Application of NAT10 and related biomolecules thereof in diagnosis and treatment of deep vein thrombosis

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and relates to application of NAT10 and related biomolecules thereof in diagnosis and treatment of deep vein thrombosis. Specifically, research finds that NAT10 is an important starting factor of endothelial cell ferroptosis, and the expression of NAT10 is remarkably increased in DVT. The result shows that the endothelial ferroptosis is relieved by inhibiting the NAT10 to reduce the ac4C level in the DVT. However, reduction of expression of NAT10 leads to reduction of stability of HMOX1, reduction of release of Fe < 2 + > and inhibition of lipid peroxidation, so that targeting NAT10 / HMOX1 axis may be a potential method for treating DVT, and the application has good potential practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of NAT10 and related biomolecules in the diagnosis and treatment of deep vein thrombosis. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Deep vein thrombosis (DVT) is a common peripheral vascular disease, with an estimated annual incidence of more than 1 / 1000 people. The incidence of DVT is increasing year by year, seriously affecting the health and quality of life of patients. The most common causes of DVT are endothelial injury, blood stasis, and hypercoagulable state. The underlying mechanism of endothelial injury is crucial in the occurrence of DVT. Although great efforts have been made to explore the related underlying molecular mechanisms, there are still no feasible therapeutic targets for reducing endothelial injury in clinical practice.

[0004] More and more evidence indicates that high iron is associated with thrombosis, suggesting that iron homeostasis plays an important role in thrombosis. Iron accumulation significantly accelerates thrombosis after vascular injury and increases vascular oxidative stress. Recent studies have shown that ferroptosis is a form of regulated cell death characterized by iron overload and lipid peroxidation, which is closely related to vascular injury. In addition, excessive free ferrous iron promotes the occurrence of ferroptosis by aggravating intracellular oxidative stress. Here, RNA modification may play an important role in DVT, but its etiology is still unclear and further research is needed to identify new prevention and treatment targets.

[0005] N4-acetylcytidine (ac4C) modification, as a newly discovered mRNA modification, is considered a universal epigenetic marker of mRNA and plays an important role in regulating mRNA stability and mRNA translation efficiency. N-acetyltransferase 10 (NAT10) is the only known ac4C modifying enzyme in mammals, catalyzing the acetylation of cytidine residues of mRNA. In addition, NAT10 is found in the nucleolus and regulates telomerase activity, ribosomal RNA transcription, and cytokinesis. Recent studies have found that nat10-mediated ac4C acetylation is involved in various physiological and pathological processes, including aging, apoptosis, autophagy, and ferroptosis. However, the changes and roles of NAT10 in DVT ferroptosis are still unclear.

[0006] Heme oxygenase 1 (HMOX1) is a ferroptosis activator that can decompose heme into carbon monoxide, biliverdin, and iron ions (Fe 2+), reactive oxygen species are generated through the Fenton reaction, promoting the accumulation of lipid peroxidation. Research reports have shown that HMOX1 has a cytoprotective function, but more and more evidence indicates that when the expression level of HMOX1 in cells exceeds a certain threshold, HMOX1 will produce cytotoxic effects. More importantly, overexpression of HMOX1 promotes Fe 2+ overload, an increase in iron content, ROS generation, aggravates lipid peroxidation, and leads to ferroptosis of endothelial cells. However, the current research on the mechanism of HMOX1 involvement in DVT is not sufficient. Summary of the Invention

[0007] Aiming at the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide the application of NAT10 and its related biomolecules in the diagnosis and treatment of deep vein thrombosis. Specifically, through research, the present invention finds that NAT10 is an important initiator of ferroptosis of endothelial cells, and its expression is significantly increased in DVT. The research results of the present invention show that by inhibiting NAT10 to reduce the ac4C level in DVT, ferroptosis of endothelial cells can be alleviated. However, the decrease in the expression of NAT10 leads to a decrease in the stability of HMOX1, reduces the release of Fe 2+ and inhibits lipid peroxidation. Therefore, targeting the NAT10 / HMOX1 axis may be a potential method for treating DVT. Based on the above research results, the present invention is completed.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] In the first aspect of the present invention, there is provided the application of a reagent for detecting the expression level of NAT10 and its related biomolecules in the preparation of a deep vein thrombosis detection product.

[0010] Furthermore, the deep vein thrombosis detection product can be used for the screening, (auxiliary) diagnosis, monitoring or prognosis of deep vein thrombosis.

[0011] Specifically, the expression level of NAT10 and its related biomolecules is positively correlated with the occurrence and development of deep vein thrombosis. Specifically, the expression of NAT10 is significantly up-regulated in venous vascular tissues and is positively correlated with the severity of DVT disease. Therefore, it can be used for the screening, (auxiliary) diagnosis, monitoring or prognosis of deep vein thrombosis.

[0012] Among them, the related biomolecules of NAT10 include ac4C modification and HMOX1.

