Application of USP21 in the diagnosis and treatment of abdominal aortic aneurysms

Abstract

This invention discloses the application of USP21 in the diagnosis and treatment of abdominal aortic aneurysms, belonging to the field of biomedicine. This invention discovers that the level of ubiquitin-specific proteinase 21 (USP21) in abdominal aortic aneurysm tissue is significantly upregulated compared to normal abdominal aortic tissue, and is closely related to the occurrence and development of abdominal aortic aneurysms (AAA). Furthermore, knocking down USP21 can reduce the tumor formation rate of abdominal aortic aneurysms, decrease the diameter of the abdominal aorta, and improve elastic fiber rupture. Therefore, USP21 can serve as a diagnostic molecular marker for abdominal aortic aneurysms, and also as a potential therapeutic target. This invention provides a more advantageous means for the diagnosis and treatment analysis of abdominal aortic aneurysms, which is of great significance for the research and treatment of abdominal aortic aneurysms. It also lays an experimental foundation and provides new perspectives for the development of highly effective drugs for the treatment of abdominal aortic aneurysms, thus possessing good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving the application of USP21 in the diagnosis and treatment of abdominal aortic aneurysms. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Abdominal aortic aneurysm (AAA), a common vascular disease, is characterized by localized dilation of the abdominal aorta. In the clinical treatment of AAA, pharmacological interventions are increasingly important, particularly the management of risk factors such as hypertension and high cholesterol. Current treatments include, but are not limited to, antihypertensive drugs such as β-adrenergic receptor antagonists and angiotensin-converting enzyme (ACE) inhibitors, which reduce intravascular pressure to alleviate mechanical stress on the aortic wall and theoretically inhibit aneurysm progression. Meanwhile, the application value of antiplatelet drugs, such as aspirin, in reducing the risk of thrombosis has also attracted attention. It is important to note that currently, there is a lack of officially approved drugs for the direct treatment of AAA in clinical practice, highlighting the urgent need to develop novel drug targets and innovative treatment regimens.

[0004] RNA interference (RNAi) is an effective method for gene function research by specifically inhibiting the expression of target genes. The molecular mechanism involves small interfering RNA (siRNA) guiding the RNA-induced silencing complex (RISC) to specifically recognize and degrade target mRNA, thereby silencing gene expression. Therefore, the design and optimization of siRNA (including sequence selection and chemical modification) are key factors in ensuring the high efficiency and targeted specificity of RNAi. How to effectively apply siRNA to reduce the expression level of target genes is crucial for studying the biological function of these genes in specific cell phenotypes and also provides important theoretical support for the development of related targeted drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of USP21 in the diagnosis and treatment of abdominal aortic aneurysms. This invention has discovered that the level of ubiquitin-specific proteinase 21 (USP21) in abdominal aortic aneurysm tissue is significantly upregulated compared to normal abdominal aortic tissue, and is closely related to the occurrence and development of abdominal aortic aneurysms (AAA). Furthermore, knocking down USP21 can reduce the tumor formation rate of abdominal aortic aneurysms, decrease the diameter of the abdominal aorta, and improve elastic fiber rupture. Therefore, USP21 can serve as a diagnostic molecular marker for abdominal aortic aneurysms, and also as a potential therapeutic target for abdominal aortic aneurysms, thus completing this invention.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A first aspect of the invention provides the use of a substance for detecting USP21 in the preparation of any one or more of the following products:

[0008] (a1) Products for the treatment of abdominal aortic aneurysms;

[0009] (a2) Diagnostic or auxiliary diagnostic products for abdominal aortic aneurysm.

[0010] It is understood that the substance may be a substance for detecting the expression level of the USP21 gene (mRNA, DNA) or for detecting the expression level of the USP21 protein.

[0011] The nucleotide sequence of the USP21 mRNA is NM_013919.4, and its length is 2200 base pairs. It is derived from the NCBI database.

[0012] This study found that USP21 expression was significantly higher in abdominal aortic aneurysm tissue than in normal tissue, suggesting that USP21 plays a role in the progression of abdominal aortic aneurysms. Inhibiting USP21 expression can modulate signaling pathways related to the formation and progression of abdominal aortic aneurysms, thereby effectively slowing aneurysm growth and improving patient prognosis. Therefore, USP21 mRNA can serve as a novel diagnostic or auxiliary diagnostic biomarker for abdominal aortic aneurysms, providing a basis for early diagnosis.

