A total of 77 samples of BC tumor tissue and paired adjacent tissue were collected from patients with BC at the First People's Hospital of Yibin (Yibin, China). Tissue specimens were collected from patients (age range, 30–82 years; all patients were female) with primary BC who underwent surgical resection between September 2010 and December 2012. All patients were histologically diagnosed with invasive ductal breast cancer after surgery. Patient information on age, tumor size, clinical stage, lymph node metastasis, distant metastasis, and information on the expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 was collected (17,18). This study was conducted in accordance with the Helsinki Declaration and was approved by the Ethics Review Committee of the Yibin First People's Hospital and the Ethics Review Committee of Southwest Medical University (Luzhou, China). Written informed consent was obtained from all patients for the use of their tissue in this study.

Five BC cell lines (MCF-7, SKBR-3, MDA-MB-231, MDA-MB-468 and BT-20) and a normal human mammary epithelial cell line (MCF-10A) were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences). All cell lines were cultured in DMEM containing 10% fetal bovine serum (FBS) and incubated at 37°C in 5% CO₂.

MCF-7 and MDA-MB-231 cells (3×10⁵) were transfected with pre-miR-425-5p (miR-425-5p), mimic negative control (NC), miR-425-5p inhibitor, inhibitor NC or pcDNA-PTEN (Shanghai GenePharma Co., Ltd.). pcDNA plasmid without PTEN was used as a negative control. The sequences of the miRNA mimic, inhibitor and controls are presented in Table SI. The concentrations used for mimic, inhibitor and pcDNA were 50, 100 nM and 1000 ng/ul, respectively) Cells were transfected using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequent experiments were performed 24–72 h after transfection.

Total RNA was extracted from BC tissues, paired adjacent tissue and cell lines using TRIzol reagent (Takara Bio, Inc.). PrimeScript RT Reagent Kit (Takara Bio, Inc.) was used for the reverse transcription of cDNA. The temperature protocol for RT was as follows: 35°C for 5 min, followed by 42°C for 40 min and 75°C for 5 min. Next, qPCR was performed with SYBR Premix Ex Taq II (Takara Bio, Inc.) and a LightCycler system (Roche Diagnostics GmbH). The results were analyzed using the 2^−ΔΔCq method (19). U6 was used as an internal control for microRNA, and GAPDH was used for mRNA. The sequences of the primers used for each gene are presented in Table SII. The thermocycling conditions for qPCR were as follows: Initial activation step at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec.

Total protein from BC cell lines, a normal human mammary epithelial cell line (1×10⁶) and tissues was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology Co., Ltd.). Protein was quantified using the Bradford protein assay (Bio-Rad Laboratories, Inc.) with a Nanodrop spectrophotometer, and an equal amount (30 µg) was added to each well of 12% gels and resolved by SDS-PAGE. Next, proteins were transferred to PVDF membranes and were blocked by incubation for 1 h at room temperature with 5% non-fat powdered milk. The membranes were probed at 4°C overnight with antibodies against PTEN (1:5,000; ab32199; Abcam) and anti-GAPDH (1:5,000; ab181602; Abcam). Subsequently, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; ab6721; Abcam) at room temperature for 1 h. Protein detection was performed using incubation with an enhanced chemiluminescence solution (EMD Millipore), and imaged using a ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.). GAPDH was used as the internal loading control in the western blot. The protein bands were quantified using QuantityOne software v4.6.7 (Bio-Rad Laboratories, Inc.), and the values are expressed relative to GAPDH. Each experiment was repeated three times.

Following transfection, cells were seeded onto 96-well plates and incubated at 37°C for 24, 48 or 72 h before the addition of CCK-8 reagent. Next, 10 µl CCK-8 reagent was added to each well, and after 2 h of incubation at 37°C, absorption was determined at 450 nm was determined by microplate spectrophotometer.

