MTFR2 regulated the proliferation, apoptosis, migration, and invasion of endometrial cancer cells through the Drp1/MFN1 signaling pathway mediated mitochondrial fission

Introduction

Endometrial cancer (EC), one of the most common malignant tumors in the female reproductive system, has shown a global annual increase in incidence, posing a serious threat to women’s health (1,2). In recent years, both the incidence and mortality rates of EC have been rising worldwide, particularly evident in developed countries (1). Although some progress has been made in the traditional treatment methods, such as surgery, radiotherapy, and chemotherapy, the prognosis of patients with advanced EC is still poor, with a high recurrence rate and a low 5-year survival rate (3). Therefore, it is of great significance to explore the molecular mechanism of the occurrence and development of EC and find new therapeutic targets and biomarkers for improving the treatment effect and prognosis of patients with EC.

Mitochondria, as pivotal organelles within cells, serve not only as the central hub for cellular energy metabolism but also participate in numerous biological processes, including cell proliferation, apoptosis, and oxidative stress (4,5). Recent studies have increasingly demonstrated a strong association between mitochondrial dysfunction and the onset and progression of tumors (6,7). Mitochondrial fission and fusion are critical processes for maintaining mitochondrial homeostasis, and disruptions in these processes can lead to aberrant mitochondrial morphology and function, thereby impacting cellular physiological functions (8). Dynamin-related protein 1 (Drp1) is a key mediator of mitochondrial fission, facilitating this process by recruiting to the mitochondrial surface (8). Research has shown that Drp1 is overexpressed in various tumor cells and is closely linked to tumor cell behaviors such as proliferation, apoptosis, migration, and invasion (9-11). Moreover, studies indicate that the mitochondrial fusion protein mitofusin 1 (MFN1) is crucial in various cancer types, with its expression levels showing strong correlations to tumor growth and progression. Studies have demonstrated that MFN1 is also crucial in high-glucose-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. Under high-glucose conditions, increased MFN1 expression promotes cellular proliferation and invasion, while autophagy inhibition can reverse this abnormal EMT (12). Additionally, MFN1 participates in ferroptosis by enhancing reactive oxygen species production and lipid peroxidation through mitochondrial fusion, thereby increasing pancreatic cancer cell sensitivity to ferroptosis (13). Therefore, the Drp1/MFN1 signaling pathway may play an important role in the regulation of mitochondrial fission in tumor cells.

Mitochondrial fission regulator 2 (MTFR2) is a newly discovered mitochondrial protein which plays an important role in the regulation of mitochondrial fission (14,15). Studies have shown that MTFR2 can interact with Drp1 and promote the recruitment of Drp1 to the mitochondrial surface, thereby enhancing mitochondrial fission (14). However, little is known about the role of MTFR2 in EC and its molecular mechanisms.

The aim of this study was to investigate the expression of MTFR2 in EC and its relationship with clinicopathological features, and to clarify the molecular mechanism of MTFR2 regulating the proliferation, apoptosis, migration, and invasion of EC cells through the Drp1/MFN1 signaling pathway-mediated mitochondrial fission. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2793/rc).

Methods

Clinical bioinformatics analysis

RNA sequencing data, clinical information, and survival data pertaining to EC were procured from The Cancer Genome Atlas (TCGA) database, encompassing 548 EC samples and 35 normal samples. The raw data were standardized using R software, version 3.6.3. The “ggplot2” package in the R programming language was employed to visualize MTFR2 messenger RNA (mRNA) expression levels within the TCGA, allowing for a comparison between EC tissues and normal endometrial tissues. Furthermore, the expression of MTFR2 across different pathological stages (G1/G2 vs. G3) was analyzed in paired cancer and paracancerous tissues. Using data from the TCGA database, the Kaplan-Meier plotter platform was employed to assess the correlation between MTFR2 expression levels and patient survival outcomes. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell culture and treatment

