RNF112 mediates immunosuppression to inhibit the proliferation of cervical cancer by Foxm1

Introduction

Cervical cancer is a malignant tumor originating from the cervix, and its development is closely associated with persistent infection with high-risk carcinogenic types of human papillomavirus (HPV) (1). Epidemiological data indicate that cervical cancer is the fourth most common cancer in women globally. In 2022, there were approximately 660,000 new cases of cervical cancer and 350,000 related deaths worldwide, with the highest incidence and mortality rates occurring in low- and middle-income countries (2). The clinical manifestations of cervical cancer are diverse, and there may be no obvious symptoms in its early stage (3). With the progression of the disease, patients may experience abnormal vaginal bleeding, increased secretions, and postcoital bleeding, among other symptoms (4). According to the American Cancer Society, approximately 70% of patients with cervical cancer are already at a locally advanced stage or beyond at the time of diagnosis. Therefore, early diagnosis is crucial for improving the survival rate in this patient population (5).

Cervical cancer screening is primarily conducted via cytological examination, colposcopy biopsy, and high-risk HPV testing (6,7). However, the examination of many patients is often delayed due to the difficulty of sample collection (8). With the rapid development of bioinformatics technology, in-depth investigations into the signal transduction, metabolic mechanisms, and related biomolecular targets of cervical cancer have become more frequent (9).

Despite the availability of prophylactic HPV vaccines, cervical cancer remains one of the most common malignancies in women. Notably, the incidence of cervical cancer in China has been increasing annually (10). The principal approaches in the treatment of cervical cancer typically include surgery, radiotherapy, and chemotherapy (11). Studies have shown that although chemotherapy drugs can provide a degree of efficacy in the treatment of cervical cancer, their acute hematological toxicity remains a concern; for instance, chemotherapy drugs can lead to a decline in patients’ immune function, thereby affecting the long-term curative effect (12). Immunosuppression is a mechanism of tumor-cell drug resistance and escape, in which tumor cells secrete chemokines to recruit immunosuppressive cells and thus downregulate the body’s antitumor immune response (13). Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), the two most important types of immunosuppressive cells, are highly expressed in a variety of tumors, and their expression levels are closely related to disease progression (14).

Ferroptosis is a type of an iron-dependent programmed cell death induced by lipid peroxidation. Recent studies have demonstrated that ferroptosis is closely associated with tumor occurrence, development, and treatment, exerting a critical function in tumor immune regulation. Examining this process in the context of the tumor microenvironment (TME) may provide insights into cancer immunity and promote the development of targeted cancer therapy (15). Ferroptosis is characterized by the iron-dependent accumulation of lipid peroxides (LPOs) to lethal levels (16). In recent years, ferroptosis has been proven to effectively kill various cancer cells, and progress has been made in sensitizing cancer cells to ferroptosis (17). Inhibition of system Xc^– or glutathione peroxidase 4 (GPX4) is a classic pathway for inducing ferroptosis, and research has shown that it can effectively kill drug-resistant cancer cells (18). Other work has examined how immune cells in the TME adapt to lethal levels of LPOs (18,19). The TME is an environment that promotes the occurrence of ferroptosis via fatty acids, redox stress, and cystine competition, among other factors (20). Characterizing the different responses of immune cells after treatment with ferroptosis inducers and clarifying the complex ferroptosis-related crosstalk between tumor cells and immune cells may yield novel strategies for the treatment of cancer (21).

Forkhead box protein M1 (Foxm1), a member of the forkhead box family, features a characteristic winged-helix DNA-binding domain and plays a critical role in the occurrence and progression of various malignant tumors (22). It is significantly associated with poor clinical prognosis in patients with cancer. Foxm1 controls the G1/S and G2/M phase transitions by transcriptionally activating cell cycle-related genes (including cyclin B1, centromere protein A, and centromere protein B) (23). Recent studies have shown that excessive activation of Foxm1 can maintain low levels of reactive oxygen species in gastric cancer, ensuring the survival of gastric cancer stem cells and promoting chemoresistance in gastric cancer cells (23,24). Additionally, Foxm1 can mediate ferroptosis resistance in melanoma cells, ensuring tumor cell survival (25). On the other hand, pharmacological inhibition of Foxm1 expression significantly reduces glucose metabolism levels and weakens the proliferative capacity of tumor cells. Given its pivotal role in tumorigenesis and development, Foxm1 is regarded as a promising therapeutic target for cancer (26).

