Novel N⁶-methyladenosine (m⁶A) writer METTL16 promotes the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis

Abstract

Iron-dependent programmed cell death (known as ferroptosis) is closely linked to cervical cancer progression. Here, the present research identified that novel N⁶-methyladenosine (m⁶A) writer methyltransferase-like 16 (METTL16) was a ferroptosis-related gene in cervical cancer, and more exploration was performed to investigate its function and mechanism. Results showed that METTL16 was elevated in the cervical cancer cells, and METTL16 functionally repressed the ferroptosis-related characteristic. Mechanistically, ferritin heavy chain 1 (FTH1) was identified as a direct target of METTL16 via the m⁶A-dependent manner. In vivo, METTL16 silencing repressed the cervical cancer tumor growth. Therefore, these findings revealed that novel m⁶A writer METTL16 promoted the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis, which providing distinct insights for cervical cancer.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-026-04403-8.

Introduction

Cervical cancer is the fourth most common cancer among women in the world [1]. The main treatment strategies for cervical cancer are hysterectomy, radiotherapy, chemotherapy and chemoradiotherapy [2]. However, the high recurrence and metastasis rate remains a major predicament faced in the treatment of cervical cancer. In view of the characteristics of cervical cancer, new treatment methods are also under development, including targeted therapy [3]. Therefore, targeted therapy based on the true pathogenesis of cervical cancer is more effective.

N⁶-methyladenosine (m⁶A) methylation is the most common type of mRNA alteration, which has been proven to promote or inhibit tumorigenesis in various tumor types, but research on cervical cancer is still relatively scarce [4]. Therefore, it is necessary to further explore the role of m⁶A methylation in cervical cancer and its influence on its tumor microenvironment [5]. Furthermore, the effect of m⁶A methylation on ferroptosis in cervical cancer tumors is not yet very clear. Therefore, this study explored the expression of the novel m⁶A RNA “writer” METTL16 in cervical cancer and its influence on ferroptosis.

Ferroptosis is a novel iron-dependent programmed cell death pattern, characterized by lipid peroxidation accumulation and closely related to the inhibition of glutathione peroxidase (GPX4) activity [6–8]. In recent years, studies have found that ferroptosis plays a key role in tumors, neurodegenerative diseases, ischemia-reperfusion injury and infectious diseases [9–11]. The regulatory mechanism involves pathways such as System Xc⁻-GSH-GPX4 and FSP1-CoQ10 [12]. Targeted ferroptosis (such as GPX4 inhibitors and iron chelators) has become a potential therapeutic strategy, but its molecular mechanism and tissue specificity still need to be further explored to provide a new direction for disease treatment.

Here, this research investigated the relationship between m⁶A methylation and cervical cancer ferroptosis. We thoroughly investigated the roles of METTL16 and investigate its mechanism in cervical cancer. Moreover, ferritin heavy chain 1 (FTH1) was identified as a direct target of METTL16 via the m⁶A-dependent manner. Therefore, these findings revealed that novel m⁶A writer METTL16 promoted the cervical cancer tumorigenesis via FTH1-dependent ferroptosis, which providing distinct insights for cervical cancer.

Materials and methods

Cell culture

Human cervical cancer cells (CaSki, HeLa) were provided by Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), as well as and human keratinocytes (HaCaT). Cervical cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) with 5% CO₂ at 37 °C. Erastin (10 µmol/L) was utilized for the ferroptosis analysis.

Single-cell RNA sequencing (scRNA-seq)

Samples (GSE208653, cervical tissue samples were collected from colposcopy examination and analyzed using scRNAseq) were dissociated using a combination of enzymatic digestion (Collagenase IV, 1 mg/mL) and mechanical disruption. Cell viability was assessed by Trypan Blue staining (> 90% viability required). Single-cell suspensions were then loaded onto a microfluidic chip (10x Genomics Chromium System) for droplet-based encapsulation and barcoding, followed by cDNA synthesis and library construction according to the manufacturer’s protocol. The final libraries were quantified by Qubit and sequenced on an Illumina NovaSeq platform (150 bp paired-end reads).

