A human embryonic kidney cell line, HEK293 and a human breast cancer cell line, MCF7, were obtained from the Cell Bank of Central South University (Changsha, China). MCF7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. MCF7 cells were treated with E2 for 3 h, then cell viability and migration rates were analyzed.

An MTT assay was used to examine cell viability. MCF7 cells (5,000 per well) were cultured in a 96-well plate. Each well contained 100 µl fresh serum-free DMEM with 0.5 g/l MTT. Following an incubation at 37°C for 0, 12, 24, 48 or 72 h, the medium was removed by aspiration and 50 µl dimethyl sulfoxide (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well. After incubation at room temperature for 10 min, formazan production was detected by measuring the optical density at 570 nm using an AIA 600II Automated Enzyme Immunoassay Analyzer (Tosoh Corporation, Tokyo, Japan).

A wound healing assay was used to examine cell migration. MCF7 cells were cultured at 37°C with 5% CO₂ to full confluence in 6-well plates. Wounds of ~1 mm width were created with a plastic scriber. After that, cells were washed with phosphate-buffered saline (PBS) and incubated at 37°C with 5% CO₂ for 48 h. Then, the wounds were observed and photographed under an Eclipse Ti-E inverted microscope (Nikon Corporation, Tokyo, Japan).

MCF7 cells were transfected with a scrambled miR mimic (Yearthbio, Changsha, China) as a negative control (miR-NC), an miR-148a mimic (Yearthbio), a negative control inhibitor (Yearthbio), an miR-148a inhibitor (Yearthbio), ERα siRNA (Yearthbio) or pcDNA3.1-ERα ORF plasmid (Yearthbio) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocols. Transfection efficiency was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting as follows.

Total RNA from MCF7 cells was isolated using Trizol reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Total RNA was reverse transcribed using the RevertAid Reverse Transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, qPCR was conducted using the SYBR-Green miScript PCR kit (Qiagen, Inc., Valencia, CA, USA) and a Roche LightCycler 480 PCR machine (Roche Diagnostics, Basel, Switzerland). All primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The primers for ERα were: Forward, 5′-CCCACTCAACAGCGTGTCTC-3′ and reverse, 5′-CGTCGATTATCTGAATTTGGCCT-3′. The primers for GAPDH were: Forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. The reaction conditions were: 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 30 sec. The relative mRNA expression of ERα was normalized against that of GAPDH. The relative expression of miR-148a was normalized against that of U6. The relative expression was analyzed by the 2^−ΔΔCq method (21).

Cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). The concentration of protein was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). After that, proteins (50 µg) were separated using 10% SDS-PAGE gels (Beyotime Institute of Biotechnology) and blotted onto a polyvinylidene difluoride (PVDF) membrane (Life Technologies, Grand Island, NY, USA), which was then blocked with 5% non-fat dried milk (Mengniu, Beijing, China) in PBS with Tween (PBST; Beyotime Institute of Biotechnology) overnight at 4°C. The PVDF membrane was then incubated with rabbit monoclonal anti-human ERα antibody (1:100; ab32063; Abcam, Cambridge, MA, USA), or rabbit monoclonal anti-human GAPDH antibody (1:100; ab9485; Abcam) antibody at room temperature for 3 h. After being washed with PBST three times, the PVDF membrane was incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; ab7090; Abcam) for 40 min at room temperature. Chemiluminescent detection was performed with an ECL kit (Thermo Fisher Scientific, Inc.). The relative protein expression was determined using Image-Pro plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.

TargetScan 3.1 online software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) was used to analyze the putative target genes of miR-148a (22).

A wild type (WT)-or mutant type (MT)-ERα 3′UTR was inserted downstream of the luciferase reporter gene in a pMIR-REPORT vector (Yearthbio), generating a WT-ERα-3′UTR reporter vector or MT-ERα-3′UTR reporter vector, respectively. HEK293 cells were co-transfected with miR-148a mimic (Yearthbio) or miR-NC (Yearthbio), a WT-ERα-3′UTR or MT-ERα-3′UTR reporter vector, and pRL-SV40 (Promega Corporation, Madison, WI, USA) expressing Renilla luciferase using Lipofectamine^® 2000 according to the manufacturer's protocol. Following incubation at 37°C with 5% CO₂ for 48 h, the luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corporation), according to the manufacturer's instructions.

Data are expressed as the mean ± standard deviation of three independent experiments. The Student's t-test was used to compare differences between two groups. One-way analysis of variance was used to analyze differences among more than two groups. Statistical analysis was conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a statistically significant difference.

It has been well established that MCF7 is an ERα-positive breast cancer cell line (23). In the current study, MCF7 were treated with E2 (1 mM) for 3 h, followed by analyses of cell viability and migration. MTT assay data indicated that MCF7 cell viability was significantly increased following treatment with E2 for 48 h (P<0.05) or 72 h (P<0.01), as compared with the control group (Fig. 1A). Furthermore, wound healing assay data showed that E2 treatment also upregulated the migration of MCF7 cells at 48 h, when compared to the control group (P<0.01; Fig. 1B). These results suggest that E2 activates ERα-mediated viability- and migration-related signaling pathways in MCF7 cells.

