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Introduction

Of all malignant tumors, esophageal cancer has the seventh highest incidence and sixth highest mortality worldwide in 2020 (1). The predominant pathological classifications of esophageal cancer include esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma, with ESCC being the prevailing pathological subtype (2). China has a high incidence of esophageal cancer and ESCC. Moreover, China holds the top position globally in terms of both annual incidence (324,422, 53.7%) and mortality rates (301,135, 55.3%) of esophageal cancer in 2020 (1). Although comprehensive treatment, which is primarily based on surgery, has been applied in clinical practice, the poor prognosis, rapid progression and early metastasis of esophageal cancer lead to a 5-year survival rate of 20% (3), and the primary factor affecting the prognosis of patients is lymph node metastasis. Moreover, molecular biomarkers such as PD-L1 have been identified to predict prognosis and applied as therapeutic targets, which improves the prognosis of ESCC (4). Therefore, identifying novel molecular biomarkers associated with lymph node metastasis and the underlying molecular mechanism are highly important for patients with ESCC.

The solute carrier subfamily 25 member 1 (SLC25A1), identified as the mitochondrial citrate/isocitrate carrier or citrate transport protein, comprises solute carrier proteins embedded in the inner mitochondrial membrane (5,6). The SLC25A1 protein facilitates transport of citrate either from the cytoplasm into the mitochondria to participate in the tricarboxylic acid cycle reaction as a substrate, producing ATP or, conversely, from mitochondria into the cytoplasm as precursors for fatty acid, cholesterol and triglyceride synthesis by exchanging for malate (7,8). Thus, SLC25A1 serves an important role in cell energy metabolism and lipid synthesis. Lipid metabolism reprogramming is one of the hallmarks of malignancy and can promote cancer progression and metastasis in multiple ways: It not only supplies the substrates and energy for rapid proliferation but can also induce epithelial-mesenchymal transition, resistance to ferroptosis, immune escape and the activation of oncogenic pathways such as the Hedgehog and mTOR signaling pathways as signal messengers (9-15). Zhou also confirmed that fatty acid 2-hydroxylase promotes the metastasis of ESCC by regulating lipid metabolism (16). Upregulation of SLC25A1 has been discovered in lung and colon cancer and demonstrates an association with tumor development via the regulation of lipid metabolism (17,18). Nevertheless, SLC25A1 expression in ESCC and its role in ESCC development require further exploration.

It was hypothesized that SLC25A1 may promote the progression of ESCC by regulating lipid metabolism. Therefore, the present study aimed to investigate the expression of SLC25A1 in ESCC and determine whether its expression is correlated with clinical and pathological attributes. Furthermore, the role of SLC25A1 in lipid metabolism and oxidative phosphorylation in ESCC and potential underlying mechanisms were explored to provide potential novel targets and theoretical foundations for the treatment of ESCC.

Materials and methods

Patients

A total of 97 cancer tissue samples were obtained from patients (age, 42-77 years; 75 male, 22 female) with ESCC who underwent esophageal cancer resection at Shandong Provincial Hospital affiliated with Shandong First Medical University (Jinan, China) in January-December 2017, along with corresponding non-cancerous tissue samples (distance, 5 cm). All patients met the following criteria: i) Postoperative pathological confirmation of ESCC; ii) absence of preoperative radiotherapy treatment; iii) postoperative pathological confirmation of negative cancer tissue margins; iv) no severe preoperative complications and v) all patients were followed-up for ≥3 year. The present study was approved by the Ethics Committee of Shandong Provincial Hospital, affiliated with Shandong First Medical University (approval no. SZRJJ: NO.2022-015). All procedures were performed in accordance with Guidelines for the Work of Ethics Review Committees in China (19).

Cell lines and culture

Human esophageal cancer cell lines (KYSE150, 30, 450 and 510) and HeLa cervical cancer cells were obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cells were cultured in RPMI-1640 medium comprising 10% fetal bovine serum (FBS; both Gibco, US) with L-glutamine and maintained in an incubator with 5% CO2 at 37°C. CTPI-2, a specific blocker of SLC25A1 protein, was used to inhibit activity of SLC25A1 protein. A stock solution was prepared by dissolving CTPI-2 (MedChemExpress) powder in DMSO. The stock solution was diluted with phosphate buffer (Gibco; Thermo Fisher Scientific, Inc.) to achieve the required concentration (30 μM) before being added into the culture medium for further use. In the lentivirus transfected group, the cells without lentivirus transfection were the blank group. In the dosing group, cells were cultured in the medium without DMSO and CTPI-2 as the blank group.

