CN109810981A - Polynucleotide and its application in cardiovascular disease diagnosis and treatment - Google Patents

Abstract

A kind of polynucleotide has the sequence as shown in SEQ ID No 1.Polynucleotide or carrier provided by the invention carry the RIDs core fragment in NCoR1 or NCoR1, after transducer cell, can significantly improve the cardiovascular diseases such as myocardial hypertrophy caused by Pressure Overload-induced and impaired cardiac function.Verified, NCoR1 expresses increase in the cardiac muscular tissue of myocardial hypertrophy patient and mouse, can be used as the biomarker of diagnosis myocardial hypertrophy.

Description

Polynucleotide and its application in cardiovascular disease diagnosis and treatment

Technical field

It is the present invention relates to a kind of polynucleotide and its polypeptide/albumen of expression, in particular to a kind of auxiliary using nuclear receptor Polypeptide/the albumen for helping inhibiting factor 1 (NCoR1) and its part polynucleotide sequence and its expression, examines cardiovascular disease Disconnected and therapeutic effect.

Background technique

Nuclear receptor corepressor 1 (NCoR1) is a transcriptional co-factor, is usually repaired with transcription factor and chromatin Decorations enzyme plays a role together, participates in the expression of regulation lots of genes.Existing animal experimental data shows NCoR1 in fat, 2 types It plays a significant role in the metabolic diseases such as diabetes.However, function of the NCoR1 in cardiovascular cell and numerous cardiovascular diseases Can be unclear, also have no the report for carrying out gene therapy to disease using NCoR1.

Summary of the invention

It is an object of the present invention to provide a kind of polynucleotides, interact with MEF2a, have and inhibit its turn Record active function.

It is another object of the present invention to provide a kind of polynucleotides, and there is regulation and myocardial hypertrophy to have correlation gene The effect of the transcriptional activity of Acta1 and Nppa promoter, so that myocardial hypertrophy is improved.

It is yet a further object of the present invention to provide a kind of carriers, load polynucleotide, are used to prepare for treating The composition of cardiovascular disease.

Yet another object of the invention is that a kind of polynucleotide is provided, as active constituent in preparation for treating Application in cardiovascular disease medicine.

Yet another object of the invention is that the albumen or polypeptide of a kind of polynucleotide and its translation are provided, as diagnosis The biomarker of myocardial hypertrophy is preparing the application in Cardiovascular agents box.

A kind of polynucleotide provided by the invention comprising sequence shown in SEQ ID No 1.

A kind of polynucleotide provided by the invention, is NCoR1.

In normal myocardial cells, NCoR1 and MEF2a and HDAC4 form compound.The compound inhibits turning for MEF2a Record activity simultaneously makes DNA methylase inhibitor with inhibition of gene expression.In the absence of NCoR1, which is destroyed, more MEF2a, which is raised, arrives its binding site, a large amount of acetylations of histone, and raises more rna plymerase iis, leads to myocardium fertilizer The expression of thick related gene is raised.

Polynucleotide provided by the invention interacts with MEF2a, inhibits the transcription of Acta1 and Nppa promoter Active raising.

Polynucleotide provided by the invention, the corresponding protein/polypeptide sequence of SEQ ID No 1 such as 20 institute of SEQ ID No Show.

A kind of inhibitor comprising sequence shown in NCoR1 or SEQ ID No 1 inhibits Actal and Nppa promoter Transcription.

Polynucleotide provided by the invention is preparing the composition for treating cardiovascular disease as active material Application in (such as: drug).

A kind of carrier provided by the invention, such as: plasmid, including serum cardiac troponin T promoter and NCoR1-RIDs (SEQ Shown in ID No 1).

A kind of active material for gene therapy provided by the invention, (such as: adeno-associated virus serum with adeno-associated virus Type 9, AAV9) it is carrier, it further include serum cardiac troponin T promoter and NCoR1-RIDs.

Carrier provided by the invention, as active material, in preparation for treating the composition of cardiovascular disease (such as: medicine Object) in application.

The so-called cardiovascular disease of the present invention, the especially disease of myocardial hypertrophy caused by Pressure Overload-induced and impaired cardiac function Disease.

