HUVECs were obtained from the China Center for Type Culture Collection (Wuhan, China) and cultured in Dulbecco's modified Eagle medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an incubator (HF-90; Shanghai Lishen Scientific Equipment Co., Ltd., Shanghai, China) containing 5% CO₂. The culture medium was supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA).

HUVECs were plated at a density of 5×10³ cells/well onto 96-well plates and cultured for 24 h at 37°C. Following this, cells were incubated at 37°C with various doses of MgCl₂ (1.25, 2.5, 5, 6.25, 12.5, 25, 50 and 100 mM) or ZnCl₂ (1, 25, 50, 100, 300 and 500 µM; both Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Untreated HUVECs served as a control. Following 24 h treatment, MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to the cells and they were incubated at 37°C for 4 h. Subsequently, the supernatant was discarded and 200 µl dimethylsulfoxide (Sigma-Aldrich; Merck KGaA) was added to dissolve the formazan crystals. The absorbance was recorded at 490 nm using an ELX-800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The upper chamber of a Transwell chamber (Corning Life Sciences, Tewksbury, MA, USA) was coated with 45 µl diluted Matrigel (BD Biosciences, San Jose, CA, USA) and placed in a 24-well plate for the invasion assay. HUVECs were cultured in DMEM supplemented with 10% FBS. At 90% confluence, the medium was replaced with serum-free DMEM and cells were incubated with 10 µg/ml mitomycin C (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Following this, cells were digested with 0.25% trypsin and resuspended in serum-free DMEM to prepare a single cell suspension. Cells were seeded into the upper chamber uncoated (8,000 cells in 200 µl cell suspension for migration assay) or coated with Matrigel (2×10⁴ cells in 200 µl cell suspension for invasion assay) and DMEM supplemented with 20% FBS was added into the lower chamber. Subsequently, the cells were incubated with 12.5 mM MgCl₂ or 100 µM ZnCl₂. After 24 h cell culture at 37°C, the cells on the upper side of the filters were wiped with cotton swabs. The migrated or invaded cells were fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 20 min at room temperature and stained with crystal violet (Amresco, LLC, Cleveland, OH, USA) for 5 min. The migrated or invaded cell number was counted in five random fields under an AE31 inverted microscope (Motic, Xiamen, China) at magnification, ×200, and the average was calculated.

HUVECs were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1% phenylmethanesulfonyl fluoride protease inhibitor (ST506; Beyotime Institute of Biotechnology) on ice for 5 min and centrifuged at 10,005 × g for 10 min at 4°C. The supernatant was collected and protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (40 µg/lane) were separated by 8 or 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h. Subsequently, membranes were incubated with antibodies against matrix metalloproteinase (MMP)-2 (1:400; BA0569; Wuhan Boster Biological Technology, Ltd., Wuhan, China), MMP-9 (1:400; BA0573; Wuhan Boster Biological Technology, Ltd.), vimentin (1:500; bs-8533R; BIOSS, Beijing, China), Snail (1:500; bs-1371R; BIOSS), N-cadherin (1:400; BA0673; Wuhan Boster Biological Technology, Ltd.), Wnt1 (1:200; sc-5630; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-catenin (1:400; BA0426; Wuhan Boster Biological Technology, Ltd.) overnight at 4°C. Subsequent to washing with Tris-buffered saline-Tween-20, membranes were incubated with goat anti-rabbit horseradish peroxidase-labeled immunoglobulin G (1:5,000; A0208; Beyotime Institute of Biotechnology) for 45 min at 37°C and visualized using a high sensitivity enhanced chemiluminescence reagent kit (WLA003; Wanleibio Co., Ltd., Shenyang, China). The experiment was repeated three times and results were quantified using Gel-Pro Analyzer v.4 (Media Cybernetics, Inc., Rockville, MD, USA).

