Enhancement of circular RNA 0002577 in serum exosomes in patients with endometrial cancer accelerates disease progression via general transcription factor II-I repeat domain-containing 1 (GTF2IRD1)

Abstract

Objective

Uterine corpus endometrial carcinoma (UCEC) is a common gynecologic malignancy with poor prognosis in advanced stages. Circular RNA (circRNA) and exosomes have been documented as significant contributors to the advancement of tumor cells, but the specific regulatory mechanisms between them is unclear. Therefore, our study attempts to explore the mechanism between them.

Methods

Firstly, we isolated and identified exosomes, and then validated their role in UCEC progression by experiments in vivo and in vitro. Secondly, a human competing endogenous RNA (ceRNA) array was used to identify the circRNA with the most significant differences in expression from serum of UCEC patient, and validated its role in UCEC progression by experiments in vitro. Then, we find the target gene of this circRNA by RNA sequencing, and further clarify the correlation between those and their role in tumor cell progression through experiments in vitro.

Results

Serum exosomes in patients with UCEC can promote the progression of UCEC. The human ceRNA array identified that circRNA 0002577 (circ_0002577) was up-regulated and was the most significantly altered circRNA. Moreover, the up-regulated circ_0002577 in exosomes derived from UCEC patients promote proliferation and migration of UCEC. Based on RNA sequencing results, general transcription factor II-I repeat domain-containing 1 (GTF2IRD1) gene was identified as being highly correlated with circ_0002577. Additionally, a positive correlation between circ_0002577 and GTF2IRD1 was confirmed by experiments in vitro.

Conclusion

Exosomes promote UCEC progression through circ_0002577 mediated regulation of GTF2IRD1, highlighting the potential therapeutic targets in treatment for UCEC.

Synopsis

Exosome mediate uterine corpus endometrial carcinoma (UCEC) progression via circular RNA (circRNA) regulation. CircRNA 0002577 is upregulated in patient-derived exosomes. GTF2IRD1 is a therapeutic target in UCEC treatment.

INTRODUCTION

Uterine corpus endometrial carcinoma (UCEC), originating from the endometrium, is the most prevalent gynecologic malignancy globally [1]. UCEC is marked by the unrestrained growth of endometrial cells, and its occurrence is on the rise, with around 417,000 new cases and 97,000 deaths in 2020 [2]. Advances in molecular biology have led to a deeper understanding in pathogenesis of UCEC, which is influenced by genetic mutations, hormonal imbalances, and environmental factors. Among which, the genetic alterations are noteworthy, including mutations in the PTEN, PIK3CA and TP53 genes, which are key drivers of tumorigenesis. In addition, alterations in estrogen and progesterone signaling pathways contribute to disease progression [3,4].

Although some progress has been made, the clinical management of UCEC remains challenging, especially in advanced UCEC. In addition, the primary treatment for localized UCEC involves surgical intervention, typically total abdominal hysterectomy with bilateral salpingo-oophorectomy, which requires removal of the fallopian tubes, ovaries, and uterus [5]. Moreover, the survival rate for advanced UCEC remains unsatisfactory, patients with stage III–IV disease typically have five-year survival rates of 20%–30% [6]. The probable reasons for this phenomenon are that the major challenges in UCEC treatment include the heterogeneity of the disease, therapeutic resistance, and there is a deficiency of operational biomarkers for primary detection and personalized treatment [7]. So, it is vital to investigate the molecular mechanisms of UCEC and improve ground-breaking therapeutic approaches to enhance treatment efficacy and patient survival.

Exosomes are small extracellular vesicle, typically between 30 and 150 nm in diameter, is a pivotal mediator for intercellular communication. These vesicles are produced by many cell types, and are involved in numerous biological processes, particularly in the field of cancer [8,9,10]. In UCEC, tumor-derived exosomes have been identified as the important contributors to the metastasis and progression of tumor. These exosomes encompass a wide range of biomolecules, which can modify the tumor microenvironment and affect the behavior of recipient cells [11]. Specifically, UCEC-derived exosomes promot tumor growth [12,13], angiogenesis and stimulate cell proliferation [14].

Thus, understanding the intricate mechanisms by which exosome influence UCEC progression is crucial for developing targeted therapeutic approaches and identification of innovative biomarkers for primary recognition and therapeutic effectiveness.

Non-coding RNAs are important regulators of gene expression and various biological processes though they do not encode proteins [15,16]. Among them, circular RNAs (circRNAs) have garnered significant interest due to their covalently closed loop configurations, which provide protection against degradation by exonucleases and enable them to play a regulatory molecular role within cells [17]. CircRNAs are formed mainly by reverse splicing, where the downstream splicing donor site links to the upstream splicing acceptor site, forming circRNAs molecules [18]. These molecules have been reported to involve in diverse biological functions, including interacting with RNA-binding proteins, regulating transcription, serving as microRNA (miRNA) sponges [19,20]. In particular, circRNAs have been identified as having dual roles in cancer, either enhancing or suppressing tumor progression based on the specific circRNA and the cancer type [20,21]. For example, circRNAs have been reported to modulate key signaling pathways and gene expression profiles for UCEC [22,23]. Due to the wide diversity of the identified circRNAs, it is necessary to elucidate their specific roles in UCEC to figure out the molecular mechanisms that propel this malignancy and seek out potential therapeutic targets.

As it is mentioned above, though exosome and circRNA have been demonstrated to significantly impact tumor cell progression, the specific regulatory mechanisms between those is unclear. Thus, our study attempts to explore the molecular mechanism between them by experiments.