[0013] A second aspect of the present invention provides a product comprising a substance for detecting USP21, the product having any one or more of the following uses:

[0014] (a1) Treatment of abdominal aortic aneurysm;

[0015] (a2) Diagnosis or auxiliary diagnosis of abdominal aortic aneurysm.

[0016] Substances for detecting USP21 include those used in RT-PCR, real-time quantitative PCR, in situ hybridization, gene chips, and gene sequencing to detect the expression level of USP21.

[0017] The products include primers, probes, chips, nucleic acid membrane strips, formulations, or kits for detecting the expression level of USP21 in the sample to be tested.

[0018] A third aspect of the invention provides the use of USP21 as a target in the treatment and / or screening of drugs for abdominal aortic aneurysms.

[0019] A fourth aspect of the present invention provides a method for screening drugs for abdominal aortic aneurysm, comprising:

[0020] c1) Treat the system expressing and / or containing USP21 with the candidate substance; set up a parallel control without the candidate substance treatment;

[0021] c2) After completing step c1), detect the expression level of USP21 in the system; if the expression level of USP21 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate drug for abdominal aortic aneurysm.

[0022] A fifth aspect of the invention provides the use of a substance that inhibits the expression of USP21 in the preparation of a product;

[0023] The product has any one or more of the following functions:

[0024] (b1) Reduce the tumor formation rate of abdominal aortic aneurysms;

[0025] (b2) Reduce the diameter of the abdominal aorta in the abdominal aortic aneurysm;

[0026] (b3) Improves elastic fiber rupture in abdominal aortic aneurysms;

[0027] (b4) Reversing cell phenotype transformation;

[0028] (b5) Treatment of abdominal aortic aneurysm;

[0029] The reversal of cell phenotype transformation is manifested as the transformation of cells from a secretory phenotype to a contractile phenotype;

[0030] The substances that inhibit USP21 expression levels include RNA interference molecules or antisense oligonucleotides targeting USP21, siRNA, shRNA, microRNA, USP21 gene chemical inhibitors, and gene knockout or knockdown substances.

[0031] The reversal of vascular smooth muscle cell phenotypic transformation specifically involves an increase in the contractile phenotype-related protein SM22α and / or a decrease in the secretory phenotype-related protein MMP9.

[0032] The cell types mentioned include three types: human aortic smooth muscle cells (HASMC), human umbilical vein endothelial cells (HUVEC), and mouse bone marrow-derived macrophages (BMDM). In this embodiment of the invention, human aortic smooth muscle cells (HASMC) are preferred. Reversing the phenotypic transformation of vascular smooth muscle cells is manifested as the transformation of vascular smooth muscle cells from a secretory phenotype to a contractile phenotype.

[0033] The siRNA is si-USP21, and the nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2:

[0034] Sense 5'-CUCUGCGGGAUUGUUUCAATT-3'(SEQ ID NO.1);

[0035] Anti-sense 5'-UUGAAACAAUCCCGCAGAGTT-3' (SEQ ID NO. 2).

[0036] The product is a medicine.

[0037] The USP21 gene chemical inhibitor can be disulfiram.

[0038] In a sixth aspect, the present invention provides a product whose active ingredient is a substance for inhibiting the expression level of USP21, said product having any one or more of the following functions:

[0039] (b1) Reduce the tumor formation rate of abdominal aortic aneurysms;

[0040] (b2) Reduce the diameter of the abdominal aorta in the abdominal aortic aneurysm;

[0041] (b3) Improves elastic fiber rupture in abdominal aortic aneurysms;

[0042] (b4) Reverse the phenotypic transformation of vascular smooth muscle cells;

[0043] (b5) Treatment of abdominal aortic aneurysm;

[0044] Specifically, the reversal of vascular smooth muscle cell phenotype transformation is manifested as the transformation of vascular smooth muscle cells from a secretory phenotype to a contractile phenotype;

[0045] The substances that inhibit USP21 expression levels include RNA interference molecules or antisense oligonucleotides targeting USP21, siRNA, shRNA, microRNA, USP21 gene chemical inhibitors, and gene knockout or knockdown substances.

[0046] The reversal of vascular smooth muscle cell phenotypic transformation specifically involves an increase in the contractile phenotype-related protein SM22α and / or a decrease in the secretory phenotype-related protein MMP9.

[0047] The siRNA is si-USP21, and the nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2:

[0048] Sense 5'-CUCUGCGGGAUUGUUUCAATT-3'(SEQ ID NO.1);

[0049] Anti-sense 5'-UUGAAACAAUCCCGCAGAGTT-3' (SEQ ID NO. 2).

[0050] The product is a medicine.