The ring finger protein family is a group of proteins containing a ring finger domain, and ring finger protein 112 (RNF112), an important member of this family, plays crucial roles in various cellular biological processes (27). Previous studies have found that certain RNF gene family members, including RNF181, RNF182, and RNF146, exert a variety of effects in different cancers, either promoting or inhibiting cancer (27,28). RNF112 has been found to be frequently methylated in gastric, pancreatic, and liver cancers, but research on esophageal cancer remains sparse (29). RNF112 is a key member of the ring finger protein family, and other family members have also been shown to play important roles in the occurrence and development of a variety of tumor types (30). RNF112 methylation is significantly associated with tumor stage, degree of differentiation, lymph node metastasis, vascular tumor thrombus, and distant metastasis and serves as an independent risk factor for poor prognosis in patients with esophageal cancer (31). A recent study found that combined detection of SEPTIN9 and RNF112 methylation in plasma can achieve the early diagnosis of gastric cancer (32). Other work has revealed that RNF112 participates in DNA damage repair by downregulating RAD51 (32). In our study, we investigated the effects of RNF112 in cervical cancer and the related molecular mechanisms. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1325/rc).

Methods

Patients and quantitative real-time polymerase chain reaction

This study included 24 patients with cervical cancer (median age 56 years; age range 50–65 years) and 6 healthy controls (median age 54 years; age range 50–65 years). All cervical cancer tissues were confirmed to be positive for high-risk HPV (specifically HPV 16 and HPV 18). Serum and tissue samples were collected and immediately stored at 80 ℃. Total RNA was isolated via RNA isolator total RNA extraction reagent (Takara Bio, Kusatsu, Japan), and complement DNA (cDNA) was synthesized with PrimeScipt RT Master Mix (Takara Bio). Quantitative real-time polymerase chain reaction was performed with the Applied Biosystems 7500 sequence detection system (Thermo Fisher Scientific, Waltham, MA, USA). Relative levels of the sample messenger RNA (mRNA) expression were calculated and expressed as 2^△△Ct.

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments, and was approved by the Ethics Committee of the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University (Key Laboratory of Embryo Original Diseases; approval No. 2022JWKT-001). Written informed consent was obtained from all patients.

Cell culture and mice model of cervical cancer

H8 (cat. no. PCS-480-011; American Type Culture Collection; Manassa, VI, USA), HeLa (cat. no. SCSP-504; Cell Bank of the Chinese Academy of Sciences, Shanghai, China), SiHa (cat. no. TCHu113; Cell Bank of the Chinese Academy of Sciences), CaSki (HPV16; cat. no. CRM-CRL-1550; American Type Culture Collection), and HT-3 (cat. no. ml096567; mlBio, Shanghai, China) cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco). All animal experiments were approved by the Ethics Committee of The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University (Key Laboratory of Embryo Original Diseases; approval No. 2022JWKT-002) and were implemented strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice were inoculated with CaSki cells (1×10⁷ cells). Subsequently, 200 µL of negative or RNF112-knockdown (sh-RNF112) lentivirus (10⁹ PFU/mL) was injected into mice via the tail vein (12 mice per group). Mice were anesthetized via the intraperitoneal injection of 50 mg/kg pentobarbital sodium and then killed via cervical dislocation.

In vitro co-culture

Jurkat cells were transfected with negative, RNF112, small interfering RNA negative control (si-NC), or si-RNF112 via Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific). Jurkat cells were stimulated with anti-CD3/CD28 antibodies (1 µg/mL each) and interleukin (IL)-2 (50 U/mL) for 48 hours. CaSki cells were seeded into 24-well plates (2×10 cells/well) and cultured for 24 hours until 70% confluence was attained. The activated Jurkat cells were added to CaSki wells until a ratio of 1:2 (CaSki:Jurkat) was reached. CaSki cells were seeded in the lower chamber, and activated Jurkat cells were seeded in the upper chamber. The co-culture was continued for 24–72 hours.

Electron microscopy, histological, immunohistochemical, and immunofluorescence analyses

For immunohistochemical and immunofluorescence analyses, mouse tissue samples were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) as described in previous work (33). Tissue samples were observed under a fluorescence microscope (Axio Observer A1, Zeiss, Oberkochen, Germany) and a transmission electron microscope (80 kV) (H-7650, Hitachi, Tokyo, Japan) in a manner described in a previous study (34).

