HepG2, HuH-7, SMMC-7721, QGY-7703 and HL-7702 cell lines were obtained from Shanghai Cell Bank. The cells were maintained under a humidified atmosphere of 5% CO₂ at 37°C in DMEM medium (HyClone), supplemented with 10% fetal bovine serum (FBS; HyClone).

Set of four Rap1-miRNA oligonucleotides were designed and synthesized by Invitrogen for Rap1 gene knockdown studies. The Rap1-miRNA expression vector (pcDNATM 6.2-GW/EmGPF-miR; Invitrogen; catalog no. K4936-00) was used to construct a Rap1 miRNA plasmid by inserting the miRNA-coding sequence. HepG2 cells were transfected with selected Rap1-miRNA plasmid or control miRNA plasmid.

HepG2 cells were seeded on 6-well plates at a density of 5×10⁵ cells/well, and grown for 48 h to reach 80% confluence before transfection. Control miRNA and Rap1 miRNA were transfected respectively into cells using Lipofectamine 2000 (Invitrogen; catalog no. 11668-027) according to the manufacturer’s instructions. The cells were then harvested and analyzed by flow cytometry, electron microscopy, reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis at 24–72 h after the transfection. All measurements were performed in triplicate.

The transfected cells were cultured in the medium with Blasticidin S HCl (Invitrogen; catalog no. R210-01) at 2–10 μg/ml. After 8 weeks, surviving colonies arising from stably transfected cells were selected and individually amplified.

At 48 h after transfection, cell survival was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The transfected and control cells were seeded at a density of 5,000 cells/well in 96-well plates and incubated at 37°C in humidified 5% CO₂ for 24 h. Serially diluted 5-FU (Sigma) was added to give the intended final concentrations. Cells were then incubated an additional 72 h, and the MTT assay was performed according to the manufacturer’s instructions. Absorbance values were determined at 570 nm on a SpectraMax 250 spectrophotometer. The assays were performed three times in triplicate. The 50% inhibitory concentration (IC₅₀) values were defined as the drug concentrations required to reduce cellular proliferation to 50% of the untreated control well.

The HepG2 cells were harvested for evaluation of apoptosis at 48 and 72 h after the transfection. Firstly, the cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min. Then, the cells were re-suspended in PBS supplemented with 0.1% Triton X-100, and incubated at 4°C for 2 min. TUNEL solution (50 μl) was added to each tube, and incubated in the dark at 37°C for 60 min. After that, the cells were washed with PBS, and re-suspended in 250 μl PBS for flow cytometry using the FACSCalibur system.

The cells were harvested in PBS for evaluation of apoptosis with electron microscopy at 48 h after the transfection. Firstly, the cells were subsequently fixed in 3.5% glutaraldehyde and 1% OsO₄, then, washed with PBS, dehydrated in ethanol and acetone series, and embedded in Epon 618. Thin sections were cut with a Leica Ultracut UCT microtome, stained with 2% uranyl acetate and lead citrate. Observation followed using JEM-1011 transmission electron microscopy.

To elucidate the mechanism by which Rap1 induces cell growth and inhibits apoptosis in HCC, we examined the expression of NF-κB p65 and IκB using quantitative real-time PCR at 48 h after the transfection. Total RNA was isolated from the Rap1 miRNA transfected and control cell lines with TRIzol (Invitrogen; catalog no. 15596-026). Then, the RNA was subjected to real-time PCR analysis with the use of a One Step SYBR PrimeScript RT-PCR kit (Bio-Rad Laboratories) and superscript pre-amplification system (Invitrogen Life Technologies). NF-κB p65 was amplified using forward primer 5′-GGGAAGGAACG CTGTCAGAG-3′ and reverse primer 5′-TAGCCTCAGGG TACTCCATCA-3′. IκB was amplified using forward primer 5′-GGATACCTGGAGGATCAGATTA-3′ and reverse primer 5′-CCACCTTAGGGAGTAGTAGATCAAT-3′. PCR amplification was performed with 32 cycles of 45 sec denaturing at 94°C, 45 sec annealing at 55°C and 10 min extension at 72°C. A melting curve analysis was conducted to control for the specificity of the amplification products. The expression values were normalized by the β-actin expression. Moreover, the amplified gene fragments were separated in a 2% agarose gel and visualized by ethidium bromide staining and UV light.

