Archived formalin-fixed, paraffin-embedded undifferentiated NPC tissue blocks were prepared from 81 patients with NPC who underwent biopsy surgery between January 2012 and November 2014 at the Radiation Oncology Department of Fudan University Cancer Hospital (Shanghai, China). Firstly, a pathologist marked the areas of tumor on the tissue block subsequent to reviewing a hematoxylin and eosin-stained section from each specimen. Single 2-mm diameter core tissues were taken from each paraffin-embedded tissue and placed into a recipient block. A total of 81 tissue sample cores were arrayed in each single block. A retrospective chart review of all patients was performed to collect data on clinical characteristics, including age, sex, pathological features and disease stage (T and N) according to the 2010 TNM classification of the American Joint Committee on Cancer Staging system (12). The histological type of all samples was undifferentiated. Approval was obtained from the Ethics Committee of Fudan University Cancer Hospital and all patients provided written prior informed consent.

For IHC analysis, TMA sections were deparaffinized in xylene, and endogenous peroxidase activity was blocked with methanol and 3% H₂O₂ for 15 min. For antigen retrieval, 4-µm thick TMA sections were boiled in a pressure cooker at ~120°C in citrate buffer (pH 6.0) (Beyotime Institute of Biotechnology, Haimen, China) for 3 min. Tissues were incubated for 1 h at room temperature with the following primary antibodies: Monoclonal rabbit anti-ROCK1 (cat. no. ab45171; 1:200; Abcam, Cambridge, MA, USA) or monoclonal rabbit anti-PIK3CA (cat. no. ab152155; 1:200; Abcam). The secondary antibody used was goat anti-rabbit horseradish peroxidase (HRP) (cat. no. K500711; ready-to-use; Dako; Aligent Technologies, Inc., Santa Clara, CA, USA). Immunostaining of tissues was evaluated independently by two trained pathologists who were unaware of the clinical background of the samples. For each case, five random fields at 400x magnification were captured by an Olympus IX73 microscope (Olympus Corporation, Tokyo, Japan) and digital camera (Olympus Corporation) using CellSens Dimension software (version 1.9; Olympus Corporation).

ROCK1 and PIK3CA immunoreactivity were analyzed using a semi-quantitative scoring system in which only cytoplasmic membrane staining was considered. Based on the percentage of positive cells, the level of ROCK1 and PIK3CA expression was classified as (+++) if ≥76% of cells were stained, (++) if 26–75% of cells were stained, (+) if ≤25% of cells were stained, and (−) if cells completely lacked membranous staining. Thus, (−) and (+) cases were classified as exhibiting low protein expression, whereas (++) and (+++) cases were classified as exhibiting high protein expression (Table I).

The human nasopharyngeal epithelial cell line NP69 and the NPC cell line 5–8F were donated by the Sun Yat-sen University Cancer Center (Guangzhou, China). Following passage, cells were grown for 48 h in RPMI-1640 medium (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc.) at 37°C in an atmosphere containing 5% CO₂. Following passage, the NP69 cell line was maintained in keratinocyte serum-free medium supplemented with human epidermal growth factor and bovine pituitary extract (cat. no. 17005-042; Gibco; Thermo Fisher Scientific Inc.) in a humidified atmosphere containing 5% CO₂ at 37°C for 72 h.

Cells were washed twice with ice-cold PBS and resuspended in 1 ml lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific Inc.). Cell debris was removed by centrifugation (12,000 × g; 4°C; 10 min) and the protein concentration of the supernatant was subsequently determined using the Bradford method (13). Aliquots that contained 30 µg total protein were separated by SDS-PAGE (6–10% gradient gels) and proteins were transferred onto polyvinylidine diflouride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% bovine serum albumin (Biosharp, Hefei, China) and incubated at 4°C for 18 h with the following monoclonal antibodies: Rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam), rabbit anti-PIK3CA (cat. no. ab152155; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. ab8227; 1:1,000; Abcam). Membranes were subsequently washed three times with 0.1% Tween-20 in TBS and incubated for 2 h at room temperature with HRP-conjugated secondary antibody (cat. no. sc2301; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Immunoreactive protein bands were visualized using an electrochemiluminescence system (GE Healthcare Life Sciences, Chalfont, UK). To quantify the protein expression by Western blotting analysis, densitometric analyses were performed using ImageJ version 1.46 software (imagej.nih.gov/ij/). Experiments were performed at least three times.

The associations between clinicopathologic variables and ROCK1 and PIK3CA protein expression were examined using the χ² test. Continuous data were compared using a Student's t-test if the distribution was normal, or a one-way analysis of variance if distribution was asymmetrical. All data were analyzed using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. All P-values were based on two-tailed tests.

Expressions of ROCK1 and PIK3CA in clinical NPC tissues were evaluated using IHC. Representative images of the immunohistochemical staining patterns are displayed in Fig. 1. ROCK1 and PIK3CA expression was observed in the cytoplasm, with high ROCK1 expression detected in 28.40% (23/81) of the NPC specimens and high PIK3CA expression detected in 62.96% (51/81) of the NPC specimens.

To assess the association between expressions of ROCK1 and PI3KCA and NPC, NP69 and 5–8F cells were used. ROCK1 and PIK3CA protein levels were demonstrated to be significantly higher in 5–8F cells compared with NP69 cells (Fig. 2). Expression of ROCK1 (relative to β-actin) in NP69 and 5–8F cells was 0.326±0.078 and 1.191±0.114, respectively (P<0.001). Expression of PIK3CA (relative to β-actin) in NP69 and 5–8F cells was 0.696±0.134 and 1.159±0.144, respectively (P=0.015). Similar results were obtained from at least three independent western blots.

The associations between ROCK1 and PIK3CA expression and the clinicopathological features of NPC are displayed in Table I. High expression of ROCK1 was significantly associated with advanced N stage (P=0.032). High expression of PIK3CA was significantly associated with advanced N stage (P=0.027) and TNM cancer stage (P=0.019). Furthermore, χ² and correlation analyses demonstrated that ROCK1 expression was significantly positively correlated with PIK3CA expression, (P=0.01; r=0.313; Table II). No statistically significant associations were observed between ROCK1 and PIK3CA expression and patient gender, age or T stage.