WO2022102885A1 - Technique de diagnostic du stress associé à la dermatite atopique utilisant des miarn dérivés d'exosomes - Google Patents

Technique de diagnostic du stress associé à la dermatite atopique utilisant des miarn dérivés d'exosomes Download PDF

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WO2022102885A1
WO2022102885A1 PCT/KR2021/005258 KR2021005258W WO2022102885A1 WO 2022102885 A1 WO2022102885 A1 WO 2022102885A1 KR 2021005258 W KR2021005258 W KR 2021005258W WO 2022102885 A1 WO2022102885 A1 WO 2022102885A1
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mmu
seq
mir
exosome
derived
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Korean (ko)
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우정민
성민경
서민수
강경구
성수은
최주희
이시준
김길수
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재단법인 대구경북첨단의료산업진흥재단
경북대학교 산학협력단
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a composition for diagnosing stress using the fact that the expression pattern of exosome-derived miRNA changes when stress is induced by atopic dermatitis.
  • stress refers to a non-specific biological response occurring in the body to various injuries or stimuli applied to the living body.
  • Response to external temporary stress may be a natural phenomenon, but if the response to stress persists for a long time, mental or physical damage may occur.
  • stress is not always perceptible to the subject to which the stress is applied, and in the case of animals, it is sometimes difficult to recognize that they are in a state of stress.
  • exosomes are small extracellular vesicles with a diameter of about 200 nm or less that are formed inside the cell and secreted out of the cell through multivesicles. Exosomes, which contain protein and genetic information of parental cells, have recently been suggested to be useful as biomarkers for various diseases.
  • Another object of the present invention is to provide a kit for diagnosing stress induced by atopic dermatitis, comprising the composition.
  • Another object of the present invention is to provide an information providing method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a specific miRNA derived from an exosome of an individual with the expression level of an exosome-derived miRNA of a control group will provide
  • the present invention provides mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3) , mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu -miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu -miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 1
  • the exosome may be a nerve-derived exo.
  • the agent may include a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
  • the present invention also provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.
  • the present invention also comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • the biological sample may be blood.
  • the exosome may be a nerve-derived exo.
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), when the expression level of one or more miRNAs selected from the group consisting of mmu-miR-466i-5p (SEQ ID NO: 8) and mmu-miR-5128 (SEQ ID NO: 9) is higher than that of the control group, the subject is in a stress state It may further include the step of determining that it is.
  • mmu-let-7i-5p SEQ ID NO: 10
  • mmu-miR-130a-3p SEQ ID NO: 11
  • mmu-miR-140-3p SEQ ID NO: 11
  • the present invention comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • the present invention analyzed miRNAs derived from exosomes of mice induced by stress due to atopic dermatitis, and then identified a number of miRNAs whose expression patterns change before and after stress treatment, and derived that they can be used as biomarkers. , it can be used in various fields related to the relationship between skin inflammation and stress and the stress diagnosis project.
  • FIG. 1 schematically shows the experimental design for the method and each group setting for inducing atopic dermatitis in Balb/c mice.
  • Figure 2 shows whether atopic dermatitis is induced after DNCB treatment is confirmed through skin tissue.
  • 3 is a graph confirming the expression pattern of specific proteins in the hippocampus of the brain at 2 days, 6 days, and 14 days after induction of atopic dermatitis.
  • Figure 4 shows the morphological confirmation of atopic dermatitis-induced animal serum-derived exosomes (A), the size distribution of the exosomes (B), and the results of confirming the exosome surface-specific expression protein.
  • Figure 5 confirms the expression of nerve-specific markers in some serum-derived exosomes and nerve-derived exosomes.
  • FIG. 6 shows the results of comparative analysis through NGS of the miRNA expression pattern of the nerve-derived exosomes in the atopic dermatitis-induced group compared to the normal group.
  • the present invention provides mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-let-7e-5p ( SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu-miR-466i-5p (SEQ ID NO: 7) 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 11) 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-mi
  • miRNA refers to an approximately 22 nt untranslated RNA that acts as a post-transcriptional repressor through base binding with the 3' untranslated region (UTR) region of mRNA.
  • exosome miRNAs can be carried out by extracting exosomes from a sample and detecting exosome miRNAs in the extract. Detection of these exosomes miRNAs can be measured by hybridization and amplification reactions, but is not limited thereto and can be easily carried out using various techniques known in the art.
  • the exosome may be a nerve-derived exosome.
