Application of ZC3H13 in the preparation of products for monitoring and preventing endothelial injury-induced venous thrombosis

Abstract

The present invention provides the application of ZC3H13 in the preparation of products for monitoring and preventing endothelial injury venous thrombosis, and belongs to the field of biomedical technology. The present invention establishes an endothelial injury venous thrombosis model, and through research, it is found that the decrease in the expression level of ZC3H13 has an important effect on venous endothelial damage. By constructing a ZC3H13 knockdown endothelial cell model, it is found that ZC3H13 downregulation causes endothelial damage by increasing DYRK1B expression and releases a series of procoagulant and prothrombotic molecules, such as TF, PAI‑1 and VWF, which accelerates venous thrombosis. It was subsequently found that after ZC3H13 was overexpressed in mice, the release of procoagulant and prothrombotic molecules after venous endothelial damage could be significantly improved, and the tendency of thrombosis could be reduced. The above studies reveal the mechanism of action and therapeutic effect of ZC3H13 in venous endothelial cells, and provide new targets and strategies for the diagnosis and prevention of venous endothelial injury venous thrombosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of ZC3H13 monitoring products in the preparation of screening, diagnosis, auxiliary diagnosis and / or monitoring products for venous thrombosis, and the application of ZC3H13 overexpression reagents in the preparation of drugs for the prevention and treatment of endothelial injury-induced venous thrombosis. Background Art

[0002] Venous thrombosis is a disorder of venous return caused by abnormal blood coagulation, which can lead to serious complications such as pulmonary embolism in the acute phase and post-thrombotic syndrome in the late phase. Venous thrombosis can also complicate the course of other diseases and impose a considerable burden on the global healthcare system and economy. Vascular endothelial damage, as one of the three factors affecting thrombosis, greatly affects the thrombosis process.

[0003] The venous endothelium not only serves as a barrier between blood and tissues, but is also a dynamic, heterogeneous, and distributed organ with important secretory, synthetic, metabolic, and immune functions. Under physiological conditions, the endothelium provides a natural anticoagulant and antithrombotic environment. When exposed to repeated or long-term stimulation, the venous endothelium becomes dysfunctional and eventually loses its protective function after sustained damage. Factors such as blood flow disturbances and internal and external stimulations can damage and activate the venous endothelium. Vascular endothelial injury may be at the core of thrombotic events and is considered an early predictor of subsequent cardiovascular events. Injured venous endothelial cells will transform to a prothrombotic phenotype by increasing the expression of procoagulants and prothrombotic molecules. In addition, although current anticoagulant therapy can effectively prevent venous thrombosis, there is still a significant risk of bleeding. Therefore, based on the biological role of the venous endothelium in venous thrombosis, exploring its potential molecular mechanisms and finding new potential therapeutic targets are of great scientific significance and clinical value.

[0004] M6A methylation is a new epigenetic modification and the most common and abundant modification in RNA post-transcriptional modification. It is involved in almost all stages of eukaryotic metabolism and plays a role in mRNA stability, degradation, splicing, nuclear export and translation efficiency. M6A methylation is a reversible process that requires a series of proteins for complex and delicate regulation. Methyltransferases and demethylases help catalyze and remove m6A modifications on RNA to maintain the dynamic balance of m6A modifications. m6A binding proteins play a role by recognizing the dynamic m6A modifications of RNA.

[0005] ZC3H13 was recently identified as a new m6a regulator that interacts with WTAP to fix the methyltransferase complex in the nucleus. ZC3H13 loss leads to an approximately 80% reduction in m6A modification levels in mammalian cells. ZC3H13 is abnormally expressed in cancer, and its role in cancers such as breast cancer, pancreatic cancer, papillary thyroid cancer, and cervical cancer has been studied in recent years. Recently, some studies have found that abnormal m6A modification is involved in mediating endothelial inflammation and pyroptosis in atherosclerosis. However, the role of m6A modification involved in ZC3H13 in regulating endothelial damage-induced venous thrombosis remains unknown. Summary of the invention

