Application of lncRNAs Foxo6os in preparation of medicine for detecting, preventing, relieving and / or treating heart failure

Abstract

The invention relates to an application of lncRNAs Foxo6os in preparation of a medicine for detecting, preventing, relieving and / or treating heart failure. Compared with the prior art, the method has the advantages that the serum sample data of the patient at the early stage of heart failure is included, the exosomes are extracted from the serum in the queue for verification, auxiliary serological indexes are provided for the early diagnosis of the patient at the mental failure, and meanwhile, a novel and powerful biological detection target is provided for the early diagnosis of the heart failure. The result can assist clinicians in early accurate diagnosis and prevention of the heart failure phenotype and pertinently intervene the occurrence of bad outcomes, and also provides an important theoretical basis for the development of targeted drugs.

Description

Technical Field The present invention relates to the field of biotechnology, and in particular to the application of lncRNAs Foxo6os in the preparation of drugs for detecting, preventing, alleviating and / or treating heart failure. Background Art Heart failure is a serious disease caused by various cardiovascular diseases. According to the "Report on Cardiovascular Health and Diseases in China 2022", there are approximately 8.9 million heart failure patients in China, and the 5-year survival rate is less than 50%. The annual mortality rate of patients with severe heart failure is as high as 60%, comparable to that of common malignancies. Despite the continuous progress of treatment methods, heart failure remains a major public health challenge. Therefore, in-depth research on the pathological mechanism of heart failure and the discovery of new therapeutic targets are crucial for preventing the deterioration of heart failure and reducing readmission. In particular, activating the endogenous protection mechanism in the early stage of myocardial injury and exploring the related molecules and signaling pathways of the transformation from myocardial hypertrophy to heart failure at the molecular level have important theoretical and clinical significance for improving the prognosis of heart failure. Long non-coding RNAs (lncRNAs) are a class of endogenous regulatory RNA molecules with a length greater than 200 bp nucleotides. They play key regulatory roles in physiological and pathological processes. They are widely expressed in various body fluids and tissues and have become diagnostic markers and therapeutic targets for the early onset of cardiovascular diseases. Current studies have found that many non-coding RNAs play important roles in the pathological processes of various cardiovascular diseases [1] : miR-1-3p is elevated in the peripheral blood of patients with myocardial infarction and animal models. In rodents, coronary artery ligation can lead to a rapid increase in serum miR-1-3p [2] In addition, it has been found that the expressions of serum exosomal IncRNANEAT1 and matrix metalloproteinase-9 (MMP-9) in patients with ST-segment elevation myocardial infarction are significantly higher than those in patients with unstable angina and non-myocardial infarction. Spearman test found that IncRNA NEAT1 was positively correlated with the level of MMP9, and logistic regression analysis showed that both IncRNANEAT1 and MMP-9 were independent predictors of ST-segment elevation myocardial infarction [3] The above studies indicate that lncRNAs in peripheral blood can be used as biomarkers for human cardiovascular diseases. However, there are still relatively few potential lncRNAs biomarker candidates that have been identified and reported for the diagnosis of cardiovascular diseases. The references are as follows:

[0001] WANG Y,SUN X.The functions ofLncRNAin the heart[J].Diabetes Res ClinPract,2020,168:108249.

[0002] KOUTALIANOS D, KOUTSOULIDOU A, MYTIDOU C, et al. miR-223-3p and miR-24-3p as novel serum-based biomarkers for myotonic dystrophy type 1[J]. Mol Ther Methods Clin Dev, 2021, 23: 169-83.

