Detection of oxidative stress in cells using epigenetic means

Abstract

The present invention relates to a method of identifying oxidative stress (OS) in a test cell, comprising (a) determining the methylation status of at least one CpG site in a DNA sample obtained from the test cell, (b) comparing the methylation status of the CpG site from (a) to the methylation status of a control without OS, where the methylation status of the CpG site from (a) is determined as compared to the CpG site in the control, and (b) comparing the methylation status of the CpG site from (a) to the methylation status of the control without OS. The difference in methylation status of the CpG site in the test cell is indicative of the test cell having an OS.

Description

Field of the Invention

[0001] The present invention relates to a method for detecting oxidative stress (OS) in cells using epigenetic markers. Specifically, the method can identify OS in cells by determining the methylation state of CpG sites in test cells and comparing the resulting methylation state with a reference methylation state of control cells without OS. Differential methylation of CpG sites in test cells indicates that the test cells have OS. Background of the Invention

[0003] Living organisms are constantly subjected to various stresses from the external environment. In order to resist these stresses, they maintain their homeostasis through various regulatory systems. Oxidative stress refers to a serious imbalance between the level of reactive oxygen species (ROS) in cells and their antioxidant defense mechanisms. In order to survive under this stress, living organisms have a system called redox regulation to cope with stress, so as to maintain their homeostasis by regulating the redox status. This system functions to adapt to many external stress factors such as radiation, ultraviolet (UV) rays, environmental pollutants, high fever, low temperature, hypoxic conditions and infectious diseases, as well as oxidative stress from lifestyle-related diseases (such as cancer, diabetes, arteriosclerosis, hypertension and obesity). However, if this regulatory mechanism is destroyed for some reason or other, oxidative stress (OS) occurs. OS can cause cell damage, DNA fragmentation, apoptosis and cell death. Early detection of OS can prevent further damage in living organisms, prompting organisms to receive early treatment or start using protective measures.

[0004] Some methods for early detection of OS are known in the art. However, none of these methods known in the art have been formally used to detect OS in cells using genomic samples of the body.

[0005] Human skin is constantly exposed to oxidative stress and free radicals, such as large amounts of ROS, which are derived not only from common metabolic reactions, but also from continued exposure to air, radiation and UV rays, environmental pollutants, and physical and / or chemical agents (e.g., cosmetics). Under some conditions, the production of ROS may become so abundant that it may contribute to the pathogenesis of, for example, psoriasis or skin cancer. Oxidative damage caused by free radicals such as ROS is also a major cause of body aging in general, and skin aging in particular. Accordingly, there is a need in the art to detect OS in cells, such as skin cells, to prevent further damage to the cells.

[0006] Some methods for early detection of OS are known in the art. However, none of these methods known in the art have been formally used to detect OS in cells by using genomic samples of the body.

[0007] Human skin is constantly exposed to oxidative stress and free radicals, such as large amounts of ROS, which are derived not only from common metabolic reactions, but also from continued exposure to air, radiation and UV rays, environmental pollutants, and physical and / or chemical agents (e.g., cosmetics). Under some conditions, the production of ROS may become so abundant that it may contribute to the pathogenesis of, for example, psoriasis or skin cancer. Oxidative damage caused by free radicals such as ROS is also a major cause of body aging in general, and skin aging in particular. Accordingly, there is a need in the art to detect OS in cells, such as skin cells, to prevent further damage to the cells.

[0008] Description of the invention

[0009] The present invention attempts to solve the above-mentioned problems by providing a method for detecting oxidative stress (OS) in a test cell by comparing the methylation status of at least one CpG site in the test cell with the corresponding CpG site in a control cell without OS, wherein the presence of hypomethylation or hypermethylation at the CpG site in the test cell indicates that the test cell has OS.

[0010] Since environmental factors / factors such as ultraviolet exposure, aging, diet, etc. may trigger OS, the OS can further induce changes in the promoter CpG methylation state of genes by recruiting DNA methyltransferases (DNMTs) and TET enzymes to various promoters, so the biomarkers of differential methylation in cells with OS are crucial to overcome the problems mentioned above. In particular, CpG sites can be used as biomarkers for detecting OS in cells. Compared with the corresponding CpG sites in cells without OS, the CpG sites in cells with OS are differentially methylated (i.e., hypomethylated or hypermethylated). Accordingly, these CpG sites can be effectively used to determine whether a cell has OS. This is particularly advantageous because the use of epigenetics provides a means for predicting the onset of OS in cells, thus allowing OS to be treated as early as possible before causing even more damage to cells. Further, epigenetic markers are a long-term biomarker, that is, it is heritable and, if necessary, can also be used to detect OS in the next generation.

