CN116004814A - Medical application of miR-3154 and its downstream target gene Pax7 in diseases related to phenotypic transformation of VSMCs - Google Patents

Abstract

The present invention relates to the use of miR-3154 and its downstream Pax7, in particular to the use of miR-3154 as a biomarker in abdominal aortic aneurysm, and the preparation of miR-3154 inhibitors and its downstream Pax7 nucleic acid molecule for the prevention and/or The invention relates to a medicine for treating abdominal aortic aneurysms and diseases related to VSMCs phenotype conversion, and belongs to the field of biomedicine. The inventors found through experiments that miR-3154 was highly expressed in the serum and tissues of abdominal aortic aneurysms of humans and mice; overexpression of Pax7 could reverse the regulation of miR-3154 on VSMCs phenotype conversion; in vivo/in vitro studies found that tail vein injection Pax7 intervention virus can reduce the occurrence and development of abdominal aortic aneurysm, and has a certain antihypertensive effect. miR‑3154 has the potential of a biomarker in abdominal aortic aneurysm, and the intervention of its downstream target gene Pax7 can be used to prevent or treat abdominal aortic aneurysm, which has great clinical significance.

Description

Medical application of miR-3154 and its downstream target gene Pax7 in diseases related to phenotypic transformation of VSMCs

technical field

The invention belongs to the field of biomedicine and relates to the medical application of miR-3154 downstream target gene Pax7 intervention, in particular to the medical application of miR-3154 downstream target gene Pax7 intervention for preventing and/or treating abdominal aortic aneurysm.

Background technique

Abdominal aortic aneurysm (AAA) is a common aortic disease of unknown etiology, with an insidious onset and high morbidity and mortality rates. Increasing age is the most important risk factor for abdominal aortic aneurysm, and the incidence rate is as high as 8% in men over 65 years old. The New England Journal of Medicine defines an abdominal aortic aneurysm as having an aortic diameter >3 cm. Predisposing factors for abdominal aortic aneurysm include advanced age, family history, past or current smoking, hypercholesterolemia, and hypertension; diabetes is associated with a reduced risk. The main risks posed by abdominal aortic aneurysms are the risk of aneurysm rupture and patient death from hemorrhage. The prevalence of abdominal aortic aneurysm accounts for 63% to 79% of aortic aneurysms. Most abdominal aortic aneurysms are caused by atherosclerosis. They are generally located at the distal end of the renal artery and extend to the bifurcation of the abdominal aorta. Involving the iliac artery, occasionally located above the renal artery, also known as thoracoabdominal aortic aneurysm, mostly invades the inferior mesenteric artery branch. Pathological examination of abdominal aortic aneurysms showed rupture of elastic fibers in blood vessels, decreased elastin content, inflammation of the media and adventitia, infiltration of B lymphocytes and plasma cells with phenotype transformation of smooth muscle cells. For the prevention of abdominal aortic aneurysms, the guidelines recommend that the occurrence of atherosclerosis should be actively prevented first. If it has occurred, it should be actively treated to prevent the development of the lesion and strive for its reversal. If complications have occurred, they should be treated in time to prevent their deterioration and prolong the patient's life. The goal of treatment is to surgically repair the aneurysm before it ruptures. Although several factors influence the timing and type of repair, the most important predictor of aneurysm rupture is aneurysm diameter, with larger aneurysms associated with greater risk of rupture. In a prospective observational study of patients with abdominal aortic aneurysms who were ineligible for surgery, the risk of rupture was 1% per year in men with aneurysms 5.0 to 5.9 cm in diameter and 1% in men with aneurysms ≥ 6 cm in diameter. The risk was 14.1% per year; the corresponding analyzes for female patients were 3.9% and 22.3%, respectively, and there is a lack of effective treatment options for patients with abdominal aortic aneurysms who are not suitable for surgery. In summary, abdominal aortic aneurysm is a disease with high incidence, lack of preventive measures, single treatment and serious consequences of rupture. New interventions to prevent and treat the disease.

Vascular smooth muscle cells (VSMCs) are important cellular components of the arterial wall, mainly located in the middle layer of the arterial wall, and play an important role in maintaining the structure of the blood vessel wall. The phenotypic transformation of VSMCs plays an important role in the formation of abdominal aortic aneurysm. Inhibition of VSMC phenotypic transformation has been shown to slow the progression of abdominal aortic aneurysms. MiRNA (microRNA, miR) is a short-chain non-coding RNA containing 22 nucleotides that is highly conserved in evolution and has a wide range of biological functions. Recent studies have shown that microRNA is involved in various biological processes of VSMCs, including the proliferation and apoptosis of VSMCs. For example, miR-23 can promote the proliferation of VSMCs and inhibit the apoptosis of VSMCs. miR-155-5p can promote the apoptosis of VSMCs and aggravate abdominal aortic aneurysm by targeting FOS and ZIC3. Studies have confirmed that miR-3154 can be used as a prognostic marker for cervical cancer and leukemia. Pax7 is a transcription factor closely related to the process of skeletal muscle cell differentiation. However, the effects of miR-3154 and Pax7 on abdominal aortic aneurysms have not been reported, and their medicinal potential has not been protected by intellectual property rights.

Contents of the invention

In view of the above problems, the present invention provides the medical application of miR-3154 downstream target gene Pax7 intervention for preventing and/or treating abdominal aortic aneurysm.

In order to achieve the above object, the present invention provides the following technical solutions.

The present invention provides an application of a reagent for detecting the expression level of miR-3154 in the preparation of a kit for auxiliary diagnosis and/or prognosis assessment of diseases related to VSMCs phenotype conversion, characterized in that the VSMCs phenotype conversion related diseases The kit uses miR-3154 as a marker.

The present invention also provides a method for screening potential substances for preventing and/or treating diseases related to VSMCs phenotype transformation, characterized in that the method comprises the following steps:

Step 1, treating the system expressing miR-3154 with a candidate substance; and

Step 2, detecting the expression of miR-3154 in the system;

Wherein, if the candidate substance can reduce the expression of miR-3154, it indicates that the candidate substance is a potential substance for preventing and/or treating diseases related to phenotypic conversion of VSMCs.

Further, the step 1 includes: in the test group, adding candidate substances to the system expressing miR-3154; and/or

Step 2 includes: detecting the expression of miR-3154 in the system of the test group, and comparing it with the control group, wherein the control group is a system expressing miR-3154 without adding the candidate substance;

If the expression of miR-3154 in the test group is statistically higher than that in the control group, it indicates that the candidate is a potential substance for the prevention and/or treatment of diseases associated with phenotypic transformation of VSMCs.

The present invention also provides the application of an inhibitor of the expression level of miR-3154 or an agonist of the expression level of the downstream target gene Pax7 of miR-3154 in the preparation of medicines for treating diseases related to VSMCs phenotype conversion.

