CN107904239B

CN107904239B - siRNA for inhibiting ADAMTS-5 gene and application thereof

Info

Publication number: CN107904239B
Application number: CN201711239584.8A
Authority: CN
Inventors: 张必良; 米其·托尔托雷; 王喆; 杨秀群; 王秋云
Original assignee: Guangzhou Ribobio Co ltd
Current assignee: Agna biopharmaceutical Co.,Ltd.
Priority date: 2014-12-25
Filing date: 2014-12-25
Publication date: 2020-11-27
Anticipated expiration: 2034-12-25
Also published as: CN104560999A; CN107904239A; CN104560999B

Abstract

The invention discloses siRNA for inhibiting ADAMTS-5 genes and application thereof. The invention discloses a chemically modified double-stranded siRNA molecule, which is formed by complementing at least one strand of siRNA molecules shown in the following (1) to (13) through any chemical modification shown in the following (A). The siRNA disclosed by the invention can be used as a therapeutic drug for arthritis and related inflammation, and can realize the treatment of the arthritis by inhibiting the expression of inflammatory factors through local injection of the siRNA and a preparation thereof in a bone joint cavity.

Description

siRNA for inhibiting ADAMTS-5 gene and application thereof

The present application is a divisional application having an application number of 201410828587.5, an application date of 2014.12.25, and an invention creation title of "s iRNA inhibiting ADAMTS-5 gene and use thereof".

Technical Field

The invention relates to siRNA for inhibiting ADAMTS-5 gene and application thereof, belonging to the technical field of biology.

Background

Osteoarthritis (OA) is a chronic degenerative osteoarthropathy that seriously harms human health and currently lacks an effective treatment means, and a new method for effectively preventing and treating Osteoarthritis is urgently needed to be researched. One of the clinical pathological features of osteoarthritis is cartilage destruction and hFLS extracellular matrix degradation, which ultimately results from proteolysis of the extracellular matrix. Abnormal degradation of the secondary extracellular matrix (ECM) caused by increased activity of various proteolytic enzymes is therefore a direct cause of cartilage degeneration, ultimately leading to cartilage destruction overlying the articular bone surface. Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have the effects of promoting the catabolism of hFLS cells and degrading extracellular matrix, and studies have shown that high concentrations of IL-1 and TNF-alpha are found in synovial fluid of arthritis patients, and are considered as proinflammatory cytokines playing a key role in the pathogenesis of arthritis.

The disaggregated protein-like metalloprotease family (ADAMTS) containing type I platelet binding protein motifs consists of a group of structurally similar secreted zinc binding proteases, 19 members of which have been found so far. The protein structures of the protease and the protease have high similarity, the proteins are all in a proprotein domain structure after an amino-terminal signal peptide sequence, and the proprotein needs to be subjected to post-translational cleavage in cells to become active protease. And they all contain at least one conserved TSP 1-like repeat motif. The TSP-like repeat motif located at the carboxy-terminus of the protease is an important part of the binding to extracellular matrix components. The ADAMTS family uses a variety of extracellular matrix components as substrates in vivo, and they have important functions in physiological processes such as embryonic development, angiogenesis, coagulation, inflammatory reaction, and the like.

ADAMTS-5 (Deploymerin-like metalloprotease family 5 containing type I platelet binding protein motifs) is a degrading enzyme of the cartilage matrix protein proteoglycan aggrecan. Articular cartilage contains a large amount of aggrecan, which plays an important role in the physical properties of the joint such as stress resistance and tension. Substantial degradation destruction of aggrecan is found in osteoarthritis and rheumatoid arthritis patients. There are two major cleavage sites in the agrecen domain, one of which is the MMPs cleavage site, located at Asn341 and Phe 342; and the other is a proteoglycan enzyme aggrecanase cleavage site, located at Glu373 and Ala 374. ADAMTS-5 was localized to human 21q21-q 22. The discovery of the proteoglycan enzyme, which is a new target for treating arthritis, enables the treatment of arthritis to be expected to make a new breakthrough, and provides a foundation for fundamentally treating and preventing arthritis. Adipose tissue can also produce ADAMTS-5. The change of the expression level of ADAMTS-5 and its substrate aggrecan can be seen in the formation and differentiation of the adipocyte, and the aggrecan can stimulate the differentiation and maturation of the adipocyte in vitro and in vivo, which suggests that ADAMTS-5 plays an important role in the formation of the adipocyte. In malignant glioma, the expression level of ADAMTS-5 is obviously increased, and invasion and metastasis of glioma are mediated by degrading agrecen. In addition, the proteolytic enzymes are known to play a role in other diseases in which extracellular protein degradation or destruction occurs, such as cancer, asthma, chronic obstructive pulmonary disease, atherosclerosis, age-related macular degeneration, myocardial infarction, corneal ulceration and other ocular surface diseases, hepatitis, aortic aneurysm, tendonitis, central nervous system diseases, abnormal wound healing, angiogenesis, restenosis, liver cirrhosis, multiple sclerosis, glomerulonephritis, graft versus host disease, diabetes, inflammatory bowel disease, shock, disc degeneration, stroke, osteopenia and periodontal disease.

ADAM17 (depolymerin-metalloprotease 17) is a member of the ADAMs family (depolymerin and metalloprotease) which has been discovered in recent years and is a family of cell membrane surface glycoproteins with various functions, and is commonly involved in various physiological processes such as adhesion between cells and matrix, cell fusion, degradation of extracellular matrix, and signal transduction, and migration of leukocytes. In addition, they are involved in pathological processes such as tumor formation, proliferation and metastasis [ Mochizuki S ]. ADAM17 is called TNF-. alpha.converting enzyme (TACE) because it cleaves membrane-bound TNF-. alpha.to generate free TNF-. alpha.. Free form of TNF- α causes excessive secretion of inflammatory cytokines, apoptosis, disorder of intracellular signaling, etc., resulting in various diseases including Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), multiple sclerosis, acute infectious diseases, asthma, atopic dermatitis, psoriasis, etc. In addition to TNF- α as a substrate, ADAM17 has been regulated by ADAM17 as macrophage colony stimulating factor or chemokine FKN. Therefore, a compound inhibiting ADAM17 is considered to be promising as a therapeutic agent for inflammatory diseases. However, the metalloprotease family is highly conserved, and the development of selective small molecule inhibitors has proven to be a very great challenge. Previous trials using broader-spectrum metalloprotease inhibitors have been shown to be tissue toxic, and therefore the development of highly selective ADAM17 inhibitors (Moss, 2008) is a problem to be solved.

In 1998, gene silencing was discovered by Kreger Merlow and Andrew Fall, and then Tuschl and his colleagues discovered chemically synthesized 19-25 base small interfering RNA (siRNA) in mammalian cells, which specifically and efficiently silences target mRNA. Since then, siRNA is widely used for gene function research and disease treatment. The siRNA can specifically bind to and degrade a target mRNA that is complementary to the sequence. The double-stranded RNA of the long fragment is cut into short fragment RNA of 21-23 bases by Dicer enzyme. Of the two strands, the strand that binds to the target mRNA is called the antisense strand, and the other strand is called the sense or messenger strand. After entering cells, siRNA chemically synthesized in vitro also plays an RNA interference role, and effectively reduces immune response caused by long-chain RNA. But various siRNAs can be designed aiming at different fragment positions of the same gene, and the silencing effect has obvious difference.

Disclosure of Invention

The invention aims to provide siRNA for inhibiting ADAMTS-5 genes and application thereof.

The invention provides a chemically modified double-stranded siRNA molecule, which is formed by complementing at least one strand of siRNA molecules shown in the following A after any chemical modification shown in the following (1) to (13):

A. an siRNA molecule which is shown in the following 1) or 2):

1) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 2;

2) the double-stranded siRNA molecule is formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 1;

or, the double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 2;

or, the double-stranded siRNA molecule is formed by complementing an RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.2 and an RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No. 1;

(1) phosphorothioate modification of the phosphate backbone;

the thiophosphoric acid is modified by replacing a P-OH bond with a P-S bond of a phosphate skeleton;

(2) 2' -methoxy modification of ribose or deoxyribose;

(3) 2' -fluoro modification of ribose or deoxyribose;

(4) modifying locked nucleic acid;

the locked nucleic acid is modified into a 2 '-O site and a 4' -C site of ribose or deoxyribose to form a ring structure through the action of shrinkage;

(5) open loop nucleic acid modification;

the ring-opened nucleic acid is modified into a C-C bond break between 2 '-C and 3' -C of ribose or deoxyribose;

(6) modifying indole;

the indole is modified to replace a base by an indole;

(7) 5-methylcytosine modification of a base;

(8) 5-ethynyluracil modification of the base;

(9) single-stranded 5' terminal cholesterol modification;

(10) single-chain 3' end galactose modification;

(11) single chain 5' end polypeptide modification;

the polypeptide is specifically a polypeptide with the sequence from the N end to the C end being Arg-Gly-Asp;

(12) single-stranded 5' end phosphorylation modification;

(13) modifying the single-chain 5' end by fluorescent labeling;

the fluorescent label is specifically a Cy series fluorescent label;

the double-stranded siRNA molecule formed by complementing the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.2 and the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.1 is specifically a double-stranded siRNA molecule formed by complementing the single strand shown in SEQ ID No.3 and the single strand shown in SEQ ID No. 4.