Immunohistochemistry

All tissues were fixed with 4% paraformaldehyde for 24 h at 25°C, embedded in paraffin and sectioned into 5-μm slices. Following deparaffinization, washing with xylene for 45 min) and rehydration with ethanol (75, 85, 95, 100%) for five minutes each), sections were treated with 0.01 mol/l citrate buffer for 15 min at 100°C for antigen retrieval. Then the sections were put into 3% hydrogen peroxide solution for 30 min at 37°C for quenching. The sections were then treated with 10% goat serum (G1208-5ML, Servicebio, China) for 10 min at 25°C. The sections were exposed to anti-SLC25A1 (1:500, 15235-1-AP, Proteintech) or anti-FGFBP1 (1:500, bs-1768R, Bioss antibodies) antibody at 4°C overnight. The sections were subsequently incubated for 30 min with a horseradish peroxidase (HRP)-conjugated secondary antibody from the Immunohistochemistry kit (1:200, G1215-200T, Servicebio, China) at 25°C and then stained (25°C, 3 min) with DAB and hematoxylin. Proportion of positive cells was scored as follows: 0-5, 0; 6-25, 1; 26-50, 2; 51-75, 3 and 76-100%, 4. Positive staining intensity scoring was as follows: 0, negative, 1 weak, 2 moderate and 3 strong staining. The total immunohistochemistry staining score (IHS) was calculated by multiplying the proportion of positively stained cells score by the positive staining intensity score. A total score of 0-7 represented low expression, whereas a score of 8-12 represented high expression. The samples were observed under a light microscope (200×) and independently scored by two pathologists.

Hematoxylin-Eosin staining: All tissues were fixed in formalin, embedded in paraffin and sectioned into 5-μm slices. Following deparaffinization and rehydration, the sections were stained with hematoxylin dye for 3 min, treated with 1% hydrochloric acid alcohol for 30 sec, stained with eosin dye for 2 min, dehydrated with ethanol (75, 85, 95, 100%), washed with xylene, and finally sealed with neutral resin. The results were observed under light microscope.

Lentiviral infection

SLC51A1 RNA-interfering (5′-CCAUCAAGGUGAAGUUCAU-3′) and negative control lentivirus were obtained from Beijing Tsingke Biotech. The sequences were negative control: sense 5′-UUCUCCGAACGUGUCACGU-3′. sh)RNA was subcloned into the pLKO.1-puro vector. The generation system is the second system. Subsequently, pLKO.1-puro-shRNA plasmid (20 μg) and virus packaging plasmids (pMD2.G, 5 μg; psPAX2, 10 μg) were cotransfected into 293T cells (China Center for Type Culture Collection, Wuhan, China) using Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Medium was replaced with fresh DMEM (Thermo Fisher Scientific, Inc.) containing 10% FBS and incubated at 37°C for 48 h. The cell supernatant was collected, then filtered through a 0.45-μm filter (Pall Life Sciences, Port Washington, NY, USA). MOI for lentivirus transfection was 20. KYSE150 and KYSE30 cells in the exponential growth phase were plated in 6-well plates and cultured for 24 h. SLC51A1-interfering and negative control lentivirus were inoculated into the cells. Following 24 h incubation, the cell medium was replaced with complete medium (RPMI-1640 medium comprising 10% FBS, Gibco, US). The monoclonal cells were stably and continuously expressed after screening with purinomycin (5 μg/ml). The time interval between transduction and follow-up experiment was 10 days. The purinomycin concentration for maintenance was 0.25 μg/ml.

Reverse transcription-quantitative (RT-q)PCR

Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific, US), followed by RT via Evo M-MLV RT Kit with gDNA Clean for qPCR., Accurate Biology) according to the manufacturer's instructions. qPCR was performed using SYBR Green Real-time PCR Master Mix (Takara Biotechnology Co., Ltd.) on a LightCycler 480 (Roche, Switzerland). Thermocycling conditions: Initial denaturation: 95°C, 10 sec. Denaturation: 95°C, 5 sec. Annealing and extension: 60°C, 30 sec, 40 cycles. The relative mRNA expression was measured by the ∆∆Cq method (20). The internal reference gene was GAPDH. The primer sequences were as follows: SLC25A1 forward, 5′-CCAUCAAGGUGAAGUUCAU-3′ and reverse, 5′-AUGAACUUCACCUUGAUGG3′; FGFBP1 (Fibroblast Growth Factor Binding Protein 1) forward, 5′-CTTCACAGCAAAGTGGTCTCA-3′ and reverse, 5′-GACACAGGAAAATTCATGGTCCA-3′ and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′.

RNA-seq analysis and bioinformatics analysis

RNA seq data for human ESCC cells were acquired from The Cancer Genome Atlas database. (TCGA, ualcan.path.uab.edu/cgi-bin/TCGAExResultNew2.pl?genenam=SLC25A1&ctype=ESCA). Following transfection of KYSE150 cells with SLC25A1-interfering lentivirus or nonsense lentivirus, the total RNA was extracted using TRIzol (Cat. No. 15596026, Thermo Fisher) and treated with DNase to remove genomic DNA contamination. The NEBNext® Poly (A) mRNA Magnetic Isolation Module and NEBNext® Ultra™ II mRNA Library Prep kit (cat. no. NEB #E7770S/L, Cat. No. #E7775S/L, New England Biolabs, Inc.,) for Illumina® were used for mRNA isolation and library construction following the manufacturer's protocols. And then the RNA-seq library was sequenced using an Illumina NovaSeq 6000 PE150 instrument (Illumina, Inc.) by Haplox Genomics Center. DESeq2 (1.18.1) and edegR (3.209.) were used for Difference analysis (21,22), and the ClusterProfier (4.8.2) was used for Reactcome enrichment analysis (23).

