Dilated cardiomyopathy zebra fish model and construction method and application thereof

Abstract

The invention provides a dilated cardiomyopathy zebrafish model and a construction method and application thereof, and belongs to the technical field of biology.The dilated cardiomyopathy zebrafish model is constructed by overexpressing miR-16-5p in myocardial cells of zebrafish, and the sequence of miR-16-5p is shown as SEQ ID NO.1; the invention further provides a construction method of the dilated cardiomyopathy zebra fish model. The invention constructs the dilated cardiomyopathy zebrafish model of overexpression miR-16-5p in myocardial cells for the first time, and the dilated cardiomyopathy zebrafish model can be used for screening drugs for resisting dilated cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a zebrafish model of dilated cardiomyopathy and a construction method and application thereof. Background Art

[0002] Dilated cardiomyopathy (DCM) is a heterogeneous cardiomyopathy characterized by ventricular enlargement and decreased myocardial systolic function, with a wide range of clinical manifestations. Patients often experience symptoms such as left ventricular failure, arrhythmias, and thromboembolism. The incidence of DCM is increasing worldwide, with an annual incidence of approximately 1 in 2,500. As a cardiovascular disease that poses a serious threat to public health, DCM poses a serious threat to patients' health and life. However, current clinical medications primarily focus on symptomatic treatment, such as angiotensin-converting enzyme inhibitors (ACEIs), beta-blockers, and aldosterone receptor antagonists, which alleviate symptoms and improve cardiac function but cannot completely cure the disease. Some drugs can also have significant side effects during treatment. For example, while inotropic drugs can enhance myocardial contractility, they may increase myocardial oxygen consumption, potentially inducing arrhythmias. Long-term use can also increase cardiac workload and adversely affect patient prognosis. Therefore, identifying innovative drugs for the treatment of DCM has become an urgent clinical need.

[0003] Traditional mammalian models, such as mice, are subject to a series of challenges, including high experimental costs, demanding technical skills, and long experimental cycles. While in vitro cell models can evaluate the activity of a small number of compounds, they cannot accurately reflect the compounds' metabolic processes and mechanisms of action in vivo. These limitations make them difficult to meet the needs of large-scale early screening of anti-DCM drugs. There is an urgent need to develop new activity evaluation technologies that are both consistent with the characteristics of DCM and capable of efficiently screening compounds in the early stages of new drug development, thereby improving the efficiency of lead compound screening.

[0004] The zebrafish, a model animal, shares a high degree of cardiac structure and function with mammals. Both hearts are composed of atria and ventricles, and their diastolic and systolic mechanisms are highly conserved. Zebrafish share a high degree of genetic homology with humans, with approximately 87% of human genes having corresponding orthologs in zebrafish. These include many key genes implicated in dilated cardiomyopathy (DCM), such as cardiac sarcomeric proteins (MYH7 and TNNT2). The transparency of zebrafish embryos allows for the specific labeling of cardiac cells using fluorescent protein tagging. Combined with microscopy and image processing software, precise analysis of pericardial area, sinus venosus-bulbus arteriosus (SV-BA) distance, and cardiac function parameters such as stroke volume, fractional shortening, and ejection fraction can be performed. Furthermore, the high-throughput nature of zebrafish makes them ideal for large-scale drug screening and gene function studies. Large numbers of embryos or larvae can be cultured and processed simultaneously, enabling rapid screening of drugs that affect cardiac development and function. This accelerates the discovery of new therapeutic targets and drugs, providing a wider range of potential drug options for the clinical treatment of DCM. These significant advantages make the zebrafish model play an important role in the study of the pathogenesis of DCM and drug screening, providing a powerful tool for heart disease drug research.

[0005] The etiology of dilated cardiomyopathy (DCM) is complex, primarily involving multiple factors, including genetics, infection, autoimmunity, alcohol, and medication. Genetic mutations and genetic factors are key intrinsic contributors to DCM. Numerous studies have demonstrated that mutations in various genes are closely associated with the pathogenesis of DCM, with mutations in genes such as titin (TTN), lamin A (LMNA), MYH7, MYH6, and TNNT2 being particularly common. In addition to these gene mutations, recent studies have also revealed that microRNA-16-5p (miR-16-5p) plays a crucial regulatory role in the development and progression of dilated cardiomyopathy (DCM). In normal cardiomyocytes, miR-16-5p expression is relatively stable, regulating the expression of a range of genes involved in cell survival and apoptosis. However, the application of miR-16-5p in establishing a zebrafish DCM model has not been reported. Summary of the Invention

[0006] In response to the deficiencies of the existing technology, the present invention provides a zebrafish model of dilated cardiomyopathy and a construction method and application thereof.

[0007] The technical solutions of the present invention are as follows: A zebrafish model of dilated cardiomyopathy is constructed by overexpressing miR-16-5p in zebrafish cardiomyocytes, wherein the sequence of miR-16-5p is shown in SEQ ID NO. 1; SEQ ID NO. 1: UAGCAGCACGUAAAUAUUGGCG.

[0008] Preferably, according to the present invention, the dilated cardiomyopathy zebrafish model is constructed by overexpressing miR-16-5p in the cardiomyocytes of wild-type AB zebrafish.

[0009] Further preferably, the dilated cardiomyopathy zebrafish model is constructed by introducing the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRSv40 recombinant plasmid into AB strain zebrafish; The nucleotide sequence of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid is shown in SEQ ID NO.2.

