The study was approved by the institutional review board of Liaoning Medical University (Jinzhou, China). Primary tumor specimens were obtained from 135 patients diagnosed with astrocytoma who underwent resection in the First Affiliated Hospital of Liaoning Medical University between 2000 and 2005. The histological diagnosis was evaluated for sections stained with hematoxylin and eosin according to the World Health Organization classification guidelines. Clinical and histopathological data, including histopathological diagnosis and tumor grade, were extracted from medical records.

The U87 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and the A172 cell line was from the Shanghai Cell Bank (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen Life Technologies), 100 IU/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/ml streptomycin (Sigma-Aldrich). Cells were grown on sterilized culture dishes and were passaged every 2 days with 0.25% trypsin (Invitrogen Life Technologies).

On-TargetPlus SMARTpool CIP2A siRNA (#L-014135-01) and On-TargetPlus siControl (D-001810-01-20) were purchased from Dharmacon (Thermo Fisher Scientific, Waltham, MA, USA). For transfections, cells were seeded in plates 24 h prior to the experiment. Cells were transfected with siRNA using DharmaFECT 1 (0.20 μl/well; Thermo Fisher Scientific) according to the manufacturer’s instructions.

Sections (4-μm thick) were prepared from paraffin-embedded tissues. Immunostaining was performed by the streptavidin-peroxidase method (Ultrasensitive; MaiXin, Fuzhou, China). Sections were deparaffinized in xylene, rehydrated with graded alcohol and then boiled in citrate buffer (pH 6.0) for 2 min in an autoclave. Next, 0.3% hydrogen peroxide was applied to block endogenous peroxidase activity and the sections were incubated with normal animal serum to reduce nonspecific binding. Tissue sections were incubated with CIP2A rabbit polyclonal antibody (1:300 dilution; Novus Biologicals, LCC, Littleton, CO, USA) for 2 h at room temperature. Rabbit immunoglobulin (at the same concentration as the antigen-specific antibody) was used as a negative control. Staining was followed by incubation with biotinylated secondary antibodies. The peroxidase reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride. Counterstaining was performed lightly with hematoxylin and the sections were dehydrated in alcohol prior to mounting.

Immunostaining of CIP2A was scored on a semiquantitative scale by evaluating the intensity and percentage of tumor cells as described previously (10). We counted 400 tumor cells and calculated the percentage of positively stained cells. The intensity of CIP2A staining was scored as 0 (no signal), 1 (weak), 2 (moderate) and 3 (marked). Percentage scores were assigned as 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The scores of each tumor sample were multiplied to produce a final score of 0–12 and the tumors were finally determined as negative (−), 0; lower expression (+), ≤4; moderate expression (++), 5–8; and high expression (+++), ≥9. Tumor samples that scored (+) to (+++) were considered to overexpress CIP2A.

Quantitative real-time PCR was performed using the SYBR-Green PCR master mix in a total volume of 20 μl on a 7900HT Fast Real-Time PCR System (both Applied Biosystems, Bedford, MA, USA) as follows: 95°C for 30 sec and 40 cycles at 95°C for 5 sec and 60°C for 30 sec. A dissociation step was performed to generate a melting curve to confirm the specificity of the amplification. Expression levels of the analyzed genes were normalized against β-actin expression. Relative levels of gene expression were determined using the following formula: ΔCt = Ct_gene − Ct_ref and the fold change of gene expression was calculated by the 2^−ΔΔCt method. The primer sequences were as follows: CIP2A, 5′-ATACTTCAGGACCCACGTTTGAT-3′ (forward) and 5′-TCTCCAAGTACTAAAGCAGGA AAATCT-3′ (reverse); β-actin, 5′-ATAGCACAGCCTGGA TAGCAACGTAC-3′ (forward) and 5′-CACCTT CTACAATGAGCTGCGTGTG-3′ (reverse). Experiments were repeated in triplicate.

Total protein from tissue and cells was extracted in lysis buffer (Pierce Biotechnology, Inc., Rockford, IL, USA) and quantified using the Bradford method. Samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4°C with antibodies against CIP2A (1:1,000; Novus Biologicals, LCC), caspase-3, cleaved caspase-3, c-Myc, phospho-Akt, Akt, Bcl-2 (1:800, Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following incubation with peroxidase-coupled anti-mouse IgG (Santa Cruz Biotechnology, Inc.) at 37°C for 2 h, bound proteins were visualized using an ECL detection system (Pierce Biotechnology, Inc.) and detected using a BioImaging System (UVP Inc., Upland, CA, USA). Relative protein levels were quantified using β-actin as a loading control.

For the colony formation assay, A172 and U87 cells were transfected with siRNA for 48 h and plated into three 6-cm cell culture dishes (1,000 cell/dish). Cells were incubated for 12 days in medium containing 10% FBS. The plates were washed with phosphate-buffered saline (PBS) and stained with Giemsa. The number of colonies with >50 cells was counted manually using a microscope.

