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J Biomol Tech. 2007 February; 18(1): 86.
PMCID: PMC2291871

SP21 Transcriptional Profiling Including miRNA Expression in Human Keratinocytes Undergoing Differentiation in Organotypic Cultures


A novel paradigm of gene regulation in which multiple genes are simultaneously regulated by microRNA (miRNA) is beginning to emerge. We have addressed the importance of miRNA in cell differentiation using human keratinocytes as a model in a systems-level investigation. Keratinocytes are highly structured and organized into several strata, including basal, spinous, granular, and cornified layers. The continuous process of self renewal and barrier formation is dependent on a homeostatic balance achieved among keratinocytes, involving proliferation, differentiation, and cell death. To determine the transcriptional profile responsible for initiating and maintaining a cornified epidermis, organotypic cultures comprised entirely of human keratinocytes maintained on a matrix-coated membrane were raised from a submerged state to an air/liquid interface. Both mRNA and miRNA were isolated from the cultures and were subjected to array-based analyses. Compared to the array profile of submerged cultures containing only keratinocytes in a proliferative (relatively undifferentiated) state, the organotypic cultures displayed a remarkably consistent and distinct profile mainly for mRNAs. miRNA expression changed with time, but not to the extent of mRNA. Raising the cells to an air/liquid interface triggered the induction of groups of genes that regulate proliferation, differentiation, and cell death, and several distinct mRNA species. We identified mRNA expression clusters and regulatory pathways, and compared with skin expression data previously published by us. These results demonstrate the importance of mRNA and miRNA during cell differentiation.

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