The human laryngeal cancer cell line Hep-2 was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and originated from a metastatic peidermoid carcinoma of the larynx (12). The cell was cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a 5% CO₂ incubator. Upon reaching 60–70% confluence, the cells were divided into two new dish or seeded in six-well plates at a density of 1×10⁵ cells per well for further experiments. The Hep-2 cells were transfected with short hairpin RNA (shRNA) control or S100A4 shRNA expression plasmids. Hep-2-shRNA cells expressed an shRNA, specifically targeting S100A4 mRNA, while Hep-2 negative control (Hep-2-shNC) cells expressed an unrelated control sequence. shRNA were transfected into Hep-2 cells using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. Total RNA was prepared 24 h post transfection and the results of gene knockdown were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cells were incubated for 72 h and then harvested for further experiments.

The quantification of S100A4 expression levels was performed using an SYBR-Green I-based real-time fluorescence detection method (13). Total RNA was isolated from Hep-2 cells using an mRNA isolation kit (Qiagen, Valencia, CA, USA), and subsequently reverse transcribed using the Reverse Transcription PCR kit kit (Takara Bio Inc., Shiga, Japan) with Oligo-dT primers. cDNA was amplified using an SYBR Premix Ex Taq kit (Takara Bio Inc.) on an ABI 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH served as a reference gene and samples without cDNA or without oligonucleotides were used as negative controls. The control GAPDH fragment was amplified using the following primer sequences: GAPDH, forward 5′-ATCATCAGCAATGCCTCC-3′ and reverse 5′-CATCACGCCACAGTTTCC 3′; S100A4, forward 5′-CCCTGGATGTGATGGTGTC-3′ and reverse 5′-CTCGTTGTCCCTGTTGCTG-3′. The 25 μl PCR included 0.5 μl cDNA, SYBR^® Premix EX Taq™ 12.5 and 2 μl of primers. The reaction was run at 95°C for 1 min, followed by 45 cycles at 95°C for 5 sec and 60°C for 20 sec. The Ct data was determined using default threshold settings. The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence passes the fixed threshold. All measurements were performed at least three times.

To determine the levels of protein expression, soluble proteins were isolated using radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.2% NP40) containing complete protease inhibitor followed by centrifugation at 12,000 × g at 4°C. The protein concentration was determined from the supernatant using a bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China) and the results were produced by an ELISA-plate reader (Sunrise RC; Tecan, Theale, UK). Protein extracts were diluted with 5X sodium dodecyl sulfate (SDS) loading buffer, boiled and resolved on a 12% w/v SDS-PAGE. Protein extract (40 mg) was loaded. Following electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Roche Diagnostics, Mannheim, Germany) by semidry blotting. Detection was performed using rabbit polyclonal anti-S100A4 antibody (Thermo Fisher Scientific, Waltham, MA, USA; 1:1,000) and a mouse monoclonal anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:1,000) was used as a loading control. Secondary antibodies anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase (Beyotime Institute of Biotechnology) were used at dilutions of 1:2,000. Blots were developed with Western Lightning Chemiluminescence Reagent Plus (Thermo Fisher Scientific) and chemiluminescence detection film (Beyotime Institute of Biotechnology).

S100A4-shRNA Hep-2 cells and control cells between 12 h and 5 days after transfection, were seeded in a 96-well flat-bottomed plate at a density of 2×10³ cells in 190 μl of the medium per well. Cell proliferation was determined using the Cell Counting kit-8 (CCK-8; Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. After 10 μl CCK-8 was added to each well, the cells were incubated at 37°C for 2 h and the absorbance was monitored using a microplate reader (Sunrise RC) at a wavelength of 490 nm. Each experiment was performed in triplicate and repeated three times. Data are presented as the mean ± standard deviation.

The anchorage-independent growth of cells was determined by assaying colony formation in soft agar. Following transfection, single cell suspension of transfected Hep-2 cells was prepared by trypsinization and homogenization. Cells were plated onto each well of a six-well plate at a density of 1,000 cells/well in RPMI-1640 containing 10% fetal bovine serum and 0.3% low melting point agarose (Amresco, Solon, OH, USA) on a base layer of 0.6% low melting point agarose. The number of colonies >50 μm was counted and images were captured following 2 weeks of incubation at 37°C. The experiments were performed three times independently.

