Comparative Proteomic And Metabonomic Study On The Evolution Of Experimental Colorectal Carcinoma

Objective: To research the proteins and metabolic products differently expressed during evolution of experimental colorectal carcinoma (normal mucosa→adenoma→carcinoma→liver metastasis) so as to find the early diagnostic biomarker of colorectal cancer as well as to understand its pathogenesis.Methods: 90 male Wistar rats were selected, 80 were injected with 1, 2-dimethylhydrazine intraperitoneally once a week for consecutive 10 weeks and 4-6 rats were sacrificed every 2 weeks to establish the experimental colorectal tumor models (from normal mucosa to liver metastasis). The remaining 10 rats were injected with normal saline as control. Samples of these different stages were collected and divided into 4 groups (1. normal mucosa 2. adenoma 3. carcinoma 4. liver metastasis). The proteins of these 4 groups were extracted to conduct 2-dimensional gel electrophoresis and then screen the differential protein spots to perform mass spectrometry as well as bio-information analysis. Protein spot of special importance, transgelin, was subjected to western blot and immunohistochemistry analysis so as to explore the rule of its expression alteration. Meanwhile, the serum and urea of rats were collected to undergo mass spectrometry or magnetic resonance spectroscop study.Results: 8 rats died during the experiment. 68 out of 72 rats were found bearing colorectal tumor. Adenomas were found in 17rats mainly in 14th-18th week with an incidence of 55%; adenomas were found in 17rats mainly in 14th-18th week with an incidence of 55%; adenocarcinomas were observed in 51 rats mainly after 22nd week with an incidence of 92.3% and those with liver metastasis were detected in 11rats mainly after 32nd week with an incidence of 36.3%. 10 differential proteins were identified by 2-dimensional gel electrophoresis and mass spectrometry includingα-enolase, cardiacα-actin (CA), transgelin, Myosin regulatory light chain smooth muscle isoform (MRLC), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), haptoglobin, Disulfide Isomerase (DI), Creatine Kinase mitochonbrial (CK-m), heat shock protein 8 (HSP 8) and Keratin complex-2 (KC-2). Among them, western blot and immunohistochemistry proved that transgelin sequentially down-regulated through the evolution of rat colorectal carcinoma. Metabonomic research revealed that compared with normal mucosa group, level of L-threonine increased while 5-oxo-L-Proline decreased in adenoma group and compared with adenoma group, level of L-sarcosine, L-phenylalanine, cholesterol significantly increased and 2-hydroxy-1,2,3-Propanetricarboxylic acid dramatically decreased in adenoma group. Besides, some small metabolites also changed among 4 groups.Conclusion: there are differential proteins and metabolites in tissues, serum and urine during the evolution of colorectal carcinoma. These proteins and metabolites may be the candidate biomarkers for the early diagnosis of colorectal carcinoma while transgelin was of most importance due to its sequentially reduced expression through the evolution of rat colorectal carcinoma.