[0006] In view of the problem that the existing anticoagulant therapy still has a significant risk of bleeding when treating venous thrombosis and the shortcomings of the prior art, the purpose of the present invention is to provide a new therapy targeting endothelial cells for the prevention and treatment of endothelial injury-induced venous thrombosis. The present invention establishes an endothelial injury-induced venous thrombosis model, and through research, it is found that the decrease in the expression level of ZC3H13 has an important effect on venous endothelial injury. By constructing an endothelial cell model with ZC3H13 knockdown, it is found that ZC3H13 downregulation causes endothelial injury by increasing the expression of DYRK1B and releasing a series of procoagulant and prothrombotic molecules, such as TF, PAI-1 and VWF, which accelerates venous thrombosis. It was subsequently found that after ZC3H13 overexpression in mice, the release of procoagulant and prothrombotic molecules after venous endothelial injury can be significantly improved, reducing the tendency of thrombosis. The above study reveals the mechanism of action and therapeutic effect of ZC3H13 in venous endothelial cells, and provides new targets and strategies for the diagnosis and prevention of venous endothelial injury-induced venous thrombosis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide the use of a monitoring product of ZC3H13 in the preparation of a screening, diagnosis, auxiliary diagnosis and / or monitoring product for venous thrombosis.

[0009] Furthermore, the venous thrombosis is endothelial damage venous thrombosis.

[0010] Furthermore, in the application, downregulation of ZC3H13 accelerates endothelial injury-induced venous thrombosis, and overexpression of ZC3H13 reduces the tendency of thrombosis.

[0011] The second object of the present invention is to provide the use of a monitoring product of DYRK1B in the preparation of a product for screening, diagnosis, auxiliary diagnosis and / or monitoring endothelial injury-induced venous thrombosis, wherein DYRK1B is a downstream expression product of ZC3H13.

[0012] Furthermore, downregulation of ZC3H13 increases DYRK1B expression, while overexpression of ZC3H13 inhibits DYRK1B expression. Therefore, overexpression of DYRK1B accelerates endothelial injury-induced venous thrombosis, while downregulation of DYRK1B reduces the tendency of thrombosis.

[0013] The third object of the present invention is to provide the use of ZC3H13 overexpression reagent in the preparation of drugs for preventing and treating endothelial injury-induced venous thrombosis.

[0014] The fourth object of the present invention is to provide the use of a DYRK1B inhibitory agent in the preparation of a drug for preventing and treating endothelial injury-induced venous thrombosis.

[0015] Furthermore, the Z3H13 overexpression reagent and the DYRK1B inhibition reagent include a vector that overexpresses ZC3H13.

[0016] The beneficial effects of the present invention are:

[0017] 1. In addition to the existing means of detecting vascular endothelial function, the present invention proposes ZC3H13 as a new indicator for detecting vascular endothelial damage, thereby achieving the purpose of assisting in the comprehensive assessment of vascular endothelial function.

[0018] 2. Existing anticoagulant therapy still faces the risk of bleeding when treating venous thrombosis. The present invention proposes that overexpression of ZC3H13 can significantly improve venous endothelial function and reduce the tendency of thrombosis. Therefore, the present invention provides a new target and strategy for the clinical prevention and treatment of endothelial injury-induced venous thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression of TF, PAI-1 and VWF in different tissues, cells and patient plasma in each treatment group in Example 1 of the present invention;

[0020] Figure 2 The expression of ZC3H13 in different tissues, cells and patient plasma in each treatment group in Example 1 of the present invention;

[0021] Figure 3 This is the process of inferring that DYRK1B is a potential target molecule of ZC3H13 in Example 1 of the present invention;

[0022] Figure 4 The expression of DYRK1B in different tissues, cells and patient plasma in each treatment group in Example 1 of the present invention;

[0023] Figure 5 This is the effect of endothelial-targeted ZC3H13 overexpression on venous endothelial injury in vivo in Example 2 of the present invention. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention are within the scope of the present invention. The reagents, products and instruments used in the following examples can all be obtained commercially, and the methods used are consistent with the conventional methods unless otherwise specified.

