HCC tissues, paired adjacent tissues (distance from tumor tissue is >5 cm) and peripheral blood samples were collected from 175 patients with HCC who underwent surgical resection at The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China) between October 2011 and May 2013 (mean age, 64 years; age range, 32–81 years; 94 males and 81 females) (Table I). In addition, 27 hepatic hemangioma (HH) and 27 healthy control (HC) samples (the HC tissues were not from the same patients) were collected at The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). The corresponding clinicopathological data were obtained from The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). Patients with HCC were followed up to 5 years post-surgery to evaluate their survival rate. Patients were followed up by phone every 3 months and the total duration of follow up was 97 months. The present study was approved by the Ethics Committee of The Second Affiliated Hospital of Chongqing Medical University and was conducted in accordance with the Declaration of Helsinki.

The human HCC Hep3B, MHCC-97H, MHCC-97L and Huh-7 cell lines, and the normal liver THLE-2 cell line were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. HCC cell lines were cultured in high-glucose DMEM with 10% FBS, and the normal liver THLE-2 cell line was cultured in Bronchial Epithelial Cell Growth Medium (BEGM) with 10% FBS (all Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and streptomycin All cells were cultured in a humidified incubator at 37°C with 5% CO₂.

Blood samples (5 ml blood was collected) were centrifuged. The whole blood was kept for 30 min and centrifuged at 2,000 × g for 20 min at room temperature, and serum and plasma components were stored as aliquots at −80°C until further use. An ELISA kit (cat. no. ELH-GPC1; RayBiotech, Inc.) was used to measure the levels of serum GPC1. According to the manufacturer's protocol, a monoclonal antibody specific for GPC1 was coated onto the wells of the microtiter strips. Subsequently, diluted samples, including standards of known GPC1 content, control specimens and unknowns, were pipetted into these wells, followed by the addition of a second biotinylated monoclonal antibody. Next, streptavidin-peroxidase was added to complete the four-member sandwich. After incubation at room temperature for 30 min and washing steps, the 3,3′,5,5′-tetramethylbenzidine substrate solution was added, which reacted with the enzyme antibody-target complex to produce measurable signals. The optical density values were measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Total RNA from HCC tissues, adjacent tissues, cell lines and HH tissues was extracted using TRIzol^® reagent (Takara Bio, Inc.). PrimeScript RT reagent (Takara Bio, Inc.) was used for RT to cDNA, and RT-qPCR was performed using SYBR Premix Ex Taq II (Takara Bio, Inc.) using a LightCycler system (Roche Diagnostics). The temperature protocol for RT was as follows: 30°C for 10 min, followed by 47°C for 40 min and 75°C for 30 min. The primer sequences of GPC1 were as follows: Forward, 5′-CGGCCCCGCCATGGAGCTCC-3′ and reverse, 5′-GGCAGTTACCGCCACCGGGG-3′. GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 92°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 65°C for 30 sec, and a final extension step at 72°C for 30 sec. GAPDH was used as an internal reference, and the mRNA expression levels of GPC1 were analyzed using the 2^−ΔΔCq method (10).

Total protein from HCC cell lines, a normal liver cell line (1×10⁶) and tissues (Hep3B, MHCC-97H, MHCC-97L, Huh-7, THLE-2 cell lines, HCC and paired adjacent tissues) was extracted using RIPA lysis buffer. Protein was quantified using the Bradford protein assay (Bio-Rad Laboratories, Inc.) with a Nanodrop spectrophotometer and an equal amount (35 µg) was added to each well of 10% gels and resolved by SDS-PAGE. The samples were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk powder at room temperature for 1 h. Next, the membranes were probed at 4°C overnight, using the following primary antibodies: GPC1 (1:1,500; cat. no. ab199343; Abcam) and β-actin (1:5,000; cat. no. ab8227; Abcam). Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence solution (EMD Millipore) and a ChemiDoc Imaging system (Bio-Rad Laboratories, Inc.). Protein expression was semi-quantified using the Quantity One v4.6.6 software (Bio-Rad Laboratories, Inc.). β-actin was used as an internal reference.

The collected 175 HCC tissues were made into tissue microarrays. The tissue was fixed in 4% paraformaldehyde at room temperature for 24 h and embedded in paraffin. Sections were deparaffinized in xylene I for 15 min and xylene II for 15 min at 37°C, and rehydrated in a graded ethanol series (100, 95, 80 and 75% ethanol for 5 min each). Subsequently, the sections were incubated with 3% H₂O₂ for 30 min at 37°C and for 15 min at 37°C with 5% goat serum (Origene Technologies, Inc.) to block non-specific binding, followed by incubation with a rabbit monoclonal anti-GPC1 antibody (1:1,000; cat. no. ab55971; Abcam) at 4°C overnight. Next, the sections were incubated with an anti-rabbit secondary biotin labelled IgG antibody (1:100; cat. no. SAP-9100; Origene Technologies, Inc.) at 37°C for 30 min. After washing with PBS, the visualization signal was detected using 3,3′-diaminobenzidine (Boster Biological Technology) and counterstaining was performed using hematoxylin at room temperature for 5 sec.

