WO2009039743A1 - Sirnas useful for inhibiting expression of tyrosinase gene, the compositions comprising the sirnas and their uses - Google Patents

Abstract

siRNAs useful for inhibiting expression of tyrosinase gene are provided, wherein the siRNA has the nucleotide sequence as shown in TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8, or has the chemically modified nucleotide sequence derived from the nucleotide sequence as shown in TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8. Cosmetic compositions and the uses of siRNAs in the preparation of a cosmetic composition for inhibiting melanin production and deposit or eliminating melanin are provided. The siRNAs have high activity for inhibiting the expression of tyrosinase gene, and can effectively inhibit melanin production and deposit.

Description

 siRNA, composition and application thereof for inhibiting tyrosinase gene expression

 The present invention relates to an siRNA for inhibiting tyrosinase gene expression and a cosmetic composition containing the same and use of the siRNA for preparing a cosmetic composition for inhibiting or eliminating melanin production and deposition. Background technique

 (1) About melanin

 Melanin is a biopolymer synthesized by melanocytes produced by melanocytes of the skin, hair follicles, iris and retina. When stimulated by ultraviolet light and other factors, melanocytes can synthesize a large amount of tyrosinase necessary for melanin biosynthesis. Under the catalyzed by tyrosinase, tyrosine is converted into dopaquinone, and dopaquinone is further oxidized to form more. Bae pigment, finally forming melanin.

 Melanin can be divided into two categories, one is true melanin, contains no sulfur atom, is brown or black; the second is demelamine, containing sulfur atoms, yellow or reddish brown. Animal and human skin and hair color are directly related to the number, size, and distribution of melanosomes. Excessive melanin darkens the skin, and non-uniform distribution of melanin causes chloasma and ecchymoses.

 Melanin is composed of multiple genes that are coordinated with each other and synthesized through a series of complex physiological and biochemical processes. These genes involved in pigmentation are involved in the synthesis, transport and distribution of melanin. Among them, the tyrosinase gene is an extremely important gene in the process of melanin biosynthesis, and therefore, the expression of the tyrosinase gene is specifically inhibited by RNA interference, thereby silencing the activity of the tyrosinase gene, It is an effective way to inhibit melanin synthesis and improve skin color.

 (2) About RNA interference technology

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) molecules shut down the expression of the corresponding gene at the mRNA level or silence the gene. RNA interference technology, also known as knock-down or gene silencing, is a typical post-transcriptional gene regulation method, also known as post-transcriptional gene silencing (PTGS). ). The earliest reports of RNA interference occurred in 1990, and two different research groups reported on RNA interference in transgenic plants, which were later observed in almost all eukaryotes such as nematodes, fruit flies, zebrafish, and mice. Arrived RNA interference phenomenon. In 1999, Hamilton and Baulcombe detected RNA fragments of 21-25 nucleotides in length in plants with RNA interference. These RNA fragments have been shown to be required for RNA interference, called small interfering RNA (siRNA). Double-stranded siRNA forms an RNA-induced silencing complex (RISC) with cell-derived related enzymes and proteins. In the process of RNA interference, the sense strand in the double-stranded siRNA is excluded from the complex, the antisense strand directs RISC to bind to the corresponding site of the target mRNA, and then the target mRNA is degraded by ribonuclease III in the complex, thereby shutting down the target. Gene expression.

 Since RNA interference is directed to gene silencing in the post-transcriptional phase, the design of the entire process is simpler and more rapid, and the effect is obvious compared to traditional gene therapy methods such as antisense nucleic acid technology or the transfer of non-functional mutants to compete with the gene. . This feature of RNA interference opens up new and powerful tools for gene therapy.

 For example, US 2005/0137151 A1 discloses a composition for treating pigmentation, which composition contains one or more siRNAs for inhibiting tyrosinase gene expression, the siRNA being of the following nucleotide sequence One or several of the siRNAs:

 C1 ) 5 '-UAGGACCUGCCAGUGCUCUdTdT-3 '

 3, -dTdTAUCCUGGACGGUCACGAGA-5,;

 C2) 5' -UCCUGGAAACCAUGACAAAdTdT-3 '

 3, -dTdTAGGACCUUUGGUACUGUUU-5 ';

 C3) 5 '-CACACCUGUCUUUGUCUUAdTdT-3 '

 3 ' -dTdTGUGUGGACAGAAACAGAAU-5 '.

 However, the siRNA has a problem in that the inhibitory activity against tyrosinase gene expression is low. Therefore, development of a siRNA having a high inhibitory activity on tyrosinase gene expression has become an urgent problem to be solved. Summary of the invention

 The first object of the present invention is to overcome the shortcomings of the conventional siRNA having low inhibitory activity against tyrosinase gene expression, and to provide an siRNA having high inhibitory activity against tyrosinase gene expression.

 A second object of the present invention is to provide a cosmetic composition for inhibiting melanin production and depositing or removing black pigment.

