CN104988216A - Serum miRNA relevant to chronic heart failure and application of serum miRNA - Google Patents
Serum miRNA relevant to chronic heart failure and application of serum miRNA Download PDFInfo
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Abstract
The invention provides serum miRNA relevant to a chronic heart failure and application of the serum miRNA. The invention provides application of peripheral blood miRNAs to the chronic heart failure as biomarker risk assessment/diagnosis and prognosis. Hsa-miR-4491, hsa-miR-1285-3p, hsa-miR-665, hsa-miR-660-3p, hsa-miR-206, hsa-miR-1268b, hsa-miR-130a-3p and hsa-miR-330-3p have differential expressions in peripheral blood of heart failure patients. It is proved that the peripheral blood hsa-miR-665 and the like can perform specific diagnosis on the chronic heart failure, the expression level of the hsa-miR-665 and the like is relevant to ejection fraction, and the hsa-miR-665 has an assessment and prognosis effect. Thus, by detecting the expression level of the miRNAs, the existing chronic heart failure can be predicted and coordinately diagnosed, or prognosis of the chronic heart failure can be estimated.
Description
Technical field
The present invention relates to the new medicine use of multiple endogenic non-coding tiny RNA, more specifically to the purposes of 8 kinds of microRNAs in the diagnosis and prognosis assessment of chronic heart failure disease, belong to the diagnosis of cardiovascular disorder, prevention and therapy field.
Background technology
Chronic heart failure is the clinical syndrome that different cardiovascular disordeies develops into the end stage eventually, main pathophysiological feature is ventricular filling and penetrates blood capability deteriorates, finally cause ventricular pump blood hypofunction, its prognosis mala, mortality ratio is high, threaten one of human health and main cause of disease of causing medical burden to increase, for particularly important the China that aging population increases the weight of day by day.Up to the present, the medicine based on diuretic(s), ACEI and beta-blockers combines invalid to some patients or effect is undesirable, and new remedy measures is urgently excavated; On the other hand, the Hazard Factor of chronic heart failure are not illustrated completely, therefore need to find new Hazard Factor and adopt more effective methods of risk assessment.
Microrna (microRNA, miRNA) is the newfound non-coding RNA in multiple biological processes with important regulating and controlling effect of a class, is the single stranded RNA s that the length deriving from endogenous hairpin structure transcript is approximately 22 Nucleotide.Usual miRNA, as guiding molecule, is combined with 3 ' UTR base complementrity of said target mrna, mediated gene post-transcriptional control.In most cases, after miRNA is combined with said target mrna, arrestin matter translated and activate Exonucleolytic enzyme liberating mRNA, if miRNA and the complementation of said target mrna height combine, can endonuclease activity be activated.In the pathophysiological process of complexity, miRNA or miRNAs bunch collaborative, and to participate in Multi-regulation be one of important biological property of miRNA; Also be one of theoretical basis of the application for the treatment of for miRNA.Compared with treating with traditional single target drug, miRNA treatment when single core thuja acid molecular target is treated, from multiple level modulation disease pathology physiological status, can reach more effective result for the treatment of.
There are some researches show that miRNAs can by emiocytosis to Peripheral Circulation, and stable existence, can be used as the new diagnosis marker of various diseases.And RNA perturbation technique is as the biotechnology with breakthrough potential applicability in clinical practice, just there is multiple product introduction clinical trial between the short several years from occurring, obtain huge success.But whether there is a class miRNAs, in diagnosis of chronic congestive heart failure and prognosis evaluation, there is biomarker effect, new strategy can be provided to be still challenge to this field scientific research personnel for diagnosis of chronic congestive heart failure and prognosis by detecting this kind of miRNA.Investigator is had to carry out examination (Circulation.2014 to Patients with Chronic Heart Failure peripheral blood miRNAs express spectra; 129 (9): 1009-21; Proc Natl Acad Sci U S A.2014; 111 (30): 11151-6) investigator, is also had to carry out detection (Eur J Heart Fail.2013 to peripheral blood miRNAs express spectra before and after Patients with Chronic Heart Failure treatment; 15 (11): 1277-88), but the miRNAs of same patient cardiac muscular tissue and peripheral blood do not detected simultaneously and compared, and lacking corresponding epidemiology follow-up investigation.In the present invention, applicant's originality the miRNAs of same patient cardiac muscular tissue and peripheral blood is detected simultaneously and is compared, and epidemiology follow-up investigation has been carried out to the PD of some patients.Determine peripheral blood miRNAs express spectra as the effect in chronic heart failure disease diagnosis and prognosis biomarker.
