Triple-negative breast cancer (TNBC) cell lines MDA-MB-231 (ATCC, HTB-26) and the non-tumorigenic mammary epithelial cell line Hs578Bst (ATCC, HTB-126) were obtained from the American Type Culture Collection (ATCC, USA). TNBC cells and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Catalog No. 11965092) supplemented with 10 % fetal bovine serum (FBS; Gibco, Catalog No. 10082147), 1 % penicillin-streptomycin (Gibco, Catalog No. 15140122), and 2 mM L-glutamine (Gibco, Catalog No. 25030081). All cell lines were maintained at 37 °C in a humidified atmosphere containing 5 % CO₂. Cells were passaged at 70–80 % confluence using 0.25 % trypsin-EDTA (Gibco, Catalog No. 25200072).

For overexpression of LncRNA-NONHSAT192404.1 and miR-518a-3p, full-length sequences of LncRNA-NONHSAT192404.1 and precursor miR-518a-3p were cloned into the pcDNA3.1(+) vector (Invitrogen, Thermo Fisher Scientific, Catalog No. V79020). For knockdown experiments, small interfering RNAs (siRNAs) targeting LncRNA-NONHSAT192404.1 and antisense oligonucleotides (ASOs) targeting miR-518a-3p were synthesized by GenePharma (Shanghai, China). Cells were transfected with overexpression constructs, siRNAs, or ASOs using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Catalog No. L3000015) according to the manufacturer’s protocol. The sequences of the siRNAs used for knockdown are listed in Supplementary Table 1.

Transfection efficiency was confirmed by quantitative real-time PCR (qRT-PCR) 48 h post-transfection. Scrambled siRNA or ASO was used as a negative control.

Expression levels of LncRNA-NONHSAT192404.1, NONHSAT148701.1, and miR-518a-3p were measured in TNBC cell lines (MDA-MB-231) and compared with the normal mammary epithelial cell line (Hs578Bst). Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Catalog No. 15596026). miRNA was extracted with the mirVana miRNA Isolation Kit without phenol (Thermo Fisher Scientific, AM1561). RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Catalog No. ND-2000). cDNA was synthesized from 1 µg total RNA using the PrimeScript RT Reagent Kit (Takara Bio, Japan, Catalog No. RR037A). qRT-PCR was performed with SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, Catalog No. 4309155) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Catalog No. 4485690). Expression levels of LncRNA-NONHSAT192404.1, miR-518a-3p, and other genes of interest were normalized to GAPDH or U6 snRNA, respectively, and calculated by the 2^-ΔΔCt method. Primer sequences are provided in Supplementary Table 2.

Cell proliferation was assessed with the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Japan, Catalog No. CK04-11). Cells were seeded in 96-well plates at 2,000 cells/well in 100 µL complete medium. After overnight adhesion they were transfected as described. At 24, 48, 72, and 96 h post-transfection, 10 µL CCK-8 solution was added per well and incubated for 2 h at 37 °C. Absorbance was read at 450 nm on a microplate reader (Bio-Rad, USA, Catalog No. 168-1000EDU). Experiments were done in triplicate.

Cell-cycle distribution was analyzed by flow cytometry after propidium iodide (PI) staining. Briefly, MDA-MB-231 cells (transfected as indicated) were harvested 48 h post-transfection, washed twice with ice-cold PBS, and fixed in 70 % ethanol at 4 °C overnight. After washing, cells were incubated with PI (50 µg/mL) and RNase A (100 µg/mL) in PBS for 30 min at room temperature in the dark, and DNA content was measured on a flow cytometer. Percentages of cells in G₀/G₁, S, and G₂/M phases were determined with appropriate software.

Apoptotic cells were quantified by Annexin V-FITC/PI dual staining followed by flow cytometry. Forty-eight hours after transfection, MDA-MB-231 cells were harvested by trypsinization, washed twice with PBS, and resuspended in 100 µL 1× binding buffer. Annexin V-FITC (5 µL) and PI (5 µL) from the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN BioTECH, China; Cat. No. KGA107) were added and incubated for 10 min at room temperature in the dark. Samples were analyzed within 1 h. Cells were classified as viable (Annexin V⁻/PI^-), early apoptotic (Annexin V⁺/PI^-), and late apoptotic/necrotic (Annexin V⁺/PI⁺).

