The cancer-testis lncRNA AC007271.3 acts as a decoy of MBP-1 and promotes chemotherapy resistance by inhibiting ferroptosis via c-Myc/GPX4 pathway in oral squamous cell carcinoma

Abstract

Background

Oral squamous cell carcinoma (OSCC) is the most common subtype of oral cancer, and its poor prognosis is largely attributed to uncontrolled proliferation, invasion, and therapeutic resistance. Our recent transcriptomic profiling identified that the non-coding RNA (lncRNA) AC007271.3 was markedly upregulated in OSCC compared with paired normal mucosal tissues. This lncRNA may serve as a prognostic biomarker for OSCC, which accelerates tumor progression. However, its underlying mechanisms remain unclear. In this study, we investigated the regulatory mechanism by which cancer-testis long noncoding RNA (CT-lncRNA) AC007271.3 mediates cisplatin resistance through ferroptosis.

Methods

GTEx database was used to reveal that AC007271.3 is a CT-lncRNA. Bioinformatic analysis was employed to analyze differential gene expression and associated pathways after knocking down AC007271.3. Transmission electron microscopy and flow cytometry were used to detect mitochondrial reactive oxygen species (ROS). Wound healing, CCK-8, colony formation, invasion, migration and cell line-derived xenograft (CDX) models were used to assess the proliferation, migration and invasion abilities of cancer cells in vitro and in vivo. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) assays were conducted to identify and validate the underlying regulatory mechanisms of AC007271.3.

Results

Bioinformatics analysis suggested that AC007271.3 is a CT-lncRNA, and transcriptome sequencing confirmed that AC007271.3 may promote OSCC progression by modulating ferroptosis. Mechanistically, AC007271.3 acts as a decoy for MBP-1 to block the binding of MBP-1 to the c-Myc promoter, and thus activating c-Myc transcription, leading to upregulated expression of the ferroptosis regulator GPX4, thereby suppressing ferroptosis and facilitating G1/S transition, which ultimately accelerates tumor progression and chemoresistance. This study identified AC007271.3 as a critical CT-lncRNA that inhibits ferroptosis and leads to cisplatin resistance in OSCC cells, thereby accelerating tumor advancement via the MBP-1/c-Myc/GPX4 axis.

Conclusions

To our knowledge, this is the first study to report that this CT-lncRNA promotes carcinogenesis and that c-Myc directly binds to the GPX4 promoter, thus triggering GPX4 transcription to inhibit ferroptosis in OSCC. These findings highlight AC007271.3 as a potential therapeutic target and provide new insight into the molecular pathogenesis of OSCC.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04281-8.

Introduction

Oral squamous cell carcinoma (OSCC) accounts for approximately 380,000 new cases annually worldwide, with the highest incidence in Asia, and it remains a major public health burden [1]. Despite advances in surgery, radiotherapy and chemotherapy, the patient prognosis is far from optimistic [2, 3]. One of the major challenges is the development of therapeutic resistance to cisplatin, which involves diverse mechanisms including DNA repair, epigenetic regulation, drug efflux, regulated cell death pathways, and tumor microenvironmental interactions [4]. Therefore, uncovering the molecular mechanisms driving OSCC progression and cisplatin resistance is crucial for improving therapeutic efficacy.

Long non-coding RNAs (lncRNAs) are defined as non-coding RNAs with transcript lengths over 200 nucleotides that usually lack open reading frames (ORFs) and have limited or no coding ability. It is well known that lncRNAs serve as crucial regulatory molecules, playing significant roles in a wide range of biological processes. Plenty of studies have shown that lncRNAs play crucial roles in the development and progression of various cancers, indicating that lncRNAs have emerged as important therapeutic targets for malignant diseases. Our recent transcriptomic profiling further identified that AC007271.3, a 382-nucleotide lncRNA located on chromosome 2, was markedly upregulated in OSCC compared with the paired adjacent oral tissues. Its elevated expression is also detectable in patient serum and predicts an unfavorable prognosis for OSCC patients [5]. Further research confirmed that AC007271.3 promotes cell proliferation, invasion and migration via the Wnt/β-catenin signaling pathway and regulates epithelial-mesenchymal transition (EMT) by miR-125b-2-3p/Slug/E-cadherin axis in OSCC [6, 7]. Cancer-testis long noncoding RNAs (CT-lncRNAs) represent a subclass of lncRNAs which were ultraconserved, highly expressed in tumors and specifically expressed in the testis. For example, THOR, a testis-specific lncRNA, exhibits expression exclusively in testis and a broad range of human cancers [8]. Their restricted expression pattern and oncogenic potential highlight them as both biomarkers and therapeutic targets. To our knowledge, however, CT-lncRNAs in OSCC have not yet been investigated.

Ferroptosis is an iron-dependent and regulated form of cell necrosis [9]. Research has demonstrated that ferroptosis, as a therapeutic approach for tumors, can inhibit tumor progression by modulating the tumor suppressors p53 and BAP1 [10]. Ferroptosis is regulated in tumor cells through multiple tumor-related signaling pathways. In ferroptosis, cells experience oxidative stress that leads to iron-dependent lipid peroxidation, forming toxic oxidized lipids. This further leads to cell membrane disruption and cell death. Emerging evidence has revealed that ferroptosis serves as a key determinant of chemotherapeutic response in various cancers. Numerous studies have shown that lncRNAs enhance tumor chemotherapy resistance by regulating ferroptosis in tumor cells. For example, lncRNA-HMG protects colorectal cancer (CRC) cells from ferroptosis upon chemotherapy, thus enhancing the drug resistance of CRC cells [11]. However, no report has shown taht lncRNA promotes OSCC chemotherapy resistance by regulating ferroptosis.

