Diagnostic Biomarkers of Oxidative Stress

Abstract

The present invention relates to a diagnostic biomarker for oxidative stress. The present invention relates to a method for identifying oxidative stress (OS) caused by senescence and / or ultraviolet (UV) light exposure in a test cell, comprising: (a) determining the methylation status of at least one CpG site in protein tyrosine phosphatase receptor type N2 (PTPRN2) and / or a regulatory region of said PTPRN2 in a DNA sample from a test cell; and (b) comparing the methylation status of the CpG sites of PTPRN2 from (a) with the methylation status of a control that does not contain OS caused by aging and / or ultraviolet (UV) light exposure; wherein a difference in the methylation status of at least one CpG site in the PTPRN2 and / or regulatory region of the PTPRN2 in the test cell compared to a CpG site in the control indicates that the test cell is suffering from OS caused by aging and / or ultraviolet (UV) light exposure.

Description

[Technical field]

[0001] The present invention relates to a method for diagnosing cells with oxidative stress (OS), in particular by determining differential methylation occurring at CpG sites in the body of specific genes and in the regulatory regions of specific genes, the method being able to identify OS in cells that occurs specifically due to exposure of the cells to UV light and / or due to cellular senescence. [Background technology]

[0002] Living organisms are constantly exposed to various stresses from the external environment. They maintain homeostasis and resist the stresses through various regulatory systems. Oxidative stress refers to a serious imbalance between the levels of reactive oxygen species (ROS) in a cell and its antioxidant defense mechanisms. To maintain its homeostasis, living organisms have a system called redox regulation, which regulates the redox state to deal with and resist stress. This system functions to adapt to oxidative stress from many external stressors, such as radiation, ultraviolet light, environmental pollutants, high heat, low temperature, hypoxic conditions, and infectious diseases, as well as lifestyle-related diseases, such as cancer, diabetes, arteriosclerosis, hypertension, and obesity. However, when this regulatory mechanism is disrupted due to some cause, oxidative stress (OS) occurs. OS can lead to cell damage, DNA fragmentation, apoptosis, and cell death. Early detection of OS can prevent further damage in the organism, which causes the organism to receive early treatment or start using protective mechanisms. There are several methods known in the art for the early detection of OS, however, none of the methods known in the art formally use a genomic sample of the body to detect OS in cells. Summary of the Invention [Problem to be solved by the invention]

[0003] Human skin is constantly exposed to large amounts of oxidative stress and free radicals, such as ROS, from normal metabolic reactions, as well as from continuous exposure to air, radiation and ultraviolet light, environmental pollutants, and physical and / or chemical agents (e.g., cosmetics). Under certain conditions, the production of ROS can be so significant that it can be the cause of, for example, psoriasis or skin cancer. Oxidative damage caused by free radicals such as ROS is also the main cause of body aging in general, and skin aging in particular. Therefore, there is a need in the art to detect OS in cells, such as skin cells, to prevent further damage to cells. [Means for solving the problem]

[0004] The present invention solves the above problems by providing differentially methylated gene biomarkers that can be used to detect OS in cells. Environmental factors / agents such as UV light exposure, aging, diet, etc. can induce OS, which can further induce changes in promoter CpG methylation status by recruiting DNA methyltransferases (DNMTs) and TET enzymes to various promoters, so a biomarker that results in differential methylation in cells with OS is essential to overcome the above challenges. In particular, a biomarker for detecting OS in cells is differential methylation of the gene protein tyrosine phosphatase receptor type N2 (PTPRN2). The CpGs of PTPRN2 in cells with OS are differentially methylated (i.e., hypomethylated or hypermethylated) compared to the corresponding CpGs in cells without OS. In particular, the CpGs of PTPRN2 are differentially methylated based on the source of OS. Thus, PTPRN2 can be effectively used to determine whether a cell has OS. Different external factors can cause OS in a cell, and a cell can have a different DNA methylation signature for each external factor that causes OS. More specifically, the OS in cells caused by UV light exposure and / or aging can result in a specific group of CpGs that are differentially methylated compared to cells without OS and / or cells without OS caused by UV light exposure and / or aging. Even more specifically, the group of CpGs is one or more preselected CpG sites that are differentially methylated in cells with OS specifically caused by UV light exposure and / or aging compared to cells without OS or cells with OS caused by another external factor. This is particularly advantageous in that it provides a means of predicting the onset of OS in cells using epigenetics, and therefore allows for earlier treatment of OS before the cells are further damaged. Furthermore, epigenetic markers are long-term biomarkers, i.e., they are heritable, and can be used to detect OS in subsequent generations, if necessary. In one aspect of the invention, there is provided a method for identifying oxidative stress (OS) caused by senescence and / or ultraviolet (UV) light exposure in a test cell, comprising: (a) determining the methylation status of at least one CpG site in protein tyrosine phosphatase receptor type N2 (PTPRN2) and / or a regulatory region of said PTPRN2 in a DNA sample from a test cell; and (b) comparing the methylation status of the CpG sites of PTPRN2 from (a) with the methylation status of a control that does not contain OS caused by aging and / or ultraviolet (UV) light exposure; wherein a difference in the methylation status of at least one CpG site in the PTPRN2 and / or regulatory region of the PTPRN2 in the test cell compared to a CpG site in the control indicates that the test cell is suffering from OS caused by aging and / or ultraviolet (UV) light exposure.

