Application of LncRNA Gm20257 in the preparation of drugs for the prevention and treatment of pathological myocardial hypertrophy

Abstract

This invention relates to the field of biomedical technology, and in particular to the application of lncRNA Gm20257 in the preparation of drugs for the prevention and treatment of pathological myocardial hypertrophy. The invention first determines the changes in lncRNA Gm20257 in pathological myocardial hypertrophy and its inhibitory effect on it, finding that the expression of lncRNA Gm20257 is elevated in pathological myocardial hypertrophy, a stress-induced increase. Inhibition of lncRNA Gm20257 expression can induce cardiomyocyte hypertrophy; in vitro overexpression of lncRNA Gm20257 can significantly inhibit cardiomyocyte hypertrophy; and in vivo overexpression of lncRNA Gm20257 can significantly inhibit pathological myocardial hypertrophy in mice. This invention then uses lncRNA Gm20257 as a novel drug or drug target for the prevention and treatment of pathological myocardial hypertrophy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of LncRNA Gm20257 in the preparation of drugs for the prevention and treatment of pathological myocardial hypertrophy. Background Technology

[0002] Pathological myocardial hypertrophy is a common phenotype of many cardiovascular diseases (such as hypertension, diabetes, myocardial infarction, and myocarditis). However, with social development and increasing aging, the incidence of cardiovascular diseases is rising year by year. Cardiovascular diseases and other underlying conditions not only seriously affect people's quality of life but also exacerbate the severity of other diseases. Therefore, the prevention and treatment of cardiovascular diseases is an important task in the medical field, and inhibiting pathological myocardial hypertrophy can contribute to the treatment of many cardiovascular diseases.

[0003] However, current medical treatments cannot significantly inhibit the development of pathological myocardial hypertrophy. As the condition worsens, the patient's health deteriorates rapidly, eventually leading to heart failure or even sudden death. Therefore, there is an urgent need to find new therapeutic targets to develop corresponding drugs and implement effective treatments to inhibit this irreversible malignant remodeling.

[0004] The pathogenesis of pathological myocardial hypertrophy is complex, with intracellular energy deficiency being a key factor in its development. Gradual decompensation of intracellular energy metabolism leads to the death of numerous cardiomyocytes, resulting in irreversible malignant damage to the heart. Therefore, correcting energy production disorders plays a crucial role in the treatment of pathological myocardial hypertrophy. Although some currently used drugs have some influence on cellular energy metabolism, their effects are not significant. In many cardiovascular diseases, lncRNA expression is altered and widely involved in the development and progression of these diseases; some lncRNAs have even become targets for the diagnosis and treatment of cardiovascular diseases. Increasing evidence shows that lncRNAs also play an irreplaceable regulatory role in pathological myocardial hypertrophy. Although more and more lncRNAs capable of regulating pathological myocardial hypertrophy have been discovered, lncRNAs that can significantly and stably inhibit pathological myocardial hypertrophy remain unidentified. Therefore, finding lncRNAs that can effectively and stably inhibit pathological myocardial hypertrophy remains a top priority in pharmaceutical research and development. Summary of the Invention

[0005] To address the aforementioned issues, the present invention aims to provide the application of LncRNA Gm20257 in the preparation of drugs for the prevention and treatment of pathological myocardial hypertrophy, representing a pharmaceutical use of an endogenous long non-coding RNA. The invention first demonstrates that LncRNA Gm20257 is significantly elevated in mouse models of pathological myocardial hypertrophy and in isolated cardiomyocytes, exhibiting a stress-induced increase, which is crucial for its application as a diagnostic biomarker. Further, it is found that inhibiting the expression of LncRNA Gm20257 in isolated cardiomyocytes using si-Gm20257 significantly induces pathological myocardial hypertrophy-related phenotypes. Finally, in vivo overexpression of LncRNA Gm20257 via adeno-associated virus-9 (AAV-9) encapsulation or in vitro overexpression via plasmid transfection significantly inhibits pathological myocardial hypertrophy, confirming that LncRNA Gm20257 possesses a significant and stable anti-hypertrophy effect. This invention confirms that LncRNA Gm20257, as a novel drug or drug target, can be applied to the prevention and treatment of pathological myocardial hypertrophy.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The first object of the present invention is to provide the use of LncRNA Gm20257 as a biomarker for the diagnosis of pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0008] In one embodiment of the present invention, lncRNA Gm20257 is upregulated in pathological myocardial hypertrophy; inhibiting the expression of lncRNA Gm20257 can induce pathological myocardial hypertrophy.

[0009] In one embodiment of the present invention, in vitro overexpression of LncRNA Gm20257 can significantly inhibit cardiomyocyte hypertrophy; in vivo overexpression of LncRNA Gm20257 can significantly inhibit pathological myocardial hypertrophy.

[0010] A second objective of this invention is to provide a diagnostic kit for pathological myocardial hypertrophy, comprising the aforementioned LncRNAGm20257.

