KR20230036088A - Atopic dermatitis-associated stress diagnosis technology using exosome-derived miRNA - Google Patents

Abstract

The present invention analyzed miRNAs derived from exosomes of mice from which atopic dermatitis stress is induced, and then identified a number of miRNAs of which the expression patterns changed before and after stress treatment, and found that the miRNAs could be used as biomarkers, and thus, the miRNAs can be used in various fields related to the relationship between skin inflammation and stress, stress diagnosis business and the like.

Description

Atopic dermatitis-associated stress diagnosis technology using exosome-derived miRNA}

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.

In general, stress refers to a non-specific biological response that occurs in the body to various injuries or stimuli applied to the living body. A response to external temporary stress may be a natural phenomenon, but if the response to stress lasts for a long time, it may cause mental or physical damage. In particular, stress is not always conscious of the subject to which stress is applied, and in the case of animals, it is sometimes difficult to recognize that they are in a stressed state, so a method for objectively diagnosing whether or not they are in a stressed state is needed.

On the other hand, exosomes are small extracellular vesicles with a diameter of about 200 nm or less that are formed inside cells and secreted out of cells through multiple vesicles. Exosomes, which contain parental cell proteins and genetic information, have recently been suggested to be used as biomarkers for various diseases.

However, technology for diagnosing atopic dermatitis-related stress using exosomes has not yet been studied, and in-depth research on this is required.

Republic of Korea Patent Publication No. 10-2016-0042703

An object of the present invention is to provide a composition for diagnosing stress induced by atopic dermatitis, including an agent for measuring the expression level of a specific miRNA derived from exosomes.

Another object of the present invention is to provide a kit for diagnosing stress caused by atopic dermatitis, including the above composition.

Another object of the present invention is to provide information for diagnosing stress induced by atopic dermatitis, comprising the step of comparing the expression level of a specific exosome-derived miRNA of an individual with the expression level of a control group's exosome-derived miRNA. is to provide

In order to achieve the object of the present invention, the present invention 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: 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: 17) 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 21) 23), an agent for measuring the expression level of at least one exosome-derived miRNA selected from the group consisting of mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) , A composition for diagnosing stress induced by atopic dermatitis is provided.

In one embodiment of the present invention, the exosome may be a nerve-derived exosome.

In one embodiment of the present invention, the agent may include primers or probes that specifically bind to the one or more exosome-derived miRNAs.

In addition, the present invention provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.

In addition, the present invention comprises the steps of (a) isolating exosomes from a biological sample of an individual; and

(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 exosomes 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-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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured in the corresponding exo of the control sample. Provided is an information providing method for diagnosing stress induced by atopic dermatitis, comprising the step of comparing the expression level of some derived miRNA.

In one embodiment of the present invention, the biological sample may be blood.

In one embodiment of the present invention, the exosome may be a nerve-derived exosome.

In one embodiment of the present invention, in 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 high compared to the control group, the subject is in a stress state The step of determining that is may be further included.

In one embodiment of the present invention, mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 10) in step (b) 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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o Determining that the subject is in a stress state when the expression level of one or more miRNAs selected from the group consisting of -3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) is lower than that of the control group may further include.

The present invention analyzed miRNAs derived from the exosomes of mice induced by atopic dermatitis stress, and then identified a number of miRNAs whose expression patterns changed before and after stress treatment, and found that they could be used as biomarkers. It can be used in various fields, such as the relationship between skin inflammation and stress and stress diagnosis business.

Figure 1 schematically shows the method of inducing atopic dermatitis in Balb/c mice and the experimental design for each group setting.
Figure 2 confirms whether or not atopic dermatitis is induced after DNCB treatment through skin tissue.
Figure 3 confirms the expression pattern of specific proteins in the hippocampus region of the brain on days 2, 6, and 14 after induction of atopic dermatitis.
Figure 4 shows the results of confirming the morphology of exosomes derived from animal serum derived from atopic dermatitis (A), confirming the size distribution of exosomes (B), and confirming proteins expressed on the surface of exosomes.
5 confirms the expression of nerve-specific markers in serum-derived exosomes and nerve-derived exosomes.
Figure 6 shows the results of comparative analysis of miRNA expression patterns of nerve-derived exosomes in the atopic dermatitis-induced group compared to the normal group through NGS.
7 shows miRNAs whose expression is elevated in the atopic dermatitis-induced group.
8 shows miRNAs whose expression is down in the atopic dermatitis-induced group.

