WO2013155980A1 - Microarn associé à une maladie auto-immune et son utilisation - Google Patents

Microarn associé à une maladie auto-immune et son utilisation Download PDF

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WO2013155980A1
WO2013155980A1 PCT/CN2013/074423 CN2013074423W WO2013155980A1 WO 2013155980 A1 WO2013155980 A1 WO 2013155980A1 CN 2013074423 W CN2013074423 W CN 2013074423W WO 2013155980 A1 WO2013155980 A1 WO 2013155980A1
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mirna
mir
seq
expression
microrna
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钱友存
沈南
朱书
潘文
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中国科学院上海生命科学研究院
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention is in the field of biotechnology and medicine, and in particular, the present invention relates to microRNAs related to autoimmune diseases and uses thereof. Background technique
  • Autoimmune diseases are diseases caused by the body's immune response to autoantigens and cause damage to their own tissues. It is generally believed that abnormal inflammatory reactions occur in tissue damage caused by various autoimmune diseases, such as rheumatoid arthritis ( Rheumatoid arthriti s , RA), mult iple scleros is (MS) and systemic lupus erythematosus (SLE).
  • RA rheumatoid arthritis
  • MS mult iple scleros
  • SLE systemic lupus erythematosus
  • the main features of RA are synovial inflammation accompanied by articular bone and cartilage damage.
  • MS is an inflammatory lesion characterized by demyelination of the central nervous system.
  • SLE is a deposition of immune complexes. Chronic activation of the immune system causes multiple systemic autoimmune diseases, often accompanied by nephritis.
  • Collagen-induced rheumatoid arthritis (CIA) and experimental allergic encephalomyelitis (EAE) are mouse models of autoimmune diseases induced by autoantigens. , corresponding to RA and MS respectively.
  • the leg L/r mouse is a mouse model that spontaneously produces SLE.
  • MicroRNAs are a class of single-stranded RNA molecules of approximately 18-26 bases in length that are found in higher eukaryotes. It can specifically bind to target sites on some mRNAs through base pairing principles, causing target mRNA degradation or translational inhibition, and then regulating target genes at the post-transcriptional level.
  • the microRNA is derived from a long-chain RNA initial transcript (Pri_miRNA) of about 1000 bp in length, and the Pri_miRNA molecule is cleaved by the Drosha enzyme in the nucleus to form a miRNA precursor having a stem-loop structure of about 60-80 nt in length.
  • a miRNA precursor having a stem-loop structure of about 60-80 nt in length.
  • the miRNA is transported to the cytoplasm, it is further cleaved into a double-stranded miRNA of about 18_26 nt.
  • the mature miRNA enters the RNA-induced si lencing complex (RISC), which is completely or incompletely paired with the complementary mRNA, degrades the target mRNA or represses its expression.
  • RISC RNA-induced si lencing complex
  • microRNAs account for a small proportion of total RNA in cells, because it can efficiently regulate all mRNAs with target sites, microRNAs cannot play a role in the development of organisms and even the occurrence and development of tumors. Small vision.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective one or more active ingredients selected from the group consisting of: (a) MicroRNAs of the miRNA-23 family, the microRNAs of the miRNA-23 family include: miRNA_23 or a modified miRNA-23 derivative, and the core sequence is UCCAUAU, the length is 18_26nt, and the function is the same or substantially the same as miRNA-23b MicroRNA or modified miRNA derivative;
  • a precursor miRNA capable of being processed into a microRNA as described in (a) in a host (b) a polynucleotide which can be transcribed by a host (b) a precursor miRNA as described, and processed to form the microRNA described in (a);
  • the miRNA-23b is miRNA-23b_3p.
  • the core sequence described in (a) refers to the nucleotide sequence of positions 2-8 of the microRNA; and/or the "function is identical or substantially identical to miRNA-23b" means retention 40% of miRNA-23b_3p, and 500% of autoimmune function.
  • the inhibition of autoimmunity refers to inhibition of inflammatory factor-mediated signaling pathways and gene expression.
  • the microRNA is derived from a human or a non-human mammal; preferably, the non-human mammal is a rat or a mouse.
  • the core sequence described in (a) refers to the nucleotide sequence of positions 2-8 of the microRNA.
  • the "function is identical or substantially identical to miRNA-23b" as described in (a) means that 40%, 50%, 60%, 70%, 80%, 90% of miRNA-23b_3p is retained. , ⁇ 100%, and 500% of the function of inhibiting autoimmunity.
  • the host described in (b) is: human or rodent (e.g., rat, mouse).
  • the pharmaceutical composition is in the form of an injection, an oral preparation (tablet, capsule, oral solution), a transdermal agent, and a sustained release agent.
  • the pharmaceutical composition further has a miRNA selected from the group consisting of miRNA-23a-5p and miRNA_23b_5p.
  • the precursor miRNA described in (b) further comprises a sequence corresponding to the miRNA selected from the group consisting of miRNA -23a-5p and miRNA-23b-5p.
  • the miRNA-23 is a miRNA selected from the group consisting of miR A-23a_3p, miRNA_23b_3p, miRNA_23c, miRNA_23a_5p, or miRNA_23b_5p.
  • the miRNA-23 is a miRNA selected from the group consisting of miR A-23a_3p, miRNA_23b_3p, or miRNA_23c.
  • the miRNA-23 described in (a) comprises miRNA-23a_3p having the sequence of SEQ ID NO.: 1, miRNA-23b_3p as shown by SEQ ID NO.: 2, or SEQ. ID NO.: miRNA_23c shown in 3.
  • the modified miRNA derivative has a modification selected from one or more modifications of the group consisting of: a sugar group modification of a nucleotide; a modification of a linkage between nucleotides, Cholesterol modification, lock nucleotide modification, peptide modification, lipid modification, halogen modification, hydrocarbyl modification, and nucleic acid modification.
  • the glycosyl modification of the nucleotide comprises a 2-0-methyl glycosyl modification, a 2-0-methoxyethyl ester glycosyl modification, a 2-0-fluorenyl sugar a base modification, a 2-fluoro sugar group modification, a sugar ring modification, a locked nucleotide modification; and/or a modification of the linkage between the nucleotides includes a phosphorothioate modification, a phosphorylation modification; / or the nucleic acid modifications described include "TT" modifications.
  • the modified miRNA derivative described in (a) is a compound monomer having the structure of formula I or a multimer thereof:
  • Each X is a microRNA as described in (a);
  • Each Y is independently a modification that promotes the stability of administration of microRNAs
  • n is a 1-100 (preferably 1-20) positive integer (preferably n is 1, 2, 3, 4 or 5);
  • n is a 1-1000 (preferably 1-200) positive integer
  • Each "-" indicates a linker, a chemical bond, or a covalent bond.
  • the linker is a nucleic acid sequence of 1-10 bases in length.
  • the Y includes, but is not limited to, cholesterol, steroids, alcohols, alcohols, organic acids, fatty acids, esters, monosaccharides, polysaccharides, amino acids, polypeptides, single nucleotides, polynucleotides.
