Etomoxir (ETX) was purchased from MedChemExpress Chemical Ltd. (Middlesex County, NJ, USA). Rabbit polyclonal antibodies against ACSL1 (cat: DF9605), phosphorylated AMPK (cat: AF3423, phosphorylation site: Thr172), AMPK (cat: AF6423), CPT1C (cat: DF12150), PCNA (cat: AF0239), Ki67 (cat: AF0198), E-cadherin (cat: R868), and vimentin (cat: I444) were purchased from Affinity Biosciences (Changzhou, China). Our lab acquired a rabbit polyclonal antibody against actin (cat: BS6007M) from Bioworld Technology, Inc. (St Louis Park, MN, USA). Takara Biotechnology Co., Ltd. (Beijing, China) provided the PrimeScript RT Reagent Kit and TRIzol®. The Cell-Light EdU Apollo567 In Vitro Kit was obtained from RiboBio Biotechnology Co., Ltd. (Guangzhou, China).

This research was carried out using specimens from 31 female patients (mean age: 55.45 $\pm$ 7.46 years) who were diagnosed with endometrial cancer following endometrial curettage between April 2019 and November 2019 at the Affiliated Hospital of Xuzhou Medical University, Xuzhou, China, as well as samples from 33 healthy female volunteers (mean age: 36.45 $\pm$ 5.57 years). Endometrial cancer patients were enrolled by the Department of Obstetrics & Gynecology, and healthy participants were selected by the Health Screening Center at the Affiliated Hospital of Xuzhou Medical College. Samples of venous blood were collected after an overnight fast. Serum was promptly separated by centrifugation and stored at –20 °C for subsequent analysis. Similarly, six pairs of cancer and paracancerous tissues were harvested from patients with EC in the Department of Obstetrics and Gynecology at Xuzhou Central Hospital. The female subjects in our study were not treated with systemic hormone therapy prior to sample collection. The research followed to the principles of the Declaration of Helsinki and was registered with the Chinese Clinical Trial Register on January 26, 2018, under Registration No. Chi-CTR-1800014658. Each participant provided their written agreement in advance of the investigation.

Female BALB/c mice (10–12 g) were obtained from Charles River Co., Ltd. (Beijing, China) and were housed under conditions of regulated temperature, humidity, and lighting. Ethical approval for all the animal experiments was obtained from the Animal Ethics Committee of Xuzhou Medical University, and the study was conducted in accordance with the principles outlined in the Declaration of Helsinki. All 24 mice that were used in the study were fed a normal diet. ACSL1-knockout Ishikawa cells and ACSL1-overexpressing Ishikawa cells were subcutaneously injected into mice to establish a xenograft tumor model. The mice were injected with 0.2 mL of a cell suspension (approximately 3 $\times$ 10${}^5$ cells). The mice were divided into four distinct groups: the ACSL1 knockdown negative control (SI-NC) group, ACSL1 knockdown (SI) group, ACSL1 overexpression negative control (OE-NC) group, and ACSL1 overexpression (OE) group. Each group included six mice. We measured the long side (L) and short side (W) of the tumor every four days for four weeks and calculated the tumor volume with the following formula: L $\times$ W${}^2$/2. Serum and tumor tissues were collected from the animals at the end of the experiment and either stored at –80 °C or fixed overnight in 10% neutral-buffered formalin. The paraffin-embedded tissues were used for subsequent histological and immunohistochemical analyses, as detailed below.

Two hundred microliters of each sample was added to a 1.5 mL centrifuge tube after all the samples had been carefully thawed on ice and vortexed to mix them. Subsequently, 200 µL of water was added, followed by the sequential addition of 240 µL of precooled methanol and 800 µL of MTBE. The solutions were agitated, subjected to 20 minutes of sonication in a chilled water bath, and subsequently incubated at room temperature for 30 minutes. The samples were then centrifuged at 14,000 $\times$g for 15 min at 10 °C. The upper organic phase of the samples was harvested, subjected to nitrogen drying, and stored at –80 °C. The resulting supernatant was then collected for subsequent liquid chromatograph-mass spectrometer (LC-MS) analysis.