[0011] According to one aspect of the present invention, there is provided a method for identifying oxidative stress (OS) in a test cell, comprising

[0012] (a) determining the methylation status of at least one CpG site in a DNA sample obtained from a test cell,

[0013] (b) The methylation status of the CpG sites from (a) was compared with that of the control without OS,

[0014] Wherein a difference in the methylation status of the CpG site in the test cell compared to the CpG site in the control indicates that the test cell has OS.

[0015] As used herein, the term "cell" refers to a naturally occurring or modified intact living cell. Cells can be isolated from other cells, mixed with other cells in culture, or in a tissue (partial or complete) or organism. In particular, cells can be eukaryotic cells. More particularly, cells can be mammalian cells. The term "mammalian cell" refers to any cell derived from a mammalian subject. Cells can also be cells derived from cultures and amplifications of cells obtained from a subject. Cells can also be genetically modified to express recombinant proteins and / or nucleic acids. Mammalian cells can come from humans and other primates, including non-human primates, such as chimpanzees and other ape and monkey species; 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 game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, etc. In particular, the subject is a mammal. More particularly, the mammal is selected from the group consisting of mouse, rat, guinea pig, dog, miniature 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.

[0016] As used herein, "CpG sites" or "methylation sites" are nucleotides within nucleic acids (DNA or RNA) that are susceptible to methylation, either by naturally occurring events in vivo or by events that chemically methylate nucleotides in vitro. Some of these sites may be hypermethylated and some may be hypomethylated in cells with OS compared to cells without OS.

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

[0018] As used herein, "CpG island" describes a DNA sequence segment containing a functionally or structurally deviated CpG density. For example, Yamada et al. have 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 have less strictly defined CpG islands as sequences of at least 200 nucleotides in length, with 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, the terms "methylation profile", "methylation pattern", "methylation status" or "methylation state" are used herein to describe the status, situation or condition of methylation of a genomic sequence, and such terms refer to the characteristics of a DNA segment at a specific genomic locus associated with methylation. Such properties include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, for example, differences in allelic origin.

[0019] The term "methylation state" refers to the state of a specific methylation site (i.e., methylation relative to non-methylation), 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. Accordingly, the term "methylation profile" or "methylation pattern" refers to the relative content or absolute content of methylated C residues or unmethylated C residues at any specific residue segment in the genomic material of a biological sample. For example, if a cytosine (C) residue that is not usually methylated in a DNA sequence is methylated, it can be referred to as "hypermethylated"; and if a cytosine (C) residue that is usually 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 different individual (e.g., relative to a standard nucleic acid of a normal nucleic acid or a reference sequence), the sequence is considered to be hypermethylated compared to another sequence. Alternatively, if the cytosine (C) residues within a DNA sequence are non-methylated compared to another sequence from a different region or different individual, the sequence is considered to be hypomethylated compared to the other sequence. These sequences are said to be "differentially methylated". The measurement of differential methylation levels can be accomplished 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 each queried CpG site 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 a recognized typical nucleotide base. 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 may not be considered a methylated nucleotide, while 5-methylcytosine may 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 may not be considered a methylated nucleotide when present in DNA. Typical nucleotide bases for DNA are thymine, adenine, cytosine and guanine. Typical bases for RNA are uracil, adenine, cytosine and guanine. Accordingly, a "methylation site" is a location in a target gene nucleic acid region where methylation has the possibility of occurring. For example, a location containing CpG is a methylation site where cytosine may be methylated or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached to a position of the nucleotide accessible for methylation. These methylated nucleotides are commonly found in nature, and to date, the methylated cytosines that occur mainly in the context of the dinucleotide CpG, and 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 considered for any aspect of the present invention.

[0021] In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation status" or "methylation state" are used herein to describe the condition, situation or situation of methylation of a genomic sequence, and such terms refer to the properties of a DNA segment at a specific genomic locus that are associated with methylation. Such properties include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the percentage of methylated C at any particular residue stretch, and allelic differences in methylation due to, for example, differences in allelic origin.

[0022] The term "hypermethylation" refers to an average methylation status that corresponds to an increased presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides within a normal control DNA sample. In particular, a control refers to cells without an OS indicator.

