Esophageal tissue samples from 25 patients with ESCC (age, 64.1±7.49) and 10 esophageal tissues from healthy controls (age, 63.58±8.76) were acquired from Tianjin Medical University General Hospital (Tianjin, China) between January 2021 and December 2022. Prior to the surgery, none of the patients had received any cancer treatment therapies. The last follow-up date was 31 October 2021. The 8th American Joint Committee on Cancer staging method was used to categorize the tumor stage and grade of the tissue samples (36). The present study was conducted according to the principles set out in The Declaration of Helsinki of the World Medical Association. Written informed consent was provided by each patient, and the Ethics Committee of Tianjin Medical University General Hospital approved the study (ethics no. IRB2021-WZ-134). The clinicopathological characteristics of the patients are presented in Table I.

Paraformaldehyde (4%) was used to fix the tissue specimens at room temperature for 24 h. The fixed paraffin-embedded tissue sections (4-µm thick) were subsequently dewaxed using xylene and hydrated through graded concentrations of ethanol in water. Sodium citrate buffer (0.01 mol/l) was used for antigen retrieval and the endogenous peroxidase activity was blocked by Enhanced Endogenous Peroxidase Blocking Buffer (cat. no. P0100A; Beyotime Institute of Biotechnology) at 37°C for 20 min. The primary antibodies used for the immunohistochemical analysis were anti-TAGLN (1:100; cat. no. ab233971; Abcam), anti-GPX4 (1:200; cat. no. DF6701; Affinity Biosciences, Ltd.), anti-ACSL4 (1:400; cat. no. ab155282; Abcam), anti-p53 (1:800; cat. no. 60283-2-Ig; ProteinTech Group, Inc.), anti-cyclooxygenase-2 (COX2; 1:200; cat. no. abp51035; Abbkine Scientific Co., Ltd.), anti-transferrin (TF; 1:400; cat. no. 17435-1-AP; ProteinTech Group, Inc.) and anti-ferritin heavy chain 1 (FTH1; 1:800; cat. no. abp54401; Abbkine Scientific Co., Ltd.). These primary antibodies were added to the tissue sections and incubated at 4°C overnight. Following a washing step using Immunol Staining Wash Buffer, Beijing Beyotime company, the tissue sections were incubated for 30 min at 37°C with the following HRP-conjugated secondary antibodies: Goat anti-mouse (1:500; cat. no. ab6789; Abcam) and goat anti-rabbit (1:1,000; cat. no. ab6721; Abcam). Horseradish peroxidase was then added, and the samples were further incubated at 37°C for 20 min. The sections were scanned using a PRECICE 510B digital slice scanning system (Unic Technologies, Inc.), and observed by iViewer software (version 7.2.7.2; Unic Technologies, Inc.). The staining results were graded according to the staining intensity and percentage of positive cells. The cell staining intensity was scored as follows: 0, non-staining; 1, light yellow; 2, brownish yellow; and 3, yellowish brown. The percentages of positive cells were scored as follows: 0, <5%, 1, 5-25%; 2, 26-50%; 3, 51-75%; and 4, >75%. The positive intensity is the product of two scores: 0-2, negative; 3-5, weakly positive; 6-8, positive; and 9-12, strongly positive. Two pathologists assessed the findings independently, and the average values of five fields of view were used.

The mRNA expression levels of TAGLN between the ESCC samples and normal tissues were compared using data acquired from the Gene Expression Omnibus (GEO) records (GSE161533, GSE45670 and GSE100942; http://www.ncbi.nih.gov/geo).

GSEA was completed using ESCC data obtained from The Cancer Genome Atlas (TCGA) project to identify the signaling pathways and biological phenomena that exert a role in the advancement of ESCC. The RNA-seq transcriptome data of ESCC patients in fragment per kilobase of transcript per million mapped reads format were extracted from TCGA. The average TAGLN transcript level was used to divide the patients with ESCC into high- and low-expression groups. For each assessment, gene set permutations were set to 1,000. The pathways enriched for each phenotype were classified according to the false discovery rate, nominal P-value and normalized enrichment score (NES). The DESeq algorithm for differential gene expression analysis was used, and the significance of DEGs was evaluated by P<0.05 and |log FC|>1 (where FC is fold change) and were used as screening criteria. The R programming language was then used to generate volcano plots of the DEGs and to conduct clustering analysis. Volcano plots were generated using ggplot2, a data visualization package in the R programming language. Specifically, the geom_point function was used to plot the log FC values against the negative logarithm of the adjusted P-values. Similarly, heatmaps were created using the R package heatmap, using the heatmap.2 function to display the relative expression levels of genes across different conditions. To investigate the biological roles of DEGs, biological process (BP) gene ontology (GO) enrichment analysis and molecular function (MF) GO term enrichment analysis based on the GO database (http://geneontology.org; accessed on April 5, 2022) was performed. Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp/kegg; accessed on April 5, 2022) analysis was performed for further assessment of the signaling pathways.