The invasive capacity of TNBC cells was evaluated in Transwell chambers (Corning, USA, Catalog No. 3422) with 8-µm-pore polycarbonate membranes coated with Matrigel (BD Biosciences, USA, Catalog No. 354234) diluted 1:8 in serum-free DMEM. Hs578Bst was included as a negative control. Cells (5 × 10⁴) in serum-free medium were seeded in the upper chamber; DMEM with 10 % FBS was placed in the lower chamber as chemoattractant. After 24 h, non-invading cells were removed, and invading cells were fixed with 4 % paraformaldehyde, stained with 0.1 % crystal violet, and counted under a microscope (Olympus, Japan, Catalog No. CX23) in five random fields. Experiments were done in triplicate.

Transfected TNBC cells were seeded at 5 × 10⁵ cells/well in 6-well plates and grown to 90 % confluence. A linear scratch was made with a 200 µL pipette tip, debris was removed with PBS (Gibco, Thermo Fisher Scientific, Catalog No. 10010049), and cells were cultured in serum-free DMEM. Images were captured at 0, 24, and 48 h with an inverted microscope. Wound width was measured in ImageJ and quantified as previously described31.

To validate the interaction between LncRNA-NONHSAT192404.1 and miR-518a-3p, the pmirGLO dual-luciferase vector (Promega, USA; Cat. No. E1330) was used. A fragment of the TLR4 3′-UTR containing the predicted miR-518a-3p site was cloned downstream of firefly luciferase; a mutant site was generated with the QuikChange II kit (Agilent Technologies, USA; Cat. No. 200523). MDA-MB-231 cells were co-transfected with wild-type, mutant, or empty pmirGLO plus miR-518a-3p mimics (GenePharma, China) using Lipofectamine 3000. After 48 h, luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, USA; Cat. No. E1910). Experiments were done in triplicate.

Direct interaction between LncRNA-NONHSAT192404.1 and miR-518a-3p was tested by a biotin-labeled RNA pull-down assay. Wild-type and mutant transcripts of LncRNA-NONHSAT192404.1 were in vitro transcribed with 3′-biotin and incubated with streptavidin-magnetic beads using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA; Cat. No. PI20164). After incubation with MDA-MB-231 lysates, bound RNA was eluted and miR-518a-3p enrichment quantified by qRT-PCR. Scrambled biotin-RNA served as negative control.

MDA-MB-231 cells were transfected as indicated. Total protein was extracted with RIPA buffer (Cell Signaling Technology, USA, Catalog No. 9806S) plus protease/phosphatase inhibitors (Thermo Fisher Scientific, Catalog No. 78440). Protein concentration was determined with the BCA kit (Thermo Fisher Scientific, Catalog No. 23225). Equal protein amounts (30 µg) were resolved by SDS-PAGE on 4–20 % gradient gels (Bio-Rad, USA, Catalog No. 4561094) and transferred by wet transfer to PVDF membranes (Millipore, USA, Catalog No. IPVH00010). Membranes were blocked with 5 % non-fat milk in TBST (Cell Signaling Technology, Catalog No. 9997S) for 1 h and incubated overnight at 4 °C with primary antibodies (1:1000): TLR4 (Proteintech, 19811-1-AP), AKT (Proteintech, 10176-2-AP), PI3K (Proteintech, 27921-1-AP), PPP2R1A (Proteintech, 15882-1-AP), and GAPDH (Proteintech, 10494-1-AP). After washing, membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Catalog No. 7074S; 1:2000) for 1 h at room temperature. Bands were visualized with ECL substrate (Bio-Rad, Catalog No. 1705061) and imaged on a ChemiDoc XRS+ system (Bio-Rad, USA, Catalog No. 1708265). Densitometry was performed with Image Lab.

Potential miR-518a-3p target genes were predicted with TargetScanHuman 7.2. Functional enrichment (GO terms, KEGG pathways) was analyzed in DAVID v6.8. Protein-protein interaction networks were generated with STRING v11.0 and visualized in Cytoscape v3.

Data are presented as mean ± SD. Two-group comparisons used Student’s t-test; multiple groups used one-way ANOVA with Tukey’s post-hoc test. p < 0.05 was considered significant. Analyses were performed in GraphPad Prism.