Cell viability and enzyme-linked immunosorbent assays

CaSki cells were seeded in 24-well plates at a density of 2×10⁴ cells per well (or in 96-well plates at a density of 5×10³ cells per well) and cultured for 24 hours until approximately 70% confluence was achieved. Jurkat cells were stimulated with anti-CD3/CD28 antibodies (1 µg/mL each) and IL-2 (50 U/mL) for 48 hours to simulate T-cell priming, following transfection with the respective plasmids via Lipofectamine 3000 or treatment with a Foxm1 inhibitor. The primed Jurkat cells were then co-cultured with CaSki cells at a ratio of 1:2 (CaSki:Jurkat). After co-culture for 0, 12, 24, 48, and 72 hours, cell viability was assessed via Cell Counting Kit-8 assay (cat. no. C0037; Beyotime Biotech Inc., Shanghai, China) in a manner described in a previous study (34). Additionally, the culture supernatant was collected following a 48-hour co-culture for subsequent enzyme-linked immunosorbent assay (ELISA) and detection of cytokine secretion. Detection methods of malonaldehyde (MDA) (A003-1-2, Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China), superoxide dismutase (SOD) (A001-3-2, Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China), glutathione (GSH) (A006-2-1, Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China), and glutathione peroxidase (GSH-PX) (A005-1-2, Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) was performed as described in a previous study (35). Absorbance was measured on a BioTek microplate reader (Agilent Technologies, Santa Clara, CA, USA). EdU kit (cat. no. C0075S, Beyotime), lactate dehydrogenase (LDH) activity kit (cat. no. C0016; Beyotime), and a commercial reagent kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) were used for the quantitative detection of caspase 3 (G01513), caspase 7 (H080), and caspase 9 (G01811). Absorbance was measured at 450 nm with a BioTek Synergy H1 fluorescent reader (Agilent Technologies).

Western blotting and immunofluorescence

Protein extracted from cells and animals were used for Western blotting detection, and cells were subjected to immunofluorescence analysis (34). RNF112 (cat. no. ab169757; Abcam, Cambridge, UK), Foxm1 (cat. no. ab81298), GPX4 (cat. no. ab125066), β-actin (1:10,000; cat. no. AC028; ABclonal Technology, Woburn, MA, USA), and anti-rabbit IgG (1:5000; cat. no. GB23303; Servicebio Technology Co., Ltd., Wuhan, China) were used in this study. Protein was measured with an BeyoECL Plus kit (cat. no. P0018S; Beyotime) and analyzed with Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNF112 (1:100; Abcam) and Foxm1 (1:100; Abcam) were used for immunofluorescence analyses.

Microarray hybridizations

Microarray hybridizations were executed using Applied Biosystems Human Genome Survey Arrays V2.0. DIG-UTP labelled cRNA was generated and linearly amplified from total RNA using the Chemiluminescent RT-IVT Labeling Kit v 2.0 (Applied Biosystems, Foster City, CA).

Statistical analysis

Significance was determined with the Student t-test or one-way analysis of variance followed by Tukey post hoc test, with a P value <0.05 being considered significant. Data are expressed as the mean ± standard deviation (SD).

Results

RNF112 expression in cervical cancer patients and cells

Microarray hybridizations were used to clarify the disease targets for the occurrence and progression of cervical cancer via DepMap Portal and Gene Expression Profiling Interactive Analysis 2 (GEPIA 2) platform. In patients with cervical cancer, RNF112 expression levels were downregulated (Figure 1A,1B), and the survival rate of those with CD4⁺ T high expression was higher than that of those with CD4⁺ T low expression (Figure 1C). Patients with a high expression of RNF112 had a better survival rate within 100 months (Figure 1C). However, after 100 months, the survival rate of patients with a high expression of RNF112 significantly decreased (Figure 1C). We examined 24 patients with cervical cancer and found that RNF112 mRNA expression was downregulated in patients with cervical cancer as compared with the healthy controls (Figure 1D). RNF112 mRNA and protein expression in cervical cancer cell lines was reduced as compared with that in H8 cells (immortalized human cervical epithelial cells) (Figure 1E,1F).