The human ESCC cell lines (KYSE-150 and Eca-109) were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium containing 10% FBS (Gibco^®; Thermo Fisher Scientific, Inc.) and maintained at 37°C in an atmosphere of 5% CO₂.

For cell transfection experiments, the Eca-109 and KYSE-150 cells were added at a density of 5×10⁵ cells/well into a 6-well plate. When confluency was 70-90%, cells were transfected using Lipofectamine 3000™ Transfection Reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's guidelines. The TAGLN-overexpression plasmid (cat. no. HG14991-NY; TAGLN cDNA ORF Clone; human, pCMV3-N-HA as vector; Sino Biological) and an empty control plasmid (vector-NC; cat. no. CV017; pCMV3-N-HA as negative control vector; Sino Biological) were transfected into the KYSE-150 and Eca-109 cells. The final transfection concentration of plasmid was 100 nmol/l. After 6-8 h of culture in 10% serum medium without penicillin-streptomycin at 37°C, the culture was replaced to 10% serum complete medium for 24-48 h.

To avoid off-target results, two different gene-specific siRNAs against TAGLN (si-TAGLN; one from Santa Cruz Biotechnology, Inc. (cat. no. sc-44163) and the other from Shanghai GenePharma Co., Ltd. (sense, 5′-GCU GAA GAA UGG CGU GAU UTT-3′; antisense, 5′-AAU CAC GCC AUU CUU CSG CTT-3′), and the corresponding siRNA negative control (si-NC; cat no. sc-37007; Santa Cruz Biotechnology, Inc.) were transfected into KYSE-150 and Eca-109 cells, as aforementioned (using Lipofectamine 3000™; the final transfection concentration of siRNAs was 10 nmol/l). After culturing the cells at 37°C in 10% serum medium without penicillin-streptomycin for 6-8 h, the medium was replaced with complete medium containing 10% FBS (cat. no. 10099141C; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.), and the cells were incubated for an additional 24-48 h prior to the follow-up experiments.

Experimental cells were separated into the following groups according to the nature of the transfections: si-NC, si-TAGLN-1, si-TAGLN-2, vector-NC (empty plasmid) and TAGLN overexpression plasmid. Subsequently, the cells were examined by Cell Counting Kit 8 (CCK-8) viability assay, EdU-labeling proliferation assay, colony formation assay, wound healing assay, and Transwell invasion and migration assays within 72 h after the completion of cell transfection.

KYSE-150 and Eca-109 cells were seeded into a 96-well plate at a density of ~5,000 cells/well and cultured at 37°C. After 24, 48 and 72 h, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well, and the cells were incubated at 37°C for 1 h. A Tecan Infinite M200 Pro microplate analyzer (Tecan Group, Ltd.) was used to measure the absorbance of each well at 450 nm to calculate cell viability. Each experiment was repeated three times.

KYSE-150 and Eca-109 cells were seeded into 12-well plates at a density of 5×10⁴ cells/well and incubated in RPMI-1640 medium containing 10% FBS at 37°C. The BeyoClick™ EdU-488 kit (Beyotime Institute of Biotechnology) was used to determine cell proliferation by examining DNA synthesis in the cells. EdU reagent (1 ml at 10 µM) was added to the cells and incubated at 37°C for 60 min. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and then treated with 1% Triton X-100 at room temperature for 15 min. Subsequently, 200 µl Click reaction solution was added to each well, and the cells were incubated in dark at room temperature for 30 min. Nuclei were stained with Hoechst (Beyotime Institute of Biotechnology). A Leica DM5000B fluorescence micro-scope (Leica Microsystems GmbH) was used to examine and capture images of the cells. Images were analyzed using LAS AF software v2.6.0.7266 (Leica Microsystems GmbH). The percentages of EdU-positive (DNA-replicating) and Hoechst-positive cells (total cells) were determined.

