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Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma

  • Authors:
    • Zhengjia Kang
    • Gaoqin Liu
  • View Affiliations / Copyright

    Affiliations: Department of Ophthalmology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China
    Copyright: © Kang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 226
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    Published online on: April 8, 2026
       https://doi.org/10.3892/ol.2026.15582
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Abstract

This study investigated the potential role of ferroptosis‑associated competing endogenous RNA (ceRNA) networks in retinoblastoma (RB). In total, two independent RB gene expression datasets (GSE97508 and GSE208143) and a microRNA (miRNA) expression dataset (GSE208677) were retrieved from the Gene Expression Omnibus database. Differential expression analysis was conducted to identify ferroptosis‑related (FR) differentially expressed genes (DEGs) in RB. A protein‑protein interaction network for FR‑DEGs was constructed using the STRING database, whereas hub genes were determined using the CytoHubba plugin. Reverse transcription‑quantitative PCR (RT‑qPCR) was used to verify the expression of IDH2 and CDKN2A. Immune cell infiltration in RB was assessed using the CIBERSORT algorithm. Additionally, miRNA‑targeting hub genes were predicted, where an FR miRNA‑mRNA regulatory network was constructed. Finally, a ceRNA network and a drug‑gene interaction network were constructed. A total of 584 FR‑DEGs were identified from the GSE97508 dataset, revealing 135 upregulated and 449 downregulated genes. The intersection of FR‑DEGs with FerrDb‑extracted FRGs yielded 23 genes that were defined as core FR‑DEGs for subsequent analysis. Functional and pathway enrichment analyses highlighted their involvement in iron and transition metal ion homeostasis, response to hypoxic and ischemic conditions and apoptotic processes. Hub genes (CAV1, CDKN2A, EPAS1, IDH2, RB1 and SLC2A3) were identified and validated in an independent dataset, with RT‑qPCR confirming IDH2 and CDKN2A upregulation. Immune cell infiltration analysis indicated altered immune cell populations in RB, where an association analysis revealed associations between hub genes and immune cells. A comprehensive FR miRNA‑mRNA regulatory network and a ceRNA network were established, shedding light on additional layers of regulatory mechanisms in RB. Furthermore, the drug‑gene interaction network consisted of 20 potential candidate drugs. In conclusion, the present integrated analysis offers a comprehensive understanding of the potential role in RB, providing valuable insights into the molecular mechanisms and potential therapeutic targets for this childhood ocular tumor.
View Figures

Figure 1

Workflow of the present study. DEGs,
differentially expressed genes; PPI, protein-protein interaction;
GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes;
CTD, Comparative Toxicogenomics Database; ceRNA, competing
endogenous RNA; DEMs, differentially expressed miRNAs.

Figure 2

Identification of ferroptosis-related
FR-DEGs. (A) Volcano plot showing the gene expression differences
between two groups in the GSE97508 dataset. Blue nodes represent
downregulated genes, red nodes represent upregulated genes and gray
nodes represent genes that are stable in comparison to controls.
(B) Venn diagram showing the intersection of DEGs in GSE97508 with
FRGs from the ‘FerrDb’ database. (C) Heatmap of the identified 23
FR-DEGs in GSE97508. (D) Location of the 23 FR-DEGs on the 22
chromosomes. DEGs, differentially expressed genes; FRGs,
ferroptosis-related genes; FR-DEGs, ferroptosis-related DEGs;
adj.P.Val, adjusted P-value; RB, retinoblastoma

Figure 3

Enrichment analysis of FR-DEGs. (A)
Gene Ontology analysis of the FR-DEGs. (B) Kyoto Encyclopedia of
Genes and Genomes analyses of the FR-DEGs. (C) Metascape bar chart
of top 20 non-redundant enrichment clusters. BP, biological
processes; CC, cellular components; MF, molecular functions;
FR-DEGs, ferroptosis-related differentially expressed genes.

