VSMC isolation and cell culture have been previously described (21). The present study was approved by the Ethics Committee of the Department of Laboratory Animal Science, Central South University (Changsha, China) prior to the experiments. Briefly, VSMCs were isolated from the thoracic aorta of SD rats. A total of 6 male Sprague-Dawley rats aged 5-6 weeks and weighting 160-220 g were purchased from Human SJA Laboratory Animal Co., Ltd. (Changsha, China). All animals were housed in micro-isolator cages with free access to food and water in a light-controlled room under a 12/12 h light/dark cycle and controlled temperature (23-25°C). Aortic VSMCs were then cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Merck KGaA, Darmstradt, Germany) supplemented with 10% Gibco fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 µg/ml penicillin-streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C and 5% CO₂. VSMCs of passage 5 (young) and passage 15 (aging) at 70-90% confluence were used. All cells were divided into four groups: Young VSMCs, young VSMCs with rapamycin, aging VSMCs, and aging VSMCs with rapamycin. For the groups treated with rapamycin, VSMCs were treated with 20 nM rapamycin (Sigma-Aldrich; Merck KGaA) for 12 h. The untreated cells were used as controls.

The miR-30a mimics and negative control (NC) were chemically synthesized by GenePharma Co., Ltd. (Shanghai, China). The sequences of miR-30a mimics and miR-NC were 5′-UGUAAACAUCCUCGACUGGAAG-3′ and 5′-UUCUCCGAACGUGUCACGUTT-3′, respectively. The aging cells were pre-transfected with miR-30a mimics or miR-NC with a final concentration of 50 nM for 48 h, prior to treatment with rapamycin for 12 h. Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect oligonucleotides and constructs into cell lines, according to the manufacturer's instructions. The infection efficiency was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or western blotting 48 h post-transfection.

For cell cycle analysis, cells were fixed with 70% cold ethanol overnight at 4°C, washed with PBS, and then stained with 5 mg/ml propidium iodide in the presence of RNase A (10 µg/ml) for 30 min. Cell cycle phase distribution was analyzed by flow cytometry as reported elsewhere (22).

Measurement of cellular senescence was conducted by SA-β-gal staining. Briefly, cells were fixed with 4% formaldehyde for 15 min at room temperature, washed with PBS, and then incubated at 37°C overnight in SA-β-gal staining solution (1 mg/ml of X-gal; 40 mmol/l citric acid/sodium phosphate buffer, pH 6.0; 5 mmol/l potassium ferrocyanide; 5 mmol/l potassium ferricyanide; 150 mmol/lNaCl; and 2 mmol/l MgCl₂). Next day, the slides were washed twice in PBS, mounted in glycerol, and observed in five optical fields per sample using a light microscope.

Immunofluorescence was conducted to evaluate the expression of LC3B. The cells were fixed with 4% formaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and then incubated with anti-LC3B rabbit antibodies (cat. no. ab48394, 1:200; Abcam, Cambridge, MA, USA) overnight at 4°C. The cells were then incubated with Alexa Fluor^® 594-conjugated goat anti-rabbit IgG antibodies (cat. no. ab150080; 1:1,000; Abcam) for 1 h at room temperature. The nuclei of ells were then counterstained with DAPI for 10 min at room temperature. A Leica TCS-SP laser scanning confocal microscope was used to capture the photomicrographs in five optical fields per sample.

For the dual luciferase reporter assay, the wild type (WT) or a mutant (MUT) 3′-untranslated region (UTR) sequence of Beclin1 was amplified and subcloned into the pGL3-basic luciferase vector (Promega Corporation, Madison, WI, USA). VSMCs were co-transfected with either the WT or MUT 3′-UTR sequence of Beclin1 (1 µg), together with 25 nM miR-30a mimics or negative control. After 48 h of transfection, relative luciferase activity was measured with the Bright-Glo Luciferase Assay System (Promega Corporation).

