CN102793910A - New application of casein kinase2-interacting protein-1 (CKIP-1) protein and coding gene thereof - Google Patents

Abstract

The invention discloses a new application of CKIP-1 protein and its coding gene. The present invention provides the application of CKIP-1 protein, the coding gene of the CKIP-1 protein or the plasmid containing the coding gene of the CKIP-1 protein in the preparation of drugs for preventing and/or treating cardiac hypertrophy; the CKIP- 1 The protein is shown in sequence 1 of the sequence listing. The present invention discovers the regulatory function of CKIP-1 protein and its coding gene in cardiac hypertrophy, so as to find new targets and methods for the diagnosis and treatment of cardiac hypertrophy, and lay the foundation for the clinical diagnosis and treatment of related diseases. The invention has great value for the treatment and prevention of cardiac hypertrophy.

Description

New application of CKIP-1 protein and its coding gene

technical field

The invention relates to a new application of CKIP-1 protein and its coding gene. the

Background technique

Cardiovascular disease is one of the main diseases that endanger human health. The annual medical expenses of major cardiovascular diseases amount to 130 billion yuan, causing a huge economic burden to the society. In-depth study of the pathogenesis and molecular mechanism of cardiovascular diseases and the establishment of new prevention strategies and measures on this basis to reduce the mortality and disability rates of cardiovascular diseases are major basic scientific issues that life sciences need to solve. the

Cardiomyocyte hypertrophy is a pathological change associated with a variety of clinical cardiovascular diseases, and it is a major response of the heart to biomechanical stretch and neurohumoral stimulation. Although early myocardial hypertrophy is a compensatory mechanism for the heart to maintain effective cardiac output, persistent myocardial hypertrophy will lead to the heart entering a decompensated stage, re-expression of fetal genes ANP, BNP, β-MHC, etc., and subsequent irreversible events. Reversed myocardial hypertrophy and dilation, decreased myocardial contractility, leading to heart failure. The response to trigger cardiac hypertrophy is related to the activation of multiple signaling pathways, including calcium ion/calmodulin-dependent protein kinases (CaMK)/HDACs/MEF2, calcineurin, silk Mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), protein kinase B (protein kinase B, PKB)/AKT, mammalian target of rapamycin protein (mammalian target of rapamycin, mTOR) and nuclear factor-κB (nuclear factor kappa B, NF-κB), etc. Thus, the cardiomyocyte hypertrophy signaling pathway is a complex regulatory network. Finding new genes and signaling pathways involved in the regulation of cardiac hypertrophy is a research hotspot in the cardiovascular field. the

In 1999, He Fuchu's laboratory cloned the CKIP-1 gene from human fetal liver at 22 weeks old, and expressed CKIP-1 protein consisting of 409 amino acids. The N-terminus of CKIP-1 protein contains a PH (pleckstrin homology) domain, which mediates its plasma membrane localization by binding phospholipids, and the C-terminus contains a leucine zipper, which mediates CKIP-1 and c-Jun, JunD and other AP- 1 Interaction of family members. The Litchfield laboratory obtained the same gene when screening the binding protein of the kinase CK2 through yeast two-hybrid screening, and named it casein kinase2-interacting protein-1 (CKIP-1). the

Contents of the invention

The purpose of the present invention is to provide a new application of CKIP-1 protein and its coding gene. the

The present invention provides the application of CKIP-1 protein, the coding gene of the CKIP-1 protein or the plasmid containing the coding gene of the CKIP-1 protein in the preparation of drugs for preventing and/or treating cardiac hypertrophy; the CKIP- 1 protein (ckip-1 protein) is shown in sequence 1 of the sequence listing. the

The gene encoding the CKIP-1 protein (also known as CKIP-1 gene or ckip-1 gene) is the DNA molecule described in any of the following 1)-4):

1) The DNA molecule shown in the sequence 2 from the 43rd to the 1266th nucleotide at the 5' end in the sequence listing;

2) The DNA molecule shown in sequence 2 in the sequence listing;

3) A DNA molecule that hybridizes to the DNA molecule shown in 1) or 2) under stringent conditions and encodes the protein;

4) A DNA molecule that has more than 90% homology with the gene in 1) or 2) or 3) and encodes the protein. the

The above-mentioned stringent conditions can be hybridized and washed in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C. the

The plasmid containing the gene encoding the CKIP-1 protein can specifically be a recombinant plasmid obtained by inserting the gene encoding the CKIP-1 protein into the pJG/ALPHA MHC plasmid. the

The preventive and/or therapeutic effect of the CKIP-1 protein, the gene encoding the CKIP-1 protein or the plasmid containing the gene encoding the CKIP-1 protein on cardiac hypertrophy can be embodied as follows (a) to (e) At least one of: (a) promotes cardiomyocyte size reduction and myocardial fibrosis; (b) reduces cardiac index; (c) reduces left ventricular index; (d) reduces the expression level of fetal period genes in cardiac tissue; (e) Increased left ventricular ejection fraction and ventricular fractional shortening. the

The present invention also provides the application of the CKIP-1 protein, the coding gene of the CKIP-1 protein or the plasmid containing the coding gene of the CKIP-1 protein in the preparation of products; the product has the following (a) Products with at least one function in (e): (a) promote myocardial cell size reduction and myocardial fibrosis; (b) reduce cardiac index; (c) reduce left ventricular index; (d) reduce cardiac tissue Expression levels of prenatal genes; (e) increased left ventricular ejection fraction and ventricular fractional shortening. the

