| ObjectiveUsing immunohistochemistry(IHC) to investigate the expression of E-cadherin, vimentin, β-catenin and TGF-β1 in Oral squamous cell carcinomas(OSCC) tissue, combined with using immunofluorescence assay(IFA) and Transwell chamber invasion assay to investigate the correlation between the expression of these four markers and the invasiveness of SCC-9 and Tca8113, two kinds of oral squamous cancer cell in vitro, and discussing the basic performance of oral squamous cell carcinoma epithelial- mesenchymal transition, in order to further explore the epithelial- mesenchymal transition phenomenon and provide a theoretical basis of studying the prevention of oral squamous cell carcinoma.Methods(1) Histology experiment: we had collected 89 cases of OSCC and 20 cases of normal oral mucosa. The 89 cases of OSCC were classified as I, II, III grades, and stained E-cadherin, Vimentin, β-catenin and TGF-β1 by immunohistochemistry(IHC) respectively. We calculated the positive intensity and positive rate by the semiquantitative method. Then we analyzed the correlation between these four biomarkers and OSCC differentiated degree and the correlation between the four biomarkers.(2) Cytology experiments: ①we made SCC-9 and Tca8113 growing on the glasses, and stained the cells these four biomarkers by immunofluorescence assay(IFA). Selecting five visions randomly each glass to take pictures by the fluorescence microscope, and calculating the mean density of each picture, then analysing the difference of the expression of these four biomarkers between SCC-9 and Tca8113 by statistical method. ②Culturing SCC-9 and Tca8113 hungrily for 12 hours, and then planting in the upper chamber. We added 10% fetal bovine serum medium in the lowerchamber, and culture for 36 hours. After that, we stained the bottom of the chambers by crystal violet, and calculate the cells which passed through the chamber.Results(1)The results of histology experiment: ①Every group of data had been tested by S-W test. The results showed that the data did not conform to a normal distribution, so we used median±interquartile range to describe the data. The median of E-cadherin in the normal tissue was9.00, which in OSCC I, II and III were 9.00, 6.00 and 6.00 respectively. There were differences statistically in comparison of the expression result between the normal group and summation of OSCC I, II and III grades groups(Z=-4.211, P=0.000), and that was related with the differentiated degree(P=0.000). The median of vimentin in the normal tissue was 0.00, which in OSCC I, II and III were 0.00, 0.00 and 4.00 respectively. There were differences statistically in comparison of the expression result between the normal group and summation of OSCC I, II and III grades groups(Z=-3.675, P=0.000), and that was related with the differentiated degree(P=0.000).The median of β-catenin in the normal tissue was 9.00, which in OSCC I, II and III were 3.00, 4.00 and 3.00 respectively. There were differences statistically in comparison of the expression result between the normal group and summation of OSCC I, II and III grades groups(Z=-6.300, P=0.000), and that was related with the differentiated degree(P=0.017). The median of TGF-β1in the normal tissue was 2.00, which in OSCC I, II and III were 3.00, 4.00 and 6.00 respectively. There were differences statistically in comparison of the expression result between the normal group and summation of OSCC I, II and III grades groups(Z=-3.329,P=0.000), and that was related with the differentiated degree(P=0.000). ② The correlation between these four biomarkers: using the Spearman’s correlation test, E-cadherin expression was significant positively correlated to β-catenin expression(r=0.327, P=0.002), significant inverse correlated to Vimentin expression(r=-0.386, P=0.001) and significant positively correlated to TGF-β1 expression(r=-0.304, P=0.004). Vimentin expression was significant positively correlated to TGF-β1 expression(r=0.401, P=0.000).(2) The results of the cytology experiments: ① In the part of immunofluorescence of cell culture in vitro, the expression of E- cadherin in SCC-9 was not observed fluorescence. The means of vimentin, β-catenin and TGF-β1 meandensity were 0.0531, 0.0570 and 0.05750.; the means of E-cadherin, vimentin, β-catenin and TGF-β1 mean density in Tca8113 were 0.00145, 0.0421, 0.0500 and 0.04990. The expression level of E-cadherin in SCC-9 and Tca8113 was not statistically different(t=-1.395, P=0.176); The expression level of Vimentin in SCC-9 and Tca8113 was statistically different(t=3.125, P=0.003); The expression level of β-catenin in SCC-9 and Tca8113 was statistically different(t=2.581, P=0.014); The expression level of TGF-β1 in SCC-9 and Tca8113 was statistically different(t=2.495, P=0.016). ②In the part of Transwell chamber invasion assay, the means of SCC-9 and Tca8113 passed through the Matrigel to the bottom of chambers were 57.93 and 42.47 respectively, with statistically difference(t=3.309,P=0.003).Conclusions(1) E-cadherin and β-catenin in OSCC had a down-regulated expression, while the Vimentin has an up-regulated expression and TGF-β1 had a trend of increased expression, which suggested that there was EMT in the occurrence and development of OSCC;(2) There was significant positive correlation between β-catenin and E-cadherin on the OSCC cell membrane, and was negative correlation trend between Vimentin; there was significant negative correlation between TGF-β1 and E-cadherin, and was significant positive correlation between Vimentin, which suggested that β-catenin and TGF-β1 played important roles in the EMT process of OSCC;(3) Compared with Tca8113, the expression of Vimentin, TGF-β1 and β-catenin in nucleus and cytoplasm of SCC-9 increased, the expression of E-cadherin decreased. Combining the result that SCC-9 passed through the Matrigel was more in the Transwell chamber invasion assay suggested that SCC-9 had a more significant mesenchymal phenotype than Tca8113, which provide a favourable cytological model for further study EMT phenomenon in OSCC. |
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