TW201239097A - MiRNAs in joint disease - Google Patents

Abstract

The present invention concerns microRNAs (miRNA), in particular mir-199a, as an indicator of a tissue status or a disease such as osteoarthritis (OA) and as a target molecule for the discovery of a substance for the treatment of a joint disease. Further provided are methods and compounds for investigation, analysis and/or treatment of a joint disease, especially OA.

Description

201239097 VI. Description of the Invention: [Technical Field] The present invention relates to microRNAs which are indicators of tissue state or disease such as osteoarthritis (OA) and molecules which are found as substances for treating joint diseases ( MicroRNAs, miRNAs, especially mir-199a. The invention further provides methods and compounds for the study, analysis and/or treatment of joint diseases, particularly 〇A. [Prior Art] Arthritis is the most common joint disease in humans and is characterized by joint pain, inflammation, redness and swelling. Several different types of arthritis are known. Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that mainly invades the synovial joint, while ankylosing spondylitis (AS) is also a chronic inflammatory arthritis, the main violation. The joints of the spine and the pelvis. Juvenile idiopathic arthritis (JIA) is characterized by inflammation of the synovial membrane and affects children under 16 years of age. Gout is characterized by recurrent acute inflammatory arthritis due to an increase in the concentration of uric acid in the blood. Septic arthritis is caused by joint infections, and psoriatic arthritis may occur in patients with psoriasis. Osteoarthritis (OA) is characterized by mechanical abnormalities of the joints, such as the degradation of articular cartilage and subchondral bone. 〇A is the most common disease in the Western world. In 2 years, more than 21 million people were diagnosed in the United States, Japan, and Germany. Because the ethnic group is aging, the population of OA patients must continue to increase, and their quality of life is seriously affected. In addition, 0A imposes a significant socio-economic burden on direct and indirect expenditures. Current treatments focus on controlling OA-related pain, but there is still no need to find a pharmaceutical treatment that slows, stops, or even reverses the course of OA and its effects. 〇 A can be viewed as a clinical and pathological result of a structural and functional decline in synovial joints. It occurs when the dynamic balance between joint tissue damage and repair is compromised. The structural decline of articular cartilage may be due to abnormal mechanical stretching that damages healthy cartilage and a pathologically impaired joint deterioration due to physiological and mechanical stretching. OA is characterized by progressive deterioration of articular cartilage, which ultimately leads to dysfunction of the affected joint. During the chronic progression of 〇A, the articular cartilage is destroyed and the underlying bone tissue is emptied. Finally, the affected joint needs surgical replacement. In addition to cartilage destruction, pathological deterioration of the synovium and ligaments also occurs. Inflammation that occurs in OA is a secondary effect. The type changes observed in OA include cartilage erosion and varying degrees of synovial inflammation. These changes are attributed to the biochemicals of complex networks, including proteolytic enzymes that cause the breakdown of cartilage macromolecules (especially type II, type X collagen, and aggrecan). Cytokines such as activated synoviocytes, monocytes, or IL-1 and TNF-α produced by articular cartilage itself can significantly upregulate matrix metalloproteinase (MMP) and cytokine gene expression and enable compensatory synthesis The route becomes insensitive. 4 201239097 The exact pathogen of ◦A is still pending, but some contributing factors are related to the onset and progression of the disease. The most important factor is age, but cyclical burdens, obesity, joint relaxation, and genetically susceptible diseases also play an important role. Decomposition of cartilage matrix components is generally accepted because extracellular proteases (mainly MMPs) and the synthesis and activation of cytokines that amplify the degenerative process are increased. According to clinical tests, the joints of patients with symptoms and signs are gradually diagnosed and diagnosed with X-rays. Typical changes that can be observed via X-rays include joint space narrowing, marginal bone spurs, subchondral sclerosis, subchondral formation of cysts, bony remodelling, and bone effusion. Laboratory tests without diagnostic OA are available because the commonly used markers remain normal in OA. These tests can only be used to distinguish OA from other forms of arthritis, particularly inflammatory types of arthritis. Therefore, it can only be diagnosed as 〇A when the joint deterioration has progressed to a stage that can be seen by X-rays. It is still impossible to detect the early onset of the disease and prevent timely treatment of joint damage. Thus, there is still a need for improved tools that can identify 〇A early. It is not possible to see the etiology of pharmacological therapy to reduce degenerative symptoms because there is no 〇A drug (DM〇AD) that can improve the disease. Therefore, existing disease control is forced to administer painkillers (primarily non-steroidal anti-inflammatory drugs (NSAIDs)) to relieve pain and thereby improve quality of life. Because of the shortcomings of the state of the art, there is a great need for an improved diagnostic method that is capable of identifying the early stages of the disease and thus preventing and/or early treatment of 〇A. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide an improved method for identifying a disease and a substance and composition suitable for enhancing the treatment. This can be reversed by various aspects of the invention: methods and materials based on miRNAs that differentially express during the differentiation process of chondrocytes and cultured bone marrow stem cells (BMCS) are provided. The following inventors of the present invention discovered the following foundations: the inhibition of mir-199a in cartilage, 'month 1 will increase the cartilage standard (chondrogenic coffee (4), while excessive expression of mir-199a in chondrocytes will reduce cartilage In the t-th aspect, the present invention provides a use of miM99a as an indicator of tissue status or disease, and a second aspect as an indicator of tissue status or disease. In the second aspect The present invention provides a method for identifying a tissue state change or a disease (preferably a joint disease) risk and/or identifying the presence of a disease and/or monitoring disease progression in an individual, comprising 4 test samples Mir-199a content. In the fourth aspect, the present invention provides a method for determining a dosage of a pharmaceutical product for modifying a tissue state or preventing or treating a disease, preferably a joint disease, in an individual, comprising the following steps: (4) Determining the mir-199a content in the sample of the individual, and (b) determining the pharmaceutical dosage based on the mir-199a content in the test sample. In the fifth aspect, the present invention provides an adjustment for A method of altering the state of the tissue 6 201239097 or a pharmaceutical dosage for preventing or treating a joint disease, comprising the steps of: (a) determining the mir-199a content in the sample, (b) determining the mir_199a content in one or more reference samples, The test sample σσ is whether the mjr_i99a content in the sample is different from the content of one or more reference samples ten, and is adjusted according to whether the mir-199a content in the test sample is different from the content in one or more reference samples. Pharmaceutical dosage. In a sixth aspect, the invention provides a method of determining the beneficial and/or adverse effects of a substance on the tissue state of a joint disease or the onset of a joint disease, comprising the steps of: (a) determining a test sample The mir_199a content, (b) determining the mir-199a content in one or more reference samples, and (c) verifying whether the test sample has a diir-199a content in the sample that is different from one or more reference samples Included, wherein the test sample is exposed to the substance different from the one or more reference samples. In the seventh aspect, the present invention provides mir_199a at Use in any of the three to six aspects. In an eighth aspect, the present invention provides a kit for use in any of the methods of the third to sixth aspects, the 纟 comprising one or more detection mir The member of -199a. In the nine aspect, the present invention provides the use of a H-like kit in any of the methods of the second to sixth samples. In the tenth aspect, the present invention provides one or more uses. The nucleic acid of the miM99a gene, the gene product or the functionally active variant thereof of 201239097 is speculated in any of the third to sixth aspects. In the eleventh aspect, the present invention provides a method for A peptide, polypeptide or protein that detects a gene, a gene product, or a functionally active variant thereof, in any of the third to sixth aspects. In a twelfth aspect, the invention provides the use of a nucleic acid according to the tenth aspect, or a peptide, polypeptide or protein according to the eleventh aspect, in any one of the third to sixth aspects. In a twelfth aspect, the invention provides a method of screening for a mir_199a agonist, wherein the method comprises the steps of: (4) providing a mir-199a or mir-199a gene, (b) providing a test compound, and (c) measuring Or to detect the effect of test compounds on the mir l99a or mir_199a genes. In the fourteenth aspect, the present invention provides a mir l99a agonist for changing the tissue state of a joint disease or preventing or treating a joint disease. In a fifteenth aspect, the present invention provides a pharmaceutical product comprising the mir-199a agonist as in the fourteenth aspect. In the sixteenth sample, the present invention provides a method for changing a joint disease, a woven state or a rib or a joint disease, wherein the fifteenth & medicinal product is administered to the joint Individuals at risk of developing or at risk of joint disease. DETAILED DESCRIPTION OF THE INVENTION Before the present invention is described in detail below, it should be understood that the invention may be modified and not limited to the particular methodology, steps, or reagents described herein. It is also to be understood that the terminology used herein is for the purpose of describing the particular embodiments, and is not intended to limit the scope of the invention. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined. Preferably, the terms used herein are as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Defined in Basel, Switzerland. Several documents are cited throughout the text of this manual. The documents cited in this article (including all patents, patent applications, scientific publications, manufacturer's instructions, operating instructions, GenBank) The accession number sequence is submitted to the present specification, whether hereby or below, and is hereby incorporated by reference in its entirety. This disclosure is not to be construed as a There is a need, unless the words "comprise" " An integer or a step or a group of integers or steps. A "nucleic acid" molecule is understood to mean a polymeric or recruitment macromolecule composed of nucleomonomers. It consists of a nucleobase, a pentacyclic ruthenium (such as but not limited to ribose or 2'-deoxyribose), and 1 to 3 phosphate groups. In general, 'polynucleotides are permeabilized in separate nucleomonomers. Formed by a phosphodiester bond between 201239097. In the context of the present invention, a nucleic acid molecule is meant to include, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA mixtures. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein. Nucleic acids can be chemically synthesized 'for example, according to the sulphuric acid triacetate method (see 'eg Uhlmann, E. &

Peyman, A. (1990) Chemical Reviews, 90, 543-584). Nucleic acids can be broken down by endonucleases or exonucleases, particularly DNase and RNase found in cells. Therefore, it is advantageous to modify the nucleic acid to make the nucleic acid more stable against decomposition, thereby ensuring that the high concentration of nucleic acid is maintained in the cells for a long time (Beigelman et al. ¢ 1995) Nucleic Acids Res. 23: 3989-94; WO 95/11910; WO 98/37240; WO 97/29116). In general, such stabilization can be achieved by introducing one or more internucleotide groups or by introducing one or more non-nucleotide internucleotides. These modified nucleotides are in accordance with Uhlmann and

Peyman (1990), supra (see also Beigelman et al. (1995) Nucleic Acids Res. 23: 3989-94; WO 95/119l; w〇 98/37240; WO 97/29116). The intranucleotide modified internucleotide phosphate group and/or the non-bridge may be used in one of the uses of the present invention, for example, decyl phosphate, phosphorothioate, phosphoniumamine, dithionate And/or phosphates, but not interphosphoric acid analogs, such as, for example, a sulphur bridge, a carbonate bridge, a carboxymethyl ester, an acetamide bridge, and/or a sulfur bridge. This modification also intends to enhance the durability of the pharmaceutical composition, which can be used in one of the uses of the present invention. The 201239097 nucleic acid can be selected from the group consisting of a polynucleotide probe, a primer (eg, a primer pair, preferably for polymerase chain reaction (PCR), reverse transcription (RT) reaction, or DNA sequencing). Primers), peptide nucleic acids (PNA), locked nucleic acids (LNA), glycolic acids (GNA), threose nucleic acids (TNA), microRNAs (miRNAs), and small interfering RNAs (siRNAs). The term "probe" as used herein, refers to a single-stranded oligonucleotide that is generally used to detect a target RNA and/or DNA sequence that is complementary to a probe sequence. The probe and its nucleotide sequence are probed and labeled. The complementarity between sequences allows hybridization of single-stranded nucleic acids (DNA or RNA) with nucleotide pairing. The length of the probe depends on the desired use of the probe and the specificity required. In general, the probe is 20- 500 (eg 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 11 〇, 12 〇, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400) 500) nucleotides long, preferably 20-100 nucleotides, more preferably 2〇_5〇. For detecting microRNAs, the probe is between 12 and 30 nucleotides. Needles are used in different experimental groups and are not limited to southern ink dots and northern ink dots, such as real-time PCR or in situ hybridization (ISH) and microarray experiments. Probes can be unmarked, directly labeled or indirectly labeled. Biotin that can be bound, such as after use of the streptavidin complex. The marker can be spectrally, photochemically, biochemically, immunochemically Molecules detected by chemical or other physical methods. For example, suitable markers include 32P, fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin or haptens. And other entities detectable or made detectable. The marker can be incorporated at any position of the nucleic acid, such as 201239097 at the 3' end, at the 5' end or inside. The term "probe" also encompasses A nucleic acid having a different skeleton composition, such as, but not limited to, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), and a threose nucleic acid (TNA). The term "bend scorpion" as used herein means A single-stranded oligonucleotide that is generally the starting point for DNA replicase. The primer binds to the DNA template or hybridizes to the DNA template and generally comprises a sequence that is complementary to the DNa sequence to which it should bind. The tick can also contain additional sequences, such as sequences that are cleavage sites for nucleases (e.g., Bam HI, Hind III, etc.). The length of the primer is selected according to the intended use. For example, primers used to amplify DNA in polymerase chain reaction (PCR) typically have a length of at least 1 nucleotide 'preferably between 1 and 50 (eg 15, 16, 16, 18, 19) , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50) Further, it is preferably between 15 and 30 nucleotides. A shorter primer with at least 5 nucleosides is used for the sequencing of the DNA template. "degenerate primers" also included in the term, primer, which may be a mixture of similar but not identical primers. The primers can be labeled or labeled using spectrally, photochemically, biochemically, immunologically, chemically, or otherwise physically detected. As used herein, the term "microRNA" and variants such as "miRNA" and "miR" include human miRNA, mature single-stranded miRNA, precursor miRNA (pre-miR) and variants thereof, which may be natural In some cases, the term "miRNA" also includes primary r mRNA transcripts and double-stranded miRNAs. Unless otherwise stated, when used in 2012-039797, the name of a particular miRNA means a mature miRNA. For example, Mir-199a means the mature miRNA sequence derived from pre-mir-199a. The sequences of miRNAs are reported in the following databases, including human maturation and precursor sequences (also known as stem-loop sequences): miRBase-microRNA library, available as follows Linked to: http://microrna.sanger.ac.uk or http://www.mirbase.org/ Public publications containing miRNA sequences include Griffiths-Jones et al., Nucleic Acids Research, 2008, 36, Database Issue, D154-D158; Griffiths-Jones et al., Nucleic Acids Research, 2006, 34, Database Issue, D140-D144;

Griffiths-Jones, Nucleic Acids Research, 2004, 32, Database Issue, D109-D111, Griffiths-Jones et al., Nucleic Acids Research, 2011, Vol. 39, Database Issue, D152-D157).

Lagos-Quintana et al. ["New microRNAs from mouse and human" Lagos-Quintana M, Rauhut R, Meyer J, Borkhardt A, Tuschl T RNA. 9:175-179 (2003)·] from human osteoblast sarcoma The cells were colonized with miR-199. They also reported the identification of miRNAs from the opposite arm of mouse cells. This sequence was named miR-199-s and the sequence from the 3' arm of the homologous mouse precursor was named miR-199-as. Lim et al. ["Vertebrate microRNA genes" Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP Science. 299:1540 (2003).] independently using the conservation of mouse and red fin puffer sequences The miRNA is predicted by the computer ["Vertebrate microRNA 13 201239097 genes" Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP Science. 299:1540 (2003).]. The performance of the miR cut by the 5' arm was evaluated in zebrafish and its ends were depicted by colonization. The excised miR sequence was renamed miR-199a (to avoid confusion with miR-199b (MI0000282) identified later) and miR-199a* (from 3' arm). The two putative hairpin precursors correspond to chromosome 19 (mir-199a-l, MI0000242) and chromosome 1 (mir-199a-2, MI0000281) in the human map. Landgraf et al. showed that two mature products were expressed, prompting the renaming of miR-199a-5p and miR-199a-3p. [ΠΑ mammalian microRNA expression atlas based on small RNA library sequencing" Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foa R, Schliwka J, Fuchs U, Novosel A, Muller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M Cell· 129:1401-1414 (2007).]. (See also "Identification of human fetal liver miRNAs by a novel method" Fu H, Tie Y, Xu C, Zhang Z, Zhu J, Shi Y, Jiang H, Sun Z, Zheng X FEBS Lett. 579:3849-3854 (2005) The sequence of the wise man mirl99a (MI identification code is the access number in the mirbase database, see below): 1) hsa-mir-199a-1 stem loop sequence (MI0000242)

GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGG

CUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUA 201239097 GGC(SEQIDNO: 1) 2) Stem loop sequence of hsa-mir-199a-2 (MI0000281) AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCA GUGUUCAGACUACCUGUUCAGGACAAUGCCGUUG UACAGUAGUCUGCACAUUGGUUAGACUGGGCAAG GGAGAGCA (SEQ ID NO: 4) 3) Mature sequence hsa-miR-199a-5p (MIMAT0000231) CCCAGUGUUCAGACUACCUGUUC (SEQ ID NO: 2) hsa-miR-199a-3p (MIMAT0000232) ACAGUAGUCUGCACAUUGGUUA (SEQ ID NO: 3) hsa (smart person) mirl99-al has the same mature sequence as mirl99-a-2. This miRNA sequence is based on a confirmed miRNA with a mouse ["Identification of novel genes coding for small expressed RNAs" Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T Science. 294: 853-858 (2001), &quot The homology of Michael MZ, O' Connor SM, van Holst Pellekaan NG, Young GP, James RJ Mol Cancer Res. 1:882-891 (2003)] was predicted by the reduced accumulation of specific microRNAs in colorectal neoplasia. Its performance was later confirmed in humans ["Kaposi's sarcoma-associated herpes virus expresses an array of viral microRNAs in latently infected cells" Cai X, Lu S, Zhang Z, Gonzalez CM, Damania B, 15 201239097

