KR20180044617A - Biomarker composition for diagnosing cell senescence comprising UHRF1 and DNMT1 - Google Patents

Abstract

The present invention relates to a cell aging diagnosing biomarker composition comprising UHRF1 and DNMT1. Specifically, the present invention relates to a biomarker composition for diagnosing cell aging, a kit for diagnosing cell aging using the same, and a method for diagnosing cell aging, wherein the biomarker composition comprises: a UHRF1 gene or a protein encoded with the UHRF1 gene; and a DNMT1 gene or a protein encoded with the DNMT1 gene. In addition, the present invention provides a pharmaceutical composition for preventing or treating cell aging, wherein the pharmaceutical composition comprises a UHRF1 protein and a DNMT1 protein, or an expression promoter or activator of the UHRF1 protein and DNMT1 protein as an active ingredient; and a method for screening a cell aging therapeutic agent using the UHRF1 protein and DNMT1 protein. Therefore, the UHRF1 or DNMT1 of the present invention can be useful in diagnosing and treating cell aging.

Description

Biomarker composition for diagnosing cell senescence comprising UHRF1 and DNMT1 [

The present invention relates to a biomarker composition for diagnosing cellular senescence comprising a PHD-containing ubiquitin-like and PHM and Ring Finger Domains 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) .

Cell senescence is a cellular fate that gradually loses its ability to replicate in mitotic cells and eventually stops cell division. Cell senescence has also been reported for various aging-related diseases, including cancer, Alzheimer's disease and cardiovascular disease, as well as the cause of aging itself. In addition, senescent cells have been shown to have an expanded and flattened cell shape with low elasticity, increased reactive oxygen species (ROS) generation, senescence-associated beta-galactosidase (SA-beta -gal) activity and senescence-associated secretory phenotypes (SASP).

In eukaryotic cells, DNA methylation is an important welfare genetic modification that regulates gene expression and silencing. This often occurs at the C5 position of cytosine, typically among CpG islands rich in gene promoter sites. CpG island-hypermethylation is frequently associated with gene silencing, while hypo-methylation is associated with gene transcription activation. The DNA methylation pattern can vary depending on the life span of an organism and can often change to adapt to environmental conditions. Aging tissue exhibits loss of DNA methylation throughout the entire genome (promoter, intergenic region, intron region and exon region). However, over-methylation in a variety of tumor suppressor genes and Polycomb target genes has also been reported to be associated with senescence. This indicates that excessive DNA hypomethylation is closely related to the induction of large expression of various genes that accelerate the aging process. Correspondingly, the reversibility of hypomethylation may be essential to the viability of the organism.

Humans exhibit two types of DNA methylation activity produced by DNA methyltransferases (DNMTs): newly synthesized ( de novo ) methylation and maintenance methylation. The newly synthesized ( de novo ) methylation produced by DNMT3a and DNMT3b forms an early DNA methylation pattern during the embryogenesis process. On the other hand, retention methylation restores and preserves the DNA methylation pattern after each cell DNA replication cycle. DNMT1 replicates DNA methylation patterns in daughter strands during DNA replication, which may be associated with senescence-related welfare genetic alterations.

Although DNMT1 has important enzymatic functional groups for maintenance methylation, DNMT1 activity is sensitively controlled by physical interactions with various proteins and post-translational modifications. Binding of PCNA to DNMT1 increases methylation activity, while binding of UHRF1 to DNMT1 promotes recognition of hemi-methylated DNA. DNMT1 is acetylated by KAT5, deacetylated by HDAC1, phosphorylated by casein kinase 1δ / ε and AKT1, and methylated by SETD7. In DNMT1, the overall integration of the DNMT1-interacting proteins (DIPs) contributes to maintaining methylation activity and related cell phenotypes. However, it is still unclear how DNMT1 and its interacting proteins are involved in aging-associated gene reprogramming.

U.S. Published patent application US2013-0324479 (December 31, 2013)

It is an object of the present invention to provide a biomarker composition for diagnosing cell senescence including UHRF1 and DNMT1 and a method for diagnosing cell senescence using the same.

The present invention relates to a UHRF1 gene or a protein encoded by the UHRF1 gene; And a biomarker composition for diagnosing cellular senescence comprising a DNMT1 gene or a protein encoded by the DNMT1 gene.

