Strand-specific functions of miR-301a-3p and -5p drive opposing roles in pancreatic cancer via ferroptosis and pyroptosis.

Pancreatic ductal adenocarcinoma is among the most lethal malignancies, underscoring the urgent need for a deepened molecular insights and targeted strategies. While microRNAs (miRNAs) are known to regulate key oncogenic pathways in PDAC, most studies overlook the functional heterogeneity between the 3p and 5p strands derived from the same miRNA precursor. Here, we systematically investigate the strand-specific roles of miR-301a-3p and miR-301a-5p in PDAC progression. Using a multidimensional approach, including clinical tissue-based FISH, serum analysis, cell-based assays, and xenograft models, we revealed a consistent pattern of miR-301a-3p upregulation and miR-301a-5p downregulation in PDAC tissues and fluids. Importantly, the ratio of 5p to 3p is negatively correlated with TNM stage, and its combination with CA19-9 significantly improves prognostic prediction. Mechanically, the two strands exert opposing effects on tumor progression via distinct cell death pathways. miR-301a-3p promotes tumorigenesis by targeting ACSL4 to suppress ferroptosis, while miR-301a-5p induces pyroptosis by activating NF-κB pathway through CEACAM6-TNFR1 axis. These findings challenge the prevailing "dominant strand" paradigm and uncover a previously unrecognized antagonism between miRNA strands. By highlighting the risks of indiscriminate miRNA targeting, our work supports the development of strand-specific diagnostic and therapeutic strategies in PDAC. This study advances the concept of chain-level regulation as a foundation for precision oncology.