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DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

Journal of Translational Medicine volume 22, Article number: 1167 (2024) Cite this article

Abstract

Background

The RAR-related orphan receptor alpha (RORA), a circadian clock molecule, is highly associated with anti-oncogenes. In this paper, we defined the precise action and mechanistic basis of RORA in ESCC development under hypoxia.

Methods

Expression analysis was conducted by RT-qPCR, western blotting, immunofluorescence (IF), and immunohistochemistry (IHC) assays. The functions of RORA were assessed by detecting its regulatory effects on cell viability, motility, invasion, and tumor growth. DNA pull-down assay and proteomic analysis were employed to identify proteins bound to the RORA promoter. The promoter methylation level of RORA was detected by DNA pyrosequencing. RNA-seq analysis was performed to explore the downstream mechanisms of RORA, and the transcriptional regulation of RORA on SLC2A3 was verified by ChIP-qPCR and dual-luciferase reporter assay. Glycolysis was assessed by detecting the consumption of glucose and the production of lactic acid and ATP.

Results

In vitro, RORA was shown to suppress ESCC cell viability, motility, and invasion under hypoxic condition. In vivo, increased RORA expression in mouse xenografts impeded tumor growth. DNMT1 was identified to widely exist in the RORA promoter, increasing DNA methylation and reducing RORA expression in hypoxia-induced KYSE150 ESCC cells. Mechanistically, RORA was found to inactivate the transcription of glucose transporter protein SLC2A3 by interacting with its promoter F1 region. Furthermore, rescue experiments revealed that RORA-mediated suppressive effects on ESCC cell migration and invasion were largely based on its negative regulation of SLC2A3 and glycolysis.

Conclusion

DNMT1-driven methylation of RORA promotes ESCC progression largely through affecting SLC2A3 transcription and glycolysis. These findings turn RORA into potential target of anti-cancer therapeutic agents.

Highlights

RORA works as a tumor suppressor in ESCC under hypoxia.

DNMT1 downregulates RORA by induction of methylation.

RORA inactivates SLC2A3 transcription through its promoter F1 region.

RORA inactivates SLC2A3 transcription to inhibit ESCC cell glycolysis and metastasis.

Introduction

Esophageal squamous cell carcinoma (ESCC) is the most prevalent subtype of esophageal cancer and is recognized as one of the most aggressive malignancies [1]. The pathogenesis of ESCC is influenced by a multitude of complex factors, including genetic predispositions and environmental stimuli [2,3,4]. Despite substantial research efforts in recent years, the molecular determinants of ESCC remain largely elusive, leading to a lack of effective targeted therapies and poor clinical outcomes. Enhancing awareness and deepening the understanding of the molecular underpinnings of ESCC are essential for improving disease management strategies.

Accumulating evidence have suggested that dysregulation of the circadian clock is pivotal in cancer initiation and progression, including ESCC. The transcription factor (TF) RAR-related orphan receptor alpha (RORA), which functions as a circadian clock molecule, is highly associated with anti-oncogenes and has been highlighted as a potential drug target in cancer management [5, 6]. The abundance of RORA can be increased by various stimuli, including hypoxia, thereby contributing to the cellular stress response [7, 8]. Researches across various cancer types have elucidated the inactivation and significant implications of RORA. In lung cancer, reduced expression of RORA suggests its potential utility as a biomarker for this disease, and its biological function depends on the modulation of target protein activation and oncogenic signaling [9]. In hepatocellular carcinoma, comprehensive researches have uncovered the anti-cancer property of RORA, indicating its potential as a novel prognostic biomarker for patients with hepatocellular carcinoma [10]. Furthermore, a previous study reported that circRNA-induced upregulation of RORA is responsible for the repression of gastric cancer cell growth and metastasis [11]. Similarly, dysregulated RORA has been reported to be relevant to ESCC tumorigenesis [12, 13]. However, the mechanism underlying RORA dysregulation in ESCC remains unclear.

Gene expression is regulated by DNA methylation [14]. Generally, abnormal methylation levels at gene promoters result in the inactivation of tumor suppressor genes and other genes associated with tumorigenesis, thereby contributing to the progression of human cancers [15]. This study investigated whether the dysregulation of RORA expression in ESCC is associated with DNA methylation. DNMT1, a key member of the DNA methyltransferase family, is crucial for maintaining DNA methylation by mediating inheritance from maternal cells to offspring cells [16]. Inhibitors targeting DNMT1 have been proposed as promising anti-tumor agents due to their ability to restore anti-tumor gene function [17], including ESCC [18, 19]. Hence, we investigated whether DNMT1 was involved in the regulation of RORA expression by regulating the DNA methylation of its promoter.

In addition to gene targets, aerobic glycolysis may be a new potential target for cancer therapy due to the increased dependence of tumor cells on glycolysis. Cancer cells undergo metabolic reprogramming to facilitate survival, growth, and metastasis, characterized by increased glucose uptake and conversion of glucose to lactate to support elevated demands for cell proliferation. The upregulation of glycolysis not only meets energy requirement but also plays a critical role in generating metabolic intermediates essential for macromolecule synthesis in cancer cells [20]. Targeting glycolytic changes has been reported to be a promising therapeutic strategy [21]. The glucose transporter (GLUT) family, also known as the solute carrier 2 A (SLC2A) family, exerts a vital function in glucose transport across the plasma membrane, which is the initial step of glycolysis [22].

Here, we address these questions-what drives RORA dysfunction and what are the consequences of dysregulated RORA activity in ESCC and the possible working mechanism by exposing ESCC cells to hypoxia, an important feature of solid cancers [23], with the hope that this work may provide a new opportunity for targeted drug development in ESCC.

Materials and methods

Analysis of clinical samples

Paired carcinomatous and para-carcinomatous tissues were collected from five ESCC patients (demographic information could be found in Supplementary Table 1) and were subjected to IHC assay to measure RORA and DNMT1 levels. Briefly, samples were fixed with 4% paraformaldehyde, paraffin-embedded with 100% liquid paraffin, and sliced into 5 μm-thick sections. A commercial VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used for IHC assay. Scale bar: 20 μm.

