The human breast cancer cell lines, MCF-7 and SKBR3, were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultivated in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C with 5% CO₂.

DOX was purchased from the First Affiliated Hospital of Kunming Medical University (Kunming, China). MCF-7 and SKBR3 cells were cultured in DOX-free Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) until cells were in the logarithmic growth phase, at which point the culture medium was replaced with DMEM medium containing a low dose of DOX (0.2 µg/ml). Subsequently, the concentration of DOX was increased from 0.2 to 0.6 µg/ml in 0.04 µg increments every 3 weeks. The DOX-resistant MCF-7 and SKBR3 cell lines were considered to have been successfully established when cells were able to survive under high DOX treatment (0.6 µg/ml). Prior to subsequent experimentation, cells were cultured in the drug-free DMEM medium containing 5% CO₂ at 37°C for 2 days.

LncRNA-HOTAIR small interfering RNAs (siRNAs or siR; siR-HOTAIR1, 3′-GAACGGGAGUACAGAGAGAUU-5′; siR-HOTAIR1-2, 3′-CCACAUGAACGCCCAGAGAUU-5′) were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Negative control siRNA (3′-CUACAACAGCCACAACGUCdTdT-5′) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Briefly, 500 ng siR-HOTAIR1 was transfected into MCF-7 cells and DOXR-MCF-7 cells using 1 µl Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were incubated at 37°C in an atmosphere containing 5% CO₂ for 6 h. Subsequently, transfected cells were incubated at 37°C for 72 h. Cells were then divided into the following experimental groups: Untransfected MCF-7 cells, negative control siRNA-transfected MCF-7 cells, siR-HOTAIR1 transfected MCF-7 cells; untransfected DOXR-MCF-7 cells, negative control siRNA-transfected DOXR-MCF-7 cells and siR-HOTAIR1 transfected DOXR-MCF-7 cells.

Total RNA was extracted from MCF-7 and DOXR-MCF-7 cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was then reverse-transcribed into cDNA using an RT kit (Takara Biotechnology Co., Ltd., Dalian, China) following the manufacturer's protocol. qPCR was performed using the SYBR Premix Ex Taq (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling conditions: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. Relative expression levels were calculated using the 2^−ΔΔCq method (18). GAPDH was used as the reference gene. The following primers were used for amplification: GAPDH forward, 5′-ATTGATGGATGCTAFGAGTATT-3′ and reverse, 5′-AGTCTTCTGGGTGGCAGTGAT-3′; lncRNA-HOTAIR forward, 5′-CGGAGTGAGTTTATCGCAG-3′ and reverse, 5′-GGCGACCGGAGCTCATCTTACC-3′.

Following 72 h incubation, cell proliferation analysis was performed using an MTT assay kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). MCF-7 and DOXR-MCF-7 cells (5×10⁵ cells/well) were seeded into 96-well plates. Subsequently, cell proliferation was detected at different time points (24, 48 and 72 h). MTT (5 mg/Ml, 20 µl) was added to cells and incubated for 4 h at 37°C. The precipitate was dissolved in 100 µl dimethyl sulfoxide and absorbance at 490 nm was detected using a microplate spectrophotometer.

A colony formation assay was performed following transfection. Cells (500 cells/well) were seeded into 6-well plates and incubated for 10 days in 5% CO₂ at 37°C to form colonies. Cells were fixed in 95% methanol for 15 min at 4°C and stained with 1% crystal violet solution for 30 min at room temperature. Images were captured under a light microscope (magnification, ×100). Colony number was quantified using Alpha-View Software 3.1 (FluorChem Q; ProteinSimple, San Jose, CA, USA).

Cell apoptosis was assessed using a fluorescein isothiocyanate (FITC) apoptosis detection kit (Oncogene Research Products, La Jolla, CA, USA) in accordance with the manufacturer's protocol. Cells were harvested, washed twice with pre-cooled PBS and stained successively with propidium iodide (10 µl) and Annexin-V-FITC (10 µl; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Following 15 min of incubation at room temperature, a flow cytometer (FACSVantage SE; BD Biosciences, San Jose, CA, USA) was used for sample analysis using the FlowJo software package (version 10.0.7; Tree Star, Inc., Ashland, OR, USA). The number of cells in each classification was presented.

