A total of 60 peripheral blood samples (2 ml per individual) from 60 patients with atherosclerosis (age range, 47–78; male:female, 2:1), as well as 60 peripheral blood samples from 60 healthy volunteers (age range, 45–79; male:female, 2:1) were collected at Wuhan Central Hospital (Wuhan, China) between April 2016 and April 2017. Clinicopathological characteristics of patients were shown in Table I. The protocols were approved by the Ethics Committee of Wuhan Central Hospital and informed consent was obtained from each patient.

The human acute monocytic leukemia cell line THP-1 was purchased from the American Type Culture Collection (cat. no. TIB-202). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin mixed solution. Cells were incubated at 37°C with 5% CO₂.

To differentiate THP-1 monocytes into macrophages, THP-1 monocytes were treated with 10 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich; Merck KGaA) for 48 h (31). Atherosclerotic cell model of macrophages was established as previously described by treating differentiated THP-1 cells with 25 µg/ml ox-LDL for 24 h (31); the significant formation of THP-1 foam cells suggested that the model was successfully established.

miR-217 inhibitor (5′-UACUGCAUCAGGAACUGAUUGGA-3′; Shanghai GenePharma Co., Ltd.), inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd.), control-siRNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), SIRT1-siRNA (cat. no. sc-40986; Santa Cruz Biotechnology, Inc.) or miR-217 inhibitor + SIRT1-siRNA was transfected into THP-1 macrophages (ox-LDL untreated) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol at 37°C for 48 h. Transfection efficiency was measured at 48 h post-transfection using reverse transcription-quantitative PCR (RT-qPCR).

TargetScan bioinformatics software (www.targetscan.org/vert_71) was used to predict the potential targets of miR-217, and results identified binding sites between miR-217 and 3′UTR of sirtuin 1 (SIRT1). Dual luciferase reporter assay was performed to determine whether miR-217 directly binds to SIRT1. Wild-type (WT-SIRT1) and mutant (MUT-SIRT1) 3′UTR of SIRT1 were cloned into pmiR-RB-Report™ dual luciferase reporter gene plasmid vectors (Guangzhou RiboBio Co., Ltd.) following the manufacturer's instructions. THP-1 cells were co-transfected with WT-SIRT1 or MUT-SIRT1 and miR-217 mimic or mimic control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. At 48 h post-transfection, luciferase activity was assessed by the Dual-luciferase Assay System (Promega Corporation) and normalized to Renilla luciferase activity.

The levels of TG and TC in the cell lysates of THP-1 macrophages were determined using the TG Assay kit (cat. no. ab65336; Abcam) and Cholesterol Assay kit (cat. no. ab133116; Abcam) following the manufacturer's instructions.

Transfected and ox-LDL-treated THP-1 cells were used in the apoptosis assay. The Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (cat. no. 70-AP101-100; Hangzhou MultiSciences Biotech Co., Ltd.) was used to analyze apoptotic rates. Briefly, cells (1×10⁶ cells) were dyed with 5 µl Annexin V-FITC and 5 µl PI for 30 min at room temperature in the dark. Apoptotic rates were analyzed using a flow cytometer (BD Biosciences) with FlowJo software (version 7.6.1; FlowJo LLC).

The levels of the tumor necrosis factor α (TNF-α) (cat no. PT518), interleukin (IL)-6 (cat no. PT330) and IL-1β (cat no. PT305) in the supernatant (100 µl) of transfected and ox-LDL-treated THP-1 cells were determined using sandwich ELISA kits from Beyotime Institute of Biotechnology following the manufacturer's protocol of each kit. Cell supernatant was collected through centrifugation (500 g, 5 min).

Total RNA from blood samples (1 ml) and cells (1×10⁶ cells) was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. For cDNA generation, reverse transcription was performed using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). cDNAs were analyzed by qPCR assay with SYBR Premix Ex Taq™ II (TliRNaseH Plus) kit (Takara Bio, Inc.). U6 for miRNA and GAPDH for mRNA were used as internal controls. Primer sequences for qPCR were as follows: miR-217, forward 5′-TACTGCATCAGGAACTGACTGGA-3′, reverse 5′-GTGCAGGGTCCGAGGT-3′; SIRT1, forward 5′-AATCCAGTCATTAAAGGTCTACAA-3′, reverse 5′-TAGGACCATTACTGCCAGAGG-3′; U6, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′; GAPDH, forward 5′-CTTTGGTATCGTGGAAGGACTC-3′, reverse 5′-GTAGAGGCAGGGATGATGTTCT-3′. Expression levels were normalized to the respective internal controls and calculated using the 2^−ΔΔCq method (32).

Proteins from blood samples (1 ml) and cells (1×10⁶ cells) were extracted by using RIPA lysis buffer (cat. no. P0013E; Beyotime Institute of Biotechnology) following the manufacturer's instructions. Bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific; Inc.) was used to quantify the protein samples. Equal amounts of protein samples (30 µg/lane) were separated on 12% SDS-PAGE and transferred onto PVDF membranes (Merck KGaA). Following blocking in 5% skimmed milk at room temperature for 1.5 h, the membranes were incubated with primary antibodies against SIRT1 (120 kDa; 1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase α (AMPK-α; 62 kDa; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (62 kDa; 1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), p-NF-κB p65 (65 kDa; 1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), p65 (65 kDa; 1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.) and β-actin (45 kDa; 1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, the membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Protein bands were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories Inc.). Expression was normalized to β-actin.