Western blot analysis

Tissue and cellular proteins were isolated using PMSF-containing RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was evaluated via a BCA protein concentration assay and the loading quantity of the samples was calculated. The protein samples were added to 10% PAGE (20 μg/lane) for electrophoretic separation and electrotransferred to a PVDF membrane. PVDF membrane at 25°C was blocked with 10% skimmed milk powder for 2 h to prevent specific antibody binding. The membrane was incubated with SLC25A1 (1:2,000, 15235-1-AP, Proteintech), AKT (1:1,000, db14689, diagbio), phosphorylated (p)-AKT (1:1,000, db12718, diagbio), FGFBP1 (1:1,000, bs-1768R, Bioss antibodies), GAPDH (1:1,0000, bs-10900R, Bioss antibodies) and β-actin (1:1,000, bs-0061R, Bioss antibodies) primary antibodies in a shaker at 4°C overnight, followed by rinsing with TBST (Tris Buffered Saline with 0.1% Tween-20). The membrane was exposed to HRP-conjugated goat anti-rabbit IgG polyclonal secondary antibody (1:5,000, HA1008, Huabio, China) for 1 h at room temperature on a shaker and rinsing with TBST. Finally, the PVDF membranes were treated with visualisation reagent (Immobilon ECL Ultra Western HRP Substrate, Millipore) and detected with Amersham Imager 680 (GE HealthCare, US). ImageJ (National Institutes of Health) was used to analyze the gray values of each blot.

EdU cell proliferation assay

A total of 5,000 transfected or untransfected cells in the logarithmic growth phase were inoculated into each well of a 96-well plate. After 24 h, the cells were incubated in CTPI-2-containing or CTPI-2 free medium at 37°C for 2 days. The cells were fixed with 4% paraformaldehyde at 25°C for 30 min and stained with an EdU fluorescence staining kit (Cell-Light EdU Apollo In Vitro Kit; ribobio) according to the manufacturer's guidelines. Images were captured by inverted fluorescence microscope (200×) and ZEN 3.3 blue edition software (Zeiss, Germany).

Colony formation assay

Following 24 h inoculation in 6-well plates, the untransfected or transfected KYSE150 cells and KYSE30 cells were cultured with CTPI-2-containing or CTPI-2 free medium. The medium was changed every 3 days, and the cells were maintained at 37°C with 5% CO2 for 10 days. The cells were washed with PBS, fixed with 4% paraformaldehyde at 25°C for 30 min and stained with 0.1% crystal violet at 25°C for 3 min. The number of cells in a single clone exceeding 50 is called a colony, and the results were detected by ImageJ software (ImageJ 1.50b, National Institutes of Health).

Cell Counting Kit (CCK)8 assay

A total of 5,000 cells in the exponential growth phase were inoculated in 96-well plates. At 24 h post-inoculation, the medium was changed to CTPI-2-containing or CTPI-2 free medium. After 24, 48, 72 and 96 h incubation at 37°C, CCK8 reagent (MedChemExpress, US) was added for 1 h at 37°C. A microplate reader (Multiskan Go, Thermo) was used to measure the absorbance of each well at 450 nm.

Wound healing assay

Untransfected KYSE150 cells and KYSE30 cells were incubated with serum-free medium containing CTPI-2 in 6-well plates, the transfected KYSE150 cells and KYSE30 cells were incubated with serum-free medium in 6-well plates. At 90-95% confluence, a scratch was made in using a pipette tip (200 μl), and the cells were incubated in serum-free medium with or without CTPI-2 for 24 h. Then, the scratch was imaged under a light microscope at 0 and 24 h. The wound area at the same location was subsequently measured via ImageJ. The cell migration rate was calculated as follows: Cell migration rate (%)=(initial wound area-wound area after 24 h)/initial wound area ×100%.

Cell migration and invasion assay

Transwell upper chambers coated with Matrigel (BD Science, US) at 37°C for 1 h were used to determine the invasive ability of cells, whereas upper chambers lacking Matrigel coating were used to determine migratory ability. FBS-free RPMI-1640 medium mixed with 50,000 cells was added to the upper chambers. For untransfected cells, CTPI-2 reagent (30 μM) was added to the upper chambers. RPMI-1640 Medium with a 15% FBS concentration was added to the lower chambers. Following incubation for 24 or 48 h at 37°C in a 5% CO2 incubator, the cells on the lower surface were fixed with 4% paraformaldehyde at 25°C for 30 min, stained with crystal violet at 25°C for 10 min and sealed. The slides were observed under a light microscope (200×) and images were captured in three randomly selected fields of view.

Apoptosis assay

Apoptosis was detected via flow cytometry using an Annexin V-PE/7-AAD Apoptosis Detection kit (cat. no. MA0429, Meilun Biotechnology Co., Ltd.). The transfected or untransfected KYSE150 cells and KYSE30 cells were cultured in 6-well plates until the cell density reached 85%. The cells were digested with EDTA-free trypsin and centrifuged (1,000 g, 5 min) to collect the cell pellets, which was washed with PBS (Gibco, US) solution precooled at 4°C. Binding buffer working fluid was added to the cell pellets and the cell concentration was suspended to 1×10^6/ml. 100 μl cell suspension (the total number of cells was 1×10^5) was absorbed, 5 μl Annexin V-PE and 7-AAD dye were added to the cell suspension, mixed and incubated at 25°C for 15 min without light. BD LSRFortessa (BD Biosciences) and BD FACSDiva 7.0 software (BD Biosciences, US) were used to determine degree of apoptosis. The apoptosis rate was the sum of early and late apoptotic cells.