A third object of the present invention is to provide an application of siRNA in the preparation of a cosmetic composition for inhibiting melanin production and deposition or removing melanin. The siRNA for inhibiting tyrosinase gene expression provided by the present invention has the TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8 Nucleotide sequence, or chemical modification of the nucleotide sequence indicated by TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8 The obtained nucleotide sequence, wherein

 TYRO-1 justice chain: 5 ' -GCAACUUCAUGGGAUUCAAdTdT-3 '

 Antisense chain: 5 '-UUGAAUCCCAUGAAGUUGCdTdT-3,;

 TYRO-2 justice chain: 5 ' -GCACAGAGAGACGACUCUUdTdT-3 '

 Antisense strand: 5 '-AAGAGUCGUCUCUCUGUGCdTdT-3,;

 TYRO-3 justice chain: 5 ' -GGAGGAGUACAACAGCCAUdTdT-3 '

 Antisense chain: 5 '-AUGGCUGUUGUACUCCUCCdTdT-3,;

 TYRO-4 justice chain: 5 ' -GCCAUCAGUCUUUAUGCAAdTdT-3 '

 Antisense chain: 5 '-UUGCAUAAAGACUGAUGGCdTdT-3,;

 TYRO-5 justice chain: 5 ' -CCAUCAGUCUUUAUGCAAUdTdT-3 '

 Antisense chain: 5 '-AUUGCAUAAAGACUGAUGGdTdT-3,;

 TYRO-6 justice chain: 5 ' -GCGUAAUCCUGGAAACCAUdTdT-3 '

 Antisense strand: 5 '-AUGGUUUCCAGGAUUACGCdTdT-3,;

 TYRO-7 justice chain: 5 ' -CCAGGUUCCCAGAGAAUAUdTdT-3 '

 Antisense chain: 5 '-AUAUUCUCUGGGAACCUGGdTdT-3,;

 TYRO-8 justice chain: 5 ' -GGUAAUGAGGAACUGUUAUdTdT-3 '

 Antisense strand: 5 '-AUAACAGUUCCUCAUUACCdTdT-3 '.

 The present invention provides a cosmetic composition comprising the siRNA provided by the present invention as an active ingredient.

 The present invention provides the use of an siRNA for the preparation of a cosmetic composition for inhibiting melanin production and deposition or removal of black pigment.

The present invention provides an siRNA capable of inhibiting the expression of a tyrosinase gene, which has high inhibitory activity against tyrosinase gene expression and is capable of effectively inhibiting melanin production and deposition. For example, the siRNAs TYRO-1 to TYRO-8 provided by the present invention can efficiently inhibit the expression of the tyrosinase gene, in particular, the siRNA TYRO-1, TYRO-4 and TYRO-5 inhibit the tyrosinase gene, respectively. 81%, 82%, and 87%, while the reference siRNA C1-C3 inhibited the tyrosinase gene by 65%, 58%, and 52%, respectively, indicating that it is used to inhibit cheese. In contrast to the expressed siRNA, when the siRNA provided by the present invention is used for inhibiting the expression of the tyrosinase gene, the inhibitory activity against tyrosinase gene expression is greatly improved. detailed description

 The present invention provides an siRNA for inhibiting tyrosinase gene expression, wherein the siRNA has TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 Or a nucleotide sequence as shown by TYRO-8, or having a nucleus as shown for TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8 a nucleotide sequence obtained by chemically modifying a nucleotide sequence, wherein

 TYRO-1 justice chain: 5 '-GCAACUUCAUGGGAUUCAAdTdT-3 '

 Antisense chain: 5 '-UUGAAUCCCAUGAAGUUGCdTdT-3,;

 TYRO-2 justice chain: 5 '-GCACAGAGAGACGACUCUUdTdT-3 '

 Antisense strand: 5 '-AAGAGUCGUCUCUCUGUGCdTdT-3,;

 TYRO-3 justice chain: 5 '-GGAGGAGUACAACAGCCAUdTdT-3 '

 Antisense chain: 5 '-AUGGCUGUUGUACUCCUCCdTdT-3,;

 TYRO-4 justice chain: 5 '-GCCAUCAGUCUUUAUGCAAdTdT-3 '

 Antisense chain: 5 '-UUGCAUAAAGACUGAUGGCdTdT-3,;

 TYRO-5 justice chain: 5 ' -CCAUCAGUCUUUAUGCAAUdTdT-3 '

 Antisense chain: 5 '-AUUGCAUAAAGACUGAUGGdTdT-3,;

 TYRO-6 justice chain: 5 ' -GCGUAAUCCUGGAAACCAUdTdT-3 '

 Antisense strand: 5 '-AUGGUUUCCAGGAUUACGCdTdT-3,;

 TYRO-7 justice chain: 5 ' -CCAGGUUCCCAGAGAAUAUdTdT-3 '

 Antisense chain: 5 '-AUAUUCUCUGGGAACCUGGdTdT-3,;

 TYRO-8 justice chain: 5 ' -GGUAAUGAGGAACUGUUAUdTdT-3 '

 Antisense strand: 5 '-AUAACAGUUCCUCAUUACCdTdT-3 '.

 In order to obtain a better inhibitory effect, preferably, the siRNA has a nucleotide sequence represented by TYRO-1, TYRO-4 or TYRO-5; more preferably, the siRNA has TYRO-5 shown Nucleotide sequence.

The tyrosinase is a copper-containing oxidoreductase widely present in plants, animals and microorganisms. In the synthesis of melanin, tyrosinase is required in two steps, therefore, tyrosine Enzymes play a pivotal role in the production of melanin. The tyrosinase gene refers to a nucleotide sequence encoding a tyrosinase. The human tyrosinase gene is about 35kb-70kb long and is located on chromosome 11, which contains 5 exons and 4 introns. The lengths of 5 exons are 919bp, 135bp, 150bp, 183bp and 219bp, respectively.

