WO2016034095A1 - 在骨形成中发挥调控作用的应力敏感性microRNA - Google Patents

在骨形成中发挥调控作用的应力敏感性microRNA Download PDF

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WO2016034095A1
WO2016034095A1 PCT/CN2015/088697 CN2015088697W WO2016034095A1 WO 2016034095 A1 WO2016034095 A1 WO 2016034095A1 CN 2015088697 W CN2015088697 W CN 2015088697W WO 2016034095 A1 WO2016034095 A1 WO 2016034095A1
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mir
expression
modification
runx2
bone
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PCT/CN2015/088697
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French (fr)
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张晓玲
左斌
陈晓东
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中国科学院上海生命科学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention is in the field of biotechnology, and more particularly, the present invention relates to stress-sensitive microRNAs that exert a regulatory role in bone formation.
  • Mechanical stress stimulation is critical for the regulation of bone remodeling homeostasis through mechanical signaling pathways.
  • mechanical stress stimuli are stimulated by mechanical signals such as bone cells and osteoblasts and transduced into biological response signals to the main effector cells (osteoblasts and osteoclasts) on the bone surface to complete the relevant response.
  • Osteoblast differentiation can be mediated by mechanical stress stimulation and cause a cascade of hormones, growth factors, transcription factors, etc., which in turn affect cell proliferation and differentiation.
  • the loss of load such as long-term bedridden patients, long-term in the absence of gravity, such as astronauts can lead to bone loss and rapidly progress to disuse osteoporosis.
  • Long-term overload conditions such as sports athletes, will lead to trabecular microfractures and progress to stress fractures or fatigue fractures.
  • MicroRNAs are a class of endogenous single-stranded, small-molecule non-coding RNAs of approximately 22 nucleotides in length that play important regulatory roles in many biological processes. Through post-transcriptional regulation, miRNAs inhibit target gene expression by binding to the 3'UTR seed region of the target gene mRNA to degrade or inhibit translation of the target gene. miRNAs regulate about one-third of human protein-coding genes, suggesting a key role in regulating gene expression. Multiple miRNAs have been shown to regulate the process of osteogenic differentiation by targeting the expression of specific genes involved in osteogenic differentiation at in vitro levels.
  • a down-regulator of miR-103a for the preparation of a medicament for preventing or treating a bone metabolic disease (a stress caused by a stress-related bone metabolic disease such as an astronaut in a long-term space microgravity environment or a prolonged bedridden patient)
  • a bone metabolic disease a stress caused by a stress-related bone metabolic disease such as an astronaut in a long-term space microgravity environment or a prolonged bedridden patient
  • the miR-103a inhibitor comprises: chemically synthesized miR-103a inhibition a viral and non-viral product that inhibits miR-103a using an expression plasmid; a nucleic acid sequence or sequence fragment complementary to miR-103a.
  • the down-regulator of miR-103a is selected from the group consisting of: antagomir-103a, the nucleotide sequence of which is set forth in SEQ ID NO: 49; or inhibitor-103a, the nucleotide sequence of which is SEQ ID. NO: 47.
  • the down-regulating agent of miR-103a is a modified down-regulating agent, including but not limited to: methoxylation modification, thio modification, cholesterol modification, alkyl modification (such as an alkyl modification at the 2' position of ribose), a locked nucleic acid modification, a peptide nucleic acid modification, and/or an antisense nucleotide having a phosphate backbone replaced by a phospholipid linkage; preferably, the modification of the antagomir-103a These include: cholesterol modification at the 3' end, two thio skeleton modifications at the 5' end, four thio skeleton modifications at the 3' end, and full chain methoxy modification.
  • the medicament is further used to:
  • miR-103a for screening for a medicament for preventing or treating a bone metabolic disease.
  • the bone metabolic disease comprises: osteoporosis, abnormal osteogenic differentiation, and loss of bone mass.
  • a medicament for preventing or treating a bone metabolic disease which is a down-regulator of miR-103a, is selected from the group consisting of: antagomir-103a, and the nucleotide sequence thereof is SEQ ID NO: 49 Shown; or inhibitor-103a, the nucleotide sequence of which is shown in SEQ ID NO:47.
  • a method of screening for a potential substance for preventing or treating a bone metabolic disease comprising:
  • the candidate substance can reduce the expression of miR-103a, it indicates that the candidate substance is a potential substance for preventing or treating a bone metabolic disease.
  • system further expresses Runx2 protein, the method further comprising: detecting expression of Runx2 protein in the system;
  • the candidate substance is increased by down-regulating the expression of miR-103a (preferably, a significant increase, such as an increase of 20% or more, preferably an increase of 50% or more; a more preferable increase of 80% or more) of Runx2 protein expression, This indicates that the candidate substance is a potential substance for preventing or treating a bone metabolic disease.
  • step (1) comprises: adding a candidate substance to a system expressing miR-103a or co-expressing miR-103a and Runx2 proteins in a test group; and/or
  • Step (2) comprises: detecting expression of miR-103a and/or Runx2 protein in a system of the test group, and comparing the expression to miR-103a and/or the addition of the candidate substance to the control group.
  • Runx2 protein system
  • miR-103a in the test group is statistically lower (preferably significantly lower than, eg, less than 20% lower, preferably lower than 50%; more preferably lower than 80%), or still make Runx2 A significant increase in protein expression indicates that the candidate is a potential substance for the prevention or treatment of bone metabolic diseases.
  • the system is selected from the group consisting of a cell system (or cell culture system), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
  • the method further comprises performing further cellular experiments and/or animal tests on the obtained potential substances to further select and determine substances useful for preventing or treating bone metabolic diseases from the candidate substances.
  • Figure 1 Stress loading promotes bone remodeling and bone formation in vivo.
  • miR-103a inhibits osteoblast function by targeting target Runx2 at in vitro levels.
  • qRT-PCR showed 8% CMS-mediated expression of miR-103a in hFOB 1.19 human osteoblast line differentiation (8% elongation, Sin, 0.5 Hz, 3d). The expression of miR-103a was referenced to the resting group control.
  • 8% CMS was loaded with hFOB 1.19 human osteoblast cell line for 3 days, and PANK2 and PANK3 expression levels were detected by qRT-PCR.
  • MiR-103a inhibits osteoblast activity and extracellular matrix mineralization during in vitro stress-stimulated osteoblast differentiation.
  • HU+PBS HU mice injected with PBS in the tail vein
  • HU+Antagomir-103a HU mice injected with antagomir-103a in the tail vein.
  • qRT-PCR analysis of miR-103a expression levels in the femur of HU, HU+PBS, HU+Antagomir-103a mice correcteded in WB mice.
  • Western blot analysis showed Runx2 protein levels in the femur of WB, HU, HU+PBS, HU+Antagomir-103a mice.
  • g Three-dimensional reconstruction of the distal femur microCT of WB, HU, HU+PBS, HU+Antagomir-103a mice.
  • Bone histomorphology Analysis bone formation related parameters (Ob.S/BS, MAR and N.Ob/B.Pm) in WB, HU, HU+PBS, HU+Antagomir-103a mice (k)WB, HU, HU+PBS , HU+Antagomir-103a mice distal femur TRAP staining. Scale, 100 ⁇ m
  • bone histomorphometric analysis WB, HU, HU + PBS, HU + Antagomir-103a mice bone resorption related parameters (Oc.S / B.S, N.Oc / B.Pm) detection
  • FIG 7 pmiR-RB-REPORT TM dual luciferase reporter vector map.
  • miR-103a is a new mechanically sensitive miRNA and plays an important role in osteoblast differentiation, which can be used as a prevention and treatment of bone metabolic diseases (including osteoporosis and abnormal osteogenic differentiation). , bone loss, etc.) targets.
  • the down-regulation of miR-103a improves the osteoporosis phenotype caused by stress loss.
  • the miR-103a is a small ribonucleic acid (hsa-miR-103a-3p, MIMAT0000101) having the nucleic acid sequence of SEQ ID NO: 1:
  • miR-103a is a steady-state small ribonucleic acid that has been found to promote lipid metabolism and regulate glycolipid metabolism, and its role in bone metabolism has not been reported in the prior art.
  • the present inventors verified the effects of mechanical stress loading on osteogenic differentiation and bone remodeling in vivo and in vitro by constructing a stress-loaded cell model under load and a dual hindlimb unloading mouse model in vivo and in vitro.
  • Screening of target miRNAs by bioinformatics methods and subsequent functional verification by qRT-PCR, screening and identification of miR-103a is a new mechanically sensitive miRNA and plays an important role in osteoblast differentiation.
  • miR-103a and its host gene PANK3 were down-regulated in mechanical stress-stimulated osteoblast differentiation (8% CMS, 0.5 Hz, Sin), while Runx2 protein levels were up-regulated.
  • miR-103a plays an inhibitory role.
  • the expression of miR-103a was up-regulated in hindlimb unloaded mice, which may negatively regulate bone formation by inhibiting Runx2 expression at the in vivo level.
  • the administration of long-acting inhibitor mimics by tail vein partially rescues the stress-deficient Osteoporosis phenotype.
  • miR-103a is a new marker that can be used to prevent and treat bone metabolic diseases including osteoporosis, abnormal osteogenic differentiation, loss of bone mass, and the like.
  • the present invention provides a use of a miR-103a down-regulating agent for the preparation of a composition (such as a drug) for preventing or treating a bone metabolic disease.
  • the miR-103a down-regulating agent can prevent and treat abnormal bone metabolism (such as osteoporosis) by inhibiting the expression of miR-103a.
  • the miR-103a down-regulator is also used to promote the expression of Runx2 protein; to enhance the expression levels of ALP and Ocn in osteoblast differentiation; or to enhance extracellular matrix mineralization.
  • miR-103a down-regulator includes antagonists, inhibitors, blockers, blockers and the like as long as they are capable of down-regulating the expression level of miR-103a. They can be compounds, chemical small molecules, biomolecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or a viral product that inhibits the expression of miR-103a.
  • the miR-103a down-regulation agent refers to any substance which can reduce the activity of miR-103a, decrease the stability of miR-103a, down-regulate the expression of miR-103a, and reduce the effective action time of miR-103a.
  • the invention as a substance useful for down-regulating miR-103a, can be used to improve the growth of a bone metabolic disease.
  • the down-regulator is a nucleic acid inhibitor, a protein inhibitor, a nuclease, or a nucleic acid-binding molecule as long as it can down-regulate the expression of miR-103a.
  • the down-regulating agent is selected from the group consisting of: a chemically synthesized miRNA down-regulator; a plasmid-mediated miRNA-inhibiting virus and a non-viral product; and a nucleic acid sequence or sequence fragment complementary to miR-103a. .
  • the miR-103a down-regulator is a specially modified miRNA antagonist, such as, but not limited to, a methoxylation modification, an alkyl modification (eg, at the 2' position of ribose Alkyl modification), locked nucleic acid modification, peptide nucleic acid modification, thio modification, and antisense nucleotides in which the phosphate backbone is replaced by a phospholipid linkage.
  • a specially modified miRNA antagonist such as, but not limited to, a methoxylation modification, an alkyl modification (eg, at the 2' position of ribose Alkyl modification), locked nucleic acid modification, peptide nucleic acid modification, thio modification, and antisense nucleotides in which the phosphate backbone is replaced by a phospholipid linkage.
  • the miR-103a down-regulation agent is antagomir-103a or inhibitor-103a.
  • the modification includes: cholesterol modification at the 3' end, two thio skeleton modifications at the 5' end, four thio skeleton modifications at the 3' end, and full chain methoxy modification, thereby increasing its stability and promoting its effectiveness.
  • Antagomir-103a inhibits the complementary pairing of miRNAs with their target gene mRNA and inhibits the action of miRNAs by binding to mature miRNAs in vivo.
  • miRNA antagomir has higher stability and inhibitory effects in vivo and in vitro, and can overcome the imbalance of cell membrane and tissue in vivo and concentrate on target cells.
  • Antagomir does not require transfection reagents in cell experiments, thus avoiding the complex steps of the transfection reagent packaging process and its impact on the experiment.
  • systemic or local injection, inhalation, medication, etc. can be administered for a duration of up to 6 weeks.
  • the present invention also provides a composition (e.g., a pharmaceutical) comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the miR-103a.
  • a composition e.g., a pharmaceutical
  • an effective amount e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • a formulation, as well as a pharmaceutically acceptable carrier can be used to modulate bone metabolism. Any of the foregoing down-regulators of miR-103a can be used in the preparation of the compositions.
  • the "effective amount” refers to an amount that is functional to a human and/or animal and that is acceptable to humans and/or animals.
  • the “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, or a buffer.
  • auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the vector may also contain a cell transfection reagent.
  • the modulator or pharmaceutical composition thereof can be administered to a mammal using a variety of methods well known in the art. These include, but are not limited to, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the administration mode is parenterally administered.
  • the administration method of the lowering agent may also be an injection administration of a local site, for example, a method of intra-articular administration.
  • the effective amount of the down-regulator of miR-103a according to the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the down-regulator of miR-103a such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, and the immunity of the patient Status, route of administration, etc. For example, by treatment In the case of an urgent need, several separate doses may be administered per day, or the dose may be proportionally reduced.
  • miR-103a is closely related to bone metabolic diseases including osteoporosis, abnormal osteogenic differentiation or loss of bone mass
  • substances that inhibit the expression of miR-103a can be screened based on this feature.
  • Drugs that are truly useful for preventing or treating bone metabolic diseases can be found from the substances described.
  • the present invention provides a method of screening for a potential substance for inhibiting a bone metabolic disease, the method comprising: treating a system for expressing miR-103a with a candidate substance; and detecting expression of miR-103a in the system;
  • the candidate substance inhibits the expression of miR-103a, indicating that the candidate substance is a potential substance for inhibiting bone metabolic diseases.
  • the system for expressing miR-103a may be, for example, a cell (or cell culture) system, which may be a cell that endogenously expresses miR-103a; or may be a cell that recombinantly expresses miR-103a.
  • the system for expressing miR-103a may also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal such as a mouse, a rabbit, a sheep, a monkey, etc. )Wait.
  • a control group in order to make it easier to observe a change in the expression of miR-103a, a control group may be provided, and the control group may be an expression miR-103a without adding the candidate substance.
  • the system further expresses a Runx2 protein, the method further comprising: detecting expression of a Runx2 protein in the system; wherein, if the candidate substance is capable of downregulating expression of miR-103a Increasing the expression of Runx2 protein indicates that the candidate substance is a potential substance for the prevention and treatment of bone metabolic diseases.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine substances that are truly useful for inhibiting bone metabolic diseases.
