NEMF inhibits the proliferation and migration of ovarian cancer cells by blocking the PI3K/mTOR pathway

Abstract

Background

Nuclear export mediator factor (NEMF) is considered to be a tumor suppressor in Drosophila. Based on the analysis of data from an online database, NEMF is downregulated in OV. However, the mechanism underlying its expression has not been reported thus far.

Results

In this study, the immunohistochemistry results showed that the expression of NEMF may be related to patient age and tissue differentiation. In vitro experiments indicated that overexpression of NEMF may inhibit cell proliferation and migration by inhibiting the PI3K/mTOR signaling pathway, thereby blocking cell cycle progression and promoting apoptosis (P < 0.05). In vivo xenografts showed NEMF overexpression reduced tumor volume and proliferation markers (Ki-67, PCNA).

Conclusions

We speculate that NEMF is a potential tumor suppressor gene. Targeting NEMF may be an innovative therapeutic approach for OV.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-026-05302-8.

Background

Ovarian cancer (OV) is associated with high mortality rates, posing a serious threat to women’s health and lives worldwide [1, 2]. According to data from the Global Cancer Research Agency published in 2022, the annual incidence rate of OV has reached 6.5/100,000 individuals, with approximately 320,000 new cases and 200,000 deaths recorded worldwide [3]. The incidence of OV is second only to that of cervical cancer and endometrial cancer among female reproductive system tumors. However, it has the highest mortality rate among these tumors [4, 5]. The early symptoms of this type of cancer are relatively insidious; thus, most patients are diagnosed at an advanced stage of the disease [6], resulting in a poor prognosis [7]. Therefore, exploring the biological characteristics and potential therapeutic targets of OV is essential to improve the survival rates and quality of life of patients.

Studies have shown that cell signaling pathways play an important role in tumor development and occurrence [8], particularly the phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway [9]. This pathway plays a crucial role in regulating cell proliferation, apoptosis, deformation, and migration [10, 11]. Moreover, its abnormal activation is closely related to the occurrence of numerous types of cancer, including OV [12–14]. PI3K regulates cell growth and proliferation by activating downstream AKT, which in turn affects mTOR [15]. The PI3K pathway is commonly upregulated in OV [16], and PI3K signaling is associated with improved survival and chemoresistance [17]. Thus, therapeutic strategies targeting this pathway are becoming a hotspot in research on antitumor therapy.

Nuclear export mediator factor (NEMF) is a relatively newly recognized tumor-related gene. At present, there is limited knowledge regarding this gene. NEMF encodes a component of the ribosomal quality control complex [18, 19]. The encoded protein contributes to the ubiquitin ligase Listerin recognition and ubiquitination arrested 60 S subunit [20]. Similar proteins in Drosophila act as tumor suppressors [21]. The exact role of NEMF in cancer and the underlying mechanism remain unknown. Therefore, the aim of this study was to preliminarily investigate the role of NEMF in the proliferation and migration of OV cells through the PI3K/mTOR pathway and elucidate the underlying molecular mechanism.

Methods

Subcellular localization analysis

We utilized the HPA(Human Protein Atlas,http://www.proteinatlas.org) database as a resource to investigate the subcellular localization of key genes in OV cells [22]. Differentially expressed genes (DEGs) and protein–protein interactions (PPIs) were also identified.

In this study, we screened DEGs between tumor tissue samples from TCGA(The Cancer Genome Atlas, https://xenabrowser.net/datapages/?dataset=TCGA-OV.star_counts.tsv) database using the DESeq2, edgeR, and limma software packages. In addition, we created heatmap and volcano map visualizations using the Pheatmap R package [23]. The PPIs for all common DEGs were analyzed using the search tool for retrieval of interacting genes (STRING) database [24].

Functional evaluation

We queried the biological processes annotated in the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The adjusted P-value threshold for rejecting the null hypothesis was set to 0.05. Explains the most important eight GO terms and the KEGG pathway.

