Death domain protein and application thereof in treatment of atherosclerosis

Abstract

The invention provides a death structural domain protein and application thereof in treatment of atherosclerosis. The important role of the death domain protein Daxx in regulation and control of atherosclerosis is determined through analysis of an atherosclerosis animal model and a cell model for the first time, and siRNA is constructed to carry out RNA interference on the Daxx, so that feasibility of treating atherosclerosis by using the Daxx is realized, an important supplement is provided for biological functions of the Daxx, and the application of the Daxx in treatment of atherosclerosis is realized. And a new thought is provided for early diagnosis, prevention and / or treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a death domain protein and an application thereof in treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is an inflammatory disease characterized by the gradual formation of atherosclerotic plaques in large and medium arteries. Worldwide, atherosclerosis is the main pathogenic factor for vascular diseases such as coronary heart disease and stroke, and thrombosis based on this is the main cause of death. It is well known that multiple processes are involved in the pathophysiological process of atherosclerosis. A large number of studies have shown that excessive accumulation of lipids in the cytoplasm of macrophages will cause macrophages to transform into destructive foam cells, causing instability, rupture and thrombosis of atherosclerotic plaques. Therefore, it is of great significance to study the role of macrophages in the stability of atherosclerotic plaques and the prevention and treatment of atherosclerosis.

[0003] With the development of molecular biology and genetics, more and more evidence shows that certain specific genes and proteins play a key role in the development of atherosclerosis. Among them, the discovery of death-domainassociated protein Daxx (Death-domainassociated protein) and its potential role in atherosclerosis have opened up a new perspective for the study of this disease. Daxx was originally identified as a protein involved in apoptosis signal transduction, with diverse functions, including regulating apoptosis, inflammatory response, DNA repair, gene transcription and other biological processes. Since its first discovery in 1994, the study of Daxx protein has gradually deepened, and its role in cardiovascular disease has also attracted widespread attention.

[0004] In the context of atherosclerosis, Daxx has been found to affect the progression of lesions through multiple pathways. On the one hand, Daxx can regulate the survival and apoptosis of vascular endothelial cells, smooth muscle cells, and macrophages, thereby affecting the stability and inflammatory state of plaques. On the other hand, Daxx is involved in the regulation of lipid metabolism and may indirectly promote or inhibit the formation of atherosclerotic plaques by affecting the uptake and efflux of cholesterol. In addition, Daxx may also play an important role in the inflammatory response of atherosclerosis by affecting the function of immune cells and regulating the production of inflammatory factors. Nevertheless, the exact mechanism of action of Daxx in atherosclerosis is still in the exploratory stage, and its potential as a potential diagnostic marker and therapeutic target needs to be further verified. Therefore, in-depth research on the function of Daxx protein in atherosclerosis and its regulatory mechanism is of great significance for understanding the development of the disease and developing new diagnostic methods and therapeutic strategies. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a death domain protein and its application in treating atherosclerosis. The present invention determines the important role of death domain protein Daxx in regulating atherosclerosis through analysis of atherosclerosis animal model and cell model, and constructs siRNA to interfere with Daxx, thus realizing the feasibility of using Daxx to treat atherosclerosis, providing an important supplement to the biological function of Daxx, and providing a new target for the diagnosis and / or treatment of atherosclerosis.

[0006] First, the present invention provides a death domain protein Daxx, characterized in that the sequence of the death domain protein Daxx is shown in SEQ ID NO.13.

[0007] Secondly, the present invention provides an interfering RNA of the death domain protein Daxx, characterized in that the interfering RNA is selected from one or a combination of several of SEQ ID NO.5-12.

[0008] Preferably, the interfering RNA is selected from a combination of SEQ ID NO. 5-6.

[0009] Preferably, the interfering RNA is selected from the combination of SEQ ID NO.7-8.

[0010] Preferably, the interfering RNA is selected from the combination of SEQ ID NO.9-10.

[0011] Furthermore, the present invention also provides the use of the death domain protein Daxx as a diagnostic marker and / or therapeutic target in the preparation of early diagnostic reagents, preventive and / or therapeutic drugs for atherosclerosis.

[0012] Preferably, the death domain protein Daxx is expressed at an increased level in subjects suffering from atherosclerosis.

[0013] Preferably, the early diagnosis reagent comprises qRT-PCR primers for quantitatively detecting the death domain protein Daxx gene, and the primer sequences are shown in SEQ ID NO.1-2.

[0014] Preferably, the preventive and / or therapeutic drug is an interfering RNA of the death domain protein Daxx, characterized in that the interfering RNA is selected from one or a combination of several of SEQ ID NO.5-10.

[0015] Furthermore, the present invention also provides the use of the interfering RNA in preparing a product for preventing and / or treating atherosclerosis.

[0016] Furthermore, another object of the present invention is to provide a pharmaceutical composition for treating atherosclerosis, characterized in that the pharmaceutical composition contains interfering RNA of the death domain protein Daxx.

