| Objective: This study was performed to investigate the relationship between epithelial to mesenchymal transition(EMT) and radioresistance, so that we can further study the malignant biological behavior of esophageal cancinoma and find a new target to reduce the incidence of radioresistance providing a new clue. For this purpose, we examined the expression of neurotrophin receptor p75 interacting MAGE homologue(NRAGE)in three kinds cells and its location in cells, including TE13 cells、EMT cells model、TE13R120 cells, by Real-Time PCR、Western blot and immunocytochemistry, while EMT cells model were induced by TGF-β1 and TE13R120 cells with EMT-like properties were screened frome TE13 with repeated exposue.Methods: The human esophageal squamous cell carcinoma cell lines TE13 were routinely cultured. Cells were applied to the experiment when they entered the logarithmic phase. TE13R120 cells were screened from TE13 with repeated exposure, and detected radiation resistance by cell colony formation assay. What’s more, we observed the cells morphological changes and detected the the m RNA and protein expression of epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin to validate TE13R120 with EMT-like properties. We stimulated TE13 cells with TGF-β1 at the concentration of 10ng/ml, and used invert microscope to observe the cells morphological changes. In addtion, Real-Time PCR and Western blot methods detected respectively the m RNA and protein expression of epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin to further validate the farmation of EMT in TE13 cells. Then, it was divided into three groups: TE13 cells group、EMT cells model group and TE13R120 cells group, and the m RNA and protein expression of NRAGE were detected respectively by Real-Time PCR and Western blot methods among all groups. What’s more, the expression of NRAGE in nucleus and cytoplasm among three groups were detected by Western blot. Immunocytochemistry detected the protain expression of NRAGE further. All statistical analysises were performed with SPSS 17.0 statistical software package.Results:1 Radiation-resistant cell lines TE13R120 with EMT-like propertiesAfter the construction of TE13R120 cells, we obtained the radiobiology parameters of the two kinds of cells by cell colony formation assay. The values of D0、Dq and N in TE13R120 cells were larger than that in TE13 cells. Survival fractions 2(SF2), as an important indicator of cell radiosensitivity, was 0.734 in TE13R120 and 0.538 in TE13 cells.Observeing morphological characters of TE13 cells and TE13R120 cells with invert microscope, TE13 cells showed irregular polygon and no clear boundaries between cells, while TE13R120 cells showed slender and spindle shape. The expression levels of two marker proteins, that is, E-cadherin and Vimentin, were evaluated by Real-Time PCR and Western blot. The expression of E-cadherin decreased while the expression of Vimentin rised.2 The formation of EMT induced by TGF-β1Observed morphological changes before and after TGF-β1 treatment with invert microscope, the cells form of TE13 changed from irregular polygon to spindle shape, and from tight binding to loose. The expression levels of two marker proteins, that is, E-cadherin and Vimentin, were evaluated by Real-Time PCR and Western blot. After TGF-β1 stimulation, the expression of E-cadherin decreased while the expression of Vimentin rised.3 The expression of NRAGE in three groupsWe detected the m RNA expression and total protain expression of NRAGE in TE13 model cells group、EMT model cells group and TE13R120 cells group by Real-Time PCR and Western blot methods, and the expression of NRAGE in EMT model cells group and TE13R120 cells group were higher than TE13 cells group with statistical significance, while it was no statistical significance between EMT model cells group and TE13R120 cells group. Additionally, the protain expression of NRAGE in cytoplasm showed no statistical significance among three groups. However, the protain expression of NRAGE in nucleus were higher in EMT model cells group and TE13R120 cells group than TE13 cells group, and there was no statistical significance between TE13R120 cells group and EMT model cells group. Immunocytochemistry further confirmed these.Conclusions:1 NRAGE subcellular localization may participate in the formation of radioresistant.2 EMT may participation NRAGE subcellular localization to promote the formation of radioresistant... |
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