Application of miR-300-3p detection reagent in the preparation of diagnostic products for Ang II-mediated myocardial hypertrophy.

Abstract

This invention belongs to the field of molecular diagnostics, specifically relating to the application of miR-300-3p detection reagents in the preparation of products for diagnosing Ang II-mediated myocardial hypertrophy. The sequence of miR-300-3p is shown in SEQ ID NO.1. This invention, by detecting the expression level of miR-300-3p in myocardial tissue and cardiomyocytes, and through bioinformatics analysis and dual-luciferase reporter gene analysis, identifies ACOX1 as the direct target of miR-300-3p. Therefore, this invention elucidates the role of miR-300-3p in an angiotensin II-induced myocardial hypertrophy model, and the upregulation of miR-300-3p promotes myocardial hypertrophy by targeting ACOX1, providing a theoretical basis for future molecular-level diagnosis of myocardial hypertrophy, and possessing significant theoretical importance and potential practical value.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics, specifically to the application of miR-300-3p detection reagents in the preparation of products for diagnosing Ang II-mediated myocardial hypertrophy. Background Technology

[0002] Pathological myocardial hypertrophy is a change in the structure and function of cardiomyocytes caused by stress and / or volume overload under pathological conditions, as well as excessive stimulation by various growth factors and / or hormones. It manifests as enlargement of the myocardium along the long axis of the cell, upregulation of embryonic genes such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC), ultimately leading to decreased cardiac compliance and circulatory pump function, resulting in heart failure, arrhythmias, and sudden death. Current technologies for early diagnosis of myocardial hypertrophy are insufficient, and the efficacy of drug treatments is not ideal. Although early myocardial hypertrophy is a compensatory response, under persistent inflammation, oxidative stress, and other pathological stressors, hypertrophic cardiomyocytes can experience severe energy metabolism disorders, inducing massive apoptosis of cardiomyocytes, ultimately leading to decompensation and heart failure. However, most patients are diagnosed with heart failure by the time the condition is diagnosed. Therefore, identifying specific key molecular diagnostic targets is crucial for the early diagnosis of myocardial hypertrophy.

[0003] Angiotensin II (Ang II) is a bioactive peptide derived from the renin-angiotensin-aldosterone system. Elevated Ang II levels contribute to cardiac remodeling. Prolonged high cardiac workload can lead to myocardial hypertrophy, myocardial remodeling, and even heart failure. In myocardial remodeling associated with heart failure, Ang II levels are directly proportional to the degree of myocardial hypertrophy; inhibiting its synthesis or blocking its effects can effectively reduce the degree of myocardial hypertrophy.

[0004] MicroRNAs, or miRNAs for short, are a class of endogenous non-coding RNAs approximately 20-25 nucleotides in length. They not only regulate cell development and differentiation, cell cycle, and homeostasis in the human body, but also influence the progression of various pathological states, playing a crucial role in the mechanisms of many diseases. Studies have shown that miRNA-1 and miRNA-133 are key molecules in signaling pathways associated with myocardial hypertrophy, cardiac fibrosis, and arrhythmias; overexpression of miR-142 alleviates myocardial hypertrophy by improving mitochondrial function; and miR-26a can reduce myocardial hypertrophy and dysfunction by targeting ADAM17. Research indicates that miR-300-3p participates in regulating cell proliferation, migration, and invasion in various cancers, including glioblastoma, breast cancer, and liver cancer. However, the role of miR-300-3p in Ang II-mediated myocardial hypertrophy remains unclear; therefore, there is an urgent need to investigate the role of miR-300-3p in Ang II-mediated myocardial hypertrophy and its clinical significance. Summary of the Invention

[0005] To address the above issues, this invention provides the application of miR-300-3p detection reagent in the preparation of products for diagnosing Ang II-mediated myocardial hypertrophy.

[0006] This invention is achieved through the following technical solution:

[0007] The application of miR-300-3p detection reagent in the preparation of products for diagnosing Ang II-mediated myocardial hypertrophy, wherein the sequence of miR-300-3p is shown in SEQ ID NO.1.