In the chemically modified double-stranded siRNA molecule, the sense strand and the antisense strand of the double-stranded siRNA molecule complementary after chemical modification have the following sequences B1 and C1, respectively:

B1、5'-K-LLMUUUAUGUGGGCAUPMQdTdT-3’；

C1、5'-R-MQLAUGCCCACAUAAAQPPdTdT-3’；

the K is unmodified or modified by 5' end cholesterol;

the R is a 5' terminal phosphorylation modification;

the dT is thymine deoxyribonucleotide;

l, M, P and Q are respectively guanine deoxyribonucleotide modified by 2 '-methoxy of deoxyribose, adenine deoxyribonucleotide modified by 2' -methoxy of deoxyribose, cytosine deoxyribonucleotide modified by 2 '-methoxy of deoxyribose and uracil ribonucleotides modified by 2' -methoxy of ribose;

or the like, or, alternatively,

l, M, P and Q are guanine deoxyribonucleotide modified by 2 '-methoxy modification of deoxyribose and phosphorothioate modification of phosphate skeleton, adenine deoxyribonucleotide modified by 2' -methoxy modification of deoxyribose and phosphorothioate modification of phosphate skeleton, cytosine deoxyribonucleotide modified by 2 '-methoxy modification of deoxyribose and phosphorothioate modification of phosphate skeleton, and uracil ribonucleotides modified by 2' -methoxy modification of ribose and phosphorothioate modification of phosphate skeleton respectively;

the antisense strand is a single strand that binds to the mRNA of ADAMTS-5 (Deplosin-like metalloprotease family-5 containing a type I platelet binding protein motif), and the sense strand is a single strand that binds complementarily to the antisense strand.

An siRNA molecule also belongs to the protection scope of the invention, and is shown as the following (1) or (2):

(1) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 2;

(2) the double-stranded siRNA molecule is formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 1;

the siRNA molecule is applied to preparing products for preventing and/or treating inflammation;

the inflammation is in particular inflammation of the joints, and more in particular osteoarthritis.

The carrier containing the siRNA molecule also belongs to the protection scope of the invention;

the carrier is a cationic liposome, chitosan nanoparticles, polypeptide and a polymer material.

In the siRNA molecules, the double-stranded siRNA molecule formed by complementing the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.2 and the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.1 is a double-stranded siRNA molecule formed by complementing the single strand shown in SEQ ID No.3 and the single strand shown in SEQ ID No. 4.

A DNA molecule capable of producing the siRNA molecule also belongs to the protection scope of the invention;

the siRNA molecule is specifically a double-stranded siRNA molecule formed by complementing an RNA single strand shown in SEQ ID No.1 and an RNA single strand shown in SEQ ID No. 2;

the DNA molecule is specifically a DNA molecule containing 36 th to 54 th nucleotides from the 5' end in SEQ ID No.6, and is specifically a DNA molecule containing double-stranded DNA formed by complementing a DNA single strand shown in SEQ ID No.5 and a DNA single strand shown in SEQ ID No.6, more specifically a recombinant siRNA expression plasmid obtained by replacing a sequence between BamHI and HindIII enzyme cutting sites of pGCsi-H1/Neo and keeping the rest sequences of pGCsi-H1/Neo with the molecule of the double-stranded DNA formed by complementing the DNA single strand shown in SEQ ID No.5 and the DNA single strand shown in SEQ ID No. 6.

Also included in the scope of the present invention is a chemically modified double-stranded siRNA molecule composition comprising a double-stranded siRNA molecule represented by the following (1) and (2):

(1) at least one double stranded siRNA molecule of any of the chemically modified double stranded siRNA molecules described above;

(2) at least one double-stranded siRNA molecule in the double-stranded siRNA molecules shown in the following A 'or B', wherein at least one strand of the double-stranded siRNA molecules is subjected to any chemical modification shown in the following 1) -13) and then is complemented:

a', the RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No.8 are complemented to form a double-stranded siRNA molecule;

b', the RNA single strand shown in SEQ ID No.8 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No.7 are complemented to form a double-stranded siRNA molecule;

or, the double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.7 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 8;

or, the double-stranded siRNA molecule is formed by complementing an RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.7 and an RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No. 8;

1) phosphorothioate modification of the phosphate backbone;

the thiophosphoric acid is modified by a P-S bond to replace a P-OH bond;

2) 2' -methoxy modification of ribose or deoxyribose;

3) 2' -fluoro modification of ribose or deoxyribose;

4) modifying locked nucleic acid;

5) open loop nucleic acid modification;

6) modifying indole;

the indole is modified to replace a base by an indole;

7) 5-methylcytosine modification of a base;

8) 5-ethynyluracil modification of the base;

9) single-stranded 5' terminal cholesterol modification;

10) single-chain 3' end galactose modification;

11) single chain 5' end polypeptide modification;

12) single-stranded 5' end phosphorylation modification;

13) modifying the single-chain 5' end by fluorescent labeling;

the fluorescent label is specifically a Cy series fluorescent label;

the active ingredients of the chemically modified double-stranded siRNA molecule composition are the double-stranded siRNA molecules shown in (1) and (2), the double-stranded siRNA molecules shown in (1) and (2) can be packaged separately or in a mixed manner, and the molar ratio of the double-stranded siRNA molecules shown in (1) and (2) is specifically 1: 1;

the double-stranded siRNA molecule formed by complementing the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.7 and the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.8 is specifically a double-stranded siRNA molecule formed by complementing the single strand shown in SEQ ID No.9 and the single strand shown in SEQ ID No. 10;

the chemically modified double-stranded siRNA molecule composition specifically comprises at least one siRNA molecule in the chemically modified double-stranded siRNA molecules and at least one siRNA molecule in the siRNA molecules with a sense strand shown as F1 and an antisense strand shown as G1:

F1、5'-K’-L’P’M’UCAUGUAUCUGAA P’M’M’dTdT-3’；

G1、5'-R’-Q’Q’L’UUCAGAUACAUGA Q’L’P’dTdT-3’；

the K 'is a 5' end cholesterol modification;

the R 'is no modification or 5' terminal phosphorylation modification;

the dT is thymine deoxyribonucleotide;

the L ', M', P 'and Q' are respectively guanine deoxyribonucleotide modified by 2 '-methoxyl of deoxyribose, adenine deoxyribonucleotide modified by 2' -methoxyl of deoxyribose, cytosine deoxyribonucleotide modified by 2 '-methoxyl of deoxyribose and uracil ribonucleotides modified by 2' -methoxyl of ribose;

or the like, or, alternatively,

l ', M', P 'and Q' are guanine deoxyribonucleotide, adenine deoxyribonucleotide, cytosine deoxyribonucleotide and uracil ribonucleotides respectively;

the antisense strand is a single strand that binds to ADAM17 (depolymerin-metalloprotease 17) mRNA, and the sense strand is a single strand that binds complementarily to the antisense strand.

A siRNA molecule composition also belongs to the protection scope of the invention, and comprises at least one of siRNA molecules shown as the following H and at least one of siRNA molecules shown as the following I:

h is at least one siRNA molecule in the siRNA molecules;

i is at least one siRNA molecule in the siRNA molecules shown in the following (1) and/or (2):

(1) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No. 8;

(2) the double-stranded siRNA molecule is formed by complementing the RNA single strand shown in SEQ ID No.8 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 7;

the siRNA molecule composition is applied to preparing products for preventing and/or treating inflammation;

the inflammation is in particular arthritis, and more in particular osteoarthritis;

the siRNA molecule composition specifically comprises the following double-stranded siRNA molecules 1) and 2):

1) a double-stranded siRNA molecule formed by complementing the single strand shown in SEQ ID No.3 and the single strand shown in SEQ ID No. 4;

2) the single strand shown in SEQ ID No.9 and the single strand shown in SEQ ID No.10 are complemented to form the double-stranded siRNA molecule.

The carrier containing the siRNA molecule composition also belongs to the protection scope of the invention;

A DNA molecule capable of producing the siRNA molecule composition also belongs to the protection scope of the invention;

the siRNA molecule composition specifically comprises double-stranded siRNA molecules shown in the following (1) and (2):

(2) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No. 8;

the DNA molecule is a DNA molecule composition containing DNA molecules of 36 th to 54 th nucleotides from 5 'end in SEQ ID No.6 and DNA molecules of 38 th to 56 th nucleotides from 5' end in SEQ ID No.11, and is a DNA molecule composition containing double-stranded DNA molecules formed by complementing a DNA single strand shown in SEQ ID No.5 and a DNA single strand shown in SEQ ID No.6 and double-stranded DNA molecules formed by complementing a DNA single strand shown in SEQ ID No.11 and a DNA single strand shown in SEQ ID No.12, more specifically, a DNA molecule of double-stranded DNA formed by complementing a DNA single strand shown in SEQ ID No.5 and a DNA single strand shown in SEQ ID No.6 replaces a sequence between BamHI and HindIII enzyme cutting sites of pGCsi-H1/Neo, and a recombinant siRNA expression plasmid 1 obtained by unchanging the rest sequences of pGi-H1/Neo and a double-stranded DNA molecule formed by substituting a DNA single strand shown in pGCsi-H1/Neo and complementary single-stranded DNA molecules of pGCsi-H1/Neo And the sequence between the BamHI and HindIII enzyme cutting sites and the rest sequence of pGCsi-H1/Neo are not changed to obtain the recombinant siRNA expression plasmid 2.

Also within the scope of the present invention is a kit comprising any of the chemically modified double stranded siRNA molecules described above, any of the siRNA molecules described above, the DNA molecules described above, the chemically modified double stranded siRNA molecule composition described above, the siRNA molecule composition described above, and/or the DNA molecules described above;

the kit also comprises an instruction recorded on the readable carrier, and the instruction is as follows: injecting any one of the chemically modified double-stranded siRNA molecules or the preparation thereof into a site where inflammation occurs, the siRNA molecules or the preparation thereof obtained by chemically modifying any one of the siRNA molecules, the siRNA molecules or the preparation thereof obtained by chemically modifying the RNA molecules generated by the DNA molecules, the double-stranded siRNA molecule composition or the preparation thereof obtained by chemically modifying the siRNA molecule composition, the siRNA molecules or the preparation thereof obtained by chemically modifying the siRNA molecule composition, and/or the siRNA molecules or the preparation thereof obtained by chemically modifying the RNA molecules generated by the DNA molecules.

The application of any chemically modified double-stranded siRNA molecule described in the above, any siRNA molecule described in the above, the above DNA molecule, the above chemically modified double-stranded siRNA molecule composition, the above DNA molecule and/or the above kit in the preparation of a product for preventing and/or treating inflammation also belongs to the protection scope of the invention;

or the like, or, alternatively,

use of any of the chemically modified double stranded siRNA molecule described above, any of the siRNA molecule described above, the DNA molecule described above, the chemically modified double stranded siRNA molecule composition described above, the DNA molecule described above and/or the kit described above in the manufacture of a product as described in any of W1-W5:

w1, product for inhibiting fibrosis of the articular surface;

w2, a product to inhibit cartilage erosion;

w3, a product for the prevention and/or treatment of synovitis;

w4, a product that protects cartilage and/or synovium;

w5, product for preventing and/or treating rheumatoid arthritis.

