CN118109575A - Application of NONO in slowing down vascular medial calcification by inhibiting BMP2 transcription - Google Patents

Abstract

The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of NONO in slowing down calcification of a blood vessel medium membrane by inhibiting transcription of BMP 2. The invention discovers that NONO expression is obviously reduced in calcified blood vessels of mice through research. Meanwhile, smooth muscle cell-specific NONO knockout mice are more susceptible to vascular media calcification, which is manifested by increased calcification and calcium deposition. Likewise, smooth muscle cell-specific NONO knockdown aggravates the degree of calcification of aortic annulus extracted from mice. In vitro, knockout of NONO aggravates high phosphorus-induced osteogenic differentiation and calcium deposition in vascular smooth muscle cells, while overexpression of NONO improves this phenomenon. Mechanically, NONO targets BMP2, inhibiting its gene transcription. The NONO is a novel negative regulation factor for calcification of the blood vessel media, so that the NONO has good potential practical application value.

Description

Application of NONO in slowing down vascular medial calcification by inhibiting BMP2 transcription

Technical Field

The invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of NONO in slowing down vascular media calcification by inhibiting BMP2 transcription.

Background technique

The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

99% of the calcium in the human body exists in the form of hydroxyapatite crystals in bone tissue. When hydroxyapatite crystals are deposited ectopically in the vascular system, it is called vascular calcification. With the change of people's lifestyle and the aging of the population, the incidence of vascular calcification continues to rise. It often occurs in people with diabetes, end-stage renal disease and the elderly. This type of calcification mainly occurs in the vascular media, which is called vascular media calcification. It may lead to increased vascular stiffness and increased pulse pressure, further causing reduced coronary perfusion and ventricular hypertrophy, increasing the risk of stroke, and ultimately increasing the mortality rate of cardiovascular disease. It is reported that the incidence of vascular calcification in patients with end-stage renal disease exceeds 20%, and increases with the deterioration of renal function. Adverse cardiovascular events caused by vascular calcification have become an important cause of death in patients with chronic kidney disease (CKD).

At present, it is generally believed that vascular calcification is an active pathological process that can be prevented and adjusted, involving the participation of multiple cells, such as smooth muscle cells, endothelial cells, macrophages and pericytes. Among them, the phenotypic transformation of vascular smooth muscle cells to osteoblasts is the most important intermediate link, which is mainly manifested by the decreased expression of smooth muscle cell contractile phenotype markers (such as α-SMA) and the upregulation of osteogenic phenotype markers (such as BMP2 and RUNX2). In addition, the apoptosis of smooth muscle cells and the release of matrix vesicles are all involved in the occurrence of vascular calcification.

NONO is an octamer binding protein without a POU domain. It is mainly located in the cell nucleus and is an important component of the parafollicle of the nuclear subunit. It has both DNA and RNA binding domains in structure. As an RNA and DNA binding protein, it participates in multiple steps of gene regulation, including transcriptional regulation, mRNA splicing, and DNA repair. Previous studies have found that NONO inhibits the expression of P4Hα1, a key enzyme in collagen maturation and secretion, indicating that NONO plays an important role in the remodeling of the extracellular matrix. Considering that vascular media calcification is closely related to the remodeling of the extracellular matrix, the relationship between NONO and vascular media calcification deserves further discussion.

Summary of the invention

In view of the deficiencies in the prior art, the present invention aims to provide an application of NONO in alleviating vascular media calcification by inhibiting BMP2 transcription.

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

The first aspect of the present invention provides the use of a reagent for detecting NONO gene and its expression product in the preparation of a product for (auxiliary) diagnosis, detection, monitoring or prediction of the progression of vascular medial calcification.

In the present invention, the expression product of the NONO gene may obviously be the corresponding NONO protein.

The product may be a test kit, a test device or an apparatus.

A second aspect of the present invention provides a system for (assisting) diagnosis, detection, monitoring or prediction of the progression of vascular medial calcification, the system comprising:

i) an acquisition module, the acquisition module comprising: a detection substance for determining the expression level of NONO in a subject, and;

ii) Analysis module: The analysis module comprises: analyzing and judging the disease condition of the subject according to the NONO expression level determined in i).

