JP6324597B1 - Melanin production inhibitor, whitening agent, gene expression inhibitor, cosmetic composition for inhibiting melanin production, and cosmetic composition for whitening - Google Patents

Abstract

The present invention provides a novel melanin production inhibitor, a whitening agent, a cosmetic composition for inhibiting melanin production, and a cosmetic composition for whitening. A melanin production inhibitor comprising, as an active ingredient, one or more selected from the group consisting of miRNA-141, miRNA-200a, a precursor thereof, and a DNA construct encoding them. , Whitening agents, cosmetic compositions for inhibiting melanin production, and cosmetic compositions for whitening. [Selection] Figure 2

Description

  The present invention relates to a melanin production inhibitor, a whitening agent, a gene expression inhibitor, a cosmetic composition for inhibiting melanin production, and a cosmetic composition for whitening.

  It is considered that pigmentation such as skin spots causes excessive melanin to be deposited in the skin due to hormonal abnormalities, ultraviolet rays, and local inflammation of the skin. This melanin, which causes skin pigmentation, is produced in organelles called melanosomes in pigment cells (melanocytes) in the basal layer of the epidermis.

  In order to prevent and improve pigmentation in the skin, substances that have a whitening effect and substances that suppress melanin production are used. Examples of methods for preventing and improving pigmentation include a method of orally ingesting vitamin C and the like, a method of locally applying vitamin C, kojic acid, hydroquinone and the like to the skin. Substances such as vitamin C, kojic acid and hydroquinone have an inhibitory effect on the activity of tyrosinase, an enzyme involved in melanin synthesis.

  On the other hand, miRNA is a non-coding RNA of about 20 to 25 bases that is endogenous to cells. miRNA is first transcribed from a miRNA gene on genomic DNA as a primary transcript (pri-miRNA) having a length of about several hundred to several thousand bases, and then processed to a hairpin structure of about 60 to 70 bases. It becomes pre-miRNA which has. Then, it moves from the nucleus into the cytoplasm and further undergoes processing to become a mature miRNA of a dimer (guide strand and passenger strand) of about 20 to 25 bases. In mature miRNA, the guide strand (antisense strand) of the mature miRNA forms a complex with a protein called RISC (RNA-Induced Silencing Complex) and acts on the mRNA of the target gene, thereby inhibiting the translation of the target gene. It is known to do.

  Patent Document 1 discloses that miRNA-141 and miRNA-200a both inhibit the differentiation of mesenchymal cells through binding to the 3 ′ untranslated region of Dlx5 gene, and miRNA-208 targets the Ets1 gene. Inhibiting differentiation, it is described that these miRNA inhibitors can be used as pharmaceuticals for preventing / treating diseases such as osteoporosis as differentiation promoting agents that promote the differentiation of mesenchymal cells.

  Development of a pharmaceutical that can suppress the production of melanin and prevent or improve pigmentation such as spots using miRNA having a function of inhibiting translation of a target gene is desired.

International Publication No. 2010/058824

  The present invention has been made in view of the above problems, and an object of the present invention is a novel melanin production inhibitor, whitening agent, and gene expression inhibitor using miRNA having a function of inhibiting translation of a target gene. An object of the present invention is to provide a cosmetic composition for inhibiting melanin production and a cosmetic composition for whitening.

As a result of intensive studies to solve the above problems, the present inventors have found that miRNA-141 and miRNA-200a have an action of suppressing melanin production, and have completed the present invention.
More specifically, the present inventors have found that miRNA-141 and miRNA-200a inhibit gene expression of transcription factor MITF, which plays an important role in melanin production, at the translation level, thereby completing the present invention.

Therefore, according to the present invention, the subject is one kind selected from the group consisting of miRNA-141, miRNA-200a, a precursor of miRNA-141, a precursor of miRNA-200a, and a DNA construct encoding them. This is solved by a melanin production inhibitor containing two or more active ingredients.
Since the melanin production inhibitor of the present invention inhibits gene expression of the transcription factor MITF, which plays an important role in melanin production, at the translation level, it can effectively suppress melanin production.

