Application of microRNA-21-3p as target spot in preparation of medicine for treating cerebral cavernous malformation

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of microRNA-21-3p as a target spot in preparation of a medicine for treating cerebral cavernous malformation. According to the application disclosed by the invention, it is determined for the first time that the microRNA-21-3p plays an important role in cerebral hemorrhage with cerebral cavernous malformation, the microRNA-21-3p can be used as a potential treatment target spot of the cerebral cavernous malformation, and the functions of vascular endothelial cells and pericytes can be effectively improved by improving the expression level of the microRNA-21-3p through a microRNA-21-3p agonist; therefore, a good effect of treating the cerebral cavernous malformation is achieved, and a theoretical basis is provided for researching a new target spot and a new strategy for treating the cerebral cavernous malformation.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of microRNA-21-3p as a target in the preparation of a medicine for treating cerebral cavernous malformations. Background Art

[0002] Cerebral cavernous malformation (CCM), also known as cerebral cavernous hemangioma, has a prevalence of about 0.16%-0.8%, accounting for about 10%-15% of all central nervous system vascular malformations. The histological characteristics of CCM are mainly manifested by significant dilation of the vascular lumen, and the vascular wall is composed of a single layer of endothelial cells, lacking smooth muscle cells and elastic tissue. The most common clinical manifestations of CCM include epileptic seizures, intracranial hemorrhage, and focal neurological damage. At present, the clinical treatment methods of CCM include microsurgical resection, stereotactic radiotherapy, and conservative observation. Among them, surgical treatment is the most effective treatment for CCM, and stereotactic radiotherapy is mostly used for lesions located in areas with high surgical risks. At present, there is still a high treatment risk for complex CCM lesions located in the brainstem or deep parts, and there is still a lack of effective treatment options. Therefore, it is of great scientific significance to clarify the mechanism of CCM formation and development and to discover potential therapeutic drugs and strategies for CCM.

[0003] CCM is a progressive cerebrovascular disease. Studies have shown that CCM mainly appears in familial or sporadic aggregation forms. Familial CCM accounts for about 20%, which is mainly manifested as autosomal dominant inheritance with incomplete penetrance. Patients show the coexistence of multiple vascular malformations. Sporadic CCM accounts for about 80%, often manifested as a single cavernous malformation. Studies have shown that familial and sporadic CCM have similar molecular mechanisms, that is, Ccm1, Ccm2, and Ccm3 in vascular endothelial cells can form a Ccm complex and play a specific functional role. When these genes are missing and mutated, they will promote the formation and development of cerebral cavernous malformations. The signal regulation pathways that destroy the Ccm complex and the formation and development mechanism of cerebral cavernous malformations after the loss of pathogenic gene function are not yet fully understood.

[0004] After the Ccm1 gene function in vascular endothelial cells is lost, it can induce the increased expression of transforming growth factor-β and bone morphogenetic protein, promote the mesenchymal transformation of vascular endothelial cells, lead to enhanced endothelial cell invasion and proliferation ability, changes in the intercellular connection structure, and ultimately promote the formation and development of cerebral cavernous malformations. When endothelial cell-mesenchymal transformation is inhibited, the occurrence of vascular malformations caused by Ccm1 gene loss can be significantly reduced. After the Ccm1 gene is lost, it can also induce myosin light chain phosphorylation by promoting the RhoA / ROCK signaling pathway, destroy the normal adhesion between endothelial cells, increase vascular permeability, and promote the formation and development of cerebral cavernous malformations.

[0005] Loss of Ccm2 gene function in vascular endothelial cells can promote the expression of mitogen-activated protein kinase 3 (MEKK3) and its target genes Krüppel-like transcription factor (KLF) 2 and KLF4, inducing the formation of cavernous malformations. When the MEKK3-KLF2 / 4 signaling pathway is inhibited, the formation and development of cavernous malformations can be significantly inhibited. Ccm2L is a new homologous gene of Ccm2. The Ccm2L protein contains a functional phosphorylated tyrosine binding domain, binds to Ccm1 to form a signaling complex, and does not bind to Ccm3. In newborn mice with loss of Ccm2 gene function, loss of Ccm2L function can also promote the formation of cavernous malformations by upregulating the MEKK3-KLF2 / 4 signaling pathway. On the basis of the loss of Ccm2 gene, intestinal Gram-negative bacteria can activate Toll-like receptor 4 (TLR4) of vascular endothelial cells through lipopolysaccharide, activate the downstream MEKK3-KLF2 / 4 signaling pathway, and cause the formation and development of cavernous malformations.

[0006] After the loss of Ccm3 gene function in vascular endothelial cells, the wnt / β-catein signaling pathway is first activated, and then the expression of transforming growth factor-β and bone morphogenetic protein is increased, which induces endothelial cell-mesenchymal transformation and promotes the formation of cavernous malformations. After the loss of Ccm3 gene function, the exocytosis of vascular endothelial cells is regulated, which causes the increase of angiopoietin-2 secretion, promotes the progression of cavernous malformations, and the intervention of angiopoietin-2 normalizes the brain lesions caused by endothelial cell-specific Ccm3 loss. STK24 and STK25 kinases, which are closely related to Ccm3 protein, also play a key role in the development of cavernous malformations. The simultaneous loss of STK24 and STK25 kinases can aggravate the vascular defect phenotype of cavernous malformations. In addition to Ccm1, Ccm2, and Ccm3 genes, guanine trinucleotide enzyme CDC42 also inhibits the MEKK3-KLF2 / 4 signaling pathway by participating in the Ccm complex. Specific knockout of endothelial cell CDC42 can form cerebral vascular malformation lesions similar to cavernous malformations.

