The discovery of miRNAs as a novel class of gene expression regulators has provided new strategies for disease diagnostics and therapeutics. Cell cycle, cell proliferation, cell survival and tumorigenesis are all regulated by miRNAs. Altered abundance of cell survival and cell cycle regulation proteins and aberrant expression of miRNAs frequently coexist in human breast cancers . miRNAs are aberrantly expressed or mutated in cancer, acting as a novel class of oncogenes or tumor suppressor genes . In this study, we demonstrated that a set of E2-repressible microRNAs in breast cancer cell lines was associated with altered cell cycle progression and cell proliferation, which could play a causal role for miRNAs in controlling breast tumor growth.
In breast cancer, abnormalities of the cell cycle are frequently observed in response to E2 treatment. Cyclin D1 encodes a key regulator of the cell cycle transition from G1 to S phase and is overexpressed in more than 50% of breast cancers, functioning as a rate-limiting factor for human breast cancer cell proliferation in vivo and in vitro [36, 37]. The bcl-2 protein is associated with the inner mitochondrial membrane and functions to inhibit apoptosis and promote survival [38, 39]. Survivin, a member of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins, regulates the G1 checkpoint and G2/M phase of the cell cycle by associating with mitotic spindle microtubules. Survivin directly inhibits caspase-3 and caspase-7 activity, is inversely correlated with apoptosis and is positively correlated with cell proliferation [40, 41]. We demonstrated that E2 induced upregulation of cyclin D1, bcl-2 and survivin in MCF-7 cells, which played an important role in E2 stimulated cell proliferation and cell cycle transition. This result was consistent with previous studies [7, 9, 10, 23–25]. Similar results were also observed in another estrogen-responsive breast cancer cells T47D, E2 dramatically induced the expression of cyclin D1, bcl-2 and survivin as shown in Additional file 1: Figure S1.
MiRNAs have emerged as a novel regulator of gene expression at the post-transcriptional level by base-pairing interactions between miRNAs and the 3'-UTR of their target mRNAs . In order to explore whether miRNAs participate in the upregulation of bcl-2, cyclin D1 and survivin by E2, we focused on the E2-repressible miRNAs that may play a role in the modulation of cell proliferation and survival genes. Several studies have demonstrated that E2 upregulates or downregulates a variety of miRNAs by miRNA expression profilings in MCF-7 cells [18–20]. The difference of the results may be due to the cell status, treatment and stimulation time. Based on previous studies, we demonstrated miR-16, miR-143 and miR-203 were coordinately suppressed in response to E2 treatment using QPCR quantification. Therefore, we proposed that these miRNAs might be involved in the regulation of cell proliferation and survival by targeting bcl-2, cyclin D1 and survivin at the post-transcriptional level.
Some publications have provided support for our hypothesis, Cimmino et al.  have demonstrated that miR-15a and miR-16 expression is inversely correlated with bcl-2 expression in Chronic lymphocytic leukemia and that both microRNAs negatively regulate bcl-2 at a post-transcriptional level. Bonci et al.  have demonstrated that the miR-15a-miR-16 cluster targets CCND1 (encoding cyclin D1), acting as tumor suppressor genes in prostate cancer by the control of cell survival, proliferation and invasion. miR-143 has been validated to target the oncogene KRAS  and may also modulate extracellular-regulated protein kinase 5 (ERK5) [43, 44] and bcl-2 . In this study, we confirmed the previous studies that miR-16 targets bcl-2 and cyclin D1 and miR-143 targets bcl-2. However, we can also see the impairment of endogenous cyclin D1 by miR-143 as shown in Figure 3A, which may result from the interference of ERK5 expression by miR-143 , since ERK5 has been shown to regulate cyclin D1[45, 46]. TargetScan and miRbase programs predict conserved binding sites of miR-16, miR-143 and miR-203 in 3'-UTR of survivin. We further elucidated that miR-16, miR-143 and miR-203 target survivin at the post-transcriptional level by transfection of miRNA mimics and inhibitors. Luciferase activity assay indicated these miRNA directly regulated the expression of survivin by binding to the 3'-UTR of survivin.
A miRNA regulates a variety of target genes, and a gene is modulated by many miRNAs. Therefore, in response to E2 stimulation, several miRNAs are coordinately suppressed to upregulate the target genes which are involved in cell proliferation and survival. It provides a novel mechanism for regulation of genes containing ERE in the promoters. Previous studies have shown the ERα-mediated transcriptional regulation of bcl-2, cyclin D1 mRNAs by binding to the ERE of target genes. We cannot conclude that the increase of bcl-2, cyclin D1 and survivin is due solely to E2-mediated reduction of miR-16, miR-143 and miR-203, but miRNAs do play a crucial role in the post-transcriptional regulation because when actinomycin D was used to inhibit de novo RNA synthesis, E2 can still enhance the expression of bcl-2, cyclinD1 and survivin, though at a low level (Additional file 2: Figure S2). Further studies will be needed to dissect the relative contributions of ERα-mediated multiple pathways controlling bcl-2, cyclin D1 and survivin expression.
ERα is essential for E2-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ERα-positive breast cancer. Our data indicated that ICI 182, 780 and Raloxifene can abrogate E2 repressed miRNA levels and therefore attenuate the expression of target genes. It is reported that ICI 182, 780 and Raloxifene can locally alter the ERα ligand binding structure via specific hydrophobic residues and decrease its transcriptional activity . In addition, knockdown of the ERα protein also impaired E2 induced upregulation of bcl-2, cyclin D1 and survivin. In ERα negative MDA-MB-231 cells, E2 has no effect on the regulation of miRNAs and target genes as shown in Figure 4E and 4F. In ERα negative non-cancer cells MCF-10A, we also observed similar effect (data not shown). Thus, the regulation is mainly dependent on ERα protein expression and transactivation in both breast cancer cells and in a normal breast cells.
Suzuki et al.  have recently shown that a central tumor suppressor, p53, enhances the post-transcriptional maturation of several miRNAs with growth-suppressive function, including miR-16, miR-143, miR-145 and miR-203 in response to DNA damage. P53 interacts with the Drosha processing complex through the association with the DEAD-box RNA helicase p68 and facilitates the processing of primary miRNAs to precursor miRNAs. In our study, we used MCF-7 cells carrying wild type p53 genes as a model to investigate the E2-repressible miRNAs target genes involved in cell proliferation. However, in p53 mutant ERα-positive T47D cells, we observed similar regulation yet to a weaker extent. Previous studies have elucidated the interaction of ERα and p53 [49, 50]. It is likely that both ERα and p53 participated in the regulation of E2-repressible miRNAs. Further studies are needed to elucidate whether ERα-mediated induction of target genes by E2 via p53-regulated miRNA maturation.
The downregulated miRNA exhibits the growth suppressive effect in response to E2. miR-16 and miR-15 act as tumor suppressors and control cell cycle transition by targeting cyclin D1 and cyclin E [29, 51]. MiR-143 has been claimed to be a anti-oncomir in human colorectal tumors and can increase the sensitivity of chemotherapy [30, 52–54]. MiR-203 has recently been identified to inhibit cell proliferation and invasion in prostate cancer, and reverse chemoresistance in p53-mutated colon cells [55–57]. In this study, overexpression of miR-16, miR-143 and miR-203 remarkably inhibited E2 induced cell proliferation of breast cancer cells. Further studies have shown that these miRNAs were significantly higher in triple positive than in triple negative breast tissues. This suggested a potentially tumor suppressive effect of the miRNAs on cancer progression of ER positive breast cancers, which may open new avenues for therapeutic intervention in breast cancer treatment.