[0005] The term "cell" as used herein refers to a whole living cell, naturally occurring or modified. The cell may be isolated from other cells, mixed with other cells in culture, or within a tissue (partially or whole) or organism. In particular, the cell may be a eukaryotic cell. More particularly, the cell may be a mammalian cell. The term "mammalian cell" refers to any cell derived from a mammalian subject. The cell may also be a cell derived from the culture and propagation of cells obtained from a subject. The cell may also be genetically modified to express recombinant proteins and / or nucleic acids. Mammalian cells may be derived from humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species; domestic animals such as cows, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters and guinea pigs; domestic animals such as chickens, turkeys and other galliformes, ducks, geese, and birds, including wild and game birds. In particular, the subject is a mammal. More particularly, the mammal is selected from the group consisting of mouse, rat, guinea pig, dog, mini pig, human, cow, sheep, pig, goat, horse, donkey and mule. In particular, the mammalian cell may be a skin cell, a stem cell or a cell derived therefrom. More particularly, the mammalian cell may be a skin cell.

[0006] As used herein, the term "CpG site" or "methylation site" refers to a nucleotide in a nucleic acid (DNA or RNA) that is susceptible to methylation, either by naturally occurring events in vivo or by events initiated to chemically methylate the nucleotide in vitro. For example, there may be approximately 1300 CpG sites in PTPRN2. Some of these sites may be hypermethylated and some may be hypomethylated in cells with OS compared to cells without OS. In particular, the following table in PTPRN2: Table 1. Eight CpG sites in PTPRN2 that are differentially methylated in cells with OS caused by aging and / or UV light exposure.

[0007] [Table 1] The methylation status of at least one CpG site selected from the eight CpG sites described in is determined to determine whether the cell has OS caused by senescence and / or UV light exposure.

