UA was purchased from Sigma-Aldrich (Merck KGaA). RPMI-1640 medium, PBS and penicillin-streptomycin were purchased from Hyclone (GE Healthcare Life Sciences). Fetal bovine serum (FBS) and trypsin-EDTA were obtained from Gibco (Thermo Fisher Scientific. Inc.). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Beijing Solarbio Science & Technology Co., Ltd. miR-200a/b/c and U6 primers were synthesized by Takara Biotechnology Co., Ltd. RNAiso for small RNA kit, Mir-X™ miRNA First-Strand Synthesis kit and TB Green™ Premix Ex Taq II kit were purchased from Takara Biotechnology Co., Ltd. N-cadherin (cat. no. ab18203) and E-cadherin (cat. no. ab1416) antibodies were purchased from Abcam. TGF-β1 (cat. no. 3711), phosphorylated (p-) Smad2/3 (cat. no. 8828), Smad2/3 (cat. no. 8685), p-FAK (cat. no. 3284), FAK (cat. no. 71433), ZEB1 (cat. no. 3396) and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG; cat. no. 7074; and anti-mouse IgG; cat. no. 7076) were purchased from Cell Signaling Technology, Inc. The β-actin antibody (cat. no. 66009-1-Ig) was purchased from ProteinTech Group, Inc. The Transwell chambers were obtained from Corning Life Sciences and the BD BioCoat Matrigel Invasion Chamber was purchased from BD Bioscience. All the other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (Merck KGaA).

Human colon cancer HCT116 and HCT-8 cell lines were obtained from the Nanjing KeyGen Biotech. Co. Ltd. Cells were cultured in RPMI-1640 medium, supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in a 37°C humidified incubator supplemented with 5% CO₂.

UA was dissolved in DMSO and diluted with culture medium to the desired concentrations. The final concentration of DMSO in the culture medium was <0.1% throughout the study. HCT116 and HCT-8 cells were plated into 96-well plates at a 1×10⁴ cells/well and treated with 0, 10, 20, 40 µM UA for 24 h and 48 h. Treatment with DMSO was included as the vehicle control. Following treatment, 100 µl of 0.5 mg/ml MTT solution was added in each well at 37°C for 4 h. The MTT formazan precipitate was dissolved in 100 µl of DMSO. Subsequently, the resulting absorbance of the purple formazan product was determined at 570 nm with a ELX800 microplate reader (BioTek Instruments, Inc.). The cell viability was determined using the formula: Cell viability (%)=sample optical density (OD)/control OD ×100.

To evaluate the cell migration and invasion, Transwell assays were conducted using Transwell cell culture chambers with 8 µm pore filters (Corning Life Sciences). After treatment with 0, 10, 20, 40 µM of UA for 24 h, HCT116 and HCT-8 cells were harvested and resuspended in serum-free RPMI-1640 without UA. Then, ~5×10⁴ cells that survived after the indicated concentrations of UA treatment for 24 h were seeded into the upper chambers. The lower chambers were filled with RPMI-1640 media containing 10% FBS as a chemoattractant. Cells were allowed to migrate towards the complete medium for 12 h in the migration assay, the non-migrating cells in the upper chamber were wiped and the migrated cells were stained with crystal violet for 15 min at room temperature. For quantification, the average number of migrated cells per field was assessed by counting three random fields under a phase contrast microscope (Leica Microsystems GmbH) at a magnification of ×200. The cell invasion assay was similar to the migration assay, except that the upper chambers were coated with Matrigel matrix (BD Biosciences).

Following treatment with the indicated concentrations of UA for 24 h, total small RNA was extracted using RNAiso for small RNA kit. Total small RNA was reverse transcribed with an Mir-X™ miRNA First-Strand Synthesis kit following the manufacturer's protocol. The primers for miR-200a (cat. no. DHM0178), miR-200b (cat. no. DHM0179), miR-200c (cat. no. DHM0180) and U6 (cat. no. D356-03) were obtained from Takara Biotechnology Co., Ltd. The obtained cDNA was used to determine the levels of miR-200a, miR-200b and miR-200c using TB Green™ Premix Ex Taq II in an ABI 7500 Fast PCR system, according to the manufacturer's instructions. The PCR conditions were as follows: Pre-denaturation at 95°C for 2 min, then 45 cycles of 95°C for 3 sec and 60°C for 30 sec. U6 was used as an internal control. Relative quantification was performed using the 2^−ΔΔCq method (31). Each PCR amplification was carried out in triplicate.

After treatment with the indicated concentrations of UA for 24 h, total protein was extracted with cell lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) containing protease and phosphatase inhibitors. The concentration of total protein was detected using a bicinchoninic acid assay. A total of 50 µg protein was separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (EMD Millipore Corporation). The membranes were blocked with 0.5% BSA for 2 h at room temperature and then probed with primary antibodies against TGF-β1 (1:1,000), p-Smad2/3 (1:1,000), Smad2/3 (1:1,000), p-FAK (1:1,000), FAK (1:1,000), N-cadherin (1:1,000), E-cadherin (1:2,000), ZEB1 (1:1,000) and β-actin (1:5,000) overnight at 4°C, followed by 1 h incubation with the anti-rabbit IgG HRP-conjugated secondary antibodies (1:2,000) or anti-mouse IgG HRP-conjugated secondary antibodies (1:2,000) at room temperature. Subsequently, using TBS/Tween-20 to wash the membranes, the immunoreactive bands were visualized via Image Lab software (version 3.0; Bio-Rad Laboratories, Inc.) using enhanced chemiluminescence (Yuheng Biotechnology Co., Ltd.).

