Tumor samples were collected from female patients with BC (age range, 20–65) undergoing breast biopsy or breast mass resection at Lianyungang Clinical College of Nanjing Medical University (Lianyungang, China) between March 2016 and March 2018. The study protocol was approved by the Ethics Committee of Nanjing Medical University, and all patients involved provided written informed consent. Resistance to ADM was defined as no response (tumor size unchanged or increased) to treatment, or relapse within six months of discontinuing treatment as adjuvant therapy (21). In total, 40 cases of BC (20 with ADM resistance and 20 without) were included in the study. Patient clinical data, including pathological grades and stages are presented in Table I. Tumor samples were collected before chemotherapy and divided into resistant and sensitive tumor groups according to the response to chemotherapy. The samples were stored in liquid nitrogen. Cancer staging was determined as stage 1 to 4 according to the Union for International Cancer Control TNM classification system (22). Pathological grading (G1-G3) was performed according to the modified Scarff-Bloom-Richardson grading system (23). HER2 positive/negative status was determined according to the American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing in breast cancer (24).

MCF-7/S and MCF-7/A cells were purchased from iCell Bioscience Inc. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Biological Industries), 100 U/ml penicillin and 100 mg/ml streptomycin sulfate (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ in a 95% humidified atmosphere.

The half maximal inhibitory concentration (IC₅₀) of ADM in MCF-7/S and MCF-7/A cells was determined by performing MTT assays. Cells (5×10³) were seeded in 96-well plates and cultured overnight at 37°C. Next, cells were treated with increasing concentrations of ADM (Zhejiang Hisun Chemical Co., Ltd.) for 48 h. The concentrations of ADM for MCF-7/S treatment were 0.01, 0.05, 0.2, 1, 5 and 10 µM, and the concentrations of ADM for MCF-7/A treatment were 0.01, 0.05, 0.2, 1, 5, 10, 20, 50 and 100 µM. MTT reagent (neoFroxx GmbH) was added to the culture for 4 h according to the manufacturer's protocol. Subsequently, formazan was dissolved with DMSO. Cell viability was determined using a microplate reader (Thermo Fisher Scientific, Inc.) at 570 nm.

MCF-7/S and MCF-7/A cells were seeded into 6-well plates at 200 cells/well and maintained for 24 h before ADM treatment (0, 1, 2, 5 and 10 µM). The treated cells were cultured for 21 days to allow colony formation. The medium with ADM was replaced every 7 days. Colonies were stained with 1% crystal violet at room temperature for 10 min and subsequently counted.

MCF-7/S and MCF-7/A cells (2×10⁵) were seeded into 6-well plates and incubated overnight. When 70–80% confluence was reached the next day, MCF-7/S cells were transfected with 100 nM miR-16 inhibitor or control, and MCF-7/A cells were transfected with 50 nM miR-16 mimic or control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). miR-16 mimic (5′-UAGCAGCACGUAAAUAUUGGCG-3′) and corresponding negative control (mi-mNC; 5′-UUCUCCGAACGUGUCACGU-3′), as well as the miR-16 inhibitor (5′-CGCCAAUAUUUACGUGCUGCUA-3′) and corresponding negative control (mi-iNC; 5′-CAGUACUUUUGUGUAGUACAA-3′) were obtained from Shanghai GenePharma Co., Ltd. The cells were collected for 24 h after transfection for reverse transcription-quantitative PCR (RT-qPCR), and 48 h after transfection for western blot analysis.

The analysis of predicted miRNA targets was performed using miRWalk 3.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/index.html) and TargetScan 7.1 (http://www.targetscan.org/vert_71/) software.

Dual-luciferase reporter assay. To confirm that miR-16 has binding sites in the 3′UTRs of the genes encoding Wip1 (PPM1D) and Bcl-2, MCF-7/S cells were seeded into 12-well plates and co-transfected, using Lipofectamine^® 2000, with miR-16 or mi-mNC and pMIR-PPM1D-3′UTR (wild-type and mutant) or pMIR-BCL-2-3′UTR (wild-type and mutant) reporter plasmids (Nanjing Genebay Biotech Co., Ltd.). Renilla luciferase plasmid (pRL-TK vector; Nanjing Genebay Biotech Co., Ltd.) was transfected as the internal control. The lysate was prepared by adding cell lysis buffer 48 h after co-transfection. A Dual-Luciferase Reporter Assay kit (Promega Corporation) was used to measure activity, which was normalized to Renilla.