[0003] CHEN Z, YAN Y, WU J, et al. Expression level and diagnostic value of exosomal NEAT1 / miR-204 / MMP-9 in acute ST-segment elevation myocardial infarction[J]. IUBMB Life, 2020, 72(11): 2499-507. Summary of the Invention The object of the present invention is to provide an application of lncRNAs Foxo6os in the preparation of drugs for detecting, preventing, alleviating and / or treating heart failure. By incorporating the serum sample data of early heart failure patients and verifying with exosomes extracted from serum in the cohort, the present invention provides auxiliary serological indicators for the early diagnosis of heart failure patients, and at the same time provides novel and powerful biological detection targets for the early diagnosis of heart failure. The results will assist clinicians in the early and accurate diagnosis, prevention, and targeted intervention of adverse outcomes of heart failure phenotypes, and also provide an important theoretical basis for the development of targeted drugs. The object of the present invention can be achieved by the following technical solutions: The first object of the present invention is to provide an application of lncRNAs Foxo6os in the preparation of drugs for detecting, preventing, alleviating and / or treating heart failure, and the nucleotide sequence of the lncRNAs Foxo6os is shown as SEQ ID NO.18. Furthermore, the drug is a drug for improving myocardial hypertrophy. Even further, the drug is a drug for inhibiting heart failure induced by ventricular remodeling caused by myocardial hypertrophy. Furthermore, the lncRNAs Foxo6os interacts with myosin binding protein MYBPC3. Even further, the Foxo6os enhances the phosphorylation level of MYBPC3 by binding to MYBPC3 and recruiting the related protein kinase PKC-α, promotes the formation of myocardial actin cross-bridges, and increases the Ca of the myofilaments2+ Sensitivity, enhance the contractility of the myocardium, thereby slowing down the pathological process of heart failure. The second object of the present invention is to provide an application of the biomarker lncRNAs Foxo6os in the early diagnosis of heart failure. Furthermore, the lncRNAs Foxo6os is used to evaluate the expression level of lncRNAs that are functionally conserved with lncRNA Foxo6os in the serum of early-stage heart failure patients. The third object of the present invention is to provide an aortic arch constriction model based on AAV9-cTnT-mediated overexpression of lncRNAs Foxo6os, and the aortic arch constriction model is used to achieve high expression of lncRNAs Foxo6os in cardiomyocytes. The fourth object of the present invention is to provide an application of an aortic arch constriction model based on AAV9-cTnT-mediated overexpression of lncRNAs Foxo6os in preparing a research model for the pathogenesis of heart failure or a screening model for drugs for treating heart failure. The fifth object of the present invention is to provide a primary cardiomyocyte model with overexpression of lncRNAs Foxo6os, and the primary cardiomyocyte model is transfected with a plasmid vector containing lncRNAs Foxo6os. Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the analysis of the transcriptome data of heart failure samples, the present invention screens for differentially expressed lncRNAs in the myocardium. The present invention finds that long non-coding RNA Foxo6os (lncRNA Foxo6os) is specifically highly expressed in the heart, and the expression of Foxo6os is significantly downregulated in the pathological process of myocardial hypertrophy-induced heart failure. Knockdown of Foxo6os leads to an upregulation of the level of hypertrophy markers; further identification shows its interaction with myosin-binding protein MYBPC3. Foxo6os can act as a "scaffold" structure, simultaneously bind MYBPC3 and recruit protein kinase PKC-α to promote the phosphorylation of specific sites of MYBPC3 to maintain the stability of its function. In the state of heart failure, the expression level of Foxo6os decreases, resulting in a decrease in the phosphorylation level of MYBPC3 and a weakened contractility of the sarcomere. This aggravates the pathological process of heart failure. Description of the Drawings Figure 1Schematic diagram of the screening results of differentially expressed lncRNAs in the heart tissues of TAC mice; among them, (A) is the volcano plot of differentially expressed genes at week 4 and week 8 of GSE66630 and week 5 of GSE112055; (B) is the heat map of differentially expressed genes at week 4 and week 8 of GSE66630 and week 5 of GSE112055; (C) is the intersection of differentially expressed genes; (D-E) is the expression of significantly differentially expressed lncRNAs in the myocardial tissues of TAC mice. Figure 2 Schematic diagram of the result that Foxo6os has a certain delaying effect on the heart failure process in mice; among them, (A) is the schematic diagram of the construction of the aortic arch constriction model; (B-C) are the representative echocardiogram images of mice at 2 weeks, 4 weeks, 6 weeks and 8 weeks after surgery, and the quantitative analysis of ejection fraction (LVEF), fractional shortening (FS), left ventricular systolic / diastolic end-volume (LVESV / LVEDV) and left ventricular mass (LV Mass) (n = 4 in the control group, n = 5 in the empty virus group, n = 5 in the overexpression virus group, P < 0.05 was considered statistically significant); (D-E) are the representative images of WGA and Masson staining of the left ventricular myocardial tissues of mice at 8 weeks after surgery (n = 4 in the control group Sham, n = 5 in the empty virus group AAV9-NC, n = 5 in the overexpression virus group AAV9-over Foxo6os, P < 0.05 was considered statistically significant), scale bar: 200 μm. Figure 3 Schematic diagram of the results of the effect of Foxo6os on the myocardial mast cell model; among them, (A) is the change in cell area detected by immunofluorescence after transfection with the Foxo6os overexpression plasmid after inducing pmCMs hypertrophy with Ang II (10 nM) (n = 5, P < 0.05 was considered statistically significant), scale bar: 200 μm; (B) is the change in the expression levels of the myocardial cell hypertrophy markers ANP, BNP and MYH7 detected by qPCR after transfection with the Foxo6os overexpression plasmid after inducing pmCMs hypertrophy with Ang II (10 nM) (n = 5, P < 0.05 was considered statistically significant). Figure 4Schematic diagram of the results of the interaction between Foxo6os and MYBPC3 and its effect on their expression; among them, (A) is the RNA-pull down experiment to verify the interaction between Foxo6os and MYBPC3, with the antisense strand Antisense as the negative control; (B) is the RIP experiment to verify in reverse that MYBPC3 can enrich Foxo6os, with the IgG group as the negative control; (C) is the RNA-FISH&IF experiment to confirm the overlap of Foxo6os and MYBPC3 in subcellular localization; (D) is the change in the expression levels of the myocardial hypertrophy markers ANP, BNP, and MYH7 after co-transfecting the Foxo6os overexpression plasmid and MYBPC3 siRNA in cardiomyocytes compared with only transfecting the Foxo6os overexpression plasmid. Figure 5 Schematic diagram of the screening results of lncRNAs functionally related to Foxo6os; among them, (A) is the RNA sequencing data (GSE141910) of healthy and heart failure (HF) human left ventricles, using t-distributed stochastic neighbor embedding (t-SNE); (B) and (C) are volcano plots and heatmaps showing differentially expressed genes in HF patient samples; (D) is the RNA-pull down analysis of MYBPC3 and 5 lncRNAs related to function in human embryonic stem cell-derived cardiomyocytes (hESCs-CMs). Figure 6 Schematic diagram of the operation steps of the mouse aortic arch constriction surgery. Detailed implementation mode The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation modes and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments. The principle of the present invention is as follows: In the first stage of the present invention, the differentially expressed lncRNA Foxo6os during the disease process of heart failure mice is screened using existing datasets. In the second stage of the present invention, the specific effect of Foxo6os on the occurrence and development of heart failure is clarified. Observe the dynamic changes in the expression of lncRNA Foxo6os under the cardiac pathophysiological state, and observe the expression changes of Foxo6os in the myocardial hypertrophy cell model and the animal heart failure model respectively. In the third stage of the present invention, the regulatory function of the change in Foxo6os on myocardial remodeling is explored. 1) In vitro level: To study the regulatory function of Foxo6os on cardiac hypertrophy, the present invention will first conduct research using a primary cardiomyocyte hypertrophy model induced by drugs in vitro. By intervening in the expression of Foxo6os through an expression vector, the effects of changes in Foxo6os expression on indicators such as cardiac hypertrophy markers and changes in the area and morphology of cardiomyocytes will be detected. 