WO2013155980A1

WO2013155980A1 - Auto-immune disease-related microrna and use thereof

Info

Publication number: WO2013155980A1
Application number: PCT/CN2013/074423
Authority: WO
Inventors: 钱友存; 沈南; 朱书; 潘文
Original assignee: 中国科学院上海生命科学研究院
Priority date: 2012-04-19
Filing date: 2013-04-19
Publication date: 2013-10-24
Also published as: CN103372218B; CN103372218A

Abstract

Provided is an auto-immune disease-related microRNA. In particular, provided is the miR-23b generally down-regulated in the partially inflamed tissue, and further provided are members of the miR-23 family capable of inhibiting the occurrence and development of auto-immune disease after the members are introduced, comprising miR-23a, miR-23b and miR-23C. Also provided is the use of the microRNA in the treatment and prevention of auto-immune disease. The expression of miR-23b is regulated by IL-17, and targets the genes of TAB2 and TAB3, etc., in the signal pathway of inflammatory factors; by inhibiting the expression of TAB2 and TAB3, miR-23b can inhibit the occurrence and development of auto-immune disease and significantly relieve the symptoms and development of the disease.

Description

Autoimmune disease related microRNA and its application

Technical field

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). 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 (miRs or miRNAs, 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. Prerequisites After the miRNA is transported to the cytoplasm, it is further cleaved into a double-stranded miRNA of about 18_26 nt. After the double-stranded miRNA is unraveled, 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.

Although 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.

To date, little is known about the relationship between autoimmune diseases and microRNAs in the field, although some miRNAs, such as miR-155, miR_146a and miR-326, regulate the pathogenesis of autoimmune diseases by affecting T cell function, but in situ cells It is not clear how miRNAs function in autoimmune diseases. Therefore, there is an urgent need in the art to study microRNAs and their regulatory networks related to autoimmune diseases.

Summary of the invention

It is an object of the present invention to provide a class of autoimmune disease related microRNAs and uses thereof. In a first aspect of the invention, there is provided 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;

(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 a host (b) a precursor miRNA as described, and processed to form the microRNA described in (a);

(d) an expression vector comprising the microRNA described in (a), or the precursor miRNA described in (b), or the polynucleotide described in (c);

(e) An agonist of the microRNA described in (a).

In another preferred embodiment, the miRNA-23b is miRNA-23b_3p.

In another preferred embodiment, 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.

In another preferred embodiment, the inhibition of autoimmunity refers to inhibition of inflammatory factor-mediated signaling pathways and gene expression.

In another preferred embodiment, the microRNA is derived from a human or a non-human mammal; preferably, the non-human mammal is a rat or a mouse.

In another preferred embodiment, the core sequence described in (a) refers to the nucleotide sequence of positions 2-8 of the microRNA.

In another preferred embodiment, 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.

In another preferred embodiment, the host described in (b) is: human or rodent (e.g., rat, mouse).

In another preferred embodiment, 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.

In another preferred embodiment, the pharmaceutical composition further has a miRNA selected from the group consisting of miRNA-23a-5p and miRNA_23b_5p.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, the miRNA-23 is a miRNA selected from the group consisting of miR A-23a_3p, miRNA_23b_3p, or miRNA_23c.

In another preferred embodiment, 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.

In another preferred embodiment, 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. In another preferred embodiment, 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.

In another preferred embodiment, the modified miRNA derivative described in (a) is a compound monomer having the structure of formula I or a multimer thereof:

(X) n- (Y) m formula I

In formula I,

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);

m is a 1-1000 (preferably 1-200) positive integer;

Each "-" indicates a linker, a chemical bond, or a covalent bond.

In another preferred embodiment, the linker is a nucleic acid sequence of 1-10 bases in length.

In another preferred embodiment, 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.

