Blood samples were obtained from 30 healthy controls and 30 breast cancer patients at Zhejiang Cancer Hospital (Hangzhou, China). The use of the blood for the present study was approved by the Hospital's Protection of Human Subjects Committee. MCF-10A, T-47D, MCF-7, MDA-MB-231 and MDA-MB-435 cell lines were provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Science (Shanghai, China). The breast cancer cell lines (including T-47D, MCF-7, MDA-MB-231 and MDA-MB-435) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (both from Gibco, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin sulfate. The MCF-10A cell line was cultured in DMEM/F12 media supplemented with 5% horse serum, 10 µg/ml insulin (all from Gibco), 100 ng/ml cholera toxin, 20 ng/ml EGF and 0.5 µg/ml hydrocortisone (all from Sigma-Aldrich, USA) at 37°C in a humidified incubator with 5% CO₂. To study the role of miR-181b in chemoresistance, we established a doxorubicin-resistant T-47D cell line (T-47D-R) by stepwise exposure of T-47D cells to increasing concentrations of DOX (Sigma-Aldrich). Briefly, the T-47D cells were initially cultured with 0.1 µg/ml DOX for 8 weeks, and then the DOX concentration was increased every 4 weeks by 0.02 µg/ml up to a final concentration of 0.3 µg/ml. The T-47D-R cells were exposed to DOX over a time period of 12 months. Before the following experiments were performed, the T-47D-R cells were cultured in DOX-free DMEM for 2 weeks.

Human miR-181b mimics, 2′-omethyl modified miR-181b inhibitors, negative control oligonucle-otides (NCO) and Bim siRNA were purchased from GenePharma Co. (China). The sequences of RNAs were as follows: miR-181b mimics, 5′-AACAUUCAUUGCUGUCGGUGGGU-3′; miR-181b inhibitors; 5′-ACCCACCGACAGCAAUGAAUGUU-3′; NCO, 5′-AUCCCAUGGUGGGUUACAUGGUU-3′; and Bim siRNA, 5′-GACCGAGAAGGUAGACAAUUU-3′. The RNAs were transfected into cells with Lipofectamine 2000 (Invitrogen, USA) at the final concentration of 50 nM according to the manufacturer's protocols.

The expression levels of miR-181b and Bim were measured by quantitative RT-PCR (qRT-PCR), using TaqMan MicroRNA assays kit and supplies (Applied Biosystems, USA) according to the manufacturer's instructions. The expression of miR-181b was normalized to U6 snRNA, and the Bim level was normalized to GAPDH. Relative quantities of miR-181b and Bim were calculated using the 2^−ΔΔCt method (18).

The Bim 3′-UTR was cloned into the pMIR-REPORT™ miRNA Expression Reporter Vector (Life Technologies, USA) to generate the wild-type constructs. The mutant plasmid was created by mutating the seed regions of the miR-181b-binding sites (UGAAUGU to UGAUAGU) using the Site-Directed Mutagenesis kit (Takara, Japan) based on the wild-type constructs. Then, the Dual-Luciferase Reporter assay (Promega, Madison, WI, USA) was conducted to investigate the interaction between miR-181b and its predicted target gene Bim. Briefly, the T-47D-R cells were co-transfected with the miR-181b mimics or NCO and Bim 3′UTR or Bim 3′UTR-mutant. Forty-eight hours later, the firefly and Renilla luciferase activities were detected according to the manufacturer's protocol. The firefly luciferase activity was normalized to the Renilla luciferase activity.

The whole cells were lysed with RIPA buffer (Cell Signaling Technology, USA) containing 2 mM of phenylmethanesulfonyl fluoride. The samples were then subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies at 4°C overnight, and were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 h. Blots were developed using an enhanced chemiluminescence detection kit (Pierce, USA). The primary antibodies against Bim, caspase-3, caspase-9, PARP and β-actin were purchased from Cell Signaling Technology.

