Application of siRNA-OTUB1 in preparation of drugs for preventing or treating cardiomyocyte hypertrophy
By using siRNA-OTUB1, which specifically interferes with the expression of the OTUB1 gene, the problems of poor targeting and large side effects in the treatment of cardiomyocyte hypertrophy have been solved, achieving precise regulation and safe treatment of cardiomyocyte hypertrophy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF SHANDONG FIRST MEDICAL UNIV (QIANFOSHAN HOSPITAL OF SHANDONG PROVINCE)
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-05
AI Technical Summary
Existing treatments for cardiomyocyte hypertrophy lack targeting and precision. Traditional methods have significant side effects, are difficult to directly regulate at the gene expression level, and lack molecular intervention strategies centered on the deubiquitinase OTUB1.
By employing siRNA-OTUB1, which specifically interferes with OTUB1 gene expression, molecular interventions on cardiomyocyte hypertrophy are achieved by reducing OTUB1 mRNA and protein expression levels. Drug interventions targeting OTUB1 are designed.
It effectively inhibits cardiomyocyte hypertrophy and reduces the degree of cell hypertrophy, providing a new approach to the precise treatment of myocardial hypertrophy and reducing the risk of systemic adverse reactions.
Smart Images
Figure CN121868495B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the application of siRNA-OTUB1 in the preparation of drugs for the prevention or treatment of cardiomyocyte hypertrophy, and belongs to the field of biomedical technology. Background Technology
[0002] Cardiomyocyte hypertrophy is an important pathological basis for the occurrence and development of various cardiovascular diseases, such as hypertensive heart disease, heart failure, and cardiomyopathy. Currently, clinical interventions for cardiomyocyte hypertrophy mainly rely on drug therapy or surgical treatment. However, these two approaches primarily intervene at the level of overall hemodynamics or neuroendocrine regulation, which has at least two drawbacks: First, insufficient target specificity. Existing drug treatments are mostly non-specific interventions, making it difficult to precisely target the key molecular targets that induce cardiomyocyte hypertrophy, easily causing systemic adverse reactions. Second, difficulty in achieving refined regulation. Traditional methods cannot directly regulate cardiomyocyte hypertrophy-related molecules at the gene expression level, resulting in limited control over disease progression.
[0003] Small interfering RNA (siRNA) is a double-stranded RNA composed of 19-25 nucleotide pairs and is a key component in RNA interference technology. After siRNA is transfected into the cell, it binds to the RNA-induced silencing complex (RISC). Subsequently, one sense strand of the siRNA degrades, and the remaining antisense strand guides the RISC to bind to the target mRNA according to the base pairing principle, ultimately inducing the degradation of the mRNA, thereby inhibiting the expression of the target gene.
[0004] In recent years, with the development of molecular biology techniques, research on signaling pathways related to cardiomyocyte hypertrophy has gradually increased. However, existing research mainly focuses on downstream signaling molecules such as kinases or transcription factors, and research on protein homeostasis regulation, especially the role of deubiquitinating enzymes in cardiomyocyte hypertrophy and their potential as intervention targets, remains relatively limited. Currently, there is no established strategy for regulating cardiomyocyte hypertrophy with the deubiquitinating enzyme OTUB1 as the core target, a molecular intervention system targeting OTUB1 is lacking, and there is a lack of technical solutions for the prevention or treatment of cardiomyocyte hypertrophy using RNA interference technology. This limits the precision and effectiveness of cardiomyocyte hypertrophy treatments.
[0005] In other words, the existing technological solutions for treating and regulating cardiomyocyte hypertrophy are relatively limited. Traditional methods often focus on macroscopic drug interventions or surgical procedures; however, these methods have many limitations, such as unsatisfactory treatment effects and significant side effects. Although there are some studies targeting cardiomyocyte-related signaling pathways, research on the specific direction of regulating cardiomyocyte hypertrophy through small interfering siRNA targeting the deubiquitinase OTUB1 is still insufficient, and effective and precise intervention methods are lacking. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides the application of siRNA-OTUB1 in the preparation of drugs for the prevention or treatment of cardiomyocyte hypertrophy. This invention specifically reduces the expression level of OTUB1 in cardiomyocytes, intervening in the occurrence and development of cardiomyocyte hypertrophy at the molecular level. This overcomes the problems of poor targeting, significant side effects, and inability to achieve precise regulation in existing treatment methods, providing a new, safe, and effective technical solution for the prevention and treatment of cardiomyocyte hypertrophy.
