WO2021104208A1 - 预防皮肤衰老与早衰的内源性小rna分子标靶及其应用 - Google Patents

预防皮肤衰老与早衰的内源性小rna分子标靶及其应用 Download PDF

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WO2021104208A1
WO2021104208A1 PCT/CN2020/130911 CN2020130911W WO2021104208A1 WO 2021104208 A1 WO2021104208 A1 WO 2021104208A1 CN 2020130911 W CN2020130911 W CN 2020130911W WO 2021104208 A1 WO2021104208 A1 WO 2021104208A1
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mir
skin
aging
hair
stem cells
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French (fr)
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张亮
于瑶
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中国科学院上海营养与健康研究所
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR

Definitions

  • the present invention relates to the field of molecular medicine, in particular to endogenous small RNA molecular targets for preventing skin aging and premature aging and their applications.
  • Skin epithelial stem cells (mainly including hair follicle stem cells and epidermal stem cells) are the basis of skin epithelial tissue homeostasis maintenance and wound repair. Among them, epidermal stem cells maintain the dynamic balance of epithelial tissue, while hair follicle stem cells support hair growth and regeneration, and can repair epithelium under traumatic conditions.
  • the purpose of the present invention is to provide a substance and method that can effectively prevent or delay skin aging.
  • the anti-aging substance and method of the present invention target endogenous small RNA molecules (microRNA31/miR-31).
  • the first aspect of the present invention provides a use of miR-31 (ie microRNA31) inhibitor to prepare a composition or preparation for preventing or treating skin aging (or anti-skin aging).
  • miR-31 ie microRNA31
  • the composition includes a pharmaceutical composition and a daily chemical composition.
  • the daily chemical composition includes shampoo, shower gel, skin care products, hair care products, and/or cosmetics.
  • composition or preparation is used for one or more (or all) applications selected from the following group:
  • the skin aging has one or more indicators selected from the following group:
  • the epithelial stem cells are selected from the group consisting of hair follicle stem cells, epidermal stem cells, skin epithelial stem cells, or a combination thereof.
  • the skin aging further includes: an increase in one or more representative senescent cell markers or markers, and/or a decrease in one or more representative young cell markers or markers .
  • the representative senescent cell markers or markers include (but are not limited to): SA- ⁇ -galactosidase, P16, cell proliferation ability.
  • the representative young cell markers or markers include (but are not limited to): H3K9me3, collagen gene COL17A1.
  • the skin aging includes skin aging caused by DNA damage.
  • the skin aging is induced by an inducing factor selected from the following group: ionizing radiation, ⁇ -ray irradiation, ultraviolet light, radiotherapy, excessive sun exposure, chemical mutagens, or a combination thereof.
  • the skin aging is caused by age factors.
  • the miR-31 includes hsa-miR-31, more preferably hsa-miR-31-5p.
  • the miR-31 has the nucleotide sequence shown in SEQ ID NO:1: 5'-aggcaagaugcuggcauagcu-3'.
  • the miR-31 has a core sequence shown in SEQ ID NO.: 5: 5'-aggcaaga-3' (SEQ ID No.: 5).
  • the miR-31 inhibitor (or antagonist) is selected from the group consisting of miR-31 antibody, miR-31 binding protein, miR-31 small molecule inhibitor, miR-31 antisense sequence, AntagomiR-miR-31, miR-31 sponge, reagent for knocking out miR-31 encoding gene, and physiologically acceptable carrier, or a combination thereof.
  • the AntagoniR-hsa-miR-31 has the nucleotide sequence shown in SEQ ID NO: 3 or its derivative or modified nucleotide sequence.
  • the AntagomiR-mmu-miR-31 has the nucleotide sequence shown in SEQ ID NO: 4 or a derivative or modification thereof.
  • the "reagent for knocking out genes encoding miR-31" includes Cas9-based gene editing reagents.
  • the Cas9 gene editing reagent is used to perform base substitution on the DNA region corresponding to the miR-31 core sequence, or to cut the DNA region corresponding to the miR-31 core sequence, thereby reducing or Eliminate miR-31.
  • the miR-31 inhibitor (or antagonist) is a modified or unmodified nucleic acid sequence.
  • the modification is selected from the following group: methylation modification, methoxyethyl modification, hydrocarbyl modification, cholesterol modification, glycosylation modification (such as 2-methoxy-glycosyl modification, Hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification (such as fluorine modification, etc.), nucleic acid modification (such as "TT" modification, etc.), phosphorothioate modification , Locked nucleotide modification, or a combination thereof.
  • the second aspect of the present invention provides a composition or preparation that can be used to prevent or treat skin aging.
  • the composition or preparation includes: (a) a physiologically acceptable carrier; and (b) an effective amount of active Ingredients, the active ingredient is miR-31 inhibitor.
  • composition or preparation also contains (c) other anti-aging active ingredients.
  • the active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required under aseptic conditions.
  • composition or preparation further contains a skin penetration enhancer.
  • the physiologically acceptable carrier is selected from the group consisting of water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, nanogels, cellulose Its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water or combinations thereof.
  • the dosage form of the composition or preparation is a topical dosage form.
  • the dosage form of the composition or preparation includes ointment, cream, patch, lotion, spray, microinjection and the like.
  • the method of administration of the composition or formulation is selected from the group consisting of topical application, transdermal application and transdermal application.
  • the third aspect of the present invention provides a method for in vitro screening of potential substances for anti-skin aging, including the steps:
  • test group and control group wherein a test substance is added to the detection system in the test group, and a control substance is added to the detection system in the control group, and the control substance includes a negative control substance;
  • the experimental conditions of the test group and the control group are the same or substantially the same.
  • step (1) further includes the following steps:
  • step (1.1) treatment is performed by a method selected from the following group to form the DNA damage-inducing environment: irradiation treatment, chemical mutagens, high temperature, violent shaking, pathological induction Stress, or a combination thereof.
  • the irradiation treatment is selected from the group consisting of ionizing radiation, ⁇ -ray irradiation, UVA/UVB irradiation, radiotherapy, excessive sun exposure, or a combination thereof.
  • the detection system is a cell system.
  • the cells include skin cells, preferably epithelial cells, more preferably epithelial stem cells.
  • the method further includes the steps:
  • step (4) compare the DNA damage amplitude D1 in the test group with the DNA damage amplitude D0 in the control group, if the ratio of (D0-D1)/D0 is ⁇ 5%, preferably ⁇ 10 %, preferably ⁇ 20%, preferably ⁇ 50%, more preferably ⁇ 80%, and most preferably ⁇ 100%, indicating that the test substance is a potential anti-aging substance.
  • the fourth aspect of the present invention provides a method for preventing or treating skin aging, including the steps of: administering a miR-31 inhibitor, or a miR-31 inhibitor-containing composition or preparation to a subject in need.
  • composition or preparation includes: antisense nucleotides of miR-31 as an active ingredient.
  • the antisense nucleotide of miR-31 includes AntagomiR-miR-31.
  • a method for diagnosing/detecting skin aging including the steps:
  • sample to be tested is skin cells or skin tissue
  • (b) Detect the expression level L1 of miR-31 in the sample to be tested, and compare it with the reference value L0. If the expression level of miR-31 in the sample to be tested is significantly higher than the reference value, then it will be prompted to The skin aging process of the sample is fast and/or the skin aging degree is high, wherein the reference value L0 is the expression level of miR-31 in normally aging skin cells.
  • the sample to be tested is skin epithelial tissue or skin epithelial cells.
  • the "significantly higher” refers to the increase in the expression level of the corresponding miRNA-31 (that is, the ratio of (L1-L0)/L0) ⁇ 10% compared with the negative control sample , Preferably ⁇ 20%, preferably ⁇ 50%, preferably ⁇ 100%, more preferably ⁇ 150%, and most preferably ⁇ 200%.
  • the skin is human skin.
  • the miR-31 includes hsa-miR-31, more preferably hsa-miR-31-5p.
  • kit further contains instructions, and the instructions describe the method described in the fifth aspect of the present invention.
  • Figure 1 shows the establishment of a local skin premature aging model induced by local ionizing radiation:
  • Figure 1A shows a schematic diagram of the experimental procedure of a mouse model of local premature skin aging induced by local ionizing radiation (local skin premature aging mice). Yellow is the irradiated area. The mice received local irradiation at around 7 weeks of age (P50). Wax hair removal (Wax) was performed five days later to induce hair regrowth. After 28 days (4 weeks), the skin phenotype was observed.
  • Mox Wax hair removal
  • Figure 1B shows a photo of the back skin and hair of the above-mentioned localized premature aging mouse.
  • the scale is 1cm.
  • Figure 1C shows a statistical graph of the wound healing rate of the above-mentioned local premature skin aging mice.
  • Figure 1D shows the in vitro skin tissue mass (Exo-plant) of the above-mentioned local skin premature aging mouse keratinocyte migration callus rate experiment.
  • Left Microscope photo, black is the body of the skin tissue block, and the white line indicates the front scale of the migrating keratinocyte layer 500 ⁇ m.
  • Right: Statistics of the area of the migrating keratinocyte layer: **: P ⁇ 0.01, n 3, two-tailed t-test.
  • Figure 1F shows a photo of the back skin hair after the second wax depilation in the skin of the above-mentioned localized premature skin aging mouse skin. It can be seen that the newly born hair in the irradiated area is very sparse. The scale is 1cm.
  • FIG. 1 shows that Mir31 knockout can restore stem cell depletion caused by radiation damage:
  • Figure 2A shows a schematic diagram of the construction principle of Mir31 conditional knockout mice.
  • Figure 2C-G shows the phenotype analysis of localized skin premature aging induced by local ionization radiation in cKO and WT mice:
  • Figure 2C shows the white hair ratio analysis in the irradiated area;
  • Figure 2D shows the wound in the irradiated area Healing rate analysis, the measurement method is the same as Figure 1C;
  • Figure 2E shows the in vitro skin tissue mass (Exo-plant) keratinocyte migration callus rate analysis, the measurement method is the same as Figure 1D, Rad and noRad respectively represent the irradiated area and non-irradiated area
  • Figure 2F shows the relative ratio of epithelial stem cells in the skin. The measurement method is the same as Figure 1E.
  • Rad and noRad represent the irradiated and non-irradiated skin respectively;
  • Figure 2G shows the left image after the second wax depilation
  • FIG. 2H shows the Q-RT-PCR analysis of miR-31 expression in mouse epithelial cells (MK) and human epithelial cells (NHEK) after Antagomir transfection.
  • SCR shows the control Scramble Antagomir transfection
  • Figure 3 shows that conditional knockout of miR31 does not affect normal hair growth.
  • Figure 3A shows photos of back skins of cKO and littermate WT control mice 39 days after Tamoxifen injection. Tamoxifen injection induction was performed on the 21st day after birth.
  • Figure 3D shows a microscope photo of the back fur of a mouse, showing the hair regeneration process after waxing.
  • d0, d6, and d10 represent the 0th, 6th, and 10th days after waxing, respectively. All mice were induced by Tamoxifen injection at 7 weeks of age, and waxed and shaved two days later.
  • noRad means not irradiated; “Rad” means irradiated.
  • Figure 4 shows that knocking out miR-31 significantly inhibits the natural aging of the skin.
  • the left panel of Figure 4A shows a representative back photo of a 17-month-old Mir31 conditional knockout mouse cKO and a littermate same-sex wild-type mouse WT.
  • the arrows represent areas of baldness.
  • the scale is 1cm.
  • Figure 4C shows the CD34+CD49f+ hair follicle stem cell content statistics in the whole skin epithelial tissue of the same-sex cKO and WT mice at 21 months of age, based on flow cytometry analysis.
  • Figure 4F shows the in vitro callus migration ability analysis of 21-month-old same-sex cKO and WT mouse skin tissue mass (exoplant).
  • the left picture is a representative photo of the tissue block on the 6th day of culture.
  • the black part on the left is the body of the tissue block, and the dashed line is the migration front of the epidermal cell layer.
  • the area between the dotted line and the body is the migration area of epidermal cells.
  • the figure on the right shows the statistical results of the migration area of epidermal cells in the experiment represented by the figure on the left.
  • an endogenous small RNA molecule miR-31 is closely related to skin aging (especially premature aging or aging caused by irradiation), and therefore can be used to prevent skin aging.
  • Targeting endogenous small RNA molecules with premature aging have shown that on the one hand, miR-31 is significantly increased in ⁇ -ray irradiated tissues, especially dorsal epithelial stem cells, and is positively correlated with aging indicators.