A method of prediction cardiovascular patient responds therapeutic scheme, comprising:

NCoR1 the or SEQ ID of the cardiovascular patient cardiac muscle cell is detected in the cardiac muscle cell from cardiovascular patient The expression quantity of sequence shown in No 1 will test resulting expression quantity digitlization, carry out with the corresponding data stored in database Compare, will not be responded greater than the corresponding data stored in database with the cardiovascular patient when detecting resulting expression numerical quantity A possibility that applying the therapeutic scheme of sequence shown in NCoR1 or SEQ ID No 1 increases associated.

A kind of embodiment computer program product in computer-readable medium executes step when executing on computers Suddenly include:

Receive the data of the relevant expression of sequence shown in NCoR1 or SEQ ID No 1 in detection cardiac muscle cell；

Data according to acquired relevant expression calculate corresponding expression numerical quantity；

Resulting expression numerical quantity will be calculated to be compared with the corresponding data stored by database；

Finally, feeding back comparison result to terminal.

When will calculate resulting expression numerical quantity and be compared with the corresponding data stored by database, using single number The comparison of value is relatively simple mode.Such as: it is stored from being calculated in cardiac muscle cell in resulting expression numerical quantity and database A comparison from sequence expression quantity large sample statistic shown in NCoR1 or SEQ ID No 1 in cardiac muscle cell；Again Such as: from calculating this cardiovascular patient that stores in resulting expression numerical quantity and database in the cardiac muscle cell of cardiovascular patient The comparison of (average) the expression numerical quantity of sequence shown in NCoR1 or SEQ ID No 1 in previous cardiac muscle cell.

A kind of kit, the nucleotide chain including sequence expression quantity shown in detection NCoR1 or SEQ ID No 1.

Another kit further includes the computer program product embodied in computer-readable medium.

Technical solution of the present invention realize the utility model has the advantages that

Polynucleotide or carrier provided by the invention carry the RIDs core fragment in NCoR1 or NCoR1, transduction After cell, it can significantly improve the cardiovascular diseases such as myocardial hypertrophy caused by Pressure Overload-induced and impaired cardiac function.

Verified, NCoR1 expresses increase in the cardiac muscular tissue of myocardial hypertrophy patient and mouse, can be used as the diagnosis heart The biomarker of flesh plumpness.

Detailed description of the invention

Fig. 1 is that the present invention strikes cardiac myocyte hypertrophy result figure caused by low NCoR1 promotion phyenlephrinium, wherein (A) system Neonatal cardiac myocytes immunofluorescence dyeing figure, NCoR1 strike low pass and cross siRNA (siRNA) completion；Control indicates blank SiRNA is used as the solvent control of phyenlephrinium using physiological saline；It (B) is that the quantitative of neonatal cardiac myocytes area is divided Analyse result figure, * * * P < 0.001；

Fig. 2 is that the present invention knocks out myocardial hypertrophy result figure caused by NCoR1 exacerbation Pressure Overload-induced, wherein (A) is mouse Anatomical cardiac figure, with abdominal aorta constriction surgical simulation Pressure Overload-induced；(B) be mouse heart weight and weight ratio quantifies Analyze result figure, * * * P < 0.001；

Fig. 3 is that the present invention knocks out myocardial fibrosis result figure caused by NCoR1 exacerbation Pressure Overload-induced, wherein (A) is small Mouse myocardial fibrosis colored graph identifies fibrosis with sirius red stains；It (B) is that the quantitative of mouse cardiac muscle fibrosis area is divided Analysis, * * P < 0.01；

Fig. 4 is that the present invention knocks out impaired cardiac function quantitative analysis figure caused by NCoR1 exacerbation Pressure Overload-induced, wherein (A) Score quantitative analysis results figure shortens in system, is measured by mouse heart ultrasonic examination, * * * P < 0.001；It (B) is that ejection fraction is fixed Amount analysis result figure, is measured, * * * P < 0.001 by mouse heart ultrasonic examination；

