A cohort of 96 patients (57 men and 39 women) with CRC diagnosed between March 2012 and January 2014 were selected. Patient consent and approval from the Ethics Committee of Affiliated Hospital of Weifang Medical University (Weifang, China) were obtained prior to use of these clinical materials for research. Tumor and paired normal samples (10 cm from colorectal tumor) were surgically obtained from these patients. No local or systemic treatment was administered in these patients before surgery. In each selected case, pathological diagnosis was performed in the Department of Pathology, Affiliated Hospital of Weifang Medical University.

The human colorectal cancer cell lines (HCT116, SW480, SW620, LoVo, CaCo-2 and HT-29) were purchased from the American Type Culture Collection (Manassas, VA, USA). SW620 and SW480 cells were cultured in Leibovitz's L-15 media (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA); Caco-2 and HT-29 cells were maintained in RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc.) with 10% FBS, and LoVo cells were cultured in Ham's F-12 Kaighn's medium (Invitrogen; Thermo Fisher Scientific, Inc.) with 10% FBS.

Formalin-fixed, paraffin-embedded tissues were cut into 3-µm sections. The tissues were obtained from the Department of Pathology of the Affiliated Hospital of Weifang Medical University, where the formalin fixation process was carried out. The immunohistochemistry steps were performed using streptavidin-biotin peroxidase complex immunostain kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China) according to the manufacturer's protocols. The sections were incubated in a moist chamber with primary rabbit anti-human FOXM1 monoclonal antibody (cat. no., sc-501; dilution, 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 30 min at room temperature, followed by a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. SA00001-2; (dilution, 1:2,000; Proteintech Group, Inc., Chicago, IL, USA) for 30 min at room temperature. Rabbit serum (HyClone, Logan, UT, USA) served as a negative control. The images were collected and analyzed using a Leica DC200 image system (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Five random microscopic fields (magnification, ×200) were examined per slide, and 100 cells were evaluated per field. The expression of FOXM1 was classified into five groups according to the percentage of positively staining cells: 0=absent; 1=1–25%; 2=26–50%; 3=51–75%; and 4=≥76%. The staining intensity was categorized as follows: 0=negative; 1=weak; 2=moderate; and 3=strong. The proportion and intensity scores were subsequently multiplied to obtain a total score. Where the product of multiplication between staining intensity and the percentage of positive cells was ≤4, it was defined as low expression; by contrast, where the overall score was >4, it was defined as high expression.

Three FOXM1 siRNAs were designed and synthesized by Qiagen GmbH (Hilden, Germany) to generate effective FOXM1-siRNA oligonucleotides for gene knockdown studies. An siRNA with the sequence CUCUUCUCCCUCAGAUAU AdTdT was determined to exert the greatest effect on inhibition of FOXM1 expression. The LoVo CRC cells were transfected with FOXM1-siRNA oligonucleotides or control siRNA oligonucleotides (50 nmol/l; Santa Cruz Biotechnology, Inc.) with the use of Lipofectamine^® 2000 Transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols.

Total RNA was isolated from the cell lines using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration and quality of the extracted total RNA was determined by measuring the optical density at 260 and 280 nm, and then calculating the OD260:OD280 ratio. Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using a Reverse Transcription System kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Briefly, the samples were pre-incubated at 70°C for 10 min, cooled on ice and then added to a reaction mixture consisting of 10 mmol/l deoxynucleotide triphosphate, 25 mmol/l MgCl2, 15 units avian myoblastosis virus reverse transcriptase, 10X reverse transcription buffer, 0.5 units RNasin^® and 0.5 µg oligo-(dT)₁₅ primer (all provided with the Reverse Transcription System kit). A final volume of 20 l reaction mixture was incubated successively at 44°C for 15 min, 99°C for 5 min and 4°C for 5 min. The cDNA was maintained at 20°C prior to use. Then, it was reverse-transcribed from 5 µg of total RNA. RT-qPCR was performed using a SYBR Master Mix (Takara Bio Inc., Otsu, Japan) on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primer sequences of FOXM1 were as follows: Sense, 5′-GGAGGAAATGCCACACTTAGCG-3′; and reverse, 5′-TAGGACTTCTTGGGTCTTGGGGTG-3′ (designed by Guangzhou Ruibo Trading Co., Ltd., Guangzhou, China). The products of PCR were separated by 1.5% agarose gel electrophoresis, and the expression levels of the mRNA were detected, with GAPDH serving as the control. The primer sequences for GAPDH were as follows: Sense, 5′-GCAGTGGCAAAGTGGAGATT-3′; and reverse, 5′-TGAAGTCGCAGGAGACAACC-3′. The cycling conditions used were as follows: 94°C for 2 min for 2 cycles, followed by 40 cycles of 94°C for 15 sec, 58°C for 25 sec, and 72°C for 30 sec. qPCR was conducted on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each experiment was performed three times, each time in duplicate. The relative expression levels of Trop2 mRNA were calculated using the 2^−ΔΔCt method (16).

Whole cell lysates were prepared from the colon cells as described previously (17). Standard western blot analysis of the lysates was conducted with the primary rabbit anti-human FOXM1 monoclonal antibody and a second anti-IgG antibody (GE Healthcare Life Sciences, Chalfont, UK). The membranes were then stripped and blotted, and an anti-β-actin antibody (Sigma-Aldrich) served as a loading control. Blots were visualized using an enhanced chemiluminescence kit (Santa Cruz Biotechnology., Inc.) according to the manufacturer's protocol, and exposed to film (X-OMAT BT; Kodak, Rochester, NY, USA).

The effects of FOXM1 on CRC cell survival were determined using the 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Four groups of cells were seeded into 96-well plates (Nunc A/S Plastfabrikation, Roskilde, Denmark; 5×10³ cells/well) and cultured for 120 h. Following treatment, cells were incubated with MTT (20 µl/well; Sigma-Aldrich) at 37°C for 4 h, and then 200 µl dimethyl sulfoxide was added to each well. The absorbance of the cells was measured at a wavelength of 570 nm using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The percentage of residual cell viability was determined by: [(optical density (OD) of experimental group − OD of blank group)/(OD of negative group − OD of blank group)] ×100%. The assays were performed three times.

Motility in vitro was assessed using Transwell^® chambers (Corning Incorporated, Corning, NY, USA). Four groups of cells (5×10⁵) were seeded on the upper wells with Ham's F-12K Kaighn's medium. Medium (500 µl) with 20% FBS was plated in the bottom wells as chemoattractants. Following a 48-h incubation, cells were fixed with methanol and stained with 1% crystal violet (Sigma-Aldrich) for 30 min at 37°C. Cells on the upper filter of the membranes were wiped, while cells that had penetrated to the lower filter were stained with hematoxylin (Sigma-Aldrich). Cells in 15 randomized fields were counted and images were captured using an inverted microscope (magnification, ×50; Olympus BX51; Olympus Corp., Tokyo, Japan).

Data analyses were performed using SPSS version 15.0 (SPSS, Inc., Chicago, IL, USA). Patient characteristics were expressed as the mean ± standard deviation for continuous variables, and as the count and percentage for discrete variables. The data were analyzed using the Pearson's chi-square test and Fisher's exact test, and P<0.05 was considered to indicate a statistically significant difference.