Cell lines, treatment and hypoxia stimulation

Human TE-1 (#STCC11904G, Servicebio, Wuhan, China) and KYSE150 (#CL-0638, Procell, Wuhan, China) ESCC cell lines, authenticated by their providers, were used in this paper. We maintained the ESCC cells in Corning culture plates/dishes (Corning, Shanghai, China) with 10% FBS RPMI-1640 (Servicebio) in a 37℃ atmosphere of 5% carbon dioxide. For repression of DNA methylation in ESCC cells, we obtained decitabine (DCA, #S1200), a repressor of DNMT1, from Selleck (Shanghai, China). ESCC cells were seeded on 24-well sterile plastic dishes. After 24 h, DCA reagent was added at a 0.5 µM concentration and incubation was done for 48 h. Exposure of ESCC cells to hypoxia was achieved by maintaining cells for 24 h in a modular incubator containing 1% O2, 94% N2, and 5% carbon dioxide.

Cell transfection

Plasmids including pLV3-U6-RORA(human)-shRNA1 (#P37005), pLV3-U6-RORA(human)-shRNA3 (#P37004), pLV3-CMV-RORA(human) (#P37357), and matched control mocks (shNC and vector) were from Miaolingbio (Wuhan, China). Lentiviral constructs expressing shRORA or shDNMT1 sequence and non-targeting sequence were made by Tsingke (Beijing, China). ESCC cells stably expressing RORA were produced by introducing pLV3-CMV-RORA(human) in cells via Lipofectamine 3000 (Invitrogen, Bleiswijk, the Netherlands). The pLV3-U6-RORA(human)-shRNA1 and pLV3-U6-RORA(human)-shRNA3 were transfected to ESCC cells in a ratio of 1:1 to downregulate RORA expression. To stably knock down RORA, lentivirus supernatants were applied to infect ESCC cells and puromycin was utilized to select target cells after 72 h of infection.

RT-qPCR

For analysis of RORA and SLC2A3 mRNA, we carried out RT-qPCR with 100 ng of RNA prepared from ESCC cells and xenografts with RNAeasyTM Isolation Kit (#R0027, Beyotime, Shanghai, China). cDNA was Oligo(dT)-primed using SweScript RT II cDNA Synthesis Kit (#G3333, Servicebio), and SYBR-based qPCR was conducted with 2× SYBR® mix (#4472908, ABI, Foster city, CA, USA) using primers for RORA (sense: 5’-CCGTGGTCAATCATGGGTCAT-3’; anti-sense: 5’-AGTTCATCCCTTCTGGCTCC-3’), primers for SLC2A3 (sense: 5’-TGCCTTTGGCACTCTCAACCAG-3’; anti-sense: 5’-GCCATAGCTCTTCAGACCCAAG-3’) and primers for SLC2A14 (sense: 5’-TTGGTGGGTTCTTGGTCTCACT-3’; anti-sense: 5’-CCGATTGTAGCAACTGTGATGG-3’). Via the 2-ΔΔCt with logarithm transformation, we determined the fold change after normalization with β-actin (sense: 5’-GATTCCTATGTGGGCGACGA-3’; anti-sense: 5’-TCCCAGTTGGTGACGATGC-3’).

Western blotting

After transfection and hypoxia stimulation, we prepared whole cell lysate in a lysis buffer (RIPA buffer plus PMSF and phosphatase inhibitors) (all from Beyotime). For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR). Immunoreactive bands were visualized by a Hypersensitive ECL Kit (#G2020, Servicebio).

Detection of cell viability

ESCC cells maintained in a 96-well sterile plastic plate were subjected to hypoxia stimulation after the relevant transfection. After that, culture media were changed with media plus 10 µL per well of CCK-8 substrate (#C0041, Beyotime). The plate was then incubated for 2 h in a 37℃ incubator. We scored the absorbance for each well at 450 nm.

Wound-healing and transwell assays

The wound-healing assay for motility evaluation was carried out under standard procedures [24]. Briefly, ESCC cells after transfection and hypoxia stimulation were grown in Corning 6-well sterile plastic plates. Using a sterile tip, we made a vertical wound. For migration assessment, images were taken at 0 h and 24 h under light microscopy. We also performed in vitro migration assay with the aid of 8.0 μm pore size Transwells (24-well, BD Biosciences, Stockholm, Sweden). Meantime, the in vitro invasion assay was done under the use of Matrigel chambers (BD Biosciences). In brief, serum-starved ESCC cells after transfection and hypoxia stimulation (2.5 × 104 cells for migration and 1 × 105 cells for invasion) were added to each upper compartment and allowed to migrate or invade for 24 h to lower compartments containing 10% FBS RPMI-1640. The ESCC cells that attached to the undersurface were observed and quantitated under microscopy after visualization with crystal violet.

Xenograft studies

All mouse work was done following approved protocols from Henan Provincial People’s Hospital Animal Care and Use Committee. At the time of cell implantation, all BALB/c nude mice (n = 10, #VM0020, Shulaibao, Wuhan, China) were six to eight weeks of age. These mice were grouped into designated groups, and each group contained five mice. Lenti-RORA- and lenti-NC-infected KYSE150 ESCC cells were prepared as mentioned above and injected into right flanks (5 × 106 cells/mouse) by subcutaneous implantation. After 28 days, the volume and weight of xenografts were recorded, and the xenografts were acquired for RORA mRNA analysis, HIF1A protein expression analysis, IHC staining, and IF staining. For HIF1A protein expression analysis, tissue samples from the xenografts, as well as normal esophageal tissues from healthy BALB/c mice, were collected for this purpose. The normal group served as a control comparison to assess the change in HIF1A protein expression level.

Immunohistochemistry (IHC) analysis

As described earlier [25], standard IHC staining of formalin-fixed paraffin-embedded xenografts was done using rabbit anti-Ki67 pA (1:5,000, #27309-1-AP, Proteintech). After de-paraffinization and rehydration, slides of xenografts were subjected to heat-induced epitope retrieval and blocking before incubation of anti-Ki67 antibody and HRP-IgG secondary antibody (1:300, #GB23303, Servicebio). For color development, slides underwent DAB staining and haematoxylin counterstaining before microscopy examination.

Immunofluorescence (IF) staining assay

After transfection and hypoxia stimulation, expression of MMP2 and MMP9 in ESCC cells was detected by IF microscopy with rabbit anti-MMP2 pA (1:300, #10373-2-AP) or rabbit anti-MMP9 pA (1:500, #10375-2-AP) from Proteintech. Briefly, after transfection and hypoxia stimulation, ESCC cells were subjected to fixation and permeabilization in 0.5% Triton X-100. Following 3% BSA incubation, the relevant antibody was used overnight at 4℃, and followed by secondary antibody incubation with Alexa-488-linked IgG (1:500, #GB25303) from Servicebio. The cells were then subjected to three PBS washes, with the third wash containing DAPI for nuclei staining.

