| Esophageal cancer is a highly aggressive neoplasm. On a global basis, cancer of the esophagus is the sixth leading cause of cancer death worldwide, especially in Henan province of China. With advances in surgical techniques and treatment, the prognosis of esophageal cancer has slowly improved over the past three decades. However, the 5-year overall survival rate remains poor. The most important reason is the mechanism of invasion and metastasis is still unclear. To explore the mechanism of the invasion and metastasis of esophageal carcinoma has become a focus.Epithelial-mesenchymal transition (EMT) is an important mechanism for reorganizing germ layers and tissues during embryonic development. Originally EMT was defined by the formation of mesenchymal cells from epithelia in different embryonic territories, but recently, this process is also found reactivated in a variety of diseases including fibrosis and in the progression of carcinoma. Thiery describes EMTs in tumorigenesis like this : Normal epithelial cell grow along the basal membrane, then the phenotype gene change or mutation make the cell transfer to carcinoma in situ (now the basal membrane still maintains integrity); with development , cancer cells proliferation in region by Epithelial-mesenchymal transition, the basal membrane becomes crisply , cells invasion into lymph-vessels and bloodvessels and metastasis; In the planter spot, cancer cell could growth into metastatic tumor by mesenchymal- epithelial transition; For epithelial cells ,EMT involves loss of epithelial phenotype, or de-epithelialization, which would turn itself to non-epithelial, or nominal 'mesenchymal' cells, in place of what was an epithelium, and it involves a lot of remodeling of junctions, polarity, motility, adhesive properties as well. EMT includes changing of phenotypic markers, an increased capacity for migration and three-dimensional invasion, as well as apoptosis. So far, there is noreport about EMT in Esophageal Squamous Cell Carcinoma .EMT is a very complex process, including: (1) the cell-cell junction (sticking connection, tight junction, desmosome connection) disintegrates, and the cell becomes dispersible; (2) the cell skeleton rearrangement to cell - matrix attaches, cell movement ability enhancement. (3) gene change: In the EMT process, up-regulated factors involves mesenchymal cell marker such as fibronectin,Vimentin,N-cadherin and so on, as well as cell factors and transcription regulaters which can induce EMT, like snail, slug, TGF- beta(transforming growth factor- beta), FGF (fibroblast growthfactor) MMP-2 (matrix metal proteinase -2 ), MMP-9 (matrix metal proteinase -9); down-regulated factors involves E-cadherin,β-catenin,γ-catenin,cytokeratinl 8 and so on. It has found that TGF-βplay a critical role in EMT.We have not seen any report about EMT and relationship of EMT induced by TGF-P with invasion and metastasis of ESCC. In this study ,we detected the expression of TGF-βin ESCC tissue and ESCC cell line EC9706, and relationship of the expression of TGF-βwith E-cadherin and Vimentin; inhibited the expression of TGF-β1 by TGFβ1-ASODN in EC9706 to detect changes of expressions of E-cadherin and Vimentin,cell morphous, migration ability ,from these aspects to investigate EMT induced by TGF-β1 in ESCC, and investigate the effect of TGF-β1 to cell proliferation and Apoptosis.Materials and methods:1. Expressions of TGF-β1,E-cadherin and Vimentin proteins were detected in 100 cases of surgically resected esophageal squamous carcinoma specimens by the method of immunohistochemistry, which including carcinoma tissues and corresponding normal tissues.2. Expressions of TGF-β1,E-cadherin和Vimentin proteins and mRNA in the ESCC cell lines EC9706 were investigated by reverse transcription-polymerase chain reaction (RT-PCR),immunocytochemistry and flowcytometry.3. The antisense oligodeoxynucleotides (ASODN) was complementary to the TGF-β1 sequences. EC9706 was transfected with chemical synthesised TGF-β1 oligodeoxynucleotides by transfection reagents.(1) Cell morphological changes were observed with inversphase microscope.(2)The changes of TGF-β1 protein and mRNA were tested by using immunohistochemistry,flowcytometry and RT-PCR.(3) The changes of E-cadherin and Vimentin were observed by using RT-PCR, immunohistochemistry and flowcytometry.(4) Cell migration potentia were tested by scarification test.4. The apoptosis,cell cycles and proliferation of EC9706 were detected by flowcytometry and MTT.5. Statistical analysis: All the dates were analyzed by SPSS 13.