HEC-1A and Ishikawa human EC cell lines were purchased from Shanghai Jikai Gene Technology Co., Ltd. (Shanghai, China). KLE human EC cell line and human normal endometrial epithelial cell line HEEC were from Gansu Gynecological Tumor Clinical Medical Research Center (Lanzhou, China). HEC-1A, KLE, Ishikawa, and HEEC cells were cultured in McCoy’s 5A (Procell, Wuhan, China), DMEM/F-12 (HyClone, Shanghai, China), and RPMI-1640 (HyClone) medium, respectively, all containing 10% fetal bovine serum (FBS; SijiQing, Hangzhou, China) and 100 µg/mL penicillin-streptomycin. After reaching 80–90% confluence, the cells were harvested by trypsin digestion and subsequently passaged. We used cells in the logarithmic growth phase for all experiments. The study included the following experimental groups: (I) the pcDNA-normal control (Ov-NC) group and the MTFR2 overexpression plasmid (Ov-MTFR2) group; (II) the RNA knockdown control (Sh-NC) group, the MTFR2 knockdown #1 (Sh1-MTFR2) group, and the MTFR2 knockdown #2 (Sh2-MTFR2) group. Plasmids and short hairpin RNAs (shRNAs) were constructed and designed by Shanghai Jikai Gene Medical Technology Co., Ltd. (Shanghai, China), and were used for lentivirus packaging. HEC‑1A or Ishikawa cells were infected with lentiviruses carrying the indicated shRNAs or overexpression vectors, and stable cell lines were subsequently selected using puromycin (2 µg/mL, Beyotime, Shanghai, China).

Cell Counting Kit-8 (CCK-8) assay

The viability of HEC-1A and Ishikawa cells was assessed utilizing the CCK-8 (Biosharp, Hefei, China) in accordance with the manufacturer’s protocol. To quantify cell viability, absorbance at 450 nm was measured using a BioTek ELx800 microplate reader (Winooski, VT, USA).

Colony formation assay

HEC-1A and Ishikawa cell lines were plated in six-well culture dishes at a density of 1.0×10³ cells per well and maintained under standard culture conditions (37 ℃, 5% CO₂) for a duration of 2 weeks. Following the incubation period, the cells were fixed using formaldehyde and subsequently stained with 0.1% crystal violet for five minutes at ambient temperature. Colonies comprising more than 50 cells were enumerated utilizing a BX51 microscope (Olympus Corporation, Tokyo, Japan). All experiments were performed in triplicate.

Flow cytometry analysis of apoptosis

After 48 hours of culture, the cells were harvested and resuspended in phosphate-buffered saline (PBS) at a concentration of 1×10⁶ cells/mL. Annexin V-APC and propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA) were added according to the manufacturer’s protocol and incubated in the dark for 15 minutes. Apoptosis was analyzed by flow cytometry (BD Biosciences).

5-ethynyl-2'-deoxyuridine (EdU) assay

Cells were seeded into 96-well plates at 6×10³ cells per 100 µL and incubated with EdU (Abbkine, Wuhan, China) for 2 hours to label newly synthesized DNA. Following fixation with 4% paraformaldehyde (15 minutes) and permeabilization with 0.5% Triton X-100 (20 minutes), click chemistry was performed in the dark for 30 minutes using a reaction mixture containing Tris-HCl, CuSO₄, Alexa Fluor 488 azide, and sodium ascorbate. After washing, nuclei were counterstained with Hoechst. EdU-positive cells were visualized by fluorescence microscopy and quantified using ImageJ. Experiments were performed in triplicate.

Wound healing assay

Cell migration was assessed utilizing a wound-healing assay. In this procedure, 2×10⁵ cells per well were seeded into six-well plates and incubated in a complete medium at 37 ℃ for 24 hours. A uniform wound was then created at the center of the cell monolayer by scraping the well surface with a 10 µL pipette tip. We washed off any cells that had detached using PBS, and fresh serum-free medium was added to ensure that there was no cell proliferation. Images of wounds were captured at 0 and 48 hours using a phase-contrast microscope (BX51, Olympus). Cell migration was quantified by analyzing wound closure using ImageJ software.

Transwell migration and invasion assays

Cell migration and invasion assays were performed using 24-well Transwell chambers (Corning Inc., Corning, NY, USA; 8-µm pore polycarbonate membrane). For the invasion assay, the upper chamber was pre-coated with 60 µL of Matrigel (BD Biosciences) diluted 1:8 in serum-free medium and incubated at 37 ℃ for 3 hours to create a matrix barrier; the migration assay was conducted without Matrigel coating. A total of 4×10⁴ cells suspended in 200 µL of serum-free medium were seeded into the upper chamber, while complete medium was added to the lower chamber. After incubation at 37 ℃ for 24 hours, the chambers were washed with PBS. Cells that had traversed the membrane were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet in the dark for 10 minutes, and then imaged under an inverted light microscope at 200× magnification in five randomly selected fields per membrane. The number of migrated or invaded cells was counted using ImageJ software. All experiments were replicated threefold.