  • polynucleotide described in (c) has the structure represented by Formula I:
  • Seq is forward processed into the microRNA nucleotide sequence in the host; Seq reverses
  • Seq is forward to a substantially complementary or fully complementary nucleotide sequence
  • X is a spacer sequence located between the Seq forward and Seq reverse, and the spacer sequence is not complementary to the Seq forward and Seq reverse
  • Formula II The structure shown forms a secondary structure of the formula ⁇ after transfer into the host cell:
  • the agonist of miRNA-23 is selected from the group consisting of a substance that promotes expression of miRNA-23, a substance that enhances miRNA-23 activity, a substance that inhibits IL-17 expression, and a substance that inhibits IL-17 activity.
  • the substance which inhibits IL-17 activity includes an antibody against IL-17.
  • the pharmaceutically acceptable carrier is selected from the group consisting of water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanocoagulation Glue, or a combination thereof.
  • the expression vector described in (d) comprises: a viral vector and a non-viral vector.
  • the viral vector is: an adenovirus vector, an adeno-associated virus vector, a retroviral vector, or a lentiviral vector.
  • the non-viral vector is: a plasmid or a bacterium.
  • the sequence of the precursor miRNA is as shown in SEQ ID NO.: 4, SEQ ID NO.: 5, or SEQ ID NO.: 6.
  • the pharmaceutical composition is for treating an autoimmune response disease.
  • an active ingredient wherein the active ingredient is selected from the group consisting of:
  • MicroRNAs of the miRNA-23 family include: miRNA_23 or a modified miRNA-23 derivative, and a core sequence of UCCAUAU, a length of 18_26nt, and a function of the same or substantially the same as miRNA_23b RNA or a modified miRNA derivative;
  • a precursor miRNA capable of being processed into a microRNA described in (a) in a host (c) a polynucleotide, said polynucleoside The acid can be transcribed by the host to form the precursor miRNA described in (b) and processed to form the microRNA described in (a);
  • an agonist of the microRNA described in (a) the active ingredient is used for preparation to inhibit itself
  • An inhibitor of an immune response a medicament for preparing a disease for
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple cerebrosostosis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, scleroderma, chronic ulcer Enteritis, chronic atrophic gastritis, chronic lymphatic thyroiditis, insulin-dependent diabetes mellitus, Crohn's disease, Sjogren's syndrome.
  • a third aspect of the invention there is provided a method of preventing or treating an autoimmune response disease, wherein the pharmaceutical composition of the first aspect of the invention is administered to a subject in need thereof.
  • the object comprises a person.
  • a miRNA-23 antagonist for the preparation of a modulator which upregulates an autoimmune response.
  • the miRNA-23 is miRNA_23b_3p.
  • a method of screening an active ingredient for inhibiting autoimmunity comprising the steps of:
  • the method further comprises the step (c): further determining the inhibitory effect on the inflammatory factor-mediated signaling pathway or gene expression of the autoimmune-inhibiting active component obtained in the step (b) .
  • a TAB2 or TAB3 inhibitor for the manufacture of a medicament for inhibiting an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple cerebrosostosis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, scleroderma, chronic ulcer Enteritis, chronic atrophic gastritis, chronic lymphatic thyroiditis, insulin-dependent diabetes mellitus, Crohn's disease, Sjogren's syndrome.
  • a method for detecting an autoimmune response disease comprising the steps of: separately detecting a expression level of a test sample and a negative control sample miR-23b, if compared with a negative control sample, A decrease in the expression level of miR-23b in the sample to be tested is a potential sample of an autoimmune response disease.
  • the method further comprises the steps of: separately detecting the expression levels of the test sample and the negative control sample miR-30a, and/or miR-146a, and/or miR-214, if compared with the negative control sample In contrast, the expression level of miR-30a in the test sample is decreased, and/or the expression level of miR_146a is increased, and/or the expression level of miR-214 is increased, which is a potential autoimmune disease disease disease sample.
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple cerebellar sclerosis, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, chronic ulcerative enteritis , chronic atrophic gastritis, chronic lymphatic thyroiditis, insulin-dependent diabetes mellitus, Crohn's disease, Sjogren's syndrome.
  • the reduction means that the level of decrease in the expression level of the corresponding miRNA is lower than the negative control sample by a low amplitude of 1100%%, preferably 20%, preferably 50%. %, preferably 80%, optimally 100%.
  • the increase means that the expression level of the corresponding miRNA is increased by 10%, preferably 20%, preferably 50%, more preferably 80%, compared to the negative control sample. Best 100%.
  • the autoimmune response disorder is one or more selected from the group consisting of rheumatic arthritis, multiple sclerosis, or systemic lupus erythematosus.
  • a microRNA, miR-23b which is used for the preparation of a reagent or kit for detecting an autoimmune disease.
  • the above reagent is a chip, a primer, or a probe.
  • the kit described above includes instructions for use.
  • the instructions for use include a description of the steps: respectively detecting the expression level of the test sample and the negative control sample miR-23b, if the expression level of the miR-23b of the sample to be tested is decreased compared with the negative control sample, It is a sample of potential autoimmune diseases.
  • the miR-23b is miRNA-23b_3p. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Limited to the length, no longer one by one. DRAWINGS
  • Figure 1 shows that the expression of miR-23b_3p is down-regulated in local inflammatory tissues of autoimmune diseases; wherein, Figure la shows the up-regulated expression of inflammatory tissues in human autoimmune diseases and related mouse autoimmune disease models.
  • Top 15 and down-regulated miRNAs in the top 15 including comparison of four rheumatoid arthritis patients with two patients with osteoarthritis and two normal individuals (accidental injuries) of synovial tissue, comparing two collagen-induced A mouse sample of arthritis (CIA) and two untreated control mouse samples (joint tissue of six mice mixed in each sample), and eight undiagnosed erythema diagnosed with lupus nephritis Kidney biopsy specimens from patients with lupus were compared with paracancerous tissues from four patients with renal cell carcinoma, two MRL/lpr mice (kidney tissue mixed with six mice per sample) and two normal mouse samples (each sample) Experimental autoimmune encephalomyelitis with two M0G immunizations for 16 days of experimental autoimmune
  • Figure lc shows that the expression of miR-23b_3p in the inflammatory tissue of patients with rheumatoid arthritis is significantly downregulated in the synovial tissue of rheumatoid arthritis patients (in patients with osteoarthritis and traumatic controls);
  • d shows that the renal biopsy specimens of patients with lupus erythematosus (the renal adjacent renal tissue of patients with renal cancer are normal controls), the expression of miR-23b_3p in the inflammatory tissue of patients with lupus erythematosus is significantly down-regulated;
  • Figure le_ shows that in type II Collagen-immunized DBA/ 1 mouse joints (mixed with 6 mouse joint samples) miR-23b expression was significantly down-regulated;
  • Medium Figure If showed that mixed samples of miR- in MRL/lpr mouse kidney tissue at 10 and 40 weeks of age
  • the expression of 23b_3p was significantly down-regulated (Fig. If), and lg showed that the expression of miR-23b in
  • Figure 2 shows that IL-17 mediates down-regulation of miR-23b_3p in inflammatory autoimmune diseases;
  • Figure 2a shows joint slip in rheumatoid arthritis patients, osteoarthritis patients, and normal controls (accidental injuries)
  • miR-23b_3p expression was negatively correlated with IL-17 expression;
  • Figure 2b shows miR-23b_3p expression and IL-17 expression in renal biopsy specimens of patients with lupus erythematosus and adjacent tissues of renal cancer patients Negative correlation;
  • Figure 2c shows an increase in the expression of inflammatory factors in synovial tissue from patients with rheumatoid arthritis, with samples from 17 patients with rheumatoid arthritis, 19 patients with osteoarthritis and 3 normal controls ( Joint synovial tissue of accidental injury;
  • Figure 2d shows increased expression of inflammatory factors in kidney tissue of patients with lupus erythematosus, including samples from 18 renal biopsy samples from patients with lupus erythematos
  • FIG. 2f mouse primary cultured kidney cells
  • TNF a 10 ng/ml
  • IL-1 ⁇ 10 ng/ml
  • IL-17 50 ng/ml
  • IFN 50 ng/ml
  • Figure 2g shows IL-17 regulates miR-23 expression by NF_ ⁇ B, respectively, WT, Act l-z - and IKK mouse embryonic fibroblasts
  • IL-17 (50 ng/ml) stimulated miR-23b_3p levels;
  • Figure 2h shows that IL-17 regulates miR-23b_3p expression in vivo, and detects adenovirus infected with IL-17 (Ad_IL_17) and infected empty
  • Ad_IL_17 adenovirus infected with IL-17
  • Ad-EV adenovirus infected empty
  • the level of miR-23b in the mouse joint of the adenovirus of the vector (Ad-EV) was measured by qPCR as a positive control for the expression levels of IL-17 and KC (cxcl l).