2) In vivo level: To study the regulatory function of Foxo6os protein on the occurrence and development of heart failure in vivo, the present invention will construct a mouse model with conditional knockout of Foxo6os in cardiomyocytes and a mouse model with cardiac-specific overexpression of Foxo6os driven by the cardiac-specific promoter AAV9-cTNT. By inducing heart failure in mice through transverse aortic constriction (TAC), the effects of changes in Foxo6os expression on myocardial remodeling markers, cross-sectional area of cardiomyocytes, heart-to-body ratio, cardiac function indicators (ejection fraction, left ventricular shortening fraction, stroke volume, end-systolic volume, and end-diastolic volume, etc.), myocardial fibrosis, degree of collagen deposition, and degree of immune cell infiltration in heart failure mice will be detected. More specifically, the present invention will be described in detail in combination with the following examples. In the fourth stage of the present invention, the specific regulatory mechanism of the Foxo6os-MYBPC3 interaction during the occurrence of heart failure will be revealed. Through methods such as Real-time PCR, Western blotting, LC-MS phosphorylation mass spectrometry analysis, co-immunoprecipitation, and fluorescence protein imaging and localization analysis, combined with methods such as FISH, siRNA, and plasmid transfection, it will be verified that Foxo6os enhances the phosphorylation level of MYBPC3 by binding to MYBPC3 and recruiting the related protein kinase PKC-α, thereby promoting the formation of myocardial actin cross-bridges and increasing the Ca 2+ sensitivity of the myofilaments, thus enhancing the contractility of the myocardium. In the following examples, the nucleotide sequence of the lncRNA Foxo6os is as shown in SEQ ID NO.18: In the following embodiments, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw material products or conventional processing techniques in the art. The following will, with reference to the accompanying drawings, elaborate on some embodiments of the present invention. Without conflict, the following embodiments and the features in the embodiments may be combined with each other. Example 1 As Figure 1 shown, this example provides a method for constructing a mouse myocardial hypertrophy model induced by aortic coarctation, and the specific steps are as follows: 1) Preparation before surgery: Use 25 - 30 g 8-week-old C57BL / 6 mice; disinfect and sterilize all surgical instruments and normal saline one day before surgery; depilate the necks and chests of the mice with depilatory cream; cut off water and food 6 h before surgery, and replace the bedding with new ones; 2) Place the animal supine on a clean operating table, fix the four limbs with adhesive tape, and intraperitoneally inject 50 mg / kg of 1% pentobarbital sodium; press or pinch the hind leg joints of the mouse to confirm the anesthesia depth; adjust the ventilator parameters to: respiratory ratio 1:1.5, respiratory frequency 140 times / min, tidal volume 1 ml; use the light transmission method for tracheal intubation. Place a high-power cold light source closely against the mouse's neck. With the help of the light passing through the neck, use ophthalmic forceps to pull the mouse's tongue root to expose the glottis, and insert a 22G intravenous indwelling needle without the needle tip into the mouse's trachea under direct vision. 3) Remove the needle core, continue to push the outer catheter into the trachea, but the depth should not be too deep to avoid single-lung ventilation. Connect the outer catheter of the indwelling needle to the ventilator and observe the mouse's breathing condition. The chest rise and fall being consistent with the ventilator frequency indicates successful intubation. Subsequently, fix the ventilator pipeline and the indwelling needle on the operating table to prevent them from falling off during the operation. 4) Perform thoracotomy under a stereomicroscope, transect the sternum at the second rib, push aside the thymus to expose the aortic arch field of view, and fix the sternum with a retractor to ensure sufficient operating space. Gently separate the adipose tissue from the aortic arch with 45° curved fine forceps; after identifying the aortic arch and the three branch vessels, use a leader to horizontally pass a 6-0 silk thread between the brachiocephalic trunk and the left common carotid artery; tie a loose knot on the aorta and place a small section of 27G blunt needle parallel to the aorta. Quickly tie a square knot on the needle tip, and then pull out the needle to form a coarctation with a diameter of 0.4 mm. For the mice in the sham operation group, except for the aortic ligation step, the other processes are the same. 5) Carefully remove the thoracic retractor, squeeze out the excess gas in the chest cavity, suture the sternum and ribs in an interrupted suture manner using 6-0 prolene suture, and then suture the skin in a continuous suture manner using 4-0 prolene suture. 6) Pause the ventilator and remove the indwelling catheter outer tube. After the mouse resumes spontaneous breathing, place it on a warming pad (37°C). After waking up in about 20 minutes, put it back into the cage. In this example, the aortic arch (usually located between the left subclavian artery and the left common carotid artery) was surgically narrowed to increase the afterload of the left ventricle, simulate the state of pressure overload, induce compensatory hypertrophy of cardiomyocytes, and ultimately develop into heart failure, thus highly simulating the pathological processes of human pressure overload-induced myocardial hypertrophy and heart failure. The experimental results are as Figure 1-2 shown. Based on the transcriptome analysis of the hearts of mice and humans with heart failure, it was first discovered that lncRNA Foxo6os was specifically highly expressed in the heart. Overexpression of Foxo6os could reduce the cell area and delay the degree of fibrosis of the left ventricular wall. Its expression level was significantly decreased during the pathogenesis of heart failure. Overexpression of Foxo6os could delay the pathological process of heart failure induced by pressure overload after surgery in TAC mice, and the expression of lncRNA Foxo6os was significantly downregulated in the TAC mouse model. Example 2 This example provides a method for injecting adeno-associated virus type 9 into the tail vein of mice. The specific steps are as follows: 1) Storage and dilution of adeno-associated virus: AAV9 virus is stored in a -80°C refrigerator for a long time. Depending on the experimental needs, the virus can be aliquoted according to the virus titer, and the aliquot volume can be 3 - 5 μl. 2) Take out the AAV9 virus from the -80°C refrigerator, melt it in an ice bath and then dilute it. Use PBS containing 5% glycerol as the diluent. After dilution, the titer is 1×10 12 V.G. / mL, and the injection volume into the tail vein of C57BL / 6 mice is 200 μl. 3) Injection into the tail vein of mice: Put the mouse into a fixator, lock the lid and expose the tail outward. Wipe the mouse's tail with an alcohol cotton ball or soak it in 45°C hot water to dilate the blood vessels; straighten the tail and observe the red veins on both sides of the mouse tail; insert the needle at 1 / 3 from the tip of the mouse tail on the relatively obvious side tail vein. If the injection is unobstructed, it means the needle is in the blood vessel; check if there is blood return in the syringe. If there is, injection can be carried out. 4) Press the distal end of the injection site with a cotton ball for about 1 minute to stop bleeding; remove the mouse from the fixator and put it back into the cage. The injection is completed. Conclusion: The above examples can enable specific overexpression of Foxo6os in the myocardium, thus accurately exploring its cardioprotective effect. Example 3 This example provides a method for isolating and culturing primary mouse cardiomyocytes. The specific steps are as follows: (1) Day 1: Isolation of neonatal 24h mouse heart tissue 1) Prepare 50 mL of 1×PBS (without Ca 2+ and Mg 2+ ), and add 20 mM BDM (2,3-Butanedione monoxime). Divide the mixed buffer into two 10 cm sterile culture dishes and place them on ice for later use. 2) Add 30 mL of 0.04% Trypsin digestion solution to a 50 mL sterile centrifuge tube and place it on ice for later use. Prepare two pairs of sterile ophthalmic scissors (straight pointed tips) and two pairs of ophthalmic toothed forceps for later use. 3) Place neonatal 24h C57BL / 6 mice on ice for hypothermic anesthesia, and then quickly disinfect the surface in 75% ethanol solution. Grasp the back of the mouse with the left thumb and index finger, make an incision along the third and fourth intercostal spaces on the left side of the chest with a sterile scissor. Slightly pinch the upper back of the neonatal mouse with the two fingers of the left hand to apply a certain tension to the chest cavity, and the heart will automatically jump out of the chest cavity. Cut the myocardial tissue and immediately transfer it to a pre-cooled culture dish containing 1×PBS and 20 mM BDM, and then euthanize the neonatal mouse. 4) Use ophthalmic forceps in the culture dish to remove excess lung tissue, large blood vessels, and atria. Rinse the heart in 1×PBS solution to remove blood. Then, transfer the washed heart tissue from the first culture dish to the second culture dish and rinse again. 5) After sucking off the PBS buffer in the culture dish with a Pasteur pipette, use ophthalmic scissors to cut the tissue as finely as possible. The obtained tissue pieces are about 0.5 - 1 mm 3 or smaller; 6) Transfer the minced heart to a pre-cooled 50 mL centrifuge tube containing 10 mL of 0.04% Trypsin digestion solution (the amount of digestion solution for the heart tissue of 30 mice is about 10 mL). Digest overnight at 4°C. (2) Day 2: Digestion and seeding of cardiomyocytes 1) According to the number of mice and experimental design, take a certain number of cell culture plates, coat them with 1% gelatin, and incubate at 37°C for at least 1 h; (the number of cells that can be harvested from 30 mice should be at least 6×10 6 cells). 2) Prepare 50 mL of 0.5 mg / mL Collagenase II (50 mL DMEM + 25 mg Collagenase II + 20 mM BDM) as the digestion solution for later use; 3) Prepare two 50 mL centrifuge tubes and add 30 mL of high-glucose DMEM medium for later use; 4) Take out the centrifuge tube of heart tissue that has been incubated overnight. The tissue fragments are in an aggregated state. Add DMEM medium equal to or greater than the volume of the pre-digestion solution, and place it in a 37°C water bath and gently shake for 5 min to terminate digestion. Subsequently, place it on a tube rack to allow the fragments to settle to the bottom of the tube, and use a Pasteur pipette to aspirate the supernatant (to reduce tissue loss, about 1 - 5 ml can be left in the tube). 5) Add 10 ml of collagenase digestion solution to the tissue fragments. After mixing well for 30 s with the tube open, transfer the centrifuge tube containing the digested heart tissue fragments to a 37°C water bath and stir rapidly for 5 min to fully digest the heart tissue; 6) Place the centrifuge tube containing the heart tissue fragments on a tube rack to stand still, allowing the fragments to settle to the bottom of the tube. Aspirate the upper layer of supernatant containing cardiomyocytes and transfer it to a centrifuge tube containing 30 mL of DMEM medium. Then repeat steps 5) and 6) three to four times; (The digestion time depends to a large extent on the enzyme batch number and the concentration of the mixture. The total duration should not exceed 30 min. A longer incubation time or a higher enzyme concentration may reduce cell viability). 7) Place a cell strainer with a pore size of 70 μm into a new 50 mL sterile centrifuge tube. Invert and mix the 50 mL centrifuge tube containing the cardiomyocyte suspension so that the cells are evenly distributed in the suspension, and immediately filter and transfer them to a new centrifuge tube. 8) Centrifuge the centrifuge tube containing the suspended cardiomyocytes at 1000×g for 5 min, then aspirate the supernatant and resuspend all cell pellets in 15 mL of DMEM medium. 