NUCLEIC ACID MOLECULE TARGETING MITF GENE AND USE THEREOF

Abstract

Provided is a double-stranded nucleic acid molecule, in particular to a small activating nucleic acid molecule, wherein the small activating nucleic acid molecule may be a nucleic acid molecule targeting MITF gene and at least comprises a first oligonucleotide strand. Further provided are compositions or formulations comprising the small activating nucleic acid molecule and uses thereof. The small activating nucleic acid molecule, or the composition or formulation comprising the small activating nucleic acid molecule, can be used to activate or upregulate the MITF gene expression in a cell, and treat a disease or condition related to insufficient or decreased MITF protein expression.

Claims

1. A small activating nucleic acid molecule at least comprising a first oligonucleotide strand having at least 75% homology or complementarity to any consecutive fragment of 16 to 35 nucleotides in length in a promoter region of human MITF gene.

2. The small activating nucleic acid molecule according to claim 1, wherein the small activating nucleic acid molecule comprises a first oligonucleotide strand and a second oligonucleotide strand that form a double-stranded structure by complete complementarity or incomplete complementarity.

3. The small activating nucleic acid molecule according to claim 2, wherein the first oligonucleotide strand has at least 75% homology or complementarity to any consecutive fragment of 16 to 35 nucleotides in length in SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304 or SEQ ID NO: 305.

4. The small activating nucleic acid molecule according to claim 2, wherein the first oligonucleotide strand has at least 75% homology or complementarity to any nucleotide sequence selected from SEQ ID NOs: 8-104.

5. The small activating nucleic acid molecule according to claim 4, wherein the first oligonucleotide strand comprises any nucleotide sequence selected from SEQ ID NOs: 105-201, or the second oligonucleotide strand comprises any nucleotide sequence selected from SEQ ID NOs: 202-298.

6. The small activating nucleic acid molecule according to any one of claims 2-5, wherein the first oligonucleotide strand has of 16 to 35 nucleotides in length, and the second oligonucleotide strand has of 16 to 35 nucleotides in length.

7. The small activating nucleic acid molecule according to any one of claims 2-6, wherein the small activating nucleic acid molecule is a double-stranded nucleic acid, and the first oligonucleotide strand and the second oligonucleotide strand are located on two strands of the double-stranded nucleic acid.

8. The small activating nucleic acid molecule according to claim 7, wherein the first oligonucleotide strand and/or the second oligonucleotide strand has overhangs at 5′ terminus and/or 3′ terminus.

9. The small activating nucleic acid molecule according to claim 8, wherein the overhang is an overhang of 0 to 6 nucleotides in length.

10. The small activating nucleic acid molecule according to claim 9, wherein the overhang is dTdT or dTdTdT.

11. The small activating nucleic acid molecule according to any one of claims 2-6, wherein the small activating nucleic acid molecule is a single-stranded nucleic acid having a hairpin structure that can form a double-stranded region, or the first oligonucleotide strand and the second oligonucleotide strand have a complementary region that can form a double-stranded structure.

12. The small activating nucleic acid molecule according to any one of claims 2-11, wherein the first oligonucleotide strand and the second oligonucleotide strand have at least 75% complementarity.

13. The small activating nucleic acid molecule according to any one of claims 1-12, wherein the nucleotide constituting the small activating nucleic acid molecule is a natural and non-chemically modified nucleotide.

14. The small activating nucleic acid molecule according to any one of claims 1-12, wherein one or more nucleotides of the small activating nucleic acid molecule are nucleotides having a chemical modification.

15. The small activating nucleic acid molecule according to claim 14, wherein the chemical modification is one or more selected from the following modifications: modification of phosphodiester bonds of nucleotides, modification of 2′-OH of ribose in nucleotides and modification of base in nucleotides.

16. The small activating nucleic acid molecule according to claim 14, wherein the chemical modification is one or more selected from the following modifications: thiophosphate modification, boranophosphate modification, 2′-fluoro modification, 2′-oxymethyl modification, 2′-oxyethylidene methoxy modification, 2,4′-dinitrophenol modification, locked nucleic acid modification, 2′-amino modification, 2′-deoxy modification, 5′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification and 2,6-diaminopurine modification.

17. The small activating nucleic acid molecule according to any one of claims 14-16, wherein the terminus of the first oligonucleotide strand and/or the second oligonucleotide strand is linked to a lipophilic group, and the lipophilic group is one or more selected from a liposome, a macromolecular polymer, a polypeptide and cholesterol.

18. A nucleic acid molecule comprising a fragment encoding the small activating nucleic acid molecule according to any one of claims 1-13.

19. The nucleic acid molecule according to claim 18, wherein the nucleic acid molecule is an expression vector.

20. A cell comprising the small activating nucleic acid molecule according to any one of claims 1-17 or the nucleic acid molecule according to any one of claims 18-19.

21. A pharmaceutical composition comprising: the small activating nucleic acid molecule according to any one of claims 1-17 or the nucleic acid molecule according to any one of claims 18-19, and optionally, a pharmaceutically acceptable carrier.

22. A formulation comprising the small activating nucleic acid molecule according to any one of claims 1-17, or the nucleic acid molecule according to any one of claims 18-19, or the cell according to claim 20, or the pharmaceutical composition according to claim 21.

23. A kit comprising the small activating nucleic acid molecule according to any one of claims 1-17, or the nucleic acid molecule according to any one of claims 18-19, or the cell according to claim 20, or the pharmaceutical composition according to claim 21.

24. Use of the small activating nucleic acid molecule according to any one of claims 1-17, or the nucleic acid molecule according to any one of claims 18-19, or the cell according to claim 20, or the pharmaceutical composition according to claim 21 to prepare a drug or formulation for activating/upregulating the MITF gene expression in a cell.

25. A method for activating/upregulating the MITF gene expression in a cell, wherein the method comprises administering the small activating nucleic acid molecule according to any one of claims 1-17, or the nucleic acid molecule according to any one of claims 18-19, or the composition according to claim 21, or the formulation according to claim 22 to the cell.

26. A method for treating a disease or condition related to insufficient or decreased MITF protein expression, wherein the method comprises administering to an individual in need thereof the small activating nucleic acid molecule according to any one of claims 1-17, or the nucleic acid molecule according to any one of claims 18-19, or the composition according to claim 21, or the formulation according to claim 22.

27. Use of the small activating nucleic acid molecule according to any one of claims 1-17, or the nucleic acid molecule according to any one of claims 18-19, or the cell according to claim 20, or the pharmaceutical composition according to claim 21 to prepare a drug for treating a disease or condition related to insufficient or decreased MITF protein expression.

28. The use according to claim 27, wherein the disease or condition related to insufficient or decreased MITF protein expression is vitiligo, or Waardenburg syndrome type 2A or Tietze syndrome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 shows changes in the human MITF mRNA expression mediated by saRNA. 239 saRNAs targeting the human MITF promoter were transfected into HEM cells at a final concentration of 25 nM. 72 h after the transfection, the MITF mRNA expression was analyzed by one-step RT-qPCR. Shown in the drawing is the descending rank of changes in the MITF mRNA expression (log 2) relative to control treatment (Mock). The ordinate values represent the mean±SD of 2 replicates.