The present invention also provides the application of the substance for inhibiting the expression of the CKIP-1 protein coding gene in the preparation of products; the product is a product with at least one of the following functions (1) to (4): (1 ) to make animal models of cardiac hypertrophy; (2) to promote myocardial cell enlargement and myocardial fibrosis; (3) to increase cardiac index; (4) to increase the expression level of fetal period genes in cardiac tissue. the

Any one of the above fetal genes is at least one of ANF gene, BNP gene and β-MHC gene. the

The present invention has the following major discoveries: (1) CKIP-1 gene knockout can lead to the occurrence of spontaneous cardiac hypertrophy, increasing the sensitivity of cardiac hypertrophy caused by pressure overload; (2) CKIP-1 gene overexpression can obviously resist the The decline of myocardial function and the occurrence of myocardial hypertrophy caused by pressure overload; (3) The mechanism of action of CKIP-1 protein is realized through the direct interaction with HDAC4. The interaction between the two can promote HDAC4 to enter the nucleus and inhibit Transcriptional activity of MEF2C. the

The present invention discovers the regulatory function of CKIP-1 protein and its coding gene in cardiac hypertrophy, so as to find new targets and methods for the diagnosis and treatment of cardiac hypertrophy, and lay the foundation for the clinical diagnosis and treatment of related diseases. The invention has great value for the treatment and prevention of cardiac hypertrophy. the

Description of drawings

Figure 1 shows the changes in the heart tissue morphology of KO mice and WT mice. the

Figure 2 shows the changes in the ratio of heart weight to body weight in KO mice and WT mice. the

Figure 3 shows the changes in the expression of ANF gene, BNP gene and β-MHC gene between KO mice and WT mice. the

Figure 4 shows the changes in the localization of MEF2C transcriptional repressor HDAC4 in cardiomyocytes in KO mice and WT mice. the

Fig. 5 is the change of HDAC4 phosphorylation level in KO mice and WT mice. the

Figure 6 is a comparison of the phosphorylation levels of Akt/mTOR/S6K in KO mice and WT mice. the

Fig. 7 shows the changes of heart tissue morphology and structure in KO mice and WT mice 4 weeks after surgery. the

Figure 8 shows the changes in heart index of KO mice and WT mice after 4 weeks of surgery. the

Figure 9 shows the changes in the expression of ANF gene, BNP gene and β-MHC gene in KO mice and WT mice 4 weeks after surgery. the

Figure 10 is a comparison of cardiac function changes between KO mice and WT mice 4 weeks after surgery. the

Figure 11 shows the changes in cardiac tissue morphology and structure of TG mice and WT mice 4 weeks after surgery. the

Figure 12 shows the changes in heart index of TG mice and WT mice after 4 weeks of operation. the

Figure 13 shows the changes in the expression of ANF, BNP and β-MHC in heart tissue of TG mice and WT mice 4 weeks after surgery. the

Fig. 14 shows changes in cardiac function detected by echocardiography after 4 weeks of operation in TG mice and WT mice. the

Figure 15 shows the inhibitory effect of CKIP-1 on the transcriptional activity of myocardial enhancer factor MEF2C. the

Figure 16 shows the effect of exogenously expressed CKIP-1 on the intracellular localization of HDAC4. the

FIG. 17 is the PCR amplification program in Step 3-2 of Example 2. the

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. the

C57/BL mice (also known as wild-type mice or WT mice, represented by WT): purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SPF grade. the

ploxPI plasmid and pBluescript SK(+) plasmid: References (this document is also a document describing the construction of targeting vectors in detail) are as follows: Dr. Li Li's paper on gene targeting of important functional genes such as PACT derived from human fetal liver, 2005, Chinese People's Liberation Army Academy of Military Medicine. the

Mouse embryonic stem cells: References: LiL , Deng B , Xing G , Teng Y , Tian C , Cheng X , Yin X , Yang J , Gao X , Zhu Y , Sun Q , Zhang L , Yang X , He F. Proc Natl Acad Sci US A .PACT is a negative regulator of p53 and essential for cell growth and embryonic development. 2007May 8;104(19):7951-6. Epub 2007Apr 30.

pJG/ALPHA MHC plasmid: References: Gulick J, Robbins J. Cell-type-specific transgenesis in the mouse. Methods Mol Biol. 2009;561:91-104.. the

CKIP-1 expression plasmid (pCMV-Myc-CKIP-1 plasmid): References: Lu K, Yin X, Weng T, Xi S, Li L, Xing G, Cheng X, Yang X, Zhang L, He F. Target ing ww domains linker of hect-type ubiquitin ligase smurf1 for activation by ckip-1. Nat Cell Biol. 2008;10:994-1002. the

HDAC4 plasmid (Flag-epitope-tagged HDAC4 plasmid): References: Vega RB, Matsuda K, Oh J, Barbosa AC, Yang X, Meadows E, McAnally J, Pomajzl C, Shelton JM, Richardson JA, Karsenty G, Olson EN .Hi stone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis. Cell.2004Nov 12;119(4):555-66. the

