CN107881173A - A kind of small molecules of miRNA 21 and application thereof - Google Patents
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Abstract
The invention provides a kind of small molecules of miRNA 21, its nucleotide sequence such as SEQ ID NO:Shown in 1.Present invention also offers a kind of application of the above-mentioned small molecules of miRNA 21 in preparing prevention myocardial infarction Earlier period of inflammation or delaying the medicine of myocardial infarction Earlier period of inflammation disease.The miRNA 21 of the present invention can suppress the inflammatory reaction of myocardial infarction early stage, so as to play the effect of anti-inflammatory.The present invention can be used for preparing the medicine for suppressing myocardial infarction Earlier period of inflammation and suppressing myocardial infarction Earlier period of inflammation disease.
Description
Technical field
The invention belongs to biotechnology and medical domain, is related to a kind of RNA small molecules, specifically a kind of miRNA-
21 small molecules and application thereof.
Background technology
The ischemic heart disease such as myocardial infarction incidence of disease is high, and disability rate and the death rate are high, is endanger human health main
One of disease.According to statistics, for the kainogenesis patient of the annual myocardial infarction in China up to 500,000, existing patient is 2,500,000, wherein 1/2
Disability【Chinese cardiovascular disease report, Chen Weiwei etc. write, Encyclopedia of China Publishing House, 2014】.Cardiac muscle stalk
Dead patient intervenes the reperfusion as treatment such as (PCI) and thrombolysis even by direct coronary artery and given treatment to, and its later stage also tends to send out
Open up as chronic heart failure, this is the major complications of myocardial infarction, has a strong impact on its ability to act and quality of life【de
Carvalho LP. etc., Long-Term Prognosis and Risk Heterogeneity of Heart Failure
Complicating Acute Myocardial Infarction.Am J Cardiol.2015；115:872-878】.From the heart
The necrosis of myocardial ischemia apoptosis to the later stage heart failure, it is necessary to undergo multiple event procedure, its pathologic process is broadly divided into three
Period：Acute inflammation stage, repair phase and reconstruct phase【Reed GW. etc., Acute myocardial infarction.Lancet
2017；389:197-210】.The acute inflammation stage of myocardial infarction is mainly shown as that the apoptosis of infarcted region cardiac muscle is bad (within 7 days)
Extremely, the infiltration of a large amount of inflammatory cells and the secretion of inflammatory mediator.Initially enter within 5-7 days after heart infarction reparation phase, infarcted region necrosis
Tissue is gradually absorbed, and substrate degradation and granulation tissue are formed, collagen deposition and myocardial fibrosis and marginal zone angiogenesis.
Enter the reconstruct phase after heart infarction after several weeks, be mainly shown as that infarcted region scar is gradually stablized, the cardiac myocyte hypertrophy of non-infarcted region and
Myocardium reactive fibre, ventricular dilatation and heart function decline, and develop into bad cardiac remodeling, its final result is exactly heart failure.
In these pathologic processes, myocardial inflammation damage is vital Node Events, and excessive inflammatory reaction can promote marginal zone
The apoptosis of cardiac muscle cell, strengthen the degraded of extracellular matrix, cause cardiac rupture；Lasting inflammatory reaction can reduce collagen and sink
Product, causes the tensile strength of cicatricial tissue to weaken, so as to cause ventricular dilatation, therefore the response intensity imbalance of myocardial inflammation damage
The incidence of bad cardiac remodeling and heart failure can be significantly improved【Frangogiannis NG. etc., The inflammatory
response in myocardial injury,repair,and remodelling.Nat Rev Cardiol 2014；11:
255-265】。
Inflammation after MI is mainly the endogenous danger signal material discharged by necrotic myocardium tissue and cell
(DAMPs) activate the innate immunity system in inflammatory cell and trigger.Necrotic myocardium cell caused by myocardial infarction with
And extracellular matrix can discharge a variety of endogenous danger signal molecules, including HMGB1 (High Mobility Group Box-
1), heat shock protein (Heat Shock Proteins, HSPs) 60 and 70 and nucleic acid compositions etc., what they can be infiltrated
Immunoinflammatory cell, mainly macrophage and BMDC etc.), endothelial cell and cardiac muscle cell expression Toll-like by
Body (Toll-like receptors, TLRs) is identified【Timmers L. etc., The innate immune response in
reperfused myocardium.Cardiovasc Res.2012；94:276-283；Frantz S. etc., Cardiac
macrophages and their role in ischaemic heart disease.Cardiovasc Res.2014；
102:240-248】.Mainly including TLR2, TLR3, TLR4, TLR7 and TLR9 etc., they identify accordingly these Toll-like receptors
Endogenous a bad actor after, downstream signaling pathway is activated by MyD88 adaptor proteins, and then activate NF- κ B and MAPKs etc.
