Method for determining autoimmune disease and / or inflammatory disease, and preventive and / or therapeutic agent for inflammatory disease and / or autoimmune disease

Abstract

The purpose of the present invention is to provide a novel method which, in the described circumstances, can determine likelihood of morbidity, severity, risk of onset and / or aggravation, treatment effects, etc., of inflammatory disease including Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, or other autoimmune diseases. A further purpose of the present invention is to provide a novel therapeutic method for inflammatory disease including Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, or other autoimmune diseases.　The present invention is a method for determining autoimmune disease and / or inflammatory disease in a subject which targets at least one type of microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-628-3p, miR-548ac, miR-193b-5p, miR-204-3p and miR-3535 in a biological sample. Further, the present invention includes a preventive and / or a therapeutic agent of autoimmune disease and / or inflammatory disease which has a miR-455-3p inhibitor as the effective component.

Description

[Technical field]

[0001] The present invention relates to a method for determining an autoimmune disease and / or an inflammatory disease, and a preventive and / or therapeutic agent for an inflammatory disease and / or an autoimmune disease. [Background technology]

[0002] miRNA (microRNA) is a non-coding RNA consisting of approximately 17 to 24 nucleotides that is not translated into protein. More than 2,500 types of miRNA have been discovered in humans to date. These miRNAs work to suppress the translation of various genes that have complementary target sites to themselves, and control basic biological functions such as cell development, differentiation, proliferation, and cell death. As microRNAs thus control the expression of many genes within cells, changes in their expression are thought to be related to the development and progression of diseases, and research is being conducted to utilize microRNAs for the diagnosis and early detection of diseases.

[0003] For example, regarding the relationship between cancer and miRNA, Calin et al. reported that miR15 and miR16 are frequently downregulated in lymphocytic leukemia (see Non-Patent Document 1; Calin GA, Dumitru CD, Shimizu M et al., Proc Natl Acad Sci USA, 2002, vol. 99, p. 15524-9). In addition, Michael et al. reported that the expression of miR-143 and miR-145 is decreased in human colon cancer (see Non-Patent Document 2; Michael MZ, SM OC, van Holst Pellekaan NG, Young GP, James RJ, Mol Cancer Res, 2003, vol. 1, p. 882-91). There are also reports that specific miRNAs exist in tissue cancer cells (see Patent Document 1; JP 2009-100687 A). Thus, miRNAs are deeply related to diseases such as cancer, and it is believed that they may be useful for diagnosing and predicting the prognosis of these diseases.

[0004] On the other hand, there are no reports that miRNAs can be used for the diagnosis or prognosis prediction of Stevens-Johnson syndrome (SJS), an intractable ocular surface disease. There is a strong demand for a method to diagnose and predict the prognosis of inflammatory diseases such as SJS and other autoimmune diseases with high accuracy and in a simple manner. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2009-100687 A [Non-patent literature]

[0006] [Non-Patent Document 1] Calin GA, Dumitru CD, Shimizu M et al., Proc Natl Acad Sci USA, 2002, vol.99, p.15524-9 [Non-Patent Document 2] Michael MZ, SM OC, van Holst Pellekaan NG, Young GP, James RJ, Mol Cancer Res,2003,vol.1,p.882-91 Summary of the Invention [Problem to be solved by the invention]

[0007] In light of the above-mentioned circumstances, the present invention aims to provide a novel method capable of determining the possibility of contracting inflammatory diseases, including Stevens-Johnson syndrome (SJS), a refractory ocular surface disease, and other autoimmune diseases, the risk of onset and / or aggravation, and the effectiveness of treatment. Another object of the present invention is to provide a novel therapeutic agent for inflammatory diseases including Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, and other autoimmune diseases. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have conducted various studies and found that the expression pattern of specific microRNAs in conjunctival epithelial tissues of conjunctival tissues extracted during surgery from patients with Stevens-Johnson syndrome (SJS), an intractable ocular surface disease, is significantly different from that of healthy individuals. Using these microRNAs as indicators, the inventors have completed a novel method for determining the possibility of disease onset, severity, risk of onset and / or aggravation, treatment effect, etc. Furthermore, results have been obtained showing that inhibitors of specific microRNAs among these are effective in disease treatment, strongly suggesting their potential as novel therapeutic agents. That is, the gist of the present invention is as follows.

[0009] [1] A method for determining whether a subject has an autoimmune disease and / or an inflammatory disease, using at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-628-3p, miR-548ac, miR-193b-5p, miR-204-3p and miR-3535 in a biological sample as an indicator. [2] The method of determining the possibility of a subject suffering from an autoimmune disease and / or an inflammatory disease, as described in [1]. [3] The method of determining the severity of an autoimmune disease and / or an inflammatory disease in a subject, as described in [1]. [4] The method of [1] for determining the risk of onset and / or aggravation of an autoimmune disease and / or an inflammatory disease in a subject. [5] The method of determining the therapeutic effect of an autoimmune disease and / or an inflammatory disease in a subject, comprising the steps of: [6] The method according to any one of [1] to [5], wherein the biological sample is at least one selected from the group consisting of epithelial cells, blood (serum, plasma, whole blood, etc.), and tears of a subject. [7] A preventive and / or therapeutic agent for inflammatory diseases and / or autoimmune diseases, comprising an miR-455-3p inhibitor as an active ingredient. [8] The preventive and / or therapeutic agent according to [7], wherein the miR-455-3p inhibitor inhibits the expression and / or function of miR-455-3p in a living body. [9] The preventive and / or therapeutic agent according to [7] or [8], wherein the miR-455-3p inhibitor is at least one selected from the group consisting of a nucleic acid oligonucleotide, a low molecular weight compound, and an Hsa-miR-455-3p antibody.