The present invention 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: 6) 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: 10) 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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o- 3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) Stress induced by atopic dermatitis, including an agent for measuring the expression level of one or more exosome-derived miRNAs selected from the group consisting of A composition for diagnosis is provided.

In the present invention, "miRNA (microRNA)" refers to an untranslated RNA of about 22 nt that acts as a post-transcriptional repressor through base binding with the 3' untranslated region (UTR) of mRNA.

Detection of exosome miRNAs can usually be performed by extracting exosomes from a sample and detecting exosomes miRNAs in the extract. Detection of these exosome miRNAs can be measured by hybridization reaction and amplification reaction, but is not limited thereto and can be easily performed using various techniques known in the art.

In the present invention, the exosome may be a nerve-derived exosome.

The agent for measuring the expression level of the exosomal miRNA of the present invention is an exosomal miRNA, a marker whose expression level is reduced in a sample of a biological sample in a state of stress induced by atopic dermatitis, and an exosomal miRNA, a marker whose expression level is increased It refers to a molecule that can be used for detection of a marker by confirming the expression level of.

In the present invention, the agent may include primers or probes that specifically bind to the one or more exosome-derived miRNAs.

That is, detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers that hybridize to a nucleic acid molecule or a complement of the nucleic acid molecule. For example, detection of exosome miRNAs using primers can be performed by amplifying a 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 with a short free 3' hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. means Primers can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In the present invention, PCR amplification is performed using sense and antisense primers that specifically bind to the one or more miRNAs, and expression levels are checked to diagnose stress. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

A probe refers to a nucleic acid fragment such as RNA or DNA corresponding to as short as several bases to as long as several tens of bases 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. In the present invention, stress can be diagnosed by performing hybridization using one or more of the above miRNAs and a complementary probe to check the expression level. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.

Such primers or probes can be appropriately designed by those skilled in the art based on known sequences.

For example, primers or probes can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

The expression level of miRNA can be measured according to methods commonly used in the art, such as reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), and real-time reverse transcriptase polymerase reaction. (Real-time RT-PCR), RNase protection assay (RPA; Northern blotting) or gene chip, etc. are included, but are not limited thereto.

As another aspect of the present invention, the present invention provides a kit for diagnosing stress caused by atopic dermatitis comprising the above composition.

The kit may include not only agents for measuring the expression level of the exosome miRNA, but also tools and reagents commonly used in the art to be suitable for use as a stress diagnosis kit.

Examples of such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance 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, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly polymers such as acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, latex-coated magnetic microparticles, other paper, glass, metal, agarose, and combinations thereof.

Since the kit of the present invention includes the above-described composition as a configuration, redundant information is omitted to avoid excessive complexity of the present specification.

According to another aspect of the present invention, (a) isolating exosomes from a biological sample of an individual; and

(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 exosomes 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-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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured in the corresponding exo of the control sample. Provided is an information providing method for diagnosing stress induced by atopic dermatitis, comprising the step of comparing the expression level of some derived miRNA.

The “comparison with the expression level of the corresponding exosome-derived miRNA in the control sample” means that the object of comparison between the individual and the control group is the same type of miRNA derived from the same type of exosome.

In step (b) of the present invention, 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- Determining that the individual is in a stress state when the expression level of one or more miRNAs selected from the group consisting of miR-466i-5p (SEQ ID NO: 8) and mmu-miR-5128 (SEQ ID NO: 9) is higher than that of the control group may further include.

In step (b) of the present invention, 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) number 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 23) No. 24) and mmu-miR-93-5p (SEQ ID NO: 25), when the expression level of one or more miRNAs selected from the group consisting of is lower than that of the control group, determining that the subject is in a stressed state. can

In the present invention, the term "underexpression" used while referring to the expression level of the exosomal miRNA is a biomarker (in the present invention, exosome-derived miRNA) indicates an abnormal process, disease, or other condition in an individual, or a symptom thereof, It refers to a value or level of a biomarker in a biological sample that is lower than the value or level range of a biomarker detected in a biological sample obtained from a healthy or normal individual or a comparison subject.

