Figure 5 confirms the SASP induction according to the inhibition of expression of βPIX, 5A is siRNA (siPIX1, siPIX2) and control siRNA (siCtrl) for βPIX after treatment in HDF cell lines, respectively, human inflammatory antibody array for each experimental group It shows the image confirming SASP by performing, 5B shows the result of measuring the levels of IL-6, MCP-1 and TIMP-2, 5C is performed immunoblotting to confirm the expression level of βPIX and p16 one result is shown.
Figure 6 confirms the inhibition of cellular senescence according to overexpression of βPIX, 6A is a schematic diagram of the experimental procedures of 6B and 6C, and 6B is a lentiviral vector (GFP-βPIX) for inducing overexpression of βPIX in 12-month-old senescent mice. And after each injection of the control lentiviral vector (GFP), 4 weeks later, the expression levels of aging index factors (SA-β-Gal, p16, pγH2AX) in the lung tissue of each mouse were stained with immunohistochemistry. is shown, and 6C is a quantitative bar graph showing the expression levels of aging index factors (SA-β-Gal, p16, pγH2AX).
7 shows a cleavage map of a recombinant expression vector prepared for overexpression of βPIX in an embodiment of the present invention.

The present invention is characterized in that the senescence-regulating function of βPIX was first identified. βPIX (PAK1-interacting exchange factor beta) is a guanine nucleotide exchange factor and has a function of regulating cell differentiation and adhesion. It is known, but nothing is known about the contents of research related to aging so far.

On the other hand, in the present invention, βPIX is related to the aging of tissues or cells of an individual, and when the expression of βPIX is reduced or inhibited, aging is promoted, whereas when the expression of βPIX is increased, aging is inhibited. Through this, it was possible to confirm the possibility of using βPIX as a new aging index that can control aging.

Specifically, according to an embodiment of the present invention, the expression of βPIX protein in lung, kidney, spleen, heart and skin tissues obtained from 3, 15, and 24 month old mice in order to confirm the change in the expression level of βPIX according to aging. As a result of measuring the level, the expression level of βPIX decreased in each tissue as the age of the mouse increased and aging progressed, and it was confirmed that this was related to aging by the increase in the amount of p16 expression (see FIG. 1A ).

Accordingly, the present inventors found that βPIX is a protein associated with aging, and to further confirm this, mouse lung tissue according to the increase in age as an in vivo model, young or old human lung tissue, and cell passage as an in vitro model The expression level of βPIX and the expression level compared to the aging indicators were analyzed in young HDF cells (young passage human dermal fibroblasts cells) and old passage human dermal fibroblasts cells in different cultures.

As a result, it was found that the expression level of βPIX in aged HDF cells was significantly decreased compared to that of young HDF cells, as was the result in the lung tissue of mice, whereas the expression level of p16, known as an aging marker, increased. and SA-β-gal (senescence-associated β-galatosidase) activity was also increased compared to young cells (see FIGS. 1 and 2 ).

Based on these results, the present inventors first analyzed the effect of inhibiting the expression of βPIX on the progression of aging in order to confirm the effect of the expression level of βPIX in relation to cellular aging. To this end, after treatment with βPIX siRNA for inhibition of βPIX expression in the lung tissue of a 3-month-old mouse, the expression patterns of aging indicators compared to the control group not treated with βPIX siRNA were analyzed. As a result, it was found that the expression of p16, SA-β-gal and pγH2AX, known as aging indicators, was significantly increased in the group in which the expression of βPIX was inhibited compared to the group in which the expression of βPIX was not inhibited (see FIG. 3). . Through these results, the present inventors found that when the expression of βPIX is reduced, aging can be accelerated.

Furthermore, the effect of decreasing the expression of βPIX on the cell cycle and cell proliferation in relation to the promotion of aging or the induction of aging was analyzed. Cellular senescence refers to a state in which cell division and proliferation are permanently stopped, unlike normal cells. Accordingly, in one embodiment of the present invention, as a result of measuring the expression levels of cell cycle-related factors in HDF cells treated with βPIX siRNA and HDF cells not treated with βPIX siRNA, p19, cyclin E in the group in which βPIX expression was inhibited and CDK2 expression was decreased, and expression of p16, cyclin D and CDK4 was increased (see Fig. 4A), and analysis using flow cytometry showed that cell cycle progression was stagnant in G1 phase. (See Figure 4B). In addition, the effect on cell proliferation when the expression of βPIX was suppressed was analyzed, and it was observed that the cell proliferation was inhibited in the group with reduced expression of βPIX (see Fig. 4C). It was confirmed that cell senescence can be induced by controlling their expression and arresting the cell cycle in the G1 phase to inhibit cell proliferation.

In addition, in another embodiment of the present invention, when suppressing the expression of βPIX, SASP (Senescence-associated secretory phenotype) change, which is known as another indicator of aging, was analyzed (FIG. 5). The SASP (Senescence-associated secretory phenotype) is a generic term for a total protein secreted by senescent cells, and the secretion of SASP substances promotes aging. As a result of the analysis, the amount of cytokines, such as IL-6, MCP-1, and TIMP-2, known as senescence promoting factors, was increased in the cell group in which the expression of βPIX was suppressed. It was found that the secretion of SASP proteins was increased in the cells in which senescence was promoted due to the decrease in the expression of βPIX.