MATERIALS AND METHODS

1. Cell culture

Human endometrial carcinoma cell lines, Ishikawa and HEC-1-A, were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Waltham, MA, USA) and McCoy’s 5A medium (Gibco) media, respectively. Both media were supplemented with 10% fetal bovine serum (FBS; Vazyme, Nanjing, China) and 1% penicillin-streptomycin (Servicebio, Wuhan, China). The culture medium was refreshed every 2–3 days, and cells were passaged upon reaching approximately 80% confluence. Detachment of the cells was achieved using trypsin-ethylenediaminetetraacetic acid (Gibco). All cell cultures were incubated in a humidified atmosphere at 37°C with 5% CO₂.

2. Lentiviral transduction

All the cell cultures were placed in an environment with 37°C and 5% CO₂, under humidified conditions for incubation. Stable over-expression of circRNA 0002577 (circ_0002577) and stable knockdown of general transcription factor II-I repeat domain-containing 1 (GTF2IRD1) were established by lentiviral transduction. Cells were infected with lentiviral vectors carrying the respective constructs along with a puromycin resistance gene. After lentiviral transduction, puromycin (from Sigma-Aldrich, St. Louis, MO, USA) was employed to select the cells that had been successfully infected. Subsequently, single clones were isolated. The lentiviral vectors were produced by Jinsirui Biological Science and Technology Co., LTD (Nanjing, China).

3. Clinical sample collection

Blood and tumor samples were collected from 10 patients diagnosed with UCEC at Wuhan Children’s Hospital in China between 2023 and 2024. All patients provided written informed consent for the use of their samples in this study. The study was conducted in accordance with the Declaration of Helsinki, and ethical approval was obtained from the Medical Ethics Committee of Wuhan Children’s Hospital (approval number 2024R045). Ethical approval was granted from May 6, 2024, to May 5, 2025. Specific patient information is provided in Table S1.

4. Exosome extraction

Exosomes were obtained from patient serum through differential ultracentrifugation. Collection of 10 mL of whole blood occurred, followed by clotting and centrifugation at 1,200 ×g for 10 minutes at 4°C. Dilution of the resulting serum with an equivalent volume of phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) was performed, followed by filtration using a 0.22 μm filter. Sequential centrifugation of the serum involved an initial step at 300 ×g for 10 minutes at 4°C to eliminate cells, a subsequent step at 2,000 ×g for 10 minutes at 4°C to remove larger vesicles, and a final step at 10,000 ×g for 60 minutes at 4°C to clarify the sample. Ultracentrifugation of the supernatant was carried out at 100,000 ×g for 90 minutes at 4°C with a Beckman Coulter Optima L-100K Ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA). Resuspension of the resulting pellet in cold PBS took place, followed by repetition of the ultracentrifugation process. Storage of the final exosome pellet occurred at −80°C for subsequent analysis. Execution of all procedures adhered to sterile conditions.

5. Transmission electron microscopy (TEM) assay

Preparation of exosome samples for TEM imaging involved negative staining. Application of exosome samples onto 200-mesh copper grids occurred, followed by incubation with 1% phosphotungstic acid (Aladdin, Shanghai, China) for 1 minute. Subsequent washing of the grids with PBS was conducted twice, with each wash lasting 1 minute. Air drying of the samples ensued. Imaging via TEM was carried out using a Hitachi HA7100 transmission electron microscope, with an accelerating voltage set at 80 kV.

6. Flow cytometry

Analysis via flow cytometry occurred using a Sony ID7000 flow cytometer (Sony, Tokyo, Japan). Utilization of fluorochrome-conjugated antibodies targeting exosome markers, namely CD63 (catalog No: 557288; BD, Franklin Lakes, NJ, USA) and CD81 (catalog No: 551108; BD), was implemented. Incubation of samples with these antibodies followed manufacturer instructions, succeeded by washing to eliminate unbound antibodies. Assessment of exosome marker expression levels ensued through flow cytometry. Nanoparticle flow cytometry was conducted with a NanoFCM flow nanoanalyzer (NanoFCM, Xiamen, China). Calibration of particle size relied on S23M-SEV (NanoFCM) as the reference standard, while concentration calibration utilized the Quality Control Nanospheres Series (NanoFCM). Direct loading of exosome samples into the system facilitated measurement of particle size and concentration.

7. Sequencing and microarray analysis

Storage of collected samples occurred at −80°C until further processing. The competing endogenous RNA (ceRNA) expression profiling was conducted using the Agilent-078298 human ceRNA array. The microarray experiments were outsourced to Beijing Qingke Biotechnology Co., Ltd. (Beijing, China). Raw data were extracted using Agilent Feature Extraction software. Quality control involved filtering out probes with low intensity or high background signals to ensure data reliability. Normalization was performed using the quantile normalization method to adjust for technical variations and ensure comparability across samples. Differential expression analysis was carried out using the limma package in R software (version 4.1.0; R Foundation for Statistical Computing, Vienna, Austria). Significantly differentially expressed ceRNAs were identified based on an adjusted p-value <0.05 and an absolute log2 fold change (|logFC|) >1. The RNA sequencing was performed to profile messenger RNA (mRNA) expression. Total RNA was extracted, and sequencing libraries were prepared following standard protocols. Sequencing was carried out on an Illumina HiSeq platform by Beijing Qingke Biotechnology Co., Ltd. Raw reads were subjected to quality control using FastQC to assess sequencing quality, and adapter sequences along with low-quality bases were trimmed using Trimmomatic. The cleaned reads were aligned to the human reference genome (hg38) using HISAT2. Gene expression levels were quantified using HTSeq to obtain read counts, and differential expression analysis was conducted with the edgeR package in R software (version 4.1.0; R Foundation for Statistical Computing). Genes with an absolute log2 fold change (|logFC|) >1 and a p-value <0.05 were considered significantly differentially expressed.