The Hep-2-shRNA transfected cells and control cells were harvested, fixed and resuspended in phosphate-buffered saline. Apoptotic cells were measured using an annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7AAD) kit (Nanjing KeyGen Biotech., Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Briefly, annexin V-PE and 7AAD were added to a mixture containing 100 μl of cell resuspension in binding buffer (BD Biosciences, Franklin Lakes, NJ, USA). The cells were vortexed and incubated for 15 min at room temperature in the dark and 400 μl of binding buffer was added to the mixture for flow cytometric analysis using a Becton Dickinson FACScan Flow Cytometer (FACScan; Becton Dickinson, Franklin Lakes, NJ, USA).

The activity of caspase-3, -8 and -9 was determined using the caspase-3, -8 and -9 activity kit (Beyotime Institute of Biotechnology). To evaluate the activity of caspase-3, -8 and -9, Hep-2-shRNA transfected and control Hep-2 cells were homogenized in 100 ml reaction buffer [1% NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM Nicotinamide adenine dinucleotide and 10% glycerol] containing 10 ml caspase-3, -8 and -9 substrate (Ac-DEVD-pNA; 2 mM) following all treatments. Lysates were incubated at 37°C for 2 h. Samples were measured with an ELISA reader (Sunrise RC) at an absorbance of 405 nm.

All the animals (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) in the present study were housed in a pathogen-free facility and the animal experiments were performed in accordance with the institutional animal welfare guideline of The Center for Laboratory Animal Research, China Medical University (Shenyang, Liaoning, China). Cells were injected subcutaneously into BALB/c nude mice (6–8 weeks). The tumor volume (TV) was calculated using the following formula: TV (mm³) = (Length × Width²) / 2.

Unless otherwise stated, each experiment was performed a minimum of three times. Data were subjected to statistical analysis using Statistical Package for the Social Sciences software (version 13.0; SPSS, Inc., Chicago, IL, USA) and presented as the mean ± standard error of the mean. The independent Student’s t-test or analysis of variance was used to compare the continuous variables among groups. The Kaplan-Meier estimate was used for survival analysis. P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression of S100A4 in cultured Hep-2 cells 72 h after shRNA transfection was detected by RT-qPCR and western blot analysis. Hep-2-shNC cells were also transfected as a control (Fig. 1A). The results of the analysis of S100A4 are shown in Fig. 1. Following transfection with S100A4 shRNA, the Hep-2 cells showed significant downregulation of the S100A4 gene at the mRNA and protein levels (Fig. 1B and C; P<0.05).

As determined by the CCK-8 assay, inhibition of S100A4 in Hep-2 cells resulted in a significant decrease in cellular proliferation at 2, 4, 6 and 8 days after transfection (P<0.05). The results indicated that S100A4 suppression correlated with decreased proliferation in Hep-2 cells (Fig. 2A).

The anchorage-independent growth of cells was determined by assaying colony formation in soft agar. Our results demonstrated that, compared with Hep-2-shNC cells, the colonies in the Hep-2-shRNA cells were significantly decreased in number and notably smaller in size (Fig. 2B and C).

Apoptosis of Hep-2-shNC and Hep-2-shRNA cells was examined by flow cytometry. As shown in Fig. 3, after 3 days, 4.4 and 4.92% of the control cells and Hep-2-shNC cells were apoptotic, while 11.7% of Hep-2-shRNA cells were apoptotic.

Caspase proteins are cysteine proteases that act downstream of the B-cell lymphoma 2 family by initiating cellular breakdown during apoptosis. Among the effector caspases, caspase-3 is the most frequently involved in apoptosis. To determine whether caspase-3, -8 and -9 are activated following S100A4 shRNA treatment, caspase-3, -8 and -9 activity was measured by the caspase-3, -8 and -9 activity kit. In the present study, the results demonstrated that compared with the control cells, treatment of cells with S100A4 shRNA caused a significant increase in caspase-3, caspase-8 and caspase-9 activation (P<0.05). The activity of caspase-3, caspase-8 and caspase-9 in cells treated with S100A4 shRNA increased >12% (P<0.05), compared with cells treated with control shRNA (Fig. 4). These results suggested that the downregulation of S100A4 was able to significantly increase the activity of caspase-3, caspase-8 and caspase-9 in Hep-2 cells.

Since inhibition of S100A4 reduced Hep-2 cell proliferation in vitro, whether these in vitro data have in vivo relevance was examined. To accomplish this goal, Hep-2 cells generated tumors when 2×10⁵ cells were injected into the flank of nude mice. Visible tumors had developed at the injection sites within 2 weeks (Fig. 5A and B). The S100A4 knockdown Hep-2 cells significantly reduced the tumor volumes and prolonged the survival rate of nude mice (Fig. 5C and D). Our data indicated that downregulation of S100A4 decreased the tumorigenicity of laryngeal cancer in vivo.