This study conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects and has been approved by the Institutional Review Board of Nagoya University, Aichi, Japan (no. 2013–0295). Written informed consent for usage of clinical samples and data, as required by the Institutional Review Board, was obtained from all patients.

Nine HCC cell lines (Hep3B, HepG2, HLE, HLF, HuH1, HuH2, HuH7, PLC/PRF/5 and SK-Hep1) were obtained from the American Type Culture Collection (Manassas, VA, USA). Primary HCC tissues and corresponding non-cancerous tissues were collected from 151 consecutive patients undergoing liver resection for HCC at Nagoya University Hospital between January, 1998 and January, 2012. Treatment after recurrence generally included the following options: surgery, radiofrequency ablation, transcatheter arterial chemoembolization, and chemotherapy according to tumor status and liver function.

Tissue samples were collected, immediately flash frozen in liquid nitrogen and stored at −80°C until RNA extraction (28 days on average) was performed. Tumor samples ~5 mm² in size that did not contain a necrotic component and were confirmed to contain >80% tumor cells by definition were used for RNA extraction. Corresponding non-cancerous liver tissue samples were collected >2 cm away from the edge of the tumor, were obtained from the same patient and did not contain any regenerative or dysplastic nodules.

Sample collection, RNA extraction, and Affymetrix HG-U133A and HG-U133B GeneChip (Affymetrix, Inc., Santa Clara, CA, USA) gene expression arrays were performed as previously described (22–24).

The BTG1 mRNA expression levels were analyzed by RT-qPCR. Total RNA (10 μg per sample) was isolated and used to generate complementary DNA. The primer sequences used in this study are listed in Table I. RT-qPCR was performed on nine HCC cell lines and 151 pairs of clinical samples with a SYBR-Green PCR Core Reagents kit (Perkin-Elmer/Applied Biosystems, Inc., Foster City, CA, USA) and included no-template samples as a negative control. Real-time detection of the SYBR-Green emission intensity was conducted with an ABI StepOnePlus Real-Time PCR system (Perkin-Elmer/Applied Biosystems, Inc.). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was quantified in each sample for standardization. For cell lines, biological replicates were tested in triplicate. Technical replicates were performed in triplicate for both cell lines and HCC tissues. The expression levels for each sample are shown as the BTG1 value divided by the GAPDH value. BTG1 mRNA expression was considered to be downregulated in tumor tissues when its level was <40% of the level in the corresponding non-cancerous tissues.

The BTG1 gene consists of two exons. Mutational surveillance of HCC cell lines was performed in exon 1 and 2 of the BTG1 gene by high resolution melting (HRM) analysis. HRM is known to be a reliable and concise technique for the detection of genetic alterations (25–27). Genomic DNA obtained from HCC cell lines was amplified with specific primer pairs according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). All samples were tested in triplicate. Eight wells of each PCR plate were allocated to wild-type control DNA, and one well contained a non-template control to validate the PCR. HRM was conducted using a StepOnePlus instrument (Life Technologies) with a melting temperature range set between 60 and 98°C. Scanning data were analyzed by HRM software v3.0.1 (Life Technologies). The primers used for mutational analysis are listed in Table I.

The BTG1 gene contains a CpG island near the promoter region; thus, we hypothesized that aberrant methylation is responsible for regulating BTG1 transcription in HCC. Genomic bisulfite-treated DNA from HCC cell lines was sequenced to ascertain the levels of DNA methylation. The bisulfite treatment and sequencing procedures were performed as previously reported (28,29).

IHC was performed to investigate BTG1 protein localization in 48 representative sections of well-preserved HCC tissue as described previously (30). Sections were incubated for 1 h at room temperature with a rabbit antibody directed against BTG1 (PA5-25035; Thermo Fisher Scientific, Inc., Rockford, IL, USA) diluted 1:100 in Antibody Diluent (Dako, Carpinteria, CA, USA) and then developed for 2 min using liquid 3,3′-diaminobenzidine as the substrate (Nichirei Corp., Tokyo, Japan). The staining patterns were compared between HCC tissue and the corresponding non-cancerous tissue. The intensity of BTG1 protein expression was graded depending on the percentage of stained cells as follows: no staining, minimal (<30%); focal (30–70%); and diffuse (>70%) (31). To avoid subjectivity, specimens were randomized and coded before analysis by two independent observers blinded to the status of the samples. Each observer evaluated all the specimens at least twice within a given time interval to minimize intra-observer variation.