Figure 1 RNF112 expression in the mice model of cervical cancer or cervical cancer cells. RNF112 expression levels in patients with cancer (A: DepMap Portal; B: GEPIA 2), the cumulative survival in patients with cancer (C), the RNF112 expression levels in patients with cervical cancer (D), and the RNF112 mRNA and protein expression in cervical cancer cell lines (E,F). **, P<0.01; ***, P<0.001. GEPIA2, gene expression profiling interactive analysis 2; mRNA, messenger RNA; N, normal; RNF112, ring finger protein 112; T, tumor; TPM, transcripts per million.

sh-RNF112 promoted tumor cell growth in the mouse model of cervical cancer

The study investigated the effects of sh-RNF112 on a mouse model of cervical cancer. It was found that sh-RNF112, as compared to the control condition, significantly increased tumor weight (P<0.001; Figure 2A) and volume (P<0.001; Figure 2B,2C). Furthermore, sh-RNF112 treatment led to a reduction in the activity levels of caspase 3, 7, and 9 (P<0.001, P<0.001 or P<0.001; Figure 2D-2F), indicating reduced apoptotic activity in tumor tissues. Additionally, there was a significant upregulation in the mRNA expression of Cox2 (P<0.001, Figure 2G), MYC (P<0.001, Figure 2H), and tumor necrosis factor-alpha (TNF-α) (P<0.001, Figure 2I), along with a significant downregulation of TP53 mRNA expression (Figure 2J) (P<0.001).

Figure 2 sh-RNF112 promoted tumor cell growth in the mouse model of cervical cancer. Tumor weight (A), volume (B), and tumor organization (C). Caspase 3, 7, and 9 activity levels (D-F). Cox2, MYC, TNF-α, and TP53 mRNA expression (G-J). **, P<0.01, ***, P<0.001. Cox2, cyclooxygenase-2; mRNA, messenger RNA; MYC mRNA, c-MYC messenger RNA; sh-RNF112, short hairpin RNA targeting RNF112 gene; TNF-α, tumor necrosis factor-alpha; TP53, tumor protein p53.

Co-cultivation with RNF112 suppressed the cervical cancer cell proliferation

In the in vitro model of cervical cancer, upregulation of RNF112 significantly increased RNF112 mRNA expression (P<0.001; Figure 3A). This upregulation was associated with a significant increase in cell proliferation (P<0.001; Figure 3B), migration (P<0.001; Figure 3C), and EdU positivity (P<0.001; Figure 3D). Meanwhile, si-RNF112 treatment resulted in a significant decrease in RNF112 mRNA expression (P<0.01; Figure 3E), leading to reduced cell proliferation (P<0.001; Figure 3F), migration (P<0.001; Figure 3G), and EdU positivity (P<0.001; Figure 3H).

Figure 3 Cocultivation of RNF112 suppressed the proliferation of cervical cancer cells. RNF112 mRNA expression (A), cell proliferation (B), migration (C), and EdU-positive cells (D) were detected in the vitro model by RNF112. RNF112 mRNA expression (E), cell proliferation (F), migration (G), and EdU-positive cells (H) were detected in the vitro model by si-RNF112. Staining method: EdU staining. *, P<0.05, **, P<0.01, ***, P<0.001. EdU-positive cells, 5-ethynyl-2’-deoxyuridine-positive cells; mRNA, messenger RNA; RNF112, ring finger protein 112; si-RNF112, small interfering ring finger protein 112.

RNF112 suppressed immunosuppression in model of cervical cancer

In the mouse model of cervical cancer, sh-RNF112 reduced the number of CD4⁺ T cells and CD8⁺ T cells, inhibited IFN-γ expression in CD4⁺ T and CD8⁺ T cells, and increased the number of Tregs, CD4⁺ T cells, CCR5⁺ Tregs, and CCR5⁺ CD8⁺ cells (Figure 4A-4G). In the in vitro model of cervical cancer, RNF112 upregulation increased transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ), and IL-10 levels in Jurkat cells (Figure 4H-4J), while si-RNF112 reduced them (Figure 4K-4M).

