CN114377130A - Endogenous small RNA molecular target for regulating and controlling skin and hair regeneration and application thereof - Google Patents
Endogenous small RNA molecular target for regulating and controlling skin and hair regeneration and application thereof Download PDFInfo
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- CN114377130A CN114377130A CN202011112587.7A CN202011112587A CN114377130A CN 114377130 A CN114377130 A CN 114377130A CN 202011112587 A CN202011112587 A CN 202011112587A CN 114377130 A CN114377130 A CN 114377130A
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Abstract
The invention relates to an endogenous small RNA molecular target for regulating the regeneration of skin and hair and application thereof. Specifically, the invention provides an application of an active component for inhibiting miRNAs, preferably an active component for inhibiting miR-24, or a preparation containing the active component, namely the application of the active component in preparing medicines for promoting skin and hair growth.
Description
Technical Field
The field relates to the medical field, in particular to an endogenous small RNA molecular target for regulating and controlling the regeneration of skin and hair and application thereof.
Background
The skin epithelial tissue is the outermost barrier of the human body and mainly comprises epidermis, hair follicles, sebaceous glands, sweat glands and the like. Normal skin follicles are driven by their stem cell/precursor cell activation-resting cycle and undergo continuous cyclic hair regeneration including the resting phase-anagen phase-catagen phase, referred to as the hair cycle. Stem/precursor cells in hair follicles mainly include bulge stem cells (Bu SCs) located in the bulge region and Hair Germplasm (HG) cells located between the bulge and the dermal papilla. Wherein the activation of proliferation of HG cells by stimulation with an exogenous growth signal is the initial step in the transition of the hair follicle from the resting phase to the anagen phase.
Disorders of hair follicle regeneration ability are closely related to common skin diseases such as alopecia. For example, androgenic alopecia (AGA) is a common alopecia and is characterized clinically by a large reduction in hair regeneration capacity, atrophy of hair follicles, and a significant decrease in the number of HG cells. In addition, other pathological factors such as chemotherapy and anxiety can also lead to alopecia. In addition to the scalp, fine hair is also widely present in human skin in other areas. At present, the problem that the hair of the wound surface is difficult to regenerate is faced in the clinical repair and reshaping process of the large-area wound surface. Promoting the regeneration of these hairs is of great importance for restoring the normal appearance of the skin.
Currently, there are only two drugs approved by the U.S. Food and Drug Administration (FDA) for the clinical treatment of AGA, topical minoxidil and oral finasteride, respectively. Clinically, 2% minoxidil is used to treat female AGA and 5% concentration is used to treat male AGA. Finasteride is only used in men and its dose is 1mg per day. However, these drugs all have significant side effects. Minoxidil may cause erythema, desquamation, hair loss, etc., while finasteride may cause male sexual dysfunction and feminization. Therefore, if the regeneration ability of hair follicle stem cells/precursor cells can be promoted to replace the above-mentioned drugs or the dosage of the drugs can be reduced to reduce side effects, the drug has an important practical value in the treatment of alopecia. On the other hand, there is also a need in the cosmetic and toiletry field to remove or inhibit hair growth in a specific area. The mainstream method at present is depilation by physical or chemical means. The short-term alopecia method mainly uses methods such as depilatory cream, wax depilatory cream, shaving cream, etc. to directly remove the hair in the target area. However, this hair removal method activates the regenerative function of the hair follicle itself to cause rapid hair regeneration. The long-term hair removal method directly destroys hair follicles mainly by laser, but has high cost and certain damage to skin. It is suitable only for removing thick hair in a small area, and is difficult to be used for inhibiting fine hair growth in a large area of skin.
The micro RNAs play an important role in the development, physiology, disease and evolution of animals
Therefore, there is an urgent need in the art to develop an effective target for micrornas, and the use of inhibitors or analogs thereof in the preparation of a medicament for promoting or inhibiting the regeneration of skin and hair thereof.
Disclosure of Invention
The invention aims to provide an effective micro RNAs target and application of an inhibitor thereof in preparing a medicament for promoting skin and hair regeneration.
In a first aspect of the invention, the application of an active ingredient targeting miR-24 or a preparation containing the active ingredient is provided, and the active ingredient is used for preparing a medicament for regulating the growth of skin and hair thereof.
In another preferred embodiment, the skin is derived from a mammal.
In another preferred embodiment, the mammal includes, but is not limited to, a human, a rodent (e.g., mouse, rat).
In another preferred example, the active ingredient targeting miR-24 is used for up-regulating (or increasing) the amount and/or activity of miR-24; or for down-regulating (or reducing) the amount and/or activity of miR-24.
In another preferred embodiment, the active components targeting miR-24 comprise: a miR-24 inhibitor or antagonist.
In another preferred embodiment, the miR-24 inhibitor or antagonist is used for preparing a preparation or composition for promoting the growth of skin and hair thereof.
In another preferred embodiment, the active components targeting miR-24 comprise: a miR-24 agonist or enhancer.
In another preferred embodiment, the miR-24 agonist or promoter is used for preparing a preparation or composition for inhibiting the growth of skin and hair thereof.
In another preferred embodiment, the composition comprises: a cosmetic composition, or a pharmaceutical composition.
In another preferred embodiment, regulating skin and its hair growth comprises:
a) inhibiting the growth of skin and hair; and/or
b) Promoting skin and hair growth.
