Medical application of MiR-378a-5p in prevention and treatment of abdominal aortic aneurysm

Abstract

The invention discloses application of miR-378a-5p, and belongs to the technical field of molecular biomedical treatment. Mainly relates to application of intervention of miR-378a-5p expression in preparation of drugs for preventing and treating abdominal aortic aneurysm. A large number of experiments prove that in a mouse abdominal aortic aneurysm model, the expression of miR-378a-5p in aortic tissues of abdominal aortic aneurysm is obviously reduced. The low expression of the miR-378a-5p inhibits the differentiation of the VSMCs, increases the migration of the VSMCs, and aggravates the occurrence and development of the abdominal aortic aneurysm. The miR-378a-5p is overexpressed in a mouse, so that the occurrence and development of abdominal aortic aneurysm are delayed, the differentiation of VSMCs is promoted, and the migration of VSMCs is inhibited. The invention provides a new target for diagnosis and treatment of abdominal aortic aneurysm, and 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 vascular degenerative disease characterized by aortic diameter expansion greater than 30 mm or greater than 50%. It is a common and life-threatening disease in the elderly. Currently, large, asymptomatic AAA or symptomatic or ruptured AAA of any size require AAA repair. Therefore, it is crucial to find early prevention and treatment targets for AAA.

[0003] Studies have shown that the occurrence and development of AAA involves a variety of pathological processes, including extracellular matrix (ECM) decomposition, inflammation, vascular smooth muscle cell (VSMC) phenotypic transformation and oxidative stress, etc. These pathological processes are jointly involved in the occurrence and development of AAA. VSMCs are the main component of the vascular wall and are essential for maintaining the integrity of the vascular wall and vascular homeostasis. In the normal vascular system, VSMCs are located in the tunica media and are in a quiescent state. VSMCs can dedifferentiate under pathological and physiological stimulation, leading to vascular damage. In AAA, the homeostasis of VSMCs is disturbed, and VSMCs undergo phenotypic transformation, resulting in VSMS migration and apoptosis. It can also promote the occurrence and development of AAA by promoting the production of inflammatory factors and elastin-degrading matrix metalloproteinases. Therefore, targeted improvement of the biological function of VSMCs will be of great significance for finding new prevention and treatment targets for AAA.

[0004] MicroRNA (miRNA) is a class of conserved endogenous small non-coding single-stranded RNA with about 20 nucleotides. MiRNA regulates target genes by partially complementary targeting mRNA. MiRNAs are involved in a variety of cell biological functions, including proliferation, differentiation, apoptosis, migration, invasion and angiogenesis. Increasing evidence shows that miRNA plays an important role in the occurrence and development of AAA, such as miRNA-29b, miRNA-33b, miRNA-21 and miRNA-24. Studies have shown that miR-378a-5p inhibits the proliferation of colon cancer cells by targeting cyclin-dependent kinase 1 (CDK1); miR378a-5p regulates VSMCs proliferation by targeting CDK1, and may play an important role in in-stent restenosis. However, the role and mechanism of miR378a-5p in AAA are still 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 effect 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 invention discloses application of a reagent for detecting the expression level of miR-378a-5p in preparing 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 the preparation of 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: (1) miR-378a-5p, or a modified miR-378a-5p derivative, or a miR-378a-5p analog; (2) a polynucleotide encoding a miR-378a-5p gene (3) an expression construct, wherein the expression construct comprises the miR-378a-5p described in (1) or the polynucleotide described in (2); (4) An agonist of miR-378a-5p as described in (1); (5) miR-378a-5p nucleic acid molecule or its recombinant vector or recombinant cell; (6) An agent capable of upregulating the expression of miR-378a-5p or its activity.

[0013] Furthermore, the pharmaceutical composition is used in the preparation of a drug for treating abdominal aortic aneurysm.

[0014] Furthermore, the drug is in any pharmaceutically therapeutically acceptable dosage form.

