Medical applications of miR-378a-5p in the prevention and treatment of abdominal aortic aneurysms

Abstract

The present invention discloses the use of miR-378a-5p, which belongs to the field of molecular biomedical technology. It mainly relates to the use of intervening in the expression of miR-378a-5p for the preparation of drugs for preventing and treating abdominal aortic aneurysms. The present invention has proved through a large number of experiments that in the mouse abdominal aortic aneurysm model, the expression of miR-378a-5p in the aortic tissue of abdominal aortic aneurysm is significantly downregulated. Low expression of miR-378a-5p inhibits the differentiation of VSMCs and increases the migration of VSMCs, aggravating the occurrence and development of abdominal aortic aneurysms. Overexpression of miR-378a-5p in mice delays the occurrence and development of abdominal aortic aneurysms, promotes the differentiation of VSMCs and inhibits the migration of VSMCs. The present invention provides a new target for the diagnosis and treatment of abdominal aortic aneurysms, which has important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to use of intervening in miR-378a-5p expression for preparing a drug for preventing and treating abdominal aortic aneurysm. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a degenerative vascular disease characterized by dilation of the aorta greater than 30 mm or greater than 50%. It is a common and life-threatening condition in the elderly. Currently, large, asymptomatic AAAs, as well as symptomatic or ruptured AAAs of any size, require AAA repair. Therefore, identifying early prevention and treatment targets for AAA is crucial.

[0003] Research has shown that the development and progression of AAA involves multiple pathological processes, including extracellular matrix (ECM) breakdown, inflammation, phenotypic transitions of vascular smooth muscle cells (VSMCs), and oxidative stress. These processes collectively contribute to the pathogenesis and progression of AAA. VSMCs are a major component of the vascular wall and are crucial for maintaining vascular wall integrity and vascular homeostasis. In the normal vascular system, VSMCs reside in the tunica media and remain quiescent. Under pathophysiological stimulation, VSMCs can undergo dedifferentiation, leading to vascular damage. In AAA, VSMC homeostasis is disrupted, causing VSMCs to undergo phenotypic transitions, leading to VSMC migration and apoptosis. VSMCs can also promote AAA development by promoting the production of inflammatory factors and elastin-degrading matrix metalloproteinases. Therefore, targeted improvements in the biological functions of VSMCs are crucial for identifying new targets for the prevention and treatment of AAA.

[0004] MicroRNAs (miRNAs) are a class of conserved, endogenous, small, noncoding, single-stranded RNA molecules of approximately 20 nucleotides. They regulate target genes by partially complementing mRNAs. MiRNAs participate in a variety of cellular biological functions, including proliferation, differentiation, apoptosis, migration, invasion, and angiogenesis. Accumulating evidence indicates that miRNAs, such as miRNA-29b, miRNA-33b, miRNA-21, and miRNA-24, play a crucial role in the development and progression of AAA. Studies have shown that miR-378a-5p inhibits the proliferation of colorectal cancer cells by targeting cyclin-dependent kinase 1 (CDK1). Furthermore, miR-378a-5p regulates the proliferation of VSMCs by targeting CDK1 and may play a key role in in-stent restenosis. However, the role and mechanism of miR-378a-5p in AAA remain unclear. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide the diagnostic function of miR-378a-5p in AAA, and the medical use of intervening miR-378a-5p in the prevention and treatment of AAA.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The present invention discloses the use of a reagent for detecting the expression level of miR-378a-5p in the preparation of an abdominal aortic aneurysm detection kit.

[0008] Furthermore, the kit is used for diagnosis and / or prognosis assessment of abdominal aortic aneurysm; compared with normal tissue, the miR-378a-5p expression level is downregulated in abdominal aortic aneurysm cells or tissues.

[0009] The present invention also discloses the use of the primers for amplifying the miR-378a-5p described above in preparing an abdominal aortic aneurysm detection kit.

[0010] Furthermore, the kit is used for diagnosis and / or prognosis assessment of abdominal aortic aneurysm; compared with normal tissue, the FHL2 expression level is downregulated in abdominal aortic aneurysm cells or tissues.

[0011] Furthermore, the sequences of the primers are: forward: 5'-CTCCTGACTCCAGGTCCTGTGT-3'; reverse: 5'-CGGCGTCACCGGGTGTAAATC-3'.

[0012] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more active ingredients selected from the following group:

[0013] (1) miR-378a-5p, or a modified miR-378a-5p derivative, or a miR-378a-5p analog;

[0014] (2) A polynucleotide encoding the miR-378a-5p gene

[0015] (3) an expression construct comprising the miR-378a-5p described in (1) or the polynucleotide described in (2);

[0016] (4) Agonists of miR-378a-5p described in (1);

[0017] (5) miR-378a-5p nucleic acid molecule or its recombinant vector or recombinant cell;

[0018] (6) Reagents that can upregulate the expression of miR-378a-5p or its activity.