The third aspect of the present invention provides the use of a substance that inhibits the NONO gene and its expression product and/or reduces its activity in at least one of the following a1)-a5):

a1) Promoting aortic calcium deposition and osteogenic differentiation of vascular smooth muscle cells and/or preparing products that promote aortic calcium deposition and osteogenic differentiation of vascular smooth muscle cells;

a2) promoting high phosphorus-induced aortic ring calcification or preparing a product for high phosphorus-induced aortic ring calcification;

a3) Promoting osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus or preparing products that promote osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus;

a4) promoting the transcription of BMP2 gene, increasing the expression of BMP2 mRNA and protein, or preparing products that promote the transcription of BMP2 gene, increasing the expression of BMP2 mRNA and protein;

a5) Constructing vascular media calcification model cells and/or animals.

The fourth aspect of the present invention provides the use of a substance that promotes NONO gene and its expression product and/or increases its activity in at least one of the following b1)-b3):

b1) Alleviating high phosphorus-induced osteogenic differentiation and calcium deposition of vascular smooth muscle cells or preparing a product for alleviating high phosphorus-induced osteogenic differentiation of vascular smooth muscle cells;

b2) inhibiting BMP2 gene transcription, reducing the expression of BMP2 mRNA and protein, or preparing products that inhibit BMP2 gene transcription, reducing the expression of BMP2 mRNA and protein;

b3) preparing a drug for preventing and/or treating vascular media calcification.

A fifth aspect of the present invention provides a method for preventing and/or treating vascular medial calcification, the method comprising: administering to a subject a substance that promotes the NONO gene and its expression product and/or increases its activity.

Beneficial technical effects of one or more of the above technical solutions:

Through research, the above technical solution found that NONO expression was significantly reduced in the calcified blood vessels of mice. At the same time, smooth muscle cell-specific NONO knockout mice were more susceptible to vascular media calcification, as manifested by increased calcification and calcium deposition. Similarly, smooth muscle cell-specific knockout of NONO aggravated the calcification of aortic rings extracted from mice. In vitro, knockout of NONO aggravated high phosphorus-induced osteogenic differentiation and calcium deposition of vascular smooth muscle cells, while overexpression of NONO improved this phenomenon. Mechanistically, NONO targets BMP2 and inhibits its gene transcription, indicating that NONO is a new negative regulator of vascular media calcification.

In summary, the above technical solutions provide new mechanism research for the occurrence of vascular calcification and related diseases, and provide promising prevention and treatment strategies for vascular calcification, and therefore have good potential practical application value.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

Figure 1 shows that the expression level of NONO is reduced under calcification conditions in an embodiment of the present invention. a: Western blot was used to detect the protein expression of NONO in the aorta of mice in the sham operation (Sham) group and the 5/6 nephrectomy (5/6Nx) group. b: Western blot was used to detect the protein expression of NONO in the aorta of mice fed with a normal diet (Chow diet) group and the adenine diet (Adenine diet) group. c: Western blot was used to detect the protein expression of NONO in the aorta of mice injected with a solvent control (Vehicle) group and the vitamin D (VD) group. d: Western blot was used to detect the protein expression of NONO, α-SMA, BMP2, and RUNX2 in vascular smooth muscle cells under high phosphorus stimulation. e: qPCR was used to detect the mRNA expression of BMP2 in vascular smooth muscle cells under high phosphorus stimulation. f: qPCR was used to detect the mRNA expression of RUNX2 in vascular smooth muscle cells under high phosphorus stimulation. g: qPCR was used to detect the mRNA expression of α-SMA in vascular smooth muscle cells under high phosphorus stimulation. h: qPCR detection of NONO mRNA expression in vascular smooth muscle cells under high phosphorus stimulation. **P<0.01, ***P<0.001.

Figure 2 shows that the smooth muscle cell-specific knockout of NONO in the embodiment of the present invention aggravates the aortic calcium salt deposition in calcified mice. a, b: Alizarin red staining and Von kossa staining were used to detect the aortic calcium salt deposition in the control (Control) mice and NONO ^SMKO mice after sham surgery (Sham) and 5/6 nephrectomy (5/6Nx), respectively. c, d: Alizarin red staining and Von kossa staining were used to detect the aortic calcium salt deposition in the control (Control) mice and NONO ^SMKO mice after feeding the Chow diet group and the adenine diet (Adenine diet), respectively. e, f: Alizarin red staining and Von kossa staining were used to detect the aortic calcium salt deposition in the control (Control) mice and NONO ^SMKO mice after injection of the vehicle control group and vitamin D (VD), respectively. ***P<0.001.