The present invention also provides a pharmaceutical composition for treating diseases related to VSMCs phenotype conversion, characterized in that the pharmaceutical composition includes an inhibitor of miR-3154 expression level or a carrier and reagent for overexpressing Pax7.

Further, the inhibitor of the expression level of miR-3154 is selected from one or more active ingredients in the following group:

(1) miR-3154, or modified miR-3154 derivatives, or miR-3154 analogs;

(2) Pre-miRNA, which can be processed into miR-3154 in the host;

(3) polynucleotide, the polynucleotide can be transcribed by the host into the precursor miRNA described in (2), and processed to form miR-3154, or can be processed into miR-3154 in the host;

(4) An expression construct, which contains the miR-3154 described in (1), or the precursor miRNA described in (2), or the polynucleotide described in (3);

(5) Inhibitors of miR-3154 described in (1);

(6) The nucleic acid molecule of miR-3154 or its recombinant vector or recombinant cell;

(7) Reagents capable of down-regulating the expression of miR-3154 or its activity.

Further, the vector and reagent for overexpressing Pax7 are selected from one or more active ingredients in the following group:

(1) Pax7, or modified Pax7 derivatives, or Pax7 analogs;

(2) polynucleotide, said polynucleotide can encode Pax7 gene

(3) an expression construct, which contains the Pax7 described in (1) or the polynucleotide described in (2);

(4) An agonist of Pax7 as described in (1);

(5) The nucleic acid molecule of Pax7 or its recombinant vector or recombinant cell;

(6) Reagents capable of up-regulating the expression of Pax7 or its activity.

Further, the diseases related to the transformation of VSMCs phenotype include aortic aneurysm, abdominal aortic aneurysm, thoracic aortic aneurysm, thoracoabdominal aortic aneurysm, aortic dissection, and atherosclerosis.

The present invention also provides a pharmaceutical composition, which is characterized in that the pharmaceutical combination includes a pharmaceutically acceptable carrier and one or more active ingredients selected from the following group:

(1) Pax7, or modified Pax7 derivatives, or Pax7 analogs;

(2) polynucleotide, said polynucleotide can encode Pax7 gene

(3) an expression construct, which contains the Pax7 described in (1) or the polynucleotide described in (2);

(4) An agonist of Pax7 as described in (1);

(5) The nucleic acid molecule of Pax7 or its recombinant vector or recombinant cell;

(6) Reagents capable of up-regulating the expression of Pax7 or its activity;

The pharmaceutical composition promotes the expression level of Pax7 and inhibits the expression level of miR-3154 through the above active ingredients, thereby achieving the effect of lowering blood pressure.

Compared with the prior art, the present invention has beneficial effects.

The inventors have found through a large number of experiments that miR-3154 is highly expressed in serum and tissues of abdominal aortic aneurysms of humans and mice. Further studies found that the expression level of miR-3154 was the highest in VSMCs, and the expression level of miR-3154 increased after the application of angiotensin II to stimulate VSMCs. After miR-3154 mimic was transfected in VSMCs, the pathological phenotype transformation increased, and the results of dual luciferase reporter gene detection confirmed that miR-3154 could inhibit the luciferase activity of Pax7, which confirmed that Pax7 was the downstream of miR-3154 and interacted with miR -3154 binds directly. Later, gain-of-function studies found that overexpression of Pax7 could reverse the regulation of miR-3154 on phenotypic switching of VSMCs. In vivo studies have found that tail vein injection of Pax7 intervention virus can reduce the occurrence and development of abdominal aortic aneurysm, and has a certain antihypertensive effect. Pathological examination found that Pax7 can protect the mouse vascular elastic plate from damage. The above results indicate that the intervention of miR-3154 downstream target gene Pax7 can be used to prevent or treat abdominal aortic aneurysm, and has potential medical applications.

Description of drawings

Figure 1. Association of miR-3154 with abdominal aortic aneurysms. Among them, A is the expression change of miR-3154 detected by qRT-PCR in the serum of the control population and AAA patients; B is the expression change of miR-3154 detected by qRT-PCR in the vascular tissues of AAA mice.

Figure 2. miR-3154 can promote the conversion of Ang II-induced VSMCs to a synthetic phenotype. Among them, A is the expression change of α-SMA and SM22α detected by Western blot; B is the quantitative map of α-SMA and SM22α expression detected by Western blot; C is the expression change of α-SMA detected by immunofluorescence staining.

Figure 3. miR-3154 directly regulates Pax7. A is the downstream target gene predicted by bioinformatics software; B-C is the RT-PCR detection of the change in the transcription level of the downstream predicted target gene; D is the change of the Pax7 protein level detected by Western blot; E-F is the quantitative map of the Pax7 protein level detected by Western blot; G is the target scan prediction and Pax7 binding site H is a dual-luciferase reporter gene to verify the direct binding of miR-3154 to Pax7.

Figure 4. Regulation of Pax7 on phenotypic switching of VSMCs. Among them, A-B are VSMCs transfected with overexpressed Pax7 plasmid in vitro, Western blot detection of α-SMA, SM22α, PCNA expression changes and their quantification diagrams; C-D are Western blot detection of α-SMA, SM22α, PCNA expression after VSMCs transfected with si-Pax724h in vitro Changes and their quantification plots. n=3, *P<0.05, **P<0.01, ***P<0.001 compared to NC.

Figure 5. Pax7 inhibits the effect of miR-3154 on promoting the conversion of VSMCs to a synthetic phenotype. Among them, A-B are VSMCs transfected with miR-3154 mimic for 24 hours, and then overexpressing Pax7 for 24 hours. Western lot detection of Pax7, α-SMA, SM22α, expression changes and their quantification diagrams. C, immunofluorescence staining detection of VSMCs morphology and Pax7 expression changes; D, immunofluorescence staining detection of VSMCs morphology α-SMA expression changes.

Figure 6. The interventional effect of Pax7 on Ang II-induced abdominal aortic aneurysm. Among them, A is the abdominal aortic tissue specimen diagram of each group of mouse abdominal aortic aneurysm modeling and intervention experiment; B is the diameter of blood vessel in mouse abdominal aortic aneurysm modeling and intervention experiment; C is the mouse abdominal aortic aneurysm modeling and the tumor formation rate of the intervention experiment; D is the survival curve after 28 days of the mouse abdominal aortic aneurysm modeling and intervention experiment; E-G are the blood pressure changes of the mouse abdominal aortic aneurysm modeling and intervention experiment.