 The siRNA is a double-stranded RNA molecule, including a sense strand and an antisense strand, and the sense strand and the antisense strand are 21 nucleotides in length, and each of the sense strand and the antisense strand has two 3' ends. For one continuous deoxythymidylate, the sense strand and the antisense strand are complementary to form a double strand by eliminating 19 nucleotides other than the two consecutive deoxythymidyl acids at the 3' end.

 According to the invention, the chemical modification is at least one of the following modifications:

 (1) a modification of a phosphodiester bond of a linked nucleotide in a nucleotide sequence of the siRNA;

(2) modification of the 2'-OH of ribose in the nucleotide sequence of the siRNA;

 (3) Modification of a base in the nucleotide sequence of the siRNA.

 The chemical modification is well known to those skilled in the art, and the modification of the phosphodiester bond refers to modification of oxygen in the phosphodiester bond, including phosphorothioate modification, as shown in Formula 1; and boron bismuth phosphate Salt modification, as shown in Formula 2. Both modifications stabilize the siRNA structure, maintaining high specificity and high affinity for base pairing.

The ribose modification refers to a modification of 2'-OH in a nucleotide pentose, gp, which introduces a certain substituent at the hydroxyl position of the ribose, for example, a 2'-fluoro modification, as shown in Formula 3; - oxymethyl modification, as shown in Formula 4; 2,-oxyethylene methoxy modification, as shown in Formula 5; 2,4,-dinitrophenol modification, as shown in Formula 6; LNA), as shown in Formula 7; 2'-amino modification, as shown in Formula 8; 2'-deoxy modification, as shown in Formula 9.

,0 0"CH ₂ "CH ₂ "0"CH _:i DHP

The base modification refers to modification of a base of a nucleotide, for example, 5'-bromouracil modification, as shown in Formula 10; 5'-iodouracil modification, as shown in Formula 11; N-A The quinolidine modification, as shown in Formula 12; 2,6-diaminopurine modification, as shown in Formula 13.

Preferably, the modification enhances the ability of the modified siRNA to resist nuclease hydrolysis in the cell.

 Further, in order to promote entry of the siRNA into the cell, a lipophilic group such as cholesterol may be introduced at the end of the siRNA sense strand on the basis of the above modification to facilitate interaction with the intracellular mRNA by the cell membrane composed of the lipid bilayer.

 The preparation method of the siRNA provided by the present invention includes the design of the siRNA sequence and the synthesis of the siRNA sequence.

The design of the siRNA sequence means: a 19 bp nucleotide sequence is selected within the range of l-2082 bp of the tyrosinase gene cDNA sequence (Genbank accession number NM- 000372). The principle of selecting the 19 bp nucleotide sequence is: (1) the total siRNA double bond energy <1; (2) the 5' end binding energy of the antisense strand 3-9; (3) the 5' end binding energy of the sense strand 5-9; (4) GC content is between 36-53%; (5) The energy difference between the 5' end of the antisense strand and the sense strand 5' is between -1 and 0. After selection according to the above principles, the candidate siRNA target site is combined with the human gene sequence by BLAST analysis. Sequence homology alignment, excluding sequences with higher sequence homology (16 or more bases) than human gene sequences other than the tyrosine gene, to ensure that the candidate siRNA target site does not affect other human genes. Inhibition occurs, and only a specific inhibitory effect on the tyrosinase gene is observed.

 Finally, the 3' end of the obtained 19 bp nucleotide sequence was added with two deoxythymidine nucleotides as the sense strand of the siRNA sequence, and two deoxythymidines were added to the 3' end of the complementary sequence of the 19 bp nucleotide sequence. Nucleotides serve as the antisense strand of the siRNA sequence.

 The synthesis of the siRNA sequence can be carried out by chemical synthesis or by a biotechnology company specializing in nucleic acid synthesis, such as commissioning by Shanghai GenePharma.

 In general, the method of chemical synthesis includes the following four processes: (1) synthesis of oligoribonucleoside acid; (2) deprotection; (3) purification separation; (4) desalting.

 For example, the specific steps for chemical synthesis of siRNA having the nucleotide sequence shown by TYRO-1 are as follows:

 (1) Synthesis of oligoribonucleotides: 1 millimolar RNA was synthesized on an automated DNA/RNA synthesizer (for example, Applied Biosystems EXPEDITE 8909), and the coupling time per cycle was set to 10-15. Minutes, the starting material is a solid phase-linked 5'-0-p-dimethoxy-thymidine support, the first cycle is linked to a base on the solid support, and then at the nth (19 ≥ n ≥ 2) In the cycle, one base is ligated to the base to which the n-1th cycle is ligated, and this cycle is repeated until the synthesis of all nucleic acid sequences is completed.

 (2) Deprotection

 The solid support to which the siRNA was attached was added to the test tube, and 1 ml of ethanol/ethylamine (1:3 by volume) was added to the test tube, which was then sealed and placed in a 55-70 ° C incubator. After incubation for 2-30 hours, the solid support to which siRNA was attached was taken out and rinsed twice with double distilled water (1 ml each time), the eluate was collected, and dried at room temperature for 30 minutes. Then, 1 ml of a tetrabutylammonium fluoride solution in tetrahydrofuran (1 M) was added, and the mixture was allowed to stand at room temperature for 4 to 12 hours, and then 2 ml of ethanol was added thereto to collect a precipitate to obtain a crude product of siRNA.

 (3) purification separation

 The obtained crude product of siRNA was dissolved in 2 ml of an aqueous solution of ammonium acetate having a concentration of 1 mol/ml, and then separated by C18 high pressure liquid chromatography to obtain a purified siRNA product.

