Figure 3. ADAMTS9-AS2 binds to miR-27a-3p and inhibits its expression. (A) ADAMTS9-AS2 was preferentially enriched in Ago2-containing beads compared to beads harboring the IgG antibody, whereas β-actin was not detectably enriched, as analyzed by RIP assay. (B) Schematic diagrams of the mutual interactions between miR-27a-3p and ADAMTS9-AS2. The calculated ΔG values (kcal/mol) are presented. (C) Overexpression of ADAMTS9-AS2 contributed to an obvious reduction of miR-27a-3p expression while knockdown led to an evident increase of miR-27a-3p expression according to qRT-PCR analysis. (D) Overexpression or knockdown of miR-27a-3p had no obvious effect on ADAMTS9-AS2 expression at the mRNA level, according to qRT-PCR analysis. (E) The luciferase reporter gene vector containing ADAMTS9-AS2 3′UTR WT or MUT with miR-27a-3p was respectively transfected into caki-1 cells. MiR-27a-3p notably suppressed the activity of the luciferase reporter harboring full length ADAMTS9-AS2 in the WT group but had no significant effect in the MUT group. (F) A pull-down assay was performed in caki-1 cells transfected by ADAMTS9-AS2 with the presence or absence of miR-27a-3p binding sites. Three independent experiments were performed and data shown are mean ± SD. Statistically significant differences are indicated as *, P<0.05, **, P<0.01; ns, no significance; Student’s t-test among two groups; ANOVA among multiple groups. ADAMTS9-AS2, ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2; miR-27a-3p, microRNA-27a-3p; RIP, RNA immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild type; MUT, mutant type; SD, standard deviation.