KR20180078870A - Skin aging-specific Epigenetic gene markers and detection of skin aging using them - Google Patents

Abstract

The present invention relates to a skin aging-specific biomarker consisting of a profilin-1 (PFN1) gene, a protein encoded by the gene, and a methylated promoter of the PFN1 gene. The present invention further relates to a detection composition for skin aging or skin aging progression stages, containing a preparation for measuring a level of the biomarker.

Description

[0002] Skin aging-specific Epigenetic gene markers and detection of skin aging using the same [

The present invention relates to a skin aging-related haplotype gene marker and its use for detecting skin aging. More particularly, the present invention relates to a skin aging-specific biomarker comprising a PFN1 gene, a protein encoded by the gene, or a methylation promoter of a PFN1 gene And a composition for measuring the level of the biomarker.

Heterogeneous DNA methylation is a phenomenon in which a methyl group binds to the cytosine of the CpG island of the gene promoter region, and binding of the gene promoter of the transcription factor is inhibited by the DNA methylation of the gene, Lt; / RTI >

The tumor suppressor gene is known to be inhibited in many cancer cells. In addition, as one of the tumor suppressor gene expression inhibiting mechanisms, the DNA methylation of the progeny gene of the tumor suppressor gene has been studied.

Therefore, methods for diagnosing cancer by measuring the methylation of the progeny DNA of a specific gene are suggested.

Korean Patent Laid-Open Publication No. 2014-0122529 discloses a method for detecting the methylation of the CpG island region of a bladder cancer biomarker gene. In Korean Patent Registration No. 10-1597812, the methylation level of the CpG region of the ZFP37 gene promoter is To provide predictive information of responsiveness to platinum-based anticancer drugs in ovarian cancer patients.

However, there is no known method for diagnosing skin aging using the correlation between gene related to skin senescence and epigenetic gene DNA methylation of skin aging.

The technique of using the progeny gene DNA methylation of a gene related to skin aging as a skin aging diagnostic marker can be applied to the monitoring of a lesion site in the course of treatment of a specific skin, and development of the technique is required.

It is an object of the present invention to provide a skin senescence-related progeny marker comprising a PFN1 (Profilin 1) gene, a protein encoded by the gene, or a methylation promoter of a PFN1 gene.

A further object of the present invention is to provide a PFN1 gene, a protein encoded by the gene, or a PFN1 gene comprising a methylation promoter of the PFN1 gene, or an agent capable of measuring the expression of a protein encoded by the gene, And a kit comprising the same, which comprises an agent capable of measuring the methylation of the methylation promoter of the skin.

A further object of the present invention is to provide a PFN1 gene, a protein encoded by the gene, or a PFN1 gene comprising a methylation promoter of the PFN1 gene, or an agent capable of measuring the expression of a protein encoded by the gene, Which is capable of measuring the methylation of the methylation promoter of the plant.

A further object of the present invention is to provide a screening method of a skin aging inhibiting drug using a PFN1 protein, a gene encoding a PFN1 protein, or a methylation promoter of a PFN1 gene, which are skin aging-specific biomarkers.

The present invention relates to a method for measuring the methylation of PFN1 (Profilin 1) gene or the expression of PFN1 gene or PFN1 protein to provide information necessary for diagnosing skin aging or skin aging progression, It can be used to monitor areas, develop biomarkers to diagnose skin aging, and develop anti-aging skin cosmetics.

When the skin is aged or aging of the skin progresses, methylation of the CpG island of the PFN1 gene promoter in the cell increases.

In addition, when the skin ages or aging of the skin progresses, expression of the PFN1 gene or PFN1 protein in the cell decreases.

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

An example of the present invention provides a skin aging-specific biomarker comprising a PFN1 gene, a protein encoded by the gene, or a methylation promoter of a PFN1 gene.

It is known that PFN1 (Profilin 1) binds to actin and affects the cytoskeleton structure. It is known that when the concentration of PFN1 is high, the polymerization of actin is inhibited.

The PFN1 protein may be derived from a mammal such as a primate including a human (Homo sapiens), a rodent including a mouse (Mus musculus), a rat (Rattus norvegicus), an amphibian such as a frog (Xenopus laevis) . For example, PFN1 may be, but is not limited to, human PFN1 (e.g., NP_005013.1. (Gene: NM_005022.3.)).

The protein encoded by the PFN1 gene may include the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

The PFN1 gene may include the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

The PFN1 gene promoter to be methylated may include the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

Wherein the methylated PFN1 gene promoter comprises a nucleotide sequence corresponding to -1319 from an ATG sequence that is a start codon of PFN1 to an ATG sequence that is a start codon and wherein the methylation in the methylation promoter is 81 to 262 nucleotides in the polynucleotide sequence of SEQ ID NO: But not limited to, the CpG island cytosine present on the nucleotides corresponding to the CpG islands.

The region from -1239 to -998 from the start codon of SEQ ID NO: 1 is the portion of methylation specific PCR, and the region of -1241 to -997 from ATG is the corresponding portion of unmethylation specific PCR.

The methylation promoter of the PFN1 gene may mean that methylation may occur on the CpG island of the promoter controlling the expression of the PFN1 gene. The promoter may be present outside the PFN1 gene, And preferably exists outside the PFN1 gene.

The term " skin aging " is often caused by external and internal processes associated with increased wrinkling, sagging and sagging, and since external aging is mainly caused by repeated exposure to ultraviolet rays (UV) Is called "photoaging". Naturally aged skin is smooth, pale, and has fine wrinkles, but photoaged skin has thick wrinkles and causes pigmentation and capillary dilatation. Damage to collagen, a major structural component of the skin, is considered to be a major factor in skin aging and is found in both natural aging and photoaging skin. Collagen damage is partially associated with the induction of matrix metalloproteinase (MMP). MMP is a structurally related family of matrix-degrading enzymes and plays an important role in various degradation processes such as inflammation, tumor invasion and skin aging. The level of MMP is increased by various stimuli such as ultraviolet light, oxidative stress and cytokine, and ultraviolet irradiation promotes DNA binding of activator protein-1 (AP-1), collagenase (MMP- 1), stromelysin (MMP-3), and gelatinase (MMP-9). Once collagen is degraded by MMP-1, it is further degraded by MMP-3 and MMP-9.

In the present invention, the skin aging may mean aging of the skin or aging of the skin caused by UV, and the aging of the skin may be characterized by an increase in the wrinkle formation or an increase in the thickness of the skin epithelium, May mean optic skin aging.

The skin aging step refers to a step in which skin aging occurs due to an external and internal process, and specifically occurs through the following process. In the case of epidermis, keratin synthesis of keratinocyte is reduced, depletion of keratinocyte, keratohylanin, and various enzymes is worsened, and dead keratinocytes do not fall off and stagnate, thickening of keratinocytes, It can be confirmed that the phenomenon is deepened and the phenomenon of moisture transfer to the base layer is slowed down. Next, the production of sebum is reduced and the distribution of melanin pigment becomes irregular, and the skin becomes a whole face dull. In the case of the dermis, collagen and elastic fibers become hardened and insoluble, and the skin elasticity-related factors are cross-linked. Next, as the aging progresses, the wrinkles of the skin are increased by the decrease of the ground sugstance, and the amount of hyaluronic acid is gradually decreased. The skin aging step may mean all steps including the step of increasing the keratinocyte layer and lowering the skin elasticity, but the present invention is not limited thereto.

