The Regulation Of Cellular EMT,Migration And Invasion By TAB182 Through EGFR Signaling Pathway In Human Lung Cancer

Objective:establishing TAB182-knockdown stable lines by lentivirus infection using human lung cancer cells,to perform following works: 1)to explore the effect of TAB182 on DNA damage response proteins;2)to discover the influence of TAB182 on EGFR signaling;3)to elucidate the regulatory mechanism of TAB182 on cellular EMT,migration and invasion via EGFR signaling pathway.Method: 1)the establishment of shRNA-mediated TAB182-knockdown stable lung cancer cell lines: after infecting human lung cancer A549 and H1299 cells with lentiviral particles,the proportion of infected cells expressing GFP was observed under fluorescence microscope,then the knockdown efficacy of TAB182 was validated at mRNA and protein levels.Control-shRNA presented normal TAB182 expression level(hereinafter termed control cells),while TAB182-shRNA#1 and TAB182-shRNA#2were referred as TAB182-knockdown cells.2)to verify the involvement of TAB182 in DNA double-strand breakages repair: A549 TAB182-knockdown stable lines were given4 Gy 60Co γ irradiation,and protein levels of DSB marker γ-H2 AX were detected at 0 h,1 h,2 h and 4 h after IR;to validate the knockdown efficacy of HUWE1-shRNA Hela cell lines,and observe the numbers of γ-H2 AX foci at 1 h,2 h and 4 h after 10 Gy IR in Hela cells.3)to explore the translational regulation of TAB182 for DDR proteins including ATM,DNA-PKcs,Ku86,Ku70,Ct IP,EGFR,p21 and p16.4)to uncover the regulatory effect of TAB182 on EGFR signaling: the mRNA and protein levels of EGFR were detected in A549 TAB182-knockdown stable lines.5)to verify the translational modulation of TAB182 for EMT markers: E-cadherin,N-cadherin,Vimentin,MMP-2and Snail were detected in A549 TAB182-knockdown stable lines.6)to validate EGFRoverexpression efficacy of pc DNA3.1-EGFR plasmid: at 24 h after transfecting EGFRoverexpression plasmid into normal A549 cells,the levels of EGFR and EMT-related markers such as E-cadherin,N-cadherin,Vimentin and α-SMA were analyzed.7)to confirm whether EGFR mediates the regulation of TAB182 for molecular EMT phenotype: at 24 h after transfecting EGFR-overexpression plasmid into A549 TAB182-knockdown stable lines,protein expression of EGFR and EMT-associated markers comprising E-cadherin,N-cadherin,Vimentin and MMP-2 were detected.8)to validate the radiation-responsiveness of EGFR protein: normal A549 cells were irradiated by 4Gy IR,and EGFR level was detected at 1 d and 4 d post-IR.9)to validate radiation dosedependence of EGFR protein: normal A549 cells were delivered a single dose of 0.5,1,2,4,6,8 and 10 Gy IR,and EGFR protein levels were detected at 7 d after IR.10)to verify the radiation-responsiveness of TAB182 protein: A549 TAB182-knockdown stable lines were given 4Gy IR,and TAB182 level was examined at 3 d after IR.11)to investigate the influence of TAB182 on IR-induced EGFR upregulation and molecular EMT progression: A549 TAB182-knockdown stable lines were exposed to 4Gy IR,and expression levels of EGFR and EMT markers including E-cadherin,N-cadherin and Vimentin were analyzed at 0 h,24 h,48 h and 7 d after IR.12)to reveal the effect of TAB182 on IR-enhanced cancer cell invasion: A549 TAB182-knockdown stable lines were given 4Gy IR,and cell invasion was evaluated by Transwell invasion assay at 7 d after IR.13)to demonstrate the influence of TAB182 on EGFR protein stabilization:A549 TAB182-knockdown stable lines were administrated with 10 μg/m L CHX and sampled at 0 h,2 h,4 h,8 h,12 h and 24 h after CHX-treatment to detect EGFR levels;and in the context of IR exposure,A549 TAB182-knockdown stable lines were pretreated with 10 μg/m L CHX and subjected to 4Gy IR,and EGFR levels were examined at above-mentioned time points post-IR.14)to reveal the effect of TAB182 on various tumor phenotypes: CCK-8 assay,clonogenicity assay,scratching assay and Transwell invasion assay were performed to evaluate cell proliferation,colony-formation,migration and invasion in A549 TAB182-knockdown stable lines.15)to explore whether EGFR mediates the regulation of TAB182 for migration and invasion: at 24 h after transfecting EGFR-overexpression plasmid into A549 TAB182-knockdown cells(shRNA#1),migrative and invasive activity were assessed by Transwell migration/invasion assays.Results: 1)TAB182-knockdown stable lines were generated using human lung cancer A549 and H1299 cells.2)TAB182-knockdown delayed the retention time of γ-H2 AX after IR,and raised the protein levels of ATM,DNA-PKcs,p21 and p16 in lung cancer cells;at 1 h,2 h and 4 h after 10 Gy IR,silencing HUWE1 significantly raised the numbers of γ-H2 AX foci.3)Silencing TAB182 decreased the mRNA and protein levels of EGFR without affecting EGFR stability.4)Depleting TAB182 increased E-cadherin and decreased mesenchymal markers N-cadherin and MMP-2.5)Overexpressing EGFR reduced E-cadherin while upregulating N-cadherin,Vimentin and α-SMA in normal A549 cells.6)Overexpressing EGFR downregulated E-cadherin and increased Ncadherin and Vimentin without changing MMP-2 level in TAB182-knockdown cells(namely TAB182-shRNA#1).7)EGFR protein level was upregulated by 4Gy IR.8)EGFR upregulation was triggered by 2Gy～10Gy IR in a dose-independent manner.9)4Gy IR upregulated TAB182 level in control cells without affecting that in TAB182-kockdown cells(TAB182-shRNA#1 and-shRNA#2).10)Silencing TAB182 repressed4 Gy IR-triggered EGFR upregulation,and relieved IR-induced molecular EMT alterations at 48 h post-IR.11)4Gy IR significantly increased invasion activity of both control cells and TAB182-knockdown cells(TAB182-shRNA#1 and-shRNA#2).12)4Gy IR stabilized EGFR protein,and TAB182-knockdown obviously suppressed IRenhanced EGFR stabilization.13)Depleting TAB182 remarkably inhibited proliferation,clonogenicity,migration and invasion in lung cancer cells.14)Overexpressing EGFR reversed migrative and invasive activity of TAB182-knockdown cells(TAB182-shRNA#1).Conclusion:1.TAB182 participates in DNA double-strand breakages repair,and negatively regulates DSB sensor kinases ATM and DNA-PKcs as well as checkpoint regulator p21 and p16 in A549 and H1299 cells.2.TAB182 raised EGFR expression level,and promotes proliferation and clonogenicity in A549 cells.3.TAB182 drives molecular EMT,migration and invasion through EGFR signaling pathway in A549 cells.4.TAB182 is involved in IR-induced EGFR upregulation,which may be contributed to the enhancement of TAB182 for IR-triggered EGFR stabilization.