Figure 4 RNF112 inhibited immunosuppression in the model of cervical cancer. The number of CD4⁺ T cells and CD8⁺ T cells (A,B), number of IFN-γ-producing cells in CD4⁺ T and CD8⁺ T cells (C,D), ratio of Treg⁺ CD4⁺ cells (E), ratio of CCR5⁺ Treg cells/CCR5⁺ CD8⁺ cells (F,G) in the mouse model of cervical cancer. TGF-β, IFN-γ, and IL-10 levels in Jurkat cells treated with RNF112 (H-J). TGF-β, IFN-γ, and IL-10 levels in Jurkat cells treated with si-RNF112 (K-M). *, P<0.05; **, P<0.01, ***, P<0.001. CCR5⁺ CD8⁺ cells, chemokine receptor 5-positive cluster of differentiation 8-positive T cells; CCR5⁺ Treg cells, chemokine receptor 5-positive regulatory T cells; CD4⁺ T, cluster of differentiation 4-positive T cells; CD8⁺ T, cluster of differentiation 8-positive T cells; IFN-γ, interferon-gamma; IL-10, interleukin-10; RNF112, ring finger protein 112; si-RNF112, small interfering ring finger protein 112; TGF-β, transforming growth factor-beta.

RNF112 reduced oxidative stress in T cells in the model of cervical cancer

In the mouse model of cervical cancer, knockdown of RNF112 significantly reduced MDA levels (P<0.01; Figure 5A) and significantly increased those of SOD and GSH-PX (P<0.01; Figure 5B,5C), indicating a reduction in oxidative stress. In the in vitro model of Jurkat cells, upregulation of RNF112 led to a decrease in reactive oxygen species (ROS) production and MDA levels (Figure 5D,5E) but an increase in SOD and GSH-PX levels (P<0.01; Figure 5F,5G). Conversely, si-RNF112 increased ROS production and MDA levels (P<0.01; Figure 5H,5I) while decreasing those of SOD (P<0.05; Figure 5J) and GSH-PX (P<0.01; Figure 5K).

Figure 5 RNF112 reduced oxidative stress in T cells in the cervical cancer model. MDA (A), SOD (B), and GSH-PX (C) levels in the mouse model. ROS (D), MDA (E), SOD (F), and GSH-PX levels (G) in the RNF112 in vitro model. ROS (H), MDA (I), SOD (J), and GSH-PX levels (K) in the si-RNF112 in vitro model. *, P<0.05, **, P<0.01. GSH-PX, glutathione peroxidase; MDA, malondialdehyde; RNF112, ring finger protein 112; ROS, reactive oxygen species; SOD, superoxide dismutase.

RNF112 reduced oxidative stress-induced mitochondrial damage in T cells in the cervical cancer model

RNF112 upregulation in Jurkat cells reduced mitochondrial CoCl2 levels and JC-1 assay levels, suggesting a decrease in mitochondrial damage (P<0.001; Figure 6A,6B); conversely, si-RNF112 increased these levels (P<0.01; Figure 6C,6D). Furthermore, RNF112 upregulation reduced the extracellular acidification rate and increased the oxygen consumption rate, whereas si-RNF112 had the opposite effect (Figure 6E-6H). Electron microscopy further confirmed that RNF112 upregulation reduced mitochondrial damage (Figure 6I,6J).

Figure 6 RNF112 reduced oxidative stress-induced mitochondrial damage in T cells of the mice model of cervical cancer. Mitochondria CoCl2 levels (A) and JC-1 assay findings (B) for the RNF112 in vitro model. Mitochondria CoCl2 levels (C) and JC-1 assay findings (D) for the si-RNF112 in vitro model. ECAR levels (E) and OCR levels (F) for the RNF112 in vitro model. ECAR levels (G) and OCR levels (H) for the si-RNF112 in vitro model. Mitochondrial damage under electron microscopy (I) and schematic of the description (J). **, P<0.01, ***, P<0.001. CoCl2, cobalt(II) chloride; DAPI, 4',6-diamidino-2-phenylindole; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-(trifluoromethoxy) phenylhydraz one; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide is a cationic, lipid-soluble fluorescent dye; OCR, oxygen consumption rate; RNF112, ring finger protein 112; ROS, reactive oxygen species; si-RNF112, small interfering ring finger protein 112; TCA, tricarboxylic acid.

RNF112 reduced ferroptosis in T cells in the cervical cancer model

In Jurkat cells, RNF112 upregulation increased LDH activity and iron concentration but decreased GSH activity and GPX4 protein expression (P<0.001, P<0.001, P<0.001, and P<0.001; Figure 7A-7D), while si-RNF112 reversed these effects (P<0.001, P<0.001, P<0.001 and P<0.001; Figure 7E-7H). In the mouse model, sh-RNF112 reduced FeRhNOX-1 and ferrous iron levels and increased GSH levels and GPX4 protein expression (P<0.001, P<0.001, P<0.001 and P<0.001; Figure 7I-7L).

