The experimental protocol of the present study was approved by the ethics committee of the Hebei Medical University (Shijiazhuang, China). In addition, the present study was approved by the Institutional Review Board for Human Studies, and written informed consent was obtained from all patients.

Plasma samples were taken from 175 healthy subjects and 150 patients with UA at the Tangshan Workers' Hospital (Tangshan, China) between January 2012 and June 2013, in order to establish seven plasma pools for the healthy subjects and six pools for the patients with UA. Each plasma pool consisted of 25 cases (100 µl plasma/case). UA patients were eligible to participate in the present study if they met the American College of Cardiology/American Heart Association criteria for UA (16).

The 6th generation of the miRCURY LNA™ microRNA Array (Exiqon A/S, Vedbaek, Denmark) contains >1,891 capture probes, covering all human, mouse and rat microRNAs annotated in the miRBase 16.0 (http://www.mirbase.org/), as well as all viral miRNAs associated with these species. In addition, the array contains capture probes for 66 novel miRPlus™ human miRNAs. Exiqon gene-chips are able to detect specific hybridization and to perform a rigid statistical analysis in order to reduce the rate of false-positive signals (17).

All plasma samples were subjected to RNA extraction. Briefly, total RNA was isolated from the plasma samples using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and an miRNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's protocols. This RNA extraction step efficiently recovered 90% RNA species, including miRNAs. The concentration and purity of the total RNA samples were assessed by measuring the absorbance at 260, 280 and 230 nm in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). RNA integrity was determined by agarose gel electrophoresis (using 1% gels). Briefly, agarose (0.5 g; Zhongshan Jinqiao Biology & Technology Co., Ltd., Beijing, China) was dissolved in electrophoresis buffer (50 ml, Zhongshan Jinqiao Biology & Technology Co., Ltd.), following mixing. The sample was then added to the wells, and the voltage was adjusted to 100 v, with the RNA electrophoresis from anode to cathode. The gel was dyed for 5 min with EB dyeing liquid (Applygen Technologies Inc. Beijing, China).

Following RNA isolation from the plasma samples, RNA was labeled using the miRCURY LNA™ microRNA Array Hy3™/Hy5™ Power labeling kit (Exiqon A/S). Briefly, 1 µg of each sample was 3′-labeled with Hy3 fluorescent dye, using the T4 RNA ligase (provided in the kit), according to the following procedure: RNA in 2.0 µl water was combined with 1.0 µl calf intestinal phosphatase buffer (Exiqon A/S), after which the mixture was incubated for 30 min at 37°C, followed by termination of the reaction by incubating for 5 min at 95°C. Subsequently, 3.0 µl labeling buffer, 1.5 µl Hy3 and 2.0 µl labeling enzyme (both Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Beijing, China) were added to the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.

Following termination of the labeling reaction, the Hy3-labeled samples were hybridized to the miRCURY LNA™ microRNA Array, according to the manufacturer's protocol. Briefly, the total 25 µl mixture consisting of 5 µl Hy3-labeled samples and 20 µl hybridization buffer were denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay NimbleGen Hybridization System (Roche Diagnostics, Basel, Switzerland), which provides an active mixing action and constant incubation temperature in order to improve hybridization uniformity and enhance signals. Following hybridization, the slides were obtained, washed three times using Wash buffer kit (Roche Diagnostics GmbH, Mannheim, Germany; cat no. 5188–5327) and finally dried by centrifugation for 5 min at 400 × g and 4°C. Subsequently, the slides were scanned using the Axon GenePix^® 4000B Microarray Scanner (Molecular Devices, LLC, Sunnyvale, CA, USA).

Microarray data analysis was performed using a series of models in the limma package (version 3.22.7; https://bioconductor.org/packages/release/bioc/html/limma.html). Scanned images were then imported into GenePix^® Pro 6.0 Microarray Acquisition and Analysis software (http://axon-genepix-pro.software.informer.com/6.0/) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs with intensities ≥30 in all samples were selected for normalization. Data was normalized using the Median normalization method and ComBat software (version 1.1.4; http://www.bu.edu/jlab/wp-assets/ComBat/Download.html) was used to adjust the normalized intensity to eliminate batch effects. Following normalization, significantly differentially expressed miRNAs were identified via volcano plot filtering, with P-value value between single fluorescent chip data group and a fold change value established by Volcano plot using R Software (version 5.50; MathSoft, Windows). Hierarchical clustering was performed using Multiexperiment Viewer software, version 4.6 (http://www.tm4.org/mev.html).