Cullen BR Proc Natl Acad Sci USA. 102:5570-5575(2005)., "A mammalian microRNA expression atlas based on small RNA library sequencing" Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foa R, Schliwka J, Fuchs U, Novosel A, Muller RU, Schermer B, Bissels U, Inman J , Phan Q, Chien M Cell. 129:1401-1414 (2007).]. MicroRNAs (miRNAs) are endogenous small RNA molecules that mean regulation of gene expression at the translational level. In addition, they are part of the cellular RNA interference (RNAi) mechanism. miRNAs are encoded by genes transcribed from DNA but are not translated into proteins (non-protein-encoding RNA). They are first discovered in nematodes and are present in both plants and animals. The sequence of many miRNAs is highly conserved among different species, suggesting that the miRNA pathway is part of a relatively old and important regulatory mechanism (Grosshans et al. J Cell Biol 156: 17-21 (2002)). miRNAs have been found to regulate approximately 30% of mammalian genes (Czech, NEJM 354: 1194-1195 (2006); Mack, Nature

Biotech. 25: 631-638 (2007); Eulalio, et al., Cell 132: 9-14 (2008)). Recently, miRNAs have been found to regulate gene expression by blocking translation or decomposing transcripts to inhibit protein production'. A single miRNA can determine 250-500 different mRNAs, demonstrating that such RNA is a very important regulator of a wide range of cellular functions. A gene regulated by miRNA 201239097 These genes are pathogenic genes. Therefore, any miRNA-regulatory function has great therapeutic potential. In animals, miRNAs are first expressed by the genome as RNA transcripts, called primary miRNAs (pri-miRNAs). They are transcribed by rna polymerase II and form a hairpin structure. In the nucleus, the dsRNA-specific nuclease Drosha processes the pri-miRNA into a shorter, approximately 70 to 1 nucleotide long stem-loop structure (known as the pre-miRNA), which is then shipped out. Enter the cytoplasm, as by exporting protein-5 (Exp5) (Yi, et al. Genes Dev. 17: 3011-3016 (2003)). In the cytoplasm, Dicer, a member of the RNase III nuclease family, cleaves the pre-miRNA into a double-stranded guide/passenger (miRNA/miRNA) double strand with a 3' cantilever at both ends. Two strands of miRNA double strands usually have mismatches and imperfect complementarity in their sequences' and when they are separated, mature miRNAs (in many cases between about 19 and 23 nucleotides long) It is bound by an RNA-induced silencing complex (RISC) or a similar protein complex. RISC is also a protein complex that is mediated by small or short interfering RNA (siRNA) that affects target-specific mRNA breakdown. At present, once the RISC catalytic component (argonaute) is selected as the lead vector, the mature miRNA is incorporated into the RISC complex and binds to the messenger despite the apparent but often imperfect complementary sequence. RNA (mRNA) molecule. The passenger miRNA* can be decomposed. In turn, inhibition of mRNA binding by the miRNA-RISC complex is translated, resulting in reduced performance of the corresponding gene. In some cases, the bound mRNA is cleaved or de-adenylated and decomposed. For certain miRNAs, a single precursor contains more than one mature miRNA sequence. In other cases, the 'multiple precursor miRNAs contain the same mature sequence. The term "protein" or "polypeptide" is used interchangeably herein and refers to a peptide linker of any amino acid, regardless of length or post-translational modification. The proteins (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) useful in the present invention can be further modified by chemical modification. This suggests that such a chemically modified polypeptide contains two chemical groups other than the naturally occurring amino acid. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amine guanidines. In addition, the chemical modification may provide more advantageous properties than the parent polypeptide, for example, one or more of increased growth, increased biological half-life, or increased water solubility may be applied to the chemical modification package of the variant used in the present invention. It is not limited to the glycosylation of a glycolated, non-glycosylated parent polypeptide, or a glycosylation pattern present in a parent polypeptide. - As used herein, the term ", variant" is understood to mean a polypeptide or a protein or nucleic acid variant derived from a polynuclear (tetra) or protein in its length or sequence. Multinuclear (4) is also known as a parent polypeptide or a multinuclear allogeneic "fragment, or, derivative"_ and 5, fragment " is smaller in length or size than the parent molecule, , in its sequence, showed one or more 201239097 differences compared to the parent molecule. Also included are modified molecules such as, but not limited to, post-translationally modified proteins (eg, glycosylated, biotinylated, acidified, ubiquitinated, palmitated, or proteolytically cleaved proteins), and Modified nucleic acids (such as methylated DNA). Mixtures of different molecules, such as, but not limited to, RNA-DNA mixtures, are also encompassed by the term "variant." In general, variants are artificially constructed, preferably by genetic techniques and the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, it should be understood that naturally occurring variants are also encompassed by the term "variant" as used herein. Furthermore, variants useful in the present invention are also derived from homologs, orthologs or paralogs or artificially constructed variants of the parent molecule, provided that the variant exhibits at least the parent molecule A biological activity, that is, functionally active. Additionally or alternatively, a "variant" as used herein is characterized by a certain degree of sequence identity to a parent polypeptide or a parent polynucleotide derived therefrom. More specifically, in the context of the present invention The protein variant exhibits at least 80% sequence identity to its parent polypeptide. The polynucleotide variants in the context of the invention exhibit at least 80% sequence identity to their parent polynucleotide. The term "at least 80% sequence identity" Is used throughout the specification for polypeptide-to-polynucleotide sequence alignment. This term preferably means that the sequence identity to an individual reference polypeptide or individual reference polynucleotide is at least 80%, at least 81%, at least 82%. At least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% 19 201239097 or at least 99%. The similarity of nucleotides to amino acid sequences (i.e., the percentage of sequence identity) can be determined by sequence alignment. These comparisons can be calculated using several techniques It is preferred to use Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877) mathematical algorithm, hmmalign (HMMER package, http://hmmer. Wustl.edu/), or for example at http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2/index.html or http:/ /npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/N PSA/npsa_clustalw.html Get the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) dcz· And es. 22, 4673-80). The preferred parameters used are at http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2 The default parameters set by /index.html. The degree of sequence identity (sequence pairing) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). A similar algorithm is incorporated into Altschul et al. (1990) J. Mol Biol. 215: 403-410 in the BLASTN and BLASTP programs. BLAST polynucleotide studies were performed using the BLASTN program, score = 100, wordlength = 12 to obtain a polynucleotide sequence homologous to the nucleic acid encoding mir-199a. BLAST protein studies were performed using the BLASTP program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to mir-199a. To obtain a gapped alignment for comparison, use 201239097

Gap BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When using BLAST and the gap BLAST program, the preset parameters of the individual programs are used. Sequence pairing analysis complements established homology mapping techniques such as Shuffle-LAGAN (Brudno Μ., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are referred to in this application, these percentages are calculated as the total length of the longer sequence, unless otherwise specified. "Hybridization" can also be used as a measure of sequence identity or homology between two nucleic acid sequences. A nucleic acid sequence encoding a miRNA or a portion thereof can be used as a "hybridization probe" according to standard hybridization techniques. Hybridization of the probe to the DNA of the test source or RN A indicates the presence of mir-199a DNA or RNA in the test source. Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 6.3.1-6.3 6 1991 found. "Medium hybridization conditions" is defined as equivalent to hybridization in 2X sodium/sodium citrate (SSC) at 3 〇t: followed by 5 (rc) 1χ SSC, 0.1% SDS wash.” Highly stringent conditions, defined as equivalent to hybridization at 6 ° C gasification sodium/sodium citrate (ssc) at 45 ° C, followed by 0.2 X SSC at 65 ° C, 0.1% SDS Wash. The term "tissue" as used herein, refers to all cells of the same source that perform a particular function consistently. Examples of tissues include, but are not limited to, bone, cartilage, connective tissue, muscle tissue, neural tissue And epithelial tissue. Organizations form "organs" together to perform specific functions. Examples of 2012 21,039,897 include (but are not limited to) joints, bones, muscles, blood, brain, heart, liver, kidneys, stomach, and skin. For example, joints It is formed by several different tissues, such as, but not limited to, bone, cartilage, synovium, muscle, ligament, and tendon. The term ''joint' as used herein means where two or more bones are in contact. The term may mean a movable joint or an immovable joint. In addition, the joint may be provided with a dense connective tissue containing collagen attached to the fibrous joint of the bone, or a cartilage joint of the cartilage connecting the bone. The joint may also be a synovial joint An articular cartilage sac with a synovial cavity and attached to an auxiliary ligament or tendon. The synovial joint includes, but is not limited to, a ball-and-wolf-joint, such as a hip or shoulder joint; an elliptical joint and Flat joints, such as wrist joints; joints of the joint, such as elbows, knees, ankles or joints of the fingers and toes; saddle joints, such as the hallux joint; and frame-axis joints, such as the first neck The joint of the vertebra and the second cervical vertebra. The terms "tissue status,", "state of the tissue,", "tissue state" "and, the state of the tissue" are used interchangeably herein. Thinking of the condition of the tissue (C0nditi0np tissue state is characterized by a specific pattern or characteristic of the tissue may be characterized by the expression of one or more specific molecules such as, but not limited to, peptides, proteins and nucleic acids, or a combination thereof. It is assumed that the tissue state is similar to when Organizational status can be considered "healthy," or "normal" when there is no disease or injury and its specific function is effectively performed. If an organization is unable to perform its function due to illness or injury, the state of the organization can be considered as "degenerate rickets' or 'abnormalities. In addition or in addition, if the tissue type or its molecular expression pattern is "changed" or changed, compared to the normal tissue, group 22 201239097, ''qi... can be regarded as "degraded"," Rickets or "abnormal". = The expression pattern of a given molecule in an organization may be tissue = 'target. Examples of tissue states include, but are not limited to, tissue degradation, such as cartilage degradation, bone degeneration, and degeneration; inflammatory, inflammatory or synovial inflammation; tissue reorganization, such as bone remodeling or cartilage weight. Amelioration, fluid accumulation; or proliferative tissue, such as a shot, a capsule, or a cancer during wound healing. ^ Disease or disease is used interchangeably herein, meaning a condition, especially an abnormal medical condition such as a disease: or ^, a tissue, an organ or an individual can no longer effectively perform its function. Typically, 'but not necessarily' the disease is associated with a particular symptom or sign indicative of the presence of the disease. Therefore, the presence of such symptoms or signs may indicate ', and the loom g or the individual's rickets. These changes in symptoms or signs may indicate Disease progression. Disease progression is usually characterized by an increase or decrease in symptoms or signs; it may indicate that the disease is "deteriorating, or,, getting better,". Disease, deterioration@ characterizes the reduced ability of a tissue, organ or organism to perform its function effectively, and the disease "gets better", usually characterized by an increased ability of the tissue, organ or individual to perform its function effectively. In disease, risk of disease , under the woven, $ official or individual is in a healthy state but shows the susceptibility of the disease. Generally speaking, at the risk of the disease is related to the early or weak signs or symptoms of the disease. In this case, still Treatment to prevent the occurrence of diseases. Examples of diseases include, but are not limited to, traumatic diseases, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, TB diseases, diseases, joint diseases, genetic diseases, autoimmune diseases 23 201239097 ο Disease and various types of cancer terminology • 'Joint disease' as used herein means an abnormal condition of the joint, especially an abnormal condition caused by injury, trauma, degeneration, inflammation, infection or autoimmune factors. Joint diseases include, but are not limited to, arthritis, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), juvenile idiopathic arthritis (JIA), gout, septic arthritis, psoriasis joints Inflammation and osteoarthritis (OA); cancer, such as chondrosarcoma, osteosarcoma, fibrosarcoma and multiple myeloma; tendonitis, bursitis, fracture and cartilage or bone damage. The ''symptoms' of the disease are indications of the disease that can be noted by tissues, organs or individuals having the disease, and include, but are not limited to, pain, weakness, weakness, strain, stiffness, and cancer of the tissues, organs, and individuals. "symptoms," or "signals," including but not limited to changes or changes in specific indicators (such as biological or molecular markers), such as presence, absence, :: or increase, decrease, or decrease 'Or the occurrence, presence, or absence of a symptom = used in the text means monitoring the biological state of the patient or the health of the individual and the presence, absence, or absence of disease in the cell, tissue, or function. , can be this molecule in the cell -:: woven in: 2: white matter with a change in the expression content or type. Refers to:: 2:: start, onset or presence, or the disease into the body disease label It may also be a sign of risk of morbidity in an individual. 24 201239097 Indicators related to arthritic diseases (such as OA) include, but are not limited to, transcription factors such as Sox-5, Sox-6, Sox-9, Nfatl, pitxl, Fox 〇, HIF2A, S AF-1, RUNX-2; cytokines, such as IL_1 β, IL-2, IL_7, IL-12, IL-18, GM-CFS, TNF_a, NF_kB, and INF-γ; phosphatase, such as alkaline phosphatase (ALP); protease, such as metalloproteinase (eg MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-13, MMP-14); aggrecanase (eg ADAM-8, ADAM-12, ADAM-TS4, ADAM-TS5) And cysteine proteases (eg, cathepsin B, cathepsin κ, cathepsin S, calpain, cysteine protein _3, thiocysteine protein -9); tissue inhibitors of metalloproteinases (eg, τΐΜΡ-2, TIMP-3, TIMP-4); extracellular matrix components such as collagen (eg collagens II, VI, IX, X, XI), proteoglycans (eg e-sulfate) Heparin, chondroitin sulfate, keratan sulfate, aggrecan, elastin, hyaluronic acid, fibronectin, laminin, CD-RAP, CDMP, chondromodulin and multi-effect protein (Plei〇tr〇Phin) Generally speaking, in tissues, organs, or wealth that suffer from arthritis (especially 0A), these sputum-inducers are compared with _, organs, or Body performance is either activated to an older or lower content. For example, t, in Luodong Qianjian

