Diagnostic biomarkers of oxidative stress

Abstract

The present invention relates to a method for identifying oxidative stress (OS) caused by aging and / or ultraviolet (UV) exposure in a test cell, comprising: (a) determining the methylation status of at least one CpG site in a protein tyrosine phosphatase receptor of type N2 (PTPRN2) and / or a regulatory region of PTPRN2 in a DNA sample obtained from said test cell, (b) comparing the methylation status of the CpG site of PTPRN2 from (a) to a control that does not cause OS by aging and / or ultraviolet exposure wherein the methylation status of the CpG site of PTPRN2 and / or a regulatory region of PTPRN2 in the test cell differs compared to the CpG site in the control, the test cells are indicated to have OS resulting from exposure of the cells to ultraviolet rays and / or aging.

Description

Technical Field

[0001] The present invention relates to a method for diagnosing cells with oxidative stress (OS). Specifically, the method can identify OS in cells caused specifically by cell exposure to ultraviolet light and / or caused by cell senescence by determining differential methylation occurring at CpG sites located on specific gene bodies and specific gene regulatory regions. Background Art

[0002] Living organisms are constantly affected by various stresses from the external environment. In order to resist such stresses, they maintain their homeostasis through various regulatory systems. Oxidative stress refers to a serious imbalance between the level of intracellular reactive oxygen species (ROS) and its antioxidant defense mechanism. In order to survive under this stress, living organisms have a system called redox regulation to cope with stress, to maintain homeostasis by regulating the redox state. This system functions to adapt to many external stress factors, such as radiation, ultraviolet (UV) rays, environmental pollutants, high fever, low temperature, hypoxia and infectious diseases, and oxidative stress from lifestyle-related diseases (such as cancer, diabetes, arteriosclerosis, hypertension and obesity). However, if this regulatory mechanism is destroyed for some reason, oxidative stress (OS) will occur. OS can cause cell damage, DNA fragmentation, apoptosis and cell death. Early detection of OS can prevent further damage in living organisms, so that the organism receives early treatment or begins to use protective measures.

[0003] Some methods for early detection of OS are known in the art. However, none of these methods known in the art has been formally used to detect OS in cells by using a genomic sample of the body.

[0004] Human skin is constantly exposed to oxidative stress and free radicals, such as large amounts of ROS, not only from ordinary metabolic reactions, but also from continued exposure to air, radiation and UV rays, environmental pollutants, and physical and / or chemical agents (e.g., cosmetics). In some cases, the production of ROS can become so intense that it can contribute to the pathogenesis of, for example, psoriasis or skin cancer. Oxidative damage caused by free radicals (e.g., ROS) is also a major cause of general body aging, and in particular skin aging. Therefore, there is a need in the art to detect OS in cells (e.g., skin cells) to prevent further damage to the cells.

[0005] Description of the invention

[0006] The present invention attempts to solve the above problems by providing a gene biomarker, the differential methylation of which can be used to detect OS in cells.

[0007] Since environmental factors / agents (e.g., UV exposure, aging, diet, etc.) can induce OS, and OS can further cause changes in promoter CpG methylation status by recruiting DNA methyltransferases (DNMTs) and TET enzymes to various promoters, biomarkers that cause differential methylation in cells with OS are essential to overcome the above problems. Specifically, the biomarker used to detect OS in cells is the differential methylation of the gene N2-type protein tyrosine phosphatase receptor (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. Specifically, the CpGs of PTPRN2 are differentially methylated based on the source of OS. Therefore, PTPRN2 can be effectively used to determine whether a cell has OS. Different external agents can cause OS to occur in cells, and for each external agent that causes OS, cells may have different DNA methylation characteristics. More specifically, OS caused by UV exposure and / or aging in cells can result in differential methylation of specific CpG groups compared to cells without OS and / or cells without OS caused by UV exposure and / or aging. Still more specifically, the CpG group is one or more preselected CpG sites that are differentially methylated in cells specifically causing OS by UV exposure and / or aging compared to cells without OS or cells causing OS by another external agent. This is particularly advantageous because the use of epigenetics provides a method for predicting the occurrence of OS in cells, thereby allowing OS to be treated earlier before more damage is caused to the cells. Further, the epigenetic mark is a long-term biomarker, that is, it is heritable and can also be used to detect OS in the next generation if necessary.

