Patients (84 HCC tissues and pair-matched adjacent normal liver tissues) who had not undergone radiofrequency ablation or chemoembolization in the present study were enrolled at The First Affiliated Hospital of Xi'an Medical University. Samples were pathologically confirmed and rapidly put into liquid nitrogen after surgical operation. Informed consent was signed by each patient before the clinical specimens were collected and used. Details of the clinicopathological data are shown in Table I. The protocols involved in the use of the clinical specimens in the present study were according to the Research Ethics Committee of Xi'an Medical University.

Human HCC cell lines including Hep3B, Huh7, MHCC97H and HCCLM3, and the human immortalized normal hepatocyte cell line (LO2) (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) were cultured under standard conditions. hsa-miR-196b mimics (HmiR0103-MR04), inhibitors (HmiR-AN0286-AM04) and their control fragments (NC, CmiR0001-MR04 and CmiR-AN0001-AM04) were produced by GeneCopoeia (Guangzhou, China). FOXP2 siRNA and FOXP2 expression plasmid (pcDNA3.1-FOXP2) were designed and synthesized by GenePharma (Shanghai, China). All vectors were then transfected into HCC cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers protocol.

Paraffin sections from HCC tissues underwent deparaffination and then rehydration. Antigen retrieval, suppression of endogenous peroxidase activity and 10% skim milk blocking were performed before primary antibody incubation. FOXP2 primary antibody (; ab16046; Abcam, Cambridge, MA, USA) was used for incubation overnight at 4°C. The slides were subsequently incubated with peroxidase conjugated secondary antibody (ZSGB BIO, Beijing, China) for 90 min, and a peroxidase-labeled polymer, DAB solution was used for signal development for 5 min. The sections were counterstained with hematoxylin followed by dehydrating and mounting. Staining intensity was scored as no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3. Staining quantity was graded as <25%, 1; 25–75%, 2; and >75%, 3. IHC score was manually confirmed by two independent experienced pathologists using the formula: IHC score = staining intensity × staining quantity.

RNA was extracted and prepared for qRT-PCR as previously described (13). Total RNA was reverse-transcribed to cDNA with PrimeScript Reverse Transcriptase kit (Takara, Dalian, China) according to the manufacturer's protocol. By using SYBR Green chemistry, qRT-PCR was implemented with the ABI 7900HT sequence detection machine (Bio-Rad, Hercules, CA, USA). The target and reference genes (U6 and GAPDH) were amplified in separate wells in triplicate. Gene expression was calculated with the comparative threshold cycle (2^−ΔΔCt) approach. The primers used for miR-196b, U6, FOXP2 and GAPDH were synthesized and purchased from Sangon Biotech (Shanghai, China).

Transwell chambers (Corning Costar, Cambridge, MA, USA) were employed to evaluate the migratory and invasive abilities of HCC cells. HCC cells were resuspended in serum-free DMEM and subsequently seeded in the upper chambers. To induce the migration and invasion of HCC cells, the lower chambers were filled with 600 µl DMEM supplemented with 20% FBS. Forty-eight hours after cell seeding, HCC cells that migrated or invaded through the membranes (the membranes were covered with 70 µl Matrigel) were stained with crystal violet for cell counting under a microscope.

Total cell lysates were prepared in a 1X sodium dodecyl sulfate buffer and quantified with a BCA protein assay kit (Pierce, Bonn, Germany). Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Bio-Rad, USA). After incubation with the antibody specific for FOXP2 (ab16046, Abcam) overnight, the blots were incubated with secondary antibodies (#7074 and #7076; Cell Signaling Technology, Beverly, MA, USA) and detected using a chemiluminescent detection system (Bio-Rad). GAPDH (sc-47724; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control for western blots. The immunoreactive bands were quantified by the densitometry with ImageJ software (NIH, USA).

BALB/c nude mice aged 4 weeks were subjected to a pulmonary metastasis model. HCCLM3 cells that were transfected with the miR-196b inhibitor or NC inhibitor were injected through the tail vein of nude mice and cultivated for 9 weeks. After euthanasia, the lungs were harvested and fixed, paraffin-embedded, sectioned and stained for hematoxylin and eosin (H&E) (14), and the metastatic nodules were counted. All animal experiments were approved by the Ethics Committee of Xi'an Medical University.

The sequences of FOXP2-3′UTR were cloned into the pmiR-RB-Report™ vector (RiboBio, Guangzhou, China), and its corresponding mutant (mt) 3′UTR sequences were subsequently generated using overlap extension PCR and cloned into the pmiR-RB-Report™ vector. Pmir-RB-FOXP2 or pmir-RB-FOXP2-mt was transfected into Hep3B cells with miR-196b mimic or NC mimic by Lipofectamine-mediated gene transfer. The relative luciferase activity was normalized to Renilla luciferase activity 48 h after transfection.

Data are presented as mean ± SD and analyzed by GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). Chi-squared test, Student's t-test, ANOVA, Spearman correlation analysis, Kaplan-Meier method and log-rank test were performed for statistical analysis. A P-value <0.05 was considered statistically significant. *P<0.05.

