To induce cerebral aneurysms, male wild-type mice (C57BL/6; 6–8 weeks; 30–32 g; n=20) were housed in a temperature (25±1°C) and a humidity (55±5%) and maintained under 12 h light and 12 h dark cycle with free access to food and water. All mice were purchased from Shanghai SLAC Laboratory Animals Co., Ltd. (Shanghai, China). Under intraperitoneal Zoletile^® anesthesia (30 mg/kg) with xylazin (10 mg/kg), the left common carotid artery and the posterior branches of the two renal arteries were ligated to initiate cerebral aneurysms. Following the surgery, 1% normal saline was substituted for the drinking water to promote the degree of hypertension. The mice were divided into a control group with no surgery (n=10) and an aneurysm group (n=10) with ligation on the left common carotid artery and the posterior branches of the two renal arteries. The Institutional Animal Care and Use Committee of the Affiliated Hospital of Hebei University of Engineering (Hebei, China) approved all procedures prior to the initiation of the study.

In order to evaluate the effect of miR-29a on IA, the chemically modified agomir was used to increase miRNA expression in vivo. miRNA agomir is a chemically modified, cholesterylated, stable miRNA mimic, and its in vivo delivery resulted in target silencing similar to the effects induced by the overexpression of endogenous miRNA. Agomir-29a was synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequence of agomir-29a is 5′-ACUGAUUUCUUUUGGUGUUCAG-3′. The mice received agomir-29a (100 µl) via tail vein injection (40 mg/kg body weight; n=10) for 3 consecutive days. The mice in the no surgery group (n=10) and the aneurysm group (n=10) with ligation were treated as mentioned above. Expression of miR-29a was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR; data not shown). At 200 days following the induction procedure, all mice were euthanized with CO gas. Cerebral arteries were dissected and stripped from brains under a surgical microscope. Samples from the regions of aneurysmal dilation on left posterior communicating artery in the aneurysm group and samples from the circles of Willis in the control groups were obtained.

Human brain VSMCs (HBVSMCs; cat. no. CSC-7824W) were purchased from Creative Bioarray (Shirley, NY, USA). 293-T cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, which were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin (100 U/l) and streptomycin (100 µg/ml). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded at 10×10⁶ cells/dish in ten 15-cm dishes 1 day prior to transfection. The miR-29a mimics, miR-29a inhibitor, and the corresponding controls [mimics negative control (NC) or inhibitor NC] were obtained from GeneCopoeia, Inc. (Rockville, MD, USA). Transfection was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Cells were transfected with 50 nM miR-29a mimics, miR-29a inhibitor or corresponding controls for 2 h, followed by treatment with 200 µM H₂O₂ for 6 h, and cells were harvested at different times (24, 36 and 48 h) for subsequent experiments. Cells in the control group were treated with the same medium without H₂O₂ (blank group).

Written informed consent was obtained from patients, according to a protocol approved by the Ethics Committee of the Affiliated Hospital of Hebei University of Engineering. In total, 2 ml peripheral blood samples were obtained from 24 pairs of patients with IA and healthy volunteers from the Affiliated Hospital of Hebei University of Engineering (recruited from March 2014 to April 2015). Twenty-four patients met the inclusion criteria and were enrolled. Inclusion criteria (25) were as follows; age ≤70 years (1) and planned microsurgery or endovascular surgery for unruptured IA (2). Exclusion criteria (25) were as follows; preoperative intelligence quotient <80 (n=2) (1); initial modified Rankin scale ≥1 (n=1) (2); and loss to follow-up (n=3) (3). There were 28 males and 20 females, and the age range was 35–78 years.

TissueLyser II (Qiagen, Inc., Valencia, CA, USA) was used to homogenize the aneurysm and control samples obtained from mice. Total RNA from tissue, cells and blood samples were isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Low molecular weight RNA (<200 nucleotides) was isolated from the total RNA by mirVana miRNA purification columns (Ambion; Thermo Fisher Scientific, Inc.) for microarray analysis and RT-qPCR, according to the manufacturer's protocol. RNA quality and quantity were determined using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.).