[10] The preventive and / or therapeutic agent according to [9], wherein the miR-455-3p inhibitor is a nucleic acid oligonucleotide.

[11] The preventive and / or therapeutic agent according to

[10] , wherein the nucleic acid oligo is an oligo consisting of natural or non-natural RNA or DNA.

[12] The preventive and / or therapeutic agent according to

[11] , wherein the nucleic acid oligo is an antisense oligo of miR-455-3p.

[13] The preventive and / or therapeutic agent according to any one of

[10] to

[12] , wherein the nucleic acid oligo is modified. Effect of the Invention

[0010] According to the present invention, it is possible to determine the possibility of suffering from inflammatory diseases such as Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, and other autoimmune diseases, the severity, the risk of developing and / or becoming severe, and the therapeutic effect. Specifically, microRNAs whose expression changes specifically in SJS disease are medically useful biological indicators, and microRNAs that correlate with disease severity enable evaluation of therapeutic effects. The present invention leads to the provision of a new medical technology, namely, the establishment of a non-invasive diagnostic technique by disease-specific microRNA expression profiling targeting the conjunctival epithelial tissue of intractable ocular surface diseases and the use of it in selecting a therapeutic method. Furthermore, according to the present invention, it is also possible to provide a new therapeutic agent for inflammatory diseases such as Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, and other autoimmune diseases. [Brief description of the drawings]

[0011] [Figure 1-1] The results of comparing miRNA expression in conjunctival epithelial cells of SJS patients and healthy controls (quantitative PCR) are shown. [Figure 1-2]The results of comparing miRNA expression in conjunctival epithelial cells of SJS patients and healthy controls (quantitative PCR) are shown. [Figure 1-3] The results of comparing miRNA expression in conjunctival epithelial cells of SJS patients and healthy controls (quantitative PCR) are shown. [Diagram 2] The results of a comparison of miRNA expression in conjunctival epithelial cells of SJS patients and healthy controls using a human miRNA measurement system (quantitative PCR) are shown. [Figure 3-1] The results of quantitative PCR of ANKRD1, CXCL8, CXCL2, GEM, PTGS2, and RNASE8 in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed are shown. [Figure 3-2] The results of quantitative PCR of IL6, CXCL1, CCL20, and KPRP in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed are shown. [Figure 4] 1 shows the results of quantitative PCR of SLITRK6 in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed. [Diagram 5] The results of quantitative PCR of PPP1R3C, CTGF, CCL20, CXCL2, PTGS2, and CXCL8 in cultured human conjunctival epithelial cells with enhanced expression of miR-455-3p are shown. [Figure 6] The results of quantitative PCR of IL1RL1, CTGF, PPP1R3C, ADM, and SERPINB2 in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed are shown. [Figure 7-1] The results of quantitative PCR for ANKRD1, CXCL2, PTGS2, IL-6, CXCL1, and CCL20 in a mouse contact dermatitis model are shown. [Figure 7-2] 1 shows the results of quantitative PCR of IL1RL1 and SERPINB2 in a mouse contact dermatitis model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will now be described in detail. In addition, the terms used in this specification are to be interpreted as having the meanings usually used in the technical field unless otherwise specified.

[0013] [Method for determining autoimmune disease and / or inflammatory disease] The method for determining an autoimmune disease and / or an inflammatory disease of the present invention is characterized in that at least one microRNA (hereinafter, sometimes referred to as "miRNA") selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-628-3p, miR-548ac, miR-193b-5p, miR-204-3p, and miR-3535 in a biological sample is used as an index. The method for determining the present invention is a method for determining the possibility of an autoimmune disease and / or an inflammatory disease, the severity of an autoimmune disease and / or an inflammatory disease, the risk of developing and / or becoming severe of an autoimmune disease and / or an inflammatory disease, and the therapeutic effect of an autoimmune disease and / or an inflammatory disease in a subject.

[0014] As the organism from which the microRNA of the present invention is derived, mammals such as humans, mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats, and dogs can be preferably exemplified, among which humans and mice can be more preferably exemplified, and humans can be particularly preferably exemplified. The nucleotide sequences of the microRNAs of the present invention, miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-628-3p, miR-548ac, miR-193b-5p, miR-204-3p, and miR-3535-5p, are shown in SEQ ID NOs: 66 to 74, respectively. The sequences of microRNAs are highly conserved, particularly among mammals, and are highly likely to show the same expression profile as humans in mammals other than humans.

[0015] The microRNA of the present invention also includes microRNA consisting of RNA in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 66 to 74. The above "one or more" is preferably one to five, more preferably one to three, even more preferably one to two, and particularly preferably one. It can be easily confirmed whether the expression of the RNA consisting of the above nucleotide sequence is increased or decreased in a patient sample compared to a control, for example, by a microarray method or a quantitative PCR method described later. The sequence of the microRNA of the present invention from each of the above organisms other than humans can be confirmed or identified based on information registered in a database such as GenBank.