Determination of intracellular lipid content

The lipid content in the transfected or untransfected KYSE150 cells and KYSE30 cells was determined via the Triglyceride, Free Fatty Acid and Total Cholesterol Content Assay kits (BC0625, BC0595, BC1985) (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions.

BODIPY 493/503 staining of intracellular lipids

ESCC cells were treated with BODIPY 493/503 fluorescent dye (5 μM) (MedChemExpress) at room temperature for 30 min and shielded from light, to visualize lipid distribution within the cells. Images were captured using a fluorescence microscope.

Measurement of cellular oxygen consumption rate

A total of 50,000 ESCC cells were seeded in 96-well plates in the dark. Cells were cultivated at 37°C in a glucose-free RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) for 24 h. The oxygen consumption rate was assessed via fluorescence microplate using Extracellular OCR Plate Assay kit (Dojindo, E297) according to the manufacturer's instructions.

Measurement of mitochondrial respiratory chain complex activity

Mitochondrial respiratory chain activity was evaluated via the Mitochondrial Respiratory Chain Complex I-V Activity Assay kit (BC0515, BC3230, BC3240, BC0945, BC1445) (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's instructions. The absorbance (340, 605, 550, 550, 660 nm) was measured via a microplate reader (Multiskan Go, Thermo Fisher Scientific, Inc.) and relative activity was calculated.

Xenograft model of ESCC

A total of 14 male BALB/c mice (age, 4-6 weeks; weight, 10-14 g, Charles river) were allocated into negative control (7 mice) and lentiviral transfection groups (7 mice) and reared under standard environmental conditions (26-28°C, and the relative humidity is 40-60%, 12/12-h light/dark cycle, with commercial rat food and water ad libitum). Subsequently, 1,000,000 lentivirus-transfected or negative control cells were subcutaneously injected into the right axilla of mice. Tumor size was measured every 5 days once the xenograft tumors reached a subcutaneous volume of 100 mm3. After 20 days, the subcutaneous tumor in nude mice reached its maximum volume of 700-800 mm3, all mice were euthanized, and the xenograft tumors were surgically excised for volume measurement and tissue weighing. No nude mice died unexpectedly during the experiment. The excised tissues were preserved in 4% paraformaldehyde (at 25°C, for 24 h) for immunohistochemical analysis. Tumor volume was calculated as follows: Volume (mm3)=maximum diameter × minimum diameter2/2 (24). Experiments were approved by the Animal Ethics Committee of the Shandong Provincial Hospital, affiliated with the Shandong First Medical University (approval no. SDNSFC 2023-0026).

Statistical analysis

SPSS 19.0 (SPSS, Inc.) was used for clinical data analysis. For continuous variables, unpaired Student's t-test was performed. The association between SLC25A1 protein expression and pathological parameters was determined via χ2 or Fisher's exact probability test (two-tailed). Survival rates were calculated via the Kaplan-Meier method and analyzed via log-rank test. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was conducted using GraphPad Prism 8 (Dotmatics). Differences between two groups were assessed via unpaired t-test. One-way ANOVA was used to analyze variations between >2 groups followed by Least Significance Difference test was used for the post hoc test. The correlation between SLC25A1 and FGFBP1 expression was determined by Pearson's correlation analysis. The outliers were removed or replaced by a median value. P<0.05 was considered to indicate a statistically significant difference.

Results

SLC25A1 overexpression is correlated with TNM stage, recurrence rate and prognosis of ESCC

Expression of SLC25A1 in esophageal squamous cell carcinoma was significantly greater than that in normal esophageal mucosa in TCGA (Fig. 1A). Mean IHS of SLC25A1 in 97 ESCC tissue samples was 7.4±3.5; that of 97 non-cancerous tissue samples was 3.7±2.9. These findings indicate a significant increase in SLC25A1 expression in ESCC relative to normal tissues (Fig. 1B and C). Moreover, the IHS in the lymph node metastasis-positive group was 9.6±1.6, whereas that in the negative group was 4.3±1.9. These findings suggested significant upregulation of SLC25A1 expression in the tissues of patients with ESCC metastasis relative to those without lymph node metastasis (Fig. 1D). Western blot analysis further revealed significant upregulation of SLC25A1 expression in ESCC tissue (Fig. 1E). χ2-test revealed a significant association between positive SLC25A1 overexpression and lymph node metastasis, T stage and postoperative regional lymph node recurrence in patients with ESCC (Table I). Kaplan-Meier analysis revealed that patients with positive SLC25A1 expression had a significantly lower disease-free (45.5 vs. 74.2%) and 3-year overall survival rate (60.6 vs. 80.6%) than patients with negative SLC25A1 expression (Fig. 1F).

Figure 1 Expression of SLC25A1 in ESCC. (A) Differences in SLC25A1 expression between tumor and non-cancerous tissues in The Cancer Genome Atlas database. (B) SLC25A1 expression in ESCC and non-cancerous tissue samples. Cancer cells are arranged in a nest-like pattern, with large and deeply stained nuclei and notable atypia compared with non-cancerous cells and SLC25A1 mainly located in the plasma. Arrow points to the representative location for immunohistochemical staining. (C) IHS of SLC25A1 in tumor was significantly greater than that in non-cancerous tissues. (D) IHS of SLC25A1 in patients with ESCC and lymphatic node metastasis was significantly greater than that in patients without lymphatic node metastasis. (E) SLC25A1 expression in pairs of ESCC and non-cancerous tissue samples detected via western blot analysis. (F) Kaplan-Meier analysis showed that the patients with SLC25A1 positive expression had lower 3-year disease-free and overall survival than patients with negative expression. *P<0.05, **P<0.01, ****P<0.0001. IHS, immunohistochemistry staining score. SLC25A1, solute carrier family 25 member 1; ESCC, esophageal squamous cell carcinoma.