[0007] The technical solution of the present invention is as follows:
[0008] Application of OTUB1 gene as a target in the preparation of drugs for the prevention or treatment of cardiomyocyte hypertrophy.
[0009] According to a preferred embodiment of the present invention, the nucleotide sequence of the OTUB1 gene is shown in SEQ ID NO.1.
[0010] According to a preferred embodiment of the present invention, the drug for preventing or treating cardiomyocyte hypertrophy uses the OTUB1 gene as an intervention target, which can efficiently and specifically downregulate the expression level of the OTUB1 gene.
[0011] Application of siRNA-OTUB1, which specifically interferes with OTUB1 gene expression, in the preparation of drugs for the prevention or treatment of cardiomyocyte hypertrophy.
[0012] According to a preferred embodiment of the present invention, the target nucleotide sequence of the siRNA-OTUB1 that specifically interferes with the expression of the OTUB1 gene is shown in SEQ ID NO.2 or SEQ ID NO.3.
[0013] A drug for the prevention or treatment of cardiomyocyte hypertrophy, said drug containing siRNA-OTUB1 that specifically interferes with the expression of the OTUB1 gene.
[0014] According to a preferred embodiment of the present invention, the target nucleotide sequence of the siRNA-OTUB1 that specifically interferes with the expression of the OTUB1 gene is shown in SEQ ID NO.2 or SEQ ID NO.3.
[0015] Beneficial effects:
[0016] This invention innovatively proposes a strategy to prevent or treat cardiomyocyte hypertrophy by intervening in the expression of the deubiquitinating enzyme OTUB1. In experiments with neonatal rat cardiomyocytes, the siRNA-OTUB1 designed in this invention effectively reduced the expression levels of OTUB1 mRNA and OTUB1 protein, thereby alleviating the degree of cardiomyocyte hypertrophy. Based on this discovery, the OTUB1 gene can be developed as a novel therapeutic target for cardiomyocyte hypertrophy, enabling the design of targeted drugs and providing a new technical approach for the precision treatment of cardiomyocyte hypertrophy. Attached Figure Description
[0017] Figure 1 It represents the expression levels of a series of deubiquitinating enzymes.
[0018] Figure 2 It represents the expression level of OTUB1 protein after treatment with hypertrophic factor.
[0019] Figure 3 This is the inhibitory effect of OTUB1-siRNA on cardiomyocyte hypertrophy.
[0020] In the figure, A shows the efficient knockdown effect of siRNA on OTUB1 in NRVCs; B shows the quantitative PCR analysis of Otub1 mRNA in NRVCs after siRNA transfection; C shows phalloidin staining (red) and quantitative analysis; D shows the surface area of PE-treated NRVCs after siRNA transfection; E shows the protein synthesis level of NRVCs assessed by puromycin incorporation assay; and F shows the quantitative PCR analysis of myocardial hypertrophy-related gene mRNA in NRVCs after siRNA transfection. Detailed Implementation
[0021] The technical solution of the present invention will be further described below with reference to specific experimental examples, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and materials involved in the examples are all commercially available products.
[0022] In the examples, SD rats were available from Shandong Pengyue Experimental Animal Technology Co., Ltd.
[0023] The two siRNAs that specifically interfere with OTUB1 gene expression in the examples are siRNA-OTUB1-a and siRNA-OTUB1-b, and their nucleotide sequences are as follows:
[0024] siOTUB1-a Justice Chain: 5'-GCAAGGAACUGCAGCGGUUdTdT-3';
[0025] siOTUB1-a antisense chain: 5'-AACCGCUGCAGUUCCUUGCdTdT-3'.
[0026] siOTUB1-b Justice Chain: 5'-CGAUAUCCUCUACAAAUGAdTdT-3';
[0027] siOTUB1-b antisense chain: 5'-UCAUUUGUAGAGGAUAUCGdTdT-3'.
