Application of miRNA-147b-3p in preparation of angiogenesis inhibition drugs

Abstract

The invention discloses an application of miR-147b-3p in preparation of angiogenesis inhibition drugs, the miR-147b-3p has an angiogenesis inhibition effect, directly acts on mRNA of vascular endothelial cells Rap2B, and inhibits expression of Rap2B protein, thereby promoting proliferation and migration of the endothelial cells, inducing apoptosis of the endothelial cells, and inhibiting angiogenesis. And a theoretical basis is provided for clinical treatment of abnormal angiogenesis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of miRNA-147b-3p in the preparation of drugs for inhibiting angiogenesis. Background Art

[0002] Angiogenesis is the physiological process of forming new blood vessels from existing ones. In this process, each link is tightly regulated by numerous active molecules. However, under certain disease conditions, such as tumor growth and metastasis, and wet age-related macular degeneration (AMD), the angiogenesis process is dysregulated, leading to the development of neovascularization pathology.

[0003] Although strategies to inhibit angiogenesis have been successful in the treatment of certain cancers and eye diseases, there are still challenges, including the development of treatment resistance, individual differences in treatment, and treatment-related side effects. Therefore, there is still a need to develop more precise therapeutic targets and explore new treatment strategies to overcome resistance, improve efficacy, and reduce side effects. Summary of the invention

[0004] The purpose of the present invention is to provide the application of miRNA-147b-3p in the preparation of angiogenesis-inhibiting drugs, so as to obtain angiogenesis-inhibiting drugs prepared by using miR-147b.

[0005] The present invention adopts the following technical scheme: application of miR-147b-3p in preparing drugs for inhibiting angiogenesis.

[0006] Furthermore, the miR-147b-3p inhibits angiogenesis by inhibiting the proliferation and migration of vascular endothelial cells.

[0007] Furthermore, the miR-147b-3p inhibits angiogenesis by promoting apoptosis of vascular endothelial cells.

[0008] Furthermore, the miR-147b-3p binds to the mRNA of Rap2B in vascular endothelial cells, resulting in downregulation of Rap2B protein expression, thereby inhibiting endothelial cell proliferation and migration and promoting endothelial cell apoptosis.

[0009] Furthermore, the miR-147b-3p comes from exosomes produced after co-culture of vascular endothelial cells and circulating fibroblasts.

[0010] A method for obtaining exosomes containing miR-147b-3p,

[0011] Cultivating endothelial cells;

[0012] extracting circulating fibrocytes and culturing the circulating fibrocytes;

[0013] Vascular endothelial cells and circulating fibroblasts were co-cultured, and exosomes containing miR-147b-3p were extracted from the supernatant of the co-culture.

[0014] Furthermore, when culturing the circulating fibroblasts, high-glucose DMEM containing 10% fetal bovine serum is used for culturing, and the high-glucose DMEM containing 10% fetal bovine serum is replaced every 5 to 7 days.

[0015] A use of exosomes containing miR-147b-3p, which is used to bind to Rap2B mRNA in vascular endothelial cells, resulting in downregulation of Rap2B protein expression, thereby inhibiting endothelial cell proliferation and migration and promoting endothelial cell apoptosis.

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

[0017] The miR-147b-3p of the present invention has the function of inhibiting angiogenesis, directly acts on the mRNA of Rap2B in vascular endothelial cells, inhibits the expression of Rap2B protein, thereby promoting endothelial cell proliferation and migration, and inducing endothelial cell apoptosis, providing a theoretical basis for the clinical treatment of abnormal angiogenesis diseases;

[0018] After the circulating fibroblasts are extracted, they are cultured using high-glucose DMEM containing 10% fetal bovine serum, and the time for replacing the high-glucose DMEM containing 10% fetal bovine serum is 5 to 7 days. At this time, the cell amount is the largest and the adhesion rate is high. At this time, replacing the high-glucose DMEM containing 10% fetal bovine serum can make the cells in the best state and facilitate culture.

[0019] The miRNA-147b-3p of the present invention can be used as a potential target or a drug itself. By downregulating or inhibiting the expression of miRNA-147b-3p, the expression of its target gene Rap2B can be affected, thereby inhibiting abnormal angiogenesis. Therefore, miRNA-147b-3p shows new therapeutic potential in the development of drugs for inhibiting angiogenesis, opening up new pathways and strategies for treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the effect of the medium replacement time of CF culture on the growth of CF in the present invention;

[0021] Figure 2 This is a diagram of co-culturing VEC and CF in Traswell in the present invention;

[0022] Figure 3 This is the CCK-8 proliferation test result after VEC was added to the co-cultured exosomes in the present invention;

