Rat aortic smooth muscle cells SMCs were isolated from the medial layer of the thoracic aorta of female SHRs and WKY rats (10 weeks old) and cultured in Dulbecco's modified Eagle's medium (DMEM; Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin (HyClone, Logan, UT, USA). The cells of passage 4–6 were employed in the experiment. The experimental protocol was approved by the Ethics Committee of Shougang Hospital, Peking University, Beijing, China. Human aortic SMCs (HASMCs) were cultured in SmGM-2 growth medium (both from Lonza) supplemented with 5% FBS (HyClone) following the manufacturer's instructions.

Six patients who underwent open aortic arch reconstruction that required aortic dissection at Shougang Hospital were recruited in this study. Their demographic and clinical data are presented in Table I. The study protocol was approved by the Medical Ethics Committee of Shougang Hospital. Informed consent was obtained from all patients prior to enrollment. The SMCs were isolated as previously described (18). Briefly, resected vascular tissue was placed in DMEM with penicillin/streptomycin (5/500 ml) and transferred to a super-clean bench. The vascular tissue was rinsed 3 times with phosphate-buffered saline (PBS) and the intima was removed. The tunica media was finely cut into 2–3-mm-thick sections in another 100-mm culture dish, and 5 ml of 0.1% type I collagenase (Invitrogen, Carlsbad, CA, USA) was added to the culture dish. Subsequently, the dish was placed in an incubator for 2 h at 37°C. Digestion media were collected and filtrated using a cell strainer to remove the undigested explants and then centrifuged (1,000 rpm, 5 min, 4°C). The aforementioned procedures were repeated 3 times in order to harvest more cells.

Total RNA was isolated using the miRNeasy Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions, and the quality of the isolated RNA was evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose electrophoresis. The PrimerScript RT reagent kit (Takara, Dalian, China) was used to synthesize the cDNA. The SYBR-Green real-time detection system was purchased from Bio-Rad Co., Ltd. (Hercules, CA, USA). U6 was used as an internal control to normalize the miR-1 and IGF1 mRNA expression levels. All fold changes were calculated using the ΔΔCt method. The primer sets used for amplification are listed in Table II.

miR-1 mimics or anti-IGF1 siRNA (Ambion, Austin, TX, USA) were used in the experiments, and Lipofectamin 2000 (Invitrogen) was employed for the transfection of the HASMCs. Fluorescently labeled control oligo was first transfected and the transfection efficiency was evaluated by visual fluorescence microscopic analysis and flow cytometry. When the transfection efficiency was >85%, the transfection was considered successful. The in vitro experiments described below included cohorts transfected with 30 and 100 pmol of miR-1 mimics and 100 pmol of anti-IGF1 siRNA or the fluorescently labeled scrambled control oligo (negative control) known to have no effect on any human miRNA in order to minimize the non-specific effect.

We searched two major online microRNA databases: TargetScan (http://www.targetscan.org) and miRDB (http://www.mirdb.org) to identify the candidate target genes of miR-1. Among all the candidate genes, we further narrowed down a short list of candidates based on the phyiopathological function of the genes.

Transwell invasion assay was carried out in 24-well fitted inserts with membranes (8 mm pore size; Costar, Cambridge, MA, USA). As previously described (20), cell invasion was examined using a polycarbonate membrane cell culture insert (Costar, Corning Inc., Corning, NY, USA) coated with growth factor-reduced Matrigel (BD Biosciences, Bedford, MA, USA). The cells transfected with the control, miR-1 mimics and anti-IGF1 siRNA were treated with 10 mg/ml mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) for 2 h and placed into the top of the wells at a density of 2.5×10⁵ cells/well.

A modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to examine HASMC viability. The HASMCs transfected with miR-1 mimics or anti-IGF1 siRNA were cultured in 96-well plates for 24 h and 10 μl of MTT AB solution (Millipore, Billerica, MA, USA) were then added, followed by incubation for 4 h. The formazan product was dissolved by the addition of 100 μl of acidic isopropanol, and the absorbance was determined at 570 nm (reference wavelength 630 nm) using an xMark Microplate Absorbance Spectrophotometer (Bio-Rad Co., Ltd.).

Programmed cell death rates were assessed using a commercially available apoptosis assay (Sigma-Aldrich). The HASMCs were transfected with miR-1 mimics or anti-IGF1 siRNA, and 1×10⁵ cells were harvested and stained with 10 μl FITC Annexin V and 10 μl propidium iodide and sorted by FACS within 1 h (BD Biosciences, San Jose, CA, USA).

The transfected HASMCs were harvested and lysed, and the cell lysates were subjected to 10% PAGE. The separated proteins were transferred onto PVDF membranes blocked with TBST (10 mM Tris-Cl pH 8.0, 150 mM NaCl, and 0.05% Tween-20) containing 5% non-fat dry milk powder at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies directed against human IGF1 (1:2,000) and β-actin (1:1,500), and an HRP-conjugated goat-anti-rabbit secondary antibody (1:1,000) (both from Cell Signaling Technology, Danvers, MA, USA). Exposed films were scanned and fluorescence signals were detected using an ECL kit (Pierce, Rockford, IL, USA).