MEF2C plasmid (Myc-tagged MEF2C plasmid): References: Arnold MA, Kim Y, Czubryt MP, Phan D, McAnally J, Qi X, Shelton JM, Richardson JA, Bassel-Duby R, Olson EN.MEF2C transcription factor controls chondrocyte hypertrophy and bone development. Dev Cell. 2007 Mar;12(3):377-89). the

pRL-TK plasmid: purchased from Promega (E2241). the

293T cells: purchased from the cell bank of the Basic Medical Cell Center of Peking Union Medical College. the

C2C12 cells: purchased from the cell bank of the Basic Medical Cell Center of Peking Union Medical College. the

pEGFP-N1-HDAC4 plasmid: References: Wang AH, Kruhlak MJ, Wu J, Bertos NR, Vezmar M, Posner BI, Bazett-Jones DP, Yang XJ. Regulation of histone deacetylase 4 by binding of 14-3-3proteins.Mol Cell Biol. 2000 Sep;20(18):6904-12. the

pIRES-DsRed-CKIP-1 plasmid: References: Zhang L, Xing G, Tie Y, Tang Y, Tian C, Li L, Sun L, Wei H, Zhu Y, He F. Role for the pleckstrin homology domain-containing protein CKIP-1in AP-1 regulation and apoptosis.EMBO J.2005Feb 23;24(4):766-78.

Both pIRES-DsRed plasmid and pEGFP-N1 plasmid were purchased from Clontech. the

Embodiment 1, the acquisition of CKIP-1 knockout mice

1. Obtaining CKIP-1 knockout mice

1. Construction of targeting carrier

The mouse CKIP-1 genome is a double-stranded DNA molecule shown in GENBANK ACCESSION NO.67220 (Gene ID: 67220, updated on 11-May-2012). the

(1) The mouse CKIP-1 genome was double digested with restriction endonucleases NotI and XhoI, and a fragment of about 1.4 kb (fragment A) was recovered. the

(2) Digest the ploxPI plasmid with restriction endonucleases NotI and XhoI to recover the vector backbone. the

(3) Ligate the fragment recovered in step (1) with the vector backbone of step (2) to obtain a recombinant plasmid. the

(4) The mouse CKIP-1 genome was double digested with restriction endonucleases EcoR I and SspI, and a fragment of about 9.6kb was recovered. the

(5) Digest the pBluescript SK(+) plasmid with restriction endonucleases EcoR I and SspI to recover the vector backbone. the

(6) Ligate the fragment recovered in step (4) with the vector backbone in step (5) to obtain a recombinant plasmid. the

(7) Digest the recombinant plasmid obtained in step (6) with restriction endonucleases EcoRI and Clal, and recover a fragment (fragment B) of about 9.6 kb. the

(8) Digest the recombinant plasmid obtained in step (3) with restriction endonucleases EcoRI and Clal to recover the vector backbone. the

(9) Ligate the fragment recovered in step (7) with the vector backbone in step (8) to obtain the targeting vector pTV-CKIP-1. Fragment A and Fragment B on the targeting vector can undergo homologous recombination with mouse genomic DNA, thereby knocking out the CKIP-1 gene. the

2. Transfect the targeting vector into mouse embryonic stem cells

After the targeting vector was linearized by Not I, it was electroporated (600V, 25μF) into mouse embryonic stem cells. 24 hours after electroporation, target cells were screened with 500 μg/ml G418 and 300 μg/ml hygromycin B. the

Embryonic stem cells containing the neomycin gene (neomycin) can be obtained by G418 screening, and the insertion of the targeting vector has occurred in this cell, but G418 screening cannot exclude cells that randomly integrate the targeting vector into the genome and acquire resistance; hygromycin B It is used to screen cells that do not contain thymidine kinase (tk), and the killed cells are cells in which the targeting vector is randomly integrated into the genome to obtain resistance. the

4. The target cells are injected into blastocysts, and then transplanted into the embryos of WT female mice. The mice produced by the female mice are chimeric mice of CKIP-1 (offspring generation). the

5. Mate the chimeric mice with WT mice, and the offspring mice obtained are heterozygous mice (second generation). the

6. Mating heterozygous mice to obtain offspring mice (three generations). the

2. Genotype identification of CKIP-1 knockout mice

Each of the third-generation mice obtained in step 1 was subjected to the following experimental steps:

1. Extract the genomic DNA of the mouse tail. the

2. Using the genomic DNA extracted in step 1 as a template, perform PCR amplification using primer pair A and primer pair B respectively. If a target sequence of about 500 bp is obtained with primer pair A and a target sequence of about 443 bp is obtained with primer pair B, the genotype of the mouse to be tested is CKIP-1+/-. If the target sequence of about 500 bp is obtained by using primer pair A and the target sequence of about 443 bp is not obtained by using primer pair B, the genotype of the mouse to be tested is CKIP-1-/-. If the target sequence of about 500 bp is not obtained by using primer pair A and the target sequence of about 443 bp is obtained by using primer pair B, the genotype of the mouse to be tested is CKIP-1+/+. the

Primer pair A consists of primer KO-1 and primer KO-2, used to identify knockout mutant mice, and the target sequence is about 500bp. Primer pair B consists of primer WT-1 and primer WT-2, used to identify wild-type mice, and the target sequence is about 443bp. the

KO-1: 5'-CCA GAC TGC CTT GGG AAA AGC GCC TCC CCT ACC-3';

KO-2: 5-Ttc ccc ctt tgt gaa gcc cca act ctt gac tc'-3'. the

WT-1: 5'-GTT CTG CTT TTG TCA CTA GAC ACT TGT TTT CTG CC-3';

WT-2: 5'-tgg ttt ccc ctc gga cct gta gga ag-3'. the

PCR reaction system (15μl): 0.5μl each of two primers (0.1μg/μl), 1.5μl 10×Buffer, 1.2μl dNTP (2.5mmol/L), 0.3μl Taq DNA polymerase (5U/μl), 1μl genomic DNA , add ddH ₂ O to make up to 15 μl.

