Laser Capture Microdissected Mucosa versus Whole Tissue Specimens for Assessment of Radiation-Induced Dynamic Molecular and Pathway Changes in the Small Intestine

Abstract

BackgroundThe intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis.Methodology/Principal FindingsRNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples.Conclusions/SignificanceLCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution.

Emerging Roles for Noncanonical NF-κB Signaling in the Modulation of Inflammatory Bowel Disease Pathobiology

“…This results in the processing of p100 and is negatively regulated by molecules such as cIAP1, A20, and TRAF3 (Figure 3). Although XEDAR has not been investigated in IBD, it has been shown to be greatly upregulated in the gastrointestinal mucosa of nonhuman primates and mice after radiation exposure (71, 72). This may have functional implications in IBD, where mucosal epithelial cell turnover and barrier integrity are critical events in the inflammatory process.…”

Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy

“…This is consistent with the results from Zheng et al, who, when studying irradiated mice, found activated pathways mediating repair, apoptosis, coagulation and cell cycle control. 23 Interestingly, when looking at negative and positive dose-correlated probes separately, the positively correlated pathways (demethylated) had a lower FDR, suggesting that demethylation of genes is more prominent after irradiation. We found that irradiation resulted in demethylation of pathways either involved in the immune system response or in cell proliferation and survival.…”

“…Within the dose‐dependent gene list, a significant change in methylation pattern for pathways associated with cellular immune response, cell cycle regulation and apoptosis was found, representing mechanisms known to be important in radiation response. This is consistent with the results from Zheng et al ., who, when studying irradiated mice, found activated pathways mediating repair, apoptosis, coagulation and cell cycle control . Interestingly, when looking at negative and positive dose‐correlated probes separately, the positively correlated pathways (demethylated) had a lower FDR, suggesting that demethylation of genes is more prominent after irradiation.…”

Site‐specific gene expression analysis from archived human intestine samples combining laser‐capture microdissection and multiplexed color‐coded probes

“…Hence, assessment of tissue layer profiles is crucial to correlate identified molecular patterns with clinical outcomes. In fact, laser-capture microdissection (LCM) coupled with gene expression analysis [4][5][6][7][8][9][10][11][12][13] has therefore been established. LCM enables separation of tissue sub-regions providing histologically pure enriched cell populations.…”