CN108339119A - The application of miR-146a and its target spot in treating osteoarthritis - Google Patents

Abstract

Disclose the application of miR 146a and its target spot in treating osteoarthritis.Specifically, the combination for providing selected from the following a kind of or arbitrary plurality of reagents is preparing the drug for promoting cartilage synthesis or the application in preparing the drug for the treatment of or relief from osteoarthritis：(a) expression of miR 146a or active reagent are lowered；(b) expression of up-regulation Camk2d or active reagent；(c) expression of up-regulation Ppp3r2 or active reagent；(d) expression of the molecule in NAFT signal paths or active reagent are raised.

Description

The application of miR-146a and its target spot in treating osteoarthritis

Technical field

The present invention relates to the application of miR-146a and its target spot in treating osteoarthritis.

Background technology

Osteoarthritis (osteoarthritis, OA) is a kind of most common degenerative joint disease, relates generally to cartilage And perienchyma.In addition to the damage of articular cartilage is lost, subchondral bone structural remodeling (remodeling), joint margins spur shape At, laxity of ligament, periarticular muscles reduction etc. be all OA essential characteristic；Synovitis also happens occasionally.OA typically results in trouble Person's arthralgia, swelling, stiffening, the serious work for interfering patient are one of the main reason for leading to the elderly's lower limb disability.

Up to the present, the purpose for treating OA is only the pain alleviated OA and brought, rather than radical cure OA.Current OA treatments Guilding principle suggestion synthesis uses the therapeutic scheme of drug and non-drug.Drug therapy includes using paracetamol, oral or office Portion's non-steroidal anti-inflammatory drugs, opioid drug and steroid injection.Non-drug therapy include physical therapy, acupuncture therapy with And use the means such as walk helper.But intra-articular injection corticosteroid would generally cause it is short-lived；And non-steroidal anti-inflammatory drugs and Ah Opiates have apparent side effect, especially in elderly population.Therefore, still lack intrinsically safe treatment OA pains at present The means of pain.Operation is to alleviate the last therapy of OA end-stage patients' pain.Operation is typically with referred to as " prosthese " Joint prosthesis replaces the joint being affected.Joint prosthesis can generally maintain 10-15 even longer.However, these prostheses Limited service life makes it that can not meet the needs of growing patient OA especially young patient.

Microrna (miRNA) plays an important role in adjusting body development and atomization；However, people are to it It is maintained in cartilage homeostasis and the developmental Pathological Physiology effects of OA is known little.

Invention content

The combination that the application first aspect provides selected from the following a kind of or arbitrary plurality of reagents is preparing promotion cartilage conjunction At drug or prepare treatment or relief from osteoarthritis in drug in application：

(a) expression of miR-146a or active reagent are lowered；

(b) expression of up-regulation Camk2d or active reagent；

(c) expression of up-regulation Ppp3r2 or active reagent；With

(d) expression of the molecule in NAFT signal paths or active reagent are raised.

In one or more embodiments, the reagent is nucleic acid molecules, carbohydrate, lipid or small molecule chemical combination Object.

In one or more embodiments, the reagent of the expression for lowering miR-146a is the miR- knocked out in cell The targeting vector of 146a.

In one or more embodiments, the reagent of the expression for lowering miR-146a is miR-146a inhibitor, Such as express the virus of the reverse complements of miR-146a.

In one or more embodiments, the expression vector for raising the reagent that Camk2d is expressed and being Camk2d.

In one or more embodiments, the reagent of the expression for raising Ppp3r2 is the expression vector of Ppp3r2.

In one or more embodiments, the NAFT signal paths molecule is selected from NAFT1 and NAFT2.

In one or more embodiments, the reagent of the expression of the molecule in the up-regulation NAFT signal paths is described The expression vector of molecule.

In one or more embodiments, the osteoarthritis be spontaneous osteoarthritis, joint caused by aging not Stablize the osteoarthritis of induction, the osteoarthritis of tears of anterior cruciate ligament induction or sections inner side Meniscectomy and inside pair is tough Osteoarthritis with cross-section induction.

What the application second aspect provided in a kind of following reagent one or more is preparing the synthesis of monitoring cartilage or bone closes Save the application of scorching kit：

(a) it is used to detect the reagent of miR-146a expressions；

(b) it is used to detect the reagent of Camk2d expressions；

(c) it is used to detect the reagent of Ppp3r2 expressions；With

(d) it is used to detect the reagent of the expression of NAFT signal path molecules.

In one or more embodiments, the reagent for detecting Camk2d expressions includes detection Camk2d The reagent used during the reagent and detection Camk2d protein contents that are used during mRNA level in-site.

In one or more embodiments, the reagent for detecting Ppp3r2 expressions includes detection Ppp3r2 The reagent used during the reagent and detection Ppp3r2 protein contents that are used during mRNA level in-site.

In one or more embodiments, the reagent for detecting NAFT signal paths developed by molecule level includes The reagent and detection NAFT signal path molecule protein contents used during detection NAFT signal path molecules mRNA level in-site The reagent used in the process.

The application third aspect provide it is a kind of promotion cartilage synthesis or treatment osteoarthritis method, the method includes with The combination of any one or any number of steps down：

(a) expression of miR-146a in object or the step of activity are lowered；

(b) expression of Camk2d in object or the step of activity are raised；

(c) expression of Ppp3r2 in object or the step of activity are raised；With

(d) expression of the molecule in object in NAFT signal paths or the step of activity are raised.

In one or more embodiments, the method includes giving the object or less one kind or arbitrary plurality of reagents Combination the step of：

(a) expression of miR-146a or active reagent are lowered；

(b) expression of up-regulation Camk2d or active reagent；

(c) expression of up-regulation Ppp3r2 or active reagent；With

(d) expression of the molecule in NAFT signal paths or active reagent are raised.

In one or more embodiments, the reagent is nucleic acid molecules, carbohydrate, lipid or small molecule chemical combination Object.

In one or more embodiments, the reagent of the expression for lowering miR-146a is the miR- knocked out in cell The targeting vector of 146a.

In one or more embodiments, the reagent of the expression for lowering miR-146a is miR-146a inhibitor, Such as express the virus of the reverse complements of miR-146a.

In one or more embodiments, the expression vector for raising the reagent that Camk2d is expressed and being Camk2d.

In one or more embodiments, the reagent of the expression for raising Ppp3r2 is the expression vector of Ppp3r2.

In one or more embodiments, the NAFT signal paths molecule is selected from NAFT1 and NAFT2.

In one or more embodiments, the reagent of the expression of the molecule in the up-regulation NAFT signal paths is described The expression vector of molecule.

The application fourth aspect provides a kind of method for diagnosing osteoarthritis or monitoring osteoarthritic condition development, the side Method includes one kind or more in the molecule detected in object body fluid in miR-146a, Camk2d, Ppp3r2 and NAFT signal path The expression step of kind.

In one or more embodiments, the detection Camk2d expressions include detection Camk2d mRNA level in-sites With detection Camk2d albumen.

It is described to include detection Ppp3r2 mRNA for detecting Ppp3r2 expressions in one or more embodiments Horizontal and detection Ppp3r2 protein contents.

It is described to include detection for detecting NAFT signal path developed by molecule levels in one or more embodiments NAFT signal path molecule mRNA level in-sites and detection NAFT signal path molecule protein contents.

In one or more embodiments, the mRNA is detected using Northern hybrid methods, RT-PCR or RNA-seq Level.

In one or more embodiments, the content of albumen is detected using Western blot or ELISA.

In one or more embodiments, the body fluid is peripheral blood.

Description of the drawings

Fig. 1：MiR-146a up-regulated expressions in mouse OA cartilages.

Fig. 2：The expression of TNF-α, IL-1 β in mouse OA cartilages significantly increases.

Fig. 3：TNF-α, IL-1 β and IL-17 induction miR-146a expression." Mock " stimulates for the acellular factor.

Fig. 4：IL-1 β induce the expression of miR-146a by NF- к B and ERK accesses.

Fig. 5：MiR-146a KO inhibit spontaneity OA.Wherein, F indicates that femur, T indicate shin bone.

Fig. 6：MiR-146a KO alleviate the OA regressions of DMM inductions.

Fig. 7：MiR-146a KO alleviate the OA regressions of ACLT and PMM inductions.

Fig. 8：MiR-146a inhibits cartilage anabolic.

Fig. 9：MiR-146a targets the anabolism that multiple genes inhibit cartilage.

Figure 10：Intra-articular injection miR-146a has therapeutic effect to OA.

Figure 11：Camk2d and Ppp3r2 is the function target that miR-146a adjusts OA.