Western blot analysis was performed as previously described at 24, 48 and 72 h after the transfections. The antibodies included Rap1 monoclonal antibody (Santa Cruz Biotechnology, 60 kDa, mouse), β-actin monoclonal antibody (Abmart; 43 kDa, mouse), goat anti-rabbit and mouse IgG-HRP (Abmart). Cells were collected by trypsin digestion and washed twice with PBS. RIPA lysis buffer (100 μl) (Soledad) was added. Then, protein was extracted, and protein concentrations were determined using BCA protein assay kit (Tiangen) based on the manufacturer’s instructions. Protein samples (30 μl) were loaded onto a NuPAGE 8% TA gel (Invitrogen) and run at 80 V. Next, the proteins were transferred to PVDF membranes (Millipore) using an iBlot Gel Transfer Device (Bio-Rad Mini Protein 3) at 200 mA for 1.5 h. The membranes were blocked in 3% BSA blocking buffer for 2 h, exposed to primary antibody overnight at 4°C, and secondary antibody for 2 h at room temperature. After that, the membranes were washed with 1% PBST, and rinsed with Rap1 three times (5 min each time) and with β-actin three times (3 min each time). Excess reagent was removed. The membranes were covered with transparent plastic wraps. Images were acquired using darkroom development techniques.

The animal protocol was approved by the the Institutional Animal Care and Use Committee of Kunming Medical University. Four-week-old BALB/c nude mice were obtained from Vital River Laboratories (Beijing, China) and maintained in a specific pathogen-free facility. These mice were randomly divided into four groups with 10 mice each group: i) control; ii) 5-FU treated; iii) Rap1 miRNA; and iv) Rap1 miRNA + 5-FU treated group. The mice were implanted subcutaneously into the right flank with Rap1 miRNA stable transfected and control HepG2 cells (1.0×10⁷ cells in 0.2 μl PBS per mouse). When palpable tumors developed (average volume 100 mm³), the mice in 5-FU treated and Rap1 miRNA + 5-FU treated groups were administered intraperitoneally with 5-FU at a dose of 30 mg/kg every 3 days. Those in control and Rap1 miRNA groups were injected with the same volume of 0.9% saline. Tumor size was measured at 3-day intervals with a caliper (calculated volume = shortest diameter² × longest diameter/2). Tumor growth was followed for 25 days from the first injection.

At the termination of the experiments, the tumors were removed for histological examination. The tumor samples were fixed in 10% formaldehyde, embedded in paraffin and then cut into 3-μm thick sections. Immunohistochemistry was conducted using streptavidin-peroxidase method with the EnVision Kit (Maixin Bio, Fuzhou, China). The sections were dewaxed in xylene and rehydrated in a graded series of ethanol. The tissue sections were treated by high temperature and pressure in 10 mmol/l sodium citrate (pH 6.0) for 2 min for antigen retrieval. Next, the primary anti-Rap1 antibody (1:100 dilution; Santa Cruz Biotechnology; 60 kDa) was added to the slides. The slides were incubated at 4°C overnight. After that, the slides were stained with secondary antibody, followed by incubation with diaminobenzidine kit for 10 min, hematoxylin staining, vitrification with dimethylbenzene, and coverslip with mounting medium. The Rap1 and caspase-3 expression were assessed at the center and the invasive front of the tumor in each section. A positive Rap1 expression was defined as nuclear staining at both the cancer central and invasive front. A positive caspase-3 expression was defined as cytoplasmic staining.

Data are presented as the means ± SD. Statistical analysis was performed using the SPSS version 14. The differences were evaluated by t-test. P-values <0.05 were considered to indicate a statistically significant result.

To determine the effect of Rap1 expression on the apoptosis in HCC cells, we first determined the expression of Rap1 in four HCC cell lines and normal liver cells by quantitative RT-PCR. Quantitative RT-PCR revealed that expression of Rap1 was significantly higher in HCC cell lines HuH-7, SMMC-7721, QGY-7703 and HepG2 as compared with normal liver HL-7702 cells. Given the relative high expression of Rap1 in HepG2 cells, this cell line was selected for subsequent experiments to determine whether inhibition of Rap1 expression by miRNA would enhance cells apoptosis.