[0008] Preferably, the diagnostic test sample is animal heart tissue or cardiomyocytes.

[0009] Preferably, the detection reagent is a detection reagent for quantitatively detecting the expression level of miR-300-3p.

[0010] Preferably, the detection reagent includes a specific primer pair for amplifying miR-300-3p: a forward primer and a reverse primer; the forward primer is shown in SEQ ID NO.2; and the reverse primer is shown in SEQ ID NO.3.

[0011] Preferably, the method for quantitatively detecting miR-300-3p is qRT-PCR.

[0012] Preferably, the specific steps of the qRT-PCR are as follows:

[0013] (1) RNA extraction: Extracting total RNA from cells or tissues.

[0014] (2) Reverse transcription: RNA is reverse transcribed into cDNA using reverse transcriptase.

[0015] (3) qRT-PCR detection of miR-300-3p expression: PCR amplification was performed on Forward Primer and Reverse Primer using miR-300-3p specific primers, and changes in fluorescence signal were monitored.

[0016] Preferably, the reaction system for detecting miR-300-3p expression by qRT-PCR is 10 μL of TB Green Premix ExTaqII, 0.8 μL of Forward Primer, 0.8 μL of Reverse Primer, 2 μL of template cDNA, and 6.4 μL of ddH2O, with a total volume of 20 μL.

[0017] Preferably, the reaction system for detecting miR-300-3p expression by qRT-PCR is 10 μL of TB Green Premix ExTaqII, 0.8 μL of Forward Primer, 0.8 μL of Reverse Primer, 2 μL of template cDNA, and 6.4 μL of ddH2O, for a total volume of 20 μL.

[0018] Preferably, the reaction program for detecting miR-300-3p expression by qRT-PCR is 95℃ for 30s, 95℃ for 5s, 60℃ for 34s, and the reaction is performed for 45 cycles.

[0019] Preferably, the reaction program for detecting miR-300-3p expression by qRT-PCR is 95℃ for 30s, 95℃ for 5s, 60℃ for 34s, and the reaction is performed for 45 cycles.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides the application of a miR-300-3p detection reagent in the preparation of products for diagnosing Ang II-mediated myocardial hypertrophy, the sequence of which is shown in SEQ ID NO.1. This invention is the first to discover that miR-300-3p is upregulated in Ang II-induced myocardial hypertrophy, and that miR-300-3p promotes Ang II-induced myocardial hypertrophy. By detecting the expression level of miR-300-3p in cardiac tissue and cardiomyocytes, and finding that overexpression of miR-300-3p promotes myocardial hypertrophy by targeting ACOX1, this provides a basis for the diagnosis of myocardial hypertrophy caused by excessive pressure load and a theoretical basis for future molecular-level diagnosis of pathological myocardial hypertrophy, possessing significant theoretical importance and potential practical value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the HE staining results of myocardial cell morphology in SD rats after chronic infusion of Ang II according to the present invention; the left image is the normal control group (Sham group), and the right image is the experimental group (Ang II infusion group);

[0024] Figure 2 This is a schematic diagram of Masson staining observation of intercellular collagen fibers in SD rats after chronic infusion of Ang II according to the present invention; the left image is the normal control group (Sham group), and the right image is the experimental group (Ang II infusion group);

[0025] Figure 3 This is a schematic diagram of the heart weight / body weight ratio (HW / BW) results in SD rats according to the present invention.

[0026] Figure 4 A schematic diagram showing the heart weight / tibia length ratio (HW / TL) results for SD rats;

[0027] Figure 5 This is a schematic diagram of the ultrastructural changes of SD rat cardiomyocytes observed by transmission electron microscopy according to the present invention; the left image is the normal control group (Sham group), and the right image is the experimental group (Ang II infusion group);

[0028] Figure 6 This is a schematic diagram illustrating the expression of ANP and BNP proteins in the cardiac tissue of SD rats according to the present invention.

[0029] Figure 6In the diagram, A represents the results of the Western Blot experiment; B represents the statistical graph corresponding to the results of the Western Blot experiment.

