CN105617398A - MicroRNA-342-3p and microRNA-210 and applications of inhibitors of microRNA-342-3p and microRNA-210 - Google Patents

Abstract

The invention relates to microRNA-342-3p and microRNA-210 and applications of inhibitors of microRNA-342-3p and microRNA-210. According to the invention, the research proves that microRNA-342-3p and microRNA-210 play important roles in generating and developing processes of rheumatoid arthritis diseases, and can be taken as markers for diagnosing and prognosing rheumatoid arthritis. In addition, the invention further discloses the applications of antagonism over the expression of microRNA-342-3p and microRNA-210 in alleviating the symptoms of rheumatoid arthritis.

Description

The purposes of microRNA-342-3p and microRNA-210 and inhibitor thereof

Technical field

The invention belongs to biomedical sector, more particularly it relates to microRNA-microRNA-342-3p and miR-210 and inhibitor purposes in the medicine preparing rheumatoid arthritis disease and diagnostic reagent thereof.

Background technology

In humans and animals tissue, long-term evolution makes the various cells of body define the Hypoxia adaptation mechanism of elaborate, and in most health tissues, oxygen concentration maintains between 2.5%��9%. And under some pathological states, such as the hypoxemia that respiratory system or cardiovascular system diseases cause, the disease such as septicemia, autoimmune disease and tumor all also exists the hypoxia (��1%O of general or local organization₂). This long-term or irreversible hypoxia can cause that the Hypoxia adaptation of the histiocyte of body, vascular endothelial cell and immunocyte occurs losing compensatory phenomenon, and then it is abnormal to cause multiple inflammatory factor and cytokine to produce, cell behaviors occurs abnormal, the dynamic equilibrium ultimately resulting in body immune system occurs abnormal, and disease is prolonged not to heal. Rheumatoid arthritis (RA) makes the oxygen concentration of affected joints intracavity be substantially reduced because of inflammation, thus having promoted that in articular cavity, pannus is formed, articular cartilage matrix is degraded, and inflammatory cell release inflammatory factor has promoted the further development of inflammation. Therefore, research hypoxia is machine-processed to the regulation and control of immunocyte biological function and the Hypoxia adaptation of immunocyte, and to finding the key signal molecule controlled by hypoxia, diagnostic reagent and the treatment new drug of exploitation rheumatoid arthritis have potential clinical value.

Dendritic cell (Dendriticcells, DCs) is the bridge that body connects the natural immunity and adaptive immunity, and DCs is the antigen presenting cell that the function being currently known is the strongest, it is possible to stimulation T cells (Tcells) activation and propagation, thus starting and regulation and control specific immune response. Different DC subgroups, the DC in different differentiation and development stage, under different PAMP stimulate, type and intensity to acquired immune response all have different regulating and controlling effects, and the functional status of DC is subject to the impact of microenvironment residing for it, and some the endogenous inflammatory factors such as C5a produced in microenvironment can promote the ripe activation of DC, and promote that ripe DC induces the differentiation of Th1 or Th2 cell. At present a lot of evidences show, at rheumatoid arthritis, chronic inflammatory bowel disease, and in the disease such as systemic lupus erythematosus (sle), the active and regulatory T cells of generating that there is Th1 and Th7 generates reduce unbalance. Recent study finds, DCs participation is many has regional hypoxia feature advancing of disease process, and the regional hypoxia condition such as tumor is likely to participate in inducing substantial amounts of toleration DCs, with body, the Mechanism of immunotolerance of tumor is relevant; Arteriosclerosis plaque, the activity of rheumatoid arthritis localized clusters DCs and changes of function affect advancing of disease and prognosis. These research phenomenon promptings, DCs has oxygen impression and adaptation mechanism, and this oxygen is experienced and adapts to be likely to affect the biological behaviour of DCs, and then affects generation and the type of immunne response. Therefore, the research of the Hypoxia adaptation mechanism of DCs is contributed to the treatment of clinical hypoxia-related diseases and the preparation research of efficient DCs vaccine.

Complement system is the important component part of body inherent immunity opposing pathogenic microorganism, and the multiple active chronic inflammation disease of wide participation, such as autoimmune disease, septicemia, acute lung injury, ischemical reperfusion injury and asthma etc. Complement activation can not only directly play the effect of dissolved cell, antibacterial, virus, a series of active fragment can also be produced, play conditioning phagocytosis, remove apoptotic cell, chemotactic immunocyte and participate in regulating the effector function of panimmunity cell, thus maintaining immunity homeostasis. Complement activation products c5a anaphylatoxin has powerful biological activity, it it is important inflammatory mediator, inflammatory cell such as neutrophilic granulocyte, mononuclear cell and T lymphocyte etc. there is strong chemotactic activity, can activated macrophage promote the release of its lysozyme and superoxide ion, thus participating in autarcetic regulation and control or promoting tissue injury. Research finds, C5a is by being combined with its receptor C5aR1 (CD88) and C5L2 and play biological effect, C5aR1 belongs to g protein coupled receptor superfamily member, has wide expression at panimmunity cell surface, is the major receptors of mediation C5a. Having research to point out, the overactivity at septicemia their early stage C5a can cause the further release of inflammatory mediator, causes tissue injury, the further deterioration of disease. As hypoxia-related diseases, rheumatoid arthritis is also a kind of known C5 relevant disease, C5a level in RA patients serum and joint fluid is significantly raised, its CIA inductivity of the mice of C5 deficiency and disease severity, significantly lower than wild-type mice, illustrate that C5a/C5aR participates in the pathology damage mechanism of RA. Therefore prevention and the treatment of inflammatory diseases there is important scientific meaning by the research of C5a/C5aR signal path regulation and control.

To sum up, this area still need based on rheumatoid arthritis disease pathogeny mechanism, find for treatment or the effective medicine of diagnostics classes rheumatic arthritis relevant disease.

Summary of the invention

It is an object of the invention to provide the purposes of microRNA-342-3p and microRNA-210 and inhibitor thereof.

In a first aspect of the present invention, it is provided that adjust under a kind of miR-342-3p or adjust under miR-210 and treat the purposes in the medicine of rheumatoid arthritis disease in preparation.

In a preference, described lower adjustment includes: inhibitor, blocker, antagonist etc. Such as, described lower adjustment is to carry the spongy body viral vector in combinations with miR.

In another preference, the nucleotide sequence of the lower adjustment of described miR-342-3p such as shown in SEQIDNO:5 or its modified after product; It is preferred that its modified outcome is on the basis of nucleotide sequence shown in SEQIDNO:5, skeleton part phosphate group is carried out thio-modification, 2 ' nucleic acid carry out methoxyl group modification and 3 ' end coupling cholesterol.

In another preference, the nucleotide sequence of the lower adjustment of described miR-210 such as shown in SEQIDNO:8 or or its modified product; It is preferred that its modified outcome is on the basis of nucleotide sequence shown in SEQIDNO:8, skeleton part phosphate group is carried out thio-modification, 2 ' nucleic acid carry out methoxyl group modification and 3 ' end coupling cholesterol.

In another preference, described medicine is additionally operable to treat the disease with hypoxia pathological characters:

The expression of cell surface C5aR1 is blocked under tissue hypoxia microenvironment; Or

Expression and/or the phosphorylation of STAT5 and/or STAT6 is promoted under tissue hypoxia microenvironment; Or

The expression of USP13 is improved in tissue hypoxia microenvironment; Or

The expression of PTPN1 is reduced in tissue hypoxia microenvironment.

In another aspect of this invention, it is provided that the purposes of miR-342-3p or miR-210, for the mark as diagnosis or prognosis rheumatoid arthritis disease; Or for preparing the reagent of diagnostics classes rheumatic arthritic diseases.

In a preference, described diagnosis or prognosis rheumatoid arthritis disease include: miR-342-3p or the miR-210 expression of detection experimenter, expression is more high, then it represents that the extent of rheumatoid arthritis disease is more serious.

In another preference, the reagent of described diagnosis or prognosis rheumatoid arthritis disease is specific recognition or the reagent (such as probe or primer) of amplification miR-342-3p or miR-210.

In another aspect of this invention, it is provided that the purposes for diagnosing or in the test kit of prognosis rheumatoid arthritis disease prepared by the reagent of a kind of specific recognition or amplification miR-342-3p or miR-210.

In a preference, the reagent of described specific amplification miR-342-3p is the primer of nucleotide sequence such as SEQIDNO:23 and SEQIDNO:24; Or

The reagent of described specific amplification miR-210 is the primer of nucleotide sequence such as SEQIDNO:20 and SEQIDNO:21.

In another aspect of this invention, it is provided that a kind of method of potential material screening treatment rheumatoid arthritis disease, described method includes:

(1) candidate substances being contacted with the system comprising miR-342-3p and/or miR-210, described system is cell culture system; With

(2) filter out notable downward (as significantly lowered more than 20%, it is advantageous to downward more than 50%; Downward more than 80% more preferably) miR-342-3p and/or miR-210 express material, described material be treatment rheumatoid arthritis disease potential material.

In another preference, step (1) including: adds candidate substances in the system comprise miR-342-3p and/or miR-210; With

Step (2) including: the expression of detection miR-342-3p and/or miR-210, and compares with matched group, and wherein said matched group is without described candidate substances, the system expressing miR-342-3p and/or miR-210;

If candidate substances lowers the expression of miR-342-3p and/or miR-210 statistically, then this candidate substances is the potential material for the treatment of rheumatoid arthritis.