 (4) Desalting

The purified siRNA product was washed 2-4 times (2 ml each time) with a 75% by weight aqueous solution of ethanol, the salts were removed, and dried at room temperature. Then the oligoribonucleic acid of the sense strand and the antisense strand Mix and dissolve in 1-2 ml of buffer (10 mM Tris, pH = 7.5-8.0, 50 mM NaCl), heat the solution to 95 ° C, then slowly cool the solution to room temperature, and maintain room temperature 16-22 In hours, a solution containing siRNA was obtained.

 A cosmetic composition for inhibiting melanin production and depositing or removing melanin according to the present invention, which comprises the siRNA provided by the present invention as an active ingredient.

 According to the invention, the cosmetic composition further comprises a carrier.

 In the present invention, the content of the siRNA and the carrier may vary within a wide range. Preferably, the carrier is contained in an amount of from 1000 to 10,000,000 parts by weight relative to 100 parts by weight of the siRNA; further preferably, relative to 100 The siRNA is contained in an amount of from 2,000 to 100,000 parts by weight.

 In the present invention, the carrier is not particularly limited and may be any carrier for a cosmetic composition, and may be, for example, one or more of animal and vegetable oils, hydrocarbon oils, esters, higher fatty acids, and higher fatty alcohols; The animal and vegetable oil may be one or more of eucalyptus oil, eucalyptus oil, soybean oil, sesame oil, corn oil, rapeseed oil, cottonseed oil, olive oil and castor oil; the hydrocarbon oil may be paraffin oil, One or more of isotrimidine and petrolatum; the ester may be isopropyl palmitate, butyl stearate, hexyl laurate, isodecyl isononanoate, 2-ethylhexyl palm One or more of an acid ester, 2-hexyldecyl laurate, and 2-octyldodedodecyl myristate; the higher fatty acid may be palmitic acid, stearic acid, linoleic acid, and flax One or more of the acids; the higher fatty alcohol may be cetyl alcohol and/or octadecyl alcohol.

 According to the invention, the cosmetic composition further contains other ingredients for the cosmetic composition. In the present invention, the content of the siRNA and other ingredients for the cosmetic composition may vary within a wide range, and preferably, the content of the other ingredients for the cosmetic composition is relative to 100 parts by weight of the siRNA. It is preferably from 1000 to 10,000,000 parts by weight; more preferably, the content of the other component for the cosmetic composition is from 2,000 to 100,000 parts by weight with respect to 100 parts by weight of the siRNA.

 In the present invention, the other ingredients for the cosmetic composition are not particularly limited, and for example, may be one of a moisturizing agent, a coloring agent, a preservative, a thickener, an antioxidant, a surfactant, and an adjuvant or Several.

In the present invention, the humectant is not particularly limited, and may be various humectants for use in a cosmetic composition. For example, the humectant may be propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol, or sorbitol. , one of hexanediol, 1,3-butanediol, glycerin, seaweed extract and hyaluronic acid Kind or several.

 In the present invention, the coloring agent is not particularly limited, and may be various coloring agents for use in a cosmetic composition. For example, the coloring agent may be carmine, talc, mica, magnesium carbonate, calcium carbonate, silicic acid. One or more of magnesium, silica, zinc oxide, and ultramarine.

 In the present invention, the thickener is not particularly limited and may be various thickeners for use in a cosmetic composition. For example, the thickener may be beeswax, candelilla wax, cetyl wax, or microcrystals. One or more of wax, starch, xanthan gum, gum arabic, guar gum, pectin and bentonite.

 In the present invention, the preservative is not particularly limited, and may be various preservatives for use in a cosmetic composition. For example, the preservative may be benzoic acid, salicylic acid, methyl paraben, p-hydroxyl One or more of propyl benzoate, resorcinol and diimidazolium urea.

 In the present invention, the antioxidant is not particularly limited, and may be various antioxidants for use in a cosmetic composition. For example, the antioxidant may be ascorbic acid, vitamin bismuth, β-carotene, vitamin Β3, and cysteine. One or more of amino acid, glutathione, catalase, citric acid, and diphenol.

 In the present invention, the surfactant is not particularly limited and may be various surfactants for use in a cosmetic composition. For example, the surfactant may be a fatty acid glyceride, a polyoxyethylene hydrogenated castor oil, or a hydrazine. One or more of sodium benzenesulfonate, dinonyl sulfosuccinate, decanoylglutamic acid, and sodium polyoxyethylene sulfonate.

 The kind of the adjuvant is not particularly limited and may be various adjuvants which can be used in a cosmetic composition, for example, the adjuvant may be mannitol, glucose, albumin, arginine, azone and trehalose. One or several of them.

 The cosmetic composition may be in the form of various conventional cosmetic compositions, for example, an emulsion, a paste, a liquid, an aerosol or a powder. The cosmetic composition is an external preparation which can be applied to skins of various parts of the body such as the face, neck, arms, trunk (chest, abdomen and back), legs, hands and feet.

 The present invention also provides the use of an siRNA for the preparation of a cosmetic composition for inhibiting melanin production and deposition or for removing melanin, wherein the siRNA is an siRNA provided by the present invention.

