Effect Of JUP On Migration And Invasion Of Gastric Cancer Cells And Its Molecular Mechanism

Part 1 Effect of JUP on Biological Behavior of Gastric Cancer CellsObjective To investigate the effect of knockdown or overexpression of JUP on cell biological function in gastric cancer cells.Methods The lentiviral vector sequences knocked down and over-expressed JUP gene and their respective negative control viruses were constructed by sh RNA lentivirus-mediated manner.The expression of JUP m RNA and protein was verified by quantitative real-time PCR(q RT-PCR)and Western blot after transfected into NUGC-3 and MGC-803 cells.Level of silence and overexpression;using CCK-8,cell scratch healing experiments and Transwell chamber experiments were detected in each stable knockdown or overexpression of cell proliferation,migration and invasion ability changes.Results The NUGC-3,and MGC-803 cell lines stably knocked down by JUP and the NUGC-3 and MGC-803 cell overexpressing JUP cells and their respective negative control strains were successfully constructed.Compared with the control group,knockdown JUP had no significant effect on the proliferation of gastric cancer cells;scratch healing experiments and Transwell chamber experiments showed that NUGC-3 cells can promote cell migration and invasion can see,in MGC-803 cells can inhibit cell proliferation Migration and invasion ability;JUP overexpression in NUGC-3 and MGC-803 cells significantly inhibited cell migration and invasion.Conclusion In gastric cancer cells,silence of JUP had no significant effect on the proliferation of gastric cancer cells,and promoted the invasion and migration of cells in JUP cells with high expression in the cell membrane or cytoplasm.In the cells with high expression of JUP in the nucleus,Migration and invasion ability;overexpression of JUP,can reduce the migration and invasion ability of gastric cancer cells.Part 2 Molecular Mechanism of JUP on Invasion of Gastric Cancer CellsObjective To investigate the molecular mechanism of silencing and overexpression of JUP on gastric cancer cell migration and invasion.Methods Immunofluorescence was performed to detect the expression and localization of b-catenin in the JUP knock-down NCL-87/NUGC-3/MGC-803 and the JUP-overexpressing NUGC-3 and MGC-803 and their control cells.Levels of phosphorylated b-catenin(p-b-catenin),nuclear b-catenin(n-b-catenin),total b-catenin(T-b-catenin)and MMP7 were determined by Western blot in the indicated cells.Find the protein that interacts with JUP using the Protein Interaction Network (https://string-db.org/)and our previous LC-MS / MS data.Pearson correlation coefficients between JUP and interacting proteins were analyzed by GEPIA database(http://gepia.cancer-pku.cn/).Immunofluorescence co-localization and Co-IP were performed to detect the interaction between JUP and interacting proteins.The expressions of p-EGFR,T-EGFR,p-AKT,T-AKT,p-GSK3b,T-GSK3b were detected by Western blot.Results Knockdown of JUP in NCL-87,NUGC-3 cells resulted in increased p-b-catenin,reduced n-b-catenin and MMP7.However,loss of JUP led an increase of p-b-catenin in cytoplasm,and decrease of n-b-catenin and MMP7 in MGC-803.Over-expression of ectopic JUP in NUGC-3 and MGC-803 cells,p-b-catenin was dramatically increased,n-b-catenin and MMP7 were notably reduced.Protein interaction network found 12 proteins interacting with JUP;Pearson's correlation coefficient showed that EGFR was closely related to JUP;co-localization of JUP and EGFR was confirmed by immunofluorescent co-localization on the cell membrane;Co-IP also proved JUP and EGFR have a correlation,p-EGFR,p-AKT and p-GSK3b were increased after NCL-87 knocked down.While decreased after overexpression of JUP in NUGC-3 and MGC-803 cells.Conclusion Knockdown of JUP in cells that express JUP in the plasma membrane or cytoplasm can activate the EGFR / AKT / GSK3b signaling pathway,resulting in decreased expression of p-catenin and increased intracytoplasmic b-catenin translocation to the nucleus,promoting its invasion ability.In contrast,JUP overexpression in gastric cancer NUGC-3 and MGC-803 cells inhibited the EGFR / AKT / GSK3b signaling pathway,the degradation of p-b-catenin elevated,MMP7 decreased,thus inhibited cell migration and invasion.