Transfected cells were seeded into 6-well plates and cultured for 14 days. At that time, the colonies that had formed were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature. Finally, the cell colonies were counted with a light microscope (Olympus Corporation; magnification, ×40).

For the invasion assay, Tranwell inserts were precoated with Matrigel (1 mg/ml) at 37°C for 30 min. Transwells were uncoated for the migration assay. Transfected cells were seeded into the upper chamber with serum-free media. In the bottom chamber, 500 µl DMEM containing 10% FBS functioning as a chemoattractant was added. After 48 h of incubation, the migrated or invaded cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature. Finally, the migrated or invaded cells were counted with a Leica DM IL LED inverted light microscope (Leica Microsystems, Inc.; magnification, ×100). Five microscopic fields of each well were randomly selected and the number of cells in microscopic fields was counted.

Following transfection, cells were seeded into 96-well plates and incubated at 37°C until the cells reached 30% confluence. An EdU assay was performed using a Cell-Light EdU Apollo 567 in vitro kit (cat. no. 100T; Ruibo Biotechnology Co., Ltd.). According to manufacturer's instructions, the cells were incubated with EdU (50 µM) for 120 min, 0.5% Triton X and ApolloR reaction cocktail (100 µl) for 30 min, and Hoechst 33342 (100 µl) for 30 min sequentially. Cell proliferation was analyzed using the mean number of the cells in three fields for each sample using a fluorescence microscope (Lionheart; BioTek Instruments, Inc.; magnification, ×100).

The online miRNA databases Oncomir (http://www.oncomir.org/), MiRanda (http://www.microrna.org/microrna/home.do), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and TargetScan (http://www.targetscan.org) were used to identify downstream target genes of miR-425-5p. The wild-type (WT) PTEN 3′ 3′UTR and mutant (MUT) PTEN 3′UTR oligonucleotides containing the putative binding site of miR-425-5p were cloned into the firefly luciferase-expressing pMIR-REPORT vector (Obio Technology Corp., Ltd.). These constructs were co-transfected with inhibitor NC or miR-425-5p inhibitor into MCF-7 and MDA-MB-231 cells. After 48 h of transfection, luciferase activity was determined using the Dual-Luciferase Reporter Assay kit (Promega Corporation) according to the manufacturer's protocol. The ratio of Renilla luciferase activity to firefly luciferase activity was calculated.

The statistical data were analyzed using SPSS version 22.0 software (IBM Corp.) and GraphPad Prism version 6.0 software (GraphPad Software Inc.). The differences between groups were analyzed using paired or unpaired t test and one-way analysis of variance, followed by the Newman-Keuls test. Kaplan-Meier and log-rank tests were used to assess recurrence-free survival (RFS) and disease-specific survival (DSS) times. For Kaplan-Meier curves, patients were divided into high and low expression groups using the mean expression (0.2) as the cut-off value. Correlation analysis was performed using Spearman's rank correlation test. The χ² test was used to analyze the association of miR-425-5p expression with the clinicopathological characteristics of BC. Univariate and multivariate Cox regression analyses were performed to analysis the prognostic significance of miR-425-5p. P<0.05 were considered to indicate a statistically significant difference.

RT-qPCR was performed to detect the expression of miR-425-5p in BC tumor tissue, paired adjacent tissue and cell lines. The results showed that miR-425-5p was higher in BC tissues compared with in paired adjacent tissue (Fig. 1A). The expression of miR-425-5p was also higher in BC cell lines compared with human mammary epithelial cells (Fig. 1B). To further explore the role of miR-425-5p in BC, MCF-7 and MDA-MB-231 cells were transfected with pre-miR-425-5p to increase the expression of miR-425-5p (Fig. 1C), and miR-425-5p inhibitor to knockdown the expression of miR-425-5p (Fig. 1D).