[0025] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0026] Example 1

[0027] 1. Establishment of Endothelial Injury Venous Thrombosis Model

[0028] Animal model: All animal experiments were approved by the Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology (

[2023] S3644). A well-established model of inferior vena cava stenosis was established in 8-week-old male C57BL / 6J mice. Mice were anesthetized continuously with an isoflurane mixture and placed in the supine position. After a midline laparotomy, the intestines were placed on sterile gauze and soaked in sterile saline to prevent drying during the entire procedure. After the inferior vena cava was slightly separated from the surrounding tissue and aorta, the inferior vena cava was ligated with 7-0 polypropylene suture on a 30-gauge needle. The needle was then removed and blood flow was restored, resulting in inferior vena cava stenosis (~90%). All renal vein side branches were ligated. The inferior vena cava was then clamped twice with a neurovascular clamp for 15 to 20 seconds each time to induce endothelial injury. The peritoneum and skin were closed separately. Mice were allowed to recover from the surgery. After 48 hours of treatment, the mice were euthanized, and thrombi and 2-mm vascular tissue sections at the ligation site of the inferior vena cava were collected.

[0029] Cell model: HUVECs were treated with thrombin (8 IU / ml) for 12 h to induce an endothelial injury model.

[0030] 2. Methods used to detect differences in m6A levels between different groups of tissues and cells

[0031] M6Adot blot: RNA samples were boiled in a 95°C metal bath for 3 minutes, placed on ice for 1 minute, cooled quickly, and the samples were dotted on a nylon membrane. Then, the RNA was bound to the nylon membrane using UV crosslinking. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated with the mA primary antibody at 4°C overnight. Finally, the membrane was incubated with the secondary antibody for 1 hour at room temperature, and the signal was detected using an ECL kit. Another membrane was stained with methylene blue as a control. Methylene blue stained membranes were used to ensure consistency between groups.

[0032] Total RNA m6A quantification: The overall m6A level of mRNA was assessed using the EpiQuik m6A RNA methylation quantification kit according to the kit instructions. Briefly, 200 ng of RNA sample was applied to the analysis wells. Then, the capture antibody solution and the detection antibody solution were added to the measurement wells at appropriate dilution concentrations. The m6A level was quantified by measuring the absorbance at 450 nm and calculating based on the standard curve.

[0033] 3. Methods used in the confirmation of ZC3H13 downstream targets

[0034] Transcriptome sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq): A total of 6 samples, 3 replicates in each group for RNA extraction. RNA-seq and MeRIP-seq were performed by Yunxu Biotechnology. For RNA-seq, rRNA was removed from the total RNA. Then, according to the reagent instructions, the Low Input RNAlibrary Prep Kit was used to construct the library for the rRNA-removed samples. The library was quality controlled and quantified using the BioAnalyzer 2100 system. Library sequencing was performed on the IlluminaNovaseq instrument with an end read pairing length of 150bp. For MeRIP-seq,: RNA was randomly fragmented into fragments of approximately 200nt. ProteinA / G magnetic beads were incubated with m6A antibodies at room temperature for 1 hour with rotation to allow the antibody to bind to the magnetic beads. The RNA fragments were then incubated with the antibodies bound to the magnetic beads at 4 degrees for 4 hours with rotation to allow the RNA to bind to the antibody. The bound complex was washed several times, and the RNA was further eluted from the complex. Use The RNA sequencing library was constructed using the Low Input Whole RNA Library Prep Kit. The constructed library was quality controlled using an Agilent 2100 bioanalyzer and then subjected to high-throughput sequencing on an Illumina NovaSeq sequencer.

[0035] 4. Detection method of ZC3H13 and its downstream molecule expression levels

[0036] The changes in the expression levels of ZC3H13 and its downstream molecules in different tissues, cells, and patient plasma were detected by qRT-PCR, WB, immunohistochemistry, immunofluorescence and ELISA.