Another example of the present invention provides a composition or kit for detecting a skin aging or skin aging progressive stage comprising an agent for measuring the expression level of the PFN1 gene or an agent for measuring the methylation of the PFN1 gene promoter.

The agent for measuring the expression level of the PFN1 gene may include, but is not limited to, a primer, a probe, an extramammer or an antisense oligonucleotide that specifically binds to the PFN1 gene.

The agent for measuring the expression level of the PFN1 gene may include at least one selected from the group consisting of small molecule chemicals, proteins, peptides, nucleic acid molecules, and antibodies that specifically bind to the PFN1 protein. It is not.

The detection of the agent for measuring the expression level of the PFN1 gene may be carried out by detecting the expression level of PFN1 gene selected from the group consisting of reverse transcriptase polymerase, competitive reverse transcriptase polymerase, real-time reverse transcriptase polymerase, RNase protection assay, Northern blotting, But the present invention is not limited thereto.

The detection of the agent for measuring the expression level of the PFN1 gene may be performed by Western blotting, ELISA or mass spectrometry, for example, by means of protein determination of PFN1, but is not limited thereto.

The agent for measuring the methylation of the promoter may be a pair of PCR primers for amplifying SEQ ID NO: 1 including the methylated CpG isoform of the methylation promoter of the PFN1 gene, and the pair of PCR primers for amplifying the SEQ ID NO: 1 is SEQ ID NO: To 11, but is not limited thereto.

The detection of the agent that measures the methylation of the PFN1 gene promoter can be carried out by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA But is not limited to, a method selected from the group consisting of chip, pyrosequencing, and bisulfite sequencing.

In the present invention, the skin aging may mean aging of the skin or aging of the skin caused by UV, and the aging of the skin may be an increase in the wrinkle formation or an increase in the thickness of the skin epithelium, It can mean aging.

In another aspect, the present invention relates to a microarray for detecting skin aging or skin senescence progression stage comprising a primer, a probe, an umbrella, or an antisense oligonucleotide that specifically binds to the PFN1 gene.

The term " agent for measuring the expression level of a gene " of the present invention refers to an agent that directly or indirectly expresses the gene or a protein encoded by the gene in a sample of a subject, And measuring the degree of expression. For example, the agent may comprise a primer, a probe, an umbrella, or an antisense oligonucleotide capable of specifically binding to the PFN1 gene. In addition, the agent may include at least one selected from the group consisting of small molecule chemicals, proteins, peptides, nucleic acid molecules, and antibodies that specifically bind to the PFN1 protein, but the present invention is not limited thereto.

In the present invention, the expression level of the PFN1 gene can be determined by confirming the expression level of the mRNA of the PFN1 gene or the expression level of the protein encoded by the gene. The mRNA expression level can be determined by measuring the amount of mRNA in the sample, and determining the presence or absence of the mRNA and the expression level of the marker gene. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), and reverse transcriptase-polymerase chain reaction RNase protection assay, Northern blotting, DNA chip, and the like.

The term " primer " of the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers can incorporate additional features that do not alter the primer properties of primers that serve as a starting point for DNA synthesis, as sense and antisense nucleic acids with 7 to 50 nucleotide sequences. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary so long as it can hybridize with the template. The position of the primer or the primer binding site refers to a target DNA fragment to which the primer hybridizes.

The term " probe " in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to mRNA, and is labeled, Can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, hybridization is carried out using a probe complementary to the marker polynucleotide of the present invention, so that the aging of the skin or the progress of skin aging can be predicted or verified through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The term " Aptamer " of the present invention refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and capable of binding to a target molecule with high affinity and specificity. aptus ", which has the meaning of" fitting ", has been unearthed many digestamers that can bind to a variety of target molecules, including peptides and membrane proteins. In addition, because of its low molecular organic nature, it is often compared with monoclonal antibodies, and it is also known as a "

The antisense oligonucleotides, primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, " measurement of protein expression level " refers to a process of confirming the presence and the degree of expression of a protein encoded by a marker gene in a subject sample. Using the antibody specifically binding to the protein of the gene, You can check the amount. Examples of the assay method include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis , Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.

The term " antibody " as used herein in the present invention means a specific protein molecule indicated for an antigenic site as a term known in the art. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the marker of the present invention, and the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, Part thereof is included in the antibody of the present invention and includes all the immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The kit of the present invention can detect skin aging markers by confirming mRNA expression level or protein expression level of PFNl gene. In the present invention, the kit may comprise a primer, a probe, or an antibody that selectively recognizes a marker for measuring the expression level of the PFN1 gene whose expression is increased according to skin aging, as well as one or more other component compositions suitable for the assay, Solutions or devices may be included.

As a specific example, in the present invention, the kit for measuring the mRNA expression level of the PFN1 gene may be a kit containing elements necessary for performing RT-PCR. The RT-PCR kit can be used to detect the presence of enzymes such as test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNases, RNase inhibitors , DEPC-water (DEPC-water), sterilized water, and the like.

In addition, the kit of the present invention may be a gene detection kit including essential elements necessary for performing a microarray. A substrate on which a cDNA corresponding to a microarray, a gene, or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. More specifically, it may be a DNA microarray in which oligonucleotides or complementary strand molecules corresponding to 10 or more genes selected in the microarray of the present invention, or fragments thereof, are integrated. The oligonucleotide or its complementary strand molecule comprises 18 to 30 nucleotides of the gene, preferably 20 to 25 nucleotides.

In addition, the kit for measuring the level of protein expression in the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The chromogenic enzyme may be peroxidase, Alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline- ), Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

Another example of the present invention provides a method for providing information necessary for diagnosis of a skin aging or skin aging progress stage including a step of measuring an expression level of a PFN1 gene.

The expression level of the gene may be measured by mRNA, or protein.

The expression level of the mRNA may be measured by reverse transcriptase polymerase chain reaction, nucleic acid hybridization, electrophoresis, or Northern blotting, but is not limited thereto.

The expression level of the gene may be measured by obtaining RNA from a sample, reverse-transcribing the RNA to obtain cDNA, attaching a detectable label to the cDNA, and performing a polymerase chain reaction to measure the gene, no.

The detectable label may be, but is not limited to, an enzyme, a radioactive isotope, a fluorescent substance, a chemiluminescent molecule, a radioactive substance, a liposome, or a hapten molecule.

The hapten is a low molecular organic compound that can not induce an immune reaction by itself but induces an immune reaction once it is attached to a carrier molecule. An antibody that binds to a hapten is developed and used as a reference antibody to be used for accurate quantitative measurement of a sample have.