Specific implementation mode

Herein by building joint instability OA models caused by spontaneous OA animal models and wound in mouse (mouse anterior cruciate ligament-transection (ACLT), mouse medial meniscus go to stablize (DMM) and mouse sections inner side meniscectomy (PMM)) effects of the miR-146a in OA diseases, is disclosed.It finds herein, expression of the miR-146a in the cartilage of damage is aobvious It writes and increases, this trend is related with proinflammatory cytokines IL-1 β and TNF-α that the cartilage of damage is secreted.Experiment in vitro shows IL-1 β It is mainly logical by NF- к B and ERK signals with the expression of miR-146a in the TNF-α different degrees of Induction of chondrocytes of energy Road.By spontaneity OA models and traumatic animal model caused by aging, find herein miR-146a knock out (Knockout, KO) cartilage degeneration of mouse has the variation significantly alleviated compared to wild type (WT) mouse, shows in OA growth courses, miR- The presence of 146a may play the role of promoting OA.By experiment external in vivo, find herein：MiR- is knocked out in cartilage cell 146a significantly enhances the anabolism of cartilage；Conversely, miR-146a up-regulations inhibit the anabolism of cartilage；It is dry in OA early stages The expression of pre- miR-146a influences the process of OA, specifically, being overexpressed miR-146a in OA modeling mouse makes mouse OA symptoms obviously aggravate, and the mouse OA symptoms for knocking out miR-146a are relieved.Go out miR- herein by experimental identification 146a inhibits cartilage anabolic by targeting the genes such as Camk2d, Ppp3r2.The functional study of its target spot is shown in cartilage High expression Camk2d and Ppp3r2 promotes cartilage synthesis in cell；And it lowers Camk2d and Ppp3r2 expression and then cartilage is inhibited to close At.Further, deeper into the study found that the NFAT signal paths in the downstreams Camk2d and Ppp3r2 are small in miR-146a KO It is activated in mouse, the transcription of this signal path and cartilage synthetic gene SOX9 and COL2A1 is closely related, shows inflammation- This central axes miR-146a-Camk2d＆Ppp3r2-NFAT-SOX-9＆COL2A1 are thus complete in the developmental key effects of OA At herein.

Therefore, present document relates to regulation and control object cartilage synthetic method, this method include regulation and control miR-146a, Camk2d, The step of one or more expression or activity in Ppp3r2 and NFAT pathway proteins.

Herein, " object " can be mammal, preferably people.MiR-146a, Camk2d, Ppp3r2 and NFAT access Albumen is usually the corresponding molecule of mammal especially people.

The protein sequence and its coded sequence of these molecules can be found from corresponding database such as Genbank.For example, The sequence of miR-146a can be obtained from miRbase databases, and the number of logging in is MIMAT0000158；Camk2d、Ppp3r2、 The sequence of NFAT1 and NFAT2 can be obtained from ncbi database, and the number of logging in is respectively 108058,19059,18019 and 18018.It answers Understand, miR-146a, Camk2d, Ppp3r2 and NFAT pathway protein from different plant species source are in amino acid sequence And/or there may be certain differences on nucleotide sequence, it may also even from these molecules of same species Different Individual There are certain sequence difference, but as long as the biological function of these molecules is identical as biological function described herein or class Seemingly, this kind of molecule is included within the scope of the present invention.In certain embodiments, NFAT pathway proteins of the invention include NFAT1 and NFAT2 albumen.

The synthesis of " regulation and control " cartilage includes inhibiting cartilage synthesis and cartilage being promoted to synthesize.For the feelings for needing that cartilage is inhibited to synthesize Condition, expression that can be by up-regulation miR-146a or activity, lower expression or the activity of Camk2d, lower the expression or work of Ppp3r2 Property, and/or lower NAFT expression or activity and realize.It the case where for needing to promote cartilage synthesis, can be by lowering miR- The expression of 146a or activity raise expression or the activity of Camk2d, raise expression or the activity of Ppp3r2, and/or up-regulation NAFT Expression or activity and realize.

In general, miR-146a, Camk2d, Ppp3r2 and NFAT expression or activity can be realized by giving suitable reagent Up-regulation or downward.For example, lowering or inhibiting the reagent of miR-146a, Camk2d, Ppp3r2 and NFAT gene expression dose can Including but not limited to：The reagent for promoting miR-146a degradations inhibits for the siRNA of Camk2d, Ppp3r2 and NFAT gene The reagent of Camk2d, Ppp3r2 and NFAT mRNA translations, knocks out the examination of miR-146a, Camk2d, Ppp3r2 and NFAT gene The guiding nucleic acid of agent and specific recognition Camk2d, Ppp3r2 and NFAT gene is simultaneously sheared it to reduce its expression water Flat reagent etc..In certain embodiments, can by giving targeting vector by miR-146a, Camk2d, Ppp3r2 and/or NFAT full genomes knock out, to realize the reduction of expression.In certain embodiments, the examination of the expression of miR-146a is lowered Agent is miR-146a inhibitor.In certain embodiments, miR-146a inhibitor is the antisense complementarity sequence for expressing miR-146a The viral vectors (such as slow virus carrier) of row.

It is the miR-146a that those can specifically bind intracellular mature to be suitable for the invention reverse complements, to Check the reverse complements of the effect of miR-146a.Its antisense complementarity sequence can be designed according to the miR-146a expressed by object Row.It is suitable for the invention the mammal that is suitable for that viral vectors (such as slow virus carrier) can be well known in the art, especially It is the viral vectors of human administration.

It can be the respective specificity of these albumen to lower or inhibit the reagent of Camk2d, Ppp3r2 and NFAT protein active Antibody.

Herein, the activity for reducing Camk2d, Ppp3r2 or NFAT albumen includes keeping host cell and control cell (wild Type cell) compare, Camk2d, Ppp3r2 or NFAT albumen activity reduce at least 30%, for example, at least 40%, at least 50%, At least 60%, at least 70%, at least 80%, at least 90%, or even do not express these albumen completely or can't detect these completely The activity of albumen.

Also its activity can be made to reduce by introducing mutation in Camk2d, Ppp3r2 or NFAT albumen.Herein, Camk2d, Ppp3r2 or NFAT albumen activity especially its in inflammation-miR-146a-Camk2d＆ described herein The promotion or the activity for inhibiting cartilage to synthesize that this central axes Ppp3r2-NFAT-SOX-9＆COL2A1 play.Therefore, certain In embodiment, is introduced in the functional domain of Camk2d, Ppp3r2 or NFAT albumen and cause the prominent of its corresponding reduced activity or forfeiture Become.Mutation can be 1 or several even more (such as 10 or more, 20 or more, 30 or more) a amino acid insertion, Missing or substitution.Can by donation in the reagent of Camk2d, Ppp3r2 or NFAT gene, make its encoded Camk2d, There is the mutation for causing its related biological activities to weaken or lose in the functional domain of Ppp3r2 or NFAT albumen.This kind of reagent can The sequence for changing Camk2d, Ppp3r2 or NFAT gene causes encoded Camk2d, Ppp3r2 or NFAT albumen to exist corresponding Mutation, to weaken activity or loss of activity.For example, can by homologous recombination technique be mutated Camk2d, Ppp3r2 or NFAT genes replace Camk2d, Ppp3r2 or NFAT gene in wild-type cell, and weak work is expressed so as to cause it Property or inactive Camk2d, Ppp3r2 or NFAT albumen.

The up-regulation that miR-146a, Camk2d, Ppp3r2 and NFAT are respectively expressed can pass through the mistake in host or host cell It expresses these molecules and realizes its up-regulation expressed.For example, being suitable in host cell using this field conventional technique structure It is middle to express the expression vector of these molecules, and be transferred in host cell by conventional method so that expression vector is in host cell The middle expression molecule, to realize the up-regulation of its expression.In certain embodiments, the upstream gene of these controllable molecules Expression, to improve the expression of this kind of molecule.For example, in certain embodiments, can give object representation miR-146a at The viral vectors (such as slow virus carrier) of ripe sequence, to improve expressions of the miR-146a in subject cell.

In general, the raising of protein active refers to compared with wild type, protein active has at least 20%, at least 30%, at least 40%, at least 50%, at least 60% raising；Or compared with wild-type cell, the protein active in host cell has at least 20%, at least 30%, at least 40%, at least 50%, at least 60% raising.

Therefore, it includes giving the object one or more of reagent of needs that the present invention, which inhibits cartilage synthetic method,：

(1) expression of up-regulation miR-146a or active reagent；

(2) expression of Camk2d or active reagent are lowered；

(3) expression of Ppp3r2 or active reagent are lowered；With

(4) expression of NAFT or active reagent are lowered.

It includes giving the object one or more of reagent of needs that the present invention, which promotes cartilage synthetic method,：

(a) expression of miR-146a or active reagent are lowered；

(b) expression of up-regulation Camk2d or active reagent；

(c) expression of up-regulation Ppp3r2 or active reagent；With

(d) expression of up-regulation NAFT or active reagent.

The reagent of this paper can be nucleic acid molecules, carbohydrate, lipid, micromolecular compound and albumen.Herein, core Acid molecule can be such as siRNA, expression vector or targeting vector.Micromolecular compound is often referred to using being chemically synthesized The lower chemical synthesis compound of molecular weight.Albumen includes specific antibody.For example, reagent can be and Camk2d, Ppp3r2 Or the antibody that NFAT protein-specifics combine.Antibody can be polyclonal antibody, monoclonal antibody or scFv.This field can be used Known method prepares the antibody of Camk2d, Ppp3r2 or NFAT albumen, especially monoclonal antibody.Commercially available antibody can also be used. In certain embodiments, the above method of the invention include will be overexpressed miR-146a viral vectors (such as adenovirus vector) or The viral vectors (such as adenovirus vector) of reverse complements for being overexpressed miR-146a is injected into articular cavity, to inhibiting or Promote cartilage synthesis.