Figure 4

Construction of PPI network and
identification of hub genes. (A) PPI network of ferroptosis-related
differentially expressed genes. Each node in the network represents
a protein. The small 3D structural models inside the circles are
schematic representations of the protein conformations. TF,
Transferrin; CP, Ceruloplasmin; SLC11A2, Solute Carrier Family 11
Member 2; SLC2A3, Solute Carrier Family 2 Member 3; SLC2A14, Solute
Carrier Family 2 Member 14; IDH2, Isocitrate Dehydrogenase 2;
EPAS1, Endothelial PAS Domain Protein 1; PPARA, Peroxisome
Proliferator-Activated Receptor Alpha. (B) Venn diagram
illustrating the hub genes identified by five algorithms
(BottleNeck, Degree, EPC, MCC and MNC) using Cytoscape software and
CytoHubba. (C) Validation of hub genes in the GSE208143 dataset.
The box represents the interquartile range, the line inside the box
denotes the median and the dots represent individual sample data
points. (D) ROC curve of hub genes in the GSE208143 dataset.
**P<0.01, ***P<0.001 and ****P<0.0001. ns, no
significance; PPI, protein-protein interaction; EPC, Edge
Percolated Component; MCC, Maximal Clique Centrality; MNC, Maximum
Neighborhood Component; ROC, receiver operating characteristic;
AUC, area under the ROC curve; RB, retinoblastoma.

Figure 5

Landscape of immune cell
infiltration. (A) Infiltration levels of each immune cell type
between control and RB samples. (B) Correlation between hub genes
and immune cells in RB. *P<0.05, **P<0.01 and ***P<0.001.
RB, retinoblastoma; cor, correlation coefficient.

Figure 6

Construction of the miRNA-mRNA
network. (A) Volcano plot showing the gene expression differences
between two groups in the GSE208677 dataset. (B) Heatmap of the
identified 23 ferroptosis-related differentially-expressed genes in
GSE97508. (C) Venn diagram showing the intersection of DEMs in
GSE208677 with predicted miRNAs from muitimir. (D) miRNA-mRNA
regulatory network. miRNA, microRNA; DEM, differentially expressed
miRNAs; adj.P.Val, adjusted P-value.

Figure 7

ceRNA networks, drug-gene
interactions and PCR validation. (A) The ceRNA-regulating networks
illustrate protein-coding genes (red circles), miRNAs (blue
diamonds) and lncRNAs (green hexagons), with black lines indicating
interactions among lncRNA, miRNA and mRNA, where ceRNA refers to
ceRNAs. (B) Validation through reverse transcription-quantitative
PCR analysis confirms that CDKN2A and IDH2 expression
levels are significantly higher in Y79 cells compared to ARPE-19
cells. Data were normalized to GAPDH expression using the
2−ΔΔCq method, with each experiment performed in
triplicate. ***P<0.001 and ****P<0.0001. (C) The drug-gene
interaction analysis shows the linkage map of two hub genes,
CDKN2A and IDH2, along with their potential target
drugs, including bisphenol A (code: C006780), sodium arsenite
(code: C017947), docetaxel (code: D000077143) and
hexabromocyclododecane (code: C089796). ceRNA, competing endogenous
RNAs; miRNA, microRNA; lncRNA, long non-coding RNA; RB,
retinoblastoma.
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Copy and paste a formatted citation
Spandidos Publications style
Kang Z and Liu G: Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma. Oncol Lett 31: 226, 2026.
APA
Kang, Z., & Liu, G. (2026). Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma. Oncology Letters, 31, 226. https://doi.org/10.3892/ol.2026.15582
MLA
Kang, Z., Liu, G."Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma". Oncology Letters 31.6 (2026): 226.
Chicago
Kang, Z., Liu, G."Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma". Oncology Letters 31, no. 6 (2026): 226. https://doi.org/10.3892/ol.2026.15582
Copy and paste a formatted citation
x
Spandidos Publications style
Kang Z and Liu G: Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma. Oncol Lett 31: 226, 2026.
APA
Kang, Z., & Liu, G. (2026). Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma. Oncology Letters, 31, 226. https://doi.org/10.3892/ol.2026.15582
MLA
Kang, Z., Liu, G."Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma". Oncology Letters 31.6 (2026): 226.
Chicago
Kang, Z., Liu, G."Exploring multiple biomarkers and constructing ferroptosis‑associated competing endogenous RNA networks as dual targets in retinoblastoma". Oncology Letters 31, no. 6 (2026): 226. https://doi.org/10.3892/ol.2026.15582
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