In the present invention, the expression level of the protein can be measured by an immunological assay, Western blotting, ELISA, or mass spectrometry, but is not limited thereto.

In the present invention, the skin aging may mean aging of the skin or aging of the skin caused by UV, and the aging of the skin may be an increase in the wrinkle formation or an increase in the thickness of the skin epithelium, It can mean aging.

Another example of the present invention provides a method for providing information necessary for diagnosis of a skin aging or skin aging progress stage including a step of measuring methylation of a methylation promoter of PFN1 gene.

The methylation promoter may be methylated at the cytosine of a CpG island (CpG island) present in nucleotides 81-262 of the polynucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The methylation can be measured by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, umi, pyrosequencing, Fate sequencing, but the present invention is not limited thereto.

The step of measuring the methylation may be carried out by the presence or absence of sequence amplification or sequence analysis using a PCR primer for amplifying SEQ ID NO: 1 including the methylated CpG island of the methylation promoter of the PFN1 gene, It is not.

In the present invention, the skin aging may mean aging of the skin or aging of the skin caused by UV, and the aging of the skin may be an increase in the wrinkle formation or an increase in the thickness of the skin epithelium, It can mean aging.

The present invention relates to a skin aging-related haplotype gene marker and its use for detecting skin aging. More particularly, the present invention relates to a skin aging-specific biomarker comprising a PFN1 gene, a protein encoded by the gene, or a methylation promoter of a PFN1 gene And an agent for measuring the level of the biomarker. The present invention relates to a composition for detecting skin aging or a skin aging progression stage, and can be used for the development of cosmetics or the provision of information necessary for diagnosis of skin aging by detecting the biomarker Do.

FIG. 1 schematically illustrates a method of manufacturing a skin photoaging animal model.
FIG. 2 is a graph showing changes in skin aging when UVB irradiation is increased in a photoaging mouse model according to an embodiment of the present invention, and cacao mover (CP) or pycnogenol (Pyc) Indicating that it has decreased. CL of FIG. 2 is data from a tissue sample of a mouse in which CP was taken at 39.1 mg / kg / day, and CH is data of a sample that consumed CP at 156.3 mg / kg / day.
FIG. 3 is a graph showing a change in the thickness of epithelial cells during UVB irradiation in a photoaging mouse model according to an embodiment of the present invention, and when the cyanocouple (CP) or anti-aging pycnogenol (Pyc) The decrease in thickness was confirmed by H & E staining.
FIG. 4 is a graph showing changes in epithelial cell thickness when irradiated with UVB in a photoaging mouse model or with a photoaging mouse model and treated with cacao mudder (CP) or anti-aging agent pycnogenol (Pyc) according to an embodiment of the present invention .
FIG. 5A shows the selection of 17 genes with increased methylation upon UVB irradiation in a photoaging mouse model according to an embodiment of the present invention. FIG. 5B shows a group of genes satisfying the condition of FIG. 5A among the genes group in which the amount of expression of mRNA is changed upon UVB irradiation and recovered upon CP treatment, in Heatmap. The Ct group indicates that the UVB is not irradiated, the UV is the UVB irradiation, and the CP indicates the UVB irradiation and the CP treatment.
FIG. 6 shows the change of DNA methylation when the PFN1 gene was treated with UVB or UVB and treated with cacao powder (CP) in a photoaging mouse model.
FIG. 7 shows the change in the expression of PFN1 mRNA when UVB treatment and UVB treatment and CP treatment were observed in the photoaging mouse model.
Fig. 8 shows the result of immunoblotting of the model of the photoaging mouse. CTL1 and 2 are control mice not irradiated with UVB, Cacao1 and 2 are mice treated with UVB and treated with CP, and UV1 and 2 irradiated with UVB alone.
FIG. 9 shows the results of RNA sequencing analysis of the PFN1 gene in a photoautotized mouse model. Control is a control group not treated with UVB and CP, and UV is mRNA analysis data obtained from mouse tissue treated with UVB alone. CL was given at a dose of 39.1 mg / kg / day over a period of 8 weeks, and CH was given at a dose of 156. 25 mg / kg / day. Finally, Pyc was an experimental group receiving 625 mg / kgday of pycnogenol. The significance is 0.05 or more.
Fig. 10 shows changes in protein expression of PFN1 by UV treatment in photo-induced human-derived skin cells.
Fig. 11 shows the results of methylation-specific PCR of PFN1 in photo-induced human-derived skin cells.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1 . PFN1  Gene selection

1-1: Skin Photoaging  Making of mouse model

(1) Manufacturing method

In order to select markers for diagnosing skin photo-aging, a skin photo-aging mouse model was prepared as follows.

Specifically, as shown in FIG. 1, cacao powder (cacao powder, baba Callebaut, Lebbeke-Wieze, Belgium) was fed at a dose of 156.25 mg / kg / day five times a week to female albino hairless mice (Skh- And irradiated 100 to 200 mJ / cm < 2 > of UV light three times a week using TL20W / 12RS UV lamps (Philips, Eindhoven, The Netherlands). In the control group, the mice were raised under the same conditions for 8 weeks, but no UVB irradiation and CP ingestion were performed. The mice that did not eat CP but UVB only were included in the experimental group as UV mice. The UV intensity was gradually increased to 100 mJ / cm2 in the first week, 150 mJ / cm2 in the second and third week, and 200 mJ / cm2 in the fourth and 8th weeks. The photoautotized mouse model was obtained through biopsy at 8 weeks.

(2) Confirmation of skin photo-aging of manufactured mouse

In order to measure whether the skin was photographed or not, the wrinkle formation was measured by the following method, and the tissue was observed by H & E staining.

Specifically, to measure wrinkle formation, the degree of wrinkle formation was measured using Bissett's visual wrinkle scale method, and skin aging of photoaging mouse model was measured. Photo-realistic mouse model with Silicone rubber (Silflo Dental Impression Materials, Potters Bar, UK) Photographs were taken using a coupling charge system video camera modeled as skin wrinkles. The degree of wrinkles was measured with skin-Visiometer SV 600 software (CK Electronic GmbH, Germany) (0, none; 1, minimal; 2, mild; 3; moderate; 4; secere; 5, very severe).

As a result, as shown in FIG. 2, in the case of the mouse in which the photoaging was induced in comparison with the mouse in which the photoaging was not induced, it was found that the number of the wrinkle formation sites was increased twice or more, and the concentration of the cacao powder (Pycnogenol), which is a strong antioxidant (antioxidant), was administered to a photoinduced mouse to induce a wrinkle-forming site Was decreased. From the above results, it was confirmed that the photoaging induced mice were induced by UVB.

In addition, since the thickness of epithelial cells of the skin in which photoaging has progressed generally is thicker than that of the control, the epithelial cell thickness was observed by H & E staining as follows to determine whether skin photoaging occurred in the skin photoaging model.

