miRNA-34b as a tumor suppressor in estrogen-dependent growth of breast cancer cells

Specimens from 47 breast cancer patients were used for miRNA analysis. The study was approved by the Institutional Review Board of the National Taiwan University Hospital, Taipei, Taiwan. Written informed consent was obtained from all patients. Frozen sections of breast cancer tissue were obtained when patients underwent surgical resection of breast tumors at the National Taiwan University Hospital between December 2003 and December 2006. The 47 clinical samples were grouped according to pathological classification, which included the ER, PR and HER2 status. Of these specimens, 26 samples were ER+ and 21 samples were ER-negative (ER-). The clinical characteristics of the breast cancer patients are listed in Table 1. None of these patients had received adjuvant chemotherapy.

MCF-7 cells were purchased from America Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in 10% fetal bovine serum (FBS) as prescribed. MDA-MB-361, MDA-MB-435, T47D and OVCAR4 cells were purchased from among the NCI-60 cell lines (Developmental Therapeutics Program, National Cancer Institute, Frederick, MD, USA) and cultured in RPMI 1640 with 10% FBS as prescribed. 293T cells were purchased from ATCC and maintained in 10% FBS as prescribed. The ligands 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), diethylstilbestrol (DES) and zeranol (ZEA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Before MCF-7 cells were treated with ligands, the medium was replaced with phenol red free α-MEM (Life Technologies, Rockville, MD, USA) containing 10% dextran charcoal-stripped FBS (Thermo Fisher Scientific Inc, Waltham, MA, USA) for 3 days. Cells were treated with 10 nM E2, 100 nM 4-OHT, 10 nM DES or 10 nM ZEA for 24 hours prior to analyses.

A 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay was performed to determine cell proliferation. Briefly, MCF-7 cells were plated in 96-well plates at a density of 5 × 10³/well. After 24-hour incubation, cells were serum-starved overnight. The cells were then treated with different concentrations of estrogen for indicated times, then culture medium containing 0.5 mg/ml MTT solution was added to each well. After 1.5-hour incubation, the medium was removed and dimethyl sulfoxide was added to the wells. The color intensity of solubilized formazan was measured at 570 nm with an ELISA plate reader (VICTOR X3 Multilabel Plate Reader; PerkinElmer, Waltham, MA, USA).

Six-well plates were used for seeding 100 cells in α-MEM supplemented with 10% FCS. E2 and 4-OHT were added to the plate at different concentrations every 2 days. Plates were incubated in a 37°C incubator for 2 weeks. The number of colonies was counted after they were stained with 0.05% crystal violet (Sigma-Aldrich) for 1 hour and washed extensively with PBS.

The human miRNA TLDA contains eight sample-loading lines, each of which is connected by a microchannel to 48 miniature reaction chambers for a total of 384 wells per card. It contains 365 different human miRNA assays plus two carefully selected small nucleolar RNAs (snRNAs) that function as endogenous controls for data normalization. The human miRNA TLDA was used in conjunction with Multiplex reverse transcriptase (RT; Applied Biosystems) consisting of eight predefined RT primer pools containing up to 48 RT primers each. All 365 miRNA targets were reverse-transcribed in eight separate RT reactions, and each RT reaction was pipetted into one of the eight filling ports on the TaqMan array. To perform PCRs, we used a 7900HT Fast Real-Time PCR System (Applied Biosystems), and SDS version 2.3 software (Applied Biosystems) was used for comparative differential expression (ΔCt) analysis.

Total RNAs were extracted from cells using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA). miRNAs were quantified using TaqMan miRNA assays (Applied Biosystems). U6B was used for normalization of miRNA expression. The PCR was run in a 7900HT Fast Real-Time PCR System, and SDS version 2.3 software (Applied Biosystems) was used for comparative ΔCt analysis. Experiments were performed three times in triplicate.

