Written informed consent was obtained from each patient recruited for the present study for the use of materials. The consent procedures and all experimental protocols were approved by the Medical Institutional Ethical Committee of Shandong Provincial Hospital Affiliated to Shandong University (Jinan, China; approval no. 2017-231), according to the Declaration of Helsinki.

A total of 73 patients with HCC were recruited between February 2012 and January 2013 at Shandong Provincial Hospital Affiliated to Shandong University. The inclusion criteria were as follows: i) Patients with pathologically confirmed HCC; ii) patients who underwent curative surgical resection; and iii) patients >18 years old. The exclusion criteria were as follows: i) Patients who received preoperative chemotherapy or radiotherapy; and ii) patients with two or more primary tumors, asynchronously or synchronously. For each patient, paired HCC tissues and adjacent non-tumor tissues (as a control) were fresh-frozen in liquid nitrogen immediately following surgical resection and stored at −80°C. Patients with HCC were divided into metastasis (n=24) and non-metastasis (n=49) groups, and complete capsule (n=45) and incomplete capsule (n=28) groups, according to their clinicopathological features.

The human immortalized normal hepatic cell line LO2 and HCC cell lines (Huh7, SMMC-7721 and Bel-7402) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The Bel-7402 cell line was derived from a surgical specimen obtained in 1974 from a 53-year-old male patient with HCC and a positive serum α-fetoprotein status (21). The Huh7 cell line is a well-differentiated hepatocyte-derived cellular carcinoma cell line, originally obtained from a liver tumor from a 57-year-old Japanese male in 1982 (22). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin G (100 U/ml) and streptomycin (100 µg/ml), at 37°C in a humidified atmosphere containing 5% CO₂. The PPARγ antagonist GW9662 (10 µM) and the PPARγ agonist pioglitazone (10 μM) (MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) were dissolved in dimethyl sulfoxide (DMSO; as a vehicle) and added to the cell culture medium of Huh7 and Bel-7402 cells, respectively. Following lncRNA Ftx overexpression, Huh7 cells were treated with GW9662 or vehicle for 24 h, whereas following lncRNA Ftx downregulation, Bel-7402 cells were treated with pioglitazone or vehicle for 24 h.

Transfections of lentivirus (LV)-Ftx and its negative control LV-CON220 (Ubi-MCS-SV40-EGFP-IRES-puromycin) and LV-Ftx-RNA interference (RNAi) and its negative control LV-CON077 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) were performed using a lentivirus (Shanghai GeneChem Co., Ltd., Shanghai, China), according to the manufacturer's protocol. To obtain cell lines stably expressing lncRNA Ftx, Huh7 cells were transfected with the LV-Ftx, polybrene and enhanced infection solution (Shanghai GeneChem Co., Ltd.) and selected with puromycin (2 μg/ml) for 24 h. LV-CON220 was used as a control. To produce cell lines with stably interfered expression of lncRNA Ftx, Bel-7402 cells were transfected with LV-Ftx-RNAi, polybrene and enhanced infection solution (Shanghai GeneChem Co., Ltd.) and selected with puromycin (2 μg/ml) for 24 h. LV-CON077 was used as a control. The stably overexpressing or interfered cell lines were validated by RT-qPCR. Huh7 clones with ~12 times increased lncRNA Ftx expression levels compared with the normal control were chosen as Ftx, and Bel-7402 clones with ~74.68% decreased lncRNA Ftx expression levels compared with the normal control were chosen as short hairpin (sh)-Ftx. Their negative controls were termed Ftx-NC and sh-NC, respectively.

The transfected Bel-7402 and Huh7 cells were seeded onto 96-well plates (Corning Incorporated, Corning, NY, USA) at densities of ~3,000 and 5,000 cells/well, respectively. Following an overnight incubation, 10 μl Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) solution was added to each well and incubated at 37°C for 3 h. Subsequently, the optical density values were measured at 450 nm using a Multiskan Go spectrophotometer (Thermo Fisher Scientific, Inc.).

Cell invasion and migration were assayed using Transwell chambers (Corning Incorporated) with and without Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), respectively. For the invasion assay, each Transwell chamber was coated with 60 μl Matrigel and placed into a 24-well plate. Following lentiviral transfection, Bel-7402 cells and Huh7 cells (1×10⁵) were seeded into each chamber in serum-free medium, and the lower chambers were loaded with DMEM supplemented with 10% FBS. A total of 48 h subsequently, non-migrated cells in the upper chambers were removed with cotton swabs. For the migration assay, HCC cells (5×10⁴) were seeded in the upper chambers in serum-free medium without a Matrigel membrane, and the lower chambers were loaded with DMEM supplemented with 10% FBS. A total of 36 h subsequently, HCC cells in the upper chambers that had not migrated were removed with cotton swabs, and the migrated cells were fixed in 100% methanol at room temperature for 30 min. The cells on the bottom surface of the membrane were stained with hematoxylin (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) at room temperature for 20 min. Cell images were obtained in high-power (x400 magnification) fields using a phase-contrast microscope (Leica DM4000B; Leica Microsystems, GmbH, Wetzlar, Germany).

