A high-content screen profiles cytotoxic microRNAs in pediatric and adult glioblastoma cells and identifies miR-1300 as a potent inducer of cytokinesis failure
Authors: Boissinot Marjorie , King Henry , Adams Matthew , Higgins Julie , Ward Thomas A. , Steele Lynette P. , Tams Daniel , Morton Ruth , Polson Euan , da Silva Barbara , Droop Alastair , Hayes Josie L. , Martin Heather , Laslo Peter , Morrison Ewan , Tomlinson Darren C. , Wurdak Heiko , Bond Jacquelyn , Lawler Sean E. , Short Susan C. 
Published: October 2019

Background

MicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.

Methods

To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3’UTR luciferase assays and Western Blotting.

Results

Our screen identified ∼100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.

Conclusion

MiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.

Key points (2 or 3 key points 85 characters plus spaces each)

70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cells

MiR-1300 expression is restricted to endomitosis within megakaryocyte differentiation

MiR-1300’s ectopic expression is a potent and promising therapeutic tool in cancer

Importance of Study

Previous functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.

Not only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.