Signal, the expression of inflammation inducing cell factor, chemotactic factor (CF), adhesion molecule and growth factor, further raises inflammatory cell
It is local to damage, injury tissue and dead cell are removed, promotes angiogenesis etc., but also trigger local organization inflammation damnification simultaneously
【Ha T. etc., Toll-like receptors:new players in myocardial ischemia/reperfusion
injury.Antioxid Redox Signal.2011；15:1875-1893】.Therefore, the inflammation damnification organized after heart infarction is controlled
Intensity, for preventing bad cardiac remodeling and improving heart function, there is key effect【Arslan F. etc., Innate
immune signaling in cardiac ischemia.Nat Rev Cardiol.2011；8:292-300】.
MiRNAs is a kind of feature non-coding small molecule being made up of 21~23 nucleotides, can be by with reference to target gene
MRNA carrys out the expression of regulatory gene, so as to play biology regulatory function, including regulation innate immune response, cardiovascular disease
Occurrence and development of disease etc..The expression of disease related gene is adjusted by miRNAs, is current so as to reach the purpose of disease preventing and treating
The emphasis and focus of research work.Increasing evidence shows：MiRNAs is in regulation and control innate immune response and inflammatory reaction
Play key effect【Sheedy, FJ. etc., Adding fuel to fire: microRNAs as a new class of
mediators of inflammation.Ann Rheum Dis.2008；67suppl: iii50-iii55】.One is found recently
A little miRNAs, as miR-1, miR-133, miRNA-21, miR-320 and miR-199a participate in the occurrence and development of angiocardiopathy
Process【Belevych, AE. etc., MicroRNA-1 and-133 increase arrhythmogenesis in heart
failure by dissociating phosphatase activity from RyR2 complex.PLoS One.2011；
6:e28324；Han, M. etc., MicroRNAs in the cardiovascular system. Curr Opin
Cardiol.2011；26:181-189；Bauersachs, J. etc., miRNA-21:a central regulator of
fibrosis not only in the broken heart.Cardiovasc Res.2012；96:227-229】.miRNA-
21 are reported in the pathological change process such as myocardial fibrosis and cardiac muscle cell apoptosis and play important regulating and controlling effect.MiR-21
Promote ERK-MAP kinase activities by suppressing target molecule Spry1 expression, so as to promote the survival of cardiac fibroblast and growth
Cytokine secretion, so as to aggravate the degree of interstitial fibrosis and cardiomegaly in heart failure model mouse【Thum T. etc.,
MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase
signalling in fibroblasts.Nature.2008；456:980-984】.MiR-21 has to cardiac muscle cell apoptosis to be protected
Shield acts on, on the one hand, miRNA-21 can suppress FasL expression by PTEN/Akt signal paths, so as to suppress myocardial ischemia
The apoptosis of cardiac muscle cell after reperfusion injury；On the other hand, miRNA-21 lowers PDCD4 expression so as to suppressing H2O2Induction
Cardiomyocyte cell death and apoptosis【Tu Y. etc., Ischemic postconditioning-mediated miRNA-21
protects against cardiac ischemia/reperfusion injury via PTEN/Akt
pathway.PLoS One.2013；8:e75872；Sayed D. etc., MicroRNA-21 is a downstream
effector of AKT that mediates its antiapoptotic effects via suppression of
Fas ligand.J Biol Chem.2010；285:20281-20290；Cheng Y. etc., MicroRNA-21 protects
against the H(2)O(2)-induced injury on cardiac myocytes via its target gene
PDCD4.J Mol Cell Cardiol.2009；47:5-14】.But after so far, there is not been reported miRNA-21 is to heart infarction
The regulating and controlling effect of Earlier period of inflammation.