Breast cancer cells were transfected with miR-181b mimics or miR-181b inhibitors at a final concentration of 50 µM. Forty-eight hours after transfection, the medium was replaced with fresh medium containing DOX at different concentrations. After culturing for 48 h, the cells were subjected to cell viability assay. DOX concentrations leading to 50% cell death (IC₅₀) were calculated by the viability curve determined by the MTT assay. To determine cell proliferation, the ³H thymidine incorporation assay was used to determine the cell proliferation during the last 6 h of incubation as previously described (19).

Breast cancer cells were trans-fected with miR-181b mimics or inhibitors for 48 h. Before being seeded, the undersurface of the upper chamber of the Transwell was coated with collagen I overnight at 4°C, and then 1×10⁵ cells were seeded in serum-free media on the upper chambers of Transwells with a porous transparent polyethylene terephthalate membrane having a pore size of 8-micron (Corning Costar Corporation, USA), and hydroxyurea (Sigma-Aldrich) was added to stop cellular proliferation. After 24 h, cells on the undersurface of the upper units were fixed, stained and then counted under a phase-contrast microscope.

Apoptosis was assessed using the Annexin V-FITC apoptosis detection kit (Sigma-Aldrich) according to the manufacturer's protocol. After transfection and DOX treatment, T-47D-R cells were harvested and rinsed in cold phosphate-buffered saline (PBS), followed by resuspension in 1X Annexin binding buffer at 1×10⁶ cells/ml. Annexin V (5 µl) and 0.1 µg of propidium iodide (PI) were then added to the cells. Samples were incubated at room temperature for 15 min in the dark and were analyzed using flow cytometry (Becton-Dickinson, USA). ΔΨ_m was detected using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbo-cyanine iodide (JC-1; Molecular Probes, USA) as an indicator (20). After treatment, the T-47D-R cells were collected and resuspended with PBS containing JC-1 at a final concentration of 5 µM. Following a 20-min incubation period at 37°C in the dark, MMP was determined by flow cytometric analysis.

Data are represented as mean ± SE of three independent experiments. The Student's t-test was conducted with SPSS 14.0 software to assess the statistical significance between treatments. p<0.05 was considered to indicate a statistically significant result.

The expression of miR-181b was analyzed in the serum of breast cancer patients and the healthy controls using RT-qPCR. miR-181b was significantly upregulated in the cancer patient serum compared with that in the normal controls (Fig. 1A). Furthermore, we found that the expression level of miR-181b was significantly higher in all of the four breast cancer cell lines compared with the level in the normal breast cell line MCF-10A (21) (Fig. 1B). These results suggest that miR-181b may function as a tumor promoter in breast cancer.

To study the role of miR-181b in the tumor progression of breast cancer cells, miR-181b mimics or inhibitors were transfected to change the level of miR-181b in the T-47D and MCF-7 cells. Briefly, the miR-181b level was increased ~10.22-fold after the miR-181b mimics were introduced, and the miR-181b level was decreased ~4.27-fold after the miR-181b inhibitors were introduced (data not shown). Results of ³H thymidine incorporation assays demonstrated that the proliferation of both T-47D and MCF-7 cells was significantly enhanced in the miR-181b-overexpressing group compared with the control group. In contrast, the proliferation was significantly impaired in the miR-181b-knockdown cells (Fig. 2A). Furthermore, we evaluated the effect of miR-181b on cell migration using a Transwell system. We observed that the overexpression of miR-181b significantly promoted the migration in breast cancer cells, which could be obviously inhibited by knockdown of miR-181b (Fig. 2B). These results suggest that miR-181b may function as a novel oncogene in breast cancer.