[0007] The latest research results show that in sporadic cerebral cavernous malformations, PIK3CA and MAP3K3 somatic mutations play a more important role than other genes. Loss-of-function mutations in the Ccm complex and somatic mutations in PIK3CA enhance the endothelial cell mTOR signaling pathway, forming a complex mechanism similar to cancer occurrence that promotes excessive vascular growth and leads to the formation of cerebral cavernous malformations.

[0008] Studies on the pathogenesis of cerebral cavernous malformations suggest that specific molecular dysfunction is closely related to the occurrence and development of vascular malformations, and specific inhibitor intervention may have an effective therapeutic effect on cerebral cavernous malformations. In animal models of cerebral cavernous malformations and in vitro cell studies, it was found that RhoA kinase inhibition by fasudil and simvastatin in mouse models can improve the increase in vascular permeability and reduce the number of vascular malformation lesions. Targeted inhibitors of bone morphogenetic protein in the endothelial-mesenchymal transition signaling pathway can also effectively improve the phenotype of cavernous malformations. In addition, the combined use of HMG-COA reductase inhibitors and valerylation inhibitors can effectively reduce neurovascular damage in mouse models, reduce lesion volume, and prolong the survival time of cavernous malformation mouse models. Recent studies have found that the mTORC1 inhibitor rapamycin can effectively inhibit the formation of cavernous malformations in mouse models. Whether the effectiveness of the above-mentioned targeted inhibitors also exists in clinical patients has not yet been evaluated by clinical trial results.

[0009] MicroRNA (microRNA) is a class of endogenous non-coding small RNA molecules, which are about 20-22 nucleotides long. MicroRNA mainly regulates the post-transcriptional expression of target genes by inducing mRNA degradation or inhibiting mRNA translation through base complementary pairing with the non-coding region of target gene mRNA. Recent studies have found that non-coding RNA plays an important role in the mechanism of cerebral vascular malformations, and it also plays a potentially important role in the occurrence and development of cerebral cavernous malformations. However, its role in the development of CCM and cerebral hemorrhage lesions is still unclear. Summary of the invention

[0010] The purpose of the present invention is to overcome the defects and shortcomings in the prior art and provide the application of microRNA-21-3p as a target in the preparation of drugs for treating cerebral cavernous malformations. By clarifying the function of microRNA-21-3p in cerebral cavernous malformations, an important theoretical basis is provided for the development of effective disease treatment strategies, drug screening and other research.

[0011] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0012] In a first aspect, the present invention provides use of a microRNA-21-3p agonist in the preparation of a medicament for treating cerebral cavernous malformations.

[0013] Currently, there is still a high treatment risk for complex cerebral cavernous malformation (CCM) lesions located in the brainstem or deep parts, and there is still a lack of effective treatment options. Therefore, it is of great scientific significance to clarify the mechanism of CCM formation and development and discover potential therapeutic drugs and strategies for CCM.

[0014] The present invention first determined the important function of microRNA-21-3p in CCM. In the human brain microvascular endothelial cell and pericyte model with Ccm2 gene deletion, up-regulating microRNA-21-3p can reduce the abnormal increase of endothelial cell reactive oxygen and permeability, reduce the migration and lumen formation ability of endothelial cells, reduce the abnormal increase of pericyte reactive oxygen and permeability, and inhibit the abnormal increase of pericyte proliferation ability. In the zebrafish model with Ccm2 gene down-regulation, up-regulating microRNA-21-3p can reduce the cerebral hemorrhage changes of zebrafish embryos, reduce cerebral vascular permeability, and reduce abnormal vascular dilation and angiogenesis. Therefore, microRNA-21-3p plays an important role in cerebral hemorrhage of cerebral cavernous malformation. Increasing the expression level of microRNA-21-3p by microRNA-21-3p agonist can effectively improve the function of vascular endothelial cells and pericytes, thereby achieving a good effect of treating cerebral cavernous malformation, and providing a theoretical basis for studying new targets and new strategies for treating cerebral cavernous malformation.

[0015] Preferably, the cerebral cavernous malformation includes a cerebral hemorrhagic lesion of a cerebral vascular malformation.

[0016] The present invention investigates the relationship between the expression level of microRNA-21-3p and the manifestation of cerebral hemorrhage in CCM. Experimental investigations reveal that compared with the CCM group without cerebral hemorrhage on MR imaging, the expression of microRNA-21-3p in the vascular endothelial cells and pericytes of the lesions with cerebral hemorrhage is reduced. Therefore, microRNA-21-3p is a potential therapeutic target for cerebral hemorrhage lesions of cerebral cavernous malformations.

[0017] Preferably, the agonist comprises at least one of a gene sequence, a small molecule compound and a polypeptide capable of increasing the expression level of microRNA-21-3p.

[0018] Preferably, the agonist is a gene sequence capable of increasing the expression level of microRNA-21-3p, the nucleotide sequence of the sense strand of the gene sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand of the gene sequence is shown in SEQ ID NO: 2.