[0010] The method for constructing the above-mentioned dilated cardiomyopathy zebrafish model comprises the following steps: (1) Constructing a Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid with a nucleotide sequence as shown in SEQ ID NO.2; (2) The recombinant plasmid Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 and Tol2 transposase mRNA constructed in step (1) were introduced into AB zebrafish embryos by microinjection, and zebrafish fry that specifically expressed green fluorescence in the heart were cultured and selected to obtain the F0 generation; (3) The F0 generation zebrafish selected in step (2) are raised to adulthood and then hybridized with AB zebrafish to obtain the F1 generation. The zebrafish that specifically express green fluorescence in the heart are cultured and selected. The genome of the F1 generation zebrafish embryos is extracted for verification to confirm that the F1 generation zebrafish has successfully inherited the target gene; (4) The F1 generation zebrafish selected in step (3) were raised to adulthood and then self-fertilized to obtain the F2 generation. The zebrafish that specifically expressed green fluorescence in the heart were cultured and selected. The genome of the F2 generation zebrafish embryos was extracted for verification to confirm that the F2 generation zebrafish successfully inherited the target gene. The F2 generation zebrafish and their offspring were all transgenic zebrafish Tg( cmlc2: miR-16-5p, EGFP ), which is a zebrafish model of dilated cardiomyopathy.

[0011] Preferably, according to the present invention, in step (3) or step (4), the verification method is to perform PCR amplification on the zebrafish genome using primers Primer-F and Primer-R, and sequence and identify the amplified products; the sequence of Primer-F is shown as SEQ ID NO.3, and the sequence of Primer-R is shown as SEQ ID NO.4.

[0012] According to the present invention, preferably, in step (4), the transgenic zebrafish Tg ( cmlc2: miR-16-5p, EGFP ) were cultured to 2-3 dpf as a zebrafish model of dilated cardiomyopathy.

[0013] Use of the above-mentioned zebrafish model of dilated cardiomyopathy or the zebrafish model of dilated cardiomyopathy constructed by the above-mentioned method in screening drugs for preventing or treating dilated cardiomyopathy.

[0014] The beneficial effects of the present invention include at least the following: 1. The inventors discovered for the first time that overexpression of miR-16-5p in zebrafish cardiomyocytes resulted in typical dilated cardiomyopathy phenotypes in zebrafish at 2-3 dpf, including pericardial edema, increased SV-BA distance, and significantly decreased stroke volume, fractional shortening, and ejection fraction.

[0015] 2. In view of the current problems of numerous cardiovascular disease targets and the difficulty in establishing experimental animal models suitable for screening complex and trace natural products, this paper first used miR-16-5p to construct a zebrafish model of dilated cardiomyopathy Tg (cmlc2: miR-16-5p, EGFP) , established a new technology for evaluating the protective activity of compounds against dilated cardiomyopathy based on whole zebrafish animals, provided a theoretical basis and experimental basis for the screening of new drugs against dilated cardiomyopathy, and solved the problem of the lack of an efficient evaluation model for the activity of lead compounds in the early stage of new drug development against dilated cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the map of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid.

[0017] Figure 2 This is the identification diagram of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid; In the figure: A is the Sanger sequencing result of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid, in which the blue box is the cmlc2 partial promoter sequence, the red box is the miR-16-5p sequence, and the green box is the IRES2 partial DNA sequence; B is the electrophoresis band of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid double enzyme digestion.

[0018] Figure 3 The electrophoresis and sequencing images of F1 generation miR-16-5p overexpressing zebrafish; In the figure: A is a fluorescence microscopic observation of 48 hpf miR-16-5p-overexpressing zebrafish; B is an agarose electrophoresis diagram of PCR products, in which 1 is a DNA marker (250, 1000, 5000, 7500, 10000, 15000 bp from bottom to top), 2 is a negative control without template, 3 is a positive control with plasmid template, and 4 is a PCR amplification product of F1 genomic DNA fragment; C is a PCR sequencing result diagram, in which the blue box is the cmlc2 partial promoter sequence, the green box is the miR-16-5p sequence, and the black box is the IRES2 partial DNA sequence.

[0019] Figure 4 The electrophoresis and sequencing images of F2 generation miR-16-5p overexpressing zebrafish; In the figure: A is the agarose electrophoresis diagram of PCR products; B is the PCR sequencing results.

[0020] Figure 5 The zebrafish heart morphology and statistical diagram; In the figure: A is the morphological diagram of zebrafish in each group; B is the statistical diagram of zebrafish pericardial area; C is the statistical diagram of zebrafish SV-BA distance; Compared with the blank control group, ** p<0.01, *** p<0.001, **** p<0.0001.

[0021] Figure 6 This is a statistical chart of zebrafish cardiac function; In the figure: A is a statistical diagram of zebrafish stroke volume; B is a statistical diagram of zebrafish ejection fraction; C is a statistical diagram of zebrafish short axis shortening rate; Compared with the blank control group, * p<0.05, ** p<0.01, **** p<0.0001.

[0022] Figure 7 A diagram of zebrafish heart pathology.

[0023] Figure 8 This is an effect diagram of the expression levels of dilated cardiomyopathy-related genes in 2-3 dpf zebrafish; In the figure: compared with the blank control group, **** p<0.0001.

[0024] Figure 9 This is a diagram showing the effects of clinical drugs on the cardiac morphology of zebrafish with dilated cardiomyopathy; In the figure: A is the morphological diagram of zebrafish in each group, the red dotted box is the heart area, and the orange arrow indicates pericardial edema; B is the statistical diagram of zebrafish pericardial area; C is the statistical diagram of zebrafish SV-BA distance; compared with the blank control group, ** p<0.01, **** p<0.0001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0025] Figure 10 The effects of clinical drugs on cardiac function in zebrafish with dilated cardiomyopathy; In the figure: A is the statistical graph of the stroke volume of zebrafish in each group; B is the statistical graph of the ejection fraction of zebrafish; C is the statistical graph of the short axis shortening rate of zebrafish; compared with the blank control group, *** p<0.001, **** p<0.0001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0027] In the examples, the contents without specific conditions were carried out under conventional conditions; the reagents or instruments used without specifying the manufacturer were all common commercial products.

