An Actin Binding Protein Girdin In Migration And Invasion Of Human Glioblastoma Cells

Objectives:Girdin was first discovered by Japanese researchers in 2005 which is involved in the remodeling of the actin cytoskyeleton.As we all known,cell migration is not only play an important role in physiological functions,but also the key point of pathological processes such as tumorigenesis and development.Girdin is an actin binding protein that plays an essential role modulates key signaling pathways during a diverse set of biological processes,e.g.cell motility,tumor angiogenesis,vascular repair,and cell autophagy.To investigate the function of Girdin on glioblastoma cells provides insights into molecular mechanisms of cell proliferation and motility,which represents an active target site for therapeutic purpose.Methods:Part one:1.We construct the recombinant lentivirus vectors with Girdin.Lentiviruses were produced by transfection into the retroviral packaging HEK-293T cell line.2.Glioma cells LN229 were infected with Girdin lentiviruses to enhance Girdin expression,which was identified by Western Blotting analysis.3.Growth curve assay were applied to detect the effect of Girdin on glioma cells proliferation.4.Using scratch assay to detect the influence of Girdin up-regulation on migration of glioma cells.5.Adhesion assay detected the influence of Girdin Girdin up-regulation on the adhesion of glioma cells.6.The invasion ability of gliloma cells was examined using matrigel invasion assay in vitro.Part two:1.Glioma cells LN229 and U87 were respecti-vely transfected with Girdin small RNA interference plasmids and transient transfection to disrupt Girdin expression which was identified by Western Blotting analysis.2.Using scratch assay,chemotaxis assay and chemokinesis assay to detect the influence of Girdin down-regulation on migration of glioma cells.3.F-actin polymerization assay to explain the role of Girdin down-regulation in actin polymerization stimulated by EGF in different time.4.Adhesion assay detected the influence of down-regulated Girdin on the adhesion of glioma cells.5.Using Western Blotting assay to detected the expression of phospho-integrin ?1,phospho-FAK,MMP-2 and MMP-9 in Girdin down-regulation glioma cells and control glioma cells.6.We used the invasion in vitro assay to validate the role of Girdin in glioblastoma invasion.Results:Part one:1.Although the expression level of Girdin was increased,no significant associations were identified between the proliferation ability of Girdin overexpression cells and the control cells.2.The scratch assay showed that the migration ability of Girdin overexpression LN229 cells was increased compared with the control cells(P<0.05).3.In the adhesion assay,the number of adherent cells in Girdin overexpression cells is significantly higher than that in control cells(P<0.05).4.Invasion assay showed the ability of invison is significantly increased after Girdin overexpression in LN229 cells(P<0.01).Part two:1.After Girdin was down-regulated in LN229 cells,migration ability and chemotaxis ability was impaired compared with control cells(P<0.05).2.In the Girdin down-regulated LN229 cells,the ability of actin polymerization was significantly impaired.(P<0.05).3.In the Girdin down regulated LN229 cells,the number of adherent cells was significantly decreased and the expression of phospho-integrin ?1 and phospho-FAK were down-regulated compared with control cells(P<0.05).4.The invasion assay in vitro demonstrated that the invasion ability of Girdin-reduction cells was significantly impaired,the expression level of MMP-2 and MMP-9 were also decreased compared with control cells(P<0.05).Conclusions:1.Girdin participated in EGF-induced glioma cells migration by regulating the cytoskeletal rearrangement.2.Girdin accommodated the activation of integrin ?1 and FAK to influence the adhesion ability in glioblastoma cells.3.Girdin mediated the glioma cells invasion by regulating the expression of MMP-2 and MMP-9.