[0019] Furthermore, the pharmaceutical composition is used in the preparation of a drug for treating abdominal aortic aneurysm.

[0020] Furthermore, the drug is in any therapeutically acceptable dosage form.

[0021] Furthermore, the drug is any drug in a therapeutically acceptable dose.

[0022] Compared with the prior art, the present invention has the following beneficial effects.

[0023] Through extensive experiments, the present invention established a mouse AAA model using an angiotensin II (AngII) implantation pump method and found that miR-378a-5p expression was significantly downregulated in the aorta tissue of AAA-bearing mice. In vivo models of miR-378a-5p underexpression or overexpression were established by tail vein injection of miR-378a-5p antagomiR and angomiR. AAA models were then established. Overexpression of miR-378a-5p in mice alleviated AAA formation, while underexpression of miR-378a-5p aggravated AAA formation. Cytological analysis revealed that tumor necrosis factor alpha (TNFα) stimulation inhibited VSMC differentiation and promoted VSMC migration, while simultaneously downregulating miR-378a-5p expression in VSMCs. Overexpression of miR-378a-5p inhibited TNFα-induced VSMC migration and dedifferentiation, whereas underexpression of miR-378a-5p increased TNFα-induced VSMC migration and dedifferentiation. The above results indicate that miR-378a-5p plays an important regulatory role in the occurrence of AAA, and intervention of miR-378a-5p may be a target for the prevention and treatment of AAA. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 In animal and cell models of AAA, miR-378a-5p expression is downregulated. Panel A shows quantitative PCR analysis of miR-378a-5p expression in mouse aorta tissue following implantation of an AngII microosmotic pump (n = 3). Panel B shows quantitative PCR analysis of miR-378a-5p expression in VSMCs following TNFα stimulation (n = 3). **p < 0.01, ***p < 0.001 vs. saline or control groups.

[0025] Figure 2Overexpression of miR-378a-5p in mice delayed the development of AAA. Figure A shows a gross image of the aorta in each group of mice (saline group, n=5; Ang II group, n=10); Figure B shows the AAA tumorigenesis rate in each group of mice (saline group, n=5; Ang II group, n=10); Figures C and D show the maximum abdominal aortic aneurysm diameter measured by ultrasound (saline group, n=5; Ang II group, n=10); Figure E shows HE, Sirius red, and elastin staining of the aorta in each group of mice (n=3); Figures F and G show the statistical results of Sirius red and elastin staining (n=3); Figures H and H show the expression of VSMC differentiation markers in the aorta tissue of each group of mice detected by western blot (n=3). *P<0.05, ***P<0.001vs.Angomir-NC+saline; #P<0.05, ##P<0.01vs.Angomir-NC+AngII; &P<0.05, &&P<0.01vs.Angomir-378a-5p.

[0026] Figure 3 Low expression of miR-378a-5p in mice exacerbates the development of AAA. Figure A shows a gross image of aortic aneurysm formation in each group of mice (saline group, n=5; Ang II group, n=15); Figures BC show the maximum abdominal aortic aneurysm diameter measured by ultrasound (saline group, n=5; Ang II group, n=10); Figure D shows HE, Sirius red, and elastin staining of the aorta in each group of mice (n=3); Figures EF show the statistical analysis of Sirius red and elastin staining (n=3); and Figures GH show the expression of VSMC differentiation markers in the aorta tissue of each group of mice as assessed by western blot (n=3). *P<0.05, **P<0.01, ***P<0.001vs.Antagomir-NC+saline; #P<0.05, ##P<0.01vs.Antagomir-NC+AngII; &P<0.05, &&P<0.01vs.Antagomir-378a-5p.

[0027] Figure 4Overexpression of miR-378a-5p promotes VSMC differentiation and inhibits VSMC migration. Panel A shows the effect of mimics-378a-5p on VSMC differentiation markers after TNFα stimulation by western blot (n=3); Panel B shows the effect of mimics-378a-5p on VSMC differentiation markers after TNFα stimulation by quantitative PCR (n=3); Panel C shows the effect of mimics-378a-5p on VSMC migration after TNFα stimulation by transwell assay (n=3). **P<0.01, ***P<0.001 vs. Mimics-NC; ##P<0.01 vs. Mimics-NC+TNFα; &&P<0.01 vs. Mimics-378a-5p.

[0028] Figure 5 Down-regulation of miR-378a-5p inhibits VSMC differentiation and increases VSMC migration. Figure A shows the effect of inhibitor-378a-5p on VSMC differentiation markers after TNFα stimulation by western blot (n=3); Figure B shows the effect of inhibitor-378a-5p on VSMC differentiation markers after TNFα stimulation by quantitative PCR (n=3); Figure C shows the effect of inhibitor-378a-5p on VSMC migration after TNFα stimulation by transwell assay (n=3). **P<0.01, ***P<0.001 vs. Inhibitor-NC; ##P<0.01 vs. Inhibitor-NC+TNFα; &&P<0.01 vs. Inhibitor-378a-5p. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0030] Unless otherwise specified in the examples, the experiments were conducted under conventional conditions or those recommended by the manufacturer. All reagents and instruments used, for which the manufacturer is not indicated, are commercially available. The experimental data presented in this invention are all presented as percentages. The chi-square test was used to compare the two sample rates. Statistical analysis was performed using the GraphPad Prism 8.0 software package, with P < 0.05 considered statistically significant.