PCR reaction program: 94°C for 3min; 94°C for 1min, 61°C for 50s, 72°C for 1min, cycle 38 times; 72°C for 7min. the

The mice whose genotype is CKIP-1-/- are selected as homozygous CKIP-1 knockout mice (also known as KO mice, denoted by KO). the

3. Acquisition of empty vector control mice

Use the ploxPI plasmid instead of the targeting vector to perform step 1 and 2 to obtain the empty vector control mouse A. the

Embodiment 2, KO mice and the comparison of parameters such as heart morphology and heart function of WT mice

1. Changes in cardiac tissue morphology

The hearts of 2-month (or 8-month) KO mice and WT mice were taken respectively, and the myocardial structure and fibrosis degree were detected by HE staining, WGA staining and MTT staining after tissue sections. the

The results are shown in Figure 1. Figure 1A is a photograph of HE staining, Figure 1B is a partially enlarged view of Figure 1A, Figure 1C is a photograph of MTT staining, and Figure 1D is a photograph of WGA staining. Compared with WT mice, heart volume and cardiomyocytes in KO mice were significantly larger at 2 months, and KO mice showed myocardial fibrosis at 8 months. the

The morphological changes of the hearts of the empty vector control mice A were consistent with those of the WT mice. the

2. Changes in cardiac index

Take 3 KO mice of 2 months, 3 KO mice of 8 months, 3 WT mice of 2 months and 3 WT mice of 8 months, and weigh them (measured in g), Then take the heart and weigh it (measured in mg), and calculate the cardiac index, which is the ratio of heart weight to body weight (mg/g). the

The results are shown in Figure 2 (average of 3 mice), ##P<0.01. The results showed that the cardiac index was significantly increased in KO mice compared with WT mice. the

The cardiac index of the empty vector control mice A was consistent with that of the WT mice. the

3. Expression of genes related to cardiac hypertrophy

Take 3 KO mice of 2 months, 3 KO mice of 8 months, 3 WT mice of 2 months and 3 WT mice of 8 months, and carry out the following steps respectively:

1. Take heart tissue and extract total RNA. the

2. Using total RNA as a template, carry out RT-PCR to detect the expression of each embryonic gene (ANP gene, BNP gene or β-MHC). the

RT-PCR reaction system: 5×Reaction buffer 5μl, substrate dNTP (10mmol/l) 0.5μl, MgSO4 (25mmol/l) 1μl, AMV reverse transcriptase (5U/μl) 0.5μl, Tfl DNA polymerase (5U /μl) 0.5μl, upstream primer (25pmol/μl) 1μl, downstream primer (25pmol/μl) 1μl, total RNA about 100ng, add RNase-free water to 25μl. the

The RT-PCR reaction program is shown in Figure 17. the

The primer pair used to detect the atrial natriuretic peptide gene (ANP gene) is as follows (the target sequence is 142bp):

Upstream primer: 5'-TTCGGGGGTAGGATTGACAG-3';

Downstream primer: 5'-CACACCCACAAGGGCTTAGGA-3'. the

The primer pair used to detect the brain natriuretic peptide gene (BNP gene) is as follows (the target sequence is 185bp):

Upstream primer 5'-TGTTTCTGCTTTTCCTTTATCTG-3';

Downstream primer 5'-TCTTTTTGGGTGTTCTTTTGTGA-3'. the

The primer pair used to detect the myosin heavy chain gene (β-MHC gene) is as follows (the target sequence is 110bp):

Upstream primer 5'-TCCCCAACCGCATTCTCTAT-3';

Downstream primer 5'-CAGTTTCTCAGCCCCCTTTCC-3'. the

Taking the expression level of genes in WT mice in the same month as 1, the relative expression levels of genes in each embryonic stage of KO mice in each month were calculated, and the results are shown in FIG. 3 . The results showed that compared with WT mice, the expression of ANF gene, BNP gene and β-MHC gene in the heart tissue of KO mice showed a tendency to increase at 2 months, and this difference was more obvious at 8 months. the

The expression levels of each gene in the empty vector control mice were consistent with those in the WT mice. the

4. Echocardiographic testing of cardiac function

Take 8 KO mice of 2 months, 4 KO mice of 8 months, 9 WT mice of 2 months and 5 WT mice of 8 months, and carry out the following experimental steps respectively:

Mice were intraperitoneally injected with chloral hydrate (450mg/kg), using an ultrasonic diagnostic instrument (the probe was 15W, the Doppler detection frequency was 14.0MHz, the instrument gain was fixed at 65dB in the test, the image depth was adjusted to 30mm, and the probe was placed on the left side of the sternum On the left side, at 10°-30° to the midline of the sternum, M-mode scans were performed at the level of the left ventricular long-axis and short-axis chordal tendons) to measure left ventricular function-related indicators. the

The results are shown in Table 1. the

Table 1 Echocardiographic test results of cardiac function (mean ± standard deviation) 

^A P<0.05, ^B P<0.01 compared to WT mice.