The siRNA provided by the invention can be used as a therapeutic drug for arthritis and related inflammation, and can realize the treatment of the arthritis by locally injecting the siRNA into a bone joint cavity or inhibiting the expression of an inflammatory factor by using a preparation of the siRNA.

Drawings

FIG. 1 is a screen of effective siRNA for inhibiting ADAMTS-5 gene.

FIG. 2 is an immunoblot experiment of siRNA-RB-04.

FIG. 3 shows siRNA-RB-04 down-regulates inflammatory factors.

FIG. 4 shows the effect of siRNA structure on the silencing effect of a target gene.

FIG. 5 is a schematic diagram showing the modification position of phosphorothioate (P-S bond).

FIG. 6 shows that chemical modification enhances the serum stability of oligonucleotide.

FIG. 7 is a histopathological section analysis of rats.

FIG. 8 shows the contents of inflammatory factors in rat synovial fluid.

FIG. 9 is a screen of effective siRNAs inhibiting ADAM17 gene.

FIG. 10 shows siRNA-AD-08 immunoblot assay.

FIG. 11 shows siRNA-AD-08 down-regulates inflammatory factors.

FIG. 12 is a graph showing the effect of siRNA structure on the silencing effect of a target gene.

FIG. 13 shows that chemical modification enhances the serum stability of oligonucleotide.

FIG. 14 is a histopathological section analysis of rats.

FIG. 15 shows the contents of inflammatory factors in rat synovial fluid.

FIG. 16 shows the down-regulation of inflammatory factors by the combination of siRNA.

FIG. 17 is a rat histopathological section analysis.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

hFLS cells (human fibroblast-like synoviocytes) are the Cell Applications products, catalog # 408-05 a.

293T cells are ATCC products with catalog number CRL-3216.

MCF-7 cells were ATCC product, catalog number HTB-22.

The Human IL-1 beta immunoassay detection kit is an AssayPro product, and the catalog number of the product is EI 2200-1.

pGCsi-H1/Neo in the literature, "Quzhou nationality, Zhang invention, yellow eosin et al. construction and identification of Smad4/DPC4 Gene Small hairpin RNA plasmid expression vector [ J ]. proceedings of medical research, 2006, 19 (11): 973-.

The Lipofectamine2000 kit is Invitrogen.

Male SD rats (220 + -20 g) are central products of medical laboratory animals in Guangdong province.

The siRNA in the following examples are double-stranded siRNA molecules, and the solvent of each injection solution injected into rats is PBS.

Example 1 screening of effective oligo-nucleic acids inhibiting the expression of mRNA of ADAMTS-5 Gene

First, siRNA design was performed to determine siRNA targeting ADAMTS-5, and bioinformatic screens were performed to ensure that the sequences were specific for ADAMTS-5 sequences and not specific for sequences from any other gene. The target sequence was checked against the sequences in GenBank using the BLAST search engine provided by NCBI and 8 effective siRNAs were screened out by preliminary experiments, named siRNA-RB-01, siRNA-RB-02, siRNA-RB-03, siRNA-RB-04, siRNA-RB-05, siRNA-RB-06, siRNA-RB-07, and siRNA-RB-08, respectively. The siRNA is designed aiming at different positions of ADAMTS-5 gene sequences.

Second, cell transfection

The experiment is divided into 10 groups, namely an siRNA-RB-01 to siRNA-RB-08 experiment group, a No Target (NTC) negative control group and an NC blank control group.

The setting method of the siRNA-RB-01 to siRNA-RB-08 experimental groups is as follows:

the hFLS cells were digested with 0.25% pancreatin and prepared in DMEM medium to a concentration of 1X 10⁴One/ml of cell suspension was seeded into 12-well plates at 500ul per well, and when hFLS cells grew to logarithmic growth phase (i.e., grew up to 80% confluent as a pellet), each corresponding siRNA was transfected into hFLS cells at a final concentration of 50nM according to the instructions of the Lipofectamine2000 kit.

No Target (NTC) negative control group: the siRNA of the experimental group is replaced by random non-specific siRNA, and the rest steps are unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed aiming at a target gene (ADAMTS-5 gene), and the sequence is as follows:

5 '-AGAUCGUUAGUUAGGUUGC dTdT-3' of sense strand;

the antisense strand is 5 '-GCAACCUAACGAUCUdTdT-3'.

NC blank control group: the remaining steps were identical to the experimental groups without siRNA.

Thirdly, collecting the hFLS cells of each group after 24h of transfection, centrifuging at 1000rpm for 5min, removing supernatant, and extracting RNA of each group by a Trizol method.

And fourthly, carrying out reverse transcription on the RNA of each group into cDNA, carrying out real-time fluorescence quantitative PCR by taking the cDNA of each group as a template and F and R as primers, wherein the detection result is shown in figure 1, and beta-actin is taken as an internal reference gene.

The primers are as follows:

F:5’-CTGCTCCCAGAAACAACG-3’；

R:5’-ATTCAGTGCCATCGGTCA-3’。

FIG. 1 shows that among 8 effective siRNAs obtained by previous screening, siRNA-RB-04 has the best silencing effect on ADAMTS-5 gene, and inhibits 90% of gene expression level.

Wherein the sequence of the sense strand of the siRNA-RB-04 is shown as SEQ ID No.1, and the sequence of the antisense strand is shown as SEQ ID No. 2.

siRNA-RB-04 sense strand: 5'-GGAUUUAUGUGGGCAUCAU-3' (SEQ ID No.1)

siRNA-RB-04 antisense strand: 5'-AUGAUGCCCACAUAAAUCC-3' (SEQ ID No.2)

Fifth, Western blot detection

Taking hFLS cells of the siRNA-RB-04 experimental group, discarding cell culture solution, washing the cells for 2 times by PBS, pouring off the PBS, adding a proper amount of precooled 2 XLysis Buffer, scraping the cells by a cell scraper, fully lysing the cells on ice for 30min, centrifuging for 15min at 12000g at 4 ℃ of a low-temperature centrifuge, taking supernatant, determining protein concentration by a Bradford method, finally adjusting the final concentration of sample protein to 2 mu g/mu l, and storing in a refrigerator at-80 ℃ for later use. Samples of 12. mu.g total protein were each added to an equal volume of 2X loading buffer. Mixing the two solutions, boiling in boiling water for 10 min, and storing at 4 deg.C. Preparing gel (10% SDS-PAGE separation gel and 5% concentrated gel) with corresponding concentration according to the molecular weight of the target protein, after the gel is prepared, pulling out a comb, washing sample loading holes by using electrophoresis buffer solution, loading the prepared sample, adding a protein sample into each hole, and carrying out electrophoresis. After the electrophoresis was completed, the protein was transferred to a PVDF membrane by electrotransfer for 2 hours at a constant current of 400mA at 4 ℃ using a transfer electrophoresis apparatus, followed by color development and exposure analysis.

The above experiments were performed with NC blank control and No Target (NTC) negative control as controls.

The results are shown in FIG. 2.

In FIG. 2, Control is NC blank Control group, No target is No Target (NTC) negative Control group, and siRNA is siRNA-RB-04 experimental group.

FIG. 2 shows that siRNA-RB-04 significantly inhibits the protein expression of ADAMTS-5, and siRNA-RB-04 was subsequently selected for further analysis.

Example 2 inhibition of inflammatory Agents by oligonucleic acids

Firstly, the experiment is divided into the following groups:

hFLS-siRNA-RB-04 Experimental group: primary culture of hFLS cells to 6-well plates, when cell density was about 50%, hFLS cells were transfected with siRNA-RB-04 at a final concentration of 50nM according to Lipofectamine2000 kit instructions.

293T-siRNA-RB-04 Experimental group: 293T cells were primary cultured in 6-well plates and, when the cell density was about 50%, the cells were transfected with siRNA-RB-04 at a final concentration of 50nM, according to the Lipofectamine2000 kit instructions.

hFLS-No Target (NTC) negative control group: the siRNA of the hFLS-siRNA-RB-04 experimental group is replaced by random non-specific siRNA, and the rest steps are consistent with the hFLS-siRNA-RB-04 experimental group. Wherein, the random non-specific siRNA is not siRNA specifically designed aiming at a target gene (ADAMTS-5 gene):

5 '-AGAUCGUUAGUUAGGUUGC dTdT-3' of sense strand;

the antisense strand is 5 '-GCAACCUAACGAUCUdTdT-3'.

293T-No Target (NTC) negative control group: the siRNA of the 293T-siRNA-RB-04 experimental group was replaced with random non-specific siRNA, and the rest of the procedure was identical to that of the 293T-siRNA-RB-04 experimental group. Wherein, the random non-specific siRNA is not siRNA specifically designed aiming at a target gene (ADAMTS-5 gene):

5 '-AGAUCGUUAGUUAGGUUGC dTdT-3' of sense strand;

the antisense strand is 5 '-GCAACCUAACGAUCUdTdT-3'.

hFLS-NC blank control group: the hFLS-siRNA-RB-04 experimental group is not added with siRNA, and the rest steps are consistent with the hFLS-siRNA-RB-04 experimental group.

293T-NC blank control group: no siRNA was added to the 293T-siRNA-RB-04 experimental group, and the remaining steps were identical to those of the 293T-siRNA-RB-04 experimental group.

And secondly, after 24 hours of transfection, replacing serum-free starvation culture cells of each group for 24 hours.

And thirdly, adding IL 1-alpha into each group of cells to ensure that the final concentration is 10ng/ml, and stimulating for 24 hours.

And fourthly, extracting RNA of each group of cells and performing reverse transcription to obtain cDNA, performing real-time fluorescence quantitative PCR by using the cDNA of each group as a template, TNF-F and TNF-R as primers, COX2-F and COX2-R as primers and IL-1 beta-F and IL-1 beta-R as primers, correspondingly detecting the expression levels of TNF, COX-2 and IL-1 beta genes, and using beta-actin as an internal reference gene.

