Construction Of DUSP10 Knockdown T98G Cell Line And Its Effect On Cell Function

Objective:In the brain glioma is one of the most common malignant tumor in the nervous system,the traditional treatment is given priority to with surgery and radiation and chemotherapy,but poor treatment effect,easy to relapse,the median surial in patients with short,especially the high level of invasive glioblastoma,treatment has been no breakthrough,to solve this problem,research the pathogenesis of glioma is of great significance.Bispecific phosphatase 10(DUSP10),also known as mitogen-activated protein kinase phosphatase 5(MKP-5),belongs to silk/threonine and tyrosine bispecific phosphatase,and is a member of the DUSP family.It is often mediated by dephosphorylation of mitogen-activated protein kinase(MAPK)to regulate cell proliferation,differentiation,cell survival and cell death,which is closely related to the occurrence and development of tumors.The purpose of this study was to explore the difference in the expression of DUSP10 in glioma and normal brain glioma cells,and to analyze the effects of DUSP10 on the proliferation,migration and apoptosis of glioma cells T98G by silencing the expression of DUSP10 in glioma cells by lentivirus.Methods:1.Based on relevant bioinformatics knowledge,the expression difference of DUSP10 in glioma cells and normal cells was preliminarily explored,and the correlation between DUSP 10 and glioma was studied by using the survival curve analysis of TCGA and CGGA database.2.The expression level of HAC/T98G/U251/U87 cells was detected by RT-Q PCR and Western blot to verify the difference in the expression level of DUSP 10 in glioma cells from the level of gene and protein.3.A plasmid vector PLKO.1 with the function of silencing DUSP10 was constructed by means of enzyme digestion,ligation,transformation and extraction,etc.Lentivirus transduction technology was used to interfere with DUSP10 RNA of T98G cell line.and fluorescent positive control group was set to verify virus packaging and transfection.T98G cell lines stably-transfected with lentivirus were screened with purinomycin.RT-Q PCR and Western blot were used to verify the knockdown effect of T98G cells after interference from gene and protein levels,respectively.4.Glioma cells T98G were divided into shRNA-blank group,shRNA-NC group,shRNA-DUSP10-1 group and shRNA-DUSP10-2 group.Cell Counting Kit 8(CCK-8)reagent detection method,Transwell chamber migration detection,flow cytometry detection and other experimental technologies were used to detect the biological functions of the above groups of cells,and the effects of DUSP10 on proliferation,migration and apoptosis of glioma cells T98G were analyzed.5.SPSS 18.0 and Graph Pad Prism7 software were used for statistical analysis and mapping,and P<0.05 indicated that the results were statistically significant.Results:The expression level of DUSP10 was different in different glioma WHO classification.With the increase of WHO classification,the expression level of DUSP10 was also increased(P<0.05).In survival analysis of patients,compared with the low expression of DUSP10,the high expression of DUSP10 showed a trend of low survival rate(P<0.05).T98G cells were transfected with lentivirus,and then the cells were screened by purinomycin and the fluorescence control group.The results showed that the cells were successfully constructed.Real-timePCR and Western blot analysis showed that the expression of DUSP10 in shRNA-DUSP10-1 group and shRNA-DUSP10-2 group was decreased compared with shRNA-blank group and shRNA-NC group(P<0.05).The results showed that the effect of RNA interference was successful.CCK8 assay was used to detect and plot the growth curve of cells in each group.It was found that the proliferation activity of T98G decreased significantly in the second and third days after silencing DUSP10(P<0.05).Transwell method was used to detect the migration ability of T98G in different groups,and we found that shRNA-blank group and shRNA-NC group,shRN A-DUSP10-1 group and shRNA-DUSP10-2 group all had significant transmitability through the ventricular cells.There were 64±4,61±5,21±4 and 19±4 cells per field in each group,respectively.Compared with the blank control group and the negative control group,the number of migrated cells in the two interference groups of DUSP10 was significantly decreased.with statistical significance(P<0.01).Flow cytometry was used to detect the apoptosis rate.The late apoptotic rate of shRNA-Blank group,shRNA-NC group and shRNA-DUSP10-1 group were(4.79±0.68)%,(5.13±1.03)%and(8.94±0.7)%,(8.98±0.85)%,respectively.The rate of late apoptosis in DUSP10 interference group was significantly higher than that in blank group and negative control group,and the difference was statistically significant(P<0.05).Conclusion:The expression of DUSP10 varies among patients at all levels of classification,which increases with the increase of WHO classification and is closely related to the survival rate of patients.The higher the expression level of DUSP10,the lower the survival rate.Through cell test,we can conclude that the expression of DUSP10 in glioma cells is higher than that in normal brain glioma cells.Silencing DUSP10 can inhibit the proliferation and migration ability of glioma T98G cells,and induce cell apoptosis.