Primary HCC specimens and their matched normal specimens were obtained from patients undergoing hepatic resection at the Department of Hepatopancreatobiliary Surgery, The First Hospital, Jilin University (Changchun, China), and were snap-frozen in liquid nitrogen and stored at −80°C until use. None of the patients received radiotherapy, chemotherapy, or other anticancer treatment prior to surgery. All patients provided written informed consent according to the protocol approved by the Ethics Committee of Jilin University.

Four human HCC cell lines (Huh-7, SMMC7721, HepG2 and HCCLM3) and a normal human hepatocyte cell line HL-7702, were obtained from the Shanghai Institute for Biological Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal calf serum (FCS) (all from Gibco, Grand Island, NY, USA) at 37°C in a humidified chamber supplemented with 5% CO₂.

Total RNA was extracted from the clinical specimens and cultured cells with TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's instructions. The purity and concentration of RNA were measured by using a dual-beam ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). Ten nanograms of total RNA was transcribed into cDNAs using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. To detect miR-154 expression, real-time PCR was performed with a TaqMan MicroRNA Assay kit on the ABI 7900 Fast system (both from Applied Biosystems). U6 small nuclear RNA was used as an internal control.

To measure ZEB2 mRNA expression, qRT-PCR was performed with SYBR Green Premix Ex Taq (Takara) on ABI 7900 Fast system. The primer sequences of ZEB2 and GAPDH used were: ZEB2 sense, 5′-AGGAGCAGGTAATCG-3′ and antisense, 5′-TGGGCACTCGTAAGG-3′; GAPDH sense, 5′-GAAGGTGAAGGTCGGAGTC-3′ and antisense, 5′-GAAGATGGTGATGGGATTTC-3′. GAPDH was used as an internal control. All reactions were run in triplicate, and fold changes in gene expression were calculated by the 2^−ΔΔCt method.

miR-154 mimics and corresponding controls (miR-NC) were purchased from Ribobio Co. (Guangzhou, China). The plasmids carrying ZEB2 (pCDNA3-ZEB2) were a gift from Dr Lin (Jilin University, Changchun, China). These molecular products were transiently transfected into HepG2 cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Transfection efficiencies were evaluated in every experiment at 48 h after transfection.

The cell proliferation was determined by the MTT assay. Briefly, 5×10³ transfected cells were seeded into 96-well culture plate, and cultured for 24, 48 and 72 h, respectively. Then, 20 µl of MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added, and cultured for 4 h at 37°C. Then, the medium was removed and the residue was dissolved in 150 µl DMSO (Sigma-Aldrich). The absorbance of each well was read at 570 nm with a microplate reader.

Transfected cells were digested, and a single-cell suspension was prepared. Then, the cells (1,000 cells/well) were added to 6-well plates followed by incubation under a standard condition for 24 h. Non-adherent cells were removed. After culturing for 10–14 days, the cells were stained with 0.5% crystal violet for 30 min. The percentage of colony formation was calculated by adjusting the control (miR-NC group) to 100%.

Cell cycle distribution and apoptosis were examined by flow cytometry at 48 h post-transfection. For the cell cycle assay, 2×10⁴ transfected cells were fixed in 10 µl ice-cold ethanol for at least 2 h. and then washed twice in PBS and incubated with 500 µl RNase (0.25 mg/ml) at 37°C for 30 min. The cells were pelleted by centrifugation at 1,000 × g for 5 min, resuspended in 50 µg/ml propidium iodide (PI; KeyGen, Nanjing, China), and incubated at 4°C for 30 min in the dark. The PI signal was examined using a FACSCalibur flow cytometer (BD Biosciences, Mansfield, MA, USA).

The cell apoptosis assay was performed using cell staining with PI and FITC-labeled Annexin V (KeyGen) to detect phosphatidylserine externalization as an endpoint indicator of early apoptosis, according to the manufacturer's instructions.

Transfected cells were seeded at 2.0×10⁴ cells/well in 6-well culture plates. After the cells had grown to confluency, the confluent monolayer in each well was scratched using a 10-µl pipette tip in order to evaluate cell migration by testing the capability of cells to migrate into the wounded area. The cells were photographed at 0 and 24 h; the scratch area was measured using Image J software (National Institutes of Health, Bethesda, MD, USA).

