Application of substances that reduce the content or activity of RCAN1 in preventing and treating aging and osteoarthritis

Abstract

The present invention discloses the use of reduced RCAN1 content or active substances in the prevention and treatment of aging and osteoarthritis. The present invention finds that knocking down the RCAN1 gene can delay aging and reduce aging-related proinflammatory factors, and that knocking down the RCAN1 gene can also slow the progression of osteoarthritis. Therefore, the present invention provides new insights into the development of preventive and therapeutic drugs for delaying aging or osteoarthritis. Furthermore, the present invention's findings are particularly applicable to the prevention or treatment of osteoarthritis in the elderly.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a substance that reduces the content or activity of RCAN1 in preventing and treating aging and osteoarthritis. Background Art

[0002] Cellular senescence is one of the fundamental mechanisms driving aging. Cellular senescence refers to the gradual decline in cell proliferation, differentiation, and physiological functions as cells accumulate chronic stress during their life activities. Senescent cells secrete a large number of inflammatory and oncogene-related factors, known as the senescence-associated secretory phenotype (SASP). The SASP includes proinflammatory cytokines (such as IL-1α, IL-1β, IL-6, and IL-8), growth factors (such as HGF, TGF-β, and GM-CSF), chemokines (such as CXCL-1 / 3 and CXCL-10), and matrix-remodeling enzymes (such as metalloproteinases). The production of the SASP worsens the tissue microenvironment. Senescent cells (SNCs) accumulate in aging tissues or organs. These accumulated senescent cells lose their normal physiological functions, affecting organ and tissue function and potentially causing or exacerbating age-related diseases. Using drugs or gene therapy to delay cell senescence, eliminate senescent cells or the SASP they secrete, can delay the aging of the body or aging-related degenerative changes.

[0003] Existing drugs that eliminate senescent cells, such as BCL2 inhibitors, have certain cytotoxic properties and can cause side effects such as neutropenia and thrombocytopenia. Drugs targeting the p53-MDM2 combination, because they do not specifically recognize senescent cells, can cause side effects on normal cells. Furthermore, some gene therapies targeting senescent genes, while delaying cellular senescence, can cause senescent cells to repopulate. Because senescent cells accumulate significant amounts of DNA damage and have relatively unstable genomes, these repopulated cells carry the potential risk of tumor formation. Therefore, it is necessary to explore and develop safer and more reliable drugs or therapies to delay cellular senescence.

[0004] Osteoarthritis (OA) is an aging-related degenerative joint disease that severely impacts patients' later years. Symptoms of OA include gradual loss of joint cartilage, distorted bone growth, joint inflammation, and pain. Treatment options include symptomatic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid injections, and in later stages, mechanical joint replacement. Currently, there is a lack of treatments that can improve or reverse OA in its early stages. Summary of the Invention

[0005] In order to develop safer and more reliable drugs or therapies to delay aging, the purpose of the present invention is to provide an aging-related target and a preventive or therapeutic method for the target. In addition, the target of the present invention is related to osteoarthritis and can also serve as a target for the prevention or treatment of osteoarthritis.

[0006] In accordance with the above technical problems and objectives, the present invention provides any of the following applications:

[0007] 1. Use of a substance that reduces the content or activity of RCAN1 in the preparation of a product for preventing and / or treating aging.

[0008] 2. Use of substances that inhibit RCAN1 gene expression or knock out RCAN1 gene in the preparation of products for preventing and / or treating aging.

[0009] 3. Use of substances that reduce the content or activity of RCAN1 in the preparation of products for preventing and / or treating osteoarthritis.

[0010] 4. Use of substances that inhibit RCAN1 gene expression or knock out RCAN1 gene in the preparation of products for preventing and / or treating osteoarthritis.

[0011] In the aforementioned applications, the substances include a CRISPR / Cas9 knockout system targeting the RCAN1 gene, siRNA, and an RCAN1 inhibitor. These substances can be used alone or in combination. The CRISPR / Cas9 knockout system reduces RCAN1 levels by knocking out RCAN1; siRNA inhibits RCAN1 expression by interfering with gene expression; and RCAN1 inhibitors can inhibit RCAN1 gene expression, reduce RCAN1 mRNA levels, or reduce RCAN1 protein expression. The products can be pharmaceuticals, research reagents, and the like.

