Time Sequential Changes of Epithelial-Mesenchymal Transition Signaling Induced by Ionizing Radiation using Next-Generation Sequencing in Glioblastoma

Abstract

Epithelial-mesenchymal transition (EMT) is considered as one of the mechanisms of treatment resistance in glioblastoma. We evaluated a time sequential changes of EMT signaling in glioblastoma induced by ionizing radiation. The cell line U-373 MG (human glioblastoma) was irradiated with 6Gy of high energy X-ray (6MV) with dose rate of 6 Gy/min. Next generation sequencing (NGS) was performed on the cell line before irradiation (baseline), 6 hours, 24 hours, and 48 hours after irradiation and database of essential genes (DEG) was evaluated in each period. The DEGs were compared with those included in the 128 of the GeneSets. P value from the Fisher exact test and Q value from the Benjamini Hochberg methods were calculated. All KEGG map pathways were evaluated and the EMT related KEGG pathways were assessed using the EMT gene database (dbEMT). GeneSet analysis showed that ionizing radiation up-regulated the expression of the GeneSet of the wholemark of EMT at 6 hours and 24 hours after irradiation. On KEGG pathway analysis, extracellular matrix (ECM) receptor interactions increased over time and the expression of miRNA-21 increased at 6, 24, and 48 hours after irradiation resulting in cell growth and survival pathway. Increased RTK expression suggested RTK-induced EMT process. HIF-α induced hypoxia and activated TGF-β signaling pathway, which is related with EMT. TGF- β activated Smad2/3 followed by activation of Smad4. Ionizing radiation may promote EMT in glioblastoma, resulting in treatment related resistance. miRNA-21 may play a critical role in EMT after irradiation. EMT blocking to prevent treatment resistance may be a promising strategy to increase survival in glioblastoma.