Differentially Expressed Proteins In Colorectal Tumor

Colorectal cancer is one of the most common causes of cancer related deaths worldwide and the second leading cause of cancer death in western developed countries. In China, the incidence and mortality of colorectal cancer have been rising rapidly in the past two decades, with the changes of life style and diet structure. So it is very important to research the mechanism of colorectal carcinogenesis.Colorectal carcinogenesis is well known as a multi-step process. The central paradigm for the initiation and development of colorectal carcinoma is the adenoma-carcinoma sequence. Colorectal cancer can be effectively controlled if the main premalignant lesion -adenoma was detected and removed before invasion occurred. Therefore, it is crucial to elucidate the molecular mechanism underlying colorectal adenoma.It is well known that the protein is the executant of cell function. Genetics effect will be exhibited by changes of proteome. Therefore, proteins are just the direct targets of early detection, prevention and treatment. It has been accepted the human proteome was one of the strategical resources in the new century. The detection, prevention and treatment of colorectal tumor will be greatly promoted, if the specific protein marker of colorectal tumor was discovered.Proteomics technologies are effective methods for the discovery of protein markers. The concept of proteome was brought forward by Wilkins and Williams in 1994. The word, Proteome cames from the combination of protein and genome and includes the entire protein complement expressed by a genome, by a cell or by a tissue. In this study, two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry was used to detect the differentially expressed proteins in matched colorectal normal mucosa, adenoma and cancer tissues.Total proteins from 10 cases of colorectal adenoma, carcinoma and their matchednormal mucosa were separated by 2-DE and visualized by silver staining. The digitized images then were analyzed with PDQuest software in order to establish the differential expression profiles between the adenoma, carcinoma and normal tissues. Thirty-two differentially expressed proteins that changed over 5-fold and had the same changes in at least 5 cases were detected. Compared with normal mucosa, seventeen proteins were down-regulated over 5-fold in adenoma (p<0.05), and 11 proteins were up-regulated over 5-fold in adenoma (p<0.05). Compared with cancer tissues, four proteins were down-regulated over 5-fold in adenoma (p<0.05).The differentially expressed protein spots were cut from the gels using proteomework spot cutter and subjected to in-gel digestion with trypsin. The digested peptides' separation was conducted by a MALDI-TOF or Finnigan LTQ MS coupled with a Surveyor HPLC system. And based on the mass spectrometry raw data, protein identification was performed by application of the Mascot or SEQUEST search program, respectively. Twenty-seven of 32 proteins were detected by mass spectrometry.In order to find more differentially expressed proteins in colorectal tumor, the articles that had been published about proteomics study of colorectal tumor before April of 2006 were searched from Pubmed (www.ncbi.nlm.nih.gov/pubmed). All data were summarized and analyzed. Total 213 proteins were detected, including the 27 proteins in this study. There were 44 (21%) proteins that had been reported more than twice. In the 44 proteins, many were subunit of 7 protein families. They were actin, annexin, ATP synthase, elongation, heat shock protein, hemoglobin, and keratin, respectively.To verify the results of 2-DE, two new proteins in this study which had never been reported in colorectal tumor, and two new proteins that had the confused results in two previous studies were verified by westen-blot. The results showed that mimecan highly expressed in normal mucosa, and was absent in adenoma and cancer; TXNDC5 (Thioredoxin domain-containing protein 5) were expressed in all tissues, and the expression in adenoma and cancer were significant higher than that in normal mucosa (p<0.05). This result was coincident with the result of 2-DE. RhoGDI (RhoGDP-dissociation inhibitor 1) and Cyclophilin A (Peptidyl-Prolyl Cis-Trans Isomerase A) were reported to differentially express in two studies, but their results were inconsistent. In the western-blot experiment, we found that RhoGDI significantly expressed in colorectal cancer, compared with normal mucosa (p<0.05); and there were no significant changes of Cyclophilin A expression in cancer, compared with normal mucosa (p>0.05).To observe the effect of mimecan to the shape and the growth of the tumor cells, the mRNA of mimecan was transformed into SW480 cell lines. We found that mimecan didn't change the shape of the tumor cells, but significantly inhibited the growth of them. And the cell cycle was significantly blocked at the G1 period.In conclusion, firstly, Twenty-seven differentially expressed proteins could be involved in carcinogenesis of colorectal tumor. Secondly, compared with the normal mucosa, mimecan and RhoGDI were down-regulated in colorectal tumor, and TXNDC5 was up-regulated. The roles of the three new proteins in colorectal tumor need further studies. Thirdly, mimecan significantly inhibited the growth of tumor cell, implying that mimecan might be a potential tumour suppressor gene in cancer initiation and progression.