[0023] Figure 4This is the result of cell migration detection after VEC was added to co-culture exosomes in the present invention;

[0024] Figure 5 The results of cell chemotaxis detection after VEC was added to co-cultured exosomes in the present invention;

[0025] Figure 6 The results of exosome transcriptome sequencing after co-culture in the present invention show that hsa-miR-147b-3p expression is increased;

[0026] Figure 7 The biological effects of miR-147b on vascular endothelial cells (VECs) in the present invention are: inhibiting VEC proliferation (a: BrdU staining) and migration (b: CDC42 staining), and inducing VEC apoptosis (c: Caspase 3 staining);

[0027] Figure 8 The inhibitory effect of miR-147b on endothelial cell tube formation in the present invention (the number of tubes formed in the control group was significantly greater than that in the miR-147b group);

[0028] Fig. 9 Preparation of acute wounds and subcutaneous injection of wounds in mice for the animal experiment in the present invention;

[0029] Fig.10 The results of the analysis of wound healing in mice in the present invention;

[0030] Fig.11 Masson staining of mouse wound tissue in the present invention (black arrows indicate new blood vessels);

[0031] Fig.12 Blood vessel marker staining (ICAM-1 immunofluorescence staining) of mouse wound tissue in the present invention;

[0032] Fig.13 HE staining of mouse wound tissue and statistics of epidermal thickness and hair follicle number in the present invention;

[0033] Fig.14 This is the dual luciferase binding experiment of miR-147b and Rap2B in the present invention; Fig.14 In the middle, MUT UTR represents mutant type, and WT UTR represents wild type;

[0034] Fig.15 This is the effect of miR-147b overexpression on Rap2B expression in VECs in the present invention. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] The invention discloses application of miR-147b-3p in preparing drugs for inhibiting angiogenesis.

[0037] Furthermore, the miR-147b-3p inhibits angiogenesis by inhibiting the proliferation and migration of vascular endothelial cells.

[0038] Furthermore, the miR-147b-3p inhibits angiogenesis by promoting apoptosis of vascular endothelial cells.

[0039] Furthermore, the miR-147b-3p binds to the mRNA of Rap2B in vascular endothelial cells, resulting in downregulation of Rap2B protein expression, thereby inhibiting endothelial cell proliferation and migration and promoting endothelial cell apoptosis.

[0040] Furthermore, the miR-147b-3p comes from exosomes produced after co-culture of vascular endothelial cells and circulating fibroblasts.

[0041] The present invention also discloses a method for obtaining exosomes containing miR-147b-3p, comprising:

[0042] Cultivating endothelial cells;

[0043] extracting circulating fibrocytes and culturing the circulating fibrocytes;

[0044] Vascular endothelial cells and circulating fibroblasts were co-cultured, and exosomes containing miR-147b-3p were extracted from the supernatant of the co-culture.

[0045] Furthermore, when culturing the circulating fibroblasts, high-glucose DMEM containing 10% fetal bovine serum is used for culturing, and the high-glucose DMEM containing 10% fetal bovine serum is replaced every 5 to 7 days.

[0046] The present invention also discloses a use of exosomes containing miR-147b-3p, which is used to bind to the mRNA of Rap2B in vascular endothelial cells, resulting in downregulation of Rap2B protein expression, thereby inhibiting endothelial cell proliferation and migration and promoting endothelial cell apoptosis.

[0047] Example 1

[0048] 1.1. Culture of vascular endothelial cells (VECs)

[0049] Vascular endothelial cells (VECs) were purchased from sceincell Company and cultured in ECM medium. Cells of passages 4 to 6 were used for subsequent experiments.

[0050] 1.2. Extraction of circulating fibrocytes (CF)

[0051] Add 15mL of human lymphocyte separation solution to the L tube and centrifuge at 1000g for 15 minutes (normal temperature). Take fresh human blood buffy coat, dilute it with PBS 1:1, and add 30mL to the L tube. Centrifuge at 800g for 15 minutes. After centrifugation and stratification, discard the upper plasma layer and the buffy coat layer will be visible. Pour the buffy coat layer into a 15mL centrifuge tube, dilute it with PBS at a ratio of 1:3, centrifuge at 1000g for 10 minutes, and repeat this step 3 times. Resuspend with 5mL PBS and count the cells, averaging 1×10 7 / mL was inoculated in a 10 cm culture dish, 10mL / dish.