Study on the infiltration pattern of immune cells

Using the TIMER (Tumor Immune Estimation Resource, http://timer.comp-genomics.org/) database, we analyzed the distribution patterns and functional contributions of various immune subpopulations in the tumor microenvironment, including B cells, two T-cell subtypes (CD4 + and CD8+), neutrophils, macrophages, and dendritic cells.

Cell culture and reagents

Two human OV cell lines were used, namely OVCAR3 and SKOV3. The human normal ovarian epithelial cell line IOSE80 was also used. OV cell lines were cultured in RPMI 1640 Medium (Vivacell, Shanghai, China) containing 10% fetal bovine serum (Nobimpex, Herbolzheim, Germany) at 37 °C in a 5% CO₂ atmosphere. Culture dishes were purchased from Jet Biofil (Guangzhou, China).

Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA from OVCAR3, SKOV3, and IOSE80 cells was extracted using the Total RNA Isolation Kit (Foregene, Chengdu, China). Using the Evo M-MLV RT Mix Kit with gDNA Clean for quantitative PCR (AG, Hangzhou, China), mRNA was converted into cDNA, which was used to amplify target genes through quantitative PCR. Quantitative real-time PCR was performed using the CFX96 Real-Time System (Bio-Rad). The following primer sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used: GAPDH-forward: 5′-GGACCTGACCTGCCGTCTAG-3′, GAPDH-reverse: 5′-GTAGCCCAGGAT GCCCTTGA-3′. The following primer sequences of NEMF were used: NEMF-forward: 5′-TACTTG ACACCAGGAGAACCC-3′, NEMF-reverse: 5′-CCAAGCAGCACTGTAGCAA A-3′.

Western blotting

The cells were lysed and the protein concentration was determined using the BCA protein assay kit (Tiangen Biotechnology Co., Ltd., Beijing, China). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Finally, a high-sensitivity chemiluminescent reagent (Epizyme, Shanghai, China) was used to visualize specific proteins. The following antibodies were purchased from Proteintech (Wuhan, China): NEMF (11840-1-AP; 1:1000), E-cadherin (20874-1-AP; 1:5000), N-cadherin (22018-1-AP; 1:5000), vimentin (10366-1-AP; 1:5000), β-actin (60008-1-Ig; 1:5000), GAPDH (60004- 1-Ig; 1:5000), cyclin D1 (CCND1; 26939-1-AP; 1:1000), P21 (10355-1-AP; 1:1000), B-cell lymphoma 2 (BCL-2; 60178-1-Ig; 1:1000), BCL-2-associated X (BAX; 50599−2-Ig; 1:1000), and mTOR (66888-1-Ig; 1:1000). Antibodies against phosphorylated-PI3K (p-PI3K; 17366 S; 1:1000), PI3K (4292 S; 1:1000), and p-mTOR (5536 S; 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against tubulin (AP0064; 1:5000) was obtained from Bioworld (Nanjing, China).

Tissue samples and immunohistochemical staining

Fifty human OV tissue specimens and five normal ovarian tissues were obtained from the Third Affiliated Hospital of Southern Medical University (Guangzhou, China). The use of clinical specimens was approved by the ethics committee of the Third Affiliated Hospital of Southern Medical University. Streptavidin/Peroxidase and 3,3’-diaminobenzidine (DAB) kits (purchased from ZSGB BIO, Beijing, China) were used for immunohistochemical staining. Immunohistochemical staining was performed according to the instructions provided by the manufacturer. The score was determined by multiplying the score of staining intensity by the score of the proportion of positive cells (0–12 points). Proportion score: Positive cells < 25% (1 point), 25–50% (2 points), 50–75% (3 points), > 75% (4 points). Strength score: − (0 points), + (1 points), ++ (2 points), +++ (3 points). Total scores of 0–6 and 7–12 indicate low and high expression, respectively. Staining scores were calculated with the assistance of two senior doctors in the Department of Pathology.