[0016] Examples of autoimmune diseases to which the method of the present invention can be applied include autoimmune hepatitis, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), acquired immune deficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, cardiomyopathy, celiac sprue-herpetiform dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, experimental autoimmune encephalomyelitis (EAE), fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, and the like. Inflammatory Tissue Disease (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (type 1 diabetes, type 2 diabetes), juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, nonalcoholic steatohepatitis (NASH), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, These include primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also known as progressive systemic sclerosis (PSS) or systemic sclerosis (SS)), Sjogren's syndrome, stiff-person syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0017] Examples of inflammatory diseases to which the method of the present invention can be applied include Stevens-Johnson syndrome, dermatitis (i.e., psoriasis, eczema, burns, dermatitis), arthritis (rheumatoid arthritis, spondyloarthropathy, gouty arthritis, degenerative joint disease (i.e., osteoarthritis), systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet's disease, hemolytic autoimmune anemia, multiple sclerosis, amyotrophic lateral sclerosis, hay fever, acute shoulder pain, psoriatic arthritis, juvenile arthritis, and osteoarthritis. Inflammatory bowel disease (including inflammatory bowel disease, peptic ulcer, regional enteritis, diverticulitis, gastrointestinal bleeding, Crohn's disease, gastritis, diarrhea, irritable bowel syndrome, ulcerative colitis), gastroesophageal reflux disease, eosinophilic esophagitis, gastroparesis (e.g., diabetic gastroparesis, etc.), food intolerance, food allergy, other functional bowel disease (e.g., non-ulcer dyspepsia, non-cardiac chest pain, etc.), etc. Among these, inflammatory diseases to which the method of the present invention is suitably applied are preferably Stevens-Johnson syndrome and dermatitis, and more preferably Stevens-Johnson syndrome.

[0018] The determination method of the present invention includes the following steps: (A) extracting RNA from a biological sample of a subject (epithelial cells, blood (serum, plasma, whole blood, etc.), tear fluid) and from a biological sample of a normal subject as a control; (B) measuring the expression level of a specific microRNA in the extracted RNA; (C) A step of comparing and determining the expression levels of microRNAs in the control and subject biological samples. The specific microRNA is not particularly limited as long as it has the above features. The specific microRNA is at least one type of microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-628-3p, miR-548ac, miR-193b-5p, miR-204-3p, and miR-3535.

[0019] When the expression level of at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac in a biological sample of a subject is increased compared to a control, and / or when the expression level of at least one microRNA selected from the group consisting of miR-125a-5p, miR-193b-5p, miR-204-3p, and miR-3535 is decreased compared to a control, the subject is evaluated as having an autoimmune disease and / or an inflammatory disease, that is, as possibly suffering from an autoimmune disease and / or an inflammatory disease (possibility of suffering from an autoimmune disease and / or an inflammatory disease). In addition, the severity of an autoimmune disease and / or an inflammatory disease, and the risk of developing and / or becoming severe of an autoimmune disease and / or an inflammatory disease can also be determined depending on the degree of increase and / or decrease in the expression level. Furthermore, if the expression level of at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac after treatment is lower than that before treatment, and / or if the expression level of at least one microRNA selected from the group consisting of miR-125, miR-193b-5p, miR-204-3p, and miR-3535 is higher than that before treatment, it is determined that the treatment is effective, and if there is no change or the opposite change is observed, it is determined that the treatment is ineffective or has deteriorated. Note that, in the determination method of the present invention, a biological sample from a normal subject or a subject not suffering from an autoimmune disease and / or an inflammatory disease is used as a control. In the present invention, the expression level of microRNA is preferably determined by using a relative expression level corrected with an appropriate internal standard gene (e.g., RNU44, miR-149*) or an external standard gene (e.g., miR-39) to correct for variations between samples, from the viewpoint of making a more accurate determination.

[0020] In the present invention, the biological sample is not particularly limited, but examples thereof include cells from the subject's tissue, blood, etc., and specifically, preferred biological samples include conjunctival epithelium, epidermal cells such as skin, serum, plasma, whole blood, tears, etc.

[0021] The method for extracting RNA in step (A) above is not particularly limited as long as it is a method that can extract RNA, including microRNA, from a biological sample. For example, total RNA, including microRNA, can be extracted from cells and tissues using the miRNeasy Mini Kit (QIAGEN), and from plasma and serum using the miRNeasy Serum / Plasma Kit (QIAGEN) in accordance with the attached protocols.

[0022] The method for measuring the expression level of a specific microRNA in step (B) above is not particularly limited as long as it is a method capable of identifying the amount of the specific microRNA in RNA containing a microRNA. For example, examples of the method include a microarray method in which a microarray comprising a polynucleotide or a part thereof consisting of a nucleic acid sequence complementary to the sequence of the specific microRNA is used for the determination, which can measure the expression level of the specific microRNA; a quantitative PCR method in which a primer set capable of amplifying the sequence of the specific microRNA is used for the determination; and a quantitative PCR method (fluorescent probe method) in which a primer set capable of amplifying the sequence of the specific microRNA and a fluorescent probe comprising a polynucleotide or a part thereof consisting of a nucleic acid sequence complementary to the sequence of the microRNA in question are used for the determination.