9) Transfer the cell suspension into a 10 cm cell culture dish without gelatin coating, and incubate it in a 37°C CO2 cell culture incubator for 40 min (not exceeding 1 h). Fibroblasts and endothelial cells with stronger adhesion ability can be removed by differential adhesion. 10) Take out the 10 cm culture dish that has completed adhesion, resuspend the cells by pipetting repeatedly on the culture dish, and transfer the cell suspension to a sterile centrifuge tube. 11) Centrifuge at 1000×g for 5 min, aspirate the supernatant and add an appropriate amount of medium to concentrate the cell suspension, and perform cell counting; 12) Take out the cell culture plate incubated with gelatin, aspirate the gelatin and dry it, add pre-warmed seeding medium (the seeding medium formula is shown in Table 1), and seed the cardiomyocytes into the medium at a cell density of 1×10 5 per square centimeter. After shaking the culture plate evenly, place it in a 37°C CO2 cell culture incubator and let it stand still for 12 - 24 h to allow the cardiomyocytes to adhere and spread.: Table 1 Seeding medium formula (3) On the third day: Culture of primary cardiomyocytes 1) Prepare the maintenance medium (the formula of the maintenance medium is shown in Table 2) and preheat it in a 37°C water bath. (You can directly start adding drugs or transfection experiments, or starve the cells in serum-free maintenance medium for 12 - 18 h before adding drugs); Table 2 Formula of the maintenance medium 2) After plating for 24 h, it was observed that most cardiomyocytes had adhered to the cell culture plate and began to beat rhythmically. Aspirate the inoculation medium, and then add the maintenance medium and continue culturing for 1 - 3 days. The maintenance medium needs to be replaced on time. Summary: The above examples can isolate and purify highly active primary cardiomyocytes from mouse hearts, providing key experimental materials for subsequent studies on the functions and molecular mechanisms of cardiomyocytes under pathological conditions. Example 4 This example provides a method for constructing an Ang II-induced hypertrophy model of primary mouse cardiomyocytes (pmCMs), and the specific steps are as follows: 1) Inoculate primary mouse cardiomyocytes into a cell culture dish. After adhering for 24 h, replace the serum-free maintenance medium and starve the cells for 18 h; 2) Replace the medium again, add an Ang II solution with a concentration of 10 nM to the maintenance medium to stimulate cardiomyocytes to cause hypertrophy, and add an equal volume of 1×PBS to the control group. Generally, the cell hypertrophy phenotype appears 24 - 48 h after drug addition. As Figure 3 shown, after 48 hours of Ang II treatment, the surface area of pmCMs increased by approximately 40% compared with the control group (*P < 0.01), and ANP, BNP, and MYH7 were all significantly upregulated. Example 5 This example provides a transfection method for primary mouse cardiomyocytes (pmCMs), and the specific steps are as follows: (1) Cell inoculation 1) Inoculate primary mouse cardiomyocytes or mCM cell lines at 1×10 6 per well into a 6-well plate; 2) When the cell density reaches 70% the next day, replace the maintenance medium and conduct transfection experiments; (2) Transfection process 1) Dilute 5 μL of Lipo3000 reagent in 125 μL of Opti-MEM medium. Dilute 5 μg of DNA and 5 μL of P3000 reagent in 125 μL of Opti-MEM medium. Note that when transfecting siRNA and miRNA mimic / inhibitor, P3000 does not need to be added; 2) Mix Lipo3000 and P3000 diluent, and incubate at room temperature for 10 - 15 min; 3) Replace the maintenance medium in the cell culture plate, and add the incubated DNA - liposome complex into the culture plate. Gently shake to mix well, and then place it statically in a 37°C CO₂ cell culture incubator; 4) After 6 h, replace with a brand - new medium, and continue culturing for 48 h before collecting samples for analysis. Conclusion: The above steps can achieve overexpression of Foxo6os in primary cardiomyocytes and conduct subsequent studies. Example 6 This example provides a method for obtaining cardiac tissue samples. According to different experimental purposes, after feeding the experimental mice for 4 - 12 weeks, use an echocardiogram to detect the cardiac function of the experimental mice. After analyzing multiple indicators to confirm the phenotype, the mouse heart samples can be obtained. The specific steps are as follows: 1) After weighing and euthanizing the mice, fix the four limbs of the mice on the operating table; 2) Cut open the chest cavity along the xiphoid process to expose the heart. Make an opening in the inferior vena cava, and then infuse 5 mL of normal saline at the cardiac apex to remove the blood in the heart tissue. The infusion speed should not be too fast to affect the immunohistochemical results; 3) After perfusion until the liver turns white, harvest the heart, remove the blood and other redundant tissues in pre - cooled 1×PBS buffer, and weigh the heart (the whole heart or the left and right ventricles without atria); 4) Cut the tissue into small pieces weighing 20 mg and store them in liquid nitrogen. Conclusion: This example provides a standardized method for obtaining mouse heart tissue samples. Its core process includes: after confirming the cardiac phenotype by echocardiogram, perfusing with normal saline to remove the residual blood in the heart to ensure sample quality, then precisely separating and weighing the heart tissue (the whole heart or ventricular part can be distinguished), and finally cutting it into 20 - mg small pieces and quickly freezing them in liquid nitrogen for storage; this method strictly controls the perfusion speed (to avoid tissue damage), blood clearance (to reduce immunohistochemical interference), and standardized sub - packaging (to ensure the consistency of subsequent experiments), and is applicable to multi - scenario requirements such as the study of cardiac pathological mechanisms, molecular detection (such as RNA / protein analysis), and histological morphology observation, providing a reliable basis for sample preparation in the construction of cardiac disease models. Example 7 This example provides a method for paraffin - embedding mouse tissues. The specific steps are as follows: (1) Obtaining mouse heart samples 1) As described in Example 6, after completing cardiac perfusion, hold the dorsal side of the heart with curved forceps, cut the blood vessels and immediately transfer it to pre - cooled 1×PBS buffer, and gently squeeze out the blood; (2) Place the processed clean heart samples into pre-cooled 4% paraformaldehyde (PFA) solution and fix them on a shaker at 4°C for 1 h or overnight. (2) Dehydrate with gradient ethanol (1) Prepare clean 6-well plates, 50 mL centrifuge tubes, Pasteur pipettes and absolute ethanol; (2) Prepare 50 mL of 30%, 50%, 75%, 95% and 100% (i.e., absolute) ethanol solutions respectively. Ethanol solutions are volatile, so prepare them freshly for use to avoid incomplete dehydration; (3) Take out the overnight heart samples, remove 4% PFA, transfer them to a petri dish containing 1×PBS buffer, and use fine scissors to remove the excess tissue and blood vessels on the heart samples, but the left and right auricles and aorta need to be retained for positioning during paraffin embedding; (4) Transfer the heart samples to 6-well plates, add an appropriate amount of 1×PBS to the 6-well plates with a clean Pasteur pipette, close the lid and place them on a shaker for 5 min each time, for a total of 3 washes; (5) After sucking out the PBS in the 6-well plates, take another clean pipette and add 30% ethanol solution to the plates. The liquid level must submerge the samples, and place them on a shaker for 1.5 h of dehydration. Subsequently, dehydrate in 50% and 70% ethanol for 1.5 h each. Finally, replace with 95% ethanol solution and leave on a shaker at 4°C overnight; (6) After sucking out the 95% ethanol solution, replace it with absolute ethanol and place it on a shaker for 1.5 h, twice in total. (3) Tissue permeabilization and paraffin embedding (1) Prepare small bottles corresponding to the number of samples, label them, open the fume hood, put the samples into the bottles, add xylene (determine the amount according to the tissue size, and the liquid level must completely submerge the tissue), let it stand for 45 min, and repeat once. (When changing the liquid, try to remove all the solution in the bottle as much as possible before adding new xylene. The clearing time depends on the tissue size, but the total duration should not exceed 3 h); (2) After the clearing is completed, remove the xylene in the bottle, then pour in half a bottle of new xylene and half a bottle of paraffin, tighten the bottle cap and place it in a constant temperature environment at 60°C (the temperature needs to be higher than the melting point of paraffin), and let it stand in a semi-wax state for 30 min; (3) Prepare a metal mold and a perforated embedding cassette, pour paraffin into the mold, transfer the samples to the mold, cover the embedding cassette, keep at a constant temperature of 60°C, and let it stand in a full-wax state for 30 min; (4) Suck out the paraffin, then pour in new paraffin, and perform paraffin infiltration at a constant temperature of 60°C for 1 h. This step needs to be repeated 3 times; (5) Keep the tissue sample in the mold, refill it with fresh paraffin, transfer the mold to a freezing table, and adjust the position of the tissue in the mold during the gradual solidification of the paraffin. Use the aortic arch and auricle as position references during fixation. During the adjustment process, ensure that the cut surface faces downward. Note that the sample should not completely sink to the bottom and touch the mold. After the position is adjusted, cover the embedding cassette on the mold while the paraffin is not completely solidified, and wait for it to cool and solidify rapidly. After about 10 minutes, take it out and store it at 4°C. (4) Sectioning 1) Trim the wax block, cut off the unwanted parts, and make direction marks; before sectioning, observe the knife edge under the microscope and select the part with a flat and undamaged knife edge for sectioning; 2) Fix the embedding cassette on the sample holder, ensure that the cut surface of the wax block is parallel to the cross-section of the sample holder, install the blade, set the section thickness to 8 μm on the microtome, move the knife table close to the sample, and let the knife edge slightly touch the cut surface of the wax block without hitting the knife; when sectioning, apply force evenly and rotate the crank at a constant speed to reduce the shaking of the machine body, which may cause uneven section thickness. 