Figure 4. CircRNA AFF4 acted as a sponge of miR-7223-5p. (A) The target site of miR-7223-5p on circRNA AFF4 was predicted by CircBase. (B) Luciferase reporter activity of circRNA AFF4 with miR7223-5p. (C) circRNA AFF4 levels in the calluses measured by qRT-PCR during fracture healing. (D) Agarose gel electrophoresis showing that divergent primers amplified circRNAAFF4 in complementary DNA (cDAN) but not genomic DNA (gDNA). (E) The amplified product of specific divergent primers was confirmed to be of circRNA AFF4 by sequencing. (F) Expression and location of circRNA AFF4 in MC3T3-E1 determined by fluorescence in situ hybridization (FISH). circRNA AFF4 was labelled with Cy3. (G) circRNA AFF4 and AFF4 mRNA levels in MC3T3-E1 cells with or without RNase R treatment measured by qRT-PCR. (H) MC3T3-E1 cells were transfected with biotinylated miR-7223-5p or miR-NC. circRNA AFF4 and GAPDH levels quantified by qRT-PCR, and relative ratios of immunoprecipitate to input. (I) AGO2 RNA immunoprecipitation in MC3T3-E1 cells transfected with miR-7223-5p. circRNA AFF4 levels measured by qRT-PCR, and relative ratio of IP to input. (J) RNA FISH showing co-localization of circRNA AFF4 and miR-7223-5p in the cytoplasm of MC3T3-E1 cells. (K) RNA FISH showing co-localization of circRNA AFF4 and miR-7223-5p in the cytoplasm of cells derived from bone and calluses. CircRNA AFF4 and miR-7223-5p were labelled with Cy3 and AFM, respectively. The cell nuclei were stained with DAPI.