[0023] The microarray method is not particularly limited as long as it is possible to measure the expression level of the specific microRNA. An example of the microarray method is one in which RNA extracted from tissue is labeled with a label (preferably a fluorescent label), and the RNA is brought into contact with a microarray on which a polynucleotide (preferably DNA) consisting of a nucleic acid sequence complementary to the microRNA to be identified or a probe consisting of a part thereof is fixed to perform hybridization, and then the microarray is washed and the expression level of the microRNA remaining on the microarray is measured.

[0024] The type of nucleotides in the above nucleic acid sequence is not particularly limited as long as it can specifically hybridize to the specific microRNA of the present invention, but is preferably DNA because of its high stability. The length of the part of the above polynucleotide is not particularly limited as long as it can specifically hybridize to the specific microRNA of the present invention, but is preferably 10 to 100 mer, more preferably 10 to 40 mer, from the viewpoint of ensuring the stability of hybridization. The above polynucleotide or a part thereof can be obtained by chemical synthesis or the like using a method well known in the art.

[0025] The array on which the polynucleotide or a part thereof is fixed is not particularly limited, but glass substrates and silicon substrates are suitable examples, and glass substrates are more suitable examples. The method for fixing the polynucleotide or a part thereof on the array is not particularly limited, and known methods can be used. In addition to the array, any element such as a reagent used in a microarray method, such as a reagent used in an RNA labeling reaction, a reagent used in a hybridization reaction, a reagent used for washing, or a reagent used for RNA extraction from tissue, may be further used.

[0026] The quantitative PCR method is not particularly limited as long as it is a method using a primer set capable of amplifying the sequence of the specific microRNA and is capable of measuring the expression level of the specific microRNA, and conventional quantitative PCR methods such as agarose electrophoresis, SYBR Green, and fluorescent probe methods can be used.

[0027] A primer set in quantitative PCR refers to a combination of primers (polynucleotides) capable of amplifying the sequence of the specific microRNA. The primers are not particularly limited as long as they can amplify the sequence of the specific microRNA, but an example of a primer set is one consisting of a primer (forward primer) consisting of a part of the 5'-side sequence of the specific microRNA sequence of the present invention and a primer (reverse primer) consisting of a part of the 3'-side sequence of the microRNA sequence. Here, the 5'-side means that the 5'-side is closer to the sequence corresponding to the reverse primer when compared with the sequence of the mature microRNA, and the 3'-side means that the 3'-side is closer to the sequence corresponding to the forward primer when compared with the sequence of the mature microRNA.

[0028] The above-mentioned fluorescent probe is not particularly limited as long as it contains a polynucleotide or a part thereof consisting of a nucleic acid sequence complementary to the sequence of the above-mentioned specific microRNA, and suitable examples include fluorescent probes that can be used in the TaqMan (registered trademark) probe method (including those that utilize the FRET principle) and the cycling probe method.

[0029] The above primer set and the above fluorescent probe can be obtained by chemical synthesis or the like using methods well known in the art based on the sequence information.

[0030] In the above step (C), when the expression levels of microRNA in a control and a subject's biological sample are compared and determined, the degree of increase or decrease in the expression level of the specific microRNA in the biological sample can be, for example, an increase of preferably 50% or more, more preferably 75% or more, and even more preferably 100% or more, or a decrease of 50% or more, more preferably 75% or more, and even more preferably 90% or more, relative to the control.

[0031] [Determination kit] The present invention also includes a kit for determining an autoimmune disease and / or an inflammatory disease, which includes a reagent or the like necessary for the above-mentioned method for determining the present invention. The kit for determining the present invention can have various configurations according to the measurement target or the measurement principle. The components of the kit for determining the present invention can include, for example, a microarray comprising a polynucleotide or a part thereof consisting of a nucleic acid sequence complementary to the sequence of the specific microRNA, which can measure the expression level of the specific microRNA, a primer set capable of amplifying the sequence of the specific microRNA, and a probe for hybridization. As such miRNA, miRNAs employed as indicators in the above-mentioned method for determining the present invention are preferable. Specifically, miRNAs consisting of the nucleotide sequences of SEQ ID NOs: 1 to 8.

[0032] Furthermore, the determination kit of the present invention may contain other elements or components known to those skilled in the art, such as various reagents, enzymes, buffer solutions, reaction plates (containers), etc., depending on the configuration, intended use, etc. In order to facilitate detection after the PCR reaction, any labeling substance known to those skilled in the art, such as a fluorescent substance, may be bound to at least one of the ends of these primers.

[0033] [Preventive and / or therapeutic agent for inflammatory disease and / or autoimmune disease] The present invention also includes a preventive and / or therapeutic agent for inflammatory disease and / or autoimmune disease, which contains a miR-455-3p inhibitor as an active ingredient. The miR-455-3p inhibitor is not particularly limited as long as it inhibits the expression and / or function of miR-455-3p in a living body, but the miR-455-3p inhibitor is preferably at least one selected from the group consisting of a nucleic acid oligo, a low molecular weight compound, and an Hsa-miR-455-3p antibody, more preferably a nucleic acid oligo, even more preferably an oligo consisting of natural and non-natural RNA or DNA, and particularly preferably an antisense oligo of miR-455-3p. The nucleic acid oligo is preferably modified for the purpose of suppressing degradation when administered to a living body and / or for the purpose of specifically binding to a disease site.

