Sixty patients with HCC who underwent hepatic resection at Kyushu University Beppu Hospital and Affiliated Hospitals between 2001 and 2004 were enrolled in the present study. Resected HCC tumor and paired adjacent liver tissues were immediately frozen in liquid nitrogen and kept at −80°C until RNA extraction. An intermittent follow-up was conducted after the operation, and the average follow-up period for the 60 patients was 70.1 months (range, 1.7–138.8 months). All protocols were approved by the Ethics and Indications Committee of Kyushu University. Written informed consent was obtained from all the patients.

Human HCC cells (HuH-7 and HepG2) were provided by the Cell Resource Center for Biomedical Research (Institute of Development, Aging and Cancer, Tohoku University, Japan). Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum and antibiotics. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

To generate TTF1 expression lentiviral vectors, we amplified the insert (full-length human TTF1; NM_001205296.1) by polymerase chain reaction (PCR) from human reference cDNA. Transient transfections were performed using CSII-CMV-MCS (empty) plasmid DNAs (5′-BamHI and 3′-HpaI sites) and Lipofectamine 2,000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

Total RNA from frozen tissue specimens and HCC cell lines was extracted using ISOGEN (Nippon Gene, Tokyo, Japan). The quality assessment of extracted RNA was performed by measuring absorbance, and we confirmed that all of them kept satisfactory quality. cDNA was synthesized from 8 µg total RNA with M-MLV reverse transcriptase (Invitrogen).

Gene-specific oligonucleotide primers were designed for PCR. The following primers were used: TTF1, 5′-GAAAGGTTGTATGAAATAA ATGTGGA-3′ (sense) and 5′-TTGAACGTAAGATGGAGGAACA-3′ (antisense); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TTGGTATCGTGGAAGGACTCA-3′ (sense) and 5′-TGTCATCATATTTGGCAGGTT-3′ (antisense); 28S ribosomal RNA, 5′-TTACCCTACTGATGATGTGTTGTTG-3′ (sense) and 5′-CCTGCGGTTCCTCTCGTA-3′ (anti-sense). PCR amplification was performed in a LightCycler 480 instrument using a LightCycler 480 Probes Master kit (both from Roche Applied Science Basel, Switzerland). mRNA amplification conditions consisted of initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 62°C (60°C for some genes) for 10 sec, and elongation at 67°C (65°C for some genes) for 10 sec. Melting curve analysis was performed to distinguish specific products from non-specific products and primer dimers. The relative expression levels of the gene were obtained by normalizing the amount of mRNA to that of GAPDH mRNA as an endogenous control in each sample.

Total protein was extracted from TTF1-expressing and mock-transfected cell lines. Aliquots of total protein (40 µg) were electrophoresed on 10% polyacrylamide gels and then electroblotted on nitrocellulose membranes using Trans-Blot Transfer Medium (Bio-Rad Laboratories, Hercules, CA, USA) at 0.4 A for 120 min. TTF1 protein was detected using rabbit polyclonal antibodies (ab87726; Abcam, Cambridge, UK) diluted 1:2,000. TTF1 protein levels were normalized to the level of β-actin protein, which was detected using monoclonal antibodies (Cytoskeleton Inc., Denver, CO, USA) at a 1:1,000 dilution. Blots were developed with horseradish peroxidase-linked anti-rabbit or anti-mouse immunoglobulin (Promega, Madison, WI, USA) diluted at 1:1,000. Enhanced chemiluminescence detection reagents (Amersham Biosciences, Piscataway, NJ, USA) were used to detect antigen-antibody reactions.

HCC tissues were surgically removed, embedded in paraffin and sectioned (5-µm sections). Immunohistochemical analysis was applied to determine the localization of TTF1. A polyclonal rabbit anti-TTF1 antibody (ab87726; 1:200; Abcam) was used as the primary antibody following the manufacturer's protocols.

We obtained expression data of 371 HCC cases and copy number data of 377 cases from The Cancer Genome Atlas (TCGA) from the Broad Institute's Firehose (http://gdac.broadinstitute.org/runs/stddata_2014_12_06/data/LIHC/2014_12_06/). Expression profiles of TCGA dataset were analyzed by GSEA (16). Gene sets extracted from the Broad Institute database and the Uniform Resource Locator of their source are as follows. LEE_LIVER_CANCER_SURVIVAL_DN (http://www.broadinstitute.org/gsea/msigdb/cards/LEE_LIVER_CANCER_SURVIVAL_DN.html), LEE_LIVER_CANCER_SURVIVAL_UP (http://www.broadinstitute.org/gsea/msigdb/cards/LEE_LIVER_CANCER_SURVIVAL_UP.html), RIBONUCLEOPROTEIN_COMPLEX (http://www.broadinstitute.org/gsea/msigdb/cards/RIBONUCLEOPROTEIN_COMPLEX.html), STRUCTURAL_CONSTITUENT_OF_RIBOSOME (http://www.broadinstitute.org/gsea/msigdb/cards/STRUCTURAL_CONSTITUENT_OF_RIBOSOME.html), RIBONUCLEOPROTEIN_COMPLEX_BIOGENESIS_AND_ASSEMBLY (http://www.broadinstitute.org/gsea/msigdb/cards/RIBONUCLEOPROTEIN_COMPLEX_BIOGENESIS_AND_ASSEMBLY.html), PROTEIN_RNA_COMPLEX_ASSEMBLY (http://www.broadinstitute.org/gsea/msigdb/cards/PROTEIN_RNA_COMPLEX_ASSEMBLY.html). We also acquired the expression profiles of TTF1 and the survival information in Gene Expression Omnibus database (accession code GSE14520) (17).

