Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: Characterisation of circRNA molecular functions in rhabdomyosarcoma tumour

Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules whose important role in regulating gene expression at different levels is recently emerging. Particularly, circRNAs often show a deregulated expression in several pathological processes, including cancer, contributing to the disease’s onset and progression. My PhD work aimed at investigating the molecular role of a specific circRNA, named circZNF609, in regulating cell proliferation in rhabdomyosarcoma, a paediatric skeletal muscle tumour. We found that circZNF609 is overexpressed in rhabdomyosarcoma with respect to primary myoblasts and its depletion slows down cell-cycle progression at the G1-S transition. We also characterised the impact of circZNF609 knock-down on rhabdomyosarcoma cell transcriptome and major pathways involved in proliferation, e.g. Retinoblastoma/E2F1 and PI3K/AKT pathways. Thanks to the analysis of circZNF609 molecular interactors, we discovered that the circRNA can bind to Ckap5 mRNA, a transcript encoding a microtubule polymerase with a fundamental role in controlling mitotic spindle assembly and chromosome segregation. We found that circZNF609 depletion impairs CKAP5 protein expression possibly through HUR, an RNA-binding protein promoting mRNA stability and translation. By reducing Ckap5 mRNA stability and protein levels, circZNF609 knock-down impairs microtubule dynamics and induces defective chromosome segregation. This results in the accumulation of DNA damage that could be responsible for the activation of checkpoints during the following cell cycle, blocking it at the G1-S transition. Therefore, our research unveiled a novel molecular circuit through which a circRNA regulates cell cycle progression in rhabdomyosarcoma tumour. In the last part of this work, we identified other circRNAs deregulated between myoblasts and rhabdomyosarcoma cells, and we started to elucidate the role of circHIPK3 in modulating YAP/TAZ activation in tumour cells. Our long-term aims are to unveil circRNA-dependent circuits which can be responsible for tumour onset and progression, and to investigate potential uses of these circRNAs as rhabdomyosarcoma biomarkers and/or therapeutic targets.