Western blotting was performed to assess the expression of proteins of interest. Proteins were isolated using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Proteins (20 µg/per lane) were subsequently separated via SDS-PAGE on 10% gels using polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) for protein transfer. Membranes were then blocked with 5% nonfat milk for 2 h at room temperature and incubated with the following primary antibodies obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA): Anti-Bcl-2-associated X protein (Bax; 1:1,000; cat. no. 2774), anti-B-cell lymphoma 2 (Bcl2; 1:1,000; cat. no. 2872), anti-cleaved caspase3 (1:500; cat. no. 9664), anti-PI3K (1:1,000; cat. no. 4255), anti-AKT (1:500; cat. no. 9272), anti-mTOR (1:500; cat. no. 2972), anti-phospho (p)-mTOR (ser2448; 1;1,000; cat. no. 5536), anti-p-PI3K (tyr458; 1:1000; cat. no. 4228), anti-p-AKT (ser473; 1:500; cat. no. 4060), anti-MRP1 (1:500; cat. no. 14685), anti-multidrug resistance protein 1 (MDR1; 1:500; cat. no. 13978), anti-ATP binding cassette subfamily B member 1 (ABCB1; 1:1,000; cat. no. 12683) and anti-GAPDH (1:2,000; cat. no. 5174). The membrane was then incubated with goat anti-rabbit IgG secondary antibodies (1:3,000; cat. no. ab6721; Abcam, Cambridge, UK) for 1 h at room temperature. An enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for visualization and the images were analyzed using ImageJ v1.8.0 (National Institutes of Health, Bethesda, MD, USA). The relative quantification of protein expression was analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Statistical analysis was performed using SPSS version 20.0 software (IBM Corp, Armonk, NY, USA). Each experiment was repeated in triplicate and all data are presented as the mean ± standard deviation. Differences among multiple groups were detected using one-way analysis of variance followed by the Dunnett's post hoc-test. P<0.05 was considered to indicate a statistically significant difference.

The results of RT-qPCR revealed that HOTAIR was upregulated in the drug resistant breast cancer cell line, DOXR-MCF-7, compared with the other cell lines. HOTAIR expression also significantly differed between MCF-7 and MCF-7-DOX cell lines (Fig. 1A). Therefore, MCF-7 and MCF-7-DOX cell lines were used for the subsequent experimentation. The interference efficiency of HOTAIR siRNA was determined via RT-qPCR. The results revealed that the expression of lncRNA-HOTAIR1 was significantly reduced in the siR-HOTAIR1 and siR-HOTAIR1-2 groups compared with the negative control (Fig. 1B). The siR-HOTAIR1 plasmid, which exhibited the greatest interference effect, was selected for subsequent experimentation.

An MTT assay was performed to assess the proliferation of siR-HOTAIR1-transfected MCF-7 cells using optical density values (Fig. 1C). The results indicated that proliferation was markedly decreased in the siR-HOTAIR1 group compared with the control and NC groups. Subsequently, a colony formation assay was performed to further assess the proliferation rate of MCF-7 cells (Fig. 1D). The results demonstrated that cell colony formation ability was significantly decreased in the siR-HOTAIR1 group compared with the control and NC groups. Furthermore, the results of flow cytometry revealed that the rate of apoptosis was increased in the siR-HOTAIR1 group compared with the control and NC groups (Fig. 2). These results indicate that the knockdown of lncRNA-HOTAIR may suppress the proliferation of MCF-7 cells.

DOXR-MCF-7 cells were transfected with siR-HOTAIR1 and an MTT assay revealed that proliferation was significantly decreased in the siR-HOTAIR1 group compared with the control and NC groups (Fig. 3A). Additionally, cell colony formation was significantly decreased in the Dox-siR-HOTAIR1 group (Fig. 3B and C). Furthermore, the apoptosis rate of Dox-siR-HOTAIR1 cells was significantly increased compared with the control group (Fig. 4). The results indicate that lncRNA-HOTAIR may serve a key role in the resistance to DOX in MCF-7 cells.

The activation of programmed cell death machinery is considered to be associated with the inhibition of cell proliferation. To assess the effect of siR-HOTAIR1 on cell apoptosis, the expression of apoptosis-associated proteins was determined via western blot analysis. Caspase-3, Bcl-2 and Bax have essential roles in cell apoptosis and tumorigenesis. As demonstrated in Fig. 5, the protein levels of caspase-3 and Bax were increased, and the expression of Bcl-2 was markedly decreased in the DOXR-MCF-7 siR-HOTAIR1 group compared with control group. The results indicated that HOTAIR silencing may affect proliferation and promote apoptosis by regulating the expression of apoptosis-associated proteins.

Classical multidrug-resistant proteins, MDR1, MRP1 and ABCB1 were detected to determine the effect of lncRNA-HOTAIR on the drug resistance of DOXR-MCF-7 cells. The protein levels of MDR1, MRP1 and ABCB1 were significantly decreased in DOXR-MCF-7 siR-HOTAIR1 cells compared with the siR-NC DOXR-MCF-7 cells (Fig. 6).

The PI3K/AKT/mTOR pathway serves a key role in cell proliferation, cellular metabolism, protein synthesis and in the cell cycle (14). It is considered to be one of the most frequently mutated pathways in cancer (14). To determine the effect of HOTAIR silencing on the PI3K/AKT/mTOR pathway, the phosphorylation of these proteins were analyzed via western blotting. The protein expression of PI3K, AKT and mTOR remained unchanged, while the phosphorylation of PI3K, AKT and mTOR was significantly reduced by HOTAIR silencing in DOXR-MCF-7 cells compared with the control and NC groups (Fig. 7). This series of analyses indicate that siR-HOTAIR alters the resistance of MCF-7 cells to DOX.