Positive staining of PCNA and expression of MMP2 and MMP9 in KYSE150 xenografts were also evaluated by IF microscopy with rabbit anti-PCNA mAb (1:200, #ab92552, Abcam, Cambridge, UK), rabbit anti-MMP2 pA, or rabbit anti-MMP9 pA following standard protocols. Briefly, after rehydration, the slides of formalin-fixed paraffin-embedded xenografts were subjected to heat-induced epitope retrieval and 3% BSA blocking. After incubation with a relevant antibody at 4℃, the slides were then incubated with Cy3-labeled IgG (1:500, #GB21303) before examination using microscopy.

DNA pull-down assay and proteomic analysis

The human RORA promoter sequence (positions − 2000 to -44) was retrieved from Ensembol database (http://asia.ensembl.org/index.html) and synthesized by Tsingke. Using the promoter sequence as templates, the biotin-labelled RORA promoter probe (P4:1-1956 bp) and three biotin-labelled promoter truncations (P1: 1-895 bp, P2: 550–1468 bp, P3: 1177–1956 bp) were PCR-amplified with primers for P4 (sense: 5’-AAAGGAAACCAGGGATACTT-3’; anti-sense: 5’-GAAGGAGAAGGCGACCGGCG-3’), P1 (sense: 5’-AAAGGAAACCAGGGATACTT-3’; anti-sense: 5’-CTCCAAGATGAAGGAACAATAC-3’), P2 (sense: 5’-TGGCATATGGCGGTTCCTCTG-3’; anti-sense: 5’-AAAGGAATTAGAACGGGGCTGAC-3’), and P3 (sense: 5’-ATCCAGCTTAGATAGTTGTCG-3’; anti-sense: 5’-GAAGGAGAAGGCGACCGGCG-3’). The four generated PCR products were preliminary validated by 1% agarose gel electrophoresis, and their biotin labelling efficiencies were detected by an HRP-conjugated streptavidin (1:3,000, #G3431) from Servicebio. In brief, each of PCR products was added to 0.45 μm pore size nylon membranes (#FFN10, Beyotime) before ultraviolet crosslinking and blocking. The membranes underwent color development with ECL kit after probing with HRP-conjugated streptavidin.

We performed the DNA pull-down assay with the nuclear protein extracted from KYSE150 ESCC cells exposed to hypoxia under the use of Cytoplasmic&Nuclear Protein Isolation Kit (#P0028) from Beyotime. Briefly, a mixture of each probe (10 µg) and streptavidin beads (35 µL, #SA10004, Invitrogen) rinsed with TBST was prepared for 30 min at room temperature. The extracted nuclear proteins were then added into the mixture and incubation was done for 60 min. Proteins in the co-precipitated complexes were harvested and boiled in SDS loading buffer for 5 min. The co-precipitated protein samples in each probe group were subjected to proteomics analysis by HPLC-MS/MS method with L-3000 HPLC System, which was performed by Qinglianbio (Beijing, China). Through Proteome Discoverer2.4, the Raw MS data were processed.

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was carried out to verify the relationship of DNMT1 and the RORA promoter with ChIP Assay Kit (#P2078) using standard protocols recommended by Beyotime. In brief, whole cell lysate extracted from KYSE150 ESCC cells exposed to hypoxia were subjected to ultrasonic processing to produce DNA fragments of 400–800 bp, and followed by the addition of rabbit anti-DNMT1 pA (1:500, #24206-1-AP, Proteintech) or rabbit IgG pA (1:1000, #30000-0-AP, Proteintech) and overnight incubation at 4℃ on a shaker. The next day, 60 µl of A + G Agarose/Salmon Sperm DNA was added to pull down the DNMT1-associating complexes. The RORA promoter was detected by qPCR with specific primers (sense: 5’-AAGGAGCTGAAAGCGACCTC-3’, anti-sense: 5’-GGCCAGTGGGTTTCCAATCT-3’), producing a product of 116 bp.

The binding motif of RORA and SLC2A3 promoter region was predicted by the JASPAR database (http://jaspar.genereg.net/). ChIP assay was performed to identify the binding sites between RORA and SLC2A3 promoter using the similar procedure. Rabbit anti-RORA polyclonal antibody (pA) (1:500, #10616-1-AP, Proteintech) or rabbit IgG pA (1:1000, #30000-0-AP, Proteintech) were used. Three pairs of primers targeting SLC2A3 promoter were used for qPCR detection, and the sequences could be found in Supplementary Table 2.

DNA pyrosequencing

KYSE150 ESCC cells were subjected to the indicated treatment or transfection before hypoxia stimulation. The promoter methylation level of RORA in the cells was analyzed by DNA pyrosequencing as reported [26]. For pyrosequencing-based analysis of RORA promoter methylation induced by DNMT1, the bisulfite-treated DNA was PCR-amplified with the biotin-labelled primers (shown in Supplementary Table 3), designed and synthesized by Geneland (Shanghai, China). Sequencing was performed with Pyro Q-CpG software after quantitation of cytosine methylation using PyroMark Q48 platform.

RNA-seq analysis

RNA-Seq analysis was carried out by Oebiotech (Shanghai, China). RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and assessed for integrity and abundance using a commercial Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The libraries were sequenced on the Illumina HiSeqTM 2500 platform, generating paired-end reads of either 125–150 bp. Gene expression profiles were normalized using RPKM, and differentially expressed genes (DEGs) were identified based on the criteria of q-value < 0.05 and |Log2FC|>1. GO enrichment analysis of DEGs was employed through DAVID (https://david.ncifcrf.gov/home.jsp) database.

Dual-luciferase reporter assay

To verify the binding sites between RORA and SLC2A3 promoter, dual-luciferase reporter assay was conducted using the Dual-luciferase reporter assay system (Promega, Madison, WI, USA). pGL3 plasmid carrying the wild-type SLC2A3 promoter or mutant SLC2A3 promoter region F1/F3 was constructed and named SLC2A3-WT or SLC2A3-MUT-F1 or SLC2A3-MUT-F3. HEK293T cells were transfected with the above reporter plasmids and RORA plasmid. The luciferase activities of Firefly and Renilla were analyzed by FLUOstar OPTIMA (BMG Labtech, Offenburg, German) after transfection for 48 h, and Firefly luciferase activity was adjusted using Renilla luciferase activity.

Glycolysis analysis

The uptake of glucose and the production of lactic acid and ATP were assessed by commercial glucose test kit (#S0201S, Beyotime), lactic acid test kit (GM3029, Servicebio), and ATP content test kit (#G4309-48T, Servicebio), respectively.

