CN104031987A - Application of miRNA to treat myocardial fibrosis diseases - Google Patents
Application of miRNA to treat myocardial fibrosis diseases Download PDFInfo
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Abstract
The invention aims at providing application of miRNA to treat or diagnose myocardial fibrosis diseases. The sequence of miRNA is shown as SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 or SEQ ID 6. By applying miRNA to treat myocardial fibrosis diseases, substantial effect is obtained especially when different human derived miRNA-19b sequences and non-human derived miRNA-19b sequences are mixed for application, namely a human derived miRNA-19b sequence of SEQ ID 1 or SEQ ID 2 and one or more of the non-human derived miRNA-19b sequences of SEQ ID 3, SEQ ID 4, SEQ ID 5 and SEQ ID 6 are mixed.
Description
Technical field
The invention belongs to biomedical professional domain, relate to precursor RNA, miRNA Antisensedigonucleotsequence sequence and the expression vector thereof of miRNA, miRNA, and the application in diagnosis and treatment myocardial fibrosis disease.
Background technology
Cardiovascular disorder is the serious disease of harm humans health, is murderous one of the main reasons.At present, in China resident's death notation, cardiovascular disorder has become No. second killer who is only second to malignant tumour, and separately has ten hundreds of people to cause deformity because suffering from this disease every year.This disease is of a great variety, the cause of disease is complicated.Wherein, by, narrow the caused cardiac muscle fibre persistence of coronary atherosclerotic of severe and/or the hypoxic-ischemic repeatedly increasing the weight of will cause myocardial fibrosis disease.Early stage in this disease, left ventricular diastolic function is impaired.Develop as one pleases and will lose because Ventricular Remodeling causes myocardium normal configuration, finally make ventricular systolic function also impaired, thereby cause cardiac insufficiency.In myocardial fibrosis disease progression process, in myocardial tissue structure, in collegen filament excess accumulation, heart tissue, collagen concentration significantly raises or collagen composition changes.This pathological change exists in multiple cardiovascular disorder, now thinks even closely related [the Jessup M and Brozena S:Heart fai lure.N Engl J Med2003 of sudden cardiac death of myocardial fibrosis and irregular pulse, cardiac dysfunction; 348:2007-2018; Swynghedauw B:Molecular mechanisms of myocardial remodeling.Physiol Rev1999; 79:215-262.].
The accumulation of extracellular matrix protein (ECM) in cardiac muscular tissue is the principal character of myocardial fibrosis, and Connective Tissue Growth Factor (CTGF) has been confirmed as powerful inducible factor [Koitabashi N, the et al:Increased connective tissue growth factor relative to brain natriuretic peptide as a determinant of myocardial fibrosis.[J] Hypertension2007 of ECM; 49:1120-1127; Tsoutsman T, et al:CCN2plays a key role in extracellular matrix gene expression in severe hypertrophic cardiomyopathy and heart failure.[J] J Mol Cell Cardiol.2013; 62C:164-178; Chen MM, et al:CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes:a potentiai role in heart fibrosis.[J] J Mol Cell Cardiol2000; 32:1805-1819.].Multinomial result of study shows, in heart failure progression, CTGF has played an active part in the myocardial fibrosis process of Angiotensin II (Ang II) induction.Therefore, in the multiple cardiovascular disorder that the expression of CTGF causes in myocardial fibrosis, rise and vital role [Finckenberg P, et al:Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways.[J] Am J Pathol2003; 163:355-366; Ahmed MS, et al:Connective tissue growth factor--a novel mediator of angiotens in II-stimulated cardiac fibroblast activation in heart failure in rats.[J] J Mol Cell Cardiol2004; 36:393-404; Sopel MJ, et al:Treatment with activated protein C (aPC) is protective during the development of myocardial fibrosis:an angiotensin II infusion model in mice.[J] PloS one2012; 7:e45663; Matsui Y and Sadoshima J:Rapid upregulation of CTGF in cardiac myocytes by hypertrophic stimuli:implication for cardiac fibrosis and hypertrophy.[J] J Mol Cell Cardiol2004; 37:477-481; He Z, et al:Differentiai regulation of angiotensin II-induced expression of connective tissue growth factor by protein kinase C isoforms in the myocardium.[J] J Biol Chem2005; 280:15719-15726.].