The present invention will be further illustrated by the following examples, and the reagents and culture media used in the present invention are commercially available unless otherwise specified. Example 1

 Synthesis of siRNA

 A 19 bp nucleotide sequence was selected in the range of l-2082 bp of the tyrosinase gene cDNA sequence (Genbank Accession No. NM - 000372). The principle of selecting the 19 bp nucleotide sequence is: (1) the total siRNA double bond energy < 1; (2) the 5' end binding energy of the antisense strand 3-9; (3) the 5' end binding energy of the sense strand 5-9; (4) GC content between 36-53%; (5) The energy difference between the 5' end of the antisense strand and the sense strand 5' is between -1 and 0. After selection according to the above principles, the candidate siRNA target site is homologously aligned with the human gene sequence by BLAST analysis, and the sequence homology is excluded from other human gene sequences except the tyrosine gene (16). The sequence of more than one base) ensures that the candidate siRNA target site does not inhibit other human genes, but only has a specific inhibitory effect on the tyrosinase gene.

 Finally, the 3' end of the obtained 19 bp nucleotide sequence was added with two deoxythymidine nucleotides as the sense strand of the siRNA sequence, and two deoxythymidines were added to the 3' end of the complementary sequence of the 19 bp nucleotide sequence. Nucleotides serve as the antisense strand of the siRNA sequence.

 The designed siRNA was chemically synthesized by GenePharma, and the siRNAs TYRO-1 to TYRO-8 were obtained. The nucleotide sequences of the siRNAs TYRO-1 to TYRO-8 are shown in Table 1.

 Table 1

The range of attack refers to the corresponding position of the siRNA in the sequence No. 1 of the Sequence Listing. Comparative example 1

 A reference siRNA Cl-C3 having the nucleotide sequence shown in Table 2 was chemically synthesized by Shanghai GenePharma. Table 2

 The range of attack refers to the corresponding position of the reference siRNA in the sequence No. 1 of the sequence listing. Example 2

 Detection of siRNA against tyrosinase gene expression

 (1) Culture of melanocytes

Containing 10% FBS with density, 2mM L- glutamine, 100U / ml penicillin, lOO g / ml streptomycin, MEM complete medium in six-well cell culture plates at 5x l0 ⁶ cells / well SK-MEL-1 melanoma tumor cells (Beijing Baixing Hi-Tech Development Co., Ltd.) were inoculated, cultured in an incubator at 37 ° C and a C0 ₂ content of 5%, and fresh medium was replaced every 48 hours.

 (2) Transfection of siRNA

Using the Invitrogen Lipofectamine ^TM 2000 liposomes obtained in Example 1 siRNA TYRO- 1 to TYRO-8, respectively, were transfected siRNA as control without adding. The specific steps are as follows: The siRNA was dissolved in RNase-free sterile water to prepare a solution having a concentration of 20 mol/L. SK-MEL-1 melanoma cells were seeded into 24-well plates and diluted to a concentration of 8 χ 10 ⁵ cells/ml with 500 μl per well using OptiMEM I low serum medium (Invitrogen, 31985-062). 1.5μ1 siRNA (20μηιο1/Ε) was diluted in 50μ1 Opti-MEM I low serum medium (Invitrogen, 31985-062), and Ιμΐ LipofectamineTM 2000 liposome was diluted in 50μ1 Opti-MEM I low serum medium. (Invitrogen, 31985-062), and then the above two solutions were incubated at room temperature for 5 minutes and then mixed. After the mixed solution was allowed to stand at room temperature for 20 minutes, the mixed solution was added to the cells inoculated with the cells. In the orifice plate. siRNA The final concentration is 50 nM. The cells were cultured at 37 ° C for 4 hours, and then 1 ml of MEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin was added, and then at 37 ° C Cultivate for 24 hours.

 (3) Inhibition activity detection

 The expression of tyrosinase mRNA in SK-MEL-1 melanoma cells transfected with siRNA TYR0-1 to TYR0-8 was detected by RT-PCR. The specific steps were as follows:

The SK-MEL-1 melanoma cells transfected with siRNA were lysed with 1 ml of Trizol (GIBCOL), and total RNA was extracted. The specific steps for extracting total RNA were as follows: The transfected cells were at a temperature of 37 ° C and a C _{2 2} content. The cells were cultured for 24 hours in a 5% incubator, and then the cells were collected by centrifugation and washed once with pre-cooled 2 ml of PBS; the composition of the PBS was: NaCl 137 mmol/L, KC1 2.7 mmol/L, Na ₂ HP0 ₄ 4.3 mmol/ L, KH ₂ P0 ₄ 1.4mmol / L; Add 1ml Trizol per well, let stand for 5 minutes at room temperature, lyse the cells; transfer the lysate to 1.5ml EP tube; add 200μ1 chloroform, shake vigorously for 15 seconds by hand, place at room temperature 3 Minutes; 14000 rpm, centrifuge at 15 °C for 15 minutes; take liquid phase supernatant to about 500 μl and put in a new tube, add 500 μl of isopropanol, let stand for 10 minutes at room temperature; centrifuge at 10 °C for 10 minutes at 12000 rpm, remove the supernatant. The precipitate was washed once with 1 ml of 75% ethanol; centrifuged at 7600 rpm for 5 minutes at 4 ° C; the supernatant was removed, and the RNA was dried at room temperature for 10 minutes; 20 μl of ddH ₂ 0 was added to dissolve the RNA.