In another preferred example, the active components targeting miR-24 are selected from the group consisting of:
i) an active ingredient targeted to inhibit miR-24 selected from the group consisting of: a small molecule compound, miRNA, antisense nucleic acid (e.g., antisense RNA), mRNA, antibody, gene editing agent, sponge inhibitor, or a combination thereof;
ii) an active ingredient targeted to agonistic miR-24 selected from the group consisting of: a small molecule compound, miRNA, a nucleic acid analog, or a combination thereof.
In another preferred example, the sequence of the antisense nucleic acid inhibitor targeting miR-24 is selected from the group consisting of: SEQ ID No. 3, SEQ ID No. 4, or combinations thereof.
In another preferred example, the sequence of the miR-24-targeting nucleic acid analog is selected from the group consisting of seq id no: SEQ ID No. 5, SEQ ID No. 6, or combinations thereof.
In another preferred embodiment, the formulation is selected from the group consisting of: external preparation, oral preparation, and injection.
In another preferred embodiment, the external preparation is selected from the group consisting of: cream, ointment, and lotion.
In another preferred embodiment, the formulation is selected from the group consisting of: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet, or dripping pill.
In another preferred embodiment, the formulation further comprises: minoxidil, finasteride, or a combination thereof.
In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:
A) an active ingredient targeting miR-24;
B) other pharmaceutically acceptable carriers or excipients.
In another preferred example, the active miR-24-targeting component comprises:
A1) an active ingredient targeted to inhibit miR-24 selected from the group consisting of: a small molecule compound, miRNA, antisense nucleic acid (e.g., antisense RNA), mRNA, antibody, gene editing agent, sponge inhibitor, or a combination thereof; or
A2) An active ingredient targeting agonism miR-24 selected from the group consisting of: a small molecule compound, miRNA, a nucleic acid analog, or a combination thereof.
In another preferred example, the sequence of the antisense nucleic acid inhibitor targeting miR-24 is selected from the group consisting of: SEQ ID No. 3, SEQ ID No. 4, or combinations thereof.
In another preferred embodiment, the sense strand sequence of the miR-24-targeting nucleic acid analog is selected from the group consisting of seq id no: SEQ ID No. 5, SEQ ID No. 7, or combinations thereof.
In another preferred example, the antisense strand sequence of the miR-24-targeting nucleic acid analog is selected from the group consisting of: SEQ ID No. 6, SEQ ID No. 8, or combinations thereof.
In a third aspect of the invention, a method of modulating the sensitivity of a cell to growth factor stimulation is provided by contacting the cell with a therapeutically effective amount of an active ingredient targeting miR-24.
In another preferred embodiment, the cell is selected from the group consisting of: epithelial cells, dermal cells, mesenchymal cells.
In another preferred embodiment, the miR-24 inhibition active ingredient is as described above.
In another preferred example, the sequence of the antisense nucleic acid inhibitor targeting miR-24 is selected from the group consisting of: SEQ ID No. 3, SEQ ID No. 4, or combinations thereof.
In another preferred embodiment, the sense strand sequence of the miR-24-targeting nucleic acid analog is selected from the group consisting of seq id no: SEQ ID No. 5, SEQ ID No. 7, or combinations thereof.
In another preferred example, the antisense strand sequence of the miR-24-targeting nucleic acid analog is selected from the group consisting of: SEQ ID No. 6, SEQ ID No. 8, or combinations thereof.
In a fourth aspect of the invention, there is provided a method of promoting the regulation of hair growth by administering to a patient in need thereof a medically effective amount of an active ingredient targeting miR-24 at a site in need thereof.
In another preferred embodiment, the regulating skin and hair growth thereof comprises:
a) inhibiting the growth of skin and hair; and/or
b) Promoting skin and hair growth.
In another preferred embodiment, the active ingredients targeting miR-24 are as described above.
In another preferred embodiment, the patient in need thereof is selected from the group consisting of: patients with alopecia, scald, burn, ulcer, skin transplantation, and plastic patients requiring skin and hair regeneration.
In another preferred embodiment, the site in need is selected from:
s1) epithelial tissue, scalp areas, skin of the face and other body parts;
s2) tissue engineering skin.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1: the targeted inhibition or the knockout of miR-24 can effectively promote the regeneration of skin and hair thereof.
Wherein (a): schematic of the construction of miR-24 dKO mice.
(b) The method comprises the following steps And (3) detecting the expression quantity of miR-24 cluster member miRNA in the dKO and littermate qKO control mouse primary MK by qRT-PCR. N is 3 per sample.
(c) The method comprises the following steps Representative photographs of the dorsal skin (left) of littermates qKO and dKO mice after shaving on postnatal day 55 (postnatal days 55, P55) to time points P119 and P155 and their% dorsal skin hair coverage at P120 were counted. Scale bar 1 cm.
(d) Representative photographs of dorsal skins of littermates dKO and qKO mice at day 29 (d29) after quantitative 200 hair plucking in a 5.5mm diameter area (dashed circles) (remaining hair shaved). Scale bar 1 mm. Arrow head: black hair regrowth signs.
(e) Quantitative epilation mice according to d picture described in the gross immunofluorescence staining of dorsal epithelium in the area of hair plucked 10 days after epilation (left picture) and statistics of the ratio of Ki67 staining positive HG in the area (right picture). Scale bar: 25 μm. Ki67 stained white. The arrows and dashed circles represent HG.