3) After a certain length of strip-shaped sections are formed, stop rotating with the right hand, hold a brush and gently pick up the wax ribbon, and gently place it on the water surface of the spreading machine (water temperature 40°C); 4) Transfer the spread paraffin sections to the glass slide. Insert the glass slide into the water at a 45° angle, adjust the position of the section and the glass slide, and then pull the glass slide vertically out of the water; 5) Confirm the tissue position and morphology of the section under the microscope, dry it overnight in an oven at 37°C, and then store it at 4°C. (5) Dewaxing and Hydration 1) Before dewaxing, place the tissue section at room temperature for 60 minutes or in an oven at 60°C for 20 minutes; 2) Immerse the tissue section in xylene for 20 minutes, twice; 3) Immerse it in absolute ethanol for 5 minutes, twice. Then immerse it in 95%, 90%, 85%, and 70% ethanol solutions for 5 minutes, once each. Finally, immerse it in ddH2O for 5 minutes, twice. (6) Antigen Retrieval and Immunostaining 1) Heat the 0.01M sodium citrate buffer (pH 6.0) to 95°C, put the tissue section in and heat for 10 - 15 minutes. After completion, remove the heat source and cool it at room temperature for 30 - 40 minutes; 2) After the buffer cools down, take out the section and rinse it with PBST for 5 minutes, twice. 3) Block it with 70% fetal bovine serum (FBS) at room temperature for 30 - 60 minutes; remove the excess serum, add the primary antibody (the antibody information used in this example is shown in Table 3), and incubate it at 37°C for 2 hours or overnight at 4°C. Rinse it with PBS for 5 minutes, three times in total; 4) Remove the excess serum, add the primary antibody dropwise, and incubate at 37 °C for 2 h or overnight at 4 °C. Rinse with PBS for 5 min, three times in total; 5) Add the fluorescently labeled secondary antibody dropwise (the antibody information used in this example is shown in Table 3), and incubate at 37 °C for 1 h in the dark at room temperature. Rinse with PBS for 5 min, three times in total; 6) Mount the slides with an anti-quenching mounting medium (containing DAPI), and store at 4 °C in the dark. Table 3 List of Antibody Information Conclusion: Through a standardized mouse immunohistochemical staining process (including tissue fixation, paraffin embedding, antigen retrieval, and specific antibody incubation, etc.), the precise localization and visualization analysis of the target protein in mouse heart tissue were successfully achieved in this experiment. The results showed that the staining signals were clear and highly specific (verified by positive and negative controls), which could intuitively reflect the expression level and spatial distribution characteristics of the target protein in cardiomyocytes. Combined with pathological morphological changes (such as fibrosis, inflammatory infiltration, etc.), it provided a key basis for analyzing the molecular mechanism of heart diseases. This method is both reliable and reproducible, and is applicable to the pathological phenotype research and drug intervention effect evaluation of models such as myocardial hypertrophy and ischemia-reperfusion injury. Example 8 This example provides a method for immunofluorescent staining of cells (culture plates), and the specific steps are as follows: 1) Take out the multi-well culture plate from the incubator, discard the culture medium, and wash with PBS for 3 min, twice in total; 2) Fix with 4% paraformaldehyde (200 μL for 6-well plate) for 10 min, and wash with PBS for 5 min, twice in total; 3) Add the immunostaining permeabilization solution containing 0.1% Triton X-100 (1 mL for 6-well plate) for 10 min, and wash with PBS for 5 min, twice in total; 4) Prepare a 5% BSA blocking solution, 300 μL for 6-well plate, and block for 30 min; 5) Aspirate the liquid completely and directly add the primary antibody, incubate at 37 °C for 1 h or overnight in the refrigerator at 4 °C, and wash with PBS for 10 min, three times in total; 6) Add the fluorescently labeled secondary antibody dropwise, incubate in the dark at room temperature for 1 h, and wash with PBS for 10 min, three times in total; 7) Add DAPI for nuclear staining, incubate in the dark at room temperature for 10 min, and wash with PBS for 5 min, twice in total; 8) Observe and take pictures with a fluorescence microscope. Conclusion: The cell immunofluorescence staining method established in this example realizes the visualization of target proteins in cells in the culture plate with high signal-to-noise ratio by optimizing key steps such as fixation (4% paraformaldehyde), permeabilization (0.1% Triton X-100), and blocking (5% BSA), combined with specific incubation of primary / secondary antibodies (flexibly selected at 37°C or 4°C) and DAPI nuclear staining technology. By precisely controlling the reagent dosage (such as 200 μL of fixative for a 6-well plate) and the standardized washing process (PBS gradient washing), this method effectively reduces non-specific background interference. Fluorescence microscopy imaging shows clear localization of target proteins (such as cytoplasmic, membrane, or nuclear distribution), and there is a low overlap between DAPI nuclear staining and fluorescence labeling signals. It is applicable to dynamic monitoring of in vitro cell protein expression, subcellular localization analysis, and drug / gene intervention studies, providing efficient and reproducible technical support for exploring cell biological mechanisms. Example 9 This example provides a method for extracting RNA, and the specific steps are as follows: (1) Preparation of samples For mouse heart tissue, add 1 mL of TRIzol to every 50 - 100 mg of tissue sample, and grind the tissue with a homogenizer; for adherent cells (primary mouse cardiomyocytes or MCM cell line), take the culture plate out of the incubator, aspirate the culture medium, and add 1 mL of TRIzol to every 1×10 5 -10 7 cells, and incubate at room temperature for 10 min. (2) Isolation of RNA Add 200 μL of chloroform to every 1 mL of TRIzol, immediately cover and vigorously invert the sample mixture for 15 s, and incubate at room temperature for 3 min; centrifuge the sample at 12000×g at 4°C for 15 min. After centrifugation, the sample mixture presents three layers, and RNA remains in the upper transparent aqueous phase, with a volume of approximately 60% of the TRIzol used; transfer approximately 450 μL of the aqueous phase to a new 1.5 mL centrifuge tube. (3) Precipitation of RNA 1) Add isopropanol with the same volume as the water, gently mix and incubate at room temperature for 10 min; 2) Centrifuge the sample at 12000×g at 4°C for 15 min, and the RNA precipitate forms white gel-like particles at the bottom and side of the tube. (4) Washing of RNA 1) After removing the supernatant, add 1 mL of pre-cooled 75% ethanol solution, gently blow up the precipitate with a pipette, and centrifuge the sample at 7500×g at 4°C for 5 min; 2) Repeat the washing once, and adjust the precipitation direction during centrifugation. (5) Harvest and preservation of RNA Remove the supernatant and dry the product in a well-ventilated place, taking care to avoid complete drying which may affect the yield; add 15 - 50 μL of RNase-Free water and dissolve the precipitate in a metal bath at 55 °C for 10 min; the RNA product can be stored at 2 - 8 °C for up to one week and at -20 - 80 °C for one year. Avoid multiple freeze-thaw cycles during the experiment. (6) Measurement of RNA concentration Use a spectrophotometer to measure the absorbance and quantify the RNA sample. The closer the ratio of absorbance at 260 nm to that at 280 nm is to 2, the higher the purity. Example 10 This example provides a method for real-time fluorescence quantitative polymerase chain reaction (qPCR) as follows: (1) Establishment of reverse transcription reaction system Use PrimeScriptTM RT reagent Kit Perfect Real Time (TaKaRa, RR037A) to reverse transcribe the RNA extraction product. The reverse transcription system for conventional mRNA is shown in Table 4, and the reaction solution is prepared on ice. After preparation, the reaction conditions are 37 °C for 15 min; 85 °C for 5 s; 4 °C. Table 4 mRNA reverse transcription system After the reverse transcription reaction, dilute the obtained cDNA by 10 times, which can be used for qPCR or stored at -20 °C. (2) Real-time fluorescence quantitative PCR Use qPCR SYBR Green Master Mix (No Rox) (Yeasen, 11201ES03) to perform qPCR reaction on the reverse transcription product. For the calculation of results, the relative quantification method is used. The specific reaction system is shown in Table 5, and the reaction solution is prepared on ice. The specific primer sequences are shown in Table 6. Table 5 qPCR reaction system Table 6 Primer sequence table The amplification program of qPCR uses the three-step method, and the specific reaction conditions are shown in Table 7: Table 7 qPCR reaction conditions Note: "*" indicates fluorescence signal collection After the qPCR reaction is completed, observe the amplification curve and melting curve, with GAPDH as the internal reference. Calculate the relative expression level of the target gene in the sample according to the relative quantification formula: Conclusion: In this study, through qPCR experiments, the changes in the expression level of Foxo6os were explored under normal and disease model conditions. Example 11 This example provides a method for Western Blot of proteins, and the specific steps are as follows: (1) Lysing and protein extraction 1) Prepare the lysis buffer, with the preparation ratio of RIPA:PMSF = 100:1; for animal tissues, add 200 μL of lysis buffer to every 20 mg of tissue sample after cutting it into small pieces, and use a homogenizer or mortar to break the tissue; for cells, 100 μL of lysis buffer can be added to each well of a 6-well plate, and immediately scrape the adherent cells with a cell scraper and transfer them into a 1.5 mL centrifuge tube; 2) Place it on ice for lysis for 30 - 40 min, then centrifuge at 12000×g at 4℃ for 10 min, and transfer the supernatant to a new centrifuge tube; (2) Determining protein concentration by BCA method 1) Prepare the BCA working solution, with the volume ratio of solution A to solution B being 50:1; 2) Add 200 μL of BCA working solution to each