Forty paired human HCC and adjacent non-tumoral liver tissues were obtained from the patients who underwent routine curative surgery at the Department of Thoracic Surgery, The First Hospital, Jilin University (Changchun, Jilin, China) between July, 2010 and September, 2014. None of the patients received radiotherapy or chemotherapy prior to surgery. During the surgical procedure, samples of malignant liver tissue and samples from adjacent non-tumoral liver tissue (>5 cm away from the tumor site, cirrhosis tissue was excluded) were taken. Tissue samples were immediately frozen in liquid nitrogen, and stored at −80°C until RNA extraction. Data on all the subjects were obtained from the medical records, pathology reports and personal interviews with the subjects. Data on all the subjects including age, gender, overall survival and HCC features such as tumor number, size and growth phase were collected from the medical records, pathology reports and personal interviews with the subjects. Informed consent was obtained from each patient prior to surgery. The study protocol and consent procedures were approved by the Ethics Committee of the Jilin University.

The human Huh-7 and HepG2 HCC cell lines, as well as the normal human HL-7702 hepatocyte cell line were purchased from the institute of Cell Biology of the Chinese Academy of Science (Shanghai, China), and originated from ATCC. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; HyClone, South Logan, UT, USA) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) at 37°C in a humidified chamber supplemented with 5% Co₂.

The cells were transiently transfected with 100 nmol/l of miR-494 mimics (miR-494), miR-494 inhibitors (anti-miR-494), and miRNA negative control (miR-NC), or 100 nmol/l small interfering RNA (siRNA) specific to phosphatase and tensin homolog (si-PTEN), and scrambled siRNA negative control (si-NC) (all from GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen Life Technologies, grand Island, NY, USA) according to the manufacturer’s instructions. Transfection efficiencies were evaluated in each experiment by RT-qPCR at 48 h post-transfection.

Total RNA containing miRNA and mRNA from tissue samples and cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed using a PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. The expression levels of miR-494 were quantified using a Taqman miRNA assay kit (Applied Biosystems, Foster City, CA, USA). The expression of phosphatase and tensin homolog (PTEN) mRNA was detected by quantitative PCR. The primers for PTEN mRNA were: 5′-ACCAGTGGCACTGTTGTTTCAC-3′ (sense), and 5′-TTCCTCTGGTCCTGGTATGAAG-3′ (anti-sense). Quantitative PCR was performed on an ABI 7900 Fast system (Applied Biosystems) using Real-Time PCR mixture Reagent (Takara) according to the manufacturer’s instructions. The relative expression levels of the interest gene were calculated by the 2^−ΔΔCt method. U6 and GAPDH were used as internal controls for miRNAs and mRNAs, respectively.

Cell viability was assessed using a Cell Counting kit-8 (CCK-8) assay (Dojindo, Japan), was performed. Briefly, 5×10³ cells/well were seeded in 96-well plates. The cells were transfected with miR-494 mimic, miR-494 inhibitor (anti-miR-494), or corresponding negative control (miR-NC), or siRNA-PTEN (si-PTEN) and scrambled negative control (si-NC), and cultured for 72 h. The proliferative activity was determined using a CCK-8 assay according to the manufacturer’s instructions. Absorbance was recorded at 450 nm using an enzyme-linked immunosorbent assay reader (Thermo Labsystems, Vantaa, Finland).

The migration and invasion assays were performed using Transwell insert chambers (Costar, Corning, NY, USA). For the migration assay, 1×10⁵ transfected cells were seeded in each well of the upper Transwell chamber in serum-free medium (8-μm pore size; Corning Costar Corp., Acton, MA, USA). DMEM supplemented with 20% FBS was added to the lower chamber. After incubating for 24 h at 37°C with 5% Co₂, cells migrating to the lower surface of filter were fixed in 70% ethanol for 30 min and stained with 2% crystal violet for 10 min on a glass slide. The number of cells penetrating across membrane was counted under a microscope in five random visual fields. For the invasion assay, 1×10⁵ transfected cells were seeded in upper chambers precoated with matrigel (BD Biosciences, San Jose, CA, USA) in serum-free medium in triplicate. The following steps were similar to the migration assay. The number of cells invading the Matrigel was counted under a microscope in 5 randomly selected fields.

Cell cycle and apoptosis were examined by flow cytometry. Briefly, two days post-transfection, the cells were collected for apoptosis and the cell cycle assay. For the cell cycle assay, the cells were collected and fixed in 70% ethanol at 4°C for 16 h and then stained with propidium iodide (PI; Sigma, St. Louis, USA) at 4°C for 30 min in the dark. Cell apoptosis assay was performed using an Annexin V/PI detection kit (KeyGene, Nanjing, China). The apoptotic rate and cycle distribution were measured by flow cytometry (BD Biosciences, Mansfield, MA, USA), and the data were analyzed using CellQuest software (Bd Biosciences San Jose, CA, USA).