Total RNA was isolated from cultured Huh7 cells or Bel-7402 cells, or frozen tissues, using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 1 μg RNA was reverse-transcribed into cDNA with an RT reagent kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. The conditions were as follows: 37°C for 15 min, 85°C for 5 sec and 4°C for 10 min. The amplification was detected using a SYBR Premix Ex Taq kit (Takara Bio, Inc.) and a LightCycler^® 480 Real-Time PCR system (Roche Diagnostics, Indianapolis, IN, USA). The thermocycling conditions were as follows: Pre-incubation at 95°C for 5 min; 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec, and extension at 72°C for 10 sec; a melting cycle at 95°C for 5 sec, 65°C for 1 min, and 97°C with continuous per 5°C acquisition of fluorescence; and finally cooling at 40°C for 30 sec. The primer sequences are listed in Table I. The relative gene expression values are presented according to the 2^−ΔΔCq method (23), relative to β-actin.

Total protein was extracted from tumor cells with Radioimmunoprecipitation Assay Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China) and phenylmethanesulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), and the concentrations were determined with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 40 μg protein was separated by 10 or 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes, following blocking with 10% skimmed milk at room temperature for 1 h, were probed with the following primary antibodies at 4°C overnight: Rabbit anti-PPARγ (1:1,000; cat. no. ab191407; Abcam, Cambridge, UK), rabbit anti-CS (1:1,000; cat. no. 16131-1-AP), rabbit anti-PFKL (1:1,000; cat. no. 15652-1-AP), mouse anti-TNFα (1:1,000; cat. no. 60291-1-lg) (Wuhan Sanying Biotechnology, Wuhan, China), rabbit anti-OGDH (1:250; cat. no. bs-17710R; BIOSS, Beijing, China), rabbit anti-IDH1 (1:500; cat. no. PB0632), rabbit anti-GLUT1 (1:500; cat. no. PB0439), rabbit anti-PDK1 (1:200; cat. no. BA4499), rabbit anti-leptin (1:100; cat. no. BA1231) and rabbit anti-GLUT4 (1:500; cat. no. PB0143) (Wuhan Boster Biological Technology, Ltd., Wuhan, China). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (cat. no. ZB-2301 or ZB-2305, respectively; OriGene Technologies, Inc., Beijing, China) at a dilution of 1:8,000 at room temperature for 1 h, followed by enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA). The protein bands were visualized using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). Total protein levels were normalized to tubulin-β (1:1,000; cat. no. BM1453; Wuhan Boster Biological Technology, Ltd.) expression on the same membrane, and the bands were quantified using ImageJ k 1.45 software (National Institutes of Health, Bethesda, MD, USA).

Bioinformatics analysis of predicted lncRNA targets was performed using the nucleotide BLASTn program (blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch).

The data were obtained from at least three independent experiments and are presented as the mean ± standard error of the mean (unless otherwise stated). Pearson's correlation (r) was utilized to measure correlations and logarithmic regression was used to derive the equation of the slope. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 22.0 (IBM Corporation, Armonk, NY, USA). The significance of differences was evaluated by Student's t-tests (two-tailed) for two-group comparisons, and the one-way analysis of variance and the Bonferroni post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To examine the expression of lncRNA Ftx, RT-qPCR was performed to analyze 73 HCC and adjacent non-tumorous liver samples. Compared with the non-tumorous control tissues, lncRNA Ftx expression was markedly higher in the HCC tissues (Fig. 2A). The clinicopathological characteristics, including metastasis, tumor capsule, histological grade and tumor size, are summarized in Table II.

In addition, to determine the potential role of lncRNA Ftx in HCC, correlations between the expression status of lncRNA Ftx and important clinical features associated with tumor progression and disease prognosis were analyzed. Although there was no significant association between lncRNA Ftx expression and age, sex, tumor size or histological grade, high lncRNA Ftx expression levels were positively associated with incomplete capsules and HCC metastasis (Fig. 2A).