[0034] The "nucleic acid oligo" in the present invention is an oligo consisting of natural or non-natural RNA or DNA, and means a nucleic acid oligomer that controls the function of miR-455-3p. The nucleic acid oligo in the present invention includes siRNA, shRNA, antisense nucleic acid, decoy nucleic acid, and nucleic acid aptamer.

[0035] The nucleic acid oligo in the present invention is preferably siRNA or shRNA, more preferably siRNA. siRNA means double-stranded RNA consisting of a short chain that is not toxic to cells, and can be, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. shRNA is RNA in which a single-stranded RNA forms a double strand via a hairpin structure.

[0036] siRNA and shRNA do not need to be completely identical to a target gene, but have at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology.

[0037] The double-stranded RNA portion in which RNAs in siRNA and shRNA are paired is not limited to being completely paired, and may contain unpaired portions due to mismatches (corresponding bases are not complementary), bulges (there is no corresponding base in one strand), etc. In the present invention, the double-stranded RNA region in which RNAs are paired in dsRNA may contain both bulges and mismatches.

[0038] The preventive and / or therapeutic agent (drug) for inflammatory disease and / or autoimmune disease in the present invention may contain pharmacy-acceptable materials such as preservatives and stabilizers. Pharmaceutically acceptable materials may be materials that have a preventive and / or therapeutic effect for the above-mentioned inflammatory disease and / or autoimmune disease by themselves, or materials that do not have the therapeutic effect, and refer to pharmacy-acceptable materials that can be administered together with the above-mentioned therapeutic agent. In addition, materials that do not have a therapeutic effect but have a synergistic effect or an additive stabilizing effect when used in combination with the preventive and / or therapeutic agent for inflammatory disease and / or autoimmune disease may also be used.

[0039] Examples of pharma- ceutical acceptable materials include sterilized water, physiological saline, preservatives, stabilizers, excipients, buffers, antiseptics, surfactants, chelating agents (EDTA, etc.), binders, and the like.

[0040] When the drug of the present invention is prepared as an aqueous solution for injection, it may be used in combination with, for example, physiological saline, an isotonic solution containing glucose or other auxiliary drugs (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride), a suitable solubilizing agent, for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc. If desired, it may further contain a diluent, a solubilizing agent, a pH adjuster, a soothing agent, a sulfur-containing reducing agent, an antioxidant, etc.

[0041] If necessary, the drug of the present invention can be encapsulated in a microcapsule (such as a microcapsule of hydroxymethylcellulose, gelatin, or poly[methyl methacrylic acid]) or can be made into a colloid drug delivery system (such as a liposome, albumin microsphere, microemulsion, nanoparticle, or nanocapsule). Furthermore, methods for making drugs into sustained-release drugs are also known and can be applied to the present invention. The pharmaceutically acceptable carriers used are appropriately selected from the above or in combination depending on the dosage form, but are not limited thereto.

[0042] When the prophylactic and / or therapeutic agent for inflammatory and / or autoimmune diseases of the present invention is used as a human medicine, it is possible to administer the substance directly to a patient, or to administer the substance after formulating the substance by a known pharmaceutical method. When formulating the substance, the above-mentioned pharmacopoeia-acceptable materials may be added.

[0043] All the drugs in the present invention can be administered in the form of pharmaceuticals, and can be administered orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric-coated drug, etc. can be selected, and the administration method can be appropriately selected according to the age and symptoms of the patient. The effective dosage is selected in the range of 0.000001 mg to 1 g per kg of body weight per administration. Alternatively, the dosage can be selected from 0.00001 to 100 mg, preferably 0.0001 to 50 mg per patient.

[0044] The present invention also includes a method for preventing and / or treating inflammatory diseases and / or autoimmune diseases using a miR-455-3p inhibitor. For specific explanations of the miR-455-3p inhibitor, inflammatory diseases, and autoimmune diseases, the explanations in the section on the preventive and / or therapeutic agent for inflammatory diseases and / or autoimmune diseases of the present invention described above can be applied. EXAMPLES

[0045] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0046] [Analysis of miRNAs in conjunctival epithelium related to the pathology of Stevens-Johnson syndrome (SJS)] 1. Comprehensive miRNA analysis using microarrays Conjunctival epithelium was isolated using dispase from conjunctival tissue excised during ocular surface reconstruction surgery from SJS patients, and miRNA was extracted according to the protocol of a commercially available reagent (miRNeasy mini kit (Qiagen)). Comprehensive miRNA analysis was then performed using GeneChip miRNA 4.0 Array (Affimetrix). As a control, conjunctival epithelium isolated from conjunctival tissue excised during ocular surface reconstruction surgery from a patient with conjunctivochalasis was used. The number of SJS patients and controls analyzed was n=3 for each.

[0047] Comprehensive miRNA analysis showed that, of the approximately 30,000 miRNAs, the expression of 13 miRNAs shown in Table 1 was reduced to 1 / 5 or less than that of controls in SJS patients (ANOVA p-value<0.05). In addition, the expression of 52 miRNAs shown in Table 2 was increased to 5 times or more than that of controls in SJS patients (ANOVA p-value<0.05). The fold change values ​​in the table are the average of each expression signal of the three SJS samples divided by the average of each expression signal of the three control samples.