Lentivirus infection

The full-length sequences of NEMF and FLAG-Tag were inserted into the lentivirus (Genechem, Shanghai, China), which contains green fluorescent protein (GFP). To construct cell lines with stable overexpression of NEMF(OE-NEMF group), OVCAR3 and SKOV3 cells were infected with GFP empty vector or full-length NEMF GFP vector(NC group).

Cell counting kit-8 (CCK-8) assay

Cells (2,000 cells/well) were seeded in 96-well plates, and quadruplicate wells were prepared for each condition. CCK-8 reagent (Vazyme) was added every 24 h for 5 days. Two hours after treatment with CCK8 reagent, optical density in each well was measured at 450 nm wavelength.

5-Ethynyl-2’-deoxyuridine (EdU) stainin

Cells (6000 cells/well) were seeded in 96-well plates, and triplicate wells were prepared for each condition. Cells were incubated with EdU reagent (10 µM) at 37 °C for 2 h and subsequently fixed in 4% paraformaldehyde. EdU staining was performed according to the instructions provided by the manufacturer (Cell-Light EdU Apollo567 in vitro kit; Ribobio, Guangzhou, China).

Colony formation assay

Cells (500 cells/well) were seeded in six-well plates. The cells were incubated at 37 °C in a 5% CO₂ atmosphere for 1 week. Thereafter, the colonies were gently washed twice with phosphate-buffered saline (PBS), fixed in paraformaldehyde, and stained using crystal violet.

Wound-healing assay

The transfected OV cells were seeded in six-well plates when they reached 95% confluency. When cells were full, a scratch in each well was performed using a 100 µl pipette tip, and the wells were washed thrice with PBS to remove detached cells. Cells were cultured using serum-free medium. Images were captured with a microscope at regular intervals.

Phalloidin staining experiments

1 × 10⁵ cells were placed into the dish. Following adherence, the cells were fixed with 4% paraformaldehyde, and 0.5% Triton X-100 solution was used to permeate the cells. After washing with PBS, phalloidin (200 µl) was added to each dish. Following incubation at room temperature for 1 h, DAPI was added to leave overnight.

Cell cycle analysis

After transfection, a total of 106 cells were digested and centrifuged. Subsequently, the cells were resuspended with 70% ethanol and left overnight at 4 °C. Following treatment of the cells at 37 °C and 4 °C for 30 min each with the Cell Cycle Assay Kit (DojinDo, Shanghai, China), the DNA content was measured using a FACS bore flow cytometer (BD Biosciences).

Animal experiments

OVCAR3 cells (5 × 10⁶) from either the NC or OE-NEMF groups were injected subcutaneously into the hind legs of 5-week-old female BALB/c nude mice purchased from Ruige Biotechnology(Guangzhou, China), and one week later, we measured the length and width of the tumors every 3 days. The tumor volume was calculated using the formula: volume = (length × width²)/2. The maximal permitted tumor size approved by the Southern Medical University was a maximum tumor diameter of 15 mm and a tumor burden not exceeding 10% of the animal’s body weight.

After 12 days, the mice were sacrificed and the length, width and weight of the tumors were recorded. Tumors were collected in paraformaldehyde and embedded in paraffin for HE staining and IHC staining, and the expression of PCNA and Ki-67 was used to detect the proliferation of tumor cells.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0 software. Data are expressed as the mean±standard deviation from three independent experiments.For normally distributed data, comparisons were performed using a Student’s t-test for two groups, one-way analysis of variance (ANOVA) for multiple groups.The association between NEMF expression and clinicopathological features was analyzed using the chi-square test.Survival analysis was performed using the Kaplan-Meier method. Univariate Cox regression analysis was conducted to evaluate prognostic factors. A P value < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Results

