Expression And Roles Of Ski In Activated Astrocytes Of Rats

Objective Ski,as an evolutionary conserved protein,is a versatile transcriptional regulator which widely distributes in various tissues and species.Recently,we have demonstrated for the first time that Ski was strikingly up-regulated in reactive astrocytes after spinal cord injury(SCI)in vivo.In this study,we explore the change of Ski protein expression level in rat activated astrocytes and its role in astrocyte proliferation and migration.Methods The astrocytes were obtained from rats cerebral cortex and cultured in vitro.The astrocytes were treated with LPS and scratch injury for activation.Western blot analysis was used to determine GFAP,PCNA CDK4 and CyclinD1 protein levels in activated astrocytes at a series of time points.The indirect immunofluorescent staining method was performed to detect the location of Ski protein in the astrocytes.To further explore the role of Ski,Ski specific small interfering RNA(siRNA)was used.This section was divided into five groups: Ski-siRNA-1 group,Ski-siRNA-2 group,Ski-siRNA-3 group,negative control group and control group.Firstly,we used a fluorescein-conjugated siRNA consisting of a scrambled sequence to detect the efficiency of transfection.Next,the efficiency of Ski-siRNA was confirmed by Western blot analysis.The protein of GFAP,PCNA,CDK4 and CyclinD1 expression level were determined by Western blot analysis after transfection with Ski-siRNA-3.After transfection,the changes in proliferation of astrocyte were measured by CCK8 assay and EdU staining.The effect of Ski-siRNA-3 on the cell cycle of astrocytes was analyzed with flow cytometer.At last,the changes in migration of astrocytes after transfection with Ski-siRNA-3 were measured by wound scratch assay and Transwell cell migration assay.Results The proteins of GFAP and PCNA were natural expressed in the astrocytes,beginning to increase after treated with LPS and scratch injury.Little protein expression of Ski in the normal astrocytes was observed,and began to increase after treated with 1 ?g/mL LPS,and peaked at 4 d(P<0.05)and then deceased,but the Ski level was stills higher than the normal cells in sustainable times.Meanwhile,up-regulation of Ski was accompanied with the increase in proliferation-related proteins including PCNA,CDK4 and CyclinD1.The protein expression level of Ski in scratch injury was highly consistent with above mentioned.Furthermore,immunofluorescent staining analysis also demonstrated a highly positive relationship between Ski and GFAP,PCNA in astrocytes.These results indicated that Ski might play an important role in astrocyte proliferation.To further explore the role of Ski,astrocytes were transfected with Ski-siRNA.The transfection efficiency was more than 80% after 72 h,and Ski expression in astrocytes significantly decreased after transfection with Ski-siRNA-3 compared with other groups.CCK8 assay and EdU staining assay have demonstrated that the primary activated astrocytes' proliferation decreased significantly after transfection with Ski-specific siRNA.The results demonstrated that,compared to the control group and the negative control group,the cells in the G1 phase increased while the cells in the S phase deceased in the Ski-siRNA-3 group.Surprisingly,wound scratch assay and Transwell cell migration assay have demonstrated that Ski knockdown also weakened the primary astrocyte migration.Conclusion The protein expression of Ski is expressed in the astrocytes,and the expression level is increased in activated astrocytes mainly located in the nucelus.We had shown that Ski expression was positively associated with proliferation index of PCNA in reactive astrocyte for the first time,which suggested that Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.Moreover,Ski knockdown decreased astrocytes' proliferation and migration.It's a reminding that Ski might play an essential role in the process of astrocytes' activation,proliferation and migration.