KYSE-150 and Eca-109 cells were seeded into a 6-well plate, at a range of densities between 2×10² and 10×10² cells/well, and cultivated in RPMI-1640 medium containing 10% FBS at 37°C for 24 h. The cells were treated with various concentrations of Tanshinone I (Tan-I; Selleck Chemicals), 0, 1.2, 2.4, 4.8 and 9.6 µg/ml, prior to being resuspended in RPMI-1640 medium containing 10% FBS and cultured at 37°C for 15 days in an atmosphere containing 5% CO₂ to stimulate colony formation; the medium containing 10% FBS was replaced every three days. The plate was washed using cold PBS and fixed with 4% paraformaldehyde solution. The fixed colonies were subsequently stained with 0.1% crystal violet for 30 min at room temperature. The number of visible colonies, those with a diameter >0.05 mm, was calculated using FIJI/ImageJ software (v2.1.0/1.53c; National Institutes of Health).

To prevent cell proliferation upon migration, the cells were allowed to rest overnight in serum-free medium prior to wound scratching. Digested cells were harvested and cultured (at a density of 2×10⁵ cells/well). Once the confluency of the cells had reached to 100%, wounds were made using a pipette tip. The wound closures were subsequently measured at 0, 12, 24 and 36 h to assess the degree of cell migration using a Nikon ECLIPSE Ts2 microscope (Nikon Corporation) and calculated using FIJI/ImageJ software (v2.1.0/1.53c; National Institutes of Health). Experiments were performed three times.

Matrigel™ (BD Biosciences) was diluted in RPMI-1640 medium containing 10% FBS after dissolution overnight at 4°C. The mixture (50 µl) was added to the upper and the lower compartments of Costar^® 24-well plates (cat. no. 3422; Corning, Inc.), which were then incubated at 37°C for 30 min. Eca-109 and KYSE-150 cells in the exponential growth phase were collected; serum-free medium was used to wash and resuspend the cells to 5×10⁴ cells/ml. The bottom part of the Matrigel insert was merged in RPMI-1640 medium with 20% FBS, and cells were gently added to the upper compartment. Culture plates were then incubated at 37°C with 95% relative humidity and in the presence of 5% CO₂ for 24 h, after which, the upper Transwell chambers were removed. The cells on the lower side of the insert were stained with 0.1% crystal violet for 15-30 min at room temperature, and images were captured for cell counting. The number of cells was counted in 10 fields, which were randomly selected using a Nikon ECLIPSE Ts2 microscope (Nikon Corporation), and the average numbers of cells were recorded. The Transwell migration assay was conducted as described above for the Transwell invasion experiment, but without Matrigel. Each experiment was repeated three times.

Four groups of female BALB/c nude mice (aged 4-6 weeks; n=24/group) were acquired from the SPF Beijing Biotechnology Co., Ltd. The mice were kept under pathogen-free conditions and provided sterile food and water ad libitum. The mice were raised in an environment with constant temperature of 20-26°C, moderate humidity of 50-60% and a 12-h light/dark cycle. A total of 5×10⁶ cells in 100 µl PBS containing the TAGLN-overexpression vector, vector-NC, si-TAGLN or si-NC were subcutaneously inoculated into the right flank of the mice. Tumor growth was observed every 4 days. Following the procedure outlined in previous studies (37,38), transfection experiments were performed 10, 13 and 16 days after the injection of the tumor cells (designated as day 0). On days 10 and 13, 14 µg vector-NC or TAGLN-overexpression plasmids were combined with 21.7 µl Lipofectamine 3000™ Transfection Reagent (Thermo Fisher Scientific, Inc.); in addition, 434 pmol si-NC or si-TAGLN was mixed with 50 µl siRNA-Oligofectamine (Thermo Fisher Scientific, Inc.) to form a complex. The solutions are mixed with 5% dextrose water to a total volume of 100 µl. Following a period of incubation (10-15 min), an insulin syringe with a permanently attached needle was used to inject these reagent mixtures above into the tumor. On day 16, double doses of the siRNA and plasmid complexes were injected, and the amount of Oligofectamine was increased accordingly. This procedure was performed in accordance with the guidelines of the Animal Ethics Committee of Tianjin Medical University. The weight of the tumors in mice was not allowed to exceed 10% of their body weight, and the average tumor diameter never exceeded 15 mm. Mice and tumors were examined every 2-3 days; when the tumor exceeded 15 mm in diameter, the mice were euthanized. In case of ulceration, infection or necrosis, the experiment was terminated, and the mice were euthanized at that stage. On the 18th day of the experiment, one of the mice in the TAGLN knockdown group died; H&E staining was performed on their lungs and liver, and it was found that they had systemic metastasis. The remaining mice survived until the end of the experiment. At the end of the experiment, the mice were sacrificed and the tumors were removed. Mice were euthanized using the CO₂ method: Mice were placed into a cage, and 100% CO₂ was introduced at a flow rate of 30% CO₂ volume displacement/min. The CO₂ added to the chamber was allowed to reach equilibrium in the gas mixture to quickly realize the goal of animal unconsciousness and reduce animal pain. Animal death was confirmed by cardiac arrest, respiratory arrest, animal stiffness and dilated pupils. A 4% paraformaldehyde solution was used to fix the tumors at room temperature for 12-24 h, which were then submerged in paraffin and sectioned (4-µm thick) for later observation. The animal study was approved by the Ethics Committee of Tianjin Medical University General Hospital (ethics no. IRB2019-DWFL-414).