[0008] As used herein, the term "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more nucleotides that are methylated. "CpG island" as used herein describes a segment of DNA sequence that contains functionally or structurally deviant CpG density. For example, Yamada et al. report a set of standards for determining CpG island, which must be at least 400 nucleotides long, have a GC content higher than 50%, and an OCF / ECF ratio higher than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266). Other researchers define CpG island as a sequence at least 200 nucleotides long, with a GC content higher than 50% and an OCF / ECF ratio higher than 0.6, but are less strict (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745). In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation state" or "methylation status" are used herein to describe the state, condition or status of methylation of a genomic sequence, which refers to the characteristics of a DNA segment at a particular genomic locus related to methylation, including but not limited to whether any of the cytosine (C) residues in this DNA sequence are methylated, the position of the methylated C residues, the ratio of methylated C in any particular stretch of residues, and allelic differences in methylation due to, for example, different origins of the alleles. The term "methylation state" refers to the state of a particular methylation site (i.e., methylated vs. unmethylated), meaning that the residue or methylation site is methylated or unmethylated. A methylation profile can then be determined based on the methylation state of one or more methylation sites. Thus, the term "methylation profile" or "methylation pattern" refers to the relative or absolute concentration of methylated or unmethylated C residues in any particular stretch of residues in the genomic material of a biological sample. For example, if a cytosine (C) residue that is normally unmethylated in a DNA sequence is methylated, it may be referred to as "hypermethylated"; whereas, if a cytosine (C) residue that is normally methylated in a DNA sequence is unmethylated, it may be referred to as "hypomethylated". Similarly, if a cytosine (C) residue in a DNA sequence (e.g., DNA from a sample nucleic acid from a test subject) is methylated compared to other sequences from different regions or from different individuals (e.g., compared to a standard nucleic acid of a normal nucleic acid or reference sequence), the sequence is considered to be hypermethylated compared to other sequences. Alternatively, if a cytosine (C) residue in a DNA sequence is unmethylated compared to other sequences from different regions or different individuals, the sequence is considered to be hypomethylated compared to other sequences. The sequence is said to be "differentially methylated". Measuring the level of differential methylation can be done by various methods known to those skilled in the art. One method is, as a non-limiting example, measuring the methylation level of each interrogated CpG site as determined by bisulfite sequencing. The term "methylated nucleotide" or "methylated nucleotide base" as used herein refers to the presence of a methyl moiety on a nucleotide base, which is not normally present in recognized normal nucleotide bases. For example, cytosine in its normal form does not contain a methyl moiety on the pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5-position of the pyrimidine ring. Thus, cytosine in its normal form cannot be a methylated nucleotide, and 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may have a methyl moiety at the 5-position of the pyrimidine ring, but for the purposes of this specification, thymine may not be considered a methylated nucleotide when present in DNA. The normal nucleotide bases of DNA are thymine, adenine, cytosine, and guanine. The normal bases of RNA are uracil, adenine, cytosine, and guanine. Correspondingly, a "methylation site" is a position in a target gene nucleic acid region where methylation can occur. For example, a position containing CpG is a methylation site regardless of cytosine methylation. In particular, the term "methylated nucleotide" refers to a nucleotide with a methyl group attached to a position of the nucleotide accessible for methylation. Such methylated nucleotides are usually found in nature and to date can be considered as most common with methylated cytosine occurring mostly in the context of the dinucleotide CpG, but also in the context of CpNpGand CpnPn sequences. In principle, other naturally occurring nucleotides can also be methylated, but they are not considered in relation to any aspect of the present invention. The terms "methylation profile", "methylation pattern", "methylation state" or "methylation status" in the context of the present invention are used herein to describe the state, condition or status of methylation of a genomic sequence, which refers to the characteristics of a DNA segment at a particular genomic locus related to methylation, including but not limited to whether any of the cytosine (C) residues in this DNA sequence are methylated, the position of the methylated C residues, the ratio of methylated C in any particular stretch of residues, and allelic differences in methylation due to, for example, different origins of the alleles.

[0009] The term "hypermethylation" refers to an average methylation state that corresponds to an increased presence of 5-mCyt at one or more CpG dinucleotides in a DNA sequence of a test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample. In particular, the control refers to cells that are not indicative of OS. The term "hypomethylation" refers to an average methylation state that corresponds to a decreased presence of 5-mCyt at one or more CpG dinucleotides in a DNA sequence of a test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample. In particular, the control refers to cells that are not indicative of OS. As used herein, the term "gene" refers to each genomic DNA sequence, including any promoter and regulatory sequences (e.g., enhancers and other gene sequences involved in regulating the expression of a gene) of the gene and / or the body of the gene itself. A gene sequence may be an expressed sequence (e.g., expressed RNA, mRNA, cDNA). Furthermore, if SNPs within a gene are known, the term should be construed to include all sequence variants thereof. As used herein, the term "genomic material" refers to a nucleic acid molecule or fragment of the genome of a subject or group of subjects. In particular, the nucleic acid molecule or fragment is DNA or RNA or a hybrid thereof, and most preferably is a molecule of the DNA genome of the subject or group of subjects. As used herein, the term "promoter" or "gene promoter", which is used interchangeably with the term "regulatory region" or "regulatory sequence", refers to each contiguous gene DNA sequence extending from 1.5 kb upstream to 1.5 kb downstream relative to the transcription start site (TSS), or a contiguous portion thereof. In particular, "regulatory region" refers to each contiguous gene DNA sequence extending from 1.5 kb upstream to 0.5 kb downstream relative to the TSS. In one example, "regulatory region" refers to each contiguous gene DNA sequence extending from 1.5 kb upstream to the downstream end of a CpG island that overlaps with the region 1.5 kb upstream to 1.5 kb downstream from the TSS (and thus may extend even more than 1.5 kb downstream in that case), and contiguous portions thereof. In particular, with respect to PTPRN2, any CpG dinucleotide of a gene that is coordinately methylated with the "regulatory region" of the gene has substantial diagnostic / classification utility as disclosed herein.