IVSd ventricular septal end-diastolic thickness; IVSs ventricular septal end-systolic thickness; LVPWd left ventricular end-diastolic posterior wall thickness; LVPWs left ventricular end-systolic posterior wall thickness; LVDd left ventricular end-diastolic diameter; LVDs left ventricular end-systolic diameter; EF stands for left ventricle Ejection fraction, FS stands for fractional shortening of left ventricle. the

The results showed that knockout of CKIP-1 gene at 2 months could increase the thickness of the ventricular septum and wall in mice, and the compensatory enhancement of cardiac systolic function, and the short-axis shortening rate and Left ventricular ejection fraction was significantly lower than in WT mice. the

The relevant indicators of the empty vector control mouse A were consistent with those of the WT mice. the

5. Observation of HDAC4 localization changes by tissue immunofluorescence

The hearts of 8-month-old KO mice and WT mice were taken respectively, and frozen sections were made, dried in the air, soaked in -20°C frozen acetone for 5 minutes, and then sealed with 5% fetal bovine serum at 37°C 30 minutes, then incubated with HDAC4 antibody (Santa Cruz) and CKIP-1 antibody (Cell Signaling), and then detected with FITC-labeled anti-rabbit secondary antibody and rhodamine-labeled anti-goat secondary antibody respectively, laser co- Focusing microscope observation. the

The results are shown in Figure 4. The results showed that in the cardiomyocytes of WT mice, HDAC4 was localized in the cytoplasm and nucleus, and in the cardiomyocytes of KO mice, HDAC4 was mainly localized in the cytoplasm. the

6. Western Blotting test to detect changes in phosphorylation levels of proteins related to cardiac hypertrophy in myocardial tissue

Extract the myocardial tissue proteins of 8-month-old KO mice and WT mice, add protease and phosphatase inhibitors, electrophoresis in 10% polyacrylamide gel, transfer to nitrocellulose membrane, and incubate with different antibodies After hybridization with GAPDH as an internal reference, it was incubated with a secondary antibody labeled with horseradish peroxidase and detected with a chemiluminescence kit. the

The results are shown in Figures 5 and 6. In the myocardial tissue of KO mice, the phosphorylation level of HDAC4 was significantly higher than that of WT mice, and the phosphorylation level of Akt/mTOR/S6K was significantly higher than that of WT mice. the

The results of steps 1 to 6 showed that: compared with wild-type mice, the cardiomyocytes of 2-month-old CKIP-1 gene knockout mice were significantly larger, the cardiac index was significantly increased, and fetal period genes (ANP gene, BNP gene, CKIP-1 gene and β-MHC gene) expression levels were significantly up-regulated, cardiac left ventricular function was enhanced compensatoryly, ejection fraction and left ventricular short-axis shortening rate were increased; compared with wild-type mice, 8-month-old CKIP-1 gene In addition to the further enlargement of myocardial cells in mice, the further increase of cardiac index, and the further significant up-regulation of fetal gene expression, however, the ejection fraction and cardiac short-axis shortening rate showed a significant decrease, and the above indicators were in line with the symptoms of pathological cardiac hypertrophy; CKIP-1 The phosphorylation levels of HDAC4, AKT, mTOR and S6K in the myocardium of knockout mice were significantly increased; the above results indicated that CKIP-1 knockout mice showed spontaneous cardiac hypertrophy with age. the

Example 3. Confirmation of the role of CKIP-1 gene through the mouse cardiac hypertrophy model (KO mice and WT mice)

1. Making a mouse model of myocardial hypertrophy (arch arterial constriction)

KO mice and WT mice were subjected to the following experiments:

Model group (mouse cardiac hypertrophy model, represented by TAC): 8-week-old mice were anesthetized with tribromoethanol, connected to a small animal ventilator through tracheal intubation, and artificially ventilated. -3 intercostals to open the thorax, separate the aortic arch and thread a thread under it, make the aorta constriction (Transverse aortic constriction, TAC) according to the uniform standard, and close the thorax. the

Sham operation group (indicated by Sham): except that TAC was not performed, the rest of the operation procedures were completely the same as the model group. the

Arch artery constriction is a routine method, and the literature describing arch artery constriction is: deAlmeida,A.C.,van Oort,R.J.,Wehrens,X.H.Transverse Aortic Constriction in Mice.J.Vis.Exp.(38),e1729,DOI : 10.3791/1729 (2010). the

2. Changes in cardiac tissue morphology

The hearts of KO mice and WT mice 4 weeks after the operation in Step 1 were taken, photographed, and then tissue sections were performed for HE staining and MTT staining respectively. the

The results are shown in Figure 7. Figure 7A is a photograph of the heart, Figure 7B is a photograph of HE staining, Figure 7C is a partially enlarged view of Figure 7B, and Figure 7D is a photograph of MTT staining. The results showed that CKIP-1 gene knockout exacerbated the occurrence of myocardial hypertrophy induced by arch artery constriction, showing significant enlargement of the myocardium, accompanied by severe fibrosis. the

