Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent
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The epidermal compartment is complex and organized into several strata composed of keratinocytes (KCs), including basal, spinous, granular, and cornified layers. The continuous process of self-renewal and barrier formation is dependent on a homeostatic balance achieved amongst KCs involving proliferation, differentiation, and cell death. To determine genes responsible for initiating and maintaining a cornified epidermis, organotypic cultures comprised entirely of stratified KCs creating epidermal equivalents (EE) were raised from a submerged state to an air/liquid (A/L) interface. Compared to the array profile of submerged cultures containing KCs predominantly in a proliferative (relatively undifferentiated) state, EEs raised to an A/L interface displayed a remarkably consistent and distinct profile of mRNAs. Cultures lifted to an A/L interface triggered the induction of gene groups that regulate proliferation, differentiation, and cell death. Next, differentially expressed microRNAs (miRNAs) and long non-coding (lncRNA) RNAs were identified in EEs. Several differentially expressed miRNAs were validated by qRT-PCR and Northern blots. miRNAs 203, 205 and Let-7b were up-regulated at early time points (6, 18 and 24 h) but down-regulated by 120 h. To study the lncRNA regulation in EEs, we profiled lncRNA expression by microarray and validated the results by qRT-PCR. Although the differential expression of several lncRNAs is suggestive of a role in epidermal differentiation, their biological functions remain to be elucidated. The current studies lay the foundation for relevant model systems to address such fundamentally important biological aspects of epidermal structure and function in normal and diseased human skin.
KeywordsKeratinocytes Differentiation mRNA Transcriptome miRNA
We would like to thank Laura Brovold and Ally Perlina from GeneGo, Inc. (http://www.genego.com) for assistance with systems level network mapping and the Affymetrix chip design team for making the custom miRNA array and protocols. We also would like to thank the Invitrogen/Life Technologies epigenetic gene regulation team for miRNA and lncRNA NCode array support and Dr. Brian Nickoloff for providing RNA for expression analysis and Fig. 1 for EE differentiation. Debbie McFadden provided assistance in formatting the manuscript.
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