Transcriptional Landscape of Oral Mucosal Differentiation

Objectives: Normal oral mucosa consists of a highly proliferating layer of epithelial cells covered by multiple layers of stratified squamous epithelium. Differentiation is the process by which progenitor cells, from the basal layer, acquire functional capabilities; i.e., undergo distinct changes as they differentiate from the basal layer to the spinous, granular, and cornified layers. Our objective was to develop a comprehensive transcriptomic compendium of the normal human oral mucosa as cells undergo normal differentiation.
Methods: Oral tissues biopsied from the cheeks of three healthy subjects were put in OCT medium. A Leica Cryostat was used to cut 20 μm thick sections, followed by H&E staining. Slides were prepared for Laser Capture Microdissection (LCM); zones 1 (stratum basale), 2 (intermediate) and 3 (differentiated) were micro-dissected using an Arcturus 1000 LCM system. Approximately 30 captures per zone were collected and pooled into a single sample, RNA was isolated, and used for Affymetrix Clariom S Human expression microarray.
Results: The principal components analysis (PCA) of transcriptional profiles of the three captured regions, particularly Zone 1 and 3 ,were found to be distinctly different from each other, with marginal deviation among the three biological replicates, demonstrating the usefulness of this approach to separate out the basal from the differentiated layers. Our bioinformatics analyses identified that, in addition to genes associated with epithelial cell differentiation, Zone 3 is enriched with genes associated with positive regulation of defense responses and negative regulation of endopeptidase activity. Zone 1, on the other hand, is enriched in genes associated with the regulation of sequence specific DNA binding transcription factor activity, favoring selective transcriptional regulation in the stratum basale.
Conclusions: To our knowledge, this study is the first to combine LCM with transcriptional profiling of different regions of the oral mucosa. It will provide a unique basis of comparison for future work that will use LCM to examine specific gene markers of disease states where neoplastic or other disease specific changes occur.