The study was approved by the Human Ethics Committee Review Board of Wuxi Taihu Hospital (Wuxi, China), and informed consent was obtained from each patient. In total, primary breast cancer tissues and paired adjacent normal tissues were obtained from 30 females aged between 35 to 65 years (mean age, 50 years) who underwent resection surgery of breast tumors in the Wuxi Taihu Hospital between September 2015 and June 2016. None of the patients had received treatment prior to surgery. All patients recruited for the current study were diagnosed with invasive ductal carcinoma according to the morphological criteria detailed in the World Health Organization histologic classification of breast tumors (17). Of the 30 patients, 3, 7 and 20 patients were diagnosed with stage III, II and I invasive ductal carcinoma, respectively. All tissue samples were assessed and diagnosis was confirmed by pathologists. Tumor and adjacent normal tissues were snap-frozen in liquid nitrogen and then stored at −80°C until further use.

The MCF-7 cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS). Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂, and passaged every 2–3 days.

At 24 h before cell transfection, MCF-7 cells (5×10⁴ cells per well) were plated into 6-well plates and incubated at 37°C with 5% CO₂. Next, miR-34a mimic (item no. P4185-250UCI), mimic control (cat. no. M-03-S), miR-34a inhibitor (item no. P4185-250UCI) or inhibitor control (cat. no. M-04; GenePharma Co., Ltd., Shanghai, China) were transfected into the MCF-7 cells using 30 µl Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as per the manufacturer's protocol. Cells without any treatment were considered as the negative control (NC) group. After incubation for 48 h, these cells were subjected to subsequent experiments. The transfection efficiency was assessed by detecting miR-34a expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

At 48 h after transfection, cell viability was detected using an MTT assay. Briefly, MCF-7 cells were harvested, reseeded into a 96-well culture plate (5,000 cells per well) and cultured at 37°C for 24, 48 or 72 h. Subsequently, MTT solution (Amresco, LLC, Solon, OH, USA) was added into each culture well and incubated for another 4 h. Finally, the optical density value was measured at 570 nm by using a SynergyTM 2 Multi-function Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). Each experiment was repeated at least three times.

At 48 h after transfection, flow cytometry was applied to analyze the MCF-7 cell cycle distribution. Briefly, the transfected MCF-7 cells were collected by trypsinization and then fixed with ethanol overnight at 4°C. Subsequently, the MCF-7 cells were washed by PBS and stained with ribonuclease A (100 µg/ml) and propidium iodide (PI; 50 µg/ml; both Beyotime Institute of Biotechnology, Shanghai, China) in the dark at 4°C for 30 min. Finally, a flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the cell cycle distribution. Tests were repeated at least three times.

MCF-7 cells were transfected with miR-34a mimic, mimic control, miR-34a inhibitor or inhibitor control, and at 48 h after the transfection, cells were labeled with Annexin V-FITC and PI (Cell Signaling Technology, Inc., Danvers, MA, USA) as per the manufacturer's protocol. Flow cytometry (BD Biosciences) was then applied to analyze cell apoptosis. Tests were repeated three times.

Transwell assay was used to detect the cell invasion ability. Briefly, MCF-7 cells were transfected with miR-34a mimic, mimic control, miR-34a inhibitor or inhibitor control. After 48 h, the cells were harvested, re-suspended in serum-free medium, and then seeded into the upper chamber with a Matrigel-coated membrane matrix. Cell culture medium containing 20% FBS was added to the lower chamber as a chemoattractant, and the cells were incubated for 48 h at 37°C with 5% CO₂. Finally, the non-invading cells on the upper surface were removed, while the invasive cells on the bottom surface of the membrane were fixed with methanol for 30 min at room temperature and then stained with hematoxylin for 5 min at room temperature. A microscope was then used to observe the stained cells. Tests were repeated three times.

At 48 h after transfection, cells were harvested by cell scrapers and washed with PBS. The total cell proteins were extracted using a lysis buffer (Cell Signaling Technology, Inc.) according to the manufacturer's protocol. The concentration of protein samples was determined with a BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amount of the protein samples (30 µg/lane) were resolved by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Subsequent to blocking with 5% skim milk at room temperature for 2 h, the membranes were blotted overnight at 4°C with primary antibody against Notch1 (cat no. 3608; Cell Signaling Technology, Inc.; dilution, 1:1,000), and then incubated with anti-rabbit horseradish peroxidase-linked IgG secondary antibody at room temperature for 2–3 h (cat no. 7074; Cell Signaling Technology, Inc.; dilution, 1:2,000). GAPDH antibody (cat no. 5174; Cell Signaling Technology, Inc.; dilution, 1:5,000) was also used as the internal control and incubated at room temperature for 2–3 h. Finally, the protein bands were observed using an enhanced chemiluminescence detection system (Super Signal West Dura Extended Duration Substrate; Thermo Fisher Scientific, Inc.).