[0052] 1.3. Cultivation of circulating fibroblasts (CF)

[0053] The cells were cultured in high-glucose DMEM with 10% fetal bovine serum. Figure 1 As shown, from Figure 1 It can be seen that the cell amount was small when the medium was changed on the 4th day, the cell amount was large and the cell state was better when the medium was changed on the 7th day, and the cell amount decreased and a large number of apoptotic cells appeared when the medium was changed on the 9th day; therefore, the time of each medium change after inoculation is controlled at 5 to 7 days. Most preferably, each medium change is on the 7th day, that is, the first medium change is performed on the 7th day, and the second medium change is performed on the 14th day, and high-glucose DMEM with 10% fetal bovine serum is replaced during the medium change.

[0054] This experiment found that the CF attachment process is relatively long, and changing the medium every 2 to 4 days will easily cause a large number of CFs that are not firmly attached to the wall to be lost, affecting the cell recovery rate. If the medium is changed for more than 8 days, the time is too long, the cells are insufficient in nutrients, and a large number of cells will cause apoptosis.

[0055] Table 1 Relationship between medium replacement time, adhesion rate and harvested cell number

[0056]

[0057] Note: 1×10 8 CFs were inoculated per dish

[0058] 1.4. Co-culture of VECs and CFs

[0059] like Figure 2 As shown, VECs were seeded in the upper chamber of Transwell, and CFs were seeded in the lower chamber of Transwell. After co-culture for 24 hours, exosomes in the co-culture supernatant were extracted.

[0060] 1.5. Extraction of Exosomes

[0061] The Beibo exosome extraction kit was used for extraction and purification, specifically: collect 4 mL of supernatant, centrifuge at 3000g for 15 minutes at 4°C, discard the precipitate, and collect the supernatant. Transfer the supernatant to another clean centrifuge tube. Centrifuge again at 4°C, centrifuge at 10000g for 20 minutes, discard the precipitate, and collect the supernatant. Transfer the supernatant to another clean centrifuge tube. Take 4 ml of supernatant and add 1 ml of extraction solution A, cover the centrifuge tube cap tightly, and mix upside down for 1 minute to mix the liquid thoroughly. Let stand overnight at 2-8°C. Centrifuge at 11000g for 60 minutes at 4°C, carefully remove the supernatant, and collect the precipitate. Take 100 μl of exosome preservation solution to resuspend the precipitate to obtain the exosome sample.

[0062] 1.6. Incubation of vascular endothelial cells (VECs) using exosomes

[0063] Take VEC cells in logarithmic growth phase and in good growth condition, and use 1×10 5 Each well was inoculated into a 96-well plate and cultured overnight in a 5% CO2, 37°C cell culture incubator. VECs were incubated with 100 μg / ml exosomes.

[0064] 1.7 Biological effects of exosomes on vascular endothelial cells (VECs)

[0065] Cell proliferation detection: CCK-8 kit was used to detect the effect of exosomes on VEC proliferation. The culture plates were taken out on days 1, 2, 3, 4, and 5, and 10 μL of CCK8 solution was added to each well (avoid bubbles). The cells were incubated in a 37°C cell culture incubator for 4 h, and the absorbance at 450 nm was measured using an ELISA reader.

[0066] like Figure 3 As shown, the results showed that the absorbance values ​​of the exosome group (Exosome) cells at 490nm decreased at 5 time points, 1, 2, 3, 4, and 5 days, indicating that the proliferation number of the exosome group (Exosome) cells was lower than that of the conventional culture group (Control), indicating that the exosomes co-cultured with VEC and CF had a significant inhibitory effect on the proliferation of VEC.

[0067] Cell migration assay:

[0068] Three groups were set up, namely control group (Ctrl group) (Endothelial Cell Medium, ECM), supernatant group (Co-supernatant), and exosome group (CO-Exosomes). The control group cultured VECs with conventional ECM culture medium; the supernatant group cultured VECs with the supernatant of co-culture of CFs and VECs; and the exosome group cultured exosomes extracted from the supernatant of co-culture of CFs and VECs in ECM culture medium.

[0069] Oris TM Cell migration was detected using the Cell Migration Assay Kit. When cells formed a confluent monolayer, the seeding plug was removed. The culture plate was dynamically imaged for 24 hours in a CytationTM3 microplate imager at 37°C and 5% CO2. Random images were taken within the well every 6 hours.

[0070] like Figure 4 As shown, the cells in the central vacancy of the control group were almost fused at 20 hours, while the migration to the center of the supernatant group and the exosome group was inhibited to varying degrees, and the inhibitory effect of the exosome group on VEC was more significant.

[0071] Cell chemotaxis assay:

[0072] Transwell assay was used to detect the effect of Exosomes on VEC chemotaxis. Two groups were set up, namely the control group (Ctrl group) (ECM conventional culture) and the exosome group (co-cultured exosomes added). In the exosome group (CO-Exosomes), VEC was inoculated in the upper chamber, and the ECM culture medium containing exosomes was added to the lower chamber, while the control group added the ECM conventional culture medium to the lower chamber. After culturing for 24 hours, the cells in the upper chamber were wiped off with a cotton swab, and the surface of the lower chamber of the membrane was stained with 0.1% crystal violet, and photographed and counted under a light microscope.

