The human colorectal carcinoma cell lines, Caco2 (Boster, Wuhan, China), HT29, RKO and HCT116 (R&S Biotechnology Co., Ltd., Shanghai, China) were routinely cultured in DMEM containing 100 ml/l fetal bovine serum (FBS; ExCell Bio, Shanghai, China) and incubated at 37°C with 5% CO₂.

Each of the cell lines was divided into 3 groups as follows: the control group (N group), the SKI-II (an inhibitor of SphK1) intervention group (SK group) and PF-562271 (an inhibitor of FAK) intervention group (PF group). The cells in the SK group were cultured for 24 h and then incubated with SKI-II (Selleck, Houston, TX, USA) at 20 µM (22) for 48 h. The cells in the PF group were also cultured for 24 h and then incubated with PF-562271 (Selleck) 5 µM (23) for 48 h.

The cells were cultured in serum-free medium to produce suspension, and the cell density was adjusted to 5×10⁵/ml. Subsequently, 200 µl cell suspension were added to the upper chamber of the Transwell (Corning, Inc., Corning, NY, USA), while 600 µl medium including 10% FBS were added to the bottom chamber, followed by incubation for a period of 24 h at 37°C. The assay was terminated as a result of the removal of the medium from the upper well and the filter was fixed with methanol for 10 min. The cells in the upper chamber were wiped off and the cells migrating to the lower side of the upper chamber were stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 30 min. Random fields were scanned (6 fields/filter) under a fluorescent inverted phase contrast microscope (magnification, ×200; TS100-F; Nikon, Tokyo, Japan) for the presence of the cells at the lower membrane side only.

RNA isolation was performed using a RNA extraction kit (Tiangen, Beijing, China) according to the manufacturer's instruction. cDNA synthesis was performed using a reverse transcription kit (Takara, Dalian, Japan). Fluorescence-based qPCR was performed using 2 µl of cDNA and SYBR-Green (Takara) in a total volume of 20 µl and using the ABI 7500 Real-time PCR system (Applied Biosystems, Rockford, IL, USA). The cycling parameters were: 95°C for 30 sec followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec, and a dissociation program that included 95°C for 15 sec, 60°C for 1 min,and 95°C for 15 sec ramping up at 0.2°C/sec. Gene expression levels were determined with the 2^−ΔΔCq method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference and the non-stimulated condition was set to 1. The forward and reverse primers (Takara) of the genes are listed in Table I.

The cells were collected using cell scrapers and lysed with RIPA lysis buffer supplemented with 1% protease and 1% phosphatase inhibitor for 30 min following 2 washes with cold phosphate-buffered saline (PBS). The cells were subjected to centrifugation at 12,000 × g/min for 15 min at 4°C to remove the cell debris. Protein concentrations were measured by bicinchoninic acid (BCA) assay (Solarbio, Beijing, China) according to the standard protocol.

Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto 0.45 nitrocellulose membranes. The membranes were subsequently blocked in non-fat milk [dissolved in Tris-buffered saline with 5% Tween-20 (TBST)] buffer at room temperature for 30 min to block non-specific binding and incubated with antibodies diluted by western blot antibody diluents overnight at 4°C, and subsequently incubated with secondary fluorescent antibodies (IRDye^® 680RD Goat anti-Rabbit 925-68071; C60329-11; dilution, 1:10,000; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature following 3 washes with TBST. The membranes were scanned using an Odyssey infrared imaging system (LI-COR Biosciences). Relevant signal intensity was determined using LI-COR imaging software (LI-COR Biosciences). The strips were analyzed using Quantity One software (Bio-Rad, Shanghai, China). Antibodies against E-cadherin (3195S; 1:1,000) and p-FAK (Tyr397; 8556P; 1:1,000) were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Antibodies against SphK1 (10670-1-AP; 1:500), FAK (12636-1-AP; 1:1,000), Slug (12129-1-AP; 1:500), N-cadherin (66219-1-1g; 1:500), vimentin (10366-1-AP; 1:500) and GAPDH (10494-1-AP; 1:1,000) were purchased from Proteintech Group (PTG, Rosemont, IL, USA).

Each experiment was repeated at least 3 times. Data were presented as the means ± SD. The results of Transwell chamber assay and western blot analysis were analyzed using the t-test. The Mann-Whitney U test was used to analyze the results of PCR. Results were considered statistically significant at P<0.05.

To examine the effect of SphK1 on the migration of human CRC cells, the cells were exposed to 20 µM SKI-II for 48 h, in order to inhibit SphK1 expression. The migrating cells were observed under an inverted microscope. The results revealed that the number of migrating cells in the SK group was significantly lower than that of the cells in the N group in all 4 CRC cell lines (P<0.05; Fig. 1), indicating that SphK1 plays an important role in CRC cell migration.

In order to investigate the signaling pathways involved in the regulatory effects of SphK1 on the migratory abitlity of CRC cells, some EMT-related markers were examined in the present study. EMT is known to play a critical role in the metastasis of cancer cells (24,25). The mRNA expression of EMT-related makers was detected, including the expression of the transcription factor, Slug (26), the mesenchymal cell markers, vimentin and N-cadherin, and the epithelial cell marker, E-cadherin (27–29). As shown in Fig. 2, the mRNA levels of Slug, vimentin and N-cadherin decreased, whereas that of E-cadherin increased (P<0.05). This indicated that the suppression of SphK1 suppressed Slug, vimentin and N-cadherin gene expression, while it promoted E-cadherin gene expression.

The results revealed that the expression of Slug, vimentin and N-cadherin decreased, whereas that of E-cadherin increased following the suppression of SphK1 (P<0.05; Figs. 3 and 4). These results were consistent with those obtained by PCR.

Compared to the N group, although in the SK group FAK mRNA and total FAK protein expression exhibited no significant difference (P>0.05; Fig. 5B and D), p-FAK protein expression was markedly decreased (P<0.05; Figs. 3 and 5E). This indicated that SphK1 regulated the expression of p-FAK, suggesting that SphK1 is involved in modulating the FAK pathway.

To date, some researchers have demonstrated that FAK is involved in regulating the EMT process in cancer cells (30). Moreover, FAK affects the expression of Slug (31). In this study, our results revealed that the numbers of migrating cells in the PF group were lower than those in the N group in the Caco2, HT29, RKO and HCT116 cells (P<0.05; Fig. 1 and 6A). In addition, compared with the N group, the mRNA expression levels of FAK, Slug, vimentin and N-cadherin in the PF group were decreased, whereas the mRNA expression level of E-cadherin was increased (P<0.05; Fig. 6B). Moreover, the protein expression levels of FAK, p-FAK, Slug, vimentin and N-cadherin in the PF group were lower than the levels in the N group (P<0.05; Figs. 3 and 6C). Similar with the mRNA expression, the protein expression of E-cadherin in the PF group was higher than that in the N group (P<0.05; Figs. 3 and 6C). Thus, the results revealed that FAK may affect the mRNA and protein expression levels of Slug, vimentin, N-cadherin and E-cadherin, as well as the cell migratory ability.

On the whole, according to our data, it can be suggested that SphK1 modulates the expression of Slug, E-cadherin, N-cadherin and vimentin, as well as CRC cell metastasis by regulating the expression of p-FAK in Caco2, HT29, RKO, HCT116 CRC cell lines.