In this study, we report that AC007271.3 is an oncogenic CT-lncRNA that functions as an RNA decoy for MBP-1, thereby relieving MBP-1-mediated repression of c-Myc transcription and leading to upregulated expression of GPX4, which suppresses ferroptosis and facilitated G1/S transition, ultimately accelerating tumor progression and chemotherapy resistance. To our knowledge, this study is the first to report that the CT-lncRNA promoted carcinogenesis of OSCC and c-Myc directly binds to the GPX4 promoter which triggers GPX4 transcription, inhibiting OSCC ferroptosis and contributing chemotherapy resistance. In summary, these findings provide direct evidence that AC007271.3 may serve as a promising therapeutic target and offer new insight into the molecular pathogenesis of OSCC.

Methods and materials

TCGA and GTEx databases

AC007271.3 expression in normal human tissues was obtained from the GTEx database, which contains RNA-seq data from 17,382 samples across 54 tissue types derived from 948 donors. The expression profiles of AC007271.3 in tumor and matched normal tissues were retrieved from The Cancer Genome Atlas (TCGA) to assess its cancer-specific expression patterns.

RNA sequencing

SCC9 cells (5 × 10⁶) were transfected with control or shRNA1. Total RNA was extracted, quantified, and quality assessed prior to library construction. Poly(A) + RNA was enriched using oligo(dT) magnetic beads, fragmented, and reverse-transcribed to generate double-stranded cDNA. After end repair, A-tailing, adaptor ligation, and PCR amplification, sequencing libraries were generated and subjected to high-throughput sequencing on the Illumina HiSeq 2500 platform.

Raw read counts were normalized to fragments per kilobase of transcript per million mapped reads. Differentially expressed genes were identified relative to the corresponding control using a false discovery rate threshold of < 0.05.

Cell culture

Human tongue squamous cell lines (SCC9, SCC15) and human embryonic kidney cell lines (HEK293T) were obtained from the Institute of Antibody Engineering, Southern Medical University (Guangzhou, China). All cells were cultured in DMEM (Gibco, NY, USA) supplemented with 10% FBS (ExCell Bio, Shanghai, China) and penicillin/streptomycin solution (Beyotime, China) at 37℃ in a humidified atmosphere containing 5% CO₂.

Expression plasmid, RNA oligonucleotides, and transfection

pcDNA-AC007271.3 overexpression vector (pcDNA3.1(+)-AC007271.3), the human c-Myc overexpression and its corresponding control vector (Control) were constructed by Obio Technology Corp., Ltd. (Shanghai, China). siRNAs against AC007271.3 (si-AC007271.3) and c-Myc (si-c-Myc), a scramble control siRNA and the AC007271.3 antisense oligonucleotide (ASO) were all synthesized by Tsingke (Guangzhou, China) (Supplementary Table 1). The transfection procedure was carried out following the manufacturer’s guidelines using the Lipofectamine 3000 reagent (Invitrogen, CA, USA).

Lentivirus-NC and lentivirus-AC007271.3 were transfected into SCC9 according to the operating instructions. Puromycin (MCE, Monmouth Junction, NJ, USA) was used to select the AC007271.3 stably expressed cells and the control cells.

Cell proliferation assay

Cell proliferation was evaluated using the CCK-8 assay and colony formation assay. For the CCK-8 assay, cells were seeded at 1 × 10³ cells per well in 96-well plates (Corning, New York, USA) and incubated at 37 °C with 5% CO₂. CCK-8 reagent (10%) was added at 0, 24, 48, and 72 h, and absorbance at 450 nm was measured after 2 h. For colony formation assays, 1 × 10³ cells were plated in 12-well plates and cultured for 7 days, followed by fixation with 4% paraformaldehyde and crystal violet staining. Colonies were visualized and counted under a microscope (Olympus, Japan).

Cell cycle assay by flow cytometry

Cells were collected and fixed in 70% ethanol overnight at 4℃. Subsequently, we labeled single-cell suspensions with 50 µg/ml PI (Beyotime, Shanghai, China) and analyzed them using flow cytometry. Using FlowJo software (Treestar, Inc., Ashland, OR), 1 × 10⁴ cells were evaluated, and the percentage of cells in the G2 phase was denoted in the 2 N population phase.

Double luciferase gene detection

The DNA sequences corresponding to the GPX4 and c-Myc promoters were cloned into the pGL3-basic vector. Additionally, the complementary DNA sequence of the c-Myc promoter, as well as the deleted P2 promoter within this sequence, was also cloned into the pGL3-basic vector. The luciferase activity of all constructs was measured using the dual-luciferase reporter assay system, following the manufacturer’s protocol.

RNA pull-down assay and mass spectrometry

Purified RNAs were biotin-labelled using Pierce RNA 3′End Desthiobiotinylation Kit (Thermo Fisher Scientific, CA, USA). Biotin-labeled wild-type AC007271.3 (AC007271.3-Bio) and antisense control (AS-Bio) were incubated with SCC9 cell lysates overnight at 4℃. Magnetic beads were added to each mixture for 1 h at room temperature. The eluted proteins were detected by Fast Silver Stain Kit (#BL620A, Biosharp Ltd, China). The different parts gel were cut for the mass spectrometry analysis by GeneCreate (Hubei, China).

RNA immunoprecipitation assay

We performed RIP experiments using Magna RNA immunoprecipitation (RIP) RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. In brief, the SCC9 cells transfected with His-MBP-1 for 48 h were collected (1 × 10⁷) and lysed with lysis buffer. Then, the lysates were divided into two groups and were separately incubated with anti–His-tag (#12698, CST) and anti-IgG (#A7016, Beyotime) antibodies, which had already been incubated with protein A/G Magna beads. The RNA was extracted for further qRT-PCR analysis.