[0053] FIG. 2 shows hot spot regions where saRNAs are on the human MITF promoter. 239 saRNAs targeting the human MITF promoter were transfected into HEM cells at a final concentration of 25 nM for 72 h. After the transfection was completed, the MITF mRNA expression was analyzed by one-step RT-qPCR. Shown in the drawing is changes in the MITF expression relative to control treatment (Mock) ranked from −500 to −0 according to the target positions of the saRNA in the MITF promoter. The black filled dots represent functional saRNAs, the white hollow dots represent non-functional saRNAs, and the dotted frames represent hot spot regions (H1-H4) and warm spot regions (W1-W3) of functional saRNAs. The ordinate values represent the mean±SD of 2 replicates.

[0054] FIG. 3 shows high-throughput screening results validated by two-step RT-qPCR. saRNAs shown in FIG. 3 were transfected into HEM cells at a final concentration of 25 nM for 72 h. After transfection was completed, RNA was extracted using Qiagen RNeasy kit. After reverse transcription, an ABI 7500 FAST real-time PCR system was used to perform qPCR amplification. At the same time, HPRT1 gene was amplified as an internal reference. Shown in the drawing is relative MITF mRNA expression value after treatment of cells with a single saRNA. Mock, dsCon2 and RAG4-618i are blank transfection, non-specific control duplex RNA transfection and small interfering RNA control transfection, respectively. The ordinate values represent the mean±SD of 2 replicates.

[0055] FIG. 4 shows high-throughput screening results validated by two-step RT-qPCR. saRNAs shown in FIG. 4 were transfected into NHEK cells at a final concentration of 25 nM for 72 h. After transfection was completed, RNA was extracted using Qiagen RNeasy kit. After reverse transcription, an ABI 7500 FAST real-time PCR system was used to perform qPCR amplification. At the same time, HPRT1 gene was amplified as an internal reference. Shown in the drawing is relative MITF mRNA expression value after treatment of cells with a single saRNA. Mock, dsCon2 and RAG4-618i are blank transfection, non-specific control duplex RNA transfection and small interfering RNA control transfection, respectively. The ordinate values represent the mean±SD of 2 replicates.

[0056] FIG. 5 shows that saRNAs promote MITF protein expression in human NHEK cells. saRNAs shown in FIG. 5 were transfected into NHEK cells at a final concentration of 25 nM for 5 days. After cells were collected and the total cell protein was extracted, the MITF protein expression level was detected using western blot. At the same time, Tubulin (α/β-Tubulin) was detected as an internal control. Shown in the drawing is relative MITF protein expression value of single saRNA-treated cell. Mock, dsCon2 and RAG4-618i are blank transfection, non-specific control duplex RNA transfection and small interfering RNA control transfection, respectively.

[0057] FIG. 6 shows that saRNAs promote MITF protein expression in human HEM cells. saRNAs shown in FIG. 6 were transfected into HEK cells at a final concentration of 25 nM for 72 h. After cells were collected and the total cell protein was extracted, the MITF protein expression level was detected using western blot. At the same time, Tubulin (α/β-Tubulin) was detected as an internal control. Shown in the drawing is relative MITF protein expression value of single saRNA-treated cell. Mock, dsCon2 and RAG4-618i are blank transfection, non-specific control duplex RNA transfection and small interfering RNA control transfection, respectively.

DETAILED DESCRIPTION

[0058] In the present invention, the related terms are defined as follows.

[0059] The term “complementarity” as used herein refers to the capability of forming base pairs between two oligonucleotide strands. The base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing).

[0060] Complementarity includes complete complementarity and incomplete complementarity. “Complete complementarity” or “100% complementarity” means that each nucleotide from the first oligonucleotide strand can form a hydrogen bond with a nucleotide at a corresponding position in the second oligonucleotide strand in the double-stranded region of the double-stranded oligonucleotide molecule without “mispairing”. “Incomplete complementarity” means that not all the nucleotide units of the two strands are bonded with each other by hydrogen bonds. For example, for two oligonucleotide strands each of 20 nucleotides in length in the double-stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 10%. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 90%. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% complementarity.

[0061] The term “oligonucleotide” as used herein refers to polymers of nucleotides, and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides. The oligonucleotide for activating target gene transcription described herein is a small activating nucleic acid molecule.

[0062] The terms “oligonucleotide strand” and “oligonucleotide sequence” as used herein can be used interchangeably, referring to a generic term for short nucleotide sequences having less than 35 bases (including nucleotide strands including deoxyribonucleic acid DNA or ribonucleic acid RNA, and also including mixed oligonucleotide strands formed collectively from one or more deoxyribonucleotides and one or more ribonucleotides). In the present invention, an oligonucleotide strand may have any of 16 to 35 nucleotides in length.

[0063] As used herein, the term “first oligonucleotide strand” may be a sense strand or an antisense strand. The sense strand of a small activating RNA refers to a nucleic acid strand contained in a small activating RNA duplex which has identity to the coding strand of the promoter DNA sequence of a target gene, and the antisense strand refers to a nucleic acid strand in the small activating RNA duplex which is complementary to the sense strand.

[0064] As used herein, the term “second oligonucleotide strand” can also be a sense strand or an antisense strand. If the first oligonucleotide strand is a sense strand, the second oligonucleotide strand is an antisense strand; and if the first oligonucleotide strand is an antisense strand, the second oligonucleotide strand is a sense strand.

[0065] The term “gene” as used herein refers to all nucleotide sequences required to encode a polypeptide chain or to transcribe a functional RNA. “Gene” can be an endogenous or fully or partially recombinant gene for a host cell (for example, because an exogenous oligonucleotide and a coding sequence for encoding a promoter are introduced into a host cell, or a heterogenous promoter adjacent to an endogenous coding sequence is introduced into a host cell). For example, the term “gene” comprises a nucleic acid sequence consisting of exons and introns. Protein-coding sequences are, for example, sequences contained within exons in an open reading frame between an initiation codon and a termination codon, and as used herein, “gene” can comprise such as a gene regulatory sequence, such as a promoter, an enhancer, and all other sequences known in the art for controlling the transcription, expression or activity of another gene, no matter whether the gene comprises a coding sequence or a non-coding sequence. In one case, for example, “gene” can be used to describe a functional nucleic acid comprising a regulatory sequence such as a promoter or an enhancer. The expression of a recombinant gene can be controlled by one or more types of heterogenous regulatory sequences.

[0066] The term “target gene” as used herein can refer to nucleic acid sequences naturally present in organisms, transgenes, viral or bacterial sequences, can be chromosomes or extrachromosomal genes, and/or can be transiently or stably transfected or incorporated into cells and/or chromatins thereof. The target gene can be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene and a long non-coding RNA gene). The target gene generally contains a promoter sequence, and the positive regulation for the target gene can be achieved by designing a small activating nucleic acid molecule having sequence identity (also called homology) to the promoter sequence, characterized as the upregulation of expression of the target gene. “Sequence of a target gene promoter” refers to a non-coding sequence of the target gene, and the reference of the sequence of a target gene promoter in the phrase “complementary to the sequence of a target gene promoter” of the present invention refers to a coding strand of the sequence, also known as a non-template strand, i.e., a nucleic acid sequence having the same sequence as the coding sequence of the gene. “Target” or “target sequence” refers to a sequence fragment in the sequence of a target gene promoter that is homologous or complementary with a sense oligonucleotide strand or an antisense oligonucleotide strand of a small activating nucleic acid molecule.

[0067] As used herein, the terms “sense strand” and “sense nucleic acid strand” can be used interchangeably, and the sense oligonucleotide strand of a small activating nucleic acid molecule refers to the first oligonucleotide strand having identity to the coding strand of the sequence of a target gene promoter in the small activating nucleic acid molecule duplex.