Table I Association between SLC25A1 expression and clinical characteristics of patients with esophageal squamous cell carcinoma).

Table I

Association between SLC25A1 expression and clinical characteristics of patients with esophageal squamous cell carcinoma).

Clinical characteristic	Total cases (n=97)	SLC25A1-positive (n=66)	SLC25A1-negative (n=31)	P-value
Age, years				0.432
≥60	59	30	29
<60	38	26	12
Sex				0.591
Male	75	50	25
Female	22	16	6
Pathological T stage				0.006
T1 + T2	40	21	19
T3 + T4	57	45	12
Lymphatic node metastasis				0.021
Positive	51	40	11
Negative	46	26	20
Weight loss				0.146
Yes	55	35	20
No	42	31	11
Recurrence				0.008
Yes	44	36	8
No	53	30	23
Differentiation				0.199
Well/moderate	53	39	14
Low	44	27	17

[i] SLC25A1, solute carrier family 25 member.

Silencing or blocking SLC21A1 inhibits the proliferation, invasion and migration of ESCC cells, and promotes the apoptosis of ESCC cells. Expression of SLC25A1 in ESCC and HeLa cells was validated via RT-qPCR (Fig. 2A). Compared with KYSE510 cell line, Hela cell line and KYSE450 cell line, SLC25A1 expression was elevated in KYSE150 and KYSE30. Given the increased expression of SLC25A1 in ESCC cell lines, shRNA lentivirus targeting SLC25A1 gene was constructed to silence SLC25A1 expression in ESCC cells (Fig. 2B). SLC25A1 protein function in ESCC cells was specifically blocked by CTPI-2. CCK8 assay revealed that cell proliferation rate was considerably lower in the shSLC25A1 and CTPI-2 groups than in the blank or DMSO groups (Figs. 2C and 3A). EdU cell proliferation and colony formation assays indicated that silencing or blocking SLC25A1 significantly decreased the proliferation and colony formation abilities of KYSE 150 and 30 cells (Figs. 2D and E and 3B and C).

Figure 2 Silencing SLC25A1 inhibits proliferation, migration and invasion but induces apoptosis of ESCC cells. (A) SLC25A1 expression in KYSE 30, 150, 450 and 510 and HeLa cell lines was detected by RT-qPCR. (B) Transfection efficiency of the SLC25A1-silencing lentivirus was detected via western blotting and RT-qPCR. (C) KYSE 150 and 30 cell proliferation. (D) Colony formation assay (1×). (E) EdU staining assay. (F) Apoptosis rates of KYSE 150 and 30 cells were analyzed via PE-7AAD staining. (G) Migration ability of ESCC cells was detected via a wound healing assay. (H) Migration and invasion abilities of ESCC cells were detected via Transwell assay. **P<0.01, ***P<0.001, ****P<0.0001. SLC25A1, solute carrier family 25 member 1; ESCC, esophageal squamous cell carcinoma; RT-q, reverse transcription-quantitative; NC, Negative control; sh, short hairpin; ns, not significant; RFP, Red Fluorescent Protein; OD, optical density.

Figure 3 Inhibiting SLC25A1 inhibits proliferation, migration and invasion but induces apoptosis of ESCC cells. (A) Proliferation of KYSE 150 and 30 cells treated with CTPI-2. (B) Colony formation assay (1×). (C) EdU staining assay. (D) Apoptosis rates of KYSE150 and KYSE30 cells treated with CTPI-2 were analyzed via PE-7AAD staining. (E) Migration ability of ESCC cells treated with CTPI-2 (30 μM) was detected via a wound healing assay. (F) Migration and invasion abilities of ESCC cells treated with CTPI-2 (30 μM) were detected via Transwell assay. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. SLC25A1, Solute carrier family 25 member 1; ESCC, esophageal squamous cell carcinoma; OD, optical density; ns, not significant; RFP, Red Fluorescent Protein.

Flow cytometry assay was conducted to assess whether silencing SLC25A1 or blocking its protein function promotes apoptosis in ESCC cells. The proportion of apoptotic KYSE 150 and 30 cells significantly increased when SLC25A1 was silenced or SLC25A1 protein function was inhibited (Figs. 2F and 3D).

Silencing or inhibiting SLC25A1 significantly decreased the wound healing ability of ESCC cells in vitro (Figs. 2G and 3E). Transwell assay demonstrated that both silencing and inhibiting SLC25A1 could inhibit the in vitro migration and invasion of KYSE 150 and 30 cells (Figs. 2H and 3F).