[0015] Furthermore, the drug is any drug in a therapeutically acceptable dose.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] The present invention has found through a large number of experiments that the mouse AAA model is established by the method of angiotensin II (AngII) implantation pump, and it is found that the expression of miR-378a-5p is significantly downregulated in the aortic tissue of AAA. After the miR-378a-5p low-expression or over-expression model in mice is constructed by tail vein injection of miR-378a-5p antagomiR and angomiR, the AAA model is established, and it is found that over-expression of miR-378a-5p in mice reduces AAA formation; low-expression of miR-378a-5p aggravates AAA formation. Cytological level found that tumor necrosis factor alpha (TNFα) stimulation can inhibit VSMCs differentiation and promote VSMCs migration, while down-regulating miR-378a-5p expression in VSMCs. MiR-378a-5p overexpression can inhibit VSMCs migration and dedifferentiation caused by TNFα; low-expression of miR-378a-5p increases VSMCs migration and dedifferentiation caused by TNFα. The above results indicate that miR-378a-5p plays an important regulatory role in the occurrence of AAA, and intervention in miR-378a-5p may be a target for the prevention and treatment of AAA. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In animal models and cell models of AAA, miR-378a-5p expression is downregulated. Figure A is a quantitative PCR test for the expression of miR-378a-5p in mouse aorta tissue after implantation of AngII micro-osmotic pump (n=3). Figure B is a quantitative PCR test for the expression of miR-378a-5p after TNFα stimulation of VSMCs (n=3). **p<0.01, ***p<0.001vs.Saline or Control group.

[0019] Figure 2Overexpression of miR-378a-5p in mice delayed the occurrence and development of AAA. Figure A is a general picture of the aorta of each group of mice (saline group n=5; AngII group n=10); Figure B is the statistics of AAA tumor formation rate of each group of mice (saline group n=5; AngII group n=10); Figure CD is the maximum abdominal aortic aneurysm diameter measured by ultrasound (saline group n=5; AngII group n=10); Figure E is HE staining, Sirius red staining and elastin staining of the aorta of each group of mice (n=3); Figure FG is the statistics of Sirius red staining and elastin staining (n=3); Figure HI is the expression of VSMCs differentiation indicators 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.

[0020] Figure 3 Low expression of miR-378a-5p in mice aggravates the occurrence and development of AAA. Figure A is a general picture of aortic aneurysm formation in each group of mice (saline group n=5; AngII group n=15); Figure BC is the maximum abdominal aortic aneurysm diameter measured by ultrasound (saline group n=5; AngII group n=10); Figure D is HE staining, Sirius red staining and elastin staining of the aorta of mice in each group (n=3); Figure EF is the statistics of Sirius red staining and elastin staining (n=3); Figure GH is the expression of VSMCs differentiation indicators in the aortic tissue of each group of mice detected 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.

[0021] Figure 4Overexpression of miR-378a-5p promoted VSMCs differentiation and inhibited VSMCs migration. Figure A shows the effect of Mimics-378a-5p on VSMCs differentiation after TNFα stimulation detected by western blot (n=3); B shows the effect of Mimics-378a-5p on VSMCs differentiation after TNFα stimulation detected by quantitative PCR (n=3); Figure C shows the effect of Mimics-378a-5p on VSMCs migration after TNFα stimulation detected by transwell assay (n=3). **P<0.01, ***P<0.001vs.Mimics-NC; ##P<0.01vs.Mimics-NC+TNFα; &&P<0.01 vs. Mimics-378a-5p.

[0022] Figure 5 The low expression of miR-378a-5p inhibited VSMCs differentiation and increased VSMCs migration. Figure A is the effect of Inhibitor-378a-5p on VSMCs differentiation after TNFα stimulation detected by western blot (n=3); B is the effect of Inhibitor-378a-5p on VSMCs differentiation after TNFα stimulation detected by quantitative PCR (n=3); Figure C is the effect of Inhibitor-378a-5p on VSMCs migration after TNFα stimulation detected by transwell experiment (n=3). **P<0.01, ***P<0.001vs.Inhibitor-NC; ##P<0.01 vs. Inhibitor-NC+TNFα; &&P<0.01 vs. Inhibitor-378a-5p. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0024] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products available on the market. The experimental data of the present invention are all percentages, and the comparison of the two sample rates is performed using the chi-square test. The statistical analysis is performed using the GraphPad Prism 8.0 software package, and P < 0.05 is considered statistically significant.

[0025] Example 1: miR-378a-5p expression is downregulated in animal models and cell models of AAA.

[0026] 1. Establishment of mouse AAA model.

[0027] 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 with a room temperature of (22±2)°C, a humidity of 45%-70%, a 12-h light cycle, and free access to food and water.