In addition, according to the present invention, the subject is one or more selected from the group consisting of miRNA-141, miRNA-200a, a precursor of miRNA-141, a precursor of miRNA-200a, and a DNA construct encoding them. This is solved by a whitening agent containing two or more active ingredients.
At this time, the whitening agent may be used for preventing or treating spots.
At this time, the stain may be sparrow egg spot, sunlight mole, liver spot or bilateral delayed Ota nevus-like pigment spot.
The whitening agent of the present invention inhibits the gene expression of the transcription factor MITF, which plays an important role in melanin production, at the translation level, and therefore can effectively suppress the production of melanin, and is suitable for the prevention and treatment of spots. Can be used.

In addition, according to the present invention, the subject is one or more selected from the group consisting of miRNA-141, miRNA-200a, a precursor of miRNA-141, a precursor of miRNA-200a, and a DNA construct encoding them. It is solved by a gene expression inhibitor for MITF, a cosmetic composition for suppressing melanin production, and a cosmetic composition for whitening, which contains two or more kinds as active ingredients.

ADVANTAGE OF THE INVENTION According to this invention, the novel melanin production inhibitor, a whitening agent, a gene expression inhibitor, the cosmetic composition for melanin production suppression, and the cosmetic composition for whitening can be provided.
The melanin production inhibitor, whitening agent, gene expression inhibitor, melanin production-suppressing cosmetic composition and whitening cosmetic composition of the present invention have a translation level of the gene expression of the transcription factor MITF that plays an important role in melanin production. Inhibiting with can effectively suppress the production of melanin, and can be suitably used for the prevention and treatment of spots.

It is explanatory drawing which showed a part of process in which melanin produces | generates from tyrosine. In Test 2, it is a graph of the amount of melanin which shows the result of having investigated the influence on the melanin production in the mouse melanoma B16-4A5 cell which introduce | transduced miRNA-141 and miRNA-200a (there is a statistically significant difference between different alphabets) Is shown). In Test 2, it is a photograph of the cell deposit which shows the result of having investigated the influence on the melanin production in mouse melanoma B16-4A5 cell which introduced miRNA-141 and miRNA-200a. It is a figure which shows the influence on the melanin production by introduction | transduction of miRNA-141 and miRNA-200a by the expression level (Western blotting) of MITF and tyrosinase.

Hereinafter, embodiments of the present invention will be described with reference to FIGS.
This embodiment is selected from the group consisting of miRNA-141, miRNA-200a, which is a miRNA that binds to the 3 ′ untranslated region of the MITF gene, a precursor thereof, and a DNA construct that holds the DNA encoding them in a transcribable manner. The present invention relates to a melanin production inhibitor, a whitening agent, a gene expression inhibitor, a cosmetic composition for inhibiting melanin production, and a cosmetic composition for whitening.

<Generation of melanin in melanocytes>
Melanin, which causes pigmentation in the skin, is produced in melanosomes, which are organelles within melanocytes. Melanocytes are also called melanocytes and pigment cells.
As shown in the process of melanin production in FIG. 1, melanin is made from the tyrosine amino acid. In this process, enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2) are involved. The production of these enzymes involves the microphthalmia-related transcription factor (MITF), which is a pigment cell-specific transcription factor.

  Microphthalmia-associated transcription factor (hereinafter referred to as MITF) is a master transcription factor that controls the differentiation and proliferation of melanocytes, and tyrosinase, which is a melanin synthase, has 40% similarity to the amino acid sequence of tyrosinase. It specifically promotes the expression of TRP-1 and TRP-2 / dopachrome tautomerase (DCT).

  When stimulated by ultraviolet light or α-melanocyte stimulating hormone (hereinafter referred to as α-MSH), tyrosinase, which is a melanin synthase, is activated, and the enzyme reaction using tyrosine in melanocytes as a substrate causes dopa. Convert to Tyrosinase further acts on dopa and is converted to dopachrome and indolequinone (indole-5,6-quinone), and gradually becomes black by oxidative polymerization reaction to produce a melanin pigment.