(f) As shown in the upper schematic, a representative (n-3) dorsal skin photograph of P50 mice on day 21 (d21) after topical application of high or low doses of minoxidil (MioLo or MioHi) or solvent control (Veh) after dorsal skin shaving. Scale bar: 1 cm. A dotted line frame: and (4) applying a medicine area.
(g) Representative photographs of the dorsal skin at the d21 time point after topical application of MioLo or Veh to littermate qKO and dKO mice (left panel) and% coverage of regenerated hair by its dotted box painted area (right panel) as described in panel f above. Scale bar: 1cm, each sample n 3.
(h) The miR-24 expression amount is analyzed by fluorescent quantitative PCR. The samples were qKO or dKO MK (d or q) stably infected with scramble or miR-24 overexpressing lentivirus (-scr/-m 24). Cells were pre-treated in CNT-Basal media for 16 hours. Each sample n is 4.
(i) Growth rate statistics for four MK cells described in the above h plot in different percentages of growth medium (horizontal axis, percentage of diluted CNT-PR in CNT-basal). Longitudinal axis: growth fold measured by CCK-8 method for 5 days (day 5 was measured versus the starting point of measurement). Prior to the experiment, the cells were pre-treated in CNT-Basal medium for 16 hours. Each sample n is 3.
(j) The miR-24 expression amount is analyzed by fluorescent quantitative PCR. The samples were WT MK cells 24 hours after transfection with antagomir-Scramble (SCR) or antagomir-24(A-m 24). Each sample n is 3.
(k) MK cells as described in the above graph j were counted by the 2-day amplification factor measured by the CCK-8 method (measurement of day 2 vs. measurement start). The starting point of the measurement is 12 hours after transfection. Each sample n is 3.
The P values in all the above statistical plots were calculated by the two-tailed t-test, with Error bars (Error Bar) representing Standard Error and scatter points representing individual data points.
FIG. 2: the miR-24 is overexpressed, so that the skin and hair regeneration can be effectively inhibited.
Wherein (a) K14-rtTA, Tre-Mir24 Double Transgene (DTG) mouse model schematic diagram.
(b) And (3) analyzing the expression quantity of the miR-24 and the miRNA nearby by fluorescent quantitative PCR. The sample was tail epithelium isolated from dispase from P52 (Dox-induced from P49) DTG/WT littermates. Each sample n is 3.
(c) Representative (n-3) photographs of dorsal skin and hair coverage statistics of P150 WT/DTG littermates. no Dox/+ Dox: mice were either not induced or induced with doxycycline from P52. Scale bar: 1 cm. Each sample n is 3.
(d) And (3) analyzing the miR-24 expression quantity by fluorescent quantitative PCR. Samples were dKO MK cells 6 hours after transfection with agomir-scramble (Ago-SCR) or agomir-24 (Ago-M24). Each sample n is 3. (e) MK cells as depicted in panel d above were counted by the 3-day amplification factor measured by the CCK-8 method (measurement of day 3 vs. measurement start). The starting point of the measurement is 12 hours after transfection. Each sample n is 3.
The P values in all the above statistical plots were calculated by the two-tailed t-test, with Error bars (Error Bar) representing Standard Error and scatter points representing individual data points.
Detailed Description
The present inventors have made extensive and intensive studies and, as a result, have surprisingly found for the first time that inhibition of a specific microrna, preferably miR-24, is effective in regulating the regeneration of skin and hair thereof, and have completed further inventions.
Specifically, the invention finds that the inhibition of the expression of miR-24 does not influence the development of skin while promoting the regeneration of skin and hair thereof. Further, the embodiment of the invention shows that the inhibition of the expression of miR-24 can improve the hair regeneration sensitivity caused by physical epilation stimulation and a hair growth drug minoxidil. On the basis, the invention provides an antisense nucleic acid inhibitor of miR-24, an miRNA sponge inhibitor or a specific small molecule drug targeted inhibition miR-24, and application of the antisense nucleic acid inhibitor, the miRNA sponge inhibitor or the specific small molecule drug targeted inhibition miR-24 in preparation of drugs for promoting skin and hair regeneration.
In addition, the invention also discovers that the expression of miR-24 is up-regulated, so that the skin and hair thereof can be inhibited from regenerating, and the development of the skin is not influenced. On the basis, the invention provides miR-24 analogues and miR-24 targeted agonism small molecular compounds, and application of the miR-24 analogues and the miR-24 targeted agonism small molecular compounds in preparation of medicines for inhibiting skin and hair growth.
Term(s) for
As used herein, an "active ingredient" of the invention is an active ingredient that inhibits miRNAs, preferably miR-24, which comprises an inhibitor targeting miR-24.
In another preferred example, the active component inhibiting miR-24 is selected from the group consisting of: a small molecule compound targeted to inhibit miR-24, a miRNA targeted to miR-24, an antisense nucleic acid (such as antisense RNA) inhibitor targeted to miR-24, mRNA, an antibody, a gene editing reagent, a sponge inhibitor targeted to miR-24, or a combination thereof.
In another preferred example, the sequence of the antisense nucleic acid inhibitor targeting miR-24 is selected from the group consisting of: SEQ ID No. 3, SEQ ID No. 4, or combinations thereof.
