The human hepatocyte cell lines HepG2, Hep3B and normal human hepatocyte cell line L02 were purchased from the American Type Culture Collection (Manassas, VA, USA); in addition, the human hepatoma cell lines, SMMC-7721 and Huh7, were provided by Shanghai Cell Collection, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Rockville, MD, USA), as previously reported (19). A hairpin small interfering RNA (siRNA) was used to knock down the ANXA2 gene in SMMC-7721 cells. The target sequences for ANXA2 were: siANXA2a, 5′-GCGGGATGCTTTGAACATT-3′, and siANXA2b, 5′-CGACGAGGACTCTCTCATT-3′. The siRNAs were obtained from Shanghai GenePharma Co., Ltd (Shanghai, China), and were inserted into the pSilencer 4.1-CMV-neo vector (Life Technologies) to generate the p-siANXA2a, p-siANXA2b and p-siNC knockdown plasmids. Negative pSilencer 4.1-CMV-neo vector (Life Technologies) that expresses a hairpin siRNA with limited homology to any known sequences in human, mouse, and rat genomes was used as the negative control (siNC). For the overexpression of ANXA2, total RNA was extracted from SMMC-7721 cells with the RNeasy Plus Mini Kit (Qiagen, Chatsworth, CA, USA), following the manufacturer’s instructions. Then, cDNA was synthesized from total RNA using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA), and the ANXA2 gene was amplified from this cDNA using the PCR Master Mix (Promega, Madison, WI, USA) and the following cycling conditions: One cycle at 95°C for 2 min followed by 30 cycles of 95°C for 45 sec, 55°C for 45 sec, and 72°C for 90 sec, followed by an extension cycle of 72°C for 5 min. The amplified cDNA was purified by QIAquick Gel Extraction Kit (Promega), and then introduced into the pcDNA3.1(−) expression vector (Life Technologies) to generate the ANXA2 expression plasmid. All transfections were performed using Invitrogen™ Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Cells were lysed using SDS sample buffer [62.5 mM Tris-HCl, pH 6.8; 2% (wt/vol) SDS; 10% glycerol; 50 mM dithiothreitol, 0.1% (wt/vol) bromphenol blue] obtained from Sangon Biotech Co.,. Ltd. (Shanghai, China). Lysates were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham, Piscataway, NJ, USA). The membranes were blocked with 5% nonfat milk and then incubated with rabbit anti-human polyclonal antibodies targeting ANXA2, β-catenin, cyclin D1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), as indicated by the manufacturer. After washing in tris-buffered saline with Tween 20 (0.5% v/v), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies for 1 h, and visualized using the Enhanced Chemiluminescence Plus kit (Amersham). Band intensities were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). All the experiments were independently repeated at least three times.

Cells were plated on 6-well culture dishes in the presence of 500 μg/ml of G418 (Sigma-Aldrich, St. Louis, MO, USA) after treatment. The DMEM medium was replaced every three days with fresh medium containing G418. In certain experiments, the cells were also incubated with various concentrations of 5-FU, purchased from Sigma-Aldrich. Colonies were stained using crystal violet and counted 2 weeks following transfection under an inverted microscope (Olympus, Tokyo, Japan). All the experiments were performed in triplicate wells three times.

The viability of the cells was assessed using an MTT-based Cell Growth Determination kit (Sigma-Aldrich). Cells at the logarithmic growth phase were plated in 96-well dishes in triplicate wells. Seventy-two hours following treatment, MTT (500 mg/ml) was added to the cells and cells were left to incubate for an additional 4 h. The absorbance of the formazan product was measured on an enzyme-linked immunosorbent assay reader (Molecular Devices, Sunnyvale, CA, USA). Each assay was repeated three times.