[0011] A third objective of this invention is to provide the application of LncRNA Gm20257 in screening diagnostic drugs for pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0012] A fourth objective of this invention is to provide the application of LncRNA Gm20257 in a diagnostic kit for screening pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0013] A fifth objective of this invention is to provide the use of LncRNA Gm20257 in the preparation of drugs for the prevention or treatment of pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0014] A sixth objective of this invention is to provide the application of LncRNA Gm20257 in the preparation of a kit for pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0015] A seventh object of the present invention is to provide the use of LncRNA Gm20257 activator in the preparation of drugs for the prevention or treatment of pathological myocardial hypertrophy, wherein the nucleotide sequence of LncRNA Gm20257 is shown in SEQ ID NO.11.

[0016] The eighth object of the present invention is to provide the use of LncRNA Gm20257 activator in the preparation of a kit for pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0017] In one embodiment of the present invention, the expression of LncRNA Gm20257 in isolated cardiomyocytes was inhibited by si-Gm20257, which significantly induced the pathological myocardial hypertrophy-related phenotype.

[0018] In one embodiment of the present invention, in vivo overexpression of LncRNA Gm20257 via adeno-associated virus-9 (AAV-9) encapsulation or in vitro overexpression of LncRNA Gm20257 via plasmid transfection can significantly inhibit pathological myocardial hypertrophy.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a new pathophysiological mechanism for the occurrence and development of pathological myocardial hypertrophy, confirms that lncRNAGm20257 has a significant and stable anti-hypertrophy effect, and provides a long non-coding RNA that can be used for the prevention and treatment of pathological myocardial hypertrophy. Attached Figure Description

[0021] Figure 1This is an example of detecting changes in lncRNA Gm20257 during pathological myocardial hypertrophy in Example 1. Figure 1 A is a representative figure showing the myocardial contractility and the thickness of the left ventricular posterior wall in mice in the TAC surgery group and the Sham group detected by ultrasound in small animals. Figure 1 B is a statistical graph of the thickness of the posterior wall of the left ventricle in mice; Figure 1 C represents the heart weight / body weight ratio, lung weight / body weight ratio, and heart weight / tibia length ratio, n = 3, *P < 0.05 vs. Sham; Figure 1 D represents the relative mRNA expression levels of hypertrophy markers ANP, BNP, and β-MHC in mouse hearts; Figure 1 E represents the protein content of β-MHC in Sham and TAC mice, with corresponding statistical figures. GAPDH was used as an internal control. n=3. *P<0.05 vs. Sham. Figure 1 F represents the difference in lncRNA Gm20257 expression between the TAC group and the Sham group detected by Real-time PCR, n=3, *P<0.05 vs. Sham.

[0022] Figure 2 In Example 2, inhibiting the expression of lncRNA Gm20257 can induce pathological myocardial hypertrophy. Figure 2 A represents the expression of lncRNA Gm20257 in the si-lncRNA Gm20257 and si-NC groups detected by Real-time PCR, n=6, *P<0.05 vs. si-NC; Figure 2 B represents α-actinin-labeled cardiomyocyte cytoskeleton to visualize cardiomyocyte size. Cardiomyocytes were cultured for 48 h and then transfected with si-lncRNA Gm20257 or si-NC for 48 h before incubation with α-actinin-labeled cardiomyocyte cytoskeleton to visualize cardiomyocyte size. Both cell groups were incubated with α-actin-stained cytoskeleton and DAPI-stained nuclei (blue: nucleus, red: α-actinin, magnification 400×; *P<0.05 vs. si-NC, n=10 cells per group). Figure 2 C represents the relative mRNA expression levels of hypertrophy markers ANP, BNP, and β-MHC in cardiomyocytes transfected with si-lncRNA Gm20257 or si-NC by Real-time PCR. Figure 2 D is a Western blot diagram showing the protein content of β-MHC in cardiomyocytes transfected with si-lncRNA Gm20257 or si-NC and the corresponding statistical graph. GAPDH was used as an internal control. n=6. *P<0.05 vs. si-NC.

