Research Paper

Figure 4. Circ-DNAH1 sponges miR-663a in OS cells. (A) Four potential binding sites for miR-663a in a conserved locus of Circ-DNAH1 were identified. In the construct cire-DNAH1-MUT, the four putative binding sites were mutated, (B) Luciferase reporter analysis of MG63 and U2OS cells transfected with wild-type (circ-DNAH1 WT) and circ-DNAH1 MUT. Note that circ-DNAH1-MUT completely reversed the inhibitory effect of circ-DNAH1 on miR-663a expression. (C, D) Immunoprecipitation with the anti-Ago2 antibody from MG63 and U2OS cell extracts, followed by qPCR analysis of circ-DNAH1 and miR-663a. (E, F) RNA pull-down analysis of the homogenates of MG63 and U2OS cells using a biotin-labeled circ-DNAH1 and streptavidin detection, followed by qPCR analysis of miR-663a. (G, H) The biotinylated miR-663a and biotinylated miR-663a-MUT were transfected in MG63 and U2OS cells. RNA pull-down experiment was performed, followed by qPCR analysis of circ-DNAH1. (I) FISH dialysis of circ-DNAH1 (red) and miR-663a in MG63 cells, with DAP I (blue) counterstaining the nucleus.