[0073] like Figure 5 As shown, the number of VECs migrating to the lower chamber in the control group was significantly greater than that in the exosome group, indicating that exosomes significantly inhibited the chemotactic behavior of VECs to the lower chamber.

[0074] 1.8. Exosome transcriptome sequencing

[0075] Total RNA of exosomes was extracted, and the highly sensitive Agilent 2100pic600 was used to accurately detect the total amount of RNA and the distribution of fragments. The Small RNA Sample Pre Kit was used to construct the library. The 3'-terminal hydroxyl group and the complete 5'-terminal phosphate group of Small RNA were used. The total RNA was used as the starting sample, and the adapters were directly added to both ends of the Small RNA, and then reverse transcribed to synthesize cDNA. Subsequently, PCR amplification, PAGE gel electrophoresis, separation of target DNA fragments, and cutting and recovery were performed to obtain the cDNA library. After the library was constructed, Qubit 2.0 was used for preliminary quantification, the library was diluted to 1ng / μl, and the insert size of the library was detected using Agilent 2000. After the insert size met the expectations, Q-PCR was used to accurately quantify the effective concentration of the library (library effective concentration>2nM) to ensure the quality of the library. After the library inspection was qualified, different libraries were pooled according to the effective concentration and the target data volume requirements for HiSeq / MiSeq sequencing. The animal small RNA reference analysis process was used for bioinformatics analysis. The analysis results were used to screen differential miRNAs, and a volcano plot was drawn to analyze the overall distribution of differential miRNAs to screen differential genes.

[0076] like Figure 6 As shown, differentially expressed genes with log2FC greater than 5 were selected for analysis and preliminary experiments, and hsa-miR-147b-3p was finally selected for subsequent research based on the preliminary experimental results.

[0077] Table 2 Ordering hsa-miR-147b-3p mimics and their control group NC mimics

[0078]

[0079] 1.9 Cell transfection

[0080] Place a sterile coverslip on the bottom of a 12-well plate. Take VEC cells in the logarithmic growth phase with good growth status and add 1×10 5 12-well cell culture plates were inoculated with 100 μL / well and cultured overnight in a 37°C, 5% CO2 incubator. Two hours before transfection, the medium was changed to serum-free ECM medium. 5 μL miR-147b mimics (concentration of 20 μM) was diluted with 50 μL serum-free opti-MEM and gently mixed with a pipette tip and allowed to stand at room temperature for 5 minutes. Lipofectamine was gently mixed before use. TM 2000, then take 2μL of Lipofectamine TM2000 was diluted in 50 μL opti-MEM, and allowed to stand at room temperature for 5 minutes before mixing with Lipofectamine TM 2000 and miR-147b mimics dilutions (total volume of 100 μL), gently mix and let stand at room temperature for 20 minutes; add 100 μL of the mixed solution to each culture well, and gently shake the cell culture plate back and forth to mix the mixed solution with the culture medium in the culture plate; culture the cells in a 37°C CO2 incubator, and after 6 hours, aspirate the mixed solution and replace it with normal culture medium; culture in a 37°C, 5% CO2 incubator.

[0081] 1.10 Biological effects of miR-147b-3p on vascular endothelial cells (VECs)

[0082] Cell proliferation, migration and apoptosis: Three groups were set up, namely control group VEC, VEC+NC, and VEC+miR-147b. The control group VEC was cultured in ECM conventionally, VEC+NC was ECM culture medium with mimics NC added, and VEC+miR-147b was ECM culture medium with has-miR-147b-3p mimics added. The VECs that had been spread on the culture plate were washed with PBS three times, each time for 3 minutes. They were fixed with 4% paraformaldehyde for 15 minutes and washed with PBS three times, each time for 3 minutes. They were permeabilized with 0.5% Triton X-100 at room temperature for 20 minutes and washed with PBS three times, each time for 3 minutes. PBS was dried with absorbent paper, and normal goat serum was added to the slides and blocked at room temperature for 30 minutes. The blocking solution was absorbed with absorbent paper, and a sufficient amount of diluted primary antibody was added to each slide and placed in a humidified box and incubated overnight at 4°C. The primary antibodies were BrdU, CDC42, and Caspase 3. Among them, BrdU was first denatured with 2N HCl for 30 minutes, then neutralized with 0.1M sodium borate, washed with PBS, and then added with BrdU antibody. The next day, the slides were washed with PBS three times, and the diluted fluorescent secondary antibody was added. The slides were incubated at 37°C in a wet box for 1 hour, and then washed with PBS three times, each time for 3 minutes. DAPI was added and incubated in the dark for 8 minutes to stain the nucleus, and then washed with PBS three times, each time for 3 minutes. The slides were sealed with a sealing solution containing an anti-fluorescence quencher, and images were collected under a fluorescence microscope.