In a preference, described system is selected from (but not limited to): cell system (or cell culture system), subcellular fraction system, solution system, animal system or organizational framework.

In another preference, described candidate substances is selected from (but not limited to): for the disturbing molecule of miR-342-3p and/or miR-210 design, nucleic acid inhibitor, binding molecule (such as antibody or part), micromolecular compound.

In another preference, described method also includes: the potential material obtained is carried out further cell experiment and/or animal experiment, to select further from candidate substances and to determine the material useful for treatment rheumatoid arthritis disease.

The other side of the present invention, due to this disclosure, is apparent to those skilled in the art.

Accompanying drawing explanation

Fig. 1, hypoxia condition mononuclear origin dendritic cell high expressed C5aR1 to C5a and endogenous derivant thereof-remove arginic C5a (C5a_desArg) there is high response.

A mononuclear cell that () GM-CSF and IL-4 stimulates is cultivated when different oxygen concentrations respectively, including omnidistance normal oxygen (21%), omnidistance hypoxia (respectively 9,, or sequential hypoxia (9%, 3 days 5,2%), 5%, 2 days, 2%, 1 day, and 1%, 1 day), detect monokaryon dendritic cells derived under normal oxygen and hypoxia condition after 7 days and express C5aR1.

(b) C5a and derivant C5 thereof_adesArgMediate the chemotactic activity of monokaryon dendritic cells derived under normal oxygen and hypoxia (2%) condition. Represent with chemotactic index: have C5a or C5_adesArgThe mononuclear origin dendritic cell number of lower room/without C5a or C5 is entered under existence condition_adesArgMononuclear origin dendritic cell number (free diffusing) of lower room is entered under existence condition.

(c) C5a and derivant C5 thereof_adesArgUnder the normal oxygen stimulated and hypoxia (2%) condition, monokaryon dendritic cells derived expresses the level of costimulatory molecules-CD86 and HLA-DR.

(d) C5a and derivant C5 thereof_adesArgThe level of monokaryon dendritic cells derived secretion inflammatory factor under the normal oxygen stimulated and hypoxia (2%) condition.

(e) C5a and derivant C5 thereof_adesArgUnder the normal oxygen stimulated and hypoxia (2%) condition, monokaryon dendritic cells derived induces the level of CD4+T cell proliferation of the same race. CD4+T cell uses CFSE labelling in advance, after hatching 5 days altogether with dendritic cell, and the dilution situation of detection CFSE.

(f) C5a and derivant C5 thereof_adesArgUnder the normal oxygen stimulated and hypoxia (2%) condition, monokaryon dendritic cells derived induces the level of CD4+T cell activation of the same race.

(g) C5a and derivant C5 thereof_adesArgThe level that under the normal oxygen stimulated and hypoxia (2%) condition, monokaryon dendritic cells derived induces CD4+T cell differentiation of the same race to be secretion IFN �� and IL-17.

(h) C5a and derivant C5 thereof_adesArgUnder the normal oxygen stimulated and hypoxia (2%) condition, monokaryon dendritic cells derived induces CD4+T cell differentiation of the same race to be the level of regulatory T cells.

Fig. 2, hypoxia raise mononuclear origin 1 expressed by dendritic cells miR-210 and miR-342-3p.

Normal oxygen (N) and hypoxia (H, 2%O₂, below test and all adopt 2%O₂) person monocytic cell induces the expression of miR-210 (a) and miR-342-3p (b) in MoDC atomization at GM-CSF and IL-4 under condition.

MiR-210 and the miR-342-3p that Fig. 3, hypoxia raise promotes that MoDC expresses C5aR1.

Under normal oxygen condition, mononuclear cell is transfected analogies (mimic) and negative control (a), the inhibitor (inhibitor) transfecting miR-210 or miR-342-3p under low oxygen conditions and the negative control (b) thereof of miR-210 or miR-342-3p, adding GM-CSF and IL-4 after 48h in the medium induces it to break up, the change that after induction 96h, detection cell surface C5aR1 expresses.

Fig. 4, miR-210 and the miR-342-3p expression in collagen-induced experimental arthritis model (CIA).

In the DBA/1 arthritis mouse model (CIA) of II Collagen Type VI induction, the expression of morbidity different time serum miR-210 (a) and miR-342-3p (b). Ctrl: comparison (not inducing CIA).

Fig. 5, miR-210 and miR-342-3p analogies and mortifier are on the CIA impact being in progress.

The DBA/1 arthritis mouse model of II Collagen Type VI induction is carried out the antagonist (antagomir) of independent miR-210 or miR-342-3p of tail vein injection or the negative control (NC) of the two associating or antagonist respectively, Per-Hop behavior is once, started within every 2-3 days, to observe its arthritic situation in the 3rd week, including arthritic incidence rate (a), clinical score (b), representative inflammation swelling degree (c) of affected joints of each group, damaged articular cartilage situation (d) and pathology (e).

The downward of the mononuclear origin dendritic cell of STAT5 and the STAT6 activation induction that Fig. 6, low oxygen quenching GM-CSF and IL-4 trigger C5aR1 in atomization.

(a) mononuclear cell respectively under normal oxygen and hypoxia condition (2%), with and without GM-CSF, IL-4 or the two combine and exist in situation, the expression of C5aR1 after cultivating 7 days.

B () mononuclear cell is after transfection RNA interfering (negative control, STAT5 or STAT6), under there is situation in GM-CSF+IL-4, and the expression of C5aR1 after cultivating 4 days.

(c) mononuclear cell respectively in advance at normal oxygen and lower 36 hours of hypoxia condition (2%), then adding GM-CSF+IL-4, the expression of STAT5 and STAT6 before and after 8 hours.

D () mononuclear cell respectively in advance at normal oxygen and lower 36 hours of hypoxia condition (2%), is then adding the level of STAT5 and STAT6 phosphorylation before and after GM-CSF+IL-4, different time.

Fig. 7, miR-342-3p and miR-210 suppress STAT5 and STAT6 signal pathway.

A mononuclear cell is transfected the analogies of miR-342-3p and negative control, the inhibitor transfecting miR-342-3p under low oxygen conditions and negative control thereof by () under normal oxygen condition, add GM-CSF and IL-4 in the medium, detect the constitutive expression of STAT5 and STAT6 after 8 hours and the phosphorylation of STAT5 and STAT6 after 30min after 48h.

B mononuclear cell transfects the analogies of miR-210 and negative control thereof by () under normal oxygen condition, transfect under low oxygen conditions miR-210 inhibitor and and negative control, add GM-CSF and IL-4 after 48h in the medium, detect the constitutive expression of STAT5 and STAT6 after 8 hours and the phosphorylation of STAT5 and STAT6 after 30min.

Fig. 8, miR-342-3p are directly targeted STAT5b and suppress the constitutive expression of STAT5b.

(A) the miR-342-3p binding site of prediction is contained in 3 ' the UTR districts of people STAT5bmRNA.

(B) under normal oxygen condition, the luciferase reporter plasmid in carrier STAT5bmRNA3 ' UTR district and internal reference Hemicentrotus seu Strongylocentrotus kidney plasmid and miR-342-3p analogies or analogies compare cotransfection HEK293 cell; Under hypoxia is readjusted prices, the luciferase reporter plasmid in carrier STAT5bmRNA3 ' UTR district and internal reference Hemicentrotus seu Strongylocentrotus kidney plasmid and miR-342-3p mortifier or mortifier compare cotransfection HEK293 cell; Then the activity of relative fluorescence element enzyme is detected.

Fig. 9, miR-342-3p targeted inhibition USP13 expresses and then suppresses STAT5,6 signal paths.

A the miR-342-3p binding site of prediction is contained in 3 ' the UTR districts of () people USP13mRNA.

B (), under normal oxygen condition, the luciferase reporter plasmid in carrier USP13mRNA3 ' UTR district and internal reference Hemicentrotus seu Strongylocentrotus kidney plasmid and miR-342-3p analogies or analogies compare cotransfection HEK293 cell; Under low oxygen conditions, the luciferase reporter plasmid in carrier USP13mRNA3 ' UTR district and internal reference Hemicentrotus seu Strongylocentrotus kidney plasmid and miR-342-3p mortifier or mortifier compare cotransfection HEK293 cell; Then the activity of relative fluorescence element enzyme is detected.

C (), under normal oxygen condition, transfects miR-342-3p analogies or in comparison with mononuclear cell, or transfect miR-342-3p mortifier under low oxygen conditions or in comparison with mononuclear cell, detects the expression of USP13 after 48 hours.

D () is under low oxygen conditions by transfecting siRNA in CD14⁺Human peripheral blood mononuclear cell disturbs the expression of USP13, cultivates 4 days under GM-CSF and IL-4 exists, and collects cell FACS and detects the expression of cell C5aR1.

E () is under low oxygen conditions by transfecting siRNA interference USP13 in CD14⁺Human peripheral blood mononuclear cell, after 48h, supplementing GM-CSF and IL-4 stimulates 40min, collects cell and carries out the expression of STAT5, STAT6, p-STAT5, p-STAT6 in WesternBlot detection cell.

Figure 10, miR-210 targeting promotes PTPN1 expression and then the phosphorylation of suppression STAT5,6.

(A) the miR-210 binding site of prediction is contained in 3 ' the UTR districts of people PTPNmRNA.