SLC5A1 promotes lipid synthesis and affects oxidative phosphorylation of ESCC cells

Citrate directly provides precursor lipids for intracellular fatty acid synthesis in the cytoplasm. SLC25A1 is the exclusive citrate transporter in the mitochondrial membrane (7). Silencing or specific blockade of SLC25A1 resulted in a notable decrease in intracellular fatty acid staining levels (Fig. 4A). Intracellular lipid content assay experiments confirmed that silencing or inhibition of SLC25A1 protein resulted in decreased free fatty acid, triglyceride, and cholesterol contents in KYSE150 and KYSE30 cells (Fig. 4B-D). These findings suggested that SLC25A1 was involved in lipid synthesis in ESCC cells.

Figure 4 SLC5A1 promotes lipid synthesis and affects oxidative phosphorylation of ESCC cells. (A) Intracellular lipids were stained with fluorescent lipophilic dye BODIPY 493/503. Levels of (B) free fatty acids, (C) triglycerides, and (D) cholesterol. Following glucose starvation, (E) oxygen consumption rate and (F) activity of mitochondrial respiratory chain complexes in ESCC cells was determined. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. SLC5A1, solute carrier family 25 member 1; ESCC, esophageal squamous cell carcinoma; ns, not significant; NC, Negative control; sh, short hairpin.

To determine the effect of SLC25A1 on energy metabolism of ESCC cells, the oxygen consumption rate was measured after 24 h starvation. Silencing or inhibiting SLC25A1 led to a significant decrease in oxygen consumption in KYSE 150 and 30 cells compared with that in the negative control and blank groups (Fig. 4E) and activity of the mitochondrial respiratory chain complex was reduced in cells in which SLC25A1 was silenced or specifically blocked (Fig. 4F). These findings collectively suggest that SLC25A1 can facilitate oxidative phosphorylation in ECSS cells under starvation.

SLC25A1 silencing downregulates FGFBP1 expression and inhibits the AKT signaling pathway in ESCC cells

The aforementioned results indicated that SLC25A1 may serve a key role in promoting malignant biological behaviors of ESCC cells, particularly through its regulatory influence on lipid and energy metabolism processes. High-throughput transcriptome sequencing was performed on KYSE150 cells to identify the potential molecular mechanism by which SLC25A1 modulates malignant biological behavior of ESCC cells. Sequencing and Reactome pathway enrichment analysis showed that several pathways, including those associated with 'signaling by interleukins', 'interferon signaling', 'PI3K cascade: FGFR3', 'FGFR2b ligand binding and activation', 'PI3K cascade', 'downstream signaling of activated FGFR3' and 'FGFR1 mutant receptor activation', were significantly enriched in the shSLC25A1 ESCC cells compared with the control cells (Fig. 5A-C). As numerous pathways were associated with FGFR activation and activation of FGFR could also activate the PI3K/AKT pathway, the significant downregulation of FGFBBP1, a key gene in the FGF signaling pathway (25), in shSLC25A1 ESCC cells (Fig. 5D) was investigated. Western blotting suggested that silencing or inhibiting SLC25A1 led to a significant decrease in FGFBP1 expression and downstream activation of the AKT signaling pathway in KYSE 150 and 30 cells (Fig. 5E and F). Furthermore, the expression FGFBP1 was assessed by immunohistochemistry (Fig. 5G). These results demonstrated SLC25A1 regulated expression of FGFBP1 and activation of the AKT signaling pathway in ESCC cells.

Figure 5 Silencing or inhibiting SLC25A1 downregulates expression of FGFBP1 and suppress the AKT pathway. (A) Volcano plot and (B) cluster analysis of differential genes in shSCL25A1 compared with the NC group, red color clusters represent genes up-regulated while green represents genes down-regulated, and connecting lines represent clustering result. (C) Reactcome enrichment analysis result of differential genes in shSCL25A1 compared with the NC group, pathways with number of differential genes ranking top 20 were showed. (D) Reverse transcription-quantitative PCR confirmed the downregulation of FGFBP1 in SLC25A1-silenced ESCC cells. After SLC25A1 was (E) silenced and (F) inhibited, the expression of FGFBP1 and the phosphorylation of AKT in KYSE 150 cells and 30 cells were detected via western blotting. (G) SLC25A1 and FGFBBP1 expression in ESCC samples was detected via immunohistochemistry. Arrow points to the representative location for immunohistochemical staining. **P<0.01, ***P<0.001, ****P<0.0001. SLC25A1, solute carrier family 25 member 1; FGFBP1, fibroblast Growth Factor Binding Protein 1; p-, phosphorylation; NC, Negative control; sh, short hairpin; FC, Fold change.

SLC25A1 silencing inhibits tumor growth in vivo

To ascertain the impact of SLC25A1 silencing on ESCC cell proliferation in vivo, a tumor xenograft model was developed by subcutaneously inoculating nude mice with KYSE 150 cells transfected with either SLC25A1-interfering or negative control lentivirus. Tumors formed by KYSE 150 cells transfected with SLC25A1-interfering lentivirus exhibited a significantly decreased size and growth rate (Fig. 6A and B). IHC and western blotting revealed a reduction in the expression of both SLC25A1 and FGFBP1 in the SLC25A1 silenced group (Fig. 6C and D). These findings indicated that silencing SLC25A1 expression effectively inhibited the proliferation of ESCC cells in vivo.

