CN107693535A - A kind of microRNA application - Google Patents
A kind of microRNA application Download PDFInfo
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- CN107693535A CN107693535A CN201710790361.4A CN201710790361A CN107693535A CN 107693535 A CN107693535 A CN 107693535A CN 201710790361 A CN201710790361 A CN 201710790361A CN 107693535 A CN107693535 A CN 107693535A
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention provides a kind of microRNA application, microRNA can be used for preparing the medicine for suppressing osteoclast differentiation and osteoclasia, and the microRNA is selected from miR 17, miR 20a and miR 92a, the miR 17 sequence such as SEQ ID NO:Shown in 1, the sequence such as SEQ ID NO of the miR 20a:Shown in 2；The sequence of the miR 92a such as SEQ ID NO:Shown in 3.Differentiation of the up-regulation of miR 17, miR 20a or miR 92a in the microRNA for osteoclast is inhibited, thus can be as the new therapy target of regulation Bone m etabolism.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of microRNA application, more particularly to one kind
MicroRNA prepare suppress osteoclast differentiation and osteoclasia medicine in purposes, and screening prevention, alleviate or
Treat the purposes in the potential material of osteoclasia.
Background technology
Bone information caused by osteoclast plays the physiological action of key in the maintenance of mineralization of skeleton stable state.Bone information
Enhancing is one of notable feature of many diseases, such as osteoporosis, the Bone tumour of cancer etc..
Osteoclast is the multinucleate giant cell in myelomonocyte source, and bone marrow macrophage first is divided into anti-tartaric acid
The positive osteoclastic precursor of acid phosphatase (TRAP), then in macrophage colony stimulatory factor (M-CSF) and Nuclear factor kappa
Under B receptor activations factor ligand (RANKL) stimulates, osteoclastic precursor, which merges into each other, to be formed the maturation with multiple nucleus and breaks
Osteocyte, by secretory tissue protease (cathepsin) K, the substrate enzyme such as metalloproteinases (MMP) and TRAP is come bone of degrading
Tissue.Finally, osteoclast is attached at bone tissue surface and is finally divided into functional osteoclast.
MicroRNA (miRNA) is the single-stranded non-coding RNA molecule of a kind of 20 to 23 nucleotides composition, is adjusted after transcription
Play the part of important role in section.MiRNA is the small molecule non-coding RNA that cellular endogenous produces regulatory gene protein expression, and it is adjusted
The effect of section is typically inhibition of gene expression.The expression of some genes in MicroRNA (miRNA) regulation bodies, they participate in thin
Nearly all life processes such as born of the same parents' propagation, differentiation, apoptosis and metabolism, in angiocardiopathy, the nervous system disease, hemopoietic system disease
Also played a significant role in the pathologic process of disease, diabetes and various tumours.But it there is no find microRNA to osteoclastic thin at present
The mechanism of action of born of the same parents.
The content of the invention
For drawbacks described above of the prior art, it is a primary object of the present invention to provide a kind of microRNA application,
Differentiation of the up-regulation of miR-17, miR-20a or miR-92a in the microRNA for osteoclast is inhibited,
Thus can be as the new therapy target of regulation Bone m etabolism.
In order to achieve the above object, the present invention adopts the following technical scheme that：MicroRNA is preparing suppression osteoclast point
Purposes in the medicine of change and osteoclasia, the microRNA are selected from miR-17, miR-20a and miR-92a, the miR-17
Sequence such as SEQ ID NO:Shown in 1, the sequence such as SEQ ID NO of the miR-20a:Shown in 2；The sequence of the miR-92a
Such as SEQ ID NO:Shown in 3.
Purposes of the above-mentioned microRNA upper adjustment in the medicine for suppressing osteoclast differentiation and osteoclasia is prepared.
As further preferably, upper adjust is the upper adjustment through modification, and described modification includes：Methoxylation is repaiied
Decorations, thio-modification, cholesterol modification, alkyl modified, lock nucleic acid modification, peptide nucleic acid modification, and/or phosphate backbones are connected by phosphatide
Instead of GEM 132.