Figure 7. Tail vein injection of Pax7 alleviates aortic fibrosis and reduces collagen deposition. Among them, A is the observation of vascular tissue staining after 28 days of tail vein injection of Pax7 to Ang II embedded pump; B-C is Western blot detection of vascular tissue Collagen I, MMP2, Pax7, α- SMA protein expression level change and its quantification diagram.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

The experimental data of the present invention are all percentages. The chi-square test was used for the comparison of the two sample rates, and the SPSS 24.0 software package was used for statistical processing. P<0.05 means statistical difference.

Example 1. miR-3154 is highly expressed in abdominal aortic aneurysm serum and aortic tissue.

(1) Serum expression level detection of miR-3154: collect serum and basic information, past history, imaging and other relevant data of the control group and patients with abdominal aortic aneurysm; use Tirzol method (Invitrogen) to extract miRNA in serum (200 µL), reverse transcribe After generating cDNA, RT-PCR analysis was performed to detect the expression of miR-3154. The results confirmed that the expression of miR-3154 in the serum of patients with abdominal aortic aneurysm was significantly higher than that of controls (Fig. 1A).

(2) Detection of miR-3154 tissue expression level: Apoe ^-/- mice fed with HFD/Western Diet were treated with angiotensin-II pump to establish an abdominal aortic aneurysm model. Male Apoe ^−/− (8–10 weeks old) were purchased from Nanjing Model Animal Center (Nanjing, China). This study was approved by the Northern Theater Command General Hospital Institutional Animal Care and Use Committee. All animal experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Mice were fed high-fat diet (HFD) or Western diet (Western Diet) for at least 12 weeks, and then treated with angiotensin-II or saline pump for 4 weeks and divided into two groups. After modeling, mice were sacrificed under isoflurane anesthesia, aortic tissue was isolated and mRNA was extracted. Detecting the changes of miR-3154 in aortic tissue, it was found that the expression of miR-3154 was significantly increased in the aortic tissue of abdominal aortic aneurysm model mice induced by angiotensin-II (Fig. 1B).

Example 2. miR-3154 promotes the conversion of Ang II-induced VSMCs from a contractile phenotype to a synthetic phenotype.

Western blot detected the effect of miR-3154 on the differentiation of VSMCs, that is, the expression of α-SMA and SM22α. The results showed that miR-3154 could inhibit the expression of α-SMA and SM22α (Fig. 2A-B); immunofluorescence staining showed that miR-3154 overexpressed Expression can reduce the expression of α-SMA (Fig. 2C). In order to induce VSMCs to transform into a dedifferentiated phenotype, after miR-3154 mimic was transfected for 24 hours in vitro, Ang II 1 μmol was given to stimulate 24 hours.

Example 3. miR-3154 directly regulates Pax7.

(1) Bioinformatics prediction of downstream target genes of miR-3154: By using Target scan, miRDB, miRWalk and other bioinformatics software to predict the downstream target genes of miR-3154, the Venn diagram was intersected to obtain 17 related downstream target genes ( KMT2B, C20orf96, SCAMP5, URM1, HOXB8, ERH, SLC2A14, DCAF5, PPP1R10, IFNLR1, IDH3A, ZFP91, PRELP, CYP4A11, Pax7, ZC3H11A and MKLN1). Then, the four genes ERH, ZFP91, URM1, and Pax7 related to the phenotype conversion of vascular smooth muscle cells were searched through the literature (Figure 3A), and then quantitative PCR was performed to detect the changes in the transcription level of miR-3154 (Figure 3B-C), so that The identified downstream target genes of miR-3154 were obtained. The results suggested that the expression of Pax7 gene changed most significantly.

(2) In vitro cell experiments proved that miR-3154 regulates Pax7: after transfection of miR-3154 mimic and inhibitor in VSMCs in vitro for 24 hours, the expression level of Pax7 was detected by Western blot. The results showed that transfection of miR-3154 mimic could inhibit the expression level of Pax7 protein, and the expression level of Pax7 protein could be increased after transfection of inhibitor miR-3154, suggesting that miR-3154 may directly regulate Pax7 (Figure 3D-F). Then by querying the Target scan bioinformatics software, the Pax7 3'UTR binding site was obtained (Figure 3G), the wild-type Pax7 3'UTR vector was constructed, and the Pax7 3'UTR and miR-3154 mimic were co-transfected into HEK293 cells, and the results were obtained after 48 hours showed that overexpression of miR-3154 significantly inhibited the luciferase activity of Pax7 3'UTR. Then the Pax7 3'UTR vector with miR-3154 binding site mutation was constructed, and the mutant Pax7 3'UTR and miR-3154mimic were also co-transfected into HEK293 cells. After 48 hours, the results showed that the Pax7 3'UTR vector luciferase There was no significant difference in activity (Fig. 3H). The above results suggest that miR-3154 directly binds to the Pax7 3'UTR and regulates the expression of Pax7.

Example 4. Pax7 can inhibit the phenotypic conversion of VSMCs to the synthetic type.

(1) Pax7 can promote the contractile differentiation of VSMCs and inhibit the synthesis of collagen: VSMCs were given overexpression plasmid pcDNA3.1 Pax7, and the Western blot results showed that the expression levels of α-SMA and SM22α proteins increased, PCNA did not change significantly, collagen I Protein levels decreased, suggesting that overexpression of Pax7 could promote VSMCs differentiation and inhibit collagen synthesis (Fig. 4A-B). After silencing Pax7, Western blot results showed that the protein expression levels of α-SMA and SM22α decreased, but PCNA did not change significantly. The above results suggest that Pax7 can directly regulate the differentiation of VSMCs, but may not directly regulate cell proliferation (Fig. 4C-D).

(2) Overexpression of Pax7 can reverse the phenotypic transformation of VSMCs induced by miR-3154 to the synthetic phenotype: VSMCs were transfected with miR-3154 mimic and then given pcDNA3.1 Pax7 overexpression plasmid. Compared with the 3154 mimic intervention group, overexpression of Pax7 could significantly improve miR-3154-induced decrease in α-SMA and SM22α protein expression levels, suggesting that Pax7 could reverse the dedifferentiation of VSMCs caused by miR-3154 (Fig. 5A-B). To confirm whether the morphological results were consistent, we used immunofluorescence staining to detect α-SMA morphology. The results were consistent with Western blot, suggesting that miR-3154 mimic could reduce the expression of α-SMA, while overexpression of Pax7 could increase the expression of α-SMA (Fig. 5C-D).

Experimental Example 5: Tail vein injection of Pax7 group mice improved tumor formation rate and lowered blood pressure.

(1) Apoe ^-/- mice fed with HFD/Western Diet were used to establish an abdominal aortic aneurysm model. The establishment method of abdominal aortic aneurysm mouse model was the same as before. After successful modeling, the mice were randomly divided into two groups, the experimental group was AAV-control+AngII buried pump group and tail vein injection AAV-Pax7+AngII buried pump group. On the day when the pump was buried, the adeno-associated virus Pax7 was injected into the caudal vein, and the dose was 2x10 ¹¹ per mouse according to the instructions.

