Tissues samples from patients were collected at the First Affiliated Hospital of Guangxi Medical University (Guangxi, China) from March 2013 to October 2013 (24). There were 71 male and 43 female patients included in the present study, all of whom were newly diagnosed and had not received any chemotherapy or radiotherapy previously, and the age of the patients ranged between 25 and 83 (56.58±13.47) years old (24). Based on the National Comprehensive Cancer Network classification, 19 patients were diagnosed with stage I CRC, 44 with stage II CRC, 30 with stage III CRC and 21 with stage IV CRC (24). The study was approved by the Institutional Ethics Committee of Guangxi Medical University under full consideration of the Helsinki declaration of human rights. Written informed consent was obtained from all patients.

Immunohistochemistry staining was performed on deparaffinized 4-μm thick sections according to the manufacturer’s protocol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at room temperature for 10 min. Subsequently, the sections were blocked with 10% normal goat serum (Beyotime Institute of Biotechnology, Haimen, China) in PBS for 1 h, incubated with primary antibody for overnight 4°C, and finally incubated with undiluted horseradish peroxidase-conjugated secondary antibody for 30 min at 37°C. The secondary antibody was the B reagent in an immunohistochemistry kit (Zhongshan Golden Bridge Biotechnology, Beijing, China; cat. no. PV-9000). Visualization of the immunohistochemical reaction was performed using DAB as the chromogen. The slides were evaluated by two pathologists independently. Protein expres sion was examined by using five random fields on each slide at using a light microscope (magnification, ×100; PH50-3A43L-PL optical microscope; Phmias2008 Computer Image Analysis System; Phoenix Optical Co., Ltd., Jiangxi, China). Polyclonal antibodies against SphK1 (cat. no. 10670-1-AP; 1:400) and FAK (cat. no. 12636-1-AP; 1:800) were from ProteinTech Group, Inc. (Chicago, IL, USA). Polyclonal antibodies against phosphorylated (p)-FAK (Tyr397; cat. no. 8556P; 1:1,200) were from Abcam (Cambridge, UK). Polyclonal antibodies against E-cadherin (cat. no. bs-1519R; 1:200) and vimentin (cat. no. bs-8533R; 1:200) were from BIOSS (Beijing, China). The degree of staining was determined by semi-quantitative evaluation and categorized by the extent and intensity of staining as follows: i) The percentage of positively stained cells <5% was scored as 0, 5-25% scored as 1, 26-50% scored as 2, >50% scored as 3; and ii) The intensity of staining was scored as: 0, achromatic; 1, light yellow; 2, yellow; and 3, brown. The two scores were added for the final score: 0-2, negative (−) and >3, positive (+).

The human adenocarcinoma cell line RKO and HT29 (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (ExCell Biology, Inc., Shanghai, China) and penicillin/streptomycin (100 U/ml) at 37°C with 5% CO₂. The Lenti-FAK-EGFP-miR (5×10⁷ TU/ml) and Lenti- SPHK1-EGFP-miR (5×10⁷ TU/ml) (Shanghai R&S Biotechnology Co., Ltd., Shanghai, China) were introduced into RKO cells respectively via lentiviral transfection [multiplicity of infection (MOI) =30]. RKO cells were additionally transfected with the blank vector (Lenti-EGFP-miR; 2×10⁸ TU/ml) (negative control; NC) as control (MOI=30). Lentiviral vector encoding human SphK1 (Lenti-SPHK1-IRES-EGFP; 9×10⁷ TU/ml) was introduced into HT29 cells via lentiviral transfection (MOI=20). HT29 cells were additionally transfected with the blank vector (Lenti-EGFP; 10⁹ TU/ml) [negative control (NC); MOI=20]. The POLOdeliverer™ 3000 Transfection Reagent was purchased from Shanghai R&S Biotechnology Co., Ltd. Stably transfected cells were selected for subsequent experiments. The time interval between transfection and subsequent experimentation was 14 days.