The organization under the risk, n official or a wealth, the 0A day 4 ID rainbow, the extracellular matrix group is still low. In addition, in the second: = wind: = 25 201239097 or a fortune, turn _ child a _ = inhibitor TMM and TIMP_4 containing = _ or _ under the occurrence of OA New Zealand, or from the risk of occurrence 0A = also 匕: In the cytokine IL], IL_6 * gamma, · woven, beneficial g or individual IM, green 2, MMp_3 and ΜΜρ·9α === enzyme content is less than 罹 or not in the occurrence of 〇Α and phosphorus _ ALP organs or individuals increase. The organization under the risk of 0A, the wire ^ (four) difficult indicator is mir- (10) you) trace 21, ‘22, · 14〇, such as joint $ ruins 1993. , tearing in the body is particularly good as a disease indicator of p inflammation (especially 〇A). Breastfeeding 3: ’ '""" means any of the four (4) senses or genus that can benefit from the present invention. Preferably, the individual is selected from the group consisting of a lower π object r' such as a guinea pig, a rabbit, a horse, a Yue, a cow, a sheep, a tortoise, a snake or a lizard, or a primate (such as a chimpanzee or a pheasant). Chimpanzees, gorillas and humans). More preferably, the individual is a human. The term "sample" or ', a sample of interest, as used interchangeably herein, means a part or small piece of tissue, organ or individual, Typically smaller than tissue, organ or individual, it is intended to represent the entire tissue, organ or individual. The sample provides information about the state of the tissue or the organ or disease state during analysis. Examples of samples include (but are not limited to): 26 201239097 Such as blood, serum, blood, liquid, or solid samples, saliva and lymphatic soft kidney membrane, sac and connective cartilage, bone, synovium, cells, chondrocytes trr) cartilage, bone, smooth, Cultures of bone marrow osteocytes, chondrocytes, and sputum cells. Enchanted cell stem cells and/or mesenchymal stem cells are based on visual or chemical basis - including but not limited to tissue, M 4 analysis Scan to allow imaging or radiography of the individual to speculate on specific indicators; chemical analysis includes (but is not limited to, in or absence, or its quantity or amount of modified reference samples) As used herein, it refers to the standard of the information obtained from the sample 1 which is substantially compared with the product. The loss of k is estimated by the sample of interest, sputum, or normal tissue, organ _ reference sample State body ^ = disease onset '; disease or reference sample can be derived from abnormal or rickets, thus providing the purchase of sudden - less, when ^ or a (four) ~ discuss the individual's 罹 recording standard > test sample status and interest The difference between the state of the sample:,: Luna 27 201239097 = the low-ranking Lange or the money-faced improvement of the official or ί3 self-proclaimed _ sample of the same tissue, the shape of the fish 41 earlier than the fish. The difference between the states of the sample of interest of the reference sample obtained earlier may be an indicator that the disease time is improved or worsened. The reference sample was taken earlier, after obtaining the reference sample and obtaining the sample of interest ^, , & over & time. This time can be equivalent to several years (for example, 0 8〇5; Γ15'2〇'25'3〇'35'4〇'45-°-〇^7〇, 80, 90, 100 years), months ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and December), weeks (for example, 2, 3, 4 5, 6 7, 8 weeks), several days (for example, 35 , 40, 45, 50, 60, 70, 80, 90, 100, 200, 3, 400, Tongtian), several hours (1, 2, 3, 45, 6, 789, 10, 1 b 12 hours) , a few minutes (for example, 1, 2, 3, 4, 5, 1〇, 15, 2〇, 25, 3〇, 35, 40, 45, 5〇, 60 minutes) or a few seconds (for example, Bu 2, 3, 4, 5, 1〇, 15, 2〇, 25, 3〇35, 4〇45, 50, 60 seconds). The term "decrease" or "reduced" in the term indicator (e.g., miM99a) means that the indicator is present in the sample at a lower level than the reference or reference sample. The term "elevated" or "increased" in terms of miM99a) means that the index is in the sample containing the reference nucleus reference 4 high in the sputum than the sputum sputum sputum Content. In the case of miM"a, a 1 increase in the sample (28 201239097/month liquid) indicates an increased susceptibility or increased likelihood of the onset of 〇a*〇a. If the two samples are in addition to a single factor The treatment is performed in a substantially identical manner, ie the reference sample is compared to the sample of interest via a differential treatment or a differential storm. Such single factors include, but are not limited to, exposure time, exposure concentration or exposure temperature for a particular substance. Therefore, the sample of interest may be exposed to a substance that is not related to the reference sample, or may be exposed to a different time than the reference sample or may be exposed to a different temperature than the reference sample. The differential doses exposed by the sample of interest include, but are not limited to, increasing or decreasing the doses exposed by the reference sample by 2, 5, 10, 20, 30, 40, 5, 1 and / or 1000 times the dose. The differential exposure time exposed by the sample of interest may include, but is not limited to, 2 times, 5 times, 1 time, 20 times, 30 times, 40 times, 5 times, 1 time longer than the reference exposure time. Double and, or 1 times. The differential exposure temperature to which the sample of interest is exposed may include, but is not limited to, 2 times, 5 times, 1 time, 2 times, 30 times, 40 times, 5 times, and then times the exposure temperature of the reference. And / or coffee times temperature. In a non-limiting example, the sample of interest may be exposed to a substance that is increased by one 〇, 倍 concentration than the reference sample. The sample is then analyzed in substantially the same way: the sample is analyzed to determine the effect, i.e., the concentration of the substance is added to the sample of interest or has an advantageous or adverse effect. Those skilled in the art will appreciate that the present embodiments can be applied to different ranges of concentrations, different storms = time, and/or different temperatures upon exposure, as appropriate. "Terminal" agonist" as used herein, means a substance that acts in tissues, organs, or a cell, such as receptors, signal transduction, gene expression, egg j, and protein breakdown. The agent is caused by the active site or the heterologous position of the two receptor molecules, thereby triggering the generation of the genus. Examples of the _ include: (but secret) nucleic acid fraction =, mRHmiRNA; or protein, such as hormones, a serotonin 'growth factor' neurotransmitter and a transcription factor. & used to mean a barrier to the action of an agonist. Generally, a dysplasia is bound to an activated or ectopic position of a receptor molecule, or It interacts with a specific knot that does not affect the activity of the receptor. Typically, the antagonist and the agonist compete for structural constraints. (4) The binding position or the promotion of (4) does not make it usually because of binding. The manner of changing the binding site of the agonist. The activity of the antagonist is reversible or irreversible, and the end-effect antagonist-receptor complex: the interaction time of the interaction is long. Examples of riding antagonists include, but are not limited to, nucleic acid molecules. , such as siRNA or miRNA Or a protein, such as a hormone, a cytokine, a growth factor, or a neurotransmitter; an antibody; or a transcription factor. ', term "mimic, as used herein, means imitation of a target knife (eg, such as tissue, organ) Or a substance of an agonist or an antagonist in an individual, such as receptor-signaling, gene expression, protein synthesis, and protein breakdown. Thus, a mimetic may be structurally different from the target molecule (eg, an agonist or antagonist) that mimics but still has the same specific response (eg, by binding to the activated or ectopic position of the acceptor molecule) (ie, by exhibiting at least one activity of the stimulating agent 201239097 agent or antagonist). In general, the mimetic performs the same function as the individual agonist or antagonist, i.e., functionally the same. For example, the mimetic of mir-199a is a substance that exhibits at least one activity of mir-199a, has the function of mir-199a, and/or mimics the action of mir-199a. The term "receptor" as used herein means a molecule such as a protein or polynucleotide to which one or more specific signal-conducting molecules are bound. Signaling The conducting molecule can function as an agonist or antagonist, including but not limited to nucleic acid molecules such as siRNA or miRNA; or proteins such as hormones, cytokines, growth factors or neurotransmitters; antibodies or transcription factors. The receptor may be located at the cell membrane of the cell, within the cytoplasm, and/or within the intracellular compartment. As used herein, "treat,", "treating" or "treatment" a disease or condition means achieving one or more of the following: (a) reducing the severity of the condition; (b) limiting or preventing the development of the symptomatic features of the condition to be treated; (c) inhibiting the deterioration of the symptomatic characteristics of the condition to be treated' (d) limiting or preventing the recurrence of a condition in an individual previously having the condition; and (e) limiting or Preventing the recurrence of symptoms of an individual who has previously had symptoms of the condition. As used herein, "prevent,", "prevendng",,, (prevemi〇n) or, (pr〇phyiaxis) A disease or condition means that the prevention of a disease or condition occurs in a patient. The terms "pharmaceutical", "pharmaceutical," and "drug" are used interchangeably herein to mean a combination of substances and/or substances used to identify, prevent or treat a tissue state or disease. 31 201239097 , "Pharmaceuticals " Acceptable" means that it is approved by the federal or state government or the United States Pharmacopoeia or other recognized pharmacopoeia for use in animals, and in particular humans. Terminology, active ingredient, means pharmaceutical composition or blending A substance that is medicinal, that is, a person who provides medical value. A combination of pharmaceutical products = 2 - or a plurality of active ingredients - can act in combination with each other or independently of each other. 'The tongue component can be formulated into a neutral or salt form. The pharmaceutically acceptable salt includes salts formed with free silk based® (such as from hydrochloric acid, phosphorus-methyl acetonide, oxaloquinone, ochreic acid, etc.), and free radical groups ( Salts such as, but not limited to, sodium, m-iron hydroxide, isopropylamine, diethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. The term "preparation" and "Composition" is intended to include The sealing material is used as a carrier to provide a formulation of the active compound of the capsule 'where the active group t ^ is associated with the carrier associated with the carrier in the presence or absence of other carriers, "carriers, as used herein A pharmaceutically pharmacologically inactive substance, such as, but not limited to, a diluent, excipient or vehicle, together with a therapeutically active ingredient. Such pharmaceutical carriers can be liquid or solid. Not limited to) sterile liquids such as aqueous saline solutions and oils (including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Saline aqueous solution and aqueous dextrose The glycerin solution can also be used as a liquid 32 201239097 carrier, especially for injection solutions. If the pharmaceutical composition is administered intravitably, a saline solution is a preferred carrier. The solid preparation is suitable for oral administration. And includes powders, spinners, pills, capsules, lozenges, cachets, suppositories and dispersible granules. Solid excipients may be one or more a diluent, a flavoring agent, a binder, a preservative, a tablet dispersing agent or a substance of an encapsulating material. In a powder, the excipient is preferably a finely divided solid which is in the form of a finely divided active ingredient. In the form of a mixture, the active ingredient of the present invention is mixed with a carrier having the necessary binding characteristics in an appropriate ratio and pressed to a desired shape and size. The powder and the binder preferably contain 5% to 80%. % 'more preferably 20% to 70% of active compound. Suitable pharmaceutical excipients" including house powder, glucose, lactose, Yan sugar, gelatin, pectin, dextrin, malt 'rice, flour, white peony, Silicone' magnesium carbonate, sodium stearate, magnesium stearate, glyceryl monostearate, talc, sodium chloride, gum tragacanth, methylcellulose, sodium carboxymethylcellulose, skimmed milk powder, glycerin, Propyl alcohol, water, ethanol, low-point butterfly, cocoa butter and the like. For the preparation of the test agent, the low-dise, such as a mixture of fatty acid glycerin or cocoa butter, is first melted and the active components are dispersed therein by stirring. The solubilized homogeneous mixture is then poured into a suitably sized mold, cooled and thus cured. Examples of suitable pharmaceutical carriers are also described in E. % Manin # "Remington's Pharmaceutical Sciences" 中 〇 33 201239097 The term "adjuvant" means to amplify, stimulate, activate, enable or modulate the activity of a composition at the cellular or hormonal level. The immune response of the component, such as an immunological adjuvant, stimulates the immune system's response to the actual antigen, but does not itself have an immune effect. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (eg, inorganic metal salts such as acid or oxyhydrin | g), organic adjuvants (eg, saponin or squalene), oil-based adjuvants (eg, Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (such as IL-ip, IL_2, IL-7, IL-12, IL-18, GM-CFS and INF-γ), granular adjuvant ( For example, immunostimulating complexes (ISCOMS), liposomes or biodegradable microspheres, viral particles, bacterial adjuvants (eg, monophosphoryl lipid A or cell wall peptide), synthetic adjuvants (eg, nonionic Sexually block copolymers, cell wall purine peptide analogs or synthetic lipids A) or synthetic polynucleotide adjuvants (eg, polyarginine or polylysine). '• Dosing, as used herein, includes administration in vivo and direct administration to tissues (such as vein grafts) in vitro. In the context of the present invention, it is preferably oral, topical, percutaneous, and/or intraluminal (eg, intravenous) Administration: The π therapeutically effective amount "is a number I of a therapeutic agent sufficient to achieve the desired purpose. The effective amount of a particular therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to which the therapeutic agent is to be administered, and the purpose of administration. In individual cases, the effective amount can be determined empirically by a skilled practitioner in accordance with established methods in the art. Inventive Example In a first aspect, the present invention provides mir-199a for use as an indicator of tissue status or disease. The inventors have unexpectedly discovered that the presence or absence of mir_199a 34 201239097 affects the content of known Herzos (such as transcription factors, extracellular matrix components and proteases), demonstrating the content of Qin tear and the presence of tissue or disease. , * existence or progress related. Therefore, mir-199a indicates the state of the tissue or disease. The presence, absence or quantity change of mir l99a& is a sign that the state of the tissue or disease is present, absent or progressive. In the context of the present invention, the characteristic of tissue state change is preferably that the tissue exhibits a different mir-199a content than the normal tissue or the same tissue at an earlier time point. The condition of the disease state is preferably that the g or the individual exhibits a different mir 199a content than the normal organ or individual or at the earlier time point than the same g or the individual. In the first aspect, the present invention relates to the use of mir_丨99a as an indicator of a tissue state or disease. Because of the differential expression of mir_199a during disease onset and/or progression, mir_丨99a is used as an indicator of tissue status or disease. In more specific embodiments of the first and/or second aspects of the invention, the mir-199a content is expressed in the individual (a) tissue state or disease, and/or (b) tissue state change or disease occurs. Risk, and/or (c) an individual suffering from a tissue state change or disease, and/or (d) a tissue state or progression or stage of the disease. The mir-199a content is the expression level of miM99a or the amount of mir-199a present. Thus, the amount of performance and/or the amount of mir-199a present in the individual indicates (a) tissue state or disease, and/or (b) risk of developing tissue state changes or disease, and/or (c) individual suffering A change in tissue status or disease, and/or (d) a state or progression of a tissue or disease. In a more preferred embodiment, the mir-199a content is 35 201239097 mir-199a in an individual or in a sample of an individual. In more specific examples, a change in the mir-199a content indicates a change in tissue state or disease, such as a deterioration or improvement in the state of the tissue or disease. Preferably, a decrease in the mir-199a content indicates a deterioration in tissue state or disease. An increase in the mir-199a content indicates an improvement in the state of the tissue or the disease. A change in the mir-199a content also indicates a risk of tissue state change or disease, and more preferably, a decrease in the mir-199a content indicates a risk of degenerative tissue state or disease. A change in the mir-199a content, preferably a decrease in the mir-199a content, also indicates that the individual is suffering from a tissue state or disease. In addition, a change in the mir-199a content (e.g., an increase or decrease in miM99a content) indicates the progression or stage of the tissue state or disease in the individual. Preferably, a decrease in the mir-199a content indicates a deterioration in tissue state or disease. More preferably, the mir-199a content indicates the state of the tissue, wherein the tissue is selected from the group consisting of bone, cartilage, connective tissue, meat tissue, nerve tissue, and epithelial tissue. Preferably, the tissue state is tissue degradation such as, but not limited to, cartilage degradation, bone degeneration and synovial degeneration; tissue inflammation such as cartilage inflammation or synovial inflammation; tissue reorganization, such as bone remodeling or cartilage recombination; sclerosis; Fluid accumulation; or proliferative tissue, such as hyperplasia during wound healing, cyst formation, or cancer. Particularly preferred are tissue states associated with tissue degeneration or tissue inflammation, preferably cartilage degradation, synovial deterioration, cartilage inflammation, and/or synovial inflammation. More preferably, the 'mir-199a content indicates a disease in which the disease is a joint disease such as, but not limited to, arthritis, such as rheumatoid arthritis 36 201239097 (RA), ankylosing spondylitis (AS), juvenile Arthritis (JIA), gout, septic arthritis, psoriatic arthritis and osteoarthritis (OA); cancer, such as chondrosarcoma, osteosarcoma, fibrosarcoma and multiple myeloma; tendonitis, bursitis, Fracture and / or cartilage or bone damage. In a particularly preferred embodiment, the disease is arthritis, and more preferably osteoarthritis. In a third aspect, the invention provides a method of identifying the following in an individual: (a) a change in the state of the tissue or the presence of a disease, and/or (b) a risk of developing a tissue state change or disease, and/or (c) Monitoring the progress or stage of tissue status or disease, the method comprising detecting mir-199a levels. Preferably, 'mir-199a is detected at the RNA or DNA level, preferably via known methods such as, but not limited to, antibodies, primers that are capable of specifically binding to mir-199a RNA or cDNA' and/or hybridizing to mir_i99aRNA or cDNA. Or nucleic acid probes) detect mir_199a. Preferably, the mir-199a content is the amount of mir-199a in the individual or in the sample of the individual. Preferably, the mir_i99a content is the mir-l99a expression level or the presence of mir-199a in the individual or in the individual's sample. Still more preferably, the individual is a mammal, a reptile or a bird. More preferably, the individual is selected from the group consisting of experimental animals (eg, mice, rats, or rabbits), domesticated animals (including, for example, guinea pigs, rabbits, horses, donkeys, cows, sheep, goats, pigs, baboons, ducks, ducks) , collaterals, tracing, dogs, turtles, tortoises, snakes or lizards, or groups of primates (including macaques, chimpanzees, bonobos, gorillas, and humans). Particularly preferably, the individual is a human. 37 201239097 4 body-like ασ疋- or multiple solid sample tissue extracts, cartilage, bone H is not limited to) tissue; or - or multiple liquid samples:; capsule, skin and knot::: medium = two two = = The sample is cartilage, sputum, serum and/or ride. ^ In the system, 9.2 is used to detect the known method of detecting nucleic acid by a known method, for example, by combining specific nucleic acids or polymorphisms of the miRNA to be described. Methods include, but are not limited to, polymerase chain reaction (PCR) techniques (such as reverse transcriptase pcR or quantitative real-time PCR) followed by complementary strands, hybridization techniques (such as northern blotting, in situ hybridization, or wafer hybridization). ), immunohistochemistry or immunohistochemistry or immunocytochemistry techniques (such as Western blots, immunofluorescence or ELISA), mass spectrometry (MS) (such as LC/MS), high performance liquid chromatography (HPLC, especially ion exchange HPLC) ). The manner in which the mir-199a content present in a sample of an individual or an individual is measured includes, but is not limited to, those generally used in the above methods. These include, but are not limited to, one or more primer sets that are capable of specifically detecting mir-199a RNA or cDNA (eg, for quantitative RT PCR), one or more capable of specifically interacting with mir- under standard conditions 199a RNA or cDNA hybridized nucleic acid probes (eg, for use in northern blot or wafer hybridization techniques), one or more antibodies that specifically detect mir-199a (eg, for immunohistochemistry or immunohistochemistry or immunochemistry) In the technique, Example 38 201239097, for example, mir-199a in a tissue section or mir-199a immobilized on a suitable carrier such as a membrane, a wafer ELISA plate, or the like is detected. In a more preferred embodiment of the invention, the mir_l"a content in the sample of the individual or individual is compared to the mir-content in one or more reference and/or sample samples. Preferably, the reference is selected from the group consisting of a healthy individual, a diseased individual, or the same as the test subject, but the time point is earlier or later == outside or another 'reference is representative of the _19-known content in the tissue-like L-modified or disease-free The presence or absence of a change in the state of the tissue or the presence or absence of a disease or an increase or decrease in the risk of a disease. The reference sample is preferably an individual from the health, H disease, or from the same individual as the sample of interest. While the reference sample is taken from the disk ===, the reference sample is preferably taken at a later time than the earlier of the sample of interest 4. Time lapse between the samples of interest (4) (eg 1, 2, 3, 4, 5, 10, 15, 20, 25; 30, 35, two years 45, 50, 60, 70, 80) , 9〇, 1〇〇), months (Bu 2, 34, 5, 6, 7, 8, 9, 10, 11, and December), weeks (for example, 1, 2, 3, ^, ..., Called for several days (10) such as Bu ^^ Xiao Yu... 20, 25, 30, 35, 40, 45, 5〇, 6〇, 7〇, 8〇9〇1〇〇, 200, 300, 400, 500 days), Hours (Bu 2, 3, 4, 5, 6, 7, 8, 9, 10, U, 12 hours), minutes (for example, Bu 2, 3, 4, 5 ' 10 > 15 U5 ”, 40, 45, 50, 60 minutes) or seconds (eg, 2, 3, 4, 5, 10, 15, 2, 25, 3, 35, 40, 45, 5G, 6G seconds). Additionally or additionally, Reference sample is