GPC1 immunostaining was scored and examined by two double-blinded pathologists. All tissues observed using a light microscope at a magnification of ×200 were manually scored based on the percentage of positive cells and the intensity to determine the final staining scores. The scoring parameters included staining intensity (according to the color development degree of the positive markers; Light yellow, indicating weak positive; brown yellow, medium positive; and brown black, strong positive) (range, 0–3: 0, no staining; 1, weak; 2, moderate; and 3, strong) and the percentage of positive cells (range, 0–4: 0, <5; 1, 6–25; 2, 26–50; 3, 51–75; and 4, 76–100%). Slides with a total score <4 were defined as low GPC1 expression, while slides with a score ≥4 were defined as high GPC1 expression.

The data were analyzed using SPSS v22.0 (IBM Corp.) and GraphPad Prism v6.0 (GraphPad Software, Inc.). GPC1 expression was presented as the mean ± SD. All experiments were repeated 3 times. Statistical differences among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. The significance of GPC1 was examined using paired or unpaired Student's t-test. The association between clinicopathological parameters and GPC1 expression was analyzed using χ² test. Kaplan-Meier, log-rank tests and Cox regression for univariate and multivariate analysis were used to analyze the prognostic significance of GPC1 expression. RFS was recorded as the time from liver tumor resection removal to liver tumor recurrence. DSS was recorded as the time from cancer diagnosis to death from cancer or to the follow-up deadline. P<0.05 was considered to indicate a statistically significant difference.

GPC1 expression in HCC tissues and cell lines was analyzed via RT-qPCR. The results revealed that GPC1 expression was significantly higher in HCC tissues compared with that in paired normal adjacent tissues (P<0.01; Fig. 1A). Furthermore, GPC1 expression was significantly increased in aggressive HCC tissues (tumors with distant metastasis) compared with that in non-aggressive HCC tissues (tumors with no distant metastasis) (P<0.05; Fig. 1B). However, there was no significant difference in the expression levels of GPC1 between HH and HC tissues (P>0.05; Fig. 1C). Western blotting results revealed that GPC1 protein expression was higher in HCC tissues compared with that in their paired normal adjacent tissues (Fig. 1D and E).

In addition, GPC1 expression was higher in HCC cell lines compared with that in the normal liver cell line and GPC1 expression was higher in cells with high malignancy (MHCC-97H cells had high metastatic ability compared with the other cell lines tested) (Fig. 2A and B).

Subsequently, the serum levels of GPC1 were analyzed in HCC, HH and HC samples. As shown in Fig. 3A, the serum levels of GPC1 in HCC samples were significantly higher than in HC samples (P<0.05). However, there was no significant difference in the serum levels of GPC1 between HH and HC samples (P>0.05; Fig. 3B). Furthermore, higher serum levels of GPC1 were detected in stage III/IV HCC samples than in stage I/II HCC samples (P<0.05; Fig. 3C). The serum levels of GPC1 were higher in tumors >5 cm in size than in tumors <5 cm in size. There was no significant difference between stage III/IV or >5 cm in HC samples (P<0.05; Fig. 3D).

The present results revealed that GPC1 expression was high in 124/175 HCC samples (70.9%) and low in 51/175 HCC samples (29.1%) (Table I), and GPC1 was mainly expressed in the cytoplasm and cell membrane (Fig. 4A and B). As shown in Table I, GPC1 expression was positively associated with tumor size (P=0.011) and Tumor-Node-Metastasis (TNM) stage (P=0.033). However, there were no significant differences between GPC1 expression and age, sex, α-fetoprotein levels, liver cirrhosis, hepatitis B virus surface antigen, vascular invasion, multiplicity and intrahepatic metastasis (all P>0.05; Table I).

Kaplan-Meier survival analysis revealed that the RFS time was significantly shorter in patients with HCC with high GPC1 expression compared with that in patients with low GPC1 expression in HCC (P=0.003; Fig. 5A). In addition, the univariate analysis revealed that tumor size [hazard ratio (HR)=1.561; P=0.041], TNM stage (HR=1.609; P=0.019) and GPC1 expression (HR=1.579; P=0.009) were significantly associated with RFS in patients with HCC (Table II). The multivariate analysis revealed that tumor size (HR=1.773; P=0.047), TNM stage (HR=1.473; P=0.025) and GPC1 expression (HR=1.311; P=0.007) were independent prognostic factors for RFS in patients with HCC (Table II).

Kaplan-Meier survival analysis revealed that the DSS time was significantly shorter in patients with HCC with high GPC1 expression compared with that in patients with low GPC1 expression (P=0.002; Fig. 5B). In addition, the univariate analysis revealed that tumor size (HR=1.204; P=0.039), TNM stage (HR=1.342; P=0.022) and GPC1 expression (HR=1.770; P=0.017) were significantly associated with DSS in patients with HCC (Table III). Similarly, the multivariate analysis revealed that tumor size (HR=1.119; P=0.031), TNM stage (HR=1.554; P=0.028) and GPC1 expression (HR=1.883; P=0.014) were independent prognostic factors for DSS in patients with HCC (Table III).