Actinic keratosis (AK) is a carcinoma in situ precursor of cutaneous squamous cell carcinoma (cSCC), the second most common cancer affecting the Caucasian population. AK is frequently present in the sun-exposed skin of the elderly population, UV radiation being the main cause of this cancer, and other risk factors contributing to AK incidence. The dysregulation of microRNAs (miRNAs) observed in different cancers leads to an improper expression of miRNA targets involved in several cellular pathways. The TaqMan Array Human MicroRNA Card assay for miRNA expression profiling was performed in pooled AK compared to healthy skin scraping samples from the same patients. Forty-three miRNAs were modulated in the AK samples. The expression of miR-19b (p < 0.05), -31, -34a (p < 0.001), -126, -146a (p < 0.01), -193b, and -222 (p < 0.05) was validated by RT-qPCR. The MirPath tool was used for MiRNA target prediction and enriched pathways. The top DIANA-mirPath pathways regulated by the targets of the 43 miRNAs are TGF-beta signaling, Proteoglycans in cancer, Pathways in cancer, and Adherens junction (7.30 × 10−10 < p < 1.84 × 10−8). Selected genes regulating the KEGG pathways, i.e., TP53, MDM2, CDKN1A, CDK6, and CCND1, were analyzed. MiRNAs modulated in AK regulate different pathways involved in tumorigenesis, indicating miRNA regulation as a critical step in keratinocyte cancer.

Background The β3 human papillomavirus (HPV)49 induces immortalization of primary keratinocytes through the action of E6 and E7 oncoproteins with an efficiency similar to alpha high risk (HR)-HPV16. Since HR-HPV oncoproteins are known to alter microRNA (miRNA) expression and extracellular vesicle (EV) production, we investigated the impact of HPV49 E6 and E7 proteins on miRNA profile and EV expression, and their involvement in the control of cell proliferation. Methods The miRNA expression was evaluated by a miRNA array and validated by RT-qPCR in primary human keratinocytes immortalized by β3 HPV49 (K49) or α9 HR-HPV16 (K16), and in EVs from K49 and K16. The modulation of miRNA target proteins was investigated by immunoblotting analyses. Results By comparing miRNA expression in K49 and K16 and the derived EVs, six miRNAs involved in HPV tumorigenesis were selected and validated. MiR-19a and -99a were found to be upregulated and miR-34a downregulated in both cell lines; miR-17 and -590-5p were upregulated in K49 and downmodulated in K16; miR-21 was downregulated only in K16. As for EV-carried miRNAs, the expression of miR-17, -19a, -21 and -99a was decreased and miR-34a was increased in K49 EVs. In K16 EVs, we revealed the same modulation of miR-19a, -34a, and -99a observed in producing cells, while miR-21 was upregulated. Cyclin D1, a common target of the selected miRNAs, was downmodulated in both cell lines, whereas cyclin-dependent kinase 4 was down-modulated in K49 but upregulated in K16. Conclusion These data suggest that E6 and E7 proteins of β3 HPV49 and α9 HR-HPV16 affect key factors of cell cycle control by indirect mechanisms based on miRNA modulation.