Taken together, the results indicated that the upregulation of lncRNA Ftx expression is associated with poor prognosis-associated clinicopathological features, suggesting that lncRNA Ftx is involved in HCC tumorigenesis.

To choose suitable cell lines for functional research, lncRNA Ftx expression was screened in a panel of HCC cell lines and a non-neoplastic hepatic cell line (LO2). It was identified that the expression levels of lncRNA Ftx were significantly increased in the HCC cell lines compared with the LO2 cell line. Additionally, the expression level of lncRNA Ftx was the highest in Bel-7402 cells and the lowest in Huh7 cells (Fig. 2B).

The present study aimed to overexpress and knock down lncRNA Ftx and investigate the general role of lncRNA Ftx. Thus, lncRNA Ftx was overexpressed in Huh7 cells as the base level of lncRNA Ftx was low, and it was knocked down in Bel-7402 cells as the base level of lncRNA Ftx was high. Bel-7402 cells were transfected with LV-Ftx-RNAi (sh-Ftx) and its negative control LV-CON077 (sh-NC), while Huh7 cells were transfected with LV-Ftx (Ftx) and its negative control LV-CON220 (Ftx-NC). RT-qPCR was used to confirm the transfection efficiency. lncRNA Ftx expression in the overexpressing Huh7 cells was ~12 times higher compared with the normal control cells, while the inhibition rate of lncRNA Ftx in Bel-7402 cells was ~74.68% (Fig. 2C).

Tumor cell proliferation, invasion and migration are pivotal steps in tumorigenesis. First, CCK-8 assays were performed to measure cell viability and determine whether lncRNA Ftx has an impact on tumor cell proliferation. Cell proliferation was significantly increased in lncRNA Ftx-overexpressing Huh7 cells (via LV-Ftx transfection) compared with negative control cells at the 48, 72 and 96 h time-points, whereas decreased Bel-7402 cell viability occurred following endogenous lncRNA Ftx knockdown (via LV-Ftx-RNAi transfection), even at 24 h (Fig. 2D). These results suggested that lncRNA Ftx supports HCC cell proliferation.

In addition, to assess the effect of lncRNA Ftx on HCC cell invasion and migration ability, cell invasion and migration assays were performed using stably transfected cells. As presented in Fig. 2E and F, significant migration and invasion decreases were found in lncRNA Ftx-knockdown Bel-7402 cells compared with negative control cells, and cell migration and invasion increases were observed in lncRNA Ftx-overexpressing Huh7 cells; these findings suggested that lncRNA Ftx enhances the invasion and migration ability of HCC cell lines in vitro.

Collectively, the present data demonstrated that the overex-pression of lncRNA Ftx exerts a promoting effect on HCC cell proliferation, migration and invasion.

The measurement of the Warburg effect comprises three parts: The analysis of glucose consumption, the measurement of the glycolytic pathway, and the detection of the Krebs cycle by analyzing the activity and expression of the involved enzymes and the assessment of relative products in carbohydrate metabolism (Fig. 3). Bel-7402 cells exhibited increased GLUT1, PFKL and PPARγ mRNA expression levels and decreased IDH1, CS and OGDH mRNA expression levels compared with Huh7 cells (Fig. 4A), indicating that lncRNA Ftx may promote glycolytic metabolism in HCC cells.

The majority of cancer cells generate more ATP by increasing glucose utilization (24). To investigate whether lncRNA Ftx may affect aerobic glycolysis in HCC, a glucose assay kit was used. lncRNA Ftx-overexpressing Huh7 cells had significantly increased glucose consumption compared with negative control cells, while lncRNA Ftx-knockdown Bel-7402 cells had reduced glucose utilization (Fig. 4B); these findings suggested that lncRNA Ftx may be involved in aerobic glycolysis in HCC. GLUTs, as facilitative-type glucose transporters, are highly expressed in a number of types of cancer and are involved in cellular glucose consumption (25). To gain an understanding of the mechanisms through which lncRNA Ftx promotes glucose utilization in HCC cells, the GLUT1 and GLUT4 mRNA and protein expression levels were measured following lentiviral transfection. As presented in Fig. 4C and D, the mRNA and protein expression levels of GLUT1 and GLUT4 were significantly decreased following the downregulation of lncRNA Ftx in Bel-7402 cells. Consistently, the opposite results occurred with the upregulation of lncRNA Ftx in Huh7 cells. Taken together, the present findings suggested that GLUT1 and GLUT4 may be involved in lncRNA Ftx-mediated HCC glucose uptake.

