Aortic specimens were collected from 10 patients who underwent ascending aortic aneurysm surgery (mean age, 63.25±6.24; men, 7; women, 3) and from 10 control patients who underwent coronary artery bypass graft with non-aneurysmal aortas (mean age, 57.80±6.56; men, 8; women, 2) between January 2017 and December 2018 at Tianjin Chest Hospital. Patients with bicuspid aortic valves or hereditary connective tissue disorders were excluded from the control group. The obtained aortic specimens were split into two parts, which were snap-frozen in liquid nitrogen for RNA isolation or primary VSMCs isolation. All patients signed written informed consent, and ethical approval was obtained from the Institutional Review Board of Tianjin Chest Hospital.

Primary VSMCs were isolated from the aforementioned 10 clinical aortic specimens who underwent ascending aortic aneurysm surgery as previous described with modifications (16). Following removal of fat and connective tissue, the tissues were cut into 3-mm long sections and rubbed to remove the endothelium, mixed the 10 samples together, followed by incubation with 0.2% collagenase at 37˚C and centrifugation at 120 x g for 15 min at 4˚C to obtain VSMCs. Following washing with PBS, the obtained pelleted VSMCs were seeded into six-well plates containing medium 231 (cat. no. M231500; Life Technologies; Thermo Fisher Scientific, Inc.) containing 5% Smooth Muscle Growth Supplement (cat. no. S00725; Life Technologies; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and streptomycin (cat. no. 15140122; Life Technologies; Thermo Fisher Scientific, Inc.), and incubated in under 5% CO₂ at 37˚C for 24 h before transfection.

All the mimics and vectors were purchased from GeneCopoeia, Inc. For transfections, the miR-7 mimics (5'-UGGAAGACUAGUGAUUUUGUUGU-3'), miR-negative control (mimics NC, 5'-CAGUACUUUUGUGUAGUACAA-3'), miR-7 antisense oligonucleotide (miR-7 ASO, 5'-ACAACAAAAUCACUAGUCUUCCA-3'), ASO NC (5'-CAGUACUUUUGUGUAGUACAA-3'), small interfering RNA (si)-NC (5'-UUCUCCGAACGUGUCACGUTT-3'), si-CDR1as (5'-CCAAUAAGGCCAGUUCAUUTT-3') were used at 50 mM. pcDNA3 (abbr. NC), pcDNA3-CDR1as (abbr. CDR1as), pcDNA3-CKAP4 were used at 25 ng/ml in six-well plates seeded with 3x10⁵ VSMCs cells. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for all transfections according to the manufacturer's instructions. The transfected cells were used for subsequent experiment at 48 h post-transfection.

TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA extraction from aortic specimens or VSMCs. Briefly, 1 µg of the RNA was reverse transcribed using the High-Capacity cDNA reverse transcription kit (cat. no. 4368813; Thermo Fisher Scientific, Inc.). The temperature protocol for reverse transcription was as follows: Incubation at 25˚C for 5 min, 37˚C for 120 min and 85˚C for 5 min. qPCR was then performed using SYBR Green Master mix (Thermo Fisher Scientific, Inc.) using the following parameters: 95˚C for 5 min; 40 cycles of 10 sec at 95˚C, 20 sec at 55˚C and 15 sec at 72˚C, followed by a melting curve of 10 sec at 95˚C, 60 sec at 60˚C and a continuous increase up to 95˚C at a rate of 0.15˚C/sec for 15 sec with a continuous reading of the fluorescence. The 2^-ΔΔCq method was used to quantify the target gene expression (17). The primers used were as follows: miR-7 forward, 5'-CCACGTTGGAAGACTAGTGATTT-3' and reverse, 5'-TATGGTTGTTCTGCTCTCTGTCTC-3'; CDR1as forward, 5'-GTGTCTCCAGTGTATCGGCG-3' and reverse, 5'-TACTGGCACCACTGGAAACC-3'; GAPDH forward, 5'-GACTCATGACCACAGTCCATGC-3' and reverse, 5'-AGAGGCAGGGATGATGTTCTG-3'; U6 forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'.

StarBase v3.0 (http://starbase.sysu.edu.cn) was used for predicting the potential long ncRNA (lncRNA) that can bind miR-7. TargetScan 7.1 (http://www.targetscan.org/vert_72/) was used to predict the downstream target genes of miR-7.