[0068] As used herein, the terms “antisense strand” and “antisense nucleic acid strand” can be used interchangeably, and the antisense oligonucleotide strand of a small activating nucleic acid molecule refers to the second oligonucleotide strand complementary with the sense oligonucleotide strand in the small activating nucleic acid molecule duplex.

[0069] The term “coding strand” as used herein refers to a DNA strand in the target gene which cannot be used for transcription, and the nucleotide sequence of this strand is the same as that of an RNA produced from transcription (in the RNA, T in DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0070] The term “template strand” as used herein refers to the other strand complementary with the coding strand in the double-stranded DNA of the target gene, i.e., the strand that, as a template, can be transcribed into RNA, and this strand is complementary with the transcribed RNA (A to U and G to C). In the process of transcription, RNA polymerase binds to the template strand, moves along the 3′.fwdarw.5′ direction of the template strand, and catalyzes the synthesis of the RNA along the 5′.fwdarw.3′ direction. The template strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0071] The term “promoter” as used herein refers to a sequence which plays a regulatory role for the transcription of a protein-coding or RNA-coding nucleic acid sequence by associating with them spatially. Generally, a eukaryotic gene promoter contains 100 to 5000 base pairs, although this length range is not intended to limit the term “promoter” as used herein. Although the promoter sequence is generally located at the 5′ terminus of a protein-coding or RNA-coding sequence, the promoter sequence may also exist in exon and intron sequences.

[0072] The term “transcription start site” as used herein refers to a nucleotide marking the transcription start on the template strand of a gene. The transcription start site may appear on the template strand of the promoter region. A gene can have more than one transcription start site.

[0073] The term “identity” or “homology” as used herein means that one oligonucleotide strand (a sense or an antisense strand) of a small activating RNA has similarity to a coding strand or a template strand in a region of the promoter sequence of a target gene. As used herein, the “identity” or “homology” may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99% or 100%.

[0074] The term “overhang” as used herein refers to non-base-paired nucleotides at the terminus (5′ or 3′) of an oligonucleotide strand, which is formed by one strand extending out of the other strand in a double-stranded oligonucleotide. A single-stranded region extending out of the 3′ terminus and/or 5′ terminus of a duplex is referred to as an overhang.

[0075] As used herein, the terms “gene activation”, “activating gene expression”, “gene upregulation” and “upregulating gene expression” can be used interchangeably, and mean an increase in transcription, translation, expression or activity of a certain nucleic acid as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, “gene activation”, “activating gene expression”, “gene upregulation” or “upregulating gene expression” refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, the nucleic acid sequence plays a regulatory role as a regulatory sequence, the nucleic acid sequence is transcribed into RNA and the RNA is translated into a protein, thereby increasing the protein expression.

[0076] As used herein, the terms “small activating RNA”, “saRNA”, and “small activating nucleic acid molecule” can be used interchangeably, and refer to a nucleic acid molecule that can upregulate target gene expression and can be composed of the first nucleic acid fragment (antisense nucleic acid strand, also referred to as antisense oligonucleotide strand) containing a nucleotide sequence having sequence identity or homology with the non-coding nucleic acid sequence (e.g., a promoter and an enhancer) of a target gene and the second nucleic acid fragment (sense nucleic acid strand, also referred to as sense oligonucleotide strand) containing a nucleotide sequence complementary with the first nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex. The small activating nucleic acid molecule can also be composed of a synthesized or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, wherein the first region comprises a nucleotide sequence having sequence identity to the target sequence of a promoter of a gene, and the second region comprises a nucleotide sequence which is complementary to the first region. The length of the duplex region of the small activating nucleic acid molecule is typically about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, and about 28 to about 32 base pairs, and typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. In addition, the terms “saRNA”, “small activating RNA”, and “small activating nucleic acid molecule” also comprise nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.

[0077] As used herein, the term “hot spot (H region)” refers to a gene promoter region of at least 25 bp in length. The gathering of targets of functional small activating nucleic acid molecules appears in these hot spot regions, wherein at least 30% of the small activating nucleic acid molecules targeting these hot spot regions can induce 1.1 folds or more change in the mRNA expression of a target gene; the term “warm spot (W region)” refers to a gene promoter region of at least 25 bp in length. The gathering of targets of functional small activating nucleic acid molecules appears in these warm spot regions, wherein 8% to 30% of the small activating nucleic acid molecules targeting these warm spot regions can induce 1.1 folds or more change in the mRNA expression of a target gene.

[0078] As used herein, the term “synthesis” refers to a method for synthesis of an oligonucleotide, including any method allowing RNA synthesis, such as chemical synthesis, in vitro transcription, and/or vector-based expression.

[0079] According to the present invention, the MITF gene expression is upregulated by RNA activation, and a related disease (particularly vitiligo) is treated by increasing the MITF protein expression level. The MITF gene in the present invention is sometimes also called a target gene.

[0080] The method for preparing the small activating nucleic acid molecule provided by the present invention comprises sequence design and sequence synthesis.

[0081] The synthesis of the sequence of the small activating nucleic acid molecule of the present invention can be performed by adopting a chemical synthesis or can be entrusted to a biotechnology company specialized in nucleic acid synthesis.

[0082] Generally speaking, the chemical synthesis comprises the following four steps: (1) synthesis of oligomeric ribonucleotides; (2) deprotection; (3) purification and isolation; and (4) desalination and annealing.

[0083] For example, the specific steps for chemically synthesizing the saRNA described herein are as follows.

[0084] (1) Synthesis of Oligomeric Ribonucleotides

[0085] Synthesis of 1 μM RNA was set in an automatic DNA/RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909), and the coupling time of each cycle was also set as 10 to 15 min. With a solid phase-bonded 5′-O-p-dimethoxytriphenylmethyl-thymidine substrate as an initiator, one base was bonded to the solid phase substrate in the first cycle, and then, in the n.sup.th (19≥n≥2) cycle, one base was bonded to the base bonded in the n-1.sup.th cycle. This process was repeated until the synthesis of the whole nucleic acid sequence was completed.

[0086] (2) Deprotection

[0087] The solid phase substrate bonded with the saRNA was put into a test tube, and 1 mL of a mixed solution of ethanol and ammonium hydroxide (volume ratio: 1:3) was added to the test tube. The test tube was then sealed and placed in an incubator, and the mixture was incubated at 25-70° C. for 2 to 30 h. The solution containing the solid phase substrate bonded with the saRNA was filtered, and the filtrate was collected. The solid phase substrate was rinsed with double distilled water twice (1 mL each time), and the filtrate was collected. The collected eluent was combined and dried under vacuum for 1 to 12 h. Then the solution was added with 1 mL of a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M), let stand at room temperature for 4 to 12 h, followed by addition of 2 mL of n-butanol. Precipitate was collected to give a single-stranded crude product of saRNA by high-speed centrifugation.

[0088] (3) Purification and Isolation

[0089] The resulting crude product of saRNA was dissolved in 2 mL of triethylamine acetate solution with a concentration of 1 mol/L, and the solution was separated by a reversed-phase C18 column of high pressure liquid chromatography to give a purified single-stranded product of saRNA.

[0090] (4) Desalination and Annealing

[0091] Salts were removed by gel filtration (size exclusion chromatography). A single sense oligomeric ribonucleic acid strand and a single antisense oligomeric ribonucleic acid strand were mixed in a 1 to 2 mL of buffer (10 mM Tris, pH=7.5-8.0, 50 mM NaCl) at a molar ratio of 1:1. The solution was heated to 95° C., and was then slowly cooled to room temperature to give a solution containing saRNA.