[0083] like Figure 7 As shown in (a), after transfection with miR-147b-3p, BrdU expression decreased, indicating that VEC proliferation was significantly inhibited; Figure 7 As shown in (b), CDC42 expression was significantly reduced, indicating that cell migration was also significantly inhibited; Figure 7 As shown in (c), the expression of Caspase 3 increased, indicating that miR-147b-3p promoted VEC apoptosis.

[0084] Tube Formation experiment: there are control group and miR-147b group. The control group was cultured with conventional ECM culture medium, and the miR-147b group was cultured with has-miR-147b-3p mimics added to the ECM culture medium. Matrigel was purchased from BD Company and was taken out from -20℃ and thawed in a 4℃ refrigerator the day before use. Pre-cool the pipette tip and 96-well plate, draw 50μL Matrigel and spread it on the bottom of the 96-well plate. Avoid bubbles during the entire operation and perform it on ice. After laying the 96-well plate, incubate the plate in an incubator at 37℃ and 5% CO2 for 30 minutes to allow the Matrigel to solidify. VEC uses Vybrant TM CFDA SE cell tracer was used to label the cells. 4 VECs were seeded in 96-well plates at a cell density of 100 cells / well. The culture plates were removed after 12 hours and photographed under a fluorescence microscope to record the tube formation.

[0085] like Figure 8 As shown in the figure, after transfection with miR-147b-3p, the ability of VEC to form tubes in the miR-147b group was significantly weakened, while the VEC in the control group formed a reticular lumen-like structure. This indicates that miR-147b-3p has an inhibitory effect on VEC tube formation.

[0086] 2. Animal experiments

[0087] Table 3 Ordering mmu-miR-147b-3p agomir and its control group NC agomir

[0088]

[0089] Balb / c mice (male) aged 6-8 weeks were purchased from Hubei Academy of Preventive Medicine. Each group had 12 mice + 1 spare, for a total of 39 mice.

[0090] Balb / c mice were adaptively raised for 3 days and divided into the following groups:

[0091] Group A: blank control group (12 rats + 1 spare rat): 0.9% sterile sodium chloride was injected subcutaneously at multiple points in situ (equal volume);

[0092] Group B: negative control group (12 rats + 1 spare rat): agomir NC group (n=12), agomir NC was injected subcutaneously at multiple points in situ;

[0093] Group C: Experimental group (12 mice + 1 spare): miR-147b-3p agomir group (n=12), miR-147b-3p agomir was injected subcutaneously at multiple points in situ.

[0094] The three groups of mice were anesthetized with an anesthesia machine and fixed in a prone position. The hair on the back was shaved, and full-thickness excision was performed on the mouse skin with a 1-cm diameter punch to create acute skin defect wounds. The mice were housed individually after surgery.

[0095] After wound modeling, mice in groups B and C were subcutaneously injected with agomir NC and miR-147b-3p agomir at multiple points in situ on days 0, 3, 6, and 9, respectively, with 2 nmol / mouse / time per injection. Mice in group A were subcutaneously injected with an equal volume of 0.9% sterile sodium chloride at multiple points in situ, such as Fig. 9 shown.

[0096] On days 0, 3, 6, 9, and 12, mice in each group were anesthetized with isoflurane, and the wounds on the back were observed and photographed. Fig.10 As shown in the figure, the wound area of ​​group C (miR-147b-3p agomir injection group) on the 6th, 9th and 12th day after modeling was significantly larger than that of groups A and B. This indicates that miR-147b-3p agomir has a significant inhibitory effect on wound healing and significantly delays the healing time of wounds.

[0097] On the 3rd, 6th, 9th and 12th days, mice in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate at 330 μL / 100 g, and skin tissues were collected from 3 mice in each group. The specimens were fixed with 4% paraformaldehyde.

[0098] Tissue dehydration: Dehydrate the tissue in sequence with gradient alcohol, 75% alcohol (4h) - 85% alcohol (2h) - 90% alcohol (1.5h) - 95% alcohol (1h) - anhydrous ethanol I (0.5h) - anhydrous ethanol II (0.5h).