[0031] Example 1: miR-378a-5p expression is downregulated in animal and cell models of AAA.

[0032] 1. Establishment of mouse AAA model.

[0033] The experiment used 8-week-old male ApoE - / - Mouse. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Mice were housed in a specific pathogen-free animal room at a room temperature of (22 ± 2)°C, a humidity range of 45%–70%, and a 12-h light cycle with free access to food and water.

[0034] ApoE - / - Mice were randomly divided into a control group and an experimental group. The experimental group received a 28-day-old subcutaneous microosmotic pump containing angiotensin II (AngII, 1200 ng / kg / min), while the control group received a 28-day-old microosmotic pump containing an equal volume of saline. AngII was purchased from APExBIO Technology (Houston, US). After 28 days of pump implantation, the mice were killed and aortic tissue was obtained.

[0035] 2. Quantitative PCR was used to detect the expression of miR-378a-5p in aortic tissue of mice with AAA induced by AngII.

[0036] (1) RNA extraction from vascular tissue.

[0037] A. Place the mouse aorta in an RNase-free EP tube, add 1 mL of Trizol, add RNase-free grinding beads, and grind in a tissue grinder homogenizer for 120 s at 70 Hz.

[0038] B. Incubate at room temperature for 5 minutes, add 200 μL of chloroform, mix by inversion, and let stand at room temperature for 15 minutes.

[0039] C.12000rpm / s, 4℃, centrifuge for 15min.

[0040] D. Aspirate the supernatant, add an equal volume of isopropanol, invert and mix thoroughly, and let stand at room temperature for 10 minutes.

[0041] E. Centrifuge at 12000 rpm / s, 4°C for 15 min and discard the supernatant.

[0042] F. Add 1 mL of 75% ethanol to resuspend the precipitate.

[0043] G. Centrifuge at 12000 rpm / s, 4°C for 15 min and discard the supernatant.

[0044] H. Dry at room temperature and become transparent.

[0045] I. Add 25 μL of enzyme-free water to dissolve RNA.

[0046] (2) Reverse transcription reaction.

[0047] ①Poly(A)Tailing: Prepare the reaction system on ice and prepare the required reaction system according to the following proportions:

[0048] .

[0049] ②Reverse transcription reaction

[0050] A. Prepare the reverse transcription reaction system on ice. Prepare the required reaction system according to the following proportions:

[0051] ;

[0052] B. Mix the reaction system, incubate at 42°C for 1 hour, and then incubate at 72°C for 10 minutes.

[0053] C. After the reaction is completed, the obtained cDNA is placed on ice for later use or stored at -20℃.

[0054] ③ Quantitative PCR reaction

[0055] A. Prepare the reaction system on ice. Prepare the required reaction system according to the following proportions:

[0056] ;

[0057] B. Reaction Procedure

[0058] ;

[0059] C. Primer sequences

[0060] .

[0061] The results showed that compared with the saline group, the expression of miR-378a-5p in aorta tissue of the AngII group was significantly decreased ( Figure 1 A).

[0062] 3. Extraction and Culture of Primary Mouse VSMCs.

[0063] Six to eight eight-week-old male C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China) were sacrificed and disinfected in 75% alcohol. The aorta was isolated and excised, placed in ice-cold sterile phosphate-buffered saline (PBS) containing 1× antibiotics. Forceps were used to gently remove the aortic adventitia, and ophthalmic scissors were used to cut the aortic media into approximately 2 mm long pieces. The pieces were then resuspended in 3 mL of 0.15% type II collagenase digestion solution. The pieces were then transferred to a six-well plate and incubated at 37°C in a 5% CO2 incubator for digestion. After 2-3 hours, growth medium (DMEM supplemented with 20% fetal bovine serum) was added to terminate the digestion. Cells were centrifuged at 1000 rpm for 5 minutes to obtain a cell pellet. An appropriate amount of medium was added to disperse the cell pellet, and the cell suspension was plated in a six-well plate at 2 mL per well. After 3 days of culture, the medium was changed for the first time to remove any unadhered cells or tissue fragments. The medium was then changed every 3-5 days depending on cell growth. Cells were passaged when the cell density reached 90%. VSMCs from passages 6-10 were used for experiments. VSMCs were stimulated with TNFα (20 ng / mL) for 24 hours and then harvested.

[0064] 4. Quantitative PCR was used to detect the expression of miR-378a-5p in VSMCs after TNFα stimulation.