  • by inhibiting miR-31 it can delay the skin (especially skin epithelial cells). Or epithelial tissue) aging.
  • skin aging and premature aging can be prevented and/or delayed.
  • the present invention has been completed.
  • miR-31 is significantly increased in ⁇ -ray irradiated tissues, especially dorsal epithelial stem cells, and is positively correlated with a variety of aging indicators: loss of skin stem cells (especially epithelial stem cells), hair albinism, decreased hair regeneration ability, and epidermal growth. The callus function declines.
  • mice showed anti-aging effects, such as reducing hair albino caused by anti-skin aging, and promoting hair regeneration. Promotes wound healing ability.
  • antisense nucleic acids are used to inhibit miR-31 in human and mouse epithelial stem cells, similar anti-aging effects can be achieved.
  • the term "about” may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • Sequence identity compares two alignments along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein) Sequence, and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein Sequence.
  • skin aging and “skin aging” are used interchangeably to refer to the aging of skin epithelial tissues.
  • the term includes normal skin aging, as well as premature or too rapid skin aging (ie, premature skin aging).
  • Skin epithelial stem cells (mainly including hair follicle stem cells and epidermal stem cells) are the basis of skin epithelial tissue homeostasis maintenance and wound repair.
  • Epithelial stem cells include hair follicle stem cells, epidermal stem cells, other stem cells belonging to skin epithelial tissues, or a combination thereof.
  • epidermal stem cells maintain the dynamic balance of epithelial tissue, while hair follicle stem cells support hair growth and regeneration, and can repair epithelium under traumatic conditions.
  • stem cells There are some poorly defined stem cells in the skin epithelial tissue, such as the stem cells at the junction of hair follicles and epidermis, collectively referred to as skin epithelial stem cells.
  • skin epithelial stem cells As a lifelong self-renewing tissue, the aging of skin epithelial tissue is closely related to the failure of its stem cells, but the specific molecular mechanism leading to this type of stem cell failure is not fully understood.
  • the skin aging has one or more indicators selected from the group consisting of: (1) loss of skin epithelial stem cells; (m) albinism of hair; (n) decrease in hair regeneration ability; and ( o) Decreased epithelial callus function.
  • skin aging also includes: an increase in one or more representative senescent cell markers or markers, and/or a decrease in one or more representative young cell markers or markers.
  • Said representative markers or markers of senescent cells include (but are not limited to): SA- ⁇ -galactosidase, P16, cell proliferation ability; said representative markers or markers of young cells include (but Not limited to): H3K9me3, collagen COL17A1.
  • DNA damage is generally considered to be a major factor leading to tissue aging/premature aging.
  • tissue aging/premature aging In skin tissues, both exogenous and endogenous DNA damage inducing factors can cause significant premature aging.
  • typical skin aging phenotypes including hair loss and albinism will appear early.
  • ultraviolet light (UV) in the sun is a well-known cause of DNA damage, and it can also significantly accelerate skin aging, leading to the common symptoms of photoaging in the skin.
  • ionizing radiation commonly used in tumor radiotherapy is also a strong inducer of DNA damage, which can induce premature aging of skin cells in the radiotherapy area, and cause long-term skin callus dysfunction, which can lead to the development of refractory ulcers.
  • the risk of cancer is one of the most common complications in tumor radiotherapy.
  • DNA damage is the direct cause of skin epithelial and epithelial stem cell failure. Inhibiting the failure of skin epithelial stem cells caused by DNA damage has broad application prospects in skin anti-aging beauty and the prevention and treatment of radiation skin damage. However, there is no specific treatment that has entered the clinical stage.
  • the present invention provides a technical approach for inhibiting the aging effect of DNA damage on skin epithelial stem cells by targeting miR-31 inhibitors, thereby effectively combating skin aging and premature aging.
  • MicroRNA also known as microRNA (miRNA), as used herein, the term “miRNA” refers to a type of RNA molecule that is processed from transcripts that can form miRNA precursors. Mature miRNAs usually have 18-26 nucleotides (nt) (more specifically about 19-22 nt), and miRNA molecules with other numbers of nucleotides are not excluded. miRNA can usually be detected by Northern blotting.
  • miRNA can be processed from precursor miRNA (Precursor miRNA, Pre-miRNA), the precursor miRNA can be folded into a stable stem-loop (hairpin) structure, the length of the stem-loop structure is generally 50-100bp Sometimes longer.
  • the precursor miRNA can be folded into a stable stem-loop structure, and both sides of the stem of the stem-loop structure contain two substantially complementary sequences.
  • the precursor miRNA can be natural or artificially synthesized.
  • the precursor miRNA can be sheared to generate miRNA, and the miRNA can be substantially complementary to at least a part of the sequence of the mRNA encoding the gene.
  • substantially complementary means that the sequence of nucleotides is sufficiently complementary to interact in a predictable manner, such as forming a secondary structure (such as a stem-loop structure).
  • two "substantially complementary" nucleotide sequences have at least 70% of the nucleotides complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; more preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • two sufficiently complementary molecules can have up to 40 unmatched nucleotides; preferably, up to 30 unmatched nucleotides; more preferably, up to 20 unmatched nucleosides Acid; More preferably, there are at most 10 unmatched nucleotides, such as 1, 2, 3, 4, 5, 8, 11 unmatched nucleotides.
  • the "stem-loop” structure is also referred to as the "hairpin” structure, which refers to a nucleotide molecule that can form a secondary structure including a double-stranded region (stem).
  • the double-stranded region is formed by two regions (located on the same molecule) of the nucleotide molecule, the two regions are arranged on both sides of the double-stranded portion; it also includes at least one "loop” structure, including non-complementary nucleotides Molecules, that is, single-stranded regions.
  • the double-stranded portion of the nucleotide can maintain the double-stranded state.
  • insertions, deletions, substitutions, etc. can lead to non-complementarity in a small region or the small region itself forms a stem-loop structure or other forms of secondary structure.
  • the two regions can still be substantially complementary, and in the foreseeable Interaction occurs in the way to form the double-stranded region of the stem-loop structure.
  • the stem-loop structure is well-known to those skilled in the art. Generally, after obtaining a nucleic acid with a nucleotide sequence with a primary structure, the skilled in the art can determine whether the nucleic acid can form a stem-loop structure.
  • the miRNA in the present invention refers to miR-31, which is an endogenous microRNA that is highly conserved in mammalian evolution.
  • the sequence is shown in SEQ ID NO.:1: 5'-aggcaagaugcuggcauagcu-3', miRbase named hsa-miR-31-5p, accession number: MIMAT0000089, which is the MIR31 gene (hg38chr9:21,512,115-21,512,185, Gencode Gene :ENSG00000199177.1) expression product.
  • mouse miRbase is named mmu-miR-31-5p, accession number: MIMAT0000538, and the corresponding gene is Mir31(mm10chr4:88,910,557-88,910,662 ).
  • Mouse miR-31 only has one more nucleotide at the 3'end than human miR-31, and it is known that the biological function of miRNA mainly depends on its 5'end 8nt seed sequence, that is, its core sequence is 5'at the 5'end.
  • miR-31 can play an important regulatory role in multiple biological processes such as embryo implantation, organ development, bone and muscle formation, and immune system homeostasis maintenance. However, miR-31 plays an important role in skin aging/premature aging. The possible effect has not yet been reported.
  • miR-31 is a key endogenous factor that mediates DNA damage-related skin aging and premature aging. Taking the phenomenon of premature skin aging induced by ionizing radiation in mice as a model, the inventors found that miR-31 is significantly up-regulated in skin epithelial stem cells stimulated by DNA damage, which is closely related to the loss of epithelial stem cells and subsequent premature skin aging.
  • miR-31 is a small RNA molecular target that can be used to prevent DNA damage-related skin aging and premature aging.
  • the genetic/epigenetic intervention methods developed on this basis to inhibit its endogenous expression, as well as antisense nucleotide drugs or other methods that inhibit its function, can be used to prevent skin aging and premature aging symptoms related to DNA damage , Has a wide range of application value.
  • the microRNA is derived from a human or non-human mammal; preferably, the non-human mammal is a rat, a mouse, a mouse, or a human whose family sequence is completely identical.
  • the core sequence refers to the nucleotide sequence at positions 1-8 of the microRNA.
  • the "function identical or substantially the same as miR-31" means that miR-31 retains ⁇ 40%, ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90% of the functions of miR-31.
  • the invention also includes miRNA variants and derivatives.
  • miRNA derivatives in a broad sense can also include miRNA variants.
  • Those of ordinary skill in the art can use general methods to modify miR-31, including but not limited to: methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification) , Hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification), etc.
  • MicroRNA31 refers to mouse miR-31 gene DNA
  • MIR31 refers to human miR-31 gene DNA
  • the "microRNA31”, “microRNA31/miR-31”, “miRNA-31”, “miR-31” and “microRNA-31” mentioned in the present invention are interchangeable and can refer to one or more of the following active ingredients :
  • the microRNA of the miRNA-31 family, the microRNA of the miRNA-31 family includes: miRNA-31 or a modified miRNA-31 derivative, and the core sequence is 5'-aggcaaga-3' (SEQ ID NO.: 5), microRNAs or modified miRNA derivatives with the same or substantially the same function as miRNA-31;
  • precursor miRNA which can be processed in the host into (a) The miRNA-31;
  • the miRNA-31 is derived from human or non-human mammals.
  • the present invention also provides an antisense nucleic acid targeting miR-31 or a precursor thereof, or a derivative thereof.
  • the modified miRNA derivative (especially an antisense nucleic acid targeting miR-31 or a precursor thereof) is a compound monomer having the structure shown in formula I or a compound thereof. Aggregate:
  • each X is an antisense sequence targeting miR-31; each Y is independently a modification that promotes the stability of the antisense administration of miR-31 targeting; n is 1-100 ( Preferably 1-20) a positive integer (preferably n is 1, 2, 3, 4 or 5); m is a positive integer of 1-1000 (preferably 1-200); each "-" represents a joint, Chemical bond or covalent bond; in another preferred example, the linker is a nucleic acid sequence with a length of 1-10 bases.
  • the Y includes (but is not limited to): cholesterol, steroids, sterols, alcohols, organic acids, fatty acids, esters, monosaccharides, polysaccharides, amino acids, polypeptides, mononucleotides, polynucleotides.
  • a polynucleotide targeting miR-31 has the structure shown in Formula II:
  • Seq forward is an antisense nucleotide sequence that can be processed in the host into miRNA-31
  • Seq reverse is a nucleotide sequence that is substantially or completely complementary to Seq forward
  • X is The spacer sequence between the Seq forward and the Seq reverse, and the spacer sequence is not complementary to the Seq forward and Seq reverse; and the structure shown in formula II is transformed into the host cell to form the structure shown in formula III secondary structure:
  • Seq forward , Seq reverse and X are defined as above, and
  • the present invention also provides non-human mammal models with high expression of miR-31 or low expression of miR-31, which can be used to assess the relationship between miR-31 and skin aging or to screen or verify whether a test substance has passed miR-31. The effect of delaying skin aging.
  • a representative low expression of miR-31 includes the low expression (or no expression) of miR-31 knock-down and knock-out types.
  • two tools mouse Sox9CreER tool mouse and miR-31 conditional knockout mouse are used, and the target transgenic mouse Sox9CreER and miR of Mir31 LoxP/LoxP are obtained by crossing the F2 generation.
  • -31 Conditionally induces knockout mice.
  • a Tamoxifen injection model was performed.
  • miR-31cKO mice and littermate wild-type control mice (WT) are both treated with a single intraperitoneal injection of 40 mg/kg of Tamoxifen (sigma, T564) for miR-31 conditional knockout .
  • each group of mice is subjected to ⁇ -ray local irradiation treatment, the specific steps are as in Example 1, and then, in order to test the hair regeneration ability of mice , Wax treatment was used to induce hair regrowth, and then the skin phenotype was observed for about 28 days (4 weeks).
  • the experimental flow chart is shown in Figure 1A.
  • conditional knockout induced by Tamoxifen injection is performed 2 days before the irradiation.
  • active ingredient or “active ingredient for skin anti-aging” refers to an antagonist or inhibitor that targets miR-31.
  • Representative inhibitors include (but are not limited to): antibodies targeting miR-31, binding proteins, small molecule inhibitors, antisense sequences, AntagomiR, micro-RNA sponges, and micro-RNA coding genes Reagents, or combinations thereof.