FIG. 3 shows that the smooth muscle cell-specific knockout of NONO in the embodiment of the present invention aggravates the high phosphorus-induced aortic ring calcification. a: Schematic diagram of the process of isolating and culturing aortic rings and inducing calcification. b: Detection of calcium ion content in aortic rings isolated from control mice and NONO ^SMKO mice under high phosphorus stimulation. ce: Alizarin red staining and Von Kossa staining to detect calcium salt deposition in aortic rings isolated from control mice and NONO ^SMKO mice under high phosphorus stimulation. ***P<0.001.

Figure 4 shows that knocking out NONO in the embodiment of the present invention aggravates the osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus. a-e: Western blot detection of NONO, α-SMA, BMP2, and RUNX2 protein expression in the control (Control) and NONO knockout (NONO KO) groups under high phosphorus stimulation. f, g: qPCR detection of BMP2 and RUNX2 mRNA expression in the control (Control) and NONO knockout (NONO KO) groups under high phosphorus stimulation. h, i: Alizarin red staining detection of calcium salt deposition in the control (Control) and NONO knockout (NONO KO) groups under high phosphorus stimulation. *P<0.05, **P<0.01, ***P<0.001.

Figure 5 shows that overexpression of NONO aggravates osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus in an embodiment of the present invention. a-e: Western blot detection of NONO, α-SMA, BMP2, and RUNX2 protein expression levels in the control (Ad-GFP) and NONO overexpression (Ad-NONO) groups under high phosphorus stimulation. f, g: qPCR detection of BMP2 and RUNX2 mRNA expression levels in the control (Ad-GFP) and NONO overexpression (Ad-NONO) groups under high phosphorus stimulation. h, i: Alizarin red staining detection of calcium salt deposition in the control (Ad-GFP) and NONO overexpression (Ad-NONO) groups under high phosphorus stimulation. *P<0.05, **P<0.01, ***P<0.001.

Figure 6 shows that NONO binds to the BMP2 promoter through its carbon end (C-terminus) in an embodiment of the present invention to inhibit the transcription of the BMP2 gene. a: Dual luciferase reporter gene experiment to detect the effect of NONO overexpression on BMP2 transcription. b: Construction pattern diagram of NONO domain deletion mutant plasmid. c: Dual luciferase reporter gene experiment to detect the effect of NONO domain deletion mutant on BMP2 transcription. d: Dual luciferase reporter gene experiment to detect the effect of BMP2 promoter CATAAAT sequence deletion on NONO inhibition of its transcription. e: CHIP experiment to detect the binding of NONO to BMP2 DNA. *P<0.05,***P<0.001.

Detailed ways

It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. 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, it indicates the presence of features, steps, operations, devices, components and/or combinations thereof.

The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

In a typical embodiment of the present invention, there is provided the use of a reagent for detecting the NONO gene and its expression product in the preparation of a product for (auxiliary) diagnosis, detection, monitoring or prediction of the progression of vascular medial calcification.

The present invention has found through research that in calcification model mice, the expression of aortic NONO shows a decreasing trend. At the same time, in vitro, vascular smooth muscle cells are given high phosphorus stimulation, and the expression of NONO is detected by Western blot and qPCR. The results show that the expression level of NONO decreases in a time gradient manner; and through biological verification, it is shown that mice with NONO knockout are more prone to vascular media calcification, indicating that NONO can be used as a biomarker for vascular calcification.

In the present invention, the expression product of the NONO gene may obviously be the corresponding NONO protein.

The product may be a test kit, a test device or an apparatus.

In one or more specific embodiments of the present invention, a system for (assisting) diagnosis, detection, monitoring or prediction of the progression of vascular medial calcification is provided, the system comprising:

i) an acquisition module, the acquisition module comprising: a detection substance for determining the expression level of NONO in a subject, and;

ii) Analysis module: The analysis module comprises: analyzing and judging the disease condition of the subject according to the NONO expression level determined in i).

The subject may be a human or a non-human mammal, and the non-human mammal includes mice, rats, guinea pigs, gorillas, monkeys, etc.; in a specific embodiment of the present invention, the non-human mammal is a mouse.

