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FOXO1 mediates miR-99a-5p/E2F7 to restrain breast cancer cell proliferation and induce apoptosis

BMC Cancer volume 25, Article number: 747 (2025) Cite this article

Abstract

Background

FOXO1 is known to act as a tumor suppressor gene in breast cancer, but its exact mechanism of action remains unclear.

Objective

This study aimed to clarify how FOXO1 suppresses breast cancer cell proliferation and induces apoptosis.

Methods

Breast cancer cell lines were generated with stable knockdown or overexpression of FOXO1. RT-qPCR and western blot assays were conducted to confirm transfection efficiency. CCK-8 and colony formation assays were used to assess cell proliferation, while flow cytometry measured apoptosis. The cells were subcutaneously injected into nude mice, and the volume and mass of the resulting tumors were evaluated. Immunohistochemistry was used to analyze Ki-67 expression in the tumors. A TUNEL assay examined apoptosis in the tumor cells. We performed bioinformatic analysis to identify FOXO1-targeted miRNAs and their downstream target mRNAs.

Results

Overexpression of FOXO1 inhibited breast cancer cell proliferation and promoted apoptosis. In contrast, knockdown of FOXO1 enhanced cell proliferation and reduced apoptosis. Among the downstream miRNAs we identified, miR-99a-5p was found to be downregulated in breast cancer tissue. FOXO1 binds to the miR-99a promoter, facilitating its transcription. Inhibition of miR-99a-5p partially reversed the effects of FOXO1 overexpression on cell proliferation and apoptosis. E2F7, a target mRNA of miR-99a-5p, showed a negative correlation with FOXO1 expression in breast cancer mRNAs we screened. Silencing E2F7 partially mitigated the inhibitory effects of miR-99a-5p on proliferation and apoptosis in FOXO1-overexpressing cells. E2F7 binds to the FOXO1 promoter, thus suppressing its transcription and reducing its expression.

Conclusion

FOXO1 suppresses breast cancer cell proliferation and promotes apoptosis by enhancing the transcription and expression of miR-99a-5p, while inhibiting its target gene E2F7. E2F7, in turn, represses the transcription of FOXO1, lowering its expression.

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Introduction

Breast cancer is the most common cancer among women and a leading cause of cancer-related deaths globally, accounting for about 24.5% of all new cancer cases [1]. Risk factors include pregnancy-related factors, obesity, smoking, age, and genetic mutations [2]. Despite significant advances in breast cancer treatment, outcomes and prognosis for patients with advanced disease remain poor. This is attributed to factors such as unclear pathogenesis, inadequate early screening, limited treatment options, complex disease phenotypes, and high recurrence rates [3]. Thus, exploring the molecular mechanisms behind breast cancer is essential.

Transcription factors play critical roles in various physiological processes by regulating DNA transcription and gene expression through specific DNA binding. Dysregulation of these factors in tumors can contribute to tumor-related characteristics [4]. Forkhead box (FOX) proteins form a vital family of transcription factors with distinctive winged helical DNA binding domains [5]. FOXO proteins represent a subgroup of these factors, with FOXO1—also known as FKHR, FKH1, or FOXO1A—being the most extensively studied. Located on chromosome 13q14.11, FOXO1 influences cell cycle arrest, apoptosis, DNA repair, and cell growth by regulating specific genes [6, 7]. Various studies have indicated that FOXO1 inhibits breast cancer progression. For instance, one study found that FOXO1 promotes the transcription of CYP1B1-AS1, which binds to the E1 subunit of the NEDD8 activating enzyme to regulate protein ubiquitination, thus inhibiting breast cancer cell proliferation and inducing apoptosis in vitro [8]. Another study demonstrated that FOXO1 can inhibit the Wnt/β-catenin signaling pathway and prevent the formation of the hnRNPK/β-catenin complex by promoting LYPLAL1-DT transcription, thereby limiting the proliferation, migration, and epithelial-mesenchymal transition of triple-negative breast cancer cells [9]. However, the detailed mechanisms by which FOXO1 inhibits breast cancer development require further exploration.