Quantification of intracellular ferrous ions

The collected cells (1 × 10⁶ cells) were lysed by Reagent I (0.2 mL) and incubated on ice for 10 min. The cellular Fe²⁺ level was tested using Cell Ferrous Iron Colorimetric Assay Kit (Elabscience). The absorbance was detected using a spectrophotometer at 593 nm.

FerroOrange measurement

FerroOrange fluorescent assay was performed to test the Fe²⁺ in cancer cells FerroOrange fluorescent probe (Servicebio, Cat. G1727). After the cellular collection, the probe was added to each group serum-free medium according to an instruction to prepare working solution (1 µM). Lastly, the images were obtained using a fluorescence microscope.

Glutathione (GSH) quantification and malondialdehyde (MDA) determination

The total cellular glutathione level was detected using GSH and GSSG Assay Kit (Beyotime) in accordance with the manufacturer’s instructions, and the absorbance at 412 nm was measured using a spectrophotometer. For MDA detection, the cells were lysed and treated with Lipid Peroxidation MDA Assay Kit (Beyotime). The absorbance at 532 nm was measured.

Transmission electron microscopy (TEM)

Cancer cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 2 h, followed by post-fixation in 1% osmium tetroxide for 1 h. After dehydration through a graded ethanol series (50–100%), the samples were embedded in Epon 812 resin and polymerized at 60 °C for 48 h. Ultrathin Sect. (70 nm) were cut using a diamond knife (Leica Ultracut), mounted on copper grids, and stained with uranyl acetate and lead citrate. Images were acquired under a Hitachi HT7700 transmission electron microscope operated at 80 kV.

RT-qPCR

Total RNA was extracted from cervical tumor cells using TRIzol reagent, and cDNA was synthesized with 1 µg RNA via reverse transcription (PrimeScript RT Reagent Kit). PCR amplification was performed using primers (METTL16, F-5′ TTCTGTCAAGGTCGGACAATG-3′, R-5′-CAGCACCACGAATGTTATGGG-3′; FTH1, F-5′-TCCTACGTTTACCTGTCCATGT-3′, R-5′-GTTTGTGCAGTTCCAGTAGTGA-3′) under the following conditions: 95 °C for 30 s, 55–60 °C for 30 s, and 72 °C for 30 Sect. (35 cycles). GAPDH served as an internal control.

Immunoprecipitation

Cervical cancer cells were lysed by immunoprecipitation buffer (protease, phosphatase inhibitors, MCE) containing magnetic beads conjugated to specific antibodies (anti-IGF2BP2, anti-METTL16, anti-m⁶A). For the control, the lysate was incubated with mouse IgG antibodies (Proteintech) overnight at 4 °C, followed by incubation with protein A/G magnetic beads. The beads were washed, eluted, and analyzed using PCR.

Immunofluorescence

Cervical cancer cells were fixed with 4% paraformaldehyde and treated with 0.3% Triton X-100 (15 min). Then, the cells were blocked with 5% bovine serum albumin at room temperature. Anti-METTL16 antibody (1:2000, 19924-1-AP, Proteintech), anti-FTH1 antibody (1:1000, 83428-1-RR, Proteintech), anti-IGF2BP2 antibody (1:1000, 11601-1-AP, Proteintech) acted as the primary antibody for incubation. FITC-conjugated goat anti-mouse IgG (ZF-0312) acted as the secondary antibody. The nuclei were labeled by DAPI staining solution.

RNA stability assay

Cervical cancer cells were seeded in 24-well plates and treated with actinomycin D (5 µg/mL, Act D) for 0, 4, 8, 12 h. RNA was extracted from cells at specified time points and determined by quantitative PCR. The half-life (t_1/2) of the FTH1 mRNA was calculated by linear regression analysis.

Animal experiments

BALB/c nude mice (female, 10, 4–5 weeks old) were purchased from Slac Laboratory Animal Center (Shanghai, China) and kept at Yichang Central People’s Hospital. All the animal experiments protocols had been approved by the Ethics Committee of Yichang Central People’s Hospital. The number of 5 × 10⁶ CaSki cells was injected subcutaneously into the right flank of the mice. The tumor volumes were calculated every 3 days. Finally, mice were sacrificed for the tumor weights and immunohistochemical staining. Our IRB permits a maximum tumor size and this ethical requirement was strictly adhered to throughout the study. All the animal studies were performed in accordance with the guidelines of the International Council for Laboratory Animal Science (ICLAS).

