CN112852721A - Experimental method for long non-coding RNA up-regulated gene in myocardial hypertrophy - Google Patents

Abstract

The invention discloses an experimental method of a long non-coding RNA up-regulated gene in myocardial hypertrophy, wherein the levels of lncRNATUG1, miR-497 and myocardial cell enhancement factor 2CmRNA are evaluated through qRT-PCR, Western blot assay is carried out to determine the expression of MEF2C protein, and the endogenous interaction between TUG1, miR-497 and MEF2C is confirmed through a dual-luciferase reporter gene and an RNA immunoprecipitation experiment. Overexpression of MiR-497 mediates the protective role of TUG1 knockdown in AngII-induced myocardial hypertrophy. In addition, TUG1 regulated MEF2C expression by sponges of miR-497. The knockdown of TUG1 rescues AngII-induced myocardial hypertrophy, at least in part, by targeting the miR497/MEF2C axis, highlighting a new promising therapeutic target for the treatment of myocardial hypertrophy.

Description

Experimental method for long non-coding RNA up-regulated gene in myocardial hypertrophy

Technical Field

The invention relates to the technical field of gene coding, in particular to an experimental method of a long non-coding RNA up-regulated gene in myocardial hypertrophy.

Background

Cardiac hypertrophy is a common physiological compensatory response of the heart to a variety of stressors to maintain normal cardiac function. However, cardiac enlargement due to myocardial injury, hypertension stress or excessive neurohumoral activation is associated with maladaptive remodeling and cardiac dysfunction and is classified as pathologic hypertrophy. Pathologic cardiac hypertrophy is a major risk factor for sarcoidosis, heart failure and sudden cardiac death. Although the understanding of the pathological modulators of cardiac hypertrophy has improved, the molecular mechanisms of cardiac hypertrophy remain unclear. Long non-coding RNAs (lncrnas) are RNA molecules of 200 or more nucleotides that perform various functions in a series of important biological processes. They have been found to be associated with human diseases, including cardiac hypertrophy. Such as Wang, et al. It is reported that myocardial hypertrophy-associated factor (CHRF) regulates cardiac hypertrophy by acting as a sponge of microrna (mirna) -489. Wang and colleagues found that epigenetic regulators associated with cardiac hypertrophy (Chaer) are epigenetic checkpoints for cardiac hypertrophy. Recent literature suggests that taurine upregulation gene 1(TUG1) is involved in the pathogenesis of myocardial hypertrophy by spongification of miRNA-29b-3 p.

miRNAs are known to be a class of endogenous, small, non-coding RNAs with

Nucleotides, present in the RNA-induced silencing complex (RISC), silence gene expression. It has now been found that dysregulation of mirnas is associated with human diseases including cardiac hypertrophy. By inhibiting the TGF β pathway, miR-497, a member of the miR-15 family, was identified as a novel modulator of cardiac hypertrophy and fibrosis. Recent literature indicates that by targeting sirtuin 4(Sirt4), increased expression of miR-497 improves cardiac hypertrophy in vitro and in vivo. Previous studies reported that TUG1 exerts a regulatory function in human disease through a competitive endogenous rna (cerana) network by spongiform mirnas. However, between TUG1 and miR-497The role of the interactional interactions in cardiac hypertrophy remains uncertain.

Disclosure of Invention

The invention aims to provide an experimental method for long non-coding RNA up-regulation of genes in myocardial hypertrophy, which aims to solve the problems in the background technology by targeting miR-497/myocyte enhancer factor 2C (MEF2C) axis and reducing in vitro myocardial hypertrophy through TUG1 knock-down.

In order to achieve the purpose, the invention provides the following technical scheme:

an experimental method of a long non-coding RNA up-regulated gene in myocardial hypertrophy comprises the following experimental steps:

step 1: all rats were kept at constant temperature (22 ± 2 ℃), 60% humidity and 12 hours light and dark cycle and were fed at least one standard daily diet prior to the experiment;

step 2: dividing the experimental rats of the animal model into two groups, namely a sham operation group (n ═ 8) and a transverse abdominal aorta coarctation (TAC) group (n ═ 8);

and step 3: separating, culturing and treating primary myocardial cells, namely separating the primary myocardial cells from the heart of a newborn SD rat which is 1-3 days old;