In another preferred embodiment, the polynucleotide described in (c) has the structure represented by Formula I:

Seq forward one X-Seq reverse

Formula I I

In formula II, 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; and Formula II The structure shown forms a secondary structure of the formula 转 after transfer into the host cell:

Seq is positive ―,

X

Seq reverse

I I I,

In the formula I I I, the Seq forward, Seq reverse and X are defined as above, | | represents the base complementary pairing relationship formed between the Seq forward and the Seq reverse.

In another preferred embodiment, 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.

In another preferred embodiment, the substance which inhibits IL-17 activity includes an antibody against IL-17.

In another preferred embodiment, 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.

In another preferred embodiment, the expression vector described in (d) comprises: a viral vector and a non-viral vector.

In another preferred embodiment, the viral vector is: an adenovirus vector, an adeno-associated virus vector, a retroviral vector, or a lentiviral vector.

In another preferred embodiment, the non-viral vector is: a plasmid or a bacterium.

In another preferred embodiment, the sequence of the precursor miRNA is as shown in SEQ ID NO.: 4, SEQ ID NO.: 5, or SEQ ID NO.: 6. In another preferred embodiment, the pharmaceutical composition is for treating an autoimmune response disease.

In a second aspect of the invention, there is provided the use of an active ingredient, wherein the active ingredient is 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 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; (b) 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); (d) an expression vector containing the microRNA described in (a) Or the polynucleotide described in (b), or the polynucleotide described in (c); (e) 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 preventing or treating an autoimmune reaction.

In another preferred embodiment, 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.

In 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.

In another preferred embodiment, the object comprises a person.

In a fourth aspect of the invention, there is provided the use of a miRNA-23 antagonist for the preparation of a modulator which upregulates an autoimmune response.

In another preferred embodiment, the miRNA-23 is miRNA_23b_3p.

In a fifth aspect of the invention, there is provided a method of screening an active ingredient for inhibiting autoimmunity, comprising the steps of:

(a) administering the candidate substance to the cells or animals of the test group, and determining the expression level of miRNA-23 in the test group after administration;

(b) comparing the expression levels of the miRNA-23 of the test group with the miRNA-23 of the control group in which the candidate substance was not administered;

Among them, when the expression level of miRNA-23 in the test group was significantly higher than that in the control group, it indicated that the candidate substance was an active ingredient for inhibiting autoimmunity.

5倍倍, (1. 5-5倍,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 5-3倍)。 Preferably, 1. 5-3 times).

In another preferred embodiment, 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) .

In a sixth aspect of the invention, there is provided a use of a TAB2 or TAB3 inhibitor for the manufacture of a medicament for inhibiting an autoimmune disease.

In a seventh aspect of the present invention, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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%.

In another preferred embodiment, 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%.

In another preferred embodiment, the autoimmune response disorder is one or more selected from the group consisting of rheumatic arthritis, multiple sclerosis, or systemic lupus erythematosus.

In an eighth aspect of the invention, there is provided the use of a microRNA, miR-23b, which is used for the preparation of a reagent or kit for detecting an autoimmune disease.

In another preferred embodiment, the above reagent is a chip, a primer, or a probe.

In another preferred embodiment, the kit described above includes instructions for use. Preferably, 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.

In another preferred embodiment, 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

The following drawings are used to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the appended claims.

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 meningitis (Experimental Autoimmune) Encephalomye l it is, EAE) mice with two untreated control mouse samples (synthesis of spinal cord tissue of four mice per sample); Figure lb shows screening for general regulation in inflammatory tissues of autoimmune diseases The process of the mi croRNA lists two commonly up-regulated miRNAs and two commonly down-regulated microRNAs. 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 the mixed spinal cord samples of C57BL/6 mice immunized with 14 MOGs for 14 days or 28 days was significantly down-regulated.

