Antisense oligonucleotide for inhibiting tyrosinase expression and application thereof
阅读说明：本技术 抑制酪氨酸酶表达的反义寡核苷酸及其应用 (Antisense oligonucleotide for inhibiting tyrosinase expression and application thereof ) 是由 费嘉 苏睿 于 2021-08-06 设计创作，主要内容包括：本发明公开一种抑制酪氨酸酶表达的反义寡核苷酸及其应用,属于生物医药领域。本发明利用RNA Structure v6.2设计了靶向人类酪氨酸酶(TYR)的反义寡核苷酸,筛选出能有效抑制TYR表达进而降低黑色素产生的反义寡核苷酸TYR-2、TYR-5。该反义寡核苷酸具有较高的递送效率和特异性靶向性；可以高效靶向人酪氨酸酶基因、进而降低黑色素合成,淡化色斑,避免了化学美白产品带来的有害化学物质损伤细胞、重金属沉积等副作用。为美白化妆品的研发提供了有效数据。(The invention discloses an antisense oligonucleotide for inhibiting tyrosinase expression and application thereof, belonging to the field of biological medicines. The invention designs antisense oligonucleotides targeting human Tyrosinase (TYR) by utilizing RNA Structure v6.2, and screens the antisense oligonucleotides TYR-2 and TYR-5 which can effectively inhibit the expression of TYR and further reduce the generation of melanin. The antisense oligonucleotide has higher delivery efficiency and specific targeting; can efficiently target human tyrosinase gene, further reduce melanin synthesis, lighten color spots, and avoid the side effects of harmful chemical substances damaging cells, heavy metal deposition and the like brought by chemical whitening products. Provides effective data for the research and development of whitening cosmetics.)
1. An antisense oligonucleotide that inhibits tyrosinase expression, comprising: the antisense oligonucleotide is shown as TYR-2 or TYR-5:
TYR-2：5′-AGGTCAGGCTTTTTGGCCCT-3′；
TYR-5：5′-TGGCTGCTTTTCTTCAGGAA-3′。
2. an antisense oligonucleotide that inhibits tyrosinase expression according to claim 1, characterized in that:
the antisense oligonucleotide for inhibiting tyrosinase expression is modified by thiophosphorylation.
3. An antisense oligonucleotide for inhibiting tyrosinase expression according to claim 2, characterized in that:
the antisense oligonucleotide for inhibiting tyrosinase expression is subjected to phosphorothioate modification at the tail of the 5 'end by more than 3 continuous basic groups and at the tail of the 3' end by more than 3 continuous basic groups.
4. Use of an antisense oligonucleotide according to any one of claims 1 to 3 for inhibiting the expression of tyrosinase in the preparation of a cosmetic composition for inhibiting or removing the formation and deposition of melanin.
5. Use of an antisense oligonucleotide according to any one of claims 1 to 3 for inhibiting tyrosinase expression in the manufacture of a medicament for the treatment of melanin-related disorders.
6. Use according to claim 5, characterized in that: directly administering to the pigmented plaque in the subject.
7. A composition characterized by: the composition contains the antisense oligonucleotide for inhibiting tyrosinase expression according to any one of claims 1-3.
8. The composition of claim 7, wherein:
the composition is a pharmaceutical composition or a cosmetic composition.
9. The composition of claim 8, wherein:
the pharmaceutical composition further comprises any pharmaceutically acceptable auxiliary;
the cosmetic composition also contains conventional cosmetic ingredients.
10. The composition of claim 9, wherein:
the cosmetic composition is emulsion, ointment, cream, aqua, aerosol or powder.
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to antisense oligonucleotide for inhibiting tyrosinase expression and application thereof.
Background
With the improvement of living standard, the requirement of consumers on whitening products is increasing, and the safety and whitening effect of the products are highly concerned by the consumers. This also puts higher demands on the development of whitening agents, and therefore, it is important to select ingredients that are highly effective and safe in enhancing the whitening effect.
Melanin is a biological pigment synthesized and secreted by melanocytes in the human body. Melanin can absorb and dissipate 99.9% of ultraviolet[1]And the body is protected from being damaged by ultraviolet rays. The mechanism of melanin production is: tyrosine gradually forms intermediate products of dopa, dopaquinone and dopachrome under the oxidative catalysis of tyrosinase, finally forms skin melanin, and causes the skin to turn black[2]. Tyrosinase has a critical role in this synthetic pathway. Therefore, the synthesis of melanin can be reduced by inhibiting the expression of tyrosinase gene.
