Promoter Hypermethylation Downregulates MiR-125b-5p and MiR-199b-5p Targeting of ΔNp63, Resulting in PI3K/AKT/mTOR Pathway Activation and Keratinocyte Differentiation

Background: Normal keratinocyte differentiation is important for epidermal wound healing. ΔNp63 is a major gene regulating epidermal formation and differentiation. We identified miRNAs targeting ΔNp63 and studied the association between the miRNAs and DNA methylation in keratinocyte differentiation. Aims: This study aimed to explore the mechanisms regulating ΔNp63 expression during keratinocyte differentiation. Methods: Bioinformatics analysis was performed to screen the miRNAs targeting ΔNp63 and uncover potential pathway mechanisms. The differentiation model of keratinocytes was established by CaCl2 treatment. Furthermore, the effects of the miRNA transgenic technique on Δ Np63 and keratinocyte differentiation were studied. In addition, the RNA FISH experiment was conducted to detect the location of different miRNAs. A double luciferase reporter experiment was carried out to verify the potential bindings between the miRNAs and ΔNp63. A rescue experiment was also performed to assess the effects of different miRNAs targeting ΔNp63 on keratinocyte differentiation. We analyzed the methylation patterns of the promoter regions of miRNAs using keratinocytes treated with 5-Aza-2’-deoxycytidine. Finally, we designed a methylation rescue experiment to verify the effects of miRNA promoter methylation on keratinocyte differentiation. Results: Bioinformatics analysis showed that the miR-125b-5p and miR-199b-5p binding to the ΔNp63 3’UTR region decreased during skin development. Moreover, such binding may downregulate the PI3K/AKT/mTOR pathway. The expression levels of CK10, Inv, TG1, ΔNp63, and PI3K/AKT/mTOR were all significantly increased during keratinocyte differentiation. Both miR- 125b-5p and miR-199b-5p were localized in the cytoplasm. Luciferase assay results showed that both miR-125b-5p and miR-199b-5p can bind to the 3’UTR region of ΔNp63. Overexpression of ΔNp63 can significantly counteract the inhibitory effect of miRNA mimics on keratinocyte differentiation. Moreover, the promoter regions of both miR-125b-5p and miR-199b-5p had methylation sites, and the methylation levels in those promoter regions were significantly increased during keratinocyte differentiation. 5-Aza-2’-Deoxycytidine treatment increased the expression of miR- 125b-5p and miR-199b-5p and inhibited the differentiation of keratinocytes. Finally, miRNA inhibitors reversed the inhibitory effects of 5-Aza-2’-deoxycytidine on keratinocyte differentiation. Conclusion: Promoter hypermethylation in miR-125b-5p and miR-199b-5p seem to promote keratinocyte differentiation via upregulation of ΔNp63 expression and the activation of the PI3K/AKT/mTOR pathway. The findings of this study are helpful for future research on skin development and clinical scar-free healing.