[0028] ApoE - / - Mice were randomly divided into a control group and an experimental group. The experimental group mice were subcutaneously implanted with a 28-day micro-osmotic pump containing angiotensin II (AngII, 1200ng / kg / min), and the control group mice were implanted with a 28-day micro-osmotic pump containing an equal volume of saline. AngII was purchased from APExBIO Technology (Houstun, US). After 28 days of implantation, the mice were killed and the aortic tissue was obtained.

[0029] 2. Quantitative PCR was used to detect the expression of miR-378a-5p in aortic tissue of mice with AAA established by AngII.

[0030] (1) RNA extraction from vascular tissue.

[0031] A. Place the mouse aorta in an RNase-free EP tube, add 1 mL of Trizol, put in RNase-free grinding beads, and grind in a tissue grinder homogenizer for 120 s at 70 Hz.

[0032] B. Let stand at room temperature for 5 minutes, add 200 μL of chloroform, mix by inverting, and let stand at room temperature for 15 minutes.

[0033] C.12000rpm / s, 4℃, centrifuge for 15min.

[0034] D. Aspirate the supernatant, add an equal volume of isopropanol, invert and mix, and let stand at room temperature for 10 minutes.

[0035] E. Centrifuge at 12000 rpm / s, 4°C for 15 min and discard the supernatant.

[0036] F. Add 1 mL of 75% ethanol to resuspend the precipitate.

[0037] G. Centrifuge at 12000 rpm / s, 4°C for 15 min and discard the supernatant.

[0038] H. Dry at room temperature and become transparent.

[0039] I. Add 25 μL of enzyme-free water to dissolve RNA.

[0040] (2) Reverse transcription reaction.

[0041] ①Poly(A)Tailing: Prepare the reaction system on ice and prepare the required reaction system according to the preparation ratio in the following table: .

[0042] ②Reverse transcription reaction A. Prepare the reverse transcription reaction system on ice and prepare the required reaction system according to the following proportions: ; B. Mix the above reaction system, react at 42℃ for 1h, and then incubate at 72℃ for 10min; C. After the reaction is completed, the obtained cDNA is placed on ice for later use or stored at -20℃.

[0043] ③Quantitative PCR reaction A. Prepare the reaction system on ice and prepare the required reaction system according to the following proportions: ; B. Reaction Procedure ; C. Primer sequences .

[0044] The results showed that compared with the saline group, the expression of miR-378a-5p in aortic tissue of the AngII group was significantly decreased ( Figure 1 A).

[0045] 3. Extraction and culture of primary mouse VSMCs.

[0046] Six to eight 8-week-old male C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China) were killed and disinfected in 75% alcohol. The aorta of the mouse was isolated and cut off, placed in precooled sterile phosphate buffer containing 1× antibiotics, and the aortic adventitia was gently removed with forceps. The aortic media tissue strips were cut into small pieces of about 2 mm long with ophthalmic scissors, and then resuspended in 3 mL of 0.15% type II collagenase digestion solution. Then it was transferred to a six-well plate and placed in a 37°C, 5% CO2 cell culture incubator for digestion. After 2-3 hours, growth medium (DMEM medium containing 20% ​​fetal bovine serum) was added to terminate the digestion. After centrifugation at 1000 rpm / s for 5 minutes, the cell pellet was obtained, and an appropriate amount of culture medium was added to blow the cell pellet away. The cell suspension was inoculated in a 6-well plate, 2 mL per well. After 3 days of culture, the first medium change was performed to remove non-adherent cells or tissue blocks. After that, the medium was changed every 3-5 days according to the cell growth, and the cells were passaged when the cell growth density reached 90%. VSMCs of passage 6-10 were used for the experiment. VSMCs were stimulated with TNFα (20 ng / mL) for 24 hours and then the cells were collected.

[0047] 4. Quantitative PCR was used to detect the expression of miR-378a-5p in VSMCs after TNFα stimulation.

[0048] (1) Cell RNA extraction: After collecting cells, add 1 mL of Trizol and the remaining steps are the same as tissue RNA extraction.

[0049] (2) The steps of miRNA reverse transcription and quantitative PCR are the same as those in the histology part, and the specific method is the same as method 2 in Example 1.

[0050] The results showed that compared with the control group, TNFα significantly reduced the expression of miR-378a-5p in VSMCs ( Figure 1 B).