[0030] Figure 7 This is a schematic diagram illustrating the expression of ANP and BNP proteins in cardiomyocytes according to the present invention;

[0031] Figure 7 In the diagram, A represents the results of the Western Blot experiment; B represents the statistical graph corresponding to the results of the Western Blot experiment.

[0032] Figure 8 This is a schematic diagram showing the mRNA expression results of miR-300-3p in SD rats according to the present invention;

[0033] Figure 9 This is a schematic diagram showing the mRNA expression results of miR-300-3p in cardiomyocytes according to the present invention.

[0034] Figure 10 This diagram illustrates the expression of ANP and BNP in cardiomyocytes treated with Ang II and transfected with miR-300-3p mimic, miR-300-3p inhibitor, or miR-NC according to the present invention.

[0035] Figure 10 In the diagram, A represents the results of the Western Blot experiment; B represents the statistical graph corresponding to the results of the Western Blot experiment.

[0036] Figure 11 This is a schematic diagram of the bioinformatics analysis results of the binding site between ACOX13'UTR and miR-300-3p in this invention;

[0037] Figure 12 This is a schematic diagram of the results of dual-luciferase reporter gene detection using the WT-ACOX13'UTR reporter gene and the Mut reporter gene in the presence of miR-300-3p mimic in this invention.

[0038] Figure 13 This is a schematic diagram of ACOX1 protein expression in the heart of SD rats according to the present invention;

[0039] Figure 13 In the diagram, A represents the results of the Western Blot experiment; B represents the statistical graph corresponding to the results of the Western Blot experiment.

[0040] Figure 14 This is a schematic diagram of ACOX1 protein expression in cardiomyocytes according to the present invention;

[0041] Figure 14 In the diagram, A represents the results of the Western Blot experiment; B represents the statistical graph corresponding to the results of the Western Blot experiment.

[0042] Figure 15 This is a schematic diagram showing the expression results of ACOX1 in cardiomyocytes transfected with miR-300-3p mimics or miR-300-3p inhibitors according to the present invention.

[0043] Figure 15 In the figure, A is the Western Blot experimental result graph; B is the statistical graph corresponding to the Western Blot experimental result graph. Detailed Implementation

[0044] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0045] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0046] The inventive concept of this invention is as follows:

[0047] In the current technology, there are still shortcomings in the early diagnosis of myocardial hypertrophy, and the drug effects of treatment are not ideal. Although myocardial hypertrophy is a compensatory response in the early stage, under the stimulation of various pathological stresses such as persistent inflammation and oxidative stress, hypertrophic cardiomyocytes can experience severe energy metabolism disorders, induce a large number of cardiomyocyte apoptosis, and eventually decompensate and lead to heart failure. However, most patients have already developed heart failure when the disease is diagnosed. Therefore, finding specific key molecular diagnostic targets is of great significance for the early diagnosis of myocardial hypertrophy.

[0048] miR-300-3p is an endogenous non-coding RNA of approximately 20–25 nucleotides in length, involved in regulating cell proliferation, migration, and invasion in various cancers, including glioblastoma, breast cancer, and liver cancer. However, the role of miR-300-3p in Ang II-mediated myocardial hypertrophy remains unclear. Therefore, there is an urgent need to investigate the role of miR-300-3p in Ang II-mediated myocardial hypertrophy and its clinical significance.

[0049] Based on this, the present invention provides the use of miR-300-3p detection reagent in the diagnosis of Ang II-mediated myocardial hypertrophy. The sequence of miR-300-3p is shown in SEQ ID NO.1. By detecting the expression level of miR-300-3p in myocardial tissue and cardiomyocytes, and through bioinformatics analysis and dual-luciferase reporter gene analysis, ACOX1 was identified as the direct target of miR-300-3p. Therefore, the present invention elucidates the role of miR-300-3p in an angiotensin II-induced myocardial hypertrophy model, and the upregulation of miR-300-3p promotes myocardial hypertrophy by targeting ACOX1, providing a theoretical basis for future molecular-level diagnosis of myocardial hypertrophy, and possessing significant theoretical significance and potential practical value.