  • Figure 3 shows that the miR-23 family is capable of inhibiting inflammatory factor-mediated signaling as well as gene expression;
  • Figure 3a shows HeLa cell blanks (Mocks) that were transfected with the miR-23b_3p mimetic and mock control at each specific time point, IL-17 (50 ng/ml), TNF a (10 ng/ml), IL-1 ⁇ (10 ng/ml) stimulation, collection of cell lysates for immunoblot analysis with anti-pI B , 1 B , p-p65 , p_p38 Antibodies to TAB2, TAB3, and -actin were tested, indicating that miR-23b_3p inhibits inflammatory factor-mediated signaling;
  • Figure 3b shows that HeLa cells were transfected with miR-23b_3p mimic and mock control, given at specific time points Mock, IL-17 (50 ng/ml), TNF a (10 ng/ml) and IL-1 ⁇ (10 ng/ml) stimulation
  • Fig. 3c shows in primary Renal epithelial cells were transfected with miR-23a-3p mimic and their mimetic controls, and stimulated with IL-17 (50 ng/ml), TNF a (10 ng/ml), IL_1 ⁇ (10 ng/ml) alone or in combination for 6 hours. Normalization was performed with reference to the housekeeping gene Rpl l3a to detect the expression of KC and IL-6.
  • Figure 4 shows that the miR-23 family can inhibit the onset of autoimmune diseases in a mouse model; wherein, Figure 4a shows that miR-23b_3p inhibits the onset of CIA, and the control group of ad hoc adenovirus is injected into the joint of DBA/1 mice, respectively.
  • Ad_EV carrying an adenovirus encoding miR-23b
  • Ad-sponge carrying an adenovirus encoding the miR-23b_3p adsorbate
  • Ad-sponge carrying an adenovirus encoding both miR_23b and its adsorbate
  • Figure 4c and Figure 4d show that miR-23b-3p inhibits spontaneous lupus nephritis in mice, and intravenously injects adenovirus (Ad-EV) every three weeks during 6 to 24 weeks of MRL/lpr mice, carrying the coding miR -23b adenovirus (Ad_23b) and adenovirus (Ad-sp) carrying miR-23b_3p adsorbate, 24 weeks old MRL mice were injected with Ad_EV as control, and the degree of damage of kidney tissue in the treated group was according to the experimental method. The criteria were graded (Fig.
  • Figure 4d shows that the 24-week-old mice treated in Figure 4c were made into H&E-stained kidney sections, and glomerular cell hyperplasia (upper row, middle row) and periphery were seen. Monocyte infiltrating fissures (lower row);
  • Figure 4e shows that induction of local inflammatory cytokines in the kidney is significantly inhibited in mice receiving Ad_23b adenovirus.
  • Figure 4f shows that miR-23b_3p inhibits the onset of EAE, and different adenoviruses are injected into the tail vein of c57 mice, including the control group with adenovirus (Ad-EV), adenovirus carrying miR-23b_3p (Ad_23b), and the coding miR -23b_3p
  • Adsorbent adenovirus (Ad_sp) and adenovirus (Ad-23b+sp) carrying both miR-23b_3p and its adsorbate, M0G was used to induce the onset of EAE in mice at the time indicated in the figure, and EAE was performed. Clinically score and calculate the incidence.
  • Figure 4g shows that miR-23a_3p inhibits the pathogenesis of EAE, and the c57 mouse tail vein injection control group is empty (Ad_EV), carrying the virus encoding miR-23a (Ad_23a), and M0G-induced mice are used at the time points indicated in the figure.
  • Ad_EV c57 mouse tail vein injection control group
  • Ad_23a carrying the virus encoding miR-23a
  • M0G-induced mice are used at the time points indicated in the figure.
  • the incidence of EAE the EAE clinical score, and calculate its incidence.
  • Figure 5 shows that the miR-23 family can effectively treat autoimmune diseases; wherein, Figure 5a shows that miR-23b_3p can effectively treat CIA, and that type 2 collagen is administered from DBA/1 mice 25 days after primary immunization/4 days after secondary immunization. After the onset of the disease, 50 nM/Agomir-miR-23b-3p and the control were injected intra-articularly every two weeks for 3 times, and the arthritis score (sem) was performed every 5 days, *P ⁇ 0.05; 5b showed that miR_23b_3p can effectively treat EAE, M0G immunized c57 mice after the onset, 11 days and 15 days of tail vein injection of ⁇ / only
  • Agomir-miR-23b-3p and the control were injected twice, and the encephalomyelitis score was performed daily (Shi s. e. m. ).
  • Figure 6 shows that miR-23b_3p controls multiple genes in the inflammatory factor pathway; wherein, Figure 6a shows that the human primary fibroblast-like synovium transfected with the miR-23b_3p mimic and the mock control was transfected by gene chip comparison analysis.
  • the differentially expressed genes of cells (FLS) and the associated TargetScan bioinformatics analysis found 11 possible inflammatory response-related genes regulated by miR-23b_3p, Rpl l3a as a negative control, gradient color and number representing 11 genes Relative expression levels in FLS cells transfected with miR-23b_3p mimic and transfected mock control;
  • Figure 6b shows specific 3' UTR reporter plasmid transfected into 293T cells, The results of luciferase activity assay were transfected with miR-23b_3p or control mimic (NC).
  • Figure 7 shows that in autoimmune diseases, TAB2 and TAB3 are targets of miR-23b_3p; wherein, Figure 7a shows immunoblotting in detecting synovial membranes in normal people, patients with osteoarthritis, and patients with rheumatoid arthritis.