[0092] It was discovered in this study that after being introduced into a cell, the aforementioned saRNA could effectively increase the MITF mRNA and protein expression.

[0093] The present invention will be further illustrated with reference to specific examples and drawings below. It should be understood that these examples are merely intended to illustrate the present invention rather than limit the scope of the present invention. In the following examples, study methods without specific conditions were generally in accordance with conventional conditions, such as conditions described in Sambrook, et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer.

[0094] Embodiments of the present disclosure will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. If specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturers shall be adopted. Reagents or instruments without specified manufacturers used herein are conventional products that are commercially available.

EXAMPLE 1

Design and Synthesis of saRNA Targeting Human MITF Promoter

[0095] To screen a functional small activating RNA capable of activating the MITF gene expression, with a MITF promoter sequence of 500 bp in length as a template, a target with a length of 19 bp was selected from −500 bp upstream of TSS. The target sequences were then filtered to keep those which met the following criteria: (1) GC content between 40% and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeat sequences; (4) with 3 or less trinucleotide repeat sequences. After the filtration, the remaining 239 target sequences entered the screening process as candidates. Corresponding double-stranded small activating RNAs were chemically synthesized based on these candidate sequences.

[0096] Each of the sense strand and antisense strand in the double-stranded small activating RNA used in the study had 21 nucleotides in length. The 19 nucleotides in the 5′ region of the first ribonucleic acid strand (sense strand) of the double-stranded saRNA had 100% identity with the target sequence of the promoter, and the 3′ terminus of the first ribonucleic acid strand contained a TT sequence. The 19 nucleotides in the 5′ region of the second ribonucleic acid strand were complementary with the first ribonucleic acid strand sequence, and the 3′ terminus of the second ribonucleic acid strand contained a TT sequence. The aforementioned two strands of the double-stranded saRNA were mixed at a molar ratio of 1:1. After annealing, a double-stranded saRNA was formed.

[0097] The sequence of the human MITF promoter is shown as follows, which corresponds to position 1 to position 500 from 5′ to 3′ of SEQ ID NO: 1 in the sequence listing:

TABLE-US-00001 -500 gcgaaggaaa gttcttcctc gttgttccaa tccgaggaca agctgatatg -450 tcgcagcagc ccagggaagc atgcgagctg ataggaagtc cttttatttt -400 aagacaggct cgaatgctaa aactttcttg tgccaaaacc cttgactatt -350 ttatttttaa aataagcact tggcgtgccc tcgcagatgt ctgagctgag -300 aggtcggggc gatggtagaa gagcagtcag tgtccattct tattcatatt -250 aagtagccaa gtctgtaccc ttgaagcaag tggggagaga ggagggagag -200 gagctgctga cattgacaat gaatccaaac aggagttgca ctagcggtgt -150 ccaccacgtt gcctctcccc cgcctggcct tctgggagct gtagttttcg -100 tgggagcggc tccccaggcg agctgggaat gccccgcccg ggccgaacta  -50 cagatcccag gcggcgctcg gccgccagcc cctcccgccc gggtgcgagt

EXAMPLE 2

High-Throughput Screening of saRNAs Targeting Human MITF Promoter

[0098] (1) Cell Culture and Transfection

[0099] Human epidermal melanocytes (HEMs) (purchased from Beina Chuanglian Biotechnology Co., Ltd (Beijing), BNCC350795) and normal human epidermal keratinocytes (NHEKs) (purchased from Beina Chuanglian Biotechnology Co., Ltd (Beijing), BNCC340593) were cultured in DMEM media (Gibco) containing 10% newborn calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco). The cells were cultured at 5% CO.sub.2 and 37° C. The HEM cells were plated at 2000 cells/well into a 96-well plate; according to the instructions provided by the manufacturer, RNAiMax (Invitrogen, Carlsbad, Calif.) was used to transfect small activating RNAs at a concentration of 25 nM (unless otherwise specified) for 72 h, and 2 replicate wells were used for each treatment.

[0100] (2) One-Step RT-qPCR

[0101] After transfection, the media were discarded, and each well was washed with 150 μL of PBS once. After PBS was discarded, each well was added with 50 μL of cell lysis solution, and incubation was performed at room temperature for 5 min. 1 μL of cell lysis solution was taken from each well and subjected to qPCR analysis on an ABI 7500 fast real-time PCR system (Applied Biosystems) using a one-step TB Green™ PrimeScrip™ RT-PCR kit II (Takara, RR086A), and each sample was repeatedly amplified in 3 replicate wells. PCR reaction conditions are shown in Table 1 below.

TABLE-US-00002 TABLE 1 PCR reaction preparation Reagent Volume 2× One-step TB Green RT-PCR buffer 4 2.5 μl PrimeScript 1 step enzyme Mix 2 0.2 μl Forward and reverse primers Mix (5 μM) 0.4 μl No RNase dH.sub.2O 1.4 μl Crude lysate (RNA) 0.5 μl Sum 5 μl

[0102] Reaction conditions were as follows: reverse transcription reaction (stage 1): 5 min at 42° C., 10 s at 95° C.; PCR reaction (stage 2): 5 s at 95° C., 20 s at 60° C., 45 cycles of amplification. HPRT1 and TBP were used as internal reference genes. PCR primers used by MITF, HPRT1 and TBP are shown in Table 2, wherein MITF was amplified using the MITF F1/R1 primer pair.

TABLE-US-00003 TABLE 2 Primer sequences for RT-qPCR analysis Primer SEQ ID NO Sequence (5′-3′) MITF F1 SEQ ID NO: 2 CGACAGAAGAAACTGGAGCAC MITF R1 SEQ ID NO: 3 CCCGTGGATGGAATAAGGGAAA HPRT1 F SEQ ID NO: 4 AAAGATGGTCAAGGTCGCAAG HPRT1 R SEQ ID NO: 5 TAGTCAAGGGCATATCCTACAAC TBP F SEQ ID NO: 6 TGCTCACCCACCAACAATTTAG TBP R SEQ ID NO: 7 TCTGCTCTGACTTTAGCACCTG

[0103] To calculate the expression value (Erel) of MITF (target gene) of a saRNA-transfected sample relative to control treatment (Mock), the Ct values of the target gene and the two internal reference genes were substituted into formula 1 for calculation.

E.sub.rel=2.sup.(CtTm−CtTs)/((2.sup.(CtR1m−CtR1s)*2.sup.(CtR2m−CtR2s)).sup.(1/2))   (formula 1)

[0104] wherein CtTm was the Ct value of the target gene from the control treatment (Mock) sample; CtTs was the Ct value of the target gene from the saRNA-treated sample; CtR1m was the Ct value of the internal reference gene 1 from the Mock-treated sample; CtR1s was the Ct value of the internal reference gene 1 from the saRNA-treated sample; CtR2m was the Ct value of the internal reference gene 2 from the control treatment sample; and CtR2s was the Ct value of the internal reference gene 2 from the saRNA-treated sample.

[0105] (3) Screening of Functional saRNAs

[0106] To obtain saRNAs capable of activating MITF transcription, the aforementioned 239 saRNAs were individually transfected into HEM cells at a transfection concentration of 25 nM. 72 h after the transfection, according to the same method as described above, the cells were lysed and subjected to one-step RT-qPCR analysis to obtain the relative (compared to the control treatment group) MITF gene expression value of each saRNA-treated sample. As shown in Table 3, 29 (12.1%) saRNAs showed high activation (≥1.5 fold), 68 (28.5%) saRNAs showed mild activation (≥1.1 fold), and 142 (59.4%) saRNAs had no upregulating effect on MITF expression. The maximum activation was 2.75 fold, and the maximum inhibition was 0.38 fold, and the saRNAs with activation activity were referred to as functional saRNAs.

