Function Of Girdin In Migration And Apoptosis Of Glioblastoma Cells

ObjectivesGirdin was first discovered by Japanese researchers in 2005.The phosphorylated Girdin plays an essential role in both the remodeling of the actin cytoskeleton and tumor progression including migration,invasion,metastasis and angiogenesis.In our study,we used the siRNA technology to knock down the expression of Girdin,and then to investigate the function of Girdin down-regulation in migration,invasion,apoptosis,and the related molecular mechanisms.MethodsPart one:1.Glioma cells LN229 and U87 were respectively 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 β1,phospho-FAK,MMP-2 and MMP-9 in glioma cells and control glioma cells.6.We used the invasion in vitro assay and subcutaneous mouse xenograft model in vivo to validate the role of Girdin in glioblastoma invasion.Part two:1.We used small interfering RNA technology to knock down Girdin expression level in glioma cells LN229.U87 cells were transfected with small interfering RNA duplex oligoribonucleotide to disrupt Girdin expression.2.Proliferation assay and MTT assay were applied to detect the effect of Girdin-reduction on LN229 cells proliferation.3.DAPI staining,flow cytometry and AnnexinV/PE staining assay were used to analyze the function of Girdin-reduction in cells apoptosis.4.Western blotting was applied to detect the expression of apoptosis related proteins cytochrome C and Bad.5.We used Nu/Nu nude mice to set up xenograft animal model to explore the role of Girdin in glioblastoma tumors growth in vivo.6.Immunohistochemistry staining was used to analyze the expression of Cyt-C and Bad.ResultsPart one:1.After transfected LN229 cells and U87 cells by small interference RNA,the expression of Girdin significantly decreased.2.Girdin-reduced glioma cells showed decreased chemotaxis ability compared with control cells(P<0.05).3.In the Girdin down regulated glioma cells,the actin polymerization in response was significantly reduced.(P<0.05).4.The adherent Girdin-reduced cells was significantly decreased and the expression of phospho-integrin β1 and phospho-FAK were down regulated compared with control cells(P<0.05).5.The invasion assay in vitro demonstrated that the invasion ability of Girdin-reduced cells was significantly reduced,the expression of MMP-2 and MMP-9 were also down regulated compared with control cells(P<0.05).6.The in vivo further demonstrated that the invasion frequency of Girdin down regulated mice group was significant lower than the control group(P<0.05).Part two:1.Compared with the control cells,Girdin-reduction impaired the proliferation in LN229 cells(P<0.05).2.The results of DAPI staining assay,flow cytometry and AnnexinV/PE staining assay showed that the experssion of Girdin-reduction induced the apoptosis ability in LN229 and U87 cells.3.Compared with the control cells,the expression of Cyt-C and Bad were increased in siGirdin/LN229 cells by Western blotting analysis.4.Animal experiments indicated that reduction of Girdin inhibited tumor growth in vivo(P<0.05).5.Immunohistochemistry staining showed that the content of Cyt-C and Bad in cytoplasm were up-regulated in siGirdin/LN229 group,compared with the control cells(P<0.05).Conclusions1.Girdin participated in EGF-induced glioma cells migration by regulating the cytoskeletal rearrangement.2.Girdin accommodated the activation of integrin β1 and FAK to influent the adhesion ability in glioblastoma cells.3.Girdin mediated the glioma cells invasion by regulating the expression of MMP-2 and MMP-9.4.Reduction of Girdin in glioma cells inhibited cell growth and induced cell apoptosis through regulating the activation of Bad and Cyt-C.