  • the protein expression levels of TAB3, TAB2 and ⁇ were determined as ⁇ -actin as an internal reference;
  • Figure 7b shows that the level of miR-23b_3p in the above samples was negatively correlated with the protein expression levels of TAB3, TAB2 and IKK ⁇ ;
  • Immunoblotting method was used to detect the expression levels of ⁇ 3, ⁇ 2 and ⁇ in kidney specimens of patients with lupus erythematosus and its control group, using ⁇ -actin as an internal reference;
  • Figure 7d shows the level of miR-23b_3p and TAB3, TAB2 in the above samples.
  • Figure 7e_g shows that the expression levels of TAB2, TAB3 and ⁇ are up-regulated in the in situ inflammatory tissues of autoimmune disease mice by immunoblotting, including comparison of these three proteins.
  • Expression in DBA/1 mice after 30 weeks of type II collagen immunization and in untreated control mice Fig. 7e
  • Comparison Expression of these three proteins in kidney tissues of 10 weeks old, 40 week old MRL/lpr and control MRL mice Fig. 7f
  • Comparison of these three proteins in the spinal cord of M0G immunized 14 days and untreated control mice Expression Fig. 7g
  • these results show that these three proteins are significantly up-regulated in both human and mouse inflammatory tissues as targets for miR-23b_3p.
  • Figure 8 shows that inhibition of the expression of TAB2 and TAB3 can inhibit the development of autoimmune diseases; wherein, Figure 8a shows the siRNA sequences for screening TAB2 and TAB3; Figure 8b shows the effect of using shRNA to reduce the expression levels of TAB2 and TAB3; Figure 8c and Figure 8d It was shown that decreasing the expression levels of TAB2 and TAB3 inhibited the onset of CIA: Figure 8c shows that in DBA/1 mice immunized with type II collagen, lentiviral-mediated intravenous ligation of TAB2 and TAB3 expression was performed at the time indicated by the arrow.
  • FIG. 8d shows the results of H&E staining of the ankle section 50 days after the completion of the second immunization
  • Figure 8e shows that re-expression of TAB2 and TAB3 reverses the effect of miR-23b_3p on the expression of inflammatory cytokine genes
  • TAB2 and TAB3 plasmids transfected with miR-23 binding site mutations in human primary fibroblast-like synoviocytes (FLS) or Empty plasmids were infected with empty or miR_23b-encoding adenovirus and stimulated with TNF a (10 ng/ml) plus IL-17 (50 ng/ml) for 6 hours.
  • Figure 9 shows the method and effect of preparing an adenovirus overexpressing miR-23b or preparing an adenovirus overexpressing the miR-23b_3p adsorbate.
  • Figure 9a shows a schematic diagram of the preparation of an adenovirus overexpressing miR-23b (Ad_miR_23b) or an adenovirus (Ad-miR-23_sponge) overexpressing the miR-23b_3p adsorbate, Ad_miR_23b
  • Ad_miR_23b Ad_miR_23b
  • the sequence containing mmu-miR-23b was used as described in the experimental method; the sequence of a single fragment of the miR-23b_3p adsorbate is shown in Figure 9a.
  • Figure % shows the GFP-expressing fluorescent display of an adenovirus containing an empty plasmid (Ad-EV) containing an adenovirus encoding miR-23b (Ad_miR-23b) and an adenovirus containing an adsorbent encoding miR-23b_3p (Ad-miR-23) -sponge)
  • Ad-EV empty plasmid
  • Ad_miR-23b an adenovirus encoding miR-23b
  • Figures 9c and 9d show the detection of miR-23b_3p expression in specific tissues after intra-articular injection (Figure 9c) or after intravenous injection (Figure 9d), microRNA levels normalized to the expression of snoRNA202;
  • Figure 9e shows the use of miR- containing The 23b-3p binding site luciferase reporter plasmid (miR_23b reporter) detects the overexpression of miR_23b_3p or inhibits the inhibition of its site of action and the efficiency of de-inhibition.
  • miR_23b is generally down-regulated in local inflammatory tissues in a large number of microRNAs.
  • the present invention further finds that the introduction of miR-23 family members can inhibit the development of autoimmune diseases, including miR-23a, miR_23b, and miR_23c; for the autoimmune diseases that have already occurred, the miR-23 family members can be introduced. Significantly relieve the symptoms and progression of the disease.
  • the present invention also found that the cytokine IL-17 regulates the expression of miR-23b; miR_23b targets the genes such as TAB2 and TAB3 in the inflammatory factor signaling pathway; inhibition of the expression of TAB2 and TAB3 can inhibit the onset and progression of autoimmune diseases. .
  • the present invention has been completed on this basis. miRNA and its precursors
  • miRNA refers to a class of RNA molecules that are processed from transcripts that form miRNA precursors. Mature miRNAs typically have 18-26 nucleotides (nt) (more specifically about 19-22 nt), and miRNA molecules with other numbers of nucleotides are not excluded. miRNAs are usually detected by Northern blotting.
  • Human-derived miRNAs can be isolated from human cells.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • miRNAs can be extracted from precursor miRNAs (Pre- miRNAs), which can be folded into a stable stem-loop (hairpin) structure, generally having a length of 50- Between 100bp.
  • the precursor miRNA can be folded into a stable stem-loop structure, and the stem of the stem-loop structure comprises two sequences that are substantially complementary on both sides.
  • the precursor miRNA can be natural or synthetic.
  • the precursor miRNA can be cleaved to generate a miRNA that is substantially complementary to at least a portion of the sequence encoding the mRNA of the gene.
  • substantially complementary means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure).
  • At least 70% of the nucleotides of the two "substantially complementary" nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • a “stem loop” structure also referred to as a "hairpin” structure, refers to a nucleotide molecule that forms a secondary structure comprising a double-stranded region (stem), said double
  • the chain region is formed by two regions of the nucleotide molecule (on the same molecule), the two regions are flanked by double-stranded portions; it also includes at least one "loop” structure, including non-complementary nucleotide molecules , that is, a single-chain area. Even if the two regions of the nucleotide molecule are not completely complementary, the double-stranded portion of the nucleotide can remain in a double-stranded state.
  • insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself, however, the two regions may still be substantially complementary and are foreseeable Interaction occurs in the manner, forming a double-stranded region of the stem-loop structure.
  • Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid is capable of forming a stem-loop structure.
  • the miRNA of the present invention refers to: a microRNA of the miRNA-23 family, and the microRNA of the miRNA-23 family includes: miRNA-23 or a modified miRNA_23 derivative, and a core sequence of UCCAUAU, a length of 18_26nt, a function and a miRNA -23b The same or substantially the same microRNA or modified miRNA derivative.
  • the miRNA-23 family comprises: miRNA_23b, miRNA_23a, and miRNA-23c ; preferably, the nucleotide sequence of miRNA_23b is as shown in SEQ ID NO.: 2; the nucleotide sequence of miRNA_23a As shown in SEQ ID NO.: 1; the nucleotide sequence of miRNA-23c is shown in SEQ ID NO.: 3.
  • the microRNA is derived from a human or a non-human mammal; preferably, the non-human mammal is completely identical in the 23 family sequences of the rat, mouse, mouse and human.
  • the core sequence refers to the nucleotide sequence of positions 2-8 of the microRNA.
  • the "function is the same or substantially the same as miRNA-23b" means that 40%, 50%, 60%, 70%, 80%, 90% of the miRNA-23b_3p retains the autoimmune function.
  • miRNA derivatives in a broad sense may also include miRNA variants.