 Two units of DNase I (RNase-free) (TakaRa) were added to the above-mentioned RNA-dissolved DEPC water, and allowed to stand at 37 ° C for 30 minutes to remove residual DNA in the total RNA. After treatment with DNase I, the total RNA was purified by Invitrogen PureLink Micro-to-Midi Total RNA Purification Kit. The specific steps of purification were as follows: 20 μl of 70% ethanol was added to the total RNA, and the mixture was shaken evenly. The mixture was transferred to a purification column, centrifuged at 12,000 rpm for 15 seconds at room temperature, the filtrate was discarded, 700 μl of Wash Buffer I (TakaRa) was added, and the mixture was centrifuged at 12,000 rpm for 15 seconds at room temperature, and the filtrate was discarded, and 500 μl of Wash Buffer II (TakaRa) was added. Centrifuge at 12000 rpm for 15 seconds at room temperature, discard the filtrate, add 500 μl of Wash Buffer II (TakaRa), centrifuge at 12,000 rpm for 15 seconds at room temperature, discard the filtrate, centrifuge at 12000 rpm for 1 minute at room temperature, and transfer the purification column to the RNA collection tube. 30 μl of DEPC water was added, allowed to stand at room temperature for 1 minute, centrifuged at 13,000 rpm for 2 minutes at room temperature, and the RNA sample was stored at -80 ° C.

The reverse transcription reaction is carried out on the total RNA obtained after purification. In the reverse transcription reaction, the reverse transcriptase used is Promega's M-MLV reverse transcriptase. The specific steps of the reverse transcription reaction are: the total RNA after purification of lug and 0.5ug of Oligo dT is mixed in test tubes with DEPC water The total volume was made up to 16.25 μ1, and the tube was heated under heating conditions of 70 ° C for 5 minutes; then the tube was rapidly cooled to 0 ° C and buffer (5 x MLV buffer 5 ΐ, lOmMDntp 1.25 1, RNasin0.5 l, M-MLV 1μ1), incubated at 42 °C for 1 hour to obtain cDNA.

The obtained cDNA was used as a template for a PCR reaction, and a Real-time PCR reaction was carried out. The Real-time PCR reaction system is: ddH ₂ 017.5μ1, 10mM Dntp 0.5μ1, lO Taq buffer 2·5μ1, Taq 0.5 1, F primer 0.5 ΐ, R primer 0.5 1, Syber Green I 1μ1, οϋΝΑ2 1; PCR reaction The conditions were: 94 ° C for 2 minutes, 94 ° C for 15 seconds, and 60 ° C for 30 seconds for a total of 40 cycles. The inhibitory activity of siRNA was calculated according to the following formula, and the results are shown in Table 3.

 siRNA inhibitory activity = [1- ( copy number of tyrosinase gene after siRNA transfection / GAPDH copy number after siRNA transfection:) I (copy number of control tyrosinase gene / control well GAPDH copy number) )]χΐοο%.

 GAPDH: glyceraldehyde-3-phosphate dehydrogenase gene. The glyceraldehyde-3-phosphate dehydrogenase gene is a housekeeping gene in cells that is stably expressed in cells and is not affected by other additional factors, and thus serves as an internal reference for fluorescence quantitative PCR reactions. Comparative example 2

The inhibitory activity of siRNA against tyrosinase mRNA expression was examined by the method shown in Example 2, except that the siRNA used was the reference siRNACl-C3 obtained in Comparative Example 1, and the results are shown in Table 3.

从表 3可以看出， 本发明提供的 siRNA TYRO-1至 TYRO-8能够高效 的抑制酪氨酸酶基因的表达， 特别是 siRNA TYRO- 1、 TYRO-4和 TYRO-5 对酪氨酸酶基因的抑制活性达到 81 %、 82%和 87% , 而参比 siRNA C1-C3 对酪氨酸酶基因的抑制活性分别为 65 %、 58%和 52 % , 说明与现有的用于 抑制酪氨酸酶基因表达的 siRNA相比， 本发明提供的 siRNA用于抑制酪氨 酸酶基因的表达时， 对酪氨酸酶基因表达的抑制活性较高。 实施例 3

As can be seen from Table 3, the siRNAs TYRO-1 to TYRO-8 provided by the present invention can efficiently inhibit the expression of tyrosinase genes, particularly siRNAs TYRO-1, TYRO-4 and TYRO-5 to tyrosinase. The inhibitory activity of the gene reached 81%, 82% and 87%, while the inhibitory activity of the reference siRNA C1-C3 on the tyrosinase gene was 65%, 58% and 52%, respectively, indicating that it is used to inhibit cheese. When the siRNA provided by the present invention is used for inhibiting the expression of the tyrosinase gene, the siRNA for expressing the tyrosinase gene has a higher inhibitory activity against tyrosinase gene expression. Example 3

 Detection of tyrosinase protein inhibition by siRNA

 (1) Culture of melanocytes

Inoculation at a density of ⁵ x 106 cells/well in a six-well cell culture plate using MEM complete medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin SK-MEL-1 melanoma tumor cells (Beijing Baixing Hi-Tech Development Co., Ltd.) were cultured in an incubator at 37 ° C and a C0 ₂ content of 5%, and passaged and replaced with fresh medium every 48 hours.

 (2) Inhibition activity detection

The inhibitory activities of siRNA TYRO-1 to TYRO-8 on tyrosinase protein were detected, and SK-MEL-1 melanoma cell suspension (8× ^{10 5} cells/ml) was added to a 96-well culture plate at a volume of lOOul per well. siRNA was then added and the liposome Lipofectamine ^TM 2000 (Invitrogen) were transfected as a control siRNA without adding. The final concentration of siRNA was 50 nM. The cells were collected by centrifugation 72 hours after transfection and washed twice with PBS of pH 7.4. The composition of the PBS was NaCl 137 mmol/L, KC1 2.7 mmol/L, Na ₂ HP0 ₄ 4.3 mmol/L, KH ₂ P0 ₄ 1.4 Methyl/L; 50 L of 1% by weight of Triton X-100 solution per well, the composition of the Triton X-100 solution is 99 ml of water and 1 ml of Triton X-100; quickly placed at -80 ° C, frozen After 30 minutes; then the cells were completely ruptured by melting at room temperature. After pre-warming at 37 °C, L-dopa solution with a concentration of 2 mg/ml was added, and reacted at 37 ° C for 2 hours to measure the absorbance at 475 nm. As shown in Table 4.