MCF-7 cells were treated with the chemicals described above. Cells were washed twice with ice-cold PBS and collected for protein extraction. Protein was extracted by using mammalian protein extraction reagent (Pierce Biotechnology, Inc, Rockford, IL, USA) containing protease inhibitors (Sigma-Aldrich). SDS-PAGE with 10% resolving gel was used to separate proteins (25 mg/lane). Mouse anti-cyclin D1 mAb (Merck KGaA, Darmstadt, Germany), mouse anti-Jagged-1 (anti-JAG1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-β-actin mAb (Santa Cruz Biotechnology) were all used according to the manufacturer's instructions. Bound antibodies were detected using an enhanced chemiluminescence system (Santa Cruz Biotechnology). Chemiluminescent signals were captured using the Fujifilm LAS-3000 system (Fujifilm, Tokyo, Japan). All experiments were performed at least three times in triplicate.

We used the pSilencer 4.1-CMV puro vector (Applied Biosystems), the pTRE-Tight vector (631059; Clontech Laboratories, Inc, Mountain View, CA, USA) and the Tet-On vector (Clontech Laboratories, Inc) in the plasmid constructs. A precursor form of miR-34b was inserted into the pSilencer 4.1-CMV puro vector, then subcloned into the pTRE-Tight vector for overexpression. Primer sequences are shown in Additional file 1, Supplementary Table 1.

MCF-7 cells were plated in six-well plates at a density of 5 × 10⁵ cells per well in Opti-MEM Reduced Serum Medium (Life Technologies) supplemented with 10% FBS. Lipofectamine 2000 transfection reagent (Invitrogen/Life Technologies) and pTRE-miR-34b and Tet-On plasmids were mixed according to the manufacturer's instructions and added to the cells. After 6 hours of incubation, the medium was changed with fresh α-MEM plus 10% FBS. After cotransfection of the two plasmids, stable clones were selected by addition of neomycin (1 mg/μl). Precursor miRNAs, antagomirs and negative controls (mock transfection) were obtained from Applied Biosystems. For precursor miRNA, cells were transfected with Lipofectamine LTX (Invitrogen/Life Technologies) with 6 nM precursor miRNA and harvested 48 or 72 hours later.

Genomic DNA was extracted from MCF-7 cells using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA). Genomic DNA (2 μg) was modified by sodium bisulfite using the EZ DNA Methylation Kit (Zymed Research Corp, Irvine, CA, USA). Methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) analysis were then performed as described previously [18]. Primers for MSP and BSP were designed by using the MethPrimer software program [19]. Primer sequences are shown in Additional file 1, Supplementary Table 1.

Upstream regions of miR-34b/c were amplified by PCR and then cloned into the pGEM-T Easy Vector System (Promega, Madison, WI, USA). Each PCR primer carried a 5' overhang that contained a HindIII recognition site. After verification of the sequences, miR-34b/c fragments were excised using HindIII and ligated into a pGL3-Basic vector (Promega). In addition, oligonucleotide fragments corresponding to p53-responsive elements were synthesized and inserted upstream of the miR-34b/c promoter in the pGL3-Basic vector. Cells (5 × 10⁴ per well in 24-well plates) were transfected with 100 ng of one of the reporter plasmids using Lipofectamine 2000 reagent (Invitrogen/Life Technologies). For cotransfection of reporter genes with ER expression plasmid, equal numbers of cells were cotransfected with 100 ng of one of the reporter plasmids, 100 ng of pcDNA 3.1-ER (Invitrogen/Life Technologies) or an empty vector. The pGL3-Basic vector without an insert served as the negative control. Luciferase activity was measured 48 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega). Primer sequences are shown in Additional file 1, Supplementary Table 1.

The 3'UTRs of human JAG1 and cyclin D1 were amplified from human genomic DNA and cloned into a pMIR-REPORT miRNA Expression Reporter Vector System (Ambion/Life Technologies) by directional cloning. The resultant plasmid was designated Luc-JAG1-3'UTR-WT and Luc-CyclinD1-3'UTR-WT. To generate the Luc-JAG1-3'UTR-Mut and Luc-CyclinD1-3'UTR-Mut construct, seed regions were mutated by removing complementary nucleotides of miR-34b. 293T cells (3 × 10⁵ per well) were cotransfected with a 0.5-μg firefly luciferase reporter vector and a 0.05-μg control vector containing Renilla luciferase, pRL-SV40 (Promega), using Lipofectamine 2000 reagent (Invitrogen/Life Technologies) in six-well Costar plates (Corning Life Sciences, Lowell, MA, USA). For each well, 200 pM miR-34b precursor molecule (Ambion/Life Technologies) or a negative control (mock) precursor miRNA (Ambion/Life Technologies) was cotransfected with the reporter constructs. Luciferase assays were performed 24 hours after transfection (Dual-Luciferase Reporter Assay System; Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.