<Pigment spots caused by excessive production of melanin>
Skin pigment spots (so-called spots) resulting from excessive production of melanin are caused by diseases associated with skin pigment abnormalities.
Here, diseases accompanied by pigment abnormalities on the skin include, for example, senile pigment spots, sparrow egg spots, liver spots, bilateral delayed Ota nevus-like pigment spots, pigmented nevus, blue nevus, Ota mother Including, but not limited to, plaques, acquired dermal melanocytosis, pigmented contact dermatitis, Addison's disease, and other pigmentations.

  Senile pigment spots are also referred to as sunburst, sunburst, or sunburst. Ultraviolet rays act on epidermal cells to promote melanocyte growth and secretion of differentiation promoting factors. In addition, ultraviolet rays directly act on melanocytes, thereby improving the expression of receptors for melanocyte activators. Senile pigment spots are pigment spots produced by repeated increase in melanocytes and promotion of melanin synthesis by ultraviolet rays for many years. In the Japanese, the frequency of senile pigment spots increases with age (aging), and almost all people have senile pigment spots in their 60s.

  Sparrow egg spot, so-called buckwheat, is a spot-like pigment spot that appears in the region from both cheeks to the nose and is known to be highly heritable. Sparrow egg spot begins to appear around the age of 3 and is most prominent around adolescence. In sparrow eggs, melanin production is promoted.

  Liver spots are pigment spots that occur frequently in women in their 30s. Symmetrical flat brown spots appear on the cheeks, around the mouth, and in the forehead. In melasma, melanin production of melanocytes in the basal epidermis is promoted. The cause of melasma is ultraviolet rays, but it is said that female hormones are involved because onset increases due to pregnancy, menopause, irregular menstruation, and the like.

  Bilateral delayed Ota nevus-like pigmentation is an acquired skin disease in which several bluish-brown pigmentation spots appear on the face. Acquired Dermal Melanocytosis (ADM) be called. In bilateral delayed Ota nevus-like pigment spots, melanocytes that produce melanin are in the dermis layer deeper than the epidermis, and are often seen in women between the ages of 20-30 and especially It is a disease common to Japanese and Chinese.

<MiRNA-141, miRNA-200a>
The melanin production inhibitor, the whitening agent, and the gene expression inhibitor according to the embodiment of the present invention are miRNA-141, miRNA-200a, a precursor thereof, and a precursor thereof, which are miRNAs that bind to the 3 ′ untranslated region of the MITF gene. One or more selected from the group consisting of DNA constructs that hold the DNA to be transferred in a transcribable manner are contained as active ingredients.

  In this specification, in miRNA, pri-miRNA, and pre-miRNA, an antisense strand (or an RNA region containing the antisense sense strand) against mRNA that finally pairs with mRNA of a target gene is used as a guide strand. The sense strand (or the RNA region containing the sense strand) for the antisense strand is referred to as a passenger strand.

(MiRNA)
The miRNA that binds to the 3 ′ untranslated region of the MITF gene is a single-stranded RNA having an action of binding to the untranslated region and suppressing translation of the MITF gene (corresponding to a guide strand of a double-stranded miRNA) Means. Such RNAs are miRNA-141 and miRNA-200a, and their mature sequences are as shown below (SEQ ID NOs: 1 to 4), respectively, and are conserved in mice, humans, and the like.

(Human)
miRNA-141: 5'-UAACACUGUCUGUGUAAGAUGGG-3 '(SEQ ID NO: 1)
miRNA-200a: 5'-UAACACUGUCUGGUAACGAUGU-3 '(SEQ ID NO: 2)
(mouse)
miRNA-141: 5′-UAACACUGUCUGGUAAAGAUGG-3 ′ (SEQ ID NO: 3)
miRNA-200a: 5'-UAACACUGUCUGGUAACGAUGU-3 '(SEQ ID NO: 4)

  These miRNAs can be produced by isolation from natural products, chemically synthesized, or in vivo or in vitro genetic engineering based on their nucleotide sequences. Can do. These miRNAs may also be analogs synthesized to mimic endogenous mature miRNAs. Such analogs are available from, for example, Ambion, and may be single stranded or double stranded with complementary strands.

  Here, as shown in Table 1, miRNA-141 and miRNA-200a (SEQ ID NOs: 1 to 4) bind to the 3 ′ untranslated region of the target gene, MITF gene, thereby mitigating MIFT gene expression at the translation level. It is expected to inhibit.

