Aberrant O-glycosylation genes and miR-21-5p targets define a molecular signature of tumor aggressiveness in triple-negative breast cancer.

Aberrant O-glycosylation and miRNA dysregulation are established features of tumor aggressiveness, particularly in triple-negative breast cancer (TNBC). Using a Cosmc-silenced 4T1 murine model (Tn+), characterized by truncated O-glycans and enhanced metastatic potential, tissue and serum miRNA profiling identified miR-21-5p as a circulating marker associated with the Tn+ phenotype. Transcriptomic analysis revealed that miR-21-5p targets multiple tumor suppressor genes, including Btg2, Spry1, Tbx2, Rhob, and Dusp8, suggesting its involvement in epithelial-mesenchymal transition and immune modulation. Integrative single-cell RNA sequencing (scRNA-seq) of the 4T1 tumor microenvironment revealed distinct cellular clusters with inverse expression patterns of GALNT enzymes (involved in Tn synthesis) and miR-21-5p target genes, defining Tn+-like and Tn--like subpopulations. Translating these findings to human breast cancer (TCGA-BRCA), a prognostic model combining clinical variables (age, metastasis, PAM50 subtype) with three-gene expression (OLR1, PCSK6, and GALNT6) significantly improved patient risk stratification (P = 0.015). By integrating multi-omics analyses (scRNA-seq, TCGA) with an aggressive Tn+ TNBC model, this study defines a novel three-gene prognostic signature that links the glyco-miRNA axis to tumor aggressiveness, offering a promising tool for advanced patient stratification and the development of precision glyco-therapeutics.