The chromatin immunoprecipitation (ChIP) assays were carried out as described previously [20]. Chromatin was immunoprecipitated for 16 hours at 4°C using 10 μl each of anti-RNA polymerase II, clone CTD4H8 (Millipore, Billerica, MA, USA), normal mouse immunoglobulin G (IgG; Millipore) and anti-ERα, clone HC-20 (Santa Cruz Biotechnology). In addition, 1/100 of the solution collected before adding the antibody was used as an internal control for the amount of input DNA. PCR was carried out in a 20-μl volume containing 1/100 of the immunoprecipitated DNA, blend Taq polymerase and 5 μM of each primer. The PCR protocol entailed initial denaturation for 3 minutes at 94°C and 33 cycles of 20 seconds at 94°C, 30 seconds at 59°C and 30 seconds at 72°C, followed by a final extension at 72°C for 2 minutes. Primer sequences are shown in Additional file 1, Supplementary Table 1.

This study was approved by the Institutional Review Board and the Animal Care and Use Committee at National Taiwan University. The survival rate of transfected MCF-7 cells was determined by counting cell numbers using trypan blue. Transfected MCF-7 cells (1 × 10⁶ live cells in 100 μl Hank's balanced salt solution) were injected into the mammary fat pads of 5-week-old NOD SCID mice (supplied by the animal center at the College of Medicine, National Taiwan University). To determine whether miR-34b could reduce orthotopic tumor growth, the mice were randomized into two groups (n = 10 for each group): (1) miR-34b Tet-On overexpression stable clone without doxycycline (Sigma-Aldrich) or (2) miR-34b Tet-On overexpression clone with doxycycline. Mice were injected with doxycycline (2 mg/ml) in water [21, 22] after tumor size reached 5 mm³. Tumor sizes were monitored every 7 days by using electronic vernier calipers, and the tumor volume (V) was calculated by using the formula V = 0.4 × ab², where a and b are the longest and shortest diameters of each tumor, respectively.

We performed immunohistochemistry by using 5-μm formalin-fixed, paraffin-embedded tissue sections, placing them on silane-coated slides and baking them at 70°C overnight. Afterward the slides were deparaffinized, hydrated, placed in 10 mM citrate buffer (pH 6) and microwaved for a total of 20 minutes for antigen retrieval. After cooling, the slides were treated with 3% H₂O₂ for 15 minutes. Appropriate blocking (3% BSA and 0.2% Triton X-100) for 1 hour and anti-cyclin D1, clone H-295 (Santa Cruz Biotechnology) rabbit pAb was used. Rabbit IgG (Millipore) was used as a negative control. We used the VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) for detection. Chromagen diaminobenzidine (Vector Laboratories) was used, and sections were counterstained with H & E.

To investigate whether estrogen regulates miRNA expression, the ER+ human breast cancer cell line MCF-7 was treated with 10 nM E2 for 24 hours, and changes in the miRNA expression profile were analyzed by performing qPCR-based TLDA miRNA assays. Table 2 lists the 26 miRNAs that were significantly downregulated and the 28 miRNAs that were significantly upregulated (greater than fivefold changes) by E2 treatment of MCF-7 cells. Among these estrogen-regulated miRNAs, miR-34b has been shown to regulate tumor progression in a variety of cancers [15‐17, 23‐25]. Because E2 markedly decreased miR-34b expression levels (72-fold), miR-34b was chosen for further study.

We next examined the expression levels of miR-34b in 47 human breast cancer patients (Table 2). We found that the ER+ tissues had lower miR-34b expression levels compared to ER- tissues (ER+: n = 26 vs ER-: n = 21; P < 0.05) (Figure 1A). Moreover, the correlation between miR-34b and ER expression levels were inversely correlated (Pearson coefficient = -0.4185, P = 0.0019) for ER+ patients (Figure 1B). However, the correlation of miR-34b and ER showed no significance for ER- patients (Pearson coefficient = 0.009, P = 0.9679) (Figure 1C). These data suggest that miR-34b expression is closely linked to ERα expression levels in ER+ human breast tumors involved in the complexity of in vivo systems.

