Evaluation method of environmental stimulus response

Abstract

The purpose of the present invention is to evaluate the influence of environmental stimulation on the skin using a new index. Provided is a method for evaluating an environmental stimulus response in skin using the amount of a specific miRNA as an index by finding out miRNA that fluctuates in the skin subjected to environmental stimulus. Also provided is a method for screening for a regulator of an environmental stimulus response in the skin, said method using fluctuations in a specific miRNA as an indicator.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the response to an environmental stimulus in the skin based on miRNA in exosomes contained in a biological sample, and a method for screening a regulator of the response to an environmental stimulus in the skin. Background Art

[0002] MiRNAs, short, single-stranded RNAs, are reported to be present not only within cells but also outside of cells in exosomes, such as in blood and tissue fluid, contributing to intercellular communication. Furthermore, disease-specific miRNAs are sometimes expressed in various diseases, including cancer, and are being studied for their development as biomarkers. Single-stranded mature miRNAs have a gene sequence that is partially complementary to the 3'-UTR of an mRNA. By binding to mRNAs with sequences that are partially complementary to their own, they inhibit the translation of the target mRNA. Exosome-encapsulated miRNAs are secreted from certain cells and transferred to other cells, where they function as signaling agents, controlling the expression of genes involved in various physiological functions.

[0003] Skin, the outermost tissue of a living organism, is exposed to environmental stimuli such as temperature, ultraviolet rays, humidity, physical stimulation, and chemical stimulation. When environmental stimuli are applied to the skin, various physiological responses occur. Skin exposed to environmental stimuli gradually accumulates damage, and this accumulated damage can cause skin problems such as roughness, hyperpigmentation, sagging, and wrinkles. To date, research on miRNA in the skin field has mainly focused on intercellular communication, with reports on exosome therapy derived from mesenchymal stem cells and interstitial cells (Non-patent document 1: Cells (2020) May 7: 9(5):157), intercellular communication between keratinocytes and macrophages (Non-patent document 2: ACS Nano. 2020 Oct 27; 14(10): 12732-12748, Non-patent document 3: J Dermatol. 2020 Mar 47(3): 265-276, Non-patent document 4: J Dermatol Sci. (2019) Mar; 93(3):159-167), intercellular communication between keratinocytes and fibroblasts (Non-patent document 5: Int J Mol Sci. 2020 Feb 4; 21(3): 1022), Non-patent document 6: J Invest Dermatol. (2019) Nov. 139(11): 2131-2323. e8.). In addition, it has been reported that exosomes released from keratinocytes regulate the pigment production of melanocytes (Non-patent document 7: Nat. Commun. (2015) Jun 24; 6: 7506).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent literature 1: Cells (2020) May 7: 9(5): 1157

[0007] Non-patent literature 2: ACS Nano. (2020) Oct27; 14(10): 12732-12748

[0008] Non-patent literature 3: J Dermatol. (2020) Mar 47(3): 265-276

[0009] Non-patent literature 4: J Dermatol Sci. (2019) Mar ;93(3): 159-167

[0010] Non-patent literature 5: Int J Mol Sci. (2020) Feb 4; 21(3): 1022

[0011] Non-patent literature 6: J Invest Dermatol. (2019) Nov. 139(11):2131-2323. e8

[0012] Non-patent literature 7: Nat. Commun. (2015) Jun 24; 6:7506)

[0013] Non-patent literature 8: Int J Mol Sci. 2017 Jul; 18(7): 1375. Summary of the Invention

[0014] Problems to be solved by the invention

[0015] The present invention aims to evaluate the effects of environmental stimuli on the skin using new indicators.

[0016] Means for solving problems

[0017] The present inventors developed a technique for analyzing miRNA secreted from the skin in response to environmental stimuli. Through extensive research on miRNA secreted from the skin in response to environmental stimuli, they discovered that several miRNAs are altered in the skin exposed to environmental stimuli, leading to the completion of the present invention. Therefore, the present invention relates to the following:

[0018] [1] A method for evaluating the response to environmental stimuli in the skin, using the amount of at least one specific miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p in a biological sample as an indicator.

[0019] [2] The evaluation method according to item 1, wherein the biological sample is selected from tissue fluid, blood, lymph, saliva, urine, tears, and sweat.

[0020] [3] The evaluation method according to item 1 or 2, wherein the specific miRNA is a skin-derived miRNA.

[0021] [4] The evaluation method according to item 3, wherein the skin-derived miRNA is a keratinocyte-derived miRNA.

[0022] [5] The evaluation method according to any one of items 1 to 4, wherein the specific miRNA is hsa-miR-4497.

[0023] [6] According to the evaluation method described in item 5, the above-mentioned environmental stimulus is a stimulus that combines dryness stimulus, low temperature stimulus, warming stimulus and cooling stimulus.

[0024] [7] The evaluation method according to any one of items 1 to 4, wherein the specific miRNA is at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0025] [8] According to the evaluation method described in item 7, the above-mentioned environmental stimulus is a stimulus that combines low temperature stimulation, warming stimulation and cooling stimulation.

[0026] [9] A method for identifying accumulated skin damage to environmental stimuli by using the method for evaluating the response to environmental stimuli in the skin according to any one of items 1 to 8.

[0027]

[10] A method for determining the responsiveness to environmental stimuli by evaluating the response of the skin to environmental stimuli according to any one of items 1 to 8.

[0028]

[11] A method for screening a regulator of environmental stimulus response in the skin, comprising the following steps:

[0029] a step of culturing a culture comprising keratinocytes in the presence of a drug candidate;

[0030] a step of measuring at least one specific miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p in the culture after culturing; and

[0031] This is a step of screening for drug candidates as modulators of environmental stimulus responses in the skin when the amount of a specific miRNA is altered compared to a control.

[0032]

[12] The screening method according to item 11, wherein the specific miRNA is hsa-miR-4497.

[0033]

[13] According to the screening method described in item 12, the above-mentioned environmental stimulation is a stimulation that combines dry stimulation, low temperature stimulation, warming stimulation and cooling stimulation.

[0034]

[14] The screening method according to item 11, wherein the specific miRNA is at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0035]

[15] According to the screening method described in item 14, the above-mentioned environmental stimulation is a stimulation that combines low temperature stimulation, warming stimulation and cooling stimulation.

[0036]

[16] An hsa-miR-4497 expression inhibitor comprising at least one extract selected from a Tokyo cherry blossom extract or a tomentosa extract.

[0037] [16-1] Use of at least one extract selected from a Tokyo cherry blossom extract or a tomentosa extract for producing an hsa-miR-4497 expression inhibitor.

[0038] [16-2] At least one extract selected from Tokyo cherry blossom extract or Isodon pubescens extract, for use in the treatment, prevention or improvement of dry skin via inhibition of hsa-miR-4497 expression.

[0039] [16-3] A method for inhibiting hsa-miR-4497 expression, comprising: applying at least one extract selected from a Tokyo cherry blossom extract or a tomentosa extract to a subject in which hsa-miR-4497 expression inhibition is desired.

[0040] [16-4] A cosmetic method for inhibiting hsa-miR-4497 expression, comprising: applying at least one extract selected from Tokyo cherry blossom extract or Isodon pubescens extract.

[0041]

[17] An hsa-miR-4497 expression enhancer comprising at least one extract selected from safflower extract, loquat extract, and ashitaba extract.