Bioinformatics and general data analysis

Publicly available UCSC XENA data sets TCGA and GTEx were applied to extract the RNA-seq data from a series of ESCC specimens (from TCGA) and normal tissues (from GTEx) at https://xenabrowser.net/datapages/. Differential expression of RORA between ESCC specimens and normal esophageal samples were analyzed by package ggplot2 [3.3.6] in R software (version 4.2.1). The diagnostic sensitivity of RORA was depicted by receiver operating characteristic (ROC) curve analysis by pROC [1.18.0] package in R. For prognosis analysis of RORA, we utilized survival [3.3.1] package in R to generate Kaplan-Meier survival curves. The GO and KEGG pathway enrichment analyses of the co-precipitated proteins were carried out using DAVID database (https://david.ncifcrf.gov/home.jsp). The gene set enrichment analysis (GSEA) by clusterProfiler [4.4.4] in R after single gene difference analysis by DESeq2 [1.36.0] was performed to observe the association of RORA and DNA methylation. Data of RNA-seq in esophageal carcinoma (ESCA) tumor specimens and normal esophageal samples were retrieved from TCGA database. R package (version 4.2.1) ggplot2 was employed to analyze SLC2A3 expression in ESCA tumor specimens and normal tissues. Data without clinical information of pathologic T stage are discarded, and sample information of squamous cell carcinoma subtype are retained. R package (version 4.2.1) ggplot2 was utilized to analyze the correlation between the expression of SLC2A3 or SLC2A14 and pathologic T stage. All data derived from ≥ 3 independent biological repeats and were analyzed either by t-test (two groups) or one-way ANOVA (≥ 3 groups). Histograms presented the mean and SD. Values of P less than 0.05 were taken as the level of significance.

Results

Association of RORA expression with ESCC diagnosis and prognosis

RNA-seq data from UCSC XENA revealed that the mRNA expression of RORA in ESCC tumor specimens was significantly reduced compared with normal esophageal samples (Fig. 1A), suggesting the potential of RORA as a biomarker for ESCC diagnosis. Moreover, IHC analysis also showed low expression of RORA in ESCC tumor tissue samples compared with adjacent normal tissues (Fig. 1B). ROC curve analysis demonstrated the diagnostic potential for RORA in ESCC, with the AUC of 0.737 (Fig. 1C). The median value of RORA mRNA expression was utilized as a threshold to classify the ESCC patients into high expression group and low expression group. Prognosis analysis indicated that in pathologic T3 stage, high RORA expression was associated with a better prognosis in ESCC patients (p = 0.032) (Fig. 1D), while it did not serve as a prognostic biomarker in pathologic T1&T2 stages (p > 0.05) (Fig. 1E).

Fig. 1
figure 1

Association analysis of RORA expression and ESCC diagnosis and prognosis. (A) Data of RNA-seq in ESCC tumor specimens and matched healthy esophageal samples were retrieved from UCSC XENA. R package ggplot2 showing the under-expression of RORA in ESCC tumor specimens versus normal tissues. (B) IHC assay was implemented to measure the expression of RORA in five pairs of ESCC tumor tissues and adjacent normal tissues. (C) ROC curve and AUC value analyzed by pROC package in R demonstrating the diagnostic potential for RORA. (D and E) Survival package in R indicating the correlation of RORA expression with overall survival of ESCC. ***P < 0.001

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RORA works as a suppressor of ESCC cell viability, motility, and invasion under hypoxia

To elucidate RORA’s precise role in ESCC, we modulated its level in two ESCC cell lines (KYSE150 and TE-1) by introducing RORA shRNA (shRORA) and RORA cDNA vector, respectively. RT-qPCR demonstrated that transfection with shRORA resulted in decreased RORA mRNA expression relative to control shRNA, and RORA cDNA vector significantly upregulated the mRNA level of RORA versus control vector (Supplementary Fig. 1A). Although RORA expression alteration was induced by shRORA and RORA vector, neither RORA knockdown (KD) nor RORA overexpression (OE) affected cell viability under normoxia (Supplementary Fig. 1B), providing the first indication that RORA did not alter the viability of ESCC cells under normoxic condition.

Hypoxia is a common feature of solid tumors [27]. Previous reports demonstrated that RORA actively participated in cellular stress response induced by various stimuli including hypoxia [7, 8], prompted us to investigate RORA’s function under hypoxia. We first validated that hypoxia induced a remarkable augmentation in RORA expression at both mRNA and protein levels (Fig. 2A and B). Previous studies have reported that the regulation of RORA expression by hypoxia depends on HIF1A [28, 29]. Our data showed that exposure to hypoxia resulted in an obvious upregulation of HIF1A and RORA protein expression (Fig. 2C). However, the introduction of shHIF1A mitigated these effects (Fig. 2C), confirming that the hypoxia-induced upregulation of RORA was contingent upon the presence of HIF1A. The high transfection efficiencies of shRORA and RORA ectopic expression plasmid were verified by western blotting under hypoxic and normoxic conditions (Fig. 2D). RORA could affect cell viability under hypoxia as evidenced by these findings that under hypoxic conditions, RORA KD strongly enhanced cell viability, whereas cells transfected with RORA OE showed reduced cell viability (Fig. 2E).

Fig. 2
figure 2

Inhibitory activity of RORA in ESCC cell viability and motility under hypoxia. (A and B) RT-qPCR and western blotting demonstrating that RORA expression increased in cells exposed to hypoxia. (C) The regulatory effect of HIF1A on RORA protein expression under hypoxia or normoxia was analyzed by western blotting. (D) The transfection efficiencies of shRORA and RORA ectopic expression plasmid in ESCC cells under hypoxia or normoxia were assessed by western blotting assay. (E) Under hypoxic conditions, CCK-8 assay revealing that RORA knockdown (KD) promoted cell viability, and RORA overexpression (OE) decreased cell viability. *P < 0.05, ***P < 0.001

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We also performed migration and invasion analyses in RORA KD cells and RORA OE cells to further illustrate the function of RORA under hypoxia. RORA KD increased the migration ability of ESCC cells as determined by wound-healing assay, and RORA OE exerted a repressive effect on cell migration (Fig. 3A). Transwell assay showed the increased ability of invasion following RORA KD compared with control group and repressed invaded rate of RORA OE cells (Fig. 3B). Furthermore, IF demonstrated that the levels of metastasis-associated factors MMP2 and MMP9 were upregulated following RORA KD and inhibited by RORA OE under hypoxic condition (Fig. 3C and D), reinforced the notion that RORA could alter cell motility under hypoxia. Epithelial-mesenchymal transition (EMT) is featured by the downregulation of epithelial molecular markers such as E-cadherin (E-cad) and the upregulation of mesenchymal molecular markers such as N-cadherin (N-cad) and Snail [30, 31]. Western blotting of these markers in RORA KD cells and RORA OE cells under hypoxia also provided the support for the negative control of RORA in cell motility and invasiveness, as indicated by E-cad downregulation, N-cad increase and Snail upregulation following RORA KD and E-cad upregulation, N-cad downregulation and Snail reduction following RORA OE (Fig. 3E). Overall, RORA was a negative regulator of ESCC cell growth, motility, and invasion under hypoxia.