[0008] According to one aspect of the present invention, there is provided a method for identifying oxidative stress (OS) caused by aging and / or ultraviolet (UV) exposure in a test cell, comprising:

[0009] (a) determining the methylation status of at least one CpG site in a protein tyrosine phosphatase receptor N2 type (PTPRN2) and / or a PTPRN2 regulatory region in a DNA sample obtained from a test cell,

[0010] (b) The methylation status of CpG sites of PTPRN2 from (a) was compared with that of controls without OS induced by aging and / or UV exposure,

[0011] A difference in the methylation status of CpG sites of PTPRN2 and / or the regulatory region of PTPRN2 in the test cell compared to the CpG sites in the control indicates that the test cell has OS caused by exposure of the cell to ultraviolet light and / or senescence.

[0012] As used herein, the term "cell" refers to a naturally occurring or modified intact living cell. The cell may be isolated from other cells, mixed with other cells in culture, or in a (partial or complete) tissue or organism. Specifically, the cell may be a eukaryotic cell. More specifically, the cell may be a mammalian cell. The term "mammalian cell" refers to any cell from a mammalian subject. The cell may also be a cell cultured and amplified from a cell obtained from a subject. The cell may also be genetically modified to express recombinant proteins and / or nucleic acids. Mammalian cells may come from humans and other primates, including non-human primates, such as chimpanzees and other apes and monkeys; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; rodents, such as mice, rats, rabbits, hamsters, and guinea pigs; birds, including poultry, wild birds, and birds for hunting, such as chickens, turkeys, and other quails, ducks, geese, and the like. Specifically, the subject is a mammal. More specifically, the mammal is selected from mice, rats, guinea pigs, dogs, mini pigs, humans, cows, sheep, pigs, goats, horses, donkeys and mules. Specifically, the mammalian cell can be a skin cell, a stem cell or a cell derived therefrom. More specifically, the mammalian cell can be a skin cell.

[0013] As used herein, a "CpG site" or "methylation site" is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation by naturally occurring events in vivo or by events performed in vitro to chemically methylate the nucleotide. For example, there may be about 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. Specifically, the methylation status of at least one CpG site selected from the following list of 8 CpG sites in PTPRN2 is determined to determine whether the cell has OS caused by aging and / or UV exposure:

[0014] Table 1: Eight CpG sites differentially methylated in PTPRN2 in cells with OS caused by aging and / or UV exposure to determine whether cells have OS caused by aging and / or UV exposure:

[0015]

[0016]

[0017] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more methylated nucleotides.

[0018] As used herein, "CpG island" describes a DNA sequence segment that contains a functionally or structurally biased CpG density. For example, Yamada et al. described a set of criteria for determining a CpG island: it must be at least 400 nucleotides in length, have a GC content greater than 50%, and an OCF / ECF ratio greater than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266). Others define CpG islands more loosely as sequences with a length of at least 200 nucleotides, a GC content greater than 50%, and an OCF / ECF ratio greater than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745). In the context of the present invention, as used herein, the terms "methylation profile", "methylation pattern", "methylation state" or "methylation status" are used to describe the state, condition or condition of methylation of a genomic sequence, and these terms refer to the methylation-related characteristics of a DNA segment at a specific genomic locus. Such features include, but are not limited to, whether any cytosine (C) residues in the DNA sequence are methylated, the positions of methylated C residues, the percentage of methylated C residues on any particular stretch of residues, and allelic methylation differences due to, for example, differences in allelic origin.