[0023] The term "hypomethylation" refers to an average methylation status corresponding to a reduced presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of the test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides within the normal control DNA sample. In particular, the control refers to cells without OS indication.

[0024] As used herein, the term "genomic material" refers to a nucleic acid molecule or fragment of the genome of a subject or subject group. In particular, 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 group.

[0025] As used herein, "DNA sample" refers to DNA extracted from cells according to any aspect of the invention using methods known in the art.

[0026] In particular, when there is detected differential methylation 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 without OS indication), the test cell has OS. More particularly, when the CpG site in the test cell shows hypomethylation compared to the corresponding CpG site in the control cell, the test cell has OS. In another example, when the CpG site in the test cell shows hypermethylation compared to the corresponding CpG site in the control cell, the test cell has OS.

[0027] In particular, in the method according to any aspect of the present invention, in step (a), at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、

[0028] The methylation status of 100 CpG sites. The skilled person will be able to determine the number of CpG sites that need to be used in step (a) according to any aspect of the present invention. Even more particularly, the methylation status of at least two CpG sites is determined in step (a) of the method according to any aspect of the present invention.

[0029] More particularly, in step (a), the CpG site is selected from the list provided in Table 2:

[0030] Probe ID, Chromosome, Location Probe ID, Chromosome, Location Probe ID, Chromosome, Location Probe ID, Chromosome, Location cg10435849,chr21,47518747 cg02339482,chr3,191068318 cg23441673, chr11, 134831429 cg24637319,chr11,88071195 cg11349857,chr19,2388952 cg01955286,chr3,125475060 cg11785465,chr1,51717051 cg09332835,chr11,784136 cg00848693,chr16,69400894 cg23268879,chr7,5359951 cg01429408,chr1,156860314 cg22425466,chr9,140115987 cg21149107,chr14,71228406 cg01944087,chr15,69110737 cg02075410,chr4,151504138 cg18117149,chr6,41746516 cg02585025,chr16,74347151 cg06544141,chr1,27320384 cg17773376,chr3,107941759 cg15268826,chr4,132897691 cg18693345,chr5,2754148 cg20076186,chr3,128372563 cg12365107,chr3,52720035 cg23636099,chr2,114359251 cg25126321,chr11,66462313 cg20906710,chr12,50065474 cg23023126,chr19,308808 cg09788788,chr4,87468529 cg22155376,chr11,17591837 cg25319564, chr13, 114214573 cg08721076,chr2,172967670 cg26163843,chr14,34944819 cg17251097,chr10,81838583 cg21598631,chr2,242154679 cg09325695,chr14,37825967 cg13864229,chr14,37051830 cg16098051,chr17,39663178 cg20393002,chr3,120909792 cg11434693, chr1, 226349343 cg17942925, chr5, 180611441 cg23153556, chr11, 64900163 cg27626141, chr8, 103876469 cg04110544, chr5, 42424942 cg18659937, chr11, 66669653 cg11271430, chr4, 187984718 cg14075645, chr21, 47552343 cg07758428, chr11, 68193549 cg03611733, chr15, 45585637 cg22545649, chr4, 96470459 cg08351474, chr20, 4666916 cg10811474, chr19, 8428787 cg13514165, chr9, 130544369 cg05736120, chr6, 170125165 cg15047751, chr3, 52395045 cg23807890, chr2, 228736357 cg04360557, chr1, 33282743 cg14417917, chr9, 140065568 cg23247704, chr1, 116518985 cg16112558, chr17, 18157478 cg11578055, chr4, 10447440 cg23526353, ch beer, 100484905 cg05791356, chr3, 11578196 cg14751914, chr18, 46477404 cg01283246, chr5, 135266135 cg02884346, chr4, 6271501 cg01728507, chr4, 3563357 cg10853608, chr16, 713101 cg21606780, chr1, 197881327 cg12528713, chr4, 81187605 cg12833948, chr16, 1384648 cg11986082, chr2, 66652819 cg09009011, chr9, 117030773 cg05943476, chr2, 70780904 cg08714407, chr2, 24307071 cg12271199, chr1, 59248280 cg11926460, chr2, 234378272 cg03379894, chr5, 141131868 cg18908499, chr1, 247712237 cg25458727,chr3,10261761 cg11718315,chr15,65579480 cg17135325,chr3,160939158 cg15709169, chr12, 125549783 cg14418275,chr11,66243281 cg22054008,chr9,131873921 cg16362899, chr10, 121554998 cg12383788,chr1,22471748 cg19936912,chr4,152330012 cg07896133,chr16,2060022 cg25546329,chr19,40175243 cg05831191,chr16,67201012 cg02318866,chr17,74723064 cg25109721,chr17,48125064 cg00532451,chr1,228528913 cg06818207,chr6,6003238 cg18129198,chr1,63249693 cg20671801,chr12,51041328