[0012] In the above application, the substance can reduce the level of aging-related pro-inflammatory factors, including IL1B, IL6 and IL1a.

[0013] Preferably, the substance can be used for the preparation of a product for simultaneously preventing and / or treating aging and osteoarthritis.

[0014] Furthermore, the senescence includes the senescence of animal cells, tissues, organs or individuals. In one embodiment of the present invention, the senescent cells are human mesenchymal stem cells.

[0015] Specifically, the osteoarthritis includes osteoarthritis caused by meniscus damage.

[0016] RCAN1 of the present invention is also known as DSCR1, Down syndrome critical region gene 1 and calcineurin regulatory factor 1, which are used interchangeably in this application; the gene number is NCBI Entrez Gene: 1827. RCAN1, Rcan1 and rcan1 are used interchangeably in this invention and all refer to the RCAN1 gene.

[0017] The advantages of the present invention are:

[0018] (1) The present invention provides a new target for preventing and / or treating aging and osteoarthritis.

[0019] (2) RCAN1 disclosed in the present invention is an endogenous regulatory factor. Reducing its content or activity or inhibiting its expression has little toxic side effects on normal cells and will not induce the re-proliferation of senescent cells, thereby avoiding the potential risk of tumor formation after the proliferation of senescent cells.

[0020] (3) RCAN1 disclosed in the present invention is both an aging target and an osteoarthritis target, and is particularly suitable for developing products for preventing and / or treating osteoarthritis in elderly patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of RCAN1 in normal and aging (or osteoarthritis) tissues or cells in Example 5 of the present invention is shown. * indicates a significant difference, p < 0.05; ** indicates a very significant difference, p < 0.01.

[0022] 1A is a comparison of RCAN1 expression levels in the joints of normal subjects (norm) and osteoarthritis patients (OA).

[0023] 1B is a comparison of the co-localization expression of p53, IL1A and RCAN1 in the synovial tissues of normal subjects (Normal) and patients with osteoarthritis (OA), with the bar value being 10 μm.

[0024] Figure 1C shows the results of Safranin O staining of articular cartilage in a mouse osteoarthritis model (DMM) and a normal control (Sham); RCAN1 expression in articular cartilage, synovium, and subchondral bone; and SA-β-Gal (SA-b-Gal in the figure) detection in the synovium; Bar value is 20 μm.

[0025] 1D is a graph showing the changes in RCAN1 expression during doxorubicin-induced aging of human mesenchymal stem cells cultured in vitro.

[0026] 1E is the ratio of SA-β-Gal (i.e., SA-b-Gal and SA-β-gal in the figure), Ki67, and RCAN1 positive cells in senescent cells and proliferating cells (non-senescent cells).

[0027] Figure 2 This figure shows the cellular senescence and related gene expression in human mesenchymal stem cells in Example 6 of the present invention, in which RCAN1 was knocked out via CRISPR / Cas9 and senescence was induced by rudorubicin. sgNTC represents a cell group infected with the sgNTC virus, sgRCNA1 (including sgRCAN1-1 and sgRCAN1-2) represents a cell group infected with the sgRCAN1-human lentivirus, and hESC-MSCs represent fourth-generation human mesenchymal progenitor cells. * indicates a significant difference, p < 0.05; ** indicates a very significant difference, p < 0.01.

[0028] 2A shows the detection results of SA-β-Gal (i.e., SA-b-Gal and SA-β-gal in the figure) and Dapi (4',6-diamidino-2-phenylindole).

[0029] 2B is the expression of the pro-inflammatory factor IL1A.

[0030] 2C shows the expression of pro-inflammatory factors IL1B, IL16, IL8 and CXCL1.

[0031] Figure 3 This figure shows the aging and related gene expression of human synovial stem cells induced by rudorubicin and then knocked down RCAN1 via siRNA in Example 7 of the present invention. NC, si01, and si03 represent: non-targeting siRNA transfection (Non-targeting control), anti-RCAN1-targeting siRNA 01 (AntiRCAN1-si01), and anti-RCAN1-targeting siRNA 03 (AntiRCAN1-si03), respectively.

[0032] 3A represents the cell surface protein level of the pro-inflammatory cytokine IL1A. * indicates significant difference, p < 0.05.