[0019] The term "methylation state" refers to the state of a specific methylation site (i.e., methylated vs. unmethylated), which means that a residue or methylation site is methylated or unmethylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined. Therefore, the term "methylation profile" or also referred to as "methylation pattern" refers to the relative or absolute concentration of methylated C residues or unmethylated C residues of any specific residue segment in the genomic material of a biological sample. For example, if a cytosine (C) residue that is not normally methylated in a DNA sequence is methylated, it can be referred to as "hypermethylated"; and if a cytosine (C) residue that is normally methylated in a DNA sequence is not methylated, it can be referred to as "hypomethylated". Similarly, if a cytosine (C) residue in a DNA sequence (e.g., DNA from a sample nucleic acid of a test subject) is methylated compared to another sequence from a different region or from a different individual (e.g., relative to a normal nucleic acid or relative to a standard nucleic acid of a reference sequence), the sequence is considered to be highly methylated compared to the other sequence. Alternatively, if the cytosine (C) residues in a DNA sequence are not methylated compared to another sequence from a different region or a different individual, the sequence is considered to be hypomethylated compared to the other sequence. These sequences are referred to as "differentially methylated". The measurement of differential methylation levels can be performed in a variety of ways known to those skilled in the art. As a non-limiting example, one method is to measure the methylation level of a single interrogated CpG site, which is determined by a bisulfite sequencing method.

[0020] As used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not generally present in recognized typical nucleotide bases. For example, cytosine in its usual form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine in its usual form would not be considered a methylated nucleotide, while 5-methylcytosine would be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring, however, for the purposes of this article, thymine is not considered a methylated nucleotide when present in DNA. Typical nucleotide bases of DNA are thymine, adenine, cytosine, and guanine. Typical bases of RNA are uracil, adenine, cytosine, and guanine. Accordingly, a "methylation site" is a position in a target gene nucleic acid region where methylation is likely to occur. For example, a position containing CpG is a methylation site, where cytosine may or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached at a position at which the nucleotide can undergo methylation. These methylated nucleotides are commonly found in nature and, by far, methylated cytosine, which occurs mainly in the context of the dinucleotide CpG, but also in the context of CpNpG- and CpNpN-sequences, can be considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but they will not be taken into account for any aspect of the present invention.

[0021] In the context of the present invention, as used herein, the terms "methylation profile", "methylation pattern", "methylation state" or "methylation status" are used to describe the state, condition or status of methylation of a genomic sequence, and these terms refer to methylation-related features of a DNA fragment at a specific genomic locus. Such features include, but are not limited to: whether any cytosine (C) residue in the DNA sequence is methylated, the position of the methylated C residues, the percentage of methylated C on any specific residue stretch, and allelic methylation differences due to, for example, differences in allelic origin.

[0022] The term "hypermethylation" refers to an average methylation state that corresponds to an increase in the presence of 5-mCyt at one or more CpG dinucleotides in the 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. Specifically, a control refers to cells without an indication of OS.

[0023] The term "hypomethylation" refers to an average methylation state corresponding to a decrease in the presence of 5-mCyt at one or more CpG dinucleotides in the 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. Specifically, a control refers to cells without an indication of OS.

[0024] As used herein, the term "gene" refers to the corresponding genomic DNA sequence, including any promoter and regulatory sequences of the gene (e.g., enhancers and other gene sequences involved in regulating gene expression), and / or the body of the gene itself. The gene sequence can be an expressed sequence (e.g., expressed RNA, mRNA, cDNA). Further, when it is known that the SNP is within a gene, the term should include all its sequence variants.

[0025] As used herein, the term "genomic material" refers to a nucleic acid molecule or fragment of the genome of a subject or subject population. Specifically, such nucleic acid molecule or fragment is DNA or RNA or a hybrid thereof, and most preferably is a molecule of the DNA genome of a subject or subject population.

[0026] As used herein, "promoter" or "gene promoter" is used interchangeably with the term "regulatory region" or "regulatory sequence", which refers to a corresponding continuous gene DNA sequence extending from 1.5 kb upstream to 1.5 kb downstream relative to the transcription start site (TSS), or a continuous portion thereof. Specifically, a "regulatory region" refers to a corresponding continuous gene DNA sequence extending from 1.5 kb upstream to 0.5 kb downstream relative to the TSS. In some examples, a "regulatory region" refers to a corresponding continuous gene DNA sequence extending from 1.5 kb upstream to the downstream edge of a CpG island, which overlaps with a region extending from 1.5 kb upstream of the TSS to 1.5 kb downstream (and in this case, the regulatory region may therefore extend further beyond 1.5 kb downstream), and a continuous portion thereof. Specifically, for PTPRN2, any CpG dinucleotide in the gene that is coordinately methylated with the "regulatory region" of the gene has a tangible diagnostic / classification utility disclosed herein.