[0050] The beneficial effects of the present invention will be illustrated below through specific embodiments:

[0051] Example 1

[0052] 1. Experimental Subjects

[0053] 1.1 Laboratory Animals

[0054] Male SD rats.

[0055] 1.2 Cell lines

[0056] The H9c2 cell line is derived from rat-derived cardiomyocytes.

[0057] 2. Instruments and Equipment and Experimental Reagents

[0058] 2.1 Main Reagents

[0059] Sterile cell culture flasks and pipettes; fetal bovine serum purchased from Invitrogen (USA); DEME medium purchased from Gibco (USA); penicillin-streptomycin and trypsin digestion solution purchased from Solarbio (China); angiotensin II purchased from MCE (USA); blood / cell / tissue RNA extraction reagent purchased from Omega (USA); reverse transcription kit purchased from Takara (Japan); qRT-PCR mix purchased from Takara (Japan); total protein extraction kit purchased from Jiangsu Kaiji (China); Glycine, SDS, and Tris purchased from Biotopped (China); SDS separating gel / staple gel purchased from Shanghai Double Helix (China); 30% acrylamide (29:1) purchased from Beijing Nobride (China). INVI DNA / RNA transfection reagent was purchased from Invitrogen (USA); HE staining solution, Masson staining solution, blocking goat serum were purchased from Bio-Rad Laboratories (China); anti-fluorescence mounting quencher was purchased from Zhongshan Jinqiao Pharmaceutical Co., Ltd. (China); ANP was purchased from Abclonal (China); BNP monoclonal antibody was purchased from Affinity (China); β-actin antibody and rabbit / mouse secondary antibody were purchased from Abclonal (China); ACOX1 monoclonal antibody was purchased from Abmart (China); dual-luciferase reporter assay kit was purchased from Promega (USA); miR-300-3p and U6 primers were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.; miR-300-3p mimic and inhibitor were provided by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0060] 2.2 Main Instruments

[0061] The constant temperature cell culture incubator was purchased from Thermo Fisher Scientific, USA; the ultra-clean workbench was purchased from Suzhou Antai Technology Co., Ltd., China; the 5415D micro-volume benchtop centrifuge was purchased from Eppendorf, Germany; the precision electronic balance was purchased from Sartorius, Germany; the ice maker was purchased from AF10 SCOTSMAN, USA; the QuantStudio5 real-time PCR instrument was purchased from Thermo Fisher, USA; the micropipette was purchased from Eppendorf, Germany; the pure water system was purchased from Heal Force, China; the fully automated microplate reader was purchased from Bio-TEK; the centrifuge was purchased from Thermo Fisher & Eppendorf, USA; the vortex mixer was purchased from Dalong, China; and the constant temperature metal bath was purchased from Kayodi.

[0062] 3. Methods

[0063] 3.1 Cell Culture

[0064] Rat-derived cardiomyocytes H9C2 were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin in an incubator at 37°C and 5% CO2. When the cell density reached 70%, the cardiomyocytes were treated with Ang II at a final concentration of 1 μM (Ang II group). The normal control group (Control group) received only an equal volume of complete culture medium. Cells were collected 24 hours after intervention for further studies.

[0065] 3.2 Animal Models

[0066] Twenty 6-week-old male SD rats, weighing 200±5g, were purchased from the Experimental Animal Center of Ningxia Medical University. After a 2-week acclimatization period, the rats were randomly divided into two groups: the Ang II infusion group (n=10) and the Sham group (n=10). Ang II was chronically infused at a dose of 500 ng / kg / day, with a subcutaneous micropump implanted for 28 days to induce myocardial hypertrophy. Rats injected with the same dose of PBS served as the sham-operated group. All animal procedures followed the US and National Institutes of Health (NIH) guidelines for laboratory animal care and use. After anesthesia with 3.0% isoflurane, the rats were euthanized by cervical dislocation, and the heart was dissected, weighed using an analytical balance, and the heart-to-body weight ratio (HW / BW) was calculated. The tibia was dissected, and its length was measured to calculate the heart-to-tibia length ratio (HW / TL). The animal experiment was approved by the Animal Care and Use Committee of Ningxia Medical University and the Animal Experiment Center of Ningxia Medical University, with ethics approval number IACUC-NYLAC-2023-043.