TABLE-US-00004 TABLE 3 High-throughput screening results of human MITF saRNA Number of saRNA activity log.sub.2 value of changes in MITF (fold) saRNAs Percentage High activation ≥0.58 (1.50)~≤1.46(2.75) 29 12.1% Mild activation ≥0.13 (1.10)~<0.58 (1.50) 68 28.5% No activation effect <0.13 (1.10) 142 59.4% Sum 239  100%

[0107] As shown in FIG. 1, it is further shown that the descending order of changes in MITF expression of human MITF saRNA. The MITF active saRNA sequence (functional saRNA sequence), the active target sequence and changes in MITF mRNA expression are shown in Table 4 (each active target sequence listed in Table 4 corresponds to SEQ ID NOs: 8-104 in the sequence listing, each saRNA sense sequence corresponds to SEQ ID NOs: 105-201 in the sequence listing, and each saRNA antisense sequence corresponds to SEQ ID NOs: 202-298 in the sequence listing). Table 5 shows different hot spot regions and warm spot regions and related sequences where functional saRNAs are located (SEQ ID NOs: 299-305).

TABLE-US-00005 TABLE 4 Functional saRNA sequences, active target sequences thereof and changes in MITF mRNA expression caused thereby Fold of changes in Active relative target sequence MITF mRNA saRNA (5′-3′) Functional saRNA sequence (5′-3′) expression Hot spot RAG4- GCGAAGGAAAGTT GCGAAGGAAAGUUCUUCCUTT (sense) 1.64 region 500 CTTCCT AGGAAGAACUUUCCUUCGCTT (antisense) RAG4- GAAGGAAAGTTCT GAAGGAAAGUUCUUCCUCGTT (sense) 1.34 498 TCCTCG CGAGGAAGAACUUUCCUUCTT (antisense) RAG4- AAGGAAAGTTCTT AAGGAAAGUUCUUCCUCGUTT (sense) 1.44 497 CCTCGT ACGAGGAAGAACUUUCCUUTT (antisense) RAG4- GAAAGTTCTTCCT GAAAGUUCUUCCUCGUUGUTT (sense) 1.10 494 CGTTGT ACAACGAGGAAGAACUUUCTT (antisense) RAG4- AGTTCTTCCTCGTT AGUUCUUCCUCGUUGUUCCTT (sense) 1.45 491 GTTCC GGAACAACGAGGAAGAACUTT (antisense) RAG4- GTTCTTCCTCGTTG GUUCUUCCUCGUUGUUCCATT (sense) 1.97 490 TTCCA UGGAACAACGAGGAAGAACTT (antisense) RAG4- TTCTTCCTCGTTGT UUCUUCCUCGUUGUUCCAATT (sense) 1.84 489 TCCAA UUGGAACAACGAGGAAGAATT (antisense) RAG4- CTTCCTCGTTGTTC CUUCCUCGUUGUUCCAAUCTT (sense) 1.15 487 CAATC GAUUGGAACAACGAGGAAGTT (antisense) RAG4- TCCTCGTTGTTCCA UCCUCGUUGUUCCAAUCCGTT (sense) 1.32 485 ATCCG CGGAUUGGAACAACGAGGATT (antisense) RAG4- CTCGTTGTTCCAAT CUCGUUGUUCCAAUCCGAGTT (sense) 1.16 483 CCGAG CUCGGAUUGGAACAACGAGTT (antisense) RAG4- TCGTTGTTCCAATC UCGUUGUUCCAAUCCGAGGTT (sense) 1.16 482 CGAGG CCUCGGAUUGGAACAACGATT (antisense) RAG4- CGTTGTTCCAATCC CGUUGUUCCAAUCCGAGGATT (sense) 1.23 481 GAGGA UCCUCGGAUUGGAACAACGTT (antisense) RAG4- TTGTTCCAATCCGA UUGUUCCAAUCCGAGGACATT (sense) 1.16 479 GGACA UGUCCUCGGAUUGGAACAATT (antisense) RAG4- TGTTCCAATCCGA UGUUCCAAUCCGAGGACAATT (sense) 1.53 478 GGACAA UUGUCCUCGGAUUGGAACATT (antisense) RAG4- GTTCCAATCCGAG GUUCCAAUCCGAGGACAAGTT (sense) 1.54 477 GACAAG CUUGUCCUCGGAUUGGAACTT (antisense) RAG4- TTCCAATCCGAGG UUCCAAUCCGAGGACAAGCTT (sense) 1.31 476 ACAAGC GCUUGUCCUCGGAUUGGAATT (antisense) RAG4- TCCAATCCGAGGA UCCAAUCCGAGGACAAGCUTT (sense) 1.30 475 CAAGCT AGCUUGUCCUCGGAUUGGATT (antisense) RAG4- CCAATCCGAGGAC CCAAUCCGAGGACAAGCUGTT (sense) 1.12 474 AAGCTG CAGCUUGUCCUCGGAUUGGTT (antisense) RAG4- CAATCCGAGGACA CAAUCCGAGGACAAGCUGATT (sense) 1.10 473 AGCTGA UCAGCUUGUCCUCGGAUUGTT (antisense) RAG4- AATCCGAGGACAA AAUCCGAGGACAAGCUGAUTT (sense) 1.93 472 GCTGAT AUCAGCUUGUCCUCGGAUUTT (antisense) RAG4- CCGAGGACAAGCT CCGAGGACAAGCUGAUAUGTT (sense) 1.39 469 GATATG CAUAUCAGCUUGUCCUCGGTT (antisense) RAG4- GAGGACAAGCTGA GAGGACAAGCUGAUAUGUCTT (sense) 1.33 467 TATGTC GACAUAUCAGCUUGUCCUCTT (antisense) RAG4- AGGACAAGCTGAT AGGACAAGCUGAUAUGUCGTT (sense) 1.14 466 ATGTCG CGACAUAUCAGCUUGUCCUTT (antisense) RAG4- GACAAGCTGATAT GACAAGCUGAUAUGUCGCATT (sense) 1.37 464 GTCGCA UGCGACAUAUCAGCUUGUCTT (antisense) RAG4- AAGCTGATATGTCG AAGCUGAUAUGUCGCAGCATT (sense) 2.10 461 CAGCA UGCUGCGACAUAUCAGCUUTT (antisense) RAG4- AGCTGATATGTCGC AGCUGAUAUGUCGCAGCAGTT (sense) 1.63 460 AGCAG CUGCUGCGACAUAUCAGCUTT (antisense) RAG4- GCTGATATGTCGCA GCUGAUAUGUCGCAGCAGCTT (sense) 1.72 459 GCAGC GCUGCUGCGACAUAUCAGCTT (antisense) RAG4- CTGATATGTCGCAG CUGAUAUGUCGCAGCAGCCTT (sense) 1.79 458 CAGCC GGCUGCUGCGACAUAUCAGTT (antisense) RAG4- GATATGTCGCAGCA GAUAUGUCGCAGCAGCCCATT (sense) 1.58 456 GCCCA UGGGCUGCUGCGACAUAUCTT (antisense) RAG4- AGCAGCCCAGGGA AGCAGCCCAGGGAAGCAUGTT (sense) 1.23 446 AGCATG CAUGCUUCCCUGGGCUGCUTT (antisense) RAG4- GCATGCGAGCTGAT GCAUGCGAGCUGAUAGGAATT (sense) 1.32 432 AGGAA UUCCUAUCAGCUCGCAUGCTT (antisense) RAG4- CATGCGAGCTGATA CAUGCGAGCUGAUAGGAAGTT (sense) 1.53 431 GGAAG CUUCCUAUCAGCUCGCAUGTT (antisense) RAG4- CGAGCTGATAGGA CGAGCUGAUAGGAAGUCCUTT (sense) 1.10 427 AGTCCT AGGACUUCCUAUCAGCUCGTT (antisense) RAG4- GAGCTGATAGGAA GAGCUGAUAGGAAGUCCUUTT (sense) 1.43 426 GTCCTT AAGGACUUCCUAUCAGCUCTT (antisense) RAG4- TTTAAGACAGGCT UUUAAGACAGGCUCGAAUGTT (sense) 1.17 403 CGAATG CAUUCGAGCCUGUCUUAAATT (antisense) RAG4- TTAAGACAGGCTC UUAAGACAGGCUCGAAUGCTT (sense) 1.46 402 GAATGC GCAUUCGAGCCUGUCUUAATT (antisense) RAG4- TAAGACAGGCTCG UAAGACAGGCUCGAAUGCUTT (sense) 1.21 401 AATGCT AGCAUUCGAGCCUGUCUUATT (antisense) RAG4- CAGGCTCGAATGC CAGGCUCGAAUGCUAAAACTT (sense) 1.65 396 TAAAAC GUUUUAGCAUUCGAGCCUGTT (antisense) RAG4- AGGCTCGAATGCT AGGCUCGAAUGCUAAAACUTT (sense) 1.12 395 AAAACT AGUUUUAGCAUUCGAGCCUTT (antisense) RAG4- GGCTCGAATGCTA GGCUCGAAUGCUAAAACUUTT (sense) 1.15 394 AAACTT AAGUUUUAGCAUUCGAGCCTT (antisense) RAG4- GCTAAAACTTTCTT GCUAAAACUUUCUUGUGCCTT (sense) 1.48 385 GTGCC GGCACAAGAAAGUUUUAGCTT (antisense) RAG4- TCTTGTGCCAAAA UCUUGUGCCAAAACCCUUGTT (sense) 1.43 375 CCCTTG CAAGGGUUUUGGCACAAGATT (antisense) RAG4- CTTGTGCCAAAAC