  • the method for detecting the expression, activity, amount or secretion of the miR-103a or Runx2 protein of the present invention is not particularly limited.
  • Conventional protein quantification or semi-quantitative detection techniques can be employed, such as, but not limited to, SDS-PAGE, Western-Blot, and the like.
  • the present invention also provides a potential substance for inhibiting a bone metabolic disease obtained by the screening method.
  • These initially screened materials can constitute a screening library so that one can finally screen out substances that can be useful for inhibiting the expression and activity of miR-103a, thereby inhibiting bone metabolism diseases.
  • C57BL6/J mice (Shanghai Shrek Laboratory Animals) were divided into three groups: basic (BS) group, hanging tail (HU) group, and static control (WB) group.
  • the HU group mice were spirally wound with the medical wide tape (15cm ⁇ 0.5cm) from the front 1/3 of the tail of the rat, and the tape was fixed on the top of the cage, so that the double hind limbs and the bottom of the cage were suspended at 30°, but did not affect.
  • Its forelimb activity allows it to drink freely for 4 weeks, and observe the blood supply of the tail to prevent ischemic necrosis. Weigh twice a week to observe its growth and development.
  • the WB group mice were blank controls without special treatment.
  • hFOB1.19 was cultured in a 10 mm culture dish containing 10% FBS, 1% double antibody and 0.3 mg/mL G418 in DMEM complete medium, incubated at 34 ° C, 5% CO 2 concentration, 95% humidity incubator, and taken P3 substituting 2.0 ⁇ 10 5 cells were seeded in six-well culture ordinary type I collagen coated plates and BioFlex TM tin plates, medium was changed every two days; the cells grow to 90% confluency, the culture tin BioFlex TM The plate was placed in a FX-5000 TM FLEXCELL TENSION PLUS stress-loading device, and the incubator temperature was set to 34 ° C, 5% CO 2 concentration, 95% humidity, and the time was 0 days. The liquid was changed every 2 days and loaded for 3 days. 7 days; with the static set as the control; the stress loading procedure was set to: 8% CMS (8% deformation, 80000 ⁇ , Sin, 0.5 Hz, CMS), where CMS: cyclic mechanical stretch.
  • CMS
  • RNA is applied to the bottom side of the tube in a white gel.
  • the culture solution was aspirated and washed three times with PBS; 4% paraformaldehyde was fixed at room temperature for 10 minutes, rinsed with PBS, air-dried; alkaline phosphatase staining kit (Biyuntian) working solution was prepared; dyeing mixture was prepared according to the operation manual. Add 1 ml of staining mixture (6-well plate) to each well, incubate at 37 ° C for 1 h; rinse with water and dry; take a picture, the enzyme activity is bright red particles.
  • the cell samples were collected and stored frozen (-20 ° C); after use, the cells were centrifuged, and the cell pellet was submerged into the bottom of the tube at 4 ° C, 2000 rpm, 2 min, and the supernatant was discarded; 100-500 ⁇ l of distilled water (added according to the amount of cells) was mixed. If necessary, use a oscillating instrument to oscillate; repeatedly freeze-thaw: freeze 10 min ⁇ 3 times, -80 ° C, melt at room temperature after each freezing, repeatedly freeze-thaw to lyse the cells, protein precipitation; mix and centrifuge, 4 ° C, 1000 rpm, 3 min, take The supernatant.
  • DEA: pnpp 2:1 mixing, add a tube of blank to adjust the background, add 150 ⁇ l mixture and 50 ⁇ l cell supernatant to each tube, add distilled water to the blank tube, mix well; 37°C water bath for 15min (yellow is positive); Add 1N NaOH 400 ⁇ l to stop the reaction, shake, centrifuge the liquid in the lower lid; take 200 ⁇ l ⁇ 2 (multiple well) into each tube to add 96-well plate; continuous wavelength scanning microplate reader, the two groups take the average value.
  • Pnpp/BCA takes a relative quantitative value.
  • 40 mM Alizarin Red staining solution 1.3692 g Alizarin Red powder (Sigma) was dissolved in 100 ml of PBS, and the pH was adjusted to 4.1-4.3, and allowed to stand at room temperature for use. Absorb the culture solution, rinse twice with PBS; fix 4% paraformaldehyde for 15 min at room temperature; rinse twice with ddH 2 O; add 1 ml/well of 40 mM alizarin red staining solution, incubate for 20 min at room temperature and shake gently; Dye, rinsed with ddH 2 O and shaken 4 times for 5 min; tilted for 2 min, aspirate excess ddH 2 O; observe the photo recording with an inverted microscope.
  • the vigorously proliferating hFOB1.19 cells (about 70-80% confluence of pre-digested cells) were collected; the cell density was adjusted by cell counting, and inoculated on a force plate and an ordinary 6-well plate at 4 ⁇ 10 3 cells/well, lightly mixed. The seeding density was consistent; the cells were incubated in a 5% CO 2 incubator at 34 ° C; AlamarBlue (10% concentration) was added at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, lightly mixed, and returned to 34 ° C.
  • PCR amplification method was used to design the amplification primers based on Runx2 (human) 3'UTR sequence information, and the 3'UTR sequence of Runx2 gene was amplified by PCR using 293T cell genomic DNA as a template, and cloned into pmiR-RB-REPORT TM double fluorescence.
  • the prime enzyme reporter vector was constructed by Guangzhou Ruibo Biotechnology Co., Ltd., and the reported fluorescence of the vector used was hRluc, and the corrected fluorescence was hluc (corrected for internal reference).
  • the target gene fragment was cloned into the vector by XhoI and NotI restriction enzymes, and the vector map is shown in Fig. 7.
  • the XhoI, NotI restriction site sequence was set, and the previous sequence was a protected base, and the total length of the primer amplification was 3798 bp. Positive clones were identified by sequencing.
  • the TRIZOL (Invitrogen) method extracts bone tissue or total RNA from cells.
  • Prepare cDNA reverse transcription system (RNA 1 ⁇ g, Oligo d (T) 1 ⁇ l, DEPC water to make up), mix, 70 ° C for 5min, immediately placed on ice for at least 1min, continue to add 5 ⁇ RevertAid buffer 4 ⁇ l, dNTP in the reaction system 2 ⁇ l of the mixture, 0.5 ⁇ l of RevertAid, and after mixing, at 42 ° C for 1 h, the sample was stored at -20 ° C. After the RT product was diluted 10 times, a Real-time PCR reaction system was prepared.
  • the cells were discarded, washed with pre-cooled PBS; RIPA lysate (containing 1% PMSF) was added and lysed on ice for 15 min.
  • the cells were collected by pipetting in a 1.5 ml EP tube and centrifuged at 12,000 rpm for 15 min. The supernatant was taken as the total protein, and stored at -80 ° C for use; BCA protein was quantified. Take 50 ⁇ g protein, add 5 ⁇ loading buffer, mix, heat at 95 °C for 10min, denature the protein, disulfide bond is opened; prepare separation gel and concentrated gel; add electrophoresis buffer in the electrophoresis tank, take protein marker 5 ⁇ l and Denatured protein, added to the sample well, 80V electrophoresis for about 30 minutes, to the bromophenol blue into the separation gel, changed to 120V voltage, electrophoresis for about 1 hour; carefully remove the SDS-PAGE gel, with PVDF membrane, qualitative filter paper and fiber The pad is placed in the electrophoresis tank transfer buffer, and is sandwiched by sandwiching (fiber mat, filter paper, gel, PVDF film, filter paper, fiber mat from bottom to top), completely removing the internal bubbles and
  • the PVDF membrane was cut according to the molecular weight of the corresponding protein, the primary antibody was incubated at 4 ° C overnight; the primary antibody was aspirated, and the membrane was washed 3 times with TBST on a shaker for 5 min each time; horseradish peroxidase-labeled Secondary antibody, room temperature on a shaker Breed for 1 hour; aspirate the secondary antibody, wash the membrane 3 times with TBST on a shaker for 5 minutes each time; color and tablet: mix the coloring solution 1:1, add to the film, and develop color at room temperature 1-5 Minutes, use X-sensitive film to compress the film at the appropriate time and punch.
  • the image was input to a computer using a scanner and the related protein was quantified using a protein strip analysis software Gel-Pro Analyzer 3.0 (Media Cybernetics, USA).
  • the BS group (6 months old), the HU group mice (7 months old) and the WB weight control group mice (7 months old) after 4 weeks of continuous hanging were sacrificed by cervical dislocation, and the bilateral hind limbs were taken to remove the attachment.
  • the RNA was extracted from the bone tissue by the automatic freezing grinder; the hind limb specimens were washed overnight with water (12h), and the components of the fixative in the specimen were completely removed; the specimen was placed in 12.5% EDTA decalcifying solution, and decalcified at room temperature for four weeks, and replaced every three days.
  • TV total tissue volume; contains both trabecular and cortical bone
  • BV/TV trabecular bone volume per tissue volume
  • Tb.Th trabecular thick-ness
  • Tb.Sp trabecular separation
  • SMI structure model index
  • Conn.Dn connectivity density
  • Second fixation pure alcohol; degreasing, decalcification: decalcification solution is ZnSO4 plus EDTA solution, decalcification device is Hisstra-DC (normal luminosity) continuous operation at 8 ⁇ 16 °C; dehydration, embedding: ETP ( Sakura Finetek Japan) treatment for 16 hours, using a hard wax embedded in a melting point of 58-60 ° C; thin cutting, drying: making 4um slices, drying at 43 ° C for 30 minutes, drying at 37 ° C; dyeing, sealing: using TRAP Dyeing kit (SIGMA, USA) staining, drying at 37 ° C, xylene dewaxing transparent 3 times each 5 min; slice Re- hydrate sections placed in 100% alcohol, 95% alcohol, 70% alcohol each 2min repeated twice Rinse with distilled water; slice the pre-prepared TRAP dye solution for 2 h at 37 ° C, rinse with distilled water; hematoxylin counterstaining for 1 min (re-dyeing time depends on the depth of
  • Agomir-103a modified in the antisense strand, cholesterol modification at the 3' end, two thio skeleton modifications at the 5' end, four thio skeleton modifications at the 3' end, and full chain methoxy modification, the sequence is as follows (where S-thio modification; Chol-cholesterol modification):
  • Antagomir-103a cholesterol modification at the 3' end, two thio skeleton modifications at the 5' end, four thio skeleton modifications at the 3' end, and full chain methoxy modification, the sequence is as follows:
  • the amplification primers were designed according to Runx2 (human) 3'UTR sequence information, and the 3'UTR sequence of Runx2 gene was PCR amplified using 293T cell genomic DNA as template, and cloned into pmiR-RB-REPORT TM double fluorescence.
  • the prime enzyme reporter vector the reported fluorescence of the vector used was hRluc, and the corrected fluorescence was hluc (internal reference correction).
  • the target gene fragment was cloned into a vector using XhoI, NotI two restriction enzymes.
  • the XhoI, NotI restriction site sequence was set on the primer, and the sequence before the restriction enzyme site sequence was the protection base, and the total length of the primer amplification product was 3798 bp. Positive clones were identified by sequencing.
  • the obtained recombinant vector was used as a template, and a seven-base mutation was introduced into the target site using QuikChange site-directed Mutagenesis kit (Stratagene, La Jolla, CA, http://www.stratagene.com) to construct a mutant target. Gene luciferase reporter gene vector. Results The desired sequence was inserted into the plasmid by restriction endonuclease digestion and DNA sequencing. AUGCUGC, which binds complementarily to the 3'UTR seed region of Runx2, was converted to UACGACG, abolishing the specific binding of the 3'UTR seed region of Runx2 to miRNA-103a.
  • HU Hindlimb unloading mouse model
  • NAA National Aeronautics and Space Administration
  • FEA finite element analysis
  • the inventors selected 8% CMS (8% deformation, 80000 ⁇ , Sin, 0.5 Hz, CMS) to be loaded into human osteoblast precursor hFOB 1.19 for subsequent experiments.
  • qRT-PCR showed specific genes related to osteoblast differentiation in the 8% CMS group compared with the static group: Alkaline phosphatase (ALP), osteocalcin (Ocn), and collagen type I alpha 1 (Col1a1). Significantly enhanced ( Figure 2b).
  • ALP staining and ALP quantification were enhanced in the 8% CMS group (Fig. 2c, d).
  • the inventors found that the cell morphology of the CMS group was stretched and the cytoskeleton was stress-centered rearranged as compared with the resting group (Fig.
  • Runx2 is the most important transcription factor in osteogenic differentiation.
  • Runx2 and a variety of proteins, transcription factors, and signaling pathways form a cascade network to regulate osteogenic differentiation.
  • the inventors found that 8% CMS significantly enhanced Runx2 protein expression compared to the resting group, while only slightly increasing its mRNA level (Fig. 2h, i).
  • the inconsistent expression of the above mRNA and protein levels of Runx2 suggests that there may be post-transcriptional regulation of miRNA on Runx2 during osteogenic differentiation induced by mechanical stress stimulation.
  • the inventors applied 8% CMS to load hFOB1.19 human osteoblast cell line for 3 days, and extracted miRNA to detect 12 miRNAs before and after stress loading in hFOB1.19. The level of expression changes. qRT-PCR showed that 7 miRNAs including miR-103a, miR-23b and miR-374b showed a significant decrease in expression levels in stress-stimulated osteoblast differentiation. In contrast, miR-107, miR-143 and miR-154 expression increased (Fig. 3b). To further verify whether the screened miRNAs are directly targeted to Runx2, the inventors constructed a wild type (WT) Runx23' UTR dual luciferase reporter vector.
  • WT wild type
  • the WT Runx23' UTR dual luciferase reporter vector was co-transfected with the screened miRNAs agonist mimics and U6 internal reference in hFOB 1.19 human osteoblasts, respectively. Luciferase results showed that all miRNAs except miR-107, miR-143 and miR-154 inhibited the activity of Runx2WT 3'UTR dual luciferase reporter vector to different extents, especially mimic-103a (Fig. 3c). .
  • the mimics of the selected miRNAs were transfected into hFOB1.19 human osteoblast cell line. After 3% CMS was loaded for 3 days, the Runx2 protein level was detected by Western blot.
  • miR-103a and miR-107 are homeobox miRNAs, which differ only in one nucleotide at the 3' tail.
  • miR-103a/miR-107 was originally found to be up-regulated in obese mice and was found to play an important role in insulin sensitivity.