[0027] As used herein, "DNA sample" refers to DNA extracted from cells according to any aspect of the invention using methods known in the art.

[0028] Specifically, when differential methylation is detected in the test cell, that is, the cell shows hypermethylation or hypomethylation at at least one CpG site compared to the control (i.e., a cell with no OS indication), the test cell has OS. More specifically, when differential methylation is detected in one of the 8 specific CpG sites listed in Table 1 above, that is, the cell shows hypermethylation or hypomethylation at at least one CpG site in the list of 8 CpG sites in Table 1 compared to the control (i.e., a cell with no OS indication or a cell with OS indication caused by external factors other than UV exposure and / or aging), the test cell has OS caused by UV exposure and / or aging.

[0029] Specifically, in step (a), the methylation status of at least 2, 3, 4, 5, 6, 7 or 8 CpG sites of Table 1 in PTPRN2 is determined.

[0030] The method according to any aspect of the present invention may further comprise the following steps:

[0031] (i) Prior to step (a), the DNA sample is subjected to bisulfite modification.

[0032] "Bisulfite treatment" of genomic DNA is used interchangeably with the term "bisulfite modification", and refers to the treatment of genomic DNA with a deaminating agent (e.g., bisulfite), which can be used to treat all DNA, whether methylated or not. Specifically, the term "bisulfite" as used herein encompasses any suitable type of bisulfite, such as sodium bisulfite, or can chemically convert cytosine (C) into uracil (U) without chemically modifying methylated cytosine, and therefore can be used for other chemical agents that differentially modify DNA sequences based on the methylation state of DNA, such as U.S. Patent Publication US2010 / 0112595. As used herein, the reagent of "differential modification" methylation or non-methylation of DNA encompasses any reagent that modifies methylation and / or non-methylation of DNA in the following method, and produces a distinguishable product from methylation and non-methylation of DNA by the process, thereby allowing identification of DNA methylation state. Such processes may include but are not limited to chemical reactions (e.g., bisulfite converts C into U) and enzyme treatments (e.g., methylation-dependent endonuclease cleavage). Thus, an enzyme that preferentially cleaves or digests methylated DNA is able to cleave or digest DNA molecules with greater efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly greater efficiency when the DNA is not methylated.

[0033] Therefore, prior to performing step (a) according to any aspect of the present invention, the genomic DNA contained in / obtained or extracted from the cell is first subjected to a bisulfite treatment.

[0034] Alternative methods available in the art may be used instead of bisulfite treatment. The skilled person will understand which other methods will be used. 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)).

[0035] The cells used according to any aspect of the present invention are obtained from a biological sample selected from blood, brain, sperm, and any other tissue or sample that provides genomic DNA to be used in the method according to any aspect of the present invention. Specifically, the biological sample can include any biological material containing DNA obtained from a subject, and can be liquid, solid, or both, can be tissue or bone, or body fluid, such as blood, lymph, etc. Specifically, the biological sample that can be used in the present invention can include biological cells or fragments thereof.

[0036] The term "test" used in conjunction with the term cell herein refers to a cell undergoing a method according to any aspect of the invention and is the basis for the analytical application of the invention. Thus, a "test cell" is a cell or group of cells being tested according to any aspect of the invention, or a profile obtained or generated in this context. Conversely, the term "reference" or "control" shall refer to an entity that is generally predetermined for comparison with the test entity. Specifically, a "test cell" refers to a cell being tested for OS, where the methylation status needs to be determined, and a "control" refers to a cell without OS, where the methylation status is known and used as a reference.

[0037] According to a further aspect of the present invention, there is provided a method for identifying oxidative stress (OS) caused by aging and / or ultraviolet (UV) exposure in at least one cell, comprising detecting differences in the expression of protein tyrosine phosphatase receptor type N2 (PTPRN2).