[0013] The substances for detecting the expression levels of NAT10 and its related biomolecules may include reagents for detecting the expression levels of the genes encoding NAT10 and its related biomolecules based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips, and gene sequencing, and / or reagents for detecting the protein (enzyme) expression levels of NAT10 and its related biomolecules based on immunoassay methods.

[0014] In the present invention, the products may be primers, probes, (gene or protein) chips, detection kits, detection devices, detection equipment, etc.

[0015] In the second aspect of the present invention, there is provided a system for detecting deep vein thrombosis, the system comprising:

[0016] An acquisition module, which is configured to: acquire the expression level conditions of NAT10 and its related biomolecules in a sample to be tested of a subject;

[0017] An analysis module, which is configured to: analyze and judge the disease condition of the subject according to the expression level conditions of NAT10 and its related biomolecules of the subject obtained by the acquisition module.

[0018] Among them, the related biomolecules of NAT10 include ac4C modification and HMOX1.

[0019] The sample to be tested may be a blood sample of the subject, and further may be a mononuclear cell sample of peripheral blood.

[0020] The detection of deep vein thrombosis may specifically be the screening, (auxiliary) diagnosis, monitoring, or prognosis of deep vein thrombosis.

[0021] In the third aspect of the present invention, there is provided the use of NAT10 and its related biomolecules as targets in the preparation and / or screening of drugs for deep vein thrombosis.

[0022] Furthermore, based on the effects of a candidate drug on NAT10 and its related biomolecules before and after use, it is determined whether the candidate drug can be used for the prevention and / or treatment of deep vein thrombosis.

[0023] Furthermore, the method for screening drugs for preventing or treating deep vein thrombosis includes:

[0024] (I) Treating a system expressing and / or containing NAT10 and its related biomolecules with a candidate substance; setting a control without treating with the candidate substance;

[0025] (II) After completing step (I), detect the expression levels of NAT10 and its related biomolecules in the system; compared with the control, if the expression levels of NAT10 and its related biomolecules in the system treated with the candidate substance are significantly down-regulated, the candidate substance can be used as a candidate drug for preventing or treating deep vein thrombosis.

[0026] Among them, the related biomolecules of NAT10 include ac4C modification and HMOX1.

[0027] More specifically, the specific content of step 2) is: if the expression levels of NAT10 and / or acetylated HMOX1 in the system treated with the candidate substance are significantly down-regulated, the candidate substance can be used as a candidate drug for preventing or treating deep vein thrombosis.

[0028] The system can be a solution system, a cell system, a tissue system, an organ system or an animal system, and no specific limitation is made here.

[0029] In the fourth aspect of the present invention, there is provided the use of a substance that inhibits the expression of NAT10 or reduces its activity in any one or more of the following:

[0030] (a) Inhibiting the expression of HMOX1 or preparing a product that inhibits the expression of HMOX1;

[0031] (b) Inhibiting ferroptosis and iron overload or preparing a product that inhibits ferroptosis and iron overload;

[0032] (c) Inhibiting the generation of lipid peroxides or preparing a product that inhibits the generation of lipid peroxides;

[0033] (d) A product for preventing and / or treating deep vein thrombosis.

[0034] Among them, the substance that inhibits the expression of NAT10 or reduces its activity includes, but is not limited to, RNA interference molecules or antisense oligonucleotides targeting NAT10, small molecule inhibitors (such as Remodelin), siRNA, shRNA, substances for implementing lentiviral infection or gene knockout, etc.

[0035] Among them, in (a)-(c), the product can act on endothelial cells, specifically, it can be venous vascular endothelial cells (such as human umbilical vein vascular endothelial cells).

[0036] The product can be a drug or an experimental reagent for non-medical use, and the experimental reagent can be used for basic research. For example, the product can be used to regulate ferroptosis of endothelial cells in vivo (in vitro), etc., so as to prepare an endothelial cell-related biological model, thereby laying a material foundation for the mechanism research of diseases such as deep vein thrombosis.

[0037] According to the present invention, when the product is a drug, the drug further comprises at least one pharmaceutically inactive ingredient.

[0038] The pharmaceutically inactive ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. At the same time, the non-drug active ingredients such as the carrier, excipient, and diluent that may be included are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0039] The drug of the present invention can be administered into the body by known methods. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, biological type or its tissue, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0040] The subjects to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees.

[0041] In a fifth aspect of the present invention, a method for preventing and / or treating deep vein thrombosis is provided, the method comprising administering to a subject the above-mentioned substance that inhibits NAT10 expression or reduces its activity and / or the corresponding product.