[0023] Figure 3In Example 3, in vitro overexpression of lncRNA Gm20257 significantly inhibited cardiomyocyte hypertrophy. Figure 3 A represents the expression of lncRNA Gm20257 in the Real-time PCR group and the Vector group, n=6, *P<0.05 vs. Vector; Figure 3 B represents the cytoskeleton labeled with α-actinin, indicating cardiomyocyte size. Cardiomyocytes were cultured for 48 h, transfected with lncRNA Gm20257 overexpression plasmid or Vector plasmid, and treated with Ang II or PBS for 48 h. α-actin staining was then applied to the cytoskeleton, and DAPI staining was applied to the nuclei (blue: nucleus, red: α-actinin, magnification 400×; n = 4 independent experiments, *P < 0.05 vs. Control). # P<0.05 vs. Ang II+Vector (n=10 cells per component); Figure 3 C represents Real-time PCR detection of cardiomyocytes transfected with lncRNA Gm20257 or Vector plasmids, treated with Ang II or PBS for 48 h, and the relative mRNA expression levels of hypertrophy markers ANP, BNP, and β-MHC in cardiomyocytes were detected (n=6, *P<0.05 vs. Control + Vector). # P<0.05 vs. Ang II+Vector); Figure 3 D represents the protein content of β-MHC, a hypertrophy marker in cardiomyocytes, after transfection with lncRNA Gm20257 or Vector plasmids followed by treatment with Ang II or PBS for 48 h. The graph shows the corresponding statistical results (n=6, *P<0.05 vs. Control + Vector). # P < 0.05 vs. Ang II + Vector.

[0024] Figure 4 In Example 4, in vivo overexpression of lncRNA Gm20257 significantly inhibited pathological myocardial hypertrophy in mice. Figure 4 A represents the expression level of lncRNA Gm20257 in mice after tail vein injection of either an adeno-associated virus-9 (AAV-9)-wrapped lncRNA Gm20257 overexpression plasmid or an AAV-9-wrapped Vector control plasmid. Figure 4 B is a representative figure of myocardial contractility and left ventricular posterior wall thickness in mice after TAC or Sham surgery following in vivo transfection with lncRNA Gm20257 overexpression plasmid or Vector plasmid. Figure 4C represents the statistical graph of the posterior wall thickness of the left ventricle in mice, n=6, *P<0.05 vs. Sham+Vector. # P<0.05 vs. TAC+Vector; Figure 4 D represents the heart weight / body weight ratio, lung weight / body weight ratio, and heart weight / tibia length ratio in mice after in vivo transfection with lncRNA Gm20257 overexpression plasmid or Vector plasmid followed by TAC or Sham surgery. n = 6, *P < 0.05 vs. Sham + Vector. # P<0.05 vs. TAC+Vector; Figure 4 E represents the cross-sectional area of ​​mouse cardiac cardiomyocytes detected by wheat germ agglutinin staining (WGA) after in vivo transfection with lncRNA Gm20257 overexpression plasmid or Vector plasmid and subsequent TAC or Sham surgery. Figure 4 F represents the relative mRNA expression levels of hypertrophy markers ANP, BNP, and β-MHC in the heart of mice after in vivo transfection with lncRNA Gm20257 overexpression plasmid or Vector plasmid followed by TAC or Sham surgery, n = 6, *P < 0.05 vs. Sham + Vector. # P<0.05 vs. TAC+Vector; Figure 4 G represents the protein content of β-MHC in mice after in vivo transfection with lncRNA Gm20257 overexpression plasmid or Vector plasmid followed by TAC or Sham surgery, along with corresponding statistical figures. GAPDH was used as an internal control. n=6. *P<0.05 vs. Sham+Vector. # P<0.05 vs. TAC+Vector. Detailed Implementation

[0025] This invention provides the application of LncRNA Gm20257 as a biomarker for the diagnosis of pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0026] In one embodiment of the present invention, lncRNA Gm20257 is upregulated in pathological myocardial hypertrophy; inhibiting the expression of lncRNA Gm20257 can induce pathological myocardial hypertrophy.

[0027] In one embodiment of the present invention, in vitro overexpression of LncRNA Gm20257 can significantly inhibit cardiomyocyte hypertrophy; in vivo overexpression of LncRNA Gm20257 can significantly inhibit pathological myocardial hypertrophy.

[0028] This invention provides a diagnostic kit for pathological myocardial hypertrophy, comprising the above-mentioned LncRNA Gm20257.

[0029] This invention provides the application of LncRNA Gm20257 in screening diagnostic drugs for pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0030] This invention provides the application of LncRNA Gm20257 in a diagnostic kit for screening pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0031] This invention provides the use of LncRNA Gm20257 in the preparation of drugs for the prevention or treatment of pathological myocardial hypertrophy, wherein the nucleotide sequence of LncRNA Gm20257 is shown in SEQ ID NO.11.

[0032] This invention provides the application of LncRNA Gm20257 in the preparation of a kit for pathological myocardial hypertrophy, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0033] This invention provides the application of LncRNA Gm20257 activator in the preparation of drugs for the prevention or treatment of pathological myocardial hypertrophy, wherein the nucleotide sequence of LncRNA Gm20257 is shown in SEQ ID NO.11.

[0034] This invention provides the application of LncRNA Gm20257 activator in the preparation of a kit for pathological myocardial hypertrophy, wherein the nucleotide sequence of LncRNA Gm20257 is shown in SEQ ID NO.11.

[0035] In one embodiment of the present invention, the expression of LncRNA Gm20257 in isolated cardiomyocytes was inhibited by si-Gm20257, which significantly induced the pathological myocardial hypertrophy-related phenotype.