[0042] [17-1] Use of at least one extract selected from safflower extract, loquat extract, and ashitaba extract for producing an hsa-miR-4497 expression enhancer.

[0043] [17-2] At least one extract selected from safflower extract, loquat extract, and ashitaba extract, for use in the treatment, prevention, or improvement of inflammatory diseases via enhanced hsa-miR-4497 expression.

[0044] [17-3] A method for enhancing hsa-miR-4497 expression, comprising: administering at least one extract selected from safflower extract, loquat extract, and ashitaba extract to a subject in whom enhanced hsa-miR-4497 expression is desired.

[0045] [17-4] A cosmetic method for enhancing hsa-miR-4497 expression, comprising applying at least one extract selected from safflower extract, loquat extract, and ashitaba extract.

[0046]

[18] A regulator of environmental stimulus response in the skin, comprising exosomes, wherein the exosomes contain at least one miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0047] [18-1] Use of exosomes containing at least one miRNA selected from hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p for producing a regulator of environmental stimulus response.

[0048] [18-2] Exosomes comprising at least one miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p, for the treatment, prevention, or improvement of inflammatory diseases by regulating responses to environmental stimuli.

[0049] [18-3] A method for regulating environmental stimulus response, comprising: applying exosomes containing at least one miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p to a subject in need of regulating environmental stimulus response.

[0050] [18-4] A method for beauty treatment by regulating environmental stimulus response, comprising: applying exosomes containing at least one miRNA selected from hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0051]

[19] A regulator of the response to a combination of dryness stimulation, low temperature stimulation, warming stimulation, and cooling stimulation in the skin, comprising exosomes, wherein the exosomes contain at least one miRNA selected from hsa-miR-4497.

[0052] [19-1] Use of exosomes containing at least one miRNA selected from hsa-miR-4497 for producing a regulator of the response to a combination of dryness stimulation, low temperature stimulation, warming stimulation, and cooling stimulation in the skin.

[0053] [19-2] An exosome containing at least one miRNA selected from hsa-miR-4497, for maintaining homeostasis by regulating the response to a stimulus comprising a combination of dryness, low temperature, warming, and cooling in the skin.

[0054] [19-3] A method for regulating the response to a stimulus that combines dryness, low temperature, warming, and cooling in the skin, comprising: applying exosomes containing at least one miRNA selected from hsa-miR-4497 to a subject whose response to a stimulus that combines dryness, low temperature, warming, and cooling in the skin needs to be regulated.

[0055] [19-4] A cosmetic method for regulating the response to a combination of dryness stimulation, low temperature stimulation, warming stimulation, and cooling stimulation in the skin, comprising: applying exosomes containing at least one miRNA selected from hsa-miR-4497.

[0056]

[20] A regulator of the response to a stimulus that combines low temperature stimulation, warming stimulation, and cooling stimulation in the skin, comprising exosomes, wherein the exosomes comprise at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0057] [18-1] Use of exosomes containing at least one miRNA selected from hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p for producing a regulator of the stimulus response in the skin that integrates dryness stimulation, low temperature stimulation, warming stimulation, and cooling stimulation.

[0058] [18-2] Exosomes comprising at least one miRNA selected from hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p, for treating, preventing, or improving the skin's response to a combination of dryness stimulation, low temperature stimulation, warming stimulation, and cooling stimulation.

[0059] [18-3] A method for regulating the stimulus response of the skin that is a combination of dryness stimulation, low temperature stimulation, warming stimulation and cooling stimulation, comprising: for an object in which the stimulus response of the skin that is a combination of dryness stimulation, low temperature stimulation, warming stimulation and cooling stimulation needs to be regulated, applying exosomes containing at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0060] [18-4] A cosmetic method for regulating the skin's stimulus response to a combination of dryness stimulation, low temperature stimulation, warming stimulation, and cooling stimulation, comprising: applying exosomes containing at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

[0061] Effects of the Invention

[0062] By measuring miRNA in biological samples, skin conditions can be evaluated. In addition, by measuring the amount of miRNA from culture, regulators of responses to environmental stimuli can be screened. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The photographs show the localization of miR-4497 in skin models subjected to dryness stimulation, low-temperature stimulation, cooling stimulation, and warming stimulation, respectively.

[0064] Figure 2 Results of screening for agents that (1) up-regulate and (2) down-regulate miR-4497 are shown.

[0065] Figure 3 The following diagram shows the configuration of an environmental stimulus response evaluation device 10 according to the present invention. The process for determining the environmental stimulus response is executed collaboratively by hardware resources such as an input unit 11, a storage unit 12, a processing unit 13, an output unit 14, and a learning unit 15, which are connected via a bus.

[0066] Figure 4 A diagram showing the configuration of an environmental stimulus response evaluation system 20 including an environmental stimulus response evaluation device 10 according to the present invention, which is located on the Internet. The miRNA analysis device 40 and the terminal device 30 are connected to the environmental stimulus response evaluation device 10 via the Internet. DETAILED DESCRIPTION

[0067] The present invention relates to a method for evaluating the response to environmental stimuli in the skin using the amount of specific miRNA in a biological sample as an indicator. Furthermore, the present invention relates to a method for screening a regulator of the response to environmental stimuli in the skin using the amount of specific miRNA in a culture as an indicator.

[0068] In this specification, at least one specific miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p can be used. These miRNAs are released from the skin in response to environmental stimuli and can be referred to as environmental stimulus-related miRNAs. These can be appropriately selected and used depending on the desired environmental stimulus. Among them, hsa-miR-4497 is used from the perspective of evaluating responses to environmental stimuli that comprehensively include desiccation, hypothermia, warming, and cooling. From the perspective of evaluating responses to environmental stimuli that comprehensively include hypothermia, warming, and cooling, at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p is used. Each miRNA can be used alone or in combination with miRNAs from the same group. The amount of miRNA can be compared with a threshold value, and its changes can be measured over time and compared. As an example, the amount of miRNA can be measured twice during a specific period to determine the response of the skin to environmental stimuli during that period. By evaluating the response to environmental stimuli, the intensity of the environmental stimulus can also be measured. Such an evaluation method does not require a doctor's judgment, excludes medical treatment, and can also be called a so-called non-diagnostic method. The method of the present invention can be used by people other than medical practitioners, such as practitioners in cosmetics stores, beauty supply stores such as beauty salons, or practitioners in testing companies.

[0069] Hsa-miR-4497 is a miRNA expressed in the skin by at least one environmental stimulus selected from dry stimulation, low temperature stimulation, cooling stimulation and warming stimulation. Therefore, when the amount of hsa-miR-4497 is high in a biological sample, it means that the skin has been subjected to at least one environmental stimulus selected from dry stimulation, low temperature stimulation, cooling stimulation and warming stimulation, and it also means that damage has been accumulated by such environmental stimulation. Although not limited by theory, it has been shown by bioinformatics analysis that hsa-miR-4497 targets autophagy-related genes (ATG9B, RAB15, etc.). Autophagy-related genes are responsible for the removal and regeneration of waste products in the cell and contribute to the homeostasis of the cell. Therefore, it can be considered that the expression of hsa-miR-4497 increases by environmental stimulation, thereby reducing the expression of autophagy-related genes and losing homeostasis. In addition, it can be considered that similarly, when the expression of hsa-miR-4497 decreases, the expression of autophagy-related genes also increases, causing excessive organelle decomposition and the removal of necessary components, and losing homeostasis. Therefore, it is believed that controlling excessive expression fluctuations in both the enhancement and reduction directions and guiding it to a stable state is important for maintaining homeostasis. Therefore, hsa-miR-4497 expression inhibitors or expression promoters can also be referred to as autophagy regulators or homeostasis maintainers, respectively.