(B) under normal oxygen condition, the luciferase reporter plasmid in carrier PTPN1mRNA3 ' UTR district and internal reference Hemicentrotus seu Strongylocentrotus kidney plasmid and miR-210 analogies or analogies compare cotransfection HEK293 cell; Under hypoxia is readjusted prices, the luciferase reporter plasmid in carrier PTPN1mRNA3 ' UTR district and internal reference Hemicentrotus seu Strongylocentrotus kidney plasmid and miR-210 mortifier or mortifier compare cotransfection HEK293 cell; Then the activity of relative fluorescence element enzyme is detected.

(C) under normal oxygen condition, transfect miR-210 analogies or in comparison with mononuclear cell, or transfect miR-210 mortifier under low oxygen conditions or in comparison with mononuclear cell, after 48 hours, detect the expression of PTPN1.

(D) the often expression of PTPN1 in the MoDC under oxygen and hypoxia condition.

(E) under low oxygen conditions by transfecting siRNA in CD14⁺Human peripheral blood mononuclear cell disturbs the expression of PTPN1, cultivates 4 days under GM-CSF and IL-4 exists, and collects cell FACS and detects the expression of cell C5aR1.

(F) under low oxygen conditions by transfecting siRNA interference PTPN1 in CD14⁺Human peripheral blood mononuclear cell, after 48h, supplementing GM-CSF and IL-4 stimulates 40min, collects cell and carries out the expression of p-STAT5, p-STAT6 in WesternBlot detection cell.

Detailed description of the invention

The present inventor is through deep research, confirm that microRNA-342-3p and microRNA-210 (being called for short miR-342-3p and miR-210) plays an important role in the generation and evolution of rheumatoid arthritis disease first, it is possible to as the mark of diagnosis and prognosis rheumatoid arthritis. Further, also disclose the expression lowering miR-342-3p and/or miR-210 first and can alleviate the symptom of rheumatoid arthritis. Complete the present invention on this basis.

MiR-342-3p or miR-210 and application thereof

MicroRNA (miRNA) is the non-coding strand tiny RNA that a class length is about 18-25 nucleotide, interact mainly through the miRNA controlling element MRE in 3 ' the UTR districts with target gene mRNA, thus translation after regulating and controlling the transcribing of target gene, have now been found that most of miRNA has by suppressing the translation of target gene or promoting the degraded of target gene mRNA and play negative regulation effect, but also have minority miRNA to have the effect promoting that target gene is translated after transcribing.

The invention discloses miR-342-3p and/or miR-210 and inhibitor thereof the purposes in the medicine and diagnostic reagent of preparation rheumatoid arthritis. Described miR-342-3p is the micro ribonucleic acid with nucleotide sequence shown in SEQIDNO:1:

5��-UCUCACACAGAAAUCGCACCCGU-3��(SEQIDNO:1)��

Described miR-210 is the micro ribonucleic acid with nucleotide sequence shown in SEQIDNO:2:

5��-CUGUGCGUGUGACAGCGGCUGA-3��(SEQIDNO:2)��

Hypoxia microenvironment (in rheumatoid arthritis the hypoxia in affected joints) raises the expression of miR-342-3p and/or miR-210 in mononuclear origin dendritic cell, miR-342-3p is by acting on its target gene STAT5b and USP13, miR-210 acts on target gene PTPN1 and suppresses inflammatory factor GM-CSF and IL-4 induced monocyte to be divided into the effect lowering C5aR1 in dendritic cell process, thus maintaining mononuclear origin dendritic cell high expressed C5aR1, maintain the high response of the mononuclear origin dendritic cell of C5a induction, thus having promoted generation and the development of rheumatoid arthritis. therefore, the level of miR-342-3p or miR-210 in detection serum, it is possible to rheumatoid arthritis disease activeness and prognosis are made evaluation, this kind of disease can be treated by the inhibitor of miRNA-342-3p or miR-210.

Described miR-342-3p and/or miR-210 can be separated from cell, or can be obtained by the mode of synthetic. After knowing the sequence of miR-342-3p and/or miR-210 or its precursor, those skilled in the art can prepare miR-342-3p and/or miR-210 or its precursor easily.

Based on the elaboration of the present invention, miR-342-3p and/or miR-210 is a new and closely-related drug target of rheumatoid arthritis. Various treatment meanss for miR-342-3p and/or miR-210 can as the novelty of the rheumatoid arthritis of preventing and treating animal (particularly people) and effective means.

Further, miR-342-3p and/or miR-210 can be used for the target spot as drug screening, screens the medicine alleviating or treating rheumatoid arthritis by lowering the expression of miR-342-3p and/or miR-210.

The therapeutic use adjusted under miR-342-3p and/or miR-210

Based on the new discovery of the present inventor, the lower adjustment of described miR-342-3p and/or miR-210 can be used for the compositions (medicine) of preparation treatment rheumatoid arthritis.

As used herein, described " lower adjustment " includes " inhibitor ", " antagonist ", " blocker " etc.

The lower adjustment of described miR-342-3p and/or miR-210 refer to any reduce miR-342-3p and/or miR-210 or its precursor activity, the stability reducing miR-342-3p and/or miR-210 or its precursor, the expression lowering miR-342-3p and/or miR-210 or its precursor, the material that reduces miR-342-3p and/or miR-210 or its precursor effective acting time, these materials are used equally to the present invention, as the material useful for lowering miR-342-3p and/or miR-210, thus can be used for alleviating or treating rheumatoid arthritis.

As the optimal way of the present invention, the nucleotide sequence of the lower adjustment of described miR-342-3p is such as shown in SEQIDNO:5 or its modified forms. The nucleotide sequence such as SEQIDNO:8 of the lower adjustment of described miR-210 or its modified forms. The inventors discovered that, adopt these lower adjustments to have the excellent effect lowering miR-342-3p and/or miR-210.

Pharmaceutical composition

Present invention also offers a kind of compositions, it contains effective dose (such as 0.000001-50wt%; Preferably 0.00001-20wt%; More preferably, 0.0001-10wt%) described miR-342-3p and/or miR-210 under adjust, and pharmaceutically acceptable carrier. Described compositions can be used for alleviating or treating rheumatoid arthritis.

As used herein, described " effective dose " refers to amount that is that people and/or animal can produce function or activity and that can be accepted by people and/or animal. Described " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent. This term refers to so some medicament carriers: themselves be not necessary active component, and does not have undue toxicity after using. Suitable carrier is well known to those of ordinary skill in the art. Pharmaceutically acceptable carrier can contain liquid in the composition, such as water, saline, buffer. It addition, these carriers there is likely to be complementary material, such as filler, lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance etc. Described carrier can also contain lipofectamine.

After the purposes adjusted under knowing described miR-342-3p and/or miR-210, it is possible to adopt multiple method well known in the art that adjustment under described miR-342-3p and/or miR-210 or their pharmaceutical composition are delivered medicine to mammal. Include but not limited to: subcutaneous injection, intramuscular injection, percutaneous give, administer locally to, implant, slow release gives.

The effective dose adjusted under miR-342-3p and/or miR-210 of the present invention can change with the order of severity etc. of the pattern of administration and disease to be treated. The selection of preferred effective dose can be determined (such as passing through clinical trial) by those of ordinary skill in the art according to various factors. Described factor includes but not limited to: the pharmacokinetic parameter such as bioavailability, metabolism, half-life etc. adjusted under described miR-342-3p and/or miR-210; The order of severity of the disease that patient to treat, the body weight of patient, the immune state of patient, administration approach etc. Generally, give with the dosage of about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals) every day when adjusting under miR-342-3p and/or miR-210 of the present invention, gratifying effect can be obtained. Such as, by an urgent demand for the treatment of situation, several times dosage separately can be given every day, or dosage is reduced pari passu.

MiR-342-3p and/or miR-210 is as the mark of Diagnosis of Rheumatoid Arthritis and prognosis

MiR-342-3p and/or miR-210 is diagnosis and the closely-related target spot of prognosis of announcement and rheumatoid arthritis first. Diagnosis and the prognosis of rheumatoid arthritis, the curative effect of earlier evaluations treating rheumatoid arthritis medicine, optimizing therapeutic regimen is may be used for for the various detectable of miR-342-3p and/or miR-210. By miR-342-3p and/or miR-210 expression height before and after medication, patient can be passed judgment on after the treatment the need of long-term prescription and formulation therapeutic regimen.

Various technology known in the art can be adopted to detect presence or absence and the expression of miR-342-3p and/or miR-210 in cell, and these technology are all contained in the present invention. Such as can sending out with existing technology such as in situ hybridization, polymerase chain reaction technology (PCR), Southern blotting, DNA sequence analysis etc., these methods may be used in combination.

Present invention also offers for detecting the presence or absence of miR-342-3p and/or miR-210 and the reagent of expression in analyte. As a kind of optimal way, it is possible to adopt the primer of specific amplification miR-342-3p and/or miR-210; Or the probe of specific recognition miR-342-3p and/or miR-210 determines the presence or absence of miR-342-3p and/or miR-210.

As miR-342-3p and/or miR-210 in the optimal way of the present invention, detection blood plasma or serum or joint fluid.