[0051] The drug also includes at least one inactive pharmaceutical ingredient.

[0052] The USP21 gene chemical inhibitor can be disulfiram.

[0053] A seventh aspect of the present invention provides a method for treating abdominal aortic aneurysm, the method comprising administering to a subject a therapeutically effective amount of siRNA, shRNA, microRNA, or a USP21 gene chemical inhibitor or a combination thereof.

[0054] One or more of the above technical solutions have the following advantages or beneficial effects:

[0055] (1) Given the current lack of approved drugs for the direct treatment of AAA, this invention uses the USP21 gene as a target to interfere with and inhibit the mRNA expression level of the USP21 gene, which can effectively inhibit the phenotypic transformation of vascular smooth muscle cells. This invention successfully inhibits the phenotypic transformation of vascular smooth muscle cells by interfering with and inhibiting the expression level of the USP21 gene mRNA, thus providing patients with a new treatment option.

[0056] (2) This invention constructed a mouse abdominal aortic aneurysm model. Compared with the control group, the tumor formation rate decreased and elastic fiber rupture improved in the USP21 knockdown treatment group. Therefore, USP21 mRNA can serve as a diagnostic molecular marker for abdominal aortic aneurysms and also as a potential therapeutic target. This invention provides a more advantageous means for the diagnosis and treatment analysis of abdominal aortic aneurysms, which is of great significance for the research and treatment of abdominal aortic aneurysms. It also lays an experimental foundation and provides new perspectives for the development of highly effective drugs for the treatment of abdominal aortic aneurysms, thus having good practical application value. Attached Figure Description

[0057] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0058] Figure 1 This invention presents the main cell types expressing USP21 in vascular smooth muscle cells in Example 1 of the present invention, and the expression level of USP21 in vascular smooth muscle cells after angiotensin II (Ang II) stimulation; wherein, A is the difference in USP21 expression among three cell types: human aortic smooth muscle cells (HASMC), human umbilical vein endothelial cells (HUVEC), and mouse bone marrow-derived macrophages (BMDM); and B is a comparison of USP21 expression levels in HASMCs without stimulation and after Ang II stimulation.

[0059] Figure 2 This is an example of detecting the expression level of USP21 after knocking down USP21 using control siRNA and three USP21 siRNAs with different knockdown efficiencies in Example 2 of the present invention; wherein, from left to right, they are control siRNA, USP21 siRNA-1 without knockdown, USP21 siRNA-2 with a knockdown efficiency of 50%, and USP21 siRNA-3 with a knockdown efficiency of 80%.

[0060] Figure 3 This invention illustrates the effects of control siRNA and knockdown USP21 siRNA on HASMC cell phenotypic transformation in Example 3. A represents the change in expression levels of the contraction phenotype-related protein SM22α in the knockdown USP21 siRNA and control siRNA stimulated by Ang II, while B represents the change in expression levels of the secretion phenotype-related protein MMP9 in the knockdown USP21 siRNA and control siRNA stimulated by Ang II.

[0061] Figure 4This is a diagram illustrating the effects of vascular smooth muscle cell (VSMC) specific knockdown of USP21 on phenotypic transformation and the improvement of AAA development in Example 4 of this invention. A shows the efficiency of VSMC-specific USP21 knockdown virus injection in mice (AAV9-KD-USP21) and the control (AAV9-NC); B shows the change in the tumorigenesis rate of abdominal aortic aneurysms after injection of VSMC-specific USP21 knockdown virus (AAV9-KD-USP21) and the control (AAV9-NC); C shows the aortic diameter of the abdominal aorta after injection of VSMC-specific USP21 knockdown virus (AAV9-KD-USP21) and the control (AAV9-NC) after saline stimulation and Ang II stimulation. Changes: D shows the rupture of elastic fibers in the abdominal aorta after injection of VSMC to knock down USP21 virus (AAV9-KD-USP21) and control (AAV9-NC) following saline stimulation and Ang II stimulation; E shows the change in the expression level of SM22α, a protein related to the contraction phenotype of the abdominal aorta, after injection of VSMC to knock down USP21 virus (AAV9-KD-USP21) and control (AAV9-NC) following saline stimulation and Ang II stimulation; F shows the change in the expression level of MMP9, a protein related to the secretion phenotype of the abdominal aorta, after injection of VSMC to knock down USP21 virus (AAV9-KD-USP21) and control (AAV9-NC) following saline stimulation and Ang II stimulation.