(MiRNA precursor)
Examples of the miRNA precursor (precursor of miRNA-141, precursor of miRNA-200a) include pri-miRNA and pre-miRNA. Specific examples thereof include pri-miRNA and pre-miRNA shown in Table 2 below for mouse and human, respectively. These miRNA precursors can also be produced by isolation from natural products, chemical synthesis, or in vivo or in vitro genetic engineering using a conventionally known method. Furthermore, the miRNA precursor may be an analog of the miRNA precursor that has been synthesized to mimic the endogenous miRNA precursor. The miRNA precursor may be single-stranded or double-stranded with a complementary strand.

(DNA constructs such as miRNA)
DNA constructs that hold miRNAs (miRNA-141, miRNA-200a) and miRNA precursors (a precursor of miRNA-141, a precursor of miRNA-200a) in a transcribable manner are in vivo or in vitro. This is a DNA construct encoding the RNA strand in such a manner that transcription is possible. For example, a construct capable of transcribing miRNA or the like in vitro includes a vector in which DNA encoding miRNA or the like is linked under the control of a viral promoter. In addition, examples of the construct capable of transferring miRNA and the like in vivo include a vector in which DNA encoding miRNA and the like is linked under the control of a promoter effective in mammalian cells. Such vectors for in vivo or in vitro transcription are commercially available, and such vectors can be easily constructed based on the base sequence such as miRNA according to the protocol.

<Melanin production inhibitor>
The melanin production inhibitor (melanin production inhibitor, melanin production inhibitor) according to the present embodiment includes miRNA-141, miRNA-200a, precursors thereof, and precursors thereof that bind to the 3 ′ untranslated region of the MITF gene. It is a melanin production inhibitor containing one or more selected from the group consisting of DNA constructs that hold the encoded DNA in a transcribable manner as an active ingredient.
Here, the “melanin production inhibitor” refers to an agent that suppresses the production of melanin in melanocytes.

<Whitening agent>
Since the melanin production inhibitor which concerns on this embodiment has the effect | action which suppresses melanin production, it can also be used as a whitening agent.
Here, “whitening” means disappearance, reduction (treatment) of pigment black spots, pigment spots such as spots and freckles, specifically skin tone and pigment change due to excessive production of melanin, and / or Alternatively, it is an action of preventing and maintaining or restoring (revitalizing) the original color tone of the skin, for example, an action of whitening the skin.

  A whitening effect can be obtained by using the melanin production inhibitor or whitening agent according to the present embodiment on the skin, and pigment spots (for example, senile pigment spots, sparrow egg spots, liver spots, Bilateral delayed Ota nevus-like pigment spots etc.) can also be treated.

  “Treatment of pigmented spots” means, for example, pigmented spots (senile pigmented spots, sparrow eggspots) compared to the state before administration of miRNA which is an active ingredient of the melanin production inhibitor or whitening agent of the present embodiment. , Liver spots, bilateral delayed onset nevus-like pigment spots, etc.).

<Gene expression inhibitor>
The miRNA-141, miRNA-200a, miRNA-200a that is a miRNA that binds to the 3 ′ untranslated region of the MITF gene according to the present embodiment, a precursor thereof, and a DNA construct that holds the DNA encoding them in a transcribable manner are expressed in the MITF gene. Can be used as a gene expression inhibitor for MITF.

<Application>
A melanin production inhibitor, a whitening agent, and a gene expression inhibitor containing miRNA-141 and / or miRNA-200a according to this embodiment as an active ingredient are patients with pigment spots caused by melanin production being promoted in melanocytes. It is administered to patients who have a definitive diagnosis of diseases associated with skin pigment abnormalities (senile pigment spots, sparrow egg spots, liver spots, bilateral delayed Ota nevus-like pigment spots, etc.).
In addition, the melanin production inhibitor, the whitening agent, and the gene expression inhibitor of the present embodiment are configured as a composition such as a cosmetic composition and a pharmaceutical composition, and those who are aware that pigment spots have occurred, Is administered prophylactically to those who are likely to experience

In general, melanin production in melanocytes is known to be caused by various factors such as exposure to ultraviolet light, aging, and genetic factors.
Therefore, the melanin production inhibitor, the whitening agent, and the gene expression inhibitor of the present embodiment can be treated with diseases (senile pigment spot, sparrow egg spot, People who are more likely to be affected (eg, melasma, bilateral delayed Ota nevus-like pigment spots) (people who are likely to be affected), for example, staying outdoors outdoors and exposed to UV rays It can be administered to people with high levels of aging, advanced aging, and family members whose family members suffer from skin pigmentation disorders.