In one or more specific embodiments of the present invention, there is provided the use of a substance for inhibiting the NONO gene and its expression product and/or reducing its activity in at least one of the following a1)-a5):

a1) Promoting aortic calcium deposition and osteogenic differentiation of vascular smooth muscle cells and/or preparing products that promote aortic calcium deposition and osteogenic differentiation of vascular smooth muscle cells;

a2) promoting high phosphorus-induced aortic ring calcification or preparing a product for high phosphorus-induced aortic ring calcification;

a3) Promoting osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus or preparing products that promote osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus;

a4) promoting the transcription of BMP2 gene, increasing the expression of BMP2 mRNA and protein, or preparing products that promote the transcription of BMP2 gene, increasing the expression of BMP2 mRNA and protein;

a5) Constructing vascular media calcification model cells and/or animals.

Wherein, the substance that inhibits the NONO gene and its expression product and/or reduces its activity may be a substance that knocks out the NONO gene;

The product may be an experimental reagent, which is used for basic research to study the mechanism of occurrence and development of vascular media calcification.

In one or more specific embodiments of the present invention, there is provided the use of a substance that promotes the NONO gene and its expression product and/or increases its activity in at least one of the following b1)-b3):

b1) Alleviating high phosphorus-induced osteogenic differentiation and calcium deposition of vascular smooth muscle cells or preparing a product for alleviating high phosphorus-induced osteogenic differentiation of vascular smooth muscle cells;

b2) inhibiting BMP2 gene transcription, reducing the expression of BMP2 mRNA and protein, or preparing products that inhibit BMP2 gene transcription, reducing the expression of BMP2 mRNA and protein;

b3) preparing a drug for preventing and/or treating vascular media calcification.

The product may be a drug or an experimental reagent, and the experimental reagent is used for basic research to study the relevant mechanisms of the occurrence and development of vascular media calcification.

The drug may also include at least one inactive drug ingredient; the inactive drug ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to common methods, the drug may be prepared into oral preparations, external preparations, suppositories, and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

The non-pharmaceutical active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards. No specific limitation is made here.

In another specific embodiment of the present invention, the substance that promotes the NONO gene and its expression product and/or enhances its activity may be a NONO overexpression adenovirus, which is not specifically limited herein.

In one or more specific embodiments of the present invention, a method for preventing and/or treating vascular medial calcification is provided, the method comprising: administering to a subject a substance that promotes the NONO gene and its expression product and/or increases its activity.

The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

Example

Materials and Methods:

1. Construction of smooth muscle cell-specific NONO knockout mice: NONO ^flox/flox mice and SM22-Cre transgenic mice were hybridized to obtain NONO ^flox/flox /Cre+ mice, i.e. smooth muscle cell-specific NONO knockout mice (NONO ^SMKO ).

2. Construction of mouse vascular media calcification model: 8-10 week old male NONO ^SMKO mice and their littermate control mice were selected, and vascular media calcification models were constructed by three methods: (1) 5/6 nephrectomy combined with high phosphorus diet: First, the upper 1/3 and lower 1/3 of the left kidney of the mouse were ligated and removed respectively; one week later, the entire right kidney of the mouse was removed; after the operation, the mouse was fed with normal maintenance feed for 1 week and then fed with feed containing 1.5% for 12 weeks. The mice in the sham operation group underwent the same surgical procedures, including kidney exposure, tissue stripping and incision suture, but the left and right kidneys were not ligated and removed, and they were fed with normal control feed throughout the whole process. (2) Adenine feeding method: The mice were fed with special feed containing different concentrations of adenine for 8 weeks. The control group mice were fed with normal control feed. (3) Subcutaneous injection of high-dose vitamin D method: The mice were given subcutaneous injection of vitamin D (500,000 IU/kg/day) for 4 days. The control group mice were injected with the same volume of control solvent.

3. Extract mouse aortic rings and induce their calcification: 8-10 week old male NONO ^SMKO mice and their littermate control mice were selected, their aortas were isolated in a sterile clean bench, and they were cut into 5 mm long aortic rings and cultured in DMEM. Subsequently, 2.6 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ high phosphorus solution was added to the culture medium to induce calcification of the aortic rings.

