HEC-1A cell line (cat. no. CL-0099), 293T cell line (cat. no. CL-0005), and McCoy's 5A medium (cat. no. PM150710) were purchased from Wuhan Punosai Life Science and Technology Co., Ltd. Fetal bovine serum (FBS) was procured from Dalian Meilun Biotechnology Co., Ltd. McCoy's 5A Medium and DMEM High Glucose Medium (Gibco; Thermo Fisher Scientific, Inc.), both containing 10% FBS, were used to culture HEC-1A cells and 293T cells, respectively. Cryopreserved cells were removed from the −80°C freezer, rapidly thawed, and then cultured in a 37°C incubator with regular cell passaging.

The study was approved (approval no. 2024-E211-01) by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Nanning, China). Both EC tissues and corresponding paracancerous endometrial tissues from a total of 45 patients diagnosed with primary EC were retrospectively collected. The age of the patients ranged from 31 to 78 years. These patients had undergone initial surgical treatment at our hospital between January 2021 and February 2022. The tissue samples were preserved by the Department of Pathology at our hospital. The inclusion criteria were as follows: i) No prior treatment related to the disease; ii) absence of concomitant malignant tumors; and iii) absence of severe systemic diseases. Paracancerous endothelial tissues were excised at a distance of 2 cm or more from the cancerous tissues. The present study was conducted in accordance with the guidelines of the Declaration of Helsinki and the ethics committee of the hospital. Additionally, all study participants provided their written informed consent prior to recruitment.

Tissues were fixed in 4% paraformaldehyde at 4°C for 24 h. After fixation, tissue samples were prepared into pathological sections through paraffin embedding and slicing. The sections were cut at a thickness of 4 µm. The slides were baked at 60°C for 2 h. Deparaffinization and rehydration were performed using xylene and a graded ethanol series. Antigen retrieval was carried out under high pressure using citrate buffer (cat. no. MVS-0066; Fuzhou Maixin Biotech Co., Ltd.). Subsequently, an SP kit (cat. no. SP-0022, BIOSS) was used according to the manufacturer's instructions: Endogenous peroxidase activity was blocked using the 3% hydrogen peroxide solution provided in the SP kit. After washing, the sections were incubated with the normal goat serum blocking solution from the same kit at room temperature for 15–20 min. The sections were sequentially incubated with a specific STAT3 antibody (1:200; cat. no. bs-1141R; BIOSS) at 4°C overnight, followed by incubation with biotinylated secondary antibody working solution (ready-to-use; from the SP-0022 kit) at room temperature for 15–20 min, and then with horseradish peroxidase-conjugated streptavidin working solution (ready-to-use; from the SP-0022 kit) at room temperature for 15–20 min. The sections were then stained with 3,3′-diaminobenzidine (DAB; cat. no. ZLI-9018, ZSGB-BIO) at room temperature for 2 min, counterstained with hematoxylin (cat. no. S0142; BIOSS) at room temperature for 1 min, differentiated in hydrochloric alcohol, dehydrated, and finally mounted with neutral balsam. Images of five random fields (magnification, ×200) were captured under an inverted optical microscope. The staining results were quantitatively analyzed using ImageJ software (version 1.51j8; National Institutes of Health). Grayscale signal values were converted into optical density (OD) values. The integrated OD (IOD) divided by the area of target protein distribution (IOD/Area) yielded the average OD (AOD).

Lentiviruses for STAT3 overexpression were purchased from Sangon (Shanghai) Biotech Co., Ltd. Lentiviruses for miR-26a/b-5p overexpression, YKL-40 overexpression, and STAT3 knockdown were obtained from GeneChem Co., Ltd. The lentiviruses used in the present study were purchased as ready-to-use preparations. The shRNA sequence for STAT3 knockdown was designed based on a validated Qiagen sequence: sh-STAT3: 5′-CAGCCTCTCTGCAGAATTCAA-3′. The negative control sh-NC was designed using the sequence of Qiagen AllStars Negative Control: 5′-CAGGGTATCGACGATTACAAA-3′ (22). Lentiviral transduction was performed according to the manufacturer's instructions. The multiplicity of infection (MOI) used was 20, and HitransG A (GeneChem Co., Ltd.) was added to enhance transduction efficiency according to the manufacturer's protocol. HEC-1A cells were seeded into 24-well plates, and transduction was carried out the following day when the cell confluence reached ~30%. Subsequently, 16 h after transduction, the transduction medium was replaced with complete culture medium for further incubation. At 48 h post-transduction, antibiotic selection was initiated using complete culture medium containing either 600 µg/ml geneticin (G418) (cat. no. MA0321; Dalian Meilun Biotechnology Co., Ltd.) for miR-26a/b-5p and YKL-40 overexpression constructs or 2.5 µg/ml puromycin (cat. no. P8230; Solarbio Life Sciences) for STAT3 overexpression and knockdown constructs.

