The present study was approved by the Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology (Luoyang, China; approval no. 2023-0147). Cervical tissue specimens were collected between July 2021 and September 2023 from 25 women (age, 35.5–60.1 years) histologically diagnosed with stage IA CC featuring lymphovascular space involvement, or classified as IA2, as detailed in our previous research (19,20). The selection criteria for patients with cervical cancer were as follows: i) Pathologically confirmed patients with cervical cancer; and (2) the patients had no history of other cancers. No patients had preoperative chemotherapy, radiotherapy, or other treatment history or other inflammatory diseases. Patient conditions were staged according to the criteria of the International Federation of Gynecology and Obstetrics (21). Additionally, samples were collected from women with high-risk (HR)-HPV-negative typical CC (n=25) and HR-HPV-positive cervical malignancies (n=25) who had undergone HR-HPV screening and ThinPrep^® cytology (Hologic, Inc.) at the First Affiliated Hospital of Henan University of Science and Technology. Written informed consent was obtained from every participant involved.

IHC was performed on cervical cancer tissue sections prepared as formalin-fixed paraffin-embedded samples. The tissues were fixed in 10% neutral-buffered formalin at room temperature for 24 h, embedded in paraffin wax, and sectioned at a 4-µm thickness. Fresh frozen tissues were snap-frozen in liquid nitrogen-cooled isopentane at approximately −150°C and sectioned at 7 µm. The sections were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature, followed by blocking with 5% normal goat serum (cat. no. 31873; Thermo Fisher Scientific, Inc.) in PBS for 1 h at room temperature. Primary antibody incubation was performed using anti-YTHDC2 (cat. no. ab220160; Abcam) at a 1:100 dilution overnight at 4°C. After washing, sections were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (cat. no. 7074; Cell Signaling Technology) at a 1:500 dilution for 1 h at room temperature. Signal detection was performed using 3,3′-diaminobenzidine chromogen (cat. no. K3468; Dako; Agilent Technologies, Inc.). Slides were counterstained with hematoxylin and examined using a bright-field microscope (Olympus BX53; Olympus Corporation).

Cell lines (purchased from ATCC), including HFF-1 (HPV-negative human epithelial foreskin fibroblasts), C33A and DoTc2 4510 (both from HPV-negative cervical carcinomas), SiHa and CaSKi (from HPV16-positive cervical squamous carcinomas), SW756 (from an HPV18-positive squamous carcinoma), HeLa (from an HPV18-positive cervical epithelial adenocarcinoma) and H8 (from HPV-positive, immortalized cervical cells), were maintained in RPMI-1640 medium (cat. no. 11875093; Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (cat. no. A5256701; HyClone; Cytiva). All cell lines were incubated at 37°C in a 5% CO₂ atmosphere with humidity.

SiHa and CaSKi cells (5×10⁶/well) were transfected with 1.5 µg YTHDC2-overexpression vectors (pCMV-YTHDC2) and/or 1.5 µg control vectors (pCMV-empty), and with 2 µg YTHDC2-specific small interfering (si)RNAs (#1, 5′-ATATAAGAGATGTGACGAGGG-3′; #2, 5′-CTTTAGTCGAAGTTCTGACTA-3′; and #3, 5′-GGAAGCTAAATCGAGCCTT-3′), and control siRNAs (5′-CACAGGGUAAGGAACUCGUCUCUCA-3′) using Lipofectamine™ 2000 (cat. no. 11668027; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 36 h according to the manufacturer's protocols. The vector and siRNA were constructed and purchased from Genscript Biotech Corporation. The effect of overexpression or knockdown was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), as described below, at 48 h post-transfection Subsequently, siRNA #1 was randomly selected for further experiments as the knockdown efficacies of all three siRNAs were similar (Fig. S1). SiHa and CaSKi cells were co-transfected with pCMV1-YTHDC2 along with either pcDNA3.1-empty or pcDNA3.1-SLC7A11 for 48 h.

Additionally, SiHa and CaSKi cells were transfected with 2 µg SLC7A11-overexpression vector pcDNA3-SLC7A11 or 2 µg SLC7A11-specific siRNA (5′-CTGGAGTTATGCAGCTAAT-3′), or co-transfected with pCMV1-YTHDC2 along with either pcDNA3.1-empty or pcDNA3.1-SLC7A11, using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) at room temperature for 36 h according to the manufacturer's instructions, to upregulate or downregulate SLC7A11 expression, respectively. Concentrations of 10 nM for vectors and 20 nM for siRNAs were used for the transfections. Subsequent experiments were performed 48 h after transfection.

