The 6 pairs of human ESCC tissues and their matched adjacent tissues were collected for high-throughput sequencing from Nanchong Central Hospital (Nanchong, China), and the data was also available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE205121. This project was supported by the Ethics Committee of Nanchong Central Hospital . All patients did not receive treatment and signed an informed consent form.
ESCC cell lines and cell culture
The human ESCC cell lines KYSE30, KYSE410 were purchased from Procell Life Science &Technology (Wuhan, China), KYSE150 and TE-1 were purchased from GENECHEM (Shanghai, China), TE-11 and KYSE510 were purchased from Shanghai XuanYi Biotechnology Service Center (Shanghai, China), and the human normal esophageal epithelial cell line HEEC was purchased from Keygen Biotech (Jiangsu, China). All of them were incubated in RPMI-1640 Medium (Gibco, USA) with 10% fetal bovine serum (Biological Industries, USA) and 1% penicillin/streptomycin (Gibco, USA), and the human normal esophageal epithelial cell line HET-1A was cultured in DMEM Medium (Gibco, USA) with 10% fetal bovine serum and 1% penicillin/streptomycin and incubation with 5% CO2 at 37℃, which were purchased from Keygen Biotech (Jiangsu, China).
Construction of plasmids, lentivirus and stable transfection into cell lines
The SCARA5 coding sequence was inserted into the pcDNA3.1 vector (pcDNA3.1-SCARA5, SCARA5-OE vector). The stable transfection ESCC cell lines were constructed from GENECHEM CO., Ltd (Shanghai, China). pcDNA3.1-SCARA5 and SCARA5-OE vector were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time PCR analysis
Total RNA of these different cells was extracted by using Trizol Reagent (Vazyme, China). Then, total RNA was reverse transcribed into cDNA using the HiScript® III RT SuperMix (Vazyme, China) with the following temperature protocol: 37℃ for 15 min, 42℃ for 5 s. Quantitative real-time PCR (qPCR) was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The primer sequences of key genes shown in the Supplementary Table 1.
The stable cell lines TE-1 and KYSE150 with overexpression SCARA5 in the logarithmic growth phase were seeded in a 96-well plate with the density of 3 × 103 cells per well and incubated. Then, the CCK8 assay (KeyGEN BioTECH, Jiangsu, China) was used to measure the proliferation abilities of stable cell lines at 0 h, 24 h, 48 h, 72 h and 96 h, each well added 10% CCK8, incubated for 1 h at 37℃, and the absorbance at 450 nm were detected in microplate reader (BioTek Instrument Inc., Winooski, VT, USA). Cell viability was measured after cells were treated with Ferrostain-1 (1 μM), Erastin (10 μM), Necrostain-1 (10 μM), Z-VAD-FMK (10 μM) and 3-MA (60 μM). All experiments were performed in quadruplicate.
EdU incorporation assay
According to the manufacturers’ instruction, the 2 × 104 cells TE-1 and KYSE150 cells were seeded in 24-well plates to determine the number of proliferation cells with an Edu kit (BeyoClick™ EdU-488, Beyotime).
Flow cytometric analysis of percentage of cell death
The TE-1 and KYSE150 cells were seeded in 24-well plates for 24 h, and the Annexin V-FITC/PI Apoptosis Detection Kit (#A211‑02, Vazyme) was utilized to detect the percentage of cell death according to the manufacturers’ instructions.
Cell cycle analysis
The stable cell lines TE-1 and KYSE150 cells were seeded in a 6-well plate with the density of 5 × 105. After incubation 24 h, these cells were harvested and washed twice with cold PBS and then fixed with 75% cold ethanol overnight at -20℃. Subsequently, the fixed cells were centrifuged and washed twice with cold PBS and resuspended in binding buffer (Cell Cycle Analysis Kit, 4A, Biotech) containing 100 μg/mL propidium iodide and 200 μg/mL Rnase A for 30 min at 37℃ in the dark. Stained cells were collected by a FACSCalibur flow cytometer (BD Biosciences), and the data was analyzed by Flow Jo software (BD Biosciences, USA).
Wound healing assay
The stable cell lines TE-1 and KYSE150 were plated into six-well plates at 4 × 105cells/well and grown to confluency. And two separate parallel wounds were generated by scratching the cell layer with a 10 μL plastic pipette tip. The numbers of migrated cells were observed and imaged under microscope (ECLIPSE TS100, Nikon) at the time points 0 h and 24 h. These experiments were performed in triplicate.
The migration or invasion assays were performed using polycarbonate Transwell filter chambers (8 μm pore size; Corning Inc.) and the inserts were coated with or without Matrigel® (BD Biosciences). A total of 2 × 104 cells were seeded in the upper transwell chamber and 1 × 105 cells were added in the top chamber containing Matrigel with 100μL serum-free medium, whereas the bottom chamber was added with 500μL medium containing 10% serum. After being cultured for 48 h, the migrated cells on the lower membrane were stained with 0.1% crystal violet (KGA229, Keygen Biotech) and counted.