Immunohistochemical staining

All tissues were obtained from the mice tumor. IHC and HE staining were performed in tissues and using protocol methods.

Statistical analysis

All statistical tests were calculated using GraphPad Prism (version v9.0) software. The differences among several groups were calculated by Wilcoxon test and one-way analysis of variance (ANOVA). The differences between two groups were calculated by student t-test. A p-value less than 0.05 was regarded statistically significant.

Results

METTL16 up-regulated in the cervical cancer

To investigate the expression profile of differentially expressed genes (DEGs) in cervical cancer, the scRNA-seq was performed in GSE208653 (Fig. 1A). In the seq data, the numerous genes were tested, including METTL16, GPX4, FTH1, SLC7A11, LPCAT3 and NCOA4 (Fig. 1B). From them, the METTL16 and FTH1 were up-regulated in cervical cancer (Fig. 1C). In several cellular subtypes, the data revealed that METTL16 was higher in tumor than that in normal group (Fig. 1D, E). In cervical cancer cells, the METTL16 level was higher in cancer cells than that in normal cells (Fig. 1F). Overall, the data demonstrated that METTL16 up-regulated in the cervical cancer.

Fig. 1.

METTL16 up-regulated in the cervical cancer. A The scRNA-seq was performed based on the raw data of GSE208653. B The expression profile of differentially expressed genes (DEGs) in cervical cancer. In the seq data, the numerous genes were tested, including METTL16, GPX4, FTH1, SLC7A11, LPCAT3 and NCOA4. C The METTL16 and FTH1 level was shown in cervical cancer. D, E The METTL16 level in several cellular subtypes. F The RT-PCR was performed to verify the METTL16 level in cancer cells and normal cells. **p < 0.01

METTL16 inhibited the ferroptosis of cervical cancer cells

To explore how METTL16 affected the tumorigenesis of cervical cancer cells, we conducted attempts in cell function experiments. After numerous trials, the results indicated that ferroptosis was a pathway closely related to the function of METTL16. We constructed METTL16 overexpression and silencing transfection in two types of cells respectively to explore the role of METTL16 in ferroptosis of cervical cancer cells from gain/loss functional assay. Firstly, transmission electron microscopy (TEM) was used to detect the cellular substructures transformation. The results indicated that overexpression of METTL16 could enhance cellular structures, especially mitochondria. In contrast, METTL16 silencing could disrupt the structures (Fig. 2A). Regarding other characteristics related to ferroptosis (including iron ions, MDA, and GSH), the experimental results indicate that overexpression of METTL16 reduced the Fe²⁺ level in cervical cancer cells (Fig. 2B, 2E, 2F), and decreased the MDA level (Fig. 2C). Moreover, METTL16 silencing could promoted the GSH level (Fig. 2D). In summary, the data demonstrated that METTL16 inhibited the ferroptosis of cervical cancer cells.

Fig. 2.

METTL16 inhibited the ferroptosis of cervical cancer cells. A Transmission electron microscopy (TEM) was performed to detect the cellular substructures transformation in cervical cancer cells with Erastin (10 µmol/L). HeLa cells were transfected with overexpression of METTL16 plasmids (ov-NC, ov-METTL16). CaSki cells were transfected with silencing of METTL16 plasmids (sh-NC, sh-METTL16-1, sh-METTL16-2). B The Fe²⁺ level in cervical cancer cells was tested by Iron Colorimetric Assay Kit. C MDA was tested in cervical cancer cells. D GSH was tested in cervical cancer cells. E, F FerroOrange fluorescent assay was performed to test the Fe²⁺ in cancer cells. *p < 0.05, **p < 0.01

FTH1 acted as the target of METTL16

In order to identify the downstream proteins of METTL16, this study screened multiple genes related to ferroptosis. Eventually, we found that FTH1 has a synergistic correlation with METTL16. The immunofluorescence assay indicated that METTL16 and FTH1 have the same subcellular localization (Fig. 3A). Since METTL16 was an RNA methyltransferase, it could regulate the expression of target genes through the RNA methylation modification pathway. Therefore, we examined the binding sites of METTL16 for genes (GGAAC) (Fig. 3B). In cervical cancer cells, the FTH1 level was higher in cancer cells than that in normal cells (Fig. 3C). RNA immunoprecipitation (RIP) assay showed that METTL16 significantly bound with FTH1 (Fig. 3D). To test the m⁶A modification on FTH1 mRNA, the MeRIP-PCR reported that METTL16 silencing significantly reduced the m⁶A modification on FTH1 mRNA (Fig. 3E). In summary, the data demonstrated that FTH1 acted as the target of METTL16.

