The main purpose of the work was to study the key exosomal factors involved in the development of hormonal resistance of breast cancer cells. The work is based on our previous data, which demonstrated the effect of exosome-mediated transferring of hormonal resistance in in vitro cultured MCF-7 breast cancer cells.
Estrogen-dependent breast cancer cells MCF-7 and the tamoxifen-resistant subline MCF-7 /T were used as an experimental model. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. MicroRNA was extracted from by PureLink RNA Micro Kit; library preparation was carried out with NEBNext® Small RNA Library Prep Set for Illumina®. Transfection of the RNA oligonucleotides was performed using Metafectene PRO (Biontex) to result in the final RNA concentration of 50 nM.
A comparative analysis of exosomal miRNAs of MCF-7 and resistant MCF-7/T cells was carried out. In total, 2588 miRNAs have been identified in the exosomes. Among them, mir-181 family, which is one of the negative regulators of estrogen-dependent growth, was identified as the group of miRs, hyperexpressed in the resistant exosomes. Following this, we analysed the role of mir-181c, one of the main members of miR-181 family, in the regulation of cell growth and hormonal response. Mir-181c transfection was found to induce the estrogen-independent growth and partial tamoxifen resistance of MCF-7 cells. The study of the signaling proteins showed that mir-181c transfection, in contrast to scrambled RNA transfection, caused the increase of the amount of Raptor, phosphorylated forms of mTOR and Akt that correlated with increased AP-1 transcriptional activity.
We have demonstrated the involvement of miR-181c in the development of hormonal resistance of breast cancer cells that allows us to consider mir-181 as the perspective target of the treatment of hormone- independent cancers. The research was supported by the Russian Science Foundation (19-15-00245, miRNA analysis) and RFBR (#18-29-09017, tamoxifen resistance).
Clinical trial identification
Legal entity responsible for the study
Russian Science Foundation, project 19-15-00245.
All authors have declared no conflicts of interest.