To investigate whether miR-181b can modulate the sensitivity of breast cancer cells to DOX which is a potent anticancer drug, we stepwisely exposed the T-47D cells to increasing concentrations of DOX to establish a DOX-resistant T-47D cell line (T-47D-R). As shown in Fig. 3A, the expression level of miR-181b in the T-47D-R cells was significantly upregulated compared with that in the parental T-47D cells, suggesting that miR-181b promotes the chemoresistance in breast cancer. To reveal the effects of miR-181b on the efficacy of chemotherapy, T-47D cells were simultaneously treated with miR-181b and DOX. It was found that the miR-181b mimics significantly increased the IC₅₀ of DOX, which was obviously decreased by the transfection of miR-181b inhibitors compared with the controls (Fig. 3B). This indicated that the downregulation of miR-181b sensitized breast cancer cells to DOX resistance. Furthermore, knockdown of miR-181b re-sensitized the T-47D-R (DOX-resistant T-47D) cells to DOX, although the antitumor effect of DOX was not influenced by the transfection of miR-181b mimics (Fig. 3C). Taken together, these results indicated that miR-181b may play an important role in chemoresistance in breast cancer, and that miR-181b silencing reverses the resistance to DOX treatment.

In order to understand the underlying mechanism for the promotion of miR-181b to DOX resistance, we used TargetScan database (http://www.targetscan.org/) and found that Bim was a putative target of miR-181b. Notably, the position 474–480 of Bim 3′-UTR was observed to be a complementary site (5′-…..UGAAUGU….-3′) for the seed region of miR-181b. Furthermore, we observed that the expression level of Bim was significantly lower in the T-47D-R cells compared with the parental T-47D cells (Fig. 4A). Therefore, we inferred that the T-47D cells became DOX resistant by downregulating Bim expression, which is a target of miR-181b. To confirm this speculation, T-47D-R cells were transfected with miR-181b mimics, miR-181b inhibitors or NCO to alter the miR-181b level. As expected, a pronounced decrease in both Bim mRNA and protein levels was observed in the T-47D-R cells by transfection of the miR-181b mimics. On the contrary, the level of Bim was significantly upregulated when the miR-181b inhibitors were introduced into the T-47D-R cells (Fig. 4B). To further investigate whether Bim is directly targeted by miR-181b, a luciferase reporter vector was constructed, containing the putative miR-181b binding sites within the Bim 3′-UTR. The results showed that the relative luciferase activity of the reporter which contained wild-type 3′UTR of Bim was significantly inhibited in the miR-181b group compared with the control group. However, the mutations in the miR-181b binding site from the Bim 3′-UTR abolished this effect (Fig. 4C). Taken together, these results suggested that the expression of Bim was negatively regulated by miR-181b, which may play an essential role in the DOX resistance of breast cancer cells.

Our preceding results showed that miR-181b inhibitors reversed the DOX resistance in T-47D-R cells and that the Bim gene was the direct target of miR-181b. We, therefore, investigated the pathway and the relationship between Bim regulation and the antitumor effect of the treatment with DOX plus miR-181b inhibitors in T-47D-R cells. As Bim is the key member of the pro-apoptotic Bcl-2 family proteins (22), we found that the downregulation of miR-181b by its specific inhibitors significantly increased the apoptotic rate of the T-47D-R cells treated with DOX, and this synergistic effect of miR-181b inhibitors was abolished when Bim siRNA (after transfecion, the expression of Bim was reduced ~5.12-fold, data not shown) was co-transfected (Fig. 5A). The results indicated that miR-181b regulated the DOX resistance via targeting the Bim gene. We next observed that although miR-181b inhibitors alone did not influence the MMP of the T-47D-R cells, they significantly promoted DOX to damage the mitochondria of the T-47D-R cells. Notably, the mitochondrial dysfunction caused by miR-181b inhibitors plus DOX was inhibited by Bim siRNA (Fig. 5B). Since a previous study indicated that intrinsic apoptosis is activated by mitochondrial dysfunction, mitochondrial-derived apoptogenic proteins are then released from mitochondria, leading to the activation of caspase-9 which finally activates caspase-3 (23). Our results demonstrated that the apoptosis induced by miR-181b inhibitors plus DOX was caspase-dependent (Fig. 5C). Finally, using MTT assay, we indicated that both the Bim siRNA and zVAD-fmk significantly inhibited the cell death induced by DOX combined with the miR-181b inhibitors in the T-47D-R cells (Fig. 5D). Our data strongly suggest the important role of the Bim pathway in reversing the DOX resistance in breast cancer.