A UHPLC Nexera LC-30A system for ultra-highperformance liquid chromatography (UHPLC) (Waters, Wilmslow, UK) was used for all chromatographic separations. For separation, an ACQUITY UPLC CSH C18 column (100 mm $\times$ 2.1 mm, 1.7 µm, Waters, Wilmslow, UK) was employed. The flow rate was set to 300 µL/min, and the column oven was maintained at a temperature of 45 °C. The mobile phase consisted of two components: (A) a 10 mM ammonium formate acetonitrile-water solution (acetonitrile:water = 6:4, v/v), and (B) a 10 mM ammonium formate acetonitrile-isopropanol solution (acetonitrile:isopropanol = 1:9, v/v). A gradient elution program was employed as follows: from 0 to 2 minutes, B was maintained at 30%; from 2 to 25 minutes, a linear increase from 30% to 100% B was performed; and from 25 to 35 minutes, B was maintained at 30%. Throughout the entire analysis, the samples were stored in an automatic sampler at a constant temperature of 10 °C.

After separation with UHPLC, samples were analyzed using a QExactive Plus mass spectrometer. Via electrospray ionization (ESI) and positive ion mode, QExactive Plus was used for detection. In positive ion mode, the capillary temperature was maintained at 350 °C, and the spray voltage was set to 3.0 kV. In negative ion mode, the capillary temperature was maintained at 350 °C, while the spray voltage was set to 2.5 kV. The positive MS1 scan ranges were 200 to 1800 and 250 to 1800 in negative mode.

The cell lines underwent authentication, which included assessments of morphology, antigen expression, growth characteristics, DNA profiles, and cytogenetic analyses, and these analyses were performed by the provider. Ishikawa cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Clark, NV, USA), 1% penicillin, and 1% streptomycin. The cells were maintained in a controlled environment at 37 °C with 95% humidity and 5% CO${}_2$. After stable transfection, the cells were exposed to 50 M etomoxir (ETX, Middlesex County, NJ, USA) for 48 h to conduct FAO inhibition experiments. ETX is an inhibitor of the CPT1C protein, which is a pivotal enzyme in the progression of fatty acid oxidation. All the cell lines tested negative for mycoplasma.

The lentiviral vector containing green fluorescent protein (GFP) was supplied by Genechem (Shanghai, China). The lentiviral vector system carrying the /textitGV gene consists of a series of GV lentiviral vectors, the pHelper 1.0 vector and the pHelper 2.0 vector. Small interfering RNAs (siRNAs) targeting the human ACSL1 gene were designed and provided by Shanghai GeneChem Co., Ltd., China. After screening with Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA) and cotransfection of a plasmid carrying human ACSL1 (NM_001995) cDNA into HEK293T cells in six-well plates, the optimal siRNA sequence (5${}^{\prime }$-CAGATAGATGACCTCTATT-3${}^{\prime }$) was cloned and inserted into the pGCL–GFP plasmid. This plasmid, which is an HIV-derived lentiviral vector, features an upstream U6 promoter and a downstream cytomegalovirus promoter–GFP cassette flanked by loxP sites. Lentivirus preparations were generated by Shanghai GeneChem Co., Ltd., China. The sequence of the resulting lentiviral vector carrying human ACSL1-specific shRNA was confirmed by polymerase chain reaction (PCR) and sequencing analysis. A negative control lentiviral vector carrying NS shRNA was constructed using a similar procedure (NS lentivirus, 5${}^{\prime }$-TTCTCCGAACGTGTCACGT-3${}^{\prime }$). To overexpress ACSL1, the full-length cDNA of human ACSL1 was amplified by subcloning PCR and cloned into the GV657 vector (Genechem, Shanghai, China). The constructed lentiviral vector was then packaged into 293T cells. Subsequently, the concentrated virus harvest was added to the cells and incubated for 72 hours. Ishikawa cells were infected with human ACSL1 lentivirus at an MOI of approximately 150, and control cells were infected with NS lentivirus. After 24 h of cell infection, the cells were incubated with 1 µg/mL puromycin for 3–5 days, and Western blotting was used to verify protein expression after ACSL1 was overexpressed or knocked down.