  • the inventors of the present invention found that miR-107 plays a diametrically opposite role in miR-103a in the Luciferse assay, whereas the mimic-107 transfected with it does not significantly downregulate the protein level of Runx2. Role ( Figure 3c, d).
  • miR-103a directly targets Runx2 in stress-stimulated osteogenesis differentiation
  • the inventors performed functional loss and functional gain-of-function experiments on miR-103a, respectively, to verify whether there is post-transcriptional regulation of Runx2.
  • miM-103a and inhibitor inhibitor-103a which are transfected with miR-103a in hFOB1.19 human osteoblasts, respectively, overexpressed and down-regulated miR-103a, and 8% CMS was loaded for 3 days, qRT-PCR was detected.
  • the expression level of the miR-103a-derived gene showed that its mimic agonist mimic-103a significantly up-regulated miR-103a levels, while its analog inhibitor inhibitor-103a significantly down-regulated miR-103a levels (Fig. 3e).
  • the inventors constructed a mutant (MUT) Runx23'UTR dual luciferase reporter vector that predicts mutations in the binding site region of miR-103a and Runx2 based on bioinformatics. (Figure 3h).
  • the MUT Runx23'UTR dual luciferase reporter vector was co-transfected with the miR-103a mimetic agonist and inhibitor mimic-103a, inhibitor-103a, respectively, in the hFOB 1.19 human osteoblast cell line.
  • Luciferase experiments showed that mimc-103a inhibited and inhibitor-103a enhanced WT Runx23'UTR dual luciferase reporter vector activity, but both had no significant effect on MUT Runx23' UTR dual luciferase reporter vector activity (Fig. 3i).
  • miR-103a directly inhibits the expression of Runx2 at the post-transcriptional level by binding to the 3'UTR seed region of the key transcription factor Runx2.
  • Example 4 miR-103a as a stress-sensitive miRNA in mechanical stress-stimulated osteoblasts Regulation in differentiation
  • the miR-103a precursor pre-miR-103a on the human genome There are two locus loci of the miR-103a precursor pre-miR-103a on the human genome.
  • the stem-loop of human miR-103a-1 is located on human chromosome 5, while the stem-loop structure of miR-103a-2 is located on human chromosome 20.
  • the miR-103a/107 homeobox miRNA is chimeric in an intron encoding the PANK (pantothenate kinase enzyme, PANK) gene.
  • Mature miR-103a-1 and miR-103a-2 were derived from their host genes PANK3 and PANK2 intron-intron 5, respectively (Fig. 4b).
  • the PANK3 and PANK2 genes are important enzymes in the synthesis of coenzyme A and glycolipid metabolism.
  • the present inventors applied 8% CMS to load hFOB 1.19 human osteoblast cell line for 3 days, and detected mRNA levels of PANK3 and PANK2 before and after stress loading.
  • qRT-PCR showed that 8% CMS selectively down-regulated the mRNA expression level of PANK3 but had no significant effect on the mRNA level of PANK2. This change is consistent with the change in miR-103a after forcing (Fig. 4c). The above results suggest that: 1.
  • Mature miR-103a is mainly cleaved by miR-103-1 in its precursor double strand, and miR-103-1 is derived from the Pank3 gene locus.
  • the expression change of miR-103a induced by afterburner is mainly derived from the miR-103-1 precursor of miR-103a chimeric to the Pank3 gene locus; 2.
  • the expression of miRNAs localized to the host gene intron It is usually consistent with its host gene expression.
  • the inventors applied 8% CMS to culture hFOB 1.19 human osteoblasts to 21 days, and tested miR- every three days.
  • the expression level of 103a The present inventors found that the level of miR-103a was significantly down-regulated (0-9 days) in the early stage of osteogenic differentiation, and then gradually decreased to 21 days (Fig. 4d, top), and Runx2 protein level was significantly up-regulated at the initiation of osteogenic differentiation (day 3) and maintain high levels of expression to the 21 day mineralization stage, then significantly decreased ( Figure 4d, bottom).
  • the mRNA levels of Runx2, ALP, Ocn were significantly upregulated during this process (Fig. 4e).
  • This inconsistent expression of Runx2 mRNA and protein levels may be due to the regulation of miR-103a on the post-transcriptional level of Runx2.
  • miR-103a and Runx2 it can be understood that in the early stage of osteogenic differentiation, the level of miR-103a is significantly decreased, and the inhibitory factor that strongly inhibits osteogenic differentiation is suddenly unlocked, so that the differentiation process is initiated; and into the end stage of differentiation, miR The expression level of -103a remained unchanged to allow the process of osteogenic differentiation to be terminated in a timely manner.
  • miR-103a and its host gene PANK3 are sensitive to stress stimuli, miR-103a It may play a role in the initiation of the regulatory process and the terminator in the process of osteogenic differentiation mediated by mechanical stress stimulation. miR-103a acts as a stress-stimulated sensitive miRNA that regulates osteoblast differentiation by targeting the transcription factor Runx2.
  • miR-103a inhibits osteoblast activity and extracellular matrix mineralization during mechanical stress-stimulated osteoblast differentiation
  • the inventors transfected the miR-103a mimic agonist mimic-103a and the inhibitor inhibitor-103a, respectively, in the hFOB 1.19 human osteoblast cell line. After 3 days of 8% CMS loading, mimic-103a significantly reduced mRNA expression levels of ALP and Ocn in osteoblast differentiation compared with the internal reference, and inhibitor-103a enhanced their expression (Fig. 5a). Similarly, overexpression of miR-103a reduced ALP activity and ALP staining in osteoblast differentiation, and inhibition of miR-103a expression enhanced ALP activity and ALP staining (Fig. 5b, c).
  • miR-103a in extracellular matrix mineralization, the inventors used long-acting agonists and inhibitors of miR-103a, agomir-103a and antagomir-103a (miRNA antagomir/agomir, specially chemically modified miRNAs). Long acting antagonists/agonists) and NC internal controls overexpressed and inhibited miR-103a expression in hFOB 1.19 cells, respectively.
  • agomir-103a and antagomir-103a were added once every three days. qRT-PCR confirmed that agomir-103a and antagomir-103a could effectively overexpress and knock out endogenous miR-103a expression levels (Fig. 5d).
  • Blockers U0126 and IWR-1 (U0126, ERK1/2 pathway blocker; IWR-1, Wnt/ ⁇ -catenin pathway blocker) were added to hFOB 1.19 human osteoblast cell line to detect miR-103a expression (Figure 5h). qRT-PCR showed that the pathway blockers U0126 and IWR-1 with ERK1/2MAPK and Wnt/ ⁇ -catenin had no significant effect on the expression level of miR-103a.
  • miR-103a is dependent on Runx2 inhibition in mechanical stress-stimulated osteoblast differentiation and extracellular matrix mineralization. This regulation of miR-103a has no interaction with ERK1/2MAPK and Wnt/ ⁇ -catenin signaling pathways.
  • miR-103a can play an important regulatory role in load-mediated osteogenic differentiation at an in vitro level, and the inventors will next verify whether miR-103a can also regulate bone formation in vivo.
  • Bioinformatics found that miR-103a is highly conserved in multiple species in vertebrates. It is suggested that the expression of miR-103a and its relationship to bone formation can be studied in mice (Fig. 6a).
  • the present inventors first examined the expression abundance of miR-103a in various major organ tissues including bone tissues in C57BL/6J mice. The results showed that the expression abundance of miR-103a in bone tissue was significantly higher than that of other major organs, suggesting that miR-103a plays an important regulatory role in bone tissue reconstruction (Fig. 6b).
  • the inventors examined the expression of miR-103a in the femur of hindlimb unloaded mice and control mice, respectively. qRT-PCR showed that the expression level of miR-103a in the femur of hindlimb unloaded mice was significantly higher than that of the BS basic group and the WB control group (Fig. 6c).
  • the present inventors separately set HU group mice (HU+Antagomir-103a, Antagomir-103a, dosage 80 mg/kg).
  • mice in the PBS group (HU + PBS, PBS, 0.3 ml) were continuously injected 3 times through the tail vein before hanging (Fig. 6d).
  • HU group mice (HU+Antagomir-103a mice) received another injection three weeks after the first injection. All mice were sacrificed on hind limb suspension for 28 days.
  • the present inventors found that compared with the HU group and the HU+PBS group, the expression of miR-103a in the femur of the HU+Antagomir-103a group was lower and the Runx2 protein expression was higher, while in the HU group and the HU+PBS group. There was no significant difference in the expression of miR-103a and Runx2 (Fig. 6e, f).
  • antagomir-103a can effectively down-regulate the expression abundance of miR-103a in bone tissue; 2. antagomir-103a partially reversed the down-regulation of Runx2 expression in bone tissue caused by hindlimb unloading. More importantly, microCT results showed that bone loss caused by unloading was partially rescued by antagomir-103a (Fig. 6g, h). Bone histomorphology correlation analysis showed that bone formation related parameters (Ob.S/BS, MAR, N.Ob/B.Pm) in HU+Antagomir-103a mice compared with HU and HU+PBS mice. Significantly elevated (Fig.
  • hFOB1.19 cells were obtained, which expressed endogenously expressed miR-103a. This kind of cell is used as a cell model for screening drugs for preventing and treating bone metabolic diseases.
  • Test group a culture of the above cells treated with a candidate substance
  • Control group A culture of the above cells treated without a candidate substance.
  • the expression of miR-103 of the cells was determined at appropriate times after treatment. If the expression of miR-103 in the test group was significantly decreased by more than 30% compared with the control group, it indicates that the candidate substance is a substance for preventing and treating bone metabolic diseases.
  • antagomir-103a significantly reduced the expression of miR-103 in the test group by more than 90%. Therefore, antagomir-103a is a useful drug candidate.
  • Mechanical stress stimulation is critical to the role of bone remodeling.
  • the invention is prompted from the clinical phenomenon, starting from the steady state of bone reconstruction, exploring the effects of mechanical stress stimulation on osteoblast differentiation and the regulation and mechanism of miRNA in mechanical stress-mediated osteogenic differentiation.
  • the invention explores and verifies load-sensitive miRNAs in osteogenic differentiation through bioinformatics methods, and explores its expression under mechanical stress regulation and its regulation and mechanism in osteogenic differentiation and bone formation mediated by mechanical stress stimulation.
  • miR-103a acts as a new undiscovered specific force-sensitive miRNA under physiological and pathological load conditions. Regulates osteogenic differentiation and bone formation.
  • miR-103a inhibits osteoblast differentiation and bone formation by inhibiting its expression at the post-transcriptional level of the key transcription factor Runx2 in osteogenic differentiation. Inhibition.
  • the in vivo results of the present inventors suggest that the regulation of miR-103a levels in vivo by therapeutic pre-dosing can partially rescue bone loss caused by stress loss, Osteoporosis type.
  • miR-103a as a new force-sensitive miRNA, provides for the first time in osteoblast precursors the regulation and mechanism of miRNAs in stress-load-mediated osteogenic differentiation of bone formation. According to. It is also suggested that miR-103a can be used as a potential drug target in clinical treatment of disuse osteoporosis caused by clinical stress loss.
  • Mechanical stress was originally reported to play an important regulatory role in a variety of physiological and pathological processes. Among them, mechanical stress is the most widely studied in the cardiovascular system, skeletal muscle system and lung physiology. However, the role of mechanical stress in the differentiation of osteoblasts in vivo remains to be determined.
  • the invention discloses in vivo and in vitro that the osteoblasts will rapidly adapt to the load when subjected to external load stimulation, specifically in the cell function and morphological structure. The present inventors have found that mechanical stress loading can significantly promote osteoblast differentiation and bone formation.
  • Runx2 When osteoblast differentiation is initiated by mechanical stress stimulation, it will lead to a cascade of transcription factors, hormones, and growth factor responses that promote osteoblast differentiation and bone formation.
  • Runx2 binds to the osteoblast-specific cis-element 2 (OSE2) of all major osteogenic-related gene promoter regions.
  • OSE2 osteoblast-specific cis-element 2
  • Runx2 knockout mice die during the embryonic period, have no osteoblasts in the body, and therefore have no bone tissue, only cartilage.
  • Runx2 half-knocked (Runx2-/+) heterozygous mice exhibited specific skeletal developmental disorders similar to human hereditary skeletal dysplasia.
  • mice with heterozygous deletion of Runx2 showed similar traits to a typical mutant mouse: Cleidocranial Dysplasia (CCD) mice. Any heterozygous mutation in the DNA binding domain of Runx2, including deletion mutations and base substitution mutations, will exhibit CCD traits.
  • CCD Cleidocranial Dysplasia
  • the inventors have found that mechanical stress stimulation can significantly upregulate Runx2 protein levels, while their mRNA levels increase only slightly. This prompted the inventors to further explore whether there is regulation at the post-transcriptional level, such as the regulation of miRNA.
  • miRNAs Since its discovery, miRNAs have received increasing attention due to their important regulatory roles in a variety of physiological and pathological processes. Several studies have demonstrated that in a variety of cell lines in vitro, mechanical stress, including shear and cyclic tension, regulates the expression of a range of miRNAs. These miRNAs are also involved in the response of cells to mechanical stress stimuli. In recent years, a number of miRNAs have been identified as important regulatory factors in bone remodeling, by regulating the expression of genes involved in bone remodeling at post-transcriptional levels in many clinical bone metabolic diseases such as osteoporosis. Important role. However, the expression of related miRNAs in the stress-mediated osteoblast differentiation and the regulation of their target genes in this process are not fully known. The present invention relates for the first time to the expression of miRNAs in association with mechanical stress stimulation and bone remodeling homeostasis.
  • miR-103a (a homologous gene with miR-107) was recruited to inhibit the highly conserved sequence pairing of Runx23'UTR under 8% CMS stress loading to inhibit osteogenic differentiation.
  • miR-103a/miR-107 has been included in the human genome by miRBase and is highly conserved among polyvertebrate/mammalian species. They were initially found to be elevated in obese mice and thus found to play a key regulatory role in insulin sensitivity, suggesting that they may be potential targets for the treatment of type 2 diabetes. In addition, it has also been reported that miR-103a is associated with chronic pain.
  • miR-103a is highly expressed in many tumor cells, suggesting that miR-103a may play an important role in the development of tumorigenesis.
  • some studies have confirmed that miR-103a may promote lipid metabolism and regulate the homeostasis of glycolipid metabolism.
  • no studies have confirmed the role of miR-103a in regulating osteoblast differentiation.
  • ERK1/2MAPK and Wnt/ ⁇ -catenin signaling pathways have been widely demonstrated.
