Human hepatocellular carcinoma (HCC) and matched adjacent non-tumor tissues were collected from 15 patients at the Department of Oncology, The First People's Hospital of Nantong. The present study was approved by the Institute Research Ethics Committee of the First People's Hospital of Nantong. Human HCC cell lines HepG2, Huh7 and normal control liver cell line LO2, as well as human embryonic kidney 293 (HEK293) cells were all obtained from Biomics Biotechnologies Co., Ltd. (Nantong, China), cultured in Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO₂.

miR-139 mimics were used for upregulation of endogenous miR-139, and a miR-139 sequence-scrambled RNA was used as a negative control miRNA (NC_miR). Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) was used for transfection of miRNAs into cells according to the manufacturer's instructions. miR-139 mimics and NC_miR were obtained from Biomics Biotechnologies Co., Ltd. (Nantong, China).

The 3 untranslated region (3′UTR) region complementary DNA sequence of human FGF18 mRNA (NCBI Reference Sequence: NM_003862.2) was amplified by polymerase chain reaction (PCR) and constructed into the pGL3-vector (Promega, Madison, WI, USA) as a dual-luciferase miRNA target expression vector (wild-type FGF18 3′UTR, wtFGF18-3′UTR) to evaluate miR-139 activity in cells. The FGF18 3′UTR mutant vector was also constructed as the negative control (mutant FGF18 3′UTR, mFGF18-3′UTR). Briefly, HEK293 cells were seeded into a 24-well plate and grown for 24 h. Then wtFGF18-3′UTR or mFGF18-3′UTR was co-tansfected with miR-139 mimics or NC_miR using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.); pRL-TK (Promega) was co-transfected as an internal control. After transfection for 48 h, cells were collected and lysed, firefly and Renilla luciferase activities in cell lysates were detected using the DLR assay system (Promega) according to the manufacturer's instructions.

After cells were treated with miRNAs as aforementioned, the total RNA of cells was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and small RNA enriched with miRNAs was isolated using mirPremier^® microRNA isolation kit (Sigma-Aldrich, St. Louis MO, USA) according to the manufacturers instructions. Detection of miRNA expression levels in tissues or cells was performed by stem-loop RT-qPCR (21). U6 small RNA or GAPDH was used as an internal control for miRNA or mRNA detection, respectively. RT-qPCR reactions were carried out using the SYBR-Green One-Step RT-qPCR kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The relative miRNA or mRNA expression levels were evaluated by 2^−ΔΔCt method (22). The primer sequences were FGF18 forward, 5′-ACCTTCGGTAGTCAAGTC-3′ and reverse, 5′-CGTGTAGTTGTTCTCCAG-3′; GAPDH forward, 5′-GAGTCCACTGGCGTCTTC-3′ and reverse, 5′-GATGATCTTGAGGCTGTTGTC-3′.

Cells were plated in a 6-well plate, after treatment for 48 h as aforementioned. The cells were collected and lysed in a cell RIPA lysis and extraction buffer (Thermo Fisher Scientific, Inc.) on ice. Centrifugation to collect the proteins, then separation by polyacrylamide gel electrophoresis (PAGE) and transfer to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA)followed. Subsequently, the membrane was incubated with a primary antibody rabbit anti-human FGF18 (1:1,000 dilution) or a mouse anti-human β-actin antibody (1:5,000 dilution) (all from Abcam, Cambridge, MA, USA) as an internal control. After being washed with Tris-buffered saline and Tween-200 (TBST) the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution) (Abcam) for 1.5 h at room temperature, and then washed in TBST. The specific proteins were detected with ECL substrate (Thermo Fisher Scientific, Inc.).

The proliferation abilities of cells were assessed using Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich). Briefly, 5×10³ cells/well were seeded on a 96-well plate before treatment and grown to ~70% confluence for 24 h. Post-treatment at 0, 24, 48, 72 and 96 h, the cells were washed with phosphate-buffered saline (PBS) three times, and replaced with 100 µl/well of PBS, and then 10 µl of FCCK working solution was added to each well. After incubation at 37°C in an incubator for 30 min, the fluorescence intensity of each well was assessed at 535 nm using a fluorescence microplate reader (BioTek, Winooski, VT, USA).

Cell apoptosis was determined by flow cytometric (FCM) analysis using Annexin V-FITC/propidium iodide (PI) double staining (Sigma-Aldrich). The cells were seeded on a 6-well plate for 24 h of growth and treated as aforementioned for 48 h. Briefly, 1×10⁵ cells/well were harvested and washed with PBS, and then re-suspended in Annexin-binding buffer, followed by incubation with FITC-conjugated Annexin V and propidium iodide (PI) for 15 min at room temperature. Finally, the cells were analyzed by FCM (BD Biosciences, Franklin Lakes, NJ, USA).

A cell wound scratch assay was used to determine the migration abilities of HCC cells. Briefly, 1×10⁴ cells/well were plated on a 12-well plate for 24 h of growth and treated as aforementioned. Cell wounds were made to confluent monolayer cells with a pipette tip post 48-h treatment and the cells were washed with DMEM twice. Cell migration was monitored at 0 and 48 h after the scratch.

