Autophagy-associated exosomal microRNAs in triple-negative breast cancer: a discovery-phase study

Triple-negative breast cancer (TNBC) is characterised by aggressive pathobiology and frequent chemoresistance, yet the mechanisms underpinning treatment failure remain incompletely understood. Chemotherapy-induced autophagy has been implicated in small extracellular vesicle (sEV) biogenesis and cargo remodelling; whether the exosomal microRNA (exomiR) landscape of TNBC is altered under autophagy-inducing chemotherapy has not been established.

sEVs were isolated from MDA-MB-231 and MDA-MB-468 cells under doxorubicin-induced autophagy-inducing conditions (10 nM, 48 h) and from matched vehicle controls, and were confirmed by CD81 and CD63 immunoblotting with Calnexin as a negative purity control. Autophagy induction was confirmed by Beclin-1 upregulation, p62/SQSTM1 turnover and LC3 puncta formation. Small RNA sequencing (12 libraries; n = 3 biological replicates per condition; DESeq2, nominal p < 0.01 with |log₂FC| > 1) identified 10 differentially expressed exomiRs in MDA-MB-231 and 7 in MDA-MB-468, of which three per cell line additionally satisfied a Benjamini-Hochberg false discovery rate threshold (adj. p < 0.05). In-silico target analysis of these 17 exomiRs identified eight convergent autophagy and apoptosis pathway nodes, most prominently BECN1 (targeted by 6 of the 17). Exploratory qRT-PCR confirmed increased hsa-miR-1468-5p in MDA-MB-231 (23.97-fold; adjusted p = 0.007) but did not confirm the remaining five candidates, one of which changed significantly in the opposite direction.

Doxorubicin treatment under autophagy-inducing conditions is associated with cell-line-specific remodelling of the sEV miRNA cargo of TNBC cells. The present data do not provide direct evidence that autophagy causes these changes; establishing causality will require pharmacological or genetic disruption of the autophagy pathway. These discovery-phase findings identify hsa-miR-1468-5p as the priority candidate for prospective functional and clinical validation, and indicate that the remaining candidates require methodological refinement before their biological relevance can be assessed.