Mentioned reagent give mode and dosage can be according to different objects, different illnesss and different medicine types It gives.For example, in certain embodiments, administration route includes but not limited to：Direct naked RNA injection methods；Liposome RNA Direct injection；Gold coating rna gene rifle blast technique；Breeding unsoundness bacterium or replication defective adenoviral carry Plasmid DNA method；Electricity Perforation method, etc..The dosage given should be that object can be made to generate the required amount for treating or preventing effect.It can be according to actual treatment Or prevent situation, divide the reagent or pharmaceutical composition for giving the present invention several times.

When being given in the form of pharmaceutical composition, pharmaceutically acceptable excipient can be also contained in pharmaceutical composition. For example, when preparing pharmaceutical composition, usually the reagent is mixed with excipient, or with figuration dilution agent or Bao Ke with In carrier existing for capsule or anther sac, form of nanoparticles.When excipient plays diluent, it can be solid, partly consolidate The medium of body or fluent material as excipient, carrier or active constituent.Therefore, pharmaceutical composition can be tablet, pill, powder Agent, solution, syrup, sterilizing injecting solution etc..The example of suitable excipient includes lactose, glucose, sucrose, sorb Alcohol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, nano particle etc..Pharmaceutical composition may be used also Contain other ingredients, such as wetting agent, emulsifier, preservative (such as methyl hydroxybenzoate and propyl ester and sweetener.It is above-mentioned each Amount of the reagent in pharmaceutical composition should treat or prevent effective amount.

In general, the reagent should be that treatment or prevention are a effective amount of in the content of pharmaceutical composition；Specific content is given Pharmaceutical quantities are contemplated that the factors such as administration route, patient health situation consider.

It is as described herein that cartilage synthetic method is promoted to can be used for treating the various diseases for benefiting from cartilage synthesis or alleviation Benefit from the various diseases of cartilage synthesis, including but not limited to osteoarthritis.Term " treatment " and " alleviation " are used with this field The meaning understood.For example, the object state of an illness has releiving to a certain extent, it is believed that having achieved the purpose that " to alleviate ".This Wen Zhong, osteoarthritis includes but not limited to spontaneous osteoarthritis caused by aging, the osteoarthritis of joint instability induction, preceding The osteoarthritis or sections inner side Meniscectomy and the Bones and joints of the cross-section induction of medial collateral ligament of ligamentaum cruciatum tearing induction It is scorching.

Therefore, in certain embodiments, the present invention provides one kind in following reagent or arbitrary a variety of combination Application in preparing the drug for promoting cartilage synthesis：

(a) expression of miR-146a or active reagent are lowered；

(b) expression of up-regulation Camk2d or active reagent；

(c) expression of up-regulation Ppp3r2 or active reagent；With

(d) expression of up-regulation NAFT or active reagent.

By detecting one kind in object body fluid in miR-146a, Camk2d, Ppp3r2 and NFAT gene or arbitrary a variety of Combination expression, it is also diagnosable that relevant disease, especially osteoarthritis are synthesized with cartilage, or monitor the disease of the disease Feelings develop.Therefore, the present invention also provides a kind of method for diagnosing osteoarthritis or monitoring osteoarthritic condition development, the method Including in detection object body fluid in miR-146a, Camk2d, Ppp3r2 and NFAT gene one kind or arbitrary a variety of combination The step of expression.

Expression described in detection object body fluid includes the level and the corresponding protein content of detection for detecting mRNA.It can be used The method of this field routine carries out the detection.For example, the detection for mRNA, can be used Northern hybrid methods, RT- PCR or RNA-seq；Western blot or ELISA can be used in detection for protein content.For example, existing examination can be used Agent box detects the content or level of GAP-associated protein GAP.When detecting protein level, corresponding antibody can be used to carry out ELISA inspections It surveys.These are all well-known to those skilled in the art.Body fluid can be such as peripheral blood.

Therefore, one kind in following reagent or arbitrary a variety of combination is also provided herein and is preparing diagnosis and monitoring cartilage conjunction At or osteoarthritis kit application：

(a) it is used to detect the reagent of miR-146a expressions；

(b) it is used to detect the reagent of Camk2d expressions；

(c) it is used to detect the reagent of Ppp3r2 expressions；With

(d) it is used to detect the reagent of the expression of NAFT signal path molecules.

Reagent for above-mentioned detection includes detecting the reagent used during its mRNA level in-site and detecting its albumen to contain The reagent used during amount.This kind of reagent for detection can be nucleic acid molecules, carbohydrate, lipid, small molecule Compound and albumen (such as antibody).For example, when being detected or being monitored using ELISA, the reagent includes but not limited to phase Answer the specific antibody of albumen.When detecting or monitoring mRNA level in-site, the reagent includes but not limited to corresponding primer sequence And/or probe sequence, also include the reagent carried out needed for PCR or other biological experiment, such as corresponding enzyme, solvent and reaction Object etc..In certain embodiments, this kind of reagent further includes for example for developing the color or other being convenient for quantitatively or qualitatively object Matter.

In certain embodiments, the disclosure also provides a kind of detection kit, and the detection kit contains following examination A kind of, arbitrary two kinds, arbitrary three kinds or four kinds of whole in agent：

(a) it is used to detect the reagent of miR-146a expressions；

(b) it is used to detect the reagent of Camk2d expressions；

(c) it is used to detect the reagent of Ppp3r2 expressions；With

(d) it is used to detect the reagent of the expression of NAFT signal path molecules.

For example, the kit at least contains (a), also contain in (b), (c) and (d) item any one, arbitrary two Kind is three kinds arbitrary.Alternatively, the kit contains the reagent described in (b) and (c) item, or contain (a), (b) and (c) item institute The reagent stated.Preferably, the reagent for these detections includes detecting the reagent used during its mRNA level in-site and detection The reagent used during its protein content, more preferably Northern hybrid methods, RT-PCR or RNA-seq detect mRNA water Used reagent, including but not limited to primer and probe when flat and Western blot or ELISA detection protein contents, with And the reagent needed for such as progress PCR.

The present invention will be hereafter illustrated in a manner of specific embodiment.The experiment side of actual conditions is not specified in the following example Method, such as Sambrook usually according to normal condition,《Molecular cloning：Lab guide》(New York, United States：Cold spring harbor laboratory Publishing house, 1989) condition described in, or carry out according to the normal condition proposed by manufacturer.For the usage and dosage of reagent, remove It is non-to be otherwise noted, otherwise used according to conventional usage and dosage.

Furthermore, it is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as embodiment) It can be combined with each other between each technical characteristic of middle specific descriptions, to constitute preferred technical solution.Term used herein, Unless otherwise stated, its meaning for usually understanding with this field.

One, experiment material and method

1.1 experiment material

1.1.1 experiment reagent

Unless otherwise indicated, various reagents box, reagent, enzyme, antibody etc. used in embodiment, available from Invitrogen (CA, the U.S.), Sigma (CA, the U.S.), Genecopoeia (CA, the U.S.), Roche, Omega bio-tek (CA, the U.S.), hundred Tyke (BeiJing, China), Abcam (CA, Britain), CST (CA, the U.S.), Peprotech (CA, the U.S.), NEB (CA, the U.S.), Promega (CA, USA), Proteintech (CA, the U.S.), Takara (DaLian, China), TOYOBO (Japan), new (China is stepped Foochow), raw work (Chinese Shanghai), Ji Ma (Chinese Shanghai), day is with (BeiJing, China) etc..

1.1.2 experimental animal

The miR-146a KO mouse of C57BL/6 backgrounds by Beijing Vital River Experimental Animals Technology Co., Ltd. from Jackson Laboratory are introduced, and animal feeding is in Shanghai Inst. of Life Science, CAS health science research institute reality Test animal center.The sequence that one section of 295bp of miR-146a precursors is encoded on the Strains of Mouse genome is removed.Animal is arrived at Afterwards, fertile to ensure with a C57BL/6J mouse hybrids at least generation first；Genotype is then identified by PCR to select KO mouse Homozygote is for continuing breeding and subsequent experiment.Identify that primer is as follows：

5 '-ACCAGCAGTCCTCTTGATGC-3 ' of forward direction primer (SEQ ID NO:1)；

Reverse primer 5 '-GACGAGCTGCTTCAAGTTCC-3 ' (SEQ ID NO:2).

Reaction condition and reaction system are shown in Table 1 and 2.

Table 1：MiR-146a Genotyping reaction systems

Table 2：MiR-146a Genotyping thermal cycle conditions

C57BL/6J Strains of Mouse, male 8-10 weeks, are purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, animal feeding In Shanghai Inst. of Life Science, CAS health science research institute Experimental Animal Center.All animals are kept at disease-free In the environment of substance, all zooperies are in Shanghai Inst. of Life Science, CAS biomedicine Ethics Committee License is lower to be executed.