  • miRNA derivatives in a broad sense may also include miRNA variants.
  • One of ordinary skill in the art can modify miRNA-23 using a general method including, but not limited to, methylation modification, hydrocarbyl modification, glycosylation modification (e.g., 2-methoxy-glycosyl modification). , hydrocarbyl-glycosylation modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification).
  • the miRNAs of the invention also include: miR-30a, miR_146a, miR-214, and variants and derivatives thereof.
  • Polynucleotide construct includes: miR-30a, miR_146a, miR-214, and variants and derivatives thereof.
  • a polynucleotide construct which can be processed into a miRNA which can affect the expression of the corresponding mRNA can be designed, that is, the polynucleotide construct can be up-regulated in vivo.
  • the amount of miRNA Accordingly, the present invention provides an isolated polynucleotide (construct) which can be transcribed into a precursor miRNA by a human cell, which can be cleaved by a human cell And expressed as the miRNA.
  • polynucleotide construct comprises the structure of formula I:
  • Seq E ft is a nucleotide sequence which can be expressed in the cell as the miRNA-23, and Seq is reversed to a nucleotide sequence substantially complementary to Seq E ft ; or, Seq ⁇ is expressed in a cell
  • the nucleotide sequence of the miRNA, Seq is a nucleotide sequence substantially complementary to Seq
  • X is a spacer sequence between Seq « and Seq, and the spacer sequence is not complementary to Seq « and Seq;
  • I I represents the base complementary pairing relationship formed between Seq « and Seq.
  • the polynucleotide construct is located on an expression vector.
  • the invention also encompasses a vector comprising the miRNA, or the polynucleotide construct.
  • the expression vector usually further contains a promoter, an origin of replication, and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as carrageenomycin, gentamicin, hygromycin, ampicillin resistance.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier or an effective amount of one or more active ingredients selected from the group consisting of: (a) a microRNA of the miRNA-23 family, said miRNA-23
  • the family of microRNAs include: miRNA-23 or a modified miRNA-23 derivative, and a microRNA or a modified miRNA derivative having a core sequence of UCCAUAU, a length of 18-26 nt, and having the same or substantially the same function as miRNA-23b; b) a precursor miRNA capable of being processed into a microRNA as described in (a) in a host; (c) a polynucleotide which can be transcribed by the host to form (b) Said precursor miRNA, and processed to form the microRNA described in (a); (d) an expression vector comprising the microRNA described in (a), or the former as described in (b) a miRNA, or a polynucleotide described in (c); (e)
  • the miRNA-23 is derived from a human or a non-human mammal.
  • the miRNA-2 nucleotide sequences of human and genus are completely identical.
  • the miRNA-23 is a miRNA whose sequence is selected from the group consisting of SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3.
  • the modified miRNA derivative is a compound monomer or a multimer thereof having the structure represented by Formula I:
  • each X is a microRNA as described in (a); each Y is independently a modification that promotes the stability of microRNA application; n is a 1-100 (preferably 1-20) positive integer (preferably n is 1, 2, 3, 4 or 5); m is a 1-1000 (preferably 1-200) positive integer; each "-" means a linker, a chemical bond, or a covalent bond; In a preferred embodiment, the linker is a nucleic acid sequence of 1-10 bases in length.
  • the Y includes, but is not limited to, cholesterol, steroids, sterols, alcohols, organic acids, fatty acids, esters, monosaccharides, polysaccharides, amino acids, polypeptides, single nucleotides, polynucleotides.
  • the polynucleotide described in (c) has the structure shown in Formula II:
  • 8 ⁇ is a nucleotide sequence that can be processed into miRNA-23 in a host;
  • Seq is a nucleotide sequence that is substantially complementary or fully complementary to Seq;
  • X is between Seq « and Seq a spacer sequence, and the spacer sequence is not complementary to Seq « and Seq ⁇ ; and the structure shown in Formula II forms a secondary structure of the formula ⁇ after being transferred into a host cell:
  • sequence of the precursor miRNA is as shown in SEQ ID NO.: 4, SEQ ID NO.: 5, or SEQ ID NO.: 6, more preferably, as SEQ ID NO. : 5 is shown.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of the active ingredient of the present invention together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the present invention is in the form of an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent.
  • it can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc.
  • pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.
  • severity of the disease to be treated by the patient body weight of the patient, immune status of the patient, administration Ways, etc.
  • a satisfactory effect can be obtained.
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Pharmaceutically acceptable carriers of the invention include, but are not limited to: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or Its combination.
  • the choice of carrier should be compatible with the mode of administration which is well known to those of ordinary skill in the art.
  • the present invention also provides the use of the pharmaceutical composition for the preparation of an inhibitor for inhibiting an autoimmune response, and for preparing a medicament for preventing or treating an autoimmune response disease.
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple cerebrosostosis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, scleroderma, chronic ulcerative enteritis, chronic atrophic gastritis , chronic lymphatic thyroiditis, insulin dependent Diabetes, Crohn's disease, Sjogren's syndrome. diagnosis method
  • the invention also provides a method of detecting an autoimmune response disease.
  • the method comprises the steps of: separately detecting the expression level of the test sample and the negative control sample miR-23b, and if the expression level of the miR-23b of the sample to be tested is decreased compared with the negative control sample, it is a potential self.
  • Immune response disease disease samples are separately detecting the expression level of the test sample and the negative control sample miR-23b, and if the expression level of the miR-23b of the sample to be tested is decreased compared with the negative control sample, it is a potential self.
  • the method further comprises the steps of: respectively detecting the expression levels of the test sample and the negative control sample miR-30a, and/or miR-146a, and/or miR-214, if compared with the negative control sample, A decrease in the expression level of miR-30a in the test sample, and/or an increase in the expression level of miR_146a, and/or an increase in the expression level of miR-214, is a potential autoimmune disease disease sample.
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, chronic ulcerative enteritis, chronic atrophic gastritis, chronic Lymphatic thyroiditis, insulin-dependent diabetes mellitus, Crohn's disease, Sjogren's syndrome.
  • the decrease means that the expression level of the corresponding miRNA is reduced by 10%, preferably 20%, preferably 50%, more preferably 80%, and most preferably 100%, compared to the negative control sample.
  • the increase means that the expression level of the corresponding miRNA is increased by 10%, preferably 20%, preferably ⁇ 50%, more preferably 80%, most preferably 100%, compared to the negative control sample.
  • the present invention provides a general down-regulation of miR-23b in local inflammatory lesions, which is introduced into the miR23 family, including miR-23a, miR_23b, and miR_23c, to prevent and treat various autoimmune diseases;
  • Cytokine IL-17 is closely related to the expression of miR-23b.
  • miR_23 can simultaneously inhibit related genes in various inflammatory factor signaling pathways, thereby inhibiting the onset of various autoimmune diseases.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Materials and Methods
  • RA and 0A Joint tissue specimens of arthritis in patients with RA or 0A were obtained during arthroplasty.
  • the diagnosis of RA and 0A was based on the criteria of the American College of Rheumatology (ACR); normal joint tissue samples were from healthy people with knee trauma; Human primary fibroblast-like synoviocytes (FLS) were isolated from the synovial tissue of the joints of RA and 0A patients described above.
