Figure 1.
Characterization of miR-200a-MB-MNPs.
(A) A schematic representation of the miR-200a-MB-MNP structure and EMT imaging probe activation. (B) Cy5 fluorescence from miR-200a-MBs. Different concentrations of a synthetic oligonucleotide target either complementary to or unmatched to miR-200a were added to the miR-200a-MBs. ** indicates p<0.01. (C) TEM images of MNPs and miR-200a-MB-MNPs. The diameters of the MNPs and miR-200a-MB-MNPs were approximately 50 nm. (D) An agarose gel electrophoresis image; MNPs and miR-200a-MB-MNPs were loaded onto 0.5% agarose gels.
Figure 2.
Reciprocal expression of E-cadherin, ZEB1, and the miR-200 family during EMT.
(A, B) Western blot and RT-PCR analyses of ZEB1 and E-cadherin in MCF-7, MDA-MB-231, and TGF-β1 treated NMuMG cells. (C) Real-time PCR analysis of the miR-200 family. The levels of miR-200a, b, and 429 in MCF-7, MDA-MB-231, and TGF-β1 treated NMuMG cells were evaluated. *, ** indicates p<0.05 or p<0.01, respectively. (D) A schematic image of the Renilla luciferase reporter and the analysis of luminescence signals for evaluating the binding availability of miR-200a-MBs. After transfection of pRL-ZEB1 plasmid, the luminescence signals in MCF-7, MDA-MB-231, and TGF-β1 treated NMuMG cells were analyzed. Data are from three independent experiments.
Figure 3.
Validation of miR-200a-MB-MNPs as an EMT imaging probe.
(A) miR-200a-MB-MNPs were introduced into NMuMG cells at 0, 2, 6, and 12 hours post-TGF-β1 treatment. Cy5 fluorescence (pink) was observed at 2 hours after TGF-β1 treatment, and the Cy5 signals increased in a time-dependent manner. (B) Cy5 signals were analyzed in either miR-200a-MB-MNPs or scrambled-MB-MNPs delivered NMuMG cells after 6 hours of TGF-β1 treatment. Immunostaining of ZEB1 was performed to confirm the mesenchymal transformation of NMuMG into mesenchymal phenotype. Scale bar, 10 µm.
Figure 4.
Analysis of the hybridization ability of miR-200a-MBs to ZEB1 mRNA using real-time PCR and immunostaining.
(A) The illustration represents the binding sites of the primers used in real-time PCR. Real-time PCR analysis showed reduced expression levels of ZEB1 mRNA in miR-200a-MB-MNPs delivered NMuMG cells compared to the cells with scrambled-MB-MNPs. ** indicates p<0.01. (B) The localization of miR-200a-MBs and the RISC subunit Ago-2 were observed within the several regions of TGF-β1 treated cell. The Cy5 signals (yellow arrows) of miR-200a-MBs were in close proximity to the Alexa-546 signals of Ago-2 (white arrows). Small boxes were marked with a & b for the indication of the magnified regions. Scale bar, 10 µm.
Figure 5.
Real-time imaging of the 7EMT process in live cells.
(A) After the delivery of miR-200a-MB-MNPs (20 µg/ml) into NMuMG cells, real-time imaging was performed following TGF-β1 (10 ng/ml) treatment. From 2 minute 30 seconds after EMT induction, NMuMG cells containing MNPs (green) in their cytoplasm showed Cy5 fluorescence (white arrows) generated by the miR-200a-MBs. (B) The Cy5 fluorescence intensity was quantified with Image J, showing increased signals generated by miR-200a-MBs. (C) During the acquisition period of real-time imaging, the NMuMG cells exhibited morphological changes and the loss of cell-cell adhesions (red arrows) after TGF-β1 treatment. Scale bar, 10 µm.