As the optimal way of the present invention, described reagent is primer, and it can go out miR-342-3p and/or miR-210 by specific amplification. It is furthermore preferred that the reagent of specific amplification miR-342-3p is the primer of nucleotide sequence such as SEQIDNO:23 and SEQIDNO:24; Or the reagent of specific amplification miR-210 is the primer of nucleotide sequence such as SEQIDNO:20 and SEQIDNO:21.

Design for the specific probe of miR-342-3p and/or miR-210 is technology well known in the art, such as, a kind of probe of preparation, it can occur specific binding with specific site on miR-342-3p and/or miR-210, and other gene specific beyond miR-342-3p and/or miR-210 is not combined, and described probe is with detectable signal.

The test kit of detection miR-342-3p and/or miR-210

Present invention also offers for detecting the presence or absence of miR-342-3p and/or miR-210 and the test kit of expression in analyte, this test kit includes: the reagent of specific recognition or amplification miR-342-3p and/or miR-210. This test kit can be used for carrying out diagnosis and the prognosis of rheumatoid arthritis.

As a kind of optimal way, described test kit is PCR detection kit, including the primer of specific amplification miR-342-3p and/or miR-210. It is furthermore preferred that the reagent of specific amplification miR-342-3p is the primer of nucleotide sequence such as SEQIDNO:23 and SEQIDNO:24; Or the reagent of specific amplification miR-210 is the primer of nucleotide sequence such as SEQIDNO:20 and SEQIDNO:21.

Alternatively, described test kit is: PCR detection kit or in situ hybridization test kit. Including the probe of specific recognition miR-342-3p and/or miR-210, and also there is detectable signal.

Additionally, described test kit may also include the various reagent required for extracting DNA, PCR, hybridization, colour developing etc., include but not limited to: extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing liquid etc.

Additionally, described test kit may also include operation instructions and/or Nucleotide Sequence Analysis Software etc.

Screening technique

New discovery based on the present inventor, the research of miR-342-3p and/or miR-210 there is many-sided purposes, described purposes includes, but is not limited to: the material of miR-342-3p and/or miR-210 is lowered in screening, and this material can be used for the medicine that preparation is alleviated or treated rheumatoid arthritis.

Therefore, the method that the invention provides the potential material of screening treatment rheumatoid arthritis, candidate substances is contacted with the system comprising miR-342-3p and/or miR-210; Filtering out the material lowering miR-342-3p and/or miR-210, described material is the potential material for the treatment of rheumatoid arthritis.

As used herein, described " downward " or " suppression " refers to " downward " or " suppression " with statistical significance. That is: significantly " downward " or " suppression ". As compared with the protein active of the matched group not giving candidate substances, protein expression, protein-interacting, notable " downward " or " suppression " more than 20%, it is advantageous to more than 50%; More than 80% more preferably.

The described system comprising miR-342-3p and/or miR-210 is selected from: cell system (or cell culture system), subcellular fraction system, solution system, animal system or organizational framework.

As the optimal way of the present invention, described method also includes: the potential material obtained is carried out further cell experiment and/or animal experiment, to select further from candidate substances and to determine the material useful for treatment rheumatoid arthritis.

When screening, it is possible to adopt various technology well known in the art to determine situation of change and the interaction situation of albumen or gene.

The technology that can adopt multiple routine transcribing or protein expression situation of gene in identification systems. These technology include but not limited to: oligonucleotide hybridization technology (such as probe), polymerase chain reaction,PCR (PCR), polyacrylamide gel electrophoresis, immunoblotting (such as western blot) etc.

The material gone out by said method Preliminary screening may make up a screening storehouse, in order to people may finally therefrom filter out material that can be actually useful for treating rheumatoid arthritis.

Compared with the prior art, the present invention has the technique effect of following uniqueness:

The present invention illustrates the relation between hypoxia-dendritic cell-C5a/C5aR1 three around microRNA-miR-342-3p and miR-210 first, and explains this three mechanism of action in promoting rheumatoid arthritis reason process with this. On this basis, it is proposed that miR-342-3p and miR-210 and inhibitor thereof the purposes in rheumatoid arthritis. In addition, be different from the medicine treating at present rheumatoid arthritis: for the monoclonal antibody of TNFa or soluble type TNFa receptor and C5aR antagonist (as, micromolecular compound PMX-53), the present invention is based on the drug target of microRNA, has the superiority of microRNA.

Below in conjunction with specific embodiment, the present invention is expanded on further. Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention. The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition such as J. Pehanorm Brooker etc. are write, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to manufacturer it is proposed that condition.

Material and method

1, cell and cell line and laboratory animal

Human peripheral blood single nucleus cell, from Shanghai Changhai Hospital. Human Embryonic Kidney HEK 293 cell strain, purchased from ATCC, by the DMEM culture medium containing 10% hyclone, is cultivated, routine passage at the standard conditions. Female DBA/1 mice (6-8w) is purchased from Beijing China Fukang animal center.

2, molecular cloning reagent, other main agents and antibody

DNA reclaim test kit QuickGelExtractionKit, plasmid little extraction reagent kit PlasmidMiniprepKit, PCR purification kit PCRCleanupKit all purchased from AXYGEN company, cell or serum RNA separating kit purchased from Invitrogen company. Cell total rna extraction agent Trizol is purchased from GIBCOBRL. PrimeScriptRT-PCR Reverse Transcription box, SYBRPremixExTaq PCR kit for fluorescence quantitative are purchased from TAKARA. Luciferase assays test kit is purchased from Promega.

RPM1640 culture medium, DMEM culture medium and hyclone (FBS) are all purchased from PAA company. Anti-human CD14 magnetic bead and corresponding screening installation are purchased from MiltenyiBiotec company. The antibody such as anti-human fluorescent antibody such as CD14 and isotype control Ab thereof (Isotype) are all purchased from BDPharmingen company. C5a and C5a_desArgPurchased from Hycult company; Anti-CD88, p-STAT5, anti-p-STAT6, anti-STAT5, anti-STAT6, anti-PTPN1 antibody are purchased from Abcam company. Protease inhibitor cocktail (Proteaseinhibitorcocktail), chicken II Collagen Type VI (CollagenII), complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) purchased from American Sigma company. People recombinant cytokine GM-CSF, IL-4 are purchased from Peprotech company.

3, the transfection of miRNA and siRNA

The inhibitor (antagomir) of miR-210, miR-342-3p of the cholesterol coupling needed for the inhibitor (inhibitors) of miR-210, miR-342-3p needed for experiment in vitro and analogies (mimics), internal transfection synthesizes by Guangzhou Rui Bo company. SiRNA and comparison RNA thereof synthesizes by Shanghai Ji Ma company. Transfection concentrations: miRmimic (20nM); MiRinhibitor (50nM); SiRNA (20nM). Transfection reagent adopts the Polyplus of INTERFERIN company.

MiR-342-3p analogies (mimic) sequence is (when using, two are added jointly with equivalent, are also the form of double-stranded RNA mixture):

5 '-ucucacacagaaaucgcacccgu-3 ' (SEQIDNO:3);

3 '-agagugugucuuuagcgugggca-5 ' (SEQIDNO:4);

MiR-342-3p inhibitor (inhibitor) sequence is shown in SEQIDNO:5, and modifies increase stability through 2 ' nucleic acid methoxyl groups:

3 '-agagugugucuuuagcgugggca-5 ' (SEQIDNO:5);

MiR-210 analogies (mimic) sequence is:

5 '-CUGUGCGUGUGACAGCGGCUGA-3 ' (SEQIDNO:6);

3 '-GACACGCACACUGUCGCCGACU-5 ' (SEQIDNO:7);

MiR-210 inhibitor (inhibitor) sequence is shown in SEQIDNO:8, and modifies increase stability through 2 ' nucleic acid methoxyl groups:

3 '-GACACGCACACUGUCGCCGACU-5 ' (SEQIDNO:8);

The special siRNA sequence of STAT5 is:

5 '-CCGCCAUAUAUUGUACAAUTT-3 ' (SEQIDNO:9);

5 '-AUUGUACAAUAUAUGGCGGTT-3 ' (SEQIDNO:10);

The special siRNA sequence of STAT6 is:

5 '-GCACCCUUGAGAGCAUAUATT-3 ' (SEQIDNO:11);

5 '-UAUAUGCUCUCAAGGGUGCTT-3 ' (SEQIDNO:12);

The special siRNA sequence of USP13 is:

5 '-CCGGAAUUCUCCUCUAACATT3 ' (SEQIDNO:13);

5 '-UGUUAGAGGAGAAUUCCGGTT-3 ' (SEQIDNO:14);

The special siRNA sequence of PTPN1 is:

5 '-GAGCCACACAAUGGGAAAUTT3 ' (SEQIDNO:15);

5��-AUUUCCCAUUGUGUGGCUCTT-3��(SEQIDNO:16)��

MiR-210 antagonist (antagomir) sequence be through chemical modification SEQIDNO:8 sequence, this modification includes: to the skeleton part phosphate group (5 ' phosphoric acid of G; Pyrimidine (includes U, C) ribose-3 '-phosphoric acid site; 5 ' and the 3 ' free hydroxyl groups held) carry out thio-modification, 2 ' nucleic acid carry out methoxyl group modification, and 3 ' end coupling cholesterol.

MiR-342-3p antagonist (antagomir) sequence is that this modification includes through chemical modification SEQIDNO:5 sequence: to the skeleton part phosphate group (5 ' phosphoric acid of G; Pyrimidine (includes U, C) ribose-3 '-phosphoric acid site; 5 ' and the 3 ' free hydroxyl groups held) carry out thio-modification, 2 ' nucleic acid carry out methoxyl group modification, and 3 ' end coupling cholesterol.