[0065] (1) Cell RNA extraction: After collecting cells, add 1 mL of Trizol and the remaining steps are the same as tissue RNA extraction.

[0066] (2) The steps of miRNA reverse transcription and quantitative PCR were the same as those in the histology part, and the specific method was the same as method 2 in Example 1.

[0067] The results showed that compared with the control group, TNFα significantly reduced the expression of miR-378a-5p in VSMCs ( Figure 1 B).

[0068] The above results showed that the expression of miR-378a-5p was significantly downregulated in mouse aorta tissue stimulated by AngII and VSMCs stimulated by TNFα, suggesting that miR-378a-5p may be involved in the occurrence and development of AAA.

[0069] Example 2: Overexpression of miR-378a-5p in mice delays the occurrence and development of AAA.

[0070] 1. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The rearing conditions were the same as those in Example 1.

[0071] 2. Angomir-378a-5p, which overexpresses miR-378a-5p, was injected into the tail vein of mice to establish an AAA model.

[0072] In order to clarify the effect of miR-378a-5p overexpression on the occurrence and development of AAA at the in vivo level, Angomir-378a-5p and its control Angomir-NC were purchased from MCE. Mice with miR-378a-5p overexpression were established by tail vein injection of Angomir-378a-5p, and the control group of mice were injected with Angomir-NC by tail vein. The injection dose of Angomir-378a-5p and Angomir-NC was 20nmol / 20g / time, 3 times a week, for a total of 4 weeks. Four weeks after the tail vein injection, AngII and saline were implanted to establish a mouse AAA model. The specific modeling method of AAA is the same as that in Example 1.

[0073] 3. Small animal ultrasound was used to measure the maximum abdominal aorta diameter of each group of mice.

[0074] The maximum abdominal aorta diameter of each group of mice was measured using a small animal ultrasound system Vevo 2100 apparatus (Visual Sonics, Toronto, Canada). Tumor formation was defined as a local dilation of the aorta exceeding 50% of its adjacent intact aorta.

[0075] The results showed that compared with the Angomir-NC+saline group, the tumor formation rate and the maximum abdominal aorta diameter of the mice in the Angomir-NC+AngII group were significantly increased; compared with the Angomir-378a-5p group, the tumor formation rate and the maximum abdominal aorta diameter of the mice in the Angomir-378a-5p+AngII group were significantly increased; compared with the Angomir-NC+AngII group, the tumor formation rate and the maximum abdominal aorta diameter of the mice in the Angomir-378a-5p+AngII group were significantly reduced ( Figure 2 AD).

[0076] 4. HE staining was used to detect the AAA formation in each group of mice.

[0077] The mice were killed 28 days after the pump was implanted, and the aortas of the mice in each group were collected and HE staining was performed to evaluate the AAA formation. The specific steps are as follows.

[0078] (1) Preparation of paraffin sections.

[0079] A. Sampling: Place aortic tissue in 4% paraformaldehyde solution and fix for 24 hours.

[0080] B. Dehydration: Dehydrate the tissue blocks in different alcohol concentrations, specifically 70% alcohol for 2 hours, 80% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol I for 4 hours, 95% alcohol II overnight, 100% alcohol I for 1.5 hours, and 100% alcohol II for 1.5 hours.

[0081] C. Transparency: Soak the tissue block in xylene I solution for 1 hour, then remove it and soak it in xylene II solution for 1 hour.

[0082] D. Wax immersion: overnight in paraffin I, 1 hour in paraffin II, and 1 hour in paraffin III.

[0083] E. Embedding: Embed the tissue blocks in paraffin and store at room temperature.

[0084] F. Sectioning: Slice the tissue block to a thickness of 3 μm using a paraffin microtome and mount the sections on glass slides.

[0085] G. Drying and baking the slides: Place the slides in a 60°C slide dryer for 1 hour, then place the slides in a 65°C oven for 48 hours.

[0086] (2) Dewaxing of sections: Place the sections in the following reagents according to the steps: xylene I for 20 minutes, xylene II for 20 minutes, 95% alcohol I for 15 minutes, 95% alcohol II for 15 minutes, 90% alcohol for 10 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally place in distilled water for 30 minutes.

[0087] (3) HE staining and photography.

[0088] A. Immerse the sections in hematoxylin solution for 20 minutes.

[0089] B. Place the paraffin sections in 1% hydrochloric acid for 30 seconds to differentiate, and then rinse with running water.

[0090] C. Place the paraffin sections in ammonia water for 30 seconds to allow the cell nuclei to turn blue, and then rinse with running water.

[0091] D. Cytoplasmic staining: Place the paraffin sections in a water-soluble eosin solution for 5 minutes, stain, and rinse with running water.

[0092] E. Clearing: Place paraffin sections in the following reagents as follows: 80% alcohol for 5 minutes, 90% alcohol for 5 minutes, 100% alcohol I for 5 minutes, 100% alcohol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes.