[0048] [Table 1]

[0049] [Table 2-1]

[0050] [Table 2-2]

[0051] 2. Quantitative PCR Quantitative PCR was performed according to standard methods for multiple miRNAs that showed significant differences in expression between SJS patients and controls in the comprehensive miRNA analysis. The miRNAs analyzed were mmu-miR-31, odi-miR-31, ola-miR-31, crm-miR-72, bma-miR-72, bfl-miR-31, oan-miR-31, xtr-miR-31b, ggo-miR-125a, mml-miR-7193-5p, mmu-miR-455-3p, hsa-miR-1285, hsa-miR-4484, mmu-miR-455, mmu-miR-204, hsa-miR-193b, and gga-miR-3535. Conjunctival epithelial samples from SJS patients (n=6) and conjunctival epithelial samples from conjunctival laxity patients (n=7) as controls. The results are shown in Figure 1.

[0052] As shown in Figure 1, the expression of miR-31, miR-72, and miR-455-3p was significantly increased in SJS patients. On the other hand, the expression of miR-125a was significantly decreased in SJS patients. These miRNAs were thought to be useful as indicators for determining the possibility of SJS, severity, risk of onset and / or aggravation, efficacy of treatment, etc. Below, quantitative PCR tests were performed on these using a human miRNA measurement system.

[0053] 3.Quantitative PCR (human) For the miRNAs for which significant differences in expression were found between SJS patients and controls in the above test, expression was confirmed using a human quantitative PCR system. Since there is no human probe for quantitative PCR analysis of miR-72, quantitative PCR was performed for hsa-miR-31, hsa-miR-455-3p, and hsa-miR-125a. The results are shown in Figure 2.

[0054] As shown in Figure 2, it was confirmed that hsa-miR-31 and hsa-miR-455-3p were significantly more strongly expressed in the conjunctival epithelium of SJS patients compared to controls. Expression of hsa-miR-125a was significantly decreased in the conjunctival epithelium of SJS patients compared to controls. These miRNAs are considered to be useful indicators for determining the susceptibility, severity, and treatment effect of SJS.

[0055] [Analysis of miR-455-3p] 1. Inhibition test To confirm whether miR-455-3p can be a target for disease treatment, we performed a suppression test by gene transfer of an miR-455-3p inhibitor into primary cultured normal human conjunctival epithelial cells (PHCjE).

[0056] Primary normal human conjunctival epithelial cells were cultured at 5 x 10 5 The cells were seeded at 12-well plate at 200 cells / well. 50 μL of Opti-MEM (Gibco) and 2 μL of Lipofectamin were added to one well, and 50 μL of Opti-MEM and 72 pmol of miR-455-3p inhibitor (mirVana (registered trademark) miRNA inhibitor) were added to another well. The liquids in both wells were then mixed and left to stand at room temperature for 15 minutes to form a complex between the inhibitor and Lipofectamin. 100 μL of the complex was added to each well (Cnt-PR 0.5 mL / well) of the primary normal human cultured conjunctival epithelial cells and mixed. After 24 hours of culture under 37°C and 5% CO2 conditions, the medium was replaced, and the sample was collected after further culture for 6 hours. It was confirmed by PCR that the expression of miR-455-3p was suppressed.

[0057] Next, a comprehensive gene expression analysis was performed using cultured human conjunctival epithelial cells in which the expression of miR-455-3p was suppressed. Specifically, gene expression profiles were examined using high-density oligonucleotide probe arrays (GeneChip®, HumanGene1.0 ST array (Affymetrix)). Total RNA was extracted using a Qiagen RNeasy kit (Qiagen, Valencia, CA). Approximately 764,885 probe sets covering more than 28,869 genes were used. The Affymetrix protocol was followed throughout the process. Scanned microarray images were acquired with the default settings of a GeneChip Scanner 3000 7G (Affymetrix). Images were visually inspected to detect hybridization artifacts. The results are shown in Tables 3 and 4. Table 3 shows genes whose expression was reduced to one-third or less of the control in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed, and Table 4 shows genes whose expression was increased to three times or more of the control.

[0058] [Table 3-1]

[0059] [Table 3-2]

[0060] [Table 3-3]

[0061] [Table 3-4]

[0062] [Table 3-5]

[0063] [Table 4-1]

[0064] [Table 4-2]

[0065] The above-mentioned comprehensive gene expression analysis showed that the expression of genes was significantly decreased in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed (Table 3). Among these genes, which are known to be involved in inflammation, ANKRD1, CXCL8, CXCL2, GEM, PTGS2, RNASE8, IL6, CXCL1, CCL20, and KPRP were subjected to quantitative PCR. The results are shown in Figure 3.

[0066] As shown in Figure 3, in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed, the gene expression of ANKRD1, CXCL8, CXCL2, GEM, PTGS2, RNASE8, IL6, CXCL1, CCL20, and KPRP was indeed reduced compared to control cells, as confirmed by quantitative PCR. This result suggests that suppressing miR-455-3p expression may be effective in preventing and treating inflammation.

[0067] The above-mentioned comprehensive gene expression analysis showed that the expression of genes was significantly increased in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed (Table 4). Among these genes, which are known to be involved in inflammation, IFI44L, SLITRK6, TNFSF10, OAS2, MX1, and OAS1 were subjected to quantitative PCR. The results of quantitative PCR for SLITRK6 are shown in Figure 4.