Fig. 5 is that NCoR1 of the present invention inhibits cardiac myocyte hypertrophy result figure caused by phyenlephrinium, wherein (A) system is newborn Rat myocardial cell immunofluorescence dyeing figure, (B) are the quantitative analysis results figure of neonatal cardiac myocytes area, * * * P < 0.001。

Fig. 6 is NCoR1 of the present invention by core fragment RIDs and MEF2a interaction immunoblotting schematic diagram, wherein (A) Indicate the NCoR1 sequential structure schematic diagram with Flag label；(B) system using co-immunoprecipitation analysis different length NCoR1 with The combination proof scheme schematic diagram of MEF2a, using liposome respectively the MEF2a with HA label and with Flag label NCoR1 is transfected into HEK293FT cell；

Fig. 7 is that NCoR1 core fragment (RIDs) of the present invention inhibits cardiac myocyte hypertrophy result figure caused by phyenlephrinium, Wherein (A) is neonatal cardiac myocytes immunofluorescence dyeing figure；(B) be neonatal cardiac myocytes area quantitative analysis knot Fruit figure, * * * P < 0.001.

Fig. 8 be myocardial hypertrophy and heart function caused by NCoR1 core fragment (RIDs) of the present invention improvement Pressure Overload-induced by Quantitative analysis figure is damaged, wherein (A) is the quantitative analysis figure of mouse heart weight Yu weight ratio, * P < 0.05, * * P < 0.01, * * * P < 0.001；(B) score quantitative analysis figure shortens in system, is measured by mouse heart ultrasonic examination, * P < 0.05, * * P < 0.01, * * * P < 0.001；(C) it is ejection fraction quantitative analysis figure, is measured by mouse heart ultrasonic examination.* P < 0.05, * * P < 0.01, * * * P < 0.001；

Fig. 9 is that NCoR1 of the present invention expresses increase result figure in myocardial hypertrophy disease human heart, wherein (A) is Diagnosis of Sghistosomiasis Mark analyzes the NCoR1 protein level gel electrophoresis result figure in normal healthy controls and Detection in Hypertrophic Cardiomyopathy heart tissue, (B) system The quantitative analysis figure of NCoR1 protein level, * P < 0.05；

Figure 10 is that NCoR1 of the present invention expresses increase result figure in the heart of myocardial hypertrophy mouse, wherein (A) is Diagnosis of Sghistosomiasis NCoR1 protein level gel electrophoresis result figure in mark mouse heart tissue, (B) is the quantitative analysis of NCoR1 protein level Figure, * P < 0.05, * * P < 0.01.

Specific embodiment

Below in conjunction with attached drawing, the technical schemes of the invention are described in detail.The embodiment of the present invention is only to illustrate skill of the invention Art scheme rather than limit, although being described the invention in detail referring to preferred embodiment, those skilled in the art It should be appreciated that can be with modification or equivalent replacement of the invented technical scheme, without departing from the essence of technical solution of the present invention Mind and range, should all cover within the scope of the claims of the present invention.

Every test method used in following embodiment of the present invention is described as follows:

1) cardiomegaly assessment and sample collection

2 weeks execution mouse after the operation of abdominal aorta constriction.Use ventricular weight and weight ratio (VW/BW, mg/g) and the heart Room weight assesses cardiomegaly with tibia length ratio (VW/TL, mg/mm).Dissection ventricle and the ventricle that will be close to heart base portion Part is fixed in 4% paraformaldehyde for histologic analysis.It separates left ventricle and is freezed in liquid nitrogen for further analyzing.

2) Analysis of echocardiography

Cardiac function is monitored using echocardiogram (Vevo2100 imaging system), similar to being previously reported.By mouse fiber crops It is liquor-saturated and in two-dimentional LV long axis view to cardiac imaging.It is recorded based on M-mode and calculates ejection fraction and score short circuit.

3) histologic analysis

The fixed cardiac samples of paraformaldehyde are embedded in paraffin, and with hematoxylin and eosin (H&E), WGA (W32464, ThermoFisher) and 0.1% picoline red stained sections.Measure the cross-sectional area (PLoS of cardiac muscle cell One.2014；9:e110950；Development.2016；143:936-949), fibrosis is dyed quantification of to being checked The percentage of the positive staining area of the gross area.