[0070] Hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p are miRNAs expressed in the skin in response to at least one environmental stimulus selected from the group consisting of low temperature stimulation, cooling stimulation, and warming stimulation.

[0071] Non-patent document 8 (Int J Mol Sci. 2017 Jul; 18(7): 1375.) shows that hsa-miR-3648 targets Adenomatous polyposis coli 2 (APC2), which is associated with ER stress. Therefore, when the amount of miR-3648 is high, it is expected that cell proliferation will become excessive. In addition, although not limited by theory, 464 genes were listed as candidate targets of miR-3648 through bioinformatics analysis. Among them, gene groups represented by RAB8A were grouped according to endocytosis terms based on pathway analysis. Therefore, when the amount of miR3648 is high, it is shown that the skin is stimulated by endocytosis.

[0072] Regarding hsa-miR-4745-5p, although not limited by theory, bioinformatics analysis identified 3,856 genes as candidate targets of hsa-miR-4745-5p. Pathway analysis revealed the possibility that genes such as Janus kinase 3 (JAK3), SMAD family member 3 (SMAD3), Wnt family member proteins, and mitogen-activated protein kinase proteins could be targets. These genes were grouped according to the term signaling pathways regulating pluripotency of stem cells. Furthermore, hsa-miR-4745-5p showed potential to target genes such as forkhead box O3 (FOXO3) and insulin-like growth factor 1 (IGF1), and was grouped into the term "EGFR tyrosine kinase inhibitor resistance." Furthermore, hsa-miR-4745-5p showed potential to target genes such as bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 8a (BMP8A), bone morphogenetic protein receptor type 1A (BMPR1A), and transforming growth factor beta 1 (TGFB1), and was grouped into the term "TGF-beta signaling pathway."In addition, hsa-miR-4745-5p showed the possibility of targeting gene groups such as CXC motif chemokine ligand 12 (CXCL12), semaphorin 3F (SEMA3F), semaphorin 4D (SEMA4D), semaphorin 4G (SEMA4G), semaphorin 6C (SEMA6C), and semaphorin 6D (SEMA6D), and was grouped according to the term "Axon guidance". Furthermore, hsa-miR-4745-5p showed potential to target genes such as cadherin 5 (CDH5), claudin family proteins, and integrin subunit beta 8 (ITGB8), and was grouped under the term "cell adhesion molecules." Furthermore, hsa-miR-4745-5p showed potential to target genes such as RAS like proto-oncogene B (RALB), RAS p21 protein activator 4 (RASA4), Ras protein specific guanine nucleotide releasing factor 1 (RASGRF1), and Ras protein specific guanine nucleotide releasing factor 2 (RASGRF2), and was grouped under the term "Ras signaling pathway."hsa-miR-4745-5p also showed potential to target genes such as Rho-associated coiled-coil containing protein kinase 1 (ROCK1), collagen family proteins, integrin family proteins, and laminin family proteins, and was grouped under the term "focal adhesion." Furthermore, hsa-miR-4745-5p also showed potential to target genes such as RAS oncogene family members (RAB11A), RAS oncogene family members (RAB4A), and RAS oncogene family members (RAB8A), and was grouped under the term "endocytosis." Therefore, high levels of miR-4745-5p indicate that skin differentiation is stimulated.

[0073] Regarding hsa-miR-6126, although not limited by theory, bioinformatics analysis identified 3,952 target candidates. Pathway analysis revealed that these genes, grouped by the terms longevity regulating pathway-multiple species, longevity regulating pathway, FoxO signaling pathway, and AMPK signaling pathway, included autophagy related 13 (ATG13), epidermal growth factor receptor (EGFR), forkhead box O3 (FOXO3), forkhead box O4 (FOXO4), insulin like growth factor 1 (IGF1), member RAS oncogene family (RAB10), and member RAS oncogene family (RAS oncogene family). The potential targets of hsa-miR-6126 include genes such as erb-b2 receptor tyrosine kinase 2 (ERBB2), erb-b2 receptor tyrosine kinase 4 (ERBB4), and heparin-binding EGF-like growth factor (HBEGF). These genes were grouped by term in the ErbB signaling pathway. ErbB receptors transduce signals through various pathways to control cell proliferation, migration, differentiation, apoptosis, and motility, and therefore appear to be involved in stimuli that affect keratinocyte differentiation.Furthermore, bioinformatics analysis revealed that hsa-miR-6126 targets genes such as the MYC proto-oncogene, bHLH transcription factor (MYC), and Wnt family member proteins. These genes were grouped according to signaling pathways regulating stem cell pluripotency. Since these genes are suggested to be involved in the maintenance of stem cell function, hsa-miR-6126 suggests that it stimulates the maintenance or differentiation of skin stem cells.

[0074] Although not limited by theory, bioinformatics analysis identified 28 target genes for hsa-miR-762. Ontological analysis revealed that Wnt family member 7A (WNT7A) and mitogen-activated protein kinase kinase kinase 2 (MAP4K2) genes were targeted. These genes are positively regulated by the JNK cascade. Since JNK signaling is involved in cell growth, differentiation, survival, and apoptosis, changes in hsa-miR-762 suggest stimulation related to cell differentiation and survival.

[0075] Regarding hsa-miR-1237-5p, while not limited by theory, bioinformatics analysis identified 791 genes as target candidates. Pathway analysis revealed potential targets for genes including Rac family small GTPase 1 (RAC1), insulin receptor (INSR), and p21 (RAC1) activated kinase 1 (PAK1). These genes were grouped within the Ras signaling pathway. The Ras signaling pathway is associated with various signaling pathways involved in cell cycle progression, cell migration, apoptosis, aging, and other biological functions, and therefore appears to be stimulated by keratinocyte differentiation. Furthermore, bioinformatics analysis revealed that hsa-miR-1237-5p may target genes such as CC motif chemokine receptor 10 (CCR10), CXC motif chemokine ligand 14 (CXCL14), and CXC motif chemokine receptor 5 (CXCR5). These genes are grouped within the chemokine signaling pathway. Therefore, since these genes are involved in cytokine production, cell proliferation / differentiation, and apoptosis, they suggest that they may be involved in stimuli that contribute to skin inflammation and tissue changes. In addition, pathway analysis revealed the possibility of targeting genes such as forkhead box O4 (FOXO4), insulin like growth factor 1 (IGF1), and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), which were grouped by terms in the longevity regulating pathway and the FoxO signaling pathway. This suggests that they are related to aging and are exposed to stimuli that promote or inhibit aging.

[0076] Regarding hsa-miR-3960, while not being bound by theory, bioinformatics analysis identified 627 genes as candidate targets. Pathway analysis revealed that genes such as RAB11 family interacting protein 3 (RAB11FIP3) and phosphatidylinositol-4-phosphate 5-kinase type 1 gamma (PIP5K1C) were likely targets. These genes were grouped under the endocytosis mechanism. Endocytosis is the process by which cells take in external substances through the plasma membrane, invaginate them, and then take them into the cell as vesicles. It contributes to changes and adaptive responses in the absorption of extracellular information, including exosomes. Furthermore, the study showed the potential of targeting genes such as CXC motif chemokine ligand 12 (CXCL12), TGF-beta activated kinase 1 (MAP3K7) binding protein 1 (TAB1), TNF receptor associated factor 3 (TRAF3), TNF receptor superfamily member 11a (TNFRSF11A), and TNF receptor superfamily member 13C (TNFRSF13C). These genes were grouped according to the NF-κB signaling pathway. In the skin, changes in the NF-κB signaling pathway indicate involvement in inflammation, and are particularly activated in atopic dermatitis. This suggests that inflammatory stimuli are affected in the skin.