TRIzol™ (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from the cells. cDNA was synthesized using a FastKing RT Kit with gDNase (cat. no. KR116; Tiangen Biotech Co., Ltd.) and amplified by qPCR using Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix (cat. no. A25780; Thermo Fisher Scientific, Inc.), precisely following the manufacturer's instructions (Thermo Fisher Scientific, Inc.). The forward and reverse primer sequences were as follows: GAPDH forward, 5′-CCC TTC ATT GAC CTC AAC TAC ATG G-3′, and reverse 5′-CAT GGT GGT GAA GAC GCC AG-3′; TAGLN forward, 5′-GGT GGA GTG GAT CAT AGT GC-3′, and reverse 5′-ATG TCA GTC TTG ATG ACC CCA-3′; GPX4 forward, 5′-ATG AAG ATC CAA CCC AAG GG-3′, and reverse 5′-AGG TCC TTC TCT ATC ACC AGG-3′; ACSL4 forward 5′-CCA AAG AAC ACC ATT GCC ATC-3′ and reverse 5′-AGC CTC AGA TTC ATT TAG CCC-3′; and p53 forward 5′-CCT CAG CAT CTT ATC CGA GTG G-3′, and reverse 5′-TGG ATG GTG GTA CAG TCA GAG C-3′. The following PCR thermocycling conditions were used: 95°C for 30 sec, 95°C for 8 sec and 60°C for 32 sec for 40 cycles; then 95°C for 1 min, 60°C for 30 sec and 95°C for 30 sec. The relative expression of each of gene was calculated using the 2^−ΔΔCq method. Each sample was run in duplicate, and each experiment was repeated three times.

Tissues and cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology) for total protein extraction. The BCA assay was used to quantify the total protein concentration. Proteins (40 µg) were separated using 12.5% SDS-PAGE gel, and the proteins were then transferred to a PVDF membrane using the wet (submersion) method (1 kDa/min). Subsequently, the membrane was blocked using 10% skimmed milk for 1 h at room temperature. After washing with TBS with 0.1% Tween-20, the membrane was incubated with the primary antibodies anti-TAGLN, anti-β-actin (cat. no. TA-09 ZSGB-BIO; OriGene Technologies, Inc.), anti-GAPDH (cat. no TA-08; ZSGB-BIO; OriGene Technologies, Inc.), anti-GPX4 and anti-ACSL4 antibodies (also see the Immunohistochemical analysis subsection above for further details) overnight at 4°C. Note that the dilution ratio of the antibodies for western blot analysis was 1:1,000. The membrane was then washed and incubated with secondary antibodies, namely HRP-conjugated goat anti-rabbit IgG (H+L) (cat. no. ZB-2301; ZSGB-BIO; ZSGB-BIO; OriGene Technologies, Inc.) and goat anti-mouse IgG (H+L) (cat. no. ZB-2305; ZSGB-BIO; ZSGB-BIO; OriGene Technologies, Inc.) for 1 h at room temperature. The proteins were visualized using an Enhanced Chemiluminescence (ECL) Western Blotting Substrate (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.). Densitometric analysis was conducted using Image Lab software version 6.1 (Bio-Rad Laboratories, Inc.); each experiment was repeated three times.

