Effect Of RhoC On Epithelial-mesenchymal Transition Process Induced By TGFβ1in Lung Adenocarcinoma Cell

Objective:Rho GTPase is a group of guanine nucleotide-binding protein. The cell motion mediated by Rho GTPase probably plays an important role in the process of malignant tumor invasion and transition. According to recent research, compared with other family members of Rho superfamily, RhoC has a stronger binding force with its downstream effector ROCK. As a result, RhoC is regarded to have stronger ability in cell motion mediated by cytoskeleton, and RhoC functions through the whole process of tumor transition. In addition, in the process of epithelial-mesenchymal transition (EMT) for tumor cells, the change of cytoskeleton has been recovered, and cells have more power in invasion and transition. As RhoC has some similarity with EMT, it is necessary to investigate the origin of this similarity, which could be interrelated or independent with other. In this thesis, the TGFβ1induced EMT process in lung adenocarcinoma cell is researched to study the relationship between RhoC and EMT in lung adenocarcinoma cell.Methods:A549cells were stimulated with5ng/ml TGFβ1. the forms of the cells are investigated with an inverted microscope. The phenotype transition of cells is evaluated by examining the level of E-cadherin and Vimentin protein using westernblot. The invasive capability of A549cells was monitored by Transwell invasive assay, while RhoC expression level and activity were measured by westernblot and Pull-down assay, respectively. RhoC shRNA is introduced into A549cells to silence RhoC gene before and after TGFβ1stimulation, the effect of RhoC down-regulation on EMT progress is evaluated by examining the change of the cell forms and the expression levels of two mark proteins, that is, E-cadherin and Vimentin.Results:After48h TGFβ1stimulation, the cell forms of A549have changed obviously, and the expression of E-cadherin decreased while the expression of Vimentin rises. The invasive experiment reveals that the invasive ability enhaced obviously, which demonstrate that EMT occurs.In addition, the RhoC expression and activity are enhanced, and the inhibition of RhoC expression can dramatically decrease the A549cells invasive capability in vitro. However, if we pretreated A549cells by RhoC siRNA with5ng/ml TGFβ1stimulation for48h, we did not observe any EMT characteristic sign, and the reverse EMT process (MET) does not occur after A549cells are pretreated by RhoC siRNA with5ng/ml TGFβ1stimulation for48hConclusion1.The EMT process of A549cells can be induced by TGFβ1stimulation.2.In the process of TGFβ1stimulation for A549cells, the increasing level of RhoC expression and activity and the inhibition of RhoC expression can impede EMT progress. However, it can not reverse the EMT process...