Research Of The Relationship Of Nm23-H1Gene And Epithelial-mesenchymal Transition Induced By Tumor Microenvironment

Metastasis was one of the malignant property of carcinoma. Metastasis was also the main reason resulting in the failure of anticancer therapy and the death in cancer patients. nm23-H1was the first metastasis suppressor gene that was detected. The mechanism by which nm23-H1inhibit metastasis was very complex and was not completely known. Epithelial-mesenchymal transition (EMT) is a critical event which enables the cancer cell to achieve the ability of invasion and migration. Although there was no report abount the relatioship of nm23-H1gene and EMT, some indirect evidance indicate that nm23-H1gene may impact on the process of EMT. To study the moleculer mechanism of metastasis suppress induced by nm23-H1, and to find new targets for anti-metastasis therapy, we investigate the impact of nm23-H1gene on EMT and explore the signal transduction pathway involved in.Part I The impact of nm23-H1gene on EMT in lung adenocarcinoma cell linesObjective:To investigate the impact of tumor metastasis suppressor gene nm23-H1on EMT in lung adenocarcinoma cell line A549and A549-99. Methods: EMT inducers were used to stimulate EMT of cells. Transfection of nm23-H1siRNA and plasmid contained nm23-H1full lenghth cDNA were applied to regulate the level of nm23-H1expression. The changes of morphology, migratory and invasive ability were study through cell observation, wound healing test and Boyden chamber test respectively. Western blot and immunofluorescence were use to detect the protein changes of biomarker related to EMT, Real-time PCR was used to detect the mRNA changes of biomarker related to EMT. The flow cytometry was also used to study the changes of lung cancer stem cell marker and cell cycle. Results:After the treatment of TGF-β1, cells shape change from polygonal typical epithelial morphology to spindly mesenchymal morphology. The ability of migration and invasion increased. The protein and mRNA level of E-cadherin dcreased obviously. The expression of mesenchymal cell marker, fibronectin and β-catenin, increased. The results of other EMT inducers, such as HGF, IL-6, serum, et al, were similar in the expression of EMT marker. When nm23-H1gene was knockdown, cells could gain more mesenchymal cells properties at the same time. In the same TGF-β1concentration, their morphology were more alike mesenchymal cells. The ability of migration and invasion increased. The expression of E-cadherin decreased, and the expression of fibronectin and β-catenin increased. Cells have some properties of lung cancer stem cell. But the impact on cell cycle was not significant. Over expression nm23-H1gene can reverse the protein expression of EMT biomarkers. Conclusion:Metastasis suppressor gene nm23-H1can suppress metastasis by inhibiting the development of EMT.Part Ⅱ Nm23-H1gene suppress the EMT of lung adenocarcinoma cell lines through transcription factor snailObjective:Transcription factor snail is improtant for the regulation of EMT. To investigate whether the transcription factor snail was involved in the EMT suppression of metastasis suppressor gene nm23-H1. Methods:After nm23-H1gene silience, western blot and Real-time PCR were use to detect the protein and mRNA changes of snail, dual-luciferase reporter assay was used to study the chang of snail promotor. Western blot was also used to detect the protein level when nm23-H1gene was over expressed. Snail siRNA and plasmid contained snail full lenghth cDNA were transfected to knockdown or over express the snail gene. Western blot was use to detect the change of EMT biomarker protein. Results:After the induction of TGF-β1following nm23-H1gene silence, the protein and mRNA level of snail increase obviously, and the activity of snail promotor increase too. When the nm23-H1gene was over expressed, the level of snail protein decreased. The snail gene silence can reverse the expression of EMT marker in part but not completely; the over expression of snail gene can not afford for all the changes of the EMT markers especial mesenchymal cell marker. The result shows that nm23-H1gene suppress the EMT of lung adenocarcinoma cell lines through transcription factor snail in part. Conclusion:Transcription factor snail was required for metastasis suppressor gene nm23-H1to suppress the EMT of lung adenocarcinoma cell line, but some other factors may play a role in this process.Part III Signal transduction pathway involved in EMT suppression of metastasis suppressor gene nm23-H1Objective:To investigate the signal transduction pathway involved in the EMT suppression of nm23-H1gene and to explore the anti-metastasis mechanism of nm23-H1gene. Methods:Some inhibitors were used to block the important signal transdution pathways involved in EMT. Western blot was use to detect the changes of EMT biomarkers. The crucial protein of TGF-β and Src pathway were also analysed by western blot at different level of nm23-H1gene expression. The changes of EMT biomarkers and the crucial protein of TGF-β pathway following STRAP(serine-threonine kinase receptor-associated protein, a nm23-H1interacted protein) siRNA transduction were detected by western blot. Results:The TGF-β pathway was activated more easily in the case of nm23-H1gene silence at the same concentration of TGF-β1. The activity of Src was increased at the same time. The over expression of nm23-H1can reverse this process. The result of signal transduction inhibitor test shows that the regulation of Src protein activity was important for nm23-H1to suppress EMT. Furthermore, nm23-H1gene can cooperate with STRAP to suppress the development of EMT induced by TGF-P1. Conclusion: Metastasis suppressor gene nm23-H1can suppress the development of EMT by negative regulation of TGF-P signal transduction pathway. Src protein plays a role in this process.