Studies On The Correlation Between VRK1 And Glioma

Objective:This study was aimed to investigate the expression of VRK1 in glioma,and to analyze the relationship between the expression of VRX1 and the prognosis of glioma patients,and to explore how VRK1 plays a role in the development of glioma.Methods:1.Western blot was employed to detect the expression of VRK1 in normal brain tissues and glioma tissues2.Immunohistochemistry(IHC)was performed to determine the VRK1 and Ki-67 exprssion of 85 paraffin-embedded specimen.3.Pearson ?2 test was used to analyze the correlation between the expression of VRK1 and several clinicopathologic features.4.The relationship between VRK1 expression and the prognosis of glioma patients was analyzed by Kaplan-Meier survival curve and online database GEPIA.5.The expression of VRK1 in different glioma cell lines was examied by western blot,and the cell lines which highly expressed VRK1 were selected for further experiments.The small interfering RNA of VRK1 were transfected into U87 and U251 cells respectively,and the best small interfering RNA with low expression of VRK1 were screened out.6.U87 and U251 cells were transfected with control-siRNA or VRK1-siRNA#4 respectively,and the expression of PCNA and cyclin D1 was evaluated by western blot,the effect of VRK1 knockdown on cell cycle was detected by Flow cytometry,and the effect of VRK1 knockdown on cell proliferation was studied by CCK-8 and plate cloning assay.7.Scratch test and transwell test were used to detect the the effect of VRK1 knockdown on cell migration ability in U87 and U251 cells.Migration related proteins,including E-cadherin,?-catenin,N-cadherin and vimentin are detected by western blot in VRK1 knockdown U87 and U251 cells.8.After silencing the VRK1 expression,FCM was employed to check the apoptosis of U87 and U251 cells,and WB was used to analyze the expression of proteins related to apoptosis.Results:1.Western blot showed that VRK1 was overexpressed in glioma tissues,and the expression level was increased along with the increased malignant degree of glioma.2.IHC showed that the expression of VRK1 and Ki-67 were positively related to WHO grade.And pearson correlation coefficient method showed that the expression of VRK1 and Ki-67 had obvious correlation.3.There was no statistical correlation between VRK1 expression and other clinical pathological characteristics such as age,sex,tumor location,size of tumor.VRK1 expression is related to WHO grade.4.Kaplan-Meier survival curve analysis and online database GEPIA analysis revealed that patients with higher VRK1 expression had a poorer outcome.5.Western blot showed that VRK1 was expressed relatively higher in U87 and U251 cells than in other cells,and the interference efficiency of VRK1-siRNA#4 was the highest after the two cells transfected with small interference of VRK1.6.Control-siRNA or VRK1-siRNA#4 were transfected into U87 and U251 cells,western blot revealed that the expression of PCNA and cyclin D1 was down-regulated after knocking down VRK1 expression;the results of FCM showed that cell numbers increased in G1 phase and decreased in S phase;CCK8 and plate cloning assay showed that cell proliferation was inhibited after knocking down VRK1 expression.7.Scratch test and transwell test showed that the migration ability of U87 and U251 cells wre decreased after knocking down VRK1 expression.Western blot showed that silencing of VRK1 leads to the upregulation of E-cadherin and ?-catenin whereas down regulation of N-cadherin and vimentin in U87MG and U251 cells.8.FCM showed that Flow cytometry showed that knocking down the expression of VRK1 could promote cell apoptosis.Western blot results showed that silencing of VRK1 could promote the activation of caspase 3 and down-regulate the expression of Bcl-2.Conclusions:1.VRK1 was highly expressed in glioma tissues and cells,and its expression is correlated with WHO grade and Ki-67.And patients with high VRK1 expression had poor prognosis.2.VRK1 can regulate the proliferation,migration,and apoptosis of glioma cells,knocking down VRK1 would inhibit cell proliferation,migration,and promote cell apoptosis.