and 4, step 4: cell transfection for TUG1 knockdown studies, cardiomyocytes were transiently introduced with siRNA targeting TUG1 or non-target siRNA;

and 5: real-time quantitative PCR (qRT-PCR) total RNA was extracted from cardiac tissue and cardiomyocytes using an RNA purification kit according to the manufacturer's instructions;

step 6: westernblot total protease inhibitor cocktail was prepared in ice-containing RIPA buffer (150mM Tris-HCl, pH 7.6, 150mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1mM phenylmethanesulfonyl fluoride, 1mM Na3VO4) and then quantified using BCA protein assay kit;

and 7: bioinformatic analysis and dual-luciferase reporter gene assays online database LncBase predictedv.2 helped identify mirnas that may bind to TUG 1;

and 8: RNA Immunoprecipitation (RIP) assay RIP assay was performed using the Magna RIP RNA immunoprecipitation kit (Millipore) and anti-Argonaute 2(anti-Ago2, Abcam) antibody;

and step 9: statistical analysis all data were analyzed using GraphPad Prism 5.0 software and expressed as mean ± Standard Deviation (SD).

Further, the rats of step 1 were housed in a pathogen-free animal housing facility.

Further, step 2 sham group (n ═ 8), rats were exposed to abdominal aorta without ligation, transverse abdominal aorta constriction (TAC) group (n ═ 8), rats were exposed to abdominal aorta and ligated.

Further, the TAC rat model of step 2 is constructed by the following steps: one is opened on the left side of the abdomen of the rat

Then the abdominal aorta was wound in two loops through a 12-gauge needle using 4-0 suture, then the needle was removed, and 8 weeks post-surgery, the size and function of the heart were analyzed using doppler echocardiography, including: left ventricular end-diastolic dimension (LVEDD), left ventricular end-diastolic systolic diameter (LVEDD), left ventricular end-diastolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and Fractional Shortening (FS), all rats were then euthanized and heart weight to body weight ratios determined and heart tissue collected for further evaluation.

Further, step 3 neonatal rats were euthanized and hearts were immediately excised and minced, heart tissue digested with 0.1% collagenase type II and 0.1% trypsin (Gibco) at 37 ℃, and after centrifugation, cardiomyocytes were collected and stored in a incubator containing Dulbecco's modified Eagle's medium/nutrient mixture F-12(DMEM/F-12, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco), 37 ℃ and 5% CO 2.

Further, step 4 for miR-497 overexpression, cardiomyocytes were transfected with either mature miR-497 mimetic (GenePharma) or scrambled oligonucleotide sequence (miR-NC mimetic, GenePharma), MiR-497 silencing was performed using miR-497 inhibitor (in-miR-497, GenePharma), and in-miR-NC (GenePharma) was used as a negative control.

Further, the quality and quantity of the RNA extract of step 5 was evaluated by a NanoDrop ND-2000 spectrophotometer.

Further, step 6 uses the following primary antibodies: anti-ANP (Abcam, Cambridge, UK; dilution 1: 1000), anti-BNP (Abcam; dilution 1: 500), anti-beta-MHC (Abcam; dilution 1: 1000), anti-MEF 2C (Abcam; dilution 1: 1000) and anti-beta-actin (Abcam; dilution 1: 3000), horseradish peroxidase-conjugated anti-rabbit (Abcam; dilution 1: 5000) or anti-mouse (Abcam; dilution 1: 5000) IgG were used as secondary antibodies.

Further, step 7 uses microT-CDS software to perform bioinformatics analysis on the miR-497 molecular target.

Compared with the prior art, the invention has the beneficial effects that: the data of the present invention support the upregulation of TAC1 in TAC rat models and angiotensin ii (ang ii) -induced cardiomyocytes. Furthermore, by targeting the miR-497/myocyte enhancer factor 2C (MEF2C) axis, TUG1 knockdown may reduce myocardial hypertrophy in vitro.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

An experimental method of a long non-coding RNA up-regulated gene in myocardial hypertrophy comprises the following experimental steps:

step 1: care and use of animals all animal experimental procedures were performed according to the guidelines for care and use of agricultural laboratory animals, female Sprague-dawley (sd) rats (8 weeks old,