As further preferably, described modification includes：3 ' ends carry out cholesterol modification, and 5 ' the two thio skeletons in end are repaiied
Decorations, 3 ' the four thio backbone modifications in end and the modification of full chain methoxyl group.
Purposes of the above-mentioned microRNA in screening prevention, the potential material alleviated or treat osteoclasia.
As further preferably, the method for the screening includes：
(1) expression miR-17, miR-20a or miR-92a system is handled with candidate substances；With
(2) expression of miR-17, miR-20a or miR-92a in the system are detected；
Wherein, if the candidate substances can improve miR-17, miR-20a or miR-92a expression, the candidate is shown
Material is prevention, alleviation or the potential material for treating osteoclasia.
As further preferably, the raising includes：Improve 20-90%.
As further preferably, the raising includes：Improve 50-90%.
As further preferably, the raising includes：Improve 80-90%.
As further preferably, the candidate substances include：Based on miR-17, miR-20a, miR-92a or regulation and control
The gene and albumen of miR-17, miR-20a, miR-92a expression and the macromolecular or small molecule that design.
As further preferably, the macromolecular is selected from peptide and over-express vector, and the small molecule includes compound.
As further preferably, described system is selected from：Cell system or cell culture system, subcellular fraction system,
Solution system, organizational framework, organ systems or animal system.
The beneficial effects of the invention are as follows：The invention firstly discloses miR-17, miR-20a, miR-92a up-regulation for broken
The differentiation of osteocyte is inhibited, and therefore, miR-17, miR-20a, miR-92a can be as the new of regulation Bone m etabolism
Therapy target, suppress energy metabolism related genes important in osteoclast.
Brief description of the drawings
Fig. 1 be miR-17~92cluster miRNA (including miR-17, miR-20a, miR-92a, miR-18a and
MiR-19a) the expression change schematic diagram in osteoclast atomization.
Fig. 2 is to be overexpressed miRNA (including miR-17, miR-20a, miR-92a) to suppress the signal of osteoclast differentiation
Figure.
Fig. 3 is that joint is overexpressed miRNA (including miR-17, miR-20a, miR-92a) to suppress osteoclast differentiation
Schematic diagram.
Fig. 4 is the binding site schematic diagram that miR-17 is applied on Ppargc1b.
Fig. 5 is expressed for interference miRNA (including miR-17, miR-20a, miR-92a) to observe osteoclast important gene
PGC-1 β mRNA expression change schematic diagram.
Embodiment
The present invention is by providing a kind of microRNA application, it was found that miR-17, miR-20a in microRNA,
The miR-92a mechanism of action to osteoclast.
In order to solve the above problems, the main thought of the embodiment of the present invention is：
The embodiment of the present invention suppress osteoclast differentiation microRNA, the microRNA be selected from miR-17, miR-20a and
MiR-92a, the miR-17 sequence such as SEQ ID NO:Shown in 1, the sequence such as SEQ ID NO of the miR-20a:Shown in 2,
It is mouse source mmu-miR-20a-5p sequence；The sequence of the miR-92a such as SEQ ID NO:Shown in 3, it is mouse source mmu-
MiR-92a-3p sequence.
Described miR-17, miR-20a or miR-92a can be separated from cell, or can pass through artificial synthesized side
Formula obtains.After miR-17, miR-20a, miR-92a or their precursors sequence is known, those skilled in the art can be easily
Prepare miR-17, miR-20a, miR-92a or their precursor.
MiR-17, miR-20a or miR-92a are new, closely related with an osteoclastic metabolism drug targets.For
MiR-17, miR-20a or miR-92a various treatment means can be as the novelties of the osteoclasia of preventing and treating animal (particularly people)
And effective means.Also, miR-17, miR-20a or miR-92a can be used for the target spot as drug screening, screening is logical
Cross the expression for improving miR-17, miR-20a or miR-92a or activity and alleviate or treat the medicine of osteoclasia.
Therefore, above-mentioned miR-17, miR-20a and miR-92a or their upper adjustment, which can be applied to prepare, suppresses osteoclastic thin
Born of the same parents break up and the medicine of osteoclasia.It can also be used for screening prevention, alleviate or treat the potential material of osteoclasia, and to obtaining
Potential material carry out further cell experiment and/or animal experiment, further to select and determine from candidate substances pair
In the material prevented or treatment osteoclasia disease is useful.