[0031] Table 2. List of 96 differentially methylated CpG sites in OS

[0032] The method according to any aspect of the present invention further comprises the following steps:

[0033] (i) performing bisulfite modification on the DNA sample prior to step (a).

[0034] 'Bisulfite treatment' of genomic DNA, which can be used interchangeably with the term 'bisulfite modification', refers to treating genomic DNA with a deaminating agent such as bisulfite, which can be used to treat all DNA whether methylated or not. In particular, as used herein, the term "bisulfite" encompasses any suitable type of bisulfite, such as sodium bisulfite, or other chemical reagents, which 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 state of DNA, such as US Patent Publication US2010 / 0112595. As used herein, the reagents for "differentially modifying" methylated or unmethylated DNA encompass any reagents that modify methylated and / or unmethylated DNA in the process, by which the product can be distinguished from methylated and unmethylated DNA, thereby allowing the identification of DNA methylation states. Such processes may include, but are not limited to, chemical reactions (e.g., C to U conversion by bisulfite) and enzymatic treatments (e.g., by cleavage of methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one that is able to cleave or digest a DNA molecule with much 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.

[0035] Accordingly, prior to performing step (a) according to any aspect of the present invention, the genomic DNA contained / obtained or extracted from the cells is first subjected to bisulfite treatment.

[0036] Alternative methods available in the art may be used instead of bisulfite treatment. The skilled person will understand which other methods are 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).

[0037] The cells used according to any aspect of the present invention are obtained from a biological sample selected from blood, brain, semen and any other tissue or sample (which provides genomic DNA to be used in the method according to any aspect of the present invention). In particular, 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 fluids such as blood, lymph, etc. In particular, the biological sample that can be used in the present invention can include biological cells or fragments thereof.

[0038] The term "test" used in conjunction with the term cell herein refers to a cell that is subjected to 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 a group of cells tested according to any aspect of the invention, or a profile obtained or generated in this context. Conversely, the term 'reference' or 'control' shall denote a (mostly predetermined) entity for comparison with the test entity. In particular, a 'test cell' refers to a cell of an OS to be tested in which the methylation status must be determined, and a 'control' refers to a cell without an OS in which the methylation status is known and used as a reference.

[0039] OS according to any aspect of the invention may be caused by or may be a result of UV exposure, aging, H2O2 exposure, and combinations thereof (i.e., UV and H2O2 exposure, UV exposure and aging, H2O2 exposure and aging, or UV and H2O2 exposure and aging).

[0040] According to a further aspect of the present invention, there is provided a method for detecting the incidence of oxidative stress (OS) in a cell, the method comprising detecting an epigenetic change in at least one CpG site in the cell, wherein the detection of the epigenetic change indicates the incidence of OS, and wherein the epigenetic change is methylation.

[0041] In particular, the methylation is hypomethylation. DNA hypomethylation profiling may be very useful for cell culture systems ranging in stratification from 1D to 3D, stem cells under stress to differentiated skin tissue models.

[0042] As used herein, the term 'epigenetic changes' refers to chemical (e.g., methylation) or protein (e.g., histone) changes that occur to the body of a gene or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can affect genes that are passed from one generation to the next. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1Ais a scatter plot showing that a large number of probes have different methylation states in cells in which artificial OS (high UV for 24 hours) was induced according to Example 1. As can be seen, there are as many hypomethylated probes as hypermethylated probes.

[0045] Figure 1B is a box plot, which confirms Figure 1A The results in Figure 1 show that a large number of probes have different methylation states in cells in which artificial OS (high UV for 24 hours) was induced according to Example 1.

[0046] Figure 2A is a scatter plot showing that a large number of probes have different methylation states in cells in which artificial OS (low UV for 72 hours) was induced according to Example 1. As can be seen, there are as many hypomethylated probes as hypermethylated probes.