Western blot

Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China). Protein obtained from cells or tissues was separated on 10% SDS–polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Bio-Rad). After blocking for 2.5 h at room temperature, the primary antibodies were used for incubation at 4℃ overnight. The secondary antibodies were incubated for one hour at room temperature. ECL reagent (Epizyme Biotech, Shanghai, China, #SQ101) was used for chemiluminescence. Antibodies for c-Myc, p21, p27, CDK2, CDK4, cyclin D1, cyclin E1, α-tubulin, ACSL4, SLC7A11, FTH1, GPX4, and GAPDH were purchased from Abclonal (Wuhan, China). α-Tubulin and GAPDH served as loading controls.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), reverse-transcribed into cDNA, and subjected to quantitative real-time PCR (qRT-PCR) with SYBR Green Master Mix (Vazyme, Nanjing, China). Reactions were performed in triplicate on a StepOnePlus system (Applied Biosystems). Primer sequences were as follows: AC007271.3.

(forward: 5’- ACGTACTTTGAGCAGCCAAG-3’; reverse: 5’- ACCTTCTGCAGAGAGTTCTTCC-3’), GPX4 (forward: 5’- GCCTTTGCCGCCTACTGAA-3’; reverse: 5’- CATGTGCCCGTCGATGTCC-3’), GAPDH (forward: 5’- CCAGGTGGTCTCCTCTGACTTC-3’; reverse: 5’- GTGGTCGTTGAGGGCAATG-3’). The relative mRNA levels were calculated by the 2^−ΔΔCt method with GAPDH as a control.

Subcutaneous transplantation tumor model in BALB/c nude mice

Female BALB/c nude mice (5 weeks old) were randomly assigned to experimental groups. A total of 1 × 10⁶ cells suspended in PBS were injected subcutaneously into the left axilla. Tumor growth was monitored every 3–5 days using digital calipers, and tumor volume was calculated as 0.5 × L × W². At the experimental endpoint, mice were euthanized, tumors were excised, and tumor wet weights were measured. Tumor tissues were further subjected to hematoxylin and eosin staining and immunohistochemical analysis of Ki67.

Chromatin immunoprecipitation (ChIP) assay

According to Merck’s manual, we performed the chromatin immunoprecipitation (ChIP) assay using the Magna ChIP™ A/G Kits (Merck, Darmstadt, Germany).

Edu assay

Cell proliferation was quantified by EdU incorporation. Cells were exposed to 50 µM EdU, fixed, permeabilized, and subjected to the kFluor488 Click-iT reaction according to KeyGEN BioTECH’s protocol. Nuclei were counterstained with DAPI, and EdU-positive cells were analyzed by fluorescence microscopy.

Transmission electron microscope (TEM) and ultrastructure observation

Cells were harvested, fixed with glutaraldehyde, and post-fixed in 1% osmium tetroxide at room temperature. After PBS washes, samples were dehydrated through a graded ethanol series, embedded, and polymerized at 60 °C. Ultrathin Sections (60 nm) were prepared, stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope.

Intracellular ROS assay

Cells were seeded in 6-well plates (60–70% confluence), washed with PBS, and incubated with 2.5 µM C11-BODIPY 581/591 in high-glucose DMEM for 30 min at 37℃ in the dark. Cells were then washed, digested, resuspended in PBS, and analyzed by flow cytometry. Data were processed using FlowJo software.

Mitochondrial membrane potential assay

Mitochondrial Membrane Potential Assay Kit with TMRE (#C2001S, Beyotime) was used as a molecular probe, and cells were observed under a fluorescence microscope.

Ferrous iron detection

Cell Ferrous Iron Colorimetric Assay Kit (Elabscience, Wuhan, China) and FerroOrange staining (Cell Signaling Technology, #36104, Danvers, MA, USA) were used to measure ferrous iron level, following the manufacturer’s protocols.

Transwell assay for cell invasion and migration

For migration assays, 8 × 10⁵ cells in 150 µL serum-free medium were seeded into the upper chambers of 8 μm Transwell inserts, with 450 µL medium containing 10% FBS in the lower chamber as a chemoattractant. For invasion assays, the upper chambers were pre-coated with a 1:2 mixture of Matrigel and serum-free medium. After 48 h incubation at 37 °C with 5% CO₂, cells on the upper surface were removed, and migrated or invaded cells were fixed, stained with crystal violet, and quantified under a microscope using ImageJ.

Statistical analysis

All experimental assays were performed in triplicate. All data were presented as mean ± standard deviation (SD) from three independent experiments and analyzed by SPSS Statistics 20.0 (Chicago, USA). All statistical charts were generated using Graphpad Prism 7.0 (CA, USA). Student’s t-test was performed to compare differences between two groups.

Result

Bioinformatic analysis revealed AC007271.3 as a cancer-testis lncRNA inhibiting ferroptosis via GPX4 in OSCC

Expression data from the GTEx (V8) and TCGA databases revealed that AC007271.3 was specifically enriched in human testicular tissue (0.45 TPM) while being very low or essentially absent in other normal tissues, indicating that it was a cancer-testis lncRNA (Fig. 1a-c). Then, transcriptome profiling was performed in SCC9 cells with knockdown of AC007271.3 expression. Sequencing analysis showed that MYC and ferroptosis-related genes were differentially expressed (Fig. 1d-e). Functional enrichment analysis of the differentially expressed genes revealed significant enrichment in the ferroptosis pathway (Fig. 1f). These findings suggested that AC007271.3 might influence the ferroptosis pathway through ferroptosis-associated proteins. To further investigate the function of AC007271.3,we confirmed the efficiency of AC007271.3 knockdown and overexpression in OSCC cells (SCC9 and SCC15) by RT-qPCR (Fig. 1g), followed by a series of ferroptosis assays. Flow cytometry revealed that lipid peroxidation levels decreased upon AC007271.3 overexpression, while knockdown produced the opposite effect (Fig. 1h). Transmission electron microscopy further showed that AC007271.3 knockdown induced mitochondrial atrophy, cristae loss, and increased membrane density, while nuclear morphology remained intact, a typical ultrastructural feature of ferroptosis (Fig. 1i). Consistently, fluorescence microscopy demonstrated that AC007271.3 overexpression reduced mitochondrial membrane potential, whereas silencing triggered mitochondrial membrane hyperpolarization (Fig. 1j). To strengthen these observations, intracellular Fe²⁺ levels were examined. The Calcein-AM assay revealed that AC007271.3 overexpression significantly decreased the labile iron pool, whereas its silencing markedly increased the accumulation of Fe²⁺ (Fig. 1k). These results provide further evidence that AC007271.3 suppresses ferroptosis by modulating iron homeostasis. Subsequently, to identify potential downstream effectors, western blot analysis of ferroptosis-associated proteins (ACSL4, SLC7A11, FTH1, and GPX4) was conducted. AC007271.3 was found to specifically upregulate GPX4 protein expression (Fig. 1l), highlighting GPX4 as a critical mediator of AC007271.3-driven ferroptosis suppression in OSCC.