In addition, a melanin production inhibitor, a whitening agent, and a gene expression inhibitor containing miRNA-141 and / or miRNA-200a as an active ingredient for humans after the age of 40, particularly for elderly people over 60 or 65 years old. Can be administered.
Humans over 30 years old, particularly elderly people over 60 years old or over 65 years old, tend to develop senile pigment spots with aging, but miRNA-141 and / or miRNA-200a has an inhibitory effect on melanin production. It is possible to suppress pigment spots that occur with aging.

In addition, it may be affected by diseases with abnormal skin pigmentation that have been pointed out to be caused by genetic factors, such as sparrow egg spot, liver spots, bilateral delayed Ota nevus-like pigment spots, etc. The melanin production inhibitor, the whitening agent, and the gene expression inhibitor according to this embodiment can be administered to humans.
Specifically, people older than 3 years old who start sparrow egg spot, people older than 30 years old who start to develop liver spot (especially having a higher frequency of liver spots than men) ), A melanin production inhibitor, a whitening agent, and a gene expression inhibitor according to the present embodiment for a person before the age of 20 years when bilateral delayed Ota nevus-like pigment spots begin to develop Agents can be administered.

  According to the melanin production inhibitor, whitening agent, and gene expression inhibitor of the present embodiment, gene expression of the transcription factor MITF that plays an important role in melanin production can be inhibited at the translation level. Application to prevention and treatment of related diseases is expected.

  The subject to which the melanin production inhibitor, the whitening agent, and the gene expression inhibitor according to this embodiment are administered is not limited to those with the above symptoms or conditions, or animals other than humans.

  Moreover, the melanin production inhibitor and the whitening agent which contain miRNA-141 and / or miRNA-200a which concern on this embodiment as an active ingredient add a pharmacologically acceptable additive, cosmetic composition, pharmaceutical composition It can be used as a composition such as a product.

(Cosmetic composition)
The melanin production inhibitor, the whitening agent, and the gene expression inhibitor according to the present embodiment can be used as a cosmetic composition.
The cosmetic composition can be applied to any form of cosmetic. For example, it can be applied to skin care cosmetics such as lotions, milky lotions, creams, and cosmetics, foundations, concealers, makeup bases, makeup cosmetics such as lipsticks, blushers, eye shadows, and eyeliners, sunscreen cosmetics, etc. .

In addition to the melanin production inhibitor, the whitening agent, and the gene expression inhibitor according to the present embodiment, the cosmetic composition according to the present embodiment includes one or two components that can be used in a normal cosmetic composition. These can be freely selected and blended.
For example, base materials, preservatives, emulsifiers, colorants, preservatives, surfactants, UV absorbers, antioxidants, moisturizers, UV absorbers, perfumes, antiseptics, extenders, colored pigments, alcohol, All additives usually used in the cosmetic field, such as water, can be included.

In the cosmetic composition according to the present embodiment, the contents of the melanin production inhibitor, the whitening agent, and the gene expression inhibitor are not particularly limited, and can be freely set according to the purpose.
In addition to miRNA such as miRNA-141 and miRNA-200a, the cosmetic composition according to the present embodiment is added with one or more other substances known to have melanin production inhibitory action and whitening. It is also possible.

