A total of 20 male Sprague-Dawley (SD) rats (Specific-pathogen-free degree, aged 4–5 weeks, weight 100–150 g) were purchased from Tongji University Laboratory Animal Center (Shanghai, China), and housed in a temperature-controlled feeding facility (22°C) with a 12-h light/dark cycle and free access to water and rodent diet. This study conformed to the Guide for Care and Use of Laboratory Animals and procedures were approved by the Animal Care and Use Committee of Tongji University School of Medicine (Shanghai, China).

Primary VSMCs were isolated from SD rat thoracic aortas, as described previously (19). In brief, animals were sacrificed by intraperitoneal injection of 2% pentobarbital sodium (50 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and perfused with saline for 5 min. The thoracic aortas were aseptically harvested with removal of the intima, longitudinally cut into ~1 mm² fragments, explanted with the lumen side down on cell culture dishes (Corning Life Sciences, Beijing, China) containing Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and maintained in a 37°C and humidified 5% CO₂ incubator for 7 days, followed by discarding the tissue fragments and further collection of sprouted VSMCs. The following experiments were performed on the VSMCs between passages 4 and 7 in DMEM with 2.5% FBS.

VSMCs were identified by their morphology and immunofluorescence detection of marker proteins, such as alpha-smooth muscle actin (α-SMA) using rabbit anti-α-SMA polyclonal antibody (ab5694; 1:100; Abcam, Cambridge, UK), and their purity was ensured by multiple fluorescent stainings with α-SMA antibody and nucleic acid dye, DAPI (2 mg/ml; Beyotime Institute of Biotechnology, Haimen, China) using a laser scanning confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany). To determine the effect of H₂O₂ on miR-92a expression in VSMCs, VSMCs were treated with 100 µM H₂O₂ (Sangon Biotech Co., Ltd., Shanghai, China) at 37°C for 24 h.

For the transfection experiments, double-stranded miR-92a mimics or miRNA negative control (miR-NC; 100 nM; Shanghai GenePharma Co., Ltd., Shanghai, China), and sirtuin 1 (SIRT1) siRNA or NC siRNA (100 nM; Shanghai GenePharma Co., Ltd.) were respectively transfected into VSMCs using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Afterwards, VSMCs were cultured for 48 h and then used for subsequent assays.

Cell migration assays were detected using a Transwell chamber with 8-µm filter inserts (Corning Life Sciences, Beijing, China) without Matrigel. Following transfection and H₂O₂ treatment, ~2×10⁴ VSMCs in 100 µl DMEM medium with 1% bovine serum albumin (BSA; Sangon Biotech Co., Ltd., Shanghai, China) were seeded into the upper chamber. The lower chamber was filled with 600 µl DMEM medium containing 10% FBS. After 24 h, VSMCs on the upper chamber were gently scraped away, and the cells that had migrated through the filter into the lower chamber were fixed with 4% paraformaldehyde (Sangon Biotech Co., Ltd.) for 15 min at room temperature and stained with DAPI (2 mg/ml). Then, the cells from 9 independent, randomly selected visual fields were counted under a laser scanning confocal microscope (magnification, ×200; Leica Microsystems GmbH) for quantitative analysis. Experiments were performed in triplicate.

Cellular protein was extracted with ice-cold radioimmunoprecipitation assay lysis buffer containing a mixture of proteasome inhibitors (Beyotime Institute of Biotechnology) for western blot analysis. The BCA method (BCA Protein Assay kit; Beyotime Institute of Biotechnology) was used to measure the protein concentrations of the supernatant by a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) according to the manufacturer's protocols. Equal amounts (30 µg) of protein were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 0.5 h and subsequently 120 V for 1.5 h at room temperature, and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) at 100 V for 1.5 h at 4°C. The membranes were then blocked with 3% BSA in phosphate-buffered saline (PBS; Sangon Biotech Co., Ltd.) for 30 min at room temperature and incubated with specific primary antibodies, including rabbit anti-MMP9 polyclonal antibody (ab38898; 1:1,000; Abcam), rabbit anti-TIMP3 monoclonal antibody (5673; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), and mouse anti-GAPDH monoclonal antibody (97166; 1:1,000; Cell Signaling Technology, Inc.), overnight at 4°C. Tris-buffered saline and Tween 20 (Sangon Biotech Co., Ltd.) was used to wash the membranes (3 times for 10 min) and the membranes were incubated with secondary antibodies, including goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) (L3012-2; 1:10,000; Signalway Antibody LLC, College Park, MD, USA), or goat anti-mouse IgG-HRP (L3032-2; 1:10,000; Signalway Antibody LLC), for 1 h at room temperature. The standard chemical luminescence method (BeyoECL Plus kit; Beyotime Institute of Biotechnology) was used to visualize the antigen by exposing the membranes to Kodak X-Omat AR film (Seebio Biotech, Inc., Shanghai, China) in accordance with the manufacturer's protocols. The resultant films were scanned on a gel imaging and analysis system and analyzed by Quantity One software version 4.4 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Relative protein expression of MMP9 and TIMP3 was normalized by the internal control, GAPDH, in the same sample.