SiRNA control sequence is:

5 '-UUCUCCGAACGUGUCACGUTT-3 ' (SEQIDNO:17); With

5��-ACGUGACACGUUCGGAGAATT-3��(SEQIDNO:18)��

4,3 ' UTR reporter genes and mutational vector thereof build

3 ' UTR gene orders design primer (seeing below) according to people PTPN1, STAT5b, USP13mRNA. With person monocytic cell cDNA for template, above-mentioned primer obtains its 3 ' UTR through pcr amplification, after adopting double digestion method enzyme action pcr amplification product respectively and pMIR plasmid, after T4 ligase digestion products and digested plasmid carrier, routine transformation recombiant plasmid, picking bacterial colony, purification also check order, and finally respectively obtain the Firefly luciferase reporter plasmid of the 3 ' UTR of PTPN1, STAT5, USP13.

5, luciferase reporter gene detection and analysis

HEK293 cell is inoculated in 96 orifice plates, and cell concentration is 1-5 �� 10⁴After cultivating 12h under standard conditions, lipo2000 transfection reagent is used with reference to description, every hole corotation 40ngPTPN1/STAT5/USP133 ' UTR luciferase reporter gene, 10ngpRL-TK-luciferase internal reference plasmid, and the mimics/inhibitors/ comparison of the miRNA of final concentration of 20nM. After transfection 24h, use luciferase reporter gene detection system detection fluorescence activity. Record Firefly fluorescent value and the ratio of internal reference Renilla fluorescent value, be 3 ' UTR reporter gene activity values.

6, flow cytometry (FACS)

Collect single cell suspension, wash once with pre-cooling PBS, be resuspended in 50 �� lPBS, fluorescein-labeled streaming antibody and isotype control Ab are diluted to suitable concn, and room temperature labeled cell 20-30min, after washing twice with 1mlPBS, being resuspended in 200 �� lPBS, upper machine testing is also analyzed; If antibody unstressed configuration element labelling such as C5aR1, then the room temperature labelling 20-30min of required detection, after washing with 1mlPBS, it is resuspended in 50 �� lPBS, anti-it is diluted to suitable concn, resuspended and upper machine after room temperature labelling 20-30min, PBS washing by fluorescein-labeled corresponding two. The immunofluorescence dyeing of intracellular molecules, first by cell through the fixing 15min of 4% paraformaldehyde, then the antibody of fluorescein coupling prepared with the penetrating fluid containing 0.1%Saponin and add in the cell after fixing, room temperature labeled cell 120min or 4 DEG C overnight, upper machine after washing with the penetrating fluid containing 0.1%Saponin. FACSLSRII flow cytometry analysis, software are FACSDivasoftware and Flojo6.7.

7��Westernblot

Cell pre-cooling PBS washes once, adds the full cell pyrolysis liquid of M-PER containing cocktail protease inhibitor, ice bath 30min, 12000rpm4 DEG C of centrifugal 30min, takes supernatant. Take trim after 5 �� l protein sample BCA protein quantification kit measurement concentration, add 6 �� sample-loading buffer (0.5MTris-HCl, PH6.8, glycerol 5ml, 20%SDS2ml, 5% bromophenol blue 0.5ml, add 100 �� l beta-mercaptoethanols in every 1ml buffer before using), boiling water bath 5min, frozen standby in-20 DEG C. During WesternBlot detection, the SDS-PAGE glue of respective concentration is prepared according to testing protein molecular size range, take after 50 �� g albumen are separated by electrophoresis, albumen on SDS-PAGE is transferred on nitrocellulose filter, after 1h closed by 5% skim milk, 4 DEG C of overnight incubation of specificity primary antibodie, TBST (Tris12.114g, NaCl8.766g, Tween200.1%, PH7.5) wash film three times, each 10min, then film (ibid) is washed with HRP ELIAS secondary antibody incubated at room 2-3h, TBST. It is eventually adding chemical luminous substrate autography.

8, capital equipment and software

Primer-design software adopts PrimerPremier5.0. Quantitative real time PCR Instrument runs software and data analysis software is Lightcycler3.

Embodiment 1, hypoxia condition mononuclear origin dendritic cell high expressed C5aR1 to C5a and derivant C5a thereof_desArgThere is high response

Hypoxia is an important pathological characters of diseases associated with inflammation, is mainly reflected in promotion inflammatory cell release inflammatory factor. As inflammatory factor most commonly seen in diseases associated with inflammation-complement c5a anaphylatoxin, how hypoxia affects the immunocyte reactivity to C5a, has not yet to see report. Experimental procedure is as follows:

(1) separation of PBMC

After 1U White Blood Cells Concentrate normal saline dilution, it is added slowly to the upper strata of density 1.077 lymphocyte separation medium, 2000rpm is centrifuged 30min, it is divided into four layers, upper strata is serum and normal saline, the second layer is mononuclearcell, third layer is lymphocyte separation medium, and orlop is erythrocyte, carefully draws the mononuclearcell of the second layer with capillary tube to 50ml centrifuge tube, and with normal saline dilution, 1500rpm is centrifuged 10min, abandons supernatant, adds 40ml brine once, 800rpm is centrifuged 5min, abandons supernatant. Using normal saline re-suspended cell, adjusting PBMC concentration is 1 �� 10⁸Individual/ml, adds 40-60 �� l people's anti-CD14 magnetic bead, and mixing, 4 DEG C of lucifuges hatch 15-20min, and once, 800rpm is centrifuged 5min to brine, abandons supernatant, and 2ml normal saline is resuspended standby. LS type MACS detached dowel is placed on the Beads enrichment frame in magnetic field by by specification, adds 2ml normal saline prewashing. 30 ��m of nylon wires are placed on detached dowel, the cell suspension of anti-CD14 marked by magnetic bead is joined nylon wire, after liquid flows to end naturally, with 2ml brine detached dowel 3 times, naturally flow to end every time. Detached dowel is withdrawn magnetic field, adds 4ml normal saline, push away most liquid with nook closing member, collect the anti-CD14 cell of marked by magnetic bead, be repeated once. The centrifugal 5min of CD14+ mononuclear cell 1000rpm that will collect, adjusts to debita spissitudo, bed board by 1640 culture medium containing 10% hyclone.

(2) induction of normal oxygen and hypoxia condition MoDC

By above-mentioned mononuclear cell, cultivate in the complete medium containing GM-CSF (final concentration 100ng/ml) and IL-4 (final concentration 10ng/ml), be individually positioned in normal oxygen (21%O₂) and hypoxia condition (2-9%O₂) under, terminate after 7 days, the expression of detection cell surface C5aR1 or with C5a and remove arginic C5a (Ca_desArg) stimulate carry out subsequent experimental.

(3) normal oxygen and hypoxia condition MoDC are to C5a and C5a_desArgReactivity

Collect normal oxygen and hypoxia condition MoDC, under normal oxygen condition, with C5a and C5a_desArgNormal oxygen and hypoxia MoDC are carried out Chemotaxis test; And at hypoxia (2%O₂) under condition, with C5a and C5a_desArgStimulate 24h respectively, then collect supernatant, the level of CBA (purchased from BDPhrmingen) method detection inflammatory factor; Collecting cell, detection phenotype and hatching altogether 7 days with CD4+T cell of the same race, detection T cell is Proliferative Activated and the differentiation of induction Th1/Th17/Treg, and above facs analysis adopts regular growth surface and Intracellular immunofluorence dyeing.

It was found that under different hypoxia conditions, the moDC of hypoxia condition expresses high-caliber C5aR1 (Fig. 1 a); And relative to normal oxygen MoDC, C5a and derivant C5a thereof_desArgThe chemotaxis of MoDC under hypoxia condition is become apparent from (Fig. 1 b); Relative to normal oxygen MoDC, C5a and derivant C5a thereof_desArgStimulation can increase under hypoxia condition MoDC significantly and express costimulatory molecules-CD86 (Fig. 1 c), secrete inflammatory factor-IL-1 ��, TNFa, IL-6 and MCP-1 (Fig. 1 d); C5a and derivant C5a thereof_desArgThe hypoxia condition MoDC stimulated has the characteristic of function maturation DC, it is embodied in Th17/Th1 mixed type cell (Fig. 1 g) that can induce CD4+T cell proliferation (Fig. 1 e) of the same race, activation (expressing CD25) (Fig. 1 f), the secretion IL-17 inducing CD4+T cell differentiation to be inflammatory and IFN-��, but suppresses that there is negative sense immunoregulation FoxP3+ regulatory T cells differentiation (Fig. 1 h). Therefore, under low oxygen conditions, mononuclear origin dendritic cell high expressed C5aR1 to inflammatory stimulus-C5a and derivant C5a thereof_desArgThere is high response.

The studies above shows, hypoxia is by promoting that DC high expressed C5aR1 promotes C5a and derivant C5a thereof_desArgThe inflammatory reaction of induction. Because 2% hypoxia condition raises the C5aR1 level expressed the most substantially, subsequent embodiment all adopts 2% hypoxia.

