miR-20a Regulates FAS Expression in Osteosarcoma Cells by Modulating Promoter Activity and Can be Therapeutically Targeted to Inhibit Lung Metastases.

The metastatic potential of osteosarcoma cells is inversely correlated to cell surface FAS expression. Downregulation of FAS allows osteosarcoma cells to escape FAS ligand-mediated apoptosis when they enter a FAS ligand-positive microenvironment such as the lung. We have previously demonstrated that miR-20a, encoded by the miR-17-92 cluster, downregulates FAS expression in osteosarcoma. We further demonstrated an inverse correlation between FAS expression and miR-20a expression. However, the mechanism of regulation by miR-20a was still unclear. The purpose of the current study was to evaluate the mechanism of regulation by miR-20a and test the effect of targeting miR-20a We investigated whether miR-20a's downregulation of was mediated by binding to the 3'-untranslated region (3'-UTR) of mRNA with the consequent induction of mRNA degradation or translational suppression. We identified and mutated two miR-20a binding sites on the mRNA 3'-UTR. Using luciferase reporter assays, we demonstrated that miR-20a did not bind to either the wild-type or mutated 3'-UTR. In contrast, overexpression of miR-20a resulted in downregulation of promoter activity. Similarly, the inhibition of miR-20a increased promoter activity. The critical region identified on the promoter was between -240 bp and -150 bp. Delivery of anti-miR-20a using nanoparticles in mice with established osteosarcoma lung metastases resulted in upregulation of and tumor growth inhibition. Taken together, our data suggest that miR-20a regulates expression through the modulation of the promoter and that targeting miR-20a using anti-miR-20a has therapeutic potential. .