On the other hand, the inventors of the present invention, when the overexpression of the βPIX protein is induced in an individual whose aging has progressed in some way, it was confirmed how it affects the aging of the individual. A lentivirus containing the βPIX gene was injected into the lungs of a 12-month-old mouse to induced overexpression of the βPIX protein, and changes in aging markers were analyzed in the lungs ( FIG. 6 ). It was confirmed that the group overexpressing βPIX had a marked decrease in SA-β-GAL activity, which is an aging indicator, and decreased expression of p16 and pγH2AX compared to the control group (the group without introducing the βPIX gene) (see FIG. 6 ). These results confirmed that even when βPIX protein is decreased due to aging, increasing the expression of βPIX protein can inhibit or delay the progression of aging. It was found that βPIX protein or an activator thereof can be used as a new therapeutic agent for aging and aging-related diseases.

Therefore, the present invention is characterized by providing a pharmaceutical composition for preventing or treating cell aging-related diseases, comprising βPIX or an activator of βPIX as an active ingredient.

In the present invention, the βPIX may have the amino acid sequence of SEQ ID NO: 1, and the amino acid may be encoded by the nucleotide sequence of SEQ ID NO: 2. In addition, the βPIX activator is capable of enhancing the expression or activity of βPIX, but is not limited thereto, and may be a protein, compound or nucleic acid specific for βPIX.

In one embodiment of the present invention, the overexpression of βPIX was induced by using an expression vector into which the βPIX gene consisting of the nucleotide sequence of SEQ ID NO: 2 was inserted to enhance the expression of the βPIX.

The vector that can be used in the present invention is not limited thereto, but refers to a plasmid, phage, cosmid, viral vector or other medium known in the art. In addition, in the present invention, the polynucleotide encoding βPIX may be isolated from nature or artificially modified synthetically, and the nucleotide sequence encoding βPIX may be modified by substitution, deletion or insertion of one or more nucleic acid bases, such as The protein expressed by the modification should not contain significant changes in its biological functionality. Such modifications include modifications to heterologous homologous genes.

The expression vector according to the present invention can be introduced into a cell using a method known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells It can be introduced into cells by a method (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

Preferably, the βPIX activator may be an expression vector into which the βPIX gene is inserted, and more preferably, a lentiviral vector containing the βPIX gene.

The lentiviral vector of the present invention may be one in which the βPIX gene encoding the βPIX protein is operably linked to a promoter.

The "operably linked" refers to a gene requiring expression and its regulatory sequences are linked to each other in a manner that enables gene expression, and the term "promoter" means that when linked to a specific sequence, a specific nucleotide sequence is transcribed into mRNA It refers to a DNA sequence that can control it. Typically, the promoter is in all cases

Although not applicable, it is 5' (ie, upstream) of the desired nucleotide sequence to be transcribed into mRNA and provides a site for specific binding of RNA polymerase and other transcription factors for transcription initiation.

The promoter of the present invention may use a constitutive or regulatable promoter, preferably a constitutive promoter. The term “constitutive,” as used in reference to a promoter, means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence without stimulation (eg, heat shock, chemicals, etc.). In contrast, a "regulatable" promoter means a promoter capable of directing the transcriptional level of an operably linked nucleic acid sequence in the presence of a stimulus (eg, heat shock, chemical, etc.) distinctly from the absence of a stimulus.

In addition, the vector may further include a gene encoding a fluorescent protein. The fluorescent protein may be for the purpose of detecting the expression of a desired gene in a transduced cell or tissue, and any protein capable of emitting fluorescence when expressed in a cell or tissue may be used, especially Without limitation, green fluorescent protein (GFP), modified green fluorescent protein (modified green fluorescent protein), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein ( ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo fluorescent protein (ECFP) ) can be used, preferably green fluorescent protein (GFP).

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, and these carriers are commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, Gum Acacia, Calcium Phosphate, Alginates, Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Cellulose, Water, Syrup, Methyl Cellulose, Methylhydroxybenzoate, Propylhydroxybenzoate, Talc, Stearic Acid Magnesium and mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. can be Meanwhile, the dosage of the pharmaceutical composition of the present invention is preferably 0.0001-100 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. . The concentration of the active ingredient contained in the composition of the present invention may be determined in consideration of the therapeutic purpose, the patient's condition, the required period, etc., and is not limited to a concentration within a specific range.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Or it can be prepared by introducing into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

In the present invention, the treatment means reversing, alleviating, inhibiting the progression, or preventing a disease or disorder to which the term applies, or one or more symptoms of the disease or disorder, unless otherwise stated. do.

In the present invention, the disease may be senescence or senescence-related disease, and the disease is SA-β-galactosidase activation promotion, senescence-associated secretory phenotype (SASP) induction, cell cycle and cell delayed proliferation; Or it may be a senescence-related disease that may be caused by aging caused by the enhancement of expression of p16INK and pγH2AX and the progression of the aging.

The aging-related diseases are not limited thereto, but may include atherosclerosis, skin aging, osteoporosis, rheumatoid arthritis, degenerative osteoarthritis, alopecia, wrinkles and humpbacks.

In addition, the present invention can provide a composition for inhibiting cellular aging comprising the βPIX protein or the βPIX gene encoding the same as an active ingredient.

That is, as described above, when the expression level of βPIX is increased in the target cell or target tissue, it was confirmed that aging can be inhibited.

In addition, the present invention can provide a composition for regulating cell aging, comprising a substance that modulates the expression of the βPIX gene or the activity of the βPIX protein.

The present inventors have confirmed that the regulation of cellular aging is possible by regulating the expression of the βPIX gene. Specifically, it was confirmed through experiments that inhibiting the expression of βPIX can promote cellular aging, and increasing the expression of βPIX can inhibit cellular senescence. Therefore, aging can be controlled by regulating the expression level of βPIX.

In the present invention, the term “senescence” has the same meaning as aging, and thus “aging control” refers to any phenomenon that controls aging.

The substance regulating the expression of the βPIX gene may be a substance inhibiting the expression of the βPIX gene or a substance overexpressing the βPIX gene.