[0028] 1 Materials and Instruments 1.1 Experimental Reagents PCS-tp plasmid (Yao Shun Yu Biotechnology); TAE Buffer (Shanghai Bioengineering, Cat. No. B548101-0500), DL2000 Plus DNA Marker (Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. MD101-01), UltraGelRed (Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. GR501-01), 2 × Rapid Taq Master Mix (Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. P222-01), Agarose (Yisheng, Cat. No. 10208ES60), FastDigest NotI (Thermo Fisher Scientific, Cat. No. FD0594), Potassium chloride (Shanghai Bioengineering, Cat. No. A610440-0500), Calcium chloride dihydrate (Shanghai Bioengineering, Cat. No. A610050-0500), Magnesium chloride hexahydrate (Shanghai Bioengineering, Cat. No. A601336-0500), Methylene blue (Sigma-Aldrich, Cat. No. 5477-0500), 1,2-Dihydroquinone ... Aldrich, Product No. M9140), 1-phenyl-2-thiourea (Sigma-Aldrich, Product No. P7629), MS-222 (Sigma-Aldrich, Product No. A5040), 10× DNA Loading Buffer (Nanjing Novozymes Biotechnology Co., Ltd., Product No. P022-01), YSY Buffer (Nanjing Yaoshunyu Biotechnology Co., Ltd., Product No. K-101-100), 1kb DNA Marker (Beijing Biolab Technology Co., Ltd., Product No. M20024); PCR Clean-up Kit (Axygen, Product No. AP-PCR-50), mMESSAGE mMACHINE™ SP6 transcription kit (Thermo Fisher Scientific, Catalog No. AM1340); restriction endonuclease ApaI (NEB, Catalog No. R0114L), restriction endonuclease XhoI (NEB, Catalog No. R0146M); dapagliflozin (AbMole, Catalog No. 461432-26-8); empagliflozin (AbMole, Catalog No. 98418-47-4).

[0029] 1.2 Instruments and equipment used in the experiment Zebrafish embryo microinjection instrument IM-300 (Narishige, Japan), Biori New XP gene amplification instrument TC-XP-G (Bori, Hangzhou), zebrafish breeding system (Beijing Aisheng Technology Co., Ltd.), horizontal electrophoresis instrument HE-120 (Tanon), Tanon 1600 series multifunctional gel image analysis system Tanon 1600 (Tanon), fluorescence microscope MZX81 (Mingmei), electric constant temperature ancient style drying oven DH6-903385-III (Xin Miao), desktop centrifuge Sorvall Legend Micro 17 (ThermoFisher), low-temperature centrifuge 5428000295 (Eppendorf), Aosheng Nano-100 micro-spectrophotometer Nano-100 (Aosheng, Hangzhou), desktop centrifuge (Sorvall Legend Micro 17, ThermoFisher); constant temperature water bath 20L (DWB20-P, SCILOGEX); stereo fluorescence microscope SZX16 (Olympus, Japan), Zeiss fluorescence microscope AXIO-V16 (Carl Zeiss Optics GmbH, Germany), real-time fluorescence quantitative PCR instrument Light Cycler@ 96 (Roche GmbH, Switzerland).

[0030] 1.3 Experimental Animals The wild-type AB zebrafish (abbreviated as AB zebrafish) and the heart-marked green fluorescent transgenic zebrafish were used in this experiment. Tg (cmlc2: EGFP) , which can be purchased from the National Zebrafish Resource Center. Commercially available products can be used. Adult zebrafish were maintained in separate tanks, sex-specific, in a zebrafish culture system. The water temperature was maintained at a constant 28°C, with a 2:10 light-dark cycle. Zebrafish were fed twice daily, with brine shrimp in the morning and solid feed in the afternoon. For experimental eggs, male and female zebrafish were placed on opposite sides of the ovulation tank (separated by a partition, which was removed at 8:00 the following day), with a male-female ratio of 2:2. The following morning, the collected zebrafish embryos were sterilized in zebrafish culture water containing methylene blue and then incubated in a 28°C temperature-controlled light incubator. The culture water was changed regularly to ensure normal embryonic development and smooth experimental progress.

[0031] 2 Experimental methods 2.1 Construction of a dilated cardiomyopathy model in zebrafish with miR-16-5p overexpression 2.1.1 Design and acquisition of Tol2 recombinant plasmid The Cmlc2 promoter can drive high, specific expression of exogenous genes in the heart. IRES2 (Internal Ribosome Entry Site 2) is an internal ribosome entry site that recruits ribosomes and initiates protein translation independently of the traditional 5′ cap structure. Linking multiple genes to an IRES allows for simultaneous expression of multiple genes from the same mRNA molecule, which is crucial for the expression of genes with complex structures or specialized regulatory requirements. Enhanced Green Fluorescent Protein (EGFP), a reporter gene, offers enhanced fluorescence intensity and a wider range of applications, enabling studies of biological processes such as gene expression, protein localization, and cell differentiation. The Tol2 transposon system is a mobile genetic element consisting of a Tol2 transposon helper plasmid and a Tol2 transposase synthesis plasmid. The Tol2 transposon helper plasmid is flanked by specific inverted repeat sequences (Tol2-L and Tol2-R), which serve as recognition and action sites for the transposase, enabling stable gene delivery and expression. After the Tol2 recombinant plasmid and transposase mRNA are co-transfected into the host cell, the exogenous gene can be integrated into the genome in a "cut-and-paste" manner.

[0032] The independently designed Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid was based on the Tol2 auxiliary plasmid, into which the cmlc2 promoter, miR-16-5p, IRES2 and EGFP sequences were inserted in sequence ( Figure 1 ), this transgenic expression vector can simultaneously express miR-16-5p and EGFP under the drive of the same promoter cmlc2.

[0033] The recombinant plasmid map of Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 is as follows Figure 1 As shown, the sequence is shown in SEQ ID NO.2, wherein the yellow highlighted font identifies the cmlc2 promoter sequence, the red highlighted font identifies the miR-16-5p sequence, the cyan highlighted font identifies the IRES2 sequence, and the green highlighted font identifies the EGFP sequence.