[0028] The nucleotide sequence targeted by siOTUB1-a is: 5'-GCAAGGAACTGCAGCGGTTCAA-3' (SEQ ID NO.2).
[0029] The nucleotide sequence targeted by siOTUB1-b is: 5'-CGATATCCTCTACAAATGA-3' (SEQ ID NO.3).
[0030] In the embodiment, siLuci justice chain 5'-CGUACGCGGAAUACUUCGAdTdT-3';
[0031] siLuci antisense chain 5'-UCGAAGUAUUCCGCGUACGdTdT-3'.
[0032] In this invention, siLuci, siRNA-OTUB1-a, and siRNA-OTUB1-b were all artificially synthesized by Nanjing GenScript Biotech Co., Ltd. according to their sequence information.
[0033] Example 1: Discovery of OTUB1 as a target
[0034] 1. Based on GSE95140 data (NatCommun 2018) from RNA sequencing of myocardial tissue from patients with dilated myocardium, the inventors of this application analyzed the relationship between deubiquitinase and cardiomyopathy.
[0035] The specific analysis method was as follows: Based on publicly available information from the GSE95140 (Nat Commun 2018) database, RNA sequencing data from cardiac tissues of two groups of patients with dilated cardiomyopathy (DCM) were analyzed using R language. The results are as follows: Figure 1 As shown.
[0036] Depend on Figure 1 It was found that a series of deubiquitinating enzymes showed abnormal expression levels compared with the control group (CTL), among which the deubiquitinating enzyme OTUB1 was the most obvious, with an upregulation of expression level of ~3 times. This reveals that abnormal expression of OTUB1 may be related to the occurrence of dilated cardiomyopathy.
[0037] 2. Take 1-3 day old SD rats, quickly remove the heart and separate the ventricular tissue. Under aseptic conditions, mince the tissue and digest it with 0.025% trypsin and 0.08% type II collagenase. Combine the digestive fluids and remove fibroblasts using differential adhesion method, collecting the suspended cardiomyocytes. Then, add 10 μM isoproterenol (ISO), norepinephrine (NE), and phenylephrine (PE) to the cardiomyocytes, respectively. After 24 h of stimulation and culture, extract cell proteins and detect the expression level of OTUB1 protein in cardiomyocytes by Western blotting. The results are as follows: Figure 2 As shown.
[0038] Depend on Figure 2 It is known that stimulation with isoproterenol (ISO), norepinephrine (NE), and phenylephrine (PE) can induce OTUB1 protein expression and increase the expression level of OTUB1 protein.
[0039] Example 2: Inhibitory effect of OTUB1-siRNA on cardiomyocyte hypertrophy
[0040] 1. Take 1-3 day old SD rats, quickly remove the heart and separate the ventricular tissue. Under aseptic conditions, mince the tissue and digest it with 0.025% trypsin and 0.08% type II collagenase. After combining the digestive juices, remove fibroblasts using the differential adhesion method and collect the suspended cardiomyocytes.
[0041] The cardiomyocytes were then seeded into gelatin-coated culture dishes and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin for 24 h at 37°C and 5% CO2. The culture medium was then replaced with medium containing 2% FBS for synchronization treatment to obtain neonatal rat ventricular myocytes (NRVCs).
[0042] 2. The mRNA nucleotide sequence of the OTUB1 gene was obtained from the NCBI database, as shown in SEQ ID NO.1. Based on the above sequence, siRNAs (siRNA-OTUB1-a and siRNA-OTUB1-b) were designed to specifically knock down the expression level of the OTUB1 gene. Their target sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The negative control for the above siRNAs was siLuci.
[0043] When the NRVCs confluence was approximately 60-70%, siLuci, siRNA-OTUB1-a, and siRNA-OTUB1-b were transfected into NRVCs using Lipofectamine RNAiMAX reagent. The transfection method was performed according to the reagent instructions. After 6 hours of transfection, the medium was replaced with fresh medium, and the cells were cultured for another 48 hours to obtain transfected NRVCs-siLuci, NRVCs-siRNA-OTUB1-a, and NRVCs-siRNA-OTUB1-b cells.