) All rats were kept at constant temperature (22. + -. 2 ℃), 60% humidity and kept in a pathogen-free animal breeding facility12 hours of light and dark cycles and at least one standard daily diet was fed prior to the experiment;

step 2: animal model experimental rats were divided into two groups, a sham-operated group (n ═ 8), rats exposed to the abdominal aorta without ligation, a transverse abdominal aortic stenosis (TAC) group (n ═ 8), and rats were exposed to the abdominal aorta and ligated. The TAC rat model was constructed as described previously. One is opened on the left side of the abdomen of the rat

Is cut longitudinally. The abdominal aorta was then wound in two loops through a 12 gauge needle using 4-0 suture, and the needle was then removed. At 8 weeks post-surgery, the size and function of the heart was analyzed using doppler echocardiography, including: left ventricular end-diastolic dimension (LVEDD), left ventricular end-diastolic systolic diameter (LVEDD), left ventricular end-diastolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and Fractional Shortening (FS). Subsequently, all rats were euthanized and the ratio of heart weight to body weight was determined and heart tissue was collected for further evaluation.

And step 3: separating, culturing and treating primary myocardial cells, namely separating the primary myocardial cells from the heart of a newborn SD rat which is 1-3 days old; neonatal rats were euthanized and hearts were immediately excised and minced. Heart tissue was digested with 0.1% collagenase type II (Gibco, Rockville, MD, usa) and 0.1% trypsin (Gibco) at 37 ℃. After centrifugation, cardiomyocytes were collected and stored in Dulbecco's modified Eagle's medium/nutrient mixture F-12(DMEM/F-12, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco). 37 ℃ and 5% CO 2. To establish an in vitro model of cardiomyocyte hypertrophy, approximately 70% of the cardiomyocytes were treated with 1mMAng II for 48 hours.

And 4, step 4: cell transfection for TUG1 knockdown studies, cardiomyocytes were transiently introduced with siRNA targeting TUG1 or non-target siRNA; for miR-497 overexpression, cardiomyocytes were transfected with either the mature miR-497 mimetic (GenePharma) or scrambled oligonucleotide sequences (miR-NC mimetic, GenePharma). MiR-497 silencing was performed using miR-497 inhibitor (in-miR-497, GenePharma), and in-miR-NC (GenePharma) was used as a negative control. For overexpression experiments, cardiomyocytes were introduced into pcDNA-based TUG1 or MEF2C overexpression plasmids (pcDNA-TUG1 or pcDNA-MEF2C, GenePharma) or negative control plasmids (pcDNA-NC, GenePharma). All transfections were performed using Lipofectamine 2000 transfection reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's protocol.

And 5: real-time quantitative PCR (qRT-PCR) total RNA was extracted from cardiac tissue and cardiomyocytes using an RNA purification kit according to the manufacturer's instructions; the quality and quantity of RNA extracts were assessed by a NanoDrop ND-2000 spectrophotometer. Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), β -myosin heavy chain (β -MHC), TUG1, and MEF2C mRNA levels were detected by qRT-PCR. Total RNA (1. mu.g) was reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen) and qRT-PCR was performed using a PowerUp SYBRTM Green PCR Master Mix (Applied Biosystems) using an ABI 7900HT sequence detector (Applied Biosystems). The protocol of the manufacturer. The indicated gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The level of mature miR-497 was determined by qRT-PCR using TaqMan reverse transcription kit and TaqMan MicroRNA assay kit (Applied Biosystems) using snRNARNNU 6B as internal control. The amplification curve was denatured at 95 ℃ for 10 min, followed by 20s at 95 ℃ and 40 cycles at 60 ℃ for 1 min. Relative gene expression was calculated based on the 2- Δ Δ Ct method.

Step 6: westernblot total protease inhibitor cocktail was prepared in ice-containing RIPA buffer (150mM Tris-HCl, pH 7.6, 150mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1mM phenylmethanesulfonyl fluoride, 1mM Na3VO4) and then quantified using BCA protein assay kit; protein extracts were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, bedford, massachusetts, usa). The following primary antibodies were used: anti-ANP (Abcam, Cambridge, UK; dilution 1: 1000), anti-BNP (Abcam; dilution 1: 500), anti-beta-MHC (Abcam; dilution 1: 1000), anti-MEF 2C (Abcam; dilution 1: 1000) and anti-beta-actin (Abcam; dilution 1: 3000). Horseradish peroxidase-conjugated anti-rabbit (Abcam; dilution 1: 5000) or anti-mouse (Abcam; dilution 1: 5000) IgG was used as the secondary antibody. Protein bands were detected using an enhanced semiluminescence (ECL) detection kit (Immobilon Western chemiluminescence HRP substrate, millipore) and analyzed using ImageJ software.