To verify that miR-34b expression is linked to tumor growth, MCF-7 cells were treated with different concentrations of E2 (0.1, 1.0, 10 and 100 nM), and cell proliferation rates were determined. E2 increased anchorage-dependent growth in a concentration-dependent manner (Figure 2A), but the expression levels of miR-34b decreased concentration-dependently. E2 (10 nM) treatment reduced miR-34b expression by 88% in MCF-7 cells (Figure 2B). The inhibitory effect of estrogen on miR-34b expression was apparent within 2 hours and was sustained for at least 24 hours in MCF-7 cells (Figure 2B). Transfection of precursor miR-34b or antagomir-34b also showed significant decreases or increases in cell proliferation (Figure 3A). These data suggest that miR-34b may serve as a tumor suppressor that regulates tumor progression, as has been reported in a variety of cancers [15‐17, 23‐25].

Previous studies have shown that high expression of cyclin D1 and Jagged-1 correlate with poor prognosis in breast cancer [26‐28]. According to miRNA target gene prediction software, including PicTar, miRanda and miRNAMap, miR-34 could target key regulators of cell proliferation, including cyclin D1 and JAG1 (Figure 3B). To investigate the possible link between miR-34b and these possible downstream targets, the protein expression levels of cyclin D1 and JAG1 were examined. As shown in Figure 2C, both cyclin D1 and JAG1 were upregulated by treatment with E2 in a concentration- and time-dependent manner. Moreover, overexpression of precursor miR-34b leads to downregulation of JAG1 and cyclin D1 (Figure 3C).

To further confirm the interaction between miR-34b and its putative target genes, we cloned the JAG1 and cyclin D1 3'UTR sequence and inserted it downstream of the firefly luciferase coding region of the pMIR-REPORT vector (Figures 3D and 3E). Mutants with the putative binding sites were prepared as described above (see Materials and methods). Our results show that the luciferase activity of the wild-type JAG1 and cyclin D1 3'UTR construct was significantly inhibited after the introduction of miR-34b into 293T cells, but not by the negative control. Mutations of the 3'UTR-binding sites completely abolished the ability of miR-34b to regulate luciferase expression (Figures 3D and 3E). These results demonstrate that JAG1 and cyclin D1 are potential targets of miR-34b. Furthermore, these data suggest that miR-34b may suppress tumor growth through the suppression of cyclin D1 and JAG1 expression.

Since we observed that miR-34b was downregulated in ER+ breast cancer cells (MCF-7 cells), we speculated that overexpression of miR-34b might inhibit cell proliferation. To test this hypothesis, we established a miR-34b Tet-On overexpression system. In this system, miR-34b is conditionally turned on by doxycycline treatment (Figure 2D). Addition of doxycycline significantly decreased cell proliferation (Figure 2E) and cyclin D1 expression (Figure 2F) in MCF-7/miR-34b Tet-On stable clones. Doxycycline did not exert significant effects on cell growth and cyclin D expression in parental and mock transfection of MCF-7 cells (Additional file 2, Figure S1). To test whether miR-34b suppresses tumor growth in vivo, 1 × 10⁶ MCF-7/miR-34b Tet-On stable clones were injected into the mammary fat pads of female NOD SCID mice. Tumor size increased significantly with time. Doxycycline was applied to induce miR-34b expression after tumor size reached 5 mm³. Seven days after induction, the induced miR-34b group showed significant reduction in tumor size compared to the control group (P < 0.05) (Figure 4A). After 28 days, the mice were killed and the tumor volumes were compared. As shown in Figure 4B, the tumor volumes in the induced miR-34b group were significantly lower than in the control group (P < 0.01). H & E staining of the fat pads confirmed that miR-34b induction led to a decrease in tumor size (Figure 4C). The miR-34b expression level in Tet-On group was increased 2.76-fold compared to the noninduced miR-34b group (Figure 4D). In agreement with these findings, the expression levels of cyclin D1 were significantly reduced in the doxycycline induced group as shown by immunohistochemical staining (Figure 4E).