(2) Follow-up and pathological examination: 28 days after the pump was buried was the follow-up period. During the follow-up period, body weight changes were recorded on a weight scale every week, and the body weight of each mouse was measured at least 3 times. Tail cuff sphygmomanometer (BP210) was used to measure blood pressure. When measuring the blood pressure of the mice, keep the environment as quiet as possible. When the mice were in a stable state, the blood pressure data appeared and recorded. The blood pressure of each mouse was measured at least 3 times. At the end of the follow-up period, the experimental mice were killed, and the aortic lesions of the mice were observed and counted. The mouse aorta was fixed, dehydrated, and pathologically stained, including HE staining, Elastin staining, Masson staining, and immunohistochemical staining.

(3) The results showed that compared with the mice in the control group, the tumor formation rate of the mice in the AAV-Pax7 group injected into the tail vein of the Ang II implanted pump for 28 days was reduced (no statistical significance), the diameter of the blood vessels was significantly reduced, and the survival rate had no significant change (Fig. 6A-D). Blood pressure (systolic, diastolic) tended to decrease (Fig. 6E-G). The results of Masson staining showed that compared with the control group mice, the degree of vascular tissue fibrosis in the Ang II embedded pump group was more serious, while the vascular tissue fibrosis in the mice in the tail vein injection AAV-Pax7 group was alleviated. The results of EVG staining showed that compared with the control mice, the elastic plate of the tumor tissue in the Ang II-embedded pump mice was severely damaged, while the elastic plate of the vascular tissue in the tail vein injection AAV-Pax7 group was not significantly damaged. The results of immunohistochemical staining showed that compared with control mice, the expression of Pax7 in the tumor tissues of Ang II tumor-forming mice decreased, while the expression of Pax7 in the vascular tissues of mice in the tail vein injection AAV-Pax7 group increased (Figure 7A). The results of Western blot showed that compared with the mice in the AAV-control group, the expression of Collagen I in the vascular tissue of the mice in the tail vein injection AAV-Pax7 group decreased, and the expression of Pax7 increased. Staining morphology and Western blot results consistently indicated that the expression of Pax7 in the tail vein injection AAV-Pax7 group was increased and the fibrosis was alleviated (Fig. 7B-C).

The experimental materials and experimental methods used in the above examples are as follows.

1. Experimental materials.

(1) Experimental cells: Mouse primary VSMCs were incubated at 37°C in an incubator with 10% fetal bovine serum (FBS) and 1% antibiotics.

(2) Experimental animals: SPF grade ApoE-/- mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

Second, the experimental method.

(1) Selection of research population.

This study screened patients who were diagnosed with abdominal aortic aneurysm in the Department of Cardiology, General Hospital of the Northern Theater Command, from November 2019 to April 2021. Inclusion criteria: (1) Patients with AAA confirmed by vascular ultrasound and CT. The diagnostic criteria are that the diameter of the aortic vessel is greater than 3 cm or more than 1.5 times the normal diameter. Exclusion criteria: (1) aortic dissection or pseudoaneurysm; (2) combined with Marfan syndrome and other connective tissue diseases; (3) liver and kidney diseases, other organic diseases, blood system diseases, old myocardial infarction or Cardiac insufficiency; (4) Case information and materials are incomplete. The above research has been approved by the Hospital Ethics Committee. (Ethics number: Lun Shen No. Y (2021) 002), in addition, this study has obtained informed consent from all patients. The serum of all patients was stored at -80°C after obtaining, and the serum should avoid repeated freezing and thawing, so as not to affect the experimental results.

(B) Cell transfection.

1. When the cell density reaches 70%-80%, miR-3154 mimic and inhibitor are transfected into the cells through RNA IMAX and Lipofectamine 2000, and the cell culture medium is replaced with serum-free DMEM; when the cell density is 70%-80% , pcDNA3.1Pax7 was transfected into cells by Lipofectamine 2000, miR-3154 mimic was simultaneously transfected into cells by RNA IMAX, and silence-Pax7 and inhibitor miR-3154 were simultaneously transfected into cells by RNA IMAX, and the cell culture medium was changed to serum-free DMEM.

2. Observe the state of the cells after 24 hours, rinse with PBS twice, scrape the cells off gently, and collect the cells in EP tubes.

(3) Protein immunoblotting.

1. Use RIPA to lyse on ice for 30 minutes to collect cells, centrifuge at 12,000 rpm for 15 minutes at 4°C, and take the supernatant to obtain protein samples.

2. Determine the protein concentration by BCA method, and then add 4×loading buffer and protein lysate to prepare protein samples.

3. For tissue protein extraction, grind the tissue with a tissue grinder and centrifuge at 4°C for 15 min, then take the supernatant and use the BCA method to measure the protein concentration, and then add 4×loading buffer and protein lysate to prepare protein samples.

4. Load 20 μg of protein sample, and adjust to 80 V for 2 h of electrophoresis. The PVDF membrane was first activated with methanol solution, and then put into the membrane transfer apparatus for 1 h, at this time, the protein gel was transferred to the polyvinylidene fluoride (PVDF) membrane.

5. Incubate at room temperature for 1 h in a blocking solution containing 5% skimmed milk powder, dilute primary antibodies (PCNA, CCND1, α-SMA, SM22α, MMP2, and Pax7) at 1:1000, and incubate overnight at 4°C in a refrigerator.

6. Wash the membrane with TBST after rewarming for 30 minutes every day, once every 10 minutes, and wash three times in total.

7. Dilute the secondary antibody 1:1000 and incubate for 1 h at room temperature on a shaker.

8. Wash the membrane with TBST, once every 10 min, for a total of three times.

9. ECL kit A solution and B solution are prepared at a ratio of 1:1 and added to the PVDF membrane, and developed under the exposure machine.

(4) Real-time fluorescent quantitative RT-PCR.

Extract cellular RNA.

1. Extract RNA from cells by Trizol reagent method, add 1 mL Trizol to lyse at room temperature for 15 min.

2. Add 200 μL of chloroform and let stand at room temperature for 10 minutes.

3. Centrifuge at 12,000 g in a centrifuge at 4°C, then take the supernatant aqueous phase and add an equal volume of isopropanol to stand at room temperature for 10 min.

4. Centrifuge at 12,000 g at 4°C for 15 minutes, discard the supernatant, and a white precipitate of cells can be seen at this time.

5. Add 1 mL of 75% ethanol and centrifuge at 12,000 g at 4°C for 15 min to wash.

6. Discard the supernatant, air-dry at room temperature, add 20 μL enzyme-free water to dissolve.

Serum RNA extraction.