Micro RNAs
microRNAs (miRNAs) are endogenous short-chain non-coding RNAs of about 22 nucleotides, and play an important role in development, physiology, disease and evolution of animals. As an important endogenous small nucleic acid factor, miRNAs can be targeted and inhibited by corresponding antisense small nucleic acid inhibitors (Antogomir, Anti-miR and the like) or sponginess inhibitors (miRNA sponge) enriching miRNA target sites or specific small molecule drugs, and the miRNAs are important therapeutic targets.
miR-24
miR-24 is a miRNA that is highly expressed in normal skin epithelial tissue. Its sustained overexpression in mouse skin can lead to defects in skin and hair follicle development and to death of the mouse within days after birth. These studies claim that miR-24 may have a role in promoting epidermal cell terminal differentiation, but does not relate to its association with the skin and its hair regeneration process.
Pharmaceutical composition
The invention also provides a pharmaceutical composition, which comprises:
A) an active ingredient inhibiting miRNAs selected from the group consisting of: small molecule compounds, mirnas, antisense nucleic acids (e.g., antisense RNAs), mrnas, antibodies, gene editing agents, miRNA sponge inhibitors, or combinations thereof.
B) A drug selected from the group consisting of: minoxidil, finasteride, or a combination thereof.
C) A pharmaceutically acceptable carrier or excipient.
In another preferred example, the miRNAs inhibiting active ingredient is a miR-24 inhibiting active ingredient.
In another preferred example, the active component inhibiting miR-24 is selected from the group consisting of: a small molecule compound targeted to inhibit miR-24, a miRNA targeted to miR-24, an antisense nucleic acid (such as antisense RNA) inhibitor targeted to miR-24, mRNA, an antibody, a gene editing reagent, a sponge inhibitor targeted to miR-24, or a combination thereof.
In another preferred example, the sequence of the antisense nucleic acid inhibitor targeting miR-24 is selected from the group consisting of: SEQ ID No. 3, SEQ ID No. 4, or combinations thereof.
In another preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the present invention may also be used with other therapeutic agents.
When using the pharmaceutical composition, a safe and effective amount of the composition of the present invention is administered to a mammal, wherein the administration dose of the active ingredient of the present invention is usually 0.001mg/kg to 1000000mg/kg body weight.
Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention are:
a) the invention firstly proposes that the inhibition of the expression of miR-24 can effectively promote the regeneration of skin and hair thereof.
b) The invention discovers that miR-24 knockout can promote the regeneration of skin and hair thereof and does not influence the development of the skin.
c) According to the invention, the inhibition of the expression of miR-24 is found for the first time, so that the hair regeneration sensitivity caused by physical hair removal stimulation and a hair growth drug minoxidil can be improved, and the improvement of the hair growth rate is facilitated.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Materials and methods
First, embodiment for knocking out miR-24
1) Animal model
1.1) K14-Cre mice: the K14 promoter promotes the over-expression Cre protein, is given by Qinjin researchers of Shanghai Nutrition and health institute of Chinese academy of sciences, has a genetic background of C57BL/6, and is a tool mouse for conditional gene knockout on the skin.
1.2) miR-24-1-flox mice: we entrusted southern model organism Limited with CRISPR-Cas9 technology to position the upstream and downstream 300bp around the C57BL/6 wild-type mouse Mir24-1 gene region (Chr13:63301208-63301275)The LoxP site was spotted. Obtaining Mir24-1LoxPMouse (FloxP for short), theoretical knockout region chr13: 63300966-63301497 completely covered the region of mouse Mir24-1 gene and did not affect any known peripheral genes.
1.3) miR-24-2-ko mice: we entrust southern model organism Limited to perform site-directed knockout of 35 base pairs containing the miR-24 mature body sequence in the C57BL/6 wild-type mouse Mir24-2 gene region (Chr8: 84208815-84208208921) by CRISPR-Cas9 technology, respectively. Obtaining Mir24-2KOIn mice (KO for short), the theoretical knockout region is Chr8:84208873-84208907, completely covers the mouse miR-24 mature body sequence region, and does not affect any known peripheral genes.
To achieve miR-24 conditional knockdown in skin epithelial cells, we crossed the FloxP mice described above with KO mice and K14-Cre mice. The genotype of the obtained gene is K14-Cre, Mir24-1LoxP/LoxP,Mir24-2KO/KOThe miR-24 conditional knockout mouse (dKO for short).
2) Nucleic acid sequence: all microrna sequences used in this example are shown in table 1 below:
TABLE 1 sequence Listing of microRNAs used in miR-24 expression examples
Note: 1. for SEQ ID nos. 3 and 4, the s subscript represents a thio backbone modification, Chol represents a cholesterol modification, a full chain methoxy modification.
miR-24 core sequence: GGCUCAG.
3) Total RNA extraction and fluorescent quantitative PCR
Extracting cell RNA: after the medium was aspirated from the 6-well plate, adherent cells were washed once with DPBS, and after the DPBS was aspirated, 500 μ L Trizol (invitrogen) was added to each well, and after lysis at room temperature for 10 minutes, RNA extraction was performed using Zymo # R2070 kit. Alternatively, 500. mu.L of RZ (heaven root) was added to each well and lysed at room temperature for 10 minutes before RNA extraction using heaven root # DP 419. The specific steps refer to the product specification.