Cells were seeded in a 96-well plate at a density of 5000 cells per well. After 24 hours of incubation, 10 µL of CCK8 solution (Dojindo Laboratories, Kumamoto, Japan) per well was added to 96-well plates and incubated for 1 hour. The culture medium was replaced with FBS-free high-glucose DMEM in the dark. The cells were then incubated at room temperature for 37 °C, and the optical density(OD) values at 450 nm were measured.

Ishikawa cells were seeded in 96-well plates at a density of 4 $\times$ 10${}^3$ cells per well. The cells were then treated with the appropriate reagents for 24 hours. The cells were subjected to EdU treatment (RiboBio, Guangzhou, China) for an additional two hours prior to being harvested, and they were subsequently fixed with 4% paraformaldehyde. Then, cell staining was performed using Apollo®567 (C10310-1, RiboBio, China) and 4,6-diamidino-2-phenylindole (DAPI) in accordance with the manufacturer’s instructions. An imaging fluorescence microscope was used to view the staining results.

Ishikawa cells were plated in six-well plates and grown to 90% confluence. A scratch was generated in the cell monolayer using a sterile 10-L pipette tip, which led to the development of a wound. The progression of wound healing was recorded at a magnification of 200$\times$ at certain time intervals. The wound was examined and imaged at 0 and 24 hours under a microscope (OLYMPUS).

Ishikawa cells (1 $\times$ 10${}^5$ cells in 200 µL of serum-free DMEM) were reseeded in the upper chamber of a Boyden chamber insert (Corning Inc., Corning, NY, USA) that was precoated with 300 µg/mL Matrigel (BD Bioscience, San Jose, CA, USA), and 600 µL of media supplemented with 10% FBS was added to the lower chamber. The Ishikawa cells were incubated for 20 to 24 hours, and then, the cells that had passed through the filter were fixed using 4% paraformaldehyde and stained using 0.1% crystal violet from Solarbio Science & Technology Co., Ltd., Beijing, China. We used an inverted microscope (OLYMPUS) to view the migrated cells in various fields of view.

Each well of a 96-well microplate was seeded with 100 µL of a cell suspension, followed by the addition of 50 µL of mammalian cell lysis solution before the cells were plated. The microplate was subjected to agitation at 700 rpm for five minutes using an orbital shaker. ATP was stabilized, and the cells were lysed. After adding 50 µL of substrate solution to the wells, the microplate was agitated at 700 rpm for five minutes using an orbital shaker, and after ten minutes of adaptation to the dark, the brightness of the plate was measured. ATP levels were measured using the ATP LITE Assay Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s guidelines.

Cells from different groups were seeded at a density of 1 $\times$ 10${}^5$ cells per well in 6-well plates. Subsequently, after reaching a cell count of 1 million, each sample was lysed using 0.1 mL of lysis buffer. Following cell lysis, supernatants were collected by centrifugation at 12,000 $\times$g for 10 minutes. The samples were thoroughly mixed after adding the MDA detection working solution and then heated in a boiling bath for 15 minutes. The samples were equilibrated to ambient temperature in a water bath and then subsequently subjected to centrifugation at 1000 $\times$g for 10 minutes at room temperature. A 96-well plate was filled with 200 microliters of supernatant, and the optical density was measured at 532 nm using a microplate reader. We utilized a lipid peroxidation (MDA) detection assay kit (Beyotime, China) to analyze the MDA levels in accordance with the manufacturer’s instructions.

Ishikawa cells were cultured in XF96 plates and subjected to a 48-hour treatment with 50 µM etomoxir (ETX), which inhibits FAO. The original cell culture medium (in a pH 7.4 environment, the culture medium contained 1640 media with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine) was replaced with the test solution after the tissues had been incubated for 1 h at 37 °C without CO${}_2$. Then, each well was treated with 1.5 µM oligomycin, 1.5 µM FCCP, and 0.5 µM rotenone/antimycin A. The cell oxygen consumption rate was measured every 5 min. Cell energy metabolism was detected by an XF96 Analyzer (Agilent, Santa Clara, CA, USA).

