Based on tissue data availability, 368 cases of CRC, between 2003 and 2007, were included in the present study. Clinicopathological features of the patients with CRC are shown in Table I. Formalin-fixed paraffin-embedded tissues were collected from the Second and First Affiliated Hospitals of Hunan University of Chinese Medicine (Changsha, China). This study was approved by the Ethics Committees of Hunan University of Chinese Medicine. Informed consent was obtained from all participants and the study was performed in accordance with the Declaration of Helsinki. Follow-up data were obtained from medical records and direct communication with the patients or their relatives. The follow-up period was defined as the time from the date of surgery to the date of patient mortality or the final follow-up in January 2012.

Paraffin-embedded serial sections of CRC specimens were analyzed for PPM1D protein expression using an anti-PPM1D antibody (Abcam, Cambridge, MA, USA), as described previously (9,10). Negative control sections were incubated with pre-immunized rabbit serum (Abcam).

Immunostaining was assessed by two independent, blinded pathologists and scored by multiplying the intensity of the staining by the percentage of stained cells. The staining intensity was graded on a scale of 0 to 3. The percentage of stained tumor cells was graded as follows: 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). The final scores ranged from 0 to 12. For any subsequent analysis, scores between 0 and 4 were defined as low expression and those between 5 and 12 were defined as high expression (11). Inconsistent scores were re-evaluated by two pathologists until a consensus score was established.

Total RNA was extracted from cells using an RNA extraction kit (Qiagen Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Total RNA was used for quantification using the One-Step RT-PCR kit (Invitrogen Life Technologies, Carslbad, CA, USA) in a Roche Light Cycler (Roche Diagnostics, Basel, Switzerland). The primer sequences used in the study were as follows: PPM1D, 5′-CAATTGGCCTTG TGCCTACT-3′ (forward) and 5′-TCTTTCGCTGTGAGGTTGTG-3′ (reverse); and β-actin, 5′-CCTGTACGCCAACACAGTGC-3′ (forward) and 5′-ATACTCCTGCTTGCTGATCC-3′ (reverse). The PCR cycling conditions consisted of a denaturation step at 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 1 min. Samples were normalized using β-actin mRNA expression and relative expression was calculated using the ^ΔΔCt method.

Cells were lysed in lysis buffer and protein concentrations were measured using a BCA protein assay kit (Qiagen Co., Ltd.). Total protein was separated using SDS-PAGE on a 12.5% gel and electroblotted onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). Membranes were immunoblotted overnight at 4°C with primary antibodies against human PPM1D (1:1,000 dilution; Sigma-Aldrich, St. Louis, MO, USA) or β-actin (1:2,000 dilution; Sigma-Aldrich). Following three washes with Tris-buffered saline containing Tween 20, membranes were incubated with horseradish peroxidase-conjugated IgG secondary antibodies (1:2,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 2 h. Immunoreactive signals were detected using an enhanced chemiluminescence detection reagent (Pierce Biotechnology, Rockford, IL, USA). Expression levels were quantified from the images using Quantity One^® (Bio-Rad, Hercules, CA, USA). All experiments were performed in triplicate.

HCT-116, RKO and COLO-320 CRC cell lines were purchased from American Type Culture Collection (Rockville, MD, USA). Cells were maintained in RPMI-1640 in 5% CO₂ at 37°C. PPM1D siRNA and scrambled siRNA were purchased from Invitrogen Life Technologies and transfected using Lipofectamine^® 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions.

Cell viability was assessed using an MTT assay. Following transfection, cells were plated in 96-well plates and incubated for 24, 48 and 72 h. A total of 20 μl 5 mg/ml MTT (Sigma-Aldrich) was added to each corresponding test well and incubated for 4 h at 37°C. The supernatant was then discarded and 200 μl dimethyl sulfoxide was added to each well to dissolve the formazan. Optical density was assessed by measuring the absorbance of each well at 490 nm using a spectrophotometer (SpectraMax Plus384; Molecular Devices, Sunnyvale, CA, USA). All experiments were performed in triplicate.

At 12 h after transfection, the mixed medium containing Lipofectamine 2000 was discarded and cells were maintained in serum-free RPMI-1640 medium overnight. Cell invasion was then assessed using a 24-well Cell Invasion assay (BD Biosciences, San Jose, CA, USA) with 8 μm pores according to the manufacturer’s instructions. Cells were incubated for 72 h. The upper compartment was coated with 50 μg Matrigel™ (BD Biosciences) and the lower compartment was filled with medium containing 10% fetal calf serum as a chemoattractant. Spectrophotometry was conducted using a microtiter plate reader (SpectraMax Plus384; Molecular Devices) at 540 nm. All assays were performed in triplicate.

All statistical analyses were performed using SPSS 16.0 software (SPSS, Inc, Chicago, IL, USA). The χ² test was performed for categorical data. Kaplan-Meier estimates, log-rank tests and multivariate Cox proportional hazard regression models were performed for survival analyses. A value of P<0.05 was considered to indicate statistical significance.

PPM1D expression was analyzed in 368 CRC tissue samples and paired non-cancerous colorectal tissue samples. Among the 368 CRC tissues, 68.48% (252/368) of cases demonstrated high PPM1D expression, while only 9.24% (34/368) of the matched, non-cancerous colorectal tissue samples showed high PPM1D expression (P<0.001). Immunohistochemistry revealed a predominantly nuclear localization of PPM1D (Fig. 1) and showed that PPM1D expression was significantly higher in CRC tissue than in non-cancerous, normal colorectal tissue. In addition, significant differences in PPM1D expression were observed between tumors with node metastasis (P=0.0024), distant metastasis (P<0.001) and different TNM stages (P=0.0016; Table I). No significant correlation was observed between PPM1D expression and patient age or gender or tumor location or histology (Table I).

Kaplan-Meier survival analysis revealed that the patients with low PPM1D expression had significantly longer survival than those with high PPM1D expression (log-rank, P<0.001; Fig. 2A). The correlation between metastatic lymph node PPM1D expression and patient survival was also assessed. Kaplan-Meier analysis revealed that patients with low PPM1D expression in the metastatic lymph nodes had significantly longer overall survival than patients with high metastatic lymph node PPM1D expression (Fig. 2B, log-rank P=0.009).

Furthermore, multivariate analysis was performed using the Cox proportional hazards model. High PPM1D expression was identified to be a significant independent prognostic factor for OS (hazard ratio=0.24; 95% confidence interval, 0.13–0.86; P=0.004) as shown in Table II.

PPM1D expression was analyzed in three CRC cell lines: HCT-116, RKO and COLO-320. PPM1D expression was observed to be higher in RKO and COLO-320 cells than in HCT-116 cells (Fig. 3A). Based on this finding, RKO and COLO-320 cells were used for the subsequent functional analysis.

siRNA-induced PPM1D knockdown was confirmed using qPCR and western blot analyses (Fig. 3B). PPM1D siRNA was observed to significantly reduce proliferation and invasion in RKO and COLO-320 CRC cells (Fig. 3C and D).