The expression levels of miR-30a and Beclin1 were determined by RT-qPCR. Briefly, total RNA was extracted from VSMCs with TRIzol reagent (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. The PrimeScriptOne-Step RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to convert RNA into cDNA. PCR reactions were conducted using SYBR^®-Green PCR Master Mix (Takara Biotechnology Co., Ltd.) in an ABI7500 System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers sequences for Beclin1 were: Forward 5′-GTGCTCCTGTGGAATGGAAT-3′ and reverse 5′-TGCAACACAGTCCAGAAAAGC-3′. The primers for miR-30a were purchased from Takara Biotechnology Co., Ltd., and were: Forward 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTCCA-3′ and reverse 5′-GGCGTGTAAACATCCTCGAC-3. The primers sequences for GAPDH were: Forward 5′-GGATTTGGTCGTATTGGG-3′ and reverse 5′-GGAAGATGGTGATGGGATT-3′. The primers sequences for U6 were: Forward 5′-ATTGGAACGATACAGAGAAGATT-3′ and reverse 5′-GGAACGCTTCACGAATTT G-3′. Relative fold changes in mRNA expression were calculated using the 2^−ΔΔCq formula (23). GAPDH and U6 served as internal controls for mRNA and miRNA respectively.

The protein levels of LC3, p62, Beclin1, mTOR, phosphorylated (p-) mTOR, p53, p21, p16 and SA-β-gal were determined by western blotting. Briefly, total protein was extracted from VSMCs using a RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) and was quantitated with protein assay reagent from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Then equal amount of samples (30 µg) were loaded on 10% SDS-PAGE, followed by transfer to PVDF membranes. After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were then incubated with primary antibodies (all purchased from Abcam) targeting LC3B (cat. no. ab48394; 2 µg/ml), p62 (cat. no. ab56416; 5 µg/ml), Beclin1 (cat. no. ab62557; 2 µg/ml), p-Beclin1 (cat. no. ab183335; 1:250), mTOR (cat. no. ab2732; 1:2,000), p-mTOR (S2448; cat. no. ab109268; 1:100), p-ribosomal protein S6 kinase B1 (S6K1; cat. no. ab2571; 1:500), p-eukaryotic translation initiation factor 4E binding protein 1 (4EBP1; cat. no. ab75767; 1:1,000), p53 (cat. no. ab131442; 1:1,000), p21 (cat. no. ab109520; 1:1,000), p16 (cat. no. ab51243; 1:10,000), SA-β-gal (cat. no. ab9361; 0.5 µg/ml) and GAPDH (cat. no. ab8245; 1:500) at 4°C overnight. Subsequently, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG; cat. no. ab6940 and goat anti-mouse IgG; cat. no. ab97035; both 1:500) at 37°C for 45 min. The target bands were then developed using Super Signal West Pico Chemiluminescent Substrate kit (Pierce; Thermo Fisher Scientific, Inc.) and analyzed by Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). GAPDH served as an internal control.

All experiments were performed at least three independent times in triplicate. Data were presented as mean ± standard deviation. Statistical analyses were performed with Graphpad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Comparisons were conducted using one-way analysis of variance followed by Tukey post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

SA-β-gal staining was conducted to examine the effect of rapamycin on senescence of VSMCs. As illustrated in Fig. 1A and B, the ratio of SA-β-gal-positive cells was significantly higher in aging cells compared with young cells (P<0.01). However, following rapamycin treatment, the ratio of SA-β-gal-positive cells significantly decreased in aging cells (Fig. 1B), indicating that rapamycin could inhibit senescence in VSMCs.

Flow cytometry analysis revealed that G1 arrest occurred in aging cells and this cell cycle arrest was reduced when cells were treated with rapamycin (Fig. 1C and D). Further analysis of p16, p21, p53 and SA-β-gal expression levels by western blotting demonstrated that rapamycin treatment reduced the increasing levels of senescence-related proteins in aging cells (Fig. 1E and F). These results suggested that rapamycin could inhibit senescence of VSMCs through inhibition of cell cycle arrest and senescence-related proteins.

To examine the effect of rapamycin on the expression of miR-30a and autophagy of VSMCs, the expression of miR-30a and Beclin1 was determined by RT-qPCR. The results demonstrated that in aging cells miR-30a was significantly upregulated while Beclin1 was significantly downregulated compared with young cells (P<0.01; Fig. 2A and B), indicating that both miR-30a and Beclin1 might be associated with senescence in VSMCs. However, when treated with rapamycin, miR-30a was signifi-cantly downregulated in both young and aging cells (P<0.05; Fig. 2A). Additionally, the expression of Beclin1 was significantly upregulated in both young and aging cells following rapamycin treatment (P<0.05; Fig. 2B). These results clearly demonstrated that rapamycin inhibited miR-30a expression while it induced Beclin1 expression.

