Continuous uptake of miR-181a-2 mimetic induces constitutive overexpression of cellular miR-181a-2 in MCF-7 breast cancer cells: links with progression of drug resistance

Abstract

Purpose: The aim of this study was to elucidate the mechanisms underlying the formation and maintenance of drug resistance in cancer cells. Previously, we demonstrated that prolonged treatment of estrogen-dependent MCF-7 breast cancer cells with exosomes derived from estrogen-resistant MCF-7/T cells leads to a partial loss of estrogen sensitivity in MCF-7 cells. Moreover, repeated transfection with one of the exosomal microRNAs — microRNA-181a-2 — induced an irreversible decrease in hormonal sensitivity in the recipient cells. In the present work, to further investigate the possible mechanism of miR-181a-2-induced acquired resistance, we analyzed the effect of multiple miR-181a-2 transfections on the expression of cellular miR-181a-2 and related signaling proteins. Methods: MTT-assay, transient transfection, lentiviral infection, qRT-PCR, immunoblotting, and reporter assay. Results: We found that multiple transfections with miR-181a-2 resulted in a marked increase in cellular miR-181a-2 precursor levels, whereas single transfection had no such effect. Similarly, stable transfection with miR-181a-2 led to increased levels of cellular miR-181a-2 and its host gene, MIR181A2HG, which was associated with partial resistance to tamoxifen. Analysis of the genomic DNA encoding miR-181a-2 revealed no changes in copy number in transfected cells. Furthermore, we identified the transcription factor Snail as a key mediator of miR-181a-2–induced resistance and demonstrated its role in the formation of an autoregulatory loop of miR-181a-2 and the maintenance of cell resistance. Conclusion: Overall, these results reveal a novel mechanism of resistance-associated signaling pathway rearrangement based on the formation of a miR-181a-2 autoregulatory loop.

Keywords: breast cancer, MCF-7, microRNA, miR-181a-2, Snail, tamoxifen resistance