8. Cell uptake assay

Dilution of approximately 1×10⁸ exosome particles in PBS preceded incubation with PKH26 red fluorescent dye (Sigma-Aldrich) per manufacturer guidelines. Incubation of labeled exosomes with HEC-1-A cells lasted 4 hours at 37°C. Removal of culture medium followed, with cells washed twice using PBS to eliminate unbound exosomes. Staining with 4′,6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 minutes enabled nuclear visualization. Subsequent washing with PBS occurred, followed by imaging via a confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) to evaluate exosome uptake. Capture of fluorescent signals at wavelengths suitable for PKH26 (red) and DAPI (blue) fluorescence was performed.

9. Cell Counting Kit-8 (CCK-8) assay

Determination of cell viability involved the CCK-8 (Vazyme) assay per manufacturer protocols. Seeding of human cancer cells into 96-well plates at 5×10³ cells per well occurred, followed by culture in full growth medium. Addition of 10 µL CCK-8 reagent to each well took place after 24, 48, and 72 hours of incubation. Incubation of plates at 37°C lasted 0.5 to 4 hours. Recording of absorbance at 450 nm ensued using a microplate reader (BioTek, Winooski, VT, USA).

10. Colony formation assay

Seeding of 500 cells at low density into 6-well plates occurred, followed by culture for 14 days, with medium refreshed upon discoloration. Washing of cells with PBS was performed twice, succeeded by fixation with methanol. Staining with crystal violet solution (Beyotime, Shanghai, China) followed. Manual counting of colonies utilized an Olympus light microscope (Olympus Corporation).

11. Wound scratch assay

Evaluation of cell migration involved a wound scratch assay. Seeding of cells into 6-well plates at 2×10⁵ cells per well occurred, with culture maintained until confluence in complete growth medium. Creation of a uniform scratch across the cell monolayer utilized a sterile 200 μL pipette tip, followed by washing twice with PBS to remove dislodged cells. Monitoring of cell migration into the wound area occurred at 0 and 48 hours using an Olympus phase-contrast microscope (Olympus Corporation). Quantification of wound closure relied on measurement of the area between wound edges at both time points.

12. Transwell assay

Assessment of cell migration utilized Transwell chambers (Corning, Corning, NY, USA) with 8 μm pores. Serum starvation of cells for 24 hours preceded resuspension in serum-free medium at 1,000 or 2,000 cells per well. Addition of 200 μL cell suspension to the upper chamber occurred, while 600 μL of complete medium with 10% FBS (Vazyme) filled the lower chamber as a chemoattractant. Migration of cells proceeded for 72 hours. Removal of non-migrated cells from the upper membrane surface utilized a cotton swab. Fixation of migrated cells on the lower surface with paraformaldehyde (Servicebio) preceded staining with crystal violet solution (Beyotime) for 15 minutes. Visualization and imaging of stained cells occurred using an inverted microscope (Olympus Corporation). Manual counting of migrated cells derived from the images.

13. Western blot assay

Extraction of proteins utilized radioimmunoprecipitation assay buffer (Servicebio) with protease inhibitors (Servicebio), followed by quantification via the bicinchoninic acid assay kit (Servicebio). Resolution of equal protein amounts (10–20 μg) occurred via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with transfer to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) ensuing. Blocking of membranes with 5% non-fat milk (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 (TBST) lasted 1 hour at room temperature. Overnight incubation at 4°C with primary antibodies—GTF2IRD1 (A6613), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; A19056), CD63 (A19023), CD81 (A4863), TSG101 (A1692), and Calnexin (A4846) (all from Abclonal, Wuhan, China)—followed. Washing with TBST preceded incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (AS014; Abclonal) for 1 hour at room temperature. Detection of protein bands utilized enhanced chemiluminescence reagents (Abclonal), with visualization via a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).

14. Quantitative real-time polymerase chain reaction (qRT-PCR) assay

Extraction of total RNA from cells or tissues utilized the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme) per manufacturer instructions. Reverse transcription for mRNA quantification involved the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). Treatment of total RNA with RNase R (Vazyme) for circRNA detection removed linear RNAs, enriching circRNAs, followed by reverse transcription with the HiScript III 1st Strand cDNA Synthesis Kit using random primers. Execution of qRT-PCR analysis occurred on a Bio-Rad CFX system with SYBR Green Master Mix (Vazyme). Design and synthesis of primers for circ_0002577 and GTF2IRD1, including circRNA-specific divergent primers, were handled by Tsingke Biotechnology Co., Ltd. (Beijing, China). Calculation of relative gene expression utilized the 2^−ΔΔCt method, with GAPDH as the internal control.

15. Animal model

Procurement of nude mice (6–8 weeks old, female) occurred from the Laboratory Animal Center of Wuhan University of Science and Technology (Wuhan, China). Housing under specific pathogen-free conditions provided free access to food and water. Compliance with guidelines from the Animal Care and Use Committee of Wuhan University of Science and Technology was maintained, with ethical approval obtained (2024116). Injection of 100 µL of 5×10⁶ HEC-1-A cells into the right hind limb of each mouse established the subcutaneous tumor model. Monitoring of tumor growth involved caliper measurements. Euthanasia of mice occurred upon tumors reaching 1,500 mm³ or signs of distress, adhering to humane endpoints, followed by tumor harvesting. Intravenous injection of 100 µL of 1×10⁶ HEC-1-A cells into the tail vein established the metastasis model, with euthanasia 40 days post-injection and collection of lung tissues for histopathological analysis, including hematoxylin and eosin (H&E) staining. All procedures were performed in accordance with the guidelines set forth by the Animal Care and Use Committee of Wuhan University of Science and Technology, and ethical approval was obtained (2024116).

16. Immunohistochemical (IHC) assay

Performance of IHC staining involved deparaffinization and rehydration of paraffin-embedded tissue sections, followed by antigen retrieval in citrate buffer. Blocking to prevent non-specific binding preceded overnight incubation at 4°C with primary antibodies targeting Ki67 (catalog No: A20018; Abclonal) and GTF2IRD1 (catalog No: A6613; Abclonal). Incubation with HRP-conjugated secondary antibodies (catalog No: AS014; Abclonal) followed. Visualization utilized 3,3′-diaminobenzidine as the chromogen, with hematoxylin counterstaining for nuclei. Examination of slides occurred under a light microscope. Use of known immunostaining-positive slides served as positive controls, while slides incubated with irrelevant rabbit antiserum acted as negative controls. Evaluation of expression levels relied on the percentage and intensity of positively stained cells, with total scores calculated as their product.