[0038] As used herein, the term "epigenetic marker" refers to the CpG sites of the gene itself and / or the gene body and / or the CpG sites of the gene regulatory region, which includes epigenetic modifications, wherein the epigenetic modifications make the gene usable as a marker for determining a specific disease. Specifically, the term "epigenetic marker" is defined as at least one of a DNA methylation marker, a histone modification marker, and a deacetylase marker. More specifically, the epigenetic marker of OS caused by aging and / or ultraviolet (UV) exposure in a cell is the differential methylation found in the CpG sites on the PTPRN2 gene body and / or regulatory region. Even more specifically, the epigenetic marker of OS caused by aging and / or ultraviolet exposure is the differential methylation of 8 CpG sites in Table 1 above, relative to control cells without OS or with OS caused by other external factors other than aging and / or ultraviolet exposure, the differential methylation is found in the gene body and / or regulatory region of PTPRN2 in cells with OS caused by aging and / or ultraviolet exposure. Specifically, promoter-specific high / low methylation leads to changes in different gene expressions. More specifically, differential methylation of CpG sites in PTPRN2 results in differential expression of PTPRN2. Differential expression of genes relative to controls where differential methylation does not occur may indicate OS in the test cells, particularly OS caused by aging and / or UV exposure. In one example, hypermethylation of the promoter is associated with inactivation of PTPRN2 gene expression. In another example, hypomethylation of the promoter is associated with (over) activation of PTPRN2 gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a graph showing the number of probes that are differentially methylated in different gene bodies and promoter regions of cells in which artificial OS was induced (high intensity UV for 24 hours) according to Example 1. It can be seen that the genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (receptor-like protein tyrosine phosphatase) have the highest distribution of differentially methylated probes. Other genes that are also differentially methylated in cells with OS are shown.

[0041] Figure 2 is a graph showing the number of probes that are differentially methylated in different gene bodies and promoter regions of cells in which artificial OS was induced (low intensity UV 7 for 2 hours) according to Example 1. It can be seen that the genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (receptor-like protein tyrosine phosphatase) have the highest distribution of differentially methylated probes. Other genes that are also differentially methylated in cells with OS are shown.

[0042] Figure 3is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions of cells where artificial OS was induced (high H2O2 for 24 hours) according to Example 2. It can be seen that the genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (receptor-like protein tyrosine phosphatase) have the highest differentially methylated probe distribution. Other genes that are also differentially methylated in cells with OS are shown.

[0043] Figure 4 is a graph showing the number of differentially methylated probes in different gene bodies and promoter regions of cells where artificial OS was induced (low H2O2 for 24 hours) according to Example 2. It can be seen that the genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (receptor-like protein tyrosine phosphatase) have the highest differentially methylated probe distribution. Other genes that are also differentially methylated in cells with OS are shown.

[0044] Figure 5 is a diagram showing multiple probes that are differentially methylated in different gene bodies and promoter regions of a cell, wherein Artificial OS was induced in cells. It can be seen that the genes MAD1L1 (meiotic spindle arrest component) and PTPRN2 (receptor-like protein tyrosine phosphatase) have the highest differentially methylated probe distribution. Other genes that are also differentially methylated in cells with OS are shown.

[0045] Figure 6 is a graph showing the top 20 overlapping genes under different treatments associated with UV exposure and aging. Example

[0046] The above describes a preferred embodiment, and those skilled in the art will appreciate that the design, construction or operation of the preferred embodiment may be changed or modified without departing from the scope of the claims. For example, these changes are intended to be included in the scope of the claims.

[0047] Example 1: Oxidative stress of human tissues to ultraviolet rays

[0048] Artificial oxidative stress was induced in cell culture systems and skin tissue models to analyze the methylation status of promoters.

[0049] The T-Skin model was obtained from Episkin SA, France, and is composed of reconstructed human skin. Each skin model consists of a dermis equivalent covered by a stratified well-differentiated epidermis derived from normal human keratinocytes. Upon receipt of the skin model, it was incubated overnight in T-Skin medium at 37°C in a 5% CO2 incubator to recover.