S 39 201239097 A reference sample having a mir-199a content representing a healthy individual or representing the presence or absence of a change in the state of the tissue or disease, or representing an increased or decreased risk of developing a tissue state change or disease. In the specific case where the reference or reference sample is derived from a healthy individual, or with a reduced risk of developing a tissue state or disease, or a mir-199a content representing an individual without a tissue state change or disease, compared to the reference A decrease in the mir-199a content in the sample of interest indicates that the subject has (a) tissue deterioration or disease, and/or (b) increased tissue risk or increased risk of disease, and/or (c) tissue deterioration or disease Progress. In the specific case where the reference or reference sample is derived from a healthy individual, or has a reduced risk of developing a tissue state or disease, or a mir-199a content represents an individual who does not have a tissue state change or disease, compared to the s A similar mir-i99a content in a sample of interest indicates (a) a change in tissue status or amelioration or absence of disease, and/or (b) a decrease in the risk of developing a tissue condition or disease, and/or (c) a deterioration in the state of the tissue or disease. The progress has declined. The reference or reference sample is derived from a healthy individual, or has a reduced risk of developing a tissue state or a disease or a mir-199a content representative, in the presence of an individual with a tissue state change or disease, compared to the reference 'interested' An increase in the mir_199a content in the sample indicates (a) a change in tissue state or amelioration or absence of disease, and/or (b) a deterioration in the state of the tissue or a decrease in the risk of the disease, and/or (c) a deterioration in the state of the tissue or progression of the disease. decline. 201239097 M ^ = test or reference sample is derived from a rickety individual, or has a specific increase in the risk of developing makeup, sputum or disease, or the value of the individual representing the presence of tissue, two changes or disease, mir-199a- a content phase is not in the body (4) there is a deterioration of the tissue state or disease, and/or (b) A birth " and the woven state deteriorates or the risk of the disease increases, and/or (c) the deterioration of the tissue state or the progression of the disease. In the specific case where the reference or reference sample is derived from a rickety individual, or has an increased risk of sputum _ change or disease or a value representing an individual with a change in tissue state or disease, the __199 & content reduction table is not an individual body) There is a deterioration in the state of the tissue or disease, and/or (b) aggravation of the tissue '4 worsening or the risk of the disease, and/or (4) deterioration of the tissue state or progression of the disease. In the specific case where the reference or reference sample is derived from an individual with a disease, or has an increased risk of developing a tissue, 4 changes or a disease, or a value representing an individual with a change in tissue state or disease, an increase in the content of 111匕199& (a) a change in the state of the tissue or amelioration or absence of the disease, and/or a decrease in the risk of developing a tissue condition or disease, and/or (c) a deterioration in the state of the tissue or a decrease in the progression of the disease. In the specific case where the reference or reference sample is derived from the same individual as the individual of interest but at an earlier time point, a decrease in the mir-199a-a content in the sample of interest indicates that the subject has a worsening of the tissue state or disease in the body. And/or (b) an increased risk of tissue deterioration or disease, and/or (c) deterioration of tissue status or progression of the disease. In the specific case where the reference or reference sample is derived from the same 201239097 individual as the individual of interest but at an earlier time point, the mir-199a-a containing in the sample of interest does not (a) change the state of the grade or The disease is improved or absent, and 7 or (b), the condition of the disease is deteriorated or the risk of the disease is reduced, and/or (c) the state of the tissue is deteriorated or the progress of the disease is decreased. In a specific case where the reference or reference sample is derived from the same individual as the individual of interest but at an earlier time point, the similar content of mir-199a-a in the sample of interest indicates the risk of tissue deterioration or disease in the individual (4) Similar, and/or (b) deterioration of tissue status or progression of disease, and/or (c) deterioration of tissue status or progression of disease. In a fourth aspect, the present invention provides a method for deciding a pharmaceutical dosage of a tissue state or preventing or treating a disease in an individual, comprising the steps of: (a) determining a mirl99a content in a sample of the individual 'and determine the mi r_丨9 9 a content in the reference or reference sample as compared to the mir-199a content of the sample of interest, and (b) determine the dose of the drug based on the mir-199a content of the sample of interest, Depending on the situation, the mir· 199a content in the sample of interest and the reference or reference sample is compared. In the context of the present invention, the mir-199a content is preferably measured at the rnA or DNA level, more preferably by known means such as, but not limited to, the ability to specifically bind the mir-199a gene, gene product and/or An antibody, primer or nucleic acid probe which is a functionally active variant thereof and/or hybridizes to a mir-199a gene, a gene product and/or a functionally active variant thereof. The sample may be a liquid or a solid, and the liquid sample is preferably a body fluid sample 42 201239097. However, it is not limited to blood, serum, plasma, synovial fluid, urine, lymph, and solid samples are tissue samples such as (but Not limited to = month θ, synovial membrane, perichondrium, sac, skin and connective tissue. It is preferably derived from a healthy individual, a rickety individual or an individual with a sensation. If the reference sample is taken from a sample of interest Late: the sample is preferably earlier or additionally or additionally than the sample of interest, the reference sample is representative of = no tissue state change or disease or miM99a ☆ amount or "risk of disease increases or decreases" In a preferred embodiment, the necessity to maintain or change (i.e., increase or decrease) the dose of the medical product is determined based on the other side of the assay sample (preferably compared to a reference or reference sample). = 'Yes' is the reference or reference sample that is derived from a healthy individual ii II: a dimorphic change or a reduced risk of disease or indicates absence, and changes in i or diseased miM99a content, miM99a content of interest sample t In comparison with the reference or:, in the specific case where the measurement is low, it is advantageous to determine the necessity of maintaining or increasing the agent for changing the tissue I or preventing or treating the disease in the individual. It is expected that the content of miM99a in the sample of interest will be compared with the reference or parametric q-body or the risk of a tissue state change or disease reduction or the absence of tissue state changes or the wall of the disease. For reference or 43 201239097, the specific order determined to be high is necessary for the prevention or treatment of diseases, the necessity of changing the state of the woven fabric in the body. It is also conceivable to maintain or reduce the dosage of the drug. In the reference or 夂: have, weave state change or disease ^ in the sample of interest "two amount: = in the specific case determined to be similar, threat / f in the reference or reference sample ^ ^ ^ ^ i? ά & •: Altering tissue in an individual. 2: In the case of a disease, 'Deciding to maintain or reduce the dose of the drug. The tissue or reference sample is derived from a diseased individual or has an increased risk of:: widespread or disease or Numerical value indicates the presence of woven A The corpus callosum, and the 样品mir-199a tb, #4· in the sample of interest is also determined to be a specific example in the reference or reference sample. The individual changes the tissue state or prevents or treats the disease in the body and/or maintains or It is advantageous to increase the dose of the pharmaceutical. The tissue test sample is derived from a rickety individual or has an increased risk of developing gamma-crossing or disease or a numerical value indicating the presence of a tissue, change or disease, and the mir_199a in the sample of interest. The amount of sputum is advantageous in the specific case where the reference or reference sample is determined to be higher in order to change the tissue state or prevent or treat the disease in the individual, and to maintain or lower the dose of the drug. Or the reference sample is derived from a rickety individual or has an increased risk of developing ancestor, my state change or disease, or a numerical value indicating the presence of tissue 44 201239097 compared to the body's and the miM99a in the sample of interest, as determined for the individual sample Lower specific examples are necessary to hold or increase the dose of the pharmaceutical. In 鲂 = or the reference sample is derived from the same individual but the point of the individual of interest, and in the sample of interest - the specific enthalpy in the reference or reference sample is determined to be lower, for = 2, variable tissue In terms of the state or prevention or treatment of the disease, it is advantageous to determine the ',, 2 k plus the need for a drug dose. i in the > test or reference sample is derived from the same as the individual of interest but at an earlier time point, and the mi r-19 9 a content in the sample of interest is entangled in the reference or reference sample is determined to be elevated In a specific example, it is advantageous to determine the necessity of maintaining or lowering the dose of the drug in the case where the individual body β changes the tissue state or prevents the m disease. Where the reference or reference sample is derived from the same individual as the individual of interest but at an earlier time point, and the mir-199a content in the sample of interest is determined to be similar to the reference or reference sample, for It is advantageous to determine the need to maintain or increase or decrease the dosage of the drug in order to alter the state of the tissue or prevent or treat the disease in the individual. In a fifth aspect, the present invention provides a method of adjusting a dosage of a pharmaceutical for modifying a tissue state or preventing or treating a disease, comprising the steps of: (a) determining a mir-199a content in a sample, and (b) determining The mir-199a content in one or more reference or reference samples, (c) whether the mir-199a content of the test sample is different in the sample of interest, 45 201239097 in one or more reference or reference samples, And (d) adjusting the pharmaceutical dosage based on whether the rnir-199a content in the sample of interest is different from the content in one or more reference or reference samples. The mir-199a content is preferably detected at the RNA or DNA level, and more preferably by known means such as, but not limited to, the ability to specifically bind the mir-199a gene, the gene product and/or its functional activity. A variant, and/or an antibody, primer or nucleic acid probe that hybridizes to a mir-199a gene, a gene product, and/or a functionally active variant thereof. The sample may be a liquid or solid. The liquid sample is preferably a body fluid sample such as, but not limited to, blood, moon, plasma, synovial fluid, urine, saliva and lymph, and a solid sample. . For tissue samples such as, but not limited to, cartilage, bone, synovial membrane, perichondrium, sac, skin and connective tissue. In a preferred embodiment, maintaining or changing

Maintain or alter (ie increase or decrease) the mir-199a content in the medical product (with one or more 46 201239097 individuals without tissue state changes or disease, and the mir-199a content in the sample of interest is compared to the reference or In the specific case where the reference sample is determined to be high, it is advantageous to maintain or reduce the dosage of the drug in the case of a tissue-deformed tissue or to treat the disease. The reference or reference sample is derived from a healthy individual or has occurred. A tissue state change or a reduced risk of disease or content indicates an individual in which no tissue state change or disease exists, and the m 1 r-19 9 a content in the sample of interest is determined to be similar to the reference or reference sample. It is advantageous to maintain, reduce or minimize the dosage of the drug in the context of changing the state of the tissue or preventing or treating the disease in the individual. The reference or reference sample is derived from a rickets or has an increased risk of developing a tissue state change or disease. Or numerical value indicates the presence of a tissue, change, or disease in an individual, and the amount of ❺mir-199a 3 in the sample of interest is comparable to the reference or reference sample. Specific examples that are similar: 'It is advantageous to maintain or increase the dosage of the drug for changing the state of the tissue or preventing or treating the disease in the individual. The reference or reference sample is derived from a rickety individual or has a occurrence & woven The status _ or the increased risk of disease indicates that there is an individual with a tissue change or disease, and the mir-199a sputum in the sample of interest is determined to be higher than the reference or reference sample. It is advantageous to maintain or reduce the dosage of the drug in terms of changing the state of the tissue or preventing or treating the disease. The reference or reference sample is derived from a rickety individual or has an increased risk of developing a tissue@change or disease or a numerical representation of the presence of tissue 47 201239097 „A person who is a disease or disease, and in the sample of interest ##__99&3 is compared to the reference or reference sample which is determined to be lower than the specific example 2, for changing the tissue state or preventing or treating the disease in the individual body It is advantageous to maintain or increase the dose of the drug. The reference or reference sample is derived from the same individual as the individual of interest but earlier The individual in the intervening point, the mir-199a content in the sample of interest is compared to the reference or reference sample which is determined to be lower, in the specific case of changing the tissue state or preventing the silk disease in the individual, maintaining or increasing The pharmaceutical dosage is advantageous. Where the reference or reference sample is derived from the same individual as the individual of interest but at an earlier time point, the mir-199a content in the sample of interest is determined to be elevated compared to the reference or reference sample. In a specific example, it is advantageous to maintain or reduce the dosage of the medicament for changing the state of the tissue or preventing or treating the disease in the individual. The reference or reference sample is derived from the same individual as the individual of interest but at an earlier time point. The individual, the mir_199a content in the sample of interest is compared to the reference or reference sample, which is determined to be similar, for maintaining, increasing or decreasing the dose of the drug for changing the tissue state or preventing or treating the disease in the individual. It is beneficial. In a sixth aspect, the invention provides a method of determining whether a substance is beneficial and/or not affecting the tissue state or pathogenesis of a disease, which comprises the steps of: (a) determining the mir-199a content of the sample of interest. (b) determining - or ref_199a content in a plurality of reference or reference samples, and (c) testing whether the sample of interest is present in the sample of interest in the sample of 2012 201297, whether the content of the substance mir_199a is different from - or more Reference or reference to the content of the sample wherein the sample of interest is exposed to - or a plurality of reference or reference traces (four) is preferably in the active or DNA layer: illusory, more preferably via known means, such as (but not limited to An antibody that is capable of being specifically affixed to a human mir-gene gene, a gene product, and/or its functionally active and/or hybrid with a miM99a gene, a gene product, and/or a functional and lingual variant thereof, Primer or nucleic acid probe. The sample may be a liquid or solid 'liquid sample, preferably a body fluid sample such as, but not limited to, blood, synovial fluid, urine, read and lymph, and the solid sample is a tissue sample such as, but not limited to, cartilage. , bone, strict membrane, perichondrium, sac, skin and connective tissue. In the embodiment, the substance is advantageous and/or adversely affected by the state of the tissue or the onset of the disease, based on the mosquito in the assay sample (preferably compared to a reference or reference sample). It is envisioned that the reference or reference sample is a self-healthy individual or/or has a reduced risk of developing a tissue state or disease or a mir-199a content indicating that there is no tissue state change or disease in the individual, and the sample of interest In the specific example in which the content of Qin tearing is lower than the reference or reference H, it is determined that the substance has an adverse effect on the state of the tissue or the onset of the disease. ~ The reference or reference sample is derived from a healthy individual or has a reduced tissue state or a reduction in disease or a miM99a content indicating no tissue state changes or disease in the individual, and the 49 201239097 mir-199a content in the sample of interest is compared In the specific case where the reference or reference sample is determined to be high, it is determined that the lysate has a favorable effect on the state of weave or the onset of the disease. The reference or reference sample is derived from a healthy individual or has a reduced risk of developing a tissue state or disease or the mir l99a content indicates the absence of a tissue state change or disease, and the mir-199a content in the sample of interest is compared to the reference. Or in a specific example in which the reference sample is determined to be similar, it is determined that the substance has no or only a very limited effect on the state of the tissue or the onset of the disease. The reference or reference sample is an individual derived from a rickets or has an increased risk of developing a tissue state change or disease or a numerical value indicating the presence of a tissue state change or disease, and the mir-i99a content in the sample of interest is compared to a reference or reference sample In a specific example determined to be similar, it is determined that the substance has no or only a very limited effect on the state of the tissue or the onset of the disease. The reference or reference sample is an individual derived from a rickety individual or having an increased risk of developing a tissue state change or disease or a numerical value indicating a change in tissue state or disease, and the mir-BQa content in the sample of interest is compared to a reference or reference sample In a specific example determined to be higher, 'determination of the substance has a favorable effect on the state of the tissue or the onset of the disease. The reference or reference sample is an individual derived from a rickety individual or having an increased risk of developing a tissue state change or disease or a numerical value indicating a change in tissue state or disease, and the mir-199a content in the sample of interest is compared to a reference or reference sample In the specific case 50, 201239097, which was determined to be lower, it was adversely affected by the state of the tissue or the onset of the disease. Time % ί / test sample listens to the same as the individual of interest but the quality is determined to be lower than the butterfly ^, ,,, A-like L, or the disease is adversely affected. In the reference or reference sample is an individual derived from the fish time point, and the Wei sister is a bit timid but more interesting than the clip. The mir-199a content phase substance in the mouth is determined in the case of the case where the reference sample is derived from the same individual as the individual of interest but is the owner of the car, and the miM99a content in the sample of interest is compared to the reference or reference sample. In a specific example determined to be similar, it is determined that the object has no or no effect on the state of the tissue or the onset of the disease. β In a seventh aspect, the invention provides the use of mir-199a in any of the methods as hereinbefore described. Therefore, 'the use of mir-199a in the identification of the following methods in the individual's method, the state of the tissue changes or the presence of disease ~ and / or the risk of tissue state changes or disease, and / or (c) monitoring the state of the tissue or The progression or stage of the disease, which comprises detecting the mir-199a content as detailed above. The use of mir-199a is also provided in a method for determining the dosage of a pharmaceutical for modifying a tissue state or preventing or treating a disease in an individual, the method comprising the following steps as detailed above: (measuring mir_199a in a sample of an individual) Content, and depending on the situation, refer to the mir_199a content in reference sample 51 201239097 or the reference sample to compare with the mir-199a content of the sample of interest, and (b) determine the pharmaceutical dose based on the mir-199a content in the sample of interest, as appropriate According to the comparison of interest, σ and the content of cucurbit 199a in the reference or reference sample. In addition, the use of mir-199a in the method of adjusting the dosage of the medicament for changing the state of the tissue or preventing or treating the disease is expected. The above steps include the following steps: (a) determining the mir-199a content in the sample, (b) determining the mir-199a content in one or more reference or reference samples, and (c) verifying that the test sample is present in the sample of interest. Whether the mir l99a content in the sample is different from the content in one or more reference or reference samples, and (d) whether the mir-199a content in the sample of interest is different from one or The amount of the drug is adjusted in a plurality of reference or reference samples. Also provided is the use of mir-199a in a method of determining the beneficial and/or adverse effects of a substance on the tissue state or pathogenesis of the disease, as detailed above. The following steps are included: (a) determining the mir l99a content in the sample of interest, (b) determining the amount of one or more reference or reference samples, and (c) examining the mir of the sample of interest present in the sample of interest The -199a content is different from the yttrium in one or more reference or reference samples, wherein the sample of interest is exposed to a different material than one or more reference or reference samples. mir-199a is in any of the above methods. Uses include the use of the mir-199a gene, the mir_199a gene product, and/or its functionally active variants. ^ In a preferred embodiment of any aspect of the invention, the sample of interest is a solid and/or a liquid. The sample is preferably a tissue sample, and 52 201239097 liquid sample is preferably a body fluid sample. More preferably, cartilage, sputum*, synovial membrane, perichondrium, sac, and 'tissue sample are selected from The liquid sample is selected from the group consisting of the synovial fluid of the skin and connective tissue, the urine, the blood of the saliva and lymph, the serum, the plasma, and the body. The sample is selected from the group consisting of cartilage and the group. It is composed of serum, urine and synovial fluid. The capsule, blood, plasma, and medium are cartilage, synovial membrane, and blood. The sample is better. The sample is cell culture or tissue culture. Or in addition, bone, bone, periosteum, bone marrow cells, soft d, from the soft chondrocytes, synovial cells, bone cells, face n-month mother cells, broken stem cells and / or stem cells composed of Bone cells, species t=r The present invention provides a kit for use in any of the two inventions, which comprises - or a plurality of components that are miM99a. In the preferred (four) example of green, one or more members for turning miM99a are members for determining the expression level of miM99a, and more preferably at the base S and/or RNA level. More preferably, one or more members of the secret are selected from the group consisting of nucleic acids (preferably DNA or RNA), peptides and proteins (preferably monoclonal antibodies or polyclonal antibodies). . Preferably, the means for detecting mir_199a is a naturally occurring nucleic acid (e.g., DNA or RNA) or a chemically modified nucleic acid complementary to mir-199a and/or its precursor. In still further preferred embodiments, the kit further comprises (a) a container, and/or (b) a data carrier. More preferably, the data carrier contains information such as, but not limited to, the following: (1) the risk of identifying a joint disease and / 53 201239097 or an operating guide for identifying the presence and/or monitoring of joints, and (1) use Detection of components and/or kits of mir-199a (preferably in samples, preferably in individual samples), quality information, such as components and/or sets for detecting mir-199a Group number/batch 5, manufacturing or assembly or use period or expiration date, information on proper storage or handling of the kit, (iv) buffer, diluent, reagent composition and/or reagents for detecting miM99a Information on the composition of the components of mir_199a, (v) information explaining the information obtained when the above methods are used to identify and/or monitor the progression of joint disease, (vi) when improper methods and/or improper components are implemented, Possible misinterpretation or warning of erroneous results, and/or (vii) warning of possible misinterpretation or erroneous results when improper reagents and/or buffers are used. In a ninth aspect, the invention provides the use of the kit of the eighth aspect in the method provided by any of the methods of the invention. Thus, the kit is used in a method for identifying the following in an individual: (a) a change in the state of the tissue or the presence of a disease, and/or (b) a risk of a change in tissue state or disease, and/or ( c) Monitoring the progress or stage of the tissue state or disease, the method comprising detecting the mir-199a content as detailed above. Also provided is the use of the kit in a method of determining a dosage of a pharmaceutical agent for modifying a tissue state or preventing or treating a disease in an individual, the method comprising the following steps as detailed above: (a) determining a mi Γ in a sample of the individual -19 9 a content, and depending on the situation, determine the mir-199a content in the reference or reference sample to compare with the mir-199a content of the sample of interest, and (b) determine the drug dose according to the mir_i99a content in the sample of interest 54 201239097 Depending on the situation, the mir-199a content in the sample of interest and the reference or reference sample is compared. Furthermore, the use of the kit in a method of adjusting the dosage of a pharmaceutical for modifying a tissue state or preventing or treating a disease is contemplated, the method comprising the following steps as detailed above: (a) determining the mir-199a content of the sample, (b) determining the mir-199a content in one or more reference or reference samples, (c) verifying that the mir-199a content present in the sample of interest is different from that in one or more reference or reference samples The content 'and (d) adjust the pharmaceutical dosage based on whether the mir-199a content in the sample of interest differs from the content in one or more reference or reference samples. The use of the kit in a method of determining the beneficial and/or adverse effects of a substance on the tissue state or pathogenesis of a disease is also provided, the method comprising the following steps as detailed above: determining the mir_199a content in the sample of interest, b) determining the mir_199a content in one or more reference or reference samples, and (4) testing whether the mir_i99a quinone present in the sample of interest is different from the content in one or more reference or reference samples, The sample of interest is exposed to a different substance than one or more reference or reference samples. In a tenth aspect, the invention provides one or more nucleic acids as detailed above for use in detecting a mir l99a gene, a gene product or a functionally active variant thereof, in any of the methods of the invention. In a preferred embodiment, the nucleic acid is a naturally occurring nucleic acid (such as DNA or RNA) or a nucleic acid modified by a thousand. Preferably, the nucleic acid is selected from the group consisting of a nucleic acid probe, a polyamine or peptide nucleic acid (PNA), a microRNA (miRNA),