  • One type of particularly effective inhibitors are antisense nucleotides against miR-31.
  • the miR-31 inhibitor used is AntagomiR-miR-31.
  • the AntagomiR-hsa-miR-31 was purchased from Gemma Biotech. It is an antisense oligonucleotide reagent targeting the core sequence of human hsa-miR-31, and its sequence is shown in SEQ ID NO.: 3: a s g s cuaugccagcaucuug s c s c s u s -Chol (the s subscript represents the thio skeleton modification, and Chol represents the cholesterol modification).
  • the AntagomiR-mmu-miR-31 was purchased from Gemma Biotech.
  • Antisense oligonucleotides are a kind of oligonucleotide fragments expressed by artificially synthesized or constructed antisense expression vectors. The length is mostly 15-30 nucleotides. Through the principle of base complementation, they interfere with the unwinding of genes. Replication, transcription, mRNA splicing and processing, as well as export and translation, etc., to regulate cell growth and differentiation. According to different binding sites, it can be divided into antisense DNA (asDNA), antisense RNA (asRNA), and autocatalytic ribozyme (ribozyme).
  • asDNA antisense DNA
  • asRNA antisense RNA
  • ribozyme autocatalytic ribozyme
  • antisense oligonucleotides which have the advantages of high theoretical target specificity (base complementation), easy design, Diverse and simple synthesis, high locality and pertinence. These are incomparable in the design, production and effect of conventional drugs, and therefore have great appeal and research value. Most traditional drugs are combined with proteins to modify the functions of proteins. In contrast, antisense agents work at the mRNA (DNA) level, preventing it from being translated into protein.
  • RNA interference has become a third-generation efficient method for inhibiting gene expression in mammalian cells.
  • This technology uses small interfering RNA molecules (siRNA) composed of 21-23 residues.
  • Antisense nucleotides are designed and synthesized for a specific target mRNA (DNA) sequence, with extremely high specificity; (2) Antisense nucleic acids are designed and synthesized for a target gene with a known sequence, because the target gene The sequence is known, antisense nucleic acid has only 15-30 bases, simple structure, easy to design and large-scale synthesis in vitro; (3) antisense nucleic acid into the cell has nothing to do with the cell cycle, it can enter the proliferative cell as well as the non- Cells in the proliferation phase; (4) Antisense oligonucleotides do not contain viral sequences, will not produce an immune response, and will not integrate into the host chromosome.
  • DNA target mRNA
  • a major challenge of antisense technology is the stability of oligonucleotides, because unmodified oligonucleotides are rapidly degraded by nucleases in vivo.
  • a large number of chemically modified nucleotides are used in antisense experiments.
  • the modification of nucleotides is divided into three categories: analogs of unnatural bases, modified sugars (especially the position of ribose), and altered phosphate backbones.
  • Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, and glycosylation modification (Such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification), etc.
  • a modified antisense oligonucleotide targeting miR-31 is phosphorothioate DNA Oligonucleotides (PS DNA ON).
  • Another antisense oligonucleotide targeting miR-31 contains an alkyl modification at the 2'position of ribose. 2'-O-methyl and 2'-O-ethyl are two important members of this type of modification. Oligonucleotides with this modification are less toxic than phosphorothioate DNA, and their affinity for complementary RNA has also been improved.
  • antagomir (or targeting miR-31 sponge) can be used to effectively inhibit miR-31 in epithelial tissues or cells, thereby delaying skin aging.
  • miRNA antagomir has higher stability and inhibitory effects in and outside the animal body, and can overcome obstacles such as cell membranes and tissues in vivo to be enriched in target cells.
  • Antagomir does not require transfection reagents in cell experiments, thus avoiding the complicated steps of the transfection reagent packaging process and its influence on the experiment. In animal experiments, it can be administered by systemic or local injection, inhalation, and drug feeding, and the effect can last as long as several weeks.
  • the present invention provides a composition or preparation (including cosmetics, daily chemicals, etc.) that can be used to prevent or treat skin aging.
  • the composition or preparation includes: (a) an acceptable carrier (including pharmaceutically acceptable Carrier, physiologically acceptable carrier, cosmetically acceptable carrier); and (b) an effective amount of active ingredient, said active ingredient is a miR-31 inhibitor.
  • the effective amount of the active ingredient can vary with the mode of use and the degree of skin aging.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials).
  • an effective amount or “effective dose” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • pharmaceutically acceptable ingredients are suitable for humans and/or mammals without excessive side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents.
  • physiologically acceptable carrier refers to a carrier for active ingredients, including: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, Cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate , Vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, or combinations thereof.
  • the choice of carrier should match the mode of
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the administration mode.
  • the dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, and sustained-release agent.
  • it can be prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials).
  • the factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration The way and so on.
  • the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg/kg animal body weight (preferably 0.0001 mg-10 mg/kg animal body weight), satisfactory effects can be obtained.
  • several divided doses can be given every day, or the dose can be reduced proportionally.
  • composition or product is a cosmetic or daily chemical product containing the active ingredient of the present invention (targeting miR-31 inhibition).
  • composition or preparation preferably also contains a skin penetration enhancer.
  • the dosage form of the composition or preparation is a topical dosage form.
  • the dosage forms of the cosmetic composition or preparation include (but are not limited to): solid preparations, liquid preparations, gel preparations, and semi-solid preparations. Some preferred dosage forms are ointments, creams, patches, paints, sprays, microinjections, solutions, shampoos, etc.
  • representative products include (but are not limited to): shampoo, shower gel, skin care products, hair care products, and facial masks.
  • the method of administration of the composition or formulation is selected from the group consisting of topical application, transdermal application and transdermal application.
  • miR-31 is a microRNA closely related to the aging of skin cells (especially skin epithelial cells), so it can be used as an endogenous small RNA molecular target for the prevention or treatment of skin aging and premature aging .
  • Targeted miR-31 inhibitors can effectively antagonize miR-31, thereby effectively fighting or delaying skin aging (especially premature aging).
  • the antisense nucleic acid targeting miR-31 can directly act on the skin, so the application is simple and safe.
  • mice (miR-31 conditional knockout mice) and their preparation
  • the Sox9CreER tool mouse was obtained from the Beijing College of Life Sciences, Chinese Academy of Sciences, with a genetic background of C57BL/6. This strain of mice specifically expresses Cre recombinase that can be induced and activated by tamoxifen in skin epithelial stem cells , Is a tool mouse for conditional gene knockout in skin epithelial stem cells.
  • the miR-31 conditional knockout mice were purchased from Southern Model Biology Co., Ltd., and were located at the upstream and downstream of the Mir31 gene region (mm10chr4:88910557-88910662) of C57BL/6 wild-type mice by CRISPR-Cas9 technology at about 300bp. LoxP site was knocked into the location. Obtained Mir31LoxP mice (FloxP for short), the theoretical knockout area is mm10chr4:88910180-88911062, which completely covers the mouse Mir31 gene area, and does not affect any known peripheral genes.
  • Example 1 Mouse skin premature aging model induced by local ionization radiation
  • ⁇ -ray irradiation was performed according to standard experimental procedures in the Institute of Radiation Medicine, Fudan University School of Medicine.
  • P50 7-week-old mice
  • the upper and lower lead plates are the same, and the lead plates are perpendicular to the radiation source.
  • the plates are each left with vertically aligned small holes with a diameter of 3 cm.
  • the upper part of the mouse is placed in the small hole.
  • the lead plate covers the head and lower body of the mouse.
  • the limbs of the mouse are properly fixed with tape and then irradiated.
  • the irradiation dose is 10Gy.
  • wax hair removal was performed to completely remove the existing hair, thereby activating hair regeneration. After 28 days (4 weeks), the phenotypes such as skin color and wound healing were observed. Use wax to remove hair and track and take pictures to observe its regeneration rate.
  • the method of repeating the hair regeneration ability test is to perform the second wax depilation to repeat the operation 2.1 after the above-mentioned hair regeneration is completed.
  • the results of the initial hair regrowth experiment are shown in Figure 1B. After 28 days of depilation, the newly-born hair on the unirradiated area on the back of the mouse is naturally black, while the newly-born hair on the irradiated area is light golden and almost white. .
  • the results of the secondary hair regeneration experiment are shown in Figure 1F, which are similar to the results of the first hair regeneration experiment. Newborn black hair grows from the unirradiated area on the back of the mouse, while only sparse albino hair grows from the irradiated area. .
  • ⁇ -ray irradiation shows a series of typical phenotypes of premature skin aging, mainly including the appearance of whitening of new hair and the decrease of regeneration ability.
  • mice were anesthetized with isoflurane and their hair was shaved, and the back was punched and wounded with an 8mm punch.
  • a surgical towel (3M, SP1107) to prevent the wound from deforming. Take it off after three days. Take pictures before and after 0, 4, 7, 9, 12 and 15 days after the wound. Calculate the wound area according to the scale. Within each group The ratio of the wound area of mice at different time points to the 0-day average wound area in each group was used to obtain the percentage of wounds. Each group of data was recorded until the callus was completely over.
  • mice After the mice were killed, the hair was removed with depilatory cream, and the fatty tissue was removed with a scalpel after disinfection with 75% alcohol.
  • punch a hole with a 1mm punch to obtain a skin block After washing in PBS, punch a hole with a 1mm punch to obtain a skin block, add 1-2ul matrigel (Corning, 356237) in the middle of each hole, attach the skin block and place 1mg/ml fibronectin (Millipore, FC010-1mg) to coat Cultured in the coated 24-well plate (coring, CLS3527). After 7 days of incubation, take pictures under a microscope, and use the image statistics software Imagej to measure the area of cells that migrated from the edge of the skin block with the skin block as the center point. The area of each group was ratioed to the area of the wild-type untreated part.
  • FIG. 1C The statistical results of the percentage of wound healing on the back of mice are shown in Figure 1C.
  • the wound healing speed of the back wax depilation treatment of mice is significantly slower in the irradiated area than in the unirradiated area.
  • Figure 1D The results of the exo-plant keratinocyte migration callus rate experiment of local premature skin aging mice are shown in Figure 1D: the left image is a microscope photo, the black is the skin tissue body, and the white line indicates the migration of keratinocytes At the front of the layer, the migration rate of keratinocytes in the irradiated area is significantly slower than that in the unirradiated area; the right picture shows the area statistics of the migrated keratinocyte layer, which also proves that the migration area of keratinocytes in the irradiated area is far less than that in the unirradiated area. Keratinocytes in the illuminated area.
  • ⁇ -ray irradiation showed a series of typical phenotypes of premature skin aging, mainly including decreased skin healing ability. Including the decrease of wound healing rate in vivo and the decrease of keratinocyte migration rate of exo-plant cultured in vitro.
  • mice After the mice were killed with a carbon dioxide lethal device, their hair was shaved and the back skin was removed, the dermal fat tissue was removed with a scalpel, washed with PBS, and placed in Trypsin-EDTA for overnight digestion at 4°C. Scrape the cells with a blade on the next day, collect the cell suspension, and place it in PFE (PBS+1%FBS+1Um EDTA) staining solution on ice for CD34 (1:100, eBioscience, 11-0341-82), CD49f (1:100, biolegend, 313602) staining. After staining, the cells were washed and tested on a flow analyzer (BD, CytoFLEX LX), and the results were analyzed by the flow analysis software FlowJo 10.0.
  • PFE PBS+1%FBS+1Um EDTA
  • the flow cytometric detection of CD34 + CD49f + cell population is epithelial stem cells
  • the flow cytometry software FlowJo 10.0 is used to analyze the percentage of endothelial stem cells in each group and take the ratio with the percentage of unirradiated parts of the mouse
  • the ratio of epithelial stem cells in the irradiated area of the mouse to the non-irradiated area is about 50%, which is consistent with the results of the repeated hair regeneration ability test in Example 2 ( Figure 1F). It shows that ⁇ -ray irradiation shows a series of typical phenotypes of premature skin aging, mainly including the decline in the number of epithelial stem cells in the body and the failure of repeated hair regeneration.
  • Example 5 The expression of miR-31 in mouse dorsal epithelial stem cells
  • Flow sorting was dyed according to the dyeing steps described in Example 4 and sorted on a flow sorter (BD, SORP FACSAria). The collected cellular RNA samples were stored in Trizol (Thrtmo Fisher, 3196018).