[0036] In one embodiment of the present invention, in vivo overexpression of LncRNA Gm20257 via adeno-associated virus-9 (AAV-9) encapsulation or in vitro overexpression of LncRNA Gm20257 via plasmid transfection can significantly inhibit pathological myocardial hypertrophy.

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0038] In the following examples, unless otherwise specified, all reagents are commercially available, and all detection methods and techniques used are conventional techniques and techniques in the art.

[0039] Example 1

[0040] This embodiment uses aortic arch transverse stenosis (TAC) surgery to establish a mouse model of pathological myocardial hypertrophy. Healthy adult C57BL / 6 mice aged 6-8 weeks were selected, anesthetized with 2% afomol, and underwent thoracotomy. A 27G steel needle was used as a ligation point, and 7-0 biodegradable sutures were used for ligation. After ligation, the ligation point was removed, and the chest was closed. The control group (Sham) underwent only thoracotomy without ligation. The mouse model of pathological myocardial hypertrophy was established after 4 weeks. Ultrasound examination of the left ventricular wall thickness revealed that the model group mice had significantly reduced cardiac contractility and significantly increased ventricular wall thickness. Figure 1 A, Figure 1 B). Meanwhile, the heart weight / body weight, lung weight / body weight, and heart weight / tibia length ratios in TAC model mice were significantly higher than those in the control group ( Figure 1 C). RNA was extracted and PCR was performed to detect the mRNA of myocardial hypertrophy markers ANP, BNP, and β-MHC, and Western blot was used to detect the protein expression of β-MHC. It was found that TAC model mice showed a significant increase in (…). Figure 1 D、 Figure 1 E). This indicates that the mouse model of pathological myocardial hypertrophy was successfully constructed.

[0041] Based on this, qRT-PCR detection revealed that lncRNA Gm20257 was elevated in an in vivo model of pathological myocardial hypertrophy, as shown in the following results. Figure 1 As shown in F.

[0042] The specific method is as follows:

[0043] (1) Transverse aortic arch constriction surgery (TAC) in mice

[0044] Healthy adult male C57BL / 6 mice (20-25g, 6-8 weeks old) were selected and anesthetized with 2% afomonium via intraperitoneal injection. After hair removal of the chest, the mice were fixed in a supine position on a mouse table, and the skin was disinfected with iodine. The trachea was exposed by blunt dissection of the peritracheal tissue, and a midline incision was made in the chest. The first rib was cut, and the aortic arch was located. The aortic arch was ligated with 7-0 silk suture along with a 27-gauge needle, and the needle was immediately withdrawn after ligation. The pulsation of the left and right common carotid arteries was observed. Successful ligation of the aortic arch was confirmed when the amplitude of the right common carotid artery pulsation was higher than that of the left common carotid artery. Subsequently, the pleural cavity and chest skin were sutured layer by layer until the mice regained consciousness. The control group (Sham) underwent only thoracotomy without aortic arch ligation. After surgery, both the model group and the control group mice were carefully cared for under the same conditions for 4 weeks. Echocardiography was used to detect aortic arch blood flow velocity and cardiac function to verify the successful establishment of the mouse myocardial hypertrophy model. After confirming the successful model establishment, tissue was harvested. After weighing the mice, they were anesthetized and euthanized. The hearts were removed and washed with pre-cooled PBS buffer to remove residual blood. The hearts were then weighed on an analytical balance, and the cardiac index was calculated. After weighing, the hearts were transferred to fresh ice-cold PBS, and left ventricular tissue was excised and quickly transferred to liquid nitrogen for storage at -80°C until later use.

[0045] (2) Echocardiography

[0046] C57BL / 6 mice were weighed and anesthetized with a 2% azithromycin solution (160 mg / kg). After the mice were anesthetized and their blink reflex disappeared, the hair on their chests was removed with depilatory cream. Coupling gel was applied to the chest area, and the ultrasound probe was placed on the animal's heart. The 2100 high-resolution imaging system's sensor acquired two-dimensional echocardiograms of small animals. The probe orientation was adjusted to obtain the corresponding M-mode detection curve for measurement. Left ventricular posterior wall thickness (LVPWd) was calculated from the echocardiogram images. The results showed that the model group mice exhibited significantly reduced cardiac contractility and significantly increased ventricular wall thickness. Figure 1 A, Figure 1 B).

[0047] (3) Cardiac index detection of myocardial hypertrophy indicators: The heart weight / body weight, lung weight / body weight, and heart weight / tibia length ratios in TAC model mice were significantly higher than those in the control group mice. Figure 1 C).