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [x|-±s]. The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable groups is analyzed by the Kendall correlation analysis. The level of significant difference is a=0.05.Results1.The positive rate of expressions of TGF-β1 protein were higher significantly in carcinoma tissues 85.0%than that in normal tissues 27.0% (P <0.01) . The positive rate of expressions of TGF-β1 in deeper invasion group 90.6% were significantly higher than that in superficial invasion group 75.0% (P <0.05).2. The positive rates of E-cadherin protein were significantly higher in normal mucosa tissues 85.0% than that in cancer tissues43.0% (P <0.01). The positive rates of Vimentin protein were significantly lower in normal mucosa tissues 0 than that in cancer tissues 23.0% (P <0.05).3.There were negative correlations between the positive rate of TGF-β1 protein expression and that of E-cadherin protein expression in cancer tissues (T_b=-0.257, P<0.05), positive correlations between the positive rate of TGF-β1 protein expression and that of Vimentin protein expression in cancer tissues (T_b=0.163, P<0.05 ) , and negative correlations between the positive rate of E-cadherin protein expression and that of Vimentin protein expression in cancer tissues (T_b=-0.379, P＜0.01) .4. Results of RT-PCR showed that expressions of mRNA of TGF-pl(0.43±0.09),E-cadherin (0.22±0.06)and Vimentin(0.89±0.09) can be detected in EC9706.Results of ICC showed that proteins of TGF-β1(43.57%),E-cadherin(12.53%) and Vimentin(17.97%) express in the ESCC cell line EC9706; results of flow cytometry showed that proteins of TGF-β1(41.4%). E-cadherin(12.2%) and Vimentin(17.8%) express in the ESCC cell line EC9706. 5. After transfection of TGFβ1-ASODN, TGF-β1mRNA/β-actin mRNA half quantitation shows that expressions of TGF-β1 mRNA (0.250±0.681) were obviously lower than control group (0.425±0.093) (P <0.05) . ICC results shows that the positive rates of expressions of TGF-β1 protein were lower significantly in ASODN group (35.07%) than that in control group (43.57%) (P<0.01) , and protein in cytoplasm of transfered group obviously descend. Results of flowcytometry indicated that the positive rates of expressions of TGF-β1 protein were lower significantly in ASODN group (35.4%) than that in control group (41.38%) (P <0.01) .6. After transfection, results of RT-PCR showed that expressions of E-cadherin mRNA (0.3750±0.0925) were obviously higher than that in control group(0.2183±0.0646) (P <0.05), and expressions of Vimentin mRNA in ASODN group (0.7300±0.0669) were obviously lower than that in control group (0.8933±0.0894) (P<0.05). Results of ICC showed that expressions of E-cadherin protein in ASODN group (17.13%)were obviously higher than that in control group (12.53%) (P<0.01), and expressions of Vimentin protein in ASODN group (14.15%)were obviously lower than that in control group (17.97%) (P<0.01).Results of flowcytometry showed that expressions of epithelial marker E-cadherin protein in ASODN group (17.8%)were obviously higher than that in control group (12.2%) (P<0.01), and expressions of mesenchymal marker Vimentin protein in ASODN group (14.9%)were obviously lower than that in control group (17.8%) (P <0.01).7. After transfection by TGFβ1- ASODN, transfected cells display distinct biological behaviour, such as shape becoming shrink and round , and more grains . But control group cells were fusiform shape, adherence growth, and had clear layout among cells.8. Results of scratch test showed that after transfion, migration lenth of ASODN group in24 (0.14±0.02),48 (0.30±0.06),72h (0.45±0.05 ) were significantly shorter than control group 24h (0.23±0.02) , 48h (0.69±0.12 ) ,72h (0.81±0.11 ) (P<0.05).9. After transfection, apoptosis incidence rate in ASODN group (0.69%) was lower than that in control group (0.96%),and the compare has significant difference(P<0.05). Cells in G stage of ASODN group(19.3%) was lower than those of control group (23.7%) (P<0.01); cells in S stage of ASODN group(19.3%)was lower than those of control group (23.7%) (P＜0.01); cells in G2 stage of ASODN group (14.8%) was higher than those of control group ( 9.8% ) (P<0.01); cell survival rate in ASODN group (109.4%) was higher than that in control groupConclutions1. Overexpressions of TGF-β1 in esophageal cancer tissue and in ESCC cell lines EC9706 ; in cancer tissues , negative correlations between the expression of TGF-β1 protein and that of E-cadherin protein , and positive correlations between the expression of TGF-(31 protein and that of Vimentin protein indicated that there may have TGF-β1 induced EMT in carcinogenesis and development of ESCC.2. After transfection by TGFβ1-ASODN , expressions of protein and mRNA of TGF-β1 were suppressed, which indicated that TGFβ1-ASODN can specific inhibit expressions of TGF-β1.3.After transfection by TGFβ1-ASODN, ESCC cell line EC9706 change in morphous, decrease capacity for migration , and up-regulate of E-cadherine and down-regulate of Vimentin, indicates that blockage of TGFβ1 can inhibit the EMT progress in ESCC cell line EC9706.4. After TGF-β1 inhibited by TGFβ1-ASODN , the effect of growth inhibition and Apoptosis promotion by TGF-β1 might be relieved.
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