miR-193a inhibits osteogenic differentiation of bone marrow-derived stroma cell via targeting HMGB1

Background: miR-193 alpha has been shown to be involved in a variety of biological processes, including cell proliferation, differentiation, and apoptosis. However, little is known about how miR-193 alpha regulates osteogenic differentiation. Methods: We employed RT-qPCR to determine the level of miR-193 alpha and mRNA level of HMGB1 and osteoblast-specific markers (Runx2, ALP, OSX, OCN). Besides, westernblot was used to probe protein level of phosphorylated MAPK family members and beta-catenin. Bioinformatic analysis was used to predict the putative binding sequence of miR-193 alpha to the 3'-UTR of HMGB1 and we confirmed this result by dual luciferase reporter assay. Alizarin red staining assay (ARS) and alkaline phosphatase activity (ALP) were performed to detect osteogenic differentiation. Results: miR-193 alpha was downregulated in OM (osteogenic medium)induced hBMSC. More interestingly, we found that miR-193 alpha mimic attenuated matrix mineralization and alkaline phosphatase activity, whereas miR-193 alpha inhibitor exerted the opposite phenotypes. Mechanistically, we observed that miR193 alpha played an inhibitory role in expression of osteoblast-specific markers and activation of MAPK and Wnt signaling pathways. Bioinformatic analysis and dual luciferase assay demonstrated that miR-193 alpha directly targeted 3'-UTR of HMGB1. Furthermore, we overexpressed HMGB1 in miR-193 alpha overexpressed hBMSC to establish that HMGB1 acted as downstream target of miR-193 alpha-inhibited osteogenic differentiation. Conclusions: Here, we reveal miR-193 alpha plays a suppressive role in osteogenic differentiation of hBMSC via targeting HMGB1. These findings provide a novel mechanism underlying osteogenic differentiation and offer therapeutical strategy for bone formation. (C) 2018 Elsevier Inc. All rights reserved.