RNA isolation was performed using the Total RNA Extraction kit (Tiangen Biotech Co., Ltd., Beijing, China), according to the manufacturer’s protocol. cDNA synthesis was performed using the RT kit (Takara Bio, Inc., Otsu, Japan). RT was performed using 2.0 μ 5X gDNA Eraser Buffer, 1.0 μ gDNA Eraser, 1 μ Total RNA and RNase Free dH₂O to a total volume of 10 μ! The reaction conditions were 42°C for 2 min and rapidly cooled to 4°C. Subsequently, 10.0 μ reaction solution, 1.0 μl PrimeScript RT Enzyme Mix 1, 1.0 μl RT Primer Mix, 4.0 μl 5X PrimeScript Buffer 2 and 4.0 μl RNase Free dH₂O were used. The reaction conditions were 37°C for 15 min, 85°C for 5 sec, rapidly cooled to 4°C and stored at −20°C. A fluorescence-based qPCR method was performed using 2 μl cDNA, 10 μl SYBR Green (Takara Bio, Inc.), 0.6 μl PCR forward primer (10 μM), 0.6 μ PCR reverse primer (10 μM) and 6.8 μ dH₂O in a 20 μ PCR reaction volume. The RT-qPCR reaction was run on a StepOne Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling parameters were as follows: Denaturing at 95°C for 30 sec, 40 cycles at denaturing at 95°C for 5 sec, primer annealing at 60°C for 34 sec and extension temperature at 95°C for 15 sec; final extension at 60°C for 1 min and final denaturing at 95°C for 15 sec. Gene expression levels were determined using the 2^−ΔΔCq method (25). The primers were from Takara Bio, Inc. The sequences were as follows: GAPDH, forward: 5′-GCA CCG TCA AGG CTG AGA AC-3, reverse: 5′-TGG TGA AGA CGC CAG TGGA-3′; SphK1, forward: 5′-GGC TTC ATT GCT GAT GTG GA-3′, reverse: 5′-AGG AAG GTG CCC AGA GTG AA-3′; FAK, forward: 5′-CAA CCA CCT GGG CCA GTA TTA TC-3′, reverse: 5′-CCA TAG CAG GCC ACA TGC TTTA-3′; vimentin, forward: 5′-TGA CAT TGA GAT TGC CAC CTA CAG-3′, reverse: 5′-TCA ACC GTC TTA ATC AGA AGT GTCC-3′; E-cadherin, forward: 5′-GAG TGC CAA CTG GAC CAT TCA GTA-3′, reverse: 5′-AGT CAC CCA CCT CTA AGG CCA TC-3′; MMP2, forward: 5′-GCT CCA CCA CCT ACA ACT TTG AGAA-3′, reverse: 5′-TGT CAT AGG ATG TGC CCT GGAA-3′; MMP9, forward: 5′-TGG GCT GCT GCT TTG CT-3′, reverse: 5′-GCC TGT CGG TGA GAT TGG TT-3′; and fibronectin, forward: 5′-GAC CAC ATC GAG CGG ATC TG-3′, reverse: 5′- GTC TCT TGG CAG CTG ACT CC-3′.

Total proteins were extracted using radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology). Protein concentrations were measured by a bicinchoninic acid assay (Solarbio Biotech Co., Ltd., Beijing, China) according to the manufacturer’s protocol. A total of 30 μg of protein from each sample was separated via 12% SDS-PAGE (Beyotime Institute of Biotechnology) for 1 h at 100 V, and subsequently transferred onto nitrocellulose membranes. Samples were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (Solarbio Biotech Co., Ltd.) for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies diluted in WB Antibody Diluent (Beyotime Institute of Biotechnology). Subsequently, the membranes and secondary antibodies were incubated for 1 h at room temperature. Bands were quantified by Odyssey infrared imaging (LI-COR Biosciences, Lincoln, NE, USA) and GAPDH served as the internal reference. The blots were analyzed with Quantity One 4.6.2 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Polyclonal antibodies against E-cadherin (cat. no. 3195; 1:1,000), p-FAK (Tyr397; cat. no. 8556P; 1:1,000), AKT (cat. no. 4691; 1:1,000) and p-AKT (Ser473; cat. no. 4060; 1:1,000) were from Cell Signaling Technology, Inc., Danvers, MA, USA. Polyclonal antibodies against SphK1 (cat. no. 10670-1-AP; 1:500), FAK (cat. no. 12636-1-AP; 1:2,000), MMP2 (cat. no. 10373-2-AP; 1:500), MMP9 (cat. no. 10375-2-AP; 1:500), vimentin (cat. no. 10366-1-AP; 1:500), fibronectin (cat. no. 15613-1-AP; 1:500) and GAPDH (cat. no. 10494-1-AP; 1:1,000) were purchased from ProteinTech Group, Inc. IRDy^® 680RD goat anti-rabbit (cat. no. 60329-11; 1:10,000) secondary antibodies were from LI-COR Biosciences.

