MiR-99a directly targets the mTOR signalling pathway in breast cancer side population cells.

Abstract

miR-99a has been reported to function as a tumour suppressor in breast cancer. However, its role in the regulation of breast cancer stem cell (CSC) phenotype has up to now remained unknown. In this study, we isolated the side population (SP) cells by staining cultured MCF-7 and MDA-MB-231 cells with fluorescent DNA-binding dye Hoechst 33342, then by flow cytometric sorting. Next, we detected expression of miR-99a in the SP cells compared to non-SP cells using real-time PCR, and explored effects of miR-99a on the CSC phenotype of the breast cancer cells, including sphere formation, self-renewal, tumourigenicity and cell migratory capability. We found that expression of miR-99a was down-regulated in the SP cells compared to non-SP cells. Restoration of expression of miR-99a inhibited cell migration and invasion, reduced sphere formation of breast SP cells in vitro, and suppressed tumour growth in vivo. Finally, bioinformatic prediction suggested the oncogene, mammalian target of rapamycin (mTOR) - a downstream effector of the PI3K/AKT signalling pathway, was a target gene of miR-99a in SP cells. Further, quantitative RT-PCR and western blot assays identified that overexpression of miR-99a suppressed expression of mTOR and its downstream gene, HIF-1α. Collectively, these data suggest that miR-99a reversed the breast cancer malignant CSC phenotype, probably by targeting the mTOR signalling pathway.