The values between the two groups were analyzed using the Mann-Whitney U test. The χ² test was used to analyze the association between the expression status of BTG1 and clinicopathological parameters. The strength of the correlation between two variables was assessed by Spearman’s rank correlation coefficient. Disease-specific and -free survival rates were calculated using the Kaplan-Meier method, and the difference in survival curves was analyzed using the log-rank test. We performed multivariable regression analysis to detect prognostic factors using the Cox proportional hazards model, and variables with P<0.05 were entered into the final model. All statistical analysis was performed using JMP 10 software (SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered statistically significant.

The age of the 151 patients ranged from 34–84 years (median 64 years), and the male-to-female ratio was 126:25. Thirty-seven patients presented with hepatitis B infections, and 84 patients presented with hepatitis C infections. In terms of the non-cancerous liver samples, the number of patients with normal liver, chronic hepatitis, and cirrhosis were 10, 87, and 54, respectively. When classified according to the 7th edition of the UICC classification, 84, 39 and 18 patients were in stages I, II and III, respectively.

Gene expression that was reduced further in tumor tissues than in the corresponding non-cancerous tissues was used to identify new candidate tumor suppressors in HCC. BTG1 expression was reduced in HCC compared with normal tissue, with a log₂ ratio of −1.6 and −1.5 (Table II).

Decreases in BTG1 mRNA were confirmed in eight (89%) of the nine HCC cell lines compared with the median expression level in non-cancerous liver tissues; these results demonstrate the heterogeneity of BTG1 expression in HCC cell lines (Fig. 1A). No mutations were detected by the HRM analysis of BTG1 exons 1 and 2 (Fig. 1B). Direct nucleotide sequence analysis of bisulfite-treated GC cell lines showed absence of hypermethylation of BTG1 promoter region in all GC cell lines (Fig. 1C).

The BTG1 mRNA expression levels of non-cancerous tissue samples were categorized pathologically into normal liver (n=10), chronic hepatitis (n=87), and cirrhosis (n=58). Upon evaluation of these samples, no significant differences were found, suggesting that the expression of BTG1 mRNA in non-cancerous liver was not affected by background liver fibrosis (Fig. 1D). In 129 (85%) of 151 patients, the expression level of BTG1 mRNA was lower in the HCC tissues than in the corresponding normal tissues. The HCC tissues exhibited significantly lower expression levels of BTG1 mRNA than the corresponding normal tissues (P<0.001, Fig. 1D).

Expression patterns of the BTG1 protein were evaluated by IHC. Representative cases with downregulated BTG1 mRNA expression in HCC tissues exhibited reduced expression of the BTG1 protein in the cytoplasm of the cancerous tissues compared with the adjacent non-cancerous tissues (Fig. 2A). Overall, the staining intensity (shown in Fig. 2B) of the BTG1 protein in 48 patients was consistent with the RT-qPCR data (Fig. 2C).

Fifty-four of 151 HCC patients showed substantial downregulation (<40%) of BTG1 mRNA in HCC tissues compared with non-cancerous tissues. The downregulation of BTG1 mRNA in the HCC samples was significantly associated with male gender, protein induced by vitamin K antagonists (PIVKA) II >40 mAU/ml, tumor size ≥3 cm, tumor differentiation (poorly to moderately differentiated), serosal infiltration, vascular invasion, advanced UICC stage, and extra-hepatic recurrence (Table III). The BTG1 mRNA expression levels in HCC tissues were inversely correlated with preoperative PIVKA II levels (Fig. 3A). Patients exhibiting a downregulation of BTG1 mRNA expression in the HCC samples had a significantly shorter disease-specific survival rate than the other patients (2-year survival rates, 67 and 82%, respectively, Fig. 3B). Multivariate analysis identified the downregulation of BTG1 mRNA as an independent prognostic factor for HCC (hazard ratio 2.12, 95% confidence interval 1.12–4.04, P=0.022, Table IV). In terms of recurrence-free survival rates, patients with a substantial downregulation of BTG1 mRNA in the HCC samples had significantly earlier recurrence rates after surgery than the other patients (2-year recurrence-free survival rates: 39 and 65%, respectively, P=0.032, Fig. 3C).