  • the inactivation of ⁇ -catenin blocks the differentiation of mesenchymal stem cells into osteoblasts, suggesting that ⁇ -catenin plays a key regulatory role in osteoblast differentiation in vivo.
  • the inventors have found that mechanical stress loading can significantly activate the ERK1/2 and ⁇ -catenin signaling pathways and further promote the expression of many downstream genes including Runx2.
  • the present invention did not find an interaction between miR-103a and ERK1/2MAPK and Wnt/ ⁇ -catenin signaling pathway, suggesting that the expression of miR-103a may not be in the two in osteoblast differentiation mediated by mechanical stress loading. Under the control of the passage, it exists independently.
  • Moderate strength mechanical stress stimulation can also be used as a treatment for certain bone metabolic diseases.
  • Dynamic stress loading on osteoblasts activates/activates the Wnt- ⁇ -catenin pathway and promotes osteoblast differentiation/generation. Osteoblast differentiation and development into osteoblasts promote fracture healing by loading moderately stressed mechanical stress loads. Osteoblasts and bone cells respond to stress stimuli of varying strength. Strong internal fixation after fracture completes the strain of the repaired tissue at the fracture site under physiological load, and the healing of the fracture does not result in direct healing of the epiphysis.
  • Elastic internal fixation includes indirect healing of intramembranous osteogenesis and endochondral ossification, which is characterized by osteophyte formation.
  • the results of the present invention reveal in vivo a novel mechanism by which miR-103a regulates bone formation in vivo.
  • miR-103a is highly abundantly expressed in bone tissue, suggesting that it may play an important regulatory role in bone remodeling.
  • the abnormal increase in miR-103a caused by abnormal pathological stress can be reversed by therapeutically administering the long-acting inhibitor antagomir-103a of miR-103a. Partially rescued the bone loss phenotype due to stress loss.
  • miR-103a acts as a novel stress-load-sensitive miRNA to regulate osteogenic differentiation, and miR-103a achieves its regulation in osteogenic differentiation by directly targeting Runx2 at the post-transcriptional level.
  • miR-103a can be used as a new potential drug target in vitro and in vivo to regulate bone formation by regulating its expression under physiological and pathological stress conditions.

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Abstract

提供了在骨形成中发挥调控作用的应力敏感性miR-103a，其在成骨细胞分化中起重要作用，并可作为防治骨代谢疾病（包括骨质疏松、成骨分化异常、骨量丢失等）的靶标。miR-103a的下调剂能够改善由应力缺失造成的骨质疏松表型。

Description

在骨形成中发挥调控作用的应力敏感性microRNA 技术领域
本发明属于生物技术领域，更具体地，本发明涉及在骨形成中发挥调控作用的应力敏感性microRNA。
背景技术
机械应力刺激通过力学信号传导途径对于骨重建稳态的调控作用至关重要。作为一种动力学器官，骨组织的结构和功能在很大程度上依赖于所处的力学环境。通过骨细胞，成骨细胞等力学信号感受器感受机械应力刺激并将之转导为生物学信号传递至骨表面主要的效应细胞(成骨细胞和破骨细胞)完成相关应答。成骨细胞的分化可由机械应力刺激介导并引起系列激素，生长因子，转录因子等的级联反应，进而影响细胞增殖分化。临床上，载荷缺失如长期卧床患者，长期处于重力缺失环境如宇航员可导致骨量丢失进而迅速进展为废用性骨质疏松症。而长期超载荷状态如体育运动员将导致骨小梁微骨折进而进展为应力性骨折或疲劳性骨折。
microRNAs(miRNAs)是一类内生单链，长度约为22个核苷酸的小分子非编码RNA，在许多生物进程中起重要调控作用。通过转录后调节机制，miRNAs通过与靶基因mRNA的3’UTR种子区域结合降解或抑制靶基因翻译进而抑制靶基因表达。miRNA调节着人类约三分之一的蛋白编码基因，提示其在调控基因表达中的关键作用。多条miRNAs已在体外水平被证实可通过靶标于成骨分化相关特异性基因的表达进而调控成骨分化进程。
然而，关于机械应力载荷与miRNA的相互关系以及在成骨分化中的调控作用还有待研究，有待找到适用于调控成骨分化的miRNA分子。
发明内容
本发明的目的在于提供在骨形成中发挥调控作用的应力敏感性microRNA。
在本发明的第一方面，提供一种miR-103a的下调剂在制备预防或治疗骨代谢疾病(为应力相关性骨代谢疾病，如宇航员长期太空微重力环境或长期卧床患者所导致的载荷缺失导致的废用性骨质疏松症)的药物中的用途。
在一个优选例中，所述的miR-103a抑制剂包括：化学合成的miR-103a抑制 剂；以表达质粒为载体的抑制miR-103a的病毒和非病毒产品；与miR-103a互补的核酸序列或序列片段。
在另一优选例中，所述的miR-103a的下调剂选自：antagomir-103a，其核苷酸序列如SEQ ID NO:49所示；或inhibitor-103a，其核苷酸序列如SEQ ID NO:47所示。
在另一优选例中，所述的miR-103a的下调剂是经修饰的下调剂，所述的修饰包括(但不限于)：甲氧基化修饰、硫代修饰、胆固醇修饰、烷基修饰(如在核糖的2’位置进行烷基修饰)、锁核酸修饰、肽核酸修饰、和/或磷酸骨架由磷脂连接代替的反义核苷酸；较佳地，所述的antagomir-103a的修饰包括：3’端进行胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，全链甲氧基修饰。
在另一优选例中，所述的药物还用于：
增加Runx2蛋白的表达；
增强成骨细胞分化中ALP和Ocn的表达水平；或
增强胞外基质矿化。
在本发明的另一方面，提供一种miR-103a的用途，用于筛选预防或治疗骨代谢疾病的药物。
在一个优选例中，所述的骨代谢疾病包括：骨质疏松、成骨分化异常、骨量丢失。
在本发明的另一方面，提供一种预防或治疗骨代谢疾病的药物，所述的药物是miR-103a的下调剂，选自：antagomir-103a，其核苷酸序列如SEQ ID NO:49所示；或inhibitor-103a，其核苷酸序列如SEQ ID NO:47所示。
在本发明的另一方面，提供一种筛选预防或治疗骨代谢疾病的潜在物质的方法，所述方法包括：
(1)用候选物质处理表达miR-103a的体系；和
(2)检测所述体系中miR-103a的表达；
其中，若所述候选物质可降低miR-103a的表达，则表明该候选物质是预防或治疗骨代谢疾病的潜在物质。
在一个优选例中，所述的体系中还表达Runx2蛋白，所述方法还包括：检测所述体系中Runx2蛋白的表达；
其中，若所述候选物质通过下调miR-103a的表达而增加(优选显著增加，如增加20％以上，较佳的增加50％以上；更佳的增加80％以上)Runx2蛋白的表达， 则表明该候选物质是预防或治疗骨代谢疾病的潜在物质。
在另一优选例中，步骤(1)包括：在测试组中，将候选物质加入到表达miR-103a或共表达miR-103a和Runx2蛋白的体系中；和/或
步骤(2)包括：检测测试组的体系中miR-103a和/或Runx2蛋白的表达，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达miR-103a和/或Runx2蛋白的体系；
如果测试组中miR-103a的表达在统计学上低于(优选显著低于，如低20％以上，较佳的低50％以上；更佳的低80％以上)对照组，或还使得Runx2蛋白的表达显著增加，就表明该候选物是预防或治疗骨代谢疾病的潜在物质。
在另一优选例中，所述的体系选自：细胞体系(或细胞培养物体系)、亚细胞体系、溶液体系、组织体系、器官体系或动物体系。
在另一优选例中，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以从候选物质中进一步选择和确定对于预防或治疗骨代谢疾病有用的物质。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、应力载荷促进体内骨重建、骨形成。
(a)实验小鼠模型设计BS，baseline基准组。WB，weightbearing承重组。HU，hind-limb unloading后肢去负荷组；
(b-h)BS，WB，HU组小鼠远端股骨骨重建相关参数指标分析。(b)WB组和HU组小鼠股骨大体形态改变(c)BS，WB，HU组小鼠股骨远端microCT骨结构。标尺，1mm。(d)BS，WB，HU组小鼠股骨远端microCT三维骨骨密度等指标。(e)各组小鼠中钙黄绿素双标实验反映新骨形成情况。标尺，10μm。(f)BS，WB，HU组小鼠骨组织形态学分析：骨形成相关参数指标(Ob.S/BS，MAR和N.Ob/B.Pm)检测。(g)BS，WB，HU组小鼠远端股骨TRAP染色(Tartrate-resistant acid phosphatase，TRAP，抗酒石酸酸性磷酸酶染色染色)。标尺，100μm。(h)BS，WB，HU组小鼠组织形态学分析：骨吸收相关参数指标(Oc.S/B.S，N.Oc/B.Pm)分析。
*P<0.05，**P<0.01，***P<0.001。NS，无显著性差异。P值计算基于 Student’s t test。数据以平均值±标准差表示，代表4次独立实验。
图2、CMS载荷在体外水平调控成骨细胞分化
(a)人股骨近端有限元分析模型(A three-dimensional finite element，FE，含29841有限元元素)。FE分析显示人股骨近端骨组织承载的应力载荷约为815±57με(375με～1,583με)。(b)8％CMS载荷加载hFOB1.19人成骨细胞系3天后，qRT-PCR检测Ocn，ALP，Col1a1表达水平，以静置组为参照。(c)8％CMS载荷加载hFOB1.19人成骨细胞系3天后ALP定量检测，以静置组为参照。(d)8％CMS载荷加载hFOB1.19人成骨细胞系3天后，ALP染色检测，以静置组为参照。标尺，10mm。(e)8％CMS载荷加载hFOB1.19人成骨细胞系3天后，细胞骨架免疫荧光染色显示较之静置组细胞骨架微丝发生向心性重排列。(f)8％CMS载荷加载hFOB1.19人成骨细胞系3天，细胞增殖无显著性差异，以静置组为参照。(g)8％CMS载荷加载hFOB1.19人成骨细胞系3天后，Western blot检测Wnt/β-catenin和Erk1/2MAPK信号通路活化程度。(h)8％CMS载荷加载hFOB1.19人成骨细胞系3天后，Western blot检测Runx2蛋白表达水平变化，以静置组为参照。(i)8％CMS载荷加载hFOB1.19人成骨细胞系3天后，qRT-PCR检测Runx2的mRNA表达水平变化，以静置组为参照。
*P<0.05，**P<0.01，***P<0.001。NS，无显著性差异。P值计算基于Student’s t test；数据以平均值±标准差表示，代表3次独立实验。
图3、miR-103a在体外水平通过靶标于Runx2抑制成骨细胞功能。
(a)Target Scan，miRDB，miRanda和miRWalk miRNA靶基因预测筛选软件生物信息学分析筛选Runx2标靶。(b)8％CMS加载hFOB1.19人成骨细胞系3天后，qRT-PCR检测预筛选miRNAs的表达水平变化。miRNAs的表达变化比率比例倍数以静置对照组为参照。(c)hFOB1.19人成骨细胞系中，12条筛选miRNAs及U6内参对WT Runx23’UTR双荧光素酶报告载体活性的影响。(d)在hFOB1.19人成骨细胞系中，Western blot检测12条筛选miRNAs及U6内参对Runx2蛋白表达水平的影响。(e)在hFOB1.19人成骨细胞系中分别转染mimic-103a，inhibitor-103a及其对应NC参照(mimic-NC，inhibitor-NC)后，qRT-PCR检测miR-103a的表达水平变化。(f)在hFOB1.19人成骨细胞系中分别转染mimic-103a，inhibitor-103a及其对应NC参照(mimic-NC，inhibitor-NC)后，Western  blot检测Runx2蛋白水平表达变化。(g)在hFOB1.19人成骨细胞系中分别转染mimic-103a，inhibitor-103a及其对应NC参照(mimic-NC，inhibitor-NC)后，qRT-PCR检测Runx2的mRNA水平表达变化。(h)WT Runx23’UTR双荧光素酶报告载体及Mutant Runx23’UTR双荧光素酶报告载体示意简图。hRluc，human Renilla luciferase人海肾萤光素酶。(i)在hFOB1.19中分别转染mimic-103a，inhibitor-103a及其对应NC参照后，检测WT Runx23’UTR，MUT Runx23’UTR双荧光素酶报告基因活性。
