Real-time quantitative PCR analysis of microRNAs in a mice model of Neurogenic Heterotopic Ossifications (NHOs)

Neurogenic heterotopic ossifications are intramuscular bone formations developing following central nervous system injury. The pathophysiology is poorly understood and current treatments for this debilitating condition remain unsatisfying. Here we explored the role of miRNAs in a clinically relevant mouse model that combines muscle and spinal cord injury (SCI), and in patients’ cells. We found an osteo-suppressive miRNAs response in injured muscle that was hindered when the spinal cord injury was associated. In isolated fibro-adipogenic progenitors from damaged muscle (cells at the origin of ossification), spinal cord injury induced a downregulation of osteo-suppressive miRNAs while osteogenic markers were overexpressed. The overexpression of selected miRNAs in patient’s fibro-adipogenic progenitors inhibited mineralization and osteo-chondrogenic markers in vitro. Altogether, we highlighted an osteo-suppressive mechanism involving multiple miRNAs in response to muscle injury that prevents osteogenic commitment and which is ablated by the neurologic lesion in heterotopic ossification pathogenesis. This provides new research hypotheses for preventive treatments. These data were generated from RT-qPCR miRNA arrays on pooled samples (n=3 independant biological replicates) from control (SHAM+PBS), damaged (SHAM+CDTX), spinalized (SCI+PBS) or NHOs developping (SCI+CDTX) muscles from mice gastrocnemius biopsy at 2 or 4 days after induction. MicroRNAs screening was performed using miRNome Mouse&Rat panel I+II (V5, 339322, Qiagen). These results were subsequently validated in RT-qPCR for each individual donor (n=6 independant biological replicates) in the linked manuscript.