Embodiment 2, hypoxia strengthen the expression of miR-210 and miR-342-3p

The present inventor utilizes miRNA high flux chip detection to cultivate respectively and induces the miRNA express spectra change of 72h at normal oxygen and hypoxia condition resulted in monocyte at GM-CSF and IL-4. The inventors discovered that, hypoxia can affect a series of miRNA expression and change. Find that the fluorescence intensity of miR-210 and miR-342-3p is higher than 2000 after normalization, and they have the obvious rise of statistical significance. It follows that the expression enhancing whether the two miRNA is participated in regulation and control hypoxia inducible C5aR1 by the present inventor has been studied. Experimental procedure is as follows:

Adopt real-time quantitative RT-PCR (quanlitativeRT-PCR, qRT-PCR) method checking miRNA chip results. With TRIzol reagent extracting cell total rna. With PrimeScriptRT-PCR test kit with reference to description reverse transcription synthesis cDNA, with cDNA for template, with SYBRPremixExTaq test kit and on LightCycler real-time PCR the mRNA level in-site of detection by quantitative molecules of interest. The change relatively of sample amplification all uses 2-�� �� Ct method to calculate.

(1) reverse transcription (RT) primer of miR-210:

5 '-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGC-3 ' (SEQIDNO:19);

(2) quantification PCR primer of miR-210:

5 '-AGGAGCTGTGCGTGTGACAGCG-3 ' (forward) (SEQIDNO:20);

5 '-GTGCAGGGTCCGAGGT-3 ' (reversely) (SEQIDNO:21);

(3) miR-342-3p reverse transcription primer is:

5 '-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGGG-3 ' (SEQIDNO:22);

(4) quantification PCR primer of miR-342-3p:

5 '-AGGAGTCTCACACAGAAATCGCA-3 ' (forward) (SEQIDNO:23);

5 '-GTGCAGGGTCCGAGGT-3 ' (reversely) (SEQIDNO:24).

(5) relative quantification of miRNA uses U6 small nuclear rna to be internal reference, the reverse transcription primer of U6:

5 '-AACGCTTCACGAATTTGCGT-3 ' (SEQIDNO:25),

(6) U6 small nuclear rna quantification PCR primer:

5 '-CTCGCTTCGGCAGCACA-3 ' (forward) (SEQIDNO:26);

5 '-AACGCTTCACGAATTTGCGT-3 ' (reversely) (SEQIDNO:27);

(6) quantitative pcr amplification system is 20 �� l, wherein:GreenMix:10 �� l; CDNA template: 4 �� l; Forwards/reverse primer (10 ��Ms): each 0.5 �� l; DdH2O: polishing is to 20 �� l.

(7) reaction condition: 94 DEG C, 15s; 40 circulations: 94 DEG C, 20s; 55 DEG C, 10s; 72 DEG C, 10s; 72 DEG C, 10min.

It was found that miR-210 and miR-342-3p significantly raises under low oxygen conditions, such as Fig. 2.

Embodiment 3, miR-210 and miR-342-3p raise MoDC and express C5aR1

Further, present inventors studied miR-210 and miR-342-3p and MoDC cell under hypoxia condition is expressed the impact of C5aR1. Experimental procedure is as follows:

(1) person monocytic cell is separated as previously mentioned.

(2) under normal oxygen condition, mononuclear cell is transfected analogies mimics and the mimics comparison of miR-210 or miR-342-3p, the inhibitor (inhibitor) transfecting miR-210 or miR-342-3p under low oxygen conditions and inhibitor comparison respectively, adding GM-CSF and IL-4 in the medium induces it to break up, the change that after induction 96h, detection cell surface C5aR1 expresses.

MiRmimic uses final concentration of 20nM; The final concentration of 50nM of miRinhibitor. The purification CD14+ mononuclear cell of fresh separated is incubated in 6 orifice plates, and concentration is adjusted to 1 �� 10⁶Every hole, with reference to the transfection of INTERFERin reagent description.

Found that, after transfecting miR-210 or miR-342-3p analogies under normal oxygen condition, MoDC expresses C5aR1 significantly raised (Fig. 3 a), and after transfecting the inhibitor of miR-210 or miR-342-3p under low oxygen conditions, MoDC expresses C5aR1 then substantially reduction (Fig. 3 b).

Therefore, hypoxia expresses miR-210 and miR-342-3p by induced monocyte, participates in hypoxia and maintains high expressed C5aR1 in MoDC atomization.

Embodiment 4, miR-210 and the miR-342-3p expression in collagen-induced experimental arthritis model (CIA)

It is known that during the rheumatoid arthritis of the mankind (RA) affected joints chamber and synovial tissue exist complement activation and be in the inflammatory microenvironment of a kind of hypoxia, illustrate that RA relevant to hypoxia and C5a all has closely-related disease. In view of above result of study, present inventor considered that miR-210 and miR-342-3p likely with RA disease association, participate in the process of RA disease, and the inhibitor for them likely has therapeutic effect. Therefore, it follows that the mice model of rheumatoid arthritis (CIA) that the present inventor is induced by II Collagen Type VI, a good model of a kind of reflection mankind RA generally acknowledged at present, the supposition of the present inventor is verified. Experimental procedure is as follows:

(1) induction of CIA

Powdered inactivates tubercule bacillus be dissolved in the completely not formula adjuvant (CFA) of 2mg/ml and make the final concentration of 1mg/ml of tubercule bacillus, with isopyknic 2mg/ml chicken mutual emulsifying 30-60min of II Collagen Type VI (CollagenII) solution, in DBA/1 (6-8 week old) mouse tail subcutaneous injection, every 100 �� l. After 21 days, by still afterbody subcutaneous injection after incomplete Freund's adjuvant (IFA) and the mutual emulsifying of equal-volume CollagenII. Generally fall ill in 4-6 week after first time injection. 3 weeks after first time immunity start assessment mouse disease situation weekly, including: CIA sickness rate; Mouse ankle joint diameter; Clinical score; And do tissue HE dyeing. Clinical score standard: 0 point, normally; 1 point, mild swelling; 2 points, moderate swelling; 3 points, all arthroncuss; 4 points, joint deformity and (or) dysfunction; Extremity are assessed respectively, and score adds up to the clinical score of a mice.

(2) serum collection and PCR detection

After the first time induction of CIA arthritis model mice, after 1 week (w), their early stage i.e. first time induction, 4 weeks and morbidity stable phase have detected the expression of serum miR-210 and miR-342-3p for 8 weeks respectively after namely inducing for the first time. PCR method detection as described in Example 2.

It was found that in CIA pathogenic process, mice serum miR-210 (Fig. 4 A) and miR-342-3p significantly raised (Fig. 4 B).

The above results is pointed out, and miR-210 and miR-342-3p and CIA progression of disease have dependency. Therefore, miR-210 and miR-342-3p can be used to indicate the activeness of RA disease and therapeutic effect and prognosis.

Embodiment 5, miR-210 and miR-342-3p mortifier on the CIA impact being in progress

It follows that the antagonist that the present inventor utilizes miR-210 and miR-342-3p observes they impacts on CIA disease degree. Experimental procedure is as follows:

(1) antagonist of miR-210 and the miR-342-3p treatment to CIA

Latter 1 day of the 2nd immunity in induction CIA process respectively, tail vein injection mode vivo medicine-feeding, the antagonist (antagomir) of miR-210, miR-342-3p of cholesterol coupling is available from Guangzhou Rui Bo company, every 50nm, within every 1 week, being administered once, continuous 5 times, its arthritic situation is observed at interval for 2-3 days, assessment disease severity, terminates to from first time immunity for latter 60 days.

(2) detection of the CIA membranous type vivo immunization index of the antagonist for treating of miR-210 and miR-342-3p

In observing terminal, gather each group of mice affected joints picture, serum, morbidity joint tissue, nest lymph node, spleen. Wherein serum is used for detecting inflammatory cytokine TNF-�� and IL-6, soluble type C5aR1, adopts CBA method (as described in Example 1) and ELISA method detection; Joint tissue 10% formalin is fixed, and carries out H&E dyeing pathological analysis and micro-CT detection; Nest lymph node and spleen cell, fix through 4% paraformaldehyde, after the infiltration of the Permeat liquid of 0.1%Saponin, and Intracellular immunofluorence dyeing, FACS detect the percentage ratio of Th17/Th1 and Treg.

Found that, the antagonist of miR-342-3p can substantially delay arthritic generation, and the antagonist of miR-210 can slightly delay arthritic generation (Fig. 5 a), clinical score (Fig. 5 b), alleviates inflammation swelling degree (5c) of affected joints, articular cartilage damage (5d) and arthritis infiltration (5e). Therefore, miR-210 and miR-342-3p raises the morbidity and progress that promote CIA, and individually injects miR-342-3p or miR-210 antagonist and can delay and alleviate morbidity and the progress of CIA disease.

STAT5 and the STAT6 activation that embodiment 6, GM-CSF/IL-4 trigger is lowered C5aR1 and is expressed

Next step, the present inventor's Primary Study mechanism of hypoxia condition moDC high expressed C5aR1. Experimental procedure is as follows:

Prepare person monocytic cell as described in Example 1, be divided into 8 groups, 1 �� 10⁶Each 4 groups of/ml/ group, normal oxygen and hypoxia condition, 4 groups to be respectively as follows: the cultivation being not added with cytokine basis set, GM-CSF (50ng/ml) group, IL-4 (10ng/ml) group, GM-CSF+IL-4 group; After 7 days, FACS detects the expression (Fig. 6 a) of C5aR1.

