In an earlier gene expression study of hepatocellular carcinoma, CDC25B was upregulated in tumor compared to non-tumor samples. We used quantitative real-time PCR assays to validate the observed upregulation of CDC25B in hepatocellular carcinoma using 24 pairs of HCC tissues and adjacent non-tumor tissues. CDC25B was found to be expressed at significantly higher levels in HCC (median of 0.443) compared to adjacent non-tumors (median of 0.149) (P < 0.001) (Fig. 1A). Further statistical analyses showed no significant correlations between CDC25B transcript over-expression and clinicopathological features of the specimens.

Immunohistochemistry of liver tissue arrays further confirmed the over-expression of CDC25B protein in HCC tissues compared to non-tumor liver tissues (P < 0.001) (Table 1, Fig. 1B). Whereas only 1/42 (2.4%) non-tumor tissues showed greater than intermediate signal, 142/248 (57.3%) of HCC tissues showed intermediate or strong signal. Staining of CDC25B was observed in the cytoplasm. The lack of detailed clinical information from these archived pathology specimens precluded further analyses of associations between CDC25B protein over-expression and other clinicopathological features.

To validate the potential of CDC25B as a therapeutic target for HCC, our approach was to use siRNA oligos to deplete CDC25B expression in HCC cells, and to study the biological effects of this suppression. Three CDC25B-specific siRNA (siRNA1-CDC25B, siRNA2-CDC25B, and siRNA3-CDC25B) and negative control siRNA-N (all at 50 nM) were transfected into Hep3B and Hep40 cells (2 tumorigenic cell lines that over-express CDC25B based on Western blot results). Western blotting confirmed suppression of CDC25B expression in CDC25B siRNA transfected cells, with greatest suppression by siRNA2-CDC25B. Cells transfected with siRNA-N or RNAiMAX alone had no effects on CDC25B expression (data not shown). We therefore chose to use siRNA2-CDC25B for all subsequent experiments.

Transfection with siRNA2-CDC25B caused a dose-dependent decrease in CDC25B mRNA levels 48 hours post-transfection (Fig. 2), with greatest suppressions at 50 nM and 100 nM concentrations in both Hep3B (Fig. 2A) and Hep40 cells (Fig. 2B). At 50 nM siRNA2-CDC25B, mRNA levels were suppressed by up to 86% in Hep3B cell lines and 90% in Hep40 cell line by siRNA2-CDC25B. Since 100 nM did not cause greater suppression than 50 nM, we used 50 nM siRNA2-CDC25B for all subsequent experiments.

We next studied the effects of CDC25B suppression on the growth and invasive properties of HCC cell lines. Hep3B and Hep40 cells were transfected with siRNA-N or siRNA2-CDC25B, and cell growth was assessed daily over 4 days. Both cell lines transfected with siRNA2-CDC25B showed significantly slower growth rates than untransfected or siRNA-N transfected cells (P < 0.01 at 72 hours and 96 hours; Fig. 3A). This suggests that CDC25B mediates cell proliferation in HCC cells, and that its suppression led to growth inhibition (Fig. 3A).

Additionally, Hep3B and Hep40 cells transfected with siRNA2-CDC25B showed significantly reduced ability to migrate across non-Matrigel coated control membranes (P < 0.01; Fig. 3B) and to invade through Matrigel coated membranes (P < 0.01; Fig. 3B). After siRNA2-CDC25B transfection, migration of Hep3B and Hep40 cells were decreased by 4 and 2-folds respectively, and invasion were decreased by 4 and 5-folds respectively, relative to cells transfected with control siRNA-N. These findings indicate that silencing of CDC25B has significant inhibitory effects on cell invasion and migration.

To monitor the effect of CDC25B siRNA targeting on the cell cycle, we analyzed the DNA content of siRNA-transfected Hep3B and Hep40 cells by FACS at 48 hours after transfection. After siRNA2-CDC25B transfection, the percentage of cells in G2 or M phase was increased by 1.6 and 1.9 folds in Hep3B or Hep40 cells respectively (Fig. 4). Cells transfected with siRNA-N showed no changes in cell cycle distribution. The results demonstrated that RNAi-mediated reduction of CDC25B level leads to a delay in the G2/M transition.

We next studied the in vivo effects of CDC25B suppression on tumor growth. Hep40 xenografts were grown in nude mice, and mice were given intratumoral injections of siRNA2-CDC25B and RNAiMAX (n = 5) or RNAiMAX alone (n = 5) every 2 days. All mice in the siRNA2-CDC25B group had a significantly smaller tumor size compared with that observed in RNAiMAX group (P < 0.05 from day 7 onwards, P < 0.01 from day 15 onwards) (Fig. 5).

The human HCC cell lines Hep3B and Hep40 were purchased from ATCC (Manassas, VA). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC, Manassas, VA), supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 100 μg/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO².

Quantification of CDC25B transcript (from both patient specimens and cell lines) was performed using the MX3000P Real-Time PCR System (Stratagene, La Jolla, CA) as previously described 48. Human CDC25B (Assay ID: Hs01550934_m1) and 18s rRNA (normalization control) primers and probe reagents were purchased as Pre-Developed TaqMan Gene Expression Assay reagents from Applied Biosystems (Foster City, CA, USA). Transcript quantification was performed in at least duplicate for every sample. The amount of CDC25B was normalized with 18s rRNA to control for RNA amount variation. Paired (HCC and adjacent non-tumor liver) tissues were obtained from 24 patients who underwent liver resection for HCC. This study was approved by the Institutional Review Board for the use of human subjects in medical research, and informed consent was obtained from patients prior to liver resection.

