# Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

## TL;DR

This study uses a new imaging system to show how miR-30a and the Notch pathway interact in aging human skin models.

## Contribution

The RIFES system enables spatiotemporal visualization of miR-30a activity in 3D epidermal models.

## Key findings

- miR-30a is expressed in the suprabasal layers of the epidermis using the RIFES system.
- Notch1 inhibition increases miR-30a expression and redistributes fluorescence to the basal layers.
- miR-30a overexpression leads to NOTCH1 downregulation, suggesting a negative feedback loop.

## Abstract

MicroRNAs are short noncoding 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 3-dimensional (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 2-dimensional 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 Notch1 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 NOTCH1 downregulation, suggesting a negative feedback loop between miR-30a and Notch. Because the Notch pathway was found downregulated in aged epidermis biopsies, we propose that Notch downregulation contributes to miR-30a induction during aging. Therefore, the RIFES system appears as a powerful tool to visualize the expression of microRNAs in 3D epidermis and to identify their potential upstream regulators.