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) In membrane tissue, 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 erythematosus and 9 adjacent renal cancer patients Samples; Figure 2e and Figure 2f show that IL-17 stimulation down-regulates miR-23b_3p expression and detects human primary fibroblast-like synoviocytes (Fig. 2e) And mouse primary cultured kidney cells (Fig. 2f), receiving human or mouse recombinant TNF a (10 ng/ml), IL-1β (10 ng/ml), IL-17 (50 ng/ml, respectively) , IL-6 (20 ng/ml) and IFN (50 ng/ml) stimulated miR_23b levels; Figure 2g shows IL-17 regulates miR-23 expression by NF_κB, 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 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; wherein, 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 β (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 β (10 ng/ml) stimulation, EMSA experiments verify that miR-23b-3p inhibits the activation of NF-κΒ Figure 3c - Figure 3e shows transfection of miR-23b_3p mimics in primary fibroblast-like synoviocytes (Figure 3c), primary renal epithelial cells (Figure 3d), primary astrocytes (Figure 3e), miR-23b_3p inhibitor and its mimetic control, respectively, alone or in combination with IL-17 (50ng/ml), TNF a (10ng/ml), IL-1 β (10ng/ml) to stimulate 6 small , The reference housekeeping gene to standardize Rpl 13a, The expression of KC and IL-6 was detected; the data was integrated four times (Fig. 3c) or three times (Fig. 3d, Fig. 3e). The independent experiment showed that miR-23b_3p inhibited inflammatory factor-mediated gene expression; Fig. 3f 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 β (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_23b), carrying an adenovirus encoding the miR-23b_3p adsorbate (Ad-sponge), and carrying an adenovirus encoding both miR_23b and its adsorbate

(Ad-23b+Ad-sponge), the type I collagen was used to induce arthritis in mice at the time points indicated in the figure, and the average clinical score (standard deviation) and the incidence rate were calculated. Figure 4b shows that the mice in Figure 3a were subjected to hind paw micro-CT scans 80 days after the second immunization. The results showed that the expression of miR-23b_3p inhibited bone damage in the pathogenesis of CIA, while the miR-23b_3p adsorbate promoted Bone damage in the onset of CIA. 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. 3c); 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. 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 ΙΟΟηΜ / 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). The results showed that transfection of miR-23b-3p down-regulated the expression of IKK α, ΤΑΒ 2 and ΤΑΒ 3, indicating that ΙΚΚ α, ΤΑΒ 2 and ΤΑΒ 3 are Target gene for miR-23b_3p action; Figure 6c shows transfection of 293T with wild-type (wt UTR) or point-mutated 3' UTR (mut UTR) reporter plasmid into miR-23b_3p or control mimic (NC) for fluorescence detection Results of prime enzyme activity; Figure 6d shows that miR-23b_3p can reduce the protein level of its acting genes, detecting primary fibroblast-like synoviocytes (FLS) and 293 that were untransfected and transfected with control mimic and miR-23b_3p The levels of endogenous TAB2, TAB3 and ΙΚΚα in the cells, β-act in are internal parameters, s represents short exposure, and 1 represents long exposure.

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 ΙΚΚα were determined as β-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 IKKα; Immunoblotting method was used to detect the expression levels of ΤΑΒ3, ΤΑΒ2 and ΙΚΚα in kidney specimens of patients with lupus erythematosus and its control group, using β-actin as an internal reference; Figure 7d shows the level of miR-23b_3p and TAB3, TAB2 in the above samples. There is a negative correlation between the protein expression levels of ΙΚΚα and ΙΚΚα; Figure 7e_g shows that the expression levels of TAB2, TAB3 and ΙΚΚα 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. shRNA (LV_shTAB2/3) and control shRNA (LV_shNC), the average clinical score (score standard deviation) of the onset of arthritis and the incidence rate; Figure 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. qPCR was used to detect gene expression of KC and IL-6, which was normalized with reference to the housekeeping gene Rpl l3a; Figure 8f shows that re-expression of TAB2 and TAB3 reversed the inhibitory effect of miR-23b_3p in CIA progression; DBA/1 was small in type II collagen immunization Intravenous injection of miR-23b-containing adenovirus (Ad_miR_23b) or adenovirus carrying control plasmid (Ad-EV) and coding for TAB2, TAB 3 but miR_23b binding site mutation plasmid (LV-TAB2, LV-TAB3) or control The plasmid (LV-EV) lentivirus, an average clinical score (score standard deviation) and an assessment of the incidence of arthritis. The injection process is done in several steps, as indicated by the arrows.