Immunohistochemistry was performed using DAKO Envision Plus Kit. (Dako, Carpinteria, CA, USA). Rabbit polyclonal affinity-purified antipeptide antisera against CDC25B were generated by Applied Genomics Inc. (Sunnyvale, CA, USA). We used four tissue microarrays for this study, which consisted of archived tissues retrieved from surgical pathology files. In total, there were 42 normal livers and 248 malignant hepatocellular carcinomas, among other control tissues. Tissue arrays were constructed as previously described 49 with core sizes ranging from 0.6 to 2 mm on different arrays. Arrays were scored using a four-tier scale: 0 – negative (no staining), 1 – weak signal (weak staining in < 50% of cells), 2 – intermediate signal (weak staining in ≥ 50% or strong staining in < 50% of cells), 3 – strong signal (strong staining in ≥ 50% of cells).

CDC25B-specific siRNAs and Silencer negative control #1 (siRNA-N) were purchased from Ambion, Inc. (Austin, TX). The sequences for CDC25B-specific siRNAs are:

siRNA1-CDC25B (GCCGGAUCAUUCGAAACGATT),

siRNA2-CDC25B (GGAAAAGGACCUCGUCAUGTT),

siRNA3-CDC25B (GCUCUUACUCUUUCCUAUUTT).

The transfection reagent RNAiMAX (Invitrogen, Carlsbad, CA) was used to transfect these siRNAs into Hep3B and Hep40 cells, using the reverse transfection method according to the manufacturer's instructions.

Transfected cells were harvested and lysed in PARIS lysis buffer purchased from Ambion, Inc. (Austin, TX). Immunoblotting was done with rabbit polyclonal antibody against CDC25B at 250 fold dilution. (Applied Genomics, Inc., Burlingame, CA).

For the assessment of cell growth, Hep3B and Hep40 cells transfected with 50 nM siRNA2-CDC25B or siRNA-N were plated on a 96-well plate in triplicate wells. Cell growth was determined by the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's protocol. Optical density (OD) was read at 490 nm at various time points using a SAFIRE microplate reader (TECAN, Research Triangle Park). Once cells have attached (about 4 hours after transfection), a background OD value was obtained; the corresponding background values were then subtracted from data obtained from each well. Three independent experiments were done.

Invasion assays were done using the BD Biocoat Matrigel chamber in 24-well plates (BD Biosciences Labware, Bedford, MA). Transfected cells were added to coated filters (5 × 10⁴ cells/filter) in 500 μl of serum-free DMEM in triplicate wells. In the lower compartments of the chambers, a volume of 750 μl of chemoattractant (DMEM with 20% FBS) was added. After 48 hours incubation at 37°C in a 5% CO² incubator, the non-invading cells on the upper surface of the filter were wiped off using a cotton swab. Cells that invaded through the filters were fixed, stained with Diff-Quik Stain Set (Dade Behring Inc., Newark, DE, USA), and counted under the microscope by randomly selecting 6 fields per filter (×100 magnification). The migration assay was done in a similar manner but without the Matrigel coating on the filters. Two independent experiments were done for each assay.

Cells were transfected with Silencer negative control siRNA-N and siRNA2-CDC25B and then plated on 12-well chamber. Two days after transfection, the floating cells were collected and the adherent cells were detached by trypsin treatment. The floating and detached cells were combined, washed twice with PBS containing 1% BSA, and then were exposed to 70% ethanol at 4°C overnight. After resuspending in PBS containing 1% BSA, cells were subsequently digested by RNase in PBS (100 μg/mL) for 30 min on 37°C, and then stained with propidium iodide (PI) (50 μg/ml in PBS). After incubation for 30 min on ice in the dark, cells were analyzed by flow cytometry (BD FACScan). Cell cycle data were analyzed by Flowjo software.

All animal experiments were approved by the Administrative Panel on Laboratory Animal Care of Stanford. Ten nude mice (Athymic nu/nu, Taconic, NY), ages 4 to 6 weeks (about 20 g of weight) were randomly divided into two groups. Hep40 cells were harvested and mixed with an equal volume of Matrigel (BD Biosciences, Bedford, MA). Cells (10 million) were inoculated into the flank of nude mice. After 50 days, when the average tumor volume (V) reached 200 mm³ in each group, the mice were given intratumoral injections of 5 μg siRNA2-CDC25B and RNAiMAX in 30 μl PBS or RNAiMAX alone in 30 μl PBS (as control) every two days. Growth curves were plotted using average tumor volume within each experimental group at the set time points. The tumor dimensions were measured before each injection using a digital caliper, and the tumor volume calculated using the formula: V = π/6 × larger diameter × (smaller diameter)².

The statistical analyses were aided by the SPSS version 15.0 software package (SPSS Inc., Chicago, IL, USA). Statistical significance was determined by one-way ANOVA, independent-samples T-Test, or Fisher's Exact Test. P values of less than 0.05 and 0.01 were considered statistically significant and highly significant, respectively.