  • ACR American College of Rheumatology
  • FLS Human primary fibroblast-like synoviocytes
  • the present invention collects lupus nephritis specimens from 18 SLE patients and simultaneously controls 9 paracancerous tissues of renal cancer patients as controls. These SLE patients cover various types of classification criteria of the American College of Rheumatology.
  • the SLE clinical activity index (SLEDAI) was used to assess the clinical activity of SLE, and the study excluded the effects of concurrent infection.
  • mice Mouse C57BL/6, DBA/1 J, MRL/MpJ and leg L/MpJ- 3 ⁇ 4 ⁇ j (MRL/ ⁇ or) mice were purchased from the Shanghai Slack Laboratory Animal Center of the Chinese Academy of Sciences. All mice were housed under conditions of no specific pathogen (SPF). All animal experiments follow the standards and welfare of experimental animals and are approved by the Biomedical Ethics Committee of the Shanghai Institute of Biological Sciences (Chinese Academy of Sciences).
  • 293T cells HeLa cells, human primary fibroblast-like synoviocytes (FLS cells), mouse embryonic fibroblasts (MEFs), mouse primary kidney cells, etc., were purchased from ATCC in the United States or purified using methods common in the art. Cultured in DEM medium containing 10% (vol/vol) FBS (Hyclone), 100 g/ml penicillin and 100 g/ml streptomycin.
  • CFA complete Freund's adjuvant
  • mice The incidence of the mice was checked daily, and the grading criteria were used to score according to the severity of the disease: grade 0, no clinical features; grade 1, lower tail tension; grade 2, mild paralysis (weakness, 1 to 2 hind limbs incomplete) ⁇ ); Level 3, lower body squat (double hind limbs completely paralyzed); Grade 4, hind limb paralysis with forelimb weakness or paralysis; Level 5: Sudden death or death.
  • mice Take 8 ⁇ 10 weeks old commercial C57BL / 6 mice, the 100 ⁇ 8 chicken type II collagen (purchased from the company Sigma-Aldich) emulsified in CFA in, respectively, on days 1 and day 21 in the base of the tail of mice selected Several intradermal injections were performed. Male DBA/1J mice of 8 to 10 weeks old were taken, and 100 ⁇ 8 of chicken type II collagen emulsified in CFA was intradermally injected on the first day, and emulsified in incomplete Freund's adjuvant (IFA) on the 21st day ( Collagen in Difco).
  • IFA incomplete Freund's adjuvant
  • Joint inflammation was examined every 2 to 5 days and scored according to the following criteria: 0, normal no redness; grade 1, mild swelling inside the ankle; grade 2, mild swelling, extending from the ankle to the metacarpophalangeal or ankle Joint; Grade 3, moderately swollen, extending from the ankle to the metacarpophalangeal or metatarsophalangeal joint; Grade 4, severe swelling, extending from the ankle to the finger or toe, visible joint stiffness joint movement disappeared.
  • Grade 4 is the highest grade for each foot arthritis score, with a maximum score of 16 points per mouse.
  • Specimens for histochemical analysis were taken from M0G-immunized C57 mice, CI I-immunized DBA mice or MRL/r mice, and the specimens were fixed with 4% paraformaldehyde immediately after collection.
  • Spinal cord specimens were embedded in paraffin and 5 ⁇ thick sections were stained with hematoxylin-eosin ( ⁇ & ⁇ ) and Luxol fast blue staining, and observed under an optical microscope.
  • the mouse ankle specimen was first decalcified in a 10% formic acid aqueous solution for 2 weeks, and then embedded in paraffin. 5 ⁇ thick sections were subjected to ⁇ & ⁇ staining.
  • Renal Morphology Referring to the general classification criteria for human lupus nephritis, lupus nephritis is divided into four grades (normal, mild, moderate, and severe) according to the degree of damage to the kidney of the mouse. From the perspective of kidney morphology, murine lupus nephritis can be classified into grade 0 to grade 3 according to activity and severity according to human lupus nephritis (normal, mild, moderate, and severe, respectively).
  • At least 10 visual fields containing glomerular tubules and their interstitial were included in each mouse and were based on glomerular cell structure, lymphocytic infiltration, mesangial matrix extension, crescent formation, cortex and medulla Individual nuclear cell infiltration, transparent sediment and other indicators to calculate the score in each field of view, wherein the partial coefficient of necrosis and crescent formation is 2.
  • Non-destructive 3D imaging of the ankle joint using the GE Healthcare eXplore CT 120 MicroCT scanner Scan the specimen at medium resolution (43. 5 ⁇ ⁇ pixel resolution on the x, y, and z axes). Correction was made using water, air, and standard bone during the scan to give the observed micro-CT image a consistent grayscale. After scanning the image of the ankle bone of each mouse, the median sagittal section image was formed using the relevant software. MicroCT results were observed and analyzed using MicroView software.
  • FIG. The construction method and effect of the adenovirus carrying miR-23b or its adsorbate are shown in FIG. Specifically, in order to construct an adenoviral vector (Ad-miR-23b), the precursor sequence of mmu-miR-23b and the sequence of about 100 bp each before and after the genome sequence (sequence shown in SEQ ID NO.: 7) The virus was packaged by restriction enzyme digestion into a plasmid of pAdEasy vector (purchased from Qbiogen) and transfected with commercially available 293A cells. The mice were diluted with PBS to 10 1 ⁇ - 10 11 viruses per ml, and the mice were infected by intravenous or intra-articular injection. In order to build an adenoviral vector (Ad-miR-23b), the precursor sequence of mmu-miR-23b and the sequence of about 100 bp each before and after the genome sequence (sequence shown in SEQ ID NO.:
  • Ad-miR-23b-spice 7 replicate adsorbate sequences synthesized (sequences as shown in SEQ ID NO.: 8): were constructed into the same vector. Constructed Ad-miR-23a, Ad_miR_23a and Ad-IL-17 o using the same principles and methods
  • MiR-23b-3p mimic, miR-23a_3p mimic and miR-23b_3p inhibitor were purchased from Dharmacon (Thermo Fi sher Scientifi c).
  • the MiRNA mimetic is a double-stranded RNA oligonucleotide
  • the miRNA hairpin-shaped inhibitor is a single-stranded oligonucleotide.
  • the cel-miR-67 sequence in Caenorhabditis elegans is used as a negative control, and the sequence has been confirmed to have the lowest sequence in human, mouse and rat. Source.
  • Ago-miR-23a_3p, Ago-miR-23b_3p, and Ago-miR-23c_3p are cholesterol-modified microRNAs purchased from Guangzhou Ruibo Biotechnology Co., Ltd.
  • the sequences of these RNAs are shown in SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, respectively, and their modified forms are: All nucleotides of the glycosyl group are modified by 2_0_methylation, The 3' end is linked to cholesterol, and the 5' end two sites (linkage between nucleotides) are modified with thiophosphoric acid (PS) and modified at 4 positions at the 3' end.
  • PS thiophosphoric acid
  • the amount of cytokines and chemokines in the serum was measured using an ELISA kit of IL-6 and KC (purchased from R&D Systems, Inc.) according to the method described. A standard curve was drawn using a known concentration of pure recombinant mouse cytokine or chemokine.