[0051] The above results showed that the expression of miR-378a-5p was significantly downregulated in mouse aortic tissue stimulated by AngII and VSMCs stimulated by TNFα, suggesting that miR-378a-5p may be involved in the occurrence and development of AAA.

[0052] Example 2: Overexpression of miR-378a-5p in mice delays the occurrence and development of AAA.

[0053] 1. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The feeding conditions were the same as those in Example 1.

[0054] 2. Angomir-378a-5p overexpressing miR-378a-5p was injected into the tail vein of mice to establish the AAA model.

[0055] 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 mice in the control group 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 implantation pumps were started to establish a mouse AAA model. The specific modeling method of AAA is the same as that in Example 1.

[0056] 3. Small animal ultrasound was used to measure the maximum abdominal aorta diameter of each group of mice.

[0057] 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), and tumor formation was defined as a local dilation of the aorta exceeding 50% of its adjacent intact aorta.

[0058] 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).

[0059] 4. HE staining was used to detect AAA formation in each group of mice.

[0060] 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.

[0061] (1) Preparation of paraffin sections.

[0062] A. Sampling: Place aortic tissue in 4% paraformaldehyde solution and fix for 24 hours.

[0063] 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.

[0064] C. Transparency: Soak the tissue block in xylene I solution for 1 hour, then take it out and soak it in xylene II solution for 1 hour.

[0065] D. Wax immersion: overnight in paraffin I, 1 hour in paraffin II, and 1 hour in paraffin III.

[0066] E. Embedding: Embed the tissue blocks in paraffin and place at room temperature.

[0067] F. Sectioning: Slice the tissue block with a paraffin slicer to a thickness of 3 μm and mount the sections on glass slides.

[0068] G. Drying and baking the slides: Place the slides in a 60℃ slide dryer for 1 hour, then place the slides in a 65℃ oven for 48 hours.

[0069] (2) Dewaxing of sections: Place the sections in the following reagents according to the steps: xylene I for 20 min, xylene II for 20 min, 95% alcohol I for 15 min, 95% alcohol II for 15 min, 90% alcohol for 10 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and finally place in distilled water for 30 min.

[0070] (3) HE staining and photography.

[0071] A. Immerse the sections in hematoxylin solution for 20 minutes.

[0072] B. Place the paraffin sections in 1% hydrochloric acid for 30 seconds for differentiation and rinse with running water.

[0073] C. Place the paraffin sections in ammonia water for 30 seconds to turn the cell nuclei blue, and then rinse with running water.

[0074] D. Cytoplasmic staining: Place the paraffin sections in a water-soluble eosin solution for 5 minutes, stain, and rinse with running water.

[0075] E. Transparency: Place the paraffin sections in the following reagents according to the steps: 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.

[0076] F. Sealing: Place the paraffin sections in a fume hood to dry and use neutral resin to seal the sections.

[0077] G. Observe the HE staining results under a microscope and take photos.

[0078] 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 widening degree of the mice in the Angomir-378a-5p+AngII group was reduced ( Figure 2 E).

[0079] 5. Sirius red staining was used to evaluate the fibrosis of aortic tissue in each group of mice.

[0080] The mice were killed 28 days after the pump was implanted, and the aortas of the mice in each group were collected and picrosirius red staining was performed to evaluate the fibrosis of the aortic tissue of the mice in each group. The specific steps are as follows.

[0081] (1) The preparation and dewaxing procedures of paraffin sections are the same as those for HE staining.

[0082] (2) Sirius red staining for 1 hour.

[0083] (3) Wash with water for 30 seconds.

[0084] (4) 3-5 min in anhydrous ethanol I and 5-10 min in anhydrous ethanol II.

[0085] (5) Transparency: 10 min in xylene I, 10 min in xylene II.

[0086] (6) Seal the slides and take photos.

[0087] 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 of mice in the Angomir-378a-5p+AngII group was reduced ( Figure 2 EF).

[0088] 6. Elastin staining.

[0089] (1) Dewaxing of sections: Same as HE staining.

[0090] (2) Vascular elastin staining was performed according to the procedure of the Verhoef van Gibson Elastic (Sigma, T25A) kit.

[0091] The results showed that compared with the Angomir-NC+saline group, the aorta elastic fiber rupture of the mice in the Angomir-NC+AngII group was obvious; 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 rupture of the aorta elastic plate in the Angomir-378a-5p+AngII group was alleviated ( Figure 2 E, G).