The substance inhibiting the expression of the βPIX gene may include a substance capable of inhibiting or reducing the expression of the βPIX gene, or inhibiting the activity of βPIX, but is not limited thereto, but small interfering RNA (small interfering RNA) for βPIX. interfering RNA: siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or an antisense oligonucleotide.

Preferably, it may be an siRNA for at least one βPIX selected from the group consisting of SEQ ID NOs: 3 to 6.

The material for overexpressing the βPIX gene may include any of those capable of promoting or enhancing the expression of the βPIX gene, preferably an expression vector containing the βPIX gene, and the expression vector is the same as described above. .

In the present invention, the substance inhibiting the expression of the βPIX gene promotes SA-β-galactosidase activity; induction of senescence-associated secretory phenotype (SASP); delay of cell cycle and cell proliferation; and by increasing the expression of p16INK and pγH2AX to increase the level of cellular senescence.

In addition, the substance overexpressing the βPIX gene may inhibit SA-β-galactosidase activity; inhibition of senescence-associated secretory phenotype (SASP); And it may be to reduce the level of cellular senescence by suppressing the expression of p16INK and pγH2AX.

Furthermore, in the present invention, as it is confirmed that βPIX protein can be used as a new aging indicator for aging, the present invention can provide a biomarker composition for measuring cellular aging, including βPIX protein or βPIX gene.

In the present invention, the term "biomarker" refers to a substance that can distinguish senescent cells or tissues from non-senescent or less advanced cells or tissues. For the purpose of the present invention, the biomarker for measuring cellular senescence is a nucleotide (including a fragment thereof) of the βPIX gene or a protein (including a fragment thereof) encoded therein, and is a gene with reduced expression in senescent cells or tissues. These markers may use either mRNA for any one gene or any one protein encoded by the gene, and may be a composite marker including two or more of these markers.

Preferably, the βPIX protein may consist of the amino acid sequence of SEQ ID NO: 1, and the βPIX gene may consist of the nucleotide sequence of SEQ ID NO: 2.

The present invention may also provide a method for screening a cellular senescence inhibitor through the process of detecting the expression level of βPIX, the method comprising the steps of: treating a biological sample with any candidate; detecting the expression level of βPIX protein or βPIX gene from the biological sample; and comparing the expression level with the expression level of the same gene or the same protein in a control group not treated with any candidate substance.

In addition, the method may further include the step of determining the candidate substance as a cellular senescence inhibitor when the expression level of the βPIX protein or βPIX gene is increased in the group treated with the candidate substance compared to the control group.

The biological sample may be a tissue or a cell.

Methods for detecting or measuring the expression level of the βPIX protein include, but are not limited to, Western blotting (immunoblotting), ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion ), Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter, FACS ) and protein chips.

Measurement of the expression level of the βPIX gene, but is not limited thereto, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA Microarray usage and Northern blot.

In addition, the present invention may provide a method for inhibiting cellular senescence, comprising treating the isolated tissue or cell with an expression vector containing βPIX protein or βPIX gene.

That is, by introducing the expression vector containing the βPIX protein or the βPIX gene into the tissue or cell, it is possible to suppress the aging of cells through the increase in the expression of the βPIX.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

< preparation example >

Reagents and test methods

Reagents and experimental methods used in the experiments of the following Examples are as follows.

reagent preparation

Invivofectamine, Lipofectamine 2000, Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), OPTI-MEM, Alexa Fluor 594 Conjugated Transferrin and Alexa Fluor Conjugated Secondary Antibodies were purchased from Thermo Fisher Scientific (Waltham, MA).

shRNA lentiviruses for p16 and p53 were purchased from Santa Cruz Biotechnology (Dallas, TX), and lentiviruses for si-rPIXs were provided by Dr. Sung (KRIB, Cheongju, Korea) and used. In addition, lentiviruses for GFP (LVP690) and GFP-βPIX (LVP718951) were purchased from Abm Inc (Richmond, Canada) and used.

SA-β-gal staining solution and other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used.

Human inflammatory antibody arrays were purchased from Abcam (Cambridge, UK) and used.

siRNAs for βPIX were purchased from Thermo Fisher Scientific and used.

In addition, the sequences of the siRNAs used in the experiments below are as follows.

siPIX1: 5′-UCAACUGGUAGUAAGAGCAAAGUUU-3'

siPIX2: 5'-UUGAGCUGCAGAUCCUGACGGAAGC-3'

siCtrl: 5′-CCUACGCCACCAAUUUCGU-3'

siPIXm1: 5'-CCAACUGGUAGUACGAGCCAAGUUU-3'

siPIXm2: 5′-GAGGACCUAGGAGAGUUCAUGGAAA-3'

Experimental animal preparation

All experiments were performed on the animals used in the experiment according to the approved animal protocol and guidelines set by the Animal Experiment Ethics Review Committee (CBNUA-901-15-01) of Chungbuk National University. Mice were obtained from Dahan Biolink (Seoul) and used.

Tissue sample preparation

Lung cells and lung tissue collected from pneumothorax patients who underwent surgery at Chungbuk National University Hospital (Cheongju City) were used. In addition, the patients did not show any other pathology in the lungs, and all studies were conducted under the review and approval of the Chungbuk National University Hospital Clinical Trial Review Committee (2014-02-009-009).

