Public database and bioinformatics
The Cancer Genome Atlas (TCGA) database (https://tcga-data.nci.nih.gov/tcga/) and Gene Expression Omnibus (GEO) datasets (GSE 33630) were used to obtain NGEF expression data in tumor tissues, and the TCGA Data Portal was used for typing and clinical correlation analyses. Disease-free survival curves were generated in GEPIA (http://gepia.cancer-pku.cn/). RNA-sequencing expression (level 3) for NGEF was downloaded from TCGA dataset (https://portal.gdc.com). R software GSVA package was used for further analysis, following parameter selection as method= ‘ssgsea’. The correlation between genes and pathway scores was analyzed using Spearman’s Rank correlation coefficient. Based on the JASPAR (http://jaspar.binf.ku.dk/) and human TFDB databases (http://bioinfo.life.hust.edu.cn/HumanTFDB), NGEF candidate transcription factors were determined, including AP-1. TCGA was also used to identify AP-1 and co-expressed genes. All analysis methods and R packages were implemented by R version 4.0.3. P < 0.05 was considered to indicate a statistically significant difference.
Thyroid tumor samples
Fresh tissue samples were obtained from patients with thyroid cancer admitted to The First Affiliated Hospital of Guangxi Medical University, and were collected between April 2020 and November 2020. Written informed consent was obtained from all patients, and ethics approval was obtained from the Human Ethics Committee of The First Affiliated Hospital of Guangxi Medical University. The Tissue Stabilizer RNA Keeper (R501-01; Vazyme Biotech Co., Ltd.) was used to preserve thyroid samples. Moreover, samples were preserved in liquid nitrogen for long-term storage, and stored at 80°C for short-term storage. Following fixation in formalin, specimens were embedded in paraffin wax. Total RNA was extracted using Trizol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and total protein was extracted using RIPA lysis buffer.
Immunohistochemical (IHC) analysis
For IHC analysis, 1X Antigen Retrieval Buffer (pH 9.0; Abcam) was added to samples at 95°C for 15 min for heat-induced antigen retrieval. Following blocking of endogenous peroxidase using 3% H2O2 solution at 4°C overnight, the primary antibody, anti-NGEF (1:100; cat. no. PA5-52521, ProteinTech Group, Inc.), was added to tissues. Following mixing, samples were incubated with an HRP-conjugated secondary antibody (ProteinTech Group, Inc.) for 1 h at room temperature. Samples were subsequently incubated in DAB substrate for 2 min. Protein expression was quantified using Image-Pro Plus 6.0 (Media Cybernetics, Inc.). Staining sections were evaluated using consistent brightness, magnification and exposure intensity conditions.
Cell culture, drug and genetic treatments
Cell lines, such as Nthy-ori 3 − 1, KTC-1, TPC-1, 8305C, K1, NIH3T3 and 293T were purchased by Procell Life Science & Technology Co., Ltd. All cell lines were cultured in RPMI 1640 or DMEM containing 10% fetal bovine serum at 37°C. Control small interfering (si)RNA and siRNA targeting human NGEF were obtained from Guangzhou RiboBio Co., Ltd. When 60–70% confluence was reached, 50 nM siRNA was subsequently transfected into cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Recombinant NGEF and negative control lentiviruses were obtained from Jikai. When 30% confluence was reached, cells were infected with the aforementioned lentivirus at a multiplicity of 20–60, and screened with 2 g/ml puromycin. Moreover, cells were treated with 500 nM MEK1/2 inhibitor (GSK120212; MedChemExpress), and 30 uM AP-1 binding site inhibitor (T-5224; MedChemExpress) for 24 hours or 48 hours. Dimethylsulfoxide (DMSO) was used to dissolve all inhibitors, which were aliquoted and stored at 80°C until further use. In addition, an equal volume of DMSO was used as the control. Plasmids expressing wild-type BRAF and a BRAFV600E mutation, and an empty vector (pEFm6) were purchased from Jikai. Plasmids were transfected into cells using Lipofectamine 3000 and P3000 (Invitrogen; Thermo Fisher Scientific, Inc.).
Reverse transcription-quantitative (RT-q)PCR
Total RNA was extracted using a NucleoZOL kit (Macherey-Nagel GmbH), following the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix (R323-01; Vazyme Biotech Co., Ltd.), following the manufacturer’s instructions. qPCR was subsequently performed using SYBR green (Q711-02; Vazyme Biotech Co., Ltd.) on an Applied ThermoFisher 7500 Real-time PCR machine. mRNA levels were quantified using the 2−ΔΔCq method and normalized to the internal reference genes GAPDH or 18S RNA. Primer sequences are displayed in Table I. All experiments were performed in triplicate.
