The SMMC-7721, HepG2 and Hep3B liver cancer cell lines, and the HL-7702 normal liver cell line were provided by the Key University, Ministry of Education (Xi'an, China). These cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C in a humidified chamber containing 5% CO₂. Tumor tissues and matched normal tissues were obtained from 24 HCC patients (19 males, 5 females; age, 21–72 years). Patients were enrolled between September 2011 and January 2013 at the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China). All patients provided written informed consent. The present study was approved by the Medical Ethical Committee of the College of Medicine, Xi'an Jiaotong University (Xi'an, China).

Total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) from the cells or tissues according to the manufacturer's protocol. cDNA synthesis was performed using the Prime-Script RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. qPCR was performed on cDNA using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. PCR amplification was performed on a FTC-3000TM system (Funglyn Biotech Inc., Toronto, ON, Canada). Thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec. U6 was used for normalization in miRNA detection. The 2^−ΔΔCq method was used to quantify the relative expression levels of miR-214 (11). The primer sequences are listed in Table I.

EcoRI and HindIII sites were inserted into the multiple cloning sites of the pcDNA6.2-GW/EmGFP vector (Invitrogen; Thermo Fisher Scientific, Inc.). The primary miR-214 sequence was amplified by PCR from genomic DNA, as aforementioned, and then cloned into the EcoRI and HindIII sites of the pcDNA6.2-GW/EmGFP vector. The wild-type (wt) and mutant (mut) Wnt3a 3′ untranslated region (UTR) sequences with SacI and XhoI restriction enzymes were synthesized by Sangon Biotech. Co., Ltd. (Shanghai, China) and cloned between the SacI and XhoI sites of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, WI, USA). The 3′UTR sequences are listed in Table I. Wnt3a was cloned into a GV230 vector (Shanghai GeneChem Co., Ltd., Shanghai, China).

Potential target genes for miR-214 were predicted using RegRNA online software version 2.0 (http://regrna2.mbc.nctu.edu.tw). A potential miR-214-binding site in the 3′-UTR of Wnt3a mRNA was identified. To determine whether Wnt3a was a direct target of miR-214, HepG2 cells were co-transfected with miR-214 and wt Wnt3a 3′UTR or mut Wnt3a 3′UTR pmirGLO plasmid, using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent, according to manufacturer's protocol. HepG2 cells co-transfected with miR-214 and pmirGLO vector were used as the control. After 48 h, luciferase activity was detected using the Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocol.

Cells were seeded into 96-well plates at a density of 5,000 cells/well. Cells were transfected with miR-214, miR-214 inhibitor, small interfering RNA(si) Wnt3a, or their respective controls using Lipofectamine 2000 as the transfection reagent, according to manufacturer's protocol. The miR-214 inhibitor was synthesized by Sangon Biotech Co., Ltd. The siWnt3a and the corresponding control (ctrl) siRNA were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cell Counting kit-8 (CCK8; 7Sea Biotech, Shanghai, China) assay was performed according to the manufacturer's protocol. Optical density at 450 nm was measured 24, 48 and 72 h post-transfection using the FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Ortenberg, Germany), in order to measure cell proliferation.

HepG2 and Hep3B cells were transfected with miR-214, miR-control (ctrl), miR-214 inhibitor, inhibitor-ctrl, siWnt3a or si-ctrl (Shanghai GenePharma Co., Ltd.) using Lipofectamine 2000 according to the manufacturer's instructions. At 48 h post-transfection, 1×10⁶ cells were harvested and washed in PBS, then fixed with 70% ice-cold ethanol at 4°C overnight. The cells were washed in PBS and then incubated at 4°C with 0.1 mg/ml RNase A and 0.05 mg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min. Fluorescence-activated cell sorting was performed using a BD FACSort flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). ModFit LT version 4.0 (Verity Software House, Inc., Topsham, ME, USA) was used to analyze cell cycle distributions.

Formaldehyde-fixed, paraffin-embedded tissue samples were sectioned at 5 µm, after which the samples were deparaffinized in xylene and hydrated using graded alcohol. Antigen retrieval was performed with citrate buffer 0.01 M (pH 6.0), followed by blocking endogenous peroxidases with 3% H₂O₂ for 20 min, and blocking of non-specific binding with normal goat serum (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) at room temperature for 20 min. The slides were then incubated with anti-Wnt3a primary antibodies [FT20][a21](1:100; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China, bs-1700R) at 4°C overnight, followed by incubation with the HRP-conjugated secondary antibody (Beijing ZhongShan-Golden Bridge Biological Technology Co., Ltd., Beijing, China) [FT22][a23] for 30 min at 37°C. Detection was performed using 3,3′-diaminobenzidine [FT25][a26] for 3 min at room temperature (Beijing ZhongShan-Golden Bridge Biological Technology Co., Ltd., Beijing, China) and Harris hematoxylin [FT27][a28] for 30 sec at room temperature. Finally, a Leica Photo Microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used to obtain digital images.