[0034] Based on SEQ ID NO. 2, the company used artificial double-stranded DNA technology to synthesize the cmlc2-miR-16-5p-IRES2-EGFP tandem sequence in vitro. This tandem sequence was then inserted into the auxiliary plasmid of the Tol2 transposon system via homologous recombination to construct the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid. The constructed Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid was sequence verified using Sanger sequencing and double enzyme digestion techniques.

[0035] 2.1.2 Synthesis of Tol2 transposase mRNA The original plasmid pCS-tp (Yao Shun Yu Biotechnology) was digested with the single restriction site NotI to linearize the DNA. The linearized DNA was purified using a nuclease-free PCR cleanup kit. The linearized pCS-tp was used as a template for transcription using the mMESSAGE mMACHINE™ SP6 Ultra Transcription Kit to generate capped Tol2 transposase mRNA. After in vitro transcription, the DNA template was removed and the mRNA was purified. The Tol2 transposase mRNA concentration was determined using a microspectrophotometer.

[0036] 2.1.3 Zebrafish Embryo Microinjection Harvest AB zebrafish fertilized eggs according to standard procedures and transfer them to the grooves of an agarose plate. Remove excess water while avoiding excessive drying. Adjust the injection time, pressure, and other parameters of the microinjector. Mix the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid at a final concentration of 60 ng / μL with Tol2 transposase mRNA at a final concentration of 200 ng / μL and microinject 1 nL per embryo into one-cell stage zebrafish fertilized eggs. The injection solution contains 0.05% phenol red solution as an indicator. Perform the injection as quickly as possible to minimize mechanical damage to the embryo.

[0037] 2.1.4 Detection of EGFP reporter gene expression in F0 generation zebrafish The injected zebrafish were placed in zebrafish culture water (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) containing 0.003% phenylthiourea (PTU) to inhibit melanin production and facilitate fluorescence observation. Zebrafish were reared to 48 and 72 hpf, and fluorescence was observed under an inverted fluorescence microscope. Zebrafish that exhibited cardiac fluorescence were selected, designated as the F0 generation, and cultured in a constant temperature incubator until adulthood.

[0038] 2.1.5 Screening and identification of miR-16-5p-F1 transgenic zebrafish filter: After the F0 generation zebrafish selected in the previous step are raised to adult zebrafish, they are hybridized with AB line zebrafish and the offspring zebrafish embryonic hearts are observed under a fluorescence microscope to see if they have EGFP green fluorescence. If the offspring zebrafish embryonic hearts have green fluorescence, then the zebrafish are transgenic zebrafish, that is, F1 generation transgenic zebrafish.

[0039] Identification: (1) Genome extraction of miR-16-5p overexpressing zebrafish F1 Five F1 embryos were collected to prepare genomic DNA template. 10 μL of YSY Buffer (Nanjing Yaoshunyu Biotechnology Co., Ltd., Cat. No. K-101-100) was added to the wall of each PCR tube containing the zebrafish embryo (taking care not to touch the tissue with the pipette tip). After addition, the PCR tube was quickly centrifuged to ensure that the solution was at the bottom of the tube. The PCR tube was placed in a thermal cycler to prepare the genomic template for subsequent PCR amplification. The PCR program is shown in Table 1.

[0040] Table 1 Reaction procedures for genome extraction

[0041] (2) PCR amplification of genomic DNA fragments The prepared template genomic DNA was amplified. The reaction components are shown in Table 2 , the reaction conditions are shown in Table 3 , and the PCR primer sequences are shown in Table 4 . Table 2 PCR reaction system for template genomic DNA amplification

[0042] Table 3 PCR reaction conditions for template genomic DNA amplification

[0043] Table 4 PCR primer sequences for genotyping of transgenic zebrafish

[0044] (3) Electrophoresis detection Take an appropriate amount of PCR amplification product and mix it with loading buffer, add the mixed sample to the 1% agarose gel electrophoresis well, and perform electrophoresis at an appropriate voltage to separate the DNA fragments in the gel according to size.

[0045] (4) Sequencing verification The PCR products were sent to Generic Biotechnology for Sanger sequencing. The sequencing primers used were Primer-F and Primer-R, and their sequences are shown in Table 4.

[0046] 2.1.6 Screening and identification of miR-16-5p-F2 transgenic zebrafish filter: The F1 generation zebrafish selected in the previous step are raised to adult zebrafish and then self-fertilized. The offspring zebrafish embryonic hearts are observed under a fluorescence microscope to see if they have EGFP green fluorescence. If the offspring zebrafish embryonic hearts have green fluorescence, then the zebrafish are transgenic zebrafish, that is, F2 generation transgenic zebrafish, that is, transgenic zebrafish whose cardiomyocytes overexpress miR-16-5p. Tg (cmlc2: miR-16-5p, EGFP) .

[0047] Identification: (1) Genome extraction of miR-16-5p overexpressing zebrafish-F2 Five F2 embryos were collected to prepare genomic DNA template. 10 μL of YSY Buffer was added to the wall of each PCR tube containing the zebrafish embryos. After addition, the tubes were quickly centrifuged to ensure that the solution remained at the bottom of the tubes. The tubes were then placed in a thermal cycler to prepare the genomic template for subsequent PCR amplification. The PCR protocol is shown in Table 1. (2) PCR amplification of genomic DNA fragments The prepared template genomic DNA was amplified. The reaction components are shown in Table 2, the reaction conditions are shown in Table 3, and the PCR primer sequences are shown in Table 4.

[0048] (3) Electrophoresis detection Mix an appropriate amount of PCR amplification product with loading buffer and load the mixture onto a 1% agarose gel. Run the gel at an appropriate voltage to separate the DNA fragments by size. From left to right, the first lane is the DNA marker, the second lane is the DNA loading buffer (a no-template negative control), and the third lane is the PCR amplification product of the F2 genomic DNA fragment.