[0099] Tissue transparency: Tissue blocks must be transparent after being dehydrated with alcohol. The transparent agent (xylene) can be mixed with the dehydrating agent and paraffin at the same time. After it replaces the dehydrating agent, the paraffin can smoothly penetrate into the tissue. Anhydrous ethanol: xylene (1:1) (10min) - xylene I (10min) - xylene II (10min).

[0100] Tissue wax immersion: The transparent tissue block is waxed in three cylinders of paraffin (60°C) in sequence.

[0101] Ⅰ(60℃)(1h)-paraffin Ⅱ(60℃)(1h)-paraffin Ⅲ(60℃)(1h).

[0102] Tissue embedding, slicing and baking: Wrap the wax-soaked tissue block in a paraffin block. The temperature of the embedding wax should be slightly higher than the wax soaking temperature to ensure that the tissue block and the embedding paraffin are completely integrated. Use a Leica pathology slicer to slice the embedded wax block. Put the sliced ​​tissue slices into a 40°C water bath for spreading. Insert the anti-slip slide into the water surface at an angle to pick up the slices, so that the slices are attached to the appropriate position of the slide. Bake the slices in a 60°C oven for 3 hours.

[0103] Dewaxing of sections: Place the paraffin sections in xylene Ⅰ (15 min) - xylene Ⅱ (15 min) - xylene Ⅲ (15 min) - anhydrous ethanol Ⅰ (5 min) - anhydrous ethanol Ⅱ (5 min) - 95% alcohol (5 min) - 90% alcohol (5 min) - 80% alcohol (5 min) - 70% alcohol (5 min) in sequence, and then soak in distilled water for 5 min.

[0104] Masson staining: circle the slices with a histochemical pen and place them in a wet box (with a certain amount of distilled water to prevent the slices from drying up), add potassium dichromate staining solution, cover the tissue overnight, and wash with distilled water 3 times the next day for 3 minutes each time. Add Weigert iron hematoxylin staining solution (A solution and B solution are prepared in a 1:1 ratio for immediate use) to stain the nucleus for 5 minutes, and wash with distilled water 3 times. Differentiate with 1% hydrochloric acid alcohol for 3 seconds, and wash with distilled water 3 times. Differentiate with 1% phosphomolybdic acid solution for about 1 minute, spin dry, and directly use aniline blue staining solution for 1 minute without washing with water, and quickly rinse the slices with 1% glacial acetic acid for 1 minute. 95% alcohol for 30 seconds; 95% alcohol for 1 minute; anhydrous ethanol I, II for 5 minutes each; xylene I, II for 5 minutes each. After air drying, seal the slides with neutral gum and finally examine under a microscope.

[0105] like Fig.11As shown, the number of new blood vessels in group A and group B was roughly the same at 6 days and 9 days, but increased significantly at 12 days; while the number of new blood vessels in group C (miR-147b-3p transfection group) was significantly less than that in groups A and B at 6 days, 9 days and 12 days, and the number of new blood vessels at 12 days did not increase significantly compared with 6 days and 9 days. Therefore, it can be seen that miR-147b-3p also has the effect of inhibiting angiogenesis in animals. Immunofluorescence staining: For antigen repair, the dewaxed and hydrated tissue sections were placed on the high-temperature plastic slice rack in the repair box, and an appropriate amount of repair solution (0.01M Tris-EDTA repair solution pH9.0) was added to the beaker. The liquid level should be immersed in the slice tissue to a certain height. The microwave oven was first heated at high temperature to make the liquid boil. When it was heated to boiling, it was adjusted to medium temperature. At this time, the timing started. The repair time was 15 minutes. During this process, the tissue was prevented from drying (the repair solution should be sufficient). After the time is up, take the beaker out of the microwave oven and let it cool naturally. When the repair solution drops to room temperature, take out the slide and rinse it with TBST (PH7.4) three times, 3 minutes each time (avoid breaking the tissue during the rinsing process). Wipe the slide dry with absorbent paper, draw circles around the tissue with an immunoblot brush, add diluted normal goat serum, and block at room temperature for 30 minutes to reduce nonspecific staining. Shake off the excess liquid, do not wash, then add diluted primary antibody (ICAM-1) and incubate in a 4°C wet box overnight. Rinse the sections with TBST three times, 3 minutes each time, wipe the sections dry with absorbent paper, add diluted fluorescent secondary antibody FITC-goat anti-rabbit IgG, incubate in a wet box at 37°C for 1 hour, and rinse the sections with TBST 4 times, 3 minutes each time. Add DAPI and incubate in the dark for 5 minutes, stain the specimens, and wash off the excess DAPI with TBST 5min×4 times. Wipe the liquid on the sections dry with absorbent paper, seal the sections with sealing solution containing anti-fluorescence quencher, and observe and collect images under a fluorescence microscope.

