Male SD rats (4 W, 60-80 g), provided by Shanghai Jasper Laboratory Animal Co., Ltd. and housed under specific pathogen-free (SPF) conditions, received standard chow, with free access to water. They were randomly divided into the normal group (drinking tap water, n=8) and model animals (n=8), which were administered β-aminopropionitrile monofumarate (BAPN, Sigma, USA) in drinking water at 0.4 g/100 g diet for 28 days as previously proposed. 
Establishment of the animal model of carotid artery dissection
(a) Anesthesia: Before sampling, the animals were fasted for 4 hours and intraperitoneally administered pentobarbital sodium (3 ml/Kg body weight) for anesthesia. The experimental design and anesthesia had approval from the Animal Room Ethics Committee of Fudan University.
(b) Modeling: After local disinfection of the neck, the neck’s skin along the midline was longitudinally incised, and the left and right carotid arteries were carefully separated. A carotid artery was randomly selected, and two small edentulous microhemostatic clips were used to clamp the carotid artery from both sides of the head and tail in the opposite direction of the handle end (the distance between the two hemostatic clips was 1 cm). Then, both hemostatic clips were rotated at 180° in the opposite direction and fixed (Figure 2A); the other internal carotid artery was left untreated and used as a control.
(c) Specimen collection and storage: After 20 min, the microvascular clamp was gently removed, and the skin was sutured and disinfected. Carotid arteries were taken 24 hours later for subsequent experiments.
(d)All rats dying before the expected study end time (4 weeks) were necropsied right away.
Animal experiments were performed at the Institute of Neurology, Huashan Hospital, Fudan University. The animal experiments followed the Guide for the Care and Use of Laboratory Animals, and had approval from the Institutional Animal Care and Use Committee of Huashan Hospital, Fudan University.
Cell culture, grouping and transfection
A7R5 smooth muscle cells (SMC), provided by Shanghai Cell Bank, Chinese Academy of Sciences, were maintained in Dulbecco's modified Eagle's medium (DMEM, 190040; Gibco) with 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C.. Cells were then seeded in 6-well plates at 105/well and underwent incubation at 37°C in a 5% CO2 incubator, with the medium refreshed at 2 to 3-day intervals. At 70-80% confluency, cell sub-culturing was carried out. This was followed by two PBS washes, a 1-2-minute incubation with 0.25% trypsin, and resuspension and subculture in DMEM with 10% FBS.
Actively growing A7R5 SMC were assigned to blank (A7R5 SMC; transfection with no sequence), negative control (NC), miR‐144-3p mimic(transfection with miR‐144-3p mimic),miR‐144-3p inhibitor (transfection with miR‐144-3p inhibitor),siRNA‐FBN1(transfection with siRNA‐FBN1)and miR-144-3p inhibitor + siRNA-FBN1 (co-transfection with miR‐144-3p inhibitor and siRNA‐FBN1) groups, respectively. Transfection was carried out on 6‐well plates using the RNAimax reagent (Invitrogen, USA) as directed by the manufacturer at 70% to 80% confluency. The miR‐144-3p mimic, siRNA‐FBN1, miR‐144-3p inhibitor, miR‐144-3p inhibitor+siRNA‐FBN1 and NC were from Genepharma Gene Co., Ltd. (Shanghai, China), and RNase-free water was utilized for solubilization.
Next, 250 μL of Opti‐MEM (Gibco) without serum was added to 5 μL of RNAimax reagent and incubated for 5 min at ambient. The two abovementioned solutions were added to cell culture wells following a 20-minute incubation at ambient. After 6-8 hours of culture at 37°C in 5% CO2 incubator, the medium was changed to complete medium for further culture for 24-48 hours. A fluorescence microscope (Wanheng Precision Instrument, Shanghai, China) was used for analysis. RNA and protein extractions from cells were carried out for subsequent assays.
Hematoxylin - eosin staining and EVG staining
Carotid artery tissue specimens from normal animals and carotid artery dissection tissue samples from model animals were collected. Upon fixation with 4% formalin for 24 h, the tissue specimens underwent dehydration with graded ethanol and N‐butanol and paraffin embedding at 60°C, followed by 5‐μm serial sectioning. Dewaxing was carried out at 60°C for 1 h with xylene.
For H&E staining, incubation was first performed with hematoxylin (3–5 min). After incubation with acid and ammonia solutions for 40 s each, the samples underwent staining with eosin (2 min) and were soaked in xylene (5 min). An inverted microscope (Olympus, Tokyo, Japan) was utilized for analysis.
In EVG staining, a 30-min incubation was carried out with EVG solution (hematoxylin, iodine solution and ferric chloride at 5:2:2) followed by washing. Ferric chloride differentiation solution was utilized for background. These steps were repeated until a grey white background was obtained. This was followed by staining with VG solution (saturated picric acid and Fuchsin solution at 9:1), multiple washes and dehydration with 100% ethanol. After neutral gum sealing, an inverted microscope (Olympus) was utilized for analysis.