[0047] Figure 2B is a box plot, which confirms Figure 2A The results in Figure 1 show that a large number of probes have different methylation states in cells in which artificial OS (low UV for 72 hours) was induced according to Example 1.

[0048] Figure 3A is a scatter plot showing that a large number of probes have different methylation states in cells in which artificial OS (low H2O2 for 24 hours) was induced according to Example 2. As can be seen, there are a large number of hypomethylated probes in cells with OS compared to cells without OS.

[0049] Figure 3B is a box plot, which confirms Figure 3A The results in Figure 2 show that a large number of probes have different methylation states in cells in which artificial OS (low H2O2 for 24 hours) was induced according to Example 2.

[0050] Figure 4A is a scatter plot showing that a large number of probes have different methylation states in cells in which artificial OS (high H2O2 for 24 hours) was induced according to Example 2. As can be seen, there are a large number of hypomethylated probes in cells with OS compared to cells without OS.

[0051] Figure 4B is a box plot, which confirms Figure 4A The results in Figure 2 indicate that a large number of probes have different methylation states in cells in which artificial OS (high H2O2 for 24 hours) was induced according to Example 2.

[0052] Figure 5Ais a scatter plot showing a number of probes in which artificial OS was used according to Example 3 The cells induced in the cells have different methylation status. As can be seen, there are a large number of hypomethylated probes in cells with OS compared to cells without OS.

[0053] Figure 5B is a box plot, which confirms Figure 5A The results in which a large number of probes were used in the artificial OS according to Example 3 The cells induced in the cells had different methylation status. Example

[0054] The foregoing describes preferred embodiments, which, as will be appreciated by those skilled in the art, may be subject to changes or modifications in design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be covered by the scope of the claims.

[0055] Example 1

[0056] Oxidative stress on human tissue using UV

[0057] Artificial oxidative stress was induced in cell culture systems and skin tissue models to analyze the methylation status of promoters.

[0058] 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 with a stratified, well-differentiated epidermis derived from normal human keratinocytes. Upon receipt of the skin model, it was recovered by incubation in T-Skin medium overnight at 37°C in a 5% CO2 incubator.

[0059] To induce oxidative stress on human tissue systems, the skin model (5x repetitions) was exposed daily to UV radiation (UVA 24 J / cm 2 +UVB 50mJ / cm 2 )(High UV) for 24 hours.

[0060] In another group, the skin model (5x repetitions) was exposed daily to UV radiation (UVA 12 J / cm 2 +UVB25mJ / cm 2 )(Low UV) for 72 hours.

[0061] Exposure to UV radiation leads to the generation of ROS, which ultimately leads to the development of oxidative stress within the cells. The skin models of the control group (5x replicates) were maintained for 72 hours without any exposure to 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 for quantification.

[0062] Using EZ DNA Methylation-Gold TM Kit (Zymo Research), genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the genome at single nucleotide resolution.

[0063] Quality control and data processing

[0064] 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. A quality check was performed on the samples to retain probes with a detection P value < 0.01 in one or more samples or an average detection P value < 0.05 across all samples. Samples were then normalized using functional normalization (implemented by the preprocessFunnorm function in minfi) for type bias correction and background correction.

[0065] 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 the normalized and filtered samples were calculated using the getBeta and getM functions, respectively, and the samples were then subjected to further downstream analysis.

[0066] Differential methylation analysis

[0067] Differential methylation analysis was performed using software packages limma version 3.50.1 and DMRcate version 2.8.5. A comparison matrix was established by comparing each corresponding treatment group and control group, and the empirical Bayesian algorithm was used to fit the M value based on the design and comparison model. Probes with adjusted P values ​​lower than 0.05 were considered as differential methylation positions (DMPs). Annotations were performed using IlluminaHumanMethylationEPICkanno.ilmn12.hg19 and annotatr packages (1.20.0).

[0068] like Figure 1A As can be seen in 2A and 2B, there are many differentially methylated probes in cells with OS compared to control cells without OS. The results also show that there are as many hypomethylated probes as hypermethylated probes.

[0069] Example 2

[0070] Oxidative stress in human tissues using H2O2

[0071] Another method to induce oxidative stress in human tissues is through hydrogen peroxide treatment, which leads to intracellular ROS generation. 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. Skin models of the control group (5x replicates) were maintained for 24 hours without any treatment with hydrogen peroxide. 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 for quantification.