[0106] The expression of ICAM is closely related to the proliferation and migration of endothelial cells. ICAM restores vascular integrity and function by promoting the proliferation and migration of endothelial cells.

[0107] like Fig.12 As shown, on day 6, the expression of ICAM-1 in groups A and B was high, while that in group C (transfected with miR-147b-3p) was significantly decreased; on day 9, the expression of ICAM-1 in groups A and B was still high, while that in group C (transfected with miR-147b-3p) was almost not expressed. Therefore, it can be seen that miR-147b-3p significantly inhibited the angiogenesis of VEC.

[0108] HE staining: Slices were stained with Mayer's hematoxylin (the staining background is clean, no differentiation is required) for 5 minutes, washed with tap water and turned blue. Slices were stained with 1% water-soluble eosin stain for 5 minutes, washed with tap water for 30 seconds. 95% alcohol for 30 seconds - 95% alcohol for 1 minute - anhydrous ethanol I5 minutes - anhydrous ethanol II 5 minutes - xylene I5 minutes - xylene II 5 minutes, air-dried and sealed with neutral gum, and finally examined under a microscope.

[0109] like Fig.13 As shown in the figure, the epidermal thickness of group A and group B gradually increased on the 6th, 9th and 12th day, while the epidermal thickness of group C (transfected with miR-147b-3p) remained thin. The recovery of epidermal thickness helps to enhance the elasticity and support of the skin. In addition, the number of hair follicles in each group was counted on the 12th day. Fig.13 As shown, the number of hair follicles in groups A and B was significantly higher than that in group C (transfected with miR-147b-3p). As an important component of the skin, the restoration of the number of hair follicles helps maintain the integrity and functionality of the skin. Therefore, the results show that miR-147b-3p inhibits the formation of epidermis on the wound surface and inhibits the formation of hair follicles during the wound recovery period. Therefore, miR-147b-3p can be used as a drug target during the wound healing period.

[0110] 3. Interaction between miR-147b-3p and Rap2B

[0111] 3.1 Dual luciferase binding assay

[0112] Take 293T cells in the logarithmic growth phase and in good growth condition, and use 1×10 5 Each well was inoculated into a 12-well cell culture plate and cultured overnight at 37°C;

[0113] Cells were treated in the following groups:

[0114] Group A: RAP2B-3'UTR wild-type luciferase + mimics NC;

[0115] Group B: RAP2B-3'UTR wild-type luciferase + hsa-miR-147b-3p mimics;

[0116] Group C: RAP2B-3'UTR mutant luciferase + mimics NC;

[0117] Group D: RAP2B-3'UTR mutant luciferase + hsa-miR-147b-3p mimics;

[0118] Action time: 24h.

[0119] (1) After the cells have been cultured for the required time, the cell culture medium is aspirated and then rinsed with PBS, and 300 μl of reporter gene cell lysis buffer is added;

[0120] (2) After sufficient lysis, centrifuge at 12,000 rpm for 3 min and take the supernatant for determination;

[0121] (3) Dissolve the firefly luciferase assay reagent and Renilla luciferase assay buffer and bring them to room temperature. Place the Renilla luciferase assay substrate (100X) in an ice bath or ice box for later use;

[0122] (4) According to the amount of 100 μl required for each sample, take an appropriate amount of Renilla luciferase assay buffer and add Renilla luciferase assay substrate (100X) at a ratio of 1:100 to prepare Renilla luciferase assay working solution;

[0123] (5) Take 100 μl of the sample, add 100 μl of firefly luciferase detection reagent, mix well and measure the RLU;

[0124] (6) Add 100 μl of Renilla luciferase assay working solution, mix well and measure RLU;

[0125] (7) When Renilla luciferase is used as an internal reference, the RLU value obtained by the firefly luciferase assay is divided by the RLU value obtained by the Renilla luciferase assay. The activation degree of the target reporter gene between different samples is compared based on the obtained ratio.

[0126] like Fig.14 As shown, there was no significant difference between group D and group C, while there was a significant difference between group B and group A. Therefore, it can be seen that the overexpression of miR-147b-3p significantly inhibited the Rap2B-wt (wild type) reporter plasmid, but did not affect the luciferase activity of the Rap2B-mut (mutant) reporter plasmid, which reflects that miR-147b-3p binds to the mRNA of Rap2B.