Fig. 3
figure 3

Suppressive function of RORA in ESCC cell migration and invasion under hypoxia. (A) Under hypoxic conditions, wound-healing assay showing that RORA KD enhanced cell motility, and RORA OE hindered migration. (B) Under hypoxic conditions, transwell assay demonstrating that cell invasiveness was facilitated by RORA KD and repressed by RORA OE. (C and D) IF microscopy revealing that RORA KD increased the levels of MMP2 and MMP9, which the two factors were downregulated following RORA OE. (E) Western blotting demonstrating the expression alteration of E-cad, N-cad and Snail following RORA KD and RORA OE. *P < 0.05, **P < 0.01, ***P < 0.001

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Enforced RORA expression in mouse xenografts impedes tumor growth

To evaluate if RORA impeded tumorigenicity of ESCC cells in vivo, we established a stable RORA OE KYSE150 cell line using a lentiviral delivery system (lenti-RORA) and subsequently implanted the cells into nude mice to construct a subcutaneous xenograft tumor model. RORA OE significantly reduced tumor volume and weight compared to the Lenti-NC control, indicating obvious inhibition on xenograft growth (Fig. 4A and D). RT-qPCR confirmed increased RORA level in xenografts derived from KYSE150 cells infected with Lenti-RORA (Fig. 4E). Since HIF1A is a well-known biomarker of hypoxia, we examined HIF1A expression to test whether the xenograft tumors were under hypoxic stress or not. Normal esophageal tissue from healthy mice was used as a control group. We observed that HIF1A protein expression was upregulated in both the Lenti-NC and Lenti-RORA groups compared to the Normal group. However, there was no significant difference in HIF1A protein expression between the Lenti-NC and Lenti-RORA groups (Fig. 4F), indicating that the xenograft tumors resided in a relatively hypoxic microenvironment. RORA OE downregulated the ratios of the Ki67-positive cells and PCNA-positive cells in xenografts (Fig. 4G and H), offering an explanation for RORA’s inhibitory activity in xenograft growth. It is noteworthy that the expression levels of MMP2 and MMP9 in KYSE150 xenografts were downregulated by RORA OE (Fig. 4I and J), implying the inhibitory role of RORA in tumor metastasis.

Fig. 4
figure 4

Suppression of RORA in KYSE150 xenograft growth. (A and B) Images of mice and xenografts showing the inhibitory role of RORA in KYSE150 xenograft growth. (C and D) Reduced tumor volume and weight after RORA OE indicating the repression of RORA in KYSE150 xenograft growth. (E) RT-qPCR indicating the upregulation of RORA in lenti-RORA-transduced xenograft tumors. (F) Western blotting was employed to detect the expression of HIF1A, a biomarker of hypoxia, in the two groups of xenograft tumors. (G) IHC revealing that RORA OE reduced the ratio of the Ki-67-positive cells in KYSE150 xenograft tumors. (H) IF microscopy indicating that RORA OE repressed the growth of xenografts as measured by the reduced ratio of the PCNA-positive cells. (I and J) IF microscopy showing that RORA OE downregulated the levels of MMP2 and MMP9 in KYSE150 xenografts. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no statistically significant difference

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DNMT1 induces methylation of RORA in ESCC cells exposed to hypoxia

GSEA analysis after single gene difference analysis predicted a significant association between RORA and DNA methylation in ESCC (Fig. 5A). Next, we observed the DNA methylation profiling of the RORA promoter in esophagus cancer (ESCA) via Ualcan database TCGA (https://ualcan.path.uab.edu/). The promoter methylation level of RORA was significantly elevated in primary esophagus tumors relative to normal esophagus samples (Fig. 5B), and it was positively related to tumor lymphatic metastasis (N) and pathological grade (Fig. 5C and D). Therefore, we aimed to explore the upstream regulators that affected the DNA methylation of RORA. We first generated a biotin-labelled RORA promoter probe (positions − 2000 to -44, called P4:1–1956 bp) and three biotin-labelled RORA promoter truncations (P1: 1–895 bp, P2: 550–1468 bp, P3: 1177–1956 bp) (Fig. 5E). Figure 5E also showed that five fragments in the RORA promoter (F1: 1–549 bp, F2: 550–895 bp, F3: 896–1176 bp, F4: 1177–1468 bp, and F5: 1469–1956 bp) were segmented by four biotinylated promoter probes. We preliminarily proved the validity of the four biotin-labelled PCR products by 1% agarose gel electrophoresis (Supplementary Fig. 2A), which were further confirmed by sequencing. The biotin labelling efficiencies were also demonstrated by HRP-conjugated streptavidin (Supplementary Fig. 2B). Subsequently, we performed DNA pull-down experiment on hypoxia-exposed KYSE150 cells using four biotin-labelled RORA promoter probes and identified their co-precipitated proteins by HPLC-MS. Relative to bead control co-precipitates, four specific bands were observed in P1-P4 probe groups, as presented by silver staining (Supplementary Fig. 2C). HPLC-MS showed multiple protein co-precipitation in four biotinylated RORA promoter probes (Supplementary Table 4). As shown by Venn diagram (Fig. 5F and H), 69 proteins were co-precipitated by P1 and P4 instead of P2 and beads, which we called specific bound proteins in the F1 fragment (Fig. 5F); 97 proteins that overlapped among P1, P2 and P4 not beads were considered as specific bound proteins in the F2 fragment (Fig. 5F); 20 proteins were pulled down by P2 and P4 instead of P1, P3 and beads, which we called specific bound proteins in the F3 fragment (Fig. 5G); 119 proteins that overlapped among P2, P3 and P4 not beads were designated as specific bound proteins in the F4 fragment (Fig. 5H); 101 proteins co-precipitated with P3 and P4 instead of P2 and beads were designated as specific bound proteins in the F5 fragment (Fig. 5H).