Reverse transcription of miRNA and fluorescent quantitative PCR: reverse transcription and fluorescent quantitative PCR were performed on miR-24/miR-205/miR125b using the # E22007 kit for the giga gene. Reverse transcription was performed on miR-23a/miR-23b/miR27a/miR27a using TAKARA #638315 kit, and fluorescence quantitative PCR was performed using abm # MasterMix-mS. The specific steps are shown in respective specifications. In both methods, U6 was used as an internal reference gene, and 2 was used-△△CTThe method calculates the relative expression amount. All samples were normalized to the control sample level illustrated in the figure.
4) Primary isolation and culture of mouse epidermal cells (MK)
Newborn mice within 4 days after the birth were taken out, cleaned with 75% ethanol, and then their heads, limbs and tails were cut off in DPBS, and their dorsal skins were separated along both sides of their trunk. After washing in clean DPBS, the cells were placed in 2.5mg/mL dispase (Roche #4942078001) overnight at 4 ℃. The following day, the dorsal skin was washed with clean DPBS and placed in Trypsin-Versene (Lonza #17-161F) and digested for 15 minutes at room temperature. The digested dorsal skin was minced and digestion was stopped with an equal volume of PFE (DPBS + 5% V/V FBS +1mM EDTA). The mixture was transferred to a new 50mL centrifuge tube and repeatedly blown through a 70 μm cell sieve 30 times. Centrifuge at 300g for 5 min at 4 ℃ and discard the supernatant. The DPBS resuspended the cell pellet, centrifuged at 300g at 4 ℃ for 5 minutes and the supernatant discarded. The cell pellet was resuspended in Growth medium (CNT-PR + 1% Pen-Strep +10 μm Y-27632) and plated onto cell culture dishes.
5) Antagomir transfection of MK
Taking a transfection 6-well plate as an example, a Qiagen Effect #301427 transfection kit is selected, the final concentration of antagomir is 200nM, and the steps are as follows:
1. 400pmol (. about.2.3. mu.g) of antagomir was mixed with buffer EC in a total volume of 100. mu.L.
2. 18.4. mu.L of Enhacer (8:1) was added, gently mixed, and then allowed to stand at room temperature for 3 minutes.
3. 6.9. mu.L of Effectene (3:1) was added, gently mixed, and then allowed to stand at room temperature for 10 minutes.
4. After replacing MK with fresh medium, the transfection complex was added dropwise. Downstream experiments were performed 12 hours after transfection.
6) CCK-8 method for detecting cell proliferation capacity
Two methods are mainly used for detecting the proliferation capacity of different MK:
1) CCK-8 (Biyuntian # C0039) measures absorbance on different days to calculate the relative proliferation fold of the cells.
2) The relative competitive power of the target cells is detected by a double-color competition experiment method.
The CCK-8 process operates as follows:
1. MK were plated in 96-well plates, 100. mu.L of medium was added to each well, leaving 4-6 wells without cells and medium only as blank wells.
2. After 16 hours of plating, 10. mu.L of CCK-8 was added to each well to be tested and placed in an incubator at 37 ℃ for 1.5 hours.
3. Absorbance at 450nm was measured using a microplate reader.
4. The sample wells minus the blank wells were recorded as absorbance a1 on the first day.
5. Repeating the steps 2-4 about the nth day after the plate is paved, and recording the absorbance An of the nth day.
An/A1 was scored as the n-day fold proliferation of the cells.
8) Detection of MK sensitivity to growth stimulation
The next day after MK plating, Basal/Growth was mixed at the following ratio for 16 hours with Basal medium CNT-Basal (# CnT-PR-BM.1) without any Growth factors: 0%, 12.5%, 25%, 50%, 100%. After five days, the absorbance was measured by the CCK-8 method. The absorbance value of each proportion/0% absorbance value is the relative proliferation multiple of the cells under the proportion. Cells with higher relative proliferation fold are more sensitive to growth stimulation than cells with the same proportion.
9) Quantitative dehairing experiment
The mouse was shaved with a razor on its back to circle a circular area of 5.5mm in diameter, and 200 hairs were plucked as evenly as possible in the designated area with forceps under a dissecting microscope, noting that only one hair was plucked at a time. In order to accurately determine the hair plucking area in the future, the small scissors are used for shearing the hairs in the whole circular area to the shortest length after the hairs are plucked. The plucked area was photographed with a dissecting microscope and a skin microscope on day 1 and day 29 after plucking, respectively.
10) Minoxidil induced Hair regrowth threshold test
Using absolute ethyl alcohol: water: 1, 2-isopropanol ═ 5: 3: 2 as a solvent, the minoxidil powder of MCE # HY-B0112 was dissolved to prepare solutions of minoxidil at 0.01g/mL (1%) and 0.02g/mL (2%), respectively. After shaving the back of the mouse, the dorsal skin of the mouse was divided into four areas by crosses at the midpoint of the dorsal midline of the mouse, each area was coated with 100 μ L of minoxidil solution for 14 consecutive days. Application of 0.01g/mL solution was designated MioLo and application of 0.02g/mL solution was designated MioHi. Photographic analysis was performed 7 days after the end of the application.