[0077] Regarding hsa-miR-4281, while not intending to be bound by theory, bioinformatics analysis identified 2,559 genes as candidate targets. Pathway analysis revealed the possibility of targeting genes such as AKT serine / threonine kinase 1, 2, 3 (AKT1, 2, 3). These genes were grouped within the AMPK signaling pathway. This suggests a downstream stimulus that affects collagen synthesis and epidermal cell proliferation. Furthermore, the FoxO signaling pathway, insulin signaling pathway, and longevity regulating pathway, all of which are thought to be involved in this pathway, also showed changes, suggesting a stimulus related to aging.

[0078] Although not limited by theory, bioinformatics analysis identified 1,427 genes as candidate targets for hsa-miR-4488. Pathway analysis revealed that these genes, including claudin 19 (CLDN19), claudin 3 (CLDN3), claudin 5 (CLDN5), integrin subunit alpha L (ITGAL), integrin subunit alpha M (ITGAM), and syndecan family proteins, are likely to be targeted. These genes are grouped as cell adhesion molecules. Therefore, hsa-miR-4488 appears to be regulated by stimulation of tight junctions and cell motility in the skin. Furthermore, bioinformatics analysis revealed that hsa-miR-4488 may target genes such as calmodulin 3 (CALM3) and mitogen-activated protein kinase 1 (MAPK1), which are grouped according to circadian rhythm regulation. Therefore, hsa-miR-4488 appears to fluctuate depending on stimuli that affect the skin's circadian rhythm. Furthermore, hsa-miR-4488 also appears to target genes such as notch receptor 2 (NOTCH2) and nuclear receptor corepressor 2 (NCOR2), which are grouped according to the Notch signaling pathway. The Notch signaling pathway is known to be important for maintaining fibroblast homeostasis. Therefore, hsa-miR-4488 appears to fluctuate depending on stimuli that contribute to skin homeostasis.Furthermore, hsa-miR-4488 showed the potential to target gene groups such as apolipoprotein C2 (APOC2), lysophospholipase 1 (LYPLA1), phospholipase A2 group IIC (PLA2G2C), phospholipase A2 group IVE (PLA2G4E), phospholipase A2 group VI (PLA2G6), phospholipase D family member 3 (PLD3), and phospholipid transfer protein (PLTP), which are involved in glycerophospholipid metabolism and cholesterol metabolism. Therefore, hsa-miR-4488 was shown to fluctuate depending on stimuli that affect lipid metabolism in the skin.

[0079] Regarding hsa-miR-6784-5p, although not limited by theory, bioinformatics analysis identified 1,478 genes as target candidates. Pathway analysis revealed the potential for targeting genes such as the VPS37B subunit of ESCRT-I (VPS37B), the VPS37C subunit of ESCRT-I (VPS37C), and the VPS37D subunit of ESCRT-I (VPS37D). These genes were grouped under the endocytosis mechanism. Endocytosis is the process by which cells take in external substances through the plasma membrane, invaginate them, and then take them into the cell, forming vesicles. This process contributes to changes and adaptive responses in the absorption of extracellular information, including exosomes. Furthermore, the study showed the possibility of targeting genes such as TNF receptor associated factor 1, 3 (TRAF1, 3), which were grouped according to the TNF signaling pathway. TNFα is known to be associated with chronic inflammation, and hsa-miR-6784-5p was shown to fluctuate depending on stimuli that affect chronic inflammation in the skin. Furthermore, it is expected that the AMPK signaling pathway, the MAPK signaling pathway, and the longevity regulating pathway will also fluctuate, suggesting that these are also stimuli involved in aging.

[0080] hsa-miR-10396a-5p and hsa-miR-10400-5p are miRNAs for which bioinformatics analysis is difficult and for which prior knowledge is lacking. Prediction of their functions awaits future analysis.

[0081] As the biological sample, any biological sample in which miRNA can be detected, such as tissue fluid, blood, lymph fluid, saliva, urine, tears, sweat, etc., can be used.

[0082] For body fluid samples taken from areas away from the skin, such as blood, lymph, saliva, urine, and tears, skin-derived exosomes can be selectively recovered from the sample to measure skin-derived miRNA. Exosomes derived from the skin can be selectively recovered based on the type and density of membrane proteins presented on the exosome surface and / or the size of the exosomes. The miRNA contained in the recovered exosomes can be quantified based on common methods. From the perspective of being able to detect in body fluids, hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, and hsa-miR-6126 are preferred.

[0083] Environmental stimuli can be selected from dryness, low temperature, warming, and cooling, but may also be a combination of these. Dryness refers to the skin surface being exposed to a low-humidity environment. Low temperature refers to the skin surface reaching a temperature as low as it can be, approximately 22°C lower than normal skin temperature. Warming refers to the skin surface experiencing a temperature change from low to high within a specified period, for example, a temperature increase of 2-13°C within 60 minutes. Cooling refers to the skin surface experiencing a temperature change from high to low within a specified period, for example, a temperature decrease of 2-13°C within 60 minutes. Using miR4497 levels as an indicator allows for comprehensive determination of the skin's response to environmental stimuli, including dryness, low temperature, warming, and cooling. Environmental stimuli set in the laboratory may differ from those set for actual human skin. In real life, environmental stimuli can be exemplified by conditions where the temperature and humidity of a room fluctuate over short periods of time due to the use of air conditioners such as heaters and air conditioners. Low temperature stimulation, warming stimulation and cooling stimulation are all temperature-related stimulations, and appropriate stimulation can be selected according to the condition of the subject.

[0084] Until now, the response of the skin to environmental stimuli has been able to be measured by the amount of moisture in the skin, the barrier function (e.g., TEWL value), the degree of skin roughness, etc. However, on the other hand, these vary greatly depending on the subject and cannot be said to be sufficient as indicators that can be compared with other indicators. By measuring miRNA in a biological sample, it can be provided as an indicator that can compare the response to environmental stimuli. The response to environmental stimuli can reflect the damage accumulated in the skin in response to the environmental stimulus. Therefore, the damage accumulated in the skin can predict the future skin condition. The method for evaluating the response to environmental stimuli in the skin of the present invention can also be referred to as a method for evaluating the damage accumulated in the skin in response to environmental stimuli or a method for predicting the future skin condition.

[0085] Another embodiment of the present invention also relates to a method for screening a modulator of the skin's response to environmental stimuli. Such a screening method comprises the following steps:

[0086] The step of culturing a culture comprising keratinocytes in a medium comprising a drug candidate;

[0087] a step of measuring specific miRNA in the culture after culturing; and

[0088] This is a step of screening for drug candidates as modulators of environmental stimulus responses in the skin when the amount of a specific miRNA is altered compared to a control.

[0089] This screening method can be used to screen regulators of environmental stimulus responses.

[0090] The culture containing keratinocytes may be a tissue culture of a skin tissue slice obtained from the skin, or a cell culture inoculated with keratinocytes. Preferably, keratinocytes are used alone, or a 3D skin model in which the skin is constructed in three dimensions by including other cells. Other cells may include melanocytes, Langerhans cells, dermal fibroblasts, etc. The 3D skin model may be a commercially available product or may be newly prepared.