TNF-F:5’-CGAGTGACAAGCCTGTAGCC-3’；

TNF-R:5’-TGAAGAGGACCTGGGAGTAGAT-3’。

cox2-F：5'-CAGGGTTGCTGGTGGTAGGA-3'；

cox2-R：5'-GCATAAAGCGTTTGCGGTAC-3'。

IL-1β-F：5'-ACGAATCTCCGACCACCA-3'；

IL-1β-R：5'-GGACCAGACATCACCAAGC-3'。

The results are shown in FIG. 3A.

The NTC groups in FIG. 3A each represent the NTC group to which the IL 1- α was added in a subsequent step.

Collecting the supernatant of each group of cells, and detecting the secretion level of IL-1 beta of each group of cells by using a Human IL-1 beta immunoassay detection kit.

Wherein, the NTC groups are respectively provided with the following groups:

hFLS-No target (NTC +) negative control group: the hFLS-No Target (NTC) negative control group was added with the above IL 1-. alpha.in the subsequent step.

hFLS-No target (NTC-) negative control group: the hFLS-No Target (NTC) negative control group was not added with the above IL 1-. alpha.in the subsequent steps.

293T-No target (NTC +) negative control group: 293T-No Target (NTC) negative control group the above IL 1-. alpha.was added in a subsequent step.

293T-No target (NTC-) negative control group: 293T-No Target (NTC) negative control group in the following steps without the addition of IL 1-. alpha.as described above.

The result is shown as B in fig. 3.

FIGS. 3A and B show that compared with NTC or NTC +, respectively, the siRNA-RB-04 experimental group can effectively inhibit the gene expression of TNF, COX-2 and IL-1 beta multiple inflammatory factors and the secretion of IL-1 beta in 293T or hFLS cells, wherein the gene inhibition rate of IL-1 beta in hFLS cells reaches 89%.

Example 3 verification of ADAMTS-5 Gene inhibitory Effect of homologous oligo-nucleic acids

To verify the effect of homology ratio on the effect of siRNA-RB-04 in inhibiting ADAMTS-5 gene, the following three sets of experiments were performed:

first, first set of experiments

The first group of siRNA antisense strands are all "5'-AUGAUGCCCACAUAAAUCC-3'"

The sense strand is the homologous sequence of "5'-GGAUUUAUGUGGGCAUCAU-3'", as shown in Table 1.

TABLE 1 antisense strand set

Note that S is the sense strand and AS is the antisense strand. The sense strand was selected from 15nt, 11nt, 23nt, 27nt, and mismatches.

Each siRNA shown in Table 1 was transfected into hFLS cells according to the method of example 1, and the suppression efficiency of ADAMTS-5 gene mRNA expression was examined.

Second and third set of experiments

The sense strand of the second group of sirnas was "5'-GGAUUUAUGUGGGCAUCAU-3'" and the antisense strand was the homologous sequence of "5'-AUGAUGCCCACAUAAAUCC-3'", as shown in table 2.

TABLE 2 sense chain set

Note that S is the sense strand and AS is the antisense strand.

Mouse hFLS cells were transfected with each siRNA shown in Table 2 according to the method of example 1, and the suppression efficiency of ADAMTS-5 gene mRNA expression was examined.

Third, third group experiment

The third group of sirnas, the sense strand and the antisense strand, are combinations of the above two groups, as shown in table 3.

TABLE 3 combination set

Note that S is the sense strand and AS is the antisense strand.

Each siRNA shown in Table 3 was transfected into hFLS cells according to the method of example 1, and the suppression efficiency of ADAMTS-5 gene mRNA expression was examined.

In each of the above experiments, a No Target (NTC) negative control group and an NC blank control group were set in the same manner as in example 1.

The results are shown in FIG. 4.

FIG. 4 shows that three groups of designed siRNAs play a role in silencing the mRNA expression of a target gene ADAMTS-5, and the RNA single strand shown in SEQ ID No.2 and the RNA single strand with homology of more than 60% with the RNA single strand shown in SEQ ID No.1 are complemented to form a double-stranded siRNA molecule; or, the double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 2; or, the double-stranded siRNA molecule formed by complementing the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.1 and the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.2 can interfere the expression of the ADAMTS-5 gene. The 21nt siRNA-RB-13 added with the dangling bases has the best interference effect, and the inhibition efficiency of the 21nt siRNA-RB-13 on the mRNA expression of the ADAMTS-5 gene is 91%; the siRNA-RB-20 complementary to only 11nt has the worst interference effect, the inhibition efficiency on the mRNA expression of the ADAMTS-5 gene is 22%, but the siRNA also plays a role in interfering the mRNA expression of the target gene ADAMTS-5.

Example 4 Effect of plasmid target Gene silencing Effect

First, based on the full-length ADAMTS-5 sequence, a double-stranded oligonucleotide sequence containing siRNA-RB-04 sequence was designed, as shown in Table 4.

TABLE 4 double strands containing siRNA-RB-04 sequences

Note: the underlined parts of SEQ ID No.5 and SEQ ID No.6 are base complementary regions, and the 10 th to 28 th nucleotides from the 5' end in SEQ ID No.5 are DNA sequences corresponding to the RNA single strand (siRNA-RB-04 sense strand) shown in SEQ ID No. 1. The 36 th to 54 th nucleotides from the 5' end in SEQ ID No.6 are DNA sequences corresponding to the RNA single strand (siRNA-RB-04 antisense strand) shown in SEQ ID No. 2.

Secondly, annealing the oligonucleotides in the table 4 to form double chains, replacing the sequence between the BamHI enzyme cutting sites and the HindIII enzyme cutting sites of the siRNA expression vector pGCsi-H1/Neo vector, keeping the rest sequences unchanged to obtain an interference fragment expression vector 1 (recombinant siRNA expression plasmid 1), and sequencing the interference fragment expression vector 1 to obtain a correct result.

Thirdly, the experiment is divided into the following groups:

experimental groups: one day before infection, good growth status of hFLS cells were inoculated in 6-well plates for transfection, according to the Lipofectamine2000 kit instructions, the interference fragment expression vector 1 according to 50nM final concentration transfection, 48h after transfection collected cells. The inhibition efficiency of ADAMTS-5 gene mRNA expression was examined according to the method of step four in example 1.

No Target (NTC) negative control group: the remaining steps are unchanged, except that the interfering fragment expression vector 1 of the experimental group is replaced by the annealed double strand of the unrelated sequence A and AS.

5 '-AGAUCGUUAGUUAGGUUGC dTdT-3' of sense strand;

the antisense strand is 5 '-GCAACCUAACGAUCUdTdT-3'.

NC blank control group: the interfering fragment expression vector 1 was not added and the remaining steps were identical to those of the experimental group.

The results are shown in Table 5.

TABLE 5 relative expression levels of mRNAs of ADAMTS-5 genes in each group

Table 5 shows that transfection of cells with DNA that transcribes the siRNA-RB-04 sequence also interferes with the expression of the target gene ADAMTS-5 mRNA.

Example 5 Effect of chemical modification on ADAMTS-5 inhibitory Effect

Different chemical modifications and combined modifications are carried out on the siRNA-RB-13 so as to improve the stability of the siRNA and improve the interference effect. The chemical modification includes halogen modification (2 '-F modification), methoxy modification (2' -OMe), thio modification, cholesterol modification and the like of ribose, the modification types are shown in Table 6, and the modified sequences are shown in Table 7.

TABLE 6 modified species

In Table 6, the modification positions of phosphorothioate (P-S bond) are shown in FIG. 5, Locked Nucleic Acid (LNA) is modified to form a cyclic structure by the glycidyl action at the 2 '-O position and 4' -C position of ribose, polypeptide is RGD (sequence from N terminal to C terminal is Arg-Gly-Asp, which is a sigma product), and A represents a certain nucleotide.

TABLE 7 Effect of chemical modifications on siRNA silencing Effect

Note that S is the sense strand and AS is the antisense strand.

Mouse hFLS cells were transfected with each siRNA shown in Table 7 according to the method of example 1, and the suppression efficiency of ADAMTS-5 gene mRNA expression was examined. When the siRNA modified by cholesterol, polypeptide and galactose is used for transfection, the transfection is directly carried out without adding a transfection reagent.

The results are shown in Table 7.

Table 7 shows that all siRNA-RB-13 modifiers obtained by various appropriate chemical modifications play a role in silencing the expression of the target gene ADAMTS-5.

Example 6 Effect of chemical modification on serum stability of oligo-nucleic acids

Serum stability assays were performed on several of the chemically modified nucleic acid molecules of example 5, as follows:

after each siRNA molecule was diluted to 5. mu.M with RNase-free water, an equal volume of fresh rat serum (a product of Biotech Co., Ltd., Yuanmu, Shanghai) was added, and the mixture was incubated at 37 ℃ for 30 minutes, and samples were taken for electrophoresis to observe the integrity of different siRNAs.

The results are shown in FIG. 6.

FIG. 6 shows that unmodified siRNA-RB-13 was significantly degraded after 30 minutes, while the modified nucleic acids siRNA-RB-41, siRNA-RB-40, and siRNA-RB-35 were not significantly degraded within 30 minutes.

Example 7 pathological section experiment in osteoarthritis model rats

First, construction of inflammation model rat

The bovine type II collagen is used as an inducer to promote the formation of arthritis. Type II bovine collagen (Sigma product) was injected into the articular cavity of male SD rats at a concentration of 4mg/mL, at a concentration of 100. mu.L/leg, at 200. mu.L in one dose.

Secondly, the following groups are arranged:

after injection of bovine type ii collagen for 3d, the inflammation model rats were randomly divided into 2 groups of 8 rats each. One group was PBS group, and one group was siRNA-RB-40 experimental group, in which siRNA-RB-40 experimental group was injected with 10nmol of siRNA-RB-40 solution per rat per time, injection volume was 100. mu.L, 50. mu.L/leg, PBS group was injected with equal volume of PBS per rat per time, each group was administered 2 times per week for 2 weeks, and administration time was consistent.