[0010] As used herein, the term "DNA sample" refers to DNA extracted from cells according to any embodiment of the present invention using methods known in the art. In particular, the test cell has OS if differential methylation is detected in the test cell, i.e., the cell exhibits hypermethylation or hypomethylation at at least one CpG site compared to a control (i.e., a cell that does not show signs of OS). More specifically, the test cell has OS caused by UV light exposure and / or aging if differential methylation is detected at one of the eight specific CpG sites listed in Table 1 above, i.e., the cell exhibits hypermethylation or hypomethylation at at least one CpG site from the list of eight CpG sites in Table 1 compared to a control (i.e., a cell that does not show signs of OS or a cell that shows signs of OS caused by external factors other than UV light exposure and / or aging). In particular, in (a) the methylation status of at least 2, 3, 4, 5, 6, 7, or 8 CpG sites from Table 1 in PTPRN2 is determined. The method according to any aspect of the present invention may further comprise, prior to (a), (i) bisulfite-modifying the DNA sample.

[0011] "Bisulfite treatment" of genomic DNA, which is used interchangeably with the term "bisulfite modification," refers to the treatment of genomic DNA with a deaminating agent such as bisulfite, which can be used to treat all DNA, whether methylated or not. In particular, the term "bisulfite" as used herein includes any suitable bisulfite, such as sodium hydrogen sulfite, or other chemical agents that can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosine, and thus can be used to differentially modify DNA sequences based on the methylation status of the DNA (e.g., U.S. Patent Application Publication No. 2010 / 0112595). As used herein, a reagent that "differentially modifies" methylated or unmethylated DNA includes any reagent that modifies methylated and / or unmethylated DNA in a process that results in products that can be distinguished from methylated and unmethylated DNA, thereby allowing identification of DNA methylation status. Such processes may include, but are not limited to, chemical reactions (e.g., C to U conversion with bisulfite) and enzymatic treatments (e.g., cleavage with methylation-dependent endonucleases). Thus, enzymes that preferentially cleave or digest methylated DNA can cleave or digest DNA molecules with much higher efficiency when the DNA is methylated, while the efficiency of enzymes that preferentially cleave or digest unmethylated DNA is significantly higher when the DNA is unmethylated. Therefore, prior to (a) above according to any aspect of the invention, the genomic DNA contained in / obtained from or extracted from the cell is first bisulfite treated. Alternative methods available in the art may be used instead of bisulfite treatment. Those skilled in the art will understand which other methods to use. In one example, TET-assisted pyridine borane sequencing (TAPS) can be used to detect 5mC and 5hMC (Yibin Liu, et al., Nature Biotechnology, 37: 424-429 (2019)). The cells used in any aspect of the invention are obtained from a biological sample selected from the group consisting of blood, brain, sperm and any other tissue or sample that provides genomic DNA for use in the method according to any aspect of the invention. In particular, the biological sample may include any biological material obtained from a subject that contains DNA, and may be a liquid, solid, or both, such as tissue or bone or a body fluid such as blood, lymph, etc. In particular, the biological sample useful in the present invention may include biological cells or fragments thereof. The term "test" in conjunction with the term "cell" herein refers to a cell that is subjected to a method according to any embodiment of the present invention and is the basis for an analytical application of the present invention. Thus, a "test cell" is a cell or group of cells that is tested according to any embodiment of the present invention, or a profile obtained or generated in this context. Conversely, the term "reference" or "control" refers to a mostly predefined entity that is used for comparison with a test entity. In particular, a "test cell" refers to a cell that is tested for an OS whose methylation status is to be determined, and a "control" refers to a cell in which the methylation status is already known and no OS is used as a reference.