[0042] The beneficial technical effects of the above one or more technical solutions:

[0043] The above technical solution reveals that NAT10 is an important regulator of ferroptosis in DVT. In particular, NAT10 increases the stability of HMOX1 by mediating ac4C modification, leading to iron overload and lipid peroxidation, thus forming a positive feedback loop and exacerbating DVT. The research results show that targeting NAT10 may be a promising therapeutic strategy for enhancing endothelial cell ferroptosis, and therefore has good potential practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0045] Figure 1Ferroptosis is involved in the formation of DVT in the embodiments of the present invention; (A) mRNA microarray analysis of peripheral blood mononuclear cells, and the top 10 pathways enriched by KEGG enrichment analysis. (B) The top 10 KEGG pathways enriched in DVT mice (with thrombus) according to 4D Label Free. (C) Transmission electron microscope showing the mitochondrial morphology and cristae (red arrows) in vascular tissues. Scale bar = 1 μm. (D-F) Levels of ferrous ions (Fe 2+ ), malondialdehyde (MDA), and glutathione (GSH) in DVT mice. (G, H) Rats were treated with Ferrostatin-1 (Fer-1), and the levels of plasma endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) were detected by ELISA. (I, J) Representative images of thrombi in each group detected by HE staining (magnification, ×100) and vascular ultrasound after Fer-1 treatment. Scale bar = 200 μm. ***P < 0.001.

[0046] Figure 2 Knockdown of NAT10 significantly alleviates ferroptosis in DVT mice in the embodiments of the present invention; (A) Dot blotting shows that the total ac4C level in the vascular tissues of DVT mice is higher (n = 5). (B) The relative expression level of NAT10 mRNA in DVT mice (n = 10) was detected by real-time fluorescence quantitative PCR. (C) The volcano plot shows the increased expression of NAT10 in DVT mice. (D) Western blotting was used to detect the NAT10 protein level. (E, F) Representative images of thrombi in NAT10-knockdown DVT mice detected by HE staining and vascular ultrasound. Scale bar = 200 μm. (G-I) Detection of Fe 2+ , MDA, and GSH levels in DVT mice with NAT10 knockdown. ***P < 0.001.

[0047] Figure 3 Deletion of NAT10 inhibits ferroptosis of HUVECs in the embodiments of the present invention; (A) Cell Counting Kit-8 was used to detect cell viability. (B) HUVECs were treated with RSL3 (4 μM) / FINO2 (40 μM) or remodeling (20 μM) for 12 h (magnification, x100). (C, E) After inhibiting NAT10 with BODIPYTM 581 / 591C11 probe to detect lipid peroxidation, HUVECs were treated with RSL3 (4 μM) or FINO2 (40 μM) for 12 h (magnified, ×600). The green image represents oxidized lipids, while the red image represents non-oxidized lipids. Scale bar = 10 μm. (D, F) After inhibiting NAT10 in HUVECs with FerroOrange probe, and then treated with RSL3 or FINO2 for 12 h, Fe 2+(Magnification, ×600). Red in the image indicates Fe 2+ ; blue represents the cell nucleus. Scale bar = 10 μm. (G, H) MDA and GSH levels in HUVECs. *P<0.05, **P<0.01, ***P<0.001.

[0048] Figure 4 In this example of the present invention, NAT10 mediates the acetylation of HMOX1 mRNA in vitro; (A) acRIP-seq analysis of highly enriched motifs of ac4C. (B) Venn diagram showing the prediction of mRNA by acRIP-seq, 4D Label Free and FerrDb V2 Database. (C) Detection of the mRNA levels in the vascular tissues of DVT mice (n = 10) and the control group (n = 10) by gRT-PCR. (D) Detection of the mRNA levels of NAT10 and HMOX1 in si-NAT10s by gRT-PCR. (E) Detection of the HMOX1 protein level after inhibiting NAT10 by Western blot. (F) Detection of the ac4C modification level of HMOX1 mRNA by acRIP-qPCR. (G) NAT10RIP-qPCR analysis of HMOX1 mRNA in HUVECs. (H) Detection of the stability of HMOX1 after treatment with actinomycin D. (I-L) Detection of the mRNA and protein levels of NAT10 and HMOX1 in HUVECs and C166 cells treated in each group by gRT-PCR and Western blotting. (M, N) MDA and GSH levels in HUVECs and C166 cells. *P<0.05, **P<0.01, ***P<0.001.

[0049] Figure 5 In this example of the present invention, the downregulation of NAT10 alleviates ferroptosis by reducing the expression of HMOX1. (A, B) Representative images of thrombi in each group detected by color ultrasound of blood vessels after treating DVT mice with HE staining (magnification, x100) and ZnPP (HMOX1 inhibitor). Scale bar = 200 μm. (C-E) Fe 2+ , MDA, GSH levels in DVT mice. (F) Detection of the cell viability of HUVECs by CCK8 kit. (G) Fe 2+ levels in HUVECs after inhibiting NAT10 and treating with CoPP for 12 h. Red in the figure represents Fe 2+, blue represents the cell nucleus (magnification ×600). Scale bar = 10 μm. (H, I) Protein levels of HMOX1 and GPX4 in si-NAT10 were detected by Western blotting. (J, K) MDA and GSH levels were measured in si-NAT10. **P<0.01, ***P<0.001.