[0019] Experimental investigations have found that the present invention uses the above-mentioned gene sequence as an agonist to significantly increase the expression of microRNA-21-3p, thereby reducing the abnormal increase in endothelial cell reactive oxygen species and permeability, reducing the endothelial cell migration and lumen formation ability, and reducing the abnormal increase in pericyte reactive oxygen species and permeability, inhibiting the abnormal increase in pericyte proliferation ability, thereby exerting a good therapeutic effect on cerebral cavernous vascular malformations.

[0020] Preferably, the microRNA-21-3p agonist targets and inhibits the expression level of NOX4 and / or VEGFA, thereby exerting the effect of treating cerebral cavernous malformations.

[0021] Studies have shown that knocking out the Ccm1 gene in vascular endothelial cells can cause a significant increase in NOX4 expression, increase the production of reactive oxygen species (ROS), a product of oxidative stress, and increase the permeability of vascular endothelial cells, leading to endothelial cell dysfunction. At the same time, studies have shown that low expression of the zebrafish Ccm2 gene can cause a significant increase in KLF2 expression, which in turn increases VEGFA expression, leading to increased angiogenesis. The present invention has found through experimental research that NOX4 and VEGFA are direct target genes of microRNA-21-3p, and microRNA-21-3p can target and inhibit the expression of NOX4 and VEGFA, thereby exerting the effect of treating cerebral cavernous malformations.

[0022] In a second aspect, the present invention provides the use of microRNA-21-3p as a target in the preparation of a drug for treating cerebral cavernous malformations.

[0023] In a third aspect, the present invention provides the use of microRNA-21-3p as a drug target for screening and treating cerebral cavernous malformations.

[0024] In a fourth aspect, the present invention provides a drug for treating cerebral cavernous malformations, wherein the drug comprises a microRNA-21-3p agonist.

[0025] Preferably, the drug is used in at least one of the following aspects:

[0026] a. Reduce the abnormal increase of reactive oxygen species and / or permeability of vascular endothelial cells;

[0027] b. Reduce the migration ability and / or lumen formation ability of vascular endothelial cells;

[0028] c. Reduce the abnormal increase of reactive oxygen species and / or permeability of vascular pericytes;

[0029] d. Inhibit the abnormal increase of vascular pericyte proliferation ability.

[0030] Preferably, the agonist comprises a gene sequence capable of increasing the expression level of microRNA-21-3p, the nucleotide sequence of the sense strand of the gene sequence is shown as SEQ ID NO: 1, and the nucleotide sequence of the antisense strand of the gene sequence is shown as SEQ ID NO: 2.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention first determined the important function of microRNA-21-3p in CCM. In the human brain microvascular endothelial cell and pericyte model with Ccm2 gene deletion, up-regulating microRNA-21-3p can reduce the abnormal increase of endothelial cell reactive oxygen and permeability, reduce the migration and lumen formation ability of endothelial cells, reduce the abnormal increase of pericyte reactive oxygen and permeability, and inhibit the abnormal increase of pericyte proliferation ability. In the zebrafish model with Ccm2 gene down-regulation, up-regulating microRNA-21-3p can reduce the cerebral hemorrhage changes of zebrafish embryos, reduce cerebral vascular permeability, and reduce abnormal vascular dilation and angiogenesis. Therefore, microRNA-21-3p plays an important role in cerebral hemorrhage of cerebral cavernous malformation. Increasing the expression level of microRNA-21-3p by microRNA-21-3p agonist can effectively improve the function of vascular endothelial cells and pericytes, thereby achieving a good effect of treating cerebral cavernous malformation, and providing a theoretical basis for studying new targets and new strategies for treating cerebral cavernous malformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The expression of microRNA-21-3p, NOX4, and VEGFA in endothelial cells and pericytes in CCM lesions in Example 1;

[0034] Figure 2 This is a graph showing the effect of microRNA-21-3p on 3'UTR NOX4 luciferase activity in Example 2;

[0035] Figure 3 The figure shows the changes in the expression of microRNA-21-3p (A), NOX4 (B) and VEGFA (C) in the human brain microvascular endothelial cell model with Ccm2 gene deletion after microRNA-21-3p intervention in Example 3;

[0036] Figure 4 This is a diagram showing the effect of microRNA-21-3p intervention on reactive oxygen species in Ccm2 gene-deficient vascular endothelial cells in Example 4;

[0037] Figure 5 This is a diagram showing the effect of microRNA-21-3p intervention on the permeability change of vascular endothelial cells with Ccm2 gene deletion in Example 5;

[0038] Figure 6 This is a diagram showing the effect of microRNA-21-3p intervention on the proliferation of Ccm2 gene-deficient vascular endothelial cells in Example 6;

[0039] Figure 7 The effect of microRNA-21-3p intervention on angiogenesis of Ccm2 gene-deficient vascular endothelial cells in Example 7 is shown in FIG. Figure 7 A is the result of the migration ability of human brain microvascular endothelial cells. Figure 7 B is the result diagram of the tube-forming ability of human brain microvascular endothelial cells;

[0040] Figure 8 The results of the intervention of microRNA-21-3p in Example 8 on the expression of microRNA-21-3p (A), NOX4 (B) and VEGFA (C) in the pericyte model with Ccm2 gene deletion are shown;

[0041] Fig. 9 This is a graph showing the effect of microRNA-21-3p intervention on reactive oxygen species in Ccm2 gene-deficient pericytes in Example 9;

[0042] Fig.10 This is a graph showing the effect of microRNA-21-3p intervention on the permeability changes of Ccm2 gene-deficient pericytes in Example 10;

[0043] Fig.11 This is a graph showing the effect of microRNA-21-3p intervention on the proliferation of Ccm2 gene-deficient pericytes in Example 11;

[0044] Fig.12 The results of the intervention of microRNA-21-3p in Example 12 on the expression of microRNA-21-3p (A), NOX4 (B) and VEGFA (C) in the brain tissue of zebrafish with low expression of the Ccm2 gene are shown in FIG.

