Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC). We performed functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for therapeutic delivery of miR-34a. In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic , which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3'-untranslated region of and mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of and , the transcription factor for and the direct target of p53, in TNBC cell lines, whereas overexpression of and rescued the effects and signaling pathways mediated by miR-34a. Moreover, therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis. Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. .

No abstract

In the past years, several methods have been developed to determine cell viability in cell culture (Cook and Mitchell, 1989). Among these methods tetrazolium salt-based assays are widely used in order to measure cytotoxicity or cell proliferation (Mosmann, 1983;Berridge et al., 1996).The principle of the MTT [3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay is the reduction of water-soluble yellow tetrazolium salt by the dehydrogenase system of metabolically active/live cells into water-insoluble blue/magenta (MTT) formazan crystals (Morgan et al., 1998). In this way, the concentration of dissolved formazan crystals can be quantified using a spectrophotometer and it is in direct correlation to the number of metabolically active cells (Gabrielson et al., 2002;Tunney et al., 2004;Wang et al., 2010). The MTT assay represents a simple and rapid colorimetric assay and yields quantitative data (Alley et al., 1988). This assay is carried out entirely in 96-well microtiter plates; thus, large numbers of experiments examining a number of variables can be readily performed (Cole, 1986).On the other hand, the MTT assay has some disadvantages that are dependent on the cell ability to overcome cell death. One remarkable disadvantage is that damaged mitochondria may be still able to reduce MTT to formazan crystals (Mosmann, 1983;Page et al., 1988;Sieuwerts et al., 1995). Loveland et al. (1992) showed that cells with inactivated mitochondria were also able to produce formazan crystals as well as cells with active mitochondria. Furthermore, many nonmitochondrial dehydrogenases and flavin oxidases are able to reduce MTT (Altman, 1976;Burdon et al., 1993). Besides, different conditions and some chemicals/phytochemicals can also lead to changes in metabolic activity (Plumb et al., 1989;Hsu et al., 2003). The MTT compound may interact with some chemicals/phytochemicals, resulting in false results in viability (