Liver gene expression profiles of rats treated with clofibric acid: comparison of whole liver and laser capture microdissected liver

Abstract

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor alpha, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci.

Figure 1.

Schematic outline of the experiment. Summary of preparation of the different sample types.

Figure 2.

Paraffin section of liver (HES stain). a: Liver section of control animal. b: Liver section of 0.54% CLO-treated animal. Note the diffuse hepatocellular hypertrophy characterized by enlarged hepatocytes with a glassy eosinophilic appearance. Bars, 100 μm

Figure 3.

Analysis of RNA quality and size. a: Agilent 2100 Bioanalyzer profiles of total RNA extracted from: W, whole liver; S, stained slices; D, dehydrated slices; L, LCM time-reference liver samples; M, microdissected liver tissue. b: Agilent 2100 Bioanalyzer electrophoregram gel of cRNA obtained after labeling of the total RNA of the samples W, S, D, L, and M.

Figure 4.

Scatter plot after PCA of raw data. Raw data intensities were reduced to the top 1000 most differentially expressed genes as determined by a χ ² = 0.05 test and were submitted to a PCA. The initial 1000 dimensions of the data set were reduced into three dimensions accounting for most of the differences, before visualizing it by a scatter plot using Spotfire. Each symbol of the graph represents a sample. The process stage is color-coded and each treatment group is represented by a specific form. Treatment controls clustered together in an orange circle. CLO-treated samples clustered together in a green circle.

Figure 5.

Heat map of genes representative of CLO exposure after each step of the LCM procedure, grouped by biochemical categories. The complete data set can be accessed at the website http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE691. Genes were selected if their fold change was statistically significant (P value <0.05) in the 0.29% CLO versus treatment control group for whole liver sample. The 98 most significant genes obtained were manually classified into biochemical functions. The 0.29% CLO gene list was then mapped to the 0.54% CLO fold-changes. The fold-changes between CLO-treated and treatment controls were color-coded. The green color indicates a transcriptional down-regulation of the gene by CLO treatment and the red an up-regulation, whereas the black color represents no modulation. The color intensity represents the magnitude of the change in gene expression. These fold-changes have been calculated for three stages of the process: whole liver samples, stained, and microdissected/LCM time reference samples (considered as replicates).