To investigate the role and prognostic value of the DTNBP1 gene in triple negative breast cancer (TNBC).

RNA sequencing expression profiles of TNBC patients were collected in the TCGA database from the establishment, yielding 621 TNBC patient samples and 32 normal samples. For accuracy, the top 497 TNBC patient samples with the most significant differential expression compared with normal samples were selected for analysis. Differentially expressed genes were screened using bioinformatics methods. The impact of DTNBP1 gene expression on the prognosis of 497 TNBC patients was assessed through Kaplan-Meier survival analysis. Principal component analysis (PCA), GO, and KEGG enrichment analyses were employed to explore the function of the DTNBP1 gene. Cox univariate and multivariate regression analyses were conducted to identify prognostic factors for TNBC and construct a prognostic model. Real-time quantitative PCR (qRT-PCR) was used to detect DTNBP1 expression in normal human mammary epithelial cells (MCF-10A), hormone receptor-positive breast cancer cells (MCF-7), and TNBC cells (HCC1937, MDA-MB-231, MDA-MB-361, MDA-MB-468). Transfection experiments were performed on MDA-MB-231 and MDA-MB-468 cells to construct DTNBP1 low-expression TNBC cells. Clonogenic assays and flow cytometry were used to assess the proliferation and cell cycle of DTNBP1 low-expression cells and control cells.

Compared with normal breast tissue, TNBC tissue sample exhibited 3 196 differentially expressed genes, with 2 056 upregulated and 1 140 downregulated. Using a median DTNBP1 mRNA expression level of 8.12 as a threshold, 497 TNBC patients were divided into high expression (248 cases) and low expression (249 cases) groups. Survival analysis indicated median DFS of 9.8 years for the high expression group and 18.2 years for the low expression group, with a statistically significant difference (t=3.824, P<0.001). PCA identified 1 138 differentially expressed genes between high and low DTNBP1 expression groups, with 647 upregulated and 491 downregulated. GO enrichment analysis suggested that DTNBP1 may be involved in different biological processes (such as leukocyte migration, extracellular matrix formation, and extracellular matrix organization), cellular components (including collagen-containing extracellular matrix, cell-matrix junctions, and focal adhesions), and molecular functions (such as extracellular matrix structural constituent, antigen binding, and cell adhesion molecule binding). KEGG pathway enrichment analysis indicated potential involvement of DTNBP1 in cell cycle, PI3K-Akt signaling pathway, MAPK signaling pathway, Hippo signaling pathway and TNF signaling pathway. Cox univariate analysis identified age (HR=1.099, 95%CI: 1.080-1.135, P=0.004), clinical stage (HR=2.885, 95%CI: 1.743-4.284, P<0.001), tumor stage (HR=12.576, 95%CI: 6.514-26.583, P<0.001), distant metastasis (HR=1.676, 95%CI: 1.477-2.201, P=0.034), node stage (HR=1.922, 95%CI: 1.567-2.756, P=0.006), and DTNBP1 gene expression (HR=2.934, 95%CI: 1.904-4.513, P<0.001) as factors related to TNBC prognosis. Multivariate analysis indicated that age (HR=1.168, 95%CI: 1.126-1.214, P<0.001), tumor stage (HR=3.771, 95%CI: 2.731-6.682, P=0.002), and DTNBP1 gene expression (HR=1.563, 95%CI: 1.315-1.961, P<0.001) were independent prognostic factors for TNBC. A clinical prognostic model for TNBC patients was constructed using age, tumor stage, and DTNBP1 gene expression, with its calibration curve closely matching the ideal curve. The mRNA expression levels of the DTNBP1 gene in normal human mammary epithelial cells (MCF-10A), hormone receptor-positive breast cancer cell lines (MCF-7), and TNBC cell lines (HCC1937, MDA-MB-231, MDA-MB-361, MDA-MB-468) were 1.00±0.28, 1.71±0.41, 3.25±0.42, 6.81±0.55, 2.43±0.21 and 5.57±0.26, respectively, with significant differences between groups (F=7.250, P=0.032). Compared with MCF-10A cells, DTNBP1 expression increased in MDA-MB-231 (t=-0.947, P<0.001) and MDA-MB-468 cells (t=-0.978, P=0.021). After transfection with sh-NC and sh-DTNBP1 plasmids, the mRNA expression levels of DTNBP1 in MDA-MB-231 cells were 1.00±0.05 and 0.33±0.04, respectively, with significant differences between groups (t=0.078, P=0.031). For MDA-MB-468 cells, the mRNA expression levels were 1.00±0.10 and 0.18±0.07, respectively, with significant differences between groups (t=0.080, P<0.001). The number of cell colonies in MDA-MB-231 cells transfected with sh-NC and sh-DTNBP1 plasmids were 100.00±10.00 and 24.00±7.00, respectively, with significant differences between groups (t=158.771, P<0.001). For MDA-MB-468 cells, the number of colonies were 100.00±7.00 and 17.00±4.00, respectively, with significant differences between groups (t=169.778, P<0.001). Flow cytometry cell cycle analysis showed that the numbers of MDA-MB-231 cells in G₀/G₁, S, and G₂/M phases transfected with sh-NC plasmid were 31.94±4.50, 25.23±1.20, and 42.83±1.80, respectively. For cells transfected with sh-DTNBP1 plasmid, the numbers of MDA-MB-231 cells were 52.39±3.10, 20.11±1.90, and 27.25±2.40, respectively, with significant differences between groups (t=-74.063, P=0.026). For MDA-MB-468 cells transfected with sh-NC plasmid, the numbers of cells in G₀/G₁, S, and G₂/M phases were 43.15±2.50, 31.26±2.90, 25.59±3.60, respectively. For cells transfected with sh-DTNBP1 plasmid, the numbers of cells were 64.70±3.00, 23.24±3.10, and 12.06±2.30, respectively, with significant differences between groups (t=-64.992, P=0.037).

DTNBP1 may serve as a potential therapeutic target for TNBC, with its expression level closely related to the prognosis of TNBC patients.