Specifically, to observe epithelial cell thickness by H & E staining, mouse tissues were fixed with 4% (v / v) paraformaldehyde (PFA: Sigma, USA) and dehydrated to make paraffin blocks. The paraffin block was chopped to a size of 5 um using a microtome (Leica, USA). The slides were placed in xylene (SAMCHUN, Korea), deparaffinized for 3 to 10 minutes, and reduced by 5% in 100% ethanol (Millipore, USA) The slurry was reacted with a slide to remove xylene. The slides were then washed by immersion in distilled water, stained with a Harris Hematoxylin solution for 2 minutes, washed with distilled water and methanol containing 0.5% (v / v) HCl. It was then washed once more with distilled water and neutralized by washing with ammonia water 0.5% (v / v) (JUNSEI, Japan) more than 20 times. Eosin is dyed for 2 minutes, washed, dehydrated with increasing the ratio of ethanol from 70% to 100% by 5%, transparentized with xylene, and mounted with Canada valsam. Finally, photographs were taken using an optical microscope (Nikon, eclipse 50i, Japan) and the thickness of epithelial cells was measured using the I Solution x64 (image & Microscope Technology, CANADA) program.

As a result, as shown in FIG. 3 or 4, the epithelial cell thickness of the prepared photoautotated mouse tissue was about twice as thick as that of the mouse not inducing photoaging, but the skin aging prevention Treatment of cacao powder (CP) or pycnogenol, which is a substance, decreased the thickness of the epithelium increased by photoaging.

1-2: Skin aging As a marker PFN1  Gene selection

(1) Extraction of genomic DNA or total RNA

In order to select a skin aging marker gene, tissues were taken from a photoaging mouse produced in the same manner as in Example 1-1, and the following experiment was conducted.

Mouse tissues were biopsied in a photoautotated mouse prepared in the same manner as in Example 1-1, and then rapidly cooled using liquid nitrogen. Then, genomic DNA was isolated using G-spinTM Total DNA Extraction Kit (Intron, Gyeonggi-do, Korea) , Or total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia, USA). The extracted total RNA was electrophoresed on 1% (w / v) agarose gel to confirm the 18S and 28S RNA bands, and high quality whole RNA was extracted.

(2) Selection of PFN1 gene

To isolate the skin senescence marker gene by analyzing the methylation pattern of the extracted genomic DNA, the extracted genomic DNA was subjected to bisulfite treatment to convert unmethylated cytosine residues into Uracil residues. Then, gene methylation analysis was performed using MeDIP (Methylated DNA immunoprecipitation), and the result was shown in the graph shown in FIG. 5 through Heatmap.

Specifically, unmethylated cytosine residues were converted to Uracil residues by bisulfite treatment using the EZ DNA Methylation-Lightning ™ Kit (Zymoresearch, CA, USA) on the genomic DNA in 1-2 (1). Only methylated genes whose cytosine residues were converted to uracil residues were selected by MeDIP (Methylated DNA immunoprecipitation) using the methylation specific antibody. The amount of methylation increased from green to red, and the amount of methylation decreased as the color elongated from green.

As a result, as shown in FIG. 5A, methylation was increased by more than 1.3 times and 17 genes having a p value of 0.05 or less were selected when UVB alone was irradiated compared to UVB irradiation and CP treatment.

Then, RNA-sequencing (RNA-Seq) data analysis (TheragenEtex, Korea) was performed using the total RNA extracted from 1-2 (1) to examine the UVB of 17 genes selected through the methylation change or UVB And the amount of mRNA expression by CP treatment was changed after irradiation. It indicates that the amount of RNA increases from green to red, and the amount of RNA decreases with increasing color in green.

As a result, as shown in Fig. 5B, the mRNA level of the 17 genes selected in Fig. 5A was reduced by 1.3 times or more when the UVB was irradiated, the p value was 0.05 or less, the UVB , The amount of mRNA increased by more than 1.3 times when treated with UVB compared to the amount of mRNA, and the value of p-value was less than 0.05. UVB and CP treatment were performed compared to the control without UVB treatment MRNA levels were varied within 1.3 times, and genes with a p value of 0.05 or less were selected.

Among the genes selected by FIGS. 5A and 5B, the PFN1 (Profin 1) gene, which has been previously excluded from genes known to function in skin cells, was selected as a new skin aging marker.

(3) Analysis of methylation pattern of PFN1 promoter or exon

It has been found that gene methylation, which is one of the in vivo expression control mechanisms of genes, can be generally regulated by UVB, and it is generally known that gene expression is regulated by methylation of the promoter region of the gene. Therefore, in order to determine the site of methylation of PFN1 selected by the skin aging marker, methylation of the exon and promoter region of PFN1 was analyzed using WashU genome browser as follows.

Specifically, the base sequence of the gene was analyzed using Hiseq 2000 (illumina, USA) and the mouse genome map analysis was performed using the bowtie2 program. Next, methylation sequence analysis was performed on the whole gene sequence through mode-based analysis of Chip-seq (MACS) by referring to the article of feng J (geng J et al., Nat Protoc. 2012, 7, 1728-40) . After that, standardization, statistical analysis, and cluster analysis of methylated gene sequences were performed using GeneSpring 7.3 (silicon. Genetics, USA). Finally, the results of gene methylation analysis were analyzed by WashU genome browser.

As a result, as shown in Fig. 6, methylation occurred at the promoter region of PFN1 located on chromosome 11, and the methylation was increased by UV irradiation, but it was confirmed that the methylation was decreased upon treatment with CP after UV irradiation.

Example  2. PFN1  Analysis of gene expression level

2-1. In vivo PFN1  Analysis of gene expression level

(1) Analysis of expression level of PFN1 mRNA using RT-PCR

In order to analyze the changes in gene expression (mRNA) due to skin aging of PFN1 selected by skin aging markers, experiments were conducted as follows.

Specifically, 1.5 .mu.g of total RNA extracted by the method 1-2 (1) was dissolved in DEPC-treated water to a mouse prepared by the method of 1-1 (1), and then 1 uL of oligo (dT) 12- 18 and 10 mM dNTP were added, reacted at 70 ° C for 5 minutes, and immediately cooled on ice. 5 μl of 5 × reaction buffer and 0.5 μl of RNase inhibitor (Invitrogen, USA) were added to the sample, reacted at 42 ° C for 1 minute, and 1 μl of AMV reverse transcriptase (Intron, No. 27021) The mixture was incubated at 42 DEG C for 60 minutes and reacted at 70 DEG C for 10 minutes to inactivate the residual protein to synthesize cDNA. 1 uL of the synthesized cDNA, 1 pmol each of the primer pair (10 pmol) and 171 uL of distilled water were added to AccuPower® PCR PreMix (Bioneer, Gyonggi-do, Korea), reacted at 95 ° C for 5 minutes, 30 sec at 55 [deg.] C for 1 min, and 30 sec at 72 [deg.] C for 30 cycles. Elongated at 72 ° C for 5 minutes and then confirmed on 2% (w / v) agarose gel. At this time, RPLPO was used as a loading control to quantify the expression of the PFN1 gene. The primer pairs used in RT-PCR are shown in Table 1 below.

denomination The sequence (5 '> 3') SEQ ID NO: Tm (占 폚) Cycle mPFN1_F GAT GGG CAA ACA AGG TGT C 4 55.2 30 mPFN1_R ATG TGT GTG TAT GGA AAG AAG G 5 55.4 30 mRPLPO_F CAA AGG AAG AGT CGG AGGA 6 55.1 30 mRPLPO_R CAA AGA TGC CAA CAC AAA GRAYA 7 54.8 30

As a result, as shown in Fig. 8, the mRNA expression of PFN1 was reduced by about 8.9 times compared with the case where UV irradiation was not applied to the mouse by UV irradiation. However, when UV irradiation and CP treatment were performed, PFN1 And the amount of water was increased again.