*P<0.05，**P<0.01，***P<0.001。NS，无显著性差异。P值计算基于Student’s t test；Real-time PCR以GAPDH为内参。*P<0.05；**P<0.01；数据以平均值±标准差表示，代表3次独立实验。
图4、miR-103a在体外CMS介导的成骨细胞分化过程中对成骨分化起负调控作用
(a)qRT-PCR显示8％CMS介导的hFOB1.19人成骨细胞系分化中miR-103a的表达水平(8％elongation，Sin，0.5Hz，3d)。miR-103a的表达以静置组对照为参照。(b)miR-103a和miR-107在基因组中定位示意简图。PANK基因的外显子以长方形表示，内含子以曲线标识。(c)8％CMS加载hFOB1.19人成骨细胞系3天后，qRT-PCR检测PANK2和PANK3表达水平。(d)8％CMS加载hFOB1.19人成骨细胞系21天，qRT-PCR检测成骨细胞分化成熟胞外基质矿化过程中miR-103a的表达水平(上)；Western blot检测Runx2蛋白表达水平(下)。(e)8％CMS加载hFOB1.19人成骨细胞系21天，qRT-PCR检测成骨细胞分化成熟胞外基质矿化过程中Ocn，ALP，Runx2mRNA表达水平。
*P<0.05，**P<0.01，***P<0.001。NS，无显著性差异P值计算基于Student’s t test。Real-time PCR以GAPDH为内参。*P<0.05；**P<0.01；所有数据以平均值±标准差表示，代表3次独立实验。
图5、miR-103a在体外应力刺激介导的成骨细胞分化过程中抑制成骨细胞活性及胞外基质矿化
(a)分别转染miR-103a的模拟物mimic-103a，inhibitor-103a及NC参照后，8％CMS加载hFOB1.19人成骨细胞系3天，qRT-PCR分别检测Ocn，ALP，Runx2mRNA水平。(b)分别转染miR-103a的模拟物mimic-103a，inhibitor-103a及NC 参照后，8％CMS加载hFOB1.19人成骨细胞系3天，检测ALP活性。(c)分别转染miR-103a的模拟物mimic-103a，inhibitor-103a及NC参照后，8％CMS加载hFOB1.19人成骨细胞系3天，检测ALP染色。标尺，10mm。(d)分别转染miR-103a的长效模拟物agomir-103a，antagomir-103a及NC参照后，8％CMS加载hFOB1.19人成骨细胞系21天，qRT-PCR检测miR-103a表达水平。(e)分别转染miR-103a的长效模拟物agomir-103a，antagomir-103a及NC参照后，8％CMS加载hFOB1.19人成骨细胞系21天，检测茜素红染色。标尺，10mm。(f)Runx2特异性siRNA(siRNA-Runx2)的敲除效率检测，以siRNA-NC为参照。(g)共转染siRNA-Runx2，mimic-103a，inhibitor-103a以及NC参照后，8％CMS加载hFOB1.19人成骨细胞系3天，qRT-PCR检测Ocn，ALP mRNA水平以及ALP定量。(h)分别应用Wnt/β-catenin信号通路抑制剂IWR-1和Erk1/2MAPK信号通路抑制剂U0126阻断两条通路后，8％CMS加载hFOB1.19人成骨细胞系3天，qRT-PCR检测miR-103a表达水平。
*P<0.05，**P<0.01，***P<0.001。NS，无显著性差异。P值计算基于Student’s t test。Real-time PCR以GAPDH为内参。*P<0.05；**P<0.01；所有数据以平均值±标准差表示，代表3次独立实验。
图6、通过体内干预性给予miR-103a长效抑制剂antagomir-103a部分挽救了由于应力缺失导致的骨量下降骨质疏松表型
(a)Hsa-miR-103a前体二级结构示意简图，以及成熟miR-103a的序列在哺乳/脊椎动物中序列比较示意简图。(b)qRT-PCR分析在C57BL/6J小鼠骨及其他各主要器官内miR-103a表达丰度。(c)qRT-PCR分析WB和HU小鼠中股骨内miR-103a表达水平(以BS组小鼠为校正)。(d)实验设计示意简图(每组实验C57/BL6J小鼠，n＝6)。HU+PBS，尾静脉注射PBS的HU小鼠；HU+Antagomir-103a，尾静脉注射antagomir-103a的HU小鼠。(e)qRT-PCR分析在HU，HU+PBS，HU+Antagomir-103a组小鼠股骨内miR-103a表达水平(以WB组小鼠为校正)。(f)Western blot分析显示WB，HU，HU+PBS，HU+Antagomir-103a小鼠股骨内Runx2蛋白水平。(g)WB，HU，HU+PBS，HU+Antagomir-103a小鼠远端股骨microCT三维重建。标尺，1mm。(h)WB，HU，HU+PBS，HU+Antagomir-103a小鼠远端股骨microCT三维重建骨形成相关参数。(i)新骨形成率检测：WB，HU，HU+PBS，HU+Antagomir-103a小鼠钙黄绿素双标检测。标尺，10μm。(j)骨组织形态学分 析：WB，HU，HU+PBS，HU+Antagomir-103a小鼠中骨形成相关参数(Ob.S/BS，MAR and N.Ob/B.Pm)检测(k)WB，HU，HU+PBS，HU+Antagomir-103a小鼠远端股骨TRAP染色。标尺，100μm(l)骨组织形态学分析：WB，HU，HU+PBS，HU+Antagomir-103a小鼠中骨吸收相关参数(Oc.S/B.S，N.Oc/B.Pm)检测
*P<0.05，**P<0.01，***P<0.001。NS，无显著性差异。P值计算基于Student’s t test。Real-time PCR以GAPDH为内参。*P<0.05；**P<0.01；所有数据以平均值±标准差表示，代表3次独立实验。
图7、pmiR-RB-REPORTTM双荧光素酶报告载体图谱。
具体实施方式
本发明人经过深入的研究，意外地发现，miR-103a是一个新的力学敏感miRNA并在成骨细胞分化中起重要作用，其可以作为防治骨代谢疾病(包括骨质疏松、成骨分化异常、骨量丢失等)的靶标。miR-103a的下调剂能够改善由应力缺失造成的骨质疏松表型。
miR-103a及其用途
本发明中，所述的miR-103a是具有SEQ ID NO:1所示核酸序列的小核糖核酸(hsa-miR-103a-3p，MIMAT0000101)：
Figure PCTCN2015088697-appb-000001
miR-103a是一种已发现可能能够促进脂代谢进而调控糖脂代谢的稳态的小核糖核酸，其在骨代谢方面的作用在现有技术中尚无报导。
本发明人通过在体内外分别构建载荷下应力加载细胞模型和双后肢去负荷小鼠模型，验证机械应力载荷在体内和体外对于成骨分化及骨重建作用。通过生物信息学方法筛选标靶miRNA并用qRT-PCR进行后续功能验证，筛选并鉴定获得miR-103a是一个新的力学敏感miRNA并在成骨细胞分化中起重要作用。miR-103a及其宿主基因PANK3在机械应力刺激介导的成骨细胞分化中均明显下调(8％CMS，0.5Hz，Sin)，而Runx2蛋白水平上调。过表达miR-103a显著降低Runx2蛋白水平，抑制miR-103a下调Runx2蛋白水平，提示miR-103a在成骨细胞中抑制Runx2表达。将miR-103a与Runx2的3’UTR结合位点突变后废除了miR-103a对Runx23’UTR双荧光素酶报告基因载体的活性抑制作用，提示miR-103a通过 与Runx2的3’UTR种子区域结合抑制其表达。成骨分化相关marker基因检测及成骨分化表型显示miR-103a在机械应力刺激介导的成骨细胞分化中起负调控作用。在成骨分化及胞外基质矿化进程中，miR-103a起抑制作用。在后肢去负荷小鼠中miR-103a表达明显上调，其可能通过抑制Runx2表达在体内水平对骨形成起负调控作用，通过尾静脉给予其长效抑制剂模拟物可部分挽救由应力缺失造成的骨质疏松表型。
因此，miR-103a是一个新的可以作为防治骨代谢疾病(包括骨质疏松、成骨分化异常、骨量丢失等)的标志物。
miR-103a下调剂及其用途
基于本发明人的上述新发现，本发明提供了一种miR-103a下调剂的用途，用于制备预防或治疗骨代谢疾病的组合物(如药物)。所述的miR-103a下调剂藉由抑制miR-103a的表达，从而实现对骨代谢异常(如骨质疏松)的防治作用。miR-103a下调剂还用于促进Runx2蛋白的表达；增强成骨细胞分化中ALP和Ocn的表达水平；或增强胞外基质矿化。
如本文所用，所述的“miR-103a下调剂”包括了拮抗剂、抑制剂、阻滞剂、阻断剂等，只要它们能够下调miR-103a的表达水平。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是抑制miR-103a表达的病毒产品。
所述的miR-103a下调剂是指任何可降低miR-103a的活性、降低miR-103a的稳定性、下调miR-103a的表达、减少miR-103a有效作用时间的物质，这些物质均可用于本发明，作为对于下调miR-103a有用的物质，从而可用于改善骨代谢疾病的生长。例如，所述的下调剂是：核酸抑制物，蛋白抑制剂，核酸酶，核酸结合分子，只要其能够下调miR-103a的表达。
作为本发明的一种优选方式，所述的下调剂选自：化学合成的miRNA下调剂；以表达质粒为载体的抑制miRNA的病毒和非病毒产品；与miR-103a互补的核酸序列或序列片段。
作为本发明的更优选的方式，所述的miR-103a下调剂是经过特殊修饰的miRNA拮抗剂，例如包括但不限于：甲氧基化修饰、烷基修饰(如在核糖的2’位置进行烷基修饰)、锁核酸修饰、肽核酸修饰、硫代修饰以及磷酸骨架由磷脂连接代替的反义核苷酸。
作为本发明的更优选的方式，所述的miR-103a下调剂是antagomir-103a或inhibitor-103a。其修饰包括：3’端进行胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，全链甲氧基修饰，从而增加了其稳定性，促进了其有效性。antagomir-103a通过与体内的成熟miRNA强竞争性结合，阻止miRNA与其靶基因mRNA的互补配对，抑制miRNA发挥作用。与普通抑制剂相比，miRNA antagomir在动物体内外具有更高的稳定性和抑制效果，且能克服体内细胞膜、组织等障碍富集于靶细胞。antagomir在细胞实验中不需要转染试剂，从而避免了转染试剂包装过程的复杂步骤及其对实验的影响。在动物实验中可用全身或局部注射、吸入、喂药等方法进行给药，作用效果持续时间可长达6周。
本发明还提供了一种组合物(如药物)，它含有有效量(如0.000001-50wt％；较佳的0.00001-20wt％；更佳的，0.0001-10wt％)的所述的miR-103a下调剂，以及药学上可接受的载体。所述的组合物可用于调节骨代谢。任何前述的miR-103a的下调剂剂均可用于组合物的制备。
如本文所用，所述“有效量”是指可对人和/或动物产生功能的且可被人和/或动物所接受的量。所述“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。
在得知了所述miR-103a下调剂的用途后，可以采用本领域熟知的多种方法来将所述的调节剂或其药物组合物给药于哺乳动物。包括但不限于：皮下注射、肌肉注射、经皮给予、局部给予、植入、缓释给予等；优选的，所述给药方式是非肠道给予的。所述的下调剂的给药方式也可以是局部部位的注射给药，例如可采取关节内给药的方式。
本发明所述的miR-103a的下调剂的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的miR-103a的下调剂的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状 况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
药物筛选
在得知了miR-103a与骨代谢疾病(包括骨质疏松、成骨分化异常或骨量丢失)的密切相关性后，可以基于该特征来筛选抑制miR-103a的表达的物质。可从所述的物质中找到对于预防或治疗骨代谢疾病真正有用的药物。
因此，本发明提供一种筛选抑制骨代谢疾病的潜在物质的方法，所述的方法包括：用候选物质处理表达miR-103a的体系；和检测所述体系中miR-103a的表达；若所述候选物质可抑制miR-103a的表达，则表明该候选物质是抑制骨代谢疾病的潜在物质。所述的表达miR-103a的体系例如可以是细胞(或细胞培养物)体系，所述的细胞可以是内源性表达miR-103a的细胞；或可以是重组表达miR-103a的细胞。所述的表达miR-103a的体系还可以是亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选非人哺乳动物的动物模型，如鼠、兔、羊、猴等)等。
在本发明的优选方式中，在进行筛选时，为了更易于观察到miR-103a的表达的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的表达miR-103a的体系。
在本发明的优选方式中，所述的体系中还表达Runx2蛋白，所述方法还包括：检测所述体系中Runx2蛋白的表达；其中，若所述候选物质可通过下调miR-103a的表达而增加Runx2蛋白的表达，则表明该候选物质是防治骨代谢疾病的潜在物质。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于抑制骨代谢疾病真正有用的物质。
本发明对于miR-103a或Runx2蛋白的表达、活性、存在量或分泌情况的检测方法没有特别的限制。可以采用常规的蛋白定量或半定量检测技术，例如(但不限于)：SDS-PAGE法，Western-Blot法等。
另一方面，本发明还提供了采用所述筛选方法获得的抑制骨代谢疾病的潜在物质。这些初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出能够对于抑制miR-103a的表达和活性，进而抑制骨代谢疾病有用的物质。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料和方法
1.实验动物模型构建
6月龄C57BL6/J小鼠(上海史莱克实验动物公司)分别分为3组：基础(BS)组，吊尾(HU)组，静置对照(WB)组。HU组小鼠用医用宽胶带(15cm×0.5cm)自鼠尾前1/3起向后螺旋形缠绕，胶带固定于笼顶，使小鼠双后肢与笼底呈30°悬空，但不影响其前肢活动，使其可自由饮水进食，持续4周，每日观察鼠尾血供情况，防止缺血坏死。每周称重两次观察其生长发育情况。WB组小鼠为空白对照，无特殊处理。
2.hFOB1.19人成骨细胞系培养及不同强度下机械应力载荷诱导成骨细胞分化模型建立