All data were expressed as mean ± standard deviation. Data were analyzed using SPSS software (version 16.0; SPSS Inc.). Statistical analysis was performed using one-way analysis of variance and least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the inhibitory effect of UA on the growth of HCT116 and HCT-8 cells, MTT assays were performed. UA treatment significantly inhibited cell viability in both a dose- and time-dependent manner (Fig. 1A and B). After 24 h of treatment, the half maximal inhibitory concentration (IC₅₀) values of UA for HCT116 and HCT-8 cells were calculated to be 37.2 and 25.2 µM, respectively. Similarly, after 48 h of treatment, the IC₅₀ values of UA were determined to be 28.0 and 19.4 µM for HCT116 and HCT-8 cells, respectively. Cells exhibiting condensed and fragmented nuclei are considered as growth inhibited. Phase-contrast microscopy was used to examine the effect of UA on HCT116 and HCT-8 cell morphology. Untreated control cells were observed in a confluent monolayer, healthy and attached to the culture plate, whereas UA treatment significantly decreased the confluence of these two cell lines and resulted in condensed, fragmented and detached cells, in a dose-dependent manner (Fig. 1C). Taken together, these results indicated that UA significantly inhibited the growth of HCT116 and HCT-8 cells.

Transwell assays were performed to evaluate the effect of UA on the migration of HCT116 and HCT-8 cells. As shown in Fig. 2, the results demonstrated that treatment with 10–40 µM UA significantly decreased the migratory rate of HCT116 and HCT-8 cells by 23.3±4.2–91.3±1.2% and 25.5±2.4–98.8±0.2%, respectively, when compared with untreated cells. Furthermore, UA was demonstrated to inhibit the invasive abilities of HCT116 and HCT-8 cells. The results revealed that the invasion rate of HCT116 and HCT-8 cells following UA treatment was 36.2±1.8–67.9±1.6% and 33.8±3.7–98.2±0.2%, respectively, compared with that in the untreated cells (Fig. 3). Taken together, these results suggest that UA exhibited an inhibitory effect on the migration and invasion properties of HCT116 and HCT-8 cells in a dose-dependent manner.

EMT has been shown to be associated with the metastasis of tumor cells (32). TGF-β1 signaling pathways, including the canonical TGF-β1/Smad pathway and the non-canonical TGF-β1/FAK signaling pathway, can trigger EMT (10,33). To better understand whether UA inhibited TGF-β1 signaling pathways, the expression of several pivotal mediators of TGF-β1/Smad and TGF-β1/FAK signaling pathways was assessed in HCT116 and HCT-8 cells. As shown in Fig. 4, western blot analysis revealed that UA treatment (0, 10, 20, and 40 µM) dose-dependently decreased the expression levels of TGF-β1, and the expression levels of the TGF-β1 target gene ZEB1. Activation of Smad2/3 and FAK is mediated by their phosphorylation, and UA treatment also significantly reduced the phosphorylation levels of both Smad2/3 and FAK (Fig. 4). Inhibition of the TGF-β1/Smad and TGF-β1/FAK signaling pathways by UA led to a decrease in the expression of the mesenchymal marker N-cadherin compared with that in the control cells (Fig. 4). However, no difference was observed between the control cells and UA-treated cells regarding the protein expression levels of the epithelial marker E-cadherin (Fig. 4). These results indicated that the antitumor properties of UA may be mediated by the inhibition of the TGF-β1 signaling pathways.

miR-200a/b/c maintains the epithelial phenotype and inhibits cell metastasis via downregulation of its target gene ZEB1 (34). In this process, TGF-β1 signaling increases the DNA methylation of miR-200a/b/c, thereby negatively regulating its expression (17). Thus, to further investigate whether UA inhibited colorectal cell invasion via regulation of the TGF-β1/ZEB1/miR-200a/b/c feedback loop, the expression levels of miR-200a/b/c were determined via RT-qPCR analysis. As shown in Fig. 5, the expression levels of miR-200a and miR-200c in HCT116 and HCT-8 cells were significantly increased following UA treatment compared with that in the untreated control cells. While the expression levels of miR-200b did not significantly change after UA treatment in HCT116 cells compared with the untreated control, HCT-8 cells exhibited a significant increase in miR-200b levels following treatment with 40 µM UA (Fig. 5). These findings are consistent with the observation that UA inhibited the TGF-β1 pathway and the expression of ZEB1 (Fig. 4). As shown in Fig. 5, the expression levels of miR-200c exhibited the highest increase following UA treatment. Therefore, the present results suggested that UA may inhibit cell growth and invasion in HCT116 and HCT-8 cells via regulation of the TGF-β1/ZEB1/miR-200c feedback loop.