Relationship Between Epithelial Mesenchymal Transition And Tumor Resistance In Cholangiocarcinoma

Objective To study the relationship between epithelial mesenchymal transition?EMT?and the mechanism of drug resistance in cholangiocarcinoma.Method LY294002,a specific inhibitor of PI3K/Akt pathway,was used to reverse the important pathway of EMT in vitro using QBC939/WT and QBC939/5-FU cell line of human cholangiocarcinoma.Part 1:Detection of drug sensitivity of QBC939/WT and QBC939/5-FU cells.Observation of morphological difference between two cell lines under the inverted microscope.The expression levels of mRNA and protein such as E-cadherin,N-cadherin and Vimentin were detected by RT-PCR and western blot.The migration ability of two cells was determined by Woundhealing test and Transwell experiment.The CCK8 experiment was used to compare the proliferation ability of the two cells.Part 2:To add QBC939/5-FU cells with LY294002,a specific inhibitor of PI3K/Akt pathway,then to detect the drug sensitivity of QBC939/5-FU?LY294002?and QBC939/5-FU.To observe the morphological changes of QBC939/5-FU cells under microscope.The expression levels of mRNA and protein such as E-cadherin,N-cadherin and Vimentin were detected by RT-PCR and western blot.Woundhealing and Transwell assay were used to detect the cell migration ability and proliferation ability.The CCK8experiment was used to compare the proliferation ability of the two cells.Results Part1:QBC939/WT and QBC939/5-FU were 37.42±0.25?mol·L^-11 and83.78±3.54?mol·L^-11 for 5-FU IC₅₀0 respectively?P<0.05?.Compared with QBC939/WT cells,the QBC939/5-FU cells changed from tightly connected paving stone to fusiform with multiple protuberances and distance between cells was estranged showed by Microscopicand.RT-PCR and western blot assay showed that the expression of E-cadherin was down-regulated and the expression of N-cadherin and Vimentin was up-regulated.The migration ability of QBC939/5-FU cells is stronger than that of QBC939/WT cells?P<0.05?.The proliferative ability of QBC939/5-FU cells was significantly higher than that of QBC939/WT cells.The proliferative ability of QBC939/5-FU cells was 1.87±0.10 times,1.65±0.09 times,1.95±0.11 times than that of QBC939/WT cells at the same number of cells cultured for 48 h,72 h and 96 h,respectively?P<0.05?.Part 2:When added LY294002 to QBC939/5-FU,We found that the EMT was reversed obviously,cells changed from fusiform to paving stone,distance between cells shortened and junction tight.RT-PCR and western blot assay showed that the expression of E-cadherin was up-regulated and the expression of N-cadherin and Vimentin downward trend obviously.QBC939/5-FU?LY294002?IC₅₀0 resistance to 5-FU decreased to 42.52±0.57?mol·L^-1?P<0.05?.The ability of cell migration was significantly decreased compared with QBC939/5-FU?P<0.05?.The ability of proliferation of QBC939/5-FU?LY294002?cells decreased to 0.72±0.03?0.45±0.05 and0.35±0.04 compared with that of QBC939/5-FU?P<0.05?.Conclusion The occurrence of EMT can enhance the resistance of cholangiocarcinoma cells and significantly increase the degree of malignancy.To reverse the incidence of EMT,can make the sensitivity of cholangiocarcinoma cells to chemotherapeutic drugs increased and drug resistance decreased.