[0092] 7. Western blot was used to detect the differentiation indexes and MMP2 expression of VSMCs in each group of mice.

[0093] (1) Protein extraction from mouse aorta tissue.

[0094] The aorta tissues of mice in each group were collected, and an appropriate amount of protein lysis buffer was added. After lysis on ice for 30 minutes, the supernatant was collected as the total tissue protein at 4°C, 12000rpm / s for 20 minutes. The protein concentration in the lysate was determined using a BCA colorimetric kit. After the sample was prepared according to the total amount of protein of 25μg, it was boiled in a 100°C water bath for 5 minutes.

[0095] (2) Western blot steps: Prepare SDS-PAGE gel of corresponding concentration, add 15 μL of protein sample to each sample well, turn on the power, and start electrophoresis. Perform the electrophoresis according to the following voltage and time: 80V voltage, 30min, 120V voltage, 60min, and turn off the power after bromophenol blue electrophoresis to the bottom of the glass plate. Transfer the sample to the PVDF membrane at 90V voltage 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 detection was performed using 1:1000 anti-MMP2 (Abcam), 1:1000 anti-CNN1 (Abcam), 1:1000 anti-SM22-α (Abcam), 1:1000 anti-α-SMA (Abcam), and 1:1000 anti-β-actin (Cell Signaling Technology) as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody as secondary antibody. ECL kit (Amersham) was used for luminescence development. ImageJ 1.51 software was used to measure the gray value of the bands and perform statistical analysis.

[0096] The results showed that compared with Angomir-NC+saline group, the expression of MMP2 in the aorta of mice in Angomir-NC+AngII group was significantly increased, and the expression of CNN1, SM22-α and α-SMA proteins was significantly decreased; compared with Angomir-378a-5p+saline group, the expression of MMP2 in the aorta of mice in Angomir-378a-5p+AngII group was significantly increased, and the expression of CNN1, SM22-α and α-SMA proteins was significantly decreased; compared with Angomir-NC+AngII group, the expression of MMP2 in the aorta of mice in 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).

[0097] The above results show that overexpression of miR-378a-5p can inhibit the occurrence and development of AAA.

[0098] Example 3: Low expression of miR-378a-5p in mice aggravates the occurrence and development of AAA.

[0099] 1. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The feeding conditions were the same as those in Example 1.

[0100] 2. Antagomir-378a-5p, which lowly expresses miR-378a-5p, was injected into the tail vein of mice to establish the AAA model.

[0101] 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 is 50nmol / 20g / time, 3 times a week, for a total of 4 weeks. Four weeks after the tail vein injection, AngII and saline implantation pumps were started to establish a mouse AAA model. The specific modeling method of AAA is the same as that in Example 1.

[0102] 3. Small animal ultrasound imaging was used to measure the maximum abdominal aorta diameter of mice in each group.

[0103] For details of the small animal ultrasound method, see Method 3 in Example 2.

[0104] 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).

[0105] 4. HE staining was used to detect AAA formation in each group of mice.

[0106] The specific steps of HE staining are the same as method 4 in Example 2.

[0107] 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 widened more significantly ( Figure 3 D).

[0108] 5. Sirius red staining was used to evaluate the fibrosis of aortic tissue in each group of mice.

[0109] The specific steps of Sirius red staining are the same as method 5 in Example 2.

[0110] The results showed that compared with the Antagomir-NC+saline group, the aortic fibrosis of the mice in the Antagomir-NC+AngII group was significantly increased; compared with the Antagomir-378a-5p+salin group, the aortic fibrosis of the mice in the Antagomir-378a-5p+AngII group was increased; compared with the Antagomir-NC+AngII group, the fibrosis of the mice in the Antagomir-378a-5p+AngII group was more obvious ( Figure 3 DE).

[0111] 6. Elastin staining.

[0112] The specific steps of elastin staining are the same as method 6 in Example 2.

[0113] 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 rupture of the aortic elastic plates in the Antagomir-378a-5p+AngII group was more obvious ( Figure 3 D, F).

[0114] 7. Western blot was used to detect the differentiation indexes and MMP2 expression of VSMCs in each group of mice.

[0115] The mouse aorta tissue protein extraction and western blot method are the same as method 7 in Example 2.