The upper adjustment refers to any activity for improving microRNA or its precursor, raising microRNA or its precursor
Stability, up-regulation microRNA or its precursor expression, increase microRNA or the material of its precursor effective acting time, these
Material is used equally for the present invention, as the material useful for raising miR-17, miR-20a or miR-92a, so as to for delaying
Solution or treatment osteoclasia.Such as：MiR-17, miR-20a or miR-92a analogies (mimics) or activator (Agomir),
It has with miR-17, miR-20a or miR-92a identical effect, or can improve miR-17, miR-20a or
MiR-92a expression or activity.
Ripe osteoclast is pasted on bone tissue surface, secreting acidic phosphatase, can corrode sclerotin.And osteoclast
Excessive activation is the first cause of destruction of bone.MiR-17, miR-20a or miR-92a can suppress the differentiation of osteoclast, suppress
Important energy metabolism related genes in osteoclast, with miR-17, miR-20a or miR- in osteoclast atomization
92a expression is lowered.
In order to which above and other purpose, feature and the advantage of the present invention can be become apparent, number cited below particularly is implemented
Example, to illustrate the microRNA of suppression osteoclast differentiation of the present invention and its application.
Osteoclast induction differentiation
RAW264.7cells (American Type Culture Collection, ATCC) is containing 10% hyclone
(FBS) culture amplification in improvement Eagle culture mediums (DMEM).
RAW264.7 cells induce to osteoclast to be broken up：Cultivated 2 days in the induction liquid containing RANKL (50ng/ml), can
To obtain the positive osteoclastic precursors (preOC) of TRAP, it is cultivated for 4 days, obtains the mature osteoclast of multinuclear
(mOC).Change liquid once within every 2 days.
Cell is dyed with leucocyte acid phosphatase kit (Sigma), apocyte positive TRAP is
Ripe osteoclast.
Ripe miRNA quantitative analyses
1. the stem ring primer of reverse transcription is designed according to ripe miRNA sequence, and quantification PCR primer (as shown in table 1).
2. stem ring primer is diluted with water to 1 μM of final concentration.
3. reverse transcription reaction：
4.PCR, U6 are internal reference.
Table 1, RT primers and Real-time PCR primer sequences
Osteoclastic related gene PCR primer
Cell transfecting
1. the day before transfection, cell culture fluid changes the nutrient solution of antibiotic-free into, by taking 24 orifice plates as an example, per hole 400ul.
2. by 25pmol siRNA (or 25pmol miR mimic or 50pmol miR inhibitor) and 50ul without
Serum free culture system liquid is well mixed.MiR mimics are double-stranded RNAs, and miR inhibitors are single stranded RNAs, and universal NC is cel-
MiR-67 sequence, and confirm that there is minimum similitude with the miRNA sequence of people, mouse.
3. 1ul lipofectamine 2000 are dissolved in 50ul serum-free mediums, it is gently mixed uniformly, room temperature is put
Put 5min.
4. two pipe liquid are well mixed, room temperature places 25min.
5. aforesaid liquid is added in cell culture fluid, and it is well mixed.
6. changing serum-free medium after 7h into, handled after staying overnight with different cytokines.
MiRNA analogies, activator and mortifier
MiRNA analogies micrONTMMiRNA mimic and micrONTMIt is sharp rich that miRNA agomir are purchased from Guangzhou
Bio tech ltd.
MiRNA mimic are directed to ripe miRNA sequence particular design, and the miRNA prepared by chemical synthesis process is simulated
Thing, ripe miRNA expression can be significantly improved.
MiRNA agomir specificity is directed to ripe miRNA sequence, is prepared by chemical synthesis process, and uses sharp rich life
The ripe miRNA double-stranded RNAs of the special stable sex modification of thing, stability is higher, can dramatically increase cell infiltration rate, improve it
Anti- RNase activity, is appropriate for miRNA zooperies.
micrONTM miRNAMimic Negative Control and micrONTM miRNA agomir Negative
That Control is selected is C.elegans two miRNA, by bioinformatic analysis show its with people, mouse, rat
In genome, and miRBase databases all miRNA have minimum homology, are suitable as people, mouse, rat
MiRNA tests negative control.