[0017] The advantages of the present invention are as follows: the present invention selects ox-LDL to treat macrophages to simulate the pathological conditions of atherosclerosis in vitro, and through Western Blot and qRT-PCR experiments, in the animal model, compared with the control group (ND), the ApoE - / - Daxx expression is upregulated in mice. In the cell model, Daxx expression was upregulated in the ox-LDL-induced RAW264.7 atherosclerosis model compared with the control group. Therefore, the important role of Daxx in regulating atherosclerosis was discovered. The present invention constructs siRNA to perform RNA interference on Daxx in macrophages, and uses Western Blot and Oil Red O experiments to achieve the use of Daxx to treat atherosclerosis in a cell model. Therefore, the present invention provides an important supplement to the biological function of Daxx, provides a new intervention target for the treatment of atherosclerosis, and also provides a new technical means for the treatment of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .Results of Daxx mRNA expression in animal models of atherosclerosis.

[0019] Figure 2 .The results of Daxx mRNA expression in the RAW264.7 atherosclerosis model induced by ox-LDL.

[0020] Figure 3 .The results of Daxx protein expression in the RAW264.7 atherosclerosis model induced by ox-LDL.

[0021] Figure 4 .Representative graph of the relative expression levels of Daxx detected by Western Blot of three groups of siRNA in the RAW264.7 atherosclerosis model induced by ox-LDL.

[0022] Figure 5 .Representative graph of the relative expression of Daxx detected by Western Blot using si001 in the RAW264.7 atherosclerosis model induced by ox-LDL.

[0023] Figure 6 .The results of observing lipid accumulation in RAW264.7 macrophages in the ox-LDL-induced atherosclerosis model and after transfection with si001 through Oil Red O experiment. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0025] Example 1

[0026] Construction of atherosclerosis mouse model: 8-week-old male ApoE - / - Mice were divided into two groups according to body weight, with 5 mice in each group. Among them, the high-fat feeding group (HFD) was given a pro-atherosclerotic diet, and the control group (ND) was fed a normal basal diet for 12 weeks. After the modeling was completed, the mice were killed and blood and aorta were taken. RNAiso PLUS (Takara, Japan) was used to extract total RNA from aortic tissue, and RNA was reverse transcribed using a reverse transcription reagent (ABclonal, China) to obtain cDNA, and qRT-PCR was used to detect the expression of Daxx gene. Among them, the primer sequences used for qRT-PCR are as follows:

[0027] Daxx Gene:

[0028] The forward primer was 5′-ACCCAGACTCCTCGTATTTGC-3′ (SEQ ID NO. 1);

[0029] The reverse primer is 5'-TTCGCTGCTCTATGACCCG-3' (SEQ ID NO.2),

[0030] GAPDH gene:

[0031] The forward primer was 5′-AGGTCGGTGTGAACGGATTTG-3′ (SEQ ID NO. 3);

[0032] The reverse primer was 5'-TGTAGACCATGTAGTTGAGGTCA-3' (SEQ ID NO. 4).

[0033] The qRT-PCR amplification procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, extension at 72°C for 10 s, 40 cycles; and extension at 72°C for 10 min.

[0034] The results are as follows Figure 1 As shown: qRT-PCR experimental data showed that compared with the control group (ND), the ApoE - / - The expression of Daxx in mice was significantly upregulated (p value less than 0.001), which directly reflects that Daxx is a key regulatory gene involved in the process of atherosclerosis.

[0035] Example 2

[0036] To further analyze the role of Daxx in the development of atherosclerosis, the inventors continued to construct a cell model, the method steps included:

[0037] Mouse macrophage RAW264.7 cells (purchased from Pronocell) were cultured in complete medium containing 10% FBS (NEWZERUM, New Zealand) and 1% penicillin-streptomycin (Servicebio, China) at 37°C and 5% CO. 2 The cell model group (ox-LDL) was treated with ox-LDL (75ug / mL) and the control group (Control) was not treated with any drug. After 24 hours of incubation, RNA was extracted from each group, and the qRT-PCR detection system in Example 1 was used to analyze the expression of Daxx gene in the cell model.

[0038] At the same time, Western Blot was used to analyze the expression of Daxx protein in the RAW264.7 atherosclerosis model induced by ox-LDL (75ug / mL). The detailed method steps include: taking some cells 24 hours after transfection to extract protein for Western Blot experiment, lysing the cell samples with RIPA lysis buffer (containing 1% phosphatase regulator and 1% protease regulator), and separating the protein samples by electrophoresis in SDS-PAGE gel, and then performing conventional membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and finally developing with chemiluminescence, and using ImageJ software to analyze the gray value of the target protein. The antibodies used are as follows: anti-Daxx (#4533, CST, United States, 1:500), anti-GAPDH (A19056, ABclonal, China, 1:1000000)

[0039] qRT-PCR results Figure 2 As shown in Figure 2, compared with the control group, Daxx expression was upregulated in the ox-LDL-induced RAW264.7 atherosclerosis model (p value less than 0.001). Figure 3 As shown: Daxx was upregulated in the ox-LDL-induced atherosclerosis model (p value less than 0.001). The results of this cell model were consistent with those of the mouse model, which once again confirmed that Daxx is a key regulatory gene involved in the process of atherosclerosis.

