The Study For The Roles Of ITSN1-S In Glioma Cells Invasion And Apoptosis

This study includes two parts:The first Part:To explor the function of ITSN1-S in glioma cells migration and invasion and the possible molecular mechanisms.The second Part:To explor the function of ITSN1-S in glioma cells apoptosis and the possible molecular mechanisms.ITSN1(intersectin-1) is an evolutionarily conserved adaptor protein; recent research has indicated that ITSN1participates in both endocytosis and exocytosis, and regulates different intra-cellular signaling transduction pathways. ITSN1protein has two isoforms:ITSN1-L and ITSN1-S. Our previous report indicated that ITSN1-L was highly enriched in neurons; while ITSN1-S was mainly detected in astrocytes and microglia. These results suggest that the expressions of ITSN1-L and ITSN1-S are strictly regulated in different cell types, and their unique cellular distribution should correspond to their functions. In the present study, we focused on the ITSN1-S and tried to explore the unique function of this isoform in glioma cells invasion and apoptosis, and the possible related molecular mechanisms.Abstract of the first PartObjectives:To investigate the role of ITSN1-S in glioma cells migration and invasion, and illustrate the possible related mechanisms.Methods:Glioma cells LN-229and U87were transfected with ITSN1-S small RNA interference plasmids and transient transfection to disrupt ITSN1-S expression. To detect the ITSN1-S protein levels by Western blotting. To detect the migration of gliloma cells using scratch assay and chemotaxis assay. To examine the invasion ability of of gliloma cells using Matrigel invasion assay in vitro. To detect the adhesion ability of of gliloma cells with adhesion assay. F-actin content of glioma cells was analyzed with F-actin polymirazation assay. Western Blotting was used to explore the level of phosphorylated PAK/LIMK/cofilin, FAK/integrinβ1, Akt and the expression level of N-cadherin, β-catenin, MMP-2and MMP-9. To explore the transplanted tumors invasion ability of gliloma cells in vivo by animal xenograft model.Results:Western blotting results showed significant decrease of ITSN1-S protein levels in LN-229and U87after ITSN1-S siRNA transfection. The ITSN1-S down regulated glioma cells showed decreased chemotaxis ability compared with the control cells (P<0.05). When a scratch was created in the fluent monolayer cells, it took the ITSN1-S down regulated glioma cells a longer time to fill the gap (P<0.05). Adhesion assay showed the number of the adhering cells of ITSN1-S down regulated group was decreased at5and15minutes in adhesion assay compared with the control group (P<0.05). The invasion assay showed prominent differences between the ITSN1-S down regulated glioma cells and the control cells (P<0.05). In the ITSN1-S down regulated glioma cells, the actin polymerization in response was significantly reduced. Western blotting results showed that the level of phosphorated PAK/LIMK/cofilin, phosphorated FAK/integrinβ1and phosphorated Akt was inhibited in the ITSN1-S down regulated cells. The N-cadherin, β-catenin and MMP-9exspression in ITSN1-S down regulated glioma cells was lower than control cells. The in vivo invasion assay showed that the invasion frequency of ITSN1-S down regulated mice group was significant lower than the control group (p<0.05).Conclusions:ITSN1-S is required in glioma cells migration and invasion. ITSN1-S mediated the EGF-induced activation of PAK/LIMK/cofilin, which was involved in the actin polymerization; ITSN1-S mediated cell adhesion signal pathway directly through phosphoration of FAK/integrinβ1and the expression of N-cadherin/β-catenin; ITSN1-S mediated glioma cells invasion by regulating the expression of MMP-9; therefore ITSN1-S plays an important role in glioma cells migration and invasion.Abstract of the second PartObjectives:To explore the ITSN1-S function in glioma cells apoptosis and the possible molecular mechanisms.Methods:We used small interfering RNA technology to knock down ITSN1-S expression level in glioma cells LN-229. Growth curve were applied to detect proliferation; MTT assay, flow cytometry and TUNEL assay were performed to detect the glioma cells apoptosis. Western blotting was applied to detect the expression of apoptosis related proteins. Furthermore, we used Nu/Nu nude mice to set up xenograft animal model to explore the tumors growth in vivo.Results:Knocking down ITSN1-S inhibited LN-229cells growth (P<0.05). Results of MTT assay, flow cytometry and TUNEL assay suggested that glioma cells were induced to apoptosis after reduction of ITSN1-S expression (P<0.05). Animal experiments indicated that reduction of ITSN1-S inhibited tumor growth in vivo (P<0.05). Based on above results, we further detected the expression of apoptosis related proteins in ITSN1-S knocking-down cells. We found that the expression leve of Bcl-2was down-regulated; The expression leve of Bad protein and Cytochrome C in cytoplasm were up-regulated.Conclusion:Reduction of ITSN1-S expression in glioma cells inhibited cell growth and induced cell apoptosis. Mitochondrial signaling pathways may be involved in ITSN1-S regulating apoptosis in glioma cells.