[0045] Fig.13 This is a graph showing the effect of microRNA-21-3p intervention on cerebral hemorrhage in zebrafish with low Ccm2 gene expression in Example 13;

[0046] Fig.14 This is a graph showing the changes in brain vascular permeability of zebrafish with low Ccm2 gene expression as a result of microRNA-21-3p intervention in Example 14;

[0047] Fig.15 This is a graph showing the effect of microRNA-21-3p intervention on vasodilation (A) and angiogenesis (B) in zebrafish with low Ccm2 gene expression in Example 15. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0050] All data in the embodiment were statistically analyzed using SPSS25.0 software. All measurement data were expressed as mean ± standard deviation. The single sample KS test was used to determine whether the measurement data was normally distributed; the data that met the normal distribution were statistically analyzed using one-way ANOVA; the non-normal distribution data were analyzed using the Mann-Whitney nonparametric test. The count data were analyzed using Fisher's exact test or chi-square test. When the P value was less than 0.05, it was considered that there was a significant difference.

[0051] The miR-21-3p in the example is the microRNA-21-3p described in the present invention.

[0052] The sequences of Ccm2 siRNA and microRNA-21-3p agonist involved in the examples are shown in Table 1.

[0053] Table 1 CCM2 siRNA and microRNA-21-3p agonist sequences

[0054]

[0055] Example 1. Expression of miR-21-3p, NOX4, and VEGFA in endothelial cells and pericytes in CCM lesions

[0056] In this example, fluorescent in situ hybridization was used to detect the expression of miR-21-3p, NOX4, and VEGFA in vascular malformation lesions of 20 CCM patients, and 5 cases of temporal lobe epilepsy tissues that underwent initial surgery during the same period were used as the control group. The expression of miR-21-3p, NOX4, and VEGFA in endothelial cells / pericytes of vascular malformations and their relationship with the manifestations of cerebral hemorrhage in CCM patients were analyzed.

[0057] 1. Expression of miR-21-3p, NOX4 and VEGFA in vascular endothelial cells and pericytes in CCM lesions

[0058] Fluorescence in situ hybridization results showed that compared with normal vessels in temporal lobe epilepsy (TLE), the expression of miR-21-3p was decreased in CCM lesion vascular endothelial cells (P<0.001), the expression of NOX4 was increased in CCM lesion vascular endothelial cells (P<0.01), and the expression of VEGFA was increased in CCM lesion vascular endothelial cells (P<0.01). Compared with normal vessels in temporal lobe epilepsy, the expression of miR-21-3p was decreased in CCM lesion pericytes (P<0.001), the expression of NOX4 was increased in CCM lesion pericytes (P<0.001), and the expression of VEGFA was increased in CCM lesion pericytes (P<0.001). The results are shown in Figure 1 A.

[0059] 2. Correlation between miR-21-3p, NOX4 and VEGFA expression levels and CCM intracerebral hemorrhage

[0060] Compared with the CCM group without cerebral hemorrhage on MR imaging, the expression of miR-21-3p in vascular endothelial cells of lesions with cerebral hemorrhage was decreased (P<0.01), NOX4 expression was increased (P<0.001), and VEGFA expression was increased (P<0.01). Compared with the CCM group without cerebral hemorrhage on MR imaging, the expression of miR-21-3p in pericytes of lesions with cerebral hemorrhage was decreased (P<0.01), NOX4 expression was increased (P<0.01), and VEGFA expression was increased (P<0.001). The results are shown in Figure 1 B.

[0061] Example 2. Study on the relationship between miR-21-3p, NOX4 and VEGFA

[0062] This example uses dual luciferase activity technology to clarify the relationship between NOX4 and miR-21-3p. First, human NOX4 3'UTR and human miR-21-3p were predicted and analyzed for potential binding sites using bioinformatics tools such as TargetScan and miRDB to analyze whether there is a potential binding site for miR-21-3p at the site of NOX4 3'UTR. Secondly, dual luciferase activity technology was used to determine whether NOX4 is a direct target gene of miR-21-3p. HEK 293T cells were transfected with miR-21-3p agonists and wild-type and mutant NOX4-3'UTRs carrying luciferase, respectively, and the effect of miR-21-3p on the activity of the NOX4 luciferase reporter gene was observed. The specific method is as follows.

[0063] 1. Co-transfection of microRNA and plasmid into cells

[0064] 24 hours before transfection, 293T cells were seeded in 96-well plates, with 6 replicates per group. Each well contained 0.01 μg of Renilla luciferase vector (Renilla), 0.2 μg of Firefly luciferase vector (Firefly), and 0.25 μl of Lipofectamine 3000 when transfecting plasmids. The miR-21-3p agonist was diluted to a final concentration of 100 nM, and 0.25 μl of Lipofectamine 3000 was added to each well. The diluted miR-21-3p agonist and DNA (including NOX4) were mixed with Lipofectamine 3000 and allowed to stand at room temperature for 20 minutes. The culture medium was discarded, and 25 μl of DNA and Lipofectamine 3000 mixture and 25 μl of miR-21-3p agonist were added to each well. After 12 hours of transfection, new complete culture medium was replaced.