[0093] F. Sealing: Place the paraffin sections in a fume hood to dry and seal the sections with neutral resin.

[0094] G. Observe the HE staining results under a microscope and take photos.

[0095] The results showed that compared with the Angomir-NC+saline group, the aorta of the mice in the Angomir-NC+AngII group was significantly widened; compared with the Angomir-378a-5p+saline group, the aorta of the mice in the Angomir-378a-5p+AngII group was significantly widened; compared with the Angomir-NC+AngII group, the aorta of the mice in the Angomir-378a-5p+AngII group was less widened ( Figure 2 E).

[0096] 5. Sirius red staining was used to evaluate the fibrosis of aortic tissue in each group of mice.

[0097] After 28 days of implantation, the mice were killed and the aortas of the mice in each group were collected. The fibrosis of the aorta tissue of the mice in each group was evaluated by picrosirius red staining. The specific steps are as follows.

[0098] (1) The preparation and dewaxing steps of paraffin sections are the same as those for HE staining.

[0099] (2) Sirius red staining for 1 hour.

[0100] (3) Wash with water for 30 seconds.

[0101] (4) 3-5 min in anhydrous ethanol I, 5-10 min in anhydrous ethanol II.

[0102] (5) Transparency: 10 min in xylene I, 10 min in xylene II.

[0103] (6) Seal the slides and take photos.

[0104] The results showed that compared with the Angomir-NC+saline group, the aortic fibrosis of mice in the Angomir-NC+AngII group was significantly increased; compared with the Angomir-378a-5p+saline group, the aortic fibrosis of mice in the Angomir-378a-5p+AngII group was increased; compared with the Angomir-NC+AngII group, the degree of fibrosis in mice in the Angomir-378a-5p+AngII group was reduced ( Figure 2 EF).

[0105] 6. Elastin staining.

[0106] (1) Dewaxing of sections: Same as HE staining.

[0107] (2) Vascular elastin staining was performed according to the procedure of the Verhoef van Gibson Elastic kit (Sigma, T25A).

[0108] The results showed that compared with the Angomir-NC+saline group, the aorta elastic fiber of the mice in the Angomir-NC+AngII group was significantly ruptured; compared with the Angomir-378a-5p+saline group, the aorta elastic plate of the mice in the Angomir-378a-5p+AngII group was ruptured; compared with the Angomir-NC+AngII group, the degree of rupture of the aorta elastic plate in the mice in the Angomir-378a-5p+AngII group was reduced ( Figure 2 E, G).

[0109] 7. Western blot was used to detect the differentiation indexes and MMP2 expression of VSMCs in each group of mice.

[0110] (1) Protein extraction from mouse aorta tissue.

[0111] Aortic tissue was collected from each group of mice and added with an appropriate amount of protein lysis buffer. The tissue was lysed on ice for 30 minutes and then centrifuged at 4°C, 12,000 rpm / s, for 20 minutes. The supernatant was collected as total tissue protein. Protein concentration in the lysate was determined using a BCA colorimetric assay. Samples were prepared to a total protein concentration of 25 μg and then boiled in a 100°C water bath for 5 minutes.

[0112] (2) Western blot procedure: Prepare SDS-PAGE gel of the appropriate concentration, add 15 μL of protein sample to each sample well, turn on the power supply, and start electrophoresis. Follow the following voltage and time: 80 V for 30 min, 120 V for 60 min, and turn off the power supply after bromophenol blue has electrophoresed to the bottom of the glass plate. Transfer the sample to a PVDF membrane at 90 V for 2 h. Place the PVDF membrane in 5% milk blocking solution for 1 h, then add the primary antibody and incubate overnight at 4°C. Western blot analysis was performed using anti-MMP2 (Abcam) at a 1:1000 dilution, anti-CNN1 (Abcam) at a 1:1000 dilution, anti-SM22-α (Abcam) at a 1:1000 dilution, anti-α-SMA (Abcam) at a 1:1000 dilution, and anti-β-actin (Cell Signaling Technology) at a 1:1000 dilution as primary antibodies, respectively. Horseradish peroxidase-conjugated goat anti-mouse (or anti-rabbit) antibodies were used as secondary antibodies. The cells were visualized using an ECL kit (Amersham). The grayscale values ​​of the bands were measured and statistically analyzed using ImageJ 1.51 software.

[0113] The results showed that compared with the Angomir-NC+saline group, the expression of MMP2 in the aorta of mice in the Angomir-NC+AngII group was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased; compared with the Angomir-378a-5p+saline group, the expression of MMP2 in the aorta of mice in the Angomir-378a-5p+AngII group was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased; compared with the Angomir-NC+AngII group, the expression of MMP2 in the aorta of mice in the Angomir-378a-5p+AngII group was significantly decreased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly increased ( Figure 2 HI).

[0114] The above results show that miR-378a-5p overexpression can inhibit the occurrence and development of AAA.

