Abstract
microRNAs (miRNAs) are a class of non-coding, small RNAs that play an important role in diverse biological processes and diseases. By regulating the expression of eukaryotic genes post-transcriptionally in a sequence-specific manner, miRNAs are widely used to design synthetic RNA switches. However, most of the RNA switches are often dependent on the corresponding ligand molecules, whose specificity and concentration would affect the efficiency of synthetic RNA circuits. Here, a fused transcriptional repressor Gal4BD-Rluc based gene-switch system Gal-miR for miRNA visualization and gene regulation is described. By placing a luciferase downstream gene under the control of endogenous miRNA machinery, the Gal-miR system makes the conversion of miRNA-mediated gene silencing into a ratiometric bioluminescent signal, which quantitatively reflected miRNA-206 activity during myogenic differentiation. Moreover, it demonstrates that this gene-switch system can effectively inhibit breast cancer cell viability, migration and invasion under the control of specific miRNAs by replacing the downstream gene with melittin functional gene. The study proposes a powerful modular genetic design for achieving precise control of transgene expression in a miRNA responsive way, as well as visualizing the dynamics of miRNA activity.
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