Figure 7 RNF112 reduced ferroptosis in T cells in the cervical cancer model. LDH level (A), iron content (B), GSH activity level (C), and GPX4 protein expression (D) in the RNF112 in vitro model. LDH level (E), iron content (F), GSH activity level (G), and GPX4 protein expression (H) in the si-RNF112 in vitro model. FeRhNOX-1 (I) and ferrous iron level (J), GSH activity level (K), and GPX4 protein expression (L) in the mouse model. **, P<0.01, ***, P<0.001. FeRhNOX-1, FerroOrange-like rhodamine-based nitroxide probe for oxidative stress and iron detection-1; GPX4, glutathione peroxidase 4; GSH, glutathione; LDH, lactate dehydrogenase; RNF112, ring finger protein 112; si-RNF112, small interfering ring finger protein 112.

RNF112 suppressed Foxm1 expression in T cells in the cervical cancer model

The effects of RNF112 on oxidative stress-induced mitochondrial damage in T cells of the cervical cancer model were examined via gene chip analysis. Foxm1 expression was upregulated by sh-RNF112 in the mouse model of cervical cancer (Figure 8A). RNF112 upregulation induced RNF112 protein expression (P<0.001) and reduced Foxm1 protein expression in the tumor tissue of mice (P<0.001; Figure 8B). Immunohistochemistry showed that Foxm1 expression was upregulated by sh-RNF112 in the cancer tissue of cervical cancer mice model (Figure 8C). In Jurkat cells of the cervical cancer model, RNF112 upregulation induced RNF112 protein expression (P<0.001) and reduced Foxm1 protein expression (P<0.001; Figure 8D). Meanwhile, si-RNF112 suppressed RNF112 protein expression and induced Foxm1 protein expression (P<0.001; Figure 8E).

Figure 8 RNF112 suppressed Foxm1 expression in T cells of the cervical cancer model. Heatmap, KEGG analysis and GO-BP enrichment analysis (A), RNF112/Foxm1 protein expression in the mouse model (B), immunohistochemical image of Foxm1 expression (C), RNF112/Foxm1 protein expression in the RNF112 in vitro model (D), and RNF112/Foxm1 protein expression in the RNF112 in vitro model (E). ***, P<0.001. Foxm1, forkhead box protein M1; GO-BP, Gene Ontology Biological Process; KEGG, Kyoto Encyclopedia of Genes and Genomes; RNF112, ring finger protein 112; ROS, reactive oxygen species; sh-RNF112, short hairpin RNA targeting RNF112 gene.

Foxm1 inhibition reduced the effects of sh-RNF112 on tumor cell growth in the mouse model of cervical cancer

Inhibition of Foxm1 with thiostrepton (20 mg/kg) significantly reduced the effects of sh-RNF112 on tumor growth in the mouse model of cervical cancer. Foxm1 inhibition led to a significant decrease in Foxm1 and GPX4 protein expression (P<0.01; Figure 9A). Furthermore, there was a marked reduction in tumor weight (P<0.01) and volume (P<0.001) (Figure 9B-9D), with accompanying decreases in caspase 3, 7, and 9 activity levels (P<0.001, P<0.001 and P<0.001; Figure 9E), indicating reduced apoptosis. Additionally, the mRNA expression of Cox2, TNF-α, MYC, and TP53 was significantly altered (P<0.001, P<0.001, P<0.001 and P<0.001; Figure 9F).

Figure 9 Foxm1 inhibition mitigated the effects of sh-RNF112 on tumor cell growth in the mouse model of cervical cancer. Foxm1/GPX4 protein expression (A). Tumor volume (B), weight (C), and tumor organization (D). Caspase 3, 7, and 9 activity levels (E). Cox2, TNF-α, MYC, and TP53 mRNA expression (F). The number of CD4⁺ T cells and CD8⁺ T cells (G,H). The number of IFN-γ-producing CD4⁺ T and IFN-γ-producing CD8⁺ T cells (I,J). Ratio of CCR5⁺ CD8⁺ (K). Ratio of CCR5⁺ Treg cells and Tregs/CD4⁺ T cells (L,M). FeRhNOX-1 level (N), ferrous iron level (O), GSH activity level (P), and GPX4 protein expression (Q) in the mouse model of cervical cancer. *, P<0.05, **, P<0.01, ***, P<0.001. CCR5⁺ CD8⁺ cells, chemokine receptor 5-positive cluster of differentiation 8-positive T cells; CD4⁺ T, cluster of differentiation 4-positive T cells; CD8⁺ T, cluster of differentiation 8-positive T cells; Cox2, cyclooxygenase-2; FeRhNOX-1, FerroOrange-like rhodamine-based nitroxide probe for oxidative stress and iron detection-1; Foxm1, forkhead box protein M1; GPX4, glutathione peroxidase 4; GSH, glutathione; IFN-γ, interferon-gamma; IL-10, interleukin-10; mRNA, messenger RNA; MYC, c-MYC messenger RNA; sh-RNF112, short hairpin RNA targeting RNF112 gene; TNF-α, tumor necrosis factor-alpha; TP53, tumor protein p53.