Total proteins were extracted using radioimmunoprecipitation assay lysis buffer (Xi'an Wolsen Biotechnology Co., Ltd., Xi'an, China). Protein concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Equal amounts (30 µg) of extracted protein samples were separated by 10% SDS-PAGE and were then transferred onto a polyvinylidene fluoride membrane, which was blocked with 5% non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. Subsequently, the membrane was washed 3 times with TBST and incubated with secondary goat anti-rabbit antibody (cat no. 111-035-144; 1:1,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 2 h at room temperature. Protein bands were visualized using Immobilon Western Chemiluminescent HRP substrate (EMD Millipore, Billerica, MA, USA). Blots were semi-quantified by densitometry using Quantity One imaging software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis was performed with SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). Student's t-test or one-way analysis of variance followed by a post hoc Tukey test were used to analyze the data from three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

To explore the expression of miR-214 in liver cancer development, the expression levels of miR-214 were detected in 24 pairs of HCC and matched normal tissue samples using RT-qPCR. miR-214 was significantly downregulated in HCC tissue samples compared with in matched non-tumor tissues, as presented in Fig. 1A. In addition, miR-214 was downregulated in the examined liver cancer cells (SMMC-7721, Hep3B, HepG2) compared with in normal HL-7702 hepatocytes (Fig. 1B). These findings suggested that miR-214 was downregulated in HCC tissues and liver cancer cell lines; therefore, miR-214 may act as a potential anti-oncogenic miRNA in liver cancer.

The prediction of miR-214 targets was acquired using RegRNA. Wnt3a was identified as a potential target gene of miR-214. A miR-214 binding site was identified in the 3′UTR of Wnt3a (Fig. 1C). Immunohistochemistry demonstrated that Wnt3a protein expression was increased in HCC samples compared with in normal tissues (Fig. 1D). The protein expression levels of Wnt3a were also measured by western blotting in HepG2 cells transfected with miR-214 or miR-ctrl, and the results demonstrated that miR-214 overexpression was able to reduce Wnt3a protein expression (Fig. 1E). Dual-luciferase reporter assay was employed to determine whether Wnt3a was a direct target of miR-214. Luciferase activity was significantly reduced following co-transfection with miR-214 and wt Wnt3a 3′UTR compared with the control; however, luciferase activity was not altered following co-transfection with miR-214 and mut Wnt3a 3′UTR (Fig. 1F). These results provided direct evidence that Wnt3a was a target of miR-214.

To explore the role of miR-214 in liver cancer cell growth, HepG2 and Hep3B cells were transfected with miR-214, miR-ctrl, siWnt3a or si-ctrl. Overexpression of miR-214 suppressed the growth of HepG2 and Hep3B cells following transfection for 24, 48 and 72 h compared with control vector-transfected cells (Fig. 2A and B). The results of Wnt3a silencing were consistent with those of miR-214 overexpression. Based on CCK8 assays (Fig. 2C and D), siWnt3A inhibited the growth of HepG2 and Hep3B cells. To further demonstrate that miR-214 suppressed the growth of HepG2 and Hep3B by targeting Wnt3a, Wnt3a overexpression vector and miR-ctrl or miR-214 were co-transfected into HepG2 and Hep3B cells. The results demonstrated that overexpression of Wnt3a can mitigate the growth inhibition caused by overexpression of miR-214 (Fig. 2E and F).

HepG2 and Hep3B cells were transfected with miR-214 or miR-ctrl, and siWnt3a or si-ctrl. Cell cycle distribution, as detected by flow cytometry, revealed an accumulation of HepG2 and Hep3B cells in G₁ phase in the miR-214 overexpression groups compared with the miR-ctrl groups (Fig. 3A and B). In addition, the number of cells in G₁ phase was increased following transfection with siWnt3a compared with the control groups (Fig. 3C and D). These results suggested that overexpression of miR-214 or Wnt3a silencing may induce G₁ cell cycle arrest.

miR-214 inhibitor was used to further investigate the effects of miR-214 silencing on liver cancer cell proliferation. HepG2 and Hep3B cells were transfected with miR-214 inhibitor or inhibitor-ctrl, and the expression levels of miR-214 were detected (Fig. 4A and B). Cell viability was measured using the CCK8 assay; transfection with the miR-214 inhibitor increased the proliferation of HepG2 cells compared with the control group (Fig. 4C and D). The number of cells in G1 phase was reduced in the Hep3B cells transfected with miR-214 inhibitor compared with the control group (Fig. 4E and F).