  • the Q-RT-PCR of mRNA is carried out according to standard procedures. First, use Fastking cDNA synthesis kit (Tiangen, KR118-02) to reverse transcribed total RNA samples to obtain cDNA. Then use BrightGreen 2X qPCR MasterMix-ROX (Abm, MasterMix-R) for quantitative PCR reaction. Follow the steps in the manual.
  • Q-RT-PCR of miRNA first uses miScript II RT Kit (Qiagen, 218161) to reverse transcribe total RNA samples to obtain cDNA, and then uses miScript SYBR Green PCR Kit (Qiagen, 218073) to perform quantitative PCR reaction.
  • miScript II RT Kit Qiagen, 218161
  • miScript SYBR Green PCR Kit Qiagen, 218073
  • Example 6 Mir31 conditional knockout mice significantly resist skin aging/premature aging
  • an epithelial stem cell-specific miR-31 knockout mouse model (Sox9CreER, Mir31 LoxP / LoxP for short cKO) that can be induced by Tamoxifen drug injection was constructed through genetic engineering.
  • Sox9CreER Mir31 LoxP / LoxP for short cKO mice
  • WT littermate wild-type control mice
  • gamma-ray irradiation and material extraction experiments were performed according to the operations described in Examples 1-5.
  • Antisense nucleotides can endogenously inhibit miR-31 in human and mouse epithelial cells
  • artificially synthesized antisense nucleotides introduced exogenously are also common methods to inhibit the expression and function of endogenous miRNAs.
  • human and mouse epithelial cells were cultured, and the exogenously introduced artificially synthesized antisense nucleotide—antagomiR was successfully transfected. The specific steps are as follows:
  • Human normal epithelial keratinocyte cells were purchased from Promocell (C-12001) and cultured in a cell incubator at 37°C using primary keratinocyte culture medium CnT-Prime (celllntec, cntpr); mouse cells were isolated from newborn mice On the back, the epithelium and the dermis were digested overnight with Dispase II (Roche, 4942078001), and then digested with Trypsin-Versene at room temperature to obtain a single cell suspension, and then cultured in a cell incubator at 37°C using the primary keratinocyte culture medium CnT-Prime.
  • CnT-Prime celllntec, cntpr
  • mice were isolated from newborn mice On the back, the epithelium and the dermis were digested overnight with Dispase II (Roche, 4942078001), and then digested with Trypsin-Versene at room temperature to obtain a single cell suspension, and then cultured in a cell incubator at
  • AntagomiR-Scr (GenePharma, B05001), AntagomiR-hsa-miR-31 (GenePharma, B05001) and AntagomiR-mmu-miR-31 (GenePharma, B05001)
  • enzyme-free water (Ambion, am9932) to prepare a 20 ⁇ M mother liquor
  • the working concentration is 5nM, and samples are collected 48 hours after adding the medium proportionally to detect changes in gene expression levels.
  • Example 8 Conditional knockout of miR-31 does not affect normal hair growth and regeneration
  • the miR-31 conditional knockout can resist gamma-ray irradiated mice showing a series of typical skin premature aging phenotypes, mainly including the appearance of whitening of newborn hair and the decline of regeneration ability. In this example, it was further verified whether the conditional knockout of miR-31 would affect hair growth and regeneration under normal physiological conditions.
  • mice Young cKO and littermate control mice were injected with Tamoxifen at the same time on the 21st day after birth, and the hair appearance, hair density, and hair follicle stem cell ratio of the two groups of mice were followed up for about 6 weeks.
  • conditional knockout of miR-31 did not affect normal hair growth and regeneration.
  • conditional knockout of miR-31 is mainly reflected in specifically inhibiting the aging effect of DNA damage on skin epithelial stem cells, rather than inhibiting or promoting normal hair growth and regeneration itself.
  • Example 9 An endogenous small RNA molecular target for the prevention and treatment of skin aging and premature aging related to DNA damage
  • ⁇ -Gal staining was performed in accordance with the instructions of Yeasen (40754ES60) kit. After staining, the nuclei were counter-stained with nuclear fast red staining solution (Shenggong, E670101).
  • Knockout of miR-31 significantly inhibits the natural aging of the skin.
  • the specific manifestations are as follows: 1) The signs of senile alopecia are significantly reduced, which is reflected in the improvement in the appearance of the hair visible to the naked eye and the significantly reduced area of hair removal (Figure 3B). 2) The aging and exhaustion of hair follicle stem cells is significantly reduced, which is reflected in the significant increase in the proportion of K14+CD34+ staining double positive cells in the immunofluorescence staining of hair follicle sections, and the significant increase in the proportion of CD34+CD49f+ hair follicle stem cells in the flow cytometric analysis of skin epithelial tissue high.
  • the study of the present invention shows that the significant increase in the expression of miR-31 is positively correlated with the severity of skin aging/premature aging.
  • the expression of miR-31 in the epithelial stem cells of skin aging mice is significantly increased, and the local ionization radiation energy It specifically induces the expression and secretion of miR-31 in mouse epithelial stem cells.
  • the reduction of miR-31 expression can promote the anti-aging/premature aging effect of the skin: the hair albino ratio is significantly reduced .
  • the skin callus decline phenomenon is significantly alleviated (including the wound healing rate in the body and the keratinocyte migration rate of the skin tissue mass cultured in vitro), the number of epithelial stem cells in the body is significantly alleviated, and the repeated hair regeneration failure phenomenon is significantly alleviated.