The term "high expression" used herein while referring to the expression level of the exosomal miRNA is a biomarker that indicates or is a symptom of an abnormal process, disease, or other condition in an individual, from a healthy or normal individual or a comparable individual. It refers to a value or level of a biomarker in a biological sample that is higher than the value or level range of the biomarker detected in the obtained biological sample.

In the present invention, "biological sample" means any sample obtained from an individual in which expression of the miRNA of the present invention can be detected.

In the present invention, "individual" may correspond without limitation to any subject showing a miRNA expression similar to the atopic induction group of the present invention when stress is induced by atopic dermatitis, and preferably may be a mammal.

According to a preferred embodiment of the present invention, 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. Preferably, it is blood, and it can be prepared by treating it by a method commonly used in the art of the present invention.

In the present invention, the exosome may be a nerve-derived exosome.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

<Example 1: Materials and Methods>

Example 1-1. laboratory animal

Experiments were performed with 8-week-old male balb/c mice. The mice were housed under conditions of temperature, humidity, and controlled room lighting (24±2° C., 50±20% relative humidity, 12/12 hour light/dark cycle with lights on at 07:00). In addition, mice were allowed unrestricted access to food and water. The mice and reagents used in the present invention can be purchased commercially.

Example 1-2. DNCB-induced atopic dermatitis model

An atopic dermatitis model was induced using 1-Chloro-2,4-dinitrobenzene (DNCB) (Sigma). The protocol for inducing atopic dermatitis in each group is as shown in FIG. 1 . First, mice were divided into control groups, DNCB 2 days, DNCB 6 days, and DNCB 14 days, and then the dorsal hair was shaved. DNCB Day 2 group mice were treated once with 200 μL of 1% DNCB diluted acetone and olive oil (4:1) and sacrificed 24 hours later. In the 6-day DNCB group, mice were treated with 200 μL of 1% DNCB twice on days 0 and 3 and then sacrificed on day 6. Finally, mice in the 14-day DNCB group were sensitized with 200 μL of 1% DNCB on days 0 and 3, then treated with 2% DNCB daily from days 6 to 13, and sacrificed on day 14.

Example 1-3. Histopathology to confirm neuroinflammation in the hippocampus

Brains were dissected and then fixed in 10% neutral buffered formalin. Tissues were then processed and embedded in paraffin. Brain paraffin block sections were cut at the level of the hippocampus at 4 μm thickness.

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 slices were stained with anti-BDNF (dilution 1:500; Abcam, Abcam, Cambridge, UK), COX2 (dilution 1:250; Abcam, Abcam, Cambridge, UK), and 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. After staining, brain sections were scanned using Pannoramic SCAN II (3DHISTECH Kft., Budapest, Hungary). Micrographs were taken with CaseViewer (3DHISTECH Kft., Budapest, Hungary). Finally, neuroinflammation markers in the hippocampus were quantified with Imaged J software (NIH, Bethesda, MD, USA).

Example 1-4. Isolation of exosomes from serum

Exosomes were isolated from serum using ExoQuick solution (System Biosciences, Palo Alto, CA, USA) with modifications to the manufacturer's instructions. First, 3 mL serum was pelleted by centrifugation at 3,500 × g for 15 minutes at 4 °C to remove cell debris. Next, 3 mL of debris-deleted serum and 885 μL of ExoQuick solution were mixed. After the mixture was centrifuged at 3,500 × g for 10 minutes, the pellet was resuspended in 200 μl PBS.

Example 1-5. Isolation of neuronal exosomes from total exosomes isolated from serum

Neuronal exosomes were isolated by referring to 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 μL PBS were incubated with 4 μL anti-CD171 antibody (Bioss Antibodies, Beijing, China) for 1 hour at 4° C. on a rotating mixer. After adding 15 μL Pierce™ streptavidin + Ultralink™ resin (Thermo Fisher Scientific) and 25 μL PBS, the mixture was incubated on a rotating mixer for 1 hour at 4 °C, then centrifuged at 500 x g for 10 minutes at 4 °C. The supernatant was removed from the sample and the pellet was resuspended in 200 μL 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 15 μL Tris-HCl (Biosesang, Seongnam, Korea) and 25 μL PBS were added.

Example 1-6. Transmission electron microscopy (TEM)

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.