To investigate how E2 regulates miR-34b, we screened the miR-34b promoter for the presence of ER-responsive elements. However, we could not find any possible binding sites of ER in the miR-34b promoter. This result showed that E2 regulation of miR-34b does not occur through classical ER signaling. Recent studies showed that p53 could bind to miR-34b/c and regulate its expression [16]. Furthermore, the interaction between p53 and ER at the promoter site plays a pivotal role in the regulation of miR-34b expression [29‐31].

We first demonstrated that miR-34b promoter activity was significantly inhibited by 10 nM E2 treatment of 293T cells cotransfected with ER (Figure 5A). In additional experiments, we used breast cancer cell lines bearing different p53 and ER status (MCF-7, T47D and MDA-MB-435). Only cells expressing both wild-type p53 and ERα responded to estrogen repression of miR-34b (Figure 5B). These data suggest that both ER and p53 activity are required for E2 to repress miR-34b. We next examined whether both proteins bind to the two p53 binding sites in the miR-34b promoter. ChIP assays confirmed that both p53 and ER bind to the miR-34b/c promoter in the absence of E2 (Figure 5C). Binding of p53 to the two p53RE binding sites of the miR-34b promoter region were disrupted by treatment of cells with E2 (Figure 5C). These data support the notion that E2's binding to ER may affect p53's binding to miR-34b promoter through p53-ER interaction.

Previously presented evidence indicated that miR-34b/c expression may be regulated by DNA methylation [18]. To investigate whether E2 could regulate miR-34b/c through DNA methylation, we examined the methylation status of the miR-34b/c promoter. Treatment of MCF-7 cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), increased the expression level of miR-34b (Figure 5D) and reduced the methylation status in the CpG islands of the miR-34b promoter region as demonstrated by MSP and BSP (Figure 5E). However, treatment of cells with E2 did not cause significant differences in DNA methylation (Figure 5F). These data suggest that E2 did not regulate miR-34b/c expression through DNA methylation in MCF-7 cells.

Some xenoestrogens may play a causative role in breast cancer tumorigenesis [32‐35]. To investigate whether xenoestrogen could promote cancer progression by regulating miR-34b (similarly to E2), we examined miR-34b and its downstream target's expression level after xenoestrogen treatment. Our results show that the xenoestrogens DES and ZEA downregulate miR-34b expression in MCF-7 cells (Figure 6A). TAM, a SERM that exhibits antiestrogenic effects in breast tissue, did not affect miR-34b expression levels in MCF-7 cells (Figure 6A). In agreement with this finding, the protein levels of cyclin D1 and JAG1 were upregulated by treatment with DES and ZEA, whereas TAM had no significant effect (Figures 6B and 6C). Importantly, combined estrogen and TAM treatment reversed the effect of estrogen on miR-34b (Figures 6D and 6E).

To investigate whether the regulation of miR-34b expression is tissue-specific, we studied human OVCAR4 ovarian cancer cells and primary culture of mouse endometrial cells. Both of these cells are p53+/+ and E2-responsive. As shown in Figure 6F, E2 downregulated miR-34b/c expression in both OVCAR4 and endometrial cells. These data suggest that the E2 regulation of miR-34b/c maybe a universal event and might also contribute to the proliferation potential in these tissues.

To evaluate this possibility by using miR-34b/c as potential therapeutic targets, we obtained four breast cancer cell lines with different ER and p53 status and overexpressed miR-34b/c. Among the four cell lines, MCF-7 expresses ER+ and wild-type p53 (ER+/p53^wt), both T47D and MDA-MB-361 are ER+ and p53-mutant (ER+/p53^mut), and MDA-MB-435 is ER- and p53-mutant (ER-/p53^mut). As shown in Figure 7, despite the different status of ER and p53, all four cell lines showed a significant decrease in growth after 48 hours with the expression of miR-34b/c. This result demonstrates that the induction of miR-34b/c could suppress cell growth and that the effect was not influenced by ER or p53 status.