[0065] 2. Dual luciferase reporter gene assay

[0066] Detect luciferase activity according to the operating steps of Promega's dual luciferase detection kit. After 48 hours of transfection, discard the original culture medium and rinse 3 times with PBS buffer. Add PLB (Passive Lysis Buffer) to the wells to lyse the cells. Add 10μL of cell lysis buffer to the white transparent 96-well plate, and then add 50μl of pre-mixed luciferase detection reagent II to each well, shake and mix, and read the value after standing for 2 seconds. Then add 50μl of Stop&Glo Reagent to each well sample, shake and mix, put it into the luminescence detector, stand for 2 seconds, and measure the data.

[0067] 3. Determination of the direct target gene NOX4 regulated by miR-21-3p

[0068] Bioinformatics tools such as TargetScan and miRDB were used to predict potential binding sites of human NOX4 3'UTR and human miR-21-3p. The results showed that there was a potential binding site for miR-21-3p at sites 162-177 of NOX4 3'UTR.

[0069] The dual luciferase reporter gene technique was used to detect the inhibitory effect of miR-21-3p agonist on NOX4 3'UTR, and the site-directed mutagenesis technique was used to determine the action site of miR-21-3p and NOX4 3'UTR. Figure 2 ), in the wild-type NOX4 3'UTR, miR-21-3p agonist can significantly inhibit the luciferase activity of wild-type NOX4 3'UTR tandem expression (0.52±0.02vs.1.00±0.02, P<0.001). In the mutant NOX4 3'UTR, miR-21-3p agonist can still partially inhibit the luciferase activity of mutant NOX4 3'UTR tandem expression (P=0.008), but the inhibitory ability is significantly weakened (P<0.001), the results show that NOX4 is a direct target gene of human miR-21-3p.

[0070] At the same time, dual luciferase experiments revealed that VEGFA is a direct target gene of miR-21-3p, and miR-21-3p can exert its regulatory effect by acting on specific sites of VEGFA 3'UTR.

[0071] Example 3. Changes in miR-21-3p, NOX4 and VEGFA expression in the Ccm2 gene-deficient human brain microvascular endothelial cell model by miR-21-3p intervention

[0072] 1. Experimental Grouping

[0073] In the in vitro model of human brain microvascular endothelial cells with Ccm2 gene deletion, the mechanism of action of miR-21-3p intervention was clarified. The experiment was divided into 3 groups, including blank control group, Ccm2 siRNA group, and Ccm2 siRNA + miR-21-3p agonist group.

[0074] 2. Establishment of an in vitro model of human brain microvascular endothelial cells with Ccm2 gene deletion

[0075] The Ccm2 gene was knocked out in human brain microvascular endothelial cells using siRNA technology to establish an in vitro model of human brain microvascular endothelial cells with Ccm2 gene deletion.

[0076] Ccm2 siRNA and miR-21-3p agonist powder (sequences in Table 1) were dissolved in enzyme-free sterile water to a final concentration of 20 μM. 5 Cells were inoculated into each well. After 24 hours, the cells reached 50%-70%. After washing 3 times with PBS solution, 1.5 mL of new endothelial cell culture medium was added. Five 1.5 mL centrifuge tubes were prepared, and 250 μL of Opti-MEM culture medium, 5 μL of corresponding Ccm2 siRNA, and 5 μL of miR-21-3p agonist group were added to each well. 30 μL of Lipofectamine 3000 was added to 1.5 mL of Opti-MEM culture medium and mixed well. Let stand for 5 minutes, and the diluted Lipofectamine3000 and Opti-MEM culture medium were mixed and let stand for 20 minutes. Finally, 500 μL of the mixture was transferred to each well of the 6-well plate, and the 6-well plate was shaken to mix well.

[0077] After overnight incubation, the mixed solution in the 6-well plate was removed, the cells were washed with PBS solution for 3 times, and the endothelial cell culture medium was replaced. The cells were then cultured in a 37°C incubator for 48 hours for subsequent cell experiments.

[0078] The cells stored at -80°C were taken out, 1 mL of Trizol was added to each 50-100 mg of tissue, total RNA was extracted, and the expression level of miR-21-3p was detected using TaqMan stem-loop RT-PCR. After the cDNA was synthesized from the RNA of the sample, all cDNA samples were configured with the Realtime PCR reaction system for PCR amplification of miRNA and result analysis. The designed and synthesized miR-21-3p and U6 primers were purchased from Guangzhou Ruibo Biotechnology Co., Ltd., and the mRNA primer sequences are shown in Table 2 below. All samples were repeated 3 times, and the results were quantified using the comparative cycle threshold (CT) method, and the calculation method was the ΔΔCt method.

[0079] Table 2 Primer sequences used in fluorescence quantitative PCR

[0080]

[0081] miR-21-3p expression: qRT-PCR results showed that compared with the blank control group, miR-21-3p was significantly decreased after siRNA Ccm2 gene knockdown (P<0.001). Compared with the Ccm2 siRNA group, miR-21-3p agonist intervention significantly increased the expression of miR-21-3p (P<0.001).

[0082] NOX4 expression: qRT-PCR results showed that compared with the blank control group, NOX4 mRNA expression was significantly increased after siRNACcm2 gene knockdown (P<0.001). Compared with the Ccm2 siRNA group, miR-21-3p agonist intervention significantly inhibited the up-regulated expression of NOX4 mRNA (P=0.001).