Fig. 1.

Bioinformatic analysis revealed AC007271.3 as a cancer-testis lncRNA inhibiting ferroptosis via GPX4 in OSCC. (a) AC007271.3 tissue-specific expression in the gene expression GTEx database (V8). (b) AC007271.3 is specifically enriched in human testicular tissue. (c) The AC007271.3 expression profile across all tumor samples and paired normal tissues in TCGA database (Dot plot) Red: Tumor tissue. Green: Normal tissue. Volcano plot (d) and heatmap (e) show the differentially expressed genes in SCC9 cells with AC007271.3 knockdown, where group_1 represents the control group and group_2 represents the knockdown group. The cutoff was set as |log2(fold change)| > 1.5. (f) Enriched pathways of genes differentially expressed upon AC007271.3 knockdown. Ferroptosis-related pathways are indicated in red. (g) AC007271.3-overexpressing cells and control cells were subjected to qRT-PCR to validate the efficiency. (h) Flow cytometry was performed to detect the effect of AC007271.3 on lipid peroxidation. (i) Transmission electron microscopy was performed to detect mitochondrial atrophy and cristae loss on mitochondrial morphology after knocking down AC007271.3, indicated by red triangle and scale bar = 500 nm. (j) Mitochondrial membrane potential assay in OSCC cells with the knockdown or overexpression of AC007271.3. (k) The Calcein-AM assay in OSCC cells with knockdown or overexpression of AC007271.3. (l) Western blot was performed to detect whether AC07271.3 affects the expression of ferroptosis-related proteins. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

CT-lncRNA AC007271.3 promotes OSCC progression by suppressing ferroptosis through the GPX4 pathway

To determine whether AC007271.3 drives oral squamous cell carcinoma progression via ferroptosis, functional assays, including wound healing, CCK-8, colony formation, invasion, and migration, were conducted. The results demonstrated that AC007271.3 promoted OSCC cell proliferation, migration, and invasion, while the ferroptosis inhibitor Ferrostatin-1 (Fer-1) partially mitigated these effects (Fig. 2a-d). To further validate these findings in vivo, tumorigenesis assays were performed. Overexpression of AC007271.3 significantly promoted the growth of OSCC xenografts. However, co-treatment with the ferroptosis inducer RSL3, a known inhibitor of GPX4 [12], suppressed tumor proliferation and partially reversed the tumor-promoting effect of AC007271.3 (Fig. 2e-f). To further confirm the involvement of ferroptosis, cell viability was assessed in SCC9 and SCC15 cells treated with inhibitors of ferroptosis (Fer-1), autophagy (3-MA), and pyroptosis (Z-VAD-FMK). AC007271.3 knockdown significantly reduced cell viability, an effect partially rescued by Fer-1 but not by 3-MA or Z-VAD-FMK. Conversely, AC007271.3 overexpression increased viability, which was attenuated by the ferroptosis inducer RSL3 (Fig. 2g). Collectively, these results confirm that AC007271.3 promots OSCC progression by suppressing ferroptosis via the GPX4-related pathway.

Fig. 2.

CT-lncRNA AC007271.3 promotes OSCC progression by suppressing ferroptosis through the GPX4 pathway. (a-d) Fer-1 rescued the proliferation, migration, and invasion abilities of OSCC cells with AC007271.3 knockdown. (e-f) Representative images and growth curves of xenograft mouse models showing tumor growth. (g) CCK-8 assay was used to detect whether the addition of Fer-1, Z-VAD-FMK, 3-MA, and RSL3 affected cell viability. Data are presented as mean ± SD from three independent experiments. * p < 0.05, * p < 0.01, *** p < 0.001, ns, not significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

AC007271.3 acts as an MBP-1 decoy, preventing its binding to the c-Myc promoter and thereby activating c-Myc transcription

Our previous studies established that lncRNA AC007271.3 promotes proliferation in OSCC [6, 7]. To further investigate the role of AC007271.3 in OSCC, RNA pull-down assay and mass spectrometry (MS) were used to identify potential proteins interacting with AC007271.3 in SCC9 cells. Proteins captured by biotin-labeled sense probes were visualized by silver staining (Fig. 3a) and identified by MS; the antisense probes served as a negative control. The MS analysis revealed six differentially enriched proteins, and MBP-1 was identified because of its inhibitory effect on c-Myc transcription. The interaction was confirmed by independent co-immunoprecipitation in SCC9 cells (Fig. 3b). Loss or cytoplasmic mislocalization of MBP-1, together with ENO1 upregulation, hypoxia-driven translational shifts, or ncRNA-mediated inhibition, results in sustained c-Myc activation, metabolic reprogramming, and increased tumor aggressiveness across multiple malignancies, including breast, gastric, colorectal, liver, esophageal, pancreatic, and oral squamous cell carcinomas [13, 14]. In OSCC, dysregulated ENO1/MBP-1 balance, with cytoplasmic ENO1 upregulation and loss of nuclear MBP-1, leads to sustained c-Myc activation and enhanced proliferation, invasion, and chemoresistance [15].

Fig. 3.