It is based on above research background, the present invention have studied miRNA-21 after mouse heart infarction in Earlier period of inflammation reaction
Regulating and controlling effect, it is found that miRNA-21 can suppress the inflammatory reaction of early stage after heart infarction, so as to play antiinflammatory action in heart infarction early stage.
The content of the invention
It is described the invention provides a kind of miRNA-21 and application thereof for above-mentioned technical problem of the prior art
This miRNA-21 and application thereof will solve treatment and prevention effect of the medicine of the prior art for Earlier period of inflammation after heart infarction
Bad technical problem.
The invention provides a kind of miRNA-21 small molecules, its nucleotide sequence such as SEQ ID NO:Shown in 1 (5 '-
UAGCUUAUCAGACUGAUGUUGA-3’)。
Present invention also offers a kind of above-mentioned miRNA-21 to prepare prevention myocardial infarction Earlier period of inflammation or delay cardiac muscle
Application in the medicine of infarct Earlier period of inflammation disease.
Present invention also offers a kind of pharmaceutical composition, contains above-mentioned miRNA-21 sequences.
Further, above-mentioned pharmaceutical composition, also containing pharmaceutically acceptable carrier or auxiliary material.
Present invention also offers a kind of above-mentioned pharmaceutical composition to prepare prevention myocardial infarction Earlier period of inflammation or delay the heart
Application in the medicine of flesh infarct Earlier period of inflammation disease.
The miRNA-21 of the present invention is expressed in the cardiac muscular tissue of myocardial infarction early stage heart infarction marginal zone to be dramatically increased；In addition,
The miRNA-21 of the present invention can suppress the inflammatory reaction of myocardial infarction early stage, so as to play the effect of anti-inflammatory.The present invention's
MiRNA-21 can be used for preparing the medicine for suppressing myocardial infarction Earlier period of inflammation and disease.
Brief description of the drawings
Fig. 1：(result is shown as flat for miRNA-21 expression in the cardiac muscular tissue of heart infarction marginal zone after murine myocardial infarction
Means standard deviation, n=5；* *, P<0.001).
Fig. 2：(result is shown for the generation of inflammatory cytokine in the cardiac muscular tissue of miRNA-21 analogies suppression heart infarction marginal zone
For mean+SD, n=5；*, P<0.05；*, P<0.01).
Fig. 3：Inflammatory is thin in the CD11b positive cells separated in the cardiac muscular tissue of miRNA-21 analogies suppression heart infarction marginal zone
(result is shown as mean+SD, n=5 for the generation of intracellular cytokine；*, P<0.05).
Fig. 4：The analogies for being overexpressed miRNA-21 suppress the production of the inflammatory cytokine that DAMPs is induced in mouse cell
It is raw.Wherein Fig. 4 A are that the analogies for being overexpressed miRNA-21 suppress the inflammatory cytokine that rmHMGB1 is induced in mouse cell
Produce；Fig. 4 B are overexpressed the generation of the inflammatory cytokine that rmHSP60 is induced in miRNA-21 analogies suppression mouse cell
(result is shown as mean+SD, n=6；*, P<0.01).
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al.《Molecular cloning：Lab guide》(New York：Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and
Number is calculated by weight.
Unless otherwise defined, anticipated known to all specialties used in text and scientific words and one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.Described in text
Preferable implementation only present a demonstration and be used with material.