[0049] (4) Sequencing verification The PCR products were sent to Universal Biotechnology for Sanger sequencing. Primer-F and Primer-R were used as sequencing primers. Their sequences are shown in Table 4.

[0050] 2.2 Observation of cardiomyopathy progression in zebrafish with miR-16-5p overexpression 2.2.1 Observation of zebrafish heart morphology The development of 2- and 3-dpf zebrafish embryos and larvae was observed using a stereofluorescence microscope. The morphology of each group of zebrafish was photographed and recorded, and any abnormalities, such as pericardial edema and increased SV-BA distance, were observed. Image-ProPlus 6.0 software was used to measure pericardial area and SV-BA distance (an increased SV-BA distance reflects cardiac enlargement).

[0051] 2.2.2 Cardiac function test The clinical manifestations of dilated cardiomyopathy are arrhythmia, ventricular dilatation, and cardiac systolic dysfunction. The stroke volume, short-axis shortening rate, and ejection fraction of zebrafish can reflect the ventricular dilatation and cardiac systolic function. Zebrafish at 2 dpf and 3 dpf were fixed with 4% methylcellulose. After fixation, the prone heartbeat video of the zebrafish was recorded under an inverted fluorescence microscope, and the end-diastolic and end-systolic images of the ventricles were extracted. The long and short axis lengths of the zebrafish at end-diastolic and end-systolic were measured using Image-Pro Plus 6.0 software, and the stroke volume, ejection fraction, and short-axis shortening rate were calculated. The specific calculation formulas are shown in (1), (2), (3), and (4).

[0052]

[0053]

[0054]

[0055] 2.2.3 H&E staining Randomly select 10 2 dpf and 3 dpf Tg (cmlc2: EGFP) and Tg (cmlc2: miR-16-5p, EGFP)Zebrafish were stained with hematoxylin and eosin. After washing three times with phosphate buffer saline (PBS), the zebrafish were anesthetized and fixed with 4% paraformaldehyde. The zebrafish were then dehydrated in a gradient of ethanol and immersed in xylene until transparent. The sections were then embedded in paraffin and sectioned. The sections were dewaxed in xylene, then in ethanol, and then dehydrated and mounted. Tissue sections were observed under a stereofluorescence microscope (Olympus, SZX2-ILLTQ, Tokyo, Japan), and cardiac tissue was photographed and recorded.

[0056] 2.3 RT-qPCR detection of changes in expression levels of dilated cardiomyopathy-related genes F2 generation of cardiac fluorescent transgenic zebrafish developed to 48 hpf and 72 hpf Tg (cmlc2: EGFP) and miR-16-5p overexpression in zebrafish Tg (cmlc2: miR-16-5p, EGFP) 90 fish were randomly selected from each group, washed three times with enzyme-free water, and collected for sampling. After homogenization, RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China). cDNA was reverse transcribed from each sample and RT-PCR was performed using ChamQUniversal SYBR qPCR Master Mix. RT-PCR amplification reaction conditions were as follows: 1 cycle of pre-denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s; and 1 cycle of 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. β-actin The results were analyzed by relative quantitative analysis using the internal reference gene. β-actin PCR primers for the target genes were synthesized, purified, and quality-tested by Boshang Biotechnology Co., Ltd. (Shanghai, China). The primer sequences are shown in Table 5.

[0057] Table 5 RT-qPCR related primer sequences

[0058] 2.4 Verification of the applicability of miR-16-5p overexpression in dilated cardiomyopathy zebrafish using clinical drugs To further validate the successful construction of a zebrafish model of dilated cardiomyopathy (DCM) caused by miR-16-5p overexpression, the inventors introduced the clinically approved drugs dapagliflozin and empagliflozin for validation. Both dapagliflozin and empagliflozin are sodium-glucose cotransporter 2 (SGLT2) inhibitors, sharing similar mechanisms of action against DCM. By inhibiting glucose reabsorption in the proximal convoluted tubule of the kidney, they increase urinary glucose excretion while producing an osmotic diuretic effect, reducing cardiac preload and alleviating edema symptoms in patients with DCM. They also promote a shift in cardiomyocyte metabolism from glucose to fatty acid metabolism, optimizing myocardial energy supply and enhancing contractile function, potentially improving myocardial function in patients with DCM. Furthermore, they exhibit anti-inflammatory and antioxidant properties, alleviating oxidative damage and inflammatory responses in cardiomyocytes, thereby protecting them and delaying disease progression.

[0059] 2.4.1 Drug Grouping Select normal development at 48 hpf and Tg (cmlc2: EGFP) Zebrafish were placed in a 24-well plate, with 15 juveniles per well. A blank control group ( Tg (cmlc2: miR-16-5p, EGFP) + zebrafish culture water), model group ( Tg (cmlc2: EGFP) + zebrafish culture water), drug-treated group ( Tg (cmlc2: miR-16-5p, EGFP) + dapagliflozin / empagliflozin), three replicate wells were set up in each group and incubated in a light incubator for 24 h.

[0060] 2.4.2 Effects of clinical drugs on cardiac morphology in zebrafish with dilated cardiomyopathy Twenty-four hours after drug administration, zebrafish were anesthetized with 0.3% tricaine and fixed in 4% methylcellulose. Cardiac morphology was recorded and photographed from the side under a stereofluorescence microscope. Pericardial area and SV-BA distance were measured using Image-Pro Plus 6.0 software.

[0061] 2.4.3 Effects of clinical drugs on cardiac function in zebrafish with dilated cardiomyopathy Twenty-four hours after administration, zebrafish were fixed with 4% methylcellulose and heartbeat videos were recorded in the prone position under an inverted fluorescence microscope. Ventricular end-diastolic and end-systolic images were extracted. Image-Pro Plus 6.0 software was used to measure the major and minor axis lengths at end-diastolic and end-systolic times, and stroke volume, ejection fraction, and fractional shortening were calculated.