CUUGUGCCAAAACCCUUGATT (sense) 1.27 374 CCTTGA UCAAGGGUUUUGGCACAAGTT (antisense) RAG4- TGTGCCAAAACCC UGUGCCAAAACCCUUGACUTT (sense) 1.26 372 TTGACT AGUCAAGGGUUUUGGCACATT (antisense) RAG4- GTGCCAAAACCCT GUGCCAAAACCCUUGACUATT (sense) 1.43 371 TGACTA UAGUCAAGGGUUUUGGCACTT (antisense) RAG4- GCCAAAACCCTTG GCCAAAACCCUUGACUAUUTT (sense) 1.11 369 ACTATT AAUAGUCAAGGGUUUUGGCTT (antisense) RAG4- AAAATAAGCACTT AAAAUAAGCACUUGGCGUGTT (sense) 1.21 342 GGCGTG CACGCCAAGUGCUUAUUUUTT (antisense) RAG4- AAATAAGCACTTG AAAUAAGCACUUGGCGUGCTT (sense) 1.15 341 GCGTGC GCACGCCAAGUGCUUAUUUTT (antisense) RAG4- CCCTCGCAGATGT CCCUCGCAGAUGUCUGAGCTT (sense) 1.32 323 CTGAGC GCUCAGACAUCUGCGAGGGTT (antisense) RAG4- CCTCGCAGATGTCT CCUCGCAGAUGUCUGAGCUTT (sense) 1.19 322 GAGCT AGCUCAGACAUCUGCGAGGTT (antisense) RAG4- TCGCAGATGTCTG UCGCAGAUGUCUGAGCUGATT (sense) 1.30 320 AGCTGA UCAGCUCAGACAUCUGCGATT (antisense) RAG4- GCAGATGTCTGAG GCAGAUGUCUGAGCUGAGATT (sense) 1.89 318 CTGAGA UCUCAGCUCAGACAUCUGCTT (antisense) RAG4- CAGATGTCTGAGC CAGAUGUCUGAGCUGAGAGTT (sense) 1.17 317 TGAGAG CUCUCAGCUCAGACAUCUGTT (antisense) RAG4- AGATGTCTGAGCT AGAUGUCUGAGCUGAGAGGTT (sense) 1.40 316 GAGAGG CCUCUCAGCUCAGACAUCUTT (antisense) RAG4- GTCGGGGCGATGG GUCGGGGCGAUGGUAGAAGTT (sense) 1.49 298 TAGAAG CUUCUACCAUCGCCCCGACTT (antisense) RAG4- TCGGGGCGATGGT UCGGGGCGAUGGUAGAAGATT (sense) 1.24 297 AGAAGA UCUUCUACCAUCGCCCCGATT (antisense) RAG4- CGGGGCGATGGTA CGGGGCGAUGGUAGAAGAGTT (sense) 1.75 296 GAAGAG CUCUUCUACCAUCGCCCCGTT (antisense) RAG4- GGGGCGATGGTAG GGGGCGAUGGUAGAAGAGCTT (sense) 1.39 295 AAGAGC GCUCUUCUACCAUCGCCCCTT (antisense) RAG4- GGCGATGGTAGAA GGCGAUGGUAGAAGAGCAGTT (sense) 1.10 293 GAGCAG CUGCUCUUCUACCAUCGCCTT (antisense) RAG4- GCGATGGTAGAAG GCGAUGGUAGAAGAGCAGUTT (sense) 1.20 292 AGCAGT ACUGCUCUUCUACCAUCGCTT (antisense) RAG4- GATGGTAGAAGAG GAUGGUAGAAGAGCAGUCATT (sense) 2.37 290 CAGTCA UGACUGCUCUUCUACCAUCTT (antisense) RAG4- ATGGTAGAAGAGC AUGGUAGAAGAGCAGUCAGTT (sense) 1.33 289 AGTCAG CUGACUGCUCUUCUACCAUTT (antisense) RAG4- TGGTAGAAGAGCA UGGUAGAAGAGCAGUCAGUTT (sense) 1.41 288 GTCAGT ACUGACUGCUCUUCUACCATT (antisense) RAG4- TAGAAGAGCAGTC UAGAAGAGCAGUCAGUGUCTT (sense) 1.35 285 AGTGTC GACACUGACUGCUCUUCUATT (antisense) RAG4- AGAAGAGCAGTCA AGAAGAGCAGUCAGUGUCCTT (sense) 2.68 284 GTGTCC GGACACUGACUGCUCUUCUTT (antisense) RAG4- GAAGAGCAGTCAG GAAGAGCAGUCAGUGUCCATT (sense) 1.61 283 TGTCCA UGGACACUGACUGCUCUUCTT (antisense) RAG4- AAGAGCAGTCAGT AAGAGCAGUCAGUGUCCAUTT (sense) 1.58 282 GTCCAT AUGGACACUGACUGCUCUUTT (antisense) RAG4- AGAGCAGTCAGTG AGAGCAGUCAGUGUCCAUUTT (sense) 1.19 281 TCCATT AAUGGACACUGACUGCUCUTT (antisense) RAG4- GAGCAGTCAGTGT GAGCAGUCAGUGUCCAUUCTT (sense) 1.71 280 CCATTC GAAUGGACACUGACUGCUCTT (antisense) RAG4- TGAATCCAAACAG UGAAUCCAAACAGGAGUUGTT (sense) 1.61 181 GAGTTG CAACUCCUGUUUGGAUUCATT (antisense) RAG4- GAATCCAAACAGG GAAUCCAAACAGGAGUUGCTT (sense) 2.28 180 AGTTGC GCAACUCCUGUUUGGAUUCTT (antisense) RAG4- AATCCAAACAGGA AAUCCAAACAGGAGUUGCATT (sense) 1.29 179 GTTGCA UGCAACUCCUGUUUGGAUUTT (antisense) RAG4- CCAAACAGGAGTT CCAAACAGGAGUUGCACUATT (sense) 2.75 176 GCACTA UAGUGCAACUCCUGUUUGGTT (antisense) RAG4- CAAACAGGAGTTG CAAACAGGAGUUGCACUAGTT (sense) 2.54 175 CACTAG CUAGUGCAACUCCUGUUUGTT (antisense) RAG4- AAACAGGAGTTGC AAACAGGAGUUGCACUAGCTT (sense) 1.44 174 ACTAGC GCUAGUGCAACUCCUGUUUTT (antisense) RAG4- ACAGGAGTTGCAC ACAGGAGUUGCACUAGCGGTT (sense) 1.49 172 TAGCGG CCGCUAGUGCAACUCCUGUTT (antisense) RAG4- AGGAGTTGCACTA AGGAGUUGCACUAGCGGUGTT (sense) 1.14 170 GCGGTG CACCGCUAGUGCAACUCCUTT (antisense) RAG4- GTTGCACTAGCGG GUUGCACUAGCGGUGUCCATT (sense) 1.73 166 TGTCCA UGGACACCGCUAGUGCAACTT (antisense) RAG4- TTGCACTAGCGGT UUGCACUAGCGGUGUCCACTT (sense) 1.50 165 GTCCAC GUGGACACCGCUAGUGCAATT (antisense) RAG4- AGCGGTGTCCACC AGCGGUGUCCACCACGUUGTT (sense) 1.55 158 ACGTTG CAACGUGGUGGACACCGCUTT (antisense) Warm RAG4- AGTAGCCAAGTCT AGUAGCCAAGUCUGUACCCTT (sense) 1.10 region 249 GTACCC GGGUACAGACUUGGCUACUTT (antisense) spot RAG4- CTGTACCCTTGAA CUGUACCCUUGAAGCAAGUTT (sense) 1.18 238 GCAAGT ACUUGCUUCAAGGGUACAGTT (antisense) RAG4- GTACCCTTGAAGC GUACCCUUGAAGCAAGUGGTT (sense) 1.47 236 AAGTGG CCACUUGCUUCAAGGGUACTT (antisense) RAG4- AGAGGAGGGAGA AGAGGAGGGAGAGGAGCUGTT (sense) 1.35 213 GGAGCTG CAGCUCCUCUCCCUCCUCUTT (antisense) RAG4- AGGAGGGAGAGG AGGAGGGAGAGGAGCUGCUTT (sense) 1.25 211 AGCTGCT AGCAGCUCCUCUCCCUCCUTT (antisense) RAG4- CCTTCTGGGAGCT CCUUCUGGGAGCUGUAGUUTT (sense) 1.69 123 GTAGTT AACUACAGCUCCCAGAAGGTT (antisense) RAG4-1 GGAGCTGTAGTTTT GGAGCUGUAGUUUUCGUGGTT (sense) 1.17 16 CGTGG CCACGAAAACUACAGCUCCTT (antisense) RAG4-1 GAGCTGTAGTTTTC GAGCUGUAGUUUUCGUGGGTT (sense) 1.38 15 GTGGG CCCACGAAAACUACAGCUCTT (antisense) RAG4-1 AGCTGTAGTTTTCG AGCUGUAGUUUUCGUGGGATT (sense) 1.36 14 TGGGA UCCCACGAAAACUACAGCUTT (antisense) RAG4-1 GCTGTAGTTTTCGT GCUGUAGUUUUCGUGGGAGTT (sense) 1.14 13 GGGAG CUCCCACGAAAACUACAGCTT (antisense) RAG4-1 TGTAGTTTTCGTGG UGUAGUUUUCGUGGGAGCGTT (sense) 1.36 11 GAGCG CGCUCCCACGAAAACUACATT (antisense) RAG4- TAGTTTTCGTGGGA UAGUUUUCGUGGGAGCGGCTT (sense) 1.30 109 GCGGC GCCGCUCCCACGAAAACUATT (antisense) RAG4- GTTTTCGTGGGAG GUUUUCGUGGGAGCGGCUCTT (sense) 1.49 107 CGGCTC GAGCCGCUCCCACGAAAACTT (antisense) RAG4- CGGGCCGAACTAC CGGGCCGAACUACAGAUCCTT (sense) 1.36 62 AGATCC GGAUCUGUAGUUCGGCCCGTT (antisense) RAG4- GGGCCGAACTACA GGGCCGAACUACAGAUCCCTT (sense) 1.29 61 GATCCC GGGAUCUGUAGUUCGGCCCTT (antisense) RAG4- AACTACAGATCCC AACUACAGAUCCCAGGCGGTT (sense) 1.64 55 AGGCGG CCGCCUGGGAUCUGUAGUUTT (antisense) RAG4- ACTACAGATCCCA ACUACAGAUCCCAGGCGGCTT (sense) 1.21 54 GGCGGC GCCGCCUGGGAUCUGUAGUTT (antisense)