[0067] 3.3 Histological analysis

[0068] After a brief rinse with cold PBS, the excised rat hearts were immediately fixed with 4% paraformaldehyde at room temperature for 24 hours, then dehydrated and embedded in paraffin. Left ventricular sections of 4 μm were cut and stained with hematoxylin and eosin (H&E) and Masson's solution (Masson's solution).

[0069] 3.4 Transmission Electron Microscopy

[0070] Ventricular samples were fixed with 2% glutaraldehyde (v / v), washed three times with sodium bicarbonate buffer for 10 min each time, fixed with 1% osmium tetroxide (v / v) for 1 h, and then dehydrated by increasing the concentration gradient of ethanol and propylene oxide. The samples were then embedded, cut into 50 nm sections, and stained with uranyl acetate and lead citrate solutions. Pathological changes were detected using the JEOLTEM method.

[0071] 3.5 Western Blot

[0072] Proteins were extracted, and the protein samples were mixed with 5× loading buffer at a ratio of 1:4 and boiled at 100℃ for 5 min, followed by SDS-PAGE electrophoresis. After electrophoresis, the samples were electrotransferred to PVDF membranes using a semi-dry or wet transfer method. The membranes were blocked with 5% skim milk powder or PBST buffer at room temperature for 2 h. After washing away the residual blocking solution with PBST, the primary antibody was added, and the membranes were incubated overnight at 4℃ with shaking. The membranes were washed with PBST for 10 min each time × 3 times. Horseradish peroxidase-labeled secondary antibody from the same source as the primary antibody was added and incubated at room temperature for 2 h. The membranes were washed with PBST for 10 min each time × 3 times. The membranes were covered with ECL luminescent solution for 1 min, and the gel was developed using a gel imaging system. The optical density of the bands was measured using ImageLab analysis. β-actin was used as an internal control, and the ratio of the optical density of the target gene to that of β-actin represented the protein expression level.

[0073] 3.6 qRT-PCR detection of miR-300-3p expression

[0074] 3.6.1 RNA Extraction

[0075] RNA was extracted according to the kit instructions. 500 μL of TRK lysis buffer was added to heart tissue and cardiomyocytes. Before use, 20 μL of β-hydroxyethanol was added to each 1 mL of TRK lysis buffer, and the mixture was homogenized. The lysed sample was then transferred to a 1.5 mL RNase-free centrifuge tube. The sample was sterilized under UV light in a clean bench for 30 min, and then allowed to stand for approximately 5 min to lyse. The time could be extended as needed depending on the lysis progress to ensure complete separation of the nucleic acid-protein complex. The sample was centrifuged at 12,000 rpm for 5 min at 4°C to remove proteins and fats. The supernatant was transferred to a new 1.5 mL RNase-free centrifuge tube. 50% anhydrous ethanol / sample was added, and the mixture was vortexed. The sample was transferred to a Hibind mini column and centrifuged at 12,000 rpm for 1 min, discarding the waste liquid. The column was placed in a new 2 mL collection tube, and 300 μL of RNAwash buffer I was added. The column was centrifuged at 12,000 rpm for 1 min, discarding the waste liquid. 500 μL of RNAwash buffer was added. Centrifuge at 12,000 rpm for 1 min using buffer II, and discard the waste liquid. Add 500 μL of RNAwash buffer II again, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid. Centrifuge again for 2 min, remove the adsorption column from the collection tube, and let it stand at room temperature for 5-10 min to remove any remaining wash solution. Prepare a new 1.5 mL RNase-free centrifuge tube, label it, place the dried adsorption column into the centrifuge tube, suspend it vertically in the center of the adsorption membrane, add 40 μL of DEPC water, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min at 4℃ to obtain RNA. To prevent RNA degradation, it should be stored at -80℃.

