All the cell lines used in the present study were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HEK 293T cells, HL-7702 (normal hepatocellular cells), and the HCC cell lines SMMC-7721, BEL-7402 and HepG2, were cultured in Dulbecco's modified Eagle's medium (DMEM). All media were supplemented with fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) to a final concentration of 10% and with antibiotics; the cells were incubated at 37°C with 5% CO₂.

This study was approved by the Ethics Review Committees of the Second Hospital of Longyan, Longyan, Fujian, China), and written informed consent was obtained from all patients. A total of 35 patients (23 males and 12 females) with HCC underwent routine surgery (complete resection) at the Second Hospital of Longyan between January 2013 and September 2014. The mean age of the patients was 52.7 years (range, 41–67 years). HCC samples and matched normal liver tissues (located ~2 cm apart) taken from these 35 patients were snap-frozen in liquid nitrogen for further quantitative polymerase chain reaction (qPCR) analysis.

Total RNA and microRNA fractions were isolated from tissues samples and the HL-7702, SMMC-7721, BEL-7402 and HepG2 cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA (1 µg) was reverse transcribed into cDNA using PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China), which contained PrimerScript reverse transcriptase, RNase inhibitor, deoxynucleotide mixture and reaction buffer.. MicroRNA extraction was performed using the microRNA Extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). RT-qPCR was performed with SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), under the following cycling conditions: 40 cycles of 58°C for 20 sec and 75°C for 10 sec. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for the mRNA, and RNU6B was used as the microRNA reference. Primers for miR-320a (forward, 5′-GGGCTAAAAGCTGGGTTGA-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′) and β-catenin (forward, 5′-AAAATGGCAGTGCGTTTAG-3′ and reverse, 5′-TTTGAAGGCAGTCTGTCGTA-3′) were obtained from RiboBio (Guangzhou, China). The expression of mRNA and microRNA was normalized to its relative control by the comparative ∆Cq method (20). The experiment was performed in triplicates.

Plasmid LV3-pGLV-H1-GFP+Puro, with hsa-miR-320a mimics or hsa-miR-320a inhibitor, or their respective control oligonucleotides, namely miR-320a and miR-negative control (NC), and anti-miR-320a and anti-miR-NC, were purchased from GenePharma (Shanghai, China). Lentivirus transfection was performed as per the manufacturer's instructions to establish miR-320a-expressing stable clones (HepG2/miR-320a) and anti-miR-320a-expressing stable clones (HepG2/anti-miR320a) in HepG2 cells. The relative control clones (HepG2/miR-NC and HepG2/anti-miR-NC) were constructed by similar methods.

Prediction of miR-320a binding sites was performed using TargetScan software (http://www.targetscan.org). Bioinformatics analysis revealed a potential miR-320a binding site for miR-320a in the 3′-UTR region of β-catenin. HEK 293T cells at 60% confluence were transfected with 200 ng DNA from the β-catenin-wild-type (WT)-UTR plasmid (firefly luciferase reporter vector containing the β-catenin 3′-UTR) or β-catenin-WT-UTR DNA (firefly luciferase reporter vector containing the β-catenin 3′-UTR mutant) and 2 ng pRL-TK vector (Promega Corporation, Madison, WI, USA) in combination with miR-320a mimics (final concentration of 100 nM; GenePharma) or miR-NC (100 nM). Transfection was performed with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.). The firefly and Renilla luciferase activities were measured by consecutively using Dual Glo Luciferase assays (Promega Corporation). Firefly l uciferase activity was normalized to Renilla luciferase for each transfected well.

For cell cycle analysis, the cells were harvested after transfection for 24 h. The cells were then fixed with 75% ethanol at 4°C overnight, washed with cold phosphate-buffered saline and treated with RNase I, followed by a 30-min staining with propidium iodide in the dark. Cell cycle distributors were analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (Abcam, Cambridge, MA, USA) and analyzed by flow cytometry as per the manufacturer's instructions.

Cell proliferation was examined using a water-soluble tetrazolium salt assay via Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies Inc., Kumamoto, Japan). Briefly, the cells were seeded in 96-well culture plates and incubated at 37°C with 5% CO₂ for 4 days. Once the assay began, 10 µl CCK-8 solution was added to the medium and then incubated at 37°C for 2 h. Cell numbers were estimated by measuring the absorbance at 450 nm using a 96-well plate reader. For the colony formation assay, 5,000 cells were plated in a 6-well plate for 9 days. Colonies were fixed with methanol/acetone (1:1) and stained with crystal violet.