[0062] Figure 5 This is a diagram showing the changes in the occurrence and development of AAA caused by the USP21 inhibitor disulfiram in Example 5 of the present invention; wherein, A is the change in the tumor formation rate of abdominal aortic aneurysm after disulfiram administration, B is the change in the aortic diameter of the abdominal aorta after disulfiram administration, and C is the rupture of elastic fibers in the abdominal aorta after disulfiram administration. Detailed Implementation

[0063] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not described in the examples are generally performed according to conventional conditions or the conditions recommended by the reagent company. Unless otherwise specified, the reagents, consumables, etc., used in the following examples are conventional reagents, methods, and equipment used in this invention and are commercially available.

[0065] Those skilled in the art should understand that the term "expression level" refers to the amount of a gene product present in an organism or sample at a specific point in time. Expression levels can be measured / quantified / detected, for example, by the amount of protein or mRNA expressed by the gene. Expression levels can be quantified, for example, by normalizing the amount of the target gene product present in the sample to the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (e.g., a sample obtained from the same organism at the same time or a portion of the same size (weight, volume) of the same sample), or by determining the amount / defined sample size (weight, volume, etc.) of the target gene product. Expression levels can be measured or detected by any method known in the art, such as methods for the direct detection and quantification of the target gene product (e.g., mass spectrometry), or methods for the indirect detection and measurement of the target gene product that typically work by binding the target gene product to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) that are specific to the target gene product. It is also known to those skilled in the art to determine the level of gene copy, which also includes determining the absence or presence of one or more fragments (e.g., by means of nucleic acid probes or primers, such as quantitative PCR, multiplex ligation dependent probe amplification (MLPA) PCR).

[0066] The terms "indicator" and "signature" are used interchangeably in this invention and refer to signs or signals of a symptom or for monitoring a symptom. Such a "symptom" refers to the biological state of a cell, tissue, or organ, or the health and / or disease state of an individual. An indicator can be the presence or absence of molecules, including but not limited to peptides, proteins, and nucleic acids, or it can be a change in the expression level or pattern of such molecules in a cell, tissue, organ, or individual. An indicator can be a sign of the occurrence, development, or presence of a disease in an individual, or a sign of further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0067] The terms "upgraded," "elevated," or "improved" in a terminology refer to an increase in the level of such a term in the sample compared to a reference or reference sample.

[0068] The terms “lower,” “reduced,” or “decreased” refer to a decrease in the level of such an indicator in the sample compared to a reference or reference sample.

[0069] Terminology Explanation:

[0070] USP21: Ubiquitin-Specific Proteinase 21.

[0071] HASMC: Human aortic smooth muscle cells.

[0072] VSMC: Vascular smooth muscle cells.

[0073] HUVEC: Human umbilical vein endothelial cells.

[0074] BMDM: Macrophages derived from mouse bone marrow.

[0075] GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

[0076] LPS+IFN-γ: Lipopolysaccharide combined with interferon.

[0077] SM22α: smooth muscle 22α protein.

[0078] MMP9: Matrix metalloproteinase-9.

[0079] Ang II: Angiotensin II.

[0080] AAV9-NC: HBAAV2 / 9-SM22α-vector refers to the smooth muscle 22α protein vector of HBAAV2 / 9 adeno-associated virus.

[0081] AAV9-KD-USP21: HBAAV2 / 9-SM22α-mir30-2-m-USP21-vector refers to the HBAAV2 / 9-smooth muscle 22α protein-mir30-2-m-USP21 vector.

[0082] Saline + AAV9-NC: Saline and adeno-associated virus type 9 negative control.

[0083] Saline + AAV9-KD-USP21: Saline and type 9 adeno-associated virus knockdown of ubiquitin-specific peptidase 21.

[0084] Ang II+AAV9-NC: Negative control for angiotensin II and adeno-associated virus type 9.

[0085] Ang II+AAV9-KD-USP21: Angiotensin II and adeno-associated virus type 9 knockdown of ubiquitin-specific peptidase 21.

[0086] Ang II + Disulfiram: Angiotensin II and disulfiram.

[0087] Knockdown: This involves reducing the expression level of a target gene through methods such as RNA interference, thereby inhibiting its function. In this invention, the knockdown efficiency ranges from 0% to 80%, specifically 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, etc.

