Figure 1.
Experimental design.
Figure 2.
Differential expression of six selected genes in normal colonic epithelial samples, adenoma, and tumor samples.
Expression of the tumor suppressor gene CDKN2B is shown for comparison. Probe set identification codes and gene names are indicated on the right. The six columns indicate microarray results obtained from six individual samples for each type of tissues. Black squares indicate that gene expression was unchanged in that experiment. The degree of intensity of red or green indicates the level of gene expression high or low, respectively.
Figure 3.
The expression of GCG, NMES-1, LRMP, and FAM161B genes, tested on independent laser microdissected colonic and also on demethylated HT-29 cells by real-time PCR.
Data analysis was carried out with the comparative Cp method (see Materials and Methods). Genes were considered to be downregulated with values lower than 0.5 (50% decrease, horizontal dotted line), and upregulated with values higher than 2 (two-fold increase). GAPDH was used as a control housekeeping gene. Light grey and dark grey columns show gene expression levels in adenoma and in tumor samples relative to normal samples, respectively. Hatched columns indicate the comparison of 5-Aza treated and control cells. Standard deviations of the measured transcript levels were calculated and are indicated for each transcript. Housekeeping gene intensities were averaged for each group.
Figure 4.
Heterogeneity of PTGDR mRNA expression levels in laser microdissected adenoma and tumor samples.
Real-time PCR and data analysis was carried out as described in Materials and Methods. Genes were considered to be downregulated with values lower than 0.5 (50% decrease, horizontal dotted line) (A) Expression value distribution of the 215894_at probeset (for PTGDR gene) in the GSE8671 (B) and GSE18105 (C) GEO data sets. P values: Normal vs. Adenoma P = 0.000712; CRC vs. Normal P = 0.0003797; LCM CRC vs. Normal P = 0.0000004; LCM CRC vs. CRC P = 0.834.
Figure 5.
The normalized melting curves of the HRM analyses.
Dashed lines represent the standard dilution series from artificially methylated DNA (0%, 25%, 50%, 75% and 100%). The solid black line represents results obtained from a normal sample. Solid grey lines represent results obtained from 5 independent tumorous tissue samples. The HRM analysis can distinguish methylation of the normal and tumorous tissues based on the different G:C contents in the bisulfite-converted promoter sequence. Promoter regions containing more methylated cytosines, which cannot be converted to uracil by the bisulfite treatment, has higher melting temperatures. The melting temperature is proportional to the ratio of non-converted cytosines.
Figure 6.
Immunhistochemical staining (20× magnification) of the prostaglandin D2 receptor (PTGDR) protein (left panel) on normal colon (top), adenoma (middle) and tumorous (bottom) colonic tissues illustrating the progressive decrease of the intraepithelial PTGDR protein expression (arrows) in the adenoma-carcinoma sequence.
In the normal samples (top), predominantly strong, diffuse cytoplasmic staining was detected, which moderately decreased in the adenoma samples (middle), and only weak cytoplasmic staining was observed in the colorectal cancer samples (bottom). The right panel shows the association plots which represent tendentiously decreasing PTGDR protein expression along the adenoma carcinoma sequence. To measure the association of two variables (expression and disease stage), the Chi-square test was used. The height (and color depth) of the boxes is proportional to the difference between the observed and expected frequency of scores. The downward red colored boxes indicate that the observed frequency is lower than expected. The upward, blue items represent the opposite. PTGDR immunostaining scores: −2 = no staining; 0 = weak staining; 1 = moderate staining; 2 = strong diffuse epithelial cytoplasmic immunostaining.
Figure 7.
The first 2 principal components (PC) for our LCM (GSE15960) (A), the GSE18105 (B) and GSE8671 sample sets (C).
The principal components were calculated from the log2-expression values of the 154 selected probe sets for the normal-CRC samples in our LCM set, and then all 3 sets were projected into the principal component coordinate space. Note that this method can be considered as an unsupervised classification, since we did not use explicitly the categories in the data analysis process. Figure 7.A shows that PC transformation separates very well the normal and LCM CRC samples and places the adenoma samples between normal and CRC samples. To validate our list of potential marker genes, we transformed the data of two other independent studies into the same PC coordinates. The two studies from the Gene Expression Omnibus microarray archive were GSE18105 with normal, CRC and LCM CRC samples and GSE8671 with normal and adenoma samples. For both sets the separation of the categories is good except for one outlier point in B marked with an arrow (see discussion in text).