And 7: bioinformatic analysis and dual luciferase reporter gene assays online database LncBase predictedv.2 helped identify mirnas that could potentially bind to TUG 1. Bioinformatics analysis was performed on the miR-497 molecular target using the microT-CDS software. Luciferase reporter plasmids (TUG1-WT and MEF2C 3'UTR-WT) harboring miR-497 binding site and site-directed mutagenesis of the seed region (TUG1-MUT and MEF2C 3' UTR-MUT) were obtained from GenePharma. Cardiomyocytes were co-transfected with each reporter construct and either the miR-NC mimetic or miR-497 mimetic. Luciferase activity was determined 48 hours after transfection using a dual luciferase reporter assay system (Promege, madison, wisconsin, usa) according to the manufacturer's instructions. RNA Immunoprecipitation (RIP) assay RIP assay was performed using the Magna RIP RNA immunoprecipitation kit (Millipore) and anti-Argonaute 2(anti-Ago2, Abcam) antibody. Cell lysates were prepared using ice-cold RIPA buffer and then incubated with anti-Ago2 or negative control IgG antibody for 4h at 4 ℃ and then protein a/G sepharose for 4 h. The beads were collected by centrifugation and washed 3 times with ice-cold PBS. Next, total RNA was extracted and enrichment levels of TUG1 and MEF2C mRNA were detected by qRT-PCR. Statistical analysis all data were analyzed using GraphPad Prism 5.0 Software (GraphPad Software, inc., San Diego, CA, USA) and expressed as mean ± Standard Deviation (SD). All experiments were repeated three times. Differences between the two groups were compared by student's t-test or analysis of variance (ANOVA). A probability value of P <0.05 was considered significant.

As a result:

up-regulation of TUG1 levels in TAC rat models and Ang II induced cardiomyocytes to study the relationship of TUG1 to myocardial hypertrophy, a myocardial hypertrophy model was first established by TAC in vivo and Ang II treatment. In contrast, TAC resulted in significant increases in LVEDD, LVESD and LVEDP, significant decreases in LVESP and FS, and strong elevation of heart weight and body weight radio. Moreover, TAC caused significant increases in the levels of the hypertrophy-associated genes ANP, BNP and β -MHC, demonstrating the successful establishment of a TAC rat model. Furthermore, Ang II treatment significantly increased the levels of ANP, BNP and β -MHC in cardiomyocytes. Interestingly, TUG1 levels were significantly upregulated in TAC model and Ang II treated cardiomyocytes as shown by qRT-PCR.

Knock down of TUG1 attenuated Ang II-induced myocardial hypertrophy to further investigate the function of TUG1 in myocardial hypertrophy, a loss of function experiment was performed using si-TUG 1. Transient transfection of si-TUG1, rather than scrambled control sequences, resulted in significantly reduced levels of TUG1 in Ang II-treated cardiomyocytes. Subsequent qRT-PCR and Westernblot analysis showed that TUG1 knockdown triggered significant reductions in ANP, BNP and β -MHC expression at both mRNA and protein levels in Ang II-treated cardiomyocytes compared to the negative control. To study the role of TUG1 in myocardial hypertrophy, a myocardial hypertrophy model was first established by TAC in vivo and Ang ii treatment in vitro. In contrast, TAC resulted in significant increases in LVEDD, LVESD and LVEDP, significant decreases in LVESP and FS, and strong elevation of heart weight and body weight radio. Moreover, TAC caused significant increases in the levels of the hypertrophy-associated genes ANP, BNP and β -MHC, demonstrating the successful establishment of a TAC rat model. Furthermore, Ang II treatment significantly increased the levels of ANP, BNP and β -MHC in cardiomyocytes. TUG1 levels were significantly upregulated in TAC model and Ang II treated cardiomyocytes as shown by qRT-PCR.