Total miRNA or mRNA was extracted from HEC-1A cells in healthy growth conditions using RNAiso Plus (cat. no. 9108, Takara Bio, Inc.). RNA was separated by adding chloroform, precipitated by adding isopropanol, washed with 75% ethanol, and finally dissolved in an appropriate volume of RNase-free water. miRNA was reverse transcribed using a tailing method (Mir-X^™ miRNA First Strand Synthesis Kit; cat. no. 638315; Takara Bio, Inc.). The resulting cDNA was diluted 10-fold with RNase-free water and used for subsequent PCR amplification. For mRNA, genomic DNA removal and reverse transcription were performed using the PrimeScript^™ RT Reagent Kit (cat. no. RR047; Takara Bio, Inc.). All cDNA amplifications were performed using TB Green Premix Ex Taq II (cat. no. RR820A; Takara Bio, Inc.) on an ABI 7500 Fast qPCR system under the following reaction conditions: Pre-denaturation at 95°C for 30 sec (1 cycle); denaturation at 95°C for 5 sec, annealing and extension at 60°C for 34 sec (40 cycles). The relative expression levels of target genes were calculated using the 2^−ΔΔCq method (23). The primer sequences are listed in Table I. The U6 primer was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primers for GAPDH, miR-26a-5p, miR-26b-5p, STAT3, and YKL-40 were designed and synthesized by Takara Biotechnology (Beijing) Co., Ltd.

Briefly, 5,000 HEC-1A cells per well were inoculated into a 96-well plate, with the experiments conducted in triplicate or more for each group. A blank control group was established. Cell Counting Kit-8 (CCK-8) reagent (cat. no. MA0218-10; Dalian Meilun Biotechnology Co., Ltd.) was added at 0, 24, 48, 72, and 96 h after inoculation and the wells were incubated at 37°C for 1 h (24). The OD was recorded at 450 nm using an enzyme-labeler and the cell proliferation curve was generated using the measurements.

The prepared cell suspension was incubated in a 6-well plate with a concentration of 2.5×10⁶ HEC-1A cells per well. Before inoculation, the bottom surface of the plate was marked with horizontal lines. Cells were serum-starved for 8 h prior to scratching. When the cell confluence reached over 90%, a scratch was created using a 200-µl pipette tip. Subsequently, the scraped cells were aspirated and 2 ml of serum-free medium was added. Images of the cells were captured immediately and at 24-h intervals using an inverted light microscope (Leica Microsystems GmbH), with the wound healing rate at 48 h calculated (25). Wound healing was quantified by measuring the wound area using ImageJ software (version 1.51j8). The wound closure ratio was calculated as (area at 0 h-area at 48 h)/area at 0 h.

In the migration assay, 600 µl of McCoy's 5A medium supplemented with 20% FBS was added to each well of a 24-well plate. A small chamber with an 8-µm pore size (Corning, Inc.) containing 5×10⁴ cells in 100 µl of serum-free McCoy's 5A medium was placed into each well. The cells were incubated at 37°C for 48 h in a CO₂ incubator. After 48 h, the non-migrated cells on the upper surface of the membrane were discarded and those that migrated were fixed using 4% paraformaldehyde (cat. no. P1110; Solarbio Life Sciences) at room temperature for 15 min and stained using 0.1% crystal violet at room temperature for 10 min. The cells were then observed and counted using an inverted light microscope equipped with a 10X objective lens. A total of five random fields per well were counted. The invasion assay followed a similar procedure, except for the addition of diluted Matrigel (cat. no. 356234; Corning, Inc.) before the cells were seeded (25). Matrigel was diluted 1:16 in serum-free McCoy's 5A medium, and 100 µl of the diluted Matrigel was added to the upper chamber and incubated at 37°C overnight. The Matrigel simulated the extracellular matrix, allowing for the evaluation of the invasive potential of the cells. ImageJ software (version 1.51j8) was used for analysis.