[0048] (4) RT-PCR detection of mRNA levels of myocardial hypertrophy markers ANP, BNP, and β-MHC

[0049] (4.1) Extract total RNA from cells or tissues

[0050] After grinding the tissue block with TRIzol until the tissue block disappeared, let it stand for 5 minutes. Add 200 μL of chloroform to the EP tube, shake vigorously for 20 seconds, and let it stand at room temperature for 10 minutes. After standing, centrifuge the EP tube at 13500 rpm for 15 minutes at 4°C. After centrifugation, remove the EP tube; the liquid in the tube will be divided into three phases: an uppermost aqueous phase (RNA phase), a middle milky white, ribbon-like protein phase, and a bottommost TRIzol phase (DNA phase). Carefully pipette approximately 500 μL of the uppermost (aqueous) supernatant and transfer it to a new 1.5 mL EP tube. Be careful not to aspirate the protein or TRIzol layer when aspirating the uppermost supernatant to avoid protein or DNA contamination. Add an equal volume of isopropanol to the EP tube, slowly invert the EP tube 10 times to precipitate the RNA, and let it stand at room temperature for 10 minutes. Centrifuge the EP tube at 13500 rpm for 10 minutes at 4°C. Remove the EP tube; a white RNA precipitate is observed forming on the side of the bottom. Carefully discard the liquid, being careful not to discard the precipitate. Add 1 mL of 75% ethanol prepared with DEPC water and gently agitate to wash the RNA. Place the EP tube in a centrifuge at 4°C and centrifuge at 10600 rpm for 5 minutes. Remove the 75% ethanol, being careful not to discard the precipitate. Invert the EP tube onto clean filter paper and air dry at room temperature for 15 minutes. Dissolve the RNA in 20 μL of DEPC water (Note: When drying the RNA, avoid over-drying it, otherwise the white color will disappear and it will be difficult to dissolve). Measure the RNA concentration and purity.

[0051] (4.2) Preparation of cDNA

[0052] In this embodiment, reverse transcription of ordinary RNA was performed using the ABM 5×Allinone Reverse Transcription kit, and the reverse transcription operation was performed according to the instructions. The internal control was GAPDH for relative normalization. The primers for Gm20257 used in this embodiment were designed using Primer 2.0, and the specific primer sequences for ANP, BNP, β-MHC, and GAPDH are as follows (Table 1).

[0053] Table 1. List of Real-time qPCR Primers

[0054]

[0055] (4.3) Real-time qPCR

[0056] This experiment used the SYBR Green I dye method and the Applied Biosystems SYBR Green PCR Master Mix kit to quantify the mRNA levels in the samples. SYBR Green Real-time PCR reactions were performed on the target RNAs (ANP, BNP, β-MHC) and internal control (GAPDH) of each sample, as well as on the target miRNAs (miR-214) and internal control (U6) of each sample. The reaction mixture was prepared in 96-well plates. 10 μL of SYBR Green reagent mixture, 7 μL of nuclease-free water, 1 μL each of forward and reverse primers (R, F), and 1 μL of cDNA were added to each well to a final volume of 20 μL. The plates were centrifuged at 1000 rpm for 1 min to ensure sufficient reaction mixture was collected at the bottom of the tube. The 96-well plates were then placed in a PCR instrument, and the program was edited. For the first detection, a melting curve of the PCR products needs to be established. The specific reaction program is shown in Table 2.

[0057] Table 2. Green PCR Master Mix fluorescent dye Real-time RT-PCR reaction program

[0058]

[0059]

[0060] (4.4) Data Analysis

[0061] The relative amount of the target gene was calculated using the cyclic value (Ct value) method. -ΔΔCT .

[0062] ΔΔCt=(Ct 目标基因 -Ct 管家基因 ) 实验组 -(Ct 目标基因 -Ct 管家基因 ) 对照组 .

[0063] (5) Western blot detection of β-MHC protein expression

[0064] (5.1) Extraction of cell or tissue proteins

[0065] Add tissue lysis buffer (RIPA:10% SDS:protease inhibitor = 100:50:1) to the tissue block at a volume of 10 mg / 100 μL and grind until the tissue block disappears. Sonicate cell or tissue protein samples for 10 seconds each time, for a total of 3 times, with 10-minute intervals between each sonication. Incubate the protein samples at 4°C for 2 hours to allow for complete lysis. After lysis, centrifuge at 13500 rpm for 25 minutes at 4°C, transfer the supernatant to a new 1.5 mL EP tube, and freeze at -80°C for later use.

[0066] (5.2) Determination of protein concentration

[0067] Protein concentration was determined using the BCA method: Fresh BCA working solution (solution A:solution B = 50:1) was prepared and added to 96-well plates at a ratio of BCA working solution:RIPA lysis buffer:protein sample solution = 200:19:1. The plates were gently tapped to mix thoroughly, and then incubated at 37°C for 30 minutes to allow the BCA working solution to fully react with the protein. After incubation, the plates were placed in a microplate reader, and absorbance values ​​were read at 562 nm. The absorbance values ​​of each sample were then substituted into the protein concentration standard curve equation to calculate the protein concentration of the sample.