1.2 experimental method

1.2.1 miR-146a knocks out the identification of musculus cdna type

The extraction of rat-tail genomic DNA illustrates to carry out according to hundred Imtech's related kits.Identify that primer is as follows：

5 '-ACCAGCAGTCCTCTTGATGC-3 ' of forward direction primer (SEQ ID NO:3)；With

Reverse primer 5 '-GACGAGCTGCTTCAAGTTCC-3 ' (SEQ ID NO:4).

Reaction condition and reaction system are shown in Table 1 and 2.

1.2.2 the preparation of the osteoarthritis of mouse operation induction

It is prepared by mouse anterior cruciate ligament-transection (ACLT) model：Take the male miR-146a KO and wild type C57 of 12 week old Mouse prepares ACLT models.It is anaesthetized in short, chloraldurate (400mg/kg) is first injected intraperitoneally in mouse, about needs 250 microlitres Mouse can be made fully to anaesthetize in 5 minutes.Under aseptic condition, with scissors the longitudinal sectional about 5mm or so of the right knee of mouse notch. After opening capsular ligament under disecting microscope, gently the fat pad being covered in above anterior cruciate ligament is pushed on one side, with a 1ml The syringe needle of syringe is cross-section anterior cruciate ligament, is careful not to be in direct contact cartilaginous tissue；It is wiped later with the cotton balls for being moistened with Iodophor Wound tissue is wiped, is used in combination silk sutures wound surrounding skin to avoid infection.After operation, by mouse be placed on plus On the instrument of temperature setting, it is transferred in rearging cage again after mouse releases narcosis substantially.Sham (sham-operation) group is additional It is prepared on mouse, by anesthesia, after opening capsular ligament program, direct sewing-up cut surrounding skin waits mouse to release narcosis After be transferred in rearging cage.De- neck puts to death mouse within postoperative eight weeks, takes out knee joint and carries out subsequent analysis.

Mouse medial meniscus goes to stablize the preparation of (DMM) model：Take the male miR-146a KO and wild type of 12 week old C57 mouse prepare DMM models.Mouse is first injected intraperitoneally chloraldurate (400mg/kg) and is anaesthetized.Under aseptic condition, scissors is used In the notch of the longitudinal sectional about 5mm or so of the right knee of mouse.After opening capsular ligament under disecting microscope, carefully removed with microforceps in Fat pad above the meniscus of side, with the cross-section connection medial meniscus of the syringe needle of a 1ml syringe and lower section tibial plateau Ligament is careful not to be in direct contact the cartilage of tibial plateau surfaces；Later with the cotton balls wiping wound tissue for being moistened with Iodophor, it is used in combination Silk sutures wound surrounding skin is to avoid infection.After operation, mouse is placed on the instrument with heating setting, It is transferred in rearging cage again after mouse releases narcosis substantially.Postoperative surrounding takes off neck and puts to death mouse, after taking out knee joint progress Continuous analysis.

It is prepared by mouse sections inner side meniscectomy (PMM) model：Take the male miR-146a KO of 12 week old and wild Type C57 mouse prepare PMM models.Mouse is first injected intraperitoneally chloraldurate (400mg/kg) and is anaesthetized.Under aseptic condition, with cutting Notch of the knife in the longitudinal sectional about 5mm or so of the right knee of mouse.After opening capsular ligament under disecting microscope, cross-section medial collateral ligament is simultaneously Remove the craniofacial region point of medial meniscus；Later with the cotton balls wiping wound tissue for being moistened with Iodophor, silk sutures are used in combination Wound surrounding skin is to avoid infection.After operation, mouse is placed on the instrument with heating setting, waits for that mouse releases substantially It is transferred in rearging cage again after narcosis.Postoperative surrounding takes off neck and puts to death mouse, takes out knee joint and carries out subsequent analysis.

It is prepared by mouse osteoarthritis treatment model：12 weeks big C57BL/6 hero mouse are randomly divided into four groups, every group of 8-9 only, Arthritis model is prepared by DMM operations.Postoperative one week, miR-146a analogies or inhibition will be carried with microinjector Agent, and the lentiviral particle of corresponding reference material are injected into mouse knee joint.Virus titer is 109/ml, every mouse injection 5 μ L, injection is primary week about, and continuous injection is three times.Postoperative surrounding takes knee joint to carry out subsequent analysis.

1.2.3 the preparation of mouse spontaneity osteoarthritis

Using the miR-146a KO of all ages and classes (3,6,9,12 months) and wild type C57 mouse as object, after taking off neck execution It collects knee joint sample and carries out subsequent analysis.

1.2.4 histology and immunohistochemistry

1.2.4.1 paraffin embedding and slice

1) fixed：The knee joint sample of materials is fixed in 4% paraformaldehyde, 12h or more is placed at room temperature for, is no more than 24h.Ensure the fixer covering tissue for having enough, fixer volume is about 5-10 times of tissue volume.2) it rinses：It will fix Sample be placed in embedding frame in, flowing water rinse overnight, remove extra fixer.3) decalcification：By sample put into a 15ml from In heart pipe, about 10ml decalcifying Fluids are added, are placed on decalcification on shaking table.A decalcifying Fluid was replaced every 3 days, about needs can complete within two weeks Decalcification program；It can show that decalcification is complete without apparent resistance when penetrating tissue with syringe needle at this time.4) it rinses：It will take off The complete sample of calcium is trimmed to suitable for size and is placed in embedding frame, and sample labeling is carried out, and flowing water rinses overnight, removes extra take off Calcium liquid.5) it is dehydrated：Serial dehydration is carried out by following procedure：80% alcohol is primary, each 2h；95% alcohol three times, each 2h； 100% alcohol twice, each 1h.6) transparent：Dimethylbenzene twice, 15 minutes every time.7) waxdip：Sample is dipped in by following procedure In 60 DEG C of paraffin：Paraffin I, 30 minutes；Paraffin II, 90 minutes；Paraffin III, 90 minutes (can stay overnight).8) embedding and slice：Often After the embedding of rule method, it is switched to 5-10 μ m thicks and is transferred in 40-45 DEG C of water-bath.Slice is picked up with glass slide, 37 DEG C It dries overnight.

1.2.4.2 slice dewaxing and aquation

1) it dewaxes in dimethylbenzene twice, 10 minutes every time.2) 100% alcohol twice, 5 minutes every time.3) 95% alcohol Twice, every time 5 minutes.4) 85% alcohol is primary, 5 minutes every time.5) it is placed 5 minutes in 75% alcohol, is dipped in distilled water later In.

1.2.4.3 the fast green dyeing of sarranine-

1) paraffin section routine dewaxing aquation.2) tap water rinses 30 seconds.3) fast green dyeing 5 minutes.4) tap water rinses 1 Minute, remove extra fast green dye liquor.5) sarranine dyes 5 minutes.6) tap water rinses 1 minute, removes extra sarranine dye liquor. 7) 1% glacial acetic acid color separation 1 second.8) 95% ethanol dehydration 1 minute, 2 times.9) 100% ethanol dehydration 2 minutes, 2 times.10) dimethylbenzene Transparent 5 minutes, 2 times.11) neutral gum sealing.

1.2.4.4 immunohistochemistry

1) routinely after dewaxing and aquation, PBST (is added Tween-20 to wash to final concentration of 0.1%) paraffin section in PBS Three times, every time 3 minutes.2) absorb the moisture around tissue with dustless paper handkerchief, according to the requirement of different antibodies, to tissue antigen into Row is corresponding to be repaired.3) (30%H2O2 and methanol are according to 1 for the enough 3%H2O2 solution of every slice dropwise addition：9 ratio is prepared), It is incubated 10 minutes at room temperature, to block the activity of endogenic peroxidase in tissue.PBST is washed three times, 5 minutes every time. 4) moisture around tissue is removed, is drawn a circle around tissue with immunohistochemistry pen.5% lowlenthal serum room temperature is closed 30 minutes, serum It is diluted with PBST.5) confining liquid is removed, according to the requirement of different antibodies, with 2.5% lowlenthal serum dilution primary antibody Mmp13 (Abcam,1:200, ab39012), X-type collagen (Abcam, 1:1000,ab58632),GFP(Abcam,1:1000,ab290), Camk2d(Proteintech,1:100,20667-1-AP),Ppp3r2(Proteintech,1:100,14005-1-AP), Nfatc1(Abcam,1:50,ab175134),Nfatc2(Proteintech,1:100,22023-1-AP).Every slice plus 50 μ l primary antibodies, 4 DEG C overnight.6) primary antibody is removed, PBST is washed three times, 5 minutes every time.Every slice plus 1 drop poly enzyme conjugates (step New immunohistochemical kit secondary antibody), it is incubated at room temperature 15 minutes.7) secondary antibody is removed, PBST is washed three times, 5 minutes every time.Every is cut Piece adds the DAB solution of 50 μ l Fresh, is placed at room temperature for 3-5 minutes.8) extra DAB solution is removed, tap water slightly rinses, and revives Lignin is redyed 10 seconds, after basket is returned in tap water flushing, is broken up 1 second with 1% hydrochloride alcohol.9) indigo plant, 95% ethyl alcohol are returned in tap water flushing Dehydration 5 minutes, 2 times.10) 100% ethanol dehydration 5 minutes, 2 times.11) transparent 5 minutes of dimethylbenzene, 2 times.12) neutral gum seals Gu.