4. Extract mouse primary vascular smooth muscle cells and induce their osteogenic differentiation: 4-6 week old male NONO ^SMKO mice and their littermate control mice were selected, their thoracic aorta was isolated in a sterile clean bench and the adventitia was peeled off. The isolated thoracic aorta was then cut into 1 mm tissue blocks and cultured in DMEM. After smooth muscle cells grew out, 2.6 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ high phosphorus solution was added to induce osteogenic differentiation. The expression of vascular smooth muscle cell contractile marker α-SMA and osteogenic differentiation markers BMP2 and RUNX2 was detected by Western blot and qPCR.

5. Detection of calcium deposition: Alizarin red staining, Von Kossa staining and detection of calcium ion content show the calcium deposition in aortic tissue and vascular smooth muscle cells.

6. Dual luciferase reporter gene experiment: First, construct a plasmid containing the BMP2 promoter and the luciferin reporter gene, and then co-transfect it with the NONO overexpression plasmid or the empty vector plasmid. After 48 hours, detect the luciferase activity.

7. Chromatin immunoprecipitation (CHIP) experiment: First, collect the cells in the 15cm cell culture dish, add 1% formaldehyde for cross-linking, and then lyse them using CHIP lysis buffer. Add NONO antibody to the lysate and incubate overnight, then add protein G magnetic beads and incubate for 2 hours. Finally, use the extraction column to extract the precipitated DNA, and then use PCR combined with agarose gel electrophoresis to detect the binding of NONO and BMP2 DNA.

result:

1. The expression level of NONO decreases under calcification conditions: In vivo, we used 5/6 nephrectomy and adenine feeding to induce a mouse vascular media calcification model caused by chronic renal failure, and used subcutaneous injection of high-dose vitamin D to induce an acute aortic calcification model in mice. After the model was successfully induced, the mouse aortic tissue protein was extracted and the expression of NONO was detected by Western blot. The results showed that the expression of NONO in the mouse aorta in the three calcification models showed a decreasing trend. In vitro, vascular smooth muscle cells were stimulated with high phosphorus, and the expression of NONO was detected by Western blot and qPCR. The results showed that the expression level of NONO decreased in a time gradient manner.

2. Smooth muscle cell-specific knockout of NONO aggravates aortic calcium salt deposition and osteogenic differentiation of vascular smooth muscle cells in calcified mice: We constructed smooth muscle cell-specific NONO knockout mice and induced three models of vascular media calcification. Alizarin red and Von kossa staining results showed that the elastic fibers in the vascular media of the model group mice were broken and there was obvious calcium salt deposition, and this phenomenon was aggravated in smooth muscle cell-specific NONO knockout mice. Subsequently, the aortic tissue protein of mice was extracted and Western blot was performed. The results showed that the expression of α-SMA in the aorta of the model group mice decreased, and the expression of BMP2 and RUNX2 increased. This phenomenon was more obvious in the smooth muscle cell-specific NONO knockout group. The above results show that smooth muscle cell-specific NONO knockout mice are more likely to develop vascular media calcification, which is manifested by aggravated calcium deposition and aggravated osteogenic differentiation of vascular smooth muscle cells.

3. Smooth muscle cell-specific knockout of NONO aggravates high-phosphate-induced aortic ring calcification: We further extracted mouse aortic rings, cultured them in vitro, and induced calcification by high phosphorus. After 7 days of high-phosphate stimulation, the calcium ion content of the aortic ring tissue was detected. The results showed that high phosphorus increased the calcium ion content of the aortic ring, and NONO knockout further aggravated this phenomenon. Alizarin red and Von kossa staining results also showed that NONO knockout aggravated high-phosphate-induced calcium salt deposition in aortic ring tissue. The above results indicate that NONO knockout aggravates high-phosphate-induced aortic ring calcification.

4. NONO knockout aggravates high phosphorus-induced osteogenic differentiation and calcium deposition in vascular smooth muscle cells: Primary vascular smooth muscle cells from NONO ^SMKO mice and their littermates were extracted and stimulated with high phosphorus. Western blot results showed that high phosphorus reduced the expression of α-SMA and increased the expression of BMP2 and RUNX2. NONO knockout further reduced the expression of α-SMA and increased the expression of BMP2 and RUNX2. Alizarin red staining was performed further, and the results showed that NONO knockout aggravated high phosphorus-induced calcium salt deposition, as shown by the increase in the positive area and density of alizarin red in the NONO knockout group. The above results show that knocking out NONO in vitro aggravates high phosphorus-induced osteogenic differentiation and calcium deposition in vascular smooth muscle cells.