[0033] 3B shows the expression of pro-inflammatory cytokines IL1B, IL16, and IL8. * indicates a significant difference compared to the senescent cell transfection group with non-targeted siRNA sequences, p < 0.05.

[0034] 3C shows the results of SA-β-Gal detection. * indicates significant difference, p < 0.05; Bar value is 20 μm.

[0035] 3D shows the pathway analysis results of transcriptome sequencing of RCAN1 knockdown group cells and non-targeted control group cells.

[0036] Figure 4 This is the effect of targeting Rcan1 by siRNA on the progression of osteoarthritis in a mouse DMM model in Example 8 of the present invention, wherein:

[0037] 4A shows the localization of Cy3 fluorescently labeled siRNA in the joint cavity.

[0038] 4B shows the results of Safranin O staining of articular cartilage after treatment with non-targeting siRNA (siRNA-Ctrl) and siRNA targeting Rcan1 (siRNA-Rcan1) in a mouse osteoarthritis model (DMM).

[0039] 4C is the score based on the Osteoarthritis Research Society International (OARSI) grading criteria for cartilage tissue. * indicates a significant difference, p < 0.05.

[0040] 4D is the scoring result of synovial inflammation. * indicates significant difference, p<0.05.

[0041] 4E is a comparison of RCAN1 expression levels in articular cartilage, subchondral bone and synovial tissues of the control group (siRNA-Ctrl) and the Rcan1 inhibition treatment group (siRNA-Rcan1).

[0042] 4F is a comparison of the expression levels of Col2, Col10, Mmp13 and Adamts5 in articular cartilage tissue between the control group (siRNA-Ctrl) and the Rcan1 inhibition treatment group (siRNA-Rcan1), where sham represents the sham operation group. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0044] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and can be implemented using existing technologies. The experimental instruments and materials used in the examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] Example 1 Knockout of the RCAN1 gene by lentiCRISPRv2

[0046] (1) The sgRNA sequence targeting RCAN1 is as follows:

[0047] sg RCAN1-huamn-F1: 5′-CACCGTCGCGTGCCAGTTCAGCTG-3′;

[0048] sgRCAN1-human-R1:5′-AAACCAGCTGAACTGGCACGCGAC-3′;

[0049] sg RCAN1-huamn-F2: 5′-CACCGAGCTGAACTGGCACGCGACG-3′;

[0050] sgRCAN1-human-R2: 5′-AAACCGTCGCGTGCCAGTTCAGCTC-3′;

[0051] (2) The oligonucleotide sequence targeting the RCAN1 gene was synthesized at Sigma, and sg RCAN1-huamn-F1 was annealed with sg RCAN1-human-R1 to obtain sg RCAN1-human-1. The oligonucleotide sequence was then ligated with T4 ligase (NEB) to the vector backbone obtained by digesting lenti-CRISPRv2 (Addgene product, #52961) with FastDigest_Esp3I (NEB). The resulting recombinant vector with the correct sequence was designated as recombinant vector sgRCAN1-human-1. The lenti-CRISPRv2 vector backbone contains a Cas9 endonuclease encoding gene and can express the Cas9 endonuclease, as well as an insertion site for an exogenous DNA fragment used to guide Cas9 to a specific site in the genome and encoding DNA for the gRNA backbone. The recombinant vector sg RCAN1-human-1 can encode sgRNA directed to the RCAN1 gene.

[0052] (3) Following the above method, sg RCAN1-huamn-F2 was annealed with sg RCAN1-human-R2 to generate sg RCAN1-human-2. This sg RCAN1-human-2 was then ligated with T4 ligase (NEB) to the lenti-CRISPRv2 (Addgene product, #52961) vector backbone that had been digested with FastDigest_Esp3I (NEB). The resulting recombinant vector with the correct sequence was designated as sgRCAN1-human-2. The recombinant vector sg RCAN1-human-2 encodes the sgRNA targeting the RCAN1 gene.

[0053] (4) According to the above method, sgNTC-F1 and sgNTC-R1 were annealed to obtain sgNTC, which was then ligated with T4 ligase (NEB) to the vector backbone obtained by digesting lenti-CRISPRv2 (Addgene product, #52961) with FastDigest_Esp3I (NEB). The resulting recombinant vector with the correct sequence was the control vector.