  • exosomal miRNA a marker whose expression level is decreased in a sample of a biological sample
  • exosome miRNA which is a marker whose expression level is increased, in a stress state induced by atopic dermatitis. It refers to a molecule that can be used for detection of a marker by checking the expression level of
  • the agent may include a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
  • the detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers hybridized to a nucleic acid molecule or a complement of the nucleic acid molecule.
  • detection of exosome miRNAs using primers can be performed by amplifying the gene sequence using an amplification method such as PCR and then confirming whether the gene is amplified by a method known in the art.
  • a primer is a nucleic acid sequence having a short free 3' hydroxyl group.
  • a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.
  • stress can be diagnosed by performing PCR amplification using the sense and antisense primers specifically binding to the one or more miRNAs to check the expression level. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.
  • the probe refers to a nucleic acid fragment, such as RNA or DNA, corresponding to several bases to several tens of bases as long and is labeled.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.
  • stress can be diagnosed by performing hybridization using one or more of the above miRNAs and complementary probes and checking the expression level. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
  • primers or probes can be appropriately designed by those skilled in the art based on known sequences.
  • primers or probes can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods.
  • Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoros. amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
  • the expression level of miRNA can be measured according to a method commonly used in the art, for example, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting), or gene chip, and the like are included, but are not limited thereto.
  • RT-PCR reverse transcriptase polymerase reaction
  • Competitive RT-PCR competitive reverse transcriptase polymerase reaction
  • Real-time RT-PCR real-time reverse transcriptase polymerase reaction
  • RNase protection assay RNase protection assay
  • Northern blotting Northern blotting
  • gene chip and the like are included, but are not limited thereto.
  • the present invention provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.
  • the kit may include not only an agent for measuring the expression level of the exosome miRNA, but also tools, reagents, etc. commonly used in the art that are used to be suitable for use as a stress diagnosis kit.
  • the tool or reagent examples include, but are not limited to, a suitable carrier, a labeling material capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like.
  • the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.
  • the carrier includes a soluble carrier and an insoluble carrier
  • an example of the soluble carrier is a physiologically acceptable buffer known in the art, for example, PBS
  • an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.
  • kit of the present invention includes the above-described composition as a component, redundant descriptions are omitted in order to avoid excessive complexity of the present specification.
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • the “comparison with the expression level of the corresponding exosome-derived miRNA in the control sample” means that the comparison target between the subject and the control group is the same type of miRNA derived from the same type of exosome.
  • mmu-let-7a-5p SEQ ID NO: 1
  • mmu-let-7b-5p SEQ ID NO: 2
  • mmu-let-7c-5p SEQ ID NO: 3
  • mmu-let-7e-5p SEQ ID NO: 4
  • mmu-miR-126a-5p SEQ ID NO: 5
  • mmu-miR-3473b SEQ ID NO: 6
  • mmu-miR-3473e SEQ ID NO: 7
  • determining that the subject is in a stress state may further include.
  • mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16) ), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 19) No.
  • the term "low expression” used while referring to the expression level of the exosome miRNA is a biomarker (exosome-derived miRNA in the present invention) indicating an abnormal process, disease, or other condition in an individual or a symptom thereof.
  • a biomarker exosome-derived miRNA in the present invention
  • high expression used while referring to the expression level of the exosomal miRNA in the present specification is when the biomarker indicates or is a symptom of an abnormal process, disease, or other condition within the subject, from a healthy or normal subject or a subject for comparison Refers to a value or level of a biomarker in a biological sample that is higher than a value or level range of a biomarker detected in the obtained biological sample.
  • biological sample refers to any sample obtained from an individual in which the expression of miRNA of the present invention can be detected.
  • the "individual” may correspond without limitation as long as it is a subject showing a similar pattern to the atopic induced group of the present invention when stress is induced by atopic dermatitis, and may preferably be a mammal.
  • the biological sample is any one selected from the group consisting of blood, saliva, biopsy, skin tissue, liquid culture, feces and urine, but is not particularly limited thereto.
  • it is blood, and it can be prepared by processing by a method commonly used in the art.
  • the exosome may be a nerve-derived exosome.
  • the present invention comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • Example 1-1 laboratory animal
  • mice were housed in conditions of temperature, humidity, and controlled room lighting (24 ⁇ 2 °C, 50 ⁇ 20 % relative humidity, 12/12 h light/dark cycle with lights on at 07:00). In addition, mice were given unrestricted access to food and water.