17. H&E staining

Execution of H&E staining involved deparaffinization and rehydration of paraffin-embedded tissue sections through graded alcohols. Staining with hematoxylin for 5–10 minutes occurred, followed by differentiation in acid alcohol. Rinsing in running tap water removed excess hematoxylin, succeeded by eosin staining for 1–2 minutes to highlight cytoplasm and extracellular components. Dehydration through increasing alcohol concentrations, clearing in xylene, and mounting with resinous medium followed. Capture of images ensued after tissue clarification and mounting.

18. Statistical analysis

Utilization of public datasets from The Cancer Genome Atlas (TCGA) facilitated evaluation of gene expression levels and their correlation with patient prognosis. Preprocessing and normalization of data adhered to standard methods, with statistical analyses conducted using R software (version 4.1.0; R Foundation for Statistical Computing) and relevant bioinformatics packages. Presentation of graphical data involved mean ± standard deviation from at least three independent experiments. Statistical comparisons between two groups utilized the Student’s t-test, while one-way analysis of variance applied to comparisons of multiple groups. Performance of post-hoc tests, such as Tukey’s test, identified specific group differences where applicable. Definition of statistical significance corresponded to a p-value <0.05. Execution of all statistical analyses occurred using GraphPad Prism (version 8.0.2; GraphPad Software Inc., San Diego, CA, USA) software.

RESULTS

1. Exosome extraction from UCEC patient serum

As depicted in Fig. 1, exosomes were effectively isolated from the serum of UCEC patients via differential ultracentrifugation (Fig. 1A). Analysis by electron microscopy demonstrated that the exosomes presented a typical cup-shaped appearance and possessed a bilayer membrane structure, with a diameter of about 100 nm (Fig. 1B). Nanoflow cytometry analysis further validated that the size distribution of the isolated exosomes spanned from 70 to 130 nm, and the average diameter was 97.1±20.4 nm (Fig. 1C). The outcomes indicated high expression levels of both markers CD63 and CD81 on the exosome surface, which was in line with exosomal traits (Fig. 1D). Moreover, Western blot analysis confirmed the presence of exosomal positive markers CD63, CD81, and TSG101 in the isolated exosome samples, while the negative control biomarker Calnexin, detected in proteins extracted from cells (used as the control sample for exosomes), was absent in the exosome fraction (Fig. 1E). These findings align with the standard protocols for exosome isolation recommended by the International Society for Extracellular Vesicles, confirming the successful isolation of exosomes from serum samples via differential ultracentrifugation.

Fig. 1. Isolation and characterization of exosomes from the serum of UCEC patients. (A) Schematic workflow for the isolation of exosomes from serum samples of UCEC patients. (B) Representative TEM image of isolated exosomes, captured at an accelerating voltage of 80 kV. (C) Size distribution of the exosomes as determined by nanoflow cytometry. (D) Flow cytometry analysis of the membrane expression of exosomal markers CD81 and CD63. (E) Western blot analysis detecting the expression of exosomal markers CD63, CD81, TSG101, and calnexin. The isolation and characterization procedures were applied to serum samples from 10 UCEC patients, with the figure presenting a representative result from one patient.

TEM, transmission electron microscopy; UCEC, uterine corpus endometrial carcinoma.

2. Exosome-mediated promotion of tumor cell migration and proliferation in vitro

To assess the properties of exosomes derived from the serum of UCEC patients on cancer progression, a cell uptake assay was performed to demonstrate that PKH26-labeled exosomes were successfully internalized by tumor cells (Fig. 2A). Subsequently, UCEC tumor cell lines were treated with exosomes from the serum of UCEC patients for a long period of time to assess their effect on tumor cell function. Both colony formation assays and CCK-8 showed a significant increase in optical density at 450 nm values and colony counts of tumor cells co-cultured with exosomes from UCEC patient serum, indicating a marked enhancement in the proliferative capacity of the UCEC cell lines (Fig. 2B-D). Furthermore, exosome treatment notably augmented the migratory potential of tumor cells (Fig. 2E-H). These findings implied that exosomes sourced from the serum of UCEC patients might propel tumor progression.

Fig. 2. Promotion of tumor proliferation and migration by exosomes from UCEC serum. Three exosome samples were randomly selected from 10 UCEC patients for further experiments. Each sample was tested in triplicate (technical replicates), and the results represent the average of three technical replicates from each of the three samples. (A) Confocal microscopy was used to assess exosome uptake in Ishikawa cells after 4 hours of co-culture with PKH26-labeled exosomes (appeared exosomes in DAPI: blue and PKH26: red). (B) Cell viability was measured using the CCK-8 assay following co-culture of exosomes with tumor cells for 24, 48, and 72 hours, with absorbance at optical density at 450 nm as an indicator of cell proliferation. (C) Clonogenic assays in vitro were performed to evaluate the clonogenic potential of tumor cells after co-culture with exosomes for 7 days. (D) Quantification of the clonogenic assay results. (E) Transwell migration assays were performed to assess the migratory ability of tumor cells after 72 hours of co-culture with exosomes. (F) Quantification of Transwell migration assay results. (G) After 72 hours of co-culture with exosomes, a scratch wound healing assay was used to evaluate the migration ability of tumor cells. (H) Statistical analysis of the scratch wound-healing assay results. Data are presented as mean ± standard deviation.