[0054] sgNTC-F1: 5′-CACCGACGGAGGCTAAGCGTCGCAA-3′;

[0055] sgNTC-R1: 5′-AAACTTGCGACGCTTAGCCTCCGTC-3′.

[0056] (5) Using the Lipo3000 transfection kit (ThermoFisher), the lentiviral plasmid sg RCAN1-human, the lentiviral packaging vector psPAX2, and pMD2G were co-transfected into 293T cells (ratio: 1 10 cm dish of 293T cells: 9 μg lentiviral plasmid sg RCAN1-human, 6 μg psPAX2, and 3 μg pMD2G) and cultured for 8 hours.

[0057] Replace with fresh 293T cell culture medium and continue culturing for 48-54 hours.

[0058] The supernatant was collected, filtered using a 0.22 μm filter membrane, and the filtrate was collected.

[0059] Centrifuge at 4°C and 19,400 rpm for 2 hours, discard the supernatant, and resuspend in culture medium to obtain the virus solution containing the sg RCAN1-human recombinant lentivirus, referred to as sg RCAN1-human virus solution.

[0060] (6) The lentiviral plasmid sg RCAN1-human was replaced with the control vector, and other steps remained unchanged to obtain the control virus solution, which was recorded as sgNTC virus solution.

[0061] (7) sgRCAN1-human lentivirus infection of mesenchymal progenitor cells: hESC-MSCs mesenchymal progenitor cells at passage 4 were used as test cells and infected with sgNTC and two sg RCAN1-human viruses, respectively. The specific method was as follows: 2 μL sgRCAN1-human lentivirus (or sgNTC virus) and 2 μL Polybrene were added to the culture well (one well of a 6-well plate) seeded with hESC-MSCs mesenchymal progenitor cells at passage 4. The medium was changed the next day, and then the cells were cultured normally and passaged.

[0062] (8) After infection with sgNTC or sg RCAN1-human lentivirus, the resulting cells were serially passaged to passage 3-4. The cells were collected, and the efficiency of RCAN1 protein knockdown was tested. The cell senescence marker SA-β-gal staining was performed, and the mRNA levels of IL6, cell proliferation molecular marker (Ki67), and senescence-related genes (Il1B, IL6, IL8, CXCL1) were detected.

[0063] Example 2 SiRNA knockdown of RCAN1

[0064] (1) siRNA sequences were synthesized at Guangzhou Ruibo Biotechnology Co., Ltd. The siRNA sequences targeting human RCAN1 are as follows;

[0065] AntiRCAN1-si01:5′-GGACGTATGACAAGGACAT-3′ (Cat. No. siB13516183302)

[0066] AntiRCAN1-si03:5′-GTGGTCCATGTATGTGAGA-3′ (Cat. No. siG000001827A)

[0067] (2) The non-targeting control siRNA sequences are as follows;

[0068] Non-targeting control:5′-TTCTCCGAACGTGTCACGT-3′ (Cat. No. siN0000001-1-5)

[0069] (3) Digestion of cells: First, cells in good growth condition are digested with trypsin and centrifuged;

[0070] (4) Cell counting: remove the supernatant, add 1 mL of fresh culture medium to resuspend, aspirate 20 μL and add it to the cell counting plate, and count the cells using a cell counter (Count Star).

[0071] (5) Replating: Cells were evenly seeded on a 12-well plate at a density of 5 × 104 cells / well and cultured overnight;

[0072] (6) Doxorubicin-induced aging: discard the old culture medium and add culture medium containing doxorubicin, and culture for 24 h;

[0073] (7) Medium change: After 24 h, replace with fresh medium without PS;

[0074] (8) Transfection: First, 100 μL opti-MEM (reduced serum medium, Gibico, #31985070) was mixed with 2.5 μL 50 nM siRNA; 100 μL opti-MEM and lipo2000 (ThermoFisher), and then incubated at room temperature for 5 min. Mix the two in a 1:1 ratio and then let it stand at room temperature for 20 min. 200 μL of each reagent was added to the corresponding well plate for transfection for 8-12 hours. Fresh complete medium was replaced.