Western blot analysis

Total proteins from HEC-1A and Ishikawa cells were extracted using a standard radioimmunoprecipitation assay (RIPA) lysis buffer (Biosharp). Protein concentrations were measured with a bicinchoninic acid (BCA) assay kit (Boster, Wuhan, China). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Solarbio, Wuhan, China) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% nonfat milk with Drp1 (BF8306; Affinity, Changzhou, China; 1:800), phospho-Drp1 (p-Drp1; Ser616, BF8306; Affinity; 1:1,000), MFN1 (DF7543; Affinity; 1:1,000), MTFR2 (A305-773A-T; Thermo Fisher, Waltham, MA, USA; 1:1,000), primary antibodies overnight at 4 ℃, followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:8,000; Servicebio, Wuhan, China). Signals were developed using a fully automatic chemiluminescence image analysis system (Tanon 5200 Multi, Tanon Company, Shanghai, China).

In vivo tumorigenesis test

Specific-pathogen-free (SPF) BALB/c-nu female nude mice (4–6 weeks old, weighing 16–18 g) were purchased from Jiangsu Huachuang Biological Company (Taizhou, China). All animal experiments were performed in strict accordance with the Regulations for the Administration of Laboratory Animals. It was reviewed and approved by the Ethics Committee of The First Hospital of Lanzhou University (No. LDYYLL2023-01). Twenty-four female nude mice (SPF grade, 4 weeks old) were housed under controlled environmental conditions, with a temperature of 24±1 ℃, relative humidity of 55%±5%, and a 12-hour light/dark cycle. The mice had ad libitum access to food and water.

HEC-1A or Ishikawa cells transfected with Sh-NC or Sh-MTFR2 were collected and resuspended in medium at a concentration of 1×10⁶ cells/mL. The HEC-1A or Ishikawa cells (2×10⁶ cells) were then mixed with Matrigel and subcutaneously injected into mice. The nude mice were maintained under standard conditions, with their weight, diet, behavior, and tumor morphology assessed every 3 days. Tumor diameter was measured using a vernier caliper. After 36 days, the mice were anesthetized with sodium pentobarbital (50 mg/kg) and euthanized via carbon dioxide exposure. Tumor tissue samples were subsequently collected.

Immunohistochemistry (IHC) staining

Following dewaxing, rehydration, and antigen retrieval (95–99 ℃, 40 minutes), sections were washed with PBS and incubated with primary antibodies against proliferating cell nuclear antigen (PCNA; ab29, Abcam, Cambridge, UK; 1:200) and Ki-67 (ab92742; Abcam; 1:200) overnight at 4 ℃. Sections were then incubated with secondary antibody (30 minutes) and HRP-streptavidin (Zhongshan Jinqiao, Beijing, China; 30 minutes, 37 ℃). DAB (Beyotime, Shanghai, China) staining was performed, followed by hematoxylin counterstaining (Solarbio). After dehydration and clearing, sections were mounted. Whole-slide images were scanned (Pannoramic MIDI II, 3D Histech, Budapest, Hungary), and mean optical density was quantified from three random fields per section using Image software.

Statistical analysis

The data from the TCGA database were processed using R software (version 3.6.3), while the experimental data were analyzed with GraphPad Prism 9.0. Independent samples t-tests or one-way analysis of variance (ANOVA) were conducted for intergroup comparisons, with Tukey’s honestly significant difference (HSD) test applied as a post hoc procedure. The relationship between MTFR2 and clinicopathological parameters was evaluated using the Kruskal-Wallis test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Measurement data are presented as mean ± standard deviation (SD), and categorical data are reported as the number of cases. A P value of less than 0.05 was considered indicative of statistical significance.