The present invention also includes a primer or a probe specifically binding to the UHRF1 gene and the DNMT1 gene, an antibody specifically binding to the UHRF1 gene and a DNMT1 gene-encoded protein, or a peptide having a binding domain specific to the protein The present invention provides a kit for diagnosing cell aging.

The present invention also provides a method for providing information necessary for diagnosis of cell senescence comprising the steps of measuring mRNA expression levels of UHRF1 gene and DNMT1 gene or expression levels of proteins encoded by UHRF1 gene and DNMT1 gene from separated samples to provide.

The present invention also relates to UHRF1 protein and DNMT1 protein; Or an expression promoter or activator of the UHRF1 protein and the DNMT1 protein as an active ingredient.

In addition, the present invention provides a screening method for cell senescence comprising the step of measuring the expression or activity level of the UHRF1 protein and the DNMT1 protein in the isolated senescent cell line.

The present invention relates to a biomarker composition for diagnosing cell senescence comprising UHRF1 and DNMT1, and more particularly to a biomarker composition for UHRF1 gene or a protein encoded by the UHRF1 gene; And a DNMT1 gene or a protein encoded by the DNMT1 gene, a cell aging diagnostic kit and a cell aging diagnostic method using the biomarker composition. The present invention also relates to UHRF1 protein and DNMT1 protein; Or an expression promoter or activator of the UHRF1 protein and the DNMT1 protein as an active ingredient, and a method for screening a therapeutic agent for cell senescence using the UHRF1 protein and the DNMT1 protein. Therefore, UHRF1 or DNMT1 of the present invention can be usefully used for diagnosis and treatment of cell senescence.

Figure 1 shows that a decrease in DNMT1-mediated DNA methylation activity is associated with aging of HDF. (A) hydrogen peroxide (H ₂ O ₂ ) -induced aging (HS) in primary HDF. (a) Western blot analysis results for DNMT1 and DNMT3A (left) and their quantitative results (right). (b) by messenger RNA (qRT-PCR) and representative image (c) for SA-β-gal analysis. ** p < control primary HDF by Student t test. (B) HDFs of DT2 (black bars) and DT14 (white bars) were used, indicating replica aging (RS) progressing in primary HDF. (a) Western blot analysis results (left) and their quantitative results (right). (b) and SA-beta-gal analysis (c) by qRT-PCR. The right panel of (c) shows representative images of SA-β-gal analysis. ** p < DT2 by Student t test. (C) HDF (DT2) was exposed to the indicated concentrations of 5-aza-2'-deoxycytidine (5-AzC, Sigma, St. Louis, MO) for 5 days. (a) Representative images (upper panel) and quantification results (lower panel) for SA- [beta] -gal analysis. (b) Western blot analysis results. (D) HDF (DT2) was transfected with siRNA against DNMT1 for 5 days. (a) Representative images (upper panel) and quantification results (lower panel) for SA- [beta] -gal analysis. (b) Western blot analysis results. ** p < siRNA for negative control or control by Student t test.
Figure 2 shows the expression of DNMTl-interacting proteins that are commonly regulated in both RS and HS of HDF. (A) Time-continuous HDFs from HS models were applied to cDNA microarrays. Time-continuous gene expression profile. (B) Upwardly regulated genes (HS_UP, 310 genes) and downregulated genes (HS_DOWN, 404 genes) correspond to four different genes (G1 to G4) identified from the previously reported time series RS model Respectively. (C) The enrichment score was expressed as log10-transformed P values, calculated from the Gene set enrichment analysis. (D) The Venn diagram showed the number of overlapping genes in the DIPs, RS, and HS models. Seven genes were found to be commonly regulated in two HDF aging models. Shows a heat map of a time-sequential gene expression profile of 7 DIPs (upper panel) and SA-beta-gal analysis (lower panel) in two HDF aging models. Protein expression levels of (E) 7 DIPs were verified by western blot analysis.
Figure 3 shows that UHRF1 is an upstream regulator of DNMTl expression. (AC) HDF (DT2) was transfected with siRNAs for the indicated target for 3 days. (A) Western blot analysis results. (B) Western blot analysis results (a) and their quantitative results (b). (C) indicates the level of messenger RNA by qRT-PCR. ** p < siNC. (D) HDF (DT7) was infected with a recombinant retrovirus (rRV) with the indicated target cDNA for 3 days. The messenger RNA level (a) and Western blot analysis (b) by qRT-PCR are shown. ** p < RFP by Student t test.
Figure 4 shows that UHRF1 knockdown effectively induces senescence through inhibition of DNMT1. (AC) HDF (DT2) was transfected with siRNAs for the indicated target for 5 days. (A) Quantitative results (upper panel) and representative image (lower panel) of SA-β-gal analysis. (b) The knockdown level of the targets was confirmed by Western blot analysis. ** p < siNC by Student t test. (B) Western analysis results. (C) Intracellular ROS levels were observed by flow cytometry after staining cells with DCF-DA fluorescent dye. (D, E) HDF (DT2) was transfected with siRNAs for the indicated target and then infected for 4 days with rRV with the indicated cDNA of the next day. (D) Cell growth rate (a) and number of intracellular ROS levels (b) using DCF-DA fluorescent dye. ** p < siNC / GFP and ## p &lt; siUHRF1 / GFP. (E) Western blot analysis results.