[0012] In a further aspect of the invention, there is provided a method for identifying oxidative stress (OS) caused by senescence and / or ultraviolet (UV) light exposure in at least one cell, comprising detecting differential expression of protein tyrosine phosphatase receptor type N2 (PTPRN2). The term "epigenetic marker" as used herein refers to the CpG sites of the gene itself and / or gene body and / or the CpG sites of the regulatory region of the gene, which contain epigenetic modifications, and the gene can be used as a marker for determining a certain disease due to epigenetic modifications. In particular, the term "epigenetic marker" is defined as at least one of DNA methylation markers, histone modification markers and deacetylase markers. More specifically, the epigenetic marker of OS that occurs in cells with aging and / or ultraviolet (UV) light exposure is the differential methylation found in the CpG sites on the gene body and / or regulatory region of PTPRN2. Even more specifically, the epigenetic marker of OS caused by aging and / or UV light exposure is the differential methylation of the eight CpG sites in Table 1 above found in the gene body and / or regulatory region of PTPRN2 in cells with OS caused by aging and / or UV light exposure compared to control cells without OS or with OS caused by another external factor other than aging and / or UV light exposure. In particular, promoter-specific hyper / hypomethylation leads to changes in the expression of different genes. More specifically, differential methylation of CpG sites in PTPRN2 leads to differential expression of PTPRN2. This differential expression of genes compared to a control in which there is no differential methylation is indicative of OS, in particular OS caused by aging and / or UV light exposure in the tested cells. In one example, promoter hypermethylation is associated with inactivation of gene expression of PTPRN2. In another example, promoter hypomethylation is associated with (hyper)activation of gene expression of PTPRN2. An embodiment of the present invention will be described with reference to the drawings. [Brief description of the drawings]

[0013] [Figure 1]1 is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions in cells induced with artificial OS (high UV light for 24 hours) in Example 1. As can be seen, genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (similar to receptor-like protein tyrosine phosphatase) have the highest differential methylation probe distribution. Other genes are similarly differentially methylated in cells with OS. [Diagram 2] 1 is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions in cells induced with artificial OS (low UV light for 72 hours) in Example 1. As can be seen, genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (similar to receptor-like protein tyrosine phosphatase) have the highest differential methylation probe distribution. Other genes are similarly differentially methylated in cells with OS. [Diagram 3] 1 is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions in cells induced with artificial OS (high H2O2, 24 hours) in Example 2. As can be seen, genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (similar to receptor-like protein tyrosine phosphatase) have the highest differential methylation probe distribution. Other genes are also shown to be differentially methylated in cells with OS. [Figure 4] 1 is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions in cells induced with artificial OS (low H2O2, 24 hours) in Example 2. As can be seen, genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (similar to receptor-like protein tyrosine phosphatase) have the highest differential methylation probe distribution. Other genes are also shown to be differentially methylated in cells with OS. [Diagram 5]1 is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions in cells in which artificial OS was induced in Example 3 using Medox® in cells. As can be seen, genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (similar to receptor-like protein tyrosine phosphatase) have the highest differential methylation probe distribution. Other genes are similarly differentially methylated in cells with OS. [Figure 6] FIG. 1 is a graph showing the top 20 overlapping genes in different treatments related to UV light exposure and aging. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The above describes preferred embodiments, and as will be understood by those skilled in the art, the present invention may be subject to changes or modifications in design, structure, or operation without departing from the scope of the claims. Such variations are intended to be covered, for example, by the claims. EXAMPLES

