Breast cancer (BC) resistance to antiestrogens is one of the main problems that limit the efficacy of chemotherapy. Exosomes, microvesicles secreted and absorbed by cells, play an important role in the development and transmission of resistance of tumor cells. Exosomes are enriched with microRNAs taking part in the regulation of target genes. The aim of this work was to identify the microRNAs involved in the development of resistance and to study the effects of their transfection into BC cells.
Experiments were performed on the in vitro cultured estrogen-dependent MCF-7 cells and tamoxifen-resisitant MCF-7/T subline. Exosomes were prepared by ultracentrifugation. Data on microRNA expression in exosomes were obtained by NGS. Transient multiple transfections of microRNAs were performed in the cells. Protein expression was determined by immunoblotting, reporter analysis was used to study the transcriptional activity of estrogen receptor (ERa). Cells growth rates were assessed by MTT test.
The microRNAs profile of the MCF-7 and MCF-7/T exosomes was studied, and 6 microRNAs - ERa suppressors were found to be overexpressed in the exosomes of resistant cells. It was revealed that the transfection of identified microRNAs into MCF-7 cells causes a slight decrease in ERa transactivation and practically does not affect the expression of ERa protein. At the same time, transient multiple transfection (totally, 20 rounds of transfection) of one of these microRNAs, miR-181a-2, into MCF-7 cells induces the tamoxifen resistance in the latter. The transfected cells maintained the resistance to tamoxifen within at least 3 months of cultivation after the last transfection. The acquired tamoxifen resistance was associated with the alteration of DNA methylation as well as with a decrease in the expression of DNMT3, an important methylation regulator, the suppression of which may be characteristic of resistant cells.
The transient multiple transfection of miR-181a-2 into MCF-7 cells induces the irreversible tamoxifen resistance demonstrating the important role of this microRNA in the formation of the resistant phenotype.
Legal entity responsible for the study
Russian Science Foundation, project 19-15-00245.
All authors have declared no conflicts of interest.