Molecular sponge of TUG1 acting as miR-497 to further understand the function of TUG1 in cardiac hypertrophy, an online database LncBase Predicted v.2 was used to help identify mirnas that may bind TUG 1. Among these candidates, several miRNAs (miR-93, miR-142-3p, miR-103, miR-631-5p, miR-16-5p and miR-497) that are associated with cardiomyocyte hypertrophy and decreased were selected. Regulating myocardial hypertrophy. The results show that miR-497 is the miRNA most significantly upregulated in cardiomyocytes silencing TUG1, so miR-497 was selected for further study. These data reveal the putative target sequence of miR-497 within TUG 1. To verify this, a dual luciferase reporter assay was performed. When the TUG1 fragment with the miR-497 binding sequence was cloned into a luciferase reporter, co-transfection of the TUG1 wild-type reporter and miR-497 mimetic into cardiomyocytes resulted in luciferase activity lower than cells co-transfected with miR-NC mimetics. However, when the target site is mutated, no reduction reaction of luciferase is observed in the presence of the miR-497 mimic. Ago2 is a core component of RISC and plays a crucial regulatory role in the maturation process of miRNA. Thus, RIP experiments were performed using anti-Ago2 antibody. The data show that miR-497 overexpression obviously improves the enrichment degree of TUG1, so that potential endogenous interaction between TUG1 and miR-497 is triggered. The data from qRT-PCR also show that miR-497 is significantly down-regulated in TAC model and Ang II treated cardiomyocytes. Moreover, miR-497 expression was significantly reduced by the TUG1 overexpression plasmid compared to the counterpart, whereas miR-497 expression increased significantly when TUG1 was depleted in cardiomyocytes. MiR-497 overexpression mediates the protective effect of TUG1 knock-down in Ang II-induced myocardial hypertrophy to determine whether TUG1 modulates myocardial hypertrophy by MiR-497, the expression of MiR-497 in si-TUC1 transfected myocardium was reduced prior to Ang II treatment. The qRT-PCR results showed that si-TUG 1-mediated miR-497 upregulation was significantly reversed by in-miR-497 transfection. Furthermore, restoration of miR-497 levels in Ang II-treated cardiomyocytes significantly abrogated the reduction of ANP, BNP and β -MHC expression by TUG1 knockdown. TUG1 regulates MEF2C expression by sponges of miR-497, and exerts biological functions by regulating its target genes. In the invention, in order to further understand the potential mechanism of miR-497 for regulating cardiac hypertrophy, the molecular target of the miR-497 is analyzed in detail. Using the microT-CDS software, predicted data show the putative complementary sequence of miR-497 in MEF2C mRNA 3' -UTR. To confirm whether MEF2C is a direct target for miR-497, a dual luciferase reporter assay was performed using MEF2C 3'UTR reporter (MEF2C 3' UTR-WT) with miR-497 binding sequence and site-directed mutagenesis. Seed region (MEF2C 3' UTR-MUT). Transfection of the miR-497 mimetic significantly reduced the luciferase activity of MEF2C 3' UTR-WT compared to the negative control. However, site-directed mutation of the miR-497 binding region significantly abolishes the inhibitory effect of miR-497 on the expression of the reporter gene. RIP analysis shows that miR-497 overexpression obviously improves the enrichment level of MEF2C mRNA compared with negative control. In addition, qRT-PCR analysis showed significant upregulation of MEF2C mRNA expression in TAC model and Ang II treated cardiomyocytes. Furthermore, MEF2C protein expression was significantly reduced by miR-497 overexpression and increased significantly with inRi-497 transfection compared to its counterpart. It was further determined whether TUG1 regulated expression of MEF2C in cardiomyocytes. As expected, MEF2C levels were significantly elevated by overexpression of TUG1, whereas miR-497 mock transfection has largely eliminated this effect. MEF2C is a functional target of miR-497 in regulating Ang II-induced myocardial hypertrophy to further understand the connection of miR-497 and MEF2C in myocardial hypertrophy, the miR-497 mimetic and pcDNA-MEF2C are co-transfected into myocardial cells for Ang II induction. In contrast to the negative control, co-transfection of pcDNA-MEF2C reversed the inhibitory effect of miR-497 overexpression on MEF2C expression