hFOB1.19培养于10mm培养皿含10％FBS，1％双抗及0.3mg/mL G418的DMEM完全培养基中，于34℃、5％CO2浓度、95％湿度培养箱中孵育培养，取P3代细胞以2.0×105密度接种于普通六孔培养板及被覆I型胶原的BioFlexTM六孔培养板，每2天换液一次；细胞长至90％融合后，将BioFlexTM六孔培养板置于FX-5000TM FLEXCELL TENSION PLUS应力加载装置内培养，设置培养箱温度为34℃、5％CO2浓度、95％湿度，并计时0天，每2天换液一次，分别加载3天、7天；以静置组为对照；应力加载程序设定为：8％CMS(8％形变，80000με，Sin，0.5Hz，CMS)，其中，CMS：cyclic mechanical stretch。
3.细胞总RNA的提取
向细胞中加入适量的TRIZOL(Invitrogen，USA)，至澄清为宜，冻于-80℃或直接处理；每1ml的TRIZOL试剂加入0.2ml的氯仿，剧烈震荡试管15秒，静置EP管2-3min；12000rpm，4℃离心15min。转移上层水相溶液到新EP管中，每1ml的TRIZOL试剂加入0.5ml的异丙醇，混匀；冰上孵育样品30min。12000rpm，4℃，10min。RNA呈白色胶状贴在管底一侧。弃上清液，加入1ml 75％乙 醇，上下轻颠倒2-3次；7500rpm，4℃离心5min；RNA样品干燥5-10min；用DEPC水(20-50μl)溶解样品后，紫外分光光度仪A260/280测出抽提RNA的浓度，-80℃保存，或直接进行qRT-PCR。
4.ALP碱性磷酸酶染色
吸去培养液，PBS洗3次；4％多聚甲醛室温固定10分钟，PBS冲洗，空气晾干；碱性磷酸酶染色试剂盒(碧云天)工作液配制；按操作手册制成染色混合液；每孔加入1ml染色混合液(6孔板)，37℃孵育1h；流水冲洗，晾干；摄片，酶活动处呈亮红颗粒。
5.碱性磷酸酶活性测试(ALP定量)
细胞样品收集，冻存(-20℃)；使用时融解后离心，将细胞团沉入管底，4℃，2000rpm，2min，弃上清；蒸馏水100-500μl(视细胞量加入)混匀，必要时使用振荡仪振荡；反复冻融：冻10min×3次、-80℃，每次冻后室温融化，反复冻融使细胞裂解，蛋白析出；混匀离心，4℃，1000rpm，3min，取上清。以A液每管200μl，B液每管4μl取液。计算总量混匀，取200μl×2(复孔)加入96孔板，另有一个空白(blank)孔加蒸馏水；每孔中加入20μl细胞上清，37℃30min孵育，有蛋白处呈紫色；连续波长扫描酶标仪测定BCA，两组取均值。DEA：pnpp＝2：1混合，加一管blank调背景，每管加入150μl混合液及50μl细胞上清，blank管中加蒸馏水，混匀；37℃水浴15min(黄色为阳性)；每管中加入1N NaOH 400μl停止反应，摇匀，离心甩下盖中液体；每管中取200μl×2(复孔)加入96孔板；连续波长扫描酶标仪测定，两组取均值。Pnpp/BCA取得相对定量值。
6.机械应力刺激对hFOB1.19细胞形态及细胞骨架排列的影响检测
8％CMS应力加载3天后，莱卡倒置显微镜观察细胞排列方向，形态变化，与静置组对照；BioFlexTM六孔培养板8％CMS应力加载3天后，PBS洗三次；以4％多聚甲醛(PBS配制)室温固定细胞1h，PBS洗3次；细胞以新鲜透膜液(0.1％Triton X-100，0.1％柠檬酸钠)冰上通透5min，PBS洗3次；以3％BSA封闭细胞2h；以鬼笔环肽孵育；PBS清洗3次；以Horchest染核；PBS清洗3次；封片剂封片；激光共聚焦显微镜下观察细胞骨架排列情况，静置组为对照。
7.茜素红钙结节染色
40mM茜素红染色液(Alizin Red)：1.3692g茜素红粉末(Sigma)溶于100ml PBS中，调节PH值到4.1-4.3，放于室温备用。吸去培养液，PBS冲洗两遍；4％多聚甲醛室温固定15min；ddH2O冲洗两遍；加入1ml/孔的40mM的茜素红染色液，室温孵育20min并轻微振荡；吸取未结合的染料，用ddH2O漂洗并振荡5min重复4次；倾斜放置2min，吸取多余的ddH2O；倒置显微镜观察拍照记录。
8.机械应力刺激对hFOB1.19增殖能力检测
收取处于旺盛增殖的hFOB1.19细胞(消化前细胞约70-80％融合)；通过细胞计数调整细胞密度，按4×103个/孔接种于加力板及普通6孔板，轻混匀使接种密度一致；细胞于34℃、5％CO2培养箱中孵育培养；分别于12h，24h，36h，48h，60h，72h时加入AlamarBlue(10％浓度)，轻混匀，放回34℃、5％CO2培养箱中孵育培养2h后收取上清于96孔板中；酶标仪测定570nm/650nm吸光值，静置组为对照，记录数据，绘出标准曲线。
9.Runx2载体构建
利用PCR方法，根据Runx2(human)3’UTR序列信息设计其扩增引物，以293T细胞基因组DNA为模板PCR扩增Runx2基因的3’UTR序列，将其克隆到pmiR-RB-REPORTTM双荧光素酶报告载体由广州锐博生物有限公司构建中，所用载体的报告荧光为hRluc，校正荧光为hluc(做内参校正)。对基因序列及载体序列分析，利用XhoI，NotI两个限制性内切酶将目的基因片段克隆到载体中，载体图谱如图7。
设计扩增引物如下：
RUNX2-3UTR-F：
Figure PCTCN2015088697-appb-000002
Figure PCTCN2015088697-appb-000003
RUNX2-3UTR-R：
Figure PCTCN2015088697-appb-000004
Figure PCTCN2015088697-appb-000005
设置XhoI，NotI酶切位点序列，之前序列为保护碱基，引物扩增总长度为3798bp。测序鉴定阳性的克隆。
10.Real-time PCR(实时定量PCR)
TRIZOL(Invitrogen)法抽提骨组织或细胞总RNA。配制cDNA反转录体系(RNA 1μg，Oligo d(T)1μl，DEPC水补足)，混匀后，70℃5min，立即置于冰上至少1min，反应体系中继续加入5×RevertAid buffer 4μl，dNTP混合液2μl，RevertAid 0.5μl，混匀后，42℃1h，样品放于-20℃保存。RT产物稀释10倍后，配制Real-time PCR反应体系。反应程序：95℃变性10sec→95℃10sec，60℃30sec，持续40个循环→进入溶解曲线检测步骤(按照仪器说明)；计算方法以GAPDH作为内参。其余数据分析按照Ct值(2-ΔΔCt)方法，图中显示为相对于对照的表达量改变的倍数值；所用引物序列如下：
GAPDH：正向：
Figure PCTCN2015088697-appb-000006
       反向：
Figure PCTCN2015088697-appb-000007
Col1a1：正向：
Figure PCTCN2015088697-appb-000008
        反向：
Figure PCTCN2015088697-appb-000009
Runx2：正向：
Figure PCTCN2015088697-appb-000010
       反向：
Figure PCTCN2015088697-appb-000011
OCN：正向：
Figure PCTCN2015088697-appb-000012
     反向：
Figure PCTCN2015088697-appb-000013
OPN：正向：
Figure PCTCN2015088697-appb-000014
     反向：
Figure PCTCN2015088697-appb-000015
OPG：正向：
Figure PCTCN2015088697-appb-000016
     反向：
Figure PCTCN2015088697-appb-000017
RANKL：正向：
Figure PCTCN2015088697-appb-000018
       反向：
Figure PCTCN2015088697-appb-000019
PANK3：正向：
Figure PCTCN2015088697-appb-000020
       反向：
Figure PCTCN2015088697-appb-000021
PANK2：正向：
Figure PCTCN2015088697-appb-000022
       反向：
Figure PCTCN2015088697-appb-000023
Figure PCTCN2015088697-appb-000024
11.Western blot
细胞弃上清，预冷PBS洗一遍；加入RIPA裂解液(含1％PMSF)，冰上裂解15min。
吹打收集细胞于1.5ml EP管，12,000rpm离心15min；取上清即为总蛋白，-80℃保存备用；BCA法蛋白定量。取50μg蛋白，加入5×上样缓冲液，混匀，95℃加热10min，将蛋白变性，二硫键打开；制备分离胶和浓缩胶；在电泳槽内加入电泳缓冲液，取蛋白marker 5μl及变性的蛋白，加入上样孔，80V电泳约30分钟，至溴酚兰进入分离胶，改为120V电压，电泳约1小时；小心取下SDS-PAGE凝胶，与PVDF膜，定性滤纸以及纤维垫放在电泳槽转膜缓冲液中，以夹三明治方式(由下至上依次是纤维垫、滤纸、凝胶、PVDF膜、滤纸、纤维垫)夹牢，彻底赶除内部气泡，放入装有转膜缓冲液的转移槽内，放入冰盒，以300A恒流转膜1.5小时；将转有蛋白的PVDF膜取出，标记方向，浸入封闭液，摇床上室温孵育1小时以封闭非特异性蛋白结合位点；将PVDF膜依照相应蛋白的分子量剪开，一抗孵育，4℃过夜；吸去一抗，用TBST于摇床上洗膜3次，每次5min；加入辣根过氧化物酶标记的二抗，室温于摇床上孵育1小时；吸去二抗，用TBST于摇床上洗膜3次，每次5min；显色和压片：将显色液1:1混合后，加到膜上，室温显色1-5分钟，用X-感光胶片压片适当时间，冲片。用扫描仪将图像输入计算机再用蛋白条带分析软件Gel-Pro Analyzer 3.0(Media Cybernetics，USA)来对相关蛋白进行定量。
12.小鼠的组织形态学检测
将BS基础组(6月龄)，连续吊尾4周后的HU组小鼠(7月龄)及WB承重对照组小鼠(7月龄)颈椎脱臼法处死，取双侧后肢，剔除附着肌肉软组织；用PBS缓冲液体冲洗干净标本表面的凝血块；取材组织固定于4％多聚甲醛中12h；右后肢保存在多聚甲醛中进行microCT检测，钙黄绿素双标；左后肢用SPEX 6770全自动冷冻研磨机提取骨组织内RNA；后肢标本经流水冲洗过夜(12h)，彻底去除标本中的固定液成分；标本放于12.5％EDTA脱钙液中，室温下脱钙四周，每三天更换一次脱钙液。当以针头可以无明显阻力的穿过标本时，表明脱钙已基本完成；将标记的标本经流水冲洗过夜(12h)，彻底去除标本中的脱钙液成分；按照以下过程梯度脱水：(1)75％酒精：12h；(2)85％酒精：16h；(3)95％酒精I：4h；(4)95％酒精II：4h；(5)100％酒精I：2h；(6)100％酒精II：2h；(7)100％酒精III：2h；按以下步骤：(1)二甲苯I：7min，(2)二甲苯II：7min，在透明过程中随时观察标本的透明程度，直到标本观察呈完全透明状时取出；透明后标本浸于60℃蜡中，时间如下：(1)苯蜡I：4h；(2)苯蜡II：4h；(3)石蜡I：4h；(4)石蜡 II：4h；(5)石蜡III：12h；常规方法包埋后，修整蜡块，分组标记；做5μm的连续切片，捞片至载玻片上，于烤片台上37℃烤片过夜；SCANCO MedicalμCT(SCANCO Medical AG，Switzerland)预装64位图像处理软件行骨组织形态学相关指标检测。TV(total tissue volume；contains both trabecular and cortical bone)，BV/TV(trabecular bone volume per tissue volume)，Tb.Th(trabecular thick-ness)，Tb.Sp(trabecular separation)，SMI(structure model index)，Conn.Dn(connectivity density)。
13.钙黄绿素双标检测
钙黄绿素100mg以PBS避光配制，0.22uM无菌滤器过滤，现配现用，小鼠编号，尾静脉注射；小鼠处死取材前10天及前3天分别给予腹腔注射10μg/g体重的钙黄绿素；麻醉小鼠，颈椎脱臼处死，解剖分离两侧股骨胫骨，置于70％乙醇中固定24小时，避光固定保存；行硬组织包埋和超薄切片(具体步骤见硬组织包埋)，切片避光保存，立即摄片；新鲜的组织切片，在荧光显微镜下观察，采集图像；用IPP软件进行图像定量分析，定量指标包括：矿物质沉积率(Mineral apposition rate，MAR)。MAR＝两次钙黄绿素沉积线宽度/两次注射时间间隔天数。用SPSS15.0统计软件进行分析。
14.Trap染色
材料收集：取小鼠股骨剔除软组织；第一次固定：4％多聚甲醛、10％福尔马林溶液。
第二次固定：纯酒精；脱脂、脱钙：脱钙液为ZnSO4加上EDTA溶液，脱钙装置为Histra-DC(普通光度)在8～16℃下连续操作；脱水、包埋：ETP(Sakura Finetek Japan)处理16小时，使用熔点为58～60℃硬石蜡包埋；薄切、干燥：制作4um切片，在43℃展开30分钟后，在37℃下干燥；染色、封片：用TRAP染色试剂盒(SIGMA，USA)染色，37℃干燥后，二甲苯脱蜡透明3次每次5min；切片Re-hydrate sections置于100％酒精，95％酒精，70％酒精各2min重复两次，蒸馏水冲洗；切片置预配好TRAP染液37℃孵育2h，蒸馏水冲洗；苏木素复染1min(复染时间取决于着色深度)，蒸馏水漂洗；封片、镜检、染色分析。
15.miRNAs激动剂模拟物mimics和抑制剂
hsa-miR-7
Figure PCTCN2015088697-appb-000025
hsa-miR-22
Figure PCTCN2015088697-appb-000026
hsa-miR-23b
Figure PCTCN2015088697-appb-000027
hsa-miR-103a (mimic-103a)
Figure PCTCN2015088697-appb-000028
hsa-miR-107
Figure PCTCN2015088697-appb-000029
hsa-miR-143
Figure PCTCN2015088697-appb-000030
hsa-miR-154
Figure PCTCN2015088697-appb-000031
hsa-miR-221
Figure PCTCN2015088697-appb-000032
hsa-miR-320d
Figure PCTCN2015088697-appb-000033
hsa-miR-374b
Figure PCTCN2015088697-appb-000034
hsa-miR-375
Figure PCTCN2015088697-appb-000035
hsa-miR-384
Figure PCTCN2015088697-appb-000036
U6内参：
Figure PCTCN2015088697-appb-000037
抑制剂inhibitor-103a：
Figure PCTCN2015088697-appb-000038
Figure PCTCN2015088697-appb-000039
agomir-103a：在反义链进行修饰，3’端进行胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，全链甲氧基修饰，序列如下(其中，S-硫代修饰；Chol-胆固醇修饰)：
Figure PCTCN2015088697-appb-000040