[0083] VEGFA expression: qRT-PCR results showed that compared with the Ccm2 siRNA group, miR-21-3p agonist intervention significantly inhibited the up-regulated expression of VEGFA mRNA (P=0.001).

[0084] Example 4. Study on the effect of miR-21-3p intervention on reactive oxygen species in Ccm2 gene-deficient vascular endothelial cells

[0085] The experimental grouping and establishment of an in vitro model of human brain microvascular endothelial cells with Ccm2 gene deletion were the same as in Example 3.

[0086] The results of detecting hydrogen peroxide expression in each group by DCFH-DA probe showed that compared with the blank control group, the expression of hydrogen peroxide increased significantly after Ccm2 siRNA intervention (P<0.001); compared with the Ccm2 siRNA group, the up-regulated expression of hydrogen peroxide was significantly inhibited after miR-21-3p agonist intervention (P<0.001). The results of detecting superoxide anion expression in each group by DHE probe showed that compared with the blank control group, the expression of superoxide anion increased significantly after Ccm2 siRNA intervention (P<0.001); compared with the Ccm2 siRNA group, the up-regulated expression of superoxide anion was significantly inhibited after miR-21-3p agonist intervention (P<0.001). Figure 4 .

[0087] Example 5. Study on the effect of miR-21-3p intervention on the permeability changes of vascular endothelial cells with Ccm2 gene deletion

[0088] The experimental grouping and establishment of an in vitro model of human brain microvascular endothelial cells with Ccm2 gene deletion were the same as in Example 3.

[0089] The transmembrane permeability experiment measured the absorption value of sodium fluorescein in each group through the upper chamber of Transwell to clarify the effect of overexpression of miR-21-3p on the permeability of human brain microvascular endothelial cells with Ccm2 gene deletion. The permeability of endothelial cells in each group showed that compared with the blank control group, the permeability of endothelial cells was significantly increased after Ccm2 siRNA intervention (P<0.001); compared with the Ccm2 siRNA group, the up-regulation of endothelial cell permeability was significantly inhibited after miR-21-3p agonist intervention (P<0.001). The results are shown in Figure 5 .

[0090] Example 6. Study on the effect of miR-21-3p intervention on the proliferation of vascular endothelial cells with Ccm2 gene deletion

[0091] The experimental grouping and establishment of an in vitro model of human brain microvascular endothelial cells with Ccm2 gene deletion were the same as in Example 3.

[0092] The CCK8 kit was used to detect the absorbance of endothelial cells in each group at 450nm to clarify the effect of overexpression of miR-21-3p on its proliferation ability. The results showed that compared with the blank control group, the proliferation ability of endothelial cells in the Ccm2 siRNA group was significantly increased (P<0.001); compared with the Ccm2 siRNA group, the miR-21-3p agonist significantly inhibited the abnormal increase in the proliferation ability of endothelial cells (P=0.002). Figure 6 .

[0093] Example 7. Study on the effect of miR-21-3p intervention on angiogenesis of vascular endothelial cells with Ccm2 gene deletion

[0094] By counting the number of endothelial cells in each group that passed through the upper chamber of Transwell, the effect of overexpression of miR-21-3p on their migration ability was clarified. The results showed that compared with the blank control group, the migration ability of endothelial cells in the Ccm2 siRNA group was significantly increased (P<0.001); compared with the Ccm2 siRNA group, the miR-21-3p agonist significantly inhibited the abnormal increase in the migration ability of endothelial cells (P=0.006). The above results suggest that miR-21-3p plays an important role in regulating the migration ability of human brain microvascular endothelial cells with Ccm2 deficiency. Figure 7 A.

[0095] By counting the number of tubules formed by endothelial cells in each group, the effect of overexpression of miR-21-3p on their ability to form tubes was clarified. The results showed that compared with the blank control group, the ability of endothelial cells to form tubes in the Ccm2 siRNA group was significantly increased (P<0.001); compared with the Ccm2 siRNA group, the miR-21-3p agonist significantly inhibited the abnormal increase in the ability of endothelial cells to form tubes (P<0.001). These results suggest that miR-21-3p plays an important role in regulating the ability of human brain microvascular endothelial cells to form tubes without Ccm2. Figure 7 B.

[0096] Example 8. Study on the changes of miR-21-3p, NOX4 and VEGFA expression in the Ccm2 gene-deficient pericyte model after miR-21-3p intervention

[0097] 1. Experimental Grouping

[0098] In the in vitro model of pericytes with Ccm2 gene deletion, the mechanism of action of miR-21-3p intervention was clarified. The experiment was divided into 3 groups, including blank control group, Ccm2 siRNA group, and Ccm2 siRNA+miR-21-3p agonist group.

[0099] 2. Establishment of an in vitro pericyte model with Ccm2 gene deletion

[0100] The Ccm2 gene was knocked out in pericytes using siRNA technology to establish an in vitro pericyte model with Ccm2 gene deficiency.

[0101] miR-21-3p expression: qRT-PCR results showed that compared with the blank control group, miR-21-3p was significantly decreased after siRNA Ccm2 gene knockout (P=0.015). Compared with the Ccm2 siRNA group, miR-21-3p agonist intervention significantly increased the expression of miR-21-3p (P<0.001). Figure 8 A.