Antisense oligonucleotides (ASOs) are short-chain DNA or RNA molecules that bind to a target RNA by the base complementary pairing principle and inhibit its function or induce its degradation. Wherein the antisense oligonucleotide fragment length typically consists of ten to twenty-several bases. To overcome the degradation problem of nucleases, antisense oligonucleotides are subjected to a series of chemical modifications, such as: phosphorothioate modifications[3]2' -O-alkyl modification[4]And the like. Based on the chemical modification of existing antisense nucleic acids, in order to improve the biological effect and stability, researchers have synthesized a chimeric antisense nucleic acid called "gap-mer" strategy[5]. In addition, based on the knowledge of the mechanism of ASOs uptake by cells, a large number of delivery methods have been studied mainly in terms of increasing the amount of ASOs taken by cells, changing the manner in which ASOs are taken by cells, enhancing the release of ASOs from endosomes, and the like, in order to address the problems in ASOs delivery. For example: cationic liposome[6]Polymeric nanoparticles[7]And GalNAC technology[8]And the like.
In the existing literature reports, the common whitening components for inhibiting the tyrosinase activity are phenols and acids. But the use of the whitening product is limited due to the complex extraction process, the unobvious whitening effect, the cytotoxicity and other adverse factors. For inhibiting melanin synthesis, researchers have selected from the list of genes involved in inhibiting melanin formationThe angle reached starts. Development of small RNA whitening products against Tyrosinase (TYR), tyrosine-related protein (TYRP) and microphthalmia-related transcription factor (Mitf)[9]. With the update of antisense oligonucleotide design software and the improvement of synthesis process, the specificity is increased and the toxic side effect is reduced. Therefore, it is very necessary to develop whitening products based on antisense oligonucleotides.
Reference to the literature
[1]MEREDITH P.RIESZJ.Radiative relaxation quantum yields for syntheticeumelanin[J].Photochemistry&Photobiology,2010,79(2):211-216.
[2] Wuchang machine, Fangyihong, Zhang Lianping, research progress of plant active whitening ingredients [ J ] science and technology Square, 2021,44(4):49-52.
[3]MilliganJF,MatteucciMD,MartinJC.Current concepts in anti-sense drug design[J].Med Chem,1993,36(14):1923.
[4]LesnikEA,GuinossoCJ,KawasakiAM,etal.Oligonucleotides containing 2′-O-modified adenosine:synthesis and effects on stability of DNA:RNAduplexes[J].Biochem,1993,32:7832.
[5]Altmann KH,DeanNM,FabbroD,etal.Second generation of antisense oligonucleotides:from nuclease resistance to biological eff-macy in animais[J].Chimia,1996,50:171.
[6]Tari A M.Preparation and application of liposome-incorporated oligodeoxynucleotides.MethodsEnzymol,2000,313:372-388.
[7]Chavany C,Le Doan T,CouvreurP,et al.Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonculeotides[J].Pharm Res,1992,9(4):441-449.
[8]Rosie Z.Yu,Rudy Gunawan,Noah Post,et al.Disposition and Pharmacokineticsof a GalNAc3-Conjugated Antisense OligonucleotideTargeting Human Lipoprotein(a)in Monkeys[J].Nucleic Acid Therapeutics.2016,26(6):372-380.
[9] A whitening and spot-removing cream of small nucleic acid for yellow soldier and Yinliwei is prepared from CN 201210369781.2P 2012.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the present invention aims to provide an antisense oligonucleotide for inhibiting tyrosinase expression.
Another objective of the invention is to provide an application of the antisense oligonucleotide for inhibiting tyrosinase expression.
The purpose of the invention is realized by the following technical scheme:
an antisense oligonucleotide that inhibits tyrosinase expression, as represented by TYR-2 or TYR-5:
TYR-2：5′-AGGTCAGGCTTTTTGGCCCT-3′；
TYR-5：5′-TGGCTGCTTTTCTTCAGGAA-3′；
the antisense oligonucleotide for inhibiting tyrosinase expression is modified by phosphorothioation, and particularly, the modification of phosphorothioation is carried out on more than 3 continuous basic groups at the tail of a 5 'end and more than 3 continuous basic groups at the tail of a 3' end.