The association between miR-425-5p expression and the clinicopathological characteristics of patients with BC was analyzed. High miR-425-5p expression was significantly associated with tumor size, clinical stage, lymph node metastasis and distant metastasis (P<0.05; Table I). Kaplan-Meier analysis revealed that patients with high miR-425-5p expression had significantly shorter RFS and DSS times compared with those with low miR-425-5p expression (Fig. 2). Univariate analysis for RFS in patients with BC is presented in Table II. Subsequent multivariate analysis demonstrated that tumor size (HR, 1.184; P=0.017), clinical stage (HR, 1.418; P=0.033), lymph node metastasis (HR, 1.524; P=0.020), distant metastasis (HR, 0.887; P=0.009) and miR-425-5p level (HR, 1.771; P=0.013) were independent prognostic factors for RFS in patients with BC (Table II).

In addition, univariate analysis was performed for DSS in patients with BC, and results are presented in Table III. Subsequent multivariate analysis showed that tumor size (HR, 1.190; P=0.037), clinical stage (HR, 1.938; P=0.030), lymph node metastasis (HR, 1.619; P=0.019), distant metastasis (HR, 1.473; P=0.019) and miR-425-5p level (HR, 1.074; P=0.015) were independent prognostic factors for DSS in patients with BC (Table III).

Among the BC cell lines, the expression of miR-425-5p was lowest in MCF-7 cells and highest in MDA-MB-231 cells (Fig. 1B). Therefore, these two cell lines were selected for subsequent experiments. To investigate the biological role of miR-425-5p on proliferation of BC cells, CCK-8 assays showed that knockdown of miR-425-5p expression significantly inhibited MDA-MB-231 and MCF-7 cell proliferation (Fig. 3A and B). Colony formation assays also revealed that reducing the expression of miR-425-5p resulted in reduced colony number in MDA-MB-231 and MCF-7 cells (Fig. 3C). Transwell assays indicated that knockdown of miR-425-5p inhibited migration in MDA-MB-231 and MCF-7 cells (Fig. 3D). However, knockdown of miR-425-5p had no significant effect on the invasive ability of MDA-MB-231 and MCF-7 cells (Fig. S1). Moreover, decreased miR-425-5p expression significantly inhibited EdU uptake, representing the proliferation of cells, in MDA-MB-231 and MCF-7 cells (Fig. 3E).

Using an online target prediction tool, it was found that PTEN may be a direct target of miR-425-5p (Figs. 4A and S2). To further verify that miR-425-5p biologically targets PTEN, a luciferase reporter assay was performed. The results indicated that co-transfection with a miR-425-5p inhibitor significantly promoted luciferase activity in cells transfected with WT PTEN 3′UTR. However, increase no luciferase activity was observed in cells co-transfected with MUT PTEN 3′UTR (Fig. 4B). Additionally, in cells with decreased expression of miR-425-5p, the expression of PTEN was significantly increased (Fig. 4C and D).

Furthermore, PTEN mRNA was lower in BC tissues compared with that in paired adjacent tissue (Fig. 5A). PTEN mRNA expression levels inversely correlated with expression of miR-425-5p (Fig. 5B). Similarly, the expression of PTEN was found to be lower in BC cells compared with normal mammary epithelial cells (Fig. 5C).

To further confirm that PTEN is a functional target gene of miR-425-5p, the expression of PTEN was restored by transfecting MDA-MB-231 and MCF-7 cells with a PTEN-containing plasmid (pcDNA-PTEN). Overexpression of PTEN compared to controls (untransfected cells and empty vector) was confirm by RT-qPCR(Fig. S3). Co-transfection with pre-miR-425-5p and pcDNA-PTEN resulted in increased PTEN protein levels (Fig. 6A). Overexpression of miR-425-5p significantly promoted cell proliferation and migration in MDA-MB-231 and MCF-7 (Fig. 6B-E). Overexpression of PTEN significantly inhibited the promotional effects of miR-425-5p on proliferation and migration in MDA-MB-231 and MCF-7 cells (Fig. 6B-E). These results suggest that miR-425-5p plays a biological role through PTEN.