Cardiovascular disease incidence rate is high, disability rate is high, mortality ratio is high, recurrence rate is high, complication is many, the serious threat mankind's life and health.Therefore, countries in the world are all attached great importance to the research of cardiovascular and cerebrovascular diseases, prevention and treatment.In the therapeutic process of cardiac fibrosis disease, how to effectively reduce CTGF expression amount in myocardial tissue structure, and then reduce accumulation in ECM cardiac muscular tissue, reduce collagen concentration in heart tissue, will become the key of the multiple cardiovascular disorder that treatment cardiac fibrosis causes.
Microrna (miRNA or microRNA) is that a kind of length is the little RNA of endogenous non-coding of 18～25 nucleotide single-chains, is to be generated under the processing of enzyme by precursor, and the sequence of precursor is generally 70～120 Nucleotide.By interacting and express in transcriptional level regulatory gene with target-gene sequence specificity, participate in regulate several biological processes, evolutionary process is conservative.The initial endogenous miRNA finding is lin24 and let27, and they participate in genetic expression and transcribe rear adjusting, by 3 ' the end non-coding region with said target mrna or coding region (3 '-UTR), is combined and is brought into play negativity regulating effect.Identified in recent years and surpassed 400 kinds of miRNA, but still have a variety of [the Berezikov E that are not yet found, Thuemmler F, van Laake LW, et al.Diversity of microRNAs in human and chimpanzee brain[J] .Nat Genet, 2006,38 (12): 1375-1377.].Systematic Analysis to miRNA space expression shows, a lot of miRNAs express [Wienholds E in tissue specificity mode, KloostermanWP, Miska E, et al.MicroRNA expression in zebrafish embryonic development[J] .Science, 2005,309 (5732): 310-311.].Each miRNA can be positioned a variety of mRNA, it is transcribed suppresses or degraded, therefore a variety of transcriptons may be subject to the meticulous adjusting of miRNA, make to transcribe-translation system effectively turns round, and then cell is able to control fast and effectively the dependent cell event of threshold value.The function that generally believes at present miRNA is some primary processes of participating in life as grown, organ formation, hematopoiesis, Cell apoptosis and proliferation, stress reaction and tumour generation etc.
Initial scientists finds that the canceration of miRNA and cell has extremely close relation, and the miRNA that tumor suppressor gene is worked declines or disappearance can be led oncogenic generation.Found miRNA and the kinds cancer such as lung cancer, mammary cancer, colorectal carcinoma, chronic lymphocytic leukemia closely related.The year ends 2006, scientist starts vital role [the Van Rooij E that notices that miRNA is risen in heart disease generation evolution, Sutherland LB, L iu N, et a1.A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure[J] .PNAS, 2006,103:18255-18260.], and in the research of the diseases such as irregular pulse, heart failure, obtained a series of impressive progresses, make miRNA become the focus in international cardiovascular research field.
The discovery of the relevant miRNA of cardiovascular disorder may produce great impact to the genes involved treatment of this disease.By the miRNA Differential expression analysis to cardiovascular patient and normal population, can identify the responsive miRNA that obtains being correlated with, and then by introducing the antisense nucleotide inhibition of specific miRNA complementation or crossing the method for expression by virus vector or plasmid vector, regulate and control the expression amount of relevant miRNA or its precursor, thereby reach the object of Cardiovarscular.Therefore, find that specific expressed miRNA has very important significance for diagnosis and the treatment of cardiovascular disorder.
Summary of the invention
The object of the invention is to find by experiment in vitro a kind of new purposes of miRNA-19b.
The invention provides miRNA-19b as the pharmacological agent application of preparation treatment myocardial fibrosis disease.