[0044] 3. Transfected NRVCs-siLuci, NRVCs-siRNA-OTUB1-a, and NRVCs-siRNA-OTUB1-b were lysed using RIPA lysis buffer containing protease inhibitors, and total protein was collected. Protein concentration was determined using the BCA method. An equal amount of protein (25 μg) from each group was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 h, then incubated overnight at 4°C with OTUB1 primary antibody (1:1000). The next day, it was incubated with HRP-labeled secondary antibody (1:5000), and the signal was detected using the ECL colorimetric system. Tubulin was used as an internal control, and the band gray values were quantitatively analyzed using ImageJ software. The results are shown below. Figure 3 As shown in A in the diagram.
[0045] Depend on Figure 3 As shown in A, both siRNA-OTUB1-a and siRNA-OTUB1-b can effectively knock down the OTUB1 protein level.
[0046] 4. Total RNA was extracted from transfected cells using the TRIzol method for NRVCs-siLuci, NRVCs-siRNA-OTUB1-a, and NRVCs-siRNA-OTUB1-b. Using the total RNA as a template, cDNA products were synthesized via reverse transcription using the LunaScript™ RTSuperMix Kit (NEB, E3010). Quantitative real-time PCR was performed on a PCR instrument using the Power SYBR® Green MasterMix Kit (Thermo Fisher Scientific, 4367659). GAPDH mRNA was selected as an internal control. The reaction was carried out according to a 2... -ΔΔCT The formula calculates the relative expression level of OTUB1 mRNA, and the results are as follows: Figure 3 As shown in B in the diagram.
[0047] Depend on Figure 3 As shown in B, both siRNA-OTUB1-a and siRNA-OTUB1-b can effectively knock down OTUB1 expression at the mRNA level.
[0048] 5. siRNA-OTUB1-a and siRNA-OTUB1-b were transfected into NRVCs using Lipofectamine RNAiMAX reagent to obtain transfected NRVCs-siOTUB1 cells. Phenylephrine (PE, final concentration 10 μM) was then added to the transfected NRVCs-siLuci and NRVCs-siOTUB1 cells, and the cells were cultured for another 24 h to induce myocardial hypertrophy. Cells without PE served as a control, and were designated as the siLuci group, siLuci+PE group, siOTUB1 group, and siOTUB1+PE group, respectively. After stimulation, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, they were incubated with phalloidin (labeled with Alexa Fluor 594, red) at room temperature in the dark for 30 min, and the cell nuclei were stained with DAPI. Then, multiple fields of view were randomly photographed using a fluorescence microscope. ImageJ software was used to delineate cell boundaries and calculate the surface area of individual cardiomyocytes. Each group analyzed at least 100 cells. The results are as follows: Figure 3 As shown in C and D.
[0049] Depend on Figure 3 As shown in C, compared with the siLuci+PE group, the cardiomyocytes in the siOTUB1+PE group were significantly hypertrophied and irregular in shape, while the cardiomyocytes in the siOTUB1+PE group were more regular in shape and relatively smaller in size. The two groups formed a sharp contrast, which also supports the inhibitory effect of siRNA-OTUB1-a and siRNA-OTUB1-b on cardiomyocyte hypertrophy from a morphological perspective.
[0050] Depend on Figure 3 As shown in D, compared with the siLuci+PE group, the surface area of cardiomyocytes in the siOTUB1+PE group was significantly reduced, indicating that siRNA-OTUB1-a and siRNA-OTUB1-b can effectively inhibit phenylephrine-induced cardiomyocyte hypertrophy.
[0051] 6. To further evaluate the effect of OTUB1-siRNA on the protein synthesis level of cardiomyocytes, a puromycin incorporation experiment was used.
[0052] Phenylephrine (PE, final concentration 10 μM) was added to transfected NRVCs-siLuci and NRVCs-siOTUB1 cells, respectively, and the cells were cultured for 24 h. Puromycin (Puromycin, final concentration 1 μg / mL) was then added, and the cells were cultured for another 30 min. Cells were then collected, lysed using RIPA lysis buffer containing a protease inhibitor, and total protein was collected. Protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. PVDF membranes were blocked with 5% skim milk powder at room temperature for 1 h, and then incubated overnight at 4°C with puromycin primary antibody (1:1000). The next day, the membranes were incubated with HRP-labeled secondary antibody (1:5000), and the signal was detected using an ECL colorimetric system. Tubulin was used as an internal control, and the band gray values were quantitatively analyzed using ImageJ software. The results are shown below. Figure 3 As shown in E in the figure.