3. Changes in cardiac index

Take 3 KO mice 4 weeks after the operation of Step 1 (model group), 3 KO mice 4 weeks after the operation of Step 1 (sham operation group), and 3 WT mice 4 weeks after the operation of Step 1 Rats (model group), 3 WT mice (sham operation group) 4 weeks after the operation in Step 1 were weighed (the measurement unit is g), and then the heart was taken out and weighed (the measurement unit is mg), Calculate the cardiac index, which is the ratio of heart weight to body weight (mg/g). the

The results are shown in Figure 8 (average of 3 mice), **P<0.01 compared to sham, #P<0.05, ##P<0.01 compared to WT mice. The results showed that the cardiac index of KO mice was significantly larger than that of WT mice after arch artery constriction. the

4. Expression of genes related to cardiac hypertrophy

Take 3 KO mice 4 weeks after the operation of Step 1 (model group), 3 KO mice 4 weeks after the operation of Step 1 (sham operation group), and 3 WT mice 4 weeks after the operation of Step 1 The mice (model group) and 3 WT mice (sham-operated group) 4 weeks after the operation in Step 1 were subjected to the experiment in Step 3 of Example 2, respectively. the

The results are shown in Figure 9, TAC- represents the sham operation group, and TAC+ represents the model group. It was found that after surgery, the expression ratios of ANF gene, BNP gene and β-MHC gene in myocardial tissue of KO mice were significantly higher than those of WT mice. the

5. Echocardiographic testing of cardiac function

Take 9 KO mice 2 weeks after the operation of Step 1 (model group), 9 KO mice 2 weeks after the operation of Step 1 (sham operation group), 9 WT mice 2 weeks after the operation of Step 1 Mice (model group), 9 WT mice 2 weeks after the operation of step 1 (sham operation group), 9 KO mice 4 weeks after the operation of step 1 (model group), 9 mice of 4 weeks after the operation of step 1 KO mice after 1 week (sham operation group), 9 WT mice 4 weeks after operation in step 1 (model group), 9 WT mice 4 weeks after operation in step 1 (sham operation group), respectively Carry out the test of step 4 of embodiment 2. the

Table 2 shows the echocardiographic results of cardiac function of WT mice and KO mice 4 weeks after surgery. the

The echocardiographic results of cardiac function of KO mice in the model group and WT mice in the model group after 2 or 4 weeks of operation are shown in Figure 10 ( ^* P<0.05, ^** P<0.01, ^*** P<0.001, compared with sham Compared with the operated group; ^#P <0.05, ^## P<0.01, ^### P<0.001, compared with WT mice).

Please review, as mentioned in the description above, Table 2 shows the results of WT mice and KO mice

The results showed that KO mice exhibited significant heart failure. the

Embodiment 4, the acquisition of trans CKIP-1 gene mouse

1. Construction of myocardial specific expression vector

1. Synthesize the CKIP-1 gene shown in sequence 2 of the sequence listing, use the CKIP-1 gene as a template, and perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product. the

F1: 5'-CCC AAGCTT ATGGACTACAAAGACGATGACGACAAGATGAAGAAGAGCGGCTCCGGCAAG-3',

R1: 5'-CCC AAGCTTT CACATCAGGCTCTTCCGGTATT-3'.

Flag tags were introduced in F1. the

2. Digest the PCR amplified product in step 1 with restriction endonuclease HindIII, and recover the digested product. the

3. Digest the pJG/ALPHA MHC plasmid with restriction endonuclease HindIII, and recover the vector backbone (about 4500bp). the

4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain the recombinant plasmid α-MHC-CKIP-1 (cardiac-specific expression vector). the

2. Acquisition of transgenic CKIP-1 mice

1. Digest the recombinant plasmid α-MHC-CKIP-1 with the restriction endonuclease NotI, and recover the linearized plasmid. the

2. Microinject the linearized plasmid into the male pronucleus of fertilized eggs of WT mice, and transplant the viable fertilized eggs into the fallopian tubes of WT surrogate mice. the

3. After the birth of the surrogate mice, the offspring mice obtained (the first generation) were identified by PCR using a primer pair composed of F2 and R2, and the mice with the target band of about 330 bp were positive mice for PCR identification. the

F1: 5'–GACTAACTAGAAGCTTATGGACTA-3';

R2: 5'-CCAGGGTGAACTTGCTGTGAT-3'. the

4. Mate the PCR-identified positive mice obtained in step 3 with WT mice to obtain offspring mice (second generation), and use the primer pair composed of F2 and R2 for PCR identification, and the mice with the target band of about 330bp Positive mice for PCR identification. the

5. Mate the PCR-identified positive mice obtained in step 4 with WT mice to obtain offspring mice (three generations), and use the primer pair composed of F2 and R2 for PCR identification. The mouse with a target band of about 330bp is The positive mice identified by PCR were the TG mice used in Example 5. the

3. Acquisition of Empty Vector Control Mouse B

The pJG/ALPHA MHC plasmid was used to replace the recombinant plasmid α-MHC-CKIP-1 for the experiment in step 2, and the empty vector control mouse B was obtained. the

Example 5. Confirmation of the role of CKIP-1 gene through the mouse cardiac hypertrophy model (TG mice and WT mice)

