The study was approved by the Ethics Committee of Xiangya Hospital and carried out in accordance with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health. Extensive efforts were made to minimize the number and suffering of the included animals.
Establishment of angiotensin II (Ang II)-induced mouse AAA models
C57BL/6 J male ApoE−/− mice (aged 6–8 weeks and weighing 18–23 g; n = 12 in each group) were selected in this study. A small incision was made where osmotic micro-pump (Alzet, 2004; DURECT Corporation, Cupertino, CA, USA) was subcutaneously implanted into the back of mouse neck following anesthesia. Ang II (A9525, Sigma, St. Louis, MO, USA) or normal saline (0.9% NaCl) was injected through micropump (1 μg/kg per min). On the 28th day after Ang II infusion, the surviving mice were euthanized and the aortic tissues were extracted for further analysis, among which six of them were used for pathological examination and six for molecular biology detection .
Mice were injected intravenously via caudal vein with (2 × 1012 virus genome particles; n = 12 in each group) lentiviruses carrying negative control (NC), overexpression (oe)-EZH2, short hairpin RNA against GAS5 (sh-GAS5), sh-EZH2 and oe-RIG-I (Shanghai Genechem Co., Ltd., Shanghai, China) using an insulin syringe (BD Biosciences, Franklin Lakes, NJ, USA). After 30 days, the injected mice were used to develop AAA models . After 28 days of modeling, the rats were euthanized for follow-up detection, including six for pathological detection and six for molecular biology detection.
Six mice in each group were euthanized and abdominal incision was made to detect the presence of aortic aneurysm. To expose the aorta, we injected 10 mL of phosphate-buffered saline (PBS) from the left ventricle and overflowed from the incision in the right atrium. Under a dissecting microscope, the abdominal aorta was separated from the surrounding connective tissues and the connective tissues remaining on the adventitia of the abdominal aorta was removed with tweezers, with a full-length abdominal aorta photographed by a digital camera. The outer diameter of the abdominal aorta above the bifurcation of the renal artery was directly measured using images.
The maximum outer diameter of the abdominal aorta above the bifurcation of the renal artery was measured with Image-Pro Plus software . The maximum outer diameter was measured at least three times and the average value was taken. The outer diameter of the aorta that increased by over 50% relative to that of the aorta injected with saline was regarded to be indicative of AAA.
Aortic specimens were fixed, embedded, and cut into 5-µm-thick sections. The paraffin sections were then heated in a 60 °C oven for 30 min, routinely dewaxed, hydrated for 5 min each, and washed with water for 2 min. Next, the sections were subjected to antigen retrieval using 1 mM Tris-ethylene diamine tetraacetic acid (pH 8.0), washed with PBS for three times (5 min/time), immersed in 3% H2O2-formaldehyde at ambient temperature for 10 min, and washed with PBS for two times (5 min/time). Next, the sections were probed with primary antibody to EZH2 (1:100, ab191080, Abcam Inc., Cambridge, UK) overnight at 4 °C, washed with 0.1% Tween-20 for three times (5 min/time), and re-probed with poly-enhancer (PV-9000, ZSGB-Bio, Beijing, China) at ambient temperature for 20 min, followed by three washes using PBS containing 0.1% Tween-20 (5 min/time). Subsequently, the sections were further incubated with enzyme-labeled anti-mouse/rabbit polymer (PV-9000, ZSGB-Bio) at ambient temperature for 30 min. After washing with 0.1% Tween-20 for three times (5 min/time), the sections were developed using 3,3′-diaminobenzidine (DAB), counterstained and blued. At last, the sections were dehydrated, cleared, mounted, and observed under an inverted microscope (CX41, Olympus Optical Co., Ltd., Tokyo, Japan). Six non-overlapping fields were randomly selected (40 ×). Image J software was used to count the number of positively-stained cells and total cells. Every six tissue sections were selected from each group to calculate the percentage of cells positive for EZH2, followed by data analysis .
Primer cell culture and grouping
Mouse primary vascular SMCs were cultured as previously described . Briefly, mouse aorta (except endothelium and perivascular connective tissue and fat) was isolated and the vascular tissues were cut into 1-mm-thick pieces, treated with 1.42 mg/mL collagenase II for 4–6 h, and then cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, streptomycin (100 μg/mL) and penicillin (100 U/mL) at 37 °C. Thereafter, the cells were treated with Ang II (10–7 mol/L) 24 h after lentivirus treatment at 37 °C. All cells were then incubated at 37 °C for subsequent experiments.
The cells were transfected with oe-NC, oe-EZH2, oe-EZH2 + oe-RIG-I, oe-GAS5 and oe-GAS5 + oe-EZH2 vectors (GenePharma Co., Ltd., Shanghai, China) and then treated with Ang II. Moreover, the cells transfected with only oe-NC were used as the controls.
Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay
The abdominal aorta of mice was immersed in 0.85% sodium chloride solution, washed, and fixed with 3.4% paraformaldehyde or PBS solution containing 10% formalin for 15 min. Next, the samples were incubated in 100 μL protease K working solution (20 μg/mL) composed of 50 mmol/L TRIS–CL (pH 8.0) + 1.5 mmol/L calcium acetate + proteinase K (final concentration 20 mg/mL) for 10–30 min. After washing, the samples were fixed with 7.4% paraformaldehyde or PBS solution containing 10% formalin and then added with 100 μL balanced buffer solution. Next, the samples were put on ice to remove excess liquid and added with 100 μL TdT enzyme reaction solution for incubation for 60 min. Thereafter, the samples were immersed in PBS containing 0.3% H2O2 to neutralize the endogenous peroxidase. After PBS washing, the samples were added with 100 μL Streptavidin horseradish peroxidase for 30 min of reaction and treated with 100 μL DAB until the samples turned light brown. The samples were rinsed with deionized water, sealed, and observed under a microscope.
Cell apoptosis and necrosis were evaluated using flow cytometry after staining with phycoerythrin-conjugated annexin V and 7-Aminoactinomycin D (BD Pharmingen, San Diego, CA, USA). The cells were then analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) using the Cell Quest software (Becton Dickinson) and expressed as the percentage of positive cells.
RNA binding protein immunoprecipitation (RIP) assay
The binding between GAS5 and EZH2 was evaluated using a Magna RIP TM RIP kit (Millipore, Billerica, MA, USA). Mouse SMCs were washed with pre-cooled PBS and scraped in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, China) on ice for 5 min. The cells were then centrifuge at 14,000 rpm for 10 min at 4 °C, and part of the cell extract was absorbed as input, and the others were incubated with antibodies. For each co-precipitation reaction system, 50 µL magnetic beads were resuspended in 100 µL RIP Wash Buffer (EHJ-BVIS08102 Xiamen Jiahui Biotechnology Co., Ltd., China), and incubated with 5 µg antibody against EZH2 (#5246, 1:300, Cell Signaling Technology, Beverly, MA, USA) or immunoglobulin G (IgG) (1:100, ab109489, Abcam). After the magnetic bead-antibody complex was washed, the complex was resuspended in 900 µL RIP Wash Buffer, and interacted with 100 µL cell extract at 4 °C overnight. The magnetic bead-antibody complex and Input were digested with proteinase K, from which RNA was extracted for subsequent polymerase chain reaction (PCR) detection.
Co-immunoprecipitation (Co-IP) assay
SMCs were lysed using cell lysis solution on ice for 30 min, after which the cell lysate was collected into a 1.5 mL Eppendorf tube and centrifuged at 12,000×g and 4 °C for 15 min, with the supernatant collected. Then 50 μL of protein A and protein G beads were extracted to the tube and suspended to form a mixture of 100 μL beads, 10 μL of which was added to the cell lysate with EZH2 antibody (ab191080, 1:1000, Abcam) and incubated at 4 °C overnight for coupling. After IP reaction, centrifugation was carried out at 3000 rpm and 4 °C for 3 min, with the agarose beads to the bottom of the tube. At last, 15 μL of 2 × sodium dodecyl sulfate (SDS) loading buffer was added to the precipitate, and boiled for 5 min. After cooling, the precipitate was analyzed by Western blot analysis to detect the expression RIG-I.
RNA isolation and quantitation
The total RNA was extracted using TRIzol reagent (16096020, Thermo Fisher Scientific Inc., Waltham, MA, USA). Then 5 µg of the extracted RNA was reversely transcribed into complementary DNA (cDNA) using Reverse Transcription Kit (K1622, Fermentas Inc., Ontario, CA, USA). Primer sequences for GAS5, EZH2 and RIG-I are shown in Additional file 1: Table S1. With β-actin as internal reference, reverse transcription quantitative PCR (RT-qPCR) was then performed using TaqMan MicroRNA Assay and TaqMan® Universal PCR Master Mix and TaqMan Gene Expression Assays protocol (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the loading control and the expression was calculated using 2−△△Ct method). The result was normalized to mRNA expression of β-actin.
Western blot analysis
The total protein was extracted using RIPA lysis buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The total protein was quantified using a bicinchoninic acid protein concentration detection kit (GBCBIO Technologies Inc., Guangzhou, China). Next, 40 μg protein was separated by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The membrane was blocked with 5% bovine serum albumin at room temperature and probed overnight at 4 °C with primary rabbit antibodies to EZH2 (1:1000, ab191080), RIG-I (1:1000, ab45428), cleaved Caspase3 (1:1000, ab2302), B-cell lymphoma-2 (Bcl-2) (1:1000, ab182858), Bcl-2 associated protein X (Bax) (1:1000, ab32503), and β-actin (1:10,000, ab8226). The aforementioned antibodies were purchased from Abcam. The following day, the membrane was re-probed with secondary goat anti-rabbit IgG (1:2000, ab97051, Abcam) at room temperature. The immunocomplexes on the membrane were visualized using enhanced chemiluminescence, followed by photograph using Image Quant LAS 4000C software (GE, USA). With β-actin as internal reference, the blots were scanned, quantified for pixel density by the optical density method, and analyzed by Image lab software.
All data were processed using SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA) and presented as mean ± standard deviation. Data between two groups were analyzed using the unpaired t-test and data among multiple groups were analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc tests. p < 0.05 was considered to be statistically significant.