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Updated: Aug 24, 2026
Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model
Published on: March 20, 2020
Autophagy-associated exosomal microRNAs in triple-negative breast cancer: a discovery-phase study
Ananya Choudhary1, Simran Tandon1, Adhiraj Roy1
1Amity Institute of Molecular Medicine and Stem Cell Research, Amity University, Noida, Uttar Pradesh, India.
Background:
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.
Methods And Results:
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.
Conclusions:
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.
Insights
Chemotherapy alters microRNA cargo in extracellular vesicles from triple-negative breast cancer cells, potentially impacting treatment resistance. Hsa-miR-1468-5p is a key candidate for further study.
Area of Science:
- Molecular Oncology
- Cancer Cell Biology
- Extracellular Vesicles
Background:
- Triple-negative breast cancer (TNBC) exhibits aggressive behavior and chemoresistance, with underlying mechanisms poorly understood.
- Chemotherapy can induce autophagy, a cellular process influencing small extracellular vesicle (sEV) biogenesis and cargo.
- The impact of autophagy-inducing chemotherapy on the exosomal microRNA (exomiR) profile of TNBC remains uninvestigated.
Purpose of the Study:
- To investigate whether autophagy-inducing chemotherapy alters the exomiR landscape of TNBC cells.
- To identify specific exomiRs that are differentially expressed in TNBC cells under doxorubicin treatment.
- To explore potential targets of these differentially expressed exomiRs within autophagy and apoptosis pathways.
Main Methods:
- Isolation of small extracellular vesicles (sEVs) from TNBC cell lines (MDA-MB-231, MDA-MB-468) treated with doxorubicin or vehicle control.
- Confirmation of autophagy induction via Beclin-1 upregulation, p62/SQSTM1 turnover, and LC3 puncta.
- Small RNA sequencing to identify differentially expressed exomiRs, followed by in-silico target analysis and qRT-PCR validation.
Main Results:
- Doxorubicin treatment induced autophagy in TNBC cells, confirmed by molecular markers.
- Small RNA sequencing identified several differentially expressed exomiRs in both cell lines, with three per line meeting stringent statistical thresholds.
- In-silico analysis revealed BECN1 as a prominent target of the identified exomiRs; qRT-PCR confirmed increased hsa-miR-1468-5p but failed to validate other candidates.
Conclusions:
- Doxorubicin treatment associated with autophagy induction remodels the sEV miRNA cargo in a cell-line-specific manner in TNBC.
- The study identifies hsa-miR-1468-5p as a priority candidate for future functional and clinical validation.
- Further methodological refinement is needed to assess the biological relevance of other identified exomiR candidates.