Three, 4 rats were taken from each group, and the animals were sacrificed the day after the 4 th dose to take materials: the skin was cut, the knee joint was immersed in tissue preservation solution, fixed, decalcified, paraffin embedded, sectioned, stained with hematoxylin-eosin (HE), and the histopathological manifestations were observed under a microscope, the results are shown in fig. 7.

In FIG. 7, the model group was PBS group, and the administration group was siRNA-RB-40 experimental group.

FIG. 7 shows that the PBS group of diseased tissues exhibited (1) degradation of meniscal cartilage (A) after 2 weeks; (2) severe fibrosis of the joint cavity (C); (3) calcification of cartilage layer, and disorder of cell arrangement (E). Compared with the PBS group, the tissues of the siRNA-RB-40 experimental group have the corresponding appearance that (1) the calcification degree of the meniscus is obviously reduced, and the structure of the meniscus is kept intact (B); (2) the cartilage layer has complete morphology, and the fibrosis degree is obviously reduced (D); (3) the cells of the cartilage layer are arranged regularly, and the cells have the characteristics of higher activity (F) and the like.

The results indicate that siRNA-RB-40 can inhibit the disease process of rats suffering from osteoarthritis, including joint surface fibrosis, cartilage erosion, synovitis and the like, and can be used as a potential arthritis therapeutic drug for improving diseases.

Example 8 measurement of inflammatory factor content in rat synovial fluid

First, a PBS group, an siRNA-RB-35 experimental group, an siRNA-RB-40 experimental group, and an siRNA-RB-41 experimental group were established according to the method of example 7. Wherein, the siRNA-RB-35 experimental group and the siRNA-RB-41 experimental group only replace the siRNA-RB-40 with the siRNA-RB-35 and the siRNA-RB-41, wherein the siRNA-RB-35 is wrapped by chitosan nanoparticles, and the rest steps are the same.

Secondly, the animals are sacrificed the next day after the 4 th administration treatment, the knee joints are taken after the skin and the tissues are peeled off, the bones are fully ground into powder in a mortar poured with liquid nitrogen, and RNA is extracted and is reversely transcribed into cDNA according to the specification of an Rneasy Mini kit (product of Guangzhou Jitai new deduction Biotechnology Co., Ltd., product catalog number 217004). Using each cDNA as a template, the relative expression level of ADAMTS-5 gene was determined according to the fourth step in example 1, and the relative expression levels of TNF, COX-2, and IL-1. beta. genes were determined according to the fourth step in example 2.

The above experiments were also performed with healthy male SD rats as controls.

The results of statistics of the relative expression amounts of ADAMTS-5 and inflammatory factors are shown in FIG. 8.

In fig. 8, the healthy group was the healthy male SD rat group and the model group was the PBS group.

FIG. 8 shows that the expression of inflammation-associated genes ADAMTS-5, TNF, COX-2, and IL-1. beta. was increased in the PBS group compared to the healthy male SD rat group. The siRNA-RB-40, the siRNA-RB-35 and the siRNA-RB-41 can obviously reduce the expression of ADAMTS-5, TNF, COX-2 and IL-1 beta in rat inflammatory diseases, and play roles in protecting cartilage and synovium and improving inflammation, thereby indicating that the siRNA molecule is a potential drug capable of preventing or treating inflammation.

Example 9 cell proliferation assay

Group of sirnas: the hFLS cells are prepared into 4X 10 by using a DMEM culture medium containing 10% fetal bovine serum⁴The concentration of each cell per ml was 100 ul/well, each well was transfected with siRNA-RB-13 at a final concentration of 50nM according to the instructions of CCK-8 kit (product of Yeasen), synchronization was performed by changing to serum-free and double-antibody-free DMEM medium 24 hours after transfection, to complete medium 24 hours after culture in serum-free and double-antibody-free DMEM medium and IL-1a stimulation (to induce inflammation) was added to a part of wells, and proliferation of cells was detected by CCK-8 kit 48 hours after IL-1a stimulation was not added to another part of wells.

NTC group: the siRNA-RB-13 in the siRNA group is replaced by random non-specific siRNA, and the rest steps are unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed aiming at a target gene (ADAMTS-5 gene):

5 '-AGAUCGUUAGUUAGGUUGC dTdT-3' of sense strand;

the antisense strand is 5 '-GCAACCUAACGAUCUdTdT-3'.

The results are shown in Table 8.

TABLE 8 cell proliferation following siRNA interference

(in Table 8, IL-1a + represents stimulation with IL-1a, and IL-1 a-represents stimulation without IL-1 a)

The results showed that the number of cells to which siRNA-RB-13 was added was slightly proliferated compared to NTC group, indicating that siRNA of the present invention (siRNA-RB-13) was not cytotoxic; and may have a repairing cellular effect.

Example 10 screening of effective oligo-nucleic acids inhibiting the expression of mRNA of ADAM17 Gene

First, siRNA design was performed to determine sirnas targeting ADAM17, and bioinformatic screens were performed to ensure that the sequences were specific for ADAM17 sequences and not specific for sequences from any other gene. The target sequence was checked against the sequences in GenBank using the BLAST search engine provided by NCBI and screened through preliminary experiments for 8 effective siRNAs, named siRNA-AD-01, siRNA-AD-02, siRNA-AD-03, siRNA-AD-04, siRNA-AD-05, siRNA-AD-06, siRNA-AD-07, and siRNA-AD-08, respectively. The above siRNA is designed for different positions of ADAM17 gene sequence.

Second, cell transfection

The experiment is divided into 10 groups, namely an siRNA-AD-01 experiment group to an siRNA-AD-08 experiment group, a Notarget (NTC) negative control group and an NC blank control group.

The setting method of the experimental groups from siRNA-AD-01 to siRNA-AD-08 is as follows:

No Target (NTC) negative control group: the siRNA of the experimental group is replaced by random non-specific siRNA, and the rest steps are unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed for a target gene (ADAM17 gene):

5 '-AGUAUGCCACAUAAGCAUC dTdT-3' of sense strand;

antisense strand 5 '-GAUGCUUAUGUGGCAUACU dTdT-3'.

Fourthly, the RNA of each group is reversely transcribed into cDNA, real-time fluorescence quantitative PCR is carried out by taking the cDNA of each group as a template and ADAM17-F1 and ADAM17-R1 as primers, and the detection result is shown in figure 9 and takes beta-actin as an internal reference gene.

ADAM17-F1:5’-GGACCAGGGAGGGAAATA-3’

ADAM17-R1:3’-TTGCTGTGGACGACGTTG-5’

FIG. 9 shows that among the 8 effective siRNAs obtained by previous screening, siRNA-AD-08 has the best silencing effect on ADAM17, and the gene expression level is suppressed by 86%.

Wherein the sequence of the siRNA-AD-08 sense strand is shown as SEQ ID No.7, and the sequence of the antisense strand is shown as SEQ ID No. 8.

siRNA-AD-08 sense strand: 5'-GCAUCAUGUAUCUGAACAA-3' (SEQ ID No.7)

siRNA-AD-08 antisense strand: 5'-UUGUUCAGAUACAUGAUGC-3' (SEQ ID No.8)

Fifth, Western blot detection

Taking hFLS cells of the siRNA-AD-08 experimental group, discarding cell culture solution, washing the cells for 2 times by PBS, pouring off the PBS, adding a proper amount of precooled 2 xLysis Buffer, scraping the cells by a cell scraper, fully lysing the cells on ice for 30min, centrifuging for 15min at 12000g at 4 ℃ of a low-temperature centrifuge, taking supernatant, determining protein concentration by a Bradford method, finally adjusting the final concentration of sample protein to 2 mug/muL, and storing the sample protein in a refrigerator at-80 ℃ for later use. Samples of 12. mu.g total protein were each added to an equal volume of 2X loading buffer. Mixing the two solutions, boiling in boiling water for 10 min, and storing at 4 deg.C. Preparing gel (10% SDS-PAGE separation gel and 5% concentrated gel) with corresponding concentration according to the molecular weight of the target protein, after the gel is prepared, pulling out a comb, washing sample loading holes by using electrophoresis buffer solution, loading the prepared sample, adding a protein sample into each hole, and carrying out electrophoresis. After the electrophoresis was completed, the protein was transferred to a PVDF membrane by means of an electrophoresis transfer apparatus by electrotransformation at 4 ℃ for 2 hours under a constant current of 400 mA. Followed by color development and exposure analysis.

The above experiments were performed with NC blank control and No Target (NTC) negative control as controls. The results are shown in FIG. 10.

In FIG. 10, Control is NC blank Control group, No target is No Target (NTC) negative Control group, and siRNA is siRNA-AD-08 experimental group.

FIG. 10 shows that siRNA-AD-08 significantly inhibited the protein expression of ADAM17, and siRNA-AD-08 was subsequently selected for further analysis.

Example 11 inhibition of inflammatory Agents by oligonucleic acids

Firstly, the experiment is divided into the following groups:

hFLS-siRNA-AD-08 experimental group: primary culture of hFLS cells to 6-well plates, when cell density was about 50%, hFLS cells were transfected with siRNA-AD-08 at a final concentration of 50nM according to Lipofectamine2000 kit instructions.

MCF-7-siRNA-AD-08 Experimental group: MCF-7 cells were primary cultured in 6-well plates and, when the cell density was about 50%, the MCF-7 cells were transfected with siRNA-AD-08 at a final concentration of 50nM, according to the Lipofectamine2000 kit instructions.

hFLS-No Target (NTC) negative control group: the siRNA of the hFLS-siRNA-AD-08 experimental group is replaced by random non-specific siRNA, and the rest steps are unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed for a target gene (ADAM17 gene):

5 '-AGUAUGCCACAUAAGCAUC dTdT-3' of sense strand;

antisense strand 5 '-GAUGCUUAUGUGGCAUACU dTdT-3'.

