Use of qki gene in preparation of drugs for preventing and treating vascular calcification

Abstract

The present application relates to the application of QKI gene in the preparation of drugs for preventing and treating vascular calcification. The present application discloses the important role of QKI gene in regulating vascular calcification by constructing smooth muscle specific QKI knockout mice. Compared with the control mice, the QKI knockout mice significantly reduce the occurrence of vascular calcification, including the decrease of Runx2 gene expression level, the significant reduction of vascular calcium salt deposition and the decrease of vascular tissue calcium ion. In the in vitro test, it is found that QKI siRNA makes the alizarin red staining of mouse vascular smooth muscle cells under high phosphorus stimulation weaken, the calcium content decreases, and the vascular smooth muscle cell calcification is reduced. It is found that the present application takes QKI gene as the action target to prepare or screen the drug for slowing down the vascular calcification, and the drug has important significance in the treatment of vascular calcification.

Description

TECHNICAL FIELD

[0001] The application relates to application of a QKI gene in preparation of a drug for preventing and treating vascular calcification, and belongs to the field of biological medicines. BACKGROUND

[0002] Vascular calcification is the deposition of hydroxyapatite mineral in the arterial wall, which is a serious vascular disease and is related to atherosclerosis, chronic kidney disease (CKD) and metabolic diseases. Coronary artery calcification accelerates the occurrence of serious cardiovascular events. Epidemiological surveys have found that more than 70% of people over 60 years old in China have different degrees of aortic calcification. Vascular calcification involves multiple mechanisms. It was once considered to be a passive process, but now it is considered to be a dynamic process similar to bone mineralization. Multiple signaling pathways are involved in the development of vascular calcification. No effective drug for preventing and treating vascular calcification has been found in clinical practice. Therefore, it has great scientific significance to further clarify the mechanism of vascular calcification and find potential intervention targets.

[0003] Smooth muscle cells exist in various tissue types, including blood vessels, bronchi, irises, bladders and digestive tracts, and have plasticity. Under normal circumstances, vascular smooth muscle cells are of a contractile phenotype and express a series of contractile proteins such as alpha-smooth muscle actin, smooth muscle myosin heavy chain, SM22 alpha and calponin. However, after being stimulated, the vascular smooth muscle cells will undergo a phenotypic switch, de-differentiate from the contractile type to the synthetic type and can maintain a series of phenotypes, which can show the characteristics of osteoblasts, chondrocytes, adipocytes and foam cells. This phenotypic switch is considered to be an important pathogenesis of cardiovascular diseases such as vascular calcification, abdominal aortic aneurysm and atherosclerosis. During the osteogenic transdifferentiation of vascular smooth muscle cells, transcription factors such as Runx2 can increase the expression of bone-related proteins, including alkaline phosphatase, osteocalcin, osteonectin and bone sialoprotein, so as to promote the differentiation of vascular smooth muscle cells into osteogenic chondrocytes and thus promote vascular calcification.

[0004] QKI belongs to the STAR family and has three main subtypes: QKI-5, QKI-6 and QKI-7. They have the same N-terminal which is encoded by exons 1 to 6, and the C-terminal which is encoded by exons 7 and 8. The nuclear localization signal is located at the C-terminal. QKI can bind to target RNA by recognizing specific RNA sequences and plays a role in post-transcriptional regulation such as pre-mRNA alternative splicing, RNA stability and transport, and protein translation. In recent years, research has found that QKI plays a role in diseases such as myelin disease, cancer and schizophrenia. QKI can also regulate cardiac myofiber generation and contraction function, but its role in vascular calcification is not clear.

[0005] Some genes are knocked out systemically, which leads to embryonic or early postnatal lethality, and cannot be followed up, while conditional gene knockout can be used to study the function of genes in specific cell types of adult mice. The target gene is inserted into a recombinase recognition (loxP) site, and the loxP sites are placed in the intron region in the same direction, so that they are located on both sides of at least one exon of the target gene. Tamoxifen is injected to induce Cre-ER T2 recombinase activity, Cre mediates the excision of the loxP flanking region, and the coding fragment is deleted from the genome of the cell expressing the recombinase, after the excision of the exon, the mutant gene becomes a non-functional protein, and subsequent studies are carried out. SUMMARY

[0006] In view of the deficiencies of the prior art, the application provides the application of QKI gene in the preparation of drugs for preventing and treating vascular calcification.

[0007] The technical scheme of the application is as follows:

[0008] The application of QKI gene in the preparation of drugs for preventing and treating vascular calcification, the nucleotide sequence of the QKI gene is shown in SEQ ID No. 1.

[0009] According to the application, the application of QKI gene in the preparation of drugs for preventing and treating vascular calcification includes the following two aspects:

[0010] (1) QKI gene is used as a target for the preparation of drugs for preventing and treating vascular calcification;

[0011] (2) QKI gene is used as a target for the screening of drugs for preventing and treating vascular calcification.

