br host gene but also
host gene, but also a target gene of miR-3614-3p, and that IGF2BP3 competes with the miR-3614 2-Deoxy-D-glucose within the 3′-UTR of TRIM25 mRNA and prevents miR-3614-mediated translation silencing.
Previous studies have shown that E2-induced upregulation of TRIM25 expression significantly promotes BC cell proliferation . As expected, knock-down of TRIM25 in BC cells markedly decreased cell vi-ability and colony formation (Fig. 5a–d), and led to a decrease in num-ber of cells in the G1 phase, owing to the decreased levels of Cyclin D1 and CDK4 (Fig. 5e and f). These data suggested that TRIM25 can act as oncogene to promote BC cell proliferation and G1-S cell cycle transition via regulation of Cyclin D1 and CDK4.
Meanwhile, the overexpression of miR-3614 inhibited E2-induced TRIM25 expression at both the mRNA and protein levels (Fig. 3c and d), and impaired cell growth and colony formation, whereas miR-3614–3p inhibitor moderately increased BC cell growth, perhaps due to the low expression levels of miR-3614-3p in MCF-7 and MDA-MB-
231 cells (Fig. 6a and b). Moreover, the overexpression of miR-3614 led to an accumulation of BC cells in the G1-phase compared to the neg-ative control, and induced G1-phase arrest via Cyclin D1 and CDK4 sig-naling pathways. However, the inhibition of miR-3614-3p promoted the transition from the G1 to the S phase (Fig. 6c and d). Additionally, xenograft assays showed that tumor nodules derived from miR-3614-overexpressing cells grew slower than the control cells. The average vol-ume of the miR-3614-treated tumors was markedly less than that of the controls (Fig. 6e). These results demonstrate that miR-3614-3p inhibits cell growth by inducing cell cycle arrest in BC cells, and may act as a tumor suppressor in BC. The overexpression of miR-3614 caused a sim-ilar effect on BC cell growth with silencing of TRIM25. We further examined the functional effect of IGF2BP3 on BC cells. First, we assessed the level of IGF2BP3 in BC tissues and non-tumorous
tissues by qRT-PCR and IHC. As shown in Fig. S5A and B, significantly higher levels of IGF2BP3 were observed in BC tissues compared to that of non-tumor tissues (p b 0.05, n = 30). Next, we used lentivirus to gen-erate IGF2BP3-depleted cell lines (LV-si-IGF2BP3). The expression levels of IGF2BP3 and TRIM25 were markedly reduced in the IGF2BP3-depleted cells (Fig. S6). Moreover, IGF2BP3 silencing inhibited growth and colony formation in MCF-7 and MDA-MB-231 cells (Fig. 7a and b). In vivo, xenografts infected with LV-si-IGF2BP3 grew slower than the controls (Fig. 7c). We also found that BC tumors with IGF2BP3-depleted cells expressed less TRIM25 at the protein level (Fig. 7d). Im-portantly, co-treatment with miR-3614-3p mimics, siIGF2BP3, and TAM synergized to markedly reduce the proliferation of BC cells
(Fig. 7e). Collectively, these findings indicate that the TRIM25-miR-3614-IGF2BP3 axis plays an important role in regulating the growth of BC cells.
Multiple examples of cooperative and competitive interplay be-tween RBPs and miRNAs in the regulation of gene expression have been previously described [30–33]. In this study, we identified a novel mechanism of mRNA translational regulation mediated by an antago-nistic interplay between a microRNA and an RBP. We found that intra-genic miR-3614 inhibits the expression of its host gene TRIM25 by binding to its 3′-UTR, and that IGF2BP3 can competitively occupy this binding site to inhibit miR-3614 maturation, thereby protecting TRIM25 mRNA from miR-3614-mediated degradation.
TRIM25, also known as estrogen-responsive finger protein, is upreg-ulated in response to estrogen. The TRIM25 mRNA was expressed in human breast tumors and the estrogen-induced expression of the TRIM25 was found in MCF-7 human breast cancer cells. Moreover, TRIM25 promoter activity was enhanced through the estrogen-