Patients and thyroid tissue sample
Thyroid cancer tissues (42 pairs) and corresponding normal tissues were collected from patients in the thyroid surgery department of the First Affiliated Hospital of Wenzhou Medical University, China, and stored in liquid nitrogen. Two pathologists performed the histological examinations of all specimens to confirm the histological diagnosis. All patients signed a letter of consent, and the patient’s information was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (approval no. 2012‐57).
Information of 502 thyroid cancer patients with complete clinical case characteristics (age, sex, LNM, tumor size, clinical stage, etc.) was downloaded from the TCGA database for analysis. The public gene chip dataset was downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). The GSEA software (GSEA v3.0, http://www.broadinstitute.org/gsea) was used to perform a single GSEA between the low S100A6 and high S100A6 expression groups in TCGA.
RNA isolation and real‐time reverse transcription/polymerase chain reaction (qPCR)
Total RNA of TC and normal tissues was extracted using TRIzol reagent（Thermo Fisher Scientific，Waltham，USA）according to the manufacturer’s instructions. cDNA synthesis was performed using the RerverTra Ace qPCR RT kit(Toyobo ,Osaka, Japan). The cDNA was stored at -20°C for further processing. To detect the expression level of S100A6, qRT-PCR was performed using The Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).The primer sequences used for quantitative PCR were as follows：GAPDH forward 5′-GTCTCCTCTGACTTCAACAGCG-3’/ reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3'；S100A6 forward 5′-GAAATTGCAAGGCTGATGGAAG-3'/ reverse 5′-GGCTTCATTGTAGATCAAAGCC-3'.
Human TC cell lines (FTC, KTC-1, and BCPAP) were provided by Professor Mingzhao Xing of Johns Hopkins University School of Medicine (Baltimore, Maryland, USA). The Htorti-3 cell line was purchased from the Stem Cell Bank, Chinese Academy of Sciences. TPC‐1 and BCPAP cell lines were cultured in RPMI‐1640 containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA, USA); FTC was cultured in DMEM medium (Gibco, Invitrogen, Carlsbad, CA, USA). Three cell lines were incubated at 37°C with 5% CO2 in a standard cell culture incubator (Thermo Fisher Scientific).
Thyroid cells were seeded in a six-well plate and transfected for 24 h. S100A6 was silenced by siRNA using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Grand Island, NY, USA). Both Si-S100A6 and Si-NC were purchased from GenePharma (Shanghai, China). 48 hours after transfection, cells were subjected to the next experiment. The sequences were as follows: Si-S100A6, forward 5'-AAGCTGCAGGATGCTGAAATT-3' ;Si-NC, forward 5-UUCUCCGAACGUGUCACGUTT-3 and reverse 5-ACGUGACACGUUCGGAGAATT-3′.
Cell proliferation and colony formation assay
Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China)and clone formation experiments were used to evaluate the proliferative ability of the cells. Transfected cells were spread (1000 cells per well) in a 96-well plate. Approximately 100 µL of cck8 were added to each well, and the cells were incubated at 37 °C for 3 h. The absorbance of each well at 450 nm was measured for four consecutive days.
Clone formation experiment
The transfected TC cells were seeded into a 6-well plate and incubated under the same conditions for 5-8 days. Cells were then fixed with 4% paraformaldehyde at room temperature for 15 min, and stained with crystal violet. Pictures were taken under a strong light by a digital camera. Each experiment was repeated three times.
Cell migration and invasion assays
Transwell assays were performed to evaluate the migration and invasion capabilities of the cells. Transfected and control cells were collected using trypsin. The Transwell chamber (#3422, Corning, NY, USA) and Matrigel Invasion Chamber (#354480; Corning Biocat, New York, USA)was used in the assays. First, 600 µL of medium containing 10% FBS were added to the lower chamber and the cells (35000 cells per well) were transferred to the upper chamber and incubated at 37 °C for 20 h. Cells migrating from the upper chamber to the lower chamber were fixed and stained with crystal violet for 15 min. Three fields of view were randomly selected, and images were taken using a microscope at ×5 magnification. Each experiment was repeated 3 times.
Annexin V-FITC apoptosis kit (#556,547; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used to detect cell apoptosis. Cells were collected, and stained with added Annexin V-FITC and PI in the dark for 15 min. The cells were analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, USA). The apoptosis rate was equal to the percentage of late apoptosis (Q2) and the percentage of early apoptosis (Q3).
Western blot analysis
Total protein was extracted 48 h after transfection, and protein concentrations were determined using bicinchoninic acid assay (BCA). Protein lysates were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The PVDF membranes were blocked with skim milk for 2 h at room temperature and then washed three times with TBST. Cells were incubated with primary antibodies overnight at 4°C. The primary antibodies are as follows:BCL-2(Abcam, Cambridge, Massachusetts, USA); S100A6, p-AKT, p-PI3K, p-MTOR, BAX and β-Actin (Proteintech, Wuhan, China). Membranes were rewashed with TBST three times and incubated with secondary antibodies (anti-mouse IgG or anti-rabbit IgG) for 1 h at room temperature. A chemiluminescence kit (Thermo Scientific, USA) was used to expose the films. Image Lab software was used to quantitatively analyze the bands.
Statistical analysis was performed using SPSS 23.0, and GraphPad Prism was used to create charts. Student's t-test or one-way ANOVA was used to assess differences between groups. Data in the figure are the mean ± standard deviation. P<0.05, represents statistical significance.