阅读说明：本技术 沉默小眼畸形转录因子mRNA表达的siRNA分子 (siRNA molecule for silencing microphthalmia transcription factor mRNA expression ) 是由 季爱民 岑柏宏 黎权辉 黄文� 于 2019-07-16 设计创作，主要内容包括：本发明公开了一组沉默小眼畸形转录因子靶基因mRNA表达的小干扰RNA分子。本发明提供的siRNA的反义链由19-29个核苷酸组成,正义链由15-21个核苷酸组成；正义链与反义链之间有15-19个核苷酸互补组成双链结构。本发明提供的siRNA中的所有核苷酸或部分核苷酸等2’-核糖上可以进行修饰,防止核苷酶降解。本发明提供的siRNA分子或同源性大于60%的分子结构,可单独或联合使用,对靶基因MITF mRNA表达的沉默效率达到40%以上,可治疗MITF突变或过表达引起的疾病或皮肤色素沉着。(The invention discloses a group of small interfering RNA molecules for silencing microphthalmia transcription factor target gene mRNA expression. The antisense strand of the siRNA provided by the invention consists of 19-29 nucleotides, and the sense strand consists of 15-21 nucleotides; 15-19 nucleotides are complementary between the sense strand and the antisense strand to form a double-stranded structure. All nucleotides or part of nucleotides and other 2' -ribose in the siRNA provided by the invention can be modified to prevent nucleosidase degradation. The siRNA molecule or the molecular structure with homology of more than 60 percent can be used independently or jointly, the silencing efficiency on the mRNA expression of the target gene MITF reaches more than 40 percent, and the siRNA molecule or the molecular structure with homology of more than 60 percent can treat diseases or skin pigmentation caused by MITF mutation or overexpression.)

1. an siRNA sequence for silencing microphthalmia transcription factor mRNA expression, wherein the siRNA sequence is a nucleotide sequence of a double-strand structural molecule consisting of a sense strand and an antisense strand, and is characterized in that: the target sequence, the sense strand and the antisense strand of the siRNA sequence are respectively one of the following varieties:

siMITF-m-2:

target sequence: CTGGAAATGCTAGAATATA the flow of the air in the air conditioner,

sense strand: 5 '-CUGGAAAUGCUAGAAUAdTdT-3',

Antisense strand: 5 '-UAUAUUCUUGCAUUCCAGdTdT-3';

siMITF- m-5：

Target sequence: CCTAGAATCAAGTTATAAT the flow of the air in the air conditioner,

Sense strand: 5 '-CCUAGAAUCAAGUUAAAUdTdT-3',

Antisense strand: 5 '-AUUAUAACUUGAUCUAGGdTdT-3';

siMITF- m-8:

target sequence: GAACGAAGAAGAAGATTTA the flow of the air in the air conditioner,

sense strand: 5 '-GAACGAAGAAGAAGAUUUADDT-3',

Antisense strand: 5 '-UAAAUCUUCUUCUUCUUCGUUCGDUTdT-3';

siMITF- m-9:

Target sequence: GAAGAAGATTTAACATAAA the flow of the air in the air conditioner,

Sense strand: 5 '-GAAGAAGAUUAACAAAAdTdT-3',

Antisense strand: 5 '-UUAUGUUAAUAUCUUCUUCdCtdt-3';

siMITF- m-10:

Target sequence: CCACTTTAGCAAATAAACA the flow of the air in the air conditioner,

sense strand: 5 '-CCACUUAGCAAAUAAACAdTdT-3',

antisense strand: 5 '-UGUUAUUGCUAAAGUGGdTdT-3'.

2. The siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 1, wherein: the method is characterized in that a Western blot method is adopted to test one of the following siRNA sequences consisting of a target sequence, a sense strand and an antisense strand with higher silencing efficiency:

siMITF- m-5：

Target sequence: CCTAGAATCAAGTTATAAT the flow of the air in the air conditioner,

sense strand: 5 '-CCUAGAAUCAAGUUAAAUdTdT-3',

Antisense strand: 5 '-AUUAUAACUUGAUCUAGGdTdT-3';

siMITF- m-8:

target sequence: GAACGAAGAAGAAGATTTA the flow of the air in the air conditioner,

Sense strand: 5 '-GAACGAAGAAGAAGAUUUADDT-3',

antisense strand: 5 '-UAAAUCUUCUUCUUCUUCGUUCGDUTdT-3';

siMITF- m-9:

Target sequence: GAAGAAGATTTAACATAAA the flow of the air in the air conditioner,

Sense strand: 5 '-GAAGAAGAUUAACAAAAdTdT-3',

Antisense strand: 5 '-UUAUGUUAAAAUCUUCUUCDdTdT-3'.