MCF-7-No Target (NTC) negative control group: the siRNA of the MCF-7-siRNA-AD-08 experimental group is replaced by random non-specific siRNA, and the rest steps are unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed for a target gene (ADAM17 gene):

5 '-AGUAUGCCACAUAAGCAUC dTdT-3' of sense strand;

antisense strand 5 '-GAUGCUUAUGUGGCAUACU dTdT-3'.

hFLS-NC blank control group: the hFLS-siRNA-AD-08 experimental group is not added with siRNA, and the rest steps are consistent with the hFLS-siRNA-AD-08 experimental group.

MCF-7-NC blank control group: the MCF-7-siRNA-AD-08 experimental group does not contain siRNA, and the rest steps are consistent with those of the MCF-7-siRNA-AD-08 experimental group.

The results are shown in FIG. 11, A.

The NTC groups in FIG. 11A each represent an NTC group to which the above IL 1- α is added in a subsequent step.

Wherein, the NTC groups are respectively provided with the following groups:

MCF-7-No target (NTC +) negative control group: MCF-7-No Target (NTC) negative control group the above IL 1-. alpha.was added in a subsequent step.

MCF-7-No target (NTC-) negative control group: MCF-7-No Target (NTC) negative control group was not added with the above IL 1-. alpha.in the subsequent steps.

The result is shown as B in FIG. 11.

FIG. 11 shows that siRNA-AD-08 can effectively inhibit the gene expression level of COX-2 and IL-1 beta inflammatory factor and inhibit IL-1 beta secretion in both MCF-7 and hFLS cells compared with NTC or NTC +, respectively, wherein the gene inhibition rate of IL-1 beta in hFLS cells reaches 88%. In MCF-7 cells, the gene expression level of TNF after siRNA-AD-08 transfection is higher than that of the NTC group, and the gene expression level is probably caused by the relatively complex cell functions of TNF.

Example 12 verification of ADAM17 Gene inhibitory Effect of homologous oligo-nucleic acids

To verify the effect of homology ratio on the effect of siRNA-AD-08 in inhibiting ADAM17 gene, the following three sets of experiments were performed:

first, first set of experiments

The first group of siRNA antisense strands are all "5'-UUGUUCAGAUACAUGAUGC-3'"

The sense strand is the homologous sequence of "5'-GCAUCAUGUAUCUGAACAA-3'", as shown in Table 9.

TABLE 9 antisense strand sets

Note that S is the sense strand and AS is the antisense strand. The sense strand was selected from 11nt, 15nt, 23nt, 27nt, and mismatches.

The hFLS cells were transfected with each siRNA shown in Table 9 according to the method of example 10, and the suppression efficiency of the expression of mRNA of ADAM17 gene was examined.

Second and third set of experiments

The sense strand of the second group of sirnas was "5'-GCAUCAUGUAUCUGAACAA-3'" and the antisense strand was the homologous sequence of "3 '-CGUAGUACAUAGACUUGUU-5'", as shown in table 10.

TABLE 10 sense chain set

Note that S is the sense strand and AS is the antisense strand.

Mouse hFLS cells were transfected with each siRNA shown in Table 10 according to the method of example 10, and the inhibitory efficiency of the siRNA on the mRNA expression of ADAM17 gene was examined.

Third, third group experiment

The third group of sirnas, the sense strand and the antisense strand, are combinations of the above two groups, as shown in table 11.

TABLE 11 combination set

Note that S is the sense strand and AS is the antisense strand.

Mouse hFLS cells were transfected with each siRNA shown in Table 11 according to the method of example 10, and the inhibitory efficiency of the siRNA on the mRNA expression of ADAM17 gene was examined.

In each of the above experiments, a No Target (NTC) negative control group and an NC blank control group were set in the same manner as in example 10.

The results are shown in FIG. 12.

FIG. 12 shows that the three groups of designed siRNAs play a role in silencing the mRNA expression of the target gene ADAM17, and the RNA single strand shown in SEQ ID No.8 and the RNA single strand with homology of more than 60% with the RNA single strand shown in SEQ ID No.7 are complemented to form a double-stranded siRNA molecule; or, the double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.7 and the RNA single strand which has more than 60 percent of homology with the RNA single strand shown in SEQ ID No. 8; or, the double-stranded siRNA molecule formed by complementing the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.7 and the RNA single strand with more than 70% of homology with the RNA single strand shown in SEQ ID No.8 can interfere the expression of the ADAM17 gene. The 21nt siRNA-AD-13 added with the dangling bases has the best interference effect, and the inhibition efficiency of the 21nt siRNA-AD-13 on the mRNA expression of the ADAM17 gene is 88%; the siRNA-AD-20 with only 11nt complementary has the worst interference effect, the inhibition efficiency of the siRNA-AD-20 on the mRNA expression of the ADAM17 gene is 20%, and the siRNA-AD-20 also plays a role in interfering the expression of a target gene ADAM 17.

Example 13 Effect of plasmid target Gene silencing

First, based on the full-length sequence of ADAM17, a double-stranded oligonucleotide sequence containing siRNA-AD-08 sequence was designed, as shown in Table 12.

TABLE 12 double strands containing siRNA-AD-08 sequences

Note: the underlined parts of SEQ ID No.11 and SEQ ID No.12 are base complementary regions, and nucleotides 38 to 56 from the 5' end in SEQ ID No.11 are DNA sequences corresponding to the RNA single strand (siRNA-AD-08 antisense strand) shown in SEQ ID No. 8. The 8 th to 26 th nucleotides from the 5' end in SEQ ID No.12 are DNA sequences corresponding to the RNA single strand (siRNA-AD-08 sense strand) shown in SEQ ID No. 7.

Secondly, annealing the oligonucleotides in the table 12 to form double chains, replacing the sequence between the BamHI enzyme cutting sites and the HindIII enzyme cutting sites of the siRNA expression vector pGCsi-H1/Neo vector, keeping the rest sequences unchanged to obtain an interference fragment expression vector 2 (recombinant siRNA expression plasmid 2), and sequencing the interference fragment expression vector 2 to obtain a correct result.

Thirdly, the experiment is divided into the following groups:

experimental groups: one day before infection, good growth status of hFLS cells were inoculated in 6-well plates for transfection, according to the Lipofectamine2000 kit instructions, interference fragment expression vector 2 according to 50nM final concentration transfection, 48h after transfection collected cells. The inhibition efficiency of the ADAM17 gene on mRNA expression was examined according to the method of step four in example 10.

No Target (NTC) negative control group: the siRNA of the experimental group was replaced with an unrelated sequence:

5 '-AGUAUGCCACAUAAGCAUC dTdT-3' of sense strand;

antisense strand 5 '-GAUGCUUAUGUGGCAUACU dTdT-3'.

The remaining steps were unchanged.

NC blank control group: the interfering fragment expression vector 2 was not added and the remaining steps were identical to those of the experimental group.

The results are shown in Table 13.

TABLE 13 relative expression levels of mRNAs of ADAM17 genes in respective groups

Table 13 shows that transfection of cells with DNA that transcribes the siRNA-AD-08 sequence also interferes with the expression of the target gene ADAM17 mRNA.

Example 14 Effect of chemical modification on ADAM17 inhibitory Effect

Different chemical modifications and combined modifications are carried out on the siRNA-AD-13 so as to improve the stability of the siRNA and improve the interference effect. The chemical modification includes halogen modification (2 '-F modification), methoxy modification (2' -OMe), thio modification, cholesterol modification and the like of ribose, the modification types are shown in Table 6, and the modified sequences are shown in Table 14.

TABLE 14 Effect of chemical modifications on siRNA silencing Effect

Note that S is the sense strand and AS is the antisense strand.

Mouse hFLS cells were transfected with each siRNA shown in Table 14 according to the method of example 10, and the inhibitory efficiency of the siRNA on the mRNA expression of ADAM17 gene was examined. When the siRNA modified by cholesterol, polypeptide and galactose is used for transfection, the transfection is directly carried out without adding a transfection reagent.

The results are shown in Table 14.

Table 14 shows that all siRNA-AD-13 modifications obtained by various appropriate chemical modifications have the effect of silencing the expression of the target gene ADAM 17.

Example 15 Effect of chemical modification on serum stability of oligo-nucleic acids

Serum stability assays were performed on several of the chemically modified nucleic acid molecules of example 14, as follows:

The results are shown in FIG. 13.

FIG. 13 shows that unmodified siRNA-AD-13 was significantly degraded after 30 minutes, while the modified nucleic acids siRNA-AD-26, siRNA-AD-39, and siRNA-AD-40 were not significantly degraded within 30 minutes.

Example 16 pathological section experiment in osteoarthritis model rats

First, the construction of the inflammation model rat was the same as in example 7.

Secondly, the following groups are arranged:

after injection of bovine type ii collagen for 3d, the inflammation model rats were randomly divided into 2 groups of 8 rats each. One group is PBS group, and the other group is siRNA-AD-26 experimental group, wherein the siRNA-AD-26 experimental group is injected with 10nmol of siRNA-AD-26 solution into each rat at each time, the injection volume is 100 mu L and 50 mu L/leg, the PBS group is injected with PBS with equal volume into each rat at each time, each group is administrated 2 times per week for 2 weeks continuously, and the administration time is consistent.

Thirdly, animals are sacrificed on the next day after the 2 nd and 4 th administration treatments, the skin is cut off, knee joints are taken and soaked in tissue preservation solution, the tissues are fixed, decalcified, embedded in paraffin, sliced, stained by hematoxylin-eosin (HE) and Toluidine Blue (TB), and the histopathological manifestations are observed under a microscope, and the results are shown in figure 14.

In FIG. 14, the siRNA group is the siRNA-AD-26 experimental group. 1W and 2W represent one and two weeks after modeling the inflammation model rats, respectively.

