Long-Read RNA Sequencing Reveals an Isoform Switching Pattern in Healthy Microglial Cell Lines Exposed to Radiotherapy

Background/Objectives: Radiotherapy (RT) is a fundamental treatment for brain tumors but often leads to long-term neurotoxicity, partly caused by microglial dysfunction. In this study, we used long-read RNA sequencing (LR-RNA-seq) to map the isoform-specific transcriptome of human microglial HMC3 cells after RT exposure. Methods: The HMC3 human microglial cell line was treated with standard radiotherapy and, after RNA extraction and library preparation, LR-RNA-seq was performed. Further bioinformatic analyses allowed to investigate on the isoform switching pattern after radiotherapy exposure. Results: Gene-level analysis found 591 differentially expressed genes, with positive enrichment of PI3K/AKT-related pathways and negative enrichment of translation-associated processes. Isoform-level analysis uncovered 827 significant isoform switching events across 420 genes, often involving shifts from protein-coding transcripts to non-coding variants. Functional annotation showed that most isoform switches resulted in the loss of open reading frames and protein domains, especially in ribosomal proteins and translation machinery components. Exploratory proteomic analysis identified 191 differentially abundant proteins, including a significant reduction in RPL7A, providing partial protein-level support for translation-associated transcriptomic remodeling. Conclusions: RT-induced stress causes qualitative transcriptomic remodeling beyond changes in overall gene expression. These findings indicate that radiotherapy induces coordinated gene-, transcript-, and isoform-level changes in HMC3 cells. The alterations observed in translation-associated genes, together with partial proteomic concordance, identify candidate mechanisms for future functional investigation.