  • the mice and reagents used in the present invention are commercially available.
  • Example 1-2 Atopic dermatitis model induced by DNCB
  • Atopic dermatitis model was induced using 1-Chloro-2,4-dinitrobenzene (DNCB) (Sigma).
  • DNCB 1-Chloro-2,4-dinitrobenzene
  • FIG. 1 The protocol for inducing atopic dermatitis in each group is shown in FIG. 1 .
  • mice were divided into control groups, DNCB 2 days, DNCB 6 days and DNCB 14 days, and then the dorsal hair was removed.
  • DNCB day 2 group mice were treated once with 200 ⁇ L of 1% DNCB diluted acetone and olive oil (4:1) and sacrificed 24 hours later.
  • mice were treated twice with 200 ⁇ L of 1% DNCB on days 0 and 3, followed by sacrifice on day 6.
  • mice in the DNCB 14-day group were sensitized with 200 ⁇ L of 1% DNCB on days 0 and 3, then treated with 2% DNCB daily from days 6 to 13, and sacrificed on day 14.
  • Brains were dissected and fixed in 10% neutral buffered formalin. The tissue was then processed and embedded in paraffin. Brain paraffin block sections were cut to a thickness of 4 ⁇ m at the level of the hippocampus.
  • Hematoxylin and eosin (H&E) staining was performed using a DAKO CoverStainer (Agilent, Santa Clara, CA, USA). Immunohistochemistry (IHC) was performed to confirm the expression of the neuroinflammatory marker protein. Brain sections were prepared for anti-BDNF (dilution 1:500; Abcam, Abcam, Cambridge, UK), COX2 (dilution 1:250; Abcam, Abcam, Cambridge, UK), GFAP (dilution 1:500; Abcam, Cambridge, UK). Stained with primary antibody. Next, the labeled polymer DAKO EnVisionTM + System-HRP (Agilent, Santa Clara, CA, USA) was sectioned immunohistochemically according to the manufacturer's instructions.
  • IHC Immunohistochemistry
  • brain sections were scanned using a Pannoramic SCAN II (3DHISTECH Kft., Budapest, Hungary). Micrographs were taken with a CaseViewer (3DHISTECH Kft., Budapest, Hungary). Finally, hippocampal neuroinflammatory markers were quantified with Imaged J software (NIH, Bethesda, MD, USA).
  • ExoQuick solution (System Biosciences, Palo Alto, CA, USA) was used to isolate exosomes from serum by modifying the manufacturer's instructions. First, 3 mL serum was pelleted through centrifugation at 3,500 ⁇ g at 4 °C for 15 min to remove cell debris. Next, 3 mL of debris-deleted serum and 885 ⁇ L of ExoQuick solution were mixed. After centrifugation of the mixture at 3,500 ⁇ g for 10 min, the pellet was resuspended in 200 ⁇ l PBS.
  • Neuronal exosomes were isolated by referring and modifying the conventional literature (Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes. Front Neurosci. 2017 May 22;11:278.). Total exosomes containing 200 ⁇ L PBS were incubated with 4 ⁇ L of anti-CD171 antibody (Bioss Antibodies, Beijing, China) for 1 hour at 4° C. in a rotating mixer. After addition of 15 ⁇ L PierceTM streptavidin + UltralinkTM resin (Thermo Fisher Scientific) and 25 ⁇ L PBS, the mixture was incubated in a rotating mixer at 4° C. for 1 hour and then centrifuged at 500 ⁇ g at 4° C. for 10 minutes.
  • Anti-CD171 antibody Bioss Antibodies, Beijing, China
  • the supernatant was removed from the sample and the pellet was resuspended in 200 ⁇ L 0.1M Glycine-HCl (Biosesang, Seongnam, Korea). After mixing for 10 seconds and vortexing for 30 seconds, the mixture was pelleted by centrifugation at 4,500 x g for 10 minutes at 4 °C. The supernatant was transferred to a new tube and then 15 ⁇ L Tris-HCl (Biosesang, Seongnam, Korea) and 25 ⁇ L PBS were added.
  • Exosomes isolated from serum were resuspended in cold distilled water. After loading the exosome suspension onto a formvar carbon coated grid (Ted Pella Inc.), the suspension was fixed in 2% paraformaldehyde for 10 minutes, the solution was removed, and the sample was dried. Grids were recorded using a bioTEM (Hitachi HT7700).
