Purpose: Exposure of living cells to ionizing radiation has different consequences, depending on the dose and cell type. Changes in gene expression at the level of transcription and translation, including those regulated by microRNAs (miRNAs), play a role in the intrinsic radiosensitivity of different cells and define their fate, survival or death. The aim of our work was to examine how ionizing radiation may influence the expression of genes regulated by different miRNAs and miRNA biogenesis.
Materials and methods: The work was performed on cultured human melanoma Me45 cells, transiently transfected with plasmids containing Renilla luciferase reporter gene targeted by miRNAs Let-7, miR-21 or miR-24. The levels of reporter mRNAs and mRNAs coding for proteins participating in miRNA biogenesis were assayed at different time points in irradiated and non-irradiated cells using RT-qPCR, and reporter protein by luciferase activity assays. MiRNA-targeted motifs in mRNAs coding for proteins engaged in miRNA biogenesis were extracted from the miRTarBase database.
Results: Messenger RNA and protein levels of transfected luciferase genes fluctuated in time in patterns that depended on the type of miRNA regulation and changed upon irradiation of the cells. The average levels of reporter mRNAs were higher in irradiated cells, whereas the levels of proteins changed in either direction. Radiation also influenced the levels of miRNAs and the expression of genes engaged in their biogenesis suggesting that the changes in gene expression following ionizing radiation result mainly from these changes in expression of genes regulating miRNA biogenesis and the influence of miRNA on mRNA translation.
Conclusions: Currently, the responses of cells to ionizing radiation are mainly ascribed to changes in their redox conditions and increased intracellular levels of ROS, but the experiments described here suggest that a further important factor is modulation of translation through changes in biogenesis and levels of miRNAs.
Keywords: Me45 cells; ionizing radiation; mRNA and miRNA expression; miRNA biogenesis; reporter genes.