10.4225/53/5A1B3688156E3
doctoral thesis
Ooi, Chi Yan
Chi Yan
Ooi
Identification and characterization of microRNAs that play novel functional roles in MYCN-driven neuroblastoma
UNSW Sydney
2017
MYCN
microRNA
Neuroblastoma
Cancer
transcriptome
nervous system
non-coding RNA
gene regulation
Childhood cancer
Bioinformatics
Human
Mouse
molecular biology
tumorigenesis
UNSW Sydney
UNSW Sydney
UNSW Sydney
EN
http://hdl.handle.net/1959.4/58944
Neuroblastoma is a childhood cancer derived from embryonic neural crest cells of the peripheral sympathetic nervous system, and is the most common extracranial solid tumours in children. The MYCN proto-oncogene is amplified in around a quarter of primary neuroblastoma tumours and this remains a major factor that predicts poor prognosis. MYCN is also a major oncogenic driver through pleiotropic effects and is regulated by multiple protein coding genes and non-coding RNAs such as microRNAs (miRNAs) in neuroblastoma. However, the complete miRNA regulatory network that contributes to MYCN-driven neuroblastoma has not been fully explored. To explore this regulatory network, we investigated the global miRNA and mRNA expression profiles of the Th MYCN neuroblastoma mouse model during early tumorigenesis and inferred a statistically significant interaction network between miRNAs and mRNAs. Of these miRNAs, miR-204 expression is significantly reduced in ganglia of Th MYCN homozygotes compared to wild-type mice. High miR-204 expression is strongly associated with better overall and event-free survival and lack of MYCN amplification in neuroblastoma patients. In addition, miR-204 expression is increased with MYCN siRNA knockdown in MYCN-amplified human neuroblastoma cells, suggesting MYCN may regulate miR-204 expression. Indeed, chromatin immunoprecipitation assay revealed MYCN binding to DNA encoding the miR-204 precursor sequence, showing MYCN can transcriptionally regulates miR-204 expression in neuroblastoma. Moreover, miR-204 over-expression significantly reduced MYCN mRNA and protein expression. A biotin pulldown assay confirmed miR-204 binding to MYCN mRNA. Importantly, miR-204 over-expression significantly reduced neuroblastoma cell proliferation and colony forming capacity. Doxycycline-induced miR-204 over-expression in two subcutaneous xenografts models of stable neuroblastoma cell lines also prolonged mouse survival. For the first time, our data demonstrate that miR-204 inhibits neuroblastoma growth in vitro and in vivo and directly represses MYCN, while MYCN transcriptionally represses miR-204 to form a double-negative regulatory feedback loop. Together these findings describe miR-204 as a tumour suppressor microRNA that functions in regulation of MYCN expression in neuroblastoma tumorigenesis.