Total RNA from samples from IA and sham mice (3/group) was sent to LC Sciences, LLC (Houston, TX, USA) for miRNA microarray analysis (miRCURY LNA™ miRNA Array kit; Exiqon A/S, Vedbaek, Denmark). Data were analyzed by one-way analysis of variance (ANOVA) using Tukey's multiple comparison test. Expression normalization was performed using a cyclic LOWESS method (26).

RNA was reverse transcribed to cDNA from 100 ng total RNA with a TIANScript II RT kit (Tiangen Biotech Co., Ltd., Beijing, China) at 37°C for 1 h. qPCR was performed with miScript SYBRGreen PCR kit (Tiangen Biotech Co., Ltd.). Primer sequences were as follows: U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; miR-29a forward, 5′-TGCGCTAGCACCATCTGAAAT-3′ and reverse, 5′-CAGTGCAGGGTCCGAGGT-3′; β-actin forward, 5′-GGGAAATCGTGCGTGACATTAAG-3′ and reverse, 5′-TGTGTTGGCGTACAGGTCTTTG-3′; caspase-3 forward, 5′-AGAGGAATGATTGGGGGTG-3′ and reverse, 5′-TTGCTAGGCAGTGGTAGCG-3′; caspase-9 forward, 5′-CGGAATCACCAATCATTACAT-3′ and reverse, 5′-AGAAACGCCCACAACTGC-3′; caspase-8 forward, 5′-CAGCATTAGGGACAGGAATC-3′ and reverse, 5′-CAGTTATTCACAGTGGCCAT-3′; myeloid cell leukemia 1 (Mcl-1) forward, 5′-TGAAATCGTTGTCTCGAGTGATG-3′ and reverse, 5′-TCACAATCGCCCCAGTTT-3′; and Cyt-c forward, 5′-CAGTGCCATACTGTGGAAAAGG-3′ and reverse, 5′-TGACCTGTCTTTCGTCCAAACA-3′. qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), with each reaction performed in triplicate. The PCR amplification protocol was as follows: An initial 95°C for 5 min and 40 cycles of 94°C for 15 sec; 55°C for 30 sec; and 70°C for 30 sec, followed by a final extension step of 72°C for 10 min. The RT-qPCR assays were performed in triplicate and the alteration in expression level was normalized to the expression of U6 using the 2^−ΔΔCq method (27).

Bioinformatics TargetScan 7.0 (http://www.targetscan.org/) (28) and miRanda (http://www.microrna.org/) (29) target gene prediction software selected Mcl-1 as a target gene of miR-29a.

For TUNEL analysis of mouse vascular tissues, the Roche In Situ Cell Death Fluorescein Detection kit (Roche Diagnostics, Basel, Switzerland) was used, according to the manufacturer's protocol. Vascular tissues were fixed in 4% paraformaldehyde for 3 h at 4°C, paraffin embedded and cut into 4 µm-thick sections. Tissue sections were subsequently de-waxed in xylene, rehydrated in graded alcohols, and placed in dH₂O. The slides were incubated for 15 min at room temperature with 20 µg/ml Proteinase K (Gibco BRL; Thermo Fisher Scientific, Inc.). The slides were rinsed twice with PBS prior to incubation in TUNEL reaction mixture for 60 min at 37°C. The Nucleotide Mix was on ice and sufficient rTdT incubation buffer was prepared for all experimental reactions, 50 µl rTdT incubation buffer was added to each slide. The sections were covered with plastic coverslips to ensure even distribution of the reagent. The DNA fragments were labeled with the rTdT at 37°C for 1 h in the dark. The sections were stained with 10 nM DAPI (Beyotime Institute of Biotechnology, Shanghai, China) at 37°C for 3 min and observed using an inverted fluorescence microscope (Olympus 1X51; Olympus Corporation, Tokyo, Japan; magnification, ×100; number of fields, n=5/tissue). TUNEL-positive cells were counted using ImageJ software version 1.33–1.34 (http://rsbweb.nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA) for subsequent statistical analysis.