Summary of the invention
Technical problem to be solved by this invention is the deficiency overcoming existing theory and technology, determines the peripheral blood miRNAs playing the effect of diagnosis and prognosis biomarker in chronic heart failure.
The present inventor is found by a large amount of experiments, not only obtains and as biomarker for diagnosing chronic heart failure patient peripheral blood miRNAs express spectra, can have also obtained Patients with Chronic Heart Failure cardiac muscle miRNAs express spectra.Meanwhile, the present inventor finds miRNAs express spectra in same Patients with Chronic Heart Failure peripheral blood miRNAs express spectra and its cardiac muscle and not quite identical, and normal people is compared merely in prompting and peripheral blood in patients miRNAs will obtain non-specific biological mark.On the other hand, the present inventor finds portion perimeter blood miRNAs, such as hsa-miR-665 or hsa-miR-660-3p, is remarkable dependency with heart function of patients with chronic heart failure, not only can be used for diagnosis, also can be used as biomarker for pointing out prognosis.
Concrete, the invention provides a kind of miRNAs biomarker that can be used for diagnosing chronic heart failure patient.
Preferably, described peripheral blood miRNAs biomarker is hsa-miR-4491, hsa-miR-1285-3p, hsa-miR-665, hsa-miR-660-3p, hsa-miR-206, hsa-miR-1268b, hsa-miR-130a-3p and/or hsa-miR-330-3p, and its sequential structure is respectively as shown in SEQ ID No.1-8.
The present invention also provides a kind of peripheral blood miRNAs biomarker that can be used for assessing chronic heart failure prognosis.
Preferably, described miRNAs biomarker is miR-1285-3p, hsa-miR-665 and/or hsa-miR-660-3p, and its sequential structure is as shown in SEQ ID No.2,3 and 4.
The present invention also provides a kind of above-mentioned miRNAs biomarker for assessment of the purposes of chronic heart failure prognosis.
The present invention also provides a kind of reagent for detecting above-mentioned miRNAs sequence, and wherein, described sequence comprises SEQ ID No.1, and 2,3,4,5,6,7 and/or 8.
Preferably, described miRNAs sequence is SEQ ID No.2,3 and/or 4.
Preferably, described reagent is reverse transcriptase primer and the amplimer of described miRNAs sequence.
Preferred, described primer obtains according to following design of primers principle: 1) based on stem bulge loop structure design specific reverse transcriptase primer be combined with miRNA 3 ' terminal sequence, the specific reverse transcriptase carried out under reversed transcriptive enzyme effect for specific miRNA reacts; 2) based on the specificity forward PCR primer of reverse transcriptase primer sequences Design, general reverse primer.
The present invention also provides a kind of mentioned reagent diagnose in preparation or assess the purposes in the test kit of Patients with Chronic Heart Failure prognosis.
Preferably, the chronic heart failure that the cause of disease such as myocarditis, myocardosis that described chronic heart failure comprises congenital heart disease, coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, irregular pulse, the various cause of disease cause causes.
The present invention also provides the test kit of a kind of diagnosis or the prognosis of assessment Patients with Chronic Heart Failure, it is characterized in that, wherein, described test kit comprises above-mentioned arbitrary described reagent.
Preferably, the chronic heart failure that the cause of disease such as myocarditis, myocardosis that described chronic heart failure comprises congenital heart disease, coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, irregular pulse, the various cause of disease cause causes.