1.2.5 the separation and identification of mouse articular chondrocytes

Mouse cartilage cell maintains culture medium：2mM L-GIn, 1%penicillin- are added in DMEM low sugar culture mediums Streptomycin (Hyclone) and 10%FBS.Collagenase digesting liquid：75mg clostridiopetidase A D are weighed, 25ml is dissolved in and maintains training It supports in base, makes its final concentration of 3mg/ml, 0.22 μm of membrane filtration.

1.2.5.1 the separation of mouse articular chondrocytes

1) one week big C57BL/6 mouse is taken, after taking off neck execution, is soaked in 75% ethyl alcohol 10 minutes.2) by mouse face It is positioned on superclean bench, is stripped down back leg upward with scissors, tweezers, be eliminated as much as the soft tissues such as muscle.3) it uses Aseptic operation knife chips away the cartilage (visually seeing for translucent sphere) of condyle of femur, tibial plateau and femoral head surfaces, It is dipped in 1X PBS and (contains 1%penicillin-streptomycin), be careful not to cut bone tissue (brown).4) with containing The 1X PBS washing cartilages block of antibiotic is twice.5) it is positioned in six orifice plates with cartilage block, 4ml collagenase digesting liquid is added per hole (3mg/ml) digests 45 minutes in 37 DEG C of cell incubators.6) cartilage block is taken out, is positioned in new hole, step 5 is repeated. 7) 2.5ml collagenase digesting liquid is taken, the digestive juice (0.5mg/ml) that 12.5ml maintains culture medium to be made into low concentration is added.Use 10ml Pipette blow and beat cartilage block, remove soft tissue.Cartilage block carefully is removed with tweezers, is put in six new orifice plates and 4ml is added Collagenase digesting liquid (0.5mg/ml) is digested overnight in 37 DEG C of cell incubators.8) pipette of 10ml is used to blow and beat cartilage block, to the greatest extent Cartilage cell may be made to be dissociated from cartilage block；Cell can be blown and beaten with 10ml syringes to obtain single cell suspension.9) with 70 μm Membrane filtration cell, room temperature 400g are centrifuged 10 minutes.10) maintenance culture medium appropriate is added, by cell inoculation in culture dish, It can cover within about one week.Typical cartilage cell is rounded or polygon, cytoplasm endoparticle sense are strong.

1.2.5.2 the identification (II Collagen Type VIs immunofluorescence) of mouse articular chondrocytes

1) cartilage cell is inoculated in the density of every hole 2x104Cells on 24 orifice plates.2) wait for cell growth to degree of converging About 70%-80% removes culture solution, twice with PBS washings cell.3) 4% paraformaldehydes of 1ml are added per hole, room temperature fixes 10 Minute.4) cell is quickly first embathed twice with PBS, 1ml PBS washings 5 minutes is then added per hole, twice.5) confining liquid (5% BSA, 0.3%triton) room temperature close 1 hour.6) 4 DEG C of II Collagen Type VIs antibody (1：100 thinner ratios, with 1%BSA, 0.1% Triton dilutes) it is incubated overnight.7) primary antibody is removed, PBS is washed three times, each 5 minutes.8) secondary antibody is incubated at room temperature 2 hours (1：1000 Thinner ratio, PBS dilutions), it slowly rocks on shaking table, is both needed to be protected from light after this step.9) secondary antibody is removed, PBS is washed three times, each 5 points Clock.10) Hoechst 33342 (1 μ g/ml) is dyed 5 minutes.11) PBS is washed three times, each 5 minutes.12) it is clapped under fluorescence microscope According to chondrocyte cell matter has strong II expression of collagen.

1.2.6 real-time fluorescence quantitative PCR

SYBR Green I dye methods are used herein；And for microRNA gene expression detections, TaqMan is used herein Sonde method is accordingly detected.

1.2.6.1 Total RNAs extraction

Illustrate that instruction extracts total serum IgE from Mouse cartilage/cartilage cell according to Invitrogen company's T RIzol reagents.

1.2.6.2 the first chain cDNA synthesis

A. takara companies cDNA the first chain synthetic agent box is used, 1 μ g total serum IgEs is taken to carry out reverse transcription, synthetic product is used In detection mRNA gene expressions.

B. Applied Biosystems companies are used MicroRNA Reverse Transcription The stem ring primer (Applied Biosystems Inc) of the microRNA specificity for including in kit synthesizes the first chain CDNA, the cDNA templates of generation are only used for expanding the microRNA.

1.2.6.3 Real time PCR are detected

A.mRNA gene expression detections：Use takara companies real-time PCR detection reagents (CAT:RR420A).

B.miR-146a gene expression detections：Use Applied Biosystem companies Universal Master Mix II reagents detect (CAT：4440040).

1.2.8 expression vector establishment is interfered with siRNA

1.2.8.1 Tgif1, Camk2d, Ppp3r2 expression vector are built

In order to study function of Tgif1, Camk2d, Ppp3r2 gene in cartilage cell, we have cloned mouse Tgif1, Camk2d and Ppp3r2 gene.Used herein is pEGFP-N1 carriers, which inserts after multiple cloning sites EGFP segments, convenient for assessment transfection efficiency.When carrier construction, the terminator codon of target gene need to be removed with obstruction free EGFP Expression.

Tgif1 coding region sequences expand

1) following primer is synthesized, first round amplification is started：

Forward direction primer：5’-AACTAAATGCACTCTTCGC-3’(SEQ ID NO:5)；

Reverse primer：5’-TTCGTCTGAGCAATCCC-3’(SEQ ID NO:6).

2) PCR reaction reagents are prepared on ice, and system is as follows：

3) soft mixing runs following procedure by reaction tube by being transferred on ice on grads PCR instrument：

4) 1 μ l pcr amplification products are taken, are added spare after 99 μ l sterile waters dilute.

5) following primer, the full length coding region sequence for expanding mouse Tgif1 genes are synthesized.Forward direction primer：5’- CCGCTCGAGGCCACCATGGAGATGGTGCTCTCG-3’(SEQ ID NO:7), wherein CCG is protection base, and CTCGAG is I restriction enzyme sites of Xho, GCCACC are Kozak sequences；Reverse primer is 5 '-GGTCCCCCGGGAAGCTGTGAGTTTGGCCT-3 ' (SEQ ID NO:8), wherein GGTCC is protection base, and CCCGGG is I restriction enzyme sites of Sma.

6) PCR reaction reagents are prepared on ice, and system is as follows：

7) soft mixing, runs following procedure in PCR instrument：

8) it by amplified production whole loading, is analyzed using 1% agarose gel electrophoresis and EB (ethidium bromide) dyeing.

Camk2d coding region sequences expand

Step is identical as Tgif1 coding region sequence amplifications, difference lies in：

Step 1) uses following primer：Forward direction primer：5’-GGGAAGCCAGGTCTGTTCG-3’(SEQ ID NO:9)；Instead To primer：5’-TGGTGCCTTGAGGACAGAATG-3’(SEQ ID NO:10)；

Step 5) synthesizes following primer, the full length coding region sequence for expanding mouse Camk2d genes.Forward direction primer： 5’-CCGCTCGAGGCCACCATGGCTTCGACCACCAC-3’(SEQ ID NO:11)；Reverse primer：5’- GGTCCCCCGGGAGTTGATGGGTACTGTGGG-3’(SEQ ID NO:12)；And

Amplimer in step 6) reaction system is the primer of step 5) synthesis.

Ppp3r2 coding region sequences expand

Using following primer, the full length coding region sequence for expanding mouse Ppp3r2 genes：

Forward direction primer：5’-CCGCTCGAGGCCACCATGGGAAATGAGGCCAG-3’(SEQ ID NO:13)；

Reverse primer：5’-GGTCCCCCGGGAAGCTTTTAAGTCTTCTTGACC-3’(SEQ ID NO:14).

PCR system is prepared on ice, and system is as follows：

PCR programs are as follows：

By amplified production whole loading, analyzed using 1% agarose gel electrophoresis and EB dyeing.

Recycling, digestion and the target fragment of target fragment and the connection of carrier are carried out using conventional method.

Conversion

1) it from -80 DEG C of taking-up DH5 α competent cells, is placed in and thaws on ice.2) 10 μ l connection liquid is taken to add to 30-50 μ l senses In by state cell, ice bath 30 minutes.3) 42 DEG C of water-bath heat shocks 1 minute, then be positioned over 3 minutes on ice.4) it is sterile not that 600 μ l are added Antibiotic LB culture mediums, 37 DEG C of shaken cultivations 60 minutes make bacterium restore normal growth state, and express ammonia benzyl resistance base Cause.5) 50-100 μ l bacterium solutions are taken to be coated on the LB tablets containing Kanamycin, face up placement 30 minutes, waits for that bacterium solution is complete It is cultured after base absorbs and is inverted culture dish, 37 DEG C are cultivated 12-16 hours.