1. Take the patient's serum out of -80°C and store it at room temperature to wait for natural thawing.

2. Take 200 μL serum and add 1 mL Trizol to fully lyse at room temperature for 15 min. At this time, a white flocculent substance appears, and the white flocculent substance can disappear after repeated blowing with the tip of the gun.

3. Prepare 1 μmol cell 39 mimic standard according to the instruction manual, add 5 μL cell 39 mimic standard to the lysed serum, then add 200 μmol chloroform, mix it well and place it at room temperature for 15 minutes, then you can Shake vigorously on a vortex shaker.

4. Centrifuge the sample in a 4°C centrifuge at 12000 rpm/s for 15 min at 4°C.

5. At this point, the samples are separated, transfer the supernatant to an RNase-free EP tube, add 200 μL of isopropanol, blow it with a pipette tip to mix it, and place it at room temperature for 10 minutes.

6. Centrifuge the sample in a centrifuge at 4°C, 12000 rpm/s, for 10 min.

7. Discard the supernatant at this time, and prepare 80% ethanol, add 1 mL of 80% ethanol to the EP tube, and mix by pipetting.

8. Centrifuge the sample in a centrifuge at 12000 rpm/s at 4°C for 10 min.

9. Discard the supernatant at this time, and dry in air for 10 minutes until no liquid remains on the tube wall.

10. Add 10 μL enzyme-free water to fully dissolve the RNA.

11. At this time, measure the RNA concentration of the extracted RNA on a UV spectrophotometer.

qRT-PCR.

Follow the instructions of the miDETECT A Track miRNA qRT-PCR Starter Kit (Guangzhou Ruibo Biotechnology Co., Ltd.).

1. Poly(A) tailed preparation system on ice: Total RNA 1 μg (3 μL for serum RNA), 5xPoly(A) Polymerase Buffer 2 μL, Poly(A) Polymerase 1 μL, enzyme-free water 7 μL. Mix the above system and react at 37°C for 1 h.

2. Reverse transcription cDNA preparation system on ice: RTase mix 4 μL, 5×RTase Buffer 4 μL, miDETECT A Track Uni-RT Primer 2 μL, the above Poly(A) Tailing product 10 μL. 1 hour at 42°C, 10 minutes at 72°C.

3. qPCR preparation system on ice: Forward Primer (10 μmol) 0.5 μL, Uni-Reverse Primer (10 μmol) 0.5 μL, 2 x SYBR Green Mix 10 μL, cDNA 2 μL, enzyme-free water 7 μL. 1 cycle of pre-denaturation at 95°C for 10 min, 2 s at 95°C, 20 s at 60°C, 10 s at 70°C.

4. U6 was used as an internal reference, and miR-39 was also used as an external reference for serum.

Takara kit for reverse transcription of mRNA.

1. Reverse transcription.

Prepare the system on ice: 5 x DNA Eraser Buffer 2 μL, GDNA Eraser 1 μL, Total RNA 1 μL, RNase Free water to 10 μL system.

2. 10 μL of the reaction solution from step 1.

3. Prime Script RT Enzyme Mix 1 μL, RT Prime Mix 1 μL, 5 × PrimeScript Buffer 4 μL, enzyme-free water 4 μL. Place at room temperature for 30 min.

4. qRT-PCR.

TB Green Premix Ex TaqII 10 μL, PCR Forward Primer 1 μL, Reverse Primer 1 μL, cDNA solution 2 μL, sterilized water 6 μL, total system 20 μL, denatured and annealed.

Table 1 Primer sequences

.

(5) Detection of EdU cell proliferation.

1. Spread cell slides in a 24-well plate.

2. Plant the vascular smooth muscle cells into a 24-well plate, and prepare 50 μmol EdU medium after 24 hours of attachment. EdU medium: EdU incubation solution and DMEM containing 10% serum are diluted 1:1000.

3. Add 300 µL of EdU medium to each well of the 24-well plate and incubate for 2 h at 37°C in a 5% CO ₂ incubator.

4. Observe the state of the cells after 2 hours, then rinse twice with PBS, 3 minutes each time, try to rinse as clean as possible, add 500 μL paraformaldehyde fixative to fix the cells at room temperature for 30 minutes.

5. Prepare 2 mg/mL glycine solution. The preparation method is: Weigh 2 mg glycine on an electronic scale and dissolve it in 1 mL distilled water to obtain a 2 mg/mL glycine solution. Add the prepared 300 μL 2 mg/mL glycine to each well of a 24-well plate and incubate on a horizontal shaker for 5 min.

6. Prepare 0.5% Triton x-100 solution. The specific preparation method is: take 5 µL Triton x-100 solution and add it to 1 mL PBS to get 0.5% Triton x-100 solution. Rinse with PBS for 5 min, then discard the PBS, add 300 μL of 0.5% Triton x-100 solution to each well to infiltrate the cells and incubate on a horizontal shaker for 10 min, then wash with PBS once.

7. Prepare 1×Apollo staining solution according to the kit instructions, 1×Apollo staining solution: deionized water: 469 μL, Apollo reaction buffer: 25 μL, Apollo catalyst solution: 5 μL, Apollo fluorescent dye solution: 1.5 μL, Apollo Buffer additive: 5 mg, you can get 1xApollo staining solution. Add 300 μL of 1×Apollo staining solution to each well of a 24-well plate and stain at room temperature for 30 min.

8. Prepare Hoest4432 solution, the specific method is: Dilute Hoest4432 solution and deionized water 1:100. Add 300 μL of Hoest4432 solution to each well of a 24-well plate and incubate for 30 min in the dark.

9. Rinse twice with PBS, seal the slide with anti-fluorescence quencher, apply nail polish along the four sides of the slide after air drying, take pictures under an optical microscope and store at 4°C.

(6) TRANSWELL.

1. Seed vascular smooth muscle cells in a six-well plate.

2. After waiting 24 hours to adhere to the wall, the cells were gently blown and digested with the prepared trypsin, and placed in a 15 mL centrifuge tube.

3. Centrifuge at 3000 bpm for 5 min in a room temperature centrifuge.

4. Discard the supernatant, add 2 mL serum-free DMEM to each sample centrifuge tube and mix by pipetting.

5. Take a 12-well plate, add 500 μL 10% serum DMEM to each well, remove the small chamber, add 200 μL serum-free cell suspension to the upper chamber of each well, and incubate the lower chamber with 500 μL 10% serum DMEM.

6. After 24 hours, first observe the cell state, remove the chamber from the 12-well plate, wash the chamber twice with PBS, 5 min each time, then add 1 mL of paraformaldehyde solution to each well, and put the chamber into paraformaldehyde Fix in formaldehyde solution for 30 min.