The immune response was also modulated, as evidenced by the significant changes in the numbers of CD4⁺ T cells (P<0.05), CD8⁺ T cells (P<0.01), and IFN-γ-producing cells within these populations (P<0.001) (Figure 9G-9J). There was a significant decrease in the ratio of Tregs/CD4⁺ T cells, CCR5⁺ Tregs cells, CCR5⁺ CD8⁺ cells (P<0.001, P<0.001 and P<0.001; Figure 9K-9M). Importantly, indicators of ferroptosis, such as FeRhNOX-1, ferrous iron levels, and GSH activity, were significantly affected (P<0.001, P<0.001, P<0.001 and P<0.001; Figure 9N-9Q). These results highlight the potential of Foxm1 inhibition to mitigate tumor growth and immune response in this model.

RNF112 promoted Foxm1 ubiquitination to reduce Foxm1 activity expression

In addition, we discovered that RNF112 regulated the effects of Foxm1 on mitochondria-dependent ferroptosis in the mouse model of cervical cancer. In the Jurkat cells mouse model of cervical cancer, RNF112 upregulation induced RNF112 expression and reduced Foxm1 expression (Figure 10A). Three-dimensional model prediction indicated that the Foxm1 protein interacts with the RNF112 protein (Figure 10B). Immunoprecipitation analysis demonstrated that the Foxm1 WT protein interacts with the RNF112 WT protein, the Foxm1 WT protein does not interact with the RNF112 Mut protein, and the RNF112 Mut protein does not interact with the Foxm1 WT protein (Figure 10C). RNF112 upregulation promoted the ubiquitination of the Foxm1 protein in Jurkat cells of cervical cancer (Figure 10D). si-RNF112 reduced the ubiquitination of Foxm1 protein in Jurkat cells of cervical cancer (Figure 10D).

Figure 10 RNF112 promoted Foxm1 ubiquitination to reduce Foxm1 expression. (A) RNF112 and Foxm1 expression on immunofluorescence. (B) Three-dimensional model depicting the interaction of RNF112 protein with Foxm1 protein. (C) Interaction of RNF112 protein with Foxm1 protein. (D) Ubiquitination of Foxm1 protein. DAPI, 4',6-diamidino-2-phenylindole; Foxm1, forkhead box protein M1; IB, immunoblotting; IP, immunoprecipitation; RNF112, ring finger protein 112.

Discussion

Among women, cervical cancer has the fourth highest incidence and mortality among malignancies in the world (36), with approximately 570,000 new cervical cancer cases and 311,000 related deaths occurring annually worldwide (37). With the popularization and implementation of HPV vaccination and early cervical screening, the incidence of cervical cancer has decreased, but the overall prognosis of patients remains unsatisfactory (38). Early-stage cervical cancer (stages IB–IIA) is typically treated with surgery and cisplatin-based adjuvant radiotherapy and chemotherapy, yet a portion of patients still die from tumor recurrence or metastasis (39). Therefore, accurately predicting disease progression and evaluating patient prognosis are critical to the clinical management of cervical cancer (40). We found that RNF112 expression levels in the patients with cervical cancer or cervical cancer cells were downregulated, sh-RNF112 promoted tumor cell growth in the mouse model of cervical cancer, and co-cultivation with RNF112 suppressed the proliferation of cervical cancer cells. Li et al. reported that RNF112 inhibits colorectal cancer growth (28). Summarizing these results, we found that RNF112 is involved throughout the process of cervical cancer.