Figure 6 Silencing SLC25A1 inhibits growth of esophageal squamous cell carcinoma) xenograft tumors in mice. (A) Nude mice were sacrificed 20 days after being inoculated subcutaneously. (B) Tumor volume and weight. SLC25A1 and FGFBP1 expression was detected by (C) IHC and (D) western blotting. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. SLC25A1, Solute carrier family 25 member 1; FGFBP1, Fibroblast Growth Factor Binding Protein 1; IHC, Immunohistochemical staining; NC, Negative control; sh, short hairpin; p-, phosphorylation.

Discussion

The incidence and mortality of esophageal cancer is increasing, and China accounts for half of incidence and mortality of EC: The number of EC cases worldwide increased from 319,969 in 1990 to 534,563 in 2019, while the number of incident cases of EC in China increased from 173,687 in 1990 to 278,121 in 2019. The number of EC deaths worldwide increased from 319,332 in 1990 to 498,067 in 2019, and the EC mortality in China increased from 176,602 in 1990 to 257,316 in 2019, with ESCC emerging as the predominant histological subtype (26). ESCC is highly malignant and prone to metastasis in the early stage leading to a high mortality : the 5-year relative survival rate is only 20%, which is the second lowest survival rate after pancreatic cancer (27,28). The limited efficacy of conventional antitumor drugs and lack of effective molecular targets and drugs make treatment challenging for patients with advanced esophageal cancer (1,29,30). The present study explored the expression of SLC25A1 in ESCC, revealing its impact on malignant biological behavior of ESCC cells and the underlying mechanisms.

Citrate is an important substance in the cell. In mitochondria, citrate, one of the key reaction substrates of the tricarboxylic acid cycle, generates ATP for cell use through the mitochondrial electron transport chain (31). Upon transportation into the cytoplasm facilitated by the mitochondrial citrate carrier SLC25A1, citrate undergoes cleavage into oxaloacetate and acetyl-CoA, a process catalyzed by ATP-citrate lyase (ACLY) (28). Acetyl-CoA is a key precursor for intracellular synthesis of fatty acids and cholesterol. SLC25A1, which belongs to the ionic protein transporter family, regulates levels of mitochondrial and cytoplasmic citrate, which is associated with various physiological metabolic processes such as lipid metabolism in the liver, cancer and aging (32,33). Abnormal distribution and regulation of SLC25A1 are associated with various cancers: High expression of SLC25A1 is observed in non-small cell lung (17) and colon cancer (18). KRAS mutant gene KRASG12D induces high expression of SLC25A1 in human pancreatic cancer cells via glioma-associated oncogene homolog 1) and promotes pancreatic carcinogenesis in mice (34). In the present study, immunohistochemistry and western blot analysis revealed upregulation of SLC25A1 expression in ESCC tissues and cell lines. Moreover, high expression of SLC25A1 in ESCC was associated with T stage, lymph node metastatic status, postoperative local lymph node recurrence and poor prognosis. Thus, upregulation of SLC25A1 expression may be associated with development of ESCC. Therefore, SLC25A1 may serve as a specific molecular marker to predict prognosis of patients with ECSS.

To confirm the involvement of SLC25A1 in the onset and progression of ESCC, lentiviral transfection was executed to silence expression of the SLC25A1 gene. In addition, CTPI-2, a specific inhibitor of the SLC25A1 protein, was used to bind to the functional site of the SLC25A1 protein to inhibit its function. Inhibition and silencing of SLC25A1 expression suppressed the proliferation, invasion and migration of the ESCC cell lines KYSE 150 and 30 in vitro and induced apoptosis. Downregulation of SLC25A1 suppressed the in vivo tumorigenic ability of ESCC cells. These findings demonstrated the critical role of SLC25A1 in the malignant biological behavior of ESCC cells.

SLC25A1, a key gene that regulates cellular metabolism, may be involved in the metabolism of ESCC cells. Silencing and specific blockade of SLC25A1 resulted in a significant decrease in lipid synthesis in KYSE 150 and 30 cells, suggesting the vital role of SLC25A1 in lipid synthesis in ESCC cells. Lipids are key substances for cell metabolism and survival. They form crucial components of cell and organelle membranes and actively participate in formation of cell signaling molecules (35). As a form of cellular energy storage, lipids serve a pivotal role in supplying energy for proliferation of tumor cells (36,37). Owing to the rapid growth and high metabolic level, the nutritional requirements of tumor tissue often exceed the supply by the microenviorment (38). Oligotrophic blood vessels in the early stage of tumor growth cannot provide sufficient nutrition to tumor tissues. In this case, tumor cells need to regulate their own metabolic pathways to survive. Lipid metabolism is involved in the developmental process of numerous types of tumor, such as lung cancer, colon cancer and breast cancer, especially in metastasis, and lipid metabolism reprogramming is a hallmark of malignancy (39,40). Therefore, overexpression of SLC25A1 may promote the aggressive biological behavior of ESCC cells via regulation of lipid metabolism. Citrate is involved in multiple metabolic pathways including lipogenesis, glycolysis and gluconeogenesis: Citrate is the key substrate of acetyl-CoA for fatty acid and sterol biosynthesis; it is an allosteric regulator of enzymes that control glycolysis and gluconeogenesis, such as 1,6-bisphosphatase (41-43). Thus, SLC25A1 may also promote the progression of ESCC via other metabolic pathways, including glycometabolism, which needs further exploration.