4) gene expression analysis

Total serum IgE is separated using Trizol (15596026, Life Technologies), and uses Reverse Transcriptase kit (RR037A, Takara) synthesizes cDNA.QRT-PCR is carried out on ABI7900HI (Applied Biosystems), SYBR Green (Applied Biosystems) is for detecting PCR product.The relative expression of each gene passes through to ventricle sample The 18s or GAPDH of GAPDH or cell are standardized to determine.

5) Western Blot is analyzed

Total protein (ArteriosclerThrombVasc is extracted by the RIPA buffer containing protease inhibitors Biol., 2016,36,874~885；Diabetes., 2017,66,1535~1547).Sample is separated by SDS-PAGE, so After be transferred on pvdf membrane.After primary antibody and secondary antibody are incubated for, ECL western blot substrate (Thermo Fisher is used Scientific) visualize protein expression, and quantitative using Image J software.Primary antibody be anti alpha-Actinin (A7811, Sigma), anti-Flag (F3165, Sigma), anti-HA (H3663, Sigma or 3724, Cell Signaling), anti-MEF2a (sc-17785, Santa Cruz), anti-NCoR1 (5948, Cell Signaling) and anti-HDAC4 (ab12172, Abcam).Two Anti- is sheep anti mouse (SA00002-1, Proteintech), goat-anti rabbit (SA00002-2, Proteintech) or goat-anti people (SA00002-8, Proteintech).

6) chromatin imrnunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP)

Ventricle sample is clayed into power, keeps cooling down by liquid nitrogen.Then 1% formalin solution is used at room temperature Sample is crosslinked 15 minutes, is then quenched with 10 × glycine.It is carried out using Ez-ChIP kit (17-371, Millipore) ChIP experiment.The anti-NCoR1 antibody of 5 μ g (5948, Cell Signaling), anti-MEF2a antibody (sc-17785, the Santa of 2 μ g Cruz), the anti-HDAC4 of 10 μ g (ab12172, Abcam), the 5 anti-Acetylated histone H3s of μ g (06-599, Millipore), 5 μ g are anti- Acetylated histones H4 (06-598, Millipore), the 10 anti-rna plymerase iis of μ g (05-623B, Millipore), mouse IgG (12-371B, Millipore) and rabbit igg (2729, Cell Signaling) are used for immunoprecipitation.It is produced using PCR analysis ChIP Object.When being ChIP using NCoR1 anti-MEF2a or anti-HDAC4 antibody, following PCR primer is used:

Actal F1 CAGCGGTATAAATAGAACCC,

Actal R1 CCTCCCAGGCAGACTCATCT；With

Nppa F1 CATCCTGTTGGCACCTTGGACAC,

Nppa R1 CGTGTAAACACCAAGGGATG；

When being ChIP using Acetylated histone H3 acetylated histones H4 antibody, following PCR primer is used:

Actal F2 CAGGCTGAGAACCAGCCGAAGGAA,

Acta1 R2 CCTGTCCCCTTGCACAGGTT；With

Nppa F2 CGTGACAAGCTTTGCCGAAC,

Nppa R2 ATTCTGTCACTTGCAGCGATAAAG；

When being ChIP using rna plymerase ii antibody, following PCR primer is used:

Actal F3 GAAGACGAGACCACCGCTCTTG,

Actal R3 ATGGATGGGAACACAGCCCTGG,

Nppa F3 TCCATCACCCTGGGCTTCTTCCT, and

Nppa R3 CCTTGAAATCCATCAGATCTGTG。

Co-IP experiment is carried out according to bibliography.It is cracked by ventricle sample broke and by IP lysis buffer.It will always split Solve object and anti-IgG and anti-MEF2a antibody respectively with albumin A/G pearl (sc-2003, Santa Cruz Biotechnology) one It rises and is incubated overnight.Finally, carrying out Western blot analysis to immune complex.