Accompanying drawing explanation
Fig. 1. Patients with Chronic Heart Failure peripheral blood miRNAs chip express spectra and myocardium miRNAs chip expression pattern analysis; Figure 1A is cardiac muscular tissue miRNAs chip express spectra scatter diagram; Figure 1B is peripheral blood miRNAs chip express spectra scatter diagram; Fig. 1 C is cardiac muscular tissue miRNAs chip express spectra volcano figure; Fig. 1 D is peripheral blood miRNAs chip express spectra volcano figure.
Fig. 2. peripheral blood miRNAs chip express spectra and cardiac muscular tissue miRNAs chip express spectra comparative result; All express the changing value of the miRNAs of rising when Fig. 2 A is heart failure in cardiac muscular tissue's chip, and the expression in peripheral blood chip changes situation; All express the changing value of the miRNAs of decline when Fig. 2 B is heart failure in cardiac muscular tissue's chip, and the expression in peripheral blood chip changes situation.
Fig. 3. adopt Real-time PCR method proofing chip screening results, be wherein eight kinds of Microrna (microRNA, miRNA) in chronic heart failure, (* represents compared with control group, p<0.05 with the relative level in normal control population's blood plasma; * represents compared with control group, p<0.01).
Fig. 4. the sensitivity of 8 kinds of miRNAs diagnosing chronics heart failure.Fig. 4 A is ROC value, the sensitivity of blood plasma miRNA s diagnosing chronic heart failure; Fig. 4 B is the result compared with ultrasonic test heart function, blood plasma miRNA s content and cardiac ultrasonic ejection fraction dependency.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1. Patients with Chronic Heart Failure peripheral blood and cardiac muscular tissue miRNAs screening results
1. collect 10 routine chronic heart failures (take from 2007-2014 at Vasculocardiology Deparment inpatient, each case makes a definite diagnosis classification by two or more clinicist) and 10 routine normal control's peripheral blood samples.After drawing materials, the centrifugal 6min of room temperature 3,500rpm, gets upper plasma, is stored in-80 DEG C of refrigerators.Add 1ml TRIZOL LS (Invitrogen company) in every 0.25ml peripheral blood blood plasma, extract RNA, RNasey Mini Kit (Qiagen) processing sample.Use
nD-1000 detects RNA quality.MiRCURY
tMafter Array Power labeling kit (Exqion) mark, carry out hybrid experiment at hybridization station.Axon GenePix 4000B microarray scanner scans and obtains chip image.GenePix pro V6.0 software analysis obtains data, utilizes microarray technology high flux screening to go out and composes at the miRNA of middle differential expression.This chip detection miRNAs totally 3100 kinds, filters out differential expression miRNAs 599, and wherein 347 miRNAs express rising in heart failure patient peripheral blood, and 252 miRNAs express decline in heart failure patient peripheral blood.
2. collect the cardiac muscular tissue of Patients with Chronic Heart Failure (take from 2007-2014 at Vasculocardiology Deparment inpatient, each case makes a definite diagnosis classification by two or more clinicist) in 1, and 10 routine normal control's peripheral blood samples.After drawing materials, the centrifugal 6min of room temperature 3,500rpm, gets upper plasma, is stored in-80 DEG C of refrigerators.Add 1ml TRIZOL (Invitrogen company) in every 0.25ml peripheral blood blood plasma, extract RNA, RNasey Mini Kit (Qiagen) processing sample.Use
nD-1000 detects RNA quality.MiRCURY
tMafter Array Power labeling kit (Exqion) mark, carry out hybrid experiment at hybridization station.Axon GenePix 4000B microarray scanner scans and obtains chip image.GenePix pro V6.0 software analysis obtains data, utilizes microarray technology high flux screening to go out and composes at the miRNA of middle differential expression.This chip detection miRNAs totally 3100 kinds, filters out differential expression miRNAs 74, and wherein 51 miRNAs express rising in myocardium of congestive heart failure, and 23 miRNAs express decline in myocardium of congestive heart failure.