Specifically, human miRNAs-19b provided by the present invention (has-miR-19b) and mouse miRNAs-19b (mmu-miR-19b and rno-miR-19b) all comprise following sequence:
5’-UGUGCAAAUCCAUGCAAAACUGA-3’(SEQ?ID7)。
The precursor RNA of human miRNAs-19b provided by the present invention has 2 kinds, comprises following sequence:
hsa-mir-19b-1(MI0000074)：
5’-CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG-3’(SEQ?ID1)
hsa-mir-19b-2(MI0000075)：
5’-ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGUAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAUAAUGU-3’(SEQ?ID?2)
The precursor RNA of mouse mouse miRNAs-19b provided by the present invention has 2 kinds, comprises following sequence:
mmu-mir-19b-1(MI0000718)：
5’-CACUGGUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUAUAAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUGGUG-3’(SEQ?ID3)
mmu-mir-19b-2(MI0000546)
5’-ACUUACGAUUAGUUUUGCAGAUUUGCAGUUCAGCGUAUAUGUGAAUAUAUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGGGA-3’(SEQ?ID4)
The precursor RNA of rat miRNAs-19b provided by the present invention has 2 kinds, comprises following sequence:
rno-mir-19b-1(MI0000847)
5’-CACUGGUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUAUAAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUGGUG-3’(SEQ?ID5)
rno-mir-19b-2(MI0000848)
5’-ACAUUGCUACUUACGGUUAGUUUUGCAGAUUUGCAGUUCAGCGUAUAUGUGGAUAUAUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAUGAUGU-3’(SEQ?I?D6)
Human miRNAs-19b provided by the present invention (has-miRNA-19b), the antisense oligonucleotide of mouse miRNAs-19b (mmu-miR-19b and rno-miR-19b) all comprises following sequence:
5’-UCAGUUUUGCAUGGAUUUGCACA-3’(SEQ?ID8)
The precursor hsa-mir-19b-1 of human miRNAs-19b provided by the present invention is positioned 13q31.3 on human chromosome, and precursor hsa-mir-19b-2 is positioned human chromosome Xq26.2.
The present invention be take the myocardial cell of newborn SD rat and is carried out the clonal analysis of miRNA molecule as material.Adopt conventional Protocols in Molecular Biology, from cell and tissue, extract total RNA, through the primer of property specially, carry out reverse transcription and pcr amplification.Fluorescence real-time quantitative PCR (qRT-PCR) detected result shows, miRNA-19b its expression amount under the myocardial fibrosis pathological state of Angiotensin induction obviously reduces; Use the synthetic miRNA-19b precursor RNA of specificity to increase the expression of miRNA-19b in cell, can effectively reduce Connective Tissue Growth Factor (CTGF) expression amount in myocardial tissue structure.
The patient who suffers from myocardial fibrosis disease, because CTGF expression amount in myocardial tissue structure increases, in heart tissue, collagen concentration content is high.Early stage in this disease, left ventricular diastolic function is impaired.Develop as one pleases and will finally make ventricular systolic function also impaired, thereby cause cardiac insufficiency.Under the myocardial fibrosis pathological state of Angiotensin induction, the expression amount of miRNA-19b in myocardial cell obviously reduces, can be using it as the indication of diagnosing myocardial fibrosis with this distinctive performance; Because miRNA-19b has obvious regulating effect for the expression amount of CTGF in myocardial tissue structure, therefore can use virus vector or plasmid vector to cross the method for expression, miRNA-19b expression amount in patient myocardial cell is increased, can effectively suppress the expression of CTGF in myocardial cell, and then collagen concentration in reduction heart tissue, this will effectively delay the pathology process of myocardial fibrosis, controls the development of the myocardial fibrosis state of an illness, thereby reaches therapeutic purpose.The discovery of this function of miRNA-19b has extremely important meaning for diagnosis and the treatment of the cardiovascular disordeies such as myocardial fibrosis.
The present invention is by human miRNAs-19b sequence SEQ ID1 and SEQ ID2, or non-human miRNAs-19b sequence SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6, independent or mixing makes an addition in the myocardial cell's nutrient solution that contains Angiotensin (AngII), sequence of the present invention can significantly suppress the expression amount of Connective Tissue Growth Factor (CTGF) in myocardial cell, especially will preferably different people miRNAs-19b sequence be mixed to application with non-human miRNAs-19b, obtain especially surprising effect.
According to the present invention, the application of a kind of miRNA in treatment or diagnosis myocardial fibrosis disease is provided, it is characterized in that, described miRNA is miRNA-19b.
The application of a kind of miRNA as above in treatment or diagnosis myocardial fibrosis disease, preferred described miRNA derives from people, mouse or rat.
The application of a kind of miRNA as above in treatment or diagnosis myocardial fibrosis disease, preferred described miRNA sequence is SEQID1, SEQ ID2, SEQ ID3, SEQ ID4, SEQ ID5 or SEQ ID6.
The application of a kind of miRNA as above in treatment or diagnosis myocardial fibrosis disease, preferably different people miRNAs-19b sequence is mixed to application with non-human miRNAs-19b, be about to human miRNAs-19b sequence SEQ ID1 or SEQID2, mix with one or more in non-human miRNAs-19b sequence SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6.