miR-16 mimic and inhibitor were transfected into MCF-7/A and MCF-7/S cells, respectively, in a 60-mm dish. Additionally, mi-mNC and mi-iNC were transfected into MCF-7/A and MCF-7/S cells, respectively, as corresponding negative controls. After incubation for 48 h, cell lysates were prepared by adding radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing 1 mM PMSF. Protein concentration was determined using a bicinchoninic acid kit (Nanjing KeyGen Biotech Co., Ltd.). A total of 50 µg protein were loaded and separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene difluoride membranes. The membranes were blocked in 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 2 h at room temperature, and then washed three times using TBST buffer. Membranes were subsequently incubated overnight at 4°C with primary antibodies (dilution, 1:1,000) against Wip1 (cat. no. 11901), Bcl-2 (cat. no. 4223) and β-actin (cat. no. 4970), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; dilution, 1:2,000; all Cell Signaling Technology, Inc.) at room temperature for 2 h. Bands were visualized with enhanced chemiluminescence on a ChemiDoc™ XRS+ imaging system (Bio-Rad Laboratories, Inc.). Quantity One software v4.6.6 (Bio-Rad Laboratories, Inc.) was used to quantify the blot intensities, which were normalized to that of β-actin.

MCF-7/A and MCF-7/S cells (2×10³; no prior treatment) were grown on a 15-mm confocal dish for 2 days and fixed in 4% paraformaldehyde at room temperature for 15 min. Subsequently, cells were washed with PBS and permeabilized with ice-cold PBS containing 0.5% Triton X-100 for 20 min at room temperature. The samples were incubated with the appropriate primary antibodies (Bcl-2, cat. no. 15071; and Wip1, cat. no. 11901; Cell Signaling Technology, Inc.) at 4°C overnight, and probed with Alexa Fluor^® 647 Conjugated (cat. no. 4410; Cell Signaling Technology, Inc.) or Alexa Fluor^® 488 conjugated (cat. no. ab150077; Abcam) secondary antibodies (all dilutions, 1:1,000) for 20 min at room temperature. The nuclei were counterstained with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at room temperature. Fluorescence was visualized and captured using a confocal microscope (magnification, ×600; Leica TCS SP5 MP; Leica Microsystems, Inc.).

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from the tissues or MCF-7/S and MCF-7/A cells treated with miR-16 inhibitor or miR-16 mimic, respectively, in 6-well plates (2×10⁵ cells/well). Then RNA was reverse transcribed into cDNA for 1 h at 37°C and 85°C for 5 min using a Mir-X miRNA First-Strand Synthesis kit (cat. no. 638315; Takara Bio, Inc.). qPCR was performed using a SYBR Premix Ex Taq II kit (Takara Bio, Inc.) on a LightCycler system (Roche Molecular Diagnostics). The thermocycling conditions were as follows: 95°C for 10 sec, 95°C for 5 sec and 60°C for 20 sec for 40 cycles, followed by 95°C for 60 sec, 55°C for 30 sec and 95°C for 30 sec. miR-16 mRNA expression was quantified by normalizing to U6. Primer sequences were as follows: miR-16 forward, 5′-TAGCAGCACGTAAATATTGGCG-3′. The U6 forward and reverse primers along with miR-16 reverse primers were included in the Mir-X miRNA First-Strand Synthesis kit. The 2^−ΔΔCq method was used to analyze the RT-qPCR data (25).

MCF-7/S cells were transfected with miR-16 inhibitor or mi-iNC, and MCF-7/A cells were transfected with miR-16 mimics or mi-mNC, as aforementioned. After 48 h, cells were treated with 10 µM ADM for 48 h to induce apoptosis. The attached and floating cells were harvested, and flow cytometry analysis was performed using an Annexin V-FITC/propidium iodide staining kit (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol. Apoptotic cells were detected using a BD Accuri C6 Plus flow cytometer (BD Biosciences) and the data were analyzed by BD CSampler analysis software (cat. no. 653123; version 1.0.23.1; BD Biosciences). The percentage of apoptotic cells was calculated by dividing the number of proapoptotic and apoptotic cells by the total number of cells.

Data are presented as the mean ± standard deviation of at least three replicates. Unpaired two-tailed Student's t-test was used to analyze the significance between two groups. For multiple comparisons, one-way analysis of variance followed by Dunnett's or Bonferroni's post-hoc test was performed using GraphPad Prism v5.01 (GraphPad Software, Inc.). Pearson's correlation analysis and calculation of the IC₅₀ for ADM were also performed using GraphPad Prism. P<0.05 was considered to indicate a statistically significant difference.

It has been reported that miR-16 is overexpressed in multiple types of cancer, including BC (26). To determine whether miR-16 was associated with BC drug resistance, tumor tissues were collected from patients with BC, and miR-16 expression was quantified by RT-qPCR. The clinicopathological characteristics of the patients are presented in Table I. As shown in Fig. 1A, miR-16 expression was decreased in drug-resistant tumor samples compared with that in drug-sensitive samples. To clarify the potential mechanism of the association between miR-16 and BC drug resistance, the expression of Wip1 and Bcl-2 was determined by western blotting. The expression of these two proteins in ADM-resistant tumors was higher compared with that in drug-sensitive tumors (Fig. 1B). Pearson's correlation analysis showed that miR-16 expression was negatively correlated with the expression of Wip1 and Bcl-2 (Fig. 1C and D), suggesting that miR-16 may contribute to BC ADM resistance.

