Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300

Marjorie Boissinot ,Jacquelyn Bond ,Josie Hayes ,Ewan E Morrison ,Peter Laslo ,J William Higgins ,Ruth Morton ,Euan S Polson ,Daniel Tams ,Henry King ,Matthew Adams ,Lynette P Steele ,Alastair Droop ,Thomas A Ward ,Heather L Martin ,Gary Shaw ,Darren C Tomlinson ,Sean E Lawler ,Heiko Wurdak ,Bárbara Da Silva ,Susan Short

doi:10.1038/s41388-020-1360-y

Abstract

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. 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 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in 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 GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Highlights

MicroRNAs are small 22–24 nt single-stranded noncoding RNAs that function by reducing the translation of target mRNAs
We applied the conditions of the primary screen to four established GBM cell lines (U251, KNS42, LN229, and U373), confirming statistically significant cytotoxicity following transfection of all seven mimic-microRNAs (Supplementary Fig. 3a)
MicroRNAs are well-known as important regulators of cellular functions [3,4,5] and several have been shown to have therapeutic potential, either by overexpression or inhibition by antisense oligonucleotides

Summary

Introduction

MicroRNAs are small 22–24 nt single-stranded noncoding RNAs that function by reducing the translation of target mRNAs. Extended author information available on the last page of the article increasing number of microRNA mimics and inhibitors in preclinical and early clinical development as cancer therapies, including one for patients with solid tumors using an oligonucleotide mimic of microRNA-34 (MRX34, NCT01829971) [7,8,9,10]. Recent preclinical studies showed efficacy of a microRNA expressing therapeutic vector in GBM [11]. MicroRNA-10b expression has been measured in a clinical trial (NCT01849952) to assess its use as a prognostic and diagnostic biomarker. An inhibitor of miR10b is currently at the preclinical development stage (Regulus Therapeutics Inc. and [12])

Methods

Results

Conclusion