MicroRNAs (miRNAs) are highly conserved across species. These are non-protein-coding, single-stranded RNA molecules encoded by endogenous genes, typically consisting of 18–25 nucleotides. miRNAs play crucial roles in various cancers by functioning as oncogenes and tumor suppressor genes [10]. A previous study has shown that FOXO1 regulates the expression of miRNAs. For instance, FOXO1 can enhance the expression of miR-148a in hepatocytes by promoting the transcription of MIR148A [11]. Additionally, FOXO1 increases the expression of miR-502-3p while downregulating its target gene CDK6 in colorectal cancer cells, thereby inhibiting the growth of these cancer cells [12]. Another study has exhibited that FOXO1 suppresses the expression of its target gene ETS1 by upregulating miR-506 expression, which in turn inhibits the invasion and migration of glioblastoma multiforme cells and increasing their sensitivity to temozolomide [13]. Nevertheless, the mechanism by which FOXO1 mediates miRNAs to inhibit breast cancer development remains to be clarified.

In this study, we screened for miRNAs targeted by FOXO1 using bioinformatics and further validated the inhibitory effect of FOXO1-mediated miRNAs and their downstream target genes on breast cancer cell proliferation and apoptosis through in vitro and in vivo experiments. The aim of this study is to elucidate the specific mechanism by which FOXO1 acts as a tumor suppressor in breast cancer, potentially establishing it as a new target for breast cancer diagnosis and treatment.

Materials and methods

Cell culture and transfection

MCF7 (CL-0149), MDA-MB-231 (CL-0150) and HEK-293T cells (CL-0005) were purchased from Wuhan Pricella Biotechnology Co., Ltd., Wuhan, China. All cells were confirmed by STR. The cells were cultured in MEM (11090081, Gibco, USA), Leibovitz’s L-15 (11415064, Gibco) or DMEM medium (11965092, Gibco) containing 10% fetal bovine serum (10100147 C, Gibco) and 1% penicillin-streptomycin (SV30010, Hyclone, USA) in a cell culture incubator at 37 °C with 5% carbon dioxide. Transfection was performed following the experimental protocol, using a lentivirus transfection kit (Genepharma, China) to transfect FOXO1 knockdown, FOXO1 overexpression, E2F7 knockdown, E2F7 overexpression, and the corresponding control lentiviruses. Puromycin was utilized to select stably transfected cells. The Lipofectamine 3000 transfection kit (L3000150, Invitrogen, USA) instructions were strictly followed to transfect miR-99a-5p mimics, miR-99a-5p inhibitor, E2F7 siRNA, and the corresponding controls.

Real-time fluorescent quantitative PCR (RT-qPCR)

The transfected cells were collected and total RNA was extracted using the Trizol method (12183555, Invitrogen). Total RNA was reverse transcribed into cDNA using the HiScript III RT SuperMix for qPCR kit (R323-01, Vazyme, China), and subjected to PCR amplification with Taq Pro Universal SYBR qPCR Master Mix (Q712-02/03, Vazyme) or miRNA Unimodal SYBR qPCR Master Mix kit (MQ102-01/02, Vazyme) for mRNA and miRNA detection, respectively. For miRNA detection, reverse transcription primers were added during the reverse transcription step. GAPDH or U6 served as internal references, and the relative expression of the target gene was calculated by the 2−ΔΔCT method. The primer sequences used for RT-qPCR are listed in Table 1.

Table 1 Primer sequences of RT-qPCR
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Western blot assay

The transfected cells were collected and total protein was extracted using RIPA lysis buffer (P0013B, Beyotime, China). The protein concentration was determined using the BCA protein concentration determination kit (P0009, Beyotime) and the protein samples were prepared at a concentration of 2 µg/µL. After boiling, 20 µg of protein sample was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to a PVDF membrane. The membrane was incubated overnight at 4 °C with primary antibodies against FOXO1 (1:2000, ab179450, Abcam, UK), E2F7 (1:1000, 82887-2-RR, Proteintech, USA) and GAPDH (1:2500, ab9485, Abcam, UK). After washing with PBST, the membrane was incubated with the HRP conjugation goat anti-rabbit secondary antibody (1:5000, ab6721, Abcam, UK) at room temperature for 1 h. Following another wash, the membrane was developed using the BeyoECL Plus Kit (P0018M, Beyotime). ImageJ software was used for semi-quantitative analysis of the results.