[0050] Figure 6 In the embodiment of the present invention, knocking out NAT10 inhibits HMOX1 and reduces the formation of DVT in vivo.

[0051] (A, B) NAT10 f / f Cdh5-Cre + (NAT10 knockout) Representative images of thrombi in DVT mice were detected by HE staining (magnification, x100) and vascular color ultrasound in each group. Scale bar = 200 μm. (C-E) Relative expression levels of Fe 2+ , MDA and GSH. (F) Expression of HMOX1 in different treatment groups was detected by gRT-PCR. (G) Expressions of HMOX1 and GPX4 in different treatment groups were detected by Western blot. (H-K) Expressions of plasma ET-1, eNOS, TNF-α, TGF-β1 in each group were detected by ELISA. Detailed implementation manners

[0052] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0053] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0054] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0055] The present invention will be further explained and illustrated below by means of examples, but this does not constitute a limitation on the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions noted in the following examples are generally carried out under conventional conditions.

[0056] Example

[0057] 1. Methods and Materials

[0058] 1.1 Microarray Analysis

[0059] Genome-wide analysis of mRNA expression in peripheral blood mononuclear cells of 6 DVT patients and 6 controls was performed using Human mRNA (4 * 180K, Design ID: 084410). Sample labeling, microarray hybridization, and washing were carried out by OE Biotech.

[0060] 1.2 Proteomics Analysis

[0061] For proteomics, vascular tissues of DVT mice (with thrombus group, n = 6), DVT mice (without thrombus group, n = 6), and control group (n = 6) were collected, proteins were extracted by centrifugation, and then 4D LFQ proteomics analysis was performed, including liquid chromatography - MS / MS analysis and data analysis.

[0062] 1.3 Cell Culture and Transfection

[0063] Human umbilical vein endothelial cells (HUVECs) and C166 cells were from Procell life Technology Co., Ltd. (Wuhan, China). Cells were transfected with siRNA or negative control (GenePharma, Shanghai, China) using Lipofectamine RNAIMAX (Invitrogen, Carlsbad, USA) according to the instructions.

[0064] 1.4 Iron Content Determination

[0065] The relative content of Fe in vascular tissues was analyzed using a ferrous ion content detection kit (BC5415, Solarbio, Beijing, China). 2+ in

[0066] 1.5 Determination of Malondialdehyde (MDA) Level

[0067] The treated cells and venous blood vessels were lysed by ultrasound and homogenized in an ice bath, and then centrifuged at 8,000 xg for 10 min at 4°C. The relative MDA level was detected using an MDA content detection kit (BC0025, Solarbio, Beijing, China).

[0068] 1.6 Glutathione (GSH) Determination

[0069] The treated cells and venous blood vessels were lysed by ultrasound and homogenized in an ice bath, and then centrifuged at 8,000 xg for 10 min at 4°C. The relative glutathione level was measured using a reduced glutathione content assay kit (BC1175, Solarbio, Beijing, China).

[0070] 1.7 Determination of lipid peroxidation (LPO)

[0071] The pretreated cells were incubated with the BODIPYTM 581 / 591 C11 probe (Invitrogen, Carlsbad, USA) at 37°C for 30 min. The cells were seeded in confocal dishes and treated with ferroptosis activators (with or without NAT10 inhibitor) for 12 h. After washing three times with PBS, the nuclei were stained with Hoechst (Beyotime, Shanghai, China). LPO fluorescence was detected using a scanning confocal microscope (LSM 880, Carl Zeiss AG, Oberkochen, Germany).

[0072] 1.8 Immunofluorescence Fe 2+ analysis

[0073] The level of Fe was detected by immunofluorescence staining. The cells were seeded on confocal dishes and treated with RSL3 or FINO2 for 12 h, with or without the addition of NAT10 inhibitor. The cells were washed 3 times with PBS and stained with Hoechst. Finally, pictures were taken with a scanning confocal microscope. 2+ The level of Fe was detected by immunofluorescence staining. The cells were seeded on confocal dishes and treated with RSL3 or FINO2 for 12 h, with or without the addition of NAT10 inhibitor. The cells were washed 3 times with PBS and stained with Hoechst. Finally, pictures were taken with a scanning confocal microscope.

[0074] 1.9 Cell viability assay

[0075] The cells were seeded in 96-well plates at 8 × 10 3 cells per well. After each treatment, the medium was changed to 100 μl of DMEM and 10 μl of CCK8 solution (Beyotime, Shanghai, China) per well according to the experimental requirements. After incubation at 37°C for 1 h, the absorbance was measured at 450 nm.

[0076] 1.10 Real-time fluorescence quantitative PCR (qRT-PCR)

[0077] Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, USA) according to the specifications. Approximately 1 μg of RNA was reverse transcribed into cDNA, and qRT-PCR analysis was performed using SYBR Green (Invitrogen, Carlsbad, USA), and the relative mRNA level was calculated according to the 2 -ΔΔCt formula.