To investigate the anti-apoptotic role of Rap1 in HCC cells, we knocked down the expression of Rap1 in HepG2 using RNA interference (RNAi) techniques with miRNA. Firstly, Rap1 protein levels were evaluated in transfected and control HepG2 cells by western blot analysis to test RNAi efficiency. The level of Rap1 expression in Rap1 miRNA-transfected cells was reduced significantly compared with those in empty plasmid transfected group, and control group at 24, 48 and 72 h after the transfections (Fig. 1). The results suggested that miRNA transfection led to specific suppression of Rap1 protein expression.

The anti-apoptotic effect of Rap1 in HepG2 cells was subsequently confirmed by TUNEL assay and flow cytometric analysis. The flow cytometric analysis revealed that of Rap1 knockdown cells had a significantly higher rate of apoptosis at 48 and 72 h after the transfection when compared with empty plasmid group and control group (P<0.05). At 48 h, 15.8±0.5% of adherent cells were apoptotic in Rap1 miRNA group, whereas 1.1±0.2% and 0.3±0.1% of adherent cells were apoptotic in empty plasmid group and control group, respectively. At 72 h, Rap1 miRNA induced apoptosis in ~33.0±2.3% of the cells, empty plasmid induced apoptosis in only 8.2±1.6% of the cells compared with 0.6±0.1% cell apoptosis in control group (Fig. 2). To gain further information about the morphological changes, the cells from the three groups were evaluated of apoptosis with electron microscopy at 48 h after the transfection. Electron microscopy revealed that some shrinking cells with a ring of condensed chromatin at the interior surface of the nuclear envelope in Rap1 miRNA group (Fig. 3), which was consistent with the result of flow cytometric analysis. Take together, these results suggested Rap1 miRNA increased apoptosis in HepG2 cells.

Next, we investigated whether Rap1 miRNA would enhance chemosensitivity to 5-FU in HepG2 cells. HepG2 cells were treated with 0, 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 μM 5-FU for 24 at 48 h after the transfection. The cell viability was evaluated using the MTT assay. MTT assays revealed the Rap1 miRNA-transfected cells showed increased sensitivity to 5-FU as compared with the empty plasmid-transfected, and the control cells (Fig. 4).

We next investigated the mechanism responsible for the anti-apoptotic effect of Rap1 in HepG2 cells. We examined the expression of NF-κB p65 and IκB at mRNA level at 48 h after the transfection. Quantitative real-time RT-PCR analysis revealed that the NF-κB p65 mRNA was downregulated significantly, whereas the IκB mRNA was greatly upregulated in Rap1 miRNA-transfected cells compared with those in control cells (P<0.05) (Figs. 5 and 6), suggesting that NF-κB was suppressed in Rap1 miRNA-transfected HepG2 cells. These results indicate that Rap1 plays an anti-apoptotic role via the NF-κB/IκB pathway.

To determine the efficacy of Rap1 miRNA alone or combined with 5-FU in vivo, we used a BALB/c murine xenograft tumor model with Rap1-deleted and control HepG2 cells. The size of the tumors was measured in three dimensions twice a week for 3 weeks, and the tumor volume was calculated. Mean tumor volume in Rap1-miRNA group was 851.2±32.6 mm³ compared to the average tumor volume of 1494.7±126.2 mm³ in control group at the termination of the experiment (P<0.05). Rap1 miRNA combined with 5-FU treatment led to a reduction of tumor growth even further as compared with 5-FU alone (624.9±45.3 mm³ vs. 1424.0±108.6 mm³; P<0.05) (Fig. 7). The results suggested that Rap1 miRNA inhibited liver cancer progression and enhanced sensitivity of HepG2 cells to 5-FU in vivo.

We also investigated the expression of Rap1 and caspase-3 expression using immunohistochemical staining in harvested tumors. Immunohistochemical staining analyses revealed that Rap1 expression was significantly inhibited in Rap1 miRNA tumors, whereas Rap1 overexpression was observed in control tumors (Fig. 8). Caspase-3 expression was positive in Rap1 miRNA treated tumors, and negative in the controls (Fig. 9). The results demonstrated that knockdown of Rap1 dramatically enhanced caspase-mediated apoptosis in the mouse model of HCC.