Inhibitory activity of tyrosinase protein = [1 - [(well absorbance value after siRNA transfection - blank hole absorbance value) / (control hole absorbance value - blank well absorbance value)]] X 100 % Comparative example 3

 The siRNA inhibited the activity of the tyrosinase protein according to the method shown in Example 3, except that the siRNA used was the reference siRNA Cl-C3 obtained in Comparative Example 1, and the results are shown in Table 4.

 Table 4

 As can be seen from Table 4, the siRNAs TYRO-1 to TYRO-8 provided by the present invention are capable of efficiently inhibiting the activity of tyrosinase proteins, particularly tyrosinase by TYRO-1, TYRO-4 and TYRO-5. The inhibitory activities of the proteins were 46%, 42%, and 56%, respectively, while the inhibitory activities of the reference siRNAs C1-C3 on tyrosinase proteins were 21%, 18%, and 20%, respectively, indicating that the siRNA provided by the present invention is effective. Inhibition of tyrosinase activity. Example 4

 Inhibition of melanin content in melanocytes by siRNA

 (1) Culture of melanocytes

Inoculation at a density of ⁵ x 106 cells/well in a six-well cell culture plate using MEM complete medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin SK-MEL-1 melanoma tumor cells (Beijing Baixing Hi-Tech Development Co., Ltd.) were cultured in an incubator at 37 ° C and a C0 ₂ content of 5%, and passaged and replaced with fresh medium every 48 hours.

 (2) Melanin content detection

 The inhibitory effects of siRNA TYRO-1 to TYR0-8 on melanin content were examined separately. The specific steps are as follows:

Inoculate a 6-well culture plate with 1.6×10 ⁶ cells per well, and add siRNA to each well. LipofectamineTM 2000 liposomes (Invitrogen) were transfected with no siRNA added as a control. The final concentration of siRNA was 50 nM. The cells were collected by centrifugation 72 hours after transfection, and washed twice with 2 ml of PBS of pH 7.4. The composition of the PBS was: NaCl 137 mmol/L, KC1 2.7 mmol/L, Na ₂ HP0 ₄ 4.3 mmol/L. , KH ₂ P0 ₄ 1.4mmol / L; then add 500μΙ ^ concentration of 1% by weight of TritonX-100 solution per well, the composition of the TritonX-100 solution is 99ml water, 1ml TritonX-100; quickly placed at -80 ° In C, it was frozen for 30 minutes; then the cells were completely ruptured by melting at room temperature, centrifuged, and the precipitate was washed once with 10% by weight of trichloroacetic acid (2 ml) and absolute ethanol (2 ml), and centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded, 2N NaOH (containing 20% by weight of DMSO) was added to the precipitate, and the absorbance was measured at 475 nm after 2 hours in a water bath at 80 ° C, and the inhibition rate of melanin content was calculated. The results are shown in Table 5.

 Inhibition rate of melanin content = [1 - [(well absorbance value after siRNA transfection - blank hole absorbance value) / (control hole absorbance value - blank well absorbance value)]] χ ΐοο% Comparative Example 4

 The siRNA was used to inhibit the melanin content in SK-MEL-1 melanoma tumor cells according to the method described in Example 4, except that the siRNA used was the reference siRNA C1-C3 obtained in Comparative Example 1, and the results are shown in Table 5.

 table 5

As can be seen from Table 5, the siRNAs TYRO-1 to TYRO-8 provided by the present invention can significantly increase the inhibition rate of melanin content, particularly by inhibition of TYRO-1, TYRO-4 and TYRO-5, in melanocytes. The inhibition rate of melanin content reached more than 35%, and the inhibition rate of melanin content in melanocytes was 19%, 18% and 21% by the inhibition of reference siRNA C1-C3, respectively, indicating that the siRNA provided by the present invention can be remarkable. Increase the inhibition rate of melanin content. Example 5

 Whitening effect test of human skin

 In order to enhance the stability of siRNA, Shanghai Gemma Synthetic Chemically Modified siRNA TYRO-1 to siRNA TYRO-8 was entrusted, among which, chemically modified siRNA TYRO-1 to siRNA TYRO-8 all strands of U and C nucleotides The 2'-OH of the pentose sugar was subjected to 2'-fluoro modification, and the 2'-OH of all U and C nucleotide pentose sugars of the antisense strand was subjected to 2'-oxymethyl modification. Then, the components were mixed according to the composition shown in Table 6, and then uniformly stirred to obtain Compositions 1 to 8 to evaluate the clinical whitening effect of the composition containing the siRNA provided by the present invention as an active ingredient.