standard well of the microplate, prepare protein standards of 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5 mg / ml, and pay attention to mixing well during each dilution; 3) Add 200 μL of BCA working solution to each sample well of the microplate, add 10 μL of the sample to be tested and 10 μL of PBS (if the concentration is too high, it needs to be further diluted); 4) After mixing well, place it at 37℃ for 30 min, and measure the OD value with a microplate reader at a wavelength of 562 nm; 5) Draw the standard protein curve. The standard curve takes the protein content (μg) as the abscissa and the absorbance as the ordinate, and find the corresponding protein content on the standard curve according to the absorbance of the sample; 6) Balance the concentrations of each protein sample, add 5×Loading buffer, with the ratio of sample:LD = 4:1; 7) Boil the sample dilution solution in a boiling water bath for 10 min, let it cool to room temperature, and then store it at -20℃. (3) Preparation of SDS-PAGE gel 1) Select glass plates with a thickness of 1.5 mm, rinse them thoroughly with tap water and then with distilled water, clamp the glass plates into the glass plate clamp, ensure that the bottom is flush, and then clip on the gel preparation rack after clamping; 2) Prepare the separating gel for the lower layer. Since the Klf15 protein molecule is 44 kDa, an 8% separating gel is selected. Quickly add the prepared separating gel between the glass plates to about 1 cm below the comb, and cover the gel layer with alcohol or water up to the top of the glass plates to accelerate the polymerization of acrylamide. Let it stand at room temperature until a distinct boundary appears; 3) After the separating gel has polymerized, pour out the upper alcohol or water. Carefully add the mixed stacking gel along the wall of the glass plate on top of the separating gel up to the top of the glass plates, gently insert the comb to prevent air bubbles, and let it stand at room temperature for 20 - 30 min until completely polymerized. (4) Electrophoresis 1) Place the gel rack into the electrophoresis tank, pour the electrophoresis buffer to fill the inner tank. After filling, the electrophoresis solution will overflow automatically and fill the outer tank, then pull out the comb and load the samples into the comb holes; 2) The sample loading amount is 5 μg or 10 μg per well. If there are remaining wells, add an equal volume of 1× Loading Buffer to prevent the samples from running crookedly. The Marker is generally loaded on the leftmost side; 3) Turn on the electrophoresis instrument, set the parameters to S1 constant voltage 80V for pre - electrophoresis for 30 min until the front of the bromophenol blue reaches the separating gel, then change the parameters to S2 constant voltage 120V and continue electrophoresis for about 70 min until the bromophenol blue reaches the bottom of the separating gel, taking care not to let the bromophenol blue run out of the separating gel. (5) Blotting 1) After electrophoresis, immediately perform blotting to prevent protein dispersion. Cut a PVDF membrane with dimensions of 6 cm × 8 cm, 2 pieces of sponge, and 6 pieces of filter paper; 2) Activate the PVDF membrane with methanol for about 3 min, then place the membrane, gel, sponge, and filter paper in a tray containing transfer buffer and equilibrate for 15 min; Remove the gel, cut out the required part, with both ends bounded by the Marker and the loading edge. Rinse the gel several times with transfer buffer. Place a sponge, three filter papers on the black side of the wet transfer plate, then place the gel (pay attention to the front and back). Use a glass rod to expel air bubbles from each layer; 3) Take out the activated PVDF membrane from methanol, rinse it in the transfer buffer, place it on the gel, then place three filter papers and one sponge. Use a glass rod to expel air bubbles from each layer, and finally clamp the transfer clip. (The black side faces the gel, and the white side faces the membrane); The positive plate (white) of the transfer clip faces the red side of the electrophoresis tank, place it in the electrophoresis tank and conduct electro - blotting at a constant voltage of 72V or a constant current of 300 mA. Determine the blotting time according to the molecular weight of the protein. The blotting time for MYBPC3 is 90 min; (6) Blocking 1) After blotting, carefully remove the gel and the membrane. The bands of the prestained Marker can be observed on the membrane; 2) Prepare a 5% skim milk or BSA solution, that is, add 5 g of skim milk or BSA to every 100 mL of TBST, mix well and filter; 3) Place the membrane into the blocking agent and block it on a shaker at room temperature for 1 h (it can be appropriately extended) or overnight at 4 °C. Note: Coomassie brilliant blue can be used for gel staining to judge the transfer situation, and the gel cannot be reused; after membrane transfer, the membrane can be placed in Ponceau S staining solution and stained at room temperature for 5 min to observe and judge the transfer situation. After rinsing the dye with distilled water, it does not affect the subsequent experiments on the membrane. (7) Primary antibody incubation 1) Dilute the primary antibody of MYBPC3 with the blocking agent according to the recommended concentration at a ratio of 1:1000; the specific dilution ratios are: GAPDH (1:5000), MYBPC3 (1:1000), PKC-α (1:1000), Pan Phospho-serine / threonine / tyrosine Antibody (1:500). 2) Cut the PVDF membrane according to different molecular weight bands, add the prepared primary antibody solution respectively, and incubate overnight at 4 °C or for 2 h at 37 °C on a shaker. 3) After incubation, take out the membrane, and the primary antibody solution can be recovered and stored at 4 °C; wash the membrane 3 times with TBST on a shaker for 10 min each time to remove the residual primary antibody. (8) Secondary antibody incubation 1) Dilute it at a ratio of 1:10000 with the secondary antibody buffer. 2) Add a secondary antibody of the same species as the primary antibody and incubate at room temperature on a shaker for 1 h. 3) After incubation, take out the membrane and wash the membrane 3 times with TBST for 10 min each time to remove the residual secondary antibody. (9) Exposure and identification 1) Add Luminal A and Luminal B to a 15 mL centrifuge tube and mix them at a ratio of 1:1. 2) Place the rinsed membrane face up and evenly drip the luminescent solution on the PVDF membrane. 3) Achieve the best effect through automatic or manual exposure. The specific information of the antibodies used is shown in Table 8: Table 8 List of antibody information Conclusion: In this example, under normal and disease model conditions, it is explored how Foxo6os affects the expression level of MYBPC3 and further regulates heart failure. Example 12 This example provides a method for RNA-pulldown experiment, and the specific steps are as follows: 1) Collect and wash the cells, and rinse them with PBS buffer to remove the residual culture medium. Resuspend the cells in the cell lysate containing protease inhibitor and lyse the cells on ice. 2) Mix the biotin-labeled Foxo6os probe with the cell lysate to ensure consistent Foxo6os concentration in each sample. 3) Incubate the lysate with the Foxo6os probe at room temperature to allow it to bind fully to the protein and form a complex. 4) Pre-incubate the streptavidin beads with the wash buffer to remove possible non-specific binding. 5) Add the pre-treated streptavidin beads to the Foxo6os-protein complex in step 3). Incubate with gentle rotation at 4 °C to allow the streptavidin beads to bind to the biotin-labeled probe. 6) After incubation, collect the streptavidin beads by low-speed centrifugation. Wash the streptavidin beads multiple times with the wash buffer to remove unbound proteins. 7) Subject the eluted protein samples to SDS-PAGE separation. Verify by the method described in Example 11. Determine the binding relationship between Foxo6os and MYBPC3. The experimental results are as Figure 4 (A) shown. In this example, the interaction relationship between Foxo6os-MYBPC3 molecules was deeply explored, and it was first confirmed by RNA pull-down combined with mass spectrometry analysis that Foxo6os directly binds to the MYBPC3 protein. Example 13 This example provides a method for RNA Binding Protein Immunoprecipitation Assay (RIP), and the specific steps are as follows: 1) When the cell density grows to about 90% confluence, perform cell counting and collect approximately 1×10 7 cells. 2) Wash the cells twice with 1× pre-cooled PBS, then resuspend with an equal volume of 1× PLB lysis buffer and incubate on ice for 5 minutes. The lysis buffer can be stored at -80 °C for several months. 3) Vortex the Protein A / G agarose beads, and pipette 40 μl of the beads into 1.5 mL centrifuge tubes according to the number of samples. Wash twice with 0.5 ml NT-2. 4) Resuspend the Protein A / G agarose beads with 100 μl NT-2, add 5 μg of the target antibody or IgG antibody, and incubate with gentle inversion at room temperature for 1 hour. 5) After centrifugation to remove the supernatant, resuspend the complex with NT-2 and wash 6 times. Finally, resuspend the Protein A / G and antibody complex with 900 μl NET-2. 6) Thaw the cell lysate, take 10 μl of the lysate supernatant as input after centrifugation, and store at -80 °C. 7) Incubate the cell lysate with Protein A / G and antibody complex by inversion overnight at 4°C. 8) After incubation, centrifuge to remove the supernatant, resuspend the complex with NT-2, and wash 6 times. 9) Resuspend the complex with Proteinase K buffer and incubate at 55°C for 30 minutes. 10) After incubation, place the EP tube on a magnetic stand and aspirate the supernatant into a new tube. 11) Add a phenol:chloroform:isoamyl alcohol mixture, vortex, and then centrifuge. 12) Aspirate the upper aqueous phase, add absolute ethanol to precipitate RNA, centrifuge at 4°C, and wash the precipitate with 80% ethanol. 13) After air-drying, dissolve the RNA with DEPC water and store at -80°C. 14) Reverse transcribe the extracted RNA into cDNA for subsequent PCR analysis. The experimental results are as Figure 4 shown in (B). The RIP experiment verified in reverse that MYBPC3 can enrich Foxo6os. Example 14 This example provides a method for fluorescence in situ hybridization analysis (RNA-Fluorescence in situ hybridization, FISH) of the subcellular distribution of Foxo6os and its co-localization with MYBPC3. The specific steps are as follows: 1) Cell washing and fixation: First, wash the cultured primary cardiomyocytes (pmCMs) three times with phosphate-buffered saline (PBS), and then fix them with 4% paraformaldehyde. 