Result shows that in myocardium in rats with chronic heart failure, miRNAs expression changes.
Embodiment 2. Patients with Chronic Heart Failure peripheral blood miRNAs chip express spectra and myocardium miRNAs chip expression pattern analysis
Bioinformatic analysis method is adopted to carry out treatment and analysis to the chip data obtained.
1. scatter diagram has reacted the repeatability between chip chamber or group, and repeatability is better the closer to diagonal lines.Use Pearson correlation coefficient to weigh repeatability in addition, relation conefficient is more better close to 1 repeatability.Figure 1A and 1B result shows: chip difficulty action accomplishment is reliable, reproducible.
2. situation about comparing between group is shown in Fig. 1 C and 1D.With-log (Pvalue) for ordinate zou, log2 (Fold change) maps to each miRNA for X-coordinate, intuitively can arrive the miRNAs (miRNAs that Grey Point representative is screened by Fold change and P-value) of significant difference.Fig. 1 C and 1D result show: miRNAs can be used as the candidate biomarker thing of a class diagnosis of chronic congestive heart failure.
Embodiment 3. peripheral blood miRNAs express spectra and cardiac muscular tissue miRNAs express spectra comparative result
The variation tendency of all miRNAs of the candidate biomarker thing as diagnosis of chronic congestive heart failure of differential expression peripheral blood chip and cardiac muscular tissue's chip examination obtained is classified.Result shows: express in cardiac muscular tissue there is significant difference (comprise raise or reduce) have 74 miRNAs, and express in peripheral blood there is significant difference (comprise raise or reduce) have 599 miRNAs, 25 expression of miRNAs in two kinds of chips are had all to change, wherein express all raise have 6 kinds of (Fig. 2 A) (hsa-miR-4491, hsa-miR-1285-3p, hsa-miR-665, hsa-miR-660-3p, hsa-miR-206 and hsa-miR-1268b), what expression all declined has 2 kinds (Fig. 2 B) (hsa-miR-130a-3p and hsa-miR-330-3p), these 8 kinds of miRNAs are pointed out all to can be used as the diagnosis of candidate's biological markers for chronic heart failure.Owing to circulating miRNAs and organize expression and distribution between miRNAs by various factors, comprise: tissue generation, tissue degradation, tissue picked-up and tissue secretion etc., therefore may there is circulation miRNAs and organize miRNAs to express changing inconsistent situation (Oncotarget.2015 Mar 10. [Epub ahead of print], PMID:25860935).And because chronic heart failure is the disease that the whole body multiple organ caused by many reasons is got involved, other organs function also changes in various degree, circulation miRNAs expression amount is not only closely related with myocardium miRNAs expression amount, and other organs function also may affect expression (the Clin Chem.2013Dec of peripheral blood miRNAs; 59 (12): 1742-52; J Hum Hypertens.2014 May; 28 (5): 288-91).Therefore these 8 kinds of miRNAs are only had to be that most probable is relevant with chronic heart failure specificity.
Embodiment 4. chip results Real-time PCR verifies
In second independent crowd, collect 200 routine Patients with Chronic Heart Failure and 200 routine normal control population's peripheral bloods respectively in addition, extract RNA in the manner described above.
Real-time PCR detection is carried out in the expression of the 8 kinds of miRNAs adopting the expression in two kind chip of Guangzhou Rui Bo company miRNA detection kit to said chip primary dcreening operation all to change in second independent crowd:
MiRNAs reverse transcription:
Reverse transcriptase primer (RT Primer Mix) configures:
miRNA RT Primer 1μl
U6RT Primer 1μl
RNase free H
2O 78μl
Reverse transcription reaction system:
RNA template 2μg
RT Primer Mix 4μl
RNase free H
2O up to 19μl
After the mixing of above system, brief centrifugation, 70 DEG C hatch 10min after, ice educates 2min, then adds following reagent:
2×TS reaction buffer 25μl
TS enzyme 2.5μl
RNase free H
2O 3.5μl
Reverse transcription reaction program:
42 DEG C of 60min, 70 DEG C of 10min; Stop rear 4 DEG C for subsequent use, product is stored in-20 DEG C.
miRNAs real-time PCR：
Reaction system: 2 × SYBR Green Mix 9 μ l
RT product 2μl
miRNA Forward Primer 2μl
miRNA Reverse Primer 2μl
RNase-free H
2O 5μl
Response procedures:
95℃30sec--(95℃10sec--60℃20sec--70℃1sec)×40 cycles--Melting Curve
Result shows: wherein 8 kinds of miRNAs, namely miR-4491, miR-1285-3p, miR-665, miR-660-3p, miR-206, miR-1268b, miR-130a-3p are consistent with chip detection result with the result that miR-330-3p expresses, and in the peripheral blood of two Patients with Chronic Heart Failure crowds, miRNAs variation tendency comes to the same thing (Fig. 3).Show that 8 kinds of miRNAs can be used as the candidate biomarker thing of a class diagnosis of chronic congestive heart failure, real-time PCR detection method reliable results.
The sensitivity of embodiment 5. differential expression miRNAs diagnosing chronic heart failure
Collect 200 routine Patients with Chronic Heart Failure (take from 2007-2014 Hospitals in Wuhan inpatient, each case makes a definite diagnosis classification by two or more clinicist) and 200 routine normal control population's peripheral bloods.Extract RNA according to preceding method, and detect the expression of the 8 kinds of miRNAs filtered out in embodiment 4, and carry out ROC analysis.
Result shows: in peripheral blood in patients, miR-4491, miR-1285-3p, miR-665, miR-660-3p, miR-206 and miR-1268b are increased to about 50 times, 40 times, 100 times, 800 times, 45 times and 50 times of control group crowd respectively, and miR-130a-3p and miR-330-3p drops to about 50% (Fig. 3) of control group crowd respectively.And miR-4491, miR-1285-3p, miR-665, miR-660-3p, miR-206 and miR-1268b have highly sensitive (AUC>0.9) (Fig. 4 A) to diagnosing chronic heart failure, the expression amount of its Myocardial abundant miR-1285-3p, miR-665 and miR-660-3p and ejection fraction significant correlation (P<0.05) (Fig. 4 B).
At present, cardiac ejection fraction is the most frequently used for assessment of cardiac function clinically, and cardiac ejection fraction lower prompting patient prognosis is poorer.Show miR-1285-3p, miR-665 and miR-660-3p can be used as the biomarker of a kind of diagnosis and assessment chronic heart failure prognosis.
Embodiment 6. prepares the test kit detecting chronic heart failure risk assessment
Kit components: detection kit comprise for the specific reverse transcriptase primer of 8 kinds of miRNAs (miR-4491, miR-1285-3p, miR-665, miR-660-3p, miR-206, miR-1268b, miR-130a-3p and miR-330-3p) of increasing and real-time PCR primer pair each a set of; For contrast RNA (U6) of increasing specific reverse transcriptase primer and real-time PCR primer pair is a set of and related reagent, composition and content following (100 times), be stored in-20 degree.Described primer obtains according to following design of primers principle: 1) based on stem bulge loop structure design specific reverse transcriptase primer be combined with miRNA 3 ' terminal sequence, the specific reverse transcriptase carried out under reversed transcriptive enzyme effect for specific miRNA reacts; 2) based on the specificity forward PCR primer of reverse transcriptase primer sequences Design, general reverse primer.
Above reagent provides by each source company, commercialization.Concrete detection method and correlated response parameter are with reference to embodiment 1.
