No abstract

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.

This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3βP) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3βP and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.