MiR-17 is with SEQ ID NO:The micro ribonucleic acid of nucleotide sequence shown in 1：
5’-CAAAGUGCUUACAGUGCAGGUAG-3’。
micrONTMmiRNA mimic(miR-17mimic)：Positive CAAAGUGCUUACAGUGC AGGUAG；Reversely
ACCUGCACUGUAAGCACUUUGUU。
micrONTMmiRNA agomir(Agomir-17)：Positive CAAAGUGCUUACA GUGCAGGUAG；Reversely
ACCUGCACUGUAAGCACUUUGUU.Modified in its antisense strand, 3 ' end cholesterol modifications, full methoxy modification, 3 ' two, ends
Thio-modification, 5 ' 4 thio-modifications in end.
micrONTM miRNAmimic Negative Control：Positive UUCUCCGAACGUGUC ACGUdTdT；Instead
To ACGUGACACGUUCGGAGAAdTdT.
micrONTM miRNAagomir Negative Control：Positive UUCUCCGAACGUGU CACGUdTdT；Instead
To ACGUGACACGUUCGGAGAAdTdT.
miR-17inhibitor：Positive CUACCUGCACUGUAAGCACUUUG.
miR inhibitor NC：UCUACUCUUUCUAGGAGGUUGUGA.
micrONTMmiRNA mimic(miR-20a mimic)：Positive UAAAGUGCUUAUAGUGCAGGUAG；Reversely
ACCUGCACUAUAAGCACUUUAUU
miR-20a inhibitor：Positive CUACCUGCACUAUAAGCACUUUA.
micrONTMmiRNA mimic(miR-92a mimic)：Positive UAUUGCACUUGUCCCGGCCUG；Reversely
GGCCGGGACAAGUGCAAUAUU
miR-92a inhibitor：Positive CAGGCCGGGACAAGUGCAAUA.
MiR-17~92cluster miRNA expression change in osteoclast atomization
MiR-17~92cluster miRNA (including miR-17, miR-20a, miR- in osteoclast atomization
92a, miR-18a and miR-19a) expression change as shown in figure 1, RAW264.7 cell lines in luring containing RANKL (50ng/ml)
Cultivated 2 days in drain, osteoclastic precursor (preOC) can be obtained, be cultivated for 4 days, obtain the osteoclast of maturation
(mOC), in quantitative PCR detection cell miRNA expression.Compared with RAW264.7 cells, * P<0.05, * * P<0.01
(student’s t test)。
QPCR detections RAW264.7 cell lines are distinguished after being induced in the nutrient solution containing RANKL (50ng/ml) 2 days and 6 days
Obtain osteoclastic precursor and mature osteoclast, osteoclastic precursor and mature osteoclast compared with RAW264.7 cells,
MiR-17, mir-20a and miR-92a expression are significantly lowered, and mir-18a and miR-19a expression have no significant change.
It is overexpressed the differentiation that miRNA inhibits osteoclast
Fig. 2 is to be overexpressed miRNA (including miR-17, miR-20a, miR-92a) to suppress the signal of osteoclast differentiation
Figure.After RAW264.7 cell transfectings miRNA, cultivated in the induction liquid containing RANKL (50ng/ml).Quantitative PCR detection is each osteoclastic
The expression of related gene.Compared with control group, * P<0.05(student’s t test).
In order to prove the effect of miRNA that these lower played in osteoclast differentiation, applicant is thin in cartilage precursor
These miRNA are overexpressed in born of the same parents, then carry out induction differentiation.Quantitative PCR result display be overexpressed miR-17, mir-20a or
MiR-92a can significantly inhibit the expression (Fig. 2) of multiple osteoclast specific genes.