Example 1-8. flow cytometry

50 μl of total exosomes were incubated with 10 μl of aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) for 15 minutes at room temperature, and 3% BSA supplemented with 500 μl of PBS was added. Samples were incubated overnight on a rotating mixer. Bead-bound exosomes were centrifuged at 3,000 x g for 10 minutes and washed with 500 μl PBS. After washing, the samples were centrifuged at 3,000 x g for 10 minutes. The supernatant was discarded from the sample and the pellet was resuspended in 50 μl of PBS containing anti-CD9 and CD81 antibodies (BioLegend, San Diego, CA, USA) for 1 hour at room temperature. Samples were centrifuged at 3,000 x g for 10 minutes and the pellet was resuspended in 200 μl of PBS. Exosomal markers were detected using a flow cytometer Galios (Beckman Coulter) and analysis was performed with Kaluza software.

Example 1-9. western blot

Total exosomes (tEV) and neuronal exosomes (nEV) isolated from serum were lysed using M-PER, Halt™ Protease and Phosphatase Inihhitor Cocktail (Thermo Scientific), respectively. The concentration of exosome lysates was determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Next, 20 μg of exosome lysate was separated on a BoltTM 4-12% Bis-Tris Plus gel (Invitrogen, Carlsbad, CA, USA) and coated on a polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA). CA, USA). Then, the membrane was blocked with 5% skim milk supplemented with TBS-T for 1 hour at room temperature. After blocking, TSG101 (NovusBio, USA), CD171 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TUJ1 (Abcam, Cambridge, UK), NSE (Abcam, Cambridge, UK) and NeuN (Abcam, Cambridge, UK) Incubated overnight at 4°C with primary antibody (diluted 1:1000). After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution) at room temperature for 1 hour and washed with TBS-T. Finally, bands were detected with EzWestLumi plus (ATTO, Japan) and analyzed using Image QuantTM LAS 4000 (GE Healthcare, UK).

Example 1-10. Next-generation sequencing (NGS) for differential expression analysis of exosomal miRNAs

Neuronal exosomes isolated from serum were collected to prepare a sample of 600 μL. 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 group) and 2.34 mg/mL (DNCB 14 days). Exosomal smRNA isolation and library preparation were performed by 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 the HiSeq 2500 system according to the HiSeq 2500 System Instruction Manual document # 15035786 v02 HCS 2.2.70.

Example 1-11. statistical analysis

All statistical analyzes were performed using Student's t-test, and p<0.05 was considered a significant difference.

<Example 2: Confirmation of atopic dermatitis in the skin induced by DNCB>

Through pathological analysis of the skin, it was confirmed whether DNCB caused atopic dermatitis. First, it was confirmed through hematoxylin and eosin (H&E) staining that an increase in the thickness of the stratum corneum and epidermis, which are typical lesions of atopic dermatitis, was confirmed (A in FIG. 2; control = 12.05 ± 2.51%; DNCB 2 days = 80.6 ± 31.46%, p = 0.008; DNCB 6 days = 108.54 ± 43.89%, p = 0.022; DNCD 14 days = 125.74 ± 19.25%, p = 0.00016). Next, toluidine blue staining was performed to confirm mast cell infiltration in the dermis. As a result, as DNCB treatment was repeated, it was observed that mast cells increased and infiltration increased (Fig. 2B; control = 6.75 ± 1.5%, DNCB 2 days = 12.75 ± 2.87%, p = 0.017; DNCB 6 days = 13.75 ± 1.89%, p = 0.0014; DNCD 14 days = 26.5 ± 4.80%, p = 0.0022).

<Example 3: Hippocampal pathological analysis of atopic dermatitis mouse model>

H&E staining and IHC were performed to analyze the expression level of neuroinflammatory markers caused by atopic dermatitis-induced stress. As a result of histopathological staining, no significant difference was found in the hippocampus of DNCB 2 days, DNCB 6 days, and DNCB 14 days in micrographs compared to the control group (Fig. 3A). And when IHC staining was performed and quantified, no significant change in BDNF expression was observed in the hippocampus (Fig. 3B; control = 0.55 ± 0.11 %, DNCB 2 days = 0.80 ± 0.20 %, p = 0.150; DNCB 6 days = 0.70 ± 0.077%, p = 0.130; DNCD 14 days = 0.90 ± 0.20%, p = 0.0706). In addition, there was no significant change in the expression level of COX2 (Fig. 3 C; control = 0.71 ± 0.084 %, DNCB 2 days = 0.97 ± 0.21 %, p = 0.150; DNCB 6 days = 0.90 ± 0.35 %, p = 0.448; DNCD 14 days=1.28±0.49%, p =0.181). However, the GFAP expression level was significantly changed at DNCB day 2, DNCB day 6 and DNCB day 14 (Fig. 3D; control = 1.01 ± 0.0526%, DNCB day 2 = 1.10 ± 0.11%, p = 0.327; DNCB day 6 = 1.74 ± 0.016%, p = 0.0102; DNCD 14 days = 3.42 ± 0.077%, p = 0.00000547).