5. Overexpression of NONO reduces osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus: Primary vascular smooth muscle cells of wild-type mice were extracted and transfected with NONO overexpression adenovirus (Ad-NONO). Western blot detection was performed 3 days after high phosphorus stimulation. The results showed that high phosphorus reduced the expression of α-SMA and increased the expression of BMP2 and RUNX2, while NONO overexpression could increase the expression of α-SMA and decrease the expression of BMP2 and RUNX2. Further Alizarin red staining was performed, and the results also showed that NONO overexpression reduced calcium salt deposition induced by high phosphorus. The above results show that overexpression of NONO in vitro reduces osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus.

6. Mechanistically, NONO targets BMP2 and inhibits the transcription of the BMP2 gene: The results of the dual luciferase reporter gene experiment showed that the luciferase activity of the NONO overexpression group was significantly reduced, indicating that NONO inhibited the transcription of the BMP2 gene. Further studies have shown that NONO binds to the CATAAAT sequence of the BMP2 promoter through its carbon end (C-terminus), thereby inhibiting the transcription of the BMP2 gene, reducing the expression of BMP2 mRNA and protein, further inhibiting the osteogenic differentiation of vascular smooth muscle cells, and improving the pathological process of vascular calcification. Our results indicate that NONO is a new negative regulator of vascular media calcification, providing a potential target for the prevention and treatment of vascular media calcification.

The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Use of a reagent for detecting the NONO gene and its expression products for the preparation of a product for (aiding in) diagnosis, detection, monitoring or prediction of the progress of membrane calcification in a blood vessel.

2. The use according to claim 1, wherein the product is a test kit, a test device or an apparatus.

3. A system for (assisting) diagnosis, detection, monitoring or prediction of progression of membrane calcification in a blood vessel, the system comprising:

i) An acquisition module, the acquisition module comprising: a test substance for determining a level of expression of a NONO in a subject, and;

ii) an analysis module: the analysis module comprises: determining the disease condition of said subject based on said analysis of NONO expression levels determined in i).

4. The system of claim 3, wherein the subject is a human or non-human mammal.

5. Use of a substance that inhibits the NONO gene and its expression products and/or reduces its activity in at least one of the following a 1) to a 5):

a1 Promoting aortic calcium deposition and vascular smooth muscle cell osteogenic differentiation and/or preparing a product for promoting aortic calcium deposition and vascular smooth muscle cell osteogenic differentiation;

a2 Promoting or preparing a product of high phosphorus-induced aortic annular calcification;

a3 Promoting the osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus or preparing a product for promoting the osteogenic differentiation and calcium deposition of vascular smooth muscle cells induced by high phosphorus;

a4 Promoting the transcription of BMP2 gene, improving the expression of BMP2 mRNA and protein or preparing products for promoting the transcription of BMP2 gene and improving the expression of BMP2 mRNA and protein;

a5 A) constructing a vascular media calcification model cell and/or an animal.

6. The use according to claim 5, wherein the substance which inhibits the NONO gene and its expression product and/or reduces its activity is a substance which knocks out the NONO gene.

7. The use according to claim 5, wherein the product is an experimental reagent for use in basic research.

8. Use of a substance that promotes the NONO gene and its expression products and/or increases its activity in at least one of the following b 1) -b 3):

b1 Relieving the osteogenic differentiation of the vascular smooth muscle cells induced by high phosphorus and calcium deposition or preparing a product for relieving the osteogenic differentiation of the vascular smooth muscle cells induced by high phosphorus;

b2 Inhibiting BMP2 gene transcription, reducing BMP2 mRNA and protein expression, or preparing a product that inhibits BMP2 gene transcription, reducing BMP2 mRNA and protein expression;

b3 For the preparation of a medicament for the prevention and/or treatment of vascular intimal calcification.

9. The use according to claim 8, wherein the product is a pharmaceutical or experimental agent for use in basic research.

10. The use as claimed in claim 8, wherein the substance which promotes the expression of the NONO gene and its expression products and/or increases its activity is a NONO over-expressing adenovirus.