Cervical cancer is the most common gynecological malignant tumor, ranking second in incidence among Chinese women. The research into immunotherapy for cervical cancer has recently intensified (41,42). Studies have shown that, as with most other tumors, cervical cancer can inhibit the proliferation, differentiation, and activation of immune effector cells by secreting immunosuppressive factors, blocking cell signaling pathways, and reducing the body’s immune response, thereby enabling rapid tumor proliferation and metastasis (43-45). TGF-β1, IL-10, and PGE2 protein are the primary immunosuppressive factors (46,47). In this study, RNF112 suppressed immunosuppression in a mice model of cervical cancer, while RNF112 reduced the oxidative stress-induced mitochondrial damage to T cells. Yang et al. found that RNF112 is one prognostic biomarker for tumor immunity in colorectal cancer (48). Therefore, RNF112 appears to function as a reparative factor for the oxidative stress-induced mitochondrial damage associated with immunosuppression in cervical cancer.

Ferroptosis, a form of regulated cell death characterized by iron metabolism imbalance and lipid peroxidation, figure prominently in various pathological processes (49). Studies have shown that the occurrence of ferroptosis is closely associated with the progression of cervical cancer (50). Ferroptosis is involved in regulating lipid metabolism, iron ion homeostasis, mitochondrial metabolism, and redox processes in cervical cancer, playing a critical role in tumor immunity. It affects tumor immune escape and progression by modulating the phenotype and function of different cells in the immune microenvironment (51). LPO and the oxidative stress induced by ferroptosis can promote the polarization of M1-type macrophages, enhance tumor proinflammatory responses, and inhibit the functions of immunosuppressive cells such as MDSCs and Tregs, thereby disrupting immune suppression (52). The expression regulation of ferroptosis-related molecules such as GPX4 and solute carrier family 7 member 11 (SLC7A11) not only affects the sensitivity of tumor cells to immunotherapy but also directly acts on the activity and survival of effector cells such as T lymphocytes and dendritic cells, further enhancing or weakening the antitumor immune response (53). Targeting ferroptosis may hold clinical value in the treatment of cervical cancer. Inducing ferroptosis through nanomedicine and molecular targeting strategies can not only directly kill tumor cells but also enhance the antitumor immune response (54). In our experiments, RNF112 promoted ferroptosis in T cells in mice model of cervical cancer.

Foxm1 is an important member of the forkhead box transcription factor family and is involved in multiple aspects of tumor progression, including tumor cell proliferation, growth, angiogenesis, and epithelial-mesenchymal transition. The m6A methyltransferase KIAA1429 promotes cisplatin (diamminedichloroplatinum) resistance in gastric cancer cells by stabilizing Foxm1 expression (55). Studies have found that Foxm1 acts as a transcription factor to promote carcinogenesis in cervical cancer and that silencing Foxm1 can inhibit DDP resistance in cervical cancer cells (55,56). HNRNPA2B1-mediated m6A modification of Foxm1 promotes DDP resistance and inhibits ferroptosis in endometrial cancer cells by regulating LCN2 (56). We found that RNF112 suppressed Foxm1 expression in T cells in the mouse model of cervical cancer. Foxm1 inhibition reduced the effects of Sh-RNF112 on tumor cell growth in mice, while RNF112 promoted Foxm1 ubiquitination to reduce Foxm1 activity expression. Zhang et al. found that RNF112 suppresses the proliferation and invasion of gastric cancer through Foxm1 ubiquitination (29). Therefore, it is plausible that RNF112 can suppress the Foxm1 signaling axis to promote oxidative stress-induced mitochondrial damage-dependent ferroptosis in T cells of cervical cancer.

Conclusions

RNF112 promoted immunosuppression to suppress the proliferation of cervical cancer through oxidative stress-induced mitochondrial damage-dependent ferroptosis in T cells via the enhancement of Foxm1 ubiquitination (Figure 11). RNF112 may thus be an effective therapeutic strategy for patients with cervical cancer or other cancers.

Figure 11 RNF112 mediated immunosuppression to suppress the proliferation of cervical cancer via Foxm1. Foxm1, forkhead box protein M1; GPX3, glutathione peroxidase 3; RNF112, ring finger protein 112; ROS, reactive oxygen species.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1325/rc

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Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1325/coif). The 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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University (Key Laboratory of Embryo Original Diseases; approval No. 2022JWKT-001) and written informed consent was taken from all the patients. All animal experiments were approved by the Ethics Committee of The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University (Key Laboratory of Embryo Original Diseases; approval No. 2022JWKT-002) and were implemented strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

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