<Example 4: Identification of total exosomes isolated from serum>

Before isolating neuronal exosomes, the characteristics of total exosomes isolated from serum were confirmed. First, the shape and size of exosomes were confirmed using a transmission electron microscope (TEM). TEM images showed that the exosomes were spherical bilayer membranes and were 30-150 nm in diameter (Fig. 4A). Next, as a result of using nanoparticle tracking analysis (NTA), it was confirmed that the exosome size was in the range of 30-150 nm (FIG. 4B). Finally, flow cytometry using an immunolabeling method was performed to confirm the presence of exosome markers such as CD9 and CD81. As a result, 98.08% of CD9-positive and 80.16% of CD81-positive particles were found in the total exosomes isolated from serum (FIG. 4C).

<Example 5: Characterization of isolated neuronal exosomes >

After isolation of total exosomes from serum, isolation of neuronal exosomes was performed using an anti-L1 cell adhesion molecule (L1CAM; CD171) biotinylated antibody for immunoprecipitation. Identification of neuronal exosomes was performed using western blotting. As a result, it was confirmed that CD171 was contained more in neuronal exosomes (nEV) than in total exosomes (tEV), and that neuronal markers such as Tuj1, NSE, and NeuN were presented in neuronal exosomes but not in total exosomes. . Finally, the exosome marker TSG101 was identified in both total and neuronal exosomes (Fig. 5).

<Example 6: Verification of exosomal miRNA expression altered by atopic dermatitis-induced stress>

Changed miRNA expression patterns caused by atopic dermatitis were confirmed using NGS (Next-generation sequencing). As a result, it was confirmed that 9 miRNAs were significantly up-regulated and 16 miRNAs were significantly down-regulated in DNCB for 14 days compared to the control group (FIG. 6). The miRNAs upregulated in DNCB for 14 days compared to control 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). The downregulated miRNAs were 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 sequences of the miRNAs are shown in Table 1 below. And some of these miRNAs have been identified as miRNAs that show expression changes in depression-related studies. According to a previous study that atopic dermatitis induces psychological stress and stress induces depression, NGS data imply that these miRNAs can be used as psychological stress biomarkers.