Plasmid extraction

The extraction of Plasmid DNA uses the Endo-free Plasmid Mini Kit II of Omega bio-tek companies, operation Refer to manufacturers instruction.

1.2.8.2 siRNA

SiRNA is synthesized by Ji Ma companies, devises three siRNA sequences herein for each gene, refers to following table (SEQ ID NO:15-32)：

1.2.9 3 ' UTR clones and luciferase detection

Used herein is the psiCHECKTM-2 carriers of promega companies.Renilla in carrier Luciferase (renilla luciferase) is main reporter gene, and target gene can be cloned into renilla luciferase translation eventually The only multiple cloning sites in codon downstream.Since the combination of miRNAs and target gene is mostly in 3 ' UTR of target gene mRNA sequence, After 3 ' UTR are inserted on carrier, overexpression miRNAs can make 3 ' UTR sequences on miRNAs identification carriers, startup pair Target the degradation of sequence mRNA.MiRNAs and target gene can be easily distinguished by detecting the renilla luciferase vigor reduced Between relationship.

1.2.9.1 3 ' UTR vector constructions

First candidate targets and the possible bound sites of miR-146a are searched by websites such as TargetScan, miRNAorg Point expands the region segments containing the site then in the binding site both sides design primer.For expanding Tgifl, Camk2d With primer sequence (the SEQ ID NO as follows of 3 ' the UTR segments of Ppp3r2:33-38)：

1) following reagent is added in order on ice：

2) mixing, executes following procedure in PCR instrument, and the annealing temperature of different genes is shown in Table 2.5.

3) scalpel cuts purpose band, recycles amplified fragments, measured concentration.

4) by recovery product and psiCHECK^TM- 2 carriers are digested using Not I and I restriction enzymes of Xho, then Gel-purified recycles segment, measures the concentration of carrier and each target gene amplified fragments.

5) psiCHECK by digestion after purification^TM- 2 carriers press 0.01pmol with target gene fragment:The ratio of 0.1pmol Example is connected 30 minutes for 16 DEG C using T4DNA ligases.

6) bacillus coli DH 5 alpha is converted, 100 μ l bacterium solutions is taken to be cultivated 12-16 hours for 37 DEG C on Amp resistance LB tablets.

7) sterilizing toothpick picking monoclonal colonies are used under superclean bench, are seeded in the LB culture mediums that 3ml contains Amp, 37 DEG C of shaken cultivations 12-16 hours.

8) plasmid, I double digestion recombinant plasmid of Not I and Xho, the presence or absence of electrophoresis detection Insert Fragment are extracted.

9) picking contains the recombinant plasmid of Insert Fragment, sequencing.

1.2.9.2 being mutated 3 ' UTR carriers

In order to further verify the associativity of miRNA and 3 ' UTR region of target gene, we are by the miRNA of prediction and target base Because the core bit point of combination is mutated, to prevent miRNA from 3 ' UTR sequences after identifying mutation.By using Agilent The QuikChange Lightning Site-Directed Mutagenesis Kit of Technologies companies, to Tgif1, 3 ' UTR regions of Camk2d and Ppp3r2 genes are accordingly mutated.

1) 3 ' UTR mutant primers, sequence (SEQ ID NO as follows are synthesized:39-46)：

2) following reagent is added in order on ice：

3) 1 μ lQuikChange Lightning Enzyme are eventually adding.

4) following procedure is executed in PCR instrument：

5) it is directly added into the Dpn I restriction endonucleases to PCR reaction products carried in 2 μ l kits.

6) soft mixing, brief centrifugation, 37 DEG C digest 5 minutes, remove unmutated template.

7) bacillus coli DH 5 alpha is converted, is cultivated 12-16 hours for 37 DEG C on Amp resistance LB tablets.

8) picking monoclonal colonies, shake bacterium, and whether 3 ' UTR site mutations of sequence verification are completed.

1.2.9.3 luciferase detects

Transfection

1) 293T cells are diluted to 30000 cells/ml, are seeded on 96 orifice plates per 100 μ l of hole.

2) after being incubated overnight, 10 μ l transfection composites (3 ' the UTR recombinant vectors containing 20ng, 80ng miRNA mistakes are added per hole Expression/control vector, 0.4 μ l lipofectamin 2000).

3) after being incubated 1 hour, 100 μ l serum free mediums are added per hole.

4) 48 hours after transfecting, luciferase vitality is measured.

Luciferase vitality measures

1) culture medium is removed, PBS washed once.

2) 50 μ l 1X PLB are added per hole, culture plate is placed on shaking table, are incubated at room temperature 15 minutes.

3) detection parameters are set on photometer, often 100 μ l LAR II solution are added in pipe.

4) it takes 20 μ l PLB pyrolysis products to be added in detection pipe, measures firefly luciferase vitalities.

5) 100 μ lStop＆ are added in same test tubeMeasure Renilla luciferase vitalities.

1.2.10 statistical analysis

Statistical analysis has used 19.0 softwares of SPSS of SPSS Inc., data to be showed in a manner of mean+SD. Significant difference between two groups is measured with two-tailed Student ' s t-test or Mann-Whitney test.P <0.05 is considered having significant difference.

1.2.11 mRNA genetic chips

Sample

The concentration and quality of RNA is measured with NanoDrop ND-1000, and the integrality of RNA is solidifying with the denaturing agarose of standard Gel electrophoresis is assessed.

DNA chip

Using Agilent companies Mouse 4x44K Gene Expression Array Platform Analysis, 39 are comprised more than, The gene and transcription of 000 standard name.

RNA is marked and hybridization array

Sample labeling and hybridization array are according to Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) are executed.In short, total serum IgE linearisation amplification Afterwards, Cy3-UTP is marked, the cRNAs after label is purified with RNeasy Mini Kit (Qiagen).Mark cRNAs (pmol Cy3/ μ g cRNA) concentration and special Sexual potency with NanoDrop ND-1000 measure.Then, 11 μ l are added in the cRNA of every 1 μ g labels 10 × Blocking Agent and 2.2 μ 25 × Fragmentation of l Buffer, 60 DEG C of heating are allowed to fragmentation in 30 minutes； 55 μ l2 × GE Hybridization buffer are eventually adding to dilute the cRNA of label.The hybridization buffer of 100 μ l is added Enter onto gene expression chip slide, 65 DEG C of 17 hours of incubation in Agilent hybridizes case, then washs, it is fixed, it is used in combination Agilent DNA Microarray scanner scannings (part number G2505C).

Data analysis

Agilent Feature Extraction softwares (version 11.0.1.1) are for analyzing obtained array Image.Quantile standardizes and data processing uses GeneSpring GX v11.5.1 software packages (Agilent Technologies).The gene of difference variation identifies that hierarchical clustering passes through GeneSpring GX by doubling variation V11.5.1 softwares execute.GO is analyzed and signal path analysis is with the enrichment algorithm performs of standard.

Two, experimental result

1, inflammatory signals regulate and control the expression of miR-146a in Mouse cartilage cell

We construct traumatic mouse OA models, by taqman sonde methods we have found that miR-146a is soft in mouse OA Expression in bone is significantly higher than the normal cartilage (Fig. 1) for being derived from sham-operation group.We guess the cartilage damaged in OA models certainly The cell factor that body generates may be related with up-regulations of the miR-146a in OA cartilages.Real time PCR results show TNF-α Significantly increase with expression of the IL-1 β in mouse OA cartilages；The expression of IFN-γ decreases, but has no statistical variations.This It is also outside to be apparently higher than control group with expression of relevant gene M MP-3, MMP-13 of arthritis in OA cartilages, shows creating Really local inflammatory environment (Fig. 2) is produced in wound property OA models.

In order to study the regulating and controlling effect that inflammatory signals express cartilage cell miR-146a, we are with different inflammatory factors Stimulate cartilage cell 24 hours.QPCR results show the table of the notable 2 induction miR-146a of IL-1 β, TNF-α and IL-17 energy It reaches, wherein the strongest with the effect of IL-1 β；And IL-6, IFN-γ and TGF-β 1 make miR-146a without apparent regulation and control With (Fig. 3).We have further found that the activation of NF- к B for inhibiting IL-1 beta induced, ERK and p38 signal paths can inhibit miR- The expression (Fig. 4) of 146a, it is especially the most notable with NF- к B and ERK signal paths, illustrate IL-1 β to miR- in cartilage cell The inducing action of 146a depends on NF- к B and ERK signal paths.

