Blood samples were obtained by EDTA vacutainer blood collection tubes (BD Biosciences, Franklin Lakes, NJ, USA) and immediately stored at −80°C from 43 patients with breast cancer (aged 18–80 years) at the First Affiliated Hospital of Zhejiang University (Hangzhou, China) between April 2013 and April 2015. The samples were immediately stored at −80°C until use. All of the patients were female and >18 years old. A total of 26 of the patients were identified to have well differentiated breast cancer (grade I–II) and 17 had poorly differentiated breast cancer (grade III). A total of 20 healthy adults were recruited from the First Affiliated Hospital of Zhejiang University to participate in the study as the control group. The Ethics Committee of The First Affiliated Hospital of Zhejiang University approved the present study. Written informed consent was obtained from all participants included in the study.

Two breast cancer cell lines, MCF-7 and MDA-MB-231, were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in Dulbecco's modified Eagle's medium (DMEM) with high glucose (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 µg/ml streptomycin, at 37°C in 5% CO₂.

Breast cancer cells were seeded in a 24-well plate with a density of 4×10⁴ cells/well and cultured for 24 h at 37°C prior to transfection. Subsequently, the cells were starved for 1 h at 37°C using FBS-free medium without antibiotics. Transfection with the negative control oligonucleotides (NC mimic), miR-194, antagomiR-194 or miR-194/antagomiR-194 was performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Briefly, 0.5 µg miRNA was diluted with 100 µl Opti-MEM (Thermo Fisher Scientific, Inc.) without FBS, which was mixed with 1.25 µl Lipofectamine^® LTX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The mixture (containing the transfection reagents) was incubated for 25 min at room temperature, and then added to wells containing cells and incubated at room temperature for 4 h. Following 48 h transfection at 37°C, the cells from the various groups were seeded (1×10⁴ cells/ml) onto 96-well plates in triplicate, and then cultured at 37°C for 24 h.

Total RNA was isolated from breast cancer cell lines using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), chloroform, isopropanol and ethanol, following the manufacturer's instructions. The isolated RNA, including miRNA, was dissolved in 30 µl diethyl pyrocarbonate-treated (high temperature sterilization at 121°C for 20 min) water. In order to detect the serum levels of miR-194, the total RNA was transcribed into cDNA using the miScript II RT kit (Qiagen GmbH, Hilden, Germany). cDNA was synthesized with the Prime-Script RT reagent kit (Takara Bio Inc., Otsu, Japan) from 500 ng of total RNA. RT-PCR was performed in triplicate using Fast SYBR^® Green Master Mix (Thermo Fisher Scientific, Inc.), according to the manufacturer instructions of the ABI Prism^® 7900HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primer pairs for miR-194 were as follows: Forward, 5′-CTAAGCTTAGTGGGCATGGGACACTCT-3′ and reverse, 5′-CTGAATTCACCTGCCTCTCCTTCTTCGT-3′. U6 was used as an internal control. The primers for U6 were as follows: Forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The PCR reaction conditions were as follows: 95°C for 5 min heating; 40 cycles of 95°C for 10 sec; 59°C for 40 sec; and 72°C for 1 min for application. The 2^−∆∆Cq method was used for quantification of the relative expression levels of miR-194 in the samples of various groups (28).

Total protein was extracted from breast cancer cell lines following a 48-h transfection using the ProteoPrep^® Total Extraction Sample kit (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The protein concentrations were evaluated using an enhanced Bicinchoninic Acid assay kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's instructions. Equal concentrations of the extracted proteins and the 5X SDS-PAGE sample loading buffer were then heated at 95°C for 10 min. Equal quantities of the protein samples (25 µg/lane) were loaded onto the gel for 10% SDS-PAGE. Subsequently, the proteins were transferred to a polyvinylidene fluoride membrane. Following washing with TBS and Tween-20, the membranes were blocked with 5% nonfat milk and 1% Tween-20 in TBS at room temperature for 2 h, and subsequently incubated with the following primary antibodies: Anti-Fbxw-7 (cat. no. ab74054; 1:1,000), anti-cyclin D (cat. no. ab7958; 1:500), anti-cyclin E (cat. no. ab7958; 1:500) and β-actin (cat. no. ab8227; 1:5,000). All primary antibodies were purchased from Abcam (Cambridge, UK). Following washing with TBST three times, the membranes were incubated with the corresponding anti-rabbit IgG (cat. no. A0545; 1:20; Sigma-Aldrich; Merck KGaA) conjugated with horseradish peroxidase (OriGene Technologies, Inc., Rockville, MD, USA) for 1.5 h at room temperature. The membranes were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).

