ΔNp63 intronic miR-944 is implicated in the ΔNp63-mediated induction of epidermal differentiation.

Kyu-Han Kim,Tae Ryong Lee,Eun-Gyung Cho,Yoon-Jin Kim,Seok Jong Yu,Sang Hoon Kim,Hyojin Kang

doi:10.1093/nar/gkv735

Abstract

ΔNp63 is required for both the proliferation and differentiation of keratinocytes, but its role in the differentiation of these cells is poorly understood. The corresponding gene, TP63, harbors the MIR944 sequence within its intron. However, the mechanism of biogenesis and the function of miR-944 are unknown. We found that miR-944 is highly expressed in keratinocytes, in a manner that is concordant with that of ΔNp63 mRNA, but the regulation of miR-944 expression under various conditions did not correspond with that of ΔNp63. Bioinformatics analysis and functional studies demonstrated that MIR944 has its own promoter. We demonstrate here that MIR944 is a target of ΔNp63. Promoter analysis revealed that the activity of the MIR944 promoter was markedly enhanced by the binding of ΔNp63, which was maintained by the supportive action of AP-2 during keratinocyte differentiation. Our results indicated that miR-944 biogenesis is dependent on ΔNp63 protein, even though it is generated from ΔNp63 mRNA-independent transcripts. We also demonstrated that miR-944 induces keratin 1 and keratin 10 expression by inhibiting ERK signaling and upregulating p53 expression. Our findings suggested that miR-944, as an intronic miRNA and a direct target of ΔNp63, contributes to the function of ΔNp63 in the induction of epidermal differentiation.

Highlights

The protein p63, which belongs to the p53/p63/p73 family of transcription factors, plays an important role in normal epidermal stratification, the maintenance of pluristratified epithelia, and the proliferative potential of epithelial stem cells [1,2]
We demonstrated that MIR944, which is located in the intron of ΔNp63 has its own promoter: an mimic)- and miR-944 mimic-transfected keratinocytes. (B) The mRNA expression levels of early differentiation markers (KRT1 and keratin 10 (KRT10)) in keratinocytes that had been transfected with 20 nM miR-944 mimic or NC mimic or with 50 nM miR-944 inhibitor or negative control inhibitor (NC inhibitor; right panel) were analyzed using RT-qPCR
Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus NC mimic or NC inhibitor, unpaired Student’s t-test. (C) The protein expression levels of keratin 1 (KRT1), KRT10, and ␤-actin were examined through western blotting after keratinocytes had been transfected with a miR-944 mimic or a miR-944 inhibitor. (D) The mRNA expression levels of KRT1 and KRT10 in keratinocytes that had been treated with PD98049 for 2 days were analyzed using RT-qPCR

Summary

Introduction

The protein p63, which belongs to the p53/p63/p73 family of transcription factors, plays an important role in normal epidermal stratification, the maintenance of pluristratified epithelia, and the proliferative potential of epithelial stem cells [1,2]. This protein exists as tissue-specific isoforms, transcribed from alternative promoters, which give rise to TAp63 containing the transactivation (TA) domain and an N-terminal truncated isoform ( Np63) lacking the TA domain. Expression of Np63 is restricted to the proliferative basal epidermal cells and plays a role in the maintenance of the proliferative capability of cells, and its expression is decreased in differentiated layers [3,4]. The molecular mechanism by which Np63 governs epidermal proliferation has been relatively well studied [5,7]; it is not clear how Np63 could induce epidermal differentiation

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Conclusion