| IntroductionColorectal cancer (CRC) is a common malignancy, a serious threat to human health, the incidence rate showed a gradual upward trend. Metastasis is one of the difficulty things for cancer treatment, and leads to the death of patients, so to explore the molecular mechanism of colorectal cancer metastasis and to find effective means of prevention and treatment of colorectal cancer are important contents of the study.Epithelial-mesenchymal transition (EMT) refers to the epithelial cells in a particular physiological and pathological conditions transfer to the mesenchymal cells. EMT is currently seen as the pathological process leading to tumor progression, invasion and metastasis. In the process of EMT, the cell-cell adhesion system function down, reduced interaction with the surrounding cells and stromal cells. The most prominent feature of EMT is the complete loss of epithelial traits, such as the keratin filament, E-cadherin, and acquisition of mesenchymal characteristics, such as Vimentin, fibronectin, N-cadherin, a-SMA and so on. E-cadherin reduction can enhance cell-cell adhesion, inhibit invasion and metastasis characteristics, the loss of E-cadherin has been considered to be the most significant features of EMT. Currently considered the process of EMT is a precise mechanism of intracellular signal transduction regulation, and is determined by the micro-environmental factors specific receptor on the cell, inducing changes in cellular pathways, eventually leading to gene expression change. Tiaml gene (T lymphoma invasion and metastasis) was cloned from a mouse T lymphoma, full name of the T lymphoma invasion and metastasis inducing factor 1. Tiaml expressed in all tumor cells and its expression level increased in accordance with tumor cell invasion and metastasis capacity. In lung, breast, ovarian cancer, laryngeal cancer, colon cancer and other malignant tumors, Tiaml showed positive expression and had closely linked with invasion and metastasis.Tiaml can control cell morphology, adhesion and movement, and had an important role in signal transduction, and can induce tumor cell invasion and metastasis.It has reported that colon cancer cells which have high expression of Tiam1 and Vimentin are with a more metastatic phenotype, while the expression of E-cadherin reduced. Our group has detected the expression of Tiaml and EMT markers in colorectal carcinoma by immunohistochemistry, indicating that Tiaml were negatively correlated E-cadherin and CK, positively corrected Vimentin.Tiaml protein expression is closely related to EMT in colorectal cancer.Tiaml possiblely promote colorectal cancer invasion and metastasis by inducing EMT. Tiaml gene silencing can reverse the EMT phenotype of Lovo cells, while weakening its ability to invasion and metastasis. So what are the relationships between the expression of Tiaml and EMT markers in different metastatic ability colorectal cancer cells lines? This will be explored in this study.TGF-β1 can affect the morphology of tumor cells to change their migration and invasion, and induced EMT of colorectal cancer cells. This is a complex process, and is adjusted by cytokines and proteins join together. MMP-2 can degrade extracellular matrix, and is closely related to the occurrence of EMT, affecting tumor cell migration. This shows that Tiaml, TGF-β1 and MMP-2 can promote tumor invasion and metastasis, and are related to the occurrence of EMT. Are there any relationships in them? It will be confirmed by the further studies.It is also reported than EMT is a process of spontaneous metastasis, and the EMT cells are responsible for degradation matrix in the surrounding cells, paving roads for invasion and metastasis of non-EMT cells. This point of view is different from the past that the EMT cells have stronger invasive than non-EMT cells. Do the EMT cells and the non-EMT have any collaborative relationships in colorectal cancer cells?This study will focus on the three aspects, and explore the relationship of EMT and its related factors Tiaml, TGF-β1 and MMP-2 with metastasis of colorectal cancer, and preliminary study the roles of the EMT and non-EMT cells in the invasion and metastasis of colorectal cancer.Purpose1 To explore the relationships between Tiaml and EMT in different metastatic ability colorectal cancer cell lines.2 To explore the relationship in Tiaml, TGF-β1, MMP-2 and EMT in colorectal cancer.3 To explore the roles of the EMT and non-EMT cells in the invasion and metastasis of colorectal cancer.Methods1,Analysis the expression of Tiaml mRNA, E-cadherin mRNA and Vimentin mRNA in the six kinds of colon cancer cell lines with Real time-RT PCR; Detecting the protein expression of Tiaml, E-cadherin, and Vimentin with immunohistochemical in the LoVo and HT29 cells; Observed LoVo and HT29 cytoskeleton with Coomassie blue staining.2,Immunohistochemistry:The expressions of TGF-β1 and MMP-2 were detected in specimens of 23 normal colorectal tissue,85 colorectal cancer and 28 lymph node metastasis by immunohistochemical (HRP) method. Analysis