3. the siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 1 or 2, wherein: the siRNA sequence consisting of the following target sequence, sense strand and antisense strand with high silencing efficiency is tested by adopting an RT-qPCR screening method and a Western blot method:

siMITF-5：

target sequence: CCTAGAATCAAGTTATAAT the flow of the air in the air conditioner,

sense strand: 5 '-CCUAGAAUCAAGUUAAAUdTdT-3',

antisense strand: 5 '-AUUAUAACUUGAUCUAGGdTdT-3'.

4. the siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 1, wherein:

the sequence of the sense strand of the siRNA molecule has more than 60 percent of homology with the sequence of the sense strand of the siRNA molecule, or the sequence of the antisense strand of the siRNA molecule has more than 60 percent of homology with the sequence of the antisense strand of the siRNA molecule; and a double-stranded structure with 15-19 nucleotide sequences as complementarity is arranged between the sense strand and the antisense strand.

5. The siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 4, wherein: the target sequence, sense strand and antisense strand of the siRNA sequence are as follows:

siMITF-5X：

Target sequence: CCTAGAATCAAGTTAT the flow of the air in the air conditioner,

Sense strand: 5'-CCUAGAAUCAAGUUAU-3' the flow of the air in the air conditioner,

antisense strand: 5 '-AUUAUAACUUGAUUUCUGUAGGCUdTdT-3'.

6. The siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 4, wherein: ribose of 3-5 nucleotides is modified by 2 '-methoxy or 2' -fluoro.

7. The siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 6, wherein: ribose in the first 3 nucleotides in the sense strand is modified by 2' -methoxyl; ribose in the 2 nd nucleotide in the antisense chain is modified by 2' -fluorine; sense and antisense strands of the resulting siRNA sequences:

siMITF-10X：

Sense strand: (mC) (mC) (mA) CUUUAGCAAAUAAA,

antisense strand: (mU) (nG) (mU) UUAUUUGCUAAAG(mU)(mG)(mG) dTdT.

8. the siRNA sequence for silencing microphthalmia transcription factor mRNA expression according to claim 1, 4 or 6, wherein: the sense strand or the antisense strand, or two or three adjacent nucleotides in the double strand are modified by phosphorothioate linkage; sense and antisense strands of the resulting siRNA sequences:

siMITF-8X：

Sense strand: GAACGAAGAAGAAGAUUU to A to dTdT,

antisense strand: u to A to AAUCUUCUUCUUCG to U to CdTdT.

9. Use of an siRNA sequence of any of claims 1 to 8, wherein: can be used for treating diseases caused by MITF mutation or overexpression.

10. Use of the siRNA sequence of claim 7 wherein: can be used for treating melanoma or skin pigmentation caused by MITF mutation or overexpression.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a siRNA sequence expressed by a silent gene or protein and application thereof.

Background

Microphthalmia-Associated Transcription Factor (MITF) has a typical helix-loop-helix-leucine zipper structure (bHLXZp) and belongs to the MiT Transcription Factor superfamily (Tachiba M, et al, MITF: a stream flowing for Transcription cells. Pigment Cell Res. 2000;13(4): 230-. MITF has been found to be expressed primarily in pigmented cells, including melanoblasts (melanoblasts) and retinal pigment epithelial cells (RPEs), as well as in other cells such as mast cells and osteoclasts (Carreira S, et al. MITF regulation of Dial control melanomas promotion and invasion. Genes Dev.2006;20(24): 3426-.

Melanocytes of the skin are derived from the neural crest, and human skin has a large number of mature melanocytes which have division-sharing and bactericidal effects and are resistant to ultraviolet light and other external stimuli, and MITF is deeply involved in the differentiation (Slominski A. Neuroendocrine activity of the melanocyte, Experimental Dermatology, 2009;18 (9) 760-763.).

The research finds that the MITF is involved in the pigmentation process and directly acts on the transcriptional regulation of genes related to the pigmentation. MITF can activate the expression of tyrosinase (tyrosinase, tyrr), tyrosinase-related protein 1(TYRP 2), tyrosinase-related protein 1(TYRP 2), and other genes in melanocytes.

melanin is a group of natural pigments derived from melanocytes under the epidermis. Melanin protects human skin from the pressure of harmful ultraviolet radiation (UVR) and environmental pollutants, toxic drugs and chemicals of diverse origins. The abnormally high production of melanin or melanin production by skin melanocytes can lead to pigmentation disorders such as chloasma, age spots, freckles, and pigmented acne scars. Melanogenesis is a multistep physiological process leading to the synthesis of a complex dark biopolymer called "melanin," a lysosome-associated organelle in melanocytes that helps protect the skin from the harmful effects of sunlight, toxic drugs and chemicals.