Cell viability was assessed using Cell Counting Kit-8 (CCK-8) reagent (cat. no. CK04; Dojindo Laboratories, Inc.) at 48 h following transfection, according to the manufacturer's guidelines. A total of 10 µl CCK-8 solution was added to each designated well (5×10⁶/well). Subsequently, the plate was incubated for 2 h and absorbance readings were then taken at 450 nm using the Tecan Infinite M200 microplate (Tecan Group, Inc.).

SiHa and CaSKi cells were cultured in a series of 12-well plates, with each well containing 3,000 cells/ml. These cells were cultured for 7–9 days at 37°C, after which they were fixed using 10% neutral-buffered formalin for ≥4 h at room temperature, stained using crystal violet for 30 min at room temperature (cat. no. C0121; Beyotime Institute of Biotechnology) and visually assessed using an advanced optical system, and counted manually (Olympus CX23; Olympus Corporation). A colony was typically defined as: i) A group containing at least 50 cells; ii) a group clearly separated from neighboring colonies; and iii) a group of cells that must be stained.

Following transfection, the cell specimens were incubated in 6-well plates for 48 h and then trypsinized and preserved in 75% ethanol at −20°C overnight. Following stabilization, these specimens were treated with 0.5 µg/ml RNase A (Thermo Fisher Scientific, Inc.) and 100 µg/ml propidium iodide (PI) for 30 min. Subsequently, apoptotic indices were evaluated using a dual staining Annexin V-FITC/PI apoptosis kit (Abcam) with analytical procedures performed using a BD FACScan flow cytometer (BD Biosciences) equipped with BD CellQuest™ software (version 5.1; BD Biosciences). Specimens unreactive to FITC/PI were utilized as negative controls in these assays.

A total of 5×10⁴ SiHa and CaSKi cells were added to the wells of a 96-well white plate optimized for luminescence studies. Following cell adhesion, cells were cultured for 24 h. After thoroughly rinsing with PBS (cat. no. 10010023; Thermo Fisher Scientific, Inc.), a solution of 20 µM carboxy-H2-DCFDA (cat. no. C400; Invitrogen; Thermo Fisher Scientific, Inc.) was added to the cellular milieu and incubated at 37°C for 1 h to facilitate ROS-associated fluorescence development. Fluorescence detection was performed using the 1420 Multi-label Counter (PerkinElmer, Inc.). Furthermore, cells were resuspended in 1 ml PBS and incubated for 1 h under identical temperature conditions after applying a second dose of 20 µM carboxy-H2-DCFDA. The cells were rinsed with PBS to remove extraneous dye, and the fluorescence intensity was quantified using a microplate reader at 485 and 535 nm excitation and emission wavelengths, respectively, to determine the ROS levels within the samples accurately.

RNA isolation from SiHa and CaSKi cell or tissue samples (10 mg) was performed using TRIzol™ Reagent (cat. no. 15596018CN; Invitrogen; Thermo Fisher Scientific, Inc.), with the quantitative assessment of yields performed using a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, Inc.) at an optical density of 260 nm. Subsequent reverse transcription to generate cDNA was performed using the SuperScript™ IV First-Strand Synthesis System (cat. no. 18091050; Invitrogen; Thermo Fisher Scientific, Inc.) with oligo(dT) 20 primers. The thermal conditions for reverse transcription PCR were as follows: 37°C for 2 min, and 55°C for 5 min. For the quantitative analysis, the prepared cDNA was subjected to amplification using SYBR™ Select Master Mix (cat. no. 4472918; Invitrogen; Thermo Fisher Scientific, Inc.), adhering to the protocols specified by the supplier. The internal standard GAPDH mRNA was used to calibrate the reaction, which was initiated with a 10 min denaturation step at 95°C, followed by 40 cycles of 15 sec denaturation at the same temperature, and a 40 sec extension phase at 60°C. The abundance of the target mRNA was quantitatively evaluated using the comparative 2^−ΔΔCq method (22). Each experimental sequence was performed in triplicate to ensure the reproducibility and reliability of the results. The sequences for primers used in this experiment were as follows: YTHDC2-forward (F), 5′-CCAGGCCGAGCAGCGTCTCC-3′; YTHDC2-reverse (R), 5′-ACAGTTAATCAGTATGGGAGCC-3′; GAPDH-F, 5′-AACAGCGACACCCACTCCTC-3′; GAPDH-R, 5′-CATACCAGGAAATGAGCTTGACAA-3′; SLC7A11-F, 5′-TCCTGCTTTGGCTCCATGAACG-3′; and SLC7A11-R, 5′-AGAGGAGTGTGCTTGCGGACAT-3′. The thermocycling conditions for qPCR were as follows: 95°C for 2 min, 95°C for 10 sec, 55°C for 30 sec and 72°C for 30 sec, for 40 cycles.