Fig. 5
figure 5

DNMT1 induces methylation of RORA in hypoxia-induced ESCC cells. (A) GSEA analysis revealing the association between RORA and DNA methylation. (B) TCGA samples from Ualcan database showing the augmentation of the promoter methylation level of RORA in esophagus cancer (ESCA). (C and D) TCGA samples from Ualcan database revealing the correlation of RORA’s promoter methylation level and tumor lymphatic metastasis (N) and pathological grade. (E) Schematic showing the four biotin-labelled RORA promoter probes and five fragments divided by the four probes. (F) Venn diagram revealing the 69 specific bound proteins in the F1 fragment and 97 specific bound proteins in the F2 fragment. (G) Venn diagram revealing the 20 specific bound proteins in the F3 fragment. (H) Venn diagram revealing the 119 specific bound proteins in the F1 fragment and 101 specific bound proteins in the F2 fragment. (I) RNAseq data of TCGA-ESCA item was downloaded from TCGA database (https://portal.gdc.cancer.gov), and DNMT1 mRNA expression in 174 cases of tumor samples and 11 cases of normal samples was analyzed. (J) IHC assay was employed to measure the expression of DNMT1 in five pairs of ESCC tumor tissues and adjacent normal tissues. (K) RT-qPCR demonstrating that in hypoxia-induced ESCC cells, decitabine (DCA) elevated RORA mRNA level, and DNMT1 upregulation decreased the mRNA level of RORA. (L) Western blotting demonstrating that in hypoxia-induced ESCC cells, decitabine (DCA) elevated RORA protein expression, and DNMT1 upregulation inhibited the protein level of RORA. (M) ChIP qPCR confirming the binding of DNMT1 and the RORA promoter in hypoxia-induced KYSE150 ESCC cells. (N and O) DNA pyrosequencing indicating the enhancement of DNMT1 in the promoter methylation level of RORA in hypoxia-induced KYSE150 ESCC cells. *P < 0.05, **P < 0.01, ***P < 0.001

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It is noteworthy that DNMT1, an essential master in maintaining DNA methylation [16], was found to widely exist in the RORA promoter (it was pulled down by all of the four RORA promoter probes) (Supplementary Table 4). DNMT1 mRNA was found to be markedly up-regulated in ESCC tumor samples compared with normal samples (Fig. 5I). Furthermore, IHC assay confirmed high expression of DNMT1 in ESCC patient tissue samples (Fig. 5J). Therefore, we aimed to uncover whether the alteration of RORA’s promoter methylation level was due to DNMT1 induction in ESCC. We first analyzed the role of DNMT1 in regulating RORA expression. RT-qPCR and western blotting demonstrated that in ESCC cells under hypoxic condition, the demethylating reagent decitabine (DCA, a repressor of DNMT1) upregulated RORA expression at both mRNA and protein versus control vehicle; however, forced DNMT1 expression by a cDNA vector introduction resulted in downregulation of RORA mRNA and protein levels (Fig. 5K and L). DNMT1 knockdown using shDNMT1 prominently upregulated the protein expression of RORA in ESCC cells (Supplementary Fig. 3A). Second, we utilized ChIP to verify the binding between DNMT1 and the RORA promoter. Relative to the anti-IgG control, anti-DNMT1 antibody upregulated the enrichment abundance of the RORA promoter in hypoxia-induced KYSE150 cells (Fig. 5M). We finally determined the effect of DNMT1 on RORA DNA methylation in KYSE150 ESCC cells under hypoxic insult through pyrosequencing, a DNA methylation detection method. The main methylation region of the RORA promoter was predicted, and primers targeting the sequence were designed for DNA pyrosequencing (Supplementary Fig. 4). Notably, forced DNMT1 expression led to augmentation in the DNA methylation level of the RORA promoter relative to the CON control (Fig. 5N and O), and DNMT1 depletion using DCA or shDNMT1 markedly reduced the DNA methylation level of the RORA promoter (Fig. 5N and O, Supplementary Fig. 3B).

RORA suppresses the transcription of SLC2A3 through its promoter F1 region

To understand the molecular mechanism underlying the anti-tumor effects of RORA under hypoxia, we explored its downstream targets identified by RNA-seq in RORA + Hypoxia and vec + Hypoxia groups. GO function annotation of down-regulated DEGs showed that the “glucose import” item was markedly enriched in the biological process group, which contained SLC2A14 and SLC2A3 (Fig. 6A). Volcano plot also indicated the significant down-regulation of SLC2A14 and SLC2A3 in RORA + Hypoxia group compared with vec + Hypoxia group (Fig. 6B). Glucose homeostasis in human is majorly mediated by the members of the glucose transporter family, also known as SLC2A, which includes 14 isoforms responsible for glucose transport across cellular membranes, including SLC2A1 to SLC2A14 [32]. Hypoxia is a common feature of solid tumors, and hypoxic tumor cells primarily use glucose to produce glycolytic energy and release lactic acid. These data implied that blocking of glycolysis might be a downstream event following RORA overexpression. SLC2A3, but not SLC2A14, was significantly up-regulated in tumor tissues compared with normal tissues, and SLC2A3 abundance was positively correlated with pathologic T stage (Supplementary Fig. 5A-5 C). In addition, RT-qPCR showed that RORA overexpression markedly reduced SLC2A3 expression, but had no significant effect on SLC2A14 (Fig. 6C). Therefore, SLC2A3 was chosen for the following assays.

Fig. 6
figure 6

The downstream targets of RORA are screened by RNA-seq. (A) GO function annotation of the significantly down-regulated genes in RORA + hypoxia group versus vec + hypoxia group was performed. (B) The differentially expressed genes were shown by the volcano plot, and blue for down-regulated genes, red for up-regulated genes. (C) The mRNA expression levels of SLC2A3 and SLC2A14 in vec + hypoxia and RORA + hypoxia groups were determined by RT-qPCR. (D) Western blot assay was employed to detect the protein level of SLC2A3 in vec + hypoxia and RORA + hypoxia groups. (E) The binding motif of RORA and the binding sequence between RORA and SLC2A3 promoter region were predicted by JASPAR database. (F) Three ChIP-qPCR primers of SLC2A3 promoter were designed based on the three predicted binding sites, and ChIP-PCR was employed to verify the binding sites between RORA and SLC2A3 promoter. (G) Dual-luciferase reporter assay was conducted to verify the binding sites between RORA and SLC2A3 promoter. *P < 0.05, **P < 0.01, ***P < 0.001, ns: non significant