There are two types of melanin: eumelanin (eumelanin) and pheomelanin. During melanin production, there are 3 major enzymes involved: tyrosinase (TYR), tyrosinase-related protein 1(Tyrp 1) and tyrosinase-related protein 2(Tyrp 2). All melanin production signaling pathways are associated with the major regulator MITF (microphthalmia-associated transcription factor) for melanogenesis, which regulates the expression of melanin-producing genes TYR, Tyrp1, and Tyrp 2. The key rate-limiting cytokine in the process of melanin generation and signal transduction pathway is MITF, which can inhibit the expression of MITF and has the effects of inhibiting skin pigmentation and whitening skin.

in humans, MITF controls the expression of various genes essential for normal melanin synthesis in melanocytes, and mutations in MITF can lead to diseases such as melanoma, Waardenburg syndrome, and Tietz syndrome (Hodgkinson CA, et al, Mutation at the mouse microbial cancer arm associated with defects in a gene encoding a novel basic-position-loop-helix-linker protein. cell.1993;74(2): 395-404.).

In addition, MITF is involved in biological activities such as cell cycle, differentiation, senescence, apoptosis, proliferation, and migration. The MITF is highly expressed in a melanoma cell line, and promotes the expression of a proliferation-related transcription factor T-box 2, thereby promoting the proliferation of the melanoma. In addition, MITF regulates cell cycle-dependent protein kinase 2 (cyclin-dependent kinase, CDK 2) and cyclin-dependent kinase inhibitor 1A/B (cyclin-dependent kinase inhibitor 1A/B, CDKN 1A/B), thereby regulating the cell growth cycle. MITF promotes tumor development by up-regulating anti-apoptotic proteins such as B cell lymphoma factor-2 (B cell lymphoma-2, Bcl-2) and C-MET. Hypoxia inducible factor-1 alpha (hypoxia inducible factor-1 alpha) has an anti-apoptotic effect in melanoma and can stimulate the production of Vascular Endothelial Growth Factor (VEGF). The research shows that the MITF can transcriptionally activate HIF-1 alpha, thereby promoting the angiogenesis of the tumor. Other cytokines such as dipeptidyl transformed for min 1, Dial, miR-211 and beta-catenin, protein-encoding genes closely related to Melanoma infiltration are also regulated by MITF (Cheli Y, et al. oncogene. 2012; 31 (19): 2461-. Therefore, the gene expression of the MITF is inhibited, so that the function of the MITF is inhibited, and the application prospect of treating the melanoma is realized.

MITF is an important transcription factor that plays an essential role in the life activity of an organism, affecting the growth, development, differentiation and various stages of death of the organism, and has important biological significance particularly on hair formation of animals. The role of MITF in the regulation of melanocyte and pigment production is currently considered to be particularly important.

Small interfering RNA (siRNA) can specifically silence the expression level of target mRNA (Nobel biomedical prize 2006), so that the siRNA molecule can treat diseases caused by gene overexpression and gene mutation by specifically silencing genes caused or promoting disease occurrence or development (Science, 2016,352(6292): 1417-.

among the siRNA duplexes, the single strand that forms an RNA Interference Silencing Complex (RISC) with the Argonaute protein (AGO protein) is called the Antisense strand (Antisense strand) or Guide strand (Guide strand), and the RISC complex formed by the strand binds to, cleaves and silences the target mRNA. While the other non-functional single strand is called the sense strand (sense strand) or the Passenger strand (Passenger strand). Using the characteristics of siRNA molecules, it is possible to design siRNA sequences for multiple silent target genes (Nature Reviews Genetics,2015,16(9): 543-552).

disclosure of Invention

The purpose of the invention is as follows:

Provides an sIRNA capable of effectively silencing the expression level of MITF mRNA expressed by melanocytes and melanoma cells and application thereof.

The technical scheme is as follows:

In the invention, an RNAi principle is adopted, an siRNA sequence for specifically silencing the MITF mRNA expression is designed and screened, and the obtained siRNA is used for inhibiting the function of the MITF, thereby realizing the effects of treating diseases caused by MITF overexpression or mutation, removing skin pigmentation and the like.

the invention discloses a siRNA (small interfering RNA molecule) sequence for silencing microphthalmia-related transcription factor mRNA expression, wherein the microphthalmia-related transcription factor (MITF) is expressed in melanoma cells and skin melanocytes, and the siRNA sequence is any one of nucleotide sequences with the following target sequence, sense strand and antisense strand:

siMITF-M-2

Target sequence: CTGGAAATGCTAGAATATA the flow of the air in the air conditioner,

Sense strand: 5 '-CUGGAAAUGCUAGAAUAdTdT-3',

Antisense strand: 5 '-UAUAUUCUUGCAUUCCAGdTdT-3'.