antagomir-103a：3’端进行胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，全链甲氧基修饰，序列如下：
Figure PCTCN2015088697-appb-000041
NC内参：
Runx2的siRNA：
RNA oligo sequences 21nt guide(5′→3′)21nt passenger(5′→3′)
Figure PCTCN2015088697-appb-000042
16.miR-103a和Runx2的结合位点区域突变的突变型(MUT)Runx23’UTR双荧光素酶报告基因载体的构建
利用PCR方法，根据Runx2(人)3’UTR序列信息设计其扩增引物，以293T细胞基因组DNA为模板PCR扩增Runx2基因的3’UTR序列，将其克隆到pmiR-RB-REPORTTM双荧光素酶报告载体中，所用载体的报告荧光为hRluc，校正荧光为hluc(做内参校正)。对基因序列及载体序列分析，利用XhoI，NotI两个限制性内切酶将目的基因片段克隆到载体中。
设计扩增引物如下：
RUNX2-3’UTR-F：
Figure PCTCN2015088697-appb-000043
Figure PCTCN2015088697-appb-000044
RUNX2-3’UTR-R：
Figure PCTCN2015088697-appb-000045
Figure PCTCN2015088697-appb-000046
引物上设置XhoI，NotI酶切位点序列，酶切位点序列之前序列为保护碱基，引物扩增产物的总长度为3798bp。测序鉴定阳性的克隆。
以获得的重组载体为模板，用QuikChange site-directed Mutagenesis试剂盒(Stratagene，La Jolla，CA，http://www.stratagene.com)在靶位点中引入七个碱基突变，构建突变型靶基因荧光素酶报告基因载体。结果经限制性内切酶酶切和DNA测序证实质粒中插入的为所需序列。与Runx2的3’UTR种子区域互补结合的AUGCUGC被转换为UACGACG，废除了Runx2的3’UTR种子区与miRNA-103a特异性结合。
17.统计分析
所有实验至少重复三次。数据以平均值±标准差(mean±standard deviation)表示。对于基因相对表达分析，对照组的平均值定义为1。Student’s t-test用于对两组数据进行统计分析。One-way ANOVA用于对多组数据进行统计分析。P<0.05 认为具有显著性差异。
实施例1、在后肢去负荷小鼠模型中载荷缺失导致骨量丢失废用性骨质疏松
为研究机械应力刺激缺失对骨重建稳态的影响，本发明人构建双下肢去负荷的小鼠模型(Hindlimb unloading，HU)，此模型最初由美国国家航空航天局(National Aeronautics and Space Administration，NASA)在1980年构建用于研究在太空失重状态对宇航员骨量的影响，随后被广泛应用于各项研究力学与运动系统的研究中(图1a)。较之基准组(Baseline group，BS)和静置对照组(Weight-bearing group，WB)小鼠，HU组小鼠尾部悬吊28天使其双后肢悬空，但双前肢可自由行走，不影响其饮水进食。在实验进程中，各组小鼠每周称重2次观察其生长发育状况，每天观察其尾部血液循环状况，如有缺血坏死征象立即处理。
实验结束后，WB和HU组小鼠基础体重及实验终末体重无明显差异(28.6±2.7g vs.30.1±2.5g；P<0.05)。HU小鼠的股骨较之WB组呈现明显细短及脆性增加易于骨折征象(图1b)。取小鼠后肢股骨进行microCT扫描进行骨组织形态计量学相关分析，结果显示，HU组小鼠股骨远端较之BS组和WB组呈现明显的骨量下降，骨质疏松表型(图1c，d)。
实施例2、CMS介导成骨细胞分化中Runx2转录后水平的调控
为探究CMS对于成骨细胞分化的调控作用，本发明人应用有限元分析(FEA)检测股骨近段载荷承载情况(图2a)。根据现有文献报导，正常人体组织水平承载的应力常小于1000με。FEA检测发现股骨近段所承载应力约为815±57με(图2a)。然而，组织水平与细胞水平的载荷并非同一概念，应力载荷经由组织水平传递至细胞水平时存在扩增机制，此即为细胞水平应力应变扩增机制。在成骨细胞，骨组织水平承载的应力被传导至细胞膜时可被扩大20至100倍。因此，本发明人选取8％CMS(8％形变，80000με，Sin，0.5Hz，CMS)加载于人成骨细胞前体hFOB1.19进行后续实验。应力加载3天后，qRT-PCR显示较之静置组，8％CMS组相关成骨细胞分化相关特异性基因：Alkaline phosphatase(ALP)，osteocalcin(Ocn)以及collagen type I alpha 1(Col1a1)表达水明显增强(图2b)。同样，8％CMS组ALP染色及ALP定量增强(图2c，d)。此外，本发明人发现较之静置组，CMS组细胞形态发生拉伸，细胞骨架发生应力向心性重排列(图2e)。然而，各组间细胞增殖并无明显差异(图2f)。Wnt/β-catenin及ERK1/2MAPK通路在机械应力方 面的重要作用已被证实。本发明中，本发明人发现8％CMS可显著激活Wnt/β-catenin及ERK1/2MAPK通路(图2g)。
鉴于8％CMS在促进成骨细胞分化中作用作为显著，在以下的实验中本发明人选取此强度载荷作为诱导成骨细胞分化的载荷强度并研究成骨分化中关键转录因子Runx2的表达变化。Runx2是成骨分化中的最为关键的转录因子，在成骨分化中，Runx2与多种蛋白，转录因子，信号通路等共同构成级联网络调控成骨分化。本发明人发现较之静置组，8％CMS可显著增强Runx2的蛋白表达，而仅使其mRNA水平轻微升高(图2h，i)。以上这种Runx2的mRNA水平和蛋白水平的表达不一致提示：在机械应力刺激诱导的成骨分化过程中可能存在miRNA对于Runx2的转录后水平调控。
实施例3、miR-103a在机械应力刺激介导的成骨细胞分化中直接靶标于Runx2
生物信息学发现Runx2的3’UTR约为3777个核苷酸长度。选用以下miRNA生物信息学预测软件：TargetScan，miRDB，miRanda及miRBase数据库分别筛选出潜在标靶于Runx2基因3’UTR区的miRNAs，取其交集。剔除文献已报道对Runx2有确定调控作用的miRNA，得到12条可结合于Runx23’UTR种子区(seed region)的miRNAs，包括：miR-7，miR-22，miR-23b，miR-103a，miR-107，miR-143，miR-154，miR-221，miR-320d，miR-374b，miR-375and miR-384(图3a)。
为了验证所筛选12条miRNAs是否受机械应力刺激调控，本发明人应用8％CMS加载hFOB1.19人成骨细胞系3天，抽提miRNA进行分别检测12条miRNAs应力加载前后在hFOB1.19中表达水平变化。qRT-PCR显示7条miRNAs包括miR-103a，miR-23b以及miR-374b在应力刺激介导成骨细胞分化中表达水平显著下降。与之相反，miR-107，miR-143和miR-154表达上升(图3b)。为了进一步验证所筛选的miRNAs是否直接靶标于Runx2，本发明人构建了野生型(WT)Runx23’UTR双荧光素酶报告基因载体。将WT Runx23’UTR双荧光素酶报告基因载体分别与所筛选的miRNAs激动剂模拟物mimics和U6内参在hFOB1.19人成骨细胞系中共转染。Luciferase结果显示除了miR-107，miR-143和miR-154以外其余miRNAs均可不同程度抑制Runx2WT 3’UTR双荧光素酶报告载体活性，其中尤以mimic-103a的抑制作用最为显著(图3c)。将筛选的miRNAs的mimics转染hFOB1.19人成骨细胞系，8％CMS加载3天后Western blot检测Runx2蛋白水 平，发现与Luciferase实验相一致的是mimic-103a使得Runx2蛋白水平下调最为显著(图3d)。在以上12条miRNA中，miR-103a和miR-107为同源盒miRNA，它们仅在3’尾端有一个核苷酸的差异。miR-103a/miR-107最初被发现在肥胖小鼠中表达上调，进而被发现在胰岛素敏感方面起重要作用。然而有趣的是，在本发明中本发明人发现miR-107在Luciferse实验中与miR-103a起截然相反的作用，而转染它的模拟物mimic-107对Runx2的蛋白水平也并无显著下调作用(图3c，d)。以上结果提示同源miRNAs有时可能在某一调控过程中起截然相反的作用。鉴于miR-103a在以上实验中可显著抑制Runx2的表达，本发明人因此在以下的研究中将研究焦点锚定于miR-103a，其序列为
Figure PCTCN2015088697-appb-000047
(hsa-miR-103a-3p MIMAT0000101；SEQ ID NO:1)。
为了验证miR-103a在应力刺激介导的成骨细胞分化中是否直接靶标于Runx2，本发明人分别进行miR-103a的功能缺失性和功能获得性实验验证其是否对Runx2存在转录后水平调控。在hFOB1.19人成骨细胞系中分别转染miR-103a的模拟激动剂mimic-103a和抑制剂inhibitor-103a分别过表达和下调miR-103a，8％CMS加载3天后，qRT-PCR检测内源性miR-103a的表达水平，发现其模拟激动剂mimic-103a可显著上调miR-103a水平，而其模拟抑制剂inhibitor-103a可显著下调miR-103a水平(图3e)。8％CMS加载3天后，两种表达方式皆可过表达miR-103a(转染mimic-103a)可显著抑制Runx2蛋白表达而下调miR-103a(转染inhibitor-103a)可增加Runx2蛋白表达水平(图3f)。然而各组中Runx2的mRNA水平无明显变化(图3g)。
为进一步验证miR-103a与Runx2mRNA的作用位点，本发明人构建了根据生物信息学预测miR-103a和Runx2的结合位点区域突变的突变型(MUT)Runx23’UTR双荧光素酶报告基因载体(图3h)。将MUT Runx23’UTR双荧光素酶报告基因载体分别与miR-103a的模拟激动剂和抑制剂mimic-103a，inhibitor-103a在hFOB1.19人成骨细胞系中共转染。Luciferase实验显示mimc-103a抑制而inhibitor-103a增强WT Runx23’UTR双荧光素酶报告载体活性，但两者均对MUT Runx23’UTR双荧光素酶报告载体活性无明显影响(图3i)。
以上结果表明在应力刺激介导的成骨细胞分化中，miR-103a通过结合于关键转录因子Runx2的3’UTR种子区域而直接靶标Runx2在转录后水平抑制其表达。
实施例4、miR-103a作为应力敏感miRNA在机械应力刺激介导的成骨细胞 分化中起调控作用
为了验证miR-103a是否为应力载荷敏感性miRNA，本发明人应用8％CMS加载hFOB1.19人成骨细胞系3天，qRT-PCR检测发现较之静置组，加力组miR-103a水平明显下降(图4a)。
在人基因组上存在两个miR-103a前体pre-miR-103a的基因座位点。人源性miR-103a-1的茎环结构(stem-loop)定位于人第5号染色体，而miR-103a-2的茎环结构定位于人第20号染色体上。对于所有已知的脊椎物种，miR-103a/107同源盒miRNA嵌合于编码PANK(pantothenate kinase enzyme，PANK)基因的内含子中。成熟的miR-103a-1和miR-103a-2分别来源于其宿主基因PANK3和PANK2内含子-intron 5(图4b)。PANK3和PANK2基因是辅酶A的合成和糖脂代谢中重要的酶。为了验证应力刺激是否通过调控miR-103a的宿主基因PANK3和PANK2的表达进而影响miR-103a的表达。本发明人应用8％CMS加载hFOB1.19人成骨细胞系3天，检测应力加载前后PANK3和PANK2的mRNA水平。qRT-PCR显示8％CMS选择性地下调了PANK3的mRNA表达水平但对PANK2的mRNA水平无明显影响。这一改变与加力后miR-103a的变化情况相一致(图4c)。以上结果提示：1.成熟miR-103a主要由其前体双链中的miR-103-1剪切而来，miR-103-1来源于Pank3基因座位。加力后引起的miR-103a的表达变化主要选择性地源于嵌合于Pank3基因座位的miR-103a的前体miR-103-1；2.定位于其宿主基因内含子的miRNAs的表达通常与其宿主基因表达相一致。
为进一步探究CMS介导下成骨细胞分化成熟全过程中miR-103a的表达情况变化，本发明人应用8％CMS加载培养hFOB1.19人成骨细胞系至21天，每三天检测miR-103a的表达水平。本发明人发现miR-103a的水平在成骨分化早期显著下调(0～9天)，继而逐渐缓慢下调至21天(图4d，上)，Runx2蛋白水平在成骨分化启动时明显上调(day 3)并维持高水平表达至21天矿化期(mineralization stage)，然后显著下降(图4d，下)。同时，Runx2，ALP，Ocn的mRNA水平在此过程中显著上调(图4e)。这种Runx2的mRNA水平和蛋白水平的表达不一致可能是由于miR-103a对于Runx2的转录后水平的调控所致。结合miR-103a和Runx2的表达，可以理解为在成骨分化初期，miR-103a的水平明显下降，强抑制成骨分化的抑制因素突然解锁，使得分化进程得以启动；而进入分化终末期，miR-103a的表达水平维持不变以使成骨分化的进程得以适时终止。
以上结果表明，miR-103a及其宿主基因PANK3对于应力刺激敏感，miR-103a 可能在机械应力刺激介导的成骨分化进程中起着调控进程启动，及终止子的作用。miR-103a作为应力刺激敏感miRNA通过靶标于转录因子Runx2在成骨细胞分化中起调控作用。
实施例5、miR-103a在机械应力刺激介导的成骨细胞分化中抑制成骨细胞活性及胞外基质矿化
为了探究miR-103a的调控成骨细胞活性的作用，本发明人在hFOB1.19人成骨细胞系中分别转染miR-103a的模拟激动剂mimic-103a和抑制剂inhibitor-103a。8％CMS加载3天后，与内参对比，mimic-103a显著降低成骨细胞分化中ALP和Ocn的mRNA表达水平，inhibitor-103a则增强它们的表达(图5a)。同样，过表达miR-103a降低了成骨细胞分化中ALP活性和ALP染色，抑制miR-103a表达增强了ALP活性和ALP染色(图5b，c)。
为了进一步探究miR-103a在胞外基质矿化中的功能，本发明人用miR-103a的长效激动剂和抑制剂agomir-103a和antagomir-103a(miRNA antagomir/agomir，经特殊化学修饰的miRNA长效拮抗剂/激动剂)以及NC内参在hFOB1.19细胞中分别过表达和抑制miR-103a表达。为了维持miR-103a的稳定表达，agomir-103a和antagomir-103a每三天补充添加一次。qRT-PCR证实agomir-103a和antagomir-103a可有效过表达及敲除细胞内源性miR-103a表达水平(图5d)。8％CMS应力加载21天后，茜素红染色显示相较空白处理对照组及NC对照组，agomir-103a处理组胞外基质矿化明显减弱，而antagomir-103a处理组胞外基质矿化增强(图5e)。