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 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) The infection efficiency in mouse B16 melanoma cells is close to 100%. 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. detailed description

The inventors have extensively and intensively studied that 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

The present invention provides a class of miRNAs involved in autoimmune correlation. As used herein, "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. As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, 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. As used herein, "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). Typically, 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%. Generally, there may be up to 40 mismatched nucleotides between two sufficiently complementary molecules; preferably, up to 30 mismatched nucleotides; more preferably, up to 20 mismatched nucleosides Acid; further preferred, having up to 10 Unmatched nucleotides, such as 1, 2, 3, 4, 5, 8, 11 mismatched nucleotides.

As used herein, 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. For example, 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.

In a preferred embodiment of the present invention, 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.

In another preferred embodiment, 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.

[!匕 c

The invention also encompasses miRNA variants and derivatives. In addition, 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

According to the miRNA sequences provided by the present invention, 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.

In a preferred embodiment of the invention, the polynucleotide construct comprises the structure of formula I:

Seq forward one X-Seq reverse

Formula II In formula II,

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;

The structure shown in Formula I forms a secondary structure of the formula 在 after being transferred into the cell:

Seq forward

I ' X

Seq reverse

I I I

In Formula III, the definitions of Seq E, Seq _fi and X are as described above;

I I represents the base complementary pairing relationship formed between Seq « and Seq.

Typically, the polynucleotide construct is located on an expression vector. Accordingly, 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. Pharmaceutical composition

The present invention provides 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) an agonist of the microRNA described in (a). In another preferred embodiment of the invention, the pharmaceutical pharmaceutical composition further comprises a TAB2 or TAB3 inhibitor.

In another preferred embodiment of the invention, 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.

In another preferred embodiment of the present invention, the modified miRNA derivative is a compound monomer or a multimer thereof having the structure represented by Formula I:

(X) n- (Y) m formula I

In 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. In another preferred embodiment of the present invention, the polynucleotide described in (c) has the structure shown in Formula II:

Seq forward one X-Seq reverse

Formula I I

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 转 after being transferred into a host cell:

Seq forward

I ' X

Seq reverse

I I I,

In the formula I I I, the Seq E direction, the Seq inverse and the X are defined as above, | | represents the base complementary pairing relationship formed between the Seq forward and the Seq reverse.

In another preferred embodiment of the present invention, the 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.

As used herein, 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.

As used herein, 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. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical 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. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, 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. For example, 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. In general, when the active ingredient of the present invention is administered at a dose of about 0.0001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, 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.

In a preferred embodiment, 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. Preferably, 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 main advantages of the invention include:

(1) 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;

(2) 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

Disease patient specimen

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.

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.

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).

MiRNA chip and DNA chip

Total RNA was extracted using TRIzol reagent (purchased from Invitrogen) and evaluated using an Agi lent 2100 bioanalyzer. Only samples with integrity greater than 8 were used. In the miRNA expression experiment, a total of 754 human miRNAs and 641 mouse miRNAs were detected by Taqman low density chip v3.0 (Appl ied Biosystems), and the experiment was performed on an Applied Biosystems real-time instrument according to related products. The steps are completed. Detection of transfection The expression of various mRNAs in miR-23b and control cells was performed by Affymetrix GeneChip® Human Genome U133 Plus 2. 0 Array, and subsequent results were processed by Agi lent GeneSpringTM GX 10 chip data analysis software.