The breast tissues (cancer and adjacent normal), MCF-10A cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) as normal breast cells and MCF-7 cells were analyzed by PCR to examine miRNA expression. Breast tissues (50–100 mg) cut into pieces were homogenized prior to total RNA extraction. Total RNA from breast tissue homogenates, and MCF-10A and MCF-7 cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in line with the manufacturer's protocol. RT was then performed to generate cDNA using a PrimeScript Reverse Transcription Reagent kit (Takara Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's protocol. Subsequently, qPCR was conducted to analyze the cDNA using a TaqMan Universal PCR Master Mix kit (Thermo Fisher Scientific, Inc.). The amplification conditions were as follows: 95°C for 10 min, followed by 38 cycles of 95°C for 10 sec and 58°C for 60 sec. miR-34a expression was normalized to U6 and Notch1 mRNA expression was normalized to GAPDH. The primer sequences for qPCR were as follows: U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; miR-34a forward, 5′-CGTCACCTCTTAGGCTTGGA-3′ and reverse, 5′-CATTGGTGTCGTTGTGCTCT-3; and Notch1 forward, 5′-GAGGCGTGGCAGACTATGC-3′ and reverse, 5′-CTTGTACTCCGTCAGCGTGA-3′. The 2^−ΔΔCq method was used to quantify the relative gene expression levels (18).

Bioinformatics analysis was initially conducted with TargetScan software (http://www.targetscan.org) in order to predict the potential targets of miR-34a, and the findings revealed that Notch1 was a potential target of miR-34a. To confirm this prediction, MCF-7 cells were seeded into each well of a 24-well plate (5×10⁴ cells/well). At 24 h later, the cells were co-transfected with Notch1 3′UTR pmirGLO plasmid (containing mutant Notch1 3′UTR or wild-type Notch1 3′UTR) and a miR-34a mimic or mimic control (NC) vector using Lipofectamine^® 2000 reagent according to the manufacturer's protocol. Following incubation for another 48 h, the luciferase activity was assessed using the dual-luciferase reporter assay system (Promega Corporation, Madison, WI, USA).

Data are presented as the mean ± standard deviation. SPSS statistical software (version 17.0; SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. Student's t-test or one-way analysis of variance followed by Tukey's test was used to compare the data between groups. P<0.05 was considered to denote a statistically significant difference.

To determine the expression levels of miR-34a in MCF-10A cells and MCF-7 cells, RT-qPCR was performed. The results demonstrated that miR-34a expression was significantly lower in MCF-7 cells compared with normal cells (Fig. 1A). Next, in order to investigate the role of miR-34a in breast cancer, the MCF-7 cell line was used. miR-34a was overexpressed or downregulated in MCF-7 cells by transfections with miR-34a mimic or miR-34a inhibitor, respectively, and the transfection efficiency was detected by RT-qPCR (Fig. 1B). Subsequently, the expression of miR-34a was analyzed by RT-qPCR in breast cancer and adjacent normal breast tissues. As shown in Fig. 1C, compared with the adjacent normal tissues, the level of miRNA-34a in breast cancer tissues was significantly reduced, indicating that miRNA-34a may be involved in breast cancer progression.

Bioinformatics analysis was conducted with the TargetScan tool to predict the potential targets of miR-34a, and the findings revealed that Notch1 is one of the target genes of miR-34a (Fig. 2A). Previous studies have demonstrated that activation of Notch1 or Notch4 in mice induced the formation of spontaneous breast tumors, demonstrating that activation of the Notch signaling pathway is an important cause of breast cancer (14–16); thus, Notch1 was selected for further investigation in the present study. To confirm this prediction, a dual-luciferase reporter assay was used, and the findings indicated that miR-34a directly targets Notch1 (Fig. 2B). To further reveal whether miR-34a was able to regulate Notch1 expression in MCF-7 cells, the protein and mRNA levels of Notch1 were detected by western blotting and RT-qPCR, respectively. The results indicated that miR-34a negatively regulated Notch1 expression in MCF-7 cells (Fig. 2C-E).

To investigate the effects of miR-34a on breast cancer cell viability, an MTT assay was performed. As shown in Fig. 3, the viability was significantly reduced in miR-34a mimic-transfected cells, while the viability of miR-34a inhibitor-transfected cells was notably increased following transfection at three time points. No statistically significant differences were detected between the NC and the mimic control or inhibitor control groups (Fig. 3).

To investigate the effects of miR-34a on breast cancer cell cycle distribution and apoptosis, flow cytometry was applied. The results demonstrated that, at 48 h after transfection with miR-34a mimic, MCF-7 cells were significantly arrested in G1 phase when compared with the NC group. In addition, miR-34a inhibitor markedly declined the G1 phase arrest in MCF-7 cells as compared with that observed in the NC group. No significant difference was identified between the NC and the mimic control or inhibitor control groups (Fig. 4A and B).

Furthermore, the data revealed that, compared with the NC group, miR-34a mimic significantly induced MCF-7 cell apoptosis (early and late apoptosis). By contrast, cell apoptosis was notably inhibited by miR-34a inhibitor. No significant differences were detected between the NC and the mimic control or inhibitor control groups (Fig. 4C and D).

At 48 h after cell transfection, the MCF-7 cell invasion ability was determined. The findings suggested that, compared with the NC group, MCF-7 cell invasion ability was significantly inhibited by miR-34a mimic, while miR-34a inhibitor notably promoted the MCF-7 cell invasion ability. However, no significant differences were observed between the NC and the mimic control or inhibitor control groups (Fig. 5).