Primary human BC and their matched normal adjacent tissues were obtained from 56 BC patients who underwent surgery at The First Hospital of Jilin University (Changchun, China) from July 2014 to July 2016. Patients with incomplete clinical data collection or lack of clinical data or patients who had undergone chemotherapy, radiotherapy or any other treatment before surgery were excluded in the present study. All samples were immediately snap-frozen following surgery in liquid nitrogen and stored at −80°C until RNA extraction. All of the samples and clinical information were harvested after the patients provided written informed consent which was approved by the Institutional Ethics Committees of Jilin University.

The non-cancerous human mammary epithelial cell line: MCF-10A, and 4 human BC cell lines (MCF-7, MDA-MB-231, BT-549 and MDA-MB-453) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF-10A cells were cultured in Dulbeccos modified Eagles medium (DMEM)-F12 (Invitrogen Corp., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, MD, USA) and antibiotics (100 U/ml penicillin or 100 µg/ml streptomycin sulfate) and 20 ng/ml epidermal growth factor (EGF), 0.1 mg/ml cholera toxin (CT), and 10 mg/ml insulin. The BC cell lines were all incubated in DMEM which contained 10% FBS, 100 U/ml penicillin plus 100 mg/ml streptomycin. All cells were grown under a humidified incubator with 5% CO₂ at 37°C. Other media supplies were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The miR-592 mimic and its negative control (miR-NC) oligonucleotides were obtained from GenePharma Co., Ltd. (Shanghai, China). TGF-β2-overexpressed plasmid was granted from Dr Tao Peng (Jilin University). Transfection was performed using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturers instructions. At 48 h post-transfection, the transfection efficiencies were determined using qRT-PCR or western blotting.

Total RNA including miRNAs were extracted from tissues and cultured cells using miRNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentrations of RNA were detected by NanoDrop 2000 (hermo Fisher Scientific, Inc., Waltham, MA, USA). The miRNA reverse transcription was performed according to the instructions of the OneStep PrimeScript^® miRNA cDNA Synthesis kit, and then was quantified using SYBR Premix Ex Taq (both from Takara Biotechnology Ltd., Dalian, China) on an ABI 7900 Sequence Detection System (Life Technologies, Grand Island, NY, USA) following the manufacturer's protocol. The primers of miR-592 and U6 were obtained from Applied Biosystems (Foster City, CA, USA). The real-time PCR for detection of TGF-β2 mRNA was performed according to the instructions of the OneStep SYBR^® PrimeScript^® PLUS RT-PCR kit (Takara Biotechnology Ltd.). The primers for TGF-β2 and GAPDH used in the present study were previously described (18). GAPDH/U6 were used as internal controls, and the relative expression of the target genes were calculated using the 2^−ΔΔCt method.

Transfected BC cells were placed into a 96-well plate, at a density of 5×10³ cells/well. After 24, 48, 72 and 96 h of culture in a CO₂ incubator at 37°C, the proliferation of BC cells was detected by MTT assay as previously described (18). The optical density (OD) at 570 nm of each well was assessed using a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA).

The clonogenic survival assay was used to investigate the colony formation ability. Briefly, the transfected cells (1×10³ cells/well) were plated into 6-well plates, and allowed to attach for 24 h. After being washed with phosphate-buffered saline (PBS), the cells were incubated for 10 days at 37°C in a humidified incubator. Finally, the images of the produced colonies were captured and counted under a light microscope (Olympus, Tokyo, Japan) after being fixed with 10% formaldehyde for 30 min and staining in 0.1% crystal violet solution (both from Sigma-Aldrich) for 10 min.

Transfected cells were incubated into a 6-well plate at 37°C. When the cells fully covered the plate bottom, the confluent monolayer in each well was created with a sterilized tip, ensuring that the width of each line was same. Following a 24-h incubation at 37°C, the cells were washed 3 times with PBS to remove any cell debris caused by the scratches. Images were captured at 0 and 24 h with an Olympus Inverted Microscope at 6 visual fields. The healing rate was calculated with the ImageTool software (Bechtel Nevada, Los Alamos Operations, USA).

