The Inhibition Of ING4 Or/and P53 Mediated By RGD-modified Adenovirus Vectors On Human Lung Adenocarcinoma Cell

Objective:To discuss the inhibition of human lung adenocarcinoma(A549)cell via constructing recombinant adenovirus vector of ING4(inhibitor of growth 4) or/and P53 modified with RGD(Arg-Gly-Asp) in vivo and in vitro and detect related molecular mechanism.Methods:Firstly,acquired P53 by PCR,and on the basis of pAdTrack-CMV-PolyA-promoter and pAdTrack-CMV-ING4-PolyA-promoter,insert P53 between NotⅠand XhoⅠ, then constructed pAdTrack-CMV-polyA-promoter-P53 and pAdTrack-CMV-ING4-polyApromoter-P53 successfully.Homologous recombination with adenovirus plasmid modified with RGD-4C,the recombinant adenovirus plasmid was packaged and amplificated in QBI-293 A cells. Observed the expression of GFP in QBI-293 A cell and A549 cell infected with adenovirus via fluorescence microscope.Detected the efficiency of adenovirus to A549 via FCM(flow cytometry).RT-PCR and Western blot method detected the expression of ING4 or/and P53 in A549 cell. Analysis cell growth, apoptosis and cell cycle of A549 by MTT and FCM.Scarification test and Tanswell method detecte the influence of the migration and invasion ability of A549 cell induced by ING4 or/and P53.Then detected the expression level of Caspase-3,Bax,Bcl-2,P21, MMP-2 and MMP-9 in A549 by Real time-PCR.Western blot method detected the expression of Bax,Bad,Bcl-2,Bcl-xl and MMP-2 and MMP-9 in A549 cell.Constructed the nude mice model of human lung adenocarcinoma(A549).Then observed the influence of anti-tumor effect of transplant tumor in nude mice through ING4 or/and P53.Detected the expression of apoptosis,cell cycle and angiogenesis related protein Caspase-3,Bax,Bcl-2,P21 and VEGF in A549 cell through immunohistochemistry analysis.Results:The result of PCR,enzyme digestion,RT-PCR and Western blot showed that the recombinant adenovirus(Ad.RGD-P53 and Ad.RGD-ING4-P53) were constructed successfully.They can infected the QBI-293 A cell and A549 cell efficiency.ING4 or/and P53 gene could significantly inhibit the growth of A549,induce its apoptosis,produce G1 arrest and inhibit the migration and invasion ability of A549 cell.In addition,the inhibition effect of the double gene adenovirus was more obvious than the signal gene adenovirus.In vivo,ING4 and P53 gene could similarly inhibit the tumor growth of A549 human lung adenocarcinoma transplanted tumor in nude mice.Moreover,the double gene adenovirus has a higher degree inhibition effect on tumor suppression than the signal gene adenovirus.The result of immunohistochemistry analysis showed that ING4 or/and P53 gene could up-regulate cell cycle related gene P21,promote the expression of apoptosis related gene Caspase-3,Bax and down-regulate Bcl-2,suppress the expression of angiogenesis related gene VEGF.Besides,there is a synergy effection on these factor between ING4 and P53 which expressed in the double gene adenovirus.Conclusion:1.The recombinant adenovirus modified by RGD-4C(Ad.RGD-P53 and Ad.RGDING4-P53) were constructed successfully.2.ING4 or/and P53 gene mediated by adenovirus could significantly inhibit the growth of A549,induce its apoptosis,produce G1 arrest and inhibit the migration and invasion ability of A549 cell.In addition,the inhibition effect of the double gene adenovirus which expressed ING4 and P53 was more obvious than the signal gene adenovirus.3.ING4 and P53 gene mediated by adenovirus could similarly inhibit the tumor growth of A549 human lung adenocarcinoma transplanted tumor in nude mice. Moreover,the double gene adenovirus has a higher degree inhibition effect on tumor suppression than the signal gene adenovirus.4.ING4 or/and P53 produced G1 arrest through up-regulating the expression of cell cycle related gene P21,induced A549 cell apoptosis through promoting the expression of apoptosis related gene Caspase-3,Bax,Bad and down-regulating the expression of Bcl-2 and Bcl-xl,inhibited the migration and invasion ability of A549 cell via suppressed the expression of MMP-2 and MMP-9.In addition,ING4 or/and P53 could inhibit angiogenesis through down-regulating the expression of angiogenesis related gene VEGF.Furthermore,the effect of these gene which influenced by the double gene adenovirus is more obvious than the signal gene adenovirus.Maybe there is a synergy effection on inhibition effect between ING4 and P53 which expressed in the double gene.