AC007271.3 functions as an MBP-1 decoy to activate c-Myc transcription. (a) Biotinylated AC007271.3 and AC007271.3-Antisense RNA pull-down assay and silver-staining indicated differential bands located at 35–55 kDa. Peptides with different scores are shown in the mass spectrometric analysis of the RNA pull-down elution proteins. (b) Western blotting was performed to verify the association of MBP-1 in SCC9 cells. (c, d) RNA-immunoprecipitation (RIP) experiments were performed using His-tag antibody for immunoprecipitation and a primer to detect AC007271.3 in SCC15 cells after transfection with pcDNA3.1 + His-MBP-1. (e) RIP assays were performed by His-tagged MBP-1 in SCC9 cells after transfected by fragmented-AC007271.3 pcDNA3.1 + plasmid. (f) Western blotting was performed to verify that MBP-1 negatively regulates c-Myc. (g) Schematic diagram showing the design of the dual-luciferase system. (h) Luciferase assays were conducted to assess the luciferase activities of the c-Myc promoter in response to MBP-1. (i) ChIP-qPCR assay revealed the direct interaction between MBP-1 and c-Myc promoter, with IgG as the negative control. (j) Schematic diagram showing that MBP-1 inhibited the c-Myc transcription. (k) Western blotting revealed that overexpressed AC007271.3 promoted c-Myc expression, which could be reversed after cotransfection with MBP-1. (l) Schematic diagram showing the design of the dual-luciferase system. (m) Luciferase assays were conducted to assess the luciferase activities of the c-Myc promoter in response to overexpressed AC007271.3, ΔAC007271.3, MBP-1, and ΔMBP-1. (n) Schematic diagram showing the hypothesis that AC007271.3 directly binds to MBP-1 and promotes transcription of c-Myc. (o) ChIP-qPCR assay revealed a lack of interaction between MBP-1 and c-Myc promoter when AC007271.3 was transfected, also in the ΔMBP-1 group, with IgG as the negative control. The experiments are representative of three independent experiments with similar results. Data are presented as mean ± SD of three biological replicates. ***, p < 0.001, ns, not significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

In comparison to IgG, the MBP-1 antibody significantly enriched AC007271.3 transcripts. Based on their interaction, we hypothesised that AC007271.3 might contribute to the proliferation of OSCC cells via MBP-1/c-Myc. MBP-1 is efficiently encoded by an alternative transcript of the ENO1 gene. Therefore, we further proved that AC007271.3 interacts with MBP-1, but not ENO1 (Supplementary Fig. 1a).

To further characterize the binding region between AC007271.3 and MBP-1, fragments of AC007271.3 cDNA were cloned into the pcDNA3.1 + plasmid [1-100 nucleotide (nt), 1-200 nt, 1-300 nt, full length] (Supplementary Table 1). Transfection of fragment-AC007271.3 plasmids and subsequent RIP analysis revealed that the 300–382 nt region of AC007271.3 was the MBP-1 binding site in SCC9 cells (Fig. 3c). According to the MBP-1 region in Uniprot and a previous study [16], the 1-139 amino acid region of MBP-1 is the c-Myc promoter binding domain. Therefore, we transfected the wildtype-MBP-1 (MBP-1) and the 1-139-depleted-MBP-1 (ΔMBP-1) plasmids with His-tag and performed RIP assays on both (Fig. 3e; plasmid efficacy verification and predicted protein structure by AlphaFold2 [17] are shown in Supplementary Fig. 1b). The RIP-qPCR assays revealed that the His-tag antibody, like IgG, enriched AC007271.3 in the MBP-1 group in SCC9 cells (Fig. 3d). Thus, the 300–382 nt of AC007271.3 binds to the c-Myc promoter binding region of MBP-1. We observed that MBP-1 overexpression suppressed c-Myc expression, whereas MBP-1 knockdown increased c-Myc levels in SCC9 cells (Fig. 2f). Consistently, luciferase reporter assays showed that MBP-1 inhibited c-Myc promoter activity (Fig. 3g-h), and Chromatin Immunoprecipitation (ChIP) assays confirmed the binding of MBP-1 to the c-Myc promoter in SCC9 cells (Fig. 3i-j). The above results indicated that MBP-1 could bind to the c-Myc promoter and inhibit its transcription. To determine whether AC007271.3 regulates c-Myc transcription by binding to MBP-1, western blot analysis showed that AC007271.3 overexpression abrogated MBP-1–mediated repression of c-Myc, resulting in increased c-Myc protein expression in SCC9 cells (Fig. 3k). Luciferase assays with c-Myc promoter constructs further demonstrated that AC007271.3 enhanced c-Myc transcriptional activity in SCC9 cells (Fig. 3l-m). To further substantiate this mechanism, ChIP-qPCR analysis revealed that the amount of MBP-1 binding to the c-Myc promoter increased in the ΔAC007271.3 group (300-382-depleted-AC007271.3) and decreased in the AC007271.3 group in SCC9 cells (Fig. 3n-o). These results establish that AC007271.3 functions as an RNA decoy to sequester MBP-1, thereby relieving its repression of c-Myc transcription and driving OSCC cell proliferation.

AC007271.3 facilitates the MBP-1/c-Myc axis to promote OSCC cell proliferation and G1/S transition

