MicroRNAs Modulate the Response of p53-MDM2 Dynamics to DNA Double-strand Breaks at Single-cell Level

Abstract

The tumour suppressor protein p53 plays a pivotal role in the response to various cellular stresses and thus undergoes complicated patterns of regulation, which has been well-studied. However, recent research exploring this topic at single-cell level has revealed that the dynamical behaviour of p53 and its regulator MDM2 in response to DNA damage was distinct from the general trends seen at population level. Moreover, these studies demonstrated that p53-MDM2 dynamics at single-cell level is correlated with cell fate determination.However, the original conclusions were obtained from the simple plasmid-based cell models, which cannot fully recreate the native behaviour of p53-MDM2 in response to stress signals. To better understand this question, we adapted and improved the previous model by introducing a BAC vector containing native p53 gene labelled with a fluorescent protein. This model has also allowed us to investigate whether miRNAs can modulate p53 dynamics and follow cell fate at the single-cell level.By using time-lapse fluorescent microscopy, our results indicated that p53-MDM2 dynamics in response to DNA double-strand breaks was indeed different in single cells when compared with the population-based results obtained in parallel in both models. Furthermore, the dynamics of p53 signalling in individual cells showed the following characteristics: 1) It was variable from cell to cell, but the mean value of some parameters remained fixed; 2) It was able to be fine-tuned by miRNAs; 3) It is involved in shaping cell fate determination with the regulation by miRNAs leading to distinct. This work identified discrepancies in different cell models, suggesting the relative importance of transgenic tools used for single-cell research.In conclusion, our research not only provided a new approach to study the interaction between miRNAs and p53, but also suggested a new dimension of p53 regulatory mechanism and cell fate determination, which further demonstrates the complexity of biological systems that respond to cellular stress in mammalian cells.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dean Jackson (Supervisor) & Michael White (Supervisor)