Radiotherapy remains a cornerstone in cancer treatment, often involving high doses of ionizing radiation. However, radiation-induced esophageal injury—a non-specific inflammatory response in the esophageal mucosa—is a frequent adverse effect, particularly among patients undergoing radiotherapy for tumors in the neck, chest, or mediastinum. This condition manifests as acute radiation esophagitis (ARE) within three months of initiating treatment, or late radiation esophagitis (LRE) if symptoms appear thereafter. ARE typically develops rapidly, most often within 2–3 weeks of the initial radiation dose. Radiation exposure disrupts esophageal tissue integrity, generating large quantities of oxygen-derived free radicals. These reactive species damage cell membranes by reducing fluidity, increasing permeability, inducing mitochondrial swelling, and promoting lysosomal enzyme release. This cascade culminates in cellular injury and triggers an inflammatory response. Clinically, ARE may initially present as a foreign body sensation during swallowing, which can progress to odynophagia, dysphagia, or persistent retrosternal pain unrelated to deglutition.

In contrast, LRE generally manifests more than three months after radiotherapy, with some cases presenting up to a year later. Pathologically, LRE is characterized by esophageal fibrosis, damage to the muscularis propria, and possible neural injury. These changes may lead to dysphagia secondary to luminal stenosis, impaired motility, or chronic ulceration. Severe cases may be complicated by life-threatening events such as esophageal perforation, esophagotracheal fistula, or aorto-esophageal fistula (1).

Notably, radiation-induced esophageal injury often necessitates treatment interruption and, in some cases, treatment discontinuation (2–4). Despite its clinical significance, no pharmacologic agent is currently approved for its prevention or treatment. Nevertheless, adjunctive therapies—such as amifostine (5) or soy isoflavone supplementation (6)—have demonstrated efficacy in reducing the incidence and severity of esophageal injury during thoracic radiotherapy.

RNA-dependent nucleic acid transferases, members of the β superfamily of DNA polymerases, include several enzymes such as mRNA poly(A) polymerase, poly(U) polymerase, CCA-adding enzyme, and terminal uridylyl transferase (TUTase) (7, 8). In mammals, the TUT family comprises three major uridyl transferases—TUT4 (Zcchc11), TUT7 (Zcchc6), and TUT1—which catalyze template-independent uridine addition to RNA molecules during post-maturation processing. Recent studies have revealed that mRNA poly(A) tails can recruit TUT4 for uridylation of specific mRNAs. Importantly, terminal uridylation functions as a regulatory signal modulating mRNA stability and expression, while exerting broader functional effects on microRNAs (miRNAs). Initially identified as key regulators of miRNA biogenesis in embryonic stem cells, TUT family enzymes—particularly TUT4—interact with the RNA-binding protein Lin28 to selectively uridylate precursor miRNAs such as pre-let-7 (9). TUT4 and related enzymes exhibit spatiotemporal and tissue-specific activity, catalyzing uridylation of diverse miRNAs including let-7, miR-10a/b, miR-26a/b, and miR-100 (7, 8, 10). Given the small size of miRNAs (~22 nucleotides), 3’-terminal uridylation can significantly alter target specificity and downstream gene regulation. For instance, under normal conditions, miR-26 represses IL-6 to modulate inflammatory responses. However, in TUT4-overexpressing cells, miR-26 variants bearing 1–3 uridine residues fail to bind the IL-6 3′ UTR, thereby impairing IL-6 repression (11).

Our prior work demonstrated elevated TUT4 expression in esophageal epithelial cells following exposure to ionizing radiation. Notably, TUT4 mediates radiation-induced esophageal injury through uridylation of miR-132/212 (12, 13). Table 1 : Top 10 downregulated mRNAs in irradiated TUT4^–/–esophageal tissues

pathways related to DNA replication, ferroptosis, and cell cycle regulation, implicating these processes in radiation-induced tissue injury (14). We further developed TUT4^−/− mice, which exhibited more severe radiation-induced esophageal injury compared to wild-type (WT) controls. Early histopathological changes included capillary congestion, epithelial thinning, and inflammatory cell infiltration, consistent with prior reports (6, 15). At later stages, TUT4−/− mice displayed muscle layer degeneration, fibrosis, and impaired tissue repair. To clarify the molecular mechanisms underlying TUT4-mediated injury, we performed RNA sequencing and downstream bioinformatics analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment identified the peroxisome proliferator-activated receptor (PPAR) and renin–angiotensin system (RAS) pathways as significantly dysregulated in TUT4^−/− esophageal tissues. These pathways may contribute to the pathogenesis of radiation-induced injury. We further propose that TUT4-dependent uridylation of miR-182 alters its regulatory activity on target genes, thereby modulating cellular radiosensitivity.