The application of the antisense oligonucleotide for inhibiting the expression of tyrosinase in preparing a cosmetic composition for inhibiting or removing the generation and deposition of melanin;
the antisense oligonucleotide for inhibiting the expression of tyrosinase is applied to the preparation of medicines for treating melanin related diseases. Such antisense oligonucleotides can be administered as a drug to a specific site, such as a pigmented plaque, in a subject, for example. Optionally, any pharmaceutically acceptable adjuvant may be included in the above pharmaceutical dosage form as long as it is suitable for the corresponding administration system and properly maintains the activity of the antisense oligonucleotide.
A composition comprising the above antisense oligonucleotide for inhibiting tyrosinase expression;
further, the composition is a pharmaceutical composition or a cosmetic composition; the pharmaceutical composition further comprises any pharmaceutically acceptable auxiliary; the cosmetic composition also contains common cosmetic ingredients;
the cosmetic composition can be emulsion, ointment, cream, aqua, aerosol or powder.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention designs antisense oligonucleotides targeting human Tyrosinase (TYR) by utilizing RNA Structure v6.2, and screens the antisense oligonucleotides TYR-2 and TYR-5 which can effectively inhibit the expression of TYR and further reduce the generation of melanin. Provides effective data for the research and development of whitening cosmetics.
(2) The antisense oligonucleotide has higher delivery efficiency and specific targeting property; can efficiently target a human Tyrosinase (TYR) gene, further reduce melanin synthesis, lighten color spots and avoid the side effects of harmful chemical substances damaging cells, heavy metal deposition and the like brought by chemical whitening products.
Drawings
FIG. 1 shows the sequence of TYR-2 designed by RNA structure v6.2 software.
FIG. 2 is the RNA structure v6.2 software design TYR-5 sequence.
FIG. 3 shows the change in the expression level of TYR mRNA in cells after transfection of different antisense oligonucleotides by qPCR (P <0.01, P <0.001, expressed as mean. + -. standard deviation; n ═ 3).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Example 1
1 materials and methods
1.1 cells and reagents
Human a375 malignant melanoma cells were purchased and stored by the laboratory.
DMEM medium, fetal bovine serum, Opti-MEM medium were purchased from GIBCO, USA; lipofectamine2000 was purchased from Thermo Fisher/Sammer Feishel technologies; trizol was purchased from Invitrogen (USA); 2xSYBR Green qPCR Master Mix was purchased from Biosharp.
1.2 primer Synthesis
The human tyrosinase TYR real-time fluorescent quantitative PCR primer and the GADPH fluorescent quantitative primer are synthesized by Biotechnology engineering (Shanghai) GmbH, and the sequences are shown in Table 1.
TABLE 1 fluorescent quantitative PCR primers
Primer name
Sequence (5 '-3')
GAPDH-Forward
CAATGACCCCTTCATTGACC
GAPDH-Reverse
GACAAGCTTCCCGTTCTCAG
TYR-Forward
TGCACAGAGAGACGACTCTTG
TYR-Reverse
GAGCTGATGGTATGCTTTGCTAA
Synthetic chemical modification of antisense oligonucleotides: synthesis of antisense oligonucleotide and modification of phosphorothioate sequence the antisense oligonucleotide sequence was completed by Biotechnology engineering (Shanghai) Ltd. The sequences are shown in Table 2:
TABLE 2 antisense oligonucleotide sequences
Sequence name
Sequence (5 '-3')
Total free energy (overhall Δ G)37)
TYR-1
TGTAGGATTCCCGGTTATGT
-21.4
TYR-2
AGGTCAGGCTTTTTGGCCCT
-21.1
TYR-3
CTGGATTTCTTGTTCCCACC
-20.9
TYR-4
ACCAAATAGCATCCTTTCCT
-19.0
TYR-5
TGGCTGCTTTTCTTCAGGAA
-19.1
SCR
CATTAATGTCGGACAACTCAAT
0
2 method of experiment
2.1 design and Synthesis of antisense oligonucleotides
Antisense oligonucleosides for designing highly effective human tyrosinase gene targeting and inhibiting its functionAcid, we obtained the full-length sequence of the human tyrosinase TYR mRNA from NCBI (national Center for Biotechnology information). And (3) predicting a secondary structure of the TYR mRNA by using RNA structure v6.2 software, and then designing an antisense oligonucleotide fragment which is targeted and combined with the TYR mRNA by using an oligowalk module on the basis of obtaining the secondary structure. 5 free energy data of antisense oligonucleotide binding to TYR mRNA are shown at the oligowalk interface, and the interface automatically shows new data for every base shift, where the total free energy (Overall. DELTA.G)37) Is a comprehensive index obtained by synthesizing other 4 free energy indexes and is also a basis for screening antisense drugs. The antisense oligonucleotide sequences (see table 2) were designed with the full length of the TYR mRNA sequence as the target, and phosphorothioate backbone modifications were performed on 3 bases consecutive to the end of the 5 'end and 3 bases consecutive to the end of the 3' end of each sequence.
