Fascin Promotes The Invasion Of Pituitary Adenoma Through Partial Dependence On Epithelial-Mesenchymal Transition

Object: This study aims to explore the role of epithelial-mesenchymal transition(EMT)in Fascin promoting the invasion of pituitary adenoma(PA),and to provide experimental evidence for the mechanism of PA invasion.Methods: 1.Experiment with mouse pituitary adenoma At T-20 cells.The experimental groups are as follows:(1)PA+blank control group(PA+BC group),(2)PA+Fascin negative control group(PA+Fascin-NC group),(3)PA+Fascin interference group group(PA+Fascin-si RNA group),using RNA interference(RNAi)technology to specifically down-regulate the expression of Fascin in mouse pituitary tumor At T-20 cells.The Transwell experiment was used to detect the invasion potential of At T-20 cells down-regulated by Fascin.Cell Counting Kit-8 and flow cytometry were used to detect the changes in cell proliferation,cycle distribution and the number of apoptosis cells in At T-20 cells after Fascin was down-regulated.Transmission electron microscope used to be observed the ultrastructural changes of At T-20 cells after Fascin downregulation.Western blot,immunofluorescence and real-time quantitative PCR were used to detect the changes of the expression of epithelial markers(E-cadherin),Fascin,mesenchymal markers(N-cadherin,vimentin)and transcription factors(Twist).2.Divide mouse pituitary adenoma At T-20 cells into(1)PA+blank control group(PA+BC group),(2)PA+E-cadherin negative control group(PA+E-cadherin-NC group),(3)PA+E-cadherin over-expression group(PA+ E-cadherin OE group),through overexpression of E-cadherin in mouse pituitary tumor At T-20 cells.Transwell experiment was used to detect the invasion potential of At T-20 cells after E-cadherin overexpression.Cell Counting Kit-8 and flow cytometry were used to detect the changes in cell proliferation and cycle distribution of At T-20 cells after overexpression of Ecadherin.Western blot and real-time quantitative PCR were used to detect the expression of Fascin,Ecadherin and transcription factor(Twist)after E-cadherin overexpression.Statistical analysis uses Pearson statistical method to detect the correlation between EMT-related markers,invasion and Fascin.Results: 1.The effect of down-regulation of Fascin expression on At T-20 cells through the EMT process.(1)CCK-8 detection of cell proliferation results: the proliferation ability of pituitary adenoma cells in the PA+ Fascin-si RNA group was lower than that in the PA+ Fascin-NC group.(2)Transwell test results of cell invasion: the invasion potential of pituitary adenoma cells in the PA+ Fascin-si RNA group was lower than that in the PA+ Fascin-NC group.(3)Cell cycle and apoptosis results: Compared with the PA+ FascinNC group,the proportion of pituitary adenoma cells in the G1 phase in the PA+ Fascin-si RNA group increased;the proportion of cells in the G2 and S phases decreased.Compared with the PA+ Fascin-NC group,the number of apoptosis cells of pituitary adenoma cells in the PA+ Fascin-si RNA group was significantly increased.(4)TEM observation of cell ultrastructure: the cell structure of the PA+ Fascin-NC group was roughly normal,and the morphological features of apoptosis appeared in the cells of the PA+Fascin-si RNA group,and the formation of apoptotic bodies could be seen.(5)Western blot and Real-Time PCR results: the expression of E-cadherin in the PA+ Fascin-si RNA group was increased compared with that in the PA+ Fascin-NC group,while the expression of Fascin,N-cadherin,vimentin and transcription factors(Twist)were decreased.(6)Immunohistochemical results: The expression of E-cadherin in the PA+ Fascinsi RNA group increased compared with the PA+ Fascin-NC group,while the expression of Fascin,N-cadherin,vimentin and transcription factor(Twist)decreased.(7)Statistical analysis showed that invasion was negatively correlated with the expression of E-cadherin,but positively correlated with N-cadherin and vimentin.Fascin expression is positively correlated with invasion.The expression of Fascin is negatively correlated with the expression of E-cadherin,while the expression of Fascin is positively correlated with the expressions of N-cadherin and vimentin.2.The effect of up-regulation of E-cadherin expression on At T-20 cells and Fascin.(1)CCK-8 detection of cell proliferation results: the proliferation ability of pituitary adenoma cells in the PA+ E-cadherin OE group was lower than that in the PA+ E-cadherin-NC group.(2)Transwell detection of cell invasion results: the invasion potential of pituitary adenoma cells in the PA+ E-cadherin OE group was lower than that in the PA+ E-cadherin-NC group.(3)Cell cycle results: Compared with the PA+ Ecadherin-NC group,the proportion of pituitary adenoma cells in the G1 phase in the PA+ E-cadherin OE group decreased,while the proportion of cells in the G2 phase increased and the proportion of cells in the S phase has not changed.(4)Western blot and Real-Time PCR results: The expression of epithelial markers(Ecadherin)in the PA+ E-cadherin OE group was increased compared with the PA+ E-cadherin-NC group,while Fascin and transcription factors Twist expression is reduced.(5)Statistical analysis showed that Ecadherin was negatively correlated with invasion,and Fascin was negatively correlated with E-cadherin.Conclusions: Fascin may be an important key factor in the invasion of PA.Inhibition of Fascin expression can alter the expression levels of various EMT markers and regulate the progression of pituitary tumor cells.Fascin may effectively promote pituitary tumor cell invasion through partial dependence on the EMT pathway.