CCK-8, Cell Counting Kit-8; DAPI, 4′,6′-diamidino-2-phenylindole; NC, negative control; OD, optical density; PBS, phosphate-buffered saline; UCEC, uterine corpus endometrial carcinoma.

Statistical significance was determined as follows: ^*p<0.05, ^***p<0.001, and >0.05 considered not significant.

3. Exosome-mediated promotion of tumor progression in vivo

We explored the effects of exosomes derived from UCEC patient serum on tumor in vivo. When the volume of tumor reached about 100 mm³, exosomes or PBS were injected at multiple points in a single injection directly into the tumor (Fig. 3A). The growth of tumor in the exosome-treated group was higher than the PBS-treated group. The tumors in the exosome-treated group were meaningfully larger than those in the PBS-treated group (Fig. 3B). Significantly, there was no momentous change in body weight between the 2 groups (Fig. 3C). Following subcutaneous tumor resection, both the weight and volume of tumors in the exosome-treated group were meaningfully greater (Fig. 3D and E). IHC analysis revealed higher relative levels of Ki67 in the exosome-treated group, suggesting increased tumor cell proliferation in this group (Fig. 3F and G). These findings confirm that exosomes from the serum of UCEC patients enhance tumor progression in vivo.

Fig. 3. Tumor progression in a nude mouse model promoted by exosomes from UCEC serum. Exosomes from the 3 patient samples analyzed in Fig. 2 were pooled and administered via intratumoral injection into a subcutaneous xenograft model in nude mice. (A) Schematic representation of the subcutaneous xenograft model in nude mice with exosome treatment (100 uL/1×10⁸/mice) (n=3). (B) Tumor volume growth curve in nude mice. Tumor volume was calculated using the formula: Volume = (Length × Width²)/2. (C) Changes in body weight of nude mice over the course of the experiment. (D) Statistical analysis of tumor mass at the study endpoint following the dissection of subcutaneous xenografts in nude mice. (E) Tumor volume measurement at the study endpoint following dissection of subcutaneous xenografts in nude mice. (F) IHC was used to assess Ki67 expression in tumors from different treatment groups. (G) Statistical analysis of IHC scores for Ki67 expression in tumor samples. Data are presented as mean ± standard deviation.

IHC, immunohistochemical; ns, not significant; PBS, phosphate-buffered saline; UCEC, uterine corpus endometrial carcinoma.

Statistical significance was determined as follows: ^*p<0.05, ^***p<0.001, and >0.05 considered not significant.

4. High expression of circ_0002577 in exosomes derived from UCEC patient serum promotes proliferation and migration of UCEC cell

To further explore the mechanisms by which exosomes in serum from UCEC patients promote tumor progression, we performed a human ceRNA array analyses to assess the expression profiles of circRNAs and mRNAs in three normal female and UCEC patient samples.

The findings showed that the changes in circRNA levels were significantly more pronounced than those in mRNAs, leading to a focus on circRNA in this study. The heatmap and volcano plot analyses identified 8,244 circRNAs with |log2FC| > 1 and adjusted p < 0.05. In this group, 4,855 circRNAs were downregulated, 3,389 circRNAs were upregulated, with the most significantly altered circRNA being circ_0002577 (Fig. 4A and B). Expression analysis of circ_0002577 revealed its significant upregulation in exosomes derived from UCEC patient serum (Fig. 4C). To further investigate the functional role of circ_0002577, we utilized lentiviral-mediated knockdown of circ_0002577 in HEC-1-A and Ishikawa cells (Fig. 4D). The knockdown of circ_0002577 expression meaningfully impaired UCEC cell proliferation and migration (Fig. 4E-G).

Fig. 4. Differential circRNAs and the biological function in the serum of UCEC patients. Three exosome samples were randomly selected from 10 UCEC patients for further experiments. (A) Differentially expressed circRNAs were identified and visualized using a clustering heatmap generated by the limma package (Agilent human ceRNA array). The logFC threshold of 1 and a p-value <0.05 were applied for selection. (B) The volcano plot illustrating the most significantly up-regulated and down-regulated circRNAs, with green representing down-regulated circRNAs and red representing up-regulated circRNAs. (C) Relative expression levels of circ_0002577 in the chip analysis. (D) qRT-PCR was performed to assess the relative expression levels of circ_0002577 in Ishikawa and HEC-1-A cell lines following stable knockdown. (E) Cell viability was measured using the CCK-8 assay in circ_0002577 knockdown cell lines and wild-type controls after 24, 48, and 72 hours. (F) Transwell migration assays were performed to assess the migration capacity of circ_0002577 knockdown cell lines compared to wild-type controls in vitro. (G) Statistical analysis of Transwell migration assay results. Data are presented as mean ± standard deviation.

CCK-8, Cell Counting Kit-8; ceRNA, competing endogenous RNA; circ_0002577, circular RNA 0002577; circRNA, circular RNA; OD, optical density; NC, negative control; PBS, phosphate-buffered saline; qRT-PCR, quantitative real-time polymerase chain reaction; UCEC, uterine corpus endometrial carcinoma.

Statistical significance was determined as follows: ^**p<0.01, ^***p<0.001, and >0.05 considered not significant.

5. Circ_0002577 expression is strongly correlated with GTF2IRD1 in UCEC

To investigate the mechanism by which circ_0002577 regulates UCEC progression, a stable HEC-1-A cell line of circ_0002577 overexpression was established (Fig. 5A). RNA sequencing was then performed, and both heatmap and volcano plot analyses revealed that GTF2IRD1 was the most significantly altered gene (Fig. 5B and C). Next, the relative expression of GTF2IRD1 and circ_0002577 were measured in 10 paired UCEC samples and their corresponding adjacent normal tissues. Correlation analysis demonstrated a notably positive correlation between the two genes. Specifically, the R value was 0.71 and the p-value reached 0.028 (Fig. 5D). These findings indicated a strong positive correlation between circ_0002577 and GTF2IRD1 expression. Furthermore, the survival rate of the group with high expression of GTF2IRD1 was lower (Fig. 5E). In addition, GTF2IRD1 levels were higher in tumor tissues than those in adjacent normal tissues (Fig. 5F), which was consistent with IHC analysis of 10 UCEC samples (Fig. 5G and H). These results suggested that circ_0002577 is closely associated with GTF2IRD1 expression in UCEC.