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WB assay validated that RORA could negatively regulate the protein expression of SLC2A3 in two ESCC cell lines under hypoxia (Fig. 6D). Previous studies have reported that RORA, as a transcription factor, drived the expression of multiple target genes through interacting with the specific DNA motif on their promoters, known as ROR response elements (ROREs) [33]. To explore whether RORA affected SLC2A3 transcription by directly binding to its promoter, we predicted their interacted regions through the JASPAR database, and the three predicted binding sequences were shown in Fig. 6E. Three ChIP-qPCR primers targeting the predicted binding sequences of SLC2A3 promoter were designed, and ChIP-PCR showed a marked enrichment of SLC2A3 promoter F1 and F3 when using the RORA antibody compared with the IgG group (Fig. 6F). To further validate the binding sites between RORA and SLC2A3 promoter, we constructed a luciferase reporter plasmid carrying either the wild-type SLC2A3 promoter or the mutant SLC2A3 promoter region F1/F3. RORA overexpression notably reduced the luciferase activity of SLC2A3-WT and SLC2A3-MUT-F3, but not SLC2A3-MUT-F1 (Fig. 6G), indicating that RORA could inactivate SLC2A3 transcription by directly binding to its promoter region F1.

The anti-tumor activity of RORA on migration and invasion of hypoxia-induced ESCC cells is largely based on its target SLC2A3

High overexpression efficiency of SLC2A3 plasmid was verified by RT-qPCR assay (Fig. 7A). To explore whether RORA functioned as a tumor suppressor in ESCC through SLC2A3, we conducted a series of rescue experiments. RORA overexpression markedly restrained cell migration, which was largely counteracted by the introduction of SLC2A3 plasmid (Fig. 7B). Transwell assay revealed that RORA accumulation blocked cell invasion, and this phenomenon was largely offset by the overexpression of SLC2A3 (Fig. 7C). Not surprisingly, IF assay showed that the abundance of metastasis-associated factors (MMP2 and MMP9) was markedly restrained by RORA overexpression, which was rescued by the addition of SLC2A3 plasmid (Fig. 7D and E). Western blotting provided the evidence that RORA overexpression-mediated suppressive effect on cell EMT was largely reversed by SLC2A3, as indicated by the down-regulation of epithelial marker E-cad and the up-regulation of mesenchymal molecular markers (N-cad and Snail) upon the addition of SLC2A3 plasmid in hypoxia-induced ESCC cells (Fig. 7F). Overall, RORA inhibited the migration and invasion of ESCC cells largely through down-regulating SLC2A3.

Fig. 7
figure 7

The anti-tumor activity of RORA on migration and invasion of hypoxia-induced ESCC cells is largely based on its target SLC2A3. (A) RT-qPCR was employed to assess the overexpression efficiency of SLC2A3 plasmid. (B-F) ESCC cells were transfected with vector, RORA, or RORA + SLC2A3 and treated with hypoxia. (E) Cell migration ability was evaluated by wound-healing assay. (C) Transwell assay was employed to assess cell invasion ability. (D and E) The abundance of cell motility-related indicators (MMP2 and MMP9) was determined by IF assay. (F) Western blotting was conducted to detect the expression of EMT-associated markers (E-cad, N-cad, and Snail) in hypoxia-induced ESCC cells. *P < 0.05, **P < 0.01, ***P < 0.001

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RORA restrains the glycolysis of hypoxia-induced ESCC cells largely by targeting SLC2A3

Glycolysis aids tumor growth by supplying ATP for tumor cell proliferation and metastasis [34]. We explored whether RORA restrained the migration and invasion of ESCC cells by negatively regulating SLC2A3 and glycolysis. RORA accumulation reduced glucose uptake and lactic acid and ATP production, and these phenomenon were largely offset by SLC2A3 overexpression (Fig. 8A and C), suggesting RORA’s role in suppressing glycolysis in hypoxia-induced ESCC cells via SLC2A3 regulation. In summary, RORA blocked ESCC cell glycolysis by down-regulating SLC2A3, thereby inhibiting the migration and invasion of ESCC cells.

Fig. 8
figure 8

RORA restrains the glycolysis of hypoxia-exposed ESCC cells largely by targeting SLC2A3. (A-C) ESCC cells were transfected with vector, RORA, or RORA + SLC2A3 and treated with hypoxia. Cell glycolysis was analyzed by measuring the uptake of glucose and the generation of lactic acid and ATP. *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

ESCC is a highly aggressive and lethal thoracic malignancy, necessitating an urgent advancement in understanding the factors contributing to its progression. Previous studies have identified the dysregulated activity of RORA as being relevant to the development of ESCC [12, 13]. Hypoxia, a critical feature of solid tumors, alters the cancer microenvironment to induce multiple malignant phenotypes, such as cell motility, metastasis, and angiogenesis, which contribute to neoplasia and cancer progression [23, 35]. This paper presents the novel finding of the anti-cancer effect of RORA in ESCC under hypoxic condition.

In prostate cancer, as a transcription factor, RORA expression is positively correlated with anti-tumor genes such as PTGS2 [5]. Under-expression of RORA has been shown in various cancers, including lung cancer and hepatocellular carcinoma [9, 10]. These reports also highlight that RORA is a beneficial factor due to its positive correlation with favorable prognostic outcomes. In line with the several cancers, via TCGA database analysis, we also found its downregulated expression in ESCC and the positive correlation between RORA abundance and favorable survival of ESCC patients in pathologic T3 stage. Notably, there was no significant association between RORA abundance with prognosis of patients in T1&T2 pathologic stage. Additionally, by analyzing GEO datasets, Dipankor and colleagues found that high RORA expression predicted a dismal prognosis of esophageal cancer [12]. These findings suggested that the inconsistent prognostic association of RORA might be due to different subtypes of esophageal cancer and different pathologic grades of ESCC. Functional analyses demonstrated that the upregulation of RORA exerted a repressive effect on glioma cell proliferation by inactivating NF-κB signaling pathways [36]. Furthermore, the stabilization of RORA, mediated by POU6F1, has been shown to suppress the progression of lung cancer [37]. The inhibition of RORA is associated with the increased growth and invasiveness of prostate cancer cells [38]. Distinct from these effects observed under normoxic condition, our study provided the evidence that in hypoxic condition in vitro, RORA worked as a negative regulator of ESCC cell viability, invasion and migration; however, RORA had no ability to affect ESCC cell viability under normoxia. Hypoxia is a critical feature of solid tumors, and RORA was activated upon hypoxia [7]. We also proved the elevation of RORA expression in hypoxia-exposed ESCC cells. The functional differences of RORA might be attributed to different tumor microenvironments. Furthermore, our findings have reinforced the anti-ESCC activity of RORA through assessing its suppressive role in xenograft tumors in vivo.