Breast cancer cells (1×10⁴ cells/well) in the various groups were seeded into a 96-well plate with 100 µl of medium/well. The cells were plated in quintuplicate each day. Following culture at 37°C for 24 h, 20 µl MTT solution (5 mg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) was added to each well and incubated at 37°C for 4 h. Subsequently, the supernatant in each well was removed and 150 µl dimethyl sulfoxide was added in order to dissolve the formazan crystals. The absorbance wavelengths were read at 570 nm with a microplate spectrophotometer. The cell growth curve in each group was drawn according to the optical density values.

The prediction of the miR-194 target genes was performed using TargetScan version 3.1 (29). The results revealed that the 3′-UTR of Fbxw-7 is complementarily base paired with miR-194. In order to confirm the prediction, a luciferase assay was performed. Briefly, 293T cells were seeded (1×10⁴ cells/ml) in a 6-well plate and incubated at room temperature for 24 h. Subsequently, the cells divided into 3 groups and co-transfected with PGL3-Fbxw-7 3′UTR, miR-194 or an NC mimic using Lipofectamine^® 2000. Following 40 h, the medium in each well was discarded and 500 µl cell lysis solution was added. Following lysis, the supernatant was centrifuged at 15,000 × g for 5 min at room temperature. A total of 100 µl luciferase assay reagent (Promega Corporation, Madison, WI, USA) was added into 100 µl supernatant from each well. The relative intensity of fluorescence was determined using a multifunctional microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland) with 2 sec measurement intervals at an absorbance wavelength of 366 nm and 10 sec measurement duration.

All data were analyzed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. The miR-194 expression levels and MTT data were statistically analyzed using a two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to explore the association between miR-194 expression levels and breast cancer, total RNA was extracted from the sera of 20 healthy adults, 26 patients with well differentiated breast cancer and 17 patients with poorly differentiated breast cancer. The results indicated that the serum expression levels of miR-194 were significantly higher in the well differentiated group (P<0.05; Fig. 1) and the poorly differentiated group (P<0.05; Fig. 1), as compared with in the control group. Additionally, the serum expression level of miR-194 was significantly higher in the poorly differentiated group, as compared with in the well differentiated group (P<0.01; Fig. 1). Therefore, the serum expression level of miR-194 was closely associated with the differentiation degree of breast cancer, suggesting that miR-194 may be involved in the progression of breast cancer.

In order to determine the effect of miR-194 expression on breast cancer cell proliferation, MCF-7 and MDA-MB-231 breast cancer cell lines were transfected with the control, miR-194, antagomiR-194 or miR-194/antagomir-194. Following a 48-h transfection, the cells from the various groups were seeded into a 96-well plate in triplicate, and then cultured for 24 h. Subsequently, an MTT assay was performed in order to determine the proliferation rate of breast cancer cells in the various groups. The results demonstrated that the overexpression of miR-194 significantly enhanced the proliferation of MCF-7 (P<0.01; Fig. 2) and MDA-MB-231 cells (P<0.01; Fig. 2). Additionally, the inhibition of miR-194 expression significantly decreased the proliferation of MCF-7 (P<0.005; Fig. 2) and MDA-MB-231 cells (P<0.005; Fig. 2). No significant change was observed in the proliferation of cells co-transfected with miR-194 and antagomiR-194 (Fig. 2).

According to the bioinformatic database (Targetscan) analysis and a previous study (30), Fbxw-7 may be a potential target of miR194. The binding sites for miR-194 were detected in the 3′-UTR of Fbxw-7, and were highly conserved and consistent between rats, mice and humans (Fig. 3A). Subsequently, the present study performed a luciferase assay to confirm the prediction made using the results of the Targetscan analysis. The assay results revealed that luciferase activity was significantly reduced in breast cancer cells with overexpressed miR-194, compared with in the control cells, following transfection of PGL3-Fbxw-7 3′UTRs (P<0.005; Fig. 3B).

It has previously been reported that the inhibition of Fbxw-7 may increase cell proliferation by increasing the number of ESCC cells in the S phase and decreasing the number of ESCC cells in the G₁/G₀ phase (31). The present study performed western blot analysis in order to determine the expression levels of the cyclin proteins, cyclin D and cyclin E. The results revealed that miR-194 expression inhibited the expression of Fbxw-7, which additionally significantly increased the expression levels of cyclin D and cyclin E in MCF-7 (P<0.05, Fig. 4A) and MDA-MB-231 cells (P<0.05, Fig. 4B).