Tumor specimens were preserved in 10% phosphate-buffered formalin, followed by paraffin embedding. Thin paraffin sections (2–3 µm) were subjected to staining, including periodic acid-Schiff (PAS) and Masson’s trichrome staining.

Paraffin-embedded xenograft tissue sections (4 µm) were deparaffinized and gradually rehydrated through a gradient of ethanol solutions. Antigen retrieval was conducted by boiling the samples in citrate buffer (0.01 M, pH 6.0) for 15 minutes. The tissue sections were treated for 10 minutes with 3% hydrogen peroxide and then blocked with goat serum for 30 minutes. Specific primary antibodies against ACSL1 (Affinity, 1:100), PCNA (Affinity, 1:100), and Ki67 (Affinity, 1:100, ready to use) were then applied to the sections. Immunohistochemical staining was conducted in accordance with the manufacturer’s guidelines utilizing the DAB method (GSK500710, Gene Tech, China).

In our experimental procedure, total tissue RNA was isolated with TRIzol® (Invitrogen), ensuring the highest quality RNA extraction. Subsequently, cDNA synthesis was performed utilizing the PrimeScript RT Reagent Kit from TaKaRa Biotechnology Co., Ltd. according to the manufacturer’s recommendations, ensuring the reliability of the process. To accurately quantify mRNA expression, real-time quantitative reverse transcriptase PCR analysis was performed using the LightCycler® 480 II system from Roche, Switzerland. This platform followed to established protocols, guaranteeing the robustness and reproducibility of our results. For the complete list of primer sequences that were used in our study, please refer to Table 1.

Total protein was extracted using ice-cold RIPA buffer, and insoluble fractions were subsequently removed from the samples. The protein concentration was quantified via bicinchoninic acid (BCA) assay. The main antibodies included antibodies against ACSL1 (Affinity, 1:1000), phosphorylated AMPK (Affinity, 1:1000), AMPK (Affinity, 1:1000), CPT1C (Affinity, 1:1000) and $\beta$-actin (Bioworld, 1:10,000).

We conducted quantitative analyses of all our experimental results using SPSS Statistics 16.0 software (SPSS Inc, Chicago, IL, USA). Experimental data are presented as the mean $\pm$ standard error of the mean (SEM). Intergroup comparisons were performed through one-way analysis of variance (ANOVA), and differences in individual parameters between two groups were assessed using Student’s t test. Statistical significance was determined at a threshold of p 0.05.

Considering the pivotal role of ACSL1 in modulating the biosynthesis of diverse acyl-CoAs, we sought to investigate whether ACSL1 is involved in endometrial tumorigenesis. Thus, we initially conducted immunohistochemical (IHC) staining of specimens from 6 endometrial cancer patients and their adjacent normal tissues. We scored the levels of ACSL1 expression based on the intensity of ACSL1 staining. The IHC scores for ACSL1 expression were significantly higher in endometrial cancer tumor tissues than in noncancerous tissues (Fig. 1A). After observing an increase in ACSL1 levels within tissues, we subsequently investigated whether its levels were increased in the serum of endometrial cancer patients. Serum ACSL1 levels serve as a potential circulating biomarker that indicates systemic changes that are associated with observed tissue alterations. This approach provides insights into the systemic implications of the observed tissue changes (Fig. 1B). In addition, Fig. 1C,D show that the protein and mRNA levels of ACSL1 were aberrantly upregulated in endometrial tissue from EC patients. The levels of PCNA and Ki67 (proliferation markers) in tumor tissues were significantly higher than those in adjacent normal tissues. In contrast to adjacent normal tissues, tumor tissues exhibited reduced E-cadherin and elevated vimentin expression, which indicated EMT (Fig. 1E). Therefore, we propose that ACSL1 is involved in the proliferation and migration of EC. Collectively, these findings indicate that increased ACSL1 expression is associated with endometrial cancer, suggesting that it has a potential oncogenic role.

Fig. 1.