Fig. 5. Screening of potential downstream target genes regulated by circ_0002577. (A) qRT-PCR was used to assess the overexpression efficiency of circ_0002577 in the HEC-1-A cell line. (B) RNA sequencing was performed on wild-type and circ_0002577-overexpressing HEC-1-A cells, followed by differential gene analysis using the limma package. A clustering heatmap of differentially expressed genes was generated, with a logFC threshold of 1 and a p-value <0.05. (C) Volcano plot illustrating the most significantly upregulated and downregulated genes, with green indicating downregulated genes and red indicating upregulated genes. (D) Correlation between the expression of circ_0002577 and GTF2IRD1. (E) Survival analysis of GTF2IRD1 expression in UCEC patients from TCGA database. Patients were grouped based on the median expression of GTF2IRD1, with red representing the high-expression group and blue representing the low-expression group. (F) Relative expression of GTF2IRD1 in adjacent non-cancerous and cancerous tissues from UCEC patients in the TCGA database. (G) IHC was performed to assess GTF2IRD1 expression levels in adjacent non-cancerous and cancerous tissues from UCEC patients, with representative IHC images shown for 1 patient. (H) Statistical analysis of IHC scores for GTF2IRD1 expression in UCEC tumor and adjacent normal tissues (n=10). Data are presented as mean ± standard deviation.

circ_0002577, circular RNA 0002577; GTF2IRD1, general transcription factor II-I repeat domain-containing 1; IHC, immunohistochemical; OE, over expression; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; TCGA, The Cancer Genome Atlas; UCEC, uterine corpus endometrial carcinoma.

Statistical significance was determined as follows: ^***p<0.001 and >0.05 considered not significant.

6. Knockdown of GTF2IRD1 expression reduces cell migration, proliferation, and tumor progression in UCEC

RNA sequencing showed that GTF2IRD1 was highly correlated with circ_0002577 expression. Moreover, TCGA data analysis suggested that GTF2IRD1 may serve as a potential prognostic factor in UCEC. Therefore, we hypothesized that the increased circ_0002577 may regulate tumor progression by its effect on GTF2IRD1. To examine the role of GTF2IRD1 in UCEC, we established a stable GTF2IRD1 knockdown UCEC cell line using the lentivirus system. As shown in Fig. 6A, GTF2IRD1 expression was significantly reduced. Subsequent functional assays revealed a striking reduction in cell proliferation of GTF2IRD1 knockdown cells. Both CCK-8 and colony formation assays showed a significant reduction in the ability to proliferate (Fig. 6C-E).

Fig. 6. Knockdown of GTF2IRD1 expression reduces proliferation and migration of UCEC cells. (A) qRT-PCR was used to measure the relative expression levels of GTF2IRD1 in a stable knockdown and wild-type Ishikawa and HEC-1-A cell line. (B) Western blot analysis was performed to evaluate the protein levels of GTF2IRD1 in a stable knockdown and wild-type Ishikawa and HEC-1-A cell line. (C) Statistical analysis of the clonogenic assay. (D) Cell viability was assessed by the CCK-8 assay in a stable knockdown and wild-type cell line at 24, 48, and 72 hours. (E) Clonogenic assay was conducted to assess the colony formation ability of a stable knockdown and wild-type cell in vitro. (F) Transwell migration assays were performed to measure the migration capacity of a stable knockdown and wild-type cell. (G) Statistical analysis of the Transwell migration assay results. (H) Wound healing assays were performed to evaluate the migration ability of a stable knockdown and wild-type cell. (I) Statistical analysis of wound healing assay results. (J) Tumor volume growth curve for subcutaneous xenograft tumors in nude mice, calculated as Volume = Length × Width × Width/2 (n=3). (K) Representative H&E staining images of lung tissue from the intravenous lung metastasis model in nude mice (n=3). (L) Statistical analysis for the number of lung metastatic nodules (n=3). Data are presented as mean ± standard deviation.

CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTF2IRD1, general transcription factor II-I repeat domain-containing 1; H&E, hematoxylin and eosin; NC, negative control; OD, optical density; PBS, phosphate-buffered saline; qRT-PCR, quantitative real-time polymerase chain reaction; UCEC, uterine corpus endometrial carcinoma.

Statistical significance was determined as follows: ^*p<0.05, ^**p<0.01, ^***p<0.001, >0.05 considered not significant.

In addition, migration assays indicated a corresponding decline in the ability of GTF2IRD1-deficient cells to migrate (Fig. 6F-I). These findings were further confirmed by experiments in vivo. In a subcutaneous xenograft model, tumors derived from GTF2IRD1 knockdown in HEC-1-A cells grew significantly slower than control group (Fig. 6J). Similarly, in a metastasis model, mice injected with GTF2IRD1 knockdown cells via tail vein showed fewer lung metastatic nodules, underscoring the reduced metastatic potential of these cells (Fig. 6K and L). Overall, the findings suggest that GTF2IRD1 is crucial for the proliferation, migration, and progression of UCEC.