significantly. Furthermore, the restored MEF2C levels significantly abolished the reduction of miR-497 overexpression to ANP, BNP and β -MHC levels in AngII treated cardiomyocytes. Pathological cardiac hypertrophy causes extracellular collagen deposition, loss of adrenergic responsiveness and metabolic disorders, resulting in heart failure. Cardiac hypertrophy can be established by Ang II induction in vitro and TAC surgery in vivo. In the present invention, cardiac hypertrophy was successfully established in vivo and in vitro as evidenced by changes in hemodynamic parameters, radioradiation of heart weight and body weight, and ANP, BNP, and β -MHC levels. To date, many lncrnas have been identified as positive or negative regulators in the cardiac hypertrophy pathway. In this study, it was first demonstrated that TUG1 reduced Ang II-induced cardiomyocyte hypertrophy by targeting the miR-497/MEF2C axis. By spongining miR-29c under hypoxic conditions, TUG1 enhanced the transformation of cardiac fibroblasts into myofibroblasts. It has been found that TUG1 regulates B-cell lymphoma 2 interacting protein 3(Binp3) in spongiform miR-145-5p, playing a key role in hypoxia-induced cardiomyocyte injury. In the current work, data indicate that TAC1 is up-regulated in TAC rat models and Ang II-induced cardiomyocytes, whereas knock-down of TUG1 attenuated Ang II-induced myocardial hypertrophy, and then, using the online database LncBase Predicted v.2 helps to identify mirnas that may bind to TUG 1. Among these candidates, miR-497 was selected for further study, and TUG1 was first validated to act as a miR-497 sponge. miR-497, a member of the miR-497 family, has been identified as a cancer-suppressing miRNA in a variety of human cancers, such as breast cancer, non-small cell lung cancer, and osteosarcoma. Moreover, miR-497 was found to be involved in postnatal quiescence and osteoblast differentiation of skeletal muscle stem cells by targeting BMP signaling. miR-497 plays a crucial regulatory role in mitotic arrest of cardiomyocytes. In the present invention, the data show that miR-497 expression is down-regulated in TAC rat model and Ang II induced cardiomyocytes. It is proved that the forced level of miR-497 can save myocardial hypertrophy in vitro. Furthermore, miR-497 was first found to be a functional mediator of TUG1 in the regulation of Ang II-induced myocardial hypertrophy. In cellular pathophysiology, mirnas are widely accepted to direct post-transcriptional inhibition of mRNA targets. Therefore, potential targets of miR-497 are predicted by using microT-CDS software. MEF2C has attracted attention in the present invention because of the key involvement of MEF2C in cardiac development, cardiomyocyte reprogramming and hypertrophic cardiomyopathy. MEF2C has been highlighted as a mediator of the signal response of cardiac transcription programs and plays a key role in the development of cardiac hypertrophy. In addition, MEF2C deficiency reduced left ventricular hypertrophy from stress overload by modulating the mTOR/S6K pathway in mice. Previous studies have shown that activation of MEF2C triggered by insulin-like growth factor-1 (IFG-1) mediates the hypertrophy-promoting function of IGF-1 on cardiac gene expression. In addition, the calcineurin-MEF 2C pathway was shown to be involved in stress-induced myocardial hypertrophy of the endoplasmic reticulum of neonatal rat cardiomyocytes. In the present invention, miF-497 was first verified to target and inhibit MEF2C directly in cardiomyocytes. The results indicate that MEF2C is a functionally important target of miR-497 in regulating Ang II-induced myocardial hypertrophy. Likewise, miR-214-3p plays an inhibitory role in Ang II-induced myocardial hypertrophy by targeting MEF 2C. It was demonstrated that high levels of miR-497 inhibited myocardial hypertrophy in vitro and in vivo by targeting Sirt 4. First, it was verified by sponge miR-497 that TUG1 regulated expression of MEF 2C. Furthermore, silencing of TUG1 can ameliorate diabetic cardiomyopathy by direct targeting of miR-499-5p, thereby causing diastolic dysfunction. Current studies indicate that TUG1 knockdown mitigates Ang II-induced myocardial hypertrophy at least in part by targeting the miR-497/MEF2C axis. The clinical significance of TUG1 and its potential value as a therapeutic target should be further explored.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (9)