CCK-8 assay

Transfected cells were evenly plated in a 96-well plate at a density of 3000 per well and cultured overnight. At the designated time points, the culture medium was discarded and replaced with serum-free culture medium containing CCK-8 reagent (C0041, Beyotime). After 1-hour incubation at 37 °C, the absorbance value at 450 nm was measured using a microplate reader (Epoch 2, BioTek, USA).

Colony formation assay

Transfected cells were evenly plated in 6-well plates at a density of 1000 per well and cultured with a complete medium for 14 days. The culture medium was discarded and residual culture medium was washed off with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min and washed with PBS. After that, the cells were stained with 0.2% crystal violet staining solution at room temperature for 1 h and leftover stain was washed off with running water. The cells were photographed after drying.

Cell apoptosis detected by flow cytometry

The Annexin V-FITC cell apoptosis detection kit (C1062L, Beyotime) was utilized to measure cell apoptosis by flow cytometry. Briefly, 2 × 106 transfected cells were collected and resuspended with 1 mL Annexin V binding buffer. A 100 µL aliquot of this suspension was taken, to which 5 µL of Annexin V-FITC mixture was added and incubated in the dark for 5 min. Following this, 10 µL of PI was added and incubated for an additional 5 min in the dark. Detection was performed using a flow cytometer (FACS Calibur, BD, USA).

Tumorigenesis in nude mice

Forty BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed in Experimental Animal Center of China Medical University. This study received approval from the Ethics Committee of China Medical University (CMUKT2024158). The transfected cells were collected and diluted to a concentration of 1 × 107 cells in 100 µL. Thirty µL of Matrigel was mixed with the cell suspension and injected subcutaneously into the armpit of nude mice. The mice were monitored for 21 days, during which time the lengths and widths of the tumors were measured every 4 days, and the tumor volume was calculated. At the end of the experiment, the mice were euthanized by cervical dislocation, and the tumors were removed and weighed. Collected tumor tissues were fixed in 4% paraformaldehyde, and paraffin sections were prepared using standard methods. Immunohistochemistry was performed to assess Ki-67 expression in the transplanted tumors. A TUNEL assay (C1086, Beyotime) was conducted to evaluate the apoptosis rate of the tumors.

Bioinformatics analysis

The GSE44124 dataset contained 50 breast cancer tissue samples and 30 normal breast tissue samples. Breast cancer tissue was designated as the experimental group, while normal breast tissue served as the control group. The miRNAs that were downregulated in breast cancer were identified based on the criteria of adj.P.Val < 0.05 and logFC<-1. The GSE156229 dataset consisted of 6 breast cancer tissue samples and 6 paracancerous tissue samples, with breast cancer tissue as the experimental group and paracancerous tissue as the control group. The mRNAs that were upregulated in breast cancer were screened based on the criteria of adj.P.Val < 0.05 and logFC > 1. The TransmiR v2.0 database (http://www.cuilab.cn/transmir) was used to predict the target miRNAs of FOXO1, while the Kaplan-Meier Plotter (https://kmplot.com/analysis/index.php?p=background) was utilized to analyze the correlation between miRNA expression and the prognosis of breast cancer patients. ENCORI (https://rnasysu.com/encori/) was employed to predict the downstream target mRNAs of miRNAs. The EPD database (https://epd.expasy.org/epd/) was used to identify E2F7 binding sites in the FOXO1 promoter region.