To clarify whether AC007271.3 regulates OSCC cell proliferation and cell-cycle progression through the MBP-1/c-Myc axis, AC007271.3 was overexpressed in OSCC cells (SCC9^OE and SCC15^OE), resulting in a marked increase in c-Myc expression (Fig. 4a). Functional assays revealed that AC007271.3 enhanced proliferation and migration, as demonstrated by increased wound healing, cell viability, and EdU incorporation, whereas silencing c-Myc produced the opposite effects (Fig. 4b-d). Flow cytometric analysis further demonstrated that AC007271.3 promoted S-phase entry, while c-Myc depletion induced G1 arrest in SCC9 cells (Fig. 4e). These findings indicate that AC007271.3 drives cell-cycle progression in OSCC cells in a c-Myc-dependent manner. Rescue experiments involving different transfection combinations (AC007271.3, ΔAC007271.3 (300–382-depleted-AC007271.3), MBP-1, ΔMBP-1 (1-139-depleted-MBP-1), and si-MBP-1) confirmed that AC007271.3 promoted proliferation and migration of SCC9 cells by interacting with MBP-1 to enhance c-Myc transcriptional activity (Fig. 4f-h). Consistent with these results, flow cytometric analysis revealed that SCC9 cells in the AC007271.3-, MBP-1-, and si-MBP-1-transfected groups accumulated in the S phase, whereas those in the ΔAC007271.3 and ΔMBP-1 groups were predominantly arrested in G1 phase (Fig. 4i). EdU assays showed a higher proportion of SCC9 cells undergoing DNA replication status in the exogenous AC007271.3, MBP-1, and si-MBP-1 groups, while the other three groups showed less (Fig. 4j). Moreover, the expression levels of CDK4, cyclin D1, CDK2, cyclin E1, and E2F1 increased, whereas p21 and p27 decreased in AC007271.3-, MBP-1-, and si-MBP-1-transfected SCC9 cells (Fig. 4k). Their expression pattern was consistent with the fact that c-Myc promotes cell cycle progression (Fig. 4l). To further verify the role of AC007271.3, antisense oligonucleotides (ASOs in Supplementary Table 1) specifically targeting AC007271.3 were introduced into SCC9^OE cells. In vivo, subcutaneous xenografts derived from SCC9^OE cells exhibited accelerated tumor growth, whereas ASO treatment markedly suppressed tumor progression (Fig. 4m-n). The level of c-Myc protein expression in xenografts was validated in Fig. 4o. HE-staining showed cancer nests of extracted xenograft tumor tissues (Fig. 4p). Ki67 expression is significantly associated with the degree of differentiation and prognosis in various tumors. Ki67 immunostaining revealed significantly less proliferation in SCC9^OE+ASO cell xenograft tumors than in the AC007271.3 group (Fig. 4q). Collectively, these findings demonstrated that AC007271.3 facilitates OSCC cell proliferation and G1/S transition by modulating the MBP-1/c-Myc signaling axis (Fig. 4r).

Fig. 4.

AC007271.3 facilitates the MBP-1/c-Myc axis to promote OSCC cell proliferation and G1/S transition. (a) The knockdown efficiency of si-c-Myc transfection in SCC9^OE and SCC15^OE cell lines was determined by western blotting. (b) Wound healing assays were conducted to measure the effect of knockdown of c-Myc in overexpressed-AC007271.3 SCC9 cells. (c-d) Cell proliferation of SCC9^OE and SCC15^OE cells following c-Myc knockdown with two independent siRNAs was evaluated by CCK-8 assays. (e) Cell-cycle analysis of overexpressed-AC007271.3 SCC9 cells after they were transfected with two different si-c-Myc. (f-h) Wound healing and CCK-8 assays were performed to evaluate the role of the AC007271.3/MBP-1/c-Myc axis in SCC9 cell proliferation. (i) Cell cycle analysis in SCC9 cells after they were transfected with overexpressed AC007271.3, ΔAC007271.3, MBP-1, ΔMBP-1, and si-MBP1. (j) EdU assays showed corresponding results with statistical analysis provided. (k) Western blotting was performed to verify the AC007271.3/MBP-1/c-Myc axis effects on the G1/S phase of the cell cycle. (l) Western blotting was performed to verify the efficacy of ASO targeting AC007271.3 in SCC9^OE cells. (m) Xenograft mice were subcutaneously implanted with SCC9^OE cells, SCC9^NC cells, and SCC9^OE cells injected with ASO. The ‘X’ symbol indicates a data point excluded from analysis due to complete tumor regression in one mouse of the SCC9^OE+ASO group. This animal was euthanized at the predefined ethical endpoint as no palpable tumor remained at the measurement time point. (n–q) Tumor growth, c-Myc expression, and histological features, including HE and Ki67 staining, were evaluated. (r) Illustrative schematic for this section. The experiments are representative of three independent experiments with similar results. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

AC007271.3 promotes OSCC proliferation and invasion through c-Myc-mediated ferroptosis suppression

As a well-established oncogenic driver, c-Myc suppresses ferroptosis to enhance the invasive potential of OSCC cells. However, the precise mechanism through which c-Myc regulates ferroptosis in OSCC remains largely undefined [18]. To clarify whether AC007271.3 modulates ferroptosis through c-Myc, rescue experiments were performed.

Knockdown of AC007271.3 markedly increased lipid peroxide accumulation, whereas c-Myc partially reversed this effect (Fig. 5a). Knockdown of AC007271.3 also elevated mitochondrial membrane potential and induced hyperpolarization, which was partially reversed by c-Myc overexpression (Fig. 5b). In parallel, GPX4 mRNA and protein levels decreased upon AC007271.3 knockdown but were rescued by c-Myc (Fig. 5c-d). These results suggest that c-Myc mediates the regulatory role of AC007271.3 in ferroptosis.

Fig. 5.