ACSL1 expression in healthy individuals and endometrial cancer patients. (A) Representative immunohistochemistry (IHC) images and scatter plot of ACSL1 protein expression in endometrial cancer tissueand adjacent normal tissues (N group: n = 6, EC group: n = 16). IOD stands for “Integrated Optical Density”. In immunohistochemistry studies, IOD quantifies staining results, reflecting the total amount of stained substance. Scale bar: 20 µm. EC group represents endometrial cancer patients and their adjacent normal tissues represent N group. (B) Serum ACSL1 levels in healthy subjects and endometrial cancer patients (N group: n = 18, EC group: n = 18). (C) Representative Western blot and bar chart showing the quantification of ACSL1 expression in endometrial cancer tissues (EC) and para-cancerous tissues (N). (D) The mRNA expression of ACSL1 in endometrial tissues from endometrial cancer tissues (EC) and para-cancerous tissues (N). (E) Representative immunohistochemistry (IHC) images and scatter plot of Ki67, PCNA, vimentin, and E-cadherin in endometrial cancer tissueand adjacent normal tissues (N group: n = 6, EC group: n = 6). Scale bar: 20 µm. Data are presented as the mean $\pm$ standard error for the sample mean (SEM), n = 6, **p 0.01 vs. the N group.

Considering that ACSL1 is elevated in endometrial cancer, we wondered whether ACSL1 promotes endometrial cancer cell proliferation and migration, and we established ACSL1-knockdown (SI) and ACSL1-overexpressing (OE) Ishikawa cell lines by lentiviral infection. Western blotting confirmed high knockdown efficiency in IK cells, which were subsequently selected for further experiments (Fig. 2A).

Fig. 2.

The proliferation and migratory potential of EC cells were assessed. (A) Western blotting validated the of ACSL1 gene overexpression and knockout in subsequent experiments. (B,C) The proliferation of Ishikawa cells was assessed by EdU assays and CCK-8. Scale bar, 100 µm. (D) Wound-healing assay was used to assess Ishikawa cell migration. Scale bar: 200 µm. (E) The migration of abilities of Ishikawa cells was determined by migration assay Scale bar, 100 µm. The data are presented as the mean $\pm$ SEM with n = 3, *p 0.05, **p 0.01 compared to the SI-NC group. ##p 0.01, ###p 0.001 compared to the OE-NC group (SI-NC, ACSL1 knockdown negative control group; SI, ACSL1 knockdown group; OE-NC, ACSL1 overexpression negative control group; OE, ACSL1 overexpression group).

Cell proliferation was assessed via EdU assays and the CCK-8 method. The findings indicate that the upregulation of ACSL1 resulted in a higher fold increase in the percentage of EdU-positive cells, while ACSL1 knockdown resulted in a lower fraction of EdU-positive cells (Fig. 2B). Moreover, ACSL1 overexpression promoted Ishikawa cell proliferation, whereas ACSL1 knockdown inhibited cell proliferation (Fig. 2C). To delve deeper into the impact of ACSL1 on cell invasion, we conducted wound healing and Transwell assays. Ishikawa cell migration was significantly decreased in the ACSL1-overexpressing (SI) group in comparison to the control (SI-NC) group (Fig. 2D). The OE group exhibited increased cell invasion, whereas the SI group exhibited decreased cell invasion (Fig. 2E).

Previous studies have revealed that ACSL1 can enhance lipid metabolism, and it is noteworthy that the progression of cancer is intricately associated with alterations in lipid metabolism [28]. Therefore, to investigate whether ACSL1 affects the progression of EC through lipid metabolism, we generated a xenograft mouse model and then carried out lipidomic profiling using the serum of EC mice from the SI group and NC group (SI group: ACSL1 knockdown group; NC group: ACSL1 knockdown negative control group). In the xenograft model, EC cells were infected with ACSL1 silencing or negative control lentivirus before injection.

