MRNA Alteration Mapping of Radiation-Induced Pulmonary Fibrosis in C57BL/6 Mice

Abstract

<h3>Purpose/Objective(s)</h3> Radiation-induced lung injury, including radiation-induced pneumonitis and pulmonary fibrosis, is a common serious dose-limiting toxicity of thoracic radiotherapy, and can be life-threatening. There is no definite conclusion of the etiology of radiation-induced pulmonary fibrosis (RIPF), and the effective interventions are still lacking. In this study, based on RIPF mouse model, we aim to identify genes responsive to irradiation, compare the difference in genome expression between normal lung tissues and the irradiated ones, and to provide the mRNA alteration mapping that can make the predictive model and potential interventions of RIPF. <h3>Materials/Methods</h3> Thirty C57BL/6 mice were randomized into 3 groups: control group (sham radiation), 16 Gy thoracic radiation group and 20 Gy radiation group to establish the RIPF mouse model. Lung tissues were harvested at 3 and 6 months after irradiation. The global gene expression of lung tissues was assessed by RNA sequencing. Differentially expressed genes were identified and subjected to function and pathway enrichment analysis. Infiltration of immune cells was evaluated by CIBERSORT. <h3>Results</h3> RIPF mouse model was successfully established in both 16 Gy and 20 Gy thoracic radiation groups. At 3 months after radiation, 317 mRNAs were significantly upregulated and 254 downregulated in the 16 Gy thoracic radiation group. 203 mRNAs were upregulated and 149 downregulated significantly in the 20 Gy group. At 6 months after radiation, 651 mRNAs were significantly upregulated and 131 downregulated in the 16 Gy group. 106 mRNAs were significantly upregulated and 4 downregulated in the 20 Gy group. Several functions and pathways including angiogenesis, epithelial cell proliferation, extracellular matrix, complement and coagulation cascades, TNF signaling pathway, NOD-like receptor signaling pathway, and HIF-1 signaling pathway were significantly enriched based on the differentially expressed genes in irradiation groups at 3 months after thoracic radiation. Cellular senescence, myeloid leukocyte activation, regulation of lymphocyte activation, mononuclear cell proliferation, immunoglobulin binding, and some other immune system associated pathways were also significantly enriched in irradiation groups at 6 months after radiation. We confirmed a dramatic increase of macrophage, neutrophil, dentric cell and natural killer cell, and a decrease of B cell in the two irradiation groups at 3 months. At 6 months, the infiltration of B cell in the 20 Gy group grew and became higher than the control group. <h3>Conclusion</h3> By RNA-seq, the irradiation responsive genes were identified. The differentially expressed genes were mainly associated with cell injury, cellular metabolism, epithelial and endothelial cell proliferation, and immune cell activation and regulation. Our results provided comprehensive clues on the mechanism of RIPF, which hopefully, may turn into the potential treatment target in preventing and reversing RIPF.