[0072] The samples here were subjected to the same quality control and data processing methods as disclosed in Example 1. Further, the data obtained from Example 2 were subjected to the same differential methylation analysis as disclosed in Example 1.

[0073] like Figure 3A and 3B As can be seen in 4A and 4B, there are many probes that are differentially methylated in cells with OS compared to control cells without OS. There are also many probes that are hypomethylated in cells with OS compared to cells without OS.

[0074] Example 3

[0075] use Oxidative stress in cell culture systems

[0076] To investigate oxidative stress in cell culture systems, mesenchymal stem cells (MSCs) were treated with Medox (Evonik, batch number: H-080719), which contains a large amount of natural anthocyanins associated with antioxidant and anti-inflammatory properties. Bone marrow-derived MSCs were cultured for 1 week in Mesencult ACF Plus Medium with two doses of Medox (4x replicates): 25 μg / ml (low) and 100 μg / ml (high). The medium with Medox was changed every other day for 1 week. As a control (4x replicates), MSCs were cultured in the absence of any The cells were cultured in Mesencult ACF Plus Medium for 1 week. Treatment produces the opposite reaction to OS.

[0077] This is followed by collection of the cell pellet and use Genomic DNA was purified from cell pellets using Blood & Tissue Kit (Qiagen). or NanoDrop TM 2000 for quantification.

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

[0079] DNA methylation profiling has been shown to be a powerful analytical tool to accurately identify tissues of origin and the effects of environmental factors. It has several advantages as a biomarker classifier because it is a stable marker and it can facilitate quantitative analysis at single nucleotide resolution.

[0080] The samples here were subjected to the same quality control and data processing methods as disclosed in Example 1. Further, the data obtained from Example 3 were subjected to the same differential methylation analysis as disclosed in Example 1.

[0081] For the MSC In the low treatment, 35,532 differentially methylated probes were identified (p<0.05), of which 15,368 probes were hypermethylated and 20,164 probes were hypomethylated.

[0082] like Figure 5A There are many differentially methylated probes in cells with OS compared to control cells without OS, as seen in A and B. There are a large number of hypomethylated probes in cells with OS compared to cells without OS.

[0083] Example 4

[0084] Oxidative stress on human tissues using UV rays

[0085] Artificial oxidative stress was induced in cell culture systems and skin tissue models to analyze the methylation status of promoters.

[0086] 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 with a stratified, well-differentiated epidermis derived from normal human keratinocytes. Upon receipt of the skin model, it was recovered by incubation in T-Skin medium overnight at 37°C in a 5% CO2 incubator.

[0087] To induce oxidative stress on human tissue systems, the skin model (5x repetitions) was exposed to UV radiation (UVA 24 J / cm 2 +UVB 50mJ / cm 2 ) (High UV) and cultured for 24 hours.

[0088] In another group, the skin model (5x repetitions) was exposed to UV radiation (UVA 12 J / cm per day). 2 +UVB 25mJ / cm 2 ) (low UV) and cultured for 24 hours.

[0089] Exposure to UV radiation leads to the generation of ROS, which ultimately leads to the development of oxidative stress within cells. The skin models of the control group (5x replicates) were maintained for 24 hours without any exposure to 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 for quantification.

[0090] Using EZ DNA Methylation-Gold TMKit (Zymo Research), genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the genome at single nucleotide resolution.

[0091] Oxidative stress in human tissues using H2O2

[0092] Another method to induce oxidative stress in human tissues is through hydrogen peroxide treatment, which leads to intracellular ROS generation. 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. Skin models of the control group (5x replicates) were maintained for 24 hours without any treatment with hydrogen peroxide. 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 for quantification.

[0093] Using EZ DNA Methylation-Gold TM Kit (Zymo Research), genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the genome at single nucleotide resolution.

[0094] Oxidative stress on human tissues using particulate matter 2.5

[0095] Another way to induce oxidative stress in human tissues is through treatment with particulate matter 2.5 (PM2.5), which, through its chemical composition and metals, leads to intracellular ROS generation. The skin model (5x replicates) was treated with two different concentrations of PM2.5 [15 μg / cm 2 (low) and 30 μg / cm 2 (high)] and maintained for 24 hours. The skin models of the control group (5x replicates) were maintained for 24 hours without any treatment with PM2.5. 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 for quantification.

[0096] Using EZ DNA Methylation-Gold TM Kit (Zymo Research), genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the genome at single nucleotide resolution.