The PCR product was then cloned into the pRL-SV40 vector (Promega, Fitchburg, WI, USA) to replace the 3′UTR of Renilla luciferase, and the QuickChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to introduce the variant. Luciferase assay was conducted on the HASMCs, and the cells were seeded at 1×10⁵ cells/well in 24-well plates. Twelve hours later, the cells were transfected using Lipofectamin 2000 (Invitrogen) according to the manufacturer's instructions. In each well, 500 ng of the wild-type or mutant construct and 50 ng of the control vector (control oligo) were co-transfected with 100 pmol miR-1 mimics or the negative control (Ambion). Twenty-four hours after transfection, the cells were harvested by the addition of passive lysis buffer (Promega). Luciferase activity in the cell lysates was determined using the Dual Luciferase assay system (Promega) with a TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA).

All data are presented as the means ± SD. One-way ANOVA was used for comparisons between samples (>2 groups), and an independent t-test was used for comparisons between 2 groups. A value of P<0.05 was considered to indicate a statistically significant difference. All experiments were performed at least 3 times. Statistical analysis was performed using the SPSS software package (version 19; SPSS, Inc., Chicago, IL, USA).

In the present study, we focused on miR-1, miR-500, miR-34b and miR-98, as these miRNAs have been previously reported to be significantly downregulated in SHR-VSMCs compared with WKY-VSMCs, based on the results of microarray-based miRNA expression profiling analysis (17). RT-qPCR was performed using the primary VSMCs isolated from the medial layer of the thoracic aorta obtained from SHRs and WKY rats, and we observed a 3.29-fold decrease in the expression levels of miR-1 in the SHR-VSMCs compared with the WKY-VSMCs (P<0.01; Fig. 1), whereas the expression levels of the miRNAs were similar between the 2 groups. Thus, miR-1 was further investigated to determine its potential biological function.

Using in silico analysis based on online predicting tools, such as TargetScan and miRDB, we identified IGF1 as a potential target gene of miR-1 (Fig. 2A), and the 'seed sequence' in the 3′UTR of IGF1 was found to be highly conserved among species (Fig. 2B), suggesting that such a 'seed sequence' may have an important function. To validate the target gene, we subcloned the 3′UTR of IGF1 into the pRL-SV40 vector to replace the 3′UTR of Renilla luciferase, and performed luciferase assay to compare the inhibitory effects between the wild-type 3′UTR and the mutant as described in the Materials and methods. The results revealed that co-transfection with the miR-1 mimic significantly suppressed luciferase activity when transfected together with the wild-type 3′UTR of IGF1, but not the mutant one (Fig. 2C).

Additionally, the HASMCs were transfected with miR-1 mimics (30 and 100 pmol) and IGF1-specific siRNA, and the IGF1 protein levels were quantified by western blot analysis. Fig. 3E shows the step-wise downregulation of IGF1 expression in the HASMCs transfected with an increasing amount of miR-1 mimics (30 and 100 pmol). The silencing effect of transfection with 100 pmol of the miR-1 mimics was comparable with that of transfection with 100 pmol of the anti-IGF1 siRNA.

The HAMSCs were transfected with the miR-1 mimics (30 and 100 pmol) and anti-IGF1 siRNA prior to the proliferation assay. MTT assay was used to measure cell proliferation. The results revealed that the exogenous overexpression of miR-1 significantly inhibited the proliferation of the HAMSCs (Fig. 3). After incubation for 48 h, significant inhibitory effects on cell proliferation were observed in the cells transfected with the miR-1 mimis or with anti-IGF1 siRNA compared with those transfected with the negative control. In the HASMCs, the inhibitory rates were 75% (anti-IGF1 siRNA), 22% (30 pmol miR-1 mimics) and 80% (100 pmol miR-1 mimics) following incubation for 48 h (Fig. 3F). This suggested that the overexpression of miR-1 inhibited the proliferation of the VSMCs in vitro. Furthermore, we performed Transwell invasion assay with the cells being treated with mitomycin C that was used to eliminate the possible effect of cell growth on cell invasion. The results revealed that the inhibition of miR-1 in the HASMCs did not affect cell invasiveness (data not shown).

We examined the SHR-VSMCs and WKY-VSMCs to determine the expression patterns of IGF1 in these cells. The IGF1 mRNA expression levels were measured by RT-qPCR, and we found that the mRNA levels of IGF1 in the WKY-VSMCs were significantly lower than those in the SHR-VSMCs (Fig. 4B). The IGF1 protein levels were also measured using western blot anlaysis. The results revealed that the protein expression levels of IGF1 in the SHR-VSMCs were substantitally higher than those in the WKY-VSMCs (Fig. 4A).

VSMCs were isolated from the vascular tissue collected from 6 patients (hypertension, 3; normal blood pressure, 3) who had undergone open aortic arch reconstruction that required aortic dissection (HTN-VSMCs vs. normal-VSMCs). The mRNA and protein expression levels of IGF1 were compared between the 2 groups of cells by RT-qPCR and western blot analysis. We found that both the protein and mRNA expression levels of IGF1 were markedly higher in the HTN-VSMCs than in the normal-VSMCs (Fig. 5B and C, respectively). Similarly, the expression levels of miR-1 were significantly lower in the HTN-VSMCs than in the normal-VSMCs (Fig. 5A).

As IGF1 functions as an anti-apoptotic protein and the exogenous expression of miR-1 causes a reduction in the enzyme (21), we examined the effects of miR-1 on the apoptosis of HASMCs by flow cytometry. We found that transfection of the cells with miR-1 mimics and anti-IGF1 siRNA promoted the apoptosis of HASMCs (Fig. 6).