Target sequence: CCTAGAATCAAGTTATAAT the flow of the air in the air conditioner,

sense strand: 5 '-CCUAGAAUCAAGUUAAAUdTdT-3',

Antisense strand: 5 '-AUUAUAACUUGAUCUAGGdTdT-3'.

target sequence: GAACGAAGAAGAAGATTTA the flow of the air in the air conditioner,

Sense strand: 5 '-GAACGAAGAAGAAGAUUUADDT-3',

antisense strand: 5 '-UAAAUCUUCUUCUUCUUCGUUCGDUCTTdT-3'.

siMITF-M-9

Target sequence: GAAGAAGATTTAACATAAA the flow of the air in the air conditioner,

Sense strand: 5 '-GAAGAAGAUUAACAAAAdTdT-3',

antisense strand: 5 '-UUAUGUUAAAAUCUUCUUCDdTdT-3'.

target sequence: CCACTTTAGCAAATAAACA the flow of the air in the air conditioner,

Sense strand: 5 '-CCACUUAGCAAAUAAACAdTdT-3',

Antisense strand: 5 '-UGUUAUUGCUAAAGUGGdTdT-3'.

Wherein A is adenine ribonucleotide; g is guanine ribonucleotide; c is cytosine ribonucleotide; u is uracil ribonucleotide; dT is thymine deoxynucleotide.

An siRNA molecule, wherein a sense strand of the siRNA molecule has more than 60% homology with a sense strand sequence in the siRNA molecule, or both strands have more than 60% homology; or has more than 60% homology with the antisense strand sequence in the siRNA molecule, or has more than 60% homology with double strands; the sense strand and the antisense strand form a double-stranded siRNA structure. Of the above sequences, the 19 nucleotide sequences after the 5 'end of the sense strand were complementary to the 19 nucleotide sequences before the 5' end of the antisense strand. Similar or better silencing efficiency can be achieved.

For example, siMITF-5X

Target sequence: CCTAGAATCAAGTTAT the flow of the air in the air conditioner,

Sense strand: 5'-CCUAGAAUCAAGUUAU-3' the flow of the air in the air conditioner,

antisense strand: 5 '-AUUAUAACUUGAUUUCUGUAGGCUdTdT-3'.

in the siRNA, ribose or deoxyribose in all nucleotides or part of nucleotides is subjected to 2 '-methoxy modification or 2' -fluoro modification (the modified group is small, so that the space occupied by molecular chains is not too large, the arrangement is disordered, and the activity of atomic groups is large, so that the silencing efficiency is improved); the internucleotide backbone can be modified by phosphorothioate (the modification increases the lipid solubility of siRNA, and the siRNA can smoothly enter cells, so that the silencing efficiency is improved).

The siRNA molecules are used independently or jointly, and the silencing efficiency on the expression of the MITF mRNA of the target gene reaches more than 40%.

Can be used alone or in combination for treating diseases caused by MITF mutation or overexpression, such as melanoma or skin pigmentation.

Using a control sequence: SimITF is a positive control siRNA sequence, and sinC is a negative control siRNA sequence.

in the siRNA sequences expressed by candidate silent MITF mRNA obtained by design and synthesis, the nucleotide sequences contained in the siRNA target sequence, sense strand and antisense strand are respectively as follows, and are shown in Table 1:

TABLE 1 siRNA sequences for candidate silencing of MITF mRNA expression

。

in the invention, the siRNA is obtained by adopting an siRNA design rule, combining with computer aided design software and screening through a comparison experiment.

On the basis, through RT-qPCR screening, the gene silencing efficiency of each candidate siRNA molecule is shown in figure 1, and siMITF-2, siMITF-5, siMITF-8, siMITF-9 and siMITF-10 with the gene silencing efficiency of more than 40 percent are obtained and used for biological function research.

drawings

FIG. 1 shows the results of the expression level of MITF mRNA in human melanocytes A375 after transfection of siRNA with lipofectamine 2000 in RT-qPCR assay. In fig. 1, the ordinate chinese meaning: MITF mRNA relative expression level. This shows how much more MITF mRNA is expressed in the cells after siRNA transfection; lower expression levels indicate more efficient silencing of MITF mRNA expression by the added siRNA molecule. The abscissa is 14 different siRNA molecular species.

FIG. 2 is a picture of Western blot detection of protein level silencing efficiency of siMITF.

Detailed Description