(2) Analysis of expression level of PFN1 mRNA using RNA sequencing

In order to further confirm the reduction of gene expression (mRNA) due to skin aging of PFN1 selected by skin aging markers, the following RNA-sequencing (RNA-seq) assay was performed.

Specifically, mRNA was extracted from mouse tissues, and the expression level of the gene was analyzed through RNA seq analysis (TheragenEtex, Korea) through FPKM (expected fragments per kilobase of transcript per million fragments sequenced) to determine expression of PFN1 mRNA A graph of the change is shown in Fig.

As a result, as shown in Fig. 10, the FPKM value was used to determine how the expression of PFN1 changed in each experimental group. FPKM is a unit for measuring the change of mRNA expression level by RNA seq analysis. It can be confirmed that the FPKM of PFN1 is reduced about 1.4 times in the UVB-irradiated group (UVB only) compared to the control group. CP (CL or CH) The amount of mRNA decreased.

(3) Analysis of PFN1 protein expression level

In order to confirm whether the expression of PFN1 protein is decreased by the decrease of expression of PFN1 mRNA by UV, the following experiment was carried out.

Specifically, the mouse tissue cells prepared by the method of 1-1 (1) were incubated with a protease inhibitor cocktail kit (P3100-005; GenDEPOT, Barker, TX) and phosphatase inhibitor (sc-45065; Santa Cruz Biotechnology, Santa Cruz , CA) (# 9803; Cell Signaling Technology, Danvers, Mass.). Partial samples containing 25 μg of cell lysate were separated by electrophoresis on SDS-polyacrylamide gel, then transferred to a buffer (25 mM Tris, 192 mM glycine, 20% [v / v] methanol [pH 8.3]) And transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK) at 75 V and 40 캜 for 180 minutes. The membranes were blocked with 3% (w / v) BSA in PBS containing 0.1% (v / v) Tween-20 for 60 min at room temperature and then incubated with anti-PFN1 (Abcam, ab118984) antibody. HRP-conjugated goat anti-mouse IgG (1: 1000; sc 2005, Santa Cruz) was used for the secondary antibody. The blots were developed using ECL lumi Femto solution A / B (Dogen, Seoul, Korea).

As a result, as shown in FIG. 9, it was confirmed that the expression level of PFN1 protein was reduced about 3 times as compared with that when UVB was not irradiated when the photoaging was induced by UVB irradiation on the mouse. However, when cucumber powder (CP), which is known to inhibit the effect of skin aging, was treated with the UVB-irradiated mouse, it was confirmed that the expression level of PFN1 protein was increased again in comparison with UBV alone.

2-2. In vitro PFN1  Analysis of gene expression level

(1) Production of human skin photoaging cell model

Immortalized human keratinocyte HaCaT cells (ATCC, USA) 5 x 105 cells were plated in a 100-mm dish containing DMEM (5% FBS) medium and cultured in a 37 ° C CO ₂ incubator for 48 hours . The cells are then removed from the incubator and replaced with serum free media. After 12 hours, wash with PBS, add 4 mL of PBS to the dish, and apply 100 mJ / cm2 of UV using a UV crosslinker (UVP, CA, USA). Dish PBS is removed, and 8 mL of DMEM (5% FBS) medium is filled and cultured for 24 hours to prepare a skin photoaging cell model. After UV irradiation, cacao powder (4 ug / mL) and cacao metabolite DHPV [5- (3 ', 4'-19 dihydroxyphenyl) -gamma-calerolactone] (10 ng / mL) ) For 24 hours. DHPV is a positive control for cacao powder. Cacao powder can be a combination of various substances. When cacao powder is ingested or treated, cacao powder changes its physical properties by metabolism in vivo, The cacao powder effect can be exhibited by the physical properties. Since HaCaT can not simulate the metabolism occurring in vivo, the treatment of DHPV, which is a product metabolized in vivo, can be a more accurate way to see the effect of cacao powder, and the metabolite of cacao powder Which can be used to make accurate judgments about which substances exhibit anti-aging effects. The control cells were not subjected to UV irradiation and the DHPV treatment on the cells.

(2) Analysis of PFN1 protein expression level in human skin photoaging cells

In order to analyze changes in the expression of PFN1 protein in the above-prepared photoinduced human skin cells, experiments were conducted as follows.

Specifically, immunoblot analysis was performed on the human skin photoautotated cells prepared in Example 2-2 (1). Cells were resolved using a lysis buffer containing a protease inhibitor cocktail kit (P3100-005; GenDEPOT, Barker, TX) and a phosphatase inhibitor (sc-45065; Santa Cruz Biotechnology, Santa Cruz, CA) (# 9803; Cell Signaling Technology, Danvers, Mass.). Partial samples containing 20 ug of cell lysate were separated by electrophoresis on SDS-polyacrylamide gel, then transferred to a buffer (25 mM Tris, 192 mM glycine, 20% [v / v] methanol [pH 8.3]) And transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK) at 75 V and 40 캜 for 180 minutes. The membranes were blocked with 3% (w / v) BSA in PBS containing 0.1% (v / v) Tween-20 at room temperature for 60 minutes and then incubated with anti-PFN1 (Abcam, ab118984) antibody. Secondary antibodies were HRP-conjugated goat anti-mouse IgG (1: 1000; sc 2005, Santa Cruz). The blots were developed and analyzed using ECL lumi Femto solution A / B (Dogen, Seoul, Korea).

As a result, as shown in FIG. 10, it was confirmed that the expression amount of PFN1 protein was reduced by about 80% when UVB treatment was applied to human skin cells when photoaging was induced. However, when the amount of the protein of PFN1, which was reduced by treatment with only UVB as described above, was treated with DHPV, a cacao metabolite known to inhibit the effect of skin aging on skin cells treated with UVB, Was recovered by the amount of PFN1 protein in the control without treatment.

Therefore, it was confirmed from the above results that the expression of PFN1 protein selectively decreased by UVB.