AC007271.3 promotes OSCC proliferation and invasion through c-Myc-mediated ferroptosis suppression. (a) Overexpression of c-Myc rescued the lipid peroxidation levels of OSCC cells following AC007271.3 knockdown. (b) Overexpression of c-Myc rescued the mitochondrial membrane potential of OSCC cells following AC007271.3 knockdown. (c) Overexpression of c-Myc rescued the GPX4 protein levels of OSCC cells following AC007271.3 knockdown. (d) Overexpression of c-Myc rescued the GPX4 mRNA levels of OSCC cells following AC007271.3 knockdown. (e-h) Overexpression of c-Myc rescued the proliferation, migration, and invasion abilities of OSCC cells following AC007271.3 knockdown. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

To determine whether AC007271.3 enhances OSCC progression via c-Myc, functional assays including wound healing, CCK-8, colony formation, invasion, and migration were conducted. These assays demonstrated that AC007271.3 consistently promoted proliferation, migration, and invasion of OSCC cells in c-Myc-dependent manner (Fig. 5e-h).

c-Myc binds to the GPX4 promoter and enhances its transcriptional activity in OSCC cells

As a well-established oncogenic driver, c-Myc suppresses ferroptosis to enhance the invasive potential of OSCC cells. However, the precise mechanism through which c-Myc regulates ferroptosis in OSCC remains largely undefined. Western blot analysis revealed that silencing AC007271.3 markedly reduced the protein levels of c-Myc and GPX4, whereas c-Myc overexpression restored their expression (Fig. 6a). To further elucidate the molecular mechanisms governing the GPX4 promoter, we used JASPAR to predict potential c-Myc binding motifs within this region (Fig. 6b). Dual-luciferase reporter assays demonstrated that c-Myc significantly enhanced transcriptional activity by binding to specific GPX4 promoter fragments (Fig. 6c). We subdivided the 1–2099 bp region of the GPX4 promoter into seven fragments, and ChIP assays revealed that c-Myc was enriched at fragments #4, #5, and #7, thereby confirming that GPX4 serves as a direct transcriptional target of c-Myc in OSCC cells (Fig. 6d-f).

Fig. 6.

c-Myc binds to the GPX4 promoter and enhances its transcriptional activity in OSCC cells. (a) Western blot detection of GPX4 protein expression. (b) The binding sequence logo of c-Myc on JASPAR database. (c) Dual-luciferase reporter assay was performed to verify that c-Myc binds to the GPX4 promoter. (d) ChIP assay schematic diagram and the sequence logo of GPX4 promoter. (e) Schematic workflow of the ChIP assay. (f) ChIP assay was performed to verify the binding between c-Myc and GPX4 promoter binding site. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

AC007271.3 accelerates OSCC growth and cisplatin resistance by inhibiting ferroptosis

Given that AC007271.3 suppresses ferroptosis through c-Myc-mediated activation of GPX4, we sought to determine whether this mechanism contributes to cisplatin resistance in OSCC. Consistent with this hypothesis, silencing AC007271.3 in SCC9 and SCC15 cells significantly enhanced cisplatin sensitivity, as indicated by reduced cell viability compared with control cells (Fig. 7a). In subcutaneous xenografts, AC007271.3 overexpression significantly promoted tumor proliferation. However, treatment with cisplatin markedly suppressed tumor growth and final tumor weight, and this inhibitory effect was partially mitigated by co-administration of ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor (Fig. 7b-e). Western blot analysis of xenograft tissues revealed that AC007271.3 overexpression increased c-Myc and GPX4 protein levels compared with control tumors. This upregulation was further increased by the addition of Fer-1. In contrast, cisplatin reduced the AC007271.3-induced upregulation of both proteins, whereas co-treatment with Fer-1 partially restored their expression (Fig. 7f). These data show that AC007271.3 drives cisplatin resistance in OSCC by inhibiting ferroptosis in vivo, which in turn enables tumors to sustain growth under chemotherapy

Fig. 7.

AC007271.3 accelerates OSCC growth and cisplatin resistance by inhibiting ferroptosis. (a) Dose-dependent cell viability assays were performed to examine the effect of knockdown of AC007271.3 on the drug sensitivity of OSCC cells treated with 0 to 200 µM cisplatin. (b) Schematic illustration of subcutaneous xenograft formation. (c) Subcutaneous xenograft assay and tumor weight measurement. (d) Tumor volume was measured in two dimensions using a vernier caliper. (e) Tumor growth curves were recorded in xenograft mouse model. (f) Western blot analysis was performed to examine the protein levels of GPX4 and c-Myc in subcutaneous xenograft tumors, and band intensities were quantified using ImageJ. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparisons

Discussion

Recently, mounting evidence indicates that CT-lncRNAs promote proliferation, invasion, and immune evasion of cancer cells and play a pivotal role in tumorigenesis [19]. A study by Xia et al. found that lnc-CTHCC, a CT lncRNA, is significantly upregulated in hepatocellular carcinoma, ultimately driving sustained tumor cell proliferation, migration, and tumor formation [20]. However, the role of CT-lncRNAs in OSCC remains elusive. We discovered that AC007271.3 is specifically enriched in human testicular tissue by analyzing expression data from the GTEx database (V8). This work provides the first evidence that AC007271.3 functions as a CT-lncRNA in OSCC, indicating that CT-lncRNAs play an important role in the development of OSCC.

OSCC serves as one of the most common oral malignancies, and the underlying mechanisms of OSCC occurrence, development and metastasis still need to be further explored. Our previous study demonstrated that AC007271.3 could promote the progression of OSCC, while the underlying mechanism requires further study. LncRNAs have been recently confirmed to act as molecular decoys that inhibit regulatory protein-DNA binding. Consequently, this decoy activity allows lncRNAs to dynamically fine-tune transcription factor networks, indicating their promise as biomarkers or therapeutic targets [21]. MBP-1 is a 37-kDa human c-Myc promoter-binding protein localized to the c-Myc P2 promoter, and it inhibits the activity of the c-Myc transcription factor and suppresses carcinogenesis in various types of tumor [22, 23]. In esophageal squamous cell carcinoma, LINC00239 [24] interacts with MBP-1 to promote c-Myc expression and the EMT process. In this study, results showed that the 300–382 nt region of AC007271.3 directly binds to the 1-139 amino acid region of MBP-1, which is the essential domain for its binding to the c-Myc promoter, and then blocks MBP-1 binding to the c-Myc promoter. It has been well established that activation of c-Myc induces transcription of the cyclin E1 gene and increases in cyclin E1 mRNA, resulting in the activation of the G1/S phase [25]. We revealed that AC007271.3 promotes the transition of OSCC cells from the G1 to the S phase via MBP-1/c-Myc regulation. Disruption of the binding site blocked the interaction between AC007271.3 and MBP-1, the G1 to S phase transition was halted, resulting in tumor cell growth suppression. Our studies showed that the MBP-1 binding level increased in the c-Myc promoter region after inhibition of AC007271.3, resulting in enhanced expression of c-Myc. These results indicate that AC007271.3 acts as a decoy for MBP-1 to regulate c-Myc expression and drive OSCC progression.