Results

MTFR2 overexpression correlated with poor prognosis and aggressive tumor behavior in EC

The expression of MTFR2 mRNA in EC tissues was significantly elevated compared to normal endometrial tissues (Figure 1A). Among patients with grade G1/G2 EC, MTFR2 expression in tumor tissues was markedly higher than in adjacent non-tumor tissues (Figure 1B). Furthermore, MTFR2 expression in tumor tissues of patients with grade G3 EC was significantly greater than in those with grade G1/G2 EC (Figure 1B). Kaplan-Meier survival analysis indicated that patients exhibiting high MTFR2 expression had a significantly reduced survival probability compared to those with low MTFR2 expression (Figure 1C). Additionally, MTFR2 expression levels were closely associated with patient age, clinical stage, pathological type, histological grade, depth of tumor invasion, and treatment response (Figure 1D). Moreover, when compared with HEC cells, the expression of MTFR2 was markedly increased in HEC-1A and Ishikawa cells (Figure 1E).

Figure 1 MTFR2 expression and its association with clinicopathological features and prognosis in EC. (A,B) The mRNA expression of MTFR2 in EC tissues and normal endometrial tissues. ***, P<0.001. (C) Kaplan-Meier survival analysis was used to analyze the survival probability of patients with high and low MTFR2 expression. (D) The association between MTFR2 expression levels and patient age, clinical stage, pathological type, histological grade, depth of tumor invasion, treatment response, etc. (E) Western blot was also used to detect the expression of MTFR2 in HEEC cells, HEC-1A cells, Ishikawa cells and KLE cells. GAPDH was the internal reference protein. *, P<0.05, **, P<0.01 vs. normal/control/HEEC. BMI, body mass index; CI, confidence interval; CR, complete response; EC, endometrial cancer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HR, hazard ratio; mRNA, messenger RNA; MTFR2, mitochondrial fission regulator 2; PD, progressive disease; PR, partial response; SD, stable disease; TPM, transcripts per million.

MTFR2 regulated cell proliferation and apoptosis in EC cells

The CCK-8 assay results indicated that the cell survival rate in the Ov-MTFR2 group was significantly higher compared to the Ov-NC group. Conversely, the cell viability in the Sh1-MTFR2 and Sh2-MTFR2 groups was significantly lower than that in the Sh-NC group (Figure 2A-2D). These findings were corroborated by the colony formation assay, which demonstrated a significantly higher colony formation rate in the MTFR2 overexpression group relative to the control group, whereas the MTFR2 knockdown group exhibited a significantly reduced colony formation rate (Figure 2E-2H). Furthermore, EdU assays revealed a significant increase in the EdU positive rate of cells in the MTFR2 overexpression group, while a significant decrease was observed in the MTFR2 knockdown group (Figure 2I-2N). Flow cytometric analysis of apoptosis was also carried out, and it was found that overexpression of MTFR2 reduced apoptosis, while knockdown of MTFR2 caused an increase in apoptosis in EC cells, indicating that MTFR2 is involved in apoptosis resistance (Figure 3).

Figure 2 Effects of MTFR2 on cell proliferation in EC cells. (A-D) The CCK-8 assay was used to measure the cell survival rate. (E-H) The colony formation assay was employed to detect the colony formation ability of EC cells (crystal violet staining). (I-N) The EdU assay was used to assess the cell proliferation rate, as indicated by the EdU positive rate. *, P<0.05, **, P<0.01, ***, P<0.001 vs. Ov-NC/Sh-NC. Ov-NC, the pcDNA-normal control; Ov-MTFR2, the MTFR2 overexpression plasmid group; Sh-NC, the RNA knockdown control group; Sh1-MTFR2, the MTFR2 knockdown #1 group; Sh2-MTFR2, the MTFR2 knockdown #2 group. CCK-8, Cell Counting Kit-8; EC, endometrial cancer; EdU, 5-ethynyl-2'-deoxyuridine; MTFR2, mitochondrial fission regulator 2; OD, optical density.

Figure 3 Effects of MTFR2 on apoptosis in EC cells. (A) Flow cytometry profile of MTFR2 overexpression in HEC-1A cells. (B) Quantitative analysis of apoptosis in HEC-1A cells following MTFR2 overexpression. (C) Flow cytometry profile of HEC-1A cells following MTFR2 knockdown. (D) Quantitative analysis of apoptosis in HEC-1A cells following MTFR2 knockdown. (E) Flow cytometry profile of MTFR2 overexpression in Ishikawa cells. (F) Quantitative analysis of apoptosis in Ishikawa cells following MTFR2 overexpression. (G) Flow cytometry profile of MTFR2 knockdown in Ishikawa cells. (H) Quantitative analysis of apoptosis in Ishikawa cells following MTFR2 knockdown. *, P<0.05, **, P<0.01. Ov-NC, the pcDNA-normal control; Ov-MTFR2, the MTFR2 overexpression plasmid group; Sh-NC, the RNA knockdown control group; Sh1-MTFR2, the MTFR2 knockdown #1 group; Sh2-MTFR2, the MTFR2 knockdown #2 group. EC, endometrial cancer; MTFR2, mitochondrial fission regulator 2.

