Luminal type A cell line (MCF-7) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Two different types of tamoxifen-resistant breast cancer cell lines were prepared. One cell line, MCF-7 Tam1 (CRL-3435TM; TAMR-H), was purchased from ATCC, and the other, a tamoxifen-resistant cell line (TAMR-V), was provided and authenticated by the University of Virginia Hospital [30, 31]. The TAMR-V has been continuously exposed to tamoxifen for more than 10 years to maintain its resistance. MCF-7 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), and TAMR-V cells were maintained in DMEM containing 10% FBS and 10− 7 mol/L TAM (Sigma-Aldrich, St. Louis, MO, USA). TAMR-H cells were maintained in DMEM containing 10% FBS, 10-µg/mL human insulin (Sigma), and 1-µM 4-hydroxytamoxifen (Sigma).

Next-generation sequencing (NGS) was performed to distinguish two different types of TAMR cell lines, and the results are presented as a heatmap (Supplementary Fig. 1). All cells were cultured for three days to verify their growth without any treatment, and it was confirmed that they proliferated well (Supplementary Fig. 2).

Profiling and confirmation of lncRNA expression via quantitative reverse transcription polymerase chain reaction (RT-qPCR).

Total RNA was isolated from breast cancer cells using RNAiso Plus (Takara, Otsu, Japan), and its concentration was measured using NanoDrop ND-2000 (Thermo Scientific, Wilmington, DE, USA). For lncRNA expression profiling, 2-µg total RNA was reverse transcribed to cDNA using Human LncProfilers™ qPCR Array Kits (System Biosciences, Mountain View, CA, USA).

To confirm the LncProfilers™ qPCR array results, the total RNA in breast cancer cells was extracted and random hexamer (ELPIS-Biotech Inc., Daejeon, South Korea) was applied, following the manufacturer’s protocols. The expression levels of specific lncRNAs, including SOX2 and SOX2OT, were further confirmed by RT-qPCR. This was performed using both TaqMan Gene Expression Master Mix and Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA).

The relative gene expression was normalized to the housekeeping genes GAPDH and β-actin, employing the 2−ΔΔCT method. Primers and TaqMan probes used were for SOX2 (5′-AAC CCC AGA TGC ACA ACT C-3′, 5′-GCT TAG CCT CGT CGA TGA AC-3′, Hs04234836-s1), SOX2OT (5′-GCT CGT GGC TTA GGA GAT TG-3′, 5′-CTG GCA AAG CAT GAG GAA CT-3′, Hs00415716_m1), GAPDH (5′-ACG GGA AGC TTG TCA AT-3′, 5′-TGG ACT CCA CGA CGT ACT CA-3′, Hs99999903_m1), and for β-actin (5’-TTG CCG ACA GGA TGC AGA A-3’ and 5’-GCC GAT CCA CAC GGA GTA CT-3’). Amplification efficiencies for both TaqMan and SYBR Green methods were tested, and all samples were evaluated in triplicate to ensure accuracy and reproducibility.

SOX2OT sequences were synthesized by Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA), and SOX2OT cDNA fragments were cloned into the pcDNA 3.1 vector to overexpress SOX2OT (Thermo Fisher Scientific). The empty pcDNA3.1 vector was used as the control and pcDNA-SOX2OT or pcDNA-vector transfection into cells was performed using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific), according to the manufacturer’s protocol.

Cells were inoculated in a six-well plate (4 × 105/well) for the wound healing assay and in a 96-well plate (5 × 103/well) for the MTT assay. Following culture overnight, cells were transfected using Lipofectamine™ RNAiMAX reagent (Invitrogen) with the transformed specific SOX2OT pcDNA3.1(+) plasmid or pcDNA3.1(+) control plasmid. At 48 h following transfection, the MTT substrate was prepared in a physiologically balanced solution, added to cells in culture typically at a final concentration of 50 µL, and incubated at 37 °C for 4 h. Following removal of the medium, 150-µL dimethyl sulfoxide was added, and the mixture was shaken for 10 min. The quantity of formazan (presumably directly proportional to the number of viable cells) was measured by recording changes in absorbance at 570 nm using microplate spectrophotometer (Agilent Technologies, Winooski, VT, USA). All of the experiments were conducted in triplicate.

To determine the effect on cell migration, TAMR-V and TAMR-H cells were seeded in six-well culture plates and transfected with control pcDNA 3.1(+) or SOX2OT-pcDNA 3.1(+). Subsequently, a line was scratched into the cell monolayer using a sterile pipette tip, and the cells were further incubated. Images were captured at 0-, 48-, and 72-h timepoints using a microscope and camera system (Olympus Co., Tokyo, Japan). Wound closure was measured using the ImageJ wound healing tool (ImageJ. Available online: https://imagej.net/Welcome). The data were representative of three independent experiments.

For the cell invasion assay, transwell chambers with 8-µm pores were coated with Matrigel (Corning Inc., Tewksbury, MA, USA) and incubated at 37 °C for 4 h, allowing it to solidify. Following 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. Following 48-h incubation, 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. Four random fields were selected for each culture well under a light microscope, and the number of cells in each view was counted.

Data were presented as means (± standard deviation, SD) of three or more independent experiments. The differences in experimental results between the two groups were analyzed using Student’s t-test, and a statistically significant difference was considered at p < 0.05 or p < 0.001.