Figure 5.

Mutant GAPDH 3′UTR regulates miR-125b levels in an Ago2-dependent manner. (A) RNA immunoprecipitation (RIP) assays were performed and the relative enrichment of the mutant (MUT) GAPDH 3′UTR RNA and miR-125b were determined using RT-qPCR in the Ago2 and IgG groups. (B) RT-qPCR and (C) western blot analysis were used to assess the efficiency of Ago2 siRNA; GUSB and β-actin were used as the internal controls, respectively. Ago2-siRNA-1 was the most effective siRNA and thus used for subsequent experiments. (D) Assessment of the stability of miR-125b in SKOV3 cells transfected with the mutant GAPDH 3′UTR construct and Ago2 siRNA-1; scramble NC was used as the negative control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; UTR, untranslated region; NC, negative control; Ago2, argonaute RISC catalytic component 2. *P<0.05, **P<0.01, ***P<0.001.