Figure 3. Long intergenic nonprotein-coding RNA 00514 (LINC00514) directly interacts with microRNA-708 (miR-708) in OS cells. (A) Distribution of LINC00514 in the cytoplasm and nucleus of HOS and MG-63 cells was analyzed by subcellular fractionation. (B) Sequences of the wild-type and mutant miR-708-binding sites in LINC00514. (C) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to assess the efficiency of miR-708 mimic transfection in HOS and MG-63 cells. (D) Luciferase reporter assay was performed to demonstrate targeted binding between LINC00514 and miR-708 in HOS and MG-63 cells. (E) Relative enrichment of LINC00514 and miR-708 in the coprecipitated RNA was examined by RNA immunoprecipitation assay. (F) RT-qPCR was performed to detect miR-708 expression in the 59 pairs of OS and adjacent normal tissue samples. (G) Spearman’s correlation analysis illustrated a negative correlation between miR-708 and LINC00514 expressions in the 59 OS tissue samples (r = −0.5731, P < 0.0001). (H) Relative miR-708 expression was measured by RT-qPCR in HOS and MG-63 cells following transfection with si-LINC00514 or si-NC. *P < 0.05 and **P < 0.01.