Alterations of microRNAs throughout the malignant evolution of cutaneous squamous cell carcinoma: the role of miR-497 in epithelial to mesenchymal transition of keratinocytes.

Skin carcinogenesis is known to be a multi-step process with several stages along its malignant evolution. We hypothesized that transformation of normal epidermis to cutaneous squamous cell carcinoma (cSCC) is causally linked to alterations in microRNAs (miRNA) expression. For this end we decided to evaluate their alterations in the pathologic states ending in cSCC. Total RNA was extracted from formalin fixed paraffin embedded biopsies of five stages along the malignant evolution of keratinocytes towards cSCC: Normal epidermis, solar elastosis, actinic keratosis KIN1-2, advanced actinic keratosis KIN3 and well-differentiated cSCC. Next-generation small RNA sequencing was performed. We found that 18 miRNAs are overexpressed and 28 miRNAs are underexpressed in cSCC compared to normal epidermis. miR-424, miR-320, miR-222 and miR-15a showed the highest fold change among the overexpressed miRNAs. And miR-100, miR-101 and miR-497 showed the highest fold change among the underexpressed miRNAs. Heat map of hierarchical clustering analysis of significantly changed miRNAs and principle component analysis disclosed that the most prominent change in miRNAs expression occurred in the switch from 'early' stages; normal epidermis, solar elastosis and early actinic keratosis to the 'late' stages of epidermal carcinogenesis; late actinic keratosis and cSCC. We found several miRNAs with 'stage specific' alterations while others display a clear 'gradual', either progressive increase or decrease in expression along the malignant evolution of keratinocytes. The observed alterations focused in miRNAs involved in the regulation of AKT/mTOR or in those involved in epithelial to mesenchymal transition. We chose to concentrate on the evaluation of the molecular role of miR-497. We found that it induces reversion of epithelial to mesenchymal transition. We proved that SERPINE-1 is its biochemical target. The present study allows us to further study the pathways that are regulated by miRNAs along the malignant evolution of keratinocytes towards cSCC.