Thus, the present inventors have found that the time-continuous gene expression profile of 53 known DIPs in two different cell senescence model systems (replicative senescence (RS) and hydrogen peroxide (H ₂ O ₂ ) -induced senescence (HS) of human diploid fibroblasts) Respectively. In addition, we have ascertained how the regulation of DIPs is involved in DNMT1 expression and DNMT1-associated senescence, and have completed the present invention.

The present invention relates to a UHRF1 gene or a protein encoded by the UHRF1 gene; And a biomarker composition for diagnosing cellular senescence comprising a DNMT1 gene or a protein encoded by the DNMT1 gene.

The term &quot; diagnosing &quot; herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).

The present invention also includes a primer or a probe specifically binding to the UHRF1 gene and the DNMT1 gene, an antibody specifically binding to the UHRF1 gene and a DNMT1 gene-encoded protein, or a peptide having a binding domain specific to the protein The present invention provides a kit for diagnosing cell aging.

The term "primer" refers to a short nucleic acid sequence capable of forming a base pair with a template complementary to a nucleic acid sequence having a short free 3 'hydroxyl group and acting as a starting point for template strand replication . Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

The term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or several hundreds of nucleotides that can specifically bind to an mRNA, and is labeled to confirm the presence or expression level of a specific mRNA . The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, the term "antibody" means a specific immunoglobulin as indicated in the art and directed against an antigenic site. Any of those prepared through the above-mentioned one or more protein injections or commercially available can be used. In addition, the antibody includes a polyclonal antibody, a monoclonal antibody, and a fragment capable of binding to an epitope. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.

The term "peptide" refers to a polypeptide that does not have the structure of the intact antibody, but has a specific antigen binding site (binding domain) directed against the antigenic site. The peptide comprises a functional fragment of an antibody molecule that is not a complete form of the antibody having two light and two heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function.

(1) measuring the mRNA expression level of the UHRF1 gene and the DNMT1 gene or the expression level of the protein encoded by the UHRF1 gene and the DNMT1 gene from the separated sample; (2) comparing mRNA expression levels of the UHRF1 and DNMT1 genes or expression levels of the UHRF1 and DNMT1 gene-encoded proteins with a control sample; And (3) determining the cell senescence if the mRNA expression level of the UHRF1 gene and the DNMT1 gene or the expression level of the protein encoded by the UHRF1 gene and the DNMT1 gene is lower than that of the control sample. Provide a method to provide.

In detail, the method for measuring the mRNA expression level may be RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) ), Northern blotting and DNA chips, but are not limited thereto.

In detail, the method for measuring the protein expression level may be Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, But are not limited to, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, Complement Fixation Assays, FACS and protein chips.

As used herein, the term &quot; sample &quot; refers to a biological marker such as a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine which is different from the control group in the expression level of the UHRF1 gene and the DNMT1 gene But are not limited to, samples.

The present invention also relates to UHRF1 protein and DNMT1 protein; Or an expression promoter or activator of the UHRF1 protein and the DNMT1 protein as an active ingredient.

Preferably, the expression promoter or activator of the UHRF1 protein and the DNMT1 protein may be a compound, a peptide, a peptide mimetic, an aptamer, an antibody or a natural substance that specifically binds to the UHRF1 protein and the DNMT1 protein, It is not.

