br responsive element dependent on estrogen and estrogen rec
responsive element dependent on estrogen and estrogen receptor . Previously, Ikeda et al. showed TRIM25 mRNA expression in 9 of 15 (60.0%) breast carcinoma tissues by using RNase protection assay, and Takashi et al. reported TRIM25 immunoreactivity in breast carcinoma cells in 22 of 30 (73.3%) cases which was significantly associated with an increased risk of recurrence and adverse clinical outcome of the pa-tients [21,29]. In this study, we observed that TRIM25 was upregulated in BC tissues and cell lines, consistent with previous literature. We showed a coordinated and strong expression of pri-miR-3614 and its host gene TRIM25; however, mature miR-3614-3p was significantly
downregulated in BC cells. Furthermore, treatment with E2 significantly increased pri-miR-3614 and TRIM25 expression in both MCF-7 and MDA-MB-231 cells, consistent with the results that TRIM25 expression can be regulated by factors independent of estrogen or estrogen signal-ing . In contrast, the expression of mature miR-3614-3p was signif-icantly decreased, and TAM blocked the effects of the estrogen-mediated induction. There has a common consensus MDA-MB-231 is a triple-negative breast carcinomas (TNBC) cell line, that lack ERα but express low levels of ERβ . In our other study, we found that MDA-MB-231 cell also express EGFR (data not shown) which is
Fig. 5. TRIM25-depleted interferes with E2-dependent BC cell proliferation. (a, b) qRT–PCR and Western blot were performed to detect the expression level of TRIM25 in MCF-7 and MDA-MB-231 cells after transfection with E2, si-TRIM25, or scrambler RNA. Data are presented as the mean ± SEM. (**P b 0.01, ***P b 0.001, ANOVA analysis). (c, d) The effects of TRIM25 expression on BC cell proliferation was determined by using the MTT assay and colony formation analysis. The absorbance of the plates was read on a microplate reader at a wavelength of 490 nm. Data are presented as the mean ± SEM. (*P b 0.05, **P b 0.01, ***P b 0.001, ANOVA analysis). (e) The Methoxy-X04 distribution was analyzed by flow cytometry after transfection with E2, si-TRIM25, or scrambler RNA. The histogram indicates the percentage of cells in the G0/G1, S, and G2/M cell cycle phases. The representative results are presented as the mean ± SD. Each experiment was repeated at least three times, and each sample was assayed in triplicate. (*P b 0.05, **P b 0.01, ANOVA analysis) (f) The expression of CDK4, CyclinD1, and β-actin analyzed by Western blot after transfection with E2, si-TRIM25, or scrambler RNA.
consistent with Samanta S′ report. Therefore, MDA-MB-231 cells cannot be considered completely triple-negative cell. In addition, estrogen not only activate the classical estrogen receptor, also can be act to RTKs and G protein-coupled estrogen receptor 1 (GPER), through the activa-tion of PI3K/AKT signaling pathways induced the expression of target genes [36,37]. The effects of estrogens binding to these classical estro-gen receptors are delayed. However, estrogens binding to GPER lead
to a fast effect on regulation of gene transcription. GPER is positive in MDA-MB-231 cell. Therefore, these may be the reason why E2 treat-ment MDA-MB-231 cell also increased the expression of TRIM25 and pri-miR-3614.
Thus, an important question was raised: What causes such a discrep-ancy between host gene TRIM25 and mature miR-3614-3p transcript levels in BC cells? Upon examining the TRIM25 mRNA sequence, we
identified putative RBP binding sites, including IGF2BP3-binding motifs. Hanane et al. identified 164 direct mRNA targets of IGF2BP3 in pancre-atic ductal adenocarcinoma cells using iCLIP and RIP analysis. IGF2BP3 may attenuate miRNA-mediated mRNA degradation in association with Ago2 . In this study, we confirmed that TRIM25 is a target gene of IGF2BP3. Using actinomycin D to interrupt TRIM25 transcrip-tion, we determined that IGF2BP3 prolonged the half-life of TRIM25. RIP and Pull-down analysis indicated that IGF2BP3 binds to the 3´-UTR of the TRIM25 stabilized transcript, and blocks the pri-miR-3614 pro-cessing. Next, we checked binding level of pre-miR-3614 and TRIM25 upon silencing of IGF2BP3 by RIP with Ago2, a RISC component. The re-sults suggest IGF2BP3 stabilized TRIM25 mRNA associated with RISC. Thus, here we identified a new mechanism whereby IGF2BP3 influences the miRNA-mRNA interaction by modulating miRNA maturation.
Furthermore, we found that TRIM25 is not only a host gene, but also a target gene of miR-3614 in BC. The overexpression of miR-3614 signif-icantly inhibited the expression of TRIM25 by binding to pri-miR-3614 at the 3′-UTR of TRIM25, suggesting that miR-3614-3p may suppress TRIM25 expression. Moreover, the IGF2BP3-binding sites overlap with the miR-3614-3p-binding site, revealing that IGF2BP3 can inhibit the binding of miR-3614-3p to its target. Thus, IGF2BP3 and miR-3614 com-pete for common binding sites within the 3´-UTR of TRIM25, thereby regulating its translation.