Cells and tissue samples
Human thyroid carcinoma cell lines TPC-1, BHT101, 8305C and CAL-62, and human normal thyroid cell lines Nthy-ori 3–1 were purchased from Procell (Wuhan, China). Cells were cultured in DMEM (Sigma Aldrich, America) supplied with 0.1% FBS (HyClone; Cytiva) and 1% streptomycin and penicillin (Sigma Aldrich, America) and maintained at 37 °C with 5% carbon dioxide (CO2). A collection of 10 pairs of human PTMC tissues and corresponding PTMC-adjacent tissues were acquired from West China Hospital of Sichuan University according to the institutional guidelines. No patients had received chemotherapy or radiotherapy prior to surgical resection. Written informed consent was obtained for use of the tissues. This study was approved by the Ethics Review Board of West China Hospital of Sichuan University.
Four-week-old nude mice were purchased from Chengdu Dossy Experimental Animals CO., LTD. Mice were housed under pathogen-free conditions and in a temperature-controlled room illuminated for 12 h daily. Following acclimation for 1 week, mice were divided into the sh-NC and the sh-TESC group (n = 6). Mice in the sh-TESC group were subcutaneously injected TESC-RNAi transfected cells (a total of 1 × 106), while mice in the sh-NC were subcutaneously injected with the same volume of negative control (NC)-RNAi transfected cells to build the xenograft model. Tumor formation was monitored by detecting tumor volumes and weights. The study was carried out in compliance with the ARRIVE guidelines. The protocol was approved by the Institutional Animal Care and Use Committee of Sichuan University. Animals received humane care in accordance with study guidelines established by the second affiliated hospital of Sichuan University.
Immunohistochemical staining (IHC)
The tissues of PTMC and non-PTMC from patients were fixed in 4% paraformaldehyde at room temperature, embedded in paraffin wax, and cut into 5 μm sections. The sections were then dewaxed with xylene and ethanol gradient and had their endogenous peroxidase activities quenched using 3% hydrogen peroxide at room temperature for 10 min. Anti-TESC antibody (1:200, cat. no. 11125–1-AP, Proteintech, USA) was incubated with the sections overnight at 4 °C before the sections were incubated with biotinylated secondary antibodies at 37 °C for 30 min. Diaminobenzidine was used for histochemical reactions and hematoxylin was used for counterstaining. Histological examination was performed on a light microscope (Olympus, Japan) and the images were analyzed using the Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA). In addition, tumor tissues of mice were collected and fixed with 4% paraformaldehyde. Then, paraffin sections (5 μm) were analyzed by IHC with anti-Ki-67 antibody (1:200, cat. no. ab16667, abcam, USA).
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cell samples using TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s specifications. The cDNA was synthesized with a PrimeScript RT reagent Kit (Takara, RR047A) with the manufacturer’s instruction. qRT-PCR was carried out by the Bio-Rad ScripTM cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences were shown as follows: TESC (Forward primer: 5′-GTCGGGAAACCCTCACATCG-3′, Reverse primer: 5′-CGAAGGTGATCCCCTCGTAC-3′), c-FOS (Forward primer: 5′-CCATTACAGCTGTGGCTACGAGT-3′, Reverse primer: 5′-GGTCTGTTCGATGTTCCTCAAG-3′), and GAPDH (Forward primer: 5′-TATCGGACGCCTGGTTAC-3′, Reverse primer: 5′-CGTTCAAGTTGCCGTGTC-3′). The qRT-PCR amplification conditions were as follows: 95 °C for 5 min, 95 °C for 15 s and 60 °C for 30 s of 40 cycles. GAPDH was the internal control. The level of genes was analyzed by the comparative threshold cycle method (2-△△CT method), where ΔΔCT = ΔCT treatment- ΔCT control and ΔCT = Ct target - Ct reference.
Three target RNAi sequences of TESC (TESC-RNAi 1: GGCCTGGCTGATGAGATCAAT; TESC-RNAi 2: CCGGAAGGAGAAGCTGAGATT; TESC-RNAi 3: CCGCATCACTCTGGAAGAATA) and the negative control sequence (NC: TTCTCCGAACGTGTCACGT) were cloned into the GV493 vector (Genechem, China). Subsequently, the constructed vector was transfected into TPC-1 and BHT101 cells using Polybrene (Sigma Aldrich, America), respectively. The relative expression level of TESC was determined using qRT-PCR after 16 h. The most significant reduction in TESC expression was selected from the three TESC-RNAi for subsequent experiments.
The sequences of TESC were synthesized and inserted into vector plasmids pcDNA obtained from Vigene Biosciences (Rockville, America), and then was transfected into TPC-1 and BHT101 cells using Lipofectamine 3000 (Invitrogen, America) for following assays. The RNAi sequences of c-Fos (sense sequence: 5′-CACCGCTTCATTCCCACGGTCACTTTCAAGAGAAGTGACCGTGGGAATGAAGTTTTTTG-3′ and the antisense sequence: 5′-GATCCAAAAAACTTCATTCCCACGGTCACTTCTCTTGAAAGTGACCGTGGGAATGAAGC-3′) were cloned into GV493 vector (Genechem, China), and then transfected into TPC-1 and BHT101 cells using Polybrene (Sigma Aldrich, America), respectively.