Person monocytic cell is first transfected under normal oxygen condition STAT5 or STAT6 siRNA (20nm), add cytokine (GM-CSF+IL-4) after 36 hours, after 96 hours FACS detect C5aR1 expression (Fig. 6 b). The special siRNA sequence of STAT5 is such as SEQIDNO:9, shown in 10, mix according to 1:1, proceed to simultaneously; STAT6 special siRNA sequence such as SEQIDNO:11, shown in 12, mix according to 1:1, proceed to simultaneously.

Person monocytic cell is first cultivated 36 hours under normal oxygen and hypoxia condition, then add cytokine (GM-CSF+IL-4), after 8 hours FACS method detection STAT5 and STAT6 expression (Fig. 6 c), as shown in fig 6d time FACS method detection STAT5 and STAT6 phosphorylation.

Result shows, normal oxygen and hypoxia condition are not added with the level cultivating basis set monocytes C5aR1 of cytokine does not have notable difference, and under normal oxygen condition, GM-CSF (50ng/ml) group, IL-4 (10ng/ml) group, GM-CSF+IL-4 group can both lower significantly C5aR1 expression but under low oxygen conditions this downward be substantially suppressed (Fig. 6 a), illustrate under normal oxygen condition, the mononuclear cell of GM-CSF and IL-4 induction is lower to the expression of C5aR1 in DC atomization, and hypoxia inhibits this effect. Because GM-CSF activates STAT5, IL-4 and activates STAT6, inventors determined that STAT5 and STAT6 signal pathway is to the C5aR1 effect expressed further, it has been found that interference STAT5 and STAT6 can alleviate the downward (Fig. 6 b) of the C5aR1 of GM-CSF and IL-4 induction; Next, detect whether that hypoxia inhibits GM-CSF to activate STAT5, IL-4 and activates STAT6 signal, as speculate, hypoxia inhibits the constitutive expression (Fig. 6 c) of STAT5 and STAT6 really, and inhibits GM-CSF to activate the phosphorylation (Fig. 6 d) of STAT5, IL-4 activation STAT6.

To sum up, hypoxia condition MoDC high expressed C5aR1 is derived from the down-regulated expression of the C5aR1 that hypoxia inhibits STAT5 and the STAT6 activation of GM-CSF induction in MoDC atomization to induce.

Embodiment 7, miR-342-3p and MiR-210 lower the expression of C5aR1 by suppressing the activation of STAT5 and STAT6 to block MoDC in atomization

Further, the present inventor has inquired into whether the expression of hypoxia condition rise miR-342-3p and miR-210 participates in suppressing the activation of STAT5 and STAT6 in MoDC atomization, thus maintaining hypoxia condition MoDC high expressed C5aR1. Experimental procedure is as follows:

Prepare person monocytic cell as described in Example 1, be divided into 6 groups, 1 �� 10⁶Each 3 groups of/ml/ group, normal oxygen and hypoxia condition; Normal oxygen condition 3 groups is respectively as follows: transfection control miRmimic group, miR-210mimic group, miR-342-3pmimic group; Hypoxia condition 3 groups is respectively as follows: transfection control miRinhibitor group, miR-210inhibitor group, miR-342-3pinhibitor group; After 24 hours, adding GM-CSF+IL-4, after 4 days, FACS detects the expression (Fig. 7 a) of C5aR1. Or above-mentioned each group 48 hours after transfection, add GM-CSF+IL-4, as shown in Figure 7b time FACS detect STAT5 and STAT6 expression (Fig. 7 a).

Result shows, after transfecting the analogies of miR-342-3p under normal oxygen condition, the expression of STAT5 and STAT6 substantially reduces; After transfecting the inhibitor of miR-342-3p under low oxygen conditions, the expression of its STAT5 and STAT6 then has significantly rise (Fig. 7 a); After transfecting the analogies of miR-210 under normal oxygen condition, the phosphorylation of STAT5 and STAT6 substantially reduces; After transfecting the inhibitor of miR-210 under low oxygen conditions, the phosphorylation of its STAT5 and STAT6 then has significantly rise (Fig. 7 b); Therefore miR-342-3p mainly suppresses the constructive expression of STAT5 and STAT6, and miR-210 mainly suppresses the phosphorylation of STAT5 and STAT6.

To sum up illustrate: the downward of the mononuclear origin dendritic cell of STAT5 and the STAT6 activation induction that hypoxia triggers by raising mononuclear origin 1 expressed by dendritic cells miR-210 and miR-342-3p, suppression GM-CSF and IL-4 C5aR1 in atomization.

Embodiment 8, miR-342-3p are directly targeted STAT5b and suppress the constitutive expression of STAT5b

It follows that the present inventor has inquired into miR-342-3p and miR-210 respectively further affects the molecular mechanism of STAT5 and STAT6 signal path activation.

First, the present inventor have selected the prediction target molecule of miR-342-3p by TargetScan bioinformatic database, wherein STAT5b is probably a direct target molecule of miR-342-3p, and according to its prediction binding site (Fig. 8 A), has carried out further checking. Experimental procedure is as follows:

The amplimer of the STAT5b3 ' UTRmRNA of binding site is predicted: 5 '-CCCAAGCTTCCTCCTTGTTCCTGCAAACCAC-3 ' (SEQIDNO:28) and 5 '-GGACTAGTGAAATAATTTCACCAAGTTTTA-3 ' (SEQIDNO:29) containing miR-342-3p; Build STAT5b3 ' UTRFirefly luciferase reporter plasmid as previously mentioned. Method as previously mentioned, at Human Embryonic Kidney HEK 293 cell cotransfection STAT5b3 ' UTRFirefly luciferase reporter plasmid and miR-342-3p analogies or inhibitor, detects luciferase reporter gene activity after 24h.

Result shows, transfection miR-342-3pmimic can suppress and transfect miR-342-3pinhibitor and can strengthen uciferase activity, therefore, miR-342-3p is directly targeted 3 ' the UTR districts acting on STAT5b thus suppressing the constructive expression (Fig. 8 B) of STAT5b.

Embodiment 9, miR-342-3p are directly targeted USP13 and suppress the constitutive expression of STAT5 and STAT6

Because miR-342-3p also lowers expression and the phosphorylation of STAT6, illustrate that miR-342-3p can also pass through other indirect mechanisms and suppress the activation of STAT6 signal path. It follows that the present inventor has inquired into miR-342-3p affects the activation of STAT5 and STAT6 signal path also by which kind of mechanism further.

By TargetScan, miRDB bioinformatic database, it has been found that the prediction target molecule of miR-342-3p all has USP13 (Fig. 9 a), a deubiquitinating enzymes. This molecule can pass through to remove the ubiquitination of some signaling molecules, maintains target molecule intracellular stable. Therefore, the present inventor speculates that miR-342-3p can pass through to be directly targeted and suppress the expression of USP13, thus reducing the expression of STAT5 and STAT6. Experimental procedure is as follows:

The amplimer of the USP13 ' UTRmRNA of binding site is predicted containing miR-342-3p: 5 '-CCCAAG CTTAGTGGTGTGGCCTTACCCAC-3 ' (SEQIDNO:30) and 5 '-GGACTAGTTGGGGAACTAATCTTCTCGAAG-3 ' (SEQIDNO:31); Build USP13mRNA3 ' UTRFirefly luciferase reporter plasmid as previously mentioned. Method as previously mentioned, at Human Embryonic Kidney HEK 293 cell cotransfection USP133 ' UTRFirefly luciferase reporter plasmid and miR-342-3p analogies or inhibitor, detects luciferase reporter gene activity after 24h.

Method as previously mentioned, mimic or inhibitor of transfection miR-342-3p is in person monocytic cell, after 48 hours, adds GM-CSF and IL-4, and after cultivating 48 hours, WB detects the expression of USP13; Mimic or inhibitor of transfection siUSP13 or miR-342-3p is in person monocytic cell, after 48 hours, adds GM-CSF and IL-4, and after cultivating 96 hours, FACS detects the expression of C5aR1; SiUSP13 is in person monocytic cell in transfection, and after 48 hours, after adding GM-CSF and IL-4,30min, WB or FACS detects the phosphorylation of STAT5, STAT6 in cell. USP13 special siRNA sequence such as SEQIDNO:13, shown in 14, mixes according to 1:1, proceeds to simultaneously.

Result shows, transfection miR-342-3pmimic can suppress and transfect miR-342-3pinhibitor and can strengthen uciferase activity, and therefore, miR-342-3p is directly targeted 3 ' the UTR districts (Fig. 9 b) acting on USP13. The down-regulated expression of USP13 after the analogies mimic of miR-342-3p is transfected under normal oxygen condition, and after transfecting the inhibitor of miR-342-3p under hypoxia condition, the expression of USP13 is significantly raised, illustrates that miR-342-3p can targeting suppress the expression (Fig. 9 c) of USP13. Further, the expression of CD14+ human peripheral blood mononuclear cell USP13 is disturbed by siRNA, express then through C5aR1 in the MoDC cell of GM-CSF and IL-4 induction differentiation and substantially raise, it was shown that suppress the expression of USP13 can promote the expression (Fig. 9 d) of C5aR1 in MoDC. Finally, under low oxygen conditions by transfecting siRNA (mixing, proceed to) interference USP13 according to 1:1 in CD14+ human peripheral blood mononuclear cell simultaneously, after 48h, supplement GM-CSF and IL-4 and stimulate 30min, it was found that interference USP13 after STAT5, STAT6, p-STAT5, p-STAT6 expression significantly raises (Fig. 9 e), and therefore the reduction of USP13 can suppress the activation of STAT5, STAT6 signal path.