 Twenty healthy female volunteers between the ages of 18 and 55 were selected to induce skin blackening on the hips of volunteers using the GS2006 solar simulator (Beijing Aohua Equipment Co., Ltd.). The time to induce blackening was After 1 hour, a blackened spot with a diameter of 1.0 cm was formed, which was obviously blackened to the skin color of the unblackened skin. Thereafter, the compositions 1-8 were applied to the blackened skin, respectively, once in the morning and evening, each time being 0.5 ml. The change in the color of the blackened skin was observed on the last day of each month after administration, and the results are shown in Table 8. Table 6

对比例 5

Comparative example 5

 After the reference siRNA Cl-C3 was chemically modified according to the method described in Example 5, the whitening effect test of human skin was carried out in the same manner. The difference was that the components were mixed according to the composition shown in Table 7 below, and then stirred uniformly to obtain reference compositions D1-D3. The results are shown in Table 8.

 Table 7

表 8

Table 8

 Whitening effect evaluation criteria:

No effect: no change; Slight effect: The skin tone of the blackened skin is slightly whiter, but it is still slightly thinner than the skin color of the unblackened skin; Significant effect: The skin color of the blackened skin is obviously white and the skin color of the unblackened skin is basically the same. As can be seen from Table 8, the composition containing the siRNAs TYRO-1 to TYRO-8 provided by the present invention has a remarkable whitening effect on the blackened skin after 5 months of use, particularly by containing TYRO-K TYRO-4. The composition with TYRO-5 has a slight whitening effect on blackened skin after 1 month of use, and has a significant whitening effect on blackened skin after 3 months of use, and a reference containing reference siRNA Cl-C3. Compositions D1-D3 showed a slight whitening effect after four months of use, and there was no significant increase in whitening effect after six months of use, indicating that the whitening effect of the composition containing the siRNA provided by the present invention was more remarkable.

Claims

Claim A siRNA for inhibiting expression of a tyrosinase gene, the siRNA having TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 Or a nucleotide sequence as shown by TYRO-8, or having a nucleus as shown for TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8 a nucleotide sequence obtained by chemically modifying a nucleotide sequence, wherein  TYRO- -1 justice chain: 5, -GCAACUUCAUGGGAUUCAAdTdT-3,  Antisense chain: 5'- -UUGAAUCCCAUGAAGUUGCdTdT-3,;  TYRO- -2 justice chain: 5, -GCACAGAGAGACGACUCUUdTdT-3 '  Antisense strand: 5'- -AAGAGUCGUCUCUCUGUGCdTdT-3,;  TYRO- -3 justice chain: 5, -GGAGGAGUACAACAGCCAUdTdT-3 '  Antisense chain: 5'- -AUGGCUGUUGUACUCCUCCdTdT-3,;  TYRO- -4 justice chain: 5, -GCCAUCAGUCUUUAUGCAAdTdT-3 '  Antisense chain: 5'- -UUGCAUAAAGACUGAUGGCdTdT-3,;  TYRO- -5 justice chain: 5, -CCAUCAGUCUUUAUGCAAUdTdT-3 '  Antisense chain: 5'- -AUUGCAUAAAGACUGAUGGdTdT-3,;  TYRO- -6 justice chain: 5, -GCGUAAUCCUGGAAACCAUdTdT-3,  Antisense strand: 5'- -AUGGUUUCCAGGAUUACGCdTdT-3,;  TYRO- -7 justice chain: 5, -CCAGGUUCCCAGAGAAUAUdTdT-3 '  Antisense chain: 5'- -AUAUUCUCUGGGAACCUGGdTdT-3,;  TYRO--8 justice chain: 5, -GGUAAUGAGGAACUGUUAUdTdT-3, Antisense chain: 5'- -AUAACAGUUCCUCAUUACCdTdT-3 ' The siRNA according to claim 1, wherein the siRNA has a nucleotide sequence represented by TYRO-1, TYRO-4 or TYRO-5. The siRNA according to claim 2, wherein the siRNA has a nucleotide sequence represented by TYRO-5. 4. The siRNA of claim 1, wherein the chemical modification enhances the ability of the modified siRNA to resist nuclease hydrolysis in the cell. The siRNA according to claim 4, wherein the chemical modification is at least one of the following modifications:  (1) a modification of a phosphodiester bond of a linked nucleotide in a nucleotide sequence of the siRNA; (2) modification of the 2'-OH of ribose in the nucleotide sequence of the siRNA;  (3) Modification of a base in the nucleotide sequence of the siRNA. A cosmetic composition for inhibiting melanin production and depositing or removing melanin, characterized in that the cosmetic composition contains the siRNA according to any one of claims 1 to 4 as an active ingredient. The cosmetic composition according to claim 6, wherein the cosmetic composition further contains a carrier selected from one or more of an automatic vegetable oil, a hydrocarbon oil, an ester, a higher fatty acid, and a higher fatty alcohol. The cosmetic composition according to claim 7, wherein the carrier is contained in an amount of from 1,000 to 10,000,000 parts by weight based on 100 parts by weight of the siRNA. 9. The cosmetic composition according to claim 7, wherein the cosmetic composition further comprises other ingredients for the cosmetic composition, the other ingredients for the cosmetic composition being selected from the group consisting of humectants, colorants, and antiseptics. One or more of a dose, a thickener, an antioxidant, a surfactant, and an adjuvant. The cosmetic composition according to claim 9, wherein the other component for the cosmetic composition is contained in an amount of from 1,000 to 10,000,000 parts by weight based on 100 parts by weight of the siRNA. The cosmetic composition according to claim 6, wherein the cosmetic composition is an emulsion, a paste, a liquid, an aerosol or a powder. Use of the siRNA according to any one of claims 1 to 5 for the preparation of a cosmetic composition for inhibiting or eliminating melanin production and deposition.