The Role And Mechanism Of MDM2 In Epithelial Mesenchymal Transition Of Lung Adenocarcinonma Cell

Objective:Lung adenocarcinoma initiation and development are closely associated with with Epithelial-Mesenchymal Transition(EMT).Murine double minute 2(MDM2)is implicated in the initiation and progression of lung adenocarcinoma,but its function and regulatory mechanism during EMT process in lung adenocarcinoma remains unclear.This study focused on the possible roles of MDM2 gene in EMT and migration and invasion processes of lung adenocarcinoma cells,as well as the underlying molecular mechanisms.Methods:(1):MDM2,E-cadherin and N-cadherin expression in paired cancer tissues and adjacent normal tissues from 30 lung adenocarcinoma patients were measured by RT-PCR,immunoblotting and immunohistochemistry.(2):MDM2,E-cadherin,Vimentin,?-catenin,Snail,Slug and N-cadherin expression in cancer cell lines PC9?H1975 and A549 were determined by RT-PCR and immunoblotting.(3):MDM2 was over-expressed in cancer cells by letivirus-meidated tranfsection with pcDNA3.0 pasmids,letivirus-meidated shRNA transfection was perform to knockdown MDM2 expression in cancer cells.Cell proliferation,Annexin V/PI double staining,Transwell and scratch tests were used to analyze cell proliferation,apoptosis,migrations and invasion were analyzed by MTT,Annexin V/PI staining,Transwell and sratch tests.(4):Smad2 and Smad3 protein abundances in cells with MDM2 overexpression or silencing were analyzed by immunoblotting.Gene silencing mediated by siRNA were done to suppress Smad2 and Smad3 expression in cancer cells,and EMT protein markers were then measured.(5):Tumorigenicity of mice transplated with MDM1-overexpressing on-silencing cells was evaluated by nude mouse tumorigenicity assay,in which transcript and protein levels of MDM2 and EMT markers were determined.Results:Expression levels of MDM2 and mesenchymal cell marker genes N-cadherin and Vimentin were greatly increased in cancerous tissues and cancer cells PC9,H1975 and A549 compared with the normal adjacent tissues and bronchial epithelium cells,while the expression levels of epithelial cell marker genes were significantly suppressed.MDM2 gene overexpression increased lung adenocarcinoma cell proliferation rate,suppressed cell apoptosis and promoted cell invasion and migration capability.The apoptosis rates of cells with MDM2 overexpression(5.6+0.23%)were significantly lower than the control(7.7±0.19%)(P<0.05).Meanwhile the expression levels of E-cadherin and ?-catenin were down-regulated and N-cadherin,Vimentin,Snail and Slug gene expressions were elevated in MDM2 over-expressed lung adenocarcinoma cells.Completely opposite changes were observed in lung adenocarcinoma cells with MDM2 gene silencing.MDM2 gene overexpression elevated the Smad2 and Smad3 protein levels and phosphorylation in lung adenocarcinoma cells,while MDM2 silencing remarkably decreased Smad2 and Smad3 protein levels and phosphorylation.Smad2 and Smad3 gene silencing suppred EMT in lung adenocarcinoma cells.At 42d,tumor volumes of the MDM2 overexpression group were significantly higher than the control group(624.59±51.22 mm3 vs 543.11±54.81 mm3,P<0.01),while tumor volumes of the MDM2 silencing group were remarkably lower then the control group(P<0.05),accompanied with significantly lowered tumor weight compared with the control group(2.57±0.163 g vs 3.47±0.14g,P<0.05).Conclusion:We disclosed in this study that high MDM2 expression in lung adenocarcinoma cells shares close correlation with EMT marker expression,and MDM2 promotes EMT and lung adenocarcinoma development by positively regulating TGF-b/Smad signaling.