Cell counting Kit-8 assay
TPC-1 and BHT101 cells were inoculated in 96-well plates with a density of 1 × 105/well and then maintained for 24 h at 37 °C in 5% CO2. Subsequently, the proliferation of cells was detected using the Cell Count Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. The absorbance was recorded at 450 nm by a microplate reader (ThermoFisher, America).
Colony formation assay
TPC-1 and BHT101 cells were inoculated in 6-well plates with a density of 1 × 104/well at 37 °C for 14 days. Then, the clone numbers were counted manually after colonies were immobilized and stained with 4% paraformaldehyde and 0.1% crystal violet (Sigma Aldrich, America) for 30 min, respectively.
Cell transwell invasion and migration assay
Cells were digested and resuspended with 1 × 106/ml after 24 h transfection. For the cell invasion, the transwell chamber was spread with the Matrigel matrix with serum-free medium dilution, and then appended with the cell suspension. Cells were hatched at 37 °C and 5% CO2. Cells were immobilized with 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma Aldrich, America) for 15 min after 24 h culture. The images were photographed and counted using microscopy (Olympus, Japan). The scratch assay was used to detect cell migration. Cell suspension was seeded onto the 6-well plate after drawing horizontal lines on the back of the plate. And the sterile spearhead was used to scratch the cells vertically relative to the lines on the back of the plate. Cells were washed with PBS, and then cultured in fresh FBS-free medium for 24 h and cell migration was observed under a microscope (Olympus, Japan).
Flow cytometry assay
TPC-1 and BHT101 cells after transfection were collected and stained with Annexin V-APC and PI (Sigma Aldrich, America) at room temperature for 20 min in the dark. The fluorescence of the cells was measured by flow cytometry (BD FACSVerse, America).
RNA-seq library preparation and Illumina sequencing
The total RNA from TPC-1 and BHT101 cells samples treated with TESC-RNAi or NC-RNAi was extracted using TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s specifications. The integrity and purity of the acquired RNA were analyzed by an Agilent BioAnalyzer 2100. The mRNA-Seq libraries were established using an Illumina TruSeq RNA Sample Prep Kit (Beijing Novogene Zhiyuan Technology Co., Ltd., China). Briefly, the mRNA was purified from total RNA by poly-T oligo-attached magnetic beads, and then the purified mRNA was reversely transcribed into cDNA. The Illumina HiSeq 2500 sequencing platform was employed to perform the sequencing in paired-end reads after cDNA was linked to the adaptor and amplified using PCR. The reads were mapped to the Homo sapiens genome using TopHat v1.4.1 with default parameters (−r 400 -p 8) after mRNA-Seq data were preprocessed. The quality of data from mRNA sequencing was evaluated using the FastQC method (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Bioinformatics analysis of mRNA-seq data
Trimmomatic (v0.30) was utilized to remove the adaptor sequences and the low-quality (< 20) bases at the 5′ and 3′ ends. Subsequently in order to obtain the clean reads ≥70 bp for subsequent analysis. The RSEM (https://www.biostat.wisc.edu/~cdewey/) was employed to analyze the gene abundances. The differentially expressed genes (DEGs) between two groups were obtained after each gene expression was quantified according to FPKM values (expected number of fragments per kilobase of transcript sequence per million base pairs sequenced) mapped to the clean reads. The DGEs from TPC-1 and BHT101 cells were screened, and then intersections were taken (control group and gene interference group|log2 fold change (FC)| ≥ 1.5). The screened differential expression bases are verified in TGCA. The DEGs in GO functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway  were analyzed using Ami GO with the default parameters and Cytoscape with the ClueGO plugin. The obtained DEGs were performed the gene enrichment analysis using DAVID or metascape software.
Western blot analysis
Cells were collected and lysed using RIPA lysis buffer (Boster, Wuhan, China) containing 1% protease inhibitors, and the concentrations of proteins were calculated using the BCA protein quantification kit (Abcam, UK) in accordance with the manufacturer’s specifications. Then, protein samples were separated with 12% SDS-PAGE and electrically transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in TBST (Sigma Aldrich, America) (containing 5% skimmed milk) at room temperature for 1 h, and then incubated with primary antibodies at 4 °C overnight. After rinsing with TBST for 3 × 5 min, the membrane was incubated with goat-anti-rabbit IgG (H + L)-HRP (1: 5000, 31,460, Thermo Fisher, diluted in TBST containing 5% skimmed milk) for 1 h at room temperature. The reaction was visualized using an enhanced chemiluminescence detection kit DAB (Bio-Rad Laboratories, Inc.). The gray value was obtained using Image-ProPlus software (Media Cybernetics, Inc., Rockville, MD, USA). The primary antibodies used were as follows: c-Fos (cat. no. 4384), ERK1/2 (cat. no. 9102), p-ERK1/2 (cat. no. 9101), E-cadherin (cat. no. 3195), N-cadherin (cat. no. 13116), Vimentin (cat. no. 5741) and Snail (cat. no. 3879), and β-actin (cat. no. 4970; Cell Signaling Technology, Inc., Danvers, MA, USA) at 1: 1000 dilutions.
Data were shown as the means ± SD. The student’s t-test was used to analyze the data with only two groups, while the one-way analysis of variance was used to analyze the differences among multiple groups by the SPSS 22.0 statistical software (IBM, Armonk, New York, USA) followed by Post-hoc Bonferroni test. The differences were considered statistically significant when p < 0.05.