Fig. 3.

FTH1 acted as the target of METTL16. A The immunofluorescence assay showed the subcellular localization of METTL16 and FTH1. B The binding motif sites of METTL16 for its target genes (GGAAC). C The FTH1 level in cervical cancer cells was test by the RT-PCR. D RNA immunoprecipitation (RIP) assay was conducted for the binding within METTL16 and FTH1 in HeLa cells. E RIP assay using anti-m⁶A conducted for the m⁶A modification on FTH1 mRNA. *p < 0.05, **p < 0.01

IGF2BP2 promoted the stability of FTH1 mRNA

Since many m⁶A methyltransferases exerted regulatory effects on target genes by binding to m⁶A recognition proteins, the possible corresponding recognition proteins were identified in the subsequent experiments. This study found that IGF2BP2 and FTH1 jointly occupied this distribution, which meant they have the same subcellular localization (Fig. 4A). In clinical samples, the IGF2BP2 was positively correlated to the FTH1 (Fig. 4B). RNA immunoprecipitation (RIP) assay showed that IGF2BP2 significantly bound with FTH1 (Fig. 4C). To test the stability of FTH1 mRNA, the RNA decay analysis reported that IGF2BP2 overexpression promoted the stability of FTH1 mRNA (Fig. 4D). Moreover, the silencing of METTL16 reduced the stability of FTH1 mRNA (Fig. 4E). Furthermore, the METTL16 overexpression could reverse the role of IGF2BP2 silencing (Fig. 4F). In summary, the data demonstrated that IGF2BP2 promoted the stability of FTH1 mRNA, and METTL16 participated in this process collaboratively.

Fig. 4.

IGF2BP2 promoted the stability of FTH1 mRNA. A The immunofluorescent staining showed the subcellular localization of IGF2BP2 and FTH1 in HeLa cells. B The positive correlation within IGF2BP2 and FTH1 in clinical samples (http://gepia.cancer-pku.cn/index.html). C RNA immunoprecipitation (RIP) assay showed the immunoprecipitated FTH1 mRNA by anti-IGF2BP2 antibody. D RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with IGF2BP2 overexpression. E RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with METTL16 silencing (sh-METTL16-1, sh-METTL16-2). F RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with IGF2BP2 silencing (si-IGF2BP2) and METTL16 overexpression. *p < 0.05, **p < 0.01

METTL16 Silencing repressed the cervical cancer tumor growth in vivo

In previous studies, the study had already discovered in cellular experiments that METTL16 had a tumor-promoting effect on cervical cancer tumor cells. In the next step, we conducted in vivo verification in animal experiments. Results reported that METTL16 silencing reduced the tumor growth of cervical cancer cells (Fig. 5A, B). For the METTL16 and FTH1, the METTL16 silencing repressed the levels of METTL16 and FTH1 (Fig. 5C, D). For the iron, the METTL16 silencing promoted the Fe²⁺ level (Fig. 5E). For the ferroptosis-related indicators, the METTL16 silencing reduced the SLC7A11 and GPX4 (Fig. 5F, G). In summary, the data demonstrated that METTL16 silencing repressed the cervical cancer tumor growth in vivo.

Fig. 5.

