Fig. 4.

The host gene TRIM25 is a direct target of miR-3614-3p. (a) miR-3614-3p binding sites within the 3′-UTR of TRIM25. (b) Dual-luciferase reporter assays showing repression of the wild-type TRIM25 3′-UTR by miR-3614-3p in HEK293T cells. Data are shown as mean ± SEM (*P < 0.05, Student's t-test) (c, d) The expression of TRIM25 was assayed by qRT-PCR and Western blot after overexpression of pre-miR-3614 by transfection with pre-miR-3614-pcDNATM6.2-GW/EmGFP or inhibition of pre-miR-3614 by transfection with antisense oligonucleotides of miR-3614 (anti-miR-3614) in MCF-7 and MDA-MB-231 cells. Data are presented as the mean ± SEM. (**P < 0.01, ***P < 0.001, ANOVA analysis). (e, f) qRT-PCR and Western blot experiments were performed to measure the TRIM25 expression in BC cells treated with pre-miR-3614/anti-miR-3614-3p plus E2 or related negative controls. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, Student's t-test). (g) Western blot analysis of protein expression of TRIM25 and IGF2BP3 in BC cells after co-transfection with siIGF2BP3, pre-miR-3614-3p, and E2. (h) IHC staining of TRIM25 (left) and IGF2BP3 (right) in tumor tissues from mice implanted with LV-miR-3614 or LV-Ctrl BC cells. Magnification ×40. Scale bar, 50 μm.