OV DEG screening

OV DEGs were screened using microarray datasets from the TCGA database. mRNA profiles of OV samples were standardized, and differential expression analysis was performed via the limma package in R, identifying 18,521 DEGs (17,768 high-confidence, 51 low-confidence; Fig. 1A). A cancer cell interaction-based central gene selection module and STRING enrichment revealed three downregulated central genes: NEMF, LTN1, and TCF25 (Fig. 1B), which are lowly expressed, interact in OV, and play key roles in protein synthesis and quality control. Linear regression showed NEMF’s genetic correlation coefficients with LTN1 and TCF25 were 0.36 and 0.26, respectively, and 0.27 between TCF25 and LTN1 (Fig. 1D).

Fig. 1.

Volcano plots, interaction, and pan-cancer analysis of genes. A Volcano plots of DEGs observed in the difference analysis dataset. B PPI network was constructed, leading to the identification of three pivotal hub genes. C Differences in the expression of NEMF in OV tissues and corresponding normal tissues from the GEPIA database. D Expression levels of NEMF, LTN1, and TCF25. E Expression of NEMF mRNA at the pan-cancer level. NEMF mRNA expression levels in 33 human tumor tissues and corresponding normal tissues from the GEPIA database (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001)

Pan-cancer analysis and gene interaction

In GEPIA, NEMF was expressed in all tissues. TCGA analysis showed NEMF was significantly downregulated in 18 cancer types (e.g., adrenal cortical carcinoma, breast invasive carcinoma) and upregulated in 12 cancer types (e.g., cholangiocarcinoma, acute myeloid leukemia; Fig. 1E).

NEMF mRNA expression and infiltration analysis in OV

Public datasets and clinical samples showed reduced NEMF mRNA/protein levels in OV vs. normal tissues (Fig. 1C). GO analysis linked enriched genes to biological processes like neuron cell body function and small GTPase signaling (Figure S-A). Cell type distribution revealed high NEMF expression in ovarian stromal cells (c-0, c-1, etc.) and some smooth muscle cells (c-9, etc.), but low expression in oocytes and granulosa cells (Figure S-B).

NEMF expression vs. tumor-infiltrating immune cells

Immune infiltration analysis (Figure S-C) and TIMER verification showed NEMF, LTN1, and TCF25 correlated with OV-infiltrating immune cells. NEMF was negatively associated with B cells, CD8 + T cells, etc. LTN1 correlated negatively with B cells but positively with CD4 + T cells. TCF25 showed opposite trends to LTN1, forming a complex gene-immune cell network (Figure S-D).

OV subcellular localization and decreased NEMF levels were associated with adverse outcomes.

The subcellular localization of NEMF in OV cells was studied using the HPA database. The NEMF gene encodes A protein that is present in the cytoplasm and nucleus (Figure S-E). The results of the overall survival and progression-free survival analysis showed that upregulation of NEMF was positively associated with the survival of patients with OV. Patients with high NEMF expression had longer overall survival (P = 0.036, hazard ratio = 0.79) (Fig. 2A) and progression-free survival (P = 0.036, hazard ratio = 0.80) (Fig. 2B) than those with lower NEMF expression. These results indicate that NEMF may be useful for predicting the survival of patients with OV.

Fig. 2.

Expression and prognostic value of NEMF protein in OV. A OS curve of high/low NEMF expression group in OV. B PFS curve of high/low NEMF expression group in OV. C Immunohistochemical staining. The expression of NEMF was higher in normal tissues versus OV tissues. D Basal expression of NEMF at the protein level (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, data are mean ± SD, n = 3)

Immunohistochemical staining was performed to further confirm the protein levels of NEMF. NEMF protein expression was significantly downregulated in OV tissues compared with normal tissues (Fig. 2C). Chi-squared test and univariate Cox proportional hazards analysis showed that the expression of NEMF may be related to age and tissue differentiation (Tables 1 and 2). In summary, the present findings demonstrated that NEMF expression is reduced in OV and may be associated with poor prognosis.