[0076] 3.6.2 Reverse transcription

[0077] This step was performed in a clean bench irradiated with UV light. RNase-free pipette tips were used throughout the procedure. 200 μL RNase-free centrifuge tubes were placed on ice, and then the components listed in Table 1 were added sequentially:

[0078] Table 1 Reverse Transcription System

[0079] reagents Dosage, in μL 5×PrimeScriptBuffer 4 PrimeScriptRTEnzymeMixI 1 OligodTPrimer 1 Random6mers 1 cDNA 1μg Nuclease-freewater Upto20 Total volume 20

[0080] After mixing the above system thoroughly, centrifuge briefly and perform a reaction at room temperature (RT). The reaction program is as follows: 37℃ for 15 min, 85℃ for 5 s, and hold at 4℃. The product after the reaction should be stored at -20℃ for a short period and at -80℃ for a longer period. Repeated freeze-thaw cycles should be avoided.

[0081] 3.6.3 qRT-PCR detection of miR-300-3p expression

[0082] Specific primer pairs, Forward Primer and Reverse Primer, were designed based on the nucleotide sequence of miR-300-3p.

[0083] The nucleotide sequence of miR-300-3p is shown in SEQ ID NO.1, which is TATGCAAGGGCAAGCTCTCTTC.

[0084] The nucleotide sequence of the Forward Primer is shown in SEQ ID NO.2, which is AATCGGCGTATGCAAGGGCAA.

[0085] The nucleotide sequence of the Reverse Primer is shown in SEQ ID NO.3, which is ATCCAGTGCAGGGTCCGAGG.

[0086] Add the systems listed in Table 2 below to 200 μL RNase-free centrifuge tubes in sequence:

[0087] Table 2 qRT-PCR reaction system

[0088] reagents Volume, in μL TBGreenPremixExTaqII 10 ForwardPrimer 0.8 ReversePrimer 0.8 template cDNA 2 <![CDATA[ddH2O]]> 6.4 Total volume 20

[0089] After all samples were added, the mixture was vortexed and briefly centrifuged before being placed in a real-time PCR instrument for reaction. The reaction program was: 95℃ for 30s, 95℃ for 5s, 60℃ for 34s, for 45 cycles. U6 was used as an internal control. The relative amount of the target gene was calculated to be 2. -△△Ct The calculation results show that the miR-133b-3p primer was designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0090] 3.7 miR-300-3p mimic and inhibitor transfection

[0091] When the cells reached 70% confluence, miR-300-3p mimic, miR-300-3p inhibitor, and corresponding control groups mimic-NC and inhibitor-NC were transfected into cardiomyocytes in good growth condition. The medium was changed 6 hours after transfection, and the cells were used for subsequent experiments 48 hours after transfection.

[0092] 3.8 Dual-luciferase assay

[0093] The target genes of miR-300-3p were predicted using the online tool TargetScan (http: / / www.targetscan.org). A dual-luciferase assay was used to verify the binding site between miR-300-3p and acyl-CoA oxidase 1 (ACOX1). Sequences containing wild-type binding sites or mutant binding sites in the 3'UTR of ACOX1 were cloned into the PmirGLO luciferase vector, purchased from Promega, CA, USA. ACOX1-WT and ACOX1-Mut plasmids containing miR-300-3p mimic or miR-NC were delivered into HEK293T cells via liposome 2000 and cultured for 48 hours. Subsequently, HEK293T cell lysates were added to renin-luciferase or firefly luciferase, and relative luciferase activity was measured using a dual-luciferase reporter assay kit on a microplate reader. The dual-luciferase reporter assay kit was purchased from Promega, CA, USA.

[0094] 4. Statistical processing

[0095] All data were quantitative. Statistical results are expressed as mean ± standard deviation. Student's t-test was used to compare the means of two samples, one-way ANOVA was used to compare the means of multiple samples, and Student-Newman-Keuls test was used to compare the pairs of groups. A p-value ≤ 0.05 was considered statistically significant.