7. Serum-derived exosomes from UCEC patients enhance UCEC cell proliferation and migration through the circ_0002577/GTF2IRD1 axis

To investigate the role of circ_0002577 in modulating tumor cell proliferation and migration through GTF2IRD1 regulation, we conducted functional validation experiments using a stable cell line with GTF2IRD1 knockdown and lentiviral-mediated circ_0002577 overexpression. qRT-PCR analysis confirmed effective overexpression of circ_0002577 in GTF2IRD1-knockdown cells (Fig. S1A). Notably, GTF2IRD1 expression in knockdown cells was reduced compared to the control group following circ_0002577 overexpression, yet it remained higher than in GTF2IRD1-knockdown cells without circ_0002577 overexpression, suggesting that GTF2IRD1 knockdown partially counteracts the upregulation of GTF2IRD1 induced by circ_0002577 (Fig. S1B). In control cells, circ_0002577 overexpression markedly enhanced proliferation and migration (Fig. S1C and D); however, these stimulatory effects were significantly attenuated in GTF2IRD1-knockdown cells, indicating that GTF2IRD1 serves as a critical mediator of circ_0002577-driven tumor cell behavior. Furthermore, to explore the influence of serum-derived exosomes from patients with UCEC on tumor progression via this pathway, both control and GTF2IRD1-knockdown cell lines were treated with UCEC-derived exosomes. Consistent with the effects of circ_0002577 overexpression, exosome-induced promotion of proliferation and migration was substantially diminished in GTF2IRD1-knockdown cells (Fig. S1E and F). Animal model experiments further corroborated these findings, demonstrating that GTF2IRD1 knockdown significantly reduced tumor growth rates; although UCEC-derived exosomes accelerated tumor progression in the control group, this effect was partially mitigated in the GTF2IRD1-knockdown group (Fig. S2). Collectively, these results establish that serum-derived exosomes from UCEC patients regulate tumor cell proliferation and migration through a circ_0002577-GTF2IRD1 signaling axis, with GTF2IRD1 acting as a pivotal downstream effector.

DISCUSSION

UCEC remains therapeutically challenging, especially in metastatic cases where treatment options are limited and patient outcome remains poor [5,24]. Because of the tumor’s heterogeneity and the complex tumor microenvironment, conventional treatments including surgery, chemotherapy, and radiation often fail to achieve the expected results in advanced stage [25,26]. Recent studies have underscored the significant role of exosomes derived from tumors in facilitating metastasis by mediating intercellular communication and promoting a pro-tumorigenic milieu [27]. Specifically, recent studies have begun to uncover the role of exosomes in disease progression, although a comprehensive understanding is still lacking in UCEC [28]. In our research, exosomes were effectively isolated from the serum of UCEC patients through differential centrifugation, and further analysis showed that these exosomes substantially increased the migration and proliferation of UCEC cells in vitro.

Additionally, intratumoral injection of patient-derived exosomes accelerated the growth of subcutaneous xenograft tumors. These outcomes emphasize the imperative role of exosomes derived from UCEC in the tumor microenvironment and provide a theoretical basis for future therapeutic strategies based on interference with their exosomes. CircRNAs have garnered significant attention for their regulatory roles in various cancers, including UCEC.

In our study, a comparative circRNA microarray analysis was performed on exosomes extracted from the serum of UCEC patients and healthy female controls, revealing substantial changes in circRNA expression profiles. CircRNAs are covalently closed, single-stranded RNA molecules that are highly stable and resistant to exonucleases. This allows them to function as microRNA sponges, interact with RNA-binding proteins, and modulate gene transcription [29].

Their role in tumorigenesis is well-documented, with numerous studies showing their ability to regulate cancer cell behavior [30,31]. Specifically, several circRNAs have been found to be crucial in UCEC. For instance, circ_POLA2 enhances microRNA-31 methylation and promotes the proliferation of endometrial cancer cells [23].

Furthermore, circRNAs can function as tumor suppressors. For example, circRNA hsa_circRNA_0001776 decreases proliferation and increases apoptosis in endometrial cancer by downregulating LRIG2 via sponging miR-182 [32]. For circ_0002577, it has begun to emerge as a potential oncogenic factor [33,34]. Notably, recent studies have elucidated the mechanisms by which circ_0002577 promotes endometrial cancer progression. For instance, Wang et al. [33] showed that hsa_circ_0002577 promotes the progression of EC by activating the IGF1R/PI3K/Akt pathway, and Wang et al. [34] identified the circ_0002577/miR-126-5p/MACC1 axis as a key regulator of proliferation, migration, invasion, and apoptosis in EC cells. Our findings showed that circ_0002577 is markedly upregulated in exosomes from the serum of UCEC patients. Functional assays, including CCK8 and Transwell experiments, demonstrated that knockdown of circ_0002577 in UCEC cell lines significantly decrease cell proliferation and migration, highlighting its potential role in promoting tumor aggressiveness. These results are consistent with the literature that has been reported, and circ_0002577 has been implicated in enhancing oncogenic pathways in other studies [33,34]. Unlike previous studies, which mainly focused on intracellular circRNA, our study highpoints the importance of exosome-mediated circRNA metastasis in the tumor microenvironment of UCEC. This suggested that circ_0002577 containing exosomes may facilitate intercellular communication that promotes tumor growth and metastasis. Consequently, circ_0002577 emerges as a promising biomarker for UCEC and a potential therapeutic target aimed at disrupting exosome-mediated tumor progression. Subsequently, GTF2IRD1 was identified as the most significantly altered gene through RNA sequencing performed on HEC-1-A cells with stable over-expression of circ_0002577. In addition, GTF2IRD1 was initially studied primarily for its association with Williams-Beuren syndrome [35,36,37]. However, recent research has indicated that GTF2IRD1 is expressed abnormally in various types of tumors. Its role in tumor progression has been increasingly recognized, and studies have shown that GTF2IRD1 can enhance oncogenic signaling pathways and enhance the growth and spread of cancer cells [38,39,40]. For example, GTF2IRD1 functions as a new oncogene that modulates the tumor suppressor gene TGFbetaR2 in colorectal cancer and has been identified as a promoter in breast tumors [38,39]. Additionally, it has been shown that reducing GTF2IRD1 expression can suppress cancer cell proliferation, invasion and colony formation in pancreatic cancer in vitro [40]. These findings illuminate the role of GTF2IRD1 in UCEC.