7) cell culture and treatment

According to disclosed scheme (Circ Res., 1982,50,101~116；Cell, 2014,156,1179~1192) point From neonatal cardiac myocytes.In short, obtaining ventricle from 1-2 age in days Sprague-Dawley rat pup.After being cut into small pieces, Ventricle is digested in the PBS containing 0.1% trypsase-EDTA and lmg/ml clostridiopetidase A II (Worthington), is obtained slender Born of the same parents' suspension.Culture dish 1mg/ml gelatin is precoated 24 hours.Neonatal cardiac myocytes (are contained 10% in culture medium The high glucose DMEM of FBS, 1% Pen .- Strep) middle culture 24 hours, then culture medium is being maintained (to contain 1% mould Element-streptomysin high glucose DMEM) in culture.For inducing cardiomyocytes hypertrophy, tieed up by neonatal cardiac myocytes It holds after being cultivated 24~48 hours in culture medium, phyenlephrinium (PE, Sigma, 200 μM) is added in culture medium.It will be newborn big Rat cardiomyocyte is coated on glass cover-slip and is fixed in paraformaldehyde for immunofluorescence dyeing.Neonate rat cardiac muscle is thin The surface area of born of the same parents is quantitative by Image J software.

8) plasmid and siRNA building

NCoR1-Flag plasmid (Cell, 2011,147,827~839) is constructed according to document.By different NCoR1 segments It is cloned into pHAGE plasmid.MEF2a-HA sequence is expanded from mouse cDNA and is inserted into pcDNA 3.1.

The sequence of siRNA is as follows:

SiNCoR1:5 ' CCAGGUCCAUGACAAGUGA3 '；

SiMEF2a:5 ' CAGUCGGAAACCAGAUCUA3 '；

SiMEF2c:5 ' CUGGCAGCAAGAACACAAU3 '；

SiMEF2d:5 ' CACCAAGUUUACUCAGCCA3 ' and

siControl 5′UUCUCCGAACGUGUCACGUTT3′。

It is used according to the explanation of manufacturer using Lipofectamine 2000 (Thermo Fisher Scientific) Negative control and siRNA transfection neonatal cardiac myocytes.

9) slow virus packaging and infection

Using Lipofectamine 2000 (Thermo Scientific) by pHAGE-NCoR1-flag plasmid and sky PHAGE plasmid is imported together with slow virus packaging plasmid in 293FT cell.It collects and contains respectively after 48 hours and 72 hours after transfection There is the culture medium of slow virus, merge, filtered by 0.22 filter, and be concentrated by ultracentrifugation, for infecting neonate rat Cardiac muscle cell.

10) it makes and delivers adeno-associated virus serotype 9 (AAV9)

AAV9-cTnT-NCoR1 (1939- is prepared using the AAV9 carrier containing serum cardiac troponin T (cTnT) promoter 2453) plasmid (Han Heng company, Chinese Shanghai).With AAV9-cTnT-NCoR1 (1939-2453) plasmid transfection AAV-293 cell (Han Heng company) is to prepare virus.It collects, purifying and concentrating virus particles.Titre is measured by qPCR.AAV9 is subcutaneously injected Into newborn mice (Circ Res., 2006,99:e3-9；Development, 2016,143.936~949).

11) luciferase assay

For luciferase assay, the 2kb segment in mouse Acta1 or Nppa promoter region is cloned into the basis pGL3 matter In grain.Then by these plasmids respectively or combined type and NCoR1-flag, MEF2a-HA and Renila plasmid co-transfection 293FT Cell.48 hours after transfection, Dual- is usedReporterAssay System (E1910, Promega) measurement Transcriptional activity.

12) it statisticallys analyze

As a result it is presented in the form of average value ± SE, and is analyzed using Prism (GraphPad software).Using two-way Then ANOVA carries out Bonferoni analysis, carry out Multiple range test.Student t is examined for comparing in pairs.If P value be less than or Equal to 0.05, then it is assumed that result is dramatically different.