In recent decades, thoracic radiation therapy has become a mainstay in the multidisciplinary treatment of breast cancer, lung cancer, Hodgkin’s lymphoma, and other thoracic malignancies, with more than half of patients receiving it (22). Its primary objective is to maximize tumoricidal effects while minimizing collateral damage to surrounding healthy tissues (23). However, radiation esophagitis remains a common and debilitating complication, arising from radiotherapy alone or in combination with chemotherapy. It compromises patient quality of life, restricts radiation dosing, and can precipitate treatment interruptions that reduce therapeutic efficacy (24–26). Clinically, patients often present with dysphagia, odynophagia, nausea, anorexia, or retrosternal burning pain (27). Despite its clinical relevance, the pathophysiological mechanisms underlying radiation-induced esophageal injury remain poorly defined. This study aimed to elucidate the molecular basis of radiation esophagitis, with a specific focus on the role of TUT4. Using transcriptomic sequencing and bioinformatic analysis, we delineated regulatory networks involving mRNAs, lncRNAs, and miRNAs. Subsequent genomic enrichment analysis identified significantly enriched pathways, allowing construction of a comprehensive KEGG network map.

Previous studies using irradiated rat esophageal tissue identified 27 differentially expressed miRNAs (7 downregulated, 20 upregulated) through RNA sequencing, implicating these miRNAs in key biological processes such as cell migration, proliferation, and lipid metabolism. Additionally, 197 differentially expressed circRNAs (87 upregulated, 110 downregulated) were detected. Functional enrichment analysis associated these circRNAs with exosomes, adhesion sites, and notably, sphingolipid metabolic processes (28).

To probe the mechanistic role of TUT4 in radiation-induced esophageal injury, we compared wild-type and TUT4 knockout (TUT4^–/–) mice subjected to thoracic irradiation. Esophageal tissues were collected at weeks 1, 2, and 3 post-irradiation and analyzed by H&E staining. Findings revealed vascular congestion, squamous epithelial thinning, and extensive inflammatory infiltration—dominated by neutrophils and lymphocytes. Notably, TUT4^–/– mice exhibited markedly exacerbated tissue damage, peaking at week 2 post-radiotherapy.

To identify molecular drivers of radiosensitivity and potential therapeutic targets, we performed RNA-seq analysis comparing irradiated esophageal tissues from wild-type and TUT4^–/– mice.

Differential expression analysis revealed 53 DEmRNAs (30 upregulated, 23 downregulated). Functional annotation and KEGG pathway analyses indicated significant enrichment in lipid metabolism, fatty acid metabolism, proteolysis, and metabolic regulation. While the cytotoxic effect of radiotherapy is classically attributed to DNA damage, it also induces lipid oxidation. Thus, targeting lipid peroxidation may represent a novel strategy to enhance radiosensitivity (29, 30).

Lipid metabolism is a critical regulator of oncogenic signaling, metastasis, and therapy resistance (31, 32). Notably, radiation has been shown to downregulate lipid metabolism pathways in skin, leading to reduced adipose tissue and altered lipid profiles, suggesting a radioprotective role for tissue lipids (31). We hypothesize that a similar protective mechanism may underlie TUT4-mediated defense against esophageal radiation injury. Our findings show significant modulation of lipid metabolism-related mRNAs during irradiation, implicating lipid metabolism as a key mediator of TUT4’s protective effects. While not all lipid pathways are necessarily TUT4-dependent, the results position lipid metabolism as a promising target for mitigating radiation esophagitis. Further enrichment analysis of TUT4^–/– DEGs highlighted involvement of additional pathways, including protein digestion, RAS signaling, PI3K-Akt signaling, and PPAR signaling. The PPAR pathway, often hyperactivated in colorectal cancer, promotes tumorigenesis, while its inhibition suppresses tumor growth and induces apoptosis (33). Importantly, PPAR signaling also contributes to radioresistance in colorectal cancer, with fatty acid metabolism playing a key modulatory role (34).