miR-29a mimics, miR-29a inhibitors and negative control (NC) miRs (mimics NC and inhibitor NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences are as follows: miR-29a mimics, 5′-TAGCACCATCTGAAATCGGTTA-3′; miR-29a inhibitor, 5′-TAACCGATTTCAGATGGTGCTA-3′; mimics NC, 5′-AAATGTACTGCGCGTGGAGAC-3′; and inhibitor NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. 293T cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were seeded into 48-well plates (2×10⁵ /well) and cotransfected with miR-29a mimics/inhibitors/NC miRs (50 nM) and NFAT luciferase reporter plasmids (200 ng; Promega Corporation, Madison, WI, USA) containing a wild-type or mutant type of the Mcl-1 3′ untranslated region (3′-UTR) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h following transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). Firefly luciferase activity was normalized to the activity of Renilla luciferase. Each transfection was performed in triplicate.

Cells were lysed using radioimmunoprecipitation buffer (cat. no. 211-40; AmyJet Scientific, Inc., Wuhan, China). Total cell protein was extracted from cells or tissues. According to the manufacturer's protocol, the protein concentration was determined by a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). A total of 2 µg per lane protein was electrophoresed with 10% SDS-PAGE. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked with 5% skimmed milk at room temperature for 1 h. Following blocking with milk, the membranes were incubated with specific primary antibodies at 37°C for 2 h. β-actin was used as the internal reference. The secondary antibody (1:10,000) was added to the membrane and incubated for 1 h with agitation at room temperature. Rabbit anti-cytochrome c (Cyt-c; 1:1,000; cat. no. 4280) and anti-caspase-3 (caspase-3; cat. no. 9665; 1:1,500) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-Mcl-1 antibody (Mcl-1; 1:1,000; cat. no. sc-819) and anti-β-actin (1:1,500; cat. no. sc-8432) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (1:2,000; cat. no. sc-2004) were purchased from Santa Cruz Biotechnology, Inc. The signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA) and autoradiography. Densitometric levels of protein signals were quantified and presented as their ratio to β-actin. Autoradiograms were quantified by densitometry using Quantity One software version 4.4.0 (Bio-Rad Laboratories, Inc.).

Cells (5×10⁵) were seeded on 96-well plates following trypsin digestion and cell counting, and five wells in a row were used for each sample. Cells were incubated at 37°C with 5% CO₂ for 5 days. An MTT stain was performed, according to the manufacturer's protocol. Formazan was dissolved by dimethyl sulfoxide. A microplate reader was used to measure the optical density 450 value of solution.

A total of 10×10⁴ HBVSMCs transfected with NC miRs, miR-29a mimics and miR-29a inhibitors, were harvested by trypsinization at 48 h following transfection. An Annexin V-Fluorescein Isothiocyanate kit (BD Biosciences, San Jose, CA, USA) was used to detect apoptotic cells, according to the manufacturer's protocol. The cells were analyzed with a flow cytometer (FACScan; BD Biosciences) equipped with CellQuest software (version 5.1; BD Biosciences). The relative ratio of early apoptotic cells was compared with control transfection from each experiment. Each sample was run in triplicate.

Data are presented as the mean ± standard deviation from at least three separate experiments. All experiments were repeated for at least three times with triplicated samples in each experiment. One-way ANOVA using Tukey's multiple comparison test was employed to evaluate the statistical differences among different groups with SPSS version 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The activation of caspase-3, −8 and −9, which are apoptotic caspases, may serve a vital role in the process of mitochondrial apoptosis. In the present study, the mRNA expression of caspases, including caspase-3, −8 and −9, was analyzed in sham control and IA mice samples by RT-qPCR. The results demonstrated that the expression levels of caspase-3, caspase-8 and caspase-9 were significantly upregulated in the IA group compared with sham controls (P<0.01; Fig. 1A), which indicate that increased apoptosis occurred in IA.