Chromatin Immunoprecipitation (ChIP)

The experiment was performed strictly according to the instructions provided in the ChIP kit (26156, Thermo Scientific, USA). Briefly, the cells were fixed in 1% formaldehyde and lysed in SDS lysis buffer. The chromatin was sonicated to obtain fragments approximately 500 bp in size, and 1 mg of protein lysate was used for ChIP analysis. After incubating the lysate with antibodies overnight at 4 °C, DNA was eluted and reverse cross-linked. The purified DNA was then analyzed by RT-qPCR.

Dual luciferase reporter gene experiment

After transfection as per the experimental protocol, the cells were cultured for 24 h. The Dual-Glo®Dual luciferase reporter gene detection system (E2920, Promega, USA) was used for testing. The culture medium was discarded and any residual medium was washed away with PBS. The supernatant was collected after the cells were lysed with PLB lysis buffer. The activities of firefly and Renilla luciferases were measured sequentially using a chemiluminescence apparatus (GloMax®20/20 Luminometer, Promega) and the ratio of the two provided the relative luciferase activity.

AGO2-RIP

The Imprint®RNA immunoprecipitation kit (RIP, Sigma-Aldrich, USA) was used to conduct RIP experiments according to the manufacturer’s instructions. After lysing the collected cells, they were immunoprecipitated with either IgG or AGO2 antibody along with magnetic beads. After extracting and purifying the precipitated RNA, E2F7 and miR-99a-5p levels were detected by RT-qPCR.

Statistical analysis

Data processing and statistical analysis were performed using SPSS 22.0 software. The measurement data that followed a normal distribution were expressed as the mean ± standard deviation. One-way analysis of variance was employed to compare multiple groups. Tukey’s post hoc test was used for pairwise comparisons. A P value of less than 0.05 was deemed statistically significant.

Results

FOXO1 restrains breast cancer cell proliferation and induces apoptosis

We first established stable knockdown and overexpression of FOXO1 in breast cancer cell lines MCF7 and MDA-MB-231, confirming transfection efficiency via RT-qPCR (Fig. 1A) and western blot analysis (Fig. 1B). Results from the CCK-8 assay (Fig. 1C) and colony formation assay (Fig. 1D) showed that FOXO1 knockdown increased proliferation activity and colony formation in both MCF7 and MDA-MB-231 cells. Conversely, FOXO1 overexpression reduced both proliferation and colony formation abilities. Flow cytometry results (Fig. 1E) indicated that FOXO1 knockdown lowered the apoptosis rate in MCF7 and MDA-MB-231 cells, while overexpression of FOXO1 increased the apoptosis rate. We further investigated the effects of FOXO1 on breast cancer cell proliferation and apoptosis in vivo. These findings revealed that FOXO1 knockdown increased the volume and mass of MCF7 and MDA-MB-231 cell transplants, whereas FOXO1 overexpression resulted in reduced volume and mass (Fig. 1F). Immunohistochemistry results (Fig. 1G) demonstrated that FOXO1 knockdown elevated Ki-67 expression in the cell transplants, while FOXO1 overexpression decreased Ki-67 expression. TUNEL assay results (Fig. 1H) showed that FOXO1 knockdown suppressed apoptosis, while FOXO1 overexpression enhanced apoptosis in the cell transplants. These results collectively indicate that FOXO1 inhibits breast cancer cell proliferation and promotes apoptosis.

Fig. 1
figure 1

FOXO1 restrains breast cancer cell proliferation and induces apoptosis. Expression of FOXO1 mRNA and protein in MCF7 and MDA-MB-231 cells after knockdown or overexpression of FOXO1 was measured using RT-qPCR (n = 3) (A) and western blot assay (n = 3) (B). Cell proliferation ability was assessed through the CCK-8 assay (n = 3) (C) and colony formation assay (n = 3) (D). Apoptosis levels were evaluated by flow cytometry (n = 3) (E). Subcutaneous injection of MCF7 and MDA-MB-231 cells into the axilla of nude mice, and the volume and mass (n = 5) (F) of xenograft were detected. Ki-67 expression in MCF7 and MDA-MB-231 tumor transplants was analyzed by immunohistochemistry (n = 5) (G). A TUNEL assay was conducted to assess apoptosis in the xenograft (n = 5) (H). *p < 0.05 vs. sh-NC group; #p < 0.05 vs. ov-NC group