1. An experimental method of a long non-coding RNA up-regulated gene in myocardial hypertrophy is characterized by comprising the following experimental steps:

step 1: all rats were kept at constant temperature (22 ± 2 ℃), 60% humidity and 12 hours light and dark cycle and were fed at least one standard daily diet prior to the experiment;

step 2: dividing the experimental rats of the animal model into two groups, namely a sham operation group (n ═ 8) and a transverse abdominal aorta coarctation (TAC) group (n ═ 8);

and step 3: separating, culturing and treating primary myocardial cells, namely separating the primary myocardial cells from the heart of a newborn SD rat which is 1-3 days old;

and 4, step 4: cell transfection for TUG1 knockdown studies, cardiomyocytes were transiently introduced with siRNA targeting TUG1 or non-target siRNA;

and 5: real-time quantitative PCR (qRT-PCR) total RNA was extracted from cardiac tissue and cardiomyocytes using an RNA purification kit according to the manufacturer's instructions;

step 6: western blot Total protease inhibitor cocktail was prepared in ice-containing RIPA buffer (150mM Tris-HCl, pH 7.6, 150mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1mM phenylmethanesulfonyl fluoride, 1mM Na3VO4) and then quantitated using BCA protein assay kit;

and 7: bioinformatic analysis and dual luciferase reporter gene assays online database LncBase predictedv.2 helped identify mirnas that could potentially bind to TUG 1.

2. The method for testing the up-regulation of genes in myocardial hypertrophy as claimed in claim 1, wherein the rat of step 1 is kept in a pathogen free animal breeding facility.

3. The method of claim 1, wherein the rat is exposed and ligated without ligation to abdominal aorta in step 2 sham group (n 8) and transverse abdominal aortic stenosis (TAC) group (n 8).

4. The method for testing the up-regulation of genes in myocardial hypertrophy of long non-coding RNA as claimed in claim 1, wherein the TAC rat model of step 2 is constructed by the following steps: one is opened on the left side of the abdomen of the rat

Then the abdominal aorta was wound in two loops through a 12-gauge needle using 4-0 suture, then the needle was removed, and 8 weeks post-surgery, the size and function of the heart were analyzed using doppler echocardiography, including: left ventricular end-diastolic dimension (LVEDD), left ventricular end-diastolic systolic diameter (LVEDD), left ventricular end-diastolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and Fractional Shortening (FS), all rats were then euthanized and heart weight to body weight ratios determined and heart tissue collected for further evaluation.

5. The method of claim 1, wherein step 3 comprises euthanizing neonatal rat and immediately excising and mincing heart, digesting heart tissue with 0.1% collagenase type II and 0.1% trypsin (Gibco) at 37 ℃, centrifuging, collecting cardiomyocytes and storing them in a Dulbecco's modified Eagle's medium/nutrient mixture F-12(DMEM/F-12, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco), 37 ℃ and 5% CO 2.

6. The method of claim 1, wherein step 4 comprises transfecting cardiomyocytes with mature miR-497 mimic (GenePharma) or scrambled oligonucleotide sequence (miR-NC mimic, GenePharma) for miR-497 overexpression, performing miR-497 silencing using miR-497 inhibitor (in-miR-497, GenePharma), and using in-miR-NC (GenePharma) as a negative control.

7. The method of claim 1, wherein the quality and quantity of the RNA extract of step 5 is evaluated by a NanoDrop ND-2000 spectrophotometer.

8. The method of claim 1, wherein the following primary antibodies are used in step 6: anti-ANP (Abcam, Cambridge, UK; dilution 1: 1000), anti-BNP (Abcam; dilution 1: 500), anti-beta-MHC (Abcam; dilution 1: 1000), anti-MEF 2C (Abcam; dilution 1: 1000) and anti-beta-actin (Abcam; dilution 1: 3000), horseradish peroxidase-conjugated anti-rabbit (Abcam; dilution 1: 5000) or anti-mouse (Abcam; dilution 1: 5000) IgG were used as secondary antibodies.

9. The method for testing the up-regulation of genes in myocardial hypertrophy of long non-coding RNA as claimed in claim 1 wherein step 7 is performed by bioinformatics analysis of miR-497 molecular target using microT-CDS software.