Cell culture

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.

EAE induction and scoring

Commercially available C57BL/6 mice, 6 to 8 weeks old, were injected subcutaneously with complete Freund's adjuvant (CFA) (Difco) containing 300 μ ₈ M0G (35_55) peptide end on the day of immunization and 48 hours later in mice. 200μ ₈ was dissolved in vivo injection of pertussis toxin in PBS. 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)瘫痪); 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.

CIA induction and scoring

Take 8~10 weeks old commercial C57BL / 6 mice, the 100μ 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 μ 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). 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.

Histochemistry and kidney morphology analysis

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 μηι thick sections were stained with hematoxylin-eosin (Η&Ε) 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 μηι thick sections were subjected to Η & Ε staining. The kidney tissue was embedded in paraffin and made into 2 μη thick slices for fast blue staining, and then observed under an optical microscope. 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.

Micro - CT

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 μ ηι 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.

Adenovirus-mediated gene expression

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 ^ΰ - 10 ¹¹ viruses per ml, and the mice were infected by intravenous or intra-articular injection. In order to build

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

MiRNA and its inhibitor

MiR-23b-3p mimic, miR-23a_3p mimic and miR-23b_3p inhibitor (hairpin inhibitor) were purchased from Dharmacon (Thermo Fi sher Scientifi c). The MiRNA mimetic is a double-stranded RNA oligonucleotide, and the miRNA hairpin-shaped inhibitor is a single-stranded oligonucleotide. Whether it is a hairpin-shaped inhibitor or a mimetic, 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- miRNA

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.

ELISA

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. Western blot

The cell lysate was transferred to the PVDF membrane after electrophoresis, for ΙΚΚα, ΤΑΒ2, ΤΑΚ1, ρ_Ι κ Βα, ρ- ρ65, ρ-ρ38 (purchased from Cell Signaling), TAB 3 (purchased from Abeam), p - ERK, TRAF6, β-actin (purchased from Santa Cruz), Flag (M2) (purchased from Sigma-Aldrich), or HA (purchased from Covance) for detection.

Lentivirus-mediated gene expression and knockdown

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.

Q-PCR

Total 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 β, IL_6, IFN γ, IL-17a, II-17f, CXCLU CXCL5, CCL2, CCL5, CCL20, GM_CSF, MMP3, MMP13, RANKL, S100A8, I κ B ζ, and human TNFa, The expression levels of IL_1β, IL_6, IFNy, IL_17a, I κ B ζ, 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. Amplification of cDNA in AbiPrism 7900 HT cycler (Applied

Completed on Biosystems). The expression of mature miR was detected using Applied Biosystems' Taqman kit.

Gel Migration Hysteresis Experiment (EMSA)

The binding sites for the binding sites of NF-κB 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-κB was detected using the Gelshift Chemiluminescence EMSA kit (Active Motif).

Statistics

Two-way Student's t-test was used for statistical analysis of the two groups of monitoring data. Two-way ANOVA was used for more complex comparisons and then analyzed by Bonferroni post hoc test. For the analysis of nonparametric statistics, the Mann-Whitney test was used. Take a P value of 0.05 or lower as a criterion for judging the significance of the results. Example 1 The expression of miRNA-23b is generally downregulated in autoimmune diseases.

In order to determine whether there is a ubiquitous miRNA functioning in the inflammatory lesions of various 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.

The results were as follows: miR-23b_3p (nucleotide sequence SEQ ID NO.: 2) in all autoimmune disease samples, including RA, SLE, CIA, MRL/7/^ and EAE, compared to the control group. Shown) and miR_30a are generally down-regulated, while miR-146a and miR-214 are generally up-regulated, while miR_23b is more pronounced than miR_30a (Fig. la-lb). Therefore, miR-23b_3p was chosen to carry out a one-step embodiment. Next, a large number of RA, SLE patients and control specimens, CIA, MRL/lpr, EAE and control mouse samples were tested by quantitative real-time PCR (qPCR), and the results confirmed that miR-23b_3p is in autoimmune diseases. It is generally downregulated in inflammatory tissues (Fig. lc_lg).

