A total of 129 HCC patients that underwent surgical resection between September 2007 and January 2014 at Xuanwu Hospital, Capital Medical University (Beijing, China) were included in the present study. None of the patients had received chemotherapy or radiotherapy prior to surgery. HCC diagnosis was based on the World Health Organization criteria (21). Tumor staging was determined according to the sixth edition of the tumor-node-metastasis classification of the International Union Against Cancer (22). The histological types were assigned according to the grading system of Edmondson and Steiner (23). All patients received a single intrahepatic arterial injection dose of 40 mg/m² epirubicin 1 month after surgery. Preoperative clinical data, including patient age, gender, pathological diagnosis, serum α-fetoprotein (AFP) levels, time after surgery to last follow-up and overall survival, were collected prospectively (Table I). The current study was approved by the ethics committee of Xuanwu Hospital, Capital Medical University (Beijing, China), and written informed consent was obtained from all patients.

HCC tissues and adjacent healthy liver tissues were resected, and the resected tissues obtained during surgery were divided into two sections: The first section was immediately snap frozen and stored in liquid nitrogen prior to RNA and protein extraction, and the second section was fixed in 10% neutral buffered formalin (OriGene Technologies, Inc., Beijing, China) for 24 h at room temperature and embedded in paraffin for subsequent immunohistochemical analysis. Liver function was assessed using the Child-Pugh scoring system (24). Only those patients who were classified as Child-Pugh class A were included in the study. Patients with portal vein thrombosis, metastasis, systemic hypertension, chronic kidney disease (creatinine >177 mmol/l and blood urea nitrogen >9 mmol/l), diabetes mellitus or aortic valve diseases were excluded from the present study.

RNA extraction and complementary DNA (cDNA) synthesis were performed as previously described (19). Total RNA was extracted from snap-frozen liver biopsy specimens using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cDNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. UII, UT and vascular endothelial growth factor (VEGF) gene expression were quantified by qPCR. The housekeeping gene GAPDH was used as the internal control for target genes. Primer sequences (Invitrogen; Thermo Fisher Scientific, Inc.) are presented in Table II. mRNA expression was measured using SYBR^® Green Real-Time PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. PCR was performed in a 20-µl reaction mixture containing 2 µg cDNA, 1 µl of each primer and 10 µl SYBR Green PCR Master Mix. The conditions for PCR amplification were as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 sec, 60°C annealing for 30 sec, and 72°C extension for 2 min, for a total of 40 cycles. qPCR analysis was performed as previously described (19,25). Comparative quantification cycle (Cq) calculations were all relative to the control group. GADPH Cq values were subtracted from gene Cq values to give a final Cq value. ΔΔCq values were achieved by subtracting the average control ΔCq value, and the expression of UII and UT relative to the control was derived by using the equation 2^−ΔΔCq (26). All experiments were performed in triplicate.

Immunohistochemical analysis was performed as previously described (19). The paraffin-embedded samples were cut into 4-µm sections and subjected to immunohistochemical staining using the EliVision™ Plus kit (Maxim Biotechnology Development, Co., Ltd., Fuzhou, China), according to the manufacturer's protocol. Tissue sections were incubated with rabbit anti-human UT antibody (1:200; catalog no. LS-A372; LifeSpan BioSciences, Inc., Seattle, WA, USA) for 18 h in a humidified chamber at 4°C and washed three times with PBS. An enhancer was added for 30 min, followed by three washes in PBS. The sections were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:1,500; catalog no. ZDR-5306; Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) for 20 min at 37°C, followed by three washes with PBS. Finally, immunoreactivity was visualized following incubation with 3,3′-diaminobenzidine (Maxim Biotechnology Development, Co., Ltd.) for 10 min. Samples were then counterstained with Mayer's hematoxylin and eosin. As a negative control, PBS was used instead of primary antibodies.

Western blot analysis was performed as previously described (18). Briefly, proteins were extracted from liver samples using the RIPA-IV type Mammalian Cell Extraction kit (catalog no. DBI-1017; Bendabio, Shanghai, China), homogenized at 7,104 × g for 15 min at 4°C and assayed using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Protein samples (40 µg) were subjected to SDS-PAGE (80 V for 40 min on a 5% acrylamide stacking gel and 120 V for 70 min on a 10% running gel) and subsequently trans-ferred to a nitrocellulose membrane (Hybond-C Extra; GE Healthcare Bio-Sciences, Uppsala, Sweden). The membranes were blocked with TBS (10 mmol/l Tris-HCl and 250 mol/l NaCl), 5% non-fat powdered milk and 0.1% Tween-20 for 2 h, followed by incubation with primary rabbit anti-human UT antibody (1:1,000; catalog no. sc-20940; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) overnight at 4°C. The blots were washed with TBS containing 0.1% Tween-20 for 10 min (three times), followed by incubation with anti-β-actin antibody (1:1,000; catalog no. ab8226; Abcam, Shanghai, China) or HRP-linked goat anti-rabbit immunoglobulin G secondary antibody (1:1,500; catalog no. GGHL-15PXSPP; Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 2 h at room temperature. Films were scanned using a Gel Doc imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate kit (catalog no. 34079; Thermo Fisher Scientific, Inc.), and bands were quantified via scanning densitometry using the Image Lab™ software version 5.1 (Bio-Rad Laboratories, Inc.). UT protein expression was normalized to β-actin expression.