MTFR2 promoted migration and invasion in EC cells

Further experiments demonstrated that MTFR2 significantly accelerates the migration and invasion of endothelial cells (Figure 4). The results of the Transwell assay showed that MTFR2 overexpression significantly enhanced the migratory capacity of EC cells (Figure 4G,4H,4I,4K), whereas MTFR2 knockdown resulted in a significant reduction in the number of migrating cells (Figure 4G,4H,4J,4L). Furthermore, MTFR2 overexpression also significantly enhanced the cells’ invasive capacity (Figure 4M,4N,4P,4Q), whilst knockdown resulted in a significant decrease in the number of invading cells (Figure 4M,4O,4P,4R).

Figure 4 Effects of MTFR2 on migration and invasion of EC cells. (A-F) The wound healing assay was utilized to evaluate the migratory ability of EC cells. (G-R) Transwell assays were employed to measure both the migration and invasion capabilities of EC cells (crystal violet staining). *, P<0.05, **, P<0.01 vs. Ov-NC/Sh-NC. Ov-NC, the pcDNA-normal control; Ov-MTFR2, the MTFR2 overexpression plasmid group; Sh-NC, the RNA knockdown control group; Sh1-MTFR2, the MTFR2 knockdown #1 group; Sh2-MTFR2, the MTFR2 knockdown #2 group. EC, endometrial cancer; MTFR2, mitochondrial fission regulator 2.

MTFR2 regulated Drp1/MFN1 signaling pathway activity

Western blot results showed that the phosphorylation levels of Drp1 in the Ov-MTFR2 group were significantly higher than those in the Ov-NC group (Figure 5A-5D). Meanwhile, the expression of MFN1 in the Ov-MTFR2 group were significantly lower than those in the Ov-NC group (Figure 5A-5D). In contrast, Drp1 phosphorylation levels were significantly lower in the Sh1-MTFR2 and Sh2-MTFR2 groups than in the Sh-NC group (Figure 5E-5H). In contrast, Drp1 phosphorylation levels were significantly lower in the Sh1-MTFR2 and Sh2-MTFR2 groups than in the Sh-NC group (Figure 5E-5H).

Figure 5 MTFR2 regulated the activity of the Drp1/MFN1 signaling pathway. (A) Western blot analysis of p-Drp1, total Drp1, and MFN1 in Ishikawa cells after MTFR2 overexpression. (B) Quantification of p-Drp1, total Drp1, and MFN1 in Ishikawa cells following MTFR2 overexpression. (C) Western blot analysis of p-Drp1, total Drp1, and MFN1 in HEC-1A cells after MTFR2 overexpression. (D) Quantification of p-Drp1, total Drp1, and MFN1 in HEC-1A cells following MTFR2 overexpression. (E) Western blot analysis of p-Drp1, total Drp1, and MFN1 in Ishikawa cells after MTFR2 knockdown. (F) Quantification of p-Drp1, total Drp1, and MFN1 in Ishikawa cells following MTFR2 knockdown. (G) Western blot analysis of p-Drp1, total Drp1, and MFN1 in HEC-1A cells after MTFR2 knockdown. (H) Quantification of p-Drp1, total Drp1, and MFN1 in HEC-1A cells following MTFR2 knockdown. Total Drp1 and MFN1 were normalized against GAPDH; p-Drp1 was normalized against total Drp1. Western blot analysis was performed to detect the expression of Drp1, p-Drp1, and MFN1. GAPDH was the internal reference protein. *, P<0.05, **, P<0.01 vs. Ov-NC/Sh-NC. Ov-NC, the pcDNA-normal control; Ov-MTFR2, the MTFR2 overexpression plasmid group; Sh-NC, the RNA knockdown control group; Sh1-MTFR2, the MTFR2 knockdown #1 group; Sh2-MTFR2, the MTFR2 knockdown #2 group. Drp1, dynamin-related protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MFN1, mitochondrial fusion protein mitofusin 1; MTFR2, mitochondrial fission regulator 2; p-Drp1, phospho-Drp1.

