MicroRNA-761 inhibits Angiotensin II-induced vascular smooth muscle cell proliferation and migration by targeting mammalian target of rapamycin

2.1 Isolation and culture of rat aortic VSMCs

All experimental procedures for animal studies were approved by the Committee for the Care and Use of Laboratory Animals, Yonsei University College of Medicine, and performed in accordance with the Committee’s guidelines and regulations for animal care. The thoracic aortas from 6- to 8-week-old Sprague–Dawley rats were removed and transferred into serum-free DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen, USA) containing 100 units/ml penicillin and 100 mg/ml streptomycin. The connective tissues were removed and the aorta was transferred to a petri dish containing 5 ml of an enzyme dissociation mixture containing DMEM with 1 mg/ml collagenase type I (Sigma, USA) and 0.5 mg/ml elastase (USB Bioscience, USA), and incubated for 30 min at 37°C. The adventitia was stripped with forceps under a microscope. The aorta was transferred into a plastic tube containing 5 ml enzyme dissociation mixture and incubated for 2 hr at 37°C. The suspension was centrifuged (1500 rpm for 10 min), and the pellet was re-suspended with 10% fetal bovine serum (FBS) containing DMEM with. Rat aortic SMCs were cultured in DMEM supplement with 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin in 75 cm² flasks at 37°C in a humidified atmosphere of 95% air and 5% CO₂ (Forma Scientific, USA).

2.2 Selection of miRNA targeting mTOR

First, miRNAs predicted to target mTOR were retrieved using a publicly available database (TargetScan, www.targetscan.org). The efficiency of miRNAs in down- regulating mTOR expression was empirically determined by Western blot using mTOR specific antibodies.

2.3 MicroRNA transfection

Transfections of miRNA mimics and anti-miRNAs were performed using siLentFect™ Lipid reagent (Life Science Research). Mature specific miRNA and miR-control (Genolution Pharmaceuticals, Inc., Korea) were used at a final concentration of 100 nM. Anti-miR was used at a final concentration of 50 or 100 nM. After 4 hr incubation in a CO₂ incubator at 37°C, the medium was changed to 10% FBS containing DMEM.

2.4 Cell proliferation assay

Rat aortic VSMCs were plated in 96-well plates at 5×10³ per well. Cells were transfected with miRNA mimics 24 hr prior to AngII (Sigma) exposure for 48 hr. After treatment, 10 ul of the WST-1 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] solution (Dojindo, Japan) was added to each well and incubated at 37°C for 2 hr to allow formation of WST-1 formazan. The Absorbance of a water soluble formazan dye was measured at 450 nm using a microplate reader (Molecular Devices, USA). Experiments were performed in triplicate.

2.5 Real-time polymerase chain reaction (PCR)

Total RNA was isolated with TRIzol^® Reagent (Life Technologies, USA). In brief, 100 ng purified total RNA was used for reverse transcription (Taqman^® MicroRNA Reverse Transcriptase Kit, Applied Biosystems, USA) in combination with Taqman^® MicroRNA Assays for quantification of specific miRNAs and U6 control transcripts, according to the manufacturer’s conditions. The threshold cycle (Ct) of each target gene was automatically defined, located in the linear amplification phase of the PCR, and normalized to control U6 (ΔCt value).

2.6 Reverse transcription polymerase chain reaction (RT-PCR)

Cellular total RNA was isolated with TRIzol^® Reagent (Life Technologies, USA). Single-stranded cDNA was synthesized from total RNA using a reverse transcription system (Promega, USA) according to the manufacturer’s protocol. The primer sequences were as follows: GAPDH, sense: 5’ – GGT GAT GCT GAG TA - 3’ and antisense: 5’ – GGA TGC AGG GAT GAT GTT CT– 3’ (369bp); mTOR, sense: 5’ – TTG AGG TTG CTA TGA CCA GAG AGA A– 3’ and antisense: 5’ – TTA CCA GAA AGG ACA CCA GCC AAT G– 3’ (540bp).