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FOXO1 facilitates miR-99a-5p transcription

To explore how FOXO1 influences miRNA activity to inhibit breast cancer cell proliferation and promote apoptosis, we screened for downregulated miRNAs in breast cancer using the GEO database (Fig. 2A), identified miRNAs targeted by FOXO1 through TransmiR online data, and utilized Venn analysis to focus on downregulated miRNAs in breast cancer among these targets (Fig. 2B). Notably, high levels of hsa-miR-99a were associated with improved overall survival rates in breast cancer patients (Fig. 2C). We then confirmed whether FOXO1 promotes miR-99a-5p transcription. ChIP assay results (Fig. 2D) revealed significant enrichment of the miR-99a promoter following precipitation with FOXO1 antibodies. Moreover, dual luciferase reporter assays (Fig. 2E) demonstrated that FOXO1 overexpression enhanced the transcriptional activity of the miR-99a promoter. We assessed the effects of FOXO1 knockdown and overexpression on the expression levels of miR-99a-5p and miR-99a-3p in breast cancer cells (Fig. 2F and Figure S1). Results showed that FOXO1 knockdown decreased the expression of both miRNAs in MCF7 and MDA-MB-231 cells, while FOXO1 overexpression increased their expression. Correlation analysis based on the TCGA database indicated that the correlation coefficient between miR-99a-5p and FOXO1 expression was 1.6 times greater than that of miR-99a-3p (Fig. 2G). Therefore, we selected miR-99a-5p for subsequent studies.

Fig. 2
figure 2

FOXO1 facilitates miR-99a-5p transcription. MiRNAs that are downregulated in breast cancer were identified using the GEO database and visualize via volcano plots (A). miRNAs targeted by FOXO1 were screened using the TransmiR online tool, and those downregulated in breast cancer were further with Venn analysis (B). Correlation between the expression of hsa-miR-99a and the overall survival of breast cancer patients was investigated using Kaplan-Meier Plotter (C). ChIP experiment was conducted to confirm the binding of FOXO1 to miR-99a promoter (D) and dual luciferase reporter assays were performed (E). Expression of miR-99a-5p in MCF7 and MDA-MB-231 cells after knockdown or overexpression of FOXO1 was measured by RT-qPCR (F). Correlation between FOXO1 with hsa-miR-99a-5p and hsa-miR-99a-3p was analyzed using the TCGA database (G). (All n = 3) *p < 0.05 vs. sh-NC group, IgG group or Vector group; #p < 0.05 vs. ov-NC group

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FOXO1 suppresses breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p

To verify whether FOXO1 inhibits breast cancer cell proliferation and induces apoptosis via upregulation of miR-99a-5p, we examined the effects of miR-99a-5p inhibition on proliferation and apoptosis in MCF7 and MDA-MB-231 cells overexpressing FOXO1. Our findings indicated that inhibiting miR-99a-5p decreased its expression in both MCF7 and MDA-MB-231 cells expressing FOXO1 (Fig. 3A). Furthermore, miR-99a-5p inhibition partially reversed the inhibitory effects of overexpressed FOXO1 on cell proliferation (Fig. 3B) and colony formation (Fig. 3C), as well as its promotion of apoptosis (Fig. 3D). These results demonstrate that FOXO1 restrains breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p.