11) Statistical analysis
Statistical differences between the two groups of samples were analyzed by two-tailed t-test, showing results presented as mean ± sem. P values greater than or equal to 0.05 are considered to be statistically non-different, values less than 0.05 are considered to be statistically different, and values less than 0.01 and less than 0.001 are considered to be statistically significant.
Second, example for agonizing miR-24 overexpression
1) Animal model
1.1) K14-rtTA mice: the K14 promoter promotes the overexpression rtTA protein, which is given by Chenting researchers of Beijing Life sciences institute, the genetic background is C57BL/6, and the promoter is a tool mouse for inducing gene overexpression on the skin.
1.2) TRE-Mir24 mice: we entrusted Shanghai's model Biotechnology, Inc. to make transgenic mice that can over-express miR-24 under the drive of doxycycline in the presence of rtTA.
To achieve miR-24-induced overexpression in skin epithelial cells, we crossed the TRE-miR24 mice described above with K14-rtTA mice. Obtaining miR-24 induced over-expression mice (DTG for short) with the genotype of K14-rtTA, TRE-Mir 24.
2) Nucleic acid sequence: the sequences of the microRNAs used in this example are shown in Table 2 below:
TABLE 2 sequence listing of microRNAs used in the example of agonizing miR-24 overexpression
Note: 1. for SEQ ID nos. 5 and 6, the s subscript represents a thio backbone modification, Chol represents a cholesterol modification, a full chain methoxy modification.
miR-24 core sequence: GGCUCAG.
3) Total RNA extraction and fluorescent quantitative PCR
Extracting cell RNA: after the medium was aspirated from the 6-well plate, adherent cells were washed once with DPBS, and after the DPBS was aspirated, 500 μ L Trizol (invitrogen) was added to each well, and after lysis at room temperature for 10 minutes, RNA extraction was performed using Zymo # R2070 kit. Alternatively, 500. mu.L of RZ (heaven root) was added to each well and lysed at room temperature for 10 minutes before RNA extraction using heaven root # DP 419. The specific steps refer to the product specification.
Reverse transcription of miRNA and fluorescent quantitative PCR: reverse transcription and fluorescent quantitative PCR were performed on miR-24/miR-205/miR125b using the # E22007 kit for the giga gene. Reverse transcription was performed on miR-23a/miR-23b/miR27a/miR27a using TAKARA #638315 kit, and fluorescence quantitative PCR was performed using abm # MasterMix-mS. The specific steps are shown in respective specifications. In both methods, U6 was used as an internal reference gene, and 2 was used-△△CTThe method calculates the relative expression amount. All samples were normalized to the control sample level illustrated in the figure.
4) Integral dyeing of mouse dorsal skin
Fresh mice were taken of full-thickness dorsal skin (2 mm or more, shaved, but without removing subcutaneous fat, the following procedure was performed:
preparing a sealing liquid: PBS + 10% v/v Normal coat Serum + 2% m/v BSA + 0.2% v/v Triton-X. Filtered through a 0.22 μm filter and stored at 4 ℃. Note that: normal coat Serum (JACKSON # 005-.
The above tissues were placed in 5mM EDTA (PBS dilution) for 6 hours at 37 deg.C (dorsal skin, shortest possible 4 hours). All subsequent manipulations were performed in 1.5mL centrifuge tubes. The epidermis and dermis were torn apart with forceps. In this case, the epithelial tissue including the epidermis and hair follicle should be separated as a whole. The isolated whole epithelial tissue was placed in 4% paraformaldehyde at room temperature for 1 hour or overnight at 4 ℃. PBS was washed three times for 5 minutes each. EdU staining was performed as needed, and the specific procedure was performed according to the thermo # C1063 kit. If not, directly into the enclosure. Blocking for 2 hours at room temperature. Primary antibody was diluted with blocking solution in 1, 4 degrees overnight. PBS was washed three times for 5 minutes each. The secondary antibody was diluted with blocking solution from 1 at room temperature for 1 hour. PBS was washed three times for 5 minutes each.
Sealing: firstly, 200 microliter of sealing liquid ebioscience #00-4959-52 is added on the glass slide
Then the outer surface of the epidermis is attached to the glass slide, the hair follicle side (or the dermis side) is attached to the cover glass, the cover glass is covered, and bubbles are removed as much as possible. Nail polish sealing sheet.
Photo analysis was performed using Zeiss Cell Observer Z-stack mode.
5) Primary isolation and culture of mouse epidermal cells (MK)
Newborn mice within 4 days after the birth were taken out, cleaned with 75% ethanol, and then their heads, limbs and tails were cut off in DPBS, and their dorsal skins were separated along both sides of their trunk. After washing in clean DPBS, the cells were placed in 2.5mg/mL dispase (Roche #4942078001) overnight at 4 ℃. The following day, the dorsal skin was washed with clean DPBS and placed in Trypsin-Versene (Lonza #17-161F) and digested for 15 minutes at room temperature. The digested dorsal skin was minced and digestion was stopped with an equal volume of PFE (DPBS + 5% V/V FBS +1mM EDTA). The mixture was transferred to a new 50mL centrifuge tube and repeatedly blown through a 70 μm cell sieve 30 times. Centrifuge at 300g for 5 min at 4 ℃ and discard the supernatant. The DPBS resuspended the cell pellet, centrifuged at 300g at 4 ℃ for 5 minutes and the supernatant discarded. The cell pellet was resuspended in Growth medium (CNT-PR + 1% Pen-Strep +10 μm Y-27632) and plated onto cell culture dishes.