To evaluate glycolytic activity, a lactate assay kit was used to measure lactate production. lncRNA Ftx knockdown significantly decreased the lactate production in Bel-7402 cells, and lncRNA Ftx overexpression enhanced the lactate production in Huh7 cells (Fig. 5A). Glycolysis is regulated by slowing down or speeding up certain steps in the glycolytic pathway, and this regulation is accomplished by inhibiting or activating the involved enzymes. Furthermore, PFKL, a kinase enzyme that phosphorylates 6-phosphofructose, is a key regulatory enzyme in glycolytic metabolism. To elucidate the regulatory mechanism of lncRNA Ftx, the enzymic activity levels of LDH and PFKL and the mRNA and protein expression levels of PFKL were evaluated in lncRNA Ftx-overexpressing Huh7 cells and lncRNA Ftx-knockdown Bel-7402 cells, respectively. A positive association was observed between the lncRNA Ftx expression level and the enzymic activity levels of LDH and PFKL, and the mRNA and protein levels of PFKL (Fig. 5B–E), indicating that lncRNA Ftx enhances the activity and expression of glycolytic enzymes to increase lactate production in HCC cells.

As the Krebs cycle is determined by the activity and expression of relative enzymes, the enzymatic activity and mRNA and protein expression levels of CS, IDH1 and OGDH were measured. As presented in Fig. 6, the activity, mRNA and protein expression levels of CS, IDH1 and OGDH were significantly enhanced with the downregulation of lncRNA Ftx in Bel-7402 cells, whereas the overexpression of lncRNA Ftx in Huh7 cells impaired the activity and expression levels. The results revealed that lncRNA Ftx promotes the Warburg effect by impairing the activity and expression of Krebs-cycle-associated molecules in HCC cells.

To determine the underlying mechanism of the lncRNA Ftx-induced promotion of HCC cell tumorigenesis and aerobic glycolysis, bioinformatics analysis was performed to predict the possible target genes or proteins of lncRNA Ftx. According to the nucleotide BLASTn program, the 940-1058 nt region of Ftx (a length of 118 nt) was highly homologous with PPARγ (Fig. 7A). The present results suggested that lncRNA Ftx may directly target the PPARγ gene and regulate the transcriptional and post-transcriptional expression of PPARγ.

To assess PPARγ expression levels and their potential association with lncRNA Ftx in HCC tissues, the mRNA expression levels of PPARγ were compared in 73 pairs of HCC and adjacent normal tissue samples using RT-qPCR. The results demonstrated a positive correlation between the expression levels of lncRNA Ftx and PPARγ in HCC tissues (Fig. 7B). To identify the PPARγ expression status in HCC cells, mRNA and protein expression levels were assessed by RT-qPCR and western blotting upon knocking down and overexpressing lncRNA Ftx in Bel-7402 cells and Huh7 cells, respectively. Consistent with the tissue results, PPARγ mRNA and protein expression levels were positively associated with lncRNA Ftx levels (Fig. 7C and D).

Together, these results suggested that PPARγ is a target gene of lncRNA Ftx and functions downstream of lncRNA Ftx in HCC cells.

To confirm whether the promotion of aerobic glycolysis by lncRNA Ftx is mediated through the PPARγ pathway, PPARγ was suppressed with its antagonist GW9662 in lncRNA Ftx-overexpressing Huh7 cells, and activated with its agonist pioglitazone in lncRNA Ftx-knockdown Bel-7402 cells. Subsequently, the glucose consumption, lactate production, LDH activity and mRNA expression levels of relative enzymes and molecules were analyzed. As presented in Fig. 8A, PPARγ activation enhanced glucose uptake, lactate production, LDH activity and the mRNA expression of GLUTs and PFKL, and suppressed the expression of CS, IDH1 and OGDH in lncRNA Ftx-knockdown Bel-7402 cells. The opposite results were observed in Huh7 cells (Fig. 8B). These results indicated that PPARγ may act as an important functional downstream mediator of lncRNA Ftx in HCC.

To further validate the underlying mechanisms, the protein and mRNA expression levels of PPARγ pathway effectors (TNFα, leptin and PDK1) were examined. It was observed that upon lncRNA Ftx overexpression in Huh7 cells, TNFα, leptin and PDK1 expression levels were decreased significantly, whereas in Bel-7402 cells, the opposite results were observed (Fig. 9A and B). As presented in Fig. 9C, pioglitazone downregulated the expression of TNFα, leptin and PDK1, while GW9662 upregulated their expression. Taken together, these data further support the hypothesis that lncRNA Ftx overexpression promotes the activation of the PPARγ pathway, thereby attenuating TNFα, leptin and PDK1 expression to maintain increased glycolysis in HCC.