(Pharmaceutical composition)
The melanin production inhibitor, whitening agent, and gene expression inhibitor of this embodiment can be used as a pharmaceutical composition.
In the pharmaceutical field, the melanin production inhibitor, whitening agent, and gene expression inhibitor of this embodiment are combined with a pharmaceutically acceptable carrier or additive together with an amount of miRNA that can effectively exert the action. A pharmaceutical composition having the action is provided. The pharmaceutical composition may be a pharmaceutical or a quasi drug.
The pharmaceutical composition may be applied externally or internally. Therefore, the pharmaceutical composition is used in a pharmaceutical form such as transdermal application agent, transmucosal application agent, intravenous injection, subcutaneous injection, intracutaneous injection, intramuscular injection and / or intraperitoneal injection, etc. be able to.
The dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, for example, liquid preparations such as liquids and suspensions, semi-solid preparations such as ointments or gels, tablets, granules, Solid preparations such as capsules, powders, powders and the like can be mentioned.

In the pharmaceutical composition according to this embodiment, one or more pharmaceutically acceptable additives can be freely selected and contained.
For example, when the pharmaceutical composition according to this embodiment is applied transdermally, for example, a base material, an excipient, a thickener, an absorption accelerator, a surfactant, a preservative, a stabilizer, a preservative, All additives that can be normally used in the field of pharmaceutical preparations, such as antioxidants, can be included. Moreover, it can also be set as the formulation using a drug delivery system (DDS).

In the pharmaceutical composition according to this embodiment, the contents of the melanin production inhibitor, the whitening agent, and the gene expression inhibitor are not particularly limited, and can be freely set according to the purpose.
In addition to miRNAs such as miRNA-141 and miRNA-200a, the pharmaceutical composition according to this embodiment may contain one or more other substances known to have melanin production inhibitory action and whitening. It is.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on a specific Example, this invention is not limited to these.
In the following examples, miRNA-141 and miRNA-200a were used to examine the influence on melanin production.

<Test 1 Introduction of miRNA into cells>
In order to examine the effect of melanin production by α-melanocyte-stimulating hormone (α-MSH) induction by miRNA-141 and miRNA-200a, simulated artificial miRNA-141 and miRNA- were introduced into mouse melanoma B16-4A5 cells (RIKEN). 200a (both manufactured by Ambion), miRNA-Negative Control (miRNA-NC), which is a miRNA having no target, was introduced by the following method.

Mouse melanoma B16-4A5 cells containing 10% fetal bovine serum (hereinafter FBS, fetal bovine serum) (DMEM, Dulbecco's Modified Eagle Medium, Hyclone) low glucose to 5 × 10 ⁴ cells / mL After adjusting with a medium, seeded with a 12-well multiwell plate (Nunc) and allowed to adhere overnight in a 95% air-5% carbon dioxide environment. Thereafter, the medium was removed by aspiration, and after washing with a phosphate buffer (PBS (−), phosphate-buffered saline), 900 μL of Opti-MEM (registered trademark) medium was added.

  Simulated artificial miRNA-141, miRNA-200a, and miRNA-NC introduced into cells are Lipofectamine (registered trademark) RNAiMAX transcription reagent 3 μL, simulated artificial miRNA 2 μL (final concentration: 20 nM) in an RNase-DNase-free 1.5 mL Eppendorf tube. , And Opti-MEM medium were added in an amount of 95 μL to prepare a mixed solution. After stirring, the mixture was allowed to stand for 30 minutes in a clean bench (stirring every 10 minutes for 30 minutes).

  After the preparation of the simulated artificial miRNA-solution was completed, the entire amount of the simulated artificial miRNA-solution in the 1.5 mL Eppendorf tube was added to the cells in an incubator prepared in a 95% air-5% carbon dioxide environment. The cells were introduced for 12 hours. After 12 hours, fresh DMEM medium containing 10% FBS was replaced.

<Test 2 Melanin Production Test>
Α-MSH (Sigma) was added to a well in which mock artificial miRNA-introduced cells or untreated cells were cultured so that the final concentration was 2 μM to induce melanin production. Thereafter, the cells were cultured for 72 hours in an incubator adjusted to a 95% air-5% carbon dioxide environment.
After 72 hours, the cell culture was detached from each well with a cell scraper and collected in a 1.5 mL Eppendorf tube. The cell suspension was immediately subjected to centrifugation (560 × g, 4 ° C., 10 minutes) to precipitate the cells. The precipitated cells were washed twice with PBS (−). After washing, 200 μL of 1N NaOH was added to the cell pellet, and the cells were lysed at 50 ° C. for 1 hour.
Cell lysate is dispensed in 50 μL aliquots into a 96-well multiwell plate (3 wells per sample), and the absorbance at 475 nm is measured with a microplate reader. The amount of melanin was calculated with a standard curve prepared in advance with artificial melanin and corrected with the protein concentration of each sample (Biorad DC ^™ Protein Assay Kit).