[0097] Oxidative stress in human tissues using glyoxal

[0098] Another method of inducing oxidative stress on human tissue systems is through glyoxal treatment, which triggers oxidative stress by increasing intracellular ROS levels via the production of advanced glycation end products. Skin models (5x replicates) were treated with two different concentrations of glyoxal [0.5 mM (low) and 1 mM (high)] and maintained for 24 hours. Skin models of the control group (5x replicates) were maintained for 24 hours without any treatment with glyoxal. 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 for quantification.

[0099] Using EZ DNA Methylation-Gold TM Kit (Zymo Research), genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the genome at single nucleotide resolution.

[0100] Oxidative stress in human tissues using aging

[0101] Another way in which oxidative stress is systematically induced in human tissues is through aging, which leads to the generation of reactive oxygen and nitrogen species (RONS) within cells.

[0102] The skin model was maintained in deep well plates with culture medium for 14 days (6x replicates), where the culture medium was renewed after 7 days to induce senescence of the skin tissue. The skin model was collected after 14 days and used Blood & Tissue Kit (Qiagen) was used to purify genomic DNA from tissue samples. or NanoDrop TM 2000 for quantification.

[0103] Using EZ DNA Methylation-Gold TM Kit (Zymo Research), genomic DNA (500 ng) from tissue samples was subjected to bisulfite conversion. Methylation levels were quantified using the Infinium Methylation EPIC v2.0 Kit (Illumina), which can quantify more than 850,000 methylation sites across the genome at single nucleotide resolution.

[0104] Quality control and data processing

[0105] A total of 103 samples from 16 different treatments and their respective controls were analyzed. The treatments can be grouped into two batches. Batch 1 consists of all 24 hour treatments at high and low concentrations. All treatments in batch 1 share the same control group. Table 1 shows sample information about batch 1.

[0106] Methylation EPIC array data processing was performed in R version 4.2.2 (2021-11-10r83330) using minfi version 1.42.0. Raw intensity data (IDAT) were imported into R (4.2.2) and processed using the minfi (1.42.0) Bioconductor package. A quality check was performed on the samples to retain probes with a detection P value < 0.01 in one or more samples or an average detection P value < 0.05 across all samples. The samples were then normalized using functional normalization (implemented by the preprocesssFunnorm function in minfi) for type bias correction and background correction.

[0107] 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 the normalized and filtered samples were calculated using the getBeta and getM functions, respectively, and the samples were then subjected to further downstream analysis.

[0108] Table 1: Sample information about batch 1

[0109]

[0110] Differential methylation analysis

[0111] Use limma package version 3.52.4 to perform paired differential methylation analysis (16 pairs in total).Batch 1 sample is analyzed together.By comparing each corresponding treatment group and control group, a comparison matrix is ​​established, and the empirical Bayes algorithm is used to fit the M value based on the design and comparison model.The probe with the adjusted P value lower than 0.05 is regarded as a differential methylation position (DMP).After this, compare the DMPS in batch 1 comparison, batch 1 high concentration comparison and batch 1 low concentration comparison, to identify the common DMP that exists and always has the same methylation state in all comparisons in each group.

[0112] The Batch 1 comparison had a total of 96 common DMPs, the Batch 1 High comparison had a total of 616 common DMPs, and the Batch 1 Low comparison had a total of 238 common DMPs. Tables 2, 3, and 4 show a list of common DMPs within each group for the Batch 1 comparison, the Batch 1 High comparison, and the Batch 1 Low comparison, respectively.

[0113] Table 2 List of common DMPs within the group for batch 1 comparison (i.e. all treatments)