[0088] Given the current lack of approved drugs for the direct treatment of AAA, this invention provides a new treatment option for patients by reducing USP21 mRNA levels. The inventors' proteomic studies showed that USP21 levels in mouse abdominal aortic aneurysm tissue were significantly upregulated compared to normal abdominal aortic tissue, and were closely related to the occurrence and development of AAA. Therefore, the inventors further clarified the important role of targeting USP21 in the treatment of AAA, exploring new drug targets by targeting USP21 to intervene in the formation and development of abdominal aortic aneurysms at the molecular level; effectively controlling the progression of abdominal aortic aneurysms, reducing the risk of death from aneurysm rupture or other complications; providing a new research direction for the cardiovascular drug field; promoting the clinical application of USP21 mRNA in the treatment of AAA; and improving patients' quality of life and survival rates.

[0089] In view of this, in one specific embodiment of the present invention, the use of a substance for detecting USP21 in the preparation of any one or more of the following products is provided:

[0090] (a1) Products for the treatment of abdominal aortic aneurysms;

[0091] (a2) Diagnostic or auxiliary diagnostic products for abdominal aortic aneurysm.

[0092] This study found that USP21 expression was significantly higher in abdominal aortic aneurysm tissue than in normal tissue, suggesting that USP21 plays a role in the progression of abdominal aortic aneurysms. Inhibiting USP21 expression can modulate signaling pathways related to the formation and progression of abdominal aortic aneurysms, thereby effectively slowing aneurysm growth and improving patient prognosis. Therefore, USP21 mRNA can serve as a novel diagnostic or auxiliary diagnostic biomarker for abdominal aortic aneurysms, providing a basis for early diagnosis.

[0093] In another specific embodiment of the present invention, a product is provided, the product comprising a substance for detecting USP21, the product having any one or more of the following uses:

[0094] (a1) Treatment of abdominal aortic aneurysm;

[0095] (a2) Diagnosis or auxiliary diagnosis of abdominal aortic aneurysm.

[0096] In another specific embodiment of the present invention, the substance for detecting USP21 includes substances used for detecting the expression level of USP21 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

[0097] The products include, but are not limited to, primers, probes, chips, nucleic acid membrane strips, formulations, or kits for detecting the expression level of USP21 in the sample to be tested.

[0098] In another specific embodiment of the present invention, the sample to be tested may be a human sample, and more specifically, the sample to be tested includes the vascular tissue of the subject.

[0099] In another specific embodiment of the present invention, the kit contains reagents expressing USP21.

[0100] Specifically, the kit contains specific PCR primers (human) for USP21 mRNA and GAPDH internal control, and the nucleotide sequences of the specific PCR primers are as follows:

[0101] USP21 mRNA (human origin):

[0102] Upstream primer sequence: 5'-AAGAAACTGGAGCTGGGACG-3' (SEQ ID NO.5);

[0103] Downstream primer sequence: 5'-TGGAACGGGCTAAGTTGGTC-3' (SEQ ID NO.6).

[0104] Human-derived specific PCR primers for GAPDH internal control:

[0105] Upstream primer sequence: 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO.7);

[0106] Downstream primer sequence: 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO.8).

[0107] In another specific embodiment of the present invention, the use of the above-mentioned USP21 as a target in the treatment of abdominal aortic aneurysm and / or screening of drugs for abdominal aortic aneurysm is provided.

[0108] In another specific embodiment of the present invention, the abdominal aortic aneurysm drug is a drug for the prevention and / or treatment of abdominal aortic aneurysm.

[0109] In another specific embodiment of the present invention, a method for screening drugs for abdominal aortic aneurysm is provided, comprising:

[0110] c1) Treat the system expressing and / or containing USP21 with the candidate substance; set up a parallel control without the candidate substance treatment;

[0111] c2) After completing step c1), detect the expression level of USP21 in the system; if the expression level of USP21 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate drug for abdominal aortic aneurysm.

[0112] In another specific embodiment of the present invention, the system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0113] In another specific embodiment of the present invention, the cells in the cell system may be vascular smooth muscle cells.

[0114] In another specific embodiment of the present invention, the tissue in the cell system can be vascular tissue.

[0115] In another specific embodiment of the present invention, the animals in the animal system can be mammals, such as rats, mice, guinea pigs, rabbits, monkeys, humans, etc.

[0116] In another specific embodiment of the present invention, the use of a substance that inhibits the expression of USP21 in the preparation of a product is provided;

[0117] The product has any one or more of the following functions:

[0118] (b1) Reduce the tumor formation rate of abdominal aortic aneurysms;

[0119] (b2) Reduce the diameter of the abdominal aorta in the abdominal aortic aneurysm;

[0120] (b3) Improves elastic fiber rupture in abdominal aortic aneurysms;

[0121] (b4) Reverse the phenotypic transformation of vascular smooth muscle cells;

[0122] (b5) Treatment of abdominal aortic aneurysm;

[0123] The substances that inhibit USP21 expression levels include RNA interference molecules or antisense oligonucleotides targeting USP21, siRNA, shRNA, microRNA, USP21 gene chemical inhibitors, and gene knockout or knockdown substances.