Lipid peroxidation was detected in Eca-109 and KYSE-150 cells using a Lipid Peroxidation MDA Assay Kit (cat. no. BC0025; Beijing Solarbio Science & Technology Co., Ltd.), following the manufacturer's instructions. The cultured and transfected cells were collected and then subjected to ultrasonic fragmentation (power, 200 W; ultrasound 3 sec; interval, 10 sec; repeated 30 times), and the supernatant is centrifuged. MDA detection working solution was added and the mixture was incubated for 60 min in a 100°C water bath; subsequently, then the mixture was centrifuged 8,000 × g at 4°C for 10 min and 200 µl supernatant was added to each well of a 96-well plate for detection using a microplate analyzer to measure the absorbance of both the protein (at wavelength 450 nm) and MDA (at wavelengths of 532 and 600 nm).

GSH was measured in Eca-109 and KYSE-150 cells using a Reduced Glutathione (GSH) Colorimetric Assay kit (cat. no. E-BC-K030-M; Elabscience Biotechnology, Inc.), following the manufacturer's instructions.

The intracellular level of ROS was calculated by means of the fluorescent 2,7-dichlorofluorescein diacetate probe (Nanjing Jiancheng Bioengineering Institute), which was added to serum-free medium at a final concentration of 10 µM. The probe and Eca-109 and KYSE-150 cells (5×10⁶) were then incubated for 30 min in the dark. The cells were washed twice with PBS to remove excess probe, and the fluorescence intensity was measured using a Tecan Infinite M200 Pro microplate analyzer (Tecan Group, Ltd.); the optimal excitation wavelength was 488 nm, and the optimal emission wavelength was 525 nm). Images of the cells were captured under a fluorescence microscope.

The level of Fe²⁺ or total iron in 5×10⁶ transfected Eca-109 and KYSE-150 cells was measured using the of Sigma-Aldrich Iron Assay kit (cat. no. MAK025; Merck KGaA) and a Tecan Infinite M200 Pro microplate analyze, following the manufacturer's instructions.

Transfected Eca-109 and KYSE-150 cells (5×10⁴) were inoculated in black polystyrene 96-well plates (cat. no. CLS3916; Corning, Inc.) and incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was removed and the cells were washed three times with serum-free RPMI-1640 medium. FerroOrange (cat. no. F374; Dojindo Laboratories, Inc.) was added at a 1 µmol/l working solution and the cells were incubated at 37°C in a 5% CO₂ incubator for 30 min. Immediately following incubation, the cells were observed under a fluorescent microscope (Leica Microsystems GmbH).

Co-IP was performed according to the instructions offered for each reagent in the process. Eca-109 cells (1×10⁷) were lysed with Cell Lysis Buffer (cat. no P0013; Beyotime Institute of Biotechnology) and centrifuged at 13,000 × g for 15 min at room temperature. The anti-p53 antibody (1:100; cat. no. 10442-11-AP; Proteintech Group, Inc.), anti-TAGLN antibody (1:100; cat. no. ab233971; Abcam) and anti-rabbit IgG antibody (negative control) were incubated with 40 µg Protein A+G-Sepharose beads (cat. no. P2105; Beyotime Institute of Biotechnology) at 4°C for 1 h. Each 1,000 µl extracted cellular protein sample was mixed with 40 µl of magnetic bead antibody mix and incubated for 2 h at room temperature. At the end of the incubation, the mixtures were placed on a magnetic stand for 10 sec to ensure separation, and the supernatant was removed. The beads were washed three times with a TBS, and 40 µg magnetic beads was added to 100 µl of protein loading buffer in a metal bath heated at 95°C for 5 min, and then placed on the magnetic rack to remove the supernatant, leaving the target protein to be analyzed by western blotting.

TAGLN- overexpressing Eca-109 cells (1×10⁴) were seeded into 24-well plates and fixed for 10 min using 4% paraformaldehyde at room temperature. PBS containing 0.1% Triton X-100 was added for cell membrane permeabilization, and the cells were incubated for 10 min at room temperature. Then PBST containing 1% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) was used for blocking at room temperature for 1 h. The cells were subsequently incubated with the primary antibodies, anti-TAGLN (1:100; cat. no. ab233971; Abcam), anti-p53 (1:100; cat. no. 10442 11 AP; Proteintech Group, Inc) and anti-HSPB1 (1:500; cat. no. ab109376; Abcam) and then carefully washed three times with PBS. Goat anti-,mouse IgG H&L (Alexa Fluor^® 488-conjugated; 1:200; ab150113; Abcam) and donkey anti-rabbit IgG H&L (Alexa Fluor^® 647-conjugated; 1:200; ab150075; Abcam) was used to stain the secondary antibodies. Nuclei were stained with DAPI (cat. no. S2110; Beijing Solarbio Science & Technology Co., Ltd.). Images of the cells were obtained using a laser-scanning confocal microscope (Leica Microsystems GmbH).