[0015] Oxidative stress on human tissues induced by UV light Artificial oxidative stress was induced in cell culture systems and skin tissue models, and the promoter methylation status was analyzed. The T-Skin models were obtained from Episkin SA (France) and consisted of reconstructed human skin. Each skin model consisted of a dermal equivalent covered by a stratified, well-differentiated epidermis derived from normal human keratinocytes. After receiving the skin models, they were harvested by incubating in T-Skin culture medium overnight at 37° C. in a 5% CO2 incubator. To induce oxidative stress in human tissue systems, skin models (5x replicates) were exposed to UV light (UVA 24 J / cm 2 +UVB 50mJ / cm 2) daily for 72 hours. Exposure to UV irradiation generates ROS and ultimately oxidative stress in cells. A control set of skin models (5x replicates) was maintained for 72 hours without exposure to UV irradiation. After treatment, skin models were harvested and genomic DNA was purified from tissue samples using DNeasy® Blood & Tissue Kit (Qiagen). Genomic DNA was quantified using PicroGreen® or NanoDrop™ 2000. Genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ kit (Zymo Research). The Infinium MethylationEPIC v2.0 kit (Illumina) can be used to quantitatively analyze over 850,000 methylation sites across the genome at single-base resolution. Quality Control and Data Processing Methylation EPIC array data processing was performed in R version 4.1.2 (2021-11-01) using minfi version 1.40.0. Raw intensity data (IDAT) were imported into R (4.1.2) and processed using minfi (1.4.0); Bioconductor package, 18. Sample quality check was performed and probes with detection P value < 0.01 in one or more samples or average detection P value < 0.05 across all samples were kept. Samples were then normalized using the function normalization (done by the preprocessFunnorm function in minfi) for type bias correction and background correction. Before differential methylation analysis, probes with non-specific binding, cross-reactive probes, probes affected by common SNPs, and probes annotated to chromosomes X and Y were also filtered out. Beta and M values ​​of normalized and filtered samples were calculated using getBeta and getM functions, respectively, and the samples were subjected to further downstream analysis. Differential methylation analysis Differential methylation analysis was performed using the packages limma version 3.50.1 and DMRcate version 2.8.5. Contrast matrices were set by comparing each corresponding treatment group with the control group, and the empirical Bayes algorithm was used to fit M values ​​based on the design and contrast model. Probes with adjusted P values ​​lower than 0.05 were considered as differentially methylated positions (DMPs). Annotation was performed using IlluminaHumanMethylationEPICanno .ilm10b2.hg19 and the annotatr package (1.20.0). As seen in Figure 1, one of the top differentially methylated genes in the promoter region is PTPRN2 (protein tyrosine phosphatase receptor type N2). PTPRN2 is a phosphatidylinositol phosphatase that functions to dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 4,5-bisphosphate, and plays an important role in lipid signaling, cell signaling and membrane trafficking. EXAMPLES

[0016] H 2 O 2 Oxidative stress on human tissues caused by Another method of inducing oxidative stress in human tissue systems is by hydrogen peroxide treatment, which leads to the generation of ROS in cells. Skin models (5x replicates) were treated with two different concentrations of hydrogen peroxide [100μM (low) and 200μM (high)] for 2 hours and maintained for 24 hours. A control set of skin models (5x replicates) was maintained for 24 hours without any treatment with hydrogen peroxide. After treatment, the skin models were harvested and genomic DNA was purified from the tissue samples using DNeasy® Blood & Tissue Kit (Qiagen). Genomic DNA was quantified using PicroGreen® or NanoDrop™ 2000. The samples in this example were subjected to similar quality control and data processing methods as disclosed in Example 1. In addition, the data obtained in Example 2 was subjected to an analysis similar to the differential methylation analysis disclosed in Example 1. The results are shown in Figures 3 and 4. EXAMPLES