SiHa and CaSKi cells were disrupted in a lysis buffer comprising HEPES (0.02 M), NaCl (0.15 M), EDTA (1 mM, pH 7.4), glycerol (10%), Triton X-100 (1%), a comprehensive protease inhibitor mixture and Na₃VO₄ (5 mM). The resulting lysate was incubated at 4°C for 1 h, followed by centrifugation at 12,000 × g at 4°C for 15 min. After separation via 5–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, proteins (10 µg/lane; determined by BCA method) were transferred onto polyvinylidene difluoride membranes. The membranes were then blocked for 2 h at 4°C in PBS supplemented with 5% skim milk powder (cat. no. LP0033B; Invitrogen; Thermo Fisher Scientific, Inc.) and 0.5% Tween 20. Subsequently, proteins were probed with appropriate primary antibodies, including anti-YTHDC2 (1:1,000; cat. no. ab220160; Abcam), anti-actin (1:5,000; cat. no. ab8227; Abcam), anti-SLC7A11 (1:2,000; cat. no. ab37185; Abcam), anti-p53 (1:2,000; cat. no. ab32389; Abcam), anti-ACSL4 (1:1,000; cat. no. ab155282; Abcam) and anti-GPX4 (1:1,000; cat. no. ab41787; Abcam) antibodies for 1 h at room temperature, and detected using horseradish peroxidase-conjugated secondary antibodies derived from rabbits or mice, including goat anti-mouse HRP antibody (1:10,000; cat. no. ab6708; Abcam) and goat anti-rabbit antibody (1:10,000; cat. no. ab6721; Abcam) for another 1 h at room temperature. Finally, protein expression was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

Fragments of SLC7A11 wild-type (−WT) or SLC7A11 mutant (−Mut) (where m6A was substituted with C) were inserted into the pRP (Exp)-Puro-EF1A-Luciferase (mSLC7A11_3′UTR_1862-4861bp: SNP) (VectorBuilder) plasmid to create pcDNA3-SLC7A11-WT and pcDNA3-SLC7A11-Mut plasmids as aforementioned. At 72 h post-transfection using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation). Relative firefly luciferase (Fluc)/Renilla luciferase (Rluc) activity was calculated by normalizing the activity of firefly luciferase to that of Renilla luciferase. Each experiment was conducted in triplicate for each group.

Data were statistically analyzed using SPSS version 18.0 software (IBM Corp.). Data are expressed as the mean ± standard deviation. Comparisons between two groups were assessed using an unpaired t-test, whereas comparisons between multiple groups were analyzed using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

CC specimens were first classified as HPV- positive or -negative. YTHDC2 mRNA levels in HPV-positive CC specimens were significantly lower than those in their HPV-negative counterparts, according to RT-qPCR, WB and immunohistochemistry results (Fig. 1A-C). To further evaluate these observations based on clinical specimens, YTHDC2 expression in HPV-negative (HFF-1, C33A and DoTc2 4510) and HPV-positive CC cell lines (SiHa, CaSKi, SW756, HeLa and H8) were assessed. At the cellular level, the significant downregulation of YTHDC2 mRNA and protein expression was detected in HPV-positive CC cells compared with levels in HPV-negative CC cells (Fig. 1D and E). These data indicate that HPV-positive CC specimens and cell lines consistently have lower YTHDC2 levels.

YTHDC2 expression was modulated in SiHa and CaSKi cells through overexpression and silencing techniques to further evaluate the function of YTHDC2 in HPV-positive CC cells. After transfection with the overexpression vector, both RT-qPCR and WB analyses revealed a significant increase in YTHDC2 mRNA and protein levels compared with those in the control group. Conversely, silencing YTHDC2 expression via siRNA transfection significantly diminished its mRNA and protein levels in both SiHa and CaSKi cells, compared with those in the control group (Fig. 2).

YTHDC2 overexpression led to a significant decrease in cell viability in both cell lines at 48 h post-treatment, compared with that in the control group. Meanwhile, YTHDC2 silencing significantly increased cell viability, compared with that in the control group, according to CCK-8 assays (Fig. 3A and B). The results of the colony formation assays indicated that the cell proliferation rate was significantly decreased after YTHDC2 overexpression and significantly elevated after YTHDC2 knockdown, compared with that in the controls groups (Fig. 3C and D). These data suggest that YTHDC2 overexpression attenuates HPV-positive cell proliferation.