Table 1.

Correlation of NEMF expression in OV with clinicopathologic characteristics

Characteristics	The expression of NEMF		Chi square value	P value
	Low expression	High expression
Age			3.944	0.047
≥ 55	25	4
<55	13	8
FIGO			2.211	0.137
Ⅰ+Ⅱ	13	7
Ⅲ+Ⅳ	25	5
Lymph metastasis			1.873	0.1711
N stage	18	3
T stage	20	9
Differentiation			5.032	0.0249
Low	29	5
High	9	7

Table 2.

Analysis of risk factors affecting the prognosis of OV based on the univariate Cox proportional hazards model

Characteristics	Total(N)	HR(95% CI)	P value
FIGO		0.774 (0.148–4.042)	0.761
Ⅰ+Ⅱ	20
Ⅲ+Ⅳ	30
Lymph metastasis		0.470 (0.112–1.971)	0.302
N stage	29
T stage	21
Differentiation		1.912 (0.447–8.183)	0.382
Low	34
High	16

OE of NEMF inhibited the proliferation and migration of OV cells

We first verified the basal expression of NEMF at the protein level. NEMF expression was significantly higher in normal ovarian epithelial cells (IOSE80) than in OV cell lines (OVCAR3 and SKOV3). This suggests that NEMF plays an important role in the physiological function of the ovary, and its decreased expression in OV cells may be related to the occurrence and development of tumors. These results were consistent with those obtained from the bioinformatics analysis (Fig. 2D).

To investigate the mechanism through which NEMF affects the proliferation and migration of OV cells, we constructed NEMF-overexpressing viruses and cultured OVCAR3 and SKOV3 cell lines with stable OE of NEMF. The transfection efficiency of GFP was observed and quantified by fluorescence microscopy (Fig. 3A). Next, we investigated the phenotype by formal validation of OE. The efficiency was verified by quantitative PCR and western blotting (Fig. 3B-C). NEMF expression was significantly higher in the OE group versus the negative control (NC) group, indicating that the OV cell lines (OVCAR3 and SKOV3) were successfully and stably transfected with the NEMF-overexpressing virus.

Fig. 3.

Expression levels of NEMF. A Green fluorescence transfection efficiency. B, C NEMF overexpression validated by WB and quantitative PCR. (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, data are mean ± SD, n = 3)

After verifying the OE efficiency, we investigated the proliferation and migration of OV cells with OE of NEMF in vitro. Firstly, CCK-8 assay results showed that the optical density values at 24, 48, 72, and 96 h were higher in the NC group versus the OE group in both OVCAR3 and SKOV3 cells (Fig. 4A). Secondly, the EdU staining results showed that the proportion of OVCAR3 and SKOV3 cells in the proliferative phase was lower in the OE group than in the NC group (Fig. 4B). The plate cloning experiment also yielded consistent results. In OVCAR3 and SKOV3 cells, fewer and smaller cell clones were formed in the OE group versus the NC group (Fig. 4C). The above results indicate that OE of NEMF can inhibit OV cell proliferation.

Fig. 4.

NEMF inhibited the proliferation of OVCAR3 and SKOV3 cells in vitro. A CCK-8 assay was used to detect the OD value of OV cells for 5 consecutive days. B EdU proliferation assay was used to detect cells in the proliferation phase in the NC and OE groups. C The number and size of cell clones in the NC and OE groups were measured by plate cloning after 7 days (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, data are mean ± SD, n = 3)

In addition, wound healing experiments showed that the healing ability of cells was significantly weakened in the OE group compared with the NC group (Fig. 5A). The phalloidin staining experiments also showed changes in the cell morphology and a reduction in the number of microfilaments in the cytoskeleton in the OE group compared with the NC group (Fig. 5B). Results of western blotting analysis of cell migration proteins indicated that the NC group expressed higher levels of N-cadherin protein and lower levels of E-cadherin protein compared with the OE group (Fig. 5C). The above results showed that OE of NEMF inhibited the migratory ability of cells.