TABLE-US-00006 TABLE 5 Hot spot and warm spot regions and sequences of functional saRNAs Hot spot region Sequence of hot spot region H1 gcgaaggaaagttcttcctcgttgttccaatccgaggacaagctgatatgtcgcagcagcccagggaagcatgc (-500/-408) gagctgataggaagtcctt H2 tttaagacaggctcgaatgctaaaactttcttgtgccaaaacccttgactatt (-403/-351) H3 aaaataagcacttggcgtgccctcgcagatgtctgagctgagaggtcggggcgatggtagaagagcagtcagtg (-342/-262) tccattc H4 tgaatccaaacaggagttgcactagcggtgtccaccacgttg (-181/-140) Warm spot region Sequence of warm spot region W1 agtagccaagtctgtacccttgaagcaagtggggagagaggagggagaggagctgct (-249/-193) W2 ccttctgggagctgtagttttcgtgggagcggctc (-123/-89) W3 cgggccgaactacagatcccaggcggc (-62/-36)

EXAMPLE 3

saRNA Promoting MITF mRNA Expression in Human Epidermal Melanocytes (HEMs)

[0108] (1) Cell Culture and Transfection

[0109] Cell culture was described in Example 2, human epidermal melanocytes (HEMs) were plated at 20×10.sup.4 cells/well into a 6-well plate, small activating RNAs were transfected at a final concentration of 25 nM for 72 h, and 2 replicate wells were used for each treatment.

[0110] (2) Two-Step RT-qPCR

[0111] After transfection, the media were discarded, each well was added with 500 μL of cell lysis solution, and incubation was performed at room temperature for 5 min. RNA was extracted using Qiagen RNeasy kit. After reverse transcription, qPCR analysis on an ABI 7500 fast real-time PCR system (Applied Biosystems), and each sample was repeatedly amplified in 3 replicate wells. PCR reaction conditions are shown in Table 6 and Table 7 below.