Fig. 3
figure 3

FOXO1 suppresses breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p. RT-qPCR were used to measure FOXO1 and miR-99a-5p expression in MCF7 and MDA-MB-231 cells following the inhibition of miR-99a-5p while overexpressing FOXO1 (A). Cell proliferation was evaluated through CCK-8 assay (B) and colony formation assay (C). Apoptosis levels were detected by flow cytometry (D). (All n = 3) *p < 0.05 vs. ov-NC group; #p < 0.05 vs. ov-FOXO1 group; $p < 0.05 vs. ov-FOXO1 + inhibitor NC group

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miR-99a-5p targeted Inhibition of E2F7 expression in breast cancer cells

miRNAs inhibit the expression of their target genes by binding to the 3’-UTR of the target gene mRNA [14]. To identify the target mRNAs of miR-99a-5p that are upregulated in breast cancer and negatively correlated with FOXO1 expression, we conducted a screen. We discovered that E2F7 was upregulated in breast cancer and negatively correlated with FOXO1 expression among the target mRNAs of miR-99a-5p (Fig. 4A-B). Dual luciferase reporter assays and AGO2-RIP confirmed the binding of miR-99a-5p to E2F7 mRNA (Fig. 4C and Figure S2). We further examined the effect of transfecting miR-99a-5p mimics or inhibitors on E2F7 expression in breast cancer cells. The results showed that transfection of miR-99a-5p mimics reduced E2F7 mRNA and protein levels in MCF7 and MDA-MB-231 cells, while transfection with miR-99a-5p inhibitors increased E2F7 mRNA and protein expression in MCF7 and MDA-MB-231 cells (Fig. 4D-E). These findings indicate that miR-99a-5p directly targets and inhibits E2F7 expression.

Fig. 4
figure 4

miR-99a-5p targeted inhibition of E2F7 expression in breast cancer cells. mRNAs that were upregulated in breast cancer were identified using the GEO database and visualized via volcano plots (A). The target mRNAs of miR-99a-5p that were upregulated in breast cancer and negatively correlated with FOXO1 expression were identified using Venn analysis (B). Dual luciferase reporter assays were used to confirm the binding of miR-99a-5p to E2F7 mRNA (C). The effects of transfecting miR-99a-5p mimics or inhibitors on E2F7 expression in breast cancer cells were measured by RT-qPCR (D) and western blot assays (E). (All n = 3) *p < 0.05 vs. inhibitor NC group; #p < 0.05 vs. mimics NC group

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FOXO1 restrains breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p to inhibit E2F7 expression

Additionally, we investigated how FOXO1 influences breast cancer cell proliferation and apoptosis through the miR-99a-5p/E2F7 pathway. We have first verified whether FOXO1 regulates E2F7 expression in breast cancer cells through miR-99a-5p. Overexpression of FOXO1 led to a decrease in E2F7 expression in MCF7 and MDA-MB-231 cells, and the inhibition of miR-99a-5p partially reversed this downregulation (Fig. 5A-B). Silencing E2F7 also partially restored proliferation (Fig. 5C) and colony formation abilities (Fig. 5D) in MCF7 and MDA-MB-231 cells overexpressing FOXO1 and mitigated the inhibition of apoptosis (Fig. 5E) produced by miR-99a-5p suppression. Moreover, we found that overexpression of E2F7 promotes the proliferation and inhibits apoptosis of MCF7 and MDA-MB-231 cells that overexpress FOXO1 (Figure S3). These results suggest that FOXO1 suppresses E2F7 expression by upregulating miR-99a-5p, thereby inhibiting breast cancer cell proliferation and inducing apoptosis.

Fig. 5
figure 5

FOXO1 restrains breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p to suppress E2F7 expression. After silencing E2F7 in the context of overexpressing FOXO1 and inhibiting miR-99a-5p, RT qPCR (A) and western blot assay (B) were used to measure E2F7 mRNA and protein expression. Cell proliferation was evaluated through CCK-8 assay (C) and colony formation assay (D). Apoptosis levels were measured by flow cytometry (E). (All n = 3) *p < 0.05 vs. ov-NC group; #p < 0.05 vs. ov-FOXO1 group; $p < 0.05 vs. ov-FOXO1 + inhibitor NC group; &p < 0.05 vs. ov-FOXO1 + miR-99a-5p inhibitor group; @p < 0.05 vs. ov-FOXO1 + miR-99a-5p inhibitor + si-NC group