[0115] Example 3: Low expression of miR-378a-5p in mice aggravates the occurrence and development of AAA.

[0116] 1. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The rearing conditions were the same as those in Example 1.

[0117] 2. Antagomir-378a-5p, which down-expresses miR-378a-5p, was injected into the tail vein of mice to establish an AAA model.

[0118] In order to further clarify the role of low expression of miR-378a-5p in the occurrence and development of AAA at the in vivo level, Antagomir-378a-5p and its control Antagomir-NC were purchased from Guangzhou Ruibo Company. Mice with low expression of miR-378a-5p were established by tail vein injection of Antagomir-378a-5p, and the control group of mice were injected with Antagomir-NC by tail vein. The injection dose of Antagomir-378a-5p and Antagomir-NC was 50nmol / 20g / time, 3 times a week, for a total of 4 weeks. Four weeks after the tail vein injection, AngII and saline were implanted to establish a mouse AAA model. The specific modeling method of AAA is the same as that in Example 1.

[0119] 3. Small animal ultrasound imaging was used to measure the maximum abdominal aorta diameter of mice in each group.

[0120] For details of the small animal ultrasound method, see Method 3 in Example 2.

[0121] The results showed that compared with the Antagomir-NC+saline group, the tumor formation rate and the maximum abdominal aorta diameter of the mice in the Antagomir-NC+AngII group were significantly increased; compared with the Antagomir-378a-5p+saline group, the tumor formation rate and the maximum abdominal aorta diameter of the mice in the Antagomir-378a-5p+AngII group were significantly increased; compared with the Antagomir-NC+AngII group, the tumor formation rate and the maximum abdominal aorta diameter of the mice in the Antagomir-378a-5p+AngII group were significantly increased ( Figure 3 AC).

[0122] 4. HE staining was used to detect the AAA formation in each group of mice.

[0123] The specific steps of HE staining are the same as those of method 4 in Example 2.

[0124] The results showed that compared with the Antagomir-NC+saline group, the aorta of the mice in the Antagomir-NC+AngII group was significantly widened; compared with the Antagomir-378a-5p+saline group, the aorta of the mice in the Antagomir-378a-5p+AngII group was significantly widened; compared with the Antagomir-NC+AngII group, the aorta of the mice in the Antagomir-378a-5p+AngII group was even more widened ( Figure 3 D).

[0125] 5. Sirius red staining was used to evaluate the fibrosis of aortic tissue in each group of mice.

[0126] The specific steps of Sirius red staining are the same as those of method 5 in Example 2.

[0127] The results showed that compared with the Antagomir-NC+saline group, the aortic fibrosis of mice in the Antagomir-NC+AngII group was significantly increased; compared with the Antagomir-378a-5p+salin group, the aortic fibrosis of mice in the Antagomir-378a-5p+AngII group was increased; compared with the Antagomir-NC+AngII group, the degree of fibrosis in mice in the Antagomir-378a-5p+AngII group was more obvious ( Figure 3 DE).

[0128] 6. Elastin staining.

[0129] The specific steps for elastin staining are the same as those of method 6 in Example 2.

[0130] The results showed that compared with the Antagomir-NC+saline group, the aortic elastic fibers of the mice in the Antagomir-NC+AngII group were significantly ruptured; compared with the Antagomir-378a-5p+saline group, the aortic elastic plates of the mice in the Antagomir-378a-5p+AngII group were significantly ruptured; compared with the Antagomir-NC+AngII group, the degree of rupture of the aortic elastic plates in the Antagomir-378a-5p+AngII group was more obvious ( Figure 3 D, F).

[0131] 7. Western blot was used to detect the differentiation indexes and MMP2 expression of VSMCs in each group of mice.

[0132] The methods for protein extraction and western blot of mouse aorta tissue were the same as those in method 7 in Example 2.

[0133] The results showed that compared with the Antagomir-NC+saline group, the expression of MMP2 in the aorta of mice in the Antagomir-NC+AngII group was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased; compared with the Antagomir-378a-5p+saline group, the expression of MMP2 in the aorta of mice in the Antagomir-378a-5p+AngII group was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased; compared with the Antagomir-NC+AngII group, the expression of MMP2 in the aorta of mice in the Antagomir-378a-5p+AngII group was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased ( Figure 3 GH).

[0134] The above results showed that low expression of miR-378a-5p aggravated the occurrence and development of AAA.

[0135] Example 4: Overexpression of miR-378a-5p promotes VSMCs differentiation and inhibits VSMCs migration.

[0136] 1. The extraction and culture methods of primary mouse VSMCs were the same as those in Method 3 of Example 1.

[0137] 2. Establish mouse VSMCs overexpressing miR-378a-5p.