The SLC25A1 protein transports citrate bidirectionally between cytoplasm and mitochondria. In addition, SLC25A1 is involved in energy metabolism in colon cancer during metabolic stress (37). Therefore, the present study investigated involvement of SLC25A1 in the regulation of energy metabolism in ESCC cells. With a sufficient supply of energy, there was a minimal difference in the oxygen consumption rate of KYSE 150 and 30 cells between the treated and untreated groups. However, following starvation, a substantial reduction in the oxygen consumption rate was observed in the SLC25A1-silenced and -inhibited groups, concomitant with a decrease in activity of the mitochondrial respiratory chain complexes. Compared with normal cells, tumor cells undergo glycolytic reactions more frequently. Compared with oxidative phosphorylation, glycolysis can produce ATP at a faster rate for use by tumor cells (38). However, when they detach from the extracellular matrix and adapt to anchorage-independent growth in a low nutrient environment, tumor cells may undergo oxidative phosphorylation to produce ATP for survival, which is key for the invasive and metastatic behavior of tumor cells (6,44). In the absence of glucose, SLC25A1 protein transports cytoplasmic citrate to the mitochondria to increase oxidative phosphorylation, thereby ensuring cell survival. The aforementioned results confirm that SLC25A1 is involved in lipid metabolism and energy metabolism in ESCC and provides material and energy for the tumor development of ESCC. The mechanism by which SLC25A1 influences the oxidative phosphorylation pathway of mitochondria may be complex and is unclear. In addition to reverse import of cytosolic citrate into mitochondria, the import of malate into the mitochondria, which leads to an increase in the tricarboxylic acid cycle flux and generation of reducing equivalents including NADH/NAD+ for the electron transport chain also participates this regulation (31).

The present results suggested that SLC25A1 may promote the malignant biological behavior of ESCC cells by regulating cellular lipid and energy metabolism. To reveal the underlying mechanism of SLC25A1 in promoting the onset and progression of ESCC, high-throughput expression profile sequencing was performed on SLC25A1-silenced KYSE 150 ESCC and control cells. The expression of FGFBP1, a key gene in the FGF signaling pathway, was significantly downregulated. The co-expression of SLC25A1 in ESCC tissue with FGFBP1 was confirmed via immunohistochemistry. FGFBP1, belonging to the FGFBP family (45), serves as a secretory chaperone protein. FGFBP1 releases FGF immobilized in the extracellular matrix and facilitates binding of FGF to its receptor (46,47). FGF is a crucial molecule associated with cell proliferation, migration and differentiation (48). FGFBP1 is highly expressed in colon and pancreatic cancer and oral squamous cell carcinoma (49,50). The AKT signaling pathway is a key signaling pathway during tumor growth and is involved in regulating the onset and progression of numerous types of tumor, such as ESCC (51-53). The activation of the AKT pathway could regulate the downstream genes to directly promote cell survival, proliferation, migration and angiogenesis; it also serves an important role in lipid metabolism in the progression of tumors (54). The AKT pathway may regulate the expression of key synthetases of lipids, such as fatty acid synthase, ACLY and acetyl-CoA carboxylase to promote lipid synthesis (55,56); however, recent studies have revealed high fat microenvironment promotes the progression of tumors by activating the AKT pathway (57,58). As a key pathway activated by FGF binding to its receptor, the AKT pathway may be inhibited upon downregulation of FGFBP1 expression. In the present study, the silencing or inhibition of SLC25A1 led to considerable downregulation of the activation of the AKT signaling pathway in ESCC cells. These findings suggested that SLC25A1 may activate the AKT signaling pathway by regulating FGFBP1 expression, facilitating tumor initiation and progression of ESCC. In addition, FGF receptor binding could activate not only the AKT pathway, but also the MAPK, JAK/STAT3 and PLCγ pathways and SLC25A1 could also regulate the TNF signaling pathway by reducing the expression of TNF-α and IL-6 (59). Therefore, upregulation of SLC25A1 may promote the progression of ESCC via signaling pathway network regulation. However, the exact mechanism by which SLC25A1 regulates transcription of FGFBP1 and other signaling pathways requires further exploration.

In summary, expression of SLC25A1 was elevated in ESCC and significantly associated with the malignant biological behavior of ESCC, particularly lymph node metastasis. SLC25A1 may contribute to the onset and progression of ESCC by regulating ESCC metabolism. SLC25A1 may promote the development of ESCC by regulating the FGFBP1-activated AKT signaling pathway. Consequently, SLC25A1 may serve as a potential novel target for ESCC treatment and a molecular biological marker for the prediction of patient prognosis.

Availability of data and materials

The data generated in the present study may be found in Figshare under accession number (10.6084/m9.figshare.28023275) or at the following URL: (https://figshare.com/s/a5ed5881f88d63947aa6).

Authors' contributions

GZ conceived the study, designed and performed experiments and wrote the manuscript. JW and MJ performed the experiments. XL and MS analyzed the data and edited the manuscript. All authors have read and approved the final manuscript. GZ and MS confirm the authenticity of all the raw data.

Ethics approval and consent to participate

All procedures involving human participants were performed in accordance with the ethical standards of the Institutional Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (approval no. SZRJJ:NO.2022-015). All patients agreed to participate in the study. All procedures involving animals were in accordance with the ethical standards of Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (approval no. SDNSFC 2023-0026).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from Natural Science Foundation of Shandong Province (81902418).

References