A previous study reported that miR-196b expression was upregulated in HCC tissues compared to non-tumor tissues (11). Consistently, the qRT-PCR analysis revealed that miR-196b expression was markedly increased in HCC tissues compared with that noted in the adjacent non-cancerous tissues from 84 patients in the present study (P<0.05, Fig. 1A). In accordance, we found that miR-196b was also significantly upregulated in 4 HCC cell lines compared with that noted in the normal hepatic cell line LO2 (P<0.05, Fig. 1B). These findings provide valid evidence that miR-196b could be implicated in the pathogenesis and development of HCC.

Different subgroups (miR-196b low/high expression) were plotted according to the cut-off values of miR-196b, which were defined as the median of the cohort. Relationship between the clinical characteristics of the HCC patients and miR-196b expression are listed in Table I. High miR-196b expression was associated with venous infiltration (P=0.023) and advanced TNM tumor stage (P=0.001). Furthermore, survival analyses indicated that miR-196b high expressing HCC patients showed a significant reduced 5-year overall survival and disease-free survival (P<0.05, respectively, Fig. 2). Thus, we suggest that miR-196b is a possible prognostic biomarker for HCC patients.

To detect the effects of miR-196b on migration and invasion of HCC cells, Transwell assays were conducted when miR-196b expression was downregulated or upregulated. HCCLM3 cells showed the highest level of miR-196b, while Hep3B cells showed the lowest level of miR-196b. Thus, HCCLM3 and Hep3B were used for loss- and gain-of-function experiments, respectively. miR-196b was obviously knocked down by miR-196b inhibitor in HCCLM3 cells (P<0.05, Fig. 3A). Transwell assays revealed that miR-196b knockdown significantly weakened the migratory and invasive abilities of HCCLM3 cells (P<0.05, respectively, Fig. 3B). In turn, overexpression of miR-196b was confirmed by qRT-PCR after miR-196b mimic transfection in Hep3B cells (P<0.05, Fig. 3C). Our data showed that upregulation of miR-196b significantly facilitated migration and invasion of Hep3B cells compared with the NC group (P<0.05, respectively, Fig. 3D). Based on the findings in vitro, we further probed the role of miR-196b during HCC progression with xenograft models. A notable decrease in metastatic nodes in the lung was noted in the miR-196b inhibitor group when compared with the NC inhibitor groups (P<0.05, Fig. 4). Thus, miR-196b exerts a pro-metastatic role in HCC.

In order to explore the molecular mechanisms underlying miR-196b, first, bioinformatic prediction (TargetScan: http://www.targetscan.org and miRanda: http://www.microrna.org/microrna/hpme.do) showed that miR-196b could bind to the 3′UTR of FOXP2 mRNA (Fig. 5A). qRT-PCR and immunoblotting results indicated that miR-196b overexpression reduced the levels of FOXP2 mRNA and protein in Hep3B cells (P<0.05, respectively, Fig. 5B and C). Then, to further confirm the binding of miR-196b and 3′UTR of FOXP2, a dual-luciferase reporter assay was implemented. Co-transfection of Hep3B cells with Pmir-RB-FOXP2 vector and miR-196b mimic significantly reduced luciferase reporter activity compared with the negative control (P<0.05, Fig. 5D). This repressive effect was abolished by directed mutagenesis of the miR-196b-binding seed region in 3′UTR of FOXP2 (Fig. 5D), which initially suggested that the miR-196b and 3′UTR of FOXP2 may combine with each other. A recent study found that FOXP2 was prominently reduced in HCC tissues and inhibited migration and invasion of cancer cells (15). Data from the GEO database (GSE45436) demonstrated that the expression of FOXP2 in HCC tissues was notably downregulated compared to that noted in normal liver tissues (P<0.0001, Fig. 5E). Moreover, IHC data revealed that the expression of FOXP2 in miR-196b low-expressing HCCs was significantly higher than that in high-expressing cases (P<0.05, Fig. 5F). Subsequently, a significant negative correlation between miR-196b and FOXP2 expression was observed in HCC tissues (r=−0.7051, P<0.001, Fig. 5G). Together, these data demonstrated that FOXP2 is a direct target of miR-196b in HCC.

Next, we tested whether FOXP2 was able to mediate the function of miR-196b in HCC cells. Co-transfection of miR-196b inhibitor + FOXP2 siRNA was able to abrogate the miR-196b silencing-induced upregulated expression of FOXP2 (P<0.05, Fig. 6A). Furthermore, FOXP2 knockdown enhanced the migration and invasion of HCCLM3 cells with miR-196b silencing (P<0.05, respectively, Fig. 6B). In accordance, co-transfection of miR-196b + FOXP2 abrogated the miR-196b-induced downregulation of FOXP2 expression and metastasis of Hep3B cells (P<0.05, respectively, Fig. 7). Therefore, these results indicate that FOXP2 may function in miR-196b-induced HCC metastasis.