Tumor cells inhabit highly vascularized microenvironments and secrete pro-angiogenic factors such as VEGF (35). The RAS pathway, primarily involved in cardiovascular and electrolyte regulation has emerged as a therapeutic target in oncology (36). Angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) inhibit tumor angiogenesis, remodel the extracellular matrix, and modulate the hypoxic tumor microenvironment. For instance, the ACEI captopril inhibits tumor angiogenesis and suppresses hepatic metastasis (37). ARBs and ACEIs have shown potential as adjuvants in chemoradiotherapy, enhancing antitumor efficacy (38). Preclinical models also demonstrate ARB-mediated suppression of VEGF signaling and inhibition of pancreatic cancer proliferation (36). RAS blockers further reduce lung cancer metastasis and augment responses to radiotherapy and chemotherapy (39). Our findings show that RAS and PPAR signaling pathways are significantly enriched in TUT4^–/– mice, suggesting that TUT4 modulates these pathways to confer protection against radiation-induced esophageal injury. We propose that TUT4 may function downstream of RAS signaling, positioning it as a putative molecular target of RAS-inhibiting therapies for esophageal radioprotection.

To further characterize the regulatory role of TUT4, we constructed ceRNA networks using Cytoscape. Notably, TUT4-mediated uridylation plays a critical role in maintaining genomic stability during early radiotherapy by regulating DNA damage response and repair pathways. Our data suggest that TUT4 protects normal esophageal cells from radiation injury. Among the differentially expressed transcripts related to radiosensitivity, TRIM67 emerged as a key modulator of apoptosis, cell cycle arrest, DNA repair, and senescence. It also suppresses colorectal tumorigenesis through activation of the p53 pathway (21). Furthermore, analysis of The Cancer Genome Atlas by Lin et al. revealed significant upregulation of the miR-182/96/183 cluster following radiotherapy, with miR-182 being the most elevated. Overexpression of miR-182-5p enhances radiosensitivity in head and neck squamous cell carcinoma by elevating intracellular ROS levels and modulating radiation-induced antioxidant responses (20).

By performing RNA-seq on TUT4^–/– and wild-type esophageal tissues, we identified key DEGs implicated in radiation-induced injury. Functional analyses highlighted lipid metabolism, RAS signaling, and PPAR signaling as central pathways in TUT4-mediated radiosensitivity. Additionally, we found that lncRNAs competitively bind to miR-182 within the ceRNA network, influencing TUT4 target gene regulation. Together, these findings uncover critical molecular pathways underlying TUT4’s radioprotective function and suggest its potential as a therapeutic target for preventing radiation-induced esophageal injury. A key limitation of this study is the exclusive use of an animal model, without validation in cellular systems. Furthermore, biochemical mechanisms of TUT4 function remain to be elucidated through wet-lab experiments. Future studies will incorporate in vitro and in vivo approaches to validate these findings and further define the molecular mechanisms of TUT4-mediated radioprotection.

Our study provides experimental evidence that TUT4 confers protection against radiation-induced esophageal injury. RNA-seq analysis revealed pathways and molecular mechanisms underlying TUT4-mediated radioprotection. Notably, TUT4 knockout esophageal tissues exhibited significant enrichment of the lipid metabolism, PPAR signaling, and RAS pathways, suggesting that TUT4 may exert its protective effects by repressing these radiation-responsive pathways, thereby enhancing radiosensitivity. Moreover, we identified a competing endogenous RNA (ceRNA) network in which lncRNAs sequester miR-182, modulating the expression of TUT4 target genes. This finding offers novel insights into the poorly understood role of miR-182 in radiation-induced esophageal damage. Bias from some unmeasured clinical variables may have weakened the validity of our study. In the future, we will verify our results in a follow-up in clinical drug practice. Meanwhile, it could be therapeutically targeted without compromising tumor control.