  • the experiment of the present invention proves that miR-31 is an effective intervention target for preventing skin aging/premature aging related to DNA damage.
  • the results suggest that targeting miR-31 does not inhibit DNA damage itself or normal hair growth and regeneration, but inhibits the aging effect of DNA damage on skin epithelial stem cells.
  • Knockout of the gene fragment corresponding to miR-31 by genetic engineering technology or by introducing antisense nucleotides targeting miR-31 can antagonize the expression and function of miR-31 in skin epithelial cells, thereby delaying skin aging.

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Abstract

提供了预防皮肤衰老与早衰的内源性小RNA分子标靶及其应用。具体地，提供了miR-31抑制剂的用途，其被用于制备预防或治疗皮肤衰老(或抗皮肤衰老)的组合物或制剂。还提供了相应抑制miR-31从而预防和治疗皮肤衰老/早衰的方法，以及抗皮肤衰老的组合物和制剂。

Description

预防皮肤衰老与早衰的内源性小RNA分子标靶及其应用 技术领域
本发明涉及分子医学领域，具体地涉及预防皮肤衰老与早衰的内源性小RNA分子标靶及其应用。
背景技术
皮肤上皮组织(包括表皮、毛囊、皮脂腺、汗腺)的衰老是人类衰老最显著的特征。主要表现为皱纹形成、愈伤/再生能力下降、毛发白化/稀疏、毛囊萎缩等。皮肤上皮干细胞(主要包括毛囊干细胞和表皮干细胞)是皮肤上皮组织稳态维护与创伤修复的基础。其中表皮干细胞维持着上皮组织动态平衡，而毛囊干细胞支持着毛发生长与再生，并能够在创伤条件下修复上皮。
作为一种终身不断自我更新的组织，皮肤上皮组织的衰老与其干细胞的衰竭密切相关，但导致这种干细胞衰竭的具体分子机制尚不完全清楚。
因此，本领域急需开发能有效防止或延缓皮肤衰老的物质。
发明内容
本发明的目的就是提供了一种能有效防止或延缓皮肤衰老的物质和方法，本发明的抗衰老物质和方法以内源性小RNA分子(microRNA31/miR-31)为标靶。
本发明的第一方面，提供了一种miR-31(即microRNA31)抑制剂的用途，用于制备预防或治疗皮肤衰老(或抗皮肤衰老)的组合物或制剂。
在另一优选例中，所述的组合物包括药物组合物、日用化学品组合物。
在另一优选例中，所述的日用化学品组合物包括洗发水、沐浴露、护肤品、护发产品、和/或化妆品。
在另一优选例中，所述的组合物或制剂用于选自下组的一种或多种(或全部)应用：
(a)降低毛发白化比率或减少白化毛发的产生；
(b)提高皮肤愈伤能力或促进创伤愈合；
(c)促进角质细胞的迁移；
(d)促进上皮干细胞功能和/或减缓上皮干细胞的减少；和
(e)提高毛发反复再生能力。
在另一优选例中，所述的皮肤衰老具有选自下组的一个或多个指标：
(l)皮肤上皮干细胞丢失；
(m)毛发白化；
(n)毛发再生能力下降；和
(o)上皮愈伤功能下降。
在另一优选例中，所述的上皮干细胞选自下组：毛囊干细胞、表皮干细胞、皮肤上皮干细胞、或其组合。
在另一优选例中，所述的皮肤衰老还包括：一种或多种代表性的衰老细胞标记物或标志的增多，和/或一种多种代表性的年轻细胞标记物或标志的减少。
在另一优选例中，所述的代表性的衰老细胞标记物或标志包括(但并不限于)：SA-β-半乳糖苷酶、P16、细胞增殖能力。
在另一优选例中，所述的代表性的年轻细胞标记物或标志包括(但并不限于)：H3K9me3、胶原蛋白基因COL17A1。
在另一优选例中，所述的皮肤衰老包括DNA损伤导致的皮肤衰老。
在另一优选例中，所述的皮肤衰老是由选自下组的诱导因素诱导的：电离辐射、γ射线辐照、紫外线、放射治疗、过度暴晒、化学诱变剂、或其组合。
在另一优选例中，所述的皮肤衰老是年龄因素导致的。
在另一优选例中，所述的miR-31包括hsa-miR-31，更佳地hsa-miR-31-5p。
在另一优选例中，所述的miR-31具有SEQ ID NO：1所示的核苷酸序列：5'-aggcaagaugcuggcauagcu-3'。
在另一优选例中，所述的miR-31具有如SEQ ID NO.:5所示的核心序列：5'-aggcaaga-3'(SEQ ID No.:5)。
在另一优选例中，所述miR-31抑制剂(或拮抗剂)选自下组：miR-31抗体、miR-31结合蛋白、miR-31小分子抑制剂、miR-31反义序列、AntagomiR-miR-31、miR-31海绵、用于敲除miR-31编码基因的试剂、及生理上可接受的载体、或其组合。
在另一优选例中，所述的AntagomiR-hsa-miR-31为具有SEQ ID NO：3所示的核苷酸序列或其衍生物或经修饰的核苷酸序列。
在另一优选例中，所述的AntagomiR-mmu-miR-31为具有SEQ ID NO：4所示的核苷酸序列或其衍生物或修饰物。
在另一优选例中，所述的“用于敲除miR-31编码基因的试剂”包括基于Cas9的基因编辑试剂。
在另一优选例中，所述Cas9的基因编辑试剂用于对miR-31核心序列所对应的DNA区域进行碱基替换、或对miR-31核心序列所对应的DNA区域进行切割，从而减少或消除miR-31。
在另一优选例中，所述的miR-31抑制剂(或拮抗剂)是修饰或未修饰的核酸序列。
在另一优选例中，所述的修饰选自下组：甲基化修饰、甲氧基乙基修饰、烃基修饰、胆固醇修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰(如氟代修饰等)、核酸修饰(如“TT”修饰等)、硫代磷酸化修饰、锁核苷酸修饰、或其组合。
本发明的第二方面，提供了一种可用于预防或治疗皮肤衰老的组合物或制剂，所述的组合物或制剂包括：(a)生理上可接受的载体；和(b)有效量活性成分，所述的活性成分为miR-31抑制剂。
在另一优选例中，所述的组合物或制剂还含有(c)其他抗衰老的活性成分。
在另一优选例中，所述的活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
在另一优选例中，所述的组合物或制剂还含有透皮促进剂。
在另一优选例中，所述生理上可接受的载体选自下组：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纳米凝胶、纤维素及其衍生物、明胶、滑石、固体润滑剂、硫酸钙、植物油、多元醇、乳化剂、润湿剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水或其组合。
在另一优选例中，所述的组合物或制剂的剂型为外用(topical)剂型。
在另一优选例中，所述的组合物或制剂的剂型包括膏剂、乳霜、贴剂、涂剂、喷剂、微针剂等。
在另一优选例中，所述的组合物或制剂的施用方式选自下组：局部施用、透皮施用和经皮施用。
本发明的第三方面，提供了一种体外筛选用于抗皮肤衰老的潜在物质的方法，包括步骤：
(1)提供测试组和对照组，其中，在测试组中向检测体系中加入测试物，在对照组中向检测体系中加入对照物,所述对照物包括阴性对照物；
(2)分别检测测试组和对照组中miR-31的表达水平；如果阴性组相比，测试 组的miR-31的表达水平显著降低，则表示所述测试物是抗皮肤衰老的潜在物质。
在另一优选例中，除了添加的测试物和对照物不同之外，所述的测试组和对照组的实验条件是相同的或基本相同的。
在另一优选例中，所述的步骤(1)之后还包括步骤：
(1.1)分别将测试组和对照组的检测体系暴露于DNA损伤诱导环境下，从而诱导细胞衰老或诱导DNA损伤。
在另一优选例中，在步骤(1.1)中，用选自下组的方法进行处理，从而形成所述的DNA损伤诱导环境：辐照处理、化学诱变剂、高温、剧烈震荡、诱导病理应激、或其组合。
在另一优选例中，所述的辐照处理选自下组：电离辐射、γ射线辐照、UVA/UVB辐照、放射治疗、过度暴晒、或其组合。
在另一优选例中，所述的检测体系为细胞体系。
在另一优选例中，所述的细胞包括皮肤细胞，较佳地为上皮细胞，更佳地为上皮干细胞。
在另一优选例中，所述方法还包括步骤：
(4)分别检测测试组和对照组中上皮干细胞耐受DNA损伤能力；如果与对照组相比，测试组的上皮干细胞耐受DNA损伤能力显著提高，则提示所述测试物是抗皮肤衰老的潜在物质。
在另一优选例中，在步骤(4)中，比较测试组中DNA损伤幅度D1和对照组中DNA损伤幅度D0,如果(D0-D1)/D0的比值≥5％，较佳地≥10％，较佳地≥20％，较佳地≥50％，更佳地≥80％，最佳地≥100％，则提示所述测试物是抗皮肤衰老的潜在物质。
本发明的第四方面，提供了一种预防或治疗皮肤衰老的方法，包括步骤：给需要的对象施用miR-31抑制剂、或含miR-31抑制剂的组合物或制剂。
在另一优选例中，所述的组合物或制剂包括：miR-31的反义核苷酸作为活性成分。
在另一优选例中，所述miR-31的反义核苷酸包括AntagomiR-miR-31。
在本发明的第五方面，提供了一种诊断/检测皮肤衰老的方法，包括步骤：
(a)提供一待测样本，所述待测样本为皮肤细胞、或皮肤组织；和
(b)检测所述待测样本中miR-31的表达水平L1，并与参考值L0进行相比，如 果待测样本的miR-31的表达水平显著高于参考值，则提示所述待测样本的皮肤衰老进程快和/或皮肤衰老程度高，其中所述参考值L0是正常衰老的皮肤细胞中miR-31的表达水平。
在另一优选例中，所述的待测样本为皮肤上皮组织或皮肤上皮细胞。
在另一优选例中，所述的“显著高于”是指：与阴性对照样本相比，相应miRNA-31的表达水平的提高幅度(即(L1-L0)/L0的比值)≥10％，较佳地≥20％，较佳地≥50％，较佳地≥100％，更佳地≥150％，最佳地≥200％。
在本发明的第六方面，提供了一种miR-31或及其检测试剂的用途，用于制备用于评估皮肤衰老的检测试剂或检测试剂盒。
在另一优选例中，所述的皮肤为人的皮肤。
在另一优选例中，所述的miR-31包括hsa-miR-31，更佳地hsa-miR-31-5p。
在另一优选例中，所述的试剂盒还含有说明书，所述的说明书中记载了本发明第五方面中所述的方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了建立局部电离辐照诱导诱导的局部皮肤早衰模型：
图1A显示了局部电离辐照诱导的小鼠皮肤局部早衰模型(局部皮肤早衰小鼠)的实验流程示意图。黄色为辐照区域。小鼠在7周龄(P50)左右小鼠接受局部辐照。五天后进行蜡脱毛(Wax)以诱导毛发再生。之后28天(4周)左右观测其皮肤表型。
图1B显示了上述局部皮肤早衰小鼠的背皮毛发照片。比例尺1cm。
图1C显示了上述局部皮肤早衰小鼠的创伤愈合速率统计图。横轴：愈伤天数，纵轴：愈合率。**：P<0.01,n＝3,双尾t-test。
图1D显示了上述局部皮肤早衰小鼠的体外皮肤组织块(Exo-plant)的角质细胞迁移愈伤速率实验。左图：显微镜照片，黑色为皮肤组织块本体，白色线指示迁移角质细胞层的前沿比例尺500μm。右图：迁移角质细胞层的面积统计：**：P<0.01,n＝3,双尾t-test。
图1E显示了上述局部皮肤早衰小鼠皮肤中的上皮干细胞相对比率统计(以noRad区为基准)。**：P<0.01,n＝3,双尾t-test。
图1F显示了上述局部皮肤早衰小鼠皮肤中二次蜡脱毛后的的背皮毛发照片。可见辐照区新生毛发明显稀疏。比例尺1cm。
图1G显示了上述局部皮肤早衰小鼠皮肤上皮干细胞中的miR-31表达量的Q-RT-PCR分析。**：P<0.01,n＝3,双尾t-test。上述所有图中的误差条(Error Bar)代表标准误(Standard Error)。
图2显示了Mir31敲除能够回复辐照损伤导致的干细胞耗竭：
图2A显示了Mir31条件性敲除小鼠构建原理示意图。
图2B显示了Q-RT-PCR分析Tamoxifen诱导后的Mir31条件性敲除小鼠(cKO)的上皮干细胞中的miR-31表达量。**：P<0.01,n＝3,双尾t-test。cKO：Mir31条件性敲除小鼠，WT：同窝野生型小鼠，下同。
图2C-G显示了在cKO和WT小鼠中进行局部电离辐照诱导诱导的局部皮肤早衰表型分析：图2C显示了辐照区域的白色毛发比率分析；图2D显示了辐照区域的创伤愈合速率分析，测量方法同图1C；图2E显示了体外皮肤组织块(Exo-plant)的角质细胞迁移愈伤速率分析，测量方法同图1D，Rad和noRad分别代表辐照区与未辐照区皮肤；图2F显示了皮肤中的上皮干细胞相对比率统计，测量方法同图1E，Rad和noRad分别代表辐照区与未辐照区皮肤；图2G显示了左边图为二次蜡脱毛后的的背皮毛发照片，右边为辐照中心区域的体外长度>5mm的毛发数量统计。**显示了P<0.01,n＝3,双尾t-test。
