The authors have declared that no competing interests exist.
Conceived and designed the experiments: MHJ SG JZ. Performed the experiments: SG JZ JW DSL. Analyzed the data: JZ SG. Contributed reagents/materials/analysis tools: JZ SG JW DSL. Wrote the paper: JZ SG MHJ.
The 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.
RNA 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.
LCM-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.
The intestine is a dose limiting organ during radiation therapy of abdominal cancers and a critical determinant of survival in radiological emergency situations. This is mainly due to the radiosensitivity of its rapidly proliferating crypt epithelium. The intestinal mucosa invariably sustains the most severe injury and undergoes the most change after exposure to ionizing radiation
Methods such as
The isolation of RNA from whole tissue is relatively quick, easy, and may reveal important information about differential gene expression patterns. However, the utility of analysis performed on whole gut RNA extracts is questionable because it is hampered both by the structural complexity of the intestine, as well as by the relative change in structure that the mucosa undergoes during the process of development of radiation toxicity. In contrast, RNA isolated by laser capture microdissection (LCM) can be used to isolate relatively pure cell populations or tissue segments of interest from fixed tissue sections, thereby avoiding both of the problems associated with whole tissue RNA extracts
LCM is widely used for gene expression analysis in both basic research and clinical applications
Here, a well-defined mouse model of total body radiation was used to compare the transcriptional profiles of LCM-separated mucosal and whole gut specimens obtained in parallel. Our results clearly revealed that it is important to use LCM for analysis of pathways related to cellular injury, cell cycle control, and DNA damage repair pathways, all pathways that are central in the radiation response. Only genes where there are substantial differences in expression levels, such as certain apoptotic and cytokine signaling pathways, can be safely analyzed in whole gut RNA extracts.
The experimental protocol was reviewed and approved by the University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System's (CAVHS) Institutional Animal Care and Use Committees (IACUC).
Experiments were carried out in a total of 40 random-bred male CD2F1 mice (Harlan Sprague Dawley, Indianapolis, IN), 6–7 weeks old on arrival and with an initial body weight of 22–25 g. Animals were housed in conventional cages under standardized conditions with controlled temperature and humidity and a 12–12-h day-night light cycle. Animals had free access to water and chow (Harlan Teklad laboratory diet 7012, Purina Mills, St. Louis, MO).
Total body γ irradiation was performed as described before
Collection of intestinal mucosa was performed by LCM as shown in
Mucosal RNA was extracted from the LCM-dissected jejunum using the RNAqueous-Micro kit (Ambion, Austin, TX) specifically designed to recover small amounts of RNA from LCM samples.
Whole gut RNA was extracted from jejunum collected at different time points following irradiation. Tissues were snap frozen in liquid nitrogen and stored at −80°C. Frozen tissue samples were homogenized in Ultraspec RNA reagent (Biotecx Laboratories, Houston, TX), according to the manufacturer's instructions. Two µg of RNA were kept for microarray analysis (including quality control analysis) and 2 µg were used for real time PCR.
DNase-treated RNA was quantitated using the Rediplate 96 Ribogreen RNA quantitation kit (Invitrogen, Carlsbad, CA).
RNA samples from LCM dissected mucosa and whole gut were screened by checking their integrity and genomic DNA contamination. Selected RNA samples were processed for microarray analysis using Illumina one-color MouseWG-6_V2 chip. The sample size for LCM and for whole gut microarray was N = 4/time point.
Microarray data was analyzed by Genespring GX 11.0 (Agilent Technologies, CA). Raw data were log2 transformed and then normalized to the 75th percentile of all values on a chip. Student's T-test was used to examine for differentially expressed genes. A list of genes with ≥2.0-fold change was generated first and tested by the Benjamini-Hochberg Multiple Testing Correction procedure. Significant genes were selected by a cut-off of p<0.05 and fold change > = 2.0.
The selected genes were subsequently analyzed using IPA 5.0 (Ingenuity Systems Inc., CA). Pathways which were predicted to be influenced by the differentially expressed genes were ranked in order of significance and further analyzed by overlaying with function, disease and canonical pathways. An overlap p-value<0.05 indicates a statistically significant, non-random association between a set of genes in the dataset and a related function. The p-value of overlap is calculated by the Fisher's Exact Test.
Verification of gene expression was carried out using different sets of total RNA samples from those that were sent for microarray. For each RNA sample, 1 µg was used as the template for cDNA synthesis by using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the protocol provided by the manufacturer. Steady-state mRNA levels were measured with real-time quantitative PCR using the following Applied Biosystems predesigned Taqman gene expression assays: Casp14, Mbl2, Mcpt1, Eda2r, CCL-9 and IL-33. PCR amplification and detection were carried out on an ABI prism 7000 Detection System (Applied Biosystems). Transcript levels were normalized to eukaryotic 18S rRNA and calculated relative to control, unirradiated jejunum, using the standard ΔΔCt method.