13) animal model

By by floxed NCoR1 mouse (Cell, 2011,147,827~839) and α MHC-Cre mouse (Circ Res., 2005,97,372~379) hybridize, generate cardiac myocytespecific NCoR1 knock-out mice, carry out abdominal aorta constriction hand Art come induced pressure Overload (J Exp Med., 2002,195,375~381；Hypertension, 2017, 70,137~147).In short, abdominal aorta is dissected, by one with 2% Isoflurane inhalation anesthesia 8-12 week old male mice It is placed in parallel on 27G syringe needle and abdominal aorta, and is ligatured abdominal aorta together with syringe needle using 6-0 silk thread, then take out needle Head.For sham-operation, we are passed beneath silk thread from abdominal aorta, but do not ligature.For Angiotensin II (AngII) Micropump (2004D, Alzet) is subcutaneously implanted in 11~12 week old mouse and compares (chlorine with delivery vector by the cardiomegaly of induction Change sodium) or AngII (750ng/kg/min) 4 weeks.It is measured and is received using BP-2000 analysis of blood pressure system (Visitech Systems) Contracting pressure and diastolic pressure (Hypertension, 1995,25,1111~1115；Circ Res., 2017,120,1584~1597).

The mouse used is in C57BL6/J background and to raise in no-special pathogen (SPF) facility.All animals Research is ratified through Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch's Institutional Review and Ethics Committee.

RIDs core fragment in embodiment 1:NCoR1 or NCoR1 is to myocardial hypertrophy caused by improvement Pressure Overload-induced With the effect of the cardiovascular diseases such as impaired cardiac function

NCoR1 struck using small interfering RNA technology low, then observed to cardiac myocyte hypertrophy caused by phyenlephrinium It influences.After immunofluorescence dyeing to cardiac muscle cell and quantitative analysis, discovery strikes low NCoR1 and remarkably promotes phyenlephrinium Caused cardiac myocyte hypertrophy (Fig. 1).

It is small by the way that floxed NCoR1 mouse is obtained cardiac myocytespecific NCoR1 knockout with α MHC-Cre mouse hybrid Mouse.After mouse is condemned to death, using ventricular weight and weight ratio (VW/BW, mg/g) come the case where assessing myocardial hypertrophy.Solution is treated others for earnest Room and the ventricular section that will be close to heart base portion are fixed in 4% paraformaldehyde for histologic analysis, separate left ventricle and It is frozen in liquid nitrogen for further analyzing.

NCoR1 defect in cardiac muscle cell exacerbates cardiomegaly caused by abdominal aorta is shunk.Mouse cardiac myocytes because It knocks out NCoR1 and has significantly aggravated pressure load, Pressure Overload-induced results in myocardial hypertrophy, and (ventricular weight significantly rises with weight ratio It is high) (Fig. 2).The fixed cardiac samples of paraformaldehyde are embedded in paraffin, are divided after making histotomy with sirius red stains Analyse the fibrosis of cardiac muscular tissue, the results showed that knock out NCoR1 in mouse cardiac myocytes and significantly aggravate the heart caused by Pressure Overload-induced Myofibrosis (Fig. 3).Cardiac function is monitored using echocardiogram (Vevo2100 imaging system), by mouse anesthesia and in two dimension To cardiac imaging in long axis of left ventricle view, shorten score and ejection fraction with recording and calculating based on M-mode.The results show that NCoR1 is knocked out in mouse cardiac myocytes and significantly aggravates impaired cardiac function caused by Pressure Overload-induced, shows as shortening score and is penetrated Blood fraction declines (Fig. 4).Conversely, exogenous NCoR1 is imported in neonatal cardiac myocytes using slow-virus infection technology, Significantly inhibit cardiac myocyte hypertrophy caused by phyenlephrinium (Fig. 5).

Different NCoR1 segments is cloned into pHAGE plasmid, expand MEF2a-HA sequence from mouse cDNA and is inserted into In pcDNA3.1, and plasmid is transferred to HEK293FT cell using liposomal transfection teclmiques.Then Immunoprecipitation is utilized, Immunoprecipitation is carried out with the antibody for Flag, then carries out immunoblotting assay with the antibody for Flag or HA.The results show that NCoR1 passes through core fragment RIDs and MEF2a interaction (Fig. 6).

Further verifying shows effect of the RIDs segment to myocardial hypertrophy.Using slow-virus infection technology newborn big RIDs segment is transferred in rat cardiomyocyte also can significantly inhibit cardiac myocyte hypertrophy caused by phyenlephrinium (Fig. 7).

