By Alejandro Gonzalez Torres, Fabien P. Chevalier, Ruth Aquino, Mélanie Aimard, Patrick Baril, Jérôme Lamartine
ABSTRACT
MicroRNAs are short non-coding RNAs that play important roles in fine-tuning genetic networks as genes post-transcriptional regulators. Monitoring the regulatory activity of microRNAs is technically challenging especially in primary cells and 3D organotypic cultures. We optimized the previously reported RILES miRNA-ON sensor system to visualize the spatial expression of miR-203 and miR-30a by fluorescence imaging in 2D and 3D cultures of human primary keratinocytes. The generated system, called RIFES (RNAi-Inducible Fluorescence Expression System), successfully imaged the expression of miR-30a-5p and miR-30a-3p in the suprabasal layers of the epidermis. This information was exploited to uncover the molecular mechanisms regulating the expression of miR-30a in human keratinocytes. We demonstrate that chemical inhibition of the Notch-1 pathway induced GFP expression in undifferentiated RIFES/miR-30a keratinocyte cells, with fluorescence redistribution in the basal layers of 3D RIFES/miR-30a epidermis. Moreover, overexpressing miR-30a in 3D epidermal models resulted in Notch-1 downregulation, suggesting a negative feedback loop between miR-30a and Notch. As the Notch pathway was found downregulated in aged epidermis biopsies, we propose that Notch downregulation contributes to miR-30a induction during aging. The RIFES system therefore appears as a powerful tool to visualize expression of microRNAs in 3D epidermis and to identify their potential upstream regulators.