Antibody preparation

Anti-pFAK (Y576) (#3281, 1:500), FAK (#3258, 1:1,000 for immunoblotting/1:200 for immunohistochemistry), p53(#2524, 1:1,000), pp53 (S15) (#9284) , 1:500), pPAK1(T423) (#2610, 1:500), ppaxillin (Y118) (#2541, 1:500), and pγH2AX (S139) (#9713, 1:200) antibodies were prepared by Cell Signaling Technology (Danvers, MA) was purchased and used. In addition, Anti-pFAK (Y397) (611806, 1:500), paxillin (610051, 1:1,000), GIT2 (P94020, 1:1,000), Cdk2 (610145, 1:500), Cdk4 (610147, 1:500) ), Cyclin D (610279, 1:500), Cyclin E (551159, 1:500), pRB (610884, 1:500), ppRB (610490, 1:500) and p19 (610530, 1:500) antibodies were It was purchased from BD Biosciences (San Jose, CA) and used. calpain-2 (sc-373966, 1:1000) and 4 (sc-30065, 1:1,000), p16 (sc28260, 1:500 <for immunoblotting analysis>/1:200 <for immunohistochemistry analysis>), p21 (sc Antibodies against -6246, 1:500), GIT1 (sc-9657, 1:500) and amphiphysin I (sc-376402 and sc-39028, 1:1,000) were purchased from Santa Cruz Biotechnology (Dallas, TX) and used. did.

In addition, GFP (NB600-308, 1:1,000 <for immunoblotting analysis>/1:200 <for immunohistochemistry analysis>) antibody was purchased from Novus Biologicals (Centennial, CO) and used. Activated integrin β1 (MAB2079Z, 1:50) and GST (A00895, 1:1,000) antibodies were purchased from Merck Millipore (Burlington, MA) and Genscript (Piscataway, NJ), respectively. Anti-β1 integrin antibody (ab30394, 1:50) and 6xHis-tag (ab18184, 1:1,000) antibodies were purchased from Abcam (Cambridge, UK) and used. Anti-βPIX antibody (1: 1,000 <for immunoblotting analysis>/1: 200 <for immunohistochemistry analysis>) was used as an antibody against the C-terminal region (439-648 aa) of βPIX.

Plasmids and DNA constructs

βPIX constructs, si-rPIX (WT) and si-rPIX (DHmt) were cloned into pHR-CMV SV40 for lentiviral expression. si-rPIX (WT) and si-rPIX variants were prepared using the QuickChange II site-directed mutagenesis kit (Agilent). N-terminal (NT, aa 1 to 351) and C-terminal (CT, 346 to 695) sites for amphiphysin I and calpain-2 were cloned into pGEX4T-1. Wild-type (WT) and mutant (MT (V392G)) in amphiphysin I were cloned into pHR-CMV SV40 for lentiviral expression. Calpain-2 was cloned into pGEX4T-1. Calpain-2 cDNA was purchased from OriGene and used. The GIT1 C-terminus (CT, aa 376-770) was cloned into the pGEX4T-1 vector for expression in bacteria and into the pHR-CMV SV40 vector for expression in lentiviruses, respectively. The cleavage map of the recombinant vector (pHR-CMV-SV-Puro-βPIX recombinant vector) for overexpression of βPIX prepared in the present invention is shown in FIG. 7 .

siRNA or lentiviral in into vivo relay

Animals Mice were anesthetized by intraperitoneal injection of avertin (2-2-2 tribromoethanol, body weight 0.45 mg/g body weight), and the incisors of the mice were fixed on a bar on a platform. A 1-inch, 22-gauge Safelet IV catheter equipped with a blunted needle was placed inside the mouth to locate the white light emitted by the trachea. After confirming the catheter located in the bronchus, the needle was removed from the catheter. Thereafter, InvivofectamineRNAi complexes (75 μl liposomes) were prepared according to the manufacturer's protocol, and siRNA or lentivirus was delivered in vivo by pipetting lentiviral particles or Alexa Fluor 594-transferrin directly into the opening of the catheter.

cell culture

HDF (human diploid fibroblast) cells were cultured in DMEM (Dulbeccos' modified Eagle's medium) medium containing 10% FBS and antibiotics in an incubator maintained at 37 °C and 5% CO2.

β- galactosidase (SA-β-gal) assay

Age-associated β-galactosidase (SA-β-Gal) activity was measured under pH 6.0 conditions with a slight modification of the method described in Proc Natl Acad Sci USA 92, 9363-9367 (1995). Specifically, cells were washed with phosphate buffered saline (PBS), fixed with 3% formaldehyde for 5 min, and then washed with PBS. Thereafter, the cells were incubated in SA-β-gal staining solution (Sigma-Aldrich) at 37° C. for 13-14 hours, and then stained with Hoechst 33258 for 30 minutes to count the cells. Cell senescence was calculated as the percentage of SA-β-gal-positive cells (stained blue) to total cell number, and for tissues, the animals were anesthetized and perfused with saline, then the tissues were flash frozen in liquid nitrogen and OCT compounds. embedded with Then, the tissue was immediately cut to a thickness of 10 μm, fixed with a PBS solution containing 1% formaldehyde, washed with PBS, and incubated in SA-β-gal staining solution at 37° C. for 13-14 hours. Nuclei were then stained with safranin-O and analyzed with Vectashield mounting medium (Vector Laboratories, Inc, Burlingame, CA).