[0068] As shown in Figure 4, quantitative PCR confirmed that SLITRK6 gene expression was significantly increased in human cultured conjunctival epithelial cells in which miR-455-3p expression was suppressed, compared to control cells. However, no significant increase in expression was observed for other genes, IFI44L, TNFSF10, OAS2, MX1, and OAS1.

[0069] 2. Expression enhancement test (Mimic test) To confirm the function of miR-455-3p, we performed a test to enhance the expression of miR-455-3p in primary normal human cultured conjunctival epithelial cells (PHCjE).

[0070] A test for enhancing expression of miR-455-3p mimic (mirVana® miRNA mimic) was carried out on primary normal human cultured conjunctival epithelial cells (PHCjE). Primary normal human cultured conjunctival epithelial cells were cultured in Cnt-PR medium at 5 × 10 5 The cells / well were seeded on a 12-well plate. 50 μL of Opti-MEM (Gibco) and 2 μL of Lipofectamin were added to one well, and 50 μL of Opti-MEM and 18 pmol of miR-455-3p mimic (mirVana (registered trademark) miRNA mimic) were added to another well, and then the liquids in both wells were mixed and left to stand at room temperature for 15 minutes to form a siRNA and Lipofectamin complex. 100 μL of miR-455-3p mimic was added to each well (Cnt-PR 0.5 mL / well) of normal human cultured conjunctival epithelial cells and mixed. After 24 hours of culture under 37 ° C, 5% CO2 conditions, the medium was replaced, and the sample was collected after further culture for 6 hours. Enhancement of miR-455-3p expression was confirmed by PCR.

[0071] Next, we performed a comprehensive gene expression analysis using cultured human conjunctival epithelial cells in which the expression of miR-455-3p was enhanced. The specific method of the comprehensive gene expression analysis was the same as that used in the inhibition test described above. As a result, we found that the expression of PPP1R3C, CTGF, CCL20, CXCL2, PTGS2, and CXCL8, which are known to be involved in inflammation, was enhanced compared to the control. These results were also confirmed by quantitative PCR (Figure 5).

[0072] 3. Comprehensive analysis of gene expression in conjunctival epithelial cells of SJS patients Comprehensive gene expression analysis was performed on conjunctival epithelial cells isolated from conjunctival tissue excised during ocular surface reconstruction surgery from SJS patients. The results were compared with the results of comprehensive gene expression analysis of cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed by treatment with an inhibitor of miR-455-3p, as shown in Table 3 above. Among the genes whose expression was increased in conjunctival epithelial cells from SJS patients compared to controls, the genes whose expression was significantly decreased in cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed are listed in Table 5.

[0073] [Table 5]

[0074] Of the genes shown in Table 5, quantitative PCR was performed on IL1RL1, CTGF, PPP1R3C, ADM, and SERPINB2 using cultured human conjunctival epithelial cells in which miR-455-3p expression was suppressed. The results are shown in Figure 6.

[0075] As shown in Figure 6, quantitative PCR confirmed that gene expression of IL1RL1, CTGF, PPP1R3C, ADM, and SERPINB2P was indeed decreased in human cultured conjunctival epithelial cells in which miR-455-3p expression was suppressed, compared to control cells. By suppressing miR-455-3p expression, expression was increased in conjunctival epithelial cells of SJS patients, and the expression of genes involved in inflammation was also reduced. This suggests that inhibiting miR-455-3p may be effective in treating SJS.

[0076] 4. Mouse Contact Dermatitis Model We analyzed the dynamics of genes whose expression is suppressed by miR-455-3p inhibition in tissues of a mouse contact dermatitis model.

[0077] 1% trinitrochlorobenzene (TNCB) was applied to the abdomen and chest of Balb / c mice. On the 7th day, 1% TNCB or solvent alone (acetone: olive oil = 4:1) was applied to the ears of the mice. The next day (8th day), the ears were collected and immersed in RNLater, removed after 6 hours, and stored at -80°C. The ears were then crushed with dry ice, dissolved in Trizol, and ethanol precipitated to extract RNA. Q-PCR was performed using the SybrGreen method with StepOne Plus.

[0078] It was found that the expression of ANKRD1, CXCL2, PTGS2, IL-6, CXCL1, CCL20, IL1RL1, and SERPINB2 was increased in the tissues of the mouse contact dermatitis model. The results are shown in Figure 7. It was found that the expression of these genes was suppressed by inhibition of miR-455-3p in the above-mentioned test using human cultured conjunctival epithelial cells. Therefore, it was suggested that substances that inhibit miR-455-3p may have a preventive or therapeutic effect on inflammation such as dermatitis, and even autoimmune diseases.

[0079] [Analysis of blood (plasma) miRNAs related to the pathology of Stevens-Johnson syndrome (SJS)] 1. Comprehensive miRNA analysis using microarrays Plasma was separated from the blood of SJS patients, and miRNA was extracted according to the protocol of a commercially available reagent (miRNeasy Serum / Plasma Kit (QIAGEN)). Comprehensive miRNA analysis was then performed using the GeneChip miRNA 4.0 Array (Affimetrix). Blood from healthy volunteers was used as a control.

