To gain comprehensive data of gene expression in breast cancer cell lines, four cell lines were used in the present study to cover three common breast cancer subtypes: the ER/PR⁺ cell lines MCF-7 and T47D; the HER2⁺ cell line SKBR3; and the TNBC cell line MDA-MB-231 (23). The breast cancer cell lines and the normal breast cell line MCF-10A were obtained from the American Type Culture Collection. The breast cancer cells were cultured in DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ environment at 37°C. The MCF-10A cell line was cultured in DMEM/F12 (1:1; Hyclone; GE Healthcare Life Sciences) supplemented with 5% FBS, 10 µg/ml insulin, 20 ng/ml epidermal growth factor (EGF), 100 ng/ml cholera toxin and 0.5 mg/ml hydrocortisone at 37°C with 5% CO₂. MCF-7 and MDA-MB-231 cells were used as representative for ER⁺ and ER^- breast cancer, respectively, for subsequent functional assays.

miR-124 mimics, miR-124 inhibitors, NEAT1 small interfering RNA (siRNA), STAT3 siRNA and the corresponding negative controls were synthesized by GenePharma Co., Ltd (the sequences of the oligonucleotides are provided in Table I). The full-length sequences of NEAT1 or STAT3 were amplified by PCR and subcloned into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate the pcDNA-NEAT1 (NEAT1) or pcDNA-STAT3 (STAT3) overexpression plasmids, respectively. NEAT1, STAT3 and miR-124 levels were overexpressed by transfection with the NEAT1-overexpressing vector, STAT3-overexpressing vector and miR-124 mimics (50 nM). NEAT1, STAT3 and miR-124 levels were depleted by transfection with NEAT1 siRNA, STAT3 siRNA and miR-124 inhibitors (100 nM). All transfections were performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total RNA and protein were extracted at 48 or 72 h post-transfection, respectively.

All clinical samples (31 pairs of matched breast cancer and normal breast tissue samples; age, 29-65) were obtained from the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between November 2016 and December 2017. The study was approved by the Ethics Committee of Xi'an Jiaotong University First Affiliated Hospital and each patient provided written informed consent. The specimens were resected and frozen in liquid nitrogen immediately after surgery. None of the patients had received any preoperative local or systemic treatment.

Reverse transcription-quantitative PCR (RT-qPCR). Total RNA was extracted from surgical specimens and cultured cells using RNAiso Plus (Takara Biotechnology Co., Ltd.) according to the manufacture's protocol. Reverse transcription was performed using PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd.) as previously described (24). PCR was conducted using SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus2X; Takara Biotechnology Co., Ltd.) on a CFX96TM Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: 30 sec at 95°C, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. β-actin was used as internal control for NEAT1 and STAT3. U6 was used as internal control for miR-124. The relative expression levels were calculated using the 2^−ΔΔCq method (25). Five normal breast tissue samples derived from breast cancer patients were used as the normal control. The PCR primers used in this study are provided in Table II.

Total protein was extracted from cells by using a Total Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's instructions. Measurement of protein concentration was conducted with a protein bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). The total protein extracts (20 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). Non-specific binding sites were blocked by incubation in 5% non-fat milk for 2 h at room temperature. Subsequently, the membranes were incubated with anti-STAT3 antibody (1:1,000; cat. no. ab68153; Abcam) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. sc2004; Santa Cruz Biotechnology, Inc.). An anti-β-actin antibody (1:5,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA) was used as the loading control. Protein signals were visualized using an enhanced chemiluminescence kit (EMD Millipore).

The putative binding sites for miR-124 were predicted using Targetscan (targetscan.org/mamm_31/), StarBase (starbase.sysu.edu.cn/) and miRcode (mircode.org/). The fragments containing the wild-type or mutant miR-124-binding sites of NEAT1 and STAT3 3'-UTRs were cloned into the pGL3-control vector (Promega Corporation) at the NheI and XhoI restriction sites to construct the luciferase reporter vectors. The putative STAT3-binding sites in the NEAT1 promoter were identified by using the University of California Santa Cruz (UCSC) Genome Browser (genome-asia.ucsc.edu/index.html) and the JASPAR database (jaspardev.genereg.net/). The NEAT1 promoter fragments containing putative STAT3-binding sites were inserted into the pGL3-basic vector (Promega Corporation). MDA-MB-231 cells were seeded in 24-well plates and cotransfected with luciferase reporter vectors, miR-124 mimics, STAT3 siRNA or negative control, and pRL-TK Renilla vector (Promega Corporation), using Lipofectamine^® 2000, according to the manufacturer's instructions. At 48 h post-transfection, the cells were lysed and the firefly and Renilla luciferase activities were detected using a Dual-Luciferase Reporter Assay System (Promega Corporation).