[0078] 1.11 RNA Immunoprecipitation (RIP)

[0079] After connecting magnetic beads with NAT10 antibody or IgG antibody, the RIP kit was used to capture the antigen, and then RNA was extracted and verified by qRT-PCR.

[0080] HUVECs were transfected using Acetylated RNA Immunoprecipitation Sequencing (acRIP-seq) and sequenced by Cloudseq Biotech (Shanghai, China). First, RNA was extracted from the samples (extracted using TRIzol), the quality of the RNA was controlled, and the concentration and purity of the RNA were detected. Total RNA was immunoprecipitated according to the manufacturer's instructions. The samples were sequenced using the NovaSeq platform (Illumina).

[0081] 1.12 ac4C-RIP

[0082] Detection was performed using the RIP kit (GS-ET-005, Cloudseq Biotech, Shanghai, China) according to the manufacturer's instructions. Briefly, total RNA (200 μg) was randomly digested into nucleotide chains of 100 - 200 bp, and a mixture of 5 μg ac4C antibody and magnetic beads was incubated with the disrupted RNA. The enrichment of HMOX1 mRNA was analyzed by qRT-PCR.

[0083] 1.13 Western Blot

[0084] Samples were lysed in RIPA buffer with protease and phosphatase inhibitors (Solarbio, Beijing, China), processed by SDS-PAGE, and transferred to nitrocellulose membranes. Then, the membranes were incubated with diluted primary antibodies and horseradish peroxidase-labeled secondary antibodies. The following antibodies were used in this study: anti-NAT10 (1:1000, ab194297, Abcam, Cambridge, USA), HMOX1 (1:1000, ab68477, Abcam, Cambridge, USA), GPX4 (1:1000, ab125066, Abcam, Cambridge, USA), and GAPDH (1:1000, ab181603, Abcam, Cambridge, USA).

[0085] 1.14 RNA ac4C Dot Blot

[0086] Total RNA was extracted from the venous blood vessels and denatured at 95 °C for 3 minutes. 5 μg of RNA was loaded onto a nylon membrane and crosslinked at 37 °C for 30 minutes. After blocking, the membrane was incubated overnight at 4 °C with anti-ac4c antibody (ab252215, 1:500, Abcam, Cambridge, USA). After incubation with horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (1:4000, zgb-bio, Beijing, China), the membrane was imaged using a Tanon 4800 multi-chemiluminescence imaging system (Tanon, Shanghai, China).

[0087] 1.15 RNA stability analysis

[0088] HUVECs were transfected with NC or si-NAT10s (sequences are shown in Table 1). Then, they were treated with actinomycin D (AbMole, Houston, USA) at a final concentration of 5 μg / ml. Cells were collected at 0, 2, 4, and 6 h respectively, and RNA was extracted for qRT-PCR.

[0089] Table 1 siRNA and NC sequences

[0090]

[0091] 1.16 Construction and generation of endothelial cell-specific NAT10 gene knockout mice

[0092] Endothelial cell-specific NAT10 gene knockout mice were purchased from Cyagen Biosciences (Suzhou, China). By mating NAT10 flox / flox Cdh5-Cre ERT2 mice with Cdh5-Cre ERT2 mice, tamoxifen (60 mg / kg) was intraperitoneally injected daily for one week. Endothelial cell-specific NAT10 conditional knockout mice (NAT10 flox / flox Cdh5-Cre ERT2 ) were established. The genotypes of NAT10 gene knockout mice were identified by PCR. The knockout efficiency of NAT10 flox / flox Cdh5-Cre ERT2 (NAT10 f / f Cdh5-Cre + ) mice was identified by Western blot.

[0093] 1.17 DVT mouse model and treatment

[0094] Wild-type mice (C57BL / 6J, 8-week-old males) were purchased from Beijing Huafukang Bioscience Co., Ltd. (Beijing, China). Animal experiments were approved by the Ethics Committee of Shandong University of Traditional Chinese Medicine. A DVT mouse model was constructed according to the inferior vena cava (IVC) stenosis method.

[0095] Ferroptosis inhibitor experiment: The mice were randomly divided into 3 groups (15 mice / group): (1) Sham operation group; (2) DVT group; (3) DVT + Ferrostatin-1 (Fer-1, ferroptosis inhibitor, aladdin) group. Fer-1 (5 mg / kg) was intraperitoneally injected continuously for 3 days before the inferior vena cava stenosis surgery in mice and continuously for 3 days after the surgery.

[0096] HMOX1 inhibition, the mice were randomly divided into 2 groups (15 / group): (1) DVT group; (2) DVT + zinc protoporphyrin IX (ZnPP, HMOX1 inhibitor, aladdin, China) group. ZnPP (10 mg / kg) was intraperitoneally injected 3 days before IVC stenosis and continuously for 3 days after the surgery.

