Ethics
Both human serum samples and animal studies were approved by the Clinical Research Committee and the Institutional Animal Care and Use Committee of General Hospital of Northern Theater Command respectively. All human studies adhered to the guidelines of the Declaration of Helsinki. All participants in the experiment provided informed consent. All animal experiments complied with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.
Animal model and samples
We used 6–8 week-old male ApoE-/- mice (weight 20–25 g) purchased from Jackson laboratories (Bar Harbor, ME, USA). Mice were infused with Ang II (Sigma Chemical Co; # a-9295, USA) at 1 µg/kg/min (the dose was based on a previous study[9]) for 4 weeks using a micro-osmotic pump (model 1004, ALZET Osmotic Pumps, Cupertino, CA, USA). AAA group mice were fed a western diet for 4 weeks before Ang II injection and continued on the same diet afterwards. Control group mice were fed a chow diet and infused with saline. The level of abdominal aorta from the left renal artery to the suprarenal area was analyzed by echocardiography. Images of the whole aorta were obtained by scanning the long and short aorta axes. Each mouse was scanned three times by an investigator blinded to the nature of treatment. Blood pressure was measured by the tail-cuff method using a Softron BP2010A Blood Pressure Meter (Softron Beijing Biotechnology Co., Ltd, Beijing, China). The tail was fixed to the sphygmomanometer, and sensors were used to record blood pressure indirectly. Blood pressure was measured at least three times in each mouse at 0, 7, 14, and 28 days. Mice that died during the experiment were dissected, and RAAAs were detected based on dilated perivascular blood clots or abdominal bleeding; these mice were included in the assessment of the tumor formation rate and risk of rupture. The surviving mice were euthanized under anesthesia at day 28, and the aorta (from the ascending aorta to the iliac artery) was dissected out. The AAA specimens were frozen at -80 ℃, transferred into centrifuge tubes, sealed on dry ice, and mailed to BioMiao Biological Technology Co., Ltd. (Beijing, China) for microarray analysis.
Microarray analysis
miRNA expression in mouse aortas was analyzed by BioMiao Biological Technology Co., Ltd (Beijing, China) using the Agilent Mouse miRNA Microarray Kit, Release 21.0, 8 × 60 K (Design ID:070155; Agilent Technologies, Santa Clara, CA, USA), which contained 1902 probes for mature miRNA.
Patient cohorts
In total, 59 patients were included in the study: 20 with RAAA, 20 with URAAA, and 19 with CAD (control group). To assess the maximal aortic diameter and the presence of an entry tear, all patients underwent computed tomographic angiography (CTA) during hospitalization, as previously described[25]. CTA was carried out using a Siemens Somatom Sensation 64 CT Scanner after administration of intravenous boluses containing 80–150 mL of nonionic contrast medium; three measurements were performed and the mean values were calculated.
miRNA isolation from blood samples and qRT-PCR
Peripheral venous blood was collected from patients into EDTA-containing tubes and centrifuged at 3000 × g for 10 min at 4 ℃, and plasma was stored at -80 ℃. Total RNA was extracted from 200 microliter plasma samples by using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) and cDNA was synthesized using a reverse transcription kit (RiboBio, Guangzhou, China) according to the manufacturer's instructions. After equal volume dilution of cDNA (20 microliters of DNase/RNase-Free Deionized water was added to 20 microliters of cDNA), the expression levels of miR-30c-1-3p, miR-432-3p, miR-3154, and miR-379-5p were evaluated by Quantitative real-time Polymerase Chain Reaction (qRT-PCR) using specific primers (Supplemental Table 3) and the miDETECT A TrackTM miRNA qRT-PCR Kit (RiboBio) following the manufacturer’s protocol; reactions were performed on a CFX96 Touch™ Real-Time PCR Detection System (Hercules, California, USA). Each reaction was performed in triplicate, the relative expression level of miRNAs were calculated based on cycle threshold (Ct) values through formula 2-∆Ct, which ∆Ct value is the Ct value of miRNAs in each patient of URAAA/RAAA group minus the average Ct value of miRNAs in the control group.
Western blot analysis
Cells and tissues were homogenized using a RIPA buffer (Thermo Fisher Scientific, Glen Burnie, MD) supplemented with protease and phosphatase inhibitors. The total protein was estimated using the BCA protein assay reagent kit (Thermo Fisher Scientific). Briefly, 40 µg total protein was loaded and detected with antibodies against MMP9 (Cell Signaling Technology, Danvers, MA, US), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology).
Functional annotation of differentially expressed miRNAs
The target genes of differentially expressed miRNAs were predicted using miRWalk, miRanda, RNA22, and Targetscan )http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/)and analyzed based on GO terms enrichment in three functional categories: molecular function, cellular component, and biological process[26]. KEGG was used for the functional classification of miRNAs according to the relevant biological pathways and for analysis of their relative enrichment.
Cell culture and transfections
We obtained RAW 264.7 cells and human embryonic kidney HEK293 cells lines from the Chinese Academy of Sciences Cell Bank. Mouse macrophage RAW 264.7 cells and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies) at 37°C in a humidified atmosphere of 5% CO2. RAW 264.7 cells were transfected with a miRNA mimic (100 nM), miRNA inhibitor (100 nM), or their controls (RiboBio, Guangzhou, China) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, collected after 48 h, and analyzed for MMP-9 expression by western blotting. HEK293 cells were used for the dual-luciferase reporter assay described below.
Dual-luciferase reporter assay
PmirGLO Dual-Luciferase miRNA Target Expression plasmids containing the MMP-9 3'-UTR or coding sequence were synthesized by GENEWIZ (Suzhou, China) and amplified it in E. coli. The plasmids were extracted using the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
HEK293 cells were co-transfected with 200 ng of pmirGLO Dual Luciferase plasmids, and miR-30c-1-3p mimic (100 nM) or mimic control (100 nM) using X-tremeGENE HP DNA Transfection Reagent (Sigma, St. Louis, MO, USA) .
Luciferase activity was analyzed by the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s protocol. Transfection efficiency was normalized by Renilla luciferase activity.
Biochemical analysis
Serum samples were analyzed for TC (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), TG (Nanjing Jiancheng Bioengineering Institute) by using the total cholesterol and triglyceride quantification kit according to the manufacturers’ instructions.
Measurement of MMP-9 plasma concentration
Plasma levels of MMP-9 were determined by enzyme linked immunosorbent assay (ELISA) using a human MMP-9 ELISA kit ( no. ab246539, Abcam, UK) according to the manufacturer’s instructions.
Statistical analysis
Continuous variables were reported as the mean ± standard deviation (SD) or medians and interquartile ranges (25th or 75th percentiles) and compared by unpaired Student’s t-test, one-way ANOVA, or Mann-Whitney U test. Categorical variables were expressed as percentages and compared by chi-square test or Fisher’s exact tests. Pearson correlation coefficient (r) was used for bivariate normally distributed data. The prediction potential of MMP-9 expression or the maximal aortic diameter was evaluated by ROC curve analysis. The optimal cutoff level was calculated by the Youden index (sensitivity + specificity - 1). GraphPad Prism 8.0 (GraphPad, La Jolla, CA, USA) was used for graphical data presentation. SPSS version 22.0 was used for statistical analysis (IBM, Armonk, New York, NY, USA). The level of significance was set at P < 0.05.