the relationships in Tiam1,E-cadherin,TGF-β1 and MMP-2 in colorectal cancer.3,TGF-β1 has stimulated colon cancer cells HT29 for a long time, causing HT29 cells occurring EMT. The expressions of Tiam1 mRNA, E-cadherin mRNA and Vimentin mRNA of HT29 and HT29/TGF-β1 cells which have stimulated by TGF-β1 for 10d were detected by fluorescence quantitative PCR. The Cytoskeleton of HT29 and HT29/TGF-β1 cells were observed by Coomassie blue staining, the green and red fluorescent protein plasmid were transfected into EMT and non-EMT cells respectively, then mixed culturing two kind cells, doing scratch experiments, observing the movement of two kinds of cells.Results1,The expressions of Tiaml and EMT markers in colorectal cancer cell lines of different metastatic characteristic.Ct values which obtained by PCR were analysised through calculating the relative quantitative, statistical analysis found that the expressions average rank of Tiaml mRNA in SW480, SW620, SW480/M5, HT29, LoVo, and LS174T were 13.00,9.67,17.00,2.33,10.00,5.00, respectively. The difference was statistically significant (χ2=14.839, P=0.011). The average rank of expression levels of the E-cadherin mRNA were 8.00,14.00,11.00,17.00,2.00,5.00, respectively, the difference was statistically significant (χ2=16.717, P=0.005). The average rank of expression levels of the Vimentin mRNA were 11.00,14.00,8.00,4.00,17.00, 3.00 respectively, the difference was statistically significant (χ2=16.225,P=0.006). E-cadherin (++) protein was moderately expressed in the cytoplasm of HT29 cells, while Tiaml (-) and Vimentin (-) proteins were not detectable in HT29 cells. Tiam1(++) and Vimentin(+++) proteins were moderately and strongly expressed in the nuclei of LoVo cells, respectively, while E-cadherin(-) protein was undetectable in LoVo cells.HT29 cells had more surface projections, and less cytoskeletal structures and spot-like actin bodies than LoVo cells.2,Expressions of TGF-β1, MMP-2 in CRC:TGF-β1 was expressed heterogeneously among the normal colorectal tissue, colorectal carcinoma and lymphatic metastasis of carcinoma (χ2=55.928, P=0.000). TGF-β1 was significantly higher expressed in carcinomas compared with normal colorectal tissue(Z=-6.913, P=0.000); TGF-β1's expression was stronger in lymphatic metastasis in comparison with colorectal carcinoma(Z=-2.822, P=0.005); But there is no significant difference between well differentiated colorectal carcinoma and poorly differentiated colorectal carcinoma (Z=-1.589, P=0.112).MMP-2 was expressed heterogeneously among the normal colorectal tissue, colorectal carcinoma and lymphatic metastasis of carcinoma (χ2= 63.079, P=0.000). MMP-2 was significantly higher expressed in carcinomas compared with normal colorectal tissue(Z=-6.978, P=0.000); MMP-2's expression was stronger in lymphatic metastasis in comparison with colorectal carcinoma(Z=-4.381, P=0.000); But there is no significant difference between well differentiated colorectal carcinoma and poorly differentiated colorectal carcinoma (Z=-1.854, P=0.064).Tiaml were positively correlated TGF-β1 and MMP-2(r=0.209, P=0.015; r=0.486, P=0.000); TGF-β1 were negatively correlated E-cadherin, positively correlated MMP-2(r=-0.196, P=0.024; r=0.445, P=0.000); But there is no significant correlation with MMP-2 and E-cadherin(r= 0.048, P=0.582).3,The roles of the EMT and non-EMT cells in the invasion and metastasis of colorectal cancer.After stimulated by TGF-β1, the expression of E-cadherin mRNA decreased with significant statistical difference(t=-2.969, P=0.041), the expression of Vimentin mRNA increased with significant statistical difference (t=3.985, P=0.016), but the expression of Tiaml mRNA is no significant difference (t=0.910, P=0.414). HT29 cells were round, confluent growth; cell-cell adhesion is stronger; after continuous stimulated by TGF-β1 for 40d, spindle cell morphology changes than before, decline cell-cell adhesion, scattered growth. Coomassie blue staining shows that HT29 cells had more surface projections, and less cytoskeletal structures and spot-like actin bodies than HT29/TGF-β1 cells. Scratch test results show that HT29 cells' migration rate is less than the HT29 cells after stimulation, mixed culture the two cells, the mixed cells to migrate faster than HT29, but slower than HT29 cells after stimulation. Cells which migrate fastest include the round and spindle cells. Using 1μg red and green fluorescent plasmid DNA transfected cells, and after 24h, transfections are the most efficient. Co-culturing two kinds of cells, scratches, after 48h, we can see the red and green cells evenly distributed.Conclusion1,There is a part of invasion and metastasis of colorectal cancer cells associated with the Tiaml inducing EMT.2,In colorectal carcinogenesis process of EMT, Tiaml, TGF-(31 and MMP-2 are likely to have a synergistic effect.3,TGF-β1 can induce colorectal cancer cells EMT.4,In colorectal cancer, the EMT cells can enhance invasion and metastasis of the non-EMT cells.
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