In the present invention, the term "Aptamer" is 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. Aptamers are comparable to monoclonal antibodies due to their inherent high affinity (usually pM levels) and their ability to bind to target molecules with specificity, and there is a high likelihood of being an alternative antibody, especially as a "chemoantibody".

In the present invention, the term "antibody" is either manufactured through UHRF1 or DNMT1 injection or commercially available. In addition, the antibody includes a polyclonal antibody, a monoclonal antibody, and a fragment capable of binding to an epitope.

Polyclonal antibodies can be produced by conventional methods of injecting the above UHRF1 and DNMT1 into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc. Monoclonal antibodies Can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology.

The pharmaceutical composition of the present invention may contain a chemical substance, a nucleotide, an antisense, an siRNA oligonucleotide and a natural product extract as an active ingredient. The pharmaceutical composition or combination preparation of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a flavoring agent may be used. The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile solutions suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Pharmaceutical dosage forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions . The pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, &Lt; / RTI &gt; The effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, the inhibitor of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg when the compound is administered once to several times a day, a polypeptide, In the case of protein or antibody, 0.1 ng / kg to 10 g / kg, antisense nucleotide, siRNA, shRNAi or miRNA can be administered at a dose of 0.01 ng / kg to 10 g / kg.

The present invention also relates to a method for producing a test cell, comprising the steps of (1) contacting a test substance to an isolated aging cell line; (2) measuring the level of expression or activity of UHRF1 protein and DNMT1 protein in an aging cell line in contact with the test substance; And (3) selecting a test substance having increased expression or activity level of the UHRF1 protein and the DNMT1 protein as compared with a control sample.

The term "test substance" used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening in order to examine whether it affects the expression amount of a gene or affects the expression or activity of a protein. do. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Cell culture and induction of aging

Primary human diploid fibroblasts (HDFs) were isolated from the foreskin of 5-year-old boys and supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY) and antibiotics (Invitrogen) Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; in a 5% CO ₂ humidified incubator. In order to induce replicative senescence (RS) of HDF, confluent cells were transferred to two new dishes evenly and subcultured continuously, and the doubling time (DT) ) And the population doublings (PD) were counted. Hydrogen peroxide HDF - to proceed with the induced aging (HS), 1 car HDF cells (DT2) every 12 hours to 150μM H ₂ O ₂ were treated twice, which add further to for as long as the inducing stable aging, and time To react.

2. Aging-related β- Galactosidase  Active dyeing

The senescence-associated β-galactosidase (SA-β-gal) activity was assayed at pH 6.0. The staining was visualized after incubation at 37 ° C for 12 hours. The number of blue cells and total cells was counted using Image J software (NIH, Bethesda, MA) and the percentage of blue stained cells for aging cell counts was determined.

3. Intracellular siRNA  Introduction

HDFs were treated with siRNA duplexes using Oligofectamine ^TM Reagent (Invitrogen). All siRNAs for the target were purchased from Bioneer (Daejeon, Korea). UGRF1 (# 1, 5'-GCUGACCAUGCAGUAUCCATT;# 2, 5'-ACUGCUUAGCGUCUGAGAUTT), DNMT1 (# 1, 5'-CGAGUCUGGUUUGAGAGUTT;# 2, 5'- GGAAUGGCAGAUGCCAACAGCTT), HELLS (# 1, 5'-GUUGUUUAUCGCCUUGUUATT; , 5'-CAGCAAAUACUAUCGAUCATT), CBX5 (5'-AGGAAUGAACAUGAGACUUAATT), EZH2 (5'-GGAUAGAGAAUGUGGGUUUAUTT), SUV39H1 (5'-ACCACAGAAACUUUGUACUAGTT), PARP1 (5'-GGCAGAGGUGAAGGCAGAGCCTT), CHK1 (5'-GGUCUUUCCUUAUGGGAUACTT) and a negative control ( NC, 5'-CCUACGCCACCAAUUUCGUTT).