The protein-protein interaction (PPI) network information for TAGLN was downloaded from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) website (version 11.0; https://string-db.org). Using the FerrDb dataset (version 1; http://www.zhounan.org/ferrdb), a total of 259 ferroptosis-associated genes were identified. Genes involved in the TAGLN and ferroptosis pathways were illustrated using a Venn diagram.

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software; Dotmatics). All measurement data are shown as the mean ± standard deviation. Comparisons between two groups were analyzed using a two-tailed unpaired t-test, and multiple groups were analyzed by one-way ANOVA followed by Tukey's post hoc test for further pairwise comparisons. Correlation analysis was performed using Pearson's correlation method, and the Kaplan-Meier followed by log-rank method was used to detect relevant associations between the expression levels of TAGLN and the subsistence prognosis of patients with ESCC. P<0.05 was considered to indicate a statistically significant difference.

The respective endoscopic presentations of the normal esophageal tissue and ESCC are shown in Fig. 1A. The changes in TAGLN expression level in normal esophageal tissues and in ESCC tissues were distinguished using immunohistochemical analysis (Fig. 1B). The results showed that relative TAGLN expression was lower in ESCC compared with normal tissues (Fig. 1C). Statistical analysis confirmed that no significant associations were identified between the relative expression level of TAGLN in ESCC and age, sex, lymph node metastasis, tumor grade, tumor size, differentiation or tumor site of the patients (Table I). However, the relative expression of TAGLN was significantly associated with tumor grade, lymphatic invasion and AJCC stage (Table I). Moreover, the expression of ferroptosis-associated proteins, ACSL4 and GPX4, in ESCC and normal tissues was confirmed using immunohistochemistry, and the differences were found to be statistically significant (Fig. 1B and C). The correlation analyses were based on IHC stating scores; the expression of GPX4 was negatively correlated with TAGLN expression (Fig. 1E), whereas that of ACSL4 was positively correlated with TAGLN expression (Fig. 1F). The expression level of TAGLN was statistically higher in ESCC tissues at the early stages of cancer (stage IIa) compared with the advanced stages (stage III) (Fig. 1D); the difference between the expression of TAGLN in stage IIa and in the control groups was not statistically significant, whereas the differences between the expression of TAGLN in stage III groups and in the control group was identified as statistically significant. The differences between the expression of ACSL4 and GPX4 in ESCC stages IIa and stages III were found not to be statistically significant (Fig. 1D); however, the differences between the expression of ACSL4 and GPX4 in these two ESCC groups compared with expression in the control group were statistically significant.

The cut-off value used to separate patients into high and low expression groups was 3.7 (median value) according to IHC staining score. Kaplan-Meier analysis of the data demonstrated a significant association between the survival prognosis of patients with ESCC and the relative expression levels of TAGLN; that is, patients with high expression of TAGLN had a longer overall survival rate compared with patients with a low expression of TAGLN (Fig. 1G).

Subsequently, the expression levels of the ferroptosis-associated proteins p53, COX2, TF and FTH1 in ESCC and normal tissues were detected by immunohistochemical analysis. The differences in the expression of p35, COX2, TF and FTH1 between the ESCC and normal tissues were found to be statistically significant (Fig. 2A and B). The differences in the expression of p53, COX2, TF and FTH1 between ESCC stage III and normal tissues were found to be statistically significant. The differences in the expression of p53 and TF between ESCC stage IIa and normal tissues were not statistically significant, whereas the differences in the expression of COX2 and FTH1 between ESCC stage IIa and normal tissues were found to be statistically significant. The differences between the expression levels of p53, COX2, TF and FTH1 in ESCC stage IIa and stages III, however, were found not to be statistically significant (Fig. 2C). FTH1 expression was negatively correlated with TAGLN expression, whereas TF expression was positively correlated with TAGLN expression (Fig. 2D). However, the correlations between p53 and COX2 expression and TAGLN expression were found not to be statistically significant.