The volcano plot visually represents the overall fold change in lipid molecules between the SI and SI-NC groups, with magenta points denoting lipids that met the criteria for differential expression [fold change (FC) $>$1.5 or FC 0.67, p value 0.05] (Fig. 3A). Next, we performed hierarchical clustering based on the qualitative expression levels of significantly different lipids [variable importance for projection (VIP) $>$1, p value 0.05] across the sample groups (VIP serves as a metric for assessing the contribution of variables (features) in multivariate datasets to model interpretation and prediction. In multivariate datasets, such as those in metabolomics, features with VIP values exceeding 1 are considered to have a significant impact on the model. These features are thought to make notable contributions to model interpretation and prediction in the context of the dataset). Lipids clustered together share similar expression patterns, suggesting potential proximity in metabolic pathways. The heatmap illustrating the clustering of differential lipids between the SI and SI-NC groups portrays samples on the horizontal axis and differential lipid molecules on the vertical axis. Color intensity corresponds to expression levels, with darker shades indicating higher expression. Lipid molecules with similar expression patterns tend to cluster together. The results indicate a clear distinction between lipid molecules from the SI and SI-NC groups (Fig. 3B). In this study, a total of 28 lipid subclasses were detected, including a total of 869 lipid molecules across these classes (Supplementary Table 1). Notably, lipid molecules exhibiting significant differences and having VIP values greater than 1 are detailed in Supplementary Table 2 (lipid molecules with VIP $>$1 are considered to make a substantial contribution to model interpretation). Subsequently, quantitative analysis was conducted on lipid subclasses within each sample. Metabolic profile comparisons indicate that comparative analysis between the SI and SI-NC groups revealed differentially regulated lipid subclasses, as outlined in Supplementary Table 3. The results indicate significant downregulation of ACCA, Cer, DG, and PI in the SI group compared to the SI-NC group, while MG and PG were significantly upregulated (Supplementary Fig. 1A). Collectively, ACSL1 markedly altered the lipid profile, demonstrating that ACSL1 might cause dramatic lipid metabolism disorder.

Fig. 3.

ACSL1 overexpression is associated with lipid metabolism disorders in endometrial cancer. (A) Volcano plot analysis was performed to identify lipid species that exhibited significant changes in relative abundance in the xenograft mouse model. Each point on the plot represents a lipid molecule. The magenta area represents molecules that met the criteria for being differentially expressed [fold change $>$1.5 or 0.67, p value 0.05, where fold change equals the experimental group’s average protein expression level (SI) divided by the control group’s average protein expression level (SI-NC)]. (B) The clustered heatmap illustrates variations in serum lipid composition between the SI and NC groups. (C) The concentration of malondialdehyde (MDA) in IK cells, which correlated with varying levels of ACSL1 expression, was quantitatively measured to assess lipid oxidation levels. (D) ATP level. The data are presented as the mean $\pm$ SEM, with n = 6, * indicating significance at p 0.05,**p 0.01, compared to the NC group. ##p 0.01 compared to the OE-NC group.

Subsequently, we assessed the MDA and ATP levels. Malondialdehyde (MDA) is a common metabolic byproduct of lipid peroxidation and is frequently utilized as a quantitative marker for assessing the extent of lipid oxidation. MDA contents serve as an indicator for evaluating lipid metabolism and oxidative lipid levels. The results are shown in Fig. 3C,D. ACSL1 increased FAO and ATP production in EC cells. These findings strongly indicate that ACSL1 modulates EC cell energy metabolism by regulating lipid metabolism.

To elucidate the molecular mechanisms underlying the role of ACSL1 and the pivotal role of FAO in EC progression, we used EC (Ishikawa) cells, including control cells (OE-NC) and ACSL1-overexpressing cells (OE); we treated these cells with ETX (inhibits the CPT1C protein) to suppress FAO. The CPT1C protein is a key enzyme in FAO.

As shown in Fig. 4A–D, ACSL1 overexpression increased FAO, induced ATP production, and enhanced cell proliferation and migration. These effects were inhibited after the administration of ETX and restored with the upregulation of ACSL1. When ACSL1 was overexpressed, EC cells showed higher basal and maximum respiratory capacity (Supplementary Fig. 1), with increased intracellular ATP levels (Supplementary Fig. 1). Moreover, ACSL1 upregulation partially attenuated the ability of ETX to suppress FAO.

Fig. 4.