The random oligonucleotide sequence, denoted as SCR, was also modified with a phosphorothioate backbone with 3 bases at the 5 'end and 3 bases at the 3' end.
2.2 subculture of malignant melanoma cells of A375
Culturing human A375 malignant melanoma cells (commercially available) in DMEM medium containing 10% fetal calf serum and 1% double antibody, and placing at 37 deg.C and 5% CO2Culturing in an incubator under saturated humidity. And selecting cells in logarithmic growth phase for experiment, and selecting cells in logarithmic growth phase for experiment.
When cells were passaged, the original medium was discarded and washed twice with PBS buffer. Adding 1mL of 0.25% pancreatin-EDTA, placing the culture bottle in an incubator for digestion for about 2min, and adding a complete culture medium into the bottle to stop digestion when the cell morphology is observed to be rounded under a microscope, so as to obtain a cell suspension for bottle-splitting passage.
2.3A375 cell transfection and grouping
A375 cells were divided into SCR and ASO groups. The SCR group was cells transfected with phosphorothioate-modified random oligonucleotide sequences (random oligonucleotides SCR, see Table 2), and the ASO group was transfected with five phosphorothioate-modified predicted antisense oligonucleotides, respectivelyCells of the sequence (antisense oligonucleotides TYR-1-5, see Table 2). The final concentration of the SCR and ASO groups was set at 0.5. mu. mol/L. By using cationic liposome LipofectamineTM2000 transfection, the transfection procedure was as follows:
(1) taking cells in logarithmic growth phase, and adjusting the concentration of the cell suspension to be 3x10 by using DMEM medium containing serum and antibiotics5Per mL, 1mL per well, was seeded in 6-well plates. The plates were incubated at 37 ℃ in 5% CO2Carrying out transfection when the cells grow to 60-70%;
(2) prior to transfection, the 6-well plate was aspirated of the original medium. Adding 1.5mL of DMEM medium without serum and double antibodies for starvation treatment;
(3) preparation of antisense oligonucleotide LipofectamineTM2000 and mixing the solution.
a. Dilution of transfection reagent LipofectamineTM2000; before use, Lipofectamine is addedTM2000 transfection reagents were shaken gently, then 5. mu.L were taken, diluted with 250. mu.L serum free optimized medium (Opti-MEM), mixed gently, and incubated for 5min at room temperature;
b. dilution of antisense oligonucleotides: mu.L of antisense oligonucleotide was diluted with 250. mu.L of serum-free optimized medium (Opti-MEM);
c. diluted LipofectamineTM2000, after incubation for 5min, gently mixing the antisense oligonucleotide with the diluted antisense oligonucleotide particles obtained in the step b, and incubating for 20min at room temperature;
(4) adding the mixed solution into a cell culture plate containing cells and a culture medium, and slightly shaking to mix the mixed solution;
(5) after culturing for 6h at 37 ℃, removing the culture medium containing the liposome mixed solution in the holes, and replacing the culture medium with a complete culture medium (containing serum and double antibodies);
(6) the plates were incubated at 37 ℃ in 5% CO2And culturing in an incubator for 48h, and then extracting RNA.