A cell invasion assay was performed using 24-well Transwell chambers (8-µm pores; BD Biosciences, San Jose, CA, USA). In short, transfected cells (1×10⁵) were seeded onto Transwell chambers with Matrigel in serum-free medium. DMEM containing 10% FBS was added to the lower chamber as the chemoattractant. After incubation at 37°C for 48 h, the noninvading cells were removed with cotton swabs, whereas the invasive cells at the bottom of the membrane were fixed with 90% alcohol and stained with 0.1% crystal violet for 5 min. The invaded cells were counted and photographed using an Olympus Inverted Microscope in at least 5 randomly selected fields.

A wild-type TGF-β2-3′-UTR, containing the miR-592 binding sites in the 3′-UTR region and a mutant-type-TGF-β2 with a mutant sequence on the miR-592 binding site were amplified from human breast cDNA using PCR, and incorporated into the downstream of the firefly luciferase gene of a the pGL3-control vector (Promega, Madison, WI, USA). For luciferase assays, 1×10⁵ cells were plated in 24-well plates and cultured for 24 h, then the cells were contransfected with 100 ng wild-type or mutant-type reporter constructs, and 50 nM miR-592 mimic or the miR-NC with Lipofectamine 3000 transfection reagent according to the manufacturer's instructions. The luciferase activity was assessed 48 h after transfection with the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was then normalized to the corresponding Renilla luciferase activity.

Total protein was extracted from cultured cells or tissues with RIPA lysis buffer containing a proteinase inhibitor (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) on ice for 30 min. Total protein concentrations were assessed using a bicinchoninic acid (BCA) kit (Wuhan Boster Biotechnology Co., Ltd.). After the addition of the loading buffer, the extracted proteins were heated at 95°C for 10 min. A total of 30 µg loading buffer were separated using 10% SDS polyacrylamide gels (SDS-PAGE), and then were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Laboratories Inc.). The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, following incubation with a primary antibody against TGF-β2 (diluted at 1:1,000) or GAPDH (diluted at 1:5,000) (both from Santa Cruz Biotechnology, Inc., Cruz CA, USA) at 4°C overnight. The membranes were incubated with horseradish peroxidase (HRP)-conjugated corresponding second antibodies (diluted at 1:6,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The protein bands were detected using the SuperSignal West Pico ECL chemiluminescence kit (Thermo Scientific, Rockford, IL, USA) and exposed on an X-ray film. The protein levels were normalized to GAPDH.

Four-week-old BALB/c female nude mice were purchased from the Experiments Animal Center of Changchun Biological Institute (Changchun, China), and kept under specific pathogen-free (SPF) conditions. To establish the BC xenografts, ~2×10⁶ MCF-7 cells were subcutaneously inoculated into the right flank of each nude mouse, respectively. On day 10, when tumors reached ~100 mm³, mice were randomly divided into control and treatment groups (n=10/group). Then, the mice were intratumorally injected with miR-592 mimic or miR-NC 3 times/week for 4 weeks. The tumor width and length were assessed every 7 days using a caliper. The tumor volume was monitored and calculated according to the formula: V (volume) = 1/2 × length × width². The mice were sacrificed at 28 days post-implantation. Xenograft tumors were excised, photographed, weighed and stored at −80°C until use.

All data were analyzed using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA) and were expressed as the mean ± standard deviation (SD) from at least 3 independent experiments. The t-test was used for comparisons between 2 groups. One-way ANOVA was applied for comparisons between multiple groups. The correlations between miR-592 and TGF-β2 were analyzed in BC tissues using Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant result.

To investigate the expression of miR-592 in BC, firstly, 56-paired BC and adjacent normal breast tissues were detected in the present study. As shown in Fig. 1A, the expression of miR-592 was significantly downregulated in BC samples compared to matched adjacent normal tissues (P<0.01). The correlation between the expression levels of miR-592 and the clinico-pathological characteristics were also investigated. As shown in Table I, decreased miR-592 was significantly associated with clinical stage, distant and lymph node metastases (P<0.01; Table I), whereas no statistical difference was found in the correlation between miR-592 expression and age, and tumorsize (P>0.05). Furthermore, we assessed miR-592 expression levels in 4 BC cell lines (MCF-7, MDA-MB-231, BT-549 and MDA-MB-453) by quantitative real-time PCR (qPCR). As shown in Fig. 1B, the expression of miR-592 was found to be downregulated in all BC cell lines in contrast to the expression level of the non-malignant breast epithelial cell line, MCF-10A. Additionally, compared with the other BC cell lines, miR-592 was expressed the lowest in the MCF-7 cell line (Fig. 1B), and was selected for subsequent study.