TABLE-US-00007 TABLE 6 RT reaction preparation Volume Reagent (RT reaction 1) 5× gDNA Eraser buffer 2 μl gDNA Eraser 1 μl Total amount of RNA (1 μg) + D.W 7 μl Final volume 10 μl 2 min at 42° C. and stored at 4° C. Reagent (RT reaction 2) 5× Prime Script buffer 2 4 μl PrimeScript RT Enzyme Mix 1 1 μl RT primer mixture 1 μl No RNase dH.sub.2O 4 μl RT reaction 1 10 μl Final volume 20 μl 15 min at 37° C., 5 s at 85° C., stored at 4° C.

TABLE-US-00008 TABLE 7 RT-qPCR reaction preparation Reagent Volume SYBR Premix Ex Taq II (2×) 5 μl ROX reference dye II (50×) 0.2 μl Forward and Reverse Primers Mix (5 μM) 0.8 μl cDNA (RT products) 4 μl Sum 10 μl

[0112] Reaction conditions were as follows: 30 s at 95° C., 5 s at 95° C., 30 s at 60° C., 40 cycles of amplification. At the same time, HPRT1 gene was amplified as an internal reference, and MITF was amplified using the MITF F1/R1 primer pair, the amplification primers are shown in Table 2.

[0113] To calculate the expression value (Erel) of MITF (target gene) of a saRNA-transfected sample relative to control treatment (Mock), the Ct values of the target gene and the one internal reference gene were substituted into formula 2 for calculation.

E.sub.rel=2.sup.(CtTm−CtTs)/2.sup.(CtRm−CtRs)   (formula 2)

[0114] wherein CtTm was the Ct value of the target gene from the control treatment (Mock) sample; CtTs was the Ct value of the target gene from the saRNA-treated sample; CtRm was the Ct value of the internal reference gene from the control treatment Mock-treated sample; and CtRs was the Ct value of the internal reference gene from the saRNA-treated sample.

[0115] FIG. 3 shows relative MITF mRNA expression value of different saRNA-treated cells. The control group (Mock) was used as a blank transfection control, the relative mRNA expression value of the siRNA (RAG4-618i) group was knocked down by 81.0% compared to the control group, indicating that the siRNA was successful as a small interfering RNA control transfection. Compared with the control group, the MITF mRNA expression was increased after the cells were treated by different saRNAs, especially the MITF expression was upregulated by 3.9 folds, 3.1 folds and 6.1 folds by RAG4-175, RAG4-176 and RAG4-290, respectively. Other saRNAs (RAG4-284, RAG4-123, RAG4-396, RAG4-461, RAG4-490, RAG4-316 and RAG4-318) increased relative MITF mRNA expression by about 2 folds. This indicated that randomly selected functional saRNAs were capable of being validated for activity in HEM cells.

EXAMPLE 4

saRNA Promoting MITF mRNA Expression in NHEK

[0116] Cell culture was described in Example 2, normal human epidermal keratinocytes (NHEKs) were plated at 20×10.sup.4 cells/well into a 6-well plate, small activating RNAs were transfected at a final concentration of 25 nM for 72 h, and 2 replicate wells were used for each treatment. Two-step RT-qPCR was as described in Example 3.

[0117] FIG. 4 shows relative MITF mRNA expression value of different saRNA-treated cells. The control group (Mock) was used as a blank transfection control, the relative mRNA expression value of the siRNA (RAG4-618i) group decreased by 88.5% compared to the control group, indicating that the siRNA was successful as a small interfering RNA control transfection. Compared with the control group, the siRNA after the treatment of the RAG4-290 had a particularly significant activation effect, and the relative MITF mRNA expression value was increased by 13.1 folds. The relative expression values of mRNA of the RAG4-175, the RAG4-176 and the RAG4-316 groups were higher than those of the control group, the relative expression values of the mRNA in the three groups were increased by 5.7 folds, 3.7 folds and 3.5 folds, respectively, and the siRNAs in the three groups had a significant activation effect. The mRNA relative expression value in the RAG4-284, RAG4-123, RAG4-396, RAG4-461, RAG4-490 and RAG4-318 groups was about 2 folds higher than that in the control group, and the siRNAs in these groups had a certain activation effect. This indicated that randomly selected functional saRNAs were capable of being validated for activity in NHEK cells.

EXAMPLE 5

saRNA Promoting MITF Protein Expression in NHEK

[0118] Cell culture was described in Example 2, normal human epidermal keratinocytes (NHEKs) were plated at 20×10.sup.4 cells/well into a 6-well plate, and small activating RNAs were transfected at a final concentration of 25 nM for 5 days. An appropriate amount of cell lysis solution containing protease inhibitors (1× RIPA buffer, Cell Signaling Technology) were used for lysis. Protein quantification was performed by using the BCA method, polyacrylamide gel electrophoresis separation was then performed, and the protein was transferred to a 0.45 μm PVDF membrane. Rabbit monoclonal anti-MITF (Cell Signaling Technology, #12590) α/β-tubulin antibody (Cell Signaling Technology, 2148s) was used as a primary antibody to detect blots. Anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology) was used as a secondary antibody. Image Lab (BIO-RAD, Chemistry Doctm MP Imaging System) was used to scan detecting signals.

[0119] FIG. 5 shows the relative MITF protein expression level in cells treated with different saRNAs. Compared with the control group, the relative MITF protein expression level in the siRNA (RAG4-618i) group was decreased by about 50%, indicating that siRNA was successful as a small interfering RNA control transfection. Compared with the control group, each saRNA could improve the MITF protein expression level, wherein the MITF protein expression level was regulated by more than 1.5 folds by the RAG4-416, the RAG4-490 and the RAG4-318, particularly the activation effect of the RAG4-318 was most significant, and the MITF protein expression level was increased by 1.71 folds. This indicated that randomly selected functional saRNA can be verified for activity at the protein level.

EXAMPLE 6

saRNA Promoting the MITF Protein Expression in Human Epidermal Melanocytes (HEMs)

[0120] Cell culture was described in Example 2, human epidermal melanocytes (HEMs) were plated at 20×10.sup.4 cells/well into a 6-well plate, and small activating RNAs were transfected at a final concentration of 25 nM for 72 h. Protein lysis and detection methods were as described in Example 5.

[0121] FIG. 6 shows the relative MITF protein expression level in cells treated with different saRNAs. Compared with the control group, each saRNA could improve the MITF protein expression level, and the protein expression level in the RAG4-396 and RAG4-490 groups was increased by 1.4 folds. The the MITF protein expression level in each group of RAG4-175, RAG4-176, RAG4-290, RAG4-284 and RAG4-123 was higher than that in the control group, the activation level was above 1.5 folds, and the activation effect was significant. This indicated that randomly selected functional saRNA can be verified for activity at the protein level.

[0122] Based on the results above, a plurality of human saRNAs capable of activating the MITF gene expression were found through high-throughput screening of saRNAs targeting human MITF gene promoter. Theses saRNAs can upregulate the MITF gene expression in the cell at mRNA and protein expression level, and can be used for a disease or symptom caused by insufficient or decreased MITF protein expression, such as vitiligo, or for preparing a methods or a drug for treating the disease or symptom.

[0123] Various embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selected terms used herein were to best explain the principles of the various embodiments, practical application, or technical improvements to the market, or to enable others of ordinary skill in the art to understand the various embodiments disclosed herein.

REFERENCES

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