2.4Real-time PCR detection of ability of antisense oligonucleotide to degrade TYR
In order to detect the degradation capability of different antisense oligonucleotides to target genes TYR after the antisense oligonucleotides are transfected into A375 cells, a Real-time PCR method is used for detecting the relative expression quantity of TYR mRNA in the cells transfected with different groups of antisense oligonucleotides modified by phosphorothioation so as to find ASO with the best action effect, and the specific experimental method is as follows:
2.4.1 extraction of Total RNA from cells
1) The experimental groups were as follows: SCR (random group) and ASO groups, 3 multiple wells per group;
2) collecting cells, centrifuging at 1500rpm for 5min, and removing supernatant;
3) adding 1mL of PBS into each group, transferring the PBS into a 1.5mL of EP tube, 1500rpm for 5min, and washing once;
4) adding 1mL of Trizol into each component, fully and uniformly mixing, and standing for 5min on ice;
5) adding 200 μ L chloroform into each component, swirling for 15s, and standing on ice for 15 min;
6) centrifuging at 12000rpm at 4 deg.C for 10 min;
7) transfer the supernatant (about 400. mu.L) to a new EP tube, add an equal volume of isopropanol, and stand at-20 ℃ for 10 min;
8) centrifuging at 12000rpm at 4 deg.C for 10 min;
9) removing supernatant, adding 75% ethanol to wash RNA precipitate, washing at 4 deg.C and 12000rpm repeatedly for three times;
10) removing the supernatant, placing the extracted total RNA on an ultra-clean workbench, and naturally drying in the air;
11) adding appropriate amount of DEPC water, and storing at-80 deg.C.
2.4.2 reverse transcription of cDNA and qPCR detection of the relative expression level of TYR-mRNA
a. Reverse transcription of cDNA
1) Determining the concentration of the extracted RNA by using a NanoDrop 2000 trace nucleic acid quantifier;
2) according to the specification of the reverse transcription reagent and the quantitative result of the RNA concentration, 1 mu g of RNA, the reverse transcription reagent and enzyme-free water are added into a 10 mu L reverse transcription system in proportion to a 200 mu L EP tube;
3) after being uniformly mixed, the mixture is subjected to instantaneous centrifugation, placed in a PCR instrument, and reverse transcription is completed according to the following procedures; storing at 42 deg.C for 15min, 82 deg.C for 2min, and 4 deg.C.
qPCR detection of TYR-mRNA relative expression
1) According to the specification of 2x SYBR Green qPCR Master Mix reagent, the qPCR reaction solution is prepared according to the following components (the preparation of the reaction solution needs to be carried out on ice)
Principal Components
Dosage per reaction (μ L)
2xSYBR Green qPCR Master Mix
10
cDNA
1
Upstream primer (10. mu.M)
1
Downstream primer (10. mu.M)
1
Deionized water
Supply to 20
Total reaction system
20
Setting three multiple holes, mixing the above components, adding into eight-connecting tube, and performing instantaneous centrifugation before detection.
2) Amplification program setup
The two-step PCR amplification conditions were as follows: stage 1: pre-denaturation for 1 cycle at 95 ℃ for 30 seconds; stage 2: qPCR reaction 40 cycles of 95 ℃ for 15 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds; stage 3: dissolution curves 1 cycle 95 ℃ for 15 seconds, 60 ℃ for 60 seconds, 95 ℃ for 15 seconds.
3) Analysis of results
GAPDH is used as internal reference, and the Ct values of the target gene and the internal reference are 2-△△CtThe relative level change of the gene of interest was calculated.
2.5 determination of melanin content
1) A375 malignant melanoma cells were selected at 3X10 in log phase of growth5The number per well was plated in 6 well plates.
2) The next day, when the cell fusion degree reaches 60-70%, using LipofectamineTM2000 transfection of phosphorothioate modified antisense oligonucleotides into A375 cells, and 72h incubation in cell culture incubator.
3) Washed twice with PBS buffer, 1mL of 0.25% trypsin was added to each well, digested in an incubator for about 2min, and digested by addition of 1mL of complete medium.
4) After collecting the cell suspension, centrifuging for 5min at 1000r/min, and discarding the supernatant. 1mL of PBS buffer was added to each well, the cells were blown off, and the plates were counted.