Fig. 5.

NEMF inhibited the migration of OVCAR3 and SKOV3 cells in vitro. A A wound healing experiment was conducted to compare the distance of migration between the NC and OE groups. B The migratory ability of cells was detected by phalloidin staining experiments. C Expression results for proteins related to cell migration (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, data are mean ± SD, n = 3)

We sought to further understand the specific mechanisms through which NEMF affects OV cell proliferation. Thus, we used flow cytometry to study the cell cycle of OV cells after OE of NEMF. Compared with the NC group, the OE group exhibited a higher proportion of cells in the G1 phase and a lower proportion of cells in the G2 and S phases following OE of NEMF (Fig. 6A). The results showed that OE of NEMF prevented the cell cycle transition from the G1 phase to the S phase, resulting in cell arrest in the G1 phase.

Fig. 6.

Results of the cell cycle, apoptosis, and PI3K/mTOR pathway analyses. A The cell cycle in the NC and OE groups was detected by flow cytometry. B Cell cycle and expression of apoptosis-related proteins. C Expression of proteins related to the PI3K/mTOR signaling pathway (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, data are mean ± SD, n = 3)

Western blotting of cell cycle-associated proteins revealed higher levels of CCND1 in the NC group versus the OE group, and lower levels of P21 protein in the NC group versus the OVCAR3 and SKOV3 cell lines (Fig. 6B-C). It is well established that CCND1 promotes the transition from the G1 to the S phase, whereas P21 inhibits this transition. The above results revealed that OE of NEMF may arrest the cell cycle of OV cells in the G1 phase by inhibiting CCND1 and promoting the expression of P21. Western blotting results of apoptosis-related proteins showed that the expression of BCL-2 protein in the NC group was higher than that recorded in the OE group. Moreover, the expression of BAX protein was lower in the NC group versus the OE group (Fig. 6B-C). This evidence indicates that NEMF may promote apoptosis in OV cells.

Finally, according to previous data, it is well established that the PI3K/mTOR signaling pathway is often activated in OV and plays an important role in cell cycle and apoptosis. Therefore, we investigated whether OE of NEMF exerts an effect on the PI3K/mTOR signaling pathway. Results from western blotting revealed significantly lower levels of PI3K/mTOR signaling pathway signature proteins (i.e., p-PI3K/PI3K and p-mTOR/mTOR) in the OE group compared with the NC group (Fig. 6D), suggesting that OE of NEMF may inhibit the PI3K/mTOR signaling pathway. Based on the above findings, OE of NEMF may affect the expression of proteins involved in the cell cycle and apoptosis by inhibiting PI3K/mTOR signaling. These effects lead to cell cycle arrest in the G1 phase, promote apoptosis, and eventually inhibit the malignant biological behavior of OV cells.

Knockdown of NEMF promoted the proliferation and migration of OV cells

NEMF knockdown via siRNA (SI-NEMF) in OVCAR3 and SKOV3 cells may augment cell proliferation: CCK-8 assay exhibits elevated OD450 nm values, EdU staining reveals an increased proportion of proliferative cells, and colony formation assay showed enhanced clone formation efficiency with more and larger clones(Fig. 7A-D). Cell cycle progression is accelerated, upregulated CCND1 and downregulated P21. It may also enhance cell migration: wound-healing assay demonstrates accelerated scratch closure, downregulated E-cadherin、Vimentin and upregulated N-cadherin(Fig. 7E-G).

Fig. 7.