Cells (10×10³) were seeded into a 96-well plate. Following treatment with the FAK pathway inhibitor (PF-562271) at concentrations of 0, 2.5, 5 and 10 μM for 24, 48 and 72 h, the cells were incubated in fresh medium containing MTT solution (2 mg/ml) and were incubated for 4 h at 37°C in the dark. Following removal of the MTT solution, 150 μ dimethyl sulfoxide was added to dissolve the formazan crystals and the absorbance (A) at 450 nm was measured using a fully automated microplate reader (EL×800™ ; BioTek Instruments, Inc., Winooski, VT, USA). Cell viability (%) = A450 (sample)/A450 (control) ×100.

The cells (1×10⁵) were cultured in fresh serum-free medium in the upper chambers of Transwell plates. The lower chambers contained fresh medium with 10% FBS. After 48 h of incubation at 37°C, cells remaining on the upper surface of the membrane were removed. The cells that migrated to the lower membrane surface were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) at 4°C for 5 min and stained with 0.1% crystal violet solution (Beyotime Institute of Biotechnology) at 37°C for 20 min. Migrating cells were counted in five random fields under a light microscope (Nikon Corporation, Tokyo, Japan; (magnification, ×200). The migrated cells were counted with Nikon microscope software (version 4.0; Nikon Corporation). Cells were seeded into a 6-well cell culture plate. When the cells reached 80-90% confluency, the cells were scratched with a pipette tip (1 mm). Images were acquired after cells were cultured in medium for 0, 24 and 48 h. The migration distance was measured with the microscope software (Nikon Corporation).

Cells were grown on cover glass slides placed in 6-well plates. Subsequent to being treated, cells were fixed with 3% glutaraldehyde at 4°C for 2 h, gradient ethanol dehydration: 50% for 10 min one time and 70% for 10 min one time. Gold was used for sputter coating. Images of RKO cells and HT29 cells were obtained using the scanning electron microscope of VEGA3, TESCAN (Brno-Kohoutovice, Czech Republic) and S4800 (Hitachi, Ltd., Tokyo, Japan).

Each experiment was performed at least three times. Statistical analysis was performed using SPSS 20.0 software (IBM, Corp., Armonk, NY, USA). The significance between SphK1, FAK, p-FAK, E-cadherin, vimentin and clinicopathological characteristics of the patients was assessed with the χ² test. Survival curves were compared using the log-rank test. The PCR was analyzed by the Mann-Whitney U test. Statistical significance was determined by unpaired Student’s t-test. Analysis of variance was performed, followed by the Least Significant Difference’s test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Immunohistochemistry staining demonstrated that the expression density of SphK1, FAK, p-FAK and vimentin in colorectal cancer tissues were higher compared with normal colonic mucosa tissues, whereas, the expression of E-cadherin was lower compared with normal colonic mucosa tissues (Fig. 1A; Tables I and II). Furthermore, the expression of SphK1, FAK, p-FAK and vimentin in metastatic cancer tissues were higher compared with non-metastatic cancer tissues, whereas, the expression of E-cadherin was lower (Fig. 1A; Tables I and II). As presented in Tables III and IV, the positive scores of SphK1, FAK, p-FAK and vimentin expression in advanced tumors (stage III and stage IV) with lymph nodes and distant metastases of colorectal cancer tissues were higher compared with those identified in less advanced tumors (stage I and stage II) without-lymph nodes and distant metastases. However, the positive rate of E-cadherin was lower in advanced tumors. There was a significant difference in E-cadherin and vimentin expression between different infiltration depths in colorectal cancer tissues; however, there was no significant difference in SphK1, FAK and p-FAK expression. These results suggested that the expression of SphK1, FAK, p-FAK, E-cadherin and vimentin was associated with the malignant invasion and metastasis of colorectal cancer.

Patients with colorectal cancer with SphKl-positive cancer cells had a significantly lower survival rate compared with patients with SphK1-negative cancer (Fig. 1B; P=0.0169). The results suggested that the prognosis of patients with colorectal cancer with SphK1-positive tumor was poorer. Therefore, SphK1 may be used as a prognostic indicator for patients with colorectal cancer.