Invasion assays were carried out in modified Boyden chambers (BD Biosciences, San Jose, CA, USA) with 8-mm pore filter inserts in 24-well plates. Twenty-four hours after transfection, 2.0×10⁴ transfected cells suspended in serum-free RPMI-1640 medium were added to the upper chamber coated with Matrigel (BD Biosciences) and incubated at 37°C for 4 h, allowing it to solidify. RPMI-1640 containing 10% FCS was added to the lower chambers as a chemoattractant. After a 24-h culture at 37°C, the non-filtered cells were gently removed with a cotton swab. Filtered cells located on the lower side of the chamber were stained with 2% crystal violet, imaged, and counted with a microscope (Olympus, Tokyo, China). All experiments were performed in triplicate.

A human ZEB2 3′UTR was amplified from cDNAs of the HCC cell line by RT-PCR, and cloned into the downstream of the firefly luciferase gene of the psiCHECK-2 vector (Promega, Madison, WI, USA). For mutagenesis of the miR-154-binding site, a QuickChange Site-Directed Mutagenesis kit (Agilent Technologies, Palo Alto, CA, USA) was used according to the manufacturer's instructions.

For the luciferase activity assay, HepG2 cells were co-transfected with wild-type (WT) or mutant (Mut) 3′UTR of psiCHECK-2-ZEB2 and miR-154 or miR-NC. The cells were incubated for 48 h, and dual-luciferase activities were assessed using the Dual-Luciferase Reporter Assay system (Promega), according to the manufacturer's instructions. Renilla luciferase activity was normalized to firefly luciferase activity.

Tissue samples and cells were harvested and homogenized with RIPA lysis buffer (Beyotime, Beijing, China) according to the manufacturer's instructions. Equal amounts of proteins (30 µg each lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with the following primary antibodies: anti-ZEB2, anti-E-cadherin, anti-N-cadherin, anti-vimentin, and anti-human GAPDH (all from Cell Signaling Technology, USA). Then the blots were incubated with the corresponding HRP-labeled secondary antibody for 1 h at room temperature. Proteins on the membranes were detected using the ECL detection system (Pierce, Cambridge, MA, USA).

Sixteen BALB/C nude male mice (20–25 g) (5-weeks old) purchased from the Experimental Animal Center of Changchun Institute for Biological Sciences (Changchun, China), were maintained under pathogen-free (SPF) conditions. All animal experiments were approved by the Animal Ethics Committee of Jilin University (Changchun, China)

For the in vivo tumor assay, 2×10⁶ HepG2 cells stably expressing the miR-154 mimic or the miR-NC were injected subcutaneously into the right rear flank of each mouse (8 per group) to establish an HCC xenograft model. Tumor volume was calculated using the formula: Volume (mm³) = 1/2 width² × length. All mice were sacrificed, and the tissues were removed and weighed 5 weeks after the injection. Partial liver tissues were snap-frozen for RNA and protein extraction.

Data are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Data were imaged with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Two-tailed Student's t-test or ANOVA was used to analyze differences. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential role of miR-154 in HCC carcinogenesis, we assessed the miR-154 expression in a panel of human HCC cell lines by real-time quantitative RT-PCR (qRT-PCR). The result showed that the expression level of miR-154 was downregulated in the HCC cell lines compared with that noted in the normal hepatic HL-7702 cell line (Fig. 1A). Additionally, the expression level of miR-154 in the HepG2 cell line was lowest, thus, we selected it for subsequent study. Consistent with the results from the cell lines, the miR-154 level in the HCC tissues was significantly lower than that in the corresponding non-cancerous tissues (Fig. 1B).

The associations of miR-154 expression with various clinicopathological parameters of HCC tissues were also analyzed (Table I). The patients were divided into two groups according to their miR-154 expression levels, using the median (0.467) of miR-154 expression in all 50 patients as a cut-off: high miR-154 expression group (n=24) and low miR-154 expression group (n=26). It was found that expression of miR-154 was significantly downregulated in HCC patients with poorly differentiated tumors (P<0.01), positive lymph node metastasis (P<0.01) and advanced TNM stage (P<0.01). No significant difference was observed between miR-154 expression and patient age and gender. These results suggest that miR-154 may be involve in the initiation and development of HCC.