Knockdown NEMF and animal experiments. A NEMF knockdown validated by WB. B CCK-8 assay was used to detect the OD value of OV cells for 5 consecutive days. C EdU proliferation assay was used to detect cells in the proliferation phase in the NC and SI groups. D The number and size of cell clones in the NC and SI groups were measured by plate cloning after 14 days. E A wound healing experiment was conducted to compare the distance of migration between the NC and SI groups. F, G Expression results for proteins related to cell cycle and cell migration. H Comparison of xenograft tumor volume and weight of nude mice in NC group and OE-NEMF group. I Images of nude mice xenograft tumors. J HE staining and IHC staining of Ki67 and PCNA in subcutaneous xenograft tumors (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, data are mean ± SD, n = 3)

Animal experiments showed tumor-suppressive effect of NEMF in ovarian cancer

To investigate the potential effect of NEMF on ovarian cancer progression in vivo, we constructed a xenograft model of female BALB/c nude mice using OVCAR3 cells. The results showed that the tumor volume was significantly reduced in the OE-NEMF group compared with the NC group (Fig. 7H-I). After 19 days, the tumor tissue was fixed and embedded in paraffin. HE staining was used to examine the histological morphology of the sections, demonstrating typical tumor histomorphology. IHC showed that Ki-67 and PCNA levels were significantly reduced in tumor tissues in the OE-NEMF group compared to the NC group (Fig. 7J). These results demonstrate the tumor-suppressive effect of NEMF in ovarian cancer.

Discussion

OV is a common type of malignant gynecological tumors. The vast majority of ovarian tumors originate from the epithelium (90%) [25], including four subtypes (i.e., serous adenocarcinoma, clear cell carcinoma, endometrial adenocarcinoma, and mucinous adenocarcinoma) [26, 27]. Because the early clinical symptoms of OV are not obvious and non-specific, most patients are diagnosed at a clinically advanced stage of the disease, thereby missing the opportunity for radical cure [28–30]. Studies have shown that approximately 75% of patients with OV are clinically diagnosed at an advanced stage due to the lack of early symptoms [31, 32]. At present, the first-line treatment of OV involves cytoreductive surgery combined with platinum-based chemotherapy [33, 34]. However, most patients with OV eventually develop platinum resistance and experience tumor recurrence [35–38]. Consequently, the overall 5-year survival rate of these patients is only 30–40% [39].

NEMF is an essential component of the ribosome quality control complex (RQC). It extracts partially synthesized nascent chains from stalled ribosomes and degrades them through ubiquitin-mediated proteasome degradation [20, 40–42]. It has been reported that NEMF may be associated with neuromuscular diseases [43], and its mutations cause damage to the central nervous system [44]. The present study is the first to investigate the mechanism underlying the effects of NEMF in cancer.

Initially, we used online databases such as TCGA, GEPIA, Kaplan–Meier plotter and R language to carry out basic expression analysis, pan-cancer analysis, enrichment analysis, and prediction of survival and prognosis in relation to NEMF. The results showed that NEMF was downregulated in OV, and its low expression was closely related to the poor prognosis of OV. Based on OV cell lines and clinical samples, we performed in vitro experiments to verify the reliability of the database. The experimental results showed that the protein levels of NEMF in OV cell lines were significantly reduced compared with those recorded in normal cells. Immunohistochemical experiments also confirmed the low expression of NEMF in OV and its prognostic significance at the tissue level.

Functional experiments using OV cell lines (OVCAR3, SKOV3) and normal ovarian epithelial cells (IOSE80) demonstrated that NEMF acts as a tumor suppressor. Basal expression analysis showed lower NEMF protein levels in OV cell lines than in IOSE80 cells. NEMF overexpression (OE) inhibited OV cell proliferation (reduced CCK-8 OD values, EdU-positive cells, and colony formation), arrested the cell cycle at the G1 phase (downregulated CCND1, upregulated P21), suppressed migration (impaired wound healing, upregulated E-cadherin, vimentin, downregulated N-cadherin), and promoted apoptosis (downregulated BCL-2, upregulated BAX) [45–48]. Mechanistically, NEMF OE reduced the phosphorylation of PI3K and mTOR (p-PI3K/PI3K and p-mTOR/mTOR ratios), inhibiting the PI3K/mTOR pathway—a well-established regulator of OV cell growth and survival [49, 50]. NEMF knockdown had opposite effects, while in vivo xenograft experiments (using BALB/c nude mice) confirmed NEMF OE reduced tumor volume and expression of proliferation markers (Ki-67, PCNA).