The supernatant of the cell culture was harvested by centrifugation (1,000 × g for 10 min) following 24 h treatment with MgCl₂ or ZnCl₂ at 37°C. The expression levels of MMP-2 and MMP-9 in the supernatant were measured using ELISA kits (DRE11368 and DRE10154; Shanghai WHB Biotech Co., Ltd., Shanghai, China), according to the manufacturer's instructions. The plates were read on a microplate reader (BioTek Instruments, Inc.) at 450 nm.

Total RNA was isolated from HUVECs using RL lysis buffer containing DNase, according to the manufacturer's protocol (BioTeke Corp., Beijing, China). RNA was then reverse transcribed into cDNA using M-MLV reverse transcriptase (BioTeke Corp.). Template RNA (1 µg) was mixed with 1 µl Oligo (dT)₁₅, 1 µl random primer, and 2 µl dNTP and ddH₂O (final volume of 14.5 µl), incubated at 70°C for 5 min and then put on ice for 2 min. The mixture was subsequently incubated with 0.5 µl RNasin, 4 µl reaction buffer and 1 µl (200 U) M-MLV reverse transcriptase (final volume of 20 µl) at 25°C for 10 min, followed by 42°C for 50 min and 95°C for 5 min. The primer sequences for qPCR were as follows: E-cadherin, forward 5′-AGAACGCATTGCCACATACA-3′ and reverse 5′-TAAGCGATGGCGGCATTGTA-3′; N-cadherin, forward 5′-CAACACACTCGCAGACGCTCA-3′ and reverse 5′-AAGACGGCTCCAGGCAGTTT-3′; and β-actin, forward 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ and reverse 5′-CTGTCACCTTCACCGTTCCAGTTT-3′. qPCR was performed using an Exicycler^™ 96 Real-Time Quantitative PCR system (Bioneer Corporation, Daejeon, Korea). The cycling conditions were as follows: 10 min at 95°C; followed by 40 cycles of 10 sec at 95°C, 20 sec at 60°C and 30 sec at 72°C (20 µl reaction volume comprising 1 µl cDNA template, 0.5 µl forward primer, 0.5 µl reverse primer, 10 µl SYBR Green PCR Mastermix and 8 µl ddH₂O). The experiment was repeated three times and β-actin was used as an internal control. The relative mRNA expression levels were normalized to β-actin, following the 2^−ΔΔCq method (27).

HUVECs were treated with MgCl₂ or ZnCl₂ for 24 h and subsequently lysed by repeated freezing and thawing. Following centrifugation of the homogenate at 10,005 × g for 10 min at 4°C, the supernatant was harvested and the protein concentration was determined by the BCA method (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg/lane) were separated by 10% SDS-PAGE containing 1 ml gelatin (10 mg/ml) (Sigma-Aldrich; Merck KGaA). Following this, the gels were washed twice with elution buffer (40 min; 2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl₂ and 1 µM ZnCl₂; pH 7.6) at room temperature and then twice with washing buffer (20 min; 50 mM Tris-HCl, 5 mM CaCl₂ and 1 µM ZnCl₂; pH 7.6). Subsequently, the gels were stained with Coomassie Brilliant Blue R-250 (Amresco, LLC) for 3 h. Gels were then destained with methanol and acetic acid, and imaged using a gel documentation system (WD-9413B; Beijing Liuyi Instrument Factory, Beijing, China).

HUVECs were seeded in coverslips and incubated with 12.5 mM MgCl₂ or 100 µM ZnCl₂ for 24 h at 37°C. Following this, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 15 min at room temperature and washed three times with PBS. Subsequently, coverslips were incubated with Acti-stain^™ 488 Fluorescent Phalloidin (F-actin staining; Cytoskeleton, Inc., Denver, CO, USA) for 1 h. Then, 4′,6-diamidino-2-phenylindole (DAPI; Biosharp, Hefei, China) was added to counterstain the nuclei of the cells. Images were captured under a fluorescent microscope (magnification, ×400; BX53; Olympus Corp., Tokyo, Japan) and analyzed using Image Pro Plus software v.6 (Media Cybernetics, Inc.).