Embodiment 1：After murine myocardial infarction in the cardiac muscular tissue of heart infarction marginal zone miRNA-21 up-regulated expression
Mouse (C57 strains, male, 8-10 week old, the western pul-Bi Kai experimental animals Co., Ltd purchase in Shanghai), according to
Literature procedure establishes myocardial infarction model【Tarnavski, O. etc., Mouse cardiac surgery:
comprehensive techniques for the generation of mouse models of human diseases
and their application for genomic studies.Physiol Genomics.2004；16；349-360】,
Sham-operation group mouse only threads when establishing myocardial infarction model and does not ligature descending anterior branch.Respectively 1 day after myocardial infarction, 2 days, 3
My god, take within 5 days, 7 days mouse heart infarction marginal zone cardiac muscular tissue.
Total tissue RNA is extracted using TRIzol (Invitrogen companies).Using Real Time RT-PCR (qRT-
PCR) expression of the method to miRNA-21 in the cardiac muscular tissue of heart infarction marginal zone is analyzed.QRT-PCR uses SYBR RT-
PCR kit (Takara companies) is simultaneously completed on LightCycler 480 (Roche companies) real-time PCR.
MiRNA-21RT primers are：5’-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG
GAT ACG ACT CAA CT-3’。
MiRNA-21 quantification PCR primers are 5 '-CGG CTA GCT TAT CAG ACT GA-3 ' (upstream) and 5 '-GTG
CAG GGT CCG AGG T-3 ' (downstream).
The reverse transcription reaction primer of internal reference U6 small nuclear rnas be 5 '-AAC GCT TCA CGA ATT TGC GT -3 ' (i.e.
For the anti-sense primer of U6 quantitative PCRs).
U6 quantification PCR primers are 5 '-CTC GCT TCG GCA GCA CA-3 ' (upstream) and 5 '-AAC GCT TCA CGA
ATT TGC GT-3 ' (downstream).
MiRNA relative quantification uses 2-ΔΔCtMethod calculates (U6 is internal reference)【Livak, KJ. etc., Analysis of
relative gene expression data using real-time quantitative PCR and the 2-ΔΔ
Ct method. Methods.2001；25：402-408】.
MiRNA-21 expression is as shown in Figure 1 in the cardiac muscular tissue of heart infarction marginal zone after murine myocardial infarction.As a result show
Show：MiRNA-21 expression significantly raises in the cardiac muscular tissue of heart infarction marginal zone after murine myocardial infarction, and 3 days after myocardial infarction arrive
Up to peak.
The result shows：In the cardiac muscular tissue of murine myocardial infarction marginal zone miRNA-21 be after myocardial infarction inflammation should
Answer gene.
Embodiment 2：MiRNA-21 analogies suppress the production of the inflammatory cytokine in the cardiac muscular tissue of mouse heart infarction marginal zone
It is raw
Mouse (C57 strains, male, 8-10 week old, the western pul-Bi Kai experimental animals Co., Ltd purchase in Shanghai), strictly according to the facts
Apply and murine myocardial infarction model is prepared described in example 1, the adenovirus that heart infarction marginal zone local injection carries miRNA-21 analogies carries
Body and control adenovirus vector (Shanghai and the synthesis of first Bioisystech Co., Ltd), respectively 3 days, 5 days, 7 days after myocardial infarction
Take the normal cardiac muscular tissue of area of mouse heart infarction and heart infarction marginal zone cardiac muscular tissue.
Total tissue RNA is extracted using TRIzol (Invitrogen companies).Using Real Time RT-PCR (qRT-
PCR) expression of the method to inflammatory cytokine in heart infarction cardiac muscular tissue is analyzed.QRT-PCR uses SYBR RT-PCR
Kit (Takara companies) is simultaneously completed on LightCycler 480 (Roche companies) real-time quantitative PCR instrument.