[0096] 5 Results

[0097] 5.1 To ensure the accuracy of the experiment, we first assessed whether the myocardial hypertrophy model was successfully established in vivo and in vitro. H&E staining was performed on the myocardial tissue, and the results are as follows: Figure 1 The model of Ang II-induced myocardial hypertrophy was replicated in SD rats. SD rats were purchased and housed at the Experimental Animal Center of Ningxia Medical University. The Sham group served as the normal control group, and the Ang II infusion group served as the experimental group. HE analysis showed that the myocardial cells exhibited disordered morphology and extensive inflammatory cell infiltration.

[0098] 5.2 Masson staining was performed on the myocardial tissue, and the results are as follows: Figure 2 The model of Ang II-induced myocardial hypertrophy was replicated in SD rats, and Masson's results showed a large number of blue collagen fibers in the intermyocardial spaces.

[0099] 5.3 such as Figure 3 As shown, after sacrificing the rats, the hearts were separated, weighed using an analytical balance, and the HW / BW ratio was calculated. The results showed that infusion of Ang II caused the rat hearts to become heavier.

[0100] 5.4 such as Figure 4 As shown, after sacrificing the rats, the hearts were separated, weighed using an analytical balance, and the tibia length was measured using a ruler to calculate the heart weight / tibia length ratio (HW / TL). The results showed that infusion of Ang II led to an increase in the heart weight / tibia length ratio in rats (*P<0.05).

[0101] 5.5 The ultrastructure of the myocardium was examined using transmission electron microscopy, and the results are as follows: Figure 5 As shown, infusion of Ang II resulted in moderate hypertrophy of cardiomyocytes, a significant increase in the number of organelles, moderate swelling, thinning and dissolution of the intracellular matrix, disordered arrangement and extensive breakage of myofibrils, and numerous breakages of sarcomeres.

[0102] 5.6 The expression of hypertrophy marker genes, including ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), was detected in the rat heart. The results are as follows: Figure 6 As shown, the expression of ANP and BNP was significantly upregulated.

[0103] 5.7 The expression of hypertrophy marker genes ANP and BNP was detected in cardiomyocytes, and the results are as follows: Figure 7 As shown, the expression of ANP and BNP was significantly upregulated (*P<0.05).

[0104] 5.8 qRT-PCR analysis of miR-300-3p expression in Ang II-injected rat hearts, results are as follows Figure 8 As shown, compared with the sham-operated group, infusion of Ang II can lead to a significant increase in the expression of miR-300-3p, *P<0.05.

[0105] 5.9 qRT-PCR analysis of miR-300-3p expression in Ang II-treated cardiomyocytes, results are as follows: Figure 9 As shown, Ang II treatment significantly increased miR-300-3p expression (*P<0.05). This result suggests that miR-300-3p may be involved in the pathogenesis of myocardial hypertrophy.

[0106] 5.10 miR-300-3p mimics or inhibitors were transfected into Ang II-treated cardiomyocytes. Western blot analysis yielded the following results: Figure 10 As shown, overexpression of miR-300-3p promotes AngII-induced upregulation of ANP and BNP, while miR-300-3p inhibitors inhibit this upregulation (*P<0.05).

[0107] 5.11 Since miR-300-3p plays an important role in myocardial hypertrophy, TargetScan was further used to predict downstream target genes of miR-300-3p. Based on this, a conserved binding site of miR-300-3p was found in ACOX13'UTR, such as... Figure 11 As shown.

[0108] 5.12 To further verify that miR-300-3p directly targets ACOX1, a luciferase construct carrying the ACOX1 3'UTR was prepared. The results are as follows... Figure 12 As shown, miR-300-3p can regulate ACOX1-WT. In addition, there is no significant difference between the ACOX1 mutant group and the NC group, indicating that miR-300-3p exerts its regulatory effect by binding to the predicted site of ACOX1-WT.

[0109] 5.13 Western blot analysis of ACOX1 expression in the hearts of rats injected with Ang II showed the following results: Figure 13 As shown, the expression of ACOX1 in the heart of rats injected with Ang II was significantly reduced compared with Sham (*P<0.05).