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E2F7 binds to the FOXO1 promoter and restrains its expression

We analyzed the E2F7 binding site in the FOXO1 promoter using bioinformatics and confirmed its presence (Fig. 6A). ChIP assays (Fig. 6B) and dual luciferase reporter experiments (Fig. 6C and Figure S4) validated that E2F7 binds to the FOXO1 promoter. Overexpression of E2F7 resulted in reduced FOXO1 expression in MCF7 and MDA-MB-231 cells, while knockdown of E2F7 led to increased FOXO1 expression (Fig. 6D-E). These findings demonstrate that E2F7 binds to the FOXO1 promoter and downregulates its expression.

Fig. 6
figure 6

E2F7 binds to the FOXO1 promoter and restrains its expression. The E2F7 binding site in the FOXO1 promoter was analyzed using bioinformatics methods (A). ChIP experiment (B) and dual luciferase reporter assay (C) confirmed the binding of E2F7 to the FOXO1 promoter. The expression of FOXO1 mRNA and protein in MCF7 and MDA-MB-231 cells after knockdown or overexpression of E2F7 was assessed using RT-qPCR (D) and western blot assay (E). (All n = 3) *p < 0.05 vs. IgG group, Vector group or ov-NC group; #p < 0.05 vs. sh-NC group

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Discussion

FOXO1 plays a crucial role in various cellular processes, including cell cycle arrest, apoptosis, DNA repair, oxidative stress, cancer prevention, treatment, and resistance to chemotherapy/radiotherapy [15]. Down-regulation of FOXO1 is associated with poor prognosis in breast cancer patients [16]. Our study initially examined the influence of FOXO1 on breast cancer cell proliferation and apoptosis, revealing that its overexpression suppressed breast cancer cell proliferation and induced apoptosis. Conversely, knockdown of FOXO1 promoted both processes. We further validated the inhibitory impacts of FOXO1 on breast cancer cell proliferation and apoptosis at the in vivo level.

Previous studies have shown that FOXO1 can regulate tumor development by binding to the promoter of downstream target miRNAs and enhancing their transcription [12, 13]. To explore how FOXO1 mediates miRNAs affecting breast cancer development, this study first predicted the downstream target miRNAs of FOXO1 using the TransmiR online database. Recognizing the inhibitory role of FOXO1 in breast cancer, we screened for downregulated miRNAs in breast cancer tissues based on the GEO database, followed by Venn analysis to identify downregulated miRNAs among the downstream target miRNAs of FOXO1. Among those screened, high levels of hsa-miR-99a was associated with better overall survival in breast cancer patients, and there was a stronger correlation between the expression of hsa-miR-99a-5p and FOXO1 in breast cancer compared with hsa-miR-99a-3p. Therefore, we further explored whether FOXO1 mediates the effect of miR-99a-5p on breast cancer development.

miR-99a-5p is dysregulated in various tumors and acts as a tumor suppressor by inhibiting the proliferation, migration, and invasion of cancer cells [17,18,19]. In breast cancer, a recent study has found that miR-99a-5p restrains the proliferation and invasion of breast cancer cells and induces apoptosis by targeting and inhibiting CDC25A expression [20]. Our study confirmed that FOXO1 binds to the miR-99a promoter, and its overexpression enhances the transcriptional activity of this promoter. We also found that knocking down FOXO1 diminished miR-99a-5p expression in breast cancer cells, while its overexpression accelerated miR-99a-5p levels. Moreover, inhibiting miR-99a-5p partially reversed the inhibitory effect of overexpressed FOXO1 on breast cancer cell proliferation and apoptosis. In summary, FOXO1 suppresses breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p.