[0017] Oxidative stress in cell culture systems To investigate oxidative stress in cell culture systems, mesenchymal stem cells (MSCs) were treated with Medox® (Evonik batch: H-080719), which contains many natural anthocyanins associated with antioxidant and anti-inflammatory properties. Bone marrow-derived MSCs were cultured for one week in Mesencult ACF Plus Medium with two doses of Medox® (4x replication): 25 μg / ml (low) and 100 μg / ml (high). The medium with Medox® was changed every two days for one week. As a control (4x replication), MSCs were cultured for one week in Mesencult ACF Plus Medium without any Medox® treatment. Medox® treatment is expected to produce an opposite reaction to OS. After this, the cell pellet was collected and genomic DNA was purified from the cell pellet using the DNeasy® Blood & Tissue Kit (Qiagen). Genomic DNA was quantified using PicroGreen® or NanoDrop™ 2000. Genomic DNA (500 ng) from cell pellets was subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ kit (Zymo Research). The Infinium MethylationEPIC v2.0 kit (Illumina) allows quantitative analysis of over 850,000 methylation sites across the genome at single-base resolution. DNA methylation profiling has proven to be a powerful analytical tool for accurately identifying tissue origin and the effects of environmental factors. It has the advantage as a biomarker classifier since it is a stable marker, facilitating quantitative analysis at single nucleotide resolution. The samples in this example were subjected to similar quality control and data processing methods as disclosed in Example 1. In addition, the data generated in Example 3 was subjected to similar analysis as the differential methylation analysis disclosed in Example 1. As seen in Figure 5, one of the top genes differentially methylated in the promoter region is PTPRN2 (protein tyrosine phosphatase receptor type N2). PTPRN2 is a phosphatidylinositol phosphatase that can dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 4,5-bisphosphate, and plays an important role in lipid signaling, cell signaling and membrane trafficking. Results for both Examples 1, 2 and 3 Two independent experimental settings to analyze the promoter methylation status during oxidative stress showed differential methylation of the PTPRN2 promoter, suggesting it to be a potential biomarker of oxidative stress. EXAMPLES

[0018] Oxidative stress on aging human tissues Another way of inducing oxidative stress in human tissue systems is through aging, which leads to the generation of reactive oxygen and nitrogen species (RONS) within cells. The skin models were maintained in deep well plates with culture medium for 7 days (4 replicates), 14 days (4 replicates) and 21 days (4 replicates), respectively, to induce aging of skin tissue. A control set of skin models (4x replicates) was collected at 0 hours for genomic DNA isolation. The skin models were harvested after 7 days, 14 days and 21 days, respectively, and genomic DNA was purified from the tissue samples using DNeasy® Blood & Tissue Kit (Qiagen). Genomic DNA was quantified using PicroGreen® or NanoDrop™ 2000. Genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ kit (Zymo Research). The Infinium MethylationEPIC v2.0 kit (Illumina) can be used to quantitatively analyze over 850,000 methylation sites across the genome at single-base resolution. Oxidative stress on human tissues induced by UV light To induce oxidative stress in human tissue systems, the skin model was exposed to UV irradiation (UVA 24 J / cm 2 +UVB 50mJ / cm 2 ) daily and incubated for 24 h (4 replicates), 48 h (4 replicates), 72 h (4 replicates), 96 h (4 replicates), and 120 h (4 replicates), respectively. Exposure to UV irradiation generates ROS, which ultimately leads to oxidative stress in cells. A control set of skin models was maintained without exposure to UV irradiation for 24 hours (4 replicates), 48 hours (4 replicates), 72 hours (4 replicates), 96 hours (4 replicates), and 120 hours (4 replicates). After treatment, the skin models were harvested and genomic DNA was purified from the tissue samples using DNeasy Blood & Tissue Kit (Qiagen). Genomic DNA was quantified using PicroGreen or NanoDrop™ 2000. Genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ kit (Zymo Research). Over 850,000 methylation sites across the genome can be quantitatively analyzed at single-base resolution using the Infinium MethylationEPIC v2.0 kit (Illumina). Quality Control and Data Processing A total of 52 samples from seven different treatments and their respective controls were analyzed (Table 2). The seven treatments can be classified into two oxidative stresses - UV and age. Five treatments were oxidative stress by UV at different time points - 24 h, 48 h, 72 h, 96 h and 120 h. Each treatment had its own control. The other two treatments were oxidative stress by aging after 7 days and 14 days, respectively. Both treatments were compared to the same control group. Methylation EPIC array data processing was performed in R version 4.2.2 (2021-11-10 r83330) using minfi version 1.42.0. Raw intensity data (IDAT) were imported into R (4.2.2) and processed using minfi (1.42.0). A quality check of the Bioconductor package samples was performed to keep probes with detection P-value < 0.01 in one or more samples or with an average detection P-value < 0.05 in all samples. Samples were then normalized using function normalization (performed by the preprocesssFunnorm function in minfi) for type bias correction and background correction. Prior to differential methylation analysis, probes with non-specific binding, cross-reactive probes, probes affected by common SNPs, and probes annotated to chromosomes X and Y were also filtered out. Beta and M values ​​of normalized and filtered samples were calculated using getBeta and getM functions, respectively, and then the samples were subjected to further downstream analysis. Table 2: Sample information for batch 2