6) Transfection of MK with agomir
Using a transfection 6-well plate as an example, a Qiagen Effect #301427 transfection kit was selected, the final concentration of agomir was 200nM, and the procedure was as follows:
400pmol (. about.2.3. mu.g) of agomir was mixed with buffer EC in a total volume of 100. mu.L.
18.4. mu.L of Enhacer (8:1) was added, gently mixed, and then allowed to stand at room temperature for 3 minutes.
6.9. mu.L of Effectene (3:1) was added, gently mixed, and then allowed to stand at room temperature for 10 minutes.
After replacing MK with fresh medium, the transfection complex was added dropwise. Downstream experiments were performed 12 hours after transfection.
7) CCK-8 method for detecting cell proliferation capacity
Two methods are mainly used for detecting the proliferation capacity of different MK: 1) CCK-8 (Biyuntian # C0039) measures absorbance on different days to calculate the relative proliferation fold of the cells. 2) The relative competitive power of the target cells is detected by a double-color competition experiment method.
The CCK-8 process operates as follows:
MK were plated in 96-well plates, 100. mu.L of medium was added to each well, leaving 4-6 wells without cells and medium only as blank wells.
After 16 hours of plating, 10. mu.L of CCK-8 was added to each well to be tested and placed in an incubator at 37 ℃ for 1.5 hours.
Absorbance at 450nm was measured using a microplate reader.
The sample wells minus the blank wells were recorded as absorbance a1 on the first day.
Repeating the steps 2-4 about the nth day after the plate is paved, and recording the absorbance An of the nth day.
An/A1 was recorded as the n-day fold proliferation of the cells.
Example 1 knockout of miR-24 enhances hair growth without affecting skin development
Method
Endogenous miR-24 in the mouse can be divided into 13 chromosomes(Mir24-1) and chromosome 8 (Mir 24-2). Both Mir24 genes are located in the Mir23-Mir27-Mir24 cluster. We directly knock out the Mir24-2 gene (Mir24-2) by genetic engineering technologyKO/KO) Simultaneously, LoxP sites are knocked in at two sides of the Mir24-1 gene (Mir24-1)LoxP/LoxP). These mice were crossed with each other and then Krt14-Cre mice (Jakson Lab) (FIG. 1a), and we obtained a skin epithelium-specific miR-24 knockout mouse [ K14-Cre, miR24-1LoxP/LoxP,Mir24-2KO/KO]Abbreviated as dKO. At the same time, we claim that only one Mir24-2 copy is missing [ Mir24-1 ] obtained during this propagation processLoxp /Loxp,Mir24-2KO/+]The mouse was qKO, which is the primary control mouse we used in the following studies.
Fluorescence quantitative PCR (qRT-PCR) analysis and hair growth analysis of the above genetically engineered mouse epidermal cells (MK).
Results of the study
By the fluorescent quantitative PCR (qRT-PCR) analysis of the above genetically engineered mouse epidermal cells (MK), it was confirmed that miR-24 expression in the skin epithelial cells of the dKO mouse was greatly down-regulated, while the miRNA members of the other miR23-miR27-miR24 clusters were unaffected (FIG. 1 b).
Through hair growth analysis of the mice, the miR-24 knockout is found to remarkably enhance the regeneration capacity of skin and hair thereof.
The natural hair regeneration rate of the dKO mice was significantly accelerated (fig. 1 c). Notably, the appearance of skin and hair in dKO mice was not visibly different from qKO control mice (fig. 1c), indicating that the miR-24 knockout had no adverse effects affecting skin development and structure.
Example 2 knockout of miR-24 increases susceptibility to hair regrowth by physical epilation stimulation
Method
The genetically engineered mouse epidermal cells (MK) were physically plucked.
Results
Previous studies have shown that quantifying the density of plucking in mouse skin requires a certain threshold to be exceeded to trigger hair regrowth. While a quantitative plucking density insufficient to trigger qKO mouse hair regrowth was still effective in triggering dKO mouse hair regrowth (fig. 1d, 1 e).
It can thus be concluded that the sensitivity of dKO mice to hair regrowth by physical epilation stimulation is significantly increased.
Example 3 knock-out of miR-24 increases sensitivity to hair regrowth triggered by the hair restorer minoxidil drug
Method
In wild type mice (WT), solutions of minoxidil at 1% and 2% concentration (concentration for clinical female alopecia treatment) were applied to the surface, respectively. The surfaces of the dKO mice are respectively coated with 1% and 2% minoxidil solution.
Results
The sensitivity of the dKO mice to hair regrowth triggered by the hair drug minoxidil was significantly increased. In wild type mice (WT), topical application of a 2% concentration (concentration used for clinical female hair loss treatment) minoxidil solution was effective in activating hair regrowth, but a 1% concentration of minoxidil solution was ineffective (fig. 1 f).
Whereas in dKO mice, a 1% concentration solution of minoxidil was effective in activating hair regrowth (fig. 1 g).
It was therefore concluded that dKO significantly increased the sensitivity of mouse epidermal cells (MK) to growth factor-induced cell proliferation, and this effect could be significantly suppressed by miR-24 overexpression (fig. 1h, 1 i).