[0124] The reversal of vascular smooth muscle cell phenotype transformation is manifested as the transformation of vascular smooth muscle cells from a secretory phenotype to a contractile phenotype; specifically, the contractile phenotype-related protein SM22α is increased and / or the secretory phenotype-related protein MMP9 is decreased.

[0125] The chemical inhibitor of the USP21 gene can be disulfiram.

[0126] In another specific embodiment of the present invention, the siRNA is si-USP21, and the nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2.

[0127] The product is a medicine.

[0128] In another specific embodiment of the present invention, a product is provided, the active ingredient of which is a substance used to inhibit the expression level of USP21.

[0129] The substances that inhibit USP21 expression levels include RNA interference molecules or antisense oligonucleotides targeting USP21, siRNA, shRNA, microRNA, USP21 gene chemical inhibitors, and gene knockout or knockdown substances. The USP21 gene chemical inhibitor is disulfiram.

[0130] In another specific embodiment of the present invention, the siRNA is si-USP21, and the nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2.

[0131] In another specific embodiment of the present invention, the product has any one or more of the following functions:

[0132] (b1) Reduce the tumor formation rate of abdominal aortic aneurysms;

[0133] (b2) Reduce the diameter of the abdominal aorta in the abdominal aortic aneurysm;

[0134] (b3) Improves elastic fiber rupture in abdominal aortic aneurysms;

[0135] (b4) Reverse the phenotypic transformation of vascular smooth muscle cells;

[0136] (b5) Treatment of abdominal aortic aneurysm.

[0137] The reversal of vascular smooth muscle cell phenotype transformation is manifested as the transformation of vascular smooth muscle cells from a secretory phenotype to a contractile phenotype, specifically by an increase in the contractile phenotype-related protein SM22α and / or a decrease in the secretory phenotype-related protein MMP9.

[0138] The product is a medicine.

[0139] According to the present invention, the concept of "treatment" means any measure applicable to the treatment of abdominal aortic aneurysm-related diseases, or preventive treatment of such diseases or symptoms, or prevention of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of diseases that have already occurred, or preemptive intervention to prevent, suppress or reduce the occurrence of such diseases or symptoms.

[0140] According to the present invention, the above-mentioned drug also includes at least one inactive pharmaceutical ingredient.

[0141] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0142] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0143] The carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0144] The drug of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0145] In another specific embodiment of the present invention, a method for treating abdominal aortic aneurysm is provided, the method comprising administering to a subject a therapeutically effective amount of siRNA, shRNA, microRNA, or a USP21 gene chemical inhibitor or a combination thereof.

[0146] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0147] Experimental materials:

[0148] The genetic and cellular information was obtained from the NCBI database. The gene number of USP21 is 27005, and the gene number of GAPDH is 2597.

[0149] The reagents and chemicals used in the examples are all available through commercial purchase channels.

[0150] The siRNA is si-USP21, and the nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2:

[0151] Sense 5'-CUCUGCGGGAUUGUUUCAATT-3'(SEQ ID NO.1);

[0152] Anti-sense 5'-UUGAAACAAUCCCGCAGAGTT-3' (SEQ ID NO. 2).

[0153] The nucleotide sequence of si-NC is shown in SEQ ID NO.3-4:

[0154] Sense 5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO.3);

[0155] Anti-sense 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 4).

[0156] Example 1: It has been confirmed that vascular smooth muscle cells USP21 in the aorta are associated with the development of AAA.

[0157] Proteomic analysis revealed that USP21 levels were significantly upregulated in mouse abdominal aortic aneurysm tissue compared to normal abdominal aortic tissue, and were closely related to the occurrence and development of AAA. Therefore, further investigation clarifies the important role of targeting USP21 in specific cells in the treatment of AAA.

[0158] The specific implementation process is as follows:

[0159] The samples used in this study were human aortic smooth muscle cells (HASMC), human umbilical vein endothelial cells (HUVEC), and mouse bone marrow-derived macrophages (BMDM). A concentration of 10 was used. -6 After stimulating HASMC and HUVEC cells with angiotensin II (Ang II) at mol / L for 24 hours, BMDM cells were treated with lipopolysaccharide combined with interferon (LPS+IFN-γ) at a concentration of 100 ng / ml for 6 hours before RNA was extracted.