FIG. 14 shows that after one week of modeling of the rats in the inflammation model, the PBS group shows fibrosis and ossification of meniscus, part of the fibrosis tissues invades into the cartilage layer, the arrangement of chondrocytes is disordered, part of collagen is lost, and the connective tissues in joints also show obvious fibrosis and inflammatory cells; the siRNA-AD-26 experimental group has smooth articular surface, ordered arrangement of cartilage layer cells, and only slight ossification degeneration of cartilage cells and extracellular collagen loss appear locally. After two weeks of modeling, the PBS group has the advantages that the articular surface is subjected to multifibrous tissue hyperplasia, a large amount of fibrous tissues in the articular cavity are subjected to meniscal ossification and covered with multiple layers of fibrous hyperplastic tissues, part of cartilage layers are ossified and broken, cartilage layer cells are disorderly denatured, and a large amount of collagen is lost; although the meniscus in the siRNA-AD-26 experimental group has local ossification and fibroplasia, the joint and the meniscus still keep normal shape, the joint surface is smooth, the cells of the cartilage layer are normal, and the loss degree of each item is obviously slight compared with that of the PBS group.

The results show that siRNA-AD-26 can inhibit the disease process of rats suffering from osteoarthritis, and can be used as a potential arthritis therapeutic agent for improving diseases.

Example 17 measurement of inflammatory factor content in rat synovial fluid

First, a PBS group, an siRNA-AD-26 experimental group, an siRNA-AD-39 experimental group, and an siRNA-AD-40 experimental group were established according to the method of example 16. Wherein, the siRNA-AD-40 experimental group and the siRNA-AD-39 experimental group only replace the siRNA-AD-26 with the siRNA-AD-40 and the siRNA-AD-39, wherein the siRNA-AD-40 is wrapped by chitosan nanoparticles, and the other steps are the same.

Second, animals were sacrificed the day after the 4 th dosing treatment for material draw: the knee joint was removed after peeling the skin and tissue, ground thoroughly in a mortar poured with liquid nitrogen until the bone tissue became a powder, and RNA was extracted and reverse-transcribed into cDNA according to the Rneasy Mini kit (product of the new deduction biotechnology ltd, jitai, guangzhou, catalog No. 217004). Using each cDNA as a template, the relative expression level of ADAM17 gene was determined according to step four in example 10, and the relative expression levels of TNF, COX-2, and IL-1. beta. genes were determined according to the method of step four in example 11.

The results of statistics of the relative expression amounts of ADAM17 and an inflammatory factor are shown in fig. 15.

In FIG. 15, Normal was the healthy male SD rat group and Model was the PBS group.

FIG. 15 shows that compared with the healthy male SD rat group, the expression of inflammation-related genes ADAM17, TNF, COX-2 and IL-1 beta in the PBS group is increased, while the expression of ADAM17, TNF, COX-2 and IL-1 beta in different processes of rat inflammation diseases can be obviously reduced by siRNA-AD-26, siRNA-AD-39 and siRNA-AD-40, so that the effects of cartilage protection and inflammation improvement are achieved, and the siRNA molecule is a potential drug capable of preventing or treating inflammation.

Example 18 cell proliferation assay

Group of sirnas: the hFLS cells are prepared into 4X 10 by using a DMEM culture medium containing 10% fetal bovine serum⁴The concentration of each cell per ml was 100 ul/well, each well was transfected with siRNA-AD-13 at a final concentration of 50nM according to the instructions of CCK-8 kit (product of Yeasen), after 24h of transfection, the cells were synchronized by changing to serum-free and double-antibody-free DMEM medium, after 24h of transfection, the cells were cultured in complete medium with IL-1a stimulation (to induce inflammation) in a part of wells, while the cells were not cultured in the other part of wells, and after 48h and 72h of IL-1a stimulation, the cells were examined for proliferation using CCK-8 kit.

NTC group: the siRNA-AD-13 in the siRNA group is replaced by random non-specific siRNA, and the rest steps are unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed for a target gene (ADAM17 gene):

5 '-AGUAUGCCACAUAAGCAUC dTdT-3' of sense strand;

antisense strand 5 '-GAUGCUUAUGUGGCAUACU dTdT-3'.

The results are shown in Table 15.

TABLE 15 cell proliferation following siRNA interference

(in Table 15, IL-1a + represents stimulation with IL-1a, and IL-1 a-represents stimulation without IL-1 a)

The results showed that the number of cells added with siRNA-AD-13 was slightly proliferative compared to NTC group, indicating that the siRNA of the present invention (siRNA-AD-13) was not cytotoxic; and may have a repairing cellular effect.

Example 19 inhibition of inflammatory Agents by combination of oligonucleotide

Firstly, the following groups are arranged:

ADAMTS-5 group: primary culture of hFLS cells to 6-well plates, when cell density was about 50%, hFLS cells were transfected with siRNA-RB-13 at a final concentration of 50nM according to the instructions of Lipofectamine2000 kit.

Group ADAM 17: the ADAMTS-5 group siRNA-RB-13 was replaced with siRNA-AD-13, and the rest of the procedure was the same.

ADAMTS-5+ ADAM17 group: the ADAMTS-5 group siRNA-RB-13 was replaced with 25nM siRNA-RB-13 and 25nM siRNA-AD-13, and the rest of the procedure was the same.

No Target (NTC) negative control group: the siRNA of ADAMTS-5 group was replaced by random non-specific siRNA, and the rest steps were unchanged. Wherein, the random non-specific siRNA is not siRNA specifically designed aiming at target genes (ADAMTS-5 and ADAM17 genes):

sense strand: 5'-UUCUCCGAACGUGUCACGU dTdT-3';

antisense strand: 5 '-ACGUGACACGUCGGAGAAdTdT-3'

NC blank control group: the remaining steps were consistent with the ADAMTS-5 group without siRNA addition.

And thirdly, adding IL 1-alpha (10ng/ml) into each group of cells to ensure that the final concentration is 10ng/ml, and stimulating for 24 hours.

And fourthly, detecting the expression level of TNF, COX-2 and IL-1 beta genes and the secretion level of IL-1 beta of each group of cells according to the method of the fourth step in the example 2.

The results are shown in FIG. 16.

In fig. 16B, control represents NC blank control group, and No target represents No Target (NTC) negative control group.

FIG. 16 shows that, compared with siRNA alone, after siRNA-RB-13 and siRNA-AD-13 are compounded, the interference effect on inflammatory factors is significantly improved, and the two have the synergistic effect of inhibiting inflammatory factors.

Example 20 pathological section experiment in osteoarthritis model rats

Secondly, the following groups are arranged:

after injection of bovine type ii collagen for 3d, the inflammation model rats were randomly divided into the following 2 groups.

AD5&17 and Control groups: a total of 12 mice were treated with the Control, PBS was administered to one hind limb as Control group, siRNA (10 nmol each of siRNA-RB-40 and siRNA-AD-26) was administered to one hind limb as AD5&17 group, injection volume was 100. mu.L, and animals were sacrificed on the second day of

treatment

2, 4, and 6 times per week for 3 weeks at a dosing frequency of 2 times per week.

Thirdly, animals are sacrificed on the next day of the 2 nd, 4 th and 6 th administration treatment respectively, the skin is cut off, the knee joints are taken and soaked in tissue preservation solution, the fixation, decalcification, paraffin embedding and slicing are carried out, hematoxylin-eosin (HE) and Toluidine Blue (TB) staining are carried out, and the histopathological manifestation is observed under a microscope, and the result is shown in figure 17.

In FIG. 17, A is the result of HE staining, and B is the result of TB staining.

FIG. 17 shows that after one week of modeling, chondrocyte alignment in Control group was disturbed, cartilage layer was thickened, meniscal ossification, cartilage collagen loss; cartilage collagen loss was not evident in the AD5&17 group, and the menisci only partially ossified. After two weeks of modeling, the cartilage layer of the Control group is locally invaded into the subchondral bone layer by the fibrosis tissue, the part of the cartilage layer close to the articular cavity and the synovial layer are both subjected to fibrosis proliferation, collagen is seriously lost, articular capsule fibrosis is serious, and meniscus ossification is realized; AD5&17 groups lost only a small amount of local cartilage layer collagen, and the meniscal portion chondrocytes degenerated. After three weeks of modeling, the joint surface of the Control group is seriously fibrotic, the joint cavity has tissue fragments and fibrotic tissues, cells of the cartilage layer are ossified and denatured, collagen is seriously lost, and the joint capsule is obviously fibrotic and proliferated and infiltrated by a large amount of inflammatory cells; the AD5&17 groups have smooth articular surfaces, and chondrocytes basically keep the shape and activity. The results show that the pathological changes of the AD5&17 group are lighter than those of the Control group at each stage of observation. The siRNA aiming at ADAMTS-5 and ADAM17 is shown to be combined to be used for inhibiting the disease process of rats with arthritis, including fibrosis, cartilage erosion and the like, and can be used as a potential arthritis therapeutic drug for improving the disease.

Example 21 measurement of inflammatory factor content in rat synovial fluid

One, establishing an ADAMTS5-siRNA administration set, an ADAMTS 17-siRNA administration set, and an ADAMTS5-siRNA & ADAM17-siRNA administration set according to the method of example 7, wherein the siRNA of the ADAMTS5-siRNA administration set is siRNA-RB-40, the siRNA of the ADAMTS 17-siRNA administration set is siRNA-AD-26, the siRNA of the ADAMTS5-siRNA & ADAM17-siRNA administration set is siRNA-RB-40 and siRNA-AD-26, and the siRNA doses of the groups except the ADAMTS5-siRNA & ADAM17-siRNA administration set are the same and are 10 nmol/leg; the doses of each siRNA of ADAMTS5-siRNA & ADAM17-siRNA drug administration group were the same and were 5 nmol/leg. Each group was dosed 2 times per week for a consistent time period.

Healthy rat groups: male SD rats (220. + -.20 g).

Inflammation model rat group: example 7 the constructed inflammation model rat.

Two, animals were sacrificed on the day of 2, 4, 6 dose treatments, respectively, with 4 rats per time point per group: the knee joint was removed after peeling the skin and tissue, ground thoroughly in a mortar poured with liquid nitrogen until the bone tissue became a powder, and RNA was extracted and reverse-transcribed into cDNA according to the Rneasy Mini kit (product of the new deduction biotechnology ltd, jitai, guangzhou, catalog No. 217004). Using each cDNA as a template, the relative expression level of ADAMTS-5 was determined according to the method of step four in example 1, the relative expression levels of TNF, COX-2, and IL-1. beta. genes were determined according to the method of step four in example 2, and the relative expression level of ADAM17 was determined according to the method of step four in example 10.