[0012] According to the application, the application of QKI gene as a target for the preparation of drugs for preventing and treating vascular calcification means that QKI gene is used as a target of drugs or preparations to interfere with the inhibition of QKI gene, and drugs or preparations for preventing and treating vascular calcification are prepared, and the prepared drugs or preparations can efficiently and specifically interfere with the transcription or translation of QKI gene of smooth muscle cells, or can efficiently and specifically reduce the expression or activity of QKI protein of smooth muscle cells.

[0013] According to the application, the application of QKI gene as a target for the screening of drugs for preventing and treating vascular calcification means that QKI gene is used as a target of drugs or preparations to interfere with the inhibition of QKI gene, and drugs or preparations for preventing and treating vascular calcification are screened, and the screened drugs or preparations can efficiently and specifically interfere with the transcription or translation of QKI gene of smooth muscle cells, or can efficiently and specifically reduce the expression or activity of QKI protein of smooth muscle cells.

[0014] According to the application, the drug for preparing or screening the vascular calcification includes, but is not limited to, nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or viruses.

[0015] Further preferably, when the drug for vascular calcification is a nucleic acid molecule, the nucleic acid molecule includes, but is not limited to, antisense oligonucleotides, double-stranded RNA, small interfering RNA or short hairpin RNA. The nucleic acid molecule can interfere with, inhibit or silence the QKI gene, reduce the expression or activity of QKI protein in smooth muscle cells, and thus reduce the vascular calcification.

[0016] Further preferably, when the drug for vascular calcification is a virus, the virus contains a nucleotide sequence interfering with the expression of the QKI gene. The virus can interfere with, inhibit or silence the QKI gene, reduce the expression or activity of QKI protein in smooth muscle cells, and thus reduce the vascular calcification.

[0017] Technical features and advantages of the application:

[0018] The application discloses the important role of the QKI gene in regulating the vascular calcification by constructing the smooth muscle-specific QKI knockout mouse. After the vascular calcification modeling of the mouse, it is found that the QKI knockout mouse can reduce the vascular calcification, and the QKI siRNA used in the in vitro test can also inhibit the vascular smooth muscle cell calcification induced by high phosphorus, thereby providing a theoretical basis for preparing or screening the drug or preparation for inhibiting the expression of the QKI gene in smooth muscle cells as an alternative drug or preparation for preventing and treating the vascular calcification.

[0019] The application takes the QKI gene as the action target to prepare or screen the drug for preventing and treating the vascular calcification. The drug preparation is mainly based on the target gene, and the drug for preventing and treating the vascular calcification is prepared or constructed to inhibit the expression of the target gene; the drug screening is mainly aimed at unknown drugs, and the drugs are used on the target gene, and the drugs for the vascular calcification are screened according to whether the drugs can inhibit the expression of the target gene; the prepared or screened drugs have important significance in the treatment of the vascular calcification. The application prepares the smooth muscle-specific QKI knockout mouse and the QKI siRNA, and under the CKD calcification model or high phosphorus stimulation, the vascular calcification degree can be significantly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The strategy flow chart for knocking out the QKI gene of smooth muscle;

[0021] Figure 2 The QKI SMKO The expression condition diagram of the QKI protein in the mouse aortic smooth muscle cells;

[0022] In the figure, Control represents control mice, QKI SMKO represents smooth muscle-specific knockout QKI mice, and Tubulin is the internal reference protein.

[0023] Figure 3 Runx2 gene expression after calcification modeling in mice, and GAPDH is the internal reference protein.

[0024] Figure 4 Alizarin red staining after calcification modeling in mice, scale bar is 100 μm.

[0025] Figure 5 Von Kossa staining after calcification modeling in mice, scale bar is 100 μm.

[0026] Figure 6 The figure is the result of tissue calcium content determination after calcification modeling in mice.

[0027] Figure 7 The figure is the result of alizarin red staining of mouse vascular smooth muscle cells transfected with QKI siRNA after high-phosphorus stimulation.

[0028] Figure 8 The figure is the result of calcium content determination of mouse vascular smooth muscle cells transfected with QKI siRNA after high-phosphorus stimulation. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with examples, which are only used to illustrate the application, and not to limit the protection scope of the application.

[0030] QKI flox / flox Mice were prepared by SinoBiological Inc. (Guangzhou) Co., Ltd., SMMHC-CreER T2 Mice are available from SinoBiological Inc. (Guangzhou) Co., Ltd.

[0031] CTR siRNA and QKI siRNA were prepared and synthesized by Genomeditech Co., Ltd.

[0032] In the examples, the drugs and reagents involved are all ordinary commercially available products unless otherwise specified; in the examples, the experimental operations are all carried out according to the conventional operations in the art unless otherwise specified.