Example  3. PFN1  Position and level analysis of promoter methylation

In general, it is known that the expression of the gene is decreased by methylation of the promoter of the gene. Therefore, based on the result that the expression of PFN1 is decreased by the photoaging by UVB irradiation, the methylation of the promoter of PFN1 by UVB irradiation Or more. In order to confirm whether the increase of promoter methylation of PFN1 by irradiation of UVB was increased, methylation specific PCR was performed as follows in order to confirm methylation by comparing the amount of methylated DNA based on non-methylated DNA.

Specifically, human photoaged skin cells prepared in Example 2-2 (1) were harvested and DNA was extracted using a NucleoSin® Tissue Kit (Macherey-Nagel, Germany). The extracted DNA was quantified using Nano Drop and its quality was confirmed. For methylation analysis, unmethylated cytosine residues were converted to Uracil residues by Bisulfite treatment using EZ DNA Methylation-Lightning ™ Kit (Zymoresearch, CA, USA) for methylation analysis.

100 ng of the above bisulfite DNA, 1 uL of each primer pair (20 pmoles), 0.25 uL of TaKaRa EpiTaq HS (5 U / μl), 5 uL of 10X EpiTaq PCR Buffer (Mg2 + free), 5 uL of 25 mM MgCl2, ), 6 uL and final volume of 50 uL. After reacting at 98 ° C for 3 minutes, the reaction was repeated at 40 ° C for 10 seconds, at 55 ° C for 1 minute, and at 72 ° C for 30 seconds. I got it. Then, the cells were elongated at 72 ° C for 5 minutes, and the results were analyzed on a 2% (w / v) agarose gel.

Generally, when preparing primers for methylation specific PCR, at least 2 to 3 CpG sequences should be present in the primer, and the Tm values should be similarly prepared for methylation and non-methylation primers. In the bisulfite conversion process, the unmethylated cytosine is transformed into uracil. Therefore, a primer should be prepared. Under these conditions, the base sequence between methylated and unmethylated primers may differ by about 1 to 3 bp. In addition, the methylation of the gene may be 0-100% of the entire gene pool, and it is possible to accurately grasp the DNA methylation by grasping the amount of the methylated gene versus unmethylated in the same gene sequence which can be expressed there.

In consideration of the above, a primer for carrying out methylation specific PCR was prepared, and the sequence information thereof is shown in Table 2. The region from -1239 to -998 from the start codon of SEQ ID NO: 1 is the portion of methylation specific PCR, and the region of -1241 to -997 from ATG is the corresponding portion of unmethylation specific PCR.

Methylation specific PCR was carried out using the primers 8 and 9 shown in Table 2 for the sequences corresponding to 81 to 262 in the promoter of SEQ ID NO: 1, and the methylated region in the promoter of PFN1 was cleaved in the promoter of SEQ ID NO: 262, respectively.

Further, unmethylation specific PCR was performed using primers 10 and 11 shown in Table 2 in cells treated with UVB and treated with CP and DHPV to restore skin senescence, and the degree of methylation generated in the promoter was confirmed. The primer sequences and PCR conditions used in DNA methylation specific PCR are shown in Table 2 below.

denomination The sequence (5 '> 3') SEQ ID NO: Tm (占 폚) Cycle MF1 TGC GAA GAA ATT TAA AGG GTT C 8 54.6 40 MR1 ACA ATA TCT AAA TCA CCG ACC GTA 9 56.6 40 UF1 GAT TGT GAA GAA ATT TAA AGG GTT T 10 54 40 UR1 AAC AAT ATC TAA ATC ACC AAC CATE 11 53.5 40

As a result, as shown in FIG. 11, when primers corresponding to SEQ ID NOs: 8 and 9 were used in skin cells induced by UVB irradiation (Promoter 1), primers corresponding to SEQ ID NOs: 10 and 11 were used (M) in the PFN1 promoter1 was increased about 3-fold by UVB, and this methylation was drastically reduced by DHPV treatment.

Thus, the above results indicate that the methylation level of the PFN1 promoter sequence corresponding to 81-262 of SEQ ID NO: 1 is increased in the aging model by UVB irradiation, and that methylation of the PFN1 promoter can be used as a skin aging-specific marker.