MTT assay was used to evaluate the effect of miR-92a mimics on VSMC viability. Briefly, following transfection and H₂O₂ treatment, VSMCs were cultured in DMEM medium in 96-well plates at a density of 6,000 per well at 37°C under 5% CO₂. After 24 h of maintenance, 20 µl MTT (5 mg/ml in PBS; Beyotime Institute of Biotechnology) was added to each plate and incubated for 4 h at 37°C. Subsequently, the supernatant was removed and 150 µl DMSO (Sangon Biotech Co., Ltd.) was added to each well. Following 10 min of incubation at room temperature, absorbance of formazan dissolved in DMSO was immediately assessed at a wavelength of 490 nm using a microplate reader (BioTek Instruments, Inc.). Experiments were performed in quintuplicate.

Total RNA was isolated from VSMCs using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols and its concentration was determined with NanoDrop Spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription of miRNA or ordinary RNA was performed using miRcute miRNA First-Strand cDNA Synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China) or PrimeScript RT Reagent kit with gDNA Eraser (Perfect Real Time; Takara Biotechnology Co., Ltd., Dalian, China) according to the suggested protocol, respectively. Then, miR-92a expression level was measured with miRcute miRNA qPCR Detection kit (Tiangen Biotech Co., Ltd.) under the conditions: 94°C for 2 min; 40 cycles (94°C for 20 sec and 60°C for 34 sec), while the expression level of SIRT1 mRNA was evaluated with SYBR Green PCR Master Mix Kit (Takara Biotechnology Co., Ltd.) under the conditions: 95°C for 6 min; 40 cycles (95°C for 15 sec and 60°C for 34 sec), using 7900 HT Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 snRNA and GAPDH mRNA served as endogenous controls. The relative expression level was computed according to the 2^−ΔΔCq analysis method (20). Experiments were performed in triplicate and the following primers were used: Forward, 5′-ACAGGCCGGGACAAGTGCAATA-3′ and reverse, 5′-GCTGTCAACGATACGCTACGTAACG-3′ for miR-92a; forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′ for U6; forward, 5′-CCAGATCCTCAAGCCATGT-3′ and reverse, 5′-TTGGATTCCTGCAACCTG-3′ for SIRT1; and forward, 5′-TGGGCTACACTGAGCACCAG-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAAT-3′ for GAPDH.

Verification of miR-92a targeting SIRT1 was conducted by luciferase reporter assay in HEK-293T cells, as described previously (21). Briefly, wild-type 3′-UTR sequence of SIRT1 (WT-UTR) predicted to interact with miR-92a, was synthesized by GenePharma Co., Ltd and inserted into the dual-luciferase reporter vector (Promega Corporation, Madison, WI, USA), yielding the WT-UTR plasmid. Mutations (Mut-UTR) of potential miR-92a binding sites in WT-UTR were subjected to the Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA), generating the Mut-UTR plasmid. All plasmids were confirmed with DNA sequencing. HEK-293T cells were cultured in 12-well plates at 37°C under 5% CO₂ and cotransfected with the plasmids carrying 3′-UTR variants, and miR-92a mimics or miR-NC for 48 h, using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, luciferase values of cell lysates were determined with a Dual-Luciferase Reporter Assay System (Promega Corporation) according to the protocol. All data were normalized by Renilla luciferase activity. Experiments were performed in triplicate.

All experiments were repeated 3 times. All statistical analyses were performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). Data were presented as means ± standard deviation. Student's t-test was used for comparisons between two different groups. One-way analysis of variance, followed by Tukey's post hoc test was performed to compare between multiple experimental groups. P<0.05 was considered to indicate a statistically significant difference.