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.

This example 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

One common feature of inflammatory autoimmune diseases is local chronic inflammation, which is manifested by increased production of inflammatory factors such as TNF a and IL-Ι β, 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-1β, 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.

To further determine the relationship between miR-23b_3p and these cytokines, the inventors stimulated human primary fibroblast-like synoviocytes (FLS) and small with TNF a, IL-Ι β, 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-κΒ by binding to Act l and I κ B kinase (ΙΚΚ) complexes. The inventors found that the deletion of Act l or ΙΚΚγ can counteract the regulation of IL-17 on miR-23b_3p (Fig. 2g ), indicating that IL-17-mediated activation of NF-κB is important for the regulation of miR-23b_3p. Similar to the experimental results obtained in primary cells, IL-17 inhibited the expression of miR-23b_3p in mouse joints in vivo (Fig. 2h).

These results indicate that IL-17 downregulates the expression of miR-23b_3p in autoimmune diseases. Example 3 MiR-23 family inhibits inflammatory factor-mediated signaling and gene expression

Inflammatory factor-mediated activation of the NF-κB 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_1β-mediated activation of NF-κκ and IL-17-mediated activation of NF-i B (Fig. 3a, Fig. 3b). Next, the inventors found that 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-Ι β 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-Ι β 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

The above examples show that 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.

First, 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. Using adenovirus to transfer miR-23b or its adsorbate into mice, further induction of CIA, it was found (Fig. 4a, 4b): exogenous expression of miR-23b in mouse ankle significantly reduced the incidence and delay of CIA The onset time, the severity of the disease and bone damage were alleviated; while the miR-23b adsorbate counteracted the inhibition of miR-23b overexpression; the adenovirus expressing miR-23b_3p adsorbate alone significantly increased the incidence of CIA The severity and bone damage. The inventors used adenovirus to transfer miR-23b_3p or its adsorbate into MRL/r mice and found that (Fig. 4c-e) _: Overexpression of miR-23b_3p significantly reduced the degree of renal damage in MRL/r mice. 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.

Considering that the miR-23 family members are highly similar in sequence, 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. Studies have shown that the introduction of miR-23a-3p into mice by adenovirus can significantly inhibit the pathogenesis of EAE and alleviate the disease (Fig. 4g). The introduction of miR-23a-3p can also inhibit the condition of CIA and SLE. Studies have shown that the introduction of miR_23c also inhibits the development and progression of a variety of autoimmune diseases in mouse models.

The above experiments show that miR-23 can prevent the development of autoimmune diseases. Example 5 MiR-23 family treatment of autoimmune diseases

In the present example, 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.

The inventors first discovered that 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).

Considering the high similarity of the miR-23 family members in sequence, 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.

This example demonstrates that members of the miR-23 family have significant therapeutic effects on autoimmune diseases. - Example 6 MiR-23b target gene is present in a variety of pro-inflammatory factor signaling pathways

In order to confirm the possible target of miR-23b_3p, in the present example, 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 ΤΑΒ3, ΙΚΚα and ΤΑΒ2 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, IKKα and ΤΑΒ2 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 ΤΑΒ3, ΙΚΚα and ΤΑΒ2, which is consistent with the results of the reporter analysis.

This example shows that ΤΑΒ3, ΙΚΚα and ΤΑΒ2 are the sites of action of miR_23b_3p. Example 7 In autoimmune diseases, TAB2 and TAB3 are targets of miR-23b.

To verify whether miR-23b_3p can target the regulation of TAB2 and TAB3 in vivo in autoimmune diseases, the inventors tested the expression levels of TAB2 and TAB3 in RA or SLE patients. Compared with normal or OA patients, the levels of TAB2 and TAB3 in synovial tissue of RA patients 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). In a mouse model of autoimmune disease, the levels of TAB2, TAB3, and ΙΚΚα 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).