[0160] USP21 primer information (human source):

[0161] Upstream primer sequence: 5'-AAGAAACTGGAGCTGGGACG-3' (SEQ ID NO.5);

[0162] Downstream primer sequence: 5'-TGGAACGGGCTAAGTTGGTC-3' (SEQ ID NO.6).

[0163] Information on GAPDH primers for the internal reference gene (human origin):

[0164] Upstream primer sequence: 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO.7);

[0165] Downstream primer sequence: 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO.8).

[0166] Information on GAPDH primers for the internal reference gene (mouse source):

[0167] Upstream primer sequence: 5'-TGCACCACCAACTGCTTAG-3' (SEQ ID NO.9);

[0168] Downstream primer sequence: 5'-GGCATGGACTGTGGTCATGAG-3' (SEQ ID NO.10).

[0169] USP21 primer information (mouse source):

[0170] Upstream primer sequence: 5'-CCGAGTGGGAGCCAAGATAC-3' (SEQ ID NO.11);

[0171] Downstream primer sequence: 5'-TGGGAGGCAAAGGTCGTAAC-3' (SEQ ID NO.12).

[0172] Reverse transcription reaction system: Total reaction volume: 20 µL, reverse transcriptase: 1 µL, 5× reverse transcription buffer: 4 µL, dNTP mixture: 1 µL (10 mM for each dNTP), primers: 1 µL, RNA sample: 1 µg, RNase inhibitor: 0.5 µL, water: to 20 µL. Reaction conditions: 25°C, 10 min (primer binding), 42°C, 60 min (reverse transcription), 70°C, 15 min (enzyme inactivation).

[0173] Real-time quantitative PCR reaction program: PCR reaction system: Total reaction volume: 20 µL, 2× PCR MasterMix: 10 µL, primers (GAPDH or USP21): 0.5 µL each, cDNA template: 1 µL, water: to 20 µL. Real-time PCR program: Initial denaturation: 95°C, 10 min, cycles (40 times), denaturation: 95°C, 15 sec, annealing: 60°C, 30 sec, extension: 72°C, 30 sec, final extension: 72°C, 5 min.

[0174] Reverse transcription-real-time quantitative PCR revealed that vascular smooth muscle cells (corresponding cell line HASMC) are the main cell type expressing USP21 in vascular tissue. Figure 1A in the middle.

[0175] Furthermore, Western blot analysis showed increased USP21 expression in HASMCs after Ang II stimulation. Figure 1 B in the middle.

[0176] Example 2: Design and fabrication of USP21 siRNA sequence

[0177] Based on the mRNA nucleotide sequence of the USP21 gene, si-USP21 was designed to specifically knock down the expression level of USP21 mRNA. The nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2.

[0178] The specific implementation process is as follows:

[0179] Human aortic smooth muscle cells (HASMCs) were selected and cultured in a suitable medium for HASMCs, typically SMCM (Smooth Muscle Cell Medium), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to provide necessary nutrients and antibiotics. Approximately 1-2 × 10⁶ cells were seeded per well. ^5 To ensure sufficient cell growth during culture, place the 6-well plate in a 37°C, 5% CO2 incubator for 24 hours, until the cell density reaches approximately 60%. At this point, the cells should adhere well to the plate with adequate spacing between them.

[0180] Choose a suitable transfection reagent (such as Lipofectamine 3000) and prepare two siRNAs: si-USP21 (siRNA targeting the USP21 gene) and si-NC (negative control siRNA).

[0181] Ensure the siRNA concentration is suitable for the transfection experiment, typically 20-100 nM. When cells reach 60% confluence, perform transfection as follows: Take appropriate amounts of transfection reagent, si-USP21, and si-NC, and mix according to the manufacturer's instructions, usually under aseptic conditions. Gently add the mixture to each well, ensuring even distribution. Gently shake the 6-well plate to ensure full contact between the transfection solution and cells, and incubate for 24 hours. During this period, cells will absorb siRNA, initiating the inhibition of USP21 expression. Extract total cell protein after 24 hours. Detect USP21 expression levels using Western blot, selecting the sequence with the highest knockdown efficiency (see [link to relevant documentation]). Figure 2The results showed that USP21 siRNA-1 had no knockdown effect, USP21 siRNA-2 had a knockdown efficiency of 50%, and USP21 siRNA-3 had a knockdown efficiency of 80%; therefore, USP21 siRNA-3 was selected for subsequent experiments.

