Increased expression of miR-758 inhibits migration of metastatic breast cancer cells

Introduction: Metastatic breast cancer is the most advanced form of breast cancer. Although advances in medicine have improved outcomes and provided more treatment options, patients with advanced stages remain incurable and have poor survival rates. MicroRNAs are post-transcriptional regulators and previous work by our research group identified miR-379 as a potent tumour suppressor in breast cancer. miR-379 is part of a large miR-379/656 cluster and this study aimed to investigate the role of another microRNA within this cluster, miR-758, in the breast cancer setting.

Methodology: MDA-MB-231 cells were stably transduced with a lentiviral vector carrying non-targeting control (NTC) sequence or mature miR-758 along with green fluorescent protein (GFP) to generate MDA-NTC and MDA-758 cells. Transduction success was confirmed by fluorescent imaging and analysis of miR-758 expression by quantitative real-time PCR (qPCR). Transwell membranes with a pore size of 8 μm were employed to assess the migration capacity of MDA-NTC, MDA-758 and wild type cells in response to chemoattractants. Migration of MDA-NTC and MDA-758 cells suspended in basal media (without fetal bovine serum (FBS)) towards chambers containing 10% FBS as a chemoattractant was assessed. The membranes were fixed, stained with crystal violet and mounted on glass slides. Images were captured from five fields of view of each membrane using an Olympus BX60 microscope. The migrated cells were then counted using Image J. MTS assay was performed on MDA-NTC and MDA-758 cells to investigate the impact of miR-758 on cell proliferation. In addition, an initial tubule formation assay was employed to investigate the impact of miR-758 overexpression on the ability of MDA-MB-231 cells to stimulate angiogenesis.

Results: MDA-MB-231 transfected with NTC or miR-758 vectors successfully expressed GFP fluorescent signal, and qPCR analysis revealed a 180-fold increase in miR-758 sequence expression in MDA-758 compared to MDA-NTC. There was no significant change in proliferation between the miR-758 overexpressing group versus control. Transwell results showed that the number of migrated cells in the MDA-NTC group (294 cells ± 10 cells) was higher than that in the MDA-758 group (193 cells ± 26 cells). MDA-758 cells demonstrated a 34% reduction in migration (P=0.02) towards chemoattractants across transwell inserts in comparison to MDA-NTC cells. The initial results of the tubule formation assay suggest a trend towards a reduced ability to stimulate tubule formation of conditioned media of MDA-758 cells compared to the conditioned media of control cells.

Conclusion: MDA-MB-231 cells were successfully engineered to stably express miR-758. Although no impact on cell proliferation was observed, enrichment with miR-758 resulted in a significant decrease in migratory capacity of this highly invasive cell population. Combined with results suggesting decreased capacity to stimulate angiogenesis, this exciting data supports a potential tumour suppressor role for miR-758. Further studies will be performed to determine the specific mechanism of action of this microRNA.