To further detect whether the mitochondrial pathway is involved in the apoptosis observed in IA mice, the protein expression of Cyt-c and Mcl-1 was detected by western blotting. The results demonstrated that the protein expression of Cyt-c was activated, while Mcl-1 expression was decreased, in the IA group compared with the sham controls (Fig. 1B). Additionally, pathological analysis demonstrated that the apoptosis of VSMCs, accompanied with decreased numbers of VSMCs, occurred in the IA group (Fig. 1C). The apoptosis of vascular tissue between the two groups was detected by a TUNEL assay (Fig. 1C). Quantification of TUNEL staining demonstrated that apoptosis was significantly increased in the IA group compared with the sham group (P<0.01). These results indicated that mitochondrial apoptosis may participate in the process of IA.

miRNAs have been reported to regulate the mitochondrial apoptosis pathway (30,31). In the present study, to detect the miRNAs that may be involved in the IA process, an miRNA array was used. Significantly different expression levels of miRNAs were identified between the IA group and the sham controls, with 31 miRNAs significantly upregulated and 20 miRNAs significantly downregulated (fold change value >2; P<0.05) in the IA tissues compared with the control group (Fig. 2A).

For validation of the microarray data, the most dysregulated miRs, including miR-29a, miR-233, miR-433, miR-489 and miR-126 were selected for RT-qPCR analysis. The relative expression alterations of these miRNA analyzed by RT-qPCR were consistent with the microarray analysis results, with the exception of miR-126, which was not significantly altered between the sham control and IA mice (P<0.05; Fig. 2B). In order to determine whether miR-29a expression in IA mice models exhibits a similar pattern to clinical patients, a clinical experiment was performed to detect miR-29a expression in patients with IA and healthy volunteers. The blood samples obtained from the case group included 24 patients with IA, while the control group included 24 healthy volunteers. miR-29a expression was detected and compared between the two groups. The results of RT-qPCR demonstrated that the expression of miR-29a in patients with IA was significantly higher compared with healthy controls (P<0.01; Fig. 2C).

miR-29a has been reported as a potential biomarker in the development of IA (31). In addition, it has been reported that upregulation of miR-29a may be implicated in cell apoptosis by targeting Mcl-1 expression in rat germ cell death (32). Previous studies have demonstrated that miR-29a is able to induce cell apoptosis by activating caspase proteins (33,34). For example, miR-29a was reported to be responsible for the activation of caspase-3-induced K562 cell apoptosis (35). Therefore, miR-29a was selected for further investigation in the present study.

An in vitro H₂O₂-induced apoptosis model was established to mimic IA (36). HBVSMCs were treated with H₂O₂ (0–300 µM) and miR-29a expression levels were significantly increased in a concentration-dependent manner, peaking at 200 µM, compared with the 0 µM group (Fig. 2D). Based on these results, the concentration of 200 µM H₂O₂ was selected for subsequent experiments.

HBVSMC cells were transfected with miR-29a mimics or inhibitor. Fig. 3A demonstrates that the expression of miR-29a was significantly upregulated in the miR-29a mimic treatment group compared with the mimics NC group (P<0.01), while miR-29a expression was significantly decreased in cells with miR-29a inhibitor treatment compared with the inhibitor NC group (P<0.01). Following transfection with miR-29a inhibitor for 24, 36 and 48 h, the cell viability of VSMCs treated with H₂O₂ was significantly increased, compared with the inhibitor NC treatment group (P<0.01; Fig. 3B). However, the H₂O₂-induced reduction of HBVSMC viability was significantly enhanced following transfection with miR-29a mimics, compared with the mimics NC treatment group (P<0.01; Fig. 3C). These results indicated that the miR-29a inhibitor reversed H₂O₂-induced cell viability inhibition, while miR-29a mimics promoted H₂O₂-induced cell viability inhibition.