(Result of Test 2)
The results are shown in FIG. FIG. 2 shows, in order from the left, (1) α-MSH treatment, (2) α-MSH treatment, (3) α-MSH treatment + miRNA-NC treatment, (4) α-MSH treatment + miRNA-141, (5) Results of α-MSH treatment + miRNA-200a, (6) α-MSH treatment + 200 μM arbutin.

As shown in FIG. 2, when α-MSH was added, melanogenesis in mouse melanoma B16-4A5 cells was induced ((1) and (2) in FIG. 2).
When miRNA-141 or miRNA-200a was introduced, it was confirmed that melanin production in mouse melanoma B16-4A5 cells induced by α-MSH was suppressed ((4) and (5) in FIG. 2).

It was confirmed that miRNA-141 or miRNA-200a suppresses melanin production to the same extent as arbutin, which is a whitening agent ((6) in FIG. 2).
When miRNA-NC having no target was introduced, melanin production in mouse melanoma B16-4A5 cells induced by α-MSH was promoted without being suppressed ((3) in FIG. 2).

  The above results were also confirmed from a photograph of the cell precipitate shown in FIG. Specifically, when miRNA-141 or miRNA-200a is introduced ((4) and (5) in FIG. 3), when melanin production is not induced ((1) in FIG. 3), or when arbutin is added When it was done ((6) of FIG. 3), it was exhibiting white. On the other hand, when only α-MSH treatment was performed ((2) in FIG. 3), or when miRNA-NC without a target was introduced ((3) in FIG. 3), black color was generated due to melanin generation. It was.

  From the above, it was found that miRNA-141 and miRNA-200a have an action of suppressing the production of melanin.

<Test 3 Target protein expression analysis>
(3.1. Cell preparation and cell recovery after stimulation of melanogenesis)
Cells were prepared and cultured according to Test 1, and α-MSH was added to each well so that the final concentration was 2 μM (the same amount of dimethyl sulfoxide was added to untreated wells). After 72 hours of culturing, the cells were washed twice with PBS (−), and 2% Protease inhibitor, 2% Phosphatase inhibitor-containing RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% 100 μL of sodium deoxycholate (0.1% sodium dodecyl sulfate (SDS)) was added to lyse the cells. Then, it peeled with the cell scraper and collect | recovered to the 1.5 mL Eppendorf tube. The collected cell lysate is stored on ice for 30 minutes, and then disrupted using an ultrasonic crusher (10 seconds x 3 times). After centrifugation at 16,200 xg for 20 minutes at 4 ° C, the top The supernatant was collected as a sample solution. The protein concentration in the sample solution was measured with a Bio Rad DC protein assay kit (Bio Rad, Hercules, CA, USA). A sample buffer containing 10% mercaptoethanol (0.125 M Tris-HCl (pH 6.8), 10% Glycerol, 4% SDS, 0.004% bromophenol blue) is added to the sample solution with the adjusted protein concentration, and 5% at 98 ° C. The sample was heated for minutes and used as a sample for SDS electrophoresis.

(3.2. SDS-PAGE)
A 10-15% polyacrylamide gel corresponding to the target detection protein was set in the electrophoresis tank, and electrophoresis buffer (Bio Rad Tris / Glycine buffer) was added to about 1/3 of the electrophoresis tank. 10 μL (sample concentration: 5-20 μg / lane) of each sample was applied to each lane of the gel, an equal amount of protein molecular weight marker was applied to the first lane, and an equal amount of Laemmli sample buffer was applied to the unused lane. The sample protein was separated by electrophoresis at 120 V for 90 minutes. After the electrophoresis, the gel was removed from the glass plate, and unnecessary gel was cut out and transferred onto a filter paper soaked in a blotting buffer (BioRad Tris / Glycine buffer 10%, methanol 20%, ultrapure water 70%).

