The hormonal therapy is among the most effective treatment of the hormone-dependent breast cancer however its efficiency is limited by the acquired resistance to the hormonal drugs. Previously we have shown the exosomes involvement in the transferring of the hormonal resistance from the resistant to the sensitive cells, and here the study of the features of the exosomes of the resistant cells was performed.
Experiments were performed on the MCF-7 breast cancer cells, MCF-7/T resistant subline developed under long-term tamoxifen treatment, and MCF-7/exoT resistant subline developed under resistant exosomes treatment. Exosomes were prepared from the conditioned medium by the differential ultracentrifugation, and exosome imaging was carried out by transmission electron microscope. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. Library preparation was carried out with NEBNext® Small RNA Library Prep Set for Illumina® (E7330S). More than 2500 miRNAs were identified in the exosomal samples. DNA methylation was evaluated by the RRBS (Reduced Representation Bisulfite Sequencing) method.
The analysis of the exosomal microRNAs revealed 471 microRNAs over-expressed in the exosomes of the resistant cells. Among them, three DNMT1/3-targeting microRNAs - miR-148b-3р, miR-193a and miR-383, were identified. The subsequent analysis of the cellular proteins revealed the decreased expression of DNMT1/ DNMT3 both in the primary-resistant MCF-7/T cells and in the exosome-treated MCF-7/exoT cells. The suppression of DNMT1/3 correlated with the hypomethylation of particular CpG islands in DNA of the resistant cells.
Taken together, the results obtained demonstrate the important role of the DNA (de)methylation in the exosome-mediated transferring and maintaining of the hormone resistance in the breast cancer cells.
The authors.
The Russian Science Foundation, project 19-15-00245.
All authors have declared no conflicts of interest.