Transient transfection )

Transfection using DNA or siRNA was performed using Lipofectamine 2000 or Lipofectamine RNAiMAX transfection reagent according to the manufacturer's instructions. Cells were aliquoted on fibronectin-coated plates or glass coverslips, and transfection with the indicated DNA was performed for 1 day, and transfection with siRNA was performed for 3-4 days.

immunohistochemistry ( Immunohistochemistry )

Tissues were fixed with 10% neutral buffered formalin, dehydrated and embedded in paraffin. Formalin-fixed, paraffin-embedded tissue blocks were sectioned (thickness 4 μm). After deparaffinization, the slides were subjected to antigen retrieval procedure in 10 mM sodium citrate buffer (pH 6.0) for 10 min using a pressure cooker (Decloaking chamber; Biocare Medical), followed by a blocking solution (0.3% Triton X-100, 1 % bovine serum albumin, 0.05% Tween 20, 0.1% cold-water fish gelatin and 0.05% sodium azide in PBS) at room temperature for 1 hour. Primary antibodies were reacted with each of the sections at 4°C overnight. Then, it was washed 5 times with PBS containing 0.1% Tween 20 and 0.1% BSA, and each slide was reacted with Alexa Fluor-conjugated secondary antibody (1:200) in the dark at room temperature for 1 hour. After washing several times, the slices were stained with Hoechst 33258, and analyzed with Vectashield mounting medium (Vector Laboratories, Inc). For DAB (diaminobenzidine-HCl) staining, the slides were reacted with methanol containing 0.3% hydrogen peroxide for 20 minutes at room temperature to block the intrinsic peroxidase activity before treatment with a blocking solution. Thereafter, the slides were reacted with biotin-conjugated secondary antibody for 30 minutes at room temperature and finally peroxidase-conjugated streptavidin for 30 minutes at room temperature. Peroxidase activity was measured using DAB as a substrate, and as a negative control, sections treated with only TBS solution without primary antibody were used. Sections were stained with H & E for histological analysis.

immunocytochemistry ( Immunocytochemistry )

Cells were fixed with 3.7% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 for 5 minutes, and blocked with PBS containing 2% BSA at 25° C. for 30 minutes. For antigen staining, cells were reacted with a primary antibody at 25° C. for 1 hour, and then with a secondary Alexa Fluor-conjugated antibody for 1 hour. For visualization of F-actin, cells were stained with Alexa Fluor 568-conjugated phalloidin at 25° C. for 30 minutes, and the expression of the stained protein was analyzed with MetaMorph software version 7.1.7 (Molecular Devices).

immunoblotting ( Immunoblotting ) and Immunoprecipitation ( immunoprecipitation )

Cells were lysed with cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 500 μM EDTA, 200 μM sodium pyruvate, 50 mM β-glycerophosphate), and the supernatant was lysed with primary antibody Immunoprecipitation was performed by reacting at 4 °C for 18 hours. Immunoprecipitates were electrophoresed by 8-10 % SDS-PAGE and transferred onto polyvinylidene fluoride membranes in Tris-glycine-methanol buffer (25 mM Tris base, 200 mM glycine and 20% methanol). After that, the membrane was blocked for 30 minutes using Tris-buffered saline (TBS-T; 50 mM Tris, 150 mM NaCl, 0.1% Tween-20) containing 3% BSA, followed by reaction with primary antibody at room temperature for 1 hour. Then, it was washed 3 times with TBS-T. Then, the membrane was reacted for 1 hour at room temperature using a secondary horseradish peroxidase-conjugated antibody, washed 3 times with TBS-T, and then the signal was detected using an enhanced chemiluminescence reagent.

SASP assay

Cell lysates treated with the same amount of siRNA were incubated with a blot for human inflammatory antibody array, and then secreted cytokines were analyzed according to the manufacturer's instructions (Abcam).

cell cycle analysis

HDF cells were transfected with siRNA for 2 days, starved for 24 h, and then stimulated with 10% FBS for 24 h. Cell cycle analysis was assessed by PI (propidium iodide) staining and flow cytometry. That is, trypsinized cells were fixed with 70% ethanol and resuspended in PI staining solution (20 μg/ml of propidium iodide, 200 μg/ml of RNase A in 1 ml of 0.1% Triton X-100), Incubated at 37°C for 30 minutes. Subsequently, flow cytometry was performed using a FACScalibur instrument (Becton Dickinson, San Jose, CA).

Cell growth curve measurement

HDF cells were aliquoted into 6-well plates and transfected with siRNA. Then, at each time point, the cells were collected using trypsin, stained with a crystal violet solution, and the number of cells was measured using a hemocytometer.

Statistical processing

All data are expressed as mean±SEM; Representative data from at least three independent experiments were analyzed. Statistical significance was assessed by Student's t-test, WilcoxonMann-Whitney test using Sigma Plot for Windows (version 12). P <0.05 was considered statistically significant.

< Example 1>

According to the aging process in mouse tissue of βpix Expression level analysis

In order to determine whether the expression change of βPIX occurs according to the aging process, the present inventors investigated the expression level of βPIX in tissues (lung, kidney, spleen, heart and skin) obtained from mice aged 3 months, 15 months, and 24 months. was confirmed by immunoblotting (FIG. 1A). As a result, it was confirmed that the expression of βPIX decreased in each tissue as the age of the mouse increased and aging progressed, and the expression of p16, an aging index, increased. In addition, to confirm this in more detail, the expression levels of βPIX and SA-β-Gal, p16 and pγH2AX, which are known as senescence indicators, in lung tissue obtained from 3 month, 15 month, and 24 month old mice were subjected to immunohistochemical staining. confirmed through. As a result, the expression of βPIX decreased with increasing age, and it was confirmed that the activity of SA-β-Gal, an aging indicator, and the expression of p16 and pγH2AX increased (see FIGS. 1B to 1H).

Therefore, through these results, the present inventors first identified that βPIX is a protein whose expression is reduced as aging progresses, and it can be seen that βPIX protein can be used as a new indicator of aging.