[0040] Example 3

[0041] To further understand the regulatory effect of Daxx on atherosclerosis, the inventors continued to design three siRNAs (si001, si002, si003) and a control siRNA (si-NC) for Daxx. Taking the cell model in Example 2 as an example, the interference efficiency of different siRNAs was analyzed. The specific method steps include:

[0042] Transfection of si-Daxx: One day before transfection, subculture RAW264.7 cells to make the cell confluence 50%-60%. 30-60 minutes before transfection, add an appropriate amount of culture medium to the well plate (6-well plate: 1 ml / well), prepare transfection complex: take 6-well plate as an example, add 1 μL siRNA master solution (20nM) and 2.4 μL PowwerfectTMreagent to each well to dilute to 100 μL Transfection Buffer, set 5 parallel replicates for each group of siRNA. After standing for 15 minutes, add to the cell culture plate. After incubation at 37℃ with 5% incubator for 6 hours, replace with original cell growth medium and give ox-LDL (75 μg / mL) for 24 hours, then extract protein.

[0043] Western Blot analysis: The cell samples in step 1) were lysed with RIPA lysis buffer (containing 1% phosphatase regulator and 1% protease regulator), and the protein samples were separated by electrophoresis in SDS-PAGE gel, followed by conventional membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and finally developed by chemiluminescence, and the gray value of the target protein was analyzed using ImageJ software. The antibodies used are as follows: anti-Daxx (#4533, CST, USA, 1:500), anti-GAPDH (A19056, ABclonal, China, 1:1000000).

[0044] Among them, the si001 sequence information is:

[0045] Sense strand: 5'-GGAUCUCAUCUACAACUUUdtdt3' (SEQ ID NO.5)

[0046] Antisense strand: 5'-AAAGUUGUAGAUGAGAUCCdtdt-3' (SEQ ID NO.6)

[0047] The sequence information of si002 is:

[0048] Sense strand: 5'-GGUCCUAGCGGAAUGGCAUdtdt-3' (SEQ ID NO.7)

[0049] Antisense strand: 5'-AUGCCAUUCCGCUAGGACCdtdt-3' (SEQ ID NO.8)

[0050] The sequence information of si003 is:

[0051] Sense strand: 5'-GGAGGAGAUCAUCGUGCUUdtdt-3' (SEQ ID NO.9)

[0052] Antisense strand: 5'-AAGCACGAUGAUCUCCUCCdtdt-3' (SEQ ID NO.10)

[0053] The si-NC sequence information is:

[0054] Sense strand: 5'-UUCUCCGAACGUGUCACGUdtdt-3' (SEQ ID NO.11)

[0055] Antisense strand: 5'-ACGUGACACGUUCGGAGAAdtdt-3' (SEQ ID NO.12)

[0056] The results are as follows Figure 4 and 5 As shown: the three siRNAs (si001, si002, si003) designed by the present invention can effectively interfere with the expression of Daxx gene in the cell model, and it is determined that they have a targeted inhibitory effect on Daxx and the transfection is effective.

[0057] Among the three siRNAs, si001 (i.e., si-Daxx) has the best interference efficiency. The inventor then continued to use si-Daxx as an example to analyze its effect on lipid accumulation in the cell model using Oil Red O staining. The specific method steps include: taking some cells 36 hours after transfection from the well plate, discarding the culture medium, and washing twice with PBS. Fix with paraformaldehyde for 20 minutes, wash twice with PBS, rinse with 60% isopropanol for 10 seconds, incubate with Oil Red O working solution at room temperature in the dark for 30 minutes, add 60% isopropanol and let stand for 30 seconds, then wash with distilled water 3 times, stain the nucleus with hematoxylin for 1-2 minutes, add 0.01% NaOH for anti-blueing, add aqueous sealing agent to seal the slices, and observe under a microscope.

[0058] The results are as follows Figure 6 The experiment showed that compared with the control, lipid accumulation in the ox-LDL-induced RAW264.7 atherosclerosis model was obvious, and compared with the ox-LDL group, transfection with si-Daxx (i.e., si001) could reduce lipid accumulation in cells, thus realizing the feasibility of using Daxx to treat atherosclerosis in cell models.

[0059] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting Daxx in the preparation of a product for diagnosing atherosclerosis, characterized in that: The amino acid sequence of the protein Daxx is shown in SEQ ID NO.

13.

2. The use according to claim 1, characterized in that: The reagent is a primer.

3. The use according to claim 2, characterized in that: The sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. Use of an agent that inhibits Daxx in the preparation of a drug for treating atherosclerosis, characterized in that: The amino acid sequence of the protein Daxx is shown in SEQ ID NO.13; the reagent for inhibiting Daxx is siRNA, and the sequence of the siRNA is shown in SEQ ID NO.5 and SEQ ID NO.6.

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