This example shows that 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

In order to study the possible physiological functions of TAB2 and TAB3, we considered the overlap of the functions of the two, and we used shRNA to express shRNA to simultaneously silence TAB2 and TAB3. We screened a range of siRNAs from mouse B16 cells for siRNAs that effectively silence TAB2 and TAB3. Finally, we selected siTAB2_4, siTAB2_5, siTAB3_l and siTAB3_3 for the next in vivo experiment (Fig. 8a). We will simultaneously introduce lentiviruses that inhibit TAB2 and TAB3 shRNA into the joints of mice (Fig. 8b). The results showed that although the effect of overexpression of miR_23b was slightly weaker, knocking down TAB2 and TAB3 significantly inhibited the incidence of CIA (Fig. 8c). The results of H&E staining showed that both lymphocyte infiltration and bone damage were alleviated (Fig. 8d).

To further demonstrate that TAB2 and TAB3 are targets for miR-23b_3p to function in autoimmune diseases, we designed a recovery experiment to use lentiviral expression to lack miR-23 expression in a CIA mouse model overexpressing miR-23b_3p The TAB2 and TAB3 of the locus (Fig. 8e). Mandatory expression of TAB2 and TAB3 significantly reduced the inhibitory effect of miR-23b_3p on CIA, indicating that TAB2 and TAB3 are miR_23b_3p An important target in immune diseases (Fig. 8f).

This example demonstrates that TAB2 and TAB3 are important targets for miR-23b_3p in autoimmune diseases, and inhibition of TAB2 and TAB3 expression can inhibit the development of autoimmune diseases. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

Claims

Rights request

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of 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;

(b) a precursor miRNA capable of being processed into a microRNA as described in (a) in a host;

(c) a polynucleotide which is transcribed by a host to form a precursor miRNA as described in (b) and which is processed to form the microRNA described in (a);

(e) An agonist of the microRNA described in (a).

The pharmaceutical composition according to claim 1, wherein the core sequence described in (a) refers to a nucleotide sequence of positions 2-8 of the microRNA; and/or the "function and miRNA" -23b is identical or substantially identical" means that 40% of miRNA-23b_3p is retained and 500% inhibits autoimmune function.

The pharmaceutical composition according to claim 1, wherein the miRNA-23 described in (a) is a miRNA selected from the group consisting of miRNA-23a_3p, miRNA-23b_3p, miRNA_23c, miRNA-23a_5p, or miRNA_23b_5p.

The pharmaceutical composition according to claim 1, wherein the miRNA-23 described in (a) comprises the miRNA-23a_3p having the sequence of SEQ ID NO.: 1, as SEQ ID NO.: 2 miRNA-23b_3p shown, or miRNA-23c as shown in SEQ ID NO.: 3.

The pharmaceutical composition according to claim 1, wherein the modified miRNA derivative is modified to be one or more modified forms selected from the group consisting of: a sugar group modification of a nucleotide, Modifications between linkages between nucleotides, cholesterol modifications, locked nucleotide modifications, peptide modifications, lipid modifications, halogen modifications, hydrocarbyl modifications, and nucleic acid modifications.

6. The pharmaceutical composition according to claim 5, wherein the glycosyl modification of the nucleotide comprises a glycosyl modification of 2-0-methyl, a glycosyl group of 2-0-methoxyethyl ester Modification, 2-0-thiol glycosyl modification, 2-fluoroglycosyl modification, sugar ring modification, locked nucleotide modification; and/or

Modifications of the manner of linkage between the nucleotides include phosphorothioate modification, phosphorylation modification; and/or the nucleic acid modifications include "TT" modifications.