To evaluate miR-16 expression in BC cells, a cell line sensitive to ADM (MCF-7/S) and a resistant line (MCF-7/A) were used. The IC₅₀ values for ADM were determined in the MCF-7/S and MCF-7/A cells by MTT assay. As presented in Fig. 2A and B, the IC₅₀ of ADM in MCF-7/S and MCF-7/A cells was 2.192 µM and 52.25 µM, respectively. The ratio of the IC₅₀ in MCF-7/A cells to the IC₅₀ in MCF-7/S cells was 23.84 (52.25/2.192), confirming that MCF-7/A cells were resistant to this drug. In addition, colony-formation assays were used to further confirm resistance to ADM in MCF-7/A cells. The results revealed that the surviving fraction of MCF-7/S cells was significantly lower compared with that in MCF-7/A cells at the same concentration of ADM (Fig. 2C and D), consistent with the MTT assay results.

miR-16 expression in ADM-sensitive and resistant BC cells was examined to illustrate the potential mechanism of ADM drug resistance. miR-16 expression was significantly decreased in MCF-7/A cells, compared with that in MCF-7/S cells (Fig. 3A). Wip1 and Bcl-2 mRNAs are potential targets of miR-16 as the 3′UTRs of these genes contain highly conserved sites for miRNA binding. To assess whether miR-16 impacted these targets in BC, the expression of Wip1 and Bcl-2 was determined in MCF-7/S and MCF-7/A cells. The results showed that the expression of these proteins was increased in MCF-7/A cells, as determined by western blotting (Fig. 3B). Higher Wip1 and Bcl-2 protein expression was also observed in MCF-7/A cells using immunofluorescence, suggesting that miR-16 may regulate the expression of these two endogenous proteins in BC cells (Fig. 3C).

To confirm that miR-16 regulates the expression of Wip1 and Bcl-2 in MCF-7 cells, the 3′UTR of the human PPM1D and BCL-2 genes were predicted. It was found that 12 nucleotides from miR-16 were complementary to target sequences in the 3′UTRs of these two genes (Fig. 4A). To further validate that PPM1D and BCL-2 were targets of miR-16, their 3′UTRs, containing the target sites of miR-16, were cloned into a firefly luciferase reporter construct. Mutant versions of the 3′UTRs were transfected as controls. Luciferase activity was detected following co-transfection of reporter constructs with miR-16 into MCF-7/S cells. In the presence of miR-16, the luciferase activity of constructs containing the wild-type PPM1D and BCL-2 3′UTR was significantly decreased by 75 and 55%, respectively (Fig. 4B and C). However, luciferase activity was unaffected in cells transfected with mutant PPM1D and BCL-2 3′UTRs and miR-16, indicating that the PPM1D and BCL-2 genes are direct targets of miR-16.

To investigate the effects of miR-16 on BC drug resistance, its expression in MCF-7/S cells was downregulated by transfecting an inhibitor of miR-16. Transfection with the miR-16 inhibitor decreased expression of miR-16 compared to untransfected MCF-7/S cells and those transfected with a non-targeting control (Fig. 5A). Expression of miR-16 in MCF-7/S cells after transfection with the inhibitor was comparable to miR-16 expression in untransfected ADM-resistant MCF-7/A cells. Downregulation of miR-16 in MCF-7/S cells resulted in increased Wip1 and Bcl-2 protein expression compared with the negative control (Fig. 5B). To investigate the effects of downregulation of miR-16 on ADM resistance, apoptosis following 10 µM ADM treatment was analyzed using flow cytometry (Fig. 5C and D). The apoptotic rate of MCF-7/S cells treated with ADM was significantly higher compared with that of MCF-7/A cells. Apoptosis in MCF-7/S cells transfected with miR-16 inhibitor and exposed to ADM was markedly decreased compared with that in the ADM-treated mi-iNC MCF-7/S group.

To test the effect of miR-16 on chemotherapy sensitization, MCF-7/A cells were transfected with an miR-16 mimic. Transfection with the miR-16 mimic resulted in a 17-fold increase in miR-16 expression compared with the mi-mNC group (Fig. 6A). Next, Wip1 and Bcl-2 protein expression were examined by western blotting. As presented in Fig. 6B, expression of these proteins was decreased in MCF-7/A cells transfected with miR-16 mimic compared with mi-mNC MCF-7/A cells. Additionally, apoptosis was assessed following exposure to ADM (Fig. 6C and D). It was found that the apoptotic rate in the ADM-treated group was significantly increased, compared with the untreated group, particularly in MCF-7/S cells. However, in MCF-7/A cells transfected with miR-16 mimic, apoptosis was further elevated, compared with the ADM-treated mi-mNC group.