The HepG2 human hepatoma cell line was obtained from the Cell Culture Engineering Center of the Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). HepG2 cells were cultured in 6 well plates with 2.0 ml/well Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin and 100 µg/ml strep tomycin (Invitrogen Life Technologies) at 37°C in a 5% CO₂ incubator. HepG2 cells were serum starved for a minimum of 16 h prior to treatment.

Control mimics, miR-28-5p mimics or inhibitors (100 nM; Invitrogen Life Technologies) were transiently transfected into HepG2 cells using Lipofectamine^® RNAi 2000 Reagent and Opti MEM I Reduced Serum Medium (Invitrogen Life Technologies) according to the manufacturer's instructions. Cells were treated with the ERK2 inhibitor PD98059 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) as described previously (13). Briefly, HepG2 cells were pre-transfected with or without the mir-28-5p inhibitor and incubated with 20 or 40 µM PD98059 overnight. Following 48 h of transfection, cells were harvested for protein analysis or were treated for further experiments. Briefly, the treated cells were washed with times with phosphate buffered saline, and lysed by radioimmunoprecipitation acid buffer (Beyotime Institute of Biotechnology, Shanghai, China) at 4°C. All transfection experiments were performed at least three times.

Treated cells were lysed in radioimmu noprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Protein extracts (40 µg/lane) were separated on a 10% SDS-polyacrylamide gel (Sigma-Aldrich, St. Louis, MO, USA) and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) using a Mini Trans-Blot^® system (Bio-Rad Laboratories, Inc., Shanghai, China). Membranes were blocked with immuno-blotting blocking buffer (Beyotime Institute of Biotechnology) for 1 h at room temperature prior to incubation with the following primary antibodies obtained from Santa Cruz Biotechnology, Inc.: Polyclonal rabbit anti-ABCA1 (1:500; cat no. sc-20794), polyclonal rabbit anti ERK2 (1:500; cat no. sc-154) or polyclonal rabbit anti-β-actin (1:1,000; cat no. sc-130656) overnight at 4°C. Following washing with Tris buffered saline with Tween-20, the membranes were incubated for 2 h at room temperature with horseradish peroxidase conjugated goat anti rabbit IgG antibodies (1:5,000; cat no. sc-2004; Santa Cruz Biotechnology, Inc.). Protein bands were visualized by chemiluminescence using the SuperSignal™ West Dura Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Signal intensity was quantified using AlphaEaseFC 4.0 software (Cell Biosciences, Inc., Santa Clara, CA, USA) and normalized to β-actin levels.

All patients with UA (n=39) in the current study were enrolled from the Department of Cardiology in Tangshan Gongren Hospital (Tangshan, China) between March 2012 and August 2013. The diagnosis of UA was confirmed using the American College of Cardiology and the American Heart Association 2007 guidelines (22). Healthy subjects (n=28) were selected if the following conditions were absent: Diabetes, hypertension, hepatitis, kidney disease, cardiovascular disease and medication history at the Tangshan Gongren Hospital. The current study was approved by the Ethics Committee of Tangshan Gongren Hospital and written informed consent was obtained from each volunteer.

The handling of blood samples was completed within 2 h of collection according to the Tangshan Gongren Hospital Biobank Handling and Storage Protocol for the collection, processing and archiving of human blood. Briefly, fasting blood samples were drawn in the morning and collected into EDTA anticoagulant tubes (Guangzhou Improve Medical Instruments Co., Ltd., Guangzhou, China) and centrifuged at 3,000 × g then 16,000 × g for 10 min at 4°C each to separate the plasma from blood cells, platelets and cellular debris. Plasma was then transferred to RNase/DNase-free 1.5 ml Cryo tubes (Sangon Biotech Co., Ltd., Shanghai, China) and stored at −80°C until required.

Total small RNAs were isolated from 500 µl plasma using the mirVana™ RNA Isolation kit (Ambion Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Extracted RNAs were eluted with 80 µl diethylpyrocarbonate treated ddH₂O (Sangon Biotech Co., Ltd.) and stored at−80°C. Quality of RNA was determined using a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific).