As the function of E2 is through activation of ERα-mediated downstream signaling, a bioinformatics search was conducted for putative miRs that directly target ERα. As indicated in Fig. 2A and B, ERα was predicted to be a direct target of miR-148a, and the targeting relationship was evolutionarily conserved. In order to examine this targeting relationship, the WT- or MT-ERα-3′UTR was inserted downstream of the luciferase reporter gene in a pMIR-REPORT vector (Fig. 2C and D). A luciferase reporter assay demonstrated that transfection with the miR-148a mimic significantly reduced the luciferase activity in HEK293 cells transfected with WT-ERα-3′UTR reporter vector, but not in cells transfected with MT-ERα-3′UTR reporter vector, when compared to the control group (P<0.01; Fig. 2E). These findings indicated that ERα was a direct target of miR-148a.

As miRs most often inhibit the expression of their target genes at the post-transcriptional level, the effects of miR-148a upregulation and downregulation on the protein level of ERα in MCF7 cells were evaluated. MCF7 cells were transfected with miR-NC, miR-148a mimic, NC inhibitor, or miR-148a inhibitor. After transfection, qPCR was used to evaluate the level of miR-148a expression in each group. As indicated in Fig. 3A and B, transfection with an miR-148a mimic significantly increased the level of miR-148a expression, while transfection with an miR-148a inhibitor significantly decreased the level of miR-148a expression in MCF7 cells as compared with the control group (P<0.01 for both).

Next, a western blot analysis was conducted to determine the protein level of ERα. Overexpression of miR-148a significantly reduced the level of ERα protein (Fig. 3C), while knockdown of miR-148a significantly increased the level of ERα protein in MCF7 cells, as compared with the control (P<0.01 for both; Fig. 3D). In addition, neither miR-148a upregulation nor downregulation affected the mRNA expression of ERα in MCF7 cells (Fig. 3E and F). These results suggested that miR-148a negatively regulated the expression of ERα at the post-transcriptional level in MCF7 cells.

As E2 functions through activation of ERα-mediated signaling, it was examined whether miR-148a could affect E2-induced MCF7 cell viability and migration. MCF7 cells with or without miR-148a overexpression were treated with E2 for 3 h. An MTT assay was then used to examine the cell viability in each group. It was found that the viability rate of miR-148a-overexpressing cells was significantly reduced at 48 and 72 h, as compared with the control group (P<0.01; Fig. 4A). A wound healing assay showed that the scratch width for miR-148a-overexpressing cells was significantly larger at 48 h compared with the control group, indicating that the migration rate of miR-148a-overexpressing cells was significantly reduced (P<0.01; Fig. 4B).

In order to investigate the underlying mechanism of miR-148a further, MCF7 cells were transfected with ERα-specific small interfering RNA (siRNA), which significantly decreased the protein level of ERα as compared with the control (P<0.01; Fig. 4C). MCF7 cells with or without ERα knockdown were treated with E2 for 3 h. An MTT assay showed that the viability of MCF7 cells was significantly decreased after knockdown of ERα for 48 or 72 h, when compared to the control group (P<0.01; Fig. 4D). A wound healing assay showed that the scratch width for MCF7 ERα-knockdown cells was significantly larger at 48 h compared with the control group, indicating that the migration rate of MCF7 cells was significantly decreased after knockdown of ERα (P<0.01; Fig. 4E). These results suggest that both miR-148a overexpression and ERα knockdown result in suppressed E2-induced viability and migration of MCF7 cells.

In order to investigate whether the effects of miR-148a on E2-induced MCF7 cell viability and migration were through directly targeting ERα, MCF7 cells were co-transfected with miR-148a mimic and ERα ORF plasmid. A western blot analysis showed that the protein level of ERα was significantly higher in cells co-transfected with miR-148a mimic and ERα ORF plasmid, when compared with cells transfected with miR-148a mimic alone (P<0.01; Fig. 5A). This indicated that transfection with ERα ORF plasmid rescued the suppressive effect of miR-148 on the protein expression of ERα in MCF7 cells. Next, MCF7 cells in each group were treated with E2 for 3 h, and MTT and wound healing assays were performed. The viability of MCF7 cells was significantly increased in cells at 24, 48 and 72 h after co-transfection with miR-148a mimic and ERα ORF plasmid, as compared with miR-148a-overexpressing cells without plasmid (P<0.01; Fig. 5B). In a wound healing assay, the scratch width of MCF7 cells at 48 h after co-transfection with miR-148a and ERα ORF plasmid was significantly smaller compared with miR-148a-overexpressing cells without plasmid, indicating that the migration rate of was significantly increased (P<0.01; Fig. 5C). These results suggest that miR-148a inhibits E2-induced MCF7 cell viability and migration via inhibition of ERα expression.

The effect of E2 treatment on the expression of miR-148a in MCF7 cells was examined using qPCR. It was found that E2 treatment decreased the miR-148a levels in MCF7 cells in a dose-dependent manner (Fig. 6). A dosage of 1 mM significantly reduced miR-148a expression as compared with the control (P<0.01). Furthermore, a dosage of 10 mM significantly reduced miR-158a expression as compared with a dosage of 1 mM. These results suggest that E2 treatment decreases miR-148a expression, which increases the ERα protein level in MCF7 cells. Through activation of ERα-mediated downstream signaling, E2 treatment induces the viability and migration of breast cancer cells.