The 3′-UTR of PTEN containing the PTEN miR-494 response element was cloned into the pGL3-control vector (Ambion, Austin, TX, USA) at the NheI and XhoI sites. A mutant 3′-UTR of PTEN was produced by PCR and cloned into the pGL3-control vector (Ambion) at the NheI and XhoI sites. HepG2 cells were seeded in a 24-well plate (5×10³ cells/well) and transiently transfected with wide PTEN-UTR-pgL3/mute-PTEN-UTR-pgL3, a Renilla luciferase control vector (20 ng) and miR-494 mimic or control-miR. After 48 h, luciferase activity was measured using a dual luciferase reporter assay system, according to the manufacturer’s instructions (Promega, Madison, WI, USA).

The relationship between miR-494 expression and cell sensitivity to sorafenib was examined. HepG2 cells were treated with miR-494 mimic, anti-miR-494 (miR-494 inhibitor) or miR-NC, and were divided into 96-well plates for 48 h. The cells were treated with indicated concentrations of sorafenib (10 μm; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Cell viability was assessed using the CCK-8 assay, and cell apoptosis was measured using the Annexin V/PI detection kit.

Tissue samples and cells were collected and homogenized with RIPA lysis buffer (Beyotime, Beijing, China). Whole extracts were prepared, and the protein concentration was detected using a bicinchoninic acid (BCA) protein assay kit (Boster, China). Equal amounts of protein lysates (30 μg each lane) were separated by 10% SDS-PAGE gel and then electrotransferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with TBST containing 5% non-fat dry milk for 2 h. The membranes were probed with mouse monoclonal antibodies specific to PTEN (1:400; Santa Cruz Biotechnology, inc.), and rabbit monoclonal antibodies of phospho-Akt (p-Akt) (Ser473) (1:500) and anti-Akt (1:500) (both from Cell Signaling, Boston, MA, USA), anti-PI3K (1:500), and anti-GAPDH (1:1,000) (both from Santa Cruz Biotechnology, inc.) which was used as an internal control for protein loading. The membrane was further probed with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000) or goat anti-rabbit IgG (1:5,000) (both from Santa Cruz Biotechnology, inc.) for 2 h at room temperature. Proteins were visualized with an ECL-chemiluminescent kit (ECL-Plus; Thermo Scientific, Rockford, lL, USA). The integrated density of the band was quantified by Quantity one software (Bio-Rad, Hercules, CA, USA).

Data are presented as mean ± standard deviation (SD) of at least three independent experiments of three samples. Statistical analysis between two samples was performed using the Student’s t-test, while for more than two groups one-way ANOVA was used followed by a Tukey’s post hoc test. The relationship between the miR-494 expression level and clinical and pathological variables was analyzed using the Pearson’s χ² test. The Graphpad Prism 6.0 software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. P<0.05 was considered to indicate a statistically significant result.

To determine the expression levels of miR-494 in HCC, RT-qPCR detection was performed in 40 pairs of HCC (T) and adjacent non-tumoral (ANT) tissues. As revealed by RT-qPCR analysis, the expression level of miR-494 was significantly upregulated in tumoral tissues compared to that in the matched non-tumoral tissues (Fig. 1A). We also examined the expression levels of miR-494 in human Huh-7 and HepG2 HCC cell lines and the normal human HL-7702 hepatocyte cell line. It was found that the miR-494 expression level was substantially upregulated according to the RT-qPCR analysis in Huh-7 and HepG2 cells compared to the HL-7702 cells. The median expression level of the miR-494 level (0.87) was used as a cut-off point to divide all 40 patients into two groups: patients who expressed miR-494 at levels less than the median value were assigned to the low expression group (n=19), while those with a miR-494 expression higher than the median value were assigned to the high expression group (n=21). The association between miR-494 expression and the clinicopathological characteristics of the patients, including age, gender, tumor size, TNM stage, differentiation and lymph node metastasis were investigated. As shown in Table I, a high miR-494 expression positively correlated with tumor differentiation (P<0.01), TNM stage (P<0.01) and lymph node metastasis (P<0.01). There was no correlation between miR-494 expression, and age and gender. These data suggested that miR-494 is involved in HCC initiation and progression.

To analyze the function of miR-494 on HCC, we transfected miR-494 mimic or anti-miR-494 into HepG2 cells, and cell proliferation, cell cycle, apoptosis, migration and invasion assays were performed. The RT-qPCR analysis confirmed that miR-494-transfected mimics caused the upregulation of miR-494 expression, while the miR-494-transfected inhibitor resulted in down-regulation of the miR-494 expression (Fig. 2A). The functional analysis demonstrated that the downregulation of miR-494 by anti-miR-494 inhibited the proliferation (Fig. 2B), migration (Fig. 2C) and invasion (Fig. 2D), and induced cell cycle arrest at G1 stage (Fig. 2E) and apoptosis (Fig. 2F) in HepG2 cells. By contrast the overexpression of miR-494 by miR-494 mimic increased the proliferation (Fig. 2B), migration (Fig. 2C) and invasion (Fig. 2D), and suppressed cell cyle arrest at G0/G1 stage (Fig. 2E) and apoptosis (Fig. 2F) in HepG2 cells.