Cells were harvested 48 h after treatment, and fixed in ice-cold 70% ethanol for 2 h. Invitrogen™ RNase A (1 mg/ml; Thermo Fisher Scientific) was added, and incubated with the cells at 37°C for 30 min. Then, propidium iodide (50 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added, and the cells were incubated at 4°C for 30 min away from light. The samples were immediately subjected to flow cytometry analysis (Navios instrument; Beckman Coulter, Miami, FL, USA). Cell cycle analysis data were analyzed using the MultiCycle for Windows software (Phoenix Flow Systems, San Diego, CA, USA). Experiments were repeated in triplicate. Average and standard deviation (SD) values were computed.

All experiments were carried out at least three times. Statistical analysis was conducted using the SPSS software (IBM, Armonk, NY, USA). Data were expressed as the means ± SD. The significance of the differences between groups was determined with Student’s t-tests. P<0.05 was considered to indicate statistically significant differences.

To study the role of ANXA2, we first studied its expression pattern in hepatoma cells by western blot. Compared to the level of ANXA2 in a the healthy hepatocyte line L02, the expression of ANXA2 was increased in the four hepatoma cell lines (Hep3B, HepG2, SMMC-7721 and Huh 7), with the highest level observed in the SMMC-7721 cell line (Fig. 1A). We next knocked down the ANXA2 gene in the SMMC-7721 cell line. Western blot analysis showed that ANXA2 expression is markedly reduced in both knockdown cell lines (siANXA2a and siANXA2b), but not in the control cells, transfected with the p-siNC plasmid (Fig. 1B). We then examined the growth of these cell lines using an MTT assay. A reduction in viability was observed in both ANXA2-knockdown cell lines compared to the control cells (Fig. 1C). Moreover, ANXA2 knockdown significantly reduced colony formation (Fig. 1D). These data suggested that knockdown of ANXA2 inhibits hepatoma cell growth.

To investigate the mechanism mediating the antiproliferative effect of the ANXA2 knockdown, we examined changes in the cell cycle by flow cytometry. This analysis showed that the ANXA2 knockdown significantly increases the proportion of cells at the G1 phase (Fig. 2A), indicating that ANXA2 knockdown may induce G1 to S phase arrest. Accordingly, we examined the expression of cell cycle regulators mediating the transition from the G1 to the S phase. Following ANXA2 knockdown, the β-catenin and cyclin D1 levels were markedly reduced, especially in the siANXA2a line (Fig. 2B). These data suggested that ANXA2 knockdown inhibits the cell cycle by regulating the expression of β-catenin and cyclin D1.

To evaluate the relationship between ANXA2 and 5-FU, the effect of 5-FU treatment on ANXA2-overexpressing and ANXA2-knockdown hepatoma cells was examined. 5-FU decreased the viability of hepatoma cells, similarly to previous studies (20,21). However, the viability of ANXA2-overexpressing hepatoma cells was not decreased by 5-FU treatment, indicating that ANXA2 overexpression antagonizes 5-FU (Fig. 3A). On the other hand, hepatoma cells where ANXA2 was silenced showed reduced viability upon 5-FU treatment, indicating that ANXA2 knockdown and 5-FU act synergistically (Fig. 3B). Similar antagonistic and synergistic effects were also observed in the colony formation assay (Fig. 3C and D). These data demonstrated that ANXA2 overexpression reduces, while knockdown enhances the inhibitory effects of 5-FU on hepatoma cell growth.

Since ANXA2 expression modulated the effects of 5-FU, we investigated the underlying molecular mechanism. Upon treatment with 5-FU, the hepatoma cells showed decreased expression of β-catenin and cyclin D1 (Fig. 4A). Given the reported roles of β-catenin and cyclin D1 in the cell cycle, these results indicated that 5-FU may inhibit hepatoma cell growth via downregulation of β-catenin and cyclin D1. Furthermore, we investigated the role of ANXA2 in the regulation of the two cell cycle-related proteins. Our data showed that ANXA2 overexpression induces, while ANXA2 knockdown decreases the expression of β-catenin and cyclin D1 under 5-FU treatment (Fig. 4B and C). Taken together, these data indicated that the ANXA2 protein modulates the effects of 5-FU by regulating β-catenin and cyclin D1 expression.