Small interfering RNA (siRNA), locked nucleic acid (LNA), primers for polymerase chain reaction (pCR) 55 201239097, primers for reverse transcription (RT) reactions, and primers for DNA sequencing group. More preferably, the nucleic acid exhibits strong binding to mir-199a. Thus, an exemplary LNA having about 8 nucleotides is suitable for detecting mir-199a, and only the sequence is unique in the entire microRNA sequence. In an eleventh aspect, the invention provides a variant as described above for detecting a mir-199a gene, a gene product, a functionally active variant thereof and/or a microRNA in any of the methods of the invention. a labeled peptide, polypeptide or protein. In a preferred embodiment, the peptide, polypeptide or protein is a protein ligand 'preferably an antibody, or a fragment or derivative thereof, darpin or an anticarinin, or a polypeptide or peptide as a probe, preferably mass spectrometry Probe. In a preferred embodiment, the protein ligand is a recombinantly produced antibody 'and, if appropriate, a modified antibody, such as a chimeric antibody, a humanized antibody, a multifunctional antibody, a bispecific antibody or an oligo specific antibody, Single-stranded antibodies and F(ab) or F(ab)2 fragments (see, for example

EP-B1-0368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213 or WO 98/24884). In addition to or in addition to a typical antibody, a protein backbone against mir-199a is used as a protein ligand, such as a lipocalin-based anti-carrier protein (Beste et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 1898-1903). The natural ligand binding site of the lipocalin (eg, retinol-binding protein or bile-triad binding protein) can be altered, for example, by way of a "combined protein design" method, such that it binds to the selected half 56 201239097 antigen (at This is the way of mir-199a) (Skerra, 2000, Biochim. Biophys. Acta, 1982, 337-50). Other known protein backbones are known as antibody replacements for molecular recognition (Skerra (2000) J. Mol. Recognit., 13, 167-187). In a twelfth aspect, the invention provides the use of a nucleic acid according to the tenth aspect of the invention and/or a peptide, polypeptide or protein according to the eleventh aspect of the invention in any of the methods of the invention . In a preferred embodiment, the nucleic acid is used in its original form, in a gene transfer vector or in a complex with liposomes or gold particles. Examples of gene transfer vectors are viral vectors, such as adenoviral vectors or retroviral vectors (Lindemann et al. (1997), Mol. Med., 3, 466-76; Springer et al. (1988) Mol. Cell. , 2, 549-58). Complexation with liposomes typically achieves very high transfection efficiencies, particularly skin cells (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 146-a79-85). At the time of lipofection, a small unilamellar vesicle composed of cationic lipids was prepared by ultrasonically treating a lipid raft suspension. The DNA is ionically bound to the surface of the liposome in a ratio that maintains a net positive charge and all of the plastid DNA is complexed by the liposome. In addition to Feigner, PL et al. (1987), Proc. Natl. Acad. Sci USA, 84, 7413 - 7414 DOTMA (1,2-bisoctadecyloxypropyl-3-trimethyl bromide) In addition to the ammonium and DOPE lipid mixtures, a large number of lipid formulations have been synthesized to date and tested for their efficiency in transfecting various cell lines (Behr et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 6982-6986; Zhao and Huang (1991), Biochim. Biophys. Acta, 1189, 57 201239097 195-203; Feigner et al. (1994) J. Biol. Chem., 269, 2550-2561 ). An example of a lipid formulation is DOTAP N-[l-(2,3-bisoctadecyloxy)propyl]-N,N,N-trimethylammonium sulphate or DOGS (dioctadecyl decylamine) Glysamine guanyl spermine). For example, a helper substance that increases the transcription of a nucleic acid into a cell is a protein or peptide that binds to DNA or is a synthetic peptide-DNA molecule that causes the nucleic acid to be transported into the nucleus (Schwartz et al. (1999) Gene Therapy 6:282; Brandon et al. (1999) Nature Biotech., 17, 784). Auxiliary substances also include molecules that allow nucleic acids to be released into the cytoplasm (Planck et al. (1994) J. Biol. Chem., 269, 12918; Kichler et al. (1997) Bioconj. Chem, 8, 213) or, for example, Liposomes (Uhlmann and Peyman (1990), supra). In addition, a particularly suitable form can be obtained by applying the above nucleic acid to gold particles and directing the particles to the tissue or cells using a so-called "gene grab; (Wang et al. (1999) J. Invest. Dermatol. 112: 775-81, Tuting et al. (1998) J. Invest. Dermatol. 111:183-8). The mir-199a or mir-199a gene or its functionally active variant can be used as a miRNA-199a agonist. a molecule which is the subject of its analogy. In a preferred embodiment, the mir-199a or mir-199a gene or a functionally active variant thereof is contacted directly or indirectly with a test compound, and the test compound is measured or cross-tested for mir- Preferably, the test compound is a chemical molecule such as, but not limited to, a non-polymeric organic compound, a lipid, a saccharide, a peptide (preferably from about 10 to about 80 amino acids) a peptide, especially 10 to 25 58 201239097 amino acids) and an oligonucleotide (preferably about 10 to about 9 nucleotides, especially 15 to 25 nucleotides). Chemicals, especially non-polymeric organic compounds (in laboratory synthesis or in nature) It has been found to have a molecular weight of from about 200 g/mol to about 15 g/mole, especially from 400 g/mol to 1000 g/mol. Preferably, the test compound changes the mir_199a content, For example, increasing or decreasing the expression content or the amount of mir_199a. In the present invention, a test compound which increases the miM99a content is particularly preferred. A method for screening a mir-199a agonist or a mimetic thereof comprises the following steps: (a) Providing the mir-199a or mir-i99a gene, (b) providing the test compound', and (c) measuring or detecting the effect of the test compound on the mir-199a or mir-199a gene. The mir-199a agonist or its mimetic is available To alter the state of the tissue or to prevent or treat joint disease. The agonist or mimetic of mir-199a may be any molecule or composition that increases or increases the mir-199a function or the expression level or amount of mir_i99a. Examples include, but are not limited to, 1. Nucleic acids, such as oligonucleotides, oligoribonucleotides, particularly dsRNA, ssRNA, siRNA, miRNA, shRNA, etc., known in the art, 2. agonist peptides or proteins 'such as stability or promotion a protein of the function of a miRNA, a agonist antibody or a fragment thereof or a transcriptional agonist of the miRNA of interest, and/or 3. a small molecule compound having an agonistic effect. Additionally or alternatively, an agonist of the invention or a mimetic thereof It may be in the form of a natural product extract (whether in crude or purified form). The extract may be manufactured according to the procedure of 59 201239097, such as water and / or alcohol and / or organic - extraction and / or & column chromatography and / or animal, plant or microbial sources (such as snake venom, leaves or microorganisms) Fermentation medium) precipitated. An agonist or a mimetic thereof can be used to treat joint diseases (especially degenerative joint diseases such as osteoarthritis). The mir-199a agonist or its mimetic can be used to treat joint pain, particularly by reducing joint pain in degenerative joint diseases. Further, mir_199aK agent or a mimetic thereof is used for the manufacture of a medicament for treating joint diseases and/or treating joint pain as described above. A pharmaceutical product may contain a mir-199a agonist or a mimetic thereof for preventing or treating a joint disease. The pharmaceutical product further contains a pharmaceutically acceptable carrier and/or excipient and, if desired, one or more additional active substances. The additional active substance is effective against the same or surprisingly different diseases or conditions or conditions. Thus, the additional active substance comprises an active substance effective to treat joint disease and/or treat joint pain as described above. In addition, additional active substances suitable for administration in combination with the active ingredients of the present invention to treat different diseases or conditions or conditions are also included. Exemplary additional active ingredients that further affect the miRNA and/or its gene product, such as agonists or antagonists of individual miRNAs, are contemplated. Additional active substances also include food supplements such as, but not limited to, vitamins, minerals, which are considered to be beneficial personal diets and/or to treat joint disorders and/or treat joint pain and/or any other disease, condition, as described above. Substance, vitamins, fatty acids or amino acids. For the manufacture of a pharmaceutical 'mir-199a agonist or its analogy, 201239097 with one or more pharmaceutically acceptable additives or auxiliary substances (eg physiological buffer solution (eg sodium vaporized solution), demineralized water, Stabilizer (such as a protein stalk or nuclease antagonist, preferably aprotinin, & acaproic acid or aprotinin) or a chelator (such as ED TA), a gel formulation (such as white petrolatum, low viscosity) Sexual paraffin and/or yellow wax, etc.) are formulated together, depending on the type of medication. More suitable additives are, for example, detergents (such as, for example, Triton X-l or sodium deoxycholate) and polyols (such as, for example, polyethylene glycol or glycerol), sugars (such as, for example, sucrose or glucose), Zwitterionic compounds (such as, for example, amino acids such as glycine or especially bovine or beet) and/or proteins such as, for example, bovine or human serum albumin. Preferred are detergents, polyols and/or zwitterionic compounds. The physiological buffer solution preferably has a pH of from about 6.0 to 8.0, especially a pH of from about 6.8 to 7.8, especially a pH of about 7.4 and/or an osmotic pressure of from about 200 to 400 osmol/ml, preferably about 290. To 310 osmolality in milligrams per liter. The pH of the drug is usually adjusted using a suitable organic or inorganic buffer, such as, for example, phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4 -(2. hydroxyethyl) piperazinyl]ethanesulfonic acid) or MOPS, buffer (3-morpholinyl-indole-propanesulfonic acid). The choice of individual buffers is usually based on the desired buffer molar concentration. Phosphate buffers are suitable, for example, for injection or infusion solutions. Regarding the manufacture of pharmaceuticals, mir-199a agonists or mimics thereof may be formulated with non-particles, liposomes or other lipid complexes, or with proteins, forms, nucleic acids (eg aptamers) or gels (eg with hyaluronic acid) Based on a combination of the following or a similar sustained release formulation. ...the pharmaceutical product is preferably in unit dosage form. - The substance can be distinguished in such a form as a unit dose containing an appropriate amount of the active ingredient. The unit dosage form can be a packaged preparation containing a discrete amount of preparation such as a packaged tablet, a capsule, and a powder in a vial or bottle. The unit dosage form can also be a capsule, an injection bottle, a lozenge, a capsule or a tablet itself, or any of these may be in the form of a package. 3 A method for altering the state of the tissue or preventing or treating joint diseases An effective amount of mir containing mir_199a agonist or its analogy is given to an individual at risk of developing joint disease or suffering from a joint disease. In a preferred embodiment, the pharmaceutical product is administered orally, administered or administered. Intracavitary administration includes, but is not limited to, intravenous and gamut. The prescription of the sputum: in a known manner, for example, by means of an oral dosage form (S# such as, for example, a lozenge) An infusion or a 4 gel (which contains a pharmaceutical product according to the invention) is administered orally or partially administered to a medicament as described above, and may be in the form of a liposome complex, if appropriate. The disease 'if (10)) is carried out in a way that it can release the mouth and mouth over time. TTS is known, for example from ΕΡ0 944398Αι ερ 0 916 336 Α1, ΕΡ 889 889 723 Α1 or Ep 852 852 493 A1. If only a relatively small amount of solution or suspension (e.g., about i to about 20 ml) is to be administered to the body, an injection solution is usually used. Intrabody administration includes 62 201239097 (but not limited to) infusion and injection. If there is a large amount (for example, liters or more than liters) of the solution or (4), the solution is infused. Therefore, relative to the infusion solution, when the injection solution is administered only a few milliliters, the small difference in the MpH and osmotic pressure of the blood or tissue fluid at the time of injection is not obvious per se or only detectable to the pain. Degree. Thus, it is generally not necessary to dilute the formulation according to the invention prior to use. However, in the case of administration of a comparable amount, the HA according to the present invention should be simply diluted before administration to obtain an example of a vaporized sodium solution having a concentration of at least about isotonic solution of 0.9%. In a preferred embodiment in which dilution is performed, for example, by using a so-called bypass tube, the tissue state is group 嚢 and (4) Μ疋 is selected from bone, cartilage, synovium, perichondrium, and 22. a group of lesions, and/or characterized by a concentration of regulatory molecules, signaling molecules, and/or degenerative molecules in the presence of =, ^ ^ ^ Ϊ from; ^ f : more? In the specific case, 'the disease is Joint disease, skin and phlegm arthritis" t bloody stagnation, rheumatoid arthritis, porcine dermatomyositis, muscle spasm; ankylosing vertebral inflammation, bursitis (arthritis (Steel's disease), 9 Into the rheumatoid arthritis, juvenile chronic pain, multiple, rheumatic scapulohumeritis, Sjogren's syndrome, systemic ===), scleroderma, group and / or characterized by physical or metabolic damage of the joint 63 201239097 k or joint degeneration), wherein the disease is preferably osteoarthritis. Further, non-limiting aspects and specific examples of the invention are described below.