7. Take the Giemsa staining kit, add Giemsa staining reagent to the 24-well plate according to the ratio of solution A:B (1:1), stain for 30 min, then add Giemsa staining solution to the 24-well plate Stain for 10 min according to solution A: solution B (1:3).

8. Then rinse with PBS twice, 3 minutes each time.

9. Use a 1 mL syringe needle to remove the round patch of the small chamber, place the small chamber upside down on the glass slide, and seal the slide with neutral resin. After air drying, apply nail polish along the four sides to ensure that the specimen will not fall.

10. Take photos and record in optical microscope photos.

(7) Scratch test.

1. Seed vascular smooth muscle cells into a 6-well plate.

2. Observe the cell density after 24 hours of cell attachment. When the cell density reaches 80%, draw a cross vertically and horizontally with a small pipette tip of 1 μL. Afterwards, the cells were washed 3 times with PBS, 2 min each time, in order to wash away the dead cells.

3. At this time, take pictures with an inverted microscope and record the initial situation at 0 h.

4. After 24 hours, wash the cells with PBS for 3 times, each time for 2 minutes, in order to wash away the dead cells. At this time, use an inverted microscope to take pictures and record the migration of 24 hours.

(8) Immunofluorescence.

1. Take a six-well plate, lay a cell slide, and plant the cells on the slide.

2. After adhering to the wall for 24 hours, rinse with PBS 3 times, 2 minutes each time. Afterwards, 500 μL of paraformaldehyde fixative solution was added to each well for 15 min.

3. Then wash the cells twice with PBS to prepare 0.2% Tritonx-100 solution. The specific method is: take 2 μL Tritonx-100 solution and add it to 1 mL PBS. Add 500 μL of 0.2% Tritonx-100 solution to each well to permeabilize the cells for 10 min, and then wash with PBS twice, each time for 2 min.

4. Take goat serum, add 500 μL goat serum to each well, infiltrate and block at room temperature for 30 min.

5. Prepare the fluorescent primary antibody. The specific method is: dilute the primary antibody (α-SMA, Pax7) at 1:100, take 1 μL of the antibody stock solution and add it to 100 μL of PBS solution to obtain the fluorescent primary antibody. Each specimen was infiltrated with 50 μL fluorescent primary antibody, and incubated at room temperature in the dark for 1 h or overnight at 4 °C.

6. Rewarm for 30 minutes the next day to prepare the fluorescent secondary antibody. The specific method is: dilute the fluorescent secondary antibody at 1:200, take 1 μL of the fluorescent secondary antigen solution and add it to 200 μL of PBS solution. Add 50 μL of fluorescent secondary antibody to each specimen, and incubate for 1 h in the dark.

7. Rinse with PBS for 30 min, and prepare DAPI solution. The specific method is: dilute DAPI at 1:1000, take 1 μL DAPI stock solution and add it to 1 mL PBS solution to obtain diluted DAPI. Each specimen was stained with DAPI for 5 min, and then washed 3 times with PBS, 2 min each time.

8. Seal the slide with anti-fluorescent quenching agent. After air-drying, apply nail polish along the four sides, and take photos and record in optical microscope photos.

(9) Plasmid extraction.

1. Take a 15 mL centrifuge tube the night before and add 10 mL of LB solution with anti-ampicillin. Prepare an alcohol lamp, first burn the puncture needle until it turns red, then cool it naturally for 2 minutes, puncture the puncture needle along the white line of the agar, put the puncture needle with the puncture bacteria into the LB for a few times, and then put it into the Incubate overnight at 37°C for 16 h at increased speed.

2. Take the sample off the shaker, take 800 μL of the puncture suspension LB and add 200 μL of glycerol to save the glycerol puncture bacteria, and store it at -80°C.

3. Put the centrifuge tube with the puncture bacteria suspension into the centrifuge and centrifuge at 3000 bpm for 10 min. Discard the LB solution, add 250 μL solution I to the precipitated bacteria, pipette and resuspend, then transfer the liquid to the EP tube.

4. Add 250 μL solution II to EP and invert by pipetting.

5. Add 350 μL solution IV to the EP tube, and invert several times by pipetting.

6. Put the lysed bacterial suspension into a centrifuge at 4°C and centrifuge at a speed of 12000 g for 10 min.

7. After 10 minutes, pipette 750 μL of supernatant into the centrifuge tube DNA column.

8. Repeat the above steps several times until all the supernatant has been collected.

9. Then add 750 μL of CWA eluent to the DNA column, put it in a centrifuge, centrifuge at 12000 g for 2 min, and discard the lower solution.

10. Repeat the above steps, that is, add 750 μL of CWA eluent to the DNA column again, put it in a centrifuge, and centrifuge at 12000 g for 2 min, then discard the lower solution.

11. Empty, without adding any liquid, put it in a centrifuge at a speed of 12000 g, and centrifuge for 1 min.

12. Transfer the DNA spin column to an Ep tube, add 30 μL enzyme-free water to the middle and incubate at room temperature for 5 minutes.

13. Put the EP tube into the centrifuge and centrifuge at 12000 g for 2 min.

14. At this time, the added enzyme-free water is centrifuged and transferred to the EP tube.

15. Measure the double-strand (dsDNA) concentration with a UV spectrometer and store at -20°C.

(10) Dual luciferase reporter gene.

1. Extract the Pax7 3'UTR plasmid: see method (9) for specific steps.

2. Take a 24-well plate and plant HEK293 cells in the 24-well plate. The experimental groups were control group: Pax7 3'UTR; experimental group: Pax7 3'UTR +miR-3154 mimic.

3. After 24 hours of cell attachment, the Pax7 3'UTR plasmid and miR-3154 mimic were co-transfected into HEK293 cells with Lipofectamine 2000.

4. After 48 h, wash the cells twice with PBS, 2 min each time. Take the lysate (1×Lysis Buffer), dilute 1×Lysis Buffer into 5×Lysis Buffer with PBS, add 100 μL 5×Lysis Buffer to each well and place it on a horizontal shaker for 15 min.

5. After 15 minutes, take the luciferase kit and dilute solution A and solution B according to the instructions, take 10 μL of the lysed cell suspension and add it to a special assay plate, first add 50 μL of solution A, and then add 50 μL of solution B. Then check the position of the plate hole on the machine to measure the luciferase activity and record the A value and B value. A/B analysis of the difference in luciferase activity between the control group and the experimental group.

(11) Establishment of abdominal aortic aneurysm model.

1. Use 8-12 w male ApoE ^-/- mice. The required Ang II dose was calculated according to the body weight, and the required Ang II dose was prepared according to the literature dose of 1200 ng/kg·min.