2, influences of the assessment miR-146a KO to spontaneity OA caused by aging

We have firstly evaluated miR-146a roles in spontaneous OA models caused by aging.As a result it sends out Existing, when age of mouse reaches 12 months, the articular cartilage surface of WT mouse becomes coarse, cartilage superficial layer occur cartilage loss and The phenomenon that proteoglycans staining power weakens.On the contrary, the articular cartilage surface of miR-146a KO mouse is not apparent Wear condition, proteoglycans staining power remain unchanged (Fig. 5, a).The OA scorings of miR-146a KO mouse are also much low In WT mouse (Fig. 5, b).In addition, the medial tibial plateau edge of the WT mouse at 12 monthly ages has apparent spur to be formed, and MiR-146a KO mouse are almost without significant spur feature (Fig. 5, c-d).We have further found that the WT mouse at 12 monthly ages Its hyaline cartilage area is more than that 80% cartilage cell expresses X-type collagen, and miR-146a KO mouse of the same age are in hyaline cartilage area X-type collagen positive rate is substantially less than WT mouse, and expression intensity is also to be substantially reduced.Expression of the MMP-13 in WT mouse be about 50%, it is far above 20% (Fig. 5, the e-g) of miR-146a KO mouse.Since IRAK-1 and TRAF6 are well-known miR- The main target genes of 146a, we have detected their distribution, find the expression of the two in WT and miR-146a KO mouse Significant difference (Fig. 5, h-j) is had no, illustrates that in OA pathogenic processes inflammation is not the adjusting target of miR-146a.

3, miR-146a KO alleviate the OA regressions of joint instability induction

Next we pass through the OA animal models of three kinds of different joint instability inductions, i.e. DMM, ACLT and PMM mould Type, further to assess influences of the miR-146a KO to the OA state of an illness.Our joint instability OA models caused by DMM first In assessed.After DMM modelings, the knee joint surface of WT mouse shows apparent OA regressions, and showing as cartilage surface becomes Coarse, hyaline cartilage loss, superficial layer cation stain weakened and subchondral bone hyperostosis are more, and bone marrow cell is reduced. Correspondingly, miR-146a KO mouse then show relatively smooth cartilage surface, and superficial layer joint wear is substantially reduced (Fig. 6, a-b).After DMM causes OA to damage, X-type collagen and MMP-13 are in the expression drastically of WT mouse zonas cartilaginous areas It rises, in contrast, expression of the X-type collagen with MMP-13 in miR-146a KO mouse zonas cartilage area is compared with sham groups Substantially do not change, show during the traumatic OA that DMM is induced, cartilage cell's hair can be significantly inhibited by knocking out miR-146a Raw pathologic phenotype migration (Fig. 6, c-e).In ALCT and PMM models, it has been found that identical trend (Fig. 7, A- F), show in the mouse osteoarthritis of wound-induced, knock out miR-146a effective protections cartilage, alleviate OA diseases Shape.

4, miR-146a inhibits cartilage anabolic

As previously mentioned, IL-1 β, TNF-α and IL-17 can promote the induction of miR-146a in Mouse cartilage cell. We next research be induced expression miR-146a cartilage homeostasis caused by inflammatory factor it is unbalance during play the part of Role.As a result, it has been found that IL-1 β, TNF-α and IL-17 significantly inhibit cartilage matrix synthesis related gene Col2a1 and Sox9 Expression, and be overexpressed miR-146a further suppress Col2a1 and Sox9 albumen expression (Fig. 8, a).In contrast, exist Do not stimulate or IL-17 stimulations under, lower miR-146a and increase considerably the protein expression (Fig. 8, b) of Col2a1 and Sox9. Experience OA operation after, in the articular cartilage of miR-146KO mouse the expression of Col2a1 and Sox9 to be significantly higher than WT mouse (figure 8, c-d).These results absolutely prove, the miR-146a that proinflammatory cytokines induce, accelerate the former mediation to cartilage matrix Anabolic inhibiting effect.

5, miR-146a targets the anabolism that multiple genes inhibit cartilage

The anabolic targeted molecular of cartilage matrix is adjusted in order to find miR-146a, we distinguish Mouse cartilage cell It is overexpressed or is lowered miR-146a expression, then total serum IgE is sent to company and carries out mRNA gene expression spectrum analysis.Based on miR- Conservative of the identification sequence of 146a and target gene between each species, we have selected 12 genes to carry out further verification (figure 9, a).Then we demonstrate chip gene expression profile result (Fig. 9, b) by qPCR.

In order to further verify the marriage relation between miR-146a and target gene, we perform 3 ' UTR luciferases Detection.It is overexpressed the relative fluorescence element enzyme activity that miR-146a significantly reduces multiple Reporter gene vectors, including Tgif1, Bag1, Camta1, Sox5, Sema3g, Camk2d, Ppp3r2 and Stim2 (Fig. 9, c).In order to reduce research range, we measure Expression situation of the above candidate targets gene in OA cartilages.After DMM modelings 4 weeks, the articular cartilage of miR-146a KO mouse The mRNA of middle Tgif1, Camk2d and Ppp3r2 expression are significantly higher than WT mouse (Fig. 9, e-g).Therefore, we are directed to these three bases Because carrying out deep verification.In the presence of wild type UTR, be overexpressed miR-146a and significantly reduce Tgif1, Camk2d and The luciferase vitality of Ppp3r2 report carriers；And after 3 ' UTR are mutated, miR-146a identifies the 3 ' of target gene due to failing It is similar with control (Fig. 9, d) to show relative fluorescence element enzyme activity to fail to play a role by UTR.

These three genes of Tgif1, Camk2d and Ppp3r2 are further studied to synthesize cartilage what miR-146a was mediated Effect during metabolic inhibition.Pass through the full length coding region sequence of PCR amplification Tgif1, Camk2d and Ppp3r2 first. Then by Tgif1, Camk2d and Ppp3r2 genes are cloned into pEGFP-N1 expression vectors by Xho I and Sma I restriction enzyme sites. Next the target gene over-express vector built is transfected to Mouse cartilage cell.After transfected expression vector, cartilage cell The mRNA expression of interior Tgif1, Camk2d and Ppp3r2 gene improves 560 ± 8.2,966 ± 26.7 and 31102 respectively ± 1917.5 again.We have found that the expression of Ppp3r2 can also be promoted simultaneously by being overexpressed Tgif1 in cartilage cell, biological value can reach As many as 305 ± 26.7 times.Western blot the result shows that, Tgif1, Camk2d and Ppp3r2 bases are overexpressed in cartilage cell Because significantly increasing the Tot Prot of transcription factor Sox9, it is significantly enhanced by the protein expression of the Col2a1 of Sox9 regulation and control, Trend is consistent (Fig. 9, h) caused by being lowered with miR-146a in cartilage cell.

Interfere the expression for lowering Tgif1, Camk2d and Ppp3r2 that can be substantially reduced Col2a1's and Sox9 by siRNA Protein expression (Fig. 9, i), this is overexpressed in cartilage cell under the protein expression of Col2a1 and Sox9 caused by miR-146a Drop is consistent.The above is the results show that miR-146a is played in turn by targeting Tgif1, Camk2d and Ppp3r2 genes Inhibit the effect of cartilage matrix synthesis.

6, intra-articular injection miR-146a has therapeutic effect to OA

As previously mentioned, knocking out miR-146a either to caused by spontaneity OA caused by the age or wound natural disposition OA All there is significant inhibiting effect.In order to study the influence for adjusting the level of miR-146a after the onset of OA and restoring for OA, I Be prepared in mouse by DMM first joint instability induction OA models.DMM modelings after a week, we by mouse with Every group 8~9 are randomly divided into 4 groups, and miR-146a is overexpressed viral (Lenti-mimic 146a), miR-146a inhibitor diseases Malicious (Lenti-inhibitor 146a) and respective comparison virus are only injected into joint with 5 μ l/ by microinjector Intracavitary.Injection is primary week about later, and a co-injection 3 times (Figure 10, a).After DMM modelings 4 weeks, after sampling is fixed, decalcification is done Continuous analysis.The fast green dyeing display of sarranine-, it is soft that injection has miR-146a to be overexpressed the joint negative control group (Lenti-mimic NC) Bone surface abrasion is apparent, especially femoral surface cartilage；Subchondral bone has apparent hardening under tibial plateau.In contrast, MiR-146a is overexpressed virus injection group its cartilage and shows to degrade even more serious, and either tibial plateau or condyle of femur surface is equal There is apparent regression；In addition, cartilage surface color substrates decline to a great extent, Subchondral bone sclerosis degree is similar with control group, shows OA early stages give the destruction that miR-146 up-regulations can aggravate articular cartilage.On the contrary, we have found that lowering cartilage in OA initial periods The expression of intracellular miR-146a can significantly alleviate cartilage degeneration.There are miR-146a inhibitor negative control groups with injection (Lenti-inhibitor NC) is compared, and miR-146a inhibitor group (Lenti-inhibitor 146a) remains relatively smooth Articular cartilage surface loses situation (Figure 10, b-c) without apparent color substrates.Showed by immune group result is in Lenti- Mimic NC groups, DMM cause the positive rate of X-type collagen in hyaline cartilage to reach 64.6 ± 11.7%；And miR-146a is injected After being overexpressed virus, hence it is evident that aggravated the phenotype of cartilage cell to loose cartilage cell differentiation, the positive rate of X-type collagen reaches As many as 87.2 ± 7.1%.The expression for striking low miR-146a significantly suppresses the character mutation of cartilage cell, relative to control group 67.5 ± 17.9% positive rate, the positive rate of the X-type collagen of Lenti-inhibitor-146a injection groups only has 44.9 ± 9.89% (Figure 10, d and e).