[0075] (9) Sample collection: Retransform and collect RNA within 24-48 hours; qPCR: Detect the expression levels of aging-related genes (IL1B, IL6, IL8).

[0076] (10) Detection of SA-β-gal (senescence-associated β-galactosidase) in senescent cells.

[0077] (11) A portion of the cells was taken and RNA was extracted using TRIZOL (gibco, 15596018) and then sent to the company (Jingneng) to build a library for transcriptome RNA-seq sequencing.

[0078] Example 3 SA-β-gal detection of cell senescence

[0079] Step 1: Mesenchymal progenitor cells were seeded into one well of a gelatin (Sigma)-coated 6-well plate at a density of 1×10 5 / well and stained on the second day.

[0080] Step 2: After step 1, fix the cells with a fixative solution [2% formaldehyde (volume percentage, v / v) + 0.2% glutaraldehyde (volume percentage, v / v) + 97.8% PBS (volume percentage, v / v)] for 3-5 minutes and wash twice with PBS.

[0081] Step 3: After step 2, add 2 mL of staining solution (40 mM citric acid / sodium phosphate buffer, 5 mM K4[Fe(CN)6], 5 mM K3[Fe(CN)6], 150 mM NaCl, 2 mM MgCl2, 1 mg / mL X-gal) to each well and incubate in a 37°C bacterial incubator in the dark overnight.

[0082] Step 4: After completing step 3, wash twice with PBS, observe under an inverted microscope, and take pictures.

[0083] Example 4 Osteoarthritis Modeling and Drug Administration in Mice

[0084] (1) siRNA sequence

[0085] Cholesterol- and methylation-modified siRNA sequences, as well as cyanine dye 3 (Cy3)-modified siRNA sequences, were synthesized at Guangzhou Ruibo Biotechnology Co., Ltd. The siRNA sequence targeting mouse Rcan1, siRNA-Rcan1: 5′-TGCTCAGACTTTACACATA-3′ (Cat. No. siG171116021722); the non-targeting control sequence, siRNA-Ctrl: 5′-TTCTCCGAACGTGTCACGT-3′ (siN0000005).

[0086] (2) DMM modeling

[0087] The surgical procedure was as follows: mice (C57BL / 6, male, 2 months old, 10 pil / g body weight) were anesthetized with an intraperitoneal injection of 0.8% sodium pentobarbital, shaved, short hair removed with depilatory cream, and rubbed with iodine; the skin was longitudinally incised at the knee joint of the mouse using a scissors and scissors, and the ligament and muscle were cut along the medial side of the ligament under a stereoscope with a scalpel. The tibialis ligament was turned sideways to expose the knee joint; the medial meniscus was cut with microscissors to avoid scratching the articular cartilage; the joint cavity, muscle, and skin were sutured layer by layer; and the mouse was placed on a heating blanket and waited for it to wake up.

[0088] (3) Administration

[0089] Two weeks after surgery, mice were injected directly into the left leg with control siRNA (non-targeting control siRNA-Ctrl) and the right leg with anti-Rcan1 siRNA (siRNA-Rcan1, targeting mouse Rcan1), with injections repeated weekly. Mice were sacrificed at 8 and 12 weeks post-injection, and joint samples were collected for subsequent processing.

[0090] (4) Sample processing and testing

[0091] Joint sampling: Cut off the entire joint of the mouse and remove the muscle tissue as much as possible with ophthalmic scissors;

[0092] Fixation: Soak the joint in 4% paraformaldehyde in the dark for more than 24 hours, then rinse with running water overnight to completely remove the fixative.

[0093] Decalcification: Place the joint in decalcification solution for 3 weeks, changing the solution 2-3 times during this period. The femur and tibia will become soft at this time, and the degree of decalcification can be judged by cutting with scissors. Rinse with running water overnight to completely rinse out the decalcification solution.

[0094] Paraffin sections were prepared and subsequently subjected to SO staining and immunohistochemical staining of rcan1, col2a, mmp13, adamts5, col10, etc.

[0095] The progression of OA in mice was assessed by OARSI scoring and positive cell quantification.