Diagnostic methods involve measuring the amount of one or more miRNAs in a patient sample and using the test results to diagnose and/or predict the best treatment for the patient. The compositions described in the present invention include mir-i99a antagonists which act as miRNA antagonists, can be introduced into a patient for treatment and reduce one or more conditions associated with joint diseases, particularly osteoarthritis. Diagnostic methods involve measuring the amount of one or more miRNAs in a patient sample and using the test results to diagnose and/or predict the best, on-the-spot treatment regimen for the patient. The composition described in the present invention comprises an antagonist of mir_199a/mir-199a, which acts as a miRNA/miRNA, and the agent/inhibitor' can be treated in vivo and reduced by one or the other. A condition associated with an inflammatory disease (which is osteoarthritis). In one or more aspects of the invention (eg, such as the use of miRNAs as biomarkers), a tool for knowing nucleic acids can be known (such as being able to bind to the miRNA of the miRNA in question) by any known method = line m_A The present invention relates to the use of a component for the production of a diagnostic enzyme or a lipid metabolism disorder using a quantity of components for the primary im RNA in a biological sample. The amount of the miRNA present in the biological sample is measured (ie, stable) A further component can be any component of the miRNA in question. This: for example, can be used exclusively for the sub-group of miRNs, for example for quantitative RTPCR. Another tool can = 64 201239097, for example, can specifically target the miRNA a nucleic acid probe that hybridizes under standard conditions, such as for Northern blot or wafer hybridization techniques. Another component can be an antibody that specifically detects the miRNA in question, such as for immunohistochemistry or immunohistochemistry or For immunochemical techniques (eg, detecting miRNAs in a tissue section or miRNAs immobilized on appropriate vectors such as membranes, wafers, ELISA plates, etc.). The present invention relates to a method for identifying an increased risk of a joint disease (such as OA) or a joint disease (such as 〇A) in an individual, which comprises testing a sample taken from an individual regarding mir- present in the sample. Whether the 199a content and/or mir-199a activity is different from one or more reference samples. An increase in the level indicates an increased risk of suffering or developing a joint disease (such as OA). The term "reduced content" or "increased" "meaning that the amount of miRNA in a sample of tissue or tissue fluid is known to have a detectable concentration in some cases. If the miRNA is being studied (such as cell or tissue extract, /month, serum or plasma) (especially The individual from the test to be tested is comparable to the generational sample in terms of the number of miRNAs, and therefore has no joint disease (especially OA). For example, in the synovial fluid of an individual suffering from OA

An increase in the amount of miRNA $ was detected in the synovial fluid of individuals who did not have OA. Regarding mir_199a, a 3 summer increase in a patient sample (such as synovial fluid) indicates an increase in OA or an increase in susceptibility or an increase in the occurrence of 〇A. In some cases, the amount of miRNA labeling is compared to the control 65 201239097 to determine a decrease or increase in content. The control can be an external control, such as a miRNA from a sample of a patient known to have no joint disease, particularly OA. In other cases, the external control can be a miRNA or a known amount of synthetic RNA obtained from a sample of tissue/tissue fluid known to have no detectable amount of the miRNA (or regardless of the disease state of the individual). The internal control can be the miRNA of the tissue/tissue fluid sample to be tested. The body of the miRNA control can be the same or different from the serum or plasma miRNA of the patient to be measured. In the context of the present invention, a miRNA antagonist can be any molecule or composition that reduces or inhibits the function or amount of the miRNA being discussed. Examples include, but are not limited to: 1. Nucleic acids, such as oligonucleotides, oligoribonucleotides, particularly dsRNA, ssRNA, siRNA, miRNA, shRNA, ssDNA, ssDNA/RNA mixtures or chemically known in the art. Modified nucleic acid, etc. 2. Antagonistic or protein, such as a protein that destabilizes or inhibits the function of a, an antagonistic antibody against a miRNA of interest or a fragment biting transcriptional antagonist thereof, 3. a small molecule compound having an antagonistic effect, 4· containing 1-4 One or more people and groups of people who may have a job agent. Thus, one of the present invention is directed to an antagonistic agent for the treatment of joint diseases, particularly degenerative joint diseases such as osteoarthritis. miRNA antagonists can also be used to treat joint pain, in particular by aug. 66 201239097 by reducing joint pain in degenerative joint diseases. In addition, miRNA antagonists can be used in the manufacture of a medicament for treating joint disorders and/or treating joint pain as described above. According to the invention, the term ''chemical molecule', encompasses non-polymeric organic compounds, fats, domains, peptides (preferably from about 1 〇 to about 8 胺 amino acids, especially from 10 to 25 amines) And an oligonucleotide (preferably from about 10 to about 90 nucleotides, especially from 15 to 25 nucleotides). Particularly preferred are small chemical molecules, especially non-polymeric organic compounds (synthesized in the laboratory or found in nature), preferably having a molecular weight of from about 2 gramg/mole to about 1500 years old, especially 400 g/m to 1000 g/mole. Additionally, the antagonists of the present invention may be in the form of a natural product extract (whether in crude or purified form). The extract may be prepared according to standard procedures, such as water and/or alcohol and/or organic solvent extraction and/or Or column chromatography and/or precipitation of animal, plant or microbial sources (eg snake venom, leaf or microbial fermentation media). The term "binding protein, or "binding peptide" means a type of protein or peptide that binds to and inhibits mir-199a' including but not limited to a plurality of antibodies or monoclonal antibodies, directed to antibody fragments against mir-199a or a protein backbone (eg, an anti-carrier protein directed against mir_199a). The step of preparing an antibody or antibody fragment according to methods well known to those skilled in the art is, for example, by immunizing a mammal (eg, a rabbit) with mir-199a (if appropriate) In the presence of, for example, Freund's adjuvant and/or aluminum hydroxide gel (see, for example, Diamond, BA et al. (1981) The 67 201239097

New England Journal of Medicine: 1344-1349) Therefore, a plurality of antibodies formed in an animal due to an immune reaction can be isolated from blood using known methods and purified, for example, by column chromatography. . For example, 'monoclonal antibodies can be prepared according to known methods of Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299). According to the invention, the term antibody or antibody fragment is also understood to mean an antibody or antigen-binding portion thereof which is recombinantly produced and, if appropriate, modified, such as a chimeric antibody, a humanized antibody, a multifunctional antibody, a bispecific antibody or an oligo Specific antibodies, single-stranded antibodies and F(ab) or F(ab)2 fragments (see, for example, EP-B1-0 368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213 or WO 98/ 24884) » As an alternative to typical antibodies, it is also possible to use, for example, a protein backbone against mir-199a, such as a lipocalin-based anti-carrier protein (Beste et al. (1999) Proc. Natl. Acad. Sci. USA , 96, 1898-1903). The natural ligand binding position of the lipocalin (eg, retinol-binding protein or biletriene binding protein) can be altered, for example, by "combined protein design, in a manner that binds to a selected hapten This is the way of mir-199a) (Skerra, 2000, Biochim. Biophys. Acta, 1982, 337-50). Other protein backbones are known which are well known for antibody recognition for molecular recognition (Skerra (2000) J. Mol. Recognit., 13, 167-187). The term "nucleic acid against mir-199a gene or mir-199a itself" 68 201239097 means double-stranded or single-stranded DNA or RNA, for example, which inhibits or activates mir-199a gene expression or mir-199a activity, and includes (but not Limited to) antisense nucleic acids, aptamers, siRNA (small interfering RNA), miRNA, shRNA (short inflammatory RNA) and ribozymes. Nucleic acids can be synthesized, for example, chemically, for example according to the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). Aptamers are nucleic acids that bind to a polypeptide (herein mir-199a) with high affinity. The aptamer can also be used by SELEX, for example. Screening methods (see, for example, Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981) from a group of different single strands Isolation from RNA molecules. Aptamers can also be synthesized and screened in mirror images, such as L-ribonucleotide (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). The form isolated in this way enjoys not being naturally The advantages of nuclease digestion and therefore higher stability. Nucleic acids can be broken down by endonucleases or exonucleases, in particular DNase and RNase which can be found in cells. Therefore, it is advantageous to modify nucleic acids so that Nucleic acids are more stable against decomposition, ensuring long-term maintenance of high concentrations of nucleic acids in cells (Beigelman et al. (1995) Nucleic Acids Res. 23: 3989-94; WO 95/11910; WO 98/37240; WO 97/ 29116). In general, such stabilization can be obtained by introducing one or more internucleotide phosphorus groups or by introducing one or more of the 69 201239097 non-phosphorus nucleotides. The interglycosides are in accordance with Uhlmann and Peyman (1990), supra (see also Beigelman et al. (1995) Nucleic Acids Res. 23: 3989-94; WO 95/11910; WO 98/37240; WO 97/29116). A nucleic acid-containing modified internucleotide phosphate group and/or a non-phosphorus bridge may be used in one of the uses of the present invention, for example, decyl phosphate, phosphorothioate, phosphoniumamine, dithiophosphate, and /or phosphate, rather than inter-phosphorus analogs, such as a siloxane bridge, Carbonate bridge, carboxymethyl ester, acetamide bridge and/or thioether bridge. This modification also intends to enhance the durability of the pharmaceutical composition, which can be used in one of the uses of the present invention. The use of suitable antisense nucleic acids is further described, for example, in Zheng and Kemeny (1995) Clin. Exp. Immunol., 100, 380-2; Nellen and Lichtenstein (1993) Trends Biochem. Sci., 18, 419-23, Stein (1992) Leukemia, 6, 697-74 or Yacyshyn, BR et al. (1998) Gastroenterology, 114, 1142). The use of siRNA and its use as a RNA interference in down-regulating or severing gene expression (herein the mir-199a gene expression) is described, for example, in Elbashir, SM et al. (2001) Genes Dev., 15, 188 or Elbashir. , SM et al. (2001) Nature, 411, 494. Ribozymes are also an appropriate tool for inhibiting nucleic acid translation (in this case, the RAK gene) because they specifically bind and cleave mRNA. They are described, for example, in Amarzguioui et al. (1998) Cell. Mol. Life Sci., 54, 1175-202; Vaish et al. (1998) Nucleic Acids 201239097

