Fig. 3.

IGF2BP3 binds to TRIM25 mRNA and inhibits miR-3614 maturation in BC cells. (a) Proposed model of IGF2BP3, HuR and miR-3614 binding sites at the TRIM25 mRNA 3′-UTR. (b–e) The levels of IGF2BP3 mRNA, TRIM25 mRNA, pri-miR-3614 and miR-3614-3p were determined by qRT–PCR after BC cells transfection with si-IGF2BP3 and related controls. Data are presented as the mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA analysis). (f) Forty-eight hours after transfection with si-IGF2BP3 and treatment with E2 (100 nM), the levels of TRIM25, IGF2BP3, and β-actin (loading control) were assessed by Western blot analysis. (g) qRT–PCR analysis of TRIM25 mRNA stability in control or IGF2BP3-depleted BC cells. Data are presented as the mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test) (h, i) RNA immunoprecipitation (RIP) combined with qRT–PCR assays of IGF2BP3 binding to TRIM25 mRNA in BC cells. Nonspecific rabbit IgG was used as a negative control. Input was used as a positive control. Western blot analysis of IGF2BP3 immunoprecipitated from control or IGF2BP3-overexpressed in MCF-7 cells or IGF2BP3-depleted in MDA-MB-231 cells (H). RT–PCR analysis of RNA precipitated with IGF2BP3 antibody or control IgG (I). (j) Schematic depiction of the TRIM25 (A-D) biotinylated probes used for biotin pull-down analysis. (k) IGF2BP3 in the biotin pull-down samples was detected by Western blot analysis. (l) Western blot of TRIM25 and AGO2 expression in control or IGF2BP3-depleted (sh-IGF2BP3) MDA-MB-231 cells. (m-o) qRT-PCR analysis of RNA precipitated with a-Ago2 (Ago2 RIP) or nonspecific rabbit IgG (IgG RIP). Relative quantification for TRIM25 and IGF2BP2 transcript was performed using β-actin mRNA as a reference gene. Relative quantification for pri-miR-3614 and miR-3614-3p were performed using U6 as a reference gene. Statistical significance was estimated for each comparison using an unpaired t-test (***P < 0.001).