4. Production of retroviral vector and production of retrovirus

To construct a retroviral vector containing the DNMT1 gene, a cDNA fragment for DNMT1 was obtained from the pcDNA3 / Myc-DNMT1 vector (Roh Tae Young; Postech, Korea) using EcoRI and NotI restriction enzymes and pMXs-IRES-GFP retrovirus Vector (Cell Biolabs, Inc., San Diego, Calif.). The cDNAs for UHRF1 and WNT5A were amplified by PCR from whole cDNAs isolated from Chang cells and pcDNAWNT5A (Addgene, Cambridge, Mass.). The primer set (Bioneer) used for cDNA amplification is as follows. UHRF1, 5'-CAGTTAACATGGGGGTTTTTGCTGTCCC and 5'-GACTCGAGTCACCGGCCATTGCCGTA; WNT5A, 5'-CAGTTAACATGAAGAAGTCCATTGG and 5'-GACTCGAGTCACTTGCACACAA. The amplified cDNA fragment was inserted into the HpaI and XhoI sites of the pCMMP-MCS-IRES-RFP vector (Addgene).

To prepare the recombinant retrovirus, the modified 293T cell line H29D cells were cultured in DMEM medium (Invitrogen) and transfected with a retroviral plasmid containing the target cDNAs using Lipofectamine Reagent (Invitrogen). Viruses containing medium (supernatant) were harvested daily for 4 days. The harvested virus supernatant was filtered through a 0.45 μm filter unit (UFC 920008, EMD Milipore Corp., Darmstadt, Germany) and stored at -80 ° C.

5. Gene expression profiling and data analysis

To obtain the biotinylated cRNA, the total RNA was amplified and separated. Briefly, for microarray hybridization, total RNA isolated from HDFs was amplified and isolated using the Ambion Illumina RNA amplification kit (Ambion, Austin, Tex.). Labeling and hybridization of human HT-12 v4 expression beadchip arrays were performed according to the manufacturer's instructions (Illumina Inc., San Diego, Calif.). Raw data was obtained using the software GenomeStudio (Illumina Inc.), filtered to detect p value (<0.05), processed by log2 transformation and quantile normalization. Analysis of gene sets for biological functions was analyzed using David software (https://david.ncifcrf.gov/). All data processing and subsequent data analysis were performed using R / Bioconductor packages.

6. Intracellular ROS  Measure level

To measure intracellular ROS levels, cells were treated with 10 μM 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular probes, Eugene, OR) Gt; 37 C &lt; / RTI &gt; The stained cells were washed and resuspended in PBS and analyzed using a flow cytometer (FACS Vantage, Becton Dickinson Corp., Franklin Lakes, NJ). The mean number of random fluorescence units for 10,000 cells was expressed as a percentage of negative control.

7. Western Blat  analysis

Western blot analysis was performed according to standard procedures. (Sc-9996), GFP (sc-9996) and beta (sc-9996), DNMT1 (sc-20701), DNMT3A (sc-20703), HELLS Antibodies to -actin (sc-1616) were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies to Suv39H1 (# 8729) and EZH2 (# 5246) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to PARP1 (ab32071) and RFP (ab62341) were purchased from Abcam (Cambidge, UK). Antibodies to UHRF1 (3A11) and CBX5 (# 05-689) were purchased from Calbiochem (San Diego, CA) and Millipore (Billerica, MA) Antibodies against WNT 5A (MAB645) and human β-galactosidase (AF6464) were purchased from R & D systems (Minneapolis, MN).

8. Quantitative Reverse transcription (Quantitative reverse transcription; qRT ) - PCR

Total RNAs were isolated using Trizol (Invitrogen) and cDNAs were prepared using AMV reverse transcriptase (Promega). PCR was performed according to the manufacturer's protocol using THUNDERBIRD® SYBR® qPCR Mix (Toyobo Co., Ltd., Osaka, Japan). Primer sets were produced by Bioneer. DNMT1, 5'-GAGCTACCACGCAGACATCA and 5'-CGAGGAAGTAGAAGCGGTTG; DNMT3A, 5'-TATTGATGAGCGCACAAGAGAGC and 5'-GGGTGTTCCAGGGTAACATTGAG; UHRF1, 5'-TGTGGACCATGGGAATTTTT and 5'-GCTATTCTTGCCACCCTTGA; HELLS, 5'-ACTGGAGGAGTGATGCGATG and 5'-GCCACAGACAAGAAAAGGTC; WNT5A, 5'-CAAGGGCTCCTACGAGAGTG and 5'-CTTCTCCTTCAGGGCATCAC; LOXL4, 5'-AACTGCCTCTCCAAGTCTGC and 5'-TGCTGTGGTAATGCCTGTGG; PLA2G4C, 5'-GACCCCGAAAGGAAAGGCTG and 5'-CGAGTGGGAAGGGAGTGTTG; PPP1R14A, 5'-GCTGGACGTGGAGAAGTGGA and 5'-CCTGGATGAAGTCCTCGACAG; SPINT2, 5'-TGTGTATGGGGGCTGTGACG and 5'-CTTCTGGGAGCACTTGGGACA; TACSTD2, 5'-GTCACGCTTCCTGATTCCTC and 5'-GGACCGAAAGGGGATACATT; β-actin, 5'-CCTTCCTGGGCATGGAGTCCTGT and 5'-GGAGCAATGATCTTGATCTTC.