[0062] 2.5 Data Analysis The experimental data were expressed as mean ± SD, and statistical differences were analyzed using T test. p <0.05 indicates a significant difference. p <0.01 represents a highly significant difference. Statistical analysis was performed using GraphPad Prism 8.0 and Image-Pro Plus 6.0 software.

[0063] 3 Experimental results 3.1 Identification of the Tol2 transposition system 3.1.1 Identification of Tol2 recombinant plasmid The Sanger sequencing results of the To12 - cmlc2 - miR-16-5p- IRES2 - EGFP - UTRsv40 recombinant plasmid are as follows Tg (cmlc2: miR-16-5p, EGFP) As shown in A, it is consistent with the expected base sequence; ApaI-XhoI double enzyme digestion identification is as shown in Figure 2 As shown in B, the sizes of the fragments after enzyme digestion were consistent with the expected sizes of 1416 bp and 3884 bp. The above results indicate that the construction of the To12-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid was completed.

[0064] 3.1.2 Identification of Tol2 transposase mRNA The final concentration of Tol2 transposase mRNA was determined to be 2154 ng / µL, and the absorbance ratio 260 / 280 was 1.91.

[0065] 3.1.3 Screening and identification results of miR-16-5p-F1 overexpressing zebrafish (1) Fluorescence screening results like Figure 2 As shown in middle A, when zebrafish were observed under an inverted fluorescence microscope at 48 hours of development, green fluorescence expression could be seen in the heart region, indicating that the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid was successfully injected into the zebrafish and expressed in cardiomyocytes.

[0066] (2) Agarose gel electrophoresis results like Figure 3As shown in center B, by comparison with DNA markers of known molecular weight (250, 1000, 5000, 7500, 10000, and 15000 bp, from bottom to top), the genome of the F1 progeny in lane 4 is consistent with the positive control band of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid template in lane 3. A clear target band of the expected size (907 bp) appears, representing the DNA fragment between the cmlc2 promoter and EGFP sequences inserted into the zebrafish genome. This preliminary indication suggests the presence of transgenic elements in the F1 zebrafish genome, but further sequencing verification is required.

[0067] (3) PCR sequencing verification results like Figure 3 As shown in C, the peak diagram obtained by Sanger sequencing of the F1 generation was analyzed. Through sequencing, it can be detected that the key elements of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid (cmlc2 promoter sequence, miR-16-5p sequence, IRES2 partial DNA sequence) are completely consistent with the expected target gene sequence. It can be determined that the F1 generation zebrafish successfully inherited the target gene and achieved stable transmission of the transgene.

[0068] 3.1.4 Results of screening and identification of miR-16-5p-F2 overexpressing zebrafish (1) Agarose gel electrophoresis results like Figure 3 As shown in middle A, by comparing with DNA markers of known molecular weight (250, 500, 750, 1000, and 1500 bp from bottom to top), a clear target band of the expected size (907 bp) appeared in the genome of the F2 offspring (the third lane), preliminarily indicating that transgenic elements may exist in the genome of the F2 generation zebrafish.

[0069] (2) PCR sequencing verification results like Figure 4 As shown in B, the peak diagram obtained by Sanger sequencing of the F2 generation was analyzed. Through sequencing, it can be detected that the key elements of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 plasmid (cmlc2 promoter sequence, miR-16-5p sequence, and IRES2 partial DNA sequence) are completely consistent with the expected target gene sequence. It can be determined that the F2 generation zebrafish successfully inherited the target gene and achieved stable transmission of the transgene. The F2 generation zebrafish is the miR-16-5p overexpression zebrafish Figure 4 Tg .

[0070] 3.2 Study of disease progression in the miR-16-5p overexpression zebrafish model 3.2.1 Effects of miR-16-5p overexpression on zebrafish cardiac morphology Changes in the size of the zebrafish pericardium can directly reflect abnormalities in the zebrafish heart. Zebrafish cardiac dilatation is caused by the stretching of the heart's S-ring, which results in a longer distance between the bulbus arteriosus and the sinus venosus (SV-BA). Therefore, the pericardial area and SV-BA distance can directly reflect whether the zebrafish heart is abnormal. (cmlc2: miR-16-5p, EGFP) As shown, zebrafish with heart-specific fluorescent transgenic Figure 5 Tg Compared with miR-16-5p overexpression in zebrafish (cmlc2: EGFP) At 2-4 dpf, the pericardial area increases significantly and the SV-BA distance becomes longer. Tg (cmlc2: miR-16-5p, EGFP) Tg (cmlc2: miR-16- The pericardial area and SV-BA distance of zebrafish were not significantly different from those of normal zebrafish, indicating that miR-16-5p overexpression can lead to abnormal cardiac morphology in zebrafish.

[0071] 3.2.2 Effects of miR-16-5p overexpression on zebrafish cardiac function The clinical manifestations of dilated cardiomyopathy are ventricular dilatation and cardiac systolic dysfunction. The stroke volume, ejection fraction, and fractional shortening of zebrafish reflect ventricular dilatation and cardiac systolic function. These indicators can be used to evaluate the effect of miR-16-5p overexpression on cardiac function in zebrafish with dilated cardiomyopathy. 5p, EGFP) As shown, zebrafish with heart-specific fluorescent transgenic Figure 6 Compared with miR-16-5p overexpression in zebrafish Tg (cmlc2: EGFP) At 2-3 dpf, stroke volume, ejection fraction and fractional shortening decrease significantly. Tg (cmlc2: miR-16-5p, EGFP) Tg Zebrafish showed decreased stroke volume, ejection fraction, and fractional shortening compared to normal zebrafish, but the changes were not significant. At 6-7 dpf, stroke volume and ejection fraction were also lower than those of normal zebrafish. This suggests that miR-16-5p overexpression can lead to cardiac damage and decreased cardiac function in zebrafish.

