A total of 147 colon cancer tissue samples and adjacent normal control samples were obtained from patients diagnosed with colon cancer who underwent radical surgery at The Second Hospital of Shandong University (Jinan, China) between January 2013 and June 2014. All patients exhibited primary tumors and none of the patients had received chemotherapy or radiation therapy prior to tumor excision. The patient cohort included 93 males and 54 females, with a median age of 62 years (range, 37–72 years). Patient clinicopathological factors are shown in Table I. The present study was approved by the Ethics Committee of the Second Hospital of Shandong University (Jinan, China) and additionally received institutional approval. The experiments were performed in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research.

Resected tissues were washed using phosphate-buffered saline (PBS), cut into sections and lysed with RIPA lysis and extraction buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The lysates were centrifuged in an ice bath to obtain the supernatant and total protein was calculated using the bicinchoninic acid assay method (Pierce BCA Protein Assay kit; Thermo Fisher Scientific, Inc.). A total of 50 µg protein was used for each sample. Following electrophoresis with 12% gel, samples were transferred to polyvinylidene fluoride membranes and incubated with FRAT1 rabbit monoclonal antibody (1:500; catalog no., ab108405; Abcam, Cambridge, UK) overnight at 4°C. Next, the membranes were incubated with goat anti-rabbit IgG-horseradish peroxidase (1:2,000; catalog no., sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 37°C for 2 h and visualized using a chemiluminescence detection reagent (Applygen Technologies, Inc., Beijing, China). The western blot gray values were quantified using ImageJ software (National Institutes of Health, Bethesda, MA, USA).

RNA was isolated from the tissue samples using mechanical homogenization and TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China). cDNA was synthesized from the total RNA using SuperScript III RNase H-Reverse Transcriptase (Takara Biotechnology Co., Ltd.). Gene expression was quantified using a One-Step SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd.). The primers were designed as described previously (17). The sequences of the primers were as follows: Forward, 5′-GCCCTGTCTAAAGTGTATTTTCAG-3′ and reverse, 5′-CGCTTGAGTAGGACTGCAGAG-3′ for FRAT1 (Invitrogen; Thermo Fisher Scientific, Inc.). GADPH was used as the internal reference gene and its primers were as follows: forward, 5′-GAAGTGAAGGTCGGAGTCA-3′ and reverse, 5′-TTCACACCCATGACGAACAT-3′. The gene expression was analyzed on the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was performed in a 10 µl reaction volume, which consisted of 5 µl One-Step SYBR buffer III, 0.2 µl Takara Ex Taq HS, 0.2 µl PrimerScript RT enzyme mix II, 0.2 µl forward primer, 0.2 µl reverse primer, 1 µl RNA and 3.2 µl RNase-free dH₂0. PCR was performed under the following conditions: Initial denaturation at 90°C for 5 min, followed by 40 cycles of denaturation at 95°C for 45 sec, annealing at 55°C for 40 sec and extension at 72°C for 50 sec (18).

The specific sequence of the shRNA targeting FRAT1 was designed as previously described (19–22) using the following primer sequences: Forward, 5′-GCAGTTACGTGCAAAGCTTTTCAAGAGAAAGCTTTGCACGTACTGC-3′ and reverse, 3′-CGTCAATGCACGTTTCGAAAAGTTCTCTTTCGAAACGTGCATTGACG-5′. The shRNA template was then added to two endonucleases, HindIII and BamH1, for digestion. The synthetic template was annealed to form a double-stranded oligo and then cloned into the vector pLV-H1-EF1a-puro (Biosettia Inc., San Diego, CA, USA). Finally, recombinant pshRNA-FRAT1 was successfully constructed and verified by Sanger dideoxy DNA sequencing. HT-29 cells were transfected with the recombinant plasmid to screen the stable cell line for FRAT1 inhibition. In addition, HT-29 cells were tranfected with empty vector pLV-H1-EF1a-puro and used as the control. To ensure efficient transfection, reverse transcription (RT)-PCR and western blot analysis were used to analyze FRAT1 gene and protein expression.

Cell proliferation was analyzed using the MTT colorimetric method. HT-29 cells (1×10⁴ cells/well) were subcultured to 80% confluence and seeded in 96-well plates. The cells were incubated for 48 h, and were cultured for 4 h with 5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO, USA). The optical density (OD) was then measured (ELx800 UV universal microplate reader; Bio-Tek Instruments, Inc., Winooski, VT, USA) and the inhibition rate was calculated according to the following formula: Inhibition rate = 1 - experimental OD / control OD (23).

Cell migration was performed using Transwell inserts (8-µm pore size; Merck Millipore, Darmstadt, Germany). The upper chamber was coated with 1 mg/ml Matrigel (Corning, Inc., Corning, NY, USA). The cells were incubated without serum for 12 h and seeded at a density of 2×10⁵/ml into the upper chamber, while 600 µl 10% serum medium was placed into the lower chamber. The cells were cultured for 36 h and subsequently the cells on the upper surface were removed gently with a cotton swab. The Transwell chambers were fixed in 95% ethanol for 15 min and washed with PBS 3 times. Eosin was used to stain the chambers for 10 min, followed by washing with PBS. Finally, the chambers were viewed under a high-powered inverted microscope (CKX41; Olympus Corp., Tokyo, Japan). Cells were counted in 6 random fields at magnification, ×200.

All data analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard error of the mean. Differences between groups were determined using Student's t-test and the correlation between FRAT1 expression, TNM stage and pathological stage was determined using χ² and Spearman's rank correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, mRNA expression of FRAT1 was significantly increased in colon cancer tissues compared with adjacent tissues at different pathological stages (P<0.05; Fig. 1A) and various Tumor-Node-Metastasis (TNM) stages (P<0.05; Fig. 1B) (24). Western blot results revealed that the expression of FRAT1 significantly increased in colon cancer tissues of various pathological (P<0.05; Fig. 1C) and TNM stages (P<0.05; Fig. 1D) compared with adjacent tissues.

In the colon cancer tissues, increased FRAT1 gene and protein expression was found to significantly correlate with increased tumor malignancy and a higher TNM stage (P<0.05) (Table II).

Small hairpin RNA (shRNA)-transfected HT-29 cells were analyzed using PCR and western blotting to determine the effects of reducing FRAT1 expression. The results revealed that levels of FRAT1 gene and protein expression were significantly decreased in transfected HT-29 cells (due to conversion of shRNA to small interfering RNA) compared with control cells (P<0.05).

To investigate the effects of FRAT1 gene regulation on the proliferation and migration of colon cancer cells, MTT and Transwell assays were performed. MTT assay revealed that reducing FRAT1 expression significantly inhibited the proliferation of colon cancer cells (Fig. 2C; P<0.05). Furthermore, Transwell assay demonstrated that in transfected HT-29 cells, the number of cells which passed through Matrigel within 12 h was significantly lower than that in normal HT-29 cells and empty vector transfected HT-29 cells (P<0.05). These results indicated that reducing FRAT1 expression significantly decreased the migration ability of tumor cells (Fig. 2D).