NCoR1 is transferred in cardiac muscle using adeno-associated virus serotype 9 (AAV9), to detect its suppression to myocardial hypertrophy Production is used.To contain serum cardiac troponin T (cTnT) promoter, AAV9-cTnT-NCoR1-RIDs plasmid is prepared for (referring to SEQ ID No 2).With AAV9-cTnT-NCoR1-RIDs plasmid transfection AAV-293 cell to generate virus, then collect, purify and Concentrating virus particles, then AAV9 is subcutaneously injected into newborn mice.Through the heart caused by Pressure Overload-induced (abdominal aorta constriction) The verifying of flesh plumpness mouse model, the importing for the exogenous NCoR1-RIDs core fragment that AAV9 is mediated significantly improve Pressure Overload-induced Caused myocardial hypertrophy and impaired cardiac function (Fig. 8) show that there are NCoR1-RIDs core fragment the means as therapeutic agent to answer For gene therapy.

Embodiment 2: myocardium NCoR1 protein expression level and the relevance that myocardial hypertrophy disease occurs are verified

The heart tissue of Detection in Hypertrophic Cardiomyopathy and the heart tissue of mouse cardiac muscle hypertrophy model are collected, using containing There is the RIPA buffer of protease inhibitors to extract total protein, sample is separated by SDS-PAGE, is then transferred on pvdf membrane. After primary antibody and secondary antibody are incubated for, protein expression level of the detection NCoR1 in the cardiac muscular tissue that myocardial hypertrophy occurs uses ECL Western blot substrate visualizes protein expression, and quantitative using Image J software.The results show that patients with cardiac hypertrophy Myocardium NCoR1 protein expression be significantly higher than normal healthy controls (Fig. 9).The mouse cardiac muscle hypertrophy model caused by Pressure Overload-induced In, either postoperative 1 week or 4 weeks postoperative, protein level of the NCoR1 in cardiac muscular tissue was all apparently higher than sham-operation group (figure 10).The grade tests show that NCoR1 expresses increase in the cardiac muscular tissue of myocardial hypertrophy patient and mouse, can be used as and examine The biomarker of disconnected myocardial hypertrophy.

Sequence table

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<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 6

cgtgtaaaca ccaagggatg 20

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 7

caggctgaga accagccgaa ggaa 24

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

cctgtcccct tgcacaggtt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 9

cgtgacaagc tttgccgaac 20

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 10

attctgtcac ttgcagcgat aaag 24

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 11

gaagacgaga ccaccgctct tg 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 12