The application of a kind of miRNA as above in treatment or diagnosis myocardial fibrosis disease, preferably in described mixing miRNA-19b sequence nucleotide sequence, described human miRNAs-19b sequence SEQ ID1 or SEQID2 containing proportional be 10%～90%.
The application of a kind of miRNA as above in treatment or diagnosis myocardial fibrosis disease, more preferably in described mixing miRNA-19b sequence nucleotide sequence, described human miRNAs-19b sequence SEQ ID1 or SEQ ID2 containing proportional be 30%～70%.
The present invention also provides a kind of pharmaceutical composition of preparing treatment or diagnosis myocardial fibrosis disease, it is characterized in that one or more in miRNA sequence SEQ ID1, SEQ ID2, SEQ ID3, SEQID4, SEQ ID5 and the SEQ ID6 that contains pharmaceutically acceptable carrier and significant quantity.
A kind of pharmaceutical composition of preparing treatment or diagnosis myocardial fibrosis disease, preferably by human miRNAs-19b sequence SEQ ID1 or SEQ ID2, mix with one or more in non-human miRNAs-19b sequence SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6.
The present invention also provides the probe for the treatment of or diagnosis myocardial fibrosis disease, and the sequence that it is characterized in that described probe is the complementary sequence of SEQ ID1, SEQ ID2, SEQ ID3, SEQ ID4, SEQ ID5 or SEQ ID6.
The present invention also provides the test kit of a kind for the treatment of or diagnosis myocardial fibrosis disease, it is characterized in that, it is characterized in that containing the probe that sequence is SEQ ID1, SEQ ID2, SEQ ID3, SEQ ID4, SEQ ID5 or SEQ ID6.
Accompanying drawing explanation
Fig. 1. in the Fibrotic process of Angiotensin (AngII) induction myocardial cell, the expression of miR-19b obviously reduces; Hypertensin inhibitor telmisartan (Telmisartan) can be blocked AngII effect, maintains miR-19b expression level.*: p<0.05, has compared statistical significance with control group
In the Fibrotic process of Fig. 2 .AngII induction myocardial cell, the mrna expression of CTGF obviously increases.AngII inhibitor Telmisartan can block AngII effect, maintains the mrna expression level of CTGF.A qRT-PCR detects the mrna expression amount of CTGF; B agarose gel electrophoresis detects the mrna expression amount of CTGF.*: p<0.05, has compared statistical significance with control group
In the Fibrotic process of Fig. 3 .AngII induction myocardial cell, the protein expression level of CTGF obviously increases.AngII inhibitor Telmisartan can block AngII effect, maintains the protein expression level of CTGF.A.Western-blot detects the protein expression of CTGF in myocardial cell; B. the quantitative Western-blot of gray analysis detects CTGF result.*: p<0.05, has compared statistical significance with control group
3 ' non-coding region (3 '-UTR) bindingsite assay of Fig. 4 .A.miR-19b and CTGF mRNA.B. transfection miR-19b precursor (pre-miR-19b) can obviously increase the expression level of miR-19b in myocardial cell.*: p<0.05, has compared statistical significance with control group
Fig. 5. transfection miR-19b precursor (pre-miR-19b) can obviously reduce the expression level of CTGF mRNA in myocardial cell.A.qRT-PCR detects the mrna expression amount of CTGF; B. agarose gel electrophoresis detects the mrna expression amount of CTGF.*: D<0.05, has compared statistical significance with control group
In Fig. 6 .AngII induction myocardial cell fibrotic processes, pre-miR-19b can effectively reduce the mrna expression level of CTGF in myocardial cell.*: p<0.05, has compared statistical significance with control group
In Fig. 7 .AngII induction myocardial cell fibrotic processes, pre-miR-19b can effectively reduce the protein expression level of CTGF.A.Western-blot detects the expressing quantity of the interior CTGF of myocardial cell under different states; B. the quantitative Western-biot of gray analysis detects CTGF expression of results.*: p<0.05, has compared statistical significance with control group
Embodiment
The present invention is further elaborated by the following examples.
RNA sequence disclosed in this invention all can obtain with method or other biological method of synthetic.