In this paper, we uncover a novel molecular determinant driving RORA dysfunction in ESCC under hypoxic condition by identifying DNMT1 as a strong upstream modulator of RORA. Hypermethylation of gene promoter can lead to inactivation of anti-tumor factors and consequently induces human tumorigenesis [15]. DNMT1 mediates epigenetic regulation by maintaining gene methylation level [16]. DNMT1 has also been reported to play a critical role in cancer by mediating DNA methylation. For instance, in liver cancer, enhanced promoter methylation level of BEX1 induced by DNMT1 activated the Wnt/β-catenin pathway, thereby contributing to liver tumorigenicity [39]. DNMT1 downregulated TRAF6 through epigenetic regulation and thus facilitated prostate tumorigenesis and metastasis by elevating EZH2 stability [40]. In addition, DNMT1 was found to be upregulated in various cancers, such as prostate cancer and breast cancer, making it a potential target for cancer treatment [40]. In ESCC, DNMT1 was highly expressed and contributed to cancer progression through hypermethylation-mediated gene silencing [18, 41]. Inhibition of DNMT1 led to increased expression of the tumor suppressor PTEN, thereby enhancing the radiosensitivity of ESCC [42]. Our study presented novel insights into the epigenetic regulation by DNMT1, which elevated the promoter methylation of RORA and subsequently downregulated its expression in ESCC cells under hypoxic condition. A previous report also showed the methylation-silencing regulation of DNMT1 in RORA by binding to the RORA promoter in C3H10T1/2 mesenchymal stem cells [43]. These findings suggested that this epigenetic regulation mechanism might exist not only in the two cell lines but also in other types of cells.

The above results provided the evidence that DNMT1-mediated methylation of RORA promoter was responsible for the dysregulation of RORA in ESCC. However, the exact molecular mechanism underlying the anti-tumor activity of RORA remains to be illustrated. Hypoxia is a vital characteristic of solid tumors, and glycolysis is a complementary pathway for cancer cells to product ATP under hypoxia [20, 44]. Glucose transport across the plasma membrane in mammalian cells is facilitated by the SLC2A family, commonly known as the GLUT family, which includes SLC2A1 through SLC2A14. Elevated expression levels of glucose transport facilitators lead to enhanced glucose uptake and support oncogenic growth [30]. Kuang et al.. demonstrated extensive metabolic reprogramming in anti-angiogenic therapy-resistant tumors, which attributed to enhanced SLC2A3 abundance [45]. Here, RNA-seq analysis indicated that RORA overexpression down-regulated the level of glucose transport factor SLC2A3 under hypoxia. Mechanically, RORA inactivated the transcription of SLC2A3 by interacting with its promoter F1 region. Glycolysis is known to facilitate tumor growth and metastasis through providing ATP [46], and we hypothesized that RORA exerted its anti-tumor effects in hypoxia-induced ESCC by affecting SLC2A3 expression and blocking glycolysis and thereby inhibiting tumor metastasis, which was further validated by the following rescue experiments.

Taken together, our study elucidated the inhibitory role of RORA in ESCC cell growth, invasion and migration under hypoxic condition. The epigenetic regulation by DNMT1 leads to RORA silencing in ESCC through methylation modulation. In addition, RORA restrained ESCC metastasis largely through down-regulating the level of glycolysis-associated protein SLC2A3, thereby inhibiting glycolysis. This study is the first to report the precise biological function of RORA in ESCC development. We propose that RORA acts as an anti-cancer factor in ESCC and represents a potential therapeutic target for this malignancy.

Data availability

The data and material presented in this manuscript is available from the corresponding author on reasonable request.

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Acknowledgements

I would like to express my gratitude to all those who have helped me during the writing of this thesis. I gratefully acknowledge the help of Key Science and Technology Projects in Henan Province(212102310669, 222102310045), Henan Province medical science and technology research plan joint construction project(LHGJ20210043)&23456“Talent Project” of Henan Provincial People’s Hospital that funded this research. Also, I would like to thank Mr. Wenjian Yao, Linlin Shang, Yinghao Wang, Lei xu, Yu bai, Mingyu Feng, who contributed to the research work.

Funding

This research was supported by Key Science and Technology Projects in Henan Province(212102310669, 222102310045), Henan Province medical science and technology research plan joint construction project(LHGJ20210043)&23456“Talent Project” of Henan Provincial People’s Hospital.

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Author notes

  1. Wenjian Yao and Linlin Shang equal contribution.

Authors and Affiliations

  1. Department of Thoracic Surgery, School of Clinical Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Henan University, No.7, Wei Wu Road, Jinshui District, Zhengzhou, Henan, 450003, China

    Wenjian Yao, Lei Xu, Xiangbo Jia & Sen Wu

  2. Department of Thoracic Surgery, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, No.7, Wei Wu Road, Jinshui District, Zhengzhou, Henan, 450003, China

    Linlin Shang

  3. Department of Thoracic Surgery, Henan University People’s Hospital, Henan Provincial People’s Hospital, No.7, Wei Wu Road, Jinshui District, Zhengzhou, Henan, 450003, China

    Yinghao Wang

  4. Department of Pathology, Xinxiang Medical University, No.601 Jinsui Avenue, Hongqi District, Xinxiang, Henan, 453003, China

    Yu Bai

  5. Department of Education, Xuanwu Hospital, Capital Medical University, No.45 Changchun Street, Xicheng District, Beijing, 100053, China

    Mingyu Feng

Authors
  1. Wenjian Yao
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  2. Linlin Shang
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  3. Yinghao Wang
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  4. Lei Xu
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  5. Yu Bai
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  6. Mingyu Feng
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  7. Xiangbo Jia
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  8. Sen Wu
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Contributions

Wenjian Yao and Linlin Shang designed and performed the experiments, wrote the manuscript. Yinghao Wang, Lei Xu, Yu Bai, Mingyu Feng conducted the experiments. Sen Wu and Xiangbo Jia revised the manuscript. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Xiangbo Jia or Sen Wu.

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Ethical approval

All animal experiments were performed with the approval of the Henan Provincial People’s Hospital Animal Care and Use Committee. and the procedures for Care and Use of Laboratory Animals in cancer research.

Conflict of interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influencethe work reported in this paper.

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Yao, W., Shang, L., Wang, Y. et al. DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3. J Transl Med 22, 1167 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05960-8

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05960-8

Keywords

Journal of Translational Medicine

ISSN: 1479-5876

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