[0050] To induce oxidative stress in the human tissue system, the skin model (5x replicates) was exposed to UV radiation (UVA 24 J / cm 2 +UVB 50mJ / cm 2 ). Exposure to UV radiation leads to the generation of ROS, which ultimately leads to the development of intracellular oxidative stress. The control skin model (5x replicates) was kept free of any UV radiation for 72 hours. After treatment, the skin models were collected and used Blood & Tissue Kit (Qiagen) was used to purify genomic DNA from tissue samples. or NanoDrop TM 2000 pairs of genomic DNA were quantified.

[0051] Using EZ DNA Methylation-GoldT M Kit (Zymo Research) was used to bisulfite convert genomic DNA (500 ng) from tissue samples. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the entire genome at single-nucleotide resolution.

[0052] Quality control and data processing

[0053] 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 the minfi (1.4.0) Bioconductor package, 18. Samples were quality checked to retain probes with detection P values ​​< 0.01 in one or more samples or average detection P values ​​< 0.05 across all samples. Samples were then normalized using function normalization (implemented by the preprocessFunnorm function in minfi) for type bias correction and background correction.

[0054] Before differential methylation analysis, probes with nonspecific binding, cross-reactive probes, probes affected by common SNPs, and probes annotated to chromosomes X and Y were also filtered out. Beta values ​​and M values ​​of normalized and filtered samples were calculated using getBeta and getM functions, respectively, and the samples were then subjected to further downstream analysis.

[0055] Differential methylation analysis

[0056] Differential methylation analysis was performed using the limma package version 3.50.1 and the DMRcate package version 2.8.5. A contrast matrix was established by comparing each corresponding treatment group with the control group, and the M values ​​were fitted according to the design and control models using the empirical Bayes algorithm. Probes with adjusted P values ​​less than 0.05 were considered differentially methylated positions (DMPs). Annotation was performed using Illumina HumanMethylation EPICkanno.ilmn12.hg19 and annotatr packages (1.20.0).

[0057] like Figure 1 As shown, one of the front genes that was differentially methylated in the promoter region was PTPRN2 (protein tyrosine phosphatase receptor N2). PTPRN2 is a phosphatidylinositol phosphatase that has the ability to dephosphorylate phosphatidylinositol-3-phosphate and phosphatidylinositol-4,5-bisphosphate, and plays an important role in lipid signal transduction, cell signal transduction, and membrane trafficking.

[0058] Example 2: Oxidative stress of human tissues to H2O2

[0059] Another way to induce oxidative stress in human tissue systems is through hydrogen peroxide treatment, which leads to the generation of intracellular ROS. Skin models (5x replicates) were treated with two different concentrations of hydrogen peroxide [100 μM (low) and 200 μM (high)] for 2 hours and maintained for 24 hours. Control skin models (5x replicates) were maintained without any hydrogen peroxide treatment for 24 hours. After treatment, the skin models were collected and used Blood & Tissue Kit (Qiagen) was used to purify genomic DNA from tissue samples. or NanoDrop TM 2000 pairs of genomic DNA were quantified.

[0060] The samples here were subjected to the same quality control and data processing methods as those disclosed in Example 1. Furthermore, the data obtained in Example 2 were subjected to the same differential methylation analysis as in Example 1.

[0061] The results are as follows Figure 3 and Figure 4 shown.

[0062] Example 3: Oxidative stress in cell culture systems

[0063] To study oxidative stress in cell culture systems, a culture medium containing high amounts of natural anthocyanins associated with antioxidant and anti-inflammatory properties was used. Mesenchymal stem cells (MSCs) were treated with Evonik (Batch: H-080719). Bone marrow derived MSCs were cultured for 1 week in Mesencult ACF Plus medium containing two doses of (4x replicates): 25μg / ml (low) and 100μg / ml (high). The culture medium was changed every two days for 1 week. As a control (4x replicates), MSCs were cultured in Mesencult ACF Plus medium for 1 week without any Process. Expect Processing produces the opposite reaction to OS.

[0064] The cell pellets were then collected and used Blood & Tissue Kit (Qiagen) was used to purify genomic DNA from cell pellets. or NanoDrop TM 2000, and genomic DNA was quantified.

[0065] Using EZ DNA Methylation-Gold TM Kit (Zymo Research) was used to convert genomic DNA (500 ng) from cell pellets. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the entire genome at single-nucleotide resolution.