图2H显示了Q-RT-PCR分析Antagomir转染之后的小鼠上皮细胞(MK)和人类上皮细胞的(NHEK)中的miR-31表达量。SCR显示了对照Scramble Antagomir转染，A-M31显示了miR-31Antagomir转染。**显示了P<0.01,n＝3,双尾t-test。
图3显示了miR31条件性敲除不影响正常毛发生长。
图3A显示了Tamoxifen注射诱导39天后的cKO和同窝WT对照小鼠背皮照片。Tamoxifen注射诱导在出生后第21天进行。
图3B显示了上述A中小鼠背皮毛发相对密度分析(以WT为基准)。计数体外长度>5mm的毛发密度。n＝3,双尾t-test。
图3C显示了上述A中小鼠背皮上皮中的毛囊干细胞相对比率分析(以WT为基准)。n＝3,双尾t-test。
图3D显示了小鼠背皮毛发显微镜照片，展示蜡去毛后的毛发再生过程。 d0，d6，d10分别代表蜡去毛后第0，6，10天。所有小鼠在7周龄进行Tamoxifen注射诱导，并在两天后蜡去毛。
图3E显示了上述D中小鼠的背皮毛发平均相对长度(以WT d6为基准)分析。n＝3,双尾t-test。
图3F显示了上述D中小鼠在蜡去毛后第四周的背皮上皮中毛囊干细胞相对比率统计分析(以WT为基准)。n＝3,双尾t-test。
上述所有图中的误差条(Error Bar)代表标准误(Standard Error)。
在各图中，“noRad”表示未辐照；“Rad”表示辐照。
图4显示了敲除miR-31显著抑制皮肤自然衰老。
图4A左图显示了17月龄的Mir31条件性敲除小鼠cKO与同窝同性别野生型小鼠WT的代表性背部照片。箭头代表秃发区域。比例尺1cm。右图：左图所代表小鼠在17月龄/21月龄的的背皮脱毛面积统计。纵轴为相对于WT的百分比。**：P<0.01，双尾t-test，n＝5每组。
图4B显示了21月龄同窝同性别cKO和WT小鼠毛囊上皮组织中的KRT14+CD34+细胞百分比统计，基于切片免疫荧光分析。**：P<0.01，双尾t-test，n＝5每组。
图4C显示了21月龄同窝同性别cKO和WT小鼠整体皮肤上皮组织中的CD34+CD49f+毛囊干细胞含量统计，基于流式细胞分析。纵轴代表相对于WT的倍率。*：P<0.05，双尾t-test，n＝3每组。
图4D显示了21月龄同窝同性别cKO和WT小鼠毛囊上皮组织中的γH2AX+(代表DNA损伤)细胞百分比统计，基于切片免疫荧光分析。**：P<0.01，双尾t-test，n＝5每组。
图4E显示了21月龄同窝同性别cKO和WT小鼠毛囊上皮组织中的β-Gal染色阳性(代表衰老细胞)细胞百分比统计，基于切片染色分析。**：P<0.01，双尾t-test，n＝5每组。
图4F显示了21月龄同窝同性别cKO和WT小鼠皮肤组织块(exoplant)的体外愈伤迁移能力分析。左图为组织块培养第6天的代表性照片，左侧黑色部分为组织块本体，虚线为表皮细胞层迁移前沿，比例尺500μm。虚线和本体之间为表皮细胞迁移面积。右图为左图所代表实验中的表皮细胞迁移面积的统计结果。纵轴为相对于WT的百分比。**：P<0.01，双尾t-test，n＝6每组。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次发现一种内源性小RNA分子miR-31与皮肤衰老(尤其是早衰或辐照引起的衰老)密切相关，因此可作为预防皮肤衰老与早衰的内源性小RNA分子标靶。实验表明，一方面miR-31在γ射线辐照组织中尤其是背皮上皮干细胞显著升高且与衰老指标正相关，另一方面，通过抑制miR-31,可以延缓皮肤(尤其是皮肤上皮细胞或上皮组织)的衰老。通过抑制miR-31，可预防和/或延缓皮肤衰老和早衰。在此基础上，完成了本发明。
实验表明，miR-31在γ射线辐照组织中尤其是背皮上皮干细胞显著升高且与多种衰老指标正相关：皮肤干细胞(尤其是上皮干细胞)丢失、毛发白化、毛发再生能力下降、表皮愈伤功能下降。
此外，在miR-31被敲除的小鼠模型中，当miR-31被抑制时，模型小鼠表现出抗皮肤衰老的效果，例如减少了抗皮肤衰老导致的毛发白化，促进了毛发再生，促进了创伤愈合能力。此外，用反义核酸来抑制人类和小鼠上皮干细胞中的miR-31时，同样可取得类似的抗皮肤衰老效果。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
皮肤衰老与DNA损伤
如本文所用，术语“皮肤衰老”、“皮肤老化”可互换使用，指皮肤上皮组 织的衰老。此外，所述术语包括正常的皮肤衰老，以及过早或过快速的皮肤衰老(即皮肤早衰)。
皮肤上皮组织(包括上皮、毛囊、皮脂腺、汗腺)的衰老是人类衰老最显著的特征。主要表现为皱纹形成、愈伤/再生能力下降、毛发白化/稀疏、毛囊萎缩等。皮肤上皮干细胞(主要包括毛囊干细胞和表皮干细胞)是皮肤上皮组织稳态维护与创伤修复的基础。上皮干细胞包括毛囊干细胞、表皮干细胞、其它属于皮肤上皮组织的干细胞、或其组合。其中表皮干细胞维持着上皮组织动态平衡，而毛囊干细胞支持着毛发生长与再生，并能够在创伤条件下修复上皮。皮肤上皮组织里面还存在一些不好明确定义的干细胞，例如毛囊和表皮交界处的干细胞，统称皮肤上皮干细胞。作为一种终身不断自我更新的组织，皮肤上皮组织的衰老与其干细胞的衰竭密切相关，但导致这种干细胞衰竭的具体分子机制尚不完全清楚。
在本发明的一个优选例中，所述的皮肤衰老具有选自下组的一个或多个指标：(l)皮肤上皮干细胞丢失；(m)毛发白化；(n)毛发再生能力下降；和(o)上皮愈伤功能下降。
另外，皮肤衰老还包括：一种或多种代表性的衰老细胞标记物或标志的增多，和/或一种多种代表性的年轻细胞标记物或标志的减少。所述的代表性的衰老细胞标记物或标志包括(但并不限于)：SA-β-半乳糖苷酶、P16、细胞增殖能力；所述的代表性的年轻细胞标记物或标志包括(但并不限于)：H3K9me3、胶原蛋白COL17A1。
DNA损伤被普遍认为是导致组织衰老/早衰的一个主要因素。在皮肤组织中，外源和内源性的DNA损伤诱导因素都能够导致显著的早衰现象。在基因组稳定性相关基因突变的早衰症患者及其对应的小鼠遗传模型中，包括毛发脱落与白化在内的典型皮肤衰老表型都会提早出现。另一方面，阳光中的紫外线(UV)是众所周知的DNA损伤诱因，同时也能够显著加速皮肤衰老，导致皮肤中常见的光老化症状。除了紫外线之外，肿瘤放疗中常用的电离辐射(IR)也是很强的DNA损伤诱因，能够诱导放疗区域皮肤细胞早衰，并导致长期性的皮肤愈伤功能障碍，进而带来发生难愈性溃疡的风险，是肿瘤放疗中最常见的并发症之一。近年来，有研究进一步证实了DNA损伤是促使皮肤上皮与上皮干细胞衰竭的直接诱因。抑制DNA损伤所导致的皮肤上皮干细胞衰竭在皮肤抗衰老美容与放射性皮肤损伤的防治中都有着广阔的应用前景。但目前尚无进入临床阶段的特效治疗手段。
本发明提供了一种通过靶向miR-31的抑制剂，来抑制DNA损伤对皮肤上皮干细胞的的促衰老效应从而有效对抗皮肤衰老和早衰的技术途径。
微小RNA与miR-31
微小RNA，又称microRNA(miRNA)，如本文所用，所述的“miRNA”是指一类RNA分子，从可形成miRNA前体的转录物加工而来。成熟的miRNA通常具有18-26个核苷酸(nt)(更特别的约19-22nt)，也不排除具有其它数目核苷酸的miRNA分子。miRNA通常可被Northern印迹检测到。
miRNA可从前体miRNA(Precursor miRNA，Pre-miRNA)加工而来，所述的前体miRNA可折叠成一种稳定的茎环(发夹)结构，所述的茎环结构长度一般在50-100bp之间或更长。所述的前体miRNA可折叠成稳定的茎环结构，茎环结构的茎部两侧包含基本上互补的两条序列。所述的前体miRNA可以是天然的或是人工合成的。
前体miRNA可被剪切生成miRNA，所述的miRNA可与编码基因的mRNA的至少一部分序列基本上互补。如本文所用，“基本上互补”是指核苷酸的序列是足够互补的，可以以一种可预见的方式发生相互作用，如形成二级结构(如茎环结构)。通常，两条“基本上互补”的核苷酸序列互相之间至少有70％的核苷酸是互补的；优选的，至少有80％的核苷酸是互补的；更优选的，至少有90％的核苷酸是互补的；进一步优选的，至少有95％的核苷酸是互补的；如98％、99％或100％。一般地，两条足够互补的分子之间可以具有最多40个不匹配的核苷酸；优选的，具有最多30个不匹配的核苷酸；更优选的，具有最多20个不匹配的核苷酸；进一步优选的，具有最多10个不匹配的核苷酸，如具有1、2、3、4、5、8、11个不匹配的核苷酸。
如本申请所用，“茎环”结构也被称作“发夹”结构，是指一种核苷酸分子，其可形成一种包括双链区域(茎部)的二级结构，所述的双链区域由该核苷酸分子的两个区域(位于同一分子上)形成，两个区域分列双链部分的两侧；其还包括至少一个“环”结构，包括非互补的核苷酸分子，即单链区域。即使该核苷酸分子的两个区域不是完全互补的，核苷酸的双链部分也可保持双链状态。例如，插入、缺失、取代等可导致一个小区域的不互补或该小区域自身形成茎环结构或其它形式的二级结构，然而，该两个区域仍可基本上互补，并在可预见的方式中发生相互作用，形成茎环结构的双链区域。茎环结构是本领域技术人员所熟知的，通常在获得了一条具有一级结构的核苷酸序列的核酸后， 本领域技术人员能够确定该核酸是否能形成茎环结构。
本发明所述的miRNA是指miR-31，miR-31是一个在哺乳动物进化中高度保守的内源性微小RNA。在人类中序列如SEQ ID NO.:1所示：5'-aggcaagaugcuggcauagcu-3'，miRbase命名为hsa-miR-31-5p，登录号：MIMAT0000089，是MIR31基因(hg38chr9:21,512,115-21,512,185，Gencode Gene:ENSG00000199177.1)的表达产物。
在小鼠中序列如SEQ ID NO.:2所示：5'-aggcaagaugcuggcauagcug-3'，miRbase命名为mmu-miR-31-5p，登录号：MIMAT0000538，对应的基因为Mir31(mm10chr4:88,910,557-88,910,662)。小鼠miR-31仅比人类miR-31在3'端多出一个核苷酸，而已知miRNA生物学功能主要依赖于其5'端8nt的种子序列，即其核心序列为5'末端的5'-aggcaaga-3'(SEQ ID NO.:5)，因此可以认为小鼠和人类的miR-31具有功能等价性。同理，因miRNA 3'端序列重要性较低，内源性miRNA表达产物的3'末端往往具有不严谨性。因此，人类细胞中来自MIR31基因位点的，其5'末端具有5'-aggcaaga-3'序列的内源性18-24nt小RNA分子均可被视为具有等价功能的miR-31。
之前的研究显示miR-31能够在胚胎着床、器官发育、骨骼和肌肉形成以及免疫系统稳态维持等多个生物学过程中发挥重要的调控作用，但miR-31在皮肤衰老/早衰中的可能作用尚未有报道。
在本发明中，实验显示，miR-31是介导DNA损伤相关的皮肤衰老与早衰的关键内源性因子。以电离辐射的诱导小鼠皮肤早衰现象为模型，本发明人发现miR-31在DNA损伤刺激下的皮肤上皮干细胞中显著上调，与上皮干细胞的丢失和后续的皮肤早衰现象密切相关。通过体内基因敲除手段，证明了从皮肤上皮干细胞中移除miR-31能够显著抑制DNA损伤相关的皮肤衰老/早衰表型，主要包括：皮肤上皮干细胞丢失、毛发白化、毛发再生能力下降、上皮愈伤功能下降。因此，研究发现miR-31是一个能够用于预防DNA损伤相关的皮肤衰老与早衰的小RNA分子标靶。以此为基础研发的抑制其内源性表达的遗传学/表观遗传学干预手段，以及抑制其功能的反义核苷酸药物或其它手段均可用于预防DNA损伤相关的皮肤衰老与早衰症状，具有广泛的应用价值。
在另一优选例中，所述的微小RNA来源于人或非人哺乳动物；较佳地所述的非人哺乳动物为大鼠、小鼠，鼠和或人的家族序列完全一致。核心序列指微小RNA第1-8位的核苷酸序列。所述的“功能与miR-31相同或基本相同”是指保留了miR-31的≥40％、≥50％、≥60％、≥70％、≥80％、≥90％的功能。
本发明还包括miRNA变体和衍生物。此外，广义上的miRNA衍生物也可包括miRNA变体。本领域的普通技术人员可以使用通用的方法对miR-31进行修饰，修饰方式包括(但不限于)：甲基化修饰、烃基修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰、核酸修饰(如“TT”修饰)等。
本文中，“Mir31”指代的是小鼠源miR-31基因DNA，“MIR31”指代的是人源miR-31基因DNA。本发明中提到的“microRNA31”、“microRNA31/miR-31”“miRNA-31”、“miR-31”、“微小RNA-31”可以互换，可以指代以下一种或多种活性成分：(a)miRNA-31家族的微小RNA，所述miRNA-31家族的微小RNA包括：miRNA-31或经修饰的miRNA-31衍生物、和核心序列为5'-aggcaaga-3'(SEQ ID NO.:5)、功能与miRNA-31相同或基本相同的微小RNA或经修饰的miRNA衍生物；(b)前体miRNA，所述的前体miRNA能在宿主内加工成(a)中所述的miRNA-31；(c)多核苷酸，所述的多核苷酸能被宿主转录形成(b)中所述的前体miRNA，并加工形成(a)中所述的微小RNA；(d)表达载体，所述表达载体含有(a)中所述的miRNA-31、或(b)中所述的前体miRNA、或(c)中所述的多核苷酸；(e)(a)中所述的微小RNA的激动剂。
