A total of 90 samples of CRC and matched tumor-adjacent tissues were collected from the Department of Pathology, The Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between January 2007 and January 2009. The demographic features and clinicopathological parameters are presented in Table I. All specimens had a confirmed pathological diagnosis and were classified according to International Union Against Cancer and American Joint Committee on Cancer criteria (7th edition) (17). Patients did not receive preoperative chemotherapy, biotherapy or molecular targeted therapy. All samples were used after obtaining informed consent from the patients. The Xi'an Jiaotong University Ethics Committee approved all protocols, according to the Declaration of Helsinki (as revised in Tokyo, 2004).

The tissue of interest was fixed by immersing it in 10% neutral buffered formalin for 4–24 h at room temperature. The tissue was subsequently embedded in paraffin and the blocks were stored at 4°C prior to sectioning. The formalin-fixed and paraffin-embedded tissues from postoperative patients were cut into 5 µm sections. Sections were deparaffinized in xylene and rehydrated using a series of graded alcohols. Subsequently, tissue slides were subjected to antigen retrieval using 0.01 M sodium citrate (pH 6.0) at a sub-boiling temperature for 10 min and were blocked with 10% goat serum (ZSGB-Bio, Beijing, China). The samples were then incubated overnight at 4°C with anti-FOXA1 (1:100; cat. no. sc-101058; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibody. Immunostaining was performed using a SPlink Detection kit (cat. no. SP-9002; ZSGB-Bio) according to the manufacturer's protocol. Staining intensity was scored as follows: 0, negative; 1, weak; or 2, strong; and the extent of staining was based on the percentage of positive tumor cells: 0, negative; 1, 1–25%; 2, 26–50%; 3, 51–75%; or 4, 76–100% (18). Therefore, each case was ultimately considered negative if the final score was 0 (negative expression) or 1–2 (low expression), and positive if the final score was 3–5 (medium expression) or 6–8 (high expression). The final immunohistochemistry score for each sample (negative or positive) was calculated by multiplying the intensity score with the percentage of positive cells.

Two CRC cell lines, HCT116 and SW480 (The Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), were purchased for use in the present study. Cells were routinely cultured in complete Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 37°C in a humidified incubator containing 5% CO₂.

The targeted sequences for FOXA1 small interfering (si)RNA (sense 5′-GCACUGCAAUACUCGCCUU-3′) or a nonspecific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Cells were transfected with the aforementioned siRNAs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

The tissue was snap-frozen in liquid nitrogen and diced into 1 mm pieces with a scalpel in a mortar on dry ice. Total proteins were extracted using radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc.) and the protein concentrations were measured using the bicinchoninic acid assay. The proteins (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto a polyvinylidene fluoride membrane (Roche Diagnostics, Indianapolis, IN, USA). The membrane was then blocked with 5% skimmed milk at room temperature for 15 min and incubated with the following appropriate primary antibodies at 4°C overnight: FOXA1 (1:1,000), yes-associated protein (YAP; 1:1,000; cat. no. PA1-46189; Thermo Fisher Scientific, Inc., Rockford, IL, USA), caspase-3 (1:1,000; cat. no. 9662; Cell Signaling Technology, Inc., Danvers, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000; cat. no. sc-25778; Santa Cruz Biotechnology, Inc.). Blots were then incubated with horseradish peroxidase-conjugated goat anti-mouse/rabbit immunoglobulin G secondary antibodies (1:5,000; cat. nos. CW0102/103; Cwbiotech, Shanghai, China) at room temperature for 2 h. Blots were detected using enhanced chemiluminescence regents (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and semi-quantified by ImageJ software (1.46; National Institutes of Health, Bethesda, MD, USA).

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA extraction following tissue homogenization. cDNA synthesis was performed using PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan) at 37°C for 15 min, following treatment with DNase I (Invitrogen; Thermo Fisher Scientific, Inc.). Amplification and detection of FOXA1 and GAPDH mRNA were performed using an ABI PRISM 7300 Sequence Detection system (Applied Biosystems, Inc., Foster City, CA, USA) and a One Step SYBR^® PrimeScript™ RT-PCR kit II (Takara Bio, Inc.) according to the manufacturers' protocols. The following primers (Sangon Biotech (Shanghai) Co., Ltd.) were used: FOXA1, sense primer 5′-AAT CATTGCCATCGTGTG-3′, antisense primer 5′-CGCGGCTTAAAATCTGGTAT-3′; and GAPDH, sense 5′-CAAGCTCATTTCCTGGTATGAC-3′, and antisense 5′-CAGTGAGGGTCTCTCTCTTCCT-3′. The thermocycling conditions were as follows: 95°C for 60 sec; followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. GAPDH was measured as an internal control for mRNA. All samples were normalized to internal controls and fold changes were calculated based on relative quantification using the 2^−ΔΔCq method (13).

