The role of RNAs and extracellular vesicles in the radiation response

The bystander effect (BE) occurs when cells that have not been directly traversed by ionizing radiation exhibit DNA damage effects as though they had been. It is established that signalling molecules such as cytokines and reactive oxygen species (ROS) signal to surrounding cells to mediate BE, however more recently it has been demonstrated that extracellular vesicles (EVs) might be involved. In addition, following RNase treatment, the EVs are no longer able to cause DNA damage in unirradiated recipient cells, suggesting that an RNA molecule in association with the EVs is involved with BE. As the literature documenting differential regulation of RNA in EVs released from irradiated cells is scarce, the work described in this thesis has aimed to better understand the role of RNA in the radiation response. Firstly the miRStress database was developed and used to identify novel miRNA candidates involved in response to radiation by meta-analysis of the published literature. Characterisation of the EVs released was also performed using transmission electron microscopy (TEM), sucrose gradient centrifugation, sizing and the determination of EV concentration released from cells. Next-generation sequencing was performed to identify the mRNA, non-coding RNA and microRNA candidates involved. General characterisation of EVs derived from unirradiated and 2 Gy irradiated MCF7 cells was also performed to gain a better understanding of the populations of vesicles released following irradiation. The results herein suggest that EVs from irradiated cells have specific characteristics when compared to those from unirradiated cells. An increase in the number and a decrease in the size of EVs released from 2 Gy irradiated cells were observed compared to unirradiated cell EVs. In addition to the changes in size and release of EVs from 2 Gy cells, EVs released from irradiated MCF7 cells contained specific mRNAs, non-coding RNAs and miRNAs. Furthermore the miRStress database identified microRNA candidates predicted to be involved in the radiation response. Following RNA Seq analysis a functional study of the genes ANP32B, MALAT1, NET1, HSP90AA1 and NCL was performed based upon their upregulation in 2 Gy EVs. Knockdown of some of these genes resulted in changes in the DNA damage response observed in directly irradiated and bystander cells, suggesting that the RNAs carried in irradiated cell EVs do indeed have a functional role in transmission of BE. In summary, this study has identified diverse RNA species in EVs released from irradiated cells that appear to play functional roles in the mediation of the bystander effect. Further investigation would help to elucidate the mechanisms by which these RNAs function in recipient cells in order to better understand the BE mechanism