The SRA01/04 human LEC line was purchased from Shanghai SXBIO Biotechnology Co., Ltd. (Shanghai, China). SRA01/04 cells were cultured in the Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12; 1:1) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified 5% CO₂ incubator at 37°C. SRA01/04 cells were grown to 80–90% confluence, digested with a 0.25% trypsin-0.02% ethylenediamine-N,N,N',N'-tetraacetic acid solution (Gibco; Thermo Fisher Scientific, Inc.), centrifuged at 100 × g for 5 min at room temperature and transferred into a new culture dish for passage. SRA01/04 cells were stored in frozen stock solution at −80°C containing 10% dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 40% FBS and 50% DMEM/F12.

Human E-cadherin siRNA and negative control (NC) siRNA was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNA sequences were as follows: E-cadherin siRNA forward, 5′-GAGGCUGUAUACACCAUAUTT-3′ and reverse, 5′-AUAUGGUGUAUACAGCCUCTT-3′; NC siRNA forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′. SRA01/04 cells (3×10⁵) were seeded into a 6-well plate with 2 ml antibiotic-free DMEM/F12 medium 24 h prior to transfection at 37°C. Healthy, well-adhered 70–90% confluent cells were used for transfection. E-cadherin and NC siRNAs were diluted in 250 µl Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) to make a concentration of 20 µM stock solution; 3.3 µl stock solution was subsequently added to a 6-well plate to a final concentration of 66 nM and mixed gently 3–5 times. Lipofectamine 2000 transfection reagent (10 µl; Invitrogen; Thermo Fisher Scientific, Inc.) was diluted in 250 µl Opti-MEM, mixed and incubated for 5 min at room temperature. The diluted siRNA and transfection reagent were combined and incubated at room temperature for 20 min. Subsequently, 500 µl of the mixture was added to each well of the 6-well plate and mixed. Cells were transfected with the E-cadherin siRNA (siRNA group), the NC siRNA (NC group) or with Lipofectamine 2000 (blank group) and were cultured at 37°C in a 5% CO₂ incubator for 48 h; the medium was replaced with fresh antibiotic-free medium following 12 h of transfection.

Total RNA was extracted from 1×10⁶ cultivated cells using the RNAiso Plus kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Briefly, cells were lysed in 1 ml RNAiso Plus for 5 min at room temperature. The lysate was centrifuged at 12,000 × g for 5 min at 4°C, and the aqueous phase containing RNA was separated from the organic phase via incubation with 200 µl chloroform for 5 min at room temperature. The solution was subsequently centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was transferred into a new tube and incubated with 700 µl isopropanol for 10 min at room temperature. RNA was precipitated via centrifugation at 12,000 × g for 10 min at 4°C. The RNA pellet was washed with 75% ethanol, air dried for 5–10 min and redissolved in 30 µl diethyl pyrocarbonate (DEPC)-treated water. To assess the quality of RNA, A260/A280 and A260/A230 ratios of the RNA samples were measured using an SMA4000 Ultraviolet spectrophotometer (Merinton Instruments, Ltd., Beijing, China). An A260/A280 ratio between 2.0 and 2.3 was considered high quality RNA (17) and was used for the subsequent experiments.

cDNA was synthesized using a PrimeScript RT-PCR kit (Takara Bio, Inc.) according to the manufacturer's protocol. Primers for E-cadherin, N-cadherin, β-catenin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China; Table I). qPCR reactions were set up as follows: 10 µl SYBR Premix Ex Taq (2X; Takara Bio, Inc.), 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 2 µl cDNA (adjusted to the same concentration) and 6 µl double-distilled H₂O. qPCR was performed using a ViiA 7 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following conditions: 50.0°C for 3 min and 95.0°C for 3 min; followed by 40 cycles of 95.0°C for 10 sec and 60.0°C for 30 sec; followed by melting curve construction and analysis. GAPDH was used as a reference gene. Each sample was repeated three times, and calculation of gene expression was performed by the 2^−ΔΔCq method (18).

A total of 5×10⁶ SRA01/04 cells were washed three times with pre-cooled phosphate-buffered saline (Gibco, Thermo Fisher Scientific, Inc.) and lysed at 4°C for 30 min with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (Sigma-Aldrich; Merck KGaA). The supernatant without cell debris was obtained by centrifugation at 19,000 × g for 10 min at 4°C. Protein concentration was determined by Bicinchoninic Acid Protein Assay kit (Sangon Biotech, Co., Ltd., Shanghai, China) according to the manufacturer's instruments.

A total of 30 µg protein was loaded in each well and separated by 10% (w/v) SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were transferred onto a polyvinylidene fluoride membrane (Merck KGaA) and blocked with 5% non-fat dry milk for 1 h at room temperature. The membrane was probed overnight at 4°C with the following primary antibodies: rabbit anti-GAPDH (1:5,000; BM1623; Wuhan Boster Biological Technology, Ltd., Wuhan, China), rabbit anti-E-cadherin (1:500; 20874-1-AP; Wuhan Sanying Biotechnology, Wuhan, China), rabbit anti-N-cadherin (1:100; BA0673) and rabbit anti-β-catenin (1:100; BM3905) (both from Wuhan Boster Biological Technology, Ltd.). Following 3 washes with TBS + 0.05% Tween-20 (TBST), the membranes were incubated with the secondary horseradish peroxidase-conjugated goat-anti-rabbit immunoglobulin G antibody (1:5,000; 111-035-045; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h. Membranes were washed with 1X TBST buffer, and the formed immunocomplexes were visualized by Enhanced Chemiluminescence reagent (Merck KGaA). Densitometric analysis was performed with Quantity One software (version 4.5.2) on the ChemiDoc XRS Gel Imaging system (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA). Western blot analysis was repeated three times.

Statistical analysis was conducted using SPSS software version 11.0 (SPSS, Chicago, IL, USA). The data are presented as the mean ± standard error of the mean. One-way analysis of variance (19) and the post-hoc Duncan's multiple range tests (20) were used to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of E-cadherin, N-cadherin and β-catenin in the E-cadherin siRNA, NC siRNA and Blank groups were detected by RT-qPCR (Fig. 1). Following 48 h E-cadherin siRNA transfection, the expression level of E-cadherin was significantly downregulated in the E-cadherin siRNA group compared with those in the NC siRNA and Blank groups (P<0.05; Fig. 1A). The mRNA expression level of N-cadherin in the E-cadherin siRNA group was also significantly reduced (P<0.05; Fig. 1B). However, the expression level of β-catenin was unaltered compared with those in the NC siRNA and Blank groups (P>0.05; Fig. 1C).

To investigate the protein levels of the E-cadherin, N-cadherin and β-catenin, western blotting was conducted (Fig. 2). Consistent with the RT-qPCR results aforementioned, the protein expression levels of E-cadherin and N-cadherin were reduced in the E-cadherin siRNA group compared with the NC siRNA and Blank groups, and the protein expression level of β-catenin had no notable changes.