This study examined regulatory T cell (Treg) dynamics in radiation-induced lung injury (RILI), focusing on miRNA/mRNA-mediated differentiation mechanisms to identify therapeutic targets. Temporal Treg changes in irradiated mouse lungs were analyzed via single-cell RNA sequencing (scRNA-seq) and flow cytometry. Treg ablation via anti-CD25 antibodies assessed their regulatory roles in neutrophil and monocyte infiltration. High-throughput sequencing identified miRNAs/mRNAs governing Treg differentiation. Plasma miRNA profiles from GSE147242 were enrolled to validate the differential expression of miR-223-3p in RILI. Additionally, machine learning methods were used to construct most predictive miRNA signature post- radiotherapy alterations. Dual-luciferase assays validated miR-223-3p binding to leukemia inhibitory factor (Lif). In vivo/in vitro experiments evaluated METTL3’s impact on miR-223-3p maturation and Treg differentiation. Treg infiltration increased in RILI and bleomycin-induced fibrosis, interacting with epithelial, endothelial, and fibroblast cells. miR-223-3p directly targeted Lif to regulate Treg differentiation, while METTL3 suppressed miR-223-3p maturation via m6A modification. METTL3 inhibition reduced Treg accumulation and alleviated lung injury. Additionally, plasma miRNA analysis confirmed marked differential expression of miR-223-3p post-radiotherapy. A predictive model, integrating miR-223-3p, miR-150-5p, and miR-34a-5p via JMI and SVM-RFE machine learning methods, achieved an AUC of 0.997 (five-fold cross-validation AUC: 0.973), highlighting its high predictive efficacy. scRNA-seq revealed resting Tregs differentiating into Th2-like subsets (marked by KLRG1 and GATA3) with altered m6A levels, implicating epitranscriptional control of Treg plasticity. Tregs mediate multicellular interactions in irradiated lungs. METTL3 promotes miR-223-3p maturation via m6A and drives Treg differentiation through Lif. The METTL3/miR-223-3p/Lif axis regulates Treg-mediated fibrosis, highlighting METTL3 as a therapeutic target for RILI.
Peng et al. (Wed,) studied this question.