In order to explore the involvement of miR-592 in BC development and progression, miR-592 mimic or miR-NC were transfected into MCF-7 cells to restore its expression level as assessed by qPCR (Fig. 2A). The effect of miR-592 on cell proliferation was analyzed using an MTT assay. As shown in Fig. 2B, the cells transfected with the miR-592 mimic significantly inhibited cell proliferation compared to cells transfected with miR-NC from day 2 until 4, in a time-dependent manner. Consistent with these results, restored expression of miR-592 also significantly inhibited colony formation in MCF-7 cells (Fig. 2C).

To investigate the effect of miR-592 on the migration and invasion ability of BC cells, wound healing and Transwell invasion assays were performed, respectively. It was found that restoration of miR-592 expression significantly inhibited cell migration and invasion capabilities in MCF-7 cells (Fig. 3A and B), suggesting that miR-592 was able to inhibit the migration and invasion of BC cells in vitro.

TargetScan Human (http://www.targetscan.org) predicted that TGF-β2 is the target gene of the miR-592 (Fig. 4A). To investigate whether TGF-β2 is the target gene of miR-592, we transfected the miR-592 mimic and miR-NC into MCF-7 cells. Consequently, TGF-β2 expression at both the mRNA level and protein level was significantly suppressed in cells transfected with the miR-592 mimic compared to cells transfected with miR-NC (Fig. 4B and C). Meanwhile, luciferase reporter assay further revealed that MCF-7 cells transfected with the miR-592 mimic significantly inhibited the wild-type-TGF-β2-3′-UTR reporter activity (P<0.01; Fig. 4D), while it had no inhibitory effect on the mutant-type-TGF-β2-3′-UTR reporter activity (Fig. 4D). These data revealed that TGF-β2 was a target of miR-592 in BC.

We also evaluated the expression of TGF-β2 in 56-paired BC and adjacent normal breast tissues by qRT-PCR. The data revealed that the average level of TGF-β2 mRNA was significantly increased in BC tissues when compared with adjacent normal tissues. Moreover, TGF-β2 mRNA levels in BC cases were inversely correlated with miR-592 expression (r=−0.529; P<0.0001). In addition, TGF-β2 expression was upregulated at the mRNA level and protein level in 4 BC cell lines compared to non-malignant breast epithelial cell line, MCF-10A (Fig. 5C and D).

To investigate the functional relevance of TGF-β2 targeting by miR-592 in BC, we assessed whether TGF-β2 overexpression rescued the inhibitory effects of miR-592 on BC cell proliferation, migration and invasion. We restored TGF-β2 expression by transfection with TGF-β2 overexpression plasmid in miR-592 mimic transfected MCF-7 cells (Fig. 6A and B). In addition, we also found that the forced expression of TGF-β2 rescued the inhibition of cell proliferation, colony formation, migration and invasion in the presence of the miR-592 mimic in MCF-7 cells (Fig. 6C-F). These findings inferred that miR-592 exerted it suppressive roles in BC by repressing TGF-β2.

Finally, BC nude mice were set-up to investigate the role of miR-592 on cell growth in vivo. It was found that the tumor growth was slower in the MCF-7/miR-592 group compared to the MCF-7/miR-NC group (Fig. 7A). The mice were sacrificed at 28 days post-implantation, and the tumor tissues were stripped and weighed. We found that the size and weight of the tumors of the MCF-7/miR-592 group were significantly decreased compared to the MCF-7/miR-NC group (Fig. 7B and C). Furthermore, increased expression of miR-592 (Fig. 7D), and decreased TGF-β2 expression (Fig. 7E) were found in tumor tissues in the MCF-7/miR-592 group relative to the MCF-7/miR-NC group. These data implied that miR-592 suppressed tumor growth in vivo.