The fresh-frozen human HCC tissues and adjacent normal tissues were obtained from eight patients who underwent surgery without preoperative systemic chemotherapy between 2003 and 2011 at the Department of Surgery, the Affiliated Hospital of Nantong University. All tissues were from patients with newly diagnosed HCC who had received no therapy before sample collection, and were snap-frozen in liquid nitrogen immediately after surgery and stored at −80°C until use. The liver cancer tissue microarrays containing 65 matched primary HCC and adjacent non-tumorous tissues were obtained from the Department of Pathology of the Affiliated Hospital of Nantong University. The study population consisted of 40 males and 25 females, and the age ranged from 21 to 69 years. The total cases of HCC patients had been approved by the Ethics Committee of the Affiliated Hospital of Nantong University. The main clinical and pathologic variables are summarized in Table I.

In order to analyze PCBP2 and Ki67, serial sections (5 µm thick) were mounted on glass slides coated with 10% polylysine. These sections were dewaxed in xylene and rehydrated through graded alcohol. Immunoreactivity was enhanced by high temperature and pressure, and these sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min in an autoclave to retrieve the antigen. Thereafter, endogenous peroxidase activity was blocked using hydrogen peroxide (0.3%). Anti-PCBP2 antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C and anti-Ki67 antibody (1:500; Millipore Corp., Bedford, MA, USA) for 4 h at room temperature were incubated in the sections after rinsing in phosphate-buffered saline (PBS) (pH 7.2). Negative control slides were incubated in parallel using a nonspecific immunoglobulin IgG (Sigma-Aldrich, St. Louis, MO, USA) at the same concentration as the primary antibody. All slides were processed using the peroxidase-anti-peroxidase method (Dako, Hamburg, Germany). Finally, slides were counterstained with hematoxylin, dehydrated, and mounted in resin mount. Stained sections were observed under a microscope (24).

The immunostaining results were evaluated by two independent pathologists to avoid possible technical errors. Five high-power fields were randomly chosen for assessment of PCBP2, and at least 500 cells were counted per field. Each tumor section was assigned a score according to the intensity of the cytoplasmic staining and the proportion of stained tumor cells. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The extent of staining was scored based on the percentage of positive tumor cells: 0 (<10%), 1 (10–30%), 2 (>30–50%), 3 (>50–70%), and 4 (>70%). The immunostaining score was counted as the percentage positive score × the staining intensity score and ranged from 0 to 12. A score of 0 was considered negative; 1–4, weak; 5–9, moderate; and 10–12, strong. For statistical analysis, 0–4 was counted as low expression, while 5–12 was counted as high expression.

The human HCC cell lines, HepG2, Huh7, SMMC-7721 and Hep3B and the normal liver cell line (LO2) were obtained from Shanghai Institute of Cell Biology, Academia Sinica, and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin 100 µg/ml, and penicillin 100 U/ml at 37°C in 5% CO₂. The siRNA oligos of PCBP2 were synthesized by GeneChem Co., Ltd. (Shanghai, China). The targeted sequences of PCBP2 siRNA were as follows: siRNA#1, CATCACTATTGCTGGCATT; siRNA#2, CACTAATGCCATCTTCAAA; siRNA#3, CGGATTCAGT GGCATTG; and control, TTCTCCGAATGTCACGT. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Western blot analysis was conducted as previously described (25). Briefly, frozen liver tissues and harvested cells were promptly homogenized in a homogenization buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl₂, 60 mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF) and then centrifuged at 10,000 × g for 30 min to collect the supernatant. Protein concentrations were determined using a Bio-Rad BCA protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) filter membranes (Millipore Corp.). The membranes were blocked with 5% dried skim milk in TBST (20 mM Tris-Cl, 150 mM NaCl, 0.05% Tween-20) and incubated with primary antibodies overnight at 4°C. The primary antibodies used in the study were as follows: anti-PCBP2 (1:1,000), anti-PCNA (1:1,000), cyclin D1 (1:500), anti-p27 (1:1,000), anti-Bax (1:500); anti-Bcl-2 (1:500), anti-active caspase-3 (1:500) (all from Santa Cruz Biotechnology, Inc.), and anti-GAPDH (1:1,000; Sigma-Aldrich). The membranes were washed with TBST for 5 min for three times. Then, horseradish peroxidase-linked IgG was used as the secondary antibody (1:5,000; Pierce Biotechnology, Inc., Rockford, IL, USA) for 2 h at room temperature according to the manufacturer's instructions. The detection of immunoreactive bands was performed using an enhanced chemiluminescence system (NEN Life Science Products, Inc., Boston, MA, USA).

Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer's instructions. Briefly, cells were plated into a 96-well plate at a density of 1×10⁴ cells/well in a volume of 100 µl. For proliferation measurement, 10 µl of CCK-8 reagent plus 90 µl of DMEM complete medium was added to each well for a 2-h incubation at 37°C. The absorbance of cells was read in a microplate reader (Bio-Rad Laboratories, Inc.) at 450 nm with a reference wavelength of 650 nm. The detection of cell absorbance was performed every 24 h. The experiments were repeated at least three times (26).

After transfection, HepG2 and Huh7 cells were plated into a 6-well plate (500 cells/well). Fifteen days later, the colonies were washed with PBS, fixed with paraformaldehyde for 20 min, and then stained with 0.5% crystal violet for 30 min. Cell colonies (0.5 mm in diameter) were counted after staining.

The apoptosis of HCC cells was evaluated by FITC-Annexin V and propidium iodide (PI) assay using a commercial kit (BD Biosciences, Shanghai, China) as previously described (27). The upper right quadrant (UR) indicates the late apoptotic and necrotic cells, the lower right quadrant (LR) represents the early apoptotic cells, the upper left quadrant (UL) represents the debris and damaged cells, while the lower left quadrant (LL) represents the negative control cells. In this study, we consider UR and LR as apoptotic cells.

The results are expressed as mean ± SEM. Statistical analysis was performed using the Stata 7.0 software package. The association between PCBP2 expression and clinicopathological features was analyzed using the χ² test. For analysis of survival data, Kaplan-Meier curves were constructed, and the log-rank test was performed. For multivariate analysis, Cox's proportional hazards model was used and p<0.05 was considered to be statistically significant.

To determine the expression of PCBP2 in HCC tissues, we first studied the expression of PCBP2 in HCC tissues and the adjacent non-tumor liver tissues by western blot analysis. As shown in Fig. 1A, we found that the expression of PCBP2 was significantly elevated in the HCC tissues, compared with that in the adjacent normal ones. Moreover, the expression profile of PCBP2 was detected in the HCC cell lines (HepG2, Huh7 and SMMC-7721) and the normal liver cell line (LO2). Similarly, we found that HCC cell lines had a higher PCBP2 level compared with that noted in the normal liver LO2 cells (Fig. 1B). Then, we conducted immunohistochemical staining to confirm the expression of PCBP2 and Ki67 in the HCC specimens. Following the different histological stage, representative samples of PCBP2 and Ki67 are shown in Fig. 1C. In most specimens, PCBP2 was highly expressed in the cytoplasm and nucleus of the HCC tissues, whereas an obvious low signal of PCBP2 was observed in the adjacent non-tumor tissue samples (Fig. 1C-a and -b), which is in line with the expression of Ki-67 (Fig. 1C-c and -d). These findings indicated that PCBP2 may contribute to malignant progression of HCC.

To further investigate the significance and prognostic value of PCBP2, we analyzed the correlations between clinicopathological characteristics and the expression of PCBP2 in 65 patients with HCC. The results are summarized in Table I. We found that the expression of PCBP2 was correlated with tumor differentiation (p=0.028), HBsAg (p=0.031) and Ki67 expression (p=0.042), but not with other clinicopathological factors, such as age, gender, tumor size, tumor number, tumor metastasis, serum AFP level, cirrhosis, microvascular invasion and capsular formation.