Data are presented as the mean ± SD. SPSS 17.0 software (SPSS, Inc.) was used to perform statistical analysis. Differences between groups were determined using Student's t-test or one-way analysis of variance with a Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Expression levels of miR-217 were determined in the blood of 60 patients with atherosclerosis and 60 healthy volunteers using RT-qPCR. The results indicated that, compared with healthy volunteers, the level of miR-217 in the blood of patients with atherosclerosis was significantly increased (Fig. 1A). In addition, the level of miR-217 was significantly increased in ox-LDL-treated THP-1 macrophages (atherosclerosis model) compared with untreated THP-1 macrophages (Fig. 1B). These data suggested that miR-217 may be upregulated in atherosclerosis.

TargetScan identified SIRT1 as a potential miR-217 target (Fig. 2A). Results from the dual-luciferase reporter assay demonstrated that luciferase activity was significantly reduced in THP-1 macrophages co-transfected with SIRT1-WT reporter plasmids and miR-217 mimic compared with SIRT1-WT and mimic control; no differences were observed between co-transfections with SIRT1-MUT reporter plasmids and miR-217 mimic or mimic control (Fig. 2B). miR-217 significantly enhanced the level of miR-217 in THP-1 macrophages (Fig. 2C). These data indicated that miR-217 may directly target SIRT1.

SIRT1 mRNA expression levels were examined in the blood of 60 patients with atherosclerosis and 60 healthy volunteers using RT-qPCR. Compared with the healthy volunteers, SIRT1 mRNA expression level in the blood of patients with atherosclerosis was significantly decreased compared with healthy volunteers (Fig. 3A). In addition, ox-LDL treatment significantly decreased SIRT1 mRNA level in THP-1 macrophages compared with untreated cells (Fig. 3B). ox-LDL treatment markedly decreased SIRT1 protein expression in THP-1 macrophages compared with the untreated cells (Fig. 3C). These data suggested that SIRT1 may be downregulated in atherosclerosis.

To determine the role of miR-217 in atherosclerosis, miR-217 inhibitor, inhibitor control, control-siRNA, SIRT1-siRNA or miR-217 inhibitor + SIRT1-siRNA was transfected into THP-1 macrophages for 24 h prior to treatment with 25 µg/ml ox-LDL for 24 h. Transfection efficiency was determined by RT-qPCR and/or western blot analysis. miR-217 inhibitor significantly downregulated the miR-217 level in THP-1 macrophages compared with the untreated cells (Fig. 4A), and SIRT1-siRNA significantly decreased the SIRT1 mRNA level in THP-1 macrophages compared with untreated cells (Fig. 4B). The protein level of SIRT1 was also reduced in THP-1 macrophages by SIRT1-siRNA treatment (Fig. 4C). Additionally, miR-217 inhibitor significantly increased the mRNA levels of SIRT1, and the enhancement was reversed by SIRT1-siRNA (Fig. 4D). The miR-217 inhibitor markedly enhanced the expression of SIRT1, and this was reversed by SIRT1-siRNA (Fig. 4E).

The effect of miR-217 on TG and TC levels in the atherosclerosis cell model was also determined. Compared with the untreated control group, 25 µg/ml ox-LDL treatment significantly enhanced the levels of TG and TC, whereas miR-217 inhibitor significantly reduced TG and TC levels induced by ox-LDL (Fig. 5). In addition, co-treatment with SIRT1-siRNA reversed the effects of miR-217 inhibitor on the levels of TG and TC in THP-1 macrophages (Fig. 5).

The effect of miR-217 on THP-1 cell apoptosis was analyzed. Ox-LDL treatment significantly induced THP-1 macrophage apoptosis (Fig. 6), which was notably inhibited by miR-217 inhibitor transfection, and this inhibition was reversed by co-transfection with SIRT1-siRNA.

The levels of inflammatory factors were detected by ELISA. The results demonstrated that treatment with ox-LDL increased TNF-α, IL-6, and IL-1β content in THP-1 macrophage culture, which was significantly decreased by miR-217 inhibitor transfection (Fig. 7). The effects of miR-217 inhibitor were reversed by SIRT1 silencing (Fig. 7).

The SIRT1/AMPK-α/NF-κB pathway in ox-LDL-treated THP-1 macrophages was analyzed. Ox-LDL treatment markedly decreased SIRT1 protein level, reduced p-AMPK-α protein level, and increased the phosphorylation level of NF-κB p65 protein in THP-1 cells (Fig. 8A). Ox-LDL treatment significantly decreased the level of SIRT1 mRNA (Fig. 8B), reduced the p-AMPK-α/AMPK-α ratio (Fig. 8C), and increased the ratio of p-p65/p65 (Fig. 8D) in THP-1 cells. Compared with the ox-LDL-only treatment group, co-treatment with the miR-217 inhibitor markedly enhanced SIRT1 and p-AMPK-α expression, and reduced p-p65 protein level. SIRT1 silencing significantly reversed the effects of miR-217 inhibitor on the expression of SIRT1, p-AMPK-α and p-p65 in THP-1 macrophages.