HT-29 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in a monolayer culture in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), containing 100 µg/ml streptomycin, 100 µg/ml penicillin and 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences). Cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C. HT-29 cell culture was performed according to the manufacturer's protocol and as previously described by Miao et al (18). MC-LR (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was dissolved in sterile water to make a 0.25 nM stock solution and then added in the medium to reach different doses (0, 1, 5, 12.5, 25 and 50 nM).

After 48 h treatment with MC-LR of 25 nM, cells grown on coverslips were washed with PBS, fixed by immersion at room temperature with 4% polyformaldehyde for 15 min and permeabilized with 0.1% Triton-X-100 in PBS at 4°C for 15 min. Slides were then washed with PBS and blocked with blocking buffer consisting of 4% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in PBS for 30 min at room temperature. The cells were then incubated with a mouse anti-human CDH11 monoclonal antibody (cat. no. MAB1790; 1 µg/ml; R&D Systems, Inc., Minneapolis, MN, USA) in blocking buffer overnight at 4°C, followed by incubation with a goat anti-mouse phycoerythrin-labeled antibody (cat. no. sc-3738; dilution, 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in blocking buffer at room temperature for 2 h. Then cells on coverslips were captured and analyzed by fluorescence microscopy (magnification, ×400; Axio Observer A1; Carl Zeiss AG, Oberkochen, Germany). Cellular immunofluorescence was performed according to the manufacturer's protocol and as previously described by Zhang et al (20).

The CDH11 and negative control small interfering RNAs (siRNAs) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The sequences of each siRNA were as follows: CDH11 number 1, 5′-AGGAAGUAGGAAGAGUGAAAGCUAA-3′; CDH11 number 2, 5′-CAACAUCACUGUCUUUGCAGCAGAA-3′; and CDH11 number 3, 5′-CAUCGUCAUUCUCCUGGUCAUUGUA-3′ (21), these three siRNA sequences were mixed at a ratio of 1:1:1 prior to the experiment. A random sequence control was used as the negative control (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′). Cells were plated at a density of 8×10⁴ cells/well in six-well plates. Following pre-incubation of the siRNA with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in serum-free Opti-MEM medium (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min, cells were transfected with CDH11 or negative control siRNA oligo duplexes for 6 h, then cells were switched into fresh medium and incubated at 37°C in a humidified atmosphere of 5% CO₂. Following 24 h, cells were collected and used for migration or invasion assays, and 48 h later for western blot analysis. All transfections were performed according to the manufacturer's protocol and as previously described (18).

To investigate CDH11 expression, a standard western blotting analysis was conducted. Following 48 h culture with MC-LR, or being transfected with CDH11 or negative control siRNA oligoduplexes, the cells were lysed with RIPA cell lysis buffer 1 (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethylsulfonyl fluoride. The total cellular protein (30 µg/lane) was then boiled for 5 min in SDS-sample buffer (Beyotime Institute of Biotechnology) before being subjected to SDS-PAGE (8%) and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were then blocked for 2 h with nonfat dry milk in sterile water at room temperature and then incubated overnight at 4°C with a mouse anti-human CDH11 monoclonal antibody (cat. no. 32–1700; 2 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.). Following four washes in TBS-Tween, membranes were incubated with a horse anti-mouse secondary antibody (cat. no. 7076; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h. The blots were visualized by Enhanced Chemiluminescence (Thermo Fisher Scientific, Inc.) and imaged using aTanon-5200 imaging system machine (Tanon Science and Technology, Co., Ltd., Nanjing, China) and the membrane was probed with an anti-GAPDH antibody (cat. no. 5174; dilution 1:2,000; Cell Signaling Technology, Inc.) to confirm equal loading. The specific western blot assay method was performed according to the manufacturer's protocol and as described by Miao et al (18), andthe quantitative analysis was performed usingGel Image System version 4.00 (Tanon Science and Technology, Co., Ltd., Shanghai, China).