The results are shown in Table 16.

TABLE 16 expression level of inflammatory factor in rat

Table 16 shows that compared with the healthy rat group, the inflammation-related genes ADAMTS-5, ADAM17, TNF, COX-2 and IL-1 in the inflammation model rat group are increased in expression, and compared with the single administration of ADAM17-siRNA (siRNA aiming at ADAM 17) or ADAMTS5-siRNA (siRNA aiming at ADAMTS-5), the combined administration of ADAM17-siRNA and ADAMTS5-siRNA can obviously reduce the expression of ADAMTS-5, ADAM17, TNF, COX-2 and IL-1 beta in the inflammation diseases of rats 1-3 weeks after the administration, and has the effects of protecting cartilage and synovium and improving inflammation. The combined siRNA molecule is probably a high-curative-effect inflammation treatment drug.

Sequence listing

<110> Guangzhou biomedical and health research institute of Guangzhou institute of China academy of sciences, Guangzhou City Ruibo Biotechnology, Inc

<120> siRNA for inhibiting ADAMTS-5 gene and application thereof

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ggauuuaugu gggcaucauu cau 23

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ggauuuaugu gggcaucauu cauguga 27

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gaugcccaca uaaau 15

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<220>

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ugcccacaua a 11

<210> 19

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augaaugaug cccacauaaa ucc 23

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<211> 27

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<220>

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ucacaugaau gaugcccaca uaaaucc 27

<210> 21

<211> 13

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<220>

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uuaugugggc att 13

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<211> 13

<212> DNA/RNA

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<220>

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ugcccacaua att 13

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<212> DNA/RNA

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<220>

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ggauuuaugu gggcaucauu caugugatt 29

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<212> DNA/RNA

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<220>

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ucacaugaau gaugcccaca uaaaucctt 29

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<213> Artificial sequence

<220>

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cauguaucug a 11

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aucauguauc ugaac 15

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uggcaucaug uaucugaaca acg 23

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<220>

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ccuggcauca uguaucugaa caacgac 27

<210> 29

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<213> Artificial sequence

<220>

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ucagauacau g 11

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guucagauac augau 15

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<211> 23

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<213> Artificial sequence

<220>

<223>

<400> 31

gucguuguuc agauacauga ugc 23

<210> 20

<211> 32

<212> RNA

<213> Artificial sequence

<220>

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<400> 32

gucguuguuc agauacauga ugccagg 27

<210> 33

<211> 13

<212> DNA/RNA

<213> Artificial sequence

<220>

<223>

<400> 33

cauguaucug att 13

<210> 34

<211> 13

<212> DNA/RNA

<213> Artificial sequence

<220>

<223>

<400> 34

ucagauacau gtt 13

<210> 35

<211> 29

<212> DNA/RNA

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<220>

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ccuggcauca uguaucugaa caacgactt 29

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Claims

1. A chemically modified double-stranded siRNA molecule is a double-stranded siRNA molecule formed by at least one strand of siRNA molecules shown as the following A through complementation after any chemical modification shown as the following (1) to (13):

A. an siRNA molecule, which is represented by any one of 1) to 13) as follows:

2) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 13;

3) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 14;

4) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 15;

5) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 16;

6) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 17;

7) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 18;

8) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 19;

9) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 20;

10) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.21 and the RNA single strand shown in SEQ ID No. 22;

11) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.23 and the RNA single strand shown in SEQ ID No. 24;

12) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.15 and the RNA single strand shown in SEQ ID No. 17;

13) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.13 and the RNA single strand shown in SEQ ID No. 19;

(1) phosphorothioate modification of the phosphate backbone;

(2) 2' -methoxy modification of ribose or deoxyribose;

(3) 2' -fluoro modification of ribose or deoxyribose;

(4) modifying locked nucleic acid;

(5) open loop nucleic acid modification;

(6) modifying indole;

(7) 5-methylcytosine modification of a base;

(8) 5-ethynyluracil modification of the base;

(9) single-stranded 5' terminal cholesterol modification;

(10) single-chain 3' end galactose modification;

(11) single chain 5' end polypeptide modification;

(12) single-stranded 5' end phosphorylation modification;

(13) modifying the single-chain 5' end by fluorescent labeling;

the sense strand and the antisense strand of the double-stranded siRNA molecule formed by the complementary chemical modification are not sequences shown in B1 and C1 respectively:

B1、5'-K-LLMUUUAUGUGGGCAUPMQdTdT-3’；

C1、5'-R- MQLAUGCCCACAUAAAQPPdTdT -3’；

the K is unmodified or modified by 5' end cholesterol;

the R is a 5' terminal phosphorylation modification;

the dT is thymine deoxyribonucleotide;

or the like, or, alternatively,

l, M, P and Q are guanine deoxyribonucleotide modified by 2 '-methoxyl modification of deoxyribose and phosphorothioate modification of phosphate skeleton, adenine deoxyribonucleotide modified by 2' -methoxyl modification of deoxyribose and phosphorothioate modification of phosphate skeleton, cytosine deoxyribonucleotide modified by 2 '-methoxyl modification of deoxyribose and phosphorothioate modification of phosphate skeleton, and uracil ribonucleotides modified by 2' -methoxyl modification of ribose and phosphorothioate modification of phosphate skeleton, respectively.

2. An siRNA molecule, which is shown as any one of the following (1) to (12):

(1) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 13;

(2) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 14;

(3) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 15;

(4) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.2 and the RNA single strand shown in SEQ ID No. 16;

(5) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 17;

(6) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 18;

(7) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 19;

(8) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 20;

9) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.21 and the RNA single strand shown in SEQ ID No. 22;

10) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.23 and the RNA single strand shown in SEQ ID No. 24;

11) a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.15 and the RNA single strand shown in SEQ ID No. 17;

12) the double-stranded siRNA molecule is formed by complementing the RNA single strand shown in SEQ ID No.13 and the RNA single strand shown in SEQ ID No. 19.

3. A DNA molecule capable of producing the siRNA molecule of claim 2.

4. A chemically modified double-stranded siRNA molecule composition, which comprises double-stranded siRNA molecules shown in the following (1) and (2):

(1) at least one double stranded siRNA molecule of the chemically modified double stranded siRNA molecule of claim 1;

(2) at least one strand of the double-stranded siRNA molecule shown as A' is subjected to any chemical modification shown as the following 1) -13) and then is complemented to form at least one double-stranded siRNA molecule:

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.8 and the RNA single strand shown in SEQ ID No. 25;

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.8 and the RNA single strand shown in SEQ ID No. 26;

or, the double-stranded siRNA molecule formed by the RNA single strand shown in SEQ ID No.8 and the RNA single strand shown in SEQ ID No.27 through complementation;

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.8 and the RNA single strand shown in SEQ ID No. 28;

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No. 29;

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No. 30;

or, the double-stranded siRNA molecule formed by the RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No.31 in a complementary way;

or, the double-stranded siRNA molecule formed by the RNA single strand shown in SEQ ID No.7 and the RNA single strand shown in SEQ ID No.32 in a complementary way;

or; a double-stranded siRNA molecule formed by complementing the RNA single strand shown in SEQ ID No.9 and the RNA single strand shown in SEQ ID No. 10;

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.33 and the RNA single strand shown in SEQ ID No. 34;

or, the double-stranded siRNA molecule formed by the RNA single strand shown in SEQ ID No.35 and the RNA single strand shown in SEQ ID No.36 in a complementary way;

or, the double-stranded siRNA molecule formed by the RNA single strand shown in SEQ ID No.27 and the RNA single strand shown in SEQ ID No.30 in a complementary way;

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.26 and the RNA single strand shown in SEQ ID No. 31;

1) phosphorothioate modification of the phosphate backbone;

2) 2' -methoxy modification of ribose or deoxyribose;

3) 2' -fluoro modification of ribose or deoxyribose;

4) modifying locked nucleic acid;

5) open loop nucleic acid modification;

6) modifying indole;

7) 5-methylcytosine modification of a base;

8) 5-ethynyluracil modification of the base;

9) single-stranded 5' terminal cholesterol modification;

10) single-chain 3' end galactose modification;

11) single chain 5' end polypeptide modification;

12) Single-stranded 5' end phosphorylation modification;

13) and (3) carrying out fluorescence labeling modification on the 5' end of the single strand.

5. An siRNA molecule composition, comprising at least one of the siRNA molecules shown as the following H and at least one of the siRNA molecules shown as the following I:

h is at least one siRNA molecule of the siRNA molecules of claim 2;

i is at least one siRNA molecule in the siRNA molecules shown in the following (1):

or, the double-stranded siRNA molecule formed by the complementary RNA single strand shown in SEQ ID No.26 and the RNA single strand shown in SEQ ID No. 31.

6. A DNA molecule capable of producing the siRNA molecule composition of claim 5.

7. A kit comprising the chemically modified double stranded siRNA molecule of claim 1, the siRNA molecule of claim 2, the DNA molecule of claim 3, the chemically modified double stranded siRNA molecule composition of claim 4, the siRNA molecule composition of claim 5, and/or the DNA molecule of claim 6.

8. Use of the chemically modified double stranded siRNA molecule of claim 1, the siRNA molecule of claim 2, the DNA molecule of claim 3, the chemically modified double stranded siRNA molecule composition of claim 4, the siRNA molecule composition of claim 5, the DNA molecule of claim 6 and/or the kit of claim 7 for the manufacture of a product for the prevention and/or treatment of osteoarthritis.

9. Use of the chemically modified double stranded siRNA molecule of claim 1, the siRNA molecule of claim 2, the DNA molecule of claim 3, the chemically modified double stranded siRNA molecule composition of claim 4, the siRNA molecule composition of claim 5, the DNA molecule of claim 6 and/or the kit of claim 7 for the preparation of a product according to any one of the following W1-W5:

w1, product for inhibiting fibrosis of the articular surface;

w2, a product to inhibit cartilage erosion;

w3, a product for the prevention and/or treatment of synovitis;

w4, a product that protects cartilage and/or synovium;

w5, product for preventing and/or treating rheumatoid arthritis.