To evaluate the expression levels of LncRNA-NONHSAT192404.1 and NONHSAT148701.1 in triple-negative breast cancer (TNBC), we conducted quantitative real-time PCR (qRT-PCR) analyses in the TNBC cell line MDA-MB-231 and compared them with normal mammary epithelial cells (Hs578Bst). The results revealed a significant downregulation of LncRNA-NONHSAT192404.1 in TNBC cells relative to Hs578Bst cells (Figure 1A). Similarly, LncRNA-NONHSAT148701.1 was found to be significantly downregulated in TNBC cells (Supplementary Fig. 1A). These findings imply a potential involvement of LncRNA-NONHSAT192404.1 and NONHSAT148701.1 in the oncogenic processes driving TNBC. We then analyzed survival data from TNBC patients. Kaplan–Meier survival curves were generated to assess the relationship between LncRNA expression levels and patient prognosis. The analysis revealed a significant positive correlation between the expression levels of LncRNA-NONHSAT192404.1 and patient survival, indicating that higher levels of this LncRNA are associated with better outcomes in TNBC patients (Figure 1B). Similarly, increased expression of LncRNA-NONHSAT148701.1 was also found to be positively correlated with patient survival, further supporting its potential role as a prognostic biomarker in TNBC (Supplementary Fig. 1B).

Figure 1

The influence of LncRNA-NONHSAT192404.1 on TNBC cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Overexpression of LncRNA-NONHSAT192404.1 in MDA-MB-231 cells led to a significant decrease in proliferation (Figure 2A-C). In contrast, knock-down of LncRNA-NONHSAT192404.1 resulted in a marked increase in cell proliferation (Figure 2A-C), indicating that LncRNA-NONHSAT192404.1 plays a crucial role in inhibiting TNBC-cell proliferation. To understand the mechanistic basis of LncRNA-NONHSAT192404.1’s effects, we examined its impact on cell-cycle progression. Flow-cytometry analysis revealed that overexpression of LncRNA-NONHSAT192404.1 led to an increase in the proportion of cells in the S phase, with a concomitant decrease in the M phase, suggesting cell-cycle arrest at S (Figure 2D, E). In addition, overexpression significantly increased apoptosis, as evidenced by a higher percentage of Annexin-V-positive cells, whereas knock-down reduced apoptosis rates (Figure 2F, G).

Figure 2

The wound-healing assay further supported these findings, demonstrating that cells overexpressing LncRNA-NONHSAT192404.1 exhibited slower wound closure, while knock-down significantly increased wound-closure ability, suggesting a role in cell migration (Figure 3A, B). To further investigate invasion, Transwell assays were performed. Overexpression of LncRNA-NONHSAT192404.1 significantly inhibited invasion of MDA-MB-231 cells. Conversely, knock-down resulted in a significant elevation in cell invasion (Figure 3C, D).

Figure 3

Based on bioinformatics predictions using TargetScan, which identified a putative binding site for miR-518a-3p within LncRNA-NONHSAT192404.1, we hypothesized a direct interaction (Figure 4A). miR-518a-3p was significantly up-regulated in MDA-MB-231 relative to normal mammary epithelial cells (Figure 4B). Dual-luciferase assays showed a significant reduction in luciferase activity. Importantly, mutating the miR-518a-3p binding site abolished this reduction, confirming direct binding (Figure 4C). An RNA pull-down assay corroborated these findings: miR-518a-3p was specifically enriched in complexes with LncRNA-NONHSAT192404.1 but not with control RNA (Figure 4D, E). These results demonstrate that LncRNA-NONHSAT192404.1 directly interacts with, and likely sponges, miR-518a-3p in TNBC cells.

Figure 4

To explore how LncRNA-NONHSAT192404.1 regulates PI3K/AKT signaling, we quantified TLR4, AKT, PI3K and PPP2R1A expression. PI3K, AKT and PPP2R1A were significantly up-regulated in MDA-MB-231 versus Hs578Bst cells, whereas TLR4 was down-regulated (Figure 5A). Western-blotting showed that overexpression of LncRNA-NONHSAT192404.1 increased TLR4 and decreased AKT, PI3K and PPP2R1A (Fig. 5B). Notably, miR-518a-3p expression reversed the PI3K/AKT activation induced by LncRNA-NONHSAT192404.1 (Figure 5B).

Figure 5