Embodiment 1
Neonatal cardiac myocytes is cultivated and the total RNA of myocardial cell and total protein extracting
L. former culture neonatal cardiac myocytes
1.1 rat hearts of getting newborn 1-3 days are placed in the Hanks balanced salt solution (HBSS, Invitrogen) of precooling.
The cardiac muscular tissue of 1.2 about 1/3 positions of separated ventricular lower extremities, is cut into small pieces it with operating scissors.
1.3Jiang fritter cardiac muscular tissue puts into 37 ℃ of digestion solutions that 5mL contains 75U/mL col lagenase II (Worthington) hatches after 20 minutes and collects Digestive system and add fresh digestion solution to continue digestion.This digestive process repeats 6 times.
After centrifugal 5 minutes of 1.4 cell 283g with containing 10% foetal calf serum, 1% antibiotic DMEM-F12 cell culture fluid (1: 1, Invitrogen) resuspended.
1.5 on cell cultures dish culturing cell 1 hour, the separated non-myocardial cell of method who uses differential velocity adherent.Do not have adherent cell to cultivate in the culturing bottle of pre-coated Gelatin (Invitrogen) with DMEM-F12 cell culture fluid.
1.6 myocardial cells are by adherent in the DMEM-F12 nutrient solution that contains 0.1% bovine serum albumin at this 48 hours.
2. cell total rna extracts (TRIzol method)
2.1. harvested cell 1-5 * 10
7, move in 1.5ml centrifuge tube, add lml Trizol, mix the standing 5min of room temperature.
2.2. add 0.2ml chloroform, vibrate 15 seconds, standing 2min.
2.3. centrifugal 15 minutes of 12000g in 4 ℃ of whizzers of precooling.Get supernatant.
2.4. add 0.5ml Virahol, liquid in pipe is mixed gently, the standing 10min of room temperature.
2.5. centrifugal 10 minutes of 12000g in 4 ℃ of whizzers of precooling.Abandon supernatant.
2.6. add 1ml75% ethanol, gently washing precipitation.In 4 ℃ of whizzers of precooling, 7500g is centrifugal 10 minutes, abandons supernatant.
2.7. dry, add appropriate DEPC H
2o dissolves (65 ℃ of dissolution 10-15min).Measure 0D260 and 0D280 value, tentatively conclude RNA quality.
2.8. total RNA extracts the DNA enzyme I processing adopting without RNA enzyme, the total RNA of QIAGEN RNeasy test kit purifying, and principle of operation and method are shown in test kit specification sheets in detail.
3. total protein of cell extracts (RIPA cracking process)
3.1. from attached cell culture plate, carefully suck nutrient solution.
3.2. the PBS of precooling cleans adherent cell 2 times, carefully sucks PBS.
3.3. configuration is containing the protein extraction agent RIPA lysate (adding 5 μ l proteinase inhibitor mixed solutions in 1ml lysate, 5 μ 1PMSF and 5 μ 1 inhibitors of phosphatases mixed solutions) of inhibitor.
3.4. the RIPA lysate containing inhibitor that adds precooling in Tissue Culture Plate, is placed in 5 minutes on ice.
3.5. with the cell of a precooling, scrape attached cell on culturing bottle wall is scraped off, transitional cell suspension, in centrifuge tube, shakes under ice bath and within 15 minutes, carries out cracking.
3.6. lysate 14000g in the whizzer of precooling, 4 ℃ centrifugal 15 minutes.Discard precipitation, supernatant liquor be transferred at once in new centrifuge tube, preserve stand-by.
Embodiment 2
MiR-19b precursor (pre-mir-19b) transfection myocardial cell and Angiotensin Ang II stimulate induction myocardial cell fibrosis
1.pre-mir-19b the extraction of total RNA and total protein after transfection myocardial cell
1.1 cells are inoculated in 24 well culture plates, every porocyte number 4 * 10
4.
1.2 mixing of the OPTI-MEM I nutrient solutions (Invitrogen) by the transfection reagent siPORT NeoFX (AM4510, Ambion) of 1ul and 25ul are positioned over incubated at room 10 minutes.
1.3 by OPTI-MEM I nutrient solution (Invitrogen) dilution for pre-mir-19b (AMl7100, Ambion), and both are positioned over incubated at room 10 minutes after mixing.Use non-functional miRNA sequence pre-miR
tM-Negative Control (AM17110, Ambion) processes cell (Control) as a control group.