HUVECs were plated on slides, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Subsequent to blocking with goat serum (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) for 15 min at room temperature, the slides were incubated with antibody against E-cadherin (1:200; BA0474; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight and then with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature (1:200; A0562; Beyotime Institute of Biotechnology). Following this, the slides were stained with DAPI and imaged under a fluorescent microscope (magnification, ×400; BX53; Olympus Corp.).

Results were presented as the mean ± standard deviation. The differences were analyzed by one-way analysis of variance followed by Bonferroni post hoc tests using GraphPad Prism v.5 software (GraphPad Software, Inc., La Jolla, CA, USA) or Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

HUVECs were incubated with various doses of MgCl₂ (0, 1.25, 2.5, 5, 6.25, 12.5, 25, 50 and 100 mM) and ZnCl₂ (0, 1, 25, 50, 100, 300 and 500 µM) for 24 h. The cytotoxicity of MgCl₂ or ZnCl₂ was measured by MTT assay. The results demonstrated that the cell viability of HUVECs was not significantly affected following exposure to all concentrations of MgCl₂ and ZnCl₂ (Fig. 1). Therefore, 12.5 mM MgCl₂ and 100 µM ZnCl₂ were selected for further experiments.

Cell migration and invasion assays using Transwell chambers were performed to investigate the effect of MgCl₂ and ZnCl₂ on the migration and invasion capabilities of HUVECs. The results demonstrated that both MgCl₂ and ZnCl₂ significantly enhanced the migration and invasion abilities of HUVECs compared with the control group (Fig. 2A). The extracellular matrix and basement membrane provide the major physical barriers to cell invasion. MMPs are important proteolytic enzymes able to degrade the extracellular matrix and basement membrane and serve critical roles in the invasion process (28). MgCl₂ and ZnCl₂ treatment significantly increased the expression of MMP-2 and MMP-9 proteins, as determined by western blotting (Fig. 2B) and ELISA (Fig. 2C). RT-qPCR determined that the expression of MMP-2 and MMP-9 mRNA was significantly increased (Fig. 2D). The gelatin zymography assay demonstrated that MMP-2 and MMP-9 activity was significantly enhanced by MgCl₂ and ZnCl₂ (Fig. 2E).

Subsequently, the effects of MgCl₂ and ZnCl₂ on cytoskeletal reorganization in HUVECs were investigated. The results demonstrated that MgCl₂ and ZnCl₂ treatment promoted cytoskeletal reorganization, with an increased long axis/short axis dimension ratio compared with the control (Fig. 3).

To further investigate whether MgCl₂ and ZnCl₂ were able to modulate EMT, the expression of several EMT-related genes were measured using RT-qPCR, western blotting and immunofluorescence staining assays. The results demonstrated that the expression of E-cadherin was downregulated following MgCl₂ and ZnCl₂ stimulation; however, this difference was only significant following ZnCl₂ stimulation (Fig. 4A). N-cadherin mRNA and protein expression was significantly upregulated following MgCl₂ and ZnCl₂ stimulation compared with the control (Fig. 4A and B). Furthermore, the results demonstrated that MgCl₂ and ZnCl₂ treatment significantly increased the expression of vimentin and Snail at both mRNA and protein levels (Fig. 4A and B). The immunofluorescence staining assay further demonstrated that E-cadherin was downregulated following stimulation with MgCl₂ and ZnCl₂ (Fig. 4C).

HUVECs were incubated with MgCl₂ and ZnCl₂ for 24 h and the expression of Wnt1 and β-catenin were measured by western blotting analysis. The expression of Wnt1 and β-catenin protein were significantly increased in MgCl₂- and ZnCl₂-treated HUVECs compared with the control (Fig. 5).