Because these miRNA may suppress the differentiation of osteoclast or function by acting on different mRNA,
Applicant is combined to observe the inhibition that osteoclast breaks up using what different miRNA were overexpressed.As a result show miR-17 and
There was no significant difference with the effect of single overexpression for the effect of mir-20a joint overexpression suppression osteoclast differentiation, prompts
MiR-17 and mir-20a may have identical action target spot.And miR-17 combine with miR-92a overexpression or mir-20a with
MiR-92a joints, which are overexpressed, then has stronger inhibition, illustrates different from miR-17, miR-92a, miR-92a may have
Other action target spots (Fig. 3).
Fig. 3 is that joint is overexpressed miRNA (including miR-17, miR-20a, miR-92a) to suppress osteoclast differentiation
Schematic diagram.After RAW264.7 cell transfectings miRNA (each miRNA concentration is 1pM), in the induction liquid containing RANKL (50ng/ml)
Middle culture.The positive cell numbers of TRAP are counted after dyeing.*P<0.05(student’s t test).
Identify the action target spot of miRNA in osteoclast differentiation
Osteoclast needs high-energy to maintain and contains abundant mitochondria.Ppargc1b coding peroxidases body is bred
Activated receptor γ (PPAR- γ or PPARG) helper activity factor -1 β (PPARG coactivator 1 β, PGC-1 β), PGC-
1 β up-regulated expressions during bone marrow macrophage (BMM) changes to osteoclastic precursor, and to osteoclast, end is divided eventually
Change plays an important roll.CAMP response element binding proteins (CREB) are attached to Ppargc1b promoter region so as to activate
PGC-1 β expression.Ppargc1b missing can suppress osteoclast differentiation and the biosynthesis of mitochondria, while can cause small
The increase of mouse bone amount.Fig. 4 is the binding site schematic diagram that miR-17 is applied on Ppargc1b.Bioinformatics software is predicted
Ppargc1b is miR-17 target gene (Fig. 4).Fig. 5 is interference miRNA (including miR-17, miR-20a, miR-92a) expression
To observe osteoclast important gene PGC-1 β mRNA expression change schematic diagram.RAW264.7 cell transfecting miRNA mimic
Or after miRNA inhibitor, cultivated in the induction liquid containing RANKL (50ng/ml).Quantitative PCR detection PGC-1 β mRNA's
Expression.Compared with miRNA mimic or miRNA inhibitor, * P<0.05(student’s t test).
By disturbing miRNA expression, it is found that Ppargc1b expression can be significantly inhibited by being overexpressed miR-17 and mir-20a.
The experimental method of other unreceipted actual conditionses in embodiment, generally according to normal condition such as J. Pehanorm Brookers
Etc. writing, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or built according to manufacturer
The condition of view.
Drug screening
Mouse cartilage cell is taken, the cell can endogenous expression miR-17, miR-20a or miR-92a.This kind of cell is made
For the cell model for the medicine for screening prevention, alleviating or treating osteoclasia.
Test group：The culture of the above-mentioned cell handled with candidate substances；
Control group：The culture of the above-mentioned cell handled without candidate substances.
Appropriate time after treatment, determine miR-17, miR-20a or miR-92a of the cell expression.If with it is right
Compared according to group, the expression of miR-17, miR-20a or miR-92a in test group significantly improve 20% or more than 50%, then illustrate
The candidate substances are potential preventions, alleviate or treat the material of osteoclasia.
With miR-17mimic, Agomir-17, miR-17inhibitor, miR-20a mimic or miR-92a mimic
Respectively as candidate substances, above-mentioned cell is transfected.As a result find that Agomir-17 can improve the expression of the miR-17 in test group
More than 80%, miR-20a mimic can make the expression of the miR-20a in test group improve more than 50%, miR-92a mimic can
The expression of the miR-92a in test group is set to improve more than 50%, therefore, Agomir-17, miR-20a mimic or miR-92a
Mimic can be as useful drug candidate.
Technical scheme in above-mentioned the embodiment of the present application, at least has the following technical effect that or advantage：
The invention firstly discloses differentiation of miR-17, miR-20a, miR-92a up-regulation for osteoclast to have suppression
Make and use, therefore, miR-17, miR-20a, miR-92a can suppress osteoclastic thin as the new therapy target of regulation Bone m etabolism
Important energy metabolism related genes in born of the same parents.