[0080] Comprehensive miRNA analysis revealed that miR-628-3p and miR-548ac were significantly increased in the blood of SJS patients compared to controls. Quantitative PCR also confirmed that miR-628-3p and miR-548ac were expressed in SJS patients (data not shown).

[0081] 2. Analysis of the correlation between the severity of SJS and the expression levels of miR-628-3p and miR-548ac in the patient's blood The amounts of miR-628-3p and miR-548ac in the blood of healthy individuals, patients with mild SJS, and patients with severe SJS were compared. The results are shown in Tables 6 and 7.

[0082] [Table 6]

[0083] [Table 7]

[0084] As shown in Tables 6 and 7, there is a correlation between the severity of SJS and the expression levels of miR-628-3p and miR-548ac in the patient's blood, and blood miR-628-3p and miR-548ac can be used as indicators for determining the severity of SJS. Therefore, they are considered to be useful as indicators for determining the possibility of developing SJS, its severity, and the effectiveness of treatment. [Industrial Applicability]

[0085] According to the present invention, it is possible to determine the possibility of contracting inflammatory diseases such as Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, and other autoimmune diseases, the risk of developing and / or becoming severe, and the effectiveness of treatment. Specifically, microRNAs whose expression changes specifically in SJS disease are medically useful biological indicators, and microRNAs that correlate with disease severity enable evaluation of treatment effectiveness. The present invention leads to the provision of a new medical technology, namely, the establishment of a non-invasive diagnostic technique by disease-specific microRNA expression profiling targeting the conjunctival epithelial tissue of intractable ocular surface diseases and the use of it in selecting treatment methods. Furthermore, the present invention can also provide a new method of treating inflammatory diseases such as Stevens-Johnson syndrome (SJS), which is an intractable ocular surface disease, and other autoimmune diseases.

Claims

1. The present invention uses at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-193b-5p, miR-204-3p and miR-3535 in conjunctival epithelial tissue derived from a subject, and miR-628-3p and miR-548ac in plasma derived from a subject as an index; When the expression level of at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac is increased compared to a control, and / or when the expression level of at least one microRNA selected from the group consisting of miR-125a-5p, miR-193b-5p, miR-204-3p, and miR-3535 is decreased compared to a control, the possibility of suffering from Stevens-Johnson syndrome (SJS) is indicated. wherein, as the control, conjunctival epithelial tissue derived from a normal subject is used for at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac, and plasma derived from a normal subject is used for at least one microRNA selected from the group consisting of miR-628-3p and miR-548ac.

2. A method for detecting a subject's conjunctival epithelium in a subject using at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-193b-5p, miR-204-3p, and miR-3535 in the conjunctival epithelium, and miR-628-3p and miR-548ac in the subject's plasma, When the expression level of at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac is increased compared to a control, and / or when the expression level of at least one microRNA selected from the group consisting of miR-125a-5p, miR-193b-5p, miR-204-3p, and miR-3535 is decreased compared to a control, Stevens-Johnson phenotype in the subject is determined based on the degree of increase and / or decrease. and determining the severity of Stevens-Johnson syndrome (SJS) in a subject, wherein the control comprises conjunctival epithelial tissue from a normal subject for at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac, and plasma from a normal subject for at least one microRNA selected from the group consisting of miR-628-3p and miR-548ac.

3. A method for detecting a subject's conjunctival epithelium in a subject using at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-125a-5p, miR-72-5p, miR-193b-5p, miR-204-3p, and miR-3535 in the conjunctival epithelium, and miR-628-3p and miR-548ac in the subject's plasma, When the expression level of at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac is increased compared to a control, and / or when the expression level of at least one microRNA selected from the group consisting of miR-125a-5p, miR-193b-5p, miR-204-3p, and miR-3535 is decreased compared to a control, the occurrence of Stevens-Johnson syndrome (SJS) in a subject is indicated depending on the degree of the increase and / or decrease. and determining the risk of onset and / or aggravation of Stevens-Johnson syndrome (SJS) in a subject, wherein as the control, conjunctival epithelial tissue derived from a normal subject is used for at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac, and plasma derived from a normal subject is used for at least one microRNA selected from the group consisting of miR-628-3p and miR-548ac.

4. A method for detecting a patient having Stevens-Johnson syndrome (SJS), comprising: detecting a patient having Stevens-Johnson syndrome (SJS) and a subject ... A method for assisting in assessment of the effectiveness of treatment of Stevens-Johnson syndrome (SJS) in a subject, comprising: determining that the treatment is effective when the expression level of at least one microRNA selected from the group consisting of miR-455-3p, miR-31-5p, miR-72-5p, miR-628-3p, and miR-548ac after treatment is decreased compared to before treatment, and / or determining that the expression level of at least one microRNA selected from the group consisting of miR-125a-5p, miR-193b-5p, miR-204-3p, and miR-3535 after treatment is increased compared to before treatment; determining that the treatment is ineffective when there is no change; and determining that the condition has worsened when the opposite change is observed.

5. A preventive and / or therapeutic agent for Stevens-Johnson syndrome (SJS), comprising an antisense oligonucleotide of miR-455-3p as an active ingredient.

6. The preventive and / or therapeutic agent according to claim 6 , wherein the antisense oligonucleotide is modified.

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