RT-qPCR was performed in order to validate the microarray data. RT-qPCR was conducted using the RNA samples used in the microarray analyses. Single-strand cDNA synthesis was performed using a PrimeScript II 1st strand Cdna Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instructions using dNTPs (0.15 µl), enzyme mix (1 µl), primer mix (3 µl), RT buffer (1.5 µl), RNAase inhibitor (0.19 µl), RNase free H₂O (4.16 µl), RNA (5 µl). Primer sequences targeting specific genes were designed using Primer Express^® software (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to sequences published by GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The primers designed in the present study were as follows: Forward, 5′-CGCGGTATGGCACTGGTAGA-3′, and reverse, 5′-AGTGCAGGGTCCGAGGTATTC-3′ for miRNA-183-5p; forward, 5′-CCGGAATTCCCTCAACTCCACTCGTGTCC-3′, and reverse, 5′-ATTGCGGCCGCTGGGACTGTGACTCCTACCTG-3′ for miRNA-9-3p; forward, 5′-GGGAGCTGGTGTGTGAAT-3′, and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′ for miRNA-138-5p; forward, 5′-AACCUGAUCCCGUCUGAGAUUG-3′, and reverse, 5′-CCGGAUCAAGAUUAGUUCGGUU-3′ for miRNA-204-3p; forward, 5′-ACACTCCAGCTGGGTAAGGCACGCGGTGAAT-3′, and reverse, 5′-CTCAACTGGTGTCGTGGA-3′ for miRNA-124-3p; and sense, 5′-TCCACCACCCTGTTGCTGTA-3′ and antisense, 5′-ACCACAGTCCATGCCATCAC-3′ for GAPDH. GAPDH was used as an internal control. RT-qPCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions were as follows: i) 50°C for 2 min; ii) 95°C for 15 min; iii) 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec; and iv) final fluorescence detection at 95°C for 15 sec. miRNA expression levels were normalized against an endogenous reference gene (GAPDH) and relative to a control. The relative expression levels for each mRNA were determined using the 2^−ΔΔCq method (18).

Statistical analyses were performed with the SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard error of the mean. Differences in the mean values were evaluated by Student's t-test (two means comparison), and one-way analysis of variance was conducted in order to compare the means among groups. Differences in the mean values were evaluated by Student's t-test (two means comparison). P<0.05 was considered to indicate a statistically significant difference.

The concentration and purity of the RNA was determined using a NanoDrop ND-1000 spectrophotometer. The A260/A280 ratio was >2.0 and the A260/A230 ratio was >1.7. In addition, the integrity of RNA was assessed by denaturing agarose gel electrophoresis. Intact total RNA run on a denaturing gel will exhibit sharp 28S and 18S ribosomal RNA bands, as is shown in Fig. 1. Therefore, the concentration, purity and integrity of the RNA samples in the present study were suitable for microarray experiments.

A total of 1,891 miRNAs were detected in each group using the Exiqon gene-chip. In order to identify miRNAs that were significantly differentially expressed between the two groups, volcano plot filtering was performed. The cut-off threshold for significantly differentially expressed miRNAs was a fold-change ≥2.0 and P<0.05. A total of 212 miRNAs were shown to be differentially expressed between the UA and control groups. Of these, 82 were upregulated (Table I) and 130 were downregulated (Table II). Functional annotation using GO analyses suggested that the majority of the differentially expressed miRNAs were associated with cardiovascular development or cholesterol metabolism (Table III).

In order to validate the microarray results, RT-qPCR was performed. A total of 5 miRNAs from the differential gene expression profile were selected to validate the gene-chip results. As is shown in Fig. 2, RT-qPCR demonstrated the expression levels of these miRNAs were markedly downregulated, which was consistent with the gene-chip results. In addition, statistical analyses identified a conformity between the RT-qPCR analysis and microarray results.