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P015 Exploring the barrier; RNA sequencing of laser microdissected epithelium from IBD patients

A. van Beelen Granlund*1, 2, 3, S. Thorsvik1, 2, 3, A.E. Østvik1, 2, 3, V. Beisvåg1, A. Flatberg1, I. Bakke1, 3, A.K. Sandvik1, 2, 3

1Norwegian University of Science and Technology, Department of Cancer Research and Molecular Medicine, Trondheim, Norway, 2Norwegian University of Science and Technology, Centre of Molecular Inflammation Research, Trondheim, Norway, 3St. Olav's University Hospital, Department of Gastroenterology and Hepatology, Trondheim, Norway

Background

Studies of the gene expression in IBD colon samples have contributed greatly to the current understanding of IBD pathophysiology. However, all these studies have been performed on full thickness pinch biopsies. Here, the differential expression is dominated by the contribution of inflammatory cells infiltrating the lamina propria. In this study we present an RNA expression sequencing done on laser capture microdissected (LCM) epithelium from ulcerative colitis (UC) and normal control (N) pinch biopsy specimens. By isolating the gene expression to the epithelial monolayer (EM), we identify epithelial processes important in IBD pathophysiology without needing to take contribution from infiltrating inflammatory cells into consideration.

Methods

Pinch biopsies from colonic mucosa of six UC patients and N subjects were collected as previously described[1]. An area corresponding to approx. 10 000 cells was isolated from cryosections of each sample using CellCut Plus LCM microscope. RNA was isolated using RNeasy Plus Micro kit, followed by sequencing using the TruSeq RNA Access kit (Illumina, CA, USA). Reads were aligned to the human genome using Tophat2 and sample quality control was performed using RSeQC. A gene count matrix was built using featureCounts from aligned reads and UCSC gene annotations revised through cufflinks assembly. Differential gene expression analysis was performed using Limma-Voom within the edgeR Bioconductor package.

Results

The analysis resulted in > 4000 differentially expressed (DE) genes between UC and N samples. The resulting list of DE genes agreed with previously published Results from analysis of full thickness pinch biopsies[1]. Genes previously identified as highly expressed in the epithelium of UC colon are also found to be DE between the LCM samples[2]. Enrichment of epithelial cells appears to have been successful. E.g., IL8, a chemokine produced by macrophages infiltrating the lamina propria in IBD, showed in whole biopsies the 3rd highest fold change and a p value <0.001 in the contrast UC vs. N. The present study showed IL8 as the 283rd gene (p value = 0.08) sorted for fold change.

Conclusion

The colon epithelial monolayer has an important role in maintaining homeostasis. A leading hypothesis regarding IBD initiation is that it is result of a loss of epithelial integrity and a breakdown in homeostasis. In this study, LCM of the epithelial monolayer from colonic pinch biopsies followed by expression profiling has been demonstrated to be feasible, resulting in biologically interpretable data. This opens for a targeted interpretation of the role of the epithelial monolayer in IBD pathophysiology.

References:

[1] Granlund A vB et al. , (2013), Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demonstrates lack of major differences between Crohn's disease and ulcerative colitis

[2] Granlund A vB et al, (2011), Activation of REG family proteins in colitis