[0096] Example 5 CAN1 specifically upregulates expression levels in senescent cells

[0097] We observed that the expression level of RCAN1 in OA patients' joints was higher than that in normal controls ( Figure 1 A). We obtained normal human and OA synovial tissues and co-localized senescent cell markers such as p53 and IL1A with RCAN1, and found that RCAN1 was upregulated in p53 and IL1A positive cells ( Figure 1 B). In a mouse OA model, we found that the number of RCAN1-positive cells in the cartilage, synovium, and subchondral bone of OA joints was significantly higher than that in normal controls ( Figure 1 C). Subsequently, we used doxorubicin to induce cell senescence in human mesenchymal stem cells cultured in vitro and observed that RCAN1 expression gradually increased with the progression of cell senescence ( Figure 1 DE). This indicates that RCAN1 is specifically upregulated in senescent cells, and RCAN1-positive senescent cells accumulate during the course of OA disease.

[0098] Example 6: Delaying cell senescence by knocking out RCAN1 through CRISPR / Cas9

[0099] We constructed RCAN1 knockout human mesenchymal stem cells using the lentiCRISPRv2 system and induced senescence with doxorubicin. We found that knockout of RCAN1 significantly inhibited the classic senescent cell marker: SA-β-gal positive cell staining, but the cell number did not increase significantly ( Figure 2 A). Further testing of SASP-related gene levels in senescent cells revealed that RCAN1-knockout senescent cells expressed reduced levels of pro-inflammatory factors IL1B and IL6, and that the cell surface localization of IL1a, a classic pro-inflammatory SASP upstream gene, was significantly downregulated ( Figure 2 B and C). This suggests that RCAN1 regulates the secretion of the pro-inflammatory SASP in senescent cells and may serve as a potential therapeutic target.

[0100] Example 7 Knockdown of RCAN1 by siRNA to Delay Cell Senescence and Regulate Cell Metabolism

[0101] We designed RNAi sequences targeting human RCAN1 and knocked down RCAN1 in doxorubicin-induced senescent cells. Similarly, we found that inhibition of RCAN1 could reduce the number of SA-β-gal positive cells, cellular DNA damage, and the levels of pro-inflammatory cytokines ( Figure 3AC). Through high-throughput transcriptome sequencing, we found that inhibition of RCAN1 inhibited IL1 signaling and aging-related pathways, while the degradation of aging-related metabolites was enhanced and cellular responses were suppressed ( Figure 3 D) This indicates that RNAi can be used as a means to inhibit RCAN1, confirming its function in inhibiting the secretion of the pro-inflammatory SASP in senescent cells.

[0102] Example 8 Inhibiting Rcan1 levels with siRNA can delay OA progression in a mouse DMM model

[0103] In the OA model established by mouse DMM, siRNA can be effectively delivered into the joint cavity and significantly inhibit the level of Rcan1 ( Figure 4 A, 4E). After 2 months of treatment, compared with the non-targeted siRNA group, the Rcan1-targeted group showed significantly enhanced extracellular matrix staining in the articular cartilage, weakened matrix degradation, and significantly alleviated subchondral bone sclerosis and synovial inflammation ( Figure 4 BD). At the same time, the increase in articular cartilage matrix degrading enzymes mmp13 and Adamts5 caused by OA was significantly alleviated in the joints of mice in the Rcan1 targeting group ( Figure 4 F). These results indicate that siRNA can be used as an effective means to target and inhibit Rcan1 in vivo, and that reducing Rcan1 levels can delay OA progression.

[0104] The above embodiments are only for the purpose of illustrating the present invention clearly and are not intended to limit the scope of protection of the present invention. Any modifications made based on the technical solution of the present invention in accordance with the technical ideas proposed by the present invention are within the scope of protection of the present invention.

Claims

1. Use of a substance that reduces the content or activity of RCAN1 in the preparation of a product for preventing and / or treating osteoarthritis, characterized in that: The substance is siRNA, and the sequence of the siRNA is: 5'-TGCTCAGACTTTACACATA-3'.

2. Use of a substance that inhibits RCAN1 gene expression or knocks out RCAN1 gene in the preparation of a product for preventing and / or treating osteoarthritis, characterized in that: The substance is siRNA, and the sequence of the siRNA is: 5'-TGCTCAGACTTTACACATA-3'.

3. The use according to any one of claims 1 to 2, characterized in that The osteoarthritis includes osteoarthritis caused by meniscus injury.

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