PTEN has been identified as a direct target of miR-494 in several types of cancer (22,23). However, the association between miR-494 and PTEN in HCC remains to be clarified. To verify whether PTEN is a direct target of miR-494 in HCC, a fragment of the 3-UTR of PTEN containing the conserved putative miR-494 target site was fused to a lucif erase reporter vector, and co-transfected with a control Renilla luciferase reporter construct into HepG2 cells together with miR-494 mimic or control miRNA. It was found that co-transfection with miR-494 mimic and pGL3-PTEN plasmid showed a significant reduction of reporter activity in comparison with cells co-transfected with the control miRNA and pgL3-PTEN plasmid (Fig. 3A). By contrast, the cells co-transfected with anti-miR-494 and pGL3-PTEN-mut plasmid showed no significant difference in reporter activity as compared with cells co-transfected with control miRNA and pGL3-PTEN-mut plasmid (Fig. 3A). To determine whether miR-494 expression suppressed endogenous PTEN expression, synthetic miR-494 mimic, anti-miR-494 or miR-NC were transfected into HCC cells. RT-qPCR and western blot analysis revealed that the overexpression of miR-494 significantly inhibited PTEN expression at the mRNA (Fig. 3B) and protein (Fig. 3C) levels in HepG2 cells, while the underexpression of miR-494 markedly increased PTEN expression at the mRNA (Fig. 3B) and protein (Fig. 3C) levels in Hepg2 cells.

In addition, we determined PTEN protein expression in HCC tissue and cell lines by western blotting. The results of western blotting showed that PTEN protein expression was obviously upregulated in HCC tissue (Fig. 3D) and HCC cell lines (Fig. 3E) as compared to the adjacent non-tumoral tissue and normal human hepatocyte cell line. These results indicated that PTEN is also a miR-494 target in HCC.

To assess whether downregulation of PTEN affect HCC cell proliferation, migration and invasion in miR-494 depletion cells, we inhibited PTEN expression with siRNA in miR-494 depletion cells. The RT-qPCR analysis confirmed that transfected si-PTEN significantly inhibited PTEN expression in miR-494 depletion cells (Fig. 4A). Our results showed that the downregulation of PTEN significantly increased the proliferation (Fig. 4B), migration (Fig. 4C) and invasion (Fig. 4D), and suppressed cell cycle arrest at G1 stage (Fig. 4E) and apoptosis (Fig. 4F) compared to si-NC in miR-494-depleted cells, further suggesting that miR-494 affects the proliferation, migration, invasion, cell cycle arrest and apoptosis of HCC cells through the PTEN-mediated signaling pathway.

Sorafenib is the only oral multi-kinase inhibitor recently approved by the FDA with demonstrated efficacy in enhancing the overall survival of advanced HCC. It is known that some miRNAs can improve or increase the sensitivity of cancer cells to conventional drugs and chemotherapeutic agents. For this reason, we examined whether miR-494 increased the effect of sorafenib on HCC cells. For this purpose, HepG2 cells were transfected with miR-494 mimic or anti-miR-494, and then treated with sorafenib (10 μM), cell viability and apoptosis were determined. The CCK-8 assay showed that sorafenib treatment decreased the viability of HepG2 cells, and HepG2 cells transfected with miR-494 mimic were more resistant to sorafenib treatment than cells transfected with miR-NC (Fig. 5A, P<0.05). By contrast, the cells transfected with anti-miR-494 were more susceptible to the sorafenib treatment than cells transfected with miR-NC (Fig. 5A, P<0.05). In addition, we assessed the effect of expressing miR-494 with the sorafenib on cell apoptosis. The Annexin V assay revealed that the combination of miR-494 mimic with sorafenib decreases HepG2 cell apoptosis compared to sorafenib alone (P<0.05, Fig. 5B), whereas the combination of anti-miR-494 with sorafenib induced cell apoptosis compared to sorafenib alone (P<0.05, Fig. 5B). These findings suggested that elevated miR-494 enhanced HCC cell resistance to sorafenib.

To determine whether miR-494 promotes the proliferation, migration and invasion of HCC cells via PTEN inhibition, the downstream genes of the PTEN signaling pathway, phosphatidylinositide 3-kinases (PTEN/PI3K)/AKT, were analyzed in miR-494 mimic and anti-miR-494-transfected HepG2 cells. As shown in Fig. 6, after the knockdown of miR-494 in the HepG2 cell line, PTEN gene expression levels increased significantly, and the downstream expression of PI3K and phosphate-AKT (p-Akt) was reduced, while the reverse correlation was found in miR-494 mimic-transfected Hepg2 cells. These findings suggested that miR-494 promotes cell proliferation, migration and invasion via activation of the PTEN/PI3K/AKT signaling pathway.