TAGLN expression level is decreased in ESCC and is associated with various cancer cell signaling pathways (39). The GEO databank is a publicly accessible genomics data repository for high-throughput sequencing. The databank contains sequence and array based gene profile data, and was used in this study to profile the expression of genes in ESCC. Three datasets, GSE161533, GSE45670 and GSE100942, were used to examine the differential expression of TAGLN in ESCC and normal tissues. A significant decrease in the level of TAGLN expression in ESCC compared with expression in normal tissue was observed in these datasets (Figs. 3A-C and S1). Using the median TAGLN mRNA level as a cutoff value, the patients with ESCC in TCGA were split into high and low TAGLN subgroups. The signaling pathways and cellular processes that were significantly associated with the high TAGLN subgroup compared with the low TAGLN subgroup were identified by GSEA (Fig. 3D-I). Genes upregulated in the high TAGLN subgroup were mostly enriched in 'focal adhesion' (NES=2.01; P<0.0001), 'ECM receptor interaction' (NES=1.94; P=0.002), 'Gap junction' (NES=1.73; P=0.004), 'TGF-β signaling pathway' (NES=1.65; P<0.05) and 'Cell adhesion molecules (CAMS)' (NES=1.74; P<0.05). These pathways have been associated with tumor proliferation, invasion and other malignant progressions (40,41), suggesting that TAGLN may serve an important role in ESCC.

To determine whether TAGLN exerts a role in the malignant progression of ESCC, TAGLN was overexpressed in Eca-109 and KYSE-150 cell lines. The efficiency of mRNA and protein overexpression was confirmed using RT-qPCR and western blot analyses, respectively, and the mRNA and protein expression levels in TAGLN group were found to be significantly higher compared with the vector-NC (Fig. 4A and B). The CCK-8, EdU and colony formation assay results revealed that the overexpression of TAGLN in ESCC cell lines led to a significant reduction in the capability of the ESCC cells to proliferate (Fig. 4C-E). Compared with the control group, the number of migrating and invading cells was also significantly decreased (Fig. 4F) following the overexpression of TAGLN in both cell lines. The ability of the cells to migrate was also significantly reduced in the wound healing assay following TAGLN overexpression (Figs. 4G and S2). Taken together, these results demonstrated that the overexpression of TAGLN may attenuate the proliferation, colony formation, invasion and migration of the two ESCC cell lines.

To investigate whether TAGLN serves a role in ESCC in vivo, a xenograft model was constructed. The inoculation of the mice with vector-NC or vector-TAGLN transfected Eca-109 cells revealed that TAGLN overexpression caused a marked reduction in tumor size, volume and weight after one month of growth compared with the mice inoculated with Eca-109-Vector-NC cells, whereas the whole-body weight of the mice remained the same (Fig. 5A-D). The TAGLN expression level of the subcutaneous tumors was subsequently assessed using western blot, RT-qPCR and immunohistochemical analyses, and the differences between vector-NC group and vector-TAGLN group were found to be statistically significant (Fig. 5E, F and I, respectively). Changes were also observed in the expression of the ferroptosis marker proteins, including GXP4 and ACSL4, in the subcutaneous tumors according to RT-qPCR and immunohistochemical analyses. GPX4 expression decreased, whereas that of ACSL4 increased, in the TAGLN-overexpression model mice compared with the control group (Fig. 5G, 5H, J and K). Taken together, these results suggested that the overexpression of TAGLN may cause a significant suppression of the progression of ESCC in vivo.

The injection of si-NC- or si-TAGLN-transfected Eca-109 cells into mice resulted in a significant increase in tumor volume and weight after one month of growth following TAGLN knockdown compared with the control Eca-109 cells (Fig. 6A, C and D, respectively), whereas the whole-body weight of the mice remained unchanged (Fig. 6B). Multiple tumor metastases were identified (and confirmed by H&E staining) in the livers of nude mice that were injected with si-TAGLN-transfected Eca-109 cells; the number of metastases was found to be significantly higher compared with the control group (Fig. 6E and F). The expression level of TAGLN in the subcutaneous tumors was detected by western blot, RT-qPCR and immunohistochemical analyses, and the expression was found to be significantly decreased compared with the control group (Fig. 6G-I and L). In addition, changes were also observed in the expression of the ferroptosis marker proteins, GXP4 and ACSL4, in the subcutaneous tumors using RT-qPCR and immunohistochemical analyses. According to RT-qPCR, compared with the control group, the expression of GPX4 was increased, whereas that of ACSL4 was decreased, following the knockdown of TAGLN (Fig. 6J and K). However, according to IHC staining, the difference of GPX4 expression between si-NC and si-TAGLN was not statistically significant, whereas that of ACSL4 was decreased in si-TAGLN group compared with the control group, following the knockdown of TAGLN. (Fig. 6M and N).