Next, we performed Kaplan-Meier analysis to examine whether PCBP2 could serve as an independent indicator to predict the prognosis of HCC patients. Our data showed that patients with high PCBP2 expression had significantly worse prognosis compared with that of patients with low PCBP2 expression (Fig. 2). Based on the results of univariate analyses, we identified that PCBP2 expression (p=0.016), Ki67 (p=0.018), tumor differentiation (p=0.007) and HBsAg (p=0.036) were associated with patient survival. Furthermore, multivariate Cox proportional hazard analysis showed that PCBP2 (p=0.043) could be an independent prognostic indicator of overall survival (Table II).

Based on our data that PCBP2 expression is associated with PCNA and Ki67, we considered that PCBP2 may promote HCC development by regulating the proliferation of HCC cells. Therefore, we analyzed the expression of PCBP2 during cell cycle progression in HCC cells. HepG2 and Huh7 cells were arrested in G1 phase by serum deprivation for 72 h. After serum-refeeding, the cells were released from G1 phase and entered into the S phase (Fig. 3A and B). Western blot analysis showed that the expression of PCBP2 increased gradually with time following the release of cells from serum starvation in both cell lines. At the same time, we found that the expression of cyclin D1 was upregulated (Fig. 3C-F). These results suggest that PCBP2 may play a key role in regulating HCC cell proliferation.

To determinate the effect of PCBP2 in HCC cell proliferation, we firstly transfected HepG2 and Huh7 cells with PCBP2 siRNA oligos to knockdown endogenous PCBP2. Western blot analysis was carried out at 48 h after transfection, which confirmed a significant reduction in PCBP2 expression in the PCBP2-siRNA#3-transfected cells (Fig. 4A). Therefore, PCBP2-siRNA#3 was used for further experiments. At the same time, we discovered that the silencing of PCBP2 expression obviously downregulated the levels of cyclin D1 and PCNA, with the upregulated levels of cyclin-dependent kinase inhibitor p27 (Fig. 4B). Furthermore, CCK-8 assay was used to confirm the effect of PCBP2 on HCC cell proliferation. The HepG2 and Huh7 cells transfected with PCBP2 siRNA#3 exhibited a marked decreased in the cell proliferation, as compared with the control siRNA-transfected ones (Fig. 4C). Likewise, colony formation assay revealed that depletion of PCBP2 markedly inhibited the colony formation capacity of the HepG2 and Huh7 cells (Fig. 4D). According to the above results, we further demonstrated whether PCBP2 expression could affect the cell cycle distribution in HCC cells by flow cytometric analysis. The results showed that depletion of PCBP2 caused G0/G1 phase arrest, accompanying a marked reduction in the cell population in the S phase (Fig. 4E). These results suggest that depletion of PCBP2 inhibits the proliferation of HCC cells.

Sorafenib (Nexavar), a multiple kinase inhibitor, has shown survival benefits in patients with advanced HCC and has become the first approved drug for HCC (28,29). However, the clinical response of sorafenib was seriously limited by drug resistance and thus we tested the possibility that high expression of PCBP2 may be involved in sorafenib resistance in HCC. We firstly investigated the sensitivity of HCC cells to sorafenib. The results showed that sorafenib induced the apoptosis of HCC cells in a dose-dependent manner (Fig. 5A). Then we transfected HepG2 cells with PCBP2 siRNAs#3 or control siRNA, and exposed the cells to sorafenib (50 µM) or DMSO. The flow cytometric analysis showed that depletion of PCBP2 significantly increased the percentage of apoptotic cells in the absence of sorafenib treatment (33.56 vs. 55.64%). Following sorafenib treatment, PCBP2-depleted cells exhibited a markedly increased level of apoptosis (59.01 vs. 78.38%), compared with the mock-transfected group (Fig. 5B). The bar chart shows the ratio of apoptotic cells by densitometry (Fig. 5C). These results revealed that interference of PCBP2 could potentiate HCC cells to sorafenib-induced apoptosis. Furthermore, western blot analysis showed that transfection with PCBP2 siRNA#3 significantly increased the level of active caspase-3, Bax and inhibited the level of Bcl-2, especially after sorafenib treatment (Fig. 5D). The bar chart shows the ratio of PCBP2, active caspase-3, Bax and Bcl-2 expression to GAPDH (Fig. 5E). These results indicate that high expression of PCBP2 may contribute to sorafenib resistance by regulating Bcl-2 proteins in HCC cells.