Despite these findings, this study has several limitations. First, the clinical validation cohort was relatively small, and multi-center, large-sample studies are needed to confirm NEMF’s prognostic value. Second, the interaction between NEMF [51, 52] and other RQC components (LTN1, TCF25) in OV remains unexamined, leaving their potential synergistic effects unclear. Third, causality between NEMF and the PI3K/mTOR pathway was not fully validated due to the lack of rescue experiments; whether NEMF directly or indirectly regulates this pathway remains to be further verified. Fourth, current research on NEMF is extremely limited, and no existing bioinformatic or literature evidence supports a clear link between NEMF and the PI3K/mTOR pathway; our preliminary data only suggested a potential regulatory relationship, so the detailed crosstalk could not be fully elucidated. Finally, the immunodeficient nude mouse model cannot reflect the impact of NEMF on the tumor immune microenvironment, which is critical for ovarian cancer progression. Addressing these limitations will help refine our understanding of NEMF’s role in OV and facilitate its clinical translation.

Conclusions

In summary, the present results revealed that NEMF is downregulated in OV and can inhibit the proliferation and migration of OV cells. NEMF also inhibits the cell cycle and promotes apoptosis of OV cells by inhibiting PI3K/mTOR, thereby impairing the malignant biological behavior of OV cells. This evidence may provide new insight into the prognostic evaluation and molecular therapy of OV.

Limitations

This study has several limitations. First, the single-center clinical cohort was small, which may limit the generalizability of our findings; large-scale, multi-center studies are needed to validate the prognostic value of NEMF. Second, rescue experiments were not performed, so the direct causal link between NEMF and the PI3K/mTOR pathway remains to be confirmed. Third, immunodeficient nude mice were used in vivo, which cannot reflect the crosstalk between NEMF and the tumor immune microenvironment.

Author contributions

**WQ**: Data Curation; Formal Analysis; Methodology; Validation; Writing–Original Draft Preparation. **ZY**: Data Curation; Investigation; Software; Writing–Original Draft Preparation. **CZ**: Formal Analysis; Investigation; Project Administration; Writing–Original Draft Preparation. **FJ**: Investigation; Software; Validation. **RF**: Investigation; Supervision; Methodology. **SS**: Conceptualization; Funding Acquisition; Methodology; Writing–Review & Editing. **ZK**: Data Curation; Supervision; Writing–Review & Editing. Qian Wang and Ying Zhu contributed equally to this work.

Funding

This study was supported by Guangdong Yiyang Health Charity Foundation (JZ2024045),Guangdong Provincial Medical Research Fund (A2024755).

Data availability

All databases analyzed in this study are publicly accessible and the keyword searched is “NEMF” : The Human Protein Atlas (http://www.proteinatlas.org), The Cancer Genome Atlas, (https://xenabrowser.net/datapages/?dataset=TCGA-OV.star\_ counts. tsv), STRING (https://string-db.org/), TIMER (http://timer.cistrome.org/), Kaplan-Meier Plotter (www.kmplot.com), GEPIA (http://gepia.cancer-pku.cn/).The data used to support the findings of this study are available from the corresponding author upon request.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committees of the Third Affiliated Hospital of Southern Medical University(protocol code 2025-ER-075 and July 24, 2025 of approval).In vivo experiments were approved by the Animal Care and Use Committee of Southern Medical University and were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The participants’ right to be informed about the study was ensured and written informed consent was obtained from all individual participants included in the study.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.