[0097] NAT10 inhibition treatment experiment: The mice were randomly divided into 3 groups (15 mice / group): (1) Sham operation group; (2) DVT group; (3) DVT + Remodelin (NAT10 inhibitor, aladdin, China) group. The NAT10 inhibitor (10 mg / kg) was injected via the tail vein continuously for 3 days before the inferior vena cava stenosis and continuously for 3 days after the surgery.

[0098] In the HMOX1 overexpression experiment, the mice were randomly divided into 3 groups (15 mice / group): (1) DVT group (NAT10 f / f Cdh5-Cre + mice); (2) DVT nat10 knockout group (NAT10 f / f Cdh5-Cre + mice); (3) DVT nat10 gene knockout + cobalt protoporphyrin IX (CoPP, HMOX1 activator, aladdin, China) group. CoPP (15 mg / kg) was intraperitoneally injected continuously for 3 days to establish the inferior vena cava stenosis model and continuously for 3 days after the surgery.

[0099] Fresh specimens 2 mm below the inferior vena cava ligation site were sectioned for hematoxylin-eosin (H&E) analysis.

[0100] 1.8 Transmission electron microscopy (TEM) analysis

[0101] For the morphological characteristics of ferroptosis in vascular tissues, TEM images were taken with a Hitachi HT-7800 transmission electron microscope (Hitachi, Ibaraki, Japan).

[0102] 1.19 Mouse Doppler ultrasound

[0103] Mice were subjected to vascular Doppler ultrasound examination under isoflurane-oxygen mixed anesthesia. Thrombosis images were obtained using a small animal ultrasound imaging system (VINNO6 LAB, VINNO, Suzhou, China). After paraffin embedding of the sections, HE staining was performed according to the standard procedure.

[0104] 1.20 ELISA

[0105] Mouse ELISA kits were used to detect the protein expression of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1).

[0106] 1.21 Statistical analysis

[0107] Results are expressed as mean ± SEM, and comparisons were made using two-tailed Student's t-test or one-way ANOVA. Statistical analysis was performed using GraphPad Prism 8.0 software. Unless otherwise stated, all experiments had at least three independent replicates. A P value < 0.05 was considered statistically significant.

[0108] 2 Experimental results

[0109] 2.1 Inhibition of ferroptosis improves the formation of DVT

[0110] The correlation between differentially expressed genes upregulated by CHIP (P < 0.05, FC > 1.5) and 4D Label Free (P < 0.05, FC > 2) was analyzed by KEGG to clarify the physiological functions and pathogenesis of DVT. Among the top 10 significantly enriched pathways, we found that the ferroptosis-related pathway was significantly differentially enriched ( Figure 1 A, B). Similarly, the results showed mitochondrial shrinkage and reduced cristae in vascular endothelial cells of DVT mice ( Figure 1 C). The levels of Fe 2+ and MDA were significantly increased in DVT mice (Figure ID, E). In addition, the expression of GSH was decreased in DVT mice ( Figure 1 F). To further confirm the dominant role of ferroptosis in deep vein thrombosis, we treated mice by intraperitoneal injection of the ferroptosis inhibitor (Fer-1) before establishing the deep vein thrombosis model. As expected, the thrombus size was significantly reduced after Fer-1 treatment, and vascular endothelial injury was alleviated ( Figure 1 G-J). These results suggest that ferroptosis plays an important role in the formation of DVT.

[0111] 2.2 NAT10 exacerbates vascular endothelial ferroptosis and ac4C RNA modification in DVT

[0112] Total RNA ac4C acetylation quantified by dot blot was significantly increased in the vascular endothelial tissues of DVT ( Figure 2 A). Similarly, in the 4D Label Free experiment, NAT10, as the "writer" of ac4C synthesis, was significantly highly expressed in the vascular endothelial tissues of DVT mice ( Figure 2 B). The sample size was expanded for verification, and NAT10 was significantly increased in DVT mice ( Figure 2 C,D). To verify this hypothesis, we pretreated mice with a NAT10 inhibitor, which alleviated thrombosis (Figure S2A,B). To investigate the role of NAT10 in endothelial ferroptosis, by crossing NAT10 f / + mice with Cdh5-Cre ERT2 mice, endothelial cell-specific NAT10 knockout mice were established. As expected, NAT10 silencing significantly alleviated thrombosis ( Figure 2 E,F). Meanwhile, inhibiting NAT10 alleviated ferroptosis of endothelial cells in DVT mice ( Figure 2 G-I). Taken together, these results indicate that inhibiting NAT10 helps to alleviate the formation of ferroptosis in DVT.