在本发明的另一优选例中，所述的miRNA-31来源于人或非人哺乳动物。
本发明还提供了靶向miR-31的反义核酸或其前体，或其衍生物。
在本发明的另一优选例中，所述的经修饰的miRNA衍生物(尤其是靶向miR-31的反义核酸或其前体)是具有式I所示结构的化合物单体或其多聚体：
(X)n-(Y)m
式I
在式I中，各X为靶向miR-31的反义序列；各Y独立地为促进所述的靶向miR-31的反义施药稳定性的修饰物；n为1-100的(较佳地1-20)正整数(较佳地n为1、2、3、4或5)；m为1-1000的(较佳地1-200)正整数；各“-”表示接头、化学键、或共价键；在另一优选例中，所述的接头是长度为1-10个碱基的核酸序列。所述的Y包括(但不限于)：胆固醇、类固醇、甾醇、醇、有机酸、脂肪酸、酯、单糖、多糖、氨基酸、多肽、单核苷酸、多核苷酸。
在本发明的另一优选例中，一种靶向miR-31的多核苷酸具有式II所示的结构：
Seq 正向-X-Seq 反向
式II
式II中，Seq正向为能在宿主中被加工成靶向miRNA-31的反义核苷酸序列；Seq反向为与Seq正向基本上互补或完全互补的核苷酸序列；X为位于Seq正向和Seq反向之间的间隔序列，并且所述间隔序列与Seq正向和Seq反向不互补；并且式II所示的结构在转入宿主细胞后，形成式III所示的二级结构：
Figure PCTCN2020130911-appb-000001
式III中，Seq 正向、Seq 反向和X的定义如上述，||表示在Seq 正向和Seq 反向之间形成的碱基互补配对关系。
模型动物
本发明还提供了miR-31高表达或miR-31低表达的非人哺乳动物模型，它们可用于评估miR-31与皮肤衰老的关系或用于筛选或验证测试物质是否通过miR-31起到延缓皮肤衰老的作用。
一种代表性的miR-31低表达包括miR-31knock-down和knock-out类型的低表达(或不表达)。
在本发明的一个优选例中，使用了2种工具小鼠Sox9CreER工具小鼠和miR-31条件性敲除小鼠，并杂交在F2代获得了目的转基因小鼠Sox9CreER，Mir31 LoxP/LoxP的miR-31条件性诱导敲除小鼠。
疾病模型构建
在本发明中，为了诱导目的小鼠表现出皮肤上皮干细胞中的miR-31条件性敲除，进行了Tamoxifen注射造模。在本发明的一个优选实施例中，miR-31cKO小鼠与同窝野生型对照小鼠(WT)均采用40mg/kg剂量Tamoxifen(sigma，T564)单次腹腔注射进行miR-31条件性敲除。
在本发明的一个优选实施例中，为了模拟射线导致的皮肤衰老和DNA损伤，对各组小鼠进行γ射线局部辐照处理，具体步骤如实施例1,然后，为了检验小鼠毛发再生能力，使用了蜡脱毛(Wax)处理以诱导毛发再生，之后28天(4周)左右观测其皮肤表型实验流程表如图1A所示。
本发明的一个优选例中，Tamoxifen注射诱导的条件性敲除在辐照前2天进行。
抑制剂与反义核苷酸
在本发明中，术语“活性成分”或“用于皮肤抗衰老的活性成分”指靶向miR-31的拮抗剂或抑制剂。
代表性的抑制剂包括(但并不限于)：靶向miR-31的抗体、结合蛋白、小分子抑制剂、反义序列、AntagomiR、micro-RNA海绵、用于敲除micro-RNA编码基因的试剂、或其组合。
一类特别有效的抑制剂(或拮抗剂)是针对miR-31的反义核苷酸。
在本发明的一个优选例中，使用的miR-31抑制剂为AntagomiR-miR-31。所述的AntagomiR-hsa-miR-31购自吉玛生物公司，是一种针对人hsa-miR-31核心序列的反义寡核苷酸试剂，其序列如SEQ ID NO.:3所示：a sg scuaugccagcaucuug sc sc su s-Chol(s下标代表硫代骨架修饰，Chol代表胆固醇修饰)。所述的AntagomiR-mmu-miR-31购自吉玛生物公司，是一种针对小鼠hsa-miR-31核心序列的反义寡核苷酸试剂，其序列如SEQ ID NO.:4所示：c sa sgcuaugccagcaucuug sc sc su s-Chol(s下标代表硫代骨架修饰，Chol代表胆固醇修饰)。
反义寡核苷酸是一类经人工合成或构建的反义表达载体表达的寡核苷酸片段，长度多为15-30个核苷酸，通过碱基互补原理，干扰基因的解旋、复制、转录、mRNA的剪接加工乃至输出和翻译等各个环节，从而调节细胞的生长、分化等。根据结合部位的不同分为反义DNA(asDNA)、反义RNA(asRNA)、自催化性核酶(ribozyme)，后者为具有酶活性的反义分子，可裂解与其互补的mRNA及在DNA内插入DNA片段构成三链结构，但最常用的为反义寡脱氧核苷酸(反义寡核苷酸)，其优点在于其理论上的高度靶特异性(碱基互补)、设计容易、多样且合成简单及高度的局部性和针对性。这都是常规药物设计、生产和作用所不可比拟的，因而具有巨大的吸引力和研究价值。传统药物大多与蛋白质结合，从而修饰蛋白质的功能。相比之下，反义试剂在mRNA(DNA)水平上发挥作用，阻止其翻译成蛋白质。最近几年来，新的用于保护寡聚核苷酸免遭酶解、提高靶标亲和性的化学修饰技术的发展，使得反义技术也获得巨大进步。另外，RNA干扰(RNA interference)成为第三代抑制哺乳动物细胞基因表达的高效方法，该技术采用了21-23个残基组成的小干扰RNA分子(siRNA)。
反义技术具有明显的优点：
(1)反义核苷酸是针对特定的靶mRNA(DNA)的序列设计合成，具有极高的特异性；(2)反义核酸是针对已知序列的靶基因设计合成的，由于靶基因序列已 知，反义核酸仅有15-30个碱基，结构简单，容易设计和体外大量合成；(3)反义核酸进入细胞内与细胞周期无关，既可进入增殖期细胞又可进入非增殖期细胞；(4)反义寡核苷酸不含病毒序列，不会产生免疫反应，也不会整合入宿主染色体内。
反义技术的一个重大挑战是寡聚核苷酸的稳定性，因为未经修饰的寡聚核苷酸在生物体内会被核酸酶迅速降解。反义实验中应用到大量的化学修饰的核苷酸。一般来说，核苷酸的修饰分为三类：非天然碱基的类似物、经过修饰的糖(尤其是核糖的位置)、改变的磷酸骨架。
本领域的普通技术人员可以使用通用的方法对本发明的靶向miR-31的反义寡核苷酸进行修饰，修饰方式包括(但不限于)：甲基化修饰、烃基修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰、核酸修饰(如“TT”修饰)等。
通过修饰，可提高靶标亲和性、核酸酶抗性和药物动力学性质。
一种修饰的靶向miR-31的反义寡核苷酸是硫代磷酸酯寡聚脱氧核糖核酸(Phosphorothioate DNA Oligonucleotides，PS DNA ON)。
另一种靶向miR-31的反义寡核苷酸在核糖的2'位置含有烷基修饰。2'-O-甲基和2'-O-乙基是这类修饰的两个重要成员。具有这种修饰的寡聚核苷酸比硫代磷酸酯DNA的毒性低，并且与互补RNA的亲和性也得到了一些提高。
在本发明中，可使用了antagomir(或靶向miR-31海绵)，从而有效地抑制上皮组织或细胞中的miR-31，从而延缓皮肤的衰老。与普通抑制剂相比，miRNA antagomir在动物体内外具有更高的稳定性和抑制效果，且能克服体内细胞膜、组织等障碍富集于靶细胞。antagomir在细胞实验中不需要转染试剂，从而避免了转染试剂包装过程的复杂步骤及其对实验的影响。在动物实验中可用全身或局部注射、吸入、喂药等方法进行给药，作用效果持续时间可长达数周。
组合物或制剂
本发明提供了一种可用于预防或治疗皮肤衰老的组合物或制剂(包括化妆品、日化用品等)，所述的组合物或制剂包括：(a)可接受的载体(包括药学上可接受的载体、生理上可接受的载体、化妆品上可接受的载体)；和(b)有效量活性成分，所述的活性成分为miR-31抑制剂。
在本发明中，所述的活性成分的有效量可随使用的模式和皮肤衰老程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定 (例如通过临床试验)。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
术语“生理上可接受的载体”指用于活性成分的载体，包括：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2020130911-appb-000002
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约0.00001mg-50mg/kg动物体重(较佳的0.0001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
在本发明中，另一类组合物或产品是含本发明活性成分(靶向miR-31的抑制)的化妆品或日化用品。
在本发明中，所述的组合物或制剂还优选地含有透皮促进剂。
在另一优选例中，所述的组合物或制剂的剂型为外用(topical)剂型。
在本发明中，所述的化妆品组合物或制剂的剂型包括(但并不限于)：固体制剂、液体制剂、凝胶制剂、半固体制剂。一些优选的剂型膏剂、乳霜、贴剂、涂剂、喷剂、微针剂、溶液剂、洗发等。
在本发明中，代表性的产品(或日化用品)包括(但并不限于)：洗发水、沐浴露、护肤品、护发产品、面膜。
在另一优选例中，所述的组合物或制剂的施用方式选自下组：局部施用、透皮施用和经皮施用。
本发明的主要优点包括：
(a)本发明首次意外地证实，miR-31是一种与皮肤细胞(尤其是皮肤上皮细胞)衰老密切相关microRNA，因此可作为预防或治疗皮肤衰老与早衰的内源性小RNA分子标靶。
(b)靶向miR-31抑制剂可有效地拮抗miR-31，从而有效对抗或延缓皮肤的衰老(尤其是早衰)。
(c)靶向miR-31的反义核酸可直接作用于皮肤，因此施用简便，且安全性高。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实验方法和材料
核酸序列：
表1.
Figure PCTCN2020130911-appb-000003
Figure PCTCN2020130911-appb-000004
注：1.对于SEQ ID No.:3和4，s下标代表硫代骨架修饰，Chol代表胆固醇修饰。
2.cKO小鼠(miR-31条件性敲除小鼠)及其制备
(1)Sox9CreER工具小鼠获自中国科学院北京生命科学学院，遗传背景为C57BL/6,该品系小鼠在皮肤上皮干细胞中特异性表达可被他莫昔芬(Tamoxifen)诱导激活的Cre重组酶，是在皮肤上皮干细胞中进行条件性基因敲除的工具小鼠。
(2)miR-31条件性敲除小鼠购自南方模式生物有限公司，通过CRISPR-Cas9技术分别在C57BL/6野生型小鼠Mir31基因区域(mm10chr4:88910557-88910662)的上下游300bp左右的位置定点敲入LoxP位点。得到Mir31LoxP小鼠(简称FloxP)，理论敲除区域为mm10chr4：88910180-88911062，完全覆盖了小鼠Mir31基因区域，且并未影响到任何已知的周边基因。
(3)为了实现皮肤上皮干细胞中的miR-31条件性敲除，将上述FloxP小鼠与上皮干细胞特异性的Sox9CreER小鼠多次杂交，得到基因型为Sox9CreER，Mir31 LoxP/LoxP的miR-31条件性诱导敲除小鼠(简称cKO)。该品系杂交小鼠在进行Tamoxifen注射后，可诱导实现miR-31的条件性敲除，从而得到时空可控的miR-31条件性敲除小鼠。
实施例1 局部电离辐照诱导的小鼠皮肤局部早衰模型
γ射线辐照在复旦大学医学院放射医学研究所按标准实验步骤操作。进行局部辐照时，将麻醉后的7周龄(P50)左右小鼠置于辐照仓中的上下两层铅板中间的空隙，上下铅板相同，铅板垂直于放射源，两块铅板各自留有垂直对齐的直径3厘米的小孔，将小鼠上半部置于小孔中，铅板覆盖小鼠头部和下半身，用胶带适当固定小鼠四肢后进行辐照。辐照剂量10Gy。
实施例2 小鼠毛发再生能力测试
2.1如实施例1局部辐照处理五天后进行蜡脱毛(Wax)完全移除现有毛发，以此激活毛发再生。之后28天(4周)左右观测其皮肤毛色、创伤愈合等表型。用蜡去毛的方法并追踪拍照观测其再生速率。
2.2毛发反复再生能力测试的方法是在上述毛发再生完成后，进行二次蜡脱毛以重复2.1操作。
2.3结果
初次毛发再生实验结果如图1B所示，脱毛28天后小鼠背部未辐照区长出的新生的毛发为小鼠天生的黑色，而辐照区新生的毛发则表现为浅金色，几乎为白色。而二次毛发再生实验结果如图1F所示,与初次毛发再生实验结果相似，小鼠背部未辐照区长出的新生的黑色毛发，而辐照区却仅长出了更稀疏的白化毛发。
以上说明γ射线辐照表现出一系列典型的皮肤早衰表型，主要包括新生毛发明显白化，以及再生能力下降。
实施例3 小鼠创伤愈合能力实验
3.1小鼠体内创伤愈合实验
如实施例1局部辐照处理五天后小鼠用异氟烷麻醉后剃去毛发，背部用8mm打孔器进行打孔造创。贴手术巾(3M，SP1107)防止伤口变形,三天后取下，分别在造创前、造创后0、4、7、9、12和15天进行拍照，根据比例尺计算伤口面积，每组内不同时间点的小鼠创口面积与各自组内造创0天面积平均值取比值，得到愈伤百分比。各组数据记录至愈伤完全结束。
3.2体外培养的皮肤组织块(Exo-plant)的角质细胞迁移愈伤实验
小鼠处死后用脱毛膏去除毛发，75％酒精消毒后用手术刀去除脂肪组织。于PBS中清洗后用1mm打孔器打孔获得皮肤块，每孔中间加入1-2ul matrigel(Corning,356237)，贴附皮肤块并置于1mg/ml fibronectin(Millipore,FC010-1mg)包被过的24孔板(coring,CLS3527)包被中培养。培养7天后于显微镜下拍照，以皮肤块为中心点，用图像统计软件Imagej测量从皮肤块边沿迁出的细胞面积。各组面积分别与野生型未处理部位面积取比值。