Sequence table:
MiR-4491, miR-1285-3p, miR-665, miR-660-3p, miR-206, miR-1268b, miR-130a-3p and miR-330-3p
Hsa-miR-4491 nucleotide sequence:
Sequence ID No.1:5`AAUGUGGACUGGUGUGACCAAA 3`
Hsa-miR-1285-3p nucleotide sequence:
Sequence ID No.2:5`UCUGGGCAACAAAGUGAGACCU 3`
Hsa-miR-665 nucleotide sequence:
Sequence ID No.3:5`ACCAGGAGGCUGAGGCCCCU 3`
Hsa-miR-660-3p nucleotide sequence:
Sequence ID No.4:5`ACCUCCUGUGUGCAUGGAUUA 3`
Hsa-miR-206 nucleotide sequence:
Sequence ID No.5:5`UGGAAUGUAAGGAAGUGUGUGG 3`
Hsa-miR-1268b nucleotide sequence:
Sequence ID No.65`CGGGCGUGGUGGUGGGGGUG 3`
Hsa-miR-130a-3p nucleotide sequence:
Sequence ID No.7:5`CAGUGCAAUGUUAAAAGGGCAU 3`
Hsa-miR-330-3p nucleotide sequence:
Sequence ID No.8:5`GCAAAGCACACGGCCUGCAGAGA 3` 。
Claims (10)
1. the peripheral blood miRNAs biomarker of a diagnosing chronic heart failure or assessment chronic heart failure prognosis, it is characterized in that, described biomarker comprises hsa-miR-4491, hsa-miR-1285-3p, hsa-miR-665, hsa-miR-660-3p, hsa-miR-206, hsa-miR-1268b, hsa-miR-130a-3p and/or hsa-miR-330-3p, and its sequential structure is respectively as shown in SEQ ID No.1-8.
2. biomarker as claimed in claim 1, it is characterized in that, described biomarker comprises hsa-miR-1285-3p, hsa-miR-665 and/or hsa-miR-660-3p, and its sequential structure is respectively as shown in SEQ ID No.2,3 and 4.
3. for detecting a reagent for following miRNAs sequence, it is characterized in that, described sequence comprises SEQ IDNo.1, and 2,3,4,5,6,7 and/or 8.
4. reagent as claimed in claim 3, it is characterized in that, described reagent is reverse transcriptase primer and the amplimer of described miRNAs sequence.
5. the reagent as described in claim 3 or 4, is characterized in that, described miRNAs sequence is SEQ ID No.2,3 and/or 4.
6. the arbitrary described reagent of claim 3-5 is diagnosed in preparation or is assessed the purposes in the test kit of Patients with Chronic Heart Failure prognosis.
7. purposes as claimed in claim 6, it is characterized in that, described chronic heart failure comprises congenital heart disease, chronic heart failure that the cause of disease such as myocarditis, myocardosis that coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, irregular pulse, the various cause of disease cause causes.
8. a test kit for diagnosis or the prognosis of assessment Patients with Chronic Heart Failure, is characterized in that, described test kit comprises the arbitrary described reagent of claim 3-5.
9. test kit as claimed in claim 8, it is characterized in that, described chronic heart failure comprises congenital heart disease, chronic heart failure that the cause of disease such as myocarditis, myocardosis that coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, irregular pulse, the various cause of disease cause causes.
10. test right requires a method for the miRNAs biomarker described in 1, and it is characterized in that, the reagent arbitrary with claim 3-5 detects described miRNAs biomarker.
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| CN105903036B (en) * | 2015-07-15 | 2019-12-17 | 浙江大学 | application of miR-130a antisense nucleic acid and derivative thereof in Hippo-YAP signal pathway inhibitor |
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| CN110878348B (en) * | 2019-11-19 | 2023-03-28 | 南京启医科技有限公司 | High-specificity lnc RNA biomarker for heart failure and detection method and application thereof |
| CN110946872A (en) * | 2019-12-30 | 2020-04-03 | 北京大学深圳医院 | Application of miR-4491 in preparation of medicine for treating breast cancer |
| CN110946872B (en) * | 2019-12-30 | 2022-11-04 | 北京大学深圳医院 | Application of miR-4491 in preparation of medicine for treating breast cancer |
| CN111440862A (en) * | 2020-05-19 | 2020-07-24 | 华中科技大学同济医学院附属同济医院 | Primer group, kit and method for evaluating prognosis of chronic heart failure patient |
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