It is well known that miRNAs can bind to their target mRNAs to inhibit translation, thereby regulating oncogenes and tumor suppressor genes, and they are involved in various physiological and pathological processes in tumor cells [21] Based on bioinformatics analysis, we identified E2F7 as a target mRNA of miR-99a-5p. E2F7 is negatively correlated with FOXO1 expression among mRNAs upregulated in breast cancer. A dual luciferase reporter assay confirmed the binding of miR-99a-5p to E2F7 mRNA. We also demonstrated that transfecting miR-99a-5p mimics reduced E2F7 expression in breast cancer cells, while transfecting miR-99a-5p inhibitors increased E2F7 expression. The E2F transcription factor family is a key regulator of the eukaryotic cell cycle, apoptosis, and differentiation [22]. E2F7, a member of this atypical E2F family, contains an evolutionarily conserved DNA-binding domain that binds to the 5’-TTT[CG][CG]CGC-3’ sequence of target promoters, acting as a transcriptional repressor [23]. Previous studies have shown that E2F7 is upregulated in ER-positive breast cancer, which induces tamoxifen resistance in these cells [24, 25]. Additionally, another study has found that E2F7 promotes the development of triple-negative breast cancer by inhibiting ferroptosis [26]. In this study, overexpressing FOXO1 diminished E2F7 expression in breast cancer cells, while inhibiting miR-99a-5p partially reversed the downregulation of E2F7 by FOXO1. Furthermore, silencing E2F7 partially countered the effects of miR-99a-5p inhibition on the proliferation and apoptosis in breast cancer cells overexpressing FOXO1. Our results demonstrate that FOXO1 inhibits breast cancer cell proliferation and induces apoptosis by upregulating miR-99a-5p expression, which in turn inhibits E2F7 expression. Given that E2F7 acts as a transcriptional repressor, we analyzed potential binding sites for E2F7 in the FOXO1 promoter region and confirmed its binding, which suppresses FOXO1 transcription. We also demonstrated the inhibitory effect of E2F7 on FOXO1 expression.

In conclusion, FOXO1 restrains breast cancer cell proliferation and induces apoptosis by promoting the transcription of miR-99 A-5p, leading to the downregulation of its target gene E2F7. E2F7 suppresses FOXO1 expression by binding to its promoter, creating a positive feedback loop involving FOXO1, miR-99a-5p, and E2F7 that inhibits breast cancer development. However, there are currently no approved FOXO1 inhibitors for breast cancer treatment. AS1842856 is a widely used FOXO1-specific inhibitor, and in vitro studies have confirmed its ability to inhibit breast cancer cell proliferation [27]. Further clinical trials are needed to verify the potential of targeting FOXO1 in breast cancer treatment and to develop targeted drugs. This study has limitations, including the absence of patient-derived xenografts and clinical sample validation, which should be addressed in future research.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

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Funding

This study was supported by Science and Technology Joint Plan Project of Liaoning Province (2023011730-JH3/4500).

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Author notes

  1. Ying Zhang and Haili Wang contributed equally to this work and should be considered co-first authors.

Authors and Affiliations

  1. Department of Thyroid and Breast Surgery, the People’s Hospital of Liaoning Province, Shenyang, Liaoning, 110016, China

    Ying Zhang

  2. Department of Nursing, the People’s Hospital of Liaoning Province, No.33, Wenyi Road, Shenhe District, Shenyang, Liaoning, 110016, China

    Haili Wang & Bo Ma

  3. Department of Anesthesiology, the First Hospital of China Medical University, No.155, Nanjing North Street, Heping District, Shenyang, Liaoning, 110001, China

    Yiliang Wang

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  1. Ying Zhang
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Contributions

YZ and HLW completed experiment; YZ, HLW and YLW analyzed data; YZ, YLW and BM was a major contributor in writing the manuscript.

Corresponding authors

Correspondence to Yiliang Wang or Bo Ma.

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The procedures of mice handling and care were approved by the Ethics Committee for Animal Experiments of China Medical University (CMUKT2024158).

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Zhang, Y., Wang, H., Wang, Y. et al. FOXO1 mediates miR-99a-5p/E2F7 to restrain breast cancer cell proliferation and induce apoptosis. BMC Cancer 25, 747 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-025-14111-1

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-025-14111-1

Keywords

BMC Cancer

ISSN: 1471-2407

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