[0127] 3.2 Immunofluorescence experiment

[0128] Place a sterile coverslip on the bottom of a 12-well plate. Take VEC cells in the logarithmic growth phase with good growth status and add 1×10 5 12-well cell culture plates were inoculated with 100 μL / well and cultured overnight in a 37°C, 5% CO2 incubator. Two hours before transfection, the medium was changed to serum-free ECM medium. 5 μL miR-147b mimics (concentration of 20 μM) was diluted with 50 μL serum-free opti-MEM and gently mixed with a pipette tip and allowed to stand at room temperature for 5 minutes. Lipofectamine was gently mixed before use.TM 2000, then take 2μL of Lipofectamine TM 2000 was diluted in 50 μL opti-MEM, and allowed to stand at room temperature for 5 minutes before mixing with Lipofectamine TM 2000 and miR-147b mimics dilutions (total volume of 100 μL), gently mix and let stand at room temperature for 20 minutes; add 100 μL of the mixed solution to each culture well, and gently shake the cell culture plate back and forth to mix the mixed solution with the culture medium in the culture plate; culture the cells in a 37°C CO2 incubator, and after 6 hours, aspirate the mixed solution and replace it with normal culture medium; culture in a 37°C, 5% CO2 incubator.

[0129] Grouping:

[0130] Group A: VEC is conventional ECM culture.

[0131] Group B: VEC+NC, i.e., mimics NC were added to ECM culture medium.

[0132] Group C: VEC+miR-147b, i.e., miR-147b-3p mimics were added to the ECM culture medium.

[0133] The VECs that have been mounted on the culture plate were washed with PBS three times, each time for 3 minutes. They were fixed with 4% paraformaldehyde for 15 minutes and then washed with PBS three times, each time for 3 minutes. They were permeabilized with 0.5% Triton X-100 at room temperature for 20 minutes and then washed with PBS three times, each time for 3 minutes. The PBS was blotted with absorbent paper, and normal goat serum was added to the slides and blocked at room temperature for 30 minutes. The blocking solution was removed with absorbent paper, and then a sufficient amount of diluted primary antibody was added to each slide without washing, and the slides were incubated in a wet box at 4°C overnight. The primary antibody was Rap2B (1:100). The next day, the mounted slides were washed with PBS three times, and the diluted fluorescent secondary antibody was added. The slides were incubated in a wet box at 37°C for 1 hour, and then washed with PBS three times, each time for 3 minutes. DAPI was added and incubated in the dark for 8 minutes to stain the nuclei, and then washed with PBS three times, each time for 3 minutes. The slides were sealed with a sealing solution containing an anti-fluorescence quencher, and images were collected under a fluorescence microscope.

[0134] like Fig.15 As shown, the expression of Rap2B in groups A and B was high, while the expression of Rap2B protein in VECs of group C was significantly reduced. This indicates that miR-147b-3p has a significant inhibitory effect on Rap2B in VECs. Fig.14 , the results demonstrated that miR-147b-3p bound to Rap2B mRNA in VECs, ultimately inhibiting the expression of Rap2B protein.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of miR-147b-3p in the preparation of drugs for inhibiting angiogenesis.

2. The use of miR-147b-3p according to claim 1 for preparing angiogenesis inhibiting drugs, characterized in that: The miR-147b-3p inhibits angiogenesis by inhibiting the proliferation and migration of vascular endothelial cells.

3. The use of miR-147b-3p according to claim 1 for preparing angiogenesis inhibiting drugs, characterized in that: The miR-147b-3p inhibits angiogenesis by promoting apoptosis of vascular endothelial cells.

4. The use of miR-147b-3p according to claim 2 or 3 for preparing angiogenesis inhibiting drugs, characterized in that: The miR-147b-3p binds to the mRNA of Rap2B in vascular endothelial cells, resulting in downregulation of Rap2B protein expression, thereby inhibiting endothelial cell proliferation and migration and promoting endothelial cell apoptosis.

5. The use of miR-147b-3p for preparing angiogenesis inhibiting drugs according to claim 1, characterized in that: The miR-147b-3p comes from exosomes produced after co-culture of vascular endothelial cells and circulating fibroblasts.

6. A method for obtaining exosomes containing miR-147b-3p, characterized in that: Cultivating endothelial cells; extracting circulating fibrocytes and culturing the circulating fibrocytes; Vascular endothelial cells and circulating fibroblasts were co-cultured, and exosomes containing miR-147b-3p were extracted from the supernatant of the co-culture.

7. The method for obtaining exosomes containing miR-147b-3p according to claim 6, characterized in that: When culturing circulating fibroblasts, high-glucose DMEM containing 10% fetal bovine serum is used for culturing, and the time for replacing high-glucose DMEM containing 10% fetal bovine serum is 5 to 7 days.

8. A use of exosomes containing miR-147b-3p, which is used to bind to Rap2B mRNA in vascular endothelial cells, resulting in downregulation of Rap2B protein expression, thereby inhibiting endothelial cell proliferation and migration and promoting endothelial cell apoptosis.

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