atggatggga acacagccct gg 22

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 13

tccatcaccc tgggcttctt cct 23

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 14

ccttgaaatc catcagatct gtg 23

<210> 15

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 15

ccagguccau gacaaguga 19

<210> 16

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 16

cagucggaaa ccagaucua 19

<210> 17

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 17

cuggcagcaa gaacacaau 19

<210> 18

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 18

caccaaguuu acucagcca 19

<210> 19

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 19

uucuccgaac gugucacgut t 21

<210> 20

<211> 357

<212> PRT

<213> Artificial Sequence

<400> 20

Ile Thr Ala Ala Asn Phe Ile Asp Val Ile Ile Thr Arg Gln Ile Ala

1 5 10 15

Ser Asp Lys Asp Ala Arg Glu Arg Gly Ser Gln Ser Ser Asp Ser Ser

20 25 30

Ser Ser Leu Ser Ser His Arg Tyr Glu Thr Ala Ser Asp Ala Ile Glu

35 40 45

Val Ile Ser Pro Ala Ser Ser Pro Ala Pro Pro Gln Glu Lys Pro Gln

50 55 60

Ala Tyr Gln Pro Asp Met Val Lys Ala Asn Gln Ala Glu Asn Glu Ser

65 70 75 80

Thr Arg Gln Tyr Glu Gly Pro Leu His His Tyr Arg Ser Gln Gln Glu

85 90 95

Ser Pro Ser Pro Gln Gln Gln Pro Pro Leu Pro Pro Ser Ser Gln Ser

100 105 110

Glu Gly Met Gly Gln Val Pro Arg Thr His Arg Leu Ile Thr Leu Ala

115 120 125

Asp His Ile Cys Gln Ile Ile Thr Gln Asp Phe Ala Arg Asn Gln Val

130 135 140

Pro Ser Gln Pro Ser Thr Ser Thr Phe Gln Thr Ser Pro Ser Ala Leu

145 150 155 160

Ser Ser Thr Pro Val Arg Thr Lys Thr Ser Ser Arg Tyr Ser Pro Glu

165 170 175

Ser Gln Ser Gln Thr Val Leu His Pro Arg Pro Gly Pro Arg Val Ser

180 185 190

Pro Glu Asn Leu Val Asp Lys Ser Arg Gly Ser Arg Pro Gly Lys Ser

195 200 205

Pro Glu Arg Ser His Ile Pro Ser Glu Pro Tyr Glu Pro Ile Ser Pro

210 215 220

Pro Gln Gly Pro Ala Val His Glu Lys Gln Asp Ser Met Leu Leu Leu

225 230 235 240

Ser Gln Arg Gly Val Asp Pro Ala Glu Gln Arg Ser Asp Ser Arg Ser

245 250 255

Pro Gly Ser Ile Ser Tyr Leu Pro Ser Phe Phe Thr Lys Leu Glu Ser

260 265 270

Thr Ser Pro Met Val Lys Ser Lys Lys Gln Glu Ile Phe Arg Lys Leu

275 280 285

Asn Ser Ser Gly Gly Gly Asp Ser Asp Met Ala Ala Ala Gln Pro Gly

290 295 300

Thr Glu Ile Phe Asn Leu Pro Ala Val Thr Thr Ser Gly Ala Val Ser

305 310 315 320

Ser Arg Ser His Ser Phe Ala Asp Pro Ala Ser Asn Leu Gly Leu Glu

325 330 335

Asp Ile Ile Arg Lys Ala Leu Met Gly Ser Phe Asp Asp Lys Val Glu

340 345 350

Asp His Gly Val Val

355

Claims (10)

1. a kind of polynucleotide, it is characterised in that have the sequence as shown in SEQ ID No 1.

2. a kind of polynucleotide is preparing the application in the composition for treating cardiovascular disease as active material, described Polynucleotide include sequence shown in SEQ ID No 1.

3. purposes according to claim 2, it is characterised in that the polynucleotide is NCoR1

4. a kind of carrier, it is characterised in that including sequence shown in serum cardiac troponin T promoter and SEQ ID No 1.

5. carrier according to claim 4, it is characterised in that the carrier is plasmid or adeno-associated virus.

6. a kind of carrier described in claim 4 or 5 is preparing the composition for treating cardiovascular disease as active material In application.

7. a kind of inhibitor for inhibiting Acta1 and Nppa promoter transcription, it is characterised in that including NCoR1 or SEQ ID No 1 Shown in sequence.

8. a kind of method of prediction cardiovascular patient response therapeutic scheme, comprising:

NCoR1 the or SEQ ID No of the cardiovascular patient cardiac muscle cell is detected in the cardiac muscle cell from cardiovascular patient The expression quantity of sequence shown in 1 will test resulting expression quantity digitlization, be compared with the corresponding data stored in database Compared with being applied when detecting resulting expression numerical quantity and being greater than the corresponding data that stores in database and will not be responded with the cardiovascular patient A possibility that adding the therapeutic scheme of sequence shown in NCoR1 or SEQ ID No 1, increases associated.

9. a kind of computer program product of embodiment in computer-readable medium executes step when executing on computers Include:

Receive the data of the relevant expression of sequence shown in NCoR1 or SEQ ID No 1 in detection cardiac muscle cell；

Data according to acquired relevant expression calculate corresponding expression numerical quantity；

Resulting expression numerical quantity will be calculated to be compared with the corresponding data stored by database；

Finally, feeding back comparison result to terminal

10. a kind of kit, characterized by comprising:

Detect the nucleotide chain of sequence expression quantity shown in NCoR1 or SEQ ID No 1；With

The computer program product of embodiment as claimed in claim 9 in computer-readable medium.