[0091] The culture in the culture medium containing the candidate drug can be carried out by adding a predetermined amount of the candidate drug to a culture medium containing keratinocytes that has been cultured in advance, or can be carried out by replacing the culture medium with the candidate drug containing a predetermined amount. In the culture medium containing the candidate drug, the culture containing the keratinocytes can be cultured for a predetermined period, for example, 1 hour to 5 days. The culture conditions can use usual conditions, as an example, they can be cultured at 37°C and 5% CO2 in a humidified atmosphere. As a control, culture is carried out under conditions that are different only in that the candidate drug is not contained. The control experiment can be carried out at the same time, can be implemented in advance, or can be carried out afterwards. In addition, a threshold value can be appropriately set by the control value to perform threshold processing.

[0092] Specific miRNAs can be measured using conventional methods. For example, specific miRNAs in exosomes recovered from the culture medium can be quantified using PCR using sequence-specific primers. When the amount of specific miRNA changes compared to a control, candidate drugs can be screened for modulators of the environmental stimulus response in the skin. Modulators of the environmental stimulus response screened in this manner can either upregulate or downregulate the environmental stimulus response.

[0093] As a specific miRNA, hsa-miR-4497 can be used. In this case, a regulator that responds to environmental stimuli that include drying stimulation, low temperature stimulation, warming stimulation, and cooling stimulation can be screened. Regulators that can downregulate hsa-miR-4497 include Tokyo cherry blossom extract and hairy leaf tea extract. Regulators that can upregulate hsa-miR-4497 include safflower extract, loquat extract, and tomorrow leaf extract. Regulators that can downregulate and upregulate hsa-miR-4497 contribute to the maintenance of skin homeostasis by promoting the expression of autophagy-related genes. Therefore, regulators that can downregulate and upregulate hsa-miR-4497 can also be called skin homeostasis maintainers and skin problem inhibitors.

[0094] The plant extracts described in this specification can be obtained by conventional methods, for example, by immersing the flowers, fruits, stems, leaves, roots, seeds and other parts of the plant from which the extracts are derived with an extraction solvent at room temperature or under heating or reflux, and then filtering and concentrating to obtain the extracts. As the extraction solvent, any solvent commonly used for extraction can be used, and for example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as ethanol, propylene glycol, 1,3-butanediol, glycerol and other alcohols, aqueous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane and the like can be used alone or in combination. As the solvent, a mixed solvent of water and an alcohol such as 1,3-butanediol is preferably used. The extract obtained by extraction using the above-mentioned solvent can be used directly, or the extract obtained by extraction using the above-mentioned solvent can be concentrated by, for example, freeze-drying. In addition, as needed, the following can be used: an extract from which impurities have been removed by adsorption methods such as ion exchange resins; an extract obtained by extracting a porous polymer (such as After adsorption on a column (XAD-2), the extract was eluted with a desired solvent and further concentrated.

[0095] The so-called Tokyo cherry blossom extract is an extract of the flowers, wood, leaves, or roots of the Tokyo cherry blossom, for example, an extract of the leaves. It can be prepared by extracting the leaves of the Tokyo cherry blossom with any solvent, such as water, alcohol, or a mixed solution thereof. As the alcohol, ethanol, propylene glycol, or butylene glycol can be used. More preferably, the extraction can be carried out using a mixture of water and ethanol or water and 1,3-butylene glycol in any ratio, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and more preferably a mixture of 50:50. In particular, the leaves can be extracted with a mixture of water and 1,3-butylene glycol. The Tokyo cherry blossom extract is mixed at a concentration of 0.01% to 0.1%, more preferably 0.1%.

[0096] The so-called hairy leaf tea extract is hairy leaf tea ( ) (Alias: Fragrant Tea )) of the extract. It is an extract of the leaves, stems, flowers or roots of Camellia sinensis. As an example, it can be prepared by extracting the stems and leaves with any solvent such as water, alcohol or a mixed solution thereof. As alcohol, ethanol, propylene glycol or butylene glycol can be used. More preferably, the extraction can be carried out by a mixture of water and ethanol or water and 1,3-butylene glycol in any ratio, such as a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and more preferably a mixture of 50:50. The Camellia sinensis extract is mixed at a concentration of 0.01% to 0.1%, more preferably 0.1%.

[0097] Safflower extract is an extract of safflower (Carthamus tinctorius) of the Asteraceae family. It can be prepared by extracting the petals of safflower with any solvent, such as water, alcohol, or a mixture thereof. Ethanol, propylene glycol, or butylene glycol can be used as the alcohol. More preferably, extraction can be performed using a mixture of water and ethanol or water and 1,3-butylene glycol in any ratio, such as a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and more preferably 50:50. In particular, the petals can be extracted with a mixture of water and 1,3-butylene glycol. The safflower extract is mixed at a concentration of 0.01% to 0.1%, more preferably 0.1%.

[0098] The so-called loquat extract is an extract of loquat (Eriobotrya japonica) of the genus Eriobotrya in the Rosaceae family. It can be prepared by extracting loquat fruit with any solvent, such as water, alcohol, or a mixed solution thereof. As alcohol, ethanol, propylene glycol, or butylene glycol can be used. More preferably, extraction can be performed using a mixture of water and ethanol or water and 1,3-butylene glycol in any ratio, such as a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and more preferably a mixture of 50:50. The loquat extract is mixed at a concentration of 0.01% to 0.1%, more preferably 0.1%.

[0099] Angelica keiskei extract is an extract of Angelica keiskei, a plant of the Apiaceae family. It can be prepared by extracting the leaves of the Angelica keiskei plant with water, alcohol, or a mixture thereof. Ethanol, propylene glycol, or butylene glycol can be used as the alcohol. More preferably, extraction can be performed using a mixture of water and ethanol or water and 1,3-butylene glycol in any ratio, such as a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50. Rosehip extract is blended at a concentration of 0.001% to 0.05%, more preferably 0.005% to 0.01%. Angelica keiskei extract is blended at a concentration of 0.01% to 0.1%, more preferably 0.1%.

[0100] Another embodiment of the present invention also relates to an environmental stimulus response modulator comprising exosomes containing specific miRNAs. The specific miRNAs include at least one miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p. The specific miRNAs are expressed in the skin in response to environmental stimuli and can therefore improve skin resistance to these stimuli. The environmental stimulus may be at least one stimulus selected from the group consisting of drying, low temperature, warming, and cooling, or may be a combination of multiple stimuli. The specific miRNA may be contained alone or in combination with other specific miRNAs. Exosomes containing the specific miRNA are released into the culture in response to the environmental stimulus by applying the environmental stimulus to a culture containing keratinocytes. Exosomes containing the specific miRNA may be purified from the recovered culture medium and used, or the recovered culture medium may be used directly. In other embodiments, the present invention may relate to the use of exosomes containing the specific miRNA for the manufacture of an environmental stimulus response regulator. It may relate to exosomes containing the specific miRNA for use in environmental stimulus response regulation. Furthermore, it may relate to a cosmetic method involving environmental stimulus response regulation, comprising applying exosomes containing the specific miRNA to a subject exposed to the environmental stimulus.