2) Cell permeabilization: Permeabilize the fixed cells with 0.5% Triton-100 for 10 minutes. 3) Pre-hybridization and hybridization: The permeabilized cells are pre-hybridized at 55°C for 30 minutes, and then incubated with the Foxo6os-FISH probe in hybridization buffer at 55°C for 1 hour. The sequence of the probe is shown in Table 9. 4) Incubation with signal probe: After hybridization, add the signal probe and incubate at 42°C for 3 hours. Washing steps: Wash with 2×SSC at 37°C for 10 minutes, then wash twice with 1×SSC at 37°C for 5 minutes each time, all under dark conditions. 5) Washing and blocking for immunofluorescence: Wash the hybridized sections three times with PBST, and then block them with 3% bovine serum albumin (BSA) at room temperature for 2 hours. 6) Primary antibody incubation: The blocked sections were incubated overnight at 4°C with primary antibodies (anti-MYBPC3, dilution ratio 1:200; anti-α-actin, dilution ratio 1:500; #11313-2-AP, Proteintech, Wuhan, China). 7) Secondary antibody incubation: After washing with PBST, the corresponding fluorescently labeled secondary antibodies were added and incubated at room temperature for 1 h. 8) Nuclear staining and imaging: Finally, DAPI was added for nuclear staining for 5 min, followed by imaging under a confocal microscope. Table 9 Sequences of Foxo6os signal probes The experimental results are as Figure 4 shown in (C) and (D). This example provides a method for studying the spatial localization of the interaction between Foxo6os and MYBPC3, and deeply explores the overlap of their subcellular localization; after co-transfecting the Foxo6os overexpression plasmid and MYBPC3 siRNA in cardiomyocytes, the expression levels of the myocardial hypertrophy markers ANP, BNP, and MYH7 are higher than those transfected with only the Foxo6os overexpression plasmid. Example 15 This example provides a method for extracting exosomes from the plasma of heart failure patients, and the specific steps are as follows: (1) Sample collection: Inpatients diagnosed with chronic heart failure caused by dilated cardiomyopathy in the past five years in the Dongfang Hospital Affiliated to Tongji University, the Tenth People's Hospital Affiliated to Tongji University, the Tongji Hospital Affiliated to Tongji University, and the Shibei Hospital of Jing'an District, Shanghai were selected as the research objects. All hospitals have established an electronic medical record management system, which can conveniently retrieve, browse, and collect cases relying on the electronic medical record management system. Finally, peripheral blood samples of ten heart failure patients were included, and at the same time, peripheral blood of three normal people was matched as a control. (2) Sample collection: 15 ml of whole blood from each person in the selected sample group and normal control group was collected. For the extraction of plasma exosomes, after centrifuging the whole blood at 4000×g for 10 min, the upper plasma was transferred to a new centrifuge tube, and after centrifuging again at 4000×g for 10 min, the plasma was transferred to a cryotube, and then the specimen was placed in a -80°C refrigerator for storage. (3) Exosome extraction: 1) Preparation before experiment: Frozen plasma, 1×PBS, centrifuge tubes, filter membranes, etc. 2) Take out the frozen plasma from the -80°C refrigerator and quickly dissolve it in a 37°C water bath. 3) Transfer the plasma to a new centrifuge tube, centrifuge at 2000×g at 4°C for 30 min, and then transfer the supernatant to another new centrifuge tube to remove cells and cell debris in the plasma, etc. 4) Centrifuge at 12,000×g for 45 min at 4°C. After centrifugation, transfer the supernatant to a new centrifuge tube and filter it through a 0.45-μm filter membrane. Collect the filtrate. 5) Transfer the filtrate to a new centrifuge tube and centrifuge at 110,000×g for 70 min at 4°C. 6) After removing the supernatant, add 10 mL of pre-cooled 1×PBS to resuspend the exosomes in the pellet. Then centrifuge again at 110,000×g for 70 min at 4°C. 7) After removing the supernatant, add 100 μL of pre-cooled 1×PBS to resuspend the exosomes in the centrifuged pellet. Transfer the extracted exosome resuspension to two 50-μL EP tubes and store them at -80°C in a refrigerator for later use. (3) Identification of exosome size and morphology 1) Observation of exosome morphology by transmission electron microscopy (TEM): Take 10 μL of the exosome resuspension extracted by ultracentrifugation, dilute it to 10 μL, and then drop it onto a copper grid for electron microscopy and let it precipitate for 1 min. Then suck off the floating liquid with filter paper; drop 10 μL of phosphotungstic acid and let it precipitate for 1 min, and suck off the floating liquid with filter paper; dry it at room temperature for 5 - 10 min, and then observe the morphological structure of exosomes with a transmission electron microscope. 2) Detection of the size distribution of exosome particle size by nanoparticle tracking analysis (NTA): Inject the extracted exosome resuspension into a nanoparticle tracking analyzer. After laser irradiation, use an optimized automatically set camera to capture the scattered light generated by each particle, and the supporting software of the analyzer generates data on the size of exosome particles. 3) Real-time fluorescence quantitative PCR (qRT-PCR): For the detection of the expression level of ELF3-AS1 in exosomes, this example uses qRT-PCR technology to verify it. The specific steps are as follows: 3-1) Preparation before the experiment: Fluorescent quantitative reagents, PCR reagents, 96-well plates, PCR instruments, ice makers, etc. 3-2) Place the RNA sample and reagents on ice and prepare the reverse transcription first-step template-prime reaction system with 10 μl per tube on ice: First, add 1.0 μl of gDNA Eraser to a DNase / RNase-free EP tube, then add 2.0 μl of 5×gDNA Eraser Buffer, and then add total RNA. The volume of RNA is adjusted according to its concentration (concentration × volume = 1000 ng), and the remaining volume is made up with RNase Free dH2O to make the total volume reach 10 μl. Shake well. 3-3) Place the system in a PCR instrument, and the reaction conditions are 42°C for 2 min, and end when it cools down to 4°C. 3-4) Prepare the second-step reverse transcription system with 20 μL per tube on ice: First, add 10 μL of the reaction solution from the previous step to a nuclease-free EP tube, then add 1.0 μL of PrimeScript Enzyme Mix 1 and RT Primer Mix*4, and finally add 4 μL each of 5×PrimeScript Buffer 2 and RNase Free dH2O to make the total volume 20 μL. Vortex and mix well. 3-5) Place the sample in a PCR instrument with the reaction conditions: 25°C for 10 min, 50°C for 30 min, 85°C for 5 min, and 4°C for 60 min. After the reaction, store the obtained cDNA in a -20°C refrigerator. 3-6) Prepare the qPCR system with 10 μL per tube on ice: Add Premix Ex, 5 μL, then add 0.2 μL each of the PCR Forward Primer and Reverse Primer for the target gene, 0.8 μL of the cDNA of the target gene, and finally add 3.8 μL of ddH2O to make the total volume of the system reach 10 μL. 3-7) Add the corresponding 8 μl reaction systems for the target gene and the internal reference reaction system to the reaction wells of a standard 96-well reaction plate, and make 3 replicates for each cDNA sample. Place the 96-well plate on a real-time fluorescence quantitative PCR instrument for qPCR reaction. The reaction is divided into two stages: pre-denaturation and amplification: 95°C for 1 min for 1 cycle; 95°C for 10 s, 60°C for 30 s, 72°C for 1 min for 42 cycles. 3-8) After the reaction, perform data analysis using the software provided with the instrument. Calculate the relative expression level of ELF3-AS1 using the 2-ΔΔCt method, where ΔCt = Ct target gene - Ct internal reference gene, and ΔΔCt = ΔCt case group - ΔCt control group. 3-9) In this example, cel-39 is added exogenously as an internal reference for the target lncRNA in exosomes. The corresponding primer sequences are shown in Table 10. Table 10 Primer Sequences This example provides a method for extracting exosomes from the plasma of heart failure patients. By optimizing a multi-step fractionation strategy combining ultracentrifugation and kit purification, efficient enrichment of exosomes is achieved. This method provides a standardized technical path for subsequent studies on the functions of exosomes related to heart failure. As Figure 5 shown, the present invention screened high-throughput data of heart failure patients and found the lncRNA ELF3-AS1 that is functionally conserved with Foxo6os. In summary, based on the transcriptome analysis of mouse and human heart failure hearts, the present invention first discovers the lncRNA Foxo6os that is specifically highly expressed in the heart, and its expression level is significantly decreased during the pathogenesis of heart failure. Overexpression of Foxo6os can delay the pathological process of heart failure caused by pressure overload after TAC surgery in mice. The expression of lncRNA Foxo6os is significantly downregulated in the TAC mouse model and the cardiomyocyte hypertrophy model; knockdown of Foxo6os leads to an upregulation of the expression level of hypertrophy markers; further identification shows its interaction with MYBPC3. It is known that the phosphorylation of MYBPC3 plays an important role in the formation of myocardial actin cross-bridges, the calcium sensitivity of myofilaments, and cardiac contractility. In addition, the present invention screens the high-throughput data of heart failure patients and finds the lncRNA ELF3-AS1 that is functionally conserved with Foxo6os. Based on the serological samples of heart failure patients, the functional conserved homologous lncRNA ELF3-AS1 of lncRNA Foxo6os is further explored as a potential clinical detection index for heart failure. Multimodal deep learning is performed in combination with the expression level of ELF3-AS1 in heart failure patients, the hospitalization records and clinical data of patients (structured management data of electronic health records), etc., and a model is constructed. This model provides more accurate and timely diagnosis and decision support for the disease progression of heart failure patients. The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of lncRNAs Foxo6os in the preparation of drugs for detecting, preventing, alleviating, and / or treating heart failure, characterized in that, The nucleotide sequence of the lncRNAs Foxo6os is shown in SEQ ID NO.