[0114] Probe ID, Chromosome, Location Probe ID, Chromosome, Location Probe ID, Chromogen, Location Probe ID, Chromosome, Location cg10435849,chr21,47518747 cg02339482,chr3,191068318 cg23441673, chr11, 134831429 cg24637319,chr11,88071195 cg11349857,chr19,2388952 cg01955286,chr3,125475060 cg11785465,chr1,51717051 cg09332835,chr11,784136 cg00848693,chr16,69400894 cg23268879,chr7,5359951 cg01429408,chr1,156860314 cg22425466,chr9,140115987 cg21149107,chr14,71228406 cg01944087,chr15,69110737 cg02075410,chr4,151504138 cg18117149,chr6,41746516 cg02585025,chr16,74347151 cg06544141,chr1,27320384 cg17773376,chr3,107941759 cg15268826,chr4,132897691 cg18693345,chr5,2754148 cg20076186,chr3,128372563 cg12365107,chr3,52720035 cg23636099,chr2,114359251 cg25126321,chr11,66462313 cg20906710,chr12,50065474 cg23023126,chr19,308808 cg09788788,chr4,87468529 cg22155376,chr11,17591837 cg25319564,chr13,114214573 cg08721076,chr2,172967670 cg26163843,chr14,34944819 cg17251097,chr10,81838583 cg21598631,chr2,242154679 cg09325695,chr14,37825967 cg13864229,chr14,37051830 cg16098051,chr17,39663178 cg20393002,chr3,120909792 cg11434693,chr1,226349343 cg17942925,chr5,180611441 cg23153556,chr11,64900163 cg27626141,chr8,103876469 cg04110544,chr5,42424942 cg18659937,chr11,66669653 cg11271430,chr4,187984718 cg14075645,chr21,47552343 cg07758428,chr11,68193549 cg03611733,chr15,45585637 cg22545649,chr4,96470459 cg08351474,chr20,4666916 cg10811474,chr19,8428787 cg13514165,chr9,130544369 cg05736120,chr6,170125165 cg15047751,chr3,52395045 cg23807890,chr2,228736357 cg04360557,chr1,33282743 cg14417917,chr9,140065568 cg23247704,chr1,116518985 cg16112558,chr17,18157478 cg11578055,chr4,10447440 cg23526353,chr4,100484905 cg05791356,chr3,11578196 cg14751914,chr18,46477404 cg01283246,chr5,135266135 cg02884346,chr4,6271501 cg01728507,chr4,3563357 cg10853608,chr16,713101 cg21606780,chr1,197881327 cg12528713,chr4,81187605 cg12833948,chr16,1384648 cg11986082,chr2,66652819 cg09009011,chr9,117030773 cg05943476,chr2,70780904 cg08714407,chr2,24307071 cg12271199,chr1,59248280 cg11926460,chr2,234378272 cg03379894,chr5,141131868 cg18908499,chr1,247712237 cg25458727,chr3,10261761 cg11718315,chr15,65579480 cg17135325,chr3,160939158 cg15709169,chr12,125549783 cg14418275,chr11,66243281 cg22054008,chr9,131873921 cg16362899,chr10,121554998 cg12383788,chr1,22471748 cg19936912,chr4,152330012 cg07896133,chr16,2060022 cg25546329,chr19,40175243 cg05831191,chr16,67201012 cg02318866,chr17,74723064 cg25109721,chr17,48125064 cg00532451,chr1,228528913 cg06818207,chr6,6003238 cg18129198,chr1,63249693 cg20671801,chr12,51041328

[0115]

[0116]

[0117]

[0118]

Claims

1. A method for identifying oxidative stress (OS) in a test cell, comprising (a) determining the methylation status of at least one CpG site in a DNA sample obtained from a test cell, (b) The methylation status of the CpG sites from (a) was compared with that of the control without OS, Wherein a difference in the methylation status of the CpG site in the test cell compared to the CpG site in the control indicates that the test cell has OS. 2 . The method of claim 1 , wherein the difference in methylation status is hypomethylation or hypermethylation of a CpG site in the test cell, and the hypomethylation or hypermethylation of the CpG site indicates OS in the test cell.

3. The method according to claim 1 or 2, wherein the CpG site is selected from the CpG site list in the following table:

4. The method according to any one of the preceding claims, wherein the methylation status of at least 2 CpG sites is determined in step (a).

5. The method according to any one of the preceding claims, wherein the methylation status of at least 3 CpG sites is determined in step (a).

6. The method according to any one of the preceding claims, wherein the methylation status of at least 5 CpG sites is determined in step (a).

7. The method of claim 3, wherein the methylation status of all listed CpG sites is determined in step (a).

8. The method according to any one of the preceding claims, further comprising the steps of: (i) performing bisulfite modification on the DNA sample prior to step (a).

9. The method according to any one of the preceding claims, wherein the cell is obtained from a biological sample selected from blood, brain, semen and any other tissue or sample providing genomic DNA.

10. The method of any one of the preceding claims, wherein the cell is a eukaryotic organism.

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

12. The method of claim 11, wherein the mammal is a mouse, rat, guinea pig, dog, miniature pig, human, cow, sheep, pig, goat, horse, donkey, and mule.

13. 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.

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