In addition, we also found that antisense nucleic acid inhibitors (antagomir) transfected with miR-24 can effectively reduce miR-24 levels in skin epithelial cells (FIG. 1j) and promote their growth (FIG. 1 k).
In conclusion, by constructing and analyzing a miR-24 conditional knockout mouse model specific to skin epithelial tissues, the knockout of the mouse model is found not to influence normal skin development and structure, but can obviously accelerate the hair cycle rate. More importantly, this knockout significantly increases the sensitivity of the skin to hair regrowth stimulating factors (e.g., minoxidil or epilation), allowing us to effectively induce hair regrowth using significantly lower than normal doses of hair-growth drugs.
Through cell experiments, the miR-24 knockout remarkably improves the sensitivity of skin epithelial cells to growth factor stimulation. In addition, the antisense nucleic acid inhibitor of miR-24 can be used for remarkably reducing the level of miR-24 in skin epithelial cells and promoting the growth of the skin epithelial cells.
Therefore, the miR-24 is estimated to be targeted and inhibited through an antisense nucleic acid inhibitor, a miRNA sponge inhibitor or a specific small molecule drug, or the miR-24 is knocked out through a genetic engineering method, so that the effective new way for promoting the regeneration of the skin and the hair thereof is realized.
Example 4miR-24 overexpression inhibits Hair growth
Method
To avoid the lethality problem of constitutively over-expressing miR-24, we constructed a Tre-miR24, K14-rtTA Double Transgene (DTG) mouse model (FIG. 2a) by mimicking the previously established tetracycline-inducible miRNA over-expression transgene system.
In these mice, doxycycline (doxycline, Dox) strongly induced skin epithelium-specific overexpression of miR-24 without interfering with other adjacent mirnas of the Mirc22 cluster (including three members: miR23b, miR27b, miR24-1) and Mirc11 cluster (including three members: miR23a, miR27a, miR24-2) (fig. 2 b).
W T litter group, DTG mouse control group without Dox induction, and DTG mouse experimental group induced by Dox were set. And hair growth analysis was performed on the mice.
Results
The inventor finds that the over-expression of miR-24 obviously inhibits the regeneration capacity of skin and hair thereof.
First, our data show that Dox-induced DTG mice had significantly delayed hair growth in the third follicular cycle compared to WT littermates or DTG mice not induced by Dox (fig. 2 c).
Secondly, the inhibition effect of miR-24 on the proliferation of skin epithelial precursor cells is also detected in vitro. We found that nucleic acid analogs (agomir) transfected with miR-24 were effective in increasing miR-24 levels in skin epithelial cells (FIG. 2d), and inhibiting their growth (FIG. 2 e).
Therefore, it can be concluded that an agonist of miR-24, or a method of overexpressing miR-24 by transfecting a nucleic acid analog of miR-24, can increase miR-24 levels in cells, thereby inhibiting skin and hair growth thereof.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence Listing
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Claims (10)
1. The application of the active ingredient targeting miR-24 or the preparation containing the active ingredient is characterized in that the active ingredient is used for preparing a medicine for regulating the growth of skin and hair thereof.
2. The use according to claim 1, wherein regulating the growth of skin and its hair comprises:
a) inhibiting the growth of skin and hair; and/or
b) Promoting skin and hair growth.
3. The use of claim 1, wherein the miR-24-targeting active ingredient is selected from the group consisting of:
i) an active ingredient targeted to inhibit miR-24 selected from the group consisting of: a small molecule compound, miRNA, antisense nucleic acid (e.g., antisense RNA), mRNA, antibody, gene editing agent, sponge inhibitor, or a combination thereof;
ii) an active ingredient targeted to agonistic miR-24 selected from the group consisting of: a small molecule compound, miRNA, a nucleic acid analog, or a combination thereof.
4. The use of claim 3, wherein the sequence of the miR-24-targeted antisense nucleic acid inhibitor is selected from the group consisting of SEQ ID NO: SEQ ID No. 3, SEQ ID No. 4, or combinations thereof.
5. The use of claim 3, wherein the sequence of the miR-24-targeting nucleic acid analog is selected from the group consisting of SEQ ID NO: SEQ ID No. 5, SEQ ID No. 6, or combinations thereof.
6. The use of claim 1, wherein said formulation is selected from the group consisting of: external preparation, oral preparation, and injection.
7. The use of claim 1, wherein said formulation further comprises: minoxidil, finasteride, or a combination thereof.
8. A pharmaceutical composition, wherein said pharmaceutical composition comprises:
A) an active ingredient targeting miR-24;
B) other pharmaceutically acceptable carriers or excipients.
9. A method of modulating the sensitivity of a cell to growth factor stimulation, comprising contacting the cell with a therapeutically effective amount of an active ingredient targeting miR-24.
10. A method of promoting regulation of hair growth, comprising administering to a patient in need thereof a medically effective amount of an active ingredient targeting miR-24 at a site in need thereof.
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US20080069791A1 (en) * | 2003-12-29 | 2008-03-20 | Universitaetsklinikum Muenster | Means for stimulation and activation of hair growth by il-15 |
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CN103736102A (en) * | 2013-11-14 | 2014-04-23 | 黄兵 | Small RNA used for psoriasis treatment, and derivatives and medicinal preparations thereof |
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