Res., 26, 5237-42; Persidis (1467) Nat. Biotechnol., 15, 921-2 or Couture and Stinchcomb (1996) Trends Genet·, 12, 510-5. The nucleic acid preferably means an antagonist used as or as mir_199a. Regarding the manufacture of a pharmaceutical product, the mir_199a antagonist of the present invention is usually combined with one or more pharmaceutically acceptable additives or auxiliary substances such as a physiological buffer solution (for example, a sodium vapor solution), demineralized water, a stabilizer (such as a protease or a nucleic acid). An enzyme antagonist, preferably aprotinin, ε-amine hexanoic acid or aprotinin) or a chelating agent (such as ED ΤΑ), a gel formulation (such as white petrolatum, low-viscosity sarcophagus and/or yellow wax) ), etc. - from the deployment, depending on the type of medication. More suitable additives are, for example, detergents (such as, for example, Τηωηχ_100 or sodium deoxycholate) and polyols (such as, for example, polyethyl alcohol or glycerol), sugars (such as, for example, sucrose or glucose), and sexes. Green: Compounds (such as, for example, amino acids such as glycine or especially bovine or beet) and/or proteins (such as cattle or humans are innocent). Preferred are detergents, polyols and/or zwitterionic compounds. The physiological buffer solution preferably has a pH of about 6 〇 to 8 ,, especially 7·8 @ PH ', especially a pH of about 7.4 and/or an osmotic pressure of about n, r, and a houser/liter. Preferably, it is about 290 to the right gram molecule per liter. The yang of the drug is usually adjusted using an appropriate machine or inorganic buffer, such as, for example, a stone HS salt buffer, a tris buffer (tri(methyl)aminocarbamate 201239097 HEPES buffer). ([4-(2-Hydroxyethyl)piperazinyl]ethanesulfonic acid) or MOPS buffer (3-morpholinyl-1-propanesulfonic acid). The choice of individual buffers is usually based on the required buffer Depending on the molar concentration, the phosphate buffer is suitable, for example, for injection or infusion solutions. The drug may be administered in a conventional manner, for example by means of an oral dosage form such as, for example, a lozenge or capsule, by mucosal Means (for example, nasal cavity or oral cavity), implanted under the skin in the form of a depot, by injection, infusion or gel (which contains a drug according to the invention). The drug may also be administered locally or partially to treat the above Said specific joint disease, if appropriate in the form of a liposome complex. Furthermore, the treatment can be carried out by means of a transdermal therapeutic system (TTS), which is capable of controlling the release of the drug over time. TTS is known, for example From EP0 944 398 Al, EP0916 336 Al, EP 0 889 723 A1 or EP 0 852 493 A1. If only a relatively small amount of solution or suspension (eg, from about 1 to about 2 〇ml) is to be administered to the body, 'injectable solutions are usually used. If large amounts (eg 1 liter or more) 1 liter) solution or suspension to be administered, usually using an infusion solution. Therefore, compared to the infusion solution, in the case of only a few milliliters of the injection solution, the small difference in pH and osmotic pressure of the blood or tissue fluid at the time of injection is Not obvious or only to the extent that the pain is detectable to inconspicuous. Therefore, it is generally not necessary to dilute the formulation according to the invention with hydrazine. However, in the case of relatively large doses, the formulation according to the invention The degree of at least about isotonic solution should be obtained by simply diluting before administration. An example of an isotonic solution is a 9% solution of sodium sulphate. Dilution is carried out in the case of infusion, for example, by 72 201239097 by so-called bypass The tube is administered as sterile water. It can also be used in its original form, in a gene transfer vector or in combination with liposomes (especially cationic lipids) or gold particles. Nucleic acids, which may be formulated as liposomes (wherein the nucleic acid is carried as a cargo in a membrane lipid layer such as a lipid bilayer), or may be a nanoparticle (eg, dextrose if, peptide or other polymerization) An example of a gene transfer vector is a viral vector, such as an adenoviral vector or a retroviral vector (Lindemann et al. (1997), Mol. Med., 3, 466-76; Springer et al. (1988). Mol· Cell., 2, 549-58). Complexation with liposomes usually results in extremely high transfection efficiency, especially in skin cells (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4:146-a79- 85). In liposome transfection, a small monolayer vesicle composed of cationic lipids is prepared by ultrasonically treating a liposome suspension. The DNA is ionically bound to the lipid raft surface in a ratio that maintains a net positive charge and all plastid DNA is complexed through the liposome. In addition to Feigner, PL et al. (1987), Proc. Natl. Acad. Sci USA, 84, 7413 - 7414 DOTMA (1,2-bisoctadecyloxypropyl-3-trimethyl bromide) In addition to the ammonium and DOPE lipid mixtures, a large number of lipid formulations have been synthesized to date and tested for their efficiency in transfecting various cell lines (Behr et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 6982-6986; Zhao and Huang (1991), Biochim. Biophys. Acta, 1189, 195-203; Feigner et al. (1994) J. Biol. Chem., 269, 2550-2561). An example of a lipid formulation is DOTAP N-[l-(2,3-bisoctadecyloxy)propyl]-N,N,N-three 73 201239097 methylammonium sulphate or DOGS (double octadecyl) Amidinoglycine hydrazinidine). For example, an auxiliary substance that increases the transcription of a nucleic acid into a cell is a protein or peptide that binds to DNA or a synthetic peptide-DNA molecule that causes the nucleic acid to be transported into the nucleus (Schwartz et al. (1999) Gene Therapy 6.282, Branden et Al. (1999) Nature Biotech., 17, 784). Auxiliary substances also include molecules that allow nucleic acids to be released into the cytoplasm (Planck et al. (1994) J. Biol. Chem., 269, 12918; 1 (^11^6131_(1997) 31〇〇〇11..0: 116111, 8, 213) or 'eg liposomes (Uhlmann and Peyman (1990), supra) 〇 In addition, these nucleic acids can be obtained by applying the above nucleic acids to gold particles and using so-called "gene grabs" to direct these particles to tissues or cells. A particularly suitable heart type (Wang et al. (1999) J. Invest. Dermatol. 112.775-81, Tuting et al. (1998) J. Invest. Dermatol. 111: 183-8). Another subject of the invention is The mir_199a or mir_199a gene is used as a mir-199a antagonist for the treatment of joint diseases (especially degenerative joint diseases such as osteoarthritis) or joint diseases such as rheumatoid arthritis, and/or for the treatment of joint pain (especially reduction) Use of a joint pain of a degenerative joint disease. Preferably, the mir. 199a antagonist is used in the form of a drug as described above. Thus, the present invention also relates to a method of screening for an antagonist of mir_199a, wherein the method comprises the following steps: (4) Providing mir-199a Or mir-199a gene, 74 201239097 (b) to provide test compounds and (C) to measure or detect the effects of mir-ship genes. M-mouth for *1 or for example to provide mir_199a or library in an analytical system And directly or indirectly connected to a compound 4 (particularly a biochemical or chemical lysine compound), for example, in the form of a chemical compound: its measurement, the test compound is tested for the influence of mir-199a or Luguang & Afterwards, appropriate antagonists can be analyzed and/or isolated. For screening chemical human 3 ^ pre- sigmatism stocks, the car is well known or familiar with the skilled artisan (4) high-throughput analysis. According to the invention, the term "chemical compound library" means a plurality of chemical compounds consisting of any of a variety of sources, including chemically synthesized molecules and day 4 products, or those which have been produced by combinatorial chemistry techniques. For example, the effect of a test compound on the miM99a or mir_199a gene is measured or detected in a heterologous or homology analysis. As used herein, a heterologous assay is an assay comprising one or more washing steps and a washing step. In the same It is not necessary in the sex analysis. The reagents and the compounds are only mixed and measured. The functional analysis of each is based on the gene expression of mir-199a. In particular, the present invention relates to the following aspects: L. State or disease indicator of mir_199a. 2. The use of mir-199a as an indicator of tissue status or disease. 3. Use of mir-199a as in Aspect 1 or 75 201239097 in mir-199a as in Aspect 2, wherein mir-199a content is expressed in the body (a) tissue state or disease, and/or (b) tissue development A change in status and risk of disease, and/or (c) an individual suffering from a change in tissue status or disease, and/or (d) a state or progression of the disease or stage of the disease. 4. A method for identifying the following in an individual: (a) a change in the state of the tissue or the presence of a disease, and/or (b) a risk of developing a tissue state or disease, and/or (c) monitoring the state of the tissue or the progression of the disease. Or stage, which contains speculative mir-199a content. 5- The use of mir-199a or mir-199a as in the case 3 or the method of the method 4, wherein the mir-199a content is the mir-199a content in the sample in the individual or in the individual. 6. Use or method of mir-199a or mir-199a according to aspect 5, which further comprises comparing the mir-199a content of the individual or sample to the mir-199a content of one or more reference or reference samples. Use or method of mir-199a or mir-199a as in the case of 'the reference' is selected from the group consisting of: a healthy individual, a rickety individual or the same as the test subject but at an earlier or later time point Late, or mir-199a content values indicate individuals who have no tissue state changes or disease, have a change in tissue status, or have an increased or reduced risk of developing a tissue state or disease. 76 201239097 8* The use or method of mir_j99a or mir-199a according to aspect 7, wherein the reference sample is selected from the group consisting of: a reference sample derived from a healthy individual, a reference sample derived from a ricket individual a reference sample derived from the same individual as the sample of interest but taken at an earlier or later time point, and having a mir_199a content indicating a healthy individual or indicating the presence or absence of a tissue state change or disease or indicating a change in tissue state or disease Reference samples with increased or decreased risk. 9. The use or method of mir_i99a or mir-199a as in the case of '7' where the reference is for a healthy individual or has an increased risk of developing a tissue state or disease or a mir-199a content indicating that no tissue state or disease exists, or as Use or method of mir-199a or mir-199a of the aspect 8, wherein the reference sample is derived from a healthy individual or derived from a risk of having a tissue state change or disease reduced or having a mir-199a content indicating a healthy individual or no disease The state or the individual whose tissue state changes or the risk of the disease is reduced, wherein the decrease in mir-199a content is expressed in the individual: (a) the presence of tissue deterioration or disease, and/or (b) the risk of tissue deterioration or disease Increase, and / or (c) deterioration of the state of the organization or progression of the disease. 10. The use of mir-199a or mir-199a in the form 7 or the method of the method of the method of the method of the singularity of the singularity of the singularity of the singularity of the singularity of the disease, or the disease, or the disease Or the use or method of gamma 99a or mir-199a, wherein the reference sample is derived from a rickety individual or has an increased risk of developing a tissue state change or disease or has a mir-199a content or quantity indicating a ricket individual or presence An individual with a disease state or an increased risk of a change in tissue status or a disease in which the mir-199a content is similarly expressed in the individual: (a) a tissue state deterioration or disease, and/or (b) a tissue state deterioration or disease Increased risk, and / or (c) deterioration of organizational status or progression of the disease. 11. The use or method of mir-199a or mir-199a as in the case of 'the reference is the same individual at an earlier time point, or the use or method of mir_199a or mir-199a as in the aspect 8' Is derived from an individual that is identical to the sample of interest and taken at an earlier time point, where: (i) a decrease in the mir-i99a content in the sample of interest indicates in the individual (a) a tissue-like deterioration or disease, and / or (b) an increased risk of tissue deterioration or disease, and / or (c) deterioration of tissue status or progression of the disease, 78 201239097 (ii) An increase in the mir-199a content in the sample of interest indicates: (a) Changes in tissue status or disease improvement or absence, and/or (b) deterioration of tissue status or risk of disease, and/or (c) deterioration of tissue status or progression of disease, (iii) mir in samples of interest A similar amount of -199a is expressed in the individual (a) a similar risk of developing tissue deterioration or disease, and/or (b) a deterioration in tissue status or progression of the disease, and/or (c) a persistent deterioration in tissue status or disease. - A method of determining the dosage of a pharmaceutical substance that changes the state of tissue or prevents or treats a disease in an individual, comprising the steps of: (a) determining the mir-199a content in an individual sample and, where appropriate, determining the reference or reference sample The mir-199a content is compared to the mir-199a content in the sample of interest, and (b) the pharmaceutical dose is determined based on the mir_199a content of the sample of interest, depending on the sample of interest and the reference or reference sample, as appropriate The mir-199a content is relatively high. 13 - A method of modulating a dosage of a medicament for altering tissue status or preventing or treating a disease, comprising the steps of: (a) determining the mir-199a content of the sample, and (b) determining one or more references or references Mir-199a content in the sample, 79 201239097 in the sample in the sample of interest, in one or more references or references (d) depending on whether the miM99a content in the sample of interest is different = in a reference or reference sample The method of adjusting the drug to determine the mir-199a content in the sample of interest; _-- or more The mir-l99a content in the reference or reference sample, and (c) whether the mir-199a content present in the sample of interest is different from the content in one or more reference samples, > The sample of interest is exposed to the substance different from the one or more reference or reference samples. And the method of any one of the aspects 5 to 14, wherein the sample of interest is a tissue and/or a liquid. 16. The method of mir_i99a or mir_i99a according to aspect 15, wherein the tissue sample is selected from the group consisting of tissue extract, synovial tissue, and cartilage, and the body fluid sample is selected from the group consisting of synovial fluid, A group consisting of serum plasma and urine. 17. Use of mir-199a in a method as in any of the methods 4 to 16. 18. A kit for use in any of the methods of any of aspects 4 to 16, the package S having one or more components for detecting mir-199a. 19. A kit according to aspect 18, wherein the one or more members for detecting mir-199a are members for determining the expression level of mir l99a, preferably at the gene and/or RNA level. A set of 18 or 19, wherein the one or more members for the group mir-199a are selected from the group consisting of nucleic acids, preferably DNA or RNA, DNA and RNA. The delta or chemically modified nucleic acid purine peptide, and the protein/preferably a monoclonal antibody or a plurality of antibodies. The kit of any one of 18 to 2, wherein the kit (a) container, and/or the material carrier, wherein the data carrier contains, for example, the following y regarding the risk of joint disease and/or clock The operation of the disease and/or monitoring the progress of joint disease refers to the operation guide of the components and/or kits of mir_i99a (preferably in the sample, better in the sigma), For example, the components of the Russian mir-199a = 3⁄4 sets of the number / reduction, manufacturing or assembly or use of the government information, about the correct storage or operation kit 201239097

Information on V, (iv) Information on the composition of buffers, diluents, reagents used to detect mir-199a or to detect the composition of mir_199a, (v) Interpretation When performing the above methods to identify and/or monitor joints Information on the information obtained when the disease progresses, (vi) warnings about possible misinterpretation or erroneous results when improper methods and/or improper components are implemented, and/or (vii) when using inappropriate reagents and/or buffers At the time, warning about possible misinterpretation or wrong results. 22. Use of a kit according to any of the aspects 18 to 21 in the method of any of the aspects 4 to 16. 23. Or a plurality of nucleic acids for use in detecting a mir-199a gene, a gene product or a functionally active variant thereof, as in any of the methods 4 to 16. 24. The nucleic acid according to aspect 23, wherein the nucleic acid is selected from the group consisting of a locked nucleic acid (LNA), a nucleic acid probe, a poly- or a nucleic acid (pNA), a microRNA (miRNA), a small interference R>fA (siRNA) ), a polymerase chain reaction (PCR) primer, a reverse transcription (RT) reaction primer, and a DNA sequencing primer. 25. A nucleic acid as in the form 24 which comprises a nucleotide sequence of at least 8 nucleotides. 26. A peptide, polypeptide or protein for use in a method of detecting a mir-199a gene, a gene product or a functionally active 82 201239097 variant thereof, as in any of the methods 4 to 16. 27. A peptide, polypeptide or protein according to aspect 25, wherein the protein or polypeptide is a protein ligand, preferably an antibody, a fragment or derivative thereof, a darpin or an anti-carrier, or a polypeptide or peptide thereof The needle is preferably a mass spectrometer probe. 28. The use of mir-199a or mir-199a according to any one of the aspects 5 to 11, or the method of any of the aspects 5 to 14 and 35 to 36, the use of the set of the aspect 22, The use of the nucleic acid of any one of clauses 23 or 25, the peptide, polypeptide or protein of the form 26 or 27, or the nucleic acid, peptide, polypeptide or protein of the aspect 28, wherein the tissue state is tissue damage, preferably Selected from a group consisting of damage to bone, cartilage, synovium, perichondrium, sac, and connective tissue, and/or characterized by changes in the content of regulatory molecules, signaling molecules, and/or degenerative molecules present in the tissue . 29. The use of mir-199a or mir-199a according to any one of the aspects 5 to 11, or the method of any of the aspects 5 to 14 and 35 to 36, the use of the set of the aspect 22, The use of the nucleic acid of any one of the aspects 23 to 25, the peptide, polypeptide or protein of the form 26 or 27, or the nucleic acid, peptide, polypeptide or protein of the aspect 28, wherein the disease is a joint disease, preferably Selected from osteoarthritis, rheumatoid arthritis, psoriatic arthritis, septic arthritis, ankylosing spondylitis, bursitis (inflammation), dermatomyositis, fibromyalgia, gouty arthritis, juvenile 83 2〇1239〇97^Sexual arthritis (Stil's disease), mixed connective tissue wet polymyalgia, polymyositis, reactive joints, scleroderma syndrome, scleroderma, scapularitis, Schroder's group, group of systemic lupus erythematosus and/or characteristic physical or metabolic damage to the f joint (expressed as joint inflammation or joint degeneration), wherein the disease is preferably osteoarthritis. The following are more aspects of the invention: 1. [Prognostic method] A method for identifying an increased risk of joint disease in an individual comprising: a) measuring the mir-199a content of the biological sample of the individual; and, b The mir_199a content in the sample and the mir-199a content in the appropriate control were compared, wherein the mir-199a content in the sample was determined to be decreased when compared to the control, thereby identifying the individual with an increased risk of joint disease. 2. [Diagnostic method] A method for diagnosing joint disease in an individual, the method comprising: a) measuring the mir-199a content in the biological sample of the individual; and b) comparing the mir-199a content in the sample with an appropriate control The mir-199a content in the sample, wherein the mir-199a content in the sample is determined to be decreased when compared with the control, thereby identifying the individual having a joint disease. 84 201239097 3. [Patient level] A method for treating an individual with a joint disease, the method comprising: a) measuring the mir-199a content in the biological sample of the individual; b) comparing the mir-199a content in the sample with The mir-199a content of the appropriate control; and c) when the mir-199a content in the sample is determined to be decreased as compared with the control, the patient is administered a pharmaceutical composition for treating a joint disease, thereby treating the individual. 4. [Dose Optimization] A method of determining the dosage of a pharmaceutical for treating an individual having a joint disorder, the method comprising: a) measuring the mir-199a content in the biological sample of the individual; b) comparing the mir in the sample -199a content and mir-199a content in an appropriate control; and c) if the mir-199a content in the sample is increased as compared with the control, the dosage of the pharmaceutical composition is lowered, or if the mir-199a content in the sample is When the comparison is measured as a decrease, the dose of the pharmaceutical composition is increased to determine the dosage of the individual's pharmaceutical. 5. [Screening analysis] A method of determining the effect of a substance or test compound on a joint disease, the method comprising: a) measuring the content of mir-199a in a sample treated with the substance or test compound; b) comparing the treated sample The mir-199a content in the appropriate control and the mir-199a content in the appropriate control; and 85 v v201239097 c) if the mir-199a content in the sample is determined to be reduced as compared to the control, the substance is identified as having an adverse effect, or if When the mir-199a content is measured as an increase compared to the control, the substance is identified to have a beneficial effect, thereby determining the effect of the substance or test compound on joint disease. 6. The method of any of aspects 1 to 4, wherein one or more nucleic acid probes are used to detect the mir-199a content. 7. The method of any of aspects 1 to 6, wherein the appropriate control is a biological sample of the healthy individual. 8. The method of aspect 6, wherein the appropriate control is a sample that has not been treated with the substance. 9. The method of any of the preceding aspects, wherein the joint disease is osteoarthritis. 10. [Treatment method using mir-199a antagonist] A method for treating a joint disease of an individual comprising administering to the individual a mir-199a agonist to treat a joint disease of the individual. 11. The method of aspect 10, wherein the mir-199a agonist is selected from the group consisting of siRNA, shRNA, antisense RNA, and ribozyme. 12. The method of aspect 10, wherein the mir-199a agonist is substantially complementary to at least a portion of the mir-199a precursor form (SEQ ID NO: 1). 13. The method of aspect 10, wherein the mir-199a agonist is substantially 86 201239097 and at least a portion of the mature form of mir-199a (SEq ID NO: 2) at least a portion of the mature form of miM99aA ( SEQ ID NO: 3) is complementary. 14. The method of aspect 10, wherein the miRNA146aK agent is a nucleic acid molecule of from about 6 to about 20 nucleotides in length. 15. The method of sadness 10, wherein the miRNA-146a agonist is a nucleic acid molecule modified with LNA or modified with pNA. 16. The method of g-like 1G, wherein the joint disease is osteoarthritis. 17. The method of 1, 10, wherein (5) the administration of the agonist increases the number of cartilage cells in the individual or the cartilage markers. 18. The method of aspect 17, wherein the one or more cartilage markers are selected from the group consisting of type 11 collagen, aggrecan, S〇X9 type X collagen, ALP, CD-Rap, MMP- 1 and the group formed by MMP-3. 19. Method of Aspect 11 2〇. A set containing one or more components for detecting Qin 199a and appropriate controls. Wherein the mammal is a human, the present invention, the examples and the examples are as follows: 1. In the culture of chondrocytes and stem cells, cartilage fines 87

V V201239097 mir-199a in cells and stem cells behaved in the culture of primary chondrocytes and mesenchymal stem cells derived from bone marrow (BMSC) during the determination of mir-199a in primary chondrocytes and mesenchymal stem cells derived from bone The performance of the mir-199a in these cells for a predetermined period of time. The chondrocytes were cultured in the form of pellets supplemented with 2 mM L-face valeric acid (Sigma-Aldrich), 1 χ non-essential amino acid (Sigma-Aldrich), 10 η 地 dexamethasone (Sigma-Aldrich), 1 〇pg/ml insulin, 5.5 pg/ml transferrin, 5 ng/ml sodium selenite (Sigma-Aldrich), 44 pg/ml ascorbic acid (Sigma-Aldrich) and 10 ng/ml TGF-βΙ (R&D Systems were cultured in DMEM medium for 0, 1, 4, 7 or 14 days. BMSCs were cultured in the same medium in pellet culture for 1, 2, 7, 14, or 21 days. Cartilage cells and BMSCs were harvested and each pellet was treated separately for RNA isolation. For the analysis of secreted proteins, the supernatants of the entire culture period were collected and pooled. Regarding: RNA extraction, pellets were collected after 7 days of culture and homogenized individually by Mixermill (Retsch). The homogenized cells were subjected to protein || K digestion. Total RNA containing miRNA was eluted by the Qiagen miRNeasy Plus Micro kit. Reverse transcription into cDNA using the reverse transcriptase kit and RNase inhibitor (Life Technologies) according to the manufacturer's protocol. The mir-199a expression pattern in the soft bone cells and BMSCs was determined by miRNA-specific quantitative real-time PCR using Taqman probes according to procedures well known to those skilled in the art. Using eight of 88 201239097