[0033] Example 1 Construction of mouse model

[0034] QKI flox / flox Mice were bred with smooth muscle-specific Cre recombinase-expressing mice SMMHC-CreER T2 Mice were bred with smooth muscle-specific Cre recombinase-expressing mice SMMHC-CreER flox / flox / Cre + Mice were bred with smooth muscle-specific Cre recombinase-expressing mice SMMHC-CreER flox / flox / Cre + Mice were injected intraperitoneally with 1 mg tamoxifen to induce Cre recombinase into the nucleus, and QKI flox / flox / Cre + The QKI gene in the smooth muscle of mice was knocked out to inhibit the function of the QKI gene, thereby obtaining a smooth muscle-specific QKI gene-knockout mouse, which was denoted as QKI SMKO . Figure 1 A flow chart of the strategy for smooth muscle-specific QKI gene knockout.

[0035] After intraperitoneal injection of tamoxifen for two weeks, the aortas of the control and QKI SMKO mice were collected, and 15 μg of the sample was subjected to Western blot after BCA determination of the protein concentration to detect the expression of QKI, as shown in Figure 2 It was found that the QKI SMKO gene was almost completely removed from the aortas of the mice, providing a good animal model for subsequent studies.

[0036] Example 2 Role of smooth muscle QKI in vascular calcification in mice

[0037] Vascular calcification is common in CKD patients, so we studied the effect of QKI gene knockout on the calcification of the aortas of CKD mice. The QKI SMKO mice obtained in Example 1 were divided into four groups, and CKD vascular calcification modeling was performed at 12 weeks of age. The treatment of the four groups of mice was as follows:

[0038] First group: control mice + sham operation (CTR + Sham);

[0039] Second group: QKI SMKO mice + sham operation (QKI SMKO + Sham);

[0040] Third group: control mice + calcification model (CTR + CKD);

[0041] Fourth group: QKI SMKO mice + calcification model (QKI SMKO + CKD).

[0042] After the vascular calcification modeling was completed, the aortas of the mice were collected, and 15 μg of the sample was subjected to Western blot after BCA determination of the protein concentration to detect the expression level of the Runx2 gene, as shown in Figure 3As shown, the expression level of Runx2 gene was significantly reduced after QKI gene knockout, indicating that the vascular calcification was reduced. The mouse aorta was sectioned, and alizarin red staining and Von Kossa staining were performed to detect calcium deposition, and the results are shown in Figure 4 and Figure 5 As shown, compared with the control, the alizarin red staining was lighter, and the Von Kossa staining calcium salt deposition was significantly reduced after QKI gene knockout, indicating that the vascular calcification was reduced. At the same time, the calcium content in the vascular tissue of the mouse was detected, and the results are shown in Figure 6 As shown, it was found that the calcium content in the blood vessels was reduced after QKI gene knockout, further indicating that the vascular calcification was reduced.

[0043] Example 3 Effect of QKI on vascular smooth muscle cell calcification

[0044] CTR siRNA and QKI siRNA (CTR siRNA and QKI siRNA were prepared and synthesized by Genomed) were constructed, and the sequences are as follows:

[0045] CTR siRNA sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3'

[0046] CTR siRNA antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'

[0047] QKI siRNA sense strand: 5'-CUAUGACCUUCUGACCUCUGAAUU-3'

[0048] QKI siRNA antisense strand: 5'-UUCAGAGGUCAGAAGGUCAUAGUU-3'

[0049] Mouse vascular smooth muscle cells were cultured and transfected with CTR siRNA or QKI siRNA, respectively, and 24 hours later, high-phosphorus stimulation was given, and 7 days later, alizarin red staining was performed, and the results are shown in Figure 7 As shown, compared with the control, the alizarin red staining was lighter, and the vascular smooth muscle cell calcification was reduced. At the same time, the calcium content in the vascular smooth muscle cells of the mouse was detected, and the results are shown in Figure 8 As shown, compared with the control, the calcium content in the vascular smooth muscle cells was reduced after transfection of QKI siRNA, indicating that the vascular smooth muscle cell calcification was reduced.

[0050] The above results confirmed the key role of QKI gene in regulating vascular calcification, smooth muscle-specific knockout QKI gene in CKD mouse calcification model can reduce the expression of Runx2 gene, reduce calcium deposition and reduce the content of calcium ions in vascular tissue, inhibit the occurrence of vascular calcification. In vitro test, using QKI siRNA can make the alizarin red staining of mouse vascular smooth muscle cells under high phosphorus stimulation lighter, the calcium content is reduced, and the vascular smooth muscle cell calcification is reduced in vitro.

Claims

1. Application of small interfering RNA for interfering with QKI gene in preparation of drugs for preventing and treating vascular calcification, wherein the nucleotide sequence of the QKI gene is shown as SEQ ID No. 1; The small interfering RNA is QKI siRNA sense strand and QKI siRNA antisense strand, and the sequences are as follows: QKI siRNA sense strand: 5'-CUAUGACCUUCUGACCUCUGAAUU-3'; QKI siRNA antisense strand: 5'-UUCAGAGGUCAGAAGGUCAUAGUU-3'.

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