Western blot analysis was performed using anti-cyclin D1 (rabbit monoclonal; 1:4,000 dilution; catalog no. ab137875; Abcam), anti-c-myc (rabbit monoclonal; 1:5,000 dilution; catalog no. ab109416; Abcam), anti-dickkopf-1 (DKK-1; rabbit monoclonal; 1:2,500 dilution; catalog no. ab109416; Abcam) and anti-β-catenin (rabbit polyclonal; 1:3,000 dilution; catalog no. ab6302; Abcam) antibodies, with β-actin (mouse monoclonal; 1:3000 dilution; catalog no. ab20272; Abcam) used as a loading control. The band intensities of the western blotting were analyzed using Image Analysis Software v2.0 (Thermo Fisher Scientific Inc.).

All statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA, USA). The differences among groups were analyzed by a one-way analysis of variance followed by Bonferroni's multiple comparison tests or a t-test, as appropriate. All data are expressed as the mean ± standard error of the mean. P<0.05 was used to indicate a statistically significant difference.

The expression level of miR-320a in 35 HCC tissues and their paired adjacent normal liver tissues was quantitatively analyzed by RT-qPCR. The expression of miR-320a in the HCC tissues was found to be lower than that in the normal tissues (P=0.0003; n=35; Fig. 1A). The expression level of miR-320a was also examined in a panel of human liver cancer cell lines. The results of the RT-qPCR showed that the miR-320a expression level was decreased in all three live cancer cell lines examined compared with the HL-7702 normal liver cells (P=0.02; n=3; Fig. 1B).

To evaluate the efficiency of miR-320a and its inhibitors, an RT-qPCR assay was performed to determine the expression level of miR-320a in HepG2 cells transfected with miR-320a or its inhibitors or their relative negative controls. Fig. 2A shows that the level of miR-320a increased significantly following transfection with mimics and that it decreased significantly following transfection with inhibitors (both P=0.01; n=3; Fig. 2A). The role of miR-320a in cell growth was also assessed. As indicated in Fig. 2B, expression of miR-320a in HepG2 markedly inhibited cell growth at day 4 compared with in the HepG2/miR-NC cells. By contrast, expression of anti-miR-320a in HepG2 increased the growth ability when compared to its negative control (both P=0.03; n=3; Fig. 2B). In the apoptosis assay, compared with their respective controls, the expression of miR-320a in the HepG2 cells increased the cell apoptosis rates, and expression of anti-miR-320a in the HepG2 cells decreased the cell apoptosis rates (both P=0.03; n=3; Fig. 2C). The impact of miR-320a on the cell cycle was further assessed. Results showed that the expression of miR-320a increased cell populations at the G₀/G₁ phase, with an associated reduction of cell populations at the G₂/M phase, while the expression of anti-miR-320a caused a reduction of cell populations at the G₀/G₁ phase, with an associated increase of cell populations at the S phase, compared with their respective negative controls (both P=0.02; n=3; Fig. 2D). The colony formation study also showed that the expression of miR-320a decreased the number of colonies, while the expression of anti-miR-320a increased the number of colonies, compared with their respective negative controls (both P=0.01; n=3; Fig. 2E and F). These results indicated that miR-320a expression suppresses cell proliferation by affecting cell cycle distribution.

To assess miR-320a binding to the 3′-UTR, luciferase reporters were constructed with the β-catenin 3′-UTR. The relative luciferase activity of the construct with WT 3′-UTR was significantly repressed by ~35% following miR-320a transfection, and the expression of anti-miR-320a significantly increased the luciferase activity by ~20%, when compared with their respective controls (both P=0.03; n=3; Fig. 3). Site-directed mutagenesis of the miR-320a binding site within the β-catenin 3′-UTR completely abolished the effect of miR-320a or anti-miR-320a transfection.

RT-qPCR was first performed to detect the expression levels of β-catenin mRNA. The expression of miR-320a downregulated the expression levels of β-catenin, while the expression of anti-miR-320a upregulated the expression levels of β-catenin mRNA (both P=0.02; n=3; Fig. 4A). Next, western blotting was performed to detect the expression levels of β-catenin, c-myc, cyclin D1 and DKK-1. The expression of miR-320a in the HepG2 cells downregulated the expression levels of β-catenin, c-myc, cyclin D1 and DKK-1, while the expression of anti-miR-320a upregulated the expression levels of β-catenin, c-myc, cyclin D1 and DKK-1, when compared with their respective negative controls (all P=0.04; Fig. 4B-F).