RIPA lysis buffer (cat. no. PC101; EpiZyme) was used to lyse the HEC-1A cells. Protein concentration was determined using a BCA kit. The cell lysate was collected and mixed with loading buffer (cat. no. 1610747; Bio-Rad Laboratories, Inc.), followed by incubation at 100°C for 10 min. Equal amounts of protein (50 µg per lane) were loaded onto a 10% SDS-PAGE gel. SDS-PAGE was performed at 150 V for 60 min, and the proteins were then transferred onto a PVDF membrane at 200 mA for 1 h (26). The membrane was blocked with a protein-free rapid blocking buffer (cat. no. PS108; EpiZyme) at room temperature for 15 min, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies used were: STAT3 (1:3,000; cat. no. 9139S; Cell Signaling Technology, Inc.), YKL-40 (1:1,000; cat. no. PA5-43746; Invitrogen; Thermo Fisher Scientific, Inc.), β-tubulin (1:10,000; cat. no. 66240-1-Ig; Proteintech Group, Inc.), β-actin (1:10,000; cat. no. MA5-15739; Invitrogen; Thermo Fisher Scientific, Inc.), and GAPDH (1:5,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.). Subsequently, the membranes were incubated with corresponding species-specific fluorescent goat anti-rabbit IgG (H+L) secondary antibodies, DyLight^™ 800 4X PEG (1:10,000; cat. no. SA5-35571, Invitrogen; Thermo Fisher Scientific, Inc.) in the dark at room temperature for 45 min. Finally, PVDF membranes were scanned using an Odyssey infrared imaging system. ImageJ software (version 1.51j8) was used to analyze the band intensity of the collected band images, and normalization was performed based on the ratio of the target protein to the internal reference protein.

The potential binding sites between miR-26a-5p/26b-5p and the STAT3 3′-UTR were predicted using RNAhybrid (http://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). The 3′-untranslated region (3′-UTR) of STAT3 containing the miR-26a/b-5p binding site was synthesized and inserted into the pmirGLO luciferase vector by Genepharma Co., Ltd. to construct wild-type (STAT3-WT) and mutant-type (STAT3-MUT) STAT3 reporter plasmids [cat. no. C09005; Genomeditech (Shanghai) Co., Ltd.] Corresponding mimics (miR-26a-5p, miR-26b-5p, and miR-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′) were also synthesized (cat. no. B02001; Genepharma Co., Ltd.) 293T cells were prepared as single-cell suspensions and seeded into 12-well plates at a density of 5×105 cells per well, followed by incubation for 24 h. A mixture of reporter vector and mimics was pre-prepared. Subsequently, Lipofectamine 2000 transfection reagent (cat. no. G04004; Genepharma Co., Ltd.), pre-diluted in serum-free medium, was added and mixed gently, and the transfections complexes were allowed to stand for 20 min. The transfection complexes were then added to the cells cultured in 12-well plates and incubated for 6 h. After 6 h, the transfection mediume was replaced with complete culture medium, and the cells were further cultured for 48 h. Cells were processed using a dual luciferase reporter assay kit (cat. no. G06001; GenePharma Co., Ltd.). First, PLB lysis buffer (part of the luciferase assay kit) was added, and the resulting cell lysate was transferred into wells of a microplate reader plate. Subsequently, Firefly and Renilla luciferase reaction reagents were added sequentially. Luminescence values were measured using a BioTek multimode microplate reader (Agilent Technologies, Inc.) equipped with Gen5 software. Finally, Firefly luciferase activity was normalized to Renilla luciferase activity (Firefly/Renilla ratio). The aforementioned steps were performed in accordance with the manufacturer's instructions.

Total protein was extracted through lysis of HEC-1A cells using NP-40 cell lysis buffer (cat. no. P0013F; Beyotime Institute of Biotechnology). The lysate was centrifuged at 12,000 × g for 12 min at 4°C to remove debris. A total of 35 µl of Protein A/G Magnetic Beads (cat. no. HY-K0202; MedChemExpress) were collected into EP tubes and washed twice with PBST, then mixed with STAT3 antibody (12 µg; from a 30-µg/ml stock; cat. no. 9139S; Cell Signaling Technology, Inc.) or IgG antibody (12 µg; from a 30-µg/ml stock; mouse; cat. no. B900620; Proteintech Group, Inc.) in a total volume of 400 µl. The mixture was incubated at 25°C with gentle rotation for 30 min to allow antibody-bead coupling. The beads were retained and washed with PBST to remove unbound antibodies, and then 400 µl of the extracted total protein was added and mixed by rotation at 4°C for 2 h. The beads were then retained and unbound protein was thoroughly washed. Finally, the magnetic beads were eluted to obtain protein samples and subjected to western blot analysis using the primary antibodies at the following concentrations: STAT3 (Cell Signaling Technology, Inc.) and YKL-40 (1:1,000; rabbit; cat. no. 12036-1-AP; Proteintech Group, Inc.) (27).