Transcriptome analysis results demonstrated significant differential normalized transcripts per million and gene expression in the vector-NC and TAGLN-overexpression Eca-109 cells (Figs. 7A and B, and S3), and the significant differential gene expressions are presented in Tables S1 and S2. To screen the differentially expressed genes, GO and KEGG enrichment analyses were performed (Fig. 7C). The analysis of GO enrichment showed that the BP and MF subontology were differentially regulated in TAGLN-overexpressing Eca-109 cells. For GO-BP, this regulation was associated with 'metal ion transport', 'dynamin family protein polymerization related to mitochondrial fission', 'epithelial cell apoptotic process', 'regulation of amino acid import across plasma membrane', and 'blood vessel endothelial cell proliferation in angiogenesis'. The overexpression of TAGLN induced the regulation of GO-MF gene categories associated with 'dynein intermediate chain binding', 'divalent inorganic cation transmembrane transporter activity', 'transition metal ion transmembrane transporter activity', 'ion transmembrane transporter activity' and 'protein serine/threonine phosphatase activity'. KEGG analysis showed that the overexpression of TAGLN regulated various pathways, including 'oxidative phosphorylation', 'transcriptional misregulation in cancer', 'TGF-β signaling pathway', 'signaling pathways regulating pluripotency of stem cells', 'pathways in cancer' and the 'PI3K-Akt signaling pathway'. These results indicated that TAGLN may contribute to the processes of oxidative phosphorylation, metal iron ion transport and cell apoptosis, leading to the occurrence of ferroptosis in ESCC cells and the inhibition of cancer proliferation, invasion and metastasis, together with associated signaling pathways. Moreover, the STRING tool was used to analyze the PPI networks of the TAGLN protein to determine their interaction in the progression of ESCC (Fig. 7D and E). As the results show, the crossover gene between PPI network and ferroptosis-related genes was GPX4, an essential protein of ferroptosis. These data suggested that TAGLN may regulate ferroptosis.

Western blot analysis was used to determine the expression levels of the ferroptosis marker proteins, GPX4 and ACSL4, following TAGLN overexpression. The protein expression level of ACSL4 was significantly increased, whereas that of GPX4 was significantly decreased, in Eca-109 and KYSE-150 cell lines following TAGLN overexpression compared with the vector-NC group (Fig. 8A). Furthermore, RT-qPCR results showed that the mRNA expression level of GPX4 in both ESCC cell lines was significantly decreased, whereas the expression level of ACSL4 was significantly increased (Fig. 8B). The overexpression of TAGLN caused a significant increase in the intracellular concentrations of MDA and lipid ROS (Fig. 8C and D, respectively), and a decrease in the concentration of GSH (Fig. 8E), suggesting that the overexpression of TAGLN may induce sensitivity to and promote ferroptosis. Levels of intracellular total iron (Fig. 8F) and Fe²⁺ iron (Fig. 8G and H) increased in Eca-109 and KYSE-150 cells overexpressing TAGLN compared with the vector-NC transfected groups. Taken together, these results suggested that TAGLN may serve a crucial oncogenic function in cancer progression by regulating ferroptosis in ESCC cells.

p53, one of the main factors that regulate ferroptosis, was identified from the intersection of the TAGLN interacting gene. Therefore, the functional interaction between TAGLN and p53 was selected for further investigation to unravel the molecular mechanism and metabolism of the malignancy in ESCC. RT-qPCR analysis revealed that the mRNA expression level of p53 significantly increased with an upregulation of TAGLN expression in Eca-109 cells (Fig. 9A). To investigate a putative interaction of TAGLN with p53, Co-IP and fluorescence co-localization experiments were conducted in Eca-109 cells (Fig. 9B and C, respectively). As Fig. 9C shown, TAGLN and p53 were both found to be abundant in the vector-TAGLN Eca-109 cell cytoplasm. These results indicated that TAGLN interacts with p53 and suggested that TAGLN may regulate ferroptosis through interacting with p53 in ESCC cells. Taken together, these results suggested that TAGLN inhibits the malignant progression and regulates ferroptosis by interacting with p53 in ESCC cells (Fig. 9D).