2.7 Western Blot Analysis

VSMCs cultured in 60-mm dishes were treated with or without miRNA mimics that were predicted to target mTOR and stimulated with AngII. Proteins were separated in a 10% SDS-polyacrylamide gel and transferred to PVDF membrane (Millipore, USA). After blocking the membrane with TBS-T (TBS-tween 20, 0.1% tween 20) containing 5% (w/v) non-fat dried skimmed milk powder for 1 hr at room temperature, membranes were washed twice with TBS-T and incubated with primary antibodies for 1 hr at room temperature or overnight at 4°C. The membrane was washed three times with TBS-T for 5 min and incubated with HRP (horseradish peroxidase)-conjugated secondary antibody for 1 hr at room temperature. After extensive washing, the bands were detected using ECL^® (enhanced chemiluminescence) reagent (AbClon, Republic of Korea). The band intensities were detected using a Photo-Image System (Molecular Dynamics, Canada). The primary antibodies were from Cell Signaling (mTOR:2972, p-mTOR:2971, P70S6K:9202, p-P70S6K:9206, 4EBP1:9452, p-4EBP1:9459, cyclin D1:9262), except the β-actin (Abcam, ab8227).

2.8 Luciferase reporter assay

We synthesized the 3′-UTR of rat mTOR containing the predicted binding sites for corresponding miRNA. The 3’-UTR fragments were then cloned into the pmirGLO vector (pmTOR-3UTR). HeLa cell was plated at 1×10⁵ in 12 well plates. After 24 hr, the cells were co-transfected with pmTOR-3UTR vector, negative control miRNA mimic (NC), miR-761, or anti-miR-761 using siLentFect™ Lipid reagent. The Renilla luciferase was used for normalization. Luciferase activity was measured by the Dual Luciferase assay (Promega, USA) according to the manufacturer’s instructions after 24 hr. Each assay was repeated three times.

2.9 Migration assay

VSMCs (8×10³ cells) were seeded onto the upper chamber of a Transwell filter with 8 μm pores (Costar Corning, USA) coated with 10 μg/ml fibronectin. The cells were deprived of serum for 24 hr, and AngII-containing stimulating medium (50 nM) was added to the lower chamber. Transwell chambers were incubated at 37°C for 24 hr. After incubation, the cells migrated through the pores of the filter were stained with 0.25% crystal violet. Non-migrating cells on the upper side of the filter were removed with cotton swabs.

2.10 Wound healing assay

VSMCs were plated at a density of 8×10⁴ cells/well in six-well plates. After the cells had reached 90% confluence, cells were deprived of serum for 24 hr. After incubation, wounds were produced by scratching with 200 μL pipette tips. The leading edge of the wounds was marked as a baseline. The medium was replaced with or without serum-deprived medium-containing AngII (50 nM), and the cells were incubated for 0, 6, 12, and 24 hr. Images were captured using an Axiovert 40 C inverted microscope (Carl Zeiss, Germany) equipped with a Powershot A640 digital camera (Canon, Japan).

2.11 Ring assay

The rat aorta was isolated and cleaned of perivascular adipose tissue. The isolated rat aortas were endothelial cell (EC)-denuded by elastase treatment in the lumen and cut into segments of 1 mm long aortic rings that were placed in Matrigel (BD Biosciences, USA). The aorta rings were transfected with miRNA mimics prior to treatment with media containing AngII. Over the next 7 days, the aorta rings were monitored for the outgrowth of SMCs using microscope once per day.

2.12 Statistical analysis

Quantitative data were expressed as the means±SEM. For statistical analysis, one-way ANOVA with Bonferroni correction was performed using the OriginPro 8 SR4 software (ver. 8.0951, OriginLab Corporation, Northampton, MA, USA) if there were more than 3 groups. For two group comparison, student’s t-test was used. A p value of less than 0.05 was considered to be statistically significant.

3.1 AngII-induced rat aortic VSMC proliferation is mediated by mTOR

We first confirmed that AngII-induced VSMCs proliferation. AngII significantly increased VSMC proliferation at the concentrations of 50 nM or higher (Fig. 1A). However, when the cells were pretreated with a potent mTOR inhibitor rapamycin, AngII-induced VSMC proliferation was suppressed indicating that mTOR was involved in the AngII-induced proliferation of VSMCs (Fig. 1B). Since 50 nM (or higher) AngII induced VSMC proliferation, 50 nM of AngII was used to stimulate VSMC proliferation for further experiments.

3.2 miR-761 directly targets mTOR and is down-regulated during AngII-induced VSMC proliferation

We have hypothesized that AngII-induced proliferation of VSMCs may be mediated by miRNAs, leading to increased mTOR activity. To test the hypothesis, first, we have selected 16 miRNAs based on miRNA database (www.TargetScan.org) (Table 1). Next, we transfected HeLa cells with selected miRNAs, and then evaluated mTOR expression using Western blot (Fig. 2A). Four miRNAs (miR-143/150/214/761) significantly repressed mTOR expression. However, three of them (miR-143/150/214) induced significant cell death (data not show), thus consequently excluded from further experiments. The expression of miR-761 was significantly decreased as the VSMCs proliferated in response to AngII treatment (Fig. 2B).