[0102] NOX4 expression: qRT-PCR results showed that compared with the blank control group, NOX4 mRNA expression was significantly increased after siRNA Ccm2 gene knockdown (P<0.001). Compared with the Ccm2 siRNA group, miR-21-3p agonist intervention significantly inhibited the up-regulated expression of NOX4 mRNA (P<0.001). Figure 8 B.

[0103] VEGFA expression: qRT-PCR results showed that compared with the blank control group, the expression of VEGFA mRNA was significantly increased after siRNA Ccm2 gene knockdown (P<0.001). Compared with the Ccm2 siRNA group, the up-regulated expression of VEGFA mRNA was significantly inhibited after miR-21-3p agonist intervention (P<0.001). Figure 8 C.

[0104] Example 9. Effect of miR-21-3p intervention on reactive oxygen species in pericytes with Ccm2 gene deletion

[0105] The results of DHE probe detection of superoxide anion expression in each group showed that compared with the blank control group, the expression of superoxide anion was significantly increased after Ccm2 siRNA intervention (P<0.001); compared with the Ccm2 siRNA group, the up-regulated expression of superoxide anion was significantly inhibited after miR-21-3p agonist intervention (P<0.001). Fig. 9 Example 10. Study on the effect of miR-21-3p intervention on the permeability changes of pericytes in Ccm2 gene-deficient mice

[0106] The transmembrane permeability test was used to measure the absorption value of sodium fluorescein in each group through the upper chamber of Transwell to clarify the effect of overexpression of miR-21-3p on the permeability of pericytes with Ccm2 gene deletion. Compared with the blank control group, pericyte permeability was significantly increased after Ccm2 siRNA intervention (P=0.001); compared with the Ccm2 siRNA group, the up-regulation of pericyte permeability was significantly inhibited after miR-21-3p agonist intervention (P=0.015), as shown in Figure 2. Fig.10 .

[0107] Example 11. Study on the effect of miR-21-3p intervention on the proliferation of pericytes with Ccm2 gene deletion

[0108] In the pericyte model with Ccm2 gene deletion, the absorbance value at 450nm of each group of pericytes was detected by using CCK8 kit to clarify the effect of overexpression of miR-21-3p on its proliferation ability. The experiment was divided into 3 groups. The results showed that compared with the blank control group, the proliferation ability of pericytes in the Ccm2 siRNA group was significantly increased (P<0.001); compared with the Ccm2 siRNA group, the miR-21-3p agonist significantly inhibited the abnormal increase of pericyte proliferation ability (P<0.001). Fig.11 .

[0109] Example 12. Changes in miR-21-3p, NOX4 and VEGFA expression in zebrafish brain tissues with low Ccm2 gene expression induced by miR-21-3p intervention

[0110] 1. Experimental Grouping

[0111] The experiment was divided into three groups: MO negative control group (Ctrl MO), Ccm2 MO+miR-21-3pMO group (Ccm2MO+miR-21MO) and Ccm2 MO+miR-21-3p agonist group (Ccm2MO+miR-21mimic).

[0112] 2. Establishment of a zebrafish model with low expression of the Ccm2 gene

[0113] Ccm2 MO was injected into zebrafish embryos by microinjection to establish a zebrafish model with low expression of Ccm2 gene.

[0114] MO was designed and synthesized by Gene Tools, USA. Because MO is a white lyophilized powder, it needs to be dissolved in sterile distilled water to prepare the mother solution. The specific steps are as follows: the finished MO is heated in a 65°C water bath for 10 minutes, dissolved in sterile distilled water to a concentration of 1mM, and stored at room temperature in a humid environment. Referring to previous literature, when using, the MO mother solution needs to be diluted with sterile distilled water to the use concentration: Ccm2 MO is 200μM, miR-21-3pMO is 100μM, and miR-21-3p agonist is 10μM. The sequence information of the MO and miR-21-3p agonist used is shown in Table 3 below.

[0115] Table 3 Sequence information of zebrafish MO, miR-21-3p agonist and negative control

[0116] name sequence sequence name MO negative control 5'CCTCTTACCTCAGTTACAATTTATA-3' SEQ ID NO: 21 Ccm2 MO 5'-GAAGCTGAGTAATAACCTTAACTTCC-3' SEQ ID NO: 22 miR-21-3p MO 5'-ACAGCCAACACCAGTCTGATAAGCT-3' SEQ ID NO: 23 miR-21-3p agonists 5'-UAGCUUAUCAGACUGGUGUUGGC-3' SEQ ID NO: 24

[0117] The qPCR and WB methods were the same as those in Example 3 above. The designed and synthesized miR-21-3p and U6 primers were purchased from Guangzhou Ruibo Biotechnology Co., Ltd. The mRNA primer sequences are shown in Table 2. All samples were repeated 3 times, and the results were quantified using the comparative cycle threshold (CT) method, and the calculation method was the ΔΔCt method.

[0118] qRT-PCR results, miR-21-3p expression: Compared with the MO negative control group, the Ccm2MO+miR-21-3p MO group significantly reduced the expression of miR-21-3p (P<0.001). Compared with the Ccm2MO+miR-21-3p MO group, the miR-21-3p agonist significantly increased the expression of miR-21-3p (P<0.001), see Fig.12 A.

