CB-03IDENTIFICATION AND CHARACTERISATION OF MicroRNAs INVOLVED IN GLIOBLASTOMA CELL PROLIFERATION AND SURVIVAL USING HIGH-THROUGHPUT SCREENING

INTRODUCTION MicroRNAs, single-stranded non-coding RNAs that function by reducing translation or causing degradation of target mRNAs, have been shown to play roles in multiple hallmarks of GBM, suggesting they may be of therapeutic importance. METHODS: The miRIDIAN mimic library (Dharmacon) that encompasses all human microRNAs annotated in miRBase v16.0 was used with a high-throughput imaging platform (Operetta) to identify microRNAs with potent effects on GBM cell proliferation and survival. Screens were performed in duplicate on U251 (adult) and KNS42 (paediatric) GBM cell lines. Cell number (nuclei count) was assessed 72h post-transfection and expressed as a z-score for each microRNA. MicroRNAs were considered significant candidates if their mean z-score was below zero and if it differed from that of the negative control by at least two standard deviations. Validation included RTqPCR, imaging and flow cytometry based assays. RESULTS: The functional screens resulted in approximately 100 candidates per cell line with 70% overlap between them. Based on available knowledge and potential relevance for GBM therapy, seven initial microRNAs candidates were chosen for further validation in a panel of four adult and two paediatric GBM cell lines: miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-449a-5p, miR-449b-5p which all share a common seed sequence; miR-340-5p and miR-X. Of these candidates miR-X was identified as having the most potent, robust effects. MiR-X consistently caused a G2M cell cycle arrest followed by apoptosis in all cell lines and has predicted targets involved in the induction of the cytoskeleton rearrangement and non-canonical Wnt pathways following the G2M phase transition. This phenotype was specific to mimic-miR-X transfected cells. CONCLUSION: We have identified a novel potent pro-apoptotic microRNA in GBM that may be relevant for therapy. Work is ongoing to characterise the mechanism responsible for this effect and test for efficacy in vivo.