1. Making a mouse model of myocardial hypertrophy (arch arterial constriction)

TG mice were used instead of KO mice, and the others were the same as Step 1 of Example 3. the

2. Changes in cardiac tissue morphology

TG mice were used instead of KO mice, and the rest were the same as Step 2 of Example 3. the

The results are shown in Figure 11. Figure 11A is a photograph of the heart, Figure 11B is a photograph of HE staining, Figure 11C is a partially enlarged view of Figure 11B, and Figure 11D is a photograph of MTT staining. The results showed that the overexpression of CKIP-1 gene attenuated the occurrence of myocardial hypertrophy induced by arch artery constriction, and compared with WT mice, TG mice showed a smaller myocardium and less fibrosis. The morphological changes of the hearts of the empty vector control mice B were consistent with those of the WT mice. the

3. Changes in cardiac index

Take 5 TG mice 4 weeks after the operation of step 1 (model group), 6 TG mice 4 weeks after the operation of step 1 (sham operation group), 6 WT mice 4 weeks after the operation of step 1 Rats (model group), 8 WT mice (sham operation group) 4 weeks after the operation in Step 1 were weighed (the measurement unit is g), and then the heart was taken and weighed (the measurement unit was mg), and the The left ventricle was weighed (measured in mg). Calculate the cardiac index, which is the ratio of heart weight to body weight (mg/g). Calculate the left ventricular index, which is the ratio of left ventricular weight to body weight (mg/g). the

The results are shown in Figure 12 (average of 3 mice), **P<0.01 compared with sham, #P<0.05, ##P<0.01 compared with WT mice. The results showed that after arch artery constriction, the cardiac index and left ventricular index of TG mice were significantly smaller than those of WT mice. The change of cardiac index in empty vector control mice B was consistent with that in WT mice. the

4. Expression of genes related to cardiac hypertrophy

Take 4 TG mice 4 weeks after the operation of Step 1 (model group), 4 TG mice 4 weeks after the operation of Step 1 (sham operation group), 5 WT mice 4 weeks after the operation of Step 1 The mice (model group) and 5 WT mice (sham-operated group) 4 weeks after the operation in Step 1 were subjected to the experiment in Step 3 of Example 2, respectively. the

The results are shown in Figure 13. It was found that after surgery, the expression ratios of ANF gene, BNP gene and β-MHC gene in myocardial tissue of TG mice were significantly lower than those of WT mice. The expression of each related gene in the empty vector control mice B was consistent with that in the WT mice. the

5. Echocardiographic testing of cardiac function

Take 5 TG mice 4 weeks after the operation of step 1 (model group), 6 TG mice 4 weeks after the operation of step 1 (sham operation group), 6 WT mice 4 weeks after the operation of step 1 The mice (model group) and 8 WT mice (sham operation group) 4 weeks after the operation in Step 1 were respectively subjected to the test of Step 3 of Example 2, and the test of Step 4 of Example 2 respectively. the

The results are shown in Figure 14. The results showed that the ejection fraction and short-axis shortening rate of TG mice were significantly higher than those of WT mice. The ejection fraction and short-axis shortening rate of the empty vector control mice B were consistent with those of WT mice. the

Example 6 Fluorescence double reporter experiment confirms the inhibitory effect of CKIP-1 on MEF2C

293T cells were cultured in DMEM containing 10% FBS and cultured in a 24-well plate. When the cell density reached 60%, the following parallel transfection treatments were carried out:

The first group: blank control group;

The second group: co-transfect MEF2C luciferase reporter plasmid and pRL-TK plasmid, the transfection dose is 0.4 μg MEF2C luciferase reporter plasmid and 0.4 μg pRL-TK plasmid per well;

The third group: transfection with CKIP-1 expression plasmid, the transfection dose is 0.4 μg of CKIP-1 expression plasmid per well;

The fourth group: co-transfect MEF2C luciferase reporter plasmid, pRL-TK and CKIP-1 expression plasmids, the transfection dose is 0.4 μg MEF2C luciferase reporter plasmid, 0.4 μg pRL-TK plasmid and 0.4 μg per well CKIP-1 expression plasmid;

The fifth group: co-transfect MEF2C luciferase reporter plasmid, pRL-TK and CKIP-1 expression plasmid, the transfection dose is 0.4 μg MEF2C luciferase reporter plasmid, 0.4 μg pRL-TK plasmid and 0.4 μg per well CKIP-1 expression plasmid;

Group 6: Co-transfect MEF2C luciferase reporter plasmid, pRL-TK and CKIP-1 expression plasmids, the transfection dose is 0.4 μg MEF2C luciferase reporter plasmid, 0.4 μg pRL-TK plasmid and 0.4 μg per well CKIP-1 expression plasmid. the

After 18 hours of transfection, it was observed under a fluorescent microscope, and the results are shown in FIG. 15 . The results of reporter gene experiments showed that exogenous transfection of CKIP-1 gene can significantly inhibit the transcriptional activity of MEF2C in a dose-dependent manner. the

Example 6. In vitro co-transfection experiment to detect the effect of CKIP-1 on the intracellular localization of HDAC4

The C2C12 cells were subjected to the following parallel transfection treatments in a 6-well plate:

The first group: transfection with pEGFP-N1-HDAC4 plasmid, the transfection dose is 2 micrograms of pEGFP-N1-HDAC4 plasmid per well;

The second group: transfection with pIRES-DsRed-CKIP-1, the transfection dose is 2 micrograms of pIRES-DsRed-CKIP-1 plasmid per well;

The third group: co-transfection of pGFP-N1-HDAC4 and pIRES-DsRed, the transfection dose is 2 micrograms of pEGFP-N1-HDAC4 plasmid and 2 micrograms of pIRES-DsRed plasmid per well;

The fourth group: co-transfect pEGFP-N1 and pIRES-DsRed-CKIP-1, the transfection dose is 2 micrograms of pEGFP-N1 plasmid and 2 micrograms of pIRES-DsRed-CKIP-1 plasmid per well;

The fifth group: co-transfect pEGFP-N1-HDAC4 and pIRES-DsRed-CKIP-1, the transfection dose is 2 μg pEGFP-N1-HDAC4 plasmid and 2 μg pIRES-DsRed-CKIP-1 plasmid per well. the

The effect of CKIP-1 expression on HDAC4 localization was observed by confocal laser microscopy, and the results are shown in FIG. 16 . In C2C12 cells, exogenously transfected RFP-tagged CKIP-1 protein was mainly localized in the cytoplasm, and GFP-tagged HDAC4 was mainly localized in the cytoplasm. When the two were co-transfected, CKIP-1 affected the localization of HDAC4, making it mainly localized in the nucleus. The results showed that CKIP-1 could promote HDAC4 to enter the nucleus from the cytoplasm, and enhance the inhibitory effect of intracellular HDAC4 on MEF2C. the

Claims (7)

1. Application of the CKIP-1 protein, the coding gene of the CKIP-1 protein or the plasmid containing the coding gene of the CKIP-1 protein in the preparation of a drug for preventing and/or treating cardiac hypertrophy; the CKIP-1 protein As shown in sequence 1 of the sequence listing. 2. The application according to claim 1, characterized in that: the gene encoding the CKIP-1 protein is the DNA molecule described in any one of the following 1)-4): 1) The DNA molecule shown in the sequence 2 from the 43rd to the 1266th nucleotide at the 5' end in the sequence listing; 2) The DNA molecule shown in sequence 2 in the sequence listing; 3) A DNA molecule that hybridizes to the DNA molecule shown in 1) or 2) under stringent conditions and encodes the protein; 4) A DNA molecule that has more than 90% homology with the gene in 1) or 2) or 3) and encodes the protein. 3. application as claimed in claim 1 or 2, is characterized in that: described CKIP-1 albumen, the encoding gene of described CKIP-1 albumen or the plasmid that contains the encoding gene of described CKIP-1 albumen have effect on myocardial hypertrophy. The preventive and/or therapeutic effect is reflected in at least one of the following (a) to (e): (a) promote the reduction of cardiomyocytes and the reduction of myocardial fibrosis; (b) reduce cardiac index; (c) decrease left ventricular index; (d) reducing the expression level of a prenatal gene in cardiac tissue; (e) Increased left ventricular ejection fraction and ventricular fractional shortening. 4. Application of the CKIP-1 protein, the gene encoding the CKIP-1 protein or the plasmid containing the gene encoding the CKIP-1 protein in the preparation of products; the CKIP-1 protein is shown in sequence 1 of the sequence listing ; The product is a product with at least one of the following functions (a) to (e): (a) promote the reduction of cardiomyocytes and the reduction of myocardial fibrosis; (b) reduce cardiac index; (c) decrease left ventricular index; (d) reducing the expression level of a prenatal gene in cardiac tissue; (e) Increased left ventricular ejection fraction and ventricular fractional shortening. 5. The application according to claim 4, characterized in that: the gene encoding the CKIP-1 protein is the DNA molecule described in any one of the following 1)-4): 1) The DNA molecule shown in the sequence 2 from the 43rd to the 1266th nucleotide at the 5' end in the sequence listing; 2) The DNA molecule shown in sequence 2 in the sequence listing; 3) A DNA molecule that hybridizes to the DNA molecule shown in 1) or 2) under stringent conditions and encodes the protein; 4) A DNA molecule that has more than 90% homology with the gene in 1) or 2) or 3) and encodes the protein. 6. The application of the substance used to inhibit the expression of the gene encoding CKIP-1 protein in the preparation of products; the CKIP-1 protein is shown in sequence 1 of the sequence listing; the product has the following (1) to (4) Products with at least one feature in: (1) Make an animal model of cardiac hypertrophy; (2) Promote myocardial cell enlargement and myocardial fibrosis; (3) Increase cardiac index; (4) Increase the expression level of fetal period genes in heart tissue. 7. The application according to claim 6, characterized in that: the gene encoding the CKIP-1 protein is the DNA molecule described in any one of the following 1)-4): 1) The DNA molecule shown in the sequence 2 from the 43rd to the 1266th nucleotide at the 5' end in the sequence listing; 2) The DNA molecule shown in sequence 2 in the sequence listing; 3) A DNA molecule that hybridizes to the DNA molecule shown in 1) or 2) under stringent conditions and encodes the protein; 4) A DNA molecule that has more than 90% homology with the gene in 1) or 2) or 3) and encodes the protein.

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