[0116] 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).

[0117] The above results showed that low expression of miR-378a-5p aggravated the occurrence and development of AAA.

[0118] Example 4: Overexpression of miR-378a-5p promotes VSMCs differentiation and inhibits VSMCs migration.

[0119] 1. The extraction and culture methods of primary mouse VSMCs were the same as those in method 3 of Example 1.

[0120] 2. Establish mouse VSMCs overexpressing miR-378a-5p.

[0121] In order to clarify the effect of overexpression of miR-378a-5p on differentiation and migration in mouse VSMCs, VSMCs overexpressing miR-378a-5p were established. Specific methods: VSMCs were plated in six-well plates, and Mimics-378a-5p and its control Mimics-NC were transfected when the cell density reached 80%. The transfection system was configured, and after adding an appropriate amount of Opti-MEM medium to the EP tube, the transfection reagent Lipofectamine RNAiMAX and Mimics-378a-5p (final concentration of 50nM) or its control Mimics-NC (final concentration of 50nM) were added respectively. The transfection system mixture was incubated at room temperature for 15min and then added to the 6-well plate. After 6h of culture in a 37℃5% CO2 cell culture incubator, it was replaced with DMEM medium containing 20% ​​fetal bovine serum and 1% antibiotics, and cultured for 24h, and then TNFα (20ng / ml) was stimulated for 24h.

[0122] 3. Western blot was used to detect the effect of overexpression of miR-378a-5p on MMP2 and differentiation indexes in VSMCs. The specific steps of Western blot were the same as those of method 7 in Example 2.

[0123] The results showed that compared with Mimics-NC, the expression of MMP2 in Mimics-NC+TNFα group was significantly increased, and the expression of CNN1, SM22-α and α-SMA proteins was significantly decreased; compared with Mimics-378a-5p group, the expression of MMP2 in Mimics-378a-5p+TNFα group was increased, and the expression of CNN1, SM22-α and α-SMA proteins was decreased; compared with Mimics-NC+TNFα group, the expression of MMP2 in 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).

[0124] 4. Quantitative PCR was used to detect the effect of overexpression of miR-378a-5p on MMP2 and differentiation indicators in VSMCs.

[0125] (1) The steps for extracting cellular RNA are the same as those in method 4 (2) of Example 1.

[0126] (2) Using Takara Reverse Transcription Kit A. Removal of genomic DNA ; B. Reverse transcription reaction ; Reaction conditions: 37°C, 15 min-85°C, 5 s.

[0127] (3) Quantitative PCR reaction A. Primer sequences ; B. Fluorescence quantitative PCR .

[0128] According to the volume in the table above, prepare 20 μL qPCR reaction system, and set up 3 replicate wells for each sample group. Reaction conditions: 95℃5min, (95℃5s, 60℃30s, 72℃30s)×40 cycles, 72℃5s, 95℃15s. －△△Ct The expression of each sample was analyzed.

[0129] The results showed that compared with Mimics-NC, the expression of MMP2 in Mimics-NC+TNFα group was significantly increased, and the transcriptional expression levels of CNN1, SM22-α and α-SMA were significantly decreased; compared with Mimics-378a-5p group, the transcriptional expression level of MMP2 in Mimics-378a-5p+TNFα group was increased, and the transcriptional expression levels of CNN1, SM22-α and α-SMA were decreased; compared with Mimics-NC+TNFα group, the transcriptional expression level of MMP2 in 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).

[0130] 5. The Transwell method was used to detect the effect of overexpression of miR-378a-5p on VSMCs migration.

[0131] (1) VSMCs from mice transfected with Mimics-378a-5p and Mimics-NC were seeded into the upper chamber of the Transwell chamber (approximately 2.5×10 4 The upper chamber medium was replaced with serum-free medium.

[0132] (2) Add 500 μL of culture medium containing 10% serum to the lower chamber.

[0133] (3) After 24 hours, the cells in the upper chamber were washed and fixed with 4% paraformaldehyde.

[0134] (4) The cells in the upper chamber were stained with 0.1% crystal violet solution (Solabo, Beijing, China) at room temperature for 20 min.

[0135] (5) Take pictures under a microscope and count the number of cells in the field of view to quantify the number of migrated cells.

[0136] 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).