The proteins were first prepared using Discovery Studio software 4.5 (https://www.3ds.com/products/biovia/discovery-studio). The processed protein structures were then visualized using PyMOL 2.5 (https://pymol.org). The 3D structures of STAT3 and YKL-40 were obtained from UniProt (https://www.uniprot.org) and subsequently imported into Discovery Studio for protein-protein docking analysis, which was performed using the ZDOCK module.

The expression levels of YKL-40 in EC tumor samples and non-tumor samples were analyzed using the GEPIA2 database (http://gepia2.cancer-pku.cn/). Protein-protein interactions between STAT3 and CHI3L1 were predicted using the STRING database (https://string-db.org/).

Results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 23.0 software (IBM Corp.). Data with a normal distribution were compared using unpaired Student's t-test, whereas one-way ANOVA was used to compare three or more groups, followed by Tukey's HSD post hoc test (equal variances) or Tamhane's T2 test (unequal variances). Non-normally distributed data were analyzed using Mann-Whitney U test (for two groups) or Kruskal-Wallis H test with Dunn's post hoc test (for three or more groups). All experiments were conducted in triplicate. Notably, P<0.05 was considered to indicate a statistically significant difference. All the diagrams were drawn using Adobe Illustrator and GraphPad Prism 10 (Dotmatics).

The overall experimental design and workflow are shown in Fig. 1.

The protein STAT3 has been identified as a proto-oncogene (28), and to elucidate its role in EC, IHC staining for STAT3 was performed on EC tissues collected from 45 patients and on paracancerous tissues from 40 patients, as shown in Fig. 2A. All representative images are shown in the Fig. S1. Notably, five paracancerous tissue samples were not included in the analysis because they were located less than 2 cm from the cancerous tissues. Before analysis, statistical tests were conducted to confirm that there was no difference in age among patients from the cancerous and paracancerous tissue groups. The staining results were quantified and statistically analyzed. As shown in Fig. 2B, STAT3 expression levels were significantly higher in EC tissues than in adjacent normal tissues. Various clinicopathological parameters of these 45 patients were collected and statistically analyzed, with the analysis indicating that the STAT3 level was higher in cancer tissues characterized by lower differentiation rates and aggressive infiltration of the muscularis propria; additionally, a high level of STAT3 was associated with the formation of pulvinar cancer thrombus in patients (Table II). Notably, STAT3 was upregulated in EC and was associated with malignant progression of EC. While evaluating STAT3 expression in tumor cells, it was noticed that the expression of total STAT3 in tumor-infiltrating lymphocytes (TILs) was heterogeneous; that is, scattered STAT3-positive lymphocytes were observed in a few samples, whereas most samples showed negative TILs, which may be related to the immunosuppressive tumor microenvironment (29).

HEC-1A cell models overexpressing and silencing STAT3 were constructed, and the levels of STAT3 after silencing or overexpression were confirmed using WB (Fig. 3A and B). The CCK-8 proliferation and scratch assays demonstrated that overexpression of STAT3 promoted proliferation and healing of HEC-1A cells (Fig. 3C and E), whereas silencing of STAT3 achieved the opposite results (Fig. 3D and F). Transwell experiments showed that overexpressed STAT3 levels markedly enhanced cell migration and invasion (Fig. 3G and H), whereas its downregulation decreased the number of migrated and invasive cells (Fig. 3I and J). Therefore, it was concluded that STAT3 promotes EC cell progression in vitro.

Research has identified miRNAs as factors of interest in tumor suppression (30). The predicted binding sites between STAT3 and miR-26a/b-5p are shown in Fig. 4A. Subsequently, dual luciferase tests were performed to verify these interactions. miR-26a/b-5p mimics and STAT3-WT or STAT3-MUT vectors were transfected into 293T cells. Notably, the incorporation of miR-26a/b-5p suppressed firefly luciferase activity following STAT3-WT vector transfection (Fig. 4B). Moreover, STAT3 mRNA and protein expression were decreased following miR-26a/b-5p overexpression (Fig. 4C and D). These findings suggest that miR-26a/b-5p targeting of STAT3 suppresses STAT3 transcription.