5 µm sections were mounted on glass slides. TUNEL assay was performed using the Dead End fluorometric TUNEL kit (Promega, Madison, WI) following the manufacturer's instruction. Image acquisition was performed on a Zeiss Imager Z1 Epifluorescence Microscope (Zeiss, Goettingen, Germany).
5 µm sections were mounted on slides, deparaffinized in xylene, and rehydrated in graded solutions of ethanol. After blocking with 5% normal serum, sections were incubated with primary antibody overnight at 4°C, followed with Alexa Fluor conjugated secondary antibody incubation (Molecular Probes, Eugene, OR; 1∶400) for 1 h at room temperature. Slides were mounted with Vectashield Mounting Medium with DAPI for nuclear staining (Vector Laboratories, Burlingame, CA). Image acquisition and intensity measurements were performed on a Zeiss Imager Z1 Epifluorescence Microscope (Zeiss, Goettingen, Germany).
Data are presented as mean ± standard deviation. Statistical analysis was performed by t-test. p<0.05 was considered to be significant. For the microarray, a list of genes with > = 2-fold change was generated by t-test and filtered by the “Benjamini and Hochberg false discovery test”. Genes with a false discovery of less than 0.05 were used to for the final list of differentially expressed genes. For IPA, the p-value of overlap is calculated by the Fisher's Exact Test.
To verify radiation-induced cellular injury/death, TUNEL staining was applied on the intestinal tissue at each time point.
TUNEL staining to demonstrate DNA damage in mouse small intestine at baseline and 4 h, 24 h, and 3.5 d after irradiation. Green: TUNEL positive, Blue: DAPI. 10×/20×: the magnifying power of the objectives. rTdT: recombinant Terminal Deoxynucleotidyl Transferase enzyme.
The gene list with a cut-off of 2-fold and p-value<0.05 was generated at each time point. In LCM samples, the number of genes that were altered ≥2-fold was 615 at 4 h, 461 at 24 h, and 613 at 3.5 d post radiation. In the whole gut, the number of genes that altered ≥2-fold was 799 at 4 h, 784 at 24 h, and 1624 at 3.5 d post radiation. Venn diagrams (
The change of selected genes of Casp14, Mbl2, Mcpt1, CCL-9, IL-33 and Eda2r at 4 different time points were validated by real-time RT-PCR using Applied Biosystems predesigned Taqman gene expression assays (
The left panels (black bars) demonstrate expression of the selected genes at different time points plotted by raw signals from the microarray. The right panels (gray bars) show the relative expression of the same selected genes normalized by 18S rRNA at different time points by real-time RT-PCR. The opposite direction of Mcpt1 demonstrates its down-regulation after radiation.
The lists of altered genes were imported into IPA for pathway analysis.
Stacked bar charts demonstrate IPA-generated activated cellular injury and DNA damage repair pathways in mucosa and whole gut at 4 h, 24 h, and 3.5 d after irradiation. The height of the bars indicates the percentage of genes that changed in the particular pathway. Red bar: up-regulated. Green bar: down-regulated. Pathways with p-value (yellow dot) above the threshold (dashed line) are significantly activated. Heatmaps demonstrate the change of the genes in the selected signaling pathway before (left column) and after radiation (right column). Blue: decreased, red: increased.
In the whole gut, there were no significantly activated cellular injury pathways at 4 and 24 h. At 3.5 d, up-regulation of DNA damage repair was not significant. Instead, there was a down-regulated HIF1α signaling.
Corresponding to cellular injury and DNA damage repair,
Stacked bar charts demonstrate IPA-generated activated cell cycle control pathways in mucosa and whole gut at 4 h, 24 h, and 3.5 d after irradiation. The height of the bars indicates the percentage of genes that changed in the particular pathway. Red bar: up-regulated. Green bar: down-regulated. Pathways with p-value (yellow dot) above the threshold (dashed line) are significantly activated. Heatmaps demonstrate the change of the genes in the selected signaling pathway before (left column) and after radiation (right column). Blue: decreased, red: increased.
In the whole gut, there was no significantly activated pathway at 4 and 24 h. At 3.5 d, among the highly up-regulated pathways in the mucosa, only BTG was significantly changed (
As a rapidly renewing tissue, the intestine undergoes extensive cell death in response to radiation. To study the global impact of gene expression profiles on cell death in mucosa and whole gut, regulation of apoptosis pathways was analyzed by IPA.
Stacked bar charts demonstrate IPA-generated activated apoptosis pathways in mucosa and whole gut at 4 h, 24 h, and 3.5 d after irradiation. The height of the bars indicates the percentage of genes that changed in the particular pathway. Red bar: up-regulated. Green bar: down-regulated. Pathways with p-value (yellow dot) above the threshold (dashed line) are significantly activated. Heatmaps demonstrate the change of the genes in the selected signaling pathway before (left column) and after radiation (right column). Blue: decreased, red: increased.