[0066] DNA methylation analysis has been demonstrated to be a powerful analytical tool for accurately identifying tissues of origin and the influence of environmental factors. It has several advantages as a biomarker classifier because it is a stable marker and can facilitate quantitative analysis at single nucleotide resolution.

[0067] The samples here were subjected to the same quality control and data processing methods as those disclosed in Example 1. Furthermore, the data obtained in Example 3 were subjected to the same differential methylation analysis as in Example 1.

[0068] like Figure 5 As shown, one of the front genes that was differentially methylated in the promoter region was PTPRN2 (protein tyrosine phosphatase receptor N2). PTPRN2 is a phosphatidylinositol phosphatase that has the ability to dephosphorylate phosphatidylinositol-3-phosphate and phosphatidylinositol-4,5-bisphosphate, and plays an important role in lipid signal transduction, cell signal transduction, and membrane trafficking.

[0069] Results of Examples 1, 2 and 3

[0070] Two independent experimental setups for analyzing the methylation status of promoters during oxidative stress have shown differential methylation of the PTPRN2 promoter, suggesting it as a potential biomarker for oxidative stress.

[0071] Example 4: Oxidative stress in aging human tissues

[0072] Another way in which oxidative stress is induced in human tissue systems is through aging, which leads to the intracellular generation of reactive oxygen and nitrogen species (RONS).

[0073] The skin models were cultured in deep well plates with culture medium for 7 days (4x replicates), 14 days (4x replicates), and 21 days (4x replicates) to induce senescence in skin tissue. The control skin models (4x replicates) were collected at 0 hours to isolate genomic DNA. The skin models were collected after 7 days, 14 days, and 21 days and used Blood & Tissue Kit (Qiagen) was used to purify genomic DNA from tissue samples. or NanoDrop TM 2000, and genomic DNA was quantified.

[0074] Using EZ DNA Methylation-Gold TM Kit (Zymo Research) was used to convert genomic DNA (500 ng) from tissue samples. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the entire genome at single nucleotide resolution.

[0075] Oxidative stress in human tissues to ultraviolet rays

[0076] To induce oxidative stress in the human tissue system, the skin model was exposed to UV radiation (UVA 24 J / cm 2 +UVB 50mJ / cm 2 ) and cultured for 24 h (4x replicates), 48 h (4x replicates), 72 h (4x replicates), 96 h (4x replicates) and 120 h (4x replicates).

[0077] UV radiation exposure leads to the generation of ROS, which ultimately leads to the development of intracellular oxidative stress. The control skin models were maintained for 24 hours (4x repetitions), 48 hours (4x repetitions), 72 hours (4x repetitions), 96 hours (4x repetitions), and 120 hours (4x repetitions) without any UV radiation. After treatment, the skin models were collected and used Blood & Tissue Kit (Qiagen) was used to purify genomic DNA from tissue samples. or NanoDrop TM 2000, and genomic DNA was quantified.

[0078] Using EZ DNA Methylation-Gold TM Kit (Zymo Research) was used to bisulfite convert genomic DNA (500 ng) from tissue samples. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the entire genome at single-nucleotide resolution.

[0079] Quality control and data processing

[0080] A total of 52 samples from 7 different treatments and their respective controls were analyzed (Table 2). The 7 treatments can be divided into 2 types of oxidative stress - 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 2 treatments were oxidative stress by aging (after 7 days and 14 days, respectively). Both treatments were compared with the same control group. Methylation EPIC array data processing was performed in R version 4.2.2 (2021-11-10r83330) using minfi version 1.42.0. The raw intensity data (IDAT) were imported into R (4.2.2) and processed using the minfi (1.42.0) Bioconductor package. The samples were quality checked to retain probes with detection P values ​​< 0.01 in one or more samples or average detection P values ​​< 0.05 across all samples. Then, samples were normalized using functional normalization (implemented by the preprocess Funnorm function in minfi) to correct for type bias and background.

[0081] Before differential methylation analysis, probes with nonspecific binding, cross-reactive probes, probes affected by common SNPs, and probes annotated to chromosomes X and Y were also filtered out. Beta values ​​and M values ​​of normalized and filtered samples were calculated using getBeta and getM functions, respectively, and the samples were then subjected to further downstream analysis.