In the present study, the expression level of miR-92a was decreased in VSMCs with H₂O₂ treatment for 24 h (Fig. 1A). This result intimated that miR-92a exerted a potential protective effect in VSMCs. Following transfection and H₂O₂ treatment, the expression of miR-92a in VSMCs was detected by qPCR (Fig. 1B). The outcomes revealed that the miR-92a expression level in the miR-92a mimic group was significantly higher than that in the miRNA negative control (miR-NC) group.

To investigate the potential effect of miR-92a on VSMC viability, cell viability was detected via MTT assay. It was found that H₂O₂ remarkably attenuated the viability of normal VSMCs. In contrast, cell viability in miR-92a mimic-transfected VSMCs was identified to be improved compared with miR-NC-transfected VSMCs following H₂O₂ treatment for 24 h (Fig. 1C).

In addition, cell migration assays were performed to evaluate whether miR-92a overexpression inhibited H₂O₂-induced VSMC migration. The Transwell migration assay demonstrated that the number of VSMCs in the H₂O₂ group that migrated through the membrane was greater compared with the control group. Notably, the miR-92a mimic group markedly decreased the number of VSMCs that migrated through the membrane when compared with the H₂O₂ group or the miR-NC group (Fig. 2A-E). These results illustrate that upregulated miR-92a expression by mimics suppresses H₂O₂-induced VSMC migration in the absence of adverse effects on cell viability.

miR-92a may target MMP9 and TIMP3, and influence their expression in certain types of cancer cells (22). However, the mechanism between miR-92a and MMP9/TIMP3 in H₂O₂-induced VSMCs remains unclear. MMP9, inhibited by TIMP3, is involved in the progression of ECM degradation and VSMC migration. In the current study, the expression levels of MMP9 and TIMP3 in VSMCs following the above-mentioned transfection and H₂O₂ treatment were evaluated by western blot assay. The results demonstrated that H₂O₂ clearly caused the upregulation of MMP9 and the downregulation of TIMP3 in VSMCs. Nevertheless, transfection of miR-92a mimics markedly decreased the expression level of MMP9, but clearly increased the expression level of TIMP3 in comparison with transfection of miR-NC in H₂O₂-treated VSMCs (Fig. 3A-C). These data suggest that the expression levels of MMP9 and TIMP3 are modulated by miR-92a in VSMCs.

To verify whether miR-92a directly targets the SIRT1 gene, dual-luciferase reporter vectors harboring a wild-type or mutant 3′-UTR region of SIRT1 were constructed and transfected into HEK-293T cells. Dual-luciferase reporter assay demonstrated that miR-92a mimics significantly weakened the luciferase activity of the reporter vector containing a wild-type SIRT1 3′-UTR sequence (WT-UTR) compared with miR-NC. However, this inhibitive effect of miR-92a mimics was eliminated when the reporter vector contained a mutant SIRT1 3′-UTR sequence (Mut-UTR), which was unable to interact with miR-92a (Fig. 4A and B). These results indicate that miR-92a directly binds to SIRT1.

The above-mentioned findings suggested that miR-92a may regulate the expression levels of MMP9 and TIMP3 to inhibit VSMC migration by targeting SIRT1. To ascertain whether this influence can be indicated by silencing SIRT1, SIRT1 siRNA was transfected into VSMCs before H₂O₂ treatment. The transfection efficiency of SIRT1 siRNA and its influence on the expression levels of MMP9 and TIMP3 were demonstrated by qPCR and western blot analysis, respectively. The data revealed that the overexpression of SIRT1 mRNA resulting from H₂O₂ stimulation was markedly reduced in the SIRT1-siRNA group compared with the NC-siRNA group (Fig. 5A). As presented in Fig. 5B-D, SIRT1 siRNA decreased the expression level of MMP9, and increased the expression level of TIMP3 in H₂O₂-treated VSMCs. Notably, the Transwell migration assay demonstrated that the quantity of VSMCs in the SIRT1-siRNA group that migrated via the membrane was lower than that in the H₂O₂ group or the NC-siRNA group (Fig. 6A-E). Collectively, these outcomes demonstrated that miR-92a regulates the expression levels of MMP9 and TIMP3 via SIRT1 signaling, and further interferes with H₂O₂-induced VSMC migration.