The expression levels of miR-28-5p and miR-423-3p were determined by RT-qPCR. Briefly, small RNA was reverse transcribed into DNA using the TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems Life Technologies, Foster City, CA, USA). In brief, the RT reaction mixture contained 0.8 µl 10X reverse transcription buffer, 4 µl small RNA (~20 ng/µl), 0.1 µl 100 mM dNTPs, 0.1 µl RNase inhib itor (20 U/µl), 1.5 µl RT primer, 0.5 µl MultiScribe™ Reverse Transcriptase (50 U/µl) and 1 µl RNase/DNase free ddH₂O. The reaction mixture was incubated at 16°C for 60 min, 42°C for 60 min and 85°C for 5 min. RT qPCR was performed using an Applied Biosystems^® 7500 Real time PCR System (Applied Biosystems Life Technologies). The PCR reac tion mixture contained 4 µl RT product, 10 µl 2X TaqMan Universal PCR Master Mix, 1 µl TaqMan probe (2.5 µM) and 5 µl RNase/DNase free ddH₂O. The cycling parameters were as follows: Denaturation at 95°C for 10 min followed by 40 cycles each of denaturation at 95°C for 15 sec and annealing and extension at 60°C for 1 min. miRNA levels were determined using the 2-[Ct(target miRNA) Ct(reference miRNA)] method. Hsa-miR-423 was used as a stable internal reference in the plasma samples according to the guidelines of ABI miRNA profiling (Applied Biosystems Life Technologies) in serum/plasma and as previously reported (23).

miRNA target interactions were predicted using the miRwalk database (http://www.umm.uniheidelberg.de/apps/zmf/mirwalk). miRNA sets for ERK2 were obtained based on the miRwalk prediction algorithm.

Data were presented as the mean ± standard error unless otherwise stated. Comparisons between two groups were performed using the Mann Whitney U test or Student's t test as appro priate. A Fisher's exact test or χ² test was used to compare categorical variables. Correlation analysis was performed using Spearman's correlation analysis. Statistical analysis was performed with GraphPad Prism software, version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To validate the fact that miR-28-5p targets ERK2, the protein levels of ERK2 in HepG2 cells were measured following transfection with the miR-28-5p mimic or inhibitor. Western blotting indicated that transfection with the miR-28-5p mimic significantly reduced the expression levels of ERK2 in HepG2 cells, whereas the miR-28-5p inhibitor resulted in elevated levels of ERK2 following 48 h of transfection (Fig. 1). These results indicate that miR-28-5p is a key mediator in the regulation of the translation of ERK2.

In a previous preliminary study (10), the transfection of miR-28-5p mimics was observed to result in an increase in ABCA1 expression levels in HepG2 cells and THP 1 derived macrophages. In the current study, miR-28-5p inhibitors were transfected into HepG2 cells to further investigate the miR-28-5p mediated upregulation of ABCA1. As presented in Fig. 2, transfection with the miR-28-5p inhibitor significantly reduced the expression levels of ABCA1 (P<0.05) compared with control and miR-28-5p mimics (Fig. 2).

To investigate whether ERK2 alters the expression of ABCA1, HepG2 cells were incubated with an ERK2 inhibitor, PD98059. As previously reported, the induction of ABCA1 expression is semi dependent on the concentration of ERK2 inhibitors, with the maximal induction effect dose for macrophage ABCA1 expression by PD98059 being 20 µM (14). As presented in Fig. 3, PD98059 increased the expression levels of ABCA1 in HepG2 cells.

To confirm that the miR-28-5p-mediated upregulation of ABCA1 is dependent on ERK2, HepG2 cells were transfected with the miR-28-5p inhibitor prior to incubation with the ERK2 inhibitor (PD98059) overnight. As presented in Fig. 4, HepG2 cells stimulated with 20 µM PD98059 antagonized the miR-28-5p inhibitor mediated ABCA1 reduction. Taken together, these data indicate that miR-28-5p mediates ABCA1 expression through the ERK2 signaling pathway.

To explore the potential miRNAs with the ability to upreg ulate ABCA1 through ERK2 inhibition, the predicted dataset of miRNA and ERK2 interactions were retrieved from the miRwalk database. The top 30 predicted miRNAs are shown as Table II, and all have potential roles in ERK2-mediated ABCA1 upregulation.

Circulating levels of miR-28-5p in healthy subjects (n=28) and patients with UA (n=39) were measured to investigate the association between the circulating levels of miR-28-5p and clinical parameters in patients with UA. Clinical parameters of patients with UA are presented in Table I. There were no significant differences between the groups in age, gender or risk factors except for total cholesterol and HDL C. The circulating levels of miR-28-5p were not significantly associated with age or gender or with diabetic status. However, the circulating levels of miR-28-5p in patients with UA were observed to be positively correlated with HDL-C levels (R= −0.327; P=0.042; Fig. 5), supporting the role of miR-28-5p in the upregulation of ABCA1 in vitro.