Long Noncoding RNA snaR Regulates Proliferation, Migration and Invasion of Triple-negative Breast Cancer Cells

Materials and Methods

Human breast cancer cell lines. The normal breast epithelial cells MCF10A and the breast cancer cell lines MDA-MB-231 (basal type, TNBC), MCF-7 (luminal-A type, estrogen receptor-positive breast cancer), SKBR3 (HER2-positive breast cancer), T47D (luminal-A type, estrogen receptor-positive breast cancer), BT474 (estrogen receptor-positive breast cancer), and BT20 (TNBC) were purchased from the American Type Culture Collection (Manassas, VA, USA) (6, 7). MCF10A was maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 10 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 μg/ml insulin. MDA-MB-231, MCF7, and SK-BR3 were maintained in DMEM (Gibco) supplemented with 10% FBS. T-47D, BT-20, and BT-474 cells were cultured in RPMI (Gibco) supplemented with 10% FBS.

RNA extraction and lncRNA profiling. Total RNA was isolated from cells using RNAiso Plus (Takara, Otsu, Japan), in accordance with the manufacturer's instructions. RNA concentration was measured using NanoDrop ND-2000 (Thermo Scientific, Wilmington, DE, USA). RNA was treated with DNA-free™ DNase Treatment and Removal Reagents (Life Technologies, Carlsbad, CA, USA). For lncRNA expression profiling, 2 μg of total RNA was reverse-transcribed to cDNA using Human LncProfilers™ qPCR Array Kits (System Biosciences, Mountain View, CA, USA), in accordance with the manufacturer's recommendations.

Confirmation by quantitative reverse transcription-polymerase chain reaction (RT-PCR). In order to confirm the LncProfilers™ qPCR Array results, the total RNA in breast cancer cells was extracted using RNAiso Plus (Takara) and was treated with DNA-free™ DNase Treatment and Removal Reagents (Life Technologies). Reverse transcription was performed using Superscript III RT (Invitrogen, Carlsbad, CA, USA) and random hexamer (ELPIS-Biotech Inc., Daejeon, South Korea), following the manufacturer's protocol. Real-time PCR was performed for relative quantification of the expression levels of each lncRNA using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCT method was used to determine relative gene expression, and the level of expression was normalized to that of β-actin (ACTB). The primer sequences for real-time PCR were as follows: snaR: 5’-ATT GTG GCT CAG GCC GGT T-3’ and 5’-TTT TTC CGA CCC ATG TGG ACC-3’; urothelial cancer associated 1 (UCA1): 5’-CCC AAG GAA CAT CTC ACC AA-3’ and 5’-GAT GGT CCA AGG GGC TTC-3’; antisense noncoding RNA in the INK4 locus (ANRIL): 5’-TTG TTA GAA ACC AGG CTG CAC-3’ and 5’-TTC TCT CTT TCT GTG GTT TCT CAA T-3; 200-nucleotide-long non-messenger RNA (BC200): 5’-GCC CAG GAG TTC GAG ACC-3’ and 5’-GCT TTG AGG GAA GTT ACG CTT AT-3’; small nucleolar RNA host gene 5 (SNHG5): 5’-GAG CAG CTC TGA AGA TGC AA-3’ and 5’-TTT TAA CCA AGC GAT TTT CCA-3’; ACTB: 5’-TTG CCG ACA GGA TGC AGA A-3’ and 5’-GCC GAT CCA CAC GGA GTA CT-3’.

siRNA transfection. siRNAs specific to snaR and control RNA were synthesized by Cosmogentech (Seoul, South Korea). The sequence of si-snaR was 5’-CCA CAU GGG UCG GAA AAA AUU-3’, and the sequence for the negative control (NC), not showing homology with the human genome, was 5’-UUC UCC GAA CGU GUC ACG UTT-3’ (12).

Cells were inoculated in a six-well plate (2×10⁵/well) for the wound-healing assay or a 96-well plate (5×10³/well) for the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After culture overnight, cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen) with the synthesized specific snaR or control siRNA. At 48 h after transfection, total RNA was extracted and the silencing effect was determined by real-time RT-PCR.

MTT assay. The effect of siRNA interference for snaR on the proliferation of breast cancer cells was determined by the MTT assay (Sigma, St. Louis, MO, USA). Cells at a total volume of 200 μl were inoculated in a 96-well plate, with 5×10³ cells in each well. At each time point, 50 μl of MTT (2 mg/ml) was added to the wells and the cells were cultured at 37°C for 4 h. After removal of the medium, 100 μl of dimethyl sulfoxide was added and the mixture was shaken for 10 min. The optical density was then determined at 570 nm. All of the experiments were conducted in triplicate.

Wound-healing assay. In order to determine effect of siRNA interference for snaR on cell migration, MDA-MB-231 cells were seeded in six-well culture plates and transfected with negative control siRNA or si-snaR. Next, a line was scratched into the cell monolayer using a sterile pipette tip, and the cells were further incubated. Images were then captured with the aid of a microscope after 0, 24, 48 h of incubation. The data are representative of three independent experiments.

Cell invasion assay. For the invasion assay, transwell chambers with 8-μm pores were coated with Matrigel (Corning Inc., Tewksbury, MA, USA) and incubated at 37°C for 2 h, allowing it to solidify. After transfection, cells were resuspended in DMEM containing 1% FBS and plated in the upper chamber at a density of 1×10⁴ cells. The lower chamber contained complete medium supplemented with 10% FBS. After incubation for 48 h, the cells on the internal surface of the chamber bottom were wiped with a cotton swab, fixed with 2% paraformaldehyde, stained with 0.5% crystal violet, and rinsed with phosphate-buffered saline. Cells were counted in four random fields for each well chambers under a light microscope.

Statistical analysis. Data are presented as the mean±standard deviation (SD) of three or more independent experiments. The differences in experimental results between two groups were analyzed using Student's t-test and a statistically significant difference was defined at p<0.05.