Intracellular chelable iron was determined using the fluorescent indicator Phen green SK (#P14312), the fluorescence of which is quenched by iron. 5 × 104 cells were inoculated into a 24-well plate respectively, and climbing slides were added, and then transfection reagent was added after incubating at 37 °C for 24 h. After 48 h of transfection, the culture medium was discarded, the slides were washed with PBS three times, and the 20 μM Phen green SK probe was added with incubated at 37 °C for 20 min, and the slides were washed with PBS three times, and then photographed by ordinary fluorescence microscope. Meanwhile, 3 × 105 cells were inoculated into a 6-well plate, incubated at 37 °C for 24 h, and then added transfection reagent, respectively. After 48 h of transfection, the cells were collected and added 20 μM phen green SK probe, incubated at 37 °C for 20 min, washed with PBS three times. The intracellular chelable iron was measured by a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).
Measurement of ROS
The peroxide-sensitive fluorescent probe DCFH-DA (#CA1410) was used to detect intracellular total ROS and the peroxide-sensitive fluorescent probe C11-BODIPY (#D3861) was used to detect the lipid ROS according to the manufacturer’s instruction. 3 × 105 cells were inoculated into a 6-well plate respectively, incubated at 37 °C for 24 h, and then added transfection reagent. After 48 h of transfection, the cells were collected and incubated at 37℃ for 30 min with 10 μM DCFH-DA probe and incubated at 37℃ for 20 min with 5uM C11-BODIPY probe. Cells were then washed with PBS three times and were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ).
Malondialdehyde (MDA) assay
MDA, as a major indicator of lipid peroxidation, was detected using MDA Assay Kit (#A003-4–1) according to the manufacturer’s instructions, which was purchased from Nanjing Jiancheng. Protein concentration was assayed using a Beyotime BCA Protein Assay Kit according to the manufacturer’s instructions.
The total quantities of glutathione were measured using a GSH Assay Kit (#KTB1600), which was purchased from Abbkine. Then, the GSH of each of group was detected by referring to the manufacturer’s instructions.
The cell pellets of ESCC cells TE-1 and KYSE150 were lysed, the protein concentration was determined by the BCA assay, and the expression levels of SCARA5 and FTL were detected. Then, each group was added 200ul of Protein A + G agarose beads, and added FTL antibody to the IP group, and rabbit IgG to the IgG group, and bonded for 2 h at 4 °C. Add cell lysate to each group and rotate overnight at 4 °C. Wash with PBS 3 times, add SDS loading buffer for elution. The elution proceeded with standard western blotting.
Western blot analysis
Cells were harvested and then lysed with RIPA lysis buffer on ice. The BCA Protein Assay Kit was utilized to determine the protein concentration was determined using. Protein samples were separated on 10% or 12% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were cut prior to hybridization with antibodies during blotting. The membranes were blocked with 5% non-fat milk, and incubated overnight with primary antibody at 4℃. After washing with PBS, the membrane was incubated with secondary antibody at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence reagents (Thermo Scientific). GAPDH and β-tubulin were used as a housekeeping control protein. Primary antibodies against SCARA5 (#ab118894), FTL (#ab69090), FTH1 (#ab75973, #ET1705-55), TFR1 (#136,800, Invitrogen).
Expression and distribution of SCARA5 in 5 μm thick paraffin section of ESCC patients and adjacent precancerous tissues was detected by immunohistochemistry staining. Immunohistochemical staining was estimated on the basis of previous studies in our laboratory . Firstly, sections were dewaxed, rehydrated and then antigen retrieval with 0.01 M sodium citrate buffer (pH 6.0). After sections were blocked with 3% hydrogen peroxide for 10 min, and incubated with 0.1% Triton X- 100 for 10 min, and then blocked with 3% BSA, followed by incubation with primary antibodies (37 °C, 2 h). Subsequently, the slides were detected with antirabbit HRP secondary antibody for 1 h at room temperature. Finally, the sections were developed with 3,3ʹ-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. Meanwhile, detecting the expression of SCARA5/FTL/FTH1 in the subcutaneous tumor nude mice in the control and overexpression SCARA5 group by immunohistochemistry.
In vivo tumor model
Male BALB/C mice (5-6 weeks old) were purchased from the Beijing Huafukang Biotechnology Co., Ltd. The stable overexpression SCARA5 or vector KYSE150 cells (1 × 106 cells in 200μL PBS) were injected subcutaneously into the dorsal region of each mouse. The mice weight and tumor size were measured every three days after 4 days of injection. All the mice were sacrificed after 24 days. Tumor volume (mm3) was calculated as follows: volume = length × width2 × 0.5. All the tumor was placed in 4% paraformaldehyde and then embedded with paraffin. H&E staining was performed to evaluate the tissue morphology.
GraphPad prism 8.0 was used to performed Statistical analysis. All data were presented as mean ± standard deviation (SD) of at least three independent experiments. One way analysis of variance (ANOVA) was used for comparison differences between groups, and the statistical significance of the difference of mean values between two group was determined using Student’s t test. Data were considered statistically significantly when P < 0.05.