Real-Time Functional Bioimaging of Neuron-Specific MicroRNA Dynamics during Neuronal Differentiation Using a Dual...

The capability of monitoring the neuronal differentiation process in living cells is crucial to the understanding of neuronal development and the practical application of cell therapy for the treatment of neurodegenerative disorders. Current research methods, including Northern blot and real-time PCR analysis, have been extensively employed to quantify miRNA expression during cellular processes. However, these methods require cell destruction and could not provide dynamic information on miRNA expression and function in living organisms. In the present study, we developed a dual luciferase reporter to monitor the expression pattern of neuron-specific miRNA-9 and miRNA-124a during neuronal differentiation in vitro and in vivo. The miRNA-responsive reporter was designed to encode a firefly luciferase (Fluc) gene containing miRNA target sequences and a Renilla luciferase (Rluc) gene for normalization. These two genes were independent modules and transcribed by two different promoters, which enables precise sensing miRNA activity without mutual transcription interference. We demonstrated that the functional activation of miRNA-9 and miRNA-124a during neurogenesis was visualized by the reduction of Fluc bioluminescence signal in P19 cells and nude mice without Rluc signal change, suggesting that miRNA-9 and miRNA-124a specifically downregulated their targets in accordance with their expression. Our dual luciferase-based miRNA imaging system provides a useful tool to quantitatively and continuously monitor miRNA activity during various biological processes.