To investigate the cellular function of miR-154 in HCC, HepG2 cells were transfected with the miR-154 mimic or miR-NC, and miR-154 expression was examined by qRT-PCR. The expression of miR-154 was markedly elevated in the miR-154 mimictransfected cells compared with the miR-NC-transfected cells (Fig. 2A). Cell proliferation and colony formation were then measured using MTT and colony formation assays. Our results showed that restoration of miR-154 led to a significant decrease in cell proliferation (Fig. 2B) and colony formation (Fig. 2C) of the HepG2 cells. As proliferation is directly linked to cell cycle distribution, the effect of miR-154 on cell cycle progression was analyzed. As expected, the percentage of S phase cells was reduced, while the percentage of G1 phase cells was increased in the HepG2 cells upon transfection with the miR-154 mimic (Fig. 2D). Next, we used flow cytometry to test the role of miR-154 in apoptosis. Our results showed that restoration of miR-22 significantly induced cell apoptosis in the HCC cells (Fig. 2E). Thus, restoration of miR-154 expression inhibited cell growth in HCC by inhibiting cell proliferation and inducing cell apoptosis.

As it has been shown that miR-154 expression is associated with lymph node metastasis in patients suffering from HCC, we aimed to ascertain whether miR-154 affects cell migration and invasion in HepG2 cells transfected with the miR-154 mimic or miR-NC by wound healing and Transwell chamber assays. Consistent with the clinical data, restoration of miR-154 significantly decreased migration and invasion capacities of the HepG2 cells (P<0.05; Fig. 3A and B).

Since EMT is closely related to cancer cell metastasis ability, we next examined EMT markers in HCC cells transfected with the miR-154 mimic or miR-NC by western blot analysis. We found that overexpression of miR-154 led to increased expression of E-cadherin and decreased expression of N-cadherin and vimentin (Fig. 3C), suggesting that miR-154 inhibits EMT.

To understand the molecular mechanism of miR-154 action in HCC, we searched for miR-154 targets by using TargetScan, miRanda, and miRWalk algorithms. Among the common predicted targets of miR-154, ZEB2 was selected since ZEB2 has been reported to play an important role in cell proliferation and invasion in various types of cancers including HCC (20). Using prediction tools, we predicted that ZEB2 may be a target of miR-154 (Fig. 4A). To further confirm that miR-154 directly targets ZEB2, luciferase reporter assays were performed. Our results showed that HepG2 cells transfected with miR-154 significantly decreased wild-type ZEB2-3′UTR reporter activity compared with the cells co-transfected with miR-NC (P<0.01), while miR-154 had no inhibitory effect on mutant ZEB2-3′UTR reporter activity (Fig. 4B). To further validate the association between miR-154 and ZEB2, we detected endogenous ZEB2 expression in the HepG2 cells transfected with the miR-154 mimic or miR-NC. Our results showed that the miR-154 mimic markedly decreased the expression levels of ZEB2 mRNA (Fig. 4C) and protein (Fig. 4D). Taken together, these data indicate that ZEB2 is a target of miR-154.

To determine whether the role of miR-154 in HCC is mediated by ZEB2, HepG2 cells were transfected with the pCDNA3.0-ZEB2 plasmid or the vector. Western blot assay confirmed that the pCDNA3.0-ZEB2 plasmid significantly increased ZEB2 expression in the HepG2 cells (Fig. 5A). HepG2 cells were co-transfected with miR-154 and pcDNA3-ZEB2, or the blank pCDNA3 vector, and then cell proliferation, migration and invasion were assessed at the indicated times. MTT assay showed that overexpression of ZEB2 markedly reversed the tumor-suppressive effect of miR-154 on cell proliferation (Fig. 5B). Wound-healing migration and invasion assays demonstrated that overexpression of ZEB2 reversed the inhibitory effects of miR-154 on HCC cell migration and invasion (Fig. 5C and D). These data suggest that miR-154 acts as a tumor suppressor in HCC by targeting ZEB2.

To evaluate the effect of miR-154 in HCC in vivo, we examined the potential effect of miR-154 on tumorigenesis using a HepG2 xenograft model. Tumors grew slower in the HepG2/miR-154 group compared with the rate in the HepG2/miR-NC group (Fig. 6A). A significant decrease in tumor size (Fig. 6B) and weight (Fig. 6C) was observed in the HepG2/miR-154 group compared to the HepG2/miR-NC group after the mice were sacrificed. Furthermore, we also determined ZEB2 expression in the tumor tissues by western blot analysis. ZEB2 expression was markedly decreased in the HepG2/miR-154 group compared to the level in the HepG2/miR-NC group (Fig. 6D). These data indicate that miR-154 suppresses tumor growth of HCC in vivo by targeting ZEB2.