[0019] [Table 2] Differential methylation analysis Pairwise differential methylation analysis (total of 7 pairs) was performed using the limma package version 3.52.4. UV and aged samples were analyzed separately. Contrast matrices were set up comparing each corresponding treatment group with the control group, and an empirical Bayes algorithm was used to fit M values ​​based on the design and contrast model. Probes with adjusted P values ​​lower than 0.05 were considered as differentially methylated positions (DMPs). Annotation was performed using IlluminaHumanMethylationEPICanno .ilm10b2.hg19. Genes that overlapped across all treatments were then found and their DMPs were analyzed to identify DMPs that were present in all comparisons. As seen in Figure 6, one of the top differentially methylated genes in all comparisons is PTPRN2 (protein tyrosine phosphatase receptor type N2). PTPRN2 is a phosphatidylinositol phosphatase that can dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 4,5-bisphosphate, and plays an important role in lipid signaling, cell signaling, and membrane trafficking. The PTPRN2 gene had a total of 670 unique DMPs, of which 8 DMPs were found in all comparisons (Table 3). Table 3: Eight DMPs of PTPRN2 that were common across all treatments and their methylation status in each treatment

[0020] [Table 3]

Claims

1. A method for identifying oxidative stress (OS) caused by aging and / or exposure to ultraviolet (UV) light in test cells, the following: (a) To determine the methylation status of at least one CpG site in the protein tyrosine phosphatase receptor type N2 (PTPRN2) and / or the regulatory region of PTPRN2 in a DNA sample derived from test cells, (b) The methylation state of the CpG site of PTPRN2 is compared with the methylation state of a control that does not contain OS and is caused by aging and / or exposure to ultraviolet (UV) light. Including, here, A method for indicating that the test cells have aging and / or UV-induced OS if there is a difference in the methylation state of at least one CpG site in the PTPRN2 and / or regulatory region of the PTPRN2 in the test cells compared with the CpG site in the control.

2. In (a) above, the following table in PTPRN2: Table 1 The method according to claim 1, wherein the methylation state of at least one CpG site selected from the eight CpG sites described is determined.

3. The method according to claim 2, wherein in (a) above, the methylation state of at least five CpG sites selected from the eight CpG sites in the PTPRN2 in the table described in claim 2 is determined.

4. The method according to claim 2, wherein, in (a) above, the methylation states of all eight CpG sites in the PTPRN2 in the table described in claim 2 are determined, and the methylation states of at least five CpG sites selected from are determined.

5. Furthermore, prior to (a) above, (i) Modifying the DNA sample with bisulfite, The method according to claim 1, including the method described in claim 1.

6. The method according to claim 1, wherein the test cells are obtained from a biological sample selected from the group consisting of blood, brain, sperm, and any other tissue or sample providing genomic DNA.

7. The method according to claim 1, wherein the test cells are eukaryotes.

8. The method according to claim 1, wherein the test cells are derived from a mammal.

9. The method according to claim 8, wherein the mammal is selected from the group consisting of mice, rats, guinea pigs, dogs, miniature pigs, humans, cattle, sheep, pigs, goats, horses, donkeys, and mules.

10. The method according to claim 1, wherein the test cells are skin cells, stem cells, or cells derived therefrom.

11. A method for identifying oxidative stress (OS) caused by aging and / or exposure to ultraviolet (UV) light in at least one cell, comprising detecting differences in the expression of protein tyrosine phosphatase receptor type N2 (PTPRN2).

12. The method according to claim 11, wherein the cells are obtained from a biological sample selected from the group consisting of blood, brain, sperm and any other tissue or sample providing genomic DNA.

13. The method according to claim 11, wherein the cells are derived from a mammal selected from the group consisting of mouse, rat, guinea pig, dog, miniature pig, human, cattle, sheep, pig, goat, horse, donkey, and mule.

14. The method according to claim 11, wherein the cells are skin cells, stem cells, or cells derived therefrom.

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