Furthermore, treatment with 200 µM H₂O₂ indicated that transfection of miR-29a inhibitor protected against H₂O₂-induced HBVSMC apoptosis, while miR-29a mimics worsened the H₂O₂-induced HBVSMC apoptosis (P<0.05; Fig. 3D and E). Western blot analysis was performed to observe alterations in the protein expression of apoptosis-associated proteins in H₂O₂-induced HBVSMCs transfected with miR-29a inhibitor or mimics. In H₂O₂-treated cells with miR-29a inhibitor treatment, the expression levels of the proapoptotic proteins caspase-3 and Cyt-c were decreased, while Mcl-1 was increased, compared with the inhibitor NC group (P<0.01; Fig. 3F). Conversely, in cells treated with miR-29a mimics, the expression levels of the proapoptotic proteins caspase-3 and Cyt-c were increased, while Mcl-1 was decreased, compared with the mimics NC group (P<0.01; Fig. 3G). These results indicated that miR-29a may regulate cell apoptosis during IA.

miR-29a has been reported to directly target Mcl-1 in intestinal epithelial cells (37). In the present study, bioinformatics tools, TargetScan and miRanda, were used to identify the target genes of miR-29a. Fig. 4A demonstrates that Mcl-1 is a theoretical target gene of miR-29a. The wild-type 3′-UTR of the Mcl-1 gene was cloned and inserted it into the downstream region of a luciferase reporter gene. Subsequently, miR-29a mimics/inhibitors were cotransfected with different luciferase 3′-UTR constructs into 293T cells. The results of luciferase assays demonstrated that miR-29a mimics significantly weakened the relative luciferase activity of the luciferase reporter containing the wild-type 3′-UTR of Mcl-1 mRNA compared with mimics NC group, whereas the knockdown of miR-29a increased luciferase activity (P<0.01; Fig. 4B). To determine whether Mcl-1 mRNA is directly targeted by miR-29a, the predicted binding site of miR-29a in the 3′-UTR of Mcl-1 was muted. By contrast, the luciferase activities of luciferase reporters containing mutant 3′-UTRs of Mcl-1 luciferase activity remained unaltered in miR-29a mimic/inhibitor-transfected 293T cells compared with the respective mimics/inhibitor NC groups (Fig. 4B).

Results from the western blot analysis demonstrated that the protein expression of Mcl-1 was markedly decreased following miR-29a mimic treatment and increased following miR-29a inhibitor treatment, compared with the NC group (Fig. 4C).

As miR-29a overexpression in vitro enhanced VHBSMC apoptosis (Fig. 3B) and Mcl-1 was determined as a direct target of miR-29a (Fig. 4), the present study aimed to determine whether miR-29a may regulate apoptosis by regulating Mcl-1 expression in vivo. The miR-29a function in IA injury was detected by administration of the agomir-miR-29a to mice. As demonstrated in Fig. 5A, the expression of miR-29a in IA ligated mice was significantly higher compared with sham mice, while the level of miR-29a is further significantly increased in agomir-miR-29a + IA ligated mice. RT-qPCR results demonstrated that genes involved in mitochondrial apoptosis pathways, including Cyt-c, caspase-3 and caspase-9, were markedly increased, while Mcl-1 was decreased, in the IA ligated mice compared with sham controls (Fig. 5B). In agomir-miR-29a + IA ligated mice, Cyt-c, caspase-3 and caspase-9 were significantly increased, while the anti-apoptosis gene Mcl-1 was decreased, compared with the IA ligated group (Fig. 5B), indicating that miR-29a overexpression promoted mitochondrial apoptosis in vivo. TUNEL staining confirmed that apoptosis in tissue sections was significantly increased in IA + miR-29a agomir mice, compared with IA ligated mice (P<0.01; Fig. 5C).

Taken together, these results indicated that miR-29a overexpression may stimulate mitochondrial apoptosis pathways, subsequently contributing to the injury caused by IA.