Western blot analysis
Total proteins were extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with phosphatase inhibitor (cat. no. P0100; Beijing Solarbio Science & Technology Co., Ltd.) and PMSF. Total protein was quantified using a BCA assay (cat. no. P0012S; Beyotime Institute of Biotechnology). Lysates were denaturized using SDS-lysis buffer [4% SDS, 20% glycerol, 100 mM Tris-HCl (pH 6.8), 100 mM DTT and 100 mM Tris-HCl (pH 6.8)]. Proteins were separated via electrophoresis and subsequently transferred to PVDF membranes (MilliporeSigma). Membranes were blocked with 5% skimmed milk/TBS-Tween-20 (TBST) for 1 h at room temperature, and subsequently incubated with the primary antibodies against NGEF (cat. no. 13271-1-AP; ProteinTech Group, Inc.), BRAF (cat. no. 20899-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-ERK (cat. no. 28733-1-AP; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-c-fos (cat. no. 44-280G; Thermo Fisher Scientific, Inc,), c-fos (cat. no. 66590-1-Ig; ProteinTech Group, Inc.), p-c-jun (cat. no. 28891-1-AP; ProteinTech Group, Inc.), c-jun (cat. no. T55290; Abmart Pharmaceutical Technology Co., Ltd.), MMP2 (cat. no. 10373-2-AP), MMP9 (cat. no. 10375-2-AP), E-cadherin (cat. no. 20874-1-AP; ProteinTech Group, Inc.), N-cadherin (cat. no. 22018-1-AP; ProteinTech Group, Inc.), Vimentin (cat. no. 10366-1-AP; ProteinTech Group, Inc.), Tubulin (cat. no. 11224-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight. Following washing with TBST, membranes were incubated with secondary antibodies (ProteinTech Group, Inc.) for 1 h at room temperature. Following washing three times, protein bands were visualized using an ECL kit (Thermo Fisher Scientific, Inc.). Protein expression was quantified using ImageJ (version no. 1.8.0; National Institutes of Health).
Wound healing assay
Cells were used for wound healing assays when 70–80% confluence was reached. Briefly, K1 and 8305C cells were seeded in 12-well tissue culture plates following treatment. In a growth medium containing 0.2% serum, cells were starved for 24 h. Using a sterile 10-µl pipette tip, a scratch was created on the confluent monolayer and medium was replenished. Images were obtained at 0 and 24 h, and ImageJ software was used to determine the mean and standard deviation of the wounds. Each group was compared with the control group. Cell migration was expressed using the following equation: (Original scratch width − 24-hr scratch width) /original scratch width x 100. Each experiment was repeated three times.
Transwell assay
Cells were placed in serum quiescence for the duration of the invasion experiment, and 3x104 cells were seeded in the upper chambers of Transwell plates with 8.0-mm wells. Transwell membranes were precoated in Matrigel (M8370; Beijing Solarbio Science & Technology Co., Ltd.). Briefly, K1 cells were incubated for 12 h and 8305C cells were incubated for 14 h. For migration, 3x104 cells were seeded in the upper chambers of uncoated membranes with 8.0-mm wells. K1 cells were incubated for 12 h and 8305C cells were incubated for 14 h. Cells were plated in the upper chamber in medium without serum, and subsequently transferred to complete growth medium. Following fixing with 4% paraformaldehyde, cells were mounted on glass slides, and stained with hematoxylin. Stained cells were counted in five randomly selected fields using an optical microscope. All experiments were performed in triplicate.
Immunofluorescence
Following incubation in a medium containing siRNA-NGEF, cells were fixed in 4% paraformaldehyde (Leagene) for 30 min. Subsequently, cells were washed in phosphate-buffered saline incubated with 0.1% Triton-X 100 (Jiayan Biotech) for 10 min, and blocked in 3% bovine serum albumin for 30 min. Cells were incubated with a Vimentin mouse polyclonal antibody (1:200) and an E-cadherin rabbit polyclonal antibody (1:100) overnight. Following primary incubation, cells were incubated with the corresponding fluorescent secondary antibody for 1 h at room temperature in the dark. Goat anti-mouse IgG (H + L; cat. no. A0423; 1:50; Beyotime Institute of Biotechnology), was used to detect green fluorescence, indicative of E-cadherin. Goat anti-rabbit IgG (H + L)-labeled Alexa Fluor 555 was used to detect red fluorescence, indicative of Vimentin (cat. no. A0460; 1:50; Beyotime Institute of Biotechnology). Cell nuclei were counterstained with DAPI (Beijing Solarbio Science & Technology Co., Ltd.) for 5 min and visualized using confocal microscopy (Leica Microsystems GmbH).
Dual-luciferase reporter assay
To determine whether the promoter regions of NGEF genes are directly associated with c-fos/c-jun, promoter regions were cloned into pGL3-Basic luciferase vectors (Promega Corporation). Sanger sequencing was used to create and verify the constructs. To determine the regulatory role of AP-1 in the NGEF promoter, pGL3-Basic or pGL3-CYP2S1-Luc reporter plasmids (Promega Corporation) were co-transfected with either 293T cells expressing c-fos or c-jun, or control cells. A dual-luciferase reporter assay (Promega Corporation) was used to detect luciferase activity 48 h following transfection under hypoxic or normoxic settings.
Statistical analysis
GraphPad Prism 8 (GraphPad Software, Inc.) and SPSS 22.0 (IBM Corp) were used for statistical analysis. The R ‘ggpubr’ package (https://cran.r-project.org/web/packages/ggpubr/index.html) was used to create boxplots. Data are presented as the mean ± standard deviation. Biological processes associated with NGEF expression in thyroid cancer were determined using GSEA (https://www.broadinstitute.org/gsea/). Differences between two groups were analyzed using Student’s paired t-tests, and differences between multiple groups were analyzed using one-way or two-way ANOVA. All of the aforementioned experiments were repeated in triplicate. P < 0.05 was considered to indicate a statistically significant difference.