Knockdown of MTFR2 can inhibit the occurrence of EC

MTFR2 shRNA was transfected into HEC-1A and Ishikawa cell lines to establish subcutaneous xenograft tumor models in nude mice, with the aim of investigating the in vivo effects of MTFR2. Silencing of MTFR2 resulted in the inhibition of tumor growth in vivo. Mice with MTFR2 knockdown exhibited a significant reduction in tumor weight (Figure 6A-6F). In addition, IHC results showed that MTFR2 knockdown significantly reduced the expression levels of Ki-67 and PCNA in tumor tissues (Figure 6G-6N).

Figure 6 Effects of MTFR2 knockdown on EC development in vivo. (A-F) MTFR2 shRNA was transfected into HEC-1A and Ishikawa cell lines, and subcutaneous xenograft tumor models were established in nude mice. Tumor growth was monitored and tumor weight was weighed. (G-N) IHC staining was employed to detect the expression levels of Ki-67 and PCNA in the tumor tissues. **, P<0.01 vs. Sh-NC. Sh-NC, the RNA knockdown control group; Sh2-MTFR2, the MTFR2 knockdown #2 group. EC, endometrial cancer; IHC, immunohistochemistry; MTFR2, mitochondrial fission regulator 2; PCNA, proliferating cell nuclear antigen; shRNA, short hairpin RNA.

Discussion

MTFR2 has been identified as a crucial regulator that facilitates tumor cell proliferation, migration, and invasion across various cancer types. Research indicates that increased expression of MTFR2 in breast cancer, gastric cancer, lung adenocarcinoma, hepatocellular carcinoma, and other malignancies is significantly associated with adverse patient outcomes (14,16-18). In the context of breast cancer, MTFR2 overexpression is significantly correlated with clinical staging, T classification, N classification, M classification, human epidermal growth factor receptor 2 (HER2) expression, and molecular subtypes, with a particularly strong association observed in HER2-positive and triple-negative breast cancer (TNBC) subtypes (16). Additionally, MTFR2 is regarded as a potential biomarker for immunotherapy targeting cancer stem cells (18). In gastric cancer, elevated MTFR2 expression is linked to a poorer prognosis, whereas its downregulation leads to a reduction in cancer cell proliferation, migration, and invasion (19). Niu et al. observed that MTFR2 expression was elevated in endometrial carcinoma and positively correlated with poor patient prognosis, consistent with the findings of this study. Additionally, they demonstrated that MTFR2 promotes endometrial carcinoma cell proliferation and growth via the miR-132-3p/PI3K/Akt signaling pathway. This suggests that the PI3K/Akt pathway could be a promising target for regulating the progression of endometrial carcinoma mediated by MTFR2 (20).

Moreover, MTFR2 is associated with immune cell infiltration, indicating its potential involvement in the immune microenvironment (18). In hepatocellular carcinoma, MTFR2 facilitates cell proliferation, migration, and invasion by modulating the PI3K/AKT signaling pathway (16). Research has further demonstrated that MTFR2-dependent mitochondrial fission accelerates tumor progression and is associated with immune cell infiltration and immune checkpoint expression within the tumor microenvironment (18,21). These findings imply that MTFR2 not only contributes to the malignant behavior of cancer cells but may also influence tumor progression by modulating the tumor immune microenvironment. In the current study, it was observed that the mRNA expression level of MTFR2 was significantly elevated in EC tissues compared to normal endometrial tissues. The expression level of MTFR2 increased concomitantly with tumor malignancy. Furthermore, the expression level of MTFR2 is closely associated with the patient’s age, clinical stage, pathological type, histological grade, tumor invasion depth, and treatment response. These data suggest that MTFR2 is not only a critical molecular marker but may also play a pivotal role in the diagnosis, prognostic evaluation, and development of individualized treatment strategies for EC.