[0138] To determine the effects of miR-378a-5p overexpression on the differentiation and migration of mouse VSMCs, VSMCs overexpressing miR-378a-5p were established. VSMCs were plated in six-well plates and transfected with Mimics-378a-5p or its control, Mimics-NC, when the cell density reached 80%. To prepare the transfection system, Opti-MEM medium was added to an EP tube, followed by the transfection reagent Lipofectamine RNAiMAX and Mimics-378a-5p (final concentration of 50 nM) or its control, Mimics-NC (final concentration of 50 nM). The transfection mixture was incubated at room temperature for 15 minutes before being added to the six-well plate. The cells were cultured in a 37°C, 5% CO2 incubator for 6 hours. The medium was then replaced with DMEM supplemented with 20% fetal bovine serum and 1% antibiotics and cultured for an additional 24 hours. The cells were then stimulated with TNFα (20 ng / ml) for 24 hours.

[0139] 3. Western blot analysis of the effect of overexpression of miR-378a-5p on MMP2 and differentiation markers in VSMCs. The specific steps of Western blot were the same as those in Method 7 of Example 2.

[0140] The results showed that compared with Mimics-NC, the expression of MMP2 in the Mimics-NC+TNFα group was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased; compared with the Mimics-378a-5p group, the expression of MMP2 in the Mimics-378a-5p+TNFα group was increased, and the expression of CNN1, SM22-α, and α-SMA proteins was decreased; compared with the Mimics-NC+TNFα group, the expression of MMP2 in the Mimics-378a-5p+TNFα group was significantly decreased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly increased ( Figure 4 A).

[0141] 4. Quantitative PCR was used to detect the effect of overexpression of miR-378a-5p on MMP2 and differentiation indicators in VSMCs.

[0142] (1) The steps for extracting cellular RNA are the same as those in method 4 (2) of Example 1.

[0143] (2) Using Takara reverse transcription kit

[0144] A. Removal of genomic DNA

[0145] ;

[0146] B. Reverse transcription reaction

[0147] ;

[0148] Reaction conditions: 37°C, 15 min-85°C, 5 s.

[0149] (3) Quantitative PCR reaction

[0150] A. Primer sequences

[0151] ;

[0152] B. Fluorescence quantitative PCR

[0153] .

[0154] Prepare 20 μL qPCR reaction system according to the volume in the table above, and set up 3 replicate wells for each sample group. Reaction conditions: 95℃ for 5 min, (95℃ for 5 s, 60℃ for 30 s, 72℃ for 30 s) × 40 cycles, 72℃ for 5 s, 95℃ for 15 s. －△△Ct The expression of each sample group was analyzed.

[0155] The results showed that compared with Mimics-NC, the expression of MMP2 in the Mimics-NC+TNFα group was significantly increased, and the transcriptional expression levels of CNN1, SM22-α, and α-SMA were significantly decreased; compared with the Mimics-378a-5p group, the expression of MMP2 in the Mimics-378a-5p+TNFα group was increased, and the transcriptional expression levels of CNN1, SM22-α, and α-SMA were decreased; compared with the Mimics-NC+TNFα group, the expression of MMP2 in the Mimics-378a-5p+TNFα group was significantly decreased, and the transcriptional expression levels of CNN1, SM22-α, and α-SMA were significantly increased ( Figure 4 B).

[0156] 5. The Transwell method was used to detect the effect of overexpression of miR-378a-5p on VSMCs migration.

[0157] (1) VSMCs from mice transfected with Mimics-378a-5p and Mimics-NC were seeded into the upper chamber of a Transwell chamber (approximately 2.5×10 4 The upper chamber medium was replaced with serum-free medium.

[0158] (2) Add 500 μL of 10% serum culture medium to the lower chamber.

[0159] (3) After 24 hours, the cells in the upper chamber were washed and fixed with 4% paraformaldehyde.

[0160] (4) The cells in the upper chamber were stained with 0.1% crystal violet solution (Solabo, Beijing, China) at room temperature for 20 min.

[0161] (5) Take pictures under a microscope and count the number of cells in the field of view to quantify the number of migrated cells.

[0162] The results showed that compared with Mimics-NC, the cell migration in the Mimics-NC+TNFα group increased; compared with the Mimics-378a-5p group, the cell migration in the Mimics-378a-5p+TNFα group increased; compared with the Mimics-NC+TNFα group, the cell migration in the Mimics-378a-5p+TNFα group was significantly reduced ( Figure 4 C).

[0163] The above results showed that miR-378a-5p overexpression promoted VSMCs differentiation and inhibited VSMCs migration.

[0164] Example 5: Low expression of miR-378a-5p inhibits VSMCs differentiation and increases VSMCs migration.

[0165] 1. The extraction and culture methods of primary mouse VSMCs were the same as those in Method 3 of Example 1.

[0166] 2. Establish mouse VSMCs overexpressing miR-378a-5p.