[0068] (5.3) Processing of protein samples

[0069] Based on the protein concentration determined in the previous step, use RIPA to fill the concentration difference in the samples, and add an equal volume of 5× loading buffer to the samples. Place the processed protein sample EP tubes in a heater and boil at 100°C for 7 minutes, then centrifuge briefly in a small centrifuge and freeze at -80°C for later use.

[0070] (5.4) Protein electrophoresis

[0071] The formulations of 12% separating adhesive and 5% building block adhesive are shown in Table 3:

[0072] Table 3. 12% separating adhesive and 5% stacking adhesive

[0073]

[0074] Load the sample into a pre-prepared SDS-PAGE gel, and add a marker on each side to indicate the position of the proteins during electrophoresis. Electrophoresis is performed at a constant voltage of 70V for 30 minutes. Once the sample has passed through the stacking gel and formed a straight line, the voltage is increased to 110V to separate proteins of different molecular weights. Electrophoresis is stopped when the bromophenol blue indicator reaches the appropriate position.

[0075] (5.5) Transfer membrane

[0076] Equilibrate the appropriately sized NC membrane and filter paper in pre-cooled transfer buffer for 15 minutes. After opening the transfer clamp, place the filter paper, SDS-PAGE gel, NC membrane, and filter paper in sequence on the black clamp side, taking care to avoid air bubbles between the NC membrane and gel. Place the assembled clamp into the transfer tank and place the transfer tank in an ice bath. Transfer at a constant current of 300 mA for 1-1.5 hours (depending on the molecular weight of the protein).

[0077] (5.6) Blocking and incubating primary antibody

[0078] After the transfer was complete, the NC membrane was rinsed three times in PBST for 5 minutes each time to remove excess transfer buffer. Then, the NC membrane was placed in 5% skim milk prepared with PBS and blocked at room temperature for 2 hours on a horizontal shaker. After blocking, the NC membrane was placed face up in the primary antibody solution diluted with blocking buffer and incubated overnight at 4°C on a shaker.

[0079] (5.7) Incubation of secondary antibodies

[0080] After the primary antibody incubation is complete, the NC membrane is washed three times in PBST for 10 minutes each time. Then, it is incubated with the fluorescent secondary antibody at room temperature in the dark for 50 minutes. After the incubation, the membrane is washed three times with PBST for 10 minutes each time.

[0081] (5.8) Sweeping the film

[0082] The NC membrane was placed face down on the Odessey infrared scanning imager, the NC membrane was scanned, and the relative gray values ​​of the protein bands were analyzed using ImageJ software.

[0083] Example 2

[0084] C57BL / 6 suckling mice within 3 days of birth were harvested, and cardiomyocytes were digested with trypsin. After 48 hours of stable culture, the cardiomyocytes were transfected with either si-NC or si-Gm20257. PCR detection showed that lncRNA levels in the si-Gm20257 group were significantly lower (…). Figure 2 A). Examination of cardiomyocyte area revealed that transfection with si-Gm20257, which inhibited endogenous Gm20257 expression, significantly increased cardiomyocyte area compared to the si-NC group. Figure 2 B). PCR detection of myocardial hypertrophy markers ANP, BNP, and β-MHC revealed a significant increase in the si-Gm20257 group. Figure 2 C). Simultaneously, the expression of the hypertrophy marker β-MHC protein was significantly increased ( Figure 2 D). The above experiments confirm that knocking down lncRNA Gm20257 (nucleotide sequence as shown in SEQ ID NO.11) can induce cardiomyocyte hypertrophy.

[0085] The specific method is as follows:

[0086] (1) Culture of primary suckling rat cardiomyocytes

[0087] Newborn 1-3 day old C57BL / 6 suckling mice were disinfected in a laminar flow hood by immersion in 75% alcohol. The hearts were then removed via decapitation and thoracotomy and placed in pre-cooled sterile DMEM culture medium to allow residual blood to be expelled. After removing the atrial appendages and aorta in DMEM culture medium, the heart tissue was transferred to another fresh, pre-cooled sterile DMEM culture medium. Each heart was cut into two identical tissue pieces. Once all hearts were trimmed, they were transferred to 15 mL centrifuge tubes. 0.25% trypsin solution was added, and the heart tissue pieces were rinsed with shaking to remove residual blood until the trypsin solution became clear. Fresh 0.25% trypsin solution was then added to digest the heart tissue pieces. The turbid supernatant was aspirated and placed in DMEM culture medium containing 10% fetal bovine serum to terminate digestion. The above trypsin digestion steps were repeated until the heart tissue pieces disappeared. The collected cells were filtered through a 200-mesh filter and centrifuged at 1500 rpm for 6 minutes. After centrifugation, the supernatant was slowly discarded, and fresh DMEM containing 10% fetal bovine serum was added to the cells. The cells were gently and repeatedly pipetted until they were evenly dispersed and suspended in the DMEM culture medium. The cells were transferred to culture flasks and incubated at 37°C for 2 hours. Fibroblasts adhered to the bottom of the culture flasks, while cardiomyocytes remained suspended in the culture medium. The cell suspension was then transferred to new culture flasks, and DMEM culture medium was added to the required volume. 1% BrdU was added to inhibit the proliferation of non-cardiomyocytes, and cardiomyocytes were seeded onto cell culture plates. After 48 hours, the cardiomyocyte status was observed. If the cells showed good adhesion and rhythmic beating, subsequent experiments were performed.

