The transcription factor Foxm1 controls pro-stemness microRNAs in cerebellar neural stem cells (NSCs)

Background: Cerebellar neural stem cells (NSCs) maintenance is of great interest since NSCs can be used to treat impaired cells and tissues or improve regenerative power of degenerating cells in neurodegenerative diseases or spinal cord injuries. Under maintenance conditions, NSCs express a number of Hedgehog-Gli (Hh-Gli) linked and stemness genes (e.g. Nanog, Oct4, Sox2) whose mechanisms of regulation have been under investigation. However, the interplay between transcription factors and microRNAs in NSCs is still being charted. Aim: Identification of new molecular players involved in NSCs’ maintenance with particular interest in the major regulatory pathway Hedgehog-Gli. Materials and Methods: Cells used for the study were NSCs isolated from postnatal day 4 (P4) wild type (C57BL/6) mice cultured both as neurospheres in selective medium and as differentiated NSCs when cultured in medium with serum. NSCs and their differentiated counterparts were analysed by high-throughput technologies. Bioinformatics analysis was used for the identification of the Foxm1-regulated miRNAs; knock-down experiments and clonogenic assays were used for functional studies. Chromatin immunoprecipitation experiments (ChIP) were used to investigate the binding between Foxm1 and its targets and between Foxm1 and its regulators. Results: NSCs and their differentiated counterparts were analysed using next-generation mRNA- and miRNA-sequencing. The transcriptional analysis allowed the identification of Foxm1 as one of the highest transcripts in NSCs and the miRNA-sequencing provided a number of highly expressed miRNAs. The use of bioinformatics analysis resulted in the Foxm1-regulated miRNAs, miR-15 ~ 16 cluster, miR-17 ~ 92 cluster, miR- 130b and miR-301a. Functional experiments, such as knock-down experiments and clonogenic assays enabled the identification of Foxm1 as a downstream mediator of the Hh-Gli signalling and with the ability to regulate the above mention miRNAs. Conclusion: The study presented reveals a new Foxm1-microRNAs network with a major role in the maintenance of NSCs. These results add a previously unidentified important molecular aspect that could be used in future neurodegenerative disease studies, thus enriching the field of translational medicine.