| ObjectiveIn this study,the expression,correlation of TAp73α/TAp73β and DLC2 in glioma specimens,glioma cell lines were detected to explore the mechanism by which DCL2 participates the pathology of glioma.Methods1.WB(Western Blot)was employed to detecte the expression of TAp73α and TAp73β in different grades of glioma samples and glioma cell lines(A172,T98 G,U251,Shg44),and verified the relation between their their expression and glioma grades.2.After overexpressing the expression of TAp73β in U251 and Shg44 cells,flow cytometry and WB were employed to detect cell apoptosis and the expression of apoptosis-related proteins,like Caspase3 and Bax.After overexpressing TAp73α and TAp73β in A172 and T98G cells,clonogenic assay was used to evaluate their influence on cell colony formation,WB was used to detect the expression of Caspase3 and Bax.3.WB and q PCR were used to detect the expression of DLC2 in protein and m RNA level in different grades of glioma samples and normal brain tissues.The expression of DLC2 protein and m RNA in different grades of gliomas were analyzed based on REMBRANDT and TCGA datasets.At the same time,the correlation between DLC2 and TAp73α,TAp73β TAp73α/TAp73β ratio were analyzed.4.WB was employed to detecte the expression of DLC2 in glioma cell line(A172,T98 G,U251,Shg44).After over-expressing DLC2 in U251 and Shg44 cells,flow cytometry was used to detect the change of cell apoptosis.U251 and Shg44 cells which over-expressing DLC2 were injected subcutaneously into nude mice to construct a glioma ectopic tumor model.The growth of tumors in the control group and DLC2 overexpression group were compared.After knocking down the expression of DLC2 in A172 and T98 G cells,cell colony formation was detected by clonogenic assay.5.The expression of Tap73α,TAp73β,Caspase3 and BAX were detected by WB in U251 and Shg44 cells which overexpressing DLC2.After knocking down the expression of DLC2,the expression of TAp73α and TAp73β protein and the change of TAp73α m RNA in A172 and T98G cells were detected by WB and q PCR.U251 and Shg44 cells were treated with MG132 and different amounts of Myc-DLC2(0,0.5,1.0,1.5 μg),then the ubiquitination degradation of endogenous TAp73α by DLC2 was detected by IP.MG132,His-Ub,Flag-TAp73α and different amounts of Myc-DLC2 were transfected into 293 T cells,then IP was employed to detect the ubiquitination degradation of exogenous TAp73α by DLC2.6.Myc-DLC2 was transfected into U251 and Shg44 cells,then we employed IP to detect the interaction between DLC2 and endogenous TAp73α,TAp73β.Then Myc-DLC2,Flag-TAp73α,His-TAp73β were transfected into 293 T cells respectively or jointly,IP was used to detect the Binding of DLC2 and exogenous TAp73α,TAp73β.After transfecting 293 T cells with Myc-DLC2,Myc-DLC2-d SAM and TAp73α,IP was used to detect the interaction between them.After treating with MG132,Myc-DLC2 and Myc-DLC2-d SAM,IP was employed in U251 and Shg44 cells to detect the effect of DLC2 and DLC2 mutant on ubiquitination of endogenous TAp73α.293 T cells were transfected with MG132,His-Ub,Flag-TAp73α and Myc-DLC2 or Myc-DLC2-d SAM,IP was used to detect the ubiquitination degradation of DLC2 and DLC2-d SAM to exogenous TAp73α.7.U251 and Shg44 which overexpress DLC2-d SAM or DLC2 were injected subcutaneously into nude mice to construct a glioma ectopic tumor model,the growth of tumors was compared.At the same time,WB was used to detect the protein expression levels of Caspase3 and Bax in tumor tissues derived from the ectopic tumor model.TUNEL was used to detect the apoptosis of glioma cells in tumor tissues.Results1.WB showed that the expression of TAp73α in glioma tissues was significantly higher than that in normal brain tissues,but there was no significant change in TAp73β protein levels.The expression of TAp73α protein in U251 and Shg44 was higher than in A172 and T98G cells.Experiments confirmed that U251 and Shg44 cells formed more colonies than A172 and T98 G cells.2.Flow cytometry confirmed that overexpression of TAp73β promoted U251 and Shg44 cell apoptosis,and apoptosis-related proteins such as Caspase3 and Bax were also increased.While,overexpression of TAp73β in A172 and T98 G inhibited cell proliferation and promoted the expression of Caspase3 and Bax.But,overexpression of TAp73α in A172 and T98 G can promote cell proliferation and inhibit the expression of Caspase3 and Bax.3.The expression of DLC2 protein and m RNA decreased in glioma tissues than in normal brain tissues,and it was negatively correlated with TAp73α expression and the ratio of TAp73α/TAp73β(p<0.001),but had no correlation with TAp73β expression(p=0.442).4.WB suggested that the expression of DLC2 was higher in U172 and T98G cells than in U251 and Shg44 cells.Overexpression of DLC2 in U251 and Shg44 inhibited cell apoptosis and tumor growth.knockdown of A172 and T98 G in DLC2 promoted cells clonal formation.5.Overexpression of DLC2 in U251 and Shg44 can inhibit the expression of TAp73α protein,but there is no significant change in TAp73α m RNA expression.The expression of caspase3 and Bax were also raised,while the expression of TAp73β had no change.DLC2 inhibited the ubiquitination of endogenous and exogenous TAp73α in glioma cells 23 T cells.6.After transfecting Myc-DLC2 into U251 and Shg44,IP can detect co-precipitation of DLC2 and endogenous TAp73α,while DLC2 and TAp73β are not co-precipitated.In 293 T cells which transfected with Myc-DLC2 and Flag-TAp73α or His-TAp73β,IP detected DLC2 could co-precipitate with exogenous TAp73α but not TAp73β.However,DLC2 with SAM domain deleted could not co-precipitate with TAp73α,the function on ubiquitination degradation of TAp73α also attenuated.7.After constructing nude mouse ectopic glioma model with U251 and Shg44 cells which had been transfected with DLC2-d SAM or DLC2,we found that the weight and volume of DLC2 overexpression group were larger than that of mutant transfection group.WB showed that the expression of Caspase3 and Bax in the tumor tissue of the mutant group were lower than DLC2 group.TUNEL suggested that cell apoptosis in mutant group was also less.ConclusionsThe expression of DLC2 is relatively lower,while TAp73α is higher in glioma tissues than in normal brain tissues.The expression of them were negatively correlated both in glioma tissues and glioma cells.DLC2 could interact with TAp73α through the SAM region and regulate its ubiquitination degradation,by which the downstream target genes of TP73 were modulated.Therefore,DLC2 plays a role of tumor suppressor in glioma. |
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