In whole gut, p53 signaling was significantly up-regulated at both 4 h and 24 h. At 3.5 day, there was wide down regulation of various genes involved in apoptosis such as Calcium-induced T lymphocyte apoptosis signaling, Aryl hydrocarbon receptor signaling, Nur77 signaling in lymphocytes, and CD27 signaling in lymphocytes.
Gene microarray revealed that radiation induced a striking increase of casp-14, both in LCM samples from the intestinal mucosa and in samples from the whole gut. For example, while expression of casp14 mRNA was very low under normal conditions in mucosa, it increased 67-fold at 4 h, 33-fold at 24 h and then dropped to baseline levels at 3.5 d (
LCM is a technique by which pure cell populations or tissue segments of interest can be procured from frozen or fixed tissue sections. Since the first development of LCM in 1996, it has become widely used in the basic research and clinical areas for the study of
A single dose (8.0 Gy) of total body γ radiation to CD2F1 mice did not cause significant 30-day lethality but considerable histological damage and, as shown by TUNEL staining coincident with upregulation of CD27 signaling, a wave of apoptosis in mucosa
At 24 h post radiation, the most significantly changed pathways were tight junction and p53 signaling pathways. The disrupted tight junction signaling in mucosa could possibly explain a response to the breakdown of the integrity of epithelial tight junction. There was significant up-regulation of p53 signaling both in the mucosa and the whole gut. The up-regulated p53 signaling in mucosa and whole gut at 24 h while TUNEL positive nuclei were significantly decreased at this time could repudiate the direct link of p53 to cell death in the intestine, as documented in other models
A broad up-regulation of cell cycle control and DNA damage repair pathways occurred at 3.5 d in mucosa coinciding with downregulation of pathways that control apoptosis in both mucosa and whole gut. As expected, up-regulated DNA damage repair and cell cycle control signaling were mainly evident in the mucosa, i.e., the compartment where most DNA damage occurs. ATM, a key regulator of multiple signaling cascades in response to radiation-induced DNA strand breaks
It is interesting to note that up-regulation of pathways that control the coagulation system, lymphocyte apoptosis, tight junction, cell cycle control, and DNA damage repair was largely only found in the LCM-isolated mucosa but not in the whole gut extract. The sequential activation of these pathways after radiation exposure provides important information for understanding the mechanism of the injury. Thus, inclusion of the non-mucosal layers may seriously dilute the altered mucosal genes and disguise activation of relevant pathways.
A novel and unexpected finding was the marked increase of Casp14 and Eda2r expression, present in both mucosa and whole gut samples. The sharp and robust increase of these genes at the early time points suggests their important roles in the regulation of acute intestinal radiation injury.
Casp14 belongs to a conserved family of aspartate-specific proteinases. It is expressed in the suprabasal layers of the epidermis and is associated with protection against UVB-induced apoptosis and water loss
EDA2R is a type III transmembrane protein of the TNFR (tumor necrosis factor receptor) superfamily and has been identified as a p53 target which regulates p53-mediated anoikis
One concern of this study is the comparability of the gene expression profiles from LCM and whole gut. To address this problem, RNA samples for microarray processing were screened by checking their integrity and genomic contamination. The parallel expression pattern of Casp14 at both mucosa and the whole gut revealed the comparability of the expression profiles from LCM and whole gut. The analysis result of activated cellular injury, apoptosis, and DNA damage repair pathways in mucosa but not in the whole gut is consistent to the pattern of cellular injury demonstrated by TUNEL staining. Taken together, these data demonstrate the reliability of the analysis in this paper.
In conclusion, the present investigation revealed that alterations of many of the pathways known to undergo changes in response to radiation exposure are obscured if RNA extracts are obtained from whole gut samples as opposed to by LCM of the mucosa only. Hence, our data strongly suggest that RNA for analysis of pathways related to the coagulation system, lymphocyte apoptosis signaling, tight junction signaling, cell cycle control, and DNA damage repair signaling should be procured by LCM. This study also suggests that the newly identified genes Casp14 and Eda2r could provide novel research objectives and potential therapeutic targets to protect against or mitigate intestinal radiation injury. Future study will investigate RNA expression related to the position or differentiation state of the cells in the mucosa during intestinal radiation injury.
We thank Jennifer D. James (from Experimental Pathology Core Laboratory, Winthrop P. Rockeffeler Cancer Institute, University of Arkansas for Medical Sciences) for assistance with tissue processing. We also thank Neurobiology and Developmental Sciences Core, University of Arkansas for Medical Sciences, for providing us with the Leica AS LMD microdissection system.