< Example  1> Reduced DNMT1 expression leading to loss of retentive DNA methylation leading to cell senescence

The present inventors induced aging in HDF using H ₂ O ₂ at a subcytotoxic concentration (250 μM). In this regard, the expression of DNMT1, a major DNA maintenance methyltransferase, was measured. DNMT1 protein and mRNA levels were significantly reduced in the HS HDF model. Conversely, no change could be detected in protein or mRNA levels of DNMT3A, a methyltransferase important for newly synthesized ( de novo ) DNA methylation (FIG. 1A). Similar results were obtained with the RS HDF model (Fig. 1B), indicating that cell senescence is particularly associated with impaired DNA methylation retention due to decreased DNMT1 mRNA expression. Next, in order to block the cellular DNA methylation activity (mainly the retinomethyltransferase activity), the present inventors used young HDF (cells having a doubling time of 2 days: DT2) with 5-aza-2'-deoxycytidine (5-aza-2'-deoxycytidine; 5-AzC) for 5 days. HDF exhibited senescence-associated β-galactosidase activity (SA-β-gal) activity and exhibited aging phenotypes such as induction of major cell cycle inhibitors, p21 and p16 1C). In addition, DNMTl alone siRNA-mediated knockdown was sufficient to induce HDF aging (Fig. 1D). These results support that the specific damage of retention DNA methylation by DNMT1 inhibition in aging induction is important.

< Example  2> In the process of aging DNMT1 and  Reduced expression of 7 DIPs at the same initial time point

To determine when DNMT1 expression declines in the progression of aging, the inventors have carefully examined time-sequentially in HS and performed gene expression profiling assays. Gene expression changes were normalized except for expression levels in control experiments and filtered for altered genes representing median absolute deviation greater than 0.3. After H ₂ O ₂ treatment, the genes showing more than 2-fold difference in expression at any time were differentially expressed by H ₂ O ₂ treatment (HS signature). By the above definition, 714 HS signature genes were obtained, 310 were upregulated (HS_UP) and 404 were downregulated (HS_DOWN) (FIG. 2A). Next, we compared the signature genes with the four previously reported RS signature gene modules. G1, early-downward-regulated genes (earlier than DT3); G2, down-regulated genes after mid-stage (DT3-DT7); G3, genes up-regulated in the intermediate and developmental stages (DT10-DT20); And G4, up-regulated genes (over DT30) in the developmental stage and in the very developmental stage. Overall, 77.8% of the HS signature genes were identical to the RS signature genes. Of the 310 HS_UP genes, most were identified in the G3 (25) or G4 (213) RS modules. Of the 403 HS_DOWN genes, most were identified in the G1 (213) or G2 (105) RS modules (Fig. 2B). This gene expression profile similarity between two different cell senescence models supports that the gene profile is suitable for the mechanistic study of aging. In the gene set enrichment analysis for the HS signature, the number of cell cycle-related genes among HS_DOWN genes was considerably high (FIG. 2C).

Interestingly, DNMT1 expression began to decline at the initial time (Fig. 2D, top and bottom of the heatmap) before proceeding to a clear aging phenotype such as SA-β-gal. Next, we analyzed the expression levels of previously reported DIPs (n = 53). Of the DIP genes, seven genes were common in RS and HS signature genes: UHRF1, EZH2, CHEK1, SUV39H1, CBX5, PARP1 and HELLS. Unexpectedly, each of the seven DIPs showed gradual down-regulation in a pattern similar to DNMTl (Figure 2D). In additional experiments, we verified the expression levels of the protein (Figure 2E). In conclusion, these results demonstrate that diminished DNMTl -mediated DNA methyltransferase activity observed during the aging process reduces the expression of DNMTl itself and reduces the expression of DIPs, which is presumed to play an essential or auxiliary role in promoting proper DNMTl activity .