[0053] Depend on Figure 3 As can be seen from E, compared with the siLuci group and the siLuci+PE group, the puromycin incorporation level in the siOTUB1 group and the siOTUB1+PE group was significantly reduced. This indicates that siRNA-OTUB1-a and siRNA-OTUB1-b can inhibit the protein synthesis level of cardiomyocytes, thus further confirming their inhibitory effect on cardiomyocyte hypertrophy.
[0054] 7. The inventors further investigated the effect of siOTUB1 on the regulation of PE-induced myocardial hypertrophy-related genes (Nappa, Nappb, Sercaα and SM22α).
[0055] Phenylephrine (PE, final concentration 10 μM) was added to transfected NRVCs-siLuci and NRVCs-siOTUB1 cells, respectively, and the cells were cultured for 24 h to induce myocardial hypertrophy. These were designated as the siLuci group, siLuci+PE group, siOTUB1 group, and siOTUB1+PE group, respectively. Total RNA was extracted using the TRIzol method. Using total RNA as a template, cDNA products were synthesized by reverse transcription using the LunaScript™ RT SuperMix Kit (NEB, E3010). Quantitative real-time PCR was performed using the Power SYBR® Green Master Mix (Thermo Fisher Scientific, 4367659) kit. GAPDH mRNA was used as an internal control. -ΔΔCT The formula calculates the relative mRNA expression levels of genes related to myocardial hypertrophy (Nappa, Nappb, Sercaα, and SM22α), and the results are as follows: Figure 3 As shown in F in the diagram.
[0056] Depend on Figure 3 As shown in F, compared with the siLuci group and the siLuci+PE group, the relative mRNA expression levels of myocardial hypertrophy-related genes (Nappa, Nappb, Sercaα, and SM22α) in the siOTUB1 group and the siOTUB1+PE group were significantly reduced. This indicates that siRNA-OTUB1-a and siRNA-OTUB1-b can not only inhibit the hypertrophic phenotype of cardiomyocytes, but also regulate molecular markers related to myocardial hypertrophy at the gene expression level, further confirming the effectiveness of OTUB1 as a potential target for the treatment of myocardial hypertrophy.
Claims
1. The application of siRNA-OTUB1, which specifically interferes with OTUB1 gene expression, in the preparation of drugs for the prevention or treatment of cardiomyocyte hypertrophy, characterized in that, The siRNAs that specifically interfere with OTUB1 gene expression are siRNA-OTUB1-a and siRNA-OTUB1-b, and their nucleotide sequences are as follows: siOTUB1-a Justice Chain: 5'-GCAAGGAACUGCAGCGGUUdTdT-3'; siOTUB1-a antisense chain: 5'-AACCGCUGCAGUUCCUUGCdTdT-3'; siOTUB1-b Justice Chain: 5'-CGAUAUCCUCUACAAAUGAdTdT-3'; siOTUB1-b antisense chain: 5'-UCAUUUGUAGAGGAUAUCGdTdT-3'.
2. A drug for preventing or treating cardiomyocyte hypertrophy, characterized in that, The drug for preventing or treating cardiomyocyte hypertrophy contains siRNA-OTUB1 that specifically interferes with the expression of the OTUB1 gene; the siRNA-OTUB1 that specifically interferes with the expression of the OTUB1 gene are siRNA-OTUB1-a and siRNA-OTUB1-b, and their nucleotide sequences are as follows: siOTUB1-a Justice Chain: 5'-GCAAGGAACUGCAGCGGUUdTdT-3'; siOTUB1-a antisense chain: 5'-AACCGCUGCAGUUCCUUGCdTdT-3'; siOTUB1-b Justice Chain: 5'-CGAUAUCCUCUACAAAUGAdTdT-3'; siOTUB1-b antisense chain: 5'-UCAUUUGUAGAGGAUAUCGdTdT-3'.