< Example 2>

elderly person lung tissue and in cells of βpix Expression reduction assay

In order to verify whether the results for the mouse lung tissue used in Example 1 are also applicable to human lung tissue, βPIX and p16 proteins were targeted for lung tissue obtained from young (10s) or elderly (70s) people. The same result was obtained from the result of analyzing the expression level of βPIX by immunohistochemical staining method, and it was found that the expression of βPIX was decreased in the lung tissue of elderly people, whereas the level of p16 protein was increased (see FIGS. 2A and 2B). ). In addition, to confirm that these results are the same in cells other than tissues, βPIX and p16 expression levels in young passage human dermal fibroblasts cells and old passage human dermal fibroblasts cells were measured. As a result, the expression of βPIX in aged HDF cells was decreased compared to that of young HDF cells, while the expression of p16 was increased, and SA-β-Gal ( senescence associated-β-galactosidase) was also found to be increased in senescent HDF cells (see FIGS. 2C to 2E ).

Through these results, the present inventors found that, as in Example 1, the βPIX protein is related to aging in human lung tissues and cells, and is a protein whose expression level is reduced by aging in particular.

< Example 3>

of βpix Aging-promoting activity analysis according to expression inhibition

To investigate the effect of βPIX expression on cellular senescence, siRNA was used to reduce βPIX expression in HDF cells and mouse lung tissue, and then the expression levels of cellular senescence markers were analyzed. Specifically, HDF cells were treated with siRNA for βPIX, and after 3 days, an increase in the activity of SA-β-Gal, a senescence indicator, and an increase in the expression of pγH2AX and p16 were investigated (see FIGS. 3A to 3C). In addition, the lung tissue of a 3-month-old mouse was treated, respectively, and immunohistochemical staining was performed on each sample after 4 weeks. At this time, βPIX siRNAs treated in mouse lung tissue were siPIXm1 and siPIXm2, and βPIX siRNAs treated in HDF cells were siPIX1 and siPIX2.

siPIX1: 5′-UCAACUGGUAGUAAGAGCAAAGUUU-3' [SEQ ID NO: 3]

siPIX2: 5'-UUGAGCUGCAGAUCCUGACGGAAGC-3' [SEQ ID NO: 4]

siPIXm1:5′-CCAACUGGUAGUACGAGCCAAGUUU-3′ [SEQ ID NO: 5]

siPIXm2: 5′-GAGGACCUAGGAGAGUUCAUGGAAA-3′ [SEQ ID NO: 6]

As a result, when the expression of βPIX was suppressed in HDF cells, SA-β-Gal activity was increased and the expression of p16 was also increased (see FIGS. 3A to 3C ). In addition, the expression of βPIX in the lung tissue of mice was decreased by siRNA treatment for βPIX, the activity of SA-β-Gal, an aging indicator, was increased, and the expression of p16INK and pγH2AX was also increased. was found to exist (see FIGS. 3D to 3F).

Therefore, these results suggest that inhibition of βPIX expression can accelerate aging.

< Example 4>

of βpix Analysis of the effect of delayed induction of cell cycle and cell proliferation according to expression inhibition

In order to determine whether cellular senescence induced by βPIX affects the cell cycle and cell proliferation, changes in the expression of cell cycle regulatory proteins were observed in HDF cells in which βPIX expression was inhibited using siRNA. For the analysis of expression changes, the expression levels of p19, cyclin E, CDK2, p16, cyclin D, and CDK4, which are factors involved in the cell cycle and cell proliferation, were analyzed through immunoblotting, and cell cycle changes were analyzed through flow cytometry. analyzed. In addition, in order to analyze whether the growth curve of cells changes when βPIX expression is suppressed, cells were collected using trypsin, stained with crystal violet solution, and the number of cells was measured using a hemocytometer.

As a result, when the expression of βPIX was suppressed, the expression of the cell cycle regulatory proteins p19, cyclin E, and CDK2 was decreased, whereas the expression of p16, cyclin D, and CDK4 was increased (see FIG. 4A ). . This can be said to mean that the cell cycle can be changed by suppressing the expression of βPIX. In addition, as a result of analyzing the cell cycle through flow cytometry (FACS), it was found that the cell cycle was delayed in the G1 phase due to inhibition of βPIX expression (see FIG. 4B ). In addition, as a result of cell proliferation analysis, in the case of cells in which βPIX expression was suppressed, cell proliferation was significantly reduced compared to control siCtrl-treated cells and untreated HDF cells (none) (see FIG. 4C ).

These results suggest that cell senescence is induced by delaying the cell cycle and inhibiting cell proliferation by inhibiting βPIX expression.

< Example 5>

of βpix according to expression inhibition SASP Induction Effect Analysis

In order to more specifically confirm the cellular senescence phenomenon induced by inhibition of βPIX expression, a change in senescence-associated secretory phenotype (SASP), another indicator of senescent cells, was observed. In SASP, the expression of various types of aging-related cytokines is induced as aging progresses. Accordingly, the present inventors analyzed whether the induction of senescence by inhibition of βPIX expression could be confirmed through SASP analysis.

As a result, SASP expression was investigated in cells in which βPIX expression was suppressed by specific siRNA treatment, and increased expression of cytokines such as IL-6 and MCP-1 and TIMP-2 protein, which are related to the promotion of cellular senescence. Expression was shown to be increased (see FIGS. 5A-5C ).

These results can be regarded as confirming evidence that more firmly proves that inhibition of βPIX expression induces cellular senescence.

< Example 6>

in a mouse animal model of βpix Analysis of anti-aging effect by overexpression

Through the experiments of the previous examples, it was confirmed that inhibition of the expression of βPIX induces aging. Accordingly, the present inventors performed the following experiment to determine whether aging can be inhibited when the expression of βPIX is increased.

After introducing the lentivirus-made βPIX gene into the lungs of elderly mice (12-month-old mice), βPIX was overexpressed for 4 weeks, and changes in aging markers were analyzed by SA-β-Gal assay and immunohistochemical analysis. confirmed through.