IL-1 β quantification PCR primers be 5 '-GGTGTGTGACGTTCCCATTAGAC-3 ' (upstream) and 5 '-
CATGGAGAATATCACTTGTTGGTTGA-3 ' (downstream).
IL-6 quantification PCR primers be 5 '-TAGTCCTTCCTACCCCAATTTCC-3 ' (upstream) and 5 '-
TTGGTCCTTAGCCACTCCTTC-3 ' (downstream).
TNF-α quantification PCR primer be 5 '-AAGCCTGTAGCCCACGTCGTA-3 ' (upstream) and 5 '-
GGCACCACTAGTTGGTTGTCTTTG-3 ' (downstream).
Internal reference GAPDH quantification PCR primers be 5 '-AGGTCGGTGTGAACGGATTTG-3 ' (upstream) and 5 '-
TGTAGACCATGTAGTTGAGGTCA-3 ' (downstream).
MRNA relative quantification uses 2-ΔΔCtMethod calculates (GAPDH is internal reference)【Livak, KJ. etc., Analysis of
relative gene expression data using real-time quantitative PCR and the 2-ΔΔ
Ct method. Methods.2001；25：402-408】.
MiRNA-21 analogies are as shown in Figure 2 to the production of the inflammatory cytokine in mouse heart infarction cardiac muscular tissue.
As a result show：MiRNA-21 analogies can suppress the generation of IL-1 β, IL-6, TNF-α in the cardiac muscular tissue of mouse heart infarction marginal zone.
The result shows：MiRNA-21 analogies can suppress inflammatory cytokine in the cardiac muscular tissue of mouse heart infarction marginal zone
Produce.
Embodiment 3：It is scorching in the CD11b positive cells separated in the cardiac muscular tissue of miRNA-21 analogies suppression heart infarction marginal zone
The generation of property cell factor
Mouse (C57 strains, male, 8-10 week old, the western pul-Bi Kai experimental animals Co., Ltd purchase in Shanghai), strictly according to the facts
Apply and murine myocardial infarction model is prepared described in example 1, the adenovirus that heart infarction marginal zone local injection carries miRNA-21 analogies carries
Body and control adenovirus vector (Shanghai and the synthesis of first Bioisystech Co., Ltd), are divided for 3 days after myocardial infarction with magnetic cell
Select the CD11b positive cells in the cardiac muscular tissue of method (Miltenyi Biotec companies) separation heart infarction marginal zone.
CD11b positive cells total serum IgE is extracted using TRIzol (Invitrogen companies).Using real-time quantitative reverse transcription
Expression of PCR (qRT-PCR) methods to inflammatory cytokine in the CD11b positive cells in the cardiac muscular tissue of heart infarction marginal zone is carried out
Analysis.QRT-PCR is using SYBR RT-PCR kits (Takara companies) and in LightCycler 480 (Roche companies)
Completed on real-time PCR.
IL-1 β quantification PCR primers be 5 '-GGTGTGTGACGTTCCCATTAGAC-3 ' (upstream) and 5 '-
CATGGAGAATATCACTTGTTGGTTGA-3 ' (downstream).
IL-6 quantification PCR primers be 5 '-TAGTCCTTCCTACCCCAATTTCC-3 ' (upstream) and 5 '-
TTGGTCCTTAGCCACTCCTTC-3 ' (downstream).
TNF-α quantification PCR primer be 5 '-AAGCCTGTAGCCCACGTCGTA-3 ' (upstream) and 5 '-
GGCACCACTAGTTGGTTGTCTTTG-3 ' (downstream).
Internal reference GAPDH quantification PCR primers be 5 '-AGGTCGGTGTGAACGGATTTG-3 ' (upstream) and 5 '-
TGTAGACCATGTAGTTGAGGTCA-3 ' (downstream).
MRNA relative quantification uses 2-ΔΔCtMethod calculates (GAPDH is internal reference)【Livak, KJ. etc., Analysis of
relative gene expression data using real-time quantitative PCR and the 2-ΔΔ
Ct method. Methods.2001；25：402-408】.
