HCT116 and DLD1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle medium (DMEM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) and RPMI 1640 medium (Wako Pure Chemical Industries, Ltd.), respectively, supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaille, France) and antibiotics. Cells were authenticated by short tandem repeat analysis using GenePrint^® 10 System (Promega Corporation, Madison, WI, USA) in 2014. HEK293T cells (RIKEN BioResource Center, Tsukuba, Japan) were used to produce recombinant retroviruses and were maintained in DMEM with 10% FBS. The anti-TRIP13 antibody (cat. no. A303-605A) was obtained from Bethyl Laboratories (Montgomery, TX, USA) and the anti-Flag antibody (cat. no. 1E6) from Wako Pure Chemical Industries, Ltd. Cells were incubated with the antibodies for 1 h at room temperature for immunoblot analysis.

Full-length TRIP13 RNA was extracted from HCT116 cells using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). RNA was amplified by polymerase chain reaction (PCR) following reverse-transcription using PrimeScript Reverse Transcriptase (Takara Bio, Inc., Otsu Japan) and was cloned into the pQCXIP retroviral vector (Clontech Laboratories, Inc., Mountainview, CA, USA) with an N-terminal Flag tag. The following primer sequences were used to clone TRIP13: Forward, 5′-ACTATCTCGAGATGGACGAGGCCGTGGGCGAC-3′ and reverse, 5′-TCGATAGCGGCCGCTCAGATGTAAGCTGCAAGCTTC−3′. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio, Inc.). PCR was performed under the following conditions: Denaturation at 94°C for 10 sec, annealing at 55°C for 30 sec and extension at 72°C for 1 min. To generate a recombinant retrovirus, HEK293T cells were transfected with pQCXIP-Flag-TRIP13 together with pVPack-GP and pVPack-Ampho vectors (Promega Corporation) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Following 48 h, the supernatants were added to HCT116 or DLD1 cells in the presence of 2 µg/ml polybrene (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), and the infected cells were selected with 1 µg/ml puromycin for 3 days.

When the cells had reached 80% confluence, they were lysed using Laemmli sample buffer and boiled for 5 min. The protein concentrations of the lysates were measured using the RC-DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal protein quantities of the lysates were then separated on 0.01% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Merck Millipore). Next, the membranes were blocked with 0.5% skim milk followed by incubation with anti-TRIP13 (1:1,000 dilution) and anti-β-actin antibody (1:3,000 dilution; cat. no. A1978; Sigma-Aldrich; Merck Millipore) primary antibodies for 1 h at room temperature. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution; cat. nos. 7074 and 7076; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Blots were visualized using Chemi-Lumi One Super (Nacalai Tesque, Inc., Kyoto, Japan).

The siRNA sequences used to deplete TRIP13 were 5′-GCUGGUAACCAAGAUGUUUTT-3′ (siTRIP13-1), 5′-CCCAUCGAUUUGAGUGCAUTT-3′ (siTRIP13-2) and 5′-GGAUGCAUAAUGCCAGCAATT-3′ (siTRIP13-3). The sequence of the control siRNA targeting luciferase was 5′-CUUACGCUGAGUACUUCGATT-3′. A total of 20 nM of siRNAs were transfected into the cells using RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. All siRNAs were purchased from Hokkaido System Science Co., Ltd. (Sapporo, Japan).

To measure cell invasion using Boyden chambers, a filter was pre-coated with Matrigel, and 2×10⁵ HCT116 or DLD1 cells transfected with the aforementioned siRNAs were seeded onto the upper surface of the chamber. Cells were fixed with 70% methanol 24 h later and stained with 0.5% crystal violet. The cells that had invaded the lower surface of the filters were counted in five randomly selected fields. Three independent experiments were performed, and the data were represented as the mean ± standard deviation (SD).

Cell migration was evaluated using Boyden chambers. siRNA-transfected cells (5.0×10⁴) were seeded onto the upper surface of the chamber. The lower surface of the filter was coated with fibronectin (Sigma-Aldrich; Merck Millipore). Cells were fixed with 70% methanol 6 h later and stained with 0.5% crystal violet. The cells that had migrated to the lower surface of the filters were counted in five randomly selected fields in three independent experiments.

CRC tissue samples and normal colorectal tissue sections were obtained from patients who had undergone surgery at Nagoya University Hospital (Nagoya, Japan) between May 2010 and October 2014. The study was approved by the institutional review board of Nagoya University Hospital and conformed to the standards set by the Declaration of Helsinki. All participants provided written informed consent to participate in the study.

RNA was extracted from CRC samples and cells using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), and cDNA was generated using PrimeScript Reverse Transcriptase (Takara Bio, Inc.). The CRC tissue samples were obtained from patients at Nagoya University Hospital who had provided informed consent. qPCR was performed using the SYBR Premix Ex Taq™ II (Takara Bio, Inc.), and the Thermal Cycler Dice Real Time System TP800 (Takara Bio, Inc.) was used for the analysis. PCR was performed under the following conditions: 95°C for 10 sec and 60°C for 30 sec. The relative messenger RNA (mRNA) expression levels were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using LightCyclerR^® Nano software 1.0 (Roche Diagnostics, Tokyo, Japan). The sequences of the primers used to amplify each gene were 5′-AGGTGGAGGAGTGGGTGTCGCTGTT-3′ (forward) and 5′-CCGGGAAACTGTGGCGTGATGG-3′ (reverse) for GAPDH, and 5′-CTGTCTCTGGCAGTGGACAAG-3′ (forward) and 5′-TTGGTTTGCAGAAGGGATTC-3′ (reverse) for TRIP13.