For the anchorage-independent colony growth assay ~2,000 cells/well were seeded in medium containing 0.5% agarose on top of bottom agar containing 1% low-melting agar in regular medium. After 14–21 days, colonies were stained with Giemsa and counted using a microscope.

For the MTT assay, cells were plated in 96-well plates in medium containing 10% FBS at ~3,000 cells/well 24 h following transfection. For quantification of cell viability, 20 μl MTT (5 mg/ml) solution was added to each well and incubated for 4 h at 37°C. The medium was removed from each well and the resulting MTT formazan was solubilized in 150 μl DMSO. Each solution was measured spectrophotometrically at 490 nm.

Cells (5×10⁵) were seeded into 6-cm tissue culture dishes. After 12 h, the cells were transfected with siRNA using DharmaFECT 1 (0.20 μl/well). For the detection of apoptosis, adherent cells were collected and resuspended in cold PBS for analysis. The cells were stained with the Annexin V-FITC Apoptosis kit (BD Pharmingen, San Diego, CA, USA) to monitor apoptotic cells and propidium iodide (PI) to detect dead cells. Data were collected using a BD FACSCalibur flow cytometer (San Jose, CA, USA).

SPSS version 11.5 for Windows was used for all statistical analyses (SPSS, Inc., Chicago, IL, USA). A χ² test was used to examine the possible correlations between CIP2A expression and clinicopathological factors. The Student’s t-test was used to compare densitometry data on focus numbers between control and CIP2A-transfected cells. All P-values are based on a two-sided statistical analysis and P<0.05 was considered to indicate a statistically significant difference.

In normal brain tissues, negative expression of CIP2A in astrocytes and positive cytoplasmic CIP2A expression in neurons was observed (Fig. 1A). Positive cytoplasmic CIP2A staining was observed in 75/135 (55.6%) human astrocytomas (Fig. 1B-E), while no staining was detected in sections from the same samples subjected to immunohistochemical analysis using non-immune rabbit immunoglobulin (Fig. 1F). The correlations between CIP2A protein expression and clinicopathological factors was investigated and no relationship was found between CIP2A expression and age and gender. The positive rates of CIP2A overexpression in grades I (Fig. 1B), II (Fig. 1C) and III astrocytomas (Fig. 1D) and grade IV astrocytoma/glioblastoma (Fig. 1E) were 11.1 (1/9), 47.0 (31/66), 76.2 (32/42) and 61.1% (11/18), respectively (Table I). The positive rate of CIP2A was identified to be significantly higher in high-grade astrocytomas than in those of low-grade (P<0.001).

To determine whether CIP2A enhances the proliferation of astrocytoma, CIP2A expression levels were analyzed in several astrocytoma cell lines and A172 and U87 cells were found to exhibit high levels of CIP2A expression (Fig. 2A). siRNA-mediated knockdown of CIP2A was performed in these cell lines. As demonstrated in Fig. 2B, CIP2A siRNA decreased the levels of CIP2A protein and mRNA in the A172 and U87 cells. The proliferation rate of the cells was determined by MTT assay. A172 and U87 cells treated with CIP2A siRNA exhibited a significantly slower growth rate than the vector control cells (Fig. 3A). Consistent with MTT results, the colony formation assay revealed that CIP2A-knockdown in A172 and U87 cells led to a marked decrease in focus numbers (A172: control 292±17 vs. CIP2A siRNA 157±26, P<0.05; U87: control 207±19 vs. CIP2A siRNA 119±6, P<0.05; Fig. 3B). To examine the impact of CIP2A on anchorage-independent cell growth, a soft agar colony formation assay was performed. CIP2A-knockdown reduced the colony numbers in soft agar (A172: control 257±22 vs. CIP2A siRNA 133±16, P<0.05; U87: control 199±21 vs. CIP2A siRNA 109±11, P<0.05)

Annexin V/PI analysis was employed to characterize the rate of apoptosis. As demonstrated in Fig. 3D, the populations of cells with CIP2A-knockdown that were observed to be undergoing early and late apoptosis (A172: 8.7%; U87: 13.2%) were significantly larger compared with scramble controls (A172: 4.6%; U87: 3.9%), demonstrating that CIP2A-knockdown results in the apoptosis of astrocytoma cells.

To investigate the underlying mechanism by which CIP2A affects proliferation and apoptosis, the effect of CIP2A-knockdown on several potential molecular targets was analyzed. As revealed in Fig. 4, western blot analysis demonstrated that the knockdown of CIP2A decreased the expression levels of c-Myc protein in the two cell lines. In addition, the levels of apoptosis-related caspase-3 and Bcl-2 expression were determined and it was observed that CIP2A depletion led to reductions in caspase-3 and Bcl-2 protein levels and increased the levels of cleaved caspase-3. In addition, Akt phosphorylation levels were markedly lower in the cells treated with CIP2A siRNA. Together, these results indicate that CIP2A regulates cell proliferation and apoptosis through modulation of c-Myc, Bcl-2 and Akt levels.