Fig. 4

MiR-5047 regulates OS stemness and malignancy by targeting SOX2. A. ENCORI, TargetScan and miRDB prediction of potential targets binding to miR-5047. Expression of indicated target genes was detected after transfecting the indicated miR-5047 mimics. B. Expression of SOX2 was detected in WAC-AS1-, miR-5047- or control-transfected cells. C. Hypothetical wild-type and mutant binding sites in miR-5047 for SOX2 3’UTR. Luciferase reporter assay detecting the binding of miR-5047 to SOX2 in OS cells. D. RIP with anti-AGO2 antibody was performed to detect WAC-AS1, miR-5047 and SOX2 mRNA. E. CCK8 assay showing the proliferation of OS cells following transfection of SOX2, miR-5047 + SOX2 or control. F. Colony formation of OS cells following transfection of SOX2, miR-5047 + SOX2 or control. G. Transwell assay showing the migration of miR-5047- or SOX2-transfected or control OS cells (scale bar, 50 μm). H. Wound healing assay the migration of SOX2 transfected, miR-5047-transfected or control OS cells (scale bar, 100 μm). I. Western blots showing stemness-related proteins in miR-5047-transfected, miR-5047 + SOX2-transfected or control cells. J. Changes in tumorsphere-forming ability of SOX2-transfected, miR-5047-transfected or control OS cells (scale bar, 50 μm). K. Percentage of CD133 + cells in populations of miR-5047-transfected, miR-5047 + SOX2-transfected or control OS cells was determined by flow cytometry. Error bars represent three independent experiments, *p < 0.05, **p < 0.01, *** p < 0.001