Flunarizine changes microRNA expression in cell cultures and in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, leading to reduced SMN protein levels and widespread disruption in RNA metabolism. Despite of disease-modifying therapies, which remarkably improve patient outcomes, the long-term effects remain unknown. Dysregulated small non-coding RNAs, including microRNAs, have been reported in SMA, but their contribution to the disease remains unclear. We previously showed that flunarizine could improve the phenotype of an SMA mouse model; however its mode of action is incomplete. Here, we showed that flunarizine modulates the expression of numerous microRNAs. Using small-RNA sequencing of a flunarizine-treated SMA patient fibroblast cell line, we identified several microRNAs, which are also dysregulated in the brains and/or spinal cords of SMA mouse models at early disease stages and corrected with flunarizine. Transfection of the miR-128-3p inhibitor interferes with the flunarizine-induced neurite outgrowth in the murine neuronal NSC34 cells. Among the mRNAs modulated by flunarizine, homeodomain interacting protein kinase 2 (Hipk2) transcripts were revealed as novel miR-128-3p targets using either the mimic or its inhibitor. Our findings suggest that an early microRNA accumulation in spinal cords of SMA models can contribute to molecular dysfunctions and may represent an initiating event in pathogenesis.