  • Example 1-7 Nanoparticle tracking analysis (NTA)
  • NTA was performed using a PMX120 (Particle Metrix) nanosight instrument according to the manufacturer's instructions.
  • tEV Total exosomes
  • nEV neuronal exosomes isolated from serum were each lysed using M-PER, HaltTM Protease and Phosphatase Inihhitor Cocktail (Thermo Scientific). Concentrations of exosome lysates were determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Next, 20 ⁇ g of exosome lysates were separated on a BoltTM 4-12% Bis-Tris Plus gel (Invitrogen, Carlsbad, CA, USA) and polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA).
  • BoltTM 4-12% Bis-Tris Plus gel Invitrogen, Carlsbad, CA, USA
  • PVDF polyvinylidene fluoride
  • the membrane was blocked with 5% skim milk supplemented with TBS-T for 1 h at room temperature.
  • TSG101 NovusBio, USA
  • CD171 Santa Cruz Biotechnology, Santa Cruz, CA, USA
  • TUJ1 Abcam, Cambridge, UK
  • NSE Abcam, Cambridge, UK
  • NeuN Abcam, Cambridge, UK
  • HRP horseradish peroxidase
  • a 600 ⁇ L sample was prepared by collecting neuronal exosomes isolated from serum. The concentration of exosomes was measured using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. As a result of BCA analysis, the concentrations of each sample were 1.59 mg/mL (control) and 2.34 mg/mL (DNCB 14 days). Exosome smRNA isolation and library preparation were performed in Macrogen (Seoul, Korea) using the SMARTer smRNA-Seq Kit (Clontech Laboratories Inc., Mountain View, USA) according to the manufacturer's instructions. MiRNA sequencing was then performed using a HiSeq 2500 system according to HiSeq 2500 System Instruction Manual Document # 15035786 v02 HCS 2.2.70.
  • H&E staining and IHC were performed to analyze the expression level of neuroinflammatory markers due to stress induced by atopic dermatitis.
  • Histopathological staining no significant difference was found in the hippocampus of DNCB 2 days, DNCB 6 days, and DNCB 14 days in the photomicrograph compared to the control group (FIG. 3A).
  • IHC staining was performed and quantified, no significant change in BDNF expression was observed in the hippocampus (Fig.
  • NGS Next-generation sequencing
  • miRNAs upregulated in DNCB for 14 days compared to controls were mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7c-5p, mmu-let-7e-5p, mmu-miR- 126a-5p, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-466i-5p and mmu-miR-5128 (FIG. 7).
  • Down-regulated miRNAs are mmu-let-7i-5p, mmu-miR-130a-3p, mmu-miR-140-3p, mmu-miR-142a-3p, mmu-miR-16-5p, mmu-miR-17 -5p, mmu-miR-185-5p, mmu-miR-19b-3p, mmu-miR-24-3p, mmu-miR-27a-5p, mmu-miR-29a-3p, mmu-miR-301a-3p , mmu-miR-451a, mmu-miR-669a-3p, mmu-miR-669o-3p and mmu-miR-93-5p (Fig. 8).
  • the sequence of the miRNA is shown in Table 1 below. And some of these miRNAs were identified as miRNAs showing expression changes in depression-related studies. According to previous studies that atopic dermatitis induces psychological stress and stress induces depression, NGS data suggest that these miRNAs can be used as psychological stress biomarkers.
  • the present invention analyzed miRNAs derived from exosomes of mice induced by stress due to atopic dermatitis, and then identified a number of miRNAs whose expression patterns change before and after stress treatment, and derived that they can be used as biomarkers. , it can be used in various fields related to the relationship between skin inflammation and stress and the stress diagnosis project.

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  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
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Abstract

La présente invention comprend l'analyse des miARN dérivés des exosomes d'une souris présentant un stress induit par une dermatite atopique, puis la confirmation que les profils d'expression d'une pluralité de miARN ont changé après le traitement induisant le stress par rapport à avant le traitement, ce qui permet de déduire que les miARN peuvent être utilisés comme biomarqueur, et donc que la présente invention peut être utilisée dans divers domaines connexes tels que la corrélation entre l'inflammation cutanée et le stress, et les activités de diagnostic du stress.
PCT/KR2021/005258 2020-11-13 2021-04-26 Technique de diagnostic du stress associé à la dermatite atopique utilisant des miarn dérivés d'exosomes WO2022102885A1 (fr)

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KR10-2020-0152203 2020-11-13

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KR20230036088A (ko) 2023-03-14
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