For the proliferation assay, CRC cells were seeded into 96-well plates at a density of 5,000 cells/well for 24 h and were assessed using Cell Proliferation enzyme-linked immunosorbent assay, 5-bromodeoxyuridine (BrdU) (chemiluminescent) (Roche Diagnostics) according to the manufacturer's protocol. Flow cytometric analysis was conducted using fluorescence-activated cell sorting (FACS)Calibur (BD Biosciences, San Jose, CA, USA) and Cell Quest Pro software (BD Biosciences). An Annexin-V-FLUOS Staining kit (Roche Diagnostics) was used to analyze the level of apoptosis according to the manufacturer's protocol. The percentage of apoptotic cells was calculated using the flow cytometry software.

Results are expressed as the mean ± standard error of the mean. All experiments were repeated three times. All statistical analyses were performed using SPSS statistical package for Windows version 13 (SPSS, Inc., Chicago, IL, USA) or GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). The quantitative data were compared between groups using the Student's t-test. Categorical data were analyzed using the Pearson chi-squared test. The Kaplan-Meier method and log-rank test were used to compare the cumulative recurrence and survival rates. The independent factors influencing the survival and recurrence of patients with CRC were determined using the Cox proportional hazards model. Correlation analysis was conducted using the Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

Immunohistochemical staining was performed to investigate differences in FOXA1 protein expression between CRC and adjacent noncancerous tissues. As shown in Fig. 1, FOXA1 staining was negative in the adjacent noncancerous tissue (Fig. 1A), whereas positive FOXA1 staining with nuclear location was observed in CRC tissues (Fig. 1B–D). FOXA1 expression was detected in 57.8% (52/90) of the CRC samples, whereas only 37.8% (34/90) of the noncancerous specimens exhibited a positive FOXA1 signal (P<0.05; Fig. 2A). Furthermore, 20 randomly selected cases were subjected to RT-qPCR in order to determine FOXA1 mRNA expression. The mRNA expression levels of FOXA1 were significantly increased in CRC tissues compared with in the adjacent noncancerous tissues (P<0.05; Fig. 2B). There results indicate an oncogenic role for FOXA1 in CRC.

To elucidate the clinical significance of FOXA1 expression in CRC, the present study investigated the association between FOXA1 expression and clinicopathological features of patients with CRC. As shown in Table I, positive FOXA1 expression in CRC tissues was associated with poor tumor differentiation (P=0.005), large tumor size (P=0.004), lymph node metastasis (P=0.040) and advanced tumor-node-metastasis stage (P=0.023). These results indicate that FOXA1 may promote the development and progression of CRC.

To further investigate the prognostic value of FOXA1 expression, the overall survival (OS) and recurrence-free survival (RFS) rates were compared between the FOXA1-positive group (n=52) and the FOXA1-negative group (n=38). Kaplan-Meier survival curves indicated that the positive expression of FOXA1 in CRC was significantly correlated with reduced OS (P=0.0191; Fig. 3) rate and RFS rates (P=0.0155; Fig. 3). Notably, a multivariate Cox regression analysis demonstrated that FOXA1 expression was an independent factor for predicting the OS and RFS of patients with CRC (P=0.005 and 0.017, respectively; Table II). These data indicate that FOXA1 expression is a promising predictor for patient prognosis in CRC.

To determine the functional role of FOXA1 in CRC, FOXA1 siRNA was used to inhibit the expression of FOXA1 in SW480 and HCT116 cells, and the proliferation and apoptosis of CRC cells was subsequently investigated. FOXA1 knockdown was confirmed by immunoblotting (P<0.05; Fig. 4A). Subsequently, BrdU incorporation assays demonstrated that the proliferation of SW480 and HCT116 cells was significantly decreased following FOXA1 knockdown (P<0.05; Fig. 4B). In addition, the percentage of apoptotic SW480 and HCT116 cells was significantly increased following suppression of FOXA1 (P<0.05; Fig. 4C). Western blot analysis indicated that FOXA1 knockdown evidently increased the protein expression of cleaved caspase-3 in SW480 and HCT116 cells (P<0.05; Fig. 4D).

Hippo-YAP signaling has been confirmed to have a fundamental role in the pathogenesis of CRC (19,20). Furthermore, inhibiting YAP expression has been reported to lead to a marked decrease in the proliferation and apoptosis of CRC cells (21). A recent study reported that FOXA1 could regulate YAP expression in HCC cells (13). Therefore, the present study investigated whether the functional effects of FOXA1 in CRC cells were mediated via the modulation of YAP expression. As expected, FOXA1 knockdown resulted in a significant decrease in YAP expression in SW480 and HCT116 cells (P<0.05; Fig. 5). These data indicate that FOXA1 may regulate YAP expression in CRC cells.