  • the cell lysate was transferred to the PVDF membrane after electrophoresis, for ⁇ , ⁇ 2, ⁇ 1, ⁇ _ ⁇ ⁇ ⁇ , ⁇ - ⁇ 65, ⁇ - ⁇ 38 (purchased from Cell Signaling), TAB 3 (purchased from Abeam), p - ERK, TRAF6, ⁇ -actin (purchased from Santa Cruz), Flag (M2) (purchased from Sigma-Aldrich), or HA (purchased from Covance) for detection.
  • the sequences encoding TAB2 and TAB3 in the mouse were cloned into pLVX-IRES_ZsGreenl (purchased from Clontech) plasmid.
  • the two sequences used to knock down the TAB2 gene are shown in SEQ ID NO.: 9 and SEQ ID NO.: 10; the two sequences used to knock down the TAB3 gene are SEQ ID NO.: 11 and SEQ ID NO. : 12 is shown.
  • These shRNA sequences were cloned into the commercially available pLSLG lentiviral plasmid.
  • These plasmids and helper plasmids were separately transfected into 293FT cells to package the virus. After 60 hours of transfection, the virus was collected and concentrated, and 10 pg/ml polybrene (Sigma-Aldrich) was added to infect the cells or mice.
  • RNA in cells and mouse tissues was extracted using TRIzol (Invitrogen) reagent according to the manufacturer's instructions.
  • the cDNA was obtained from PrimeScript RT reagent kit (manufactured by Takara Co., Ltd.).
  • Mouse TNFa, IL-1 ⁇ , IL_6, IFN ⁇ , IL-17a, II-17f, CXCLU CXCL5, CCL2, CCL5, CCL20, GM_CSF, MMP3, MMP13, RANKL, S100A8, I ⁇ B ⁇ , and human TNFa The expression levels of IL_1 ⁇ , IL_6, IFNy, IL_17a, I ⁇ B ⁇ , p65, and pri_miR_23b were determined by quantitative real-time PCR using SYBR Premix ExTaq kit (manufactured by Takara). All gene expression was normalized to the level of the housekeeping gene Rpll3a.
  • the binding sites for the binding sites of NF- ⁇ B on the KC (cxcll) and IP-lO (cxcllO) genes were selected.
  • the 5' end of the synthesis contains a biotin-labeled double-stranded oligonucleotide (synthesized by Takara Corporation).
  • Nuclear extracts were obtained using a nuclear extraction kit (Active Motif).
  • Activation of NF- ⁇ B was detected using the Gelshift Chemiluminescence EMSA kit (Active Motif).
  • Example 1 The expression of miRNA-23b is generally downregulated in autoimmune diseases.
  • this example takes RA and SLE patients and related mice (CIA corresponds to RA, MRL/r corresponds to SLE, and EAE corresponds to MS). Inflammatory tissues were compared for miRNA microarray analysis.
  • the human miRNA microarray contains 754 miRNAs, including the synovial tissue of RA patients and control groups, the renal biopsy of SLE patients and the control group; the mouse miRNA chip includes 641 miRNAs, which are tested for CIA and control mice, respectively. Joint sample, Kidney tissue of female MRL/r and control mice and spinal cord specimens of EAE and control mice.
  • miR-23b_3p nucleotide sequence SEQ ID NO.: 2
  • miR-146a and miR-214 are generally up-regulated
  • miR_23b is more pronounced than miR_30a (Fig. la-lb). Therefore, miR-23b_3p was chosen to carry out a one-step embodiment.
  • the inventors also tested other members of the miR-23 family, including miR-23a_3p, miR_23c, expression in RA, SLE patients, and control specimens, CIA, MRL/lpr, EAE, and control mouse samples, showing miR -23a_3p (SEQ ID NO.: 1) and miR_23c (SEQ ID NO.: 3) also showed significant down-regulation in inflammatory tissues of certain autoimmune diseases.
  • Example 2 demonstrates that the miR-23 family exhibits a downregulated trend in inflammatory tissues of autoimmune diseases.
  • Example 2 IL-17 down-regulates the expression of miR-23b
  • inflammatory autoimmune diseases are local chronic inflammation, which is manifested by increased production of inflammatory factors such as TNF a and IL- ⁇ ⁇ , both of which regulate miRNA expression levels in cell culture.
  • MiR-23b-3p was significantly decreased in local inflammatory tissues of RA and SLE patients, and inflammatory factors including TNF a, IL-1 ⁇ , IL-17, IL-6 and IFNy were significantly increased in these tissues (Fig. 2c, 2d).
  • the inventors performed a linear correlation analysis between the transcription levels of miR-23b_3p and these cytokines, and the results showed that the expression level of miR-23b_3p was inversely related to IL-17 levels, both in RA patients and their control groups (P ⁇ 0. 001), or between SLE patients and their control group (P ⁇ 0.05) (Fig. 2a, 2b), suggesting that IL-17 expression in inflammatory tissues may regulate the down-regulation of miR-23b_3p.
  • the inventors stimulated human primary fibroblast-like synoviocytes (FLS) and small with TNF a, IL- ⁇ ⁇ , IL-17, IL_6 and IFN Y , respectively.
  • Mouse primary kidney cells were then subjected to qPCR detection of miR-23b_3p levels (Fig. 2e, 2f).
  • the results showed that IL-17 stimulation significantly down-regulated the expression of miR-23b_3p in both cells.
  • IL-17 mediates the activation of NF- ⁇ by binding to Act l and I ⁇ B kinase ( ⁇ ) complexes.
  • IL-17 inhibited the expression of miR-23b_3p in mouse joints in vivo (Fig. 2h).
  • MiR-23 family inhibits inflammatory factor-mediated signaling and gene expression
  • Inflammatory factor-mediated activation of the NF- ⁇ B signaling pathway plays an important role in the pathogenesis of autoimmune diseases.
  • the inventors found that overexpression of miR-23b_3p significantly inhibited TNFa and IL_1 ⁇ -mediated activation of NF- ⁇ and IL-17-mediated activation of NF-i B (Fig. 3a, Fig. 3b).
  • miR-23b_3p mimics can inhibit inflammatory factors in human FLS cells (Fig. 3c), mouse primary kidney cells (Fig. 3d), and mouse primary astrocytes (Fig. 3e).
  • TNF a, IL- ⁇ ⁇ and IL 17 are tempted by downstream inflammatory genes such as KC and IL_6 Expression is expressed, and the miR-23b_3p inhibitor enhances this effect. These results indicate that miR-23b_3p inhibits inflammatory factor-mediated signaling and gene expression.
  • the inventors further examined the inhibitory effects of miR-23a_3p and miR_23c on inflammatory factor-mediated signaling pathways in other members of the miR-23 family.
  • the results showed that miR-23a-3p (Fig. 3f) and miR-23c also inhibited the expression of KC and IL-6 induced by inflammatory factors (TNF a, IL- ⁇ ⁇ and IL-17), indicating that the miR-23 family has Inhibition of inflammatory factor-mediated signaling pathways and gene expression.
  • Example 4 MiR-23 family inhibits the development of autoimmune diseases
  • miR-23 is generally downregulated in various autoimmune diseases, and down-regulation of miR-23 leads to the promotion of inflammatory factor-mediated signaling pathways and gene expression, thereby aggravating the inflammatory response.