[0088] (1.1) Cell drug administration

[0089] Cardiac cells at 2 × 10 per well 5 The cells were seeded at a density in 6-well plates and incubated at 37°C for 48 h. After confirming that the neonatal rat cardiomyocytes were in good condition and had rhythmic beating, transfection was performed. At the same time, or angiotensin II (Ang II, 1 μmol / L) was added directly for 48 h (adding the drug once every 12 h, for a total of 4 times). Cell proteins or RNA were then collected for later use.

[0090] (1.1) Cell transfection

[0091] Cardiac cells at 2 × 10 per well 5Cells were seeded at a density of [insert density here] in 6-well plates and incubated at 37°C for 48 hours. After confirming good cell growth, transfection was initiated. First, the cell culture medium was replaced with serum-free and antibiotic-free DMEM. Then, the transfection system was prepared: the oligomer or plasmid was diluted in an appropriate amount of Opti-MEM low-serum medium and gently mixed with a sterile pipette tip; simultaneously, lipo-2000 Transfection Reagen was mixed with an appropriate amount of Opti-MEM low-serum medium, gently mixed, and incubated at room temperature for 5 minutes. Finally, the diluted oligomer or plasmid and the diluted lipo-2000 Transfection Reagen were mixed and incubated at room temperature for 20 minutes to form a stable complex. The complex was then added to the culture plate, and the plate was gently shaken to mix thoroughly. The plate was incubated at 37°C, 5% CO2 for 8 hours, then replaced with complete culture medium and cultured for another 40 hours before further processing.

[0092] (2) Detection of myocardial cell area

[0093] Primary neonatal rat cardiomyocytes were seeded in 24-well plates with coverslips and cultured to a density of just a monolayer. After 48 hours of cell culture and adherence, cells were transfected and treated with drugs, then harvested for fluorescent staining. First, the culture medium in the 24-well plates was discarded, and the cells were gently washed five times with PBS. Then, 1 mL of 4% paraformaldehyde was added to each well, and the plates were fixed on a shaker at room temperature for 15 min. After washing five times with PBS, the cells were penetrated for 1 hour at room temperature using PBS buffer containing 1% BSA and 0.4% Triton. After 1 hour, the cells were washed five times with PBS, and then 1 mL of goat serum was added to each well, and the plates were blocked at 37°C for 30 min. Finally, the cells were washed five times with PBS, the PBS was aspirated, and the cells were incubated overnight at 4°C with the primary fluorescent antibody (α-actinin, 1:200). The following day, after washing five times with PBS, secondary antibody (Dylight 594, 1:200) was added, and the cells were incubated at 37°C in the dark for 1 hour. Then, the cells were washed five times with PBS, and DAPI (1:50) was added to the cells and incubated at room temperature for 5 minutes. Finally, the cells were washed five times with PBS to thoroughly remove unbound dye. Randomly selected fields of view were photographed and saved under a fluorescence microscope. The surface area of ​​cardiomyocytes was delineated using Image-Pro Plus 6.0 software, and the relative area of ​​cardiomyocytes was read using arbitrary units, thus serving as a basis for assessing the degree of cardiomyocyte hypertrophy.

[0094] (3) PCR detection of myocardial hypertrophy markers ANP, BNP, and β-MHC. The method is the same as step (3) in Example 1.

[0095] (4) Western blot detection of β-MHC protein expression in cardiomyocytes. The method is the same as step (4) in Example 1.

[0096] Example 3

[0097] Hearts were harvested from C57BL / 6 suckling mice within 3 days of birth, and cardiomyocytes were digested using trypsin. After 48 hours of stable culture, the cardiomyocytes were transfected with Vector (control plasmid) and lncRNA Gm20257 overexpression plasmid. PCR was used to detect the expression level of lncRNA Gm20257. PCR results showed that the overexpression group had a significantly higher expression level of lncRNA Gm20257 compared to the Vector group. Figure 3 A). Further, angiotensin II (Ang II) was used to treat cardiomyocytes for 48 hours to induce cardiomyocyte hypertrophy. Simultaneously, cardiomyocytes were transfected with a Vector (control plasmid) and an lncRNA Gm20257 overexpression plasmid, resulting in four groups: normal culture medium + Vector group; normal culture medium + lncRNA Gm20257 overexpression plasmid; Ang II + Vector group; and Ang II + lncRNA Gm20257 group. Ang II induction significantly increased cardiomyocyte area, while overexpression of lncRNA Gm20257 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Figure 3 B). Ang II treatment for 48 hours significantly increased the expression of hypertrophy marker genes (ANP, BNP, β-MHC) in cardiomyocytes, while overexpression of lncRNA Gm20257 significantly inhibited the Ang II-induced increase in hypertrophy gene expression. Figure 3 C). β-MHC protein expression also showed the same trend ( Figure 3 D).