[0072] In patients with dilated cardiomyopathy, the ventricular wall becomes thinner, the ventricular cavity becomes larger, the myocardial cells are arranged in disorder, and the myocardium is often fibrotic. HE staining can be used to observe the morphological changes under a microscope to diagnose whether cardiomyopathy has occurred. (cmlc2: miR-16-5p, EGFP) As shown, 2 and 3 dpf groups Figure 7The zebrafish atria and ventricles are of normal size, the intercellular spaces are normal, and the myocardial cells are closely arranged. Tg (cmlc2: EGFP) The atria and ventricles of zebrafish in the 44th group were significantly enlarged, the intercellular spaces were larger, and the arrangement of cardiomyocytes was disordered, which was consistent with the typical pathological characteristics of DCM.

[0073] Effects of miR-16-5p overexpression on the expression levels of dilated cardiomyopathy-related genes in zebrafish To further investigate the mechanism of miR-16-5p overexpression on zebrafish cardiac injury, RT-qPCR was used to detect the expression of miR-16-5p at 2-3 dpf. Tg (cmlc2: miR-16-5p, EGFP) and Tg (cmlc2: EGFP) Dilated cardiomyopathy-related genes in zebrafish ( Tg (cmlc2: miR-16-5p, EGFP) ) mRNA expression levels. Troponin I ( ttn.1, ttn.2, myh6, myh7, mybpc3, tnnt2a, mypn, lmna ) and troponin T ( ttn.1 ) is an important regulatory protein in myocardial cells and is closely related to the regulation of myocardial contraction. Its reduced expression will affect the contractile properties of the myocardium. ttn.2 ) and myosin heavy chain 7 ( myh6 ) is a key protein for myocardial contraction. Its abnormal expression may affect the structure and function of the myocardium, leading to decreased myocardial contractility and possibly causing dilated cardiomyopathy. myh7 ) interacts with myosin to regulate the strength and speed of myocardial contraction. Reduced expression of cardiac troponin T (CTT) disrupts the normal regulation of myocardial contraction and causes dilated cardiomyopathy. mybpc3 ) is an important component of myocardial contractile regulatory proteins, which participates in the excitation-contraction coupling process of myocardial cells. Its downregulation can lead to myocardial cell contractile dysfunction, thereby causing dilated cardiomyopathy. tnnt2a ) is a crucial cytoskeletal protein in cardiomyocytes, playing a crucial role in maintaining their structure and elasticity. Lamin A / C (LMNA) is a component of the nuclear envelope and is crucial for maintaining the structural and functional integrity of the nucleus. Reduced LMNA expression can cause nuclear envelope structural abnormalities, affecting various cellular processes in cardiomyocytes, including signal transduction and gene expression regulation, leading to dilated cardiomyopathy.

[0074] like mypn As shown, at 2 dpf, miR-16-5p overexpressing zebrafish showed a significantly higher expression compared to the blank control group. Figure 8 Tg (cmlc2: middle miR-16-5p, EGFP) The expression level of miR-16-5p was significantly reduced. The results were consistent at 3 dpf. This suggests that overexpression of miR-16-5p leads to downregulation of the expression levels of genes associated with dilated cardiomyopathy in zebrafish, triggering dilated cardiomyopathy.

[0075] 3.4 Verification of the applicability of miR-16-5p overexpression in dilated cardiomyopathy zebrafish using clinical drugs 3.4.1 Effects of clinical drugs on cardiac morphology in zebrafish with dilated cardiomyopathy like ttn.1, ttn.2, myh6, myh7, mybpc3, tnnt2a, mypn, lmna As shown, after 24 hours of administration, the Figure 9 + zebrafish culture water) compared with the model group ( Tg (cmlc2: EGFP) + zebrafish culture water) zebrafish pericardial edema was obvious and the SV-BA distance increased, indicating that the zebrafish dilated cardiomyopathy model was successfully established. Compared with the model group, the zebrafish pericardial edema was alleviated in the low and medium concentrations (1.25, 2.5 μM) of dapagliflozin and medium and high concentrations (5, 10 μM) of empagliflozin (EMPA) administration groups; there was no significant change in the pericardial area of ​​zebrafish in the high concentration (5 μM) of dapagliflozin and low concentration (2.5 μM) of empagliflozin administration groups; the SV-BA distance of zebrafish hearts was significantly reduced in the dapagliflozin (1.25, 2.5, 5 μM) and empagliflozin (2.5, 5, 10 μM) administration groups. This shows that dapagliflozin (1.25, 2.5 μM) and empagliflozin (5, 10 μM) have the effect of alleviating Tg (cmlc2: miR- 16-5p, EGFP) Tg (cmlc2: The role of pericardial edema and reduced SV-BA distance in zebrafish.

[0076] 3.4.2 Effects of Clinical Drugs on Cardiac Function in Zebrafish with DCM like miR-16-5p, EGFP) As shown, after 24 hours of administration, the Figure 10 + zebrafish culture water) compared with the model group ( Tg (cmlc2: EGFP) + zebrafish culture water) zebrafish stroke volume, ejection fraction, and fractional shortening rate decreased significantly, indicating that the zebrafish dilated cardiomyopathy model was successfully established. Compared with the model group, the stroke volume of zebrafish in the dapagliflozin (1.25, 2.5, 5 μM) and empagliflozin (2.5, 5, 10 μM) treatment groups increased; the ejection fraction of zebrafish in the low and high concentration (1.25, 5 μM) dapagliflozin and low and medium concentration (2.5, 5 μM) empagliflozin treatment groups increased; the short axis shortening rate of zebrafish in the low and high concentration (1.25, 5 μM) dapagliflozin and low concentration (2.5 μM) empagliflozin treatment groups increased; there was no significant change in the ejection fraction of zebrafish in the medium concentration (2.5 μM) dapagliflozin and high concentration (10 μM) empagliflozin treatment groups; there was no significant change in the short axis shortening rate of zebrafish in the medium concentration (2.5 μM) dapagliflozin and medium and high concentration (5, 10 μM) empagliflozin treatment groups. It showed that dapagliflozin (1.25, 5 μM) and empagliflozin (5 μM) had Tg (cmlc2: miR-16-5p, EGFP) Zebrafish cardiac function has a restorative effect.