5) Centrifuging at 1000r/min for 5min, removing supernatant, air drying the sample every 10 times6The cells were dissolved in 500mL of a 1mol/L NaOH solution containing 1% DMSO.
6) After heating at 80 ℃ for 1h, cooling and reading the absorbance value A on a spectrophotometer by selecting the wavelength of 475 nm. Relative melanin content inhibition ═ random absorbance value-experimental absorbance value)/random absorbance value%.
2.6 statistical treatment
Data are all expressed as mean ± SD of at least three biological replicates and were statistically analyzed using GraphPad Prism software version 8.0 (sysstat software, san jose, CA, USA). Unpaired t-test (Student's two-tailed unpaired t test) was used to analyze the significance of differences between groups of data, and p-values <0.05 were considered statistically significant.
3 results of the experiment
3.1 design of antisense oligonucleotides
The key to antisense oligonucleotide design is the accurate simulation of the secondary structure of the target sequence and the coverage of different binding sites of the designed antisense oligonucleotide on the target sequence. The entire sequence of human tyrosinase TYR mRNA (NM-000372.5) was selected for a total of 2062 bases. Secondary structure prediction was performed on this sequence using RNA structure v 6.2.
All possible sites in this secondary structure to which antisense oligonucleotides bind were subsequently analyzed by Oligowalk function, and part of the design results are shown in FIGS. 1 and 2. At the interface of the figure, for every base position moved, the program will take the position as the starting point, calculate antisense oligonucleotide fragment Gibbs free energy and annealing temperature parameters. Binding of a rationally designed antisense oligonucleotide to a target sequence will tend to result in lower system free energy under thermodynamic equilibrium conditions, by the principle of minimum Gibbs free energy. Therefore, we selected antisense oligonucleotides with relatively low gibbs free energy as predicted sequences and verified them experimentally. Through screening, a total of 5 different potential functional sequences were designed. A total of 5 TYR antisense oligonucleotide sequences were designed, and the numbers were assigned from TYR-1 to TYR-5, respectively. The TYR targets corresponding to the 5 antisense oligonucleotide sequences are located in exons exon4, exon5, exon1, exon1 and exon5 respectively.
3.2 detection of the ability of different antisense oligonucleotides to degrade TYR mRNA
To verify whether the antisense oligonucleotides we designed inhibited the expression of TYR mRNA, different sets of phosphorothioate modified antisense oligonucleotides (0.5 μ M) were transfected into a375 malignant melanoma cells using lipo2000 and total RNA was extracted from the cells after 48h of culture. And detecting the relative expression quantity of TYR mRNA in different groups of cells by adopting real-time fluorescent quantitative PCR. The results are shown in FIG. 3, where the antisense oligonucleotides TYR-1, TYR-2 and TYR-5 inhibited TYR mRNA expression to different degrees. Wherein, the inhibition effects of TYR-2(P <0.001) and TYR-5(P <0.001) are good and are very significant differences, and the inhibition effect of TYR-1(P <0.01) is inferior and is significant difference. Because TYR-2 and TYR-5 have obvious TYR inhibition expression, they are screened out as effective sequences and are the subsequent experimental study objects. The sequences of TYR-2 and TYR-5 are shown in Table 2.
3.3NaOH cracking method for detecting melanin content
To further demonstrate the inhibition of tyrosinase gene expression by the above-mentioned effective sequences, we determined the melanin content in a375 cells by NaOH lysis. As shown in Table 3, the melanin content of the group transfected with the phosphorothioate-modified antisense oligonucleotides TYR-2 (0.5. mu.M) and TYR-5 (0.5. mu.M) was significantly reduced compared to the random group (0.5. mu.M), and the percentage of reduction was 14% (P <0.05) and 15% (P <0.05), respectively, which were statistically different. Therefore, the antisense oligonucleotides TYR-2 and TYR-5 both had the effect of inhibiting the expression of TYR mRNA and thus reducing melanin synthesis.
TABLE 3 determination of melanin content in A375 cells by NaOH lysis
Grouping
A375
Percentage of Down Regulation
Blank control
0.212±0.003
SCR
0.205±0.007
0
TYR-2
0.177±0.004*
14％
TYR-5
0.175±0.005*
15％
Note: p <0.05 compared to SCR group; blank control refers to the a375 cell group that was not transfected with oligonucleotide.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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