Transwell assays were conducted to investigate the motility and invasiveness of HT-29 cells. Cell migration assays were performed in 24-well Transwell chambers with 8.0-µm pore size transwell inserts (Costar; Corning Incorporated, Corning, NY, USA,). Cell invasion assays were investigated using an 8.0-µm filter coated with 30 µl of 0.5% Matrigel (Sigma-Aldrich; Merck KGaA). Transwell assays were performed according to the manufacturer's protocol and as previous described (18). Briefly, 4×10⁴ cells treated with MC-LR or transfected with siRNA were added to the upper chamber of DMEM, and DMEM with 10% FBS was added to the lower chamber. Following incubation for 48 or 72 h (migration assay) and 72 or 96 h (invasion assay) at 37°C in a humidified atmosphere of 5% CO₂, cells on the upper surface were removed completely by gentle wiping with a cotton swab. Cells that penetrated through pores and adhered to the underside of the filters were then fixed with 4% paraformaldehyde and stained with 0.02% crystal violet solution containing 20% ethanol. Then cells were observed and counted under light microscopy (magnification, ×100) (CKX41 microscope; Olympus Corporation, Tokyo, Japan). For each replicate (n=3), cells in three randomly selected fields per well were counted and averaged. Data are expressed as a ratio to the control (no MC-LR treatment but negative control siRNA treatment). Tumor cell motility and invasiveness was defined as the mean number of the treated cells.

All statistical analyses were carried out by two-tailed Student's t-test or a one-way analysis of variance. Data are presented as the mean ± standard deviation from three different experiments. P<0.05 was considered to indicate a statistically significant difference. Computer-based calculations were conducted using Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA, USA).

To investigate the motility and invasiveness of the HT-29 cells following MC-LR treatment, Transwell chamber assays were performed. In the present study, MC-LR at 25 nM promoted migration and invasion of HT-29 cells and significantly increased the number of translocated cells compared with the control group (Fig. 1). MC-LR treatment resulted in a 1.4-fold increase in cell migration (P<0.01; Fig. 1A), suggesting that MC-LR induces migration of HT-29 cells. The results from the invasion assay in HT-29 cells demonstrated that MC-LR exposure led to a 1.5-fold increase in cell number compared with the control group (P<0.05; Fig. 1B).

To confirm the effect of MC-LR treatment on CDH11 expression, the protein levels of CDH11 in HT-29cancer cells treated with different concentrations of MC-LR were investigated. MC-LR treatment resulted in a significant increase in CDH11 expression in HT-29cancer cells as measured by cellular immune fluorescence. Compared with the control, cell treatment with 25 nM of MC-LR produced a 1.9-fold raise in fluorescence intensity of CDH11 (P<0.01; Fig. 2A). Similar to the results of immunofluorescence assays, western blot analysis demonstrated a marked increase in protein levels with MC-LR treatment, particularly at 12.5, 25, 50 nM compared with GAPDH expression (Fig. 2B). The results from cellular immunofluorescence and western blotting revealed that the protein level of CDH11 is upregulated by MC-LR in HT-29 cells, which may mediate the effects of MC-LR on cell migration and invasion.

To further evaluate the association between MC-LR-induced CDH11 overexpression, and tumor migration and invasion, an RNA interference assay was performed. Western blot analysis demonstrated that CDH11-siRNA markedly reduced the expression of CDH11 in HT-29 cells (Fig. 3A). Following treatment with CDH11-siRNA, cells exposed to MC-LR (25 nM; Group C) exhibited significantly decreased motility (0.52-fold; P<0.05) and invasiveness (0.42-fold; P<0.01) compared with the control group (Group A, negative control siRNA), whereas treatment with MC-LR in the presence of the negative control siRNA (Group B) significantly increased cell migration (0.43-fold; P<0.05) and invasion (0.73-fold; P<0.01) compared with the control group (Group A), and in the presence of MC-LR, the knockdown of CDH11 reduced cell migration and invasion ability by approximately0.67-fold (Fig. 3B and C). These data revealed that MC-LR-induced migration and invasion decreases following CDH11 knockdown, suggesting that CDH11 serves an important role in MC-LR-induced migration and invasion of HT-29 cells.