18.

2. The application according to claim 1, characterized in that, The drug is a drug for improving myocardial hypertrophy.

3. The application according to claim 2, wherein The drug is a drug for inhibiting heart failure induced by ventricular remodeling caused by myocardial hypertrophy.

4. The application according to claim 1, characterized in that, The lncRNAs Foxo6os interacts with myosin binding protein MYBPC3.

5. The application according to claim 4, characterized in that, The Foxo6os enhances the phosphorylation level of MYBPC3 by binding to MYBPC3 and recruiting the related protein kinase PKC-α, promotes the formation of myocardial actin cross-bridges, increases the Ca 2+ sensitivity, enhances the contractility of the myocardium, and thus slows down the pathological process of heart failure.

6. Application of a biomarker lncRNAs Foxo6os in the early diagnosis of heart failure.

7. The application according to claim 6, wherein The lncRNAs Foxo6os is used to evaluate the expression level of lncRNAs that are functionally conserved with lncRNA Foxo6os in the serum of early-stage heart failure patients.

8. An aortic arch constriction model based on AAV9-cTnT-mediated overexpression of lncRNAs Foxo6os, characterized in that, The aortic arch constriction model is used to achieve high expression of lncRNAs Foxo6os in cardiomyocytes.

9. Application of an aortic arch constriction model based on AAV9-cTnT-mediated overexpression of lncRNAs Foxo6os in preparing a research model for the pathogenesis of heart failure or a screening model for drugs for treating heart failure.

10. A primary cardiomyocyte model with overexpression of lncRNAs Foxo6os, characterized in that, The plasmid vector containing lncRNAs Foxo6os was transfected into the primary cardiomyocyte model.

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