[0098] The specific method is as follows:

[0099] (1) Cell culture, drug administration, and transfection with lncRNA Gm20257 overexpression plasmid. The method is the same as step (1) in Example 2.

[0100] (2) Detection of cardiomyocyte area. Same as step (2) in Example 2.

[0101] (3) PCR detection of myocardial hypertrophy markers ANP, BNP, and β-MHC. The method is the same as step (3) in Example 1.

[0102] (4) Western blot detection of β-MHC protein expression in cardiomyocytes. The method is the same as step (4) in Example 1.

[0103] Example 4

[0104] Healthy adult C57BL / 6 mice aged 6-8 weeks were selected, and in vivo overexpression of lncRNA Gm20257 was achieved by tail vein injection of adeno-associated virus-9 (AAV-9)-encapsulated lncRNA Gm20257 overexpression plasmid. Three weeks after viral injection, TAC surgery was performed, with Sham as the control. The experiment consisted of four groups: Sham + Vector group; Sham + lncRNA Gm20257 group; TAC + Vector group; and TAC + lncRNA Gm20257 group.

[0105] LncRNA Gm20257 overexpression mice showed significantly increased expression of it. Figure 4 A). After TAC surgery, cardiac contractility is significantly reduced and ventricular wall thickness is significantly increased. Figure 4 B Figure 4 C); heart weight / body weight; lung weight / body weight; heart weight / femur length ratio were significantly increased in TAC mice. Figure 4 D), overexpression of lncRNA Gm20257 can significantly inhibit the above-mentioned cardiac function and phenotypic changes induced by TAC. Figure 4 B- Figure 4 D). Wheat germ lectin staining showed that TAC induced an increase in cardiomyocyte area, while overexpression of lncRNA Gm20257 significantly inhibited the TAC-induced increase in cardiomyocyte area. Figure 4 E). After TAC surgery, the expression of hypertrophic marker mRNA (ANP, BNP, β-MHC) and protein (β-MHC) was significantly increased, and overexpression of lncRNA Gm20257 significantly inhibited the increase in hypertrophic marker gene expression. Figure 4 F, Figure 4 G).

[0106] The specific method is as follows:

[0107] (1) Tail vein injection of adeno-associated virus 9 (AAV-9)

[0108] An in vivo model of lncRNA Gm20257 overexpression was established by intravenous injection of adeno-associated virus (AAV9) encapsulating lncRNA Gm20257 into C57BL / 6 mice, with AAV9-Vector serving as the control group. C56BL / 6 mice were randomly divided into two groups, receiving intravenous injections of AAV9-lncRNA Gm20257 and AAV9-Vector, respectively, for 3 weeks. Mice in each group were then randomly assigned to the Sham group and the TAC group. Samples were collected 4 weeks post-surgery for analysis.

[0109] (2) Transverse aortic arch stenosis surgery (TAC). Same as step (1) in Example 1.

[0110] (3) Echocardiography. Same as step (2) in Example 1.

[0111] (4) Cardiac index detection of myocardial hypertrophy indicators. Same as step (3) in Example 1.

[0112] (5) Wheat germ lectin staining

[0113] Prepare the sliced ​​paraffin wax and bake in a 61℃ oven for 30 minutes (start timing from when it is placed on the oven).

[0114] (5.1) Dewaxing

[0115] a. Pure xylene I, 15 min;

[0116] b. Pure xylene II, 10 min;

[0117] c. 100% ethanol, 10 min;

[0118] d. 96% ethanol, 3 min;

[0119] f. 70% ethanol, 3 min;

[0120] g.ddH2O, 3 min;

[0121] (5.2) Wash the slides twice with PBS;

[0122] (5.3) Incubate the WGA-FITC antibody prepared with PBS for 2 hours;

[0123] (5.4) After incubation, wash twice with PBS to remove unbound WGA-FITC antibody;

[0124] (5.5) Incubate DAPI;

[0125] (5.6) Seal the film with anti-quenching sealing agent;

[0126] (5.7) Store at 4℃ until use / take photos with a fluorescence microscope.

[0127] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of LncRNA Gm20257 in the preparation of drugs for treating pathological myocardial hypertrophy, characterized in that, The nucleotide sequence of the LncRNA Gm20257 is shown in SEQ ID NO.

11. In vivo overexpression of LncRNA Gm20257 inhibits pathological myocardial hypertrophy.

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