Moreover, flow cytometry was used to assess the role of YTHDC2 in cell apoptosis, especially ferroptosis in SiHa and CaSKi cells. The numbers of apoptotic cells for both cell lines overexpressing YTHDC2 were significantly elevated, whereas cells with YTHDC2 knockdown exhibited a significantly decreased cell apoptosis rate, compared with that of controls (Fig. 4A and B). Ferroptosis is a newly identified type of apoptosis, and excessive ROS production is a predominant marker of this process (23). Therefore, the present study assessed the ROS levels in cell lines after overexpressing or silencing YTHDC2. Compared with that of controls, increased ROS levels were observed in cells overexpressing YTHDC2, whereas they were significantly reduced in cells with silenced YTHDC2 expression (Fig. 4C and D). Furthermore, the protein expression of the ferroptosis markers SLC7A11, p53, acyl-CoA synthetase long chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4) in cells with varying YTHDC2 expression levels were assessed. WB analyses demonstrated that GPX4 and ACSL4 levels were negatively associated with the expression of YTHDC2 in SiHa and CaSKi cells, whereas SLC7A11 and p53 were positively associated with YTHDC2 levels in these cells (Fig. 4E and F). These findings indicate that YTHDC2 promotes ferroptosis in SiHa and CaSKi cells.

Based on previous publications (24,25), we hypothesized that SLC7A11 could be modulated by YTHDC2 (an RNA m6A reader) in an m6A-dependent manner. Therefore, the present study aimed to assess this hypothesis. The results revealed that, compared with controls, SLC7A11 mRNA expression was significantly downregulated after YTHDC2 overexpression and significantly upregulated after YTHDC2 silencing in both cell lines, according to RT-qPCR analysis (Fig. 5A and B). Subsequently, luciferase reporter assays were performed to assess SLC7A11 mRNA 5′-UTR methylation and gene expression. SiHa and CaSKi cells demonstrating either upregulated or downregulated YTHDC2 expression were transfected with reporter plasmids that included the full-length SLC7A11 5′-UTR immediately upstream of the luciferase gene and incubated for 36 h. Compared with in controls, luciferase activity was significantly diminished in cells with elevated YTHDC2 levels, whereas cells with suppressed YTHDC2 expression exhibited a significant increase in luciferase activity (Fig. 5C and D). Subsequently, the G residue within the SLC7A11 5′-UTR consensus sequence was mutated (5′-GGCUGC-3′ to 5′-AACUAC- 3′ in mRNA), and wild-type and mutant reporter-driven luciferase activities were compared using luciferase assays. The A residue mutation reduced the luciferase activity by ~70% in SiHa and CaSKi cells (Fig. 5E and F). Collectively, these data suggest that YTHDC2 contributes to SLC7A11 mRNA 5′-UTR m6A methylation, thereby inhibiting SLC7A11 expression and protein translation.

Subsequently, the present study evaluated the function of SLC7A11 in YTHDC2-regulated cell proliferation and ferroptosis in SiHa and CaSKi cells by overexpressing it in these cell lines, both with and without YTHDC2 overexpression. Compared with the controls, SLC7A11 mRNA and protein levels were significantly upregulated in SiHa and CaSKi cells, independent of YTHDC2 overexpression (Fig. 6). Subsequently, proliferation and ferroptosis in SiHa and CaSKi cells was assessed following YTHDC2 overexpression and/or SLC7A11 overexpression. Compared with the controls, increased SLC7A11 expression was associated with a significant increase in cell viability and colony formation in SiHa and CaSKi cells (Fig. 7).

Finally, regarding ferroptosis, SLC7A11 overexpression was demonstrated to significantly reduce apoptosis in SiHa and CaSKi cells with silenced YTHDC2 expression, compared with controls (Fig. 8A and B). Moreover, the generation of ROS in cells overexpressing YTHDC2 with elevated SLC7A11 levels was significantly reduced compared with the control group (Fig. 8C and D). Meanwhile, SLC7A11 and p53 markedly promoted GPX expression, and ACSL levels were notably reduced by SLC7A11, according to WB analysis (Fig. 8E). These results suggest that SLC7A11 overexpression is associated with YTHDC2-mediated proliferation and ferroptosis in SiHa and CaSKi cells.