As a result, the enzyme activity of SA-β-Gal, known as an aging index, was significantly reduced by overexpression of βPIX, and the expression of p16INK and pγH2AX was also decreased (see FIGS. 6A to 6C ). These results mean that aging can be suppressed by overexpressing βPIX in aged tissues.

Combining the above experimental results, it can be seen that βPIX can function as a regulator capable of regulating aging. Specifically, inhibiting the expression of βPIX can promote or induce aging, while the expression of βPIX When increasing, it can be seen that aging can be inhibited or delayed.

Therefore, it can be seen that βPIX according to the present invention can be used not only as a new indicator of aging, but also that βPIX protein or its activator can be used as a therapeutic agent for aging and aging-related diseases.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Chungbuk National University <120> Novel use of beta PIX having cellular senescence modulating activity <130> NPDC-80182 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 646 <212> PRT <213> Artificial Sequence <220> <223> betaPIX amino acid sequence <400> 1 Met Thr Asp Asn Thr Asn Ser Gln Leu Val Val Arg Ala Lys Phe Asn 1 5 10 15 Phe Gln Gln Thr Asn Glu Asp Glu Leu Ser Phe Ser Lys Gly Asp Val 20 25 30 Ile His Val Thr Arg Val Glu Glu Gly Gly Trp Trp Glu Gly Thr His 35 40 45 Asn Gly Arg Thr Gly Trp Phe Pro Ser Asn Tyr Val Arg Glu Ile Lys 50 55 60 Pro Ser Glu Lys Pro Val Ser Pro Lys Ser Gly Thr Leu Lys Ser Pro 65 70 75 80 Pro Lys Gly Phe Asp Thr Thr Ala Ile Asn Lys Ser Tyr Tyr Asn Val 85 90 95 Val Leu Gln Asn Ile Leu Glu Thr Glu His Glu Tyr Ser Lys Glu Leu 100 105 110 Gln Ser Val Leu Ser Thr Tyr Leu Arg Pro Leu Gln Thr Ser Asp Lys 115 120 125 Leu Ser Ser Ala Asn Thr Ser Tyr Leu Met Gly Asn Leu Glu Glu Ile 130 135 140 Ser Ser Phe Gln Gln Val Leu Val Gln Ser Leu Glu Glu Cys Thr Lys 145 150 155 160 Ser Pro Glu Ala Gln Gln Arg Val Gly Gly Cys Phe Leu Ser Leu Met 165 170 175 Pro Gln Met Arg Thr Leu Tyr Leu Ala Tyr Cys Ala Asn His Pro Ser 180 185 190 Ala Val Ser Val Leu Thr Glu His Ser Glu Asp Leu Gly Glu Phe Met 195 200 205 Glu Thr Lys Gly Ala Ser Ser Pro Gly Ile Leu Val Leu Thr Thr Gly 210 215 220 Leu Ser Lys Pro Phe Met Arg Leu Asp Lys Tyr Pro Thr Leu Leu Lys 225 230 235 240 Glu Leu Glu Arg His Met Glu Asp Tyr His Pro Asp Arg Gln Asp Ile 245 250 255 Gln Lys Ser Met Thr Ala Phe Lys Asn Leu Ser Ala Gln Cys Gln Glu 260 265 270 Val Arg Lys Arg Lys Glu Leu Glu Leu Gln Ile Leu Thr Glu Pro Ile 275 280 285 Arg Ser Trp Glu Gly Asp Asp Ile Lys Thr Leu Gly Ser Val Thr Tyr 290 295 300 Met Ser Gln Val Thr Ile Gln Cys Ala Gly Ser Glu Glu Lys Asn Glu 305 310 315 320 Arg Tyr Leu Leu Leu Phe Pro Lys Pro Ser Val Met Leu Ser Pro Ser 325 330 335 Pro Arg Met Ser Gly Phe Ile Tyr Gln Gly Lys Leu Pro Thr Thr Gly 340 345 350 Met Thr Ile Thr Lys Leu Glu Asp Ser Glu Asn His Arg Asn Ala Phe 355 360 365 Glu Ile Ser Gly Ser Met Ile Glu Arg Ile Leu Val Ser Cys Thr Ser 370 375 380 Gln Gln Asp Leu His Glu Trp Val Glu His Leu Gln Lys Gln Thr Lys 385 390 395 400 Val Thr Ser Val Ser Asn Pro Thr Ile Lys Pro His Ser Val Pro Ser 405 410 415 His Thr Leu Pro Ser His Pro Leu Thr Pro Ser Ser Lys His Ala Asp 420 425 430 Ser Lys Pro Val Ala Leu Thr Pro Ala Tyr His Thr Leu Pro His Pro 435 440 445 Ser His His Gly Thr Pro His Thr Thr Ile Arg Trp Gly Pro Leu Glu 450 455 460 Pro Pro Pro His Pro Lys Ala Trp Gly Leu Ser Trp Leu Trp Thr Ala 465 470 475 480 Pro Pro Leu Arg Pro Ser Ala Ala Leu Cys Tyr Lys Glu Asp Leu Ser 485 490 495 Lys Ser Pro Lys Thr Met Lys Lys Leu Leu Pro Lys Arg Lys Pro Glu 500 505 510 Arg Lys Pro Ser Asp Glu Glu Phe Ala Val Arg Lys Ser Thr Ala Ala 515 520 525 Leu Glu Glu Asp Ala Gln Ile Leu Lys Val Ile Glu Ala Tyr Cys Thr 530 535 540 Ser Ala Lys Thr Arg Gln Thr Leu Asn Ser Ser Ser Arg Lys Glu Ser 545 550 555 560 Ala Pro Gln Val Leu Leu Pro Glu Glu Glu Lys Ile Ile Val Glu Glu 565 570 575 Thr Lys Ser Asn Gly Gln Thr Val Ile Glu Glu Lys Ser Leu Val Asp 580 585 590 Thr Val Tyr Ala Leu Lys Asp Glu Val Gln Glu Leu Arg Gln Asp Asn 595 600 605 Lys Lys Met Lys Lys Ser Leu Glu Glu Glu Gln Arg Ala Arg Lys Asp 610 615 620 Leu Glu Lys Leu Val Arg Lys Val Leu Lys Asn Met Asn Asp Pro Ala 625 