[0037] 5. Results and Analysis

[0038] (1) Changes in the expression levels of TF, PAI-1, and VWF in different tissues, cells, and patient plasma in each treatment group Figure 1As shown. (A) Representative H&E staining images of vascular cross-sections of the control group, sham operation group, and IVC model group. Scale bar, 100 μm. (B) RT-qPCR analysis of TF, PAI-1, and VWF mRNA expression in the vascular tissue of the inferior vena cava of mice in the control group, sham operation group, and IVC model group (n=6). (C) Western blot analysis of TF, PAI-1, and VWF protein expression in the vascular tissue of the inferior vena cava of each group (n=3). (D) RT-qPCR analysis of TF, PAI-1, and VWF mRNA expression in HUVECs of the control group and thrombin treatment group (n=3). (E) Western blot analysis of TF, PAI-1, and VWF protein expression in HUVECs of each group.

[0039] Figure 1 It was shown that damaged venous endothelial cells expressed increased levels of pro-coagulant and pro-thrombotic molecules.

[0040] (2) Changes in the expression levels of ZC3H13 in different tissues, cells and patient plasma in each treatment group Figure 2 As shown. (A) Dot blot analysis of m6A levels in the inferior vena cava vascular tissues of mice in the control group, sham operation group and IVC model group (n=6). (B) Quantitative analysis of the relative levels of m6A mRNA in the inferior vena cava vascular tissues of each group (n=6). (C) Dot blot analysis of mm6A levels in HUVECs in the control group and thrombin treatment group. (D) Quantitative analysis of the relative levels of m6A mRNA in HUVECs in each group (n=3). (E) RT-qPCR analysis of the levels of “writer” and “eraser” mRNA in the inferior vena cava tissues of the control group, sham operation group and IVC group (n=6). (F) RT-qPCR analysis of the mRNA levels of six m6A-related enzymes in HUVECs in the control and thrombin treatment groups (n=3). (G) Western blot analysis of ALKBH5 and ZC3H13 protein expression in HUVECs in the control and thrombin treatment groups. (H) Western blot analysis of ALKBH5 and ZC3H13 protein expression in the vascular tissue of the inferior vena cava of mice in the control group, sham operation group, and IVC model group (N=3). (I) Representative immunofluorescence staining images of the vascular tissue of the inferior vena cava of the control group, sham operation group, and IVC model group. Cell nuclei (DAPI, blue), CD31 (green), and ZC3H13 (red). Scale bar, 50 μm. (J) Expression of ZC3H13 in peripheral blood plasma of healthy adults and patients with DVT.

[0041] Figure 2 The results showed that ZC3H13 expression was downregulated in injured venous endothelial cells.

[0042] (3) The process of inferring DYRK1B as a potential target molecule of ZC3H13 Figure 3 As shown. (A) Volcano plot analysis of differentially expressed mRNAs between sh-NC and sh-ZC3H13 groups (|Fold Change|≥2, p<0.05, red dots: mRNA up-regulated, blue dots: mRNA down-regulated, gray dots: mRNA no differential expression). (B) Analysis of m6A modification peaks in sh-NC and sh-ZC3H13 groups. (C) Distribution of m6A peaks in transcripts in sh-NC and sh-ZC3H13 groups. (D) Consensus m6A motifs identified by HOMER in HUVECs with or without ZC3H13 knockdown. (E) GO-BP enrichment analysis showed that m6A site methylation levels were downregulated after ZC3H13 knockdown. (F) The intersection of m6A peaks downregulated after ZC3H13 knockdown and differentially expressed genes identified by RNA sequencing revealed that DYRK1B is a target molecule of ZC3H13. (G) The m6A modification of DYRK1B mRNA after ZC3H13 knockout was visualized and analyzed by IGV software.

[0043] Figure 3 Merip-seq and RNA-seq identified DYRK1B as a potential target molecule of ZC3H13.