Cells were cultured in 96-well plates, and the number of viable cells at the indicated times was evaluated using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan).

CRC cells were transfected with siRNAs, and EdU incorporation assays were performed 48 h later using a Click-iT^® Plus EdU Alexa Fluor^® 594 Imaging kit (Thermo Fisher Scientific, Inc.). Briefly, siRNA-transfected cells were incubated with 20 mM EdU for 24 h and fixed with formaldehyde. The cells were permeabilized with 0.5% Triton X-100 and stained with Hoechst in a reaction cocktail prepared according to the manufacturer's protocol. The cells were then imaged by fluorescence microscopy (BX60; Olympus Corporation, Tokyo, Japan), and the percentage of EdU-positive cells was evaluated.

Data were expressed as the mean ± SD. Comparisons between groups were performed using unpaired t-tests. P<0.05 was considered to indicate a statistically significant difference.

To examine whether TRIP13 has any role in CRC, the mRNA level of TRIP13 in CRC tissue specimens was first examined by RT-qPCR. As indicated in Fig. 1A, higher expression of TRIP13 was observed in multiple CRC samples compared with normal tissues. Next, the expression of TRIP13 in several CRC cell lines was examined. Immunoblot analyses demonstrated that all the cell lines expressed a similar amount of TRIP13 (Fig. 1B).

To determine whether TRIP13 has any tumor-promoting functions, TRIP13 expression was depleted in DLD1 and HCT116 cells using siRNAs that targeted different regions of TRIP13 mRNA. Transfection of either siRNA sufficiently knocked down TRIP13 expression (Fig. 2A). In the absence of TRIP13 expression, the proliferation of both cell lines was significantly suppressed (Fig. 2B). Reduced cell proliferation can be induced by a delay in cell cycle progression or by the promotion of cellular apoptosis (21). To determine how TRIP13 depletion suppressed cell proliferation, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and an EdU incorporation assay were performed. The TUNEL assay detects fragmented DNA induced by apoptosis, while the EdU incorporation assay evaluates cell cycle progression (21). Cells that progress through the S phase incorporate the thymidine analog EdU; thus, proliferating cells can be detected based on the level of EdU incorporated (21). As shown in Fig. 2C, TRIP knockdown reduced the ratio of cells that incorporated EdU; however, no increase in the level of apoptosis caused by TRIP13 depletion was observed (data not shown).

The effects of TRIP13 knockdown on cell migration were next studied using a modified Boyden chamber. DLD1 and HCT116 cells were transfected with siRNAs, and 72 h later, the cells were placed on the upper surface of the filter and allowed to migrate to the bottom surface, which was coated with fibronectin. The cells that migrated to the bottom surface were counted to evaluate cell migration. As shown in Fig. 3A, TRIP13 depletion significantly suppressed the migration of both cell lines. The invasion of TRIP13-knockdown cells was also examined using Matrigel-coated Boyden chambers. The invasion of both DLD1 and HCT116 cells was clearly suppressed by TRIP13 depletion (Fig. 3B).

To confirm that TRIP13 is associated with the invasion of cancer cells, a rescue experiment was performed. HCT116 cells that constitutively expressed either Flag or Flag-TRIP13 were generated by retrovirus infection. To specifically deplete endogenous TRIP13, an additional siRNA (siTRIP13-3) that targeted the 3′-untranslated region of TRIP13 mRNA was used. Transfection of the siTRIP13-3 depleted endogenous TRIP13, but the expression level of Flag-TRIP13 was not affected by this siRNA (Fig. 3C). The invasion of siRNA-transfected cells was then examined using Matrigel-coated Boyden chambers. The invasion of Flag-expressing HCT116 cells was significantly reduced by transfection with siTRIP13-3 (Fig. 3D). Exogenous expression of Flag-TRIP13 significantly promoted cell invasion (Fig. 3D), while depletion of endogenous TRIP13 in Flag-TRIP13-expressing cells suppressed cell invasion to a level similar to that of control siRNA-transfected Flag-expressing cells (Fig. 3D). These results indicate that the reduction in cell invasion caused by the TRIP13 siRNAs was mediated by the depletion of TRIP13.

It was next examined whether the catalytic activity of TRIP13 was required for the promotion of cell invasion. Mutations in the Walker A motif are known to disrupt ATP binding, whereas Walker B motif mutations disrupt ATP hydrolysis (2). Both were shown to be essential for the function of TRIP13 in yeast (13). The present study generated TRIP13 mutants that have mutations either in the Walker A or Walker B motifs, or in both motifs, including TRIP13-GK/AA, which has glycine 184 and lysine 185 in the Walker A motif substituted with alanine; and TRIP13-DE/NQ, which has aspartic acid 252 and glutamic acid 253 in the Walker B motif substituted with asparagine and glutamine, respectively; and TRIP13-GK/AA-DE/NQ, which has both mutations. HCT116 cells that constitutively expressed Flag-tagged versions of each mutant were established by retrovirus infection. Although the expression of TRIP13-GK/AA and TRIP13-GK/AA-DE/NQ was lower than that of Flag-TRIP13, the expression level was higher than that of endogenous TRIP13 (Fig. 4A). The invasion of these cell lines was examined using Matrigel-coated Boyden chambers. As shown in Fig. 4B, the invasion of these mutant cell lines was significantly reduced compared with that of the wild type TRIP13-expressing cells. These results demonstrated that the catalytic activity of TRIP13 is required for the promotion of cell invasion.