<110> SUNGKWANG MEDICAL FOUNDATION          College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Skin aging-specific Epigenetic gene markers and detection of skin          aging using them <130> DPP20164868KR <160> 11 <170> KoPatentin 3.0 <210> 1 <211> 1270 <212> DNA <213> Homo sapiens <400> 1 cgccgcggc gctgtgagcg cacatctgac tgcgaagaaa cccaaagggc ccgaagtggg actttcccct 120 tcttgaaaaa gttgccactc ttgcaacttg agtattcttc cgcctcgaaa aatagccccg 180 cctgttaagc cccgcgggtg tccgcgctct gtgacctctg cagacacagg ccaaggggca 240 cggccggtga tccagacatt gcctgcctgg ggcgggcggg gctgggatgt ggggtgaggc 300 cctgggctag agccgatcca gagaagggac aatcctctag actgagaatg ggccctggtg 360 aacccaacct gcacgacccg agaagacgct ggggacagaa gtggccccgg tgggtgcctt 420 cagctatgag agcgactaga ggatttggcc aaactcttct ttgctttctt attacttgaa 480 taagtaaaat caagggtggt gaggcggcag cgcgcacggc ttcgcccccg ccccttccgc 540 caggcgggag acacgtcccc agcccgcctt cctttcccgc agggtccaca cgggtcgggc 600 cgggcgcgct cccgtgcagc cggctccggc cccgaccgcc ccatgcactc ccggccccgg 660 cgcaggcgca ggcgcgggca cacgcgccgc cgcccgccgg tccttccctt cggcggaggt 720 gggggaagga ggagtcatcc cgtttaaccc tgggctcccc gaactctcct taatttgcta 780 aatttgcagc ttgctaattc ctcctgcttt ctccttcctt ccttcttctg gctcactccc 840 tgccccgata ccaaagtctg gtttatattc agtgcaaatt ggagcaaacc ctacccttca 900 cctctctccc gccacccccc atccttctgc attgctttcc atcgaactct gcaaattttg 960 caataggggg agggattttt aaaattgcat ttgcaaagtt cggtgtctgg gctggcgagt 1020 gggggaggga gggaatgggg agtaggcccc gcccctaccg tcctttgcaa ataaaaatct 1080 agcggggcgg ggggggggag gagcaggaag tggcggtgcg agggctgctg cacagcgagc 1140 ggagccgcgg tccggacggc agcgcgtgcc ccgagctctc cgcctccccc cgcccgccag 1200 ccgaggcagc tcgagcccag tccgcggccc cagcagcagc gccgagagca gccccagtag 1260 cagcgccatg 1270 <210> 2 <211> 4610 <212> DNA <213> Homo sapiens <400> 2 cgccgcggc gctgtgagcg cacatctgac tgcgaagaaa cccaaagggc ccgaagtggg actttcccct 120 tcttgaaaaa gttgccactc ttgcaacttg agtattcttc cgcctcgaaa aatagccccg 180 cctgttaagc cccgcgggtg tccgcgctct gtgacctctg cagacacagg ccaaggggca 240 cggccggtga tccagacatt gcctgcctgg ggcgggcggg gctgggatgt ggggtgaggc 300 cctgggctag agccgatcca gagaagggac aatcctctag actgagaatg ggccctggtg 360 aacccaacct gcacgacccg agaagacgct ggggacagaa gtggccccgg tgggtgcctt 420 cagctatgag agcgactaga ggatttggcc aaactcttct ttgctttctt attacttgaa 480 taagtaaaat caagggtggt gaggcggcag cgcgcacggc ttcgcccccg ccccttccgc 540 caggcgggag acacgtcccc agcccgcctt cctttcccgc agggtccaca cgggtcgggc 600 cgggcgcgct cccgtgcagc cggctccggc cccgaccgcc ccatgcactc ccggccccgg 660 cgcaggcgca ggcgcgggca cacgcgccgc cgcccgccgg tccttccctt cggcggaggt 720 gggggaagga ggagtcatcc cgtttaaccc tgggctcccc gaactctcct taatttgcta 780 aatttgcagc ttgctaattc ctcctgcttt ctccttcctt ccttcttctg gctcactccc 840 tgccccgata ccaaagtctg gtttatattc agtgcaaatt ggagcaaacc ctacccttca 900 cctctctccc gccacccccc atccttctgc attgctttcc atcgaactct gcaaattttg 960 caataggggg agggattttt aaaattgcat ttgcaaagtt cggtgtctgg gctggcgagt 1020 gggggaggga gggaatgggg agtaggcccc gcccctaccg tcctttgcaa ataaaaatct 1080 agcggggcgg ggggggggag gagcaggaag tggcggtgcg agggctgctg cacagcgagc 1140 ggagccgcgg tccggacggc agcgcgtgcc ccgagctctc cgcctccccc cgcccgccag 1200 ccgaggcagc tcgagcccag tccgcggccc cagcagcagc gccgagagca gccccagtag 1260 cgcgccatg gccgggtgga acgcctacat cgacaacctc atggcggacg ggacctgtca 1320 ggacgcggcc atcgtgggct acaaggactc gccctccgtc tgggccgccg tccccgggaa 1380 aacgttcgtc aacatcacgg tactgcgagg cctgcgcggt ccggggcact gctccggttt 1440 ggaccctgag ggagggactt gggtgggggc gggcgcggtc tagggcgcgc agcgagaggc 1500 cctgactggg tgcttgccct ggaagcggcg cgaatggcgg ctgcacgtga gtgggtccct 1560 cagttgcccg taggtcctat gccccaggcg gcggcgccca tcccgcccgc taccccgctt 1620 tccgcgacgg ggcgttacat ccggggcaca gggcggggag gggcgcggcg cccggtgcgg 1680 cggcccactt ccgcctctcc cagggcgggg cggggacgcg cctcagtttt tactggggat 1740 tgggattgag aggggacccg gggatccttg gacccccagc tccccgtccg gctgccagcc 1800 cgcgtccctc taccattgtt tctcctggtg cggaagggag ccccaggcgc gtggtcttgg 1860 tcccttgccc accctctgtg ggcttctggc ccctcctaac tccgttagag agggcgaggc 1920 caccacacgc ccccccagct cagcacgttg gattcaacat tcctgcgagg ccaatcccgg 1980 aagtcccctc gccctccctt cccgccatcc ggcggggcct ggagcagggg aactttgtga 2040 gaagttctga gacagcataa gcgaaagtat attggtttac taccgctgct cccaccacca 2100 tctccctgtc cacactgccc ggaagtgggg agggggagcg gaagttcaaa agggggggga 2160 agggggtttc cgggcgggag gggaccggat gtgggggctt ggggtgaaag ggggaggggg 2220 gaaaggttcc cggggtacct agacccgaga gctggcctgc cgggtctggc ccctgtccac 2280 cttcaaaagt tttccaactt tcccggaaag cccccagggg tctgcttcct catctcagag 2340 cagagatacc tggagctaaa ctctaggtta cttggagacc caggcgtccg ggccctaaca 2400 gctcctgcag aacctttgac caaataaggg caaagttgct tctcttgcct cgcccgcccc 2460 cctcccttcc cctctccgct tctgcttttc ctgaaggaga gttctgagca tccaaaggct 2520 cctcacttac ctcactgact ccctttattt cctgggttta ctttccccat taacactgac 2580 atctaaatct ctagaggagt atgtcatccc ttcctccctg gttccatatt ctaagtttca 2640 gaatcatgat ctttttctgc actaacgtcc cacagtcctc agagtttctc acagttcatt 2700 gatgtgtact gagtatataa tttatactta gcccccacgg cggggtggtt ctcagaagtg 2760 gaagacaggt gggtctttgg agaacacggt gggaatcttg gtgcactgac taacttgatg 2820 ggcgcttggt tcctcttcca gccagctgag gtgggtgtcc tggttggcaa agaccggtca 2880 agtttttacg tgaatgggct gacacttggg ggccagaaat gttcggtgat ccgggactca 2940 ctgctgcagg atggggaatt tagcatggat cttcgtacca agagcaccgg tggggccccc 3000 accttcaatg tcactgtcac caagactgac aagagtgagt tcctgagtct tacccttcct 3060 ttagctccaa gatctctaat gtaccgcctt ctggtaatct tgagggtgct tttttgtgtg 3120 attccccaac atgaagggag tcagaccagt attgtgttcc ttgtctgctg gatgctggaa 3180 cacgggatag tccctccatt ctgtctcagt tcctcatctt gtaattcgaa gttgctgaac 3240 tgcattacct cttaaattac ttactgcctt gagctcggat tagaaactgt tttctaggag 3300 tataggtaga ttgttggggt cttgaatcat cgagttcatc gtttgagttc ttttgttaga 3360 acctggaggt ggcatgcaca tccacagccc tttctagcct gaacacatga atcaacagtc 3420 tatgagcctt tgacccctaa gtttttagtc tgttcctctc cagcgtttaa gcttgatttt 3480 cctcttttga aaagccttat tgtttcaggg tgactgacag cctgcctgtg tgtggtgggg 3540 gggcgggttg ggagagatga ggttgggtta cagcccccag ggctggacag ctgctgaaca 3600 gctggcaggg ggtggaattg tgacacctgg atcctagcct cctttctctt tcctcctcct 3660 ccagcgctag tcctgctgat gggcaaagaa ggtgtccacg gtggtttgat caacaagaaa 3720 tgttatgaaa tggcctccca ccttcggcgt tcccagtact gacctcgtct gtcccttccc 3780 cttcaccgct ccccacagct ttgcacccct ttcctcccca tacacacaca aaccatttta 3840 ttttttgggc cattacccca taccccttat tgctgccaaa accacatggg ctgggggcca 3900 gggctggatg gacagacacc tccccctacc catatccctc ccgtgtgtgg ttggaaaact 3960 tttgtttttt ggggtttttt ttttctgaat aaaaaagatt ctactaacaa ggtgctgtgt 4020 gatttgagct tgggtgtcag aatgggggat aggcactgtg acttgcctcc tttatcgtgg 4080 cttgggagca gggaggttga ggtactacaa ggtgcaggaa tttgagcttc tggccaggtg 4140 gccctgcccc tccacagtgg catttccagg cctaagtcct cagtgtgctg gtcaaattgg 4200 tggatgatgg aatagagcca tctggaccag agaatgggtg caggcctgca ggcaaggcag 4260 ggctagttga gtcatccttg tgggtggggc tcccccttct gagtcatagc aaatgccggc 4320 tggggctgtc cccatgacta taggttagtg tctctcagca ttcccttgct ccttatggct 4380 aaaggggtga ttccaccaaa gggttaagtt cacctgtgtg agaatgatct tacccttcct 4440 tagtccacaa agaaacaatt ttattagccc aagaagtgaa gacccagtcc ctctgcttgc 4500 cccagctcca ccccaaagcc cctcaaaagg ataggtgtgg gtaagggaga agcttgggat 4560 ggaatgtccc ctatccctca gaagactgga gggaagacga cggtctgtgc 4610 <210> 3 <211> 140 <212> PRT <213> Homo sapiens <400> 3 Met Ala Gly Trp Asn Ala Tyr Ile Asp Asn Leu Met Ala Asp Gly Thr   1 5 10 15 Cys Gln Asp Ala Ile Val Gly Tyr Lys Asp Ser Pro Ser Val Trp              20 25 30 Ala Ala Val Pro Gly Lys Thr Phe Val Asn Ile Thr Pro Ala Glu Val          35 40 45 Gly Val Leu Val Gly Lys Asp Arg Ser Ser Phe Tyr Val Asn Gly Leu      50 55 60 Thr Leu Gly Gly Gln Lys Cys Ser Val Ile Arg Asp Ser Leu Leu Gln  65 70 75 80 Asp Gly Glu Phe Ser Met Asp Leu Arg Thr Lys Ser Thr Gly Gly Ala                  85 90 95 Pro Thr Phe Asn Val Thr Val Thr Lys Thr Asp Lys Thr Leu Val Leu             100 105 110 Leu Met Gly Lys Glu Gly Val His Gly Gly Leu Ile Asn Lys Lys Cys         115 120 125 Tyr Glu Met Ala Ser His Leu Arg Arg Ser Ser Gln Tyr     130 135 140 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mPFN1_F <400> 4 gatgggcaaa caaggtgtc 19 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mPFN1_R <400> 5 atgtgtgtgt atggaaagaa gg 22 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mRPLPO_F <400> 6 caaaggaaga gtcggagga 19 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mRPLPO_R <400> 7 caaagatgcc aacacaaagr aa 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MF1 <400> 8 tgcgaagaaa tttaaagggt tc 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MR1 <400> 9 acaatatcta aatcaccgac cgta 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UF1 <400> 10 gattgtgaag aaatttaaag ggttt 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UR1 <400> 11 aacaatatct aaatcaccaa ccata 25