In our analysis of TCGA UCEC dataset, GTF2IRD1 expression was significantly higher in UCEC tumor tissues, and elevated GTF2IRD1 expression was significantly linked to lower patient survival rates.

IHC analysis of clinical samples further confirmed that GTF2IRD1 was markedly higher in UCEC tissues. Furthermore, CCK8 and Transwell assays revealed that silencing GTF2IRD1 in the UCEC cell line led to a notable reduction in cell proliferation and migration. Moreover, experiments in vivo using a stable GTF2IRD1 knockdown HEC-1-A cell line showed that subcutaneous tumor growth was substantially suppressed in nude mice, and the number of lung nodules was reduced in a venous metastasis model. These results fill the gap in the functional study of GTF2IRD1 in UCEC. Base on the above results, we propose that the higher levels of circ_0002577 in exosomes from the serum of UCEC patients may indirectly activate GTF2IRD1 expression and promote UCEC cell proliferation and migration. To test this hypothesis, we performed functional validation experiments by knocking down GTF2IRD1 in cell lines with over-expression of circ_0002577. The results showed that the knockdown of GTF2IRD1 not only reduced the ability of tumor cells to proliferate and migrate, but also attenuated the tumor promotion effect of circ_0002577 over-expression. Additionally, in a co-culture system of UCEC-derived exosomes and UCEC cells, knockdown of GTF2IRD1 similarly diminished the exosome-mediated enhancement of tumor aggressiveness. Together, these results suggest that the circ_0002577/GTF2IRD1 axis plays a vital role in UCEC progression and may be a possible beneficial target for reducing tumor growth and metastasis.

While this study yields valuable insights into the role of UCEC-derived exosomes in tumor progression, it faces notable limitations that warrant consideration in future research. One primary limitation is the use of nude mice, which lack a fully functional immune system, thereby restricting the evaluation of interactions between UCEC-derived exosomes and the immune microenvironment. This constraint may obscure critical tumor-immune dynamics, such as immune evasion or modulation, which are pivotal in cancer progression. To address this, future studies should employ immunocompetent animal models to comprehensively investigate the effects of these exosomes on immune cells, stromal interactions, and the broader tumor microenvironment. Another significant issue is the failure to confirm direct binding between circ_0002577 and GTF2IRD1, despite bioinformatics predictions, as luciferase reporter assays yielded negative results. This discrepancy suggests an indirect regulatory mechanism, potentially mediated by other molecular players such as miRNAs or RNA-binding proteins. For instance, circRNAs are known to act as miRNA sponges, competitively binding to miRNAs to weaken their inhibitory effects on target mRNAs, thereby indirectly upregulating gene expression [41]. In hepatocellular carcinoma, circ_0001955 was found to sponge miR-646, promoting proliferation, metastasis, and angiogenesis via the miR-646/FZD4 axis [41]. Similarly, circ_0002577 may indirectly influence GTF2IRD1 expression through a miRNA sponge mechanism, although direct evidence is currently lacking. Based on predictions from databases like circBank and CircInteractome, we attempted to validate whether miRNAs such as miR-126-5p and miR-197 mediate this regulation, but qRT-PCR and luciferase reporter assays yielded negative results. This may indicate that the mechanism extends beyond a single miRNA sponge model or that key miRNA mediators remain unidentified. Additionally, limitations in knockdown efficiency or detection sensitivity may have obscured subtle regulatory signals. To address these shortcomings, we have expanded this discussion to analyze possible indirect pathways and acknowledge the need for more comprehensive experimental approaches.

Our study elucidates the pivotal role of circ_0002577 in promoting UCEC progression by upregulating GTF2IRD1. By isolating exosomes from the serum of UCEC patients and demonstrating the enhanced proliferative and migratory capabilities of UCEC cells upon circ_0002577 overexpression, we highlight a novel exosome-mediated mechanism contributing to tumor aggressiveness. The positive correlation between circ_0002577 and GTF2IRD1, demonstrated through both in vitro and in vivo studies, highlights the potential of the circ_0002577/GTF2IRD1 axis as a therapeutic target. Moreover, the functional validation experiments confirm the significance of GTF2IRD1 in circ_0002577-driven UCEC progression, reinforcing its role in facilitating tumor growth and metastasis. These results pave the way for developing targeted therapies that disrupt exosome-mediated circRNA communication. Future research should concentrate on elucidating the detailed molecular interactions within this axis and exploring the therapeutic efficacy of targeting circ_0002577 and GTF2IRD1 in clinical settings.

SUPPLEMENTARY MATERIALS

Table S1

Patient characteristics

Fig. S1

Serum-derived exosomes from UCEC patients enhance UCEC cell proliferation and migration through the circ_0002577/GTF2IRD1 axis. (A) qRT-PCR was performed to assess the relative expression of circ_0002577 in Ishikawa and HEC-1-A cell lines. (B) Western blot was performed to evaluate the levels of GTF2IRD1 in Ishikawa and HEC-1-A cell lines. (C) CCK-8 assay was used to assess the relative cell viability of tumor cells with differential expression of circ_0002577 and GTF2IRD1 after 72 hours of treatment. (D) Transwell assays were conducted to measure the relative migratory capacity of tumor cells with differential expression of circ_0002577 and GTF2IRD1. (E) The CCK-8 assay was used to evaluate the relative cell viability of tumor cells co-cultured with exosomes and different levels of circ_0002577 and GTF2IRD1 for 72 hours. (F) Transwell migration assays were performed to assess the relative migratory ability of tumor cells co-cultured with exosomes and different levels of circ_0002577 and GTF2IRD1 for 72 hours. Data are presented as mean ± standard deviation.

Fig. S2

GTF2IRD1 knockdown attenuates exosome-accelerated tumor growth in vivo.