[0101] [Evaluation device for environmental stimulus response]

[0102] Other embodiments of the present invention may involve a device for evaluating environmental stimulus responses based on environmental stimulus-related miRNA (hereinafter referred to as an environmental stimulus response evaluation device). Such an environmental stimulus response evaluation device can determine the response to an environmental stimulus applied to the skin based on the amount of environmental stimulus-related miRNA. Such an environmental stimulus response evaluation device specifically includes the following:

[0103] an input unit 11 to which data on the amount of environmental stimulus-related miRNA in a biological sample is input;

[0104] The storage unit 12 stores the correspondence between the amount of at least one environmental stimulus-related miRNA and the environmental stimulus response:

[0105] a processing unit 13 for determining an environmental stimulus response based on the amount of at least one environmental stimulus-related miRNA in the input data and the above-mentioned correspondence relationship stored in the storage unit 12;

[0106] The output unit 14 outputs the determined environmental stimulus response.

[0107] As an example, the correspondence between the amount of at least one environmental stimulus-related miRNA and the environmental stimulus response stored in the storage unit 12 can be a correspondence table, a graph or a correlation formula, or can be the relationship between one or more thresholds and the intensity of the environmental stimulus response.

[0108] The processing unit 13 can determine the environmental stimulus response by reading the correspondence between at least one environmental stimulus-related miRNA and the environmental stimulus response stored in the storage unit 12, and determining the environmental stimulus response based on the amount of at least one environmental stimulus-related miRNA input from the input unit 11 and the correspondence. The processing unit 13 can extract the amount of at least one environmental stimulus-related miRNA from the input data related to the amount of the environmental stimulus-related miRNA. If the correspondence is a correspondence table, the environmental stimulus response can be determined by reading the degree of environmental stimulus response corresponding to the amount of the environmental stimulus-related miRNA in the analysis data from the correspondence table. If the correspondence is a correlation equation, the degree of environmental stimulus response can be determined by substituting the amount of the environmental stimulus-related miRNA in the analysis data into the correlation equation. If the correspondence is a correspondence between one or more threshold values ​​and the degree of environmental stimulus response, the degree of environmental stimulus response can be determined by comparing the values ​​with the respective threshold values. The determined degree of environmental stimulus response may be stored in the storage unit 12 , or may be output directly from the storage unit 12 or from the processing unit 13 via the output unit 14 .

[0109] In other embodiments, the environmental stimulus response evaluation device may use a learning unit to determine the environmental stimulus response based on data related to the amount of environmental stimulus-related miRNAs, rather than using the aforementioned correspondence determination. Such an environmental stimulus response evaluation device may also use a learning unit 15 that has previously learned information related to the amount of at least one environmental stimulus-related miRNA and information regarding the degree of environmental stimulus response as teacher data when determining the environmental stimulus response. Such a learning unit 15 performs pre-learning so that, upon inputting data containing the amount of at least one environmental stimulus-related miRNA, it outputs the environmental stimulus response. The information related to the amount of at least one environmental stimulus-related miRNA used by the learning unit 15 for learning may be information related to the type and amount of the environmental stimulus-related miRNA. In other words, a learning unit 15 may also be used that has previously learned information related to the type and amount of the environmental stimulus-related miRNA and information regarding the degree of environmental stimulus response as teacher data. Such a learning unit 15 performs pre-learning so that, upon inputting data containing the type and amount of the environmental stimulus-related miRNA, it outputs the degree of environmental stimulus response.

[0110] Such an environmental stimulus response evaluation device specifically includes:

[0111] an input unit 11 to which data concerning the amount of environmental stimulus-related miRNA in a biological sample is input;

[0112] A learning unit 15 that pre-learns information including the type and amount of environmental stimulus-related miRNA and information about the degree of environmental stimulus response as teacher data, and outputs the degree of environmental stimulus response when the data including the type and amount of environmental stimulus-related miRNA is input from the input unit 11;

[0113] The output unit 14 outputs the degree of response of the output object to the environmental stimulus.

[0114] Such an environmental stimulus response evaluation device may further include a processing unit 13 and a storage unit 12. The processing unit 13 may control the input of data containing the types and amounts of environmental stimulus-related miRNAs input from the input unit 11 to the learning unit 15, and control the output of the degree of environmental stimulus response output by the learning unit 15 from the output unit 14. Furthermore, the processing unit 13 may pre-adjust the input data containing the types and amounts of environmental stimulus-related miRNAs. The storage unit 12 may temporarily store the data containing the types and amounts of environmental stimulus-related miRNAs input from the input unit 11 and the degree of environmental stimulus response output by the learning unit 15.

[0115] The input unit 11 includes an interface. The interface can be connected to an operating unit such as a keyboard and mouse, a communication unit such as a LAN or a port, or an external storage device such as a CD-ROM, DVD-ROM, BD-ROM, or memory stick. Data including the type and amount of environmental stimulus-related miRNA can be input via the operating unit. Furthermore, processing instructions in the processing unit 13 can be given from the input unit 11 via the operating unit.

[0116] The storage unit 12 has storage devices such as RAM, ROM, flash memory, fixed disk devices such as hard disk drives, or portable storage devices such as floppy disks and optical disks. The storage unit 12 can store data and instructions input from the input unit 11. The storage unit 12 stores the correspondence between the type and amount of environmental stimulus-related miRNA and the degree of environmental stimulus response. Specifically, the correspondence between the type and amount of environmental stimulus-related miRNA and the degree of environmental stimulus response is stored in a corresponding table, graph, correlation formula or threshold value. In addition to storing the calculation results performed in the processing unit 13, the storage unit 12 can also store programs, databases, etc. used for various computer processing and store programs for the learning unit 15. The computer program can be installed via a computer-readable recording medium such as a CD-ROM, DVD-ROM, or the Internet. The computer program is installed in the storage unit 12 using a well-known installation program.

[0117] The processing unit 13 executes various calculations according to the programs stored in the storage unit 12. The calculations are performed by the central processing unit (CPU) included in the processing unit 13. The CPU includes functional modules that control the input unit 11, storage unit 12, learning unit 15, and output unit 14, and can perform various controls. These components can be composed of independent integrated circuits, microprocessors, firmware, etc. The information generated after each process in the processing unit 13 can be temporarily stored in the storage unit 12 or directly used for the next process.

[0118] The output unit 14 is configured to output the degree of response to environmental stimuli generated by the calculations performed by the processing unit 13. The output unit 14 may be a display device such as a liquid crystal display that directly displays the calculation results, or an output device such as a printer, or may be an interface unit for outputting to an external storage device or via a network.

[0119] The learning unit 15 uses well-known machine learning techniques, such as deep learning, to learn the relationship between input data containing the type and amount of environmental stimulus-related miRNAs and information about the degree of response to the environmental stimulus at that time. Information about the type and amount of environmental stimulus-related miRNAs and information about the degree of response to the environmental stimulus at that time can be pre-obtained for various subjects, and the learning unit 15 can use this data to learn. Deep learning is a type of machine learning that uses a multi-layered neural network consisting of an input layer, an intermediate layer, and an output layer. Each node in the input layer inputs a feature vector of the detection information. Each node in the intermediate layer outputs the sum of the values ​​obtained by multiplying the feature vectors output from each node in the input layer by a weight. Furthermore, the output layer outputs the sum of the values ​​obtained by multiplying the feature vectors output from each node in the intermediate layer by a weight. The learning unit 15 learns while adjusting the weights to minimize the difference between the output value output from the output layer and the information about the degree of response to the environmental stimulus. The input data input to the learning unit 15 is input data related to the amount of at least one environmental stimulus-related miRNA, preferably information related to the type and amount of the environmental stimulus-related miRNA.