Claims (23)

A skin aging-specific biomarker comprising a PFN1 gene, a protein encoded by the gene, or a methylation promoter of a PFN1 gene. 2. The biomarker of claim 1, wherein the PFN1 gene comprises a polynucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 3. 2. The biomarker of claim 1, wherein the PFN1 gene comprises a polynucleotide sequence encoded by the nucleotide sequence of SEQ ID NO: 2. 2. The biomarker of claim 1, wherein the methylation promoter is methylated at a CpG island present in nucleotides 81 to 262 in the polynucleotide sequence of SEQ ID NO: 1. A composition for measuring the expression level of the PFN1 gene or an agent for measuring the methylation of the PFN1 gene promoter. 6. The composition of claim 5, wherein the aging of the skin is aging of the skin, or aging of the skin by UV. 6. The composition of claim 5, wherein the aging of the skin is aging of the skin, or aging of the skin by UV. 6. The composition according to claim 5, wherein the agent for measuring the expression level of the PFN1 gene comprises a primer, a probe, an extramammer or an antisense oligonucleotide that specifically binds to the PFN1 gene. 6. The method according to claim 5, wherein the agent for measuring the expression level of the PFN1 gene comprises at least one selected from the group consisting of small molecule chemicals, proteins, peptides, nucleic acid molecules, and antibodies that specifically bind to the PFN1 protein &Lt; / RTI &gt; 6. The method according to claim 5, wherein the detection of the agent for measuring the expression level of the PFN1 gene is carried out by reverse transcriptase polymerase chain reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, Lt; RTI ID = 0.0 &gt; PFN1 &lt; / RTI &gt; 6. The composition according to claim 5, wherein the detection of the agent for measuring the level of expression of the PFN1 gene is performed by means of Western blotting, ELISA or protein determination of PFNl, which is a mass spectrometry. 6. The composition of claim 5, wherein the agent for measuring methylation of the promoter is a PCR primer pair for amplifying SEQ ID NO: 1 comprising a methylated CpG island of a methylation promoter of a PFN1 gene. 6. The method according to claim 5, wherein the detection of the agent for measuring methylation of the PFN1 gene promoter is carried out using PCR, methylation specific PCR, real time methylation specific PCR, methylated DNA- PCR, quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing. A method for providing information necessary for diagnosing a skin aging or skin aging process step comprising the step of measuring an expression level of a PFN1 gene. 15. The method of claim 14, wherein the expression level of the gene is measured by mRNA, or protein. 16. The method of claim 15, wherein the level of expression of the mRNA is measured by reverse transcriptase polymerase chain reaction, nucleic acid hybridization, electrophoresis, or Northern blotting. 16. The method according to claim 15, wherein the expression level of the mRNA gene is measured by obtaining RNA from a sample, reverse transcribing the RNA to obtain cDNA, attaching a detectable label to the cDNA, and performing a polymerase chain reaction How it is. 18. The method of claim 17, wherein the detectable label is an enzyme, a radioactive isotope, a fluorescent substance, a chemiluminescent molecule, a radiation substance, a liposome, or a hapten molecule. 16. The method of claim 15, wherein the expression level of the protein is measured by immunoassay, Western blot, ELISA, or mass spectrometry. A method for providing information necessary for diagnosis of skin aging or skin aging progression stage comprising measuring methylation of a methylation promoter of PFN1 gene. 21. The method of claim 20, wherein the methylation promoter is methylated at a nucleotide of a promoter sequence corresponding to 81-262 in the polynucleotide sequence of SEQ ID NO: 1. The method according to claim 20, wherein the measurement of the methylation is performed using PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, &Lt; RTI ID = 0.0 &gt; pyrosequencing &lt; / RTI &gt; and bisulfite sequencing. The method according to claim 20, wherein the step of measuring the methylation is performed by using a PCR primer for amplifying SEQ ID NO: 1 comprising the methylated CpG island of the methylation promoter of the PFN1 gene, How, one.

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