MiR-146a is overexpressed viral (Lenti-mimic 146a), the lucky agate corporation of miR-146a inhibitor viruses commission It is standby.Specifically, will contain miR-146a maturation body sequences or its reverse complements be cloned into slow virus carrier (Ji Ma, in State Shanghai) in, then pack.

Mmu-mir-146a maturation body sequences：5’-TGAGAACTGAATTCCATGGGTT-3’(SEQ ID NO:47)；

Mmu-mir-146a reverse complements：5’-AACCCATGGAATTCAGTTCTCA-3’(SEQ ID NO:48).

7, Camk2d and Ppp3r2 is the function target that miR-146a adjusts OA

The positive expression rates of the Camk2d in the WT mouse articular cartilages at 12 monthly ages is only 25.7 ± 14.0%, much low In in WT mouse articular cartilages of growing up 68.8 ± 15.6% positive expression rate；Expression intensity is also to be greatly lowered.With this Corresponding to be, the positive expression rate of Camk2d is 82.5 ± 7.0% in miR-146a KO adult mices, with WT mouse of the same age And there was no significant difference；Even if when entering the senescence phase, positive expressions of the Camk2d in miR-146a KO mouse zona cartilages Rate is still up to 56.7 ± 6.1%, is significantly higher than WT mouse (P=0.009) of the same age, and it is strong to maintain stronger expression Degree.The expression trend of Ppp3r2 is similar with Camk2d (Figure 11, a-c).

MiR-146a joint cavity injections experiment in, miR-146a be also by targeted inhibition Camk2d and Ppp3r2 this two A molecule functions.As shown in Figure 11 (f-h), injection miR-146a overexpression viruses significantly suppress Camk2d and exist in section The expression of mouse zona cartilaginous areas makes its positive expression rate be down to miR-146a by the 47.2 ± 19.8% of control group and is overexpressed 19.4 ± 17.6% (P=0.02) of group.MiR-146a inhibitor then significantly increases the expression of Camk2d, makes its positive expression 68.4 ± 22.2% (P=0.03) that rate increases to inhibitor group by the 40.9 ± 12.4% of control group.Similarly, miR-146a is aobvious Work reduces expression (P=0.002) of the Ppp3r2 in articular cartilage；And lower miR- by injecting miR-146a inhibitor 146a expression can make 73.2 ± 12.6 (P=that the expression of Ppp3r2 increases to inhibitor group from the 48.1 ± 9.11% of control group 0.0001)。

It is previously noted that miR-146a inhibits the anabolism gene of cartilage such as by targeting Camk2d and Ppp3r2 equimoleculars The expression of Col2a1 and Sox9；Here we deeper into discovery in the growth course of OA miR-146a be strictly pass through inhibition Camk2d and Ppp3r2 expression is to aggravate the development of OA.According to cell-signaling pathways analysis and literature search, it has been found that living Change T cell nuclear factor (nuclear factor of activated T cells, NFATs) and is likely to associated therewith.We send out WT mouse articular cartilage surfaces of the existing NFAT2 3 monthly ages is in low expression, and intensity is weaker；And in miR-146a KO mouse NFAT2 high expression in articular cartilage, positive rate are 62.8 ± 24.2%, significantly larger than 14.3 ± 12.8% (P=of WT mouse 0.01)；It is mainly distributed on hyaline cartilage and is all expressed in core, shows to be in the state of activation.In mouse aging, Expression of the NFAT2 in miR-146a KO Mouse cartilages still has 30.0 ± 9.4%, be significantly higher than WT mouse 12.3 ± 4.7% (P=0009) (Figure 11, e).We also have detected Tissue distributions of the NFAT1 in mouse.NFAT1 is small in 3 monthly age WT Positive expression rate in mouse hyaline cartilage is 33.2 ± 9.6%, and in the table of miR-146a KO mouse zonas cartilage area of the same age Up to up to 84.2 ± 10.4% (P=0.003)；Expression in 12 monthly age KO mouse 98 also has 55.5 ± 18.2%, much Higher than 17.9 ± 9.2% (Figure 11, the d) of WT mouse, the adjusting that the activation of NFATs takes part in miR-146a to OA is absolutely proved Journey.

Sequence table

<110>Shanghai Inst. of Life Science, CAS

<120>The application of miR-146a and its target spot in treating osteoarthritis

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Claims (10)

1. the combination of selected from the following a kind of or arbitrary plurality of reagents is preparing the drug for promoting cartilage synthesis or is being controlled in preparation Application in the drug for the treatment of or relief from osteoarthritis：

(a) expression of miR-146a or active reagent are lowered；

(b) expression of up-regulation Camk2d or active reagent；

(c) expression of up-regulation Ppp3r2 or active reagent；With

(d) expression of the molecule in NAFT signal paths or active reagent are raised.

2. application as described in claim 1, which is characterized in that the reagent is nucleic acid molecules, carbohydrate, lipid or small Molecular compound；

Preferably, the reagent of the expression for lowering miR-146a is to knock out the targeting vector of the miR-146a in cell, or be MiR-146a inhibitor such as expresses the virus of the reverse complements of miR-146a；

The expression vector that the reagent of the up-regulation Camk2d expression is Camk2d；

The expression vector that the reagent of the expression of the up-regulation Ppp3r2 is Ppp3r2；

The reagent of the expression of molecule in the up-regulation NAFT signal paths is the expression vector of the molecule；Preferably, described NAFT signal path molecules are selected from NAFT1 and NAFT2.

3. application as claimed in claim 1 or 2, which is characterized in that the osteoarthritis is that spontaneous bone caused by aging closes The osteoarthritis or sections inner side meniscus for the osteoarthritis, tears of anterior cruciate ligament induction that section is scorching, joint instability induces are cut It removes and the osteoarthritis of the cross-section induction of medial collateral ligament.

It is one or more in preparing the kit of the synthesis of monitoring cartilage or osteoarthritic condition progress in reagent below 4. Application：

(a) it is used to detect the reagent of miR-146a expressions；

(b) it is used to detect the reagent of Camk2d expressions；

(c) it is used to detect the reagent of Ppp3r2 expressions；With

(d) it is used to detect the reagent of the expression of NAFT signal path molecules.

5. application as claimed in claim 4, which is characterized in that

The reagent for detecting Camk2d expressions includes detecting the reagent used during Camk2d mRNA level in-sites With the reagent used during detection Camk2d protein contents；

The reagent for detecting Ppp3r2 expressions includes detecting the reagent used in Ppp3r2mRNA horizontal process With the reagent used during detection Ppp3r2 protein contents；

The reagent for detecting NAFT signal paths developed by molecule level includes detection NAFT signal path molecule mRNA water The reagent used during the reagent and detection NAFT signal path molecule proteins content that are used during flat.

6. application as described in claim 4 or 5, which is characterized in that the kit contains：

The horizontal reagent of the mRNA is detected using Northern hybrid methods, RT-PCR or RNA-seq；And/or

The reagent of protein content is detected using Western blot or ELISA.

7. a kind of detection kit, which is characterized in that the detection kit contain a kind of, arbitrary two kinds in following reagent, Arbitrary three kinds or all four kinds：

(a) it is used to detect the reagent of miR-146a expressions；

(b) it is used to detect the reagent of Camk2d expressions；

(c) it is used to detect the reagent of Ppp3r2 expressions；With

(d) it is used to detect the reagent of the expression of NAFT signal path molecules.

8. it is a kind of promote cartilage synthesis or treatment osteoarthritis method, which is characterized in that the method includes it is following any one Or the combination of any number of steps：

(a) expression of miR-146a in object or the step of activity are lowered；

(b) expression of Camk2d in object or the step of activity are raised；

(c) expression of Ppp3r2 in object or the step of activity are raised；With

(d) expression of the molecule in object in NAFT signal paths or the step of activity are raised.

9. method as claimed in claim 8, which is characterized in that a kind of or arbitrary the method includes giving the object or less The step of combination of plurality of reagents：

(a) expression of miR-146a or active reagent are lowered；

(b) expression of up-regulation Camk2d or active reagent；

(c) expression of up-regulation Ppp3r2 or active reagent；With

(d) expression of the molecule in NAFT signal paths or active reagent are raised.

10. a kind of diagnosis osteoarthritis or the method for monitoring osteoarthritic condition development, which is characterized in that the method includes inspections Survey one or more expression in the molecule in object body fluid in miR-146a, Camk2d, Ppp3r2 and NAFT signal path Horizontal steps；

Preferably, the detection Camk2d expressions include detection Camk2d mRNA level in-sites and detection Camk2d albumen；It is described It includes detection Ppp3r2mRNA levels and detection Ppp3r2 protein contents to detect Ppp3r2 expressions；The detection NAFT signals Pathway molecule expression includes that detection NAFT signal path molecule mRNA level in-sites and detection NAFT signal path molecule proteins contain Amount；

Preferably, the level of the mRNA is detected using Northern hybrid methods, RT-PCR or RNA-seq；Using Western The content of blot or ELISA detection albumen；

Preferably, the body fluid is peripheral blood.