Identification of differently expressed genes in human colorectal adenocarcinoma

Abstract

Aim: To investigate the differently expressed genes in human colorectal adenocarcinoma.

Methods: The integrated approach for gene expression profiling that couples suppression subtractive hybridization, high-throughput cDNA array, sequencing, bioinformatics analysis, and reverse transcriptase real-time quantitative polymerase chain reaction (PCR) was carried out. A set of cDNA clones including 1260 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent-labeled probes prepared from RNA of human colorectal adenocarcinoma (HCRAC) and normal colorectal tissues.

Results: A total of 86 genes were identified, 16 unknown genes and 70 known genes. The transcription factor Somult9 influencing cell differentiation was downregulated. At the same time, Heat shock protein 10 KDis downregulated and Calmoulin is up-regulated.

Conclusion: Downregulation of heat shock protein 10 KD lost its inhibition of Ras, and then attenuated the Ras GTPase signaling pathway, increased cell proliferation and inhibited cell apoptosis. Down-regulated transcription factor Somult9 influences cell differentiation and cell-specific gene expression. Down-regulated Somult9 also decreases its binding to calmodulin, accumulates calmodulin as receptor-activated kinase and phosphorylase kinase due to the activation of PhK.

Figure 1

Digestion plasmids of EcoRI/Hind/II. Lane M: DNA size markers; 1-15 lanes: Fragment of about 500 bp after EcoRI/HindIII digestion.

Figure 2

Reduction of G3PDH after PCR-select subtraction. M: DNA size markers; 1-4 lanes: Subtracted PCR products as template, G3PDH3’ and 5’ as primers at cycles 18, 23, 28, 33; 5-8 lanes: PCR products not subtracted as template, G3PDH3’ and 5’ as primers at cycles 18, 23, 28, 33.

Figure 3

Increased c-myc after PCR-select subtraction. M: DNA size markers; 1-4 lanes (left): PCR products not subtracted as template, c-myc 3’ and 5’ as primers at cycles 18, 23, 28, 33; 5-8 lanes: No PCR products subtracted as template, c-myc 3’ and 5’ as primers at cycles 18, 23, 28, 33; 1-4 lanes (right): PCR products subtracted as template, c-myc 3’ and 5’ as primers at cycles 18, 23, 28, 33.

Figure 4

cDNA microarray hybridized with fluorescent labeled probes prepared from HCRAC (A) and total RNA from normal colorectal tissue (B).

Figure 5

Results of QRT-PCR. Blue/brown/:β-actin N/β-actinT; green/bright green: CaMT/CaMN; green/red: Hsp10T/Hsp10N; blue: Sox-9N/Sox-9T.