630 635 640 Trp Asp Glu Thr Asn Leu 645 <210> 2 <211> 1800 <212> DNA <213> Artificial Sequence <220> <223> betaPIX DNA sequence <400> 2 atgactgata acaccaacag ccaactggta gtacgagcca agtttaactt ccagcagacc 60 aatgaagatg aactctcctt ctcaaagggt gatgtcatcc atgtcacacg agtggaggaa 120 ggaggctggt gggagggcac acacaatggc aggaccggct ggttccccag caactacgtt 180 cgagagatca agccaagtga gaagcccgtg tcacccaaat cagggacctt gaagagccct 240 cccaaagggt tcgatacgac tgccatcaac aagagctatt acaacgtggt gctacagaac 300 atcctggaaa cagagcatga gtattccaag gagctgcagt ctgtgctgtc cacctacctg 360 cggccactgc agaccagtga caagttgagt tcagcaaaca cttcatattt aatgggaaat 420 ctagaggaaa tatcttcctt ccagcaagtg cttgtacagt ccctagaaga atgcacgaag 480 tctcctgaag cccaacagag agttggtggc tgcttcctga gcctgatgcc gcagatgagg 540 accctgtacc tcgcttactg tgccaaccac ccatctgctg tgagcgtcct cacagagcac 600 agtgaggacc taggagagtt catggaaaca aaaggtgcca gcagccctgg gatcctggtg 660 ctgaccaccg gcctgagcaa gcccttcatg cgcctggaca agtaccccac actgctaaag 720 gagctggaga gacacatgga ggattatcat cctgatagac aagatattca gaagtctatg 780 acggccttca aaaacctttc agctcagtgt caagaagttc gcaagaggaa ggagctggag 840 ctgcagatcc tgacggagcc catcaggagc tgggaggggg atgacataaa gaccctgggc 900 agtgtcacat acatgtccca agtcaccatt cagtgtgcgg gaagcgagga gaagaatgag 960 agatacctcc tgctcttccc caaaccttct gtcatgttgt ctccaagtcc caggatgagt 1020 gggttcatct atcaggggaa gctgccaaca acaggaatga caatcacaaa gcttgaggac 1080 agtgaaaacc ataggaatgc atttgagata tcagggagca tgatcgagcg gattctggtg 1140 tcctgcacca gccagcagga cttacacgag tgggtggaac acctgcagaa gcagacgaag 1200 gtcacatctg tgagcaaccc caccatcaaa ccccactcgg tgccatcaca cacacttcct 1260 tcccatcctc tcactccatc cagcaaacac gcggacagca agcccgtggc actgacgcct 1320 gcgtaccaca cactccccca cccctctcac catggcaccc cacacaccac catcaggtgg 1380 ggacccctgg agcctccgcc gcaccccaag gcttgggggc tgagttggct gtggactgca 1440 cctcccctcc ggccctcagc tgctctctgc tacaaggagg atctcagtaa gagccccaag 1500 accatgaaaa agctgctgcc gaagcgcaag cccgagcgga agccttcgga cgaggagttc 1560 gctgtgcgca agagcacagc ggcgctggaa gaagacgctc agatcctgaa ggttatcgaa 1620 gcttattgca caagtgcaaa gacgcgccag accctgaact caagttcacg caaagaatct 1680 gctccacaag tgttgcttcc agaagaagaa aaaattatag tcgaagaaac caagagcaat 1740 gggcagacag tgatagagga aaagagcctc gtggatacgg tgtatgcgtt aaaggatgaa 1800 1800 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siPIX1 sequence <400> 3 ucaacuggua guaagagcaa aguuu 25 <210> 4 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siPIX2 sequence <400> 4 uugagcugca gauccugacg gaagc 25 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siPIXm1 sequence <400> 5 ccaacuggua guacgagcca aguuu 25 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siPIXm2 sequence <400> 6 gaggaccuag gagaguucau ggaaa 25

Claims (18)

delete delete delete delete delete delete delete delete delete delete delete delete A biomarker composition for measuring cellular senescence comprising a βPIX protein consisting of the amino acid sequence of SEQ ID NO: 1 or a βPIX gene consisting of the nucleotide sequence of SEQ ID NO: 2. delete treating the biological sample with any candidate;
detecting the expression level of the βPIX protein consisting of the amino acid sequence of SEQ ID NO: 1 or the βPIX gene consisting of the nucleotide sequence of SEQ ID NO: 2 from the biological sample; and
Comprising the step of comparing the expression level with the expression level of the same gene or the same protein in a control not treated with any candidate substance,
A method of screening for inhibitors of cellular senescence. 16. The method of claim 15,
When the expression level of the βPIX protein or βPIX gene is increased in the group treated with the candidate substance compared to the control group, the screening method of the cellular senescence inhibitor, characterized in that it further comprises the step of determining the candidate substance as a cellular senescence inhibitor. A method for inhibiting cellular senescence, comprising treating the isolated cells with an expression vector containing the βPIX protein consisting of the amino acid sequence of SEQ ID NO: 1 or the βPIX gene consisting of the nucleotide sequence of SEQ ID NO: 2. delete

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