[0044] (4) Changes in the expression level of DYRK1B in different tissues, cells and patient plasma in each treatment group Figure 4 As shown. (A) Western blot analysis of DYRK1B protein expression in HUVECs before and after ZC3H13 knockout. (B) RT-qPCR analysis of DYRKK1B mRNA levels in HUVECs in the control and thrombin-treated groups (n=3). (C) Western blot analysis of DYRK1B protein expression in HUVECs in each group. (D) Western blot analysis of DYRK1B protein expression in the inferior vena cava vascular tissue of the mouse control group, sham operation group and IVC model group (n=3). (E) RT-qPCR analysis of DYRK1B mRNA expression in the inferior vena cava vascular tissue of each group (n=6). (F) Representative immunohistochemical staining images of the inferior vena cava vascular tissue of the control group, sham operation group and IVC model group. Scale bar, 50μm. (G) The direct binding of mA antibody to DYRK1B mRNA was verified by agarose electrophoresis in HUVECs. MeRIP-qPCR detected the relative enrichment of DYRK1B mRNA MeRIP after ZC3H13 knockdown in HUVECs (n=3).

[0045] Figure 4 It was shown that DYRK1B expression was upregulated in damaged venous endothelial cells.

[0046] Example 2

[0047] The purpose of this example is to detect the therapeutic effect of overexpression of ZC3H13 on endothelial injury-induced venous thrombosis in vivo. The specific method is as follows: an adenovirus carrying the CDS region of ZC3H13 (transcript: NM_026083.2) (ADV-Tie1-ZC3H13) and an adenovirus carrying an empty vector (ADV-Tie1-null) were constructed using the endothelial cell-specific promoter tie1 and the red fluorescent protein gene mCherry as vectors. Five days before the establishment of the inferior vena cava stenosis model, 8-week-old male C57BL / 6J mice were injected with 2×10 10 PFUADV-Tie1-ZC3H13 or ADV-Tie1-null, the injection volume was 100 μl. Then, the mice were modeled. After 48 hours, the inferior vena cava vascular tissue of the mice was taken, and the expression of ZC3H13 in the mice was detected by qRT-PCR, WB, immunohistochemical staining, etc., and the length and weight of thrombus emboli were measured to compare the difference in thrombosis tendency.

[0048] Test results such as Figure 5 As shown. (A) Schematic diagram of ADV-tie1-ZC3H13 or ADV-tie1-null injection in mice. (B) Western blot analysis of the expression of ZC3H13 and DYRK1B proteins in the vascular tissue of the inferior vena cava of mice after treatment. (C) Representative immunofluorescence staining images of the vascular tissue of the inferior vena cava in each group. Cell nucleus (DAPI, blue), CD31 (green), ZC3H13 (red). Scale bar, 50μm. (D) Representative H&E staining images of blood vessels in each group. Scale bar, 100μm. (E) Images of venous thrombosis 48h after the establishment of the IVC model in each group (n=8). (F, G) The weight (F) and length (G) of the thrombus in each group were measured.

[0049] Figure 5 showed that endothelial-targeted ZC3H13 overexpression could alleviate venous endothelial injury in vivo.

[0050] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of ZC3H13 monitoring products in the preparation of screening, diagnosis, auxiliary diagnosis and / or monitoring products for endothelial injury-induced venous thrombosis.

2. The use according to claim 1, characterized in that: In the application, downregulation of ZC3H13 accelerates endothelial injury-induced venous thrombosis, and overexpression of ZC3H13 reduces the tendency of thrombosis.

3. Application of the monitoring product of the ZC3H13 target molecule in the preparation of screening, diagnosis, auxiliary diagnosis and / or monitoring products for endothelial injury-induced venous thrombosis, characterized in that: The ZC3H13 target molecule is DYRK1B.

4. The use according to claim 3, characterized in that: In the application, downregulation of ZC3H13 increases DYRK1B expression, thereby accelerating endothelial injury-induced venous thrombosis; overexpression of ZC3H13 inhibits DYRK1B expression, thereby reducing the tendency of thrombosis.

5. Application of ZC3H13 overexpression reagent in the preparation of drugs for the prevention and treatment of endothelial injury-induced venous thrombosis, characterized in that: The ZC3H13 overexpression reagent includes an adenovirus carrying the ZC3H13 CDS region constructed with the endothelial cell-specific promoter tie1 and the red fluorescent protein gene mCherry as a vector, and the ZC3H13 transcript is NM_026083.2.

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