< Example  3> DNMT1  Upstream regulator of expression UHRF1

DIPs have been reported to modulate DNMT1 activity through physical protein interactions. Thus, the present inventors further confirmed which DIP contributes to regulate DNMT1 expression. Seven DIPs were individually inhibited in HDF (DT2) using siRNA-mediated knockdown. Only UHRFl knockdown reduced DNMTl protein expression (Figure 3A). UHRF1 inhibition significantly down-regulated both mRNA and protein expression of DNMTl whereas DNMTl knockdown did not affect UHRFl expression (Fig. 3B, C), suggesting that UHRF1 plays a role as an upregulator in DNMTl transcription . To confirm the specificity of the effect of UHRF1 on DNMT1 expression, a knockdown HELLS was used as a control (Fig. 3B, C). Interestingly, cDNA microarray analysis after UHRF1 knockdown in young HDF showed downregulation of six DIPs (all except CHEK1) suggesting that UHRF1 is an up-regulator of DNMT1 and DIPs. However, UHRFl overexpression in HDF (DT5, step with reduced UHRF1 expression) did not increase DNMT1 mRNA and protein expression (FIG. 3D). These results indicate that UHRF1 as an upstream regulator is essential but not sufficient for DNMT1 expression.

< Example  4> DNMT1  Effectively induces aging through down-regulation UHRF1  control

Next, the present inventors confirmed that UHRF1 knockdown in young HDF (DT2) induces senescence phenotypes such as SA-β-gal, p21 and p16 induction, intracellular ROS increase (FIG. 4A-C). Compared with DNMT1 knockdown (FIG. 1D), UHRF1 knockdown induced aging phenotype more effectively (FIG. 4A). Unexpectedly, HELLS knockdown also weakly induced senescence phenotype, indicating that HELLS-mediated regulation of DNMT1 activity contributes in part to inducing senescence. Although not complete, the restoration of cellular DNMT1 levels through overexpression effectively reduced the senescence phenotype exhibited by UHRF1 knockdown (FIG. 4D, E). The results indicate that UHRF1 is an effective upstream regulator of DNMTl expression and, consequently, can regulate senescence.

Claims (6)

A UHRF1 gene or a protein encoded by the UHRF1 gene; And a biomarker composition for diagnosing cell senescence comprising a DNMT1 gene or a protein encoded by the DNMT1 gene. A primer or a probe specifically binding to the UHRF1 gene and the DNMT1 gene, an antibody that specifically binds to the UHRF1 gene and a DNMT1 gene-encoded protein, or a peptide having a binding domain specific to the DNMT1 gene, . (1) measuring the mRNA expression level of the UHRF1 gene and the DNMT1 gene or the expression level of the UHRF1 gene and the DNMT1 gene-encoded protein from the separated sample;
(2) comparing mRNA expression levels of the UHRF1 and DNMT1 genes or expression levels of the UHRF1 and DNMT1 gene-encoded proteins with a control sample; And
(3) If the mRNA expression level of the UHRF1 gene and the DNMT1 gene or the expression level of the protein encoded by the UHRF1 gene and the DNMT1 gene is lower than that of the control sample, it is judged as cell senescence. How to. UHRF1 protein and DNMT1 protein; Or an expression promoter or activator of the UHRF1 protein and the DNMT1 protein as an active ingredient. The method according to claim 4, wherein the expression promoter or activator of the UHRF1 protein and DNMT1 protein is selected from the group consisting of a compound specifically binding to the UHRF1 protein and the DNMT1 protein, a peptide, a peptide mimetic, an aptamer, Wherein the composition is any one selected from the group consisting of: (1) contacting the test substance to the separated aging cell line;
(2) measuring the level of expression or activity of UHRF1 protein and DNMT1 protein in an aging cell line in contact with the test substance; And
(3) A method for screening a therapeutic agent for cell senescence comprising the step of selecting a test substance having increased expression or activity level of the UHRF1 protein and the DNMT1 protein as compared with a control sample.

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