Twenty-four hours before the assay, cells were transfected with TTF1 cDNA or empty cDNA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were conducted to evaluate cell proliferation. First, 10 µl of MTT-labeling reagent (final concentration, 0.5 mg/ml) was added to each well, and the plate was incubated for 4 h in a humidified atmosphere. Next, solubilization solution (100 µl) was added to each well, and the plate was incubated for 12 h in a humidified atmosphere. After confirming that the purple formazan crystals were completely solubilized, the absorbance of each well was measured using a Model 550 series microplate reader (Bio-Rad Laboratories) at a wavelength of 570 nm corrected to 655 nm. The assay was performed using six replicates.

For continuous variables, data are expressed as means ± standard deviations, and statistical analyses were performed using paired t-tests in comparisons between corresponding tumor and normal tissues. Other analyses were performed using Welch's t-tests. Categorical variables were compared using the Chi-square or Fisher's exact tests. Overall survival (OS) was estimated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Univariate and multivariate analysis was performed using the Cox regression model to identify independent variables predictive of OS. P-values of <0.05 were considered to indicate a statistically significant result. Data analyses of clinicopathological factors were performed using JMP 9 software (SAS Institute, Cary, NC, USA), and other analyses were performed using R version 3.1.1 (The R Foundation for Statistical Computing, Vienna, Austria).

To identify whether TTF1 was critical for HCC progression, we used qRT-PCR to examine TTF1 mRNA expression in 58 clinical paired tumor and normal tissues. TTF1 mRNA expression in cancerous tissues was significantly upregulated compared with that in the matched normal tissues (P<0.001; Fig. 1A). Moreover, immunohistochemical analysis revealed that TTF1 protein levels were markedly higher in HCC tissues than in corresponding liver tissues (Fig. 1B). Staining in cytoplasm was observed in both HCC and liver tissues. In contrast, staining in nuclei was characteristically observed in tumor tissues.

To assess whether TTF1 gene copy number variations affected TTF1 gene expression, we extracted copy number data from the TCGA dataset. A strong correlation between copy number and TTF1 expression was observed in tumor tissues (R=0.583, P<0.001; Fig. 1C).

To estimate the clinical significance of TTF1 expression in HCC, TTF1 expression was analyzed by qRT-PCR in 60 samples from patients who underwent resection of primary HCC. According to TTF1 expression level, cases were divided into two groups. Cut-off values were set at the median value of TTF1 mRNA expression. There were no significant differences in clinicopathological parameters between high and low TTF1 expression groups (data not shown). On univariate analysis, the OS rate of the high TTF1 expression group was significantly lower than that of the low TTF1 expression group (P=0.027; Fig. 2A). Furthermore, multivariate analysis showed that high TTF1 expression was an independent prognostic factor for poorer OS (Table I; P=0.020). We further analyzed OS for a public dataset of 242 HCC cases from GSE14520 (17) to confirm the results of our series. In accordance with our data, the OS of the high TTF1 expression group was significantly poorer than that of the low TTF1 expression group (Fig. 2B; P=0.003).

To investigate whether the expression levels of TTF1 were associated with known gene signatures, we applied GSEA to HCC cases from TCGA datasets. GSEA revealed that TTF1 expression levels positively correlated with an unfavorable prognostic gene signature (Fig. 2C) and negatively correlated with a signature representing favorable outcomes (Fig. 2D). Moreover, GSEA showed that TTF1 expression levels were significantly correlated with activity of gene sets which were involved in ribosomal function (Fig. 3A–D).

To explore the biological role of TTF1 in HCC, we performed overexpression experiments in HuH-7 and HepG2 cells. We confirmed that TTF1 mRNA and TTF1 protein expression was significantly higher in cells transfected with TTF1 cDNA than in cells transfected with empty plasmid (Fig. 4A and B). Next, to confirm that upregulated expression of TTF1 contributed to the increased synthesis of rRNA in HCC cells, we performed quantitative analysis with qRT-PCR of 28S rRNA, which was one of the degradation products of 45S rRNA and was reported to reflect the quantity of transcribed rRNA (18). As a result, overexpression of TTF1 led to the increased expression of 28S rRNA, HuH-7 and HepG2 cells in comparison with mock-transfected cells (Fig. 4C; P=0.011 and P<0.001, respectively).

MTT assays were used to examine whether cell growth rates were altered in cancer cells transfected with TTF1 cDNA. As a result, overexpression of TTF1 promoted the proliferation of HuH-7 and HepG2 cells in comparison with mock-transfected cells (Fig. 5A and B; P=0.007 and P=0.018, respectively).