METTL16 silencing repressed the cervical cancer tumor growth in vivo. A In vivo tumor weight verification of mice experiments was performed. CaSki cells with METTL16 silencing (sh-METTL16-1) was injected subcutaneously into the right flank of the mice. B Tumor volume was calculated in each group. C Immumohistochemical staining (IHC) images showed the METTL16 and FTH1 level. D The IHC score of METTL16 and FTH1 level. E The iron ion level in each group animal. F, G IHC of ferroptosis-related indicators (SLC7A11, GPX4) and HE staining in METTL16 silencing or control. *p < 0.05

Discussion

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation and plays a dual role in the tumorigenesis of cervical cancer [13]. Ferroptosis inhibits the progression of cervical cancer because it induces tumor cell death, especially improving the sensitivity to radiotherapy and chemotherapy [14, 15]. Therefore, regulating the ferroptosis pathway (such as targeting GPX4 or iron chelators) may provide a strategy for cervical cancer treatment, but its specific mechanism still needs further study to balance the pro-cancer and anti-cancer effects [16–18].

METTL16 is an RNA methyltransferase, mainly responsible for catalyzing the modification of m⁶A on specific mRNA [19, 20]. In recent years, studies have found that METTL16 plays a key role in RNA metabolism, splicing regulation and gene expression [21]. In present research, these findings and data identified that METTL16 inhibited the ferroptosis of cervical cancer cells in vitro. Specifically, METTL16 overexpression enhanced the mitochondrial structure and quantity, reduced the iron ion (Fe²⁺) and MDA in cervical cancer cells. Besides, the silencing of METTL16 impaired the mitochondrial structure and quantity, and promoted the Fe²⁺ and MDA. Thus, we concluded that METTL16 negatively regulated the ferroptosis of cervical cancer cells. It is well known that when the ferroptosis of tumor cells decreases, the survival and proliferation abilities of tumor cells will increase, which leads to the aggravation and development of tumors. Therefore, METTL16 could inhibit ferroptosis, which means it can promote the occurrence of tumors.

In various tumors, METTL16 has been proven to have extensive regulatory effects, such as promoting drug resistance, etc. For example, METTL16 directly interacts with the eIF3a or eIF3ab as well as ribosomal RNA, thereby facilitating the assembly of the translation-initiation complex and promoting the mRNA transcripts translation [22]. In gastric cancer, elevated METTL16 lactylation could improve the therapeutic efficacy of elesclomol (a copper ionophore), revealing the significance of METTL16 lactylation on cuproptosis in human tumor [23]. In acute myeloid leukemia, the depletion of METTL16 dramatically suppresses leukemia development, and METTL16 promotes BCAT1 and BCAT2 expression in m⁶A-dependent manner and reprograms BCAA metabolism [24].

Mechanistically, to test the mechanism of METTL16 in the cervical cancer ferroptosis, the present study found that ferritin heavy chain 1 (FTH1) was identified as a direct target of METTL16 via the m⁶A-dependent manner (Fig. 6). FTH1 (Ferritin Heavy Chain 1) is a heavy chain subunit of ferritin and plays a key role in regulating intracellular iron metabolism and oxidative stress. In Ferroptosis of tumors, FTH1 participates in regulation by influencing iron homeostasis and lipid peroxidation processes and plays an important role. In cervical cancer, suppression of FTH1 significantly enhances sorafenib-induced ferroptosis and increases its anticancer efficacy, indicating that FTH1 could repress the ferroptosis [25]. In vivo, METTL16 silencing repressed the cervical cancer tumor growth.

Fig. 6.

METTL16 promoted the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis

In summary, this research revealed that METTL16 functionally repressed the ferroptosis-related characteristic. More specifically, the novel m⁶A writer METTL16 promoted the cervical cancer tumorigenesis by targeting m⁶A/FTH1-dependent ferroptosis, which providing distinct insights for cervical cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Zhisheng Huang, Youzhen Luo performed the assays. Qiaoling Gao, Lulu Wang wrote the main manuscript and prepared Figs. 1, 2, 3, 4, 5 and 6. Caihong Li was responsible for the funding. All authors reviewed the manuscript.

Funding

Not applicable.

Data availability

The datasets generated and/or analysed during the current study are available in the Gene Expression Omnibus (GSE208653) repository, [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE208653](https:/www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE208653) .

Declarations

Ethics approval and consent to participate

All the animal experiments protocols had been approved by the Ethics Committee of Yichang Central People’s Hospital and performed in accordance with the guidelines of the International Council for Laboratory Animal Science (ICLAS).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.