[0119] Expression of NOX4 mRNA: Compared with the MO negative control group, the Ccm2 MO+miR-21-3p MO group significantly increased the expression of NOX4 mRNA (P<0.001). Compared with the Ccm2 MO+miR-21-3p MO group, the miR-21-3p agonist intervention significantly inhibited the up-regulation of NOX4 mRNA (P=0.002). Fig.12 B.

[0120] VEGFA mRNA expression: Compared with the MO negative control group, the Ccm2 MO+miR-21-3p MO group (P<0.001) significantly increased the expression of VEGFA mRNA. Compared with the Ccm2 MO+miR-21-3p MO group, the miR-21-3p agonist significantly inhibited the up-regulated expression of VEGFA mRNA (P<0.001). Fig.12 C.

[0121] Example 13. Effect of miR-21-3p intervention on cerebral hemorrhage in zebrafish with low expression of Ccm2 gene

[0122] The experimental grouping and establishment of a zebrafish model with low Ccm2 gene expression were the same as in Example 12.

[0123] In order to clarify the effects of overexpression and underexpression of miR-21-3p on the cerebral hemorrhage phenotype of zebrafish with low expression of the Ccm2 gene, the area of ​​extravascular red blood cell infiltration in zebrafish was counted under a laser confocal microscope to detect cerebral hemorrhage. The results showed that compared with the MO negative control group, the cerebral hemorrhage of zebrafish in the Ccm2 MO+miR-21-3p MO group was significantly increased (P<0.001); compared with the Ccm2 MO+miR-21-3p MO group, the intervention of miR-21-3p agonist also significantly reduced the cerebral vascular permeability of zebrafish (P<0.001), see Fig.13 .

[0124] Example 14. Changes in brain vascular permeability in zebrafish with low Ccm2 gene expression by miR-21-3p intervention

[0125] To clarify the effects of overexpression and underexpression of miR-21-3p on brain vascular permeability in zebrafish with low Ccm2 gene expression, the number of DAPI-stained cells outside the blood vessels of the zebrafish head was counted under a laser confocal microscope. The results showed that compared with the MO negative control group, the brain vascular permeability of zebrafish in the Ccm2 MO+miR-21-3p MO group was significantly increased (P<0.001); compared with the Ccm2 MO+miR-21-3p MO group, the intervention of miR-21-3p agonist also significantly reduced the brain vascular permeability of zebrafish (P<0.001), see Fig.14 .

[0126] Example 15. Effects of miR-21-3p intervention on vasodilation and angiogenesis in zebrafish with low Ccm2 gene expression

[0127] By measuring the area of ​​zebrafish original midbrain channels, the effect of miR-21-3p intervention on the phenotype of cerebral vasodilation in zebrafish with low Ccm2 gene expression was clarified. The results showed that compared with the MO negative control group, the Ccm2MO+miR-21-3p MO group significantly increased the area of ​​zebrafish original midbrain channels (P<0.001); compared with the Ccm2MO+miR-21-3p MO group, the increase of zebrafish original midbrain channel area was significantly inhibited after miR-21-3p agonist intervention (P=0.001). Fig.15 A.

[0128] By measuring the branch points of the zebrafish intestinal vessels, the effect of miR-21-3p intervention on the angiogenic phenotype of zebrafish with low expression of the Ccm2 gene was clarified. The results showed that compared with the MO negative control group, the Ccm2MO+miR-21-3p MO group significantly increased the branch points of the zebrafish intestinal vessels (P<0.001). Compared with the Ccm2MO+miR-21-3p MO group, the miR-21-3p agonist significantly inhibited the increase of the branch points of the zebrafish intestinal vessels (P<0.001). Fig.15 B.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, 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 scope of the technical solution of the present invention.

Claims

1. Application of microRNA-21-3p agonist in the preparation of drugs for treating cerebral cavernous malformations.

2. Application of microRNA-21-3p as a target in the preparation of drugs for the treatment of cerebral cavernous malformations.

3. Application of microRNA-21-3p as a drug target for screening and treating cerebral cavernous malformations.

4. The use according to any one of claims 1 to 3, characterized in that: The cerebral cavernous malformation includes cerebral vascular malformation and cerebral hemorrhagic lesions.

5. The use according to claim 1, characterized in that The agonist comprises at least one of a gene sequence, a small molecule compound and a polypeptide capable of increasing the expression level of microRNA-21-3p.

6. The use according to claim 5, characterized in that The agonist is a gene sequence that can increase the expression level of microRNA-21-3p. The nucleotide sequence of the sense strand of the gene sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand of the gene sequence is shown in SEQ ID NO:

2.

7. The use according to claim 1, characterized in that The microRNA-21-3p agonist targets and inhibits the expression level of NOX4 and / or VEGFA, thereby exerting the effect of treating cerebral cavernous malformations.

8. A drug for treating cerebral cavernous malformation, characterized in that: The drug includes a microRNA-21-3p agonist.

9. The drug according to claim 8, characterized in that The drug is used for at least one of the following aspects: a. Reduce the abnormal increase of reactive oxygen species and / or permeability of vascular endothelial cells; b. Reduce the migration ability and / or lumen formation ability of vascular endothelial cells; c. Reduce the abnormal increase of reactive oxygen species and / or permeability of vascular pericytes; d. Inhibit the abnormal increase of vascular pericyte proliferation ability.

10. The drug according to claim 8, characterized in that The agonist comprises a gene sequence capable of increasing the expression level of microRNA-21-3p, the nucleotide sequence of the sense strand of the gene sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand of the gene sequence is shown in SEQ ID NO: 2.

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