1.4 mix pre-mir-19b and the transfection reagent having diluted, and are positioned over incubated at room and within 10 minutes, make it form transfection composite.
1.5 add Tissue Culture Plate by the nutrient solution that contains transfection composite, jointly hatch 48 hours with cell.
The total RNA of 1.6 collecting cell, concrete grammar is with reference to embodiment 1.
1.7 collecting cell total proteins, concrete grammar is with reference to embodiment 1.
2. Angiotensin Ang II stimulates induction myocardial cell fibrosis
2.1 cells are inoculated in 24 well culture plates, every porocyte number 4 * 10
4.
2.2 cells add after 30 minutes in the cell culture fluid that contains 100nM Angiotensin AngII (Sigma) and cultivate 48 hours with 10 μ m telmisartan (Telmisartan, Abcam) pre-treatment.Pretreated group and cellar culture liquid cultivation group cell are not as a control group.
2.3 the total RNA of collecting cell, concrete grammar is with reference to part 2 in embodiment 1.
2.4 collecting cell total proteins, concrete grammar is with reference to the 3rd part in embodiment 1.
Embodiment 3
Analyze the expression amount of the interior miRNA-19b of myocardial cell and CTGF
1.qRT-PCR detect the expression amount of the interior miRNA-19b of myocardial cell and CTGF mRNA.
1.1 using the total RNA of myocardial cell that collects as template, with miR-19b specific mirVana qRT-PCR primer (Ambion) amplification miR-19b gene.Use real-time TaqMan miRNA analyzing and testing test kit (ABI) to carry out the expression amount of miR-19b in quantitative PCR detection cell, use U6snRNA gene as the internal reference detecting.Principle of operation and method are shown in test kit specification sheets in detail.
1.2 using the total RNA of myocardial cell that collects as template, mRNA with the PCR primer amplification CTGF of CTGF gene specific, use real-time quantitative polymerase chain reaction (qRT-PCR) to detect CTGF mrna expression amount in cell, use GAPDH gene as detecting internal reference.The product of PCR carries out visual and quantitative analysis with 1% agarose gel electrophoresis.Principle of operation and method are shown in test kit specification sheets in detail.
2.Western-blott ing analyzes CTGF protein expression level in myocardial cell
2.1 carry out Western-biot detection CTGF expressing quantity to collect myocardial cell's total protein.About 30 μ g of equivalent for total protein 10% polyacrylamide gel (Bio-Rad) carry out 200 volts, after the electrophoresis of 2 hours, electricity goes to (Millipore) on pvdf membrane.
Under 2.25% bovine serum albumin room temperature condition, seal 1 hour.
2.3 add specific CTGF (dilution in 1: 500, Santa Cruz) and GAPDH (dilution in 1: 5000, a Sigma) anti-antibody, overnight incubation under 4 ℃ of conditions.
The PBS solution that 2.4 use contain 1% Tween-20 is washed 3 times, each 10 minutes.
2.5 add specificity to resist for two of primary antibodie, hatch 1 hour under room temperature condition.
The PBS solution that 2.6 use contain 1% Tween-20 is washed 3 times, each 10 minutes.
2.7 add ECL fluorography liquid reagent (Thermo), with the sensitization of x ray film, develop.
2.8 use ImageJ software to carry out the gray scale quantitative analysis of image strip.
Embodiment 4
MiRNA bindingsite assay
Utilization information biology TargetScan program (
http:// www.targetscan.org) 3 '-UTR binding site of analyses and prediction miR-19b and CTGF mRNA.
Embodiment 5
Statistical procedures
All data are with mean ± standard deviation
represent relatively with t check, with p<0.05, there is statistical significance between two samples.Each experiment at least repeats 3 times.
Experimental example
According to the experimental technique in embodiment 2, by alternative pre-mir-19b (AM17100 of the present invention, Ambion), add in cultivated myocardial cell experimental example as shown in table 1 and reference examples, measure respectively CTGF expressing quantity in myocardial cell, its result is as shown in table 1, wherein, each miR-19b sequence of the present invention is mixed with to the solution of 10ng/ml, at experimental example 7, to experimental example 46, the total amount of individual mixing miR-19b sequence solution is 10 μ 1.