The enrichment of miR-26a/b-5p was promoted in HEC-1A cells to assess their effects on the cellular characteristics of these cells. Transduction efficiency was quantified using RT-qPCR, and the results revealed that miR-26a/b-5p was successfully enriched in HEC-1A cells, with more than a 20-fold increase in its expression levels (Fig. 5A). Cell scratch, CCK-8 cell proliferation, and Transwell assays were conducted. While no significant effect on cell proliferation was observed (Fig. 5B), miR-26a/b-5p markedly inhibited the wound healing of HEC-1A cells (Fig. 5C). Concurrently, the number of migrated and invasive cells was markedly reduced (Fig. 5D and E). These observations indicated that miR-26a/b-5p inhibits the migratory and invasive behavior of EC cells in vitro.

The association between miR-26a/b-5p and STAT3 suggested that miR-26a/b-5p may influence cellular behavior by regulating the expression of STAT3. To verify this, changes in the biological properties of cells were observed by overexpressing STAT3 in the miR-26a/b-5p-treated cell group. It was found that STAT3 was successfully enriched in miR-26a-5p + pc-STAT3 and miR-26b-5p + pc-STAT3 group cells, as detected using WB (Fig. 6A). CCK-8 assays showed that both miR-26a-5p and miR-26b-5p significantly suppressed cell proliferation compared with wild-type cells (P<0.05, Fig. 6B and C). STAT3 reversed the anti-proliferative effect of miR-26b-5p (Fig. 6C). However, STAT3 did not significantly reverse the effect of miR-26a-5p (Fig. 6B). By contrast, cell scratch and Transwell assays revealed that the introduction of STAT3 strongly abolished the inhibitory effects of miR-26a/b-5p on cell healing, migration, and invasion (Fig. 6D-F). These results suggest that STAT3 is a critical component in the regulatory pathway of miR-26a/b-5p.

As a potential diagnostic marker for EC, YKL-40 was analyzed through the GEPIA2 database, and the results revealed higher levels of YKL-40 in EC tumor samples compared with those in non-tumor samples (Fig. 7A). It was revealed in our previous study that preoperative serum YKL-40 levels were elevated in patients with EC relative to patients with leiomyoma and healthy women (31). To verify whether STAT3 regulates YKL-40 expression in EC, further analysis was conducted. As shown in Fig. 7B, the STRING database analysis suggested a potential interaction between STAT3 and CHI3L1, supported by multiple lines of evidence. To further investigate this interaction at the structural level, protein-protein docking analysis was performed, and the predicted binding mode is shown in Fig. 7C. Subsequently, Co-IP experiments using a STAT3 antibody to precipitate STAT3 from total proteins suggested that YKL-40 protein was detected in the immunoprecipated complex, indicating an interaction between YKL-40 and STAT3 (Fig. 7D). Further analysis revealed that mRNA and protein expression of YKL-40 was promoted in HEC-1A cells overexpressing STAT3 (Fig. 7E and F). By contrast, overexpression of miR-26a/b-5p resulted in a decrease of YKL-40 mRNA and protein levels (Fig. 7G and H). These findings indicated that STAT3 binds to YKL-40 protein in EC cells and promotes YKL-40 expression, whereas miR-26a/b-5p, a negative regulator of STAT3, inhibits YKL-40 expression.

To verify whether the pro-carcinogenic effect of STAT3 was dependent on YKL-40, YKL-40 was upregulated in the sh-STAT3 group of cells. Prior to this, it was validated that YKL-40 could be successfully upregulated by overexpression in wild-type HEC-1A cells, as confirmed by WB (Fig. S2). Subsequently, the changes in the levels of YKL-40 and STAT3 proteins in each group were verified using WB (Fig. 8A and B). It was found that upregulated YKL-40 did not promote STAT3 protein expression. By contrast, STAT3 knockdown was observed to decrease the migratory potential, invasiveness, proliferation, and wound healing abilities of the cells. Based on these observations, YKL-40 levels were upregulated in the cells, and all these abilities were restored (Fig. 8C-F). These findings suggest that the pro-carcinogenic impact of STAT3 is significantly mediated via YKL-40.

The role of the miR-26a/b-5p-STAT3-YKL-40 axis in EC progression was investigated. As shown in the diagram of Fig. 9, downregulation of miR-26a/b-5p led to STAT3 activation, which in turn promoted malignant progression through its interaction with YKL-40.