Our previous study demonstrated that the relative protein expression of p-FAK was 0.93±0.02 in Caco2 cells, 0.71±0.03 in HT29 cells, 0.96+0.01 in RKO cells and 0.80±0.02 in HCT116 cells (26). The protein expression of p-FAK in RKO cells was the highest. Therefore, RKO cells were selected for FAK shRNA stable transfection and the FAK and p-FAK expression were successfully suppressed (Fig. 2A-C). The expression of p-AKT, MMP2/9, vimentin and fibronectin was decreased with the suppression of FAK, whereas, E-cadherin was increased, and no noticeable alteration occurred for AKT expression (Fig. 3A and B). The microvilli and pseudopodia of FAK knockdown RKO cells [FAK(−)-RKO] were decreased compared with transfection negative control (NC) and control (Con) RKO cells (Fig. 3C). These results suggested that the EMT process was inhibited by suppression of FAK by decreasing p-AKT and MMP2/9. The migrational potency of cells may be facilitated once the EMT process is reactivated in colorectal cancer cells (15). Therefore, the migrational potency of RKO cells was significantly suppressed by FAK knockdown (Fig. 4A and B; P<0.05).

RKO cells were used for SphKl shRNA stable transfection. SphKl mRNA and protein expressions were successfully suppressed in the transfected RKO cells (Fig. 2A and B). The expression of p-FAK, p-AKT and MMP2/9 was decreased with the suppression of SphK1 in RKO cells. However, there were no noticeable alterations of FAK and AKT expression levels (Figs. 2C and 3A-C). SphK1 knockdown was accompanied by an increased expression of E-cadherin and a decrease in the expression of vimentin and fibronectin (Fig. 3A and B). The microvilli and pseudopodia of SphK1 knockdown RKO cells [SphK1(−)-RKO] were decreased compared with NC and Con RKO cells (Fig. 3C). These results suggested that the EMT was repressed with the suppression of SphK1 by decreasing p-FAK, p-AKT and MMP2/9. The migrational potency of cells was facilitated once the EMT process was reactivated in colorectal cancer cells (17). Therefore, the migrational potency of RKO cells was additionally significantly decreased with the knockdown of SphK1 (Fig. 4; P<0.05).

Our previous study demonstrated that the relative protein expression of SphK1 was 0.96±0.02 in Caco2 cells, 0.61±0.07 in HT29 cells, 0.92±0.05 in RKO cells and 0.97±0.02 in HCT116 cells (22). The protein expression of SphK1 in HT29 cells was the lowest. HT29 cells were used for stable transfection of the SphK1 overexpressing vector (Fig. 5). SphK1 mRNA and protein expression levels were significantly increased in the transfected cells (Fig. 5A and C; P<0.05). In contrast to the cells with knockdown of SphK1, the negatively transfected control (NC) and Con-HT29 cells, the expression levels of p-FAK, p-AKT and MMP2/9 were significantly increased in SphKl-overexpressing HT29 cells [SphK1(+)-HT29] (Fig. 5B and C; P<0.05). However, there were no statistical differences in FAK and AKT expression levels among SphK1(+)-HT29 cells, NC-HT29 cells and Con-HT29 cells (Fig. 5B and C; P>0.05). Compared with NC-HT29 and Con-HT29 cells, the expression of E-cadherin decreased, whereas, vimentin and fibronectin increased in SphK1(+)-HT29 cells (Fig. 5B and C), along with the microvilli and pseudopodia (Fig. 5D). These results suggested the EMT was induced by the overexpression of SphK1 by increasing p-FAK, p-AKT and MMP2/9. The migrational potency of cells is facilitated once the EMT process is reactivated in colorectal cancer cells (13). The migrational potency of the cells was additionally increased with the upregulation of SphK1 (Fig. 5E).

As demonstrated in Fig. 6A, PF562271 (inhibitor of the FAK pathway) suppressed the proliferation of HT29 cells in a time- and dose- dependent manner. The protein expression of FAK was decreased when HT29 cells were treated with PF562271 at concentrations of 2.5 and 5 μM for 48 h. The protein expression of p-FAK was significantly decreased when HT29 cells were treated with PF562271 at a concentration of 5 μM for 48 h (P<0.05). The expression of FAK, p-FAK, p-AKT and MMP2/9 was decreased when SphK1(+)-HT29 cells were treated with PF562271 at a concentration of 5 μM for 48 h [PF-SphK1(+)-HT29]. However, there was no noticeable alteration in the expression level of AKT (Fig. 6B and C). In addition, the expression of E-cadherin was increased, whereas, vimentin and fibronectin expression levels were decreased in SphK1(+)-HT29 cells treated with PF562271 (Fig. 6B and C). Furthermore, the microvilli, pseudopodia and migrational potency of PF-SphK1(+)-HT29 cells were suppressed by treatment with PF562271 (Fig. 6D and E). These results suggested that the FAK pathway inhibitor suppressed the EMT and migrational potency induced by SphK1 overexpression in HT29 cells.