[0182] Example 3: Knockdown of USP21 improves the conversion of HASMCs to the secretory phenotype

[0183] HASMC cells were selected and transfected with the selected si-USP21 and si-NC, respectively, to detect the effect of USP21 knockdown on vascular smooth muscle cell phenotypic transformation.

[0184] The specific implementation process is as follows:

[0185] HASMC cells were selected and transfected with either si-USP21 or si-NC. The cell culture and transfection procedures were the same as in Example 2. After 24 hours, 10 -6 Cells were stimulated with mol / L angiotensin II (Ang II), and the cell culture process was the same as in Example 2. After 24 h, cells were collected and total cell protein was extracted. Western blot was used to detect changes in the levels of smooth muscle phenotype transformation-related proteins.

[0186] The results showed that after Ang II stimulation and USP21 knockdown, the level of the contraction phenotype-related protein SM22α recovered. Figure 3 In A), the level of the secretion phenotype-related protein MMP9 decreased ( Figure 3 (B in the text). This indicates that knocking down USP21 can cause HASMCs to change from a secretory phenotype to a contractile phenotype.

[0187] Example 4: Specific knockdown of USP21 virus in mouse vascular smooth muscle cells affects phenotypic transformation and improves the occurrence and development of AAA.

[0188] The specific implementation process is as follows:

[0189] A mouse vascular smooth muscle-specific knockdown USP21 virus (HBAAV2 / 9-SM22α-mir30-2-m-USP21-vector, abbreviated as AAV9-KD-USP21) and a control virus (HBAAV2 / 9-SM22α-vector, abbreviated as AAV9-NC) were constructed and injected into mice via the tail vein to establish an abdominal aortic aneurysm model. Mice were anesthetized with an anesthetic and placed prone on the operating table. The skin on the back was carefully bluntly dissected and a micro-pump (containing 1000 ng / kg / min angiotensin II) was implanted into the mice. After completion, the incision was sutured with sutures.

[0190] The results showed that the AAV9-KD-USP21 group could effectively reduce the expression level of USP21 in aortic tissue. Figure 4 A). Injection of AAV9-KD-USP21 reduced the rate of abdominal aortic aneurysm formation compared to injection of AAV9-NC. Figure 4 (B in the text) reduced the diameter of the abdominal aorta ( Figure 4 (C) Improved elastic fiber breakage ( Figure 4 (D in the text). Further investigation revealed that AAV9-KD-USP21 increased the expression of the contractile phenotype-related protein SM22α and decreased the expression of the secretory phenotype-related protein MMP9. This indicates that knocking down USP21 can inhibit the progression of abdominal aortic aneurysms by affecting smooth muscle phenotype transformation. Figure 4 (EF in the text).

[0191] In conclusion, knocking down USP21 specifically on vascular smooth muscle cells can improve the occurrence and development of abdominal aortic aneurysms.

[0192] Example 5: USP21 inhibitor disulfiram improves the occurrence and development of AAA

[0193] The specific implementation process is as follows:

[0194] The inventors discovered that disulfiram can inhibit USP21, and therefore treated AAA model mice with 50 mg / kg disulfiram by gavage for 28 days.

[0195] The results showed that disulfiram treatment could reduce the tumor formation rate and the widening of the abdominal aorta diameter, improve the elastic fiber rupture, and inhibit the progression of abdominal aortic aneurysms. Figure 5 (AC in the text). In summary, disulfiram improves the occurrence and development of abdominal aortic aneurysms through USP21.

[0196] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of substances that inhibit USP21 expression levels in the preparation of products for treating abdominal aortic aneurysms; The substance that inhibits the expression level of USP21 is siRNA; The siRNA is si-USP21, and the nucleotide sequence of si-USP21 is shown in SEQ ID NO.1-2; The product is a medicine.

2. The application as described in claim 1, characterized in that, The product for treating abdominal aortic aneurysms has one or more of the following functions: (b1) Reduce the tumor formation rate of abdominal aortic aneurysms; (b2) Reduce the diameter of the abdominal aorta in the abdominal aortic aneurysm; (b3) Improves elastic fiber rupture in abdominal aortic aneurysms; (b4) Reverse the phenotypic transformation of vascular smooth muscle cells; Specifically, the reversal of vascular smooth muscle cell phenotype transformation is manifested as the transformation of vascular smooth muscle cells from a secretory phenotype to a contractile phenotype.

3. The application as described in claim 2, characterized in that, The reversal of vascular smooth muscle cell phenotypic transformation specifically involves an increase in the contractile phenotype-related protein SM22α and / or a decrease in the secretory phenotype-related protein MMP9.

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