Briefly, 1.5x10⁴ cells were seeded in a 24-well plate. After 24 h, the cells were transfected with miR-7 mimics, miR-7 ASO, miR-NC or ASO NC as well as the pmirGLO vector (Promega Corporation), which was inserted with the wild-type (WT) 3'-untranslated region (UTR) of CKAP4 or a mutant (mut) form of CKAP4 3'-UTR which contained a mutated seed sequence (from 5'-GUCUUCC-3' into 5'-GUGUUGG-3'). At 48 h following transfection, the cells were used to measure the activities of firefly and Renilla luciferase using a Dual Luciferase reporter assay kit (Promega Corporation). The activity of Renilla luciferase was used as internal control.

Cells were transfected with 50 µM of biotinylated miR-7 mimic or miR-NC (as aforementioned; GeneCopoeia, Inc.) for 24 h and lysed in 500 µl lysis buffer at 4˚C for 30 min, followed by the addition of 50 µl blocked streptavidin magnetic beads and incubation for 4 h at 4˚C. TRIzol^® was used to extract the miR-7 that interacted with ncRNAs and the CDR1as expression levels were subsequently determined using RT-qPCR.

Following transfection for 48 h, the VSMCs were lysed using RIPA buffer (150 mM Tris-HCl, 50 mM NaCl, 1% Nonidet P-40 and 0.1% Tween-20), followed by determining the protein concentration using Bradford assays (Bio-Rad Laboratories, Inc.). Proteins (30 µg) were separated by 10% SDS-PAGE, followed by transferring to PVDF membranes at 250 mA for 2.5 h. After blocking for 1 h with 3% w/v skimmed milk at room temperature, the membranes were incubated with the following primary antibodies overnight at 4˚C: Anti-CKAP4 (cat. no. ab84712; 1:2,000; Abcam), anti-cleaved-caspase 3 (cat. no. 9661; 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. 2118; 1:10,000; Cell Signaling Technology, Inc.). The next day, the membranes were washed with Tris-buffered saline containing 0.1% Tween 20, three times and incubated with a secondary antibody conjugated with horseradish peroxidase (cat. no. A0208, Beyotime Institute of Biotechnology) at 1:1,000 for 1 h at room temperature. Chemiluminescence was detected with an ECL kit (GE Healthcare Life Sciences), then quantified using ImageQuant software version 5.2 (GE Healthcare Life Sciences). GAPDH was used as an internal control.

To detect cell viability, a CCK-8 assay was performed. Treated and untreated VSMCs were cultured in a 96-well plate at 3x10⁴ cells/well. After 48 h, 10% CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added to each well and incubated for 1 h at 37˚C, as per the manufacturer's instructions, followed by analysis using a microplate reader at a wavelength of 450 nm.

To detect the extent of VSMC apoptosis, LDH, ROS and caspase 3/7 assays were performed.

For the LDH assay, transfected cells were cultured at 5x10³ cells/well in 96-well plates for 48 h. After centrifuging at 350 x g for 5 min at 4˚C and discarding of the supernatant, 150 µl LDH release regent (cat. no. C0016; Beyotime Institute of Biotechnology) was added to each well and incubated for 1 h at 37˚C. The signal was detected using a microplate reader (Bio-Rad Laboratories, Inc.) at 490 nm.

Intracellular ROS was detected using 2',7'-dichlorofluorescin diacetate (DCF-DA; Sigma-Aldrich; Merck KGaA) staining as previously described (18). After discarding the cell medium, the cells (5x10³ cells/well in 96-well plates) were stained with 20 µM DCF-DA solution at 37˚C in the dark for 30 min, followed by the measurement of the signal with a fluorescence reader (Bio-Rad Laboratories, Inc.) at excitation/emission wavelengths of 488/525 nm.

Caspase 3/7 activity was quantitated using a Caspase-Glo^® 3/7 Assay kit (Promega Corporation) according to the manufacturer's instructions.

Data are presented as the mean ± standard error of the mean. When comparing the AAA aortic tissues and normal controls, data are presented using box and whiskers plots. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc.) with one-way ANOVAs followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least three times.

The results of RT-qPCR demonstrated that miR-7 was expressed at higher levels in AAA aortic tissues compared with the normal controls (Fig. 1A). The expression levels of CDR1as and CKAP4 were notably downregulated in AAA (Fig. 1B and C).