To sum up, hypoxia is by inducing miR-342-3p targeted inhibition USP13 to suppress the activation of STAT5, STAT6 signal path, thus inhibiting the activation of STAT5, STAT6 signal path to lower C5aR1, it is achieved maintain the effect of hypoxia condition MoDC high expressed C5aR1.

Embodiment 10, miR-210 targeting promote PTPN1 expression and then the phosphorylation of suppression STAT5,6

By the prediction of TargetScan bioinformatic database, the present inventor finds that PTPN1 is probably a target molecule of miR-210, Figure 10 A is seen in its prediction land. PTPN1 is belonging to the member of protein tyrosine phosphatase family, has reported the dephosphorylation participating in some signaling molecules. There are some researches show, PTPN1 suppresses the phosphorylation level of the STAT6 of STAT5 and the IL-4 induction of prolactin antagonist induction, the therefore activation of negative regulation STAT5 and STAT6 signal path. Express in view of hypoxia raises miR-210, and PTPN1 is a potential target spot of miR-210, therefore the conventional wisdom expressed according to miRNA targeted inhibition target molecule, if hypoxia inducible miR-210 suppresses the expression of PTPN1, will promote STAT5,6 activation, this and the low oxygen quenching that it was experimentally observed that this with STAT5, the phenomenon of the activation of 6 is not inconsistent, therefore to whether discussion exists this contradiction is starting point, present inventors studied the relation between miR-210 and PTPN1, and in hypoxia situation the relations of PTPN1 and STAT5,6 activation. Experimental procedure is as follows:

Method builds PTPN13 ' the UTRFirefly luciferase reporter plasmid containing miR-210 land respectively as previously mentioned. Primer sequence: PTPN1 site 1 is 5 '-CCCAAGCTTGGTGAGGTGTGGATAAGGCTTAG-3 ' (SEQIDNO:32) and 5 '-GGACTAGTCCTGATGAGTAAATATTGGC-3 ' (SEQIDNO:33); PTPN1 site 2 is 5 '-CCCAAGCTTGCCATTTTTTGAGGAGAGTG-3 ' (SEQIDNO:34) and 5 '-GGACTAGTGGTTCAAGAAAATGCTAGTGC-3 ' (SEQIDNO:35); PTPN1 site 3 is 5 '-CCCAAGCTTTAGCCTGACCCTCCTCCACTC-3 ' (SEQIDNO:36) and 5 '-GGACTAGTGACACACATCCGGGGAAGATGG-3 ' (SEQIDNO:37). Method is at Human Embryonic Kidney HEK 293 cell cotransfection PTPN13 ' UTRFirefly luciferase reporter plasmid and miR-210 analogies or inhibitor as previously mentioned, detects luciferase reporter gene activity after 24h.

Method as previously mentioned, mimic or inhibitor of transfection siPTPN1 or miR-210 is in person monocytic cell, after 48 hours, adds GM-CSF and IL-4, and after cultivating 96 hours, FACS detects the expression of C5aR1; Mimic or inhibitor of transfection miR-210 is in person monocytic cell, after 48 hours, adds GM-CSF and IL-4, and after cultivating 48 hours, WB detects the expression of PTPN1; SiPTPN1 is in person monocytic cell in transfection, and after 48 hours, after adding GM-CSF and IL-4,30min, WB or FACS detects the phosphorylation of STAT5, STAT6 in cell. PTPN1 special siRNA sequence such as SEQIDNO:15, shown in 16, mixes according to 1:1, proceeds to simultaneously.

Result shows, miR-210 can be directly targeted 3 ' the UTR districts acting on PTPN1, and wherein site 1 and 2 is likely to play Main Function (Figure 10 B). The up-regulated of PTPN1 after the analogies mimic of miR-210 is transfected under normal oxygen condition, and after transfecting the inhibitor of miR-210 under hypoxia condition, the expression of PTPN1 substantially reduces, and illustrates, miR-210 can targeting raise PTPN1 expression (Figure 10 C). Further, the present inventor have detected PTPN1 GM-CSF and IL-4 under normal oxygen and hypoxia condition and induces the expression in the MoDC of 48h, it was found that PTPN1 expresses more normal oxygen rising (Figure 10 D) under hypoxia condition. Disturbed the expression of CD14+ human peripheral blood mononuclear cell PTPN1 by siRNA, express then through C5aR1 in the MoDC cell of GM-CSF and IL-4 induction differentiation and substantially lower, it was shown that PTPN1 can promote the expression (Figure 10 E) of C5aR1 in MoDC. Finally, under low oxygen conditions by transfecting siRNA interference PTPN1 in CD14+ human peripheral blood mononuclear cell, after 48h, supplement GM-CSF and IL-4 and stimulate 30min, it was found that p-STAT5, p-STAT6 expression significantly raises (Figure 10 F) after interference PTPN1, therefore PTPN1 can suppress the activation of STAT5, STAT6 signal path.

To sum up, hypoxia suppresses the activation of STAT5, STAT6 signal path by inducing miR-210 targeting to raise PTPN1, thus inhibiting the activation of STAT5, STAT6 signal path to lower C5aR1, it is achieved maintain the effect of hypoxia condition MoDC high expressed C5aR1.

Embodiment 11, drug screening

Take person monocytic cell, its endogenous expression miR-210 and miR-342-3p. Cultivate person monocytic cell as previously mentioned under low oxygen conditions. Using this kind of cell as being used for screening the cell model of the potential drug for the treatment of rheumatoid arthritis.

Test group: with the culture of the above-mentioned cell that candidate substances processes;

Matched group: without the culture of the above-mentioned cell that candidate substances processes.

Appropriate time after treatment, adopts the expression of miR-210 and the miR-342-3p of the conventional method described cell of mensuration. If compared with matched group, the expression of miR-210 or miR-342-3p in test group is remarkably decreased more than 30%, then illustrate that this candidate substances is the material of potential treatment rheumatoid arthritis.

The antagonist adopting miR-210 and the miR-342-3p of aforementioned preparation is tested as candidate substances, and the expression of result display miR-210 and miR-342-3p is suppressed, thus it is the potential material treating rheumatoid arthritis.

The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document. In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.

Claims (10)

1. the purposes adjusted under a miR-342-3p or adjust under miR-210 in the medicine of preparation treatment rheumatoid arthritis disease.

2. purposes as claimed in claim 1, it is characterised in that the nucleotide sequence of the lower adjustment of described miR-342-3p such as shown in SEQIDNO:5 or its modified after product; It is preferred that its modified outcome is on the basis of nucleotide sequence shown in SEQIDNO:5, skeleton part phosphate group is carried out thio-modification, 2 ' nucleic acid carry out methoxyl group modification and 3 ' end coupling cholesterol.

3. purposes as claimed in claim 1, it is characterised in that the nucleotide sequence of the lower adjustment of described miR-210 such as shown in SEQIDNO:8 or or its modified product; It is preferred that its modified outcome is on the basis of nucleotide sequence shown in SEQIDNO:8, skeleton part phosphate group is carried out thio-modification, 2 ' nucleic acid carry out methoxyl group modification and 3 ' end coupling cholesterol.

4. purposes as claimed in claim 1, it is characterised in that described medicine is additionally operable to treat the disease with hypoxia pathological characters:

The expression of cell surface C5aR1 is blocked under tissue hypoxia microenvironment; Or

Expression and/or the phosphorylation of STAT5 and/or STAT6 is promoted under tissue hypoxia microenvironment; Or

The expression of USP13 is improved in tissue hypoxia microenvironment; Or

The expression of PTPN1 is reduced in tissue hypoxia microenvironment.

The purposes of 5.miR-342-3p or miR-210, for the mark as diagnosis or prognosis rheumatoid arthritis disease; Or for preparing the reagent of diagnostics classes rheumatic arthritic diseases.

6. purposes as claimed in claim 5, it is characterized in that, described diagnosis or prognosis rheumatoid arthritis disease include: miR-342-3p or the miR-210 expression of detection experimenter, expression is more high, then it represents that the extent of rheumatoid arthritis disease is more serious.

7. purposes as claimed in claim 6, it is characterised in that the reagent of described diagnosis or prognosis rheumatoid arthritis disease is specific recognition or the reagent of amplification miR-342-3p or miR-210.

8. the purposes for diagnosing or in the test kit of prognosis rheumatoid arthritis disease prepared by the reagent of a specific recognition or amplification miR-342-3p or miR-210.

9. purposes as claimed in claim 8, it is characterised in that the reagent of described specific amplification miR-342-3p is the primer of nucleotide sequence such as SEQIDNO:23 and SEQIDNO:24; Or

The reagent of described specific amplification miR-210 is the primer of nucleotide sequence such as SEQIDNO:20 and SEQIDNO:21.

10. the method screening the potential material for the treatment of rheumatoid arthritis disease, described method includes:

(1) candidate substances being contacted with the system comprising miR-342-3p and/or miR-210, described system is cell culture system; With

(2) filtering out notable miR-342-3p and/or miR-210 of the downward material expressed, described material is the potential material for the treatment of rheumatoid arthritis disease.