MicroRNA-20a regulates autophagy related protein-ATG16L1 in hypoxia-induced osteoclast differentiation

Autophagy and autophagy-related proteins (ATGs) play decisive roles in osteoclast differentiation. Emerging lines of evidence show the deregulation of miRNA in autophagic responses. However, the role of hypoxia and involvement of miRNA in osteoclast differentiation are unclear. In the present study, we demonstrate that hypoxia caused induction of autophagy and osteoclast differentiation markers in RAW264.7 cells stimulated with M-CSF and RANKL. In addition, miR-20a was significantly repressed during hypoxia and identified as the prime candidate involved in hypoxia-induced osteoclast differentiation. The results from dual luciferase reporter assay revealed that miR-20a directly targets Atg16l1 by binding to its 3'UTR end. Further, miR-20a transfection studies showed significant down regulation of autophagic proteins (LC3-II and ATG16L1) and osteoclast differentiation markers (Nfatc1, Traf6, and Trap) thus confirming the functional role of miR-20a under hypoxic conditions. Results of chromatin immunoprecipitation assay showed that HIF-1α binds to miRNA-20a. From miRNA Q-PCR results, we confirmed that shRNA HIF-1α knockdown significantly downregulated both autophagy (LC3, p62, Atg5, Atg12, Atg16l1, Atg7, Becn1, Atg9a) and osteoclast markers (Traf6, Nfatc1, Ctsk, cFos, Mmp9, Trap) in RAW264.7 cells. Thus, our findings suggest that the regulatory axis of HIF-1α-miRNA-20a-Atg16l1 might be a critical mechanism for hypoxia-induced osteoclast differentiation.