为了验证miR-103a的这种调控成骨细胞分化的作用是否是Runx2必需和依赖的，本发明人构建了Runx2的特异性siRNA干扰其表达，进而研究其对miR-103a调控成骨分化作用的影响(图5f)。本发明人发现用Runx2的siRNA将其敲除后，成骨分化中Runx2下游基因ALP，Ocn的mRNA水平及ALP定量受到显著抑制(图5g)。然而，当将Runx2的siRNA与miR-103a的模拟物mimic-103a和抑制剂inhibitor-103a共转染后，miR-103a的化学模拟物的功效被完全阻断，ALP，Ocn的mRNA水平以及ALP定量维持在低水平。以上结果提示miR-103a在机械应力介导的成骨分化过程中的调控作用是Runx2必需和依赖的(图5g)。
经典通路Wnt/β-catenin和ERK1/2MAPK通路在应力传导、应答中的关键作用已被文献广泛证实。但miR-103a与上述两条通路间是否存在相互级联调控尚 未可知。为验证在机械应力刺激介导的成骨细胞分化中miR-103a的表达是否受ERK1/2MAPK和Wnt/β-catenin通路的调控，本发明人分别用ERK1/2MAPK和Wnt/β-catenin的通路阻断剂U0126和IWR-1(U0126，ERK1/2通路阻断剂；IWR-1，Wnt/β-catenin通路阻断剂)加入hFOB1.19人成骨细胞系，分别检测miR-103a的表达(图5h)。qRT-PCR显示加入ERK1/2MAPK和Wnt/β-catenin的通路阻断剂U0126和IWR-1对miR-103a的表达水平无明显影响。
以上结果提示：miR-103a在机械应力刺激介导的成骨细胞分化及胞外基质矿化中依赖于Runx2发挥抑制作用。miR-103a的这种调控作用与ERK1/2MAPK和Wnt/β-catenin信号通路之间无互作关系。
实施例6、miR-103a的长效抑制剂可挽救后肢去负荷小鼠模型骨量丢失表型
以上本发明人已证实miR-103a可在体外水平对载荷介导的成骨分化起重要调控作用，接下来本发明人将验证miR-103a是否同样可在体内调控骨形成。通过生物信息学发现miR-103a在脊椎动物中呈现多物种高度保守性。提示可在小鼠中研究miR-103a的表达以及其与骨形成的关系(图6a)。本发明人首先检测了miR-103a在C57BL/6J小鼠体内各主要脏器组织包括骨组织中的表达丰度。结果显示miR-103a在骨组织中的表达丰度明显高于其他主要脏器，提示miR-103a在骨组织重建中起较为重要的调控作用(图6b)。
为了验证在体内水平骨组织内miR-103a的表达是否同样受到应力的影响，本发明人分别在后肢去负荷小鼠和对照组小鼠股骨中检测miR-103a的表达。qRT-PCR显示：较之BS基础组和WB对照组小鼠，在后肢去负荷小鼠股骨中miR-103a的表达水平明显上升(图6c)。为了进一步验证外源性给予补充Antagomir-103a能否有效反转抵消由于应力缺失导致的骨量丢失，本发明人分别设置HU组小鼠(HU+Antagomir-103a，Antagomir-103a，用量80mg/kg)，PBS组小鼠(HU+PBS，PBS，用量0.3ml)，在吊尾前通过尾静脉连续注射3次(图6d)。为了维持miR-103a在体内的稳定表达丰度，HU组小鼠(HU+Antagomir-103a mice)在第一次注射后第三周接受另一次注射。所有小鼠在后肢悬吊28天处死。本发明人发现较之HU组和HU+PBS组小鼠，HU+Antagomir-103a组小鼠股骨中miR-103a表达较低而Runx2蛋白表达较高，而在HU组和HU+PBS组小鼠中miR-103a和Runx2的表达无明显差异(图6e，f)。
以上结果表明：1.antagomir-103a可有效下调骨组织中miR-103a的表达丰度； 2.antagomir-103a可部分反转由后肢去负荷导致的骨组织内Runx2的表达下调。更重要的是，microCT结果显示由去负荷导致的骨量下降可被antagomir-103a部分挽救(图6g，h)。骨组织形态学相关分析显示，较之HU组和HU+PBS组小鼠，HU+Antagomir-103a组小鼠的骨形成相关参数(Ob.S/BS，MAR，N.Ob/B.Pm)明显升高(图6i，j)，而骨吸收相关参数(Oc.S/BS，N.Oc/B.Pm)在HU，HU+PBS，HU+Antagomir-103a组小鼠中无明显差异(图6k，l)。
以上结果表明：1.miR-103a和Runx2在骨组织中的表达受应力载荷调控；2.在体内水平，载荷缺失导致的Runx2蛋白水平下降和骨量丢失与miR-103a的表达增高相关；3.在HU小鼠中，通过给予miR-103a的抑制剂可部分反转由载荷缺失导致的骨量下降表型。因此，miR-103a的抑制剂(下调剂)能够显著改善骨质疏松或骨量丢失。
实施例7、药物筛选
取hFOB1.19细胞，该细胞可内源性表达miR-103a。将该种细胞作为用于筛选防治骨代谢疾病的药物的细胞模型。
测试组：用候选物质处理的上述细胞的培养物；
对照组：不用候选物质处理的上述细胞的培养物。
在处理后适当时间，测定所述细胞的miR-103的表达。如果与对照组相比，测试组中的miR-103的表达显著下降30％以上，则说明该候选物质是潜在的防治骨代谢疾病的物质。
以antagomir-103a以及agomir-103a分别作为候选物质，转染上述细胞。结果发现antagomir-103a可使得测试组中的miR-103的表达显著下降90％以上。因此，antagomir-103a为一种有用的候选药物。
讨论
机械应力刺激对于骨重建的作用至关重要。本发明从临床现象得到提示，以骨重建稳态角度入手，探索机械应力刺激对于成骨细胞分化影响以及miRNA在机械应力介导的成骨分化中的调控作用和机制。本发明通过生物信息学方法挖掘并验证成骨分化中载荷敏感性miRNA，探究其在机械应力调控下的表达及在机械应力刺激介导的成骨分化及骨形成中的调控作用和机制。本发明人发现在生理和病理载荷状态下，miR-103a作为一种新的未被发现报道过的特异性力敏感miRNA 调控成骨分化和骨形成。在体外水平，在机械应力刺激介导的成骨细胞分化和骨形成中，miR-103a通过靶标于成骨分化中关键转录因子Runx2在转录后水平抑制其表达而对成骨细胞分化和骨形成起抑制作用。在体内水平，在双后肢去负荷HU小鼠中，本发明人的体内实验结果提示通过治疗性预给药调控体内miR-103a水平可部分挽救由应力缺失导致的骨量下降，骨质疏松表型。就目前而言，miR-103a作为一种新的力敏感性miRNA在成骨细胞前体中第一次提供了关于miRNA在应力载荷介导的成骨分化骨形成中的调控作用和机制的佐据。也提示miR-103a在临床可作为潜在药靶治疗临床因应力缺失导致的废用性骨质疏松症。
机械应力最初被报道在多种机体生理及病理进程中起重要调控作用。其中，以机械应力在心血管系统，骨骼肌系统以及肺生理学方面研究的最为广泛。然而，关于机械应力在体内对于成骨细胞分化的作用仍有待明确。本发明分别在体内外揭示成骨细胞在接受外界载荷刺激时，将发生对载荷的迅速适应性，具体表现在细胞功能和形态结构上。本发明发现机械应力载荷可显著促进成骨细胞分化及骨形成。
成骨细胞分化被机械应力刺激启动后，将导致一系列转录因子，荷尔蒙激素，生长因子应答的级联反应，促进成骨细胞分化和骨形成。作为成骨分化中最为关键且必需的转录因子，Runx2可结合于所有主要的成骨相关基因启动子区的成骨细胞特异的顺式元件2(osteoblast-specific cis-element 2，OSE2)。Runx2全敲除的小鼠在胚胎期即死亡，体内无成骨细胞，因而也无骨组织，只有软骨存在。Runx2半敲除的(Runx2-/+)的杂合子小鼠呈现与人遗传性骨骼发育异常相似的特异性骨骼发育障碍症状。杂合缺失Runx2的小鼠表现的性状与一种典型的突变体小鼠：锁骨颅骨发育不良(Cleidocranial Dysplasia，CCD)小鼠非常相似。任何Runx2的DNA结合结构域的杂合突变，包括缺失突变及碱基替换突变，都会呈现CCD性状。本发明中本发明人发现机械应力刺激可明显上调Runx2蛋白水平，而其mRNA水平仅轻微上升。这促使本发明人去进一步探究其中是否存在转录后水平的调控，诸如miRNA的调控存在。
miRNAs自被发现以来因其在在多种生理，病理进程中的起重要调控作用而被日益广泛关注。一些研究证实在体外多种细胞系中，机械应力包括剪切力和循环张力可调控一系列miRNAs的表达。这些miRNAs同样参与细胞对机械应力刺激的应答。近年，多条miRNAs被证实在骨重建中作为重要调控因素存在，通过在转录后水平调控骨重建相关基因的表达在临床许多骨代谢疾病如骨质疏松中起 重要作用。然而，在机械应力刺激介导的成骨细胞分化中相关miRNA的表达及其靶标基因在此过程中的调控作用尚未完全可知。本发明首次将miRNA的表达与机械应力刺激以及骨重建稳态相联系。
如实验结果所示，8％CMS应力载荷下miR-103a(与miR-107为同源基因)被募集抑制Runx23’UTR高度保守序列配对进而抑制成骨分化。miR-103a/miR-107已被miRBase收录存在人基因组中且在多脊椎动物/哺乳动物物种中高度保守。它们最初被发现在肥胖小鼠中表达升高进而被发现在胰岛素敏感度方面起关键调控作用，提示其可能作为治疗2型糖尿病的潜在药靶。此外，也有报道miR-103a在对于慢性疼痛有关联。正如其他许多miRNAs，miR-103a在许多肿瘤细胞中呈高表达，提示miR-103a可能在肿瘤发生发展进程中起重要作用。近年，一些研究证实miR-103a可能促进脂代谢进而调控糖脂代谢的稳态。然而，尚无研究证实miR-103a在调控成骨细胞分化中的作用。
ERK1/2MAPK和Wnt/β-catenin信号通路在成骨分化中的作用已被广泛证实。β-catenin的失活阻断了间充质干细胞向成骨细胞的分化，提示β-catenin在体内成骨细胞分化中起关键调控作用。本发明中，本发明人发现机械应力载荷可明显激活ERK1/2和β-catenin信号通路并进一部促进其下游诸多基因包括Runx2的表达。然而，本发明未发现miR-103a和ERK1/2MAPK以及Wnt/β-catenin信号通路之间存在互作关系，提示在机械应力载荷介导的成骨细胞分化中miR-103a的表达可能不处于两条通路的调控之下而而独立存在。
适度强度的机械应力刺激也可作为治疗某些骨代谢疾病的治疗手段。动态应力载荷加载于成骨细胞可激活/活化Wnt-β-catenin通路进而促进成骨细胞分化/生成。通过加载适度强度的机械应力载荷可促进成骨细胞分化发育成熟为骨细胞促进骨折愈合。成骨细胞和骨细胞对于强度不同的应力刺激起应答。骨折后坚强内固定使骨折部位的修复组织在生理负荷下的应变完全消除，骨折的愈合没有出现肉眼可见的骨痂达到直接愈合。而弹性内固定包括膜内成骨和软骨内成骨的间接愈合，其特点是骨痂形成。本发明结果在体内揭示了一种新的miR-103a在体内调控骨形成的新的机制。miR-103a在骨组织中呈高丰度表达，提示其可能在骨重建中起重要调控作用。研究表明miR-103a在机械应力刺激下的表达变化可导致Runx2的蛋白水平发生相应变化。Runx2表达的变化将进一步影响其下游成骨分化相关特异基因的表达。在后肢去负荷小鼠，通过治疗性给予miR-103a的长效抑制剂antagomir-103a可反转由异常病理应力状态下导致的miR-103a的异常升高， 部分挽救由于应力缺失导致的骨量丢失表型。
总之，本发明人的研究发现并验证了miR-103a作为一种新的应力载荷敏感性miRNA调控成骨分化，miR-103a在成骨分化中通过在转录后水平直接靶标于Runx2来实现其调控功能。这提示在体内体外，可将miR-103a作为一种新的潜在药靶，通过调控其在生理和病理应力状态下的表达来达到调控骨形成的目的。这些发现不仅为研究应力载荷传导方面提供了一种新视野，而且也提供了一种将miRNA分子作为调控骨组织工程再生性药物的途径。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种miR-103a的下调剂在制备预防或治疗骨代谢疾病的药物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述的miR-103a抑制剂包括：化学合成的miR-103a抑制剂；以表达质粒为载体的抑制miR-103a的病毒和非病毒产品；与miR-103a互补的核酸序列或序列片段。
  3. 如权利要求2所述的用途，其特征在于，所述的miR-103a的下调剂选自：antagomir-103a，其核苷酸序列如SEQ ID NO:49所示；或
    inhibitor-103a，其核苷酸序列如SEQ ID NO:47所示。
  4. 如权利要求2或3所述的用途，其特征在于，所述的miR-103a的下调剂是经修饰的下调剂，所述的修饰包括：甲氧基化修饰、硫代修饰、胆固醇修饰、烷基修饰、锁核酸修饰、肽核酸修饰、和/或磷酸骨架由磷脂连接代替的反义核苷酸；较佳地，所述的antagomir-103a的修饰包括：3’端进行胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，全链甲氧基修饰。
  5. 如权利要求1所述的用途，其特征在于，所述的药物还用于：
    增加Runx2蛋白的表达；
    增强成骨细胞分化中ALP和Ocn的表达水平；或
    增强胞外基质矿化。
  6. 一种miR-103a的用途，其特征在于，用于筛选预防或治疗骨代谢疾病的药物。
  7. 如权利要求1-6任一所述的用途，其特征在于，所述的骨代谢疾病包括：骨质疏松、成骨分化异常、骨量丢失。
  8. 一种预防或治疗骨代谢疾病的药物，其特征在于，所述的药物是miR-103a的下调剂，选自：
    antagomir-103a，其核苷酸序列如SEQ ID NO:49所示；或
    inhibitor-103a，其核苷酸序列如SEQ ID NO:47所示。
  9. 如权利要求8所述的药物，其特征在于，所述的miR-103a的下调剂是经修饰的下调剂，所述的修饰包括：甲氧基化修饰、硫代修饰、胆固醇修饰、烷基修饰、锁核酸修饰、肽核酸修饰、和/或磷酸骨架由磷脂连接代替的反义核苷酸；较佳地，所述的antagomir-103a的修饰包括：3’端进行胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，全链甲氧基修饰。
  10. 一种筛选预防或治疗骨代谢疾病的潜在物质的方法，所述方法包括：
    (1)用候选物质处理表达miR-103a的体系；和
    (2)检测所述体系中miR-103a的表达；
    其中，若所述候选物质可降低miR-103a的表达，则表明该候选物质是预防或治疗骨代谢疾病的潜在物质。
  11. 如权利要求10所述的方法，其特征在于，所述的体系中还表达Runx2蛋白，所述方法还包括：检测所述体系中Runx2蛋白的表达；
    其中，若所述候选物质通过下调miR-103a的表达而增加Runx2蛋白的表达，则表明该候选物质是预防或治疗骨代谢疾病的潜在物质。
  12. 如权利要求10或11所述的方法，其特征在于，步骤(1)包括：在测试组中，将候选物质加入到表达miR-103a或共表达miR-103a和Runx2蛋白的体系中；和/或
    步骤(2)包括：检测测试组的体系中miR-103a和/或Runx2蛋白的表达，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达miR-103a和/或Runx2蛋白的体系；
    如果测试组中miR-103a的表达在统计学上低于对照组，或还使得Runx2蛋白的表达显著增加，就表明该候选物是预防或治疗骨代谢疾病的潜在物质。
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