Increased ACSL1 expression promotes EC by regulating the fatty acid oxidation (FAO) metabolism pathway. (A) Ishikawa cell proliferation was assessed with CCK-8. Scale bar: 100 µm. (B) Ishikawa cell migration was assessed via wound healing assay. Scale bar: 100 µm. (C) MDA level. (D) OCR value of cells. (E) ATP level. (F) Representative Western blot and quantitative bar chart illustrating ACSL1, p-AMPK, and CPT1C protein expression in Ishikawa cells. (G) Representative Western blot and quantitative bar chart illustrating ACSL1, p-AMPK, and CPT1C protein expression in ACSL1-overexpressing IK cells after 48 hours of treatment with 50 µM etomoxir. Data are presented as the mean $\pm$ SEM (n = 3); *p 0.05, **p 0.01, relative to SI-NC. #p 0.05, ##p 0.01, compared to OE-NC.

Research has shown that ACSL1 can modulate AMPK activity [26] and its downstream target, CPT1C, subsequently influencing FAO [27, 29, 30]. Therefore, the protein expression of ACSL1, p-AMPK, AMPK and CPT1C was measured in this study. The findings indicated a significant upregulation of these proteins after ACSL1 overexpression (Fig. 4F). These results demonstrated that ACSL1 was aberrantly expressed in EC and could further promote the proliferation and migration of EC cells via the AMPK/CPT1C/ATP pathway.

After ETX treatment, the expression of ACSL1 and p-AMPK/AMPK was unaffected; however, CPT1C was significantly downregulated. Furthermore, the overexpression of ACSL1 reversed the inhibitory effect of ETX, which led to the upregulation of CPT1C, suggesting that ACSL1 was indeed associated with the FAO-induced progression of EC (Fig. 4G). Overall, these experiments showed that ACSL1 indeed induced FAO, effectively promoting EC growth and potentially stimulating metastasis.

To explore the association of ACSL1 with EC malignancy, ACSL1-knockdown and ACSL1-overexpressing xenograft model mice were established. As shown in Fig. 5A,C, ACSL1 dramatically increased the size and promoted the growth of the tumors. More information about the tumors is shown in Fig. 5B. According to the results of H&E staining of tumor tissues (Fig. 5D), compared with the cells of the other groups, the cells of the OE group displayed a denser composition with an intact envelope, while the dioscin-treated group exhibited a looser texture with an incomplete envelope. Additionally, the central necrotic region was expanded, accompanied by inflammatory cell infiltration.

Fig. 5.

ACSL1 promotes tumorigenicity in an EC xenograft model. (A) Representative images of mouse tumor tissue. (B) Tumor information (tumor volume: a $\times$ b${}^2$/2, a: long radius and b: short diameter). (C) Tumor growth curve. (D) Hematoxylin and eosin (H&E) staining of tumor tissues to assess their appearance and structure. Scale bar, 20 µm. Immunohistochemical staining for PCNA and Ki67 in mouse tumors (n = 6). Scale bar, 20 µm. (E) Representative Western blot and quantitative bar chart illustrating ACSL1, p-AMPK, and CPT1C protein expression in tumor tissues of the endometrial cancer (EC) mouse model. Data are expressed as the mean $\pm$ SEM (n = 6); *p 0.05, **p 0.01 compared to SI-NC, #p 0.05, ##p 0.01, ####p 0.0001,compared to OE-NC.

Next, immunohistochemical staining experiments were conducted on mouse tumor tissues. We scored the expression levels of PCNA and Ki67 based on the staining intensity. In the ACSL1-overexpressing tumor tissues, IHC scores for PCNA and Ki67 were significantly higher than those of the other groups (Fig. 5D). Subsequently, we examined the expression of ACSL1, p-AMPK and CPT1C in the xenografts by Western blotting. The findings demonstrated that the xenografts derived from ACSL1-knockdown cells exhibited significantly decreased expression of ACSL1, p-AMPK and CPT1C. Conversely, the OE group reversed this phenomenon (Fig. 5E). Overall, we propose that ACSL1 could facilitate the malignant phenotype of EC.