To further investigate the effect of rapamycin on autophagy in VSMCs, the expression of LC3, Beclin1, p62, mTOR and p-mTOR was determined by western blotting. As illustrated in Fig. 2C and D, the ratio of LC3-II/LC3-I and the expression of Beclin1 and p-Beclin1 were significantly downregulated, while the expression of p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 was significantly upregulated in aging cells compared with young cells (P<0.05). These findings indicate that autophagy-related proteins and mTOR signaling were associated with cell aging. Similarly, when treated with rapamycin, the ratio of LC3-II/LC3-I and the expression of both Beclin1 and p-Beclin1 were all significantly upregulated, while the expression of p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 significantly down-regulated (Fig. 2C and D). Immunofluorescence analysis of LC3B protein expression demonstrated the same results (Fig. 2E). In both young and aging cells, when treated with rapamycin, the expression of LC3B markedly increased compared with the untreated cells (Fig. 2E), suggesting that rapamycin might promote autophagy of VSMCs to influence the cell aging process.

To investigate the relationship between miR-30a and Beclin1, aging cells were treated with rapamycin or miR-30a mimics and the expression of miR-30a and Beclin1 was determined. The results demonstrated that expression of miR-30a was significantly upregulated following transfection with miR-30a mimics and significantly downregulated following rapamycin treatment (P<0.01; Fig. 3A). This indicated that the transfection of miR-30a mimics was successful. When treated with miR-30a mimics, expression of Beclin1 significantly decreased in aging cells, and the rapamycin-induced upregulation of Beclin1 was significantly reversed at both the mRNA and protein levels (P<0.01; Fig. 3B-D), indicating that miR-30a could downregulate the expression of Beclin1.

To further examine the function of miR-30a on regulating Beclin1, a dual luciferase reporter assay was conducted. Bioinformatics analysis by software target scan 5.1 (http://www.targetscan.org) revealed that there is a potential miR-30a binding site on the 3′-UTR of Beclin1 in rats (Fig. 3E). Furthermore, the sequence of miR-30a was identical and the homology of Beclin1 was high in both rat and human, so the binding sites of miR-30a and Beclin1 were the same in rat and human (Fig. 3E). The results from the luciferase assay demonstrated that the relative luciferase activity in WT-Beclin1 was significantly downregulated by miR-30a mimics, while no significant change was observed with MUT-Beclin1 (Fig. 3F), further confirming that miR-30a directly bound to the Beclin1 3′-UTR.

At last, the present study investigated the effects of miR-30a on senescence and the cell cycle in VSMCs. Similar to the aforementioned results, the ratio of SA-β-gal positive cells was significantly upregulated when cells were transfected with miR-30a mimics (P<0.01; Fig. 4A and B). In addition, the rapamycin inhibitory effect on senescence of VSMCs was significantly reversed by overexpression of miR-30a (P<0.01; Fig. 4A and B). Notably, transfection reagent Lipofectamine 2000 alone had no significant effect on VSMC senescence (Fig. 4A and B).

Flow cytometry analysis revealed that G1 arrest was significantly promoted in aging cells following transfection with miR-30a mimics (Fig. 4C and D). Western blotting results demonstrated that overexpression of miR-30a could significantly increase the levels of the senescence-related proteins p16, p21, p53 and SA-β-gal (P<0.01; Fig. 4E and F). Furthermore, the rapamycin inhibition effects on both cell cycle arrest and senescence-related protein expression were reversed upon transfection with miR-30a mimics (P<0.05; Fig. 4E and F). These results suggested that miR-30a could promote senescence and cell cycle arrest in VSMCs, and that rapamycin might inhibit senescence of VSMCs through inhibition of miR-30a.

The effect of miR-30a on autophagy of VSMCs was then investigated by determining the protein expression of autophagy-related proteins LC3, Beclin1, p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1. As illustrated in Fig. 5A and B, the LC3-II/LC3-I ratio and Beclin1 expression levels were significantly downregulated, while the expression levels of p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 were significantly upregulated in aging cells following transfection with miR-30a mimics, suggesting that miR-30a could inhibit autophagy of aging cells. Additionally, miR-30a mimics could significantly reverse the alteration of all autophagy-related proteins induced by rapamycin (Fig. 5A and B). Similar results were also observed by immunofluorescence analysis of LC3B expression. In both control cells and cells treated with rapamycin, when transfected with miR-30a mimics, the expression of LC3B markedly decreased (Fig. 5C). The present results fully demonstrated that miR-30a could inhibit autophagy of VSMCs and that rapamycin could induce autophagy of VSMCs through inhibition of miR-30a.