The invasion abilities of cells were detected by Transwell assay. Cells/well (2×10⁵) were seeded into a 24-well plate for 24 h and treated for 48 h, then suspended in DMEM at a density of 1×10⁶ cells/ml. Transwell chambers (Corning, NY, USA) were incubated with DMEM for 1 h before treatments; the upper and lower chamber was separated by an 8-µm pore polycarbonate membrane which was coated with 50 µl of 0.5 mg/ml Matrigel (BD Biosciences). A cell suspension of 100 µl was added into each upper chamber with 600 µl DMEM containing 10% FBS or conditioned medium which was the cell supernatant with treatment as aforementioned for 48 h. The cells on the top surface of the membrane were carefully removed using cotton swabs post-treatment for 24 h. The cells on the Transwell chambers were fixed in 10% formaldehyde for 30 sec, and then stained with 0.5% crystal violet solution. After being washed with PBS, the cells on the top surface of the membrane were carefully removed again, and the cells on the bottom surface of the membrane were observed and counted under a microscope.

An in vitro angiogenesis model was used to evaluate the inhibitory effects of miRNAs on angiogenesis. Briefly, a 24-well plate was coated with 100 µl/well Matrigel (BD Biosciences) and incubated at 37°C for 30 min. Human umbilical vein endothelial cells (HUVECs) at a concentration of 1×10⁵ cells/well were re-suspended in conditioned medium (which was the supernatant of the cells treated with miRNAs for 48 h), then seeded on a Matrigel coated 24-well plate and cultured in a 37°C/5% CO₂ incubator for 24 h. The number of branching points was counted under a microscope.

All the experiments were performed independently three times. The data are shown as the mean values ± standard deviation (SD). Statistical analyses were performed using SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA), and the results were analyzed using one way ANOVA followed by post hoc test to assess statistical significance. All P-values were based on a two-side statistical analysis and P<0.05 was considered to indicate statistical significance.

The expression of FGF18 was detected by RT-qPCR and western blot assay in 15 cases of HCC tissues and matched adjacent normal tissues, as well as in HCC cell lines HepG2 and Huh7. Compared with normal tissues, the results revealed that FGF18 mRNA and protein levels were highly expressed in HCC patients (P=0.0131) (Fig. 1A and B). Moreover, the mRNA and protein levels of FGF18 were also highly expressed in HCC cell lines HepG2 and Huh7, compared with normal liver cell LO2 (P<0.05) (Fig. 1C and D).

The expression of miR-139 was detected by RT-qPCR both in HCC tissues and cell lines. The results revealed that, miR-139 was significantly decreased in HCC tissues (P=0.0091) (Fig. 2A), compared with normal tissues, it was also lowly expressed in both HepG2 and Huh7 cells, compared with that in LO2 cells (P<0.05) (Fig. 2B).

The target site of miR-139 with the position 207 of the FGF18 mRNA sequence was predicated by web online software (http://www.targetscan.org), and then the dual-luciferase reporter (DLR) assay was used to observe the site binding and inhibition effects. The reporter vectors of the wild-type wtFGF18-3′UTR and mutant-type mFGF18-3′UTR were constructed (Fig. 3A). The result of the DLR assay revealed that co-tansfection of HEK293 cells with miR-139 mimics and wtFGF18-3′UTR, resulted in a marked decrease of luciferase activities, compared with NC_miR transfected with wtFGF18-3′UTR or mFGF18-3′UTR, and miR-139 transfected with mFGF18-3′UTR (Fig. 3B).

The inhibitory effect of miR-139 on the expression of FGF18 was detected by western blot assay. Compared with NC_miR-treated cells, the results revealed that the FGF18 protein levels were suppressed by miR-139 in both HCC cells HepG2 and Huh7 (P<0.05) (Fig. 4A and B).

To investigate the inhibition effect of miR-139 on the growth of HCC cells, a CCK8 assay was performed. The results revealed that the growth of HCC cell lines, HepG2 and Huh7, were both inhibited by miR-139 at 48 and 72 h, compared with the NC_miR-treated cells (P<0.05) (Fig. 5).

The inhibitory effect of miR-139 on the apoptosis of HCC cells was determined by flow cytometric (FCM) analysis after Annexin V-FITC/PI double staining. The results revealed that the apoptosis of HCC cell lines, HepG2 and Huh7, were both inhibited by miR-139 when compared with the NC_miR-treated cells (P<0.05) (Fig. 6).

To observe the inhibitory effect of invasion and migration abilities, Transwell and cell wound scratch assays were separately used. The result of the Transwell assay revealed that the invasion abilities of HCC cell lines, HepG2 and Huh7, were decreased significantly after miR-139 treatment, compared with the NC_miR-treated cells (P<0.05) (Fig. 7A). Similarly, the result of the cell wound scratch assay revealed that the migratory abilities of HCC cell lines, HepG2 and Huh7, were also markedly decreased post-miR-139 treatment, compared with the NC_miR-treated cells (P<0.05) (Fig. 7B).

Tube formation was evaluated in a HUVEC angiogenesis model. HUVECs stimulated by the conditioned medium derived from HCC cells were transfected with miR-139 mimics and NC_miR. HUVECs treated with the conditioned medium of miR-139 mimics were significantly inhibited to form extensive and enclosed tube networks and when compared with the untreated cells, the number of branching points was less than the NC_miR-treated cells (P<0.05) (Fig. 7C).