[0120] [Environmental stimulus response evaluation system]

[0121] The environmental stimulus response evaluation device 10 of the present invention can exist on a network, forming an environmental stimulus response evaluation system 20 that includes the environmental stimulus response evaluation device 10. The environmental stimulus response evaluation system 20 is configured such that data including the type and amount of environmental stimulus-related miRNA is input to the input unit 11 of the environmental stimulus response evaluation device 10 via the network; and the degree of the determined environmental stimulus response is output from the output unit 14 via the network. Such an environmental stimulus response evaluation system 20 can further include a terminal device 30 and / or a miRNA analysis device 40 connected via the network. That is, the environmental stimulus response evaluation device 10 exists on a server, and the input unit 11 and the output unit 14 can be connected to the network via an interface unit, respectively. In addition, the learning unit 15 used by the environmental stimulus response evaluation device 10 can also be configured externally via a server, etc., to evaluate the environmental stimulus response through communication.

[0122] The miRNA analysis device 40 is a device that analyzes miRNA extracted from a biological sample. The type and amount of miRNA contained in the biological sample can be output via an output unit. The outputted miRNA data can be provided to an environmental stimulus response evaluation device via a network or directly.

[0123] The terminal device 30 connected to the network may include:

[0124] A network connection unit connected to the environmental stimulus response evaluation device 10;

[0125] A terminal output unit that outputs the degree of the environmental stimulus response of the object output from the output unit 14 of the environmental stimulus response evaluation device via the network connection unit.

[0126] Data including the types and amounts of environmental stimulus-related miRNAs can be input to the environmental stimulus response evaluation device 10 via the network connection unit.

[0127] Another embodiment of the present invention may involve a program that causes the environmental stimulus response evaluation device 10 to perform the above-mentioned processing. Such a program includes the following instructions to the processing unit 13:

[0128] Reading input data including the amount of at least one environmental stimulus-related miRNA input from the input unit 11,

[0129] Reading the correspondence between the amount of at least one environmental stimulus-related miRNA and the degree of environmental stimulus response stored in the storage unit 12,

[0130] The degree of environmental stimulus response is determined from the above input data and the above correspondence.

[0131] The determined degree of response to the environmental stimulus is output to the output unit 14 .

[0132] In the processing unit 13, instead of the process of determining the degree of environmental stimulus response from the above-mentioned correspondence, the degree of environmental stimulus response can be determined by inputting input data into a learning unit 15 that has been pre-learned in a manner such that the degree of environmental stimulus response is output if input data related to the type and amount of miRNA related to the environmental stimulus is input.

[0133] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0134] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the scope of the claims. Modifications to the present invention may be made, for example, by adding, deleting, or replacing the constituent elements of the present invention, provided that they do not exceed the scope of the present invention.

[0135] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0136] Example

[0137] Example 1: miRNA analysis in skin

[0138] 1. Skin evaluation model

[0139] In all experiments, a skin model (EPI-200 [Mattek]) was used, and the culture medium used was EPI-100NMM [Mattek].

[0140] The skin model was acclimated overnight in culture medium at 37°C and 95-98% relative humidity (RH) or 22°C and 95-98% relative humidity (RH). After acclimation, the culture medium was exchanged. A cylindrical partition was placed on the skin model to prevent the culture medium from leaking into the cylindrical interior. The skin model inside the cylindrical partition was exposed to the atmosphere and cultured for 24 hours under the following specific conditions until harvested.

[0141] (1) The control samples were incubated at 37°C and 95-98% relative humidity (RH) for 24 hours.

[0142] (2) Drying the Stimulus Samples: The atmosphere in the incubator was set to 37°C and 20% relative humidity (RH) using a humidity control device. The samples were cultured for 24 hours and then returned to 37°C and 95-98% relative humidity (RH).

[0143] (3) Cold stimulation The samples were acclimated at 22°C and 95-98% relative humidity (RH) and then cultured at 22°C and 95-98% relative humidity (RH) for 24 h.

[0144] (4) Temperature change (warming) stimulation: The samples were acclimated at 22°C and 95-98% relative humidity (RH) and then further cultured at 37°C and 95-98% RH for 24 h.

[0145] (5) Temperature change (cooling) stimulation: The samples were acclimated at 37°C and 95-98% RH and then further cultured at 22°C and 95-98% RH for 24 h.

[0146] 2. Preparation of Culture Medium

[0147] At the end of the culture, all the culture medium was recovered and stored at 4°C for exosome purification. Then, the culture medium was centrifuged at 300×g for 5 minutes at 4°C, and only the supernatant was recovered. The culture medium was further centrifuged at 1200×g for 20 minutes at 4°C, and only the supernatant was recovered. Finally, the culture medium was centrifuged at 10,000×g for 30 minutes at 4°C, and only the supernatant was recovered. 1 / 100 of the amount of V-Save (registered trademark) Extracellular Vesicle Blocking Reagent (extracellular vesicle storage stabilizer) was added to the supernatant. (Co., Ltd.), and concentrated using Vivaspin (registered trademark) 20-100K [Cytiva].

[0148] 3. Exosome Purification

[0149] Using MagCapture (registered trademark) Exosome Isolation Kit PS (co., Ltd.), and exosomes were purified according to the attached protocol. The particle size and concentration were then measured using NanoSight NS300 (Quantum Design).

[0150] 4. Measurement of Exosomal miRNA

[0151] To comprehensively analyze the purified exosome miRNAs, 3D-GENE microarray analysis was performed ( Co., Ltd. Microarray samples were sent to a contracted analysis provider, and RNA was extracted from purified exosomes. The extracted RNA was electrophoresed using an Agilent 2100 Bioanalyzer to identify the small RNA fraction. Each sample was then labeled and hybridized (32°C, 16 hours).

[0152] Compared to the control, the following miRNAs were altered in samples subjected to 24-hour drying stimulation:

[0153]

[0154] Compared with the control, the following miRNAs were altered in the samples subjected to low temperature stimulation:

[0155]

[0156]

[0157] Compared to the control, the following miRNAs were altered in the samples stimulated by temperature change (warming):

[0158]

[0159]

[0160] Compared to the control, the following miRNAs were altered in samples stimulated by temperature change (cooling):

[0161]

[0162]

[0163]

[0164] hsa-miR-4497 increased in samples stimulated by desiccation, hypothermia, temperature change (warming), and temperature change (cooling). The expression of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p increased in samples stimulated by hypothermia, temperature change (warming), and temperature change (cooling).

[0165] 5. Localization Analysis of hsa-miR-4497

[0166] The localization of miR-4497 in a skin model (EPI-200 (Mattek)) was analyzed by in situ hybridization using miRNAscope (registered trademark) HD ​​ReagentKit-RED (Advanced Cell Diagnostics, a brand of Bio-Techne Corporation). The experiment was carried out according to the accompanying protocol. Designed probe SR-hsa-miR-4497-S1, targeting 8-17 of MIMAT0019032 (Advanced Cell Diagnostics, a brand of Bio-Techne Corporation) was designed / produced and used as a probe that can detect miR-4497. Regarding the skin model, the localization of hsa-miR-4497 after dry stimulation, low temperature stimulation, temperature change (cooling) stimulation, and temperature change (cooling) stimulation was analyzed ( Figure 1 ). After dry stimulation, low temperature stimulation and temperature change stimulation (37 At 22°C, hsa-miR-4497, which was localized in the basal layer in the control, was shown to be distributed throughout the spinous layer and granular layer in response to various stimuli.

