Chenchen, Graduate School of Science and Engineering, Department of Chemistry
| Developing valuable biomarkers is helpful for early diagnosis, make molecular staging, predict prognosis and find potential therapeutic target for colon cancer. |
| Western blot is a technique to detect specific proteins expression in samples by specific antibodies. Because of its high specificity owing to specific antibodies, high sensitivity due to chemiluminescent signal (sample weight limit as low as 10-12g) and good reproducibility, the technique has been widely used in molecular biological researches. |
| Eight pairs of normal and tumor clinical samples from colon cancer patients have been recruited and whole proteins of each sample have been extracted and separated by SDS-PAGE. Up to 195 antibodies come from 12 different groups according to their cellular function have been used to against western blot membranes, indicating different expression levels between normal and tumor pairs. |
| The intensity (volume) of band was detected by analysis software ImageQuant
TL 7.0, GE company. Significant difference was defined according to paired
t-test (p value<0.05) of volumes between normal and tumor groups and
fold change should be more than 1.5 or less than 1/1.5 for each pair appearing
at least in 4 pairs. According to the criteria, sixteen out of the 195
proteins showed significant difference between normal and tumor, in which
12 proteins overexpressed in tumor tissues while other 4 were upregulated
in normal tissues (Figure1) |
|
|
Figure1. Typical results with significant expression difference between the normal and tumor tissues.
Two patterns are listed here: High expression in tumor but low in normal and on the contrary,
high expression in normal but low in tumor.
Eight pairs (16 samples) were detected on a same membrane. |
| The 195 proteins can be classified into 12 different functional groups according to the descriptions in NCBI and Uniprot databases.(Table 1) These functional groups include apoptosis, cell cycle regulation, chromatin assemble, cytoskeleton organization, DNA metabolism, multiple function protein, protein degradation, ribosome biogenesis, RNA synthesis, signal transduction pathway, transcription modulation and transportation. |
Table1 Proteins show significant difference expression distributed in 12 functional groups
| Group names |
Number in each group |
T↑(12) |
N↑(4) |
| Apoptosis |
2 |
|
|
| cell cycle regulation |
38 |
2 |
1 |
| chromatin assemble |
12 |
1 |
|
| cytoskeleton organization |
16 |
3 |
|
| DNA metabolism |
7 |
1 |
|
| multiple function |
4 |
|
|
| protein degradation |
3 |
|
|
| ribosome biogenesis, |
2 |
|
|
| RNA synthesis |
3 |
|
|
| signal transduction pathway |
70 |
2 |
4 |
| transcription modulation |
32 |
3 |
|
| transportation |
6 |
|
|
| Total |
195 |
|
|
|
T↑: high expression in tumor samples; N↑: high expression in normal samples
|
| Interestingly, 3 positive results assembled in cytoskeleton organization and other five appeared in signal transduction pathway group. Both groups play essential roles in the early stage colorectal tumorigenesis. |
| The results will be further validated by immunohistochemistry stains in clinical cohorts and analyzed with clinical parameters. If a target protein can be proved to have close relationship with colon cancer, it can be regarded as a candidate biomarker to predict or a target of chemotherapy of the colon cancer. They may help for early diagnosis and if they can be blocked by new developed medicines, the procedure of tumorigenesis could be inhibited or reversed. |
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