(3.3. Western blotting)
Polyvinylidene difluoride membrane (hereinafter PVDF membrane, GE Healthcare UK Ltd, Amersham Place, England) was immersed in methanol for 3 minutes, hydrophilized, and then immersed in a blotting buffer for equilibration. Two sets of sponge and filter paper soaked with blotting buffer were combined as a set, and the gel obtained by SDS-PAGE described in the above section 3.2 and the PVDF membrane were sandwiched. After setting in the electrophoresis tank, the sample was electrophoresed at 60 V for 90 minutes while being cooled with ice water, and the protein separated by SDS-PAGE was transferred to a PVDF membrane. The PVDF membrane was immersed in a Tris buffered saline solution (T-TBS) containing 0.05% Tween 20 containing 5% Skim milk (Becton, Dickinson and Company) in a case and shaken for 60 minutes. After washing with T-TBS (10 minutes × 3 times), it is then immersed in various primary antibody (MITF (Millipore), tyrosinase (Tyr, abcam), β-actin (Sigma)) solutions. Shake overnight at 4 ° C. The next day, after washing with T-TBS (10 minutes × 3 times), secondary antibody diluted with T-TBS solution containing 5% nonfat dry milk (ECL Anti-Rabbit IgG, Horseradish peroxidase linked whole antibody or ECL Anti-Mouse IgG, (Horseradish peroxidase linked whole antibody, GE Healthcare) and shaken for 60 minutes. Then, it was washed with T-TBS (10 minutes × 3 times), and observed with a chemiluminescence detection device (Davinch-Chemi CAS-400SM Wako Pure Chemical Industries, Osaka) using an ECL select Western blotting detection reagent (GE Healthcare). did.

(Result of test 3)
As shown in FIG. 4, miRNA-141 and miRNA-200a (manufactured by Ambion) were introduced into mouse melanoma B16-4A5 cells, and a cell lysate was prepared from the cells after stimulation of melanogenesis, and subjected to Western blotting. However, in the cell group into which miRNA-141 and miRNA-200a are introduced, it is a transcription factor that regulates the expression of tyrosinase, TRP-1, and TRP-2 involved in melanin production, compared to the α-MSH treatment and the negative control group. The protein expression of a certain MITF was suppressed. The inhibitory effect on the protein expression of MITF by miRNA-141 and miRNA-200a was comparable when α-MSH treatment was not performed. From these results, it was confirmed that microRNA-141 and -200a have MITF as a target gene.

  Moreover, in the cell group which introduce | transduced miRNA-141 and miRNA-200a, the expression of tyrosinase was suppressed compared with the alpha-MSH process and the negative control group.

  From the above results, miRNA-141 and miRNA-200a suppress the expression of MITF, which is a transcription factor that regulates the expression of tyrosinase, TRP-1 and TRP-2 involved in melanin production, thereby producing melanin. It was found to be suppressed.

Claims (7)

Inhibition of melanin production containing, as an active ingredient, one or more selected from the group consisting of miRNA-141, miRNA-200a, miRNA-141 precursor, miRNA-200a precursor and DNA constructs encoding these Agent. A whitening agent containing, as an active ingredient, one or more selected from the group consisting of miRNA-141, miRNA-200a, a precursor of miRNA-141, a precursor of miRNA-200a, and a DNA construct encoding them.   3. The whitening agent according to claim 2, wherein the whitening agent is used for preventing or treating spots.   The whitening agent according to claim 3, wherein the stain is sparrow egg spot, sunlight mole, liver spot, or bilateral delayed Ota nevus pigment spot. MITF -141 gene containing miRNA-141, miRNA-200a, miRNA-141 precursor, miRNA-200a precursor and one or more selected from the group consisting of DNA constructs encoding them as an active ingredient Expression inhibitor. Inhibition of melanin production containing, as an active ingredient, one or more selected from the group consisting of miRNA-141, miRNA-200a, miRNA-141 precursor, miRNA-200a precursor and DNA constructs encoding these Cosmetic composition. Whitening makeup containing, as an active ingredient, one or more selected from the group consisting of miRNA-141, miRNA-200a, a precursor of miRNA-141, a precursor of miRNA-200a, and a DNA construct encoding them Composition.