The concrete effect that the present invention is obtained:
1. in the Fibrotic process of Angiotensin AngII induction myocardial cell, the expression of miR-19b obviously reduces; Hypertensin inhibitor telmisartan (Telmisartan) can partly be blocked AngII effect, maintains miR-19b expression level (Fig. 1).This results suggest: in myocardial fibrosis process, miR-19b expression amount will occur significantly to reduce; Block the expression level that extraneous paathogenic factor effect can effectively maintain miR-19b.The method that detects the expression amount of miR-19b in myocardial cell can be used as the pointer of diagnosis myocardial fibrosis disease.
2. in the Fibrotic process of Angiotensin AngII induction myocardial cell, the expression of CTGF is obviously increased; With Telmisartan blocking-up AngII effect, can maintain the expression level of CTGF.No matter CTGF is all to show relatively consistent variation tendency (Fig. 2 and Fig. 3) in mRNA level or protein level.This result shows: the expression of CTGF is increased with the generation of myocardial fibrosis disease and had dependency to a certain degree.
3. information biology result of study shows: the 3 ' non-coding region (3 '-UTR) of CTGF mRNA has specificity miR-19b inhibition binding site.Transfection miR-19b precursor (pre-miR-19b) can obviously increase the expression level of miR-19b in myocardial cell.(Fig. 4).This result shows: likely by the expression level of miR-19b in regulating cell, change the expression of CTGF.
4. by the expression level of miR-19b in transfection pre-miR-19b increase myocardial cell, can obviously reduce the expression level of CTGF in myocardial cell.(Fig. 5).
5. no matter be at mRNA level or protein level, the precursor pre-miR-19b of transfection miRNA-19b can reduce the expression level of CTGF in myocardial cell, effectively suppress myocardial cell's fibrosis of being induced by Angiotensin AngII, eliminate to a certain extent the impact of fibrosis on myocardial cell's function, reach the effect (Fig. 6 and Fig. 7) for the treatment of.
6. in the myocardial cell who cultivates under various different conditions, the expression amount of CTGF albumen is as shown in table 1, result shows that SEQ ID1 of the present invention, SEQ ID2, SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6 all can suppress CTGF protein expression, by human miRNAs-19b sequence SEQ ID1 or SEQ ID2, mix with the one (or more) in non-human miRNAs-19b sequence SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6, its inhibition is more obvious.
The expression amount (arbitrary unit) of CTGF albumen in each experimental example of table 1. and reference examples
Claims (10)
1. the application of miRNA in treatment or diagnosis myocardial fibrosis disease, is characterized in that, described miRNA is miRNA-19b.
2. application according to claim 1, is characterized in that,
Described miRNA derives from people, mouse or rat.
3. application according to claim 1, is characterized in that,
Described miRNA sequence is SEQ ID1, SEQ ID2, SEQ ID3, SEQID4, SEQ ID5 or SEQ ID 6.
4. application according to claim 3, is characterized in that,
Different people miRNAs-19b sequence is mixed to application with non-human miRNAs-19b,
By human miRNAs-19b sequence SEQ ID1 or SEQ ID2, mix with one or more in non-human miRNAs-19b sequence SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6.
5. application according to claim 4, wherein,
In described mixing miRNA-19b sequence nucleotide sequence, described human miRNAs-19b sequence SEQ ID1 or SEQ ID2 containing proportional be 10%～90%.
6. application according to claim 4, wherein,
In described mixing miRNA-19b sequence nucleotide sequence, described human miRNAs-19b sequence SEQ ID1 or SEQID2 containing proportional be 30%～70%.
7. prepare the pharmaceutical composition for the treatment of or diagnosing myocardial fibrosis disease for one kind, it is characterized in that one or more in miRNA sequence SEQ ID1, SEQ ID2, SEQ ID3, SEQ ID4, SEQ ID5 and the SEQ ID6 that contains pharmaceutically acceptable carrier and significant quantity.
8. pharmaceutical composition according to claim 7, it is characterized in that, by human miRNAs-19b sequence SEQ ID1 or SEQ ID2, mix with one or more in non-human miRNAs-19b sequence SEQ ID3, SEQ ID4, SEQ ID5 and SEQ ID6.
9. treat or diagnose a probe for myocardial fibrosis disease, the sequence that it is characterized in that described probe is the complementary sequence of SEQ ID1, SEQID2, SEQ ID3, SEQ ID4, SEQ ID5 or SEQ ID6.
10. a test kit for treatment or diagnosis myocardial fibrosis disease, is characterized in that containing probe as claimed in claim 9.
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