An MTT assay was used to measure the cell viability of MCF-7 and MDA-MB-231 lines. At 24 h post-transfection, cells were seeded in 96-well plates at a density of 4×10³ cells/well and cultured for 24, 48 and 72 h. Then, the MTT assay was conducted as previously described (26).

At 24 h post-transfection, MCF-7 and MDA-MB-231 cells were seeded in 6-well plates at a density of 500 cells/well and cultured for 2 weeks with medium replacement every 3 days. Then the cells were fixed and stained with 0.1% crystal violet at room temperature. The colonies containing >50 cells were manually counted and imaged under a microscope.

MCF-7 and MDA-MB-231 cells were seeded in 6-well plates and transfected with oligonucleotides as indicated. At 48 h post-transfection, the cells were trypsinized and fixed in 70% ethanol at 4°C overnight. Then the cells were treated with RNase A and stained with propidium iodide (PI; Sigma-Aldrich; Merck KGaA) for 30 min. Flow cytometry was conducted on a BD FACSCalibur flow cytometer (BD Biosciences). The results were analyzed using the ModiFit LT V3.3.11 software (Verity Software House).

Statistical analyses were performed using SPSS software (version 20.0; IBM Corp). Data are presented as the mean ± standard deviation of at least three independent experiments. Comparisons between two groups were conducted using the Student's t-test (two-tailed). Multiple group comparisons were conducted using one-way ANOVA followed by Dunnett's or least significant difference post hoc tests. Correlation analyses between gene expression were performed with Pearson's correlation test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of NEAT1 and miR-124 in breast cancer samples and cell lines were examined by RT-qPCR. NEAT1 levels in breast cancer tissues and cell lines were significantly elevated compared with normal breast tissues and MCF-10A cells (Fig. 1A and B). By contrast, miR-124 levels in breast cancer tissues and cell lines were significantly reduced compared with normal breast tissues and MCF-10A cells (Fig. 1C and D). These results suggested that NEAT1 overexpression and miR-124 downregulation may be associated with breast carcinogenesis.

To explore the biological function of NEAT1 in breast cancer cells, MCF-7 and MDA-MB-231 cells were transfected with the pcDNA-NEAT1 plasmid or NEAT1 siRNA, respectively. The transfection efficiency was determined by RT-qPCR analysis (Fig. 2A). MTT and colony formation assays demonstrated that the proliferation of MCF-7 cells was promoted by NEAT1 overexpression, whereas proliferation of MDA-MB-231 cells was inhibited by NEAT1 silencing (Fig. 2B and C). In addition, NEAT1 overexpression accelerated MCF-7 cell cycle progression, whereas G0/G1 cell cycle arrest was observed in the NEAT1-silenced MDA-MB-231 cells (Fig. 2D). These results suggested that NEAT1 promoted cell proliferation and cell cycle progression in breast cancer cells.

Bioinformatics analysis based on the online database tools StarBase and miRcode indicated that NEAT1 contains a putative binding site for miR-124. The complementary binding region between miR-124 and NEAT1 is shown in Fig. 3A. To validate whether NEAT1 is a direct target of miR-124, a luciferase activity assay was performed in MDA-MB-231 cells. Luciferase activity of wild-type NEAT1 constructs (NEAT1-wt) was significantly reduced when cotransfected with miR-124 mimics. By contrast, the luciferase activity of the mutated NEAT1 construct (NEAT1-mut) was not affected by miR-124 mimics transfection, confirming the functionality of the miR-124 binding site (Fig. 3B). Furthermore, miR-124 levels were increased following NEAT1 knockdown in MCF-7 and MDA-MB-231 cells (Fig. 3C). In addition, there was a negative correlation between NEAT1 and miR-124 levels in breast cancer tissues (Fig. 3D). These results suggested that NEAT1 may function as a miR-124 sponge in breast cancer cells.