Although preferred embodiments of the present invention have been described, but those skilled in the art once know basic creation
Property concept, then can make other change and modification to these embodiments.So appended claims be intended to be construed to include it is excellent
Select embodiment and fall into having altered and changing for the scope of the invention.Obviously, those skilled in the art can be to the present invention
Carry out various changes and modification without departing from the spirit and scope of the present invention.So, if these modifications and variations of the present invention
Belong within the scope of the claims in the present invention and its equivalent technologies, then the present invention is also intended to exist comprising these changes and modification
It is interior.
Sequence table
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<120>A kind of microRNA application
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<213> Homo Spapiens
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caaagugcuu acagugcagg uag 23
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uaaagugcuu auagugcagg uag 23
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Claims (10)
- Purposes of the 1.microRNA in the medicine for suppressing osteoclast differentiation and osteoclasia is prepared, it is characterised in that：It is described MicroRNA is selected from miR-17, miR-20a and miR-92a, the sequence such as SEQ ID NO of the miR-17:It is described shown in 1 MiR-20a sequence such as SEQ ID NO:Shown in 2；The sequence of the miR-92a such as SEQ ID NO:Shown in 3.
- 2. microRNA as claimed in claim 1 upper adjustment suppresses the medicine of osteoclast differentiation and osteoclasia preparing In purposes.
- 3. microRNA according to claim 2 upper adjustment suppresses the medicine of osteoclast differentiation and osteoclasia preparing Purposes in thing, it is characterised in that：Upper adjust is the upper adjustment through modification, and described modification includes：Methoxylation modification, Thio-modification, cholesterol modification, alkyl modified, lock nucleic acid modification, peptide nucleic acid modification, and/or phosphate backbones connect generation by phosphatide The GEM 132 replaced.
- 4. microRNA according to claim 3 upper adjustment suppresses the medicine of osteoclast differentiation and osteoclasia preparing Purposes in thing, it is characterised in that：Described modification includes：3 ' ends carry out cholesterol modification, and 5 ' hold two thio backbone modifications, 3 ' the four thio backbone modifications in end, the modification of full chain methoxyl group.
- Purposes of the 5.microRNA in screening prevention, the potential material alleviated or treat osteoclasia, it is characterised in that：It is described MicroRNA is selected from miR-17, miR-20a and miR-92a, the sequence such as SEQ ID NO of the miR-17:It is described shown in 1 MiR-20a sequence such as SEQ ID NO:Shown in 2；The sequence of the miR-92a such as SEQ ID NO:Shown in 3.
- 6. use of the microRNA according to claim 5 in screening prevention, the potential material alleviated or treat osteoclasia On the way, it is characterised in that：The method of the screening includes：(1) expression miR-17, miR-20a or miR-92a system is handled with candidate substances；With(2) expression of miR-17, miR-20a or miR-92a in the system are detected；Wherein, if the candidate substances improve miR-17, miR-20a or miR-92a expression, it is pre- to show candidate substances Potential material that is anti-, alleviating or treat osteoclasia.
- 7. use of the microRNA according to claim 6 in screening prevention, the potential material alleviated or treat osteoclasia On the way, it is characterised in that：The raising includes：Improve 20-90%.
- 8. use of the microRNA according to claim 6 in screening prevention, the potential material alleviated or treat osteoclasia On the way, it is characterised in that：The candidate substances include：Based on miR-17, miR-20a, miR-92a or regulation and control miR-17, miR- The gene and albumen of 20a, miR-92a expression, and the macromolecular or small molecule designed.
- 9. use of the microRNA according to claim 8 in screening prevention, the potential material alleviated or treat osteoclasia On the way, it is characterised in that：The macromolecular is selected from peptide and over-express vector, and the small molecule includes compound.
- 10. use of the microRNA according to claim 6 in screening prevention, the potential material alleviated or treat osteoclasia On the way, it is characterised in that：The system is selected from：Cell system or cell culture system, subcellular fraction system, solution system, tissue System, organ systems or animal system.
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