3.3结果
小鼠背部创口愈伤百分比统计结果如图1C所示，小鼠背部蜡脱毛处理的创伤愈合速度，辐照区相较未辐照区极其显著变慢。局部皮肤早衰小鼠的体外皮肤组织块(Exo-plant)的角质细胞迁移愈伤速率实验结果如图1D所示：其中左图为显微镜照片，黑色为皮肤组织块本体，白色线指示迁移角质细胞层的前沿，辐照区角质细胞相较未辐照区其迁移速率极其显著变慢；右图为迁移角质 细胞层的面积统计，也证明辐照区角质细胞的迁移面积远远少于未辐照区角质细胞。
γ射线辐照表现出一系列典型的皮肤早衰表型，主要包括皮肤愈伤能力减退。包括体内创伤愈合速率下降和体外培养的皮肤组织块(Exo-plant)的角质细胞迁移速率下降。
实施例4 小鼠背皮上皮干细胞的分析
小鼠用二氧化碳致死装置处死后剃毛并取背皮，用手术刀去除真皮面脂肪组织后用PBS清洗，并置于Trypsin-EDTA中4℃过夜消化。第二天用刀片刮取细胞，收集细胞悬液，置于PFE(PBS+1％FBS+1Um EDTA)染色液中于冰上进行CD34(1：100，eBioscience，11-0341-82),CD49f(1：100，biolegend，313602)染色。染色结束后清洗细胞并在流式分析仪(BD,CytoFLEX LX)上检测，并利用流式分析软件FlowJo 10.0分析结果。
如图1E所示，流式检测CD34 +CD49f +细胞群为上皮干细胞，用流式分析软件FlowJo 10.0分析每组内上皮干细胞所占的百分比，并与小鼠未辐照处理部位的百分比取比值，小鼠辐照区上皮干细胞相对未辐照处理部位的比值为50％左右，与实施例2中毛发反复再生能力测试(图1F)结果一致。说明γ射线辐照表现出一系列典型的皮肤早衰表型，主要包括体内上皮干细胞数量下降，毛发反复再生能力衰竭。
实施例5 小鼠背皮上皮干细胞的miR-31表达情况
流式分选按照如实施例4所述的染色步骤染色并在流式分选仪(BD,SORP FACSAria)上进行分选。收集后的细胞RNA样品保存在Trizol(Thrtmo Fisher，3196018)中。
采用Trizol裂解液与Direct-zol RNA MiniPrepPlus试剂盒(Zymo,R2070)提取总RNA进行，完全按说明书标准步骤操作。
mRNA的Q-RT-PCR按标准步骤进行。首先用Fastking cDNA synthesis kit(Tiangen,KR118-02)对总RNA样本进行逆转录得到cDNA。然后用BrightGreen 2X qPCRMasterMix-ROX(Abm，MasterMix-R)进行定量PCR反应。均按说明书步骤操作。
miRNA的Q-RT-PCR首先用miScript II RT Kit(Qiagen，218161)对总RNA样本进行逆转录得到cDNA，然后用miScript SYBR Green PCR Kit(Qiagen， 218073)进行定量PCR反应。均按说明书步骤操作。
结果图1G所示。流式细胞分选出辐照处理与未处理的局部皮肤早衰小鼠皮肤上皮干细胞中的miR-31表达量的Q-RT-PCR比较(图1G)，早衰的上皮干细胞显著上调了内源性miR-31表达，提示了皮肤上皮细胞内源性miR-31在皮肤衰老/早衰中的重要性。
实施例6 Mir31条件性敲除小鼠显著对抗皮肤衰老/早衰
6.1实验设计
如图2A所示，通过基因工程手段构建了可被Tamoxifen药物注射所诱导的上皮干细胞特异性的miR-31敲除小鼠模型(Sox9CreER，Mir31 LoxP/LoxP简称cKO)。为了实现条件性敲除，miR-31cKO小鼠与同窝野生型对照小鼠(WT)均采用40mg/kg剂量Tamoxifen(sigma，T564)单次腹腔注射进行miR-31条件性敲除。Tamoxifen处理2天后，按照实施例1-5所述的操作进行γ射线辐照和取材实验。
6.2结果
如图2B所示，通过Q-RT-PCR分析，确认miR-31表达水平在Tamoxifen诱导后的cKO小鼠的上皮干细胞中显著下降。
如图2C-G所示，在cKO和WT小鼠中进行局部电离辐照诱导诱导的局部皮肤早衰表型分析。cKO小鼠相对WT小鼠表现出显著对抗皮肤衰老/早衰效果：1)毛发白化比率显著下降(图2C)；2)皮肤愈伤能力减退现象显著缓解，包括体内创伤愈合速率(图2D)，和体外培养的皮肤组织块(Exo-plant)的角质细胞迁移速率(图2E)；3)体内上皮干细胞数量耗竭现象显著缓解(图2F)；4)毛发反复再生能力衰竭现象显著缓解(图2G)。
实施例7 反义核苷酸能够内源性抑制人类和小鼠上皮细胞中的miR-31
除了利用基因工程手段进行基因敲除之外，外源性导入的人工合成的反义核苷酸也是抑制内源性miRNA表达与功能的常见手段。在本实施例中培养了人类和小鼠的上皮细胞，并成功进行外源性导入的人工合成的反义核苷酸—antagomiR转染，具体步骤如下：
人类正常上皮角质细胞细胞(NHEK)购买自Promocell(C-12001)，使用原代角质细胞培养基CnT-Prime(celllntec,cntpr)于37℃细胞培养箱中培养；小鼠细胞分离自新生小鼠背部，用DispaseII(Roche,4942078001)过夜消化之后 分开上皮和真皮，并用Trypsin-Versene室温后消化得到单细胞悬液，然后使用原代角质细胞培养基CnT-Prime于37℃细胞培养箱中培养。AntagomiR-Scr(GenePharma,B05001)及AntagomiR-hsa-miR-31(GenePharma,B05001)及AntagomiR-mmu-miR-31(GenePharma,B05001)使用时用无酶水(Ambion,am9932)配置成20μM母液，工作浓度为5nM，按比例加入培养基后48小时收取样本检测基因表达水平变化。
Q-RT-PCR分析结果如图2H所示，Antagomir转染之后的小鼠上皮细胞(MK)和人类上皮细胞的(NHEK)中的miR-31表达量均显著下降。这说明antagomir的导入能够有效降低皮肤上皮细胞内源性miR-31的表达水平(图2H)，是靶向内源性miR-31的有效潜在途径。
实施例8 miR-31条件性敲除不影响正常毛发生长与再生
miR-31条件性敲除能对抗γ射线辐照小鼠表现出一系列典型的皮肤早衰表型，主要包括新生毛发明显白化，以及再生能力下降。在本实施例中，进一步验证miR-31条件性敲除是否会影响正常生理状态下的毛发生长与再生。
8.1实验方法
对出生后第21天左右的青年cKO与同窝对照小鼠同时注射Tamoxifen诱导，并追踪观察两组小鼠的毛发外观、毛发密度、毛囊干细胞比例，持续6周左右。
此外，对7周龄左右的成年cKO与同窝对照小鼠同时注射Tamoxifen诱导，并在2天后通过蜡脱毛以启动毛发再生，观察两组小鼠的毛发再生速率。
8.2结果
如图3所示，miR-31条件性敲除不影响正常毛发生长与再生。
一方面，miR-31敲除不影响正常毛发长期稳态，cKO小鼠的毛发外观(图3A)、毛发密度(图3B)、毛囊干细胞比例(图3C)均与对照小鼠无显著差异。
另一方面，miR-31敲除不影响正常毛发再生，两组小鼠在毛发再生速率方面无显著差异(图3D-E)，且毛囊干细胞比例无显著改变(图3F)。
因此，miR-31条件性敲除的效应主要体现为特异性抑制了DNA损伤对皮肤上皮干细胞的促衰老作用，而非抑制或促进正常毛发生长与再生本身。
实施例9 一个用于DNA损伤相关的皮肤衰老与早衰防治的内源性小RNA分子标靶
材料和方法
1)切片免疫荧光分析
我们首先将切片置于4％PFA中固定10分钟，再将切片置于封闭液(2.5％normal donkey serum，2.5％normal goat serum，1％BSA，0.3％Triton X-100)中进行封闭/通透。然后将一抗稀释于封闭液中、滴加到切片上并进行4℃过夜孵育。第二天进行偶联荧光基团的二抗孵育，并用Fluoromount-G mounting media(Invitrogen)封片剂(含DAPI)封片。切片照片于Zeiss Axio Imager A2(Zeiss)显微镜下拍摄。免疫荧光染色中的抗体稀释比例：K14(rabbit，1:1000，本实验室)；CD34(rat，1:200，Biolegend)，γH2AX(rabbit，1:200，abcam)。
2)β-Gal染色分析
β-Gal染色按照Yeasen(40754ES60)试剂盒的说明书操作，染色后用核固红染色液(生工，E670101)复染细胞核。
研究结果：
敲除miR-31显著抑制皮肤自然衰老。
为了进一步检测移除miR-31对皮肤及其毛发自然衰老的影响，我们对前述Mir31条件性敲除小鼠模型cKO及其同窝同性别野生型对照小鼠WT进行了长期观测。其中Tamoxifen诱导在出生后第48天进行，与前述实验类似。我们发现老龄cKO小鼠与其同窝同龄同性别WT小鼠相比，其主要皮肤及毛发衰老指标均显著减轻。
具体表现为：1)老龄秃发现象明显减轻，体现为肉眼可见的毛发外观改善和显著缩小的脱毛面积(图3B)。2)毛囊干细胞的衰老耗竭现象明显减轻，体现为毛囊切片免疫荧光染色中的K14+CD34+染色双阳性细胞比例显著升高，以及皮肤上皮组织流式细胞分析中的CD34+CD49f+毛囊干细胞比例显著升高。3)毛囊中的DNA损伤程度减轻，体现为毛囊中的γ-H2AX染色阳性细胞比例显著降低。4)毛囊中的细胞衰老程度减轻，体现为毛囊中的衰老细胞标记物β-Gal染色阳性细胞比例显著降低。5)皮肤愈伤功能衰退现象减轻，体现为皮肤组织块愈伤迁移能力显著上升。因此，我们的研究进一步证实了在皮肤上皮组织中抑制miR-31是延缓皮肤自然衰老的有效途径。
讨论
之前的研究显示DNA损伤是皮肤上皮和上皮干细胞衰老与早衰的直接诱 因，而电离辐射是人类皮肤中常见的DNA损伤诱因之一。
本发明的研究表明，miR-31表达的显著升高与皮肤衰老/早老的严重程度呈现正向相关联，miR-31在皮肤衰老小鼠的上皮干细胞中表达显著升高，局部电离辐照能特异性诱导小鼠的上皮干细胞表达和分泌miR-31。
在他莫昔芬诱导的Mir31条件性敲除小鼠(Sox9CreER，Mir31 LoxP/LoxP)的上皮干细胞中，miR-31表达量降低能促进表现出显著对抗皮肤衰老/早衰效果：毛发白化比率显著下降，皮肤愈伤能力减退现象显著缓解(包括体内创伤愈合速率和体外培养的皮肤组织块的角质细胞迁移速率)，体内上皮干细胞数量耗竭现象显著缓解，毛发反复再生能力衰竭现象显著缓解。
本发明实验证实：miR-31是预防DNA损伤相关的皮肤衰老/早衰的有效干预靶点。结果提示，靶向miR-31并不能抑制DNA损伤本身或正常毛发生长与再生，而是抑制了DNA损伤对皮肤上皮干细胞的促衰老作用。通过基因工程技术敲除miR-31对应的基因片段或通过导入靶向miR-31的反义核苷酸，可实现在皮肤上皮细胞中拮抗miR-31表达和功能，从而延缓皮肤衰老。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种microRNA31/miR-31抑制剂的用途，其特征在于，用于制备预防或治疗皮肤衰老(或抗皮肤衰老)的组合物或制剂。
  2. 如权利要求1所述的用途，其特征在于，所述的组合物包括药物组合物、日用化学品组合物。
  3. 如权利要求1所述的用途，其特征在于，所述的组合物或制剂用于选自下组的一种或多种应用：
    (a)降低毛发白化比率或减少白化毛发的产生；
    (b)提高皮肤愈伤能力或促进创伤愈合；
    (c)促进角质细胞的迁移；
    (d)促进上皮干细胞功能和/或减缓上皮干细胞的减少；和
    (e)提高毛发反复再生能力。
  4. 如权利要求3所述的用途，其特征在于，所述的上皮干细胞选自下组：毛囊干细胞、表皮干细胞、皮肤上皮组织的干细胞、或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述的皮肤衰老包括DNA损伤导致的皮肤衰老。
  6. 如权利要求1所述的用途，其特征在于，所述的皮肤衰老是由选自下组的诱导因素诱导的：电离辐射、γ射线辐照、紫外线、放射治疗、过度暴晒、化学诱变剂、或其组合。
  7. 如权利要求1所述的用途，其特征在于，所述的miR-31具有如SEQ ID NO.:5所示的核心序列：5'-aggcaaga-3'(SEQ ID No.:5)。
  8. 如权利要求1所述的用途，其特征在于，所述miR-31抑制剂选自下组：miR-31抗体、miR-31结合蛋白、miR-31小分子抑制剂、miR-31反义序列、AntagomiR-miR-31、miR-31海绵、用于敲除miR-31编码基因的试剂、及生理上可接受的载体、或其组合。
  9. 一种可用于预防或治疗皮肤衰老的组合物或制剂，其特征在于，所述的组合物或制剂包括：(a)生理上可接受的载体；和(b)有效量活性成分，所述的活性成分为miR-31抑制剂。
  10. 一种体外筛选用于预防或治疗皮肤衰老的潜在物质的方法，其特征在于，包括步骤：
    (1)分别向检测体系中加入待检测的物质或阴性对照物；和
    (2)分别检测加入了待检测的物质的样本和加入了阴性对照物的样本中miR-31的表达水平；如果与加入了阴性对照物的样本相比，加入了待检测的物质的样本的miR-31的表达水平降低，则是潜在的预防或治疗皮肤衰老疾病的物质。
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