Research On The Mechanism Of Tumor Suppressor Protein H2AX Regulating EMT Of Lung Cancer A549 Cells

Objective H2 AX is a new tumor suppressor protein,the phosphorylation of H2AX(Ser139)site(gamma H2AX),which plays an important role in the apoptosis of tumor cells.The epithelial mesenchymal transition of tumor cells is the key link of cancer cell malignancy and metastasis.Whether H2 AX regulates the role of tumor EMT and metastasis is rarely reported.The purpose of this study was to explore whether H2 AX is involved in EMT and metastasis of lung cancer cells,and the molecular mechanism of H2 AX regulation of EMT and metastasis.Methods In non small cell lung cancer A549 cells as the research object,the experiment is divided into two groups: group A549(KD-H2 AX,gene silencing and A549(CTR),the control group(A549-vector);the cells transfected with empty vector(H2AX-wt)(A549),transfected with wild type H2AX(H2AX-139m))and A549(transfected with Ser139 phosphate of mutant H2 AX overexpression cell lines stable.)5% sera were used to stimulate the two groups of cells,and the migration ability of cells was observed by wound healing assay;used transwell method detected the level of cell migration and invasion;extracted the total protein of cells in two groups,with the characteristics of protein expression by western blot method for the detection of EMT(E-Cadherin,Vimentin);extracted the total RNA,RT-PCR detected E-Cadherin,Vimentin and transcription factor Zeb1/2,Slug,Snail,SIP1,Twist,mRNA level.In order to further study the molecular mechanism of H2 AX phosphorylation to regulate the transfer of EMT,the same method was used to cultivate two groups of cells.Extracted the total RNA,RT-PCR used to detect the expression of vascular endothelial growthfactor receptor mRNA two in lung cancer cells;extracted the total protein,protein expression detected by VEGFR-2 western blot;culture gene knockout(H2AX+/+ and H2AX-/-)cells,total protein extracted to detect the expression of VEGFR-2 by western blot method.Used Lipofectamin 2000 transfected A549 cells with VEGFR-2 si RNA,transwell method detected the effect of VEGFR-2 knockout on the migration and invasion of lung cancer cells.Each experiment was repeated 3 times independently,analyzed by SPSS22.0 software.Results H2 AX gene silencing promotes epithelial mesenchymal transition of lung cancer lung cancer cells;EMT correlate phorylation H2AX;γH2AX inhibits lung epithelial mesenchymal transition and metastasis;γH2AX inhibits the expression of VEGFR-2 gene;gene silencing of VEGFR-2 inhibits lung cancer cell A549 EMT.Conclusion γH2AX through downregulated VEGFR-2,and inhibited EMT in lung cancer cell A549.