To investigate the role of miR-124 in breast cancer, MCF-7 and MDA-MB-231 cells were transfected with miR-124 mimics. RT-qPCR analysis revealed that miR-124 mimics significantly elevated miR-124 levels (Fig. 4A). Additionally, miR-124 overexpression suppressed STAT3 expression in both the mRNA and protein levels (Fig. 4B and C). Bioinformatics analysis using Targetscan identified a putative miR-124 binding site in the 3'-UTR of the STAT3 mRNA (Fig. 4D). To investigate whether miR-124 directly targeted STAT3, luciferase reporter vectors containing a wild-type 3'-UTR fragment of STAT3 (STAT3-wt) or a mutant 3'-UTR fragment of STAT3 (STAT3-mut) were generated. The luciferase activity assay indicated that miR-124 overexpression dramatically reduced the luciferase activity of the STAT3-wt vector, but not the STAT3-mut vector (Fig. 4E). In addition, RT-qPCR analysis revealed that STAT3 mRNA expression levels were significantly elevated in breast cancer tissues compared with normal breast tissues (Fig. 4F), and negatively correlated with miR-124 levels in breast cancer tissues (Fig. 4G). These results suggested that STAT3 was a direct target of miR-124 in breast cancer.

First, the efficiency of STAT3 overexpression was confirmed by western blot analysis; STAT3 protein expression levels were significantly elevated in cells transfected with the pcDNA-STAT3 plasmid compared with the empty vector (Fig. 5A). MTT and cell cycle analysis revealed that miR-124 overexpression in breast cancer cells decreased the cell proliferation rate and resulted in a G0/G1 phase cell cycle arrest, while these inhibitory effects were abolished by STAT3 overexpression in miR-124-over-expressing breast cancer cells (Fig. 5B and C). The results suggested that miR-124 inhibits breast cancer cell proliferation and cell cycle progression by targeting STAT3.

Next, the present study investigated whether NEAT1 acts as a ceRNA to increase STAT3 expression in breast cancer cells. RT-qPCR analysis demonstrated that miR-124 overexpression reversed the NEAT1-mediated inhibitory effect on miR-124 expression in MCF-7 cells, and miR-124 depletion weakened the NEAT1 silencing-induced miR-124 upregulation in MDA-MB-231 cells (Fig. 6A). Western blot analysis further revealed that NEAT1 overexpression increased STAT3 protein expression in MCF-7 cells, while this effect was weakened by miR-124 overexpression (Fig. 6B). By contrast, STAT3 expression was decreased in NEAT1-silenced MDA-MB-231 cells, and miR-124 knockdown alleviated the inhibitory effect of NEAT1 silencing on STAT3 expression (Fig. 6B). These results suggested that NEAT1 positively regulated STAT3 expression by acting as a ceRNA that sponges miR-124 in breast cancer cell lines.

Subsequently, the present study investigated whether NEAT1 promoted the proliferation and cell cycle progression of breast cancer cells by targeting the miR-124/STAT3 axis. Functional analysis revealed that miR-124 overexpression effectively reduced NEAT1-induced cell proliferation and cell cycle progression in MCF-7 cells (Fig. 6C-E). Conversely, the inhibitory effects of NEAT1 silencing on cell proliferation and cell cycle were abrogated by miR-124 depletion in MDA-MB-231 cells (Fig. 6C-E). These results suggested that NEAT1 promoted breast cancer cell growth by targeting the miR-124/STAT3 axis.

To explore the crosstalk between NEAT1 and STAT3, their correlation in breast cancer specimens was evaluated. NEAT1 levels were positively correlated with STAT3 mRNA expression levels (Fig. 7A). Then, STAT3 expression was silenced in MCF-7 and MDA-MB-231 cells using specific siRNA (Fig. 7B). RT-qPCR analysis demonstrated that NEAT1 expression levels were decreased following STAT3 silencing (Fig. 7C). Furthermore, multiple putative STAT3 binding sites were identified in the NEAT1 promoter by bioinformatics analysis using the UCSC (genome-asia.ucsc.edu/index.html) and JASPAR (jaspardev.genereg.net/) web-based tools. Two binding sites (S1 and S2) with prediction scores >10 are shown in Fig. 7D. Subsequently, two fragments containing S1 or S2 were cloned into luciferase reporter vectors. The vectors were cotransfected into MDA-MB-231 cells with STAT3 siRNA or negative control siRNA. Luciferase activity analysis revealed that STAT3 silencing significantly reduced the luciferase activity of fragment S1, whereas the luciferase activity of fragment S2 was not affected (Fig. 7E). These results demonstrated that STAT3 positively regulated NEAT1 transcription, and thus formed a positive feedback loop in breast cancer cells.