Data are presented as the mean ± standard deviation. Statistical analysis was performed using one way analysis of variance and the Student's t test. The χ² test was used to analyze the associations between UII expression and patient clinicopathological characteristics. The Kaplan-Meier method was used for survival analysis, and differences in survival were estimated using the log-rank test. Prognostic factors were examined by univariate and multivariate analyses using the Cox proportional hazards regression model. Correlations between different mRNA expression levels were analyzed using the Pearson rank sum test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 20.0 statistical software (IBM SPSS, Armonk, NY, USA).

Patient characteristics and data are presented in Table I. The patient cohort included 80 males and 49 females, with a median age of 58.37 years (age range, 21–73 years). The median follow-up time was 84 months. Histologically, all patients exhibited evidence of HCC with clear surgical margins. None of the patients had been administered somatostatin or vasoactive drugs for 1 week prior to surgery.

UII and UT mRNA expression was evaluated in 129 HCC samples and adjacent non-cancerous hepatic tissues by RT-qPCR. The results revealed a 6-fold increase in UII mRNA levels in HCC tissues compared with adjacent non-cancerous tissues (P<0.01; Fig. 1A). Similarly, in HCC tissues, UT expression levels were increased by ~10-fold compared with adjacent non-cancerous tissues (P<0.01; Fig. 1B). These results revealed that UII and UT mRNA levels were significantly higher in HCC tissues compared with adjacent non-cancerous tissues (P<0.01).

A 7-fold increase in VEGF mRNA was observed in HCC tissues when compared with adjacent non-cancerous tissues (P<0.01; Fig. 1C). Furthermore, a significant positive correlation was identified between VEGF mRNA expression and UII expression in HCC (P<0.001; r=0.78; Fig. 1D).

Immunohistochemistry revealed that non-cancerous tissues exhibited low UT protein expression levels (Fig. 2A, lower left panel). By contrast, abundant UT protein expression was identified in HCC tissues (Fig. 2A, lower right panel). Furthermore, UT staining was observed in the cytoplasm of tumor stromal cells (Fig. 2B). Western blot analysis of six representative HCC tissues identified a significant increase in the levels of UT protein in cancerous tissues when compared with non-cancerous tissues (Fig. 2C and D). These findings are consistent with the relative UT mRNA expression levels detected by RT-qPCR (Fig. 1B).

Associations between UII expression and clinicopathological parameters in HCC patients were analyzed using the χ² test. The median mRNA expression level of UII in HCC tissues, 6.56-fold, was used as the cut-off value to divide the 129 patients into two groups: A low-expression group (UII mRNA expression level <6.56; n=53) and a high-expression group (UII mRNA expression level >6.56; n=76). As presented in Table I, a correlation was identified between UII expression and tumor size (P<0.001), histological differentiation (P<0.001) and pathological stage (P=0.026). However, no significant associations were observed between UII mRNA expression and gender (P=0.398), age (P=0.421), liver cirrhosis (P=0.295), expression of hepatitis B surface antigen (HBsAg) (P=0.139) or serum AFP levels (P=0.854). Furthermore, the association between UT expression and clinicopathological parameters was analyzed (Table III). The mean UT relative mRNA expression level of HCC tissues was 10.04, which was used as the cut-off value to divide the 129 patients into two groups. Higher UT expression was significantly associated with tumor size (P<0.001) and pathological stage (P=0.016); however, no significant association was identified between UT mRNA expression and gender (P=0.197), age (P=0.543), liver cirrhosis (P=0.193), HBsAg expression (P=0.183), serum AFP levels (P=0.724) or histological differentiation (P=0.252) (Table III).

Overall survival was analyzed using the Kaplan-Meier method, which demonstrated that the survival rates of HCC patients with high UII expression were significantly lower than those of patients with low UII expression (P<0.001; Fig. 3). Univariate analysis demonstrated a significant association between overall patient survival rates and tumor size (P=0.031), histological differentiation grade (P=0.017), pathological stage (P=0.017) and UII mRNA expression (P=0.017) (Table IV). However, no significant associations were identified between UT mRNA expression (P=0.058), patient age (P=0.432), gender (P=0.781), HBsAg expression (P=0.908), serum AFP levels (P=0.407) or patient outcomes. Multivariate analysis using the Cox proportional hazards model for all the variables included in the univariate analysis indicated that histological differentiation grade (P=0.031), pathological stage (P=0.006) and UII expression (P=0.0001) were all independent prognostic factors for overall survival in HCC patients (Table IV).