Taqman® microRNA low density arrays (TLDA, Applied Biosystems, Foster City, CA) represent 668 miRNAs and reference genes outside them. This analysis consisted of three steps: multiplex RT, pre-amplification, and single TaqMan PCR. All steps are based on the manufacturer's operating instructions. Instant PCR was performed in the AB 7900HT Sequence Detection System. The comparison Ct method is also applicable to performance analysis. Figure 1 a illustrates mir-199a expression in chondrocyte pellets during culture, while Figure 2 illustrates mir-199a expression in BMSCs during culture. In chondrocytes, mir-i99a expression was detected with a cycle threshold of approximately 16 (ct), which increased to 22 cycles during culture (ct = 16 to 22), whereas ct of BMSC was determined during the culture period. 18 increased to 20. Therefore, in both cell types, the expression level of mir-199a increased as the culture period increased. The culture conditions were selected to reflect the conditions of healthy cartilage. Therefore, an increase in the expression of mir-l99a over time indicates an increase in the expression of mir-BQa in differentiated, healthy cartilage. Therefore, mir-199a is an indicator of physiologically differentiated cartilage. Increasing the expression level with the incubation time' The relative expression of mir-22 and mir-146a was relatively stable in the parallel experiments performed under the same conditions (Fig. lb), and the two microRNAs were identified as bone by the inventors. Mark of arthritic cartilage. Furthermore, under similar conditions, a similar increase in expression of mir_14〇 over time was observed, and mir-MO is a microRNA identified by the inventors as being associated with the state of the differentiated cartilage. Thus, an increase in the performance of mbepa compared to a particular control (e.g., markers of osteoarthritic cartilage, such as mir_22 89 201239097 and/or mir- 146a) indicates a physiological cartilage state and no OA is present. Therefore, mir-199a is an indicator of differentiated cartilage. Example 2: Overexpression and knockdown of mir-199a protein The expression of type II collagen, aggrecan, alp, mmp-i and MMP-3 in BMSC is known to be altered in arthritic joints. Therefore, their individual expression levels indicate the presence or absence of OA. To study the role of mir-199a in OA, after cartilage differentiation in BMSC, mRNA (type II collagen, aggrecan) or protein (MMP-1 and MMP-3) or active (ALP) Take measurements to analyze their over-expression or attenuate the effects on these molecules. In three independent experiments (V15, V18, V20), pseudoviral particles (containing DNA encoding mir-199a, more herein referred to as mir-199a) or MRP (more herein referred to as empty vector) Infected with BMSC. Mir-199a was transferred to pLenti9, a lentiviral vector containing a portion of the HIV1 LTR sequence (see map below). The pseudovirus particles are manufactured by Vectalys (Toulouse, France) according to customer service. Infection with BMSC was performed with a multiplicity of infection (MOI) of 5. To increase infection, 8 pg/ml polycondensed amine was added and the plate was centrifuged at 50 ° xg for 90 minutes at 32 °C. On the next day, high-density pellets with 2.5χ105 cells were formed by centrifugation (30〇xg, 5 minutes) in a 96-well conical bottom plate” 201239097 々<tLI (8695)___^.〇.; /./'

DeltaU3-3XTR SD Ambishilin V Apall(7449)#

Prsv^LTR~ ApaLl (5474) / Eco^l (5255) / \ IRES eGFP7 Chi (4500) pLenti9 mrp IRES eGFP 9418 bp

Pstl (2271) EF-1 alpha Pstl (2776)

Monosodium erythroprotein (mrp) Pstl (3541) Cells were harvested 7 days after pellet culture and their RNA was isolated following the same procedure as provided in Example 1. cDNA synthesis was performed using a reverse transcriptase kit and an RNase inhibitor (Life Technologies) according to the manufacturer's protocol. The expression levels of the four cartilage markers (type II collagen, aggrecan, Sox9 and type X collagen) were determined by quantitative real-time PCR. The TaqMan reaction of the target genes ACAN, COL2A, COL10A1 and SOX9 was performed using TaqMan® Universal PCR Master Mix and FAMTM-specific gene specificity analysis (see table). All gene specificity analyses consisted of a TaqMan® MGB probe labeled with FAMTM dye. PCR was performed using ABI Prism 7900 (Life Technologies). The ribosomal protein L37a (RPL37a) was used as a reference gene. The following sequence was selected for RPL37a: reference forward primer: GGCACTGTGGTTCCTGCAT, reference reverse primer: ACAGCGGAAGTGGTATTGTACGT. MGB reference probe

V V201239097 VIC mark and has the sequence CCGCCAGCCACTGTCT. The comparative Ct method is also suitable for performance analysis. Target gene gene symbol access number gene specificity analysis ID Polyprotein polysaccharide ACAN ΝΜ_013227 Hs00153936_ml Collagen, π-type, αΐ COL2A1 ΝΜ_033150 Hs00264051_ml In 3 different independent experiments (ie V15, In V18, V20), the effect of mir-199a overexpression on the expression levels of two different labeled proteins (ie, type II collagen and aggrecan) is shown in Figure 3 (see Figures 3A to B). . Each experiment had 4 samples (η = 4). ELISA was used to test ALP and ΜΜΡ-1 in experiments V15 and V18 according to procedures well known to those skilled in the art (see Figures 3C to D). The protein content in the culture supernatant was quantified using an ELISA kit according to the manufacturer's instructions: MMP1 (R&D Systems). For a single pellet culture, the supernatant obtained at each change of medium was collected and pooled over the entire 3-week culture period for determination of the protein content of each individual pellet by ELISA. The alkaline phosphatase (ALp) activity level in the pellet culture supernatant was quantified by spectroscopic optics using ALP_liquid and _ silk-based salt (p_Npp) substrate concentrate _liP〇re). The culture supernatant was collected on a dry day. For each pellet, the amount of medium applied to the pellet during the culture was phase _. Excessive expression of cells in the Qin family also showed increased levels of Dijon-type collagen, aggrecan and sputum, 92 201239097 and decreased ΜΜΡ-l content. These results confirm that increased mir-199a performance may be associated with differentiated cartilage. In three different experiments (V21, V22, V23), miRIDIAN hairpin inhibitor hsa-mir-199a (Dharmacon) was used in BMSC to attenuate mir-199a (more mir-199a inh herein), and The effect on the expression of the three marker proteins type II collagen, aggrecan and ALP was compared to the control cells. BMSCs were treated with a 50 n nM miRIDIAN hairpin inhibitor hsa-mir-199a in a monolayer culture. The miRIDIAN microRNA hairpin inhibitor negative control #1 (Dharmacon) served as a negative control. Next, a high-density pellet having 2.5 x 1 〇 5 cells was formed by centrifugation (3 〇〇 Xg, 5 minutes) in a 96-well conical bottom plate (Eppendorf). The pellets were cultured for up to 3 weeks. All of the analysis steps below were performed as described above for mir-199a overexpression. In three different independent experiments (ie, v2lV22, v23), the effect of miM99a attenuation on the expression levels of the three marker proteins (ie, n-type collagen, proteoglycan, and ALP) is shown in Figure 4 to Figure C. in. Four samples were tested for each experiment (η=4). The performance levels of all three labeled proteins tested decreased when mir l99a was attenuated. Therefore, lowering the mir-199a content reduces the amount of labeled protein in physiological cartilage. Therefore, a decrease in the mir-199a content indicates the presence of 〇A. Thus, the current content of miM99a indicates the physiological or pathological state of cartilage, particularly in relation to the presence or absence of osteoarthritis. It can be further inferred that mir_199a is effective 93

V 201239097 agent (performance or function) can be used to treat or prevent OA by increasing protein expression that is representative of and beneficial to the physiological state of the cartilage. Figure 5 is a graph showing the percentage change in the expression level of the labeled protein type II collagen and aggrecan overexpressed or attenuated mir-199a compared to the untreated control. 94 201239097 Sequence Listing - Free Information SEQ ID NO : 1 Stem loop sequence of the human hsa-mir-199a-l (access number: MI0000242) SEQ ID NO : 2 hsa-miR-199a-5p (access number: MIMAT0000231) SEQ ID NO: 3 hsa-miR-199a-3p (accession number: MIMAT0000232) SEQ ID NO : 4 Stem loop sequence of the human hsa-mir-199a-2 (access number: MI0000281) Reference 1. Grosshans et al., J Cell Biol 156: 17-21 (2002). 2. Czech, NEJM 354:1194-1195 (2006); 3. Mack, Nature Biotech. 25:631-638 (2007); 4. Eulalio et al., Cell 132 :9-14 (2008) 5. Yi, et al. Genes Dev. 17: 3011-3016 (2003) 6. Leuenberger et al., Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995) 7. Uhlmann and Peyman , Chemical Reviews, 90, 543-584 (1990) 8. Jayasena, Clin. Chem., 45, 1628-50 (1969) 9. Klug and Famulok, Mol. Biol. Rep., 20, 97-107 (1994) 10. US 5,582,981 11. Nolte et al., Nat. Biotechnol., 14, 1116-9 (1996) 95 201239097 12. Klussmann et al., Nat. Biotechnol., 14,1112-5 (1996) 13. Beigelman et Al., Nucleic Acids Res. 23:3989-94 (1995) 14. WO 95/11910 15. WO 98/37240 16. WO 97/29116 17. Beigelman et al., Nucleic Acids Res. 23:3989-94 (1995) 18. Griffiths-Jones et al., Nucleic Acids Research, 36, Database Issue, D154-D158 (2008) 19. Griffiths-Jones et al., Nucleic Acids Research, 34, Database Issue, D140-D144 (2006) 20. Griffiths-Jones, Nucleic Acids Research, 32, Database Issue, D 109-Dll 1 (2004) 21. Lagos-Quintana et al., Science. 294: 853-858 (2001) 22. Michael et al., Cancer Res. 1:882-891 (2003) 23. Cai et al Proc Natl Acad Sci USA. 102:5570-5575 (2005) 24. Landgraf et al., Cell. 129:1401-1414 (2007) 25. Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90 : 5873-5877 (1993) 26. Thompson et al., Nucleic Acids Res. 22, 4673-80 (1994) 27. Altschul et al., J. Mol. Biol. 215: 403-410 (1990) 96 201239097 28 Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997) 29. Brudno, Bioinformatics 2003b, 19 Suppl 1:154-162 30. Current Protocols in Molecular Biology, John Wiley & Sons, NY, 6.3. 1-6.3.6, (1991) 31. Mart In, Remington's Pharmaceutical Sciences 32. EP 0 368 684 B1 33. US 4,816,567 34. US 4,816,397 35. WO 88/01649 36. WO 93/06213 37. WO 98/24884 38. Beste et al., Proc. Natl. Acad Sci. USA, 96, 1898-1903 (1999) 39. Skerra, Biochim. Biophys. Acta, 1482, 337-50 (2000) 40. Skerra, Mol. Recognit” 13, 167-187 (2000) 41. Lindemann Et al., Mol. Med·, 3, 466-76 (1997) 42. Springer et al., Mol. Cell., 2, 549-58 (1988) 43. Alexander and Akhurst, Hum. Mol. Genet. 4 :146-a79-85 (1995) 44. Feigner et al., Proc. Natl. Acad. Sci USA, 84, 7413 -7414 (1987) 45. Behr et al., Proc. Natl. Acad. Sci. USA, 86, 6982-6986 (1989) 97 201239097 46. Zhao and Huang, Biochim. Biophys. Acta, 1189, 195-203 (1994) 47. Feigner et al., J. Biol. Chem., 269, 2550-2561 ( 1994) 48. Schwartz et al., Gene Therapy 6:282 (1999) 49. Brand6n et al., Nature Biotech., 17, 784 (1969) 50. Planck et al., J. Biol. Chem., 269, 12918 (1994) 51. Kichler et al., Bioconj. Chem, 8, 213 (1997) 52. Wang et al., J. Invest. Dermatol. 112:775-8 1 (1999) 53. Tuting et al., J. Invest. Dermatol. 111:183-8 (1998) 54. EP0 944 398 Al 55. EP 0 916 336 Al 56. EP 0 889 723 Al 57. EP 0 852 493 Al 58. Diamond et al., The New England Journal of Medicine: 1344-1349 (1981) 59. Winter and Milstein, Nature, 349, 293-299 (1991) 60. Zheng and Kemeny, Clin. Exp. Immunol. , 100, 380-2 (1995) 61. Nellen and Lichtenstein, Trends Biochem. Sci” 18, 419-23 (1993) 62. Stein, Leukemia, 6, 697-74 (1992) 63. Yacyshyn et al., Gastroenterology , 114, 1142 (1998) 64. Elbashir et al., Genes Dev” 15, 188 (2001) 65. Elbashir et al., Nature, 411, 494 (2001) 98 201239097 66. Amarzguioui et al., Cell. Mol Life Sci·, 54, 1175-202 (1998) 67. Vaish et al., Nucleic Acids Res., 26, 5237-42 (1998) 68. Persidis, Nat. Biotechnol., 15, 921-2 (1997) 69. Couture and Stinchcomb, Trends Genet., 12, 510-5 (1996) 99 201239097 [Simplified Schematic] Figure la shows the mir_i99a expression of chondrocyte pellets during the culture period. Figure lb shows the mir_22, mir-140, mir-146a and mir-199a tables of chondrocyte pellets during the culture period. Figure 2 shows the mir-199a expression of mesenchymal stem cell pellets during the culture period. Figure 3 is the effect of n!ir-199a overexpression on the expression levels of two marker genes and two marker proteins in three different experiments: Type II collagen (A); Polyprotein polysaccharide (B); ALP (C); MMP-1 (D). Figure 4 shows the effect of mir-199a attenuation on the expression levels of two marker genes and one marker protein in three different experiments: Π-type collagen (A); aggrecan (B); ALP (C) ). Fig. 5 is a graph showing the relative changes in the expression of the two marker genes type II collagen and polyprotein polysaccharide on mir-199a over- or under-reaction. [Explanation of main component symbols] None 100 201239097 Sequence Listing <110> SANOFI <120> miRNAs for joint diseases (2) <130> DE2010/290 <160>4 <170> BiSSAP 1.0 <210&gt ; 1 <211>71 <212>RNA<213>Smarter<220>221>Source<222> 1..71 <223> / Molecular_Type="RNA"="Wisdomman"<400> 1 60 71 gccaacccag uguucagacu accuguucag gaggcucuca auguguacag uagucugcac auugguuagg c <210>2 <211>23 <212>RNA <213>Smarter<220><221>Source<222> 1..23 <223> / Molecular_Type="RNA" 201239097 /Biology="Wisdom Man"<400> 2 cccaguguuc agacuaccug uuc <210>3 <211> 22 <212> RNA <213>Wisdom Man<220>221>Source<222> 1..22 <223> / Molecular_Type="RNA"/Bio="Wisdom Man";<400>3 acaguagucu gcacauuggu ua <210>4 <211> 110 <212>RNA <213>Smarter<220><221> Source <222>1..110 <223> / Molecular_Type="RNA" /Biology=”Wisdom Man•’ <400>4 aggaagcuuc uggagauccu gcuccgucgc cccaguguuc agacuaccug uucaggacaa ugccguugua caguagucug cacauugguu agacugggca agggagagca

Claims (1)

201239097 VII. Patent application scope: Use mir-199a as an indicator of tissue status or disease. 2. The use of mir-199a as an indicator of tissue status or disease. 3. Identifying the following methods in an individual: (a) a change in the state of the tissue or the presence of a disease, and/or (b) a risk of developing a tissue state or disease, and/or (c) monitoring the state of the tissue or disease. In the progression or stage, the method comprises detecting the mir-199a content. 4. A method for determining a dosage of a pharmaceutical product for modifying a tissue state or preventing or treating a disease in an individual, comprising the steps of: (a) determining a mir_199a content in a sample of the individual, and determining a reference or reference sample as appropriate The mir_199a content is compared to the mir-199a content of the sample of interest, and (b) the dose of the medicinal agent is determined based on the mir_199a content of the sample of interest, as appropriate, based on comparing the mir- in the sample of interest with the reference or reference sample. 199a content. 5. A method of modulating a dosage of a medicament for modifying a tissue state or preventing or treating a disease, comprising the steps of: (4) determining a mir-199a content in a sample, and (b) determining one or more reference or reference samples Mir l content, (c) Verify that the test sample is present in the sample of interest differently than - or in multiple reference or reference samples, and (Φ depending on whether the mir_199a content in the sample of interest is different One or 1 201239097 The content of multiple reference or reference samples is difficult to administer. 6. The method for the favorable or/or unfavorable vocalization of the disease state or disease, which comprises the following steps: (a) Determination of interest The mir l99a content in the sample, 1 — - or the content of miM 99a in multiple reference or reference samples, is present in the - interest sample in the same amount as in one or more reference or reference samples = = the sample is exposed to With one or more reference or reference samples 7. The use of a 199a in the third to the method of the patent application scope. JI 8. - Let the listener apply for a special section 3 to 6 a set of ones comprising one or more components for detecting mir-199a. / 9. The use of the kit of item 8 of the scope of interest in any of the methods of claims 3 to 6. 10. Or to test the nucleic acid of the Qin 19 gene, gene product or its functional and active variant in any of the methods in the scope of the patent application, No. 3 to 6. 11. A peptide, polypeptide or protein which detects a mir-199a gene, a gene product or a functionally active variant thereof, in any of items 3 to 6. 12 A nucleic acid as in claim 10, or as an application Patent No. 201239097 The use of a peptide, polypeptide or protein according to item 11 of the method of any one of claims 3 to 6.