Transfection efficiency of CDR1as overexpression plasmids and siRNA, and miR-7 mimics and ASO was evaluated using RT-qPCR. The results revealed that CDR1as overexpression increased CDR1as RNA levels 2-fold, whereas si-CDR1as downregulated the expression of this RNA by 60%. miR-7 mimics upregulated miR-7 expression 1.8-fold, whereas miR-7 ASO downregulated its expression by 70% (Fig. 2A). The results of the CCK-8 assay revealed that cell viability was increased following transfection with either CDR1as or miR-7 ASO, but decreased following transfection with siRNA CDR1as or miR-7 mimics, compared with their respective controls (Fig. 2B). Similarly, Cell growth curve assay showed that cell proliferative ability was increased following transfection with CDR1as or miR-7 ASO, but decreased following transfection with siRNA CDR1as or miR-7 mimics (Fig. 2C and D). In addition, CDR1as and miR-7 ASO decreased the ROS generation and LDH activity, which was increased following transfection with siRNA CDR1as or miR-7 mimics, compared with the respective control groups (Fig. 2E and F). These results suggested that CDR1as and miR-7 exerted opposing effects on VSMCs.

The WT version of CDR1as was found to contain a potential binding site of miR-7 (Fig. 3A). To further investigate whether miR-7 can be sponged by CDR1as in VSMCs, a CDR1as luciferase reporter was constructed. The results demonstrated that miR-7 significantly reduced the luciferase intensity of CDR1as-WT, but had no significant effect on the luciferase intensity of CDR1as-mut (Fig. 3B). In addition, the biotin pull-down assay results revealed that CDR1as was pulled down in biotinylated miR-7-transfected cells by 11-fold compared with cells transfected with biotinylated miR-NC (Fig. 3C). As presented in Fig. 3D, miR-7 expression was suppressed by CDR1as in VSMCs, which could be reversed by co-transfection with miR-7 mimics. CCK-8 and cell proliferation curve assays revealed that the CDR1as-facilitated cell viability and proliferation was reversed by co-transfection with miR-7 (Fig. 3E and F). Additionally, CDR1as-mediated suppression of ROS generation and LDH activity was reversed by co-transfections with miR-7 (Fig. 3G and H). Therefore, these data suggested that miR-7 may be sponged by CDR1as and mediate the downstream effects of CDR1as in VSMCs.

Bioinformatics analysis was performed using TargetScan to predict the potential target of miR-7, with CKAP4 being identified as a target for miR-7 (Fig. 4A). To confirm whether CKAP4 was regulated by miR-7, RT-qPCR and western blot analysis were performed, which demonstrated that overexpression of miR-7 notably downregulated the expression of CKAP4 compared with mimics NC, whereas knockdown of miR-7 upregulated the expression of CKAP4 compared with ASO NC (Fig. 4B and C). In addition, luciferase reporter assays confirmed that miR-7 mimics notably decreased the luciferase activity of CKAP4-3'UTR, whereas this was not observed with miR-7 ASO. The expression of miR-7 mimics and ASO had no significant effects on the luciferase activity of the CKAP4-3'UTR mut reporter plasmid (Fig. 4D). These data confirmed that CKAP4 was a direct target of miR-7 in VSMCs.

To investigate whether CDR1as/miR-7/CKAP4 regulated the function of VSMCs, the present study investigated the impact of miR-7 on CKAP4. Firstly, pcDNA3-CKAP4 was verified to have an effective transfection efficiency (Fig. 5A). It was then confirmed that miR-7-mediated suppression of CKAP4 could be rescued by transfection with pcDNA3-CKAP4 (Fig. 5B). miR-7 mediated induction of caspase 3/7 activity can be rescued by co-transfection pcDNA3-CKAP4 with miR-7 mimics (Fig. 5C), which suggested that miR-7-induced apoptosis may be rescued by CKAP4. In addition, the results demonstrated that CDR1as upregulated the expression of CKAP4, which was reversed by co-transfection with CDR1as and miR-7 mimics (Fig. 5D). The CDR1as-reduced caspase 3/7 activity by was rescued following co-transfection with miR-7 mimics (Fig. 5E), suggesting that the CDR1as-mediated suppression of apoptosis was dependent on the downregulation of miR-7. In summary, CDR1as mediated the downregulation of miR-7, resulting in the upregulation of CKAP4 and leading to the inhibition of caspase 3/7 activity (Fig. 5F).