The pharmaceutical composition according to claim 1, wherein the nucleotide sequence of the precursor miRNA is SEQ ID NO.: 4, SEQ ID NO.: 5, or SEQ ID NO. . : 6 is shown.

The pharmaceutical composition according to claim 1, wherein the polynucleotide described in (c) has the structure represented by Formula I:

Seq forward one X-Seq reverse

Formula I I

In formula I I, Seq is a microRNA nucleotide sequence which can be processed into the host in the host;

Seq is a nucleotide sequence that is substantially complementary or fully complementary to Seq; Is a sequence of intervals between Seq « and Seq, and the interval sequence is not complementary to Seq « and Seq _&H; _s

And the structure shown by Formula I I is transferred to the host, the secondary structure shown by ι π:

Seq reverse

I I I,

In formula I I I, Seq forward, Seq reverse and X are defined as above,

I represents the base complementary pairing relationship formed between Seq « and Seq.

The pharmaceutical composition according to claim 1, wherein the expression vector described in (d) comprises: a viral vector and a non-viral vector.

The pharmaceutical composition according to claim 1, wherein the agonist of the miRNA-23 in (e) 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.

1 1. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier is selected from the group consisting of water, saline, liposome, lipid, protein, protein-antibody conjugate. , peptides, cellulose, nanogels, or a combination thereof.

The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is for treating an autoimmune disease.

13. Use of an active ingredient, wherein the active ingredient is 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, length 18_26nt, function and miRNA-23b The same or substantially the same microRNA or modified miRNA derivative;

(e) an agonist of the microRNA described in (a);

It is characterized in that the active ingredient is used for the preparation of an inhibitor which inhibits an autoimmune reaction, and a preparation of a medicament for preventing or treating an autoimmune reaction.

The use according to claim 13, wherein 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 mellitus, Crohn's disease, Sjogren's syndrome.

A method for preventing or treating an autoimmune response disease, characterized in that the pharmaceutical composition according to claim 1 is administered to a subject in need thereof.

16. The method according to claim 15, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, Psoriasis, scleroderma, chronic ulcerative enteritis, chronic atrophic gastritis, chronic lymphatic thyroiditis, insulin-dependent diabetes mellitus, Crohn's disease, Sjogren's syndrome.

17. Use of a miRNA-23 antagonist, characterized in that it is used to prepare a modulator for upregulating an autoimmune response.

18. A method of screening an active ingredient for inhibiting autoimmunity, comprising the steps of:

(b) comparing the expression level of the miRNA-23 of the test group with the miRNA-23 of the control group in which the candidate substance was not administered;

Among them, when the expression level of miRNA-23 in the test group was significantly higher than that of the miRNA-23 in the control group, it indicated that the candidate substance was an active ingredient for inhibiting autoimmunity.

19. A method for detecting an autoimmune response disease, comprising the steps of: separately detecting a expression level of a sample to be tested and a negative control sample miR-23b, if compared to a negative control sample, a miR-23b of the sample to be tested A reduced level of expression is a potential sample of an autoimmune response disease.

20. The method according to claim 19, further comprising the steps of: separately detecting expression levels of the test sample and the negative control sample miR-30a, and/or miR-146a, and/or miR-214, if A decrease in the expression level of miR-30a, and/or an increase in the expression level of miR_146a, and/or an increase in the expression level of miR-214, as compared to a negative control sample, is a potential autoimmune disease disease sample. .

21. The method according to claim 19, wherein 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.

22. The method according to claim 19, wherein said reducing means: the expression level of the corresponding miRNA is reduced by 10%, preferably 20%, preferably 50% compared to the negative control sample. %, more preferably 80%, optimally 100%; and/or said increase means that the expression level of the corresponding miRNA is increased by 10%, preferably 20%, preferably compared to the negative control sample. 50%, preferably 80%, optimally 100%.

23. Use of a microRNA, miR-23b, for the preparation of a reagent or kit for detecting an autoimmune disease.