[0137] The above results showed that miR-378a-5p overexpression promoted VSMCs differentiation and inhibited VSMCs migration.

[0138] Example 5: Low expression of miR-378a-5p inhibits VSMCs differentiation and increases VSMCs migration.

[0139] 1. The extraction and culture methods of primary mouse VSMCs were the same as those in method 3 of Example 1.

[0140] 2. Establish mouse VSMCs overexpressing miR-378a-5p.

[0141] In order to clarify the effect of low expression of miR-378a-5p on differentiation and migration of mouse VSMCs, VSMCs with low expression of miR-378a-5p were established. Specific methods: VSMCs were plated in six-well plates, and Inhibitor-378a-5p and its control Inhibitor-NC were transfected when the cells grew to 80%. The transfection system was configured. After adding an appropriate amount of Opti-MEM medium to the EP tube, the transfection reagent Lipofectamine RNAiMAX and Inhibitor-378a-5p (final concentration of 100nM) or its control Inhibitor-NC (final concentration of 100nM) were added respectively. The transfection system mixture was incubated at room temperature for 15min and then added to the 6-well plate. After 6h of culture in a cell culture incubator at 37℃ and 5% CO2, it was replaced with DMEM medium containing 20% ​​fetal bovine serum and 1% antibiotics, and cultured for 24h, and then TNFα (20ng / mL) was stimulated for 24h.

[0142] 3. Western blot was used to detect the effect of low expression of miR-378a-5p on MMP2 and differentiation indexes in VSMCs. The specific steps of Western blot were the same as those of method 7 in Example 2.

[0143] The results showed that compared with Inhibitor-NC, the expression of MMP2 protein in Inhibitor-NC+TNFα group was significantly increased, and the expression of CNN1, SM22-α and α-SMA proteins was significantly decreased; compared with Inhibitor-378a-5p group, the expression of MMP2 protein in Inhibitor-378a-5p+TNFα group was increased, and the expression of CNN1, SM22-α and α-SMA proteins was decreased; compared with Inhibitor-NC+TNFα group, the expression of MMP2 protein in Inhibitor-378a-5p+TNFα group was significantly increased, and the expression of CNN1, SM22-α and α-SMA proteins was significantly decreased ( Figure 5 A).

[0144] 4. Quantitative PCR was used to detect the effect of low expression of miR-378a-5p on MMP2 and differentiation indicators in VSMCs.

[0145] The extraction steps of cell RNA, reverse transcription reaction and quantitative PCR method are the same as method 4 in Example 4.

[0146] The results showed that compared with Inhibitor-NC, the transcriptional expression level of MMP2 in Inhibitor-NC+TNFα group was significantly increased, and the transcriptional expression levels of CNN1, SM22-α and α-SMA were significantly decreased; compared with Inhibitor-378a-5p group, the transcriptional expression level of MMP2 in Inhibitor-378a-5p+TNFα group was increased, and the transcriptional expression levels of CNN1, SM22-α and α-SMA were decreased; compared with Inhibitor-NC+TNFα group, the transcriptional expression level of MMP2 in Inhibitor-378a-5p+TNFα group was significantly increased, and the transcriptional expression levels of CNN1, SM22-α and α-SMA were significantly decreased ( Figure 4 B).

[0147] 5. The Transwell method was used to detect the effect of low expression of miR-378a-5p on VSMCs migration.

[0148] The Transwell method is the same as method 5 in Example 4.

[0149] The results showed that compared with Inhibitor-NC, 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 5 C).

[0150] The above results showed that low expression of miR-378a-5p inhibited VSMCs differentiation and increased VSMCs migration.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

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

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

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

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

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. A pharmaceutical composition, characterized in that The pharmaceutical combination comprises a pharmaceutically acceptable carrier and one or more active ingredients selected from the following groups: (1) miR-378a-5p, or a modified miR-378a-5p derivative, or a miR-378a-5p analog; (2) a polynucleotide encoding a miR-378a-5p gene; (3) an expression construct, wherein the expression construct comprises the miR-378a-5p described in (1) or the polynucleotide described in (2); (4) An agonist of miR-378a-5p as described in (1); (5) miR-378a-5p nucleic acid molecule or its recombinant vector or recombinant cell; (6) An agent capable of upregulating the expression of miR-378a-5p or its activity.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is used in preparing a medicine for treating abdominal aortic aneurysm.

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

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

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