Moreover, we found that CT-lncRNA AC007271.3 promotes tumor progression by inhibiting ferroptosis of OSCC. Interestingly, our results showed that the antioxidant enzyme glutathione peroxidase 4 (GPX4) is a critical mediator of AC007271.3-driven ferroptosis suppression in OSCC. Evidence suggests that GPX4 plays an essential role in ferroptosis, utilizing GSH as a substrate to convert lipid hydroperoxides into non-toxic lipid alcohols, thereby preventing the accumulation of lethal lipid ROS. GPX4 serves as the central enzymatic suppressor of ferroptosis [26, 27]. However, the activation of GPX4 in ferroptosis remains largely unexplored. Converging evidence showed that the protein degradation and regulation of GPX4 are tightly regulated by a network of complex mechanisms. Previous work has shown that GPX4 is a selenocysteine (Sec)-containing protein. Selenium utilization plays a crucial role in ferroptosis because the anti-oxidative activity of GPX4 depends on a selenocysteine residue at 46 (U46) [28]. Li et al. reported that the deubiquitinase USP8 removes ubiquitination from GPX4, thereby stabilizing GPX4 protein and facilitating ferroptosis [29]. In human pancreatic ductal adenocarcinoma (PDAC) cells, heat shock 70 kDa protein 5 (HSPA5) bound GPX4 and protected against GPX4 protein degradation, negatively regulating ferroptosis [30]. A study showed that mitochondrial GPX4 acetylation, probably caused by SIRT3 reduction, partially contributes to cadmium-induced renal cell ferroptosis [31]. It indicates that the ubiquitination of GPX4 is an important regulatory mechanism for its activation. In addition, phosphorylation of proteins plays a key and universal role in the signal transduction of many vital cellular processes including ferroptosis and cell cycle regulation. A study has shown that creatine kinase B (CKB) acted as a protein kinase and phosphorylated GPX4 S104, which prevented HSC70 binding to GPX4, thereby abrogating the GPX4 degradation regulated by chaperone-mediated autophagy, alleviating ferroptosis and promoting tumour growth in mice [32]. In summary, these findings indicate that post-translational modifications, including ubiquitination, acetylation, and phosphorylation, participate in regulating GPX4 activity and ferroptosis. Unlike the known results, we found that c-Myc could bind to the GPX4 promoter region and enhance its transcriptional activity, thereby suppressing OSCC ferroptosis. To our knowledge, this is a new activation method of GPX4 that has never been reported before. It is a novel ferroptosis pathway that helps elucidate the intricate mechanism of ferroptosis in tumors.

Chemotherapy resistance is a major challenge for patients with advanced or unresectable OSCC. Cisplatin is a first-line standard chemotherapy regimen for head and neck squamous cell carcinoma. Therefore, it is urgent to reveal the mechanisms driving resistance to cisplatin in OSCC and to develop feasible strategies to enhance the efficacy of cisplatin, potentially improving the prognosis for OSCC patients. Cisplatin is a well-known ferroptosis inducer that induces RCD under oxidative stress by inducing the production of ROS in cells [33]. Accumulating evidence has demonstrated that excessive induction of ferroptosis in cancer cells can effectively overcome tumor progression and chemoresistance, including cisplatin, paclitaxel, and 5-FU in diverse cancers [33]. Yet how lncRNAs regulate ferroptosis and induce chemoresistance in OSCC remains poorly defined. Our results found that CT-lncRNA AC007271.3 could inhibit ferroptosis and promote cisplatin resistance in OSCC. This work provides the first evidence that a lncRNA inhibits ferroptosis to overcome cisplatin resistance in OSCC, offering a new treatment strategy for OSCC.

Conclusion

In summary, our study reveals that the cancer-testis lncRNA AC007271.3 acts as a decoy for MBP-1 to block MBP-1 binding to the c-Myc promoter and thus enhanced c-Myc expression. Elevated c-Myc further transcriptionally upregulated GPX4 which suppressed ferroptosis and facilitated G1/S transition, ultimately driving tumor progression and chemotherapy resistance. To our knowledge, this study provides the direct evidence that a CT lncRNA plays a vital role in OSCC carcinogenesis and inhibits ferroptosis, CT lncRNA AC007271.3 promotes cisplatin resistance by inhibiting ferroptosis in OSCC, and indentified a novel ferroptosis mechanism in which c-Myc directly triggers transcriptional activation of GPX4 and thus suppresses ferroptosis. These findings provide important mechanistic insights into ferroptosis regulation and tumorigenesis in OSCC.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

ZQ, QW designed the project. ZQ and QW conducted most of the experiments. ZQ, QW, XZ and HW performed animal experiments. ZQ, ZZ and MW analyzed the data. ZQ, QW and ST. Fonseca wrote and edited the manuscript. All authors have read and agreed to the contents of the manuscript and have consented to its publication.

Funding

This study was supported by the National Natural Science Foundation of Guangdong Province (2024A1515013010), Funding by Science and Technology Projects in Guangzhou (2024A04J4873) and President Foundation of Zhujiang Hospital, Southern Medical University (yzjj2024qn43).

Data availability

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

Declarations

Ethics approval and consent to participate

The authors affirm that all methods were conducted in accordance with the relevant guidelines and regulations. All mice were housed at the Animal Center of Southern Medical University, and approval was obtained from the Animal Committee of the Institute of Zoology at Southern Medical University (reference number: L2018199, LAEC-2025-053). All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The reporting of this in vivo research adheres to the ARRIVE guidelines 2.0.

Competing interests

The authors declare no competing interests.