[0082] Table 2: Sample information of batch 2

[0083]

[0084] Differential methylation analysis

[0085] Pairwise differential methylation analysis (7 pairs in total) was performed using the limma package version 3.52.4. UV and aging samples were analyzed separately. A contrast matrix was established by comparing each corresponding treatment group to the control group, and the empirical Bayes algorithm was used to fit the M values ​​according to the design and control models. Probes with adjusted P values ​​below 0.05 were considered differentially methylated positions (DMPs). Annotation was performed using Illumina Human Methylation EPICanno.ilm10b2.hg19. Genes that overlapped in all treatments were then found, and their DMPs were analyzed to identify DMPs present in all contrasts.

[0086] like Figure 6 As shown, one of the front genes that was differentially methylated in all comparisons was PTPRN2 (N2-type protein tyrosine phosphatase receptor). PTPRN2 is a phosphatidylinositol phosphatase with the ability to dephosphorylate phosphatidylinositol-3-phosphate and phosphatidylinositol-4,5-bisphosphate, which plays an important role in lipid signal transduction, cell signal transduction and membrane transport. The PTPRN2 gene has a total of 670 unique DMPs, of which 8 DMPs are visible in all comparisons (Table 3).

[0087] Table 3: Eight DMPs of PTPRN2 shared between all treatments and their methylation status in each treatment.

[0088] cpg site chromosome Location 7-Aging 14_Aging uv_24 uv_48 uv_72 uv_96 uv_120 cg11991903 chr7 157794519 Low Low high high high high high cg17530002 chr7 157701262 Low Low high high high high high cg25330725 chr7 158180337 high high Low Low Low Low Low cg21459816 chr7 157588756 Low Low high high high high high cg03399048 chr7 158180335 high high Low Low Low Low Low cg26059287 chr7 157468443 Low Low high high high high high cg05416758 chr7 157783082 Low Low high high high high high cg21380084 chr7 157937891 high high Low Low Low Low Low

Claims

1. A method for identifying oxidative stress (OS) caused by aging and / or ultraviolet (UV) exposure in a test cell, comprising: (a) determining the methylation status of at least one CpG site in the regulatory region of protein tyrosine phosphatase receptor N2 (PTPRN2) and / or PTPRN2 in a DNA sample obtained from the test cell, (b) The methylation status of CpG sites of PTPRN2 from (a) was compared with controls without OS induced by aging and / or UV exposure. The difference in methylation status of the CpG sites of PTPRN2 and / or the regulatory region of PTPRN2 in the test cells compared with the CpG sites in the control indicates that the test cells have OS caused by cell exposure to ultraviolet rays and / or aging.

2. The method according to claim 1, wherein in step (a) the methylation status of at least one CpG site selected from the following list of 8 CpG sites in PTPRN2 is determined:

3. The method according to claim 2, wherein in step (a), the methylation status of at least 5 CpG sites in the list of 8 CpG sites in PTPRN2 is determined.

4. The method according to claim 2 or 3, wherein in step (a), the methylation status of all 8 CpG sites in the list of 8 CpG sites in PTPRN2 is determined.

5. The method according to any one of the preceding claims, further comprising the steps of: (i) before step (a), the DNA sample is subjected to bisulfite modification.

6. The method according to any one of the preceding claims, 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.

7. A method according to any one of the preceding claims, wherein the cell is a eukaryotic organism.

8. A method according to any one of the preceding claims, wherein the cell is from a mammal.

9. The method of claim 8, wherein the mammal is a mouse, rat, guinea pig, dog, mini pig, human, cow, sheep, pig, goat, horse, donkey, and mule.

10. The method according to any one of the preceding claims, wherein the cell is a skin cell, a stem cell or a cell derived therefrom.

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

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

13. The method of claim 11 or 12, wherein the cells are from a mammal selected from the group consisting of mouse, rat, guinea pig, dog, minipig, human, cow, sheep, pig, goat, horse, donkey and mule.

14. The method according to any one of claims 11 to 13, wherein the cell is a skin cell, a stem cell or a cell derived therefrom.

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