  • the inventors found that introduction of miR-23b_3p can significantly inhibit the occurrence of autoimmune diseases.
  • the inventors constructed an adenovirus carrying the miR-23b (Ad_23b) and an adenovirus carrying the miR-23b_3p adsorbate, and increased or decreased the level of miR_23b by introduction into the mouse, respectively.
  • Ad_23b adenovirus carrying the miR-23b
  • adenovirus carrying the miR-23b_3p adsorbate
  • the transfer of miR-23b_3p adsorbent increased renal damage, indicating that the introduction of miR-23b_3p can inhibit the onset of lupus erythematosus and relieve the disease.
  • the inventors used adenovirus to transfer miR-23b or its adsorbate into mice, and further induced EAE, and found that (Fig. 4f): introduction of miR-23b_3p can significantly delay the onset of EAE, reduce the clinical score, and transfer to miR The -23b_3p adsorbate aggravated the condition of EAE.
  • the above experiments demonstrate that miR-23b_3p can significantly prevent and slow the onset and progression of autoimmune diseases.
  • miR-23a-3p the other two members of the miR-23 family, miR-23a-3p and miR-23c, were further investigated for their inhibitory effects on autoimmune diseases.
  • the introduction of miR-23a-3p can also inhibit the condition of CIA and SLE.
  • miR_23c also inhibits the development and progression of a variety of autoimmune diseases in mouse models.
  • the inventors further studied whether the introduction of the miR-23 family can be an effective therapeutic effect for an autoimmune disease that has already developed disease.
  • miR-23b_3p has a remarkable effect in treating autoimmune diseases. As shown in Figure 5, introduction of Ago-miR-23b-3p significantly induced disease in mice after induction of CIA and EAE. Introduction of an adenovirus carrying miR-23b in MRL/r mice with spontaneous lupus can also have a significant therapeutic effect (Fig. 4c_e).
  • the inventors further studied the therapeutic effects of miR-23a-3p and miR-23c on CIA, EAE, and lupus erythematosus mice, and the results showed that miR_23a_3p and miR_23c It also has a significant therapeutic effect on autoimmune diseases.
  • MiR-23b target gene is present in a variety of pro-inflammatory factor signaling pathways
  • the inventors applied gene chip expression to analyze down-regulated genes in miR-23b_3p-expressing FLS cells, and simultaneously bind and bind.
  • the "target scan” software http://www.targetscan.org) and signal pathway analysis identified 11 inflammation-related genes that may be regulated by miR-23b_3p (Fig. 6a).
  • the 3' UTR reporter activity of the three genes ⁇ 3, ⁇ and ⁇ 2 was significantly inhibited by miR-23b_3p, while miR-23b_3p had little or no effect on the 3' UTR activity of the other eight genes (Fig. 6b) ).
  • Mutation of the 3' UTRs of the three genes TAB3, IKK ⁇ and ⁇ 2 confirmed that they were the site of action of miR-23b_3p (Fig. 6c).
  • Overexpression of miR-23b_3p in primary FLS or 293T cells inhibits the protein levels of endogenous ⁇ 3, ⁇ and ⁇ 2, which is consistent with the results of the reporter analysis.
  • TAB2 and TAB3 are targets of miR-23b.
  • TAB2 and TAB3 were significantly increased, while miR-23b_3p showed significant down-regulation, and another target of miR-23b_3p was also in patients with RA. Elevation in tissues (Fig. 7a, 7b) o The levels of these proteins in SLE patients were significantly higher than those in the control group, while the level of miR-23b_3p was significantly decreased (Fig. 7c, 7d).
  • TAB2, TAB3, and ⁇ were increased in the joints of CIA mice, the kidneys of URL/lpr mice, and the spinal cord of EAE mice compared with control mice. It was inversely related to the expression level of miR-23b_3p (Fig. 7e_g).
  • TAB2 and TAB3 are targets of miR-23b_3p in the pathogenesis of autoimmune diseases.
  • Example 8 Inhibition of TAB2 and TAB3 expression can inhibit the development of autoimmune diseases
  • TAB2 and TAB3 are targets for miR-23b_3p to function in autoimmune diseases.
  • the TAB2 and TAB3 of the locus (Fig. 8e).

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Abstract

L'invention concerne un microARN associé à une maladie auto-immune. En particulier, l'invention concerne le miR-23b qui est généralement régulé à la baisse dans un tissu à inflammation partielle, et l'invention concerne en outre des membres de la famille de miR-23 aptes à inhiber l'occurrence et le développement d'une maladie auto-immune après que les membres ont été introduits, comprenant miR-23a, miR-23b et miR-23c. L'invention concerne également l'utilisation du microARN dans le traitement et la prévention d'une maladie auto-immune. L'expression de miR-23b est régulée par l'IL-17, et cible les gènes TAB2 et TAB3, etc., dans la voie de signalisation de facteurs d'inflammation ; par l'inhibition de l'expression de TAB2 et TAB3, miR-23b peut inhiber l'occurrence et le développement d'une maladie auto-immune et soulage significativement les symptômes et le développement de la maladie.
PCT/CN2013/074423 2012-04-19 2013-04-19 Microarn associé à une maladie auto-immune et son utilisation WO2013155980A1 (fr)

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CN104805085A (zh) * 2014-01-29 2015-07-29 江苏命码生物科技有限公司 串联表达的siRNA及其在治疗慢性淋巴细胞白血病中的应用
CN103948940A (zh) * 2014-03-14 2014-07-30 牡丹江医学院 microRNA-23b及其拟似物在制备防治肾病的药物中的应用
CN103961719B (zh) * 2014-03-14 2018-01-23 牡丹江医学院 microRNA‑23b及其拟似物在制备防治肥胖、高血脂、高血压及其并发症的药物中的应用
CN105567794B (zh) * 2014-10-15 2020-11-20 上海产业技术研究院 一种新的强直性脊柱炎易感性检测方法和试剂盒
CN104940955B (zh) * 2015-07-17 2018-01-12 中国农业大学 一种microRNA在制备治疗流感的药物中的应用
CN106492223B (zh) * 2016-09-29 2020-01-17 哈尔滨医科大学 microRNA-34a-5p抑制剂在制备治疗类风湿关节炎药物中的应用
CN110117647B (zh) * 2018-11-30 2022-10-18 首都医科大学附属北京潞河医院 成人隐匿性自身免疫性糖尿病相关的microRNA及相关应用
CN109652526A (zh) * 2018-12-04 2019-04-19 常州市第二人民医院 将外周血microRNA-23b指标用于评估类风湿性关节炎的研究方法
CN112390855B (zh) * 2019-07-31 2022-04-29 上海交通大学医学院附属仁济医院 Pretide-146a在制备缓解或治疗自身免疫性疾病的药物中的应用
CN110791560B (zh) * 2019-11-06 2021-09-14 中国医学科学院医药生物技术研究所 一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物
CN111378742A (zh) * 2020-04-16 2020-07-07 嘉兴程瑞医药科技有限公司 microRNA生物标志物及其在制备自身免疫疾病检测试剂盒中的应用
CN112175951A (zh) * 2020-09-30 2021-01-05 深圳大学 一种miRNA-23a-5p抑制剂及其在制备治疗高盐损害疾病的药物中的应用

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