Carotid artery tissue specimens from normal animals and carotid artery dissection tissue samples from model animals underwent fixation with 10% formalin, paraffin embedding and sectioning 3-4 μm. After treatment with 3% H2O2, xylene dewaxing (10 min) and dehydration with graded ethanol were performed. This was followed by antigen retrieval by boiling for 90 seconds. The samples were blocked with 5% bovine serum albumin for 30 min at 37°C, followed by incubation with rabbit anti‐rat FBN1 antibodies (ab523076, Abcam; 1:300) overnight at 4°C. Next, the specimens underwent incubation with horseradish peroxidase (HRP)‐linked goat anti‐rabbit IgG‐secondary antibodies (ab6721, Abcam) for 30 min at 37°C. The sections were counterstained with hematoxylin for 30 s and developed with diaminobenzidine (P0202; Beyotime Biotechnology). Next, hydrochloric acid solution was utilized for dehydration, which was followed by mounting with neutral balsam and microscopic analysis. In this study, positivity was considered as >25% cells stained for fibrillin-1 (brown or tan granules in the cytosol). Totally five high‐power visual fields (40×) were randomly examined, and 200 cells were enumerated per field. Positive cells per field were counted and averaged.
Reverse transcription quantitative polymerase chain reaction (Table. 1)
Normal carotid tissue and carotid tissue samples (200 mg) were homogenized and stored at -80ºC. In in vitro assays, cells underwent 2 washes with PBS containing protein lysis buffer, followed by centrifugation for 20 min at 4°C and 12000g for protein extraction. The resulting supernatant was treated with the bicinchoninic acid Protein Assay Kit (P0012‐1; Beyotime). Equal amounts of total protein (20 μg) were resolved by 10% sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE), followed by electro-transfer onto polyvinylidene fluoride (PVDF) membranes. Upon 1-h of blocking at ambient with 5% fat-free milk, the membranes underwent incubation with rabbit anti‐Rat FBN1 (ab231094,1:500), MYH11 (ab125884,1:3000), CNN1 (ab32124, 1:1000), ACTA2 (ab7817,1:3000), MMP2 (ab92536, 1:1000), MMP9 (ab76003, 1:1000), ELN (ab21610, 1:1000), COL1A1(ab34710, 1:1000) and GAPDH (ab8245, 1:3000) primary antibodies (Abcam). This was followed by incubation with Tris Buffered Saline-Tween (TBS-T). The enhanced chemiluminescence kit was utilized for detection by autoradiography. Optical density (OD) was obtained with a gel detection system, normalized to GAPDH. The assays were repeated thrice.
Dual - luciferase reporter gene assay
The luciferase reporter assay identifies target miRNAs of the rat and human FBN1 genes via the TargetScan database, scoring them with the context + + scoring system of the database.  Dual-luciferase reporter plasmids p3’-UTR-FBN1 (comprising the wild-type FBN13’-UTR binding site in the luciferase reporter plasmid) and p3’-UTR-FBN1mut (comprising the mutant FBN1 3’-UTR; mut), miR144-3p mimic, miR-144-3p inhibitor and negative control (NC) sequences were constructed by Shanghai Hanheng Biotechnology. For luciferase assays, the completed 3’-UTR plasmids underwent co-transfection with miR144-3p mimic, miR-144-3p inhibitor, and negative control (NC) sequences into HEK-293T cells with Lipofectamine 3000 reagent (Invitrogen, Shanghai China) for 48 h. The Dual-Luciferase Reporter Assay System (Promega, China) was utilized for Luciferase activity measurement as directed by the manufacturer.
Approximately 104 cells were seeded in 96-well plates for 24 h, and underwent transfection with miR-144-3p mimic, miR-144-inhibitor, negative control (NC), siFBN1 and miR-144-3p+siFBN1 for 24 h and further incubation with medium with 0.5% FBS for 48 h. After treatment, 10 µl CCK-8 was supplemented to the culture medium for 2.5 h. At the 2.5, 24, 48 and 72-h time points, respectively, OD at 450 nm was obtained on a Synergy H1 Bio TEK (Ptotem Instruments Co., Ltd. USA).
Upon transfection for 24 h, cells were seeded in 6‐well plates at 5×105/well. At 90% confluency, a 200-µl tip was utilized for scratching, which was followed by a PBS wash. Serum‐free medium was next supplemented for another 0.5-1 h. At 0 h, 24 h and 48 h, respectively, cell imaging was performed, followed by migration distance measurement with Image‐Pro Plus Analysis (Media Cybernetics, MD). Assays were performed three times.
Cell Adhesion Assessment
Totally 50 mg/L Matrigel (356234; BD Diagnostics) was diluted 1:10 with FBS-free culture medium, and 60 μl/well was used to coat 96-well plates, followed by incubation in a 37°C/5% CO2 incubator for 4 hours. The supernatant was aspirated and discarded. Then, the transfected cells underwent seeding in 96-well plates at 5×104/well, with 5 duplicate wells per group. Meanwhile, a control group was set up, that is, a group with the supernatant not discarded after incubation. After 4 h, the culture medium was aspirated, and non-adherent cells were washed off. Totally 100 μl of fresh medium was added to each well, and 5 μl of CCK8 solution was added to each well for incubation in the dark. In the control group, 5 μl of CCK8 was directly added (counted as 0 h). After 4 hours of culture (counted as 4 h), OD at 450 nm was obtained on a Synergy H1 BioTEK (Ptotem Instruments Co., Ltd. USA).
Matrigel (356234; BD Diagnostics) diluted with serum‐free medium at 1:3 was used to coat the upper transwell chambers (50 μL/well) for 30 min at 37ºC. Totally, 105 cells/ml were added to upper chambers in medium without serum, while 10% FBS-containing medium was placed into lower chambers. Based on the amounts of cells passing through the matrigel, cell invasion was quantitated. The assays were run thrice.
SPSS 22.0 and GraphPad Prism 8 were utilized for data analysis. Data are mean ± standard deviation (SD). The t test and one-way analysis of variance were performed to assess group pairs and multiple groups, respectively. P<0.05 reflected statistical significance.