The pathogenesis of EC is intricately linked to the imbalance between cellular proliferation and apoptosis (22). Existing research underscores the necessity of maintaining this critical balance for normal cellular functions, with its disruption potentially resulting in tumorigenesis and cancer progression (23,24). In the present study, we identified that MTFR2 facilitates the proliferation of EC cells. Furthermore, overexpression of MTFR2 was observed to decrease apoptosis rates, while MTFR2 knockdown led to an increase in apoptosis. Concurrently, both overexpression and knockdown of MTFR2 induced S-phase arrest in HEC-1A and Ishikawa cell lines. Aberrant regulation of the cell cycle is pivotal in tumor cell proliferation, and S-phase arrest may disrupt cellular DNA synthesis and repair, consequently impacting normal cell proliferation and apoptosis (25). The modulation of cell proliferation and apoptosis by MTFR2 is likely mediated through its influence on cell cycle progression. Moreover, migration and invasion are important links in tumor metastasis (26,27). The results of the present study suggest that cells in the MTFR2 overexpression group had significantly increased migration and invasion abilities, while those in the MTFR2 knockdown group had significantly decreased migration and invasion abilities. Therefore, MTFR2 may promote EC cell migration and invasion by regulating mechanisms such as cytoskeletal reorganization, intercellular adhesion, and matrix degradation. In addition, the expression level of MTFR2 was closely related to the clinical stage, histological grade, and depth of tumor invasion of EC, further supporting its importance in EC metastasis. To further validate the functional role of MTFR2 in EC, a xenograft tumor model with MTFR2 knockdown was established in nude mice. The results demonstrated that tumor growth was significantly suppressed in the MTFR2 knockdown group compared to the control group.

Mechanistically, we found that MTFR2 activated the Drp1/MFN1 signaling pathway in EC cells. Concurrently, the activation of Drp1 is primarily implicated in mitochondrial fission, a critical aspect of maintaining mitochondrial homeostasis. Drp1 modulates mitochondrial morphology, distribution, and function through its role in mediating mitochondrial fission. Research has demonstrated that polyphyllin VII triggers mitochondrial apoptosis in human ovarian cancer cells by modulating the PP2A/AKT/Drp1 signaling axis, thereby reinforcing the pivotal role of the AKT/Drp1 pathway in apoptosis (28). In a model of hypoxia-induced pulmonary hypertension, NDRG1 influences cell proliferation, apoptosis, and migration by regulating the Drp1 signaling pathway (29). Similarly, aerobic exercise reduces Drp1 expression, thus inhibiting the progression of hepatocellular carcinoma (30). Furthermore, the inhibition of Cdk5 activity can reduce apoptosis in neuroblastoma through the Erk/Drp1 pathways, further substantiating the involvement of the Drp1 signaling pathway in cellular invasion (31).

Limitations

While the present study elucidates the significant role of MTFR2 in EC and its underlying mechanisms, several limitations persist. Primarily, the research is predominantly based on in vitro and animal models, necessitating further clinical investigations to validate the applicability of these findings in human subjects. Additionally, the regulatory mechanism of MTFR2 on the Drp1/MFN1 signaling pathway requires further exploration. Future research should focus on two main areas: firstly, conducting large-scale clinical studies to confirm the viability of MTFR2 as a prognostic biomarker and therapeutic target for EC; secondly, undertaking comprehensive investigations into the interactions between MTFR2 and the Drp1/MFN1 signaling pathway, as well as other related pathways, to provide a theoretical foundation for the development of more effective therapeutic strategies.

Conclusions

This study elucidated that MTFR2 modulates mitochondrial fission via the Drp1/MFN1 signaling pathway, consequently affecting the proliferation, apoptosis, migration, and invasion of EC cells. These processes are closely linked to the initiation, progression, and prognosis of EC. These findings suggest that MTFR2 could be a promising novel target for the diagnosis, prognostic evaluation, and therapeutic intervention of EC.

Acknowledgments

We thank the Natural Science Foundation of Gansu Province for their support. We appreciate the Medical Frontier Innovation Research Center at The First Hospital of Lanzhou University for providing the experimental platform. We also thank Dr. Jinmin Ma (Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, Lanzhou, China) for his expert technical assistance.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2793/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2793/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2793/prf

Funding: This study was supported by the Natural Science Foundation of Gansu Province (No. 23JRRA1259) and the Lanzhou Science and Technology Plan Program (No. 2023-2-13).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2793/coif). X.B. reports that this work was supported by the Natural Science Foundation of Gansu Province (No. 23JRRA1259) and the Lanzhou Science and Technology Plan Program (No. 2023-2-13). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed in strict accordance with the Regulations for the Administration of Laboratory Animals. It was reviewed and approved by the Ethics Committee of The First Hospital of Lanzhou University (No. LDYYLL2023-01). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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