[0077] The present invention successfully constructed a transgenic zebrafish model that specifically overexpresses miR-16-5p in the myocardium Tg (cmlc2: miR-16-5p, EGFP) Tg The stability of transgene integration was confirmed by fluorescence screening and PCR verification. (cmlc2: miR-16-5p, EGFP) Tg At 2-3 days postnatal age, zebrafish exhibit typical dilated cardiomyopathy phenotypes, including pericardial edema, increased SV-BA distance, and significantly decreased cardiac function. These phenotypes closely mirror the ventricular dilation and systolic dysfunction seen in human patients with dilated cardiomyopathy. Histological analysis revealed ultrastructural abnormalities, including disorganized cardiomyocytes, vacuolization, and loss of mitochondrial cristae, further confirming the pathological relevance of this zebrafish model. However, symptoms gradually abate after 4 days postnatal age, presumably due to early mortality in individuals with severe phenotypes, resulting in milder symptoms in those who survive after 4 days postnatal age, consistent with the progressive course of dilated cardiomyopathy in humans. Zebrafish are one of the few vertebrates with complete cardiac regeneration capacity. Their cardiomyocytes can reenter the cell cycle after injury, for example by activating the Notch signaling pathway, promoting myocardial tissue repair. Therefore, the severity of symptoms at 2-3 dpf likely stems from myocardial structural damage and dysfunction directly induced by miR-16-5p overexpression, while the alleviation of symptoms after 4 dpf is a result of the combined effects of early mortality in individuals with severe phenotypes and the zebrafish's cardiac regenerative capacity. Therefore, transgenic zebrafish at 2-3 dpf were ultimately selected as the zebrafish model for dilated cardiomyopathy.

[0078] For 2-3 dpf (cmlc2: miR-16-5p, EGFP) Tg (cmlc2: EGFP) Group and Tg (cmlc2: miR-16-5p, EGFP) The expression levels of dilated cardiomyopathy-related genes in zebrafish of the two groups were compared to clarify the mechanism of miR-16-5p in dilated cardiomyopathy. RT-qPCR results showed that compared with the blank control group, miR-16-5p overexpressed dilated cardiomyopathy-related genes in zebrafish ttn.1、ttn.2、myh6、myh7、mybpc3、tnnt2a、mypn、lmna The expression level of miR-16-5p was significantly reduced, indicating that the zebrafish model of dilated cardiomyopathy induced by miR-16-5p overexpression was successfully established.

[0079] Zebrafish model of dilated cardiomyopathy constructed by the present invention Tg (cmlc2: miR-16-5p, EGFP) It can be used to screen drugs for dilated cardiomyopathy.

Claims

1. A zebrafish model of dilated cardiomyopathy is constructed by overexpressing miR-16-5p in zebrafish cardiomyocytes, wherein the sequence of miR-16-5p is shown in SEQ ID NO.

1.

2. The zebrafish model of dilated cardiomyopathy according to claim 1, wherein The miR-16-5p construct was constructed by overexpressing miR-16-5p in the cardiomyocytes of wild-type AB zebrafish.

3. The zebrafish model of dilated cardiomyopathy according to claim 2, wherein It was constructed by introducing the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid into AB strain zebrafish; The nucleotide sequence of the Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid is shown in SEQ ID NO.

2.

4. The method for constructing a zebrafish model of dilated cardiomyopathy according to any one of claims 1 to 3, comprising the following steps: (1) Constructing a Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 recombinant plasmid with a nucleotide sequence as shown in SEQ ID NO.2; (2) The recombinant plasmid Tol2-cmlc2-miR-16-5p-IRES2-EGFP-UTRsv40 and Tol2 transposase mRNA constructed in step (1) were introduced into AB zebrafish embryos by microinjection, and zebrafish larvae expressing heart-specific green fluorescence were selected to obtain the F0 generation; (3) The F0 generation zebrafish selected in step (2) are raised to adulthood and then hybridized with AB zebrafish to obtain the F1 generation. The zebrafish that specifically express green fluorescence in the heart are cultured and selected. The genome of the F1 generation zebrafish embryos is extracted for verification to confirm that the F1 generation zebrafish has successfully inherited the target gene; (4) The F1 generation zebrafish selected in step (3) were raised to adulthood and then self-fertilized to obtain the F2 generation. The zebrafish that specifically expressed green fluorescence in the heart were cultured and selected. The genome of the F2 generation zebrafish embryos was extracted for verification to confirm that the F2 generation zebrafish successfully inherited the target gene. The F2 generation zebrafish and their offspring were all transgenic zebrafish that overexpressed miR-16-5p. Tg (cmlc2:miR-16-5p, EGFP) , which is a zebrafish model of dilated cardiomyopathy.

5. The construction method according to claim 4, wherein: In step (3) or step (4), the verification method is to use primers Primer-F and Primer-R to perform PCR amplification on the zebrafish genome, and sequence the amplified products for identification; the sequence of Primer-F is shown in SEQ ID NO.3, and the sequence of Primer-R is shown in SEQ ID NO.

4.

6. The construction method according to claim 4, wherein: In step (4), transgenic zebrafish Tg ( cmlc2: miR-16-5p, EGFP ) were cultured to 2-3 dpf as a zebrafish model of dilated cardiomyopathy.

7. Use of the dilated cardiomyopathy zebrafish model according to any one of claims 1 to 3 or the dilated cardiomyopathy zebrafish model constructed by the method according to any one of claims 4 to 6 in screening drugs for preventing or treating dilated cardiomyopathy.

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