Abstract
The epithelial-mesenchymal transition (EMT) plays important roles in tumor progression to metastasis. Thus, the development of an imaging probe that can monitor transient periods of the EMT process in live cells is required for a better understanding of metastatic process. Inspired by the fact that the mRNA expression levels of zinc finger E-box-binding homeobox 1 (ZEB1) increase when cells adopt mesenchyme characteristics and that microRNA-200a (miR-200a) can bind to ZEB1 mRNA, we conjugated molecular beacon (MB) mimicking mature miR-200a to magnetic nanoparticles (miR-200a-MB-MNPs) and devised an imaging method to observe transitional changes in the cells during EMT. Transforming growth factor-β1 treated epithelial cells and breast cancer cell lines representing both epithelial and mesenchymal phenotypes were used for the validation of miR-200a-MB-MNPs as an EMT imaging probe. The real-time imaging of live cells acquired with the induction of EMT revealed an increase in fluorescence signals by miR-200a-MB-MNPs, cell morphology alterations, and the loss of cell-cell adhesion. Our results suggest that miR-200a-MB-MNPs can be used as an imaging probe for the real-time monitoring of the EMT process in live cells.
Highlights
The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their abilities for cell-cell adhesion and gain invasive and motile properties
As the miR-200a-molecular beacon (MB) was designed to target zinc finger E-box-binding homeobox 1 (ZEB1) mRNA, the Cyanine 5 (Cy5) fluorophore is separated from black hole quencher 2 (BHQ2) and emits a fluorescence signal when miR-200a-MB hybridizes to a target sequence
We evaluated the possibility of miR-200a-MB-Magnetic nanoparticles (MNPs) as an EMT-imaging probe by delivering them to Transforming growth factor-b1 (TGF-b1) treated NMuMG cells for various incubation times (2, 6, or 12 hours); in all cases, miR-200a-MB-MNPs in the cytoplasm was confirmed by fluorescein isothiocyanate (FITC) fluorescence (Fig. 3A)
Summary
The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their abilities for cell-cell adhesion and gain invasive and motile properties. EMT is important for embryonic development and for the tissue repair process, it has been recognized as a potential mechanism of organ fibrosis and of tumor progression to metastasis [1,2]. Previous reports have identified several EMT-related genes, such as zinc finger protein SNAI1 (SNAI1; Snail), zinc finger protein SNAI2 (SNAI2; Slug), zinc finger E-box-binding homeobox 1 (ZEB1; Zeb1), Twistrelated protein 1 (TWIST1; Twist), and Cadherin-1 (CDH1; Ecadherin) [1,2,3]. The development of EMT imaging methods is necessary, and, a method to trace the time points of changes in cellular characteristics from the epithelial to mesenchymal phenotype is expected to contribute the advancement of the knowledge on EMT process in tumor progression and metastasis
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