MiRNA-21 analogies to the inflammatory cell in the CD11b positive cells that are separated in the cardiac muscular tissue of heart infarction marginal zone because
The production of son is as shown in Figure 3.As a result show：MiRNA-21 analogies can suppress to divide in the cardiac muscular tissue of mouse heart infarction marginal zone
From CD11b positive cells in IL-1 β, IL-6, TNF-α generation.
The result shows：MiRNA-21 analogies can suppress the CD11b sun separated in the cardiac muscular tissue of mouse heart infarction marginal zone
The generation of inflammatory cytokine in property cell.
Embodiment 4：The analogies for being overexpressed miRNA-21 suppress the inflammatory cytokine of DAMPs inductions in macrophage
Produce
MiRNA-21 analogies (S:5’-UAGCUUAUCAGACUGAUGUUGA-3’；A: 5’-
UCAACAUCAGUCUGAUAAGCUA-3 ') synthesized by Guangzhou Rui Bo bio tech ltd, analogies control is sharp rich by Guangzhou
Bio tech ltd provides.Mouse (C57 strains, female, 4-6 week old, the western limited public affairs of pul-Bi Kai experimental animals in Shanghai
Department's purchase) intraperitoneal injection thioglycolate salt (Thioglycollate) (being purchased from Merck companies) 2ml/, two days later with serum-free
RPMI1640 culture mediums rinse abdominal cavity, and primary peritoneal macrophage is obtained according to literature procedure【Liu, X. etc.,
Intracellular MHC class II molecules promote TLR-triggered innate immune
responses by maintaining activation of the kinase Btk.Nature Immunology.
2011；12(5):416-424.】.Macrophage (5 × 105Individual cell/1ml RPMI1640 culture mediums) use INTERFERin
Transfection reagent (Polyplus-transfection companies) transfection miRNA-21 analogies and control (final concentration of 20nM)
Into macrophage.48 hours after transfection, cell recombinates rmHMGB1 with 0.1mg/ml or rmHSP60 is (public purchased from CUSABIO
Department), after stimulating 0,4,8 hour, cells and supernatant is collected using ELISA (used kit is purchased from R＆D companies) detection inflammatories
Cytokine protein levels.
The result of elisa assay is as shown in Fig. 4 A, B.As a result show：Mouse can be reduced by being overexpressed miRNA-21 analogies
IL-1 β, IL-6, TNF-α of rmHMGB1 and rmHSP60 inductions generation in macrophage.
The result shows：MiRNA-21 analogies can suppress the production of the inflammatory cytokine that DAMPs is induced in macrophage
It is raw.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Sequence table
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Claims (5)
1. a kind of miRNA-21 small molecules, its nucleotide sequence such as SEQ ID No:Shown in 1.
2. a kind of miRNA-21 described in claim 1 is preparing prevention myocardial infarction Earlier period of inflammation or is delaying myocardial infarction early stage
Application in the medicine of inflammation disease.
A kind of 3. pharmaceutical composition, it is characterised in that：Contain miRNA-21 sequences described in claim 1.
4. pharmaceutical composition according to claim 3, it is characterised in that：Also contain pharmaceutically acceptable carrier or auxiliary
Material.
5. a kind of pharmaceutical composition described in claim 3 is preparing prevention myocardial infarction Earlier period of inflammation or is delaying myocardial infarction early
Application in the medicine of phase inflammation disease.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110279708A (en) * | 2019-07-22 | 2019-09-27 | 上海交通大学医学院附属第九人民医院 | MiR-21 small molecule promotes the application in myocardial infarction regional vessel chemical drug object in preparation |
| CN110354275A (en) * | 2019-07-22 | 2019-10-22 | 上海交通大学医学院附属第九人民医院 | Application of the MSN/miRNA hydrogel in preparation treatment myocardial infarction drug |
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