[0113] 2.3 Downregulating NAT10 can increase the ability to inhibit ferroptosis in vitro

[0114] To further emphasize the role of NAT10 in endothelial ferroptosis, we performed a loss-of-function experiment in vitro. We induced ferroptosis in HUVECs cells using two different ferroptosis inducers (RSL3, FINO2). Inhibiting NAT10 significantly alleviated the decrease in cell viability induced by RSL3 / FINO2 ( Figure 3 A). In addition, after inhibiting NAT10, the ferroptosis morphology was significantly restored ( Figure 3 B). At the cellular level, we further investigated the changes related to ferroptosis. Downregulation of NAT10 led to a significant decrease in the levels of RSL3 / FINO2-induced LPO and Fe 2+ ( Figure 3 C-F). Meanwhile, we observed that the lack of NAT10 had a direct effect on other key markers of ferroptosis. Specifically, the absence of NAT10 decreased the expression of MDA ( Figure 3 G), and increased the expression of GSH induced by RSL3 / FINO2 in vitro ( Figure 3 H). Taken together, these data indicate that the lack of NAT10 significantly inhibits the occurrence of ferroptosis in vitro.

[0115] 2.4 NAT10 stabilizes HMOX1 by inducing ac4C modification of HMOX1

[0116] To investigate the potential mechanism by which NAT10 regulates vascular endothelial cell function, we performed RIP-seq on negative control (NC) and si-NAT10 in HUVECs. Sequence analysis of ac4C modification showed that the typical CXXCXXCXX motif was highly enriched within the ac4C sites ( Figure 4 A). Consistent with previous reports, we found that in both NC and si-NAT10, ac4C peaks were mainly located in the protein-coding region (CDS) of mRNA transcripts. Since NAT10 promotes ac4C modification and RNA stability, we selected the downregulated genes from 4D Label Free (FC>2, P<0.05), FerrDb V2 Database (Driver), and acRIP-seq (FC>2, P<0.05) for Venn diagram. The results showed that AGPS, LGMN, FAR1, and HMOX1 were screened from the overlapping region ( Figure 4 B). Among these four genes, HMOX1 was significantly upregulated in DVT mice ( Figure 4 C). After knocking down NAT10, the levels of HMOX1 mRNA and protein were significantly decreased ( Figure 4 D, E). Similarly, the results of acRIP-qPCR showed that the level of ac4c acetylation modification of HMOX1 mRNA was continuously decreased in si-NAT10 HUVECs ( Figure 4 F). We observed by RIP-qPCR that NAT10 could bind to HMOX1 mRNA ( Figure 4 G). More importantly, inhibition of NAT10 promoted the degradation of HMOX1 mRNA induced by actinomycin D ( Figure 4 H). Therefore, we speculate that NAT10 regulates ferroptosis by acetylating HMOX1 mRNA.

[0117] To explore the function of NAT10-mediated inhibition of HMOX1, knocking down NAT10 inhibited the decreased expression of HMOX1 in ferroptosis ( Figure 4 I-L). In addition, inhibition of NAT10 significantly decreased the expression of MDA in ferroptosis and promoted the increase of GSH ( Figure 4 M, N). Overall, NAT10 promotes the stability of HMOX1 mRNA through ac4C modification, thereby regulating endothelial cell function.

[0118] 2.5 Inhibition of NAT10 can alleviate endothelial dysfunction by inhibiting HMOX1

[0119] It has been reported that the upregulation of HMOX1 is the cause of increased ferroptosis in endothelial cells of diabetic patients

[26] . To confirm whether HMOX1 alters the formation of DVT, we injected ZnPP into mice to neutralize the function of HMOX1. HE staining and Doppler showed that the thrombus volume of DVT mice treated with ZnPP decreased synchronously ( Figure 5 A, B), and at the same time, the levels of Fe2t and MDA in DVT mice treated with ZnPP decreased ( Figure 5 C, D), and the expression of GSH in DVT mice treated with ZnPP increased ( Figure 5 E).

[0120] In addition, to further emphasize the role of HMOX1, we used CoPP (HMOX1 agonist) to increase the expression of HMOX1. The results showed that overexpression of HMOX1 could significantly inhibit the cell viability of HUVECs ( Figure 5 F). At the same time, overexpression of HMOX1 significantly increased the Fe 2+ level in vitro ( Figure 5 G). Notably, overexpression of HMOX1 would accumulate Fe 2+ , further exacerbating ferroptosis of endothelial cells. Inhibiting NAT10 could reduce the MDA content and increase the expression of GSH and GPX4, thus reducing ferroptosis of endothelial cells ( Figure 5 H-K). In summary, these data indicate that inhibiting NAT10 can reduce the expression of HMOX1, alleviate ferroptosis, and thus mitigate endothelial function injury.

[0121] 2.6 NAT10 alleviates DVT formation by regulating HMOX14

[0122] To verify the role of NAT10 in regulating HMOX1 to alter DVT formation, NAT10 knockout mice were injected with CoPP to activate HMOX1. According to the results of HE staining and Doppler ultrasound, we found that the thrombus in NAT10 knockout DVT mice decreased ( Figure 6 A, B). NAT10 silencing significantly reduced the expression of Fe 2+ and MDA, while the activation of HMOX1 instead increased ferroptosis ( Figure 6 C, D). At the same time, NAT10 silencing also increased the expression of GSH and GPX4, while the activation of HMOX1 had the opposite effect ( Figure 6 E, G). Notably, NAT10 silencing led to a decrease in the expression of HMOX1 ( Figure 6 F, G). Knockdown of NAT10 significantly reduced the levels of ET1 and TNF-α in DVT mice and increased the levels of eNOS and TGF-β1 ( Figure 6H-K). These data indicate that inhibiting NAT10 can reduce the expression of HMOX1, inhibit ferroptosis, thereby alleviating endothelial dysfunction and the formation of thrombi in DVT mice.

