MiR-630 Inhibits Endothelial-Mesenchymal Transition By Targeting Slug In Traumatic Heterotopic Ossification

OBJECTIVES Heterotopic ossification(HO)is the abnormal formation of mature bone in extraskeletal soft tissues that occurs as a result of inflammation caused by traumatic injury or associated with genetic mutation.Despite extensive research to identify the source of osteogenic progenitors,the cellular origins of HO are controversial and the underlying mechanisms,which are important for the early detection of HO,remain unclear.In this study,we used in vitro and in vivo models of BMP4 and TGF-?2-induced HO to identify the cellular origin and the mechanisms mediating the formation of ectopic bone in traumatic HO.A matched case-control study on the filtered-out MicroRNA was studied to distinguish HO from other processes leading to bone formation.This approach offers a novel a potential mechanism of post-traumatic ectopic bone formation and a potential early indicator of HO.METHODSImmunofluorescence analysis of lesional HO tissues from operation of trauma-induced HO patients was performed compared to the sponge bone.Meanwhile,microarray analysis of serum from patients with mature HO phase and immature HO phase was performed to explore the role of microRNAs in the regulation of HO formation via EndMT.We used an in vitro model of EndMT based on the treatment of human dermal microvascular endothelial cells(HD-MVECs)with BMP4 and an in vivo model of HO to identify the role of the filtered-out microRNA and its target gene.Finally,comparative analysis of the filtered-out microRNA expression in patients with HO and gender and age matched controls without HO after arthrolysis or with healing fractures was performed.RESULTSResults indicated ectopic bone in traumatic HO is of endothelial origin,while the normal sponge bone not.MiR-630 was downregulated in patients with HO.Further analysis of the expression of miR-630 showed downregulation in patients with immature and mature HO compared to the normal person.In in vitro and in vivo models of BMP4 and TGF-?2-induced HO,the results indicate that the inhibition of endothelial-mesenchymal transition by miR-630 targeting Slug plays a role in the formation of ectopic bone in HO.A matched case-control study showed that miR-630 is specifically downregulated during the early stages of HO and can be used to distinguish HO from other processes leading to bone formation.CONCLUSIONSOur results provide evidence of the endothelial origin of ectopic bone in the early phase of HO formation via the induction of EndMT and the potential differentiation of cells via a mesenchymal cell intermediate HO formation.We showed that miR-630 is downregulated in patients with HO and inhibits EndMT viathe downregulation of its target Slug.Analysis of serum miR-630 levels revealed a distinct pattern of expression in response to trauma-related HO,providing potential biomarkers and therapeutic targets for the management of patients with post-traumatic HO.