[0167] To determine the effects of low miR-378a-5p expression on differentiation and migration in mouse VSMCs, we established VSMCs expressing low miR-378a-5p. VSMCs were plated in six-well plates and transfected with inhibitor-378a-5p or its control inhibitor-NC (final concentration: 80%) when the cells reached 80% confluence. To prepare the transfection system, Opti-MEM medium was added to an EP tube, followed by the transfection reagent Lipofectamine RNAiMAX and inhibitor-378a-5p (final concentration: 100 nM) or its control inhibitor-NC (final concentration: 100 nM). The transfection mixture was incubated at room temperature for 15 minutes before being added to a six-well plate. The cells were cultured in a 37°C, 5% CO2 incubator for 6 hours. The medium was then replaced with DMEM supplemented with 20% fetal bovine serum and 1% antibiotics and cultured for an additional 24 hours. The cells were then stimulated with TNFα (20 ng / mL) for 24 hours.

[0168] 3. Western blot analysis of the effect of low miR-378a-5p expression on MMP2 and differentiation markers in VSMCs. The specific steps of Western blot were the same as those in Method 7 of Example 2.

[0169] The results showed that compared with the Inhibitor-NC group, the expression of MMP2 protein was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased in the Inhibitor-NC+TNFα group; compared with the Inhibitor-378a-5p group, the expression of MMP2 protein was increased, and the expression of CNN1, SM22-α, and α-SMA proteins was decreased in the Inhibitor-378a-5p+TNFα group; compared with the Inhibitor-NC+TNFα group, the expression of MMP2 was significantly increased, and the expression of CNN1, SM22-α, and α-SMA proteins was significantly decreased in the Inhibitor-378a-5p+TNFα group ( Figure 5 A).

[0170] 4. Quantitative PCR was used to detect the effect of low expression of miR-378a-5p on MMP2 and differentiation indicators in VSMCs.

[0171] The extraction steps of cell RNA, reverse transcription reaction and quantitative PCR method are the same as those of method 4 in Example 4.

[0172] The results showed that compared with the Inhibitor-NC group, the transcriptional expression of MMP2 in the Inhibitor-NC+TNFα group was significantly increased, while the transcriptional expression of CNN1, SM22-α, and α-SMA was significantly decreased; compared with the Inhibitor-378a-5p group, the transcriptional expression of MMP2 in the Inhibitor-378a-5p+TNFα group was increased, while the transcriptional expression of CNN1, SM22-α, and α-SMA was decreased; compared with the Inhibitor-NC+TNFα group, the transcriptional expression of MMP2 in the Inhibitor-378a-5p+TNFα group was significantly increased, while the transcriptional expression of CNN1, SM22-α, and α-SMA was significantly decreased ( Figure 4 B).

[0173] 5. The Transwell method was used to detect the effect of low expression of miR-378a-5p on VSMCs migration.

[0174] The Transwell method is the same as method 5 in Example 4.

[0175] The results showed that compared with the Inhibitor-NC group, the cell migration in the Inhibitor-NC+TNFα group increased; compared with the Inhibitor-378a-5p group, the cell migration in the Inhibitor-378a-5p+TNFα group increased; compared with the Inhibitor-NC+TNFα group, the cell migration in the Inhibitor-378a-5p+TNFα group increased significantly ( Figure 5C).

[0176] The above results showed that low expression of miR-378a-5p inhibited VSMCs differentiation and increased VSMCs migration.

[0177] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting miR-378a-5p expression levels in the preparation of a mouse abdominal aortic aneurysm detection kit.

2. The use according to claim 1, characterized in that The kit is used for diagnosis and / or prognosis evaluation of abdominal aortic aneurysm in mice; compared with normal tissues, the miR-378a-5p expression level is downregulated in abdominal aortic aneurysm cells or tissues in mice.

3. Use of the primers for amplifying miR-378a-5p described in claim 1 in preparing a mouse abdominal aortic aneurysm detection kit.

4. The use according to claim 3, characterized in that The kit is used for diagnosis and / or prognosis evaluation of abdominal aortic aneurysm in mice; compared with normal tissues, the miR-378a-5p expression level is downregulated in abdominal aortic aneurysm cells or tissues in mice.

5. The use according to claim 3, characterized in that The sequences of the primers are: forward: 5'-CTCCTGACTCCAGGTCCTGTGT-3'; reverse: 5'-CGGCGTCACCGGGTGTAAATC-3'.

6. Use of a pharmaceutical composition for preparing a medicament for treating abdominal aortic aneurysm; the pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more active ingredients selected from the group consisting of: (1) miR-378a-5p; (2) a polynucleotide encoding a miR-378a-5p gene; (3) an expression construct comprising the miR-378a-5p described in (1) or the polynucleotide described in (2); (4) A nucleic acid molecule of miR-378a-5p or a recombinant vector or recombinant cell of its nucleic acid molecule.

7. The pharmaceutical composition according to claim 6, characterized in that The drug is in any therapeutically acceptable dosage form.

8. The pharmaceutical composition according to claim 6, characterized in that The drug is any drug in a therapeutically acceptable dose.

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