For luciferase assay, the 3’-UTR of mTOR was cloned into a pmirGLO vector using xhoI and xbaI endonuclease site to produce a pmTOR-3UTR vector. HeLa cells were transiently transfected with the pmTOR-3UTR vector (or pmirGLO control vector), in combination with miR-761 mimics, negative control mimics (scrambled miRNA; N.C.), or anti-miR-761. Transfection with miR-761 significantly decreased luciferase activity in pmTOR-3UTR group, while it had no significant effect on pmirGLO control vector transfected group. Furthermore, anti-miR-761 treatment prior to miR-761 delivery abrogated miR-761-mediated decrease of luciferase activity (Fig. 2C). Although mRNA expression of mTOR was not changed by miR-761 transfection, mTOR protein expression was decreased by miR-761 transfection (Fig. 2D), indicating the inhibitory mechanism of miR-761 on mTOR expression was to disrupt translation of mTOR from mRNA rather than degradation of mRNA itself. Such decrease of mTOR expression in miR-761 treated group, both at mRNA and protein level, was recovered by anti-miR-761 treatment performed prior to miR-761 transfection. Additionally, when the cells were transfected with increasing doses of anti-miR-761, the expressions of mTOR mRNA or proteins were not significantly changed (Fig. 2E). Although VSMC proliferation after anti-miR-761 delivery showed a tendency to increase, it was not statistically significant (Fig. 2F).

3.3 MiR-761 suppresses AngII-induced cell cycle progression and subsequent proliferation of VSMCs by inhibiting mTOR signaling pathway

To examine the effect of miR-761 the AngII-induced proliferation of VSMCs, first, we performed cell cycle analysis. Although the percentage of cells in S phage of rapamycin-treated cells was lower than that of miR-761-treated cells, the percentage of cells in S phase of miR-761-treated cells was still lower than that of AngII-stimulated cells (Fig. 3A). Furthermore, the results of cell viability assay also indicated that transfection of miR-761 suppressed AngII-induced proliferation in a dose-dependent manner (Fig. 3B). Such anti-proliferative effect of miR-761 was further demonstrated by immunocyto- chemical staining of Ki-67, a proliferating cell marker [29]. The expression of Ki-67 was significantly decreased in miR-761-transfected VSMCs compared to AngII-stimulated cells (Fig. 3C). Additionally, we examined the phosphorylation status of p70S6k and 4EBP1 that is known to be regulated by mTOR [25]. The results of Western blot indicated that the phosphorylation of p70S6k and 4EBP1 were decreased in the miR-761 transfected group. These data suggested that exogenous miR-761 delivery decreased biological activity of mTOR, subsequently suppressing activation mTOR/p70S6K/4EBP1 signal pathway (Fig. 3D). Again, anti-miR-761 treatment performed prior to miR-761 transfection abrogated all the miR-761-mediated changes, indicating those changes were actually mediated by miR-761.

3.4 MicroRNA-761 inhibits migration in Ang II-stimulated VSMCs

Migration of VSMC under pathologic condition has been reported to contribute to development of vascular diseases, including atherosclerosis and post-angioplasty restenosis [4]. Therefore, we also examined the effect of miR-761 on the AngII-induced VSMC migration. According to the results of Transwell assay, AngII treatment significantly increased the number of migrated cells, and this increase was significantly suppressed by both miR-761 and rapamycin transfected VSMCs. MicroRNA-761 also inhibited migration of Ang II-stimulated VSMCs (Fig. 4A), but such inhibitory effect of miR-761 was abrogated by anti-miR-761 pre-treatment. In wound healing assay, the distance between wound edges were shortest in the AngII-stimulated group, while the miR-761-treated group and the rapamycin-treated group showed longer distance compared to the AngII-treated group (Fig. 4B). The sprouting of SMCs from the aortic rings of miR-761-treated group was lower compared to the AngII-treated group (Fig. 4C), indicating that miR-761 suppressed not only VSMC proliferation, but also cell migration. For both wound healing and SMC sprouting, anti-miR-761 pre-treatment abrogated the inhibitory effect of miR-761.