[0167] 6. Confirmation in Body Fluids

[0168] Blood was collected after a physical examination by a doctor. The serum was then separated by centrifugation. The exosomes were stored at -80°C for purification. 1 / 25 of a Protease inhibitor (cOmplete (registered trademark), EDTA-free, protease inhibitor cocktail) and 1 / 100 of an EV-Save (registered trademark) Extracellular Vesicle Blocking Reagent were added. (co., Ltd.), centrifuged at 800×g for 20 minutes at 4°C, and only the supernatant was recovered. Finally, centrifuged at 15,000×g for 20 minutes at 4°C, and only the supernatant was recovered. The supernatant was concentrated using Vivaspin (registered trademark) 20-100K [Cytiva]. MagCapture (registered trademark) Exosome Isolation Kit PS (Exosome Isolation Kit PS) ( (co., Ltd.) and exosomes were purified according to the attached protocol. Then, the particle size and particle concentration were measured using NanoSight NS300 (Quantum Design). In order to conduct a comprehensive analysis of miRNA in purified exosomes, 3D-GENE microarray analysis was performed ( Co., Ltd. Microarray samples were sent to a contracted analysis provider, and RNA was extracted from purified exosomes. The extracted RNA was electrophoresed using an Agilent 2100 Bioanalyzer to identify the small RNA fraction. Each sample was then labeled and hybridized (32°C, 16 hours).

[0169] Comparisons were made between people in their twenties (25-29 years old), their thirties (30-39 years old), and their forties (40-45 years old). The results showed that these factors could be detected, but the levels detected in serum tended to decrease.

[0170]

[0171] Example 2: Screening method based on skin miRNA

[0172] (1) Cell culture and sample preparation

[0173] Normal skin epidermal keratinocytes (Kurabo) were cultured in a culture medium containing normal human epidermal keratinocyte proliferation supplements (insulin, hEGF, hydrocortisone, BPE, and antibacterial agents gentamicin / amphotericin B) (Kurabo) in HuMedia-KB2 (Kurabo) basal medium for normal human epidermal keratinocyte proliferation. The cells were seeded in 24-well plates for screening, and 24 hours before collection, Japanese peach leaf extract BG30 and 0.1% Tokyo cherry blossom extract BG ( (Co., Ltd.), 0.1% Isodon chinensis extract (Maruzen Pharmaceutical Co., Ltd.), 0.1% Carthamus tinctorius extract BG ( (Co., Ltd.), 0.1% loquat extract (Xiangrong Industrial Co., Ltd.), 0.1% ashitaba extract BG ( (co., Ltd.), 0.1% Amur cork extract (Koei Kogyo Co., Ltd.), 0.1% soybean extract (Koei Kogyo Co., Ltd.). When the cells were recovered, the culture medium was removed, the cells were washed with PBS, and Isogen ( Then, RNA concentration and absorbance were measured using Nanodrop according to Isogen's protocol.

[0174] (2) Quantification of miRNA

[0175] Purified RNA was converted into cDNA using the Mir-X miRNA First-Strand Synthesis Kit included in the Mir-X miRNA qRT-PCR TB Green Kit [Clontec] according to the attached protocol. Then, real-time PCR was performed using TBGreen Advantage qPCR Premix and primers specific for each miRNA to quantify the miRNA. The following primer sequences were used:

[0176] miR-4497: ctccgggacggctgggc (SEQ ID NO: 1)

[0177] The results of changes in miRNA produced by each extract are shown in the figure ( Figure 2 ).

[0178] 0.1% Tokyo cherry blossom extract and 0.1% Isodon pubescens extract inhibited the expression of miR4497, thus enabling the selection of inhibitors of responses to environmental stimuli.

[0179] 0.1% safflower extract, 0.1% loquat extract, and 0.1% ashitaba extract enhanced the expression of miR4497. Thus, these agents can be selected as enhancers of responses to environmental stimuli.

Claims

1. A method for evaluating environmental stimulus response in the skin, using the amount of at least one specific miRNA selected from hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p and hsa-miR-10400-5p in a biological sample as an indicator.

2. The evaluation method according to claim 1, wherein the biological sample is selected from tissue fluid, blood, lymph, saliva, urine, tears and sweat. The evaluation method according to claim 1 or 2, wherein the specific miRNA is a skin-derived miRNA. The evaluation method according to claim 3 , wherein the skin-derived miRNA is a keratinocyte-derived miRNA. The evaluation method according to any one of claims 1 to 4, wherein the specific miRNA is hsa-miR-4497. The evaluation method according to claim 5 , wherein the environmental stimulus is a combination of dryness stimulus, low temperature stimulus, warming stimulus and cooling stimulus.

7. The evaluation method according to any one of claims 1 to 4, wherein the specific miRNA is at least one miRNA selected from hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p and hsa-miR-10400-5p. The evaluation method according to claim 7 , wherein the environmental stimulus is a combination of low temperature stimulation, warming stimulation and cooling stimulation.

9. A method for identifying accumulated skin damage in response to environmental stimuli by using the method for evaluating the response to environmental stimuli in the skin according to any one of claims 1 to 8.

10. A method for determining responsiveness to environmental stimuli by evaluating the response of skin to environmental stimuli according to any one of claims 1 to 8.

11. A method for screening a regulator of environmental stimulus response in the skin, comprising the following steps: a step of culturing a culture comprising keratinocytes in the presence of a drug candidate; a step of measuring at least one specific miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p in the culture after culturing; and This is a step of screening for drug candidates as modulators of environmental stimulus responses in the skin when the amount of a specific miRNA is altered compared to a control.

12. The screening method according to claim 11, wherein the specific miRNA is hsa-miR-4497.

13. The screening method according to claim 12, wherein the environmental stimulus is a combination of dryness stimulus, low temperature stimulus, warming stimulus and cooling stimulus.

14. The screening method according to claim 11, wherein the specific miRNA is at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p and hsa-miR-10400-5p. The screening method according to claim 14 , wherein the environmental stimulus is a combination of low temperature stimulation, warming stimulation and cooling stimulation.

16. An hsa-miR-4497 expression inhibitor, comprising at least one extract selected from the group consisting of a Tokyo cherry blossom extract and a tomentosa extract.

17. An hsa-miR-4497 expression enhancer, comprising at least one extract selected from the group consisting of safflower extract, loquat extract, and ashitaba extract.

18. A regulator of environmental stimulus response in the skin, comprising exosomes, wherein the exosomes comprise at least one miRNA selected from the group consisting of hsa-miR-4497, hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p and hsa-miR-10400-5p.

19. A regulator of the response to a stimulus in the skin that combines dryness, low temperature, warming, and cooling, comprising exosomes containing at least one miRNA selected from hsa-miR-4497.

20. A regulator of the response to a stimulus in the skin that combines low temperature stimulation, warming stimulation, and cooling stimulation, comprising exosomes, wherein the exosomes comprise at least one miRNA selected from the group consisting of hsa-miR-3648, hsa-miR-4745-5p, hsa-miR-6126, hsa-miR-762, hsa-miR-1237-5p, hsa-miR-3196, hsa-miR-3960, hsa-miR-4281, hsa-miR-4488, hsa-miR-6784-5p, hsa-miR-10396a-5p, and hsa-miR-10400-5p.

Images