2. Choose an appropriate location on the back of the neck and disinfect it with gauze dipped in alcohol.

3. Gently lift the epidermis with tweezers, and cut a hole longitudinally with scissors. Do not make it too long or too short. If it is too long, it will affect the postoperative recovery of the mouse. If it is too short, the micro-osmotic pump cannot enter.

4. Use tweezers to slowly send the micro-osmotic pump (Alzet 1004 type) into the incision, then suture the incision, and disinfect the suture site with gauze dipped in alcohol. By implanting a micro-osmotic pump subcutaneously in the back of the neck.

5. At the same time, inject AntagomiR-3154 into the tail vein on the day when the pump is buried. According to the instructions, the dose is 50nmol/animal, and the tail vein is injected once a week for four consecutive weeks.

6. At the same time, the adeno-associated virus Pax7 was injected into the caudal vein on the day of pump embedding, and the dose was 2x10^ ¹¹ per mouse according to the instructions.

(12) Measurement of body weight and blood pressure.

Body weight changes were recorded on a weight scale every week, and the body weight of each mouse was measured at least 3 times. Tail cuff sphygmomanometer (BP210) was used to measure blood pressure. When measuring the blood pressure of the mice, keep the environment as quiet as possible. When the mice were in a stable state, the blood pressure data appeared and recorded. The blood pressure of each mouse was measured at least 3 times.

(13) Tissue sample collection and fixation.

1. After 28 days, the experimental mice were killed by neck dislocation, the abdominal epidermis was lifted with tweezers, and the abdominal epidermis was cut with scissors. Be careful not to cut the organs gently, and cut the chest epidermis sequentially from bottom to top.

2. Until the chest cavity is exposed, carefully cut open the chest cavity without cutting the heart and lungs. The heart was taken out layer by layer. After removing the esophagus and other organs, the aorta was close to the spine, and the aorta was cut close to the spine with curved scissors until it reached the infrarenal artery.

3. Fix the aorta with a fine needle under a microscope, carefully peel off the fatty tissue around the aorta with forceps and scissors, and fix a small section in paraformaldehyde solution for 24 hours.

(14) Elastin staining.

1. Dewaxing: Soak the specimen in 100% alcohol xylene I and II for 5 min each, then in 100% alcohol I and II for 5 min each, and then in 95% alcohol, 80% alcohol and 70% alcohol respectively Infiltrate for 3 min.

2. Preparation of working solution 1: Take 1 mL of "251", 150 µL of "252", 400 µl of "253", and 250 µL of distilled water from the kit and mix well to complete the preparation of working solution 1. Preparation of working solution 2: Take 150 µL of "252" in the kit and mix with 1.85 mL of distilled water to complete the preparation of working solution 2.

3. After the specimen is deparaffinized, soak it with working solution 1 for 10 minutes, and then wash it with PBS twice, 3 minutes each time. Afterwards, soak with working solution 2 for 10 s and rinse with PBS for 3 min each time. At this time, use a microscope to observe whether the black elastic fiberboard can be colored. If the color is not good, repeat the above steps.

4. Soak the specimen with 95% alcohol for a few seconds, then use the kit "254" again to soak for 3 minutes, then dehydrate and soak with 100% alcohol for 5 minutes, put the specimen in xylene and soak for 5-10 minutes to make the specimen transparent, Mount with neutral resin.

5. Observe and take pictures under an inverted microscope.

(15) Masson staining.

1. Dewaxing: Soak the specimen in 100% alcohol xylene I and II for 5 min each, then in 100% alcohol I and II for 5 min each, and then in 95% alcohol, 80% alcohol and 70% alcohol respectively Infiltrate for 3 min.

2. The specimens were soaked in hematoxylin for 5 minutes, differentiated in 0.5% hydrochloric acid ethanol for 30 seconds, and then washed with running water for 1 minute.

3. Soak the specimen in ammonia water for 1 min and then rinse with running water for 1 min.

4. Now start using the reagents in the kit. Take the lichunhong staining solution in the kit and infiltrate for 10 min, prepare 0.2% glacial acetic acid with PBS, wash with 0.2% glacial acetic acid for 1 min, infiltrate with phosphomolybdic acid for 1-2 min, and wash with glacial acetic acid for 1 min.

5. Take the specimen and soak it in 95% ethanol for 5 seconds, then soak it in 100% ethanol for 3 minutes, soak it in xylene for 5-10 minutes to make the specimen transparent, and seal it with neutral resin. Slices, try not to produce air bubbles. After the specimen is air-dried in a fume hood, nail polish can be applied along the specimen.

6. Observe and record under an inverted microscope.

(XVI) Immunohistochemistry.

1. Dewaxing: Soak the specimen in 100% alcohol xylene I and II for 5 min each, then in 100% alcohol I and II for 5 min each, and then in 95% alcohol, 80% alcohol and 70% alcohol respectively Infiltrate for 3 min.

2. Prepare the antigen retrieval solution: Take the original antigen retrieval solution and dilute the antigen retrieval solution with distilled water at 1:50 to prepare the antigen retrieval solution. Take the rice cooker and fill it with water. After the water is boiled, take the diluted antigen retrieval solution and infiltrate it for 40 minutes, and then close the lid. After 40 min, take out the specimen and wait for natural cooling, or put the specimen in cold water to accelerate cooling.

3. After the specimen is cooled, take the immune group paintbrush to circle the specimen position, in order to prevent the liquid from flowing out of the specimen. Take out the immunohistochemistry kit, add 50 µL peroxidase blocking solution (reagent A) to infiltrate at room temperature for 10 min, and then wash with PBS twice, 3 min each time.

4. Take out the reagent B in the kit, and infiltrate the specimen with 50 µL normal non-immune animal serum (reagent B) at room temperature for 30 min.

5. Remove the serum without washing with PBS, and prepare the primary antibody: the primary antibody is diluted 1:100 with PBS, and it is best to prepare the primary antibody immediately. Take 50 µL of the diluted primary antibody and infiltrate the specimen, either at room temperature for 1 hour or overnight at 4°C.

6. It is best to rewarm for 30 minutes on the next day, and rinse with PBS twice, 3 minutes each time.

7. Take reagent C in the kit, infiltrate the specimen with reagent C (50 µL) at room temperature for about 30 minutes, and then rinse with PBS twice, 3 minutes each time, and wash as clean as possible.

8. Take reagent D in the kit, infiltrate the specimen with reagent D (50 µL) peroxidase solution at room temperature for 10 min, wash with PBS 3 times, 3 min each time.

9. Prepare the DAB chromogenic solution according to the instructions, and add the DAB chromogenic solution to the specimen. DAB color develops for 10 minutes, and the specimen will develop color at this time. If no color develops, the color development time can be extended, or continue to replenish DAB color development solution, and finally rinse with distilled water to stop the color development.