[0110] 5.14 Western blot analysis of ACOX1 expression in Ang II-treated cardiomyocytes yielded the following results: Figure 14 As shown, compared with the control group, the expression of ACOX1 in Ang II-treated cardiomyocytes was significantly reduced (*P<0.05).

[0111] 5.15 Western blot analysis of ACOX1 expression in cardiomyocytes transfected with miR-300-3p mimics or miR-300-3p inhibitors, results are as follows: Figure 15 As shown, transfection with miR-300-3p mimic significantly reduced ACOX1 expression, while transfection with miR-300-3p inhibitor increased ACOX1 expression, indicating that ACOX1 is negatively regulated by miR-300-3p (*P<0.05).

[0112] 6. Conclusion

[0113] Ang II can induce pathological myocardial hypertrophy in cardiac tissue and cardiomyocytes. miR-300-3p is upregulated in the Ang II-induced myocardial hypertrophy model. Overexpression of miR-300-3p can promote the Ang II-induced myocardial hypertrophy phenotype, while knockdown of miR-300-3p can inhibit Ang II-induced myocardial hypertrophy. In addition, the contribution of the miR-300-3p / ACOX1 axis to the myocardial hypertrophy phenotype was confirmed.

[0114] This invention successfully replicated a pathological myocardial hypertrophy model using Ang II infusion in SD rats and Ang II intervention in cardiomyocytes. In this study, we investigated the expression and function of miR-300-3p in both animal and cellular models of myocardial hypertrophy, revealing a potential contribution of miR-300-3p to the progression of myocardial hypertrophy. This provides new theoretical support for the future clinical diagnosis of pathological myocardial hypertrophy and is of great significance.

[0115] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0116] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of miR-300-3p detection reagent in the preparation of products for diagnosing Ang II-mediated myocardial hypertrophy in rats, characterized in that, The sequence of miR-300-3p is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The diagnostic test sample is rat heart tissue or myocardial cells.

3. The application according to claim 1, characterized in that, The detection reagent is a reagent for quantitatively detecting the expression level of miR-300-3p.

4. The application according to claim 3, characterized in that, The detection reagent includes a specific primer pair for amplifying miR-300-3p: a forward primer and a reverse primer; the forward primer is shown in SEQ ID NO.2; and the reverse primer is shown in SEQ ID NO.

3.

5. The application according to claim 3, characterized in that, The method for quantitatively detecting miR-300-3p is qRT-PCR.

6. The application according to claim 5, characterized in that, The specific steps of the qRT-PCR are as follows: (1) RNA extraction: Extracting total RNA from cells or tissues; (2) Reverse transcription: RNA is reverse transcribed into cDNA using reverse transcriptase; (3) qRT-PCR detection of miR-300-3p expression: PCR amplification was performed on the Forward Primer and Reverse Primer using miR-300-3p specific primers, and the fluorescence signal was monitored.

7. The application according to claim 6, characterized in that, The reaction system for detecting miR-300-3p expression by qRT-PCR consisted of 9 μL–11 μL TB Green Premix Ex TaqII, 0.7 μL–0.9 μL Forward Primer, 0.7 μL–0.9 μL Reverse Primer, 1 μL–3 μL template cDNA, and 6.3 μL–6.5 μL ddH2O, for a total volume of 17.7 μL–22.3 μL.

8. The application according to claim 7, characterized in that, The reaction system for detecting miR-300-3p expression by qRT-PCR was as follows: 10 μL TB Green Premix Ex TaqII, 0.8 μL Forward Primer, 0.8 μL Reverse Primer, 2 μL template cDNA, 6.4 μL ddH2O, for a total volume of 20 μL.

9. The application according to claim 6, characterized in that, The qRT-PCR reaction program for detecting miR-300-3p expression was 94℃~96℃ for 29s~31s, 94℃~96℃ for 4s~6s, and 59℃~61℃ for 33s~35s, with 45 cycles.

10. The application according to claim 9, characterized in that, The qRT-PCR reaction program for detecting miR-300-3p expression was 95℃ for 30s, 95℃ for 5s, and 60℃ for 34s, with 45 cycles.

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