<110> Daegu-Gyeongbuk Medical Innovation Foundation Kyungpook National University Industry-Academic Cooperation Foundation <120> Atopic dermatitis-associated stress diagnosis technology using exosome-derived miRNAs <130> P200837 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> mmu-let-7a-5p <400> 1 ugagguagua gguuguauag uu 22 <210> 2 <211> 22 <212> RNA <213> mmu-let-7b-5p <400> 2 ugagguagua gguugugugg uu 22 <210> 3 <211> 22 <212> RNA <213> mmu-let-7c-5p <400> 3 ugagguagua gguuguaugg uu 22 <210> 4 <211> 22 <212> RNA <213> mmu-let-7e-5p <400> 4 ugagguagga gguuguauag uu 22 <210> 5 <211> 21 <212> RNA <213> mmu-miR-126a-5p <400> 5 cauuauuacu uuugguacgc g 21 <210> 6 <211> 20 <212> RNA <213> mmu-miR-3473b <400> 6 gggcuggaga gauggcucag 20 <210> 7 <211> 21 <212> RNA <213> mmu-miR-3473e <400> 7 gggcuggaga gauggcucgu a 21 <210> 8 <211> 20 <212> RNA <213> mmu-miR-466i-5p <400> 8 ugugugugug ugugugugug 20 <210> 9 <211> 23 <212> RNA <213> mmu-miR-5128 <400> 9 caauugggggc uggcgagaug gcu 23 <210> 10 <211> 22 <212> RNA <213> mmu-let-7i-5p <400> 10 ugagguagua guuugugcug uu 22 <210> 11 <211> 22 <212> RNA <213> mmu-miR-130a-3p <400> 11 cagugcaaug uuaaaagggc au 22 <210> 12 <211> 21 <212> RNA <213> mmu-miR-140-3p <400> 12 uaccacaggg uagaaccacg g 21 <210> 13 <211> 23 <212> RNA <213> mmu-miR-142a-3p <400> 13 uguaguguuu ccuacuuuau gga 23 <210> 14 <211> 22 <212> RNA <213> mmu-miR-16-5p <400> 14 uagcagcacg uaaauauugg cg 22 <210> 15 <211> 23 <212> RNA <213> mmu-miR-17-5p <400> 15 caaagugcuu acagugcagg uag 23 <210> 16 <211> 22 <212> RNA <213> mmu-miR-185-5p <400> 16 uggagagaaa ggcaguuccu ga 22 <210> 17 <211> 23 <212> RNA <213> mmu-miR-19b-3p <400> 17 uggcaaauc caugcaaaac uga 23 <210> 18 <211> 22 <212> RNA <213> mmu-miR-24-3p <400> 18 uggcucaguu cagcaggaac ag 22 <210> 19 <211> 22 <212> RNA <213> mmu-miR-27a-5p <400> 19 agggcuuagc ugcuugugag ca 22 <210> 20 <211> 22 <212> RNA <213> mmu-miR-29a-3p <400> 20 uagcaccauc ugaaaucggu ua 22 <210> 21 <211> 23 <212> RNA <213> mmu-miR-301a-3p <400> 21 cagugcaaua guauugucaa agc 23 <210> 22 <211> 22 <212> RNA <213> mmu-miR-451a <400> 22 aaaccguuac cauuacugag uu 22 <210> 23 <211> 23 <212> RNA <213> mmu-miR-669a-3p <400> 23 acauaacaua cacacacacg uau 23 <210> 24 <211> 23 <212> RNA <213> mmu-miR-669o-3p <400> 24 acauaacaua cacacacacg uau 23 <210> 25 <211> 23 <212> RNA <213> mmu-miR-93-5p <400> 25 caaagugcug uucgugcagg uag 23

Claims (11)

A composition for diagnosing stress induced by atopic dermatitis, comprising an agent for measuring the expression level of exosome-derived miRNA of mmu-let-7a-5p (SEQ ID NO: 1).
The composition of claim 1, wherein the exosome is a nerve-derived exosome.
The composition of claim 1, wherein the agent comprises a primer or probe that specifically binds to exosome-derived miRNA.
The method of claim 1, wherein the composition is mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), 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-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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), At least one exosome-derived miRNA selected from the group consisting of mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) Which further comprises an agent for measuring the expression level of, the composition.
A kit for diagnosing stress caused by atopic dermatitis, comprising the composition of any one of claims 1 to 4.
(a) isolating exosomes from the subject's biological sample; and
(b) comparing the expression level of mmu-let-7a-5p (SEQ ID NO: 1), a miRNA derived from the exosomes isolated in step (a), with the expression level of the exosome-derived miRNA of the control sample A method for providing information for diagnosing stress induced by atopic dermatitis, comprising:
The method of claim 6, wherein the biological sample is blood.
The method of claim 6, wherein the miRNA in step (b) is mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-miR-126a-5p (SEQ ID NO: 2) 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu-miR-466i-5p (SEQ ID NO: 8), 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: 12) 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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a ( SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24), and mmu-miR-93-5p (SEQ ID NO: 25) Which further comprises the above exosome-derived miRNA, information providing method.
The method of claim 6, further comprising determining that the subject is in a stress state when the expression level of mmu-let-7a-5p (SEQ ID NO: 1) in the subject is higher than that of the control group in step (b) , How to provide information.
The method of claim 8, wherein in step (b), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 10) 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: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o- When the expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) is lower than that of the control group, the step of determining that the subject is in a stress state Further comprising, an informational method.
The method of claim 8, wherein in step (b), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-miR-126a-5p (SEQ ID NO: 5 ), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), and mmu-miR-466i-5p (SEQ ID NO: 8), the expression level of one or more miRNAs selected from the control group Further comprising the step of determining that the subject is in a stress state when the expression is high compared to , information providing method.

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