10. Soak the specimen in hematoxylin for 5 min, then differentiate with 0.5% hydrochloric acid ethanol for 30 s, and then rinse with tap water 3 times, 3 min each time.

11. Ammonia water for 5 minutes to turn blue, rinse with tap water 3 times, 3 minutes each time. Xylene was transparent for 5 min, and the slide was mounted with neutral resin.

12. Take pictures under an inverted microscope.

(17) Protein immunoblotting.

1. For tissue protein extraction, wash the stripped aorta with PBS and cut it into pieces with scissors. Use a 1 mL syringe to transfer the cut up aorta tissue into an EP tube, put three 4 mm grinding beads, add 100 µL RIPA. After grinding with a tissue grinder, centrifuge at 4 °C for 15 min and take the supernatant to obtain tissue protein.

2. To measure protein concentration by BCA method, first prepare protein standards (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL), after taking 96-well plate, add protein supernatant in turn, according to the standard curve, Measure the protein concentration. Add 4×loadingbuffer and protein lysate to prepare protein samples.

3. Load 20 μg of protein sample, and adjust to 80 V for 2 h of electrophoresis. The PVDF membrane was first activated with methanol solution, and then placed in a membrane transfer apparatus for 1 h, at which time the protein gel was transferred to a polyvinylidene fluoride (PVDF) membrane.

4. Incubate at room temperature for 1 h in a blocking solution containing 5% skimmed milk powder, dilute the primary antibodies (CCND1, MMP2, Pax7, α-SMA and collagen I) at 1:1000, and incubate overnight at 4°C in a refrigerator.

5. Wash the membrane with TBST after rewarming for 30 minutes at daily temperature.

6. Dilute the secondary antibody 1:1000 and incubate for 1 h at room temperature on a shaker.

7. Wash the membrane with TBST.

8. ECL kit A solution and B solution were prepared at a ratio of 1:1, added to the PVDF membrane, and developed under the developer.

(18) Sirius red staining

1. Dewaxing: Soak the specimen in 100% alcohol xylene I and II for 5 min each, then in 100% alcohol I and II for 5 min each, and then in 95% alcohol, 80% alcohol and 70% alcohol respectively Infiltrate for 3 min.

2. Dip the slices washed in water into Sirius red staining solution for 1 h.

3. Alcohol decomposition and dehydration: the film is directly put into pure alcohol for decomposition and dehydration.

4. Dehydrate and make transparent in xylene for 15 minutes, and seal the slide with neutral gum.

(19) Statistical analysis.

All the above experiments were repeated three times, and SPSS 8.0 software was used for analysis and processing. The t-test was used for the difference between the two groups, and the one-way analysis of variance was used for the comparison between multiple groups. P <0.05 was considered statistically significant.

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that, according to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (9)

1. The application of the reagent for detecting the expression level of miR-3154 in the preparation of a kit for auxiliary diagnosis and/or prognosis assessment of diseases related to VSMCs phenotype conversion, characterized in that, the kit for diseases related to VSMCs phenotype conversion is as follows: miR-3154 was used as a marker. 2. A method for screening potential substances for preventing and/or treating VSMCs phenotype conversion-related diseases, characterized in that the method comprises the following steps: Step 1, treating the system expressing miR-3154 with a candidate substance; and Step 2, detecting the expression of miR-3154 in the system; Wherein, if the candidate substance can reduce the expression of miR-3154, it indicates that the candidate substance is a potential substance for preventing and/or treating diseases related to phenotypic conversion of VSMCs. 3. The method according to claim 2, wherein step 1 comprises: in the test group, adding the candidate substance to the system expressing miR-3154; and/or Step 2 includes: detecting the expression of miR-3154 in the system of the test group, and comparing it with the control group, wherein the control group is a system expressing miR-3154 without adding the candidate substance; If the expression of miR-3154 in the test group is statistically lower than that in the control group, it indicates that the candidate is a potential substance for the prevention and/or treatment of diseases associated with phenotypic transformation of VSMCs. 4. The application of the inhibitor of the expression level of miR-3154 or the agonist of the expression level of the downstream target gene Pax7 of miR-3154 in the preparation of drugs for the treatment of diseases related to VSMCs phenotype conversion. 5. A pharmaceutical composition for treating diseases related to phenotypic conversion of VSMCs, characterized in that the pharmaceutical composition includes an inhibitor of miR-3154 expression level or a carrier and reagent for overexpressing Pax7. 6. The pharmaceutical composition according to claim 5, wherein the inhibitor of the expression level of miR-3154 is selected from one or more active ingredients of the following group: (1) miR-3154, or modified miR-3154 derivatives, or miR-3154 analogs; (2) Pre-miRNA, which can be processed into miR-3154 in the host; (3) polynucleotide, the polynucleotide can be transcribed by the host into the precursor miRNA described in (2), and processed to form miR-3154, or can be processed into miR-3154 in the host; (4) An expression construct, which contains the miR-3154 described in (1), or the precursor miRNA described in (2), or the polynucleotide described in (3); (5) Inhibitors of miR-3154 described in (1); (6) The nucleic acid molecule of miR-3154 or its recombinant vector or recombinant cell; (7) Reagents capable of down-regulating the expression of miR-3154 or its activity. 7. The pharmaceutical composition according to claim 5, wherein the carrier and reagent for overexpressing Pax7 are selected from one or more active ingredients of the following group: (1) Pax7, or modified Pax7 derivatives, or Pax7 analogs; (2) polynucleotide, said polynucleotide can encode Pax7 gene (3) an expression construct, which contains the Pax7 described in (1) or the polynucleotide described in (2); (4) An agonist of Pax7 as described in (1); (5) The nucleic acid molecule of Pax7 or its recombinant vector or recombinant cell; (6) Reagents capable of up-regulating the expression of Pax7 or its activity. 8. The application according to claim 1-7, characterized in that the diseases related to the transformation of VSMCs phenotype include aortic aneurysm, abdominal aortic aneurysm, thoracic aortic aneurysm, thoracoabdominal aortic aneurysm, aortic dissection, Atherosclerosis. 9. A pharmaceutical composition, characterized in that the pharmaceutical combination comprises a pharmaceutically acceptable carrier and one or more active ingredients selected from the group consisting of: (1) Pax7, or modified Pax7 derivatives, or Pax7 analogs; (2) polynucleotide, said polynucleotide can encode Pax7 gene (3) an expression construct, which contains the Pax7 described in (1) or the polynucleotide described in (2); (4) An agonist of Pax7 as described in (1); (5) The nucleic acid molecule of Pax7 or its recombinant vector or recombinant cell; (6) Reagents capable of up-regulating the expression of Pax7 or its activity; The pharmaceutical composition promotes the expression level of Pax7 and inhibits the expression level of miR-3154 through the above active ingredients, thereby achieving the effect of lowering blood pressure.

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