[0123] In summary, the current study found that compared with sham-operated mice, the expression of NAT10 was significantly upregulated in the venous vascular tissues of DVT mice and was positively correlated with the disease severity. Notably, inhibiting NAT10 could reduce thrombus formation in DVT mice. To emphasize the significance of NAT10, we constructed mice with endothelial cell NAT10 knockout to further explore the role of NAT10. Through this mouse model, we found that silencing NAT10 might be a feasible therapeutic intervention for the prevention and treatment of DVT. In addition, silencing NAT10 could reduce the formation of DVT by reducing ferroptosis of endothelial cells. Our study demonstrated for the first time that NAT10-mediated ac4C modification regulated the stability of HMOX1 mRNA and led to the abnormal upregulation of HMOX1 in DVT. Therefore, we speculate that NAT10 promotes ferroptosis by upregulating HMOX1, leading to iron overload and inhibiting the generation of lipid peroxides, providing support for our research data. These findings provide a new explanation for how NAT10-mediated ac4C modification functions in DVT. This study also found that HMOX1 promotes the release of free iron, leading to ferroptosis and ultimately promoting thrombus formation. Among them, we further found that the expression of HMOX1 was highly upregulated in DVT, and inhibiting HMOX1 could reduce the occurrence of thrombi. Therefore, further inhibiting the expression of HMOX1 by knocking down NAT10 can not only inhibit iron but also reduce lipid peroxidation, thereby further improving ferroptosis of endothelial cells. Therefore, targeting NAT10 may be a promising therapeutic strategy to enhance ferroptosis of endothelial cells.

[0124] Matters not covered by this invention are well-known techniques.

[0125] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and shall not be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall be covered within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression levels of NAT10 and its related biomolecules in the preparation of a product for detecting deep vein thrombosis; Among them, The related biomolecules of NAT10 include ac4C modification and HMOX1.

2. The application according to claim 1, wherein The product for detecting deep vein thrombosis is used for screening, (assistive) diagnosis, monitoring or prognosis of deep vein thrombosis.

3. The application according to claim 1, wherein The substances for detecting the expression levels of NAT10 and its related biomolecules include reagents for detecting the expression levels of the encoding genes of NAT10 and its related biomolecules based on real-time fluorescence quantitative PCR, in situ hybridization, gene chip and gene sequencing, and / or reagents for detecting the protein (enzyme) expression levels of NAT10 and its related biomolecules based on immunoassay methods; The product is a primer, probe, (gene or protein) chip, detection kit, detection device and detection equipment.

4. A system for detecting deep vein thrombosis, characterized in that, The system includes: An acquisition module configured to acquire the expression level conditions of NAT10 and its related biomolecules in a sample to be tested of a subject; An analysis module configured to analyze and judge the disease condition of the subject according to the expression level conditions of NAT10 and its related biomolecules of the subject obtained by the acquisition module; The related biomolecules of NAT10 include ac4C modification and HMOX1.

5. The system according to claim 4, wherein The sample to be tested is a blood sample of the subject, and further a mononuclear cell sample of peripheral blood.

6. The system according to claim 4, wherein The detection of deep vein thrombosis specifically refers to screening, (assistive) diagnosis, monitoring or prognosis of deep vein thrombosis.

7. Use of NAT10 and its related biomolecules as targets in the preparation and / or screening of drugs for deep vein thrombosis; Among them, The related biomolecules of NAT10 include ac4C modification and HMOX1.

8. Use of a substance that inhibits the expression of NAT10 or reduces its activity in any one or more of the following: (a) Inhibiting the expression of HMOX1 or preparing a product for inhibiting the expression of HMOX1; (b) Inhibiting ferroptosis and iron overload or preparing a product for inhibiting ferroptosis and iron overload; (c) Inhibiting the generation of lipid peroxides or preparing a product for inhibiting the generation of lipid peroxides; (d) A product for preventing and / or treating deep vein thrombosis.

9. The application according to claim 8, wherein The substances that inhibit the expression of NAT10 or reduce its activity include but are not limited to RNA interference molecules or antisense oligonucleotides targeting NAT10, small molecule inhibitors (including Remodelin), siRNA, shRNA, substances for implementing lentiviral infection or gene knockout, etc.

10. The application according to claim 8, wherein, In (a)-(c) above, the product acts on endothelial cells, and further on venous vascular endothelial cells; The product is a drug or an experimental reagent for non-medical use.

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